Potassium Channels And Their Modulators From Synthesis To Clinical Experience

Potassium Channels and their Modulators: From Synthesis to Clinical Experience

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Potassium Channels and their Modulators: From Synthesis to Clinical Experience Edited by J.M.EVANS T.C.HAMILTON S.D.LONGMAN & G.STEMP SmithKline Beecham Pharmaceuticals

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Contents

Contributors Foreword Preface

viii x xiii

1

The Synthesis and Chemistry of Benzopyran Related Potassium Channel Activators D.G.SMITH

1

2

Structure-Activity Relationships of Benzopyran Based Potassium Channel Activators J.M.EVANS & G.STEMP

32

3

Syntheses and Structure-Activity Relationships of Pyridine Based Potassium Channel Activators M.N.PALFREYMAN

66

4

Conformational Analysis of Potassium Channel Activators C.M.EDGE

91

5

The Structure-Activity Relationships of Potassium Channel Blockers R.CROSSLEY & A.OPALKO

128

6

Potassium Channels: Diversity, Assembly and Differential Expression R.LATORRE & P.LABARCA

156

7

Potassium Channel Electrophysiology in Vascular Smooth Muscle Cells and the Site of Action of Potassium Channel Openers P.I.AARONSON & C.D.BENHAM

194

8

Effects of Potassium Channel Activators in Isolated Blood Vessels U.QUAST

214

9

In Vivo Vascular Effects of Potassium Channel Activators J.C.CLAPHAM

242

vii

10

Cardiac Potassium Channel Modulators: Potential for Antiarrhythmic Therapy M.C.SANGUINETTI & J.J.SALATA

273

11

Cardioprotective Properties of Potassium Channel Modulators G.J.GROSS

316

12

Potassium Channel Activators: Airway Pharmacology and Bronchial Asthma J.R.S.ARCH

336

13

Potassium Channels in Pancreatic β -Cells: Modulation, Pharmacology and their Role in the Regulation of Insulin Secretion M.J.DUNNE, J.H.JAGGAR, E.A.HARDING, C.KANE & P.E.SQUIRES

369

14

Potassium Channels and their Modulation in Urogenital Tract Smooth Muscles A.F.BRADING & W H.TURNER

406

15

Potassium Channel Modulators and the Central Nervous System H.HERDON

434

16

Potassium Channel Modulators: Clinical Experiences and Future Prospects T.J.COLATSKY & T.C.HAMILTON

460

Abbreviations

494

Index of Compounds

499

Index

503

Contributors

P.I.AARONSON, Department of Pharmacology and Medicine, UMDS Guy’s and St. Thomas’s Hospitals, Lambeth Palace Road, London SE1 7EH, UK. J.R.S.ARCH, Department of Cellular Biochemistry, SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Hertfordshire, AL6 9AR, UK. C.D.BENHAM, Department of Biophysical Sciences, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. A.F.BRADING, Department of Pharmacology, Oxford University, Mansfield Road, Oxford, OX1 3QT, UK. J.C.CLAPHAM, Department of Vascular Biology, SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Hertfordshire, AL6 9AR, UK. T.J.COLATSKY, Division of Cardiovascular and Metabolic Diseases, WyethAyerst Research, CN 8000, Princeton, New Jersey 08543, USA. R.CROSSLEY, Wyeth Research UK, Huntercombe Lane South, Taplow, Maidenhead, Berkshire, SL6 0PH, UK. M.J.DUNNE, Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. C.M.EDGE, Computational Chemistry, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. J.M.EVANS, Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. G.J.GROSS, Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA. T.C.HAMILTON, Department of Neurology, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. E.A.HARDING, Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.

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H.HERDON, Psychiatry Research Department, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. J.H.JAGGAR, Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. C.KANE, Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. P.LABARCA, Centro de Estudios Científicos de Santiago y Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 16443 Santiago 9, Chile. R.LATORRE, Centro de Estudios Científicos de Santiago y Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 16443 Santiago 9, Chile. A.OPALKO, Wyeth Research UK, Huntercombe Lane South, Taplow, Maidenhead, Berkshire, SL6 0PH, UK. M.N.PALFREYMAN, Rhône-Poulenc Rorer, Dagenham Research Centre, Rainham Road South, Dagenham, Essex, RM10 7XS, UK. U.QUAST, Department of Pharmacology, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany. J.J.SALATA, Department of Pharmacology, Merck Research Laboratories, West Point, PA, USA. M.C.SANGUINETTI, Division of Cardiology, University of Utah, Salt Lake City, UT, USA. D.G.SMITH, SmithKline Beecham Pharmaceuticals, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey, KT18 5XQ, UK. P.E.SQUIRES, Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. G.STEMP, Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. W.H.TURNER, Department of Urology, Inselspital, Bern, Switzerland.

Foreword

Ion channels have come of age. Channels have been classified, sequenced and reconstituted and recognized as one of the major classes of biological effectors and Nobel prizes have been awarded. Ion channels have gone from being a molecular black box in the transduction machinery of the cell to being an increasingly well characterized and defined piece of molecular machinery. Ion channels are, in fact, a class of pharmacological receptor. As such we expect them to have the following general properties: 1 Possess binding sites for activator and antagonist drugs with defined structure-activity relationships. 2 These drug binding sites should be coupled to the functional machinery of the channel. 3 They may be linked to members of the G protein family. 4 They will be subject to up-and down-regulation by homologous and heterologous influences. 5 Their numbers and function will be altered in disease states, both experimental and clinical. As this book illustrates these expectations have been fulfilled for K channels. That ion channels are important to the health and welfare of the cell is scarcely surprising. One of the earliest decisions that the first-formed cells had to make was that of selective transport across the cell membrane. The cell membrane was the critical stage in the evolution of the cell as machine, but if all that it achieved was to keep the inside in and the outside out it would have functioned only in a reactionary manner. The issues of transmembrane transport by the cell were solved in several ways—passive diffusion, facilitated diffusion, active transport and ion channels. It is often convenient to consider these pathways as totally distinct, but they are not. They all have the same passengers, although the fare for transport may be paid in different currencies. The underlying molecular motifs of the proteins that comprise these transport systems have common components that contribute to the determination of ion selectivity and transport rates. Some classes of device, such as the cystic fibrosis

xi

transmembrane regulator, share functional and structural properties of both ion channels and ATP-dependent transporters. Ion channels may be conveniently classified by several distinct processes. They can be classified as ligand—and voltage—gated according to their primary mode of stimulus. They can be classified according to their electrophysiological properties-the kinetics of activation and inactivation, their conductance, large or small, and by their pharmacological sensitivity to toxins and synthetic drugs. Increasingly, they are classified according to their sequences—a process that has demonstrated that ion channels exist as ‘super—families’ with considerable structural homology between members, despite very different electrophysiological and pharmacological properties. Finally, of course, ion channels can be classified according to the ions to which they are selectively and remarkably permeable—Cl−, Na+, Ca2+ and K+. This book focuses on K channels. This class of ion—selective channels has been the subject of increasing electrophysiological, pharmacological, molecular biological and therapeutic attention. This is appropriate because K channels are particularly important regulators of cell responsiveness in both electrically excitable and non—excitable cells and they may represent ancestral ion channels. This importance was indicated in the early proposal by Bernstein in 1902 that cellular excitability arose from the selective permeability of cell membranes to K+ ions and their modulation. From the pharmacological and therapeutic perspectives progress in a field depends critically upon the type and quality of the investigational tools available. K channels were hampered, relative to Na and Ca channels, by the general unavailability of potent, selective small molecule ligands. Rather, the pharmacology of K channels was dominated by exotic toxins, frequently of limited and expensive availability and purity, and small molecule ligands of the quaternary ammonium ion class that were neither potent nor selective. This situation should be compared with that for Na and Ca channels where the availability of potent and selective small molecule synthetic ligands generated major therapeutic drug categories. This picture is now changing very rapidly for K channels, particularly those of the KATP class. The hypoglycemic sulfonylureas are already widely employed in the therapy of type II diabetes and the radioligand [3H] –glibenclamide is a popular molecular tool. The corresponding K channel activators, including the prototypical benzopyran cromakalim, offer wide therapeutic possibilities for cardiovascular, noncardiovascular and central nervous system disorders. They offer, in fact, the therapeutic possibility of being complementary to the available Ca channel antagonists. These possibilities are strengthened by the recent cloning of this channel. With the increasing molecular dissection of the ion channels contributing to the cardiac action potential there is continuing interest in both activators and antagonists of the K+ currents as antiarrhythmic agents. Given the continued widespread occurrence of ‘sudden cardiac death’, continued focus in this area can only be welcome. A major, although still potential, therapeutic

xii

target for K channel modulators is in the central nervous system in disorders including neurodegenerative diseases and ischemia. No single volume of manageable size could claim to offer a comprehensive treatment of K channels. This book is no exception. It does, however, manage to embrace a remarkably broad range of topics from chemistry to structure— activity relationships and molecular modelling to molecular cloning through smooth, cardiac and neuronal physiology and pharmacology. This broad coverage reflects, in fact, the current intellectual and practical excitement in the field of K channel modulators. Long may this excitement continue. David J.Triggle, School of Pharmacy, State University of New York at Buffalo.

Preface

When two of us published an article in Chemistry in Britain describing the discovery of cromakalim, the first compound to be shown to exert its pharmacological action purely by K channel activation, we received an invitation from Ellis Horwood OBE to use the article as a basis for a book. From this initial approach came the opportunity to produce the series of articles that comprise this book, covering the theme of K channels and their modulators. In compiling this work we have drawn on the pool of experience built up within SmithKline Beecham, the pharmaceutical company that pioneered K channel activators, and have augmented it with contributions from internationally known experts in the field of structure and properties of K channels and K channel modulators. It is clear that the K channel story did not start with the fundamental discovery of the mode of action of cromakalim by Hamilton, Weston and Weir in 1986, as nicorandil was already known to possess this mode of action in addition to its propensity to activate guanylate cyclase. However, it must be said that the discovery of cromakalim’s single mechanism of action in vascular smooth muscle acted as a spur to the generation of several series of benzopyran analogues and led to the discovery that many existing compounds, such as pinacidil and RP 49356, were forerunners of extensive families of K channel activators. In addition the availability of cromakalim initiated many novel biological studies involving K channels. Perhaps one of the most important discoveries was that by workers at Beecham, Rhône Poulenc and Sandoz who demonstrated that the channel modulated by cromakalim was blocked by glibenclamide (glyburide), a sulphonylurea drug known to block ATP-sensitive K channels in pancreatic β cells. This finding brought together the more mature field of K channel blockers, where drugs are already in the healthcare market, and the new and burgeoning field of K channel activators. Such discoveries served to confound the sceptics who thought little of the ideas underpinning the mechanism of the activators, and who merely dismissed the class as just another series of vasodilators. As K channels are found in virtually every body tissue, it was to be expected that compounds that are selective for the differing tissues would emerge. This

xiv

has happened to some extent in that K channel blockers are used clinically as antiarrhythmic and antidiabetic agents. As for the activators, we are only at the start of the search for selective agents to attenuate the disease states that are associated with different tissues. Besides hypertension, the (patent) literature indicates that activators are potentially useful in the treatment of angina, myocardial ischaemia, asthma, urinary incontinence and CNS disorders such as epilepsy. The search for K channel opening selectivity encompasses several approaches. First, there is the structural modification of known compound series, for example that of the cromakalim series has produced the in vivo airways selective compound BRL 55834, the cardioselective compound BMS-180448 and the CNS active benzamides. Second, the search for different structures that modulate ATPsensitive channels in specific tissues, for example the recent ZD-6169 that is selective for the bladder in vivo. Third, there is the search for novel structures that modulate K channels other than the ATP-sensitive K channel, exemplified by NS 004 that is reported to modulate the big calcium-activated K channel and to be of use in CNS disorders. Such investigations involve many different groups of scientists including medicinal chemists, molecular modellers, pharmacologists and electrophysiologists. Each group is dependent upon the contribution of the others for data on the K channel targets that they are seeking to modulate. The central chapters in this book attempt to show how such data are generated and using the ligands described by the chemists in the early chapters, the results that are obtained for individual tissues and how they are interpreted. The importance of molecular biology in the structural determination of the plethora of K channels illustrates the widening range of techniques that are being utilised in K channel studies. Finally, building on the results of such studies, there is the experience in the clinic of the marketed K channel blockers and the potentially clinically useful activators. In conclusion we must thank the contributors to this book for their dedication in not only producing succinct reports of the areas in which they have built up their expertise but also their major efforts in unravelling the K channel story. In the future much will depend on their present and future studies and those of the other investigators responsible for the work described herein. That may lead us to the discovery of tissue selective K channel modulator molecules with the exciting potential for attenuating a variety of disease states. The opportunity has been taken to keep the information in the text as up-todate as posssible by the inclusion of the most recent literature references where applicable.

1 The Synthesis and Chemistry of Benzopyran Related Potassium Channel Activators D.G.SMITH SmithKline Beecham Pharmaceuticals, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey, KT18 5XQ, UK. 1.1 Introduction Since the disclosure of antihypertensive activity in a series of 4-amido benzopyran-3-ols (Ashwood et al., 1986), and the emergence of cromakalim (CRK 1) as the prototype (Hamilton et al., 1986) of a new class of smooth muscle relaxant offering therapeutic potential (Longman and Hamilton, 1992), the literature in the potassium channel activator (KCA) area has increased dramatically.

Not only has the scientific literature grown over the last few years but from the patent literature it is evident that a large number of organisations have been, or are still, involved in the search for similar entities, and numerous compounds based on the CRK lead have undergone further development. Thus compounds where the aromatic ring has been variously substituted or replaced by other aromatic moieties (pyranopyridines, thienopyrans) have been investigated. Similarly benzothiapyran, indane, benzoxepine, tetralin (tetralone), tetrahydroquinoline and benzoxazine analogues of the benzopyran have been explored and have given rise to active derivatives. Positional substitution of the non-aromatic ring has shown that the C-2 and C-3 positions of CRK are relatively intolerant to structural change whereas C-4 is remarkably flexible in the type of substituent it can accommodate; the cyclic lactam being just one example of a multitude of both cyclic and acyclic amide moieties, or their equivalents, which confer KCA activity. Details of structure-activity

2 K CHANNELS AND THEIR MODULATORS

relationships (SAR) brought about by such changes have been reviewed recently (Evans et al., 1992; Buckle and Smith, 1993) and have been updated in Chapter 2 of this book. The intention of this chapter is to bring together the disparate compound types which can be collectively regarded as analogues of the benzopyran series of KCAs, and to review their preparation and salient features of their chemistry which have been developed over the last few years. The review covers the chemistry, medicinal chemistry and significant patent literature from the time of the first chemical paper relating to CRK (Ashwood et al., 1986) until the end of 1993. 1.2 General Synthetic Aspects—Racemic Derivatives The standard route to 4-amido-3, 4-dihydro-2H-1-benzopyran-3-ols 6 is shown in generalised form in Figure 1.1, regiospecific, trans orientation of amide and hydroxyl functionalities usually being assured through ring opening of the precursor epoxide 5 by amide nucleophiles at C-4 (Ashwood et al., 1986, 1990). The reaction is usually carried out on the epoxide itself or, in those cases where the epoxide is either relatively unstable or is difficult to form directly, in situ generation from the precursor bromohydrin 7 is favoured (Ashwood et al., 1986). With relatively high temperatures, long reaction times and/or excess base further reaction can occur resulting in dehydration to the corresponding benzopyran 8. This can also be formed by base-induced elimination of the mesylate 9 (Buckle et al., 1990). Direct formation of the epoxide from the benzopyran 4 is usually straightforward using a peracid, but in those cases where the epoxide is unstable to the acidic conditions successful isolation has been achieved by inclusion of a peracid-KF complex (Houge-Frydrych, C.S.V., personal communication).

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 3

Figure 1.1 General synthetic approaches to 4-amidobenzopyrans

The commonly used synthesis of benzopyrans 4 involves condensation of an appropriately substituted phenol 2 with 3-chloro-3-methylbutyne followed by Claisen rearrangement of the aryl propargyl ether 3, the reaction being facilitated by the presence of the gem dimethyl group (Harfenist and Thom, 1972). (Indeed attempted use of the reaction to prepare the benzopyran precursor to the C-2 nordimethyl analogue of CRK was not satisfactory (Buckle et al., 1991c).) The rearrangements are usually carried out in high boiling solvents such as 1, 2dichlorobenzene or N, N-diethylaniline at reflux and result in good yields of the desired benzopyran 4. The generally accepted mechanism for this reaction (Rhoads and Raulins, 1975) is a 1, 5 sigmatropic shift followed by electrocyclic rearrangement of the initially formed allene intermediate 10 and usually occurs without formation of benzofuran products. However, thermal cyclisation of the pyridyl acetylene 11 gave a concentration dependent mixture of the benzopyran 12 and benzofuran 13, higher concentration and base catalysis favouring formation of the benzofuran (Attwood et al., 1991b).

4 K CHANNELS AND THEIR MODULATORS

The Claisen rearrangement, although generally successful for the preparation of 6-substituted benzopyrans, is not particularly suitable for synthesis of the corresponding 7-substituted isomers. Thus for the 3-methoxy- (Anderson and LaVoie, 1973), 3-cyano-, 3-nitro- (Evans et al., 1983) and 3-trifluoromethyl (Buckle et al., 1990) phenylpropargyl ethers mixtures of 5- and 7-isomers are obtained with the 5-isomer 14 predominating. To overcome this problem a regioselective route to the 7-trifluoromethyl derivative 15 (R=CF3) was developed (Buckle et al., 1990) whereby 4-bromo-3-fluorobenzotrifluoride was regiospecifically coupled with 2-methyl-3-butyne-2-ol under Heck conditions and the derived acetylene partially hydrogenated to the cis olefin 16 which was cyclised to the desired derivative. This route has proved satisfactory for the preparation of 2, 2-trifluoromethylbenzopyrans whose synthesis is problematic via the standard route (Fenwick, 1993).

An alternative to the propargyl ether cyclisation which has been utilised frequently to provide benzopyran intermediates is the condensation of an acetophenone with a ketone (Kabbe, 1978). Thus reaction of a range of 2hydroxyacetophenones with acetone gave the corresponding benzopyran-4-ones 17 which, after reduction and dehydration, generated the required benzopyran 4 (Buckle et al., 1990; Bergmann and Gericke, 1990). Appropriate substitution of the acetophenone mitigates against isomeric mixtures and hence problems of regiochemistry are obviated.

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 5

The benzopyran-4-one intermediates to the C-2 nor-dimethyl 18 and monomethyl 19 analogues of CRK have been prepared in high yield by alkylation of 4-cyanophenol with the appropriate lactones, followed by cyclisation of the acid intermediates 20 (Buckle et al., 1991c). This would appear to offer a general and high yielding route to this type of compound but the regiochemical cyclisation of asymmetric substrates was not investigated. A high yielding route to CRK via the benzopyran involved condensation of 4cyanophenol with isoprene under acidic conditions to give the dihydrobenzopyran 21 Bromination at C-4 and dehydrohalogenation gave the required benzopyran 4 (R=6-CN) in 82% overall yield (Timer et al., 1991). The less potent racemic cis-isomer of CRK was originally prepared (Ashwood et al., 1986) by treatment of the epoxide 5 (R=6-CN) with HBr to give the trans-4-bromo-3-ol 22. The tetrahydropyranyl (THP) ether of 22, was subsequently reacted with NaN3, reduced and deprotected to give the cisaminoalcohol which, after acylation and cyclisation furnished the cis-isomer 23. A more facile method for the preparation of cis-isomers involves isomerisation of the trans-amidoalcohols using diethylaminosulfur trifluoride (DAST) (HougeFrydrych and Pinto, 1989; Burrell et al., 1990b; Buckle et al., 1991b). This reaction is further discussed in Section 1.6.3.

The racemic dihydrobenzopyran 25 is less active in vivo than CRK and was originally prepared by catalytic hydrogenation of the corresponding benzopyran

6 K CHANNELS AND THEIR MODULATORS

(Ashwood et al., 1986). The analogous pyridone 26 has also been reported, formed in low yield (4%) from the 4-bromodihydrobenzopyran 24 and pyridone anion (Bergmann and Gericke, 1990). The C-2 position of CRK would appear optimally substituted by a gemdimethyl (Ashwood et al., 1986; Buckle et al., 1991c) or methyl, ethyl (Attwood et al., 1991a) moiety, but the synthesis of potent gem-trifluoromethyl (Fenwick, 1993) and gem-monofluoromethyl (Koga et al., 1993d) analogues have also been described. Spiro derivatives (Bergmann and Gericke, 1990; Lang and Wenk, 1988) and compounds where one of the methyl moieties has been replaced by a ketal or thioketal substituent (Yoo et al., 1992b) are also known. Mono substitution at C-2 is complicated by the presence of a further chiral centre. The 6-nitro compound 27 was reported with unassigned stereochemistry (Ashwood et al., 1986) but more recently both isomers of the 6-cyano derivative 28 have been prepared and characterised (Buckle et al., 1991c; Attwood et al., 1991a). Dehydration of the latter com-pounds, and the corresponding 2, 2–unsubstituted derivative, gave mixtures of 2, 3 and 3, 4-enes or solely the 2, 3-derivative depending on conditions used (Buckle et al., 1991c). High potency is associated with a group of compounds substituted with methyl at C-3, particularly in conjunction with an O-linked heterocycle at C–4 e.g. 29. Preparation of the intermediate benzopyran-4-one 30 was achieved either by reaction of 4-cyano-2-hydroxypropiophenone with acetone, or via reduction of the β -keto methylene analogue 31, and elaboration to the epoxide (Gericke et al., 1991a). In contrast to the relative ease of epoxide ring opening shown by the C-3 nor-methyl derivatives, the epoxides derived from this series were more resilient to attack by amide nucleophiles, several days reflux being required for complete reaction. Syntheses of CRK analogues where the C-3 hydroxyl group is replaced by other moieties have also been described (Buckle et al., 1991b; Gericke et al., 1991d) and are reviewed in Section 1.6.4.

In a novel departure from KCAs of standard structural type the synthesis and activity of a series of potent compounds where C-3 and C-4 functionalities are transposed has been described (Cassidy et al., 1992). Thus treatment of the benzopyran 4 with t-butyl N, N,-dichlorocarbamate followed by reduction with sodium metabisulfite gave regio– and stereoselectively the trans-4-chloro-3carbamate 32 from which the aziridine 33 was formed after base hydrolysis (Orlek and Stemp, 1991). Acid hydrolysis of 33 followed by deprotection gave

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 7

the aminoalcohol 34, from which a range of amido derivatives was produced. Dehalogenation and deprotection of the initially formed adduct 32 gave amine 35, from which analogous derivatives were prepared. Ring opening of the aziridine 33 (R=CN) with other nucleophiles gave further C-4 variants.

Numerous benzopyran KCAs have been prepared to explore the effect of aromatic ring substitution. Since synthesis generally follows the routes outlined above or results from simple functional group transformation of intermediate compounds (Evans et al., 1992; McCaully, 1991 and references therein) their preparation need not be elaborated further. A range of C-6-heterocyclyl derivatives has been prepared via reaction of the appropriate substrate with CRK or its 6amino, carbamoyl or formyl analogues using standard heterocyclic methodology (Bergmann and Gericke, 1990). Largely because of its ready ease of modification, but also because it would appear to occupy a critical role in the KCA pharmacophore, the C-4 position has similarly been subject to considerable variation. Whilst most of the modifications have either retained the amide or an equivalent pharmacophore, and were prepared using the syntheses described above, other groupings which are not normally regarded as amide surrogates (e.g. pyridine N-oxide and sulfoxide) have been prepared by alternative methods. The preparation of amides and ureas is normally straightforward. However, in those cases where the epoxide appears particularly recalcitrant towards ring opening, reactions have been enhanced in the presence of Lewis acids such as MgBr2 (Buckle et al., 1992a) or BF3.Et2O (Gericke et al., 1991b) or by use of tetramethyl ethylenediamine (TMEDA) to increase the nucleophilicity of the anion in the case of C-4 pyrrole derivatives (Buckle et al., 1992b). For those amides which can react as ambident nucleophiles (e.g. 2-pyridones) then mixtures of O- and N-linked compounds are generally obtained. Results of Nversus O-alkylation in the reaction of a wide variety of heterocycles with benzopyran epoxides have been documented (Bergmann and Gericke, 1990; Bergmann et al., 1990). Pinacidil 36 and CRK are both openers of the ATP-sensitive K (KATP) channel (Cook et al., 1988). This, together with the known isosterism between N-

8 K CHANNELS AND THEIR MODULATORS

cyanoguanidine and thiourea (Durant et al., 1977), probably accounts for the number of publications describing compounds designed to exploit this equivalence at C-4. Representative preparations of generalised compound 37 were by reaction of epoxide 5 with a diamine (R2R3 linked) followed by treatment with dimethyl-N-cyanodithioiminocarbonate, or by treatment of the appropriate thiourea 38 with cyanamide (Shiokawa et al., 1989, 1990; Atwal, 1991, 1992b, Atwal et al., 1990, 1992a, 1992b; Stemp et al., 1990; Stenzel et al., 1990). The N-cyano group in these compounds has been replaced by moieties known to act as amide surrogates in the H2-antagonist area and were prepared by processes analogous to those already described (Burrell et al., 1993; Butera and Bagli, 1993). N-cyanoamidines have also been prepared via epoxide opening with the anion derived from the appropriate heterocycle to give e.g. 39 (Garcia et al., 1991), or by reaction of the amino alcohol 40 with an N-cyanoimidate to give 41 (Ohtuka et al., 1991).

A number of C-4 C-linked derivatives have been prepared which retain KCA activity, seemingly by virtue of an appropriately orientated carbonyl group. Thus reversed amides of type 42 and ester 43 have been prepared from opening of the epoxide with the appropriate lithium salt (Ashwood et al., 1991). The latter compound gave the propanone 44 after hydrolysis and decarboxylation (Ashwood et al., 1991). C-linked pyridones 45 have been similarly prepared via the anion of the N-benzyl derivative (Gericke et al., 1991c). A series of C-4 amides/thioamides has been described which was prepared by reaction of the enolate derived from the 3-ketone 46 with an isothiocyanate to give 47, followed by reduction to the diastereomeric alcohols 48. Dehydration of the trans-isomers via the mesylate gave the benzopyrans 49. Alternatively the dithioester 50, formed by reaction of 46 with CS2 and Mel was aminated (Arch et al., 1991). Similar syntheses were used by others for the preparation of such compounds (Koga et al., 1993a, 1993b, 1993c, 1993d; Ishizawa et al., 1993).

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 9

Spirocyclic benzopyran imidazolones 52 have been prepared as rigid analogues of CRK by treatment of the corresponding ethoxy derivatives 51 with amines, and many of the compounds were potent KCAs despite lacking the C-3 hydroxyl (Gadwood et al., 1993). In an alternative preparation of the C-4 pyridine N-oxide derivative 54, the enol triflate 53 was treated with 2-(tributyltin)pyridine under palladium catalysis followed by oxidation (Yoo et al., 1992a). The sulfoxide group has been investigated as an N-oxide mimic and 56 was found to be as potent as CRK, although about 30 times less potent than 54. The preparation of 56 was achieved by treatment of the benzopyran-4-one 17 (R=6NO2), with the lithium salt of the cyclic sulfoxide 57 followed by dehydration (NaI/ TFAA). This also resulted in reduction of the sulfoxide to 55 but selective reoxidation using oxone gave 56 (Yoo et al., 1993).

10 K CHANNELS AND THEIR MODULATORS

1.3 Preparation of Enantiomers Standard routes to benzopyranol KCAs provide the compounds in racemic form. For CRK there is a 100-fold potency difference in vitro between the eutomer (3S, 4R) and the distomer (3R,4S) (Hof et al., 1988; Attwood et al., 1992) and at least a 30-fold difference in vivo in the spontaneously hypertensive rat (Ashwood et al., 1986). Similar potency differences exist between enantiomers of other KCAs (Buckle et al., 1990, 1991a; Bergmann et al., 1990). Individual enantiomers have been prepared by ring opening of the racemic benzopyran epoxide with a suitably substituted chiral pyrrolidinone followed by separation of the diastereomers obtained. In this way benzopyrans bearing the 5’methyl pyrrolidinone (Englert et al., 1988), and more complex derivatives (Regnier et al., 1990; Pinto et al., 1993) at C-4 have been obtained. The C-3 hydroxyl group provides a handle by which to attach chiral auxiliaries, and enantiomers have been separated via the diastereomeric β -methylbenzyl carbamates (Ashwood et al., 1986; Buckle et al., 1990; Quagliato et al., 1991), camphorsulfonates (Bergmann et al., 1990) and camphanates (Bergmann et al., 1990). Some workers have relied upon separation at an earlier stage to provide chiral ester derivatives of the bromohydrin 7 which were separated, hydrolysed and cyclised to the (3S,4S) epoxide 58. Thus N-Boc-(L)-alanine (Shiokawa et al., 1989), (-) camphanic acid (Blarer, 1988), chiral alkylcarbonylthiobutyric acids (Setoguchi et al., 1990), and (L)-proline (Yamanaka, 1991) derivatives have been used. The (3S,4S) epoxide has also been synthesised from the chiral aminoalcohol 60, prepared by resolution of the tartrate salt or derivatives thereof (Attwood et al., 1992; Genain and Pinhas, 1990; Quagliato et al., 1991). Successful separation of the (3S,4S) epoxide by entrainment crystallisation has been reported (Devant and Gericke, 1991) and a similar procedure was used for the C-3 methyl analogue 59 (Gericke et al., 1991a). Microbial oxidation of the benzopyran 4 (R=6-CN) to the (3S,4S) epoxide 58 has been attempted using a variety of organisms but the highest enantiomeric excess (ee) reported was only 10%. By contrast a number of organisms showed high enantioselectivity (>96% ee) of epoxidation to the undesired (3R,4R) enantiomer, but only in low (<5%) yield (Woroniecki et al., 1993). The preferred method for the synthesis of enantiomeric epoxides involves oxidation of the benzopyran in the presence of a chiral catalyst (Jacobsen et al., 1991). Thus treatment of the benzopyran 4 (R=6CN) with commercial bleach and 2–4 mol% of (S,S)-Mn(salen) catalyst 61 gave the (3S,4S) epoxide in 96% isolated yield with 97% ee, converting to ca. 100% ee after one recrystallisation (Lee et al., 1991). Similar catalysts have been used to epoxidise other benzopyran KCA intermediates (Katsuki et al., 1992). The enantiomer of the pyridine-N-oxide 63 has been prepared by ring opening of the enantiomeric epoxide 58 (R=6-CN) with 2-pyridinethiol followed by hydroxyl protection and sulfur oxidation to give the sulfoxide 62. Methyl Grignardinduced extrusion of sulfur, followed by deprotection and N-oxidation gave the

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 11

required compound. A similar sequence from the (3R, 4R) epoxide provided its enantiomer (Attwood et al., 1992).

The cis-enantiomers 66 and 67 of CRK have been prepared via DAST rearrangement (see Section 1.6.3) of the corresponding trans-enantiomers (Evans, J.M., personal communication). They have also been prepared by ring opening of the chiral benzopyran epoxides with TMSI to give the TMS-protected trans-4-iodo-3-ols 64 and 65 which were converted to cis-azide, reduced to amine and thence elaborated by standard methodology to 66 and 67 (Quast and Villhauer, 1993). The 4S and 4R dihydrobenzopyran enantiomers 68 and 69 have been synthesised by tributyltin hydride-induced radical deoxygenation of the thiocarbonate enantiomers of CRK 70 and 71 and the corresponding C-4 pyridine N-oxides 73 and 74 were prepared similarly (Attwood et al., 1992). The latter compounds have also been prepared by resolution of the quinine salt of the C-6 acid derivative 72, followed by functional group interchange of each enantiomer to the nitrile and oxidation to N-oxide.

12 K CHANNELS AND THEIR MODULATORS

1.4 Benzopyran KCAs with Modified Aromatic Ring The four pyridine analogues 79–82 of CRK have been prepared by ring opening of the appropriate pyranopyridine epoxides 75–78 (or N-oxide for 7-aza derivative). Dehydration of 80 gave the 3,4-ene 83 and oxidation gave the Noxide 84 (Burrell et al., 1990a). The 5-, 6- and 7-aza pyranopyridine precursors were prepared by Claisen rearrangement of propargyl ethers whilst synthesis of the 8-aza isomer was mediated via lithiation of 3,5-dibromo-2-methoxypyridine and reaction with 3-methylcrotonaldehyde, followed by dealkylation, cyclisation and debromination (Evans and Stemp, 1988). Synthesis of the trifluoromethyl substituted analogue 85 was via Kabbe condensation of acetone with 3-acetyl-6trifluoromethylpyridone and standard methodology (Lang and Wenk, 1988). Elaboration of appropriately substituted thienopyranones 86–88 gave the corresponding epoxides 89–91 from which the three thienopyran isomers of the benzopyran KCAs 92–94 have been prepared (Press et al., 1990). Thieno[3,2-b] pyran analogues 92 have also been described independently using similar routes (Sanfilippo et al., 1992, 1993) and via transformation of the unsaturated lactone 95 (Binder et al., 1991). Separation of enantiomers was accomplished via the β -

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 13

methylbenzylcarbamates (Binder et al., 1991; Press et al., 1992; Sanfilippo et al., 1992). Early work indicated that disubstitution at C-6 and C-7 of CRK was associated with high potency (Ashwood et al., 1986) and annulation of these positions into the pyranobenzoxadiazole tricyclic structure 98 retained KCA activity (Seto et al., 1989). The preparation of 98 was achieved via elaboration of the benzoxadiazole

epoxide 97, itself formed by oxidative dehydration of the 6-amino-7-nitro derivative 96 with NaOCl followed by deoxygenation of the N-oxide with (EtO) 3P. C-4 variants of this system have been produced (May et al., 1991; Atwal, 1992a; Seto et al., 1992).

14 K CHANNELS AND THEIR MODULATORS

1.5 Benzopyran KCAs with Modified Pyran Ring The only active ring-contracted analogues of the benzopyran KCAs which have been published are derived from indane. Synthesis of the generalised structures 100 and 101 was by standard elaboration of the indanones 99. The homologated compounds 102 (R=6-CN) were prepared from 6-amino-3,3-dirnethylindanone via Wittig olefination with methylene triphenylphosphorane, Sandmeyer cyanation and isomerisation of the exo double bond to give 1,3,3trimethylindane-6-carbonitrile, followed by allylic bromination and displacement with the appropriate amide anion (Buckle et al., 1991a). Indanol derivatives with the lactam moiety replaced by cyanoguanidine entities have also been prepared using essentially the same methodology (Atwal, 1992b).

Similarly the only reported active ring expanded derivatives of the benzopyranols have been benzoxepines. The nor-dimethyl derivative 105 was prepared by nitration, reduction and cyanation of benzoxepinone 103 to give the 7-cyanobenzoxepinone 104 which was further elaborated by standard methodology. The corresponding 2,2-dimethyl derivative 106 was prepared by gem-dimethylation of benzoxepine-2-one to give 107 which was functionalised by aromatic bromination followed by cyanation at C-7. Radical bromination at C-5 gave the bromide 108 which was dehydrobrominated and further elaborated to 106 (Buckle et al., 1991c). The former compound, 105, and analogues, have also been prepared by ring opening of the epoxide using N-trimethylsilylamides and tetrabutylammonium fluoride (Utz et al., 1990).

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 15

Treatment of trimethylbenzothiopyran 109 with excess mCPBA gave the epoxysulfone 110 from which the pyrrolidinone derivative 111 was prepared (Smith, 1990). Treatment of 109 with NaIO4 gave the sulfoxide which underwent bromohydrin and thus epoxide formation regio- and stereospecifically to give the cis-isomer of the epoxysulfoxide 112, from which the pyrrolidinone derivative 113 was prepared. Attempted formation of the analogous thioether 114 by a number of routes involving functionalisation of the 3,4-double bond led to benzothiophenes, but the required compound was eventually prepared by deoxygenation of the piperidinone sulfoxide 115 with (EtO)3P/I2. Oxidation of the sulfoxide with mCPBA gave the sulfone 116, and from comparison of the 1H nmr spectra of these two compounds the cis alcohol/sulfoxide geometry of 115 was determined, thus providing structural assignment of the epoxide and bromohydrin precursors.

6-Bromodihydroquinoline 117 could not be epoxidised directly but the compound was obtained as the acetyl derivative 118 (Ashwood et al., 1991). Reaction of the anion derived from pyrrolidinone with this epoxide gave a mixture of both cis 119 and trans 120 derivatives plus the dihydroquinoline 121. The trans isomer and the dihydroquinoline were converted to the 6-cyano derivatives 122 and 123 but under similar conditions the cis isomer 119 gave the indole 124. Tetrahydroquinoline derivatives with the lactam moiety replaced by cyanoguanidine entities have also been prepared using similar methodology (Atwal, 1992b).

16 K CHANNELS AND THEIR MODULATORS

The dihydronaphthalene 125 was elaborated to the di-and tetrahydronaphthalenes 128 and 126 by standard procedures (Ashwood et al., 1991) and O- and N-linked pyridone (and related) derivatives have been prepared by similar methodology (Baumgarth et al., 1990a). 1-Naphthalenone analogues 127 and 129 have been prepared via β -dimethylation of appropriately substituted tetralones followed by bromination at C-4 and elimination to the 3,4-enes (Almansa et al., 1993). Derivatives of the 1,3-benzoxazinone system have been reported (Shiraishi et al., 1992). The representative pyridine N-oxide 132 was synthesised by condensation of 5-cyanosalicylamide with 2,2-dimethoxypropane to give the 1,3benzoxazinone 130, which was converted to the iminochloride or triflate 131 and further elaborated via reaction with 2-pyridyl lithium and oxidation. Lactams (Englert et al., 1991) and other heterocyclic moieties (Baumgarth et al., 1990b) have been incorporated at C-4 using similar methodology. A series of 1,4-benzoxazine derivatives, variously substituted at C-4 has been described (Matsuhisa et al., 1991). The basic ring system was prepared either by treatment of a suitably substituted 2-aminophenol with ethyl 2-bromoisobutyrate followed by reduction of the amide 133 to 134, or by reaction of the corresponding 2-nitrophenol with 2-bromoisobutyraldehyde followed by catalytic hydrogenation. Treatment of the benzoxazine anions with 2bromopyridine N-oxide gave the N-oxides 135 and N-acylhydrazines 138 were prepared from the nitroso compound 136 via reduction to 137 and acylation. The range of compounds has been extended to include C-6, C-7 annulated derivatives (Tsuzuki, et al., 1992).

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 17

1.6 Reactions of Benzopyran KCAs General reactions of the active benzopyran KCAs have already been described in the preceding sections, but close structural analogues have been reported which have reduced KCA activity or are inactive. It is appropriate to review their preparation here, together with those reactions which, because of a combination of particular electronic and steric effects in the substrate (e.g. C-6 electronwithdrawing group– usually CN—and the C-3 neopentylic centre) have given rise to unusual chemistry or molecular rearrangements which were quite unexpected. 1.6.1 C-3 Ketone Derivatives and Aromatic SN2’ Substitution Whilst esterification (Ashwood et al., 1986; Bergmann and Gericke, 1990) and etherification (Evans, J.M., personal communication) of CRK and similar compounds is relatively straightforward, direct oxidation to the ketone 139 has proved difficult, probably due to the neopentylic nature of C-3 (Buckle et al., 1991b). Similar lack of direct oxidation was reported for the C-4 pyridone analogue (Bergmann and Gericke, 1990). However, whilst acid rearrangement of 140 successfully gave the pyridonyl ketone 141 (Bergmann and Gericke, 1990), with pyrrolidinone as C-4 substituent further reaction accompanied the

18 K CHANNELS AND THEIR MODULATORS

rearrangement and a low yield of the Baeyer-Villiger oxidation product 142 was obtained. Interestingly, rearrangement of the epoxide 140 using ammonia or (Ph3P)4Pd gave the pyridyloxy ketone 143. Attempts to prepare the bromohydrins 144 or 145 were unsuccessful, the reaction of CRK with NBS in CCl4 yielding only starting material, and the benzopyran 146 with aqueous NBS giving only the bromoketone 147, presumably as a result of hydrolysis of the required intermediate 145.

Treatment of the bromoketal 148 with the anion derived from pyrrolidinone gave none of the expected product 149 but instead a 58% yield of the 5substituted analogue 150 was obtained by SN2’ displacement of bromine. Presumably this arises through steric hindrance associated with direct displacement and is aided by complexation of the potassium cation and the presence of the electron withdrawing group at C-6 (Houge-Frydrych et al., 1989; Buckle et al., 1991b). The presence of an electron withdrawing group at this position is known to play an important role in the reaction of epoxides 5 with the 13-membered lactam 151 since 5 (R=H, 6-C1), gave the expected ring opened products 152 (R=H, Cl), whereas reaction of 5 (R=6-CN or 6-NO2) gave products 153 and 154 arising from attack of the internal (E)-enolate at C-5, the latter albeit in very low yield (Cassidy et al., 1987). The SN2’ reaction of the bromide 148 also occurred with the anion derived from N-methylacetamide to give 155 but reaction of the anion derived from acetamide gave the alcohol 156, presumably by oxygen attack at C-4 and subsequent hydrolysis of the acetimidate (Houge-Frydrych et al, 1989; Buckle et al., 1991b). The smaller azide anion reacted at C-4 to give 157 which gave the

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 19

amine 158 after reduction and thence the pyrrolidinone derivative 159. Trifluoroacetic acid deprotection of the analogous 6-ring ketal 160 gave the ketone 139 (Buckle et al, 1991b).

Keto-enol tautomerism for ketones 139 and 141 has been reported (Buckle et al., 1991b; Bergmann and Gericke, 1990), polar solvents favouring the enol, and both O-and C-alkylated derivatives of 141 have been prepared. Similar keto-enol tautomerism exists for the 3-keto 4-amide 161 and thioamide 162 derivatives, although in this case the ketones are almost fully enolised (Arch et al., 1991). Treatment of the potassium enolate of 161 (R=Ph) with Mel in DMSO gave the C-methylated derivative 163, whilst treatment with diazomethane in methanolether gave only a mixture of imidates 166 and 167, the latter being formed by insertion of carbene into the imidate ether bond. Reaction of 161 (R=Ph) with trimethyloxonium tetrafluoroborate, however, gave the enol ether 168 (13%), together with C-methylated material 163 (48%) and the methyl imidate 166 (24%).

20 K CHANNELS AND THEIR MODULATORS

Treatment of the ketoamide 161 with DAST gave 4.5% of the C-4 fluoride 164 together with the bis spiro derivative 169. Both products were envisaged as arriving from intermediate 165. The favoured C-alkylation of this system was interpreted as reflecting the greater stability of the anionic intermediate at C-4 through cross-conjugation with two carbonyl centres and with the cyanophenyl moiety (Buckle et al., 1991b).

1.6.2 Michael-type Addition at C-3 The reaction between methyl lithium and pyridone benzopyran 170 gave the 3substituted derivative 171 by Michael-type addition (Gericke et al., 1991a). It appears that the double bond is sufficiently activated by the cyanophenyl ring and by the pyridone moiety, with possible assistance from coordination of the metal ion to the amide carbonyl group, for this to occur. By contrast, reaction of 170 with a non-nucleophilic base resulted in removal of the C-3 proton and isolation of the allenic phenol 172 (R=OH) (Gericke and Lues, 1992). Thermolysis of the corresponding acetate 172 (R=OAc) in DMSO gave the noninterconvertible benzofuran products 173 (27%) and 174 (16%), whilst in toluene the ether 175 was formed. Michael-type addition reactions also occurred between pyrrolidinone anion and CRK or its ene derivative at 100°C and resulted in high yields of the dipyrrolidinyl compound 176, (Buckle et al., 1991b). Similar incorporation was seen when the epoxide 177 was reacted with excess pyrrolidinone to give 178, presumably via the 4-pyrrolidinone ene derivative (Tedder, J.M., personal communication). Attempts to produce the pyridone benzopyran 170 from the dibromo compound 179 or the vinyl chloride 180 led only to the Michael product 181 (Bergmann and Gericke, 1990).

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 21

1.6.3 Reactions of Benzopyranols with DAST Reaction of CRK with DAST resulted not in the expected cis-fluoride 182 but the cis-isomer of CRK 183 (Houge-Frydrych and Pinto, 1989; Buckle et al., 1991b). Similar reaction was observed with the 6-formyl analogue which underwent isomerisation to the cis-isomer 184 without fluorination of the carbonyl moiety. Reaction of the cis-isomer 183 with DAST did not result in isomerisation but gave instead the benzopyran 185 (44%) together with the expected trans-fluoride 186 (3%). These observations were rationalised by invoking an intramolecular displacement of the aminosulfoxy leaving group by the amide at C-4 with subsequent hydrolysis of the intermediate 187 via attack β to nitrogen to give the cis-amidoalcohol. It was argued that the unfavourable trans-fused 6,5-system would mitigate against a similar reaction of the ciscompound, and that in this case fluoride induced elimination of the leaving group

22 K CHANNELS AND THEIR MODULATORS

is the favoured reaction, with the small amount of fluoride observed obtained through the expected displacement. However, despite the normally unfavourable nature of a trans-fused 6,5 system it is possible that with two hetero atoms such an intermediate may be present, subsequently giving the benzopyran by fluoride induced proton removal at C-4 followed by elimination. Reaction of DAST with the thiolactam analogue of CRK gave a complex mixture of products from which only a low yield of unchanged starting material was recovered (Buckle, et al., 1991b). Further work with the acyclic amides 188 and 189 resulted in isolation of oxazoline intermediates 194 and 195 which with aqueous acid gave the expected cis-alcohols 190 and 191. In the case of the urea derivative 192 the oxazoline 196 was isolated together with the 4-fluoro compound 198, presumably formed via fluoride ring opening of the aziridine intermediate 199. The fact that the gemdimethyl group was not responsible for these isomerisations was shown by the isolation of the corresponding oxazoline 197 of the nor-dimethyl derivative 193 (Burrell et al., 1990b). Similar trans to cis isomerism has also been reported in the naphthalenone series (Almansa et al., 1993). 1.6.4 C-3 Carbon and Nitrogen Linked Moieties Treatment of the benzopyran 4 (R=6-CN), with chlorosulfonyl isocyanate gave the β -lactam 200 which, after reductive work up, gave the amino ester 201. Elaboration

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 23

24 K CHANNELS AND THEIR MODULATORS

of this by standard methodology gave the pyrrolidinone derivative 202 from which the acid 203 and hydroxymethyl 204 derivatives were prepared. Reaction of 204 with benzeneselenic anhydride gave the aldehyde 205, and the methyl ether 206 and fluoro 207 compounds were prepared by reaction of 204 with Mel and DAST respectively (Buckle et al., 1991c). C-3 formyl derivatives have also been prepared by treatment of the benzopyran-4-one 17 (R=6-CN), with PBr3 in DMF/CHCl3 to give 208 followed by displacement of bromide with pyridone anion to give 209. The hydroxymethyl derivative 210 was prepared by reduction of this with NaBH4 (Gericke et al., 1991d). Attempts to incorporate a nitro group at C-3 by reaction of the pyrrolidinone benzopyran 185 with fuming nitric acid in acetonitrile resulted in hydrolysis to the 3-nitro compound 211 (34%) and the benzopyranone 212 (3%). Reaction of the benzopyran 4 (R=6-CN) with nitronium iodide in ethyl acetate gave the 3,3dinitro derivative 213 (25%) and reaction with nitronium tetrafluoroborate in dichloromethane gave only low yields (7%) of the 8-nitro derivative 214. Use of the latter reagent in acetonitrile gave a mixture of cis- and trans-nitroacetamides 215, formed by trapping of the intermediate carbonium ion 216 with acetonitrile followed by hydrolysis (Buckle et al., 1991b).

1.6.5 Reactions of C-4 Pyrrole Derivatives Treatment of the bromohydrin 7 (R=6-CN) with the anion derived from 3-cyano or 3-nitropyrroles in DMF at 100°C for 20 h gave a mixture of the expected benzopyrans 217 and 218 and the benzofurans 221 and 222 (Buckle et al., 1992a; Smith et al., 1992). For pyrrole itself, or the 2-ethyl derivative (Faller, A., personal communication), only the benzopyrans 219 and 220 were isolated in high yield. Similarly only benzopyran 223 was isolated from the reaction

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 25

between the 6-ethyl bromohydrin 7 (R=6-Et) and the anion derived from 3cyanopyrrole. These results have been rationalised (Buckle et al., 1992a). Treatment of the bromohydrin 7 (R=6-CN) with anions derived from 2-acyl pyrroles 224–227 in the presence of TMEDA resulted in the formation of tetracyclic compounds, isolated either as the hemiketals 228 and 229 or their anhydro derivatives 231 and 232 (Buckle et al., 1992b). Reaction of the anion from 224 with the deuterated bromohydrin 233 gave 234 only, demonstrating that the C-3 deuterium atom migrates quantitatively to the position β -to pyrrole. Two mechanisms were advanced to rationalise the formation of these redox products but, whereas the deuteration experiment served to discriminate the postulated mechanisms from those where transfer of deuterium did not occur, it was not sufficient to distinguish between either mechanism proposed.

26 K CHANNELS AND THEIR MODULATORS

1.7 Conclusion In the last few years considerable strides forward have been made in delineating the type of molecule which can act to open ATP-sensitive potassium channels, and knowledge of SAR governing these compounds has increased many fold. Over this relatively short period of time chemists from a number of groups have explored the pharmacophore of CRK and its congeners by both chemical synthesis and by the use of modelling techniques (see Chapter 4), and pharmacophore models are now beginning to be reported (Koga et al., 1993a). In the not too distant future it is anticipated that these will be refined to further arm the chemist in the search for yet more potent and structurally distinct compounds. This, however, is not perceived as the critical issue with the KCAs since there are a variety of structural types which include compounds of very high potency. The main drawback with the current ‘first generation’ derivatives would appear to be their lack of intrinsic tissue selectivity and it will be the challenge of the medicinal chemist over the next few years to rationally design compounds which are inherently more tissue selective. That this prospect is not an impossibility is demonstrated by reports of compounds selective for the ureter (Englert et al., 1988) and for relaxation of the airways over the vasculature (Buckle et al., 1992c; Bowring et al., 1993; Koga et al., 1993b). Hence for the medicinal chemist the benzopyran derived KCA area is still rich and fertile and it will be expected to yield further compounds of interest over the forthcoming decade. The distinct arrangement of chemical entities which contribute to the pharmacophore also have a bearing on the chemical reactivity of the system, and as such we may readily expect to see more of the unusual transformations and unexpected products that have been described in the latter half of this chapter. References ALMANSA, C., GOMEZ, L.A., CAVALCANTI, F.L., RODRIGUEZ, R., CARCELLER, E., BARTROLI, J., GARCIA-RAFANELL, J. & FORM, J. (1993) J. Med. Chem., 36, 2121–2133. ANDERSON, W.K. & LAVOIE, E.J. (1973) J. Org. Chem., 22, 3832–3835. ARCH, J.R.S., BUCKLE, D.R., CAREY, C, PARR-DOBRZANSKI, H., FALLER, A., FOSTER, K.A., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G., TAYLOR, J.F., TAYLOR, S.G., TEDDER, J.M. & WEBSTER, R.A.B. (1991) J. Med. Chem., 34, 2588–2594. ASHWOOD, V.A., BUCKINGHAM, R.E., CASSIDY, F., EVANS, J.M., FARUK, E.A., HAMILTON, T.C., NASH, D.J., STEMP, G. & WILLCOCKS, K. (1986) J. Med. Chem., 29, 2194–2201. ASHWOOD, V.A., CASSIDY, F., COLDWELL, M.C., EVANS, J.M., HAMILTON, T.C., HOWLETT, D.R., SMITH, D.M. & STEMP, G. (1990) J. Med. Chem., 33, 2667–2672.

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ASHWOOD., V.A., CASSIDY, F., EVANS, J.M., GAGLIARDI, S., & STEMP, G. (1991) J. Med. Chem., 34, 3261–3267. ATTWOOD, M.R., JONES, P.S., KAY, P.B., PACIOREK, P.M. & REDSHAW, S. (1991a) Life Sciences, 48, 803–810. ATTWOOD, M.R., CHURCHER, I., DUNSDON, R.M., HURST, D.N. & JONES, P.S. (1991b) Tetrahedron Lett., 32, 811–814. ATTWOOD, M.R., BROWN, B.S.I., DUNSDON, R.M., HURST, D.N., JONES P.S. & KAY, P.B. (1992) BioMed. Chem. Lett., 2, 229–234. ATWAL, K. (1991) Patent to E.R. Squibb and Sons, Inc. EP 450415. (1992a) Patent to E.R.Squibb and Sons, Inc. EP 488107. (1992b) Patent to E.R.Squibb and Sons, Inc. EP 488616. ATWAL, K., GROVER, G.J. & KIM, K.S. (1990) Patent to E.R.Squibb and Sons, Inc. EP 401010. ATWAL, K.S., MORELAND, S., MCCULLOUGH, J.R., AHMED, S.Z. & NORMANDIN, D.E. (1992a) BioMed. Chem. Lett., 2, 87–90. ATWAL, K., KIM, K.S. & GROVER, G.J. (1992b) Patent to E.R.Squibb and Sons, Inc. EP 501797. BAUMGARTH, M., GERICKE, R., BERGMANN, R., DE PEYER, J. & LUES, I. (1990a) Patent to E. Merck EP 368160. BAUMGARTH, M., GERICKE, R., BERGMANN, R., DE PEYER, J. & LUES, I. (1990b) Patent to E. Merck DE 3840011. BERGMANN, R. & GERICKE, R. (1990) J. Med. Chem., 33,492–504. BERGMANN, R., EIERMANN, V. & GERICKE, R. (1990) J. Med. Chem., 33, 2759–2767. BINDER, D., ROVENSZKY, F., WEINBERGER, J. & FERBER, P.H. (1991) Patent to Chemisch Pharmazeutische Forschungsgesellschaft m.b.H. E.P. 405298. BLARER, S. (1988) Patent to Sandoz Ltd. GB 2204868. BOWRING, N.E., ARCH, J.R.S., BUCKLE, D.R. & TAYLOR, J.F. (1993) Br. J. Pharmacol., 109, 1133–1139. BUCKLE, D.R. & SMITH, D.G. (1993) Curr. Top. Med. Chem., 1, 291–311. BUCKLE, D.R., ARCH, J.R.S., FENWICK, A.E., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G., TAYLOR, S.G. & TEDDER, J.M. (1990) J. Med. Chem., 33, 3028–3034. BUCKLE, D.R., ARCH, J.R.S., EDGE, C., FOSTER, K.A., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G., TAYLOR, J.F., TAYLOR, S.G., TEDDER, J.M. & WEBSTER, R.A.B. (1991a) J. Med. Chem., 34, 919–926. BUCKLE, D.R., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G. & TEDDER, J.M. (1991b) J.C. S. Perkin Trans. 1, 63–70. BUCKLE, D.R., EGGLESTON, D.S., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., READSHAW, S.A., SMITH, D.G. & WEBSTER, R.A.B. (1991c) J.C. S. Perkin Trans. 1, 2763–2771. BUCKLE, D.R., FALLER, A., PINTO, I.L. & SMITH, D.G. (1992a) Tetrahedron Lett., 33, 1109–1112. BUCKLE, D.R., CONNOR, S.C., EGGLESTON, D.S., FALLER, A., PINTO, I.L., READSHAW, S.A. & SMITH, D.G. (1992b) J.C.S. Perkin Trans. 1, 769–775. BUCKLE, D.R., EGGLESTON, D.S., PINTO, I.L., SMITH, D.G. & TEDDER, J.M. (1992c) BioMed. Chem. Lett., 2, 1161–1164.

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ISHIZAWA, T., KOGA, H., OHTA, M., SATO, H., MAKINO, T., KUROMARU, K., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993) BioMed. Chem. Lett., 3, 1659–1662. JACOBSEN, E.N., ZHANG, W., MUCI, A.R., ECKER, J.R. & DENG, L. (1991) J. Am. Chem. Soc., 113, 7063–7064. KABBE, H.J. (1978) Synthesis, 886–887. KATSUKI, T., HOSOYA, N. & HATAYAMA, A. (1992) Patent to Nissan Chemical Industries Ltd. EP 535377. KOGA, H., OHTA, M., SATO, H., ISHIZAWA, T. & NABATA, H. (1993a) BioMed. Chem. Lett., 3, 625–631. KOGA, H., SATO, H., ISHIZAWA, T., KUROMARU, K., NABATA, H. IMAGAWA, J., YOSHIDA.S. & SUGO, I. (1993b) BioMed. Chem.Lett., 3, 1111–1114. KOGA, H., SATO, H., ISHIZAWA, T., KUROMARU, K., MAKINO, T., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993c) BioMed. Chem. Lett., 3, 1115–1118. KOGA, H., SATO, H., IMAGAWA, J., ISHIZAWA, T., YOSHIDA, S., SUGO, I., TAKA, N., TAKAHASHI, T. & NABATA, H. (1993d) BioMed. Chem. Lett., 3, 2005–2010. LANG, R.W. & WENK, P.F. (1988) Helv. Chim. Acta., 71, 596–601. LEE, N.H., MUCI, A.R. & JACOBSEN, E.N. (1991) Tetrahedron Lett., 32, 5055–5058. LONGMAN, S.D. & HAMILTON, T.C. (1992) Med. Res. Rev., 12, 73–148. MATSUHISA, A., ASANO, M., MATSUMOTO, Y., TAKAYAMA, K., YODEN, T., TSUZUKI, R., UCHIDA, W. & YANAGISAWA, I. (1991) Patent to Yamanouchi Pharmaceutical Co. Ltd. EP 432893. MAY, H.J., RASCHACK, M & SCHULT, S. (1991) Patent to BASF AG DE 4010097. McCAULLY, R.J. (1991) Current Drugs—Potassium Channel Modulators, KCMB-5 19. OHTUKA, K., ISHIYAMA, N., IIDA, Y., SERI, K., MURAI, T., SONAI, K. & ISHIZAKA, Y. (1991) Patent to Kaken Pharmaceutical Company Ltd., EP 412531. ORLEK, B.S. & STEMP, G. (1991) Tetrahedron Lett., 32, 4045–4048. PINTO, I.L., BUCKLE, D.R., READSHAW, S.A., & SMITH, D.G. (1993) BioMed. Chem. Lett., 3, 1743–1746. PRESS, B.J., SANFILIPPO, P.J., MCNALLY, J.J. & FALOTICO, R. (1990) Patent to Ortho Pharmaceutical Corporation EP 360621. PRESS, B.J., URBANSKI, M. & SANFILIPPO, P.J. (1992) Patent to Ortho Pharmaceutical Corporation EP 493048. QUAGLIATO, D.A., HUMBER, L.G., JOSYLN, B.S., SOLL, R.M., BROWN, E.N.C., SHAW, C. & VAN ENGEN, D. (1991) BioMed. Chem. Lett., 1, 39–42. QUAST, U., & VlLLHAUER, E.B. (1993) European J. Pharmacol., 245, 165–171. REGNIER, G., DHAINAUT, A., VILAINE, J.P., VILLENEUVE, N., JOLY, G. & DUHAULT, J. (1990) Patent to Adir et Compagnie EP 365416. RHOADS,S.J. & RAULINS, N.R. (1975) Org. React., 22,1–253. SANFILIPPO, P.J., MCNALLY, J.J., PRESS, J.B., FITZPATRICK, L.J., URBANSKI, M.J., KATZ, L.B., GIARDINO, E., FALOTICO, R., SALATA, J., MOORE, J.B. & MILLER, W. (1992) J. Med. Chem., 35, 4425–4433. SANFILIPPO, P.J., MCNALLY, J.J., PRESS, J.B., FALOTICO, R., GIARDINO, E. & KATZ, L.B. (1993) BioMed. Chem. Lett., 3, 1385–1388.

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SETO, K., MATSUMOTO, H., KAMIKAWAJI, Y., OHRAI, K., NAKAYAMA, K., SAKODA, R. & MASUDA, Y. (1989) Patent to Nissan Chemical Industries Ltd. EP 327127. SETO, K., MATSUMOTO, H., KAMIKAWAJI, Y., OHRAI, K., OHDOI, K., SAKODA, R. & MASUDA, Y. (1992) Patent to Nissan Chemical Industries Ltd. EP 466131. SETOGUCHI, S., TSURUDA, M., KITAMI, C. & YAMANAKA, T. (1990) Patent to Yoshitomi Pharmaceutical Industries Ltd EP 386640. SHIOKAWA, Y., TAKIMOTO, K., TAKENAKA, K. & KATO, T. (1989) Patent to Fujisawa Pharmaceutical Co. EP 344747. (1990) Patent to Fujisawa Pharmaceutical Co. EP 389861. SHIRAISHI, M., HASHIGUCHI, S. & WATANABE, T. (1992) Patent to Takeda Chemical Industries Ltd. EP 477789. SMITH, D.G. (1990) J.C. S. Perkin Trans. 1, 3187–3191. SMITH, D.G., BUCKLE, D.R., FALLER, A. & PINTO, I.L. (1992) BioMed. Chem. Lett., 2, 1595–1598. STEMP, G., BURREL, G. & SMITH, D.G. (1990) Patent to Beecham Group plc. EP 359537. STENZEL, W., SCHOTTEN, T. & ARMAH, B. (1990) Patent to Beiersdorf Aktiengesellschaft EP 350805. TIMER, T., ESZENYI, T., SEBOK, P., GALAMB, V., FUZEKAS, J., ISTVAN, T., KOVACH, E. & NAGY, E. (1991) Patent to Alkaloida Vegyeszeti Gyar EP 409651. TSUZUKI, R., MATSUMOTO, Y., MATSUHISA, A., YODEN, T., UCHIDA, W. & YANAGISAWA, I. (1992) Patent to Yamanouchi Pharmaceutical Co. Ltd. EP 500319. UTZ, R., ENGLERT, H.C., SCHOLKENS, B. & KLAUS, E. (1990) Patent to Hoechst Aktiengesellschaft DE 3831697. WORONIECKI, S.R., SIME, J.T., BAGGALEY, K.H. & ELSON, S.W. (1993) Biocatalysis 7, 221–226. YAMANAKA, T., (1991) Patent to Yoshitomi Pharmaceutical Industries Ltd EP 456266. Yoo, S., SUH, J.H., LEE, S.J. & JEONG, N. (1992a) BioMed. Chem. Lett., 2, 381–382. Yoo, S., YI, K.Y., JEONG, N.C., SUH, J.H., KIM, S.J., SHIN, H.S., LEE, B.H. & JUNG, K.S. (1992b) Patent to Korea Research Institute of Chemical Technology EP 514935. Yoo, S., KIM, S-J., SHIN, H-S., & HONG, K.W. (1993) BioMed. Chem. Lett., 3, 553–554.

Additional References ATWAL, K.S., GROVER, G.J., AHMED, S.Z., FERRARA, F.N., HARPER, T.W., KIM, K. S., SLEPH, P.G., DZWONCZYK, S., RUSSELL, A.D., MORELAND, S., McCULLOUGH, J.R. & NORMANDIN, D.E. (1993) Cardioselective Antiischaemic ATP-Sensitive Potassium Channel Activators. J. Med. Chem., 36, 3971–3974. BERGMANN, R. & GERICKE, R. (1994) The Influence of Substituents in 3-Position on the Activity of Chroman-Type Potassium Channel Openers. Arch. Pharm. (Weinheim), 327, 169–173.

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BURRELL, G., EVANS, J.M., HADLEY, M.S., HICKS, F. & STEMP, G. (1994) Benzopyran Potassium Channel Activators Related to Cromakalim—Heterocyclic Amide Replacements at Position 4. BioMed. Chem. Lett., 4, 1285–1290. CZIAKY, Z. & SEBOK, P. (1994) Synthesis of 2H-Pyrano [2,3-b] quinolines. Part I. J. Het. Chem., 31, 701–705. GODFREY, J.D., MUELLER, R.H., SEDERGRAN, T.C., SOUNDARARAJAN, N. & COLANDREA, V.J. (1994) Improved Synthesis of Aryl 1,1-Dimethylpropargyl Ethers. Tetrahedron Lett., 35, 6405–6408. ISHIZAWA, T., KOGA, H., SATO, H., MAKINO, T., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1994) Substituent Effects of Benzopyran-4-(N-Cyano)carboxamidine Potassium Channel Openers for Selectivity to Guinea Pig Trachealis. BioMed. Chem. Lett., 4, 1995–1998. KOGA, H., SATO, H., ISHIZAWA, T., TAKA, N. & TAKAHASHI, T. (1995) Synthesis of Key Intermediates Benzopyran-4-carboxylic Acids of New Potassium Channel Openers Benzopyran-4-amides via Palladium-catalyzed Hydroxycarbonylation. Tetrahedron Lett., 36, 87–90. PATEL, R.N., BANERJEE, A., MCNAMEE, C.G. & SZARKA, L.J. (1995) Stereoselective Acetylation of 3,4-Dihydro-3,4-dihydroxy-2,2-dimethyl-2H-1 benzopyran-6-carboni-trile. Tetrahedron: Asymmetry, 6, 123–130. PRESS, J.B., MCNALLY, J.J., SANFILIPPO, P.J., ADDO, M.F., LOUGHNEY, D., GIARDINO, E., KATZ, L.B., FALOTICO, R. & HAERTLEIN, B.J. (1993) Novel Thieno [2,3-b]— and [3,4-b] Pyrans as Potassium Channel Openers. Thiophene Systems-XVII. BioMed. Chem. Lett., 1, 423–435. PRESS, J.B., SORGI, K.L., MCNALLY, J.J. & LEO, G.C. (1994) An Unusually Selective Diels-Alder Dimerization of a [4n + 2] Electrocyclic Ring-Opened Thieno [3,2-b]pyran. J. Org. Chem., 59, 5088–5089. SATO, H., KOGA, H., ISHIZAWA, T., MAKINO, T., KUROMARU, K., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993) Vasorelaxant Activity of 2Substituted 6-Nitro-2H-l-Benzopyran-4-carbothioamide K+ Channel Openers. BioMed. Chem. Lett., 3, 2627–2630. SATO, H., KOGA, H., ISHIZAWA, T., MAKINO, T., TAKA, N., TAKAHASHI, T. & NABATA, H. (1995) Vasorelaxant Activity of 2-Fluoroalkyl-6-nitro-2H-lbenzopyran-4-carbothioamide and Carboxamide K+ Channel Openers. BioMed. Chem. Lett., 5, 233–236. SOLL, R.M., DOLLINGS, P.J., MCCAULLY, R.J., ARGENTIERI, T.M., LODGE, N., OSHIRO, G., COLATSKY, T., NORTON, N.W., ZEBICK, D., HAVENS, C. & HALAKA, N. (1994) N-Sulfonamides of Benzopyran-Related Potassium Channel Openers: Conversion of Glyburide Insensitive Smooth Muscle Relaxants to Potent Smooth Muscle Contractors. BioMed. Chem. Lett., 4, 769–773. TAKA, N., KOGA, H., SATO, H., ISHIZAWA, T., TAKAHASHI, T. & IMIGAWA, J-i. (1994) Vasorelaxant Activity of 2-FluoromethylBenzopyran K+ Channel Openers. BioMed. Chem. Lett., 4, 2893–2898. TAKAHASHI, T., KOGA, H., SATO, H., ISHIZAWA, T., TAKA, N. & IMIGAWA, J-i. (1994) Synthesis and Vasorelaxant Activity of N-Imino-2-(Benzopyran-4-yl) Pyridine K+ Channel Openers. BioMed. Chem. Lett., 4, 2899–2902.

2 Structure-Activity Relationships of Benzopyran Based Potassium Channel Activators J.M.EVANS & G.STEMP SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. 2.1 Introduction Structure-activity relationships (SARs) are derived by considering the pharmacological response to the administration of an analogous series of compounds. Thus the effects of systematic structural changes are quantified and compared, and optimal activity correlated with a specific set of structural characteristics in the molecule. These may take the form of such parameters as the nature and location of specific chemical groups and stereochemical features. As the published activity data on the differing series of ATP-sensitive potassium channel activators (KCAs), or openers, has increased, so have SARs continued to accumulate for the benzopyran, or chroman, based KCAs derived from the pioneer compound cromakalim (CRK), 1. The topic has been reviewed specifically (McCaully, 1991), and formed the major part of a chapter devoted to the SARs of all KCA structural types (Evans et al., 1992). In this review of benzopyran KCA SARs the earlier work will be included, but emphasis will be placed on more recent findings. As has been stated previously (Evans et al., 1992), exact comparisons between analogues described in different studies are not easily made, particularly because of the diversity of indications for which the KCAs are claimed. Nevertheless, compounds can be ranked for potency, because in most studies comparisons have been made with CRK or its biologically active 3S,4R enantiomer levcromakalim (LCRK) 2, or the other lead compounds described later. In the majority of studies compounds have been compared for their in vivo effects on rat vascular smooth muscle, or guinea-pig tracheal smooth muscle, or for in vivo effects following oral administration to the spontaneously hypertensive rat (SHR). In addition there have been more recent reports on studies of KCA induced relaxation of rat detrusor muscle. This confirms that there are studies in progress aimed at the generation of KCA molecules that have selective effects on smooth muscle types other than those of the vasculature and airways. It is clear that highly desirable targets for medicinal chemistry are KCAs with appropriate selectivity for the different disease states involving

K CHANNELS AND THEIR MODULATORS 33

smooth muscle and K channel dysfunction, and the study of SARs is one facet in this process. This chapter, on the most highly studied series of KCAs, is conveniently considered in four parts. The first will discuss stereochemical aspects of the benzopyran KCAs. The second will deal with the variation in the substituents located at the C(4)-C(2) positions of the pyran ring. The third will detail the ring systems that have been used to replace the benzopyran or chroman nucleus and the fourth will cover the effect of aromatic substitution in the benzopyran and related ring systems. 2.2 Stereochemistry in the Benzopyran Series The stereochemistry of the substituents located at positions 3 and 4 in the benzopyran nucleus and the equivalent positions in other ring systems is of key importance in determining the degree of potency in this series of KCAs. CRK 1 is a racemate, with the pyrrolidinone substituent at position 4 and hydroxyl group at position 3 trans to one another. Antihypertensive activity in SHR was found to be derived principally from the 3S,4R enantiomer, LCRK 2, which was about 100-fold more potent than the 3R,4S enantiomer 3 (Ashwood et al., 1986). Other studies (Buckle et al., 1992; Quagliato et al., 1991; Bergmann et al., 1990; Sanfilippo et al., 1992; Attwood et al., 1992) support this observation. CRK 1 was also shown to be tenfold more potent than the cis racemic isomer in the SHR (Ashwood et al., 1986), a finding also reported in other analogous pairs of compounds (Bergmann and Gericke 1990; Sanfilippo et al., 1992). A comparison has been made of the in vitro potencies of the enantiomers 2 and 3 of CRK with the corresponding chromans 4 and 5 lacking the 3-hydroxyl function, in terms of their ability to inhibit 20 mMKCl induced contractions in the rat isolated portal vein (see Table 2.1). There is little difference in potency between the chiral chromans 4 and 5, but the incorporation of a trans hydroxyl group, in the 4R chroman to give LCRK 2 enhanced the potency about tenfold, whereas in marked contrast the 3R hydroxyl group insertion into chroman 5 lowered the potency about tenfold. Hence it was concluded that the stereochemistry of the hydroxyl group at position 3 accounted for a major part of the chiral discrimination between the enantiomers of CRK (Attwood et al., 1992). The enantiomers of the cis isomer of CRK, compounds 6 and 7, were shown to be KCAs by their ability to stimulate 86Rb+ efflux from rat isolated aorta in a glibenclamide-sensitive manner (Quast and Villhauer, 1993). They were compared with the enantiomers of CRK in several in vitro screens including the inhibition of spontaneous contractions in the rat isolated portal vein (Table 2.1). Both cis enantiomers 6 and 7, and the trans 3R,4S enantiomer 3 were of similar potency, and 100-fold less potent than the trans 3S,4R enantiomer LCRK 2. Clearly the incorporation of cis configured 3-hydroxyl groups in benzopyrans

34 K CHANNELS AND THEIR MODULATORS

does not have the same stereochemical discriminatory effect as that of a trans configured hydroxyl group. As the stereochemistry of the substituents at positions 3 and 4 is of prime importance, with the exception of achiral chromene based KCAs discussed later, most compounds now in development are single 3S,4R enantiomers, or their equivalent in other ring systems. One notable exception to this is S 0121 8, a compound that might not be expected to lower blood pressure (BP) to any great extent, due to its less favoured 3R,4S-

Table 2.1 In vitro potencies of the stereoisomers of CRK; comparison with the isomeric chromans Compound No

Stereochemistry

Rat Portal Veina IC50 μ M

Rat Portal Veinb pD2

2 3S,4R 0.074 ±0.002 7.89 ±0.03 3 3R,4S 6.6 ±0.7 5.66 ±0.08 4 R 1.15±0.16 — 5 S 0.52 ±0.05 — 6 3S,4S — 5.80 ±0.05 7 3R,4R — 5.39 ±0.09 aRelaxation of 20 mMKCl induced contraction. Attwood et al., 1992. b Inhibition of spontaneous activity. Quasi and Villhauer, 1993.

K CHANNELS AND THEIR MODULATORS 35

stereochemistry. Compound 8 is reported to reduce the amplitude of spontaneous contractions of the guinea-pig isolated ureter, implying an ability to ease the passage of kidney stones through the ureter while not altering BP (Bartmann, 1989, Klaus et al., 1991). Additional studies, however, suggest that compound 8 may exert its relaxant activity in the ureter by a mechanism other than K channel activation (Englert et al., 1990). A subsequent publication from the Manchester smooth muscle group (Edwards et al., 1991) indicated that compound 8 is about eightfold more potent as an inhibitor of spontaneous contractions of rat isolated portal vein than of KCl induced contractions in rat isolated detrusor muscle and questions the implied selectivity of the compound. Geometric isomerism, encountered in acyclic tertiary amides, will be considered in section 2.3.1, while conformational differences in the cyclic amides or lactams will be more appropriately considered in Chapter 4.

36 K CHANNELS AND THEIR MODULATORS

2.3 Variation in Substituents on the Pyran Ring This section of the chapter is conveniently considered in terms of changes at the C(4)-C(2) benzopyran positions. Changes made at the equivalent positions in other ring systems that have been used as replacements for the benzopyran ring are assumed to produce similar effects, unless they are specifically detailed. Changes at position-1 in the benzopyran ring system are not discussed in this section as they lead to several of the alternative ring systems that will be described in section 2.4. 2.3.1 Position 4 Although novel compounds have been sought by variation at all of the positions on the benzopyran ring system, and most of the equivalent positions on other ring systems, by far the most investigated position has been the 4-position. It has also been one of the most fruitful as there have been many variations of the pyrrolidinone group of CRK. The carbonyl group of CRK was considered to be pivotal in conferring activity as lactams (Ashwood et al., 1986) were more potent in the SHR than the original cyclic amines (Evans et al., 1983). The cyclic amines show little in vitro activity, and evidence is available to show that their metabolism to lactams occurs in vivo (Evans and Stemp, 1991). Position 4 is best reviewed by considering first, lactams and substituents containing carbonyl groups in the usual – alpha—location flanking the point of attachment to the benzopyran and second, replacements that may contain a carbonyl group but not in the usual location. Lactams and other β -carbonyl containing replacements The size of the lactam ring was found to have an important effect on potency (Ashwood et al., 1986; Buckle et al., 1990), with 6-membered lactams, as in compound 9 (see Table 2.2), conferring a superior potency to 5-membered analogues, that in turn were markedly superior to 4-, 7- or 8-membered rings. This supremacy of the 6-membered ring lactam has been found in all the series of KCAs investigated thus far. When unsaturation was introduced into the 6membered lactam, as in compound 10 (see Table 2.2), it was found that potency was enhanced

K CHANNELS AND THEIR MODULATORS 37

Table 2.2 Effects of heteroatom substitution in pyrrolidinones, piperidinones and pyridones

SHR Max decrease in blood pressure Compound

X

m

n

Dose mg/kg

%a

mm Hgb

CRK

CH2

1

1

9 10 14 15 16 17 18 19 20 21 22

CH2 CH O S O NH S N N N N

1 1 0 0 1 1 1 3 2 1 0

2 2 2 2 2 2 2 0 1 2 3

0.3 1.0 0.1 1.0 0.3 0.3 0.3 0.1 10.0 1.0 1.0 1.0 1.0

39±3 ― 46±7 — 20±4 27±3 26±3 28±3 12±4 ― — — —

― 78±6 — 108±1 ― — ― — — 77±5 19±11 75±17 20±7

aAshwood

et al., 1986. bBergmann and Gericke, 1990.

over the pyrrolidinone (Bergmann and Gericke, 1990; Buckle et al., 1990). Indeed the pyridone group has been adopted as the C(4) substituent in the lead compounds UR-8225 11, bimakalim 12 and emakalim 13.

Heteroatoms have been incorporated in pyrrolidinones, piperidinones and pyridones but in general these inclusions have not increased potency (Ashwood et al., 1986; Bergmann and Gericke, 1990) in the SHR (Table 2.2). Hence the oxazolidinone 14 and thiazolidinone 15 compounds were slightly less potent than CRK, while the morpholinone 16 and piperazinone 17 compounds were less

38 K CHANNELS AND THEIR MODULATORS

potent than the piperidinone homologue 9 of CRK. As the thiamorpholinone compound 18 was about 60-fold less potent than the morpholinone analogue 16, this difference was ascribed originally to the effective ring size of the substituent surpassing the optimum ring size of six (Ashwood et al., 1986) but subsequently (Taylor, S.G., personal communication) it was found that compound 18 demonstrated good potency in relaxation of guinea pig isolated portal vein, and this suggests the intervention of pharmacokinetic factors on administration to the SHR. Replacement of the pyridone group in compound 10, the racemate of emakalim 13, by the pyridazinone group in compound 19, or the pyrazinone group of compound 21, lowered potency by about one quarter, while the pyrimidinone compounds 20 and 22 were also less potent than the parent pyridone compound 10. Thus it is not surprising that to date no lead compounds for development have emerged with additional heteroatoms in these particular cyclic groups. Unsaturation has also been introduced into the 5-membered lactam ring, the enol ether 23 being described (Genain and Pinhas, 1990) as the most potent of the series, but as the standard CRK was not included in the study, it cannot be determined if unsaturation has the same potency enhancing effect in 5as in 6-membered rings.

In a similar manner substituent attachment to the different rings described above does not generally lead to enhancement of activity, but two compounds have appeared that have merited further evaluation as development candidates. S 0121 8 has already been noted for its interesting stereochemical features (section 2.2), but its related 3S,4R,5’R/3R,4S,5’S racemate 24 (Englert et al., 1988) is also worthy of comment. Compound 24 is said to be twice as potent as CRK in lowering BP in SHR, and assuming, not unreasonably, that this enhanced potency is due to the 3S,4R,5’R-enantiomer, then as the only structural difference between this enantiomer and LCRK is the 5’R methyl group, it must be responsible for the doubling of potency. The possible effect that this methyl group has on the conformation of the 4-substituent is discussed in Chapter 4. The second compound that bears an additional substituent on the lactam is the isoindolone celikalim 25 which is characterised by the additional fused aromatic ring. Other variants of this compound contain heteroatoms in this additional ring, but little is known of their influence on SAR. Compound 25 is as potent as CRK in lowering BP in the SHR, but it possesses two interesting differences in profile

K CHANNELS AND THEIR MODULATORS 39

compared with CRK, in that it has a slow onset to maximum effect at about 4 hours and a prolonged effect of greater than 24 hours (Quagliato et al., 1991; Oshiro and Colatsky, 1991) that may be ascribed to its increased lipophilicity over CRK (Soll et al., 1991). Certainly a slow onset to maximum effect could be advantageous in man, but whether such a prolonged effect could offer an advantage remains to be seen. In comparison it was observed that the presence of the fused aromatic ring in the homologous isoquinolone 26 diminished the potency compared with the piperidinone (Ashwood and Evans, 1985).

Acyclic amido groups were found to be effective isosteric replacements for the lactam ring. In benzopyrans containing acyclic amides (Ashwood et al., 1990), optimal activity was observed for the acetamide 27 (R1=H, R2=Me). The corresponding formamide (R1=R2=H) and analogues with larger R1 and R2 alkyl groups were less potent. In those analogues bearing alkyl groups at both positions, NMR evidence was presented to show that the orientation of the amide carbonyl group, discussed in depth in Chapter 4, is important in conferring activity in this series. Thus the 1H NMR spectrum of compound 27 (R1=R2=Me) contained two sets of signals, whereas the secondary amide 27 (R1=H, R2=Me) contained only one set of signals. Variable temperature NMR showed that the two sets of signals in the tertiary amide coalesced at about 420 K, that corresponded to an energy barrier to interconversion of 18–20 kcal/mol. Since compound 27 (R1=R2=Me) was about threefold less potent than the secondary amide, it was concluded that geometric isomerism about the tertiary amide bond was responsible for the NMR effects, and that the reduced potency was due to the presence of an isomer lacking the required geometry about the amide bond. The search for KCAs with differing cardiovascular profiles of activity based on simple acetamides has resulted in the discovery of Y 27152 27 (R1=PhCH2O, R2=Me) that exerts its pharmacological effect via an active metabolite Y 26763 27 (R1=OH, R2=Me) that is formed by hepatic cytochrome P450 mediated debenzylation in vivo (Nakajima et al., 1992). The consequences of this metabolism are a slower onset to maximum antihypertensive effect and reduced incidence of tachycardia.

40 K CHANNELS AND THEIR MODULATORS

The nature and optimal location of the carbonyl group at position 4 have been considered by several groups of investigators. Conversion to the methyl ketone 28 (R=Me) or relocation of the flanking nitrogen atom on the opposite side of the carbonyl group in compound 28 (R=NHMe) gave compounds of lower potency (Ashwood et al., 1991). It was considered that the insertion of an sp3 carbon as an attachment point to the benzopyran had the effect of altering the configuration of the carbonyl oxygen by moving the substituent out of the orthogonal relationship with respect to the benzopyran nucleus (Cassidy et al., 1989). This conformational effect is considered in more detail in Chapter 4. The amide has been replaced by a ‘reversed’ thioamide in a compound 29 that combines the key pharmacophoric features of aprikalim 30 and CRK and has been shown to be a potent KCA in in hibiting spontaneous tone in guinea pig isolated trachea with an IC50 of 0.14 μ M (Arch et al., 1991). The 6-nitro analogue of compound 29 was found to be tenfold more potent than compound 29 itself in relaxing 30 mM KCl induced contractions in rat aorta (Ishizawa et al., 1993), and from these papers and others (Koga et al., 1993a, 1993b) it is clear that extension of the terminal alkyl group beyond methyl attenuates potency, a situation not unlike that seen in the aprikalim 30 series of compounds, discussed in Chapter 3. However, despite this apparently restrictive SAR, KC-399 31 (Koga et al., 1993c), with an elongated side chain, was about 500-fold more potent than LCRK in relaxing 30 mM KCl induced contraction in the rat aorta. It also possessed a slow onset to maximum effect in the SHR, although this may be a consequence of the presence of the fluorinated alkyl groups discussed in section 2.3.3.

Although, as indicated above, increasing the carbonyl-flanking alkyl substituent size attenuated activity, in a series of benzamides 32 (R=H) good antihypertensive activity was retained. The phenyl ring could be replaced by a variety of heteroaryl rings, with optimal potency in SHR found for 2-, or 3-furyl

K CHANNELS AND THEIR MODULATORS 41

and 2-pyrrolyl rings (Ashwood et al., 1990). Benzamides have also been incorporated in thieno[3,2-b]pyrans such as compound 33 (Sanfilippo et al., 1993) that is described as threefold more potent than CRK in SHR, possessing an ED30 of 0.06 mg/kg following oral administration to the SHR. The effect of a range of substituents on the phenyl ring has been examined. Electron donating groups such as methyl and methoxy attenuated activity, but the electron withdrawing nitro group maintained activity, although the trifluoromethyl group surprisingly conferred marginal activity, particularly when halo substituents such as chlorine and fluorine provided the most potent analogues. The high potency due to halogen substituents was particularly marked for the para substituted analogues. It is interesting that in this thieno[3,2-b]pyran series, heteroaryl replacement of the phenyl ring by pyridyl or furanyl groups diminished activity, in contrast to the benzopyran series where levels of activity were maintained on such substitutions. Recently benzamides such as compound 32 (R=F) have been disclosed (Blackburn et al., 1993) that possess anticonvulsant and antihypertensive activities when examined orally for their effects in the mouse maximal electroshock test and intravenously in the SHR at a dose of 10 mg/kg. It was discovered that the antihypertensive effects were associated mainly with the 3S, 4R enantiomer of compound 32 (R=F) while the anticonvulsant effects were mainly confined to the 3R,4S enantiomer. Since the antihypertensive effects are normally ascribed to the opening of KATP channels, future reports of the anticonvulsant mechanism of action of the 3R,4S enantiomer of benzamide 32 (R=F) are awaited with interest.

Urea based substituents have also been incorporated in lieu of lactams at position 4, and for example, methylurea 34 (R=Me) is of similar potency to the acetamide 27 (R1=H, R2=Me), although the corresponding thiourea is about tenfold less potent. Extension of the terminal alkyl group beyond methyl lowers potency, so that compound 34 (R=t-Bu) is at least a 100-fold less potent than the

42 K CHANNELS AND THEIR MODULATORS

parent methyl urea 34 (R=Me). Thus it was established that ureas are effective bioisosteres of the lactam and acyclic amide groups (Ashwood et al., 1990).

Lactam replacements lacking β -carbonyl groups With the knowledge that ureas are lactam isosteres, and noting the highly favourable replacement of urea and thiourea groups in the pinacidil 35 series of KCAs (Petersen et al., 1978) by the cyanoguanidine group, several investigators have independently proposed the incorporation of this latter group into benzopyran based KCAs, both in acyclic and cyclic analogues (Shiokawa et al., 1989; Burrell et al., 1990a, 1993; Atwal et al., 1992). This proposition of common pharmacophoric features presumes that the CRK and pinacidil 35 series of KCAs act at the same putative receptor. In addition to this information, use was made of two-dimensional electrostatic potential mapping that enabled N, N’ -dimethylcyanoguanidine to be compared with N,N’ -dimethylurea and Nmethylpyrrolidinone (Stemp and Evans, 1993) and prompted the synthesis and evaluation of the cyanoguanidine 36 (Table 2.3), that was found to be about one third as potent as CRK both in in vivo and in vitro assays. Table 2.3 Effect of the terminal group R1/R2 on the activity of 4-cyanoguanidinobenzopyrans SHRa Compound No

R1

R2

dose mg/kg

Max fall in BP %

Rat aortab IC50 μ M

CRK 36 37 38 39f 40 41

― H H H H H –(CH ) – 2 4

― Me H Et CHMe2 t-Bu ―

0.3 1.0 1.0 — — 10.0 —

39±3C 41d 48e ― — 10±2

0.055 0.20 — 0.38 29.8 — 0.034

aBurrell

et al., 1993. of methoxamine-induced contractions. Atwal et al., 1992. cAshwood et al., 1986. dResult from one of a group of five SHR. eResults from two of a group of five SHR. fBMS-189365, U-89232.

bRelaxation

K CHANNELS AND THEIR MODULATORS 43

Interestingly, the compound 37 lacking the terminal methyl group retained the level of potency of compound 36 in contrast to the acyclic amides discussed previously, where the formamide was markedly less potent than the acetamide. However, increase of the terminal methyl group size as in compounds 38–40 caused potency to decline, a trend that has already become apparent in simple acyclic amides and ureas. However the highest in vitro potency, greater than that of CRK, was observed when a cyclic substituent was incorporated as a terminal group as in compound 41. It has been observed that ATP-sensitive KCAs have direct cardioprotective properties that are independent of their antihypertensive vasodilatory action (Grover et al., 1989; Auchampach et al., 1992), and of particular interest was the report that compound 39 had the property of reducing infarct size in the anaesthetised rabbit occluded coronary artery model (Toombs et al., 1992), while not displaying antihypertensive effects as confirmed by its lack of in vitro activity (Table 2.3). Other KCAs have been reported to possess this cardioprotective property while displaying minimal antihypertensive potency. For example, the cyanoguanidine 42 (R=H) was shown to be equipotent with CRK as an antiischaemic agent. This was demonstrated by its ability in vitro to increase the time to contracture in rat isolated globally ischaemic heart, while being about 40-fold less potent than CRK in relaxing methoxamine-induced contractions in the rat isolated aorta (Atwal et al., 1993). Both effects were blocked by glibenclamide 43, indicating that both actions are mediated by K channel opening. Preliminary SAR work on compound 42 (R=H) indicated that the antiischaemic activity resided principally in the 3S,4R enantiomer BMS-180447, whereas, in surprising contrast to the usual SAR in benzopyran KCAs (see section 2.2), the two enantiomers of compound 42 (R=H) were of similar potency in relaxing methoxamine-induced contractions in rat isolated aorta. Unfortunately, extensive metabolism to the para-hydroxyphenyl analogue in rats precluded further progression of this compound, and so other para substituted analogues 42 were evaluated to determine whether this metabolism could be blocked. Both the para-fluoro and para-chloro analogues were slightly superior in potency as antiischaemic agents to BMS-180447 42 (R=H). On the basis of a

44 K CHANNELS AND THEIR MODULATORS

superior half-life, BMS-180488 42 (R=para-Cl) was selected for further studies in additional models of ischaemia, although no clear indication was given as to its absolute stereochemistry. It has been confirmed since (Atwal, communication at the K channel symposium, ACS meeting, San Diego, 1994) that BMS-180488 is the 3S,4R enantiomer.

Cyclic analogues of the cyanoguanidines, and their thio analogues have also been disclosed. The direct analogue 44 (X=NH) was about an order of magnitude less active than CRK in relaxing methoxamine-induced contraction in rat isolated aorta (Atwal et al., 1992), while the cyanoiminothiazolidine FR 119748 44 (X=S, 3S,4R enantiomer) was twice as potent as CRK in the SHR (Shiokawa et al., 1989). Cyanoamidines have also emerged, typified by KP-294 45 (Muraoka et al., 1991) a compound of similar potency to CRK in the SHR, together with their cyclic analogues such as SR 47063 46 (Martin et al., 1993) that is about tenfold more potent than CRK in its effect on bronchial smooth muscle. The point of attachment of the amidine group has been altered, much in the same way as the thioamide of compound 29, to give rise to compounds such as 47. This compound possesses a remarkable selectivity of greater than 200-fold for the relaxation of spontaneous tone in the guinea pig isolated trachealis compared to the inhibition of 30 mM KCl induced contractions in the rat isolated aorta (Koga et al., 1993d), and results of in vivo studies with this compound are eagerly awaited. The presence of the N-dimethyl group is a critical feature in compound 47, as the removal of one methyl group abolishes the selectivity, while the incorporation of larger alkyl groups attenuates the activity. However, mechanistic studies of compound 47 revealed that the relaxant activity in the guinea pig isolated trachealis was non-competitively inhibited by glibenclamide 43, in contrast to the usual competitive blockade of the standard KCAs such as CRK. This suggests that an alternative binding site for compound 47 exists in the trachealis and that further mechanistic studies of compound 47 are indicated, and these are awaited with interest.

K CHANNELS AND THEIR MODULATORS 45

The principle of isosteric replacement at position 4 has been extended to include replacements for ureas and thioureas used in H2 receptor antagonists. This has resulted in the evaluation of a range of compounds 48 (Burrell et al., 1993) with differing substituents on the imino group. Thus replacement of the Ncyano group of 36 (Table 2.3), by carboxamide 48 (R=N-CONH2), acetyl 48 (R=N-COMe) or sulphonamide 48 (R=N-SO2NH2) produced a potency reduction that mirrored the loss of potency in H2 receptor antagonists (Cooper et al., 1990). But replacement of the cyanoimino group in compound 36 (Table 2.3) by the nitroethene group as in compound 48 (R=CHNO2), a change that was known to maintain potency in the H2 receptor antagonists, reduced the antihypertensive effect by some 30-fold, a finding confirmed by in vitro studies in guinea pig isolated portal vein. This result was surprising since the nitroethene group is an effective replacement for the cyanoimino group of pinacidil 35 (Manley and Quast, 1992). The replacement of key structural features in one series of KCAs cannot,

46 K CHANNELS AND THEIR MODULATORS

therefore, be expected automatically to confer activity in a structurally distinct series of KCAs, and suggests that these exquisitely subtle differences in SAR between the series may be a consequence of differing KCA molecule-receptor fits. Other urea isosteres that have been used in studies of H2 receptor antagonists include aminotriazoles (Cooper et al., 1990), and they have been incorporated in benzopyran KCA studies (Stemp and Evans, 1993). The presence of an Nmethyl group in triazole 49 was found to be pivotal in enhancing antihypertensive potency in SHR by an order of magnitude over the des-methyl compound 50, and threefold over CRK. NMR studies showed that the desmethyl compound 50 exists in the tautomeric form corresponding to that found in the isomeric N”-methyl analogue 51. Compounds 50 and 51 were of equivalent potency.

Three further examples of the successful employment of this line of research are recorded in the patent literature. The cyclobutenedione analogue 52 lowered blood pressure by 24% at an oral dose of 0.05 mg/kg (Stemp and Burrell, 1992), and the aminothiadiazole 53 relaxed carbachol-induced contractions in rat bladder with an IC50 of 4.8 μ M (Buters and Bagli, 1993), while the chloropyrimidine 54 relaxed spontaneous tone in guinea pig isolated trachea with an IC50 of 0.51 μ M (Stemp et al., 1991). In these examples further SAR data are not available.

The NH bridge has also been incorporated in simple anilines such as SR 46276 55 that show potential antidepressant activity but are devoid of cardiovascular effects (Garcia et al., 1990). However no SAR data have been published for these compounds to date.

K CHANNELS AND THEIR MODULATORS 47

The oxygen atom has been successfully used as a link to heterocyclic moieties at position 4, in two series of reasonably similar structure. The first is exemplified by SDZ PCO 400 56 that is reported to be equipotent with CRK, but possessing a longer half-life in a monkey model (Fozard et al., 1990), and capable of inhibiting KATP in pancreatic insulin secreting cells (Dunne, 1990). In contrast to this observation, CRK, pinacidil and aprikalim also open such channels but only at very high concentrations of >100 μ M, suggesting that at therapeutically administered doses these compounds will relax smooth muscle cells without having effects upon the regulation of insulin secretion. The second series of ethers is exemplified by EMD 57283 57 and the closely related 2-oxo-4-pyridyl analogue 58 (Bergmann et al., 1990) that is of similar potency to the racemate of compound 57 in the SHR. Removal of the N-methyl groups does not result in any significant change in the potency that remains in the 6–24 μ g/kg dose range to elicit a 30 mm Hg fall in mean BP, five to tenfold less than required of CRK. Other isomers of both the pyridyl and pyridazinyl series showed markedly lower potencies, and served to define a very tight set of SARs in this series of KCAs. More recently certain diazabicycloalkene derivatives, exemplified by compound 59, have been disclosed (Mimura and Kubo, 1993) that are derived from the EMD 57283 57 series. These compounds, where it appears that the cyclopropyl group acts as a bioisosteric replacement for the double bond, possess ED50 values in the range 0.0023–0.40 mg/kg.

48 K CHANNELS AND THEIR MODULATORS

Three entirely different approaches to those described so far in this section have also been documented. In the first, a 2-pyridyl N-oxide has been linked directly to the chromene nucleus in Ro 31–6930 60 (Attwood et al., 1991), and this appears to be the first instance of pyridine N-oxide and lactam bioisosterism. Interestingly, there are significant differences reported between CRK and compound 60. Thus the latter was an order of magnitude more potent than CRK in rat models of hypertension, although unlike CRK it did not reduce renal vascular resistance in the anaesthetised dog (Paciorek et al., 1990). Both compounds reduced renal vascular resistance in the anaesthetised rat, but only CRK exerted a selective action on the renal vascular bed (Duty et al., 1990). The importance of the N-oxide is illustrated by its replacement by a phenolic hydroxy group as in compound 61, that resulted in an approximate 100-fold loss in potency (Attwood et al., 1988). It is also interesting to observe high potency in a benzopyran analogue where the substituent is attached by carbon to position 4, and one can speculate that unlike the carbon attached compounds 28 discussed above, the sp2 nature at the point of attachment is partly responsible for this high potency. The second approach is based on the observation that the lactam ring of CRK prefers to adopt an orthogonal relationship with respect to the benzopyran nucleus (Cassidy et al., 1989), a likely feature of other benzopyran KCAs. In this approach (Gadwood et al., 1993), the relationship is fixed in

conformationally rigid spirocyclic benzopyran imidazolones 62–67 (Table 2.4). Evaluation of the compounds was by a membrane potential assay, where hyperpolarisation—a typical effect of KCAs—was detected by a decrease in fluorescent intensity of a voltage-sensitive dye. The nature of the substituent at position 2 of the spirocyclic group affected the level of potency of the compounds, as alkoxy- 62 and alkylamino- 64–67 compounds were more potent than alkylthio compound 63. The length and branching of the alkylamino substituent exerted an influence on potency, leading to optimal potency in the n-propyl compound 66. Compound 66 was slightly more potent than CRK in this test and it lowered mean arterial blood pressure in normotensive rats by about 50 mm Hg at a cumulative dose of 0.04 mg/kg whereas CRK caused a fall of about 23 mm Hg under the same conditions. Overlap comparison of computer-generated

K CHANNELS AND THEIR MODULATORS 49

structures of CRK and the tautomeric representation used here of compound 66 indicated that the imidazolone C=N nitrogen, adjacent to the carbonyl group, occupies the same space relative to the benzopyran ring as does Table 2.4 Effect of spirocyclic 2-substituted-imidazolones on membrane potential

Membrane Potential Assaya Compound No

R

concn (μ M)

fluorescence decrease %

CRK 62 63 64 65

― EtO EtS MeNH EtNH

66

n-PrNH

1.0 1.0 1.0 1.0 1.0 0.1 1.0 0.1 1.0

22 20 6 19 25 8 27 21 10

67 aA10

i-PrNH

cell line derived from rat embryonic rat aorta. Gadwood et al., 1993.

the carbonyl group of CRK, and it was thought that this particular nitrogen atom serves as a hydrogen bond acceptor group. If this is indeed the case then these rigid analogues support the notion that CRK most likely interacts with its putative receptor in a close to orthogonal conformation. The final approach utilised an appropriately substituted pyrrole as the substituent attached via nitrogen to position 4 (Smith et al., 1992). An exemplar of the series is compound 68 that is about twice as potent as CRK in relaxing the spontaneous tone of guinea pig isolated trachea. The pyrrole ring is thought to adopt an orthogonal relationship with respect to the benzopyran nucleus, much in the same manner as that envisaged for CRK, and the potency is derived from the substituted enamine function acting as a bioisostere for the pyrrolidinone of CRK. It is also of note that a related structure 69 was also classified as a KCA, although it was slightly less potent than CRK, despite the rigidity imposed in the molecule that precludes the attainment of an orthogonal disposition. This observation contrasts sharply with that of the rigid orthogonal spirocyclic compounds described in the second approach to benzopyran KCA design above.

50 K CHANNELS AND THEIR MODULATORS

2.3.2 Position 3 The hydroxyl group at this position has been shown to be a necessary feature for good activity, as its replacement by a hydrogen atom diminished activity (Ashwood et al., 1986). However, its elimination to form chromene analogues can in some instances provide potent KCAs. The topic of chromene analogues is discussed later in this section. Esterification of the hydroxyl group to give formate, acetate and nitrate groups (Ashwood et al., 1986; Houge-Frydrych and Evans, 1989; Bergmann and Gericke, 1990), and oxidation to the ketone (Buckle et al., 1991c) furnished analogues that retained the potency of CRK. However, a range of other substituents such as carbonyl, hydroxymethyl, nitro, fluoromethyl and formyl attenuated activity (Buckle et al., 1991c). The influence of the hydroxyl group stereochemistry at position 3 on the activity of benzopyran and other KCAs was discussed in section 2.2. A methyl group has been inserted in addition to the hydroxyl group at this position in the conventional CRK series (Gericke et al., 1991) but this caused an attenuation in the potency of relaxation of acetylcholine-induced contractions in pig coronary artery. But in marked contrast, in O-linked KCAs such as compound 70, potency was enhanced, and it is notable that this methyl group is incorporated in the closely related series of compounds exemplified by compound 59 above. It is thought that the effect of this methyl group is to modify the orientation of the C(4) substituent, and this topic is enlarged upon in Chapter 4.

K CHANNELS AND THEIR MODULATORS 51

Although the replacement of the hydroxyl group by a hydrogen atom diminished activity, it can be eliminated together with the hydrogen atom at position 4, to give chromenes such as compound 71, that retain the potency of CRK (Ashwood et al., 1986). By selecting this structural type of KCA, the question of stereochemistry at positions 3 and 4, and the necessity of stereoselective synthesis or resolution, discussed in Chapter 1, can be obviated. Thus, several analogous chromenes such as Ro 31–6930 60, KC-399 31, bimakalim 12 and SR 47063 46 have been designated as development candidates. However, the advantage of choosing chromenes for development may not be entirely the result of ease of synthesis, as certain chromenes have been found to be more potent than their chromanol counterparts. Indeed the potencies of the chromenes and the chromanols do not always parallel each other, a difference first seen in a close analogue of CRK, the 4-acetamide chromene 72 (Ashwood et al., 1990) that was about tenfold less potent than the corresponding chromanol.

This difference has subsequently been observed in other series. Thus compound 12 lowers mean arterial BP in SHR by approximately 20% more than the racemate of emakalim 13, and this differential appears to be a general characteristic of the pyridone series of KCAs (Bergmann and Gericke, 1990). In contrast, in the series of O-linked KCAs, the chromanols such as the racemate of HMD 57283 57 have a similar potency level to the racemate of chromanol 13, whereas the corresponding chromene 73 did not alter significantly BP in the SHR (Bergmann et al., 1990). In the indane series (Buckle et al., 1991a) the differences were variable, but in the thieno[3,2-b]pyran series dehydration led to compounds that were markedly less potent than the thienopyranols (Sanfilippo et al., 1992). To further complicate matters, in the tetralone series the introduction of unsaturation tended to enhance potency of blood pressure lowering ability and relaxation of guinea pig isolated trachealis (Almansa et al., 1993). While limited by the conditions of comparison of results obtained from in vivo and in vitro models, discussed in the introduction, one can speculate on the reasons for the differing potencies of closely related chromenes and chromanols. Thus it might be postulated that the loss of one molecular characteristic namely the hydroxyl group, that possibly aids binding to the putative receptor, is

52 K CHANNELS AND THEIR MODULATORS

compensated in some instances, but not in others, by a closer fit of other characteristics such as the amide carbonyl group at position 4, or the dialkyl group at position 2, that may enhance its binding capability at the receptor. A most unusual development at this position has been the discovery of KCAs with transposed trans C(3) and C(4) substituents, that retain the potency of CRK in the SHR (Cassidy et al., 1992). Optimal potency in SHR was associated with the presence of a urea or thiourea group at position 3, substituted by a bulky alkyl terminal group, as in compound 74 (R=t-Bu). Some interesting differences between this series and C(4) urea substituted series have emerged. Thus the presence of a terminal methyl group, as in compound 74 (R=Me) that is normally associated with optimal activity in conventional benzopyran KCAs, leads to a decrease in potency. The parallel with pinacidil terminal group SAR, discussed in Chapter 3, is also notable. Also in contrast to the CRK series, the piperidinone 75 (R1/R2=– (CH2)4-), acetamide 75 (R1=H, R2=Me) and benzamide 75 (R1=H, R2 =para F–Ph) displayed low levels of potency in the SHR. These are substituents that confer high potency when incorporated at position 4 in benzopyrans (Ashwood et al., 1986, 1990).

2.3.3 Position 2 In the series of KCAs based on the benzopyran system, the presence of alkyl or substituted alkyl groups at position 2 has been found to be crucial in conferring activity. In a study of dihydro, mono-, or dimethyl substituted compounds, optimal activity was associated with the dimethyl group (Ashwood et al., 1986). The dihydro compound was virtually devoid of activity, and confirmation of this observation comes from the 1,1-dimethylindane series (Buckle et al., 199la), where absence of this group from compound 76 diminished its ability to inhibit spontaneous contractions in guinea-pig trachealis by at least an order of magnitude. One possibility is that conformational differences between the dimethyl- and more flexible dihydro compounds are responsible for the difference in potency, although binding to a hydrophobic site on the putative receptor may also be of importance (Evans et al., 1992). The presence of the single methyl group at this position enhanced potency over the dihydro compound, but not to the levels associated with the dimethyl group (Ashwood et

K CHANNELS AND THEIR MODULATORS 53

al., 1986). Subsequently, both isomeric monomethyl analogues 77 and 78 of CRK have been studied (Buckle et al., 1991b) by NMR and X-ray crystallographic techniques, and although differences in conformation were elicited, they were not correlated with biological activity. A second study (Attwood et al., 1991) described both isomers as relaxants of 20 mM KCl induced contractions in rat isolated portal vein with an IC50>30 μ M, compared with an IC50 for CRK of 0.24 μ M. The conformation of the pyran ring in these isomers, discussed in more detail in Chapter 4, is described as a distorted half-chair in the β -methyl isomer 77, and a half-chair in the β -methyl isomer 78.

Higher alkyl (Buckle and Smith, unpublished results) and cycloalkyl groups at position 2 (Bergmann and Gericke, 1990) in general reduce potency in benzopyran-3-ols. In tetralones (Almansa et al., 1993) the data are somewhat equivocal, but in the two chromene analogues described, the tendency was to a reduced potency. However there are two exceptions to this reduction in potency on homologation of the gem-dimethyl group. One is the racemic 2-ethyl-2methyl analogue 79 that has twice the potency of Ro 31–6930 60 in relaxing rat isolated portal vein (Attwood et al., 1989). The second exception in the benzopyran-4-carbothioamide series (Sato et al., 1993) was exemplified by a wide range of alkyl substituents in compounds 80–87 (Table 2.5) that were examined for their relaxant effects on rat isolated aorta precontracted with 30 mM KCl. Increase in size of the dimethyl group in this series enhanced the potency in compounds 81–84, but reduced potency in the methyl/n-butyl combination in compound 85. Optimal potency was observed in compounds containing the methyl/n-propyl combination 82 and the cyclobutyl 86 and cyclopentyl 87

54 K CHANNELS AND THEIR MODULATORS

groups that were about ten thousand times more potent than CRK. The presence of a single large alkyl group, the t-butyl group in compound 84, was also sufficient to produce good potency, but enlargement of the cycloalkyl group to six membered or higher attenuated potency. Functionalised alkyl groups, in particular fluoroalkyl groups, have been inserted in KCAs such as KC-399 31. This compound was shown to possess a longer duration of action than LCRK in the SHR, with reduced tachycardia and a slower onset of action (Koga et al., 1993c). This property of longer duration of action is also shared

Table 2.5 Vasorelaxant property of benzopyran-4-carbothioamides; effect of the group at position 2

Compound No

R1

R2

Rat aortaa pEC50

CRK 80 81 82 83 84 85 86 87

― ― 6.77 ±0.03 Me Me 8.87 ±0.05 Me Et 9.74±0.15 Me nPr 10.77 ±0.34 Et Et 9.43 ±0.25 H tBu 9.40 ±0.30 Me nBu 7.39±0.11 ― (CH ) ― 10.68 ±0.12 2 3 ― (CH ) ― 10.60 ±0.21 2 4 a Negative log [M] required to relax rat isolated aorta pre-contracted with 30 mM KC1 at 50% Intrinsic activity for each compound. Sato et al., 1993.

by the gem-trifluoromethyl substituted compound 88 that was prepared during studies aimed at airways selective KCAs (Fenwick, 1993). The propensity to prolong the antihypertensive effects of KCAs in the SHR has been correlated with the lipophilicity of the agents (Soll et al., 1991), and this may be an important factor in the prolonged action of compounds 31 and 88.

K CHANNELS AND THEIR MODULATORS 55

2.4 Replacement of the Benzopyran Nucleus The replacement of the benzopyran ring system by a variety of other ring systems discussed below, has led to several new series of KCAs. In general, by the judicious choice of substituents the potency of certain analogues in these new series has been enhanced over the standard compounds CRK and LCRK. It is also notable that although the SARs of these published series have much in common with the benzopyran KCAs, there are certain significant differences that will be discussed under the appropriate sections. 2.4.1 Replacement of the Pyran Ring When the benzopyran nucleus was replaced by benzothiopyran in a 6-methyl analogue 89 (X=S) of CRK, potency was maintained (Smith, 1990), but oxidation to sulphoxide and to sulphone 89 (X=SO and SO2, respectively) gave less potent compounds. Replacement by the tetrahydroquinoline 90, and tetrahydronaphthalene 91 nuclei caused a decline in potency of about tenfold in BP lowering in SHR, compared with the benzopyran series (Ashwood et al., 1991). Using the indane skeleton as a benzopyran substitute as in compound 92 produced a series of analogues only slightly less potent than the benzopyrans in relaxing guinea-pig isolated trachealis (Buckle et al., 1991a). Besides ring contraction, ring expansion to benzoxepines such as compound 93 have been described (Buckle et al., 1991b), but this modification significantly reduced potency in relaxation of spontaneous tone in guinea-pig isolated trachealis.

56 K CHANNELS AND THEIR MODULATORS

In a recent paper, the tetrahydronaphthalen-1-one nucleus has been used to provide a series of KCAs, exemplified by UR-8225 11 (see section 2.3.1), but this variation also caused overall potency to be slightly diminished compared to the benzopyran series. Modification of the carbonyl group by reduction or oxime formation only served to reduce potency further. Compound 11, having an IC50 of 0.6 μ M against noradrenaline-induced contraction in rat isolated portal vein, is about twice as potent as LCRK in lowering SHR systolic BP. This high potency is probably the result of, in part, the presence of the 4-pyridone substituent. One of the most potent compounds in a series of 1,4-benzoxazines is the pyridine N-oxide analogue 94, YM-934, that was described as about tenfold more potent than CRK in inhibiting spontaneous contractions in rat isolated portal vein (Matsumoto et al., 1991). Little SAR detail is reported for this series, but it seems likely that the pyridine N-oxide group, a key feature of compound 59 discussed previously, is probably partly responsible for the high potency of compound 94. 1,3-Benzoxazines such as compound 95 have also appeared in the patent literature (Baumgarth et al., 1990) but without pharmacological data.

K CHANNELS AND THEIR MODULATORS 57

2.4.2 Replacement of the Aromatic Segment A study of electrostatic potentials indicated that the pyridine group could be a useful cyanophenyl surrogate in benzopyran KCAs (Stemp and Evans, 1993). Of a series of the four possible isomeric pyranopyridines 96–99, the pyrano[3,2-c] pyridine isomer 96, with the nitrogen atom occupying the key 6-position (see section 2.5) was found to possess the highest potency in the SHR (Burrell et al., 1990b). The corresponding N-oxide had a similar potency. The pyrano[2,3-c] pyridine analogue 97 was at least threefold less potent than the pyrano[3,2-c] pyridine 96, while the pyrano[3,2-b]- 98 and -[2,3–b]pyridines 99 were at least 20-fold less potent than the parent compound 96.

58 K CHANNELS AND THEIR MODULATORS

Details have emerged of the SARs of thieno[3,2-b]pyrans containing cyclic amido substituents at the 7-position, (equivalent to the benzopyran 4-position). The series is exemplified by the 2-nitro analogue 100, RWJ 29009, that is reported to be tenfold more potent than CRK in the SHR, and has potential in the indication of angina and ischemia. The 2-nitro group is thought to mimic the 6cyano group of CRK, while the thiophene replaces the CRK aromatic ring. Despite the difficulty of comparing results derived from different dosing regimes, it is clear that comparison of CRK and the thiophene equivalent 101 of CRK (ED30 in SHR of 0.19 and 0.07 mg/kg administered orally, respectively), that there is a differential in potency of about threefold in favour of the thiophene compound. Other comparative data tend to support this observation, that represents the first instance where an alternative ring system has been proved to confer superior antihypertensive potency to the benzopyran ring system (Sanfilippo et al., 1992).

Thieno[3,4–b]pyrans and thieno[2,3-b]pyrans have subsequently been studied to see if this interesting differential extends to all the thienopyran regioisomers (Press et al., 1993). The thieno[3,4-b]pyran 102 and the thieno[2,3-b]pyran 103 had similar potencies to the thieno[3,2-b]pyran 104, the three isomers reducing mean arterial BP in SHR by 49, 43 and 53% respectively on administration of an oral dose of 20 mg/kg. Thus by extrapolation it appears that the three thienopyran ring systems confer higher potency than the benzopyran ring system. The impact of addition of aromatic substituents to these thienopyrans is considered in section 2.5.

K CHANNELS AND THEIR MODULATORS 59

2.5 Aromatic Substitution Studies of the aromatic substituents have indicated that their position and nature are of prime importance in influencing the level of potency of benzopyran KCAs (Ashwood et al., 1986; Burrell et al., 1990b; Buckle et al., 1990). The original work (Ashwood et al., 1986) in SHR revealed that BP lowering was optimal when a nitro substituent was sited at position 6. Relocation to position 7 lowered potency by about tenfold, and to position 8 virtually abolished activity. In subsequent studies similar observations were made (Buckle et al., 1990) while observing the relaxation of spontaneous tone in guinea pig isolated trachealis. In addition it was noted that a trifluoromethyl group conferred high potency at position 6 but when located at position 5 potency declined markedly. Taken together these data indicate a regioselective preference for position 6, followed by position 7. Since some of the most potent benzopyran KCAs possess substituents located at the 6- and 7-positions of the benzopyran nucleus (see later) it is interesting to note the introduction of the benzoxadiazole ring in compound 105, NIP 121, as a replacement for the cyanophenyl group. No SAR data are available for this series but the modification enhances the potency of compound 105 tenfold over CRK in the SHR (Arakawa et al., 1990).

