Drug Discovery Today, December 2010, Volume 15, Numbers 23 24

Authoritative reviews and expert opinion Industry focused, peer reviewed

• The future of toxicity testing • Epigenetic therapies for non-oncology indications • In silico understanding of infectious agents • Lyotropic liquid crystal systems in drug delivery

December 2010 Volume 15 Nos. 23&24/24

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CONTENTS 1

December 2010 Volume 15 · Numbers 23/24 pp. 983–1114

REVIEWS FOUNDATION

997 The future of toxicity testing: a focus on in vitro methods using a quantitative high-throughput screening platform Sunita J. Shukla, Ruili Huang, Christopher P. Austin and Menghang Xia

GENE TO SCREEN

1008 Epigenetic therapies for non-oncology indications Jonathan D. Best and Nessa Carey

1015 Pharmacoproteomics: a chess game on a protein field Angelo D’Alessandro and Lello Zolla

INFORMATICS Cover Story The leading story of this issue of Drug Discovery Today, by Sunita J. Shukla, Ruili Huang, Christopher P. Austin and Menghang Xia highlights the US Tox21 collaborative program and how it has brought about a change in approach in toxicity testing of chemical compounds. Standard in vivo testing is being superseded to an extent by higher throughput in vitro assay techniques. This approach is prioritizing compounds for further mechanism of action studies and the development of models to predict adverse events in humans.

1024 Connecting the dots: role of standardization and technology sharing in biological simulation Samik Ghosh, Yukiko Matsuoka and Hiroaki Kitano

POST SCREEN

1032 Lyotropic liquid crystal systems in drug delivery Chenyu Guo, Jun Wang, Fengliang Cao, Robert J. Lee and Guangxi Zhai

1041 Developments towards antiviral therapies against enterovirus 71 Kan X. Wu, Mary M.-L. Ng and Justin J.H. Chu

1052 PubChem as a public resource for drug discovery Qingliang Li, Tiejun Cheng, Yanli Wang and Stephen H. Bryant

1058 Heparin/heparan sulphate-based drugs Neha S. Gandhi and Ricardo L. Mancera

1070 Metal-based drugs for malaria, trypanosomiasis and leishmaniasis: recent achievements and perspectives Maribel Navarro, Chiara Gabbiani, Luigi Messori and Dinorah Gambino

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CONTENTS 2

DRUG DISCOVERY TODAY Editorial Editor: Steve Carney Assistant Editor: Kirsty Strawbridge Content Development Manager: Joanna Aldred Journal Manager: Basil Nyaku Publisher: Jaap van Harten Editorial Enquiries: Drug Discovery Today Elsevier 32 Jamestown Road, London, NW1 7BY, UK Tel: +44 (0)20 7424 4200 Fax: +44 (0)1865 853067 Email: [email protected]

EDITORIAL 983 Aneuploidy and adult neurogenesis in Alzheimer’s disease: therapeutic strategies Philippe Taupin ADVISORY EDITORIAL BOARD Jurgen Bajorath University of Bonn, Germany Walter Blackstock Experimental Therapeutics Centre, Singapore David Brayden University College Dublin, Ireland Paul Caron Vertex Pharmaceuticals, USA David Cavalla Arachnova, UK David Clark Argenta Discovery, UK Dalia Cohen Rosetta Genomics, USA Donald Daley Argenta Discovery, UK Sean Ekins ACT LLC and Collaborations in Chemistry, USA Hans-Peter Fischer GeneData, Switzerland Peter Ghazal University of Edinburgh, UK Christopher M. Hill Organon, UK Nick Hird Takeda Chemical Industries, Japan Enoch Huang Pfizer, USA Thomas Joos University of Tuebingen, Germany Vincent H.L. Lee Chinese University of Hong Kong, Hong Kong David Lewin Yale University, USA Christopher A. Lipinski Melior Discovery, USA Lorenz Mayr Novartis, Switzerland Nick Meanwell Bristol-Myers Squibb, USA BK Muralidhara Pfizer Global Biologics, St. Louis, USA Mark Murcko Vertex Pharmaceuticals, USA Fajun Nan Shanghai Institute of Materia Medica, China Pradeep Nathan Experimental Medicine, GSK and University of Cambridge Gitte Neubauer Cellzome, Germany Eric Neumann Teranode, USA Tim Peakman UK Biobank, UK Norton Peet Aurigene, USA Manuel Peitsch Novartis, Switzerland Kurt Rasmussen Eli Lilly and Co. Ltd. USA Janice Reichert Tufts Center for the Study of Drug Development, USA John Reidhaar-Olson Hoffmann–La Roche, USA Mike Romanos GlaxoSmithKline, UK Raymond C. Rowe Intelligensys, UK Andreas Russ University of Oxford, UK Esther Schmid Pfizer, UK Jonathan Sheldon Inforsense, UK Michael Snyder Yale University, USA Susie Stephens Eli Lilly and Co. Ltd., USA Robert Strausberg The J. Craig Venter Institute, USA Donny Strosberg Scripps Florida, USA Yuichi Sugiyama University of Tokyo, Japan David Szymkowski Xencor, USA Nick Terrett Ensemble Discovery, USA Vladimir Torchilin Northeastern University, Boston, USA Mark Whittaker Evotec OAI, UK Hans Winkler Johnson and Johnson, Belgium Limsoon Wong Laboratories for Information Technology (LIT), Singapore X.F. Steven Zheng Robert Wood Johnson Medical School, USA

PERSPECTIVE FEATURE

985 The MPTP marmoset model of Parkinsonism: a multi-purpose non-human primate model for neurodegenerative diseases Ingrid H.C.H.M. Philippens, Bert A. ‘t Hart and German Torres

991 Securing reliability and validity in biomedical research: an essential task Thomas Wilckens

MONITOR 1079 Cellular Delivery of Therapeutic Macromolecules (CDTM) International Symposium 2010: lessons and progress from inter-disciplinary science Mark Gumbleton and Arwyn T. Jones

ABSTRACTS 1080 Delegate abstracts

FORTHCOMING ARTICLES: Angiotensin II Receptors and Drug Discovery in Cardiovascular Disease By Chiranjib Dasgupta and Lubo Zhang Creativity, Innovation and Lean Sigma: a controversial combination? By Craig Johnstone, Garry Pairaudeau and Jonas A. Pettersson The graphical representation of ADME-related molecule properties for medicinal chemists By Timothy J. Ritchie, Peter Ertl and Richard Lewis Characteristics of orphan drug applications that fail to achieve marketing approval in the US By Harald E. Heemstra, Hubert G.M. Leufkens, R.P.Channing Rodgers, Kui Xu, Bettie C.G. Voordouw and M.Miles Braun

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Drug Discovery Today  Volume 15, Numbers 23/24  December 2010

EDITORIAL

editorial Philippe Taupin

Aneuploidy and adult neurogenesis in Alzheimer’s disease: therapeutic strategies The confirmation that neurogenesis occurs in the adult brain and neural stem cells (NSCs) reside in the adult central nervous system (CNS) of mammals reveals that the adult brain has the potential for self-repair. Neurogenesis occurs in discrete regions of the adult mammalian brain, the subventricular zone and the dentate gyrus (DG) of the hippocampus, in various species, including humans. Newly generated neuronal cells in the adult brain originate from a pool of residual NSCs. Adult NSCs contribute to the physiology and pathology of the nervous system [1]. Recent studies show that neurogenesis is enhanced in the brain of patients with Alzheimer’s disease (AD) [2]. Enhanced neurogenesis in the brain of AD patients would contribute to regenerative attempts in the CNS, to compensate for the neuronal loss. 1359-6446/06/$ - see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2010.04.001

AD is a neurodegenerative disease associated with learning and cognitive deficits, and for which aging is the main risk factor. The hippocampus is the main region of the brain affected by the disease. There are two forms of the disease: the late onset form (LOAD) diagnosed after age 65 and the early onset form (EOAD) diagnosed at a younger age. Genetic background and acquired and environmental risk factors are causative factors for LOAD, whereas EOAD is primarily an inherited disease. The presence of the apolipoprotein E varepsilon 4 allele (ApoE4) in the genetic makeup of the individuals is the best-established genetic risk factor for LOAD. Mutations in the amyloid precursor protein (APP), the presenilin-1 (PSEN-1) and the presenilin-2 (PSEN-2) genes have been identified as causative for EOAD. Amyloid plaques, composed of deposits of amyloid proteins, and neurofibrillary tangles, composed of aggregated hyperphosphorylated Tau proteins, are the histopathological hallmarks of AD [3]. AD is also characterized by neurodegeneration and aneuploidy in the adult brain [4]. The increase of aneuploid nerve cells in regions of degeneration in the AD brain contributes to the development of the disease. In regions of degeneration, cell cycle re-entry and DNA duplication, without cell division, are at the origin of aneuploid nerve cells in the brain of patients with AD [5]. These cells are fated to die and may undergo a slow death process, underlying the process of neurodegeneration in AD [6]. The ApoE, PSEN-1, PSEN-2 and TAU genes are located on chromosomes 19, 14, 1 and 17, respectively. Aneuploidy for chromosomes carrying genes involved in AD promotes the formation of amyloid plaques, neurofibrillary tangles and neurodegeneration in the brain of patients with AD, LOAD or EOAD depending on the genetic and/or risk factors involved. Dividing cells are the most likely to generate aneuploid cells [7]. Hence, neurogenesis holds the potential to generate new neuronal cells that are aneuploid in the neurogenic regions of the adult brain. Aneuploid new neuronal cells and aneuploid newly generated neuronal cells that would not proceed with their developmental program in the adult brain would be a contributing factor of the pathogenesis of AD in the neurogenic regions. Aneuploidy, for chromosomes carrying genes involved in AD, in newly generated neuronal cells of the adult brain would further promote the pathological process of AD, particularly in the hippocampus [8]. Adult neurogenesis is a relatively low frequency event in the adult brain; it is estimated that 0.004% of the granule cell www.drugdiscoverytoday.com

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population is generated per day in the DG of adult macaque monkeys [1]. The hippocampus is one of the neurogenic regions of the adult brain and one of the regions of the brain the most affected in AD. Aneuploid newly generated neuronal cells originating from the nondisjunction of chromosomes during cell division may have their lifespan shortened or may survive for extended period of time. They would contribute to the pathogenesis of AD by promoting the formation of amyloid plaques, neurofibrillary tangles, neurodegeneration and aneuploidy, locally. This suggests that, despite being a low frequency event, the generation of aneuploid new neuronal cells in the hippocampus, in particular, may play a critical contribution to the pathology of AD. Mutated forms of PSEN-1 are detected in interphase kinetochores and centrosomes of dividing cells, where they may be involved in the segragation and migration of chromosomes during cell division [9]. The hyperphosphorylation of Tau by kinases leads to the dissociation of Tau and tubulin and to the breakdown of microtubles causing the disruption in the mitotic spindle, which promotes aneuploidy during mitosis [10]. Hence, genetic and/or risk factors involved in AD would promote the generation of aneuploid new neuronal cells in the adult brain. Enhanced neurogenesis in the hippocampus of patients with AD, and more generally conditions that promote neurogenesis, would contribute to an increase of aneuploidy newly generated neuronal cells in the adult brain. This reveals that adult neurogenesis may be involved in the pathogenesis of AD. In all, adult neurogenesis may contribute not only to regenerative attempts in the nervous system, but also to the pathogenesis of neurological diseases and disorders, particularly in AD. The

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contribution of aneuploid newly generated neuronal cells of the adult hippocampus to the pathogenesis of AD opens new avenues and perspectives for our understanding of and for treating the disease. Therapeutic strategies will aim at specifically targeting aneuploid newly generated neuronal cells of the adult brain, to limit their potential deleterious effects in patients with AD, without disrupting the regenerative capacity of adult neurogenesis. References 1 Taupin, P. (2006) Neurogenesis in the adult central nervous system. C. R. Biol. 329, 465–475 2 Jin, K. et al. (2004) Increased hippocampal neurogenesis in Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 101, 343–347 3 Querfurth, H.W. et al. (2010) Alzheimer’s disease. N. Engl. J. Med. 362, 329–344 4 Kingsbury, M.A. et al. (2006) Aneuploidy in the normal and diseased brain. Cell. Mol. Life. Sci. 63, 2626–2641 5 Yang, Y. et al. (2003) Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer’s disease. J. Neurosci. 23, 2557–2563 6 Yang, Y. et al. (2007) Cell division in the CNS: protective response or lethal event in post-mitotic neurons? Biochim. Biophys. Acta 1772, 457–466 7 Torres, E.M. et al. (2008) Aneuploidy: cells losing their balance. Genetics 179, 737– 746 8 Taupin, P. (2009) Adult neurogenesis, neural stem cells and Alzheimer’s disease: developments, limitations, problems and promises. Curr. Alzheimer Res. 6, 461–470 9 Li, J. et al. (1997) Alzheimer presenilins in the nuclear membrane, interphase kinetochores, and centrosomes suggest a role in chromosome segregation. Cell 90, 917–927 10 Kim, H. et al. (1986) The binding of MAP-2 and tau on brain microtubules in vitro: implications for microtubule structure. Ann. N. Y. Acad. Sci. 466, 218–239

Philippe Taupin School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland email: [email protected]

Drug Discovery Today  Volume 15, Numbers 23/24  December 2010

PERSPECTIVE

feature The MPTP marmoset model of Parkinsonism: a multi-purpose non-human primate model for neurodegenerative diseases Aging societies face an increasing prevalence of neurodegenerative disorders for which no cure exists. The paucity of relevant animal models that faithfully reproduce clinical and pathogenic features of neurodegenerative diseases is a major cause for the lack of effective therapies. Clinically distinct disorders, such as Alzheimer’s and Parkinson’s disease, are driven by overlapping pathogenic mechanisms that converge onto vulnerable neurons to ultimately cause abnormal clinical outcomes. These similarities, particularly in the early phases of neurodegeneration, might help identify appropriate animal model systems for studying of cell pathology. While reviewing some of the cellular mechanisms of disease progression, we discuss the MPTP-induced model of Parkinsonism in marmoset monkeys as a model system for construct, face and predictive validity in neurodegenerative studies.

Overlapping pathogenic mechanisms and modeling neurodegenerative states Aging Western societies face a steadily increasing prevalence of neurological diseases caused by a progressive degenerative process within the central nervous system (CNS). Neurodegenerative disorders as diverse as Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD) have for a long time been regarded as different pathological entities because of their specific clinical symptoms, unique cell pathology and response to drug treatment. These diseases, however, exhibit overlapping pathogenic mechanisms that are conspicuously present throughout the brain parenchyma (Fig. 1). For instance, specific defects in cellular repair

mechanisms and inability to maintain neuronal (e.g. Ca2+) homeostasis can threaten cell function and viability, particularly in genetically prone individuals [1]. The etiology of AD, PD, ALS and HD seems to be woven by similar molecular threads: the aggregation and deposition of microscopically visible abnormal proteins that are causally linked to cellular stress and inflammation. It is not clear, however, how abnormal proteins lead to synaptic damage and then faulty neurotransmission. Understanding the mechanism of toxicity of aggregation-prone proteins for each of these diseases represents the most compelling research endeavor in the neurosciences. Unfortunately, it has come as an acute disappointment that most first clinical trials of therapies designed to mitigate the

1359-6446/06/$ - see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2010.08.009

toxicity of mutant or aggregation-prone proteins have failed to help patients. Furthermore, the paucity of relevant animal models that faithfully reproduce clinical and pathogenic features of neurodegenerative states have also failed to identify basic intracellular events that are causal for the human disease. An exception to this exasperating situation might be the toxininduced model of idiopathic PD (Box 1). The observation in the early 1980s of early-onset Parkinsonism in some Californian drug users who inadvertently injected themselves with 1methyl-4-phenyl-4-propionoxy-piperidine (MPPP, desmethylprodine; a synthetic opioid with effects similar to those of morphine and pethidine) led to the identification of 1-methyl-4phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) as a www.drugdiscoverytoday.com

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Ingrid H.C.H.M. Philippens1,2,*, Bert A. ‘t Hart1,3 and German Torres2

PERSPECTIVE

Drug Discovery Today  Volume 15, Numbers 23/24  December 2010

GLOSSARY 2+

2+

Features  PERSPECTIVE

Disturbed Ca homeostasis [34] Regulation of intracellular Ca is vital for proper CNS function. However, relatively high Ca2+ levels during aging might be responsible for agedependent vulnerability to cell injury. In neurodegenerative diseases, neuronal Ca2+regulation is compromised by depletion of energy supply owing to metabolic arrest and loss of mitochondrial function. This invariably leads to synaptic dysfunction, impaired protein plasticity and overall cell degeneration. Excitotoxicity [33] In excitotoxicity, cell death is initiated by the overstimulation of excitatory amino acid receptors by high glutamate concentrations, leading to high intracellular Ca2+ levels. Excitotoxicity also generates ROS accompanied with ATP depletion. Excitotoxicity has been implicated in progressive neurodegenerative disorders through a process in which otherwise healthy neurons are unable to withstand non-lethal glutamate concentrations. Glia activation [12,35] Glia cells play an active part in neurodegenerative processes. After harmful signals to neurons, microglia produce substances (such as cytokines) that keep certain neurodegenerative processes in a constant state of inflammation. There is evidence that clusters of microglia abound in the senescent brain, thus suggesting that activation and proliferation of these cell types could account for age-related neurodegenerative states. Mitochondrial dysfunction [3] Mitochondria organelles, the source for most of the cell’s adenosine triphosphate (ATP) supply, are responsible for regulating membrane potentials and cellular metabolism through Ca2+-dependent autonomous channels. Mitochondrial dysfunction contributes to protein misfolding and aberrant oxidative stress and plays a central part in age-related neurodegeneration. Oxidative stress [10,32] An imbalance in redox homeostasis causes toxic effects on proteins, lipids and DNA strands through the production of ROS. Oxidative stress is one of the key mechanisms involved in neurodegenerative diseases. The primary sources of ROS are mitochondrial dysfunction and microglia oxidative burst. Protein aggregation [5] Several age-related neurodegenerative diseases are associated with protein aggregation or misfolding. It is still unclear whether the protein aggregation is generally toxic or the result of a protective mechanism initiated by injured neurons; however, in inherited forms of neurodegeneration, misfolded proteins often lead to an earlier onset and more severe clinical phenotype than sporadic forms. The current understanding is that microscopic aggregates are protective and that monomers and/or oligomeric precursors of the aggregates are pathological. drug derivative with discrete neurotoxic properties. Subsequent studies in non-human primates (e.g. squirrel monkeys, rhesus macaques and common marmosets) confirmed that injections of MPTP lead to a gross depletion of dopamine (DA) neurons in the substantia nigra pars compacta. This depletion causes a spectrum of movement disorders, including the clinical triad of resting tremor, rigidity and bradykinesia. In general, these initial findings have led to the development of the MPTP-induced model of

Parkinsonism in marmoset monkeys (Callithrix jacchus), which is currently used as a valid preclinical model of idiopathic PD. It should be noted that rodents are a less useful model for neurodegenerative studies because rats are not sensitive to systemic MPTP treatment and DA cell death in mice usually does not lead to the full spectrum of Parkinsonian symptoms. The aim of this review is to list several pathological similarities of the marmoset MPTP-based model with a broad spectrum of neurodegenerative diseases

BOX 1

The MPTP model of neurodegeneration Since the discovery of MPTP, this drug has become the preferred toxin to induce Parkinsonism in laboratory animals. MPTP selectively damages DA neurons, which invariably leads to impaired DA neurotransmission. The toxin is highly lipophilic and after systemic administration rapidly crosses the blood–brain barrier to cause cellular havoc. Within the CNS, MPTP is converted into MPDP+ in astrocytes by the enzyme monoamine oxidase-B; it then spontaneously oxidizes into the metabolite MPP+. MPP+ is released into the extracellular space by an as yet unknown mechanism. MPP+ is taken up by DA neurons vi a the DA transporter. Inside the neuron, MPP+ can be stored either in the vesicular monoamine transporter or mitochondria. MPP+ impairs mitochondrial respiration pathways by inhibiting complex 1 of the electron transfer chain. In this context, MPTP toxicity can be efficiently counteracted by riluzole (a drug already approved for the treatment of ALS [30]) when used in models of DA neuron degeneration [29,31]. Thus, the MPTP model of neurodegeneration can be used for rapid drug discovery and as an in vivo screening assay for drugs and nutritionals that reduce the risk of excitotoxic damage to neurons. 986

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(Fig. 2) and to discuss the possibility of using this non-human primate as an alternative model system for preclinical neuroprotective drug development.

Mitochondrial defects and neurodegenerative diseases Early indications for the role of mitochondrial dysfunction (see Glossary) in neurodegenerative states [2] came from studies showing that increases in mitochondrial oxidative stress and/ or accumulation of protein aggregates could produce a devastating pathological and clinical phenotype [3]. This possibility was further supported by the discovery that MPTP inhibits the first enzyme complex of the mitochondrial electron transfer chain (complex I) in brains and platelets of patients with PD [4]. Moreover, the finding that systemic administration of the lipophilic complex I inhibitor rotenone could recapitulate many of the symptoms of PD further highlighted the interrelationship of mitochondrial proteins, oxidative stress and DA cell function [5]. Since then, mitochondrial defects have been implicated in a variety of clinical cases, commonly involving cell networks that have high energy requirements such as those found in the CNS. For example, recessive mutations in the genes encoding DJ-1 and PTEN-induced kinase 1, both localized to mitochondria (or at least to the outer mitochondria membrane), have recently been linked to familial forms of PD [6,7]. Furthermore, several pathogenic mitochondrial DNA (mtDNA) base substitution mutations and mtDNA deletions and insertions have been identified in a variety of other neurodegenerative diseases. For instance, cortical mtDNA deletion levels are elevated in both AD and HD, and AD brains show increased oxidative damage in their mtDNA [8]. Similarly, oligomerized amyloid-b peptide, a large component of plaque pathology in the AD brain, seems to trigger mitochondrial fission or fragmentation via S-nitrosylation of dynamin-related protein [9]. Collectively, these observations rekindle the debate over mechanistic theories of neurodegenerative diseases and also revive interest in oxidative stress as an underlying mechanism for the selective demise of certain neurons. It is worth noting that mitochondrial-based diseases commonly have a delayed onset and a progressive course, very much like those seen in a broad range of neurodegenerative disorders. This is now being studied in the marmoset monkey model of PD, which might provide insights into several novel mechanisms for mitochondrial pathology.

Drug Discovery Today  Volume 15, Numbers 23/24  December 2010

[(Figure_1)TD$IG]

PERSPECTIVE

Oxidative stress and brain pathology Oxidative stress (e.g. reactive oxygen species, or ROS) can damage proteins overtly prone to changes in redox-signaling pathways [10]. Fortunately, the burden of ROS production is largely [(Figure_2)TD$IG] neutralized by a complex anti-oxidant

arsenal of enzymatic scavengers including superoxide dismutase, catalase and glutathione peroxidase; however, these protective mechanisms are often weakened by chronic oxidative stress, particularly during senescence. Thus, it is generally accepted that a crucial

PD ALS AD

MPTP

MS HD Drug Discovery Today

FIGURE 2

MPTP causes brain damage with phenotypes overlapping those caused by different etiologies. Neurons are particularly vulnerable to both the toxic effects of MPTP and aggregation of misfolded proteins. Each of these toxic effects converges on pathways that cause motor deficits or dementia. The common characteristics of these neurodegenerative disorders suggest parallel approaches to drug therapy.

balance between ROS production and antioxidant defenses determines the degree of oxidative stress in the aging nervous system [10]. Neurodegenerative diseases might, therefore, represent the effects of a chronic imbalance between ROS production and ROS clearance. Is there any supporting evidence for this working hypothesis? Indeed, alterations in mitochondrial respiratory capacity and agedependent increases in oxidative damage are seen in AD, PD, HD and ALS [11]. The relative contribution of oxidative stress to neurotoxicity is not yet clear, but one possibility is that AD, PD, HD and ALS all share early common mechanisms of oxidative pathology, which later become specific to certain brain regions with different clinical end points. Nonetheless, the observation that diverse brain pathologies show oxidative damage reinforces the central role of mitochondrial metabolism and subsequent energy-dependent ROS production in neurodegenerative states. This link provides a useful framework for understanding disease progression in the marmoset monkey model of PD. www.drugdiscoverytoday.com

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FIGURE 1

Schematic diagram depicting several pathogenic features found in a broad range of neurodegenerative diseases. The features in the inner circle are responsible for the initial cellular insult that causes failure of neuronal homeostasis. The features in the outer circle are responsible for the maintenance and progression of disease pathology. In some cases, such as in inherited PD or HD, the pathogenic process can originate from the outer circle. All of these intracellular processes of disease are potential targets for drug therapy.

PERSPECTIVE

Microglia activation and neurodegenerative diseases

Features  PERSPECTIVE

For decades, glia cells were thought to have only a passive, supporting role in the CNS. It is now becoming increasingly clear, however, that glia cells, particularly microglia, have an active role in inflammatory response signaling events. For instance, microglia cells continuously survey the brain for injury and infection, both during development and during adulthood. Microglia cells promptly migrate to areas of injury and release cytokines such as tumor necrosis factora, interleukin-1b and interleukin-6, which dramatically increase the excitability of nearby neurons. Similar migratory patterns of microglia are found in AD, PD and multiple sclerosis (MS) patients, where they often interact with neurons and surrounding blood vessels. Whether these interactions are helpful or harmful in these clinical conditions is a matter of debate. Regardless, several in vitro studies have demonstrated co-localization of activated microglia cells with amyloid fibrils and a-synuclein aggregates remarkably similar to that seen in neural tissue extracted from patients afflicted with protein-folding-related diseases. This observation implies that aberrant folded proteins might directly injure the synapses and neurites of neurons, in addition to activating microglia cells. Thus, microglia activation as seen in certain neurodegenerative states might result from changes in protein metabolism that occur before widespread cell death ensues, the characteristics of which are often disease specific. Abnormally high levels of activated microglia have also been associated for decades with senescence, thus indicating that the inability to maintain glia cell homeostasis is sufficient to cause common end points in both aging and neurodegenerative states [12]. Work in nonhuman primates also implicates microglia activation as an early step for many of the underlying mechanisms that provoke neuronal death in AD, PD and MS. For example, injections of MPTP into marmoset monkeys selectively damages DA cells in the substantia nigra pars compacta, a pathogenic event that is immediately followed by the clustering of microglia around injured neurons [13]. Of interest, activated microglia in MPTP-treated monkeys remain considerably elevated in the midbrain one year after MPTP exposure [14]. This observation suggests that activation of microglia in areas of injury might be more protracted than previously thought. Furthermore, in an autoimmune model of MS in marmoset monkeys, degeneration of white matter seems to be indirectly mediated by infiltrated inflammatory 988

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cells, whereas degeneration of grey matter in the same non-human primate is ascribed to highly concentrated recruitment of microglia cells [15]. Of interest, in a superoxide dismutase mutant mouse model of ALS, neurodegeneration can be mitigated by inhibiting the actions of microglia cells, suggesting that – in this particular case, at least – degeneration could be triggered by the abnormal release of microglia-derived cytokines that bind and perturb cell-surface receptors and/ or channels rather specifically [16]. Although the ‘microglia cascade hypothesis of degeneration’ offers a broad framework to explain certain pathological features of AD, PD and MS, it is currently hampered by a lack of detailed mechanistic understanding.

Aggregation and deposition of abnormal proteins Several age-related degenerative diseases, including AD, HD, dementia with Lewy bodies and PD, are associated with aggregation and accumulation of misfolded proteins, the characteristics of which are often disease specific [5]. For instance, in AD, the aberrant deposition of amyloid-b occurs in the form of fibrils or extracellular plaques, whereas in HD, the polyglutamine-containing protein accumulates in the form of nuclear and cytoplasmic inclusions. In PD, the toxic protein is represented by a-synuclein that ‘seeds’ the brain to produce intracellular Lewy bodies [17,18]. It should be noted that the ability of proteins to form highly organized aggregates is not restricted to the few proteins associated with recognized clinical disorders but seems to be a generic propensity of all polypeptide chains. Studies in animal models of AD and PD further support the ability of proteins to change conformation and form small, soluble aggregates that assume toxic states with a wide range of cellular targets. For example, microinjections of fibrillar amyloid-b into the aged marmoset brain induces typical pathogenic aspects of AD [19]. In addition, rat studies show that intracerebral injections of misfolded asynuclein cause degeneration of vulnerable neurons that recapitulate the pathogenic features often seen in sporadic PD [20]. Finally, mice systematically treated with MPTP spontaneously develop protein aggregates that could be equally toxic to the nervous system [18]. Despite the rapid advance in the molecular dissection of protein folding and misfolding, including the identification of several pathogenic proteins, it is not yet known whether clearance of soluble aggregates correlate with disease improvement. There is still considerable work to be done, particularly in non-human primate models of

neurodegenerative diseases, to determine whether therapeutic agents can prevent aggregates from forming or dismantling those already rooted in the brain parenchyma.

Modeling certain pathogenic features of neurodegeneration Animal models offer a useful experimental platform for target identification (e.g. pathogenic mechanism) and validation (e.g. face validity) of candidate drugs (Table 1). Target identification in neurodegenerative diseases is a major challenge, however, because a ‘spider web’ of pathological events – acting separately or synergistically – exist in certain individuals who are at risk of developing a specificdegenerative phenotype (Fig. 1). Furthermore, the multiple and diverse cellular mechanisms that characterize most neurodegenerative diseases conspire to develop an all-inclusive animal model that mimics the most obvious symptoms of the brain disease. Non-human primates, in particular the marmoset monkey, bridge this gap by providing an appropriate animal model for construct, face and predictive validity (‘face validity’ refers to the perceived similarity of the symptoms observed in the model and in the human disorder). More importantly, the close genetic, anatomical, physiological and immunological synteny of marmoset monkeys with humans makes them the preferred species for replicating brain diseases. Besides the anatomical and physiological similarities with humans, behavioral and cognitive deficits, which frequently represent the main source of disability in patients, can be assessed very accurately in the marmoset monkey [21]. There are, however, substantial ethical hurdles and acute differences associated with using monkeys for mimicking neurodegeneration in humans. For instance, potential side-effects associated with invasive approaches to disease replication (e.g. intracerebral injections of pathogenic molecules such as amyloid-b or a-synuclein) considerably limit the use of marmoset monkeys for certain clinical studies. In regards to MPTP, this toxin does not produce dementia with Lewy bodies, amyloid-b or polyglutamine-containing proteins, which are common pathological features of neurodegenerative disorders. Thus, the MPTP-treated marmoset monkey can only serve as a template model for understanding disease mechanisms and potential new drug treatments, rather than predicting different clinical manifestations of a particular brain disorder. This difference in essence relates back to the difficulty of finding an all-inclusive animal

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TABLE 1

Neuroprotective compounds used in MPTP-based pathologies, which are also currently approved for preclinical phases of several neurological studies Mechanism of action

Target

Application

Status

Reference

Coenzyme Q10

Anti-oxidant Mitochondrial energy enhancer

Oxidative stress Mitochondrial dysfunction

PD, HD AD

Research

[24]

Creatine

Mitochondrial energy enhancer

Brain atrophy Inclusion formation

ALS, HD, PD

Research

[2]

Apocynin

Anti-oxidant Anti-inflammatory

Oxidative stress Microglia activation

ALS, MS, stroke, PD

Research

[25]

Minocycline

Anti-inflammatory anti-apoptotic

Microglia activation Apoptosis

PD, MS, HD, AD, ALS

Research

[24]

Lipoic acid

Anti-oxidant Anti-inflammatory

Mitochondrial decay Oxidative stress

PD, AD, HD

Research

[26]

EGCGa

Anti-oxidant Anti-inflammatory

Oxidative stress

PD, stroke, AD, ALS

Research

[27,28]

Memantine

NMDAb antagonist

Excitotoxicity

PD, AD

Approved/research

[29]

Riluzole

NMDAb antagonist Ca2+ channel blocker

Excitotoxicity

ALS PD

Approved/research

[30,31]

Rasagiline

Monoamine oxidase-B inhibitor Anti-apoptotic GDNFc activation

b-amyloid, glutamate Apoptosis

PD AD

Approved/research

[24]

a

Epigallocatechingallate N-methyl-D-aspartate c Glial-cell-line-derived neurotrophic factor

model that could point the way to common therapeutic approaches. In this regard, it is certainly conceivable that some of the therapeutic drugs listed in Table 1 could be applied [(Figure_3)TD$IG]

across multiple neurodegenerative diseases. Indeed, memantine 10 mg twice daily has been used to treat several neurological diseases, including those associated with excessive glu-

tamate release (e.g. AD and PD with dementia [22]). Both AD and PD with dementia, regardless of their respective protein aggregate profiles and anatomical lesion loci, are often accom-

FIGURE 3

Magnetic resonance spectroscopy (MRS) imaging analysis of the MPTP-treated marmoset brain [23]. MRS imaging permits in vivo analysis in a regionally specific manner of brain metabolites relevant to neuronal density (N-acetylaspartate, or NAA). In these studies, we hypothesized that in the same MPTP-treated animal, the ratios of NAA to total creatine (tCr, the standard denominator in MRS ratio analyses) would be decreased. This pattern would reflect diminished neuronal viability to MPTP toxicity. Left panel: T2-weighted axial brain section oriented to the SN and reference area in the cortex (COR). Right panel (upper): Representative spectrum from one voxel is shown with peaks identified for NAA and tCr. Right panel (lower): Relative changes in NAA/tCr ratios (means  SEM) across defined times (weeks) in the marmoset SN. Note that modafinil (already approved for narcolepsy) also minimizes MPTP-based toxicity. Significant differences compared to baseline (*) and between treatments (§) (one-way ANOVA followed by Bonferroni post hoc tests; P  0.05). www.drugdiscoverytoday.com

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b

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Features  PERSPECTIVE

panied by non-motor complications, such as dementia, sleep disturbances, depression and psychotic symptoms, which invariably worsen their prognoses. This strategy provides rationale for the use of drugs for wider applications than conditions in which cholinergic (for AD) or DA (for PD) are considerably impaired. Furthermore, as better in vivo imaging methods become more widely available in non-human primates (Fig. 3), ambiguities related to cholinergic, glutamatergic or DA function in the brain of living marmoset monkeys suffering from MPTP toxicity will probably become better elucidated [23]. Nevertheless, the well-established MPTP model of idiopathic PD in marmoset monkeys recapitulates most of the core pathogenic mechanisms of the human condition, including mitochondrial dysfunction, oxidative stress, and activation and proliferation of microglia [14]. Furthermore, the neuronal cell loss and neurodegenerative cascade of events after MPTP administration are stable over time, thus providing a window of opportunity for testing pharmacological therapies that modify the temporal and kinetic states of brain pathology. These useful characteristics of MPTP, when applied to the marmoset monkey, can provide relevant mechanistic and therapeutic information that could be used to delay or perhaps even arrest the disease before the more typical symptoms emerge and the damage caused by the neurodegenerative state becomes irreversible.

Concluding remarks Despite the advances in clinical pharmacology and state-of-the-art of animal modeling, innovative approaches to neurodegenerative states are still needed. Rather than placing a lot of effort in the creation of disease-specific animal models, we propose that research efforts should focus on the implementation of a generic model that covers core principles of pathogenetic processes. The MPTP-treated marmoset monkey, for instance, resembles human PD with respect to pathology, biochemistry, symptomatology and response to treatment. Thus, knowledge gained from this animal model will aid in the development of drug therapies for several forms of neurodegenerative diseases.

Acknowledgement The authors would like to thank Henk van Westbroek for his excellent technical assistance regarding the artwork.

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References 1 Lin, M.T. and Beal, M.F. (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787–795 2 Beal, M.F. (2003) Bioenergetic approaches for neuroprotection in Parkinson’s disease. Ann. Neurol. 53 (Suppl. 3), S39–S47 3 Petrozzi, L. et al. (2007) Mitochondria and neurodegeneration. Biosci. Rep. 27, 87–104 4 Dawson, T.M. and Dawson, V.L. (2003) Molecular pathways of neurodegeneration in Parkinson’s disease. Science 302, 819–822 5 Dauer, W. and Przedborski, S. (2003) Parkinson’s disease: mechanisms and models. Neuron 39, 889–909 6 Bonifati, V. et al. (2003) DJ-1(PARK7), a novel gene for autosomal recessive, early onset parkinsonism. Neurol. Sci. 24, 159–160 7 Valente, E.M. et al. (2004) Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 304, 1158–1160 8 Gu, G. et al. (2002) Mitochondrial DNA deletions/ rearrangements in Parkinson disease and related neurodegenerative disorders. J. Neuropathol. Exp. Neurol. 61, 634–639 9 Cho, D.H. et al. (2009) S-nitrosylation of Drp1 mediates beta-amyloid-related mitochondrial fission and neuronal injury. Science 324, 102–105 10 Alexi, T. et al. (2000) Neuroprotective strategies for basal ganglia degeneration: Parkinson’s and Huntington’s diseases. Prog. Neurobiol. 60, 409–470 11 Simonian, N.A. and Coyle, J.T. (1996) Oxidative stress in neurodegenerative diseases. Annu. Rev. Pharmacol. Toxicol. 36, 83–106 12 Streit, W.J. et al. (2008) Microglial degeneration in the aging brain – bad news for neurons? Front. Biosci. 13, 3423–3438 13 Kanaan, N.M. et al. (2008) Age and region-specific responses of microglia, but not astrocytes, suggest a role in selective vulnerability of dopamine neurons after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposure in monkeys. Glia 56, 1199–1214 14 Barcia, C. et al. (2004) Evidence of active microglia in substantia nigra pars compacta of parkinsonian monkeys 1 year after MPTP exposure. Glia 46, 402–409 15 Merkler, D. et al. (2006) Differential macrophage/ microglia activation in neocortical EAE lesions in the marmoset monkey. Brain Pathol. 16, 117–123 16 Liang, X. et al. (2008) The prostaglandin E2 EP2 receptor accelerates disease progression and inflammation in a model of amyotrophic lateral sclerosis. Ann. Neurol. 64, 304–314 17 Taylor, J.P. et al. (2002) Toxic proteins in neurodegenerative disease. Science 296, 1991–1995 18 Gibrat, C. et al. (2009) Differences between subacute and chronic MPTP mice models: investigation of dopaminergic neuronal degeneration and alphasynuclein inclusions. J. Neurochem. 109, 1469– 1482 19 Geula, C. et al. (1998) Aging renders the brain vulnerable to amyloid beta-protein neurotoxicity. Nat. Med. 4, 827–831 20 Recchia, A. et al. (2008) Generation of a alpha-synucleinbased rat model of Parkinson’s disease. Neurobiol. Dis. 30, 8–18 21 Philippens, I.H.C.H.M. (2008) Non-human primate models for Parkinson’s disease. Drug Discov. Today Dis. Models 5, 105–111

22 Burn, D.J. (2010) The treatment of cognitive impairment associated with Parkinson’s disease. Brain Pathol. 20, 672–678 23 van Vliet, S.A. et al. (2008) Exploring the neuroprotective effects of modafinil in a marmoset Parkinson model with immunohistochemistry, magnetic resonance imaging and spectroscopy. Brain Res. 1189, 219–247 24 LeWitt, P.A. (2006) Neuroprotection for Parkinson’s disease. J. Neural Transm. Suppl. 71, 113–122 25 ’t Hart, B.A. et al. (1990) Antiarthritic activity of the newly developed neutrophil oxidative burst antagonist apocynin. Free Radic. Biol. Med. 9, 127–131 26 Maczurek, A. et al. (2008) Lipoic acid as an antiinflammatory and neuroprotective treatment for Alzheimer’s disease. Adv. Drug Deliv. Rev. 60, 1463–1470 27 Mandel, S.A. et al. (2008) Simultaneous manipulation of multiple brain targets by green tea catechins: a potential neuroprotective strategy for Alzheimer and Parkinson diseases. CNS Neurosci. Ther. 14, 352–365 28 Koh, S.H. et al. (2006) The effect of epigallocatechin gallate on suppressing disease progression of ALS model mice. Neurosci. Lett. 395, 103–107 29 Lipton, S.A. (2007) Pathologically-activated therapeutics for neuroprotection: mechanism of NMDA receptor block by memantine and S-nitrosylation. Curr. Drug Targets 8, 621–632 30 Aggarwal, S. and Cudkowicz, M. (2008) ALS drug development: reflections from the past and a way forward. Neurotherapeutics 5, 516–527 31 Obinu, M.C. et al. (2002) Neuroprotective effect of riluzole in a primate model of Parkinson’s disease: behavioral and histological evidence. Mov. Disord. 17, 13–19 32 McGeer, P.L. and McGeer, E.G. (2008) Glial reactions in Parkinson’s disease. Mov. Disord. 23, 474–483 33 Salin´ska, E. et al. (2005) The role of excitotoxicity in neurodegeneration. Folia Neuropathol. 43, 322–339 34 Mattson, M.P. (2007) Calcium and neurodegeneration. Aging Cell 6, 337–350 35 Gao, H.M. and Hong, J.S. (2008) Why neurodegenerative diseases are progressive: uncontrolled inflammation drives disease progression. Trends Immunol. 29, 357–365

Ingrid H.C.H.M. Philippens Department of Immunobiology, Biomedical Primate Research Centre, Lange Kleiweg 139, 2288 GJ Rijswijk, The Netherlands Bert A. ‘t Hart Department of Immunobiology, Biomedical Primate Research Centre, Lange Kleiweg 139, 2288 GJ Rijswijk, The Netherlands German Torres Department of Neuroscience and Histology, New York College of Osteopathic Medicine of New York Institute of Technology, Old Westbury, NY 11568, USA [email protected]

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feature Securing reliability and validity in biomedical research: an essential task The buzzword ‘translational’ dominates concepts to optimize value creation from science. This article discusses the impact of ‘old’ and contemporary data on hypothesis generation in relation to human physiology and in the effort to optimally implement translational sciences. I outline how dogmas and errors, sometimes perpetuated over decades, impact contemporary research and drug discovery projects. As a consequence and to improve value creation from science, a reevaluation of old data (i.e. of the validity and reliability of research with regard to human physiology) seems necessary. In line with this, the compliance of newly generated hypotheses, assays and tools with a conceptual focus on human physiology as the gold standard seems essential. To achieve improved research success, several measures need to be initiated and guided by industrial and academic leaders in concert to have an impact on the quality of research in the very near future. There is no ‘holy grail’, but in general terms, a constructive but critical approach – not just to contemporary biomedical research – seems mandatory to avoid the errors of the past and enable solutions to evolve dynamically.

A recent article in the Financial Times proposed ‘drug research needs serendipity’ [1]. What seems rather more needed is an incentive to identify and question pre-existing errors and dogmas, some of which have evolved over decades, and to re-evaluate essential data, which build the foundation of our contemporary research with respect to their relevance to human biology. This requirement could be defined as ‘transcriptional science’ (TS). I will use this paraphrase to describe a conceptual approach analogous to translational science, simply because in biology, transcription is the rate-limiting step for translation, and, if it goes awry, it can lead to false or irrelevant products. To this author, translational science means that human physiology is positioned in the

centre of all biomedical sciences to enable the right questions to translate biomedical research into new therapeutics [2–4]. Thus, the reliability and validity (Table 1) of a given study needs to be assessed a priori. What, however, assures the reliability and validity of the data that are used to delineate translational hypotheses and generate the related experimental designs? An old Chinese saying implicates one aspect: old data should not be neglected but might require reconsideration in a new context (Fig. 1). In general, the quality and value of a study are only as good as the design of the study and, equally importantly, the previous data and related interpretations on which the new hypothesis will be built. This is where the idea of

1359-6446/06/$ - see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2010.10.004

TS steps in: TS aims to exclude data and related interpretations, which poorly relate to human conditions, whether generated in vitro or in vivo, for both hypothesis generation and the design of new studies. If possible, TS will re-evaluate data and extract the content that can be used for translational databases or hypothesis generation, a task that obviously requires manual data analysis and (re)interpretation. TS should serve as an essential prerequisite and quality control for translational research and related drug discovery projects; otherwise, errors will inevitably be perpetuated and invalidate all efforts made in translational sciences. TS requires that researchers approach their own work and the work of others (as well as the related interpretations) most critically, from ancient reports to www.drugdiscoverytoday.com

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TABLE 1

Definitions. Validity and reliability (partially adopted from Wikipedia.org and L.T.F. Gamut, Logic, Language, and Meaning: Introduction to logic, p. 115): Reliability does not imply validity. Both terms are used in test theories and relate to the logic and accuracy of a test, i.e. an experimental design and the related results, if adapted to biomedical research. A reliable measure is measuring something consistently, but one may not be measuring what is being intended to be measured. For example, while there are many reliable tests of specific biological reactions, not all of them would be valid for predicting, for example a glucose response to different stressors. In terms of accuracy and precision, reliability is analogous to precision, while validity is analogous to accuracy. An example often used to illustrate the difference between reliability and validity in the experimental sciences involves a common bathroom scale. If someone who is steps on a scale 10 times and gets readings of 25, 50, 100, 125, etc., the scale is not reliable. If the scale consistently reads ‘‘65’’, then it is reliable, but not valid. If it reads ‘‘80’’ each time, then the measurement is both reliable and valid. This is what is meant by the statement, ‘‘Reliability is necessary but not sufficient for validity.’’ Reliability requires better comparable experiments, while validity asks the question if the experiment is tailored to appropriately answer the questions being asked; i.e. if the experiment is valid in logic terms. In the dynamic context of increasing knowledge in biomedical research, both, reliability and validity of an experiment may require adjustment to the current status of science. I.e. in retrospective reliability and validity may need to be newly assessed for a given experiment, which may enable new hypothesis generation and even conclusions based on data generated earlier.

Perspective  FEATURE

modern biomedical research and contemporary work. Obviously, the quest for an approach that assures higher validity and reliability of data used for translational science implies that there are many dogmas and perpetuated errors in our scientific literature and community. Indeed, they do exist and might even have become stronger with time. Some of them seem to erode (i.e. drug-target selectivity as a predictor of desired therapeutic effects has been questioned), and the fact that most compounds are acting on multiple targets is implying new concepts [5]. Similarly, the animal models that are the cornerstones of a research field are being challenged [6].

The cortisol story Here, just one example, which I consider of major impact for our contemporary therapeutic concepts and drug discovery related to a plethora of pathological conditions, will be briefly discussed to demonstrate some interrelationships between various mechanisms that can contribute to the constitution of a dogma. Before getting into details, it should be mentioned that I am not questioning the undisputable negative effects of chronic excessive stress or hypercortisolism. However, that acute cortisol release enables coping with a variety of stressors to defend homeostasis and even enhances immunity [7] challenges the general view of the effects that ‘glucocorticoids’

[()TD$FIG]

as a drug class might have on immunity and inflammation from a teleological viewpoint. I argue that the almost standardized approach – that is, the interpolation of effects mainly generated by the use of synthetic glucocorticoids, which cannot be used in physiological concentrations per definition, or the supraphysiological use of cortisol, often in conjunction with the neglect of appropriate experimental design (there is no physiological state without cortisol present) – has perpetuated a false dogma: glucocorticoids as a drug class are, in general, considered to be immunosuppressive and anti-inflammatory, although the ‘class’ comprises compounds with highly different physiological and pharmacological profiles. Cortisone therapy fell out of favor in the 1950s because of undesired effects observed in high non-physiological doses. The voices of Hench’s contemporary colleagues, who emphasized that low, physiological replacement-like dosing regimens (i.e. lower than those currently considered low dose and using the endogenous cortisol, not prednisone or prednisolone) benefit patients and all the negative effects were due to the very high pharmacological regimens, were overheard [8,9]. When the patents for cortisone expired, new compounds were generated, which aimed to limit some of these undesired effects [10]. In this context, it is almost ironic that lowdose corticosteroid therapy with synthetic compounds, mainly prednisolone, has become a new standard [11].

Drug Discovery Today

FIGURE 1

On Ko Chi Shin, ‘The new ideas reside within the old.’ Confucius (551–479 BC). As this saying indicates, to (re-)investigate and understand old ideas is essential for innovation. 992

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Thus, in the very early days of corticosteroid research, dexamethasone (DEX) – a very potent, high-affinity synthetic steroid, which behaves completely differently to the endogenous hormone cortisol – became the gold standard. Fig. 2 summarizes how DEX differs from cortisol, with the sole exception that it also binds to the glucocorticoid receptors (GRs), but not the mineralocorticoid receptor (MR), which cortisol binds to with a higher affinity than the GRs. It was postulated that the sum of DEX’s effects on various targets would eventually reflect the physiological functions of cortisol; although the logic behind this concept has already been questioned, more than ten years ago, the wellsupported arguments had little to no impact [12,13]. The complex interactions of the GRs and a proposed regulation at the tissue level have been described recently [14]. In line with this, a variety of microarray studies have documented that the standard GR and MR agonists induce and repress an overlapping but not identical portfolio of genes: in human liver cells, for example, of a total of 300 genes that are variously regulated by cortisol or corticosterone (both binding to the MRs) and DEX (only GRs), only 25 are equivalently regulated by all three of the gold standard agonists (M. Cidlowski, personal communication). Thus, the interpolation from one compound to the other as a ‘class effect’ seems obsolete, not least because we know that targets like nuclear receptors might dynamically and highly specifically respond to different ligands [15]. A recent and elegant study, however, has demonstrated that minor changes in low physiological corticosteroid concentrations have a major impact on experimental arthritis (i.e. a decrease in local cortisol prevents bone destruction), which is in stark contrast with all clinical experiences with glucocorticoid treatment

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[()TD$FIG]

FIGURE 2

Selected differences affecting function: cortisol binding globulin; (a) metabolism by 11b-HSD; intracellular activation/recycling; binding to GR (five isoforms), MR (two isoforms); (b) GR and MR dimer formation; heterodimer formation occurs physiologically; receptor affinity; receptor–ligand interactions (conformational changes induced by ligand); receptor–ligand co-activator, repressor assembling; (c,d) transrepression/transactivation (differences between all GR ligands); liganddependent change of conformation and related effects. Endogenous cortisol differs from synthetic glucocorticoid receptor ligands (in particular, dexamethasone) at almost every step along the activation and metabolism pathways. Figure modified, with permission, from Ref. [14].

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in rheumatoid arthritis [16]. Interestingly, although this contention would have immediate consequences for cortisol research related to inflammation and immunity, the authors highlight other results as their major finding, rather than the conflict with current views. It seems there is a bias to stay within the accepted conceptual framework. Similar observations (i.e. a ‘white hat bias’) have been made in obesity research. In general, there seems to be a trend to interpret data within the mainstream framework of a research community [17]. The impact of this dramatic perpetuation of the bias in cortisol research on biomedical research and related drug discovery is best documented by the fact that the putative pro-inflammatory function and essential role of endogenous cortisol in cardiovascular disease, stroke and, possibly, many other diseases might have been overlooked for 50 years [18]. That DEX and similar synthetic glucocorticoids do not activate the MR, which is protected from occupation with cortisol in some, but not all, tissues by a cortisol-catabolizing enzyme [19], might explain why some of these proposed proinflammatory actions of cortisol via the MR [20] were overlooked for decades. The opportunities that will arise from these new discoveries could affect inflammatory conditions such as arthritis, asthma or even metabolic diseases [19,20]. It is also easy to imagine the immense consequences this observation could have had on public health if it had been discovered 40 years ago.

A dogma can evolve from various influences, including patenting interests and the standardization of mainstream thinking and experimental designs; protected by converging commercial and academic interests, it might predominate over decades of research and drug development. In the following paragraphs, some ideas are presented to avoid a similar situation and create new value from existing data.

Data integration and interpretation: improving content How could better ‘content’ in translational terms be achieved? Often, scientists will uncritically or in a biased manner extrapolate experimental conditions and related results from recent highprofile publications to design their studies because it is neither common nor appropriate to question the publications of scientific leaders. This procedure ensures that views and designs that, in retrospect, seem irrelevant from a translational, ‘human physiology first’ viewpoint are perpetuated. The new study, consequently, might result again in data with little physiological relevance, no matter how elegantly and elaborately it is performed. This consequence often seems to be overlooked by the editors and reviewers of leading journals, in which cuttingedge technologies sometimes dominate the evaluation of a manuscript over the generation and foundation of the hypothesis per se.

In conjunction with this constellation, systems biology [21], systems chemical biology [22], bioinformatics, semantics and other tools that aim to support translational science in general [23] also rely on the quality of data entered into the relevant database. As outlined above, however, these data might be confounded if they are not manually evaluated. Thus, all computational methods are certainly helpful to extract and organize data; however, they cannot replace content ranking by the human brain, which is still superior in validating complex data constellations and experimental designs. Creating transcriptional (i.e. high-confidence content) databases might seem an insurmountable obstacle in light of the exponential growth of data. However, if each project were to start with a standardized approach to ranking and interpreting data in hypothesis generation, with respect to their relevance to the in vivo dynamics of human physiology, improved validity and reliability of contemporary research seems feasible. The standards for such a content ranking approach need to be generated first, which is challenging because even defining which questions should be asked and identifying common denominators in a given field of research might seem too complex. Nevertheless, it is anticipated that even simple standards and ranking tools could improve the quality of new data considerable. There must be both academic and commercial interest in further developing

[()TD$FIG]

Identification of physiologically relevant data

Identification of physiologically irrelevant data

Definition of standardized translational/physiological experimental designs

Validated physiological relevant databases

Incorporation of semantic computational technologies with individual content ranking

Repetition of key experiments under physiological conditions

“Transcriptional Science”:

• Ensuring reliability and validity • Ensuring physiological content • Re-evaluation of existing data • Validation of existing data • Identification & elimination of dogmas Selected benefits:

• New views and theories • New uses for existing compounds • New therapeutic concepts • Optimized use of resources • Enabling open disputation? Drug Discovery Today

FIGURE 3

Transcriptional science: an essential prerequisite to enable translational sciences. If uncritically adopted, existing physiologically questionable or irrelevant data, related interpretations and dogmas will unequivocally be perpetuated and invalidate translational science a priori, including all financial investment. 994

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Experimental design and standardization: biomarkers and more When is an experiment physiologically relevant? It would be prudent to try to give a simple answer, but a direct consequence of this question is the fact that we need more standardized and truly physiologically relevant model systems to make data better comparable and, in particular, to rank them in a physiological context. First questions that come to mind are ‘When is an experiment reflecting male or female conditions?’ and ‘How relevant is this after all?’ In relation to the discussion above, small changes of cortisol dramatically impact on cellular functions (i.e. induce opposing functions in physiological concentrations) [26]. Findings like this force thought-provoking questions, such as ‘How many in vitro assays, not just in this field, are of physiological relevance at all?’ Ultimately, TS implies that experiments that built the foundation of previous drug discovery programs and studies in progress must still be analyzed with respect to their physiological relevance, but they might also need to be repeated (e.g. to include new control conditions), perhaps often. At first, repeating ‘old’ studies might have little appeal for researchers driven by their hunt for impact factor scores. If appropriately addressed, however, the identification of new, more physiological functions for any given physiological pathway could open new and unexpected avenues for innovative treatments. This would ensure progress for both science and the scientist. In addition to efforts to make data comparable, in particular for drug discovery, new standards for experiments might need to be agreed; generally defined and accepted biomarkers are one first approach. Regulatory authorities might require such initiatives sooner or later, but the consolidation of biomedical research forces a more efficacious use of resources now. If one only analyses the investments made in cortisol research since the patent expired in the 1950s (which was a major driver for new compounds at the time) and discovery projects based on data with limited translational relevance, it becomes obvious what resources could be released if our scientific approaches included a more aggressive evaluation of the existing fundaments on which

we build our views and opinions, which often are prematurely interpreted as the truth (Fig. 3).

Transcriptional science, an integral part of translational sciences and its culture In his review, titled ‘Translational research: forging a new cultural identity’, Barry Coller identified several challenges ahead, including the willingness to embrace change in general and to induce a new culture of scientific disputation [27]. Ensuring the reliability and validity of data generated in the paradigm of translational sciences will primarily require the implementation of a new culture in biomedical research, not another new terminology like ‘transcriptional sciences’, to assess data and scientific hypothesis more openly and with constructive criticism. Concepts such as endogenous angiogenic factors would not have been developed successfully if people like Judah Folkman had not resisted the opposition to their ideas and questioned dogmas throughout their careers. Scientists in the industry and academia will have to appreciate that critical comments might help to improve the impact of their work, if the criticism is conveyed in an appropriate and constructive manner, ideally before the work is started. Open discussions and converging expertises are the only measures that will ultimately ensure a higher return of investment for societies. ‘Open content ranking forums’, established by publishers for specific areas of research, might be one approach worth exploring. Within companies, specific IT solutions for in-house content ranking by the companies’ scientists, enabling them to participate proactively, could be developed. ‘Constructive devil’s advocates’ (i.e. experts in challenging designs and ideas) could be trained and become part of project teams to promote a better success (translation) rate. In general, eliminating misleading concepts and experiments at the right time with the right questions should be rewarded – for example, by progression of a project to a milestone – both in academia and the industry. In addition, negative data or data that do not fit our current views should be more appreciated. Challenging dogmas or falsifying accepted or new theories does not really exist as a research goal, although it is an equally logical and rewarding approach to generating knowledge [28]. After all, science gets exciting when things do not fit and new ground is touched. There is no holy grail to achieve a better return on investment in biomedical research related to personal effort and funding, but there are many ad hoc opportunities to improve the reliability and

validity of experimental designs and extract additional value from old knowledge. To unfold this potential and secure progress in finding new cures for unmet needs, however, will require a concerted approach that can only be initiated and guided by leaders from academia and industry in concert. References 1 Shawitz, D. and Taleb, N. (2008) Drug research needs serendipity. Financial Times 2 Littman, B.H. et al. (2007) What’s next in translational medicine? Clin. Sci. (Lond.) 112, 217–227 3 Nathan, D.G. (2005) The several Cs of translational clinical research. J. Clin. Invest. 115, 795–797 4 Fitzgerald, G.A. (2005) Opinion: anticipating change in drug development: the emerging era of translational medicine and therapeutics. Nat. Rev. Drug Discov. 4, 815–818 5 Mestres, J. and Gregori-Puigjane, E. (2009) Conciliating binding efficiency and polypharmacology. Trends Pharmacol. Sci. 30, 470–474 6 Martin, B. et al. (2010) ‘‘Control’’ laboratory rodents are metabolically morbid: why it matters. Proc. Natl. Acad. Sci. U.S.A. 107, 6127–6133 7 Dhabhar, F.S. (2009) Enhancing versus suppressive effects of stress on immune function: implications for immunoprotection and immunopathology. Neuroimmunomodulation 16, 300–317 8 Jefferies, W.M. (1955) The present status of ACTH, cortisone and related steroids in clinical medicine. N. Engl. J. Med. 253, 441–446 9 Jefferies, W.M. (1967) Low-dosage glucocorticoid therapy: an appraisal of its safety and mode of action in clinical disorders, including rheumatoid arthritis. Arch. Intern. Med. 119, 265–278 10 Jefferies, W.M. (1996) Safe Use of Cortisol. Charles C Thomas Publisher Ltd 11 Hoes, J.N. et al. (2007) EULAR evidence-based recommendations on the management of systemic glucocorticoid therapy in rheumatic diseases. Ann. Rheum. Dis. 66, 1560–1567 12 Wilckens, T. (1995) Glucocorticoids and immune function: physiological relevance and pathogenic potential of hormonal dysfunction. Trends Pharmacol. Sci. 16, 193–197 13 Wilckens, T. and De Rijk, R. (1997) Glucocorticoids and immune function: unknown dimensions and new frontiers. Immunol. Today 18, 418–424 14 Gross, K.L. and Cidlowski, J. (2008) Tissue-specific glucocorticoid action: a family affair. Trends Endocrinol. Metab. 19, 331–339 15 Margolis, R.N. et al. (2009) Chemical approaches to nuclear receptors in metabolism. Sci. Signal. 2 mr5 16 Buttgereit, F. et al. (2009) Transgenic disruption of glucocorticoid signaling in mature osteoblasts and osteocytes attenuates K/BxN mouse serum-induced arthritis in vivo. Arthritis Rheum. 60, 1998–2007 17 Cope, M.B. and Allison, D. (2010) White hat bias: examples of its presence in obesity research and a call for renewed commitment to faithfulness in research reporting. Int. J. Obes. (Lond.) 34, 84–88 18 Funder, J.W. (2009) Reconsidering the roles of the mineralocorticoid receptor. Hypertension 53, 286–290 19 Cooper, M.S. and Stewart, P.M. (2009) 11{beta}Hydroxysteroid Dehydrogenase Type 1 and Its Role in the Hypothalamus-Pituitary-Adrenal Axis, Metabolic

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this kind of knowledge base and content ranking, which is essential for informed decision making in any problem in biomedical sciences. Some aspects of the problem (i.e. too many neglected data) have been acknowledged [24], but the solution might require ‘competitive collaboration’, not only within the pharmaceutical industry [25] but also including academia.

PERSPECTIVE

PERSPECTIVE

Syndrome, and Inflammation. J. Clin. Endocrinol. Metab. 94, 4645–4654 20 Rickard, A.J. and Young, M.J. (2009) Corticosteroid receptors, macrophages and cardiovascular disease. J. Mol. Endocrinol. 42, 449–459 21 Krishna, R. et al. (2007) Effective integration of systems biology, biomarkers, biosimulation, and modeling in streamlining drug development. J. Clin. Pharmacol. 47, 738–743 22 Oprea, T.I. et al. (2007) Systems chemical biology. Nat. Chem. Biol. 3, 447–450

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23 Ruttenberg, A. et al. (2007) Advancing translational research with the Semantic Web. BMC Bioinformatics 8 (Suppl. 3), S2 24 Editorial, (2009) Data’s shameful neglect. Nature 461, 145 25 Bingham, A. and Ekins, S. (2009) Competitive collaboration in the pharmaceutical and biotechnology industry. Drug Discov. Today 14, 1079–1081 26 Lim, H.Y. et al. (2007) Glucocorticoids exert opposing effects on macrophage function dependent on their concentration. Immunology 122, 47–53

27 Coller, B.S. (2008) Translational research: forging a new cultural identity. Mt. Sinai J. Med. 75, 478–487 28 Popper, K. (1934) The Logic of Scientific Discovery, Springer, Vienna

Thomas Wilckens Jakob-Klar-Str. 7, 80796 Mu¨nchen, Germany [email protected] [email protected]

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A synopsis of the Tox21 initiative and a focus on the NIH Chemical Genomics Center’s efforts within this program using in vitro methods and quantitative high-throughput screening.

Sunita J. Shukla, Ruili Huang, Christopher P. Austin and Menghang Xia NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, USA

The US Tox21 collaborative program represents a paradigm shift in toxicity testing of chemical compounds from traditional in vivo tests to less expensive and higher throughput in vitro methods to prioritize compounds for further study, identify mechanisms of action and ultimately develop predictive models for adverse health effects in humans. The NIH Chemical Genomics Center (NCGC) is an integral component of the Tox21 collaboration owing to its quantitative high-throughput screening (qHTS) paradigm, in which titration-based screening is used to profile hundreds of thousands of compounds per week. Here, we describe the Tox21 collaboration, qHTS-based compound testing and the various Tox21 screening assays that have been validated and tested at the NCGC to date. Introduction Traditionally, the toxicological evaluation of environmental chemicals has largely relied on animal models that have been used to extrapolate to potentially harmful events in humans. These models have been developed to evaluate specific toxicological endpoints, such as oral, dermal and ocular toxicity; immunotoxicity; genotoxicity; reproductive and developmental toxicity; and carcinogenicity. Although these animal models have provided useful information on the safety of chemicals, they are relatively expensive, low throughput and sometimes inconsistently predictive of human biology and pathophysiology. Recently, several major new initiatives have begun to utilize in vitro methods and a variety of new technologies to develop in vitro signatures and computational models predictive of in vivo response. These initiatives should enable researchers to identify a battery of in vitro assays that will detect perturbations in cellular pathways that are expected to contribute to or result in adverse health effects [1]. Furthermore, these initiatives represent a welcome movement away from traditional in vivo high-dose hazard studies [1]. To appreciate the scientific and technological advancements that are shaping toxicity testing today, it is important to appreciate where this new paradigm fits in the context of historical testing.

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Foundation review: The future of toxicity testing: a focus on in vitro methods using a quantitative high-throughput screening platform Dr Sunita J. Shukla is currently a postdoctoral fellow at the NIH Chemical Genomics Center (NCGC). She is currently working under the guidance of Dr Menghang Xia in developing in vitro toxicity-based assay screening using a quantitative high-throughput screening platform. Furthermore, she is also working under the guidance of Dr Doug Auld in the area of assay development and high-content screening. Dr Shukla is the first recipient of the Humane Society/Procter and Gamble Fellowship honoring the advancement of alternatives to animal testing. Before joining NCGC, she received her Ph.D. in Human Genetics, with a focus on pharmacogenetics of anticancer agents, from the University of Chicago in the lab of Dr M. Eileen Dolan in 2007. Additionally, she received a Master of Public Health degree at Saint Louis University with a focus on Epidemiology and Environmental/ Occupational Health in 2001. She has authored or co-authored 13 peer-reviewed publications. Dr Christopher Austin is director of the NIH Chemical Genomics Center (NCGC) and senior advisor to the Director for Translational Research at NHGRI. The NCGC is an ultra-highthroughput screening, informatics and chemistry center that develops novel compounds as probes of biology and starting points for development of new drugs for rare and neglected diseases, profiles small-molecule libraries for biological and toxicological activities and develops new paradigms to increase the efficiency and genome-wide reach of assay, screening, chemistry and informatics technologies. Dr Austin received his A.B. from Princeton and M.D. from Harvard, trained in neuroscience and genetics at Harvard, and came to NIH in 2002 from Merck. Dr Menghang Xia is group leader of cellular toxicity and signaling at the NIH Chemical Genomics Center (NCGC). Dr Xia and her research group are currently focused on the target-specific and mechanism-based toxicological studies, in collaboration with the Biomolecular Screening Branch at the National Toxicology Program (NTP) and National Center for Computational Toxicology at the US Environmental Protection Agency (EPA). Her group has developed and validated a battery of in vitro toxicological assays using a quantitative high through screening platform, and investigated the mechanism of action of chemicals in multiple cellular signaling pathways. Dr Xia received her Ph.D. from State University of New York at Buffalo, did postdoctoral training at University of California at San Francisco, and joined NCGC in 2005 from Merck.

Corresponding author:. Xia, M. ([email protected]) 1359-6446/06/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.drudis.2010.07.007

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accepted vision for future toxicology testing, calling for the development and use of in vitro models in human cells of toxicological response based on automated high-throughput screening (HTS) of pathway-based cellular assays related to toxicity and computational modeling [12]. The report envisioned that initially, such less expensive and higher throughput assays could be used to evaluate the modes of action of chemicals for more comprehensive testing programs and that eventually these data would enable the rapid and mechanism-based prediction of in vivo biological responses [2,13,14]. To move this research agenda forward, the NTP partnered with the NIH Chemical Genomics Center (NCGC) in 2005 to pilot the chemical, biological and informatics processes required for the transition from predominantly in vivo to in vitro toxicology. In 2006, this partnership was expanded to include the EPA. In 2008, in recognition of successful proof-of-principle studies [3,15] and galvanized by the NRC report, the ‘Tox21’ collaboration was formally established via a Memorandum of Understanding among the agencies (http://ntp.niehs.nih.gov/) and publication of a policy paper from the senior leadership of the three organizations [16]. The Tox21 collaboration takes advantage of the complementary strengths of the three partners (Fig. 1). The NTP, a trans-Department of Health and Human Services program headquartered at the NIH National Institute of Environmental Health Sciences, has enormous experience in experimental toxicology. The NCGC, a trans-NIH program administered by the National Human Genome Research Institute, has unparalleled capacity and expertise in in vitro assays, titration-based screening and informatics. The EPA National Center for Computational Toxicology, part of the EPA’s Office of Research and Development, has deep computational

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The advent of technological innovations in molecular and cellular biology prompted the National Toxicology Program (NTP) to propose a new Roadmap in 2004, ‘A National Toxicology Program for the 21st Century’ [10], focusing on three main areas: refining traditional toxicology assays, developing rapid mechanism-based predictive screens and improving the overall utility of data for making public health decisions. This Roadmap placed an increased emphasis on the use of alternative assays for identifying key pathways and molecular mechanisms linked to disease [10]. The US Environmental Protection Agency (EPA) started its ToxCast program in 2006 to address many of the same issues [11]. When these programs were in their early stages, a 2007 report from the National Research Council (NRC) entitled Toxicity Testing in the 21st Century (Tox21) enunciated what has become a widely

y nc ge nA tio gy ec olo rot es xic tat al P To ter d S nt en nal me ite l C tio Un iron na puta v tio En Na Com for

Toxicity testing in the 21st century and the US Tox21 partnership

Na

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Since its inception, toxicity testing has relied on animal models treated at maximum tolerated dose levels, with the results extrapolated to human health outcomes at lower doses. This approach dates back to the 1950s, when the use of more specific or mechanistic animal models, and knowledge of the underlying mechanisms for any particular toxicological response, were relatively unknown [2]. Such in vivo testing is costly, time consuming and low throughput [3]. The complete toxicological profiling of one chemical in standard in vivo assays consisted of the following toxicity tests: acute, subchronic and chronic toxicity; reproductive toxicity; developmental toxicity, ocular and skin irritation, hypersensitivity, phototoxicity and toxicokinetic studies [4]. Despite the disadvantages associated with testing in animals, the majority of the understanding regarding chemical toxicity has come from data obtained in such systems [5]. Even extensive animal testing does not provide a mechanistic understanding of toxicity and knowledge concerning adverse risks to humans is still inadequate [6]. Hence, a need for more mechanistic data and a ‘theoretical framework for rational decision making’ was noted in the early 1980s [6]. More recently, there have been numerous studies highlighting intra- and inter-species differences in mammals, including humans. Williams and Weisburger [7] pointed out that intraspecies differences among different mouse strains affect the severity and incidence of neoplasms, making extrapolation of various cancers from mice to humans difficult. Inherent resistance to spontaneous and malignant tumors in nonhuman primate models has also led to variations in the manifestation of disease across these species [8]. In addition to inter- and intra-species differences in disease models, other species-specific differences that affect disease outcome and extrapolations include differences in basal metabolic rate, metabolic pathways, cancer type (sarcomas in mice versus carcinomas in humans), genetic aberrations associated with tumors, and telomere biology, especially with regard to humans and mice [9]. In addition to physiologic differences, the difference in observed high-dose toxicity in rodents and low-dose risks in humans will require knowledge of physiological differences with regard to mode, tissue of exposure, mechanism of action and knowledge of previous in vitro data regarding the agent in question.

es

Traditional toxicity testing methods

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FIGURE 1

The Tox21 collaboration. The Tox21 collaboration brings together the experimental toxicology expertise of the NTP, high-throughput screening technology at the NCGC and computational toxicology expertise at the EPA.

toxicology expertise [14]. This combined expertise has enabled rapid implementation of the NRC vision, the application of novel methodologies to evaluate a large number of chemicals in a range of in vitro assays in a short period of time [17]. Although realization of the NRC vision might ultimately require a research effort on the scale of the Human Genome Project [18], success of this effort would be transformational for toxicology testing for environmental and pharmaceutical chemicals, providing cheaper, faster and more accurate assessment of the toxicological potential of new chemicals.

Role of the NCGC in the Tox21 collaboration The NCGC was established in 2004 as the first assay development, screening, informatics and chemistry center of what was to become the NIH Roadmap Molecular Libraries Probe Production Center Network. The Molecular Libraries Initiative, a component of the NIH Roadmap for Medical Research, was born from the need for new approaches to determine function and therapeutic potential of human genes on the heels of the Human Genome Project and to accelerate the pace at which basic research is translated into small-molecule therapeutics [17] (http://nihroadmap.nih.gov/ molecularlibraries/). As part of the NCGC’s technology development program, a platform for automated testing of hundreds of thousands of compounds in titration-based format over a short period of time was developed [13,19], and this quantitative highthroughput screening (qHTS) platform has become a central aspect of the Tox21 program. Traditional biological assays have been low throughput, employing animal models and labor-intensive testing of samples. Furthermore, the growth of small-molecule collections required the development of HTS technologies to test a large number of compounds in a timely manner [20]. Although HTS has successfully enabled the screening of large chemical libraries to generate hits for medicinal chemistry optimization in the setting of drug discovery, HTS as traditionally practiced is not suitable for toxicity testing because it assays each compound at only single concentration [19] and, thus, generates large numbers of false positives (FPs) and false negatives (FNs) [21]. By contrast, the qHTS paradigm tests each compound at multiple (7–15) concentrations across an approximately four-log concentration range, thus producing concentration–response-based activity profiles of all compounds from the primary screen with greatly reduced FN and FP rates. Miniaturized assay volumes (<10 mL/well) in a 1536-well-plate format provides the throughput to generate concentration–response curves (CRCs) for every compound library member tested [22]. Curve fitting and CRC classification characterizes each curve based on parameters, such as curve fit and efficacy after the primary screening in a qHTS format, enabling the identification of structure–activity relationships (SARs). The NCGC makes its screening data publicly available through PubChem (http://pubchem.ncbi.nlm.nih.gov) and its software available on its website (http:// www.ncgc.nih.gov/pub/openhts/) to enable the scientific community to utilize the data in their own research [23]. The ability of qHTS to produce reliable activity profiles of chemicals has also enabled the NCGC to profile large libraries of chemicals for their propensity to produce assay artifacts, which would otherwise be interpreted as true biological effects [24]. NCGC has taken advantage of titration-based screening to identify

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compounds that produce a wide variety of different artifactual activities, including apparent enzyme inhibition through compound aggregation [25], compound autofluorescence [26] and firefly luciferase inhibition [27]. These profiling examples demonstrate the utility of qHTS in distinguishing true effects from artifacts for more reliable toxicity screening and efficient chemical probe development.

NCGC chemical library collection used for Tox21 assays An essential component of NCGC’s qHTS paradigm is the availability of large chemical libraries in a titration-based format. The availability of several concentrations across different plates gives the user flexibility to use concentrations relevant to the assays. In total, the NCGC has well over 400,000 compounds from the NIH Molecular Libraries Small Molecule Repository and NCGC-specific compound collections. The latter currently includes approximately 1400 compounds each from the NTP and EPA compound libraries and 2816 clinically used drugs in the NCGC Pharmaceutical Collection (NPC). Overall, compound selection is based on having a defined chemical structure and known purity, in addition to the extent of each compound’s solubility in dimethylsulfoxide (DMSO) [13]. One limitation of compound storage in DMSO is precipitation (augmented by DMSO water content and number of freeze–thaw cycles) [28]. Compound integrity studies using P450 assays and qHTS at NCGC revealed decreased compound potency over time and lower efficacy of older samples stored in DMSO [29]. For this reason, compounds are used in screening collections at the NCGC for no longer than four to six months. The current NTP compound collection consists of 1408 compounds, with more than 50 of the compounds represented twice to assess assay reproducibility. The NTP collection includes solvents, fire retardants, dyes, preservatives, plasticizers, therapeutic agents, inorganic and organic pollutants, drinking water disinfection byproducts, pesticides and natural products [3]. Selection of the 1408 compounds was partly based on the availability of toxicological data from standard tests of carcinogenicity, genotoxicity, immunotoxicity and/or reproductive and developmental toxicity [3]. Compounds were prepared as 10 mM stock solutions in DMSO and 14 plates representing 2.23-fold dilutions in 1536-well compound plates from 384-well plates [30]. The current EPA collection consists of 1462 compounds prepared similarly to the NTP compound collection. Compounds were primarily selected based on the need to screen and prioritize environmental chemicals to which humans are exposed through the environment or food. These chemicals include those known to be bioactive, those manufactured or used in large quantities and those to which humans are exposed on a routine basis [31]. In the near future, approximately 1400 additional compounds will be added to each of the NTP and EPA libraries for testing as an integrated Tox21 library [14]. The NPC collection (R.H. et al., unpublished) was prepared at the NCGC and currently contains 2816 small molecules, 52% of which are approved by the Food and Drug Administration for human or animal use in the United States. The remaining drugs are either approved for use in other countries, such as Europe, Canada or Japan, or are compounds that have been tested in clinical trials. The majority of the NPC collection is prepared as a 10 mM stock in DMSO and prepared as 15 2.23-fold dilution plates in 1536-well format [30]. Currently, an additional 1400 compounds are being www.drugdiscoverytoday.com

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added to the NPC collection. The next phase of Tox21 testing will begin later this year, using the combined NTP, EPA and NPC compound sets, totaling more than 10,000 chemicals.

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The NCGC characterizes toxicity endpoints in cell-based assays utilizing integrated robotic systems combined with batteries of in vitro assays and computational analysis [32]. The NCGC robotic platform stores large compound collections, performs distinct assay steps and measures user-defined assay outputs in an integrated manner [23]. The compound storage unit is capable of storing approximately 300,000 compounds in seven-point titrations, which correlates to more than 2.2 million compound samples [23]. A pin transfer station performs the transfer of 23 nL of compound from a 1536-well compound plate to a 1536-well assay plate, with each plate holding up to 1408 compounds (located in columns 5–48). Assay-specific controls (located in columns 1–4) are located on an additional 1536-well compound plate and transferred simultaneously with the test compounds to the assay plate [23]. Solenoid dispensers, having the capability of dispensing volumes ranging from 200 nL to 20 mL, are used for reagent and cell dispensing. Furthermore, up to eight tips can be used for dispensing in either 908 direct dispense or 458 angled head dispense with regard to the well. This allows for the modification of straight head and angled dispense, depending on the reagent type and condition of cells (i.e. if they are grown in a delicate monolayer, then it might be suitable to use the angled head dispense). For the aspiration of liquid, each dispenser comes equipped with an aspiration head made of 32 stainless-steel tubes for columnwise removal of reagents or media from the plate. The aspirator head enables cell washing and fixing in 1536-well format [33] for cell-cycle protocols or protocols involving antibody steps. There are several factors that can be optimized with the dispenser and aspirators, such as dispense volumes, aspiration depth and aspiration speed [23]. There are four different types of detectors that are currently integrated into the robotic system. These detectors essentially enable a wide variety of assays to be performed at NCGC and accommodate various assay technologies. Furthermore, these readers cover the entire spectrum of speed and information content. Although charged-coupled-device-based camera imagers are capable of very fast read times per 1536-well plate (<1 min/plate), they provide the least information regarding characteristics of individual cells. The converse is true for confocal-based imaging readers, which can provide detailed information on subcellular structures with longer read times (1 hour/plate). Specifically, the EnVision and ViewLux (PerkinElmer) are photomultiplier-tubeand charged-coupled-device-based instruments, respectively, which cover a wide range of fluorescence, absorbance and luminescence [3,15,34,35] bulk well readouts commonly used in highthroughput assays [23]. The ViewLux can be used for luminescence, fluorescence, absorbance, time-resolved fluorescence resonance energy transfer (TR-FRET) [36] and fluorescence polarization assays [23]. The EnVision is best suited for AlphaScreen assays and b-lactamase reporter assays [37,38] and can be customized for the detection of multiple wavelength regions [23] and TR-FRET assays [36]. Each assay format and readout has its own set of advantages. For example, the use of b-lactamase as a reporter has several 1000

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advantages, such as ratiometric readouts from dual emissions (460 and 530 nm), which minimizes well-to-well and plate-toplate variation caused by differences in plating density. In addition, the 530 nm fluorescent signal can be used as an indication of cell viability (and a proxy for compound cytotoxicity) and auto fluorescence [37]. For imaging assays, the user must sacrifice speed to obtain more information about individual cells and characterized cell populations. Two imaging platforms used at the NCGC are the Acumen Explorer (TTP Labtech) and the IN Cell Analyzer 1000 (GE Healthcare). The Acumen Explorer is a photomultiplier-tube-based laserscanning microplate cytometer that is equipped with three excitation and four emission lasers for enumeration and characterization of fluorescent objects [39]. The Acumen is able to provide total well and individual cell fluorescence readings. Compared to the ViewLux, where the entire plate is read in less than 1 min, Acumen read times can vary between 10 and 20 min per plate, depending on the number of wavelengths required by the assay of interest. Thus far, the Acumen has been used in several assays performed at NCGC, such as GFP-based assays [33] and multiplexing of dual fluorescent drug-sensitive and drug-resistant cell lines [40]. For higher resolution automated fluorescent imaging, the IN Cell Analyzer is designed to collect data either at the singlecell or at the subcellular level. The data collected are often complementary to those collected from the Acumen because additional orthogonal phenotypes can be identified. Furthermore, the instrument comes with own algorithm to analyze the data acquired [41]. Once data have been obtained for each assay, the CRCs for each compound are analyzed and classified as previously described [3,19,36]. Briefly, raw plate reads for each titration point are normalized relative to an assay-specific positive control (100% or 100%) and DMSO-only wells (0%), then corrected by applying a pattern correction algorithm using DMSO-only plates at the beginning and end of each stack [3]. Half-maximal inhibition or activation concentration (AC50) and efficacy values are obtained from fitting the concentration–response titration points to the Hill equation [42]. Compounds are classified as curve classes 1–4 according to the characteristics of the CRC, such as efficacy and quality of curve fit (R2). Class 1 curves display two asymptotes, whereas class 2 curves display one asymptote. Class 1 and 2 curves are further subdivided into subclasses a (efficacy  6SD) and b (efficacy < 6SD). These curves have statistically significant curve fits and are usually selected for follow-up analyses. Compounds in curve class 3 only display activity at the highest concentration tested, and compounds with class 4 curves show no concentration response and are deemed inactive. The ability to decipher curves using qHTS for every compound tested is important because many responses, such as toxicity, are measured over broad concentration ranges (typically between 0.5 nM and 92 mM), which might greatly decrease the FP and FN rates.

Assay implementation for Tox21 Various assays (Table 1) in different cell types or lines (Table 2) have been successfully developed, miniaturized and validated for 1536-well plate format at NCGC and screened against the initial Tox21 compound collection. For the majority of assays, compound incubation durations were limited to 48 hours because

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TABLE 1

Assay

Assay endpointa

Cell type

Assay readout

Refs

Cell viability Apoptosis

Intracellular ATP content Caspase-3/7

Hek293; Jurkat; HepG2; SH-SY5Y; SK-N-SH; BJ; HUV-EC-C; MRC-5; mesangial; kidney proximal tubules; N2a; H-4-II-E; NIH3T3

Luminescence

[3,15]

Membrane integrity

LDH release Protease release

Hek293; mesangial

Fluorescence Luminescence

[34]

Mitochondrial toxicity

Membrane potential

HepG2

Fluorescence

DNA damage

Micronucleus

CHO

Fluorescence

Cytokine

IL-8; TNF-a

THP-1

Homogeneous time-resolved fluorescence

Nuclear receptor

AR; ERa; FXR; PPARd; PPARg; RXR; TRb; VDR GR hPXR; AhR; rPXR

Hek293

b-Lactamase reporter

AP-1; HIF-1a; SIE; NFkB HSR; ESRE ARE/Nrf2 CREB p53 ARE/Nrf2; HSR; ESRE

ME-180 HeLa HepG2 Hek293, CHO HCT-116 HepG2

b-Lactamase reporter

Luciferase reporter

[58]

Thallium influx

U-2OS

Fluorescence

[45]

Toxicity pathway

hERG channel

HeLa HepG2

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Tox21 assays currently available at NCGC

Luciferase reporter [37,38,59,60] [61] [53]

a

AhR, aryl hydrocarbon receptor; AP-1, activator protein-1; AR, androgen receptor; ARE/Nrf2, antioxidant response element/NF-E2 related factor 2; CREB, cAMP response element binding; ERa, estrogen receptor a; ESRE, endoplasmic reticulum stress response element; FXR, farnesoid X receptor; GR, glucocorticoid receptor; HIF-1a, hypoxia-inducible factor-1a; hPXR, human pregnane X receptor; HSE, heat shock response element; IL-8, interleukin-8; LDH, lactate dehydrogenase; NFkB, nuclear factor kappa B; PPARd, peroxisome proliferator-activated receptor d; PPARg, peroxisome proliferator-activated receptor g; rPXR, rat pregnane X receptor; RXR, retinoid X receptor; SIE, sis-inducible element; TNFa, tumor necrosis factor a; TRb, thyroid hormone receptor b; VDR, vitamin D receptor.

of evaporation-induced edge effects observed in 1536-well plates [3]. Stainless-steel assay lids with rubber gaskets were also used to enable air exchange and minimize edge effects [23]. The assays described below demonstrate the adaptation of existing low-

TABLE 2

Cell lines tested Cell type

Origin

Species

BJ

Foreskin fibroblasts

Human

CHO

Chinese hamster ovary

Hamster

HCT-116

Colorectal carcinoma

Human

Hek293

Embryonic kidney cells

Human

HeLa

Cervical carcinoma

Human

HepG2

Hepatocellular carcinoma

Human

HUV-EC-C

Vascular endothelial cells

Human

H-4-II-E

Hepatoma

Rat

Jurkat

T-cell leukemia

Human

Kidney proximal tubules

Freshly isolated from kidney

Rat

MRC-5

Lung fibroblasts

Human

Mesangial

Renal glomeruli

Human

ME-180

Cervical carcinoma

Human

N2a

Neuroblastoma

Mouse

NIH3T3

Embryonic fibroblasts

Mouse

SH-SY5Y

Neuroblastoma

Human

SK-N-SH

Neuroblastoma

Human

THP-1

Monocytic leukemia

Human

U-2OS

Osteosarcoma

Human

throughput assays to higher throughput, reliable 1536-well format assays. These studies show the ability to modify existing assays to profile larger numbers of compounds for toxicity-based safety studies. To assess chemical effect on cell membrane integrity, a newly developed cytotoxicity assay that measures released intracellular proteases upon membrane damage with a bioluminescent assay readout was evaluated [34]. Although there have been similar assays developed for lower density formats, few have been miniaturized and validated in a high-throughput format with a robust assay signal [34]. This protease-release assay for membrane damage detection was miniaturized in 1536-well format and was screened against the initial NTP compound collection in HEK 293 and human renal mesangial cells [34] (Tables 1 and 2). All compounds were tested in 14-point titration series to identify compounds that disrupt membrane integrity. The assay performed well in miniaturized format, with high reproducibility of the control compound across every plate in both cell lines, high signal-to-background ratios and high Z0 values (a statistical measure of assay performance) [43]. In addition, replicate compounds within the NTP compound collection demonstrated high intra-experimental reproducibility, further indicating the reliability of the qHTS assay. The compounds identified from this assay were known membrane disrupters, including a-solanine and zinc pyrithione. The majority of compounds active in both cell lines were detergents such as digitonin, tetra-N-octylammonium bromide and p-n-nonylphenol, which are known to disrupt membrane integrity; additional non-detergent compounds known to be membrane disrupters were also identified in the assay. Furthermore, some compounds were shown to be uniquely active in one cell line or the other, thus revealing cell-line-specific membrane disruption potential. www.drugdiscoverytoday.com

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Overall, this study demonstrated the successful miniaturization of an existing cytotoxicity assay using a luminescent readout. Furthermore, the application of qHTS to this assay format validated the need to characterize the biological activities of compounds over a broad concentration range [34]. Cardiotoxicity has been commonly examined in the human ether-a-go-go-related gene (hERG) potassium channel. The hERG channel is responsible for the repolarization of cardiac action potential, which is associated with certain forms of inherited and acquired long QT syndrome (LQTS) [44,45]. LQTS can lead to sudden death through a rare ventricular arrhythmia [45]. Some drugs have been removed from the market because of their potential to induce LQTS by inhibiting the hERG channel, which warrants the need for premarketing screening of drugs to minimize the risk of sudden death in the treatment of non-life-threatening diseases [45,46]. Patch-clamp electrophysiology technique is still the gold standard for hERG activity in drug development [47,48], but this assay is low throughput and costly and requires specialized training for personnel [45]. The radioligand-binding assay is also commonly used to test compound binding to the hERG channel, but this assay gives little or no information on the functional effect of the ligand on the channel (blocker, activator or no effect) and the allosteric effect of the ligands [49]. To overcome these limitations, a functional assay was developed for the hERG channel by measuring thallium influx into the cells and was validated in a 1536-well plate format. The assay principle is shown in Fig. 2, where thallium ions enter the cells through open hERG channels after stimulation and bind to the dye, yielding an increase in fluorescence. This fluorescence signal is inhibited in the presence of hERG channel blockers [45]. The qHTS screen identified a group of known hERG inhibitors, such as pimozide, amiodarone and verapamil, from a library of 1280 pharmacologically active compounds (LOPAC1280). Furthermore, the activities of the hERG channel inhibitors in the thallium influx assay are well correlated

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with those obtained from automated patch-clamp experiments [45].

In vitro assays for cytotoxicity One of the goals of the Tox21 collaboration is to establish in vitro signatures of in vivo human and rodent toxicity. To create in vitro signatures of compound cytotoxicity across species, 1408 compounds from the initial NTP collection were profiled for cytotoxicity across 13 different human and rodent cell types [3]. These human and rodent cell types were derived from six tissue types that are common targets of xenobiotic toxicity (Tables 1 and 2); thus, this study aimed to develop species- and cell-specific cytotoxicity profiles for each compound [3]. Each compound was tested at 14 concentrations (0.5 nM–92 mM) in a luminescent cell viability assay that measures adenosine triphosphate (ATP) levels of metabolically active cells. The luminescent ATP quantitation assay worked well in 1536-well format with robust Z0 , signal-tobackground ratio and coefficient of variation values for the positive control compound (tamoxifen). Tamoxifen CRCs for each cell line were consistent across the plates; however, the response pattern was cell-line specific, with Jurkat cells being most sensitive and mesangial cells being least sensitive [3] (Fig. 3). Furthermore, the correlation of IC50 values for the 55 duplicate compounds present in the NTP compound collection across all 13 cell lines was significant (0.71, P < 0.001). There were 428 compounds that displayed cytotoxicity in at least one cell type, and clustering analysis was performed to decipher cytotoxicity profiles across cell types. Within the subgroup of active compounds, multiple effects from the compounds were identified within and across compound types, cell types and species. For example, human- and rodent-derived cells including SH-SY5Y, Jurkat, H-4-II-E, NIH 3T3, N2a, HEK293 and rat renal proximal tubule cells were most sensitive to compound-induced cytotoxicity, whereas human fibroblast and skin cells were least sensitive. Overall, the rodent cells used in

[(Figure_2)TD$IG]

FIGURE 2

Principle of the thallium flux assay. At resting state, cells expressing hERG channels are loaded with dye from the assay kit. Upon stimulation, thallium ions enter the cells through open hERG channels and bind to the dye, yielding green fluorescence upon excitation, proportional to the bound dye. 1002

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[(Figure_3)TD$IG]

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0 HEK293 HepG2 SH-SY5Y SK-N-SH Jurkat BJ HUV-EC-C MRC-5 Mesangial Proximal tubules H-4-II-E N2a NIH 3T3

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Positive control titration across human and rodent cell lines. The reproducibility of tamoxifen, used as a positive control in ATP-mediated cytotoxicity testing, is shown for each cell line for 234 plates tested. Jurkat cells were most sensitive to tamoxifen, and NIH3T3, HEK293, BJ and mesangial cells were least sensitive.

this study demonstrated more sensitivity to the compounds tested than the human cells. Compounds that showed activity in at least one cell type were clustered according to their IC50 values, revealing clusters and specific compounds that were selectively cytotoxic

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in a particular cell type and species. For example, digoxin was more cytotoxic in human HEK293 cells than rat renal proximal tubule cells. Actinomycin D, however, was much more cytotoxic in rat renal proximal tubules than in human HEK293 cells (Fig. 4). Overall, a striking finding was the lack of concordance in the patterns of compound activity in cells derived from the same tissue but from different species (there were also instances in which cells with similar tissue origin in the same species showed discordance in compound activity profiles), highlighting inter-species differences in response. Thus, an important finding from the study is that in vitro cytotoxicity in a particular cell type, even if from the same tissue and/or species, does not necessarily predict cytotoxicity in another cell type [3]. Thus, the combination of in vitro profiling with qHTS enables the generation of hypotheses related to mechanisms of toxicity and prioritization for more intense toxicological investigation related to in vivo toxicity. The application of clustering to data with multiple endpoints can help uncover underlying mechanisms involved in broad phenotypes such as cytotoxicity. To examine the mechanism of compound-induced cytotoxicity in various cell types, two different endpoints (cytotoxicity and caspase-3/7 activation) were assessed by testing the 1408 NTP compounds for both endpoints across 13 different cell types [15]. The cytotoxicity and caspase-3/7 assays performed well in a 1536-well format and the quality of the data was suitable for use in computational efforts. The overall active rate for the 13 caspase assays (0.4–3.5%) was lower than the rate for the 13 cytotoxicity assays (4–11%). Hierarchical clustering

FIGURE 4

Species-selective compounds. Compound activity patterns were obtained through hierarchical clustering of compound IC50 values. Shown are compound activity patterns for human HEK 293 cells and rat renal proximal tubule cells. Two compounds, actinomycin D and digoxin, illustrate species-specific cytotoxicity for those particular compound clusters. www.drugdiscoverytoday.com

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based on compound cytotoxicity and caspase EC50/IC50 patterns revealed similar clustering based on endpoints rather than cell type, indicating that cytotoxicity and caspase activation assays provide distinct sets of information, and that most compounds induce cytotoxicity through mechanisms other than caspase-3/7 activation. The only exception was the Jurkat cell line, where the caspase and cytotoxicity assays clustered together. One explanation might be that the cell death induced by most compounds in Jurkat cells is dependent on caspase-3/7 activation [15]. The N2a cell line seemed to have the least number of active compound overlap between the cytotoxicity and caspase assays, indicating the contribution of mechanisms outside of caspase activation for cytotoxicity. This approach will be useful for generating hypotheses for compound mechanism of action; however, hypothesis generation will be strengthened by the inclusion of more compounds and endpoints to build stronger models predictive of in vivo toxicity.

Computational modeling Because of the rapidly increasing number of environmental chemicals that need to be tested and the need for prioritization of those compounds for in vivo studies, more computational modeling is needed to complement experimental approaches to decrease the time associated with testing and accelerate prioritization of the data [50,51]. Some challenges associated with computational modeling of toxicity data include the diversity of compounds and structures that can produce the same outcome [50]. However, the production of CRCs for every compound tested provides a data-rich resource for SAR analysis, computational modeling and

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chemical prioritization for more extensive toxicological evaluation. The NCGC recently developed a weighted feature significance (WFS) algorithm, a fragment-based approach that does not rely on whole molecule similarity to model toxicity (Fig. 5), which is designed to achieve good prediction with structurally diverse sets of compounds [50]. Such approaches can be applied to generate testable hypotheses on mechanisms of compound toxicity. Starting with the structure of a compound, one could model and predict its toxicity in one assay or cell type and in multiple cell types, which essentially forms the activity pattern or signature that indicates the compound’s mechanism of toxicity. Models were developed for two aforementioned [3,15] assays, with rigorous performance evaluation of all models using receiver operating characteristic curves [52]. One advantage of the WFS approach is its ability to identify structural features responsible for toxicity [50]. For example, structural features significantly enriched in the pan-cytotoxic compounds include substituted benzenes, 1,3-dienes, heavy metals and imines [50]. Toxic features were also identified for caspase-3/7-activating compounds [15], such as cyclic alkyl ketones and alkyl halides. Thus, the significant toxic features present in compounds could be used to predict a particular mechanism of toxicity, such as caspase-3/7 activation [50]. Overall, the WFS approach can be applied to model other toxicity endpoints such as mutagenicity and hepatotoxicity and might be applicable to a larger array of compounds. Unlike other modeling methods, WFS can be used even when compound structures are highly diverse. In addition, WFS was shown to have comparable or better predictive power when compared to Native Bayesian clustering or a support vector machine approach in most

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FIGURE 5

Weighted feature significance (WFS) algorithm. The algorithm has been developed to build fragment-based models to predict various toxicity endpoints, such as cell viability, caspase-3/7 activation, mutagenicity and hepatotoxicity, based on compound structure. The sensitivity and specificity of these models have been rigorously tested using receiver operating characteristic curves. Activity or toxicity profiles can be generated by testing compounds against a battery of cell lines or assays measuring the same toxicity endpoint. The activity pattern of a compound across such a battery of assays can be viewed as the compound signature, which can be used subsequently to group compounds into different activity clusters, each representing a distinct toxicity mechanism or mode of action. The underlying assumption is that compounds exhibiting similar activity patterns or signatures are likely to share the same biological target or mode of action. Such approaches can be applied to generate testable hypotheses on mechanisms of compound toxicity. Starting with the structure of a compound, one could then model and predict its toxicity in one assay or cell type and in multiple cell types, which essentially forms the activity pattern or signature that indicates the compound’s mechanism of toxicity. 1004

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test cases [50]. An analysis of the initial Tox21 collection of 2800 compounds revealed that additional chemicals are required for enhancement of compound diversity in these collections to increase the number of robust structural predictors of the WFS, which validates the previously described initiative to expand the compound collection to more than 10,000 chemicals.

Cellular pathway assays Although attractive, target-based screens can lead to the identification of active compounds that do not retain their activity in a physiological environment [53]. Thus, cell-based assays offer an alternative assay format in which the readout is dependent on specific components acting on a single signaling pathway. Furthermore, the combination of toxicity pathways associated with adverse health events with engineered cellular assays designed to measure the perturbation of these pathways in response to a

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chemical is a crucial implementation of the National Academy of Sciences Tox21 report [54]. As an example of such an approach, a b-lactamase reporter gene assay was employed to identify compounds that inhibit [37] or induce [38] hypoxia-inducible factor1a (HIF-1a) activity (Fig. 6). Hypoxia, the reduction in the normal level of tissue oxygen tension within a tissue, is associated with several pathologies including cancer and inflammation [55]. Under hypoxic conditions, HIF-1 subunits heterodimerize and translocate into the nucleus before binding to a hypoxia-response element (HRE) upstream of target genes that activate angiogenesis and vascular endothelial growth factor (VEGF) [56]. Hypoxic conditions attenuate the degradation of HIF-1a, leading to the transcription of survival genes in many solid tumors and poor cancer prognosis [57]. Thus, compounds that inhibit HIF-1a responsive tumor hypoxia might be a valuable chemotherapeutic approach [37]. Furthermore, a separate screen for inducers of

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FIGURE 6

Reporter gene assay for HIF-1 activity. During normoxia, the HIF-1a subunit is degraded by the ubiquitin–proteosome pathway. In this case, the absence of blactamase expression leaves the fluorescent substrate molecule, which contains coumarin and fluorescein. Excitation of the coumarin results in fluorescence resonant energy transfer to the fluorescein moiety, resulting in the emission of green fluorescent signal (530 nm). Under hypoxic conditions, however, HIF-1a heterodimerizes with the HIF-1b subunit and translocates to the nucleus. Next, the HIF-1 complex binds to HRE regulatory sequences upstream of target genes. In this assay, stimulation under hypoxic conditions results in the transcription of b-lactamase, which cleaves the fluorescent substrate molecule, disrupting energy transfer. Excitation of the coumarin molecule in the presence of b-lactamase enzyme activity results in a blue fluorescence signal (460 nm). The ratio of the blue:green signals provides a normalized reporter response.

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hypoxia is also valuable to identify those compounds that can serve as hypoxia mimetics [38]. To identify inhibitors of hypoxia, 73,000 compounds from the MLMSR compound collection [37] were screened between 7 and 15 concentrations in an HRE-bla assay performed in ME-180 cells (Tables 1 and 2). In addition, to identify HIF-1a inducers, 1408 compounds from the NTP collection were screened at 14 concentrations also utilizing the HRE-bla assay. The screening for both assays performed well and indicated the suitability for qHTS to identify inhibitors and activators of HIF-1a. Three hundred and fifty inhibitors with reliable curve classes were identified and SAR analysis of these compounds yielded 18 structural series sharing a common scaffold. Several follow-up studies, such as the evaluation of compound effects on low-oxygen-induced HIF-1 signaling and VEGF secretion, were employed to ensure the specificity of compound activity in the HIF-1a assay [37]. Overall, the primary qHTS and follow-up compounds identified from SAR analysis demonstrated specificity for inhibition of HIF-1a activity and little to no cytotoxicity and thus seem to be good candidates for further testing in other cancer cell lines or animal models [37]. Conversely, ten compounds were identified and confirmed as inducers of HIF-1a activity from the primary screening using the NTP compound collection [38]. In the follow-up studies, five of ten compounds notably induced VEGF secretion in human ME-180 cells in a concentration-dependent manner. These five compounds were further tested for their dependence on HIF-1 with regard to VEGF secretion by testing them in HIF-1a wild-type and knockout mouse embryonic fibroblast cell lines. Compounds involved in VEGF secretion dependently (such as phenathroline) or independently (such as 7,12-DMBA) of HIF-1a were identified. Finally, these five compounds were tested against a battery of reporter genes driven by hypoxia-responsive gene promoters [38,58] to establish the promoter activity profiles. For example, three compounds (including phenathroline) produced promoter activity profiles very similar to those produced by standard hypoxic conditions (1% O2) used in cell-based studies [38,58]. Furthermore, this study highlights the use of biological profiling data with hierarchical clustering to group compounds that operate under a similar mode of action [58].

Concluding remarks and future directions The Tox21 collaboration is combining technology, biology and computational methods to advance in vitro testing for toxicology [12]. The NCGC is working with its Tox21 partners to develop next-generation testing methods and alternative approaches to existing methods and to model in vitro and in vivo responses.

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The examples given here are only a few of the assays that have been utilized to date to study specific endpoints or pathways in human and rodent in vitro assays; in total, the NCGC has generated more than six million data points and more than 400,000 CRCs for Tox21 chemicals in specific assays. The qHTS-driven production of CRCs for every compound tested provides a data-rich resource for SAR analysis, computational modeling and chemical prioritization for more extensive toxicological evaluation. This directly points to the advantages of using qHTS with regard to compound hazard identification; qHTS will enable a more accurate assessment of compound-induced toxicity using cell-based studies and an idea of starting doses to use in in vivo studies. SAR analysis will also enable the identification of toxic compounds with similar structures for follow-up testing. Thus, a basis of hazard characterization with regard to toxicity will emerge before in vivo studies. Furthermore, in vitro toxicity tests performed in human-derived cell lines might provide important biomarkers of exposure that can be directly tested in human populations [2]. Human risk assessment from some in vitro studies might prove difficult; thus, a step-wise approach starting with qHTS, computational modeling and carefully designed tissue- and species-specific cell-based assays will provide a stronger and mechanistically predictive approach for in vivo testing and human risk assessment [2]. Building on the solid foundation described here, future Tox21 goals include the inclusion of new platforms for qHTS (such as high-content screening and nanotechnology), assessment of genetic variation involved in human and rodent toxicity, incorporation of metabolism and biotransformation capability into the current and future assays, identification and prioritization of crucial cellular pathways and key targets for screening, expansion of the compound libraries including compounds that are DMSO or water insoluble, discerning the link between observed perturbations in vitro and pathologies in exposed humans and the creation of relational public databases and tools to interrogate the screening data. Table 1 comprises some of the current and future Tox21 assays at the NCGC, such as those for oxidative stress response [54]. Data production in Tox21 is now moving into its exponential growth phase, and over the next several years the interdisciplinary Tox21 collaboration will continue to innovate in assay biology, screening and computation, to usher in a new era of efficient, mechanistic and predictive chemical toxicology.

Acknowledgements We gratefully acknowledge Paul Shinn for compound management. We also thank Raymond Tice for crucial reading of the manuscript and Darryl Leja for illustrations.

References 1 Andersen, M.E. and Krewski, D. (2009) Toxicity testing in the 21st century: bringing the vision to life. Toxicol. Sci. 107, 324–330 2 Krewski, D. et al. (2009) Toxicity testing in the 21st century: implications for human health risk assessment. Risk Anal. 29, 474–479 3 Xia, M. et al. (2008) Compound cytotoxicity profiling using quantitative highthroughput screening. Environ. Health Perspect. 116, 284–291 4 Goldberg, A.M. and Frazier, J.M. (1989) Alternatives to animals in toxicity testing. Sci. Am. 261, 24–30 5 Zurlo, J. et al. (1993) Animals and Alternatives in Testing: History, Science, and Ethics. Mary Ann Liebert, Inc.

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6 Rowan, A.N. (1983) Alternatives: interaction between science and animal welfare. In Product Safety Evaluation (Goldberg, A.M., ed.), pp. 113–133, Mary Ann Liebert, Inc. 7 Williams, G. and Weisburger, G. (1993) Chemical carcinogenesis. In Toxicology. The Basic Science of Poisons. McGraw-Hill 8 Beniashvili, D. (1994) Experimental Tumors in Monkeys. CRC Press 9 Rangarajan, A. and Weinberg, R.A. (2003) Opinion: comparative biology of mouse versus human cells: modelling human cancer in mice. Nat. Rev. Cancer 3, 952–959 10 National Toxicology Program, (2004) A National Toxicology Program for the 21st Century: A Roadmap for the Future.

11 Dix, D.J. et al. (2007) The ToxCast program for prioritizing toxicity testing of environmental chemicals. Toxicol. Sci. 95, 5–12 12 National Research Council (NRC), (2007) Toxicity Testing in the 21st Century: A Vision and a Strategy. National Academy Press 13 Austin, C.P. et al. (2008) Tox21: Putting a Lens on the Vision of Toxicity Testing in the 21st Century. 14 Kavlock, R.J. et al. (2009) Toxicity testing in the 21st century: implications for human health risk assessment. Risk Anal. 29, 485–487 15 Huang, R. et al. (2008) Characterization of diversity in toxicity mechanism using in vitro cytotoxicity assays in quantitative high throughput screening. Chem. Res. Toxicol. 21, 659–667 16 Collins, F.S. et al. (2008) Toxicology. Transforming environmental health protection. Science 319, 906–907 17 Austin, C.P. et al. (2004) NIH molecular libraries initiative. Science 306, 1138–1139 18 Seidle, T. and Stephens, M.L. (2009) Bringing toxicology into the 21st century: a global call to action. Toxicol. In Vitro 23, 1576–1579 19 Inglese, J. et al. (2006) Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc. Natl. Acad. Sci. U. S. A. 103, 11473–11478 20 Schnecke, V. and Bostrom, J. (2006) Computational chemistry-driven decision making in lead generation. Drug Discov. Today 11, 43–50 21 Malo, N. et al. (2006) Statistical practice in high-throughput screening data analysis. Nat. Biotechnol. 24, 167–175 22 Thomas, C.J. et al. (2009) The pilot phase of the NIH Chemical Genomics Center. Curr. Top. Med. Chem. 9, 1181–1193 23 Michael, S. et al. (2008) A robotic platform for quantitative high-throughput screening. Assay Drug Dev. Technol. 6, 637–657 24 Thorne, N. et al. (2010) Apparent activity in high-throughput screening: origins of compound-dependent assay interference. Curr. Opin. Chem. Biol. 14, 315–324 25 Feng, B.Y. et al. (2007) A high-throughput screen for aggregation-based inhibition in a large compound library. J. Med. Chem. 50, 2385–2390 26 Simeonov, A. et al. (2008) Fluorescence spectroscopic profiling of compound libraries. J. Med. Chem. 51, 2363–2371 27 Auld, D.S. et al. (2008) Characterization of chemical libraries for luciferase inhibitory activity. J. Med. Chem. 51, 2372–2386 28 Oldenburg, K. et al. (2005) High throughput sonication: evaluation for compound solubilization. Comb. Chem. High Throughput Screen. 8, 499–512 29 MacArthur, R. et al. (2009) Monitoring compound integrity with cytochrome P450 assays and qHTS. J. Biomol. Screen. 14, 538–546 30 Yasgar, A. et al. (2008) Compound management for quantitative high-throughput screening. JALA Charlottesv Va 13, 79–89 31 Judson, R. et al. (2009) The toxicity data landscape for environmental chemicals. Environ. Health Perspect. 117, 685–695 32 Schmidt, C.W. (2009) TOX 21: new dimensions of toxicity testing. Environ. Health Perspect. 117, A348–A353 33 Auld, D.S. et al. (2006) Fluorescent protein-based cellular assays analyzed by laserscanning microplate cytometry in 1536-well plate format. Methods Enzymol. 414, 566–589 34 Cho, M.H. et al. (2008) A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker. Toxicol. In Vitro 22, 1099–1106 35 Tanega, C. et al. (2009) Comparison of bioluminescent kinase assays using substrate depletion and product formation. Assay Drug Dev. Technol. 7, 606–614 36 Shukla, S.J. et al. (2009) Identification of pregnane X receptor ligands using timeresolved fluorescence resonance energy transfer and quantitative high-throughput screening. Assay Drug Dev. Technol. 7, 143–169 37 Xia, M. et al. (2009) Identification of small molecule compounds that inhibit the HIF-1 signaling pathway. Mol. Cancer 8, 117

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38 Xia, M. et al. (2009) Identification of chemical compounds that induce HIF-1alpha activity. Toxicol. Sci. 112, 153–163 39 Bowen, W.P. and Wylie, P.G. (2006) Application of laser-scanning fluorescence microplate cytometry in high content screening. Assay Drug Dev. Technol. 4, 209–221 40 Brimacombe, K.R. et al. (2009) A dual-fluorescence high-throughput cell line system for probing multidrug resistance. Assay Drug Dev. Technol. 7, 233–249 41 Norton, J.T. et al. (2009) Automated high-content screening for compounds that disassemble the perinucleolar compartment. J. Biomol. Screen. 14, 1045–1053 42 Hill, A.V. (1910) The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J. Physiol. 40, 4–7 43 Zhang, J.H. et al. (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73 44 Sanguinetti, M.C. et al. (1995) A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81, 299–307 45 Titus, S.A. et al. (2009) A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel. Anal. Biochem. 394, 30–38 46 De Ponti, F. et al. (2002) Safety of non-antiarrhythmic drugs that prolong the QT interval or induce torsade de pointes: an overview. Drug Saf. 25, 263–286 47 Diaz, G.J. et al. (2004) The [3H]dofetilide binding assay is a predictive screening tool for hERG blockade and proarrhythmia: comparison of intact cell and membrane preparations and effects of altering [K+]o. J. Pharmacol. Toxicol. Methods 50, 187–199 48 Tao, H. et al. (2004) Automated tight seal electrophysiology for assessing the potential hERG liability of pharmaceutical compounds. Assay Drug Dev. Technol. 2, 497–506 49 Leishman, D. and Waldron, G. (2006) Assay technologies: techniques available for quantifying drug–channel interactions. In Voltage-Gated Ion Channels as Drug Targets (Triggle, D.J. et al. eds), pp. 37–63, Wiley-VCH 50 Huang, R. et al. (2009) Weighted feature significance: a simple, interpretable model of compound toxicity based on the statistical enrichment of structural features. Toxicol. Sci. 112, 385–393 51 Pritchard, J.F. et al. (2003) Making better drugs: decision gates in non-clinical drug development. Nat. Rev. Drug Discov. 2, 542–553 52 Schoonjans, F. et al. (1996) Presentation of receiver-operating characteristics (ROC) plots. Clin. Chem. 42, 986–987 53 Xia, M. et al. (2009) Identification of compounds that potentiate CREB signaling as possible enhancers of long-term memory. Proc. Natl. Acad. Sci. U. S. A. 106, 2412–2417 54 Simmons, S.O. et al. (2009) Cellular stress response pathway system as a sentinel ensemble in toxicological screening. Toxicol. Sci. 111, 202–225 55 Mole, D.R. and Ratcliffe, P.J. (2008) Cellular oxygen sensing in health and disease. Pediatr. Nephrol. 23, 681–694 56 Forsythe, J.A. et al. (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16, 4604–4613 57 Redell, M.S. and Tweardy, D.J. (2005) Targeting transcription factors for cancer therapy. Curr. Pharm. Des. 11, 2873–2887 58 Simmons, S.O. (2009) Hypoxia response: a model toxicity pathway for highthroughput screening. Toxicol. Sci. 112, 1–3 59 Johnson, R.L. et al. (2009) A quantitative high-throughput screen for modulators of IL-6 signaling: a model for interrogating biological networks using chemical libraries. Mol. Biosyst. 5, 1039–1050 60 Miller, S.C. et al. (2010) Identification of known drugs that act as inhibitors of NFkappaB signaling and their mechanism of action. Biochem. Pharmacol. 79, 1272– 1280 61 Hancock, M.K. et al. (2009) HTS-compatible beta-lactamase transcriptional reporter gene assay for interrogating the heat shock response pathway. Curr. Chem. Genomics 3, 1–6

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Epigenetic therapies for non-oncology indications Jonathan D. Best and Nessa Carey CellCentric, Chesterford Research Park, Little Chesterford, Cambridge CB10 1XL, UK

Chronic and degenerative disorders are a major, and growing, human health burden, and current treatments are in many cases inadequate or very expensive. Epigenetic therapies are attractive options for treating such disorders because they manipulate the processes that maintain cells in an abnormal transcriptional state. The challenges lie in identifying the most appropriate diseases and the enzymes that should be targeted. This review describes the different approaches that can be used to address this problem, focusing particularly on CNS disorders (especially mental retardation, neurodegenerative disease, psychiatric disorders and drug addiction), diabetes and diabetic complications, and autoimmunity and inflammatory diseases.

Epigenetic traits have been defined operationally as ‘stably heritable phenotypes resulting from changes in a chromosome without alterations in the DNA sequence’ [1]. Epigenetic modifications (also known as epigenetic marks) form a network of covalent alterations to DNA and histone proteins, which, in turn, interacts with other cellular proteins, typically in multi-component mediator complexes. The end result is the regulation of gene expression. This regulation can be short-term and dynamic or exceptionally stable if the chromatin modifications lead to the hypermethylated DNA state associated with the formation of transcriptionally silent heterochromatin. Several excellent reviews cover the underlying modification mechanisms [2–4], and Fig. 1 summarizes our current knowledge of these modifications. It is impossible in one figure to demonstrate the complexity of histone modifications present on even one histone molecule at a single genomic locus in a single cell. In some cases, modifications are mutually exclusive (e.g. it is not possible for a single histone H3K4 residue to be methylated and acetylated simultaneously). A single residue can be modified to varying degrees – many lysine residues can be mono-, di- or trimethylated. Different combinations of modifications can only occur in certain situations. The combination of methylation on H3K4 and H3K27 only occurs in pluripotent cells and, even then, only at the promoters of certain key regulatory genes [5].

Corresponding author:. Carey, N. ([email protected])

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There has been cosiderable progress in the development of epigenetic drugs for the treatment of human cancers [6]. DNA methyltransferase inhibitors and histone deacetylase (HDAC) inhibitors have been licensed by the US Food and Drug Administration. Several companies are now developing inhibitors for second-generation epigenetic targets, focusing on enzymes that mediate restricted histone modifications. Although oncology is the current major focus for most of these programmes, there is optimism that epigenetic drugs will have wider therapeutic applications. It might at first seem counterintuitive that processes involved in the uncontrolled proliferation and transformation that are characteristics of cancer could be useful intervention points in nonproliferative disorders, but in reality this is not so surprising. Epigenetic mechanisms control cell fate. Aberrant epigenetic processes can have several potential outcomes, depending on the enzymes and pathways involved, the specific cell type, interactions with the environment and so on. Given the large numbers of enzymes involved in epigenetic processes, a role only in cancer would be far more surprising than an involvement in multiple indications.

New therapeutic indications Perhaps the greatest problem in developing epigenetic drugs for non-oncology indications lies in identifying the most relevant diseases to target. In general, it might be helpful to think of

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FIGURE 1

Schematic representation of major chromatin modifications. (a) The chemical structures of the major modification of DNA, cytosine methylation. This modification is mediated by a family of DNMTs. (b) A schematic representation of the major modifications of the four canonical histones that create the nucleosomal octamer. The enzymes generating or removing the modifications are shown in the boxes, and their effects are indicated by the arrows. Methylation of lysine residues can be mono, di or tri, but for clarity this has not been shown. Lysine acetylation is removed by HDACs. Unlike the specific nature of demethylase enzymes, most HDACs can remove acetyl groups from any or all accessible histone lysine residues. For the sake of clarity, the HDACs have been excluded from the schematic representation. The schematic representation has been annotated with the most widely used names for the enzymes – for a more structured nomenclature, refer to Ref. [54].

epigenetic processes serving to stabilize a response to an external stimulus, such that in disease states they act to maintain a cell in an abnormal transcriptional programme. This leads to the hypothesis that epigenetic interventions might be most useful in chronic and developmental disorders, and the limited data available support this theory. Epigenetic effects are also attractive mechanisms for accounting for discordance between monozygotic twins. Within the broad category of chronic and developmental disorders, there are two complementary and intersecting approaches that have been useful in identifying human diseases that might be amenable to epigenetic therapies: biology (both human and animal models) and drug repositioning. Figure 2 lists some of the major disorders beyond oncology for which an epigenetic component or therapeutic approach has been proposed. Several Mendelian disorders have been shown to be the result of mutations in

genes encoding epigenetic enzymes or mediators. This has been particularly fruitful in the field of mental retardation. Angelman’s syndrome and Prader-Willi syndrome were recognized many years ago as associated with parent-of-origin and imprinting deficits, extreme examples of epigenetic regulation and abnormality. Rett’s syndrome, the X-linked neurodevelopmental disorder, is predominantly caused by mutations in MeCP2, a protein that binds methylated DNA residues [7]. This protein might also be implicated in human autism [8]. Elegant work from Adrian Bird’s lab has shown the reversal of the Rett phenotype in engineered mouse strains [9]. Although this cannot yet be directly replicated in human patients, because the mouse work required a genetic approach, it suggests that neurodevelopmental defects can be reversed, offering considerable encouragement to the field. PHF8 is a histone demethylase, and mutations in this gene have www.drugdiscoverytoday.com

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Major non-oncology disease areas for epigenetic interventions. Major indications for which there might be a tractable epigenetic therapeutic approach are shown. For the majority of the indications, supporting evidence is given in the text. See, in addition, Refs. [54–57].

been identified in several families with a history of X-linked mental retardation [10,11]. The majority of chronic human disease is not associated with Mendelian inheritance patterns, however, so how can the pharmaceutical industry use biology to identify other disorders that might be amenable to epigenetic therapies? One tantalizing approach is to identify epigenetic fingerprints characteristic of disease (i.e. to identify marks present in disease states that are absent in health). This is a huge undertaking, fraught with difficulties. As described above, the large numbers of epigenetic enzymes can result in an enormous number of possible combinations of modifications, and identifying those that are associated with a disorder and demonstrate a causal relationship with disease aetiology will require a step-change in detection technologies and bioinformatics. Researchers are taking steps towards this on a global scale (the Thousand Epigenomes project is one example; see http://nihroadmap.nih.gov/epigenomics), and intriguing data already exist in the literature for several disorders. Alzheimer’s disease (AD) is marked by a loss of cholinergic neurons, along with the formation of Abeta protein plaques and neurofibriliary tangles [12]. Research into therapeutics for AD has focussed on either cleavage of the amyloid precursor protein or hyperphosphorylation of the tau protein, which forms neurofibriliary tangles [13]. Developing effective therapeutics using these approaches has met with limited success, and studies have begun to investigate epigenetic changes in animal models of AD. Regional variations in methylation and acetylation of histone proteins have been seen in the brains of the familial AD mutant Tg2576 mouse, suggesting that compounds targeting methylation and acetylation might be useful [14]. Other findings have suggested that HDAC inhibitors might play a part in ameliorating learning 1010

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and memory deficits [15]. These studies are at a very early stage, however, and further work is required. This includes analyses of human samples to determine whether the same patterns of epigenetic modifications are present as in the mouse model and whether they genuinely distinguish Alzheimer’s pathology from general ageing effects. It is also necessary to investigate whether these fingerprints are causal effectors of the disease or merely markers of disease progression. In addition to neurodegenerative disorders, epigenetics is an increasing focus in the investigation of psychiatric disorders. One of the most intensively explored has been the long-term effects of childhood abuse or neglect, which is associated with several adult health deficits including increased risk of depression, drug addiction and suicide. The most commonly used rodent model of early life stress is built around periodic mother–infant separation. The offspring maintain elevated levels of glucocorticoid secretion throughout life, and this overactivation of the hypothalamic– pituitary–adrenal axis is also found in affected humans [16]. This seems to be underpinned by epigenetic misregulation at several levels. The arginine vasopressin (AVP) protein is a key stimulator of adrenocorticotrophin release from the pituitary. A specific AVP enhancer is hypomethylated in the paraventricular nucleus of mice subjected to early life stress, and this leads to persistent overexpression of the gene [17]. Other studies have demonstrated increased DNA methylation, and concomitant decreased expression, of the neuron-specific glucocorticoid receptor promoter (Nr3cl) in the same model system [18]. Because the hypothalamic–pituitary–adrenal system is usually controlled by a positive feedback loop, the combined effects of the altered (hypo- and hyper-) methylation at each ‘end’ of the axis would be continual overstimulation of this hormonal response.

There are always questions over the applicability of animal models to human psychiatric disorders. In this instance, the Nr3cl effects reported in the rodent model are also reported in human adult suicides with a history of childhood abuse [19]. The changes were not seen in suicides with no abuse history or in unaffected controls. It is unclear from the literature whether the early events establishing a hypomethylated state at the AVP enhancer occur in mitotic or post-mitotic neurons. If it is the latter, it will be important to identify which, if any, of the recently identified putative active DNA demethylases are present in these cells, because there is no opportunity for passive DNA demethylation in post-mitotic cells [20–22]. At least some of the hypomethylation at the AVP enhancer is reported to be driven by the dissociation of the repressive MeCP2 protein from the locus. MeCP2 can act to drive the continued recruitment of DNA methyltransferases to a silenced chromatin region, and hence loss of this binding protein will lead to long-term hypomethylation. MeCP2 has been reported recently to be a key protein in animal models of drug addiction. Knockdown of MeCP2 in the rat striatum led to decreased cocaine intake in an animal model of unrestricted drug access [23]. Manipulation of MeCP2 levels in the nucleus accumbens of mice altered locomotor responses to amphetamines [24]. The details of the models and the mechanisms postulated for the effects of MeCP2 vary between the two studies (for discussion, see Ref. [25]), but both are supportive of a major role of DNA methylation. Additional support for the importance of DNA methylation in response to drugs of addiction comes from the finding that manipulating levels of the DNMT3a DNA methyltransferase in the nucleus accumbens of mice markedly affected their response to cocaine [26]. DNA methylation is an exceptionally stable epigenetic modification and might be difficult to manipulate for psychiatric disorders because even the licensed DNA methyltransferase inhibitors are unlikely to have side-effect profiles that would be acceptable for these indications. Many gene responses become initially transiently stabilized via histone modifications before more permanent DNA methylation changes are established. These histone modifications might, in the future, become useful targets for the development of drugs that target acute stresses, to prevent long-lasting psychiatric disturbances such as post-traumatic stress disorder. Moving away from psychiatric disease, the HDAC inhibitors MS275 and SAHA (vorinostat) have been shown to relieve pain in the second phase of the formalin rat pain model, causing upregulation of the brain mGlu2 receptors. This suggests a role for histone acetylation in the transcriptional activity of the mGlu2 receptor gene [27]. Neurological disorders are not the only diseases in which there is an ongoing interest in epigenetic therapies. There are expected to be 285 million cases of diabetes worldwide in 2010, 90% of which will be Type 2. This number is increasing, predominantly because of lifestyle changes (http://www.diabetesatlas.org). Affected individuals have an increased risk of cardiovascular disease and a wide range of other pathological conditions, creating a major human health burden. Currently, primary management of both Type 1 and Type 2 diabetes centres on glycaemic control and insulin therapy, along with weight control strategies and the use of statins and treatments for hypertension [28–30]. Early studies have

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begun to show links to epigenetic effects, both in the insulin release and insulin response pathways themselves and in the inflammatory pathways, which are mediators of morbidity and mortality. Hyperacetylation of histone H4 under high-glucose conditions has been reported at the insulin gene promoter in the insulinoma cell line MIN6. DNA hypomethylation of the same promoter was shown in the beta cells of the pancreas in murine and human samples. Experimental methylation of this promoter suppressed expression of the insulin gene [31]. Vascular endothelial cells cultured under hyperglycaemic conditions showed persistently increased levels of the activating H3K4me1 mark in the promoter of the pro-inflammatory NF-kB-p65 gene. This was probably mediated by the SET7 methyltransferase [32]. In the same study, it was observed that levels of the inhibitory H3K9me2 and H3K9me3 marks at the same promoter were reduced. Investigations of the inflammatory aspects of diabetes demonstrated the involvement of H3 methylation in the inflammation of vascular smooth muscle [33,34], where H3K9me3 was purported to play a part in the repression of inflammatory genes [34]. High glucose levels were seen to increase the expression of inflammatory genes and the levels of H3K4me2, reducing the recruitment of the repressive histone demethylase LSD1 [33]. Several additional studies have demonstrated the association of histone H3 methylation events with diabetic end-points, indicating that histone methylation and acetylation pathways could both be potential candidates for small-molecule epigenetic therapeutics in this disease [35,36]. In addition to Type 1 diabetes, several autoimmune diseases are being investigated for underlying epigenetic mechanisms, particularly systemic lupus erythematosus, rheumatoid arthritis (RA) and multiple sclerosis [37]. Changes in histone and DNA modifications are associated with inflammatory responses [27,38,39]. Decreased DNA methylation at a specific CpG motif in the IL-6 promoter of patients’ peripheral blood mononuclear cells has been linked to RA [40]. Systemic lupus erythematosus patients have been reported to have decreased DNA methylation in the promoter of the CD5-E1B gene, a key regulator of the same interleukin [41]. In both clinical conditions, the end-point of these DNA methylation events increased the expression of IL-6, a key inflammatory cytokine. The alternative approach for identifying disorders that might be amenable to epigenetic therapies is a repositioning strategy. Drug repositioning is the process by which drugs (usually marketed drugs) are assessed in diseases for which they were not originally developed. The potential advantage is that marketed drugs have been through safety and toxicology testing in humans and thus can be fast-tracked into clinical trials in alternative indications [42]. In this instance, it involves epigenetic drugs originally developed to treat cancer – although ironically, from this review’s perspective, one of the first examples of such an approach happened the other way around. Sodium valproate was one of the earliest successful anti-convulsant drugs and has been widely used in epilepsy treatment. It is now known that valproic acid is a lowaffinity HDAC inhibitor with anti-proliferative effects in several cancer models. HDAC inhibitors are generally well tolerated, and many compounds have been made available to researchers working in nononcology areas, using model systems ranging from fruit flies to www.drugdiscoverytoday.com

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mice. They have proved particularly informative in some Mendelian disease models. For example, Huntington’s disease (HD) is an untreatable and invariably fatal dominantly inherited disorder caused by an extension of CAG repeats in the Huntingtin (Htt) gene, with consequent loss of neurons from the striatum being one of the prominent pathologies [43]. There are several mouse models of this disease, of which R6/2 is generally accepted as a useful disease phenocopier. Repressed transcription is a common feature in HD tissues, and HDAC inhibitors generally act as transcriptional activators. R6/2 HD mice treated with the HDAC inhibitor SAHA showed improved motor function and increased longevity [44]. Later papers also demonstrated symptomatic improvements in HD models using the less potent HDAC inhibitors sodium valproate and phenylbutyrate [45,46]. Surprisingly, in all cases the symptomatic improvement that followed administration of HDAC inhibitors was not accompanied by changes in underlying cellular pathology, suggesting more investigation is needed to fully understand the mechanism of action of these drugs in the model systems. EnVivo Pharmaceuticals entered an HDAC inhibitor (ENV0334) licensed from MethylGene into phase I clinical trials with HD as a named indication. The most recent statements from the company, however, suggest that this compound is being positioned as a cognitive enhancer for other neurological indications, including AD and Parkinson’s disease (http://www.envivopharma.com/template/2_18_5.html). ENV-0334 inhibits the class I and class II zinc-dependent HDACs. Recently, an inhibitor of the class III NAD-dependent Sirtuin 1 HDAC, EX-527/SEN0014196 (generated by Elixir Pharmaceuticals and partnered with Siena Biotech), has entered phase Ia clinical trials for HD (http://www.sienabiotech.com/portfolio.jsp). In addition to acetylation, recent studies have shown that the HTT protein interacts with the polycomb repressive complex 2, which possesses methyltransferase activity targeted at H3K27 [47]. The significance of this finding in the disease aetiology is unclear. The importance of understanding the biology is further demonstrated by work on dopaminergic neuronal cell lines, where treatment with the HDAC inhibitor trichostatin A (TSA) resulted in decreased cell survival and increased apoptosis [48]. This suggested that HDAC inhibition might not be appropriate for the treatment of Parkinson’s disease. However, this is in contrast to observations of Wu et al. [49], who noted that administration of valproic acid or TSA to dopaminergic neurons in rat neuron–glia cultures upregulated GDNF and BDNF and had a neuroprotective effect against the neurotoxin MPTP. Other studies investigating the effects of HDAC inhibitors have also shown positive effects. Kim et al. [50] observed that the HDAC inhibitors sodium butyrate and TSA seemed to stimulate neurogenesis in the brains of rats with induced ischaemia. The effect was mediated by the BDNF tyrosine kinase signalling pathway. AD mouse models showed restoration of contextual memory with the chronic administration of different HDAC inhibitors [51]. This study was supported by Smith et al. [52], who demonstrated that administration of valproic acid to microglial cell lines led to increased phagocytic activity for Abeta deposits. An intriguing application of epigenetic approaches has been the use of HDAC inhibitors to drive re-expression of developmentally silenced genes, to compensate for the loss of function in Mende1012

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lian disorders. Spinal muscular atrophy is caused by inactivating mutations in the SMN1 gene [53]. The human genome also contains a highly homologous gene, SMN2, which is usually epigenetically repressed in postnatal tissues. Treatment with HDAC inhibitors leads to reactivation of the SMN2 gene in model systems [54]. A parallel approach might be possible in inherited haemoglobinopathies. Some patients with inherited mutations in the bglobin gene that lead to either sickle cell disease or b-thalassemia fare better than expected clinically [55]. Frequently, this is due to the persistence of foetal haemoglobin gene (HbF) expression. The HbF gene is usually epigenetically repressed postnatally, but studies with HDAC inhibitors in model systems have shown that the gene can be de-repressed by drug treatment [56].