Optimal potency was originally associated with the presence of strong electron withdrawing groups such as nitro, trifluoromethyl, nitrile, acetyl, methoxycarbonyl, formyl and chloro, in approximate order of descending potency (Ashwood et al., 1986; Burrell et al., 1990b) in SHR. To these can be added the phenylsulphonyl group incorporated in rilmakalim (HOE 234) 106 that confers greater potency than the nitrile of CRK (Klaus et al., 1990), the trifluoromethoxy group of compound 25 that confers approximately 20-fold higher potency than the nitrile group (Soll et al., 1991), and the pentafluoroethyl group of BRL 55834 107 that confers a tenfold increase in potency over the nitrile group in relaxing spontaneous tone in guinea pig isolated trachealis (Buckle et al., 1990). Compound 107 is the first airways-selective KCA (Buckle et al., 1992). A possible source of this selectivity is its ability to open both KATP and BKCa in bovine trachealis (Ward et al., 1992), a property not shared by LCRK. In view of this requirement for electron withdrawing capability it was surprising to find that alkyl groups could also be incorporated at position 6, and

60 K CHANNELS AND THEIR MODULATORS

still retain good potency, the 6-ethyl group conferring about one third of the potency of the nitrile group in SHR (Burrell et al., 1990b). Thus it appears that an electon withdrawing group is not mandatory at position 6. The dimensions of the group have some influence on the degree of potency, an approximate order of activity being ethyl, isopropyl, t-butyl >n-propyl, cyclopentyl >methyl >phenyl. The strong electron withdrawing group requirement for optimal potency appears to hold good for the other ring systems described in the last section. However, one interesting exception, is the thieno[3,4-b]pyran nucleus. Thus insertion of bromo or acetyl groups on either side of the ring sulphur in compound 102, either individually or in combination, lowered the potency of the parent compound (Press et al., 1993). The difference between this situation and the effect of 5- and 8-substituents in benzopyran compounds which are broadly neutral is quite striking. As the highest potency in benzopyran KCAs is associated with position 6 and to a lesser extent with position 7, it prompted limited investigations of compounds bearing substituents at both these positions. Incorporation of an acetylamino, or amino substituent at position 7 in a 6-nitro compound enhanced potency, whereas the reverse combination gave rise to a reduced potency (Ashwood et al., 1986). In contrast to the pairing of an electron withdrawing and electron donating group, the combination of two electron withdrawing groups, exemplified by the 6-bromo-7-nitro- and 6-cyano-7-nitro-benzopyrans, is the most potent substitution pattern (Ashwood et al., 1990). In support of these observations, the 6,7-dichloro combination of groups was found to confer higher potency than either the 6-cyano-or 6-chloro groups in the tetralone series (Almansa et al., 1993). 2.6 Conclusions This chapter has illustrated the huge reported increase in SAR studies in compounds containing benzopyran and related ring systems that belong almost exclusively to the class of ATP-sensitive KCAs. While novel compounds are still being discovered as agents with even higher potency as antihypertensives and smooth muscle relaxants, there are studies in progress searching for compounds with differing profiles of activity and selectivity for different channels and smooth muscle types for the amelioration of a variety of disorders (Longman and Hamilton, 1992). There are tantalising glimpses of profile change and selectivity, ranging from the longer acting antihypertensive compounds such as celikalim 25, and the prodrug Y 27152 27 (R1=PhCH2O, R2=Me) that lowers blood pressure with little associated tachycardia, to the potential anticonvulsant benzamide 32 (R=F) and the enhanced airway selective compound BRL 55834 107. Fundamental to these searches are the examination of SARs that may give the first insights into profile change and selectivity that until now, with the few

K CHANNELS AND THEIR MODULATORS 61

exceptions noted above, have not been easily attained in this class of KCAs. It is clear that further SARs will be forthcoming and it is to be hoped that the discovery of suitable selectivity will enable the potential of this class of compounds to be realised. References ALMANSA, C, GOMEZ, L.A. CAVALCANTI, F.L., RODRIGUEZ, R., CARCELLER, E., BARTROLI, J., GARCIA-RAFANELL, J. & FORM, J. (1993) J. Med. Chem., 36, 2121–2133. ARAKAWA, C., YUKINORI, M., YOKOYAMA, T., KAWAMURA, N. & TANAKA, S. (1990) Jap. J. Pharmacol., 52, (suppl. 1) 311P. ARCH, J.R.S., BUCKLE, D.R., CAREY, C., PARR-DOBRZANSKI, H., FALLER, A., FOSTER, K.A., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G. & TAYLOR, S.G. (1991) J. Med. Chem., 34, 2588–2594. ASHWOOD, V.A. & EVANS, J.M. (1985) European Patent Application 158 923 to Beecham. ASHWOOD, V.A., BUCKINGHAM, R.E., CASSIDY, F., EVANS, J.M., FARUK, E.A., HAMILTON, T.C., NASH, D.J., STEMP, G. & WILLCOCKS, K. (1986) J. Med. Chem., 29, 2194–2201. ASHWOOD, V.A., CASSIDY, F., COLDWELL, M.C., EVANS, J.M., HAMILTON, T.C., HOWLETT, D.R., SMITH, D.M. & STEMP, G. (1990) J. Med. Chem., 33, 2667–2671. ASHWOOD, V.A., CASSIDY, F., EVANS, J.M., GAGLIARDI, S. & STEMP, G. (1991) J. Med. Chem., 34, 3261–3267. ATTWOOD, M.R., JONES, P.S. & REDSHAW, S. (1989) European Patent Application 298 452 to Hoffman-La Roche. ATTWOOD, M.R., JONES, P.S., KAY, P.B., PACIOREK, P.M. & REDSHAW, S. (1991) Life Sci., 48, 803–810. ATTWOOD, M.R., BROWN, B.S., DUNDSDON, R.M., HURST, D.N., JONES, P.S. & KAY, P.B. (1992) BioMed. Chem. Lett., 2, 229–234. ARAKAWA, C, YUKINORI, M., YOKOYAMA, T., KAWAMURA, N. & TANAKA, S. (1990) Jap. J. Pharmacol., 52 (suppl. 1) 3IIP. ATWAL, K.S., MORELAND, S., MCCULLOGH, J.R., AHMED, S.Z. & NORMANDIN, D.E. (1992) BioMed. Chem. Lett., 2, 87–90. ATWAL, K.S., GROVER, G.J., AHMED, S.Z., FERRARA, F.N., HARPER, T.W., KIM, K.S., SLEPH, P.O., DZWONCZYK, S., RUSSELL, A.D., MORELAND, S., MCCULLOUGH, J.R. & NORMANDIN, D.E. (1993) J. Med. Chem., 36, 3971–3974. AUCHAMPACH, J.A., MARUYAMA, M., CAVERO, I. & GROSS, G.J. (1992) Circulation, 86, 311–319. BARTMANN, W. (1989) In: Trends in Medicinal Chemistry, van der Groot, H., Domany, G., Pallos, L. & Timmerman, H. (eds). Elsevier, Amsterdam, pp. 629–657. BAUMGARTH, M., GERICKE, R., BERGMANN, R., DE PAYER, J. & LUES, I. (1990) German Patent Application DE 4 010 488 to E Merck. BERGMANN, R. & GERICKE, R. (1990) J. Med. Chem., 33,492–504.

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BERGMANN, R., EIERMANN, V. & GERICKE, R. (1990) J. Med. Chem., 33, 2759–2767. BLACKBURN, T.P., CHAN, W.N., EVANS, J.M., THOMPSON, M., UPTON, N. & VONG, A.K.K. (1993) Poster at 7th Medicinal Chemistry Symposium, Cambridge, UK. BUCKLE, D.R., ARCH, J.R.S., FENWICK, A.E., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G., TAYLOR, S.G. & TEDDER, J.M. (1990) J. Med. Chem., 33, 3028–3034. BUCKLE, D.R., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G. & TEDDER, J.M. (1991a) J. Chem. Soc. Perkin Trans. 1, 63–70. BUCKLE, D.R., ARCH, J.R.S., EDGE, C., FOSTER, K.A., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G., TAYLOR, J.F., TAYLOR, S.G., TEDDER, J.M. & WEBSTER, R.A.B. (1991b) J. Med. Chem., 34, 919–926. BUCKLE, D.R., EGGLESTON, D.S., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., READSHAW, S.A., SMITH, D.G. & WEBSTER, R.A.B. (1991c) J. Chem. Soc. Perkin Trans. 1, 2763–2771. BUCKLE, D.R., EGGLESTON, D.S., PINTO, I.L., SMITH, D.G. & TEDDER, J.M. (1992) BioMed. Chem. Lett., 2, 1161–1164. BURRELL, G., STEMP, G. & SMITH, D.G. (1990a) European Patent Application 359 537 to Beecham. BURRELL, G., CASSIDY, F., EVANS, J.M., LIGHTOWLER, D. & STEMP, G. (1990b) J. Med. Chem., 33, 3023–3027. BURRELL, G., EVANS, J.M., HICKS, F. & STEMP, G. (1993) BioMed. Chem. Lett., 3, 999–1002. BUTERS, J.A. & BAGLI, J.F. (1993) U.S. Patent 5 206 252 to American Home Products. CASSIDY, F., EVANS, J.M., SMITH, D.M., STEMP, G., EDGE, C. & WILLIAMS, D.J. (1989) J. Chem. Soc. Chem. Commun., 377–378. CASSIDY, F., EVANS, J.M., HADLEY, M.S., HALADIJ, A.H., LEACH, P.E. & STEMP, G. (1992) J. Med. Chem., 35, 1623–1627. COOPER, D.G., YOUNG, R.C., DURANT, G.J. & GANELLIN, C.R. (1990) In: Comprehensive Medicinal Chemistry: The Rational Design, Mechanistic Study and Therapeutic Application of Chemical Compounds, 3. Hansch, C., Sammes. P.O. & Taylor, J.B. (eds). Pergamon Press, Oxford, pp. 323–421. DUNNE, M.J. (1990) Potassium channels ’90—Structure, Modulation and Clinical Exploitation, IBC Conference, London. DUTY, S., PACIOREK, P.M., WATERFALL, J.F, & WESTON, A.H. (1990) Eur. J. Pharmacol., 185, 188–197. EDWARDS, G., HENSHAW, M., MILLER, M. & WESTON, A.H. (1991) Br. J. Pharmacol., 102, 679–686. ENGLERT, H.C., KLAUS, E., LANG, H.J., MANIA, D. & SCHOLKENS, B. (1988) European Patent Application 277 611 to Hoechst. ENGLERT, K.E., HROPOT, M., MANIA, D. & ZWERGEL, U. (1990) Eur. J. Pharmacol., 183, 673–674. EVANS, J.M. & STEMP, G. (1991) Chem. Brit., 27, 439–442. EVANS, J.M., FAKE, C.S., HAMILTON, T.C., POYSER, R.H. & WATTS, E.A. (1983) J. Med. Chem., 26, 1582–1589.

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EVANS, J.M., HADLEY, M.S. & STEMP, G. (1992) In: Potassium Channel Modulators: Pharmacological, Molecular & Clinical Aspects. Hamilton, T.C. & Weston, A.H. (eds). Blackwell Scientific, Oxford, pp. 341–368. FENWICK, A.E. (1993) Tetrahedron Lett., 34, 1815–1818. FOZARD, J.R., MENINGER, K., COOK, N.S., BLARER, S. & QUAST, U. (1990) Br. J. Pharmacol., 99, 7P. GADWOOD, R.C., KAMDAR, B.V., DUBRAY, L.A.C., WOLFE, M.L., SMITH, M.P., MlZSAK, S.A. & GROPPI, V.E. (1993) J. Med. Chem., 36, 1480–1487. GARCIA, G., DI MALTA, A. & SOUBRIE, P. (1990) European Patent Application 370 901 to Sanofi. GENAIN, G. & PINHAS, H. (1990) European Patent Application 377 966 to Syntex. GERICKE, R., HARTING, J., LUES, I. & SCHITTENHELM, C. (1991) J. Med. Chem., 34, 3074–3085. GROVER, G.J., MCCULLOUGH, J.R., HENRY, D.E., CONDER, M.L. & SLEPH, P.O. (1989) J. Pharmacol. Exp. Ther., 251, 98–104. HOUGE-FRYDRYCH, C.S.V. & EVANS, J.M. (1989) Patent Cooperation Treaty Application 89/05808 to Beecham. ISHIZAWA, T., KOGA, H., OHTA, M., SATO, H., MAKINO, T., KUROMARU, K., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993) BioMed. Chem. Lett., 3, 1659–1662. KLAUS, E., LINZ, W., SCHÖLKENS, B. & ENGLERT, H.C. (1990) NaunynSchmiedeberg’s Arch. Parmacol., 342, (suppl.) R17. KLAUS, E., ENGLERT, K.E., HROPOT, M., MANIA, D., RAJAGOPALAN, R. & ZWERGEL, U. (1991) Naunyn-Schmiedeberg’s Arch. Pharmacol., 344, R35. KOGA, H., SATO, H., ISHIZAWA, T., KUROMARU, K., MAKINO, T., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993a) BioMed. Chem. Lett., 3, 1115–1118. KOGA, H., OHTA, M., SATO, H., ISHIZAWA, T. & NABATA, H. (1993b) BioMed. Chem. Lett., 3, 625–631. KOGA, H., SATO, H., IMAGAWA, J., ISHIZAWA, T., YOSHIDA, S., SUGO, I., TAKA, N., TAKAHASHI, T. & NABATA, H. (1993c) BioMed. Chem. Lett., 3, 2005–2010. KOGA, H., SATO, H., ISHIZAWA, T., KUROMARU, K., NABATA, H., IMAGAWA, J., YOSHIDA, S.& SUGO, I. (1993d) BioMed. Chem. Lett., 3, 1111–1114. LONGMAN, S.D. & HAMILTON, T.C. (1992) Med. Res. Rev., 12, 73–148. MANLEY, P.W. & QUAST, U. (1992) J. Med. Chem., 35, 2327–2340. MARTIN, C.A.E., NALINE, E. & ADVENIER, C. (1993) Drug Develop. Res., 29, 63–72. MATSUMOTO, Y., TSUZUKI, R., MATSUHISA, A., TAKAYAMA, K., YODEN, T. & UCHIDA, W. (1991) Abstr. Papers Am. Chem. Soc. 202 Meet. Pt 1, MEDI 99. MCCAULLY, J. (1991) Current Drugs-Potassium Channel Modulators KCMB 5–19. MIMURA, T. & KUBO, H. (1993) European Patent Application 571 822 to Daiichi. MURAOKA, K., NAGAO, H., HORI, T., SAKAYA, S., HOSHINO, T., MIYAO, Y., MURAI, T., EDANAGA, M. & NAKANISHI, M. (1991) Jap. J. Pharmacol., 55, (suppl. 1) 341P. NAKAJIMA, T., SHINOHARA, T., YAOKA, O., FUKUNARI, A., SHINAGAWA, K., AOKI, K., KATOH, A., YAMANAKA, T., SETOGUCHI, M. & TAKARA, T. (1992) J. Pharmacol. Exp. Ther., 261, 730–736.

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OSHIRO, G.T. & COLATSKY, T.J. (1991) Current Drugs-Potassium Channel Modulators KCMB 20–28. PACIOREK, P.M., BURDEN, D.T., BURKE, Y.M. COWLRICK, I.S., PERKINS, R.S., TAYLOR, J.C. & WATERFALL, J.F. (1990) J. Cardiovasc. Pharmacol., 15, 188–197. PETERSEN, H.J., NIELSEN, C.K. & ARRIGONI-MARTELLI, E. (1978) J. Med. Chem., 21, 773–791. PRESS, J.B., MCNALLY, J.J., SANFILIPPO, P.J., ADDO, M.F., LOUGHEY, D., GIARDINO, E., KATZ, L.B., FALOTICO, R. & HAERTLEIN, B.J. (1993) BioMed. Chem., 1, 423–435. QUAGLIATO, D.A., HUMBER, L.G., JOSLYN, B.L., SOLL, R.M., BROWNE, E.N.C., SHAW, C. & VAN ENGEN, D. (1991) BioMed. Chem. Lett., 1, 39–42. QUAST, U. & VILLHAUER, E.B. (1993) Eur. J. Pharmacol.- Molecular Pharmacol. Secn., 245, 165–171. SANFILIPPO, P.J., MCNALLY, J.J., PRESS, J.B., FITZPATRICK, L.J., URBANSKI, M.J., KATZ, L.B., GIARDINO, E., FALOTICO, R., SALATA, J., MOORE, J.B. & MILLIER, W. (1992) J. Med. Chem., 35, 4425–4433. SANFILIPPO, P.J., MCNALLY, J.J., PRESS, J.B., FALOTICO, R., GIARDINO, E. & KATZ, L.B. (1993) BioMed. Chem. Lett., 3, 1385–1388. SATO, H., KOGA, H., ISHIZAWA, T., MAKINO, T., KUROMARU, K., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993) BioMed. Chem. Lett., 3, 2627–2630. SHIOKAWA, Y., TAKIMOTO, K., TAKENAKA, K. & KATO, T. (1989) European Patent Application 344 747 to Fujisawa. SMITH, D.G. (1990) J. Chem Soc. Perkin Trans. 1, 3187–3191. SMITH, D.G., BUCKLE, D.R., FALLER, A. & PINTO, I.L. (1992) BioMed. Chem. Lett., 2, 1595–1598. SOLL, R.M., QUAGLIATO, D.A., DEININGER, D.D., DOLLINGS, P.J., JOSLYN, P.L., DOLAK, T.M., LEE, S.J., BOHAN, C, WOJDAN, A., MORIN, M.E. & OSHIRO, G. (1991) BioMed. Chem. Lett., 1, 591–594. STEMP, G. & BURRELL, G. (1992) US Patent 5 147 866 to Beecham. STEMP, G., EVANS, J.M. & BURRELL, G. (1991) European Patent Application 431 741 to SmithKline Beecham. STEMP, G. & EVANS, J.M. (1993) In: Medicinal Chemistry 2nd. Edition. Ganellin, C.R. & Roberts, S.M. (eds). Academic Press Ltd, London, pp. 141–162. TOOMBS, C.F., NORMAN, N.R., GROPPI, V.E., LEE, K.S., GADWOOD, R.C. & SHEBUSKI, R.J. (1992) J. Pharmacol. Exp. Ther., 263, 1261–1268. WARD, J.P.T., TAYLOR, S.G. & COLLIER, M.N. (1992) Br. J. Pharmacol., 107, 49P.

Recent Literature ATWAL, K., GROVER, G.J., FERRARA, F.N., AHMED, S.Z., SLEPH, P.G., DZWONCZYK, S. & NORMANDIN, D.E. (1995) Cardioselective Antiischaemic ATP-Sensitive Potassium Channel Openers. 2. Structure-Activity Studies on Benzopyranylcyanoguanidines: Modification of the Benzopyran Ring. J. Med. Chem., 38, 1966–1973.

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BERGMANN, R. & GERICKE, R. (1994) The Influence of Substituents in 3-Position on the Activity of Chroman-Type Potassium Channel Openers. Arch. Pharm. (Weinheim), 327, 169–173. ISHIZAWA, T., KOGA, H., SATO, H., MAKINO, T., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1994) Substituent Effects of Benzopyran-4-(N-Cyano)carboxamidine Potassium Channel Openers for Selectivity to Guinea Pig Trachealis. BioMed. Chem. Lett., 4, 1995–1998. RUSSELL, K., BROWN, F.J., WARWICK, P., FORST, J., GRANT, T., HOWE, B., KAU, S.T., LI, J.H., MCLAREN, P.M., SHAPIRO, H.S. & TRIVEDI, S. (1993) A Highly Potent Series of Fluoroalkyl Benzoxazine Pyridine-N-oxide Potassium Channel Openers. BioMed. Chem. Lett., 3, 2727–2728. SATO, H., KOGA, H., ISHIZAWA, T., MAKINO, T., TAKA, N., TAKAHASHI, T. & NABATA, H. (1995) Vasorelaxant Activity of 2-Fluoroalkyl-6-nitro-2H-lbenzopyran-4-carbothioamide and Carboxamide K2+ Channel Openers. BioMed. Chem. Lett., 5, 233–236. SOLL, R.M., DOLLINGS, P.J., MCCAULLY, R.J., ARGENTIERI, T.M., LODGE, N., OSHIRO, G., COLATSKY, T., NORTON, N.W., ZEBICK, D., HAVENS, C. & HALAKA, N. (1994) N-Sulfonamides of Benzopyran-Related Potassium Channel Openers: Conversion of Glyburide Insensitive Smooth Muscle Relaxants to Potent Smooth Muscle Contractors. BioMed. Chem. Lett., 4, 769–773. TAKA, N., KOGA, H., SATO, H., ISHIZAWA, T., TAKAHASHI, T. & IMIGAWA, J-i. (1994) Vasorelaxant Activity of 2-FluoromethylBenzopyran K2+ Channel Openers. BioMed. Chem. Lett., 4, 2893–2898. TAKAHASHI, T., KOGA, H., SATO, H., ISHIZAWA, T., TAKA, N. & IMIGAWA, J-i. (1994) Synthesis and Vasorelaxant Activity of N-Imino-2-(Benzopyran-4-yl) Pyridine K2+ Channel Openers. BioMed. Chem. Lett., 4, 2899–2902.

3 Syntheses and Structure-Activity Relationships of Pyridine Based Potassium Channel Activators M.N. PALFREYMAN Rhône-Poulenc Rorer, Dagenham Research Centre, Rainham Road South, Dagenham, Essex, RM10 7XS, UK. 3.1 Introduction Potassium channels comprise the most diverse group of ion channels so far investigated. Amongst these, the ATP sensitive K channels (KATP channels) have been the most studied. During the last ten years compounds have been discovered which can activate or block these KATP channels. In particular K channel activators (KCAs) have been found, along with many other activities, to be smooth muscle relaxants with their main utility in hypertension and bronchodilatation. There are at least seven classes of activators (Edwards and Weston, 1990) of which the main four are shown in Figure 3.1. The benzopyrans, typified by cromakalim (CRK, shown as its (–) 3S, 4R-enantiomer levcromakalim (LCRK)), have been the class most thoroughly explored with more than thirty companies claiming patents. In comparison there have been few reports of structural modifications in the other classes of KCAs. This chapter covers the syntheses and structure-activity relationships (SARs) of the pyridine classes of KCAs: the thioformamides, cyanoguanidines and organic nitrates together with their hybrid molecules. A detailed description of SARs is complicated by the plethora of pharmacological models used to illustrate KCA-dependent potency. Where possible, potencies derived from isolated organ bath studies will be used supplemented by measurements of hypotensive effects in in vivo models of hypertension. The precise identity of the K channel responsible for the pharmacological effects of the compounds shown in Figure 3.1 is still unknown. It is therefore uncertain that all classes interact with the same site on a given channel or even with the same channel.

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 67

3.2 Thioformamides 3.2.1 Aprikalim and Isomers The discovery of aprikalim The discovery of aprikalim 1 (RP 52891), initially as the racemate RP 49356, arose from the study of a series of 2-(2-pyridyl)tetrahydrothiophene-2thiocarboxamides

Figure 3.1 Main closes of KATP Channel Activators

(Aloup et al., 1987) which had antisecretory and antiulcer activity, possibly resulting from inhibition of K+ /H+ ATP-ase and which led to the selection of picartamide 2 for further evaluation. As part of this work, a number of structural modifications were carried out including the synthesis of the isomeric 2-(3pyridyl)tetrahydrothiophene 3 (n=1). Whereas 3 (n=1) was inactive as an antiulcer and antisecretory agent, it possessed a marked blood pressure lowering activity when given orally (5 mg/kg p.o.) to spontaneously hypertensive rats (SHR). Further analogues were then synthesised (Aloup et al., 1990) and the tetrahydrothiopyran analogue 3 (n=2) was found to be more active (0.5 mg/kg) in the SHR screen. The onset of the antihypertensive effect was slow and the activity was found to be due to the sulfoxide metabolite. Aprikalim was subsequently synthesised and found to be active in the SHR screen at 0.05 mg/kg.

68 K CHANNELS AND THEIR MODULATORS

At this time it was not realized that aprikalim was exerting its pharmacological actions by K channel activation.

The synthesis of racemic RP 49356 and its cis analogue 10 is outlined in Scheme 3.1 starting from 3-chloromethylpyridine hydrochloride 4. Formation of the isothiouronium salt of 4 followed by alkylation gave the chlorosulfide 5. Oxidation Chemical class benzopyran thioformamide cyanoguanidine organic nitrates

Typical member cromakalim aprikalim pinacidil nicorandil

Origin SmithKline Beecham Rhone-Poulenc Rorer Leo Chugai

Scheme 3.1 Synthesis of RP 49356 and its cis isomer 10 The depicted stereochemistry is relative Reagents : (i) NH2CSNH2, EtOH; (ii) aq. NaOH. Br(CH2)4Cl; (iii) MCPBA, CH2CI2; (rv) KOtBu, THF; (v) a) NaNH2; b) MeNCS; (vi) a) KOtBu, THF; b) CS2, Mel; (vii) MeNH2, EtOH: (viii) P2S5, CH2CI2; (ix) MCPBA, CH2CI2; (x) a) MeNH2, EtOH; b) chromatography.

followed by cyclisation with base gave a cis/trans mixture of the thiopyran oxides 6 which could be separated by column chromatography. Treatment of the mixture or separated isomers with strong base followed by methyl isothiocyanate

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 69

gave the thioamide 1 (RP 49356). The thioamide 1 could also be synthesised indirectly from 6 via the treatment of the carbanion with CS2/MeI to give the dithioester 7 followed by treatment with methylamine. Both direct and indirect routes gave exclusively the isomer in which the thioamide is trans to the sulfoxide. The cis isomer 10 was synthesised from the dithioester 7 by reduction to the sulfide 8, which upon reoxidation gave a mixture of cis and trans sulfoxides 9. The mixture was condensed with methylamine to give, after chromatographic separation, the cis thioamide 10. The active enantiomer of RP 49356 (aprikalim) was synthesised by the route outlined in Scheme 3.2 (Aloup et al., 1990). The acid 11 was resolved via the tBu-L-prolinate amides and the R (–) enantiomer 12 converted to the thioamide 13. Conversion of 13 to the dithioester 8 was followed by oxidation to give a mixture of diastereomeric sulfoxides, from which the desired sulfoxide 7 was obtained by column chromatography. Amination of 7 with methylamine alforded aprikalim. Aprikalim is the laevo rotatory enantiomer of RP 49356 with an absolute configuration (1R, 2R) as determined by X-ray diffraction. KCA potency of aprikalim and close analogues Later studies have shown that 1 acts by activating the KATP channel (Mondot et al., 1988; Escande and Thuringer, 1989; Brown et al., 1992). A variety of screens have been described in the literature for the evaluation of KCAs. In the thioformamide series the in vitro screen is based upon the concentration (IC90) of compound required to relax by 90% the contraction induced by 20 mMKCl on rat aortic strip (Brown et al., 1992). The smooth muscle relaxation was reversed by the addition of

Scheme 3.2 Synthesis of Aprikalim The depicted stereochemistry is absolute Reagents : (i) a) SOCl2; b) tBu-L-protinate; c) cHCI; (ii) a) SOCI2; b) MeNH2; c) P2S5; (iii) a) BuLi, Mel; b) pyridine HCI, H2S; (iv) MCPBA; (v)MeNH2

the KATP channel inhibitor glibenclamide or by high concentrations of KCl (60 mM).

70 K CHANNELS AND THEIR MODULATORS

The KCA in vitro potencies of aprikalim and close analogues are collected into Table 3.1. Whereas the parent sulfide 13 is inactive, aprikalim exhibits an activity similar to cromakalim and twice that of RP 49356. The other enantiomer (1S, 2S) of RP 49356 and the racemic cis analogue 10 are both inactive. Stereochemistry and molecular modeling of aprikalim The absolute configuration of aprikalim has been established by X-ray determination (Brown et al., 1992) and the trans relationship between the thioamide and sulfoxide

Table 3.1 In vitro KCA activities of aprikalim analogues (IC90)μ M 13 1

10 cromakalim

RP 49356 aprikalim (1S, 2S) cis (racemate)

>30 0.7 0.4 >30 >30 0.2

confirmed. The authors also described conformational analysis studies with full geometry optimization using the semiempirical molecular orbital programmes MOPAC and the AM 1 Hamiltonian. It was found that several low-energy conformations could exist within a 2 kcal/mol energy band of the lowest energy structure. The global minimum energy conformation of aprikalim is depicted in Plate 1 and corresponds to that determined by X-ray crystallography. The energy difference between this conformation with an axial thioamide in contrast to an equatorial group is small (― E 1.7 kcal/mol), some stabilization coming from an internal hydrogen bond (S― O…H—N) as shown in Plate 2. Similar conformational studies on the inactive cis sulfoxide showed that the lowest energy conformation of the molecule has the thioamide group axial (Plate 3), but twisted almost 180° relative to aprikalim, so as to possibly form an internal hydrogen bond to the now equatorial sulfoxide oxygen (S― O…H—N, 2. 1 Å from MOPAC/AM1 calculations). Conformations with the orientation of the thioamide and pyridyl groups corresponding to those in aprikalim, which could be considered as representing the ‘active’ conformation, have an energy greater than 5 kcal/mol above the lowest energy structure. 3.2.2 Structural Modification of Aprikalim The syntheses and SARs of a series of aprikalim analogues have been reported (Brown et al., 1992). For purposes of discussion the structural modifications may

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 71

be conveniently divided into changes to the pyridine, thioformamide and thiopyran ring. Modification of the pyridine ring The analogues in which the 3-pyridyl group of aprikalim has been modified synthesised by routes similar to that outlined in Scheme 3.1 for RP 49356, were starting from the appropriate chloromethylaryl analogue of 4. The efficiency the conversion of the sulfoxide into its carbanion and subsequent thioacylation of dependent upon the choice of base and reaction conditions. In all cases was compounds in which the thioamide is trans to the sulfoxide were formed. only The KCA potencies of the analogues are listed in Table 3.2. Linkage of pyridyl ring at the 2- or 4- rather than the 3-position reduces potency but potency the was maintained when a 6-chloro substituent was introduced into the 3-pyridyl analogue. Whilst the 3-quinolyl analogue exhibited a tenfold increase in potency over 1, other heterocyclic replacements showed only modest potency. In the series, the enhancement of potency by the presence of an electron withdrawing aryl group in the meta position was demonstrated by the Cl, CF3, CN and substituted analogues when compared to the unsubstituted phenyl compound. F This potency is reinforced by the presence of an extra para Cl substituent. parallel with the 3-quinolyl analogue, the 2-naphthyl analogue was more potent In than the phenyl analogue, indicating either an electron withdrawing hydrophobic effect. or a Modification of the thioformamide group Analogues of aprikalim with modified thioamide groups were synthesised (Brown et al., 1992) by the methods outlined in Scheme 3.1 from the intermediate

Table 3.2 Aryl and heterocyclyl thioformamides R

IC90(μ M)

3-pyridyl 2-pyridyl 4-pyridyl 6-Cl-pyrid-3-yl 3-quinolyl 2-Ph-thiazol-3-yl 5-isothiazolyl 2-benzthiazolyl phenyl 3,4-diCl-phenyl 3,5-diCl-phenyl 3-CF3-phenyl

0.7 >30 25 0.3 0.006 4 7 29 9.5 0.3 0.4 0.8

72 K CHANNELS AND THEIR MODULATORS

R

IC90(μ M)

3-CN-phenyl 3-F-phenyl 4-Cl-phenyl 2-naphthyl

1.6 7 2.8 2.0

dithioester 7 by reaction with the requisite amine; yields were generally low. The methylformamide was synthesized directly from 1 by treatment with nitronium tetrafluoroborate. The alcohol and thiol analogues were synthesised from the nitrile intermediate. SARs are collected into Table 3.3 and the requirement for a small alkyl group, preferably ethyl, is apparent indicating the presence of a small hydrophobic pocket. The total loss of activity of the amide is striking and the very precise requirements for high activity is reflected in the results for the dithioester, nitrile, alcohol and thiol analogues. Modification of thiopyran-1-oxide The activities of compounds with a modified thiopyran-1-oxide group are collected into Table 3.4. The essential requirement of a sulfoxide group, trans to the thioamide, is indicated by the loss in activity of the sulfone, thiane, 1,3dithiane, 1,3-oxathiane, tetrahydrothiophene and tetrahydrofuran analogues. The unsubstituted cyclohexane was unexpectedly active at 3 μ M. Full synthetic routes to these analogues have been described (Brown et al., 1992). Summary of SARs A summary of the SARs for optimal KCA potency in aprikalim analogues described here is represented in Figure 3.2. Optimal biological activity requires a 3-substituted

Table 3.3 Thioformamide analogues

R

IC90μ M

CSNHMe CSNHEt CSNHPr CSNHBu CSNHPh

0.7 0.3 0.6 8 >30

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 73

R

IC90μ M

CSNHNH2 CONHMe CN CH2OH CH2SH CSSMe

>30 >30 >30 >30 >30 >30

Table 3.4 Thiopyran-1-oxide analogues X

Y

IC90μ M

SO (trans) SO (cis) SO2 S S S S O CH2 CH2

CH2 CH2 CH2 CH2 S O db db CH2 db

0.7 >30 >30 >30 >30 >30 >30 >30 3 30

db=direct bond

pyridyl or quinolyl group and a thioformamide attached to the C-2 atom of a thiopyran-1-oxide. The absolute stereochemistry at the C-2 atom and a trans relationship of the thioformamide and sulfoxide groups are crucial for good activity. 3.2.2 Cyclohexanone Analogues The initial objective in modifying the sulfoxide of aprikalim was to synthesise the trans alcohol 18 (Cook et al., 1987); the route used is outlined in Scheme 3. 3. A

74 K CHANNELS AND THEIR MODULATORS

Figure 3.2 Summary of the SARs of aprikalim analogues

Wittig reaction on pyridine-3-carboxaldehyde gave the olefin 14, which, when treated with NBS, gave the bromohydrin 15. The rearrangement of 15 to the ketone 16 was accomplished with silver perchlorate and 16 was converted to the racemic thioformamide 17 by carbanion formation followed by thioacylation with methyl isothiocyanate. Potassium borohydride reduction of 17 furnished a mixture of alcohols (9:1 trans/cis) which were separated by column chromatography to give the trans alcohol 18. Whereas 18 had disappointing KCA potency (IC90 7.5 μ M), the intermediate ketone 17 was equipotent with RP 49356 (IC90 0.8 μ M). This unexpected discovery led to the syntheses and investigation of many cyclohexanone analogues. These changes have been briefly reviewed (Chapman, 1991, 1993).

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 75

Alkene and alkane analogues The first compounds to be synthesised (Scheme 3.4) were the alkene analogues (Brown et al., 1993) and the synthesis of the phenoxy analogue 24 is illustrative of the methods employed. Lithiation of 3-bromopyridine followed by reaction with 2-methoxycyclohexanone gave the intermediate alcohol 19, which when treated with c. H2SO4 resulted in a high yield conversion to the cyclohexanone 16. Reaction of the carbanion of 16 with CS2/MeI gave the dithioester 20 which was converted to the β , β -unsaturated nitrile by the Wadsworth-Emmons modification of the Wittig reaction. The nitrile was reduced to the aldehyde 22 with DiBAL, which upon further reduction with NaBH4 gave the alcohol 23. The alcohol was converted to the phenoxy thioamide 24 by a Mitsunobu reaction followed by treatment with ethanolic methylamine. The KCA potencies of the alkene and alkane analogues are shown in Table 3.5. The β , β -unsaturated nitrile 25, aldehyde 26 and alcohol 27 had reduced activities when compared to the ketone 17. Introduction of an aromatic nucleus by functionalisation of the alcohol as phenoxy 24 or thiophenyloxy 28 led to a 500-fold increase in activity, suggesting the occupation of an extra binding site. Changing the sp2 geometry of the unsaturated nitrile 25 to sp3 as in the alkane 29 (Table 3.5) gave a threefold increase in activity. Interestingly it was subsequently

Schema 3.4 Synthesis of alkene analogues

76 K CHANNELS AND THEIR MODULATORS

Table 3.5 Alkene and alkane derivatives of ketone 17

compd

R

17

IC90μ M

compd

R

IC90μ M

0.800

29

CN

0.3

25

CN

10

30

CH2OH

1.7

26

CHO

10

31

CH2OCOCH3

0.03

27

CH2OH

30

32

CH2OCOPh

0.03

24

CH2OPh

0.065

33

CH2OCOC6H4-p-F

0.1

28

CH2SPh

0.003

34

CH=NOCH2Ph

0.03

35

CH2NHPh

0.003

36

CH=CHPh

0.1

37

CH=CHC6H4-p-F

0.01

found that the cyanomethyl side chain in 29, obtained by LiAlH4 reduction of 38 (Scheme 3.5), was trans to the thioformamide group therefore paralleling the arrangement in aprikalim. A range of substituents were synthesised similar to the alkene analogues and the increase in activity was maintained. Further functionalisation with analogues containing an extra aromatic nucleus in the form of an ester 32–33, oxime ether 34, amine 35 or styrene 36–37 gave the expected increase in activity. A low energy conformation of the nitrile 29 can be superimposed over the Xray conformation of aprikalim (Figure 3.3) and a good overlay of the axial sulfoxide of aprikalim with the cyanomethyl side chain can be obtained (Brown et al., 1993). Sulfonamide analogues As an extension of the alkane chemistry, a series of sulfonamide analogues were synthesised and the SARs were found to follow a similar pattern culminating in the synthesis of 40 (RP 66784) with a KCA IC90 of 0.3 nM; the aryl sulfonamido group contributing to the increased activity. The synthesis of 40 is outlined in Scheme 3.5 starting from ketone 17. Reaction with diethylcyanomethyl phosphonate in a Wadsworth-Emmons reaction gave the β , β -unsaturated nitrile 38. Room temperature reduction of 38 with LiAlH4 gave exclusively the saturated trans nitrile 29. This selectivity can be understood by complexation of LiAlH4 with the thioformamide group followed by delivery of hydride from the same face. The

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 77

Figure 3.3 Stereoplot of cyanomethyl analogue 29 superimposed on aprikalim

trans geometry was unambiguously established (Brown et al., 1993) by X-ray analysis of an ester derivative. Further reduction of 29 with LiAlH4 at elevated temperature gave the ethylamine 39 which was acylated with PhSO2Cl to give racemic sulfonamide 40. Oxime, hydroxylamine and amine analogues Oximes 41 and derived hydroxylamines 42 were synthesised from 17 (Cook et al., 1987) by standard procedures. In all cases the oxime was isolated as a single compound and assumed to have the anti-configuration. As in the case of the alkyl amine 39, only the trans N-substituted hydroxylamine 42 was formed upon reduction of 41 with LiAlH4. SARs for both series parallel those of the alkane series with optimal activity where R=substituted phenyl.

78 K CHANNELS AND THEIR MODULATORS

As an extension of the oxime chemistry, two asymmetric syntheses of 17 have been developed (Hart et al., 1992). In the first route (Scheme 3.6) condensation of the ketone 16 with (S)-(-)-l-amino-2-(methoxymethyl)pyrrolidine (SAMP) using Enders methodology (Enders and Eichenauer, 1976) gave the hydrazone 43. Lithiation followed by treatment with methyl isothiocyanate produced a mixture containing, for the most part, the diastereomer 44 in 80% diastereomeric excess (d.e.) as measured by hplc. Acid hydrolysis of the crude reaction mixture gave the (S)-ketone thioformamide 17 which was easily purified to homochirality by recrystallisation. Because problems were encountered in recycling the chiral auxiliary, cheaper alternatives were investigated. This led to the use of (R)-(+)-β -methylbenzylamine (Pfau et al., 1985) which when condensed with 16 afforded the Schiffs base 45. Lithiation and thioacylation gave 46 in a d.e. of 80%. Hydrolysis led

to the chirally pure (S)-ketone 17. The diastereomer 46 was also reduced with NaCNBH3 to give the chirally pure trans benzylamine 47, which was found to be an extremely potent compound (IC90 0.03 nM). Ester analogues Given the increase in KCA potency imparted by the extra phenyl group, it was decided to synthesise ester analogues of the original trans hydroxy compound 18 (Scheme 3.3). Both chiral and nonchiral esters were made (Hart et al., 1989) and the most potent compounds are collected into Table 3.6. In the benzoate ester, the activity resides in the (1R, 2S) enantiomer.

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 79

Summary In the cyclohexanone series SARs also parallel those of the sulfoxides regarding the pyridyl group and thioformamide modifications. The chirality is an important feature as is the trans relationship of the thioformamide group with adjacent substituents, the absolute configuration of eutomers being the same as that in aprikalim. The enhanced potency associated with hydrophobic derivatives provides evidence for the existence of a lipophilic binding site at the receptor. The aromatic binding group can accommodate differing substituents and a variety of linking groups, both as sp2 (alkene, oxime) and sp3 (alkane, sulphonamide, amine and ester). The most potent analogues had subnanomolar KCA potency in the primary screen. 3.2.3 Other Syntheses and Hybrid Structures An alternative approach to the synthesis of aprikalim analogues via a DielsAlder reaction on the thioketoester 48 to the carboxamide 49 has been reported (Pinto et al., 1992). The conversion of the amide to aprikalim was not described Table 3.6 Ester analogues of 18

R

IC90nM

Me nBu Ph (1R 2S) Ph (IS 2R) 4-F-Ph 3-pyridyl

100 1 0.1 3000 0.3 0.1

presumably because of synthetic difficulties. Whereas no hybrid aprikalim analogues have been described, the pyrrolidinone group in CRK has been successfully replaced by thioformamide in structures of type 50 (Ishizawa et al., 1993). The SAR of such compounds is discussed in Chapter 2.

80 K CHANNELS AND THEIR MODULATORS

3.3 Cyanoguanidines 3.3.1 Pinacidil and Close Analogues Discovery and initial SAR studies Pinacidil was developed from a series of N-alkyl-N'-pyridyl thioureas, which were known to have hypotensive activity (Petersen et al., 1978) and, as was the case with aprikalim, the KCA properties of pinacidil were unknown. Pinacidil 53 was synthesised from the 4-pyridyl isothiocyanate 51 via the thiourea 52 (Scheme 3.7). The enantiomers of pinacidil were initially synthesised by kinetic resolution of the tartrates (Arrigoni-Martelli et al., 1980). More recently the enantiomers have been synthesised via chiral reduction (Manley and Quast, 1992). Reaction between pinacolone with (R)-β -methylbenzylamine (Scheme 3.7) gave the chiral imine 54. Reduction with diborane resulted in the addition of hydrogen to the azomethine double bond from the face opposite to that occupied by the bulky phenyl group to give the diastereomer 55. Catalytic hydrogenolysis afforded optically pure amine 56, which was converted through to the (R)enantiomer of pinacidil by the route used for 51–53. The corresponding (S)enantiomer was synthesised using the corresponding (S)- β -methylbenzylamine. The initial structure/activity data on pinacidil and analogues was based upon SHR hypotensive data (Petersen et al., 1978) and is summarized in Figure 3.4. In general 3-substituted pyridyl compounds were up to 20-fold more potent than the 4-pyridyl analogues; however pinacidil was an exception in being more potent than the 3-pyridyl isomer. Further ring substitution in the 3-series was usually detrimental. In the side chain, the thiourea was more active than the urea, but the cyanoguanidine

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 81

Figure 3.4 Hypotensive SARs of pinacidil analogues

was 200-fold more potent than the thiourea. Replacement of the cyano group with, for example, COOEt, OMe, OH and CONH2 led to a reduction in activity. A branched alkyl group containing 4–5 carbon atoms was optimal at the terminal position of the side chain. KCA activity of pinacidil analogues It is now known that pinacidil relaxes blood vessels by additional non K channeldependent mechanisms (Cook et al., 1989) making blood pressure (BP) data unreliable as an indicator of KCA potency. Consequently KCA potency data based upon the inhibition of spontaneous myogenic activity in rat portal vein was generated (Manley and Quast, 1992) and is collected into Table 3.7. The data demonstrate the higher activity of the NCN, CHNO2 and to a lesser extent S analogues over the ureas 57 and 61. The 3-pyridyl analogues were invariably more active than the corresponding 4-pyridyl counterparts e.g. 62 and 59, 63 and 53, 64

Table 3.7 KCA activities of pinacidil and analogues (rat portal vein) Pyr

57 58 59 53 60 61 62 63 64

Pyr

R

X

pIC50

4-pyridyl 4-pyridyl 4-pyridyl 4-pyridyl (pinacidil) 4-pyridyl 3-pyridyl 3-pyridyl 3-pyridyl 3-pyridyl

CH2CMe3 (R) CHMeCMe3 (S) CHMeCMe3 (±) CHMeCMe3

O S S NCN

5.5 6.3 6.5 7.2

(±) CHMeCMe3 CH2CMe3 (±) CHMeCMe3 (±) CHMeCMe3 (±) CHMeCMe3

(E)-CHNO2 O S NCN (E)-CHNO2

6.0 5.7 7.3 7.8 7.5

82 K CHANNELS AND THEIR MODULATORS

65 66

Pyr

R

X

pIC50

3-pyridyl 3-pyridyl

(R) CHMeCMe3 (S) CHMeCMe3

(E)-CHNO2 (E)-CHNO2

6.0 8.0

and 60. In the nitroethene series the higher activity resided in the (S)-enantiomer 66 rather than the (R)-enantiomer 65. Stereochemistry and molecular modeling of pinacidil analogues A receptor binding model (Figure 3.5) has been developed to explain the KCA potencies of pinacidil analogues (Manley and Quast, 1992). The relative inactivity of the ureas was explained on conformational grounds since only the cyanoguanidines, nitroethenediamines and thioureas can adopt similar relatively low energy staggered conformations of type 67–69, which could correspond to the active conformation at the receptor. Interestingly an X-ray determination of pinacidil (Pirotte et al., 1993a) has the unusual conformation 70. It was also suggested that the pyridine nitrogen was acting as a hydrogen bond acceptor with the interaction being less favourable for the 4-pyridyl compound. Evidence from pKa measurements suggested that the relative acidity of the pyridyl amino group determined the ease to which it could act as a hydrogen bond donor. The disparate geometry requirements for activity between enantiomers was explained by the receptor undergoing conformational changes in order to accommodate either the bulkier CHNO2 or NCN moieties. In essence three receptor binding elements are required as shown in Figure 3.5: a H-bond donating site (pyridyl NH), flanked by a H-bonding acceptor site (pyridine N atom) and a lipophilic site (CH(Me)CMe3 group).

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 83

3.3.2 Other Pinacidil Analogues Substituted phenyl analogues of pinacidil Paralleling the replacement of the pyridine in aprikalim with substituted aryl groups (Table 3.2), a series of aryl cyanoguanidines have been synthesised by methods

Figure 3.5 A minimum energy conformation of 66 showing proposed receptor binding interactions. Copyright ACS 1992.

similar to those used for pinacidil (Atwal et al., 1992a). SARs are collected into Table 3.8 and, as expected, the presence of an electron withdrawing group is optimal for activity, although there is no clear preference for the location of the substituent at either the meta or para positions. The bulky 1,2,2-trimethyl propyl group is optimal for activity. The analogues 71 (Nishimaru et al., 1990) and 72 (Lenfers et al., 1991) have been reported to have KCA potency. The potency of 72 is surprising in that one would predict an electron donating group to reduce potency. However 72 is reported to be equipotent with pinacidil.

Aminopyridine analogues The KCA potency of a series of aminopyridine thioureas has been reported (Takemoto et al., 1994). They were synthesised by the route similar to that used for pinacidil (Scheme 3.7) and their activities are collected into Table 3.9. The activity increased with increasing bulk of the terminal alkyl group, but altering

84 K CHANNELS AND THEIR MODULATORS

the position of the amino substituent on the pyridine ring did not have a profound effect upon activity. Maximal hypotensive activity was seen in the 6-amino compound, and all further changes had this group constant. Activity increased with a 1,2,2-trimethylpropyl group (the pinacidil substituent) and was optimal as a l-methyl-2-norbornyl group. Isosteric transformation of thiourea to cyanoguanidine gave an even more active compound (pEC100 7.5). 3.3.3 Hybrid Analogues The cyanoguanidine group has been incorporated into CRK with both aryl 73 (Atwal et al., 1993) and cycloalkyl 74 (Atwal et al., 1992b) derivatives. It is claimed that 73 possesses anti-ischaemic properties without significant vasodilatation. The

Table 3.8 Phenyl analogues of pinacidil Ar

IC50μ M(rat aorta)

Ph 3-CNphenyl 4-CNphenyl 2-NO2phenyl 3-NO2phenyl 4-NO2phenyl pinacidil

0.88 0.42 0.022 0.24 0.17 5.15 0.07

Table 3.9 Aminopyridine analogues of pinacidil R'

R

pEC100 (rat portal vein)

Me nBu cyclohexyl cyclohexyl cyclohexyl

4-NH2 4-NH2 4-NH2 2-NH2 5-NH2

IA 3.0 4.0 4.0 5.0

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 85

R'

R

pEC100 (rat portal vein)

cyclohexyl 1-Me-2-norborny1 l-Me-2-norbornyl (NCN) pinacidil

6-NH2 6-NH2 6-NH2

5.0 7.0 7.5 7.0

cycloalkyl analogue 74 was equiactive with cromakalim as a vasodilator. The diazoxide hybrid 75 has also been reported (Pirotte et al., 1993b) and found to be a powerful inhibitor of insulin release. Further discussion of certain of these compounds appears in Chapter 2.

3.4 Organic Nitrates 3.4.1 Nicorandil Nicorandil 77 (Furukawa et al., 1981) was the first organic nitrate to demonstrate KCA potency and was synthesised (Scheme 3.8) in two steps from methyl nicotinate via the alcohol 76. It has also been shown that nicorandil stimulates guanylate

86 K CHANNELS AND THEIR MODULATORS

Figure 3.6 Nicorandil analogues

cyclase in vascular smooth muscle (Holzmann, 1983) and has therefore a dual mode of action and the association of KCA and organic nitrate-like potency leads to powerful vasodilators. 3.4.2 Analogues of Nicorandil Several analogues of nicorandil 78–83 have been reported and are collected into Figure 3.6. The closest analogues all retain the nicorandil side chain together with a substituted pyridine 78 (Miura et al., 1990), a pyrazine 79 (Ito et al., 1991) or a thiazole 80 (Satake et al., 1992). The amide of nicorandil can be replaced by a cyanoamidine 81 (KRN 2391; Ishibashi et al., 1992). When the nitrate group of 81 was replaced with a phenyl group as in 82, a potent vasodilator was obtained which was found to act by K channel activation only (Okada et al., 1993). The cromakalim hybrid 83 has been reported (Evans and Frydrych, 1990). 3.5 Other Pyridine KCAs 3.5.1 Anilide Tertiary Carbinols During an investigation of anti-androgen compounds, the KCA activity of a series of anilide tertiary carbinols e.g. 84 (Russell et al., 1992) was unearthed and found to be about eightfold more potent than CRK on guinea pig detrusor (bladder) strip mildly depolarized with 15 mMKCl (Table 3.10). The compounds were synthesised by the reaction between the requisite aniline and acid chloride

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 87

in the presence of DMAP. Subsequently the activity of the (S)-pyridylsulfone 85 (Grant et al., 1993) and the phenylsulfone 86 (Russell et al., 1993) have been reported. The compounds are being pursued mainly in the area of urinary urge incontinence.

Table 3.10 Anilide tertiary carbinols IC50 μ M KCA potency (g.p. detrusor strip) 84 85 cromakalim

0.07 1.6 0.57

3.6 Conclusion A wide variety of structural types exist within the pyridine based KCAs. Combining these structures with CRK, common pharmacophoric features have been suggested (Atwal et al., 1992a, 1992b) and a common pharmacophoric model proposed (Koga et al., 1993). Although there are indeed common structural features between the benzopyrans, pyridyl thioformamides and cyanoguanidines, the evidence for a common pharmacophore is not compelling. Functional studies with ET-1 and different classes of KCAs suggest that the benzopyrans interact at a different site to pinacidil and aprikalim (Lawson et al., 1992). Binding studies using a tritiated pinacidil analogue (Manley et al., 1993) suggest that differing structural types of KCA probably interact with different receptor binding sites further weakening the evidence for a common pharmacophore. Exploration of pyridine based KCAs has led to some very potent compounds; some thioformamides have subnanomolar activity. Within the thioformamide series, a clear understanding of SARs has been obtained. The secondary thioformamide is crucial for good activity and must be in a trans relationship with a sulfoxide (aprikalim analogues) or an aryl side chain (cyclohexanone analogues). The enhanced potency associated with the hydrophobic side chain provides evidence for the existence of an extra lipophilic binding site at the receptor. More limited SARs are available for pinacidil and nicorandil analogues and they possess additional properties to that of K channel activation to account for their pharmacological profiles. Aprikalim has been selected for development for angina, and at nonhypotensive doses, may prove useful in the treatment of ischaemia. Pinacidil

88 K CHANNELS AND THEIR MODULATORS

(PINDAL™) has been marketed for hypertension and antiprostatic hypertrophy. Nicorandil (SIGMART™) has been marketed for stroke and cerebral vasodilation. References ALOUP, J-C., BOUCHAUDON, J., FARGE, D., JAMES, C., DEREGNAUCOURT, J. & HARDY-HOUIS, M.J. (1987) J. Med. Chem., 30, 24–29. ALOUP, J-C, FAROE, D., JAMES, C., MONDOT, S. & CAVERO, I. (1990) Drugs of the Future, 15, 1097–1108. ARRIGONI-MARTELLI, E., NIELSEN, C.K., OLSEN, U.B. & PETERSEN, H.J. (1980) Experientia, 36, 445–447. ATWAL, K.S., MORELAND, S., MCCULLOUGH, J.R., O’REILLY, B.C., AHMED, S.Z. & NORMANDIN, D.E. (1992a) BioMed. Chem. Letters, 2, 83–86. (1992b) BioMed. Chem. Letters, 2, 87–90. ATWAL, K.S., GROVER, G.J., AHMED, S.Z., FERRARA, F.N., HARPER, T.W., KIM, K.S., SLEPH, P.O., DZWONCZYK, S., RUSSELL, A.D., MORELAND, S., MCCULLOUGH, J.R. & NORMANDIN, D.E. (1993) J. Med. Chem., 36, 3971–3974. BROWN, T.J., CHAPMAN, R.F., COOK, D.C., HART, T.W., MCLAY, I.M., JORDAN, R., MASON, J.S., PALFREYMAN, M.N., WALSH, R.J.A., WITHNALL, M.J., ALOUP, J-C., CAVERO, I., FARGE, D., JAMES, C. & MONDOT, S. (1992) J. Med. Chem., 35, 3613–3624. BROWN, T.J., CHAPMAN, R.F., MASON, J.S., PALFREYMAN, M.N., VICKER, N. & WALSH, R.J.A. (1993) J. Med. Chem., 36, 1604–1612. CHAPMAN, R.F. (1991) Current Drugs: Potassium Channel Modulators B63–69. (1993) Current Drugs: Potassium Channel Modulators Cl-19. COOK, D.C., HART, T.W., MCLAY, I., PALFREYMAN, M.N. & WALSH, R.J.A. (1987) European Patent 0321274. COOK, N.S., QUAST, U. & MANLEY, P.W., (1989) Br. J. Pharmacol, 96,181P. EDWARDS, G. & WESTON, A.H. (1990) Trends Pharmacol. Sci., 11, 417–422. ENDERS, D. & EICHENAUER, H. (1976) Angew. Chem. Int. Edn., 15, 549–551. ESCANDE, D. & THURINGER, D. (1989) Mol. Pharmacol., 36, 879–902. EVANS, J.M. & FRYDRYCH (1990) European Patent 366273. FURUKAWA, K., ITOH, T., KAJIWARA, M., KlTAMURA, K., SUZUKI, H., ITO, Y. & KURIYAMA, H. (1981) J. Pharmacol. Exp. Ther., 218, 248–259. GRANT, T., FRANK, C.A., KAU, S.T., LI., J.H., MCLAREN, F.M., OHNMACHT, C.J., RUSSELL, K., SHAPIRO, H.S. & TRIVEDI, S. (1993) BioMed. Chem. Letters, 3, 2723–2724. HART, T.W., GUILLOCHON, D., PERRIER, G., SHARP, B.W., VACHER, B. (1992) Tetrahedron Lett., 33, 5117–5120. HART, T.W., SHARP, B.W. & VACHER, B. (1989) European Patent 390693. HOLZMANN, S. (1983) J. Cardiovasc. Pharmacol., 5, 364–370. ISHIBASHI, T., HAMAGUCHI, M. & IMAI, S. (1992) Naunyn-Schmiedeberg’s Arch. Pharmacol., 346, 94–101.

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ISHIZAWA, T., KOGA, H., OHTA, M., SATO, H., MAKINO, T., KUROMARU, K., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993) BioMed. Chem. Lett., 3, 1659–1662. ITO, Y., KATO, H., ETSUCHU, E., OGAWA, N., MITANI, K., IWASAKI, N. & HOKURIKU SEIYAKU (1991) Japanese Patent 89056667-A. KOGA, H., OHTA, M., SATO, H., ISHIZAWA, T. & NABATA, H. (1993) BioMed. Chem. Letters, 3, 625–631. LAWSON, K., BARRAS, M., ZAZZI-SUDRIEZ, E., MARTIN, D.J., ARMSTRONG, M. & HICKS, P.E. (1992) Br. J. Pharmacol., 107, 58–65. LENFERS, J.B., MUSCHALEK-LETINA, V., NIEMERS, E., JANIS, R.A. SCRIABANE, A. (1991) U.S. Patent 661720. MANLEY, P.W. & QUAST. U. (1992) J. Med. Chem., 35, 2327–2340. MANLEY, P.W., QUAST, U., ANDRES, H. & BRAY, K. (1993) J. Med. Chem., 36, 2004–2010. MIURA, K., KOYAMA, H., SUGAI, T., YAMADA, H., SAKURAI, E., HORIGOME, M. (1990) European Patent 385350. MONDOT, S., MESTRE, M., CAILLARD, C.G. & CAVERO, I. (1988) Br. J. Pharmacol., 95 (Supp), 813 P. NlSHIMARU, N., IWAMOTO, T., SUKAMOTO, T., YOSHIIZUMI, K., SEKO, N. & YOSHINO, K. (1990) Japanese Patent 01657. OKADA, Y., YOKOYAMA, T., JINNO, Y., KASHIWABARA, T., IZAWA, T., FUKUSHIMA, H. & OGAWA, N. (1993) Eur. J. Pharmacol., 241, 177–181. PETERSEN, H.J., NIELSEN, C.K. & ARRIGONI-MARTELLI, E. (1978) J. Med. Chem., 21, 773–781. PFAU, M., REVIAL, G., GUINGANT, A. & D’ANGELO, J. (1985) J. Am. Chem. Soc., 107, 273–274. PINTO, I.L., BUCKLE, D.R., RAMI, N.K. & SMITH, D.G. (1992) Tetrahedron Lett., 33, 7597–7600. PlROTTE, B., DUPONT, L., DE TULLIO, P., MASEREEL, B., SCHYNTS, M. & DELARGE, J. (1993a) Helv. Chim. Acta., 76, 1311–1318. PIROTTE, B., DE TULLIO, P., LEBRUN, P., ANTOINE, M-H., FONTAINE, J., MASEREEL, B. , SCHYNTS, M., DUPONT, L., HERCHUELZ, A. & DELARGE, J. (1993b) J. Med. Chem., 36, 3211–3213. RUSSELL, K., OHNMACHT, C.J. & GIBSON, K.H. (1992) European Patent 524781. RUSSELL, K., EMPFIELD, J.R., OHNMACHT, C.J. & GIBSON, K.H. (1993) WO 9323358. SATAKE, N., KIVOTO, S., ZHOU, Q., MATSUO, M. & SHIBATA, S. (1992) Faseb J., 6, Abs. 360. TAKEMOTO, T., EDA, M., OKADA, T., SAKASHITA, H., MATZNO, S., GOHDA, M., EBISU, H., NAKAMURA, N., FUKAYA, C., HIHARA, M., EIRAKU, M., YAMANOUCHI, K. & KAZUMASA, Y. (1994) J. Med. Chem., 37, 18–25.

Recent Literature EDA, M., TAKEMOTO, T., ONO, S., OKADA, T., KOSAKA, K., GOHDA, M., MATZNO, S., NAKAMURA, N. & FUKAYA, C. (1994) Novel Potassium Channel Openers: Preparation and Pharmacological Evaluation of Racemic and Optically

90 K CHANNELS AND THEIR MODULATORS

Active N-(6-Amino-3-pyridyl)-N'-bicycloalkyl-N"-cyanoguanidine Derivatives. J. Med. Chem., 37, 1983–1990. FRANK, C.A., FORST, J.M., GRANT, T., HARRIS, R.J., KAU, ST., NAKAJIMA, T., KASHIWABARA, T., IZAWA, T. & NAKAJIMA, S. (1994) StructureActivity Studies of N-Cyano-3-pyridinecarboxamides and their Amide and Thioamide Congeners. BioMed. Chem. Lett., 4, 2485–2488.

4 Conformational Analysis of Potassium Channel Activators C.M. EDGE SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.

4.1 Introduction Potassium channel activators (KCAs) represent a varied structural class of molecules that interact with ATP-sensitive potassium channels (KATP). Their action on these ion channels results in smooth muscle relaxation and has led to KCAs being proposed as potential drugs for the treatment of asthma and hypertension, for example. A study of the conformations adopted by this class is of interest in the design of novel therapeutic agents and in the characterization of the KATP channel itself. This chapter therefore includes conformational analysis data on the three main classes of KCA—namely, cromakalim (CRK), aprikalim, pinacidil and some of their analogues. The majority of the calculations reported in this chapter are at the semi-empirical MNDO level of theory, using the AMI Hamiltonian. This seems to have been accepted as the de facto standard in this field, notwithstanding its limitations, such as low barrier heights and poor description of some sp2 nitrogens. Novel calculations made for this chapter have used this theoretical method for consistency with previously published work. 4.1 Conformational Analysis of Cromakalim 4.1.1 Structure The structure of CRK, 1, has been studied using the techniques of X-ray crystallography (Cassidy et al., 1990) and nuclear magnetic resonance (NMR) (Thomas and Whitcombe, 1990). These reports showed that the pyran ring adopts a half-chair conformation, in which the 4-pyrrolidinone is pseudoequatorial and the 3-hydroxyl is equatorial. The 3- and 4-substituents are trans to each other, with the more potent enantiomer having 3-(S), 4-(R) stereochemistry.

92 K CHANNELS AND THEIR MODULATORS

Plate 4 shows the X-ray crystal structure of this enantiomer, levcromakalim (LCRK).

4.1.2 Rotation of the 4-pyrrolidinone in Cromakalim The rotational preference of the 4-position of CRK has been the subject of some debate. NMR experiments conducted in deuteriochloroform at 270 MHz seemed to suggest that there was a single, rigid conformer, 2 (Cassidy et al., 1990). Semiempirical calculations (Cassidy et al., 1990), using the AMI Hamiltonian (Dewar et al., 1985) suggested that there was an energy difference of 2.4 kcal/mol between this minimum and a higher energy rotamer, 3, found upon rotation of the C4-N1' bond. Assuming a Boltzmann distribution, this corresponds to about a 98:2 prevalence of the lower energy form. The presence of 2% of the higher energy rotamer was not detected by the original NMR studies.

CONFORMATIONAL ANALYSIS OF KCAS 93

In contrast to the earlier studies, the NMR spectrum in deuteriomethanol showed the presence of two structural forms at -60°C (Thomas and Whitcombe, 1990). The major component was shown to be identical to that identified by Cassidy et al.—the rotamer in which the carbonyl oxygen is on the same side of the molecule as the 4-proton. (This is the lower energy structure, 2, found in the AM1 calculations.) The minor component at -60°C corresponded to the higher energy structure, 3, in which the sp2 oxygen and the 4-H are on opposite sides. The barrier to rotation was estimated to be between 11.5 and 13.5 kcal/mol. From this NMR study, the difference in energy between the two forms was calculated to be 2.2 kcal/mol, which accords with the AM1 calculation mentioned above. Figure 4.1 shows a graph of the energy of LCRK versus the C10-C4-N1'-C2' dihedral angle. The calculation, like most in this chapter, has been made using the semi-empirical AM1 Hamiltonian (Dewar et al., 1985). Since this particular method is well known for failing to maintain the planarity of sp2 amide nitrogens (Stewart, 1990), a constant improper dihedral of 180° was imposed in the Zmatrix definition of The following colour plates are referred to in Chapters 3 and 4. Plate 1 Stereoview of the X-ray conformation of aprikalim 1. Copyright ACS, 1992. Plate 2 Stereoview of an AM1 low energy conformer of 1 with equatorial thioamide and sulfoxide groups. The dashed line (orange/white) represents a possible hydrogen bond. Copyright ACS, 1992. Plate 3 Stereoview of the AM1 lowest energy conformer of the cis isomer 10. The dashed line (orange/white) indicates a hydrogen bond between the equatorial sulfoxide and axial thioamide groups. Copyright ACS, 1992. Plate 4 Orthogonal views of the X-ray crystal structure of LCRK. Plate 5 The half-chair and boat structures of LCRK, clockwise from top left 2, 6, 5, 7. Plate 6 Notional coordinate system for the flipping of the pyran ring of LCRK. Half-chair loci are in red; boat loci in blue. The Cremer and Pople β parameter runs from 0–180° from pole to pole. The β parameter runs from 0–360° longitudinally. Plate 7 Structures of the 3-(R),4-(S) enantiomer, 14, of LCRK and the 3-(R),4(R) enantiomer, 15, of cis CRK.

94 K CHANNELS AND THEIR MODULATORS

Plate 8 The proposed overlap of indanol (blue) and chromanol (red) structures. Plate 9 An overlap of benzoxepine (blue) and chromonol (red) structures. Plate 10 Electrostatic potential surface of thieno [2,3-b]pyran, thieno [3,2-b] pyran and thieno [3,4-b]pyran structures. Areas of negative potential are coloured red. Plate 11 Local minimum energy conformations of the trans diaxial and tram diequatorial structures of RP52891, reported in Table 4.4. Plate 12 Local minimum energy conformations of six twist boat structures of RP52891, reported in Table 4.4. Plate 13 The X-ray crystal structure of pinacidil. Plate 14 Contour map of AM1 energy for two driven torsion angles of pinacidil. The torsion angles are shown in yellow and pink, and the loci of the X-ray crystal structure are shown in yellow and the loci of the proposed overlap structure are shown in red.

CONFORMATIONAL ANALYSIS OF KCAS 95

96 K CHANNELS AND THEIR MODULATORS

CONFORMATIONAL ANALYSIS OF KCAS 97

98 K CHANNELS AND THEIR MODULATORS

CONFORMATIONAL ANALYSIS OF KCAS 99

100 K CHANNELS AND THEIR MODULATORS

CONFORMATIONAL ANALYSIS OF KCAS 101

102 K CHANNELS AND THEIR MODULATORS

CONFORMATIONAL ANALYSIS OF KCAS 103

104 K CHANNELS AND THEIR MODULATORS

CONFORMATIONAL ANALYSIS OF KCAS 105

106 K CHANNELS AND THEIR MODULATORS

CONFORMATIONAL ANALYSIS OF KCAS 107

108 K CHANNELS AND THEIR MODULATORS

Figure 4.1 Graph of AM1 heat of formation versus C10-C4-N1'-C2' dihedral angle for LCRK

the third atom connected to the pyrrolidinone nitrogen. The graph shows minima at -130° and at 70°, corresponding to structures 2 and 3 respectively. Full geometry optimization of these two structures (including the aforementioned dihedral angle) resulted in structures of energy -65.8 and -63.4 kcal/mol respectively. The dihedral angles C10-C4-N1'-C2' were -129° and 83° respectively. The difference in energy between the two minima is 2.4 kcal mol. The barrier to interconversion between the two minima can be estimated to be 8.2 kcal/mol from a consideration of the lower of the two maxima on the graph in Figure 4.1. The barrier to free rotation, based on the higher energy maximum, is 9.7 kcal/ mol. It must be emphasized that these are only estimates of the barrier, since the maxima are not true transition states on the reaction coordinate. Also, AM1 barriers are known to be underestimates of the true case by a factor of about 2 or 3 (Fabian, 1988; Gundertofte et al., 1991). The structure of the graph shows that the obstacle to rotation is the 5-position hydrogen on the benzopyran nucleus. This atom clashes with the 5'-hydrogens of the pyrrolidinone for the lower barrier and with the carbonyl oxygen for the higher one. The form of the barrier can also be reproduced by simple molecular mechanics forcefields, such as the one in the Sybyl molecular modelling program (Tripos Associates), also suggesting that it is governed by the steric constraints of the system. In contrast to the above, the carbonyl oxygen of the piperidinone of 4, a BRL55834 carbamate analogue, was shown to point away from the C4 proton by X-ray crystallography (Buckle et al., 1992).

4.1.3 Flipping the Pyran Ring In addition to the rotational preferences of the 4-substituent in LCRK, the conformational preferences of the pyran ring have also been investigated. The experimental evidence points toward one half-chair structure for the benzopyran

CONFORMATIONAL ANALYSIS OF KCAS 109

ring, 2. Other possible structures include the opposite half-chair, 5, and two boat structures, 6 and 7. These structures are shown in Plate 5. An inspection of the energy surface with respect to the pyran ring internal dihedrals was attempted using the AM1 Hamiltonian. The position of n ring atoms may be described using n-3 dihedral angles—3 angles in the case of LCRK. One of these angles, that of Ol-C9-C10-C4, can be assumed to be 0°, as the pyran ring is fused to a benzene ring. Thus we are left with two dihedral angles to describe the ring geometry, namely C9-C10-C4-C3 and C10-C9-O1-C2.

Plate 6 shows a diagram of LCRK on a notional rotational coordinate, using the ― and β values from the Cremer and Pople method (Cremer and Pople, 1975) of characterizing ring conformations. The Cremer and Pople method gives values of ― of 132° and 49° and values of β of 23° and 203° for the two minima 2 and 5 (a cyclohexane half-chair would give ― values of 129.2° or 50.8° and β values of 30, 90, 150, 210, 270 or 330°). These structures are shown as red points on the β , β surface. The difference in energy for these two structures is 3.5 kcal/mol. Thus we would expect to see a preponderance of the lower energy half-chair, 2. This is in accord with the NMR and X-ray studies mentioned in section 4.1.2. In addition to the two minima discussed above, two saddle points are discernible in Plate 6. These are shown as blue points on the β , β surface. The Cremer and Pople values for the pyran rings have been calculated. Structure 6 has the following values: β =93°, q3=0.0, q2=0.53, Q=0.53, β =116° and structure 7 has β =80°, q3=0.1, q2=0.52, Q=0.53, β =280°. The characteristic values for boat and twist-boat structures are β =90°, q3=0, q2=Q. Perfect boat structures have β values of 0, 60, 120, 180, 240 or 300° and perfect twist-boats have values of 30, 90, 150, 210, 270 or 330°. Thus, it can be seen that structure 6 is a true boat structure whereas structure 7 is a distorted form, between a boat and twist-

110 K CHANNELS AND THEIR MODULATORS

boat. These are stationary points on the LCRK energy surface, but are not minima. They are true transition structures, each having one calculated imaginary frequency. One can estimate the barrier to interconversion, in an analogous manner to the interconversion of rotamers above. The AM1 barrier is 4.8 kcal/mol between the lower energy half-chair, 2, and 7, the boat with the equatorial 3-OH. The barrier via the other boat, 6, with the axial 3-OH, is higher, at 6.5 kcal/mol. A full ‘pseudorotation’ of the ring would therefore require at least 6.5 kcal/mol. It is striking that the points on the β , β surface almost form a plane, suggesting that there may be a circular path, as shown in Plate 6. This is not necessarily the case and may reflect the nature of the dihedral sampling used to identify the stationary points. There are probably two other saddle points on the ‘pseudorotation’ path as there are minima close to the saddle points, but it has proven to be extremely difficult to find these. One of the main difficulties lies in the facility in finding trivial transition states due to rotation of the 2-methyl and 3-hydroxyl groups. It is not unusual to see two or three negative eigenvalues of the force constant matrix of a suspected transition state. The 2-monomethyl analogues of LCRK have been synthesized (Buckle et al., 1991a) and the structures of the two isomers were identified by NMR techniques. The 2β -monomethyl compound, 8, was shown to exist predominantly in one form. This was the 2-(R), 3-(S), 4-(R) form in which all three substituents were equatorial (or pseudo-equatorial), with the benzopyran ring in a half-chair conformation. This structure is the global energy minimum as calculated using the AM1 Hamiltonian (Table 4.1). The flipped half-chair structure, 9, is destabilized slightly, compared to the dimethyl analogue of LCRK. The energy difference between the two half-chairs is 3.9 kcal/mol rather than 3.5 kcal/mol found for LCRK. The 2β -monomethyl compound, 10, exhibited a more complicated set of nOe difference spectra, compared with that of compound 8. These were rationalized as being due to the presence of an equilibrium between two structures, 11 and 12. Semi-empirical molecular orbital calculations on these structures show that 11 is 2.4 kcal/ mol lower in energy than 12. This is a lower energy difference than that seen for LCRK, as the higher energy half-chair has an axial hydrogen at the 2position rather than a methyl group. This proton will have less of a steric repulsion from the pyrrolidinone ring than the methyl group. Interestingly, the calculations identify 13 as the global energy minimum for the 2β -monomethyl compound, rather than 11. This structure is broadly similar to 11, except that the pyrrolidinone carbonyl group is on the Table 4.1 The AM1 energies of 2-monomethyl analogues of LCRK, showing both minima upon rotation of C4-N1'. Structure

Energy (kcal/mol)

C10-C4-N1'C2' dihedral

8 9

-63.6 -59.7

-140.0° -116.5°

CONFORMATIONAL ANALYSIS OF KCAS 111

Structure

Energy (kcal/mol)

C10-C4-N1'C2' dihedral

8 9 11 12 11 12

-62.6 -59.5 -63.1 -60.7 -62.1 -64.0

91.0° 74.7° -138.9° -136.4° 90.7° 66.9°

opposite side of the ring from the 4-hydrogen. The carbonyl oxygen of 13 is 2.17 Å away from the axial 2-hydrogen, a suitable distance for an electrostatic interaction such as a hydrogen bond. However, this disagrees with the published solid state X-ray crystal structure, which is in the configuration represented by 12; it also does not accord with all the nOe data, since mutual nOes are observed between the 2-H and 5'-H protons. Although the calculations correctly predict that the 2-(S) compound, 10, is more likely to exist in a number of conformational states than the 2-(R) compound, 8, it seems that the predicted rank ordering of the minima is different from the X-ray structures. In part these results can be rationalized by considering that the AM1 calculations simulate the situation in the gas phase at zero Kelvin, which favours the formation of intramolecular hydrogen bonds. In contrast, the X-ray crystal structure contains an intermolecular hydrogen bond between the carbonyl of one molecule and the hydroxyl of another, thus determining the conformation adopted.

112 K CHANNELS AND THEIR MODULATORS

4.7.4 Stereochemistry LCRK, the more active enantiomer of CRK, has a trans 3-(S), 4-(R) stereochemistry. This structure has been examined above, in section 4.1.1. The other trans enantiomer, 14, is 100-fold less potent. Obviously, the relationship between the carbonyl group of the pyrrolidinone and the benzopyran ring is exactly reversed in the 3-(R), 4-(S) molecule. The dihedral angle across the C4N1’ bond, C10-C4-N1’-C2’, is 130°, rather than -130° found in LCRK for the global minimum structure. It is probable that this relationship between aromatic ring and hydrogen bond acceptor is not acceptable to the receptor associated with the KATP channel.

CONFORMATIONAL ANALYSIS OF KCAS 113

Both the enantiomers of cis CRK have been shown to possess potassium channel modulator properties (Quast and Villhauer, 1993). The 3-(R), 4-(R) enantiomer of cis CRK, 15, is at least twice as potent as the 3-(S), 4-(S) enantiomer, 16, and three times more potent than the 3-(R), 4-(S) enantiomer, 14 of CRK. Calculations show that the preferred structure of 15 is a half-chair similar to that of LCRK itself, but with the 3-hydroxyl group in an axial position. This interferes with the rotational characteristics of the 4-substituent, raising the energy of the opposite rotamer because of steric and electrostatic repulsions of the 2’-carbonyl oxygen and the 3-hydroxyl oxygen. The difference in AM1 energy of the two minima upon rotation of the 4-pyrrolidinone is 6.9 kcal/mol compared to 2.4 kcal/mol for LCRK. The higher energy rotamer is also pushed further round, away from the 3-position, making a dihedral angle of 47° for C10C4-N1’-C2’, rather than the more usual 70°. This is shown in Plate 7. The fact that the global minimum energy structures of LCRK and the cis 3-(R),4-(R)enantiomer of cis CRK, 15, differ only in the orientation of the 3-hydroxyl suggests that either the 3-hydroxyl is not important in KATP binding, or that a putative hydrogen bond donor (to the hydroxyl oxygen) or acceptor (from the hydroxyl proton) must be able to interact with both the axial and equatorial positions.

114 K CHANNELS AND THEIR MODULATORS

4.2 Studies on Benzopyran Ring Replacements 4.2.7 Replacements for the Pyran Ring The pyran ring of CRK has been replaced by a five-membered ring to give 3aminoindanol compounds, following molecular modelling studies (Buckle et al., 1991b). The compound, 17, was designed to have the same trans relationship between the 2-hydroxyl and the 3-amino substituent as between the 3- and 4positions of LCRK. Overlap studies showed that one could overlay these positions and keep the phenyl rings in the same plane by allowing the indane aromatic group to slide slightly toward the 1-position. This is shown in Plate 8.

The benzopyran ring system has also been replaced by a benzoxepine (Buckle et al., 1991b), to investigate the effect of increased bulk and conformational flexibility. In this series, the 2,3,4,5-tetrahydro-2,2-dimethyl-benzoxepine, 19, was of modest potency, but the di-nor-methyl compound, 18 was more potent than compound 19. Plate 9 shows a possible overlap of a chromanol and benzoxepine. The C2, C3 region of the benzoxepine, 18, occupies a region of space between the two methyl groups of LCRK and this accommodation may in part be responsible for the enhanced potency of compound 18. 4.2.2 Replacements for the Benzene Ring The electrostatic potential in the plane of the benzene ring of 6-cyanopyran was compared to that of a series of pyranopyridines (Stemp and Evans, 1993). On the basis of the similarity of the electrostatic potential maps, it was predicted that the [3,2-c]pyranopyridine ring system would most closely resemble the benzopyran. It was subsequently found that this pyranopyridine was a more potent KCA than [3,2b], [2,3-c] and [2,3-b]pyranopyridines. The benzene ring of CRK has been replaced by a thiophene ring by workers from R.W. Johnson (Sanfilippo et al., 1992, 1993; Press et al., 1993). Thieno[3,2-

CONFORMATIONAL ANALYSIS OF KCAS 115

b]-and thieno[2,3-b]pyran compounds, such as 20 and 21, with electronwithdrawing substituents have good activity that is comparable, or better than CRK. An unsubstituted thieno[3,4-b]pyran, 22, also has good activity, but the addition of the usual electron-withdrawing groups ortho to the sulphur atom reduces or abolishes activity. The authors suggest that not only is the electronwithdrawing function of the substituents important, but also the location is critical. They claim that the electrostatic potential maps calculated for typical thieno[2,3-b]pyran and thieno[3,2-b]pyran molecules have a similar structure, with what seems to be a large negative region extending over the middle of the thiophene ring and its substituent, directed away from the pyran. The thieno[3,4b]pyran series was claimed to exhibit a shift in the location of this negative region. The MNDO Hamiltonian (Dewar and Thiel, 1977a, 1977b, 1977c) was used to calculate these electrostatic potential maps. Plate 10 shows the electrostatic potential surrounding these structures. All three structures have a negative region of electrostatic potential above the carbons of the thiophene ring. This merges with the negative region due to the pyran oxygen in each case, but the cyano-substituted thieno[2,3-b]pyran and thieno[3,2–b]pyran structures have a more extended negative region, running towards the nitrile group.

4.3 Pyrrolidinone Replacements There have been many replacements for the pyrrolidinone ring of CRK reported in the literature. Chapter 2 contains an up-to-date review of these. The variety in the tolerated replacements allows us to sketch out some structural requirements for the benzopyran 4-position. The conformational properties of the 4substituents will be discussed in this section. The substituents have been arbitrarily divided into cyclic and acyclic replacements. A subsequent section will deal with attempts to build pharmacophore models, based on tolerated replacements.

116 K CHANNELS AND THEIR MODULATORS

4.3.7 Cyclic Replacements As discussed in detail above (section 4.1.2), the archetypal 4-substituent— pyrrolidinone—adopts an orthogonal orientation, relative to the benzopyran ring system. This is likely to be because of the steric repulsion suffered by the carbonyl and the 5’-hydrogens of the ring as they approach the peri-hydrogen at position 5, as discussed in section 4.1.2. Perhaps not surprisingly, many other rings of a similar size can adopt a similar orthogonal conformation. Many of these are five-or six-membered rings, with a nitrogen attached to the 4-carbon of the pyran ring. Table 4.2 lists some of the rings that have successfully replaced pyrrolidinone, along with the orientation of the internal N-C bonds of the rings, calculated using the AM1 Hamiltonian. One can see that each of these rings adopts a similar preferred conformation, with the ring approximately at right angles to the benzopyran. For all of these substituents, there are two possible minima found upon rotation of the C4-N1’ bond. The rotamer with the carbonyl oxygen on the same side of the ring as the 4-proton is the lower energy structure, usually by a few kcal/mol. Thus, all the ring structures in Table 4.2 can adopt similar orientations to pyrrolidinone, presenting a similar hydrogen bond accepting locus to the interaction site. The conformational behaviour of the piperidinone structure, 23, has been studied by NMR in a variety of solvents (Thomas and Whitcombe, 1990). A ratio of rotamers of the order of 88:12 was found in deuteriomethanol and deuteriodimethyl sulphoxide, and a ratio of 91:9 was found in D2O. These results may be compared to those for CRK mentioned in section 4.1.2.

Table 4.2 The orientation of various cyclic 4-substituents relative to the benzopyran template, derived from geometry optimized AM1 Hamiltonian calculations. R

Energy*

C10-C4N1’, -C2’, dihedral

Energy*

C10-C4N1’, -C2’, dihedral

Energy* Difference

morpholinone

-102.1

-132.4

-98.4

70.5

3.7

oxazolidinone

-59.4

-119.1

-59.9

102.1

-0.5

piperazinone

-55.8

-131.7

-52.2

69.7

3.6

pyridone

-31.2

-142.5

-26.7

74.2

4.5

thiamorpholinone

-56.3

-133.6

-51.9

64.8

4.4

thiazolidinone

-28.9

-144.2

-28.2

99.9

0.7

* AM1 geometry optimized heat of formation

It has been reported that a 5’-methyl substitution on the pyrrolidinone enhances activity (Bartmann, 1989). This structure has a slightly modified energy profile

CONFORMATIONAL ANALYSIS OF KCAS 117

upon rotation of the C4-N1’ bond, because of the extra steric influence of the 5’ methyl group. The result of this steric influence is to rotate further the pyrrolidinone substituent in its lower energy form, giving a C10-C4-N1’ -C2’ dihedral angle of -66°, rather than -129° in the equivalent CRK structure. The difference in energy between the two minima on the rotation pathway is also reduced from the usual 2.4 kcal/mol to only 1.4 kcal/mol. The change in the preferred orientation of the 4-substituent suggests that the 5’-methyl group is destabilizing the lower energy rotamer, relative to the higher one, resulting in a lower energy difference between the minima. The fact that this compound and the piperidinone, 23, mentioned above, both have this smaller difference in energy and are both more potent than CRK, suggests that the higher energy rotamers may be closer to the required binding conformation.

Gadwood et al. have replaced the pyrrolidinone ring with conformationally restricted spirocyclic structures, such as the imidazolone, 24 (Gadwood et al., 1993). The structure contains a formal orthogonal relationship between imidazolone and benzopyran due to the strictures of the spiro connection. The authors found that there were two low energy forms of the benzopyran, differing in the pyran ring pucker. The higher energy structure of the two was used in a proposed overlap with LCRK, as this was only 1.2 kcal/mol higher in energy— presumably calculated with the AM1 Hamiltonian. Using the overlap as a basis, the authors suggest that the C=N nitrogen of the imidazolone is the likely hydrogen bond acceptor, rather than the oxygen atom of the adjacent carbonyl group. Evidence in support of this is observed in the crystal structure, where the nitrogen atom accepts an intermolecular hydrogen bond. This nitrogen atom can be overlaid easily on the C=O oxygen of LCRK. Furthermore, the activity of the imidazolones correlated with the degree of electron density on this nitrogen atom, since 2-ethoxy and 2-(alkylthio) imidazolones were generally less potent than 2(alkylamino) imidazolones. 4.3.2 Acyclic Replacements Many acyclic replacements of the pyrrolidinone ring have been investigated. Acyclic amides have been prepared (Ashwood et al., 1990). Whereas the

118 K CHANNELS AND THEIR MODULATORS

acetamide, 25, possessed reasonable activity, the formamide, 26, was less potent. Also, larger alkyl group substitutions flanking the carbonyl group did not retain the level of activity of 25. Table 4.3 shows the calculated energies of some of these amides. Inspection of Table 4.3 The orientation of various acyclic 4-substituents relative to the benzopyran template, derived from geometry optimized AM1 Hamiltonian calculations.

R

Energy*

C10-C4N1’, -C2’, dihedral

Energy*

C10-C4N1’, -C2’, dihedral

Energy* Difference

-75.4

-147.5

-70.6

72.6

4.8

-69.5

-148.2

-65.0

74.8

4.5

-67.7

-166.0

-66.4

55.7

1.3

-67.3

-139.4

-63.2

69.3

4.1

-64.1

-125.8

-63.2

60.3

3.8

* AM1 geometry optimiz ed heat of formation

the table shows that the acetamide has a similar preferred orientation of the C=O oxygen to LCRK, with a C10-C4-N1’ -C2’ dihedral angle of -147.5° and a difference in AM1 heat of formation of 4.8 kcal/mol. However, when the acetamide N-H hydrogen is replaced by a methyl group, the more stable structure is the (Z)-form 27, in which the N-Me and C=O are on the same side of the amide bond for the lower energy C4-N1’ rotamer. The higher energy rotamer has equal energy (Z) and (E)-isomers.

CONFORMATIONAL ANALYSIS OF KCAS 119

4.4 The Aprikalim Series Aprikalim was discovered via general screening of a putative antiulcer compound, 28, which possessed slow onset hypotensive activity. This activity was due to the formation of the potent sulphoxide metabolite (RP49356), 29 (Aloup et al., 1990). The (1R, 2R) (-)-enantiomer (RP52891), known as aprikalim, possesses typical KCA behaviour. The detailed SAR of molecules related to aprikalim can be found in Chapter 3.

Some molecular modelling work has been reported on this compound (Brown et al., 1992). Conformational analyses of the (-)-enantiomer and the cis-isomer of aprikalim have been performed. These will be discussed below, along with other calculations.

120 K CHANNELS AND THEIR MODULATORS

4.4.1 Structure of Aprikalim X-ray crystallography studies of aprikalim, 28, (Brown et al., 1992) show an axial position for the sulphoxide oxygen in the thiacyclohexane ring. Other crystal structures are known in which thiacyclohexane sulphoxides have an axial oxygen (Robert and Gauchotte, 1977; Miler-Srenger et al., 1981; Yuasa et al., 1990), but there is also evidence for equatorial forms (Robert, 1977; Miler-Srenger et al., 1981). It appears that the location of the oxygen atom is very sensitive to other substituents on the ring and will adopt either equatorial or axial positions to accommodate the preferences of other groups. The thioamide group is trans to this oxygen, in a trans diaxial arrangement, while the pyridine ring is obviously cis to the sulphoxide. The disubstitution at position 2 of the thiacyclohexane ring causes a steric crowding and thus interactions between the ring and the two substituents influence the conformation of the ring itself and the disposition of the substituents. The X-ray crystal structure of 28 corresponds to the AM1 global energy minimum reported by Brown et al., (1992). There is some conformational freedom, however, as a series of conformers exists within a few kcal/mol of this minimum. For instance, it is reported that the difference in energy between the axial thioamide structure and an equatorial form is of the order of 1.7 kcal/mol. Table 4.4 shows calculated energies of eight conformers of 28, two chair forms, corresponding to the trans diaxial and trans diequatorial structures mentioned above and six twist boat structures. Plates 11 and 12 show these conformations and Table 4.5 shows that the rings all have distinct pucker parameters. The structures were generated using the DGEOM program and were subsequently geometry optimized, using the AM1 Hamiltonian in MOPAC and SPARTAN. Sulphur parameters were incorporated into MOPAC from Dewar’s publication (Dewar and Yuan, 1990). One can clearly see Table 4.4 The heats of formation of various conformers of RP52891 29 and its cis isomer 30, using geometry optimized AM1 Hamiltonian calculations. Structure

AM1 Energy*

Energy relative to lower chair

Chair1trans Chair2trans Twist Boat1trans Twist Boat2trans Twist Boat3trans Twist Boat4trans Twist Boat5trans Twist Boat6trans

26.2 22.0 35.4 25.4 24.3 31.4 26.5 24.1

4.2 0.0 13.4 3.4 2.3 9.4 4.5 2.1

CONFORMATIONAL ANALYSIS OF KCAS 121

Structure

AM1 Energy*

Energy relative to lower chair

Chair1cis 28.0 Chair2cis 25.7 Twist Boat1cis 30.3 Twist Boat2cis 32.9 Twist Boat4cis 29.1 Twist Boat6cis 31.2 *AM 1 energies with zero-point correction for 298 K

2.3 0.0 4.6 7.2 3.4 5.5

Table 4.5 Cremer and Pople puckering coordinates for various conformers of RP52891 29 and its cis isomer 30, using geometry optimized AM1 Hamiltonian calculations. Structure

β

q2

Q

q3

β

Chair1trans Chair2trans Twist Boat1trans Twist Boat2trans Twist Boat3trans Twist Boat4trans Twist Boat5trans Twist Boat6trans Chair1cis Chair2cis Twist Boat1cis Twist Boat2cis Twist Boat4cis Twist Boat6cis

8 175 85

0.09 0.06 0.90

0.68 0.68 0.91

0.68 -0.68 0.08

57 200 32

87

0.89

0.89

0.04

90

95

0.83

0.84

-0.08

153

96

0.94

0.95

-0.10

208

93

0.91

0.91

-0.05

268

88

0.86

0.86

0.04

335

8 168 85

0.10 0.14 0.89

0.67 0.68 0.90

0.66 -0.67 0.08

50 223 37

85

0.90

0.91

0.08

84

98

0.85

0.86

-0.11

206

83

0.80

0.81

0.10

346

that the ring could easily adopt a range of conformations, as the energy differences between conformers are low. One interesting feature of some of the structures is a possible intramolecular ‘hydrogen bond’ from the thioamide NH to the sulphoxide O. This is noticeable in the AM1 calculations, but is probably more of a non-directional charge-charge

122 K CHANNELS AND THEIR MODULATORS

interaction, rather than a true hydrogen bond. The geometry is not that of a classical hydrogen bond, as the N-H-O angle is only 117°, but it has been suggested that this interaction helps to stabilize the trans equatorial structure (Brown et al., 1992). 4.4.2 Calculations on the Cis-isomer Calculations have been reported for the cis-isomer, 30, of aprikalim, which is inactive in vitro (Brown et al., 1992). The global energy minimum structure has a chair structure for the thiacyclohexane ring, but both the thioamide and the pyridine ring are rotated relative to the lowest energy trans form. This allows the thioamide NH to interact with the sulphoxide O in an analogous ‘hydrogen bond’ to that found in the opposite chair structure of 28 mentioned above. Table 4.4 shows calculated AM1 energies for various ring conformations of the (1S,2R) form. It was not possible to find minima for all six twist boats; two structures, corresponding to Cremer and Pople β values of 150° and 270°, could not be isolated. These ring conformations are probably unstable due to steric crowding caused by the substituents at position 2 of the ring. It is also apparent that the separation in energy between the twist boat and the chair forms is greater for 30, compared with 28. It is possible that this inflexibility of 30 does not allow the compound to adopt a conformation recognized by the receptor, thus accounting for its lack of potency.

4.5 Studies on Pinacidil Pinacidil, 31 and related cyanoguanidines were developed as bioisosteric replacements for hypotensive thioureas (Petersen et al., 1978) and were discovered subsequently to be KCAs. Chapter 3 contains data on the SAR of this class of compounds. Conformational aspects of the class are covered below.

CONFORMATIONAL ANALYSIS OF KCAS 123

4.5.1 Structure of Pinacidil The (R)-isomer of pinacidil, 31, is the more potent form. Although this is a 4pyridyl-substituted molecule, it is noteworthy that most of the analogues reported by Petersen et al. (1978), were more potent as 3-pyridyl compounds. The crystal structure of 31 has been reported (Pirotte et al., 1993) and this structure is shown in Plate 13. It is interesting to note that the cyano group is cis to the pyridyl amino group.

4.5.2 Rotation of the N- and N’-substituents of Pinacidil Theoretical studies on pinacidil have been performed, to investigate its Conformational preferences. Plate 14 shows the Ramachandran diagram (AM1 energy versus torsion angle) for rotation of the bonds connecting the pyridine ring to the cyanoguanidine group for the 3-pyridyl pinacidil analogue, LY222674, 32. The two bonds were rotated through 360°, with full relaxation of the remainder of the molecule. The figure shows that there is relatively free rotation possible about the pyridine-NH bond and restricted rotation possible about the NH-C(NCN) bond of the cyanoguanidine.

Plate 14 shows the location of the corresponding torsion angle values for the Xray crystal structure of (R)-pinacidil on the Ramachandran energy surface. It also shows the location of a structure which has been reported to overlap with cromakalim. It can be seen that there is a low energy pathway between these two conformations. This suggests that if a conformation corresponding to the overlap conformation were required at the receptor, this is attainable for a low energy cost.

124 K CHANNELS AND THEIR MODULATORS

4.6 Possible Pharmacophore Models There are many different structural classes of KCAs now extant. These pose several questions. Do these diverse structures act at the same receptor? If so, do they interact in a similar manner, with the same set of amino acids in that receptor? A reasonable pharmacophore model requires that each of these questions needs to be answered in the affirmative. There is evidence that KCAs act at the same receptor, in that they are blocked characteristically by glibenclamide. This may be taken as evidence that KCAs are acting in a similar manner, though this is certainly not conclusive proof. Further evidence that KCAs are acting at the same site has been provided (Manley et al., 1993). It has been shown that thirteen structurally distinct KCAs all displaced a tritiated pinacidil analogue from the receptor in radioligand binding studies. In general, the correlation between the pD2 for inhibition of rat portal vein spontaneous activity versus the pKi for inhibition of radioligand binding is good, (r=0.96, slope=0.88, n=13). However, although there is little difference between the concentration of compound needed to inhibit either radioligand binding or spontaneous activity for pinacidil analogues, the concentrations of benzopyrans needed to inhibit radioligand binding are higher (by about half a log unit) than those necessary for inhibiting activity. This is presented as evidence that the KCAs do not all bind at the same site. If this is the case, then attempts at constructing pharmacophores are doomed to failure. Despite this evidence—or before it was published—many workers have made and tested hybrid KCAs, based on pharmacophore models. The rest of this section will describe these models.

Work in finding new hybrid structures has shown that it is possible to mix some of the structural elements of cromakalim and pinacidil and obtain potent compounds (Atwal et al., 1992, Burrell et al., 1993). Atwal et al., (1992) synthesized cyclic and acyclic cyanoguanidines, such as 33 that have antihypertensive activity. It was suggested that these hybrids provided evidence that cyanoguanidine could mimic the pyrrolidinone amide of CRK. Additionally, the benzene ring of CRK was equivalent to the pyridine of pinacidil and both compounds had lipophilic groups, namely the alkyl sidechains, to give a three

CONFORMATIONAL ANALYSIS OF KCAS 125

point pharamacophore. Burrell et al., (1993) have reported that a series of urea analogues, including the cyanoguanidine, 34, have antihypertensive activity. However, the structure-activity relationships in this series do not correlate exactly with those of the pinacidil series. Thus, replacement of the cyanoguanidine group by the urea group leads to a loss of potency in pinacidil analogues, but not in benzopyrans. Replacement of the cyanoguanidine group by the nitroethene substituent retains potency in pinacidil analogues, but, in contrast, this change reduces potency in the benzopyran series. These observations suggest that the two series of KCAs act at different receptor sites. A series of publications has appeared where the authors claim to have designed compounds based on a pharmacophore model (Koga et al., 1993a, 1993b, 1993c, 1993d; Ishizawa et al., 1993). The pharmacophore consists of four elements: two regions of lipophilic interaction and two vectors representing interaction with hydrogen bond donors. The lipophilic regions correspond to the 2, 2-dimethyl group of CRK and the benzene ring. The hydrogen bond acceptors are defined as the carbonyl oxygen and at the cyano nitrogen of CRK. The authors claim to have designed compounds such as 35 based on this pharmacophore model. 4.7 Conclusion This chapter has presented some of the calculations and analyses that have been attempted on the ATP-sensitive potassium channel activators. Work has been published on conformational preferences, analysis of X-ray crystal structures and pharmacophoric overlaps. Novel work has also been presented here on ring flipping of some KCAs and on aprikalim conformations. It can be expected that studies on KCAs will continue to be based on their structural properties, until there is information available on the structure of their molecular targets. There are still many issues that require study and clarification in this area. For instance, the SAR of the 6-position of CRK is not satisfactorily resolved—why do certain alkyl groups retain potency, when most potent compounds have strong electron-withdrawing groups? The problems associated with the universal pharmacophore models mentioned in section 4.6 also need further study. It may prove necessary to classify KCAs into small groups to be confident of reasonable overlap at the receptor. Alternatively, it may be possible to use newer threedimensional techniques, such as DISCO and CoMFA to define a generic pharmacophore model. References ALOUP, J.C., FARGE, D., JAMES, C., MONDOT, S. & CAVERO, I. (1990) Drugs of the Future, 15, 1097–1108.

126 K CHANNELS AND THEIR MODULATORS

ASHWOOD, V.A., CASSIDY, F., COLDWELL, M.C., EVANS, J.M., HAMILTON, T.C., HOWLETT, D.R., SMITH, D.M. & STEMP, G. (1990) J. Med. Chem., 33, 2667–2672. ATWAL, K.S., MORELAND, S., MCCULLOUGH, J.R., AHMED, S.Z. & NORMANDIN, D.E. (1992) BioMed. Chem. Lett., 2, 87–90. BARTMANN, W. (1989) In: Trends in Medicinal Chemistry, van der Groot, H., Domany, G., Pallos, L. & Timmerman, H. (eds). Elsevier, Amsterdam, pp. 629–657. BROWN, T.J., CHAPMAN, R.F., COOK, D.C., HART, T.W., MCLAY, I.M., JORDAN, R., MASON, J.S., PALFREYMAN, M.N., WALSH, R.J.A., WITHNALL, M.T., ALOUP, J.-C., CAVERO, I., FARGE, D., JAMES, C. & MONDOT, S. (1992) J. Med. Chem., 35, 3613–3624. BUCKLE, D.R., EGGLESTON, D.S., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., READSHAW, S.A., SMITH, D.G. & WEBSTER, R.A.B. (1991a) J. Chem. Soc. Perkin Trans I, 2763–2771. BUCKLE, D.R., ARCH, J.R.S., EDGE, C., FOSTER, K.A., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G., TAYLOR, J.F., TAYLOR, S.G., TEDDER, J.M. & WEBSTER, R.A.B. (1991b) J. Med. Chem., 34, 919–926. BUCKLE, D.R., EGGLESTON, D.S., PINTO, I.L., SMITH, D.G. & TEDDER, J.M. (1992) BioMed. Chem. Lett., 2, 1161–1164. BURRELL, G., EVANS, J.M., HICKS, F. & STEMP, G. (1993) BioMed. Chem. Lett., 3, 999–1002. CASSIDY, F., EVANS, J.M., SMITH, D.M., STEMP, G., EDGE, c. & WILLIAMS, D.J. (1990) J. Chem. Soc., Chem. Commun., 377–378. CREMER, D. & POPLE, J.A. (1975) J. Amer. Chem. Soc., 97, 1354–1358. DEWAR, M.J.S. & THIEL, W. (1977a) J. Amer. Chem. Soc., 99, 4899–4907. (1977b) J. Amer. Chem. Soc., 99, 4907–4917. (1977c) Theor. Chim. Acta, 46, 89–104. DEWAR, M.J.S. & YUAN, Y.-C. (1990) Inorg. Chem., 29, 3881–3890. DEWAR, M.J.S., ZOEBISCH, E.G., HEALY, E.F. & STEWART, J.J.P. (1985) J. Amer. Chem. Soc., 107, 3902–3909. FABIAN, W.M.F. (1988) J. Comp. Chem., 9, 369–377. GADWOOD, R.C., KAMDAR, B.V., CIPKUS DUBRAY, L.A., WOLFE, M.L., SMITH, M.P., WATT, W., MISZAK, S.A. & GROPPI, V.E. (1993) J. Med. Chem., 36, 1480–1487. GUNDERTOFTE, K., PALM, J., PETTERSSON, I. & STAMVIK, A. (1991) J. Comp. Chem., 12, 200–208. ISHIZAWA, T., KOGA, H., OHTA, M., SATO, H., MAKINO, T., KUROMARU, K., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993) BioMed. Chem. Lett., 3, 1659–1662. KOGA, H., OHTA, M., SATO, H., ISHIZAWA, T. & NABATA, H. (1993a) BioMed. Chem. Lett., 3, 625–631. KOGA, H., SATO, H., ISHIZAWA, T., KUROMARU, K., NABATA, H., IMAGAWA, J., YOSHIDA,S. & SUGO, I. (I993b) BioMed. Chem. Lett., 3, 1111–1114. KOGA, H., SATO, H., ISHIZAWA, T., KUROMARU, K., MAKINO, T., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993c) BioMed. Chem. Lett., 3, 1115–1118. KOGA, H., SATO, H., IMAGAWA, J., ISHIZAWA, T., YOSHIDA, S., SUGO, L, TAKA, N., TAKAHASHI, T. & NABATA, H. (1993d) BioMed. Chem. Lett., 3, 2005–2010.

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MANLEY, P.W., QUAST, U., ANDRES, H. & BRAY, K. (1993) J. Med. Chem., 36, 2004–2010. MILER-SRENGER, E., STORA, C. & HUGHES, N.A. (1981) Acta Crystallogr., Sect B, 37, 356–360. PETERSEN, H.J., NIELSEN, C.K. & ARRIGONI-MARTELLI, E. (1978) J. Med. Chem., 21,773–781. PIROTTE, B., DUPONT, L., DE TULLIO, P., MASEREEL, B., SCHYNTS, M. & DELARGE, J. (1993) Helv. Chim. Acta, 76, 1311–1318. PRESS, J.B., MCNALLY, J.J., SANFILIPPO, P.J., ADDO, M.F., LOUGHNEY, D., GIARDINO, E., KATZ, L.B., FALOTICO, R. & HAERTLEIN, B.J. (1993) BioMed. Chem., 1, 423–435. QUAST, U. & VILLHAUER, E.B. (1993) Eur. J. Pharmacol. Mol. Pharm., 245, 165–171. ROBERT, F. (1977) Acta Crystallogr., Sect B, 33, 3480–3484. ROBERT, F. & GAUCHOTTE, S. (1977) Acta Crystallogr., Sect B, 33, 3484–3487. SANFILIPPO, P.J., MCNALLY, J.J., PRESS, J.B., FITZPATRICK, L.J., URBANSKI, M.J., KATZ, L.B., GIARDINO, E., FALOTICO, R., SALATA, J., MOORE, J.B. & MILLER, W. (1992) J. Med. Chem., 35, 4425–4433. SANFILIPPO, P.J., MCNALLY, J.J., PRESS, J.B., FALOTICO, R., GIARDINO, E. & KATZ, L.B. (1993) BioMed. Chem. Lett., 3, 1385–1388. STEMP, G. & EVANS, J.M. (1993) In: Medicinal Chemistry, 2nd Edition. Ganellin, C.R. and Roberts, S.M. (eds). Academic Press Ltd., London, pp. 141–162. STEWART, J.J.P. (1990) J. Comp. Aided Mol. Des., 4, 1–105. THOMAS, W.A. & WHITCOMBE, I.W.A. (1990) J. Chem. Soc., Chem. Commun., 528–529. YUASA, H., TAKENAKA, A. & HASHIMOTO, H. (1990) Bull. Chem. Soc. Jpn., 63, 3473–3479.

Recent Literature OHTA, M., KOGA, H., SATO, H. & ISHIZAWA, T. (1994) Comparative Molecular Field Analysis of Benzopyran-4-carbothioamide Potassium Channel Openers. BioMed. Chem. Letts., 4, 2903–2906.

5 The Structure-Activity Relationships of Potassium Channel Blockers R.CROSSLEY & A.OPALKO Wyeth Research UK, Huntercombe Lane South, Taplow, Maidenhead, Berkshire 516 OPH, UK.

5.1 Introduction To date very few drugs have been developed as potassium channel blocker (KCBs). This may seem to be a strange introduction to a chapter concerned with the Structure-Activity Relationships (SAR) of such agents but it is nevertheless true. In the main, compounds in this chapter had originally been developed with various therapeutic goals in mind and were screened accordingly in an in vitro or an in vivo model which reflected this goal. Subsequently, the lead compounds in the various series were found to be KCBs and the responses of these lead compounds at various K channels were then examined in some detail. This distinction may seem to be rather arbitrary and inconsequential, but it means that the derived SAR of such compounds not only reflect their abilities as KCBs, but also other factors as well. The presence of lipophilic groups, for example may reflect the need for a drug to cross a membrane rather than to locate a hydrophobic pocket on a receptor and, for these compounds, it is not possible to determine a priori what the interactions at the receptor level actually are. In spite of this, the SAR discussed here will assume that it is concerned with activity at the channels. More recently, drugs are being developed from their inception with due consideration both to their primary K channel blocking effects and with their eventual use in mind. There are many different kinds of potassium channel blockers but it is not possible to deal meaningfully with certain classes of these. Some compounds, for example tetraethylammonium (TEA) and quinine, are quite promiscuous in their effects on K channel subtypes but may be used in general classification of channels. Others, for example charybdotoxin, apamin, mast cell degranulating peptide (MCDP), and dendrotoxin, are structurally complex and there is some speculation, but little hard evidence, as to which of their elements are involved in binding. A general review of these types of compound is to be found elsewhere (Cook and Quast, 1990).

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 129

5.2 ATP-Sensitive K Channel Blockers 5.2.1 Introduction The coupling of the metabolic state of a cell to its excitability is largely governed by the presence of K channels in the cell membrane which are inhibited by a rise in intracellular levels of the purine nucleotide ATP. These ATP-sensitive K channels (KATP) have been identified in cardiac (Fosset et al., 1988; Lederer and Nichols, 1989), pancreatic (Schmid-Antomarchi et al., 1987) and nerve (Bernardi et al., 1988; Mourre et al., 1989; Levesque and Greenfield, 1991) cells and in skeletal (Weik and Neumcke, 1989) and smooth muscle (Gopalakrishnan et al., 1991) cells. They thereby present significant therapeutic opportunities in all these areas, especially in the CNS (Miller, 1990), although only two of these have been extensively exploited to date (Gopalakrishnan et al., 1993). The subject of smooth muscle KATP channel activators has already been dealt with in previous chapters and an established use of pancreatic KATP channel blockers is in the control of blood sugar levels by mediating the release of insulin. In the pancreas, a rise in intracellular glucose leads to activation of mechanisms which produce an increase in ATP and this leads to inhibition of the KATP channel. This in turn alters the membrane potential to the threshold for voltage dependent Ca channels, leading to opening and influx of calcium, which in turn triggers insulin release. Drugs which have a similar effect to this in inhibiting the KATP channels in the pancreas form the main line of drug treatment for early stage diabetes. 5.2.2 Sulphonylureas and Related Molecules The hypoglycaemic sulphoriylureas have been used in the therapy for type II diabetes for many years with various speculations as to their mode of action (Rasmussen et al., 1981; Asmal and Marble, 1984; Gylfe et al., 1984), but it is only relatively recently that their effect in lowering blood glucose has been directly linked with blockade of KATP channels (Schmid-Antomarchi et al., 1987; Gaines et al., 1988; Henquin, 1988; Bernardi et al., 1989; Ashford, 1990). They were originally developed from the antibacterial sulphonamides, which were noted to produce hypoglycaemia when administered at high doses (Loubatieres, 1957). The original group of sulphonylureas are typified by the relatively simple first generation compounds such as tolbutamide, tolazamide, chlorpropamide, carbutamide, gliclazide and glibornuride (Figure 5.1). These compounds all have a simple aryl substituent and a lipophilic urea substituent. The second generation compounds (Figure 5.2) retain this arylsulphonylurea moiety but extend it significantly by the introduction of ethylaminoacylaromatic units which extend

130 K CHANNELS AND THEIR MODULATORS

the pharmacophore. This has led to the production of gliquidone, glisoxepide, glibenclamide (glyburide), glipizide and glisindamide (HOE 036). It is these second generation compounds which, because of their greater affinity, have also provided the radioligands [3H]-glyburide (Geisen et al., 1985; Bernardi et al., 1989), [125I]-glyburide (Robertson et al., 1990) and the 5-[125I]-2-hydroxy analogue of glyburide (Rajan et al., 1993). The availability of these ligands, along with a labelled pinacidil analogue (Manley et al., 1993) have led to the isolation and identification of the sulphonylurea receptor as a 150 kDa protein which has been considered to either

Figure 5.1 Structural formulae of first generation sulphonylureas

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 131

Figure 5.2 Formulae of second generation sulphonylureas

form the ion channel or is more likely closely associated with it (Bernardi et al., 1988; Aguilar-Bryan et al., 1990; Rajan et al., 1993; Manley et al., 1993). The use of these ligands has also indicated the presence of subtypes of these channels (Gopalakrishnan et al., 1991; Lazdunski et al., 1992; Rajan et al., 1993). The observed SAR of the sulphonylureas led to the initial proposal (Rufer et al., 1974) for a putative receptor site which covered all the first and second generation compounds and which can be extended to include other compounds active at this receptor (Figure 5.3). These include sulphonamides such as glymidine, thus providing a link with the antibacterial sulphonamides. The proposed model also extends (Biere et al., 1974; Rufer and Losert, 1979) to analogues with reversed amide functionality as well as to some chiral derivatives such as (-)-S-gliflumide and the less potent (-)-S-4-N-(l-(5-fluoro-2methoxyphenyl)ethyl)carbamoyl)methylbenzoic acid (1) both of which were considerably more potent than their enantiomers, with eudismic ratios of 40 and 18 respectively. There is some evidence that the positions of the aromatic and alkyl sites on the pharmacophore may exchange when the amide group is reversed and this presumably reflects a conformational change (Biere et al., 1974). Finally, some drugs such as meglitinide (HB-699) and AZ-DF 265

132 K CHANNELS AND THEIR MODULATORS

(Figure 5.3) dispense with the sulphonylurea functionality and in its place have a carboxylic acid. These compounds can also be incorporated into a pharmacophore model. The chiral AZ-DF 265 also demonstrates enantioselectivity with the (-)-enantiomer being the eutomer with a eudismic ratio of around 10 (Garrino and Henquin, 1988). The single pharmacophore model which results from combining all these compounds illustrates, amongst other things, the non-criticality of the sulphonylurea function (Figure 5.4). In this model a central aromatic moiety is extended to right and left to pick up ancillary binding sites. The various molecules in this class may occupy either the right hand end (the first generation compounds and sulphonamides), both left and right ends (the second generation compounds) or just the left hand end (benzoic acid derivatives). At the left is an aromatic ring, preferably electron rich, an alkyl group and an amide arranged as in the S configuration of gliflumide or, as in the case of

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 133

Figure 5.3 Formulae of benzoic acid and sulphonamide analogues of sulphonylureas

Figure 5.4 Pharmacophore model for sulphonylurea binding site

gliquidone (Figure 5.2), a heterocycle containing these elements. To the right there appears to be an acidic sulphonamide or sulphonylurea NH or carboxylic acid group and a lipophilic group which presumably picks up a hydrophobic interaction at the receptor site. The range and variety of the analogues which have been studied make the sulphonylurea binding site one of the best characterised there is. The proof that this forms part of the KATP receptor comes from the observation that the hyperglycaemic compound diazoxide (Figure 5.3), a cyclic sulphonamide, opens these channels (Zunkler et al., 1988), and from extensive correlations of the activities of several sulphonylureas with channel blocking activity (Schmid-

134 K CHANNELS AND THEIR MODULATORS

Antomarchi et al., 1987; Bernardi et al., 1988; De Weille et al., 1989; Amoroso et al., 1990). There is, for example, an excellent correlation, for a whole series of compounds, including most of those in Figures 5.1 and 5.2, between the inhibition of [3H]-glibenclamide binding and rubidium efflux in insulinoma cells and a similar correlation between binding in this preparation and in heart and brain microsomes. In addition, a correlation of the KATP channel inhibition by sulphonylureas and the release of GABA in the substantia nigra indicates that they may also modify the responses in the brain to diabetes, ischaemia and anoxia and hence present alternative therapeutic opportunities for these compounds (Amoroso et al., 1990). 5.2.3 Imidazolines and Related Molecules Imidazoline-containing noradrenergic β 2-receptor antagonists have been used to treat diabetes for some time and their mechanism of action was assumed to be a result of the activity at this receptor being indirectly responsible for insulin release (Mohrbacher et al., 1987). More recently, some of these compounds (Figure 5.5) have been shown to antagonise the vascular actions of cromakalim (CRK) in a concentration dependent manner with an order of potency alinidine = phentolamine > tramazoline = naphazoline (McPherson and Angus, 1989). The relative and absolute potencies of the compounds, coupled with the observation that the closely related tolazoline was inactive as were other standard β 2-receptor antagonists, is strongly suggestive that they have the additional property as KATP channel blockers in smooth and cardiac muscle. The effect on pancreatic channels is less clear as tolazoline does seem to potentiate insulin release along with phentolamine. This is still proposed to be a consequence of the imidazoline structure

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 135

Figure 5.5 Structural formulae of imidazoline derivatives

rather than β 2-receptor blockade per se as antazoline, which has only very little activity as an β 2-receptor antagonist, is also effective in promoting insulin release (Schulz and Hasselblatt, 1989). Apart from the observation that the imidazoline moiety is essential for activity (the related compound 1-benzylimidazole is inactive), there is as yet little SAR available which can be directly related to KATP channel blockade and it remains to be seen if is possible to design out the β 2-receptor properties and develop these compounds into therapeutically effective KATP channel blockers. 5.2.4 Miscellaneous Compounds KATP channels are also blocked by relatively non-specific (Cook and Quast, 1990) KCBs such as TEA, 4-aminopyridine, quinine and quinidine as well as by more specific compounds such as lidocaine and several barbiturates, although at relatively high concentrations (Ashcroft and Ashcroft, 1990). There are, however, some compounds, linogliride, ciclazindol and BRL 31660 (Figure 5.6), which may turn out to be the progenitors of relatively specific KATP channel blockers. Linogliride Linogliride is a member of a class of guanidine-based insulin secretogogues (Mohrbacher et al., 1987) and has some structural similarities with the imidazolines. It is unclear as yet if this similarity is of consequence in its interaction with a receptor or if linogliride has the same mode of action as the

136 K CHANNELS AND THEIR MODULATORS

sulphonylureas. Nevertheless, linogliride has been shown electrophysiologically to inhibit KATP channels in pancreatic β -cells, an effect which is sensitive to pretreatment with tolbutamide (Figure 5.1). It may, therefore, prove to be a lead to a new chemical class of KATP channel blockers. Other guanidine hypoglycaemics do not seen to have been examined as KATP channel blockers in any detail.

Figure 5.6 Structural formulae of miscellaneous KATP channel blockers

Ciclazindol The antidepressant drug ciclazindol, and the related compound mazindol (Figure 5.6), mediate their antidepressant effect mainly through inhibition of monoamine uptake (Oh et al., 1979) and, although there are differences between the compounds, both have some similarities to the imidazolines. Ciclazindol and mazindol do reduce blood glucose levels in a non-insulin-dependent manner, putatively by increasing uptake into human skeletal muscle (Kirby and Turner, 1977). Subsequently, ciclazindol has been shown to block the effects of K channel activators (KCAs) in rat portal vein and bladder, an effect which is associated with KATP channel blockade, but ciclazindol is not totally selective for these channels (Noack et al., 1992). Notably, there are no reports on the effects of mazindol. Because ciclazindol does not displace [3H]-glibenclamide from porcine brain, its effect is presumed to be a result of interaction at a site different from that of the sulphonylureas. This may, however, reflect the involvement of a different subtype of KATP channel. Interactions of ciclazindol with KATP channels in the ventromedial hypothalamic nucleus may be responsible for the anorectic effects of this drug (Noack et al., 1992) and this may lead to uses for KATP channel blockers by centrally mediated mechanisms.