Concluding remarks With epigenetic-focussed therapeutics being a promising avenue for non-cancer indications, the questions of how to assess prospective therapeutics in vivo become key. The majority of animal models are centred around either genetic manipulations, such as the transgenic mice used to model neurodegenerative disorders [57,58], or pharmacologically induced behavioural phenotypes, such as the pilocarpine epilepsy model [59]. In monogenic disorders such as HD, the epigenetic changes seen in the animal models are likely to reflect those seen in humans, although even here the disconnect between behavioural improvements and cellular pathology leads to questions around behavioural testing and whether this is appropriate or linked closely enough to the underlying pathology [60]. For the more complex diseases such as AD, the current animal models mainly mimic inherited diseases [57], so being able to determine effects on the sporadic elements of the disease will require understanding from different mechanisms of the pathology. There is often surprisingly little consensus within the pharmaceutical industry on the most reliable animal models for the majority of complex diseases (including AD, schizophrenia and depression), which will only add to the difficulties. One frequent issue, which has been indicated to some degree above, is the extent to which epigenetic therapies will genuinely alter disease progression or even cure a disorder entirely, rather than simply delay symptomatic presentation. For some diseases with an unequivocally grim prognosis such as HD or AD (particularly the early onset form), current therapies are so inadequate that even a few extra years of good-quality healthy life would represent a major improvement. This will affect the risk–benefit equation in favour of repositioning of broad-acting drugs with a side-effect profile that is less than optimal, such as HDAC inhibitors. This is less likely to be true of less catastrophic human disorders, especially where there are existing therapeutic options. A clear example would be a disease such as RA, which – although debilitating – is rarely directly life-threatening, and for which effective but expensive antibody-based therapeutics are available. In such cases, more precisely targeted epigenetic therapies will be required, with strong mechanism-of-action rationales and sideeffect profiles that are, at the very least, no worse than those of existing drugs. It seems to us improbable that this can be achieved by targeting promiscuous chromatin modifiers represented by the HDACs and the DNA methyltransferases. Success is more probable through the selective second-generation chromatin targets and requires extensive collaborations in basic research, disease model-

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academic laboratories and the commercial sector have the potential to bring major benefits to patients through the development of second-generation epigenetic interventions.

Acknowledgements The authors thank Neil Pegg and Will West for helpful discussions.

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28 Aschner, P. et al. (2010) Practical steps to improving the management of type 1 diabetes: recommendations from the Global Partnership for Effective Diabetes Management. Int. J. Clin. Pract. 64, 305–315 29 Palumbo, P.J. and Wert, J.M. (2010) Impact of data from recent clinical trials on strategies for treating patients with type 2 diabetes mellitus. Vasc. Health Risk Manage. 6, 17–26 30 Prato, S.D. et al. (2010) Tailoring treatment to the individual in type 2 diabetes practical guidance from the Global Partnership for Effective Diabetes Management. Int. J. Clin. Pract. 64, 295–304 31 Kuroda, A. et al. (2009) Insulin gene expression is regulated by DNA methylation. PLoS One 4, 6953 32 Brasacchio, D. et al. (2009) Hyperglycemia induces a dynamic cooperativity of histone methylase and demethylase enzymes associated with geneactivating epigenetic marks that coexist on the lysine tail. Diabetes 58, 1229–1236 33 Reddy, M.A. et al. (2008) Role of the lysine-specific demethylase 1 in the proinflammatory phenotype of vascular smooth muscle cells of diabetic mice. Circ. Res. 103, 615–623 34 Villeneuve, L.M. et al. (2008) Epigenetic histone H3 lysine 9 methylation in metabolic memory and inflammatory phenotype of vascular smooth muscle cells in diabetes. Proc. Natl. Acad. Sci. U. S. A. 105, 9047–9052 35 Miao, F. et al. (2008) Lymphocytes from patients with type 1 diabetes display a distinct profile of chromatin histone H3 lysine 9 dimethylation: an epigenetic study in diabetes. Diabetes 57, 3189–3198 36 Cavener, D.R. (2009) Sleeping Beauty, awake! Regulation of insulin gene expression by methylation of histone H3 Diabetes 58, 28–29 37 Hewagama, A. and Richardson, B. (2009) The genetics and epigenetics of autoimmune diseases. J. Autoimmun. 33, 3–11 38 Barnes, P.J. (2009) Targeting the epigenome in the treatment of asthma and chronic obstructive pulmonary disease. Proc. Am. Thorac. Soc. 6, 693–696 39 Strietholt, S. et al. (2008) Epigenetic modifications in rheumatoid arthritis. Arthritis Res. Ther. 10, 219 40 Nile, C.J. et al. (2008) Methylation status of a single CpG site in the IL6 promoter is related to IL6 messenger RNA levels and rheumatoid arthritis. Arthritis Rheum. 58, 2686–2693 41 Garaud, S. et al. (2009) IL-6 modulates CD5 expression in B cells from patients with lupus by regulating DNA methylation. J. Immunol. 182, 5623–5632 42 Ashburn, T.T. and Thor, K.B. (2004) Drug repositioning: identifying and developing new uses for existing drugs. Nat. Rev. Drug Discov. 3, 673–683 43 HDCRG, (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Group. Cell 72, 971–983 44 Hockly, E. et al. (2003) Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington’s disease. Proc. Natl. Acad. Sci. U. S. A. 100, 2041–2046 45 Zadori, D. et al. (2009) Valproate ameliorates the survival and the motor performance in a transgenic mouse model of Huntington’s disease. Pharmacol. Biochem. Behav. 94, 148–153 46 Gardian, G. et al. (2005) Neuroprotective effects of phenylbutyrate in the N17182Q transgenic mouse model of Huntington’s disease. J. Biol. Chem. 280, 556– 563 47 Seong, I.S. et al. (2010) Huntingtin facilitates polycomb repressive complex 2. Hum. Mol. Genet. 19, 573–583 48 Wang, Y. et al. (2009) HDAC inhibitor trichostatin A-inhibited survival of dopaminergic neuronal cells. Neurosci. Lett. 467, 212–216 49 Wu, X. et al. (2008) Histone deacetylase inhibitors up-regulate astrocyte GDNF and BDNF gene transcription and protect dopaminergic neurons. Int. J. Neuropsychopharmacol. 11, 1123–1134 50 Kim, H.J. et al. (2009) The HDAC inhibitor, sodium butyrate, stimulates neurogenesis in the ischemic brain. J. Neurochem. 110, 1226–1240

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ling, compound development and clinical testing. Whether successful interventions will require monotherapy or combination therapies (as seems the most promising avenue for epigenetic drugs in oncology) also remains to be established. No one sector or organization holds all the skills necessary for success in this endeavour, but effective collaborations between cutting-edge

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51 Kilgore, M. et al. (2010) Inhibitors of class 1 histone deacetylases reverse contextual memory deficits in a mouse model of Alzheimer’s disease. Neuropsychopharmacology 35, 870–880 52 Smith, A.M. et al. (2010) Valproic acid enhances microglial phagocytosis of amyloid-beta(1–42). Neuroscience 169, 505–515 53 Lefebvre, S. et al. (1995) Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155–165 54 Riessland, M. et al. (2010) SAHA ameliorates the SMA phenotype in two mouse models for spinal muscular atrophy. Hum. Mol. Genet. 19, 1492–1506 55 Thein, S.L. et al. (2009) Control of fetal hemoglobin: new insights emerging from genomics and clinical implications. Hum. Mol. Genet. 18 (R2), R216–R223

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56 Gabbianelli, M. et al. (2000) Hemoglobin switching in unicellular erythroid culture of sibling erythroid burst-forming units: kit ligand induces a dose-dependent fetal hemoglobin reactivation potentiated by sodium butyrate. Blood 95, 3555–3561 57 Elder, G.A. et al. (2010) Transgenic mouse models of Alzheimer’s disease. Mt. Sinai J. Med. 77, 69–81 58 Kumar, P. et al. (2010) Huntington’s disease: pathogenesis to animal models. Pharmacol. Rep. 62, 1–14 59 Scorza, F.A. et al. (2009) The pilocarpine model of epilepsy: what have we learned? Ann. Acad. Bras. Cienc. 81, 345–365 60 Sadri-Vakili, G. and Cha, J.H. (2006) Mechanisms of disease: histone modifications in Huntington’s disease. Nat. Clin. Pract. Neurol. 2, 330–338

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Pharmacoproteomics: a chess game on a protein field Angelo D’Alessandro and Lello Zolla Department of Environmental Sciences, Tuscia University, Largo dell’Universita` snc, 01100, Viterbo, Italy

The application of proteomics in the field of drug discovery development and the assessment of drug administration is known as pharmacoproteomics. As a branch of proteomics – perhaps the most promising and rapidly evolving field of the post-genomic era – pharmacoproteomics has inherited all the promises that pharmacogenomics has hitherto left unfulfilled. On the road to tailor-made drugs, whole protein profiles of healthy individuals have been progressively expanded, either qualitatively or quantitatively. In this review article, we provide general information about technical advancements in the field of proteomics (the pieces of this intriguing chess game) and show how this progress has furthered our understanding of biological systems. Pitfalls on the field of biomarker individuation and drug discovery and/or testing are also discussed. Functional proteomics: the pharmacoproteomic promise The application of proteomic technologies in the field of drug discovery development and assessment of drug administration is known as pharmacoproteomics [1]. Pharmacoproteomics directly stems from proteomics, the most rapidly evolving field since the end of the post-genomic era, and holds the potential to fulfill all the promises that pharmacogenomics has hitherto left unfulfilled. Although individualized medicine treatments still remain an ongoing objective, pharmacoproteomics seems to be more suited for providing valuable information in either the design or the toxicity assessment of new drugs because it sheds light on the effects of their interactions with the actual bioactive product, the protein. This is particularly evident when considering the field of biomarker discovery, in which mere genome-oriented or in silicoonly approaches have so far failed to gather conclusive information, whereas the majority of disease diagnosis-related markers have been finally ‘found in translation’, to quote Lockhart and Walther [2]. Notwithstanding the big strides made by proteomeoriented approaches, only a handful of proteins are currently used in routine clinical diagnosis, and the rate of introduction of new protein tests approved by the United States Food and Drug Admin-

Corresponding author:. Zolla, L. ([email protected]) 1359-6446/06/$ - see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2010.10.002

istration has paradoxically declined over the past two decades to fewer than one new protein diagnostic marker per year [3]. The performance of combinatorial chemistry in filling pharmaceutical pipelines has been lower than anticipated and the tide might be turning back to nature in the search for new drug candidates. Even though diversity-oriented synthesis is now producing molecules that are natural-product-like in terms of size and complexity, these molecules have not evolved to interact with biomolecules: in both cases, the mechanisms of action of these molecules are often unclear. Chemical and reverse chemical proteomics have recently contributed to speeding up the process of individuating new druggable targets and their small-molecule ligands, suggesting an alternative route to follow to overpass undesired drug market stagnancy [4]. New strategies have become necessary to tackle these difficulties. First, the whole protein profiles of healthy individuals have been progressively expanded, from a purely qualitative point of view. This is particularly true for plasma and blood components, the protein lists of which have been continuously improved during the past few years [5]. To this end, the introduction of new technologies enabling sample pre-fractionation, such as combinatorial ligand libraries [6], has allowed a whole hidden proteome to be unveiled. A paradigmatic example is the case of the red blood cell, in which the 98% of the whole cytosolic protein content is characterized by hemoglobin, and the remaining 2% has been www.drugdiscoverytoday.com

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shown to harbor approximately 1578 unique proteins [7]. This amazing list of new proteins has been exploited to update the total map of erythrocyte proteins, from approximately 508 entries to 1989 distinct gene products [8]. In parallel, newly introduced technical caveats have endowed researchers with powerful tools – such as blue and clear native gel-based approaches – to explore the more hydrophobic protein fraction [9]. These approaches are relevant because they shed light on a considerable fraction of proteins that are difficult to separate and analyze with classical investigations, and they could also provide details about the formation of protein complexes, which is a recurring feature of enzymatic machineries in biological systems. These results should be completed or accompanied by in silico modeling, prediction and evaluation of the likely effects of drug– protein interactions, or protein–protein interactions themselves, because the protein itself does not exist in the cell as an independent entity; rather, it takes part in intricate networks of interacting molecules [10]. It is worth underlining that, until recently, approaches to biomarker identification have sought to find single molecules indicative of normal or anomalous conditions (i.e. to distinguish healthy cells from diseased ones, such as progenitors or terminally committed mature cells). Indeed, the molecular complexity is an emerging property of living cells, and reductionist approaches have, so far, restrained an exhaustive understanding of the molecular processes. In this view, ‘omics’-oriented approaches have been gaining momentum in the past two decades. The foremost example is perhaps proteomics, which studies the proteome, the cell-specific protein complement to the genome, and encompasses all the proteins that are expressed in a cell at the given time and under the given conditions (normal, stress, disease and tumor) in which the experiment is performed [1]. Second, new directions have been indicated and included in the ambitious agenda of the Human Proteome Organization (HUPO), in merit of the Plasma Proteome Project, which regroups 35 collaborating laboratories worldwide. In this view, quantitative data should be gathered, along with information about posttranslational modifications, which proteins undergo through consolidated mechanisms of functioning regulation and/or control [11]. Therefore, if on the one hand the accumulation of qualitative information about which proteins are expressed in healthy tissues is fundamental, on the other hand it is also pivotal to determine the fluctuations of quantities and the variations in patterns of post-translational modifications of these proteins in specific tissues under healthy and pathological conditions, before or upon drug assumption. Being this the chessboard, in this Review article we are briefly providing general information about technical advancements in the field of proteomics (the pieces of this intriguing chess game; Fig. 1) and showing how these advancements have furthered our understanding of biological systems. Pitfalls in the field of biomarker individuation and drug discovery and testing are also discussed.

The pawn: qualitative profiles – who, where and when The first step of proteomics analyses is the determination of the specific pattern of protein expression of a given tissue. Tissue proteomic profiling becomes fundamental when it comes to comparing healthy and diseased patients, to determine markers that 1016

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could be adopted for early diagnoses. In parallel, protein fingerprints of healthy tissues could vary upon drug assumption, thus representing a valuable tool in the assessment of new drug efficiency and effectiveness or to evaluate likely drug targets [12]. The very first phase of the proteomic era has been begun by the need to build up a knowledge body of qualitative databases.

Blood plasma: technical caveats In this respect, relevant results have been obtained in the field of transfusion medicine, as it has been reviewed recently by several authors [5,13]. Indeed, blood components particularly lend themselves to protein-oriented approaches because the enucleated nature of red blood cells and platelets nullifies transcript-oriented analyses. Nonetheless, the analysis of the protein fractions of blood and blood components still represents a challenging task. The high dynamic range spread of blood and blood component proteins hampers a unitary and comprehensive view of both highand low-abundant species, including proteins involved in the most different activities (coagulation, transport, immune system and cell signaling) and protein byproduct from cellular damage of other tissues. Blood samples can be taken at a particular point in time with little burden on patients, and the constituents of the blood samples could reflect a developing or existing illness because tissue-specific proteins might be released into the blood stream from the damaged or dead cells [14]. The latter consideration suggests new scenarios because the qualitative study of minor protein populations in blood components not only is tied to transfusion medicine but could also represent a virgin forest in the field of new biomarker discovery. In this view, blood could constitute a reservoir of proteins suggestive of specific pathological conditions. Nonetheless, blood and blood components are characterized by an extreme dynamic range of proteins, the concentrations of which span more than ten orders of magnitude, from picogram to milligram quantities per milliliter in the case of plasma. Several highabundance proteins, such as albumin, typically constitute greater than 90% of total protein mass. Therefore, the detection of lower abundance proteins, which presumably represents the biologically interesting population, is interfered with by the dominant proteins. Sample preparation and pre-fractionation become pivotal when tackling the complexity of these samples, through the removal of high-abundant species through different approaches.

The bishop: sample fractionation Because of the high complexity of biological samples (especially of blood and blood components), several strategies have been proposed for cutting through the dynamic range of protein concentrations and variability spread. Basically, these methods rely on biochemical and biophysical properties of protein/peptide molecules, viz., molecular weight, density, hydrophobicity, surface change and isoelectric point, and inter-molecular affinity (e.g. protein–protein complex formation). Although samples could be split through preliminary electrophoresis pre-fractionation, most of the methods are based on the removal of high-abundant species. Immunoaffinity depletion [14], centrifugal ultrafiltration [15] and combinatorial ligand libraries [6] are three of the most widely diffused strategies to tackle dynamic concentration rangerelated analytical issues.

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[()TD$FIG]

FIGURE 1

Pharmacoproteomics as a chess game. Proteomic strategies and chess game pieces are briefly enlisted and described.

Resin-based (e.g. Cibacron blue, blue dye) and antibody-based (e.g. IgY directed towards multiple high-abundant protein species) depletion are probably the most widely diffused pre-fractionation methods in biological analyses. Commercial kits enable the removal of the 6 (albumin, transferrin, IgG, IgA, haptoglobin and antitrypsin) [16], 20 [17] and 89 [18] most abundant protein species, thus cutting the analytical noise by more than 97%. Alternatively, avian immunoglobulin yolk (IgY) kits are available, displaying higher avidity and less cross-reactivity with heterologous human proteins. High-abundant protein removal has its drawback as well, owing to the so-called ‘albumin sponge effect’, which implies that up to 210 different minor protein species (potential candidate biomarkers) are found to be associated with the six most abundant plasma proteins (in particular with albumin) [19]. For example, the removal of albumin using a resin causes a considerable loss of several cytokines. The sponge effect could be tackled through salt-

out preparation and molecular sieve filtration [20]. It might also be possible to design a non-protein binding resin or membrane containing small peptides to selectively remove albumin, but not albumin-bound proteins, from plasma [21]. Nonetheless, immunodepletion-based technology still has some considerable technical issues, which include short life span (<200 runs), progressive reduction of antibody binding capability towards specific proteins or altered specificity through high-molecular-weight compound-induced absorption through hydrophobic interactions [14], altered proportion of quantitative outputs and small sample loading capacity (25–100 ml), not to mention the elevated costs for columns and buffers. Combinatorial hexapeptide ligand libraries have emerged recently as a valuable alternative to immunodepletion [6]. The combinatorial peptide ligand library is a mixture of porous beads on which hexapeptides are chemically attached. Each bead carries billions of copies of the same peptide bait; the beads are thus www.drugdiscoverytoday.com

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different from each other, and all combinations of hexapeptides are present. Depending on the number of amino acids used, a library contains a population of millions of different ligands (e.g. 11, 24, or 64 millions starting respectively from 15, 17 or 20 different amino acids). When a complex protein extract is exposed to such a ligand library in large overloading conditions, each bead with affinity to an abundant protein will rapidly become saturated, and the vast majority of the same protein will remain unbound. By contrast, trace proteins will not saturate the corresponding partner beads but are captured in progressively increasing amounts as the beads are loaded with additional protein extract. Thus, a solidphase ligand library enriches for trace proteins, while reducing the relative concentration of abundant species [22]. Indeed, the combinatorial hexapeptide ligand library approach has contributed to dramatically delving into the complexity of a wide series of biological samples. 2-DE gels upon treatment with combinatorial peptide ligand libraries revealed a far greater number of protein species, as in the case of urine (from 184 to 385 [23]), plasma (from 115 to 790 [24]) and platelets (from 197 to 435 [25]), as well as in other biological fluids, such as milk fractions or in egg yolk and white [26]. A reduced percentage of the proteins that are visible with common methods gets lost; however, 7% in the case of urine, 5% for red blood cells and 13% for platelets [24]. The continuous expansion of the proteomes of biological samples holds that a wider basis is available for statistical and in silico analyses to determine molecular species of interest for human health (novel candidate biomarkers, drug-like molecules, anti-microbial peptides, etc.). Organellar proteomics and subcellular fractionation: A further level of complexity is introduced by the precisely controlled spatial organization of these proteins, namely where they are actually expressed, not only in terms of tissues or cells but also in terms of subcellular compartment [27]. A simple hierarchy of cellular organization would include cellular compartments, such as the cytosol and nucleus, membrane-enclosed organelles, and large or small multiprotein complexes. Large-scale approaches to organelle proteomes have been increasingly attracting a great deal of interest as Taylor et al. have recently reviewed in the literature [28]. After an organelle has been biochemically purified, it can be analyzed through proteomic approaches: in this respect, 2-DE has been almost completely substituted by approaches involving preliminary 1D-SDS-PAGE, enzymatic digestion of band of interest, chromatographic separation of the resulting peptides and subsequent identification via mass spectrometry [29]. Recently, shotgun proteomics was used to demonstrate the implication of protease inhibition and complement activation in the anti-inflammatory properties of high-density lipoprotein [30]. This workflow enables optimal protein identification, but a validation step is still required to assess whether the identified protein is a genuine component of a specific organelle. Moreover, it should be considered that the protein constituents of most organelles are in constant exchange with the rest of the cell. Several organelles – particularly nuclear subdomains – contain both resident and transient proteins. Perturbations and temporal changes in organelle proteomes could be investigated through stable isotope of amino acid in culture (SILAC) upon blockade of transcription to stabilize the cellular subproteomes at different times (up to nine). 1018

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The knight: sample preparation Sample preparation and fractionation technologies are two of the most crucial processes to delve into the complexity of solubilized samples. There are, however, considerable limitations in currently available proteomic technologies: none of them allow for the analysis of the entire proteome in a simple step. This is due to the large number of peptides and to the wide concentration dynamic range of the proteomes in clinical blood samples. As a result, it is often observed that one sample preparation approach biases the proteomic outcome towards a slightly different output; therefore, an ideal proteomic analysis would tend to reduce sample preparation steps [31]. Plasma proteins: sample preparation – challenge ups its ante. An adequate analysis generally includes the following steps: (i) sampling, in which the sample is a good statistical representation of the investigated population; (ii) specimen preservation, during which the sample is expected to be kept stable until the analysis is completed; (iii) appropriate sample handling (fractionation); and (iv) statistical analysis and bioinformatics data treatment [31]. Ideally, sample preparation should be as simple as possible to reduce time and avoid the introduction of steps that could lead to sample loss. Studies from literature seem to attribute the most relevant role in proteomic standardization to sampling strategies, whereas handling and storage conditions apparently provoke relatively minor effects [32]. Indeed, minimal changes were observed in the samples stored at room temperature within the first four hours or six hours, whereas noticeable changes were found after eight hours, especially for peaks in the m/z range at 3000 [33], and were more pronounced after 24 h [34]. Nonetheless, these parameters need adequate standardization as well, in particular for plasma- and blood component-derived proteins, to have comparable results between different laboratories [33]. Standardization has also become an urgent issue for the HUPO, which recently started an international standardization program in proteomics (the Proteomics Standardization Initiative) [11], with the declared intent of defining community standards for data representation in proteomics, which should ease data comparison, exchange and verification. In parallel, the minimum information about a proteomics experiment (MIAPE) initiative has been started to define community standards for data production and representation in proteomics, which should reduce technical variability and ease data comparison, exchange and verification [35]. Other than storage or handling-related changes, proteomic analyses are mainly affected by sample preparation procedures, which include preliminary protein solubilization steps [31].

The rooks and the queen: membrane proteins, protein complexes and protein interactions First rook: membrane proteins. Proteins in biological samples are often insoluble because in their native state they could be found in the form of molecular complexes of associated proteins or in membranes. Solubilization steps vary depending on which kind of protein population is addressed, whether protein complexes or membrane-associated proteins. When promoting solubilization of protein complexes, it is necessary to break interactions involved in protein aggregation (e.g. disulfide and hydrogen bonds, van der Waals forces, ionic and hydrophobic interactions), which enables disruption of proteins

into a solution of individual polypeptides, thereby promoting their solubilization. Sample solubilization can be improved by agitation or ultrasonification, but an increase in temperature must be avoided. The selection of the appropriate solubilization protocol and buffers has especially been facilitated by the availability of commercial kits [36], although it is somewhat more expensive than routine reagent methods. Ionic detergents are strong solubilizing agents that produce protein denaturation. High concentrations of chaotropes such as urea (5–9 M) and thiourea (2 M) help to increase the overall number of solubilized proteins by disrupting hydrogen bonds, whereas charged chaotropic agents such as guanidine hydrochloride should be avoided because they are not compatible with isoelectrofocusing (IEF). Detergents are used to solubilize proteins and consist of a polar head group and a hydrophobic tail, enabling them to solubilize membrane proteins mimicking the lipid environment. The gentleness of detergent is mainly determined by the size of its polar head group and the length of its hydrophobic acyl tail [37]. Detergents could be used at high, medium and low concentrations. When targeting separated proteins, detergents are used within a 1–4% concentration range, to prevent hydrophobic interactions. It is important to know whether the detergent should be removed and, if so, how easy it is to remove it – for example, by dialysis. In sample preparation for 2-DE, only neutral (octylglucoside, dodecyl maltoside and Triton X-100) or zwitterionic (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate, CHAPSO, SB 310, SB 3-12 and ABS-14) detergents are used, owing to their compatibility with the separation mechanisms. The anionic detergent sodium dodecyl sulfate (SDS) improves solubilization but interferes with the first electrophoretic dimension separation and must be removed if present in the preparation (e.g. ETTanTM 2D Clean Up kit, GE Health Care; ProteoSpinTM Detergent Clean UP micro and maxi kits, Norgen Biotek Corporation). To protect proteins from protease digestion, inhibitors should also be included during sample preparation phases [38]. Solubilization protocols should be adapted when handling hydrophobic proteins, such as membrane proteins. Membrane proteins represent a large population of the proteome in the form of receptors, transporters, channels and a variety of cellular mechanisms. The importance of membrane proteins is highlighted by the fact that approximately one-third of all genes in various organisms code for this class of proteins [39]. More than two-thirds of all medications exert their effects through membrane proteins [40], which makes them a major target of pharmacological interest. However, membrane proteins are still under-identified and underrepresented during whole-cell proteome analysis. Although membrane proteins with up to 12 transmembrane ahelices have been resolved and identified by 2-DE-MS [41], most membrane proteins have been resistant to this approach. Membrane proteins are often enriched by ultracentrifugation in sucrose gradient, lectin affinity chromatography in combination with centrifugation, silica beads or biotinylation and interaction with immobilized streptavidin [42]. Solubilization of membrane proteins is achieved with different detergents, such as combinations of chloroform and methanol, which were used to extract hydrophobic chloroplast membrane proteins. Alternatively, aqueous two-phase systems (which employ detergent DDM, Triton X-114 or PEG for the selective binding of one or

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more proteins of interest to one of the incompatible aqueous phases) could be used. Second rook: protein complexes. Proteins rarely function in isolation. Rather, they are organized in functional units that are different in size, number of interacting partners and stability, ranging from huge stable ribosomes or nuclear pore domains to small and transient signal transduction complexes. Studying these multiprotein complexes and microdomains provides information about the spatio-temporal organization of signal transduction or metabolic processes within a cell. Moreover, most of these protein complexes involve membrane proteins. However, a major part of this information is lost when cells are lysed and proteins digested before analysis. Isolating protein from complexes enables reduced complexity and eases the identification of low copy number proteins and their specific particular functions [43], although most of the sample pre-fractionation strategies (see the section ‘The bishop: sample fractionation’) tend to disrupt protein complexes by negatively influencing native protein–protein interactions. Protein complexes and hydrophobic protein species can be investigated through different approaches, including affinity-based methods, recombinant pull-downs, liquid chromatography, blue native gel electrophoresis (BN-PAGE), 2DE/LC/CE and FFE methods, followed by MS analysis [41]. Protein denaturing approaches (IEF, SDS-PAGE, 16-BAC-PAGE and CTABPAGE) are not suited to tackle protein complexes [41]. Nevertheless, BN-PAGE is perhaps the gel-based criterion of choice when addressing protein complexes and membrane proteins. BN-PAGE was introduced by Schagger and Von Jagow almost 20 years ago [44] and exploits Comassie G-250 to partially charge proteins to increase their electrophoretic mobility. Native gel-based approaches target protein complexes in their native form, as an alternative to immune co-precipitation [45], against which they enable separation without antibodies at the expense of detergentlabile interacting protein loss. Gel-free platforms particularly lend themselves to shotgun proteomic approaches, such as MudPIT, and minimize the problem of insolubility encountered in membrane protein studies [46].

The queen: protein–protein interactions Protein array information can be exploited to obtain complex maps of proteins involved in disease-specific molecular signatures and could, therefore, be translated to in silico data handling through elaborations of protein–protein interaction maps [47]. Biomolecule interactions are also relevant in that they might elucidate the efficiency and effectiveness of a targeted drug treatment. For example, the extent of drug binding to plasma proteins, determined by measuring the free active fraction, has a notable effect on the pharmacokinetics and pharmacodynamics of a drug. To this end, a vast array of different methods have been developed, viz., equilibrium dialysis, ultrafiltration, parallel artificial membrane permeability assay, high-performance affinity chromatography/zonal elution approach, high-performance affinity chromatography/ frontal analysis approach, affinity capillary electrophoresis, capillary electrophoresis/frontal analysis, spectroscopic assays, isothermal titration calorimetry, competition studies, titration studies, differential scanning calorimetry, surface plasmon resonance-based assays and SILAC [47]. Vice versa, it has been hypothesized that targeted drug treatments should address target protein biomarkers, www.drugdiscoverytoday.com

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which are characterized by specific properties making them eligible interactors of the designed drug (‘drug target-likeness’) [48]. In this respect, in silico modeling becomes pivotal for optimal design of drugs against, for example, specific subclasses of receptors [49]. In parallel, progresses have been made recently in the field of protein–protein interaction mapping and elaboration of networks, pathways and enrichment of gene ontologies for biological and molecular functions, especially upon the introduction of user-friendly software and updated databases [50] (Fig. 2). The ultimate goal of this kind of investigation is to use the software output to focus on specific targets as early biomarkers of pathological conditions or druggable-like targets for medical treatment. In parallel, the in silico approach could be useful in creating models or elaborating data from experimental observations about [()TD$FIG]

changes in pathways and networks upon administration of specific drugs. These methods might reveal drug effects on pathways, which represents the cornerstone of identifying mechanisms of drugs’ efficacy.

The king: quantitative profiles Pre-analytical control Although a wealth of studies are regularly being published about successful proteomic approaches to biological marker (biomarker) discovery, the discovery of new diagnostic biomarkers lags behind because of variability at every step in proteomics studies (e.g. assembly of a cohort of patients, sample preparation and the nature of body fluids, and selection of a profiling method and uniform protocols for data analysis) [51]. Most of this gap is due to

Legend Enzyme

AGTRAP

G-protein Coupled Receptor Kinase Peptidase Transcription Regulator Translation Regulator

ATG4A

ATG4B

Transporter

GABARAP

Unknown Relationship

IRGQ

GBAS ATG3*

RNF126 PANK2

LXN

ATG7 PP (1)

CIRBP

GABARAPL2 (includes EG:11345) UBA5 NCBP2

PSMF1*

YARS

EF4G1 EPPK1

KHSRP RBMX NCBP1 HNRNPH1

GSPT1 RPL35A

UBE2O

ARF6 CNBP*

HNRNPA3

IPO11

RPL12 (includes EG:6136)

RPL31

RPL26

LYZ* LTF Drug Discovery Today

FIGURE 2

Network analysis through Ingenuity Pathway Analysis of an experimental dataset. The software graphs proteins as nodes and interactions as connecting hubs (edges). Nodes could vary in color and shape to reflect experimental quantitative fluctuations or indicate different biological functions. Red nodes are overexpressed among two groups under comparison. Highly connected nodes (high-degree) represent the most biologically relevant proteins because they function as regulatory hubs for multiple pathways. Edges could be represented by continuous or interrupted lines, to indicate direct or indirect interactions between two proteins. 1020

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the lack of proper quantitative results on large cohorts of patients. As a result, most of the currently available biomarker discovery studies lack the statistical strength for the identification of highconfidence biomarkers and are thus more suited to tailor-made studies, although they lack the statistical strength of large-scale studies [52]. Often, the validation step that follows the discovery phase does not reach desired levels of sensitivity and specificity or reproducibility between laboratories [51]. Identification of the importance of preanalytical factors has implications for the use of large sample banks [53]. Such banks enable many prospective studies, which otherwise would require years for sample acquisition, to be carried out in a timely fashion, but sample banks vary in their adherence to consistent sample processing protocols over time. Many proteins, however, can be unaffected by many of the preanalytical variables. Therefore, any study using or proposing to use such banks should not be instantly condemned, but the way in which the banked samples are used should be examined. A possible, although imperfect, solution to this issue is to restrict the initial phase of biomarker discovery to specimens collected by rigorous adherence to banking protocols. To overcome this hurdle, multi-group efforts are necessary to facilitate the generation of sufficient sample sizes. This is contingent on the ability to collate and cross-compare data from different studies, which will require the use of a common metric or standard.

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be a major contributor to laboratory medicine in the near future. The potential clinical benefits for disease-specific biomarkers (to be tested for in clinical routine practice) include a more rapid and accurate disease diagnosis and a potential reduction in size and duration of clinical drug trials, which would speed up drug development. For example, proteomics in urine analysis could help identify and quantify proteins excreted in urine, which are not only key indicators of diseases associated with renal function but also indicators of the overall health of individuals. The application of biomarkers in the clinical arena of motor neuron disease should determine both whether a drug hits its proposed target and whether the drug alters the course of disease. It is interesting to note that, despite blood lacking direct contact with brain, increasing evidence suggests that there is a blood protein signature, and possibly a transcript signature, that might act to increase confidence in diagnosis and be used to predict progression in either disease or prodromal states. It might also be used to monitor disease progression [56]. These optimistic results come partly from candidate protein studies, from which it emerged that amyloid-beta measures might have value in prediction, and those studies of inflammatory markers that consistently show change in Alzheimer’s disease [56].

In theranostics

Checkmate: biomarker discovery and quantitation The biomarker field has generated various examples of individual biomarkers that can be used as indicators of disease pathophysiology, such as blood pressure, cholesterol levels and viral load in HIV [54]. The complexity of the underlying pathophysiological pathways and interactions makes it desirable to identify shortcuts through the proteome to associate meaningful biomarker-like signatures to specific protein profiles, rather than relying on independent biomolecules alone. The identification of a pattern or profile of several biomarkers (representing, e.g. a combination of genes, proteins, organic molecules, metabolites and/or a physiological response) representative of a given condition might bring a new dimension to disease diagnosis, classification and intervention and the assessment of therapeutic responses. Nonetheless, to be eligible as a valuable biomarker candidate, a protein should be detectable early and quantifiable over disease progression, and its co-occurrence with the disease should be statistically significant in a robust cohort of patients: although most scientific efforts have been put forward to address the first and last concerns, quantification has become a pivotal node in biomarker discovery and validation in recent years, especially in the field of human cancer research. To compare the abundance of proteins in different samples, several quantitative approaches have been developed (such as SILAC, isotope-coded affinity tags, exponentially modified protein abundance index and subtractive proteomic strategies), as Veenstra brilliantly reviewed [55]. Soon enough, quantitation strategies will enable biomarker discovery to meet quality criteria of clinical laboratory medicine.

In clinical laboratory medicine Clinical proteomics consists of discovery proteomics and measurement (quantitative) proteomics. Our current main focus is still discovery proteomics, clinical proteomics will undoubtedly

Non-invasive biomarker strategies based on imaging technologies – including magnetic resonance imaging, single-photon emission computed tomography, positron emission tomography and other techniques – are also progressing quickly in various therapeutic areas [57]. State-of-the-art applications of proteomics in the field of biomarker discovery involve the elaboration of specific signatures in 2DE maps, quantitative approaches [58], the individuation of marker-like post-translational modifications [59] and, recently, the measurement of varying metabolic profiles [60]. Complementary strategies are now adopted to focus on diseaserelevant converging pathways as potential therapeutic intervention points and are accompanied by the individuation of downstream biomarkers, which enable the tracking of drug targeting and seem to correlate with disease mitigation. When putting the pieces together, one is able to envision that a companion diagnostic will be codeveloped along the therapeutic compound. This ‘theranostic’ approach is perfectly positioned to align with the emerging trend toward personalized medicine [61]. In the quest for tailor-made drugs, proteomics has been contributing with previously unexpected results, involving, for example, genderspecific differences in human serum, which could be fundamental to building gender-specific protein biomarker databases to be exploited for targeted treatments.

Protein chips Once individuated, however, it is very unlikely that biomarkers will be monitored with 2-DE or mass spectrometry, which are routine techniques in the academic setting but do not lend themselves to clinical routine practice on large cohorts of samples. Further development of protein arrays might address this issue [51]. A faster approach to biomarker monitoring, using the integration of genomic and proteomic techniques, is based on the prowww.drugdiscoverytoday.com

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duction of protein chips. In this approach, cDNAs encoding tagged proteins are expressed and proteins are isolated and printed on a slide. These slides are then used to examine protein profiling, protein–protein interactions and antibody profiling [62]. These protein array techniques [63] can help not only to detect potential novel biomarkers but also to generate a greater understanding of the signaling pathways associated with the printed proteins. Protein-detecting arrays use a wide variety of capture agents (antibodies, fusion proteins, DNA/RNA aptamers, synthetic peptides, carbohydrates and small molecules) immobilized at high spatial density on a solid surface. Each capture agent binds selectively to its target protein in a complex mixture, such as serum or cell lysate samples. Captured proteins are subsequently detected and quantified in a high-throughput manner, with minimal sample consumption. Furthermore, the first multiplex immunoassay systems have been cleared by the US Food and Drug Administration, signaling recognition of the usefulness of miniaturized and parallelized array technology for protein detection in predictive and/or early diagnosis. Although genetic tests still predominate, with further development protein-based diagnosis will become common in clinical use within a few years [64].

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components, making it possible to identify protein signatures of diagnostic value and clinical importance. Alternative strategies have been created to target either protein biomarkers (often very low abundance protein) or drug-target-like molecules (often hydrophobic proteins at the membrane level) [65], both needing thorough clinical validation on large cohorts of patients before their widespread diffusion. The real deal is the limited biological variability tested in discovery studies, the current lack of quantitative information and, thus, the need for a proper validation and the absence of quality criteria in alignment to standard clinical practice. These objectives represent the very heart of the ambitious agenda and need to be accomplished in the next few years. The growing need for quantitative information is linked to the opportunity to better understand biological systems as a whole, through the integration of quantitative proteomics, metabolomics and transcriptomics auspicated by systems biology. Whether tailor-made treatments are still far from being a reality, pharmacoproteomics and in silico elaborations have contributed to make this chess game more challenging and intriguing than ever before. As huge amounts of data are being accumulated, the fulfillment of the pharmacoproteomic promises is rapidly approaching; however, the run towards the checkmate is not yet over.

Concluding remarks One square at a time, qualitative protein profiles of healthy and diseased tissues have been thoroughly investigated. Sample preparation and fractionation techniques have been gradually optimized to cut through the biological complexity of blood

Acknowledgements ADA and LZ were supported by founds from the Italian National Blood Centre (Centro Nazionale Sangue – Istituto Superiore di Sanita` – Rome, Italy).

References 1 Zolla, L. (2008) Proteomics studies reveal important information on small molecule therapeutics: a case study on plasma proteins. Drug Discov. Today. 13 (Suppl. 23–24), 1042–1051 2 Lockhart, B.P. and Walther, B. (2009) Biomarkers: ‘found in translation’. Med. Sci. (Paris) 25, 423–430 3 Anderson, N.L. and Anderson, N.G. (2002) The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics 1, 845–867 4 Piggott, A.M. and Karuso, P. (2004) Quality, not quantity: the role of natural products and chemical proteomics in modern drug discovery. Comb. Chem. High Throughput Screen. 7, 607–630 5 Liumbruno, G. et al. (2009) Blood-related proteomics. J. Proteomics 73, 483– 507 6 Righetti, P.G. and Boschetti, E. (2009) Blood proteomics and the dynamic range: some light at the end of the tunnel? J. Proteomics 73, 627–628 7 Roux-Dalvai, F. et al. (2008) Extensive analysis of the cytoplasmic proteome of human erythrocytes using the peptide ligand library technology and advanced mass spectrometry. Mol. Cell. Proteomics 7, 2254–2269 8 D’Alessandro, A. et al. (2010) The red blood cell proteome and interactome: an update. J. Proteome Res. 9, 144–163 9 Braun, R.J. et al. (2007) Two-dimensional electrophoresis of membrane proteins. Anal. Bioanal. Chem. 389, 1033–1045 10 Lee, S. et al. (2009) Structural interactomics: informatics approaches to aid the interpretation of genetic variation and the development of novel therapeutics. Mol. Biosyst. 5, 1456–1472 11 Omenn, G.S. et al. (2009) 7(th) HUPO World Congress of Proteomics: launching the second phase of the HUPOPlasma Proteome Project (PPP-2) 16–20 August 2008, Amsterdam, The Netherlands. Proteomics 9, 4–6 12 Chanda, S.K. and Caldwell, J.S. (2003) Fulfilling the promise: drug discovery in the post-genomic era. Drug Discov. Today 8, 168–174 13 Thiele, T. et al. (2007) Proteomics of blood-based therapeutics. BioDrugs 21, 179–193 14 Ichibangase, T. et al. (2009) Limitation of immunoaffinity column for the removal of abundant proteins from plasma in quantitative plasma proteomics. Biomed. Chromatogr. 23, 480–487

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15 Greening, D.W. and Simpson, R.J. (2010) A centrifugal ultrafiltration strategy for isolating the low-molecular weight (25 K) component of human plasma proteome. J. Proteomics 73, 637–648 16 Huang, L. et al. (2005) Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis. Proteomics 5, 3314–3328 17 Lin, B. et al. (2009) Deep depletion of abundant serum proteins reveals lowabundant proteins as potential biomarkers for human ovarian cancer. Proteomics Clin. Appl. 3, 853–861 18 Qian, W.J. et al. (2006) Advances and challenges in liquid chromatography–mass spectrometry-based proteomics profiling for clinical applications. Mol. Cell. Proteomics 5, 1727–1744 19 Lee, H.J. et al. (2006) Biomarker discovery from the plasma proteome using multidimensional fractionation proteomics. Curr. Opin. Chem. Biol. 10, 42–49 20 Hey, J. et al. (2008) Fractionation of complex protein mixtures by liquid-phase isoelectric focusing. Methods Mol. Biol. 424, 225–239 21 Jurgens, M. and Schrader, M. (2002) Peptidomic approaches in proteomic research. Curr. Opin. Mol. Ther. 4, 236–241 22 Righetti, P.G. and Boschetti, E. (2008) The ProteoMiner and the FortyNiners: searching for gold nuggets in the proteomic arena. Mass Spectrom. Rev. 27, 596–608 23 Castagna, A. et al. (2005) Exploring the hidden human urinary proteome via ligand library beads. J. Proteome Res. 4, 1917–1930 24 Sennels, L. et al. (2007) Proteomic analysis of human blood serum using peptide library beads. J. Proteome Res. 6, 4055–4062 25 D’Amato, A. et al. (2009) In-depth exploration of cow’s whey proteome via combinatorial peptide ligand libraries. J. Proteome Res. 8, 3925–3936 26 D’Alessandro, A. et al. (2010) The egg white and yolk interactomes as gleaned from extensive proteomic data. J. Proteomics 73, 1028–1042 27 Andersen, J.S. and Mann, M. (2006) Organellar proteomics: turning inventories into insights. EMBO Rep. 7, 874–879 28 Taylor, S.W. et al. (2003) Global organellar proteomics. Trends Biotechnol. 21, 82–88 29 Yates, J.R. et al. (2005) Proteomics of organelles and large cellular structures. Nat. Rev. Mol. Cell Biol. 6, 702–714

30 Vaisar, T. et al. (2007) Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL. J. Clin. Invest. 117, 746–756 31 Ahmed, F.E. (2009) Sample preparation and fractionation for proteome analysis and cancer biomarker discovery by mass spectrometry. J. Sep. Sci. 32, 771–798 32 Hsieh, S.Y. et al. (2006) Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling. Proteomics 6, 3189–3198 33 Rai, A.J. et al. (2005) HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples. Proteomics 5, 3262–3277 34 Marshall, J. et al. (2003) Processing of serum proteins underlies the mass spectral fingerprinting of myocardial infarction. J. Proteome Res. 2, 361–372 35 Taylor, C.F. et al. (2007) The minimum information about a proteomics experiment (MIAPE). Nat. Biotechnol. 25, 887–893 ˜ as, B. et al. (2007) Trends in sample preparation for classical and second 36 Can generation proteomics. J. Chromatogr. A 1153, 235–258 37 Reisinger, V. and Eichacker, L.A. (2008) Solubilization of membrane protein complexes for blue native PAGE. J. Proteomics 71, 277–283 38 Tan, S. et al. (2008) Membrane proteins and membrane proteomics. Proteomics 8, 3924–3932 39 Hopkins, A.L. and Groom, C.R. (2002) The druggable genome. Nat. Rev. Drug Discov. 1, 727–730 40 Plewczynski, D. and Rychlewski, L. (2009) Meta-basic estimates the size of druggable human genome. J. Mol. Model. 15, 695–699 41 Helbig, A.O. et al. (2010) Exploring the membrane proteome – challenges and analytical strategies. J. Proteomics 73, 868–878 42 Komatsu, S. et al. (2007) The proteomics of plant cell membranes. J. Exp. Bot. 58, 103–112 43 Pasquali, C. et al. (1999) Subcellular fractionation, electromigrationanalysis and mapping of organelles. J. Chromatogr. B: Biomed. Sci. Appl. 722, 89–102 44 Schagger, H. and von Jagow, G. (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199, 223–231 45 Blonder, J. et al. (2002) Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography–tandem mass spectrometry. J. Proteome Res. 1, 351–360 46 Han, D.K. et al. (2001) Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat. Biotechnol. 19, 946–951

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47 Ong, S.E. et al. (2009) Identifying the proteins to which small-molecule probes and drugs bind in cells. Proc. Natl. Acad. Sci. U. S. A. 106, 4617–4622 48 Xu, H. et al. (2007) Learning the drug target-likeness of a protein. Proteomics 7, 4255– 4263 49 Sela, I. et al. (2010) G protein coupled receptors – in silico drug discovery and design. Curr. Top. Med. Chem. 10, 638–656 50 Shannon, P. et al. (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 51 Silberring, J. and Ciborowski, P. (2010) Biomarker discovery and clinical proteomics. Trends Analyt. Chem. 29, 128 52 Chao, T.C. et al. (2010) Towards proteome standards: the use of absolute quantitation in high-throughput biomarker discovery. J. Proteomics 73, 1641–1646 53 Banks, R.E. (2008) Preanalytical influences in clinical proteomic studies: raising awareness of fundamental issues in sample banking. Clin. Chem. 54, 6–7 54 Ilyin, S.E. et al. (2004) Biomarker discovery and validation: technologies and integrative approaches. Trends Biotechnol. 22, 411–416 55 Veenstra, T.D. (2007) Global and targeted quantitative proteomics for biomarker discovery. J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 847, 3–11 56 Thambisetty, M. and Lovestone, S. (2010) Blood-based biomarkers of Alzheimer’s disease: challenging but feasible. Biomark. Med. 4, 65–79 57 Rudin, M. and Weissleder, R. (2003) Molecular imaging in drug discovery and development. Nat. Rev. Drug Discov. 2, 123–131 58 Liu, Y.F. et al. (2009) Quantitative proteome analysis reveals annexin A3 as a novel biomarker in lung adenocarcinoma. J. Pathol. 217, 54–64 59 Papini, A.M. (2009) The use of post-translationally modified peptides for detection of biomarkers of immune-mediated diseases. J. Pept. Sci. 15, 621–628 60 Wibom, C. et al. (2010) Metabolomic patterns in glioblastoma and changes during radiotherapy: a clinical microdialysis study. J. Proteome Res. 9, 2909–2919 61 Zhang, Z. et al. (2010) Systems biology and theranostic approach to drug discovery and development to treat traumatic brain injury. Methods Mol. Biol. 662, 317–329 62 Tomizaki, K.Y. et al. (2010) Protein–protein interactions and selection: array-based techniques for screening disease-associated biomarkers in predictive/early diagnosis. FEBS J. 277, 1996–2005 63 Miike, K. et al. (2010) Proteome profiling reveals gender differences in the composition of human serum. Proteomics 10, 2678–2691 64 Tomizaki, K.Y. et al. (2010) Protein–protein interactions and selection:array-based techniques for screening disease-associated biomarkers in predictive/early diagnosis. FEBS J. 277, 1996–2005 65 Lai, Z.W. et al. (2009) Membrane proteomics: the development of diagnostics based on protein shedding. Curr. Opin. Mol. Ther. 11, 623–631

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Connecting the dots: role of standardization and technology sharing in biological simulation Samik Ghosh1, Yukiko Matsuoka1,2 and Hiroaki Kitano1,3,4 1

The Systems Biology Institute, Tokyo 108-0071 Japan JST ERATO Kawaoka Infection-induced Host-response Network Project, Tokyo 108-8639 Japan 3 Okinawa Institute of Science and Technology, Okinawa, Japan 4 Sony Computer Science Laboratories, Tokyo 141-0022 Japan 2

The role of biological modeling and simulation in enhancing productivity across the drug discovery pipeline has been increasingly appreciated over the past decade by the pharmaceutical industry. However, adoption of in silico modeling and simulation techniques has been sparse due to skepticism in the associated pay-offs and knowledge gap in research. While biological simulations have been successfully applied in specific projects, a standardized, community-wide platform is imperative for making the final leap of faith across the domain. This review outlines the issues and challenges involved in fostering a private-public collaborative effort for the development of standard modeling and biosimulation platforms and concludes with insights into possible mechanisms for integrating an in silico pipeline into the drug discovery and development process.

In the spring of 2008, a group of scientists and thought leaders from across academia and industry met in Tokyo [1] to brainstorm future challenges in systems biology and its application to the pharmaceutical industry. While unanimously acknowledging the burgeoning role of systems biology in tackling complex diseases, the researchers laid out a bold objective – ‘to create over the next 30 years a comprehensive, molecule-based, multi-scale, computational model of the human (‘the virtual human’), capable of simulating and predicting, with a reasonable degree of accuracy, the consequences of most of the perturbations that are relevant to healthcare’ [2]. The scale and timeline of the project outlined in the Tokyo Declaration [2] underscore the complexity of the problem facing researchers in life sciences and pharmaceutical companies alike. With the current economic scenario, value of impending major blockbuster drug patent expirations and thinning pipelines for anticipated phase III drug approvals [3,4], the pharma industry is undergoing a paradigm shift in its efforts to develop safe and more efficacious drugs for the complex and life-threatening diseases facing humankind. Recent industry reports on future visions for Corresponding author:. Kitano, H. ([email protected])

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the industry [3,4] have highlighted the need to shift trajectories from target-based drug discovery to more system-oriented, holistic approaches that embrace knowledge from basic academic research and computational and systems engineering techniques. In particular, predictive biosimulation software – based on interconnected sets of mathematical equations with calibrated parameters to represent biological and physiological behaviors – has been successfully applied across the different stages of drug discovery and development, from target identification and validation, lead optimization and candidate selection to clinical trial design and development [5–7]. Simulation is an indispensable tool in all engineering designs and has been successfully applied in the automobile, aerospace and telecommunication industries for decades. Computational fluid dynamics (CFD), for example, is an essential design process in aircraft design, ship design and automobile design. Any highrise building has to carry out a series of structural integrity simulations even to be approved for construction; chipmakers model, modify and simulate their designs on computers before sending them to the fabrication plants; ‘virtual cars’ are driven and ‘virtual aircrafts’ flown under simulated conditions before hitting the manufacturing floor [8]. Although the application of advanced

1359-6446/06/$ - see front matter ß 2010 Published by Elsevier Ltd. doi:10.1016/j.drudis.2010.10.001

modeling and simulation techniques has resulted in immense cost savings and standardized procedures for such R&D-intensive industries, the pharmaceutical industry has historically lacked these approaches, leading to astronomical costs in drug development (25% of its revenue, almost twice that of other knowledgedriven industries [8]). Although appreciation and awareness for the potential benefits of computational approaches in biological sciences and drug design have been on an increasing trajectory in both industry and academic circles, it is important to keep in perspective the unique hurdles and cosiderable challenges of applying in silico techniques in the life sciences. Identification of the specific features desired from computational tools in the pharmaceutical industry, together with an open, collaborative mindset between all players, would form the key stepping stones in the development of safer, efficacious and cost-efficient drugs for complex diseases such as cancer, metabolic and cardiac disorders.

Issues and challenges The adoption of simulation techniques in the life sciences requires careful and detailed consideration of the unique challenges of multi-scale modeling– from cells, to tissues and organs, to whole human body and host–pathogen interactions. A series of issues have to be addressed before simulation can be accepted as normal practice in the industry. First, a set of fundamental technical issues must be solved to further improve accuracy of simulation. Different flavors of simulation technologies exist, from deterministic, differential-equation-based systems to non-deterministic, stochastic techniques, [()TD$FIG]

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agent-based and discrete-event based simulations; each presents a unique set of assumptions and system conditions that need to be considered before successful application to specific biological problems, as elucidated schematically in Fig. 1 [9]. Cellular modeling or physiological modeling with molecular details will require the integration of heterogeneous computational models that are on different spatial and temporal scales, and the basic equations still need to be defined [10]. The purpose and goal of a simulation system applied in drug design should be clearly defined: for example, in Formula 1 aerodynamics design, the goal is to design an aerodynamically optimal body with maximum down force with minimum drag. This forms a key step in defining and determining the eventual success of a biological simulation system. Merrimack Pharmaceuticals [11], for example, used computer simulation to identify a novel drug target for specific cancer sub-types that resulted in the development of a monoclonal antibody for ErbB3, now in clinical trial. Simulation models need to be designed to sufficiently capture essential features to accomplish the task defined, but features that are unlikely to affect prediction accuracy of the given task can be ignored. Sophisticated models with molecular details that can predict cellular behaviors in various conditions are crucial for elucidating system-level properties of cellular systems. Such models should be able to provide predictions on how cells and organs respond when certain perturbations, such as drug administration, are given. Although there are some successful cases of computational modeling of limited-scale biological networks, there is no established method for developing high-precision models.

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Schematic showing different simulation and modeling approaches. www.drugdiscoverytoday.com

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In establishing such practices, it might be prudent to borrow ideas from other fields with more mature and well-established practices of simulation modeling. In particular, the integration of computational modeling and experimental data acquisition has to be promoted. Delving into the design process of Formula 1 racing cars provides a picture of an iterative design cycle – several designs are tested with CFD and some of these designs are then tested using a wind tunnel, leading to the selection of one or two designs that are actually implemented and tested in a test course before one design is selected for final production. In this process, CFD models are calibrated against wind tunnel data for further improvement of accuracy, instead of data from the test course or from actual racing telemetry data. This comparison delivers two messages. First, we need to develop highly controllable experimental systems comparable to wind tunnels in aerodynamics. This means that we need to be able to precisely control exposure to chemical substances and other environmental conditions. Second, efforts need to be made to create high-precision models against well-controlled experimental systems, instead of uncontrollable systems. The identification and integration of structural, spatial and temporal dynamics of both interaction networks and cellular structures is an essential prerequisite for defining such high-precision models. The dynamics of cellular structure and interaction networks need to be quantified by taking comprehensive, high-resolution, measurements of intracellular status – such as the concentrations, interactions, modifications and localizations of molecules – and of cellular structures in different dimensions and environmental conditions. In addition, the problem of how to identify unknown interactions from such data sets still remains. These problems are fundamental and require collaborative efforts on a community-wide scale. Although various ongoing efforts exist for tackling the problems of building molecular network maps, simulation tools, data resources and web services for sharing information, an open, integrative platform for sharing and exchange of computational approaches is of fundamental importance.

Standards and technologies The sharing of knowledge accelerates progress in science. This is perhaps more true for the biological sciences, in which the complexity and size of the problem domain make it imperative for different research groups to focus on different sections of it. Computational tools have already played an important part in the collection, storage and intelligent retrieval of vast amounts of information in the life sciences. With the adoption of biosimulation tools, the ability to store and share information in a seamless, unambiguous fashion became imperative, leading to the definition of The Systems Biology Markup Language (SBML; http:// www.sbml.org), a set of standards developed to facilitate effective and efficient sharing of models defined as a set of biochemical reactions. The effort was initiated by the ERATO Kitano Symbiotic Systems Project, funded by the Japanese government but soon grew to be a global community-wide initiative. Importantly, the SBML community has evolved a proper procedure to elect editors, implemented voting procedures and formalized discussion forums. Thus, it is no longer a project belonging to any one institution but is truly a community effort. Currently, more than 180 1026

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pieces of bio-modeling and simulation software comply with SBML [12], enabling the sharing of models across them. Although the definition of a common lingua franca is an important step in the standardization of in silico technologies, biology has traditionally been a descriptive science, in which the role of pictures and diagrams cannot be overstated. Keeping in mind the need for a common graphical representation standard in the life sciences, a community-wide effort has been undertaken to define The Systems Biology Graphical Notation (SBGN; http:// www.sbgn.org/) [13]. The SBGN community is working to formulate a set of rules for human-readable visual representations of biological networks. The goal of SBGN is to define a set of visual glyphs and syntax so that anyone can understand exactly what each diagram means (Fig. 2), in the same vein as the electrical circuit diagrams used by chip designers. With SBML and SBGN, standards for computational modeling and representation languages are being defined that will have a notable impact on standardization and model sharing. For models to be informative, they must be properly annotated; sufficient information must be attached with the models to enable third parties to use them. With the objective of proper model annotation, the MIRIAM project (http://www.ebi.ac.uk/miriam/) defines the minimum information that has to be attached to a model so that model can be informative by itself. A series of standard formation efforts are now underway to cover the whole process of modeling development and analysis. Although standardization is an integral part of the process of computational systems biology, the development of a suite of software tools for model building, distribution and running simulations is another important dimension. In this direction, a plethora of modeling building and simulation tools such as Cellarator [14], Copasi [15] and Dizzy [16] in the academic community and SimBiology1 from Mathworks Inc. (http:// www.mathworks.com/products/simbiology) and PhysioLab1 (Entelos Inc; http://www.entelos.com) exist, each catering to different modeling techniques (see Ref. [12] for comprehensive coverage of systems biology tools). One of the most popular and widely used tools in this space [12] is CellDesignerTM [17] – a modeling and simulation tool to visualize, model, and simulate gene regulatory and biochemical networks. Two major characteristics embedded in CellDesigner boost its usability to create, import and export models: the solidly defined and comprehensive graphical representation (SBGN) of network models and SBML as a model-describing basis, which function as inter-tool media to import and export SBML-based models. Moreover, CellDesigner provides the ability to embed – or smoothly connect via Systems Biology Workbench (http:// www.sys-bio.org/research/sbwIntro.htm) – different simulation and analysis packages, which enable the simulation of the pathways using various simulation techniques (Copasi, SBML ODE solver, and so on), as shown in Fig. 3a. With the explosive growth in proliferation and adoption of the Internet and web services, knowledge in the life sciences has shifted to the World Wide Web with online access to scientific literature, biological databases, and knowledge-sharing and/or discussion forums. As more biological information comes online, it is important to develop an infrastructure that enables the community to leverage the vast web resources. This online, com-

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SYSTEMS BIOLOGY GRAPHICAL NOTATION REFERENCE CARD Entity Pool Nodes

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(a) The Systems Biology Graphical Notation (SBGN) glyphs and (b) sample model representation.

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(a) CellDesigner snapshot and (b) Payao web interface snapshot.

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munity collaboration paradigm motivated the development of Payao [18], a web-based biological pathway sharing and tagging service (http://www.payaologue.org). A snapshot of the interface is shown in Fig. 3b. The goal of Payao is to provide a Google Maps equivalent for biological pathways, wherein researchers can share large-scale, curated and annotated (MIRIAM-compliant) network maps (SBGN and SBML compliant) using software such as CellDesigner and publish it to the community online. With the built-in tagging and collaborative system, the community can participate in enhancing the biological entities in the map or navigate their specific areas of interest. Other important assets for online community collaboration are the databases that provide pathway models fully curated and compliant with standards. Two major online data resources providing access to biological pathway models are the BioModels.net Initiative (http://www.biomodels.net), which provides a one-stop shop for SBML-compliant simulation models of biological pathways published in various literature, and The Panther Pathways database [19], which provides a database of annotated pathways created in CellDesigner (SBML and SBGN compliant). We have provided a slice of the spectrum of activities initiated by different research groups in developing biosimulation tools and technologies. Whereas each of these tools provides a niche solution to a particular biological problem in isolation, an integrative framework leveraging the advantages of the diverse techniques holds the promise of providing a comprehensive computational pipeline.

Community collaboration and open flow model The standards and technologies in systems biology, as elucidated in the previous section, represent various pieces of a comprehensive computational pipeline for the pharmaceutical industry. Crucial to the success of such a pipeline, however, is the clarification of the promises and pitfalls of the different components and the identification of a cohesive set of strategies to add value to the drug discovery process. In June 2008, a representative group of systems biology scientists from academia, biotechnology and the pharmaceutical sector gathered in Portofino, Italy [20], with the goal of brainstorming a set of recommendations for such a strategic path to systems biology. While identifying various action items like setting data standards, modeling drug actions and toxicity, the leaders proclaimed that a ‘network solution’ [20] (i.e. community-wide collaboration, communication and outreach) was the key to solving complex biological network problems. As outlined in the ‘Issues and challenges’ section, the size and complexity of living systems present several challenges to the systems biology community. Overcoming the obstacles of simulation speed, accuracy, high-precision experiments and knowledge sharing would require the expertise of scientists and engineers from diverse backgrounds and disciplines. Thus, developing an ecosystem of communication and information sharing empowered by powerful computational simulation tools and web services can provide the right plan for advancing biosimulation approaches in the pharmaceutical domain. Concomitant with the development of a community ecosystem is the need for an open flow model of research, in which researchers from industry and academia share knowledge in a collective

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commons of data, analytical tools, information technology, biospecimens and disease models [21]. The past decade has seen several efforts in open-source biomedical science – the Cancer Genome Atlas (http://www.cancergenome.nih.gov/), Pathway Commons (http://www.pathwaycommons.org) and World Community Grid (http://www.worldcommunitygrid.org). Efforts for establishing collaboration and outreach through joint projects have also been launched in North America and Europe, including the Alliance for Cellular Signaling by the NIH (http://www.afcs.org) and the HepatoSys project (http://www.hepatosys.de) in Germany. The Open Source Drug Discovery project (http://www.osdd.net), initiated by the Council of Scientific and Industrial Research, India, is a uniquely novel effort dedicated to developing drugs for neglected tropical diseases (Mycobacterium tuberculosis) that plague developing countries and have very thin pipelines (in terms of the number of candidate drug compounds in development) in the pharmaceutical industries. An open innovation strategy has also gained traction recently in the pharmaceutical industry, fueled by a need to reconfigure and streamline the drug discovery pipeline, particularly in the early phases of target biology and biomarker development [22]. An industry–academic consortium involving researchers from The University of California at San Diego, the California Institute of Technology, the Massachusetts Institute of Technology, the University of Massachusetts, Entelos, and several research groups from Pfizer has initiated a collaborative project on insulin-resistive pathways [23]. More recently, in May 2010, London-based GlaxoSmithKline and Novartis deposited over 300,000 structures of chemical compounds active against the malaria parasite Plasmodium falciparum in an open database archive, ChEMBL Neglected Tropical Disease from the European Bioinformatics Institute (http://www.ebi.ac.uk/chemblntd) [22]. Several socioeconomic obstacles to leveraging the power of an open-source approach exist, however – participant willingness to share data, incompatibility of formats, quality control of data, intellectual property conflicts and sustainable funding avenues. To develop a generic model for collaboration, it is necessary to develop a framework that is open, providing standardized, license-free access to biological data; integrative, providing common sets of pathway curation, annotation, modeling, analysis and simulation tools; and ‘share-and-care’, an incentive system for providing pre-competitive, early access to the results and data to participants.

An open, integrative, share-and-care model The goal of an open, integrative, share-and-care framework is to provide a common set of tools, principles and practices for the application of biosimulation techniques in a systematic and cohesive manner. It would involve a standardization platform that enables the incorporation of standards and interfaces for the systems biology community; a computational bio-networking platform that provides a suite of network building tools, databases and web services for sharing information in a standards-compliant, interoperable manner; and an advanced simulation platform that incorporates the different simulation tools and technologies and is capable of encompassing information from the standard compliant biological network resources and databases. A schematic representation of such a scheme is shown in Fig. 4. www.drugdiscoverytoday.com

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An open, integrative, share-and-care model for systems biology.

Although the technical components of such a framework are all or mostly in place, the success of the model hinges on being able to foster a bi-directional flow of knowledge between academia and industry. An open access paradigm for information and data sharing would enable the fast and effective dissemination of knowledge and the identification and filling in of information gaps in crucial biological processes and pathways. A standard-driven, integrative framework would enable a plugand-play model for diverse simulation and modeling tools, providing researchers with the freedom to test various tools and techniques. The most important part, perhaps, is ensuring the flow of information between diverse research groups. In this respect, a share-and-care model would provide incentives to all participants: academic researchers would gain access to specific data on drugs and clinical trials (e.g. access to failed drugs from phase II trial databases [24]), enabling them to develop models with higher predictive powers, whereas the business imperatives of pharmaceutical companies would be respected through early, pre-competitive access to model predictions and insights. Such a system would work in a positive feedback loop, because high-precision data from the drug industry would drive the precision of biosimulation models, which, in turn, would be able to provide better decision-making conditions for the drug companies. In a recent development, a Boston, MA based startup called 1030

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EnlightBio (http://www.enlightbio.com), working on similar paradigms, started operations focused on developing breakthrough innovations through partnership with multiple drug companies. Another effort initiated in early 2010 is Sage Bionetworks (http:// www.sagebase.org), a non-profit, open-source research organization that aims to develop and share large-scale network models of diseases. Such initiatives would foster collaborative research while paving the way for systems biology to empower the life sciences industry.

Future perspectives The value of applying systems biology techniques to the different stages of the drug design and development process has been demonstrated through various academic–industry collaborations spanning projects of different scales and sizes. Although such isolated success stories further motivate the large-scale adoption of in silico biosimulation practices, the development of a structured and coherent route to facilitate and accelerate the process is imperative. As outlined in this review, collaboration is the key to achieving the goal – not only in terms of joint projects but also – in developing a suite of standards-compliant platforms for sharing domain-specific knowledge and expertise. It might not be a distant dream that the community will develop a set of recommendations for good simulation practices (GSP), as already exists for good manufacturing practices (GMP) and good engineering practices (GEP) – standards and protocols accepted and complied with in the

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application of computational simulation tools for drug design and development.

Acknowledgements This research is, in part, supported by the ERATO-SORST Program of the Japan Science and Technology Agency (JST); the Genome

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Network Project of the Ministry of Education, Culture, Sports, Science, and Technology; the NEDO Fund for International Standard Formation from the New Energy Development Organization; and the Okinawa Institute of Science and Technology.

1 Editorial, (2008) All systems go. Nat. Rev. Drug Discov. 7, 278–279 2 FCSB, (2008) The Tokyo Declaration, International Workshop on Future Challenges for Systems Biology. http://www.systems-biology.org/myukiko/FCSB2008/ doku.php?id=workshop:statement 3 PriceWaterhouseCoopers, (2008) Pharma 2020: Virtual R7D – which path will you take? http://www.pwc.com/extweb/pwcpublications.nsf/docid/ 91BF330647FFA402852572F2005ECC22 4 Futurology, P. (2007) Joined-up healthcare, 2016 and beyond. British Telecommunications http://www2.bt.com/static/i/media/pdf/ BT_Pharma_Lowres.pdf 5 Kansal, A.R. and Trimmer, J. (2005) Application of predictive biosimulation within pharmaceutical clinical development: examples of significance for translational medicine and clinical trial design. Syst. Biol. (Stevenage) 152, 214–220 6 Rullmann, J.A.C. et al. (2005) Systems biology for battling rheumatoid arthritis: application of the Entelos PhysioLab platform. Syst. Biol. (Stevenage) 152, 256–262 7 Sams-Dodd, F. (2006) Drug discovery: selecting the optimal approach. Drug. Discov. Today 11, 465–472 8 No authors listed. (2005) Models that take drugs. The Economist June, S23–S24. 9 Michelson, S. and Scherrer, D. (2003) Predictive biosimulation for lead optimization. Curr. Drug Discov. 1, 18–22 10 Kitano, H. (2010) Grand challenges in systems physiology. Frontiers in Physiology. doi:10.3389/fphys.2010.00003. 11 No author listed. (2008) Merrimack Pharmaceuticals initiates enrollment in a phase 1 study of MM-121, an ErbB3 antagonist. Medical News Today August.

12 Klipp, E. et al. (2007) Systems biology standards – the community speaks. Nat. Biotechnol. 25, 390–391 13 Le Nove`re, N. et al. (2009) The systems biology graphical notation. Nat. Biotechnol. 27, 735–741 14 Shapiro, B.E. et al. (2003) Cellerator: extending a computer algebra system to include biochemical arrows for signal transduction simulations. Bioinformatics 19, 677–678 15 Hoops, S. et al. (2006) COPASI – a complex pathway simulator. Bioinformatics 22, 3067–3074 16 Ramsey, S. (2005) Dizzy: stochastic simulation of large-scale genetic regulatory networks. J. Bioinform. Comput. Biol. 3, 415–436 17 Funahashi, A. et al. (2008) CellDesigner 3.5: a versatile modeling tool for biochemical networks. In Proceedings of the IEEE 96 pp. 1254–1265 18 Matsuoka, Y. et al. (2010) Payao: a community platform for SBML pathway model curation. Bioinformatics 26, 1381–1383 19 Thomas, P.D. et al. (2003) PANTHER: a library of protein families and subfamilies indexed by function. Genome Res. 13, 2129–2141 20 Henney, A. and Superti-Furga, G. (2008) A network solution. Nature 455, 730–731 21 Patlak, M. (2010) Open-source science makes headway. J. Natl. Cancer Inst. 102, 1221–1223 22 Strauss, S. (2010) Pharma embraces open source models. Nat. Biotechnol. 28, 631– 634 23 No authors listed. (2008) The Insulin Resistance Pathways Project. http:// www.pfizercambridge.com/home.php?id=groups/tgu/focus_insulin 24 Petsko, G.A. (2010) When failure should be the option. BMC Biol. 8, 61

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Lyotropic liquid crystal systems in drug delivery Chenyu Guo1, Jun Wang1, Fengliang Cao1, Robert J. Lee2,3, and Guangxi Zhai1, 1

Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan 250012, People’s Republic of China Division of Pharmaceutics, The Ohio State University, Columbus, OH 43210, USA 3 Center for Affordable Nanoengineering of Polymeric Biomedical Devices, an NSF Nanoscale Science and Engineering Center, The Ohio State University, Columbus, OH 43210, USA 2

Lyotropic liquid crystal systems, such as reversed bicontinuous cubic and hexagonal mesophases, are attracting more and more attention because of their unique microstructures and physicochemical properties. Various bioactive molecules such as chemical drugs, peptides and proteins can be solubilized in either aqueous or oil phase and be protected from hydrolysis or oxidation. Furthermore, several studies have demonstrated sustained release of bioactive molecules from reversed cubic and hexagonal mesophases. This article gives an overview of recent advances and current status of reversed cubic and hexagonal mesophases, especially with respect to their preparation methods and applications in the field of drug delivery. In addition, potential problems and possible future research directions are highlighted.

Introduction Lyotropic liquid crystal (LLC) systems that commonly consist of amphiphilic molecules and solvents can be classified into lamellar (La), cubic, hexagonal mesophases, and so on. In recent years, LLC systems have received considerable attention because of their excellent potential as drug vehicles. Among these systems, reversed cubic (Q2) and hexagonal mesophases (H2) are the most important and have been extensively investigated for their ability to sustain the release of a wide range of bioactives from low molecular weight drugs to proteins, peptides and nucleic acids [1–5]. Reversed cubic and hexagonal mesophases are often formed by polar lipids in an aqueous environment. The structure-forming lipids can absorb a certain amount of water and then spontaneously form gel-like phases with unique internal structures, into which drugs can be incorporated. Moreover, non-toxic, biodegradable and bioadhesive properties also contribute to their applications for drug delivery [6]. Owing to infinite swelling capability, reversed cubic and hexagonal mesophases can also be dispersed in equilibrium with excess water and form colloidal dispersions with superior thermodynamic stability [7,8]. At present, reversed cubic and hexCorresponding authors:. Zhai, Lee, R.J. ([email protected]), G. ([email protected])

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agonal mesophases are being investigated as candidates for aural, buccal, gastrointestinal, intravenous, lung, nasal, oral, rectal and vaginal administration of drug with considerable progress [1]. In the following sections, we briefly introduce the cubic and hexagonal mesophases based on recent literature, including their textures, preparation methods, phase behaviors and applications in drug delivery. This article is not meant to provide an exhaustive review but rather to present some highlights. In particular, we discuss the current status of investigations with respect to the applications of cubic and hexagonal mesophases as drug vehicles and then propose new or promising directions of research.

Structures of reversed cubic and hexagonal mesophases For reversed bicontinuous cubic and hexagonal mesophases, three macroscopic forms are typically encountered: precursor, bulk gel and particulate dispersions.

Structure of cubic mesophase The structure of cubic mesophases is unique and comprises a curved bicontinuous lipid bilayer (with an estimated thickness of 3.5 nm) extending in three dimensions and two interpenetrating, but non-contacting, aqueous nano-channels (with a fully

1359-6446/06/$ - see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2010.09.006

swollen diameter of approximately 5 nm), with a high interfacial area of 400 m2/g [1,7,8]. At present, the cubic mesophases prepared by unsaturated monoglycerides or phytantriol (PT) are the most frequently investigated liquid crystal structures for drug delivery [9–11]. The compartmentalization in cubic mesophases can be used to introduce guest drugs of hydrophilic, lipophilic or amphiphilic nature (Fig. 1a). Hydrophilic drugs will be located close to the emulsifier polar head or in the water channels, whereas lipophilic drugs will be localized within the lipid bilayer and amphiphilic drugs in the interface [12]. The bulk phase is commonly a clear, viscous, semi-solid gel that is similar in appearance and rheology to cross-linked polymer hydrogels [13]. Its high viscosity makes it difficult to handle and limits its application and, furthermore, the bulk phase can cause the irritation reaction when in contact with the biological epithelia [14]. To overcome these issues, an innovative strategy has been formulated: to disperse the bulk phase into water in the form of small particles. The dispersed cubic particles are denoted as ‘cubosomes’, which can stably exist in equilibrium with aqueous solution with the internal bicontinuous structure unchanged [15,16]. Based on X-ray crystallographic studies, three distinct reversed bicontinuous cubic phases can be identified: the double-diamond lattice (Pn3m, Q224), the body-centered cubic phase (Im3m, Q229) and the gyroid lattice (Ia3d, Q230) [6,17].

Structure of hexagonal mesophase Hexagonal mesophases are closed and extended micellar columnar structures [18], and the long-range order is two-dimensional. It has been reported that there is no direct contact between water

[()TD$FIG]

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inside and outside the hexagonal phases [19]. Likewise, the dispersed reversed hexagonal particles denoted as ‘hexosomes’ can also be obtained by dispersing the hexagonal gel into aqueous solution [15,16]. To date, the hexagonal mesophases composed of glycerate-based surfactants such as oleyl glycerate (OG) and phytanyl glycerate (PG) have shown great potential in drug delivery [20,21]. As can be seen in Fig. 1b, hydrophilic drugs will be entrapped in the internal water domain, whereas lipophilic drugs will be located within the lipid domain and amphiphilic drugs in the interface.

Preparation methods for reversed cubic and hexagonal mesophases As a rule, cubic and hexagonal gels can be prepared more easily than their dispersions. For example, liquid crystal gels could be prepared by simply blending aqueous phase with lipid phase using vortex or ultrasonication [21]. The manufacture of cubosomes or hexosomes is more complicated, however; therefore, we mainly concentrate on the preparation methods of LLC nanoparticles. The schematic diagrams are represented in Fig. 2.

Top-down approach This approach was primarily reported by Ljusberg-Wahren in 1996 [22]. The extreme viscous bulk phase is prepared by mixing structure-forming lipids with stabilizers, then the resultant is dispersed into aqueous solution through the input of high energy (such as high-pressure homogenization [HPH], sonication or shearing) to form LLC nanoparticles. At present, HPH is the most extensively used technique in the preparation of LLC nanoparticles [23]. ¨ rle et al. [24] investigated the parameters influencing the Wo properties of glyceryl monooleate (GMO)-based cubosomes. Based on the results observed, the concentration of F127 and temperature during HPH were regarded as crucially important parameters. Recently, a novel approach of shearing was proposed to fabricate LLC nanoparticles using a laboratory-built shearing apparatus [25]. Compared with the well-established ultrasonication approach, the shearing treatment could effectively prepare more stable and homogeneous cubosomes or hexosomes with high content of the hydrophobic phase (oil + lipophilic additives) within a short time (less than one minute). It seems that the preparation procedure is simple enough to be realized conveniently. In fact, the operation units in this procedure require several cycles to achieve the desired nanoparticles with appropriate characteristics, and the high-energy input is also regarded as a barrier to the temperaturesensitive ingredients [23]. In addition, the cubosomes prepared through top-down approach are always observed to coexist with vesicles (dispersed nanoparticles of lamellar liquid crystalline phase) or vesicle-like structures, which will hamper the investigations on plain cubic mesophases.

Bottom-up approach

FIGURE 1

Structures of (a) reversed bicontinuous cubic and (b) hexagonal mesophases, inspired by Sagalowicz et al. [12]. Possible localizations of drugs in the mesophases are also pointed out. Note that for simplicity, only partial lattice is represented.

The key factor in the bottom-up approach is hydrotrope, which can dissolve water-insoluble lipids to create liquid precursors and prevent the formation of liquid crystals at high concentration [26]. Compared with the top-down approach, this dilution-based approach can produce cubosomes without laborious fragmentation. In other words, it needs less energy input. Moreover, this approach is far more efficient at generating small particles. The www.drugdiscoverytoday.com

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(a)

Oil phase (melted lipid)

Aqueous phase

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High energy input (HPH, sonication, shearing, etc)

(b)

Lipid

Hydrotrope

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(c) Heat treatment (autoclaved at 121ºC for 15 min plus an equilibration time of 5 min) LLC suspension

LLC nanoparticles

prepared by approach A or B

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Schematic diagrams of preparation methods for cubosomes or hexosomes according to the literature [13,24,27–31]. (a) Top-down approach. (b) Bottom-up approach. (c) Heat treatment. (d) Spray drying.

reason for this might relate to the forming mechanism of cubosomes. The dilution-based approach can be regarded as a process of small particles forming big particles through aggregation, which is analogous to the use of precipitation processes to produce nanoparticles, whereas the top-down approach is more analogous to the attrition of big particles. In addition, cubosomes prepared through 1034

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dilution show long-term stability, which might be attributed to the homodisperse stabilizers onto the surface of cubosomes [23]. Indeed, the use of hydrotrope can simplify the preparation process and produce cubosomes possessing similar or even better properties than those fabricated by the top-down approach. It should be noted, however, that this process via dilution is a

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than pure drug when administered perorally; it is noteworthy, however, that residual solvent content is still a problem that cannot be ignored.

Phase behaviors of reversed cubic and hexagonal mesophases

Heat treatment

Generally, molecular geometry has an important role in determining mesophase behavior, thus crucial packing parameter (CPP) can be introduced to predict molecular geometry in a surfactant–water system. CPP = v/a0l, where v is the hydrophobic chain volume, a0 is the cross-sectional area of the surfactant headgroup and l is the hydrophobic chain length [32]. Depending on CPP, different selfassembly structures can be formed (Fig. 3). When CPP = 1, lamellar liquid crystalline structure forms. When CPP is smaller than 1, oil in water self-assembly structures form, such as normal micelles (L1) and normal hexagonal (H1) phases. When CPP > 1, reversed selfassembly structures form, such as reversed cubic structure, reversed hexagonal structure and reversed micelles (L2) [12]. Based on the published literature, many factors can influence the phase behaviors of cubic and hexagonal mesophases. Addition

The coexistence of cubosomes with vesicles is speculated to provide multiphasic manipulation of the sustained release of drugs [1]; hence, to better investigate the release behavior of plain mesophases, vesicles should be eliminated as much as possible. In this case, heat treatment can be regarded as a good approach. Note that in the strictest sense, heat treatment is not an integrated process for the manufacture of cubosomes because it only promotes the transformation from non-cubic vesicles to well-ordered cubic particles. The dispersed particles, therefore, can be produced by a simple processing scheme comprising a homogenization and heat-treatment step. From the reported studies, heat treatment could cause a decrease in the small particle size fraction that corresponded to vesicles and form more cubic phases with narrow particle distribution and good colloidal stability [27–29]. Taking the whole process of preparation into account, it is obvious that the transition takes place during the procedure of heat treatment. The reason for transition could be speculated as an elevated temperature giving rise to a reduction in solubility and stability. When the temperature was below cloud point, the surfactant had a high solubility and thus the particles could exist stably and the phenomenon of fusion was hardly observed. Once reaching cloud point, the solubility of surfactant decreased notably and a notable fast fusion among vesicles would occur [27]. This ¨ rle et al. [28]. Although masses hypothesis was also verified by Wo of vesicles can transform to cubic nanoparticles through heat treatment, it does not mean that all the LLC systems are suitable for this procedure – in particular, the systems loading drugs that cannot provide sufficient stability under the condition of high temperature (usually above 1208C), such as some proteins and temperature-sensitive drugs, are not suitable.

[()TD$FIG]

Spray drying To widen the applications of cubosomes in pharmaceutical field, dry powder precursors can be fabricated by spray drying and used for the preparation of oral solid formulations and inhalants. This approach was originally proposed and investigated by Spicer et al. [30]. In his research, the powder precursor could be prepared through drying a pre-dispersed aqueous solution that consisted of GMO, hydrophobically modified starch and water or contained GMO, dextran, ethanol and water, and then the colloidally stable dispersions of nano-structured cubosomes could be created by hydration of the precursors. Afterward, Shah et al. [31] prepared GMO-based cubosome precursor containing diclofenac sodium through spray drying. The precursor was proven to have more effective and prolonged anti-inflammatory and analgesic activity

FIGURE 3

Schematic diagrams of different existing surfactant self-assembly structures and their corresponding CPP, inspired by Yaghmur and Glatter [8]. Going from the top down corresponds to a decrease in the CPP. www.drugdiscoverytoday.com

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pathway by charting trajectories on the ternary phase diagram (lipid–water–hydrotrope), which requires knowledge of the full phase behavior; hence, the extent of dilution is difficult to control precisely. Owing to the addition of hydrotrope, many issues arise, such as the effects exerted by varying concentrations of hydrotrope on the physicochemical properties of LLC nanoparticles and the possible occurrence of irritation and allergic response when the mesophase formulations are administered. Finally, this bottom-up approach cannot effectively avoid forming vesicles. Through cryo-TEM, many vesicles and vesicle-like structures were also observed to coexist with cubosomes [14].

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of a third substance – such as oleic acid, triolein, diglycerol monooleate, soybean phosphatidylcholine, retinyl palmitate, and tetradecane – could modulate the textures of mesophases and even result in phase transition [25,33–35]. It was reported that increase or decrease of temperature or pressure could also induce the phase transition of mesophases and, moreover, the pressure-dependent structural transition displayed opposite trends in lipid systems as compared to the influence of temperature [36,37]. In addition, salt concentration and pH value had been proven to have an effect on the phase behavior of mesophases to a certain extent [38,39]. An intimate knowledge of phase behavior will provide original ideas for using LLC systems for drug delivery.

In vitro release behavior of drugs from reversed cubic and hexagonal mesophases Bulk cubic and hexagonal mesophases have been investigated as sustained drug delivery systems for more than 18 years [40]. It is widely accepted that release of drugs from these mesophases in most cases has been shown to follow Higuchi diffusion-controlled kinetics [41], where the cumulative amount of drug diffusion through matrix presents a linear dependence with the square root of time. The release behavior is related to many aspects, such as the properties of drugs, initial water content, type of LLC phases, swelling capacity, drug loading, electrostatic interaction between drugs, lipid bilayers and so on [2,42–48]. For dispersed mesophases, in-depth investigations on drug release were conducted by Boyd [49]. Lipophilic compounds containing diazepam, griseofulvi, propofol and rifampicin were employed as model drugs, and the pressure ultrafiltration method was used to determine the release behavior of these drugs. The results showed that cubosomes should be classified as a burst release delivery system, in which drug was released by diffusion from the cubic phase matrix. In the subsequent study, the release of irinotecan from hexosomes was also measured using ultrafiltration and an analogous phenomenon of burst release was again found [20]. The reason can be elucidated as follows: because of dividing the bulk mesophases into lots of small particles, the surface area greatly increases, and thus drugs can be transported into aqueous phase much faster from cubosomes or hexosomes.

Applications of reversed cubic and hexagonal mesophases as drug delivery carriers Cubic and hexagonal mesophases as injectable vehicles Reversed bicontinuous cubic and hexagonal phases are highly viscous, and this mechanical stiffness makes them clumsy to handle and difficult to inject [1,6,50]. To overcome this defect, some corresponding approaches have been proposed, such as application of flowable precursor forms [21,51] and use of LLC nanoparticles [20,52,53]. According to the phase diagram of structure-forming lipid, the transition from lamellar phases to cubic phases can be completed upon heating from room temperature to body temperature or swelling with water. Therefore, lamellar phases with inherently fluid properties can act as precursors of viscous cubic phases. Once injected into the body via subcutaneous or muscular approach, flowable lamellar phases will gradually absorb water from body fluid or surrounding tissues and, subsequently, convert to cubic phases, which can form the sustained release depot in situ [6]. Hexagonal phases also cannot be directly injected because of the 1036

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limitation of high viscosity. One way to circumvent this problem is using L2 phase with low viscosity as precursors. For example, OG and PG phases underwent a transition of L2 to H2 at 378C with increasing water at approximately 7  1%, and it had been demonstrated that a drug-containing L2 phase precursor prepared by OG or PG could transform to reverse hexagonal phase, which showed sustained release behavior when injected into excess aqueous solutions [21]. It should be noted, however, that losing a considerable volume of water from the topical environment can cause irritation to the body; this issue should be considered carefully when lamellar and L2 precursor systems are administered in vivo. Another limitation is that the precursor systems containing lamellar and L2 phases all show relatively rapid release [21,54], which is not conducive to the sustained release of drugs from cubic and hexagonal mesophases. Therefore, how to reduce the amount of drugs released from precursor systems and shorten the duration of transformation should be deliberated. Recently, Fong et al. [51] designed a delicate protocol to prepare an ‘on demand’ drug release delivery system, which could vary the release rate of model drugs (glucose) through the transition of Q2 to H2 induced by tuning temperature. They employed the PTbased mesophases loading 3% of vitamin E, which was proved to have a transition temperature of Q2 to H2 at approximately 378C. In a drug release experiment under dynamic temperature (308C to 408C to 308C), when the temperature was switched to 408C, the release rate was suppressed for the transition from Q2 to H2. Once the temperature was back to 308C, the release rate was immediately returned to close to the original release rate because of the structure reverting to cubic mesophase. The result obtained from a similar release study under dynamic temperature (408C to 308C to 408C) also verified that the switching of temperature stimulated the variation of release rate. In in vivo absorption studies, when injected subcutaneously at 408C, the drug released from hexagonal mesophase slowly. After switching the temperature to 308C, a phenomenon of statistically significant increase in plasma concentration was observed; furthermore, this system displayed a more sustained manner than other control formulations. Because of their small particle size, low viscosity, biocompatibility and thermodynamic stability in excess water, cubosomes and hexosomes are particularly suitable for intravenous injection. Leesajakul et al. [52] investigated the interplay between GMObased cubosomes and plasma in vitro and in vivo. In vitro study revealed that GMO would be adsorbed out of the particles by albumin that had binding sites for GMO. From in vivo study, when injected intravenously, cubosomes were disintegrated in a short period of time. However, Chol-py (a fluorescence probe) incorporated still showed the property of long-term circulation, which might be attributed to the sustained behavior of cubosome remnant particles. This study shows probable ways in which cubosomes are degraded in blood circulation, but whether the texture of the remnant particles changed and which specific forms the drugs solubilize in after disruption of cubosomes are still not clear.

Oral administration of drug-loaded cubic and hexagonal mesophases Lipid formulations such as lipid suspensions, solutions, emulsions and self-emulsifying lipid-based formulations can all increase the oral bioavailability of poorly water-soluble drugs [55,56]. To realize

the expected aim that the formulations can exhibit sustained release of oral drugs in vivo, there are still three principal factors that need to be noticed. First, the formulations must possess the inherent property of sustained release, which is a major precondition. Second, they should stably exist in the gastrointestinal fluids to provide a persistent matrix from which drugs can be slowly released. Note that this requires the formulations to resist the digestive process to a certain extent. Third, the property of bioadhesive can extend the formulations’ retention time in the gastrointestinal tract, providing more time for drug absorption [57]. According to current literature, although GMO-based mesophase formulation has been shown previously to enhance the bioavailability of co-administered poorly water-soluble drugs [58,59] and exhibits the first and third features described above, it cannot provide sustained release owing to its sensitivity to the digestive process [48]. With in-depth investigations of some novel materials (including PT and OG) that can resist the effect of digestive enzymes, some progressions have been made, especially in the aspect of bioavailability enhancement and sustained release of oral drugs, showing promising perspectives of applications. Boyd et al. [57] investigated the oral bioavailability of a poorly water-soluble drug, cinnarizine, incorporated in different types of LLC phases. Through animal experiments, the OG-based hexagonal formulation showed a considerably higher relative bioavailability that was almost 3.5 times greater than that of the control suspension of cinnarizine and 3 times greater than the GMO-based cubic formulation. Furthermore, it was intriguing that the OG matrix provided extended absorption of drug for over 120 h, which was several times longer than the other two formulations, indicating long residence of the formulation in the gastrointestinal tract and a poor sink condition in vivo inhibiting drug release. It also should be noted that OG was more resistant to the digestive process than GMO, which was proven in in vitro digestion studies, and this might also be responsible for sustained drug absorption. Recently, Lee et al. [48] employed glucose-loaded Q2 GMO, Q2 PT and H2 PT+Vit EA phases as researching objects to investigate in vivo-in vitro correlation and realized the control over absorption of hydrophilic drug in vivo through manipulation of matrix nanostructure for the first time. The oral administration of drugs incorporated into LLC nanoparticles has also been reported [60–62]. Chung et al. [60] prepared GMO-based cubosomes containing insulin and investigated the hypoglycemic effect generated by oral administration of this formulation. The blood glucose concentration–time profile showed that the insulin formulation could provide a hypoglycemic effect comparable to intravenous administration of insulin over six hours. Simvastatin incorporated in GMO-based cubosomes was administered orally and the relative bioavailability to the control drug crystal powder was 241%. Moreover, the cubosomes showed sustained release of simvastatin over 12 h in beagle dogs. The author presumed that the mechanism of enhancing bioavailability might be related to the hydrophilic surface of cubosomes, which stimulated the permeation through the stagnant aqueous layer of the intestinal mucosa [62].

Topical application of cubic and hexagonal mesophase formulations Topical drug delivery is an attractive alternative to oral administration. Its main drawback is the limited absorption of drugs

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through the skin barrier, and investigations on topical drug uptake are necessary to facilitate the design of efficient topical drug delivery systems. At present, stratum corneum (SC) is considered to be the rate-limiting barrier in transdermal drug delivery [63]. Many studies have shown that cubic and hexagonal mesophase formulations are capable of penetrating through SC and becoming candidates for topical drug delivery systems [64–72]. At present, GMO-based and PT-based mesophases are the most widely investigated LLC systems for topical drug delivery. It has been proven that these mesophases can statistically significant enhance permeation of drugs such as acyclovir [64], d-aminolevulinic acid [65], indomethacin [66], cyclosporine (Cys A) [67–69], vitamin K [70] and diclofenac salts [71,72]. There are several natural characteristics that the reversed cubic and hexagonal phases present to make them suitable for topical drug delivery: (i) sustained release of drugs incorporated, (ii) bioadhesive properties, (iii) solubilization of hydrophilic and lipophilic drugs and protecting them from physical and enzymatic degradation, and (iv) the nontoxic permeation enhancers GMO and PT as structureforming materials [10,65,68]. Bender et al. [73] used two-photon microscopy to elucidate the penetration pathway of sulphorhodamine B, a fluorescent hydrophilic model drug that was incorporated in GMO-based and PTbased cubic mesophases and then applied in human skin in vitro. The results revealed different penetration approaches for the control formulations and cubic mesophase formulations: the intercellular pathway seems to be predominant when using the water solution and the ointment, whereas the intercluster pathway seems to dominate the skin absorption for the cubic mesophases. In addition, drugs could penetrate into the deeper layer of skin by using cubic mesophases and, moreover, GMO-based cubic mesophase – compared with PT-based cubic mesophase – seemed to permeate the lipid matrix more readily. Topical applications of d-aminolevulinic acid and its methyl ester that were incorporated into GMO–water and GMO/PT–propylene glycol–water systems, respectively, showed fast penetration in comparison to the standard ointment during the studies of one hour short-term and 24 h continuous applications. The difference between GMO and PT in terms of enhancing drug permeation mainly relied on the discrimination of the swelling extent and the rheological property [65]. Lopes et al. [68] reported that Cys A incorporated in GMO-based cubic and hexagonal phases could statistically significant elevate the penetration of Cys A. Moreover, the cubic phase formulation favored retention of Cys A in the skin, whereas the hexagonal one favored its penetration into deeper skin layers and its transdermal delivery. Cubosomes and hexosomes have also been used for topical drug delivery. Compared with the cubic and hexagonal gels, the dispersions show some unique advantages. First, the good fluidity and large surface area of the dispersions provide tighter contact with the skin. Second, the dispersions can be embedded by the other formulations. Last, but not least, they does not cause skin irritation after topical application [10,66,70]. Topical applications of carbomer-indomethacin loaded cubosomes, carbomerblank cubosomes and carbomer with an indomethacin water suspension had been reported to show different drug release behavior and effects on UVB-induced erythema through human test [66]. The first formulation statistically significant prolonged www.drugdiscoverytoday.com

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anti-inflammatory activity when exposed under UVB irradiation for six hours after removing it, whereas the other two formulations exhibited decreased activities to a certain extent. Release studies also verified the persistently higher concentration of indomethacin in SC after application of the first formulation. Cys A incorporated in hexosomes comprising GMO, oleic acid and water was reported to be capable of enhancing drug permeation when applied topically [69]. In an in vitro permeation study using the drug-loaded hexosomes, the concentration of Cys A in epidermis and dermis ([E + D]) was two times higher than that after application of the control formulation (olive oil solution of Cys A). Similarly, statistically significant enhancement of drug concentration in [E + D] (2.8 times) was derived from in vivo study. Moreover, a skin irritation test demonstrated that the daily application of this formulation did not cause skin irritation. In addition, Lopes et al. [67] found that a high concentration of GMO (20–70%) could suppress the transdermal delivery of Cys A. Similar results were also obtained that the increase of GMO concentration might prevent the penetration of Vit K into the deep layer [70]. These phenomena might be caused by an intense interaction between GMO and lipophilic drugs.

both drugs were observed for over a period of 18 h in vitro [74]. However, no data involving in vivo experiment were exhibited. Lee et al. [75] reported that GMO in the cubic and lamellar mesophases could be eroded without the action of an enzyme and then penetrate across excised porcine buccal mucosa. Moreover, the flux of a [D-Ala2, D-Leu5] enkephalin incorporated from the cubic and lamellar mesophases was enhanced statistically significant compared with PBS solution during the initial three hours. Likewise, there were no in vivo results presented. Not only the bulk mesophases but also their dispersions could be utilized for mucosal drug delivery. Swarnakar et al. [77] reported that after application of progesterone loaded hexosomes on the albino rabbit mucosa for 12 h, an obviously enhanced transmucosal flux was observed and that it was fivefold higher than that of progesterone loaded gel and nearly fourfold higher than plain progesterone suspension. In addition, lipid extraction phenomena and evident pores were exhibited in the epithelium of mucosa through FT-IR and confocal laser scanning microscopy, indicating a probable intercellular ‘virtual channel’ for hexosomes permeating.

Concluding remarks and further perspectives Mucosal drug delivery using cubic and hexagonal mesophases The structure-forming materials (such as GMO, PT, OG and PG) all possess not less than two hydroxyl groups, which make them available for hydrogen bonding to mucus membranes, and therefore the cubic and hexagonal mesophases are good candidates for mucosal drug delivery [74–77]. To facilitate operation, the flowable precursor systems are employed, which can form the viscous cubic or hexagonal gels by absorption of body fluid in vivo. It was reported that GMO-based gel was used for vaginal delivery of propantheline bromide and oxybutynin hydrochloride, and sustained release behaviors of

This review mainly discusses the current applications of reversed cubic and hexagonal mesophases as drug vehicles, with a major focus on their applications in vivo. Table 1 presents some major investigations with respect to the cubic and hexagonal mesophases as drug vehicles in recent years (2004–2009). Note that animal experiments were also conducted in these studies to validate these mesophase formulations. Based on the current literature, cubic and hexagonal mesophase formulations maintain a good momentum of growth and show broad prospects for development. Although the cubic and hexagonal mesophases possess advantageous characteristics, there is still a long way to go before their

TABLE 1

Cubic and hexagonal mesophases as drug vehicles reported in recent yearsa Type of LLC phases

Lipid system

Cubic bulk phase

PT/water; PT/VitEA/water

Cubosomes

GMO/F127/water

Lipid-based liquid crystalline nanoparticle

Phosphatidylcholine/glycerol dioleate/Tween 80/water

Cubic bulk phase

Administration route

Refs

Glucose

Subcutaneous injection

[51]

Chol-py

Intravenous injection

[52]

Somatostatin

Intravenous injection

[53]

GMO/water; OG/water

Cinnarizine

Oral administration

[57]

Cubic bulk phaseHexagonal bulk phase

GMO/water; PT/water; PT/VitEA/water

GlucoseAllura RedFITC-dextran

Oral administration

[48]

Hexagonal bulk phase

OG/water

Sodium pamidronate

Oral administration

[78]

Cubosomes

GMO/water

Omapatrilat

Oral administration

[61]

Cubosomes

GMO/F127/water

Simvastatin

Oral administration

[62]

Cubic bulk phase

GMO/water; PT/water; GMO/propylene glycol/water; PT/propylene glycol/water

d-Aminolevulinic acid

Topical application

[65]

Cubosomes

GMO/F127/water

Indomethacin

Topical application

[66]

Cubic bulk phaseHexagonal bulk phase

GMO/water; GMO/oleic acid/water

Cys A

Topical application

[68]

Hexosomes

GMO/oleic acid/F127/water

Cys A

Topical application

[69]

Hexagonal bulk phaseHexosomes

GMO/water; GMO/F127/water

Vit K

Topical application

[70]

Hexosomes

GMO/oleic acid/F68/water

Progesterone

Mucosal application

[77]

a

In vivo experiments were conducted in these investigations.

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Bioactive molecule

clinical application. For injectable cubosomes and hexosomes, more approaches should be exploited to increase the effective drug loading and control sustained release actions – for instance, multi-component cubic and hexagonal mesophases are good candidates that might meet these requirements. For oral formulations, the current investigations are mainly concentrated on the cubic and hexagonal gels loading lipophilic drugs, whereas the investigations involving the transport of hydrophilic drugs and the use of LLC nanoparticles are still very limited. In addition, some new

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structure-forming materials such as PT, OG and PG have exhibited superior properties to conventional materials. At present, however, understanding of them is still insufficient, especially in safety, biological stability and appearance in vivo. For topical applications, especially for mucosal drug delivery, there are still few studies with respect to in vivo experiments; thus, more work should be performed to validate the mesophase formulations in vivo. In the future, all these aspects should be brought to the forefront and investigated further.

References 1 Drummond, C.J. and Fong, C. (1999) Surfactant self-assembly objects as novel drug delivery vehicles. Curr. Opin. Colloid Interface Sci. 4, 449–456 2 Clogston, J. and Caffrey, M. (2005) Controlling release from the lipidic cubic phase. Amino acids, peptides, proteins and nucleic acids. J. Control. Release 107, 97–111 3 Mezzenga, R. et al. (2005) Understanding foods as soft materials. Nat. Mater. 4, 729–740 4 Ubbink, J. et al. (2008) Food structure and functionality: a soft matter perspective. Soft Matter 4, 1569–1581 5 Mohammady, S.Z. et al. (2009) Oleoylethanolamide-based lyotropic liquid crystals as vehicles for delivery of amino acids in aqueous environment. Biophys. J. 96, 1537–1546 6 Shah, J.C. et al. (2001) Cubic phase gels as drug delivery systems. Adv. Drug Deliv. Rev. 47, 229–250 7 Spicer, P.T. (2005) Progress in liquid crystalline dispersions: cubosomes. Curr. Opin. Colloid Interface Sci. 10, 274–279 8 Yaghmur, A. and Glatter, O. (2009) Characterization and potential applications of nanostructured aqueous dispersions. Adv. Colloid Interface Sci. 147–148, 333–342 9 Amar-Yuli, I. et al. (2009) Solubilization of food bioactives within lyotropic liquid crystalline mesophases. Curr. Opin. Colloid Interface Sci. 14, 21–32 10 Dong, Y.D. et al. (2006) Bulk and dispersed aqueous phase behavior of phytantriol: effect of vitamin E acetate and F127 polymer on liquid crystal nanostructure. Langmuir 22, 9512–9518 11 Dong, Y.D. et al. (2008) Impurities in commercial phytantriol significantly alter its lyotropic liquid-crystalline phase behavior. Langmuir 24, 6998–7003 12 Sagalowicz, L. et al. (2006) Monoglyceride self assembly structures as delivery vehicles. Trends Food Sci. Technol. 17, 204–214 13 Spicer, P.T. and Hayden, K.L. (2001) Novel process for producing cubic liquid crystalline nanoparticles (cubosomes). Langmuir 17, 5748–5756 14 Rosen, M.R. (2006) Delivery System Handbook for Personal Care and Cosmetic Products: Technology, Applications, and Formulations. William Andrew 15 Gustafsson, J. et al. (1996) Cubic lipid–water phase dispersed into submicron particles. Langmuir 12, 4611–4613 16 Gustafsson, J. et al. (1997) Submicron particles of reversed lipid phases in water stabilized by a nonionic amphiphilic polymer. Langmuir 13, 6964–6971 17 Larsson, K. (2000) Aqueous dispersions of cubic lipid–water phases. Curr. Opin. Colloid Interface Sci. 5, 64–69 18 Laughlin, R.G. (1994) The Aqueous Phase Behavior of Surfactants. Academic Press 19 Sagalowicz, L. et al. (2006) Investigating reversed liquid crystalline mesophases. Curr. Opin. Colloid Interface Sci. 11, 224–229 20 Boyd, B.J. et al. (2006) Hexosomes formed from glycerate surfactants – formulation as a colloidal carrier for irinotecan. Int. J. Pharm. 318, 154–162 21 Boyd, B.J. et al. (2006) Lyotropic liquid crystalline phases formed from glycerate surfactants as sustained release drug delivery systems. Int. J. Pharm. 309, 218–226 22 Ljusberg-Wahren, H. et al. (1996) Dispersion of the cubic liquid crystalline phase – structure, preparation, and functionality aspects. Chim. Oggi 14, 40–43 23 Spicer, P.T. (2005) Cubosome processing industrial nanoparticle technology development. Chem. Eng. Res. Des. 83, 1283–1286 ¨ rle, G. et al. (2007) Influence of composition and preparation parameters on the 24 Wo properties of aqueous monoolein dispersions. Int. J. Pharm. 329, 150–157 25 Salentinig, S. et al. (2008) Preparation of highly concentrated nanostructured dispersions of controlled size. J. Colloid Interface Sci. 326, 211–220 26 Mezzenga, R. et al. (2005) Shear rheology of lyotropic liquid crystals: a case study. Langmuir 21, 3322–3333 27 Barauskas, J. et al. (2005) Cubic phase nanoparticles (cubosome): principles for controlling size, structure, and stability. Langmuir 21, 2569–2577

¨ rle, G. et al. (2006) Transformation of vesicular into cubic nanoparticles by 28 Wo autoclaving of aqueous monoolein/poloxamer dispersions. Eur. J. Pharm. Sci. 27, 44–53 ¨ rle, G. et al. (2006) Effect of drug loading on the transformation of vesicular into 29 Wo cubic nanoparticles during heat treatment of aqueous monoolein/poloxamer dispersions. Eur. J. Pharm. Biopharm. 63, 128–133 30 Spicer, P.T. et al. (2002) Dry powder precursors of cubic liquid crystalline nanoparticles (cubosomes). J. Nanopart. Res. 4, 297–311 31 Shah, M.H. et al. (2006) Spray dried glyceryl monooleate-magnesium trisilicate dry powder as cubic phase precursor. Int. J. Pharm. 323, 18–26 32 Israelachvili, J. (1994) The science and applications of emulsions – an overview. Colloid Surf. A 91, 1–8 33 Nakano, M. et al. (2002) Dispersions of liquid crystalline phases of the monoolein/ oleic acid/Pluronic F127 system. Langmuir 18, 9283–9288 34 Yaghmur, A. et al. (2005) Emulsified microemulsions and oil-containing liquid crystalline phases. Langmuir 21, 569–577 35 Yaghmur, A. et al. (2006) Control of the internal structure of MLO-based isasomes by the addition of diglycerol monooleate and soybean phosphatidylcholine. Langmuir 22, 9919–9927 36 Seddon, J.M. et al. (2006) Pressure-jump X-ray studies of liquid crystal transitions in lipids. Phil. Trans. R. Soc. A 364, 2635–2655 37 Yaghmur, A. et al. (2010) Effects of pressure and temperature on the self-assembled fully hydrated nanostructures of monoolein-oil systems. Langmuir 26, 1177–1185 38 Awad, T.S. et al. (2005) Formation of cubic phases from large unilamellar vesicles of dioleoylphosphatidylglycerol/monoolein membranes induced by low concentrations of Ca2+. Langmuir 21, 11556–11561 39 Okamoto, Y. et al. (2008) Low-pH-induced transformation of bilayer membrane into bicontinuous cubic phase in dioleoylphosphatidylserine/monoolein membranes. Langmuir 24, 3400–3406 ¨ m, S. and Engstro ¨ m, L. (1992) Phase behaviour of the lidocaine–monoolein– 40 Engstro water system. Int. J. Pharm. 79, 113–122 41 Higuchi, W.I. (1967) Diffusional models useful in biopharmaceutics. J. Pharm. Sci. 56, 315–324 42 Clogston, J. et al. (2005) Controlling release from the lipidic cubic phase by selective alkylation. J. Control. Release 102, 441–461 43 Shah, M.H. and Paradkar, A. (2007) Effect of HLB of additives on the properties and drug release from the glyceryl monooleate matrices. Eur. J. Pharm. Biopharm. 67, 166–174 44 Ahmed, A.R. et al. (2008) Reduction in burst release of PLGA microparticles by incorporation into cubic phase-forming systems. Eur. J. Pharm. Biopharm. 70, 765–769 45 Rizwan, S.B. et al. (2009) Liquid crystalline systems of phytantriol and glyceryl monooleate containing a hydrophilic protein: characterisation, swelling and release kinetics. J. Pharm. Sci. 98, 4191–4204 46 Tilley, A. et al. (2009) Liquid crystalline coated drug particles as a potential route to long acting intravitreal steroids. Curr. Drug Deliv. 6, 322–331 47 Rosenbaum, E. et al. (2010) A characterisation study on the application of inverted lyotropic phases for subcutaneous drug release. Int. J. Pharm. 388, 52–57 48 Lee, K.W.Y. et al. (2009) Nanostructure of liquid crystalline matrix determines in vitro sustained release and in vivo oral absorption kinetics for hydrophilic model drugs. Int. J. Pharm. 365, 190–199 49 Boyd, B.J. (2003) Characterisation of drug release from cubosomes using the pressure ultrafiltration method. Int. J. Pharm. 260, 239–247 50 Malmsten, M. (2007) Phase transformations in self-assembly systems for drug delivery applications. J. Disp. Sci. Tech. 28, 63–72 51 Fong, W.K. et al. (2009) Stimuli responsive liquid crystals provide ‘on-demand’ drug delivery in vitro and in vivo. J. Control. Release 135, 218–226

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52 Leesajakul, W. et al. (2004) Interaction of cubosomes with plasma components resulting in the destabilization of cubosomes in plasma. Colloid Surf. B 34, 253–258 53 Cervin, C. et al. (2009) A combined in vitro and in vivo study on the interactions between somatostatin and lipid-based liquid crystalline drug carriers and bilayers. Eur. J. Pharm. Sci. 36, 377–385 54 Kumar, M.K. et al. (2004) Effect of drug solubility and different excipients on floating behaviour and release from glyceryl monooleate matrices. Int. J. Pharm. 272, 151–160 55 Porter, C.J.H. et al. (2008) Enhancing intestinal drug solubilisation using lipid-based delivery systems. Adv. Drug Deliv. Rev. 60, 673–691 56 Tang, B. et al. (2008) Development of solid self-emulsifying drug delivery systems: preparation techniques and dosage forms. Drug Discov. Today 13, 606–612 57 Boyd, B.J. et al. (2007) A lipid-based liquid crystalline matrix that provides sustained release and enhanced oral bioavailability for a model poorly water soluble drug in rats. Int. J. Pharm. 340, 52–60 58 Charman, W.N. et al. (1993) Effect of food and a monoglyceride emulsion formulation on danazol bioavailability. J. Clin. Pharmacol. 33, 381–386 59 Larsson, K. (1999) Colloidal dispersions of ordered lipid–water phases. J. Dispersion Sci. Technol. 20, 27–34 60 Chung, H. et al. (2002) Self-assembled ‘nanocubicle’ as a carrier for peroral insulin delivery. Diabetologia 45, 448–451 61 Tamayo-Esquivel, D. et al. (2006) Evaluation of the enhanced oral effect of omapatrilat – monolein nanoparticles prepared by the emulsification-diffusion method. J. Nanosci. Nanotechnol. 6, 3134–3138 62 Lai, J. et al. (2009) Glyceryl monooleate/poloxamer 407 cubic nanoparticles as oral drug delivery systems: I. In vitro evaluation and enhanced oral bioavailability of the poorly water-soluble drug simvastatin. AAPS PharmSciTech 10, 960–966 63 Nemanic, M.K. and Elias, P.M. (1980) In situ precipitation: a novel cytochemical technique for visualization of permeability pathways in mammalian stratum corneum. J. Histochem. Cytochem. 28, 573–578 64 Helledi, L.S. and Schubert, L. (2001) Release kinetics of acyclovir from a suspension of acyclovir incorporated in a cubic phase delivery system. Drug Dev. Ind. Pharm. 27, 1073–1081

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65 Bender, J. et al. (2005) Lipid cubic phases for improved topical drug delivery in photodynamic therapy. J. Control. Release 106, 350–360 66 Esposito, E. et al. (2005) Cubosome dispersions as delivery systems for percutaneous administration of indomethacin. Pharm. Res. 22, 2163–2173 67 Lopes, L.B. et al. (2005) Topical delivery of cyclosporin A: an in vitro study using monoolein as a penetration enhancer. Eur. J. Pharm. Biopharm. 60, 25–30 68 Lopes, L.B. et al. (2006) Liquid crystalline phases of monoolein and water for topical delivery of cyclosporin A: characterization and study of in vitro and in vivo delivery. Eur. J. Pharm. Biopharm. 63, 146–155 69 Lopes, L.B. et al. (2006) Reverse hexagonal phase nanodispersion of monoolein and oleic acid for topical delivery of peptides: in vitro and in vivo skin penetration of cyclosporin A. Pharm. Res. 23, 1332–1342 70 Lopes, L.B. et al. (2007) Enhancement of skin penetration of vitamin K using monoolein-based liquid crystalline systems. Eur. J. Pharm. Sci. 32, 209–215 71 Cohen-Avrahami, M. et al. (2010) HII mesophase and peptide cell-penetrating enhancers for improved transdermal delivery of sodium diclofenac. Colloid Surf. B 77, 131–138 72 Yariv, D. et al. (2010) In vitro permeation of diclofenac salts from lyotropic liquid crystalline systems. Colloid Surf. B 78, 185–192 73 Bender, J. et al. (2008) Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy. J. Control. Release 129, 163–169 74 Geraghty, P.B. et al. (1996) The in vitro release of some antimuscarinic drugs from monoolein/water lyotropic liquid crystalline gels. Pharm. Res. 13, 1265–1271 75 Lee, J. and Kellaway, I.W. (2000) Buccal permeation of [D-Ala2, D-Leu5] enkephalin from liquid crystalline phases of glyceryl monooleate. Int. J. Pharm. 195, 35–38 76 Lee, J. and Kellaway, I.W. (2000) Combined effect of oleic acid and polyethylene glycol 200 on buccal permeation of [D-Ala2, D-Leu5] enkephalin from a cubic phase of glyceryl monooleate. Int. J. Pharm. 204, 137–144 77 Swarnakar, N.K. et al. (2007) Enhanced oromucosal delivery of progesterone via hexosomes. Pharm. Res. 24, 2223–2230 78 Boyd, B.J. et al. (2005) Compositions and methods of delivery of biologically active agents. (International patent WO 2005021046).

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Developments towards antiviral therapies against enterovirus 71 Kan X. Wu, Mary M.-L. Ng and Justin J.H. Chu Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, 5 Science Drive 2, National University of Singapore, Singapore

Enterovirus 71 (EV71) has emerged as a clinically important neurotropic virus that can cause acute flaccid paralysis and encephalitis, leading to cardiopulmonary failure and death. Recurring outbreaks of EV71 have been reported in several countries. The current lack of approved anti-EV71 therapy has prompted intense research into antiviral development. Several strategies – ranging from target-based chemical design to compound library screenings – have been employed, while others revisited compound series generated from antiviral developments against poliovirus and human rhinoviruses. These efforts have given rise to a diversity of antiviral candidates that include small molecules and nonconventional nucleic-acid-based strategies. This review aims to highlight candidates with potential for further clinical development based on their putative modes of action. Enterovirus 71 (EV71) is described as one of the human enterovirus A species under the genus Enterovirus in the Picornaviridae family of viruses, which includes poliovirus [1]. While efforts to eradicate poliovirus through vaccination programs have limited the number of polio-endemic countries to just four (Afghanistan, India, Nigeria and Pakistan) [2], EV71 has emerged as an important non-polio neurotropic enterovirus. EV71 was first isolated from patients with central nervous system diseases in California between 1969 and 1974 [3]. The same authors also described the EV71 prototype strain, BrCr, isolated from a twomonth-old patient who presented with aseptic meningitis. Since then, EV71 outbreaks have been reported in several countries beyond North America, including Taiwan, Australia, Malaysia and Singapore [4]. These outbreaks have mainly involved young children, with most cases displaying mild, self-limiting hand, foot and mouth disease; however, EV71 outbreaks have also been associated with a variety of severe neurological complications that can deteriorate rapidly to involve cardiopulmonary failure with high mortality rates. Transmission of EV71 can occur rapidly via the fecal–oral and droplet and/or aerosol routes. During the largest EV71 outbreak to date, in Taiwan in 1998, more than 100,000 children were affected, with 405 severe cases involving neurological or cardiopulmonary complications, of which 78 were fatal [5]. Corresponding author:. Chu, Justin J.H. ([email protected]), ([email protected]) 1359-6446/06/$ - see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2010.10.008

The spectrum of EV71-associated neurological diseases includes aseptic meningitis, brain stem encephalitis and poliomyelitis-like acute flaccid paralysis. Like poliomyelitis, severe EV71 infections can result in permanent neurological damage. In a long-term study of children who survived neurologically involved EV71 infections during the 1998 outbreak in Taiwan, patients who had a more severe infection (involving both neurological and cardiopulmonary complications) displayed signs of neurologic sequelae, impaired neurodevelopment and impaired cognitive functions [6]. There is currently no effective vaccine or antiviral against EV71. Treatments for acute EV71 infections with neurological manifestations mainly aim to alleviate symptoms. Mechanical cardiopulmonary support systems and the administration of milrinone, a positive inotropic agent, have been used to prevent cardiopulmonary failure and thus improve the clinical outcome of patients [7]. The lack of antiviral treatment options against EV71 remains a worrying situation, however, because EV71 has been found to circulate endemically with peak activity in warmer seasons (e.g. summer to fall) [8]. Considering the propensity of EV71 to cause severe neurological diseases in children, there is a need to develop effective antiviral treatment options to prevent or reduce EV71related deaths and long-term neuropathy in the next EV71 global outbreak. In this review, we focus on documenting recent developments towards an antiviral for EV71 infections. Previous reviews have www.drugdiscoverytoday.com

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covered the development of antivirals targeting picornaviruses or enteroviruses in general [9–15]; however, with the exception of poliovirus, EV71 has distinguished itself from other enteroviruses in terms of circulation and severity of associated diseases. In contrast to poliovirus, against which highly effective vaccines are available, the global population remains largely unprotected against EV71. Our review, therefore, highlights promising anti-EV71 strategies and their putative modes of inhibition.

General virology of picornaviruses An understanding of the molecular mechanisms of viral infection and replication can enable the identification of potential antiviral targets. Although the exact mechanisms for EV71 replication have not been elucidated, picornaviruses generally share a highly similar virus architecture and mode of replication. Picornaviruses are non-enveloped viruses of 30 nm in diameter with an icosahedral capsid made up of 60 protomers, each of which is, in turn, made up of four structural proteins: VP1, VP2, VP3 and VP4. The picorna-

[()TD$FIG]

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virus genome is encoded as a single-stranded, positive-sense RNA of 7500–8000 bases that consists of an open reading frame flanked by 50 and 30 untranslated regions (UTRs). Upon virus attachment and entry into the host cell, an uncapping event occurs to release the RNA genome into the cell. Covalently attached to the 50 end is a viral protein, VPg (3B), which is cleaved by a cellular enzyme during early infection, whereas the 30 end has a poly(A) tract. The 50 and 30 UTRs are highly structured regions that interact with a variety of viral and host proteins in translation and RNA synthesis events. Cap-independent translation of the viral RNA takes place through the recruitment of host replication machinery at the internal ribosome entry site located in the 50 UTR of the viral RNA. The viral polyprotein can be divided into three precursor molecules (P1, P2 and P3) (Fig. 1). P1 contains all the viral capsid proteins, VP1–4, and P2 and P3 carry the viral non-structural proteins. Functional viral proteins and precursor proteins are produced upon maturation cleavage of the polyprotein by the virusencoded proteases 2Apro and 3Cpro. Negative RNA intermediates of

FIGURE 1

Graphical overview of picornavirus replication. Virus particle first attaches to host cell surface via a cellular receptor before entering and uncoating to unveil the viral RNA genome. Viral RNA is translated by cellular translational machinery to give a polyprotein that is then cleaved by the virus-encoded proteases 2Apro and 3Cpro to give functional precursor proteins (e.g. 2BC and 3CD) and individual proteins. Within virus-induced membrane vesicles, viral RNA (+) is copied by the viral RNA polymerase, 3Dpol, to give () strand RNA intermediates, which in turn provide the template for the synthesis of (+) strand viral RNA. The (+) strand viral RNAs are used to generate more () strand viral RNAs, translated into viral proteins or packaged into progeny virions. Lysis of host cells will result in the release of progeny virions. Adapted and modified from Figure 2 in Ref. [10] and Figure 4 In Ref. [16]. 1042

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the viral genome are also generated to serve as templates for the replication of positive-sense RNA viral genomes. These events typically take place in virus-induced membrane complexes within host cells. Progeny virions are then self-assembled from the synthesized viral proteins and RNA genomes before their subsequent release from the host cell [16] (Fig. 1).

Inhibitors of virus attachment, entry and uncoating EV71 receptors Receptor binding is an essential event in virus infection. The ability to recognize and bind specific receptors typically determines the host range and tissue tropism of viruses. The recent characterizations of EV71 receptors have opened the door to the development of antiviral strategies targeting EV71 entry into host cells. At least three cellular receptors for EV71 have been reported to date. Human scavenger receptor class B, member 2 (SCARB2) was described as a receptor for EV71 strains from all three genogroups (A, B and C) [17]. SCARB2 is a type III double-transmembrane protein primarily located in endosomes, although surface expression of SCARB2 has also been demonstrated [17]. The second EV71 receptor, human P-selectin glycoprotein ligand-1 (PSGL-1/ CD162), is a sialomucin membrane protein expressed mainly in leukocytes, including dendritic cells and macrophages [18]. PSGL1 was postulated to facilitate the viremic phase of EV71 by enabling replication in circulating leukocytes [19]. Using monoclonal antibodies targeting different parts of PSGL-1, the authors identified the N-terminal region as a crucial interaction site for EV71. The sialic acid residue of PSGL-1 might also be important for EV71 interaction. In the study by Yang et al. [20], sialidase removal of sialic acid residues from plasma membrane proteins was able to protect EV71-susceptible DLD-1 intestinal cells from infection. In another study, EV71 was found to infect immature dendritic cells via DC-SIGN. Infection was reduced by up to 50% when anti-DCSIGN antibody was used to block DC-SIGN [21]. The potential for the development of antiviral strategies targeting EV71 receptor binding was demonstrated in all these studies. Anti-SCARB2 antibodies and soluble SCARB2–Fc conjugates were able to inhibit EV71 infection in a dose-dependent manner [17]. Likewise, soluble PSGL-1, monoclonal antibodies targeting the Nterminal of PSGL-1 [19] and sialylated glycans purified from human milk [20] displayed dose-dependent inhibition of EV71 in co-infection and pretreatment experiments. Unfortunately, these were unable to inhibit EV71 infection completely at the highest concentrations tested, suggesting the involvement of multiple receptors during EV71 infection. This can be addressed in the future by the combined administration of inhibitors targeting different EV71 receptors. The potential discovery of more EV71 receptors in the future (in particular, neural-specific receptors) might eventually lead to the development of effective receptor inhibitors that can prevent EV71-induced neuropathology.

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Plough (e.g. SCH 48973) [24] and R 77975-related compounds from the Janssen Research Foundation (e.g. Pirodavir) [25]. Although antiviral activity was claimed for a broad spectrum of picornaviruses, EV71 was notably absent from the list of picornaviruses tested in these studies. The characteristics and clinical developments of picornavirus capsid-binding molecules are detailed in several comprehensive reviews [9,13,15]. After the 1998 Taiwan outbreak of EV71, Pleconaril was tested against EV71 but failed to prevent virus-induced cytopathic effects in cell cultures [26]. In view of the emergence of EV71 as a clinically important picornavirus, there is a need to re-evaluate more of these compounds against EV71.

Pyridyl imidazolidones Using WIN compounds as templates, a series of imidazolidone derivatives were designed through computer modeling, synthesized and evaluated for their ability to inhibit EV71-induced cytopathic effects in rhabdomyosarcoma (RD) cells. Several lead molecules with strong inhibition against all three genogroups of EV71 (EC50 = 2.13–4.67 mM) and low cytotoxicity (CC50 > 25 mM) were discovered. A series of publications by the same group later documented the various chemical modifications that improved antiviral activity, including substitutions on the phenoxyl group (BPROZ-194, EC50 = 1.55 mM), oxime ether group addition (BPROZ-101, EC50 = 0.0012 mM) and methyl group addition (BPROZ-033, EC50 = 0.009 mM) [27]. These compounds were evaluated for anti-EV71 activity in a murine model with promising results, although a current lack of funding is impeding progress to clinical trials (S.R. Shih, pers. commun.).

Pyridanzinyl oxime ethers Oxime-ether derivatives of Pirodavir displayed improved metabolic stability and antiviral activity against a greater spectrum of picornaviruses relative to Pirodavir [28]. Most notably, one of these compounds, BTA39, inhibited EV71 with an EC50 of 0.001 mM and a reported CC50  4.588 mM [29].

Lactoferrin Bovine lactoferrin and human lactoferrin were found to inhibit EV71 infection in RD cells during early stages of infection at a mean EC50 of 10.5–24.5 mg/ml and 103.3–185.0 mg/ml, respectively [30]. Bovine lactoferrin delayed EV71-induced paralysis and death for up to two weeks post-infection in 17-day-old mice when co-injected with EV71, and interaction assays using conjugated antibodies demonstrated direct binding of lactoferrin with VP1 and cell surfaces [31]. These suggested lactoferrin’s role in preventing virus attachment by blocking cellular receptors and/or receptor-interaction sites on EV71. The exact antiviral mechanism of lactoferrin remains to be determined; however, its potency as an early inhibitor of EV71 has led to the authors of these studies to propose investigating the ingestion of milk as a prophylaxis.

Viral capsid-binding molecules The hydrophobic pocket within the viral protein, VP1, is a wellknown target for antiviral design because its occupancy by suitable compounds will stabilize the virus capsid and prevent uncoating of virus for RNA release [22]. Some of the more prominent series of capsid-binding compounds include the WIN series from SterlingWinthrop (e.g. Pleconaril) [23], the SCH series from Schering

NF449 Suramin, a polysulfonate, was shown to be an effective inhibitor of HIV replication in infected patients. Polysulfonates are known to be multi-targeting inhibitors of HIV, with viral targets including reverse transcriptase and the viral envelope gp120 glycoprotein [32]. A suramin analog, NF449, 4,40 ,400 ,4000 -(carbonylbis(iminowww.drugdiscoverytoday.com

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5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid, was identified as an early inhibitor of EV71 infection (virus uncoating or host receptor binding) with an EC50 of 6.7 mM and CC50 of >1000 mM in a screen for anti-EV71 compounds in the LOPAC1280 drug library (Sigma–Aldrich) [33]. NF449-resistant EV71 strains isolated in the same study displayed mutations in the viral capsid protein VP1, suggesting VP1 as a putative target of NF449. Reviews
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Inhibitors of protein synthesis Translation of the viral RNA is the next key step of virus replication. Because the virus essentially uses cellular machinery for protein translation, it is important to develop antiviral strategies that inhibit viral protein synthesis without affecting host cell translation events. Currently, there are no reports of viral-specific small-molecule inhibitors of protein synthesis with activity against EV71. Amantadine was found to inhibit cap-independent translation initiated by the EV71 internal ribosome entry site in a bicistronic reporter system, although direct antiviral activity was not verified [34]. A more promising but non-conventional antiviral approach targeting viral RNA translation might be RNA interference (RNAi).

RNA interference The potential of nucleic-acid-based therapy can be seen from the FDA approval of fomivirsen (Vitravene) for use against cytomegalovirus retinitis [35]. Many RNAi-based antiviral therapeutics are currently undergoing various phases of clinical trials against viruses such as HIV-1, respiratory syncytial virus and hepatitis C virus [36]. Several highly conserved sequences have been identified as targets for RNAi in the EV71 genome. Small interfering RNA (siRNA) and plasmid-encoded short hairpin RNA (shRNA) were found to effectively block replication of EV71 when targeted against 30 UTR [37] or regions encoding the structural proteins (e.g. VP1 and VP2) [38] and non-structural proteins (e.g. 2C, 3C and 3D) [37,39]. Two of these studies reported the greatest inhibitory effects in targeting the viral RNA polymerase 3D [39,40]. siRNA and plasmid-encoded shRNA targeting 3D were able to prevent EV71-induced paralysis, weight loss and death in suckling mice when delivered via the oral or intraperitoneal route [40]. The authors also observed similar antiviral effects for nucleotides delivered with or without a lipid carrier. These promising preclinical results should be followed up with clinical trials for RNAbased anti-EV71 therapeutics.