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 137

BRL 31660 The novel combined Class I and Class IV antiarrhythmic agent BRL 31660 inhibits the effects of CRK and other KCAs in guinea pig trachea, (Taylor et al., 1989; Arch et al., 1991) an effect which is associated with inhibition of K+ efflux. The compound is a sulphonamide but it is not clear if it is mediating its effects through interaction with the sulphonylurea receptor site or elsewhere. It has some structural features, such as a basic centre and naphthalene ring, which are not commonly found in the sulphonylureas but are present in the imidazolines. It may also prove to be a useful tool to elucidate the mechanisms of action of KATP channels in different tissues (Taylor et al., 1989).

5.3 Class III Antiarrhythmic Agents 5.3.1 Introduction The treatment of arrhythmias by prolonging the repolarisation in the heart has been achieved by a number of synthetic agents through a variety of pharmacological actions. The majority of these mechanisms involve the modulation of voltage gated ion channels of one type or another and these modes of action have been classified into five distinct classes (Vaughan Williams, 1984) based on the pharmacological properties of the drug. Most early antiarrhythmic drugs were Class I agents, and these compounds block the fast sodium channels, which are responsible for depressing the rapid phase of depolarisation, and thereby stabilise cell membranes. Class II agents inhibit the activity of the sympathetic nervous system; the β -adrenoceptor (beta blockers) are examples of drugs which manifest their actions by this mechanism. Class III agents prolong the action potential duration by blockade of one or more types of K channels, whereas Class IV agents block Ca channels and Class V agents slow the rate of sinus depolarisation by modulating Cl channels. There are least eight different K channels found in the heart (Colatsky and Follmer, 1989; Colatsky, 1991) which are thought to influence the action potential under normal physiological conditions. Amongst those channels, suitable targets for drug intervention, are the delayed rectifier channel (IK), which is subdivided into the rapidly (IKr) and slowly activating (IKs) subtypes (Noble and Tsien, 1969; Sanguinetti and Jurkiewicz, 1990), a voltage dependent transient outward current (IA) (Giles and Ginneken, 1985; Kenyon and Sutko, 1987; Tseng and Hoffman, 1989) and the inward

138 K CHANNELS AND THEIR MODULATORS

Figure 5.7 Analogues of clofilium

rectifier (background) current (IK1) (Sakmann and Trube, 1984). A number of Class III antiarrhythmic drugs such as d-sotalol (Figure 5.8), E-4031 (Figure 5.10) and MS-551 (Figure 5.10) have been examined for their effects on two types of cloned rat K channels, RH1 (Kvl.2) and RH10 (Kvl.4) isolated from rat heart, but they had no effect (Yamagishi et al., 1993). Some Class III agents have been identified which are more selective for distinct K channel subtypes such as the delayed rectifier channel (IK) Agents which have been shown to be selective for (IK) are risotilide (Figure 5.8), E-4031 (Figure 5.10) and dofetalide (Figure 5.9). Drugs which are less specific are d-sotalol (Figure 5.8) which blocks (IK), (IK1) and (IA.) and amiodarone and clofilium (Figure 5.7) which block (IK) and (IK1). Also, some Class Ia drugs such as quinidine and disopyramide also block potassium currents such as (IK), (IK1) and (IA) and Class Ic drugs such as encainide and flecainide block (IK) as well as sodium currents (Colatsky et al., 1990). Although a number of Class III agents have shown selectivity for (IK). there have been no systematic structure-activity studies correlating antiarrhythmic efficacy with (IK) blocking ability. Accordingly this section will concentrate on reviewing how the current agents were developed and what the structural requirements for Class III activity are.

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 139

Figure 5.8 First generation analogues

140 K CHANNELS AND THEIR MODULATORS

Figure 5.9 Second generation phenoxyalkylamines

There have been a number of reviews of Class I and Class III drugs (Arrowsmith and Cross, 1990), Class III agents (Morgan Jr. and Sullivan, 1992), KCMs (Robertson and Steinberg, 1990; Atwal, 1992) and KCBs (Cimini and Gibson, 1992). As the biological aspects of antiarrhythmic therapy will be discussed later in this book (Chapter 10) the focus of this section will be on the chemistry of Class III agents. There are three main classes of chemical structures which have served as leads, these are clofilium, a quaternary compound (Figure 5.7), sotalol and sematilide (Figure 5.8). All the series were developed initially by virtue of their actions in vivo and subsequently the connection with K channel blocking activity has been established. 5.3.2 Quaternary Compounds Clofilium (Steinberg et al., 1984), a quaternary ammonium compound, was the starting point for a number of variations to its structure (2, Figure 5.7). The first of these imposed some rigidity into the alkylamine chain by the introduction of unsaturation and this gave compounds which were just as active as clofilium

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 141

(Morgan Jr. et al., 1986). This suggests that clofilium in its active conformation is in an extended form. The conformational flexibility at the quaternary nitrogen was also examined by constraining the quaternary ammonium moiety into 2- and 3-substituted phenylalkyl quinuclidines (Morgan Jr. et al., 1987) or an imidazolium group as in CK-1649

Figure 5.10 Miscellaneous second generation analogues

(Lis et al., 1987b) (Figure 5.7). Generally the 2-substituted analogues had both Class I and III activity whereas the 3-substituted derivatives had more Class III selectivity. Following the demonstration that the β -adrenoceptor antagonist (β -blocker) sotalol (Figure 5.8) also possessed Class III activity (Singh and Williams, 1970) some of its features, such as the sulphonamide and the ethanolamine chain were combined with these quaternary compounds to give a series of imidazolium derivatives (Lis et al., 1987b), for example CK-1649 (Figure 5.7). Quaternization of the amine group in various β -blocking drugs leads to greatly diminished β -blocking activity but maintains Class III antiarrhythmic activity (Paterson et al., 1980). A series of quaternary derivatives of the β -blocker propranolol, pranolium UM272 (Eller et al., 1983), UM301 (Gibson et al., 1986) and UM424 (Figure 5.7, Gibson et al.,. 1985), were developed and found to have respectable antiarrhythmic and antifibrillatory activity. Unfortunately, the oral bioavailability of such quaternary compounds is rather poor and so clinical development has not been possible.

142 K CHANNELS AND THEIR MODULATORS

First generation analogues Class I antiarrhythmic agents such as encainide, flecainide or moricizine (Morganroth and Bigger, 1990) used to be the most frequently used agents clinically, until results from the cardiac arrhythmia suppression trial (CAST) revealed that there was an increased risk of morbidity when patients were treated with these drugs. This resulted in a change in the focus of research interest to Class III agents, where the majority of effort has revolved around sulphonamide derivatives. The methylsulphanilide moiety features in most of the agents, for example sematilide (Figure 5.8), dofetalide (Figure 5.9) and E-4031 (Figure 5.10) which are in clinical trials and the d-enantiomer of sotalol (Figure 5.8). Procainamide is a Class 1A antiarrhythmic (Figure 5.8), which is readily acetylated in humans (Karlsson, 1978) to give the N-acetyl derivative, NAPA (Figure 5.8) and this metabolite has been shown to be primarily a Class III agent (Dangman and Hoffman, 1981). Replacement of the acetamide group with the more stable and also more acidic methylsulphonylamino group, present in the structurally related molecule sotalol, resulted in sematilide (Lumma Jr. et al., 1987). Sematilide, is fifty times more potent than NAPA, as a selective Class III compound. The same group found that this moiety could be replaced with an imidazol-1-yl function (3, Figure 5.8) (Morgan Jr. et al., 1990b) to give a compound which shows similar efficacy and potency to sematilide. Various modifications of the basic sematilide structure have resulted in compounds which have combined Class I and III activity (4, Figure 5.8) (Philips et al., 1990). It was found that substitution at R1, R2 or R3 in the general formula (4) with phenyl or substituted phenyl led to more active Class III compounds than sematilide, but also resulted in the introduction of Class I activity which was generally detected at higher concentrations. Combined Class II/III antiarrhythmic agents have been obtained by combining β -blocking (Class II) activity into the sematilide structure (Philips et al., 1992). An arylpiperazine moiety (5, Figure 5.8) provides the Class II or β -blocking pharmacophore1 and this was combined with features from sematilide. A variety of linkages X such as CH2O, CH2NH and CONH were examined, but the CONH moiety used in sematilide was found to be the most potent. This compound was found to prevent electrically stimulated arrhythmias in dogs (Class III activity) and to be effective against epinephrine-induced arrhythmias in halothane anaesthetised dogs (Class II activity). Risotilide (Figure 5.8), which is in preclinical studies, and is structurally similar to sematilide was derived from a series of benzene sulphonamides (Colatsky and Follmer, 1989). Electrophysiological studies on this compound indicate that it is a selective blocker of the delayed rectifier current without effect on IKI and IA, or on Na+ currents (Follmer et al., 1989). Sotalol (Figure 5.8) was originally developed as a β -blocking agent (Larsen and Uloth, 1969), for the treatment of hypertension, and in the early seventies it

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 143

was discovered to have Class III antiarrhythmic activity (Singh and Williams, 1970). Separation of the racemate into its enantiomers resulted in the discovery that the Class II effect resided in the l-isomer, whereas the Class III effect resided in the d-isomer (Hoffmeister et al., 1991). The d-enantiomer of sotalol was given FDA approval in the beginning of 1993. The amino function of sotalol can be constrained in a cyclic form such as the imidazole (6, Figure 5.8) analogue of the quaternary compound CK-1649 (Figure 5.7) (Lis et al., 1987a). This analogue has comparable activity to its quaternary analogue CK-1649 in vitro but was ineffective in vivo. Ibutilide (Figure 5.8) can be viewed as a modified sotalol analogue in which the secondary ethanolamine chain has been extended to a tertiary butanolamine and thereby removing the Class II activity (Hester et al., 1991). In addition, ibutilide has a novel mode of action compared with the other Class III antiarrhythmic agents. Besides its Class III activity, it activates a slow inward Na+ current at subnanomolar concentrations (Lee et al., 1990) and this dual action has the effect of prolonging the action potential duration at low concentrations and shortening it at high concentrations, so producing a bell-shaped dose response curve. All the analogues described so far, have a simple pharmacophore which comprises of a para substituted aromatic ring, normally substituted with a methanesulphonamide. This aromatic ring is linked to an secondary or tertiary amine by a three to four atom linked spacer. Second generation analogues A second generation of compounds has sought to extend the pharmacophore by the incorporation of other aryl and heterocyclic moieties, see Figures 5.9 and 5.10, but the majority of compounds still retain a sulphonamide group. In view of the Class III activity observed by including an ethanolamine β -blocking pharmacophore such as in sotalol (Figure 5.8), it is not surprising that the corresponding oxypropanolamine substituent also has activity. In a similar manner to the ethanolamines, the β -blocking effect can be removed by replacing the secondary amine with a tertiary amine. Such a modification has provided WAY-123,223 (Figure 5.9) (Butera et al., 1991) and removal of the alcohol to give the deshydroxy analogue also resulted in a very potent and selective Class III agent, WAY-125,971 (Figure 5.9). Another group deliberately set out to incorporate β 1-blocking activity with Class III activity, based on the hypothesis that combined modes of action would be potentially useful against re-entrant and catecholamine-dependent arrhythmias, at doses below those which cause β -blocker-mediated hypotension and cardiac depression. This has been achieved with both an aryl piperazine 5

1 This is not the usual pharmacophore associated with β -blocking activity, see Philips et al. (1992) for examples of β -blocking activity associated with this group.

144 K CHANNELS AND THEIR MODULATORS

(Figure 5.8) using a non-classical β -blocking pharmacophore and CK-3579 (Figure 5.9) using a ‘classical’ β -blocking pharmacophore. The latter retains a secondary amine and the S-enantiomer was found to be the eutomer (Lis et al., 1990; Morgan Jr. et al., 1990a; Connors et al., 1991). Replacement of the secondary amine to give cyclic amine derivatives, also results in selective molecules (7, Figure 5.9) (Connors et al., 1991). Another phenoxyalkylamine, dofetalide (Figure 5.9) (Cross et al., 1990) is in clinical trials. It prolongs cardiac action potential duration below 5 nM and is claimed to be one of the most potent Class III agents prepared to date (Gwilt et al., 1991). Modification of risotilide (Figure 5.8) by the further addition of a benzimidazolyl group (Figure 5.10) gave WAY-123,398 (Ellingboe et al., 1992). A number of heterocyclic groups such as quinolyl or pyrimidyl have also been examined, but the 2-aminobenzimidazole group was shown to be the most potent. WAY-123,398 was shown to have good oral bioavailability and to produce a 3-fold increase in ventricular fibrillation threshold in anaesthetised open-chest dogs. Two sulphonamide analogues (Figure 5.10) which incorporate cyclic amines are UK-66,914 and E-4301 (Oinuma et al., 1990). UK-66,914 has a pyridylpiperazino moiety in place of the isopropylamino found in sotalol, and this modification diminishes β -blocking activity. The second aromatic group probably locates an extra binding site and this makes it more potent than sotalol (Gwilt et al., 1988). UK-66,914 was withdrawn from clinical studies because of toxicity considerations. There are a number of other potential therapeutic agents, being evaluated in humans (Figure 5.10), which have different electron withdrawing groups in place of sulphonamide. MS-551 was originally derived from a β -blocking agent MS-3579 (Figure 5.10) (Katakami et al., 1992) but it is devoid of the Class II effects normally associated with the β -blocking pharmacophore. It has also been reported (Endoh et al., 1993) that MS-551 was able to rescue patients in cardiac arrest which had been induced by Class I antiarrhythmic agents. Almokalant (Figure 5.10) is a selective Class III agent, even though it contains a β -blocking pharmacophore. In rabbit ventricular cells, almokalant blocks the delayed rectifier K+ current in a time-and voltage-dependent manner and so lengthens the action potential duration (Carmeliet, 1993). Both MS-551 and almokalant have replaced the methyl sulphonamide with other electron withdrawing groups. It is still possible to find leads in this area, and the development of a novel series of spiro[benzopyran-2,4’-piperidines] led to L-691, 121 (Figure 5.11) which is essentially devoid of significant activity for the β 1 receptor, a problem found with some of the series (Elliot et al., 1992). It is a selective blocker for the rapidly activating component (IKr) of the delayed rectifier current in isolated guinea pig ventricular myocytes, with no effect on the slowly activating component (IKs) current, the inward rectifier (IK1) current and the L-type Ca2+ current (Claremon

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 145

Figure 5.11 Development of spiro[benzopyran-2,4’-piperidines]

et al., 1993). The series was derived from a 5-HT3 antagonist GR-38032, which prolongs the effective refractory period with a potency similar to sotalol. A variety of tricyclic ring systems were evaluated starting with the thienothiopyran (8) which led to thienothiopyranone spiropiperidine derivative (9), and eventually benzopyran spiropiperidine (10). Subsequent introduction of a methylsulphonamide produced L-691,121. A major metabolite of this compound is the N-dealkylated product and, in order to overcome this problem, a series of conformationally restrained derivatives were examined leading to L-702,958. Reduction of the ketone to the alcohol occurs metabolically and both enantiomers were found to be equally active in vitro, but the S-enantiomer L-706,000, is six times more potent in vivo. The two second generation compounds L-706,000 and L-702,958 have good oral bio-availablity and longer duration of action in animals, than the original compound L-691,121.

146 K CHANNELS AND THEIR MODULATORS

5.3.3 Combined Class III Pharmacophore Most of the Class III antiarrhythmic agents can be accommodated by one pharmacophore outlined in Figure 5.12. An aromatic ring is substituted with an electron withdrawing group, preferably methylsulphonamide in the para position, and this helps reduce liability to Class I effects (e.g. conversion of procainamide into sematilide). Further substitution of the nitrogen NH of the sulphonamide or extending the S-methyl group into the ethylsulphonamide leads to diminished activity (Lumma Jr. et al., 1987). The quaternary compounds outlined in Figure 5.7 also fit this pharmacophore. Apart from sulphonamide the substituent can be other electron withdrawing groups, such as halogen (clofilium, Figure 5.7), nitro (MS-551, Figure 5.10), nitrile (almokalant, Figure 5.10), acetamide (NAPA, Figure 5.8) and imidazol-1-yl (3, Figure 5.8). The aromatic ring is connected to a nitrogen atom, which is presumably charged at the receptor, by a 3 to 4 atom spacer. This spacer can be a (CH2)4 moiety in which one or two of the methylenes can be replaced with O, SO2N, NH or CO or substituted with an alcohol. It has also been possible to incorporate fused rings at this

Figure 5.12 Pharmacophore Model for Class III Binding Site

position (Figure 5.11). Substitution with an alcohol in combination with a secondary amine, can lead to a molecule with β -blocking activity (Class II), as found in sotalol (Figure 5.8), but converting the secondary amine into a tertiary amine tends to abolish this activity. The amine functionality can be a quaternary group, or a secondary or tertiary amine, and this can be acyclic, cyclic or heterocyclic. Second generation compounds are distinguished by the addition of another group to the amine, normally aromatic or heterocyclic, and this is separated by a one to three atom spacer. This has the effect of increasing potency, and this enhanced activity

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 147

suggests that an additional binding site has been located in this region. Owing to the lack of information it has not been possible to correlate K blocking activity with structure. 5.3.4 Miscellaneous Class III Agents Some compounds do not fit the pharmacophore just described in all respects, and this is reflected in their K channel blocking properties. Tedisamil (Figure 5.13) has a totally different structure to all the other class III antiarrhythmic agents and this difference in structure seems to be manifested in a different profile as a KCB. Not only is it a blocker of both the fast and slow components of the delayed rectifier current, but it also blocks the transient outward current in mammalian cells and at higher concentrations inhibits Na+ currents (Dukes and Morad, 1989; Dukes et al., 1990). Ambasilide, an analogue

Figure 5.13 Miscellaneous Class III antiarrhythmic agents

of tedisamil (a symmetrical dibasic molecule), is unsymmetrical with only one basic centre and it has been shown to block the delayed rectifier current in guinea

148 K CHANNELS AND THEIR MODULATORS

pig myocytes (Zhang et al., 1992) in a dose dependent manner. A blocking effect of Na + currents in canine Purkinje fibres and ventricular muscle was observed, which indicates that ambasilide also has some Class I activity (Takanaka et al., 1992). (-)-S-Terikalant (RP-62719) is the active enantiomer of RP-58866 and is 150 times more potent than the R-enantiomer (Escande et al., 1992) in blocking the inward rectifier K+ current. This is unusual because the usual Class III agents selectively block one or both components of the delayed rectifier current. Another unusual structure which displays selective Class III activity is NE-10064 (Figure 5.13). Information on the compound is scarce; it was reported that it blocked the slowly activating component of the delayed K+ rectifier current. Surprisingly the compound also blocked the mini K channel when expressed in Xenopus oocytes (Busch et al., 1993). The piperazinoguanadine RS-87337 (Figure 5.13) is a combined Class III/IA antiarrhythmic agent (Dumez et al., 1989). Another novel structure with combined Class I and III activity is a benzodiazepine (11, Figure 5.13) and the Renantiomer which is slightly more active than the S-enantiomer was selected for further biological and toxicological evaluation (Johnson et al., 1993). 5.4 Aminopyridines Prior to the discovery of the very potent scorpion and snake toxins the aminopyridines were a useful tool in helping dissect out various K+ currents. 4Aminopyridine (4-AP) is selective for the delayed rectifier type K+ current and is particularly active against the slowly inactivating outward current (IKS). As there are already some excellent reviews (Marshall, 1982; Glover, 1982; Cook and Quast, 1990) on the effects of aminopyridines on K channel types and sub-types it is not intended to cover this aspect here. 4-AP and its derivatives have been known to block K+ currents since the early seventies (Pelhate and Pichon, 1974; Gillespie and Hutter, 1975; Yeh et al., 1976; Meves and Pichon, 1977) and some years later there was a thorough review of the pharmacological properties and an attempt at studying the SARs of the aminopyridines was reported (Glover, 1982). There have also been more systematic studies on the SARs of aminopyridines and derivatives on axonal K+ conductance (Pelhate and Malecot, 1989; Marshall, 1982). The structure of 4-AP is an extremely simple one (Figure 5.14). Nevertheless, some manipulation of this structure has been attempted (Marshall, 1982). The amino position has been switched from position 4-to 2-(2-AP) and 3-(3-AP), but these are generally found to be less active. Diamino derivatives are also active, the most potent of which is 3,4-diaminopyridine (3,4-DAP). The order of potency for the various amine analogues is 3,4-DAP>4-AP>2,3-DAP>3-AP>2AP>2-amino-3-hydroxypyridine. Replacement of amino with hydroxy leads to less active compounds as does further substitution of amino compounds with

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 149

chloro, bromo, methyl, or ethoxy substituents (Pelhate and Malecot, 1989). Replacement of pyridine with other heterocyclic groups such as piperidine, pyrazine, pyrimidine, triazine and morpholine leads to totally inactive molecules (Pelhate and Malecot, 1989). Tacrine which has been used for the treatment of Alzheimer’s disease, by nature of its anticholinesterase activity, also has K+ blocking activity (Osterrieder, 1987;

Figure 5.14 4-Aminopyridine derivatives

Schauf and Sattin, 1987; Freeman et al., 1988) which is not too surprising, owing to its strong relationship to 4-AP. Attempts have been made to improve the K+ channel blocking abilities of aminopyridines and a series of 3-substituted-4aminopyridines were examined in the hippocampal slice (Waser et al., 1988), the most interesting of these compounds was 3-methoxy-4-aminopyridine (Figure 5.14) and this was evaluated in various preparations, such as guinea pig ileum, and for effects on botulism toxin paralysis (Berger et al., 1989a). The 14C labelled form was used to examine its distribution in mice and high levels were found in cholinergically innervated secretory organs, also in the adrenal medulla, the hippocampus, the thalmic nuclei and the cortex (Berger et al., 1989b). Another aminopyridine examined for its ability to release neurotransmitters such as noradrenaline (Foldes et al., 1988) and acetylcholine, was LF-14 (Potter et al., 1989). A number of related ureas were also examined (Ohta et al., 1982), but were found to be less active. LF-14 was evaluated in a variety of other preparations and found to be a peripherally acting compound, with less central action than 4-AP (Biessels et al., 1985). The aminopyridines block K channels in both the periphery and in the CNS. The central actions are potentially interesting because they cause the release of neurotransmitters in the brain and so could be useful in improving neurotransmission. They are also convulsant with a small therapeutic ratio which means that the aminopyridines are not suitable for use in the treatment of Alzheimer patients. They have however, been used in the treatment of botulism

150 K CHANNELS AND THEIR MODULATORS

in humans, particularly 3,4-DAP, which is less able to penetrate the blood-brain barrier, and therefore has less propensity to cause convulsions. 3,4-DAP has also been shown to be effective in multiple sclerosis (Bever et al., 1990) but two of ten patients did suffer seizures. It appears that the clinical potential of the aminopyridines is limited by their toxicity and lack of potency. 5.5 Miscellaneous Blocker 5.5.1 1-(4-methoxyphenyl)indole Neurosearch has filed a patent on this indole (Figure 5.15) for use as an antidepressant or for use in Alzheimer’s disease (Olsen et al., 1992). Antidepressant

Figure 5.15 1-(4-Methoxyphenyl)indole

activity was detected in a tail suspension assay at 10 to 100 mg/kg i.p. The compound which is claimed to block the BKCa in bovine aortic smooth muscle cells as well as in pancreatic β -cells has alsox been shown to have memory enhancing effects in a social recognition model in rats. 5.6 Conclusions The examples in this chapter serve to illustrate several points. First and foremost that KCBs are useful drugs which possess the same subtleties and range of interactions of any other class of drugs. This serves to dispel their sometimes public image of being mere bungs which prevent the passage of ions through pores in membranes. The fact that the existing channel blockers were developed from the serendipitous exploitation of their eventual functional effects and not by an attack on the molecular target itself opens up two futures. First, that other existing therapeutic agents will have their mode of action shown to be K channel blockade is a probability, and second, that blockers of so far untapped channels will become important drugs is a certainty. In fact with the wide availability of different K channel types, the emphasis will switch to identifying selective blockers (and enhancers) of these individual clones. Accordingly, the eventual

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successes in this area will go to those with the greatest understanding of the subtleties of channel blockade and the ability to identify the clinical significance of the K channel subtype they are targeting. References AGUILAR-BRYAN, L., NELSON, D.A., VU, Q.A., HUMPHREY, M.B. & BOYD III, A.E. (1990) J. Biol. Chem., 265, 8218–8224. AMOROSO, S., SCHMID-ANTOMARCHI, H., FOSSET, M. & LAZDUNSKI, M. (1990) Science, 247, 852–854. ARCH, J.R.S., BUCKLE, D.R., CAREY, C., PARR-DOBRAZANSKI, H., FALLER, A., FOSTER, K.A., HOUGE-FRYDYCH, C.S.V., PINTO, I.L., DAVID, G.S. & TAYLOR, S.G. (1991) J. Med. Chem., 34, 2588–2594. ARROWSMITH, J.E. & CROSS, P.E. (1990). Ann. Rep. Med. Chem., 25, 79–88. ASHCROFT, S.J.H. & ASHCROFT, F.M. (1990) Cellular Signalling, 2, 197–214. ASHFORD, M.L.J. (1990) Potassium channels and modulation of insulin secretion. In: Potassium Channels, Cook, N.S. (ed.). Ellis Horwood Ltd., Chichester. pp. 300–325. ASMAL, A.C. & MARBLE, A. (1984) Drugs, 28, 62–78. ATWAL, K.S. (1992) Med. Res. Rev., 12, 569–591. BERGER, S.G., WASER, P.G. & HOFMAN, A. (1989a) Arzneim. Forsch., 39, 762–765. BERGER, S.G., WASER, P.G. & SIN-REN, A.C. (1989b) Neuropharmacology, 28, 191–194. BERNARDI, H., FOSSET, M. & LAZDUNSKI, M. (1988). Proc. Natl. Acad. Sci. USA., 85, 9816–9820. BERNARDI, H., BIDARD, J.N., FOSSET, M., HUGUES, M., MOURRE, C., REHM, H., ROMEY, G., SCHMIDT-ANTOMARCHI, H., SCHWEITZ, H., WEILLE, J.R.D. & LAZDUNSKI, M. (1989) Arzneim. Forsch., 39, 159–163. BEVER, C.T., LESLIE, J., CAMENGA, D.L., PANITCH, H.S. & JOHNSON, K.P. (1990) Ann. Neurol., 27, 421–427. BIERE, H., RUFER, C., LOGE, O. & SCHRODER, E. (1974) J. Med. Chem, 17, 716–721. BIESSELS, P.T.M., AGOSOTON, S. & HORN, A.S. (1985) Eur. J. Pharmacol., 106, 319–325. BUSCH, A.E., MALLOY, K.J., VARNUM, M.D., ADELMAN, J.P., NORTH, R.A, & MAYLIE, J. (1993) Circulation, 88, I–231. BUTERA, J.A., SPINELLI, W., ANANTHARAMAN, V., MARCOPULOS, N., PARSONS, R.W., MOUBARAK, I.F., CULLINAN, C. & BAGLI, J.F. (1991) J. Med. Chem., 34, 3212–3228. CARMELIET, E. (1993) Circ. Res., 73, 857–868. CIMINI, M.G. & GIBSON, J.K. (1992) Ann. Rep. Med. Chem., 27, 89–98. CLAREMON, D.A., BALDWIN, J.J., ELLIOTT, J.M., REMY, D.C., PONTICELLO, G.S., SELNICK, H.G., LYNCH, J.J.J. & SANGUINETTI, M.C. (1993) Perspect. Med. Chem. 389–404. COLATSKY, T.J. (1991) K Blockers: Synthetic Agents and their Antiarrhythmic Potential. Potassium Channel Modulators: Pharmacological, Molecular and Clinical Aspects. Blackwell Scientific Publications, Oxford, pp. 304–340. COLATSKY, T.J. & FOLLMER, C.H. (1989) Cardiovasc. Drug. Rev-, 7,199–209.

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6 Potassium Channels: Diversity, Assembly and Differential Expression R. LATORRE & P. LABARCA Centro de Estudios Cientfficos de Santiago y Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 16443, Santiago, Chile. 6.1 Introduction The wealth of evidence on K channel structure, function, diversity and differential distribution in animal tissues obtained in the past few years presents us with an amazing panorama that is only now beginning to be revealed. Yet, in spite of the formidable progress that has followed the cloning of a K+-selective channel (Baumann et al., 1987; Kamb et al., 1987; Tempel et al., 1987) our knowledge of the basic principles that govern K channel structure and function is still in its infancy. Such principles concern the most basic phenomena associated with K channel function: their ability to catalyze with high efficiency the passive flow of K+ across the cell membrane, their various selectivity and permeation profiles, and the mechanisms of interaction with the membrane electric field (reviewed in Jan and Jan, 1992; Pongs, 1992; Caterall, 1993; Hoshi and Zagotta, 1993; Bezanilla and Stefani, 1994). As a minimum, a basic understanding of K channel structure-function implies the development of a common set of rules that are sufficient to account for the occurrence of K channels exhibiting variable open conductances, different selectivity sequences and permeation mechanisms. Likewise, the development of the basic rules that give rise to K channel voltage dependence should suffice to account for the various gating properties of K channels. K channel diversity seems to have accompanied animal cells throughout evolution and some K channels are among the most conserved proteins in eukaryotes (Jan and Jan, 1990b, Rudy et al., 1991a; Salkoff et al., 1992). K channels became fundamental to animal cell physiology very early in evolution and became part of ancient transduction mechanisms which enabled eukaryotes, through K channel-mediated control of membrane potential, to couple the cell inner dynamics to the outer environment. A differential expression of K channel mRNAs is found to accompany the events of animal development and this also occurs in adult animal tissues. This differential expression of K channels is particularly noteworthy in the brain where it has been documented from

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invertebrates to mammals. It is not too risky to predict that differential expression of K channels in the nervous system will be demonstrated to be relevant to and accompany neuronal differentiation, and be associated to plastic phenomena as well as neurological deficiencies. Although for the sake of clarity, we have divided this chapter into several sections, it contains two main topics. First, we describe the varieties of the K channels that have been cloned until now and their possible topologies in the lipid bilayer membrane. Second, since we posses a wealth of information regarding the S4-related potassium channel family (Jan and Jan, 1990a), we reviewed in some detail its diversity, subunit assembly, and differential expression. 6.2 Varieties of K Channels Before the application of molecular biology into the field of electrophysiology, K channels were placed arbitrarily in five different groups (e.g. Rudy, 1988): (1) Voltage-dependent K channels which in turn can be subdivided into channels showing fast inactivation (A-type channels) and delayed rectifiers of the Hodgkin and Huxley type (Hille, 1992); (2) Ca-activated K channels that are usually classified into two different classes of high (maxi, BKCa) and small (SKCa) conductance, although they can also be dissected pharmacologically according to their sensitivity to scorpion and bee toxins (Blatz and Magleby, 1987; Latorre et al., 1989; McManus, 1991; Latorre, 1994); (3) inward rectifiers that give rise to a current that activates with hyperpolarizing voltages in contrast to A-type and delayed rectifiers channels that activate with depolarizing voltages (e.g Rudy; 1988); (4) adenosine triphosphate-dependent channels (KATP; Noma, 1983); and (5) K channels coupled to receptors, as is the case of those coupled to muscarinic receptors in the heart muscle. With the exception of the apamin-sensitive SKCa, channels of all the classes described above have been cloned and expressed in oocytes or in cell lines in culture. Structurally, channel taxonomy has become simpler because the classes described above can be grouped into the following three large superfamilies. 6.2.1 K Channels Belonging to the S4 Channel Superfamily (Jan and Jan, 1990a) As determined from hydrophatic plots, these channels have six transmembrane segments (S1–S6; Figure 6.1). The fourth (S4) segment is characterized by having several, up to eight, positively charged amino acid residues (lysine or arginine) at every third position, with non-polar or hydrophobic residues in between. K channels in this family share the S4 segment with voltage-dependent Na and Ca channels (Figure 6.1B). For example, the S4 segment of the Shaker Atype K channel has about a 50% identity with the corresponding segment in

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domain IV of the Na channel and more important these two domains contain a similar number of positive charges (7 in Shaker and 8 in domain IV of rat brain II Na channels). Because of its peculiar structure, S4 is postulated to be part of the voltage sensor (for reviews see Catterall, 1993; Bezanilla and Stefani, 1994). In the case of Na and Ca channels, Figure 6.1 A shows that the channel-forming protein is a polypeptide consisting of four domains or pseudosubunits (I–IV) each of which contains the S1 to S6 transmembrane segments. K channels belonging to this superfamily have a molecular weight that is about a quarter of that of Na or Ca channels. This suggests that K channels are tetramers.

Figure 6.1 Transmembrane folding models for voltage dependent Na and Ca channels (A), voltage-gated and calcium-activated K channels (B), inward rectifier channels (C) and min K channels (D). In some voltage-dependent K channels the N-terminal is an integral part of the inactivation gate and provides a recognition site for heteromultimeric channel assembly and prevents coassembly between subfamilies. The S1 segment is important in the coassembly of homo- and heteromultimeric channels. The S4 is supposed to be the channel voltage sensor and the S5–S6 linker (region P) forms part of the pore walls. (For detail and references see text)

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In voltage-dependent and Ca-activated K channels (Atkinson et al., 1991; Adelman et al., 1992) an hydrophobia polypeptide segment with the shape of a hairpin located between transmembrane segments S5 and S6 forms the channel pore (H5 or P region). This region is extremely well conserved in all K channels belonging to this superfamily. The isolation of a cDNA clone from ether à go-go (eag) locus of Drosophila (Warmke et al., 1991) showed that it also encodes a polypeptide that shares a S4 transmembrane domain and its S5–S6 linker has a high degree of identity with voltage-gated K channels (Figure 6.1B). The sequence also contains a putative cyclic nucleotide binding site in the carboxy terminal region, making this channel a relative to both, voltage-dependent channels and cyclic nucleotide-activated channels (Guy et al., 1991). Expression of eag cRNA in oocytes induces a voltage–and cAMP-dependent K channel (Brüggemann et al., 1993). Although eag has been identified in mouse and human sharing 71 and 48% identity, respectively, with the Drosophila protein (Warmke and Ganetzki, 1993, 1994), the physiological importance of this channel in mammals is unclear at present. A K inward rectifier channel from the plant Arabidopsis thaliana also belongs to the S4 superfamily (Schachtman et al., 1992). Apparently, this channel has a different mechanism of activation than ‘classical’ inward rectifier channels since it activates at large hyperpolarizing voltages regardless of the value of the K+ equilibrium potential. The most salient feature of ‘classical’ inward rectifier channels is that they allow current to pass only below the K+ equilibrium potential, i.e. the position in the voltage axis of their voltage activation curves varies with external K+ concentration (Hagiwara and Takahashi, 1974). 6.2.2 Channels with Monomers Containing Two Membrane Spanning Regions Cloning of three different inward rectifier channels (Kubo et al., 1993a, 1993b; Ho et al., 1993; Dascal et al., 1993) has revealed a new superfamily of K channels. In this case the proteins only feature two putative membrane-spanning segments (M1 and M2, Figure 6.1C). However, the potential P region of these channels (with one notable exception; see Suzuki et al., 1994) has a high degree of similarity with the P region of the channels belonging to the S4 superfamily. The K channel coupled to muscarinic receptors in the heart [(5) above] belongs to this superfamily (Kubo et al., 1993b; Dascal et al., 1993). The number of inwardly rectifying channels is expanding very fast. So far five different channel genes have been identified (Kir1.0–Ki5.0), and the mRNAs of the subfamilies Kir1.0, Kir2.0 and Kir3.0 undergo alternative splicing (Doupnik et al., 1995). Inward rectification arises as a consequence of a Mg2+ and/or as a consequence of a polyamine (e.g., spermine, spermidine, putrescine) voltage-dependent block. However, intrinsic channel voltage dependence can also contribute to the inward rectification in these channels (reviewed in Doupnik et al., 1995). The ATP-

160 K CHANNELS AND THEIR MODULATORS

sensitive (inhibited) type of K channels from rat heart have been cloned, sequenced and expressed in Xenopus oocytes (Ashford et al., 1994). The cloned rcKATP is inhibited by ATP and activated by intracellular nucleotide diphosphates. The amino acid sequence revealed two putative transmembrane segments which flank a pore region with a high degree of identity with the Pregion of other K channels. Thus, the rcKATP channel belongs to the inward rectifier K channel family. 6.2.3 Channels Formed by Monomers Containing a Single Membrane Spanning Region A third family is represented by a gene putatively coding for a voltagedependent K channel with a hydropathy profile showing a single membrane spanning β -helix (Takumi et al., 1988; Figure 6.1D). The protein is a relatively small polypeptide of only 130 amino acids; the small size of this channelforming protein has prompted workers in the field to name this channel ‘minK’ (minimal K; Hausdorff et al., 1991). In oocytes the minK protein induces a slowly activating voltage-dependent K+ current that is inwardly rectifying (Blumenthal and Kaczmarek, 1993). The gene has been cloned from kidney (Takumi et al., 1988), heart (Folander et al., 1990), uterus (Murai et al., 1989), and T-lymphocytes (Attali et al., 1992), but its physiological role is still unclear. In the heart, Folander et al. (1990) have suggested that minK induces the slow component of the delayed rectifier currents. 6.3 Origin of Diversity in K Channels Belonging to the S4 Superfamily K channels are ubiquitously distributed in different cells and tissues and, therefore, K channel diversity is of great importance in determining the variety of electrical responses of cells when subjected to stimuli (e.g. Hille, 1992). Below we discuss three possible mechanisms able to originate K channel diversity: 1) alternative splicing; 2) multiple genes; and 3) formation of heteromultimeric channels (for reviews see Jan and Jan, 1990b; Rudy et al., 1991a; Pongs, 1992; Salkoff et al., 1992).

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 161

6.3.1 Shaker and Mammal Genes Encode More Than One K Channel Protein by Alternative Splicing Drosophila The molecular biology era of K channels started thanks to the existence of a Drosophila mutant named Shaker, characterized by its leg-shaking phenotype under ether anesthesia. Early studies, using intracellular recording, indicated that the neuromuscular transmission in two Shaker mutants was abnormal. The postsynaptic potentials in the mutant are abnormally large and prolonged and they are a consequence of a sustained Ca2+ current at the nerve terminals. It was later demonstrated that Shaker mutants showed prolonged action potential duration and also altered A-type K+ currents (Tanouye et al, 1981; Salkoff and Wyman, 1981). Several cDNA clones were isolated from the Drosophila Shaker locus. The sequence showed the characteristic primary structure of voltage-dependent channels: S1–S6 transmembrane segments with a positively charged S4 segment (Tempel et al., 1987; Papazian et al., 1987; Iverson et al., 1988; Baumann et al., 1987, 1988; Kamb et al., 1987, 1988). Subsequently, Shaker proved to be a family of different but closely related proteins encoded by alternatively spliced transcripts and this mechanism was proposed to be a convenient one to generate K channel diversity (Schwarz et al., 1988). It was found that all the spliced variants share the central region of the channel (i.e. S1–S6; Figure 6.1) and that they differ at the amino- and carboxy-terminal (two variants). There are five alternatively spliced amino–terminal variants of Shaker (Schwarz et al., 1988; Pongs et al., 1988; Kamb et al., 1988) and two carboxyl termini (Iverson et al., 1988; Kamb et al., 1988; Schwarz et al., 1988; Timpe et al., 1988b). Moreover, three variants (ShB, ShC, and ShD; Table 6.1) inactivate rapidly (Table 6.1; time constants (β ) of tens of milliseconds or less) whereas the other two (ShD2 and ShH37) inactivate with a β in the hundreds of milliseconds (Timpe et al., 1988a, 1988b; Zagotta et al., 1989; Aldrich et al., 1990; Iverson and Rudy, 1990). ShA and ShB (also called ShH4) have the same amino-terminus but they differ in their carboxyl terminal sequence. Kinetically, these two Shaker channels differ mainly Table 6.1 Nomenclature and kinetic properties of S4 superfamily-related K channels in invertebrates and mammals1 Subfamily of K Channels Shaker subfamily

Type of Current

Species

References

ShA2

Fast Inactivation

Drosophila

ShB

Fast Inactivation

Drosophila

Tempel et al. (1987) Pongs et al. (1988) Schwarz et al. (1988) Kamb et al. (1988)

162 K CHANNELS AND THEIR MODULATORS

Subfamily of K Channels Shaker subfamily

Type of Current

Species

ShC ShD

Fast Inactivation Fast Inactivation

Drosophila Drosophila

References

Schwarz et al. (1988) Schwarz et al. (1988) Pongs et al. (1988) 3 ShD 2 Drosophila Stocker et al. (1990) ShH37 Slow Inactivation Drosophila Iverson et al. (1988) AKv1.1a/AK01a Fast Inactivation Aplysia Pfaffinger et al (1991) rKvl. 1/RCK1/BK1/RBK1 Sustained Rat Baumann et al. (1988) McKinnon (1989) Christie et al. (1989) mKv1.1/MBK1 Mouse Tempel et al. (1988) rKv1.2/RCK5/BK2/ Sustained Rat Stühmer et al. (1989) NGK1/RH1/RK2 McKinnon (1989) Yokoyama et al. (1989) Ishii et al. (1992) Po et al. (1992) mKv1.2/MK2 Mouse Chandy et al. (1990) XKv1.2 Xenopus Ribera (1990) rKv1.3/RCK3/RGK5/KV3 Slow Inactivation Rat Stühmer et al. (1989) Douglass et al. (1990) Swanson et al. (1990) mKv1.3/MK3 Sustained Mouse Grissner et al. (1990) Chandy et al (1990) hKv1.3/HPCN/HLK3/ Slow Inactivation Human Philipson et al. (1991) HGK5 Attali et al. (1992) Yun–Cai et al. (1992) rKv1.4/RCK4/RK3/ Fast Inactivation Rat Stühmer et al. (1989) RHK1/RH10 Roberds and Tamkun (1991) Tseng–Crank et al. (1990) Meyerhoff et al. (1992) Okada et al. (1992) hKv1.4/HK1/hPCN2 Fast Inactivation Human Tamkun et al. (1991) Philipson et al. (1991) rKv1.5/RCK7/KV1/RK4 Betsholtz et al. (1990) Swanson et al. (1990) Roberds and Tamkun (1991) mKv1.5 Slow Inactivation Mouse Attali et al. (1993) mKv1.5 5’ Mouse Attali et al. (1993) hKv1.5/HK2/hPCN1 Slow Inactivation Human Tamkun et al. (1991) Philipson et al. (1991)

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 163

rKv1.6/RCK2/KV2

Sustained

Rat

hKv1.6/HBK2 mKv1.7/MK6/MK4 Shab subfamily AKv2.1/AShab rKv2.1/DRK1/cDRK1

Sustained

Human Mouse

mKv2.1 Kv2.1 Shaw subfamily Kv3.1a/NGK2/KShIIIB

Sustained Sustained

Mouse Human

Sustained

Rat, Mouse Yokoyama et al. (1989) Weiser et al. (1994)

Kv3.1b/Kv4/Raw2

Sustained

Rat, Mouse

rKv3.2a/RKShIIIA Raw 1 rKv3.2b/KShIIIA3

Sustained Sustained

Rat Rat Rat

Slow Inactivation Aplysia Sustained Rat

rKv3.2c/KShIIIA.2

Rat

rKv3.3a/KShIIID.1

Slow Inactivation Rat

rKv3.3b/KShIIID.2

Slow Inactivation Rat

rKv3.4a/Raw3 hKv3.4b/KShIIIC rKv3.4c

Fast Inactivation Fast Inactivation

Shal subfamily mKv4.1/mShal rKv4.1/RShal Kv4.2/RK5

Rat Human Rat

Sustained Mouse Slow Inactivation Rat Rat

Grupe et al. (1990) Swanson et al. (1990) Grupe et al. (1990) Grissmer et al. (1990) Quattrocki et al. (1994) Frech et al. (1989) Hwang et al. (1992) Pak et al. (1991a) Albrecht et al. (1993)

Luneau et al. (1991a) Grissmer et al. (1992) Rettig et al. (1992) McComack et al. (1990b) Rettig et al. (1992) Luneau et al. (1991b) Rudy et al. (1992) Luneau et al. (1991b) Rudy et al. (1992) Vega–Saenz de Miera et al. (1992) Vega–Saenz de Miera et al. (1992) Schröter et al. (1991) Rudy et al. (1991b) Vega–Saenz de Miera et al. (1994) Pak et al. (1991b) Baldwin et al. (1991) Roberds and Tamkun (1991)

in their recovery from inactivation. For ShA, recovery from inactivation has a β of 400 ms whereas the same process in ShB has a β of 20 ms (Wittka et al., 1991).

164 K CHANNELS AND THEIR MODULATORS

Thus, the Shaker subfamily consists of several K channels with different degrees of inactivation. A digression on inactivating mechanisms The fact that Shaker K channels with different amino-terminal domains have different inactivation rates strongly suggested that this region of the protein molecule was (or was part of) the inactivation gate. Hoshi et al. (1990) studied the effect of mutations of this domain in ShB. Deletion mutations allowed the identification of the first 20 amino acids of the amino–terminal as the inactivation gate. For example, a 41 amino acid deletion (6–46) produced a channel that, once expressed in oocytes, does not inactivate with a rapid time course. Restoration of inactivation in the Sh 6–46 mutant can be obtained if a peptide consisting of the first 20 amino acids of the ShB channels is added to the cytoplasmic side of the channel (Zagotta et al., 1990). These results indicate that fast inactivation in Atype channels is the result of the occlusion of the pore by the protein N-terminal domain. Structurally, the N-terminal is hydrophobic and is followed by a stretch of positively charged amino acids. Both hydrophobicity and the net charge of the inactivating peptide are crucial in determining the degree and the kinetics of the inactivation process (Murrel-Lagnado et al., 1993; Toro et al., 1994). This type of inactivation is called N-type inactivation as opposed to the C-type of inactivation that involves amino acid residues in the S5, P and S6 segments (Hoshi et al., 1991). In contrast to N-type, C-type of inactivation is modulated by external cations and most of the mutations that affect C-type inactivation also modified ion conduction through the pore (LopezBarneo et al., 1993). Increasing external K+ slows down C-type inactivation. Mammals In mammals, K channel diversity arises, at least in part, from the expression of alternatively spliced RNA (Luneau et al., 1991a, 1991b; McCormack et al.,

1

In the case mammalian K channels, in the first column the first denomination for a given channel corresponds to the nomenclature of Chandy (1991) and is followed by the other names given to the same channel in the literature. The second column refers to the channel kinetics. ‘Fast Inactivation’ means that the channel inactivates in the order of tens of milliseconds or less. ‘Slow inactivation’ refers to channels that show inactivation processes with time contents in the 100–1000 ms range. Potassium channels with time constants larger than 1 s are referred to as ‘Sustained’. 2 For other names of the Drosophila Shaker subfamily, the interested reader should consult, for example, Iverson and Rudy (1990). In particular, ShB is also called ShH4. This alternatively spliced variant has been used in numerous biophysical studies. 3 In the case of ShD2 there is a small component of fast current decay, but at all voltages the steady state component is large.

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 165

1990b; Rettig et al., 1992; Rudy et al., 1992; Vega-Saenz de Miera et al., 1992; Attali et al., 1993; Weiser et al., 1994). In rat it has been shown that a K channel (Kv4; see Table 6.1) structurally related to the Drosophila Shaw protein (see below), is originated by alternative splicing. In rat, NGK2, the first cloned K channel of this family (Yokoyama et al., 1989), arises from alternative exon usage at a locus that also encodes Kv4. NGK2 and Kv4 have quite different carboxyl-terminal domains. On the other hand, Schröter et al. (1991) and Rettig et al. (1992) have described a family, also related to the Shaw gene, composed of three alternatively spliced variants and denominated Raw1, Raw2, and Raw3 in the rat nervous system. In the Raw subfamily alternative splicing produces K channel proteins with different Ctermini. However, the alternative C-termini appear not to play an important role in ion channel characteristics, leaving open the question about the reason and the importance for the existence of Raw K channel variants. When expressed in oocytes Raw1 and Raw2 cRNAs induce non-inactivating currents; Raw3 cRNA, on the other hand, expresses A-type inactivating currents (Ruppersberg et al., 1991a; see below). Raw3 possesses an N-terminus which is 28 amino acids longer than the N-terminus of Raw1 and Raw2. Deletion of these extra amino acids completely eliminates fast inactivation in Raw3 (Rettig et al., 1992). Raw3 also shows a marked inward rectification that is a consequence of a voltage-dependent internal Mg2+ blockade (Rettig et al., 1992). Table 6.1 shows that the Kv3 subfamily is very rich in splice variants and Vega-Saenz de Miera et al. (1992) described in detail two alternatively spliced transcripts: KSHIIID.1 (Kv3.3a) and KSHDIIID.2 (Kv3. 3b). Kv3.3a has slow activation kinetics compared with Shaker A-type K channels, but like other A-type channels the current inactivates with a time constant of about 100 ms at 50 mV. The N-terminal of the Kv3.3a channel shares a stretch of amino acids with the Raw3 channel (ser-ser-val-cys-val). The cysteine in this sequence plays an important role in the modulation of inactivation by reducing agents such as glutathione. Oxidation of the cysteine residue inhibits N-type of inactivation (ball and chain mechanism) of Raw3 channels (Ruppersberg et al., 1991b). Table 6.1 shows that several mammalian Shaker K channels have been cloned and most of them appeared to be encoded by separate intronless genes (Stühmer et al., 1989; Beck and Pongs, 1990; Chandy et al, 1990). However, Attali et al. (1993) reported the existence of at least three mRNA isoforms able to encode the mouse cardiac delayed rectifier K channel (Kv1.5). Two alternatively-spliced variants encoded for a long (602 amino acid) protein and a short form (Kv1.5 ― 5’) in which the first 200 amino acids from the N-terminus have been deleted. A third clone codes for a non-functional protein with a truncated C-terminus (Kv1. 5 ― 3’). Kv1.5 ― 3’ mRNA co-injection with mRNA coding for the long form inhibited expression of the Kv1.5 channel.

166 K CHANNELS AND THEIR MODULATORS

6.3.2 Multiple Genes Inside a subfamily like Shaker different A-type channels can originate from alternative splicing of the Shaker gene. However, it is clear that this type of mechanism cannot account for the K channel diversity found in excitable tissues of Drosophila. It is important to remember here that the Shaker mutant still has K + currents. This fact strongly suggests the presence of a family of genes coding Shaker-related K channels allowing for the variety of K channels with different voltage dependencies, Ca2+ sensitivities, and pharmacological properties. Using a Shaker complementary DNA probe and low-stringency hybridization, Butler et al. (1989) were able to clone three new genes which they dubbed Shab, Shaw, and Shal each of which coded for proteins with a considerable degree of identity (― 38%) to the Shaker protein. The identity between members of this K channel family is considerable when the transmembrane segments are considered. For example, the identity between the S6 transmembrane segment of Shaker and Shaw is 86%. When expressed in Xenopus oocytes this family of voltagedependent K channels was shown to have different rates of activation and inactivation (Wei et al., 1990). Thus, whereas Shaker and Shal K channels displayed fast inactivation processes, Shab and Shaw are of the delayed rectifier type (Figure 6.2). Soon it became clear that a member of a subfamily in mammals was more similar to one of the four members of the Drosophila family than to a mammalian member of a different subfamily. For example, the Shaker protein shares about 70% identity with mammalian homologs (e.g. RCK1 (76%); Baumann et al., 1988), but has only

Figure 6.2 Ionic currents induced by expression in Xenopus oocytes of ShakerH37, Shal2, Shab11, and Shaw2 potassium channels. Depolarizing pulses were applied from a holding voltage of –90 mV in 10 mV steps in the range of –80 to 20 mV. Data are adapted from Wei et al. (1990).

38% identity with the Shab subfamily.1 Table 6.1 shows that Shaker genes are homologs of the Kv1 subfamily of mammalian genes; Shab are homologs of the Kv2 subfamily; Shaw are Kv3 homologs; and Shal are Kv4 homologs. The nomenclature for the mammalian genes is that of Chandy (1991) where K stands for potassium-channel gene, v for voltage-dependent, the first number corresponds to the subfamily and the second number is the number of the gene.

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 167

For instance the Kv1 subfamily is at present composed of seven different genes whereas only four genes have been found in the case of the Kv3 subfamily. Alternative nomenclatures can be found in the literature; for example ShI, ShII, ShIII, and ShIV for Kv1 Kv2, Kv3, and Kv4 respectively (e.g. Rudy et al., 1991a, 1991b; Weiser et al., 1994). It is notable that the genes of a given subfamily are extremely well conserved among invertebrates and vertebrates considering that they diverged about 570 million years ago. On the other hand, the fact that the same subfamilies of K channels are found in vertebrates and invertebrates suggest that the K channel genes diverged before eukaryotes diversified into vertebrates and invertebrates. Gene duplication and variation of these precursor genes may explain the origin of the various members of the subfamilies found in mammals (Rudy et al., 1991a). The largest subfamilies of K channels in vertebrates are Kv1 (Shaker-like) and Kv3 (Shaw-like). In contrast to the Shaker subfamily where diversity is a consequence of alternative splicing of a single gene, in the Kv1 subfamily, diversity is obtained through gene duplication. In the case of the mouse, Chandy et al. (1990) showed that K channel genes coding for MK1, MK2 and MK3 exist as a single uninterrupted exon in the mouse genoma. This prevents the generation of different K channels by alternative splicing. In the rat (RCK gene family; reviewed in Pongs, 1992) six members of the Kv1 subfamily have been characterized and each protein is encoded in a separate gene. The lack of alternative splicing in this subfamily has a notable exception in Kv1.5 (see section 6.3.1; Table 6.1; Attali et al., 1993). In contrast, the Kv3 subfamily consisting, at present, of four genes (Rudy et al., 1991b; Weiser et al., 1994) that show a large number of spliced variants (Rudi et al., 1991b; Luneau et al., 1991a, 1991b; Rettig et al., 1992; Rudy et al., 1992; Vega-Saenz de Miera et al., 1992; Weiser et al., 1994). The different K channel subfamilies generate currents that can be transient or of the delayed rectifier type. Figure 6.3 shows that the different genes belonging to the same (Kv3) subfamily are also able to express transient and sustained currents. Notice that Kv3.2a and Kv3.1a transcripts express delayed-rectifier type currents while Kv3.4b and Kv3.3a give rise to transient currents, although with different time courses (fast and slow inactivating currents). In this regard both Kv3.4 and Kv3.3 can be distinguished from Kv3.2 and Kv3.1 by the presence of N-terminal inserts likely to be the structures responsible for their inactivation process (Vega-Saenz de Miera et al., 1992). These currents activate relatively slowly and at voltages larger than –20 mV. Therefore, as discussed by Vega-

1 Percentage of identical amino acids in analogous positions between pairs of K channel proteins in other subfamilies are for example: Shab/Kv2.1 72%; Shaw/Kv3.2a 52%; Shal/ Kv4.1 82% [for more information see Rudy et al. (1991a, 1991b)]. Rudy et al. (1991a, 1991b) have pointed out that some of the Sh proteins appear to be among the most conserved proteins known. For example, the human Shaker Kv1.2 shows about 99% identity with the rat Kv1.2.

168 K CHANNELS AND THEIR MODULATORS

Saenz de Miera et al. (1992), they could not be involved in subthreshold phenomena (e.g. regulation of resting potential) and it is doubtful that they activate during a single fast action potential. However, these currents may play an important role during prolonged depolarizations or in neurons showing repetitive firing. Only a few K channels belonging to the Kv2 (Shab-like) and Kv4 (Shal-like) subfamilies have been cloned. The channels of the Kv2 subfamily (rKv2.1, Frech

Figure 6.3 Current time courses induced by alternatively spliced variants of the Kv3 subfamily of K channels. Notice that the current time courses for the different alternatively spliced variants, similarly to the current kinetics seen when channels from different subfamilies are expressed, are of the sustained type (A and B) or transient [fast (C) and slow (D) inactivation]. Data from Weiser et al. (1994).

et al., 1989; Hwang et al., 1992; mKv2.1, Pak et al., 1991a; hKv2.1, Albrecht et al., 1993) activate slowly and show delayed rectifier properties. In the case of mKv2.1 a very slow inactivation process with a time constant of several seconds has been reported (Pak et al., 1991a). In contrast, the Aplysia Kv2.1, although it activates slowly like the others members of the Kv2 subfamily, it inactivates within several hundred milliseconds (Quattrocki et al., 1994). Interestingly, the inactivation process of the Aplysia Kv2.1 channel remains intact after truncation of the N-terminal. This finding suggests that in this case inactivation is not

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 169

mediated by a mechanism of the ‘ball and chain’ type. Despite the fact that the rat, mouse, and human Kv2.1 channel proteins are highly conserved, their electrophysiological and pharmacological properties differ. For example, despite the fact that there is 100% identity in the S4 segment of these three channels, rKv2.1 is more voltage-dependent than the mouse or the human Kv2.1 (Albrecht et al., 1993). Pharmacological differences are also profound. The rat Kv2.1 is about 200-fold more sensitive to tetraethylammonium (TEA) than its mouse and human counterparts. In the case of the rat Kv2.1, it was found that deletions in either the N- or the C-terminal were able to promote major changes in channel kinetics (VanDongen et al., 1990). Surprisingly, removal of both N- and Cterminal produced a channel with almost identical characteristics to those of the wild-type Kv2.1 channel. Van Dongen et al. (1990) observations indicate that although N- and C-termini are not essential, they play an important modulatory effect on activation and inactivation kinetics. This may explain the differences in the electrophysiological properties between rat, mouse, and human Kv2.1 since the only differences in these channel proteins are present outside the membrane spanning domains. In general, the properties of these channels suggest an involvement in action potential repolarization and/or broadening. In the case of the Aplysia Kv2.1 it has been shown that the slow inactivation process contributes to action potential broadening that occurs in bag cell neurons at the onset of neuropeptide secretion. So far, two genes have been found in the Kv4 (Shal-like) subfamily (Baldwin et al., 1991; Pak et al., 1991b). The rat Kv4.1 shows a complex inactivation kinetics that cannot be described with a single-exponential decay (Baldwin et al., 1991). At least three exponentials are necessary to fit the inactivation time course of the rKv4.1 channel and both the fast (β =155 ms) and the intermediate (β =688 ms) are slowed down when positively charged amino acids (35–37) are deleted from the amino-terminal. On the other hand, deletion of amino acids 2–32 of the N-terminal of the mKv4.2 has only a small effect on the inactivation process in this channel (Pak et al., 1991b). Probably positively charged amino acids, like in the case of the ShB channel (Hoshi et al., 1990), are important in determining inactivation kinetics in Kv4.1, but whether or not a ball and chain model is appropriate in this case is not clear. Salkoff et al. (1992) pointed out that it is possible that, like in the mammalian Shab, the central core region may play an important role in determining channel gating kinetics. Although the electrophysiological properties of rKv4.1 and mKv4.1 are similar, there is only 77% identity between these two channels. In contrast, the different rat and mouse members of the Kv1 subfamily share a much higher degree of identity [almost 100% for rKvl.l (RCK1) and mKv1.1 (MBK1)]. Kv4.2 was cloned from rat heart (Roberds and Tamkun, 1991) and it has a very high degree of identity with the rat brain Kv4.1 at the N-terminal and S1–S6 region, but they diverge at the C-terminal region.

170 K CHANNELS AND THEIR MODULATORS

6.3.3 Formation of Heteromultimeric Channels is Fundamental in Subunit Composition and Diversity of K Channels The K channel proteins of the S4 superfamily (Figure 6.1) resemble one of the four internal repeats of Na or Ca voltage-dependent channels suggesting that functional K channels are multimers (tetramers). If K channels are formed by aggregation of subunits, heteromultimer formation may be another molecular mechanism to generate K channel diversity. Cotransfection of HeLa cells with cDNAs or coinjection of two different cRNAs into Xenopus oocytes corresponding to the RCK1 and RCK4 proteins were found to give rise to channels with mixed RCK1 and RCK4 channel properties (Ruppersberg et al., 1990). Heteromultimeric channels have propertie s distinct from those of the homomultimeric channels as if mixing of subunits is unrestrained. For example, RCK1 channels do not inactivate and are sensitive to dendrotoxin2 and TEA. In contrast, RCK4 channels show fast inactivation and are insensitive to dendrotoxin and TEA. The result of RCK1 and RCK4 cRNAs coinjection into Xenopus oocytes is a transient K channel sensitive to dendrotoxin and TEA (Ruppersberg et al., 1990). Coinjection of RBK1 and RGK5 cRNAS into Xenopus oocytes (Christie et al., 1990), coinjection of the RNAs coding ShA. and ShB containing a truncated N-terminal (inactivation removed) or coinjection with ShB and RCK1 cRNAs into Xenopus oocytes (Isacoff et al., 1990), or coinjection of two Sh cRNAs containing either different N-terminals or different C-terminals into Xenopus oocytes (McCormack et al., 1990a), all led to the formation of heteromultimeric channels. McCormack et al. (1990a) proposed some ‘mixing rules’ in the formation of heteromultimeric channels. First, when mixing channels with different N-terminals, the channel with the N-terminal that produces more inactivation dominates the kinetics. This can be understood if in N-type inactivating channels a single inactivation gate (one N-terminal) is enough to inactivate a channel. The coinjection of ShH4 and ShH4IR (inactivation removed; the channel has also a mutation that renders this channel toxin-insensitive to a scorpion toxin) cRNAs led to the conclusion that a single gate is sufficient for inactivation (MacKinnon et al., 1993). The reasoning is as follows: coinjection of ShH4 and ShH4IR channels into Xenopus oocytes induces the expression of channels containing 0, 1, …, 4 inactivation gates. With the proviso that a single wild-type subunit is enough to induce toxin sensitivity, if one inactivation gate is enough to inactivate a channel, all toxin sensitive channels (containing 1, …, 4 inactivation gates) will inactivate. The experimental outcome exactly matched this expectation. Measuring the inactivation time constant at increasing ratios ShH4IR cRNA: ShH4 cRNA demonstrates that channels with a single gate inactivate with an inactivation time constant that is a quarter that of channels with four gates. In contrast, the rate of recovery from inactivation is independent of the number of gates. This result suggests that the gates act independently and a gate closes a channel in a

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 171

mutually exclusive manner. Second, if the heteromultimeric channel is formed by mixing and matching proteins containing different C-terminals, the resulting heteromultimeric functional channel recovers from inactivation at a rate closer to the channel containing the C-terminal that recovers faster. It is remarkable that invertebrate (ShB) and mammal (RCK1) proteins can mix in the oocyte membrane originating functional heteromultimeric K channels (Isacoff et al., 1990). This finding indicates that, despite the evolutionary differences between these two species, the tertiary structure of the ShB and RCK1 proteins is similar enough to allow functional subunit interaction. Different subfamilies express independent K channel systems The reports described above demonstrate that closely related polypeptides (i.e. belonging to the same Kv1 subfamily) are able to form functional heteromultimeric channels. However, coinjection of cRNAs of two different subfamilies into Xenopus oocytes do not form heteromultimeric channels (McCormack et al., 1990a; Covarrubias et al., 1991). Indeed, the detailed study of Covarrubias et al. (1991) showed that Shaker, Shal, Shab, and Shaw express independent channel systems. Even when the cRNAs of all subfamilies are coinjected into a single oocyte the independence of channel systems is maintained. Mixing of subunits of different subfamilies is also hindered in vertebrates (McCormack et al., 1990a). 6.4 Structural Determinants for K Channel Assembly Covarrubia et al. (1991) hypothesized that the lack of heteropolimerization between K channels of different subfamilies was due to the existence of a ‘molecular barrier’. In the case of ShB channels such a molecular barrier was restricted to the N-terminal (Li et al., 1992). When ShB is coexpressed together with Kv2.1, the macroscopic currents are well fitted by the sum of two independent currents (e.g. Figure 6.4). However, if a chimeric cDNA is constructed such that it codes for a protein containing the ShB N-terminal and the hydrophobic core and C-terminal of Kv2.1, the resultant chimeric monomer (NShB ― 6–46-TmCDRKl) is able to form functional heteromultimeric channels when mixed with ShB. The results of Li et al. (1992) have been extended by Shen et al. (1993), Hopkins et al. (1994) and Lee et al. (1994). Lee et al. (1994) showed that heteromultimeric channels between hKv1.5 and hKv1.4 cannot be formed if a deletion (― 28–283) is introduced in the N-terminal of hKv1.4. However this deletion does not preclude the formation of homomultimeric

2

Dendrotoxin is a venom peptide from the mamba snake and blocks K channels with high affinity by binding to a site located in the external mouth of the channel.

172 K CHANNELS AND THEIR MODULATORS

channels. Moreover, deletion of the N-terminal of Kv2.1 allowed formation of hybrid channels with the ― 28–283hKv1.4 mutant. In other words, N-terminal deletions allow for the mixing of monomers of distant families of K channels. Contrasting with the results of Lee et al. (1994), deletion of large domains of the N-terminal of mKv1.1 prevents formation of functional homomultimers and of heteromultimers with mKvl.3 (Hopkins et al., 1994). This last result allows the possibility that N-terminal domains of different K channels belonging to the same family have more than one role in channel assembly. Shen et al. (1993) constructed progressive deletion clones of the Aplysia Kv1.1a (Pfaffinger et al., 1991) to investigate the minimal requirements for membrane insertion of the channel-forming protein. All deletion clones containing the S1 domain are able to incorporate into the membrane. That is, a clone containing nothing but the Nterminal linked to S1 is inserted into the lipid bilayer. Therefore, S1 provides a signal for membrane incorporation. Biochemical studies showed that once inserted, subunits are able to form stable multimeric structures.

Figure 6.4 Potassium channels of different subfamilies do not mix. Mixtures of cRNAs coding for Shaker and Shaw in a 3:1 ratio (upper-right) and Shal and Shab in 1:1 ratio (lower-right) were coinjected into Xenopus oocytes. Additive effect means digital addition of individual currents. Voltage pulses were 1 s in duration and ranged from –80 to 20 mV in 20 mV steps. Data Adapted from Covarrubias et al. 1991.

Homomultimeric structures are not formed if a domain comprising amino acids 66–194 from the N-terminal is deleted. Therefore, this region is essential for subunit coassembly in homomultimeric AKv1.1a K channels and was named T1

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 173

domain (first identified tetramerization domain; Shen et al., 1993). The results of Li et al. (1992) and Shen et al. (1993) (see also Lee et al., 1994 and Hopkins et al., 1994) provide strong evidence that the cytoplasmic N-terminal domain of the Shaker-like subfamily plays an important role in determining the assembly of subunits. However, coimmunoprecipitation experiments show that there is coassembly between T1(–) and T1(+) subunits to form tetramers. Furthermore, expression in oocytes of the amino-terminal truncated Kv1.5― 5’ cRNA produces currents similar to that of wild-type Kv1.5 albeit with lower efficiency (Attali et al., 1993). The coimmunoprecipitation experiments of cotranslated Kv1.1 and Kv1.2 of Babila et al. (1994) showed that the S1 segment plays a crucial role in the coassembly of homo–and heteromultimeric K channels. They proposed that the S1 domain is essential in homo–and heteromultimerization and in the stabilization of the interaction between subunits. On the other hand, the Nterminal confers selectivity and specificity to the K channel coassembly. 6.5 K Channels are Tetramers The fact that K channel proteins of different channels of the same subfamily can form heteromultimeric channels allowed MacKinnon (1991) to show that K channels of the S4 superfamily are tetramers. In this case the experimental strategy was to coinject the cRNAs of ShH4 and of ShH4 containing a mutation that makes the binding of a toxin about 1000-fold weaker (an aspartate in position 432 is mutated to asparagine). Assuming that monomer mixing is unrestrained, the probability (Pi) of having a channel with i mutated subunits is a function of the number of monomers forming a channel (n) and the fraction of wild-type and mutated subunits present (coinjected) initially. In this case a binomial distribution determines Pi. On the other hand, the fraction U of channels that are not blocked at a given concentration [T] of toxin is given by: U = ― (i=0.n)PiUi where Ui = Ki/(Ki +[T]) and Ki is the dissociation constant of the reaction of a channel containing i mutated subunits with the toxin. If one wild-type subunit in a channel is enough to confer tight toxin binding to a channel U ― fnmutUmut since U, Umut, and fmut can be experimentally determined, the channel stoichiometry, n, can be calculated. For two different fractions (f) of coinjected mutated cRNA values for n were 3.8 and 3.7, values in good agreement with the assumption that K channels are tetramers. K channel stoichiometry was also determined for Kvl.l (Liman et al., 1992). They mixed Kv1.1 cRNA and that of a Kv1.1 mutant that does not form functional channels (a tyrosine in position 379 was mutated to a lysine). Tyrosine 379 forms part of the S5-S6 linker. When these two cRNAs are coinjected into oocytes, the expressed channels have a much lower sensitivity to TEA than the wild-type Kv1.1 channel indicating that the mutant is able to mix with the wild-type protein. Coinjection of wild-type

174 K CHANNELS AND THEIR MODULATORS

Kv1.1 trimer cRNA with mutant Kv1.1 cRNA monomers also forms functional channels with low tetraethyl ammonium (TEA) affinity. However, coinjection of wild-type tetramer cRNA with mutant monomers produces only channels with a TEA affinity equal to that of Kv1.1. In other words, tetrameric wild-type constructions are unable to incorporate mutant monomers suggesting that functional K channel structures are tetrameric. More recently, Li et al. (1994) were able to image Shaker K channels using the electron microscope. The analysis of the Shaker protein revealed a structure of 8 nm × 8 nm × 6.7 nm with a fourfold symmetry, consistent with a tetrameric assembly of monomers. 6.6 Differential Expression of K Channels A counterpart to the wide variety of K selective channels found in animal cells is their differential distribution in animal tissues and brain regions. The electrophysiological studies provided an early indication that different K channels operate in different excitable cells. The wealth of information on K channel structure and function derived by applying DNA recombinant approaches to their study, as well as the powerful methods it provided, made it possible to start gathering direct evidence that a differential expression of K channels in animal tissues represents quite a general phenomena. At least, a differential distribution of K channels offers a fundamental mechanism underlying the diversity of electrical stereotypes exhibited by excitable cells. It follows that a differential pattern of K channel expression must accompany the events of tissue differentiation during development. The methods provided by molecular biology have demonstrated that K channel diversity is paralleled by specific patterns of channel mRNA expression and channel product in tissues and in different areas of the nervous system. However, in the nervous system, the evidence that differential mRNA’s expression implies functional specific K channels is scant. Ion channel functionality needs to be demonstrated since K channel localization patterns derived from immunohistochemical analysis yield details in channel product location which are not apparent for mRNAs analyzed by in situ hybridization. The above considerations are more relevant if we take into account the lack of information regarding the detailed electrophysiological properties of the variety of cells and cell processes that occur in the animal brain. Indeed, such information would help to strengthen the notion that differential K channel patterns, revealed by in situ hybridization, immunohistochemistry and biochemistry, correlate with relevant functional differences in the electrical properties of cellular components in different regions of the brain. Although the field of differential K channel expression is new, several questions concerning this fascinating problem that have been addressed are: (1) how general is the phenomenon of differential distribution of K channels in animal

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 175

tissues?; (2) are heteromultimeric K channels formed in vivo?; and (3) what factors modulate channel expression in cells and tissues? 6.6.1 Differential K Channel Expression is a General Phenomenon The molecular cloning of the renowned Shaker gene of Drosophila, and the realization that this gene encodes a family of K channel components pertaining to the A-class (sections 6.2 and 6.3), paved the way to begin addressing the question of differential ion channel distribution in animals. As discussed earlier, through alternative splicing, multiple transcripts arise from the Shaker gene yielding a variety of A-type channels exhibiting distinctive structural and kinetic properties. This knowledge was taken advantage of by Schwarz et al. (1990) to raise antibodies to three portions of the predicted sequences of Shaker channels. Immunoblots revealed a nonuniform distribution of products in the brain of the adult fly. Products were found to be prominently associated with the optic system and the mushroom bodies, a complex structure containing different cell types and inputs. Shaker products were detected both in the neuropils and in axons. Moreover, Schwarz and collaborators provided evidence that two splicing variants, ShA. and ShB, were present in only a subset of regions that express Shaker products, indicating that Shaker-type products do not have a fixed composition in all cells. On the other hand, the immunoblot assay was unable to show the presence of Shaker products in muscle cells, which are well known to contain A currents of the Shaker type. The immunohistochemical analysis carried out by Schwarz and coworkers was not accompanied by in situ hybridization studies which would have helped to establish how Shaker mRNA’s patterns compared to those of Shaker products revealed by the immunohistochemical assay. An electrophysiological complement supporting the observations of Schwarz et al. (1990) was provided by Baker and Salkoff (1990) who found that deletion of the Shaker gene abolished Shaker-type K+ currents only in a subset of neurons in late stage Drosophila pupae. An intriguing aside to the studies of Schwarz et al. (1990) concerns their observation that Shaker products are prominent in the mushroom bodies in the brain of Drosophila. The mushroom bodies receive inputs from olfactory systems and other sensory areas and Shaker alleles have been reported to be deficient in a conditioned odor-avoidance learning and retention protocol (Cowan and Siegel, 1986). Indeed, there is evidence that mushroom bodies are implicated in odor-avoidance associative learning and retention (Davis, 1993, 1995) and, recently, it has been documented that chemical ablation of mushroom bodies in flies causes deficiencies in this form of associative learning (De Belle and Heisenberg, 1994). The observations by Schwarz et al. (1990) raise the unexpected possibility that, owing to differential expression in well defined structures of the nervous system, K channel mutations might cause specific neurological deficiencies. In support of the above idea is the evidence that synaptic transmission and synaptic plasticity

176 K CHANNELS AND THEIR MODULATORS

is abnormal in the neuromuscular junction of Shaker mutants (Jan et al., 1977; Delgado et al., 1994). Differential expression of K channels has been documented convincingly also in the brain of higher animals. Thus, the distribution of members of the Shakerrelated RCK channel family (RCK1, RCK3, RCK4 and RCK5) has been investigated in the rat nervous system (Beck and Pongs, 1990). As discussed earlier in this chapter, the RCK gene family expresses a spectrum of voltagegated delayed rectifier and transient K+ currents. RNA blot hybridization revealed a differential distribution of RCK mRNAs in the adult rat brain, as well as a differential pattern of appearance during development. RCK1 and RCK5 mRNAs were found to predominate in the adult nervous system (postnatal days 30 (P30) and P90 animals). RCK3 and RCK4 were detected throughout all developmental stages. In adult rat central nervous system (CNS; P30 and P90), maximum levels of RCK1 were found in caudal regions of the brain, lower ones in the rostral regions and in the retina. Likewise, RCK5 mRNA levels were higher in the caudal regions of the CNS but the corpus striatum, midbrain and superior colliculus exhibited lower levels than the cerebral cortex. In the case of RCK3 mRNA levels were almost undetectable in cerebellum and high in the inferior colliculus, medula-pons and olfactory bulb. RCK4 mRNA levels were higher in the midbrain and forebrain areas of the CNS, including retina, with lower expression in cerebral cortex. RCK mRNAs were monitored also in the peripheral nervous system (PNS), leading to the conclusion that RCK1 and RCK5 mRNAs are coexpressed in the PNS, although at different levels, while faint RCK3 and RCK4 mRNAs signals could be assessed only for dorsal root ganglia. The above observations seem to indicate that both in the CNS and in the peripheral nervous system RCK1 and RCK5 are most abundant in the rat. The study of the developmental time course of RCK K channel mRNAs in the CNS of the rat by Beck and Pongs (1990) indicated the detection of RCK1 mRNA around embryonic day E15, with levels increasing during the second and third postnatal weeks in midbrain and hindbrain areas with only a moderate increase in forebrain areas. The developmental profile of RCK5 is somehow similar to that of RCK1. In contrast, RCK3 mRNA is present in all CNS regions throughout all developmental stages (E10 to P90), although RKC3 mRNA levels are low. RCK4 mRNA levels are substantial at E10 increasing slightly until the end of the first postnatal week. Thereafter, levels are maintained in the forebrain and midbrain areas but decrease in the hindbrain. Concerning the PNS, a similar developmental pattern to that of the CNS was observed for RCK1 mRNA in dorsal root ganglia (E18 to P60) suggesting that, perhaps, the developmental patterns of the other mRNAs for other RCK potassium channels might be similar in central and peripheral nervous system. The expression of RCK1, a K channel exhibiting sensitivity to the mast cell degranulating peptide (MCDP) and to dendrotoxin I (DTX1) (Stühmer et al., 1989) was investigated by Sequier et al. (1990) in rat brain. These agents are convulsant, induce neurotransmitter release, and can induce the β rhythm

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 177

associated with arousal. MCDP induces long term potentiation when applied to hippocampal slices (Bidard et al., 1987; Bidard et al., 1989; Cherubini et al., 1987). Transcript signals were prominent in granule cells of the hippocampal formation and pyramidal cells of Ammon’s horn, levels of expression being conspicuous in pyramidal CA3 cells compared to CA1 and CA2 cells. Interestingly, it is in the CA3 area of the hippocampus that the epileptogenic properties of MCDP have been traced (Cherubini et al., 1988). Other brain areas exhibiting a high RCK1 hybridization signal include the cerebellum, mainly at the level of the granular layer and in the Purkinje cell layers and in layers II, III, V and VI of the telencephalon. In contrast, levels of mRNA were low for the whole hypothalamus with the exception of the ventromedial nucleus. The distribution of mRNAs corresponding to Raw channels from the rat brain (Raw 1–3), namely a class of channels pertaining to Shaw-related K channels, was pursued by Rettig et al. (1992). With respect to tissue distribution of Raw mRNAs, it was reported that Raw1 and Raw2 mRNAs are expressed predominantly, if not exclusively, in the brain as compared to kidney, heart or skeletal muscle. Raw3 was detected both in the brain and in skeletal muscle, reflecting, perhaps, the existence of closely related Raw3 mRNAs or the occurrence of alternatively spliced variants. The distribution of Raw, investigated in the rat brain by in situ hybridization, revealed that different Raw mRNAs are expressed differentially in the nervous system. For example, each Raw mRNA exhibits a distinct pattern of distribution within the hippocampus. A high level of Raw3 RNA is found in the dentate gyrus and Raw1 RNA expresses in a gradient such that CA3 exhibits higher expression than CA1. Moreover, the granulated expression pattern of Raw1 and Raw2 would suggest that, in CA1 and CA3 hippocampal areas, they are confined to particular cells. Immunocytochemical studies revealed that, in the hippocampus, Raw3 and RCK4 mRNAs are expressed both in the same neuron and in different ones, raising the possibility that in the rat central nervous system the same cell can express multiple, independently assembled K channels and that unrestrained mixing of subunits does not occur. Weiser et al. (1994) carried out an intense scrutiny of the distribution in rat brain of the four known Shaw-related genes, or ShIII, (KV1, KV2, KV3 and KV4) using Northern blot analysis and in situ hybridization. Northern blot analysis indicated that Kv3.2 and 3.3 are expressed mainly in brain, with low levels of expression of Kv.3.1 in skeletal muscle. Kv3.4 transcripts were more abundant in skeletal muscle. In brain, ShIII transcript distribution was nonhomogeneous and the expression patterns of Kv3.1, Kv3.3 were similar. Kv3.4 mRNAs, which are less prominent in the CNS, were found mainly in areas in which Kv3.1 and/or Kv3.3 occurred in high levels. In many areas, Kv3.2 transcripts did not overlap with the products of other ShIII genes. Table 6.2 summarizes the distribution of transcripts for four ShIII genes in some regions of the CNS in the rat, as reported by Weiser et al. (1994). Higher-resolution studies of hybridization patterns, achieved by microscopic analysis of emulsion-dipped sections, revealed that

178 K CHANNELS AND THEIR MODULATORS

transcripts were present only in neuronal somas and not in glial cells. Many neuronal populations exhibiting Kv3.1 transcripts expressed also Kv3.3 mRNAs. Kv3.4 transcripts, at lower levels, were observed in some neuronal populations containing Kv3.1 and/or Kv3.3 mRNAs, indicating a potential Table 6.2 Distributions of transcript from four Shlll genes in some areas of the rat CNS.

Olfactory bulb Periglomerular cells Tufted cells Mitral cells Neocortex Interneurons Pyramidal cells Pyriform conex Hippocampus CA1 pyramidal cells Str. radiatum Str. oriens CA3 pyramidal Str. radiatum Str. oriens DG granule cells Borders with str. granulosum Hilus Thalamus (dorsal) Anterodorsal nu. Anteroventral nu. Anteromedial nu. Laterodorsal nu. Parataenial nu. Reunions nu. Mediodorsal nu.

KshIIIA (KV3.2)

KShIIIB (KV3.1)

KShIIIC (KV3.4)

KShIIID (KV3.3)

–

+

–

+

–

+ ++

– –

+ ++

– +

Layers II– IV>V–VI – ±

Layers II– IV>V–VII – +

Layers II– IV>V–VI –

+

+

–

+

– +++ ++ ++ ++ –

++ ++ +++ + + +++

– – – – – ++

+ + + + + ++

–

+

+++

±

–

–

++++

+

–

±

++++

±

–

–

++++

±

–

–

+++ ++++ +++ ++++

– – ± ±

– – –

± – –

Layers V–VI

±

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 179

KshIIIA (KV3.2)

KShIIIB (KV3.1)

KShIIIC (KV3.4)

KShIIID (KV3.3)

Intermediodors + + – – – al nu. Lateral +++ + – – posterior nu. Ventral ++++ ++ – – posterolateral nu. Ventrolateral ++++ ± – – nu. ++++ ++ – + Ventral postenomedial nu. Ventro medial ++++ ± – – nu. Central medial +++ ± – – nu. Posterior nu. ++++ ± – – Dorsal lateral ++++ ++ – + geniculate Medial ++++ – – – geniculate Thalamus (Ventral) Reticular ± ++++ ± ++ thalamic nu. Ventral lateral – + – + geniculate nu. Cerebellum Molecular cell – + + + layer Purkinje cells – + + +++ Granular cell – ++++ ± ++ layer Deep cerebellar + + +++ – +++ nu. Spinal cord Dorsal horn – +++ – +++ – + ++ +++ Ventral horn The symbols indicate signal intensity as follows: - (signals undistinguishable from background); ± (very weak signals but clearly above surrounding background);+(weak);+ + (moderate);+ + +(high);+ + + + (very high). (Modified from Weiser et al., 1994) Nu=nuclei; str=stratum.

180 K CHANNELS AND THEIR MODULATORS

for heteromultimer formation between these three gene products. Of great potential significance is the observation that coinjecting Xenopus oocytes with small amounts of Kv3.4 cRNA and an excess Kv3.1 or Kv3.3 cRNA yielded channel products with fast-inactivating properties resembling those of Kv3.4 channels. Thus, as discussed in section 6.3.3, it is probable that even a single Kv3.4 subunit is competent to confer fast-inactivating properties to ShIII channels. The higher-resolution analysis also showed that, although the expression patterns of Kv3.1 and Kv3.3 in the CNS are similar, they are not identical. Thus, Kv3.1 signals in some neurons were more intense than those of Kv3.3 while the opposite was true in other cases. In several brain areas, for example the cerebral cortex, hippocampus and caudate-putamen, individual ShIII probes labeled specifically a small number of neurons which otherwise could not be distinguished from neighboring neurons in the area. Kv3.3b, an alternatively spliced form of the Kv3.3 Shaw-type K channel identified by Goldman-Wohl et al. (1994), was found to be at a high level in mouse brain, particularly in the cerebellum. There, expression was confined to Purkinje cells and deep cerebellar nuclei. Expression of Kv3.3b mRNA appeared to be developmentally regulated in cerebellar Purkinje cells beginning between P8 and P10, to be present in virtually all Purkinje cells by P12, and continue through adulthood. P8–P10 corresponds to the period at which Purkinje cells establish synapses with granule cell parallel fibers. Since Purkinje cells exhibit aberrant morphology in agranular cerebellar cortex, the observations by Goldman-Wohl et al. (1994) suggest that, perhaps, expression of this gene is regulated by mechanisms that involve the interaction between Purkinje and granule cells. It is of interest to point out that, according to Goldman-Wohl et al.’s (1994) observations, cell type specificity and developmental regulation of this gene was maintained in dissociated primary culture of mouse cerebellum. In culture, Purkinje cells were identified by immunocytochemistry for calbindin and the time course of Kv3.3b expression was monitored by in situ hybridization. Kv3.3b mRNA was undetectable in E18 Purkinje cells cultured for one week but it was expressed in calbindin-positive cells after two to three weeks in culture and expression was not dependent on neurite extension. Hwang et al. (1992, 1993) investigated in the rat brain the localization of CDRK and DRK1 channels pertaining to the Shab subfamily by immunohistochemistry. They found that both products occur predominantly in

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 181

neurons, displaying regional variations with overall levels of expression in the various brain regions being similar for both channels. However, cellular distribution of the two channels differs greatly, differences that were particularly obvious in the cortex. For example, the DRK1 immunoreactivity was restricted mostly to pyramidal cells and dendritic processes. CDRK was not evident in pyramidal cells and was restricted to a population of smaller nonpyramidal elements resembling cortical interneurons. CDRK was detected also in the processes of such cells. Differential location of CDRK and DRK1 products was also apparent in the hippocampus where the dentate gyrus was enriched in CDRK immunoreactivity with respect to DRK1. On the other hand, although in the cerebellum there was coexpression of CDRK and DRK1, both in Purkinje and granule cells, their localization at the subcellular level differed with DRK1 localized in the somata and proximal dendrites and CDRK associated with the cell body. Rudy et al. (1992) investigated the differential expression of KShIIIA (Kv3.2a), a Shaw-like gene that encodes several alternatively spliced transcripts in the rat brain. In oocytes, KShIIIA transcripts yield voltage-dependent K+ currents exhibiting slow activation at voltages above –20 mV, Because of these properties, KShIIIA channels are not expected to be relevant to subthreshold events and they are much too slow to be active during a single, fast Na+ depolarizing phase (see Section 6.3.2). Northern blot analysis with a KShIIIA probe that does not distinguish spliced transcripts revealed that hybridization was stronger in thalamus-enriched RNA than in whole-brain RNA. It indicated the presence of two major bands at 7.5 and 6.5 kb, in addition to a diffuse one around 4 kb, and the band at 6.5 kb was not obtained with thalamus RNA. The several bands obtained could not be accounted for as arising from alternatively spliced transcripts since similar bands were obtained when probes specific for KShIIIA1, KShIIIA2 and KShIIIA3 were used. These results were interpreted as indicative of the existence of differentially processed species or of subtypes of each of the transcripts. In situ hybridization confirmed that, in rat brain, KShIIIA transcripts display a differential distribution, being most prominent in the nuclei of the dorsal thalamus and in the optic layer of the superior colliculus. Less prominent hybridization signals were found in the deep layers of all cortical areas of the neocortex, the piriform cortex, the red nucleus and the CA3 region of the hippocampus. Discrete to weak labeling was obtained in the cochlear nucleus and in the neocortex. A differential expression of alternatively spliced KShIIIA transcripts could not be documented in this study and the analysis did not address the possible subcellular distribution of the products. However, the distribution pattern of the transcripts suggested that they are expressed at highest levels in neurons. Bright field images showed that in the thalamic nuclei hybridization grains were concentrated over the soma of large neuronal cells and the density of hybridization in glia appeared similar to background. In the same study, Rudy et al. (1992) compared the hybridization patterns of KShIIIA transcripts to those of NGK2-KV4, a transcript that, in Xenopus oocytes, induces

182 K CHANNELS AND THEIR MODULATORS

currents with properties similar to those of KShIIIA. The analysis demonstrated that, if NGK2-Kv4 transcripts are concentrated in certain brain areas, their pattern of distribution differs to that of KShIIIA. For example, NGK2-Kv4 mRNAs were abundant in the cerebellar cortex where KShIIIA expression is minimal. In the hippocampus both KShIIIA and NGK2-Kv4 expressed, albeit in a different fashion. Thus, KShIIIA transcripts were more prominent in the CA3 field of Ammon’s horn while NGK2-Kv4 was found in the dentate gyrus as well as in CA3. Immunofluorescence assisted Freeman and Kass (1993) in showing that an antibody directed against the minK channel reacted with a membrane antigen on adult guinea pig ventricular myocytes and sinoatrial cells, suggesting that such a channel might actually be responsible for slow-rising cardiac delayed-rectifier K + currents. This evidence is of extreme importance considering the peculiar structure of this putative minimal K channel consisting only of 130 residues and a single transmembrane domain. In Xenopus oocytes minK cRNA yields slow voltage-gated, non-inactivating outward currents. Previous to the Freeman and Kass (1993) report, minK channel activity had not been detected in mammalian cells raising the concern that minK transcript injection could yield in oocytes a regulatory protein of endogenous K+ currents rather than a K channel. In addition to showing that cardiac cells are immunoreactive to minK antibodies these authors were able to achieve the expression of minK currents in a transfected mammalian cell line (HEK 293). The currents expressed exhibited properties similar to those recorded from guinea pig ventricular myocytes strengthening the idea that minK gives rise in mammalian cells to slow-activating, noninactivating voltage-dependent channels with delayed-rectifier properties. Although the experimental evidence indicates the existence of differential expression of K channels in animal tissues, this does not mean that expression of a given K channel type is confined only to a specific tissue. For example, the delayed rectifier (IKs) K channel has been cloned from neonatal heart and ovariectomized diethylstilbestrol-primed rat uterus and expressed in oocytes. Northern blot analysis failed to document channel expression in adult heart, nonprimed uterus or brain (Folander et al., 1990). However, an identical channel (IKs) was demonstrated earlier in rat kidney (Takumi et al., 1988). The studies by Folander et al. (1990) would indicate that, at variance with the claim of Takumi et al. (1988), expression of IKs channels would not be restricted to epithelial tissue in mammals. In the human heart, the studies of Tamkun et al. (1991), using Northern blot analysis, indicated that HK2 K channels, much more abundant in ventricle than atrium, bear a great degree of identity (86%) to Kv1, a rat brain K channel. In addition, expression of channels belonging to the RCK family (rat cortex K channels) have been reported by Betsholtz et al. (1990) in insulinproducing cells. Douglass et al. (1990) reported the cloning of RGK5, a K channel derived from rat thymus which was postulated to represent n-type currents in immature thymocytes and T lymphocytes. Northern blot analysis revealed that RGK5 transcripts are found both in thymus and rat brain.

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6.6.2 Heteromultimeric Channels in vivo The occurrence of heteromultimeric K channels in the brain has also been addressed. The fact that different K channel subunits show overlapping expression patterns in the brain fulfills one of the prerequisites of in vivo heteromultimer channel products (Drewe et al., 1992). The studies by Sheng et al. (1992, 1993) indicated that Kv1.2 and Kv1.4 mRNA, two K channel subunits of the Shaker subfamily, exhibited distinct, but overlapping expression patterns in the rat brain and these authors provide convincing evidence that Kv1.2 and Kv1.4 form heteromultimers which are differentially distributed in the brain. It is worth mentioning here that Kv1.2/Kv1.4 channels combine properties of both parent subunits to form a novel A-type channel characterized by a fast inactivation, rapid recovery from inactivation and high sensitivity to the K channel blocker 4-aminopyridine. Monitoring of the distribution of Kv1.4 and Kv1.2 in rat brain, using immunohistochemistry, indicated that these channel subunits coassemble in the rat brain and that the heteromultimers are localized in axons and nerve terminals, consistent with a role in the control of neurotransmitter release. Kv1.2 immunoreactivity overlapped with Kv1.4, particularly in the neuropil of the cerebral cortex, axon tracts of the corpus callosum and in well defined terminal fields of the hippocampus. Evidence for multimer formation was supplemented by biochemical evidence indicating the cofractionation of Kv1.2 and Kve1.4 proteins. Following detergent solubilization and separation in an anion-exchange column, identical elution patterns for both products were obtained. Moreover, the use of gene-specific antibodies demonstrated that Kv1.2 and Kv1.4 polypeptides could be coimmunoprecipitated by antibodies specific for either gene. Thus, to the growing evidence for the differential expression of homomultimer K channel products in the brain, the differential expression of heteromultimeric K channels must be added. Support for heteromultimeric K channels in the brain has also been provided by Wang et al. (1993) for mKvl.l and mKvl.2 in the juxtaparanodal regions of nodes of Ranvier and in terminal fields of basket cells in mouse cerebellum. Coimmunoprecipitation of both products could be achieved with antibodies specific for either mKvl.l or mKvl.2. The studies by Wang et al. (1993), in addition to demonstrating heteromultimeric channels in vivo, showed that such heteromultimers occur in subcellular regions in the brain. In vitro translation in transfected mouse L-cells, combined with immune purification approaches, enabled Deal et al. (1994) to gather evidence for the cotranslational assembly of Kv1.1/ Kv1.4 heteromers. Glycosilation of Kv1.1 at the extracellular N207 site was not required for assembly and did not affect channel turnover or function.

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6.6.3 Regulation of K Channel Expression Little is known currently of the rules governing the striking phenomenon of K channel differential expression in the brain and even less to its relation to neurological deficiencies in higher animals. The scant information available includes the work of Tsaur et al. (1992). In the rat brain, changes in the distribution of mRNAs from K channels. Kv1.1 and Kv1.2, encoding delayed rectifier channels of the Shaker-RCK subfamily, and Kv4.2, an A-type channel of the shal subfamily, were monitored following seizure induced by the convulsant pentylenetetrazol. Kv1.1 and Kv1.2 were expressed at higher levels in the hippocampus, thalamus, cerebral cortex and cerebellum. In turn, the highest levels of Kv4.2 expression were found in the cerebellum, intermediate levels in the hippocampus and media habenula, with lower levels in the cerebral cortex and thalamus. Following seizure, a reduction of Kv1.2 and Kv4.2 mRNAs was detected only in the dentate granule cell layer of the hippocampus, a brain region in which excitatory glutamatergic neurons predominate. No brain region exhibited changes in Kv1.1 mRNA levels. Importantly, the changes in mRNA levels could not be attributed simply to stress-related activation of the hypothalami-adrenocortical axis, since the mRNA levels of the three K channels were not altered after treatment with adrenocorticotropic hormone. Moreover, the changes in mRNA levels observed after seizure would seem to be related to neuronal activity, since administration of diazepam, prior to pentylenetrazol, both protected the animal from seizure and prevented reduction in Kv1.2 and Kv4.2 mRNA levels in the dentate granule. Takimoto et al. (1993) demonstrated that dexamethasone, a glucocorticoid agonist, induced rapid Kv1.5 channel mRNA transcription in clonal pituitary cells, an effect that could not be attributed to a change in Kv1.5 mRNA turnover. Moreover, the steroid was found to increase the expression of the Kv1.5 protein, as evidenced by immunoblots, without changing its half-life. Increases in a noninactivating component of the voltage-gated K+ currents accompanied the induction of the Kv1.5 protein, supporting the notion that hormones and neurotransmitters may affect, within hours, excitability by controlling K channel gene expression. In neuroblastoma cells, which exhibit neuronal characteristics, the effects of dimethylsulfoxide (DMSO) and retinoic acid (RA), agents which cause differentiation-like events in cell culture, were investigated by Smith-Maxwell et al. (1991) using the patchclamp technique. Under control conditions, the cell line used (N2AB-1) exhibited transient, voltage-dependent Na+ currents but little delayed rectifier outward K+ currents. Treatment with RA or DMSO, under low serum conditions, caused the expression of delayed rectifier K+ currents. It was observed, however, that there was not necessarily a correlation between K channel expression and inhibition of cell division, cellular hypertrophy or the elaboration of processes. In cultured mouse hippocampal neurons there is evidence for astroglial modulation of transient K+ current development (Wu and Barish, 1994) suggesting that

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astroglial-induced plasticity could mediate long-term modulation of excitability in the hippocampus. The mechanisms by which astroglia influenced the appearance of outward currents of the A-type were most probably surface or extracellular matrix associated ones and living glial cells were required for modulation. Glial neurons treated with an inhibitor of RNA synthesis for 5–7 days had reduced A-current amplitudes and the action of the inhibitor was localized to the astroglia. The regulation of Kv1.5 by platelet-derived growth factor (PDGF) or fibroblast growth factor (FDG) was investigated by Timpe and Fanti (1994) in Xenopus oocytes by the simultaneous expression of Kv1.5 channels and either receptor. PDGF or FDG receptor activation in oocytes led to a slow decline in K channel amplitude with a half-life of about 20 minutes without any major change in channel voltage-dependence or kinetics. Depression of K+ currents was found to require activation of phospholipase C. The time course of current decline was too fast to be caused by altered gene transcription. Since K channels and PDGF and FGF receptors can occur in the same cell type, for example progenitor cells for oligodendrocytes and type 2 astrocytes, it is conceivable that the modulation of K channels investigated by Timpe and Fanti (1994) might also occur in other cell types. 6.7 Conclusion K channels can be now grouped according to their primary structure. Structurally K channels can be divided in three large superfamilies: K channels belonging to the S4 super family, K channels with subunits containing two membrane spanning regions (e.g., inward rectifiers), and K channels with a single membrane spanning region (minimal K channel). The large variety of K channels in the S4 superfamily, that include delayed rectifiers, A-type, and Caactivated K channels among many others, is a consequence of mRNA splicing, multiple genes, and formation of heteromultimeric channels. In this superfamily, K channels are tetrameres and some of the assembly rules of monomers into tetrameres have been elucidated. Inside a K channel subfamily mixing of monomers of different types of K channels is possible. However, monomers of K channels belonging to different subfamilies do not mix. The N-terminal and the S1 transmembrane segment of the K channel-forming proteins appear to be, at least in part, the structures regulating mixing and matching of monomers in this superfamily. Differential expression of K channels is a general phenomenon, although the factors that govern the expression of a given K channel in different cells and tissues are still unknown. Both homo- and heteromultimeric K channels show differential expression in vivo. Acknowledgements This work was supported by grant FNI-1940227, Human Frontier in Sciences Program, by a European Communities research contract, by institutional support to the Centre de Estudios Científicos de Santiago provided by SAREC (Sweden),

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SALKOFF, L. & WYMAN, R. (1981) Nature, 293, 228–230. SALKOFF, L., BAKER, K., BUTLER, A., COVARRUBIAS, M., PAK, M. & WEI, A. (1992). Trends Neurosci, 15, 161–166. SCHACHTMAN, D., SCHROEDER, J., LUCAS, W., ANDERSON, J. & GABER, R. (1992) Science, 258, 1654–1658. SCHRÖTER, K.-H., RUPPERSBERG, J.P., WUNDER, F., RETTIG, J., STOCKER, M. & PONGS, O. (1991). FEBS Lett., 278, 211–216. SCHWARZ, T.L., PAPAZIAN, D.M., CARRETTO, R.C, JAN, Y.N. & JAN, L.Y. (1990) Neuron, 4, 119–127. SCHWARZ, T.L., TEMPEL, B.L., PAPAZIAN, D.M., JAN, Y.N. & JAN, L.Y. (1988) Nature, 331, 137–142. SEQUIER, J.-M., BRENNAND, J., BARHANIN, J. & LAZDUNSKI, M. (1990) FEBS Lett., 263, 163–165. SHEN, V., CHEN, X., BOYER, M. & PFAFFINGER, P. (1993) Neuron, 11, 67–76. SHENG, M., TSAUR, M-L., JAN, Y.N. & JAN, L.Y. (1992). Neuron, 9, 271–284. SHENG, M., LIAO, Y.J., JAN, Y.N. & JAN, L.Y. (1993) Nature, 365, 72–75. SMITH-MAXWELL, C.J., EATOCK, R.A. & BEGENISICH, T. (1991) J. Neurobiol, 22, 327–341. STOCKER, M., STÜHMER, W., WITTKA, R., WANG, X., MULLER, R., FERRUS, A. & PONGS, O. (1990) Proc. Natl. Acad. Sci USA., 87, 8903–8907. STÜHMER, W., RUPPERSBERG, J.P., SCHRÖTER, K.H., SAKMANN, B., STOCKER, M., GIESE, K.P., PERSCHKE, A., BAUMANN, A. & PONGS, O. (1989). EMBO. J., 8, 3235–3244. SUZUKI, M., TAKAHASHI, K., IKEDA, M., HAYAKAWA, H., OGAWA, A., KAWAGUCHI, Y. & SAKAI, O. (1994) Nature, 367, 642–645. SWANSON, R., MARSHALL, J., SMITH, J.S., WILLIAMS, J.B., BOYLE, M.B., FOLANDER, K., LUNEAU, C.J., ANTANAVAGE, J., OLIVA, C., BUHROW, S.A., BENNETT, C., STEIN, R.B. & KACZMAREK, L.K. (1990) Neuron, 4, 929–939. TAKIMOTO, K., FOMINA, A.F., GEALY, R., TRIMMER, J.S. & LEVITAN, E.S. (1993) Neuron, 11, 359–369. TAKUMI, T., OHKUBO, H. & NAKANISHI, S. (1988) Science, 242, 1042–1045. TAMKUN, M.M., KNOTH, K.M., WALBRIDGE, J.A., KROEMER, H., RODEN, D.M. & GLOVER, D.M. (1991), FASEB J., 5, 331–337. TANOUYE, M.A., FERRUS, A. & FUJITA, S.C. (1981) Proc. Natl. Acad. Sci. USA., 78, 6548–6552. TEMPEL, B.L., JAN , Y.N. & JAN, L.Y. (1988) Nature, 332, 837–839. TEMPEL, B.L., PAPAZIAN, D.M., SCHWARZ, T.L., JAN, Y-N. & JAN, L.Y. (1987) Science, 237, 770–775. TIMPE, L.C. & FANTI, W.J. (1994) J. Neurosci., 14, 1195–1201. TIMPE, L.C., JAN, Y.N. & JAN, L.Y. (1988a) Neuron, 1, 659–667. TIMPE, L.C., SCHARZ, T.L., TEMPEL, B.L., PAPAZIAN, D.M., JAN, Y.N. & JAN, L.Y. (1988b). Nature, 331, 143–145. TORO, L., OTTOLIA, M., STEFANI, E. & LATORRE, R. (1994) Biochemistry, 33, 7220–7228. TSAUR, M.L., SHENG, M., LOWENSTEIN, D.H., JAN, Y.N. & JAN, L.Y. (1992) Neuron, 8, 1055–1067. TSENG-CRANK, J.C.L., TSENG, G-N., SCHWARTZ, A. & TANOUYE, M.A. (1990) FEBS Lett., 268, 63–68.

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VANDONGEN, A.M.J., FRECH, G., DREWE, J.A., JOHO, R.H. & BROWN, A.M. (1990) Neuron, 5, 433–443. VEGA-SAENZ DE MIERA, E., MORENO, H., FRUHLING, D., KRENTROS, C. & RUDY, B. (1992) Proc. R. Soc. Lond. B., 248, 8–18. VEGA-SAENZ DE MIERA, E., WEISER, M., KENTROS, C., LAU, D., MORENO, H., SERODIO, P. & RUDY, B. (1994). In: Handbook of Membrane Channels: Molecular and Cellular Physiology. Perachia, C. (ed). Academic Press, New York. pp. 41–78. WANG, H., KUNKEL, D.D., TROY, M.M., SCHWARTZKROIN, P.A. & TEMPEL, B.L. (1993) Nature, 365, 75–79. WARMKE, J.W. & GANETZKI, B. (1993) Biophys. J., 64, A340. (1994) Proc. Natl Acad. Sci. (USA), 91, 3438–3442. WARMKE, J.W., DRYSDALE, R.A. & GANETZKI, B. (1991) Science, 252, 1560–1562. WEI, A., COVARRUBIAS, M., BUTLER, A., BAKER, K., PAK, M. & SALKOFF, L. (1990) Science, 248, 599–603. WEISER, M., VEGA-SAENZ DE MIERA, E., KENTROS, C., MORENO, H., FRANZEN, L., HILL, D., BAKER, H. & RUDY, B. (1994) J. Neurosci, 14, 949–972. WITTKA, R., STOCKER, M., BOHEIM, G. & PONGS, O. (1991) FEBS Lett., 286, 193–200. WU, R.-L. & BARISH, M. (1994) J. Neurosci., 14, 1677–1687. YOKOYAMA, S., IMOTO, K., KAWAMURA, T., HlGASHIDA, H., IWABE, N., MIYATA, T. & NUMA, S. (1989) FEBS Lett., 259, 37–42. YUN-CAI, CAI, OSBORNE, P.B., NORTH, R.A. DOOLEY, B.C. & DOUGLAS, J. (1992) DNA Cell Biol, 11, 163–172. ZAGOTTA, W.N., GERMERAAD, S., GARBER, S.S., HOSHI, T. & ALDRICH, R.W. (1989) Neuron, 3, 773–782. ZAGOTTA, W.N., HOSHI, T. & ALDRICH, R.W. (1990) Science, 250, 568–571. Recent Literature HUANG, Y. & RANE, S.G. (1994) Potassium Channel Induction by the ras/raf Signal Transduction Cascade. J. Biol. Chem., 269, 31183–31189. KNAUS, H.-G., FOLANDER, K., GARCIA-CALVO, M., GARCIA, M.L., KACZOROWSKI, G.J., SMITH, M. & SWANSON, R. (1994) Primary Sequence and Immunological Characterization of a β Subunit of the High Conductance Ca2+ activated K+ Channel from Smooth Muscle. J. Biol. Chem., 269, 17274–17278. MCMANUS, O.B., HELMS, L.M.H., PALLANCK, L., GANETZKI, B., SWANSON, R. & LEONARD, R.J. (1995) Functional Role of β Subunit of High Conductance Calcium-activated Potassium Channels. Neuron, 14, 645–650. MOTTES, J.R. & IVERSON, L.E. (1995) Tissue-specific Alternative Splicing of Hybrid Shaker / lacZ Genes Correlates with Kinetic Differences in Shaker K+ Currents in vivo. Neuron, 14, 613–623. PALLANCK, L. & GANETZKI, B. (1994) Cloning and Characterization of Human and Mouse Homologues of the Drosophila Calcium-activated Potassium Channel Gene, slowpoke. Hum. Mol. Genet., 3, 1329–1243.

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RETTIG, J., HEINEMANN, S.H., WUNDER, F., PARCEJ, D.N., DOLLY, J.O. & PONGS, O. (1994) Inactivation Properties of Voltage Gated K+ Channels Altered by the Presence of Beta Subunit. Nature, 369, 289–294. SCOTT, V.S., MUNITZ, Z.M., SEWING, S., LICHTINGHAGEN, R., PARCEJ, D.N., PONGS, O. & DOLLY, J.O. (1994) Antibodies Specific for Distinct Kv Subunits Unveil a Heterooligomeric Basis for Subtypes of β -Dendrotoxin-sensitive K+ Channels in Bovine Brain. Biochemistry, 33, 1617–1623. TSENG-CRANK, J., FOSTER, C.D., KRAUSE, J.D., MERTZ, R., GODINOT, N., DICHIARA, T.J. & REINHARDT, P.H. (1994) Cloning, Expression, and Distribution of Functionally Distinct Ca2+ -activated K+ Channel Isoforms from Human Brain. Neuron, 13, 1315–1330. VIVIENNE, N. & PFAFFINGER, P.J. (1995) Molecular Recognition and Assembly Sequences Involved in the Subfamily-specific assembly of Voltage-gated K+ Channel Subunit Proteins. Neuron, 14, 625–633. WALLNER, M., MEERA, P., OTTOLIA, M., KACZOROWSKI, G., LATORRE, R., GARCIA, M., STEFANI, E. & TORO, L. (1995) Cloning, Expression of, and Modulation by a Subunit of a Maxi KCa Channel from Human Myometrium. Receptors Channels, 3, 185–199.

ADDENDUM Diversity of K Channels is More Rich than our Imagination News from Paramecium tetraaurelia The ciliate protozoan, Paramecuim, can do amazing things. It can swim forward, or backwards when it finds an obstacle to resume swimming forward some seconds later, or when bumped from the rear, it escapes very fast from the aggressor. All of these Paramecium movements are controlled by a highly efficient set of Ca2+ and various types of K channels. Paramecium tetraaurelia has a family of K channels (Pak1 and Pak2) with a very low degree of homology to the metazoan voltage-dependent K channels (Jegla and Salkoff, 1994). According to Jegla and Salkoff (1994) the uniqueness of these K channels may be a consequence of evolutionary pressures at play in single vs multicellular organisms. The Pak channels contain six transmembrane segments and a P region. Although the pore region has a high degree of identity with other K channels, the overall identity with, for example, the Shaker K channel is only 19 per cent. In particular, the S4 transmembrane segment in Pak channels contains very few positively charged residues compared with Shaker K channels. Some K channels appear to be dimers Ketchum et al. (1995), using as a hook the P domain from several K channels, fished from gene databases a novel K channel from Saccharomyces cerivisiae. This K channel, denominated TOK1, contains two pore domains in tandem within the same polypeptide. Eight potential transmembrane regions, S1–S8, were deduced from the primary sequence of the TOK1 channel, interrupted by a

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P1 region located, as with the S4 superfamily, between S5 and S6 and a P2 region between S7 and S8. The S4 domain in TOK1 channels does not have positively charged amino acid residues. However, injection of TOK1 cRNA into Xenopus oocytes induces outwardly rectifying K+ currents. Interestingly, the position in the voltage axis of the conductance-voltage relationship varies with external K+ as in ‘classical’ inward rectifying channels. In TOK1 channels current is outward at voltages larger than the potassium equilibrium potential. Moreover, TOK1 channels are blocked by external Ca2+ in a voltage-dependent fashion and once this divalent cation is removed, channels can conduct ions inwardly. Thus, as in inward rectifier channels, the voltage dependence of TOK1 channels is not an intrinsic property of the channel protein but a consquence of a voltage-dependent blockade. Above we discussed that K channels of the S4 superfamily are tetramers requiring four P domains to form the walls of the pore (see section 6.5). The particular structure of TOK1 channels implies, thus, that their functional stoichiometry is that of dimers (for a review see Salkoff and Jegla, 1995). Additional References JEGLA, T., & SALKOFF, L. (1994) A Multigene Family of Novel K+ Channels from Paramecium tetraaurelia, Receptors Channels, 3, 51–60. KETCHUM, K.A., JOINER, W.J., SELLERS, A.J., KACCZMAREK, L.K. & GOLDSTEIN, S.A.N. (1995) A New Family of Outwardly Rectifying Potassium Channel Proteins with Two Pore Domains in Tandem. Nature, 376, 690–695. SALKOFF, L., & JEGLA, T. (1995) Surfing the DNA Databases for K+ Channels Nets yet more Diversity. Neuron, 15, 489–492.

7 Potassium Channel Electrophysiology in Vascular Smooth Muscle Cells and the Site of Action of Potassium Channel Openers P.I.AARONSON1 & C.D.BENHAM2 1

Department of Pharmacology and Medicine, UMDS Guy’s and St Thomas’s Hospitals, Lambeth Palace Rd, London, SE1 7EH, UK. 2

Department of Biophysical Sciences, SmithKline Beecham Pharmaceutical, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. 7.1 Introduction The application of patch-clamp electrophysiology to single smooth muscle cell preparations is generating a bewildering wealth of information about K+currents in vascular muscle cell membranes as it is in many other excitable cells. Functional studies such as these are hampered by a lack of a comprehensive range of specific pharmacological probes to clearly identify K+ currents. Aside from specific agents such as charybdotoxin and the relatively selective glibenclamide, many studies attempt to draw conclusions from data using tetraethylammonium (TEA) and 4-aminopyridine (4-AP), compounds with a broad K channel blocking profile. The recent advances in the molecular biology of K channels now provides the opportunity to put the functional studies into a structural framework and hopefully reveal a little more order. In this chapter we will consider work using both approaches in an effort to develop an overall picture of these channels in vascular smooth muscle. This information will then be used to consider the likely sites of action of the K channel openers, a group of compounds that have attracted interest both because of their therapeutic potential and because of the insights they have provided on vascular smooth muscle function. 7.2 Structural Division of K Channels Deduced from Molecular Biology Molecular biology has defined three families of K channels (reviewed by Hoshi and Zagotta, 1993). The largest group has homology to the Shaker encoded channel in Drosophila. This gene encodes a voltage dependent, inactivating K channel that electrophysiologists had termed an A current. Four gene sub-families have been cloned from Drosophila and their mammalian homologues have been called Kvl.x, Kv2.x, Kv3.x and Kv4.x. These channels are all voltage dependent

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K channels, opening in response to membrane depolarization but showing varying rates of activation and inactivation. The classically described delayed rectifier and transient outward currents fall into this group (for review see Pongs, 1992). Recently, a Ca dependent K channel has been cloned with some homology to the Shaker group of channels. It seems likely that this group of voltage dependent channels are part of the larger Shaker superfamily. Thus, the great majority of voltage sensitive K channels that shape repolarization in excitable cells can be gathered into this one large group. The second group is unusual in being defined by a single gene, MinK, that codes for a voltage dependent K channel with very slow activation kinetics (for review see Blumenthal and Kazcmarek, 1992). This channel is distinguished by a subunit structure that contains only one putative transmembrane spanning region in the 130 amino acid subunit sequence. The number of subunits required to form a functional channel is unknown. This structure is in marked contrast to the Shaker family which share a common structural motif of six membrane spanning regions (S1–S6) in each subunit and have been shown to exist as tetramers in Xenopus oocyte membranes. The gene is expressed in a variety of glandular tissues (Takumi et al., 1988) but also in heart and smooth muscle. The first functional expression was in fact from mRNA purified from oestrogen-primed uterine smooth muscle (Boyle et al., 1987) which preceded the identification of the MinK gene. A third major family is now coming into focus. This includes K channels that can open close to the resting membrane potential, several of which behave as inward rectifiers passing inward current more readily than outward current. Also included are a group of channels that appear to be under metabolic control, regulated by the cytoplasmic concentration of ATP and other intermediary metabolites. This last group also appear to be the site of action of the K channel opening drugs such as cromakalim. So far, sequence data for only two members of this family are available. ROMK1 (Ho et al., 1993) and IRK1 (Kubo et al., 1993) conform to a putative two membrane spanning domain structural signature. ROMK1 cloned from rat kidney and expressed in Xenopus oocytes exhibits some of the biophysical properties of an ATP-sensitive K channel (KATP). However, millimolar concentrations of cytoplasmic ATP do not close the channel as seen for native channels although the channels require low ATP concentrations to prevent rundown. Thus it seems likely that these two genes code for channels related to the KATP channel and that KATP channels will turn out to have some homology to these channels but be new additions to this family. 7.2.1 Vascular Smooth Muscle Representatives of these Families As with other excitable cells, a number of the 50 or more identified voltage dependent K channels (Pongs, 1992) appear to be expressed in vascular smooth muscle. The most ubiquitous channel in excitable tissues (at least as defined by

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mRNA expression), is that coded by the DRK1 gene. This is the mammalian homologue of the Drosophila shab gene and DRK1 is abundantly expressed in rat aorta (Roberds and Tamkun, 1991). The electrophysiological properties of this channel most closely resemble the classical delayed rectifier current described by Hodgkin and Huxley (Frech et al., 1989). Nine members of a highly homologous family of genes, corresponding to the Shaker family, have been expressed from rat, designated RCK1–9, that encode channels displaying a complete range of properties from rapidly inactivating A-type currents to noninactivating delayed rectifier currents (Pongs, 1992). Of these nine, representatives of at least four types are expressed in aorta (Roberds and Tamkun, 1991). Finally, a homologue of the Shal gene is expressed in rat aorta (Roberds and Tamkun, 1991) which might be expected to have an intermediate kinetic fingerprint (Pongs, 1992). The relative abundance of these expressed mRNAs suggests that, in aorta (Roberds and Tamkun, 1991) and probably in a number of other blood vessels, the most important K channels for conferring the overall electrical properties of the cells are the delayed rectifier DRK1 and the Shaker homologue RCK7 or Kv1. This latter channel shows relatively slow inactivation over hundreds of milliseconds (Swanson et al., 1990). Functionally, we would therefore expect to see a TEA-sensitive delayed rectifier current in vascular smooth muscle cells. In rabbit portal vein the IC50 for TEA on the delayed rectifier in this cell is about 5 mM (Beech and Bolton, 1989b). This is consistent with the properties of DRK1, IC50=6 mM (Pongs, 1992). Clearly, the complex (and no doubt incomplete picture) described above provides an obvious explanation for the widespread variation in the electrophysiological characteristics of voltage dependent K channels in different vascular cells. A large number of subtly different electrophysiological phenotypes can be defined by the palette of channels available. Further work on the molecular biology of K channels in different blood vessels will be important in unravelling this complexity. In particular the functional expression of particular channel types will allow the identification of specific antagonists that can then be used to probe the functional role of these different channels in the intact tissues. 7.3 K Channels in Vascular Smooth Muscle Cells: Electrophysiological Overview As with many other excitable tissues, characterization of K channels in vascular smooth muscle has been hampered by a relative lack of specific antagonists. However, over the last ten years the use of voltage-clamp techniques to investigate currents in single cell preparations has allowed significant progress to be made. Although K channels and K+ currents with a wide range of properties have been characterized in smooth muscle cells from many sites in the vasculature, most of these fall into one of three major classes. These include delayed rectifier-type

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channels (IK), large-conductance Ca-activated (BKCa) channels, and KATP channels. While it is unlikely that a complete characterization of K channels in any one type of vascular smooth muscle cells (VSMCs) has yet been achieved, it is apparent that these three types of K channel co-exist in VSMCs from a number of blood vessels. Whether each type of channel therefore has a distinct function, and what that function is, remains unclear. In general, however, K channels are thought to be involved in setting the resting potential and suppressing or limiting membrane depolarization. 7.3.1 Ca-activated K Channels Most of the KCa channels which have been reported in VSMCs have a conductance of approximately 100 pS when the K+ gradient is set to a physiological level, this value is roughly doubled in symmetrical high K+ solutions (e.g. Benham et al., 1986; Gelband and Hume, 1992). The opening probability (P0) of these channels is sensitive both to increases in intracellular [Ca2+] and to depolarization. Single channel studies have indicated that at likely resting conditions of membrane potentials negative to –60 mV and intracellular Ca2+ of 100 nM, the probability of opening of the KCa channels will be very low (Benham et al., 1986). However, the large number of channels and their large unitary conductance means that even with a low Po, they could contribute a functionally important component of the total K + conductance under resting conditions. KCa channels appear to be virtually ubiquitous in the smooth muscle cells of the vasculature; their high conductance contributes a characteristic ‘noisy’ appearance to cell outward currents. In addition, large current oscillations, termed spontaneous transient outward currents (STOCs), are often observed at negative membrane potentials (Benham and Bolton, 1986). These are thought to be caused by stochastic Ca2+ release from the sarcoplasmic reticulum, resulting in the brief activation of a local group of channels. KCa channels are selectively blocked at submillimolar concentrations by TEA, and are also potently and selectively suppressed by charybdotoxin (Brayden and Nelson, 1992). These blockers are therefore useful in evaluating the role of KCa channels in controlling the membrane potential. Bath application of TEA causes depolarization and action potential generation in a variety of isolated blood vessels (reviewed by Bolton, 1979). In a recent study, Brayden and Nelson (1992) reported that the vasoconstriction of rabbit cerebral arteries induced by elevation of luminal pressure was potentiated by TEA and charybdotoxin; they therefore suggested that KCa channels act as a negative feedback pathway to control the myogenic response.