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(reviewed in Ref. [9]). One of these compounds, rupintrivir (AG7088, Pfizer) reached phase II clinical trials but further studies were ceased after it displayed poor efficacy in natural infection cases of HRV [44]. Kuo et al. [45] developed a series of compounds based on rupintrivir and tested against EV71 3Cpro in vitro. A compound, 10b, was identified as a potent inhibitor with EC50 of 0.018 mM while showing no toxicity (CC50 > 25 mM). Further studies are needed to evaluate the compound’s efficacy in vivo. More recently, rupintrivir was shown to inhibit EV71 with an EC50 of 0.8 mM using a real-time, cell-based fluorescence resonance energy transfer assay and plaque reduction assay [46]. Compound 1, an orally bioavailable 3Cpro inhibitor developed in parallel with rupintrivir, also showed in vitro antiviral properties against several other human enteroviruses and should also be evaluated for anti-EV71 properties [47].

Inhibitors of protein 2C The highly conserved picornavirus protein 2C is a multifunctional protein that possesses nucleoside triphosphatase activity [48] and was shown to be involved in the synthesis of the viral negativestrand RNA [49] and encapsidation of progeny virions in poliovirus [50]. EV71 2C protein was reported to recruit host-encoded reticulon 3 in forming replication complexes [51].

Metrifudil and N6-benzyladenosine Metrifudil, N-(2-methylphenyl)methyl-adenosine and N6-benzyladenosine were identified as inhibitors of EV71 in the same screen as NF449 (inhibitors of virus attachment, entry and uncoating). Reported EC50 of 1.3 mM (metrifudil) and 0.10 mM (N6-benzyladenosine) were derived against EV71 pseudovirus (structural genes replaced by firefly luciferase gene). Metrifudil and N6-benzyladenosine, both adenosine receptor agonists, also showed low cytotoxicity with CC50 of >50 mM and 3300 mM, respectively [33]. The authors managed to isolate drug-resistant EV71 with mutations in 2C proteins after three passages, thus suggesting non-conserved regions in 2C as probable targets of the compounds.

Inhibitors of protein 3A Picornavirus 3A protein is an essential, multifunctional protein that has been shown to modulate the host cell’s intracellular membrane transport [52]. Protein 3A functions primarily in its precursor form, 3AB, which has RNA-binding properties and is known to stimulate the cleavage of 3CDpro and the activity of 3D RNA polymerase 3Dpol [53,54].

Inhibitor of 3C protease

Enviroxime and its structural analogs

Aside from their roles in the maturation cleavages of the viral polyprotein, the EV71-encoded proteases 2A and 3C also target several host proteins, such as eukaryotic translation initiation factor 4G [41] and cleavage stimulation factor 64 [42], to halt host protein synthesis and induce apoptosis [43]. The essential roles of these proteases in virus replication make them attractive targets for antiviral therapeutics.

Enviroxime is a benzimidazole derivative that inhibits rhinoviruses and poliovirus in vitro by targeting protein 3A [55,56]. Recently, enviroxime was reported to have strong antiviral effects against EV71 with an EC50 of 0.15 mM [57]. Enviroxime, however, was unable to show significant clinical effect (p > 0.05) against HRV9, with poor bioavailability and gastrointestinal side-effects observed in trial subjects [58]. Vinlyacetylene analogs of enviroxime were reported to display better oral bioavailability while retaining protein 3A targeting antiviral activity against poliovirus [59] and, therefore, should be evaluated for anti-EV71 activity (Table 1).

Rupintrivir and its structural and functional analogs Several peptide aldehydes were designed to irreversibly inhibit human rhinovirus (HRV) 3Cpro by forming covalent adducts 1044

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Anti-EV71 activity of select compounds Inhibitor

Structure

Capsid-binding BPROZ-194

Br

O

N

EC50 (mM)

CC50 (mM)

Current status (reference)

1.552

>50

In vitro [27]

0.0012

>50

In vitro [27]

0.0088

>50

In vitro [27]

0.001

4.588

In vitro [29]

10.5* 6.7

N/R >1000

In vivo; mice [31] In vitro [33]

< 5nmola <25 mga

N/R N/R

In vivo; mice [40] In vivo; mice [40]

0.018

>25

In vitro [45]

[TD$INLE]

BPROZ-101

N

O

O

N [TD$INLE]

N

N

N

O

O

BPROZ-033

Cl [TD$INLE]N

O N

N

O

BTA39

N

[TD$INLE]Cl

N

O CH CH 2 3

O

N N

Bovine lactoferrin NF449

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N

H

N/A + O- Na O S O

Na+ -O O S O O O

O S O HN Na+ O-

[TD$INLE] Na+

NH O S O -O Na+

NH H N

OO S O HN

-O

O

NH O S O

O

Na+

O O S O O- Na+ Na+

Translation-inhibition siRNA (3D) shRNA (psi-3D) 3Cpro inhibitor Compound 10b

N/A N/A

O

O S O -O

[TD$INLE]

NH H N

Ar O

O N H

H O

Ar= 4-Me2NC6H4

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TABLE 1 (Continued ) Inhibitor

Structure

Rupintrivir

H

EC50 (mM)

CC50 (mM)

Current status (reference)

0.8

N/R

In vitro [46]

1.3

>50

In vitro [33]

0.10

3300

In vitro [33]

0.15

N/R

In vitro [57]

2.0

170

In vitro [33]

0.15

>100

In vitro [64]

N O

H

O O

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H

[TD$INLE]

N

H

H

O

O

N

N O

F

2C inhibitors Metrifudil

HO N [TD$INLE]

H N

N

HO

CH3

O N

HO N6-benzyladenosine

N

HO N

H N

N

H [TD$INLE] O

O N

HO

N

3A inhibitors Enviroxime

N NH2 N [TD$INLE]

HO

O S O

N

GW5074

Br OH [TD$INLE]

Br O N H

3Dpol inhibitor DTriP22

N

Br [TD$INLE]

N S

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N

N N

CH3

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TABLE 1 (Continued ) Inhibitor

Structure

Aurintricarboxylic acid

O

OH

O

EC50 (mM)

CC50 (mM)

Current status (reference)

2.9

211

In vitro [65]

65 mg/ml

>200 mg/ml

In vivo; mice [69]

<10

N/R

In vitro [70]

0.14 mg/ml

2632 mg/ml

In vitro [73]

<1.2 mMb

N/R

In vitro [74]

0.101 0.1 mg/ml

1.521 65.86 mg/ml

In vitro [77] In vitro [78]

OH OH O

HO O

OH

Nucleoside analog Ribavirin

O

N HO [TD$INLE]

HO

NH2

N N

O

OH

Antioxidant Epigallocatechin gallate

OH OH HO

O

OH

[TD$INLE]

O OH

OH

O

OH OH Interferon inducer Aloe-emodin

OH

O

OH

[TD$INLE]

OH O

Unknown mechanism Chloroquine

Cl Allophycocyanin Raoulic acid

N

HN [TD$INLE]

N

N/A

O OH [TD$INLE]

H

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TABLE 1 (Continued ) Inhibitor

Structure

Ursolic acid

[TD$INLE] Reviews
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HO

CC50 (mM)

Current status (reference)

0.5 mg/ml

100.5 mg/ml

In vitro [79]

OH

H H

EC50 (mM)

O

H

EC50, 50% effective concentration; CC50, 50% cytotoxic concentration; N/A, not applicable; N/R, not reported. a 50% reduction of EV71 RNA from extracted intestinal cells (mice). b 104-fold inhibition of EV71 RNA synthesis.

GW5074 Other scholars have reported several functional analogs of enviroxime that also target protein 3A. GW5074, 3-(3,5-dibromo-4hydroxybenzylidine-5-iodo-1,3-dihydro-indol-2-one), is a Raf-1 inhibitor identified as an inhibitor of EV71 in the same drug library screen described previously for metrifudil (inhibitors of 2C protein). GW5074 was found to have a CC50 of 170 mM and inhibited EV71 pseudovirus at an EC50 of 2.0 mM. Post-infection addition of 50 mM of GW5074 also showed 102 to 103-fold of inhibition [33]. The target of GW5074, identified in a later study, was found to be the same region targeted by enviroxime in the protein 3A [60]. GW5074 is currently being evaluated for in vivo anti-EV71 activity in a murine model (M. Arita, pers. commun.).

Inhibitor of 3D RNA polymerase The picornavirus 3D RNA polymerase is involved in several crucial replication events besides the incorporation of nucleotides during the synthesis of negative and positive viral RNA strands. Studies have shown that 3Dpol is responsible for the uridylation of VPg, which confers the priming function of VPg during RNA replication [61]. Viral protein 3CD, the precursor form of 3Dpol, is also involved in polyprotein processing [62] and promotes RNA synthesis by binding to the 50 cloverleaf structure on the viral RNA [63].

DTriP-22 DTriP-22 is a synthetic compound from a series of piperazinecontaining, pyrazolo[3,4-d]pyrimidine derivatives that was identified as a novel class of compounds with anti-EV71 activities. This compound was evaluated to have an EC50 of 0.15–0.98 mM against strains from all genotypes of EV71 and a CC50 greater than 100 mM. It was found to suppress the synthesis of both positive and negative strands of viral RNA during EV71 infection, and subsequent analyses of resistant viruses identified DTriP-22’s target as the RNA polymerase of EV71, 3Dpol [64].

Aurintricarboxylic acid Aurintricarboxylic acid (ATA) is a polyanionic compound with broad-spectrum antiviral activity reported against a variety of viruses (e.g. HIV and severe acute respiratory syndrome coronavirus). It was recently reported to be a potent EV71 inhibitor with a plaque reduction assay derived EC50 of 2.9 mM, while displaying low cytotoxicity in African green monkey kidney (Vero) cells with 1048

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a CC50 of 211 mM [65]. The authors managed to rule out ATA targeting of viral adsorption, viral RNA translation and the viral proteases (2Apro and 3Cpro) while observing ATA inhibition of RNA elongation by EV71 3Dpol.

Nucleoside analog Ribavirin Ribavirin is a broad-spectrum antiviral that has been used in treating hepatitis C virus infections (in combination with interferon-a) [66] and severe respiratory syncytial virus infections [67]. Studies have shown that the main antiviral mechanism of ribavirin is through lethal mutagenesis of viruses during RNA replication events [68]. Ribavirin was found to inhibit EV71 in RD cells with EC50 of 65 mg/ml (266 mM) while preventing EV71-induced paralysis and death in mice [69]. With continued progress in antiviral development for EV71, ribavirin might prove to be clinically useful for EV71 infections in the future, when used at lower concentrations in combination with other antivirals.

Modulators of host cell environment Antioxidant Epigallocatechin gallate. Polyphenolic compounds isolated from green tea leaves (Camellia sinensis) were tested for their antiviral properties against EV71. Epigallocatechin gallate (EGCG) was found to be the most potent of these compounds with postinfection addition of 10 mM of EGCG resulting in 54% reduction in plaque formation, while EV71-induced cytopathic effects were reduced by two-fold and viral RNA levels were significantly decreased (p < 0.05, treated vs control). EGCG’s potency as an anti-EV71 agent among the polyphenols tested correlated with its high antioxidative capacity [70]. EV71 infection was observed to result in increased oxidative stress, while cells deficient in glucose6-phosphate dehydrogenase supported more efficient EV71 replication [71]. The association between cellular redox status and EV71 replication led the authors to suggest EGCG inhibits EV71 replication by modulating oxidative stress. However, it remains unclear whether this association represents a direct causal relationship between EGCG and EV71 inhibition.

Type I interferons The induction of Type I interferons (IFNs; e.g. interferon-a/b) is an early, non-specific host immune response to viral infections that

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Compounds with unverified targets and modes of action Synthetic compound Chloroquine Chloroquine was used as an inhibitor of virus uncoating in a study of EV71-induced apoptosis whereby the experiments were not designed to investigate chloroquine’s anti-EV71 characteristics. Nonetheless, treatment with 1.2 mM of chloroquine resulted in a 104-fold reduction of EV71 RNA synthesis [74]. The antimalarial drug chloroquine has gained interest as a potent antiviral drug, with studies showing its antiviral activities against diverse viruses such as severe acute respiratory syndrome coronavirus [75] and HIV [76]. Investigations into the inhibitory mechanisms of chloroquine on these viruses showed a variety of targets and processes involved. Given the varied antiviral pathways of chloroquine, the authors’ claim of uncoating inhibition has to be verified with further experiments. However, the experience from the use of chloroquine in malaria treatment and its wide availability add to the appeal of investigating its anti-EV71 potential.

Natural compounds Allophycocyanin. Allophycocyanin is a red fluorescent protein purified from the marine algae Spirulina platensis that has been found to prevent EV71-induced apoptosis, delay viral RNA synthesis and reduce plaque formation at an EC50 of 0.1 mM and CC50 of 1.52 mM in post-infection experiments on Vero cells [77]. The specific targets for inhibition by allophycocyanin are currently unknown. Raoulic acid. Raoulic acid was purified from whole-plant extract of a New Zealand plant, Raoulia australis, and tested for antiviral activity against a wide range of viruses. It has a CC50 of 65.86 mg/ ml in Vero cells and was able to inhibit EV71 with an EC50 of less than 0.1 mg/ml, giving it a therapeutic index of greater than 656.8 [78]. Drug treatment was performed post-infection, and the targets for inhibition were not investigated in the study. Ursolic acid. Ursolic acid is a triterpenoid purified from the water extract of Ocimum basilicum, a herb commonly used in traditional Chinese medicine. Ursolic acid showed low cytotoxicity against Hep G2 (hepatoblastoma-derived) cells (CC50 = 100.5 mg/ml) and inhibited EV71-induced cytopathic effects (EC50 = 0.5 mg/ml) [79]. Time-

of-addition studies revealed only post-infection inhibition of EV71 for the lower doses of ursolic acid tested (0.125 and 0.25 mg/ml), although the exact mechanisms of inhibition remain unclear.

Concluding remarks and future prospects Because most of the antiviral agents of EV71 in the literature are only in the preliminary stages of development (i.e. in vitro studies), promising candidates were selected based on their antiviral potencies and cytotoxicity profiles. Among the compounds mentioned in this review, those that are approved for clinical use in other diseases (e.g. chloroquine) or are generally non-toxic (e.g. lactoferrin) are attractive candidates for evaluation in vivo and in clinical trials. Earlier efforts in the development of antiviral agents against HRV and poliovirus have yielded compound series with antipicornaviral activities. Research into these series, however, seems to have ceased with the highly successful implementation of oral polio vaccine. These compounds might still prove useful as antiEV71 agents or lead molecules for designing effective anti-EV71 agents, as exemplified by the development of anti-EV71 capsid binders, and should be evaluated against current EV71 strains. In fact, the extensive pharmacokinetic characterizations in various stages of clinical trials for compounds such as Pleconaril (capsid binder), rupintrivir (3Cpro inhibitor) and enviroxime (3A inhibitor) can serve to inform future developments of anti-EV71 agents. The mechanisms behind the neuropathogenesis of EV71 infection are currently unknown. Several theories have been proposed, including host immune-mediated inflammation of the central nervous system (CNS) and neural apoptotic cell death as EV71 infection spreads to the CNS [80]. A murine study reported retrograde axonal transport to be the major transmission route for EV71 neuroinvasion, in contrast to a hematogenous transmission with virus crossing the blood–brain barrier. Skeletal muscle was found to serve as an important site for viral replication and entry into the CNS via peripheral nerves innervating the infected site [81]. In fatal EV71 infection cases, patients usually present with three to five days of fever, headache, oral ulcers and vesicular rashes on hand, foot or buttocks that test positive for EV71 before rapid deterioration to severe disease [82]. The lag time between primary infection to severe neural disease might prove to be an important window of opportunity for antiviral intervention. Although the neuropathogenesis of EV71 would argue for a systemic antiviral that can cross the blood–brain barrier, other routes of administration might also prove to be useful in preventing EV71 neuroinvasion and transmission by inhibiting EV71 replication at other sites of infection (e.g. skeletal muscle, gastrointestinal lining and vesicular rashes on the skin). With no antiviral or prophylactic available for severe EV71 infections, all available antiviral options should be considered pending future progress in our understanding of disease progression in EV71 infection and future reports on the in vivo efficacies and pharmacokinetics of antiviral candidates. An effective antiviral is an essential tool in nullifying the growing threat of EV71 as a neurotopic virus. Even as the search for an EV71 vaccine continues, development of antiviral agents remain pertinent, as seen in the case of poliovirus, in which the lack of antiviral options is preventing a complete eradication of polio [56]. www.drugdiscoverytoday.com

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can lead to the induction of antiviral mechanisms. Injection of 10 mg of polyriboinosinic:polyribocytidylic acid [poly(I:C)], a potent IFN inducer, into mice 12 hours before EV71 inoculation resulted in increased serum levels of IFN-a, improved survival rate and significantly decreased tissue viral load (p < 0.05, treated vs control) and mortality [72]. Aloe-emodin. Aloe-emodin, a plant-derived anthraquinone derivative, was able to induce a 2.5-fold increase in IFN-a expression in human medullablastoma (TE-671) cells while showing low toxicity to both human promonocyte (HL-CZ) and TE-671 cell lines (CC50 of 2632 mg/ml and 2881 mg/ml, respectively). Pretreatment of cells from both cell lines with aloe-emodin resulted in lowered plaque formation when infected with EV71 (EC50 of 0.14 mg/ml and 0.52 mg/ml, respectively) [73]. The resulting high therapeutic index (CC50/EC50) values for aloe-emodin suggest its therapeutic potential as an anti-EV71 compound.

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References

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33 Arita, M. et al. (2008) Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity. J. Gen. Virol. 89, 2518–2530 34 Chen, Y.J. et al. (2008) Amantadine as a regulator of internal ribosome entry site. Acta Pharmacol. Sin. 29, 1327–1333 35 Roehr, B. (1998) Fomivirsen approved for CMV retinitis. J. Int. Assoc. Phys. AIDS Care 4, 14–16 36 Haasnoot, J. et al. (2007) RNA interference against viruses: strike and counterstrike. Nat. Biotechnol. 25, 1435–1443 37 Sim, A.C. et al. (2005) RNA interference against enterovirus 71 infection. Virology 341, 72–79 38 Wu, Z. et al. (2009) Identification of small interfering RNAs which inhibit the replication of several enterovirus 71 strains in China. J. Virol. Methods 159, 233–238 39 Lu, W.W. et al. (2004) Selective inhibition of enterovirus 71 replication by short hairpin RNAs. Biochem. Biophys. Res. Commun. 325, 494–499 40 Tan, E.L. et al. (2007) Inhibition of enterovirus 71 in virus-infected mice by RNA interference. Mol. Ther. 15, 1931–1938 41 Kuo, R.L. et al. (2002) Infection with enterovirus 71 or expression of its 2A protease induces apoptotic cell death. J. Gen. Virol. 83, 1367–1376 42 Weng, K.F. et al. (2009) Enterovirus 71 3C protease cleaves a novel target CstF-64 and inhibits cellular polyadenylation. PLoS Pathog. 5, e1000593 43 Li, M.L. et al. (2002) The 3C protease activity of enterovirus 71 induces human neural cell apoptosis. Virology 293, 386–395 44 Patick, A.K. (2006) Rhinovirus chemotherapy. Antiviral Res. 71, 391–396 45 Kuo, C.J. et al. (2008) Design, synthesis, and evaluation of 3C protease inhibitors as anti-enterovirus 71 agents. Bioorg. Med. Chem. 16, 7388–7398 46 Tsai, M.T. et al. (2009) Real-time monitoring of human enterovirus (HEV)-infected cells and anti-HEV 3C protease potency by fluorescence resonance energy transfer. Antimicrob. Agents Chemother. 53, 748–755 47 Patick, A.K. et al. (2005) In vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3C protease. Antimicrob. Agents Chemother. 49, 2267–2275 48 Rodriguez, P.L. and Carrasco, L. (1993) Poliovirus protein 2C has ATPase and GTPase activities. J. Biol. Chem. 268, 8105–8110 49 Banerjee, R. et al. (1997) Poliovirus-encoded 2C polypeptide specifically binds to the 30 -terminal sequences of viral negative-strand RNA. J. Virol. 71, 9570–9578 50 Vance, L.M. et al. (1997) Poliovirus 2C region functions during encapsidation of viral RNA. J. Virol. 71, 8759–8765 51 Tang, W.F. et al. (2007) Reticulon 3 binds the 2C protein of enterovirus 71 and is required for viral replication. J. Biol. Chem. 282, 5888–5898 52 Belov, G.A. and Ehrenfeld, E. (2007) Involvement of cellular membrane traffic proteins in poliovirus replication. Cell Cycle 6, 36–38 53 Paul, A.V. et al. (1994) Studies with poliovirus polymerase 3Dpol. Stimulation of poly(U) synthesis in vitro by purified poliovirus protein 3AB. J. Biol. Chem. 269, 29173–29181 54 Xiang, W. et al. (1995) Molecular dissection of the multifunctional poliovirus RNAbinding protein 3AB. RNA 1, 892–904 55 Heinz, B.A. and Vance, L.M. (1996) Sequence determinants of 3A-mediated resistance to enviroxime in rhinoviruses and enteroviruses. J. Virol. 70, 4854–4857 56 De Palma, A.M. et al. (2008) Potential use of antiviral agents in polio eradication. Emerg. Infect. Dis. 14, 545–551 57 De Palma, A.M. et al. (2009) Mutations in the nonstructural protein 3A confer resistance to the novel enterovirus replication inhibitor TTP-8307. Antimicrob. Agents Chemother. 53, 1850–1857 58 Phillpotts, R.J. et al. (1983) Therapeutic activity of enviroxime against rhinovirus infection in volunteers. Antimicrob. Agents Chemother. 23, 671–675 59 Victor, F. et al. (1997) Synthesis, antiviral activity, and biological properties of vinylacetylene analogs of enviroxime. J. Med. Chem. 40, 1511–1518 60 Arita, M. et al. (2009) Cellular kinase inhibitors that suppress enterovirus replication have a conserved target in viral protein 3A similar to that of enviroxime. J. Gen. Virol. 90, 1869–1879 61 Paul, A.V. et al. (1998) Protein-primed RNA synthesis by purified poliovirus RNA polymerase. Nature 393, 280–284 62 Ypma-Wong, M.F. et al. (1988) Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor. Virology 166, 265–270 63 Xiang, W. et al. (1995) Interaction between the 50 -terminal cloverleaf and 3AB/ 3CDpro of poliovirus is essential for RNA replication. J. Virol. 69, 3658–3667 64 Chen, T.C. et al. (2009) Novel antiviral agent DTriP-22 targets RNA-dependent RNA polymerase of enterovirus 71. Antimicrob. Agents Chemother. 53, 2740–2747 65 Hung, H.C. et al. (2010) Inhibition of enterovirus 71 replication and the viral 3D polymerase by aurintricarboxylic acid. J. Antimicrob. Chemother. 65, 676–683

66 de Ledinghen, V. et al. (2002) Daily or three times a week interferon alfa-2b in combination with ribavirin or interferon alone for the treatment of patients with chronic hepatitis C. J. Hepatol. 36, 672–680 67 Wyde, P.R. (1998) Respiratory syncytial virus (RSV) disease and prospects for its control. Antiviral Res. 39, 63–79 68 Crotty, S. and Andino, R. (2002) Implications of high RNA virus mutation rates: lethal mutagenesis and the antiviral drug ribavirin. Microbes Infect. 4, 1301–1307 69 Li, Z.H. et al. (2008) Ribavirin reduces mortality in enterovirus 71-infected mice by decreasing viral replication. J. Infect. Dis. 197, 854–857 70 Ho, H.Y. et al. (2009) Antiviral effect of epigallocatechin gallate on enterovirus 71. J. Agric. Food Chem. 57, 6140–6147 71 Ho, H.Y. et al. (2008) Glucose-6-phosphate dehydrogenase deficiency enhances enterovirus 71 infection. J. Gen. Virol. 89, 2080–2089 72 Liu, M.L. et al. (2005) Type I interferons protect mice against enterovirus 71 infection. J. Gen. Virol. 86, 3263–3269 73 Lin, C.W. et al. (2008) Aloe-emodin is an interferon-inducing agent with antiviral activity against Japanese encephalitis virus and enterovirus 71. Int. J. Antimicrob. Agents 32, 355–359 74 Shih, S.R. et al. (2008) Viral protein synthesis is required for enterovirus 71 to induce apoptosis in human glioblastoma cells. J. Neurovirol. 14, 53–61

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75 Vincent, M.J. et al. (2005) Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol. J. 2, 69 76 Savarino, A. et al. (2004) Anti-HIV effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors. J. Acquir. Immune Defic. Syndr. 35, 223–232 77 Shih, S.R. et al. (2003) Inhibition of enterovirus 71-induced apoptosis by allophycocyanin isolated from a blue-green alga Spirulina platensis. J. Med. Virol. 70, 119–125 78 Choi, H.J. et al. (2009) Antiviral activity of raoulic acid from Raoulia australis against picornaviruses. Phytomedicine 16, 35–39 79 Chiang, L.C. et al. (2005) Antiviral activities of extracts and selected pure constituents of Ocimum basilicum. Clin. Exp. Pharmacol. Physiol. 32, 811–816 80 Weng, K.F. et al. (2010) Neural pathogenesis of enterovirus 71 infection. Microbes Infect. 12, 505–510 81 Chen, C.S. et al. (2007) Retrograde axonal transport: a major transmission route of enterovirus 71 in mice. J. Virol. 81, 8996–9003 82 Chan, K.P. et al. (2003) Epidemic hand, foot and mouth disease caused by human enterovirus 71, Singapore. Emerg. Infect. Dis. 9, 78–85

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PubChem as a public resource for drug discovery Qingliang Li, Tiejun Cheng, Yanli Wang and Stephen H. Bryant National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA

PubChem is a public repository of small molecules and their biological properties. Currently, it contains more than 25 million unique chemical structures and 90 million bioactivity outcomes associated with several thousand macromolecular targets. To address the potential utility of this public resource for drug discovery, we systematically summarized the protein targets in PubChem by function, 3D structure and biological pathway. Moreover, we analyzed the potency, selectivity and promiscuity of the bioactive compounds identified for these biological targets, including the chemical probes generated by the NIH Molecular Libraries Program. As a public resource, PubChem lowers the barrier for researchers to advance the development of chemical tools for modulating biological processes and drug candidates for disease treatments.

PubChem [1,2] (http://pubchem.ncbi.nlm.nih.gov) is a public repository for chemical structures and their biological properties. The bioactivity results in PubChem are contributed by more than a hundred organizations, with the majority of data coming from the screening center network under the NIH Molecular Libraries Program (MLP) [3]. This program aims to expand the use of small molecules as chemical probes, which offer dynamic, reversible and tunable perturbations for biological systems [4], to study the functions of genes and proteins in physiology and pathology. Unlike the pharmaceutical industry and biotechnology companies, which primarily focus on the ‘druggable genome’ [5,6] to screen the ‘drug-like’ small molecules against limited types of targets (such as kinases, G-proteincoupled receptors, enzymes, ion channels and nuclear hormone receptors), an extensive collection of biological targets and chemical compounds are being investigated by the MLP to address a wide scope of biological issues, from identifying inhibitors of a specific enzyme to looking for small molecules that affect protein–protein interactions or modulate splicing events [3]. With the rapid growth in data capacity, PubChem is becoming a valuable resource for drug development and has Corresponding authors:. Wang, Y. ([email protected]), Bryant, S.H. ([email protected])

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attracted considerable interest from researchers in both academia and industry. PubChem consists of three interconnected databases: Substance, BioAssay and Compound. The Substance database contains the descriptions of molecules (primarily small molecules) provided by depositors; the BioAssay database contains the screening results of substances by assay providers; and the Compound database contains unique chemical structures derived by structural standardization of the records in the Substance database. Currently, more than 25 million unique chemical structures, which were derived from a collection of 70 million substances, are in the Compound database. As of April 2010, the BioAssay database comprised more than 2700 bioassays associated with more than one million compounds tested against several thousand molecular targets. In addition, several bioassays from RNAi screening experiments have been deposited in the BioAssay database. A review of this public resource will allow the community to better understand the information content and utilize the data in PubChem, which might ultimately help to advance the development of new chemical tools and drug candidates by enabling researchers to study structure–activity relationships, investigate the interaction mechanisms between small molecules and their targets [7], and gain insights into the chemical and biological space in their research area. Here, we provide a comprehensive summary

1359-6446/06/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.drudis.2010.10.003

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of the protein targets in PubChem with respect to their functional classification, availability of 3D structure and biological pathway. The potency, selectivity and promiscuity of the bioactive compounds (including the chemical probes developed by the MLP), which are associated with those protein targets, are also investigated.

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cular interest to researchers in drug discovery and the majority of bioassays in PubChem focus on enzymes or other proteins, we focus on the analysis of protein targets in this study. A collection of 2206 protein targets was compiled from PubChem at the time of this work.

Target identification is one of the key steps of drug development [8,9]. Tremendous efforts have been made in recent decades by pharmaceutical industries and biotechnology companies that focus on the druggable genome [5,6] to identify novel drug targets for drug discovery; however, only a few drug targets are successfully used in current therapies [10]. The human genome project has identified approximately 20,000–25,000 genes and an even larger number of transcripts and proteins, which provide great opportunities for drug target investigation [11]. Currently, PubChem records two major types of molecular targets for research (i.e. protein targets for small molecules and gene targets from RNAi reagents), which represent a great diversity of types of assays, including, for example, enzyme inhibitor identification, protein–protein interactions, tumor cell growth inhibition and even organismal phenotypes. Because the protein targets are of parti-

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To look into the potential functions of these bioassay targets, we performed sequence similarity search against the annotated functional domains in the NCBI Conserved Domain Database (CDD; http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [12] by using the reverse-position-specific BLAST tool [13]. We found that the 2206 protein targets fell into 671 unique protein superfamilies (Fig. 1a). Approximately 15% of them belonged to the protein kinase superfamily. Other superfamilies such as nuclear receptor, trypsin-like serine protease, src homology protein and zinc-dependent metalloprotease comprised approximately 2–3% of the bioassay targets. The rest of the superfamilies (67%, 450 out of 671) contained only one or two bioassay targets for each member. In particular, the high-throughput screening assays under the MLP contributed 450 protein targets, scattering into 312 protein superfamilies (Fig. 1c). Although the protein kinase superfamily still dominated this subset, it accounted for 5% of the MLP target set.

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Linking protein superfamilies and 3D structures to PubChem bioassay targets. (a) and (c) represent superfamily annotations; (b) and (d) indicate the availability of related 3D structures derived from homologous analysis at each sequence similarity level. (a) and (b) denote the entire set of protein targets in PubChem; (c) and (d) represent the protein targets that are involved in high-throughput screening assays from the MLP. www.drugdiscoverytoday.com

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Functional families Bioassay targets in PubChem

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The other superfamilies, such as seven-transmembrane G-proteincoupled receptors and DNA-binding domain of nuclear receptors accounted for 2–3% on average. These results suggest that the bioassay targets in PubChem represent a broader functional diversity than the known druggable targets; thus, PubChem enables researchers to study the mechanisms of protein–ligand interactions on a wider scope and to identify novel molecular targets for potential treatments.

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likely to have similar roles in regulating a specific biological process. Thus, selectively inhibiting or activating a target in the same pathway might effectively modulate a specific biological process or restore the function from a disease state back to a normal one. Thus, the wealth of bioactivity data in PubChem might facilitate research into chemical biology and drug development at the system level.

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Bioactive compounds in PubChem Three-dimensional structures The 3D structures of macromolecular targets are important to the study of the mechanisms of protein–protein and protein–ligand interactions. To link the protein targets to relevant 3D structures, we used the BLAST tool [14,15] to search against the protein sequences derived from the Protein Data Bank (PDB; http:// www.pdb.org) [16]. We found that 78% of these targets have corresponding 3D structures with 100% sequence identity in the PDB database (Fig. 1b). When looking into the possibility of inferring related structures from the similarity search, another 8% of these targets found related structures in the PDB database with sequence identity over 90%. Given the fact that protein structures tend to be highly conserved at this level of sequence identity, this analysis suggests that more than 86% of the molecular targets in PubChem have related structural information in PDB. Conversely, less than 2% of these targets could not be linked to any relevant 3D structures or were only able to be linked to the related protein structures with sequence identity below 30%. As for the 450 protein targets from the MLP, more than 60% either have corresponding 3D structures with sequence identity of 100% or can be linked to related structures with sequence identity of 90% or above (Fig. 1d).

Related pathways Most diseases occur because of the misregulation of multiple genes that are involved in mutual interactions – including genes, transcripts and proteins – in a dynamic network. During the past decade, high-throughput technologies have been widely used in biological research and generated a tremendous amount of experimental data, which make it possible to study the functions of genes or proteins at a biological system level. Drug development is inherently a complicated process because drugs and their targets are engaged in a complex system, which is far from being thoroughly understood. Moreover, approximately 35% of known drugs or drug candidates are active against more than one target [17], which makes the interactions more sophisticated. Therefore, it is essential to investigate the connections of the drug, drug target and disease in the context of a biological system. In this study, we mapped 507 (23%) of the 2206 protein targets from PubChem to 287 pathways in the KEGG database (http:// www.genome.jp/kegg/) [17–20]. We observed that some pathways, such as the mitogen-activated protein kinase signaling pathway, were related to multiple protein targets in PubChem. In addition, some bioassay targets were involved in multiple KEGG pathways. A list of top 20 pathways that contain multiple bioassay targets and top 20 targets that are involved in multiple pathways are provided in Tables S1 and S2, respectively, in the supplementary material online. Targets involved in the same pathway are 1054

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The characteristics of small molecules make them useful, not only as drugs that modulate physiological functions but also as chemical tools that interrogate the functions of novel genes, pathways and cells [3]. The purpose of the MLP is to develop chemical probes for modulating biological processes and facilitate the development of new drugs by offering the capacity of highthroughput screening to the public sector [3]. Currently, more than one million compounds have been tested against several thousand targets and deposited in PubChem. Approximately two hundred thousand of them were reported active, among which there were 116 chemical probes generated by the MLP projects at the time of this article.

Potency A large fraction of the bioactive compounds (91,022) in PubChem were assayed with a confirmed potency measurement, which were associated with 1771 out of the 2206 protein targets in total. The distribution of bioactivity potency was analyzed, with the results showing that nearly 10% of the compounds have a potency of 1 mM (Fig. 2a). These compounds were associated with more than 60% of the 1771 targets (i.e. each of these targets had at least one bioactive compound with a potency of 1 mM). We found, however, that approximately 40% of the targets had no active compound with a potency better than 10 mM (Fig. 2a), which indicates that there are great chances to develop highly potent compounds for these targets through further study by medicinal chemistry approaches. When focusing on the 116 MPL chemical probes, we found that most of them demonstrated much higher potency in the range of 0.001–1 mM (Fig. 2b). The MLP probes are discussed in detail in the section ‘Chemical probes’.

Selectivity and promiscuity It is essential to understand the selectivity and promiscuity of small molecules when fully exploiting the therapeutic potential and minimizing the toxic effects of drugs or drug candidates [17,21,22]. To evaluate these properties of a compound, a straightforward approach is to investigate the bioactivity profile by screening this compound across a broad panel of targets; however, this could be expensive when applied to a large compound library. As more data are available in PubChem, however, it will be possible to derive such bioactivity profiles for a particular chemical compound, as well as to investigate the selectivity and promiscuity against a specific target by combining the assay results contributed by many organizations. In particular, the projects under the MLP, which share a common library of more than 340,000 compounds, make it feasible to systematically derive target profiling information for many bioactive compounds. We performed an across-target activity analysis for all of the 189,807 active compounds in PubChem to identify the selective

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The distribution of the potency of the bioactive compounds and protein targets in PubChem. (a) Blue bars show the distribution of the potency for entire bioactive compounds in PubChem in seven potency groups; red bars denote the frequency of the protein targets with most potent compounds falling in respective potency group. (b) Blue bars represent the distribution of potency for chemical probes identified by the MLP; red bars show the frequency of the protein targets with most potent chemical probes falling in respective potency group.

and promiscuous compounds, following the procedure described previously [23]. As a result, 38% (71,627) of those compounds were observed as potentially selective with bioactivity outcome reported active against a single target, and the rest of them (62%) demonstrated active against multiple targets, with a portion of them hitting multiple but otherwise related targets (Fig. 3a). Many bioassay targets in PubChem are biologically related, as revealed by sequence homology analysis [1]. In particular, the MLP projects usually take a secondary screening against related

targets in the search for compounds with higher specificity. Thus, it is not surprising to often observe common hits for related targets. However, there are many other causes of the promiscuity of a compound [24]. To address this issue, the MLP has developed several profiling bioassays for evaluating aggregation effects, filtering chemical reactivity and identifying interference molecules, including screenings for luciferase inhibitors by multiple laboratories. In summary, all of the information has made PubChem a valuable resource for studying the promiscuity of chemical com-

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An overview of the selectivity property of bioactive compounds in PubChem. (a) The distribution of compounds’ across-target activity. The x axis represents the number of distinct active protein targets associated with a compound, and the y axis represents the frequency of compounds at each across-activity level. This shows that majority compounds are associated with one or a few protein targets, and a small portion of them interact with a large amount of targets. (b) The blue bars represent the distribution of the number of tested targets for selective compounds (only active to one protein target in PubChem). Compounds are divided into six selectivity groups. This suggests that the majority of the selective compounds have been tested across more than 150 targets. The red bars denote the frequency of the protein targets associated with the compounds in the respective selectivity group. www.drugdiscoverytoday.com

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The distribution of the selective compounds in PubChem. The x axis represents the potency in micromole; the y axis represents the frequency in the scale of log10. The color of the bars denotes the range of targets against which the selective compounds were tested. For example, the range of [100, 150] means that this group of compounds was tested against 100–150 targets and selectively against one of them.

pounds and investigating the polypharmacology properties of chemical compounds in system-based drug discovery [25,26]. As it would be necessary to assess the selectivity and promiscuity properties in the context of tested targets, we looked into those potentially selective compounds (71,627) and observed that approximately 80% of them were tested against at least 50 distinct protein targets, and a significant portion (60%) was highly selective, as tested against more than 150 targets (Fig. 3b). We also observed that 14% (316) of the 2206 targets were associated with at least one of these selective compounds. Among this subset of targets, more than 60% of them were associated with highly selective compounds that were tested broadly across more than 250 distinct protein targets (Fig. 3b). These results indicate that compounds with potentially high selectivity are available for a great portion of protein targets in PubChem. In addition, we evaluated the potency of these selective compounds by dividing them into several selectivity groups based on the number of targets tested (Fig. 4). This analysis provides further insights into both the selectivity and the potency of the bioactive compounds in this subset. It enables one to apply a certain selectivity threshold to identify the compounds with a desired potency and to track down the molecular target associated with the compound as well, which might serve as a starting point for a medicinal chemist to further optimize the bioactive compound towards a chemical probe or a drug candidate.

and 41 were mapped to 155 relevant conserved pathways in the KEGG database. The distribution of the bioactivity potency of these MLP chemical probes with their corresponding targets is shown in Fig. 2b. The chemical probes with potency in the range of 0.001–1 mM have been found for more than 60% of the protein targets (43 out of 67), which indicates varying quality of the probes with respect to potency. Compared to other bioactive compounds in PubChem, the MLP probes demonstrate higher potency and considerably better selectivity for the respective targets in general. As several literature-based bioactivity databases become publicly available [27–29], it is also possible to gain insights into the novelty of the MLP probes by comparing them with the prior art. Detailed information of the MLP chemical probes, including bioactivity potency, biological pathways and related 3D structures of their targets, is provided in Table S3 in the supplementary material online. Recently, there have been intensive discussions on the criteria and principles of defining a chemical probe, and some contradictory opinions have been raised [21,30]. Although only a portion of the MLP chemical probes seem to have medium or high quality based on a crowdsourcing evaluation [31] and most of them have low citation rates by the bibliometric method [30,32], it would probably take more time to find out their merits in future studies. Researchers in both academia and industry can help, however, and are highly encouraged to assess and improve the MLP chemical probes through their own research. To this end, the efforts undertaken by the MLP to further characterize the probes and make the data publicly accessible through PubChem would help make this happen.

Concluding remarks PubChem is growing rapidly with new data being deposited on a daily basis, which makes it both feasible and imperative to evaluate the properties of a particular bioactive compound, a drug candidate or even a known drug on a large scale to identify potentially new functions or off-target effects. It is starting to emerge as a valuable resource to explore the functions of genes and proteins in physiology and pathology. A summary of public services and tools are listed in Table S4 in the supplementary material online to facilitate use of the data in PubChem. As a public molecular information resource at NIH, the free availability of PubChem will undoubtedly lower the barrier for researchers from chemical biology, medicinal chemistry and drug discovery to advance the development of new chemical tools for interrogating biological functions and potential drug candidates for disease treatments. It also provides great opportunities for researchers in bioinformatics and cheminformatics to tackle the problems in those research fields with computational approaches.

Chemical probes At the time of this work, the MLP project has generated 116 chemical probes. The detailed descriptions of the characterizations of the probes are publicly available for the community to review (http://mli.nih.gov/mli/mlp-probes/). These MLP chemical probes were associated with 67 individual protein targets, which fell into 89 CDD superfamilies (some targets belonged to more than one superfamily) according to the CDD functional domain annotations. Among them, 36 protein targets had corresponding 3D structures with sequence identity of 100% in the PDB database 1056

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Acknowledgements We thank the National Institutes of Health Fellows Editorial Board for providing editorial assistance. This work is supported by the Intramural Research Program of the National Institutes of Health, National Library of Medicine.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.drudis.2010.10.003.

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1 Wang, Y. et al. (2009) An overview of the PubChem BioAssay resource. Nucleic Acids Res. 38, D255–D266 2 Wang, Y. et al. (2009) PubChem: a public information system for analyzing bioactivities of small molecules. Nucleic Acids Res. 37, W623–W633 3 Austin, C.P. et al. (2004) NIH Molecular Libraries Initiative. Science 306, 1138–1139 4 Editorial, (2009) Perfecting probes. Nat. Chem. Biol. 5, 435 5 Hopkins, A.L. and Groom, C.R. (2002) The druggable genome. Nat. Rev. Drug Discov. 1, 727–730 6 Russ, A.P. and Lampel, S. (2005) The druggable genome: an update. Drug Discov. Today 10, 1607–1610 7 Drews, J. (2006) What’s in a number? Nat. Rev. Drug Discov. 5, 975 8 Lindsay, M.A. (2003) Target discovery. Nat. Rev. Drug Discov. 2, 831–838 9 Harland, L. and Gaulton, A. (2009) Drug target central. Expert Opin. Drug Discov. 4, 857–872 10 Overington, J.P. et al. (2006) How many drug targets are there? Nat. Rev. Drug Discov. 5, 993–996 11 Mayr, L.M. and Bojanic, D. (2009) Novel trends in high-throughput screening. Curr. Opin. Pharmacol. 9, 580–588 12 Marchler-Bauer, A. et al. (2005) CDD: a Conserved Domain Database for protein classification. Nucleic Acids Res. 33, D192–D196 13 Marchler-Bauer, A. et al. (2002) CDD: a database of conserved domain alignments with links to domain three-dimensional structure. Nucleic Acids Res. 30, 281–283 14 Altschul, S.F. et al. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410 15 Altschul, S.F. et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 16 Bernstein, F.C. et al. (1977) The Protein Data Bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112, 535–542 17 Xie, L. et al. (2009) Drug discovery using chemical systems biology: identification of the protein–ligand binding network to explain the side effects of CETP inhibitors. PLoS Comput. Biol. 5, e1000387

18 Kanehisa, M. et al. (2009) KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res. 38, D355–D360 19 Kanehisa, M. and Goto, S. (2000) KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30 20 Kanehisa, M. et al. (2006) From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res. 34, D354–D357 21 Frye, S.V. (2010) The art of the chemical probe. Nat. Chem. Biol. 6, 159–161 22 Rix, U. and Superti-Furga, G. (2009) Target profiling of small molecules by chemical proteomics. Nat. Chem. Biol. 5, 616–624 23 Han, L. et al. (2009) A survey of across-target bioactivity results of small molecules in PubChem. Bioinformatics 25, 2251–2255 24 Feng, B.Y. et al. (2005) High-throughput assays for promiscuous inhibitors. Nat. Chem. Biol. 1, 146–148 25 Yildirim, M.A. et al. (2007) Drug–target network. Nat. Biotechnol. 25, 1119–1126 26 Chen, B. et al. (2009) PubChem as a source of polypharmacology. J. Chem. Inf. Model. 49, 2044–2055 27 Liu, T. et al. (2007) BindingDB: a web-accessible database of experimentally determined protein–ligand binding affinities. Nucleic Acids Res. 35, D198–D201 28 Overington, J. (2009) ChEMBL. An interview with John Overington, team leader, chemogenomics at the European Bioinformatics Institute Outstation of the European Molecular Biology Laboratory (EMBL-EBI). Interview by Wendy A. Warr.. J. Comput. Aided Mol. Des. 23, 195–198 29 Harmar, A.J. et al. (2009) IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels. Nucleic Acids Res. 37, D680–D685 30 Workman, P. and Collins, I. (2010) Probing the probes: fitness factors for small molecule tools. Chem. Biol. 17, 561–577 31 Oprea, T.I. et al. (2009) A crowdsourcing evaluation of the NIH chemical probes. Nat. Chem. Biol. 5, 441–447 32 Bologa, C. (2010) Promiscuity and PubChem: a retrospective analysis. In Proceedings of the Society for Biomolecular Screening 16th Annual Conference and Exhibition pp. 119

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Drug Discovery Today
Volume 15, Numbers 23/24
December 2010

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Heparin/heparan sulphate-based drugs Neha S. Gandhi1 and Ricardo L. Mancera1,2, 1

Curtin Health Innovation Research Institute, Western Australian Biomedical Research Institute, School of Biomedical Sciences, Curtin University, GPO Box U1987, Perth, WA 6845, Australia 2 School of Pharmacy, Curtin University, GPO Box U1987, Perth, WA 6845, Australia

Glycosaminoglycans (GAGs) are an untapped source of novel chemical entities and, therefore, offer exciting new opportunities for the development of novel drug molecules because of their unique physical and biological properties. Advances in the functional understanding of GAG–protein interactions are enabling the development of GAG mimetics for use as anti-angiogenic, anti-metastatic, anti-inflammatory, anticoagulant and anti-thrombotic agents. Many anti-thrombotic molecules, such as Fondaparinux and Idraparinux, have been successful in clinical trials, and a new generation of heparin mimetic oligosaccharides and small molecules are currently in different stages of clinical development. In particular, the recent increased activity in the development of new mimetics by altering the composition of sulphated GAGs is very encouraging. This article reviews structurally defined heparin-mimetic oligosaccharides and small molecules currently in development or clinical trials.

Sulphated glycosaminoglycans (GAGs) are glycans found inside the cell and in the extracellular matrix, which act by binding selectively to a variety of proteins and pathogens and are crucially relevant to many disease processes, such as inflammation [1–3], neurodegeneration [4], angiogenesis [5], cardiovascular disorders [6], cancer [7] and infectious diseases [8–10]. Heparin and heparan sulphate (HS) are GAGs consisting of 1–4 linked uronic acid and glucosamine and encompassing varying degrees of sulphation, and they are involved in many of these activities [11]. Heparin is a minor form of the ubiquitous HS, and the anticoagulant activity of pharmaceutical heparin is mainly accounted by fractions containing a pentasaccharide sequence with binding affinity for anti-thrombin (AT) (see below). The wide range and intricacy of glycan-mediated cellular interactions have turned glycans into novel targets for future drug development [12–14], with drugs already being developed for the treatment of metabolic disorders, cancer and infection. In recent years, there has been a renaissance in the development of carbohydrate-based therapeutics that involve the inhibition of carbohydrate–lectin interactions and carbohydrate-based anticoagulant Corresponding author:. Mancera, R.L. ([email protected])

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and AT agents [15]. The pharmacological and therapeutic value of heparin/HS and their mimetics is now recognized because of their ability to bind and cause immobilization and/or activation of a variety of proteins, such as growth factors, chemokines and metalloproteinases [16,17]. Potential strategies based on heparin/HS– protein interactions have recently been described to assist GAGbased drug discovery [18]. GAG-based drugs can act in several ways by activating (agonists) or inactivating (antagonists) protein-based receptors, competing with endogenous GAGs and/or inhibiting GAG biosynthesis. The molecular diversity of heparin/HS interactions has been exploited for the development and clinical progression of GAG mimetics [19]. The anticoagulant market has been very active recently because of the development of new compounds, including indirect factor Xa (FXa) inhibitors (such as Fondaparinux and Idraparinux and its new biotinylated form), direct inhibitors of FXa (such as Rivaroxaban and Apixaban) and direct inhibitors of thrombin (such as Dabigatran) [20,21]. The mechanism of action of these anticoagulants has been reviewed extensively [22,23]. The discovery of the mechanism of binding of heparin to AT and FXa has focused interest on the development of small, structurally defined heparin mimetics with AT activity but with reduced side-effects [24]. This

1359-6446/06/$ - see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2010.10.009

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article reviews the principles under which recently developed GAG (heparin/HS)-based inhibitors act and gives a description of the different classes of inhibitors and their development as drugs.

strong and specific interactions with AT [26]. The pentasaccharide unit only inhibits the activity of FXa mediated by AT; however, a much larger oligosaccharide is required for the AT-mediated inhibition of thrombin [27]. The structural requirements for the binding of heparin (Fig. 1a) to AT, as shown in Fig. 1, were determined by analysing the crystal structure and by determining the structure–activity relationships of a series of pentasaccharides using various combinations of sulphate and carboxylate groups [28,29]. This approach helped to establish that charged groups, as depicted in Fig. 1, are absolutely essential for the activation of AT (highlighted in the blue boxes) and required to increase the biological activity (in the red boxes). Moreover, hydrophobic interactions between the heparin pentasaccharide and AT also contribute to increasing the binding affinity [30]. Several review

Heparin/HS mimetics as anticoagulants Anticoagulants based on heparin/HS are drugs of choice in the therapy and prophylaxis of thromboembolic diseases [25]. Structural and functional studies have shown that a unique pentasaccharide (sometimes referred to as AGA*IA or DEFGH), GlcNAc/ NS6S ! GlcA ! GlcNS3S6S ! IdoA2S ! GlcNS6S (where Glc is glucosamine, IdoA is iduronic acid and GlcA is glucoronic acid, which are either sulphated or acetylated), comprises the AT-binding domain and is responsible for the anticoagulant activity of heparin. The 3-O-sulphate group at position F is responsible for

[()TD$FIG]

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FIGURE 1

Chemical structures of heparin pentasaccharide derivatives. (a) The AT-binding pentasaccharide motif. Highlighted functional groups are essential for AT activation. (b) Structure of Fondaparinux. (c) Structure of Idraparinux. (d) Structure of Idrabiotaparinux. Natural heparin and synthetic pentasaccharides differ in their substitution pattern (synthetic pentasaccharides contains –OMe substitutions). www.drugdiscoverytoday.com

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articles have been published describing the structure–activity relationship and mechanism of action of heparin mimetic anticoagulants [24,28,29,31,32]. Research on heparin mimetic anticoagulants has gained momentum since the successful clinical development programs of the 1990s. GlaxoSmithKline registered Fondaparinux (Fig. 1b) (SR90107, Org31540) as a new anti-thrombotic drug under the name Arixtra1 after being granted approval from the US FDA and the European Committee for Proprietary Medical Products [33]. SR123781 is a short-acting hexadecasaccharide analogue of heparin with N-sulphate groups replaced by O-sulphates and alkylated hydroxyl groups in the AT-binding domain. It has tailor-made FXa- and thrombin-inhibitory activities combined with more selectivity in its mode of action. Sanofi-Aventis discontinued the development of SR123781, however, after the success of heparin mimetic AVE 5026 [34].

Idraparinux (SanOrg34006, SR34006) Idraparinux (Fig. 1c) is a synthetic pentasaccharide analogue of Fondaparinux, in which the hydroxyl groups are methylated and the N-sulphate groups are replaced by O-sulphates [35]. Idraparinux (Kd of 1 nM) interacts more strongly with AT than Fondaparinux (Kd of 50 nM) through non-ionic interactions [36] and also exhibits superior anti-Xa activity (1600 versus 700 U mg1) [35,37,38]. Idraparinux can be synthesized more easily than Fondaparinux because of the presence of a ‘pseudo’-alternating sequence that can be easily prepared from glucose [39]. The crystal structures of the complexes of a pentasaccharide analogue of Idraparinux (modified by the addition of a sulphate at the H unit) [40] with a dimer consisting of activated AT and latent AT [41], and with an intermediate state [42], have been reported. Analysis of these structures explains the lower affinity of heparin for AT on the basis of induced conformational changes in AT, such as the expulsion of the hinge region and the closure of b-sheet A to the normal five-stranded form. This then leads to the activation of AT and the allosteric inhibition of coagulation factors IXa, Xa and thrombin. Idraparinux has been evaluated in clinical trials for the treatment and secondary prevention of venous thromboembolism (VTE) and the prevention of thromboembolic events associated with atrial fibrillation (AF) [43]. The pharmacokinetics, pharmacodynamics and tolerability of Idraparinux were evaluated in several phase I studies [44,45]. Idraparinux has a long half-life (80–120 h) in the bloodstream, thus enabling once-weekly administration [46]. The phase II dose-ranging PERSIST study established that subcutaneous once-weekly administration of 2.5 mg of Idraparinux, with an increased elimination half-life of approximately 600 h, was as effective as warfarin and demonstrated dose-dependent increases in major bleeding for the secondary prevention of deep vein thrombosis (DVT) [47–49]. Idraparinux was also evaluated for the long-term treatment of patients with DVT and pulmonary embolism (PE) using a subcutaneous once-weekly dose of 2.5 mg in the three van Gogh trials [50,51]. Unfortunately, the results of these trials indicated that the rate of recurrent VTE was considerably higher with Idraparinux than with conventional therapy. In addition, the elimination half-life of 60 days led to a prolonged anticoagulant effect after completion of the therapy [52]. These findings also explained the complication of increased 1060

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bleeding during a 12-month treatment period compared to a sixmonth treatment period in patients randomly treated with Idraparinux in the DVT and PE study, as carried out in the van Gogh Extension trial [53,54]. However, repeated doses of reversal agents, such as rFVIIa, are required in bleeding patients during surgery to neutralize the anticoagulant effect of Idraparinux [55]. The van Gogh and Amadeus phase III clinical trials of Idraparinux established that its pharmacokinetics were best described by a threecompartment model in patients with DVT, PE or AF at risk of thromboembolic events [56]. The terminal half-life was measured to be 66.3 days, and the half-life during steady state was determined to be 35 weeks. Idraparinux clearance was notably related to subject weight, creatinine clearance, sex and age. The phase III AMADEUS non-inferiority trial enrolled patients with AF at risk for thromboembolism to compare the efficacy and safety of Idraparinux to therapy with vitamin K antagonists [57]. Idraparinux proved as effective as vitamin K antagonists; however, the trial was cut short because of an excess of bleeding complications in Idraparinuxtreated patients and a few documented ischemic events. Idraparinux remains in the late clinical development pipeline of SanofiAventis because of its advantageous (compared to oral anticoagulants and Fondaparinux) once-a-week dosing regimen. Idrabiotaparinux (SSR126517) (Fig. 1d) is a novel synthetic anticoagulant linked to biotin at position 2 of the non-reducing end of glucose in Idraparinux [58]. Linkage of biotin at this position in the pentasaccharide prevents interaction of the pentasaccharide with AT or FXa in vitro [59]. The optimal length of the spacer was found to be a 6C-length arm. Administration of Idrabiotaparinux to rats by either the intravenous or the subcutaneous route resulted in a similar pharmacokinetic profile to that of Idraparinux. Further animal studies into Idrabiotaparinux showed that the injection of avidin triggered the immediate elimination of the molecule from the bloodstream, resulting in the complete neutralization of the anti-thrombotic activity of Idrabiotaparinux. Sanofi-Aventis has halted the development of Idrabiotaparinux in AF in phase III trials, because of its lack of potential benefit over oral anticoagulants, such as vitamin K antagonists, which are currently being evaluated in clinical trials [60].

AVE5026 AVE5026 (Sanofi-Aventis) is in clinical development for the prevention of VTE [61]. This molecule is a complex mixture of oligomeric ultra-low-molecular-weight heparin (LMWH) fragments (molecular weights 2000–3000 Da) with a polydispersity index of approximately 1.0. It is prepared by partial and controlled chemioselective depolymerization of porcine unfractionated heparin (UFH). AVE5026 primarily targets FXa and has only a minimal effect on thrombin. It exhibits a higher ratio of FXa to anti-Factor IIa activity (>30:1). In addition, it shows dose-dependent anti-thrombotic activity in a rat microvascular thrombosis disease model, suggesting that this agent might provide the optimal treatment for cancer-associated thrombosis [62]. When given subcutaneously, the half-life of AVE5026 is 16–20 h, enabling once-daily administration. AVE5026 is excreted renally and, like Fondaparinux, its anticoagulant effects are not neutralized by protamine sulphate. An elective total knee replacement surgery study demonstrated a highly statistically significant dose-dependent response with AVE5026 for the prevention of VTE in patients

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undergoing knee arthroplasty [63]. A 20 mg dose of AVE5026 was selected for further evaluation. An extensive phase III trial is currently comparing AVE5026 with the LMWH Enoxaparin for the prevention of VTE in patients undergoing hip, knee or abdominal surgery and in cancer patients receiving chemotherapy.

M118 Momenta Pharmaceuticals developed M118 (Fig. 2), a novel anticoagulant for the treatment of patients with acute coronary syndrome. It is currently being evaluated in a phase II clinical trial with patients undergoing percutaneous coronary intervention [64]. M118 is an optimized polysaccharide compound engineered from UFH using a specific enzymatic depolymerization process. It is designed to act at multiple points in the coagulation cascade by selectively binding to both AT and thrombin, two crucial factors involved in the formation of clots [65]. Preclinical and phase I studies have shown that M118 has the positive attributes of both UFH (reversibility, monitorability and broad inhibition of the coagulation cascade) and LMWH (adequate bioavailability and predictable pharmacokinetics that enable subcutaneous administration) and can thus be administered both intravenously and subcutaneously [66]. M118 exhibits clear dose-dependent inhibition of FXa and Factor IIa, with an anti-Xa:anti-IIa ratio that is constant over time [67,68]. M118 was found to be effective at preventing thrombosis in diseased arteries in a photochemical carotid artery injury model in ApoE/ mice [69]. The reduced polydispersity (the ratio of weight averaged to number-averaged molecular weight) of M118 contributes to a more predictable pharmacokinetic profile. M118 lacks drug–drug interactions when co-administered with aspirin and clopidogrel or with glycoprotein IIb/IIIa inhibitors such as Eptifibatide [70].

EP42675 and EP217609 EP42675 is the first representative of a new class of synthetic parenteral anticoagulants with a dual mechanism of action combining the properties of an indirect FXa inhibitor and a direct thrombin inhibitor [71]. EP42675 is being trialled in patients with

[()TD$FIG]

acute coronary syndrome undergoing percutaneous coronary intervention. The structure of EP42675 contains an AT-binding pentasaccharide (an indirect FXa inhibitor) coupled to a peptidomimetic a-NAPAP analogue (a direct inhibitor of the active site of both free and clot-bound thrombin). This dual mechanism imparts a unique pharmacological profile to EP42675: (i) inhibition of both fibrin-bound and fluid-phase thrombin owing to the presence of a direct thrombin-inhibiting moiety, (ii) inhibition of FXa in the presence of AT, (iii) a favourable pharmacokinetic profile that ensures prolonged anticoagulant coverage with improved control over its therapeutic window owing to the presence of the Fondaparinux pentasaccharide, and (iv) no crossreaction with platelet factor 4 antibodies. The use of anticoagulants can result in haemorrhagic adverse events and hence the availability of an antidote is highly desirable. An antidote can also be useful in case an anticoagulated patient needs to be operated on urgently. In the development of EP42675, therefore, a biotin entity was covalently linked to the spacer between the pentasaccharide portion and the direct thrombin inhibitor portion of the molecule to give EP217609 (Fig. 3), which enables it to be neutralized upon administration of avidin [72]. EP42675 has successfully completed phase I trials with 100 healthy subjects, where it was found to be well tolerated and showed predictable pharmacokinetic and pharmacodynamic profiles, with low intra- and inter-subject variabilities [73]. The half-lives of EP42675 and EP217609 in rats was determined to be approximately three hours [31]. In animals, the pharmacokinetic/pharmacodynamic profiles of EP217609 and EP42675 were found to be similar.

Non-anticoagulant heparin/HS mimetics Heparin is known to inhibit the synthesis, expression and/or function of adhesion molecules, cytokines, chemokines, proteases and viral proteins [74]. Consequently, attention has been focused recently on the non-anticoagulant properties of heparin, which are known to inhibit inflammation [63,75] and the metastatic spread of tumour cells [7,76].

PI-88 (Muparfostat) CH2OR

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FIGURE 2

Chemical structure of M118, a LMWH characterized by a weight-averaged molecular mass between 5500 and 9000 Da and a polydispersity of approximately 1.0. M118 has the structural formula C12mH14m+1O10mNmNamR3m1R1m, C12mH14m+2O10m+1NmNamR3mR1m, where n is equal to the average number of disaccharide repeats, m = 1 + n, R is H or SO3Na and R1 is SO3Na or COCH3.

Heparanase is an endoglycosidase enzyme that has vital roles in inflammation, tumour cell invasion, metastasis and angiogenesis [77,78]. Heparanase is the enzyme responsible for processing HS. Several sulphated sugar molecules such as cyclitols and glycol-split derivatives have been identified as selective inhibitors of heparanase–heparin interactions [79]. PI-88 (Progen Pharmaceuticals) (Fig. 4) is one such inhibitor. PI-88 inhibits heparanase and the cleavage of HS by binding competitively with HS, thereby preventing the release of growth factors, such as FGF-1, FGF-2 and VEGF, involved in angiogenesis [80]. PI-88 has progressed to clinical trials to treat inflammatory diseases, thrombosis, viral infections and cancer [81]. PI-88 is a phosphomannopentose sulphate (6-O-PO3H2-a-DMan-(1 ! 3)-a-D-Man-(1 ! 3)-a-D-Man-(1 ! 3)-a-D-Man-(1 ! 2)D-Man) (Fig. 4), wherein the chain length, sugar composition and glycosidic linkages a(1 ! 3) and a(1 ! 2) play important parts in its anticoagulation activity, compared with the anticoagulant activity of sulphated glucose-containing oligosaccharides with b(1 ! 4) and b(1 ! 3) linkages [82]. PI-88 is known to consistently prolong the activated partial thromboplastin time through the www.drugdiscoverytoday.com

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[()TD$FIG]

OSO3-O C 2

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FIGURE 3

Chemical structures of EP217609 and EP42675. EP42675 and EP217609 are the first representatives of a new class of synthetic, parenteral anticoagulants with a dual mechanism of action combining the properties of an indirect FXa inhibitor and a direct thrombin inhibitor. The structure of EP42675 can be inferred by deleting the biotin and lysine moieties (shown in dotted rectangles) from the structure of EP217609.

activation of the endogenous heparin cofactor II. Apart from its anticipated anticoagulant effects, PI-88 was well tolerated in animal studies. In the first phase I trial with patients with malignant disease, PI88 was administered subcutaneously for four consecutive days either bimonthly or weekly [83]. Prolongation of the activated partial thromboplastin time was seen in only 2 of 14 patients. The recommended dose of PI-88 administered daily for four days every week was established to be 250 mg. Dose-limiting toxicity occasionally resulted in thrombocytopenia (at a dose of 2.28 mg/kg/ day for 14 days) in patients with advanced malignancies, which [()TD$FIG]seemed to be immunologically mediated through the developPO3-2

RO RO

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Chemical structure of PI-88. PI-88 is primarily composed of sulphated phosphomannopentaose and phosphomannotetraose oligosaccharide units. PI-88 is a potent anti-angiogenic, anti-tumour and anti-metastatic agent because of its inhibition of the heparan sulphate-degrading enzyme heparanase. 1062

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ment of anti-heparin platelet factor 4 complex antibodies [84]. The second phase I trial evaluated the safety, toxicity, pharmacological properties and biological activity of PI-88 with fixed weekly docetaxel (chemotherapy) in patients with advanced solid malignancies [85]. Sixteen patients received docetaxel at a 30 mg/m2 dose on days 1, 8 and 15 of a 28 day cycle, with PI-88 injected subcutaneously for four days per week. Minor toxicity responses during the course of the therapy included fatigue, dysgeusia, thrombocytopenia, diarrhoea, nausea and emesis. Docetaxel and PI-88 did not alter the pharmacokinetics of each other. In another phase I trial, the recommended dose of PI-88 was reported to be 190 mg/m2 alone and 1000 mg/m2 in combination with dacarbazine every three weeks [86]. A phase I/II trial of daily PI88 alone or with dacarbazine in patients with malignant melanoma was subsequently undertaken; however, the trial was stopped owing to cases of major febrile neutropenia [87]. A phase II trial of PI-88 in patients with advanced melanoma evaluated a fixed dose of 250 mg/day given by injection for four consecutive days followed by three drug-free days in a 28 day cycle [88]. Some patients developed serious bleeding events, with hemorrhagic cerebral metastases and arterial thrombosis. Nonetheless, in patients with advanced melanoma, PI-88 demonstrated noteworthy activity, but further investigations are needed of its use in combination with chemotherapy. A phase III trial investigating PI-88 as a post-resection treatment for hepatocellular carcinoma (liver cancer) was designed to establish the efficacy and safety of PI88, but no results have been reported. The PG500 series is a collection of newly designed compounds that are anomerically pure and fully sulphated and have single entity oligosaccharides attached to a lipophilic moiety, such as aglycone, at the reducing end of the molecule [89]. Compared with PI-88, some of these compounds are more potent inhibitors of

angiogenesis and metastasis and show strong anti-tumour activity in some aggressive tumour models [90]. These compounds are believed to interfere in processes such as tumour development, namely angiogenesis via inhibition of VEGF, FGF-1 and FGF-2, and metastasis via inhibition of heparanase [91]. PG545 was selected as the lead molecule based on its efficacy, pharmacokinetics, toxicology and ease of manufacture [92,93]. This compound has been in preclinical trials and administered subcutaneously once a week in mice for the treatment of cancer.

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Bellus Health has recently initiated a phase I clinical trial of NRM8499, a prodrug of Tramiprosate for the treatment of AD. NRM8499 increases brain exposure to Tramiprosate, which might help improve the therapeutic effect on cognitive function and other clinical results in AD. This randomized, double-blind, placebo-controlled study is expected to investigate the safety, tolerability and pharmacokinetic profile of NRM8499 in a group of up to 84 young and elderly healthy subjects. Preclinical studies conducted in rodents showed that NRM8499 increased plasma and brain exposure to Tramiprosate by 1.5–3-fold.

Tramiprosate (AlzhemedTM) HS/heparin have been widely reported to be associated with neuritic plaques in Alzheimer’s disease (AD) [94]. HS has also been shown to promote the aggregation of amyloid b-peptide (Ab) and have a pivotal role in plaque formation [95]. Several molecules have been proposed to be used to prevent HS-induced aggregation of Ab: derivatives or fractions of heparin and other GAGs, sulphated compounds that act as HS mimetics (e.g. pentosan polysulphate and dextran sulphate) [96], small-molecule anionic sulphonates or sulphates [97], and amyloidophilic, sulphonated dyes, such as Congo Red and Thioflavin S. Tramiprosate (also referred to as 3-amino-1-propanesulfonic acid, 3-aminopropylsulfonic acid, 3-APS, homotaurine or NC531) is a GAG mimetic designed to interfere with the actions of Ab early in the cascade of amyloidogenic events [98–100]. Structurally, Tramiprosate is a modification of the amino acid taurine (Fig. 5). It binds preferentially to soluble Ab peptide and maintains Ab in a random-coil/a-helical rich conformation and in nonfibrillar form, thereby inhibiting aggregation and hence plaque formation and deposition [101]. It can cross the blood–brain barrier effectively [102]. Recently, it has been reported that Tramiprosate also alters tau aggregation [103]. A phase II trial demonstrated that Tramiprosate reduces Ab42 in the cerebrospinal fluid of patients with mild to moderate AD [104]. The US phase III trial involved patients with mild to moderate AD, who were randomly assigned to receive placebo or 100 mg or 150 mg twice-daily doses of Tramiprosate. Although treatment was well tolerated, the study failed to demonstrate efficacy upon long-term clinical testing of cognitive improvement [105]. The European phase III trial has been discontinued. No further reports on the drug are available except columetric magnetic resonance imaging findings, which suggested less hippocampal shrinkage upon treatment with Tramiprosate [106]. Bellus Health (formerly Neurochem Inc.) has been promoting this medication as a nutraceutical, VivimindTM, which is being put forward as protecting against memory loss [107].

HO H2N

Eprodisate sodium (NC-503, Kiacta1 and FibrillexTM) Eprodisate (1,3-propanedisulfonic acid disodium salt) is a lowmolecular-weight, negatively charged sulphonated molecule (Fig. 6) that shares certain structural similarities with HS and is known to bind to serum amyloid protein A (SAA) [108]. Eprodisate binds to the GAG-binding site of SAA and competes with naturally occurring sulphated GAGs, thus targeting amyloid fibril polymerization and inhibiting amyloid deposition in tissues [97,109]. Eprodisate inhibits the development of amyloid deposits in in vivo mouse models of amyloid protein A (AA) amyloidosis [110]. In preclinical pharmacokinetic studies, Eprodisate has good bioavailability if administered orally; it is not metabolized, it does not bind to plasma proteins, and it is excreted primarily by the kidney, although pharmacokinetics analyses in its phase I trial revealed high inter-individual variability in its plasma concentrations [111]. Although Eprodisate is eliminated by the kidney, plasma concentrations were seen to increase as renal function decreased, resulting in a considerable increase in drug systemic exposure. An approximate terminal half-life of 10–20 h was derived from a multiple rising oral dose study. The efficacy and safety of Eprodisate was tested in a single phase II/III trial in AA amyloidosis patients [112]. Eprosidate was well tolerated, and its adverse events profile was comparable to placebo. Eprodisate can also be used with other types of amyloidosis. The results of a recent trial showed that it might slow the progression of AA amyloidosis-related renal disease [113], but no effect was seen on SAA levels, progression to end-stage renal disease or death, proteinuria and amyloid content of abdominal fat [114]. Despite having previously been granted orphan and fast-track status, the FDA and the EMEA both requested an additional confirmatory phase III trial before approval [114]. Studies using a preclinical rat model of diabetes and metabolic syndrome have confirmed that Eprodisate decreases glucose, cholesterol and triglycerides in the blood of obese diabetic Zucker rats compared with the control group, while preserving 40% more

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Chemical structure of Tramiprosate, an anti-amyloidogenic agent. In the context of Alzheimer’s disease, this molecule acts by preventing and slowing the formation and the deposition of heparin/HS-induced amyloid fibrils in the brain and by binding to soluble beta-amyloid protein to reduce the amyloidinduced toxicity on neuronal and brain inflammatory cells.

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Chemical structure of Eprodisate. Eprodisate is a promising agent designed to prevent the worsening of renal function in patients with AA amyloidosis. It inhibits the polymerization of amyloid fibrils and the deposition of the fibrils in tissues by interfering with interactions between amyloidogenic proteins and heparin/HS. www.drugdiscoverytoday.com

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pancreatic islet cells compared with the control group and showing some protective effect on renal function. However, Bellus Health has discontinued the development of Eprodisate in relation to diabetes after a phase IIa clinical proof-of-concept trial because the study did not meet its primary efficacy endpoint [115]. Instead, the company has pushed preclinical development of a prodrug of Eprodisate for the treatment of Type II diabetes and related metabolic syndromes. Reviews
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Other novel GAG mimetics Substrate-optimized glycans Zacharon Pharmaceuticals has focused its research on small-molecule inhibitors of GAG biosynthesis for lysosomal storage disease. Mucopolysaccharidosis (MPS) is a form of lysosomal storage disease caused by a deficiency in the enzymes responsible for the degradation of GAGs, which makes lysosomes fill with partially degraded GAGs and resulting in serious systemic disease. Zacharon uses substrate optimization therapy, which is a novel therapeutic approach for selectively modifying glycan structure without reducing the overall amount produced or altering normal glycan function [116]. This selective modification renders the glycan molecule more readily degradable, despite the presence of specific enzyme deficiencies. In MPS, this is accomplished by selectively and favourably modifying the glycan sulphation pattern. In MPS II, an inhibitor of the biosynthetic step involving the addition of 2-O sulphates to GAGs would produce GAGs with less 2-O sulphation and increased 6-O sulphation (Fig. 7). These GAGs would be easier to degrade for an MPS II (2-sulphatase-deficient) patient. To identify potential drug candidates, a library of 74,000 drug-like small molecules for inhibitors of HS biosynthesis were screened using cell-based assays. Of the 264 hit compounds identified in the primary screen, 30 were found to inhibit HS biosynthesis in cultured cells. Four of the compounds were found to reduce GAG accumulation in primary human fibroblasts obtained from MPS patients. ZP2345 was then chosen as the starting point for the development of a GAG mimetic based on substrate optimization therapy. ZP2345 is an HS inhibitor that reduces 2-O sulphation in a

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dose-dependent manner in a cultured human cell model of MPS-II [116]. Ongoing studies are focusing on analogue design, synthesis and testing to improve the potency and efficacy of these inhibitors.

Heparin mimetics in cancer Exogenous heparin, LMWH and their mimetics have been shown to exert anti-metastatic and anti-angiogenic properties affecting cancer progression, such as inhibition of heparanase, blocking of P- and L-selectin-mediated cell adhesion, and inhibition of angiogenesis [117]. Heparanase has been targeted with several heparinrelated inhibitors such as aza sugar derivatives, glycol-split derivatives and cyclitols that block the active site of the enzyme or the heparin/HS binding sites, or both [118]. Several LMWH derivatives have been described, among which are a deoxycholic acid conjugate and the fragmentation of a periodate-oxidized heparin, which have anti-angiogenic and anti-metastatic activities in different types of cancer models [119,120]. Endotis Pharma has created a platform for the development of anti-cancer ‘small-glyco’ drugs. These molecules are short, chemically synthesized oligosaccharides with potent affinity and selective inhibition of several growth factors and proteins (VEGF-A, FGF-2, PDGF-B, SDF-1a and heparanase) involved in tumour growth and dissemination [121]. A library of more than 100 synthetic oligosaccharides of different sizes containing various substitutions has been evaluated for their affinity for specific targets and their efficacy on cell proliferation and migration and in vitro endothelial tubule formation. The structure–activity relationship indicates that affinity and selectivity of these molecules for different targets can be fine-tuned through chemical substitutions. EP80061 is the lead compound in the series, and it induced a very potent anti-metastatic effect on a disseminated tumour model in C57Bl/6 mice [122]. However, the structure of these series has not yet been disclosed. Momenta Pharmaceuticals presented preclinical data for M402, a HS mimetic containing a mixture of linear sugar chains and engineered to have potent anti-metastatic properties [123,124].

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Substrate-optimized glycans (e.g. HS in MPS).Figure modified, with permission, from Ref. [159], Zacharon Pharmaceuticals. 1064

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Both in vitro and in vivo, M402 showed reduced anticoagulant activity and inhibited tumour metastasis through modulation of multiple factors, such as P-selectin, VEGF, FGF-2 and SDF-1a [124,125]. M402 as a monotherapy or in combination with chemotherapeutics showed statistically significant survival benefits in animal models with aggressive tumours [123,125]. In combination with gemcitabine, M402 produced much prolonged survival and reduced metastasis compared to groups treated with saline solution alone or gemcitabine alone in a murine pancreatic model [126]. Mice treated with M402 showed reduced epithelial-tomesenchymal transition, a key step in the progression of tumour cells towards a more invasive phenotype.

Regenerating agents Regenerating agents (RGTAs) are large biopolymers engineered to replace HS specifically bound to matrix proteins and growth factors destroyed after chronic tissue injury [127]. These polymers protect proteins bound to the extracellular matrix from proteolysis. RGTAs can interact with many heparin-binding growth factors, such as FGF-2 [128], transforming growth factor-b [129] and VEGF [130]. In addition to their heparin-binding-growth-factor-protecting and stabilizing properties, RGTAs have been found to inhibit human leukocyte elastase [131], plasmin [132,133] and heparanase [134]. RGTA-induced matrix therapy is a possible alternative to cell or gene therapy in regenerative medicine [135]. RGTA derivatives are potent activators of tissue repair in various in vivo wound-healing models: wound [136], bone defect [137,138], infarcted myocardium [139], colic ulceration [140] and periodontitis [141]. These RGTAs have also been shown to stimulate satellite cell growth and differentiation in primary cultures [142]. RGTAs are dextran derivatives with defined amounts of substituted carboxymethyl, benzylamide and sulfonate groups (Fig. 8). By varying the relative proportion of these substitutions, a library of heparin-mimetic biopolymers was produced. RGTAs with an increased level of sulphation and benzylamidation have shown anti-prion activity by blocking the conversion of prion protein PrPC into the abnormal forms in scrapie-infected GT1 cells [143] and scrapie-infected and bovine-spongiform-encephalopathy-infected mice [144]. RGTA polymers are easier and less costly [()TD$FIG]

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to produce, store and handle than growth factors. One such molecule is OTR4120 (alternatively called RGD120 or RG1192), derived from a glycosidic polymer of dextran T40 and functionalized with a 1.13 level of substitution of sulphate residues and a 0.46 level of substitution of carboxymethyl residues, with a maximal level of substitution of 3.0 [145], rendering this molecule structurally similar to heparin but having at least ten times less anticoagulant activity than heparin [146]. Nuclear magnetic resonance (NMR) analysis has shown that this polymeric compound is composed of a 15 sugar unit sequence statistically repeated along the molecule [147,148]. OTR4120 is known to enhance tissue repair in several animal models, including peripheral nerve injury in rats [149], burned skin in rats [150], chronic skin ulcers in mice [135] and cutaneous wound repair in rats [151]. Pharmacokinetics studies performed in a muscle crush model indicated that OTR4120 could replace degraded HS-GAG after tissue injury and bind to the heparin-binding sites present on many extracellular matrix proteins that have been freed from occupation by their endogenous GAGs [152]. In a recent clinical pilot study, an OTR4120 ophthalmic solution was found to improve the healing of severe corneal ulcers and dystrophy [153]. OTR3 is currently marketing CACIPLIQ201, an active device based on RGTA for the treatment of chronic ulcers, diabetic foot ulcers, pressure ulcers, venous ulcers and burns.

Heptagonists Use of heparin, LMWH, Fondaparinux and Idraparinux in cardiovascular surgeries often leads to a high incidence of bleeding complications. Protamine and LMW protamine are antidotes employed in heparin reversal; however, they can cause severe adverse reactions. PolyMedix has developed novel small synthetic salicylamide derivatives called heptagonists, which act as universal anticoagulant-reversing agents and are active against heparin, LMWH, Idraparinux and Fondaparinux [154–157]. One of the company’s so-called ‘heptagonists’, PMX-60056 [157], can effectively neutralize the AT and anti-Xa activities of LMWH. PMX50056 has been shown to completely reverse the anticoagulant effects of heparin and normalize blood clotting time in six human subjects in less than 10 min in a phase Ib clinical trial.

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Schematic chemical structure of RGTA. Four differently substituted units, A (<1%), B (=32%), C (=0%) and D (=67%), can be present in OTR4120, as reported by titrimetry and 1H NMR [147]. R represents the proportion of substituted group in the global C3 and C4 positions arranged to define the global dextran sulphate of each group. www.drugdiscoverytoday.com

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Concluding remarks

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Past research has highlighted the drawbacks of using native heparin oligosaccharides as drugs. Their anionic nature can result in large interactions with multiple, physiologically important proteins, leading to many side-effects. In addition to their lack of affinity, heparin oligosaccharides suffer from low tissue permeability, short serum half-life and poor stability. Consequently, the pharmacodynamic and pharmacokinetic properties of heparin make it inadequate for its direct therapeutic application. In addition, the multi-step synthesis of heparin/HS oligosaccharides poses challenges for medicinal chemists, both at the drug development and the production scale [158]. Furthermore, new therapeutic applications of sulphated GAGs now include the treatment of infectious diseases and inflammation and the control of cell growth in wound healing and cancer. These new applications require the elimination of the anticoagulant activity of heparin oligosaccharides and the engineering of appropriate pharmacokinetic properties and optimal oral bioavailability. GAG mimetics are designed to overcome these shortcomings. Detailed insight into GAG–protein interactions has predominantly been provided by recent progress in NMR spectroscopy,

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X-ray crystallography and molecular modelling techniques. Identification of the bound conformation of heparin/HS to a protein enables the design of GAG mimetics and the identification of negligible and replaceable functional groups. As a consequence, the development of GAG mimetics that have improved absorption, distribution, metabolism and excretion properties can be accomplished. The development of HS/heparin-based drugs is a fertile field of research that is providing enormous opportunities for the discovery of improved treatments for many diseases. This is evidenced by the existence of many newly established companies, such as Intellihep, Zacharon Pharmaceuticals, GlycoMimetics, Endotis Pharma, Polymedix, Progen, OTR3 and Momenta, to name a few, which are beginning to exploit the untapped potential of the structural diversity of heparin/HS in various therapeutic and biomedical applications.

Acknowledgements N.S.G. is grateful for the award of an Endeavour International Postgraduate Research Scholarship. We apologize to all those authors whose work could not be cited owing to space limitations.

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38 Desai, U.R. et al. (1998) Mechanism of heparin activation of antithrombin: role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin. J. Biol. Chem. 273, 7478–7487 39 Westerduin, P. et al. (1994) Feasible synthesis and biological properties of six ‘nonglycosamino’ glycan analogues of the antithrombin III binding heparin pentasaccharide. Bioorg. Med. Chem. 2, 1267–1280 40 Basten, J. et al. (1992) Biologically active heparin-like fragments with a ‘‘nonglycosamino’’glycan structure. Part 2: a tetra-o-methylated pentasaccharide with high affinity for antithrombin III. Bioorg. Med. Chem. Lett. 2, 901–904 41 Jin, L. et al. (1997) The anticoagulant activation of antithrombin by heparin. Proc. Natl. Acad. Sci. U. S. A. 94, 14683–14688 42 McCoy, A.J. et al. (2003) Structure of b-antithrombin and the effect of glycosylation on antithrombin’s heparin affinity and activity. J. Mol. Biol. 326, 823–833 43 Prandoni, P. et al. (2008) Idraparinux: review of its clinical efficacy and safety for prevention and treatment of thromboembolic disorders. Expert Opin. Investig. Drugs 17, 773–777 44 Faaij, R.A. et al. (1999) A phase I single rising dose study to investigate the safety, tolerance and pharmacokinetics of subcutaneous SANORG 34006 in healthy male and female elderly volunteers. Thromb. Haemost. 490–491 (Abstract 1547) 45 Faaij, R.A. et al. (1999) A phase I single rising dose study to investigate the safety, tolerance and pharmacokinetics of SANORG 34006 in healthy young male volunteers. Thromb. Haemost. 853–1853 (Abstract 2709) 46 Ma, Q. and Fareed, J. (2004) Idraparinux sodium. IDrugs 7, 1028–1034 47 (2002) PERSIST investigators. A novel long-acting synthetic factor Xa inhibitor (idraparinux sodium) to replace warfarin for secondary prevention in deep vein thrombosis. A phase II evaluation. Blood 100, 301 48 Buller, H.R. et al. (2004) A novel long-acting synthetic factor Xa inhibitor (SanOrg34006) to replace warfarin for secondary prevention in deep vein thrombosis. A phase II evaluation. J. Thromb. Haemost. 2, 47–53 49 Minar, E. and Investigators, T.P. (2004) A novel long-acting synthetic factor Xa inhibitor (SanOrg34006) to replace warfarin for secondary prevention in deep vein thrombosis. A phase II evaluation. J. Thromb. Haemost. 2, 540 50 Buller, H.R. et al. (2007) Extended prophylaxis of venous thromboembolism with idraparinux. N. Engl. J. Med. 357, 1105–1112 51 Buller, H.R. et al. (2007) Idraparinux versus standard therapy for venous thromboembolic disease. N. Engl. J. Med. 357, 1094–1104 52 Harenberg, J. et al. (2008) Anticoagulant effects of Idraparinux after termination of therapy for prevention of recurrent venous thromboembolism: observations from the van Gogh trials. Eur. J. Clin. Pharmacol. 64, 555–563 53 Harenberg, J. et al. (2008) Long elimination half-life of idraparinux may explain major bleeding and recurrent events of patients from the van Gogh trials. J. Thromb. Haemost. 6, 890–892 54 Harenberg, J. et al. (2009) The anticoagulant Idraparinux: is the extensive half life of 60 days the cause of bleeding complications. Br. J. Clin. Pharmacol. 68 (Suppl. 1), 21–121 55 Bijsterveld, N.R. et al. (2004) Recombinant factor VIIa reverses the anticoagulant effect of the long-acting pentasaccharide idraparinux in healthy volunteers. Br. J. Haematol. 124, 653–658 56 Veyrat-Follet, C. et al. (2009) The pharmacokinetics of idraparinux, a long-acting indirect factor Xa inhibitor: population pharmacokinetic analysis from Phase III clinical trials. J. Thromb. Haemost. 7, 559–565 57 (2008) The AMADEUS Investigators. Comparison of idraparinux with vitamin K antagonists for prevention of thromboembolism in patients with atrial fibrillation: a randomised, open-label, non-inferiority trial. Lancet 371, 315–321 58 Harenberg, J. (2009) Development of idraparinux and idrabiotaparinux for anticoagulant therapy. Thromb. Haemost. 102, 811–815 59 Savi, P. et al. (2008) Reversible biotinylated oligosaccharides: a new approach for a better management of anticoagulant therapy. J. Thromb. Haemost. 6, 1697– 1706 60 Sobieraj-Teague, M. et al. (2009) New anticoagulants for atrial fibrillation. Semin. Thromb. Hemost. 35, 515–524 61 Viskov, C. et al. (2009) Description of the chemical and pharmacological characteristics of a new hemisynthetic ultra-low-molecular-weight heparin, AVE5026. J. Thromb. Haemost. 7, 1143–1151 62 Hoppensteadt, D. et al. (2008) AVE5026: a new hemisynthetic ultra low molecular weight heparin (ULMWH) with enriched anti-Xa activity and enhanced antithrombotic activity for management of cancer associated thrombosis. J. Clin. Oncol. 26 (15 Suppl.), 14653 (Meeting Abstracts) 63 Lassen, M.R. et al. (2009) AVE5026, a new hemisynthetic ultra-low-molecularweight heparin for the prevention of venous thromboembolism in patients after

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total knee replacement surgery – TREK: a dose-ranging study. J. Thromb. Haemost. 7, 566–572 Melloni, C. et al. (2009) Design and rationale of the evaluation of M118 in percutaneous coronary intervention (EMINENCE) trial. Am. Heart J. 158, 726– 733 Kishimoto, T.K. et al. (2009) M118-A rationally engineered low-molecular-weight heparin designed specifically for the treatment of acute coronary syndromes. Thromb. Haemost. 102, 900–906 Draganov, D. et al. (2009) Pharmacokinetics of M118, unfractionated heparin and enoxaparin sodium in normal and 5/6 nephrectomized uremic rats. Toxicol. Lett. 189 (Suppl. 1), S113–S1113 Volovyk, Z. et al. (2009) A rationally designed heparin, M118, has anticoagulant activity similar to unfractionated heparin and different from Lovenox in a cellbased model of thrombin generation. J. Thromb. Thrombolysis 28, 132–139 Fier, I. et al. (2007) A novel, rationally engineered heparin (M118) prevents thrombosis more effectively than unfractionated heparin in a canine model of deep arterial injury. J. Am. Coll. Cardiol. 49, 379A–380A Chakrabarti, S. et al. (2009) M118, a novel low-molecular weight heparin with decreased polydispersity leads to enhanced anticoagulant activity and thrombotic occlusion in ApoE knockout mice. J. Thromb. Thrombolysis 28, 394–400 Fier, I.D. et al. (2009) Lack of pharmacokinetic and pharmacodynamic interactions between M118, a novel low-molecular-weight-heparin and Eptifibatide in healthy subjects. J. Clin. Pharmacol. 49, 73 Bal Dit Sollier, C. et al. (2009) Anticoagulant activities of EP42675 – synthetic direct inhibitor and indirect factor Xa inhibitor. In Proceedings of the XXII Congress of the International Society of Thrombosis and Haemostasis De Kort, M. and Van Boeckel, C.A.A. (2010) Antithrombotic Dual Inhibitors Comprising a Biotin Residue. N.V. ORGANON (Oss, NL) Bal Dit Sollier, C. et al. (2009) Pharmacokinetics and pharmacodynamics of EP42675 a new synthetic anticoagulant with a dual mechanism of action. In Proceedings of the XII congress of the International Society of Thrombosis and Haemostasis Ludwig, R.J. (2009) Therapeutic use of heparin beyond anticoagulation. Curr. Drug Discov. Technol. 6, 281–289 Young, E. (2008) The anti-inflammatory effects of heparin and related compounds. Thromb. Res. 122, 743–752 Borsig, L. (2010) Antimetastatic activities of heparins and modified heparins. Experimental evidence. Thromb. Res. 125 (Suppl. 2), S66–S71 McKenzie, E.A. (2007) Heparanase: a target for drug discovery in cancer and inflammation. Br. J. Pharmacol. 151, 1–14 Vlodavsky, I. and Friedmann, Y. (2001) Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis. J. Clin. Invest. 108, 341– 347 Miao, H.-Q. et al. (2006) Development of heparanase inhibitors for anti-cancer therapy. Curr. Med. Chem. 13, 2101–2111 Kudchadkar, R. et al. (2008) PI-88: a novel inhibitor of angiogenesis. Expert Opin. Investig. Drugs 17, 1769–1776 Ferro, V. and Don, R. (2003) The development of the novel angiogenesis inhibitor PI-88 as an anticancer drug. Australas. Biotechnol. 13, 38–39 Wall, D. et al. (2001) Characterisation of the anticoagulant properties of a range of structurally diverse sulfated oligosaccharides. Thromb. Res. 103, 325–335 Basche, M. et al. (2006) A phase I biological and pharmacologic study of the heparanase inhibitor PI-88 in patients with advanced solid tumors. Clin. Cancer Res. 12, 5471–5480 Rosenthal, M.A. et al. (2002) Treatment with the novel anti-angiogenic agent PI88 is associated with immune-mediated thrombocytopenia. Ann. Oncol. 13, 770– 776 Chow, L.Q. et al. (2008) A phase I pharmacological and biological study of PI-88 and docetaxel in patients with advanced malignancies. Cancer Chemother. Pharmacol. 63, 65–74 Millward, M. et al. (2007) Final results of a phase I study of daily PI-88 as a single agent and in combination with dacarbazine (D) in patients with metastatic melanoma. J. Clin. Oncol. 25 (Suppl. 18), 8532 (Meeting Abstracts) Khasraw, M. et al. (2009) Multicentre phase I/II study of PI-88, a heparanase inhibitor in combination with docetaxel in patients with metastatic castrateresistant prostate cancer. Ann. Oncol. 21, 1302–1307 Lewis, K.D. et al. (2008) A phase II study of the heparanase inhibitor PI-88 in patients with advanced melanoma. Invest. New Drugs 26, 89–94 Karoli, T. et al. (2005) Synthesis, biological activity, and preliminary pharmacokinetic evaluation of analogues of a phosphosulfomannan angiogenesis inhibitor (PI-88). J. Med. Chem. 48, 8229–8236 Ferro, V. et al. (2007) PI-88 and novel heparan sulfate mimetics inhibit angiogenesis. Semin. Thromb. Hemost. 33, 557–568

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91 Dredge, K. et al. (2009) The PG500 series: novel heparan sulfate mimetics as potent angiogenesis and heparanase inhibitors for cancer therapy. Invest. New Drugs 28, 276–283 92 Bytheway, I. et al. (2009) The dual angiogenesis/heparanase inhibitor PG545, but not the tyrosine kinase inhibitor sorafenib, inhibits spontaneous metastasis in models of breast and lung cancer. In Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics, Mol. Cancer Ther. (Meeting Abstract Supplement) 93 Hammond, E. et al. (2009) The dual angiogenesis/heparanase inhibitor PG545 inhibits solid tumor progression in models of breast, prostate and liver cancer: a comparative assessment of once versus twice weekly administration schedules. In Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics, Mol. Cancer Ther. (Meeting Abstract Supplement) 94 van Horssen, J. et al. (2003) Heparan sulphate proteoglycans in Alzheimer’s disease and amyloid-related disorders. Lancet Neurol. 2, 482–492 95 McLaurin, J.A. et al. (1999) Interactions of Alzheimer amyloid-b peptides with glycosaminoglycans. Eur. J. Biochem. 266, 1101–1110 96 Leveugle, B. et al. (1994) Binding of heparan sulfate glycosaminoglycan to [beta]amyloid peptide: inhibition by potentially therapeutic polysulfated compounds. Neuroreport 5, 1389–1392 97 Kisilevsky, R. et al. (1995) Arresting amyloidosis in vivo using small-molecule anionic sulphonates or sulphates: implications for Alzheimer’s disease. Nat. Med. 1, 143–148 98 Tremblay, P. et al. (2005) Functional GAG mimetics as an approach for the treatment of amyloid diseases. Alzheimers Dement. 1 (Suppl. 1), S2–S12 99 Aisen, P.S. et al. (2007) Alzhemed: a potential treatment for Alzheimers disease. Curr. Alzheimer Res. 4, 473–478 100 Geerts, H. (2004) NC-531 (Neurochem). Curr. Opin. Investig. Drugs 5, 95–100 101 Wright, T.M. (2006) Tramiprosate. Drugs Today (Barc) 42, 291–298 102 Gervais, F. et al. (2007) Targeting soluble Ab peptide with Tramiprosate for the treatment of brain amyloidosis. Neurobiol. Aging 28, 537–547 103 Santa-Maria, I. et al. (2007) Tramiprosate, a drug of potential interest for the treatment of Alzheimer’s disease, promotes an abnormal aggregation of tau. Mol. Neurodegener. 2, 17 104 Aisen, P.S. et al. (2006) A Phase II study targeting amyloid-b with 3APS in mild-tomoderate Alzheimer disease. Neurology 67, 1757–1763 105 Rafii, M.S. and Aisen, P. (2009) Recent developments in Alzheimer’s disease therapeutics. BMC Med. 7, 7 106 Saumier, D. et al. (2009) Lessons learned in the use of volumetric MRI in therapeutic trials in Alzheimer’s disease: the AlzhemedTM (Tramiprosate) experience. J. Nutr. Health Aging 13, 370–372 107 Neugroschl, J. and Sano, M. (2010) Current treatment and recent clinical research in Alzheimer’s disease. Mt. Sinai J. Med. 77, 3–16 108 Revill, P. et al. (2006) Eprodisate sodium. Drugs Future 31, 576–578 109 Ancsin, J.B. and Kisilevsky, R. (1999) The heparin/heparan sulfate-binding site on apo-serum Amyloid A. J. Biol. Chem. 274, 7172–7181 110 Gervais, F. et al. (2003) Proteoglycans and amyloidogenic proteins in peripheral amyloidosis. Curr. Med. Chem. Immunol. Endocr. Metab. Agents 3, 361–370 111 Kisilevsky, R. (2000) The relation of proteoglycans, serum amyloid P and Apo E to amyloidosis current status, 2000. Amyloid 7, 23–25 112 Clinicaltrials.gov. (2002) A phase II/III study of the safety and efficacy of NC-503 in patients suffering from secondary (AA) amyloidosis (NCT00035334). 113 Dember, L.M. et al. (2007) Eprodisate for the treatment of renal disease in AA amyloidosis. N. Engl. J. Med. 356, 2349–2360 114 Manenti, L. et al. (2008) Eprodisate in amyloid A amyloidosis: a novel therapeutic approach? Expert Opin. Pharmacother. 9, 2175–2180 115 Bellini, R. (2010) BELLUS Health Ends NC-503 Diabetes Development Program following Results. Bellus Health Inc. 116 Brown, J. et al. (2010) Small molecule inhibitors of glycosaminoglycan biosynthesis as substrate optimization therapy for the mucopolysaccharidoses. In Proceedings of the Lysosomal Disease Network WORLD Symposium, vol. 99 pp. S12– S112 117 Casu, B. et al. (2010) Heparin-derived heparan sulfate mimics to modulate heparan sulfate–protein interaction in inflammation and cancer. Matrix Biol. 29, 442–452 118 Vlodavsky, I. et al. (2007) Heparanase: structure, biological functions, and inhibition by heparin-derived mimetics of heparan sulfate. Curr. Pharm. Des. 13, 2057–2073 119 Mousa, S.A. et al. (2006) Anti-metastatic effect of a non-anticoagulant lowmolecular-weight heparin versus the standard low-molecular-weight heparin, enoxaparin. Thromb. Haemost. 96, 816–821 120 Lee, D.Y. et al. (2009) Antiangiogenic activity of orally absorbable heparin derivative in different types of cancer cells. Pharm. Res. 26, 2667–2676

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121 Cabannes, E. et al. (2009) Heparan sulfate mimetics as anticancer small-glyco drugs. In Proceedings of the 67th Harden Conference 122 Serina, G. et al. (2010) Antitumor activity of EP80061, a small-glyco drug in preclinical studies. In Proceedings of the AACR meeting 123 Zhou, H. et al. (2010) M402 – A Novel Heparan Sulfate Proteoglycan Mimetic Targeting Tumor–Host Interactions. American Association for Cancer Research (AACR) 124 Zhou, H. et al. (2009) M-ONC 402-a non anticoagulant low molecular weight heparin inhibits tumor metastasis. In Proceedings of the 100th Annual Meeting of American Association for Cancer Research (AACR) 125 Chu, C. et al. (2009) M-ONC 402, A novel non-anticoagulant heparin, inhibits PSelectin function and metastatic seeding of tumor cells in mice. In Proceedings of the100th Annual Meeting of American Association for Cancer Research (AACR) 126 Lolkema, M.P. et al. (2010) M402, A Novel Heparan Sulfate Mimetic, Synergizes with Gemcitabine to Improve Survival and Reduce Metastasis and Epithelial-to-mesenchymal Transition (EMT) in a Genetically Engineered Mouse Model for Pancreatic Cancer. American Association for Cancer Research (AACR) 127 Barritault, D. and Caruelle, J.P. (2006) Regenerating agents (RGTAs): a new therapeutic approach. Ann. Pharm. Fr. 64, 135–144 128 Tardieu, M. et al. (1992) Derivatized dextrans mimic heparin as stabilizers, potentiators, and protectors of acidic or basic FGF. J. Cell. Physiol. 150, 194–203 129 Meddahi, A. et al. (1996) Heparin-like polymers derived from dextran enhance colonic anastomosis resistance to leakage. J. Biomed. Mater. Res. 31, 293–297 130 Rouet, V. et al. (2005) A synthetic glycosaminoglycan mimetic minds vascular endothelial growth factor and modulates angiogenesis. J. Biol. Chem. 280, 32792– 32800 131 Meddahi, A. et al. (1996) FGF protection and inhibition of human neutrophil elastase by carboxymethyl benzylamide sulfonate dextran derivatives. Int. J. Biol. Macromol. 18, 141–145 132 Meddahi, A. et al. (1995) Inhibition by dextran derivatives of FGF-2 plasminmediated degradation. Biochimie 77, 703–706 133 Ledoux, D. et al. (2000) Human plasmin enzymatic activity is inhibited by chemically modified dextrans. J. Biol. Chem. 275, 29383–29390 134 Rouet, V. et al. (2006) Heparin-like synthetic polymers, named RGTAs, mimic biological effects of heparin in vitro. J. Biomed. Mater. Res. A 78, 792–797 135 Barbier-Chassefie`re, V. et al. (2009) Matrix therapy in regenerative medicine, a new approach to chronic wound healing. J. Biomed. Mater. Res. A 90, 641–647 136 Meddahi, A. et al. (1994) New approaches to tissue regeneration and repair. Pathol. Res. Pract. 190, 923–928 137 Albo, D. et al. (1996) Modulation of cranial bone healing with a heparin-like dextran derivative. J. Craniofac. Surg. 7, 19–22 138 Blanquaert, F. et al. (1995) Heparan-like molecules induce the repair of skull defects. Bone 17, 499–506 139 Yamauchi, H. et al. (2000) New agents for the treatment of infarcted myocardium. FASEB J. 14, 2133–2134 140 Meddahi, A. et al. (2002) Heparin-like polymer improved healing of gastric and colic ulceration. J. Biomed. Mater. Res. 60, 497–501 141 Escartin, Q. et al. (2003) A new approach to treat tissue destruction in periodontitis with chemically modified dextran polymers. FASEB J. 17, 644–651 142 Papy-Garcia, D. et al. (2002) Glycosaminoglycan mimetics (RGTA) modulate adult skeletal muscle satellite cell proliferation in vitro. J. Biomed. Mater. Res. 62, 46–55 143 Schonberger, O. et al. (2003) Novel heparan mimetics potently inhibit the scrapie prion protein and its endocytosis. Biochem. Biophys. Res. Commun. 312, 473–479 144 Adjou, K.T. et al. (2003) A novel generation of heparan sulfate mimetics for the treatment of prion diseases. J. Gen. Virol. 84, 2595–2603 145 Barbosa, I. et al. (2005) A synthetic glycosaminoglycan mimetic (RGTA) modifies natural glycosaminoglycan species during myogenesis. J. Cell Sci. 118, 253–264 146 Aamiri, A. et al. (1995) Effect of a substituted dextran on reinnervation during regeneration of adult rat skeletal muscle. C. R. Acad. Sci. III. Sci. Vie 318, 1037–1044 147 Papy-Garcia, D. et al. (2005) Nondegradative sulfation of polysaccharides. Synthesis and structure characterization of biologically active heparan sulfate mimetics. Macromolecules 38, 4647–4654 148 Martelly, I. et al. (2010) Glycosaminoglycan mimetics trigger IP3-dependent intracellular calcium release in myoblasts. Matrix Biol. 29, 317–329 149 Zuijdendorp, H.M. et al. (2008) Significant reduction in neural adhesions after administration of the regenerating agent OTR4120, a synthetic glycosaminoglycan mimetic, after peripheral nerve injury in rats. J. Neurosurg. 109, 967–973 150 Garcia-Filipe, S. et al. (2007) RGTA OTR4120, a heparan sulfate mimetic, is a possible long-term active agent to heal burned skin. J. Biomed. Mater. Res. A 80, 75– 84 151 Tong, M. et al. (2009) Stimulated neovascularization, inflammation resolution and collagen maturation in healing rat cutaneous wounds by a heparan sulfate glycosaminoglycan mimetic, OTR4120. Wound Repair Regen. 17, 840–852

152 Meddahi, A. et al. (2002) Pharmacological studies of RGTA11, a heparan sulfate mimetic polymer, efficient on muscle regeneration. J. Biomed. Mater. Res. 62, 525–531 153 Chebbi, C.K. et al. (2008) Pilot study of a new matrix therapy agent (RGTA OTR41201) in treatment-resistant corneal ulcers and corneal dystrophy. J. Fr. Opthalmol. 31, 465–471 154 Jeske, W. et al. (2007) In Vitro Characterization of the Neutralization of Unfractionated Heparin and Low Molecular Weight Heparin by Novel Salicylamide Derivatives. American Society of Hematology 155 Kuziej, J. et al. (2009) Neutralization of hemorrhagic and antithrombotic activities of heparins by a novel salicylamide derivative. FASEB J. 23, 566–569 (Meeting Abstracts 1)

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156 Fareed, J. et al. (2008) Neutralization of the anticoagulant and anti-Xa effects of fondaparinux and idraparinux by a novel synthetic antagonist. Pharmacologic implications. FASEB J. 22, 1117–1118 (Meeting Abstracts 1) 157 Jeske, W. et al. (2009) Novel Antagonists for Low Molecular Weight Heparin and Heparin-like Drugs. American Society of Hematology 158 Code´e, J.D.C. et al. (2004) The synthesis of well-defined heparin and heparan sulfate fragments. Drug Discov Today: Technol. 1, 317–326 159 Brown, J. et al. (2010) Small molecule inhibitors of glycosaminoglycan biosynthesisas substrate optimization therapy for the mucopolysaccharidoses. Mol. Genet. Metab. 99, S12–S112

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Metal-based drugs for malaria, trypanosomiasis and leishmaniasis: recent achievements and perspectives Maribel Navarro1, Chiara Gabbiani2, Luigi Messori2, and Dinorah Gambino3 1

Centro de Quı´mica, Instituto Venezolano de Investigaciones Cientı´ficas, Caracas, Venezuela Laboratory of Metals in Medicine, MetMed, Department of Chemistry ‘Ugo Schiff’, University of Florence, Florence, Italy 3 Ca´tedra de Quı´mica Inorga´nica, Facultad de Quı´mica, Universidad de la Repu´blica, Montevideo, Uruguay 2

Tropical diseases today constitute a major health problem and a big challenge for drug discovery. Because of the limited arsenal of effective antiparasitic agents and the frequent appearance of chemoresistance, there is an urgent and continuous need to develop new drugs against these ailments. Metal compounds still offer excellent opportunities to find new ‘leads’ against the major protozoan diseases such as malaria, leishmaniasis and trypanosomiasis. A few metal-based drugs are already available in this therapeutic area, and others are currently being developed. Recent progress in parasite genomics and the identification of a few biomolecular targets hold great promise for the discovery of new ‘mechanism-based’ antiparasitic metallodrugs. The trends and perspectives for this exciting research field are outlined here.

Introduction Undoubtedly, tropical diseases today are a major health problem. They affect more than two billion people worldwide (about onethird of the total population) and cause nearly two million deaths per year, mostly in the poorest areas of the planet. The wide diffusion of these diseases, the small number of effective drugs and the frequent emergence of resistance make drug discovery in this therapeutic area a very urgent and challenging task. Until recently, low financial returns greatly discouraged big pharmaceutical companies from investing in drug discovery programs against tropical diseases. Over the past decade, however, this gap has (fortunately) started to reduce considerably as several international non-profit organizations have intervened heavily and efficaciously to sustain tropical disease research, paying specific attention to the clinical development of new chemotherapeutics. Among the several known tropical diseases, we will restrict our attention here to three illnesses with protozoa as their causative agent: malaria, trypanosomiasis and leishmaniasis. These three diseases are among the major tropical diseases characterized by very high morbidity and lethality and by a widespread diffusion across the world. Corresponding author:. Messori, L. ([email protected])

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This review article specifically aims to consider the several opportunities that metal-based compounds – still a relatively unexplored area of modern medicinal chemistry – might offer today as a rich source of effective chemotherapeutics against the major protozoan diseases. Remarkably, metal-based drugs have had a major role as therapeutic agents in the ancient history of medicine and in the pioneering times of modern pharmacology, especially as antiinfective agents. Afterward, they were largely abandoned because of (partially grounded) concerns about their systemic toxicity and because of the rapid advent of modern (organic) medicinal chemistry. The most notable exceptions to this trend were the use of platinum-based drugs in cancer chemotherapy after the introduction in the clinics of cisplatin (Fig. 1a) and a few success stories (e.g. auranofin; Fig. 1b) in the treatment of rheumatoid arthritis. During the past 25 years, however, this situation has changed substantially. There has been a strong resurgence of interest in metal-based drugs with potential applications in a variety of therapeutic areas, especially within the academic bioinorganic community. It has been recognized that metal compounds provide a great variety of different and peculiar chemistries that the reactivity of the metal centers toward target biomolecules can be finely tuned through an appropriate choice of metal ligands, and

1359-6446/06/$ - see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2010.10.005

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Two representative metallodrugs: (a) cisplatin and (b) auranofin.

that systemic toxic effects can be effectively countered by specific strategies (even in terms of pharmaceutical technology). Several different metal-based compounds were thus investigated, with a renewed interest, especially as anticancer agents, and some of them provided excellent results. Such a return of interest in metal-based drugs can be witnessed in several recent review articles [1–3]. Metal compounds seem to be particularly suitable for the treatment of tropical parasitic diseases because they can show a pronounced selectivity for selected parasites’ biomolecules compared to the host’s biomolecules. The application of metal compounds against parasitic diseases is basically grounded on the following considerations: (i) Metal compounds have already found extensive application in the treatment of parasitic diseases in the pioneering times of modern pharmacology, mostly based on an empirical use. Various inorganic salts were thus administered against the major tropical diseases, sometimes with very good results. Notably, as a consequence of those ancient observations, a few antimony compounds still constitute the treatment of choice for some forms of leishmaniasis. Bismuth is still used sporadically in the prophylaxis of malaria. By contrast, arsenicals, although effective, were withdrawn completely because of toxicity. However, no detailed structure/function studies were ever performed on antiparasitic metal-based compounds. These arguments open the way to new mechanistic investigations in this research area for the optimization of the identified metal leads. In particular, antimony and bismuth compounds, already in clinical use, might be further improved through the modern technologies of synthetic inorganic and coordination chemistry, upon concomitant evaluation of their respective biological profiles. Similar metal-based compounds might be developed – showing, we hope, better biological profiles. (ii) Metals might be a part of novel antiparasitic agents based on the strategies mentioned below, well documented by a few relevant examples.
The insertion of a metal center into the scaffold of an established antiparasitic drug to enhance its pharmacological actions, for instance through redox cycling (e.g. ferroquine).
The metal complexation of classical antiparasitic drugs to modulate activity and pharmacokinetic parameters (e.g. gold–chloroquine and ruthenium–benznidazole).
The use of stable metal complexes as antiparasitic agents per se (e.g. metalloporphyrins).

(iii) New antiparasitic metal compounds could be developed as prodrugs in analogy with metal-based anticancer agents. Indeed, a strict similarity was postulated between parasite and cancer biology. Typically, these substances are able to deliver highly reactive metal-containing molecular fragments (e.g. platinum compounds and ruthenium compounds) capable of hitting parasite targets. The use of metal-containing substances for the treatment of protozoan diseases was extensively described by Sa´nchez-Delgado et al. [4,5] a few years ago in a couple of comprehensive review articles. Here, we illustrate the state of the art today and report some major achievements that have occurred during the past few years. Remarkably, the knowledge of the biology of these parasites in our postgenomic era has greatly increased, and several protein targets have been identified against which novel metal compounds might be specifically developed and tested. These advancements could have an important impact on the general strategy for the discovery and development of metal-based antiparasitic agents. Whereas in the past the screening of novel compounds was usually based on the assessment of efficacy against cultured protozoa in vitro (simply by measuring inhibition of parasite growth), there is now the opportunity to screen and select the active compounds through specific in vitro assays directed against a selected biomolecular target. In the following paragraphs, we briefly describe the state of knowledge in the application of metal-based compounds against malaria, trypanosomiasis and leishmaniasis with an emphasis on the most representative and recent compounds (2005–2010). Afterward, attention is paid to a few parasitic targets for which metal-based inhibitors might be developed. In a few cases, structural information on the actual metal–target interactions is already available. The expected future trends for this fascinating research area are outlined.

Metal-based drugs for malaria Malaria is a major cause of morbidity and death in children and adults in tropical countries. Currently, half of the world’s population is at risk of malaria. An estimated 243 million malarial cases and 863,000 malarial deaths occurred in 2008, 767,000 of which were in Africa (http://www.who.int/research/en/). The fight against malaria is largely based on chemotherapy. Historically, several organic compounds have been used as antimalarial drugs, including quinine, chloroquine, hydroxychloroquine, mefloquine, primaquine, proguanil, cotrifazid, doxycycline, sulfadoxine, pyrimethamine, artemether, lumefantrine, artesunate and amodiaquine (the most important are shown in Fig. 2). Current malaria treatments are still based on the combination of two or three of these drugs [6]. Chloroquine (CQ) represents one of the most successful drugs ever used to treat an infectious disease; it was considered a ‘wonder drug’ because of its low cost, high efficacy and lack of significant side-effects. After twenty years of successful use, however, CQresistant malarial parasites started to emerge and spread from Asia to Africa and South America. The loss of activity of CQ as a first-line antimalarial drug is a major setback to the control of malaria; fortunately, effective artemisinin-based drugs are quickly replacing CQ, but the risk of insurgence of resistance even against new endoperoxide antimalarials is very high. www.drugdiscoverytoday.com

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Chemical structure of the main antimalarial drugs used in therapy: (a) artemisinin, (b) chloroquine, (c) quinine and (d) mefloquine.

Today, the available therapeutic tools for malaria are limited: new anti-malarial drugs, preferably with new structures and/or modes of action, are urgently needed. Metal–drug synergism has been exploited to obtain effective antimalarial metal agents [4,7,8]. Remarkable success came from the modification of CQ through metal-containing fragments, a strategy intensely pursued by Sa´nchez-Delgado et al. Several metal complexes were thus synthesized with encouraging antimalarial activities [8–10]. Among them, ruthenium(II) chloroquine [RuCQCl2]2 (Fig. 3a) and gold(I) chloroquine [Au(PPh3)(CQ)]PF6 (Fig. 3b) cause marked inhibition of Plasmodium berghei and are very effective against CQresistant FcB1 and FcB2 Plasmodium falciparum strains. Typically, ruthenium and gold coordination to CQ resulted in a significant [()TD$FIG]

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enhancement of the parental drug against resistant parasites. Upon pre-incubating uninfected red blood cells with [Au(PPh3)(CQ)]PF6, protection against subsequent infection was also afforded [8]. Iridium–CQ derivatives showed activity against rodent malaria [9]. Recently, a series of organo-Ru(II)-CQ complexes were synthesized and tested against several P. falciparum strains (Fig. 4); they displayed higher activities against resistant parasites than chloroquine diphosphate [10]. Thus, metal complexation of existing antimalarial drugs represents an effective option for drug improvement that merits further exploration. In particular, ruthenium(II) complexation offers the chance of contrasting effectively some of the biochemical mechanisms responsible for CQ resistance, as highlighted by Martinez et al.

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Structures of metal derivatives of chloroquine with antimalarial properties: (a) ruthenium chloroquine [RuCQCl2]2, (b) gold chloroquine [Au(PPh3)(CQ)]PF6 and (c) ferroquine. 1072

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[11,12] in two recent papers. They found that Ru–CQ complex binds to hematin in solution and inhibits aggregation to b-hematin at
Metal-based drugs for trypanosomiasis American trypanosomiasis (Chagas disease) and human African trypanosomiasis (sleeping sickness) are important health problems of the poorest tropical and subtropical regions of the planet [19– 22]. American trypanosomiasis is endemic throughout Latin America, infecting 8–14 million people and causing more deaths (14,000 per year) in this region than any other parasitic disease.

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Drugs used in American trypanosomiasis: (a) nifurtimox and (b) benznidazole.

After being eradicated in the 1960s, human African trypanosomiasis is now a resurgent disease with epidemic character in many regions of Africa [20]. Trypanosoma cruzi and Trypanosoma brucei, the etiologic agents of American trypanosomiasis and human African trypanosomiasis, respectively, are protozoan parasites that belong to the trypanosomatid genus and the kinetoplastida order. Both are transmitted to the mammalian host by insects: T. brucei by the tsetse fly through saliva and T. cruzi by hematophagus triatomine bugs through the insect feces [4,5,23]. Chemotherapy for trypanosomatid infections mostly relies on drugs that are more than 50 years old and suffer from poor efficacy, high toxicity and increasing resistance. Treatment of American trypanosomiasis is based on nifurtimox and benznidazole (Fig. 5), two unspecific nitroheterocyclic drugs that show significant activity only in the acute phase of the disease [21–24]. Conversely, the principal drugs for human African trypanosomiasis, namely pentamidine, suramin, eflornithine and melarsoprol, suffer from severe toxicity, are not universally active and have already generated conspicuous levels of resistance [20,25]. Several attempts are under way to develop new trypanocidal metal-based compounds [25–27], mostly inspired by the three following strategies: the metal coordination of trypanocidal ligands, the metal coordination of DNA intercalators and metal compounds as direct inhibitors of parasite enzymes. Examples of these approaches are given below (Figs. 6 and 7).

Metal complexes of trypanocidal ligands The design of antiparasitic compounds by combining anti-trypanosomal ligands with pharmacologically active metals follows the concept of developing new chemical entities as dual inhibitors, capable of affecting multiple targets simultaneously. Indeed, single agents acting against multiple parasitic targets might diminish host toxicity. Pioneering work by Sa´nchez-Delgado et al. [4,5] led to the discovery of metal complexes of clotrimazole and ketoconazole intended for anti-trypanosome therapy. More recently, Gambino et al. developed several ruthenium, platinum and pallawww.drugdiscoverytoday.com

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[()TD$FIG]

(a)

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FIGURE 6

A few representative metal-based drugs for trypanosomiasis: (a) metal compounds of 5-nitrofuryl-derived thiosemicarbazones. (b) 2-Mercaptopyridine N-oxide metal complexes.

dium complexes of bioactive 5-nitrofuryl and 5-nitroacroleine containing thiosemicarbazones. These ligands had shown higher in vitro activity against T. cruzi than nifurtimox based on a similar mechanism. In turn, platinum, palladium and ruthenium com[()TD$FIG]pounds had proven and conspicuous antitumor effects caused by

their ability to bind DNA. Coordination of selected bioactive ligands to these metals seemed interesting because of the postulated metabolic similarities between tumor cells and T. cruzi cells. As a proof of concept, in vitro evaluation of T. cruzi epimastigotes showed that many of these Pt(II) and Pd(II) complexes were more

FIGURE 7

Drugs used for the treatment of leishmaniasis: (a) glucantine, (b) pentostam, (c) amphotericin B, (d) paromomycin and (e) miltefosine. 1074

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active than nifurtimox and the corresponding free ligand L [28– 30], holding great promise for future drug development. A similar situation was found in the case of 2-mercaptopyridine N-oxide (mpo). Turrens et al. demonstrated several years ago that mpo can block T. cruzi growth through inhibition of NADH– fumarate reductase affecting all stages of a parasite’s cycle without affecting mammalian cells. Remarkably, [Au2(mpo-H)2(PPh3)2], [Pt(mpo-H)2], [Pd(mpo-H)2] and [VO(mpo-H)2] showed significantly increased activities compared to mpo on epimastigotes of different T. cruzi strains. In particular, Pt(II), Au(I) and Pd(II) complexes were 39-, 67- and 115-fold more active, respectively, than nifurtimox. In turn, NADH–fumarate reductase inhibition studies showed a clear correlation between parasite inhibition and enzyme inhibition, highlighting NADH–fumarate reductase as the probable main target of these complexes [31,32].

Metal complexes of DNA intercalators Compounds that efficiently interact with DNA through intercalation, beyond being potent antitumor agents, might show significant antitrypanosomal activity [33]. With this in mind, some homoleptic and heteroleptic vanadyl complexes with DNA intercalators as ligands (dppz = dipyrido[3,2-a:20 ,30 -c]phenazine and bipy = 2,2´-bipyridine) were designed and tested as potential antitrypanosomal agents. The homoleptic vanadyl complex [VIVO(SO4)(H2O)2(dppz)]2H2O showed slightly better in vitro activity than nifurtimox on T. cruzi Dm28c strain epimastigotes [34]. Mixed-ligand vanadyl complexes, [VIVO(L2-2H)(L1)], including the bidentate polypyridyl DNA intercalator (L1) and a tridentate salycylaldehyde semicarbazone derivative (L2) as ligands were also designed. Complexes including dppz as coligand showed IC50 values in the mM range against the Dm28c strain of T. cruzi, being as active as nifurtimox [35].

Metal complexes as metal inhibitors of parasite enzymes Enzyme inhibition is one of the main modes of action of inorganic drugs. Indeed, metal ions might coordinate crucial active-site residues, thus blocking enzyme interaction with substrate, or might coordinate to external residues modifying enzyme structure. According to this strategy, many efforts were made to design metal coordination compounds that could specifically bind parasitic enzymes. Cruzipain, the major cysteine protease found in T. cruzi, is a validated target for the development of chemotherapeutics against American trypanosomiasis. Cruzipain inhibitory effects of some Pd(II) and Au(III) cyclometallated complexes and Re(V) complexes were initially explored with excellent results [27].

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with sodium stibogluconate (pentostam) and meglumine antimoniate (glucantine) as the most representative drugs. This is a clear example of clinically established metal-based drugs against a parasitic disease. Although effective, these antimonials can cause severe sideeffects such as cardiotoxicity, reversible renal insufficiency, pancreatitis, anemia, leucopenia, and others. When antimonials fail, amphotericin B is the recommended second-line treatment for visceral, cutaneous and mucocutaneous leishmaniasis. Aromatic diamidine pentamidine is a traditional alternative to pentavalent antimony. Recently, miltefosine was introduced as the first oral treatment for visceral leishmaniasis [36–38]. Another group of antileishmanial compounds are the sterol biosynthesis inhibitors, including terbinafin, imidazole derivatives (ketoconazole and clotrimazole), triazoles (fluconazole and itraconazole) and azasterols. With the exception of antimonials, the use of metal-containing drugs as antileishmanial agents (according to the metal–drug synergism concept) was scarcely explored and warrants further studies. Indeed, the appearance of drug-resistant strains of Leishmania spp. justifies intense screening of new compounds in parallel with deeper investigations of Leishmania biology to identify possible targets for rational drug design. In any case, the studies reported so far on metal compounds offer valuable hints for further research efforts. Simple salts such as zinc sulfate were tested clinically against cutaneous leishmaniasis with very promising cure rates (>96.0%), using oral doses of 10 mg/kg for 45 days [39]. Several years ago, a DNA metallointercalator (2,20 :60 200 -terpyridine)platinum (II) showed remarkable antileishmanial activity, the most effective compound causing complete growth inhibition of Leishmania donovani amastigotes, at 1 mM concentration [40]. This complex exploits simultaneous DNA intercalation of terpyridine and platinum(II) binding to the enzyme active site. As the metabolic pathways of kinetoplastid parasites are similar to those of tumor cells [33], Navarro’s group designed a group of metallointercalators showing significant antileishmanial activity, probably arising from DNA intercalation [41]. Another interesting approach was proposed by Fricker et al. [27], who developed various gold(III), palladium(II) and rhenium(V) cyclometallated complexes as potential inhibitors of different cysteine proteases in the search for metal-based drugs suitable not only for American trypanosomiasis but also for leishmaniasis [18,27].

Emerging druggable targets for metal compounds Metal-based drugs for leishmaniasis Leishmaniasis is a disease with extensive morbidity and mortality. Its various forms are caused by protozoa of the genus Leishmania and range from self-healing cutaneous leishmaniasis to progressive mucocutaneous infections and fatal disseminating visceral leishmaniasis. Leishmaniasis currently affects some 12 million people worldwide with two million new cases per year. Approximately 350 million people live at risk of infection with Leishmania parasites; leishmaniases are prevalent in 88 countries, including 72 developing countries (http://www.who.int/emc). The available treatments for leishmaniases are far from ideal. The classic first-line treatment relies on pentavalent antimonials

The recent spectacular advancements in molecular biology and genomics have greatly expanded our understanding of parasite biology. Owing to such progress, a few parasite targets that are likely to be very sensitive to metal-based compounds have already been identified; some of the identified proteins indeed bear groups such as free thiols at their active sites that manifest a high propensity to react with ‘soft’ metals and are thus amenable to strong and selective inhibition by metallodrugs. A few examples will be described below. Substantial work carried out by Krauth-Siegel et al. [42,43] revealed that dithiol reductases have crucial roles in the overall redox metabolism of parasites. Thioredoxin reductase in plasmowww.drugdiscoverytoday.com

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Crystal structure of the complex of trypanothione reductase (TR) from Leishmania infantum with NADPH and Sb(III) [44].

dia and trypanothione reductase in Trypanosoma and Leishmania are excellent examples of this kind of protein: because both proteins contain active-site thiol groups, they constitute primary druggable targets for metal compounds. Indeed, it is well known

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FIGURE 9

Two cysteine proteases as emerging parasite targets: (a) falcipain and (b) cruzipain has a dotted background. 1076

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that parasites are particularly sensitive to oxidative stress arising from dithiol reductase inhibition. Within this frame, Colotti et al. recently reported the crystal structure for Leishmania trypanothione reductase disclosing the actual mechanism of enzyme inhibition by antimonials. It was shown that trivalent antimony binds to the protein active site with high affinity, strongly inhibiting enzyme activity [44]. The metal binds directly to Cys52, Cys57, Thr335 and His461, thereby blocking hydride transfer and trypanothione reduction. Details of the structure are shown in Fig. 8. The observed Sb–protein interaction is consistent with the usual modalities of cysteine binding of thiophilic metals such as As(III), Sb(III) and Bi(III). Such metaldependent inhibition of thiol reductases opens the way to combined metal therapy of leishmaniasis. It is very likely that this enzyme is similarly inhibited by other classes of metal complexes that are Lewis soft acids. Pairwise, thioredoxin reductase is a crucial redox enzyme of plasmodia. It contains an active-site dithiol group [45]. This observation implies that thiophylic metals might act as potent enzyme inhibitors; however, because the mammalian enzyme contains pairwise an active-site selenol group and is very susceptible to inhibition by metal compounds (see, for instance, the several articles by the group of Berners Price [46–49]), extreme caution must be taken in developing metal-based thioredoxin reductase inhibitors capable of differential enzyme inhibition. Cysteine proteases constitute another family of emerging parasite targets [50–52]. Fricker et al. proposed that metal-containing compounds might be developed as antiparasitic agents upon evaluating their ability to inhibit typical cysteine proteases [27]. Parasites typically contain a few important cysteine proteases, the strong inhibition of which might lead to the parasite’s death. Important examples are given by falcipain and cruzipain. Because both these enzymes bear a thiol group at their active site, they are usually susceptible to inhibition by metal compounds (Fig. 9).

Concluding remarks and future perspectives Metal-based compounds were largely neglected by the pharmaceutical industry and the medicinal chemistry community for a

long time; thus, several good opportunities for new drug discovery were probably lost. Many recent studies, however, mainly carried out in the fields of experimental oncology and bioinorganic chemistry have revealed that metal centers can be exploited pharmacologically to obtain innovative drugs, in different therapeutic areas. Metal-based drugs offer excellent opportunities for discovering new antiparasitic agents for which there is currently a great need. In particular, we have focused our attention on the major protozoan diseases of the tropical and subtropical areas. Notably, some metal compounds are already in clinical use against these diseases, whereas others are undergoing advanced clinical evaluation (e.g. FQ is now in phase II trials). Nonetheless, there are still great and unexplored opportunities in this research field for further metal-based drug discovery. In this short review, we have considered several success stories in which metal centers have been incorporated into antiparasitic agents and the underlying strategies and rationale. From these stories, it has clearly emerged that the discovery of metal-based compounds as potential antiparasitic drugs has relied on various approaches over the course of time; analysis of these various approaches enables the depiction of some future trends in this research area. In the past, the use of metal-based compounds was mainly driven by empiricism and by sporadic medical observations. Nonetheless, these activities and observations led to the discovery of important antiparasitic actions for a few metal compounds, such as bismuth in malaria prophylaxis and antimonials for leishmaniasis. In more recent times, starting from the 1980s, the design

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and discovery of metal-based antiparasitic agents were mainly governed by progress in coordination chemistry and its applications in medicine. Several metal-based compounds bearing antiparasitic activity were thus developed according to the strategies illustrated above. In the past decade, however, coinciding with the advent of the so-called ‘postgenomic era’, the discovery of new metal-based agents seems to have been mostly driven by progress in the knowledge of parasite biology and by the identification of parasite targets susceptible to metal inhibition. Selected examples have been presented, such as dithiol reductases involved in parasite redox metabolism and a few parasite proteases. The availability of these parasite proteins in purified form might enable targeted screening in vitro of the best metal-based drug candidates out of large libraries. In turn, the knowledge of the molecular structure of the target will enable the rational design of novel metal-based antiparasitic agents according to in silico docking methods. These advanced strategies hold much promise for obtaining innovative metal-based compounds and for the optimization of known leads. In conclusion, through this rapid excursus, we have tried to highlight the great potential and the real opportunities that metalbased drugs still offer today to the discovery and development of new antiparasitic agents.

Acknowledgements L.M. and C.G. thank Toscana Life Sciences foundation for generous financial support.