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7.3.2 Delayed Rectifier-type K Channels Whole-cell membrane currents carried by voltage-gated delayed rectifier K channels are also widely distributed in VSMCs (Beech and Bolton, 1989a). The single channel conductances underlying IK are relatively small, and although extensive studies have not been carried out, appear diverse (Beech and Bolton, 1989a; Gelband and Hume, 1992). IK typically becomes apparent when cells are depolarized beyond –40 or –30 mV. The activation rate is voltage-dependent. The current subsequently inactivates, although the rate of this process varies in different VSMCs, again pointing to the existence of more than one type of IK channel in the vasculature. The contribution of IK to membrane potential regulation is not well understood; this may in part be the result of the problem that no selective blockers of IK have as yet been described. 4-AP in millimolar or submillimolar concentrations was shown to suppress IK to a much greater extent than KCa in several types of VSMCs (Beech and Bolton, 1989b), and also caused membrane depolarization in single cells and isolated blood vessels (Hara et al., 1980; Gelband et al., 1993; Smirnov et al., 1994). These results are, however, ambiguous, since 4-AP also may block KATP channels (Beech and Bolton, 1989b). Several aspects of the regulation of IK in different VSMCs have recently been reported. For example, acute hypoxia greatly attenuates a 4-AP-sensitive K+ current in cultured rat pulmonary, but not mesenteric, arterial cells (Yuan et al., 1993). A K+ current in canine pulmonary artery has also been shown to be reduced in amplitude by acute hypoxia, although the channels involved were of high conductance and were Ca2+ -sensitive (Post et al., 1992, 1993). It was suggested that this latter effect involved a reduction in cell redox state. Longterm exposure of rats to hypoxia causes a chronic depolarization of pulmonary artery, and also reduces the amplitude of IK in cells isolated from pulmonary arteries after prolonged hypoxia (Smirnov et al., 1994). IK is also inhibited by rises in intracellular Ca2+ and Mg2+. The molecular characterization of IK channels is likely to result in further and rapid advances in our understanding of the regulation and role of this current in vascular cells. 7.3.3 KATP Channels and Inward Rectifier Channels in Vascular Smooth Muscle These channels can be grouped together because they show the properties of being open near the resting membrane potential, showing little inactivation, and having ohmic current voltage relationships or a preference to passing current in an inward direction.

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Inward rectifier currents An inward rectifier K+ current (a conductance preferentially passing inward current at negative membrane potentials) was first identified in intact segments of small arterioles (Edwards and Hirst, 1988). Attempts to further characterize this conductance in single cells have been thwarted until recently by an inability to identify such a current in single cell preparations. This could be due to a localization of these channels to small resistance vessels. Such a possibility is given further weight by the recent observation of this current in single cells isolated from small resistance sized cerebral arteries (Quayle et al., 1994). This conductance showed high selectivity for K+ ions and inward current through the channels was blocked by low concentrations of barium (IC50=2.2 μ M). It is of interest that increases in extracellular K+ in the 5–15 mM range hyperpolarize and dilate these vessels (McCarron and Halpern, 1990), which may be important as a mechanism of autoregulation in these vessels and as a mechanism for increasing cerebral blood flow in response to elevated neuronal function that will be signalled by elevated extracellular K+. The localization of the inward rectifier current in these cerebral vessels may underlie this unique functional response, since the activation of this current, which leads to hyperpolarization, is shifted to more positive potentials by an elevation of extracellular K+. Regulation by ATP of KATP channels Although KATP channels are defined by the inhibitory action of ATP upon their activity, it is well established that low concentrations of ATP also stimulate channel activity (reviewed by Ashcroft and Ashcroft, 1990). The presence of MgATP is necessary to prevent the disappearance of KATP activity in isolated membrane patches in a number of types of cell. Non-hydrolyzable ATP analogues are not effective in this respect, indicating that phosphorylation of the channel or an associated regulatory protein is needed to maintain its viability. Evidence for a potentiating effect of ATP in VSMCs has been presented by Noack et al. (1992) and by Pfründer et al. (1993), both of whom found that low concentrations of ATP in the presence of excess Mg2+ greatly enhanced the K+ current elicited by ATP depletion; channel run-down was however not slowed. Although persistent KATP activity has been shown in isolated patches or dialyzed cells under ATP-free conditions (Standen et al., 1989; Clapp and Gurney, 1992; Lorenz et al., 1992), this might reflect the presence of residual ATP, or the absence of cellular phosphatases. An inhibitory effect of ATP on K channels has been demonstrated in membrane patches from a number of types of VSMC, although the range of channel properties reported is diverse. Standen et al. (1989) reported that in isolated membrane patches from rat and rabbit mesenteric artery VSMCs, ATP inhibited the opening of a slightly voltage-dependent, K-selective channel with a conductance of 135 pS. In the presence of ATP, the opening of this channel was

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increased by cromakalim (CRK), and then decreased by glibenclamide (glyburide). The inhibitory effect of glibenclamide on KATP channel activity has been described in a variety of cell types, and this compound is widely used to elucidate the role of these channels in cellular responses thought to be due to KATP activation (e.g. hyperpolarization and relaxation of VSMCs) under conditions where it is difficult or impossible to directly establish channel identity. In a subsequent report (Nelson et al., 1990) the glibenclamide-sensitive K channel in these cells was reported to have a much smaller conductance (20 pS between –20 and –50 mV). A large (258 pS) conductance ATP- and glibenclamide-sensitive (but Ca insensitive) K channel was also observed in VSMCs from rabbit kidney afferent arterioles (Lorenz et al., 1992). This channel also demonstrated a marked voltage-dependence, distinguishing it from KATP channels in other types of cells. An ATP- and glibenclamide-sensitive K channel with a conductance of 30 pS has also been described in porcine coronary artery (Miyoshi et al., 1992). This channel appears, however, to be atypical in that it is activated by extracellular Ca2+, and was only weakly inhibited by ATP unless the extracellular Ca2+ concentration was reduced to 0.1 μ M. K+ currents sensitive to intracellular ATP have also been recorded in VSMCs using the whole-cell patch-clamp technique. Clapp and Gurney (1992) compared membrane potential and K+ currents in rabbit pulmonary arterial cells dialyzed with either no, or 1 mM, ATP. In the absence of cellular ATP, cells were hyperpolarized and demonstrated a glibenclamide-sensitive non-inactivating K+ current near the resting potential. In the presence of ATP, cells were more depolarized and showed no non-inactivating current. Noack et al. (1992) demonstrated similar effects when comparing rat portal vein cells dialyzed with a solution either containing or lacking glucose and Krebs cycle intermediates. Silberberg and van Breemen (1992), using the perforated patch technique in order to preserve the intracellular milieu, found that metabolic inhibition of rabbit mesenteric artery cells with iodoacetic acid and dinitrophenol also elicited a timeand voltage-independent, glibenclamide-sensitive K+ current. Other intracellular modulators of KATP Measurements of the ATP-sensitivities of KATP channels in isolated patches from VSMCs and other tissues show that channel opening is half-maximally inhibited at [ATP]i 20–100 μ M (Nichols and Lederer, 1991). Since cellular ATP has been measured to be in the millimolar range, questions have been raised concerning whether the ATP concentration ever falls enough to allow significant channel opening (Ashcroft and Ashcroft, 1990), and additional or alternative regulators of the KATP channel have been sought. There is a wealth of information which indicates that ATP itself is not the only, or indeed the primary, physiological regulator of KATP channels in VSMCs and other cell types. Regulation of these channels by nucleotide diphosphates, pH, endogenous agonists, and second

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messenger systems has been described (Ashcroft and Ashcroft, 1990; De Weille and Lazdunski, 1990). Nucleotide diphosphates (NDPs) such as ADP and GDP influence KATP channel activity in various types of cell, including VSMCs. A number of NDPs have been shown to promote the opening of KATP channels in pancreatic β -cells (Findlay, 1987) and cardiac ventricular cells (Lederer and Nichols, 1989); in both cases this effect required the presence of Mg2+. It has been proposed that this effect may involve competition at the ATP binding site (e.g. Lederer and Nichols, 1989), and also that ADP acts at a separate site (Tung and Kurachi, 1991) which may be located on a diffusable cytoplasmic component (Larsson et al., 1993). Kajioka et al. (1991) observed a 15 pS ATP-sensitive K channel in rabbit portal vein cells which opened in the presence of pinacidil in cell attached, but not isolated, membrane patches; this implied that a cytoplasmic factor was necessary for channel activation. Channel opening could be restored in isolated patches by GDP (>0.1 mM) applied to the internal surface of the patch; GTP had a similar but weaker effect, and GMP was ineffective. The channel was inhibited by ATP (IC50=29 μ M). Pfründer et al. (1993) found that 0.1 mM ADP in the absence of ATP elicited a glibenclamide-sensitive, non-inactivating conductance in guinea-pig portal vein cells, which was not present in the absence of both ATP and ADP. This current was not entirely suppressed even in the presence of 5 mM ATP, if 0.1 mM ADP was present. Beech et al. (1993a, 1993b) also found little glibenclamide-sensitive K+ current in rabbit portal vein cells dialyzed with an intracellular solution lacking any nucleotides. Glibenclamide-sensitive currents of similar magnitude were however evoked in the absence of ATP when any of several nucleotide diphosphates, including GDP, CDP, and UDP, were present. ADP also had a similar, but lesser, effect; nucleotide tri- and monophosphates were essentially ineffective. The channel underlying this current had a conductance of 24 pS. Figure 7.1 illustrates that the glibenclamide-sensitive K+ current in rat pulmonary arterial cells is also only negligibly activated in the absence of added intracellular ATP, unless a nucleotide diphosphate is also present. Panel A shows that in the absence of intracellular ATP only a minor effect of glibenclamide on the whole cell K+ current can be demonstrated. When cells are dialyzed with an ATP-free pipette solution containing 1 mM GDP however, a large glibenclamidesensitive K+ current is observed. These results were obtained in cells which were bathed in a high K+ solution in order to shift the K+ reversal potential to 0 mV. Under such conditions a large K driving force is present at the normal resting membrane potential and the lack of voltage-dependence of the glibenclamidesensitive current in the presence of GDP was clearly revealed. The potentiating effect of nucleotide diphosphates on KATP channels in rabbit portal vein cells appears to require Mg2+ (Beech et al., 1993a), as it does in ventricular myocytes and β -cells. The block of the KATP channel by ATP in portal vein cells was also relieved by Mg2+, indicating that ATP is more effective in blocking these channels than is MgATP. This characteristic has previously been

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shown to apply to KATP channels in pancreatic β -cells, but not in ventricular myocytes. pH has also been shown to regulate KATP channel activity in a number of cell types, although both increases and decreases in channel opening have been reported,

Figure 7.1 Whole cell currents recorded from pulmonary artery VSMCs, where the patch pipette contained either no nucleotide (A) or 1 mM GDP (B). The K+ solution on both sides of the membrane was set at 135 mM. Cells were held at –60 mV and 100 ms voltage ramps from –100 to +90 mV were applied every 10s. The open circles show the resulting current under control conditions, and the solid circles represent the current measured in the presence of glibenclamide (10 μ M). Mean and S.E.M. values are from 3 cells in A and 5 cells in B.

depending on cell type (Misler et al, 1989; Davies, 1990). Koyano et al. (1993) have recently shown in guinea-pig ventricular cells that acidosis both decreases the sensitivity of the channel to ATP, and also reverses channel rundown. Similar studies in VSMCs are lacking, but it is worth noting that neither agonists, nor severe hypoxia (Aalkjaer and Lombard, 1994) have been shown to cause important changes in intracellular pH in VSMCs under physiological conditions. Hypoxia has been shown to cause dilation of guinea-pig coronary arteries (Daut et al., 1990) and inhibition of myogenic reactivity of renal afferent arteries (Loutzenhiser and Parker, 1994); both effects were glibenclamide sensitive,

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implying a role for KATP channels. The mechanism by which hypoxia activates KATP channels is, however, unclear. One possibility, suggested by Daut et al. (1990), is that hypoxia leads to a fall in intracellular [ATP] or the [ATP]/[ADP] ratio sufficient to activate KATP channels. This concept, however, runs counter to a number of reports that cellular [ATP] and energy state are maintained at normal levels in the face of hypoxia, being depleted only during anoxia or severe metabolic inhibition (e.g. Buescher et al., 1991). It is also thought that the nearmembrane [ATP] is highly dependent upon glycolysis rather than oxidative phosphorylation (Weiss and Lamp, 1987). Loutzenhiser and Parker (1994) observed that the glibenclamide-sensitive inhibitory effect of hypoxia was not associated with increases in arterial NADH, implying that oxidative ATP production was not compromised. It is therefore possible that hypoxia may activate KATP channels via an ATP-independent pathway. 7.4 Are Vascular KATP Channels Open under Basal Conditions? Although it has been demonstrated that the membrane potential of isolated VSMCs can be altered by changing the intracellular ATP concentration, either directly or via metabolic inhibition (Clapp and Gurney, 1992; Silberburg and van Breemen, 1992), the extent to which KATP channels are normally contributing to the maintenance of the resting membrane potential under physiological conditions remains unclear. Quast and Cook (1989) found that the infusion of glibenclamide into rats, which would be expected to block any basal KATP channel activity and therefore lead to depolarization and vasoconstriction, caused only a transient rise in blood pressure, implying that the K+ conductance sensitive to this drug was unlikely to be exerting a significant control over the resting potential. Similarly, glibenclamide infusion had no effect upon blood pressure in dogs (Samaha et al., 1992; Imamura et al., 1992). Glibenclamide also has been shown not to increase vascular tone in a number of types of isolated blood vessels (Quast and Cook, 1989; Eltze, 1989; Yuan et al., 1990). Glibenclamide decreases basal tone in canine cerebral and basilar arteries (Zhang et al., 1991). On the other hand, glibenclamide was shown to increase resting tone in vivo in both the hamster cheek pouch and cremasler muscle microcirculalions (Jackson, 1993). In addition, infusion of high concentrations of glibenclamide into the coronary arteries of anaesthetized dogs led to significant coronary constriction under non-ischaemic conditions (Samaha et al., 1992; Imamura et al., 1992). It would therefore appear that KATP channels are open under basal conditions in some, but not all, vascular beds. Although this might reflect the influence of endogenous agonists (see below) which may be tonically activating these channels, particularly in vivo, it is also possible that the regulation and/or

204 K CHANNELS AND THEIR MODULATORS

structure of these channels may differ significantly between different sites in the vasculature. 7.4.1 KATP Channel-independent Effects of Glibenclamide An interpretation of the effects of glibenclamide on vascular tone is complicated by the fact that this drug has also been shown to suppress tension development induced by vasoconstricting prostanoids such as PGF2― (Zhang et al., 1991; Neilsen-Kudsk and Thirstrup, 1991) and U46619 (Cocks et al., 1990) in arteries from the rat and dog. This inhibitory effect is nol endothelium-dependent (Zhang et al., 1991), and appears to involve a competitive interaction al the prostaglandin receptor(s) involved (Cocks et al., 1990). The EC50 for this effect is typically in the micromolar range; similar or higher concentrations are widely used to block KATP channel activity in most in vitro studies. Glibenclamide has also been observed to suppress contractile responses to high concentrations of K+ solution in coronary arteries (Neilsen-Kudsk and Thirstrup, 1991), and to a number of different spasmogens in airways smoolh muscle (Neilsen-Kudsk and Thirstrup, 1993). Figure 7.2 illustrates that glibenclamide has a potent inhibitory effect on the PGF2― contracture in rat pulmonary artery. This effect is contrasted wilh that of

K CHANNEL ELECTROPHYSIOLOGY IN VASCULAR SMOOTH MUSCLE CELLS 205

206 K CHANNELS AND THEIR MODULATORS

Figure 7.2 Effects of KATP channel blockers BRL31660 and glibenclamide on force development In rat pulmonary artery rings precontracted with a submaximal dose (2 μ M) of PGF2― . Vertical and horizontal scale bars represent 0.25 g and 10 min respectively.

BRL 31660, a novel antiarrhythmic compound which has also been shown to block KATP channels (Taylor et al., 1989). BRL 31660 caused a concentrationdependent increase in force development in the presence of PGF2― (Figure 7.2). BRL 31660, but not glibenclamide, also induced force development over a similar concentration range in the absence of any agonist (not shown). It therefore seems that glibenclamide is quite effective at relaxing responses to vasoconstricting prostanoids, and may also have additional, although less potent, inhibitory effects on responses to other types of stimulation. The contrasting effects of glibenclamide and BRL 31660 suggest that the widespread use of glibenclamide as a supposedly selective inhibitor of the KATP channel may have led to an underestimate of the role of this channel in regulating basal tone and membrane potential under physiological conditions. 7.4.2 Agonist Mediated Modulation Regulation of KATP channel activity by agonists, acting through G proteins and protein kinases A and C, is likely to play a major role in controlling VSMC membrane potential, and thus vasoconstriction. Effects of both vasoconstrictors and vasodilators on KATP channels have been demonstrated, both directly, and indirectly using glibenclamide. In cultured VSMCs from porcine coronary artery, endothelin (20 nM) caused a marked depolarization, which could be reversed by the KATP channel opener nicorandil. Openings of a KATP channel of 30 pS conductance were detected in cell attached and isolated membrane patches, and were potently and completely suppressed by endothelin (IC50 near 1 nM). Vasopressin had a similar effect on these channels at low concentrations (Miyoshi et al., 1992; Wakatsuki et al., 1992). This channel was somewhat atypical of KATP channels, however, in that it was regulated by the extracellular Ca2+ concentration, such that the channel was mostly open at physiological [Ca2+]0, even if intracellular [ATP] was raised to 1 mM. Vasoconstrictor effects were recorded at 0.1 mM [Ca2+]0, raising questions concerning their physiological relevance. It is becoming increasingly clear that activation of KATP channels contributes to the actions of several vasodilators. Standen et al. (1989), demonstrated that glibenclamide inhibited both the opening of KATP channels in isolated patches from rat and rabbit mesenteric artery VSMCs, and the hyperpolarizations induced in intact arteries by acetycholine and vasoactive intestinal peptide. A subsequent report showed that calcitonin gene-related peptide (CGRP), a potent vasodilator, caused hyperpolarization and the opening of K channels in rat mesenteric artery VSMCs (Nelson et al., 1990). Both effects were blocked by glibenclamide, although the CGRP-induced relaxation was partially insensitive

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to this compound, suggesting a KATP independent mechanism was also present. Recent data provide good evidence that the CGRP mediated modulation of KATP activity is through activation of protein kinase A (Quayle et al., 1994). Evidence has also been presented that the vasodilation of guinea-pig coronary arteries by adenosine was mostly blocked by glibenclamide (von Beckerath et al., 1991), as was the inhibition of myogenic reactivity in rat kidney afferent arterioles caused by low concentrations of this autocoid (Loutzenhiser and Parker, 1994). Dart and Standen (1994) have recently demonstrated that adenosine, acting at A1 receptors, causes a glibenclamide-sensitive K+ current in porcine coronary artery cells. 7.5 K Channel Openers and their Site of Action Over the last several years a number of structurally diverse molecules have been identified with the common mechanism of hyperpolarizing vascular smooth muscle. The first compound with this selective action was CRK (Hamilton et al., 1986) and a comprehensive listing can be found in Edwards and Weston (1993). The key characteristics of the profile of these compounds, in addition to hyperpolarizing the membrane potential of smooth muscle cells, is that the relaxant effects can be overcome by elevation of external [K+] above 50 mM and can be blocked by sulphonylureas such as glibenclamide at submicromolar concentrations. This profile was interpreted to suggest that the compounds mechanism of relaxant action was as potassium channel openers (KCOs). Since these initial observations, a large body of experimental evidence has been accumulated to test this hypothesis and has been reviewed by Quast (1993). 7.5.1 The K Channel Opened by KCOs Following the description of the hyperpolarizing action of CRK (Hamilton et al., 1986), the next problem to resolve was the nature of the K channel that was opened. An early study by Beech and Bolton (1989b) examined the pharmacology of the CRK-activated current in VSMCs from portal vein and compared this current with the delayed rectifier and BKCa current in these cells. These studies showed a pharmacological profile similar, but not identical to, the delayed rectifier current although the KCO activated current showed no voltage dependence unlike the delayed rectifier. The whole-cell current activated by KCO also showed no apparent increase in current noise suggesting that the channels activated were not large conductance like BKCa channels. A subsequent study revealed no significant effect of levcromakalim (LCRK) on the Caactivated and delayed rectifier K+ currents in these cells (Russell et al., 1992). In the last five years a number of studies have built on these observations and indicate that the major conductance that is activated in whole cells by LCRK is a

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KATP channel whose key properties are little voltage sensitivity, no inactivation and block by micromolar glibenclamide. Noack et al. (1992) showed that LCRK (1 μ M) activated a glibenclamide-sensitive current in rat portal vein and that this had very similar properties to a current Imet that developed in the same cells when intracellular ATP was depleted. Moreover, when Imet ran down after prolonged ATP depletion (characteristic of KATP channels) then LCRK was no longer able to activate the KCO current. The unitary conductance of this current was estimated at 10–20 pS from noise analysis of the whole-cell currents. Other KCOs such as aprikalim and P1060 also seem to activate the same conductance (Ibbotson et al., 1993). In VSMCs from rabbit pulmonary artery, a whole-cell K+ current with similar properties has been identified (Clapp and Gurney, 1992), and the unitary conductance of this current has also been estimated by noise analysis to be 16 pS (Langton et al., 1993). In addition to this enhancement of the KATP current, inhibition of the delayed rectifier has been reported in rat portal vein cells (Noack et al., 1992). This effect is likely to be functionally less relevant in VSMC than the activation of a conductance at or near the resting membrane potential of the cell that will dominate the response. However it does suggest that KCOs have more than one channel target. 7.5.2 Other Actions of KCOs The major experimental evidence that challenges the straightforward hypothesis that KCOs act by opening K channels is the ability of KCOs to relax tone induced by noradrenaline in tissues where it is known that Ca antagonists are unable to reverse noradrenaline-induced tone (Bray et al., 1991). These data suggest that KCOs must be inhibiting an intracellular mechanism as the noradrenaline-induced tone clearly does not depend on voltage-gated Ca2+ influx. This could be explained either by KCO induced hyperpolarization modulating other second messenger pathways, in which case multiple sites of action do not need to be invoked, or by direct intracellular effects of the KCOs themselves on these pathways. A link between membrane potential and phospholipase C activity would provide a membrane potential dependent pathway. Itoh et al. (1992) have demonstrated that in rabbit mesenteric artery, noradrenaline induced IP3 production is inhibited by pinacidil, and inhibition is relieved by elevation of external K+ and by K channel blockers (KCBs). Such an inhibitory profile would be expected if hyperpolarization inhibited IP3 production. This neatly explains the inhibitory effects of pinacidil on tension and intracellular Ca2+ that were measured in conditions of zero extracellular Ca2+. Direct evidence for modulation of IP3 production by membrane potential has now been provided. Ganitkevich and Isenberg (1993) showed that in guinea-pig coronary artery smooth muscle cells held under voltage clamp, hyperpolarization inhibits intracellular Ca2+ transients generated by muscarinic activation of phospholipase

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C. The effect is independent of extracellular Ca2+ and not seen with caffeine induced intracellular Ca2+ transients. The potential sensitivity of phospholipase C might be explained if, as a membrane bound enzyme, it directly senses the membrane potential through an intrinsic voltage sensor. The complex effects of membrane potential on second messenger function fail to explain all the paradoxes of experimental data. In particular, experiments showing that KCBs can inhibit the effects of KCOs on membrane potential without blocking their relaxant actions clearly imply intracellular targets for KCOs (reviewed by Quast, 1993). The data seem most consistent with the idea that reuptake of Ca2+ into intracellular stores is inhibited by KCOs. Evidence to support this comes from studies on permeabilized airways smooth muscle cells (Chopra et al., 1992). Taking all this data together (Figure 7.3), it seems that the most important action of the KCOs is to open a K channel that has very similar properties to a KATP channel. Surprisingly, some of the relaxant properties of these compounds do not mimic the actions of voltage-gated Ca channel antagonists because of the recently identified effects of membrane potential on other signal transduction systems in smooth muscle. Finally, it is also possible to identify actions that must be completely independent of changes in membrane potential although, as yet, the functional significance of these effects is not clear.

Figure 7.3 Plan illustrating the identified mechanisms of action of KCOs and the functional consequences thereof.

210 K CHANNELS AND THEIR MODULATORS

7.6 Conclusions Much of this chapter has focussed on the properties of a K channel or channels that can open close to the resting membrane potential of vascular smooth muscle cells. This property makes them well suited to exert subtle control of the membrane potential and hence excitability of these cells, particularly as the voltage dependent entry of Ca2+ is likely to be mostly a graded trickle dependent on membrane potential rather than large quantal influxes associated with action potentials. The activity of these channels is modulated both by intracellular nucleotide levels and by G-proteins, indicating that both internal and external signal transduction systems couple to these channels to regulate membrane potential. In contrast, the large group of voltage dependent K channels might be predicted to have a relatively less important functional role, reflecting the smaller contribution of action potential discharges to the overall pattern of electrical activity of these tissues. We can look forward to clarifying these speculative conclusions as molecular approaches identify all the pieces in the K channel puzzle that make up the K conductance of each type of smooth muscle cell. Understanding the extent of the diversity of channels in different tissues will help to explain the tissue variation in experimental data. Selective functional expression of channels will facilitate the search for selective blockers. These agents will then in turn be used to clarify physiological function in the original vascular preparations. References AALKJAER, C. & LOMBARD, J. (1994) J. Vas. Res., 31 (Suppl 1), 1. ASHCROFT, S.J.H. & ASHCROFT, F.M. (1990) Cell Signall., 2, 197–214. BEECH, D.J. & BOLTON, T.B. (1989a) J. Physiol, 412, 397–414. (1989b) Br. J. Pharmacol., 98, 851–864. BEECH, D.J., ZHANG, H, NAKAO, K & BOLTON, T.B. (1993a) Br. J. Pharmacol., 110 (2), 573–582. (1993b) Br. J. Pharmacol., 110(2), 583–592. BENHAM, C.D. & BOLTON, T.B. (1986) J. Physiol., 381, 385–406. BENHAM, C.D., BOLTON, T.B., LANG, R.J., & TAKEWAKI, T. (1986) J. Physiol., 371, 45–67. BLUMENTHAL, E., & KACZMAREK, L.K. (1992) Neurochem. Res., 17, 869–876. BOLTON, T.B. (1979) Physiol. Reviews, 59, 606–718. BOYLE, M.B., AZHDERIAN, E.M., MACLUSKY, N.J., NAFTOLIN, F. & KACMAREK, L.K. (1987) Science, 235, 1221–1224. BRAY, K.M., WESTON, A.H., DUTY, S., NEWGREEN, D.T., LONGMORE, J., EDWARDS, G. & BROWN, T.J. (1991) Br.J. Pharmacol., 102, 337–344. BRAYDEN, J.E. & NELSON, M.T. (1992) Science, 256, 532–535. BUESCHER, P.C., PEARSE, D.B., PILLAI, R.P., LITT, M.C., MITCHELL, M.C. & SYLVESTER, J.T. (1991) J. App. Physiol., 70(4), 1874–1881.

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CHOPRA, L.C., TWORT, C.H.C. & WARD, J.P.T. (1992) Br. J. Pharmacol., 105, 259–260. CLAPP, L.H. & GURNEY, A.M. (1992) Am. J. Physiol., 262, H916-H920. COCKS, T.M., KING, S.J. & ANGUS, J.A. (1990) Br. J. Pharmacol., 100, 375–378. DART, C. & STANDEN, N.B. (1994) J. Vasc. Res., 31 (Suppl 1), 10. DAUT, J., MAIER-RUDOLPH, W., VON BECKERATH, N., MEHRKE, G., GÜNTHER, K. & GOEDEL-MEINEN, L. (1990) Science, 247, 1341–1344. DAVIES, N.W. (1990) Nature, 343, 375–377. DE WEILLE, J.R. & LAZDUNSKI, M. (1990) Ion Channels, Vol 2, Narahashi, T. (ed.). Plenum, New York. pp. 205–221. EDWARDS, F.R. & HIRST, G.D.S. (1988) J. Physiol., 404, 437–454. EDWARDS, G. & WESTON, A.M. (1993) Annu. Rev. Pharmacol. Toxicol., 33, 597–637. ELTZE, M. (1989) Eur.J. Pharmacol., 165, 231–239. FINDLAY, I. (1987) J. Physiol., 391, 611–629. FRECH, G.C., VANDONGEN, A.M.J., SCHUSTER, G., BROWN, A.M. & JOHO, R.H. (1989) Nature, 340, 643–645. GANITKEVICH, V.Y. & ISENBERG, G. (1993) J. Physiol., 470, 35–44. GELBAND, C.H. & HUME, J.R. (1992) Circ. Res., 71, 745–758. GELBAND, C.H., ISHIKAWA, T., POST, J.M., KEEF, K.D. & HUME, J.R. (1993) Circ. Res., 73, 24–34. HAMILTON T.C., WEIR, S.W. & WESTON, A.H. (1986) Br. J. Pharmacol., 88, 103–111. HARA, Y., KITAMURA, K., & KURIYAMA, H. (1980) Br. J. Pharmacol., 68, 99–106. HOSHI, T. & ZAGOTTA, W.N. (1993) Curr. Opin. Neurobiol., 3, 283–290. HO, K., NICHOLS, C.G., LEDERER, W.J., LYTTON, J., VASSILEV, P.M., KANAZIRSKA, M.V. & HEBERT, S.C. (1993) Nature, 362, 31–37. IBBOTSON, T. EDWARDS, G., NOACK, T. & WESTON, A.H. (1993) Br. J. Pharmacol., 108, 991–998. IMAMURA, Y., TOMOIKE, H., NARISHIGE, T., TAKAHASHI, T., KASUYA, H. & TAKESHITA, A. (1992) Am. J. Physiol., 263, (Heart Circ. Physiol. 32), H399-H404. ITOH, T., SEKI, N., SUZUKI, S., ITO, S., KAJIKURI, J., & KURIYAMA, H. (1992) J. Physiol., 451, 307–328. JACKSON, W.F. (1993) Am. J. Physiol., 265 (Heart Circ. Physiol. 34), H1797-H1803. KAJIOKA, S., KITAMURA, K., & KURIYAMA, H. (1991) J. Physiol., 444, 397–418. KOYANO, T., KAKEI, M., NAKASHIMA, H., YOSHINAGA, M., MATSUOKA, T. & TANAKA, H. (1993) J. Physiol., 463, 747–766. KUBO, Y., BALDWIN, T.J., JAN, Y.N. & JAN, L.Y. (1993) Nature, 362, 127–133. LANGTON, P.D., CLAPP, L.H., DART, C., GURNEY, A.M. & STANDEN, N.B. (1993) J. Physiol., 459, 254P. LARSSON, O., ÄMMALA, C., BOKVSIT, K., FREDHOLM, B. & RORSMAN, P. (1993) J. Physiol., 463, 367–389. LEDERER, W.J. & NICHOLS, C.G. (1989) J. Physiol., 419, 193–211. LORENZ, J.N., SCHNERMANN, J., BROSIUS, F.C., BRIGGS, J.P. & FURSPAN, P.B. (1992) J. Clin. Invest., 90, 733–740. LOUTZENHISER, R.D. & PARKER, M.J. (1994) Circ. Res. In press.

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MCCARRON, J.G. & HALPERN, W. (1990) Am. J. Physiol, 259, H902–908. MISLER, D.S., GILLIS, K. & TABACHARANI, J. (1989) J. Memb. Biol., 109, 135–143. MIYOSHI, Y., NAKAYA, Y., WAKATSUKI, T., NAKAYA, S., FUJINO, K., SAITO, K. & INOUE, I. (1992) Circ. Res., 70, 612–616. NEILSEN-KUDSK, J.E. & THIRSTRUP, S. (1991) Eur. J. Pharmacol., 209, 273–275. (1993) Pulmonary Pharmacol, 6(3), 185–192. NELSON, M.T., HUANG, Y., BRAYDEN, J.E., HESCHELER, J., & STANDEN, N.B. (1990) Nature, 344, 770–773. NICHOLS, C.G. & LEDERER, W.J. (1991) Am. J. Physiol, 261, H1675-H1686. NOACK, T., EDWARDS, G., DEITMER, P. & WESTON, A.H. (1992) Br.J. Pharmacol., 107, 945–955. PFRÜNDER, D., ANGHELESCU, I. & KREYE, V.A. (1993) Pflüg. Archiv., 423, 149–151. PONGS, O. (1992) Physiol. Rev., 72 (suppl 4), S69-S88. POST, J.M., HUME, J.R., ARCHER, S.L. & WEIR, E.K., (1992) Am. J. Physiol, 262, C882-C890. POST, J.M., WEIR, E.K., ARCHER, S.L., HUANG, J.M.C. & HUME, J.R. (1993) IUPS XXXII, Proceedings, p. 177 (abstract 281.11/P). QUAST, U. (1993) Trends Pharmacol. Sci., 14, 332–337. QUAST, U. & COOK, N.S. (1989) J. Pharmacol. Exp. Ther., 250, 261–271. QUAYLE, J.M., BONEV, A.D., BRAYDEN, J.E., & NELSON, M.T. (1994) J. Physiol, 475, 9–13. ROBERDS, S.L. & TAMKUN, MM. (1991) Proc. Natl. Acad. Sci. USA, 88,1798–1802. RUSSELL, S.N., SMIRNOV, S.V. & AARONSON, P.I. (1992) Br. J Pharmacol, 105, 549–556. SAMAHA, F.F., HEINEMAN, F.W., INCE, C., FLEMING, J. & BALABAN, R.S. (1992) Am. J. Physiol, 262 (Cell Physiol. 31), C1220-C1227. SILBERBERG, S.D. & VAN BREEMEN, C. (1992) Pflüg. Archiv., 420, 118–120. SMIRNOV, S.V., ROBERTSON, T.P., WARD, J.P.T. & AARONSON, P.I. (1994) Am. J. Physiol., 466, H365-H370. STANDEN, N.B., QUAYLE, J.M., DAVIES, N.W., BRAYDEN, J.E., HUANG, Y., & NELSON, M.T. (1989) Science, 245, 177–180. SWANSON, R., MARSHALL, J., SMITH, J.S., WILLIAMS, J.B., BOYLE, J.B., FOLANDER, K., LUNEAU, C.L., ANTANAVAGE, J., OLIVA, C., BUHROW, S.A., BENNETT, C., STEIN, R.B. & KACZMAREK, L.K. (1990) Neuron., 4, 929–939. TAKUMI, T., OHKUBO, H. & NAKANISHI, S. (1988) Science, 242, 1042–1045. TAYLOR, S.G., FOSTER, K.A., SHAW, D.J. & TAYLOR, J.F. (1989) Br. J. Pharmacol., 98, 881P. TUNG, R.T. & KARACHI, Y. (1991) J. Physiol., 437, 239–256. VON BECKERATH, N., CYRYS, S., DISCHNER, A. & DAUT, J. (1991) J. Physiol, 442, 297–319. WAKATSUKI, T., NAKAYA, Y., & INOUE, I. (1992) Am. J. Physiol, 263, H491–H496. WEISS, J.N. & LAMP, S.T. (1987) Science, 238, 67–69. YUAN, X.-J., GOLDMAN, W.F., TOD, M.L., RUBIN, L.J., & BLAUSTEIN, M.P. (1993) Am. J. Physiol, 264, L116–L123. YUAN, X.J., TOD, M.L., RUBIN, L.G. & BLAUSTEIN, M.P. (1990) Am. J. Physiol, 259 (Heart Circ. Physiol. 28), H281–H289.

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ZHANG, H., STOCKBRIDGE, N., WEIR, B., KRUEGER, C. & COOK, D. (1991) Eur. J. Pharmacol, 195, 27–35.

8 Effects of Potassium Channel Activators in Isolated Blood Vessels U.QUAST Department of Pharmacology, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany.

8.1 Introduction In most cells, the plasma membrane at rest is more permeable to K+ than to Na+, Cl- and Ca2+; therefore, K channels determine the resting membrane potential and, hence, the excitability of the cell. (A notable exception from this rule is skeletal muscle where the resting membrane potential is essentially governed by Cl-channels (Hille, 1992)). The opening of K channels shifts the membrane potential towards the K+ equilibrium potential which is around -90 mV. For smooth muscle, which generally has a resting membrane potential of ― -60mV, this means that opening of K channels hyperpolarizes the tissue; however, the value of -90 mV will only be reached asymptotically as the permeability to K+ becomes absolutely dominating over that of the other ions (Hille, 1992). In excitable cells endowed with depolarization-activated Ca channels (voltage-gated Ca channels, VOCs), hyperpolarization will prevent such channels from opening and, hence, Ca2+ entry via this pathway. In cells devoid of VOCs, e.g. endothelial cells, leukocytes and others, hyperpolarization will, by increasing the driving force for Ca2+ entry into the cell, promote Ca2+ influx via pathways which are active at such (hyperpolarized) membrane potentials. In view of the physiological importance of K channels it is no surprise that this class of ion channels is particularly heterogeneous (for details, see Chapters 6 and 7 of this book or Hille, 1992). Within the large group of voltage-gated K channels, the A channels (KA) open rapidly in response to depolarization and inactivate quite rapidly again if depolarization is sustained. Another, again heterogeneous, group responds to depolarization much more slowly (delayed rectifier K channels, KV); some of these KV channels do not inactivate at all. Both groups of channels let the K+ current pass only out of the cell and are both of great importance for repolarization of the cell; in addition, KA channels are involved in setting the firing frequency of pacemaker cells (Hille, 1992). Another large group of K channels, the inwardly rectifying K channels (KIR), let K+ currents pass much better into the cell than outwards. Such channels have

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been cloned recently from rat kidney (Ho et al., 1993), mouse macrophages (Kubo et al., 1993a) and rat heart (Kubo et al., 1993b). In many cells, KlR channels determine the resting membrane potential (Hille, 1992); this is particularly well documented for cardiac myocytes. The KIR channel cloned from rat heart was opened by muscarinic receptor activation and by activated Glproteins (Kubo et al., 1993b) and is abundantly expressed in the atrium. It therefore most likely represents the K channel which mediates the regulation of cardiac frequency by the vagus nerve. Regulation of K channels by G-proteins and thus by neurotransmission constitutes a very important signalling pathway (Brown, 1990). Two families of K channels have caught the particular interest of the pharmaceutical industry, e.g. the ATP-sensitive K channels (KATP) and, more recently, the large conductance Ca-dependent K channels (BKCa). BKCa channels have a conductance of 100–200 pS under quasi-physiological conditions and form a heterogeneous and almost ubiquitous class of K channels. They open at depolarized membrane potentials in response to elevated intracellular Ca2+ concentrations (for review see McManus, 1991; Hille, 1992); hence they provide an important coupling mechanism between the intracellular Ca2+ level and the membrane potential. BKCa channels are blocked with high affinity by the scorpion toxins, charybdotoxin and iberiotoxin (review: Garcia and Kaczorowski, 1992). Recently, BKCa channels have been cloned from Drosophila and from murine skeletal muscle; their core is structurally related to the voltage-gated (KA and Kv) K channels (Atkinson et al., 1991; Butler et al., 1993). The KATP channels constitute a heterogeneous class of K channels found not only in excitable tissues like pancreatic β -cells, heart, skeletal and smooth muscle, neurons (for reviews see Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993) and the rat adenohypophysis (Bernardi et al., 1993), but also in tissues like kidney epithelium (Wang et al., 1990) or the follicular cells surrounding the Xenopus oocyte (Honoré and Lazdunski, 1991, 1993). In general, KATP channels are closed by ATP binding to an intracellular binding site; they open when ATP dissociates from this site and/or when Mg salts of nucleoside diphosphates (NDP) like MgADP and MgGDP bind to an activatory site which is different from the inhibitory ATP site (Nichols and Lederer, 1991; de Weille, 1992; Edwards and Weston, 1993; in vascular smooth muscle: Pfründer et al., 1993; Beech et al., 1993a). Thus, the opening of KATP channels is regulated by the quotient of ATP/NDP; hence, these channels link the metabolic state of the cell to cellular excitability. The activity of KATP channels is also modulated by phosphorylation (reviews: Ashcroft and Ashcroft, 1990; Nichols and Lederer, 1991; Edwards and Weston, 1993). A characteristic feature of KATP channels is their inhibition by sulphonylureas like glibenclamide with widely varying affinities in different tissues (Quast and Cook, 1989a; Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993). The primary structure of a typical KATP channel is still unknown; the K channel recently cloned from rat kidney

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(Ho et al., 1993) has not been shown to be inhibited by intracellular ATP or the sulphonylureas; however, these properties could be conferred to the channel by an additional, yet unidentified subunit (Nichols, 1993). On the other hand, evidence has been presented to show that the KATP channel in rat portal vein is a dephosphorylated form of the delayed rectifier K channel, Kv (Edwards et al., 1993). In the pancreatic β -cell, KATP channels regulate insulin release in response to the plasma glucose level (which, in turn, determines the ATP concentration in the β -cell near the channel); in most other tissues, they are generally closed and open only when the tissue is metabolically compromised, i.e. the quotient of ATP/NDP falls. In some vascular beds these channels are opened by vasorelaxant neurotransmitters and hormones (see below). This article will focus on the effect of KATP channel activators in isolated vascular preparations. After a short presentation of natural and synthetic activators of vascular KATP channels, the available binding data and the recent progress made in elucidating the K channels opened by these compounds and their mode of activation will be reviewed. This is followed by an analysis of the vasorelaxant profile of the KATP channel activators (KCAs) and of the mechanisms leading to vasorelaxation. The chapter is closed with an overview of recently described activators of the BKCa channel. 8.2 Natural and Synthetic Activators of Vascular KATP Channels In several blood vessels, hormones and neurotransmitters produce a glibenclamide-sensitive hyperpolarization and/or relaxation, suggesting that these effects are mediated by opening of KATP channels, probably via the intermediate activation of a G-protein. Well documented examples are the relaxant effects of adenosine (Daut et al., 1990; Dart and Standen, 1993) and prostacyclin or its analogue iloprost (Jackson et al., 1993) in the coronary vasculature of isolated guinea-pig or rabbit heart preparations or in isolated coronary artery cells. Part of the calcitonin gene-related peptide (CGRP)-induced relaxation in rabbit mesenteric small arteries is due to the opening of KATP channels (Nelson et al., 1990). A detailed mechanistic study in this preparation has shown that CGRP activates adenylate cyclase; the subsequent phosphorylation of KATP channels by cAMP-dependent protein kinase (PKA) induces KATP channel opening (Quayle et al., 1994). The field of synthetic KCAs is described in detail in earlier chapters of this book. It started in 1981 with the discovery that the coronary vasodilator nicorandil, a pyridine with a nitro-group in its side chain, hyperpolarized vascular smooth muscle cells by increasing the K+ conductance of the cell membrane (Furukawa et al., 1981). Shortly thereafter it was found that nicorandil, in addition to its K channel opening action, also acted as an organic nitrate and increased intracellular cGMP (Holzman, 1983). This compound thus possesses two

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independent vasodilator mechanisms and it depends on the vessel under study and on the stimulus as to which of them predominates (Borg et al., 1991; Magnon et al., 1994). The observation that cromakalim (CRK), a compound structurally unrelated to nicorandil and much more potent as a vasodilator, apparently acts solely by opening K channels (Hamilton et al., 1986), represented a major advance. Subsequently, other ‘directly acting’ vasodilators like pinacidil (Bray et al., 1987; Cook et al., 1988a), diazoxide (Quast and Cook, 1989b; Winquist et al., 1989) and minoxidil sulphate, the active metabolite of minoxidil (Meisheri et al., 1988; Winquist et al., 1989) were recognized to act by K channel opening. New KCAs, structurally unrelated to the compounds mentioned above, e.g. the thioformamide aprikalim, have been synthesized (reviews: Atwal, 1992; Evans et al., 1992; Robertson and Steinberg, 1990; Edwards and Weston, 1990a,b; Chapters 1–4 of this book). 8.3 Binding Studies with KATP Channel Activators in Vascular Smooth Muscle With the introduction of the tritiated KCA, 3H-P1075 (Manley et al., 1993), binding studies with the KCAs have become feasible in intact rings of rat aorta (Bray and Quasi, 1992a, Quast et al., 1993), in rat aortic smooth muscle cells in culture and in freshly dissociated canine cardiac myocytes (Dickinson et al., 1993). Using another tritiated KCA, 3H-BAY-X-9228, Hoffmann et al. (1993) found specific KCA binding in rat insulinoma cells. In the vascular preparations, 3H-P1075 binding was found to be of high affinity (K ― 3–6 nM) and relatively D low capacity [Bmax in aortic rings ― 20 fmol/mg wet weight (Bray and Quast, 1992a), in cells ― 5000 sites/cell (Dickinson et al., 1993)]; in cardiocytes, KD was 30 nM and Bmax ― 117 000 sites/cell (Dickinson et al., 1993). 3H-P1075 binding in rat aorta was diminished by metabolic poisoning in parallel with the intracellular ATP concentration [ATP]i. It was, however, not affected by depolarization with 55 mM KCl or by lowering pH from 7.4 to 6.0 (Quast et al., 1993) and hardly influenced by interventions which activate the cAMP signalling chain (Linde et al., 1994). 3H-P1075 binding was inhibited by representatives from all major groups of KCAs and by the sulphonylureas with regular inhibition curves which gave the same rank order of potencies as that obtained in functional assays (Bray and Quast, 1992a; Quast et al., 1993; Dickinson et al., 1993). The potencies of the KCAs determined in the binding assays essentially coincide with those determined in vasorelaxation assays; however, they are about 40 times higher than those determined in the 86Rb+ efflux assay (the latter is a qualitative measure of K channel opening, see below). It was concluded from these studies that the major KCAs bind to the same target, but possibly to different sites at this target, to elicit their effects (Bray and Quast, 1992a; Manley et al., 1993); the quantitative relationship between agonist binding and K channel opening may be complex (Quast et al., 1993). The

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sulphonylurea glibenclamide binds to a site different from and negatively allosterically coupled to the 3H-P1075 binding site (Bray and Quast, 1992a). Since there has been no success in detecting binding of KCAs in broken cell preparations (Dickinson et al., 1993; Hoffmann et al., 1993; Quast et al., 1993), the biochemical characterization of the KCA receptor is still lacking and the question whether the drug receptor of the KCAs is the KATP channel itself or a different protein, remains open. Intriguing results were obtained by Meisheri and colleagues (1991, 1993a) with minoxidil sulphate (MxS). Using two radiolabelled MxS derivatives, 35SMxS and 3H-MxS, these workers found that the sulphate group of MxS remained in the tissue longer than the tritiated piperidine moiety and that MxS covalently sulphated a 116 kD protein in rings from rabbit mesenteric artery and in A7r5 cells, a cell line derived from rat aorta (Meisheri et al., 1991), and in hair follicles from human skin (Meisheri et al, 1993a). This sulphate acceptor appears to be a membrane-associated protein with (an) isoelectric point(s) of 5.2–5.7 (Meisheri et al., 1991; 1993a). The relationship between the sulphation of the 116 kD protein and the KATP channel opening effect of MxS needs further clarification. 8.4 K Channel Opening The electrophysiological effects of the KCAs are described in detail in Chapter 7 of this book; therefore, only a short account of the most recent developments is given here. 8.4.1 Recent Electrophysiological Studies Type of K Channel activated by the KCAs Studies at the single channel level have shown that the KCAs can open a variety of K channels in vascular smooth muscle cells. Several authors have reported that KCAs increased the open probability of BKCa channels in isolated patches derived from vascular smooth muscle cells (Klöckner et al., 1989; Gelband et al., 1989; Hu et al., 1990; Gelband and McCullough, 1993). However, the pharmacological profile of the KCA-induced vasorelaxation differs profoundly from that expected if it was mediated by BKCa channel activators; in particular, differences in the sensitivities to the K channel blockers charybdotoxin, glibenclamide and tetraethylammonium (Winquist et al., 1989; Quast and Cook, 1989b; Quast, 1987) suggest that the BKCa-activating effect of the KCAs is not important physiologically.

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Recent patch-clamp studies converge to show that in vascular smooth muscle cells the KCAs open a K channel with a relatively small conductance (15–20 pS, Kajioka et al., 1990;. 1991; Beech et al., 1993b); this is in contrast with the activation of a large-conductance KATP channel (135 pS) described earlier (Standen et al., 1989). Noise analysis of the K+ current induced by various KCAs in vascular smooth muscle cells also points to a small conductance (10–20 pS) K channel (Beech and Bolton, 1989; Noack et al., 1992a; Ibbotson et al., 1993a; Langton et al., 1993). The pharmacological and biophysical properties of the K+ current elicited by the KCAs in vascular smooth muscle cells are very similar to those of the current induced by depletion of intracellular ATP in these cells (Clapp and Gurney, 1992; Noack et al., 1992b; Silberberg and van Breemen, 1992; Beech et al., 1993b). The finding that the KCAs act on precisely the K channel that opens under ischaemic and hypoxic conditions may explain the therapeutic benefit found with the KCAs in animal models of cardioprotection (Escande and Cavero, 1992) and intermittent claudication (Cook et al., 1993). Mechanisms of K channel opening The mechanism by which the KCAs open the channel in vascular smooth muscle is still a matter of debate. In the absence of KCAs, the channel opens when the concentration of Mg salts of nucleoside diphosphates increases and the ATP concentration falls (Pfründer et al., 1993; Beech et al., 1993a); these changes in intracellular nucleotide concentration occur when the cell is metabolically stressed, e.g. by ischaemia and/or hypoxia. In addition, and as mentioned above in section 8.2, stimulation of cAMP-dependent phosphorylation in intact vascular smooth muscle cells isolated from rabbit mesenteric artery induces a glibenclamide-sensitive K+ current, indicating that this intervention also opens KATP channels (Quayle et al., 1994); similar observations have been made on KATP channels in follicular cells surrounding the Xenopus oocyte (Honoré and Lazdunski, 1991; 1993). The fact that KCAs are able to open KATP channels in isolated patches from vascular smooth muscle cells if appropriate conditions are met (Standen et al., 1989; Kajioka et al., 1990; 1991; Beech et al., 1993b) does, however, not necessarily mean that the KCAs act on the channel directly; the isolated patch may contain proteins (be they integral membrane proteins or tightly associated to the membrane) which might be essential for activation of the channel, e.g. kinases or phosphatases. The KCAs could indeed act on such proteins to open the channel. In recent investigations into the mechanism of activation of KATP channels by the activators in rat portal vein cells, it was found that these compounds inhibited the K+ current (IKv) flowing through the delayed rectifier channel (Kv) and that they simultaneously induced a K+ current (IKATP) flowing through KATP channels such that the sum of the two currents remained approximately constant; both effects were inhibited by glibenclamide (Noack et al., 1992a; Ibbotson et al, 1993a). These effects of the KCAs were mimicked by several measures that

220 K CHANNELS AND THEIR MODULATORS

decrease the phosphorylation state of the cell, in particular by inhibition of protein kinase A (PKA) with a specific protein inhibitor (Edwards et al., 1993). The authors suggested that the KCAs, by a yet unknown mechanism (but not by inhibiting PKA), induce dephosphorylation of the delayed rectifier channel Kv, and thereby convert KV into the activated form of KATP (Edwards et al., 1993). The finding that in rat portal vein, inhibition of PKA induces KATP channel opening is difficult to reconcile with the observation in rabbit mesenteric artery, that stimulation of PKA activates the KATP Channel (Quayle et al., 1994). We have found that in rings of rat aorta dibutyryl-cAMP (Bt2-cAMP), a membranepermeant activator of PKA, enhances the 86Rb+ efflux stimulating effect of levcromakalim (LCRK) (Linde and Quast, 1995; note that 86Rb+ efflux is a qualitative measure of K channel opening, see below). In rat portal vein, the potentiating effect of Bt2-cAMP on CRK-stimulated 86Rb+ efflux was, however, weaker (Quast, 1987), indicating differences in the modulation of KATP channels by PKA in different blood vessels. The hypothesis that dephosphorylation converts Kv into KATP has to accommodate the fact that these channels show a number of differences in their biophysical and pharmacological properties, most notably in their gating properties: Kv are gated by depolarization, due to the voltage-sensing S4 segment in their structure (Jan and Jan, 1992) whereas KATP channels in vascular smooth muscle are not (Edwards and Weston, 1993). The long awaited cloning of the KATP channels will resolve this matter. 8.4.2 Electrophysiological and Tracer Efflux Studies The direct manifestation of K channel opening is a K+ current which, in vascular smooth muscle under physiological conditions, is directed outward, leading to hyperpolarization of the cell. This K+ efflux can also be observed in tracer efflux experiments using 42K+ or 86Rb+. It has been extensively documented that the KCAs induce all these effects in various vascular preparations (whole tissue or isolated cells; for reviews see e.g. Cook and Quast, 1990; Edwards and Weston, 1990b; Edwards et al., 1992; Longman and Hamilton, 1992; Quast et al., 1994; electrophysiological studies: Chapter 7 of this book); therefore only a few comments regarding tracer efflux studies will be made here. Recent experiments show that relatively low concentrations (0.1 μ M) of LCRK or pinacidil induce sizable K+ currents in isolated rat portal vein cells (Noack et al., 1992a) and hyperpolarizations of rabbit mesenteric artery (Itoh et al., 1992). This corresponds well to the detection limit established for these compounds in tracer efflux studies in rat aorta (Quast and Baumlin, 1988; Bray and Quast, 1992b) or rat portal vein (Quast, 1987), so that earlier discrepancies between the results obtained by the two methods have collapsed. Tracer efflux studies with KCAs suffer from several shortcomings. First, they reflect the total K+ efflux from reaction of the whole tissue instead of that from a specific cell. Second, membrane potential is not clamped during the application

EFFECTS OF KCAS IN ISOLATED BLOOD VESSELS 221

of the KCA; hence, as the K+ channels open and hyperpolarization develops, the driving force for the radioisotope to leave the cell is decreased. Hence, the effect of the KCA will be quantitatively underestimated (Videbaek et al., 1990), a bias that is particularly significant at high opener concentrations which induce a substantial hyperpolarization. A further critical point is the use of 86Rb+, which is often preferred to 42K+ for its more convenient half-life and the less penetrating radiation. Since different K channels have a different permeability ratio of 86Rb+ to 42K+, this may lead to further distortions of the flux signal. There is general agreement that under basal conditions, the ratio of 86Rb+ to 42K+ efflux is about 70% in large (rat aorta: Smith et al., 1986) and small vascular preparations (rat mesenteric resistance vessels: Videbaek et al., 1988). Detailed comparison of the 42K+ and 86Rb+ fluxes elicited by CRK in rat portal vein and aorta (Quast and Baumlin, 1988; Bray and Quast, 1991a) have shown that this compound, at low concentrations, opens K channels which are more selective for K+ over Rb+ than those opened at higher concentrations. This seems, however, not to be the case for minoxidil sulphate in rat aorta, where a constant permeability ratio of K+ to Rb + was found over the whole concentration range of the compound (― 40%; Bray and Quast, 1991a); in contrast, other workers found that minoxidil sulphate opened a Rb impermeable channel in rat aorta (Newgreen et al., 1990). Despite the limitations mentioned above it is our experience that 86Rb+ efflux experiments provide useful information on the K channel opening properties of the KCAs, in particular when these compounds are used in the low concentration range. 8.4.3 Inhibitors of KATP Channel Opening Inhibitors of the KATP channel activation have been reviewed previously (Cook and Quast, 1990; see also Robertson and Steinberg, 1990; Atwal, 1992). Here, some quantitative aspects of the sulphonylurea blockers will be discussed and hitherto unpublished data presented; then recent developments in the field of nonsulphonylurea blockers will be discussed. Glibenclamide The long chain sulphonylurea, glibenclamide, is the standard inhibitor of the effects of the KCAs (Cavero et al., 1989; Quast and Cook, 1989b; Winquist et al, 1989; Wilson, 1989; for further references see Cook and Quast, 1990; Meisheri et al., 1993b). It is well established that glibenclamide blocks KATP channels in the pancreatic β -cell at nM concentrations (Zünkler et al., 1988); in vascular smooth muscle, however, concentrations β 100 nM are required to induce substantial inhibition of the vasorelaxant and 86Rb+ efflux stimulating effects of the KCAs (see e.g. Quast and Cook, 1989b and Figure 8.1 below). At higher concentrations, however, glibenclamide has numerous additional effects (e.g.

222 K CHANNELS AND THEIR MODULATORS

Villar et al., 1986; Quast and Cook, 1989b; Cocks et al., 1990; Yoshitake et al., 1991; Zhang et al., 1991; further references in Panten et al., 1989). Thus, care must be taken when it is inferred from the inhibition of a (vasorelaxant) effect of a compound by

EFFECTS OF KCAS IN ISOLATED BLOOD VESSELS 223

224 K CHANNELS AND THEIR MODULATORS

Figure 8.1 (Continued) glibenclamide at high concentrations (e.g. ― μ M), that opening of KATP channels was involved and additional data should be provided in support of this contention. Increasing concentrations of glibenclamide induce a gradual rightward shift in the concentration- 86Rb+ efflux curves obtained with CRK in rat portal vein (Quast and Cook, 1989b). Schild analysis of this data (which showed quantitative deviations from a strictly competitive behaviour) gave a pA2 value of 7.0 for glibenclamide in this preparation. Figure 8.1 shows new data obtained in rat aorta. Panel A compares the inhibition by glibenclamide of P1075-induced 86Rb+ efflux obtained at different P1075 concentrations. With increasing concentration of the agonist, the inhibition curves were shifted rightward and became steeper; the latter would not be expected for a strictly competitive mechanism of inhibition. Cheng-Prusoff analysis (Cheng and Prusoff, 1973) of the IC50 values from these curves gave an approximate Ki value of 18±19 nM for glibenclamide (Figure 8.1A, inset). Qualitatively similar results were obtained for the inhibition of CRK (1 and 3 μ M)-induced 86Rb+ efflux (Figure 8.1B); here, the (tentative) Cheng-Prusoff analysis gave a Ki value of 40 nM for glibenclamide (Figure 8.1B, inset). Since these data show quantitative deviations from the competitive model, the Cheng-Prusoff analysis has only a tentative character. However, the Ki value of 20–40 nM determined here for glibenclamide blocking the open channel (at zero concentration of opener) agrees very well with the inhibition constants (IC50 values) for glibenclamide as an inhibitor of KCA-induced vasorelaxation in rat aorta (Bray and Quast, 1992b; note that the vasorelaxation assays were conducted at low opener concentration). They are, however, 10 times lower than the Ki value of glibenclamide determined in the 3H-P1075 binding assay in rat aorta (Bray and Quast, 1992a; see Quast et al., 1993, for possible explanations). Other sulphonylureas and related insulinotropes The insulinotropic sulphonylureas (and related compounds) are generally grouped into three classes. Class A, the first generation sulphonylureas like tolbutamide and Figure 8.1 Inhibition of KCA-induced 86Rb+ efflux from rat aortic rings by glibenclamide (GBC). A: 86Rb+ efflux was stimulated by superfusion of P1075, (― ), 20 nM for 20 min; (― ), 60 nM for 20 min; (― ), 180 nM for 10 min. The curves were analyzed according to the Hill equation giving the following parameters (from left to right): IC50 (nM) 27± 3, 97±4, 180 ±22; Hill coefficients were 1.22±0.12; 1.58±0.11, 1.77±0.30. The inset shows a plot of the IC50 values versus P1075 concentration (Cheng-Prusoff analysis): Linear regression analysis gave an ordinate intercept (i.e. the affinity of glibenclamide for the open KATP channel in the absence of activator) of 18±19 nM and a slope of 1.1±0.2. B: Cromakalim (CRK) was applied as the KCA: (― ), 0.1 μ M for 20 min; (― ), 1 μ M for 20 min; (― ), 3 μ M for 10 min. The parameters of the inhibition curves were: IC50 values (nM): 40, 76±3, 155±20; Hill coefficients 1.2±0.1, 1.0±0.1. Inset: Linear regression of the

EFFECTS OF KCAS IN ISOLATED BLOOD VESSELS 225

IC50 values versus CRK concentration gives an ordinate intercept of 35±1 nM and a slope value of 0.04±0.0004. Each preparation was stimulated twice with the same concentration of agonist with a washout period of 80–100 min in between. Glibenclamide was applied 20 min before the second stimulation until the end of the experiment and the degree of inhibition was determined by comparing the areas under efflux rate constant versus time curve as described (Bray and Quast, 1991a); n=4–6.

glibornuride are relatively short molecules, class B molecules (second generation) like glibenclamide and glipizide are the long chain sulphonylureas with a greatly increased affinity to the pancreatic KATP channel, and class C compounds like meglitinide (HB 699; Garrino et al., 1985), AZ-DF 265 (Garrino and Henquin, 1988), and UL-DF 9 (Garrino et al., 1986) possess a carboxylic acid group instead of the sulphonylurea group (Rufer and Losert, 1979). Some of these compounds have been examined as inhibitors of the vasorelaxant effects of CRK (Wilson, 1989). We have examined the inhibition of CRK (3 μ M)-induced 86Rb+ efflux from rat aorta by 12 insulinotropic compounds, 4 of each class. The results are listed in Table 8.1 together with the affinity of these compounds to the KATP channel in a rat insulinoma cell line determined in a binding assay (Schmid-Antomarchi et al., 1987). Also listed is the effect of the insulinotropes on basal 86Rb+ efflux from rat aorta as a reflection of their interference with other K+ exchange systems of the cell. It is seen that all insulinotropics, also the non-sulphonylurea derivatives (group C), are able to inhibit cromakalim-induced 86Rb+ efflux, with the (S)-(-) enantiomer of AZ-DF 265 having the second highest potency (most potent: glibenclamide). Figure 8.2 compares the potency of the compounds for binding to the pancreatic KATP channel with that for blocking the channel in the aorta. Considering all compounds, a significant linear correlation (r=0.817) is Table 8.1 Effects of insulinotropes on cromakalim-stimulated and basal 86Rb+ efflux from rat aorta and inhibition of 3H-glibenclamide binding to microsomes from RINm5F cells. Substance

Classa

Inhibition CRKstimulated 86Rb+ effluxb pIC50

Increase in the 3H-GBCC rate pKi constant of basal 86Rb+ efflux, ― k (%) at –log concentr. (M)

(–) AZ-DF 265 (+) AZ-DF 265 glibenclamide glibornuride gliclazide glipizide

C C B A A B

6.17±0.06 5.43±0.09 6.80±0.03 4.90±0.05 4.40±0.08 5.50±0.05

20 at 5.5 5 at 5.0 0 at 5.5 0 at 4.0 0 at 4.0 13 at 4.5

7.8d 6.1d 9.5 7.0 6.2 9.1

226 K CHANNELS AND THEIR MODULATORS

Substance

Classa

Inhibition CRKstimulated 86Rb+ effluxb pIC50

Increase in the 3H-GBCC rate pKi constant of basal 86Rb+ efflux, ― k (%) at –log concentr. (M)

gliquidone B 5.20±0.10 6 at 4.2 9.0 glisoxepide B 5.00±0.05 0 at 4.0 7.9 glymidine A 3.30±0.20 30 at 3.0 5.2 meglitinide C 5.30±0.05 8 at 4.3 6.6d tolbutamide A 3.50±0.30 10 at 3.0 5.0 UL-DF 9 C 4.00±0.02 0 at 3.5 5.0d aClassification as A: short (first generation), B: long (second generation) sulphonylurea; C: carboxylic acid containing compound. b pIC = negative logarithm of the midpoint (IC50, M) of the inhibition curve of CRK(3 50 μ M)-stimulated 86Rb+ efflux from rat aorta. c pK = negative logarithm of the midpoint (IC , M) of the 3H-glibenclamide (3H-GBC) i 50 displacement curve in microsomes from RINm5F cells after correction for the concentration of the radiolabel (0.3 or 0.1 nM). Values from SchmidAntomarchi et al. (1987). d M. Fosset, personal communication.

EFFECTS OF KCAS IN ISOLATED BLOOD VESSELS 227

Figure 8.2 Relationship between the inhibition by insulinotropes of CRK-stimulated 86Rb + efflux from rat aorta and of 3H-glibenclamide (3H-GBC) binding to RINm5F cells. The data are taken from Table 8.1; (― ), class A compounds; (O), class B; (― ), class C. Linear correlation analysis for all compounds together (—) and for the 3 subgroups separately (…) gave the following results (n=number of compounds, s=slope, r=correlation coefficient, SL=significance level of fit). Group

n

s

r

SL

all

12

0.52±0.12

0.81

0.001

A

4

0.79±0.12

0.98

0.02

B

4

0.90±0.54

0.76

0.23

C

4

0.74±0.17

0.95

0.03

found. The slope of 0.52 in the double logarithmic correlation indicates that the changes in molecular structure affect the affinity for the pancreatic channel more than that for the vascular channel, reflecting the fact that these compounds were optimized for the channel in the β -cell. When a subgroup analysis is made, a good correlation is found for the members of class A or C, whereas the

228 K CHANNELS AND THEIR MODULATORS

correlation is less good for the class B compounds. This data suggests that the sulphonylurea receptor in the aorta is related to, but different from its counterpart in the insulinoma cell. Other blockers Symmetrical tetra-n-alkylammonium ions These are well known K channel blockers (see e.g. French and Shoukimas, 1981; for tetraethylammonium: Stanfield, 1983). We have investigated such compounds as inhibitors of cromakalim-induced 86Rb+ efflux from rat aorta (Quast and Webster, 1989). The inhibition curves (Figure 8.3) show that the potency of these compounds increased with chain length from the tetramethylhomologue (IC50 ― 20mM) up to the n-pentyl-homologue (IC50 ― 0.1 μ M). Increasing chain length further decreased potency; in addition, the hexyl- and heptyl-homologues at higher concentrations and the octyl-compound at all concentrations tested increased basal 86Rb+ efflux from the organ, probably by depolarizing the preparation. In order to minimize this effect, these experiments were conducted in the presence of the dihydropyridine Ca entry blocker, isradipine (0.1 μ M). Even then, the octyl-homologue no longer gave any block (Figure 8.3). The data show that the tetrabutyl- and pentylammonium compounds are potent blockers of the vascular KATP channel without affecting basal K+ exchange mechanisms. The data suggest that the tetraalkylammonium site of the KATP channel has a strong preference for bulky, lipophilic compounds with a sharp cut-off at chain length ― 7 (n-heptylammonium). The increase in blocking potency with increasing chain length of tetra-n-alkylammonium ions has also been observed for the K+ conductance of squid axon (French and Shoukimas, 1981).

EFFECTS OF KCAS IN ISOLATED BLOOD VESSELS 229

Figure 8.3 Symmetrical tetraalkylammonium ions (TAA) as inhibitors of CRK (3 μ Minduced 86Rb+ efflux from rat aorta. The inhibition curves are numbered according to chain length from 1 (tetramethylammonium) to 8 (tetraoctylammonium). The curves were analyzed according to the Hill equation yielding the parameters listed in the inset; plC50= -log(IC50, M) and nH=Hill coefficient. The data were obtained as described in Figure 8.1; from the hexyl- to the octyl-compound, experiments at concentrations ― 10 μ M were performed in the presence of isradipine (0.1 μ M) in order to minimize the increase in 86Rb + efflux induced by these compounds at high concentrations.

Imidazolines, guanidines and structurally related blockers Recently, a growing number of inhibitors of the KCAs has been described which contain an imidazoline or a guanidinium group, the most prominent of them being the β -adrenergic blockers, phentolamine and antazoline and the bradycardic agent, alinidine (McPherson and Angus, 1989). In the higher micromolar range, this group of compounds produces a non-competitive inhibition of the vasorelaxant effects of CRK (McPherson and Angus, 1989; Ibbotson et al., 1993b); similar concentrations (30 μ M) completely prevent the KATP channel opening in rat portal vein cells (Ibbotson et al., 1993b). Similar data were obtained for the guanidine, guanabenz (Ibbotson et al., 1993b) and the imidazoisoindole, ciclazindol (Noack et al., 1992c). The guanidinium-related compound U-37883A, an amantadine derivative containing a carboximidine moiety, also inhibits the vasorelaxant and 42K+ efflux stimulating effects of several KCAs in the μ M range (Meisheri et al., 1993c). Interestingly, this compound, at low concentrations that do not yet inhibit the KCA-induced vasorelaxation, augments the inhibitory potency of glibenclamide and vice versa. Hence, these two compounds display a functional synergy as inhibitors of the KCAs, suggesting that they interfere with different steps of the signal chain which mediates the KCA-induced vasorelaxation (Ohrnberger et al., 1993). Blockers that inhibit KCA-induced channel opening more than vasorelaxation Several chemically unrelated molecules have been found to inhibit the K channel opening effect of the KCAs in vascular tissue without much inhibition of vasorelaxation. This was first observed with the sparteine derivative, tedisamil (Bray and Quast, 1991b; 1992b), a K channel blocker (KCB) with bradycardic and class III antiarrhythmic actions (Beatch et al., 1991); a similar profile was found with the inorganic KCBs, Rb+ (Foster et al., 1992; Greenwood and Weston, 1993), and Ba2+ (Quast and Bray, 1991; Quast et al., 1995). [Note that Rb+, which is used in μ M concentrations as a convenient substitute for 42K+ in tracer flux experiments (see section 8.4.2, above), is a general KCB at mM concentrations (Greenwood and Weston, 1993)]. It appears from these studies

230 K CHANNELS AND THEIR MODULATORS

that the majority of KATP channels can be blocked by the inhibitor without substantially impairing the vasorelaxant potency and efficacy of the KCA; at higher degrees of channel block, however, the KCA-induced vasorelaxation becomes transient or even disappears (Foster et al., 1992; Greenwood and Weston, 1993; Quast et al., 1994). The preferential inhibition of the KCAinduced channel opening by these compounds is in sharp contrast with the behaviour of glibenclamide, which inhibits both the K channel opening and the vasorelaxant effects of the KCAs at similar concentrations (Quast and Cook, 1989b; note that due to the quasi-competitive inhibition pattern of glibenclamide shown in Figure 8.1, similar KCA-concentrations are used to measure channel opening and relaxation). Taken together, these data suggest that the relationship between the channel opening and vasorelaxant effects of the KCAs is not as simple as originally thought (see below, 8.6). 8.5 Vasorelaxant Properties of the KATP Channel Activators The KCAs are potent vasorelaxants with a quite characteristic profile (Longman and Hamilton, 1992; Cavero and Guillon, 1993; Cook and Quast, 1990; Edwards and Weston, 1990b; Quast, 1993). First, their vasorelaxant effect is abolished in media containing high (>50 mM) concentrations of K+ (Hamilton et al., 1986). This is a direct consequence of their mechanism of action since depolarization by a high K+ solution results in a shift in the membrane potential towards the K+ equilibrium potential (see e.g. Furukawa et al., 1981) and the opening of additional K channels by the KCA will not significantly affect membrane potential. Figure 8.4 examines the ability of LCRK to relax rat aortic rings precontracted by different concentrations of KCl in the bath. Considering only the data up to 10 μ M, it is seen that with increasing KCl the amount of tension that can be relaxed by LCRK diminishes and is almost 0 at 55 mM KCl; in addition there is a gradual rightward shift of the relaxation curve. Thus, KCl is a non-competitive inhibitor of the vasorelaxant effect of LCRK, which is entirely expected from the changes in membrane potential and of the K+ equilibrium potential with KCl concentration, and from the known voltage-dependence of the L-type Ca channels (Hille, 1992; Furukawa et al., 1981; Häusler, 1983). At high concentrations of LCRK (>10 μ M), there is another component of relaxation which we have not characterized further and which may be nonspecific (Figure 8.4). The KCAs also relax contractions to agonists which mobilize intracellular Ca2 + via the inositoltrisphosphate (IP ) pathway (Cook et al., 1988b; reviews: 3 Edwards et al., 1992; Quast 1993) with a potency approximately similar to that against low concentrations of KCl (for LCRK compare Table 8.2 and Figure 8.4). We have

EFFECTS OF KCAS IN ISOLATED BLOOD VESSELS 231

Table 8.2 Comparison of the vasorelaxant potency of KATP channel activators under polarized and depolarized conditions in rat aortic ringsa Substance

Polarized EC50, (μ M)

Depolarized EC50, (μ M)

Selectivity ratio

levcromakalim 0.03 180 6000 (+)enantiomer 6.3 >1000 >160 SDZ PCO 400 0.04 16 400 (+)enantiomer 16 >300 >19 pinacidil (racemate) 0.06 22 367 (–)enantiomer 0.04 13 310 (+)enantiomer 0.32 39 122 P1075 0.008 >100 10000 diazoxide 7 >250 >36 RP 49356 0.2 >100 >500 a Rat aortic rings were contracted by noradrenaline (0.1 μ M) under polarized (5 mM KCl) or depolarized conditions (55 mM KC1, NaCl reduced from 120 to 70 mM, in the presence of the dihydropyridine Ca2+ blocker, isradipine (0.1 μ M) to inhibit KCl-induced tension (Cook et al., 1988b)). Concentration-relaxation curves were obtained cumulatively; maximum tension was similar under either condition (1.0 to 1.2 g). EC50=midpoint of concentration relaxation curve; Selectivity ratio=EC50 (depolarized)/EC50 (polarized).

232 K CHANNELS AND THEIR MODULATORS

Figure 8.4 Relaxation of KCI-induced contractions of rat aorta by LCRK. The concentration-relaxation curves were obtained cumulatively in a buffer where NaCl was reduced from 120 mM as KCI was increased above 5 mM. The data were subjected to Hill analysis (for two components where necessary). The maximum tension (Fmax) and the parameters of the high affinity component are listed in the following table (A, maximum relaxation in % of Fmax; EC50, midpoint of relaxation curve in nM; nH, Hill slope). KCI (mM)

Fmax (mg)

A (%)

EC50 (nM)

nH

20 700 ± 150 94 ± 2 14 ± 1 1.5 ± 0.1 25 1310 ± 100 96 ± 2 28 ± 2 1.5 ± 0.1 30 1600 ± 210 81± 1 70 ± 2 1.8 ± 0.1 35 1650 ± 106 36 ± 1 69 ± 1 1.9 ± 0.1 40 1750 ± 114 16 ± 1 123 ± 7 2.3 ± 0.3 55 1800 ± 200 6±1 130 ± 100 2±1 Analysis of the low affinity component at 35 and 55 mM KCI gave the following parameters: A (%) = 50 ± 1, 87 ± 2; EC50 (μ M) = 229 ± 2, 272 ± 8; nH = 2.0 ± 0.1, 1.7 ± 0.1.

compared the potency of some KCAs against noradrenaline under normal (5 mM KCI) and depolarized (55 mM KCI) conditions, the latter in the presence of the Ca antagonist, isradipine (0.1 μ M), in order to have comparable maximum

EFFECTS OF KCAS IN ISOLATED BLOOD VESSELS 233

tension in the absence of KCA. The results are listed in Table 8.2 and allow the calculation of a selectivity ratio which indicates the concentration range over which the KCA acts solely by the K channel opening mechanism. This selectivity is lowest for pinacidil and its enantiomers which have repeatedly been shown to have vasorelaxant mechanisms in addition to KATP channel activation (Cook and Quast, 1990; Xiong et al., 1991; Itoh et al., 1991). A further characteristic trait is the inhibition of the KCA-induced vasorelaxation by the sulphonylureas, in particular glibenclamide (see above). The fact that glibenclamide acts with similar potency against structurally quite diverse KCAs (see e.g. Meisheri et al., 1993b) provides additional support for the conclusion from binding studies (Bray and Quast, 1992a) and electrophysiological investigations (Ibbotson et al., 1993a) that these compounds have a common pathway leading to KATP channel activation and vasorelaxation. 8.6 Mechanism of KATP Channel Activator-induced Vasorelaxation The mechanisms by which the KCAs induce vasorelaxation have been reviewed recently (Quast, 1993). The existing evidence suggests that these compounds relax vascular smooth muscle primarily by opening K channels. The ensuing hyperpolarization clamps the cell at sufficiently negative values to prevent depolarization-induced Ca2+ entry by voltage-sensitive Ca channels (Hamilton et al., 1986). In this respect the KCAs may be looked at as indirect Ca channel blockers. In addition, the KCAs have recently been shown to inhibit the agonistinduced accumulation of IP3 and thus to interfere with agonist-stimulated Ca2+ mobilization from intracellular stores (Ito et al., 1991; Itoh et al., 1992; Yamagishi et al., 1992); they also reduce the intracellular Ca2+ concentration in vascular smooth muscle at rest in both Ca2+ -containing and Ca2+ -free media (Ito et al., 1991; Itoh et al., 1992; Yamagishi et al., 1992) and decrease the Ca2+ -sensitivity of the contractile elements in strips of canine coronary artery (Okada et al., 1992; 1993). All these effects are abolished in depolarizing medium and reversed by glibenclamide suggesting that they are somehow linked to the ability of the KCAs to hyperpolarize the cell membrane by opening KATP channels in the plasmalemma. The precise mechanism by which hyperpolarization of the cell membrane regulates these phenomena remains to be established; however, in recent investigations in isolated cells from guinea-pig coronary artery it was clearly demonstrated that membrane potential modulates the acetylcholineinduced Ca2+ transient, and indirect but strong evidence suggested that this was due to a modulation of acetylcholine-stimulated IP3 liberation (Ganitkevitch and Isenberg, 1993). The ability of the KCA to inhibit both the agonist-induced mobilization of Ca2+ from intracellular stores and the increase in Ca2+ sensitivity of the contractile apparatus predicts that the vasodilator profile of these compounds will differ from that of the Ca2+ antagonists and explains earlier

234 K CHANNELS AND THEIR MODULATORS

findings that the KCAs relax contractions which do not depend on depolarizationinduced Ca2+ entry (Cook et al., 1988b; Quast and Baumlin, 1991, further references in Quast, 1993). The studies with the KCBs tedisamil Ba2+ and Rb+ have demonstrated that the large majority of the K channels opened by the KCAs can be blocked without much effect on vasodilatation (see section 8.4.3). Despite the fact that these compounds are relatively nonselective KCBs and have many additional actions (see references above), these studies strongly suggest that LCRK and other KCAs, but minoxidil sulphate to a lesser degree (Bray and Quast, 1992b; Greenwood and Weston, 1993) possess mechanisms of vasorelaxation, independent of plasmalemmal KATP channel opening. The characteristic profile of the KCA-induced vasorelaxation requires these mechanisms to be abolished by depolarization and to be sensitive to inhibition by glibenclamide. One such mechanism is the (partial) inhibition of intracellular Ca2+ store refilling, produced by LCRK in vascular tissues (Cowlrick et al., 1988; Bray et al., 1991; Greenwood and Weston, 1993). In this context it is important to remember that the mode of channel activation by the KCAs is not yet elucidated (see section 8.4.1 above). If the KCAs, by binding to their target, are able to interfere with phosphorylation phenomena (Edwards et al., 1993), this could open multiple pathways leading to vasorelaxation, the most important of them being the opening of KATP channels. Such an indirect mechanism of action could also resolve the paradoxical observation that the concentrations of LCRK which induce 50% vasorelaxation are generally three to five times lower than those required to detect the direct manifestations of K channel opening (see Quast, 1993, for references). 8.7 BKCa Channel Activators As mentioned in the Introduction, the BKCa channels provide an important negative feedback mechanism between membrane potential and [Ca2+]i in many excitable cells. The availability of high affinity toxins which block BKCa channels, e.g. charybdotoxin and iberiotoxin, has helped to recognize the fundamental role that these channels play in many tissues (review: Garcia and Kaczorowski, 1992); in arterioles, they are major regulators of tone (Brayden and Nelson, 1992). In addition to the gating of these channels by depolarization the activity of some BKCa channels is modulated by cAMP-dependent phosphorylation (Kume et al., 1989; Sadoshima et al., 1988), G-proteins (Gsβ ; Scornik et al., 1993), the guanine nucleotides GMP and cGMP (Williams et al., 1988) and other factors (review: Armstrong and White, 1992). In view of the physiological role of BKCa channels, tissue-selective openers of BKCa channels can be expected to be of great therapeutic interest. Recently, three classes of molecules have been described that act as activators of BKCa channels. The imidazopyrazine SCA40 has been shown to completely relax rings from

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guinea-pig trachea (Laurent et al., 1993) and rat aorta (Michel et al., 1993) contracted by 20 mM KCl, but was a much weaker and less efficacious relaxant against contractions to 80 mM; the relaxant activity in guinea-pig trachealis against 1 μ M carbamylcholine was competitively inhibited by charybdotoxin (Laurent et al., 1993). This is indeed the pharmacological profile expected for a BKCa channel activator; however, this data can also be explained on the basis of functional antagonism (Huang et al., 1993; Cook et al., 1995) and definite proof of the proposed mechanism of action has to come from electrophysiological experiments. In guinea-pig trachea SCA40 caused full suppression of spontaneous tone without a change in membrane potential and it was concluded that K channel opening was not important in this tissue (Cook et al., 1995). The situation is the opposite with the benzimidazolones, NS004 (Olesen et al., 1993;1994a) and NS1619 (Olesen et al., 1994b). Electrophysiological data clearly show that these compounds, at micromolar concentrations, are effective activators of BKCa channels in a variety of cell types (bovine aorta and coronary artery; mouse cerebellar granule cell and cortical neuron; rat pancreatic β -cell). NS1619 activates the channel in bovine aorta by shifting the voltage-activation curve towards more negative values, i.e. it mimicks the effect of an increased [Ca2+]i, but it cannot activate the channel in the virtual absence of Ca2+ (Olesen et al., 1994b). However, in rat aorta, NS004 relaxed contractions to 110 mM KCl with a similar potency (― 10 μ M) as those to phenylephrine (0.3 μ M); the relaxation was not inhibited by iberiotoxin (50 and 200 nM) (Sargent et al., 1993). This rules out a significant contribution of BKCa channel activation to the observed vasorelaxation which may be due, at least in part to the inhibition of (Ltype-) Ca channels produced by NS004 at μ M concentrations (Sargent et al., 1993; Olesen et al., 1994a). However, the complete relaxation of phenylephrineinduced contractions of rat aorta (note that these depend largely on the mobilization of intracellular Ca2+) requires the presence of a vasorelaxant mechanism other than Ca channel inhibition; but as stated above, this mechanism cannot be an opening of K channels. Most interestingly, NS004, at submicromolar concentration, is able to activate the cystic fibrosis transmembrane transport regulator (CFTR), even in its mutated form (― F508; this mutation causes the most prevalent form of the disease) (Gribkoff et al., 1994). This effect, the mechanism of which needs further clarification and which is probably unrelated to the aforementioned properties of the molecule, offers exciting therapeutic possibilities (Gribkoff et al., 1994). A third class of exogenous compounds which have been shown to activate BKCa channels are the soyasaponins (McManus et al., 1993). These compounds, isolated from a medicinal herb which is used in Ghana as a spasmolytic in the treatment of asthma, activate the channel from the inside of the cell in the presence of Ca2+ and inhibit charybdotoxin binding by a negative allosteric interaction. Some of the compounds act at quite low concentrations (from 10 nM on); however, their poor membrane permeation limits their therapeutic use

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(McManus et al., 1993) and obviously prevents a direct assessment of these compounds in functional tests. 8.8 Conclusion In recent years there has been considerable progress in the field of the KATP channel activators, in particular the long standing controversy as to which channel is the principal mediator of the effects of the KCAs in smooth muscle has been settled in favour of a KATP channel of ― 20 pS conductance. The KATP channel has eluded cloning and expression efforts up to now but, undoubtedly, this problem will be overcome. This will probably also solve the question of how the KCAs activate the channel, whether by a direct or an indirect mechanism. If the existing KCAs have not yet fully lived up to the original therapeutic expectations (often due to limited tissue specifity) they have been important tools in examining the effects of hyperpolarization on smooth muscle. The emerging field of BKCa channel activators which is again pioneered by the pharmaceutical industry deserves greatest attention. The activators already available leave ample space for improvement; hopefully compounds will be found in the area of the BKCa channel activators which serve both the clinician and the pharmacologist. Acknowledgement I am grateful to Dr Michel Fosset, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice-Sophia Antipolis, for determining the binding affinity of some sulphonylureas. Own unpublished work reported here was performed at Sandoz Pharma, Basel, Switzerland with the excellent technical assistance of Y.Baumlin and J.Dosogne. I thank C.Linde, C.Löffler and F.Metzger (Tübingen) for artwork and help with the manuscript. References ARMSTRONG, D.L. & WHITE, R.E. (1992) Trends Neurosci., 15, 403–408. ASHCROFT, S.J.H. & ASHCROFT., F.M. (1990) Cell. Signal., 2, 197–214. ATKINSON, N.S., ROBERTSON, G.A. & GANETZKY, B. (1991) Science, 253, 551–555. ATWAL, K.S. (1992) Med. Res. Rev., 12, 569–591. BEATCH, G.N., ABRAHAM, S., MCLEOD, B.A., WALKER, M.J.A. & YOSHIDA, N.R. (1991) Br. J. Pharmacol, 102, 13–18. BEECH, D.J. & BOLTON, T.B. (1989) Br. J. Pharmacol., 98, 851–864. BEECH, D.J., ZHANG, H., NAKAO, K. & BOLTON, T.B. (1993a) Br. J. Pharmacol., 110, 573–582. (1993b) Br.J. Pharmacol., 110, 583–590. BERNARDI, H., DE WEILLE, J.R., EPELBAUM, J., MOURRE, C., AMOROSO, S., SLAMA, A., FOSSET, M. & LAZDUNSKI, M. (1993) Proc. Natl. Acad. Sci. USA, 90, 1340–1344.

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SCHMID-ANTOMARCHI, H., DE WEILLE, J., FOSSET, M. & LAZDUNSKI, M. (1987) J. Biol. Chem., 262, 15840–15844. SCORNIK, F.S., CODINA, J., BIRNBAUMER, L. & TORO, L. (1993) Am. J. Physiol., 265, H1460–H1465. SILBERBERG, S.D. & VAN BREEMEN, C. (1992) Pflügers Arch., 420, 118–120. SMITH, J.M., SANCHEZ, A.A. & JONES, A.W. (1986) Blood Vessels, 23, 297–309. STANDEN, N.B., QUAYLE, J.M., DAVIES, N.W., BRAYDEN, J.E., HUANG, Y. & NELSON, M.T. (1989) Science, 245, 177–180. STANFIELD, P.R. (1983) Rev. Physiol. Biochem. Pharmacol., 97, 1–67. VIDEBÆK, L.M., AALKJÆR, C. & MULVANY, J. (1988) Br.J. Pharmacol, 95, 103–108. VIDEBÆK, L.M., AALKJÆR., C., HUGHES, A.D. & MULVANY, M.J. (1990) Am. J. Physiol.,28,H14–H22. VILLAR, A., D'ZOCON, M.P. & ANSELMI, E. (1986) Arch. Int. Pharmacodyn., 279, 248–257. WANG, W., SCHWAB, A. & GIEBISCH, G. (1990) Am. J. Physiol., 259, F494–F502. WILLIAMS, D.L. JR., KATZ, G.M., ROY-CONTANCIN, L. & REUBEN, J.P. (1988) Proc. Nat. Acad. Sci. USA, 85, 9360–9364. WILSON, C. (1989) J. Auton. Pharmac., 9, 71–78. WINQUIST, R.J., HEANEY, L.A., WALLACE, A.A., BASKIN, E.P., STEIN, R.B., GARCIA, M.L. & KACZOROWSKI, G.J. (1989) J. Pharmacol. Exp. Ther., 248, 149–156. XIONG, Z., KAJIOKA, S., SAKAI, T., KITAMURA, K. & KURIYAMA, H. (1991) Br. J. Pharmacol., 102, 788–790. YAMAGISHI, T., YANAGISAWA, T. & TAIRA, N. (1992) Naunyn-Schmiedeberg's Arch. Pharmacol., 346, 691–700. YOSHITAKE, K., HlRANO, K. & KANAIDE, H. (1991) Br. J. Pharmacol, 102, 113–118. ZHANG, H., STOCKBRIDGE, N., WEIR, B., KRUEGER, C. & COOK, D. (1991) Eur. J. Pharmacol., 195, 27–35. ZÜNKLER, B.J., LENZEN, S., MANNER, K., PANTEN, U. & TRUBE, G. (1988) Naunyn-Schmiedeberg's Arch. Pharmacol., 337, 225–230.

9 In Vivo Vascular Effects of Potassium Channel Activators J.C.CLAPHAM Department of Vascular Biology, SmithKline Beecham Pharmaceutical, The Frythe, Welwyn, Hertfordshire, AL6 9AR, UK. 9.1 Introduction This review will address the vascular changes that occur in whole animals following activation of ATP-sensitive potassium channels KATP by the potassium channel activators (KCAs). The molecular mechanisms involved have already been discussed in Chapters 6 to 8. Indeed, there have already been many comprehensive reviews on the KCAs and some have included the activity of these agents in vivo (Richer et al., 1990b; Edwards et al., 1992; Longman and Hamilton, 1992). Whilst previous reviews have tended to cover the broader issues, I intend to restrain this chapter to the purely vascular aspects of KCAs in both anaesthetised and conscious animal models. 9.2 Acute Blood Pressure Studies Studies on the antihypertensive effects of KCAs have made extensive use of the spontaneously hypertensive rat (SHR; Table 9.1). These drugs were also very effective blood pressure (BP) lowering agents in other models of hypertension such as the deoxycorticosteroid (DOCA)/NaCl rat (Aloup et al., 1990; Morin et al., 1990), the Goldblatt (renal) rat (Aloup et al., 1990; Morin et al., 1990) and the Dahl salt-sensitive rat (Hirawa et al., 1989, 1992). Efficacy was also demonstrated in models of renal hypertension in non-rodent species such as the cat (Clapham et al., 1991a) and dog (Schliep et al, 1989; Clapham et al., 1991a; Nakajima et al., 1992). The ability of the KCAs to reduce arterial pressure in several species is now well established (Buckingham, 1989; Edwards et al., 1992; Longman and Hamilton, 1992) and accepted as being the principal measurable response in an animal. Despite this apparently universal BP lowering effect, differences in time course have emerged with the introduction of more compounds of this class (Table 9.1). In the SHR, aprikalim (RP49356, Aloup et al., 1990), levcromakalim

IN VIVO VASCULAR EFFECTS OF KCAS 243

(LCRK) (Clapham et al., 1991a), NIP-121 (Masuda et al., 1991) and HOE 234 (Linz et al., 1992), for example, elicit acute antihypertensive responses that are rapid in onset, their maximum effect Table 9.1 Oral antihypertensive effects of some KCAs in conscious SHR KCA

Dose-range mg.kg–1

HR

Time to max

Duration

Ref

HOE-234 0.1–1.0 20 min ~3h [1] bimikalim 0.0361 * * ~4h [2] levcromakalim 0.038–0.15 20 min ~4h [3] NIP–I21 0.013–0.05 1.5h ~4h [4] SDZ PCO400 0.1–0.3 <15 min >5h [5] Ro 31–6930 0.01–0.1 <30 min >8h [6] 1 celikalim 0.25 2–6 h ~24 h [7] Y–27152 0.1–0.3 7h ~24 h [8] aprikalim 0.1–1.0 * <30 min >5h [9] 1 Indicates dose calculated to produce a 30 mmHg reduction in arterial pressure. * data not given,

occurring 20 min following an oral dose. Thereafter, BP returns towards control levels with the effect lasting around 3 h. Ro 31–6930 (Paciorek et al., 1990) exhibits a similarly rapid onset of action, but the antihypertensive response persists for up to 8 h. However, at least two new KCAs have emerged with strikingly different profiles in the SHR. Celikalim (WAY–120,491; Morin et al., 1990) and Y–27152 (Nakajima et al., 1992) have a very slow onset of action, taking up to 7 h to reach a maximum fall in BP with a duration of action claimed to be 24 h. It appears, from one study at least, that the duration of action of the benzopyranrelated KCAs in SHR is related to the intrinsic lipophilicity of these compounds (Soll et al., 1991). These authors demonstrated that a long duration of action was associated with increased lipophilicity, regardless of potency, in a study comparing cromakalim (CRK), LCRK, EMD–52692, Ro 31–6930, SDZPCO 400, NIP–121, celikalim and a number of analogues of celikalim. However, the slow onset of action of Y–27152 was related to the pro-drug nature of this compound (Nakajima et al., 1992). Y–27152, which is inactive in vitro, is converted in vivo to the active desbenzyl form, Y–26763, by a cytochrome P450dependent mechanism (Nakajima et al., 1992). In contrast, the antihypertensive effects of LCRK are mediated by unchanged parent compound (Clapham et al., 1993).

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In non-rodent models of hypertension the duration of action of the antihypertensive response to LCRK was slightly prolonged (~5 h) in the renal hypertensive cat and dog (Clapham et al., 1991a) such that the duration in these models is similar to that of Ro 31–6930 (Paciorek et al., 1990). In contrast, the difference in the duration of action between LCRK and Y–27152 is maintained in the conscious hypertensive dog (Nakajima et al., 1992). 9.3 Chronic Blood Pressure Studies The majority of the published haemodynamic data on KCAs report their acute actions. In addition there are a number of studies where antihypertensive effects have been monitored after repeat dosing. In the studies listed in Table 9.2 there were no signs of tolerance to the antihypertensive effects of these KCAs. Since the dosing regimen used in these studies was once daily dosing, lack of tolerance may not be altogether surprising. The short duration of action in SHR of some of the compounds could reflect the sum of a number of acute challenges. However, reproducible antihypertensive responses were obtained for the longer acting KCAs such as Ro 31–6930 (Paciorek et al., 1990) and Y–27152 (Nakajima et al., 1992) and in Dahl salt-sensitive rats with LCRK (Hirawa et al., 1989). In addition, an antihypertensive response was still evident in SHR exposed to dietary incorporation of LCRK (resulting in a drug intake of around 0.35 mg.kg– 1day–1) for up to 6 weeks (Hamilton et al., 1993). Thus, there is no evidence that tolerance develops to the antihypertensive activity induced by KATP -channel activation in animal models of hypertension. When administered to young SHR (4 weeks of age), the KCAs CRK and bimikalim (EMD52692 or SR44866), fail to prevent the development of genetic hypertension despite exhibiting favourable haemodynamic effects (Mulder et al., 1989). In the Dahl-salt sensitive rat, LCRK reduces arterial pressure concomitant with a reduction in aortic thromboxane A2 (TXA2) formation both in vivo and in vitro (Hirawa et al., 1992). Furthermore, since TXA2 may play a role in smooth muscle cell growth and proliferation (Ishimitsu et al., 1988), this would give a theoretically favourable effect with respect to vascular hypertrophy. However, studies in this area are relatively few and more will be required to establish a consensus as to whether KCAs have real potential in vascular remodelling. When the KCAs bimikalim and CRK (Mulder et al., 1989) and LCRK (Clapham et al., 1991b) are administered once daily to SHR, heart weight/body weight ratio remains unchanged indicating that the compounds do not reverse cardiac hypertrophy in the SHR. Furthermore, these data are in agreement with earlier work where pinacidil failed to reverse cardiac hypertrophy in the SHR despite an aggressive BP lowering regimen (Jespersen et al., 1986). On the other hand, there was no induction of further work-related cardiac hypertrophy.

IN VIVO VASCULAR EFFECTS OF KCAS 245

Table 9.2 Repeat oral dose studies of some KCAs in conscious hypertensive animals KCA

Dose mg.kg-1 day-1

Species

Duration days

Tolerance

Ref

cromakalim 0.2 RHD 28 no [1] HOE-234 2.0 SHR 5 no [2] levcromakal 0.05 & 0.1 SHR 28 no [3] im levcromakal 0.5 DAHL 14 no [4] im levcromakal 6 ppm in SHR 42 no [5] im diet1 NIP– 121 0.025–0.1 SHR 15 no [6] SDZ 0.1 SHR 4 no [7] PCO400 Ro 31–6930 0.03 SHR 22 no [8] Y–27152 0.1 RHD 56 no [9] 1 Equivalent to approximately 0.35 mg.kg-l day-l , ppm = parts per million [1] Buckingham et al., 1986; [2] Linz et al., 1992; [3] Clapham et al., 1991b; [4] Hirawa et al., 1989; [5] Hamilton et al., 1993; [6] Masuda et al., 1991; [7] Fozard et al., 1990; [8] Paciorek et al., 1990; [9] Nakajima et al., 1992.

9.4 Heart Rate and Plasma Renin Activity KCA-induced reductions in arterial pressure were accompanied almost invariably by tachycardia, with the possible exception of Y–27152 (Nakajima et al., 1992) which has an extremely slow onset of action which may account for the lack of tachycardia (Nakajima et al., 1992). However, tachycardia induced by KCAs is sensitive to β -adrenoceptor blockade (Clapham et al., 1991a; Masuda et al., 1991) indicative of a reflex rise in sympathetic nervous system activity in response to a reduction in total peripheral resistance (TPR). In addition, when admixed with diet, where it is assumed that drug intake occurs over time (Hamilton et al., 1993), reflex elevations in heart rate following LCRK are attenuated. This lends support to the explanation of Nakajima et al. (1992) that a slow onset of action would lead to a reduced involvement of counter regulatory mechanisms. Reflex activation of the sympathetic nervous system may also give rise to an increase in plasma renin activity (PRA) and this was shown, for example, in the renal hypertensive cat, SHR and the conscious rhesus monkey (Fozard et al., 1990; Clapham et al., 1991a; Clapham et al., 1991b). As with heart rate, Y–

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27152 did not increase plasma renin activity in the renal hypertensive dog following repeat administration (Nakajima et al., 1992). Suppression by propranolol of these counter regulatory responses to a KCAinduced reduction in arterial pressure increases the duration and magnitude of the antihypertensive response (Clapham et al., 1991a). Furthermore, in the Dahl saltsensitive rat, which has suppressed reactivity of the renin-angiotensin system, blood pressure lowering activity of LCRK was still evident 24 h post-dose (Hirawa et al., 1989). 9.5 The Venous System There are relatively few reports describing the potential venodilator effects of KCAs in whole animal models. In the anaesthetised rabbit or cat (Clapham and Buckingham, 1988; Hof et al., 1988; Clapham et al., 1991a), CRK or LCRK have no effect on, or increase, central venous pressure when TPR was reduced by these drugs. In the anaesthetised dog, cromakalim and pinacidil lower BP together with a concomitant increase in total venous return and pulmonary artery blood flow (Gotanda et al., 1989; Kaneta et al., 1993). Together these data imply that these drugs do not increase venous capacitance via a venodilator action, but act solely on the arterial side of the circulation. Indeed, direct observation of skeletal muscle microcirculation by intravital microscopy in SHR (StruykerBoudier et al., 1992) reveals that at BP lowering doses, LCRK dilated arterioles with very little effect on venules (see Figure 9.3 for example). By contrast, intravenous, but not intracoronary, administration of bimikalim to anaesthetised pigs results in a dose-related reduction in left ventricular end diastolic pressure that is independent of heart rate (Sassen et al., 1990). Although not measured directly, these data suggest that bimikalim possesses some venodilating property. KCAs possessing a nitrate moiety, however, might be expected to elicit a venodilator effect, since this is recognised as the primary mode of action of nitrovasodilators in vivo. Indeed, Kaneta et al. (1993) have shown that nicorandil and nitroglycerine elicit qualitatively similar effects on venous haemodynamics in the anaesthetised dog. These workers showed also that the nitrate containing KCA, KRN2391, appeared to have a bimodal effect on venous haemodynamics. First, KRN2391 increased venous return in a similar way to CRK. This was followed by a nitrate-like action where KRN2391 decreased venous return via an increase in venous capacitance. The sulphonylurea KATP channel blocker, glibenclamide, abolished the initial CRK-like effect on KATP channels unmasking a purely nitrate-like action of KRN2391 (Kaneta et al., 1993).

IN VIVO VASCULAR EFFECTS OF KCAS 247

9.6 Regional Haemodynamics The mechanism by which KCAs lower BP in animal models is without doubt due to a reduction in total peripheral resistance resulting from arteriolar vasodilatation. The study of the contribution of individual vascular beds to the overall reduction in total peripheral resistance, however, has been carried out largely in anaesthetised animals using flow-probe or microsphere technology. Recently, data have begun to emerge from conscious animal studies that are beginning to strengthen our knowledge of the haemodynamic profile of KCAs (see Figure 9.1). Thus, there is a consensus view regarding the effects of KCAs on the coronary and splanchnic vascular beds but data from the renal vasculature remain equivocal. On the other hand, data on the pulmonary circulation are not so abundant and further evaluation, particularly in the area of hypoxic pulmonary constriction and pulmonary hypertension, is still required. 9.6.1 The Cerebral Circulation Intravenous administration of CRK, LCRK or bimikalim increases cerebral blood flow, when measured by radioactive microspheres or [14C]-iodoantipyrine, in normotensive rabbits, rats and pigs (Hof et al., 1988; Schliep et al., 1989; Sassen et al., 1990). However, there is no increase in radioactive microspheres in the brain following oral administration of CRK in SHR (Shoji et al., 1990). In a study where stroke-prone SHR and their normotensive counterpart, the Wistar Kyoto (WKY) rat, were compared, aprikalim increased basilar artery diameter in the WKY rat but not in the stroke-prone SHR (Kitazono et al., 1993). The effect in WKY rats was glibenclamide-sensitive and it is possible that KATP channel function/density is diminished in the cerebral vasculature of genetically hypertensive rats. In a study comparing non-diabetic and streptozotocin-diabetic rats, aprikalim dilated pial arterioles in a glibenclamide-sensitive manner in nondiabetic rats (Mayhan and Faraci, 1993). However, a slight vasoconstriction occurred in the pial arterioles in streptozotocin-diabetic rats following aprikalim, nitroglycerine was effective equally in both non-diabetic and streptozotocindiabetic rats. Thus, it appears that not only can KATP channel function in cerebral blood vessels be impaired by both hypertension and streptozotocin-diabetes, but that dilatation of cerebral blood vessels does not always occur in anaesthetised normotensive animals in response to KCAs. It is possible that KATP channels are not widely distributed throughout cerebral blood vessels. Indeed, it has been reported that rat brain microvessels of the cerebral cortex lack specific [3H]glibenclamide binding sites implying a lack of KATP channels in this vessel type (Sullivan and Harik, 1993).

248 K CHANNELS AND THEIR MODULATORS

Figure 9.1 Haemodynamic profile of levcromakalim (8.7 nmol.kg–1 iv) in conscious, instrumented dogs. (Reproduced with permission from Shen and Vatner, 1993).

Despite these findings however, KCAs may still have a potentially beneficial effect in cerebral ischaemia. For example intracerebroventricular administration

IN VIVO VASCULAR EFFECTS OF KCAS 249

of LCRK, pinacidil and nicorandil (10 nmol.5 μ l–1) blocked ischaemia-induced expression of the immediate early genes, c-fos and c-jun and of the mRNAs for the 70 kDa heat shock protein and amyloid β -protein precursor genes in the rat (Heurteaux et al., 1993). In addition, these drugs protected against neuronal death and all of the anti-ischaemic events reported were sulphonylurea-sensitive indicating an involvement of KATP channels. 9.6.2 Coronary Circulation KCAs representing most chemical classes elicit marked increases in coronary blood flow (diazoxide, Rubin et al., 1962; nicorandil, Sakai et al., 1981; Sakai et al., 1983; pinacidil, Arrigoni-Martelli and Finucane, 1985; aprikalim, Aloup et al., 1990; LCRK, Clapham et al., 1991a; YM934, Uchida et al., 1994; RWJ29009, Damiano et al., 1994). Indeed, in those studies where coronary blood flow was measured in animals instrumented for full haemodynamic profiling, KCA-induced reductions in coronary vascular resistance (–52%) are much greater than reductions in resistances in other vascular beds (–17 to –28%) (Shen and Vatner, 1993; Figure 9.1). This exaggerated effect of KCAs on the coronary circulation may reflect a greater density of KATP channels in this tissue but also indicates that KATP channel mechanisms are probably of major importance in the coronary vasculature (Duncker et al., 1993). Increases in coronary blood flow in response to KCAs are not simply a consequence of metabolic vasodilatation due to the demands of increased heart rate. For example, myocardial oxygen consumption in response to coronary vasodilatation in anaesthetised dogs is unchanged by pinacidil (Dubé and Greenfield, 1991) and RWJ29009 (Damiano et al., 1994). Furthermore, coronary vasodilatation is still evident following pinacidil or LCRK when heart rate changes are suppressed by propranolol (Giudicelli et al., 1990; Drieu la Rochelle et al., 1992; Shen and Vatner, 1993) in anaesthetised or conscious dogs. Despite the apparently exaggerated response of the coronary vasculature to KCAs compared to other vascular beds (Shen and Vatner, 1993), the coronary vasodilator effects of KCAs are also glibenclamide-sensitive (Cavero et al., 1991) confirming that an action at KATP channels underlies this effect. Interestingly, the coronary vasodilator activities of nicorandil and KRN2391, that have additional nitrate-like activity producing stimulation of soluble guanylate cyclase, (Holzmann, 1983; Kashiwabara et al., 1991), were antagonised by glibenclamide, and not methylene blue (Cavero et al., 1991; Ogawa et al., 1992), suggesting that even with vasodilators of mixed character, expression of the KATP channel effects is the overriding mechanism dilating coronary resistance vessels. Furthermore, KRN2391, despite its nitrate moiety, did not induce acute tolerance, either to itself or to isosorbide dinitrate or nitroglycerine (Kaneta et al., 1992). Dilation of large coronary vessels by these compounds however is more complex (see below). The evidence that KATP

250 K CHANNELS AND THEIR MODULATORS

channel modulation and control of coronary blood flow are intimately linked is further strengthened by evidence that glibenclamide, per se, reduces basal coronary blood flow in the anaesthetised dog (Imamura et al., 1992). It has recently emerged that the effects of the KCAs, CRK and pinacidil, are not uniform throughout the coronary vascular bed. Both KCAs induced a dosedependent reduction in coronary vascular resistance and an increase in diameter of large coronary arteries in conscious dogs, a response unaffected by propranolol (Giudicelli et al., 1990). This contrasts with the effects of nitroglycerine, that is, dilatation of the large coronary arteries only. Furthermore, when blood flow through the large coronary artery is held constant, the coronary dilator effects of CRK and pinacidil, but not nitroglycerine, are markedly reduced (but not abolished). Thus, it appears that in the dog, KCAs may dilate the coronary vascular bed by both direct (resistance vessels) and indirect, flow-dependent, (large vessels) mechanisms. This flow-dependent coronary vasodilator response to CRK and pinacidil appears to be endothelium-dependent (Drieu la Rochelle et al., 1992). In contrast, however, the relaxant effects of CRK are not endotheliumdependent in rat isolated aorta (Taylor et al., 1988) and dog isolated coronary artery (Drieu la Rochelle et al., 1992). This discrepancy is attributed to a dilution effect in the large coronary artery in vivo thereby preventing CRK and pinacidil from reaching the required local concentration to elicit a pharmacological effect. Nicorandil, like CRK and pinacidil, dilated both large and small coronary arteries in the same model but, unlike CRK and pinacidil, nicorandil-induced coronary vasodilatation was not affected when flow was restricted (Berdeaux et al., 1992) and was therefore exhibiting both KCA- and nitrate-like actions. As discussed, KCA-induced increases in coronary blood flow, with concomitant reductions in coronary vascular resistance, are readily demonstrated by standard flow probe techniques. When blood flow distribution across the ventricular wall is measured by microspheres, interpretation of data is more equivocal. For example, Bache et al. (1990a) showed that low doses of pinacidil produced uniform increases in blood flow across the left ventricular wall thereby maintaining the subendocardial/ subepicardial blood flow ratio in normal hearts. When the dose of pinacidil was raised, a further increase in blood flow to the outer, subepicardial layer, with no change to the inner, subendocardial layer resulted in an apparent alteration in the ratio of blood flow distribution within the myocardium. However, absolute blood flow to both regions of the myocardium remained greater than control throughout the experiment such that there was no underperfusion. However, in order to model occlusive coronary artery disease, a severe coronary artery stenosis to restrict blood flow to ~30% above basal, was performed by Bache et al. (1990a) in the anaesthetised dog. They reported that those doses of pinacidil eliciting favourable effects in normal hearts, increased subepicardial perfusion at the expense of subendocardial perfusions; though when a moderate stenosis (flow restricted to ~60% above basal) was applied blood flow to all layers was greater than control after pinacidil. In another study by the same group (Bache et al., 1990b), in dogs with a chronically overloaded

IN VIVO VASCULAR EFFECTS OF KCAS 251

hypertrophied left ventricle, pinacidil reduced the subendocardial/subepicardial blood flow ratio from 1.06 to ~0.8. This again implied that there was a diversion of blood away from the subendocardial layers. However, absolute values of blood flow to the subendocardium following pinacidil were substantially (139%) greater than control values and, as with the previous study (Bache et al., 1990a), pinacidil did not actually underperfuse the inner layers of the hypertrophied ventricle. Further studies are clearly required in this area to determine the effects of other KCAs on the distribution of blood flow across the ventricular wall. In addition, existing studies have tended to reflect the acute effects of KCAs. Thus, investigation of chronic administration of these drugs on blood flow distribution within the ventricular myocardium would be extremely useful. 9.6.3 Splanchnic Circulation In general, KCAs increase blood flow and reduce vascular resistance through the mesenteric artery. Thus, in the anaesthetised rat, aprikalim, CRK, Ro 31–6930 and SDZ PCO 400 reduce mesenteric vascular resistance (Cavero et al., 1989; Aloup et al., 1990; Duty et al., 1990; Fozard et al., 1990). Where measured, the reduction in mesenteric vascular resistance closely reflect reductions in systemic vascular resistance and arterial BP (Cavero et al., 1989; Aloup et al., 1990). These findings are also seen in conscious animals where LCRK elicits a doserelated increase in mesenteric blood flow in Long Evans rats (Gardiner et al., 1991). Similar results have also been shown for CRK, Ro 31–6930, KRN2391 and nicorandil in anaesthetised (Paciorek et al., 1990; Ogawa et al., 1993a) and conscious (Shen and Vatner, 1993) dogs. Where microspheres have been used to measure regional haemodynamics however, the data are not as clear. In the anaesthetised pig, bimikalim elicited doserelated increases in blood flow to the small intestine whereas stomach blood flow was increased only at the highest dose used (Sassen et al., 1990). In the anaesthetised rabbit, CRK and LCRK increase blood flow markedly to both the stomach and small intestine (Hof et al., 1988) while in conscious SHR (Shoji et al., 1990) CRK has no effect on small intestine blood flow whilst increasing stomach blood flow by 78%. These differences may reflect species differences, as suggested by Richer et al. (1990b). They may also reflect possible differences in time-course between responses of each vascular bed since the microsphere technique reflects a ‘snap-shot’ at a single time point. However, in conscious dogs (Shen and Vatner, 1993; Figure 9.1) and anaesthetised cats (Clapham and Gentry, unpublished data), LCRK increases coeliac and mesenteric blood flow implying that both stomach and small intestine receive an increased blood flow.