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14 Dive, D. and Biot, C. (2008) Ferrocene conjugates of chloroquine and other antimalarials: the development of ferroquine, a new antimalarial. ChemMedChem 3, 383–391 15 Sannella, A.R. et al. (2008) New uses for old drugs. Auranofin, a clinically established antiarthritic metallodrug, exhibits potent antimalarial effects in vitro: mechanistic and pharmacological implications. FEBS Lett. 582, 844–847 16 Messori, L. et al. (2009) Outstanding plasmodicidal properties within a small panel of metallic compounds: hints for the development of new metal-based antimalarials. J. Inorg. Biochem. 103, 310–312 17 Andricopulo, A.D. et al. (2006) Specific inhibitors of Plasmodium falciparum thioredoxin reductase as potential antimalarial agents. Bioorg. Med. Chem. Lett. 16, 2283–2292 18 Rosenthal, P.J. (2004) Cysteine proteases of malaria parasites. Int. J. Parasitol. 34, 1489–1499 19 Hotez, P.J. et al. (2007) Control of neglected tropical diseases. N. Engl. J. Med. 357, 1018–1027 20 Delespaux, V. and de Koning, H.P. (2007) Drugs and drug resistance in African trypanosomiasis. Drug Resist. Updat. 10, 30–50 21 Ribeiro, I. et al. (2009) New improved treatments for Chagas disease: from the R&D pipeline to the patients. PLoS Negl. Trop. Dis. 3, e484 22 Urbina, J. (2003) New chemotherapeutic approaches for the treatment of Chagas disease. Exp. Opin. Ther. Patents 13, 661–669 23 Maya, J.D. et al. (2007) Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their interaction with the mammalian host. Comp. Biochem. Physiol. A 146, 601–620 24 Croft, S.L. et al. (2005) Chemotherapy of trypanosomiases and leishmaniasis. Trends Parasitol. 21, 508–512 25 Cavalli, A. and Bolognesi, M.L. (2009) Neglected tropical diseases: multi-targetdirected ligands in the search for novel lead candidates against Trypanosoma and Leishmania. J. Med. Chem. 52, 7339–7359 26 Moreira, D.R. et al. (2009) Approaches for the development of new anti-Trypanosoma cruzi agents. Curr. Drug Targets 10, 212–231

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27 Fricker, S.P. et al. (2008) Metal compounds for the treatment of parasitic diseases. J. Inorg. Biochem. 102, 1839–1845 28 Otero, L. et al. (2006) Novel antitrypanosomal agents based on palladium nitrofurylthiosemicarbazone complexes: DNA and redox metabolism as potential therapeutic targets. J. Med. Chem. 49, 3322–3331 29 Vieites, M. et al. (2008) Platinum(II) metal complexes as potential anti-Trypanosoma cruzi agents. J. Inorg. Biochem. 102, 1033–1043 30 Vieites, M. et al. (2009) Platinum-based complexes of bioactive 3-(5nitrofuryl)acroleine thiosemicarbazones showing anti-Trypanosoma cruzi activity. J. Inorg. Biochem. 103, 411–418 31 Vieites, M. et al. (2008) Potent in vitro anti-Trypanosoma cruzi activity of pyridine-2thiol N-oxide metal complexes having inhibitory effect on parasite-specific fumarate reductase. J. Biol. Inorg. Chem. 13, 723–735 32 Vieites, M. et al. (2009) Synthesis and characterization of a pyridine-2-thiol N-oxide gold(I) complex with potent antiproliferative effect against Trypanosoma cruzi and Leishmania sp. Insight into its mechanism of action. J. Inorg. Biochem. 103, 1300–1306 33 Kinnamon, K.E. et al. (1979) Activity of antitumor drugs against African trypanosomes. Antimicrob. Agents Chemother. 15, 157–160 34 Benı´tez, J. et al. (2009) A novel vanadyl complex with a polypyridyl DNA intercalator as ligand: a potential anti-protozoa and anti tumor agent. J. Inorg. Biochem. 103, 1386–1394 35 Benı´tez, J. et al. (2009) Design of vanadium mixed–ligand complexes as potential anti-protozoa agents. J. Inorg. Biochem. 103, 609–616 36 Blum, J. et al. (2004) Treatment of cutaneous leishmaniasis among travellers. J. Antimicrob. Chemother. 53, 158–166 37 Le Pape, P. (2008) Development of new antileishmanial drugs – current knowledge and future prospects. J. Enzyme Inhib. Med. Chem. 23, 708–718 38 Mishra, J. et al. (2007) Chemotherapy of leishmaniasis: past, present and future. Curr. Med. Chem. 14, 1153–1169 39 Minodier, P. and Parola, P. (2007) Cutaneous leishmaniasis treatment. Travel Med. Infect. Dis. 5, 150–158

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40 Lowe, G. et al. (1999) Cytotoxicity of (2,20 :6,20 0 -terpyridine) platinum (II) complexes to Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. J. Med. Chem. 42, 999–1006 41 Navarro, M. et al. (2007) Synthesis and characterization of Au(dppz)2Cl3. DNA interaction studies and biological activity against Leishmania (L) mexicana. J. Inorg. Biochem. 101, 111–116 42 Krauth-Siegel, R.L. et al. (2005) Dithiol proteins as guardians of the intracellular redox milieu in parasites: old and new drug targets in trypanosomes and malariacausing plasmodia. Angew. Chem. Int. Ed. Engl. 44, 690–715 43 Krauth-Siegel, R.L. and Comini, M.A. (2008) Redox control in trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism. Biochim. Biophys. Acta 1780, 1236–1248 44 Baiocco, P. et al. (2009) Molecular basis of antimony treatment in leishmaniasis. J. Med. Chem. 52, 2603–2612 45 Nickel, C. et al. (2006) Thioredoxin networks in the malarial parasite Plasmodium falciparum. Antioxid. Redox Signal. 8, 1227–1239 46 Hickey, J.L. et al. (2008)Mitochondria-targeted chemotherapeutics: the rational design of gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols. J. Am. Chem. Soc. 130, 12570–12571 47 Berners-Price, S.J. and Filipovska, A. (2008) The design of gold-based, mitochondriatargeted chemotherapeutics. Aus. J. Chem. 61, 661–668 48 Barnard, P.J. and Berners-Price, S.J. (2007) Targeting the mitochondrial cell death pathway with gold compounds. Coord. Chem. Rev. 251, 1889–1902 49 Barnard, P.J. et al. (2004) Mitochondrial permeability transition induced by dinuclear gold(I)–carbene complexes: potential new antimitochondrial antitumour agents. J. Inorg. Biochem. 98, 1642–1647 50 Sajid, M. and McKerrow, J.H. (2002) Cysteine proteases of parasitic organisms. Mol. Biochem. Parasitol. 120, 1–21 51 McKerrow, J.H. et al. (1999) Cysteine protease inhibitors as chemotherapy for parasitic infections. Bioorg. Med. Chem. 7, 639–644 52 Mottram, J.C. et al. (2004) Cysteine peptidases as virulence factors of Leishmania. Curr. Opin. Microbiol. 7, 375–381

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monitor Cellular Delivery of Therapeutic Macromolecules (CDTM) International Symposium 2010: lessons and progress from interdisciplinary science

Our understanding of disease processes is rapidly increasing and an unprecedented number of macromolecular entities, including biopolymers such as nucleotides, peptides and proteins as well as synthetic polymers, are under investigation as therapeutic agents. The effective delivery of many of these therapeutic macromolecules to their target is constrained by their interaction with biological barriers, be this a feature of the macromolecule absorptive or dispositional processes, or indeed the need for the macromolecule to reach an intracellular target. Challenges faced in the effective delivery of macromolecule therapeutics to tissues, cells and subcellular compartments are considerable. The science underpinning the basic mechanisms of macromolecule interactions with biological barriers, through to the clinical translation of such entities into therapeutic agents serves as the focus of the Cellular Delivery of Therapeutic Macromolecules (CDTM) biennial international symposia series. The CDTM symposia have been held in Cardiff University since 2006 and a major objective for the organizers of this series, Drs Mark Gumbleton and Arwyn Jones, is for the symposia to serve the development of Early Stage Career researchers and to promote the inter-disciplinary collaborations necessary to make real progress in this field. All three symposia in the series CDTM2006, CDTM2008 and CDTM2010 have attracted the highest quality of international speakers and provided unique opportunities for delegates from around the

world to interact with others engaged in this research area and to learn from more experienced attendees. For information on the CDTM series go to www.CDTM.cf.ac.uk. A CDTM symposium has traditionally begun with a perspective on membrane biophysics and biochemistry as it relates to macromolecule cell trafficking. For CDTM2010 Erwin London (Stony Brook University, USA) discussed the domain organisation of proteins and lipids at the plasma membrane prior to Paul Luzio (University of Cambridge, UK) discussing regulators of endocytic pathways that will eventually determine the intracellular fate of macromolecule therapeutics. Endocytic pathways while portals for macromolecule entry into the cell do not afford ready escape from within the endomembrane system. This escape remains a major hurdle to cytoplasmic delivery of macromolecules. Jörgen Wesche (University of Oslo, NO) revealed some of the crucial aspects of the endosomal escape mechanisms for fibroblast growth factor-1. Parallels between natural proteins and synthetic systems were then exposed with presentations on plasma membrane and endosomal membrane interactions of liposomes by Frank Szoka (University of California, San Francisco, USA), of polyplexes by Ernst Wagner (Ludwig Maximilian University Munich, DE) and nanoparticles by Tore-Geir Everson (University of Oslo, NO). Cameron Alexander (Nottingham University, UK) presented a talk on how architecture and bioresponsiveness can influence the cellular delivery of synthetic polymer systems. Viruses are natural delivery vectors and Andrew Baker (University of Glasgow, UK) demonstrated the value of engineered chimeric adenovirus particles as carriers of therapeutic genes in cardiovascular disease.

1359-6446/06/$ - see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2010.10.010

The design of macromolecule therapeutics able to efficiently cross epithelial and endothelial surfaces will be more efficient once the biological landscape is characterized and an improved perspective is achieved of the mechanisms through which nature overcomes such barriers. The trafficking of IgG by FcRn within, and across, endothelial barriers was the subject of the presentation by E. Sally Ward (Southwestern Medical Centre, University of Texas, USA). Jan Schnitzer (PRISM, San Diego USA) highlighted the use of endothelial proteomics to identify tissue-specific IgGs able to traverse the endothelial barrier to deliver cargo. The blood–brain barrier represents a unique endothelial network and William Banks (VAPSHCS and University of Washington, Seattle, USA) discussed the challenges and opportunities of delivering peptides and proteins to the brain across this restrictive microvasculature. Cell penetrating peptides (CPPs) can deliver themselves and associated cargo across a wide range of biological membranes and Sandrine Sagan (Université Pierre et Marie Curie-Sciences et Medécine, Fr) discussed technologies for assessing peptide uptake. In a complementary presentation Giles Divita (Centre de Recherches de Biochimie Macromoléculaire, Montpellier, Fr) expanded on CPP-mediated delivery to highlighted successful in vivo siRNA delivery strategies using specific CPP sequences. The unique challenges in the design and development of macromolecules destined for the clinic were demonstrated by presentations from industrial scientists Ted Parton (UCB Celltech, UK) on pegylated antibody fragments, and by David Rozema (Roche-Madison Inc., USA) on polymer conjugates for siRNA delivery. Underpinning much of the innovative science are cutting edge technological www.drugdiscoverytoday.com 1079

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advancements and a unique feature of the CDTM series is the integration of ‘Technical Application’ sessions. For CDTM2010 Olav Scheimann (St Andrews University, UK) presented a session on EPR spectroscopy and its value in resolving conformations and dynamics of membrane associated proteins. Werner Witke (Leica Microsystems, DE) presented a session on Total Internal Reflection Microscopy (TIRF) and its capacity to study crucial events that occur on and very close to the plasma membrane. Dries Vercauteren (Ghent University, BE) presented a session comparing pharmacological and molecular approaches to inhibit endocytic

pathways and the requirements to integrate both to gain a more accurate picture of uptake pathways. CDTM2010 welcomed over 180 registered delegates from 27 different countries and from five continents. Over 90 delegate posters were presented, with six of these also selected to highlight their work via short talks. The publication of the CDTM2010 abstracts in the journal Drug Discovery Today is a major landmark for the symposia series reflecting its international standing in the scientific community, its maturation into an important event in the Drug Delivery calendar and its ability to

consistently deliver high quality science both on the podium and through the delegate contributions. It is hoped that all who attended CDTM2010 departed with renewed energy for the scientific challenges they face. We look forward to CDTM2012.

avoid opsonization, (3) PEI moieties to complex nucleic acids and to enhance cytosolic delivery and (4) RGD sequence for active tumor targeting. Nanoparticles were formulated by double emulsion or water-in-oil-in-water method. Physical properties of such nanoparticles were assessed by dynamic light scattering (size and polydispersity index) and laser doppler electrophoresis (zeta potential). The efficiency of nucleic acid encapsulation into the carrier was determined by the Picogreen assay. Cytotoxicity and transfection capacity were assessed in an in vitro model of B16F10 melanoma cells. To date, various designs of nanoparticles were successfully formulated with appropriate size, surface charge and encapsulation efficiency. The PLGA nanoparticles did not show cytotoxic effects on cells and, although less efficient than PEI alone, allowed DNA delivery into tumor cells.

thought-out and well-designed delivery system, which should guide the nucleic acids into the desired compartment of the selected cells. However, humans and other organisms have developed natural barriers that protect their body against different kinds of pathogens or intruders. During the evolution of the human being, these barriers have become almost perfect and difficult to overcome. The nuclear membrane, one of the final barriers that protect our genes, appears to be the most important and the crucial one to overcome in non-viral gene delivery. In this work we try to avoid the need to overcome this barrier by intracellular delivery of mRNA instead of pDNA. mRNA delivery has many advantages. First, mRNA does not have to overcome the nuclear barrier and therefore mRNA can transfect also nondividing cells or dividing cells independent of their cell cycle. Second, mRNA cannot integrate in the genome. Consequently, mRNA mediated gene expression is transient and the risk of insertional mutagenesis can be excluded. Third, there is no need to select a promoter [1]. In this work we evaluate whether mRNA complexed with cationic liposomes (composed of e.g. the cationic lipid GL67) are able to transfect the respiratory tissue of mice. The efficacy of the mRNA:liposome complexes and the gene expression kinetics will be studied and compared with pDNA:liposome complexes. In this study we focus in particularly on GL67-based liposomes. GL67 is an amphiphile consisting of a cholesterol anchor lined to a spermine headgroup in a ‘T-shape’ configuration. It was proven that GL67 based liposomes are the most effective non-viral pulmonary gene delivery systems [2]. Evaluation of the

Mark Gumbleton, Arwyn T. Jones Co-organisers CDTM2010, Welsh School of Pharmacy, Cardiff University, CF10 3NB, UK e-mails: [email protected] (A.T. Jones) [email protected] (M. Gumbleton)

DELEGATE ABSTRACTS

A1 Design and development of polymeric nanoparticles for targeted delivery of nucleic acid-based therapeutics to tumor sites Aude Le Breton 1,2,∗ , Véronique Préat 1 , Olivier Feron 2 1 Louvain Drug Research Institute, Unité de Galénique, Université catholique de Louvain, Avenue Mounier, 73 - bte 7320, 1200 Brussels, Belgium 2 Institut de Recherche Expérimentale et Clinique, Laboratoire ‘Angiogenèse et Cancer’, Université catholique de Louvain, Avenue Mounier, 52 - bte 5349, 1200 Brussels, Belgium ∗

Monitor•MONITOR

Corresponding author. E-mail: [email protected] (A. Le Breton). Nucleic acids are widely used as potent therapeutics in cancer research. They can either promote gene expression by bringing a gene either not expressed or under-expressed into tumor cells (cDNA), or alternatively silence expression of genes such as oncogenes (RNAi mediators). However, before they can be efficiently translated to the clinic, this technology requires some optimization: nucleic acids and their vehicles need for instance to be protected from rapid elimination from the bloodstream (opsonization, clearance, and nuclease-mediated degradation) and the specificity of tumor addressing has to be validated. Hence a polymeric nanoparticular carrier encapsulating nucleic acids, either plasmid DNA or siRNA, was developed. Nanoparticles are composed of (1) PLGA, a well tolerated and biodegradable polymer, (2) PEG groups to 1080

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A3 Pulmonary delivery of mRNA: in vitro and in vivo evaluation Oliwia Andries ∗ , Joanna Rejman, Cindy Peleman, Tony Lahoutte, Stefaan De Smedt, Joseph Demeester, Niek N. Sanders Ghent University, Laboratory of Gene Therapy, Faculty of Veterinary Medicine, Department of Nutrition, Genetics and Ethology, Heidestraat 19, 9820 Merelbeke, Belgium ∗ Corresponding author. E-mail: [email protected] (O. Andries). Gene therapy is a very promising field of research in medicine. The success of gene based therapeutics will depend on a well

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References 1. Yamamoto A, et al. Current prospects for mRNA gene delivery. Eur J Pharm Biopharm 2009;71:484–9. 2. Lee ER, et al. Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung. Human Gene Therapy 1996;7:1701–17.

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A4 siRNA containing nanoparticles: stability of encapsulation and particle size Kevin Buyens ∗ , Kevin Braeckmans, Joseph Demeester, Stefaan C. De Smedt, Niek N. Sanders Ghent University, Faculty of Pharmaceutical Sciences, Ghent Research Group on Nanomedicins, Harelbekestraat 72, 9000 Gent, Belgium ∗

Corresponding author. E-mail: [email protected] (K. Buyens). A large effort is currently put into the development of nano-scaled carrier systems that can guide siRNA molecules to their target cells after intravenous injection. One of the main issues in this research is the integrity of the siRNA containing nanoparticles in the blood stream. The integrity of the nanoparticles comprises both the particle size and the stable encapsulation of siRNA. Techniques currently available for studying the disassembly and size distribution of siRNA containing nanoparticles are time-consuming and incompatible with biological fluids. We initially developed a fluorescence fluctuation spectroscopy (FFS) based method which allows us to monitor the integrity of siRNA-carrier complexes in less

than one minute in complex biological media and at very low siRNA concentrations. Second, while the size distribution of the complexes can be easily measured in a clear dispersion by dynamic light scattering or electron microscopy, it cannot be measured in more complex biological media such as plasma or whole blood, which contain several different interfering components. To address this issue, we have developed a novel technique, based on single particle tracking (SPT) microscopy, for studying the size distribution (and aggregation) of nanoscopic drug complexes in biological fluids. For stabilization of the particle size of cationic lipid based nanoparticles, inclusion of lipids conjugated with PEG is widely used to sterically hinder aggregate formation. We have demonstrated that in order to obtain remaining siRNA complexation to the cationic liposomes, effective encapsulation inside the liposome, or in between lipid multilayers is required, since siRNA electrostatically bound to the outer side of the liposomes is quickly pushed away by the ubiquitous albumin molecules in blood which leads to siRNA degradation and loss of effectiveness. Formation of siRNA protecting multilayers is hindered by inclusion of PEGlipids, a hurdle that needs to be overcome either by post-insertion of the PEG-lipid into multilayer containing siRNA-liposome complexes, or by efficient encapsulation of the siRNA inside the aqueous core of the PEGylated liposome. Size stabilization in buffer can be easily achieved by inclusion of minor percentages (∼1%) of PEG-lipids. In whole blood however, we demonstrate that much higher percentages of PEG–lipids (5–10%) are required to achieve size stabilization. This requirement has not been previously considered because of the lack of a suitable technique to study the aggregation phenomena in whole blood. In our work we demonstrate that assaying the physicochemical properties of siRNA encapsulating nanoparticles should always be carried out in the biological media they are designed to be employed in. Two novel microscopy based methods were developed that enable such characterization in biological fluids such as serum, plasma or even whole blood. doi:10.1016/j.drudis.2010.09.357

A5 Investigating the effects of cationic lipid-mediated toxicity and how to optimize liposomal systems for transfection purposes S.J. Soenen 1 , N. Nuytten 1 , S.C. De Smedt 2 , M. De Cuyper 1,∗ 1 Lab of BioNanoColloids, IRC, KULeuven Campus Kortrijk, Kortrijk, Belgium 2 Lab of General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, University of Ghent, Ghent, Belgium ∗

Corresponding author. E-mail: [email protected] (M. De Cuyper). For magnetic resonance imaging (MRI) of therapeutic cells, these cells are often prelabelled in culture with iron oxide nanoparticles, enabling them to be non-invasively monitored by MRI following transplantation in vivo. Magnetoliposomes (MLs) are nanosized Fe3O4cores (14 nm diameter) each surrounded by a lipid bilayer [1]. Different types of MLs have been utilised for biomedical research applications [2,3], where cationic MLs are more optimally suited for in vitro cell labelling [1]. Unfortunately, cationic lipids display several inherent properties which, to date, have not been clearly defined [4]. In the present work, cationic MLs as well as their non-iron oxide-containing vesicular counterparts were used to label NIH 3T3 fibroblasts. Using distearoyltrimethyl ammoniumpropane (DSTAP) as the cationic lipid the effects on cell physiology of the different particles was compared. Different amounts of DSTAP were used, indicating that when the cationic lipids exceed a certain safe threshold (3.33%), this affects cell viability by different mechanisms that are dependent and independent of actual nanoparticle internalization. Internalizationdependent mechanisms are closely linked to the induction of reactive oxygen species and altered Ca2+ homeostasis; the indirect mechanisms appear to indicate plasma membrane destabilization by means of transfer of the cationic lipid from the nanoparticles to the plasma membrane. The extent of cationic effects could be modified by: (1) the size of the liposome, (2) the presence of a stabilising iron oxide core, (3) the use of reactive oxygen species or Ca2+ channel inhibitors, (4) the nature of the cationic lipid and (5) the nature of the neutral matrix lipids. Based on these results, a novel cationic peptide-lipid conjugate (dipalmitoylphosphatidylethanolamine-

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in vitro luciferase gene expression in A549 lung adenocarcinoma cells and selection of the most optimal mRNA:liposome ratios are the first steps towards this goal. The efficiency of mRNA/GL67 complexes will be compared to its 4th generation plasmid counterparts–pCpGCMV-Luc/GL67 lipoplexes. This non-viral mRNA delivery system is potentially a more efficient way for delivering therapeutic genes specifically and directly to the respiratory tract. The respiratory tract is a very interesting and important target organ for gene therapy as it is affected by many acute and chronic diseases, such as cancer, cystic fibrosis, asthma, alpha-1antytrypsin deficiency or respiratory infections. It is quite a special organ with the possibility of non-invasive, topical administration of a drug through the airways.

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succinyl-tetralysine [DPPE-succ-(Lys)4]) was synthesized, which efficiently reduced cytotoxic effects and further augmented the internalization efficiency of the MLs [5]. In conclusion, the results indicate that the use of cationic lipids for transfection purposes should be carefully considered as they can induce severe cytotoxic effects. By carefully controlling the physicochemical properties of the liposomal systems used, many of the cytotoxic effects can immediately be reduced. These data highlight the need for careful optimization of cationic liposome formulations and that great advances can still be made with respect to diminished toxicity and enhanced internalization. References 1. Soenen A, et al. Biomaterials 2009;30:3691. 2. Soenen SJ, et al. Nanomedicine 2009;4:177. 3. Martina MS, et al. J Am Chem Soc 2005;127:10676. 4. Lv H, et al. Control Release 2006;114:100. 5. Soenen SJ, et al. Biomaterials 2009;30:6803.

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A6 Intracellular iron oxide nanoparticle coating stability determines nanoparticle usability and cell functionality S.J. Soenen 1 , N. Nuytten 1 , U. Himmelreich 2 , M. De Cuyper 2,∗ 1 Lab of BioNanoColloids, IRC, KULeuven Campus Kortrijk, Kortrijk, Belgium 2 MoSAIC/Biomedical NMR Unit, KULeuven Gasthuisberg, Leuven, Belgium ∗

Delegate abstracts•MONITOR

Corresponding author. E-mail: [email protected] (M. De Cuyper). Iron oxide nanoparticles are routinely exploited as T2/T2* contrast agents [1]. One of the most active topics in this biomedical research area is the non-invasive imaging of pre-labelled stem or therapeutic cells upon transplantation in vivo in [2]. To this end, commercial particles such as Endorem® are frequently employed, however, the particles display several characteristics which makes them less suitable for in vitro labelling [3]. In the present work, the effects on cell physiology of in-house produced cationic magnetoliposomes (MLs), that is, 14-nm diameter iron oxide cores each individually enwrapped by a lipid bilayer containing 3.33% of distearoyltrimethyl ammoniumpropane (DSTAP)[4] – a cationic lipid – are compared with the effects of Resovist (carboxydextran), Endorem (dextran) and VSOP (citrate) iron oxide particles. When the particles

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are incubated at high dosages, reaching high intracellular iron levels, this results in a transient decrease in cell cycle progression, actin cytoskeleton remodelling and focal adhesion formation and maturation [5]. The extent of these effects is in line with the intracellular iron concentration and appears to be common for all particles. When reaching similar intracellular iron concentrations and when verifying that the different particles are routed along the same way and are therefore exposed to similar intracellular microenvironment at fixed time points, it is shown that intracellular stability of the coating molecules is of high importance. The results in vitro show that citrate-coated particles are rapidly degraded, whereas those coated with dextran are more stable, but still less than the MLs. The degradation of the particles can be shown by the increase in free ferric ions, and the distorted r1/r2 ratio of the particles, hampering their use for long-term imaging. Labelled cells further show increases in reactive oxygen species and transferrin receptor expression in C17.2 neural progenitor cells and impeded functionality of PC12 rat pheochromocytoma cells. The extent of these effects is in line with the degradability of the particles in vitro. The MLs appear to be the most stable particles and further show a high persistence of the label in continuously proliferating C17.2 cells. In conclusion, the results indicate that the type of coating material used is highly important with regard to maintaining cell functionality and stability of the label. Further characterization of cell-nanoparticle interactions is both warranted and needed [1]. References 1. Soenen SJ, De Cuyper M. Contrast Media Mol Imaging 2009;4:207. 2. Hoehn M, et al. J Physiol 2007;584:25. 3. Wilhelm C, Gazeau F. Biomaterials 2008;29:3161. 4. De Cuyper M, Soenen SJ. Methods Mol Biol 2010;605:177. 5. Soenen, S.J. et al. (in press) Small.

doi:10.1016/j.drudis.2010.09.359

A7 Nuclear inclusion of inert and chromatintargeted polystyrene beads and plasmid DNA containing nanoparticles N. Symens ∗ , R. Walzack, J. Demeester, I. Mattaj, S. De Smedt, K. Remaut Lab. General Biochemistry and Physical Pharmacy, Ghent Research Group on Nanomedicine, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Gent, Belgium ∗

Corresponding author. E-mail: [email protected] (N. Symens). Introduction and aim: The nuclear membrane is currently one of the major cellular barriers to the effective delivery of plasmid DNA (pDNA). Cell division has a positive influence on the transfection efficiency from naked pDNA and nanoparticles containing pDNA. At the end of mitosis, the pDNA near the chromatin is probably randomly included in the nuclei of daughter cells during reassemby of the nuclear envelope around chromatin. However, very little is known on the nuclear inclusion of nanoparticles during cell division. We were interested if inert nanospheres get randomly enclosed in artificial Xenopus nuclei and in nuclei of dividing cells. We investigated nanospheres with a different size and charge, and whether the enclosure could be enhanced by the use of chromatin binding peptides such as AT-hooks. Material and methods: Non-targeted positively charged, poly-ethyleneglycol (PEG)-ylated and negatively charged green fluorescent polystyrene nanospheres (Molecular Probes) of 100, 200 or 500 nm were used. The 100 nm nanospheres were also modified with Mel28 (GPSKPRGRPPKHKAKT), mutated Mel-28 (GPSKPGGGPPGHKAKT) or HMGA2 (SPKRPRGRPKGSKNKS), containing an AT-hook or a mutated AT-hook (targeted nanospheres). Artificial nuclei were obtained with the ‘Xenopus egg extract (XEE) nuclear assembly reaction’. The enclosure of the nanospheres in the artificial nuclei and upon microinjection was visualised by confocal fluorescence microscopy. Results and conclusions: Periodically the non-targeted nanospheres were able to get enclosed in the artificial nuclei but enclosure was rather limited. The enclosure of the positively charged spheres is higher than that of the negatively charged and the PEG-ylated variants, likely as a result of aspecific interactions with the nett negatively charged chromatin. Size is also important: spheres with a diameter of

200 nm and 100 nm are better enclosed than the 500 nm variants. The enclosure of spheres modified with chromatin binding peptides is indeed higher than the enclosure of the nontargeted spheres and the spheres modified with the mutated AT-hook. When polystyrene spheres were injected in the cytoplasm of HeLa cells, initially, the nanospheres spread homogenously in the cytoplasm. Upon cell division, however, the nanospheres accumulated in a specific perinuclear region and enclosure in the nuclei of divided cells was never observed. Therefore, reaching the nucleoplasm seems to be very difficult and we question whether the chromatin binding peptides are able to target the nanospheres into the daughter nuclei of living cells. It thus seems that nuclear inclusion in the XEE assay does not represent the situation in living cells Acknowlegements N. Symens is a predoctoral fellow from the Institute for the Promotion of Innovation through Science and Technology in Flanders. K. Remaut is a postdoctoral fellow of the Research Foundation Flanders. The financial support of these institutes is acknowledged with gratitude. doi:10.1016/j.drudis.2010.09.360

A8 mRNA delivery to cervical carcinoma and mesenchymal stem cells mediated by cationic carriers Joanna Rejman ∗ , Geertrui Tavernier, Neda Bevarsad, Joseph Demeester, Stefaan C. De Smedt Ghent University, Laboratory of General Biochemistry and Physical Pharmacy, Ghent Research Group on Nanomedicines, Harelbekestraat 72, 9000 Ghent, Belgium ∗

Corresponding author. E-mail: [email protected] (J. Rejman). We complexed mRNA encoding luciferase (mLUC) to either one of the cationic lipids Lipofectamine (LF) or DOTAP/DOPE, or to linear poly(ethyleneimine), a cationic polymer (linPEI). After incubating the resulting lipo- or polyplexes with HeLa cells for different periods of time, we determined luciferase expression by a bioluminescence assay. Both extent and duration of luciferase expression were dependent on the type of complex used. With LF, mRNA expression lasted for about 9 days maximally, which is not significantly shorter than what can be achieved with pDNA polyplexes. When electroporation was used to transfer mLUC into the cells, luciferase expression lasted for 12 h only.

An important characteristic of mRNA-mediated transfection by means of all three complexes is that it could already be detected 30 min after adding the complexes to the cells. In order to estimate the number of positive cells, we transfected the cells with an mRNA encoding Green Fluorescent Protein (GFP) and compared the results with transfection by means of pDNA. With transfection by means of mRNA complexed to LF or DOTAP/DOPE a substantially larger fraction of cells (>80%) was transfected than with pDNA (40%). After establishing the characteristics of mRNA-mediated transfection by means of expression of reporter proteins, we tested the carriers for their ability to mediate expression of a functional protein in mesenchymal stem cells. For that purpose we complexed an mRNA encoding CXCR4, a receptor binding stromal derived factor 1, to the cationic lipids and the polymer. The resulting complexes were incubated with mesenchymal stem cells and CXCR4 expression was assayed. The fraction of CXCR4-positive cells was approximately 80% and 40% for mRNA-cationic lipoplexes and lin-PEI polyplexes respectively. The results of these experiments indicate that mRNA, under certain conditions, may be preferable to pDNA to achieve transfection, particularly in cases requiring transient protein expression. doi:10.1016/j.drudis.2010.09.361

A9 Cellular uptake of long-circulating pHsensitive liposomes: evaluation of the liposome and its encapsulated material penetration in cancer cells Emilie Ducat ∗ , Julie Deprez, Olivier Peulen, Brigitte Evrard, Géraldine Piel Laboratory of Pharmaceutical Technology, CIRM, Department of Pharmacy, University of Liege, Belgium ∗

Corresponding author. E-mail: [email protected] (E. Ducat). Print 3G, a peptidic antagonist of oncoprotein involved in breast cancer, could reduce the angiogenic development of breast tumors, leading to tumor dormancy. The necessity of intravenous administration of Print 3G led to the development of long-circulating liposomes as drug carriers. Pegylated liposomes, too large to be collected by fenestrated organs, accumulate passively in solid tumors thanks to the EPR effect. The strategy was to combine the protective properties of PEG with the transfection properties of pH-sensitive lipids that could promote the uptake of liposomes by

DELEGATE ABSTRACTS

cells and avoid lysosomal sequestration and degradation of entrapped materials such as peptides. In this study, we compare two formulations in terms of cellular uptake using confocal microscopy. The first one is composed of SPC:CHOL:mPEG-750-DSPE (47:47:6), used as ‘standard’ liposomes, and the second one composed of DOPE:CHEMS:CHOL:mPEG750-DSPE (43:21:30:6), used as pH-sensitive liposomes. Firstly, we evaluated the penetration of an encapsulated model molecule, calcein, in Hs578t human breast cancer epithelial cells. When calcein was encapsulated in standard liposomes, its penetration was effective only in a few cells. On the contrary, the majority of cells were fluorescent when calcein-loaded pH-sensitive liposomes were applied on cells for three hours. Secondly, we studied the penetration of liposomes themselves in Hs578t cells using 25-[(nitrobenzoxadiazolyl)methylamino]norcholesterol (NBD-CHOL) as a fluorescent marker of the phospholipid membrane. The obtained results were comparable to those obtained with calcein: a higher penetration of liposome was observed for pH-sensitive liposomes. Finally, the cellular uptake of liposomes using both NBDCHOL and rhodamine encapsulated in the inner cavity of vesicles was evaluated with Hs578t cells and compared with WI26 human diploid lung fibroblast cells. This experimental design allowed us to follow simultaneously the cell distribution of the encapsulated material and of the liposome itself. Confocal pictures obtained with pH-sensitive liposomes on both WI26 and Hs578t cells allowed us to visualize co-localized red and green of rhodamine and NBD-CHOL, with a higher degree of colocalization in an area close to the nucleus. In comparison with ‘standard’ liposomes, we observed a higher penetration of the encapsulated material and of the liposome itself in breast cancer cells. Moreover, we visualized a colocalization near the nucleus of liposomes components. From results obtained with fibroblastic cells, there was no difference in terms of cellular uptake between the two formulations. In perspective, we would like to compare these results, obtained with model molecules, with experiments performed with biotinylated Print 3G to assess its cellular distribution. Moreover, it would be interesting to correlate results obtained with confocal microscopy with a possible increase of the peptide efficacy against cancer cells when it is encapsulated in long-circulating pH-sensitive liposomes. doi:10.1016/j.drudis.2010.09.362

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A10 Role of dynamin-dependent and clathrindependent uptake pathways in nonviral gene delivery studied by chemical and genetic means Polina Ilina 1,∗ , Zanna Hyvonen 2 , Marjo Yliperttula 1 , Marika Ruponen 2 1 Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, P.O. Box 56 (Viikinkaari 5 E), FI-00014 University of Helsinki, Finland 2 University of Kuopio, Finland ∗

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Corresponding author. E-mail: polina.ilina@helsinki.fi (P. Ilina). Introduction: Endocytosis is known to be a major cell uptake mechanism for non-viral gene delivery vehicles. Several mechanisms of endocytosis have been described and it seems that not all of them are equally beneficial in terms of gene delivery efficiency. According to the literature the preferential cell uptake pathway is both carrier and cell type dependent. Rational design of effective and safe gene delivery vectors requires deeper understanding of the cellular uptake mechanisms of gene delivery vehicles. The purpose of our study was to clarify the role of dynamin-dependent cell uptake pathways, including both clathrin-dependent and caveolae-dependent endocytosis, in nonviral gene delivery. Methods: The studies were performed with three widely used non-viral gene delivery systems: cationic polymer branched polyethyleneimine (PEI), cationic lipid N-(1-(2,3-dioleoyloxy)propyl)-N,N,Ntrimethyl ammonium methylsulfate (DOTAP) and calcium phosphate (CaP) precipitates. The internalization pathways of these gene delivery vehicles were studied by using genetically modified cell lines: HeLaK44A cells with inducible block of dynamin-dependent endocytosis and BHK21-tTA cells with inducible block of clathrin-dependent endocytosis. As an alternative approach chemical blockers chlorpromazine, dansylcadaverine, nystatin and dynasore were used to inhibit specific endocytic pathways. Relevant concentration of each inhibitor was determined by MTT cell viability assay. Size of the complexes was measured, and expression of marker protein at different timepoints from 0 to 72 hours after exposure to complexes was determined in intact cells and cells with blocked endocytic pathway(s). Results: The obtained data indicated that in both HeLaK44A and BHK21-tTA cell lines for DOTAP-based nanoparticles clathrindependent endocytic pathway seemed to be

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predominantly responsible for successful gene delivery, whereas for efficient PEI-mediated transfection caveolae-mediated pathway was important. In HeLaK44A cells block of dynamin-dependent endocytosis resulted only in moderate (40–50%) decrease of transfection efficiency of both PEI and DOTAP complexes. This suggests that other pathways, not dependent of dynamin, participate in the uptake of both PEI- and DOTAP-based nanoparticles in this cell line. In HeLaK44A cells blockage of dynamin-dependent endocytosis by genetic means increased transfection efficiency of Caphosphate precipitates 4-fold whereas chemical blockage of dynamin-dependent pathway by dynasore reduced transfection efficiency of Ca-phosphate precipitates almost completely. However, in general, the results obtained by using genetic means were comparable with results obtained by using chemical inhibitors. doi:10.1016/j.drudis.2010.09.363

A11 Enhanced intracellular delivery by guanidinium functionalized ROMP-polymers A. Ozgul Tezgel 1,∗ , Janice C. Telfer 2 , Gregory N. Tew 1 1 Polymer Science and Engineering Department, University of Massachusetts, Amherst MA, 01003, United States 2 Veterinary and Animal Science Department, University of Massachusetts, Amherst MA, 01003, United States ∗

Corresponding author. E-mail: [email protected] (A.O. Tezgel). Intracellular delivery of therapeutic molecules has always been a challenge due to the poor permeability of cell membrane to large, negatively charged macromolecules and their restricted biodistribution. In the past decades, cell penetrating peptides (CPPs) are shown to improve the intracellular delivery of bioactive molecules and among the CPPs, arginine-rich peptides are highlighted as the most effective subclass. In the light of this information, we designed and synthesized guanidinium functionalized polyoxanorbornenes which can adopt cell penetrating activity and show superior uptake properties compared to peptide analogues (i.e. nonaarginine, R9). The structure–activity relationship was studied by mono-guanidinium and diguanidinium functionalized monomers and a specific trend was observed for each cell line studied. In addition to intracellular uptake pro-

files of molecules, their exceptional ability to deliver bioactive cargo, such as DNA, siRNA and intact proteins, into both adherent and suspension cell lines, as well as in primary cells has been demonstrated. A non-covalent complexation approach was utilized for the delivery of bioactive molecules, instead of covalent attachment. Non-covalent interactions are highly favored over covalent attachment of cargo, in terms of simplicity, efficiency of delivery and stability of bioactive cargo. Furthermore, structural requirements and optimal experimental conditions have been investigated for an efficient intracellular delivery agent. doi:10.1016/j.drudis.2010.09.364

A12 Engineering functional chitosan for delivery of drugs or RNAs Xiudong Liu 1,2,∗ , Yan Yang 2 , Huofei Zhou 2 , Weiting Yu 2 , Jiani Zheng 2 , Demeng Zhang 2 , Xiaojun Ma 2,∗ 1 College of Environment & Chemical Engineering, Dalian University, Dalian 116622, China 2 Laborotary of Biomedical Materials Engineering, Dalian Institute of Chemcial Physics, Chinese Academy of Sciences, Dalian 116023, China ∗

Corresponding author. E-mails: [email protected] (X. Liu), [email protected] (X. Ma). In the last decade, considerable studies on preparation of nanocarriers with cationic liposomes or polymers have been reported for intracellular delivery of DNA and siRNA [1]. Particle uptake has been proven through several kinds of endocytosis pathways, but the uptake efficiency varies depending on the property of carrier materials, particle size, and cell types. Using biocompatible and biodegradable chitosan (CTS) as carrier material, we designed and synthesized functional chitosan derivatives (such as amphiphilic CTS, ligand-targeted CTS), and then developed different technologies to prepare CTS nanoparticles for the potential application of loading, delivering and releasing anti-cancer drugs or RNA therapeutics (siRNA and microRNA). In one system, we initially conjugated a fatty acid (LA) to CTS to obtain amphiphilc CTS-LA, and then synthezised CTS-LA-TM by quaternization. Subsequently nanoparticles with size less than 200 nm can be easily formed by self-assembly of CS-LA-TM in biological solution or neutral solution [2]. These loaded PTX with encapsulation efficiency of 60–90% and showed sustained release in 1 week without burst release. Alternatively,

we formulated CTS-RNAs (siRNA or microRNA) nanoparticles by direct complexation. The nanoparticles with sizes of 120–200 nm and surface charge of ∼20 mV showed complex stability and efficiency of protecting RNAs from RNase degradation. These nanoparticles can both transfer RNAs into cells and protect entrapped intracellular RNAs, in 2–4 hours without apparent critical cytotoxicity. Moreover, cell adhesive peptide GRGDY has been grafted to CTS by photosensitive crosslinker [3], and PEGylation has been carried out for target transportation to tumor cells with overexpressed integrin receptors and for efficient delivery of drugs or RNA therapeutics. Acknowlegements The authors are thankful for the financial support from National Natural Science Foundation of China (No. 20876018), National Key Technology R&D Program in the 11th Five-year Plan of China (2006BAD27B04), Knowledge Innovation Project of the Chinese Academy of Sciences (KJCX2.YW.M02 and KJCX2-YW-21002), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China. References 1. Jeong JH, et al. Prog Polymer Sci 2007;32:1239–74. 2. Zhou HF, et al. J Nanosci Nanotechnol 2010;10:2304–13. 3. Yang Y, et al. Carbohydr Polymers 2010;80:733–9.

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A13 Incorporation of 2,3-diaminopropionic acid in linear cationic amphipathic peptides produces pH sensitive vectors Yun Lan 1 , Bérangère Langlet-Bertin 2 , Vincenzo Abbate 1 , Louic S. Vermeer 1 , Xiaole Kong 1 , Kelly E. Sullivan 1 , Christian Leborgne 2 , Daniel Scherman 2 , Robert C. Hider 1 , Alex F. Drake 1 , Sukhvinder S. Bansal 1 , Antoine Kichler 2 , A. James Mason 1,∗ 1 King’s College London, Pharmaceutical Science Division, 150 Stamford Street, London, SE1 9NH, UK 2 CNRS FRE 3087-Généthon, 1 rue de l’Internationale, F-91002, Evry, France ∗

Corresponding author. E-mail: [email protected] (A.J. Mason). Non-viral vectors that harness the change in pH in endosomes are increasingly being used to deliver cargoes, including nucleic acids, to mammalian cells. Here we present

evidence that the pKa of the ␤-NH2 in 2,3diaminopropionic acid (Dap) is sufficiently lowered, when incorporated in peptides, that its protonation state is sensitive to the pH changes that occur during endosomal acidification. The lowered pKa around 6.3 is stabilised by the increased electron withdrawing effect of the peptide bonds by inter-molecular hydrogen bonding and from contributions arising from the peptide conformation, including mixed polar/apolar environments, Coulombic interactions and inter-molecular hydrogen bonding. Changes of the charged state are therefore expected between pH 5 and 7 and large-scale conformational changes are observed in Dap rich peptides, in contrast with analogues containing lysine or ornithine, when the pH is altered through this range. These physical properties confer a robust gene delivery capability on designed cationic amphipathic peptides that incorporate Dap. Recent results investigating the link between hydrophobicity, number of charges, Coulombic interactions and side chain pKa are considered in terms of the efficiency of gene delivery. doi:10.1016/j.drudis.2010.09.366

A14 Octaarginine mediated delivery of fluorescent cargo to human smooth muscle cells Michele Sweeney 1 , Catherine L. Watkins 2 , Arwyn T. Jones 2 , Michael J. Taggart 1,∗ 1 Reproductive and Vascular Biology Group, Institute of Cellular Medicine, Newcastle University, UK 2 Welsh School of Pharmacy, Cardiff University, UK ∗

Corresponding author. E-mail: [email protected] (M.J. Taggart). The high incidence and severity of diseases involving smooth muscle dysfunction, which include cardiovascular diseases and premature labour, dictates the need for our continued search for novel therapeutic strategies to treat these conditions. Cell penetrating peptides (CPP) are a class of non-viral vectors that show considerable promise for drug delivery purposes yet their suitability for uptake, and delivery of biologically active cargo, to human native cells and tissues remains unresolved. For any new drug delivery strategy, including the use of CPPs, to reach fruition this needs to be elucidated. We have begun to explore this issue for CPPs applied to human uterine cells and tissues (including myometrium

DELEGATE ABSTRACTS

and blood vessels) obtained from biopsies collected, following LREC-approved written informed consent, from patients undergoing elective Caesarean section at the end of pregnancy. Primary cultured human myometrial cells were prepared on glass-bottomed culture dishes, grown to 80–90% confluence and exposed to serum-reduced conditions overnight before exposure to CPP (or, separately, were methanol-fixed for subsequent immunofluoresence staining of protein localisation). Cellular uptake of fluorescently labelled (Alexa 488) D-Octaarginine (R8, 2 ␮M) was assessed in the first series of experiments for 24, 48 and 72 hours (n = 2). At each time point, zsection confocal microscopy revealed punctate intracellular fluorescence (indicative of vesicular compartmentalisation) particularly dense in the perinuclear area. A second series of experiments assessed the time-course of intracellular delivery up to 24 hours. Punctate intracellular loading was observed by 4 hours. More dense perinuclear and plasma membrane-localised fluorescence was observed at later time points. Immunofluoresence labelling revealed that human myometrial cells possessed expected cytoskeletal (␣-smooth muscle actin, tubulin), plasma membranous and perinuclear localised components of endocytotic pathways (Caveolin-1, Clathrin Heavy Chain, Early Endosomal Antigen-1, Lysosomal Associated Membrane Protein-1 and 2 and Flotillins). Next, small segments of native (non-cultured), human uterine tissue were incubated with 2 ␮M DR8 and nuclear dye Hoechst 33342 (1 ␮M) for 4 hours. Confocal microscopic examination revealed peptide entry into smooth muscle cells of both the myometrium and uterine blood vessels with homogenous intracellular fluorescence in many cells but some with more punctate perinuclear/nuclear fluorescence. In uterine tissues incubated with a similar, putatively cell-impermeant, Alexa488 control peptide (GS)4 GC, no intracellular fluorescence was observed. These preliminary investigations illustrate that an octameric cationic CPP can successfully enter primary cultured and native human smooth muscle cells and tissues. This opens up a new avenue for targeted delivery of cellular therapeutics in human tissues and in particular to human smooth muscles. doi:10.1016/j.drudis.2010.09.367

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Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

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A15 Endocytic DNA and siRNA delivery mediated by pH sensitive peptides Mia S.W. So 1 , Katarzyna Witt 2 , A. Yun Lan 2 , James Mason 2 , Jenny K.W. Lam 1,∗ 1 Department of Pharmacology & Pharmacy, LKS Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong 2 Pharmaceutical Biophysics Group, Pharmaceutical Science Division, King’s College London, Franklin-Wilkins Building, SE1 9NH, UK ∗

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Corresponding author. E-mail: [email protected] (J.K.W. Lam). Amphipathic peptides have emerged as promising candidates for both DNA and siRNA delivery. Consisting of both hydrophilic and hydrophobic domains, they bind to nucleic acids and at the same time provide pH dependent membrane destabilising activity, promoting endosomal escape. The aim of this study was to investigate the efficiency of such peptides in delivering siRNA and plasmid DNA, and to improve our understanding of how the structural difference between the peptides could affect the uptake mechanism and intracellular trafficking of the system. A series of structurally related histidine-rich amphipathic peptides (LAH4-L1, LAH6-X1L, LAH6-X1-26 and LAH6-X1-W) were investigated. The LAH peptides are 25–26 amino acids in length and comprise cationic lysines to allow electrostatic interaction and complexation with the negatively charged nucleic acids. Each of the peptides also contains four or six histidine residues. With a starting pKa around 6.0, the imidazole group of histidine may allow buffering and subsequently destabilise endosomes, thus enhancing endosomal escape of the nucleic acids. The LAH peptides demonstrated pH responsive character whish is classically manifested as a conversion from an alpha helical conformation at neutral pH to a disordered conformation at acidic pH. Differences in the number of charges and the hydrophobicity in the four peptides affect the nature and pH dependence of this transformation. Luciferase reporter gene studies showed that the in vitro DNA transfection efficiency of the LAH peptides were comparable to commercially available lipofectamine in both A549 and MCF-7 cells. These peptides, in particular LAH6-X1L, also showed high resistance to serum in MCF-7 cells. In addition, both LAH4-L1 and LAH6-X1L mediated significant knockdown of GAPDH enzyme in siRNA transfection studies in the presence of serum. Live cell confocal imaging

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was carried out to study the intracellular trafficking of the peptide/nucleic acid complexes. Co-localisation experiments were performed with LAH6-X1L- DNA/siRNA complexes and dextran in A549 cells, with the nucleic acids labelled with rhodamine (red), nucleus labelled with Hoechst (blue) and dextran lablled with Alexa-fluor-488 (green). Dextran is known to be internalised through fluid-endocytosis and end up in endososmes and later lysosomes. At an early stage (within the first hour of post-transfection) there was a high level of co-localisation between LAH6-X1L complexes and dextran (shown as orange in colour). At later stages (300 minutes post-transfection), the degree of co-localisation significantly reduced as the siRNA (red) and dextran (green) was shown to be clearly separated from each other. Our results indicate that the histidinerich peptides offer great promise as siRNA delivery vectors with the ability to promote endosomal/lysosomal escape. doi:10.1016/j.drudis.2010.09.368

A16 Role of polymer architecture on polycation induced cell death: systematic study on molecular mechanism Zuzana Kadlecova ∗ , Elisa Corbacella, Florian Maria Wurm, Harm Anton Klok Ecole Polytechnique Fédérale de Lausanne (EPFL) Laboratory of Polymers, STI - IMX - LP MXD 112 (Batiment MXD), Station 12, CH-1015 Lausanne, Switzerland ∗

Corresponding author. E-mail: zuzana.kadlecova@epfl.ch (Z. Kadlecova). More than 3000 references can be found in the literature that relate to the application of polycations for drug or gene delivery. However, systematic studies that try to correlate polymer molecular weight, architecture and/or composition with polycation induced cell toxicity are scarce, and the underlying biomolecular mechanisms remain largely unknown. In this contribution new findings are presented on the mechanisms of polycation induced cell death and its correlation with the polymer architecture and degradation rate. For our studies, we firstly synthesized a polymer library based on L-lysine monomer units. The library contained linear, hyperbranched and dendritic L-lysine analogues in a broad range of molecular weights. We then investigated the effect of molecular weight (Mn), degree of branching and polydispersity on the mechanism of

short and long term cell toxicity in vitro. The molecular mechanisms underlying cell death at various stages of cell exposure to polycation were identified. The onset and extent of these specific modes of cell death were shown to be dependent on the size and degree of branching of the polycations. Simultaneously the in vitro degradation profile for analogues was assessed and correlated with the process of cell death. For the first time the factors contributing to the differential toxicity profile of the L-lysine analogues are analyzed and discussed. doi:10.1016/j.drudis.2010.09.369

A17 A novel 3D model for the study of functionalised-nanoparticle penetration into human tissue H. Child ∗ , C.C. Berry The Centre for Cell Engineering, Joseph Black Building, The University of Glasgow, Glasgow, Scotland, G12 8QQ, United Kingdom ∗

Corresponding author. E-mail: [email protected] (H. Child). The advancing field of nanotechnology is progressing rapidly towards the development of multifunctional nanoparticles for use in biomedicine. These nanoparticles benefit from functional biomolecules attached to their surface and can act as unique carrier systems. However, the impermeable nature of both the plasma and nuclear membranes hinders their potential. Two current methods to enhance uptake are using external magnetic fields to remotely control particle direction, and functionalising the nanoparticles with a cell penetrating peptide; both of which facilitate cell entry. To date, studies have largely adopted traditional 2D cell monolayers, the results of which cannot reliably be translated to a human body. This study has focused on using 3D collagen gels seeded with human fibroblast cells as a tissue equivalent model for the study of nanoparticle penetration into human tissue. Iron oxide nanoparticles were employed, which have an attached cell penetrating peptide (penetratin); are magnetic (to allow external control via magnetic fields); and are fluorescent (to allow visualisation). Various analytical techniques were used including fluorescence staining, TEM and histology to compare nanoparticle penetration into gel models both with/without penetratin attachment, and with/without the presence of a magnetic field; both of which have previously been shown to increase nanoparticle uptake in monolayer cul-

Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

doi:10.1016/j.drudis.2010.09.370

A18 Hybrid nanoparticles from cationic lipid and polyelectrolytes as antimicrobial agents Letícia D. Melo, Elsa M. Mamizuka, Ana M. Carmona-Ribeiro ∗ Caixa Postal 26077, CEP 05513-970, Universidade de São Paulo, São Paulo SP, Brazil ∗

Corresponding author. E-mail: [email protected] (A.M. Carmona-Ribeiro). Cationic lipids and polyelectrolytes with the quaternary ammonium moiety in their chemical structure are potent antimicrobial agents. In this work, cationic bilayer fragments prepared from dioctadecyldimethylammonium bromide (DODAB), carboxymethylcellulose (CMC) and polydiallyldimethylammonium chloride (PDDA), added in this sequence, produced potent antimicrobial particles that were characterized by dynamic light-scattering and tested against two bacteria species: Pseudomonas aeruginosa and Staphylococcus aureus. Two different diameters for particles were obtained depending on DODAB concentration. At 0.1 or 0.5 mM DODAB, cationic hybrid particles of DODAB/CMC/PDDA presented final mean diameters of 108 or 500 nm, respectively and zeta-potentials of 30 or 50 mV, respectively. Both particulates yielded the same activity against P. aeruginosa: 0% of cell viability at 1–2 ␮g/mL PDDA as the outermost cationic layer. For S. aureus, at 2 ␮g/mL PDDA, cell viability for larger particles was 0%, while for smaller particles, 12–15% of cell viability was still obtained. The antimicrobial effect was dependent on the amount of positive charge on particles and independent of particle size. PDDA revealed a high potency as antimicrobial agent and P. aeruginosa was more sensitive to all cationic assemblies than S. aureus. Acknowlegements FAPESP; CNPq. doi:10.1016/j.drudis.2010.09.371

A19 Novel formulations for tuberculostatic drugs based on cationic lipid Lilian Barbassa, Elsa M. Mamizuka, Ana M. Carmona-Ribeiro ∗ Caixa Postal 26077, CEP 05513-970, Universidade de São Paulo, São Paulo SP, Brazil ∗

Corresponding author. E-mail: [email protected] (A.M. Carmona-Ribeiro). Cationic bilayers in form of bilayer fragments (BF) or large vesicles (LV) provide adequate environment for solubilization and stabilization of antimicrobial drugs with the advantage of being also antimicrobial agents. In this work, BF or LV interaction with two tuberculostatic drugs, rifamicin (RIF) and isoniazide (ISO) is characterized and the assemblies tested against Mycobacterium smegmatis. Methods were employed to determine cell viability, minimal bactericidal concentration and entrapment efficiency for both drugs from dialysis experiments. The occurrence of synergism between cationic lipid and rifamicin was a major result of this investigation. The cationic lipid alone killed M. smegmatis over a range of low concentrations. Rifamicin drug particles above its solubilization limit could be solubilized by BF at 0.5 mM lipid. LV were leaky to isoniazide whereas Rifamicin could be incorporated in the cationic bilayer at high percentiles. The novel assemblies may become useful in chemotherapy against tuberculosis. Acknowlegements FAPESP and CNPq.

radioimmunoconjugates ibritumomab tiuxetan (Zevalin® ), [131I]-tositumomab (Bexxar® ) and the drug conjugate gemtuzumab ozogamicin (Mylotarg® ). Despite the clinical application of these drugs, direct drug/radionuclide conjugation has many drawbacks such as the necessity for a linker that does not inactivate the drug compound and possible hapten immunogenicity concerns that may arise from systemic administration. To circumvent these issues we have investigated the development of novel drug-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles, coated with a layer of targeting antibodies. This approach avoids direct linkage of the antibody to the drug. We have shown that the conjugation of nanoparticles to antibodies targeting the death receptor Fas can be employed for the specific targeting of colorectal carcinoma cells. Furthermore, we have demonstrated that Fas-targeted nanoparticles encapsulating camptothecin (CPT) elicit an >50-fold improvement in the IC50 of the chemotherapy alone. This improved efficacy is due to several factors including the improved uptake and internalisation of CPT and upregulation of Fas receptor expression by CPT. The ability to exploit antibodies not only for targeting of drug-loaded nanoparticles, but also to elicit therapeutic effects themselves is an exciting approach to drug delivery. The application of this methodology in cancer and other diseases, where appropriate drug and antibody combinations can be identified, has the potential to synergistically improve their efficacies. doi:10.1016/j.drudis.2010.09.373

doi:10.1016/j.drudis.2010.09.372

A20 Antibody targeting of polymeric nanoparticles for cancer therapy Christopher J. Scott ∗ , Francois Fay School of Pharmacy, Queens University Belfast, Medical Biology Centre 97 Lisburn Road Belfast BT9 7BL, United Kingdom

A21 Cationic PLGA nanoparticles loaded with DNA for gene delivery delivery Francois Fay ∗ , Christopher J. Scott School of Pharmacy, Queens University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom ∗

∗

Corresponding author. E-mail: [email protected] (C.J. Scott). Antibodies are now the most common form of therapeutic compound under preclinical and clinical development. Normally these proteins are clinically employed for their ability to bind to their cognate antigen and elicit biological effects such as receptor antagonism. However, the application of antibodies as drug delivery agents is also an area of keen interest. This strategy has successfully reached the clinic in the form of drugs such as the

Corresponding author. E-mail: [email protected] (F. Fay). Nonviral gene delivery vectors such as liposomes, dendrimers and polymeric nanoparticles have recently been developed as alternatives to virus-based vectors in order to reduce immunogenicity and toxicity risks. In most formulations, anionic nucleic acids are bound to the positively charged vector surfaces through charge–charge interactions. However, a recent in vivo study has shown that in endosomes the DNA:nanoparticles complexes can disso-

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tures. This study has provided essential insight into the biomedical potential and possible problems of functionalised-nanoparticle tissue penetration.

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ciate and whilst the nanoparticles can reach the cytoplasm, the cargo DNA is ineffectually retained in endo/lysosomal vesicles and thus unable to perform its therapeutic action. Based on these observations, we have developed a novel poly(lactic-co-glycolic acid (PLGA) nanoparticle formulation to encapsulate and deliver target DNA into the cytoplasm of target cells. Our formulation is based on combining salting out and emulsion-evaporation processes to reduce sonication steps in an attempt to overcome DNA destruction by shearing effect. Using this formulation we have produced a uniform population of 250 nm nanoparticles entrapping plasmid DNA in both supercoiled and open circular structures. Transformation assays using plasmids released from the particles demonstrated retention of DNA functionality in these formulations. As nude anionic nanoparticles particles have previously been shown to preferentially localise in late endosomes, we have also formulated nanoparticles bearing a low cationic charge to provoke their release from the endo/lysosomal pathway. Didodecyl dimethyl ammonium bromide (DMAB) coating results in only a 10% increase in size and no significant alteration of DNA release. Furthermore, study of the localisation of fluorescent DMAB coated NP demonstrated their ability to escape from endosomal compartments into the cytosol. Finally, in vitro transfection assays performed on mammalian cells using these positively charged nanoparticles entrapping a GFP coding plasmid have exhibited significantly improved transfection profiles than anionic particles or liposomal reagents. doi:10.1016/j.drudis.2010.09.374

A22 Click chemistry for the generation of cell permeable apoptotic peptides

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Thomas Fricke 1 , Robert J. Mart 1 , Catherine L. Watkins 2 , Arwyn T. Jones 2 , Rudolf K. Allemann 1,∗ 1 School of Chemistry, Cardiff University, Cardiff, UK 2 Welsh School of Pharmacy, Cardiff University, Cardiff, UK ∗

Corresponding author. E-mail: [email protected] (R.K. Allemann). The use of proteins and peptides as drug molecules has been held back by their proteolytic instability and inability to cross-cellular membranes. Proteins and long peptides are

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often produced by expression in E. coli rather than by solid phase peptide synthesis. A drawback of in vivo protein and peptide synthesis is the difficulty to selectively modify the product peptide by the attachment of fluorescent dyes or ligation to other macromolecules like polysaccharides, lipids or peptides. Here we present a facile method to modify an expressed protein or peptide to create a C-terminal alkyne group. This functionality is then used inter alia for conjugation to the cell-penetrating peptide octa-arginine. This will provide a vector for delivery across the plasma membrane of cells. To demonstrate our method, we have produced in E. coli a peptide derived from the Bak protein; one of the key regulators of apoptosis in eukaryotic cells. In the cell it is usually found bound to Bcl-xL at the outer mitochondrial membrane. If this interaction is disrupted, Bak oligomerizes and forms pores which trigger mitochondria dependent apoptosis through cytochrome c release. Small peptides derived from the BH3 helix of Bak have been shown to induce apoptosis. We have expressed such a peptide in E. coli as a fusion protein. The ketosteroid isomerase fusion protein is insoluble and readily purified from cell extracts. The peptide is then cleaved from the fusion protein by reaction with cyanogen bromide at a strategically inserted methionine residue to generate a homoserine lactone at the C-terminus of the Bak peptide. This lactone is then used for direct amide formation with inexpensive propargylamine. The resulting alkynyl peptide serves as a reagent for highly efficient ‘click’ reactions to couple to a wide range of azides. Since the Bak peptide is not able to cross the cell membrane, the well-known octa-arginine cell penetrating peptide sequence was added as a delivery vector. Here we discuss the synthesis of this semi-synthetic peptide and its interaction with, and uptake into, cancer cell lines. doi:10.1016/j.drudis.2010.09.375

A23 Protein delivery through the intestinal epithelium: a vitamin B12-mediated approach Robyn Fowler 1,∗ , Snow Stolnik 1 , Cameron Alexander 1 , Martin Garnett 1 , Helen Horsley 2 , Bryan Smith 2 1 Boots Science Building, School of Pharmacy, University Park Campus, University of Nottingham, NG7 2RD, UK 2 UCB Celltech, UK ∗

Corresponding author. E-mail: [email protected] (R. Fowler). The vitamin B12 transport pathway offers potential for enhancing the uptake of orally administered biologicals, including proteins, peptides and immunogens. The oral delivery of these large molecules is often impeded by the epithelial cell barrier and proteolysis occurring at the mucosal surfaces. Research efforts have been made to enhance oral delivery by employing carrier molecules or ligands conjugated to the pharmaceutically active component, capable of exploiting specific receptor-mediated uptake (RME) to provide their co-absorption. One of the few potential ligands available for enabling transcytosis across the epithelium is vitamin B12. There are several sites on vitamin B12 molecule that are suitable for modification to form bioconjugates. The route followed in this work examined the preactivation of the 5 -hydroxyl group on the ribose moiety by the use of carbonyldiimidazole (CDI), followed by attack of a nucleophile to furnish the hexanediamine spacer. The resultant ␣␻-aminohexylcarbamate VB12 derivative was conjugated to fluorescent carboxy-functional nanoparticles (<200 nm size), for use as a model for potential therapeutic carriers. These systems were applied to confluent Caco-2 monolayers, which characteristically form tight junctions. Although several cell lines express the IF-B12 receptor responsible for the binding, internalisation and transcytosis of VB12, the Caco-2 cell line was chosen as the preliminary in vitro model to study the potential of the VB12 transport system for the delivery of VB12conjugated nanoparticles. Immunostaining and confocal microscopy were used to verify receptor/transport protein expression by the cells, as an essential prerequisite for ligandbased transcytosis. We demonstrate that the surface modification of nanoparticles with the ␣-␻-aminohexylcarbamate derivative of vitamin B12 enables their resultant uptake and transport in the apical-basolateral direction of

Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

A27 Efficient gene delivery using acidresponsive lipid envelopes for adenovirus

doi:10.1016/j.drudis.2010.09.376

Jeroen Van den Bossche ∗ , Wafa T. Al-Jamal, Acelya Yilmazer, Kostas Kostarelos Nanomedicine Lab, Centre for Drug Delivery Research Centre, The School of Pharmacy, University of London, London WC1N 1AX, United Kingdom

A26 Four-wave mixing imaging to study protein entry and release in mammalian cells Francesco Masia, Wolfgang Langbein, Paola Borri, Peter Watson ∗ School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3AX, United Kingdom ∗

Corresponding author. E-mail: [email protected] (P. Watson). Optical microscopy is a powerful tool for tracking the binding, internalisation and subcellular trafficking of delivery vectors to mammalian cells. By exploiting multiphoton processes, subcellular structures can be imaged with intrinsic three-dimensional (3D) spatial resolution. Common fluorescent labels in multiphoton microscopy include organic fluorophores, which suffer from photobleaching, and quantum dots which are more photostable but contain cytotoxic elements (such as Cd or In). Gold nanoparticles (GNPs) are ideal optical labels in terms of photostability and bio-compatibility, but emit weak fluorescent signal. We have developed a novel multiphoton microscopy technique that exploits the thirdorder nonlinearity called four-wave mixing (FWM) of GNPs in resonance with their surface plasmon. In terms of imaging performances, FWM microscopy features a spatial resolution better than the one-photon diffraction limit and optical sectioning capabilities. We show high-contrast background-free imaging of goldlabels (down to 5 nm size) and sensitivity to the single particle level. We are also able to demonstrate a directed dissociation of the GNP from bound proteins at their surface. These results pave the way for active tracking of conjugated nanoparticles, before the controlled release of therapeutically relevant proteins to a localised site of interest. doi:10.1016/j.drudis.2010.09.377

∗

Corresponding author. E-mail: [email protected] (J. Van den Bossche). Gene therapy involves the delivery of a functional gene by a vector into target cells, resulting in a desired therapeutic effect. Adenovirus (Ad) has shown a great promise in gene therapy [1,2]. However, in vivo studies have reported an immunogenic response and an overwhelming accumulation and gene expression in the liver resulting in significant hepatoxicity. These issues currently inhibit the use of this vector for use in clinical therapies. Such limitations have been overcome by engineering artificially enveloped Ad using zwitterionic and cationic lipid bilayers [3,4]. However, this resulted in a significant reduction of gene expression in vitro. We observed that this may be due to poor release of the Ad from its lipid envelope. In the present work, we have explored the use of pH-sensitive DOPE:CHEMS lipid-envelopes to stimulate the virus release from the envelope and consequently result in higher levels of gene expression. Artificially enveloped Ad (DOPE:CHEMS:Ad) were prepared by lipid film hydration followed by sonication. The physicochemical characteristics of the resulting hybrid biomaterials were characterised by transmission electron microscopy, atomic force microscopy, dot blot, dynamic light scattering and zeta potential measurements. The enveloped viruses exhibited good stability at physiological pH (7.4) but immediately collapsed and released naked virions at pH 5.5. Furthermore, recombinant Ad encoding for beta-galactosidase (␤-gal) enveloped in DOPE:CHEMS showed comparable levels of gene expression to naked Ad in different cell lines. These transfection results were further confirmed by studying the intracellular trafficking of fluorescently labelled, Cy3-Ad using confocal laser scanning microscopy (CLSM). Interestingly, Cy-3 Ad enveloped in DOPE:CHEMS showed a uniform fluorescence distribution within the cytoplasm indicating Ad endosomal release. In addition, pH-sensitive enveloped Ad injected directly into human cer-

vical adenocarinoma (C33a) xenografts grown on the flank of nude mice showed similar levels of gene expression to naked Ad. In conclusion, this type of artificially enveloped Ad offers a promising tool in gene delivery since high level of Ad gene expression can be maintained while one can expect to dramatically improve the innate Ad immunogenicity and hepatotoxicity in vivo. References 1. Benihoud K, et al. Curr Opin Biotechnol 1999;10:440–7. 2. Kovesdi I, et al. Curr Opin Biotechnol 1997;8:583–9. 3. Singh R, et al. ACS Nano 2008;2:1040–50. 4. Singh R, et al. FASEB J 2008;22:3389–402.

doi:10.1016/j.drudis.2010.09.378

A28 In vitro silencing of TGF␤1 in a corneal epithelial cell line using nanoparticles Isabel Arranz-Valsero 1,2,∗ , Jenny E. Párraga 3 , Antonio López-García 1,2 , Laura ˜ Seijo 3 , Alejandro Contreras-Ruiz 1,2 , Begona Sánchez 3 , Yolanda Diebold 1,2 1 Ocular Surface Group, Instituto Universitario de Oftalmobiología Aplicada, University of Valladolid, Valladolid, Spain 2 Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain 3 NANOBIOFAR Group, Department of Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Santiago de Compostela, Spain ∗

Corresponding author. E-mail: [email protected] (I. ArranzValsero). Introduction: Severe ocular inflammatory disorders constitute a sight-threatening group of diseases that present treatment difficulties due to the intrinsic barriers of the ocular surface. Previous work in our group has demonstrated that epithelial cells from human cornea (HCE cell line) basally secrete TGF␤1 (a commonly detected cytokine in ocular inflammatory diseases). At present, gene therapy (including siRNA-based therapies) holds promise for the treatment of several diseases, including ocular disorders. However, the development of safe and effective delivery vehicles still remains a major challenge for its clinical application. Purpose: This work is a proof-of-concept study meant to evaluate the efficacy of the in vitro gene silencing technique for different siRNAs targeting relevant pro-inflammatory cytokines

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Caco-2 cells through exploitation of the natural receptor governed processes involved in VB12 absorption.