252 K CHANNELS AND THEIR MODULATORS

9.6.4 Renal Circulation When the haemodynamic effects of KCAs were reviewed previously (Buckingham, 1989; Richer et al., 1990b; Edwards et al., 1992) there was no real agreement expressed about the renal haemodynamic effects of these drugs. In our laboratory we have observed that CRK increases renal blood flow in both anaesthetised and conscious cats (Clapham and Buckingham, 1988; Clapham and Longman, 1989) and others have demonstrated increases in renal blood flow in anaesthetised dogs (Dumez et al., 1988). However, these effects are not seen in anaesthetised rats or rabbits (Hof et al., 1988; Shoji et al., 1990) and, in anaesthetised pigs, bimikalim increased slightly, then decreased, renal blood flow depending on the dose used (Sassen et al., 1990). The reduction in total renal blood flow observed in the pig comprises a decrease in cortical (predominant) blood flow whereas medullary blood flow is increased significantly, producing an uneven distribution of total renal blood flow (Sassen et al., 1990). In other studies using anaesthetised normotensive rats, reductions in renal vascular resistance, following bolus ascending doses of cromakalim, but not Ro 31–6930, have been reported though both drugs elicited the same absolute falls in arterial pressure (Duty et al., 1990). Thus different changes in renal haemodynamics can be observed even within the benzopyran family of KCAs. A definitive answer to this problem is an important goal since it was declared during the mid-eighties that, if new vasorelaxant drugs were to play a major role in the future treatment of hypertension, they must exhibit a salutary effect on the kidney (Struyker-Boudier et al., 1984; Hollenberg, 1987). The earlier and sometimes conflicting data for changes in renal haemodynamics has centred largely around CRK and pinacidil. Now, with the introduction of many more KCAs and published haemodynamic studies, a slightly clearer picture has emerged. More importantly, the increased number of studies in conscious animals (Clapham and Longman, 1989; Gardiner et al., 1991; Shen and Vatner, 1993; see Figure 9.1) should, theoretically, provide the strongest evidence, since the effects of anaesthesia are obviated. In animals, KCAs tend to elicit a reduction in renal vascular resistance which is occasionally accompanied by an overt increase in renal blood flow (Table 9. 3). Where increases in renal blood flow have been recorded, it is possible that increased basal tone of the renal vasculature could be responsible for this effect and this is worthy of further investigation. Hollenberg et al. (1978) have estimated that up to two thirds of patients with essential hypertension have a 20% reduction in renal perfusion associated with renal vasoconstriction. Therefore, KCAs that dilate the Table 9.3 Effects of some KCAs on renal haemodynamics in animals KCA Species Anaesthesia

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Method RBF RVR Ref aprikalim rat Yes -NS[1] cromakalim rat Yes Doppler [2,3] dog Yes EM [4] cat Yes EM

[5] cat Yes EM [6] cat Conscious Doppler [7] cat Conscious Doppler

[8] diazoxide dog Yes EM [9]

254 K CHANNELS AND THEIR MODULATORS

bimikalim pig Yes MS [10] dog Yes -NS[11] KRN2391 dog Yes EM [12] levcromakalim rabbit Yes MS [13] rat Conscious Doppler [14] cat Conscious Doppler

[15] dog Conscious Doppler

[16] dog Yes EM

[17] nicorandil

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dog Yes EM [12] cat Yes EM [6] pinacidil cat Conscious Doppler

[8] cat Yes EM [6] dog Yes EM [18] Ro 30–6930 rat Yes Doppler [2] dog Yes EM [4] YM934 dog Yes EM

[17] EM = electromagnetic flow probes, MS = microspheres, -NS- = not stated. RBF = renal blood flow, RVR = renal vascular resistance.

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[1] Aloup et al., 1990; [2] Duty et at., 1990; [3] Cavero et al., 1989; [4] Paciorek et al., 1990; [5] Buckingham et al., 1986; [6] Longman et al., 1988; [7] Clapham and Buckingham, 1988; [8] Clapham and Longman, 1989; [9] Rubin et al., 1962; [10] Sassen et al., 1990; [11] Schliep et al., 1989; [12] Ogawa et al., 1993a; [13] Hof et al., 1988; [14] Gardiner et al., 1991; [15] Clapham et al., 1991a; [16] Shen and Vatner, 1993; [17] Uchida et al., 1994; [18] Olsen and Arrigoni-Martelli, 1983.

renal vasculature in addition to lowering BP, may beneficially affect a process involved in the progressive elevation of BP. However, as yet there are no reported effects of KCAs on renal haemodynamics in models of hypertension in larger animals or after chronic administration. When peripheral vascular resistances to a number of vascular beds have been measured simultaneously, the degrees of KCA-induced reduction in the various vascular resistances are not uniform. For example, the renal circulation dilates the least of all in response to KCAs (Cavero et al, 1989; Shen and Vatner, 1993; Uchida et al., 1994). However, this is not altogether surprising since the kidney can dramatically autoregulate its own blood flow over a wide perfusion pressure window, from about 80 to 180 mmHg (Cupples et al., 1990). Nevertheless, a reflex rise in sympathetic tone to the kidney may limit the degree of vasodilatation induced by KCAs in this bed compared to other regions. This possibility is supported by a recent study (Johns, 1993) where LCRK, given by close arterial infusion, did not affect reductions in renal blood flow elicited by β 1- and β 2-adrenoceptor agonists, or by electrical stimulation of the renal sympathetic nerves, in the anaesthetised rat. Adrenergic control of the renal vasculature would therefore have occurred normally in the presence of LCRK. In contrast, an earlier study performed in pithed rats (Richer et al., 1990a) showed that intravenous infusion of CRK or bimikalim inhibited renal vasoconstriction elicited by UK 14304 (β 2-adrenoceptor agonist) and spinal cord stimulation, but that the renal vasoconstrictor effects of cirazoline (β 1-adrenoceptor agonist) were unaffected by either KCA. The sympathoinhibitory effect observed was still apparent if BP was returned to pre-infusion level with vasopressin or prostaglandin F2― (PGF2― ). However, these studies are not directly comparable, since Johns’ (1993) study was conducted in anaesthetised rats and administration of agonists and sympathetic nerve stimulation primarily targeted the kidney itself. Vasoconstrictor responses in the former (Richer et al., 1990a) study were more generalised following intravenous administration.

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9.6.5 Skeletal Muscle Circulation The measurement of hindquarters blood flow using periarterial flow probes (pulsed Doppler and electromagnetic) appears to be the favoured model used to determine the effects of KCAs on blood flow to skeletal muscle. Probes may be placed around femoral (Olsen and Arrigoni-Martelli, 1983; Buckingham et al., 1986; Paciorek et al., 1990; Ogawa et al., 1993a) or iliac (Duty et al., 1990; Shen and Vatner, 1993) arteries or, alternatively, around the lower abdominal aorta, thus encompassing both femoral and iliac blood flow (Aloup et al., 1990; Gardiner et al., 1991). In these systems however, particularly for lower abdominal flow, changes in flow to other structures such as the rectum, tail, testes and skin may have influenced the overall response. When femoral blood flow was measured in anaesthetised cats and dogs, pinacidil, CRK, NIP-121 and YM934 (Olsen and Arrigoni-Martelli, 1983; Buckingham et al., 1986; Ogawa et al., 1993a; Uchida et al., 1994) elicited only minor changes in femoral blood flow but CRK and Ro 31–6930 reduced femoral vascular resistance (Paciorek et al., 1990). This is in contrast to the Ca antagonist, nifedipine, which elicited marked increases in femoral artery blood flow in anaesthetised cats (Buckingham et al., 1986) and dogs (Ogawa et al., 1993a). Diazoxide caused a biphasic response, first decreasing then increasing femoral blood flow (Rubin et al., 1962). Nicorandil, whose effects were also biphasic on femoral blood flow in anaesthetised mini-pigs, exhibited the reverse pattern of diazoxide, namely an increase followed by a decrease (Sakai et al., 1983). In conscious dogs, LCRK increased iliac blood flow and reduced iliac vascular resistance (Shen and Vatner, 1993), whilst CRK and Ro 31–6930 reduced iliac vascular resistance in the anaesthetised rat (Duty et al., 1990). Hindquarters vascular resistance (mainly femoral and iliac) has been shown to be reduced by levels (45–50%) that are greater than the fall in systemic vascular resistance (35%) following administration of aprikalim or CRK (Cavero et al., 1989; Aloup et al., 1990). In the conscious Long Evans rat, LCRK increased hindquarters blood flow at low doses (1 μ g.kg–1 min–1); but at a higher dose (10 μ g.kg–1min–1) hindquarters blood flow remained unchanged (Gardiner et al., 1991). Using an alternative technique (radioactive microspheres), Hof et al. (1988) have shown that CRK had little or no effect on rabbit skeletal muscle blood flow whilst in anaesthetised pigs blood flow to the muscularis sternocleidomastoideus was doubled following intravenous administration of bimikalim (Sassen et al., 1990). In the latter study however, blood flow to a number of other muscle groups remained constant suggesting that there was a heterogeneous effect of KCAs in the skeletal muscle circulation.

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In general therefore, KCAs have tended to reduce hind limb vascular resistance and in some, but not all, studies increases in muscle blood flow have been observed. An indication that the KCAs may be useful in the treatment of peripheral vascular disease occurred when Angersbach and Nicholson (1988) reported that the KCAs, CRK, pinacidil and nicorandil, but not the Ca antagonists, nifedipine, verapamil or diltiazem, markedly increased red cell flux and tissue oxygenation in chronically ischaemic (femoral artery ligation for 6 weeks) muscle of the rat hind limb. This finding gained further support when Hatton et al. (1991) communicated that CRK partially attenuated ischaemia-induced muscle fatigue in the rat hind limb. In other words, there is a functional correlate to the increased tissue perfusion and reoxygenation. However, the functional benefits of CRK have been disputed in a study using a rat model of acute hind limb ischaemia (Tresize et al., 1993). They found that CRK (10–100 μ g.kg–1 iv) and LCRK (15 μ g.kg–1 iv) reduced arterial pressure and hind limb vascular resistance concurrent with an increase in iliac artery blood flow but did not attenuate the acute ischaemic muscle fatigue induced by graded occlusion of the abdominal aorta. The reasons for the differences may be related to the different methodologies used in the two studies (Hatton et al., 1991; Tresize et al., 1993). Thus, Angersbach and Nicholson (1988) concluded that during chronic ischaemia, newly formed collateral blood vessels may be selectively dilated by the KCAs to account for the increased red cell flux and oxygenation. This would not occur in the acute situation and, as indicated by Tresize et al. (1993), comparable studies using chronically ischaemic hind limb are required to assess the contribution of newly formed collaterals using their methodology. 9.6.6 Pulmonary Circulation The pulmonary circulation differs from the systemic circulation in that it distributes output from the right ventricle to the gas exchanging surface of the alveoli. It is also a low pressure system with an average mean pulmonary artery pressure of about 15 mmHg. The phenomenon of hypoxic pulmonary vasoconstriction has been known for years and is thought to be a homeostatic mechanism by which pulmonary blood flow is diverted away from hypoxic areas to optimise gas exchange. However, the homeostatic event may deteriorate to become an undesirable event leading to pulmonary hypertension. The mechanisms for hypoxic pulmonary vasoconstriction are not yet fully understood but are likely to be multifactorial (McCormack et al., 1993). Data for KCAs on the pulmonary circulation have been relatively few but data from a number of studies are now emerging indicating increased interest in this area. In anaesthetised dogs, CRK reduces pulmonary artery pressure during hypoxic pulmonary vasoconstriction (Hicks et al., 1989; Martin et al., 1990) and both CRK and pinacidil have been shown to dilate the pulmonary vasculature, at

IN VIVO VASCULAR EFFECTS OF KCAS 259

doses not affecting right ventricular contractile function (Minkes et al., 1991). The effects of CRK and pinacidil are glibenclamide-sensitive (Minkes et al., 1991) but there is no separation of pulmonary from systemic effects (Figure 9.2) (Hicks et al., 1989; Martin et al., 1990; Minkes et al., 1991). In another study in the anaesthetised cat,

Figure 9.2 Effect of cromakalim on hypoxic pulmonary vasoconstriction in the anaesthetised dog. Pulmonary resistance (― ), systemic resistance (― ) and cardiac output (― ). Hypoxia was induced using an air/N2 (2:1) mixture to reduce arterial pO2 from 92±4 mmHg to 48±8 mmHg. (Reproduced with permission from Hicks et al., 1989).

where pulmonary pressure was elevated by the thromboxane agonist U46619, LCRK elicited a dose-related reduction in intralobar pressure, when infused directly into the intralobar artery (Hood et al., 1991). This effect was also shown to be glibenclamide-sensitive (Hood et al., 1991; McMahon et al., 1992) and independent of the nitric oxide (NO) pathway (McMahon et al., 1992). Systemic vasodilatation was also evident. These data would suggest that although KCAs have clear actions on the pulmonary circulation, the effects in acute models

260 K CHANNELS AND THEIR MODULATORS

cannot be separated from systemic effects indicating that the populations of KATP channels on the respective vasculature beds are identical. When considering a role for the KCAs in pulmonary hypertension, it should be appreciated that in states of increased pulmonary vascular resistance, attempts to reverse the condition with vasodilators have been unsuccessful owing to a reduction in systemic vascular resistance (McCormack et al., 1993). In the models of acute hypoxia already discussed, the KCAs tested showed no dose separation between pulmonary and systemic dilatation. This lack of selectivity has also been observed in a chronic model of pulmonary hypertension (high altitude, chronically hypoxic rats; Oka et al., 1993). In these animals, the benzopyran based KCA, NIP-121, but not the Ca antagonist, nifedipine, reduced pulmonary artery pressure. However, as shown in acute studies with KCAs, pulmonary vascular resistance is reduced also at doses active on the systemic vasculature. In the study of Oka et al. (1993) however, the rats were acclimatised for two days under normoxic conditions, suggesting that the pulmonary dilatation was effective against a condition not associated with hypoxic vasoconstriction. The effects of NIP–121 were glibenclamide-sensitive and independent of NO release (Oka et al., 1993). Thus, the effects of KCAs in acute studies, and in one study in chronically hypoxic rats, appear to be qualitatively similar in vivo. In vitro studies would have predicted that the hypoxic pulmonary vasculature would have been more sensitive to the effects of KCAs since it is well known that the actions of KCAs can be augmented by hypoxia (Cook and Quast, 1990). In a study using rat isolated pulmonary artery precontracted with PGF2a, the potency of LCRK was enhanced significantly (IC50 0.52±0.05 μ M in normal versus 0.23±0.06 μ M in hypoxic tissue) in rats previously subjected to chronic hypoxia compared to their normoxic controls (Leach et al., 1992). This effect was not influenced by the absence or presence of endothelium. In a study of acute hypoxia, the activity of LCRK was augmented by hypoxia and inhibition of oxidative phosphorylation (Randall and Griffiths, 1993). The role of KATP channel modulation providing a means of selective pulmonary vasodilatation remains an attractive proposition (Peacock, 1993). 9.7 Microcirculation As mentioned previously, it is now widely accepted that KCAs reduce arterial pressure by a direct action on vascular smooth muscle to reduce systemic vascular resistance (Edwards et al., 1992; Longman and Hamilton, 1992). The studies discussed so far have, in the main, involved measurement of blood flow through large (accessible) conduit arteries. Together with systemic BP, the pressure/flow relationship can be calculated to give resistance to flow (or the reciprocal, conductance) as an indirect measure of the behaviour of the resistance vessels in that tissue. Resistance in this context, therefore, is the sum of all the

IN VIVO VASCULAR EFFECTS OF KCAS 261

resistance vessels in the tissue to which flow was being measured. However, it is clear that the microcirculatory responses to vasodilators are not uniform throughout the arteriolar tree. In order to study the microcirculation in conscious rats, a lightweight thermoneutral chamber has been developed by Smith et al. (1985). This ‘dorsal microcirculatory chamber’ allows direct visualisation of skeletal muscle microcirculation by intravital microscopy. Using this technique, StruykerBoudier et al. (1992) have shown that LCRK (10–100 μ g.kg–1iv) elicits a doserelated reduction in arterial pressure in conscious SHR associated with preferential dilatation of the smallest (10–35 μ m diameter) arterioles. Larger (70–120 μ m diameter) vessels were significantly dilated only by the highest dose

262 K CHANNELS AND THEIR MODULATORS

Figure 9.3 Effects of levcromakalim on arteriole (upper panel) and venule (lower panel) diameters in conscious SHR measured by intravital microscopy. Values are mean ± S.E.M. Vessels are labelled in functional branching order with A1 or V1 vessels being the largest (70–125 μ m diameter), then A2/V2 (30–75 μ m diameter) and then the pre-capillary arterioles, A3/4 and post-capillary venules, V3/4 (10–30 μ m diameter). (Reproduced with permission from Struyker-Boudier et al., 1992).

tested (Figure 9.3). A similar profile was also found for the Ca antagonists nifedipine and verapamil (Messing et al, 1991). In contrast, the β 1-adrenoceptor antagonist, prazosin (Messing et al., 1990), dilated preferentially the larger arterioles. Thus, these important studies have provided direct evidence for heterogeneity in the response to vasodilator agents in the arteriolar tree in the conscious SHR. Intravital microscopy has shown also that, in the cutaneous microcirculation of conscious normotensive rats, bimikalim (6, 12 and 24 μ g.kg–1 iv) elicited a doserelated reduction in arterial pressure concurrent with an increase in erythrocyte flow velocity (Hertel, 1992). However, capillary diameter was increased following 6 and 12 μ g.kg–1 iv bimikalim only. At 24 μ g.kg–1 iv, vasoconstriction was observed. In summary, the data support the suggestion that the smallest blood vessels are under control of factors affecting membrane potential (Struyker-Boudier et al., 1992) and that KATP channels could be an important determinant of local tissue blood flow (Hertel, 1992). 9.8 In Vivo Mechanistic Studies 9.8.1 Glibenclamide Antagonism of the cardiovascular effects of KCAs by relatively high doses of the sulphonylurea, glibenclamide is now a hallmark for actions at KATP channels. In both conscious and anaesthetised rats (SHR and normotensive), glibenclamide (10–20 mg.kg–1 iv) has been shown to inhibit BP lowering effects of CRK and nicorandil (Cavero et al., 1989), SDZ PCO 400 (Quast et al., 1990), aprikalim (Aloup et al., 1990), LCRK (Clapham et al., 1991a), NIP–121 (Masuda et al., 1991) and Y–26763 (Nakajima et al., 1992). Glibenclamide is able to prevent the antihypertensive effects of KCAs and to reverse an already established antihypertensive response (Aloup et al., 1990; Clapham et al., 1991a). Importantly, glibenclamide appears to block selectively for hypotensive responses induced by KCAs. Thus, the vasodilator responses to dihydralazine, acetylcholine, vasoactive intestinal polypeptide and the Ca antagonists diltiazem and nifedipine, are unaffected by doses of glibenclamide that abolish the hypotensive effect of KCAs (Figure 9.4) (Cavero et al., 1989; Clapham et al., 1991a; Hood et al., 1991; Ogawa et al., 1993b).

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Demonstration of the antagonism of the systemic effects of KCAs by glibenclamide has been extended to individual vascular beds in various species. Thus, glibenclamide antagonises the pulmonary (Hood et al., 1991; Minkes et al., 1991), mesenteric (Ogawa et al., 1993b) and coronary (Cavero et al., 1991; Ogawa et al., 1992) vasodilator responses to a variety of KCAs. These data provide in vivo evidence that glibenclamide is acting at the level of vascular smooth muscle. Conversely, antihypertensive doses of the KCAs, CRK or pinacidil, do not prevent nor reverse glibenclamide-induced hypoglycaemia in conscious SHR (Clapham et al., 1994). In contrast, diazoxide at equiantihypertensive doses did affect glibenclamide-induced hypoglycaemia. These data confirm, in vivo, the lack of effect of the newer KCAs on pancreatic KATP channels at BP lowering doses.

264 K CHANNELS AND THEIR MODULATORS

Figure 9.4 Interaction between intravenously administered glibenclamide on the blood pressure lowering activity of levcromakalim and nifedipine in anaesthetised SHR. a) glibenclamide (― ) or vehicle (― ) (first arrow) was administered 15 min prior to levcromakalim (second arrow), b) levcromakalim (first arrow) administered 15 min prior to glibenclamide (― ) or vehicle (― ) where indicated by second arrow, c) glibenclamide (― ) or vehicle (O) (first arrow) was administered 15 min prior to nifedipine (second arrow).

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Values are mean ± S.E.M. * indicated P<0.05 between vehicle and glibenclamide. (Reproduced from Clapham et al., 1991 a).

9.8.2 The Endothelium The vascular responses to KCAs such as LCRK are not affected by administration of inhibitors of nitric oxide- (NO) synthase or by methylene blue, an inhibitor of soluble guanylate cyclase, the receptor for NO (Cavero et al., 1991; Gardiner et al., 1991; McMahon et al., 1992; McMahon and Kadowitz, 1993; Oka et al., 1993). Those KCAs bearing a nitrate moiety are, of course, susceptible to some inhibition by methylene blue (Cavero et al., 1991). Furthermore, indomethacin and BW755C (an inhibitor of lipo- and cyclooygenase) do not inhibit the vascular responses to various KCAs (Olsen and Arrigoni-Martelli, 1983; Cavero et al., 1989; Masuda et al., 1991). These data provide evidence that the in vivo vascular responses resulting from activation of KATP channels are not dependent on NO or cyclo-oxygenase products to elicit vasodilatation. An exception to this appears to be large coronary arteries where CRK elicits a flow-dependent vasodilatation (Drieu la Rochelle et al., 1992). However, as discussed previously, the apparent endothelium dependency of this effect may actually be due to pharmacodynamic reasons. 9.8.3 Effect of Receptor Antagonists on BP Lowering Effects of KCAs The BP lowering effects of the KCAs, CRK, aprikalim and NIP–121 are reported to be resistant to β -adreno-, β -adreno-, serotonin, dopamine, histamine and platelet activating factor receptor blockade (Cavero et al., 1989; Aloup et al., 1990; Masuda et al., 1991). 9.8.4 Effect of KCAs on Stimulation of Exogenous and Endogenous Receptors Vasoconstrictor responses in vivo are generally inhibited by KCAs (Buckingham, 1989). Moreover, the inhibitory effects of KCAs in this regard are not uniform throughout the vascular tree (Richer et al., 1990a). Thus, CRK and bimikalim inhibited sympathetically-induced (exogenous and endogenous stimulation) vasoconstriction in the pithed rat in the order mesentric>renal>hind limb, with the effects of sympathetic nerve stimulation being more sensitive to inhibition than exogenous agonists (Richer et al., 1990a). Restoration of BP to

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values observed in intact (as opposed to pithed) rats by PGF2― , had no effect on the inhibitory responses to CRK or EMD52692, whilst restoration of BP with arginine vasopressin (AVP) abolished the sympathoinhibitory effects of these KCAs (Buckingham 1988; Richer et al., 1990a). The discrepancy between the effects of PGF2― and AVP is not clear mechanistically but it is possible that AVP may act as a physiological antagonist through sensitisation of the vasoconstrictor actions to the stimuli used. The inhibitory responses of these drugs in this respect are therefore not dependent upon the prevailing level of BP. Richer et al. (1990a) postulated that the effects of CRK and bimikalim were pre-junctional since the effects of nerve stimulation were more sensitive than those of exogenously applied agonists to the inhibitory effects of these KCAs. However Johns (1993) found that LCRK had little or no effect on renal vasoconstrictor responses to β adrenoceptor agonists or sympathetic nerve stimulation. Furthermore, NIP–121 elicited a dose-related inhibition of pressor responses to nondrenaline, angiotensins I and II, AVP, PGF2― and the Ca agonist, BAY K 8644 (Masuda et al., 1991). In addition CRK inhibited pressor responses to endothelin I in pithed rats (Le Monnier de Gonville et al., 1990) indicating that a substantial postsynaptic involvement cannot be excluded. Tachycardia elicited by sympathetic-nerve stimulation was also unaffected by CRK or bimikalim (Richer et al., 1990a) confirming the results of an earlier study where CRK had no effect on sustained tachycardia evoked by stimulation of the spinal cord in the pithed rat (Clapham, 1988). Thus, if KCAs exert a prejunctional inhibitory effect on sympathetic nerves, the effect would appear to be specific for the vasculature. 9.9 Conclusion A feature that seems to have emerged from the haemodynamic studies described here, is the non-uniform effect of KCAs throughout the vascular tree which show a distinct rank order of vasodilator potential depending on the vascular bed and the size of arteriole. This may be explained partly by autoregulatory mechanisms in some vascular beds that would serve to offset the degree of vasodilatation possible. Although data are limited, it is also possible that there may be an uneven distribution of KCA-sensitive KATP channels throughout the vasculature. In certain blood vessels, such as cerebral arteries, expression of KATP function appears to be adversely affected by disease states such as hypertension and diabetes in animal models such as the SHR and streptozotocin-diabetic rat. Finally, it is clear from vascular studies in animals that KCAs have potential in the treatment of hypertension and angina. Possibilities also exist for the treatment of pulmonary hypertension where manipulation of KATP channels may yet provide a selective pulmonary vasodilator (Peacock, 1993) but further studies are clearly required. The KCAs seem also to be beneficial in a model of cerebral

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10 Cardiac Potassium Channel Modulators: Potential for Antiarrhythmic Therapy M.C. SANGUINETTI1 & J.J. SALATA2 1Division

of Cardiology, University of Utah, Salt Lake City, UT, USA,

2Department

of Pharmacology, Merck Research Laboratories, West Point, PA, USA. 10.1 Introduction

Cardiac arrhythmias arise from disturbances in the normal spread of cellular excitability and/or regional refractoriness, and can result from localized tissue damage (e.g., ischemia), anatomical anomalies or drug treatment. In the United States alone, sudden cardiac death is estimated to account for over half a million deaths yearly, and about 80% are believed to be caused by ventricular tachyarrhythmias (Morganroth and Bigger, 1990; Panidis and Morganroth, 1983). Modulation of K channel activity has been shown in numerous animal models and in some clinical studies to represent beneficial therapy in the treatment of arrhythmias that arise from such disturbances. The rationale for use of these agents is rather simplistic. Activation of K+ conductance reduces electrical excitability, an effect useful in arrhythmias arising from enhanced automaticity. Block of K+ conductance delays repolarization of action potentials, a useful mechanism to terminate some forms of reentrant-based tachyarrhythmias. The key to successful termination or prevention of an arrhythmia by either approach is to match the correct drug (and dosage) with an arrhythmia of defined origin, a difficult and often impossible clinical task. For example, lengthening of action potential duration (APD) with a K channel blocker (KCB) can prolong the refractory period sufficiently to slow or prevent a tachyarrhythmia based upon a reentrant circuit. However, excessive lengthening of action potential duration can result in the triggering of early afterdepolarizations (EADs), one probable cellular mechanism of torsades de pointes arrhythmias (Janse and Wit, 1989; Surawicz, 1989) (Figure 10.1). Druginduced torsades de pointes is a polymorphic ventricular tachycardia that is preceded by excessive QT lengthening and is characterized by a sinusoidal twisting of the QRS axis around the isoelectric line, with a cycle length that varies between 5–20 beats (Keren and Tzivoni, 1991; Surawicz, 1989). Several class 1A (e.g., quinidine, procainamide, disopyramide, aprindine) and class III (e.g., amiodarone, sotalol) antiarrhythmic drugs have been reported to induce

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this arrhythmia. However, no useful correlation between plasma concentrations, or extent of QT prolongation by these drugs and their propensity to cause torsades de pointes has been established

Figure 10.1 Therapeutic and toxic effects of class III antiarrhythmic agents depend upon extent of APD prolongation.

(Surawicz, 1989). Treatment of torsades de pointes usually consists of cardiac pacing, or infusion of isoproterenol or magnesium sulfate (Keren and Tzivoni, 1991; Surawicz, 1989). In animal and tissue models, these drug-induced arrhythmias can also be terminated with an activator of ATP-sensitive K channels (KATP), e.g. cromakalim (CRK). Evidence from animal studies suggest that excessive doses of K channel openers are equally proarrhythmic, caused by an increased dispersion in refractoriness. Obviously, there is not a single mechanism that will terminate all types of arrhythmias and as discussed above, most K channel modulators (KCMs) can also induce arrhythmias when used inappropriately. This can easily be demonstrated in animal models. For example, in anesthetized rabbits infused with the β -agonist methoxamine, there is a striking correlation between doses of various class III agents required to prolong QTU interval by 20% and doses required to induce arrhythmias with features akin to torsades de pointes (Carlsson et al., 1990). Despite the obvious limitations of KCBs as antiarrhythmic agents there has been much recent effort directed towards their development. In large part this can be traced to the publishing of interim results of the Cardiac Arrhythmia Suppression Trial (CAST), where it was reported that encainide and flecainide (class IC agents)

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increased mortality relative to placebo (The Cardiac Arrhythmia Suppression Trial (CAST) Investigators, 1989). The CAST report obviously raised questions regarding the risk versus benefit of all then currently available antiarrhythmic agents, and provided great impetus to develop new drugs that acted via mechanisms other than slowed conduction. In this chapter we will review the rationale for using KCMs as antiarrhythmic drugs and discuss the underlying mechanisms of action for some of these agents. It should be stressed that other well established antiarrhythmic mechanisms exist (e.g., block of Na or Ca channels, or β -adrenergic receptors), but are not the subject of this chapter. Not all class III agents act by block of cardiac K channels. For example, ibutilide is reported to prolong action potentials by activating a slow inward Na+ current, and at high concentrations activates an unidentified instantaneous outward K+ current, thereby limiting the prolongation of APD mediated through Na channel activation (Lee, 1992). 10.2 Arrhythmogenic Mechanisms Susceptible to K Channel Modulators A broad spectrum of cellular mechanisms underly clinically important arrhythmias, many of which are poorly defined or have been studied only in animal models. Three major categories of arrhythmogenic mechanisms are usually recognized: enhanced or abnormal automaticity, triggered activity, and reentry (Task Force, 1991). Within each category exist several types of underlying mechanisms. For example, triggered activity can arise from either EADs or delayed afterdepolarizations (DADs). It is generally believed that arrhythmias arising from EAD-based, but not DAD-based triggered activity can be affected by KCMs. KCBs worsen, whereas K channel openers (KCOs) can terminate EAD-based arrhythmias. A very extensive review of the mechanisms underlying the complex ventricular arrhythmias that result from myocardial ischemia and infarction should be consulted for details on this subject (Janse and Wit, 1989). 10.2.1 Automaticity Agents that enhance outward K channel conductance can be useful in the treatment of some types of abnormal, or enhanced normal automaticity of atrial tissue. For example, opening of K channels that are activated by acetylcholine (IK (ACh)) slows phase 4 depolarization of atrial pacemaker cells and increases maximum diastolic potential of all atrial myocytes.

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10.2.2 Triggered Activity Based on EADs Early afterdepolarizations (EADs) are slow response action potentials that are activated during the beginning of the terminal phase of repolarization (Figure 10.2). These mini-action potentials arise most readily when outward current is reduced and inward Ca2+ current is enhanced, such as may occur subsequent to treatment with certain KCBs. EADs can act to trigger tachyarrhythmias, and only occur at slow stimulation rates in vitro or slow heart rates in vivo. EADs can be suppressed by increased stimulation rate, or by enhancing outward current, for example by activation of the delayed rectifier K+ current (IK) or IK(ATP), or by block of Ca2+ current. 10.2.3 Reentry Reentry can be dependent upon Na channel or Ca channel function. Na channeldependent reentry is characterized by conduction of the excitable wavefront encroaching on refractory tissue (so-called short excitable gap). Figure 10.3 shows

Figure 10.2 Induction of early afterdepolarizations (EAD) by dofetilide. Superimposed action potential recordings from an isolated guinea pig ventricular myocyte during control at a stimulation frequency of 1 Hz and after 10 min of dofetilide (30 nM). (Unpublished observation)

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Figure 10.3 Proposed mechanisms of reentry and its termination by class 1 agents (panel A) and class III agents (panels B and C). A: Area of impaired conduction and excitability is delineated by two small lines. B: Prolongation of refractoriness blocks reentrant wavefront. C: Fibrillation is caused by numerous reentrant pathways; prolongation of refractoriness terminates arrhythmias. From Task Force of the Working Croup on Arrhythmias of the European Society of Cardiology (1991) with permission.

schematics of reentrant circuits based upon long to very short excitable gaps and the changes in conduction or refractoriness that can theoretically terminate arrhythmias based upon these different reentrant circuits. Reentry that arises from long excitable gaps can be suppressed by slowing conduction with Na channel blockers, so-called class I antiarrhythmic agents. Reentry based upon short or very short excitable gaps are most effectively treated with agents that prolong refractoriness, so-called class III antiarrhythmic activity. Specific reentrant arrhythmias that are considered to be dependent on a short excitable gap include atrial flutter, atrial fibrillation, circus movement tachycardia in Wolfe-Parkinson-White syndrome, polymorphic and sustained monomorphic ventricular tachycardia, bundle branch reentry and ventricular fibrillation (Task Force, 1991). These arrhythmias can often be suppressed by prolonging APD, for example by blocking IK. 10.3 Cardiac K Channels The cellular basis for the long plateau phase of cardiac myocytes is complex, but results primarily from inward (‘anomalous’) rectification and/or very slow

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activation of multiple types of K channels. Cardiac K channels can be grouped together into either voltage-dependent, cation-activated or ligand-activated channels. Voltage-dependent types conduct delayed rectifier (IK) inward rectifier (IK1) and transient outward K+ currents (IA). Cation-activated types conduct K+ currents activated by intracellular Na+ (IK(Na)) and intracellular Ca2+ (IK(Ca)). Ligand-activated K channels conduct currents activated by acetylcholine (IK (ACh)), arachidonic acid (IK{AA)) and phosphatidylcholine (IK(PC)), or blocked by intracellular ATP (IK(ATP)). Multiple types of IK and IA currents have been identified. Three distinct types of current have been identified in isolated cardiac cells based upon differences in rate of activation, rectification properties, and pharmacology using whole-cell voltage clamp techniques. These types are: 1) slowly activating, outwardly rectifying IK (IKs, Ix2); 2) rapidly activating, inwardly rectifying IK (IKr, Ix1), and 3) rapidly activating, outwardly rectifying IK (called IRAK in rats, IKur in human myocytes). Several of the channel proteins that conduct cardiac K+ currents have been cloned and sequenced. These include Kv4.2 and Kv1.4 (IA current), Kv1.1, Kv1. 2, Kv1.5 (ultrarapid delayed rectifiers, such as IRAK and HK2), IRK1 (IK1current), GIRK1 (IK(ACh) currennt), and IsK (IKs current). 10.4 K Channel Blockers Much effort has been directed in the past decade towards the discovery and development of drugs that have antiarrhythmic properties dependent on their ability to prolong cardiac refractoriness. These drug candidates have usually been discovered at pharmaceutical companies that screen their compound libraries, or a limited series of chemicals based upon unique structural similarities to known class III agents, for appropriate biological activity in simple assays. The usual assays rely on some measurement of cardiac refractoriness in isolated cardiac tissue preparations, most commonly isolated papillary muscles, Purkinje fibers or atria that are stimulated at a constant rate during which the effects of compounds on effective refractory period (ERP) or action potential duration (APD) are determined with a paired pulse protocol. Most often the APD is measured at the point of 90% repolarization, and is referred to as APD90. If microelectrodes are used to record action potentials, then the effect on maximum upstroke velocity (Vmax) and action potential configuration can also be determined. Limited information regarding possible mechanism of action can be determined by this method; e.g., Na channel blockers will decrease Vmax; IA blockers will reduce the size of the notch characteristic of phase 1 repolarization; Ca channel blockers will decrease the amplitude of the plateau phase; blockers of the inward rectifier K+ current will slow the rate of terminal (phase 3) repolarization (Martin and Chinn, 1992). Of course, the most likely scenario is that a specific compound will have multiple activities and therefore, change the configuration of the cardiac action potential in a complex and oftentimes

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uninterpretable manner. An even simpler screening assay relies on only measuring the contractile force of papillary muscles or muscle strips using small force transducers. ERP can be determined in these preparations by observing the amplitude of contraction of the stimulated beat immediately after the paired pulse stimulation. Alternatively, compounds can be screened for their ability to displace radiolabeled drugs with known and desirable activity, such as [3H]dofetilide, a specific IKr blocker. Class III activity of some agents were determined after compounds were shown to have useful antiarrhythmic activity in standard arrhythmia models. The structures of several compounds with class III antiarrhythmic activity are shown in Figures 10.4 and 10.5. 10.4.1 Delayed Rectifier K Channel Blockers The majority of class III antiarrhythmic agents act at least in part by blocking the cardiac delayed rectifier K+ current, IK. Recent studies have shown that ‘IK’ is actually the composite of several distinct currents and that some class III drugs may be specific blockers of only one of these currents. Multiple types of IK channels The voltage-dependent outward K+ current activated during the plateau phase of the cardiac action potential, and recorded during depolarizing steps in whole-cell mode of voltage clamp is actually the composite of as many as three distinct K+ currents (IKs, IKr, IKur). The relative contribution of each of these distinct currents to net IK varies considerably among species and perhaps between different regions of the heart. For example, an IKr-like current is the largest IK component in rabbit and cat (Colatsky et al., 1990); IKs is the largest IK component in guinea pig (Sanguinetti and Jurkiewicz, 1990b); and IKur is by far the largest component of net IK in human atrial cells (Wang et al., 1993). Many recently developed class III agents block IKr at concentrations below that required to block other types of K+ currents (e.g., IKl, IA, IK(ATP)) or the other components of IK (IKs or IKur). A component of delayed outward current (‘ixl’) that displayed inward rectifying properties was originally described in Purkinje fibers by Noble and Tsien (1969). Subsequent studies (Attwell and Cohen, 1979; Cohen and Kline, 1982, Kline and

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Figure 10.4 Methanesulfonanilide class III antiarrhythmic agents. Compounds are listed in decreasing order of potency for prolonging refractory perio in isolated ferret papillary muscle, ranging from 13 nM for (1) to 44 μ M for (9).

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Figure 10.5 Class III antiarrhythmic agents not structurally related to sotalol. Compounds are listed in decreasing order of potency for prolonginj refractory period of isolated papillary muscle, ranging from 47 nM for (10) to 10 μ M for (15).

Cohen, 1984) documented experimental artifacts caused by inadequate space clamp and K+ accumulation between myocytes of this multicellular preparation, which left many investigators to question the original data (Jaeger and Gibbons, 1985). However, the original concept by Noble and Tsien that at least two components (ixl and ix2) of delayed rectifier current exist in mammalian cardiac myocytes appears to have been correct, as more recent studies have described two distinct, time-dependent components of IK in embryonic chick (Shrier and Clay, 1986), guinea pig (Balser et al., 1991; Balser and Roden, 1988; Sanguinetti and Jurkiewicz, 1990b, 1991), canine (Gintant, 1993) and human (Wang et al., 1993) cardiac myocytes. Similar to ‘ixl’, the property of IKr that distinguishes it from other cardiac delayed rectifier K+ currents (IKur and IKs) is a pronounced rectification at positive membrane potentials. IKr was originally defined in guinea pig cardiac myocytes as a component of IK that was specifically blocked by E– 4031 (Sanguinetti and Jurkiewicz, 1990b). Methanesulfonanilide class III antiarrhythmic agents such as E-4031 (4) and dofetilide (2) were shown to block net IK in a voltage-dependent manner, with block being greatest when assessed with short test pulses to potentials less than 0 mV (Carmeliet, 1992; Jurkiewicz and Sanguinetti, 1993; Sanguinetti and Jurkiewicz, 1990b). This effect of these drugs could either result from a voltage-dependent block of a single current, or from specific block of only one of two components of net IK. Several lines of evidence suggested that the latter interpretation was correct, including: (1)

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differential modulation of the two components by β -adrenergic receptor agonists (only IKs is significantly activated by isoproterenol) (Sanguinetti et al., 1991); (2) removal of extracellular K+ increases IKs, but decreases IKr (Sanguinetti and Jurkiewicz, 1992); (3) absence of extracellular Ca2+ shifts voltage-dependence of IKs activation to more positive potentials, but IKr to more negative potentials (Sanguinetti and Jurkiewicz, 1992); (4) block of IKr but not IKs by 10 μ MLa3+ (Sanguinetti and Jurkiewicz, 1990a). As defined by sensitivity to block by 5 μ M E–4031 (Figure 10.6), IKr is half-activated at –22 mV, whereas IKs (current not blocked E–4031) is half-activated at +20 mV. The slope factor for IKr and IKs was determined to be 7.5 and 12.7 mV, respectively. In guinea pig ventricular myocytes IKs requires very long pulses to reach a pseudo steady-state level of outward current (time constants for activation: ― 1=400 msec, ― 2=2.4 sec at 0 mV), whereas IKr is fully activated in less than 500 msec (― =50 msec at 0 mV) (Sanguinetti and Jurkiewicz, 1994). Based on the kinetics of IK deactivation, Chinn (1993) also proposed that two distinct channel types contribute to net IK in guinea pig ventricular myocytes. Two distinct components of guinea pig atrial IK were also described at the single channel level, with single channel conductances of 1–3 pS (IKs?) and 10 pS (IKr?) (Horie et al., 1990). IKr and IKs contribute almost equally to net IK during the plateau phase of guinea pig ventricular myocytes (Courtney et al., 1992; Sanguinetti and Jurkiewicz, 1990b). When currents recorded during very long voltage clamp pulses are compared, IKs is about 11 times greater than IKr. Rabbit ventricular and nodal cells are reported to have only one type of Ik, with properties similar to IKr, including inward rectification and sensitivity to block by E-4031 (Shibasaki, 1987; Veldkamp et al., 1993). Shibasaki (1987) studied the properties of IK in rabbit atrial pacemaker myocytes using single channel recording techniques. The current characterized in the study by Shibasaki had several properties in common with IKr of guinea pig cardiac myocytes, including: (1) rectification at potentials >0 mV, (2) rapid activation, (3) halfmaximal activation at

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Figure 10.6 Voltage-dependent block of delayed rectifier K+ current (IK) by dofetilide in isolated guinea pig ventricular myocytes. A and B: Currents recorded during 225 msec pulses to test potentials of –20, 0, +20 and +30 mV before and after exposure of cells to 1 μ M dofetilide. C and D: Magnitude of time-dependent lK (C) and lK tail current (D) before (o) and after 1 μ M dofetilide (― ). Dofetilide specifically blocks one component (lkr), having no effect on another component (lKs) of net lK. From Jurkiewicz and Sanguinetti (1993).

–25 mV and a slope factor of 7.4 mV, (4) little or no inactivation. It was not determined if IK of rabbit pacemaker cells was blocked by methanesulfonanilides, but IK of rabbit myocytes isolated from other regions of the heart are at least as sensitive to block by these agents as is IKr of guinea pig myocytes (Carmeliet, 1991, 1992, 1993). The mechanism of rectification of

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rabbit nodal cell IK was studied by recording single channel current ensembles. Based upon the kinetics of deactivation of these currents, Shibasaki (1987) proposed that IK rectification results from an ultrarapid inactivation of channels that develops faster than activation at positive test potentials. The net result of these two opposing processes is a smaller current at positive test potentials than would be predicted solely upon the progressive increase in the electrochemical driving force for K+. Specific lKr blockers Several recently developed antiarrhythmic drugs that are still undergoing clinical evaluation were synthesized to mimic the main structural features of sotalol (9), a methanesulfonanilide (Figure 10.4). These new generation class III agents are extremely potent, blocking IKr and prolonging APD at concentrations as low as 1 nM, with half-maximal effects ranging from 13 nM [for L–691 121, (Lynch et al., 1993)] to 0.5 μ M [for UK–66 914 (6), (Baskin et al., 1991)]. Concentrations required to block IKr correlate well with those needed to prolong APD in vitro, or QTC in vivo. The term ‘specific blocker’ is a relative term at best. In the context used here it is meant to distinguish compounds that block IKr (or similar current) at concentrations at least 100–fold less than that required to affect other cardiac currents. For example, dofetilide blocks only IKr in cardiac myocytes, but also blocks a Ca-activated K channel in hippocampal CA1 neurons (McLarnon and Wang, 1991). IKr blockers lengthen cardiac refractoriness in a dose-dependent manner, with increases in QTC occurring at doses parallel to those that lengthen either effective or relative refractory periods of the ventricle. E–4031, UK–66 914, and dofetilide had no effect on mean arterial blood pressure, PR interval, QRS interval or ventricular conduction time, but did decrease heart rate, and increase maximum rate of left ventricular pressure development (LV+dP/dt) in anesthetized dogs (Wallace et al., 1991). D-sotalol had the same effects as these agents on all parameters except LV+dP/dt, which was unchanged. The increase in LV+dP/dt by these agents results from a positive inotropic effect secondary to prolonged APD (Baskin et al., 1991; Sanguinetti and Siegl, 1992). In human isolated ventricular strips, almokalant prolonged APD and contractile force in a concentration-dependent manner (Carlsson et al., 1991). At 1 μ M, almokalant (10) lengthened APD90 by 62%. Similar to studies in dogs, dofetilide (Sedgwick et al., 1991, 1992) and sotalol (Nademanee et al., 1985) produce QTC prolongation in humans without effects on conduction parameters (e.g., AH, HV, PR or QRS intervals). Dofetilide, UK–66 914, L–691121 (1), E–4031, sotalol, sematilide (8), almokalant, and MS–551 (13) exhibit clear suppression of ventricular tachyarrhythmias initiated by programmed electrical stimulation or an acute ischemic insult in dogs with previous myocardial infarcts (Lynch et al., 1992, 1993). In contrast to class I agents, E–4031, d-sotalol and L–691 121 do not

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suppress the frequency of premature ventricular complexes (PVCs) early after myocardial infarction in dogs (Lynch et al., 1990, 1993). Many lethal arrhythmias develop several days after a myocardial infarction. An attempt to model this situation led to the development of a conscious (Patterson et al., 1982) and later an anesthetized canine model of sudden cardiac death. E–4031, L–691 121 and MK–499 (3) prevented death in response to a secondary acute myocardial infarction (induced by application of current to the lumen of the circumflex coronary artery) in the setting of chronic myocardial infarction in anesthetized dogs (Lynch et al., 1990, 1993, 1994). For inclusion in these studies, the dogs were required to have inducible ventricular arrhythmias in response to programmed ventricular stimulation 6 to 10 days after an anterior myocardial infarction. The initial infarction was produced by a 2 hour occlusion of the left anterior descending coronary artery followed by reperfusion. The survival of dogs administered either placebo or MK–499 for the 3 hour period after the onset of acute posterolateral myocardial ischemia is shown in Figure 10.7. IKr blockers prolong APD of isolated rabbit and cat myocytes to a greater extent than in isolated guinea pig ventricular myocytes. This reflects the fact that guinea pig myocytes have a large IKs, whereas the cat and rabbit have very little if any IKs. There is also significant variability in the extent of APD lengthening by IKr blockers in different regions of the heart. For example, as shown in Figure 10.8, dofetilide has a greater effect on dog Purkinje fibers than on ventricular muscle (Gwilt et al., 1991). Prominent age-related changes in the effect of some class III antiarrhythmic agents on cardiac APD suggest that the contribution of IKr to net repolarizing K+ current changes during development in the rat. Potent class III antiarrhythmic agents such as almokalant and dofetilide do not prolong APD of adult rat myocytes, although these compounds do block a very small outward K+ component in these cells (Abrahamsson et al., 1994). The component of current (presumedly IKr) blocked by these drugs probably has no significant effect on net outward current in these cells, and hence does not prolong action potentials. However, the action potential configuration of rat embryonic heart myocytes is more like guinea pig (prolonged plateau phase) than adult rat, due to the relative lack of a prominent IA in the fetal heart, and is markedly prolonged by these drugs. At high concentrations, these drugs induce EADs and rhythm abnormalities in the fetal heart, which may be the mechanism of the embryotoxic effects of these agents in rats (Abrahamsson et al., 1994).

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Figure 10.7 Survival of MK–499 versus vehicle-treated postinfarction dogs with baseline inducible arrhythmias, expressed as a function of time after development of acute posterolateral myocardial ischemia. Adapted from Lynch et al. (1994).

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Figure 10.8 Prolongation of action potentials recorded from canine ventricular muscle (A) and Purkinje fiber (B) by increasing concentrations of dofetilide. Adapted from Gwilt et al. (1991) with permission.

The effects of WAY–123 398 (5) on action potentials of isolated dog Purkinje fibers and membrane currents in cat ventricular myocytes have been compared with three other methanesulfonanilide compounds, dofetilide, E–4031 and dlsotalol (Spinelli et al., 1993). The concentrations required to prolong APD to –60 mV of the Purkinje fibers by 20% were 210 nM for WAY–123 398 and 6 nM, 30 nM, and 38 μ M for dofetilide, E–4031 and dl-sotalol, respectively. Unlike the other three drugs, the effects of WAY–123 398 on APD to –60 mV in muscles stimulated at 1 Hz reached a maximal value (from a control of 264 msec to 439 msec at 1 μ M). The mechanism for the plateau in effect is unknown, but is a useful property since it might limit the proarrhythmic potential of the drug. WAY–123 398 had no effect on Vmax of the canine action potential at 3 μ M, or

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on L-type ICa, IA or IK1 in voltage clamped feline myocytes at 10 μ M. Consistent with APD prolongation and IK block in vitro, WAY–123 398 prolonged cardiac refractoriness without effects on conduction in anesthetized dogs (Spinelli et al., 1992). An interesting difference exists between the manner in which IK in guinea pig and cat myocytes is blocked by these drugs. In cat myocytes high concentrations of these drugs (e.g., 1 μ M WAY-123 398) completely block the deactivating tail current measured after a depolarizing pulse, whereas the timedependent current measured during the test pulse is only reduced by approximately 40% (Spinelli et al., 1993). In guinea pig myocytes, these drugs only block a fraction of the tail current, leaving a component that has an amplitude consistent with that expected for deactivation of the large IKs measured during the test pulse (Sanguinetti and Jurkiewicz, 1990b). Two possible explanations for this difference is that either these drugs block cat IK in a voltage-dependent manner (rapid block at less depolarized levels) or that the slowly activating outward current resistant to block by these drugs in cat myocytes is not a typical IKs. The efficacy of IKr blockers may also vary as a function of plasma levels of K+ in a manner not previously recognized. Torsades de pointes caused by class III antiarrhythmic drugs is often associated with hypokalemia (Janse and Wit, 1989; Keren and Tzivoni, 1991; Surawicz, 1989). The conductance of IKr is dependent upon [K+]e; low [K +]e reduces the magnitude of IKr in guinea pig ventricular myocytes (Sanguinetti and Jurkiewicz, 1992) and rabbit Purkinje fibers (Scamps and Carmeliet, 1989). It is possible that hypokalemia reduces the conductance of IKr channels in addition to its well-documented reduction in conductance of IK1. The resultant decrease in net outward K channel conductance, combined with IKr block produced by treatment with class III drugs may prolong action potentials beyond the point of therapeutic intent, resulting in torsade de pointes. The complex and interdependent effects of these factors on repolarization of cardiac action potentials are important considerations in the development of future antiarrhythmic drugs designed to specifically prolong refractory period at the onset of tachyarrhythmias. Nonspecific blockers of lK Several compounds that possess class III antiarrhythmic activity have many different, often poorly defined cellular mechanisms of action. Agents that were synthesized or developed for treatment of diseases other than arrhythmias have subsequently been discovered to block cardiac IK and have class III antiarrhythmic activity. Examples include ketanserin and ICS 205–930 (5–HT2 blockers), melperone (a neuroleptic), and tacrine (a cholinesterase inhibitor) (Morgan and Sullivan, 1992). The best example of a drug with multiple mechanisms of action is amiodarone; it is also arguably the most effective class III antiarrhythmic agent and is currently approved for clinical use. Amiodarone is a benzofuran derivative that was originally developed as a coronary vasodilator, but was subsequently discovered to have antiarrhythmic activity,

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especially after chronic administration (Singh et al., 1989). It is one of only a few drugs (along with β -blockers) that has been reported to actually reduce mortality of patients following myocardial infarction (Ceremuzynski et al., 1992). The cellular mechanisms of action of amiodarone are quite complex, but includes block of IKs (Balser et al., 1991), INa, ICa, and β - and β -adrenergic receptors (Task Force, 1991; Singh et al., 1993). Agents with multiple activities (e.g., block of IKr channels and β -adrenergic receptors like sotalol) may have a wide spectrum of activity since arrhythmias that arise from diverse cellular mechanisms may be affected. For this reason, it was hypothesized that the complex electrophysiological actions of a compound such as amiodarone may be preferable to those that act specifically on a single channel type such as dofetilide or E–4031 (Singh et al., 1993). Racemic sotalol is both a β -adrenergic receptor blocker (β -blocker) and an IKr channel blocker. The l-isomer is a more potent β -blocker (about 50–times greater) than the d-isomer, but the two compounds lengthen APD equipotently (Kato et al., 1986). In isolated rabbit Purkinje fibers, 10 μ M sotalol lengthened APD by 41% and decreased IK by 50%, a concentration that was without significant effect on IK1, IA or INa (Carmeliet, 1985). In guinea pig ventricular myocytes sotalol blocks IKr but had no effect on IKs at a concentration of 100 μ M (Sanguinetti and Jurkiewicz, 1990b). Sotalol was shown to be more effective than six class I antiarrhythmic agents in preventing death and recurrence of ventricular arrhythmias in a multicenter study (Beta-blocker Heart Attack Trial Research Group, 1982). It is unclear whether the protection afforded by sotalol is related to block of β -adrenergic receptors, or to reduction of IKr, since β -blockers have also been demonstrated to reduce mortality hi patients with a prior myocardial infarction (Norwegian Multicenter Study group, 1981; Beta-blocker Heart Attack Trial Research Group, 1982). Quinidine (a class 1A agent) and MS–551 (a class III agent) have been reported to block IK in a time- and voltage-dependent manner. Quinidine (Roden et al., 1988) and MS–551 (Nakaya et al., 1993) delay the activation of IK in guinea pig and rabbit ventricular myocytes, respectively. This delay was quantified by measuring the amplitude of deactivating tail currents following pulses of increasing duration to a common test potential. The tail amplitude becomes progressively larger as the test pulse duration is increased, in proportion to the slowly activating current recorded during the test pulse. In the presence of quinidine or MS–551, the tail currents were decreased more following short (<200 msec) compared to longer pulses. The most straightforward interpretation of such an effect is that these drugs slow the rate of IK activation, perhaps by preferential block of IK channels in the closed state which is then relieved upon depolarization. However, in rabbit sinoatrial and atrioventricular node cells the block of IK by quinidine was enhanced with increasingly larger depolarizations, and block was increased with longer pulses (Furukawa et al., 1989). Thus, in rabbit nodal cells quinidine was proposed to block open Ik channels preferentially. An expected result of such a mechanism would be an enhanced

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block of IK during fast heart rates. An alternate interpretation of the data from these previous experiments is that quinidine preferentially blocks IKr, which activates much faster than IKs. In rabbit nodal cells the predominant component of IK is IKr, whereas IKs dominates during long test pulses in guinea pig ventricular myocytes. Thus, preferential block of IKr in guinea pig myocytes results in a slowing of net IK activation, whereas the true nature of quinidine block of IKr (open channel block) is easily detected in rabbit nodal eels where IKs is very small. This was subsequently shown to be true for quinidine, where IKr is blocked by the drug at much lower concentrations than that required for equivalent block of IKs in guinea pig ventricular myocytes (Balser et al., 1991). Open channel block of IKr by quinidine is similar to that produced by the methanesulfonanilides (see section 10.5). MS–551 (13) prolongs APD of rabbit papillary muscles at concentrations between 0.1 and 10 μ M, but without effect on the maximum upstroke velocity of action potentials. At a concentration of 10 μ M, MS–551 decreased IK by 39.8% and IK1, IA, and ICa by about 30% (Nakaya et al., 1993). In the same study it was reported that 100 μ M d-sotalol blocked IK but not IK, or IA. MS–551 (0.3 mg/kg bolus, followed by 0.05 mg/kg constant infusion) was shown to be quite effective in suppressing the induction of atrial flutter or fibrillation in dogs that was easily caused by rapid pacing in the absence of drug. This activity was associated with an increase in atrial ERP from 128 to 168 msec (Hirata et al., 1991). This compound represents an example of an antiarrhythmic drug that has multiple cellular mechanisms of action, yet still exhibits in vivo activity that is similar to agents that are relatively specific blockers of a single type of cardiac K channel. Properties and pharmacological modulation of delayed rectifier K channels expressed in heterologous systems It is oftentimes difficult to study the effects of antiarrhythmic drugs on a single K + current in cardiac cells due to the presence of multiple types of K+ currents that overlap one another in the time and voltage domains. Expression of cloned cardiac channels in non-cardiac cells represents a recent development that will aid in characterization of these channels and their modulation by drugs. Examples of channels studied using this approach are HK2 and minK (IsK). When expressed in Xenopus oocytes, HK2 induces a current similar to IKur and minK induces a current with properties like IKs. HK2 (human cardiac Kv1.5) is 605 amino acids in length and has a relative molecular weight of 66 640 (Snyders et al., 1991). HK2 has six putative transmembrane regions as determined by hydropathy analysis, and is more abundant in human atria than in ventricle. HK2 is 86% identical to its rat Kv1.5 homolog, and shares 96% identity within the putative transmembrane domains (Tamkun et al., 1991). When expressed in mouse Ltk- cells HK2 is an outward rectifier that exhibits only very slow inactivation. It activates with a time constant of about 2 ms at +50 mV and has a half-point for activation of –10 mV (Snyders et al., 1991). HK2 is

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blocked by the antiarrhythmic agent quinidine with an IC50 of 9 μ M when assayed at a test potential of 0mV. The current is also blocked by the class III antiarrhythmic agent clofilium at concentrations less than 1 μ M (Snyders et al., 1991). IKs has been well characterized in whole cell voltage-clamp recordings of freshly isolated cardiac myocytes from guinea pigs and frogs. A channel protein called minK (IsK) is believed to form cardiac IKs channels. The minK channel was originally cloned by functional expression from a rat kidney cDNA library (Takumi et al., 1988). MinK is encoded on a single exon and has a molecular weight of only 14.7kDa (129–130 amino acids), considerably less than most other K channel subunits, which range between 50–100kDa (Philipson and Miller, 1992, Swanson et al., 1993). The deduced protein sequence is unlike any other ion channel described to date. Whereas most K channel subunits contain many hydrophilic, membrane spanning domains, minK has only one such putative domain of 23 amino acids. Messenger RNA encoding minK has been detected by Northern blot analysis in mouse, neonatal rat (but not adult), guinea pig, and human heart (Folander et al., 1990; Honore et al., 1991; Swanson et al., 1990). MinK protein has been detected immunologically in ventricular and sinoatrial node cells of the guinea pig (Freeman and Kass, 1993). When minK is expressed in oocytes the induced current (IminK) exhibits characteristics that are similar to IKs recorded in bullfrog or guinea pig cardiac myocytes (Swanson et al., 1993). Both currents are highly selective for K+ (K+>Rb+>Cs+>Na+>Li+) (Hadley and Hume, 1990; Hausdorff et al., 1991), and the reversal potential for IminK shifts approximately 58 mV per decade change in [K +]e, as predicted by the Nernst equation for a perfectly K+ -selective channel (Hausdorff et al., 1991). The permeability ratio of K+/Cs+ is 16.4 for IminK expressed in oocytes, but this ratio is reduced to 4.6 when a point mutation (F55T) is introduced into the minK protein (Goldstein and Miller, 1991). This rinding has been cited repeatedly as the best evidence that minK proteins form functional channels and are not simply activators of an endogenous channel. Both IKs and IminK are blocked by mM levels of external Ba2+ (Folander et al., 1990; Honore et al., 1991), and are enhanced by increases in [Ca2+]i or intracellular cAMP (Blumenthal and Kaczmarek, 1992; Busch et al., 1992; Honore et al., 1992). The single channel conductance of IKs in guinea pig ventricular myocytes (Walsh et al., 1991) and IminK in Xenopus oocytes (Hausdorff et al., 1991) is below detectable levels (less than 1 pS). The antiarrhythmic agents, clofilium and NE10064 block IminK and guinea pig IKs with approximately equal potencies (Folander et al., 1990; Varnum et al., 1993). The bulk of evidence is consistent with the hypothesis that minK protein is the primary subunit of functional cardiac IKs channels.

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10.4.2 Inward Rectifier K Channel Blockers The conductance of inward rectifier K+ current (IK1) in atrial and ventricular cells is high at negative membrane potentials and as such ‘clamps’ the resting potential near the equilibrium potential for K+ (about–85mV). The inward rectifier K channel of cardiac and many other cell types exhibits pronounced rectification at potentials positive to EK, the equilibrium potential for K+. In a series of elegant studies, this was demonstrated to be caused by at least two distinct molecular mechanisms: a very rapid and voltage-dependent inactivation, and block of open channels by intracellular divalent cations, primarily Mg2+ (Ishihara et al., 1989; Matsuda, 1988, 1991; Matsuda et al., 1987; Vandenberg, 1987), but also Ca2+ (Delmar et al., 1991; Mazzanti and DeFelice, 1990; Mazzanti and DiFrancesco, 1989). Upon depolarization, IK1 channels close almost instantly. IK1 channels remain closed throughout the plateau phase and thus do not contribute to the repolarization process until later in the action potential cycle. Thus, it is the activation of other outward K channels during the plateau phase that initiate repolarization and return the membrane potential to a level (about –20 mV) where IK1 channels can again open and contribute to terminal (phase 3) repolarization. Block of IK1 channels is often viewed as an undesirable mechanism by which to prolong ventricular refractoriness, since excessive block of this conductance would increase membrane resistance during diastole which in turn could result in membrane depolarization. An expected consequence of membrane depolarization would be a graded decrease in conduction velocity due to a voltage-dependent inactivation of Na channels. Despite these theoretical limitations, at least one novel class III antiarrhythmic agent, terikalant (11) was recently developed that is reported to act by specific block of IK1 channels. The RP 58866 (racemate) and its active enantiomer, terikalant (the (S)(–)isomer, Figure 10.5) are benzopyran derivatives that were discovered in a screen using guinea pig papillary muscles. The mechanisms of action of these compounds and in vivo activities were described recently (Escande et al., 1992), and are summarized below. Terikalant is about 150 –times more potent in prolonging cardiac action potentials than the inactive enantiomer. Terikalant prolonged APD90 of guinea pig papillary muscles and right atria when paced at 1 Hz by 21–23% at a concentration of 0.3 μ M. Similar effects were observed in isolated human atrial tissue (37% increase in APD90 at 0.15 μ M). The compound also slowed the spontaneous firing rate of isolated guinea pig right atria by 36% at much higher concentrations (30 μ M). Resting membrane potential was unaffected by 30 μ M terikalant in an isolated guinea pig right atrium. The lack of effect on resting membrane potential by a compound that blocks IK1 by >50% suggests that only a fraction of the normal outward IK1 in this tissue is required to maintain the membrane potential near EK during diastole. Prolongation of action potential duration and effective refractory period was attributed to block of IK1. In guinea pig myocytes RP 58866 reduced IK1 measured at –40 mV by 31% and

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51% when tested at 3 and 10 μ M, respectively. At concentrations up to 30μ M, RP 58866 had no effect on IKs, IKATP, IKACh, or T- and L-type Ca2+ currents. The effect of these compounds on IKr was not determined. In anesthetized dogs, terikalant dose-dependently (0.03–1 mg/kg) prolonged atrial, nodal, and ventricular refractory periods; with a maximal increase of 52, 57, and 21%, respectively. Thus, similar to the IKr blockers, a greater prolongation of ERP was observed in atrial than in ventricular tissue. QTC was increased by 57% at 0.1 mg/kg. At doses up to 1 mg/kg terikalant had no significant effect on AH, HV, or QRS intervals, nor did it alter tissue excitability. This ‘pure’ class III action was associated with antiarrhythmic activity in several models of reentrant ventricular tachycardia or fibrillation. For example, pretreatment with terikalant at 0.3 mg/kg protected 4 of 5 micropigs from ischemia-induced ventricular fibrillation resulting from a 20 min ligation of the left ventricular coronary artery. An interesting correlation was found between the concentration of terikalant and some other class III antiarrhythmic drugs (e.g., clofilium (12)) to lengthen action potential duration of isolated Purkinje fibers and that required to displace the binding of an ― 2 opioid receptor ligand ([3H]–3–PPP) from a rat cerebral cortex preparation (Jeanjean et al., 1993). All the compounds were about 10–100 times more potent at displacing the β 2 ligand compared to their class III activity. For example, the IC50 for inhibition of [3H]–3–PPP binding by RP5866 and clofilium were 6.1 nM and 40 nM, respectively, whereas the EC20 for prolongation of APD90 for the two drugs was 14nM and 3μ M, respectively. These studies suggest that the β 2 opioid receptor is a protein very similar to one or more cardiac K channels. The clinical significance of the binding of these class III antiarrhythmic agents with β 2 opioid receptor sites in the brain is unknown. 10.4.3 Transient Outward K Channel Blockers IA activates extremely rapidly upon membrane depolarization, then inactivates quickly with a voltage-independent time constant of about 50 msec. Thus, this current contributes to net repolarizing outward current predominantly during the initial phase of action potential repolarization. Before the channels can reopen during a subsequent action potential, the channel must undergo a process referred to as recovery from inactivation. When studied in rats and rabbits, IA is characterized by a rather slow recovery from inactivation. This slow recovery translates into a reduced current magnitude at high stimulation rates in vitro, or high heart rates in vivo. Because of this property, the contribution of IA to net repolarizing K+ current would diminish during a tachycardia. Block of this current would therefore seem not to represent a useful antiarrhythmic mechanism, since less prolongation of action potential duration would result during tachycardias, a frequency-dependent profile opposite to that most desired (see

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section 10.5). However, unlike rat and rabbit, the rate of recovery from IA activation is rapid in human cardiac myocytes, such that the magnitude of IA is not diminished during rapid pacing (Fermini et al., 1992). The magnitude of IA is much greater in the epicardium than in the endocardium of some species such as the dog and cat (Furukawa et al., 1990). In the rabbit, IA is only slightly larger in epicardial than in endocardial myocytes isolated from the wall of the ventricle, however papillary muscles have a much reduced IA (Fedida and Giles, 1991). The variable expression of IA channels across the wall of the ventricle may have significant consequences with respect to the antiarrhythmic/proarrhythmic effect of compounds that block IA. This possibility needs to be explored in the future. IA channels may represent a useful antiarrhythmic target, but no specific and potent blocker of this current has been described. A few agents (e.g., tedisamil, clofilium) are relatively potent blockers of IA, but these compounds also block other K+ currents. Tedisamil (15) was developed as a bradycardic agent for treatment of angina pectoris in patients with impaired cardiac function (Grohs et al., 1989), but it also has antiarrhythmic activity. At 1–4 mg/kg, tedisamil reduced ventricular fibrillation induced by occlusion of the left anterior descending coronary artery in rats (Beatch et al., 1991). This compound prolongs APD in rat ventricular myocytes by increasing the rate of IA channel inactivation. This action was hypothesized to result from preferential block of channels in the open state, an effect that would mimic accelerated inactivation (Dukes et al., 1990). Tedisamil also prolongs APD of guinea pig ventricular myocytes. However, these cells do not express IA channels. In the guinea pig, tedisamil acts instead by blocking IK at concentrations similar to that required for block of IA in the rat (Dukes et al., 1990). Thus, this drug has class III activity in both species, but the underlying mechanisms of action are different. Other compounds such as clofilium (12) and quinidine also reduce rat IA by increasing the apparent rate of current inactivation (Castle, 1991; Imaizumi and Giles, 1987). The block of IA by clofilium is very sensitive to voltage and repetitive pulsing (‘frequency-dependent block’). The enhancement of block that occurs during repetitive stimulation (Figure 10.9A) is believed to result from a relatively slow rate of drug dissociation from the channel during the interpulse interval, whereas the voltage-dependence of block (Figure 10.9B) arises from a preferential block of open channels (Castle, 1991). The greater the depolarization, the greater the probability of open channels that become available for block. An unusual feature of clofilium is that exposure of cells for several hours is required before steady-state effects on IA magnitude are observed. Presumedly this ultraslow onset of activity by clofilium is related to its lipophilicity and quaternary ammonium group. The onset of IA block by LY97119, a des-ethyl analog of clofilium with a tertiary nitrogen group, is much more rapid than clofilium, requiring only a few minutes to reach steady-state. In addition, LY97119 also blocks IK1 with an IC50 of about 3μ M (Arena and Kass, 1988; Castle, 1991). Similar to tedisamil, clofilium also blocks IK of guinea pig

CARDIAC K CHANNEL MODULATORS: POTENTIAL FOR ANTIARRHYTHMIC THERAPY 295

ventricular myocytes (Arena and Kass, 1988), albeit at concentrations 10–times that required for a comparable decrease of IA in rat myocytes. The class 1 antiarrhythmic agents, quinidine, flecainide and propafenone block IA (IC50=3.9, 3.7 and 3.3 μ M, respectively) in addition to INa and IK when studied in isolated rat ventricular myocytes (Slawsky and Castle, 1994). Flecainide and quinidine, but not propafenone exhibit significant frequency-dependent block of IA in this preparation.

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Figure 10.9 Voltage- and use-dependent block of IA in isolated rat ventricular myocytes by clofilium. Top panel: Relative amplitude of current during repetitive pulsing at 1 Hz. Myocytes were preincubated with drug for 3–9 hours prior to applying the train of clamp pulses. Bottom panel: Voltage-dependence of use-dependent block of IA by clofilium. The

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amplitude of IA measured during a test pulse to +40 mV was dependent upon the prepulse voltage during the preceeding train of 10 pulses. From Castle (1991) with permission.

10.4.4 lK(ACh) Channel Blockers Many class I antiarrhythmic agents antagonize the actions of acetylcholine in addition to blocking INa. Quinidine, disopyramide, procainamide, pilsicainide, flecainide and propafenone all decrease the effects of acetylcholine on IK(ACh) recorded in guinea pig ventricular myocytes (Inomata et al., 1993), although pilisicainide, procainamide and disopyramide do so by blocking ACh receptors (Inomata et al., 1993; Nakajima et al., 1989). Block of atrial IK(ACh) channels causes tachycardia in innervated hearts, shortens refractory period and accelerates A-V nodal conduction time. Activation of these channels greatly shortens APD and hyperpolarizes the resting potential of all atrial cells, a possible antiarrhythmic action under conditions in which atrial APD is excessively prolonged, such as could occur after overdosing with a ‘pure’ class III antiarrhythmic agent. 10.4.5 IK(Na) Blockers A large conductance K channel activated by high levels of intracellular Na+ was first described by Kameyama et al. (1984). Conditions that lead to marked increases in [Na+]i increase the open probability of these channels, in turn causing a shortening of APD. R56865 is a benzothiazolamine derivative that was developed as an antiischemic agent, but also has antiarrhythmic activity in several in vivo and in vitro models (Koch et al., 1990). R56865 was also reported to potently protect against ischemiaand reperfusion-induced arrhythmias in anesthetized rats (Garner et al., 1990). For example, ischemia-induced ventricular fibrillation was reduced from >50% to 8% in rats administered 2 mg/kg R56865. The cellular mechanism of action of these protective effects is not exactly clear, but may relate to its ability to block the transient inward current (TI) and IK(Na) (Leyssens and Carmeliet, 1991; Luk and Carmeliet, 1990), two currents that are normally activated after treatment of cardiac cells with toxic concentrations of cardiac glycosides. The reduction of TI by R56865 was not due to direct block of Na+/Ca2+ exchange current or the nonselective cation channel, the two components of TI . Instead, it appears that the drug may somehow reduce Ca2+ overload, perhaps via interferring with Ca2+ release from the sarcoplasmic reticulum (Leyssens and Carmeliet, 1991). As expected from block of outward current through IK(Na) channels, R56865 limits the extent of APD shortening normally caused by

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ouabain intoxication (Vollmer et al., 1987). At concentrations up to 3 μ M, this compound had no effect on force of contraction or APD in isolated guinea pig papillary muscles, but markedly attenuated the APD shortening of ouabain (0.8 μ M) at concentrations as low as 1 nM. In these isolated papillary muscles, R56865 (10 nM) also prevented the extrasystoles that developed in the presence of ouabain when muscles were stimulated at a frequency of 1 Hz (Vollmer et al., 1987). In single guinea pig ventricular myocytes R56865 blocked INa, albeit at higher concentrations (IC50=1 μ M) than those required to antagonize the effects of ouabain, whereas other K+ currents (IK1, steady-state outward current) were unaffected at 10 μ M (Himmel et al., 1990). R56865 reduced IK(Na) activated by ouabain in guinea pig myocytes by 35–70% at 0.1 μ M and by 76–98% at 1 μ M. The importance of IK(Na) activation during ischemia or cardiac glycoside intoxication and the contribution of this current to net outward current during these conditions has not been quantified sufficiently to determine whether modulation of this current as an antiarrhythmic mechanism warrants further study. 10.5 Rate-dependent Effects of K Channel Blockers D-sotalol and more potent methanesulfonanilides such as dofetilide, almokalant (10), E–4031 and L–691 121 are characterized by a rate-dependent lengthening of cardiac APD or effective refractory period (ERP) in vitro (Baskin et al., 1991; Gwilt et al., 1991; Lynch et al., 1992) and of ERP or QTC interval in vivo (Lynch et al., 1992, 1993), such that diminished activity is observed as heart rate or stimulation frequency is increased. As noted above, these agents are specific blockers of a time-dependent, delayed rectifier K+ current (IKr) of cardiac myocytes (Carmeliet, 1985, 1991, 1992; Lynch et al., 1993; Sanguinetti and Jurkiewicz, 1990b). Theoretically, an ideal drug would prolong refractory period preferentially during tachyarrhythmias and thus, would block outward current (e.g., IKr) in a direct frequency-dependent manner (more block at high heart rates) (Carmeliet, 1993; Colatsky et al., 1990; Hondeghem and Snyders, 1990; Jurkiewicz and Sanguinetti, 1993). The rate-dependent efficacy of class III antiarrhythmic agents is further compromised in the presence of adrenergic stimulation. Isoproterenol functionally antagonizes the ability of E–4031 to prolong refractory period or APD in guinea pig papillary muscles or ventricular myocytes, respectively (Sanguinetti et al., 1991). The potential mechanisms for frequency-dependent drug action are many-fold, but only a few have actually been tested experimentally. Frequency-modulated actions of Ca or Na channel blocking agents have usually been attributed to either a channel-state-dependent or voltage-dependent block of channels. For example, class I antiarrhythmic drugs such as lidocaine decrease the upstroke velocity of cardiac action potential of single cells, decrease conduction velocity in multicellular cardiac preparations, and widen the QRS complex of the

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electrocardiogram more at high, relative to low heart or stimulation rates. This augmented effect is a direct result of an increase in the number of Na channels that are blocked during more rapid, repetitive membrane depolarizations (Hondeghem and Katzung, 1984). By analogy, a logical explanation for the rate-dependent effect of class III agents would be the opposite phenomenon—a decrease in the number of K channels blocked by such agents as the frequency of repetitive depolarizations is increased (Hondeghem and Snyders, 1990). This potential mechanism has been evaluated and shown not to be the case for dofetilide (Carmeliet, 1992; Jurkiewicz and Sanguinetti, 1993), almokalant (=H234/09) (Carmeliet, 1991), or sematilide, d,l–sotalol and E–4031 (Krafte and Volberg, 1993). Dofetilide prolongs APD of isolated guinea pig ventricular myocytes more at 0.5 Hz than at 4 Hz when tested at a maximal effective concentration of 1 μ M. Whole-cell voltage clamp experiments revealed that 1 μ M dofetilide completely blocks IKr, regardless of stimulation frequency (Jurkiewicz and Sanguinetti, 1993). This observation rules out the possibility that the frequencydependent effects of these drugs on APD of cardiac cells results simply from a ‘use-dependent’ or voltage-dependent unblock of IKr. Even at submaximal concentrations, dofetilide and almokalant do not exhibit decreased blocking activity at elevated stimulation frequencies. In fact, Carmeliet has reported that these agents block IK of rabbit myocytes slightly more when voltage clamp pulses are applied at fast relative to slow rates (Carmeliet, 1991, 1992). Unlike guinea pigs, rabbit myocytes have a single type of IK, which is similar to IKr with respect to its rectification properties and sensitivity to methanesulfonanilides (Carmeliet, 1992; Shibasaki, 1987). The rate-dependent increase in block of rabbit IK by dofetilide and almokalant results from preferential block of IK channels in the open (as opposed to closed) state. Unblock of IK is extremely slow during interpulse intervals at ‘diastolic’ potentials (<–75 mV), such that rate-dependent block of IK can only be observed with very slow pacing (Carmeliet, 1991, 1992). The number of IKr channels that open during a depolarization increases as the duration of the clamp pulse (or action potential) is lengthened. If methanesulfonanilides are open channel blockers of IKr, a greater number of channels should be blocked at slow heart rates since APD is prolonged as heart rate is lowered. This effect alone would seem to explain the rate-dependent actions of these drugs. However, as discussed above, rate-dependent drug action is still observed even when IKr is completely blocked with high concentrations of drug. An obvious alternative mechanism for the observed rate-dependent effects of these drugs on APD and QTC interval could be that methanesulfonanilides affect another repolarizing current(s) in a rate-dependent manner. However, these agents have no apparent effect on any other currents examined, including IKs, ICa, IA, and IK1 (Carmeliet, 1985; Sanguinetti and Jurkiewicz, 1990b). In a study of the mechanism of action of dofetilide in guinea pig ventricular myocytes, it was concluded that the diminished functional effect at high stimulation rates of this and similar agents results from a rate-dependent increase in the magnitude of IKs. As the rate of voltage clamp pulsing (or heart rate) is

300 K CHANNELS AND THEIR MODULATORS

increased there is less time for the deactivation (closure) of channels during the abbreviated interpulse (or diastolic) interval. Since the deactivation rate of IKs is relatively slow (135 msec at –85 mV), some channels activated during a prior voltage clamp pulse (or action potential) remain open if the subsequent pulse (or action potential) is initiated after a short interval. In this manner, accumulated activation of IKs can occur during rapid pulsing. The greater contribution of IKs to net outward current would partially offset the effect of IKr block. In single guinea pig myocytes the potential role of other outward currents in the rate-dependent diminuition of dofetilide activity was determined (Jurkiewicz and Sanguinetti, 1993). It was shown that rapid pulsing (0.5 versus 4 Hz) did not increase the magnitude of outward currents other than IKs (Jurkiewicz and Sanguinetti, 1993). However, in the whole heart or isolated tissue, outward currents other than IKs are modulated by the rate of repetitive depolarizations. Most notable is the ratedependent increase in the magnitude of IK1 that occurs in multicellular preparations. K+ accumulates within intercellular clefts during rapid pulsing, resulting in a localized increase in [K+] between myocytes. Elevated extracellular [K+] results in a significant increase in the magnitude of IKI (Gintant et al., 1991). At the tissue or whole organ level, the rate-dependent increase in IKI would also offset the ability of IKr blockers to significantly prolong APD at high stimulation or heart rates. The role of an augmented IKI in offsetting the functional effects of IKr block at high stimulation rates may be especially important in species such as the rabbit where IKs is not present. Thus, the cellular mechanism for the observation that many class III antiarrhythmic agents are less effective at elevated heart rates is a consequence of their mode of action: specific block of IKr. Block of IKr is actually enhanced by rapid pulsing due to preferential block of channels in the open state. However, the relative contribution of IKr to net repolarizing outward current during the plateau phase of the action potential is diminished at elevated heart rates. This results in a diminished functional response to block of IKr (prolongation of APD and QTC) upon an elevation in heart rate. This is an unfortunate feature of these drugs, since presumedly it would be more desirable to prolong cardiac action potential preferentially during brief runs of tachycardia or at high heart rates, with little or no effects at normal heart rates. 10.6 Modulators of IK(ATP) IK(ATP) blockers and activators may be either antiarrhythmic or arrhythmogenic. These apparently conflicting results may be explained largely by considering the pre-existing conditions or electrophysiological mechanism of a particular arrhythmia. Both the action (block or activation) on the channel and the electrophysiological substrate (reentry versus abnormal automaticity) contribute to the ultimate arrhythmia related outcome. The electrophysiological effects of IK (ATP) modulation have been studied extensively in the heart (Nichols and

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Lederer, 1991), and recent reviews have described the implications and controversies for the therapeutic potential of IK(ATP) modulation and its role in arrhythmias and ischemic heart disease (Cavero and Premmerieur, 1994; Siegl, 1994; Wilde and Janse, 1994). IK(ATP) is normally inactive in cardiac cells and only functions when [ATP]i is compromised, such as during myocardial ischemia. Blockers and activators of IK(ATP) will be discussed together in this section largely in the context of ischemia related arrhythmias. In cardiac cells, during acute hypoxia, ischemia or metabolic inhibition there is a profound shortening of the cardiac APD (De Mello, 1958; Carmeliet, 1978). Noma (1983) initially identified a K+ -selective channel in guinea pig ventricular myocytes that was inhibited by physiological [ATP]i and other adenine nucleotides, and proposed that these KATP channels might cause APD shortening when cardiac metabolism is compromised. In heart, half-maximal inhibition of IK (ATP) occurs at [ATP]i of 15–100 μ M (Noma and Shibasaki, 1985; Findlay, 1988). The [ATP]i during normal physiological conditions is 5–10 mM. Only during prolonged or severe ischemia does [ATP]i fall to less than 75% of control levels (Elliot et al., 1989; Deutsch et al., 1991), raising questions about the physiological and pathophysiological role of IK(ATP) in the heart. However, numerous factors other than [ATP]i modulate IK(ATP) activity. Nucleotide diphosphates positively regulate the activity of IK(ATP) and shift the set point for ATP-induced inhibition to lower [ATP]i. During ischemia, as [ATP]i falls and [ADP]i rises (Figure 10.10), the K(ATP) open channel probability (Po) increases and APD rapidly shortens (Gassner and Vaughan-Jones, 1990). Thus the primary biochemical determinant of IK(ATP) activation is the ATP/ADP ratio. Other physiologic modulators of IK(ATP) activation, include pHi, monovalent and divalent cations, lactate, G-proteins, and A1-adenosine receptor stimulation (Keung and Li 1991; Findlay, 1987; Kirsch et al., 1990; Cuevas et al., 1991; Fan and Makielski, 1993). IK(ATP) appears to be preferentially suppressed by ATP derived from glycolytic metabolism, which may localize in a sarcolemmal membrane associated pool where [ATP] may vary widely from the cytoplasmic concentration (Weiss and Lamp, 1989). In addition, due to the high density of KATP channels and their large single channel conductance β 35 pS (depending on [K+]o), activation of <1% of the channels could shorten APD by ~50%. These and other numerous considerations for physiologic modulation of IK(ATP) have been reviewed in detail recently (Nichols and Lederer, 1991). Activation of IK(ATP) may act as an intrinsic cardioprotective mechanism when cardiac metabolism is compromised (Escande and Cavero, 1992; Lynch et al., 1992; Sanguinetti, 1992). The resultant shortening of APD, reduces Ca2+ influx into the cell via ICa and possibly Na+/Ca2+ exchange, which in turn decreases contractility. This decrease in mechanical function of ischemic myocytes protects the affected cells by reducing oxygen demand, preserving [ATP]i, increasing cell survival and limiting myocardial infarct size (Gross and Auchampach, 1992), which ultimately may reduce the likelihood of arrhythmias. On the other hand, because levels of ischemia are highly variable throughout the

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heart, the resulting non-uniform decrease in APD increases the dispersion of refractoriness throughout the myocardium, delays electrical uncoupling between non-uniform regions and predisposes the heart to reentrant arrhythmias. IK(ATP) may be more pronounced in the epicardium than in the endocardium and this gradient may contribute further to the non-uniform changes in refractoriness during ischemia (Furukaw et al., 1991; Antzelevitch and Di Diego, 1992). During myocardial ischemia, an increase in K+ efflux and an accumulation of [K +] may shift E to less negative potentials producing membrane depolarization o K and also may increase outward IK1 current thereby shortening APD further (Yan et al., 1993). These primary and secondary effects of IK(ATP) activation enhance the likelihood of arrhythmia development during the early stages of myocardial ischemia when premature beats, ventricular tachycardia and ventricular fibrillation occur primarily as a consequence of reentry (Pogwizd and Corr, 1987; Janse and Wit, 1989). In this setting, blockers of IK(ATP) should be antiarrhythmic by preventing the excessive and/or non-uniform shortening of APD that leads to reentry. In cases of sub acute ischemia or in severe or prolonged ischemia, arrhythmias are commonly due to ‘abnormal automaticity’ or ‘triggered activity’ such as DAD or EAD. In situations of ischemia followed by reperfusion, the underlying electrophysiologic mechanism for reperfusion induced-arrhythmias is unclear but both reentry and triggered activity may be involved (Janse and Wit, 1989; Pasnani and Ferrier, 1992). In K+ depolarized cardiac tissue, abnormal automatic activity occurs at reduced resting membrane potentials. Activation of IK(ATP) increases K+ conductance and hyperpolarizes the membrane potential out of the range where abnormal pacemaker activity occurs (Imanishi et al., 1984; Liu et al., 1988; Spinelli et al., 1991). Prolonged ischemia leads to a rise in [Ca2+]i which may progress to Ca2+ overload and the appearance of DAD (January and Fozzard, 1988). Activation of lK(ATP) may reduce the degree of Ca2+ loading (as discussed above) and provide an hyperpolarizing effect and both actions may reduce the propensity for DAD. EAD occur from multiple influences that act to excessively prolong APD (January and Moscucci, 1992). Activation of IK(ATP) prevents or abolishes EAD because it provides a relatively large hyperpolarizing influence at plateau potentials. In general, arrhythmias arising from abnormal automaticity or triggered activity are expected to be prevented or suppressed by activators of IK(ATP) and aggravated or induced by blockers of IK(ATP). Thus, the opposing electrophysiologic and metabolic cardioprotective effects resulting from block or activation of IK(ATP) underlie the dilemma of whether block or further activation of IK(ATP) ultimately will be beneficial in preventing ischemia related arrhythmias.

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10.6.1 IK(ATP) Blockers Many of the nonspecific organic K channel blockers such as 4–aminopyridine (4AP), tetraethylammonium (TEA) quinine, quinidine, amiodarone, and verapamil block IK(ATP) (Davies et al., 1989; Haworth et al., 1989; Undrovinas et al., 1990). Sulfonylurea drugs, like glyburide (glibenclamide) and tolbutamide (Table 10.1), appear to specifically block IK(ATP) but are not tissue selective. For instance, their potent block of IK(ATP) in pancreatic β -cells accounts for their anti-diabetic action (Sturgess et al., 1985; Trube et al., 1986; Zunkler et al., 1988). Sulfonylureas also inhibit IK(ATP) in cardiac cells (Hamada et al., 1990), but less potently. Glyburide inhibits with Ki of 5–20 μ M in heart versus 4–50 nM in β -cells (Nichols and Lederer, 1991). The concentrations of tolbutamide that inhibit whole cell IK (ATP) are 7 μ M in β -cells and 400 μ M in cardiac cells, respectively (Belles etal., 1987). Sulfonylureas reduce IK(ATP) by decreasing the P° of the channel without affecting the sensitivity to [ATP]i (Figure 10.10). Sulphonylureas also are reported to affect

Figure 10.10 [ATP] dependence of KATP channel activity. Under normal conditions, [ATP], is relatively high and the open probability (Po) for the KATP channel is near 0.

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Middle curve represents ATP-sensitivity of KATP channels in the absence of drug. As ischemia proceeds, [ATP]i falls, [ADP]i rises and consequently, Po increases. Activators of IK(ATP) or potassium channel openers (PCO) decrease the sensitivity of the KATP channels to [ATP], increasing the Po at any given [ATP]i compared to control. Clyburide (sulph) does not reduce the channel’s sensitivity to [ATP], but reduces Po at all [ATP]i values. Adapted from Baghdady and Nichols (1994).

cardiac metabolism by stimulating glycolytic ATP synthesis and thereby maintaining [ATP]i (Tan et al., 1984; Schaffer et al., 1985). Recent studies demonstrate a dissociation between the effects of glyburide on cellular K+ loss and on APD during hypoxia and ischemia (Yan et al., 1993). These actions of sulphonylureas suggest that additional or alternative mechanisms apart from direct block of IK(ATP) may need to be considered. The fatty acid, 5– hydroxydecanoate (5–HD) is suggested to be a cardioselective blocker of IK(ATP) (McCullough et al., 1991; Notsu et al., 1992), but further evaluation of the actions of this compound is needed, since it appears to affect other cell types as well (Jagger et al., 1993). Specific IK(ATP) blockers do not affect cardiac APD or resting membrane potential under normal physiological conditions, reflecting the lack of contribution of KATP channels to normal cardiac electrophysiology. During acute ischemia (Gassner and Vaughan-Jones, 1990; Pasnani and Ferrier 1992), hypoxia (Sanguinetti et al., 1988; Wilde et al., 1990; Deutsch et al., 1991; Nakaya et al., 1991) or metabolic blockade (Furukawa et al., 1991; Notsu et al., 1992), however, block of IK(ATP) by glyburide or 5–hydroxydecanoate attenuates the shortening of APD and ERP in vitro in isolated ventricular myocardium (Cole et al., 1991; McCullough et al., 1991). However, when glucose is present in the hypoxic superfusate APD does not shorten and glyburide has no effect (Venkatesh et al., 1991; Yan et al., 1993). Glyburide also prevents shortening of monophasic APD during coronary artery occlusion in vivo in rabbits and dogs (Smallwood et al., 1990). Glyburide attenuates both the rise in [K+]o and delay of conduction that occur during acute ischemia following coronary artery occlusion in dogs (Bekheit et al., 1990) These actions may prevent reentrant arrhythmias (as outlined above) occurring as a consequence of excessive or non-uniform decreases in refractoriness throughout the myocardium during the acute phase of myocardial ischemia (Janse and Wit, 1989). A number of initial reports indicated that glyburide and other sulphonylureas, prevent arrhythmias and reduce the incidence of ventricular fibrillation (VF) during ischemia. Kantor et al. (1990) reported that glyburide reduced K+ accumulation and abolished VF during both regional and global ischemia in rat hearts. In similar studies during low flow ischemia in rats, both glyburide and tolbutamide decreased the rate and incidence of ventricular tachycardia and significantly reduced the occurrence of VF (Wolleben et al., 1989). In dogs with healed myocardial infarctions, glyburide also prevented VF induced by a 2 min coronary artery occlusion during exercise in 13 of 15 animals (Billman et al., 1993). In humans, glyburide was effective in reducing the frequency and severity of ventricular arrhythmias occurring during

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transient ischemia in non-insulin-dependent diabetic patients with coronary artery disease. In these patients, however, glyburide had no effect on nonischemic arrhythmias and the number or length of ischemic episodes (Cacciapuoti et al., 1991) . In general, these studies support the antiarrhythmic action of IK(ATP) blockers in the setting of acute ischemia. In other studies, especially in models of ischemia and reperfusion, IK(ATP) blockers are mostly arrhythmogenic. The effects of glyburide were studied in arterially perfused guinea pig right ventricular wall preparations in vitro, during 20 min of no flow ischemia followed by 60 min of reperfusion (Cole et al., 1991). Glyburide treatment hastened and augmented the rise in resting tension during the ischemic period and elicited arrhythmias during the reperfusion period. Mechanical function failed to recover with glyburide treatment, suggesting Ca2+ overload which might have led to the triggered arrhythmias that did not occur in control preparations. In a similar study, Pasnani and Ferrier (1992) examined the effects of glyburide on reperfusion-induced arrhythmias but differentiated between types of arrhythmias based on their underlying mechanism. Glyburide suppressed the reentrant arrhythmias but potentiated DAD and triggered arrhythmias. In anesthetized dogs during acute myocardial ischemia, the ventricular fibrillation threshold (VFT) rose in untreated dogs but was not significantly changed in the glyburide treated animals (Smallwood et al., 1990). This suggested a greater propensity toward the development of VF during block of IK(ATP), but the method used to determine VFT was questioned and the significance of a change in VFT is unclear. Block of IK(ATP) in particular circumstances of acute ischemia may prevent malignant ventricular arrhythmias that arise due to reentry. This short term but perhaps life-saving benefit may come at the cost of a longer term detriment to myocardial salvage and protection both directly on cardiac cells and through an interference with an adaptive increase in coronary blood flow to the ischemic myocardium, thereby only postponing risk of arrhythmia development. 10.6.2 K(ATP) Activators Since the discovery of the smooth muscle relaxant activity of the original potassium channel activators (KCAs), cromakalim (CRK), nicorandil and pinacidil, intense synthetic efforts have yielded a cornucopia of these agents aimed at multiple therapeutic targets. A partial list of IK(ATP) activators appears in Table 10.1. In cardiac cells it is well established that the mechanism of action of these agents, as the name implies, is activation of IK(ATP) (Arena and Kass, 1989) (Sanguinetti et al., 1988; Escande et al., 1989; Sanguinetti, 1992). IK(ATP) activators increase the Po of KATP channels at any given [ATP]1 (Figure 10.10). This action results in a relatively large increase in outward repolarizing current especially during the plateau of the action potential which produces shortening of APD and a decrease in the cardiac refractory period both in vitro and in vivo

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(Damiano et al., 1993). The decrease in APD has been observed in numerous studies in isolated multicellular preparations and isolated cardiac myocytes (see Wilde and Janse, 1994). This effect is dependent on concentration, temperature, species, tissue type (larger in atria than ventricle) and is generally greater at lower stimulation frequencies. The changes in APD are more pronounced in the subepicardium than the subendocardium (Di Diego and Antzelevitch, 1993). When the conductance of other K channels, such as IK1 is increased by other factors (e.g. elevated [K+]o) the effect of IK(ATP) activators is blunted (Imanishi et al., 1983; Liu et al., 1988). During normal conditions, these agents do not hyperpolarize the resting membrane potential of ventricular tissues which normally rest near or at EK (Hiraoka and Fan, 1989; Spinelli et al., 1991), but do hyperpolarize atrial muscle (Yanagisawa and Tiara, 1980). Activators of IK(ATP) are expected to increase the incidence of reentrant arrhythmias because they augment the shortening of APD during acute ischemia (Cole et al., 1991; Venkatesh et al., 1992), and may enhance the non-uniform changes in cardiac refractory period (Antzelevitch and Di Diego, 1992). Because activators of IK(ATP) may have extremely pronounced effects on cardiac refractoriness, they may induce arrhythmias even during normoxic conditions. In isolated guinea pig hearts in the presence of high concentrations of pinacidil (30– 50 μ M) there was a 20% incidence of spontaneous VF and induction of VF was possible in 50–100% of hearts (Padrini et al., 1992). In isolated canine Table 10.1 KATP Channel Modulators KATP Channel Blockers Sulphonylurea derivatives glyburide (glibenclamide) glipizide tolbutamide gliquidone glisoxepide Miscellaneous 5–hydroxydecanoate KATP Channel Activators Benzopyran derivatives cromakalim (BRL 34915) levcromakalim (BRL 38227) bimakalim (SR 44866; EMD 52692) celikalim (WAY 120,491) HOE 234 Cyanoguanidines derivatives pinacidil P 1075; P 1188

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LY 222675 Nicotinamides nicorandil (SG 75) KRN 2391 Thioformamide derivatives aprikalim (RP 52891) RP 49356 Miscellaneous diazoxide minoxidil sulphate

epicardium exposed to pinacidil, premature electrical stimulation evoked reentrant excitation (Di Diego and Antzelevitch, 1993). Intracoronary infusion of high doses of nicorandil induced ventricular premature beats in dogs (Kojima et al., 1990). In similar studies, during administration of pinacidil and CRK at doses that produced extreme hypotension, supraventricular arrhythmias were easily induced (Spinelli et al., 1990). During ischemia, cardiac tissues are more sensitive to activators of Ik(ATP). Accordingly, CRK and pinacidil increased the rate of ventricular tachycardia and shortened the time to development of VF during low flow ischemia in isolated perfused rat hearts (Wolleben et al., 1989). Pinacidil facilitated the induction of VF in isolated rabbit hearts perfused with low [K+] buffer (Chi et al., 1993). There was an increase in the incidence of reperfusioninduced VF with CRK in isolated rat hearts subjected to 25 min of global ischemia (Grover et al., 1990). In conscious previously infarcted dogs, a moderately hypotensive intravenous dose of pinacidil increased the incidence of lethal arrhythmias induced by a secondary acute ischemic event at a site remote from the previous myocardial infarction (Chi et al., 1990). These and a host of other studies demonstrate the arrhythmogenic action of activators of IK(ATP) especially during acute ischemia. Activators of IK(ATP) mostly are antiarrhythmic in the setting of abnormal automaticity or triggered activity. In isolated canine ventricular myocytes, pinacidil suppressed or diminished EADs induced by Bay K 8644, ketanserin or applied current, DADs induced by ouabain, and abnormal automaticity induced by barium (Spinelli et al, 1991). Similarly, pinacidil and CRK abolished EADs and triggered activity in canine Purkinje fibers exposed to quinidine, cesium or sematilide (Fish et al., 1990). In canine Purkinje fibers surviving myocardial infarction or exposed to barium or low [K+]o, CRK and nicorandil decreased pacemaker activity (Imanishi et al., 1984; Bril and Man, 1989). CRK suppressed spontaneous activity and prevented the enhancement of this automaticity by norepinephrine, barium, the cardiac glycoside strophanthidin as well as DAD induced by high [Ca2+]o (Liu et al., 1988). In vivo studies mostly support the

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findings of in vitro models. Carlsson et al. (1990, 1992) examined the ability of pinacidil and two of its analogs (P1075 and P1188) to suppress arrhythmias caused by repolarization abnormalities in a rabbit model of the long QT syndrome in vivo and in rabbit ventricular tissues in vitro. All three activators of IK(ATP) were highly effective in suppressing clofilium-induced polymorphic ventricular tachyarrhythmias in vivo as well as clofilium-induced EADs and triggered activity in vitro. Similarly, pinacidil suppresses ventricular arrhythmias induced by administration of cesium in anesthetized rabbits (Fish et al., 1990). Delayed spontaneous arrhythmias occurring 22–24 hours after initiating coronary artery ligation in the dog are suppressed by pinacidil (Kerr et al., 1985). Intracoronary administration of CRK reduced the incidence of VF in an anesthetized dog coronary occlusion/reperfusion model (Grover et al. 1990). Because of the concomitant reduction in the extent and/or severity of myocardial ischemic injury in this model the antiarrhythmic effect might have been an indirect consequence of the myocardial salvaging effect of the IK(ATP) activator. In a similar study intracoronary administration of CRK or pinacidil produced threefold increases in coronary blood flow and prevented cesium chloride induced arrhythmias in anesthetized dogs (D’Alonzo et al., 1993). Under conditions of controlled coronary blood flow, CRK and pinacidil failed to reduce the cesium induced-ectopy, suggesting that the antiarrhythmic efficacy was a consequence of increased coronary blood flow. However, the ability of IK(ATP) activators to limit myocardial injury in models of ischemia and reperfusion is a matter of controversy because of conflicting results. K(ATP) channels are found in many other tissues including, smooth muscle, skeletal muscle, pancreatic β -cells and neurons (Ashcroft, 1988; Rorsman and Trube, 1990). The ubiquitous nature of these channels makes selective antiarrhythmic therapy a complicated challenge (Robertson and Steinberg, 1990; Longman and Hamilton, 1992). The use of IK(ATP) modulators in treating arrhythmias may be overshadowed by their effects on other tissues which may predominate over the direct myocardial effects. Foremost among these are the vascular smooth muscle effects of IK(ATP) activators, which occur at concentrations 10–100 fold less than the effects on cardiac muscle. Consequently, effects on systemic blood pressure and/or coronary blood flow can limit the ability of IK(ATP) modulators to affect cardiac function directly. IK(ATP) blockers or activators, respectively, may decrease or increase insulin secretion and thereby produce either hyperglycemia or hypoglycemia. These and other non-cardiac effects may preclude the use of IK(ATP) modulators as selective antiarrhythmic agents. However, the cardiac K(ATP) channel protein has been cloned recently and the channel expressed in Xenopus oocytes is not blocked by glyburide, implying that the glyburide binding protein is a separate protein or subunit of the channel. If different isoforms of the channels exist and these are localized preferentially in specific tissues, then selective therapy may be possible. Nevertheless, appropriate therapy will still require proper identification of the mechanism of the particular arrhythmia. Even if this continuing clinical challenge were

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11 Cardioprotective Properties of Potassium Channel Modulators G.J.GROSS Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

11.1 Introduction A variety of potassium (K) channels exist in the heart and coronary circulation which subserve a number of different functions. These channels have been shown to be regulated by several factors which include the membrane potential, intracellular Na+ and Ca2+ concentrations, arachidonic acid and other fatty acid derivatives. Some of these K channels have also been shown to be modulated by membrane receptors and G proteins (Lazdunski, 1992). More recently, a time independent K channel has been identified which is activated during ischemia and results in K+ loss from the myocardium and shortening of the cardiac action potential. This outward current was shown by Noma (1983) to be regulated by the intracellular concentration of ATP in guinea pig ventricular myocytes and this channel was named the ATP-regulated or dependent K channel (KATP). Since this original study (Noma, 1983), KATP channel activity has been shown to be modulated by a number of factors such as the ATP/ADP ratio, various nucleotide diphosphates, pH, lactate and oxygen-derived free radicals (Nichols and Lederer, 1991). Noma postulated that these channels might serve an endogenous cardioprotective role during the onset of ischemia when followed by timely reperfusion via their ability to accelerate the loss of electrical and contractile activity in the ischemic zone thereby preserving cellular energy reserves and preventing the loss of adenine nucleotides. On the other hand, the loss of cellular K+ and shortening of the action potential duration as a result of opening these KATP channels may also result in an increase in electrical inhomogeneity in the myocardium and may result in reentrant-type arrhythmias. Therefore, K channel activators (KCAs) and potassium channel blockers (KCBs) may both be expected to exert beneficial or detrimental effects on the ischemic myocardium and these effects may be determined by a number of factors including drug dose, animal species and, most importantly, by the experimental model and conditions used to study the particular drug of interest.

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Therefore, the purpose of this chapter is to summarize the effects of various K channel modulators (KCMs) on the ischemic myocardium. The majority of studies to date suggest that KCAs possess potent cardioprotective properties and these aspects will be emphasized. In contrast, KCBs generally have been shown to exacerbate ischemic injury or antagonize the beneficial effects of the KCAs, however, in the setting of certain types of arrhythmias, some of these compounds have been shown to possess potent antiarrhythmic activity. In some instances, KCAs have also been shown to be antiarrhythmic but in others these compounds have been proarrhythmic. The reason for these seemingly divergent effects of KCMs on arrhythmias are discussed in Chapters 10 and 16 in this book. 11.2 Evidence for an Endogenous Cardioprotective Role of the KATP Channel—The Preconditioning Phenomenon Single or multiple short periods of coronary artery occlusion interspersed with brief periods of reperfusion have been shown to protect the myocardium from irreversible tissue injury following a more prolonged period of coronary artery occlusion. This phenomenon, which was termed ischemic preconditioning, was first demonstrated by Murry et al. (1986) in dogs and has subsequently been shown to occur in several other species including pigs (Schott et al., 1990), rabbits (Liu et al., 1991) and rats (Banerjee et al., 1993). Recent evidence suggests that this also occurs in man (Deutsch et al., 1990). In the study of Murry et al. (1986) it was demonstrated that preconditioning anesthetized dogs with four 5 min episodes of coronary artery occlusion interspersed with 5 min of reperfusion prior to a 40 min occlusion period resulted in a 75% reduction in infarct size as compared to nonpreconditioned animals. Subsequently, marked reductions in infarct size were shown to occur in dogs (Gross and Auchampach, 1992a), rabbits (Liu et al., 1991) and pigs (Schott et al., 1990) following a single 5 or 10 min period of preconditioning. These marked reductions in infarct size produced by preconditioning are unparalleled by any pharmacological agent and have led to an intense search for the mechanism (s) involved in this fascinating phenomenon. Since several studies have shown that opening of the KATP channel by various KCAs results in a cardioprotective effect (Grover et al., 1989, 1990a; Cole et al., 1991) in vitro or in vivo, it seemed reasonable to propose that this channel may have an important role in ischemic preconditioning. In support of this hypothesis, Gross and Auchampach (1992a) first demonstrated that intravenous administration of the selective KATP channel antagonist, glibenclamide 10 min prior to, or 10 min following, a single 5 min preconditioning insult completely blocked the protective effect of preconditioning in dogs (Figure 11.1) at a dose (0.3 mg/kg) which did not increase infarct size by itself. In addition, the administration of the selective KCA, aprikalim, reduced infarct size to almost the same degree as ischemic preconditioning. These results suggest that the KATP

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channel appears to play a pivotal role in the preconditioning phenomenon in dogs. More recently, Auchampach et al. (1992a) found that the intracoronary infusion of an ischemia selective KATP channel antagonist (McCullough et al., 1991), 5-hydroxydecanoate (5–HD), or the nonischemia-selective antagonist, glibenclamide, blocked the beneficial effects of preconditioning in dogs independent of any changes in blood glucose levels. Again, these two compounds had no effect on infarct size in nonpreconditioned dogs at the doses used which suggested that these two agents did not antagonize the effects of ischemic preconditioning by increasing infarct size. Similarly, Grover et al. (1992) found that glibenclamide blocked preconditioning in

Figure 11.1 Area at risk as a percent of the left ventricle (AAR/LV) and myocardial infarct size expressed as a percent of the area at risk (IS/AAR) in preconditioned (pre) and nonpreconditioned dogs treated with either vehicle (CONT), glibenclamide (0.3 mg/kg IV bolus) or aprikalim (10 µg/kg+0.1 µg/kg/min IV). In glibenclamide-treated dogs, the drug was administered either 10 min before (G+P) or immediately after preconditioning (P+G) in preconditioned dogs or 10 min before the sustained ischemic insult in nonpreconditioned dogs (GLIB). All values are the mean±S.E.M. *P<0.05 versus the control group. Reprinted with permission from Circulation Research 1992, 70, 223–233.

dogs at a dose that had no effect on infarct size alone. In all of the aforementioned studies, effects of the KCMs occurred independently of any differences in hemodynamics, coronary collateral blood flow or ischemic bed size. Taken together, these results clearly suggest an important role for the KATP channel as a mediator of ischemic preconditioning in dogs. The role of the KATP channel in ischemic preconditioning is equivocal in other species. Preliminary results obtained in pigs by Schulz et al. (1994) suggest that the KATP channel is involved in ischemic preconditioning similar to that found in the dog. Schulz et al. (1994) found that glibenclamide completely blocked the effects of preconditioning in pigs at a dose which did not increase infarct size in nonpreconditioned pigs. Similarly, Taggart et al. (1993) also presented evidence (S-T segment elevation and APD70 measurements) to suggest that the KATP

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channel may modulate preconditioning in man following multiple brief occlusions during angioplasty. Results obtained in in vivo rabbit studies are less clear and appear to depend on the anesthetic used. Initial results of Thornton et al. (1993) showed that three doses of glibenclamide did not block the effects of ischemic preconditioning in pentobarbital-anesthetized rabbits even though these same doses all produced a marked increase in infarct size. In contrast, Toombs et al. (1993) found that glibenclamide blocked the beneficial effect of ischemic preconditioning in xylazine-ketamine anesthetized rabbits at a dose that had no effect on infarct size in nonpreconditioned rabbits. It is difficult to reconcile these differences in rabbit hearts based on anesthetic alone, however, no other obvious differences are apparent between these two studies to help explain the divergent results. Tan and co-workers (1993) recently studied the efficacy of ischemic preconditioning to prevent electrical uncoupling in rabbit isolated perfused papillary muscle subjected to global ischemia and reperfusion and the role of the KATP channel in this model. They found that a single 10 min period of preconditioning ischemia markedly delayed the onset of electrical uncoupling, an effect which was blocked by glibenclamide. They also showed that cromakalim (CRK), a KCA, mimicked the effect of ischemic preconditioning. The protective effects of ischemic preconditioning and CRK were both associated with an enhanced shortening of the cardiac action potential duration during a prolonged ischemic period which suggests that the KATP channel is involved in the cardioprotective effect of both interventions in the rabbit. Finally, one in vivo study by Liu and Downey (1992), and several in vitro studies (Grover et al., 1993; Fralix et al., 1993), suggested that the KATP channel is not involved in preconditioning in intact or isolated rat hearts. Thus, it appears that there are species differences concerning the role of the KATP channel in ischemic preconditioning with a more prominent role observed in larger mammals such as dog, pig and man and a lesser role in smaller animals such as the rabbit and rat. However, in spite of the lesser role for the KATP channel in mediating ischemic preconditioning in rabbits, and in particular, rats, a number of studies which will be mentioned in the next several sections suggest that KCAs have potent cardioprotective actions in both rats and rabbits and that blocking these channels by specific KCBs antagonizes the beneficial actions of these KCAs. 11.3 Effects of KCAs and KCBs in the Ischemic Myocardium In this section, the effects of KCAs and KCBs in various in vitro and in vivo models of ischemia will be summarized.

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11.3.1 In Vitro Models A number of studies have been published concerning the cardioprotective effects of KCAs in isolated globally ischemic rat and guinea pig hearts and ventricular muscle. Initial studies by Grover and co-workers (1989) showed that two KCAs, pinacidil and CRK, resulted in significant improvements in myocardial function and compliance following reperfusion in isolated buffer-perfused rat hearts subjected to 25 min of ischemia and 30 min of reperfusion. These beneficial effects of pinacidil and CRK were blocked by glibenclamide and in the case of CRK, the combination of glibenclamide and CRK produced an exacerbation of ischemia. These authors also presented data to suggest that one mechanism by which the KCAs may be acting is via preventing or reversing ischemia-induced membrane depolarization. CRK was also shown to improve the functional contractile reserve to isoproterenol and the efficiency of oxygen utilization in rat perfused hearts (Grover et al., 1990b). In addition, CRK was not effective when administered at reperfusion which suggests that KCAs are improving reperfusion function primarily by reducing the severity of injury during the global ischemic period (Grover et al., 1990b). These data clearly suggest that the timing of treatment is very important in the beneficial actions of KCAs. In a more recent study, Grover and co-workers (1991) investigated the importance of the stereoselectivity of CRK in its cardioprotective effects and determined its effects on myocardial adenine nucleotide levels in isolated isovolumically beating rat hearts. The results indicated that the (–)-enantiomer levcromakalim (LCRK) was more potent in reducing ischemic injury than CRK or its (+)-enantiomer. These compounds all reduced lactate dehydrogenase release, improved the recovery of left ventricular developed pressure and increased the time to ischemic contracture. CRK pretreatment also preserved ATP and improved the adenylate charge during ischemia. Interestingly, all these effects of CRK occurred in the absence of any negative inotropic effects of the drug prior to ischemia which is in marked contrast to the Ca channel blockers where cardioprotection is only observed when significant negative inotropic effects have occurred in the preischemic period (Sargent et al., 1991). These results suggest that KCAs are not acting simply as indirect antagonists of voltagedependent L-type Ca channels. Sargent et al. (1991) tested the specificity of glibenclamide to block the antiischemic effects of CRK and the selectivity of CRK to act via KATP channels by using other agents which produce antiischemic effects in rat isolated perfused hearts and several blockers of K channels independent of KATP. They observed that glibenclamide selectively blocked the antiischemic actions of CRK but not those of various Ca antagonists, calmodulin antagonists and Na channel blockers. Furthermore, charybdotoxin, a Ca2+ -activated KCB and E–4031, a delayed rectifier KCB, had no effect on the antiischemic actions of CRK. These results suggest that the KCAs and KCBs used in these experiments are specific

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for the KATP channel in the rat perfused heart. In agreement, several other KCAs such as nicorandil, KRN 2391, EMD 56431 and P–1075 have also been shown to be Cardioprotective in the rat isolated heart model (Mitani et al., 1991; Ohta et al., 1991a, 1993; Gross et al., 1992; Sargent et al., 1992). In elegant in vitro studies using a buffer-perfused guinea pig right ventricular wall, the electrophysiological (Cole et al., 1991) and metabolic mechanisms (McPherson et al., 1993) by which KCMs influence the ischemic-reperfused myocardium were addressed. In this preparation, electrical and mechanical activity were recorded via an intracellular microelectrode and a force displacement transducer. Membrane potentials, action potential duration and resting and developed tension were measured during 20 or 30 min of no-flow ischemia and during a 60 min reflow period in the presence or absence of the KCA, pinacidil, or the KCB, glibenclamide. The results indicated that pinacidil accelerated the rate of action potential shortening during ischemia and enhanced the recovery of developed tension following reperfusion, whereas glibenclamide attenuated the rate and magnitude of action potential shortening during ischemia and exacerbated myocardial dysfunction following reperfusion. These results suggest that changes in action potential duration are an important element in the cardiac actions of KCMs in the ischemic myocardium. More recently, this same group (McPherson et al., 1993) studied the relationship between opening the KATP channel and preservation of high energy phosphates during no-flow ischemia and reflow in the isolated guinea pig right ventricular wall. These investigators found that the KCA, pinacidil (10 µM), enhanced the rate of action potential shortening and the loss of contractile function. These effects were associated with the prevention of ischemic contracture and attenuation of the loss of high energy phosphates during ischemia. Glibenclamide inhibited the beneficial effects of pinacidil on electromechanical function and preservation of adenine nucleolides which indicated that KATP activation was the primary target involved in the Cardioprotective effect of pinacidil. A low concentration (10 µM) of glibenclamide by itself inhibited the decrease in action potential shortening and decreased the time to ischemic contracture, however, this concentration of glibenclamide did not enhance the loss of ATP or creatine phosphate during noflow ischemia. A higher concentration of glibenclamide (50 µM) produced a greater inhibition of action potential shortening, further decreased the time to contracture and enhanced the loss of ATP. Taken together, these results from various in vitro models of ischemia-reperfusion injury support the original hypothesis of Noma (1983) which suggested that opening the KATP channel results in a cardioprotective effect. This beneficial action is at least partially related to the shortening of action potential duration and the resultant decrease in Ca2+ influx through voltage-gated channels and the preservation of high energy phosphates. Direct evidence to support this hypothesis has recently been found in guinea pig and human isolated ventricular myocytes (Jiang et al., 1994). These investigators found that the KCA, LCRK, produced a marked shortening of action potential duration and a reduction in free intracellular Ca2+ in myocytes.

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These changes were accompanied by a decrease in cell shortening. LCRK had a more pronounced effect when the intracellular pH was reduced from 7.4 to 6.8. These effects of LCRK were blocked by glibenclamide which suggests that activation of the KATP channel was the main target of the KCA. All of these observed changes would be expected to result in a cardioprotective effect in the intact ischemic myocardium. 11.3.2 In Vivo Models—Stunned Myocardium Brief periods (5–20 min) of coronary artery occlusion followed by reperfusion have been shown to produce prolonged periods of postischemic contractile dysfunction, a reduction in tissue blood flow and decreases in tissue adenine nucleotide content. These changes are reversible over several hours to days and this phenomenon has been termed the stunned myocardium (Braunwald and Kloner, 1982). The precise cellular mechanism(s) responsible for myocardial stunning is still not clear, however, the current hypothesis with the most supporting data suggests that there is an abnormality in intracellular Ca2+ homeostasis which may be related to oxygen derived free radical-induced damage (Bolli, 1990). The first studies to suggest a role for the KATP channel in myocardial stunning were performed with nicorandil, a KCA-nitrate in anesthetized dogs (Lamping and Gross, 1985). In these experiments, dogs were subjected to 15 min of coronary artery occlusion followed by 3 hours of reflow. Nicorandil was administered in a dose which produced a 15–25 mmHg reduction in mean arterial blood pressure (BP) prior to and during the occlusion period. In nicorandil-treated dogs, there was a highly significant increase in the recovery of regional wall motion expressed as percent segment shortening (%SS) in the ischemic-reperfused region as compared to the control group (Figure 11.2). An equal hypotensive dose of the nitrovasodilator, sodium nitroprusside, had no effect on the recovery of %SS (Gross et al., 1992) which suggested that opening of the KATP channel was the most likely explanation for the beneficial effect observed following nicorandil treatment. Nicorandil was also shown to enhance the recovery of %SS in the stunned myocardium of conscious dogs subjected to 10 min of coronary artery occlusion followed by 6 hours of reperfusion (Shimshak et al., 1986). Subsequently, Grover and co-workers (1990c) also found that nicorandil improved the recovery of regional wall motion in stunned myocardium of anesthetized dogs. However, their data suggested that the effect of nicorandil was not due to direct activation of myocardial KATP channels but the result of its peripheral hemodynamic effects to reduce preload and afterload since an intracoronary dose of nicorandil did not produce a protective effect. To address

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Figure 11.2 Myocardial segment shortening (% of control) in the ischemic-reperfused region of anesthetized dogs before occlusion (C), during occlusion (OCC) and at various times after reperfusion, in animals receiving vehicle, nicorandil or nitroprusside. *Significantly different from the control group (P<0.05). All values are the mean±S.E.M. (n=8 in each group). Reprinted with permission from the Journal of Cardiovascular Pharmacology 1992, 20 (Suppl. 3), S22–S28.

further the site and mechanism of action of nicorandil in stunned myocardium of anesthetized dogs, Auchampach et al, (1992a) administered nicorandil in the absence or presence of the KATP channel blocker glibenclamide. Glibenclamide, at a dose which had no effect by itself on stunning, completely blocked the beneficial effect of nicorandil (Figure 11.3). Since glibenclamide had little effect on the peripheral hemodynamic effects of nicorandil, these data suggest that nicorandil is exerting its cardioprotective effect on the stunned myocardium in dogs by directly activating the cardiac KATP channel. A number of recent studies with KCAs more selective than nicorandil also suggest that opening KATP channels attenuates myocardial stunning in anesthetized dogs (Auchampach et al., 1992b; Gross et al., 1992; D’Alonzo et al., 1992; Yao et al., 1993). Bimakalim administered as a bolus 15 min prior to coronary artery occlusion enhanced the recovery of postischemic function in dogs subjected to 15 min of coronary occlusion and 3 hours of reperfusion (Gross et al., 1992). In a similar model, a nonhypotensive dose of aprikalim administered prior to occlusion and throughout the remainder of the experiment resulted in a marked improvement in the recovery of %SS (Figure 11.4).

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However, when the same dose of aprikalim was given 1 min prior to and throughout reperfusion, no beneficial effect was observed (Auchampach et al., 1992c). The beneficial effects of aprikalim were completely blocked by prior treatment with glibenclamide. In addition, a higher dose of glibenclamide than that necessary to block the cardioprotective effects of

Figure 11.3 Percent segment shortening (%SS) of the ischemic-reperfused area before coronary artery occlusion, during occlusion and at various times during reperfusion in the saline-treated group (CONTROL) nicorandil-treated group (NIC), glibenclamide-treated group (GLIB) and nicorandil-treated group after pretreatment with glibenclamide (NIC +GLIB). All values are the mean±S.E.M. *P<0.05 versus the control group. Reprinted with permission from the Journal of Cardiovascular Pharmacology 1992, 20, 765–771.

aprikalim, and a 100–fold higher dose of tolbutamide, both exacerbated postischemic dysfunction in the anesthetized dog model. These results have several implications and suggest that KCAs need to be present at the onset of ischemia to be effective in this model, when they are likely to have the greatest effect to accelerate KATP channel opening (Escande and Cavero, 1992), and that endogenous KATP channel activation during ischemia exerts some cardioprotective effect by itself since blocking these channels makes stunning worse.

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Multiple brief periods of coronary artery occlusion have also been shown to result in myocardial stunning (Bolli, 1990). Although this model is less well characterized than the single stunning model, it appears that the pathogenesis of stunning is similar and may involve oxygen derived free radicals and alterations in Ca2+ homeostasis (Bolli, 1990, Gross et al., 1992). In addition, this model may more closely resemble stunning in patients with unstable angina or coronary vasospasm where multiple occlusion-reperfusion episodes are known to occur (Bolli, 1992). Using a multiple occlusion model in dogs, Korb et al. (1985) found that nicorandil worsened the metabolic state of the myocardium, whereas isosorbide dinitrate had a protective effect. In a similar model, Pieper and Gross (1987)

Figure 11.4 The effects of aprikalim on myocardial segment shortening (% of control) in the ischemic-reperfused region before occlusion, during occlusion and at various times after reperfusion. Aprikalim was infused intravenously at a nonhypotensive dose (10 µg/ kg+0.1 µg/kg/min) beginning either before and throughout the remainder of the experiments (APRIKALIM OCC) or immediately before and throughout the reperfusion period only (APRIKALIM REP). All values are the mean±S.E.M. *P<0.05 versus the control group. Reprinted with permission from Circulation 1992, 86, 311–319.

found that nicorandil enhanced the metabolic and functional recovery of the ischemic-reperfused myocardium, whereas isosorbide dinitrate only improved the metabolic status. Nakagawa et al. (1991) also observed that nicorandil improved the functional recovery and prevented the loss of adenine nucleotides

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in rat isolated hearts subjected to multiple periods of coronary artery occlusion and reperfusion. The reasons for the differences in the results of these various studies with nicorandil in multiple occlusion models is not apparent but may be related to a number of factors including species differences, experimental protocol, drug doses or concentrations and the indices used to assess the extent of ischemic injury. Several recent studies have addressed a possible mechanism by which KCAs are cardioprotective in the stunned myocardium (D’Alonso et al., 1992; Yao et al., 1993). In these experiments, performed in anesthetized dogs, CRK or aprikalim were administered prior to, and during, a 15 min coronary artery occlusion (D’Alonso et al., 1992) or prior to, and during, six 5 min periods of occlusion interspersed with brief periods of reperfusion (Yao et al., 1993). In both studies, the monophasic action potential duration (APD) in the center of the ischemic region was determined with epicardial electrodes and %SS was measured by a pair of piezoelectric crystals placed in the subendocardium of the ischemic-reperfused area. Both CRK and aprikalim reduced the APD by approximately 8% prior to occlusion and accelerated the shortening observed during ischemia as compared to nontreated control animals. Associated with the enhanced shortening of the APD was a better recovery of %SS during reperfusion (Figure 11.5 for aspects of aprikalim). Thus, since Ca2+ overload during the latter part of the ischemic period is thought to be involved in myocardial stunning (Bolli, 1990), the KCAs may attenuate Ca2+ overload and enhance the recovery of %SS by accelerating the shortening of the action potential and decreasing Ca2+ entry via L-type channels. Recent results from Tan et al. (1993) also suggested that enhancing the opening of the KATP channel during ischemia, or accelerating its opening by the KCA CRK during ischemia, attenuates electrical uncoupling which occurs during a prolonged ischemic period in rabbit isolated papillary muscle. That other mechanisms affecting intracellular release of Ca2+ or myofilament sensitivity to Ca2+ may be involved in the action of KCAs in the

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Figure 11.5 Percent segment shortening (A) and monophasic action potentials during 1st occlusion period (B) in the ischemic region for aprikalim (APKL)-treated dogs. All values are the mean±S.E.M. *P<0.05 versus the control group. Reprinted with permission from the American journal of Physiology 1993, 264, H495–H504.

ischemic myocardium is suggested by the recent review of Quast (1993) and work performed in vascular smooth muscle by Yanagisawa et al. (1993). Taken together, these results obtained in stunned myocardium suggest that activation of KATP channels during ischemia accompanied by action potential shortening is an important endogenous protective mechanism during brief periods of myocardial ischemia. These results also suggest that this process can be accelerated by KCAs, or inhibited by KCBs, and may be an important mechanism by which KCAs produce a cardioprotective effect in patients suffering from single or repeated episodes of myocardial ischemia. Furthermore,

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these data indicate that KCAs have selectivity for ischemic conditions and that lower doses than were previously thought to be efficacious have cardioprotective effects. 11.3.3 In Vivo Models—Myocardial Infarction Coronary artery occlusions for periods longer than 20 min result in necrosis or myocardial infarction. The ultimate size of the infarct depends on a number of factors including the area at risk, collateral blood flow, myocardial oxygen consumption and the duration of occlusion (Reimer et al., 1989). Although controversial, there is also a considerable body of evidence which suggests that the inflammatory response and the polymorphonuclear leukocyte or neutrophil is involved in the extension of the infarct following coronary artery reperfusion (Lucchesi et al., 1989). Therefore, modulation of any of these factors by pharmacological agents may result in a reduction in myocardial infarct size and a better recovery of left ventricular function. The effects of KCAs on myocardial infarct size are equivocal. In initial studies performed with nicorandil, Lamping et al. (1984) found that this agent reduced myocardial infarct size approximately 50% in dogs subjected to 2 hours of coronary artery occlusion and 30 min of reperfusion, whereas an equal hypotensive dose of nifedipine had no significant effect even though infarct size was reduced approximately 20%. Endo et al. (1988) also showed that nicorandil reduced infarct size in dogs subjected to 6 hours of total coronary artery occlusion. In these two studies, nicorandil produced a significant decrease in the rate-pressure product, an indirect index of myocardial oxygen consumption which may be one mechanism by which nicorandil exerted its antiischemic effect, however, the role of its KATP channel opening activity was not evaluated. The results obtained with other KCAs are less clear-cut than those obtained with nicorandil. Pinacidil has been shown to have no effect (Imai et al., 1988), or to increase, infarct size in conscious instrumented dogs (Sakamoto et al., 1989). However, in both of these studies, pinacidil was administered systemically at a dose which produced marked hypotension, a reflex tachycardia and a reduction in collateral blood flow. Furthermore, pinacidil was administered 40 min after coronary artery occlusion, at a time when KATP channel activity is probably not affected by the KCA to any significant degree. Based upon all these factors, it is not surprising that pinacidil did not reduce infarct size. In contrast, Grover et al. (1990a) reported that CRK and pinacidil reduced myocardial infarct size in dogs subjected to 90 min of coronary artery occlusion and 5 hours of reperfusion when given by intracoronary infusion in doses that did not affect systemic hemodynamics. Two recent studies using a protocol identical to that of Grover et al. (1990a), Kitzen et al. (1992) and Smallwood et al. (1993) found that intracoronary infusions of four KCAs, CRK, celikalim, pinacidil and 3-pyridyl pinacidil had no effect to limit infarct size in anesthetized dogs. The reasons for

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the differences observed in these three studies are not apparent, however, the control infarct sizes were considerably smaller in the two studies in which the KCAs had no effect (Kitzen et al., 1992; Smallwood et al., 1993) thus making it more difficult to demonstrate a cardioprotective effect. Auchampach et al. (1991) and Gross and Auchampach (1992a) found that intravenous administration of a nonhypotensive dose of the KCA, aprikalim, produced a significant reduction in infarct size in dogs subjected to either 60 or 90 min of coronary artery occlusion and 4–5 hours of reperfusion (Figure 11.6). The beneficial effects of aprikalim were blocked by glibenclamide which suggests that aprikalim exerted its cardioprotective effect by activating KATP channels. Interestingly, the cardioprotective effect of aprikalim was greater in the 60 min occlusion model than in the 90 min model. Similarly, Auchampach and Gross (1994) found that another KCA, bimakalim, produced a small, but significant, reduction in infarct size in anesthetized dogs subjected to 90 min of coronary artery occlusion, whereas Rohmann et al. (1993) reported a much larger reduction in infarct size in pigs subjected to 60 min of occlusion. These results suggest that the cardioprotective effects of KCAs may have a time-related limit or threshold time for occlusion associated with their beneficial effect on myocardial infarct size and may help

Figure 11.6 Area at risk as a percent of the left ventricle (AAR/LV) and myocardial infarct size (IF) as a percent of the area at risk (IF/AAR) or left ventricle (IF/LV) in

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vehicle (CONTROL) and aprikalim-treated dogs. Aprikalim was administered as an intravenous infusion at a non-hypotensive dose (10 µg/kg+0.1 µg/kg/min) beginning 15 min before coronary artery occlusion and continued throughout the experiment. All values are the mean±S.E.M. *P<0.05 versus the control group (see Auchampach et al., 1991).

explain the divergent effects obtained from various laboratories. It is possible that 90 min of ischemia is at the threshold of effectiveness for KCAs and slight differences in hemodynamics, collateral blood flow or other factors may be enough to show a protective effect of these compounds in one laboratory versus no effect in another laboratory. Studies in the anesthetized rabbit are also equivocal. Thornton et al. (1993) showed no effect of pinacidil on myocardial infarct size, whereas Toombs et al. (1992, 1993) showed that CRK reduced infarct size. Thornton et al. (1993) also showed that blocking the KATP channel with glibenclamide produced a marked increase in infarct size, a result similar to that observed in the study of Auchampach et al. (1991). Therefore, although the weight of evidence suggests that KCAs reduce infarct size and KCBs increase infarct size, more wellcontrolled studies are necessary to substantiate clearly these findings. The mechanisms by which KCAs reduce infarct size via KATP channel activation are unknown. Evidence for a direct cardiac effect is provided by the studies which show that pinacidil, CRK (Grover et al., 1990a) and aprikalim (Auchampach et al., 1991; Gross and Auchampach, 1992a) reduced infarct size independent of their effects on peripheral hemodynamics and myocardial blood flow. Several studies also suggest that the KCAs may be modifying infarct size by reducing the inflammatory response and neutrophil function (Pieper and Gross, 1992a, 1992b; Gross et al., 1992). The results of in vitro experiments indicate that nicorandil produces a concentration-dependent reduction of superoxide production by human and canine neutrophils (Gross et al., 1992; Pieper and Gross, 1992a). Bimakalim has also been shown to reduce superoxide production in canine neutrophils and this effect was blocked by glibenclamide which suggests a role for the KATP channel in this action of bimakalim. The results of in vivo experiments have also shown that aprikalim (Gross and Auchampach, 1992a) and bimakalim (Auchampach and Gross, 1994) reduce neutrophil infiltration into the viable border zone surrounding the infarct in anesthetized dogs. These data all suggest that KCAs may exert part of their beneficial effect on myocardial infarct size by inhibiting neutrophil function during the reperfusion period. 11.4 Evidence for a Cardioprotective Effect of KCAs in Other Models A number of other studies suggest that KCAs possess Cardioprotective properties in other in vivo and in vitro models. Nakamura et al. (1987) examined the direct cardiac effects of nicorandil, verapamil, nitroglycerin and propranolol

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on the metabolic changes produced by regional hypoxia in the intact canine heart. They found that nicorandil prevented the loss in ATP and attenuated the rise in tissue lactate which occurred during 5 min of hypoxia, actions which were shared by verapamil and propranolol but not nitroglycerin. The role of the KATP channel in mediating these protective effects of nicorandil were not assessed in this study. Irie (1988) and Orita et al. (1991) studied the effects of cardioplegic solutions containing nicorandil during cardiopulmonary bypass or prior to cardiac transplantation in canine hearts. Irie (1988) demonstrated that 10 mg/1 of nicorandil in the cardioplegic solution preserved sarcoplasmic reticular function during ischemia and enhanced the recovery of cardiac function during reperfusion. Similarly, Orita et al. (1991) found that a cardioplegic solution containing nicorandil resulted in an enhanced recovery of myocardial function associated with a higher blood flow and less tissue acidosis in orthoptically transplanted canine hearts as compared to control hearts. More recently, Cohen et al. (1993) showed that a cardioplegic solution containing the KCA, aprikalim, was more effective at preserving myocardial function during cardiac arrest in rabbit isolated hearts than a solution containing high K+ to produce arrest. These data obtained with nicorandil and aprikalim suggest that KCAs may be useful adjuncts in cardiac preservation during transplantation or coronary artery bypass surgery. Otsuka et al. (1990) studied the effects of endothelin alone, or in combination with nicorandil, on hemodynamic and electrocardiographic changes in anesthetized dogs. They found that endothelin produced a number of hemodynamic effects including a prolonged increase in blood pressure, a decrease in heart rate and cardiac output and an increase in the S-T segment of the electrocardiogram, an indication that ischemia may be present as the result of an intense coronary vasoconstrictor response to endothelin. That KCAs are particularly effective in blocking the vasoconstrictor effects of endothelin is suggested by the results of several studies in which CRK and pinacidil were shown to antagonize endothelin–1 induced contractions in rat (Kim et al., 1989; O’Donnell et al., 1990) and human blood vessels (Haynes and Webb, 1993) to a greater extent than several Ca channel blockers. Since endothelin–1 has been shown to possess KATP channel blocking activity in porcine coronary arteries (Miyoshi et al., 1992) these results are not totally surprising. Finally, nicorandil has been shown to reduce myocardial cell injury in chick isolated hearts subjected to the Ca paradox (Hamaguchi et al., 1990) or to prolonged isoproterenol exposure (Ohta et al., 1991b). In these studies, it was hypothesized that nicorandil reduced Ca2+ overload by several mechanisms including hyperpolarizing the cardiac membrane and shortening action potential duration, both of which would reduce Ca2+ influx via voltage-gated Ca channels, reducing Ca2+ release from the sarcoplasmic reticulum and activating a cell membrane Ca2+ ATPase extrusion pump. Evidence that many of these mechanisms operate in vascular smooth muscle in the presence of KCAs has

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recently been presented by Yanagisawa et al. (1993). Further studies are needed in cardiac myocytes to assess the importance of these various pathways in the actions of KCMs. 11.5 Summary and Conclusions Strong evidence presented by various investigators and summarized in this chapter suggests that KCAs are potent antiischemic agents in a number of experimental models although in the setting of an acute myocardial infarction, the findings are less clear. The KCBs, however, appear to block consistently the beneficial effects of the KCAs and to produce no change or exacerbate ischemic injury when administered by themselves. These effects of the KCMs are very dose dependent and it appears that the KCAs may have a more efficacious antiischemic effect and less side effects when administered in smaller doses. Finally, the observation that the KATP channel may serve an endogenous cardioprotective role in myocardial stunning and preconditioning (Gross and Auchampach, 1992b) suggests that enhancing this endogenous mechanism by KCAs may lead to important therapeutic uses of these agents in the treatment of ischemic heart disease (Escande and Cavero, 1992). That nicorandil is an efficacious agent in the treatment of angina pectoris with minimal side effects in patients supports the feasibility of this approach. Acknowledgements The author wishes to thank Carol Knapp, Anna Hsu and Jeannine Moore for their excellent secretarial and technical assistance in the preparation of this chapter. Some of the experimental data presented were supported by a National Institutes of Health Grant HL 08311. References AUCHAMPACH, J.A. & GROSS, G.J. (1994) J. Cardiovasc. Pharmacol., 23, 554–561. AUCHAMPACH, J.A., MARUYAMA, M., CAVERO, I. & GROSS, G.J. (1991) J. Pharmacol. Exp.Ther., 259, 961–967. AUCHAMPACH, J A., GROVER, G.J. & GROSS, G.J. (1992a) Cardiovasc. Res., 26, 1054–1062. AUCHAMPACH, J.A., CAVERO, I. & GROSS, G.J. (1992b) J. Cardiovasc. Pharmacol., 20, 765–771. AUCHAMPACH, J.A., MARUYAMA, M., CAVERO, I. & GROSS, G.J. (1992c) Circulation, 86, 311–319. BANERJEE, A., LOCKE-WINTER, C., ROGERS, K., MITCHELL, M.B., BREW, E.C., CAIRNS, C.B., BENSARD, D.D. & HARKEN, A.H. (1993) Circ. Res., 73, 656–670. BOLLI, R.M. (1990) Circulation, 82, 732–748. (1992) Circulation, 86, 1671–1691. BRAUNWALD, E. & KLONER, R.A. (1982) Circulation, 66, 1146–1149.

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COHEN, N.M., WISE, R.M., WECHSLER, A.S. & DAMIANO, R.J., JR. (1993) J. Thorac. Cardiovasc. Surg., 106, 317–328. COLE, W.G., MCPHERSON, C.D. & SONTAG, D. (1991) Circ. Res., 69, 571–581. D’ALONZO, A.J., DARBENZIO, R.B., PARHAM, C.S. & GROVER, G.J. (1992) Cardiovasc. Res., 26, 1046–1053. DEUTSCH, E., BERGER, M., KUSSMAUL, W.G., HIRSHFELD, J.W., JR., HERRMANN, H.C. & LASKEY, W.K. (1990) Circulation, 82, 2044–2051. ENDO, T., NEJIMA, J., KIUCHI, K., FUJITA, S., KIKUCHI, K., HAYAKAWA, H. & OKUMURA, H. (1988) J. Cardiovasc. Pharmacol., 12, 587–592. ESCANDE, D. & CAVERO, I. (1992) Trends Pharmacol. Sci., 13, 269–272. FRALIX, T.A., STEENBERGEN, C., LONDON, R.E. & MURPHY, E. (1993) Cardiovasc. Res., 27, 630–637. GROSS, G.J. & AUCHAMPACH, J.A (1992a) Cardiovasc. Res., 26, 1011–1016. (1992b) Circ. Res., 70, 223–233. GROSS, G.J., AUCHAMPACH, J.A., MARUYAMA, M., WARLTIER, D.C. & PIEPER, G.M. (1992) J. Cardiovasc. Pharmacol., 20 (Suppl 3), S22–S28. GROVER, G.J., McCutLOUGH, J.R., HENRY, D.E., CONDER, M.L. & SLEPH, P.G. (1989) J. Pharmacol. Exp. Ther., 251, 98–104. GROVER, G.J., DZVONCZYK, S., PARHAM, C.S. & SLEPH, P.G. (1990a) Cardiovasc. Drugs Ther., 4, 465–474. GROVER, G.J., SLEPH, P.G. & DZVONCZYK, S. (1990b) J. Cardiovasc. Pharmacol, 16, 853–864. GROVER, G.J., SLEPH, P.G. & PARHAM, C.S. (1990c) J. Cardiovasc. Pharmacol, 15, 698–705. GROVER, G.J., NEWBURGER, J., SLEPH, P.G., DZVONCZYK, S., TAYLOR, S.C., AHMED, S.Z. & ATWAL, K.S. (1991) J. Pharmacol. Exp. Ther., 257, 156–162. GROVER, G.J., SLEPH, P.G. & DZVONCZYK, S. (1992) Circulation, 86, 1310–1316. GROVER, G.J., DZVONCZYK, S., SLEPH, P.G. & SARGENT, C.A. (1993) J. Pharmacol. Exp. Ther., 265, 559–564. HAMAGUCHI, T., AZUMA, J., TAKIHARA, K., OHTA, H., HARADA, H., SPERELAKIS, N. & KISHIMOTO, S. (1990) Drug Dev. Res., 19, 91–97. HAYNERS, W.G. & WEBB, D.J. (1993) Am. J.Physiol, 265, H1676–H1681. IMAI, N., LIANG, C.S., STONE, C.K., SAKAMOTO, S. & HOOD, W.B., JR. (1988) Circulation, 77, 705–711. IRIE, H. (1988) Jpn. Circ. J., 52, 563–569. JIANG, C., MOCHIZUKI, S., POOLE-WlLSON, P.A., HARDING, S.E. & MACLEOD, K.T. (1994) Cardiovasc. Res., 28, 851–857. KlM, S., MORIMOTO, S., KOH, E., MIYASHITA, Y. & OGIHARA, T. (1989) Biochem. Biophys.Res. Commun., 164, 1003–1008. KlTZEN, J.M., MCCALLUM, J.D., HARVEY, C., MORIN, M.E., OSHIRO, G.T. & COLATSKY, T.J. (1992) Pharmacology, 45,71–82. KORB, H., HOEFT, A., HUNNEMAN, D.H., SCHRAEDER, R., WOLPERS, H.G. & HELLIGE, G. (1985) Naunyn-Schmiedeberg’s Arch. Pharmacol., 329, 440–446. LAMPING, K.A. & GROSS, G.J. (1985) J. Cardiovasc. Pharmacol, 7, 158–166. LAMPING, K.A., CHRISTENSEN, C.W., PELC, L.R., WARLTIER, D.C. & GROSS, G.J. (1984) J. Cardiovasc. Pharmacol., 6, 536–542. LAZDUNSKI, M. (1992) Cardiovasc. Drugs Ther., 6, 313–319.

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LIU, G.S., THORNTON, J.D., VAN WINKLE, D.M., STANLEY, A.W.H., OLSSON, R.A. & DOWNEY, J.M. (1991) Circulation, 84, 350–356. LIU, Y. & DOWNEY, J.M. (1992) Am. J. Physiol., 263, H1107–H1112. LUCCHESI, B.R., WERNS, S.W. & FANTONE, J.C. (1989) J. Mol. Cell. Cardiol, 21, 1241–1251. MCCULLOUGH, J.R., NORMANDIN, D.E., CONDER, M.L., SLEPH, P.G., DZVONCZYK, S. & GROVER, G.J. (1991) Circ. Res., 69, 949–958. MCPHERSON, C.D., PIERCE, G.N. & COLE, W.C. (1993) Am. J. Physiol., 265, H1809–H1818. MITANI, A., KlNOSHITA, K., FUKAMACHI, K., SAKAMOTO, M., KURISU, K., TSURUHARA, Y., FUKUMURA, F., NAKASHIMA, A. & TOKUNAGA, K. (1991) Am. J. Physiol., 261, H1864–H1871. MIYOSHI, Y., NAKAYA, Y. & WAKATSUKI, T. (1992) Circ. Res., 70, 612–616. MURRY, C.E., JENNINGS, R.B. & REIMER, K.A. (1986) Circulation, 74, 1124–1136. NAKAGAWA, M., ABE, Y., NAKANASHI, T., MATSUOKO, H., SAITO, T., ONO, Y. & MIURA, M. (1991) Cardiovasc. Drugs Ther., 5 (Suppl.3), 391. NAKAMURA, Y., KOJIMA, S., MORI, H., ABE, S., MIYAMORI, R., MIYAZAKI, T., SAKURAI, K., HATTORI, S. & TAKAHASHI, M. (1987) J. Cardiovasc. Pharmacol., 10 (Suppl. 8), S92–S97. NICHOLS, C.G. & LEDERER, W.J. (1991) Am.J. Physiol, 261, H1675-H1686. NOMA, A. (1983) Nature, 305, 147–148. O’DONNELL, S.R., WANSTALL, J.C. & ZENG, X.P. (1990) J. Pharmacol. Exp. Ther., 252, 1318–1323. OHTA, H., JINNO, Y., HARADA, K., OGAWA, N., FUKUSHIMA, H. & NISHIKORI, K. (1991a) Eur. J. Pharmacol., 204, 171–177. OHTA, H., AZUMA, J., TANAKA, Y., TAKIHARA, K., HAMAGUCHI, T., AWATA, N., SAWAMURA, A., SPERELAKIS, N. & KISHIMOTO, S. (1991b) Drug Dev. Res., 22, 229–237. OHTA, H., NAKAZAWA, H., JINNO, Y., HARADA, K., OGAWA, N., MIWA, A. & NISHIKORI, K. (1993) Eur. J. Pharmacol., 231, 323–330. ORITA, H., SHIMANUKI, T., FUKASAWA, M., ABE, H., KURAOKA, S., HIROOKA, S. & WASHIO, M. (1991) Cardiovasc. Drugs Ther., 5, 727–732. OTSUKA, A., MIKAMI, H., TSUNETOSHI, T., KATAHIRA, K., MORIGUCHI, A. & OGIHARA, T. (1990) Curr. Ther. Res., 47, 584–590. PlEPER, G.M. & GROSS, G.J. (1987) Circulation, 76, 916–928. (1992a) Cardiovasc. Drugs Ther., 6, 225–232. (1992b) Immunopharmacology, 23, 191–197. QUAST, U. (1993) Trends Pharmacol. Sci., 14, 332–337. REIMER, K.A., MURRY, C.E. & RICHARD, V.J. (1989) J. Mol. Cell. Cardiol., 21, 1225–1239. ROHMANN, S., BECKER, K.H., SCHELLING, P., WEYGANDT, H. & LUES, I. (1993) J. Heart Failure, 1, 844. SAKAMOTO, S., LIANG, C.S., STONE, C.K. & HOOD, W.B., JR. (1989) J. Cardiovasc. Pharmacol., 14, 747–755. SARGENT, C.A., SMITH, M.A., DZVONCZYK, S., SLEPH, P.G. & GROVER, G.J. (1991) J. Pharmacol. Exp. Ther., 259, 97–103. SARGENT, C.A., DZVONCZYK, S. & GROVER, G.J. (1992) Pharmacology, 45, 260–268.

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SARGENT, C.A., SLEPH, P.G., DZVONCZYK, S., NORMANDIN, D., ANTONACCIO, M.J. & GROVER, G.J. (1993) J. Cardiovasc. Pharmacol., 22, 564–570. SCHOTT, R.J., ROHMANN, S., BRAUN, E.R. & SCHAPER, W. (1990) Circ. Res., 66, 1133–1142. SCHULZ, R., ROSE, J. & HEUSCH, G. (1994) Am.J. Physiol., 267, H1341–H1352. SHIMSHAK, T.M., PREUSS, K.C., GROSS, G.J., BROOKS, H.L. & WARLTIER, D.C. (1986) Cardiovasc. Res., 20, 621–626. SMALLWOOD, J.K., SCHELM, J.A., BEMIS, K.G. & SIMPSON, P.J. (1993) J. Cardiovasc. Pharmacol., 22, 731–743. TAGGART, P.I., SUTTON, P.M.I., OLIVER, R.M. & SWANTON, R.H. (1993) Circulation, 88, (Abstract) 3062. TAN, H.L., MAZON, P., VERBEME, H.J., SLEESWIJK, M.E., CORONEL, R., OPTHOF, T. & JANSE, M.J. (1993) Cardiovasc. Res., 27, 644–651. THORNTON, J.D., THORNTON, C.S., STERLING, D.L. & DOWNEY, J.M. (1993) Circ. Res., 72, 44–49. TOOMBS, C.F., NORMAN, N.R., GROPPI, V.E., LEE, K.S., GADWOOD, R.C. & SHEBUSKI, R.J. (1992) J. Pharmacol. Exp. Ther., 263, 1261–1268. TOOMBS, C.F., MOORE, T.L.& SHEBUSKI, R.J. (1993) Cardiovasc. Res., 27, 617–622. YANAGISAWA, T., YAMAGISHI, T., OKADA, Y. & TAIRA, N. (1993) Int. Conf. on ATP-sens. K+ channels and sulfonylurea receptors, 1, 173. YAO, Z., CAVERO, I. & GROSS, G.J. (1993) Am. J. Physiol., 264, H495–H504.

12 Potassium Channel Activators: Airway Pharmacology and Bronchial Asthma J.R.S.ARCH Department of Vascular Biology, SmithKline Beecham Pharmaceutical, The Frythe, Welwyn, Hertfordshire, AL6 9AR, UK. 12.1 Introduction 12.1.1 Asthma Therapy Bronchial asthma is characterised by wide variations over short periods of time in the resistance to flow in intrapulmonary airways (Sears, 1991; Lenfant et al., 1992). The disease is therefore commonly treated with bronchodilators, notably β 2-adrenoceptor agonists, which relax the smooth muscle of the constricted airways. Expert opinion on the therapy of asthma has, however, undergone a transformation in recent years, with emphasis now being placed upon the early use of anti-inflammatory or anti-allergic drugs, except in the mildest manifestations of the disease (Lenfant et al., 1992). The bronchodilators still have a role, but in the opinion of some experts, β 2-adrenoceptor agonist bronchodilators should only be used for the relief of asthma symptoms and not prophylactically (Anon, 1993; Page, 1993). The main anti-inflammatory drugs employed in asthma are glucocorticosteroids, which are, except in severe cases, given by inhalation to reduce systemic side-effects. These drugs actively resolve inflammation. Some workers also include anti-allergic drugs under the anti-inflammatory heading, but others categorise them separately, arguing that they do not actively resolve inflammation but rather prevent new inflammatory responses to allergens. This allows the body’s natural mechanisms to repair pre-existing inflammation (Morley et al., 1986; Kay, 1987; Rafferty and Holgate, 1988). The main antiallergic drugs are disodium cromoglycate and the more recently introduced drug nedocromil. These are commonly, but controversially, referred to as mast cell stabilisers (Kay, 1987; Rafferty and Holgate, 1988; Church et al., 1991). Ketotifen is also classified as an anti-allergic drug and commands a large share of the asthma market in Japan, though some workers doubt its efficacy (Rafferty

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and Holgate, 1988; Dawson et al., 1989). One other major anti-asthma drug, especially in the USA, is theophylline. Theophylline undoubtedly has bronchodilator activity when used at a high enough dose, but many workers believe that it owes much of its success to an anti-inflammatory or anti-allergic activity (Pauwels and Persson, 1991; Ward et al., 1993). Two main reasons can be discerned for the trend towards the early use of antiinflammatory or anti-allergic drugs in asthma. First, it has been recognised that the airways of most asthmatics are inflamed, with the eosinophil apparently playing a key role in inflicting airways damage (Busse and Sedgwick, 1992). The generation of inflammation in the airways of animals can lead to enhanced airways constriction in response to mild insults—a cardinal feature of asthma known as bronchial hyperresponsiveness. (This term encompasses both bronchial hypersensitivity and hyperreactivity, which are more restrictive in their meanings—Orehek, 1983.) Similarly, hyperresponsiveness in asthmatics is enhanced if the inflammation of their airways is exacerbated by inhalation of allergen. Thus the dogma has developed that the underlying cause of airways constriction in asthma is inflammation of the airways. This simple view is disputed, however. Hyperresponsiveness can be dissociated from inflammation in laboratory animals (Chapman et al., 1993), and although steroids reduce asthma symptoms and appear to remove airways inflammation, many studies suggest that they have only a minor and transient influence on bronchial hyperresponsiveness (Smith, 1992; Vathenen et al., 1991). Furthermore, when the lungs of asthmatics were transplanted into non-asthmatics they were found to carry the asthma with them—in fact it seemed to worsen (Corris and Dark, 1993). This suggests that the asthma was not dependent on the generation of an inflammatory response by the immune system of the donor, although an alternative view is that a population of memory T cells is permanently present in the lungs and was transferred with them (Barnes, 1993). Some of these findings might be explained if the changes induced in the airways by inflammation are partly irreversible, but they are also consistent with the thesis, originally advanced in the 1960s, that bronchial hyperresponsiveness, possibly of genetic origin, is often a predisposing factor in the development of asthma. Presumably, this underlying condition interacts with inflammation (Hopp et al., 1990). The second reason for the move towards anti-inflammatory drugs stems from evidence that the regular use of β 2-agonists can actually exacerbate bronchial hyperresponsiveness and accelerate the decline in lung function with age in asthmatics (reviewed by Page, 1993). These conclusions are mainly derived from studies on short-acting β 2-agonists, especially fenoterol. The new long-acting β 2agonist, salmeterol, has so far escaped relatively unscathed (Beach et al., 1993; Sears and Taylor, 1993; Fuller et al., 1993). It is disputed whether the small increases in hyperresponsiveness elicited by the short-acting β 2-agonists can be of any clinical relevance.

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An important question is whether the adverse findings are features solely of β 2agonists, or whether any bronchodilator, perhaps by opening susceptible airways to allergens or by enabling asthmatics to expose themselves to allergenic environments, would have a similar effect. Exacerbation of bronchial hyperresponsiveness does appear to be β -agonist-specific, since it is not elicited by the muscarinic antagonist ipatropium bromide. Page (1993) has discussed various mechanisms by which ― -adrenoceptor stimulation might have this effect. Ipatropium bromide has, however, been reported to accelerate the decline in lung function in asthma or chronic obstructive pulmonary disease (chronic bronchitis; Van Schayck et al., 1991). Similar adverse effects of theophylline have not been discovered, possibly owing to its anti-allergic activity as well as its non-β agonist bronchodilator mechanism of action. The movement towards a new approach to asthma therapy has also been swayed by the fact that mortality and morbidity from asthma has risen over recent years in many parts of the world, despite the availability of apparently effective anti-asthma drugs (Sears, 1991; but see Horton, 1993). This is probably due in part to a rise in the incidence of asthma (Sears, 1991; Burney, 1993), but there is a suspicion that inappropriate therapy is a confounding factor (Spitzer et al., 1992; but see Mullen et al., 1993). 12.1.2 The Potential of Potassium Channel Activators Perhaps three predictions can be made in the light of the above discussion. First, bronchodilators will retain an important place in the therapy of asthma for some years to come, particularly as symptomatic therapies. Second, the adverse research surrounding β 2-agonist bronchodilators should encourage the development and use of bronchodilators that act via a novel mechanism. Third, novel bronchodilators that have additional anti-allergic, anti-inflammatory or anti-hyperresponsiveness properties will be especially well received and will command a premium price in many markets. It is into this scenario that K channel activators or openers (KCAs) enter as a potential therapeutic approach for asthma. KCAs hold the promise of therapeutic utility in a wide range of CNS, cardiovascular, gastrointestinal and skeletal muscle disorders. This derives from their ability to suppress the activity of excitable cells— a result of membrane potential being stabilised near the K+ equilibrium potential. However, those KCAs that have been discovered, show greater potency in smooth muscle and in certain neurones than in skeletal or cardiac muscle, or nervous tissue in general. Consequently, they have received most attention with regard to their potential in smooth muscle-related disorders, such as hypertension, angina, urinary incontinence and asthma (Quast, 1992; Longman and Hamilton, 1992). The potential of KCAs in hypertension has been the subject of most intense investigation, in part because of a perception that vascular smooth muscle

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relaxation might introduce side-effects that would limit the use of KCAs in other disorders. Asthma also offers a viable target, however. The first studies in man on the prototype KCA, cromakalim (CRK), suggested that vascular side-effects might not prove an obstacle to its use as an oral bronchodilator (Baird et al., 1988; Williams et al., 1990). Although the active enantiomer of CRK, levcromakalim (LCRK), failed to meet these expectations (Kidney et al., 1993, Godard et al., 1991; Picot and De Vernejoul, 1991), a newer compound, BRL 55834, which is a selective relaxant of the airways relative to the vasculature in guinea-pigs and especially rats in vivo (Bowring et al., 1993), looks far more promising. Furthermore, with anti-asthma drugs there is the possibility of direct delivery to the lungs, which generally reduces the administered dose, and hence systemic exposure, by about 20–fold. 12.1.3 KCAs that have been Evaluated for Bronchodilator Activity In this review the pharmacology of KCAs in the airways will be illustrated primarily by reference to CRK, LCRK or BRL 55834. A number of other KCAs have also been evaluated as airways smooth muscle relaxants in vitro or as bronchodilators in vivo, and those for which data are published are shown in Figure 12.1. The majority of these compounds retain the benzopyran structure present in CRK, but studies on the airways effects of cyanoguanidine, pinacidil and four derivatives (Nielsen-Kudsk and Bang, 1991; Buckle et al., 1993), and on some tetrahydrothiopyrans, epitomised by the highly potent compound RP 66471 (Raeburn et al., 1991a; Raeburn and Brown, 1991), have also been reported. The relative potencies of these compounds in our own studies on spontaneous tone in guinea-pig tracheal spirals are shown in Figure 12.2.

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Figure 12.1 Chemical structures of KCAs that have been evaluated as airways smooth muscle relaxants. CRK, pinacidil and RP 49356 are racemates, but otherwise the stereochemistry at the asymmetric centres is as shown.

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Figure 12.2 IC50 values for inhibition of spontaneous tone in guinea-pig tracheal spirals by KCAs. Unpublished data of J.Bond, D.J.Shaw, J.F.Taylor, S.C.Taylor and J.S.Ward (see Taylor et al., 1992a for methods).

12.2 Airways Smooth Muscle Relaxation in Vitro The first evidence that KCAs might have potential as bronchodilators came from studies on guinea-pig isolated trachealis. CRK was found to reduce spontaneous and, to a lesser extent, cholinergic and histaminergic tone, but to have only a weak effect on contractile responses (Allen et al., 1986). In other words, it was a better spasmolytic than antispasmogenic agent. This feature is shared with other classes of smooth muscle relaxant (Raeburn and Karlsson, 1991). Subsequent studies in guinea-pig isolated airways confirmed the poor efficacy of CRK in preventing the development of cholinergic tone, but showed that it was a potent inhibitor of tone induced by a wide range of other spasmogens, including histamine (Arch et al., 1988; Taylor et al., 1992a). LCRK shows a similar profile of activity, but twice the potency of CRK, as would be expected if most activity resides in one enantiomer of CRK. A feature of CRK, LCRK and all the other KCAs shown in Figure 12.1 which distinguishes them from most other smooth muscle relaxants is their steep concentration-response curves. It has also been shown that LCRK (and Ro 31–6930) administered to sensitised guinea-pigs in vivo can inhibit the in vitro, antigen-induced contractile response of isolated tracheal strips taken from these animals (Gater et al., 1991). That this results from functional antagonism of released mediators, and not from inhibition of mediator release, is indicated by the absence of any inhibitory effect of CRK (10

342 K CHANNELS AND THEIR MODULATORS −7

to 10–4 M) on the antigen-induced release of histamine or LTD4 from the chopped lung of sensitised guinea-pigs (Kusner et al., 1989). KCAs are also effective relaxants of tone in bovine (Longmore et al., 1991a) and, more importantly, human airways (Black et al., 1990; Cortijo et al., 1992; Miura et al., 1992; Buckle et al., 1993). In general they are as, or even more, effective and potent in human airways than guinea-pig trachea. Efficacy has been demonstrated in human airways against tone induced by histamine, leukotriene D4, neurokinin A and cholinergic agonists (see below) as well as against spontaneous tone. BRL 55834 has similar efficacy to LCRK and is about 6–fold and 24–fold more potent than LCRK against histamine (5 µM)- and carbachol (100 µM)-induced tone respectively (S.G. Taylor, unpublished). 12.2.1 Cholinergic Tone Other KCAs have in general shown similar profiles of activity to the prototype compounds (Paciorek et al., 1990a; Raeburn and Brown, 1991; Raeburn and Karlsson, 1991; Shikada et al., 1991; Chapman et al., 1992; Englert et al., 1992; Small et al., 1992a; Martin et al., 1993). An intriguing difference between various reports, however, concerns the efficacy of KCAs against cholinergic tone. Some workers find low efficacy against cholinergic tone in guinea-pig trachea (Allen et al., 1986; Shikada et al., 1991; Chapman et al., 1992; Taylor et al., 1992a, 1992b; Martin et al., 1993) whilst others report high efficacy (Martini and Black, 1990, Paciorek et al., 1990a). One reason for these differing findings is that various workers used different challenges and that, in common with other types of smooth muscle relaxant, the efficacy of KCAs increases as the concentration of the cholinergic tone decreases (Raeburn and Brown, 1991). McCaig et al. (1992) did not find an influence of acetylcholine concentration on the potency of CRK in trachea taken from normal guinea-pigs, but did find such an influence in trachea taken from guinea-pigs that had been sensitised to albumin and chronically exposed to the aerosolised antigen. This suggests that differences between laboratories in the exposure of their guinea-pigs to allergen could account for differing findings. There also appear to be differences between KCAs in their efficacy against contractions elicited by cholinergic agonists. Englert et al. (1992) found that HOE 234, rilmakalim, was almost as potent against a low concentration of carbachol (30 nM) as against spontaneous tone, whereas LCRK was about 10– fold less potent against carbachol than against spontaneous tone. Apparently, both KCAs had good efficacy against this low concentration of carbachol. In contrast, in another study, SR 47063 was about sevenfold more potent than CRK as a relaxant of spontaneous tone but slightly less potent than CRK as a relaxant of acetylcholine-induced tone, both compounds having intrinsic activities relative to theophylline of about 0.65 against spontaneous tone and 0.3 against acetylcholine (Martin et al., 1993). From these two reports one might predict that

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the efficacy of HOE 234 and SR 47063 against cholinergic tone should differ markedly. The release of acetylcholine from parasympathetic nerve endings plays a significant role in asthma and hence ipatropium bromide is employed in a small proportion of patients. The low efficacy of most KCAs against cholinergic tone in guinea-pig trachea, which is also seen in rat trachea (S.G.Taylor, unpublished), therefore raised doubts about the potential of these agents in asthma. Findings in guinea-pigs and rats appear to be of little relevance to man, however. In human bronchi, CRK, LCRK, SDZ PCO 400, pinacidil, SR 47063 and HOE 234 are all effective relaxants of cholinergic tone, although this may require higher concentrations of the KCAs than those required to relax histamine-induced tone (Black et al., 1990; Taylor et al., 1992a; Chapman et al., 1992; Cortijo et al., 1992; Martin et al., 1993; Miura et al. 1992). Furthermore, both CRK and pinacidil can induce a significant rightward shift of the carbachol (and histamine) concentration-effect curves in human bronchi, despite the poor efficacy of these KCAs as anti-spasmogenic agents in guinea-pig trachea (Taylor et al., 1992a). Another reason for dismissing the clinical relevance of the low efficacy of KCAs against cholinergic tone in guinea-pig airways is that KCAs appear to inhibit the release of acetylcholine from parasympathetic nerve endings in the airways. This is discussed further below (section 12.4). It is appropriate to note here that, reminiscent of its effects against cholinergic tone, LCRK is effective against neurokinin A in human bronchi (Black et al., 1990) but not in guinea-pig trachea (Good et al., 1992). Moreover, it inhibits the release of neurokinin A from nerves in guinea-pig airways. The clinical significance of this finding is less clear since the role of neurokinin A in asthma is not well established. 12.2.2 Histaminergic Tone Another difference between KCAs is the time course of their relaxation of histamine (5 µM)-induced tone in guinea-pig trachea. Relaxation in response to CRK, LCRK and SDZ PCO 400 is transient, whereas BRL 55834, HOE 234, pinacidil and RP 66471 have a more sustained effect. RP 52891 and bimakalim (EMD 52692, see Sassen et al., 1990 for structure) are intermediate with respect to this property (Figure 12.3). A similar difference has been noted in the time course of the anti-spasmogenic effects of LCRK and HOE 234 in vivo (Englert et al., 1992). The

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Figure 12.3 Time course for the spasmolytic effects of LCRK (BRL 38227) (10 µM, ― ) and BRL 55834 (1 µM, ― ), and for fade in control (― ) tension in histamine (5 mM)treated guinea-pig tracheal spirals. Points are means of 3 values with S.E.M. Data of S.G.Taylor and J.S.Ward.

clinical significance of these findings is unclear, however, since CRK, LCRK, pinacidil and BRL 55834 all elicit sustained relaxations of histamine-induced tone in human bronchi (S.G.Taylor, unpublished). One possible explanation for the differences in the guinea-pig is that the shorter acting compounds not only displace ATP from its inhibitory site on K channels but they also prevent its action at a phosphorylation site, and this results in channel run-down. The longer acting compounds may have only the former effect (see section 12.6). 12.2.3 Airway Selectivity The relative potencies of KCAs as relaxants of tone in smooth muscle are significantly different from their relative potencies in cardiac and skeletal muscle and in the pancreatic islet β -cell (Edwards and Weston, 1993). In one study, the order of potency for various KCAs as inhibitors of the release of [3H]-GABA from substantia nigra differed from the order for either smooth muscle or the pancreatic ― -cell (Schmid-Antomarchi et al., 1990). The 50–fold greater potency of LCRK compared to CRK in this study is surprising and has not been

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confirmed (Herdon et al., 1993). Nevertheless, it seems that the target K channel is different in smooth muscle from most other tissues and obtaining selectivity for smooth muscle should not be a problem. Indeed, apart possibly from some neuronal effects, the compounds shown in Figure 12.1 are more potent in smooth muscle than in other tissues. A more serious difficulty is that of obtaining selectivity for the airways over other smooth muscles, especially vascular smooth muscle. Our own screening programme suggested that this might be possible, since a number of compounds that in other respects appeared to be typical KCAs (section 12.6) were found to relax 30 mM KCl-induced tone in guinea-pig portal vein but not spontaneous tone in guinea-pig trachea (S.G.Taylor, D.S.Shaw and J.Ward, unpublished results). One pinacidil derivative, BRL 49074, was especially interesting, since it stimulated potassium efflux and relaxed tone in portal vein but not trachea. Furthermore, it inhibited the effects of CRK on both K+ efflux and tone in trachea (Taylor et al., 1993). These results might be explained by there being different populations of K channels in the vasculature and airways, but could also be due to there being a larger number of channels in the vasculature and BRL 49074 having low efficacy. Despite this encouragement, there are few reports of compounds that show selectivity between smooth muscles. The benzopyran S 0121 was claimed to be selective for the ureter over the vasculature, but this was not confirmed by a later study (see also Chapter 14). HOE 234 was claimed to be selective for the airways over the ureter (see Weston and Edwards, 1991). Only one compound, BRL 55834, has shown enhanced selectivity over LCRK (and other KCAs) for the airways relative to the vasculature. Bearing in mind the reservation that tone is induced in different ways in trachea and portal vein, BRL 55834 does not display absolute selectivity in vitro. In fact, it shows very similar potencies for relaxation of 30 mM KCl-induced tone in guinea-pig portal vein and tone induced by various agonists or developed spontaneously in guinea-pig tracheal spirals (Taylor et al., 1992b). LCRK differs from BRL 55834, however, in being more potent in portal vein than in tracheal spirals. Expressed in different terms, BRL 55834 is 8– to 27–fold more potent than LCRK in tracheal spirals, but only 2.8–fold more potent than LCRK in portal vein (Figure 12.4). Thus, relative to LCRK, BRL 55834 is 3– to 100–fold more

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Figure 12.4 Relative potencies of LCRK and BRL 55834 from Taylor et al (1992b). The potency ratio is IC50 LCRK/IC50 BRL 55834. Ratios are shown for tone induced in guinea-pig tracheal spirals by various means and for 30 mM KCl-induced tone in guineapig portal vein.

selective for the trachea compared to portal vein in vitro. It was preliminary indications of this difference in selectivities between BRL 55834 and LCRK that led to BRL 55834 being evaluated in vivo, but whether its in vitro profile is of any relevance to its in vivo selectivity (see below) is unclear. 12.3 Airways Smooth Muscle Relaxation in Vivo Consistent with their efficacy as spasmolytic agents in vitro, KCAs relax an established bronchoconstriction in vivo (Englert et al., 1992). In addition, they show pronounced protective effects in vivo when they are given prior to challenge. This contrasts with their weak antispasmogenic activity in vitro in guinea-pig trachea, but parallels similar in vitro and in vivo findings for β adrenoceptor agonists and xanthines. In vivo studies have, indeed, largely addressed the anti-spasmogenic rather than the spasmolytic activity of KCAs. In conscious guinea-pigs, orally administered CRK, LCRK, BRL 55834 and Ro 31–6930 inhibit the dyspnoea evoked by inhaled histamine (Arch et al., 1988; Paciorek et al., 1990a; Bowring et al., 1993). Furthermore, CRK, RP 49356 and RP 52891 have been shown to provide protection against antigen challenge (Arch et al., 1988; Raeburn and Karlsson, 1991). In anaesthetised guinea-pigs, intravenously or intraduodenally administered KCAs have been shown to inhibit bronchoconstriction in response to histamine, 5–HT and bombesin (Arch et al.,

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1988; Paciorek et al., 1990a; Chapman et al., 1992; Englert et al., 1992; Taylor et al., 1992a; Bowring et al., 1993), but not substance P (Raeburn et al., 1991a). Clinical studies on CRK and LCRK, which are the only KCAs that have been evaluated in asthmatics, are referred to in the Introduction and are described in Chapter 16. 12.3.1 Effects on Small and Large Airways Respiratory dynamics measurements in anaesthetised guinea-pigs, rats and cats have revealed that CRK, LCRK and BRL 55834 elicit similar percentage inhibitions of the histamine (guinea-pigs)- or methacholine (rats)-induced increase in airways resistance and decrease in lung compliance (De Souza et al., 1989; Drewett and Rodger, 1989; Bowring et al., 1991; 1993). In this respect the KCAs resemble theophylline rather than β -agonists. Most studies show that β agonists have a greater effect on resistance than compliance (Daly, 1974; Greenberg and Smorong, 1975; Salonen et al., 1985; Fanta et al., 1987; but see Chapman and Danko, 1985; Chapman et al., 1985; Drewett and Rodger, 1989). Since constriction of the large airways increases resistance, whereas constriction of the small airways decreases compliance, these results indicate that, compared to β -agonists, KCAs are more effective dilators of small airways for identical large airways effects. This is somewhat surprising since the density of β adrenoceptors is greater in the smooth muscle of the small than the larger airways (see Bowring et al., 1991 for references). There are other factors, however, that could influence these findings, such as the coupling between β adrenoceptor stimulation and airways relaxation in different parts of the airways. In any event, the clinical significance of differences between various bronchodilators in the relaxation of small relative to large airways is not known. 12.3.2 Cholinergic Challenge As with studies in vitro, cholinergic challenge has produced a confusing picture, with KCAs being effective in some models but not others. One group has reported that nebulised LCRK and RP 66471 are almost as potent in preventing methacholine- as histamine-induced bronchoconstriction in anaesthetised guineapigs (Raeburn et al., 1991a). In contrast, our own group found that iv LCRK was ineffective against iv acetylcholine in anaesthetised guinea-pigs, consistent with our in vitro results (Taylor et al., 1992a). Similarly Borghi et al. (1990) found no effect of CRK, pinacidil or RP 49356 against acetylcholine. Nevertheless, we did find that oral LCRK protected conscious guinea-pigs from dyspnoea in response to inhaled aceylcholine (Taylor et al., 1992a). Moreover, iv, intraduodenal and inhaled LCRK or BRL 55834 protected anaesthetised rats from bronchoconstriction in response to inhaled methacholine (Bowring et al.,

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1993; Arch et al., 1994). (Inhaled methacholine appears to be the only practicable challenge in the rat.) A number of factors may account for these disparate findings. As suggested in the section on in vitro responses to cholinergic challenge, the dose or route of administration of the challenging agent, or perhaps its efficacy (methacholine may be less effective than acetylcholine), could be a significant factor. Some models of bronchoconstriction, especially those in conscious animals, may involve a significant reflex component which is inhibited at the neural level by KCAs (see section 12.4). Finally, differences between species may occur, studies in vitro suggesting that CRK and LCRK are somewhat more effective against cholinergic tone in rat than in guinea-pig trachea (S.G.Taylor, unpublished). 12.3.3 Airway Selectivity BRL 55834 is not more potent as a relaxant of guinea-pig trachea than of guineapig portal vein in vitro, but it does show a different profile from LCRK, which is more potent as a relaxant of portal vein (see above). These findings prompted the evaluation of BRL 55834 in vivo and led to the discovery that it has surprisingly good airways selectivity in guinea-pigs and especially rats. BRL 55834 is, indeed, the only KCA for which airways selectivity has been demonstrated in vivo. In order to investigate its selectivity, BRL 55834 has been compared with LCRK in urethane-anaesthetised, freely respiring guinea-pigs and rats (Bowring et al., 1993). The anaesthetic has the effect of abolishing reflex tachycardia, so that any effect of KCAs on vascular tone is sensitively reflected as a fall in blood pressure. Blood pressure (BP) is measured just before bronchoconstrictor challenge with iv histamine (guinea-pigs) or inhaled methacholine (rats), the challenges being sufficiently spaced for them not to affect BP at the time that its change in response to KCAs is measured. The effect of the KCAs on the airways is measured as a percentage inhibition of the increase in airways resistance or decrease in lung compliance in response to the bronchoconstrictor challenge. The two measurements of lung function lead to essentially the same conclusions concerning the selectivity of BRL 55834 and discussions will be restricted to the resistance effects. By the iv route in guinea-pigs, BRL 55834 and LCRK had similar hypotensive potencies, but BRL 55834 was 4.5 times more potent than LCRK as an inhibitor of histamine-induced increases in airways resistance. By the intraduodenal route in guinea-pigs, BRL 55834 was again about fivefold more potent than LCRK as an airways relaxant and at half the dose of LCRK it had a substantially lower hypotensive effect than LCRK. From a clinical standpoint, the comparison with LCRK would be of little significance were it not that LCRK did appear to elicit some bronchodilatation in man at the highest dose evaluated (0.75 mg), (Picot and De Vernejoul, 1991; see also Chapter 16). Since this highest dose was

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chosen as that which gives an acceptable incidence of headache and since headache results from cerebral vasodilation, a small increase in airways selectivity in BRL 55834 may allow it to have clinically useful activity without causing headache. Considering data on BRL 55834 in isolation from LCRK, the separation of airways resistance and hypotensive dose-response curves in the guinea-pig is substantial. In the rat, the picture is even more favourable, since in this species iv BRL 55834 inhibited airways resistance changes at doses that had no effect on BP. Moreover, at higher doses, the airways activity of iv BRL 55834 was sustained, whilst its effect on BP rapidly waned—the exact opposite of the profile for LCRK. This marked difference in the profile of BRL 55834 and LCRK appears to be reflected in the time course of their effects when given intraduodenally in the rat: BRL 55834 (20 µg/kg) inhibited airways resistance changes for 35 min without significantly affecting BP; LCRK (100 µg/kg) elicited a marked and long-lasting fall in BP, but had no effect on airways resistance. Only at 500 µg/kg was an effect on airways resistance seen and this was short-lived (Figure 12.5). Whilst these results are encouraging, clearly a critical question is whether the in vivo selectivity of BRL 55834 in the guinea-pig and rat translates to man.

Figure 12.5 The effects of intraduodenal BRL 55834 (― , 20 µg/kg) and LCRK (― , 100 µg/kg; ― , 500 µg/kg) on methacholine-induced increases in airways resistance (Raw, continuous lines) and baseline mean blood pressure (broken lines) in anaesthetised rats. Note that the airways resistance response for LCRK was obtained using a higher dose than the blood pressure response: there was no effect of LCRK on airways resistance at

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the lower dose. Results are taken from Bowring et al. (1993). Points are means of 5 values with S.E.M.

12.3.4 Inhaled Administration Airway selectivity is particularly important if KCAs are to be given orally. This is the preferred route of administration for anti-asthma drugs in some countries (e.g. Japan) and helps patient compliance. Selectivity can also be achieved by administering drugs topically to the airways, i.e. by giving them by inhalation. Some KCAs appear to have been evaluated predominantly by inhalation to the exclusion of the oral (or intraduodenal) route, apparently since this was viewed as the only way to achieve adequate selectivity. CRK, LCRK, BRL 55834, Ro 31–6930, RP 52891, RP 66471, HOE 234 and SDZ PCO 400 have all been reported to be effective by inhalation in anaesthetised or conscious guinea-pigs against histamine or 5–HT challenges (Paciorek et al., 1990b; Bowring et al., 1991; Raeburn et al., 1991a, 1991b; Chapman et al., 1992; Englert et al., 1992; Arch et al., 1994). As mentioned above, RP 66471 was also effective against an iv methacholine challenge in the guinea-pig (Raeburn et al., 1991a). Inhaled LCRK and BRL 55834 have been shown to be effective in rats against an inhaled methacholine challenge (Arch et al., 1994). Inhaled LCRK was evaluated in anaesthetised guinea-pigs against both inhaled and iv histamine. It proved to be about eightfold more potent against the inhaled challenge. Similar results were obtained with inhaled salbutamol, whereas iv LCRK was equally effective against inhaled and iv histamine (Bowring et al., 1991). No explanation has been found for these results. The expected improvement in airway selectivity associated with the inhaled route of administration has been demonstrated for LCRK in urethaneanaesthetised guinea-pigs, using the method described above (Bowring et al., 1991). Similar reductions in BP were elicited by inhalation of a nebulised 1000 µg/ml solution or by iv injection of 12.5 µg/kg body weight, whereas similar inhibitions of respiratory responses to iv histamine were elicited by 62–125 µg/ml solutions and a 25 µg/kg iv injection. This suggests that inhalation increased selectivity for airways relative to systemic effects by about 20–fold. Inhaled BRL 55834 appears to be even more airways selective than inhaled LCRK, as might be predicted from its greater airways selectivity by the iv and intraduodenal routes. Thus, in anaesthetised rats, LCRK (250 µg/ml nebulised solution) lowered BP by 20% at 2.8 times its EC50 dose for inhibition of inhaled methacholine-induced increases in airways resistance. BRL 55834, in contrast, had no effect on BP at a dose (200 µg/ml nebulised solution) that was 21 times its EC50 dose for inhibition of increases in airways resistance (Arch et al., 1994). This indicates an improvement in selectivity over LCRK of at least sevenfold. A

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therapeutic window of this magnitude may allow large doses of BRL 55834 to be given in extreme situations. It has already been mentioned that iv and intraduodenal CRK, LCRK and BRL 55834 have similar effects on airway resistance and dynamic lung compliance, whereas β -adrenoceptor agonists have a greater effect on resistance. The KCAs also have similar effects on resistance and compliance when they are inhaled. Since the KCAs have little or no effect on BP when they are inhaled, it is unlikely that the effect on compliance is somehow related to an influence on the volume of blood in the lung or the amount of fluid in the interstitial space—factors which can affect compliance. It seems improbable that sufficient material passes through the airways to affect the vasculature when the lower, non-hypotensive doses of KCAs are inhaled. It might be argued that the KCAs influence the pulmonary vasculature but fail to reach the systemic vasculature in sufficient concentration to reduce BP. Doses of iv nifedipine that cause large falls in systemic BP, do not, however, affect compliance (Bowring et al., 1991), even though a dose of nifedipine that elicits a 30% fall in systemic BP in the rat is known to have a marked effect on pulmonary vascular resistance (Standbrook et al., 1984). In addition to allowing a smaller dose to be given and thereby reducing systemic side-effects, the inhaled route of administration has the advantage of hastening the onset of action of bronchodilators. This is important when the asthmatic faces a crisis and needs immediate relief of symptoms. On the other hand, a potential drawback of giving small doses of bronchodilators by inhalation is that, unless, like the β -agonist salmeterol, the compound is retained in the airways, duration of action is likely to be shorter than by the oral route: once the compound passes out of the airways and into the systemic circulation it will be diluted below therapeutically effective concentrations and recirculation to the airways will be without avail. A rapid onset of bronchodilator activity (within 1–2 min) has been demonstrated for CRK, LCRK, BRL 55834, RP 66471 and HOE 234 (Bowring et al., 1993). HOE 234 and BRL 55834 tended to have slightly greater effects when the challenge was delayed (by 5 min for HOE 234; 10 or 15 min for BRL 55834) than at the earliest times studied. BRL 55834 was longer-acting than LCRK in anaesthetised guinea-pigs and rats, and in conscious guinea-pigs: the highest doses of BRL 55834 used were effective when the challenge was delayed by 20 to 45 min, whereas doses of LCRK that had similar peak effects to those of BRL 55834 had a shorter duration of action, by about 20 min (Arch et al., 1994). A possible explanation for this finding is that BRL 55834 diffuses slowly through the airways epithelium, providing a steady supply of compound to the airways smooth muscle. HOE 234, which like BRL 55834 has a slightly delayed peak effect is, however, no longer-acting than LCRK (Englert et al., 1992). Furthermore, BRL 55834 also has a longer duration of bronchodilator action than LCRK when the compounds are given iv to rats. The most likely explanation for the prolonged activity of BRL 55834 is that it has a slow off rate from its receptor.

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Thus the smooth muscle relaxant effects of BRL 55834 are reduced much more slowly than those of LCRK when tissues are washed in vitro (S.G.Taylor and J.S.Ward, unpublished). 12.4 Neural Effects The neural effects of KCAs may be relevant to their potential in the treatment of asthma. Bronchoconstriction frequently has a significant reflex parasympathetic component. This may be enhanced at night, giving rise to nocturnal asthma (Dreher and Koller, 1990). Moreover, neurogenic inflammation of the lung may contribute to the pathology of asthma (Barnes et al., 1991). Most studies of central neurones show that they are affected only by very high doses or concentrations of the known KCAs (see Longman and Hamilton, 1992; Sellers et al., 1992), but there are a number of reports which suggest that KCAs are potent inhibitors of neurotransmitter release from both cholinergic, and nonadrenergic, non-cholinergic excitatory (NANCe) neurones in guinea-pig lung. Inhibition of cholinergic transmission in guinea-pig small intestine has also been claimed (Zini et al., 1991). Small et al. (1992b) have suggested that the efficacy of CRK in an early clinical study (Williams et al., 1990) was due to an influence on neurotransmitter release, since the predicted peak plasma concentration of CRK in this study was about fivefold less than its threshold concentration for relaxation of tone in human bronchi in vitro (Taylor et al., 1992a). The potency of CRK in airways smooth muscle from asthmatics is not known, however, and may be greater than in normal airways (see section 12.5). The main evidence for an effect on neurotransmitter release is that KCAs are far more effective at inhibiting cholinergically- or NANCe-mediated bronchoconstriction when these are elicited by stimulating neurotransmitter release than when acetylcholine or an exogenous NANCe neurotransmitter is supplied directly either in vitro (Hall and Maclagan, 1988; McCaig and De Jonckheere, 1989; Burka et al., 1991; McCaig et al., 1992, Yamamoto et al., 1992) or in vivo (Ichinose and Barnes, 1990; Lewis and Raeburn, 1990; Aitken and McCaig, 1991; Lei et al., 1993; Clapham et al., 1993). The NANCe neurotransmitter given exogenously in early studies was substance P, but more recently neurokinin (NK) A has been used, because studies with selective NK receptor antagonists have shown neurokinin A to be the more important bronchoconstrictor (Maggi et al., 1991). Most studies show that KCAs are, in any event, poor inhibitors of the bronchoconstrictor action of both substance P and neurokinin A in guinea-pig airways, so the argument that their effect on NANCe-mediated bronchoconstriction must be due to pre-synaptic inhibition of neurotransmitter release, rather than a direct effect on smooth muscle, remains valid. Further evidence for an effect of KCAs on neurotransmitter release comes from the finding that inhalation of SDZ PCO 400, employed at a dose that gave

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weak protection against a histamine challenge, markedly diminished bronchoconstriction in response to an antigen challenge given 15 min subsequently in anaesthetised, sensitised guinea-pigs. This effect of SDZ PCO 400 was not detected in animals that were bilaterally vagotomised (Chapman et al., 1992). Whether the KCA was inhibiting the release of neurotransmitters onto airways smooth muscle or mast cells, i.e. whether it was acting before or after mast cell degranulation, was not established. In addition to their bronchoconstrictor activity, the sensory neuropeptides released by NANCe neurones stimulate mucus secretion and the leakage of plasma from post-capillary venules, both of which are important in the pathology of asthma. These inflammatory effects of sensory neuropeptides appear to be predominantly mediated by substance P rather than neurokinin A (Kuo et al., 1990). Mucus secretion is also stimulated by acetylcholine release. Comparison of the effects of KCAs on microvascular leakage and goblet cell secretion in response to stimuli that elicit neurotransmitter release (electrical stimulation, cigarette smoke or bradykinin), and in response to stimuli that act independently of neurotransmitter release (substance P, acetylcholine, histamine, platelet activating factor, antigen) again suggests that the KCAs inhibit neurotransmitter release. Thus they are effective only against those stimuli that act by releasing neurotransmitters (Raeburn and Karlsson, 1991; Kuo et al, 1992; Martin and Advenier, 1993; Lei et al., 1993; Gallico et al., 1994; but see Chapman et al., 1992; Ichinose et al., 1992 for effects of SDZ PCO 400 and LCRK against the bronchoconstrictor effects of antigen challenge). Not all workers agree that KCAs inhibit neurotransmitter release, however. Aikawa et al., (1992) found that CRK inhibited electrical field stimulationinduced contractions of guinea-pig bronchi only at concentrations that also reduced contractions induced by exogenous substance P, though, as explained above, they would have done better to use neurokinin A. Gater et al., (1993) found that both Ro 31–6930 and LCRK were more effective against acetylcholine than vagally-induced bronchoconstriction both in vitro and in vivo in the guinea-pig. Both groups have therefore argued that KCAs do not have a pre-synaptic effect on neurotransmitter release. These disagreements will only be resolved if it can be shown directly that KCAs affect neurotransmitter release. Studies on the effects of KCAs on neurokinin A or substance P release have not been reported. There is a report of inhibitory effects of CRK and LCRK on electrically-stimulated [3H]acetylcholine release from rat trachea, but these effects occurred only under somewhat unexpected conditions. They depended on the presence of an intact epithelium and were abolished by opening the tracheal tube, suggesting that KCAs do not interact directly with lung neurones, but release an epithelial factor that inhibits neural activity (Wessler et al., 1993). CRK was found not to affect the release of [3H]-acetylcholine evoked by electrical stimulation or by stimulation of nicotinic or 5-HT3-receptors in guinea-pig intestine, though it did inhibit release in response to M1-receptor stimulation (Schwörer and Kilbinger,

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1989). Again this contrasts with indirect evidence that cholinergic transmission in this tissue is inhibited by CRK (Zini et al., 1991). 12.5 Hyperresponsiveness—Contribution of Anti-inflammatory and Neural Inhibitory Activity An almost universal characteristic of asthmatics is that their airways are hyperresponsive to a wide range of physiological and pharmacological stimuli. KCAs appear to be able to reduce hyperresponsiveness both by a direct acute effect on the airways smooth muscle after hyperresponsiveness has developed, and by inhibiting the development of the hyperresponsiveness. They have not been shown permanently to reverse an established hyperresponsiveness so that responsiveness remains normal in their absence, apart from a small effect of BRL 55834 (0.3 mg/ kg, p.o. daily for 11 days) on the response to 5–HT in lung parenchyma from Sephadex bead-injected rats (see below for description of model, B.A. Spicer, unpublished). It would, however, be remarkable if a large effect was seen, since such an effect has not been demonstrated for steroids (Piercy et al., 1993). The effects of KCAs discussed in this section are summarised in Table 12.1. An acute effect of KCAs on hyperresponsive airways was found in a model in which Sephadex beads are injected iv into rats. The beads embolise the lung vasculature and elicit a blood and lung eosinophilia. Lung parenchyma removed from the rats is hyperresponsive to carbachol, and this appears to be due to hyperresponsiveness of the airways rather than the vascular component of the parenchyma (Taylor and Spicer, 1990). The presence of LCRK or BRL 55834 in vitro, returned the responsiveness to carbachol to normal, whilst having no effect on the response to carbachol in lung parenchyma from control rats (Ward et al., 1990; S.G.Taylor and J.S.Ward, unpublished). LCRK and BRL 55834 also caused obvious and marked relaxations of resting tone in tissues from Sephadextreated rats. These relaxations were significantly greater than the gradual loss of tone that occurred in tissues from control rats (S.G.Taylor and J.S.Ward, unpublished). Attempts to identify a spasmogen present in lung parenchyma from the Sephadex-treated but not the control rats have been unsuccessful. Inhibition of the development of hyperresponsiveness has been demonstrated by at least two groups using an anaesthetised guinea-pig model. CRK, SDZ PCO 400 and Ro 31–6930, employed at doses that did not cause bronchodilatation in control animals, inhibited hyperresponsiveness induced by platelet activating factor, isoprenaline or immune complexes (Sanjar et al., 1989; Chapman et al., 1991; Chapman et al., 1992; Paciorek et al., 1993). The methods used to elicit hyperresponsiveness in these studies have been criticised as being unphysiological. It is reassuring, therefore, that SDZ PCO 400 also attenuated hyperresponsiveness in sensitised guinea-pigs 24 hours after inhalation of

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antigen (Chapman et al., 1992) – a somewhat more relevant, though by no means perfect, model of asthma. It is unclear how KCAs influence the development of hyperresponsiveness. As outlined in the introduction, there is a view that hyperresponsiveness of the airways is a consequence of their inflammation, with the eosinophil playing a key role. CRK and SDZ PCO 400 did not, however, inhibit lung eosinophilia in these experiments (Sanjar et al., 1989; Chapman et al., 1992). Furthermore, chronic administration of SDZ PCO 400 did not affect bronchial or peritoneal eosinophil numbers in guinea-pigs exhibiting idiopathic pulmonary eosinophilia (Cook and Chapman, 1993), nor have effects of LCRK or BRL 55834 on blood or lung eosinophilia been demonstrated in Sephadex bead-injected rats (B.A.Spicer, unpublished). Table 12.1 Effects of KCAs other than relaxation of smooth muscle from normal animals Effect of KCAs1

Strength of Evidence

Relevance to Asthma

Inhibition of cholinergic and NANCe transmission

Substantial indirect evidence, but disputed. Direct evidence limited

Direct inhibition of serosal cell secretion

Shown in ferret trachea

Suppression of inflammatory cell activity Acute reversal of airways hyperresponsiveness

Weak evidence

Prevention of development of hyperresponsiveness

Shown by at least two groups in anaesthetised guinea pigs and by one group in antigenchallenged guinea pig

Reversal of established hyperresponsiveness

Some evidence for a small effect of BRL–55834

Reflex bronchoconstriction important. Microvascular leakage and mucus secretion in asthma partly neurogenic Inhibition of mucus secretion of some, but limited, value May reduce airways damage Suggests especially effective as smooth muscle relaxants in asthmatics May prevent worsening of hyperresponsiveness and allow resolution of hyperresponsiveness over long period Would be a major therapeutic achievement

1

Shown in tissue from Sephadex-treated rat

References are given in the text

KCAs may inhibit other aspects of inflammation and lung congestion. In the Sephadex rat model it was often easier to prepare viable lung cells from animals that had been treated with KCAs (see Cook, 1990 for methods), raising the possibility that activation of eosinophils in the lungs of these animals was

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suppressed. Bimkalim (EMD 52692) and nicorandil have been shown to attenuate the oxidative burst in canine neutrophils in response to opsonised zymosan by a K channel-dependent mechanism (Pieper and Gross, 1992). There is little evidence that neutrophils play a role in on-going asthma, however. On the other hand, microvascular leakage and goblet cell secretion are recognised as important in asthma, and the preceding section presented evidence that KCAs inhibit these events when they are neurogenically induced. In addition, both LCRK and Ro 31–6930 have been shown to inhibit methacholine- and phenylephrine-induced secretions from submucosal glands isolated from ferret trachea, suggesting that in serous cells at least the KCAs have a direct antisecretory effect (Griffin and Webber, 1992). Moreover, electrophysiological studies in cultured ovine submucosal cells have identified a population of Ro 31– 6930–sensitive K channels which may be important in the regulation of secretion from these cells (Griffin et al., 1992). The problem in invoking these mechanisms to account for the effects of KCAs on the development of airway hyperresponsiveness is that, whilst there is evidence for a link between airway inflammation and hyperresponsiveness, there is little evidence that microvascular leakage or mucus secretion in the absence of eosinophilia plays a significant role (Hay, 1989). Many workers suspect that KCAs inhibit the development of hyperresponsiveness by inhibiting the release of one or more neurotransmitters, but this has yet to be proven. 12.6 Mechanism of Action Evidence that KCAs do indeed open K channels, arguments as to whether this is their sole mechanism of action, the nature of the K channels opened, and the intracellular events resulting from K channel activation are extensively reviewed in Chapter 8 and elsewhere (Cook and Quast, 1990; Buckle, 1992; Longman and Hamilton, 1992; Quast, 1993). Much of this work has been conducted using vascular tissue and the details will not be reiterated here. The evidence will be summarised, however, referring, as much as possible, to work on airways smooth muscle. 12.6.1 Evidence that K Channels are Opened by KCAs The Na+/K+ and Ca2+ pumps of the plasma membrane cause Na+ and Ca2+ to be present at higher concentrations outside the cell than in the cytosol, whilst the reverse is the case for K+. In the resting cell the permeability of the plasma membrane to K+ is greater than that to Na+ or Ca2+, with the result that K+ ions flowing out of the cell cause the membrane to be negatively polarised. If the cell were permeable only to K+ the membrane potential would reach the K+ equilibrium potential, at which the negative charge attracting K+ ions into the

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cell balances the concentration gradient pushing them out. In practice the resting cell is sufficiently permeable to Na+ and Ca2+ for the membrane to remain a little less polarised than the K+ equilibrium potential. The opening of K channels suppresses the activity of excitable cells by making the K+ permeability of the membrane far greater than the Na+ or Ca2+ permeability. This hyperpolarises the membrane potential towards the K+ equilibrium potential and opposes depolarisations that would otherwise result from increases in Na+ or Ca2+ opening. The ability of CRK to hyperpolarise guinea-pig trachealis cells towards the K+ equilibrium potential provided some of the first evidence that its smooth muscle relaxant activity in the airways was due to K channel opening (Allen et al., 1986; Murray et al., 1989). Similar findings have been reported for CRK, LCRK and RP 49356 in bovine trachea smooth muscle cells (Longmore and Weston, 1989; Berry et al., 1991; Longmore et al., 1991b). Patch-clamp studies demonstrate more clearly that KCAs open ion channels with reversal potentials equal to the K+ equilibrium potential. The majority of these studies have been conducted using vascular smooth muscle cells, but LCRK has been shown to open K channels in membrane patches from rabbit and bovine airway smooth muscle cells (Collier et al., 1991). Allen et al. (1986) found little evidence of separation between the log concentration-relaxation and log concentration-hyperpolarisation curves for CRK in guinea-pig trachea, suggesting that the two phenomena are closely related. In vascular tissues, however, CRK inhibits smooth muscle relaxation or spontaneously generated action potentials at concentrations that do not noticeably raise the membrane potential (Quast, 1993). Where membrane potential and action potential are measured in the same cell, this discrepancy cannot be ascribed to a selective action on pacemaker cells; therefore a selective action on K channels involved in spike repolarisation, as distinct from those that determine resting membrane potential has been proposed. Quast (1993), however, argues forcefully that KCAs elicit smooth muscle relaxation by a mechanism additional to K channel opening, both mechanisms involving interference with ATP binding. It is beyond the scope of this article to reproduce Quast’s arguments in detail. The opening of K channels by KCAs can be demonstrated by loading tissues with 42K+ or 43K+ and following its efflux into the bathing medium. Since the K+ isotopes have short half-lives, 86Rb+ is often used instead, but, being larger, it may not permeate all the channels that are permeable to K+. CRK has been shown to stimulate 86Rb+ efflux, and CRK, LCRK and BRL 55834 to stimulate 42/43K+ efflux from guinea-pig tracheal smooth muscle (Allen et al., 1986; Foster et al., 1992; Taylor et al., 1992c). CRK has also been shown to stimulate both 86Rb+ and 42K+ efflux from bovine tracheal smooth muscle (Gater, 1989). Comparison of results for Rb+ and K+ efflux has led to some intriguing findings. In both bovine and guinea-pig trachea, CRK causes a greater stimulation of K+ than Rb+ efflux, provided each tissue is loaded with only one of the labelled ions (Longmore and Weston, 1989; Foster et al., 1992). When

358 K CHANNELS AND THEIR MODULATORS

guinea-pig trachea was labelled with both 42/43K+ and 86Rb+, however, the stimulation of 42/43K+ efflux was lowered to that of the 86Rb+ efflux, the latter being the same as in the single-label experiments. The concentration of 86Rb+ used in these studies was 0.1 to 0.2 mM. Addition of higher (millimolar) concentrations of Rb+ reduced CRK-stimulated efflux of both 86Rb+ and 42/43K+ to undetectable levels, but had no effect on CRK-stimulated 86Rb+ uptake. Furthermore, in the presence of millimolar Rb+, CRK-induced relaxation of spontaneous tone, which was sustained under control conditions, became transient (Foster et al., 1992). The authors of this work proposed that CRK opens two populations of K channel in guinea-pig tracheal smooth muscle, only one of which is susceptible to blockade by Rb+. The Rb+ -blockable channel is apparently responsible for the sustained relaxation, whilst the Rb+ -insensitive channel is responsible for the transient relaxation and CRK-stimulated Rb+ uptake. Studies in other tissues suggest that the proportions of Rb+ -permeable (insensitive?) and impermeable (sensitive?) channels opened vary between tissues, between KCAs and according to the concentrations of the individual compounds (Cook and Quast, 1990; Longman and Hamilton, 1992). It is possible that the Rb+ -insensitive relaxation involves the opening of an intracellular K channel (Foster et al., 1992; Greenwood and Weston, 1993; Quast, 1993). This is consistent with the lack of detectable ion efflux in the presence of Rb+ and evidence from studies in vascular tissues that smooth muscle relaxation in response to KCAs and K+ efflux can be dissociated. For example, K+ or Rb+ efflux generally occurs at higher concentrations of KCAs than those required to elicit vasorelaxation (Quast, 1993). The proposal that there is an intracellular, Rb+-insensitive, CRKstimulated K channel does not, however, exclude the need to invoke the presence of a plasmalemmal K channel of this type, since CRK-stimulated Rb+ uptake was not inhibited by millimolar concentrations of Rb+ (Foster et al., 1992). Measurements of membrane hyperpolarisation and K+ or Rb+ efflux therefore call into question the view that KCAs relax smooth muscle exclusively by activating K channels. Firmer evidence for an exclusive role of K channels is provided by the clear failure of LCRK and other specific KCAs to relax elevated K+ -induced contractions (Allen et al., 1986; Arch et al., 1988; Raeburn and Brown, 1991; Small et al., 1992a; Taylor et al., 1992a, 1992c; Martin et al., 1993). This key feature of KCAs is predictable from their mechanism of action. As the concentration of K+ outside the cell increases, not only does the membrane potential fall, resulting in the opening of voltage-regulated Ca channels and muscle contraction, but the K+ equilibrium potential falls further and converges with the membrane potential. Consequently, KCAs have little effect on membrane potential under conditions of high external K+ concentration. Moreover, since the K+ equilibrium potential has fallen to a level at which Ca channels are open, what little effect KCAs have is insufficient to influence Ca2+ entry. Nevertheless, this criterion cannot be taken as definitive proof that KCAs act exclusively via K channel opening, since it is possible that

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depolarisation of the cell also interferes with any additional mechanism of action. The last main line of evidence that KCAs act via K channel opening is that airways smooth muscle relaxation is inhibited by sulphonylureas, notably glibenclamide. This has been demonstrated in numerous in vitro studies (see references in section 12.2) and also in vivo (Ichinose and Barnes, 1990; Buckle et al., 1993). Inhibition of the smooth muscle relaxant effect of KCAs by sulphonylureas has focused attention on the ATP-inhibited (KATP) K channel, the pancreatic version of which is well known to be blocked by sulphonylureas. Glibenclamide is, however, about 100–fold less potent as an antagonist of KCAinduced smooth muscle relaxation than as a stimulant of insulin secretion both in vitro and in vivo. Thus a very high dose (25 mg/kg iv) of glibenclamide was required to block the airways (and vascular) effects of LCRK and pinacidil in vivo (Buckle et al., 1993). Moreover, most KCAs (with the notable exception of diazoxide) lack potency in the islets (Dunne and Peterson, 1991) and SDZ PCO 400 has even been reported to block these channels (Dunne, 1990). A further reason to be wary of overinterpreting the effects of glibenclamide is that KCAs have been shown to open ATP-independent, but nevertheless glibenclamidesensitive K channels (Hu et al., 1990; Quasthoff et al., 1990). Studies on the effects of LCRK and ATP depletion on membrane potential and ionic currents in dispersed smooth muscle cells of rat portal vein have gone some way to resolving these paradoxes (Noack et al., 1992). This work suggests that LCRK modified the interaction of ATP with sites linked to more than one type of K channel. These channels include the KATP channel, which is opened by KCAs, and a delayed rectifier channel, whose opening is inhibited. The potency of glibenclamide is lower at the smooth muscle than at islet KATP channels. Reduction of intracellular ATP initially activates the KATP channels in portal vein cells but eventually they run down, suggesting that, as is known for islet channels, there is a phosphorylation site required for channel opening, as well as an inhibitory ATP site. The possibility that some but not all KCAs, may block the phosphorylation site was raised earlier in connection with the maintenance of relaxations of histamine-induced tone (section 12.2.2). 12.6.2 Intracellular Events The sequence of events subsequent to K channel activation is especially pertinent to the potential of KCAs in asthma. The original expectation was that hyperpolarisation of the plasma membrane by KCAs would do no more than close voltage-operated Ca channels. If so, KCAs would have a similar pharmacological profile to Ca entry blockers. This was not an exciting prospect, since Ca entry blockers have proved of little or no value in the treatment of asthma (Massey and Hendeles, 1987).

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Fortunately, further investigation has shown that KCAs have a broader profile of activity than Ca entry blockers both in vitro and in vivo. In both guinea-pig trachea and human bronchi, CRK and LCRK are generally more effective than nifedipine or verapamil in antagonising tone induced by a variety of stimuli and especially in antagonising spontaneous tone (Arch et al., 1988; Taylor et al., 1992a; Buckle and Arch, 1993). CRK was effective in guinea-pig trachea in the presence of a maximal concentration of nifedipine (Arch et al., 1988; Taylor et al., 1992c). The efficacy of other KCAs against spontaneous tone (see section 12.2) similarly distinguishes them from Ca entry blockers (Ahmed et al., 1985). In vivo in guinea-pigs, nifedipine given by the iv, oral or inhaled routes has little or no effect on respiratory responses to histamine, whereas KCAs have marked effects (Arch et al., 1988; Bowring et al., 1991). The poor efficacy of nifedipine shows that L-type voltage-operated Ca channels are not involved to a significant extent in histamine-induced bronchoconstriction. Consequently, the bronchodilator effects of KCAs cannot simply be due to closure of these channels. Even disregarding the poor response to nifedipine, the efficacy of KCAs as antispasmogenics in vivo is unexpected, since the initial contraction to histamine is believed to be mediated by intracellular, rather than extracellular, sources of Ca2+. These findings are partially accounted for by studies in vascular smooth muscle preparations which support the view that KCAs can inhibit the release of Ca2+ from intracellular stores. Although the Ca2+ stores affected are intracellular, the effects of the KCAs appear to depend on the presence of an intact plasma membrane and, since they are abolished in high K+ medium, on membrane hyperpolarisation (Quast, 1993). How membrane hyperpolarisation and intracellular Ca2+ release are linked is unclear. There is evidence that KCAs inhibit the refilling of intracellular Ca2+ stores by extracellular Ca2+. In contrast to results on Ca2+ release in vascular smooth muscle cells (Itoh et al., 1992), such an effect of LCRK has been reported in rabbit airways smooth muscle cells that lack an intact plasma membrane, suggesting that KCAs influence the polarisation of the sarcoplasmic reticulum as well as the plasma membrane (Chopra et al., 1992). There is also evidence that in vascular tissue KCAs inhibit spasmogen-induced intracellular Ca2+ release by reducing inositol triphosphate formation. This is a glibenclamide and KCl-sensitive mechanism (see Quast, 1993). In bovine trachealis, however, inhibition of inositol triphosphate accumulation by LCRK was delayed and mimicked by the Ca entry blocker nitrendipine, indicating that it was secondary to closure of voltage-operated Ca channels (Challiss et al., 1992). The mechanism of action of KCAs in airways smooth muscle therefore poses even more questions than that for vascular smooth muscle. Despite this evidence that KCAs can affect the release of intracellular Ca2+ they tend to lack efficacy against muscarinically-induced responses (see sections 12.2.1 and 12.3.2). There are several possible explanations for this. It has been reported that in gastric smooth muscle cells, muscarinic agonists antagonise K+ M

KCAS: AIRWAY PHARMACOLOGY AND BRONCHIAL ASTHMA 361

currents activated by β -adrenoceptor agonists (Sims et al., 1989). If they also block K+ currents opened by KCAs, the KCAs might lack efficacy against muscarinic agonists. Some K channels do, however, appear to open in acetylcholine-depolarised tracheal smooth muscle cells (Boyle et al., 1988), and so to explain the poor efficacy of not just KCAs but also other bronchodilators against muscarinic tone, one must invoke a selective inhibition of more than one but not all K channels. Another possibility is that the inositol triphosphate stores mobilised by muscarinic agonists differ from those mobilised by other spasmogens and are less susceptible to KCAs. Thus in bovine and guinea-pig trachea, there are differences between carbachol and histamine in inositol triphosphate mobilisation and the effect of inhibitory agents thereon (Hall et al., 1989, 1990; Langlands et al., 1989). Lastly, carbachol may cause the actin and myosin filaments to ‘latch’, so that a contraction is maintained with little requirement for raised Ca2+ levels (Dillon et al, 1981). It has been suggested that HOE 234 is more effective than LCRK against carbachol-induced tone in guinea-pig tracheal rings because it has a greater effect on those K channels that influence intracellular Ca2+ release (Englert et al., 1992). Although BRL 55834 is not more effective than LCRK against carbachol-induced tone, a similar hypothesis might explain its airways selectivity relative to LCRK. Thus Ca entry blockers are more effective at lowering BP than preventing bronchoconstriction. Attempts to support this hypothesis have, however, met with failure (Taylor et al., 1992c). 12.7 Conclusions and Outlook There is great concern among some workers about the current treatment of asthma. In spite of the availability of apparently effective drugs, morbidity and mortality have not improved and in some countries may be rising. This may be due to a real increase in the incidence of the disease, but there is also evidence that regular therapy with β -adrenoceptor agonist bronchodilators, whilst often essential as a symptomatic therapy, is exacerbating the progression of the disease. Asthma is an inflammatory disease of the lung. It is almost always associated with airways hyperresponsiveness. Whether the latter is entirely a consequence of inflammation is unclear. Most experts advise practitioners to prescribe antiallergic or anti-inflammatory drugs for all but the mildest asthma. This advice is often ignored, however, because symptomatic therapy appears to provide adequate control and because of worries about the side-effects of steroids. KCAs offer the potential of bronchodilatation coupled with reduced airways hyperresponsiveness, which may in turn be due to a reduction in some components of airways inflammation, but may have other explanations. Animal studies, supported by experiments on human airways in vitro, clearly indicate that KCAs should be effective as bronchodilators in man if administered at a

362 K CHANNELS AND THEIR MODULATORS

sufficient dose. Their potential stems from an ability to restrict Ca2+ influx into the cytosol not only through voltage-operated Ca channels but also apparently from intracellular stores. Animal studies further suggest that KCAs inhibit NANCe and cholinergic transmission in the lung, although this has not been adequately demonstrated by direct measurement of neurotransmitter release. Inhibition of neurotransmitter release may largely account for why KCAs inhibit microvascular leakage, mucus secretion and the development of airways hyperresponsiveness in animals. Clinical studies have presented a mixed picture. The original enthusiasm generated by the efficacy of CRK in nocturnal asthma was dampened by the failure of LCRK to show adequate efficacy in asthma trials. It has been difficult to design airways selective KCAs and many pharmaceutical companies have withdrawn from this area. Nevertheless, these clinical studies have provided sufficient encouragement to believe that by inhalation, or by using a more airways-selective compound, such as BRL 55834, useful anti-asthmatic activity may be achieved. The ultimate test of such a therapy will be whether it shows any advantage over β 2-adrenoceptor agonists in terms of progression of the disease. Acknowledgements I am indebted to my present and past colleagues at SmithKline Beecham whose results and ideas are integral to this article. In particular, I would like to thank N.E.Bowring, D.R. Buckle and S.G.Taylor. I also thank Irene Fussell for typing the manuscript. References AHMED, F., FOSTER, R.W. & SMALL, R.C. (1985) Br. J. Pharmacol, 84, 861–869. AIKAWA, T., SEKIZAWA, K., MORIKAWA, M., ITABASHI, S., SASAKI, H. & TAKISHIMA, T. (1992) Br. J. Pharmacol., 105, 609–612. AITKEN, S. & MCCAIG, D.J. (1991) Br. J. Pharmacol., 104, 130P. ALLEN, S.J., BOYLE, J.P., CORTIJO, J., FOSTER, R.W., MORGAN, G.P. & SMALL, R.C. (1986) Br. J. Pharmacol., 89, 395–405. ANON, (1993) J. Allergy Clin. Immunol., 91, 1234–1237. ARCH, J.R.S., BUCKLE, D.R., BUMSTEAD, J., CLARKE, G.D., TAYLOR, J.F. & TAYLOR, S.G. (1988) Br. J. Pharmacol., 95, 763–770. ARCH, J.R.S., BOWRING, N.E. & BUCKLE, D.R. (1994) Pulmonary Pharmacol., 7, 121–128. BAIRD, A., HAMILTON, T.C., RICHARDS, D.H., TASKER, T. & WILLIAMS, A.J. (1988) Br. J. Clin. Pharmacol., 25, 114P. BARNES , P.J. (1993) Br. Med. J. 307, 814–815. BARNES, P.J., BARANIUK, J.N. & BELVISI, M.G. (1991) Am. Resp. Dis., 144, 1187–1198. BEACH, J.R., YOUNG, C.L., HARKAWAT, R., GARDINER, P.V., AVERY, A.J., COWARD, G.A., WALTERS, E.H. & HENDRICK, D.J. (1993) Pulmonary Pharmacol., 6, 155–157.

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Recent Literature BUCKLE, D.R. (1993) Prospects for Potassium Channel Activators in the Treatment of Airways Obstruction. Pulmonary Pharmacol., 6, 161–169. EDWARDS, G., SCHNIEDER, J., NIEDERSTE-HOLLENBERG, NOAK, TH. & WESTON, A.H. (1995) Effects of BRL55834 in Rat Portal Vein and Bovine Trachea: Evidence for the Induction of a Glibenclamide-resistant, ATP-sensitive Potassium Current. Br. J. Pharmacol., 115, 1027–1037.

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FAURSCHOU, P. MIKKELSEN, K.L., STEFFENSEN, I. & FRANKE, B. (1994) The Lack of Bronchodilator Effect and the Short-term Safety of Cumulative Single Doses of an Inhaled Potassium Channel Opener (Bimakalim) in Adult Patients with Mild to Moderate Bronchial Asthma. Pulmonary Pharmacol., 7, 293–297. KIDNEY, J.C., LOTRALL, J.O., LEI, Y.H., CHUNG, K.F. & BARNES, P.J. (1995) The Effect of Inhaled Potassium Channel Openers, Levcromakalim and HOE 234, on Bronchoconstriction and Airway Microvascular Leakage. Eur J. Pharmacol. in press. MORLEY, J. (1994) K+ Channel Openers and Suppression of Airway Hyperreactivity. Trends Pharmacol. Sci., 15, 463–468. MORLEY, J. (1994) Potassium Channel Openers and Asthma. Clin. Revs in Allergy, 12, 109–120. NlELSEN-KUDSK, J.E., MELLEMKJAER, S. & THIRSTRUP, S. (1994) Inhibition by Cromakalim, Pinacidil, Terbutaline, Theophylline and Verapamil of Non-cholinergic Nerve-mediated Contractions of Guinea-pig Isolated Bronchi. Pulmonary Pharmacol., 7, 285–292.

13 Potassium Channels in Pancreatic β -Cells: Modulation, Pharmacology and their Role in the Regulation of Insulin Secretion M.J.DUNNE, J.H.JAGGAR, E.A.HARDING, C.KANE & P.E.SQUIRES Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK. 13.1 Introduction Ion channels have an integral role to play in governing the release of insulin from the β -cells of the pancreatic islets of Langerhans. These cells synthesise, store and subsequently release insulin in response to a number of nutrient, hormonal, and neural stimuli. The major physiological regulator of insulin secretion is an increase in the plasma glucose concentration, which mediates its effects through an increase in the intracellular concentration of calcium ions ([Ca2+]i). This is associated with Ca2+ influx across the plasma membrane, and not mobilisation of Ca2+ from intracellular calcium stores (Wollheim and Sharp, 1981), To adjust the rate of insulin release in response to fluctuations in the availability of glucose, the β -cell has adopted several discrete control mechanisms to regulate [Ca2+]i. The most striking of these is a complex pattern of electrophysiological events that invokes changes in the β -cell membrane potential, and the remodelling of ionic fluxes across the plasma membrane, and intracellular membrane systems (see Figure 13.1). When the concentration of glucose is below that required to elicit insulin secretion, the membrane potential is silent and generally found to rest between −60 and −70 mV. This is determined by a high resting K+ permeability, maintained by the Na+ -K+-ATPase and open K ion channels. Raising the glucose concentration above the threshold for secretion (>7 mM), promotes a slow depolarisation of the membrane, which brings the β -cell to a critical threshold potential at which electrical activity is initiated. With conventional microelectrodes the patterns of responses were first characterised in the late 1960s/early 1970s (Dean and Matthews 1968, Matthews and Sakamoto 1975; Henquin, 1978, 1979). These basic changes in cell membrane potential have been extensively reviewed elsewhere (see Henquin and Meissner, 1984). In brief, the overall pattern is one of cycles, or slow waves of depolarisation and hyperpolarisation of the membrane culminating in the generation of plateau potentials with superimposed voltage-dependent Ca2+-spikes, (Figure 13.1). The inter-burst period is not strictly ‘silent’ since the membrane undergoes a slow 5–

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10 mV depolarisation. The function of this is to bring the cell to threshold from which a rapid depolarisation can again be initiated. For cell signalling, the most

Figure 13.1 Glucose induced electrical activity in pancreatic β -cells. Data obtained using conventional glass microelectrodes. (Modified from Henquin and Meissner, 1984.)

important events occur during the quasi-sustained plateau potential since each voltage-dependent spike results from Ca2+ influx across the membrane (Dean and Matthews 1968; Wollheim and Sharp, 1981; Henquin and Meissner, 1984; Henquin, 1990). This occurs through voltage-gated Ca selective ion channels— the activities of which are closely guarded by the change in the cell membrane potential. In this manner, changes in the β -cell electrophysiology provide the mechanism for linking glucose metabolism, to an increase in [Ca2+]i (Santos et al., 1991) and the subsequent release of insulin from the cells by exocytosis. In vivo blood-glucose levels do not undergo sudden ‘stepped’ increases or decreases. The β -cell must therefore be capable of ‘fine-tuning’ glucose homeostasis by even more subtle changes in the cell electrical activity. This is largely achieved by changes in the periodicity of the plateau potential and interburst periods, which in turn increases and/or decreases the duration of action potential firing. Therefore as the concentration of glucose is elevated beyond 7– 10 mM, the duration of the plateau potential is characteristically increased, whilst the inter-burst period is decreased—thereby augmenting changes in [Ca2+] i. This pattern continues with elevations in the glucose concentration, so that at extremes of hyperglycaemia the bursting patterns themselves cease, and the membrane becomes permanently depolarised and continuously fires Ca2+ -spike potentials (for more detailed reviews see Henquin and Meissner, 1984; Petersen and Findlay, 1987; Ashcroft and Rorsman, 1989; Dunne and Petersen, 1991). Patch-clamp approaches to β -cell electrophysiology began in the early 1980s, and have allowed us to study precise details of ion channels present in insulinsecreting cells (Figure 13.2.). Many original research papers have now been published in the literature examining the role, regulation and pharmacology of ion channels in the β -cell. As a reflection on this active field of research both stimulus-secretion coupling mechanisms and the electrophysiology of pancreatic

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β -cells have been the subject of several review articles over the past few years (Petersen and Findlay, 1987; Ashcroft, 1988; Petersen, 1988; Ashcroft and Rorsman, 1989; Henquin, 1990; Rorsman and Trube, 1990; Dunne and Petersen, 1991; Ashcroft et al., 1992b; Dunne, 1992; Hellman et al., 1992a, 1992b; Henquin et al., 1992; Dunne et al., 1994a). Pharmacological interest in K channel modulation has so far been mainly targeted to the ATP-sensitive K (KATP) channel. The sudden availability of relatively high affinity ligands for this channel has led to more recent studies examining the

Figure 13.2Summary of ion channels and their principal levels of regulation in the pancreatic β -cell. Abbreviations K-DR=delayed rectifier K channel; K-A=transient outward K channel; K-1=low conductance/receptor operated K channel; K+ATP=ATPsensitive K channel; K+maxi=Ca- and voltage-gated K channel; NS=non-selective cation channel.

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mechanisms by which synthetic compounds—both openers and blockers, interact with the ion channel. On the face of it, it is somewhat surprising that there is such a wealth of literature devoted to β -cell ion channels, particularly when one considers that the endocrine portion of the pancreas is only located in 1–2% of the total mass of the pancreatic tissue; the islets of Langerhans. Part of the explanation lies in the fact that patch-clamp experiments can provide a great deal of information from relatively small amounts of experimental material, and because there are several insulin-secreting cell lines readily available (HIT T15, RINm5F, CRI-G1, MIN 6, etc.) upon which to model stimulus-secretion coupling events. Unfortunately, despite a great deal of investigation and understanding we are still unable to describe completely the complex patterns of electrical events that typify β -cell electrophysiology in terms of which ion channels are open when, and what cellular events control their gating parameters. 13.2 Ca Channels and Insulin-secreting Cells The role of the Ca channel in pancreatic β -cells is probably the easiest of all the ion channels to define. Two different types of voltage-gated Ca channels have been described in the rat β -cell with electrophysiological properties that closely match those of the L-type and T-type Ca channels described in other tissues (Satin and Cook, 1988; Hiriart and Matteson, 1988; Ashcroft et al., 1990; Hopkins et al., 1991). Selective antagonists to the L-type Ca channel completely suppress glucose-evoked insulin secretion (Wollheim and Sharp, 1981), but evidence supporting a role for the T-type Ca channel is somewhat less convincing: mouse β -cells only possess L-type Ca channels, and even in the rat the inactivation kinetics of the T-type channel are too rapid to account for a significant contribution to the spike potential (for recent reviews and further discussions see Ashcroft and Rorsman, 1989; Smith et al., 1993a). L-type Ca channels display long-lasting openings at potentials more positive than −60 mV, and show little in the way of rapid inactivation kinetics. Ca2+ currents are maximal in the β -cell at around −20 mV and are present at sufficient magnitudes in most types of β -cells to account for all the inward current associated with the generation of the spike potential. Ca2+ ions entering the cell through L-type voltage-gated Ca channels are therefore responsible for the upstroke of the action potential spike. Termination of the spike potential involves the opening of K channels (see below), and maybe the Ca-dependent inactivation of the L-type Ca channel—this could result directly from the preceding entry of Ca2+ (Smith et al., 1993a). It has also been suggested that Ca2+ channels are responsible for the generation, and the maintenance of the slow waves of potential changes— although this is open to some considerable debate. However, as the L-type Ca channel exhibits a slow voltage-dependent inactivation that takes place over

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several seconds, it could account for the ‘pacemaker-like’ activity of the cells seen in the presence of glucose. Changes in the activity of Ca channels in this manner would also influence other Ca-dependent ion currents involved in the cyclical pattern of electrical activity. Apart from the influence of the membrane potential, the open probability of Ltype Ca channels is also increased by an elevation of the internal cyclic AMP (cAMP) concentration (Ashcroft and Rorsman, 1989), suggesting that a cAMPdependent protein kinase may be involved in governing the activity of the channels. There is also good evidence from both clonal and rodent β -cells for a link between nutrient metabolism, and the opening of voltage-gated Ca channels that does not involve a change in the cell membrane potential. This was first demonstrated using the RINm5F cell line by Velasco et al. (1988) who reported increases in the activity of Ca channels in KCl-depolarised cell-attached patches exposed to D-glyceraldehyde. Similar effects were subsequently found for glucose using normal mouse pancreatic β -cells (Smith et al., 1990b), and whilst they provide important data for a novel coupling event between the metabolic status of the cell and the regulation of Ca2+ influx, the underlying mechanisms have not yet been evaluated. 13.3 K Channels and Insulin-secreting Cells K channels are the most widely studied of all the ion channels in pancreatic β cells. They are also the most varied group of ion channels, which reflects upon the number of different roles that K channels fulfil. These include: (i) control of the resting cell membrane potential, (ii) the regulation of action potential magnitude, duration and periodicity, and (iii) K channels are also the target site for a number of hormones and neurotransmitters that are able to promote or inhibit secretion by mechanisms that are in part dependent upon changes in the cell membrane potential. 13.3.1 KATP Channels Having first been described in cardiac myocytes by Noma (1983), KATP channels are now recognised as providing a vital link between cellular metabolism and control of the cell membrane potential in a number of different tissues, none more so than in the pancreatic β -cell. Cook and Hales (1984) were the first to identify KATP channels in the pancreas, and since then there have been many papers describing the role, regulation and the intriguing pharmacology of this particular K channel.

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Role of KATP channels in β -cell electrophysiology In the resting cell, open events from KATP channels are now widely recognised to be responsible for establishing the cell membrane potential (see Ashcroft, 1988). At least two forms of KATP channels have been identified, and these may only differ in their size (Findlay et al., 1985b; Dunne and Petersen, 1991). Both channels appear to be controlled by similar changes in the concentration of nucleotides at the inside face of the membrane. Overall the response of the channel to intracellular ATP predominates, but many other nucleotides, including ADP, have a major influence on the frequency of channel openings and closures (see below). When glucose is present at stimulatory concentrations, openings from these channels in intact β -cells cease (Ashcroft et al., 1984). As glucose metabolism will also alter the internal ATP/ADP availability, this has led to the widely accepted opinion that increases in the ratio of ATP: ADP couple metabolic events to ionic events (Petersen and Findlay, 1987; Ashcroft and Rorsman, 1989; Dunne and Petersen, 1991). Another possible candidate for this link is changes in the pyridine nucleotide concentrations which are altered during nutrient metabolism (Pralong et al., 1990), and have effects on KATP channels (Dunne et al., 1988a). Nevertheless, whatever the causative event, glucoseinduced closure of KATP channels would, in the presence of an inward current tend to drive the membrane potential from its resting value towards the ‘threshold potential’ from which complex changes in electrical activity is an inevitable consequence. In intact resting β -cells the only single-channel K+ current events recorded tend to be those of the KATP channel. This is an intriguing observation given that the actual proportion of open channels is probably much less than 10% of the total number of KATP channels present in the cells (Petersen and Findlay, 1987). With an estimated 90–95% of the channels already closed at rest, this would therefore suggest that closure of just a few additional channels is able to cause a highly significant change on the cell membrane potential. This idea of ‘spare channels’ was first put forward to explain an obvious discrepancy between the fact that in excised patches of membrane the sensitivity of KATP channels for ATP (IC50 value=approximately 50 µM (see Ashcroft, 1988)), is far too high to account for channel openings seen in intact cells, where estimations of the intracellular ATP concentration are in the millimolar range (Petersen and Findlay, 1987; Cook et al., 1988). Central to this hypothesis is the suggestion that KATP channels in the β -cell should show a range of sensitivities to internal ATP. However, only in cardiac myocytes has this been elegantly demonstrated to any degree of satisfaction. Unlike the β -cell, KATP channels are not open under normal conditions in cardiac myocytes. The tissue maintains a relatively high resting permeability to K+ ions, until the plateau phase of the action potential which is then characterised by a low membrane conductance. Opening at this point, possibly as a result of ischaemia, KATP channels would tend to reduce the action potential duration. In cardiac myocytes Findlay and Faivre (1990) have been able to demonstrate using a very large

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sample of excised inside-out patches (>100), that KATP channels do indeed exhibit a very wide range of sensitivities to intracellular ATP, with IC50 values ranging from 9–580 µM. These data therefore considerably widen the ranges of internal ATP concentration that one might expect to be involved with the modulation of KATP channels. Clearly a similar process may operate in insulinsecreting cells, and whilst we do not have the level of detail as has been demonstrated in cardiac myocytes, there is certainly evidence from many published records that not all KATP channels in a patch of membrane are equally sensitive to internally applied ATP. With such a reserve of spare channels, it seems somehow unlikely that the only role for this channel is in shifting the membrane potential by just a few millivolts. Additional roles for KATP channels have therefore been proposed. It is likely that they interact at some level to regulate the bursting patterns of slow waves of membrane potential change. The evidence for this has come from studies of the effects of selective inhibitors of these channels on changes in the cell electrical activity. By themselves, i.e. without glucose, compounds such as the sulphonylureas glibenclamide and tolbutamide, are unable to maintain the repetitive cycles of electrical events that so typify the response to glucose. However, they will modulate the effects of glucose in a quasi-physiological dependent manner, and this is seen even when glucose is present at sub-threshold concentrations (see Henquin, 1990; Chay et al., 1990). Evidence from other studies has implied additional role(s) for KATP channels. In particular that they are involved in the receptor-operated control of insulin secretion. The β -cell is unique in receiving signals from a number of regulatory influences: nutrient secretagogues, gastrointestinal hormones, neurohormones and neurotransmitters, and the paracrine effects of glucagon and somatostatin. Some of the details of the actions of several agonist-mediated events have been investigated. For electrophysiological studies most experiments have been carried out using clonal insulinoma cells, where there is very good evidence for the fact that several hormones and neurotransmitters/neuropeptides will promote changes in the β cell membrane potential, and do so by modulating the gating of KATP channels. For example, both somatostatin and galanin will activate KATP channels in a Gprotein-dependent manner, hyperpolarise the membrane, lower [Ca2+]i and inhibit insulin secretion (De Weille et al., 1988, 1989; Dunne et al., 1989; Homaidan et al., 1991), whilst vasopressin and purinergic receptor agonists have exactly the opposite electrophysiological and secretory effects (Martin et al., 1989; Arkhammar et al., 1990; Gao et al., 1990; Li et al., 1990; Thorn and Petersen 1991; Squires et al., 1994). Regulation of KATP channels in β -cells In insulin-secreting cells, KATP channels are not particularly sensitive to changes in the cell membrane potential, their gating is relatively unaffected by changes in the intracellular Ca2+ concentration (see Petersen and Findlay, 1987; Ashcroft,

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1988; Ashcroft and Rorsman, 1989; Dunne and Petersen, 1991), and changes in the intracellular pH do little within physiological limits (Proks et al., 1994). Internal nucleotides however do play a very important role in governing the activity of these channels. In vitro experiments using excised inside-out patches have maybe presented an all too confusing picture of the nucleotide-dependent gating of these channels; many different nucleotides can cause changes in the gating of KATP channels, some effects are Mg2+ ion dependent, others not, and all are complicated by changes in channel activity due to run-down. How these translate to effective regulators of the channel in the intact cell has not yet been fully evaluated. Broadly speaking, intracellular nucleotides can either cause inhibition or activation of channels, effects which are further complicated by the fact that some of the inhibitory nucleotides will open channels in the presence of ATP. To present some order to what may be physiologically relevant, and what is experimental observation we should consider that in vivo KATP channels do not undergo run-down (but do they inactivate? (see below)), that there will be Mg2+ available to the membrane (but at what concentration?), and that because of the high intracellular concentration, the influence of internal ATP predominates over channel gating (we make this assumption although we do not know the precise concentration of ATP at the plasma membrane). The overriding action of ATP on these channels when added to the inside of the membrane is to evoke closure (Cook and Hales, 1984). This effect is concentration-related; the greater the concentration of ATP, the greater the degree of channel inhibition. Values of ATP corresponding to 50% inhibition are available, and indicate that the IC50 is between 10 and 70 µM (see Ashcroft, 1988). But it is very evident from experimental data that not all channels are equally sensitive to ATP and even in the presence of 5 mM intracellular ATP channel openings can be seen (Dunne et al., 1986b). Estimates of the Hill coefficient for channel blockade vary from around 1–1.8 (Cook and Hales, 1984; Ohno-Shosaku et al., 1987; Ribalet and Ciani, 1987), which may indicate that several molecules of ATP are able to bind to the channel in a co-operative manner. Non-hydrolysable analogues of ATP induce channel inhibition, suggesting that protein phosphorylation is probably not required for channel closure (Ohno-Shosaku et al., 1987; Dunne et al., 1988a). This is further supported by the fact that ATP is still an effective blocker of channels in the complete absence of internal Mg2+ (Dunne et al., 1987; Ohno-Shosaku et al., 1987; Ashcroft and Kakei, 1989). Indeed the effects of Mg2+ -free solutions on KATP channels are particularly interesting. Without intracellular ATP, removing Mg2+ from the inner membrane causes an increase in channel activity (Dunne et al., 1987). This activation is characterised by an increase in the single channel conductance (Findlay, 1987; Ashcroft et al., 1989), and by an increase in the number of channel openings (Dunne et al., 1987; Findlay, 1987). In the presence of internal ATP exactly the opposite effect occurs; Mg2+ removal enhances the potency of block (Findlay, 1987; Dunne et al., 1988b; Ashcroft and Kakei, 1989). One of the implications of these experiments is that the ‘free’

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concentration of ATP (probably ATP4–) determines the degree of K channel inhibition (Dunne et al., 1987, 1988b; Ashcroft and Kakei, 1989). This aspect of channel regulation in the β -cell is different to that in other tissues; in skeletal muscle cells the free acid and Mg salts of ATP are equally effective at blocking channels (Davies, 1990), and in cardiac cells Mg-ATP2− is a more effective blocker of channels than the free ionic form of ATP (Findlay, 1988). The inhibitory effects of ATP are not membrane potential sensitive, and characteristically involve a decrease both in the mean open time of the channel, and the number of open events per burst of activity (Ashcroft and Kakei, 1989). There is also no significant effect of ATP on the unitary current event (see Ashcroft, 1988). A major difficulty encountered when studying KATP channels in these cells is the problem of channel run-down, a time-dependent loss of channel activity when either whole-cells are generated, or excised patches formed (Findlay et al., 1985c; Trube et al., 1986; Ohno-Shosaku et al., 1987). Run-down of channels is extremely variable from experiment to experiment, and seems to follow a two stage process (Kozlowski and Ashford, 1990) with near complete loss of channel openings occurring between tens of seconds (‘the β -phase’) and sometimes several minutes/ hours after establishing the recording configuration (‘the β phase’). This gradual loss of channel activity that occurs after separation of the membrane from the structural and biochemical environment of the intact cell, has also been observed for KATP channels in cardiac myocytes (Kakei and Noma, 1984; Trube and Hescheler, 1984), but the amphibian skeletal muscle cell KATP channels do not readily exhibit run-down (Spruce et al., 1987). Understanding the process of run-down has attracted a great deal of attention, because it provides us with a means to investigate the factors responsible for maintaining channel integrity in vivo. It was noted several years ago that although ATP blocked the channels, it also possessed the ability to reactivate channels after rundown had taken place—leading to the idea of channel ‘refreshment’ (Findlay and Dunne, 1986b; Misler et al., 1986). This effect is enhanced by high concentrations of ATP, and by prolonging the duration that the nucleotide is present at the membrane (Findlay and Dunne, 1986b). However, there comes a time in the experiment when no matter how long or what concentration is used, ATP will not reactivate the channels. This ATP data along with two other pieces of evidence has fronted the idea that run-down may involve channel dephosphorylation: (i) non-hydrolysable ATP analogues cannot mimic the effects of ATP on refreshing the channels (Ohno-Shosaku et al., 1987), and (ii) in the complete absence of Mg2+ channel run-down is effectively eliminated (Kozlowski and Ashford, 1990). In cardiac myocytes it has also been demonstrated that elevation of intracellular Ca2+ will enhance the rate of rundown, suggesting the process is Ca2+ -dependent (Findlay, 1988). If the dephosphorylation model is correct then there must be some endogenous event (s) occurring at the plasma membrane to alter continually the phosphorylation status of the channels; maybe phosphorylation by a protein kinase(s), and

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dephosphorylation by a protein phosphatase(s)? It could be envisaged that the activities of these molecules are modified by the removal of the membrane from the intact cell, and that this then changes the functionality of the channels. However, doubts have been raised about the validity of the dephosphorylation model. One reason for this, is that a range of inhibitors of protein phosphatases do not influence the rate of channel run-down (Williams, 1993) [although 2,3butanedione monoximine, which behaves as a chemical phosphatase, does reversibly inhibit the channels (Smith et al., 1993b)]. Second, trypsinisation of patches has been demonstrated to eliminate effectively run-down of channels in both cardiac myocytes and pancreatic β -cells (Nichols and Lopatin, 1993; Proks and Ashcroft, 1993). The premise for the trypsin experiments comes from studies carried out on voltage-gated K channels, particularly the Shaker K(A) channels of Drosophila melanogaster. These channels undergo rapid (N-type) inactivation, and one of the more widely accepted hypotheses to account for this is the concept of a ‘ball-and-chain’ (Hoshi et al., 1990; Zagotta et al., 1990). This is located in the cytosol and originates from the N-terminal region of the channel protein. The function of ball-and-chain is to occlude the flow of ions at the intracellular mouth of the channel during the change in the membrane potential. Evidence to support these ideas is convincing. First, genetically engineered Drosophila mutants that do not have the first 22 amino acids of the N-terminal region possess K+ channels that do not exhibit inactivation kinetics, but this can be restored by the intracellular addition of a synthetic peptide that corresponds to the putative ball-and-chain region – the ‘Shaker B inactivation peptide’. Second, proteolytic treatment of inside-out patches with trypsin, which presumably strips off the intracellularly disposed ball-and-chain region, prevents channel inactivation (Hoshi et al., 1990; Zagotta et al., 1990). Trypsin and other proteolytic enzymes have also been shown to remove the inactivation process of other types of channels including voltage-gated Na channels (Armstrong et al., 1973; Armstrong, 1981; Sevcik and Narahashi, 1975), Ca channels (Hescheler and Trautwein 1988; Oberjo-Paz et al., 1991) and K channels (Dubinsky et al., 1992; Mayorga-Wark et al., 1993). The significant effects of trypsin and chymotrypsin on β -cell KATP channels could imply that a similar type of channel inactivation, albeit on a much slower time scale may be involved in run-down. In a recent series of experiments we have taken these arguments one step further, and demonstrated that the synthetic ‘inactivating’ peptide ShBN22 causes a significant modulation of KATP channels in human and rat isolated insulinsecreting β -cells (Harding et al., 1994). When added to the inside face of the cell membrane, ShBN22 (50–100 µM) was found to inhibit channels and these effects were not readily reversible (Figure 13.3). The effects of ShBN22 are therefore somewhat analogous to the induction of channel run-down, and provide support for the idea that channel inactivation plays at least some part in the process of run-down, which was previously thought to be an event only driven by channel dephosphorylation. As the Shaker inactivation peptide has recently been reported to have no effect on the gating of KATP channels in rat skeletal muscle cells, this

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may indicate functional and structural diversity in the population of KATP channels (Beirao et al., 1994). Finally on the subject of channel run-down one other interesting observation has recently been made by Edwards and Weston (1993). Using the whole-cell patch-clamp

Figure 13.3 Inhibition of KATP channels in an inside-out patch (see cartoon) by the Shaker inactivating peptide ShBN22.

technique they have suggested that there is a correlation between the delayed rectifier K+ current and the KATP current in RINm5F cells. Thus dephosphorylation of a delayed rectifier K channel may confer a voltageinsensitive state on the channel, and that in this state it represents the KATP channel. However this study failed to address if dephosphorylation of channels is purely artefactual resulting from the experimental conditions. It is therefore hard to conceive how in the intact cell complete dephosphorylation of channels could take place. Under certain experimental conditions internal ATP has also been demonstrated to activate KATP channels in the β -cell; and this is not a ‘refreshment effect’ of run-down channels (Dunne et al., 1988a; Ribalet et al., 1989). To account for blocking and activation of channels by ATP, it has been suggested that a cAMP-dependent protein kinase is closely associated with the KATP channel and that the inhibitory effects of ATP on the channels can be explained by a decrease in kinase activity due to the ATP-dependent binding of a kinase inhibitor (or a protein with similar inhibitory properties to the kinase) (Ribalet et al., 1989). The model goes further to explain how the stimulatory effects of ATP may result from kinase-mediated phosphorylation of the channel (Ribalet et al., 1989). However, this hypothesis, which relies upon rapid protein phosphorylation/dephosphorylation reactions, cannot readily explain how the inhibitory effects of ATP analogues are very readily reversible (Ohno-Shosaku et al., 1987; Dunne et al., 1988b) nor how ATP can close channels in the absence of cytosolic Mg2+ (Dunne et al., 1987; Ashcroft and Kakei, 1989). Apart from the suggestion that KATP channels are regulated by protein kinase A, there may

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also be evidence that channel gating is controlled by a Ca2+- and phospholipid dependent protein kinase C (PKC). This has mainly come from studies carried out to investigate the actions of potent activators of protein kinase C such as phorbol myristate acetate (PMA) on KATP channels. In intact insulin-secreting cells PMA has been reported to both inhibit and activate KATP channels, which results in either a depolarisation or a hyperpolarisation of the cell membrane potential (Ribalet et al., 1988; Wollheim et al., 1988; De Weille et al., 1989). These actions may be explained by a dual action on the channel possibly mediated by more than one form of protein kinase C (Dunne et al., 1990b; Dunne, 1994). Besides ATP, ADP also has complex actions on KATP channels. In general, concentrations lower than 500 µM tend to activate the channels, whereas higher concentrations tend to inhibit them (Dunne and Petersen, 1986a; Kakei et al., 1986). As for the activatory effects of ADP, these may involve phosphorylation-mediated events, since ADPβ S, a non-hydrolysable analogue of ADP, only closes channels, and ADP has no activatory effects without Mg2+ (Dunne and Petersen, 1986a; Dunne et al., 1988b; Schwanstecher et al., 1994). These somewhat variable and unpredictable effects of ADP may therefore result from ADP having multiple binding sites on the channel. The most important point to note, however, is that under ‘pseudophysiological conditions’ where internal ATP is present, blocking concentrations of ADP tend to activate the channels, and this occurs even when the concentration of ADP is some 30–fold less than ATP (Dunne and Petersen, 1986a; Kakei et al., 1986; Dunne et al., 1988b; Hopkins et al., 1992). Activation of ATP-inhibited channels by ADP can also be mimicked by ADPβ S, with both ADP and ADPβ S activating KATP channels in the presence of either ATPβ S, AMP-PNP or AMP-PCP. This may indicate that competitive interactions between the two adenosine nucleotides, rather than protein phosphorylation-dependent processes, are more important in determining the ability of the channels to open in the intact cell (Dunne et al., 1988b). The actions of ADP are also governed by the availability of internal Mg2 + and by the extent of channel run-down (Dunne and Petersen, 1986a; Kakei et al., 1986; Findlay, 1987; Dunne et al., 1988b; Bokvist et al., 1991; Schwanstecher et al., 1994). The relationship between channel run-down and ADP effects have been investigated in some detail by Bokvist et al. (1991). One of their major observations was that the ability of ADP to activate channels in the presence of ATP declines with time following patch excision (due to run-down), whilst over the same time course (30–60 min in their experiments) the effects of ADP in the absence of ATP are now associated with channel inhibition. In summary therefore, the mechanisms that govern adenine nucleotide gating of these channels are highly involved, and may indeed be mediated by several discrete processes such as direct ligand binding, protein kinases and protein phosphatases. There are other internal nucleotides that also influence the activity of KATP channels. In particular the pyridine nucleotides and the guanosine nucleotides.

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What is interesting about the effects of the pyridine nucleotides NAD, NADH, NADP, and NADPH is that they all influence channel behaviour. High concentrations (>500 µM) tend to evoke inhibition, whereas lower concentrations (<500 µM) led to channel activation, effects which occur when the pyridine nucleotides are added directly to the membrane, or in the presence of ATP/ADP (Dunne et al., 1988a). Preliminary evidence suggests that in Mg2+free solutions the stimulatory effects of the pyridine nucleotides are lost, whilst the inhibitory actions are retained (Dunne, unpublished); but these need to be reaffirmed through additional studies. Intracellular concentrations of the pyridine nucleotides in unstimulated cells have been estimated, and for NADP, NADPH and NADP values of between 30 and 100 µM have been recorded (for review see Dunne et al., 1988b). The concentration of NAD on the other hand is considerably higher at 200–350 µM. Since NAD was found to inhibit KATP channels in the presence of quasiphysiological ATP/ADP concentrations, this may indicate that in the intact cell, NAD could contribute to the tonic inhibition of K channels. It has also been suggested that because glucose metabolism will alter the pyridine nucleotide status of the cell, that these nucleotides could play a role in coupling metabolic processes to ionic events (Pralong et al., 1990; Gilon and Henquin, 1992). GTP, GDP and their analogues cause activation of KATP channels in insulinsecreting cells, effects which are not directly attributable to a G-protein(s) associated with the channel. The effects of GTP and GDP are concentrationrelated (10 µM–2 mM), and are dependent upon the availability of Mg2+ (Dunne and Petersen, 1986b; Findlay, 1987; Schwanstecher et al., 1994). When Mg2+ is present at sub-micromolar concentrations at the internal face of the membrane, GTP and GDP inhibit KATP channels (Findlay, 1987; Schwanstecher et al., 1994). One piece of evidence for a GTP-binding protein involvement in the gating of the channel, is that the alumino-fluoride complex A1F4−, which is known to have effects on a number of G-protein regulated systems (Bigay et al., 1987), causes activation of KATP channels (Dunne et al., 1989). Potassium fluoride effects are abolished when A13+ is removed from the internal solution and enhanced when additional A13+ is made available (Dunne et al., 1989). In summary, internal nucleotides have numerous effects on the gating of KATP channels in the β -cell, and much of the confusion surrounding these actions comes from the fact that effects of a given nucleotide are dependent upon its concentration, the availability of Mg2+, ATP and the rate of channel run-down. Schwanstecher et al. (1994) have provided some clarity to this subject. They report that the blocking action of nucleotides in the presence of Mg2+ has the following order; ATPβ S>ATP>AMP-PNP>ADPβ S>2’deoxy-ATP>UTP, whilst in the absence of internal Mg2+ the potency series is ATP=ATPβ S>ADP>ADPβ S=AMP-PNP>2’deoxy-ATP>UTP>2’deoxyADP>GTP>GDP>UDP. Of course in vivo intracellular Mg2+ will always be available, but its concentration and subcellular localisation may be subjected to additional levels of regulation (Henquin et al., 1983). If this were the case, then

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changes in the [Mg2+]i would have marked effects on the nucleotide-dependent gating of KATP channels. Pharmacology of KATP channels in β -cells Blockers of KATP channels Arguably the most important group of pharmacological agents capable of inhibiting KATP channels in the β -cells are the antidiabetic sulphonylureas. In this review we only have space to outline the effects of the sulphonylureas on ion channels and insulin secretion, and for a detailed review we recommend Ashcroft and Ashcroft (1992). Sulphonylureas, typified by the first generation compound tolbutamide, and the more potent second generation molecule glibenclamide, are a class of hypoglycaemia-inducing drugs that have been used for a number of years to treat type II or non-insulin dependent diabetes (reviewed by Hellman and Taljedal, 1975). These compounds promote insulin secretion from the pancreas by a mechanism associated with a decrease in the K+ permeability of the β -cell membrane (Henquin, 1980; Gylfe et al., 1984); which leads to a depolarisation of the cell, and voltage-gated Ca2+ influx (Gylfe et al., 1984; Henquin and Meissner, 1982). Patch-clamp studies have shown that these effects are mediated by the sulphonylureas selectively closing KATP channels (Sturgess et al., 1985; Dunne et al., 1987; Ohno-Shosaku et al., 1987; Zunkler et al., 1988b; Ashcroft et al., 1989; Dunne, 1990b). The potency of channel block falls over three orders of magnitude: tolbutamide KI=10–17 µM, meglitinide K1=2.1 µM, glipizide KI=6.4 nM and glibenclamide KI=4–20 nM (Zunkler et al., 1988a; Ashcroft et al., 1989; Sturgess et al., 1988). Indeed channel block by glibenclamide is more potent than that of either tetrodotoxin or saxitoxin for the voltage-gated Na channel, and this has successfully helped in the biochemical identification of sulphonylurea receptors. Receptor occupancy by the sulphonylureas was first shown to be closely associated to channel blockade by Schmid-Antomarchi and collaborators (1987). Once suitable corrections have been made for albumin binding to the drug (Aguilar-Bryan et al., 1990), the specificity and density of sulphonylurea binding sites correlates very well with the ability of the sulphonylureas to elicit secretion, and inhibit KATP channels (Niki et al., 1989; Schmid-Antomarchi et al., 1987). There is also a good correlation between the estimated number of sulphonylurea receptor binding sites and the number of KATP channels per cell (Dunne and Petersen, 1990; Rorsman and Trube, 1990). Because of this there has been a great deal of speculation as to whether the sulphonylurea binding protein is an integral part of the KATP channel or not. One way to address this is to provide insights into the mechanism(s) of action of the sulphonylureas. Gylfe and collaborators (1984) originally suggested that the drugs only act from the outside of the cell. However, patch-clamp experiments have shown that sulphonylureas are effective from both sides of the plasma membrane, closing channels in whole-cells as well as excised inside-out, outside-out and cell-

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attached membrane patches (Trube et al., 1986). The cell-attached patch data are interesting in that direct access to channels from the bath solution is occluded by the patch-clamp recording pipette. These data suggest that the receptor must be reached from either the inside of the membrane or alternatively by lateral diffusion through the lipid domain of the membrane. A similar conclusion was also reached by Zunkler et al. (1989) who showed that channels are inhibited by the undissociated forms of tolbutamide and its related compounds. Inhibition of channels is not dependent upon the availability of Mg2+ (Dunne et al., 1987; Schwanstecher et al., 1994), although in the complete absence of internal Mg2+ the effectiveness of the sulphonylureas is reduced (Lee et al., 1994). There are however interactions between cytoplasmic nucleotides and the effects of tolbutamide. In particular sulphonylureas are more potent blockers in the presence of ATP, AMP-PNP, ADPβ S or ADP (Zunkler et al., 1988b; Schwanstecher et al., 1994); maybe indicating that the effects of the drugs are not directly onto the channel. The high affinity of glibenclamide for the KATP channel has meant that the drug can be used in the purification, and isolation of the putative channel protein. From porcine brain tissue, a protein of approximately 150 KDa has been isolated (Bernadi et al., 1988), whilst using a biologically active derivative of glibenclamide—5-iodo–2–hydroxyglyburide, a 140 KDa protein was subsequently isolated from the membranes of HIT insulinoma cells (AguilarBryan et al., 1990). This protein has been found to be an ADP-binding protein (Niki et al., 1990), but despite this, no sequence data have yet been reported, nor has the isolated protein been functionally reconstituted and shown to possess iontophoretic activity. This has fuelled speculation that the sulphonylurea receptor and the KATP channel are functionally and structurally distinct proteins, and aspects of patch-clamp data would tend to support this. First, in ventromedial hypothalamic neurones KATP channels are unaffected by tolbutamide in excised inside-out membrane patches but are blocked by the drug in cell-attached patches (Ashford et al., 1990); implying that the sulphonylurea receptor is not ‘tightly’ coupled to the KATP channel. Second, trypsinisation of the inside face of the cell membrane significantly reduces the effects of sulphonylureas on KATP channels in pancreatic β -cells (Proks and Ashcroft, 1993), and all but eliminates their action in cardiac myocytes (Nichols and Lopatin, 1993). In both the insulin-secreting cells and cardiac myocytes ATP-induced channel block is reduced but not eliminated by trypsin treatment, thereby indicating that proteolysis of the membrane uncouples the sulphonylurea- but not the ATP-dependent gating of the channel. Third, a subclone of the Cambridge insulinoma cell line CRI-G1 has recently been characterised—designated CRID11, and these cells have functional KATP channels, but unlike the CRI-G1 cells they are unresponsive to sulphonylureas in terms of ligand binding data, electrophysiology and insulin secretion (Ozanne et al., 1993). These cells provide another example of how the effects of sulphonylureas can be dissociated from nucleotide-dependent gating of KATP channels in insulin-secreting cells.

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Finally, unpublished data from our laboratory suggest that although sulphonylureas can block KATP channels in patches immediately upon formation of an excised patch, many minutes later the effects are lost, but the channels can still be inhibited by ATP. This may be somewhat analogous to the situation in the central medial hypothalamic neurons, but occurring over much longer periods of time because of a relatively higher affinity of the sulphonylureas for KATP channels in the β -cell. Clinically the sulphonylureas are widely used in the treatment of type II diabetes but after several years of therapy many patients develop a resistance to their effects. Since it is still not clear why this should occur, alternative compounds have been developed in the hope of finding a more permanent and effective treatment. One of these compounds linogliride, is structurally distinct from the sulphonylureas, and will both lower blood glucose levels and promote insulin secretion from the pancreas (see Ronner et al., 1991a, 1991b). The mechanism that underlies the effect of linogliride on the pancreatic β -cell has been investigated, and as for the sulphonylureas its effects are mediated by closure of KATP channels (Ronner et al., 1991a). Overall linogliride is a much less effective blocker of KATP cannels (IC50 = approximately 20 µM) than glibenclamide, but the data do indicate how therapeutic intervention of these channels could lead to the development of more novel compounds that may be effective in the management of diabetes. There is also a great deal of interest in the effects of β -adrenoceptor antagonists as potential modulators of K channels in the insulin-secreting cell. For a number of years it has been recognised that certain compounds such as phentolamine (β 1 and β 2) and efaroxan (β 2) are able to initiate insulin secretion from β -cells by mechanisms not involving the β -adrenoceptor (Malaisse et al., 1967; Schultz and Hasselblatt, 1989; Chan and Morgan, 1990). Both compounds will also alleviate the effects of diazoxide on insulin-secreting cells, and we can explain these effects by the fact that both phentolamine and efaroxan are inhibitors of KATP channels (Henquin et al., 1982; Dunne et al., 1990c; Plant and Henquin, 1990; Chan et al., 1991; Jonas et al., 1992). Concentration-response relationships from excised patches suggest that 50% blockade of channels could be achieved by approximately 12 µM efaroxan and 0.7 µM phentolamine (Chan et al., 1991; Dunne, 1991). These compounds are therefore as effective at blocking KATP channels as tolbutamide, but not glibenclamide. ATP-regulated K channels can also be blocked by the β 2-adrenoceptor antagonists yohimbine (Plant and Henquin, 1990; Dunne, 1991), antazoline and tolazoline (Dunne, 1991; Jonas et al., 1992). With the exception of yohimbine, the common structural feature of these compounds is an imidazoline group. It is now believed that several tissues express specific and functional receptors for imidazolines that have been termed ‘imidazoline preferred binding sites’ (Michel and Insel, 1989; Michel and Ernsberger, 1992). These receptors are functionally distinct from β -adrenoceptors, and may comprise at least two subtypes—designated I1 and I2, which may differ in their tissue distribution, subcellular location and

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pharmacological properties (Ernsberger, 1992; Atlas, 1991; Heible and Ruffolo, 1992; Kilpatrick et al., 1992). From electrophysiological studies we have the evidence that certain imidazolines will modulate KATP channels, but there is no indication from these types of studies that the effects are ‘receptor-mediated’. Indeed, the substituted imidazoline idazoxan (I2 receptor agonist), at low concentrations, has little effect upon insulin secretion in the absence of an adrenoceptor agonist (Ostenson et al., 1988; Chan and Morgan, 1990), but at higher concentrations (>100 µM) will partially reverse diazoxide-induced inhibition of insulin secretion (Chan and Morgan, 1990) and block KATP channels (Chan et al., 1991). If we compare the relative affinities of both efaroxan and idazoxan for the imidazoline receptor with the effects of the drugs on K channels and insulin secretion, we find that there is a very obvious negative correlation. Both efaroxan and phentolamine have a low affinity for the I2 receptor(s) (Langin and Lafontan, 1989) but are both very effective at closing KATP channels and initiating insulin secretion, whereas idazoxan has a very high affinity for the receptor but has relatively little effects upon K channels and secretion over the same concentration range. Therefore the relationship between imidazoline effects at the cellular level and inhibition of KATP channels may not be as straightforward as was originally thought. Recent experiments from our laboratory using human isolated pancreatic β -cells, indicate that imidazolines will elevate intracellular calcium levels ([Ca2+]i) through a depolarisation of the cell membrane caused by block of both the KATP channel and the large Ca- and voltage-gated Kmaxi (BKCa) channel (Dunne et al., 1994b). In summary, we believe that either the effects of imidazolines on K channels in the β -cell are not directly governed by receptor-operated events but involve direct binding with the ion channels, or that the β -cell possesses a novel subtype of imidazoline receptor that does not fall within the I1 (‘clonidine-preferring’) or I2 (‘idazoxan-preferring’) designated nomenclature (Chan et al., 1993). Concerning the mechanism (S) of channel block, we have evidence that the site of interaction on the KATP channel is distinct from that of the sulphonylureas. This is based primarily upon the fact that imidazolines block the channels after proteolytic treatment of the inside face of the cell membrane with trypsin (Dunne et al., 1994b); whereas the effects of the sulphonylureas under these conditions are markedly reduced (Proks and Ashcroft, 1993). Despite the uncertainty surrounding the nature of the imidazoline receptor expressed by the β -cell, and its relationship with the KATP channel, imidazolines may still provide an alternative therapy to the sulphonylureas in the treatment of type II diabetes (Kawazu et al., 1987). Finally, a large number of other compounds have also been found to block KATP channels in the β -cell: antiarrythmic agents such disopyramide (IC50=3.6 µM; Hayashi et al. 1993) and cibenzoline (IC50=1.5 µM; Kakei et al., 1993), the herbal extract ligustrazine (Peers et al., 1990), sparteine and the dopaminergic agonist amantadine (Ashcroft et al., 1992a), the protein kinase C inhibitor polymyxin B (Harding et al., 1994), TMB–8 (8(N,N-diethylamino)-octyl–3,4,5–

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trimethoxybenzoate) (Szewczck et al., 1992), the local anaesthetics pentobarbitone, thiopentane, secobarbitone and phenobarbitone (Kozlowski and Ashford, 1991) and certain fluorescein derivatives which are capable of both channel inhibition and activation (De Weille et al., 1992). Finally, KATP channels are also closed by a number of more conventional K channel blockers (KCBs) such as; quinine (Findlay et al., 1985c) (Kd=40 µM), aminoacridine (100 µM) and 4– aminopyridine (100 µM) (Cook and Hales, 1984). Tetraethylammonium ions (TEA) at relatively low concentrations (<2 mM) that completely block the Ca2+and voltage-activated K channel have no significant effect on the KATP channels (Findlay et al, 1985c). The IC50 for external TEA has been estimated to be approximately 22 mM (Bokvist et al., 1990). Activators of KATP channels There have been several excellent review articles that have detailed the general effects, and structure-function relations of K channel openers (KCOs) (Cook, 1988; Quast and Cook, 1989; Hamilton and Weston, 1989; Edwards and Weston, 1990). Patch-clamp studies on smooth muscle, skeletal and cardiac muscle, neurons and pancreatic β -cells indicate that most of these compounds— typified by cromakalim (CRK), nicorandil, pinacidil, P1060, RP 49356, and diazoxide, selectively activate the KATP channel, and not other types of K channels such as delayed outward- and Ca-gated K channels. KCOs therefore share a common target site in many different tissues, even though the relative effectiveness and tissue sensitivity varies quite considerably (see Cook, 1988; Quast and Cook, 1989). Through an effect on the KATP channels this leads to a hyperpolarisation of the cell membrane, and the indirect inhibition of Ca+2 influx through voltage-operated Ca channels (Dunne et al., 1990c). However, whilst opening of plasma membrane KATP channels may indeed be the first site of action of these compounds, there is certainly other evidence to suggest that KCOs prevent mobilisation of cytosolic Ca2+ from internal Ca2+ stores (recently reviewed by Quast, 1993). At the single channel current level, KCOs operate when added to either the inside (inside-out patches) or the outside (outside-out patches) face of the plasma membrane. By analogy with the sulphonylureas, the exact nature of the interaction between the ligand binding site and the KATP channel has yet to be resolved: direct gating or effects mediated by a regulatory molecule (s)? KCOs act by increasing the channel open-state probability, and do not act by increasing the number of operational channels in the membrane, nor by changes in the permeability of the channel to K+ (Standen et al., 1989; Nakayama et al., 1991). In the β -cell the mechanisms involving channel activation are heavily dependent upon the availability of cytosolic nucleotides, in particular ATP and ADP (Trube et al., 1986; Dunne et al., 1987, 1990a; Dunne, 1990a; Harding et al., 1993; Jaggar et al., 1993; Larsson et al., 1993). In the complete absence of internal ATP, KCOs do not activate channels when added to the inside face of the

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membrane, indeed under these conditions they have been shown to induce channel blockade (Dunne et al., 1987; Dunne, 1990a). This inhibition is not associated with any significant decrease in the number of channel openings, but by a reduction in the amplitude of channel conductance. Although ATP is a prerequisite for effective channel opening, increases in the ATP concentration will also reverse the actions of the compounds. This has been demonstrated for other K channel modulators (KCMs) in several different tissues, and is characterised by a marked right-ward shift in the concentration-response relationship at elevated ATP levels (Dunne et al., 1987; Findlay et al., 1989; Fan et al., 1990). One implication of this, is that the compounds will be relatively more effective at opening channels when the ATP levels are low, such as in damaged or diseased tissue. The role of internal ATP in governing the effects of these compounds is interesting. As discussed, the predominant action of ATP on KATP channels is to promote inhibition. Is ATP required only to keep the channels in a closed state in order for the KCO to act? Our evidence would suggest that this is not the case. If channels are inhibited by a sulphonylurea, diazoxide has no activatory effect on the channels unless ATP is available (Figure 13.4) (Dunne et al., 1987). These data clearly suggest that the requirement for ATP is more complex than just to close the channel. This is also supported by the fact that the effects of KCOs are lost whenever Mg2+ is removed from the inside face of the membrane, or when ATP is replaced by non-hydrolysable analogues (Dunne et al., 1987; Dunne, 1989; Kozlowski et al., 1989). Activation of KATP channels by diazoxide or CRK is also affected by the rate of channel run-down. The greater the degree of channel inactivation the less effective the compounds are at opening channels (Jaggar et al., 1993) (Figure 13.5). Despite this, we have been able to make use of these findings to our advantage. By allowing run-down to occur and then attempting to open the channels by diazoxide (or CRK) in the presence of either ATP or ADP, we have been able to assess which of the adenosine nucleotides are more effective at opening the channels. What we found was that once the ability of the KCO to activate channels in the presence of ATP was lost, we could still stimulate the channels in the presence of ADP (Figure 13.6). These data suggest that the nucleotide diphosphate is more important for channel activation than the triphosphate (Harding et al., 1993; Jaggar et al., 1993). Other nucleotide diphosphates, such as UDP, CDP, IDP etc., have also been shown to be more effective at modulating the effects of different KCOs in cardiac myocytes (Shen et al., 1991; Tung and Kurachi, 1991). Several hypothetical models have been proposed to explain the rather complex interactions between KATP channels and ATP, cytosolic nucleotides and ion channel modulators. Whilst at this stage it is not possible to present a single unifying consensus, several features of different models seem appropriate. Schwanstecher et al. (1992) suggest that KATP channels have at

388 K CHANNELS AND THEIR MODULATORS

Figure 13.4 Diazoxide-induced activation of KATP channels is dependent upon cytosolic ATP. Tolbutamide-inhibited channels in an inside-out patch are unaffected by diazoxide until ATP is added to the inside face of the membrane. Open events are shown as upward deflections from the zero current level (dotted line). (Modified from Dunne et al., 1987.)

Figure 13.5 Modulation of the effects of diazoxide (200 µM) on KATP channels by channel run-down. Data taken from a RINmSF inside-out patch. Actions of diazoxide are attenuated as the channels become progressively more inactivated.

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Figure 13.6 Modulation of KATP channels by diazoxide is more effective in the presence of ADP than ATP. Continuous current traces come from the same open-cell (see cartoon). Within 30 min of forming the patch-clamp recording configuration, the effects of diazoxide on ATP-inhibited channel are lost, but within the same time period, diazoxide was able to open KATP channels in the presence of ADP. The breaks between each of the current traces correspond to intervals of 20–30 seconds. (Modified from Jaggar et al., 1993.)

least four possible gating states: ‘ground’, ‘activated’, ‘blocked’ and ‘dephosphorylated’. In the ground state there are no ligands bound to the channel, the channel is phosphorylated and open. By contrast, when patches of membrane are removed from the cell, this eventually leads to channel run-down, and the formation of a non-conductive dephosphorylated state. The activation of KATP channels on the other hand can be promoted by a number of endogenous cytosolic nucleotides, and by synthetic channel modulators (e.g. diazoxide, CRK, etc.), whilst the blocked state results from the binding of ATP to the channel, or the presence of specific ligands such as the sulphonylureas. Tung and Kurachi (1991) suggest that the simplest way to explain transitions between these different states, is to propose that there are at least three discrete binding sites on the internal aspect of the ion channel: a unique ATP binding site (‘A’ in Figure 13.7), a ‘phosphorylation site’ (‘P’) and a nucleotide diphosphate binding site (‘NDP’). A ‘transducing element’ contains both the phosphorylation and the nucleotide binding sites, and provides a link between the ATP site and the channel open-closed gate. When the channel is phosphorylated in the absence of ligands the gate is unlocked and open (‘ground state’), but when ATP is present the channel is closed (‘blocked state’). Phosphorylation of the transducing

390 K CHANNELS AND THEIR MODULATORS

element is critical for any association between the ATP site and the gate. Tung and Kurachi (1991) further propose that both the phosphorylation and the nucleotide binding sites are located on the same transducing element, and that binding of nucleotides, particularly nucleotide diphosphates, conveys the same conformational change in the transducing portion of the channel as modulation of the phosphorylation site. This explains how nucleotide diphosphates such as ADP or GDP cause channel activation in the presence of ATP (Dunne and Petersen, 1986a, 1986b; Findlay, 1987; Bokvist et al., 1991). Taken together these hypotheses provide a framework to explain several features of the effects of synthetic KCOs. In our model (Figure 13.7) we propose that: (i) in the absence of channel activators, the receptor for either diazoxide or CRK is uncoupled from the transducing element, and thus the channel gate, and (ii) that channel activation can only occur upon establishing contact between the transducing element and the drug receptor. Conditions that promote this linkage involve modulation of either the phosphorylation or the nucleotide diphosphate binding site(s). Thus channel activation will not occur without internal nucleotides, or in the presence of ATP analogues that confer a state of dephosphorylation on the channel (Dunne et al., 1987; Dunne, 1989; Kozlowski et al., 1989). Indeed, diazoxide does not bind to the KATP channel under these conditions (Schwanstecher et al., 1992). Second, as ADP will bind to the transducing element whether phosphorylated or not (due to run-down), channel activation will result, even under experimental conditions where both diazoxide and CRK have no effect on KATP channels in the presence of ATP alone (Jaggar et al., 1993). As a working hypothesis the model explains our findings and those of several others, but does not take into account: the nature or the precise location of the different binding sites (integral part of the channel complex or regulatory molecule?), their relative associations with KATP channels, or indeed how channel phosphorylation/dephosphorylation is related to the activities of protein kinase (s), ATPases and phosphatases associated with the membrane. Kozlowski and Ashford (1992) have presented additional data to indicate that the mechanism by which diazoxide activates KATP channels can be either dependent upon phosphorylation, or can be mediated by the availability of an intracellular protein coupled with a magnesium-purine nucleotide (protein kinase? protein phosphatase? etc.). Whilst Larsson et al. (1993) propose that diazoxide-induced stimulation of KATP channel activity involves a diffusable cytoplasmic regulatory component which is not part of the KATP channel itself, the authors also proposed that the same mechanism mediates the effects of ADP on the channels.

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Figure 13.7 Hypothetical model of interactions between ion channel modulators, nucleotide diphosphates and ATP. Changes in the gating of KATP channels are shown between the ‘ground state’ and the ‘activated state’ with diazoxide bound. The model is based upon that presented by Tung and Karachi (1991). A transducing element is shown to provide the link between the channel gate and the four discrete binding sites: drug receptor, a unique ATP binding site (‘A’), a ‘phosphorylation site’ (‘P’) and a nucleotide diphosphate binding site (‘NDP’). In the ground state, in the absence of diazoxide, the

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channel is ‘unlocked’ with the potential to open. Diazoxide will only activate the channel once the transducing element has been modified (shown in black), which can occur by either phosphorylation, or by the occupancy of the NDP site. Important: for the sake of clarity the drug receptor site has been located within the plane of the membrane.

Finally, concerning the physiological significance of these studies, perhaps one of the most intriguing findings to come from recent experiments is the direct demonstration that KCOs exhibit tissue selectivity. In vascular smooth muscle myocytes the actions of CRK, nicorandil, pinacidil and RP 49356 are potent, whilst they are less impressive in cardiac myocytes (Cook, 1988; Quast and Cook, 1989) and pancreatic β -cells (Dunne and Petersen, 1991). Diazoxide, on the other hand is the most effective opener of channels in insulin-secreting cells (Dunne et al., 1987), but inhibits KATP channels in ventricular myocytes (Faivre and Findlay, 1989). Similarly, the vasodilator SDZ PCO–400 and the bronchodilator HOE 234, both of which cause smooth muscle relaxation (Chapman et al., 1992; Englert et al., 1992; Lawson et al., 1992; Linz et al., 1992; Small et al., 1992; Miura et al., 1993), inhibit KATP channels in β -cells, including human tissue (Dunne and Finch, 1993; Jaggar, James, London and Dunne, unpublished). Clearly these findings indicate that although there are evident similarities in the mechanisms by which openers modulate K channel proteins, there is a degree of diversity in the pharmacological regulation of the family of KATP channels. 13.3.2 Ca and Voltage-Gated K (KCa) Channels These K channels are activated in the β -cell as in many other cell types, by either a depolarisation of the cell membrane potential, or by an increase in the cytoplasmic Ca2+ concentration within the physiological concentration range (Cook et al., 1984; Findlay et al., 1985a). The insulin-secreting cell has two distinct types of KCa channels: a large (maxi) Ca- and voltage-gated channel (BKCa channel), and a much smaller apamin-insensitive K channel. Kmaxi channels are highly selective for K+ over Na+ and Cl― and have a conductance of between 140 and 300 pS in symmetrical K+/K+ solutions (Cook et al., 1984; Findlay et al., 1985a; Sturgess et al., 1986b; Misler et al., 1989b; Eddlestone et al., 1989; Ashcroft et al., 1989). Na+ and Mg2+ at the internal face of the membrane reduce the conductance of the channel, which may explain why the current-voltage relationship plot for this channel exhibits a slight degree of current rectification (Tabcharni and Misler, 1989). The relationship between changes in [Ca2+]i and channel open-state probability is characterised by a saturating sigmoidal curve, which is shifted to more negative potentials as [Ca2+]i increases (Cook et al., 1984; Findlay et al., 1985a). The sensitivity of the channel to [Ca2+]i is particularly high between 0.1 and 1 µM, but at positive membrane potentials increases in [Ca2+]i actually cause a

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decrease in channel open-state probability. The mechanisms that underlie this apparent inactivation of the channel have not been identified, but similar characteristic decreases in channel open-state probability at high membrane potentials have been reported for Kmaxi channels in other tissues (Latorre et al, 1983; Vergara and Latorre, 1983; Petersen et al., 1986). In comparison to the KATP channel, there has been little investigation of the pharmacological regulation of these channels; except for block by TEA and quinine (Findlay et al., 1985c; Petersen et al., 1986; Tabcharni and Misler, 1989; Bokvist et al., 1990; Mancilla and Rojas, 1990; Fatherazi and Cook, 1991). Tolbutamide has no effect on the gating of these channels (Ashcroft et al., 1989). Kmaxi channels do not operate in the intact resting cell because the [Ca2+]i is not high enough nor the membrane potential favourable enough to support open channel events. Charybdotoxin is a selective inhibitor of this type of K channel (Miller et al., 1985), and available evidence tends to suggest that scorpion toxin does not significantly alter the pattern of β -cell electrical activity induced by glucose (Kukuljan et al., 1991). However, Kmaxi channels do play an important role in rapidly repolarising the Ca-spike potential. The evidence for this is both circumstantial—a combination of the rapid depolarisation and influx of Ca2+ being favourable for channel openings, and direct—selective blockers of these channels such as TEA (Findlay et al., 1985c) increase the duration of the spike potentials (Findlay and Dunne, 1986a) (Figure 13.8). In our laboratory we have recently investigated the effects of internal nucleotides on the gating of Kmaxi channels in human and rodent β -cells (Jaggar et al., 1994). In particular these channels can be inhibited by internal ATP, ADP and AMP, but when the ADP is added simultaneously with ATP, channel activation results. We also find that intracellular guanosine nucleotides have complex effects on the channels, being able to cause inhibition immediately upon patch excision, but then activating channels later in the experiment. The mechanisms that underlie these effects have yet to be evaluated.

394 K CHANNELS AND THEIR MODULATORS

Figure 13.8 Effects of tetraethylammonium ions on action potentials in insulin-secreting cells. Data obtained using the patch-clamp whole-cell current-clamp configuration by ‘injection’ of a positive (depolarising) current pulse. TEA has been added to the bath solution. (Data modified from Findlay and Dunne, 1986a.)

The properties of the lower conductance apamin-insensitive Ca activated K channel have only recently been addressed in the laboratory of Patrik Rorsman and Per-Olf Berggren (Ammala et al., 1991, 1993). It is very clear from their studies that oscillations in [Ca2+]i and membrane potential induced by internal GTPβ S are associated with the activation of this K+ current. The individual ion channels are likely to be in the sub-pS conductance range under normal physiological conditions [this contrasts with values of approximately 250 pS for the Kmaxi channel, and 70–90 pS for the larger KATP channel (see Petersen and Findlay, 1987; Ashcroft and Rorsman, 1989; Dunne and Petersen, 1991). These channels are not inhibited by apamin, TEA or the sulphonylureas. Further analysis is required to evaluate whether this and the K1 channel (see below) are the same channel.

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13.3.3 Delayed Rectifier K (Kv) Channels As with the Kmaxi channel, the delayed rectifier K channel is activated by progressive depolarisation of the cell membrane. Under normal physiological conditions the conductance of the channel is around 10 pS (Rorsman and Trube, 1986), the channel has a non-linear current-voltage relationship and is not significantly influenced by changes in [Ca2+]i or internal pH within the physiological range (Rorsman and Trube, 1986; Bokvist et al., 1990; Satin et al., 1990; Smith et al., 1990a). The physiological role of the channel is thought to be in contributing along with the Kmaxi channel to repolarising the Ca2+-spike potential. TEA will inhibit these channels, but as with the KATP channels they are more sensitive to quinine than TEA (Bokvist et al., 1990; Smith et al., 1990a). The effects of TEA on the profile of the β -cell action potential is demonstrated in Figure 13.8. 13.3.4 Non-selective Cation Channels Under quasi-physiological cation gradients these channels are about half the size of KATP channels, and have been identified in all types of experimental β -cells, including human tissue (Sturgess et al., 1986a; Misler et al., 1989a). The channels are permeable to both Na+ and K+ ions. In many other tissues they fulfil a variety of functions; from controlling Ca2+ influx in non-excitable cells (see Partridge and Swandulla, 1988), to governing cycles of membrane potential changes in electrically active cells. Their precise functional significance to β -cell electrophysiology has been difficult to assess. One reason for this is that it has been difficult to show any significant effects of cytosolic molecules on the channels within the physiological concentration range. The channels are Ca2+ sensitive and blocked by internal nucleotides: but Ca2+ will only open the channels at >100 µM, whilst the channels are more sensitive to AMP>ADP>ATP (Sturgess et al., 1987). Recent experiments undertaken in our laboratory however indicate that the channels may be far more sensitive to the adenosine nucleotides than previously thought—especially when more than one nucleotide is available. We find that these channels are present along with KATP channels in intact resting cells, and interestingly we see these channels more frequently in human than in either rodent or clonal β -cells. This may imply that the channel is able to contribute to the resting cell membrane potential or background K+ or Na + current. By analogy with other systems it may be involved in the generation of slow-waves of potential change, but as there are no known selective agonists or antagonists to these channels, it is difficult to assess their precise contribution to electrical events in the β -cell.

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13.3.5 Transient Outward K (KA) Channels Only a short communication has described the presence of a 4–AP-sensitive K channel in mouse β -cells that is similar to one of the two transient outward K channels present in the myocardium (Smith et al., 1989). The inactivation profile of this channel in patch-clamp experiments indicates that it would be half maximally inactivated at normal resting membrane potentials, and completely inactive at membrane potentials experienced in the presence of glucose. This channel is not seen in all preparations of β -cells, and its role in the electrophysiology is unlikely to be significant. 13.3.6 Receptor-operated K (K1) Channels A K channel activated by hyperglycaemia-inducing hormones and neurotransmitters has recently been identified in mouse β -cells (Rorsman et al., 1991). This channel could only be examined through analysis of ‘noise’ associated with the receptor-mediated events, and may be the same channel as the low-conductance apamin-insensitive K channel (see above). So far this channel has been shown to be activated by adrenaline in a G-protein dependent manner, and by the adrenoceptor agonist clonidine. Activation of the K1 channel may therefore be responsible for the membrane hyperpolarisation evoked by adrenaline, and may possibly be involved in the effects of other inhibitory hormonal and neural influences on the β -cell. 13.3.7 ATP-activated K Channels A second type of ATP-regulated K channel has recently been described in the laboratory of Fran Ashcroft (Williams et al., 1993). This channel is functionally distinct from the KATP channel: it exhibits no inward current rectification, shows no sensitivity to the sulphonylureas, and is activated by internal ATP. Interestingly, this study was undertaken using β -cells isolated from a Type II diabetic human donor. ATP-activated K channels have not (so far) been reported in any other β -cell preparation, including human tissue. One important implication of these findings is that this type of channel could be a cause of (or indeed result from) the pathophysiological state of the β -cell. 13.4 Na Channels and Insulin-secreting Cells Na channels are perhaps the least well characterised of all β -cell ion channels. One possible reason for this may be that the contribution (s) of the Na+ current to

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electrical events may vary significantly in β -cells from one species to the next. Tetrodotoxin, a specific inhibitor of voltage-gated Na channels has consistently been reported to inhibit glucose-induced secretion, and to be without effect (see Dunne et al., (1990d) for further discussion and references). In terms of the properties of Na channels, one study carried out upon mouse cells demonstrated that the Na + currents are inactivated at normal membrane potentials, and therefore could not contribute to the cell electrical activity (Plant, 1988). On the other hand, in the rat inactivation potentials are around–40 mV (Hiriart and Matteson, 1988), whilst in human and canine β -cells there is good evidence that Na channel activity contributes significantly to the action potential depolarisation (Pressel and Misler, 1991). In Table 13.1 Overview of ion channels in pancreatic β -cells and their possible physiological significance to cell electrophysiology β -Cell Ion Channel Calcium Channels ― L-type voltage-gated Ca2+ channel. ― T-type voltage-gated Ca2+ channel. Potassium Channels ― ATP-sensitive K channel. (KATP channel) ― ― ― ― ― ―

Calcium- and voltage KMaxi channel. Low-conductance KCa channel. Receptor-operated KI channel. Delayed rectifier KDR channel. Transient outward KA channel. Non-selective cation KNS channel.

― ATP-activated K channel. Sodium Channels ― Voltage-gated Na channel. –rodent β -cell –human, canine ß-cell –clonal ß-cell Chloride Channels ― Uncharacterised

Possible Physiological Role? Ca2+ influx during action potential Ca2+ influx during slow waves. Not established. Initiation of membrane depolarisation. Contribution to slow waves. Receptor-mediated events. Action potential repolarisation. Generation of slow waves. Receptor-mediated events. Action potential repolarisation. Not established. Contribution to initial depolarisation? Contribution to slow waves? Only seen in diabetic tissue.

Inactive at critical membrane potentials. Generation of action potential. Maintenance of electrical activity. Contribution to initial depolarisation? Contribution to slow waves?

clonal β -cells Na channels may have a more pronounced role since without external Na+ ions, nutrient-induced electrical events and the elevation of [Ca2+]i are abolished (Dunne et al., 1990d). In these cells phorbol esters will suppress

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Na+ currents suggesting a possible role for protein kinase C in regulating their properties (Rorsman et al, 1986). 13.5 Summary With the approaches of conventional microelectrodes and patch-clamp techniques we now have a much better understanding of the sequences of events that are responsible for the complex changes in β -cell electrical activity elicited by glucose. It is obvious to relate the properties of voltage-gated Ca channels to the regulation of [Ca2+]i, but other ion channels particularly K channels are important in governing their operation. Therefore, the initiation, the maintenance and then finally the termination of glucose-induced electrical events in the β -cell involves the integrated control of several different types of ion channels (see Figure 13.1 and Table 13.1). We are also left with many more unanswered questions. For instance, what and how are cytoplasmic second messengers able to govern the opening of ion channels, and which ion channels are involved with the regulation of insulin secretion in response to the many hormones, neuropeptides and pharmacological agents that are able to influence the pancreatic β -cell. Moreover, the study of ion channel regulation in human isolated β -cells has only just begun, and whilst there is a great deal in common between rodent, clonal and human insulin-secreting cells there are some important differences in the properties of ion channels between the different species. Acknowledgements Work in our laboratory has been generously funded by project/equipment grants to MJD from The British Diabetic Association, The Wellcome Trust, The Yorkshire Cancer Research Campaign, The Medical Research Council, The British Heart Foundation, The Nuffield Foundation, The Royal Society, and the University of Sheffield. We are indebted to the continuing support of Roger James and Nick London at the University of Leicester, Department of Surgery for their collaboration on studies of ion channels and stimulus-secretion coupling mechanisms in human islets of Langerhans. References AGUILAR-BRYAN, L., NELSON, D.A., VU, Q.A., HUMPHREY, M.B., BOYD, A.E. III (1990) J. Biol. Chem., 265, 8218–8224. ÄMMÄLÄ, C., LARSSON, O., BERGGREN, P.-O., BOKVIST, K., JUNTTIBERGGREN, L., KINDMARK, H. & RORSMAN, P. (1991) Nature, 353, 849–852. ÄMMÄLÄ, C., BOKVIST, K., LARSSON, O., BERGGREN, P.-O. & RORSMAN, P. (1993) Pflugers Arch., 422, 443–448. ARKHAMMAR, P., HALLBERG, A., KINDMAK, H., NILSSON, T., RORSMAN, P. & BERGGREN, P.-O. (1990) Biochem. J., 265, 203–211.

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14 Potassium Channels and their Modulation in Urogenital Tract Smooth Muscles A.F.BRADING1 & W.H.TURNER2 1

Department of Pharmacology, Oxford University, Mansfield Road, Oxford, OX1 3QT, UK. 2

Department of Urology, Inselspital, Bern, Switzerland. 14.1 Introduction

Smooth muscles in the urogenital tract exemplify the amazing ability of these muscles to display quite different properties which fit them exquisitely to their separate functions. It is likely, for instance, that each muscle will have a unique combination of ion channels in its plasma membranes, which will govern its spontaneous electrical activity and its ability to respond to neurotransmitters and endogenous ligands. Amongst these channels will be the K channels, many types of which are expressed by smooth muscles. These channels will play an important role in determining the ‘resting’ membrane potential of the cells, the shapes and frequency of the spontaneous action potentials and the membrane response to receptor ligands. We do not yet know precisely which channels are present in which cells, but use of the increasing number of drugs and toxins that have been shown to modulate their activity, clearly demonstrates the importance of these channels. In this chapter, we will briefly describe the function and properties of urogenital smooth muscle, highlight the clinical problems, and discuss what is known about their K channels and the therapeutic potential of modifying K channel behaviour. 14.2 The Urinary Tract The smooth muscles of importance in the urinary tract are the ureters, the detrusor of the bladder and the urethral smooth muscle. 14.2.1 Ureters The ureters transmit urine from the kidneys to the bladder. This keeps renal pelvis pressure low, an essential function, since ureteric hold-up and consequent

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raised renal pelvis pressure would soon stop filtration. Action potentials are initiated in the renal pelvis and sweep down the length of the ureter through the well-coupled smooth muscle cells, initiating a wave of contraction that pushes urine into the bladder. As in the heart, it is essential that tetanic contractions are avoided, since maintained tone would stop urine transport and allow build up of renal pelvis pressure. The action potentials thus have a long plateau, the contractile responses are very phasic in nature, and the contraction strength is related to the duration of the plateau (Shuba, 1981). Again like the heart, although there is a primary pacemaker, smooth muscle cells throughout the ureter can, under the right conditions, take over pacemaking activity (Golenhofen and Hannappel, 1973). Although there is little evidence for any direct sympathetic or parasympathetic innervation of the ureteric smooth muscles, activation of capsaicin sensitive sensory-motor nerves can modulate activity (Maggi and Giuliani, 1991), as can application of catecholamines and autacoids (Shuba, 1977). K channels in ureter Early experiments using a double sucrose-gap voltage clamp technique (Shuba, 1981) suggested that there were two components of voltage-sensitive outward currents in the ureter, one of which was blocked by tetraethylammonium ions (TEA). More recent work on enzymically dispersed single smooth muscle cells from guinea-pig ureter (Imaizumi et al., 1989; Lang, 1989) confirmed the presence of two main outward current components. The outward currents were a Ca-dependent K+ current, blocked by TEA, and a transient outward current blocked by 4-aminopyridine (4–AP). Unlike many other smooth muscles, there was little evidence of any voltage-dependent delayed rectifier channels. Single channel records (Imaizumi et al., 1990) demonstrate the frequent presence of large-conductance Ca-activated K channels (130 pS at physiological K+ gradients; BKCa or maxi-K), and a much less common small conductance channel (about 17 pS) similar to channels carrying the outward transient current (IA) in other preparations. It appears that the absence of the delayed rectifier channels allows the development of the long plateau. The role of the Ca-activated K channels may be in terminating the plateau due to a slow build-up of cellular Ca2 + during the contraction. The transient outward current plays no role during the plateau. It is probably partially active at the resting potential, and Imaizumi et al. (1990) have suggested that it would oppose the Ca2+ inward current responsible for the spike. It could thus help to ensure that spontaneous action potentials are not normally evoked in the main part of the ureter. The transient outward current is inactivated rapidly on depolarization and thus under local depolarizing conditions its braking action may be reduced, allowing local production of action potentials. It is possible that this mechanism would permit the ureter to try and overcome an obstruction.

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The ureteric smooth muscle cells also seem to possess another K channel, since the K channel opening (KCO) drugs cromakalim (CRK), pinacidil, nicorandil and S 0121 have been shown to reduce the frequency and size of the K + -stimulated rhythmic activity in the rabbit and guinea-pig ureter (Klaus et al., 1990). CRK also produced a dose-dependent reduction in the size of the electrically evoked contractions of the ureter, completely abolishing the response at about 3 µM and it also suppressed the spontaneous activity of the renal pelvis. In the intact ureter in vivo, the KCO levcromakalim (LCRK) perfused through the ureter and decreased the frequency of the spontaneous contractions dosedependently (Kontani et al., 1993), but glibenclamide alone had no effect on the spontaneous activity. Microelectrode recordings (Klaus et al., 1990) and sucrose gap recordings (Maggi et al., 1994) demonstrated the ability of CRK to hyperpolarize the membrane. CRK also initially caused a shortening of the plateau before completely abolishing the action potential (Figure 14.1). These effects were reversed by application of glibenclamide, but TEA and 4AP were much less effective, and also less effective than they were on the Ca-dependent K + currents and the transient outward K+ current (Maggi et al., 1994). Although there is little evidence for excitatory neurotransmission in the ureter, it has recently been proposed that activation of sensory nerves can release calcitonin gene related peptide (CGRP) in the wall of the ureter, and that this peptide acts as an inhibitory neurotransmitter (Maggi and Giuliani, 1991). Electrophysiological recordings using the sucrose gap show that activation of intrinsic nerves, and application of CGRP cause hyperpolarization of the smooth muscle cells (Santicioli and Maggi, 1994). Interestingly, the effects of CGRP are abolished by glibenclamide, leading to the novel suggestion that this neurotransmitter may be activating the same channels that CRK can activate. Functionally this could be important in producing quiescence of the tissue when local damage and inflammation is enough to trigger the nociceptive sensory nerves and could be important in limiting retrograde transmission of action potentials and urine back to the kidney.

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Figure 14.1 Sucrose gap recording of the effects of cromakalim (CRK) on electrically evoked activity of the guinea-pig ureter. The inset shows contractile responses (upper trace) and action potentials (lower trace) on a slow time base. The electrical recordings indicated by A (control) B and C (after application of 0.3 µM CRK) are shown expanded. Note the hyperpolarization and initial shortening of the action potential after addition of cromakalim. (From Maggi et al., 1994).

14.2.2 Detrusor The detrusor is the smooth muscle in the wall of the part of the bladder which lies above the trigone. It generates the contraction which empties the bladder during voiding. In the filling phase, the bladder wall does not behave as a floppy bag, but maintains the minimum surface area:volume relationship available to it anatomically, so that if micturition is initiated, the synchronous activation of the muscle can immediately raise intravesical pressure. Thus during filling the smooth muscle cells must lengthen but maintain enough tone to keep the bladder in shape. This is achieved through spontaneous electrical and mechanical activity. It is, however, essential that this activity does not prevent the bladder filling at low pressure, and evidence is available that synchronous activity is prevented by rather sparse electrical coupling between the cells. This poor coupling has been studied in some detail in the guinea-pig bladder, using measurement of input impedance (Brading et al., 1989) and more recently with the use of two

410 K CHANNELS AND THEIR MODULATORS

microelectrodes for recording potential and passing current (N. Bramich, Figure 14.2, personal communication). The latter experiments demonstrate that current flow at right angles to the cells’ longitudinal axis rarely occurs between cells separated by more than a few cell widths. However, the time course of the voltage response to current injection in a single cell suggests that each cell is probably coupled to its near neighbours. Activation of intrinsic motor nerves generates synchronous excitatory junction potentials and spikes in all cells in the strip, even if they are not electrically coupled, since the density of innervation is very high. In isolated tissue strips spontaneous activity occurs, and the pattern of spontaneous mechanical activity also probably reflects the poor coupling between cells. Spontaneous contractions are small in comparison to the maximum force a strip can develop in response to nerve stimulation, and they rise from and fall to the basal tension. Tetanic activity is normally never seen. The individual contractions often vary in size, and it seems likely that this is determined by the number of smooth muscle groups that happen to be firing action potentials at the same time. A characteristic of the strips is their response to sudden stretch, which results in a transient increase in activity which then decreases progressively with time, towards the basal tension. A major functional bladder disorder is detrusor instability, a condition in which during bladder filling the pressure does not remain uniformly low, but phasic increases occur, which cannot be suppressed voluntarily in man. This can lead to incontinence. Detrusor instability occurs in association with bladder outflow obstruction and with neuropathic disorders, but often there is no associated abnormality, so-called idiopathic detrusor instability. Artificial outflow obstruction in animal models can lead to conditions resembling detrusor instability, particularly in the pig (Jorgensen et al., 1983; Sibley, 1985; Speakman et al., 1987) and the rat (Malmgren, 1987; Saito et al., 1993). Instability in both humans and in the animal models is frequently associated with a reduction in motor innervation (see Brading and Turner, 1994) and smooth muscle strips from unstable bladders show changes to their properties which include a supersensitivity to agonist drugs, and in the pig and human, alteration in the spontaneous activity so that fused tetanic contractions are now seen (Brading and Turner, 1994). There is also evidence of increased electrical coupling between the cells, which may allow synchronous activity to

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Figure 14.2 Simultaneous microelectrode recordings from two cells in strips of guineapig detrusor. The bathing solution contained 10−6 M nifedipine to prevent action

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potentials. Excitatory junction potentials (EJPs) evoked by transmural stimulation. In A, note the simultaneous evoked EJPs in both cells, but non-synchronous spontaneous junction potentials (SEJPs). In B, the top pair of tracings show simultaneous evoked EJPs, and the bottom trace (from the same pair of cells) shows the lack of electrical coupling between the cells, since current injected into one cell did not affect the membrane potential in the second cell.

spread across the bladder wall leading to the unstable contraction in vivo (Fujii, 1988). K channels in the detrusor: contribution to shape of action potential Micro-electrode studies of action potentials in small mammal bladders show rising phases that are normally somewhat slower than the falling phase (rate of rise 4.2 V/s, rate of fall 5.4 V/s; Kurihara, 1975), with a pronounced but variable after-hyperpolarization. At present there are no published records of action potentials recorded with micro-electrodes from detrusor of larger mammals or humans. Action potentials have, however, been recorded under current-clamp conditions using the whole-cell recording mode from isolated human bladder myocytes (Montgomery and Fry, 1992), and show a similar shape, although without the after-hyperpolarization. The comparatively rapid falling phase suggests the opening of K channels, and whole-cell voltage clamp studies on single myocytes isolated from the bladder demonstrate the presence of voltage sensitive outward currents (guinea pig: Klöckner and Isenberg, 1985; human: Montgomery and Fry, 1992). The falling phase of the spike and the afterhyperpolarization can be differentially affected by various K channel blocking drugs (KCBs). In the guinea-pig, apamin (100 nM) and 4AP (5 mM) had no measurable effect on the rate of repolarization of the spikes, TEA (10 mM) and quinidine (100 µM) caused some prolongation of the falling phase (the time for half maximal repolarization increasing to 220% and 340% of control respectively), and procaine (5 mM) prolonged the falling phase by more than tenfold. At the above concentrations, procaine and apamin completely eliminated the after-hyperpolarization, 4AP and TEA had little effect and quinidine reduced it (Figure 14.3, Fujii et al., 1990). All of the KCBs so far tested, including charybdotoxin can enhance the spontaneous mechanical activity in the guineapig detrusor (Fujii et al., 1990; Zografos et al., 1992). These results strongly suggest that more than one type of K channel is present in guinea-pig detrusor. Voltage clamp studies (whole-cell and patch) have clearly demonstrated the presence of Ca- and voltage-activated maxi K channels (Markwardt and Isenberg, 1992; Suzuki et al., 1992) in the guinea-pig detrusor smooth muscles, and evidence strongly supports their presence in human bladder myocytes (Montgomery and Fry, 1992). In some conditions spontaneous transient outward currents (STOCS) can be seen, similar to those in other smooth muscles, in which they are thought to be caused by spontaneous release of stored Ca2+

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triggering activation of maxi K channels (Bolton and Lim, 1989). The current through these channels is inhibited by cyclopiazonic acid, a specific inhibitor of the Ca2+ ATPase of the sarcoplasmic reticulum (Suzuki et al., 1992), by reduction in intracellular Ca2+ and by TEA. Markwardt and Isenberg have suggested that the rapidity of activation of these channels by intracellular Ca2+, would enable them to be responsible for the repolarization of the spike. Another possible role of the maxi-K channels is in the responses of the smooth muscles to stretch. Recently Wellner and Isenberg (1994) have demonstrated the presence of stretch activated non-selective cation channels in isolated myocytes from guinea-pig detrusor. Stretch generates net inward currents in the whole-cell mode, but this current declines spontaneously with time. Analysis shows that the stretch-activated non-selective cation channels do not themselves inactivate with time, but that the net current is reduced due to activation of the maxi-K channels,

Figure 14.3 The effect of KCBs on the action potentials from guinea-pig detrusor, recorded with microelectrodes. Action potentials on the left are controls, and on the right after addition of the blocking drug (from Fujii et al., 1990).

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probably as a result of a local increase in intracellular Ca2+ close to the membrane, entering through the stretch activated channels. Further work by these authors has demonstrated that the rundown of the inward current is accelerated by cAMP analogues, which work largely through increase in the maxiK channel activity. This appears to be through a cAMP-dependent phosphorylation of the channels, increasing their sensitivity to Ca. Enhancement of maxi-K channel activity could thus be involved in the inhibitory effects of β receptor activation in the bladder, which can be evoked in response to circulating adrenaline. Effects of K-channel opening drugs on detrusor Although the role of K channels in determining the shape of the action potential and the physiological behaviour of the bladder is of interest, more attention has been focused on the effects of the KCOs on the properties of the detrusor. This work has recently been extensively reviewed (Andersson and Wyllie, 1992). The interest stemmed from the suggestion that drugs which reduced the excitability of the detrusor might be able to suppress the unstable contractions of the bladder which cause urinary incontinence in detrusor instability (Brading et al., 1986), and the obvious possibility that drugs that open K channels should be able to achieve this. The initial studies with CRK, the first of the benzopyran KCO drugs, showed it to be a powerful blocker of the spontaneous mechanical activity of smooth muscle strips dissected from the bladder of guinea pigs, pigs and humans (Foster et al., 1989a, 1989b). If the KCO drugs were to be of use clinically, it would be desirable for the bladder still to be able to respond to its normal parasympathetic input. High doses of CRK (10−5 M) sufficient to abolish completely spontaneous activity had no significant effect on the frequency response curves of bladder strips to intrinsic nerve stimulation in any of these species. This dose also had no effect on the dose-response curve of the guinea-pig detrusor to muscarinic agonists (Foster et al., 1989a), although it shifted the curves to the right and somewhat suppressed the maximum response in strips dissected from pig and human bladders (Foster et al 1989b; de Moura et al., 1993). Similar effects were seen with rat bladder, both with CRK and with pinacidil (Malmgren et al., 1990), although in this species the KCOs also caused a dose-dependent suppression of the maximum response to intrinsic nerve stimulation. The development of models of detrusor instability in the pig and rat (see above) provided smooth muscle from unstable bladders for in vitro testing, and also allowed the in vivo effects of the KCOs to be studied, and their potential as agents for the treatment of this disorder assessed. The effectiveness of CRK in abolishing unstable contractions was first demonstrated in the obstructed pig model (Speakman, 1988; Foster et al., 1989b), and then in the obstructed rat model (Malmgren et al., 1989), in which pinacidil was also effective. In both species substantial drops in blood pressure (BP) occurred. In the mini-pig model

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however, extensive use of LCRK has shown that at medium doses of 0.02 mg/ kg, it does not invariably abolish unstable contractions (Turner, unpublished observations). So far published information on the clinical effectiveness of CRK and pinacidil comes from two preliminary trials, and the results have proved rather inconclusive (Restorick and Nurse, 1988; Nurse et al., 1991; Hedlund et al., 1991), although some improvement was noted in both trials (see Chapter 16). Experiments have also been carried out on detrusor strips from unstable bladders in rats, pigs and humans. Spontaneous activity in such strips often shows a different pattern than in normal bladders, with a larger degree of fused tetanic tone. Spontaneous activity in smooth muscle from unstable bladders is also abolished by the KCOs (Andersson et al., 1988; Restorick and Nurse, 1988; Foster et al., 1989b; Nurse et al., 1991). Mechanisms of action of the KCOs on detrusor Although it is the effects on contractile activity which will be important for the potential clinical effectiveness of this class of drugs, there has been considerable interest in bladder, as in other smooth muscles, about the mechanisms involved in these effects. The type of K channel involved and whether or not the effects are all ascribable to changes in the membrane K+ permeability, are of interest. Electrophysiological studies on guinea-pig detrusor demonstrated that the cessation of activity was accompanied by hyperpolarization of the membrane and a drop in membrane resistance, although the action potentials often stopped before significant hyperpolarization had occurred (Figure 14.4, Foster et al., 1989a). At

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Figure 14.4 Effect of increasing concentrations of cromakalim (CRK; BRL 34915) on the membrane potential of guinea-pig detrusor smooth muscle. Note that the full sized action potentials stop before significant hyperpolarization of the cell occurs. The small deflections are probably caused by action potentials in neighbouring cells, and suggest that CRK may be reducing the coupling between the cells (from Foster et al., 1989a).

higher concentrations, CRK also caused an increase in radio labelled K+ fluxes across the cell membrane. In contrast to the channel opened in many other smooth muscles, those in the detrusor seem to be relatively impermeant to Rb+ (Foster et al., 1989a), and it was noticeable that spontaneous activity was abolished by concentrations of CRK some 50 times lower than needed to alter transmembrane fluxes. Initial studies of the effects of known KCBs on the activity of CRK on vascular smooth muscle cells led to the suggestion that the drug might be activating delayed rectifier K channels (Beech and Bolton, 1989). A similar suggestion was also made for guinea-pig bladder, judging from studies on the ability of various KCBs to attenuate the inhibitory effects of CRK on spontaneous electrical and mechanical activity (Figure 14.5, Foster et al., 1988; Fujii et al., 1990). An interesting observation was that CRK at increasing doses, was able to reverse the effects of quinidine on the action potential shape (Figure 14.6; Fujii, unpublished). However the demonstration that glibenclamide could antagonize the effects of CRK on smooth muscles, focused attention on ATP-dependent K channels. It has subsequently been found that glibenclamide can antagonize most of the effects of all the KCOs that have so far been tested on bladder smooth muscles (for examples see Andersson and Wyllie, 1992; de Moura et al, 1993; Ha et al., 1993). Recently an elegant study has been published in which the effects of LCRK on isolated smooth muscle cells from the guinea-pig bladder have been examined using whole-cell and single channel voltage clamp recording techniques (Bonev and Nelson, 1993a). To demonstrate the presence of ATP-dependent K channels in the cells, the bathing solution was a Iow-Ca2+ (100 µM) high-K+ (6 mM) solution, with the voltage sensitive Ca channels blocked with nimodipine, and the Ca-activated K channels blocked with TEA (1 mM) and iberiotoxin (100 nM). The cells were held

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Figure 14.5 The effect of 5×10−6 M cromakalim on the electrical activity of guinea-pig detrusor in the presence of various K channel blocking drugs. Microelectrode recordings (from Fujii et al., 1990).

at −80 mV to prevent activation of delayed rectifier K channels. The high K+ pipette solution also contained EGTA (10 mM), GTP (1 mM) and ATP (0.1 mM), the latter concentration selected to allow some steady-state activation of the KATP channels if present. Under these conditions, activation of the KATP channels would produce inward current. The results clearly demonstrated that application of glibenclamide (10 µM) reduced the resting current, and LCRK (30 µM) increased it, an effect blocked by glibenclamide (Figure 14.7). If the pipette ATP concentration was increased to 3 mM, the glibenclamide sensitive currents were greatly reduced. Thus it appears that the detrusor smooth muscle cells do indeed contain typical KATP channels. Further experiments demonstrated that the

418 K CHANNELS AND THEIR MODULATORS

channels were voltage independent and more sensitive than the other K+ channels to Ba2 + (half block at −80 mV by 100 µM). Noise analysis of the whole-cell currents suggested that the mean single channel current under these conditions was 0.61±0.04 pA, and that the mean number of channels per cell was 425±74, giving

Figure 14.6 Interaction between cromakalim (BRL 34915) and quinidine on the electrical activity of the guinea-pig detrusor. Quinidine (10−4 M) increased the frequency of the action potentials, reduced the size of the after-hyperpolarization and prolonged the falling phase until a second spike was superimposed. Cromakalim reversed these effects. Marked action potentials from the continuous recording above are shown on an expanded time scale below.

a rather low density of about one in every 9 µm2. Using physiological K+ gradients at 0 mV, the mean single channel current was 0.29±0.04 pA. The authors also attempted single channel recordings from outside-out patches under these latter conditions (Figure 14.7B). They demonstrated the presence of a K channel with the same single channel current, and that LCRK (10 µM) increased the activity 6. 4–fold. The single channel conductance was 7.3 pS. In the absence of ATP on the internal surface, the channels had a high initial activity, which was abolished by 10 µM glibenclamide.

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Bonev and Nelson (1993b) have also demonstrated that activation of muscarinic receptors can inhibit the KATP channels, and switch off the LCRK activated current in the whole-cell recording mode. The inhibitory mechanism appears to involve activation of protein kinase C (possibly via diacylglycerol, after its production through muscarinic receptor mediated stimulation of phospholipase C) since the inhibitory effect of carbachol was blocked by inhibitors of protein kinase C, and also by perfusion of the cells with a nonhydrolysable GDP analogue which prevents G-protein activation. Thus phosphorylation of the KATP channels may cause their closing. The authors suggest that muscarinic receptor stimulation could increase smooth muscle excitability by inhibition of KATP channels, leading to depolarization of the cell membrane.

Figure 14.7 The effects of levcromakalim (LCRK, LEM) and glibenclamide on K+ currents in guinea-pig bladder. A. Whole-cell currents recorded at a holding potential of −80 mV. External and internal K+ were 60 mM and 140 mM respectively, and the external medium contained 1 mM TEA and 100 mM iberiotoxin to block maxi-K channels. Glibenclamide (GLIB, 10 µM) reduces resting whole-cell current and prevents the effects of LCRK. Levcromakalim alone (30 µM) causes a large, glibenclamide sensitive increase in inward

420 K CHANNELS AND THEIR MODULATORS

current. B. Outside-out patch recording. External and internal K+ were 6 mM and 140 mM respectively, and the pipette medium contained 0.1 mM ATP. Holding potential 0 mV. Left hand panels: single channel records. Right hand panel: single channel current-voltage relationship. Means±SE. Arrows indicate closed channel level, dotted lines 0 current level (from Bonev and Nelson, 1993a).

Clinical potential The search is now on for KCOs that display selectivity between vascular smooth muscle and detrusor. Careful comparison between the effects of K channel modulators on vascular and detrusor smooth muscles suggest that there are differences which might be exploited (Zografos et al., 1992). Several drug companies are attempting to develop selective drugs, and there are compounds in existence that show in vitro selectivity between vascular and detrusor of the right order of magnitude, although the structures have not yet been published. There are also some interesting compounds thought to act as KATP channel openers which, although not showing in vitro selectivity, nevertheless in vivo reduce bladder activity in rats and dogs at concentrations that, in contrast to CRK, have no significant effect on BP or heart rate. One such compound (ZD6169), an anilide tertiary carbinol (see section 3.5.1) is in preclinical development. Wyeth Ayerst have examined the properties of 4–indazolinonyl derivatives of benzopyrans, and again found compounds which act in vitro through glibenclamide sensitive mechanisms (enhance outward currents in myocytes and relax vascular and detrusor smooth muscles strips) which in vivo can abolish unstable contractions in the obstructed rat model at concentrations which have little haemodynamic effect (Antane et al., 1994). It will be important to examine the effects of candidate drugs not only on the unstable detrusor contraction and the systemic BP, but also on the urethral pressure, since it would be preferable that a compound which reduces unstable contractions did not also lower urethral pressure. This could paradoxically reduce urethral closure pressure and thus might predispose to persistent incontinence.

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14.2.3 Urethra Circular urethral smooth muscle is generally considered to contribute to urethral closure during filling, although the details of the mechanisms of urethral closure remain unclear (Torrens, 1987a). Measurement of the urethral pressure profile in women shows a high pressure zone of about 3 cm, some 2 cm distal to the bladder neck (Torrens, 1987b). This is also true in the female pig, and smooth muscle dissected from the pig’s high pressure zone, in great contrast to the detrusor and the ureter, shows continual spontaneous myogenic tone (Bridgewater et al., 1993). The muscle is innervated by excitatory and inhibitory nerves and it is likely that in vivo some of the urethral pressure is mediated by a tonic sympathetic activation of the smooth muscle. Sympathetic blockade however, typically only reduces maximal urethral pressure by 25–30% in the pig (Macneil et al., 1991) and in the human (Torrens, 1987a). The muscle in the pig is supplied by two types of inhibitory nerve, one nitrergic (Persson and Andersson, 1992; Bridgewater et al., 1993), mediating a rapid and transient relaxation, and the other, with an as yet unidentified transmitter, mediated a slower and prolonged inhibition (Bridgewater and Brading, 1993). In strips of human urethra precontracted with noradrenaline, field stimulation initiates relaxations (Andersson et al., 1988), as it does in rabbit urethra precontracted by exposures to prostaglandins (Ito and Kimoto, 1985). Nitrergic innervation mediating these relaxations occurs in the rabbit and probably human urethra (Andersson et al., 1992). There is little published information about electrical activity in urethral smooth muscle from humans or pigs, but in urethral smooth muscle from rabbits, dogs and wallabies (Callahan and Creed, 1985), spontaneous contractions are associated with action potentials, and in the rabbit, intrinsic nerve activation produced relaxations, associated with inhibitory junction potentials (Ito and Kimoto, 1985).

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Figure 14.8 Whole-cell recordings from cells isolated from the female pig urethra, using a nystatin perforated patch. Cells were held at -50 mV and stepped for 2 sec to test potentials between −120 and +70 mV. Note transient and sustained outward currents at depolarized potentials, and the increased noise at more positive potentials. The control steps were repeated in the presence of 10−5 and then 10−4 M LCRK. Levcromakalim increases the outward current at depolarizing potentials, except that at +70 mV, 10−4 M LCRK actually suppresses the net outward current.

K channels in urethra Recently, in our laboratory, patch-clamp techniques have been used to examine the K channels present in smooth muscle cells isolated from the pig urethra (N. Teramoto, personal communication). Using a whole cell configuration with a nystatin perforated patch with a high K+ pipette solution, and a physiological

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bathing solution (Figures 14.8 and 14.9), voltage dependent outward currents were

Figure 14.9 Current voltage relationships of the current at the end of a 200 msec test potential. A: net currents in the absence and presence of 10−5 and 10−4 M levcromakalim. B: Difference (levcromakalim sensitive) currents. Cells isolated from pig urethra, wholecell configuration, nystatin perforated patch, physiological ionic concentrations.

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Figure 14.10 Effect of levcromakalim on single channel currents in a cell attached patch on isolated pig urethra smooth muscle myocyte. Symmetrical high K+ conditions. Holding potential −50 mV. A: continuous recording with a slow time base, showing the effects of adding 10−5 M levcromakalim to the bath solution. B: amplitude histogram before and during application of levcromakalim. The smooth lines are Gaussian curves.

elicited showing a transient and sustained component. The outward currents became very noisy at large depolarizations, suggesting activation of some large conductance channels. Addition of the KCO, LCRK caused a concentration dependent outward current at the holding potential of −50 mV. Between −120 and +20 mV, the LCRK sensitive current had a linear I-V relationship, reversing near EK, but with high concentrations of LCRK (100 µM) there was a surprising reduction in the net outward currents at potentials positive to +50 mV. In cell attached patches in symmetrical high K+, Ca2+ -free conditions, at least three types of channel could be seen. So far we have studied the maxi-K channels and

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those activated by addition of LCRK. LCRK (10 µM) at a holding potential of −50 mV induced the opening of channels with an inward current (Figure 14.10). These channels were seen in about 25% of the patches, and showed evidence of clustering, since when found, more than one channel was normally present. The channels had a conductance of about 42 pS and a long mean open time (about 100–200 msec). The most common channel seen under these conditions, had the characteristics of the maxi-K channel in other smooth muscle cells (Figure 14.11). It was a Ca2+ and voltage sensitive channel with a conductance in symmetrical high K+ solutions of about 240 pS and a mean open time of a few msecs. This channel was found in virtually all patches studied. We have also studied the effects of KCOs in vivo on urethral pressure in pigs. In anaesthetised mini-pig, LCRK had no effect on maximal urethral pressure (Turner, unpublished observations). However, in these experiments, the maximum urethral pressure before LCRK was given, was 50–75% of the maximum achievable in these animals, and this may have masked any reduction due to LCRK. The normal variations in urethral pressure seen in the pig were however reduced by LCRK. 14.3 Female Genital Tract Smooth Muscles The smooth muscles in the non-pregnant female genital tract play a role in moving the released eggs into the uterus, in maintaining the uterine wall in the correct state for implantation and in helping the movement of sperm into the uterus. In the pregnant animal the smooth muscles must adapt to allow the foetus to remain in the uterus until the end of gestation, and then bring about dilation of the cervix and expulsion of the foetus at birth. The most important areas for pharmacological intervention are reducing uterine activity in dysmenorrhoea and in threatened miscarriage, and enhancing activity for the induction of labour or abortion. 14.3.1 Uterus The smooth muscle layers of the uterus, composed of an outer longitudinally arranged layer, a middle layer in which the muscle bundles run in all directions, and an inner layer which has both longitudinal and circularly arranged bundles, show spontaneous electrical and mechanical activity in both the pregnant and non-pregnant state. The activity is myogenic, the cells showing regular slow depolarizations with superimposed spike activity, that initiate contraction. The contractile activity is under hormonal control. The body of the uterus is innervated by cholinergic, adrenergic and peptidergic neurones, which innervate both the smooth muscle and the epithelial layers. Stimulation of the intrinsic nerves in strips of myometrial smooth muscle from

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Figure 14.11 Effect of changing the holding potential on single channel currents (maxi-K +) in a cell attached patch on an isolated pig urethra smooth muscle myocyte. A: Current voltage relationship, B: single records at various potentials. Symmetrical high K+ conditions.

non-pregnant nonparous women excites the smooth muscle through muscarinic receptors (blocked by atropine) and β -adrenoceptors (blocked by phentolamine). There is no evidence of a nerve-mediated inhibition of activity. Exogenously applied VIP inhibits smooth muscle activity in the non-pregnant uterus, and causes vasodilatation. It may have a role in the mechanism of menstrual bleeding. During pregnancy there is an almost total loss of the innervation to the body of the uterus, although the smooth muscle continues to show myogenic activity. Waves of contraction pass along the uterus at regular intervals, but these do not elevate the intra-uterine pressure. The size and frequency of these waves change during pregnancy as the hormonal status changes. Towards the end of pregnancy

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the smooth muscle becomes increasingly sensitive to oxytocin, which can powerfully enhance this myometrial activity. Just before delivery, there is a large increase in the number of gap junctions between the smooth muscle cells, which allows the electrical activity of the cells to be synchronized, thus initiating coordinated contractions of the smooth muscle which do elevate intra-uterine pressure, and are necessary to expel the foetus. K channels in rat uterus Most of the studies on the K channels present in the myometrial smooth muscle cells have been carried out on rat or on human cells. In the rat, work on nonpregnant myometrial cells has been reviewed thoroughly by Ludmir and Erulaker (1993), in which they point out that the hormonal status and developmental time may alter the expression of K channels. In studies on the pregnant rat uterus, Anwer et al., (1992) using whole-cell and cell attached voltage clamp of myometrial cells have demonstrated the presence of K channels which were activated by Ca2+ and by voltage, blocked by charybdotoxin and low concentrations of TEA (less than 1 mM) but not by 4AP. Single channel conductance was 143 pS in symmetrical high KCl. Inoue et al., (1993) also studying cells from pregnant rat uterus, used conditions in which the Caactivated K channels would not be activated. Whole-cell experiments demonstrated a voltage sensitive outward current with a threshold for activation of about −40 mV. This current could be dissected into a 4AP sensitive current (blocked by 2 mM 4AP) which had the characteristics of a transient outward current, and a TEA sensitive current which had the characteristics of a delayed rectifier K+ current (blocked by 5 mM TEA). Thus rat myometrium has at least three voltage activated K channels. Betaadrenoceptor agonists are known to inhibit contractions of the uterus associated with hyperpolarization of the membrane, thus implicating an effect on K channels. Anwer et al., (1992) demonstrated that the activity of the Ca-activated K channels in cell attached patches was greatly enhanced by activation of β adrenoceptors, suggesting that they may be involved in these effects. Inoue et al., (1993) demonstrated that the delayed-rectifier-like current was actually blocked by forskolin. This action, however, was independent of its ability to activate protein kinase A, since protein kinase A inhibitors did not block the forskolin effect and the addition of cAMP or non-hydrolysable GTP analogues did not affect the outward current. It seems most probable that the Ca-activated K channel is the dominant and most important K channel in the pregnant rat myometrium, and that it may be important in modulation of uterine activity. Interestingly, however, oxytocin, a potent stimulator of the uterus, did not have any effect on the K+ currents (Inoue et al., 1992). K channels in human uterus More recently results have been published of experiments on human myometrium. Erulaker and Ludmir (1993) have also demonstrated the presence of three types of voltage sensitive K+ currents, two non-inactivating and one fast

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inactivating. A large conductance Ca-activated K channel has been characterized by incorporation of plasma membrane vesicles into lipid bilayers (Perez et al., 1993). It has all the properties of maxi-K channels seen in other smooth muscles (gated by voltage and intracellular Ca2+, blocked by charybdotoxin and low concentrations of TEA). The role of these channels in modulating contractile activity through regulation of membrane potential has been investigated by Anwer et al. (1993). Block of these channels with iberiotoxin depolarized the cells, and resulted in an increase in [Ca2+]i

Figure 14.12 Effects of levcromakalim on spontaneous mechanical activity of strips of myometrium from pregnant human, treated with oxytocin (from Morrison et al., 1993).

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and initiated phasic contractions in myometrial strips. The β -adrenoceptor agonist hexoprenaline, which is used for tocolysis after the 26th week of pregnancy, was shown by Adelwoehrer and Mahnert (1993) to increase the open probability of the maxi-K channel, an effect that was independent of an increase in [Ca2+]i, and was also shared by CGRP. The CGRP effect appears to be mediated through direct coupling of a G protein to the K channels (Tritthart et al., 1992). K channel opening drugs Again, because of the clinical potential of drugs that can diminish uterine contractions, much attention has been given to the KCO drugs in the myometrium. Earlier studies were carried out on mechanical activity of the rat uterus by Hollingsworth and colleagues (Hollingsworth et al., 1987; Downing et al., 1989; Downing and Hollingsworth 1990) Micro-electrode studies showed that CRK (10 µM) caused a small hyperpolarization of these myometrial cells (Hollingsworth et al., 1987), although interestingly there were no effects on K+ or Rb+ tracer efflux from the tissues (Hollingsworth et al., 1987, 1989). The relaxant effects of the drugs were reversed by glibenclamide (Piper et al., 1990). These results have been reviewed recently (Andersson and Wyllie, 1992). Since this review, the effects of LCRK and pinacidil have been examined on mechanical activity of strips of human myometrium obtained from women during pregnancy and labour (Morrison et al., 1993), and the effects of aprikalim and LCRK have been studied on non-pregnant and pregnant human myometrial strips (Cheuk et al., 1993). As predicted, all these drugs were potent inhibitors of spontaneous activity, and activity induced by agonists such as oxytocin (Figure 14.12), phenylephrine and low (but not high) K+ concentrations. In contrast with rat uterus, a small increase in 42K efflux was seen on application of LCRK (Cheuk et al., 1993). Glibenclamide (but not tolbutamide) acted as if it were a competitive antagonist. Metabolic inhibition in the uterus, presumably resulting in a lowered ATP concentration, enhanced 86Rb efflux from rat myometrial strips (Heaton et al., 1993), an effect which was reversed by about 50% by glibenclamide, suggesting that KATP channels are involved, possibly implicating their activation in dystocia, a condition in which there is inadequate uterine activity during labour. 14.4 Male Genital Tract Smooth Muscles The smooth muscles of the male genital tract, unlike the female, show little spontaneous activity, and are under tight control of the autonomic nervous system. The smooth muscles are responsible for permitting erection, for movement of the sperm and the components of the seminal fluid into the urethra and for ejaculation. The clinical problem most likely to involve smooth muscle dysfunction is impotence.

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So far, little attention has been paid to the K channels in the smooth muscles of the male genital tract. More attention has been paid to their innervation and the transmitters involved in controlling them. However, since one of the main events occurring in penile erection is the relaxation of the smooth muscles in the trabeculae of the corpora cavernosa, the effects of the KCOS on erectile tissue have been examined (monkeys: Giraldi and Wagner, 1990; rabbit and man: Holmquist et al., 1990a, 1990b; cat: Hellstrom et al., 1992). The results have been reviewed by Andersson and Wyllie (1992) and Andersson (1993). As expected, the KCOs relaxed isolated erectile tissue, and inhibited agonist evoked contraction. Intracavernous injection of KCOS in the monkey caused tumescence or erection (Giraldi and Wagner, 1990) and erection in the cat (Hellstrom et al., 1992). So far, however, little work has been carried out on the possibilities of these drugs in clinical treatment, largely because of the discovery of the importance of NO as a neural transmitter involved in the initiation of erection. Christ et al. (1993) have recently looked at the electrical properties of cultured corporeal smooth muscle cells, using patch-clamp analysis. In common with the other smooth muscles in the urogenital tract, these authors demonstrated a transient and sustained outward current in the whole-cell mode. The outward current was enhanced by the Ca-channel agonist, BAYK 8644, and by the KCO, pinacidil, suggesting the presence of Ca-activated K channels and KATP. Single channel analysis consistently showed maxi-K channels and a putative delayed rectifer channel. This diversity of K channels again suggests that they may play an important role in modulating corporeal smooth muscle tone. 14.5 Conclusions The diversity of K channels possessed by smooth muscles in general is reflected in the smooth muscles of the urogenital tract. Modulation of these channels can lead to increased or decrease mechanical activity in the smooth muscles, and the clinical implications of this are legion. Incontinence, impotence and premature labour are three immensely important clinical problems, none of which has adequate treatment. It can only be a matter of time and resources before selective drugs are developed. The clinical benefit from this could be enormous, with corresponding rewards for the drug company that develops specific drugs for treatment in these areas. References ADELWOEHRER, N.E. & MAHNERT, W. (1993) Arch. Gynecol. Obstet., 252, 179–184. ANDERSSON, K.-E. (1993) Pharmacol. Rev., 45, 253–308.

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ANDERSSON, K.E., ANDERSSON, P.O., FOVAEUS, M, HEDLUND, H., MALMGREN, A. & SJ― GREN, C. (1988) Drugs, 36, 41–49. ANDERSSON, K.-E., PASCUAL, A.G., PERSSON, K., FORMAN, A.T. & TØTTRUP, A. (1992) J. Urol., 147, 253–259. ANDERSSON, P.-O. & WYLLIE, M.G. (1992) In: Potassium Channel Modulators. Weston, A.H. and Hamilton, T.C. (eds). Blackwell Scientific Publications, Oxford. pp. 462–485. ANTANE, S.A., BUTERA, J.A., ARGENTIERI, T.M., NORTON, W., ZEBICK, D.M., SPINELLI, W., BRIDAL, T., WOJDAN, A., OSHIRO, G. & BAGLI, J.F. (1994) Medi 227, 207th ACS National Meeting San Diego, CA, March. ANWER, K., TORO, L., OBERTI, C, STEFANI, E. & SANBORN, B.M. (1992) Am. J. Physiol., 263, C1049–C1056. ANWER, K., OBERTI, C., PEREZ, G.J., PEREZ-REYES, N., MCDOUGALL, J.K., MONGA, M., SANBORN, B.M., STEFANI, E. & TORO, L. (1993) Am. J. Physiol., 265, C976–C985. BEECH, D.J. & BOLTON, T.B. (1989) Br.J. Pharmac., 98, 851–854. BOLTON, T.B. & LlM, S.P. (1989) J. Physiol., 409, 385–401. BONEV, A.D. & NELSON, M.T. (1993a) Am. J. Physiol., 264, C190–C200. (1993b) Am. J. Physiol., 265, C1723–1728. BRADING, A.F. & TURNER, W.H. (1994) Br. J. Urol., 73, 3–8. BRADING, A.F., MOSTWIN, J.L., SIBLEY, G.N.A. & SPEAKMAN, M.J. (1986) Clin. Sci., 70, 14. BRADING, A.F., PAREKH, A.B. & TOMITA, T. (1989) J. Physiol., 417, 63P. BRIDGEWATER, M. & BRADING, A.F. (1993) Neurourol. Urodyn., 12, 357–358. BRIDGEWATER, M., MACNEIL, H.F. & BRADING, A.F. (1993) J. Urol, 150, 223–228. CALLAHAN, S.M. & CREED, K.E. (1985) J. Physiol., 358, 35–46. CHEUK, J.M.S., HOLLINGSWORTH, M., HUGHES, S.J., PIPER, I.T. & MARESH, M.J.A. (1993) Am. J. Obstet. Gynecol., 168, 953–960. CHRIST, C.G., BRINK, P.R., MELMAN, A. & SPRAY, S.C. (1993) Int. J. Impot. Res., 5, 77–96. DE MOURA, R.S., DE MELLO, R.F. & D’AGUINAGA, S. (1993) J. Urol., 149, 1174–1177. DOWNING, S.J. & HOLLINGSWORTH, M. (1990) Br. J. Pharmac., 100, 488P. DOWNING, S.J., MILLER, M. & HOLLINSWORTH, M. (1989) Br. J. Pharmacol., 96, 732–738. ERULKAR, S.D. & LUDMIR, J. (1993) Am. J. Obstet. Gynecol., 168, 1628–1639. FOSTER, C.D., FUJII, K. & BRADING, A.F. (1988) J. Muscle Res. Cell Motility, 9, 458–459. FOSTER, C.D., FUJII, K., KINGDON, J. & BRADING, A.F. (1989a) Br. J. Pharmacol., 97, 281–291. FOSTER, C.D., SPEAKMAN, M.J., FUJII, K. & BRADING, A.F. (1989b) Br. J. Urol., 63, 284–294. FUJII, K. (1988) J. Physiol., 402, 39–52. FUJII, K., FOSTER, C.D., BRADING, A.F. & PAREKH, A.B. (1990) Br. J. Pharmacol., 99, 779–785. GIRALDI, A. & WAGNER, G. (1990) Pharmacol Toxicol, 67, 235–238. GOLENHOFEN, K. & HANNAPPEL, J. (1973) Pflugers Arch., 341, 257–270.

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HA, J.H., LEE, K.Y. & KlM, W.J. (1993) J. Korean Med. Sci., 8, 53–59. HEATON, R.C., WRAY, S. & EISNER, D.A. (1993) J. Physiol., 465, 43–56. HEDLUND, H., MATTIASON, A. & ANDERSSON, K.-E. (1991) J. Urol., 146, 1345–1347. HELLSTROM, W.J.G., WANG, R., KADOWITZ, P.J. & DOMER, F. (1992) Int. J. Impot. Res., 4, 35–43. HOLLINGSWORTH, M., AMEDEE, T., EDWARDS, D., MIRONNEAU, J., SAVINEAU, J.P. & SMALL, R.C. (1987) Br. J. Pharmacol., 91, 808–813. HOLLINGSWORTH, M., EDWARDS, D., MILLER, M., RANKIN, J.R. & WESTON, A.H. (1989) Med. Sci. Res., 17, 461–463. HOLMQUIST, F. ANDERSSON, K.-E., FOVEAUS, M. & HEDLUND, H. (1990a) J. Urol., 144, 146–151. HOLMQUIST, F. ANDERSSON, K.-E. & HEDLUND, H. (1990b) Acta Physiol. Scand., 138, 463–469. IMAIZUMI, Y., MURAKI, K. & WATANABE, M. (1989) J. Physiol., 411, 131–159. IMAIZUMI, M., MURAKI, K. & VERDONCK, F. (1990) J. Physiol, 427, 301–324. INOUE, Y., SHIMAMURA, K. & SPERELAKIS, N. (1992) Can. J. Physiol. Pharmacol., 70, 1597–1603. (1993) Eur. J. Pharmacol, 240, 169–176. ITO, Y. & KIMOTO, Y. (1985) J. Physiol, 367, 57–72. JORGENSEN, T.M., DJURHUUS, J.C., JORGENSEN, H.S. & SORENSEN, S.S. (1983) Urol Res., 11,239–240. KLAUS, E., ENGLERT, H.C., HROPOT, M., MANIA, D. & ZWERGEL, U. (1990) Eur. J. Pharmacol, 183, 673–674. KLÖCKNER, U. & ISENBERG, G. (1985) Pflugers Arch., 405, 340–348. KONTANI, H., GINKAWA, M. & SAKAI, T. (1993) Jpn. J. Pharmacol, 62, 331–338. KURIHARA, S. (1975) Jpn. J. Physiol., 25, 775–788. LANG, R.J. (1989) J. Physiol., 412, 375–395. LUDMIR, J. & ERULKAR, S.D. (1993) Microsc. Res. Tech., 25, 134–147. MACNEIL, H.F., TURNER, W.H. & BRADING, A.F. (1991) Neurourol. Urodyn., 10, 351–352. MAGGI, C.A. & GIULIANI, S. (1991) Neuroscience, 43, 261–271. MAGGI, C.A., GIULIANI, S. & SANTICIOLI, P. (1994) Naunyn-Schmiedeberg’s Arch. Pharmacol., 349, 510–522. MALMGREN, A. (1987) J. Physiol., 390, 107P. MALMGREN, A., ANDERSSON, K.-E., SJÖGREN, C. & ANDERSSON, P.O. (1989) J. Urol., 142, 1134–1138. MALMGREN, A., ANDERSSON, K.-E., ANDERSSON, P.O., FOVAEUS, M. & SJÖGREN, C. (1990) J. Urol., 143, 828–834. MARKWARDT, F. & ISENBERG, G. (1992) J. Gen. Physiol., 99, 841–862. MONTGOMERY, B.S.I. & FRY, C.H. (1992) J. Urol., 147, 176–184. MORRISON, J.J., ASHFORD, M.L., KHAN, R.N. & SMITH, S.K. (1993) Am. J. Obstet. Gynecol, 169, 1277–1285. NURSE, D.E., RESTORICK, J.M. & MUNDY, A.R. (1991) Br. J. Urol., 69, 27–31. PEREZ, G.J., TORO, L., ERULKAR, S.D. & STEFANI, E. (1993) Am. J. Obstet. Gynecol., 168, 652–660. PERSSON, K. & ANDERSSON, K.-E. (1992) Br. J. Pharmacol., 106, 416–422. PIPER, I., MINSHALL, E., DOWNING, S.J., HOLLINGSWORTH, M. & SANDRAEI, H. (1990) Br. J. Pharmacol., 101, 901–907.

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RESTORICK, J. & NURSE, D. (1988) An in vitro and in vivo study. Neurourol. Urodyn., 7, 207–208. SAITO, M., WEIN, A.J. & LEVIN, R.M. (1993) Neurourol. Urodyn., 12, 573–583. SANTICIOLI, P. & MAGGI, C.A. (1994) Br. J. Pharmacol., 113, 588–592. SHUBA, M.F. (1977) J. Physiol., 264, 853–864. (1981). In: Smooth Muscle: an Assessment of Current Knowledge. Bülbring, E., Brading, A.F., Jones, A.W. and Tomita, T. (eds). Edward Arnold, London. SIBLEY, G.N.A. (1985) Br. J. Urol., 57, 292–298. SPEAKMAN, M.J. (1988) Studies on the physiology of the normal and obstructed bladder. M.S. Thesis, London. SPEAKMAN, M.J., BRADING, A.F., GILPIN, C.J., DIXON, J.S., GILPIN, S.A. & GOSLING, J. (1987) J. Urol., 138, 1461–1466. SUZUKI, M., MURAKI, K., IMAISUMI, Y. & WATANABE, M. (1992) Br. J. Pharmacol., 107, 134–140. TORRENS, M. (1987a) Chapter 9 In: The Physiology of the Lower Urinary Tract. Torrens, M. and Morrison, J. (eds) Springer-Verlag. Berlin. (1987b) Chapter 11 In: The Physiology of the Lower Urinary Tract. Torrens, M. and Morrison, J. (eds) Springer-Verlag, Berlin. TRITTHART, H.A., STARK, U., STARK, G., MAHNERT, W., STENDER, C.O. & SCHREIBMAYER, W. (1992) Ann. N. Y. Acad. Sci., 657, 216–227. WELLNER, M.C. & ISENBERG, G. (1994) J. Physiol., 480, 439–448. ZOGRAFOS, P., LI, J.L. & KAU, S.T. (1992) Pharmacology, 45, 216–230.

Recent Literature FRANK, C.A., FORST, J.M., GRANT, T., HARRIS, R.J., KAU, S.T., LI, J.H., OHNMACHT, C.J., SMITH, R.W., TRAINOR, D.A. & TRIVEDI, S. (1993) Dihydropyridine KATP Potassium Channel Openers. BioMed. Chem. Letts., 3, 2725–2726. GRANT, T., FRANK, C.A., KAU, S.T., LI, J.H., MCLAREN, P.M., OHNMACHT, C.J., RUSSELL, K., SHAPIRO, H.S. & TRIVEDI, S. (1993) Anilide Tertiary Carbinols: A New Structural Class of Potent Potassium Channel Openers. BioMed. Chem. Letts., 3, 2723–2724. HOME, B., HALTERMAN, T.J., YOCHIM, C.L., MY LINH DO, PETTINGER, S.J., STOW, R.B., OHNMACHT, C.J., RUSSELL, K., EMPFIELD, J.R., TRAINOR, D.A. et al. (1995) Zeneca ZD6169: A Novel KATP Channel Opener with in vivo Selectivity for Urinary Bladder. J. Pharmacol. Sup. Therap., 274, 884–890.

15 Potassium Channel Modulators and the Central Nervous System H.HERDON Psychiatry Research Department, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. 15.1 Introduction Potassium (K) channels are the most diverse and ubiquitous of all ion channels. They are present in virtually every eukaryotic cell, and exert major influences on cell function. In excitable cells, they play key roles in regulating action potentials, resting membrane potentials and cellular events such as learning and memory, whilst in non-excitable cells they regulate membrane transport processes and influence intracellular events. K channels have traditionally been identified and characterised on the basis of electrophysiological measurements of their kinetics, voltage-dependence and conductance. Their importance in excitable cell function was originally recognised using voltage-clamp techniques (Hodgkin and Huxley, 1952), and from the early 1980s onwards the development of patch clamp methodology (Hammill et al., 1981) has provided enormous advances in our knowledge of K channel properties. In recent years these methods have been complimented by the use of molecular biological techniques to clone, sequence and express ion channels. This has resulted in the identification of many new K channels, producing an exponential growth in information on the structural diversity of K channels and how this relates to their function. The combination of electrophysiological and molecular biological methods now provides a partial understanding of the molecular basis of some fundamental K channel properties (Pongs, 1992a). Many attempts have been made to produce a general classification of K channels, although none have so far proved totally satisfactory due to the remarkable diversity of these channels. In two recent complementary attempts, one system classified K channels into three overlapping categories: voltageregulated, voltage and ligand-regulated, and ligand-regulated (Quast and Weston, 1994), the other into four categories: voltage-regulated, Ca-activated, receptor-coupled and ‘others’ (Watson and Girdlestone, 1994). Such categories are not mutually exclusive; for example, certain K channels in the voltage-

KCMS AND THE CENTRAL NERVOUS SYSTEM 435

regulated category can be modulated by extracellular or intracellular ligands (see below). Such classifications are based largely on the electrophysiological and pharmacological properties of native channels, but many cloned β -subunits of channels corresponding to most of the main classes of voltage-regulated K channels have now been identified. These cloned channels can be divided into two ‘superfamilies’: Shaker-related (Shaker, Shab, Shaw Shal, Slo) channels, and a new family including ROMK1, IRK1 (members of the inward rectifier) and the recently discovered rcKATP1 (cloned cardiac KATP channel) (Ashford et al., 1994), which may correspond to the inner core of Shaker-related channels (Nichols, 1993; Pongs, 1993). However, other K channel families may also exist (e.g. Suzuki et al., 1994). Although such classifications are still incomplete and not wholly analogous, they serve as useful guides to this complex area until the IUPHAR recommendations on K channel nomenclature are finalised (Spedding and Vanhoutte, 1993). Most of the major types of K channels are present in the CNS. These include at least five different types of voltage-regulated channel, three types of Ca-activated channel and several ligand-gated/receptor-coupled channels (Halliwell, 1990; Aronson, 1992). A summary of the properties of some of these channels is given in Table 15.1. CNS K channels were originally characterised in terms of macroscopic currents, and subsequently by single channel analysis. Cloned K channel ― -subunits corresponding to several classes of voltage-regulated K channels have now been identified in the CNS. Some of these channels may be closely related; for example, certain KV-type delayed rectifier channels can be altered to display KA-type inactivating properties by association with a recentlydiscovered brain-specific β -subunit (Rettig et al., 1994). The K+ equilibrium potential is normally negative to the resting membrane potential, so the opening of most types of K channels generally produces hyperpolarisation and inhibition of neuronal excitability, whilst inhibition of K channels produces depolarisation. Such effects on neuronal excitability can occur as a result of not only direct effects on membrane potential but also indirect effects via changes in membrane conductance, which alter the influence other ionic currents have on membrane potential (Galvan, 1992). Some types of K channels (e.g. KV, KA) are activated mainly by depolarisation, and exert major influences on action potential characteristics. Other channels (e.g. KM) can remain open in the absence of depolarisation and influence resting membrane potentials. Still others (e.g. KIR) are Table 15.1 Summary of properties of some CNS K channels Symbol Channel type

Cloned family Modulators

KV

Delayed Rectifier

KA

A Channel

Shaker, Shaw, Blockers: TEA Shab Shaker, Shal Blockers: 4–AP, β -dendrotoxin, MCD peptide

436 K CHANNELS AND THEIR MODULATORS

Symbol Channel type

Cloned family Modulators

KD KIR KATP

Slowly Inactivating Inward Rectifier ATP-sensitive

? IRK, ROMK rcKATP (IRK, ROMK)

BKCa

Large-conductance Ca-activated

Slo

SKCa

Small-conductance ? Ca-activated Muscarinic-inactivated GIRK

KM

Blockers: 4–AP (potent) Blockers: TEA Blockers: Sulphonylureas (e.g. Glibenclamide) Openers: Cromakalim, Pinacidil, Diazoxide Blockers: Charybdotoxin, Iberiotoxin Openers: NS 004, NS 1619 Blockers: Apamin, Scyllatoxin ?

activated by hyperpolarisation and actually produce a small depolarisation, thus again regulating resting membrane potential. Many types of K channels can be regulated by neurotransmitters, second messengers etc. (see below) and produce effects independent of changes in membrane potential. It should also be borne in mind that K channels are present on glial cells as well as neurones (Bevan, 1990). Since glia have been shown to influence neuronal activity (Nedergaard, 1994), such glial K channels could produce indirect effects on neurones. These few examples serve to illustrate the diverse influences that K channels can have on neuronal function. 15.2 Endogenous Modulators of CNS K Channels Although the main subject of this chapter will be the actions of exogenous drugs and toxins which affect CNS K channels, it must be recognised that a wide variety of endogenous substances can also regulate the activity of these channels. In particular, many neurotransmitters and their receptors are known to affect K channels (Rudy, 1988; Nicoll et al., 1990; Storm, 1993). Amongst the best known is acetylcholine, which via muscarinic receptors inhibits the voltageregulated channel responsible for the M-current. This current can also be inhibited by other receptors coupled to the inositol phosphate/diacylglycerol effector system, including metabotropic glutamate, 5–HT and several peptide (e.g. bradykinin) receptors. Acetylcholine acting via muscarinic receptors can also inhibit the AHP current, which is probably carried by SKCa channels. This current is strongly inhibited by other neurotransmitters including monoamines, VIP and glutamate. Many of these effects may be mediated via actions on the cyclic AMP/adenylate cyclase effector system. Another major voltage-regulated K channel, KA, can be inhibited by both noradrenaline (via β -adrenoceptors) and acetylcholine (via muscarinic receptors); a similar K channel is activated by

KCMS AND THE CENTRAL NERVOUS SYSTEM 437

GABAB receptors (Gage, 1992) and cannabinoid receptors (Deadwyler et al., 1993). Such examples serve to illustrate the concepts of ‘convergence’, where several different neurotransmitter receptors regulate the activity of a single type of K channel, and ‘divergence’, where a single type of receptor (e.g. muscarinic) can regulate the activity of several types of K channels (Nicoll, 1988). Dopamine has received a lot of attention as a possible modulator of K channels. Dopamine D2−type receptors can hyperpolarise neurones by increasing K+ conductance (Lacey et al., 1987), and non-selective potassium channel blockers (KCBs) can inhibit D2−autoreceptor function (Cass and Zahniser, 1991; Tanaka et al., 1992). Some D2 receptor effects on K channels may be mediated by Gi-type G-proteins (Lledo et al., 1992). Stimulation of D2−type receptors has been reported to open a K channel sensitive to quinine and sulphonylurea-type KATP channel blockers (Freedman and Weight, 1989; Roeper et al., 1990; Lin et al., 1993), although evidence against this has also been described (Hicks and Henderson, 1992). Thus, the type(s) of K channels affected by dopamine receptors remains unclear. Several peptides may also modulate ATP-sensitive K channels (KATP channels). There is indirect evidence that galanin opens CNS KATP channels (Ben-Ari, 1990), and opioid μ and β receptors may modulate this type of channel as well as KIR channels (North, 1989; Edwards and Weston, 1993). Endosulphine, a peptide extracted from brain, has been proposed as an endogenous inhibitor of KATP channels (Virsolvy-Vergine et al., 1992), though its location and function in the CNS remain unknown. Many of the effects of G-protein coupled neurotransmitter receptors on K channels are likely to involve actions via second messenger systems altering protein kinase activity and thus channel phosphorylation. However, some effects may be more direct and involve interactions of G-proteins themselves with the channels. Thus, Gi has been shown to activate both KACh and KATP channels (Birnbaumer, 1992), and several brain K channels activated by Go have also been described (VanDongen et al., 1988). 15.3 Toxin Modulators of CNS K Channels Amongst the first agents recognised to act as relatively selective modulators of K channels were a variety of peptide toxins. Toxins derived from snake, scorpion or bee venom have proved to be very valuable and versatile tools in studies on the properties of K channels and their effects on neuronal function. Such toxins have been used in electrophysiological and biochemical investigations, ligand binding assays and autoradiography, as well as functional studies both in vivo and in vitro. Although these experiments have resulted in the realisation that some toxins are not as specific for particular K channel types as was originally thought, these agents have still made important contributions to our understanding of K channels.

438 K CHANNELS AND THEIR MODULATORS

15.3.1 Scorpion Venom Toxins The most well-known scorpion venom toxin is charybdotoxin. This 37 amino acid peptide was originally classified as a specific blocker of BKCa channels, at which it is active at low nanomolar concentrations. However, recent investigations have shown that charybdotoxin can also block brain KA-type channels and probably other Ca-regulated K channels. The block of K channels by charybdotoxin is thought to involve physical occlusion of the channel pore (MacKinnon et al., 1990). Charybdotoxin has a high sequence homology to noxiustoxin, a 39 amino acid peptide, which can also block KA-type and BKCa channels. Iberiotoxin, a 37 amino acid peptide with substantial sequence homology to charybdotoxin, may be a more selective blocker of BKCa channels. In contrast scyllatoxin (leiurotoxin I), a 31 amino acid peptide, also has some sequence homology to charybdotoxin and noxiustoxin but can produce a selective block of SKCa channels. Several of these scorpion toxins have been shown to produce effects on brain function at a macroscopic level. Both charybdotoxin and noxiustoxin stimulate synaptosomal [3H]-GABA release, and charybdotoxin can increase intracellular Ca levels (see Brewster and Strong, 1992). Radioiodinated derivatives of charybdotoxin and scyllatoxin have been used to study the binding sites for these peptides in brain tissue; the scyllatoxin sites appear to be identical to those for the bee venom toxin apamin (see below) and have been taken as corresponding to SKCa channels. Autoradiographic mapping of charybdotoxin binding sites has revealed high densities in mesencephalic regions and white matter areas (Gehlert et al., 1992; Gehlert and Gackenheimer, 1993); these sites probably correspond to KA-type rather than BKCa channels (Vazquez et al., 1990). 15.3.2 Bee Venom Toxins Toxins from bee venom are generally smaller molecules than those from scorpion or snake venom. There are two of major importance: apamin and mast cell degranulating (MCD) peptide. The two are related in structure but have different pharmacological properties. Apamin, an 18 amino acid peptide, is a selective blocker of SKCa channels at low nanomolar concentrations, although some types of SKCa channel (e.g. in hippocampal neurones) seem resistant to apamin (Storm, 1993). Radioiodinated apamin has been used for autoradiographic mapping of brain binding sites thought to be related to SKCa channels. These sites are present in high densities in limbic and motor areas (Gehlert and Gackenheimer, 1993), and injection of apamin into the A10 dopaminergic region (which provides an input to the limbic forebrain) produces an increase in motor activity. Intraventricular injection of apamin can produce hyperactivity and convulsions; these symptoms can also be produced by

KCMS AND THE CENTRAL NERVOUS SYSTEM 439

peripheral administration, though a much higher concentration of toxin is required (see Brewster and Strong, 1992). MCD peptide is a 22 amino acid molecule. In contrast to apamin, it appears to be a selective blocker of KA channels at low nanomolar concentrations. Autoradiographic studies with radioiodinated peptide show a high density of binding sites in cortex, hippocampus and cerebellum. The toxin can produce long-term potentiation in hippocampal slices (Cherubini et al., 1987), and in vivo central administration causes increased arousal followed by convulsions at higher doses. The channels blocked by MCD peptide seem closely related to those which are targets for the snake venom toxins, β -dendrotoxin and β bungarotoxin. 15.3.3 Snake Venom Toxins The most important snake venom toxins are β -bungarotoxin and the dendrotoxins. The latter consist of a group of 59 amino acid peptides. They are capable of blocking both Kv and KA-type channels, with the β and β dendrotoxins being more selective for KA (see Dolly et al., 1994). β bungarotoxin, a large 180 amino acid two chain peptide, is less selective as a KCB, since it also possesses phospholipase enzyme activity. However, it appears to block the same set of K channels as β -dendrotoxin. Allosterically interacting binding sites for β -bungarotoxin, β -dendrotoxin and mast cell degranulating peptide (MCDP) have been reported, and a family of brain proteins (‘DMB binding proteins’) have been identified to which all these toxins bind and the β subunits of which are related to the Shaker K channel family (Rehm and Tempel, 1991), though β -dendrotoxin-sensitive K channels also possess a β -subunit which affects channel properties but which appears to be unrelated to any known ion channel protein (Scott et al., 1994). Autoradiographic mapping of iodinated β dendrotoxin binding shows a wide distribution with highest levels in hippocampus and cerebellum. As with other K channel toxins, dendrotoxins can depolarise synaptosomes and stimulate both GABA and glutamate release, as well as causing severe convulsions after intraventricular administration (see Dreyer, 1990). 15.4 Drugs Modulating CNS K Channels The first synthetic compounds recognised as acting on K channels were simple molecules such as tetraethylammonium (TEA) and the aminopyridines. These agents are capable of blocking a variety of K channels, although they do exhibit some selectivity at lower concentrations. Thus, TEA is a much more potent blocker of Kv, Km and BKCa than of KA, SKCa or KATP. Conversely, 4– aminopyridine (4–AP) predominantly blocks KA and KD rather than Kv. Block of

440 K CHANNELS AND THEIR MODULATORS

K channels by TEA can occur at distinct extracellular and intracellular sites, and involves physical occlusion of the channel pore (Pongs, 1992b). Compounds such as quinine are also capable of blocking a range of K channels including Kv, BKCa SKCa and KATP. Excluding some of the toxins discussed above, compounds possessing highly selective direct actions have been discovered for very few types of K channels. Recent reports have described two novel benzimidazolones, the Neurosearch compounds NS 004 and NS 1619 (see Figure 15.1), which appear to be selective activators of BKCa channels in both smooth muscle cells and neurones (Olesen et al., 1994a, 1994b). A novel Bayer compound, Bay x9227 (Lenfers et al., 1993; see Figure 15.1), is reported (as the (–) enantiomer) to hyperpolarise neuronal cells with an EC50 of 3 pM (Hunnicutt et al., 1994). Although the mechanism of this effect of the (–) enantiomer is not disclosed, the (+) enantiomer of the compound is described as being a smooth muscle KATP channel opener. In addition, this (+) enantiomer is capable of blocking the effect of the (–) enantiomer (Lenfers et al., 1993). Therefore, K channels might also be involved in the mechanism of action of the (–) enantiomer. A series of benzoylamino benzopyrans related to cromakalim (CRK) have been disclosed in a SmithKline Beecham patent (Evans et al., 1992). Resolution of certain of these compounds into their 3S, 4R and 3R, 4S enantiomers (see Figure 15.1) has indicated that the 3S, 4R enantiomers possess antihypertensive (KATP channel opening?) activity, whereas the 3R, 4S enantiomers are minimally antihypertensive but do possess anticonvulsant activity (Blackburn et al., 1993). Whether this anticonvulsant activity is related to effects at K channels has not been reported. 15.4.1 KATP Channel Modulators Apart from the few novel compounds mentioned above, virtually all the agents which have been reported as direct and selective modulators of K channels belong to the class which have been described as either blockers/inhibitors or openers/ activators of peripheral ‘Type 1’ (Ashcroft and Ashcroft, 1990) KATP channels e.g. in pancreatic β -cells or smooth or cardiac muscle. The openers (KCOs) include agents such as diazoxide, pinacidil and CRK. The effectiveness of these compounds as antihypertensive agents, together with growing knowledge of their mechanisms of action, has led to the synthesis of a large number of molecules based mainly around the benzopyran, thioformamide or cyanoguanidine structures (for examples see Figure 15.2). Such compounds may differ greatly in their potency and

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Figure 15.1 Novel compounds with possible K channel-modulating properties

pharmacokinetics, but are still classified as openers of KATP channels. However, recent reports of substantial selectivity between different smooth muscle tissues shown by certain KCOs (Weston and Edwards, 1992; Edwards and Weston, 1993) indicate that such agents can also differ in their pharmacodynamics, and may act preferentially on subtypes of KATP channels present in different tissues. Indeed, evidence has been presented recently that in certain smooth muscle cells levcromakalim (LCRK) can act on Kv channels, and that KATP channels may in fact represent a voltage-independent form of this channel (Edwards et al., 1993). However, this claim has not been supported by other studies (e.g. Evans et al., 1994), and the recent report of the cloning of a cardiac KATP channel (rcKATP1) indicates that such channels are members of the inward rectifier (IRK, ROMK) family rather than the Kv family (Ashford et al., 1994). It is also important to

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remember that KCOs can show pharmacological activity independent of their KATP channel-opening abilities (Quast, 1993). Compounds classified as blockers/inhibitors of KATP channels are based mainly around the sulphonylurea structure and are typified by glibenclamide and tolbutamide

Figure 15.2 Examples of K channel opener and K channel blocker structures

(see Figure 15.2). These KCBs were developed as treatments for non-insulin dependent diabetes, and their mechanism of action in this case is thought to involve block of KATP on pancreatic β -cells. Sulphonylureas can also block smooth and cardiac muscle KATP channels, but with substantially lower potency. For example, glibenclamide is effective at concentrations of 1–10 nM on β -cells, but concentrations of 100–1000 nM are required in smooth muscle. Conversely, most KCOs (apart from diazoxide) show far greater potency on smooth muscle than on β -cells (Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993). As

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with KCOs, sulphonylurea KCBs are not totally specific and can block other types of channel at micromolar concentrations (Ashcroft and Ashcroft, 1992). Although potassium channels sensitive to ATP have been identified in the CNS, it is not clear how their pharmacology relates to that of their peripheral counterparts. Unfortunately, most studies in CNS have used such high concentrations of KCOs and KCBs that they could not be considered as selective in terms of actions on peripheral-type KATP channels. For example, diazoxide (500 μ M) and tolbutamide (500 μ M) have been reported to modulate the activity of KATP channels in cortical neurones (Ohno-Shosaku and Yamamoto, 1992). Tolbutamide (100 μ M) also blocks KATP channels in ventromedial hypothalamic (VMH) neurones, but in this case the effect is actually reversed by 0.1 μ M glibenclamide (Ashford et al., 1990). This unexpected finding might be related to the fact that second generation sulphonylureas such as glibenclamide contain a benzoic acid as well as a sulphonylurea moiety, which could interact with a second site (Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993). These VMH KATP channels are likely to differ substantially from the well-characterised peripheral KATP channels, since they have different electrophysiological properties and are insensitive to a range of KCOs including CRK, pinacidil and diazoxide (Sellers et al., 1992). The exact relationship between the sites of action of KCOs/KCBs and the KATP channels that they modulate is also uncertain. In pancreatic β -cells and some smooth muscle cells, KCOs and KCBs can alter KATP channel activity in isolated membrane patches, suggesting that their site of action is closely related to the channel itself (Ashcroft and Ashcroft, 1990, 1992; but see Khan et al., 1993). However, the cloned cardiac KATP channel (rcKATPl) can be activated by pinacidil but is unaffected by glibenclamide, suggesting that the channel and the sulphonylurea binding site are separate entities (Ashford et al., 1994). The mRNA for this channel is also expressed in several brain areas, especially hypothalamus and preoptic area (Ashford et al., 1994), and in VMH neurones tolbutamide blocks KATP channels only in cell-attached not isolated patches, in agreement with the concept of channels and sulphonylurea binding sites as separate entities (Ashford et al, 1990). In cortical neurones, on the other hand, tolbutamide (at 500 μ M) is effective in isolated as well as cell-attached patches (Ohno-Shosaku and Yamamoto, 1992). Again, this discrepancy may be due to the existence of different types of CNS KATP channels. This uncertainty as to the site of action of KCBs in relation to the channels themselves also raises questions over the interpretation of radioligand binding studies using radiolabelled sulphonylureas. Although in pancreatic β -cells there is a good correlation between affinities of sulphonylureas in such binding assays and their effects on KATP channels, the same cannot be stated for CNS tissue. Radiolabelled glibenclamide binding is of similar affinity (ca. 0.5 nM) in brain as in β -cells, and the distribution of these binding sites in brain tissue has been taken as representing that of KATP channels (e.g. Mourre et al., 1990; Gehlert et al., 1991). However, functional effects of sulphonylureas on neurones generally

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require concentrations several orders of magnitude higher than those effective at these high affinity binding sites (see next section). This has led to the suggestion that the lower affinity binding sites for radiolabelled glibenclamide (Kd 0.1–1 μ M) may also represent a site of action of sulphonylureas on neuronal KATP channels (Gopalakrishnan et al., 1991; Zini et al., 1993a). Neither high nor low affinity binding sites for radiolabelled glibenclamide are affected by KCOs except at very high (ca. 100 μ M) concentrations (Angel and Bidet, 1991; Gopalakrishnan et al., 1991; Schwanstecher et al., 1992; Zini et al., 1993a). This adds to the uncertainty over the relationship between the binding sites for KCBs, KCOs and the channels themselves. Numerous past studies have been unsuccessful in demonstrating specific binding sites for radiolabelled KCOs, but a pinacidil derivative (P1075) has now been used to label putative KATP channels in whole segments of rat aorta (Bray and Quast, 1992). Although this compound is not totally selective and can also act as a Cl channel blocker (Holevinsky et al., 1994), the pharmacology of both KCOs and sulphonylurea KCBs at this binding site correlates well with their functional effects (Quast et al., 1993). This technique has also provided evidence that the binding sites for sulphonylureas and KCOs may be allosterically coupled, at least in peripheral tissue (Bray and Quast, 1992). Unfortunately, the fact that this binding site could be detected only in intact tissue, but not in any membrane preparation (including brain), leaves the question of the functional significance of CNS sulphonylurea binding sites unresolved. Endosulfine, a peptide extracted from brain, can bind to the same sites as sulphonylureas (Virsolvy-Vergine et al., 1992). This peptide might represent an endogenous ligand for sulphonylurea binding sites (separate from KATP channels?), but its location and function in the brain remain unknown. 15.5 Effects of K Channel Modulating Drugs on CNS Activity Although there have been many studies on the effects of potassium channel modulators (KCMs) on a range of CNS activity models both in vivo and in vitro, very few have unequivocally identified the type of K channel involved. This is partly due to the use of non-selective modulators such as aminopyridines, but also to the use of ‘selective’ drugs at concentrations at which such selectivity becomes highly questionable. Setting these caveats aside, however, such studies still show the variety of actions which such drugs can produce. 15.5.1 Potassium Channel Blockers The effects of non-selective blockers such as TEA and especially aminopyridines have been widely studied. Both types of blocker have been shown to stimulate the release of several different neurotransmitters in vitro. Both basal and

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electrically-evoked [3H]-noradrenaline release from hippocampal slices can be increased by TEA, 4–AP and 3,4–diaminopyridine (3,4–DAP) (Huang et al., 1989; Hu and Fredholm, 1991; Allgaier et al., 1993). Similarly, 4–AP and TEA have been reported to enhance acetylcholine release from both striatal (Drukarch et al., 1989; Dolezal and Wecker, 1991) and hippocampal (Fredholm, 1990) slices. Glutamate release from cortical synaptosomes (Nicholls, 1993) or hippocampal slices (Herdon, 1992) can be stimulated by 4–AP or 3,4–DAP respectively. The processes involved in the action of 4–AP on synaptosomal glutamate release have been studied in detail, and it has been suggested that blocking of nerve terminal K channels by 4–AP produces repetitive transient depolarisation of the terminals resulting in transmitter release (Tibbs et al., 1989). Investigations on the effects of 4–AP on electrical activity in hippocampal slices have shown that the compound can produce spontaneous repetitive depolarisations and increases in both EPSP and IPSP amplitude, generating long-lasting depolarisation and epileptiform activity (Perrault and Avoli, 1989, 1991). 4–AP induced epileptiform activity has also been observed in cortical slices (Mattia et al., 1993), and similar effects in human cortex have been reported (Avoli et al., 1992). In vivo studies, including those in humans, have shown that aminopyridines are powerful convulsant and epileptogenic agents (see Perrault and Avoli, 1989, 1991). The exact type of K channel involved in mediating these effects of aminopyridines is not known. However, the fact that the in vitro effects on transmitter release or electrical activity can be produced at relatively low aminopyridine concentrations (10–100 μ M) suggests that the slowlyinactivating D-current, which is particularly sensitive to block by 4–AP (Storm, 1993), may be of prime importance. 15.5.2 KATP Channel Modulators KATP channels have been of particular interest in studies on CNS K channel function. This is partly because of the wide range of pharmacological agents thought to act on these channels, but also because they provide a potential link between neuronal excitability and metabolism. However, evidence for the importance of such channels in neuronal function is still largely indirect, relying principally on studies using drugs which have been classified as modulators of smooth muscle or pancreatic KATP channels. The brain areas in which the actions of such drugs have been most investigated are the substantia nigra and hippocampus. Substantia Nigra The nigral region contains a high density of radiolabelled glibenclamide binding sites, making it a prime target for functional investigations. High concentrations

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(500 μ M) of CRK or pinacidil, or intracellular ATP depletion, have been reported to hyperpolarise dopaminergic nigral neurones, with both effects being reversed by 10 μ M glibenclamide (Hausser et al., 1991). In contrast, another study has reported no effects of CRK (500 μ M), glibenclamide (30 μ M) or tolbutamide (300 μ M) on dopaminergic neurones (Hicks and Henderson, 1992). However, the same authors recently described presynaptic effects of tolbutamide (300 μ M) and ATP-depleting conditions on GABAB potentials in nigra (Hicks et al., 1994). A specific K channel has now been identified in nigral neurones which is activated by diazoxide (300 or conditions which deplete ATP, and inhibited by tolbutamide (100–1000 (Schwanstecher and Panten, 1993). Responses to cyanide or hypoxia have also been shown to be inhibited by tolbutamide (50–500 μ M) (Murphy and Greenfield, 1991, 1992). Neurochemical studies have also identified effects of KATP channel modulating drugs. KCOs including LCRK, CRK and pinacidil have been reported as potent (EC50 values 0.01, 0.3 and 0.4 μ M respectively) inhibitors of [3H]-GABA release and stimulators of 86Rb+ efflux from nigral slices (SchmidtAntomarchi et al., 1990). These effects are mimicked by conditions depleting ATP levels, and both types of action are reversed by a range of sulphonylureas, which in themselves stimulate [3H]-GABA release (Amoroso et al., 1990; Schmidt-Antomarchi et al., 1990). Certain of these results seem internally inconsistent, since LCRK is quoted as being 30–50 times more potent than CRK itself (Schmidt-Antomarchi et al., 1990). In addition, the low concentrations of KCOs required to produce these neurochemical effects (ca. 0.5 μ M for CRK or pinacidil) contrast with concentrations of these same drugs three orders of magnitude higher reported (by the same group) to produce electrophysiologically-measurable effects on nigral neurones (Hausser et al., 1991), raising questions as to whether the same mechanism of action is involved. Behavioural studies have indicated that tolbutamide and quinine can alter amphetamine-induced locomotor activity when injected directly into the substantia nigra (Levesque and Greenfield, 1991). Effects on locomotor activity have also been reported following injections of LCRK or glipizide into dorsal pallidum (Amalric et al., 1992). Effects of KATP channel modulators in key motor areas such as the nigra suggest that such drugs might be useful regulators of movement control. Hippocampus High concentrations (10–100 μ M) of CRK have been reported to hyperpolarise hippocampal neurones by increasing a voltage-dependent K+ current (Alzheimer et al., 1989; Politi et al., 1989). Conditions producing ATP depletion produced a similar effect to CRK, and both effects were blocked by 10 μ M. glibenclamide (Politi and Rogawski, 1991). In contrast, a K channel with different electrophysiological characteristics, which is opened by low concentrations (0.1–

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1 μ M) of LCRK or hypoglycaemia and inhibited by 1 μ M glibenclamide, has also been described in hippocampal neurones (Tromba et al., 1992). Several studies have described hypoglycaemia and/or hypoxia-induced changes in hippocampal slices which can be modulated by KCOs/KCBs (see below for more detailed discussion). These include both inhibition of anoxiainduced hyperpolarisation by tolbutamide or glibenclamide (Grigg and Anderson, 1989; Godfraind and Krnjevic, 1993) and potentiation of anoxia-induced depolarisation by glibenclamide (Ben-Ari, 1990). However, in contrast to findings reported in substantia nigra, no effects of KCOs on either [3H]-GABA release or 86Rb+ efflux have been detected under either normal or hypoglycaemic/ hypoxic conditions (Nelson, 1989; Herdon et al., 1993). Although a potentiation of hypoglycaemia-induced [3H]-GABA release by high concentrations(10–100 μ M) of glibenclamide has been reported (Margaill et al., 1992), this may not involve KATP channels since it is unaffected by LCRK (Herdon et al., 1993). Such high concentrations of glibenclamide can also block other types of K channels (Crepel et al., 1992; Reeve et al., 1992), providing an alternative mechanism. Other brain areas In dorsal raphe, very high concentrations (100–1000 μ M) of glibenclamide inhibit cell firing; this effect can be reversed by LCRK (1–10 μ M) or by 50 μ M aprikalim (RP 52891). These effects are suggested to be indirectly mediated via actions of the drugs on GABAergic neurones (Haj-Dahmane et al., 1993). On the other hand, direct effects of such drugs are suggested in locus coeruleus, where cell firing rates are decreased by CRK (100 µM) or Ro 31–6930 (10 μ M), with tolbutamide (300 μ M) reversing this effect and increasing cell firing (Finta et al., 1993). In cerebral cortex slices, high concentrations (20–200 μ M) of diazoxide, CRK or pinacidil reduce [3H]-noradrenaline release, with some of these effects being reversed by 1 μ M glibenclamide (Takata et al., 1992). In the same study CRK (200 µM) was reported to increase 86Rb+ efflux, although this effect was not observed at lower drug concentrations in other reports (Nelson, 1989; Herdon et al., 1993). In conclusion, KCMs have certainly been shown by both in vitro and in vivo studies to have a wide range of effects on the CNS. However, a variety of limitations including the lack of selectivity and/or the high concentrations of some of the drugs used in such studies, together with questions over the pharmacological similarity between putative types of CNS K channels and their better-characterised peripheral counterparts, have meant that the exact identification of the type of K channel by which any drug acts to produce an effect on CNS function has not been made. Such identification will require both the further pharmacological characterisation of specific CNS K channels and the development of selective drugs for different types of such channels. Alternatively, the molecular cloning and characterisation of further CNS K

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channels combined with the generation of antisense oligonucleotides for such channels (Wahlestedt, 1994) could allow another approach for examining their effects on CNS functions. Despite these limitations and unresolved questions, the pronounced effects of K channel modulating drugs in well-characterised areas such as the hippocampus and substantia nigra have led to suggestions that drugs of this type could be of use in treating CNS disorders associated with these areas such as stroke, epilepsy and movement disorders. Additionally, the overall concept of KCMs as having wide influences on neuronal excitability continues to promote their investigation in other CNS disorders. 15.6 Potential Uses of KCMs in CNS Disorders 15.6.1 Ischaemic Stroke Brain damage due to ischaemia involves a cascade of events. The initial loss of blood supply causes failure of energy-dependent processes such as ion pumps, resulting in depolarisation, massive neurotransmitter release, Na+ and Ca2+ influx etc.; this can produce rapid cell death. However, in areas in which blood supply is restored and normal homeostasis rapidly resumed, delayed neuronal death can still occur days or weeks after the initial ischaemia. This is thought to involve ‘excitotoxicity’, where the initial cell depolarisation and high levels of excitatory amino acids such as glutamate produce intracellular Ca overload, triggering a cascade of intracellular events including enzyme activation, free radical formation and gene expression. These events can enhance excitatory synaptic efficacy and weaken cells so that even normal glutamate levels cause further excitotoxicity, leading to a vicious circle of evolving neuronal damage even in areas which were unaffected by the initial ischaemia (Choi and Hartley, 1993; Hara et al., 1993). K channel opening can produce hyperpolarisation and inhibition of neuronal excitability, suggesting that drugs which act as KCOs could be of benefit in ischaemic stroke. Prophylactic administration of such drugs might limit the spread of the initial ischaemia-induced depolarisation, and administration even some considerable time after the initial ischaemia could reduce the evolving excitotoxicity and limit delayed neuronal death. This latter concept has been validated in animal models of stroke by late administration of drugs which limit excitotoxicity by other mechanisms e.g. glutamate antagonists or neuronal Ca channel blockers (Sheardown et al., 1993; Valentino et al., 1993). It should also be noted that KATP channels and their modulators have been proposed to play key roles in the protection of the heart from the effects of cardiac ischaemia (Escande and Cavero, 1992; Gopalakrishnan et al., 1993). Based on all these ideas and

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findings, it could be predicted that KCOs should be neuroprotective in stroke. Indeed, there is now some experimental evidence for this. As summarised in the previous section, there is substantial evidence from in vitro studies that changes in neuronal activity caused by hypoxia and/or hypoglycaemia can be influenced by KCMs, especially those thought to act on KATP channels. In hippocampal slices, hypoxia induces a hyperpolarisation followed by a depolarisation in CA3 neurones. The hyperpolarisation has been reported to be inhibited by glibenclamide or tolbutamide (Grigg and Anderson, 1989; Mourre et al., 1989; Godfraind and Krnjevic, 1993). Conversely, the following depolarisation is increased by glibenclamide and inhibited by diazoxide or galanin (Ben-Ari, 1990; Ben-Ari et al., 1990). The hyperpolarisation is likely to represent a direct postsynaptic effect, whilst the depolarisation probably occurs as a result of presynaptic release of glutamate (Ben-Ari, 1990). In support of this idea, hypoxia/ hypoglycaemia-induced release of glutamate is partially inhibited by LCRK, RP 52891 or galanin, but increased by glibenclamide or gliquidone (Zini et al., 1993b). Additionally, in cultured hippocampal neurones excitotoxicity due to glutamate release and activation of NMDA receptors is inhibited by CRK or diazoxide, with the effects of these drugs being reversed by glibenclamide (Abele and Miller, 1990). However, a study on hypoxic/hypoglycaemic toxicity in cultured cortical neurones has not shown any protective effects of CRK or diazoxide (Koretz et al., 1994). Cyanideinduced neurotoxicity in hippocampal neurones can also be potentiated by glibenclamide, with this effect being reduced by diazoxide (Patel et al., 1992). As summarised above, neuronal damage due to ischaemic stroke is thought to involve glutamate-induced excitotoxicity. Therefore, these in vitro findings suggest that KCOs could be useful for the treatment of stroke. Unfortunately, the testing of such drugs using in vivo animal models of stroke is limited by their poor brain penetration, and interpretation of any neuronal effects observed after systemic administration is complicated by the potent cardiovascular actions of these drugs. In particular, such drugs can produce substantial hypothermia, which itself is strongly neuroprotective (Hara et al., 1993). However, it has been reported recently that i.c.v. injection of LCRK, pinacidil or nicorandil prevents ischaemia-induced gene expression and reduces delayed neuronal death in hippocampus in a rat global ischaemia model, with the actions of these drugs being reversed by glipizide (Heurteaux et al., 1993). This is a very encouraging finding, but the major systemic effects of such KCOs means that the development of neuronally-selective CNS-penetrating drugs will probably be required before this type of therapy is suitable for clinical development. 15.6.2 Epilepsy The basic causes of epilepsy are still uncertain, but some aspects of the pathology of the disease seem related to those of ischaemic-type delayed

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neuronal damage. Thus lesions, tumours or other less clear factors can produce local neuronal hyperexcitability leading to epileptic seizures, and repeated brief seizures can produce excitotoxic-type neuronal damage and kindling phenomena in areas such as hippocampus and amygdala. These then lead to increased susceptibility to further seizures, resulting in a spreading cascade of damaging events (McNamara, 1992; Meldrum, 1993). As with ischaemic stroke, experimental seizures can be prevented by glutamate receptor antagonists (Rogawski, 1992), and many drugs used clinically for treatment of epilepsy are capable of modulating ion channel activity to reduce neuronal excitability, for example by blocking Na channels (e.g. phenytoin, carbamazepine) or potentiating GABA/C1 channels (e.g. benzodiazepines, phenobarbitone) (MacDonald and Kelly, 1993; Upton, 1994). Therefore, it is possible that KCMs might also represent a worthwhile treatment for epilepsy. In fact, the experimental evidence for the involvement of K channels in epileptic seizures is much stronger than it is for ischaemic stroke. As mentioned previously, non-selective KCBs such as 4–AP can produce epileptiform activity in brain slices and cause convulsions in vivo; relatively selective K channel blocking toxins such as apamin, MCDP and dendrotoxins are also powerful convulsant agents. Clinically-used anti-epileptic drugs such as carbamazepine, phenytoin, phenobarbitone and valproate have been reported to inhibit some aspects of 4–AP-induced epileptiform activity in hippocampal slices (Fueta and Avoli, 1992; Watts and Jeffreys, 1993), and to protect against dendrotoxininduced seizures in vivo (Coleman et al., 1992). These pro-convulsant effects of KCBs and the inhibition of their effects by clinically-effective anticonvulsant drugs suggest that agents having the opposite action, i.e. KCOs, should be beneficial in the treatment of epilepsy. Indeed, there is evidence that carbamazepine itself can enhance K+ currents at therapeuticallyrelevant concentrations (Zona et al., 1990). Therefore, a selection of KCOs, especially those classified as acting on KATP channels, have been studied in a range of in vivo and in vitro models of epileptic processes. In vitro studies in hippocampal slices have demonstrated that epileptiform activity induced by changes in ionic concentration or treatment with Ca antagonists can be inhibited by high concentrations (30–300 μ M) of CRK (Alzheimer and ten Bruggencate, 1988; Popoli et al., 1991). CRK (given i.c.v.) can also inhibit epileptic-type seizures induced by a variety of convulsant agents including pentylenetetrazole (Del Pozo et al., 1990), MCDP (Gandolfo et al., 1989a) and digoxin (Chugh et al., 1993), and decrease seizures in genetically epileptic rats (Gandolfo et al, 1989b). The actions of CRK on MCDP seizures could be mimicked by RP 49356, but neither drug could inhibit 4–AP or dendrotoxin-induced seizures (Gandolfo et al., 1989a). However, as with the potential treatment of ischaemic stroke, the poor brain penetration and major cardiovascular effects of present generation KATP channel openers limit greatly the further testing and possible clinical application of these drugs. Whether selective openers of other types of K channels might be effective as

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anticonvulsant agents remains unknown. Certain compounds related to CRK have recently been described as being active as anticonvulsants but possessing minimal hypotensive activity (Blackburn et al., 1993), but whether this anticonvulsant activity is related to effects K channels has not been reported. 15.6.3 Pain As mentioned previously, μ - and β -opioid receptors are linked to CNS K channels (North, 1989). Therefore, KCMs might be able to mimic the actions of opiate drugs in producing analgesia. Non-selective KCBs such as 4–AP and quinine can inhibit behavioural and neurochemical effects of morphine (Pei et al., 1993), and the analgesic effects of morphine or the β -opiate peptide DPDPE can be antagonised by i.c.v. glibenclamide (Ocana et al., 1990; Wild et al., 1991; Narita et al., 1992). Sulphonylureas can also antagonise the antinociccptive effects of clonidine (Ocana and Baeyens, 1993). Conversely, i.c.v. CRK or pinacidil can potentiate morphine analgesia (Vergoni et al., 1992; Narita et al., 1993), and CRK alone can also produce antinociceptive effects (Narita et al., 1993; Kamai et al., 1994). In addition, i.c.v. CRK or diazoxide can inhibit some of the behavioural effects of morphine withdrawal (Robles et al., 1994). These findings suggest that brain-penetrant KATP channel opening drugs might have clinical utility as analgesic agents as well as for the treatment of opiate withdrawal. 15.6.4 Parkinson’s Disease As reviewed earlier, studies on the substantia nigra have shown electrophysiological, neurochemical and behavioural effects of KATP channel modulating drugs. Since the nigra is a major source of dopaminergic neurones, and nigro-striatal dopaminergic degeneration produces Parkinson’s disease, KATP channel modulators might be of benefit in this disease. Based on experiments with KATP channel inhibitors, it has been suggested that openers of these channels could both increase dopamine release and mimic the post-synaptic actions of dopamine, thus reducing Parkinsonian symptoms (Levesque and Greenfield, 1991; Murphy and Greenfield, 1991; see also Amalric et al., 1992). However, direct experimental evidence for this idea is lacking. 15.6.5 Alzheimer’s Disease Neuronal degeneration producing deficits in cholinergic transmission is thought to be a cause of some of the major symptoms such as memory impairment in Alzheimer’s disease. As summarised earlier, acetylcholine can alter the activity of several types of K channels; conversely, KCMs can affect acetylcholine

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release. This reciprocal relationship suggests that KCMs could be of use in alleviating some of the effects of cholinergic deficit. In fact, non-selective KCBs such as TEA or aminopyridines have been reported to produce long-term potentiation in vitro (Aniksztejn and Ben-Ari, 1991) and to improve memory both in animal models and Alzheimer’s disease patients (see Lavretsky and Jarvik, 1992). This area is complicated by the fact that aminopyridines can also act as cholinesterase inhibitors; conversely, cholinesterase inhibitors such as tetrahydroaminoacridine (used clinically for treatment of Alzheimer’s disease) can block K channels. In fact, it has been suggested that agents with both cholinesterase-inhibiting and K channel-blocking properties would be suitable treatments for Alzheimer’s disease (Lavretsky and Jarvik, 1992). Another possible connection between K channels and Alzheimer’s disease has produced a lot of recent interest. It has been reported that a TEA-sensitive K channel present in normal fibroblasts is functionally absent in fibroblasts from Alzheimer’s disease patients (Etcheberrigaray et al., 1993). This finding obviously suggests a potential simple diagnostic test for the disease, but also raises the question of whether there is a similar defect in CNS K channel function. The fact that low concentrations of β -amyloid protein, a key factor in the CNS pathology of Alzheimer’s disease, can produce this same defect in normal human fibroblasts (Etcheberrigaray et al., 1994) certainly suggests that abnormalities in CNS K channels could be involved in producing some of the symptoms of Alzheimer’s disease. If this is the case, it could point to another role for KCMs in the treatment of this disease.

15.7 Summary and Conclusions A range of K channels are now recognised to be present in the CNS, and the electrophysiological and molecular biological properties of several types have been well characterised. In addition, the actions of neurotransmitters, toxins and nonselective blocking drugs on certain specific types of K channels have been studied in detail; such studies have helped to elucidate some of the mechanisms of regulation of K channels at molecular or single channel level. On the other hand, the CNS effects of a variety of drugs and toxins of differing selectivities have been studied at a macroscopic level on diverse functions such as cell firing, transmitter release or behaviour. However, it has proved difficult to link the two sets of findings together i.e. to determine the specific K channels on which any drug acts to produce a macroscopic functional effect. This problem has been largely a result of the lack of synthetic drugs which are highly selective for specific CNS K channels, and it is to be hoped that further efforts by the pharmaceutical industry together with an increased knowledge of CNS K channel properties will help to overcome this.

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The ubiquity of K channels and their diverse effects on neuronal activity hold out promise for the use of KCMs in the treatment of many CNS disorders. Studies in vitro or in animal models have provided specific indications for their potential use in stroke and epilepsy, and more limited evidence for their possible utility in other conditions such as pain, movement disorders, migraine and Alzheimer’s disease. However, virtually all such investigations have been performed with drugs which are non-selective or have poor brain penetration and/ or major systematic effects. There is at present a major need for studies using drugs with selective actions on specific CNS K channels in order to convince the pharmaceutical and medical communities that the promise offered by KCMs can someday become a clinical reality. Acknowledgements Thanks to Neil Upton and Andrew Parsons for very helpful discussions, and to the editors for the invitation to write this chapter. References ABELE, A.E. & MILLER, R.J. (1990) Neurosci. Lett., 115, 195–200. ALLGAIER, C., REPP, H. & HERTTING, G. (1993) Naunyn-Schmiedeberg’s Arch. Pharmacol., 347, 14–20. ALZHEIMER, C. & TEN BRUGGENCATE, G. (1988) Naunyn-Schmiedeberg’s Arch. Pharmacol., 337, 429–434. ALZHEIMER, C., SUTOR, B. & TEN BRUGGENCATE, G. (1989) NaunynSchmiedeberg’s Arch. Pharmacol., 340, 465–471. AMALRIC, M., HEUTEAUX, C. NIEOULLON, A. & LAZDUNSKI, M. (1992) Eur. J. Pharmacol., 217, 71–77. AMOROSO, S., SCHMIDT-ANTOMARCHI, H., FOSSET, M. & LAZDUNSKI, M. (1990) Science, 247, 852–854. ANGEL, I. & BIDET, S. (1991) Fund. Clin. Pharmacol., 5, 107–115. ANIKSZTEJN, L. & BEN-ARI, Y. (1991) Nature, 349, 67–69. ARONSON, J.K. (1992) Biochem. Pharmacol., 43, 11–14. ASHCROFT, S.J.H. & ASHCROFT, F.M. (1990) Cell. Signalling, 2,197–214. (1992) Biochim. Biophys. Acta., 1175, 45–59. ASHFORD, M.L.J., BODEN, P.R. & TREHEME, J.M. (1990) Brit. J. Pharmacol, 101, 531–540. ASHFORD, M.L.J., BOND, C.T., BLAIR, T.A. & ADELMAN, J.P. (1994) Nature, 370, 456–459. AVOLI, M., MATTIA, D., HWA, G.G.C. & SINISCALCHI, A. (1992) Soc. Neurosci. Abstr., 175, (3), 401. BEN-ARI, Y. (1990) Bur. J. Neurosci., 2, 62–68. BEN-ARI, Y., KRNJEVIC, K. & CREPEL, V. (1990) Neuroscience, 37, 55–60. BEVAN, S (1990) Sem. Neurosci., 2, 467–481.

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Recent Literature AGUILAR-BRYAN, L., NICHOLS, C.G., WECHSLER, S.W., CLEMENT, J.P., BOYD, A.E., GONZALEZ, G., HERRERA-SOSA, H., NGUY, K., BRYAN, J. & NELSON, D.A. (1995) Science, 268, 423–426. DURING, M.J., LEONE, P., DAVIS, K.E., KERR, D. & SHERWIN, R.S. (1995) J. Clin. Invest., 95, 2403–2408. EDWARDS, G., NIEDERSTE-HOLLENBERG, A., SCHNEIDER, J., NOACK, T. & WESTON, A.H. (1995) Br.J. Pharmacol., 113, 1538–1547. ERDEMLI, G. & KRNJEVIC, K. (1994) Br. J. Pharmacol., 113,411–418. EREDMLI, G. & KRNJEVIC, K. (1994) NeuroReport, 5, 2145–2148. HERTEAUX, C., LAURITZEN, I., WIDMANN, C. & LAZDUNSKI, M. (1995) Proc. Natl Acad. Sci., 92, 4666–4670. JIANG, C., SIGWORTH, F.J. & HADDAD, G.G (1994) J. Neurosci., 14, 5590–5602. KENNA, S., ROPER, J., HO, K., HERBET, S., ASHCROFT, S.J.H. & ASHCROFT, F.M. (1994) Molec. Brain Res., 24, 353–356.

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KOBAYASHI, T., IKEDA, K., ICHIKAWA, T., ABE, S., TOGASHI, S. & KUMANISHI, T. (1995) Biochem. Biophys. Res. Commun., 208, 1166–1173. KRAPIVINSKY, G., GORDEN, E.A., WICKMAN, K., VELIMIROVIC, B., KRAPIVINSKY, L. & CLAPHAM, D.E. (1995) Nature, 374, 135–141. LEE, K., DIXON, A.K., ROWE, I.C.M., ASHFORD, M.L.J. & RICHARDSON, P.J. (1995) Br. J. Pharmacol., 115, 385–388. LESAGE, F., DUPRAT, F., FINK, M., GUILLEMARE, E., COPPOLA, T., LAZDUNSKI, M. & HUGNOT, J.P. (1994) FEBS Lett., 353, 37–42. MORISHIGE, K.-L, TAKAHASHI, N., JAHANGIR, A., YAMADA, M., KOYAMA, H., ZANELLI, J.S. & KURACHI, Y. (1994) FEBS Lett., 346, 251–256. OCANA, M., DEL Pozo, E., BARRIOS, M. & BAEYEN, J.M. (1995) Br. J. Pharmacol., 114, 1296–1302. PONGS, O. (1995) Sem. Neurosci., 7, 137–147. ROPER, J. & ASHCROFT, F.M. (1995) Pflugers Arch., 430, 44–54. SELLERS, A.J. & ASHFORD, M.L.J. (1994) Br. J. Pharmacol., 113, 659–661. TSENG-CRANK, J., FOSTER, C.D., KRAUSE, J.D., MERTZ, R., GODINOT, N., DICHIARA, T.J. &REINHAR.P.H. (1994) Neuron, 13, 1315–1330. VERGONI, A.V., SANDRINI, M., FILAFERRO, M. & BERTOLININ, A. (1995) Neurosci. Lett., 188, 29–32. WATTS, A.E., HICKS, G.A. & HENDERSON, G. (1995) J. Neurosci., 15, 3065–3074. WIBLE, B.A. & BROWN, A.M. (1994) Drug Develop. Res., 33, 225–234. ZETTERSTROM, T.S.C., VAUGHAN-JONES, R.D. & GRAHAM-SMITH, D.G. (1995) Neuroscience, 67, 815–821.

16 Potassium Channel Modulators: Clinical Experiences and Future Prospects T.J.COLATSKY1 & T.C.HAMILTON2 1 Division of Cardiovascular and Metabolic Diseases, Wyeth-Ayerst

Research, CN 8000, Princeton, NJ 08543, USA. 2

Department of Neurology, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK.

In this chapter we have reviewed, in separate sections for potassium channel activators (KCAs) (sections 16.1 to 16.4) and blockers (KCBs) (sections 16.5 to 16.16), clinical experiences and future prospects for their use in therapeutics. This approach seems logical to us in view of the disparate applications for these two drug classes. 16.1 KCAs Extensive pre-clinical evaluation of KCAs in a diverse range of animal models representative of a number of diseases (see Chapters 9, 11, 12, 14 and 15), has led to optimistic predictions of the likely uses of this class of drug in the clinic (Hammond et al., 1991; Andersson, 1992; Atwal, 1992; Gopalakrishnan et al., 1993; Poyser and Hamilton, 1994). This optimism is reflected by the intense efforts of a large number of pharmaceutical companies in the synthesis of many structurally novel KCAs (see Chapters 1, 2 and 3). However, whilst the extensive patent literature (see Current Drugs Database) and publications in pharmacology journals, give credence to the direction of drug discovery in this field, a relatively small number of papers describe the actions of these drugs in man (see Williams, 1992). It is our goal in this chapter to examine briefly the clinical potential of KCAs, then to review the available clinical findings for benzopyran KCAs (derived from cromakalim, CRK), for other chemical classes developed subsequently (such as thioformamides like aprikalim) and in the case of the cyanoguanidine, pinacidil, clinical data appearing since the review by Friedel and Brogden (1990). Finally, our opinions regarding future prospects for KCAs are discussed.

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 461

16.2 Therapeutic Potential Table 16.1 summarises a number of possible clinical uses of KCAs based upon the premise that such drugs, by opening membrane K channels, should raise membrane Table 16.1 Perspective of some potential therapeutic uses of KCAs

Cardiovascular System Hypertension Pulmonary Hypertension Peripheral Vascular Disease Angina Cardioprotection Congestive Heart Failure Respiratory System Chronic Obstructive Airways Disease including Asthma Inflammatory Airways Other Smooth Muscle Bladder instability Impotence Irritable Bowel Syndrome Uterine Disorders Secretory Diarrhoea Nervous System Epilepsy Pain Ischaemia (stroke) Others Hyperlipidaemia Hypotrichosis + in vivo data + (in vitro data) – no data

Pre-Clinical Findings

Clinical Findings

+ + + + + +

+ – + (+) (+) (+)

+ +

+ –

+ +(in vitro) – + +(in vitro)

+ – – – –

+ + +

– – –

– +

+ +

(+) data for nicorandil

potential (hyperpolarise) and thereby reduce tissue excitability. Support for this hypothesis has come from extensive pre-clinical data in in vivo studies using drugs such as CRK, aprikalim and pinacidil. In broad terms these potential uses may be categorised according to effects of KCAs upon body systems and tissues

462 K CHANNELS AND THEIR MODULATORS

(Table 16.1). Clearly, utility in many indications depends upon inhibition of smooth muscle contractility. For some others reduction in neuronal excitability is important whilst in hyperlipidaemia and hypotrichosis, the underlying mechanism (s) are less clear. Table 16.1 also reveals that the number of clinical indications in which the efficacy of KCAs has been demonstrated is confined to a few. Moreover, as described later, the available clinical data are derived from a limited number of studies using two or three drugs. Various speculative reasons could be proffered for the scarcity of clinical data but it is suffice to state here that, with one or two known exceptions, the biological profile as exhibited by CRK and pinacidil has been difficult to modify. Thus, for example, KCAs selective for one particular smooth muscle, or for neuronal K channels, have not yet emerged. This theme will be addressed later. 16.3 Clinical Experiences 16.3.1 Hypertension KCAs are directly acting smooth muscle relaxants and thereby produce vasodilation and reduce peripheral vascular resistance (see Chapter 9). Thus, these drugs have the ability to modify the primary haemodynamic abnormality present in essential hypertension, namely elevated total peripheral vascular resistance. Evidence has been obtained in man for KCA-mediated changes in the vasculature. In healthy volunteers, orally (0.5 to 2 mg) or i.v. (0.01 to 10 μ g/min) administered CRK increased forearm blood flow (Webb et al., 1989; Fox et al., 1991) and oral levcromakalim (LCRK) (0.5 and 0.75 mg) increased vessel diameter in the eye (Eckl et al., 1992). Oral CRK did not affect forearm venous capacitance (Fox et al., 1991) or, when infused i.v. (1 μ g/min), dorsal hand vein diameter (Haynes and Webb, 1991). Also, Thomas et al., (1990) reported that i.v. CRK (15 μ g/kg) reduced systemic, and pulmonary, vascular resistance by 29 and 24% respectively, in patients with ischaemic heart disease (IHD). Thus these KCAs are arterio-selective vasodilators in man. The ability of CRK to modify vasoconstrictor responses has been examined in volunteers. Nguyen et al. (1991) found that oral CRK (1 and 2 mg) blunted the vasoconstrictor responses to noradrenaline and angiotensin II. In contrast, CRK did not affect noradrenaline-induced venoconstriction (Webb et al., 1989) but did reduce similar responses due to endothelin (Haynes and Webb, 1991). In hypertensive patients, Lebel et al. (1991) found that CRK (1.5 mg daily for 3 days) reduced blood pressure (BP) while glomerular filtration rate was unchanged and effective renal plasma flow (ERPF) was slightly elevated. In

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 463

contrast, in hypertensive patients also receiving the β -adrenoceptor blocker, atenolol, CRK (1 mg orally) did not affect hepatic blood flow or ERPF (Donnelly et al., 1990). In the latter study the minor effect on BP of CRK, given alone or in combination with atenolol, suggested that the dose and/or plasma t1/2 (ca. 24 hours; Gill et al., 1988) for the parent drug were insufficient to produce an adequate lowering of BP of prolonged duration. However this conclusion is contrary to that from other studies with CRK (Vandenburg et al., 1986, 1987) and LCRK (Jain et al., 1991; see Hamilton et al., 1993) which showed that once daily oral administration (in the range 0.5 to 3 mg for LCRK) lowered supine DBP for at least 24 hours. In particular, in the double-blind, randomised, placebo controlled, parallel group study performed with LCRK, single daily doses of 0.5, 0.75, 1 and 1.5 mg were given daily for 8 weeks and produced significant decreases from baseline in trough supine DBP, without change in heart rate (HR) in all groups (see Hamilton et al., 1993) (Figure 16.1). Thus the pharmacodynamic and pharmacokinetic properties of this drug appear to be adequate for once daily treatment in mild to moderate hypertensive patients. Interestingly, the only other benzopyran, celikalim, tested so far in hypertension, has an exceptionally long plasma half-life (about 15 days) (Lasseter et al., 1992). As may be predicted from a direct vasodilator action, the KCAs cause reflexly mediated increases in HR, cardiac output and stroke volume (Thomas et al., 1990;

Figure 16.1 Mean supine diastolic blood pressure (mmHg) at baseline (

Fox et al., 1991; Senior et al., 1993). Indeed studies with oral bimakalim in volunteers, and i.v. CRK in IHD patients, suggested that KCAs may have

464 K CHANNELS AND THEIR MODULATORS

beneficial effects on cardiac function in patients with compromised left ventricular function or IHD. However, in the treatment of hypertension, an increase in HR is undesirable at BP lowering doses. Experiences with antihypertensive doses of CRK and LCRK in patients indicated that increases in HR are modest after single doses, and not an issue in chronic studies (Vandenburg et al., 1987; Erwteman et al., 1991). The long term effects of LCRK on cardiac mass remain to be determined but, in the case of pinacidil (Steensgaard-Hansen and Carlsen, 1988), left ventricular mass was reduced (−23%) in hypertensive patients receiving concomitant bendrofluazide treatment. Thus good prospects exist that KCAs will not exacerbate left ventricular hypertrophy. Another possible consequence of the use of directly-acting arterio-vasodilators such as LCRK and pinacidil, is reflex counter-regulatory stimulation of the reninangiotension-aldosterone system with the attendant risks of elevated plasma renin activity (PRA) and angiotensin II levels leading to peripheral oedema and, if severe, to increases in body weight due to Na retention. Although raised PRA has been reported following.CRK in volunteers (Ferrier et al., 1989; Lijnen et al., 1989a,b; Singer et al., 1989), this parameter has not been extensively studied in hypertensive patients receiving this KCA. Singer et al. (1989) found that single oral doses of CRK raised PRA, without altering plasma aldosterone, in both volunteers and hypertensive patients, but Lebel et al. (1988) found no change in PRA in a short-term daily dosing study in patients. In longer term studies, peripheral oedema has been reported after CRK and LCRK but without increased body weight (Vandenburg et al., 1987; Ertwemann et al., 1991; see Hamilton et al., 1993). Support for these findings is provided by lack of an effect of CRK on renal haemodynamics in short-term studies in patients (Singer et al., 1989; Lebel et al., 1991). However these results now need to be confirmed in longer term studies with LCRK. Most interestingly, CRK, LCRK and pinacidil have a beneficial effect on plasma lipid profile in hypertensive patients. First reported for pinacidil (see Friedel and Brogden, 1990) and CRK (Lacourciere et al., 1989), LCRK has also been shown to lower plasma triglycerides and total cholesterol, and to raise HDL cholesterol, when given in single daily doses, in the range of 0.5 to 1.5 mg, for 8 weeks (see Hamilton et al., 1993). Changes in high density lipoprotein (HDL) cholesterol and triglycerides were greater in a sub-set of patients with elevated baseline cholesterol (― 6.5 mmol/1) (Figure 16.2). Similarly, in Japanese hypertensive patients, LCRK (0.5 or 1 mg daily for 12 weeks) significantly lowered total cholesterol, triglycerides and apoproteins (Sasaki et al., 1994b). In a double-blind, comparator 12 week study, Japanese hypertensive patients were randomised to receive either LCRK (0.5, 1.0 mg) or the β 1 blocker, doxazosin (0. 5, 1.0, 2.0, 4.0 mg) (Sasaki et al., 1994a). The dose was titrated such that both drugs reduced DBP from 101 to 85 mmHg. Baseline total cholesterol levels were >250 mg/dl in both groups. Doxazosin had no significant effects on serum lipids whereas LCRK decreased total, VLDL, LDL and HDL3 cholesterol and triglycerides and increased HDL2 cholesterol. Between group differences were

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 465

detected with increases in HDL2 cholesterol and decrease in LDL/HDL. The beneficial changes in lipid profile produced by LCRK and pinacidil, distinguishes KCAs from most other classes of anti-hypertensive drugs (see Table 16.2) and, in the long term, may suggest a modifying effect on the development of atherosclerosis. Thus KCAs have the potential to modify two cardiovascular risk factors. However, in view of the prevalence of the use of combination therapy in the treatment of hypertension, it will be important to demonstrate that the changes in lipid profile produced by KCAs persist in the presence of concomitant low-dose diuretic and/or β -blocker treatment. In contrast to the benzopyran KCAs, the pharmacodynamic properties of pinacidil in the treatment of mild to moderate hypertension have been studied extensively (see Goldberg, 1988; Friedel and Brogden, 1990; Longman and Hamilton, 1992). The consensus view has emerged that this KCA is efficacious and exhibits both the pharmacodynamic and adverse event profile typical of a vasodilator antihypertensive agent. Overall pinacidil is associated with a high incidence of oedema, leading to increased body weight, and these effects have led to the recommendation from the FDA that pinacidil should be co-prescribed with a diuretic. Pinacidil is also associated with an incidence of hypertrichosis, possibly related to its mechanism of action as a KCA. Minoxidil sulphate, another KCA, is already marketed as a local application to treat hypotrichosis (see section 16.3.5). However, neither CRK nor LCRK have been associated with a single case of hypertrichosis. The short plasma half-life (2–3 hours) of pinacidil led to the development of a sustained release preparation, but twice daily dosing is still required (in contrast to once daily with LCRK). The new formulation of pinacidil may also have the advantage, by allowing the slower development of the maximum plasma levels of parent drug, of minimising reflexly-mediated counter-regulatory haemodynamic effects. Despite earlier reports of tolerability problems with pinacidil, a recent ‘Quality of Life’ study (Fletcher et al., 1992) presented a favourable outcome for pinacidil in

466 K CHANNELS AND THEIR MODULATORS

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 467

Figure 16.2 Effects of levcromakalim on plasma lipids following 8 weeks administration with single daily doses of 1.0 and 1.5 mg. Changes are % from the baseline established before administration and are shown for all patients and for a subset of hypercholesterolemic patients (total cholesterol >6.5 mmol/before treatment). (Reproduced from Poyser and Hamilton (1994) with permission). Table 16.2 Anti-hypertensive agents: changes in plasma lipid profile Class

Triglycerides

Total Chol.

LDL-Chol.

HDL-Chol.

HDL:Total

KCA

――

4

―

――

――

CCB

o

o

o

o

o

ACE-I

o

o

o

―

o

β 1-blocker

―

―

―

―

―

β -blocker

―

o

o

―

―

Diuretic

―

―

―

―

―

KCA=potassium channel activator; CCB=calcium channel blocker; ACE-I = angiotension converting enzyme inhibitor Chol.=Cholesterol; LDL=Low density lipoprotein; HDL=High density lipoprotein ― Decrease ― No change ― Increase

comparison with the dihydopyridine Ca channel blocker, nifedipine, in hypertensive patients also receiving a thiazide diuretic. Both drugs were given as sustained release formulations but unfortunately no placebo group was included in this study. Both drugs caused similar falls in supine DBP at 6 weeks, the target level being achieved in 57% and 63% of patients taking pinacidil and nifedipine respectively. Of the few differences noted between the drugs in this study, pinacidil increased body hair growth and nifedipine caused facial flushing in some patients. Recently, Buoninconti et al. (1993) found, in a 10 week study, comparable effectiveness (Figure 16.3) and tolerability for pinacidil and the ACE-inhibitor, captopril, whether given as monotherapy or combined with hydro-chlorthiazide, to achieve the target BP. Patients maintained on pinacidil alone for 6 months did not show increases in body weight. The mechanism of action of KCAs has inevitably led to questions regarding their-possible inhibitory effects on insulin secretion since this glucose-stimulated event is mediated via K channel closure in pancreatic β -cells. However, both in vitro and in vivo animal studies do not indicate a potential problem in the clinic with regard to either insulin release per se or interference with the ability of the sulphonylurea drugs (such as glibenclamide) to evoke insulin release (see Chapter 13). Recent clinical data obtained with pinacidil (up to 25 mg daily) has shown that, in healthy volunteers (Nielsen-Kudsk et al., 1990) and hypertensive patients (Ligtenberg et al., 1993) (Figure 16.4), the increases in fasting blood

468 K CHANNELS AND THEIR MODULATORS

insulin and glucose during oral and i.v. glucose tolerance tests were unaffected by the KCA. These findings therefore support those from animals and other evidence (see Chapter 13), that K channels in the vasculature and pancreatic β -cells are not identical. 16.3.2 Asthma The pharmacological properties of KCAs extend to relaxation of airways smooth muscle and to inhibition of bronchoconstriction produced by stimulation of nonadrenergic, non-cholinergic excitatory nerves to guinea-pig lungs (see Chapter 12). The latter nerves may be involved in releasing inflammatory mediators in airways. Limited clinical data exist to support the potential use of KCAs as bronchodilators but none exist so far for their anti-inflammatory activity in airways.

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 469

Figure 16.3 Supine blood pressure and heart rate (HR) during 10 weeks of treatment with pinacidil or with captopril, in monotherapy or in combination with hydrochloro-thiazide. (Values are expressed as mean±SEM [I].) *p<0.05 versus baseline; p<0.01 between times. SBP/DBP=systolic/diastolic blood pressure ; HR= heart rate . (Reproduced from Buoninconti et al. (1993) with permission).

An early study in healthy volunteers with oral CRK (2 mg) (Baird et al., 1988), in which inhibition of histamine-induced bronchoconstriction was reported, and knowledge of its prolonged plasma half-life (Gill et al., 1988), led to evaluation of this KCA in patients suffering from nocturnal asthma (Williams et al., 1990a). CRK (0.25 and 0.5 mg given at bed-time), attenuated nocturnal

470 K CHANNELS AND THEIR MODULATORS

bronchoconstriction (‘morning dipping’) as judged by measurement of forced expiry volume (FEV1) following the 5th dose. Although some headache was reported, CRK generally had no effect on BP and HR in normotensive asthmatics. Additionally this drug was well

Figure 16.4 Mean (SEM) plasma glucose (upper panel), serum insulin (middle panel) and C-peptide (lower panel) levels during ivGTT in 10 hypertensive subjects; closed symbols are with, and open symbols are without pinacidil. (Reproduced from Ligtenberg et al. (1993) with permission).

tolerated in healthy volunteers when given by inhaling nebulised solutions containing 0.05 to 1 mg CRK (Williams et al., 1990b), Thus considerable optimism surrounded the initial development of CRK as a novel bronchodilator.

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 471

However, the outcome of subsequent evaluation of LCRK in asthmatics, proved less convincing regarding the positive effects of KCAs in this patient population. Thus, Kidney et al. (1993) found that histamine—and methacholineinduced bronchoconstriction were not inhibited 5 hours following 0.5 mg oral LCRK, and a high incidence of headache was reported. In this study, salbutamol (8 mg) was effective against histamine challenge. In a repeat once daily dose study (Picot and de Vernejoul, 1991), LCRK (0.125–0.75 mg orally for 28 days) produced a small beneficial effect on FEV1 at 4, but not 16, hours after the highest dose. A large percentage of these patients were taking concomitant inhaled steroids and therefore this study represented a serious challenge of the efficacy of LCRK. To date these authors are unaware of any clinical studies in asthmatics with KCAs such as pinacidil, HOE 234 and BRL 55834 (see also Chapter 12). The conclusions from published studies with CRK and LCRK in asthmatics suggest that a greater degree of selectivity for airways versus other smooth muscle is required in order to enhance efficacy and reduce systemic cardiovascular effects. This goal may be achievable by meeting two objectives— development of molecules targeted for airways tissue and the K channels present therein. Delivery of drugs by the inhalation route should be most appropriate. In addition, the potential anti-inflammatory activity of KCAs in human airways must be evaluated as a major property of relevance to slowing the pathological progression of the disease. 16.3.3 Urinary Incontinence In vivo evaluation of KCAs in animal models of bladder instability has been relatively limited (see Chapter 14). No evidence was obtained in mini-pigs with urethral obstruction, showing a selective effect of CRK on bladder contractility versus changes in BP and HR. Nevertheless the medical need for novel effective treatment for bladder instability led to preliminary clinical trials using pinacidil and CRK. In a double-blind, cross-over study in 10 patients with detrusor instability secondary to bladder outflow obstruction, pinacidil given at 12.5 mg twice daily for 2 weeks had no significant effect on urodynamic variables but lowered standing BP (Hedlund et al., 1991). In a single blind study, CRK (0.5 mg daily for 14 days, then 1 mg for a further 14 days) had beneficial effects upon mean voided volume and urinary frequency in 17 patients whose bladder dysfunction had not responded to other drugs (Nurse et al., 1991). These sparse clinical findings are sufficiently encouraging to suggest that further evaluation of KCAs in the treatment of bladder instability should be pursued particularly if compounds emerge possessing clear selectivity for detrusor, rather that vascular, smooth muscle.

472 K CHANNELS AND THEIR MODULATORS

16.3.4 Other Cardiovascular Indications Of a number of other potential indications for KCAs in the cardiovascular system (see Table 16.1), the only data available to date stem from a double-blind, controlled trial in which pinacidil, 12.5 and 25 mg single doses, were given to patients suffering from Raynaud’s disease (Dompelling and Smit, 1992). Pinacidil had no beneficial effect on finger blood flow (assessed by plethysmography) during cooling and re-warming or on trans-cutaneous oxygen tension. In contrast, nifedipine, 20 mg, had positive effects. Thus this limited clinical study failed to demonstrate any efficacy of pinacidil in this small group of patients showing symptoms associated with one type of peripheral vascular disease (PVD). However, on the basis of this exploratory trial, it would be premature to conclude that KCAs will not find a place in the treatment of PVD. The remarkable ability of these drugs, in contrast to other types of vasodilators, to improve blood flow and function in ischaemic limbs (see Chapter 12 and Cook et al., 1993) suggests that clinical evaluation is warranted in conditions such as intermittent claudication. 16.3.5 Other Indications As summarised in Table 16.1, no clinical data are available for the effects of KCAs in a number of other potential indications. However, there are two exceptions to this general statement, namely hyperlipidaemia and hypotrichosis. The former has already been described in relation to the outcome of trials in hypertensive patients (see section 16.2.1). For the latter, reports of hypertrichosis as an unwanted side effect in the use of minoxidil to treat hypertension (Campese, 1981) led to the development of a topical application for the treatment of hypotrichosis e.g. male pattern baldness (Clissold and Heel, 1987). However it remains unclear if the K channel activator mechanism of minoxidil sulphate is responsible for this activity. Another KCA, namely pinacidil, also causes a small incidence of hair growth in hypertensive patients (see section 16.2. 1). 16.4 Future Prospects for KCAs CRK was discovered as a vasodilator anti-hypertensive drug with a KCA mechanism in the mid-1980s (see Longman and Hamilton, 1992). Unlike nicorandil, a nicotinamide-ester which acts by a dual mechanism of KCA and elevating cGMP levels in smooth muscle cells, CRK appears to act specifically at K channels. Moreover nicorandil, unlike CRK, is non-selective as both an

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 473

arterial and venous dilator and as such is used clinically to treat angina pectoris (Krumenacker and Roland, 1992; Purcell and Fox, 1993) and shows promise in congestive heart failure (Galié et al., 1990: Tice et al., 1990) and percutaneous transluminal coronary angioplasty (Saito et al., 1992). Although i.v. CRK had favourable cardiac haemodynamic effects in patients with IHD (Thomas et al., 1990), the lack of effect of CRK on venous distensibility has precluded its further evaluation in angina pectoris or in congestive heart failure. Moreover this KCA, like pinacidil, produces systemic vasodilation and falls in BP, rather than selective changes in the coronary circulation. However, evidence that KCAs such as pinacidil and CRK exhibit anti-ischaemic properties in the heart, suggests a possible beneficial effect as cardioprotectants (see Chapter 11). This activity is probably due to a direct action of KCAs on cardiac KATP channels and may mimic the opening of these channels in ischaemic conditions as a homeostatic protective mechanism (Escande and Henry, 1993). Thus the potential exists for drugs like LCRK, pinacidil and aprikalim to exhibit anti-ischaemic properties in the heart. Such activity, if also shown at anti-hypertensive doses, would confer a useful additional property on the KCA class as novel anti-hypertensive agents. Extensive evaluation of CRK, LCRK and pinacidil in the treatment of mild to moderate hypertension has revealed the haemodynamic profile and tolerability issues associated with other types of vasodilator anti-hypertensive agent. The efficacy and tolerance of the benzopyran KCAs, as typified by LCRK, seem to be superior to that shown by pinacidil and may be related to differences in pharmacokinetic profile and changes in regional blood flow. Both LCRK and pinacidil share an ability to modify plasma lipid profile in an advantageous manner and this suggests a common mechanism of action possibly related to K channel activation. Thus these drugs modify two cardiovascular risk factors. Comparison with the known effects on lipids of other classes of antihypertensive agent (Table 16.2) show that KCAs possess a more advantageous profile than β -blockers, diuretics, ACE-inhibitors and Ca channel blockers whilst, in broad terms, β 1-blockers cause similar changes in lipids as KCAs. Extensive clinical trials are now required to show if KCAs modify the development of atherosclerotic lesions in man. In this regard both nifedipine and nicardipine have been found to retard the progression of the disease without benefit to pre-existing disease in patients with coronary artery disease (see Fisher and Grotta, 1993). Thus despite the fact that, unlike KCAs, these Ca channel blockers do not modify lipid profile, some beneficial effect has been observed in patients with coronary heart disease. However, data for isradipine from the MIDAS trial in hypertensive patients did not show inhibition in progression of carotid artery atheroslerosis after 12 months treatment (Anonymous, 1994). Another issue concerning the use of KCAs to treat hypertension relates to their positioning as mono-therapy or as adjuncts to other treatment. The major consideration in this regard relates to reflex activation of counter-regulatory haemodynamic mechanisms. Some trials indicate that KCA monotherapy is

474 K CHANNELS AND THEIR MODULATORS

satisfactory treatment but others suggest that combination with a diuretic and/or β -blocker may be appropriate. Combination of a KCA with an ACE-inhibitor does not appear to have been tested in the clinic despite the logic in this approach as a means of minimising the effects of elevated PRA and angiotensin II. Certainly a low dose combination of a KCA with any of these classes offers the prospect of enhanced efficacy and improved tolerability when compared with either drug given alone. As mentioned earlier, KCAs have not been extensively examined in other C.V. indications such as PVD, cerebral vasospasm, CHF or angina. The antiischaemic effects of CRK were first described in ischaemic skeletal muscle in animals (see Chapter 12). Taken together with the cardioprotective properties of KCAs in animal models (see Chapter 11), it seems likely that hypoxia-induced reductions in intracellular levels of ATP may sensitise both ischaemic cardiac and skeletal muscle to the actions of KCAs. In the case of the latter, these drugs therefore may have potential to treat peripheral vascular diseases, such as intermittent claudication, by improving collateral circulation, oxygen availability to the muscle and thereby exercise tolerance. However, clinical trials in this condition are generally difficult. So far no such studies using KCAs have been reported despite experimental evidence that KCAs, at sub-hypotensive doses, selectivity dilate collateral blood vessels in the ischaemic region (see Chapter 12 and Cook et al., 1993). These authors also suggested that a contributory factor towards these effects of KCAs may be their ability to inhibit neuronally mediated vasoconstrictor responses in collateral vessels. In various other disorders of smooth muscle, such as asthma, bladder instability, irritable bowel and uterine conditions such as premature labour, KCAs offer a novel mechanistic approach to the control of muscle contractility. However, it remains to be determined if this K channel activating mechanism is more efficacious in the treatment of these conditions than, for example, Ca channel blockade or β 2-adrenoceptor stimulation. Limited efficacy of CRK in asthma and detrusor instability suggests that further examination of KCAs in these conditions is warranted. However, a key requirement in the profile of any such KCA will be demonstration of tissue and channel specificity for the target smooth muscle and, possibly, its innervation. In this way, cardiovascular side effects should be minimised. The outcome of trials in asthma involving benzopyran derivatives, like BRL 55834 and HOE 234, should be revealing in this regard. Importantly the ability of KCAs to inhibit the release of inflammatory modulators from NANCe nerves in the lungs, indicates potential in modifying the inflammatory processes underlying the progression of asthma. In addition to seeking compounds with tissue selective properties, it should be possible to achieve target organ specifically in vivo by using appropriate drug delivery systems and taking account of pharmacokinetic considerations. Thus, for the lungs, use of inhalation technologies will deliver drugs directly to the target organ. In the case of the bladder, it may be possible to deliver drugs via

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 475

renal excretion direct to the lumen of the bladder. Such compounds would then need to be able to penetrate the epithelial layer to reach the underlying muscle. Interestingly KCAs have a negligible propensity in both volunteers and patients to cause gastro-intestinal side effects such as constipation. It is possible however that, in intestinal conditions of heightened contractility and hypersecretion, KCAs may modulate the underlying abnormality and offer new treatment. In the central nervous system, the potential exists for neuronal selective KCAs to inhibit neuronal excitability and the release of neuro-transmitters. Experimental evidence already exists that benzopyran KCAs such as CRK have anti-epileptic and analgesic properties (see Chapter 15). Retention of such activity in a brain-penetrant KCA targeted towards specific brain regions may offer novel therapeutic approaches and should avoid side effects in the periphery. In addition the anti-ischaemic properties of KCAs in muscle may extend to cerebral ischaemia (see Chapter 9) and provide novel neuroprotective agents. Of the other possible indications for KCAs, their potential as lipid lowering drugs in hypertension has already been discussed (see section 16.2). An understanding of the mechanism of action of KCAs should aid the development of KCAs with enhanced effects on lipid profile. However, in this regard, competition from other classes of lipid lowering drugs will be formidable. A demonstration of the ability of KCAs to modify the progression of atherosclerosis in animal models may stimulate further clinical studies. 16.5 KCBs To date, the therapeutic relevance of blocking K channels has been established in two major areas: the treatment of hyperglycemia by the insulin-releasing sulfonyurea drugs (e.g. tolbutamide), which appear to exert their effect by blocking ATP-sensitive K channels to depolarise β -pancreatic cells, and in the treatment of cardiac arrhythmias by agents that can selectively prolong refractoriness through an action on one or more myocardial K channels. There is also considerable discovery research underway examining whether K channel blockade is a feasible approach to other disorders, such as multiple sclerosis and for immunosuppression, but to date these efforts have not yielded any advanced clinical candidates. The current chapter focuses on the more recent clinical findings with the new ‘pure’ class III antiarrhythmic drugs, particularly the data appearing since 1992 (Table 16.3). Some background is also given on the prototypic class III agents amiodarone and d,l-

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Table 16.3 KCBs in development as class III antiarrhythmic agents Drug

Compound no.

Company

Status

Amiodarone

–

Marketed

D,l-sotalol Sematilide Dofetilide Almokalant D-sotalol – Ibutilide –

MJ–1999 CK–1752 UK–68798 H234/09 – E–4031 U70226E MK–499 (L–706,000) LU–47110 NE–10064 MS–551 – RP–66912

Sanofi Wyeth-Ayerst Berlex Berlex Pfizer Hassle Bristol Myers-Squibb Eisai Upjohn Merck Knoll Proctor & Gamble Mitsui Upjohn Rhône-Poulenc Rorer

Phase I/II Phase I/H Phase I/II Phase I/H Preclinical

Ambasilide Azimilide – Artilide Terikalant

Marketed Pre-registration Phase II/III Phase II/III Terminated Phase II Approved Terminated

sotalol, since these results continue to condition current medical and commercial views of the area. The chapter concludes with a re-examination of the general issues surrounding the class III agents, including ‘reverse use-dependence’ and proarrhythmia, as seen from the clinical perspective. For more information on the other areas where K channel blockade may have a role, the reader is referred to several recent reviews (Cahalan and Lewis, 1990; Ashcroft et al., 1993; Williams 1994). 16.6 Amiodarone Amiodarone is a non-competitive sympathetic antagonist and inhibits cardiac Na and Ca channels in addition to blocking both the rapid (IKr) (Follmer et al., 1990) and slow (IKs) (Balser et al., 1991) components of delayed rectifier K+ current. In prospective clinical trials, amiodarone has been shown to reduce mortality and cardiac events in patients with ischaemic heart disease, particularly in the first year following myocardial infarction (see recent reviews by Nora and Zipes, 1993; Nademanee et al., 1993). In the Basel Antiarrhythmic Study of Infarct Survival (BASIS; n=312) (Burkhart et al., 1990), one year mortality was 5% in patients receiving amiodarone (200 mg/day) versus 12% and 13%, respectively, in patients receiving class I drugs or placebo. After 84 months, amiodarone reduced the probability of death from 45% to 30% (Pfisterer et al., 1993), but

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appeared to be less effective in patients with ejection fractions below 40%. In the ‘Polish Amiodarone Study’ (n=613) (Ceremuzynski et al., 1992), amiodarone reduced cardiac mortality by 42%, and the incidence of serious ventricular arrhythmia from 19.7% to 7.5%. The Cardiac Arrest in Seattle: Conventional versus Amiodarone Drug Evaluation (CASCADE) study found that patients treated empirically with amiodarone had better rates of survival post-infarction and were less likely to require defibrillator shocks than patients receiving class I agents (CASCADE Investigators, 1991). After 2 years, 78% of the amiodarone group remained event-free, compared to 52% on class I drugs. This benefit was still apparent at 6 years, despite discontinuation of therapy in 29% of the patients receiving amiodarone (versus 17% on class I drugs). In a pilot study for the Canadian Amiodarone Myocardial Infarction Arrhythmia Trial (CAMIAT) (n=77), (Cairns et al., 1991), amiodarone dramatically reduced the incidence of arrhythmic sudden death at 2 years from 13.8% to 4.2%, and overall mortality from 20.7% to 10.4% compared to placebo. CAMIAT is being continued using the same patient population and dosing regimen as the pilot study (Cairns et al., 1993). Several other randomised, controlled mortality trials with amiodarone are also underway or have been recently completed, including the European Myocardial Infarct Amiodarone Trial (EMIAT) (Camm et al., 1993), the Canadian Implantable Defibrillator Study (CIDS), (Connolly et al., 1993), CHF STAT (Singh et al, 1993) and GESICA (Dorval et al., 1994). In GESICA, low dose amiodarone (300 mg/day) reduced the risk of sudden death and death due to progressive heart failure, whilst in CHF STAT, amiodarone was without effect on overall survival. 16.7 Sotalol D,l-Sotalol is a non-selective β -adrenoreceptor antagonist that blocks IKr (Carmeliet, 1984). Most of sotalol’s β -blocking activity resides in the 1-isomer. The d-isomer is currently under development as a ‘pure’ class III agent. Controlled clinical studies have established the ability of d,l-sotalol to control supraventricular and ventricular arrhythmias (see review by Singh, 1992), but data from two early secondary prevention trials have failed to support its use in post-infarction patients (Julian et al., 1982; Spielman et al., 1985). In the Electrophysiologic Study Versus Electrocardiographic Monitoring (ESVEM) trial, d,l-sotalol was more effective than six class I agents in suppressing the induction of ventricular arrhythmias by programmed stimulation (35% versus 16%) (Mason et al., 1993). In addition, over a period of 2–6 years, there was a reduced risk of arrhythmia recurrence, all cause mortality, and cardiac and arrhythmic death. However, since ESVEM lacked a placebo control group, one cannot conclude that sotalol produced an absolute reduction in mortality, since the class I agents may have actually worsened survival, as occurred in CAST. No

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significant difference was noted between amiodarone and sotalol in 22 patients with sustained monomorphic ventricular tachycardia during a 1 year follow-up. Both drugs had comparable effects on the tachycardia cycle length, and failure with one drug tended to predict failure with the other (Martinez-Rubio., 1993a, 1993b). The absence of β -blocking activity in d-sotalol should alleviate the potential adverse effects of cardiac depression and marked bradycardia associated with the racemate while retaining class III antiarrhythmic efficacy. Both drugs appear to be similar in their effects on ventricular refractoriness and QT interval, but, as expected, d,l-sotalol had a significantly greater effect on sinus node cycle length than d-sotalol, increasing it by 26% versus 11% for the d-enantiomer. Both compounds showed similar overall efficacy (35–38%) in preventing the induction of ventricular tachycardia. However, a response to one agent did not necessarily predict a response to the other (Brachmann et al., 1992a). The increase in sinus node cycle length produced by d-sotalol is independent of β blockade (Funck-Brentano et al., 1990; Yasuda et al., 1993) and most likely reflects changes in sinus node action potential duration induced by K channel block (Campbell, 1987). In another study, patients receiving amiodarone and dsotalol in combination had outcomes similar to patients receiving only d-sotalol (Weide et al., 1993). D-Sotalol produced a modest reduction in premature ventricular beat frequency in patients on long-term therapy (Brachmann et al., 1992a), and prevented induction of ventricular tachycardia during electrophysiologic study even in patients whose arrhythmias were refractory to other drugs (Brachmann et al., 1993; Koch et al., 1993a 1993b; Weide et al., 1993). Haverkamp et al. (1993) reported the results of a larger study in which 250 patients with either sustained monomorphic ventricular tachycardia or ventricular fibrillation induced by programmed electrical stimulation were treated with oral d-sotalol at 240–640 mg/day. After d-sotalol, sustained ventricular tachycardia or fibrillation was not inducible in 124 patients (49.6%) and became more difficult to induce in another 24 patients (9.6%). Ventricular fibrillation was suppressed more readily than ventricular tachycardia. Responders had a significantly shorter ventricular tachycardia cycle length at baseline compared to non-responders, suggesting that occlusion of an excitable gap may have played an important mechanistic role. At 2 and 4 years of treatment with d-sotalol, the incidences of sudden cardiac death and arrhythmia recurrence in these patients were 12% and 18%, respectively. Data on the clinical efficacy of d-sotalol in atrial arrhythmias remain limited. In one recently published study, d-sotalol was more effective against supraventricular arrhythmias due to AV nodal re-entry (55% response) than AV re-entry (22% response) (Brachmann et al., 1992a). Despite the demonstration of clinical antiarrhythmic benefit, few data support a positive effect of d-sotalol on survival. The Survival on Oral D-Sotalol trial (SWORD) was recently terminated because patients on d-sotalol had a nearly twofold greater risk of dying post-infarction than did untreated controls (3.9%

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versus 2.0%), suggesting that antiarrhythmic efficacy may not necessarily translate into a longer-term survival benefit, at least in this patient population. 16.8 Sematilide Sematilide, a methanesulphonamide analogue of procainamide, appears to be a relatively selective, though not very potent, blocker of IKr, and prolongs cardiac action potential duration in a variety of cardiac muscle preparations. The preclinical profile and early clinical data for sematilide have been recently reviewed by Argentieri (1992). In an early i.v. dose ranging study, sematilide transiently suppressed chronic nonsustained ventricular tachycardias, but also produced a significant proarrhythmic response (Wong et al., 1992a). Sematilide (390 mg/day orally) suppressed arrhythmia induction in 42% of 24 patients with clinical ventricular tachycardia or ventricular fibrillation during electrophysiologic testing (Sager, 1992a), with responders showing a larger increase in ventricular effective refractory period (― =33ms; 15%) than non-responders (― =16ms; 7%). Sematilide produced significant increases in atrial (11 %), AV nodal (20%) and right ventricular (12%) refractory periods, with no change in corrected sinus node recovery time, QRS, PR, or HV intervals, or the Wenckebach cycle length (Sager et al., 1993). Antiarrhythmic activity was demonstrated as reductions in the frequency of premature beats, and in the incidence of couplets and nonsustained ventricular tachycandia over a 24 hr period of monitoring. During electrophysiologic testing, ventricular tachycardia could not be induced in 7 patients (26%; full responders), and became more difficult to induce in 4 additional patients (15%; partial responders), yielding an overall efficacy of 41%. 16.9 Dofetilide The preclinical pharmacology and early clinical safety and pharmacokinetics for dofetilide, an extremely potent blocker of IKr, have been reviewed by Rasmussen et al. (1992). Dofetilide is extremely potent and effective in prolonging the QT interval and refractoriness in man when given by either oral (Norregaard-Hansen et al., 1992) or i.v. routes (Tham et al., 1993). The effects on the QT interval at each dose are similar at paced cycle lengths of 450 and 600 msec, whereas the changes in ventricular effective refractory period showed a slight ‘reverse’ ratedependence, becoming smaller at the shorter cycle length (increases of 8.6% versus 12.1%). There were no changes in QRS duration, sinus node cycle length, sinus node recovery time or HV interval, while AV nodal, atrial and ventricular refractory periods were increased, and small but significant increases in sinoatrial conduction time and AH interval were also observed (Fananapazir and Cropp, 1992a). The effects of dofetilide on refractoriness in this study were also

480 K CHANNELS AND THEIR MODULATORS

essentially rate-independent at pacing cycle lengths of 400–600 ms. The class III effects of dofetilide are also seen in the monophasic action potential (Sedgwick et al., 1992), with no significant increase in the dispersion of refractoriness across the right ventricle. Dofetilide also decreased the energy requirements for successful defibrillation (Gremillion et al., 1992). Van Gelder et al. (1993) compared the efficacy of dofetilide (n=10) and flecainide (n=11) in suppressing atrial flutter. Dofetilide given i.v. over 15 min at 4–8 μ g/kg was effective in 7/10 patients, terminating the flutter with only a slight increase in flutter cycle length. Flecainide was much less effective, terminating the flutter in only 1/11 patients after prolonging the flutter cycle from 280 to 420 ms. Dofetilide i.v. suppressed inducible AV nodal re-entrant tachycardia in 21/25 patients. Partial efficacy was seen as a prolongation of the tachycardia cycle length in the patients who remained inducible. Ventricular refractory periods increased dose-dependently, while changes in atrial refractoriness occurred at lower doses than were required to see effects on the ventricle (Connelly et al., 1992). A response rate of 44% was obtained in patients with inducible AV nodal re-entry by Wong et al. (1992b). In another study (Suttorp et al., 1992), 10/19 patients with atrial fibrillation converted to sinus rhythm, as did 4/5 patients with paroxysmal atrial tachycardia. No significant side effects or proarrhythmia were noted in any of these trials. In 49 patients with inducible ventricular tachycardia (Thomsen et al., 1992), dofetilide completely prevented the re-induction of the baseline arrhythmia in 6/ 40 patients at doses producing an electrophysiologic effect. QTc interval and ventricular effective refractory period increased dose-dependently to a maximum of 17% and 16%, respectively, at the highest dose used in this study (15 μ g/kg i.v.). One patient developed a brief, self-terminating episode of torsade de pointes, but there were no other side effects or incidences of proarrhythmia. Fananapazir and Cropp (1992b) studied dofetilide in 16 patients with hypertrophic cardiomyopathy. At baseline, monomorphic ventricular tachycardia was induced in 9 patients and polymorphic ventricular tachycardia in 7 patients. After the intravenous dose, 10 patients were rendered completely non-inducible, while arrhythmias were harder to induce in the remaining 6 patients, indicating a partial response to the drug. In those patients receiving oral dofetilide, no ventricular tachycardia was noted on Holter recordings. With repeat electrophysiological testing, arrhythmias could not be induced in 11/16 patients, and became more difficult to induce in 4 patients. One patient was induced more easily after dofetilide, suggesting a potential proarrhythmic effect. Brachmann et al. (1992b) studied 33 patients with inducible ventricular tachycardia who had previously failed antiarrhythmic therapy. Arrhythmia induction was suppressed in 16/33 patients, and 12/16 of these patients remained well controlled on long term treatment; 3 patients had a recurrence of their ventricular tachycardia and 1 patient died suddenly. One patient developed self-terminating torsade de pointes on the third day of treatment.

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16.10 E–4031 E–4031, a methanesulphonanilide that selectively blocks IKr with an IC50 of approximately 400 nM, is extremely effective in a variety of ventricular and supraventricular arrhythmia animal models. The clinical electrophysiology is consistent with a class III profile, i.e., no change in RR, PR, QRS durations, sinus node recovery time, or AH and HV intervals with significant increases in the corrected QT interval, and right atrial and ventricular refractory periods (Isomoto et al., 1993; Fujiki et al., 1994). The majority of clinical studies have targeted i.v. E–4031 in patients with supraventricular arrhythmias. E–4031 increases atrial effective refractory periods and produces a significant decrease in the incidence of atrial firing (from 67% to 37% after drug), suggesting a possible utility in the treatment of paroxysmal atrial tachycardia (Shimizu et al., 1993a). In patients with supraventricular tachyarrhythmia, E–4031 may reduce the dispersion of atrial refractoriness by prolonging repolarisation at the atrial sites generating repetitive atrial firing. These sites are believed to have shorter effective refractory periods associated with presence of diseased cells (Shimizu et al., 1993b). Fujiki et al. (1994) studied 15 patients with recurrent palpitations and supraventricular tachycardia. E–4031 was without effect in 3/11 patients with accessory pathways and inducible sustained orthodromic AV re-entrant tachycardia. In 3 of 4 patients with paroxysmal atrial tachycardia who had repetitive atrial firing, E–4031 abolished the irregular discharges in 1 patient, and prolonged the duration of induced right atrial firing in the remaining 2. In patients with pre-excitation syndrome, E–4031 significantly prolonged the refractory period of the antegrade accessory pathway. No significant proarrhythmia or non-cardiac effects were noted during any of these trials. Data for E–4031 in ventricular arrhythmias are more limited. Nagasawa et al. (1993) compared E–4031 with disopyramide and the investigational class III agent MS–551 (Mitsui) for their ability to suppress premature beats arrhythmias. E–4031 and MS–551 were effective against arrhythmias that were reduced by increasing heart rate, and reverse use-dependence was seen. The dispersion of the QTc interval was slightly reduced by both class III drugs, while QTc interval was prolonged. In another study (Okada et al., 1992), E–4031 showed marginal efficacy (10% response) compared to results obtained with class la (73%), Ib (60%) and Ic (100%) agents, and increased the premature beat frequency in 2 patients. These findings are consistent with the reduced efficacy of E–4031 and other class III agents in animal models of automatic arrhythmia. 16.11 Almokalant Almokalant possesses a chemical structure distinct from most of the other K channel blockers under development as class III antiarrhythmic drugs. Although

482 K CHANNELS AND THEIR MODULATORS

it lacks the methanesulphonamide substitution common to compounds like sotalol, dofetilide and E–4031, almokalant appears to have a similar mechanism of action, i.e., it prolongs cardiac repolarisation by selectively blocking IKr. Infusions of almokalant increased paced QT by 24%, with changes in QT morphology (Darpö et al., 1992). Monophasic action potential duration (APD) measured at 90% repolarisation increased by 20% (51 ms), atrial refractory period by 18% (35 ms), and ventricular refractory period by 16% (38 ms). No changes were noted in PR, HV, QRS intervals or AV nodal refractoriness, and there were no consistent changes in spontaneous HR or BP. Darpö and Edvardsson (1994) found a dose-dependent increase in the frequency of arrhythmia termination and induction in patients with paroxysmal supraventricular tachycardia. One patient developed torsade de pointes at the highest dose tested. In a randomised placebo controlled double-blind study in 10 postinfarction patients with more than 720 premature beats or greater than 10 episodes of symptomatic nonsustained ventricular tachycardia on a 24 hr Holter monitor, i.v. almokalant increased the QTc interval significantly in 9/10 patients (Wiesfeld et al., 1992). No change in signal averaged EKG was seen. In 6 patients, the coupling interval between the sinus beat and a premature ventricular beat increased from 592 to 719 ms at the end of the infusion, and there was an associated decrease in the number of premature beats during the first 15 min after infusion. One patient developed a self-terminating episode of torsade de pointes. 16.12 Ibutilide Ibutilide, a racemic n-heptyl substituted analogue of sotalol, is reputed to prolong refractoriness by activating a slow inward Na+ current at sub-nanomolar concentrations, and an outward K+ current at 100–fold higher concentrations. Because of this dual mechanism of action, APD increases at low concentrations and shortens at high concentrations, producing a bell-shaped dose-response curve that may lead to a greater margin of safety in the clinic. The bioavailability of ibutilide is extremely low (<5%), due to extensive hepatic first pass metabolism, and the elimination half-life is extremely variable, ranging from 2–10hr (Roden, 1993). Because of this pharmacokinetic profile, ibutilide is being developed by Upjohn primarily for parenteral use, with artilide being positioned as an orally active follow-up candidate with improved bioavailability. DiMarco et al. (1991) found that ibutilide administered intravenously terminated sustained (>17 days) atrial flutter in 9/10 patients. Termination was associated with increases in flutter cycle length and in the QTc interval. One patient developed a polymorphic ventricular tachycardia that required cardioversion 2 min after terminating the flutter. Ellenbogen et al. (1994) studied 98 patients with atrial fibrillation lasting up to 90 days prior to study entry, and 99 patients with atrial flutter. Ibutilide i.v. successfully converted to sinus rhythm

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 483

39/98 patients with sustained atrial fibrillation and 52/99 patients with atrial flutter. During dosing, the QTc increased from 429 to 483 ms (12.5%), with no attendant change in QRS duration. Six patients developed polymorphic ventricular tachycardia, 4 requiring cardioversion, 2 resolving spontaneously. 16.13 MK–499 MK–499 is a potent and selective blocker of IKr (Roden, 1993). Singh et al. (1994) recently reported the electrocardiographic effects of increasing oral doses of MK-499 in 25 patients with stable coronary artery disease (EF>35%); an additional 3 patients with EF<35% received a 2 mg dose or placebo. Peak plasma levels occurred 2 hrs after dosing, and there were no changes in HR, BP, and PR or QRS intervals noted. The QT interval increased significantly after the 2 mg (9. 8%) and 4 mg (12.5%) doses. The effect of MK–499 on QT interval was slightly greater in the 2 patients with left ventricular dysfuncton (― =14.4% at 2 mg). 16.14 Reverse Use-dependence A major issue in the use of class III antiarrhythmic drugs has been that their ability to prolong the cardiac action potential and refractoriness may become severely attenuated at the rapid heart rates that are typical of most tachyarrhythmias. Preclinical studies performed on isolated strips of cardiac muscle or in single cardiac cells indeed suggest that, for most class III agents that act by selectively blocking cardiac K channels, the degree of action potential prolongation is diminished at short cycle lengths and accentuated at long cycle lengths, leading some investigators to suggest that proarrhythmia and lack of efficacy will eventually limit the widespread clinical use of these drugs (e.g., Hondeghem and Snyders, 1990). The question of ‘reverse’ use-dependence has been examined in a large number of early clinical studies. Amiodarone is suggested to be unique among the class III agents because it demonstrates a positive use-dependent effect on action potential duration (Balser et al., 1991; Hondeghem and Snyders, 1990). However, this contention has not been convincingly supported by clinical electrophysiology studies. Watanabe et al. (1992) found that amiodarone increased right ventricular monophasic action potential duration more at short cycle lengths (6%) than at long (3%), whereas the effects of E–4031 appeared to be similar at both rates (9–10%). These results suggest a positive usedependency for amiodarone, but do not necessarily support a ‘reverse’ usedependency for E–4031. Since, at these doses, E–4031 produced nearly twice the effect of amiodarone, a direct comparison between the two agents may not be valid. In another study (Sager et al., 1992b) amiodarone prolonged monophasic action potential duration by similar amounts at cycle lengths of 300–600 ms,

484 K CHANNELS AND THEIR MODULATORS

whereas increases in effective refractory period were greater at slow than at fast rates. In comparison, sematilide exhibited a marked reverse use-dependence, and was completely without effect on monophasic action potential duration at a cycle length of 300 ms (Sager et al., 1992c). Pastor et al. (1993) compared the ratedependent effects of amiodarone and quinidine on effective refractory periods in 25 patients. Refractory periods were longer at cycle lengths of 600 ms versus 400 ms for each drug as well as at baseline, but the degree of shortening seen at the faster rate was greater for quinidine than for amiodarone (9.3% versus 6.7%), suggesting a lesser reverse use-dependent effect. In 21 patients undergoing electrophysiologic testing for sustained ventricular tachycardia, the ability of d-sotalol to prolong ventricular refractoriness was modestly decreased by reducing cycle lengths from 550 ms (19.9%) to 330 ms (13.4%) (Melichercik et al., 1993). Huikuri and Yli-Mayry (1992) also found that the ability of d-sotalol to prolong right ventricular monophasic action potential duration was attenuated at short cycle lengths, while the increases produced by chronic amiodarone treatment were cycle-length independent. D-sotalol increased monophasic action potential duration by 11% and 5% at cycle lengths of 700 and 350 ms, respectively, compared to changes of 12% and 11% at fast and slow rates for amiodarone. In 16 patients with hypertrophic cardiomyopathy (Fananapazir and Cropp, 1992b), dofetilide effects on ventricular effective refractory period were not ratedependent. Sedgwick et al. (1992) measured monophasic action potentials and effective refractory periods at the right ventricular apex and outflow tract in 18 patients with ischaemic heart disease. In these patients, dofetilide increased refractoriness and monophasic action potential duration by 10–16%, with no effect on the dispersion of refractoriness. The changes in action potential duration and refractoriness were also independent of pacing rate. Morgan et al. (1992) generated restitution curves in 7 patients receiving i.v. dofetilide while undergoing electrophysiologic study. Monophasic action potential duration was increased by 47.7 ms (18.0%) at a cycle length of 700 ms and 34 ms (17.2%) at a cycle length of 400 ms. Analysis of the restitution curves revealed that the maximal degree of shortening was unchanged at each frequency (23%). Fairly similar results have been obtained by other investigators using less complete analyses (Bailey et al., 1992; Sedgwick et al., 1992). O’Nunain et al. (1992) found dofetilide increased ventricular monophasic action potential duration and refractory period by similar amounts (18–22%) during steady pacing at 500 msec and with premature stimulation. At 250 msec, the effects were attenuated, and action potential duration increased by only 10%. There was no significant effect of rate on the effects of E-4031 on ventricular refractoriness (Fujiki et al., 1994; Watanabe et al., 1992) or monophasic action potential duration (Kojima et al., 1991).

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 485

16.15 Proarrhythmia The total number of patients exposed to the newer ‘pure’ class III antiarrhythmic agents remains fairly small. It is therefore difficult to assess the general prevalence of proarrhythmia during the course of therapy. Torsade de pointes has been reported for all antiarrhythmic drugs that prolong the cardiac action potential, but appears to be most common with quinidine, a potent and relatively non-selective blocker of various myocardial K channels. It is not known whether specific block of a single type of K channel is intrinsically more or less proarrhythmic, or whether nonselective block of multiple K channel types predisposes the heart to pause-dependent proarrhythmia (Colatsky et al., 1990). Another key question continues to be whether the risk of proarrhythmia is predictable early during the course of therapy, or whether, as in CAST, fatal proarrhythmia will arise later in treatment. From the early data available, it appears that some patients may be overly sensitive to the class III effects of these agents, so that a proarrhythmic response to one agent may predict a proarrhythmic response to another. For example, the presence of torsade de pointes in congestive heart failure patients treated with class I antiarrhythmic drugs was found to predict the appearance of torsade de pointes during treatment with amiodarone (Middlekauff et al., 1993). Alternatively, basic repolarisation abnormalities may exist, e.g., an excessive prolongation of the QT interval during exercise, that can predict a proarrhythmic response in the presence of drug (Weisfeld et al., 1993). A recent study by Archibald et al. (1994) suggests that there also may be gender-related factors involved, with women showing more than a twofold greater risk of developing torsade than men. Kuhlkamp et al. (1993) reported proarrhythmic events with d-sotalol in a study of 67 patients with inducible sustained ventricular tachycardia. Following treatment, induction of ventricular tachycardia was prevented in 29 patients, while 35 other patients remained inducible. Spontaneous episodes of torsade de pointes developed in the remaining 3 patients during initial oral treatment; however, there were no measurable differences in haemodynamic or electrophysiologic effects in patients in whom sotalol was effective, ineffective or proarrhythmic. However, the effects of sotalol were seen at lower doses in the patients developing torsade, and there appeared to be a trend for greater increase in QTc duration, suggesting a greater intrinsic sensitivity of these individuals to the drug. In another study, excessive QTc interval prolongation (from 451 to 808 ms; ― =+80%) in one patient appearing 5 min after the start of dofetilide infusion led to asymptomatic runs of polymorphic ventricular tachycardia and several multifocal premature beats (Tham et al., 1993). Patients exhibited a proarrhythmic response to i.v. sematilide, ranging from a twofold increase in ectopy, to excessive QT prolongation leading to a self-limited (5 beats) polymorphic ventricular tachycardia in 1 patient, and to an episode of torsade de pointes requiring cardioversion in another (Wong et al., 1992a). The patient who

486 K CHANNELS AND THEIR MODULATORS

developed torsade had a QT interval of 400 ms at baseline that increased to 600 ms following dosing, and an intermittent junctional rhythm. The proarrhythmic effect may be idiosyncratic in susceptible patients and not related to overdosage. Weisfeld et al. (1993) have reported in depth on the first case of torsade de pointes in a patient receiving almokalant at a relatively high infusion rate, and the factors that may have predisposed him to the arrhythmia. The patient had suffered an inferior infarction 13 years prior to entry into the study, and his baseline EKG showed premature beats and sinus bradycardia. The duration of the QT interval was within normal limits at rest, but increased abnormally during exercise. Early during the infusion of almokalant, extreme changes in T-wave morphology were observed, which then degenerated into ventricular tachycardia. The plasma levels of almokalant did not appear to be excessively high, and the patient later responded successfully to sotalol without event. No proarrhythmia was seen in 9 other patients given almokalant at a slower rate of infusion (4.5 mg/ 10 min). These results suggest that abnormal responses of the QT interval during exercise, such as characterised in patients with acquired long QT syndrome, may select patients susceptible to torsade de pointes during treatment with a class III agent. A different proarrhythmic mechanism has been described by Crijins et al. (1993a 1993b), who reported that dofetilide produced aberrant conduction in a patient with atrial fibrillation during stimulation of the arrhythmia by rapid pacing. During right ventricular stimulation, isolated bundle branch re-entrant beats were recorded after induction of critical retrograde conduction delays that occurred in the setting of relatively large differences in refractoriness between the right bundle branch and right ventricular myocardium, conditions favouring distal re-entry. 16.16 Summary and Future Directions There are currently a large number of selective blockers of the rapid delayed rectifier current under development as class III antiarrhythmic agents. As a group, they show a high degree of efficacy against both atrial and ventricular tachyarrhythmias, and a relatively low degree of proarrhythmia (<3–5%). Importantly, the proarrhythmia tends to appear early during dosing, suggesting that some patients may be more sensitive than others to the action potential prolonging effects of these compounds and that electrocardiographic clues may help to identify patients at risk. Apart from studies on amiodarone and d,lsotalol, there are no results published from large scale clinical trials examining the effects of these agents on mortality. The SWORD trial (Survival with Oral dSotalol), which examined the survival benefit of d-sotalol in more than 2000 post-infarction patients, was recently stopped due to excess mortality in the treatment group (3.9% versus 2.0% on placebo). As in CAST, however, the placebo death rate in SWORD was quite low. Thus, while there appears to be a

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significant therapeutic benefit from the standpoint of efficacy in programmed stimulation (see Table 16.4) and in the suppression of atrial flutter and fibrillation, and sustained Table 16.4 Comparative efficacya of newer class III agents against induction of ventricular arrhythmia during electrophysiologic testing Investigators

Drug

Number of patients under study

% of patients non-inducible

Martinez-Rubio et Amiodarone 32 22% al. (1993a) D,l–sotalol 25% Mason et al. (1993) D,l–sotalol 486 35% Martinez-Rubio et D,l–sotalol 171 52% al. (1993b) Brachmann et al. D,l–sotalol 28 35% (1992a) D-sotalol 39% Brachmann et al. D-sotalol 56 59% (1992a) Haverkamp et al. D-sotalol 250 50% (1993) Sager et al. (1993) Sematilide 247 26% Thomsen et al. Dofetilide 40 40% (1992) Fananapazir and Dofetilide 16 63% Cropp (1992b) Brachmann et al. Dofetilide 33 49% (1992b) aEfficacy defined as the inability to re-induce the arrhythmia observed at baseline (i.e. ‘full responders’). Excluded are patients in whom either the arrhythmia became more difficult to induce or the tachycardia cycle length increased.

ventricular tachycardia, whether suppression of these arrhythmias results in a true survival benefit is questionable. Because few class III antiarrhythmics selectively acting on K channels other than IKr have moved far forward into clinical development, it is not clear whether one cardiac K channel is preferable to another as a target for drug action. Molecular biology has begun to identify new K channels that may be expressed in a tissue specific manner (i.e. atrial versus ventricular). Drugs targeting, for example, a K channel expressed only in atrial cells may be highly effective in the treatment of supraventricular arrhythmias and devoid of any potential to disturb ventricular activity or produce torsades. The feasibility of such an approach remains to be demonstrated. To date, compounds acting on currents other than (or in addition to) IKr have not shown improved safety or efficacy in preclinical models. Nevertheless, from the standpoint of basic research, the availability of more specific agents should help to make possible a rigorous analysis of cardiac K channel subtypes within the

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heart, and thus lead to a clarification of the role of these channels in the genesis and/or control of cardiac arrhythmias, which can be used subsequently as a basis for designing new therapeutic strategies. Acknowledgements One of us (TCH) wishes to thank Joanna Jones for considerable assistance with literature searches and Teresa Berney and Emma Shaw for much patience and skill in typing the manuscript. Gratitude is also extended to June Rothstein for administrative and editorial support. References ANDERSSON, K. (1992) Pharmacol. ToxicoL, 70, 244–254. ANONYMOUS (1994) Scrip 5/8 April 22. ARCHIBALD, D.G., QUART, B., HARDY, S., MACNEIL, D.J. & LEHMANN, M.H. (1994) J. Am. Coll. Cardiol., 23, 273A. ARGENTIERI, T.M. (1992) Cardiovasc. Drug Rev., 10, 182–198. ASHCROFT, S.J., NIKI, I., KENNA, S., WENG, L., SKEER, K., COLES, B. & ASHCROFT, F.M. (1993) Adv. Exp. Med. Biol., 334, 47–61. ATWAL, K.S. (1992) Med. Res. Rev., 12, 569–591. BAILEY, W.M., NADEMANEE, K., EDWARD, J., DI PASQUALE, J., ADAIR, O. & FRIEDRICH, T. (1992) Circulation, 86 (4), 1–265. BAIRD, A., HAMILTON, T.C., RICHARDS, D.H., TASKER, T. & WILLIAMS, A.J. (1988) Br. J. Clin. Pharmacol., 25, 114P. BALSER, J.R., BENNETT, P.B., HONDEGHEM, L.M. & RODEN, D.M. (1991) Circ. Res., 69, 519–529. BEERAHEE, A., TAYLOR, A.C., KAYE, C.M., DIERDORF, H.D. & ECKL, K. (1992) Br. J. Clin. Pharmacol., 35, 8IP. BRACHMANN, J., BEYER, T, SCHMITT, C., SCHOLS, W., MONTERO, M., HILBEL, T., SCHWEIZER, M. & KUBLER, W. (1992a) J. Cardiovasc. Pharmacol., 20, S91-S95. BRACHMANN, J., HAVERKAMP, W., JOHNS, J., ZEHENDER, M., KINGMA, H.G. & WlECHA, J. (1992b) Circulation, 86 (4), 1–265. BRACHMANN, J., BEYER, T, SCHOLS, W., SCHMITT, C., ENDERS, B. & MONTERO, M. (1993) Eur. Heart J., 14, 89. BUONINCONTI, R., CAGLI, V., BOSSINI, A., DI VEROLI, C., DE CESARIS, R., RANIERI, G., RAPPELLI, A. & DESSI’FUGHERI, P. (1993) Curr. Ther. Res., 53, 638–647. BURKHART, R, PFISTERER, M., KIOWSKI, W., FOLLATH, F. & BURCHHARDT, D. (1990) J. Am. Coll. Cardiol., 16, 1711–1718. CAHALAN, M.D. & LEWIS, R.S. (1990) Semin. Immunol., 2, 107–117. CAIRNS, J.A., CONNOLLY, S.J., GENT, M. & ROBERTS, R. (1991) Circulation, 84, 550–557. CAIRNS, J.A., CONNOLLY, S.J., ROBERTS, R., GENT, M. & the CAMIAT Investigators. (1993) Am. J. Cardiol, 72, 87F–94F.

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KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 493

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Recent Literature FAURSCHOU, P., MIKKELSEN, K.L., STEFFENSEN, I. & FRANKE, B. (1994) The Lack of Bronchodilator Effect and the Short-term Safety of Cumulative Single Doses of an Inhaled Potassium Channel Opener (Bimakalim) in Adult Patients with Mild to Moderate Bronchial Asthma. Pulmon. Pharmacol., 7, 293–297. KOMERSOVA, K., ROGERSON, J.W., COMWAY, E.L., LIM, T.C., BROWN, D.J., KRUM, H., JACKMAN, G.P., MURDOCH, R. & Louis, W.J. (1995) The Effect of Levcromakalim (BRL 38227) on Bladder Function in Patients with High Spinal Cord Lesions. Br. J. Clin. Pharmacol., 39, 207–209.

Abbreviations

ADP AHP AMI AMP AMP-PCP AMP-PNP 4–AP APD ATP [ATP]i ATP― S BASIS BKCa BP Bt2–cAMP [Ca2+]i [Ca2+]e CAMIAT cAMP CASCADE

CAST cDNA CDP cGMP CGRP

adenosine 5’-diphosphate after hyperpolarisation austin method 1 adenosine 5’-monophosphate adenylyl(β , β -methylene)-diphosphonate 5’-adenylyl- imidodiphosphonate 4-aminopyridine action potential duration adenosine 5’-triphosphate intracellular ATP concentration adenosine–5’-O-(3-thiotriphosphate) Basel Antiarrhythmic Study of Infarct Survival large-conductance (big) Ca-activated K channel blood pressure dibutyryl-cAMP intracellular Ca2+ concentration extracellular Ca2+ concentration Canadian Amiodarone Myocardial Infarct Arrhythmia Trial adenosine 3’,5’ cyclic monophosphate Cardiac Arrest in Seattle: Conventional versus Amiodarone Drug Evaluation Cardiac Arrhythmia Suppression Trial complementary deoxyribonucleic acid cytosine diphosphate guanosine 3’,5’ cyclic monophosphate calcitonin gene-related peptide

ABBREVIATIONS 495

CHF CIDS CNS CRK DAD DAST DBF DCC d.e. DiBAL DMF DMSO DPDPE DTX EAD ECG EF EGTA EJP EK EKG EMIAT EPSP ERP ERPF ESVEM ET–1 FDG FEV1 GDP GTP GTT 5–HD HDL HR IC50 i.c.v.

congestive heart failure Canadian Implant Defibrillator Study central nervous system cromakalim delayed after depolarisation diethylaminosulphur trifluoride diastolic blood pressure dicyclohexylcarbodiimide diastereomeric excess diisobutylaluminium hydride dimethyl formamide dimethyl sulphoxide [D-Pen2,5]–Enkephalin dendrotoxin effective after depolarisation electrocardiogram (EKG) ejection fraction ethyleneglycol bis(aminoethylether)tetraacetic acid excitatory junction potential potassium equilibrium potential see ECG European Myocardial Infarct Amiodarone Trial excitatory post-synaptic potential effective refractory period effective renal plasma flow electrophysiologic study versus electrocardiographic monitory endothelin–1 fibroblast growth factor forced expiratory volume in 1 sec. guanosine 5’—diphosphate guanosine 5’—triphosphate glucose tolerance test 5–hydroxydecanoate high density lipoprotein heart rate concentration causing 50% inhibition intra-cerebroventricular (administration)

496 ABBREVIATIONS

IDP IHD IA IK

IKt I K(Na) IK(Ca) IK(Ach) IK(AA) IK(PC) IK(ATP) Imet IPSP i.v. K (channel) [K+]e KATP KCA KCB KCM KCO kDa Kv LCRK LDL LV dP/dt

mCPBA MCD peptide MIDAS MNDO MOPAC

inosine 5’—diphosphate ischaemic heart disease transient outward K+ current ion current in a corresponding K channel: includes IKs, IX2 slowly inactivating, outward K+ current; IKr, IXl rapidly inactivating, inward rectifying K+ current; IRAK, IKur rapidly inactivating, outward rectifying K+ current inward (or anomalous) rectifier K+ current also known as IR, IAR, IQ, IH in different tissues Na+ activated K+ current Ca++ activated K+ current Acetylcholine activated K+ current Arachidonic acid activated K+ current Phosphatidylcholine activated K+ current ATP Sensitive K+ current Metabolically sensitive K+ current inhibitory post-synaptic potential intravenous (administration) potassium (channel); for type, see current I description above extracellular K+ concentration ATP-sensitive K channel potassium channel activator (opener) potassium channel blocker potassium channel modulator potassium channel opener (activator) kilodalton delayed (outward) rectifier K channel levcromakalim low density lipoprotein first derivative of left ventricular pressure with time (contractile force) meta-chloro perbenzoic acid mast cell degranulating peptide Multicentre Isradipine Diuretic Atherosclerosis Study modified neglect of differential overlap a computer program

ABBREVIATIONS 497

mRNA NAD NADH NADP NADPH NANCe NDP NBS NMR pA2

PCO PGDF PKA PMA Po PR PVC PVD QRS SAR SEJP SHR SKca SAMP SPARTAN STOC SWORD TEA THF THP TMEDA TMSI UDP UTP VF

messenger ribonucleic acid nicotinamide adenine dinucleotide reduced form of NAD nicotinamide adenine dinucleotide phosphate reduced form of NADP non-adrenergic, non-cholinergic excitatory nucleotide diphosphate N-bromosuccinimide nuclear magnetic resonance negative log of the molar concentration of competitive antagonist causing a two-fold rightward shift of the agonist dose-response curve potassium channel opener platelet-derived growth factor cAMP-dependent protein kinase phorbol myristate acetate open probability of a channel interval in ECG (EKG) premature ventricular complexes peripheral vascular disease interval in ECG (EKG) structure-activity relationship non-synchronous spontaneous junction potential spontaneously hypertensive rat small-conductance Ca-activated K channel (S)–(–)–1–amino–2–(methoxymethyl)pyrrolidine a computer program spontaneous transient outward current Survival With Oral d-sotalol (trial) tetraethylammonium (ion) tetrahydrofuran tetrahydropyranyl tetramethylethylene diamine tetramethylsilyl iodide uridine 5’—diphosphate uridine 5’—triphosphate ventricular fibrillation

498 ABBREVIATIONS

VFT VIP VLDL Vmax VMH VOL VSMC

ventricular fibrillation threshold vasointestinal peptide very low density lipoprotein maximum upstroke velocity ventromedial hypothalmic neurones Voltage operated Ca Channel vascular smooth muscle cells

Index of Compounds

(-)-S-gliflumide 102 β -bungarotoxin 365 125I-glyburide 100 14C-iodoantipyrine 201 35S-minoxidil sulfate (S-MxS) 176 3H-BAY-X–9228 176 3H-dofetilide 226 3H-glibenclamide (glyburide) 100, 201 3H-minoxidil sulfate (H-MxS) 176 3H-P1075 176 4–aminopyridine (4–AP) 104, 115, 157, 246, 318, 336, 340, 353, 366, 370, 375 5–hydroxydecanoate (5–HD) 247, 258 5–iodo–2–hydroxy-glyburide 315 alinidine 103 almokalant 109, 112, 228, 231, 242, 243, 396, 401 amantidine 317 ambasilide 114, 228, 396 aminoacridine 317, 376 amiodarone 107, 221, 234, 246, 395, 396, 402, 405 antazoline 104, 185, 316 apamin 99, 340, 364, 365 aprikalim (RP 52891) 34, 57, 58, 59, 90, 91, 168, 175, 197, 201, 203, 204, 212, 214, 249, 258, 262, 263, 265, 266, 268, 269, 278, 281, 368, 372, 374, 384, 393 aprindine 221 artilide 396 AZ-DF 265 102, 182 azimide 396

bimakalim 31, 43, 198, 199, 200, 205, 206, 212, 214, 249, 268, 269, 281 BMS 180447 37 BMS 180488 37 BRL 31660 104, 105, 166, 167 BRL 49074 282 BRL 55834 50, 51, 81, 277, 278, 279, 281, 282, 287, 290, 392, 395 BW755C 214 captopril 389 carbamazepine 374 carbutamide 100, 101 celikalim 32, 51, 198, 249, 268 charybdotoxin 99, 157, 174, 177, 261, 324, 340, 353, 354, 364 chlorpropamide 100, 101 cibenzoline 317 ciclazindol 104, 105, 185 CK–1649 106, 108, 110 CK–3579 108, 111 clofilium 106, 107, 108, 113, 228, 239 cromakalim 1, 2, 4–6, 8–10, 15, 18, 27–32, 34–44, 46, 49, 57, 58, 60, 69, 72, 74, 75, 79, 80, 85–88, 95, 103, 168, 175, 180– 185, 199–201, 203–209, 212, 214, 215, 222, 248–250, 260, 261, 267–270, 277– 284, 286–290, 293–295, 297, 319–321, 336, 337, 342, 343, 366, 371, 372, 374– 376, 383–387, 390–395 dendrotoxin 99, 136, 365 diaminopyridine 370 diazoxide 102, 103, 175, 186, 203, 206, 249, 294, 318, 319, 320, 366, 374, 376

Bay K 8644 215, 250, 356 Bay x9227 366, 367 417

499

500 INDEX OF COMPOUNDS

diltiazam 208 dimethylsulfoxide (DMSO) 148 disodium cromoglycate 275 disopyramide 107, 221, 241, 317 dofetilide 107, 109, 111, 227, 229, 231, 242, 243, 396, 399, 403 doxazosin 387 E–4031 107, 111, 227, 229, 242, 261, 400, 402 eferoxan 316 emakalim (EMD 56431) 31, 261 EMD 52692 198, 214 EMD 57283 40, 43 encainide 107, 109, 222 endosulfine 370 fenoterol 276 flecainide 107,109, 222, 239 FR119748 37 galanin 309 glibenclamide (glyburide) 36, 37, 60, 100, 101, 157, 163, 165, 168, 179, 182, 201, 212, 246, 247, 258, 259, 261, 262, 294, 308, 314, 315, 337, 343, 344, 355, 368, 369, 371, 374, 375, 389 glibornuride 100, 101, 182 gliclazide 100, 101, 182 glipizide 100, 101, 182, 309, 314, 372 gliquindone 100, 101, 103, 182, 374 glisindamide (HOE 036) 100, 101 glisoxepide 100, 101, 182 glymidine 102, 182 GR–38032 113 guanabenz 185

isosorbide dinitrate 264 isradipine 187, 394 KC–399 34, 43, 45 ketanserin 234, 250 ketotifen 275 KP–294 37 KRN 2391 74, 201, 206, 249, 261 L–691, 121 112, 227, 231, 242 L–702, 958 113 L–706,000 113 levcromakalim 27–29, 32, 45–47, 50, 57, 80–86, 89, 90, 168, 178, 186–189, 197– 203, 205–214, 249, 260, 262, 277–290, 292–297, 342–346, 348–351, 354, 355, 367, 368, 371, 372, 374, 385–388, 391– 394 LF–14 116 lidocaine 104, 242 ligustrazine 317 linogliride 104, 316 LY222674 93 LY222675 249 LY97119 239 mazindol 105 meglitinide (HB–699) 102, 182, 309, 314 melperone 234 methoxamine 36, 37, 222 methylene blue 214 minoxidil (sulfate) 175, 176, 179, 188, 249, 393 MK–499 227, 231, 232 moricizine 109 MS–3579 109, 112 MS–551 107, 109, 112, 228, 231, 235

HOE 234 (see rilmakalim) iberiotoxin 174, 189, 343, 354, 364 ibutilide 107, 110, 223, 396, 401 ICS 205–930 234 idazoxan 317 iloprost 175 indomethacin 214 ipatropium bromide 276 isoproterenol 222

naphazoline103 NE–10064 114, 115 nedocromil 275 nicardipine 394 nicorandil 58, 73, 175, 200, 203, 206, 212, 247, 248, 261, 262, 267, 269, 270, 318, 323, 336, 374, 384, 393 nifedipine 207, 208, 210, 212, 389, 394 nimodipine 343

INDEX OF COMPOUNDS 501

NIP 121 50, 197, 210, 212, 214, 215, 278 nitroglycerin 269 noxiustoxin 364 NS 004 189, 366, 367 NS 1619 189, 366, 367 P–1060 168, 318 P–1075 186, 249, 261, 369 P–1188 249 pentobarbitone 317 pentylenetetrazole 375 PGF2― 165, 209, 214 phenobarbitone 317, 375 phentolamine 103, 185, 316, 353 phenytoin 374 pilisicainide 241 pinacidil 6, 35, 44, 58, 69, 93, 175, 178, 186 187, 200, 203, 206, 209, 247, 248, 250, 260, 261, 267, 269, 278, 280, 294, 318, 323, 336, 342, 355, 356, 366, 368, 369, 375, 384, 393 polymyxin B 317 pranolium (UM272) 106, 109 prazosin 212 procainamide 110, 221 procaine 340 propafenone 239 propranolol 109, 269 quinidine 104, 107, 221, 235, 239, 246, 340, 345 quinine 99, 104, 246, 317, 371 retinoic acid 148 rilmakalim (HOE 234) 50, 197, 278, 279, 280, 287, 296, 323, 392, 395 risotilide 107, 110, 227 Ro 31–6930 40, 43, 45, 198, 204, 205, 206, 278, 279, 283 RP 49356 57, 58, 186, 249, 278, 279, 283, 287, 318, 323 RP 56865 241 RP 58866 (racemic terikalant) 115, 237 RP 66471 278, 279, 281, 287 RS–87337 114, 115 RWJ 29009 48, 203

S 0121 28, 32, 282, 336 salbutamol 391 salmeterol 276 saxitoxin 309, 314 SCA40189 scyllatoxin 364 SDZ PCO 400 39, 186, 198, 212, 278, 279, 280, 289, 290, 323 secobarbitone 317 sematilide 108, 109, 110, 227, 231, 396, 398, 405 sodium nitroprusside 262 somatostatin 309 sotalol (d-sotalol) 107, 108, 110, 221, 227, 231, 235, 397, 403 sparteine 317 419 SR 44866 (see bimakalim) 199 SR 46276 39 SR 47063 37, 43, 278, 280 tacrine 115, 234 tedisamil 114, 185, 188, 228, 239 terikalant 114, 115, 228, 237, 396 tetraethylammonium (TEA) 99, 104, 157, 177, 184, 246, 318, 325, 336, 337, 342, 343, 345, 355, 376 tetrodotoxin 309, 314 theophylline 275 thiopentane 317 TMB–8317 tolazamide 100, 101 tolazoline 103, 316 tolbutamide 100, 101, 181, 182, 246, 264, 308, 314, 323, 367, 368, 371, 374, 395 tramazoline 103 U–37883 185 U46619 165, 210 UK–66, 914 109, 111, 227, 231 UL-DF9182 UM 301 106, 109 UM 424 106, 109 UR 8225 31, 47 valproate 375 verapamil 208, 212, 246, 269

502 INDEX OF COMPOUNDS

WAY–123, 223 108, 111 WAY–123, 398 109, 111, 227, 233 WAY–125, 971 108, 111 Y 26763 33, 198, 212 Y 27152 33, 51, 198, 200 YM–93447,203,206 yohimbine 316 ZD 6169 347

Index

airways smooth muscle relaxation in vitro 279 airways smooth muscle relaxation in vivo 283 alternative splicing, in Shaker proteins 127 Alzheimer’s disease 376 4–amido–3,4–dihydro–2H–l–benzopyran– 3–ols general synthesis of 2 enantiomer synthesis of 8 reactions of 14 aprikalim alkane and alkene analogues of 64 conformational analysis of 91 cyclohexanone analogues of 63 discovery of the racemate of 57 ester analogues of 68 isomers of 57 molecular modeling of 60 oxime, hydroxylamine and amine analogues of 67 the pyridine ring of, in SAR 61 stereochemistry of 60 sulphonamide analogues of 66 synthesis of 60 synthesis of the racemate of 59 the thioformamide of, in SAR 61 the thiopyran–1 –oxide of, in SAR 62

benzopyrans C–3 ketone derivatives of 15 421 C–3 carbon and nitrogen linked moieties of 18 C–4 pyrroles, reactions of 20 C-4 spirocyclic imidazolone 41, 87 Michael-type addition at C–3 of 17 reactions of 14 stereochemistry in the SAR of 28, 84 synthesis of 3 benzopyran–3, 4–epoxides Jacobsen chiral catalytic synthesis of 9 benzothiopyrans synthesis of 12 SAR of 47 1,3– and 1,4– benzoxazines SAR of 47 1,3– benzoxazinones synthesis of 13 1,4– benzoxazinones synthesis of 14 benzoxepines synthesis of 12 SAR of 47, 86 calcium channels and insulin-secreting cells 306 cardiac potassium channel modulators 221 central nervous system 361 Claisen rearrangement of aryl propargyl ethers 3 Class I-V antiarrhythmic agents 106, 221 Class III antiarrhythmic agents 395 in development 396 clinical experiences with potassium channel activators 385 in asthma 389 in hypertension 385

benzopyran nucleus replacement of, in SAR 46 aromatic ring of, substituents 10 in SAR 49 aromatic ring, replacement of, in SAR 48, 86

503

504 INDEX

clinical experiences with potassium channel activators (continued) in hypotrichosis 393 in peripheral vascular disease 392 in urinary incontinence 392 clinical experiences with potassium channel modulators 383 combined Class II/III antiarrhythmic agents 110 conformational analysis of C–4 pyrrolidinone replacements 87 of the aprikalim series CRK conformational analysis of 79 cis isomer synthesis of 4 cis enantiomers synthesis of 9 cyanoguanidines synthesis of 69 SAR of 70 molecular modeling of 71 detrusor instability 338 muscle 338 1,1–and 3,3–dimethylindanes SAR of 47 synthesis of 12 Drosophila Shaker 127 Drosophila Shaw 131 epilepsy 374, 395 enantiomer preparation in the benzopyran series 8 glibenclamide KATP channel-independent effects of 165 imidazolones, spirocyclic SAR of 41 conformational analysis of 87 K channels see potassium channels 1-(4–methoxyphenyl)indole 116 nicorandil in vivo vascular effects of 197

synthesis of 73 SAR of 74 organic nitrates 73 pain 375 pancreatic β -cells ion channels in 327 Parkinson’s disease 376 pinacidil conformation of 93 in vivo vascular effects of 197 synthesis and SAR in close analogues of 69 molecular modeling of 71 (R)-isomer of 93 plasma lipid profile, in human 387 potassium channel activation intracellular events subsequent to 295 potassium channel activators (openers) acute blood pressure studies of 197 airway pharmacology of 275 airway selectivity of 282, 285 anilide tertiary carbinol 74 benzimidazolone 189 binding studies with, in vascular smooth muscle 176 bronchial asthma and 275, 389 calcium-activated (BKCa) 189 cardioprotective properties and SAR of 36 269 calcitonin gene-related peptide 175 chronic blood pressure studies of 198 clinical potential 346 conformational analysis of 79 cyanoguanidine types, synthesis and SAR 69 effects of in the cerebral circulation 201 in cerebral cortex 372 in coronary circulation 203 in dorsal raphe 372 in hippocampus 372 in isolated blood vessels 173 in locus coeruleus 372 in the microciculation 210

INDEX 505

in pancreatic β -cells 318 in pulmonary circulation 208 in renal circulation 205 in skeletal muscle circulation 207 in splanchnic circulation 204 in substantia nigra 371 in venous system 200 on hyperresponsive airways 290 on malegenital tract smooth muscle 355 on human plasma lipid profiles 387 on plasma renin activity 200 on small and large airways 284 on stimulation of exogenous and endogenous receptors 214 on the detrusor 341 efficacy of, against cholinergic tone 280 efficacy of, against histaminergic tone 281 evaluation of, for bronchodilator activity 277, 390 heart rate effects of 200 inhaled administration of 286 in vivo mechanistic studies 212 in vivo vascular effects of 197 mechanism of action of 170, 188, 292, 342 in myocardial infarction 267 natural 175 neural effects of 288 pharmacophore models 6, 94 pyridine based 57 molecular modeling of 60 site of action of 157, 168 therapeutic potential of 383, 384 thioformamide containing 57 tracer efflux studies 178 vasorelaxant properties of ATPsensitive 185 venodilator effects of 200 potassium channel blockers 99, 225 aminopyridine 115 ATP-sensitive 100 Class III Antiarrhythmic 106, 222, 226, 396 clinical experiences of 395 combined Class II/III pharmacophore 113

future directions 405 imidazoline and related 103 in β -cells 314 in CNS 370 in detrusor smooth muscle 340 in ischaemic myocardium 260 inward rectifier 237 l-(4–methoxyphenyl)indole 116 and proarrhythmia 403 rate-dependent effects of 242 SAR of 99 sulphonylurea 100 symmetrical tetra-n-alkylammonium ion 184, 246 transient outward 238 potassium channel modulators arrhythmogenic mechanisms susceptible to 223 ATP-sensitive, of CNS channels 366 cardioprotective properties of 257 and the central nervous system 361 clinical experiences with 383 effects of, on CNS activity 370 effects of, in the ischaemic myocardium 260 endogenous, of CNS channels 363 future prospects for 383, 393 in Alzheimer’s disease 376 in epilepsy 374 in ischaemic stroke 373 in pain 375 in pancreatic β -cells 303 in Parkinson’s disease 376 potassium channels assembly of 123 ATP-activated 326 ATP-sensitive 161, 174 activators of, in pancreatic β -cells 318 cardioprotective role of 258 inhibitors of opening of 179 intracellular modulators of 162 pharmacology of, in β -cells 314 regulation of ATP by 161 regulation of, in β -cells 309 calcium-activated 159, 174, 323 cardiac 225 CNS properties of 362

506 INDEX

endogenous modulators of 363 delayed rectifier-type 160, 325 differential expression of 123, 139 diversity of 123, 124 electrophysiology of 157, 176 families of, in vascular smooth muscle 158 heteromultimeric 136, 147 in pancreatic β -cells 303 in detrusor 340 in myometrium 355 in ureter 336 in urethra 347 in uterus 353 in vascular smooth muscle cells 159 insulin-secreting cells and 307 mechanisms of opening of 177 minK 146 modulation of in urogenital tract smooth muscle 335 receptor operated 326 regulation of expression of 148 S4 channel superfamily 124, 158 origin of diversity in 127 nomenclature and kinetic properties of 128 structural division from molecular biology 157 structural determinants for potassium channel assembly 137 subunit composition of 136 tetrameric 136, 138 tracer efflux studies in 178 transient outward 238, 326 voltage-dependent 132 preconditioning phenomenon 258 pyran ring position 4 in the SAR of 30 lactams and alpha carbonyl containing replacements at 30 lactam replacements lacking alpha carbonyl groups at 35 position 3 in the SAR of 42 replacement of, in SAR 47, 85 ring flip of 82 unsaturation at position 3/4 in the SAR of 43 position 2 in the SAR of 44, 84

pyranopyridines SAR of 48, 86 synthesis of 10 pyranobenzoxadiazole synthesis of 10 SAR of 50 Re-entry mechanisms 223 RP 49356 discovery of 57 Cis analogue of 58 synthesis of 58 1R, 2R enantiomer (aprikalim) of 57 Shaker gene of Drosophila 127, 158 molecular cloning of 140 insulin-secreting β -cells of 311 Shab gene 132 Shal gene 132 Shaw gene 132 soyasaponins 190 stroke 374 stunned myocardium model 262 sulphonylurea binding site pharmacophore model for 103 tetrahydronaphthalenes SAR of 47 synthesis of 13 tetrahydronaphthalenones SAR of 47 tetrahydroquinolines SAR of 47 synthesis of 13 thienopyrans SAR of 48, 50, 86 synthesis of 10 toxin modulators of CNS potassium channels 364 bee venom 365 scorpion venom 364 snake venom 365 urethra muscle 347 uterus 351 xenopus 128 oocytes 132, 174, 236