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involved in ocular surface inflammation. We also want to determine whether the use of nanoparticulated drug delivery systems, based on cationized gelatine and chondroitin sulfate, as carriers for siRNAs improve the level of gene silencing. Methods: HCE cells were transfected with specific siRNAs against TGF␤1 and its Receptor 2 (TGFBR2) or against GAPDH as a negative control. Lipofectamine was used at 1.6 ␮l/well in 24-well plates and different siRNA concentrations from 20 to 300 nM were assayed. Silencing efficacy was tested, comparing Lipofectamine2000- or Nanoparticle-based transfection, at protein and RNA levels. Potential toxicity was evaluated by means of the XTT test. Results: TGF␤1 and TGFBR2 silencing reached 70% at the RNA level (measured by quantitative real-time-PCR) when using Lipofectamine. Lower silencing was detected at the protein level (measured by Western blotting or ELISA). However, the use of nanoparticles did not significantively improve the silencing efficacy of the evaluated siRNAs. siTGF␤1- and siTGFBR2-transfected cells showed viability percentages equivalent to those of control untransfected cells. CONCLUSION: It is possible to silence in vitro TGF␤1 and TGFBR2 expression in a corneal epithelial cell line by conventional techniques obtaining acceptable silencing levels while maintaining high cell viability. The use of nanoparticles as siRNA vehicles to improve silencing levels requires further studies Acknowlegements FEDER-CICYT MAT2007-64626-C02-01 (Ministry of Science, Spain) and Junta de Castilla y León Pre-doctoral Scholarship Program (Spain). doi:10.1016/j.drudis.2010.09.379

A29 Efficient siRNA delivery and effective gene silencing by lipoplexes Abdelkader A. Metwally, Charareh Pourzand, Ian S. Blagbrough ∗ Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK ∗

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Corresponding author. E-mail: [email protected] (I.S. Blagbrough). siRNA is double-stranded RNA typically 21–24 nucleotide base-pairs long. Gene silencing by siRNA has gained wide acceptance in genomics and is already in different phases of clinical trials as a potential therapeutic. Long chain fatty acid conjugates of spermine have previously been synthesized and evaluated in our research group for both gene and siRNA delivery [1,2]. We report the synthesis

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of two novel unsymmetrical N4,N9-difatty acid conjugates of the naturally occurring polyamine spermine with the aim of developing structure–activity relationships for their potential as non-viral, self-assembly vectors for siRNA delivery. After transfection with lipoplexes of Alexa Fluor® 647-labelled siRNA (a 24-mer from Qiagen), silencing EGFP expression, both the efficiency of delivery and the effectiveness of knock-down (gene silencing) were evaluated in HeLa cells stably expressing EGFP. Analysis was by FACS 48 hours post transfection. All transfection experiments were carried-out in DMEM containing 10% foetal calf serum. The efficiency of intracellular delivery was measured by the (normalized) fluorescence of Alexa Fluor® 647-labelled siRNA; N4,N9-dioleoylspermine (DOS) showed 150% of the delivery efficiency achieved with N4linoleoyl-N9-oleoylspermine (LOS). However, knock-down results show that LOS is more effective with a reduction of EGFP expression levels from control (100%) to 25 ± 3% at a concentration of 3 ␮g/well (N/P = 11, n = 3 and triplicate replicates). Under the same experimental conditions, DOS reduced EGFP expression to 27 ± 2% at a concentration of 6 ␮g/well (N/P = 22) and to 32 ± 2% at a concentration of 3 ␮g/well (N/P = 11). Cell viability was measured as the percentage of viable cells using the Alamar Blue® assay [3]. The results show that at 3 ␮g/well LOS cell viability is 83 ± 4%, at 6 ␮g/well LOS cell viability is 46 ± 8%, while at 6 ␮g/well DOS cell viability is only 32 ± 9%. Transfection of cells with LipofectamineTM 2000 resulted in reduction of EGFP expression to 37 ± 3%, with cell viability of 91 ± 6%. We conclude from these results that the unsymmetrical lipopolyamine LOS is an excellent transfecting agent for the delivery of siRNA producing effective gene silencing in the presence of 10% foetal calf serum. Acknowlegements We thank the Egyptian Government for a fully-funded studentship to AAM. References 1. Ghonaim HM, et al. Very long chain N4,N9diacyl spermines: non-viral lipopolyamine vectors for efficient plasmid DNA and siRNA delivery. Pharm Res 2009;26:19–31. 2. Ghonaim HM, et al. N1,N12-diacyl spermines: SAR studies on non-viral lipopolyamine vectors for plasmid DNA and siRNA formulation. Pharm Res 2010;27:17–29. 3. Asasutjarit R, et al. Effect of solid lipid nanoparticles formulation compositions on their size, zeta potential and potential for in vitro

pHIS-HIV-Hugag transfection. Pharm Res 2007;24:1098–107.

doi:10.1016/j.drudis.2010.09.380

A30 Peptide dendrimer based drug delivery system Kui Luo, Hui Yuan, Bin He, Yao Wu, Zhongwei Gu ∗ National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China ∗

Corresponding author. E-mails: [email protected] (B. He), [email protected] (Z. Gu). In the past decades, dendrimers have been extensively studied for their unique properties such as spherical nanostructure, monodistributed size and numerous peripheral functional groups. Peptide dendrimers, which were synthesized from amino acids, have been reported as biomaterials for disease diagnosis and treatment due to their excellent biocompatibility and degradability. Herein, we reported the synthesis of peptide dendrimers and their biomedical applications as molecular probes for magnetic resonance imaging (MRI) and carriers for drug/gene delivery. The synthesis of peptide dendrimers was according to a previously reported method [1]. The dendrimers with different generations were synthesized and functionalized. Targeting moieties, mPEG, Ga-DTPA complexes and anti-tumor drugs were immobilized on the peripheral groups of the dendrimers. The dendrimers immobilized Ga-DTPA complexes were used as MRI molecular probes and the relaxivity of contrast was tested on 1.5 T MRI both in vitro and in vivoin. The generations of dendrimers were 2, 3, 4, and galactosyl moiety was used as targeting ligand for liver imaging. The relaxivity of the contrasts were measured and for G4 dendrimer was 100.8 mM-1•S-1, which was much higher than that of the commercial Ga-DTPA product. The signal intensities were determined by choosing an appropriate region of interest in mouse liver tissue. After 10 minutes injection, the SI increase in liver tissue was observed with an averaged enhancement of 43% for G3T and 37% for G4T, respectively. The non-specific dendritic agents G2, G3 and G4 showed low SI increases. The dendritic probes of G2T, G3T and G4T showed 25%, 35% and 34% relative enhanced SI after 1 hour injection. The peptide dendrimers were fabricated gene vectors and gene transfections of generation 3, 4 and 5 of peptide dendrimers were compared, the

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Reference 1. Luo K, et al. Macromol Biosci 2009;9:1227–36.

doi:10.1016/j.drudis.2010.09.381

A31 Pyridylhydrazone-based PEG for pHreversible lipopolyplex shielding Yu Nie 2 , Michael Günther 1 , Zhongwei Gu 2 , Ernst Wagner 1,∗ 1 Pharmaceutical Biology-Biotechnology, Department of Pharmacy, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, D-81377, Munich, Germany 2 National Engineering Research Center for Biomaterials, Sichuan University, Wangjiang Road. 29, 610041, Chengdu, PR China ∗

Corresponding author. E-mails: [email protected] (Z. Gu), [email protected] (E. Wagner). PEGylation that is reversed after the therapeutic agent reaches the target cell presents an attractive feature for drug, protein or nucleic acid delivery. Amine-reactive, endosomal pH cleavable ␻-2-pyridyldithio poly(ethylene) glycol ␣-(butyraldehyde)-carboxypridylhydrazone N-hydroxysuccinimide ester (OPSS-PEGHZN-NHS) was synthesized and applied for bioreversible surface shielding of DNA lipopolyplexes. N1-cholesteryloxycarbonyl-1,2diaminoethane was reacted with pH-sensitive (OPSS-PEG-HZN-NHS) or the corresponding stable (OPSS-PEG-NHS) reagent. Both types of micelles remained shielded at pH 7.4 as demonstrated by size exclusion column separation after 4 hours of incubation at 37 ◦ C. But only disruption of OPSS-PEG-HZN-Chol micelles was observed at endosomal pH 5 in 30 min, while OPSS-PEG-Chol was almost stable for 8 h in the same conditions. Lipopolyplexes composed of DNA condensed with polyethylenimine (PEI),

dioleoyl phosphatidylethanolamine (DOPE) and hydrazone linked pH labile lipid Chol-HZNPEG were prepared by the ethanol injection technique, with particle size of 160 nm and zeta potential of 8 mV. Pyridylhydrazone-based PEGylated lipopolyplexes was as stable as their non-pH sensitive counterparts at physiological conditions, and had smaller size compared with non-PEGylated variants. At pH 5.4, increasing size was only detectable in pH-reversible lipopolyplexes. Both luciferase and EGFP gene transfections of pH-reversible lipopolyplexes showed an up to 40-fold enhancement in gene expression with reversibly shielded polyplexes compared to stably shielded lipopolyplexes. Investigation of cellular association and uptake by flow cytometry, together with intracellular tracking by CLSM reveal the probability of intracelluar deshielding of PEG. Incorporation of a ligand for transferrin receptor targeting further improved the transfection. doi:10.1016/j.drudis.2010.09.382

A32 The 5th generation of poly(L-lysine) dendrimer is a potential carrier for in vivo in gene delivery Gang Wang, Caixia Li, Kui Luo, Hongmei Song, Zhongwei Gu ∗ National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, PR China ∗

Corresponding author. E-mail: [email protected] (Z. Gu). Poly(L-lysine) dendrimers have been widely used as reagents for in vitro gene transfecion. Here, different generations of dendritic poly(L-lysine)s were synthesized, including G3, onium salt G3 (OG3), G4 and G5, and their characteristics for in vitro gene transfection and potentials as in vivo gene delivery carriers were evaluated. Gel retardation assays proved that the dendrimers could form complexes with plasmid DNA, and dendrimer G3 could inhibit the migration of pDNA at an N/P ratio of 0.5, G4 and G5 at N/P ratio of 1.0 and onium salt G3 at N/P ratio of 2.0. A DNase I protection assay with G5 showed acquired resistance from combining pDNA with dendrimer; this can resist the nuclease-catalyzed degradation, and the protection capacity of G5 was even stronger than that of PEI. Atomic force microscopy demonstrated that all the 4 generations of dendrimer/DNA complexes showed similar particle size within 100–200 nm. At N/P ratios from 1 to 25, zeta potentials of

the 4 dendrimer/pDNA complexes gradually changed from negative to positive with a tendency that the higher generation and higher potential value variants gave a stronger combination potency of the complex with negatively charged cell membranes. In vitro cytotoxicity evaluation showed good biocompatibility of each dendrimer within N/P ratios of 1–25. Body weight evaluation of BABL/c mice, together with tissue section observation, blood routine detection and blood biochemistry analysis (liver and kidney function, myocardial enzymes and electrolytes, etc.) of dendrimer G5 also showed good in vivo biocompatibility 2 and 7 days after tail vein injection. In vitro gene transfection comparison revealed that G5 had an obvious higher efficiency than other dendrimers. Transfection efficiencies of each dendrimer were not influenced by the presence of serum, which is a very important merit for in vivoin gene delivery. Quantitative analysis in mRNA and protein level showed that the transfection efficiency of dendrimer G5 was ∼60% of PEI’s, but PEI had obvious toxicity to cultured cells and its transfection efficiency would be greatly reduced by the presence of serum. Considering that dendrimer G5 had almost the same in vitro gene transfection efficiency as G6, we concluded that the fifth generation of poly(L-lysine) dendrimer should be a suitable carrier for in vitro gene transfection and, more importantly, a potential carrier to construct in vivo gene delivery system. doi:10.1016/j.drudis.2010.09.383

A33 Muscle-targeted HIF-1a gene expression system for therapeutic angiogenesis in ischemic limbs Hongmei Song, Caixia Li, Gang Wang, Zhongwei Gu ∗ National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, Sichuan, PR China ∗

Corresponding author. E-mail: [email protected] (Z. Gu). Therapeutic angiogenesis is expected to be a promising treatment for patients with ischemic disorders such as cardiac and limb ischemia. However, recent clinical trials failed to show much expectant benefits, largely due to suboptimal therapeutic genes and delivery strategies. Herein, we focused on the development of a hypoxia inducible factor-1a (HIF-1a) gene induced muscle-specific angiogenesis strategy that would improve safety and effi-

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results demonstrated that G5 showed the highest gene transfection efficiency both in the medium with or without serum. Peptide dendrimer based drug delivery system was with dual targeting and pH-sensitive functions. Dendrimer–doxorubicin conjugates were synthesized via a pH sensitive bond. The drug release at pH 5.0 was much faster that that at pH 7.4. The sustained release time was as long as 20 hours and more than 90% of the immobilized drugs were released at pH 5.0. The in vitro anti-tumor effects of the dendrimer drug delivery system were investigated and it showed that the peptide dendrimer was a promising carrier for drug delivery.

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ciency. (1). A muscle-specific eukaryotic gene expression plasmid, pSV40E/MCK-HIF1a, was constructed by integrating SV40-enhancer with MCK promoter to regulate HIF-1a gene expression. (2) In vitro and in vivo studies both indicated that, compared with the natural MCK promoter, the SV40E/MCK hybrid promoter Q1 significantly increased HIF-1a gene expression, while retaining a good muscle-cell specificity. Although less efficient than the nonspecific CMV promoter, the hybrid promoter provided more stable gene expression and represented a good compromise between transcriptional activity and muscle specificity. (3) In vitro biological effects of increasing HIF-1a gene expression were analyzed in myoblasts to evaluate the function of the muscle-specific gene expression system. Real-time PCR showed upregulation of several critical angiogenic genes expression, such as VEGF, ANGPT-1, MMP-2 and SDF-1, which were previously demonstrated to facilitate new blood vessel formation and/or maturation. Transwell cell migration assay revealed that pSV40E/MCK-HIF1a transfected L6 cells could recruit progenitor cells derived from bone marrow and muscle tissue. These observations suggested the muscle-specific gene expression system may be useful for stimulating new blood vessel growth and maturation in ischemic limbs while restricting the therapeutic effect to muscle tissue. (4) When reporter gene was transferred into mice limb skeletal muscles, using various nonionic natural polymers, including hyaluronic acid, alginic acid and dextran, the formulated plasmid/polyer resulted in different levels of reporter gene expression, depending upon the type and concentration of the polymers. Some of them showed better performance than naked DNA and these results indicated that the pSV40E/MCK-HIF1a combined with a suitable nonionic polymer may provide a safe and efficient gene therapy system for treatment of limb ischemia. doi:10.1016/j.drudis.2010.09.384

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A34 Amphipathic CPPs upregulate Ca in cells’ cytosol and induce lysosomal exocytosis 1,2,3

1,2,3

A. Lorents , N. Oskolkov , T. Tõnissoo 1,2,3 , Ü. Langel 1,2,3 , M. Hällbrink 1,2,3 , M. Pooga 1,2,3,∗ 1 Institute of Molecular and Cell Biology, University of Tartu, 23A Riia Street, EE51010 Tartu, Estonia 2 Institute of Technology, University of Tartu, 23A Riia Street, EE51010 Tartu, Estonia 3 Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden ∗

Corresponding author. E-mail: [email protected] (M. Pooga). Cell penetrating peptides (CPP) promote the uptake of different bioactive cargo molecules that makes the harnessing of CPPs a promising strategy for drug design and delivery. The translocation mechanism of CPPs into cells, however, has still remained elusive. Direct passage of peptides across the plasma membrane might interfere with its integrity and introduce disturbances. In our study we assessed how cells compensate the disturbances and which processes are induced in response to CPP uptake. Applying fluorometry, flow cytometry and fluorescence microscopy we demonstrate that the uptake of various CPPs enhances the calcium levels in Jurkat and HeLa cells’ cytoplasm. The elevated cytoplasmic free calcium concentration evokes downstream effects of membrane repair response and lysosomal exocytosis. Our results indicate that ten of the most commonly used CPPs can be divided into three groups based on their interaction with plasma membrane, the induction of calcium influx, and downstream responses: (1) primary amphipathic CPPs (e.g. MAP, TP) that modulate plasma membrane integrity inducing influx of calcium ions into cells and activate membrane repair and lysosomal exocytosis starting, from low concentrations; (2) arginine-rich, secondary amphipathic, CPPs (e.g. Penetratin, pVEC) that induce changes in the intracellular calcium concentration or subsequent responses at relatively high concentrations and (3) non-amphipathic CPPs (e.g. Tat, Arg9) that do not evoke changes in the intracellular calcium concentration or subsequent responses even at high concentrations. Triggering of the plasma membrane repair response may help cells to recover by replacing the misorganized or membrane active CPPs containing plasma membrane regions,

whereas non-amphipathic CPPs could infiltrate without subsequent cellular responses. doi:10.1016/j.drudis.2010.09.385

A35 Cellular delivery of oligonucleotides by PepFect Kärt Padari ∗ , Carmen Juks, Nikita Oskolkov, Raivo Raid, Ülo Langel, Margus Pooga Institute of Molecular and Cell Biology, University of Tartu, 23A Riia Street, EE51010 Tartu, Estonia ∗

Corresponding author. E-mail: [email protected] (K. Padari). PepFect (PF) series of peptide based transfection reagents have been developed for the delivery of oligonucleotides (ON) and plasmids into cells. Some PFs are also capable of nuclear delivery of oligonucleotides, for example phosphorothioate 2 -O-methyl RNA oligomers translocate into nucleus and rescue the luciferase expression in the splicing redirection assay after coupling to PF. The optimal ratio of ON with PF for obtaining the functional complexes has been described earlier, but it is not known how such particles interact with the cell surface, enter cells, and reach nucleus. In order to characterize the oligonucleotide delivery by PFs, we labelled 2 -OMe ON with 1.4 nm Nanogold (NG) particles. The membrane interaction, uptake, and intracellular traffic of ON–NG after complexing with PFs were mapped by transmission electron microscopy to unravel their internalization mechanism. PFs pack the Nanogold-labelled ON into small (∼200 nm) particles in solution. Smaller particles of ON–NG–PF complexes associate later to form bigger assemblies at the surface of HeLa cells and are taken up by cells in vesicles. The size, electron density and regularity of ON–NG–PF containing structures vary largely depending on the PepFect and its concentration. In cells the majority of the complexes locate in the endosomal/lysosomal vesicles after four hours of incubation. However, the vesicles often have a discontinuous membrane and the Nanogold-labelled oligonucleotides can be found in the cytosol. In addition, with the help of some PFs, the oligonucleotides also reach the cell nucleus. Our results demonstrate that non-covalent complexes of Nanogoldlabelled oligonucleotides with PepFects form particles that concentrate at the cell surface and enter cells by endocytotic mechanism. The finding that oligonucleotides have reached nucleus suggests that ON–PF complexes could induce the destabilization of endosomal mem-

branes, followed by the escape of ON from vesicles and translocation into nucleus. Our electron microscopy results are in line with data published earlier regarding the redirection of splicing with oligonucleotides delivered into cells by PFs. doi:10.1016/j.drudis.2010.09.386

A36 Live-cell imaging and single-particle tracking of polyplex internalization Nadia Ruthardt ∗ , Karla de Bruin, Kevin Braeckmans, Ernst Wagner, Christoph Bräuchle Ludwig-Maximilians-Universität München, Department Chemie und Biochemie, Butenandtstr. 5-13, Haus E, 81377 München, Germany ∗

Corresponding author. E-mail: [email protected] (N. Ruthardt). Systemic delivery of therapeutic genes for gene therapy or cancer gene therapy requires gene vectors that overcome several barriers. The vector has to enable tissue-selective delivery, internalize efficiently and finally release its cargo reliably within the target cell. Tissue specificity and enhanced internalization can be achieved by cell-specific ligands that bind to certain surface markers that are upregulated in, for example, solid cancers. Functionalization with pH-sensitive and redox-sensitive linkers or polymers allows the vector to ‘sense’ external stimuli that will trigger their activation in temporally and spatially controlled manner. We investigate the uptake of targeted and untargeted polymeric gene vectors (polyplexes) by highly sensitive fluorescence microscopic methods on a single cell level [1]. The epidermal growth factor receptor (EGFR) is overexpressed on a high percentage of human carcinomas and is therefore an attractive therapeutic target for tissue-specific targeting by non-viral vectors in cancer gene therapy. Comparing uptake kinetics and internalization dynamics, single particle tracking in combination with quenching experiments revealed typical three-phase dynamics of the uptake process independent of targeting. Phase I was characterized by slow, actin-cytoskeleton-mediated movement of the particles with drift and included the internalization process. During phase II, particles displayed increased velocities with confined and anomalous diffusion in the cytoplasm. Phase III was characterized by fast active transport along microtubules. Targeting of polyplexes for receptor-mediated endocytosis

by the EGF receptor resulted in shortening of phase I and strongly accelerated internalization. Targeted as well as untargeted particles were transported in early endosomes marked by Rab5–GFP and accumulated in late endosomes marked by Rab9–GFP. The endosomal release dynamics of polyplexes consisting of DNA condensed with the cationic polymers linear polyethyleneimine (LPEI), poly-(L)-lysine (PLL) or poly-(D)-lysine (PDL) were studied by photochemical release in living cells [2]. Using double-labeled polyplexes, DNA and polymer were imaged simultaneously by dual-color fluorescence microscopy. Our results demonstrate that the characteristics of the cationic polymer significantly influence the release behavior of the polyplexes. For LPEI/DNA particles, LPEI quickly spread throughout the cytosol, whereas DNA was released slowly and remained immobile thereafter. In the case of PLL particles, both DNA and polymer showed quick release. PDL particles remained condensed upon photosensitizer activation. Reference 1. de Bruin K, et al. Cellular dynamics of EGF receptor-targeted synthetic viruses. Mol Ther 2007;15:1297–305. 2. de Bruin KG, et al. Dynamics of photoinduced endosomal release of polyplexes. J Control Release 2008;130:175–82.

doi:10.1016/j.drudis.2010.09.387

A38 Vascular endothelium remodeling in human African trypanosomiasis L. Sanderson, M. Dogruel, S.A. Thomas ∗ King’s College London, Pharmaceutical Sciences Division, Hodgkin Building, Guy’s Campus, London Bridge, London SE1 1UL, UK ∗

Corresponding author. E-mail: [email protected] (S.A. Thomas). Molecule movement into the central nervous system (CNS) is restricted by the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier. Human African trypanosomiasis (HAT) or sleeping sickness, caused by the parasites, Trypanosoma brucei (T.b.) gambiense or T.b. rhodesiense, is fatal if untreated. The first disease stage is associated with trypanosome proliferation in the periphery. The second stage is when the parasites reach the CNS. HAT treatment is stage specific with drugs, which are assumed to cross the BBB, used to treat CNS stage disease. Since the treatment of CNS-stage HAT is more toxic than that of early-stage, it is vital to stage HAT

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[1]. Staging requires a CSF sample. Lumbar puncture under field conditions is difficult and invasive. Improved tests for staging HAT are required [2]. Our studies have established that T.b. brucei crosses the murine blood–CNS interfaces at ∼day 11 post-infection (p.i.) and the animals died at day 37.9 ± 1.23. At day 7, 14 and 21 p.i. no loss of barrier integrity was measurable using the inert tracer, [14 C]sucrose ˚ nor was there any (342 Da; radius 4.6 A), endothelium remodeling (including transporter up/downregulation) as measured with eflornithine, pentamidine or nifurtimox [3,4]. BBB, but not choroid plexus, dysfunction, occurred at days 28 and 35 p.i. with resultant increases in [14 C]sucrose space [3,4]. Suramin (1429 Da) brain distribution increased at day 35 p.i., suggesting considerable BBB breakdown as this molecule is highly albumin (60 kDa; radius ˚ bound [4,5]. Furthermore, the increased 35.5 A) [14 C]sucrose association with the endothelial cell at day 35 p.i. compared to the non-infected and other infected time groups suggested an increase in vesicular trafficking [3]. This loss of integrity may be a sign of terminal disease. However, perhaps there was an earlier loss of blood–CNS barrier integrity (possibly when the parasites entered the CNS) that was not measurable using [14 C]sucrose (an inert tracer with smaller molecular dimensions being needed) and/or this was a reversible process that was undetectable at the times studied. Furthermore, endocytosis may be a sensitive marker of endothelium remodeling. The characterization of vesicular expression in a murine model of HAT may be the first step towards vesicle targeted staging strategies. Overall understanding blood–CNS barriers breakdown in HAT could contribute to the development of therapeutics and therapeutic targets to control brain injury and to the characterization of biomarkers for safer staging of the disease. Funding The Wellcome Trust (grant codes: 073542; 080268). Reference 1. Kennedy PG. Trans R Soc Trop Med Hyg 2008;102:306–7. 2. Hainard A, et al. PLoS Negl Trop Dis 2009;3:e459. 3. Sanderson L, et al. J Neurochem 2008;107:1136–46. 4. Sanderson L, et al. J Pharmacol Exp Ther 2009;329:967–77. 5. Sanderson L, et al. Antimicrob Agents Chemother 2007;51:3136–46.

doi:10.1016/j.drudis.2010.09.388

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Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

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A39 Preparation of solid DNA nanoparticles for use in gene therapy Martina Hanzlíková 1,∗ , Janne Raula 3 , Juho Hautala 3 , Esko Kauppinen 3 , Arto Urtti 2 , Marjo Yliperttula 1 1 Division of Biopharmaceutics and Pharmacokinetics, Finland 2 Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, FI-00014 Helsinki, Finland 3 NanoMaterials Group, Department of Applied Physics, Aalto University, P.O. Box 15100 FI-00076 Aalto, Finland ∗

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Corresponding author. E-mail: martina.hanzlikova@helsinki.fi (M. Hanzlíková). Background: Non-viral gene therapy, based on nanosized particles, is a potential therapeutic option in various diseases. The success is mainly dependent on an efficient gene delivery vector. Aerosol synthesis can provide pure solid DNA particles with substantial high dose of DNA per particle and thereby increase the amount of DNA delivered into cells as compared to commonly used DNA polymer complexes. The purpose of this study was to test the suitability of plasmid DNA alone or in complex with cationic polymers for the preparation of solid DNA nanoparticles by an aerosol flow reactor method. Methods: The sample solutions contained either plasmid DNA (pDNA) alone or complexed at a ratio of 1:1 (w/w) with branched or linear polyethylenimine (PEI) with the molecular weight of 25 kDa. The additive agents, L-leucine and mannitol, were added to PEI/DNA complexes at a ratio of 1:8 (w/w). The aerosol flow reactor method [1] involved atomization of sample solutions to nanosized droplets, which were immediately dried in a heated flow reactor tube by the evaporation of the solvent. The dried nanoparticles were collected with a low-pressure impactor and the size distribution was determined by a differential mobility analyzer. The surface morphology was analyzed using field emission scanning electron microscopy and the structural integrity of pDNA was evaluated by agarose gel electrophoresis. Results: The produced pure pDNA nanoparticles were spherical and had a mean diameter of 125 nm. However, pDNA in such nanoparticles did not preserve its supercoiled structure due to the shearing stresses caused by the atomization process. The complexation of pDNA with PEIs before atomization allowed the maintanence of pDNA integrity. The further

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addition of either L-leucine or mannitol to initial sample solution, stabilized nanoparticles structure and prevented them from water uptake and subsequent deformation. The resulting solid nanoparticles had a mean size between 65 and 125 nm and the loading content of pDNA in a single nanoparticle was approximately 10% (w/w). Conclusions: The aerosol flow reactor method provides an effective way of producing solid DNA nanoparticles with a size optimal for cell uptake and for potential use in non-viral gene delivery Reference 1. Raula J, et al. Influence of the solvent composition on the aerosol synthesis of pharmaceutical polymer nanoparticles. Int J Pharm 2004;284:13–21.

doi:10.1016/j.drudis.2010.09.389

A40 Exploiting a bacterial toxin translocation domain for the endosomal escape of CPP-imported cargoes Arshiya F. Mohammed ∗ , Amaalia E. Broad, Jean Gariepy ∗ Department of Medical Biophysics, University of Toronto, Sunnybrook Research Institute, 2075 Bayview Avenue, M4N3M5 Toronto, Ontario, Canada ∗

Corresponding author. E-mails: [email protected] (A.F. Mohammed), [email protected] (J. Gariepy). The clinical impact of CPP-based delivery agents has yet to be realized due to a lack of delivery efficiency to the cell cytoplasm. Polycationic cell penetrating peptides are a major class of CPPs. However, upon internalization via endocytosis, these CPPs are typically trapped in endosomes and are subsequently degraded or recycled out of cells. To promote endosomal escape, we investigated the use of a bacterial protein domain derived from Pseudomonas aeruginosa, Exotoxin A (ETA253-412), capable of translocating known protein domains out of vesicular compartments. We constructed, expressed, and purified a series of CPP–ETA253412–eGFP fusion proteins. We used confocal microscopy and flow cytometry to confirm the internalization of CPP (poly-arginine or TAT)-containing constructs at 37◦ C in human cervical carcinoma (HeLa) cells. Additionally, we observed the time-dependent relocation of CPP-ETA253-412–eGFP constructs from the endosome to the cytosol. These experiments demonstrate the potential of the ETA253-412

translocation domain in relocating cargoes, such as protein therapeutics, siRNA and vaccine formulations, to the cytosol of target cells. Acknowlegements Supported by: CBCRA and CIHR. doi:10.1016/j.drudis.2010.09.390

A41 Investigation of microsphere-mediated cellular delivery R.M. Sanchez-Martın ∗ , L.M. Alexander, S. Pernagallo, A. Livigni, J.M. Brickman, M. Bradley Chemical Biology Section, School of Chemistry & Institute of Stem Cell Research, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK ∗ Corresponding author. E-mail: [email protected] (R.M. SanchezMartın). Recently we have developed a polystyrene microsphere-based system designed to efficiently deliver biological materials into a broad range of cell lines [1,2]. This versatile delivery system is capable of transporting any biological cargo from small molecules to oligonucleotides and bulky proteins into cells [3–5]. However, the specific mechanism of cellular entry is largely unknown and widely varies from study to study. As such, chemical, biological and microscopic methods have been used to elucidate the mechanism of cellular uptake for these nanoparticles in several cell lines. Additionally, gene expression profiling has been used to determine if there is a transcriptional response to ‘beadfection’ [6,7].

Reference 1. Sánchez-Martín RM, et al. ChemBioChem 2005;6:1341–5. 2. Sánchez-Martín RM, et al. In: Pignataro B, editor. Ideas in Chemistry and Molecular Sciences: Where Chemistry Meets Life. Wiley–VCH; 2010, 117–140 (Chapter 5). 3. Sánchez-Martín RM, et al. Angew Chem Int Ed 2006;45:5472–4. 4. Alexander LM, et al. Bioconjug Chem 2009;20:422–6. 5. Sánchez-Martín RM, et al. ChemBioChem 2009;10:1453–6. 6. Tsakiridis A, et al. Biomaterials 2009;30:5853–61. 7. Alexander LM, et al. Mol BioSyst 2010;6:339–409.

doi:10.1016/j.drudis.2010.09.391

A42 Cytosolic delivery of macromolecules using pH-dependent fusogenic peptide Ikuhiko Nakase ∗ , Sachiko Kobayashi, Shiroh Futaki Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

FITC–avidin effectively internalized into cells, and diffuse signals of the FITC–avidin in cytosol were observed. In the absence of these complexes, efficiency of cytosolic diffusion of the FITC–avidin was quite low. These results suggest the usefulness of our approach for intracellular delivery of macromolecules using GALA peptide and cationic lipid [2].

bulk peptide solutions, and, had no effect at 5 ␮M and above. Overall, the results suggest the presence of concentration dependent uptake mechanisms of the Tat–LK15 peptide in cells.

Reference 1. Subbarao, et al. Biochemistry 1987;26:2964–72. 2. Kobayashi, et al. Bioconjug Chem 2009;20:953–9.

doi:10.1016/j.drudis.2010.09.393

∗

Corresponding author. E-mail: [email protected] (I. Nakase). The plasma membrane plays critical roles in maintaining cellular homeostasis. It serves as a barrier against unfavorable attack on cells from an unpredictable external world. However, the membranes are also barriers to intracellular delivery of various therapeutic molecules. For improving their translocation, we developed a novel method using GALA peptide/cationic lipid complexes. The GALA peptide (amino acid sequence: WEAALAEALAEALAEHLAEALAEALEALAA) is a 30-residue amphipathic peptide with a repeat sequence of glutamic acid–alanine–leucine–alanine, and designed to mimic the function of viral fusion protein sequences that mediate escape of virus genes from acidic endosomes into cytosol [1]. The GALA peptide converts its structure from random to helical when the pH is reduced from 7.0 to 5.0, and this leads to destabilization of the membranes. When attached with bioactive cargoes, the GALA peptide may thus serve as intracellular vector bearing efficient endosomal escape function. However, the negative charges from glutamic acids (seven residues) in the GALA sequences reduce the efficiency of binding to a negatively charged cell surface. To overcome this problem, a cationic lipid was employed as an ‘adhesive’ for pasting the GALA peptide onto cell surface to accelerate its cellular uptake. We examined the ability of GALA peptide as a delivery vector using FITC as a model of membraneimpermeable low-molecular weight drugs. When FITC–GALA (1 ␮M) was administrated to HeLa cells, co-addition of cationic lipid, Lipofectamine 2000 (LF2000), significantly increased uptake efficiency. In a time-dependent manner, FITC–GALA escaped from endosomes, and diffuse fluorescent signals were observed in both cytosol and nucleus, suggesting that the cytosolic translocation proceeds along with endosomal acidification. The GALA/cationic lipid system was also applied for the intracellular delivery of FITC–avidin protein (68 kDa). When FITC–avidin (250 nM) was mixed with biotinylated-GALA (1 ␮M)/LF2000 complexes,

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doi:10.1016/j.drudis.2010.09.392

A44 A dual uptake mechanism for the peptide Tat–LK15 Myasar Alkotaji 1,2,∗ , Alain Pluen 1,2 1 School of Pharmacy, University of Manchester, UK 2 Stopford Building, Oxford Road, Manchester, UK

Reference 1. Saleh AF, et al. Improved Tat-mediated plasmid DNA transfer by fusion to LK15 peptide. J Control Release 2010;143:233–42.

A45 Visualizing the effect of integrin targeting and surface shielding on gene vector uptake by live cell imaging F.M. Koenig ∗ , N. Ruthardt, Y. Vachutinsky, M. Oba, K. Miyata, K. Kataoka, C. Bräuchle Ludwigs-Maximilian Universität München, Physikalische Chemie I, Butenandt Str 11, Haus E, 81377 München, Germany

∗ Corresponding author. E-mail: [email protected] ∗ Corresponding author. (F.M. Koenig). E-mail: [email protected] Nonviral vectors enable the safe delivery of (M. Alkotaji). transgenes into target tissues but are still less Knowing the mechanism of uptake is fundaefficient than viral gene vectors. To develop mental in developing new and refining existing novel artificial systems with enhanced efficiendrug delivery systems. Recently, Tat–LK15 cies a detailed understanding of the cellular peptide resulting from the fusion of Tat pepuptake and intracellular trafficking is essential. tide and the synthetic amphipathic LK15 has By visualizing the entire pathway of a single shown greater transfection efficiency than Tat nanoparticle, from its first contact with the alone [1]. However, its uptake mechanism is cell surface to the delivery of the DNA to the ambiguous as the influence of the amphipathic cell nucleus, detailed information about the peptide (LK15) and Tat peptide upon binding mechanisms of uptake, intracellular traffickremains unclear. To elucidate this issue, the ing and DNA release can be gained. Here we present study investigates the effect of temstudy the effect of integrin targeting and surperature and peptide concentration on the face shielding on the internalization of gene cellular uptake mechanism of TAMRA–Tat–LK15 vectors by live cell imaging with highly sensipeptide. HeLa and HT29 cell lines were incutive fluorescent microscopy. ␣v␤3 and ␣v␤5 bated with 1, 2.5 or 5 ␮M TAMRA–Tat–LK15 integrin receptors are attractive targets for and TAMRA–Tat peptide solutions at differantiangiogenic cancer gene therapy as they ent temperatures (4◦ C, 20◦ C, and 37◦ C) or in play a pivotal role in angiogenesis and prothe presence of sodium azide (a metabolic liferation of malignant tumors. A cyclic RGD inhibitor). A Zeiss LSM510 microscope was used peptide specifically binds to those receptors to monitor cellular uptake in using a thermoand thus can be used for specific targeting electric controlled temperature chamber. Our of gene vectors such as polyplex micelles. In data indicate clearly TAMRA–Tat–LK15 peptide this study the analyzed micelles consisted of uptake (diffuse distribution in the cells) at 4◦ C a thiolated PEG-block-poly(lysine) copolymer for a 5 ␮M bulk solution while images do not complexed with fluorescently labeled DNA suggest uptake in these conditions at lower [1]. To analyze the influence of shielding, two concentrations (1 and 2.5 ␮M). At higher temtypes of micelles containing a differently sized peratures (20◦ C and 37◦ C) TAMRA–Tat–LK15 PEG shell were compared. To directly compare was observed in cells at all concentrations (a the internalization of targeted and untargeted mixture of diffuse and punctuated fluoresmicelles without knowing the details of their cence in cells). Interestingly, pre-incubation internalization pathway, we simultaneously with 10 mM sodium azide did not completely added both micelle types with different fluoblock peptide uptake in cells for 1 and 2.5 ␮M rescent labels onto cells and evaluated their

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colocalization degree over time. Additionally the internalization kinetics of integrin targeted micelles was compared to EGF targeted polyplexes that are well-known for their fast uptake kinetics [2]. The internalization pathway was then studied with inhibitor experiments and by colocalization with specific marker proteins. Our results reveal a strong competition between unspecific electrostatic interactions and specific receptor–ligand interactions that determines successful targeting of the micelles. Enhanced PEG shielding of the micelles leads to the reduction of electrostatic interactions resulting in a specific and faster internalization of the targeted micelles. Additionally we observed a considerable effect of the applied micelle concentration as well as the micelle size on their internalization properties. Our data lead to a more detailed understanding of the targeting effect than can be observed by conventional bulk instruments. The gained knowledge enables to maximize the therapeutic benefit of future gene vectors for clinical application. Reference 1. Oba M, et al. Cyclic RGD peptide-conjugated polyplex micelles as a targetable gene delivery system directed to cells possessing alphavbeta3 and alphavbeta5 integrins. Bioconjug Chem 2007;18:1415–23. 2. de Bruin K, et al. Cellular dynamics of EGF receptor-targeted synthetic viruses. Mol Ther 2007;15:1297–305.

doi:10.1016/j.drudis.2010.09.394

A46 A designer biomimetic vector for breast cancer gene therapy H.O. McCarthy 1,2,∗ , A.V. Zholobenko 1 , J.A. Coulter 1 , H.D. McKeen 1 , D.G. Hirst 1 , T. Robson 1 , A. Hatefi 2 1 School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK 2 Department of Pharmaceutical Sciences, Centre for Integrated Biotechnology, Washington State University, Pullman, WA, USA Delegate abstracts•MONITOR

∗

Corresponding author. E-mail: [email protected] (H.O. McCarthy). Introduction: Gene therapy holds the potential to cure many diseases, provided that the genetic or molecular basis is understood. In cancer, the delivery of therapeutic genes via viral vectors has proven more effective than the current alternative non-viral methods. However tissue specificity, high costs of production and safety remain major concerns with viral delivery. This study examines the use of 1096

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a designer biomimetic vector (DBV), that is essentially a recombinant fusion protein, to deliver the therapeutic inducible nitric oxide synthase (iNOS) gene to breast cancer. The DBV is composed of several discrete motifs each designed with single function architecture including: (a) a DNA condensing motif (DCM) obtained from the adenovirus mu peptide, (b) a ZR-75-1 breast cancer cyclic targeting peptide (TP) for specific delivery of the nanoparticles, (c) an endosomal disruption motif (EDM) that mimics the influenza virus fusogenic peptide and (d) a nuclear localization signal (NLS), rev, obtained from the human immune-deficiency virus type-1. We now use this DBV to deliver the cytotoxic iNOS gene in vitro and the GFP reporter gene in vivo to ZR-75-1 tumours. Methods: The DBV was expressed in Escherichia coli, extracted with affinity chromatography and purified using size exclusion chromatography. The DBV was complexed to piNOS to form nanoparticles which were used either for characterisation via electrophoretic mobility shift assays, serum stability assays or dynamic light scattering analysis. ZR-75-1 breast cancer cells were transfected with DBV/piNOS nanoparticles and toxicity was quantified using the WST-1 cell toxicity and clonogenic assays. Over expression of iNOS was also confirmed via western blotting and greiss test. Finally ZR-75-1 intradermal tumours were grown using SCID models and the DBV/pEGFP-N1 nanoparticles were delivered both intratumourally and intravenously. Tumours and organs were excised and the GFP distribution was determined. Results: The DBV was effectively expressed in E. coli at approximately 3 mg/l yield. The DBV condenses piNOS into spherical nanoparticles between N:P ratios of 4–10. At a N:P ratio of 9, piNOS was fully condensed with an average size of 75.1 nm. Transfection with the DBV/piNOS nanoparticles resulted in a maximum of 62% cell kill. INOS overexpression was confirmed and total nitrite levels were in the range of 18 ␮M and comparable with lipofectamine/piNOS. Finally 48 h after i.v. injection of the DBV/pEGFP-N1 nanoparticles GFP protein was detected in all the organs. The addition of chloroquine (30 mg/kg I.P.) did not enhance the expression of GFP indicating functionality of the EDM. Furthermore the addition of nocodazole (3 mg/kg I.P.) resulted in a reduction in GFP expression again indicating NLS functionality in vivo. Conclusions: The DBV/piNOS nanoparticles gave significant cytotoxicity in ZR-75-1 breast cancer cells in vitro and with less than 20% transfection this indicates a bystander effect. Despite a lack of tumour targeting by the DBV vector in vivo, the

data indicates that the DBV/pEGFP-N1 nanoparticles do not aggregate and can travel through the bloodstream with confirmation of gene expression in all the organs. Future studies will concentrate on using the human osteocalcin promoter (hOC) to transcriptionally target the iNOS plasmid to ZR-75-1 breast tumours. doi:10.1016/j.drudis.2010.09.395

A47 Cellular delivery and biological activity of metall complex-peptide conjugates Katrin Splith ∗ , Jan Hoyer, Ines Neundorf Leipzig University, Faculty of Biosciences, Pharmacy and Psychology, Department of Biochemistry, Brüderstr. 34, 04103 Leipzig, Germany ∗

Corresponding author. E-mail: [email protected] (K. Splith). Bioorganometallic chemistry has become more and more important in several fields, especially in the development of new drugs for cancer treatment. A number of metal-based building blocks have promising features for applications in therapy and diagnosis. Introduction of a metal centre could add new features that may help to overcome some problems in cancer treatment. However the low water solubility and bioavailability of these organometallic compounds inhibits their therapeutic use in medicine. Therefore intracellular delivery of therapeutics is the challenging task in medicinal chemistry research. Recently, socalled cell-penetrating peptides (CPP) have emerged as potent tools to introduce substances into cells. CPP are an inhomogenic group of peptides that share the ability to translocate in a large number of different cell-lines without the need of any receptor or transporter molecule. Thereby they are capable to transport various cargos inside cells, like proteins, oligonucleotides, nanoparticles or small organic drugs. This work describes the coupling of metal-based building blocks to cell-penetrating peptides based on an antimicrobial peptide cathelicidin CAP18 or on the human peptide hormone calcitonin (hCT). Synthesis was achieved by solid phase peptide synthesis using standard Fmoc chemistry and activation by HOBt/DIC. Several different metal complexes have been investigated, for example, half-sandwich-complexes of different metals as iridium, manganese, rhodium or iron. To introduce the potential metal-specific activity to the bioconjugate, up to two organometal moieties were coupled either N-terminally, to a

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See references below for additional reading 1. Stewart KM, et al. Org Biomol Chem 2008;6:2242–55. 2. Neundorf I, et al. Pharmaceuticals 2009;2:49–65. 3. Neundorf I, et al. Chem Commun 2008;43:5604–6. 4. Splith K, et al. Dalton Trans 2010;39:2536–45.

doi:10.1016/j.drudis.2010.09.396

A48 Polyelectrolite complex based microspheres for colon specific anticancer drug delivery M. Glavas Dodov 1 , N. Geskovski 1,∗ , B. Steffansen 2 , S. Kuzmanovska 3 , M. Simonoska Crcarevska 1 , V. Petrovska 1 , K. Goracinova 1 1 Institute for Pharmaceutical Technology, Faculty of Pharmacy, University Ss Cyril and Methodius, Macedonia 2 Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Denmark 3 Institute for Patophysiology and Nuclear Medicine, Medical Faculty, University Ss Cyril and Methodius, Macedonia ∗

Corresponding author. E-mail: [email protected] (N. Geskovski). Localized delivery of chemotherapeutic agents has long been the aim of clinical colon cancer therapy in order to limit the indiscriminate activity of many anti-cancer drugs on rapidly dividing cells, including normal tissues. The ideal drug delivery system (DDS) is envisioned to selectively and efficiently transport the anticancer drug to the target cells. It will not only minimize side effects associated with inappropriate drug distribution, but will also enhance therapeutic efficacy by increasing local drug concentration. The goal of our study was to develop wheat-germ aglutinin (WGA) functionalized chitosan-Ca-alginate microspheres (MS) loaded with acid-resistant nanoparticles (NP) of 5-FU, as colon targeting DDS and evaluate its in vitro efficacy and in vivo biodistribution. The rationale behind the design

of the formulation is the presence of high level of polysaccharides of microbial origin in the human colon and the possibility of direct binding of MS to the mucosal surface by nonspecific or specific ligand–receptor interactions using biological molecules (WGA), thus enabling active uptake of 5-FU in the target cancer cells. A simple one-step spray drying procedure was used to produce polyanion/polycation MS loaded with acid-resistant NP of 5-FU with mean diameter of ∼14.74 ␮m, high production yield (∼50%) and encapsulation efficiency (∼72%). Using 1,1 -Carbonyl-diimidazol as a surface group activation agent, successful conjugation of WGA to MS surface was achieved (∼50%). Haemagglutination test confirmed that WGA, treated by covalent coupling procedure, still retained its specific carbohydrate binding activity on the surface of MS. In vitro efficiacy was evaluated by investigating 5-FU permeability and [methyl-3H]thimidine uptake in Caco-2 cells. The cumulative amount of transported 5-FU through Caco-2 cells was 15.1% and 6.5% for 5-FU solution and WGA conjugated MS, respectively. Cell culture studies also indicated a marked decrease in [methyl3H]thimidine uptake for WGA decorated MS compared to 5-FU solution, suggesting that immobilization of WGA onto MS surface, due to the improved interaction and enhanced tissue accumulation of 5-FU could led to improved efficacy in targeted anticancer colon therapy. In vivo biodistribution studies were conducted with oral administration of 99m Tc labeled MS on fasted male Wistar rats. The imaging was performed at different time intervals post administration. The results showed that MS traversed fairly quickly through upper part of GI tract and resided in the colon for relatively longer period of time, probably due to the particle size, pH dependent swelling and surface properties of the MS. Overall, the results of this work showed that crosslinked polycation/polyanion MS loaded with 5-FU and decorated with WGA, were able to effectively deliver 5-FU to colon region, thus affecting the transport of 5-FU into the cells and consequently improving the efficacy. doi:10.1016/j.drudis.2010.09.397

A50 Engineering macrophages to synthesize recombinant adenoviruses in hypoxic areas of human prostate tumours Munitta Muthana ∗ , A. Giannoudis, S.D. Scott, R. Mistry, C. Murdoch, S. Coffelt, L. Georgeopolous, F. Hamdy, N. Brown, N. Maitland, C.E. Lewis Tumour Targeting Group, Department of Infection and Immunity, Medical School, Beech Hill Rd, University of Sheffield, Sheffield, UK ∗

Corresponding author. E-mail: m.muthana@sheffield.ac.uk (M. Muthana). Background: Like many other forms of human malignancy, prostate carcinomas contain multiple regions of transient and chronic hypoxia. New therapies targeting the hypoxic areas of tumours need to be designed as these sites are highly resistant to conventional cancer therapies. We have recently shown that macrophages accumulate in these hypoxic areas of prostate tumours, so we investigated the possibility of using these cells to deliver therapeutic genes to these otherwise inaccessible sites. Materials and methods: We designed a novel system in which macrophages are used to deliver hypoxia-regulated therapeutic adenovirus. In this approach, macrophages are co-transduced with a hypoxically activated E1A/B plasmid and an a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, whose proliferation is restricted to prostate tumor cells using prostate-specific promoter elements from the TARP, PSA and PMSA genes. Results: When co-cultured with prostate tumour spheroids, these ‘armed’ macrophages migrated into the hypoxic centres of the 3D tumour masses where E1A/B protein expression was upregulated, resulting in replication of the latent E1A/B-deficient adenovirus. Multiple copies of the virus (∼5000/macrophage) were released and infected neighbouring prostate tumour cells, resulting in widespread gene expression. Systemic administration of cotransduced macrophages into mice bearing human prostate xenografts resulted in their subsequent trafficking into the hypoxic areas of tumours leading to viral replication and widespread infection of neighboring tumour cells, resulting in the marked inhibition of tumor growth and reduction of pulmonary metastases. Conclusions: We show for the first time that macrophages can be engineered to express high titres of a therapeutic adenovirus

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amino acid side chain or in between two amino acids. Cellular uptake of the new bioconjugates was investigated with different methods like fluorescence microscopy, atom absorption spectroscopy or flow cytometry. High accumulation could be observed in different tumour cells. Furthermore, cell viability assays showed that those organometallic peptide conjugates are very potent and possess promising cytotoxic properties.

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specifically in hypoxic areas of human prostate tumours and that expression of the gene being delivered in the adenovirus can be restricted to prostate tumour cells by placing it under the control of a prostate-specific promoter (PSA). This novel approach employs three distinct levels of tumour-specific targeting; the homing of the macrophages to tumours, the synthesis and release of therapeutic adenovirus only in hypoxia tumour areas, and the targeting of therapeutic gene expression to prostate tumour cells. doi:10.1016/j.drudis.2010.09.398

A51 Targeted nanodrug delivery systems for the treatment of tuberculosis Yolandy Lemmer 1,3,∗ , Boitumelo Semete 1 , Laetitia Booysen 1,2 , Lonji Kalombo 1 , Lebogang Katata 1 , Arwyn T. Jones 4 , Cameron Alexander 5 , Makobetsa Khati 6 , Hulda S. Swai 1 , Jan A. Verschoor 3 1 Council for Scientific and Industrial Research, Polymers and Bioceramics, Pretoria 0001, South Africa 2 Department of Pharmaceutics, North-West University, Potchefstroom Campus, Potchefstroom 2520, South Africa 3 Department of Biochemistry, University of Pretoria, Pretoria 0001, South Africa 4 Welsh School of Pharmacy, Cardiff University, United Kingdom 5 School of Pharmacy, University of Nottingham, UK 6 Council for Scientific and Industrial Research, Biosciences, Pretoria 0001, South Africa ∗

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Corresponding author. E-mail: [email protected] (Y. Lemmer). South Africa currently has the highest incidence of TB in the world at 358 per 100,000 people. In 2007 alone 112,000 people died of TB in South Africa, of which 94,000 (72%) were co-infected with HIV [1]. Although TB treatments exist, poor patient compliance and drug resistance pose a great challenge to programs worldwide. To improve the current inadequate therapeutic management of TB, a polymeric anti-TB nanodrug delivery system, for anti-TB drugs, was developed that could enable entry, targeting, sustained release for longer periods and uptake of the antibiotics in the cells, hence reducing the dose frequency and simultaneously improve patient compliance. The aim was to prepare functionalised polymeric nanodrug delivery vehicles to target TB infected macrophage cells. Successful nanoencapsu-

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lation of anti-TB drugs and a targeting agent, mycolic acids (MA) was achieved. MA (a lipid molecule on the cell wall of M.tb) was explored due to its cholesteroid properties [2] that could attract it to the infected macrophages/foam cells. The nanoparticles were characterized and subjected to in vitro analyses in THP-1 and U937 cells in order to determine their uptake and localization. Cytotoxicity in different cell lines was also analysed. In another approach targeting was achieved via attaching nucleic acid aptamers [3], onto the surface of drug-carrying PLGA nano-particles. The aptamers were prepared via the SELEX process [4], specifically against the mannose receptor (MR), which is significantly over-expressed during the activation of the macrophages in the presence of M.tb. Uptake of the MA PLGA nanoparticles was achieved where little co localization was observed with endocytic markers, indicating that they could be localized in the cytosol. Vesicles bearing these particles were also observed in the cell membrane of these cells. We will report the uptake of the aptamers to THP-1 cells illustrating the feasibility of using the nucleic acid species for active targeted drug delivery. The success of these two approaches of anti-TB drug targeting will greatly address the challenges of poor bioavailability, reduced efficacy and adverse side effects for diseases such as TB. See references below for additional reading 1. WHO (2009). Global tuberculosis control: epidemiology, strategy, financing: WHO report. WHO/HTM/TB/2009.411. 2. Benadie Y, et al. Cholesteroid nature of free mycolic acids from M. tuberculosis. Chem Phys Lipids 2008;152:95–103. 3. Stoltenburg R, et al. SELEX—–a (r)evolutionary method to generate high affinity nucleic acid ligands. Biomol Eng 2007;24:381–403. 4. Levy-Nissenbaum W, et al. Nanotechnology and aptamers: applications in drug delivery. Trends Biotechnol 2008;26:442–9.

doi:10.1016/j.drudis.2010.09.399

A52 Targeted SAINT-O-Somes, a novel type of liposomes for improved delivery of siRNA Piotr S. Kowalski 1,∗ , Niek G.J. Leus 1 , Joanna E. Adrian 1 , Henriette W.M. Morselt 1 , Marcel H.J. Ruiters 2 , Grietje Molema 1 , Jan A.A.M. Kamps 1 1 Department of Pathology and Medical Biology, Medical Biology Section, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 2 Synvolux Therapeutics B.V., Groningen, The Netherlands ∗

Corresponding author. E-mail: [email protected] (P.S. Kowalski). Gene silencing by siRNA has become a powerful technique with a great potential for therapeutic application. Increased insight in the role of endothelial cells in the pathology of cancer and inflammatory diseases has shifted the interest in the development of siRNA drug delivery devices for pharmacological intervention towards these cells. Additionally, endothelial cells are readily accessible for substances transported by the blood and their heterogeneity allows for specific drug targeting approaches. Liposomes represent a drug-carrier system for the delivery of siRNA that can be tailored on demand to introduce cell specificity. However, unlike in macrophages or in many tumor cells, in endothelial cells the processing of liposomes and subsequent release of drug content is inefficient due to the absence of adequate intracellular processing machinery which limits pharmacological efficiency. Therefore, we developed a lipid based drug delivery system with a superior intracellular release characteristic which is suitable for the in vivo delivery of siRNA. The design of the carrier is based on long circulating conventional liposomes that were formulated with a cationic amphiphile, 1-methyl-4-(cis-9-dioleyl)methylpyridinium-chloride (SAINT-18). These so-called SAINT-O-Somes have a diameter of 100 nm and showed a 10-fold higher encapsulation efficiency for siRNA compared to liposomes without SAINT and protect the siRNA from degradation for at least 6 weeks. Moreover, SAINT-O-Somes are fully stable in a biological relevant milieu (i.e. presence of serum), but are destabilized in the lower pH in endosomes of endothelial cells, enabling release of siRNA into the cytoplasm of the cell. In order to efficiently target activated endothelial cells, SAINT-OSomes were equipped with antibodies against E-selectin or VCAM-1 adhesion molecules that are (over)expressed at sites of inflammation.

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results confirm that SLO treated cells showed an increased cellular uptake of gold nanoparticles then untreated cells. We also studied the effect of poly ethylene glycol (PEG) on SLO assisted cellular uptake by increasing the PEG amount gradually and found that PEG affects the cellular uptake adversely. We are currently combining fluorescence microscopy, photothermal microscopy and transmission electron microscopy to fully understand the mechanism, localization and fate of gold nanoparticles during SLO assisted uptake. doi:10.1016/j.drudis.2010.09.401

See reference below for additional reading 1. Adrian JE, et al. Targeted SAINT-O-Somes for improved intracellular delivery of siRNA and cytotoxic drugs into endothelial cells. J Control Release 2010;(March).

doi:10.1016/j.drudis.2010.09.400

A53 Toxin assisted intracellular delivery of gold nanoparticles Umbreen Shaheen ∗ , Yann Cesbron, Raphaël Lévy School of Biosciences, University of Liverpool, Liverpool, UK ∗

Corresponding author. E-mail: [email protected] (U. Shaheen). Targeted intracellular delivery of biomolecules using nanoparticles has attracted many of the science disciplines. Nanoparticles because of their tuneable size and unique optical properties are emerging not only as imaging probes but also serving as intracellular cargo delivery carriers. Gold nanoparticles are best candidate for all these applications because of their not particularly reported cytotoxicity and ease of biofunctionalization. For intracellular cargo delivery application, it is necessary that a carrier is not only has the capacity to carry the biomolecule efficiently but also able to deliver it to the cytosol which is the main site for all physiological and chemical activities inside the cell. It is well documented that on intracellular delivery, bioconjugated gold nanoparticles are trapped by endolysosomes where their biomolecular coating degrades eventually. For avoiding this fate and for gaining access into the cytosol, we used a new approach, that is, toxin assisted delivery for gold nanoparticles. A bacterial toxin streptolysin-O is a secreted protein of 61 kDa which forms pores in plasma membrane of host cell for gaining access into the cytosol. It has been used as a simple and rapid mean of transfection for intracellular delivery of oligonucleotides and siRNA. Our

A54 Quantifying uptake and distribution of arginine rich peptides at therapeutic concentrations using fluorescence correlation spectroscopy and image correlation spectroscopy techniques Ben Staley ∗ , Egor Zindy, Alain Pluen School of Pharmacy and Pharmaceutical Sciences, The University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK ∗

Corresponding author. E-mail: [email protected] (B. Staley). Due to an apparent ability to enter cells in an energy independent manner, cellpenetrating peptides (CPPs) are increasingly being used as vectors for the delivery of macromolecules into cells. But 20 years on, their uptake and intracellular distribution are still debated [1] especially as most studies have been carried out at relatively high concentrations (micromolar), while therapeutic doses more likely to be in the nanomolar range. Thus, we hypothesised that taking advantage of fluorescence correlation spectroscopy (FCS) and image correlation spectroscopy (ICS) should help to understand the delivery mechanisms (especially the intracellular distribution) of arginine rich peptides TAMRA-Tat and TAMRA-nona-arginine (R9) at therapeutic doses. TAMRA-Tat and TAMRA-R9 peptides were incubated for one hour with both Caco-2 and HeLa cells. Initial observation of uptake was carried with a Zeiss LSM510 Meta Confocor 2. FCS and ICS were then used to measure peptide concentrations (density of particles per beam waist area) in distinctive areas and in the whole cell (cartography). ICS, implemented in parallel to FCS, was developed in house based on the work of P. Wiseman’s group [2,3]. Subcellular distribution was analysed with confocal microscopy revealing two main areas – punc-

tate and cytoplasmic regions – sampled initially with FCS to obtain diffusion times and concentration. Diffusion times in the punctate areas are very long (300 ± 50 ␮s) compared to the cytoplasm (26 ± 8 ␮s) at 500 nM, suggesting a bound component compared to free peptide. As FCS cannot sample the whole cell, ICS provided a more complete view of the distribution of TAMRA-Tat and TAMRA-R9 in which large areas of the cells behave as the ‘cytoplasmic’ area used in FCS. Our results indicate that arginine rich peptides are observed at nanomolar concentrations in all areas sampled. At concentrations below 500 nM, punctate and discrete areas are clearly labelled suggesting a possible entry via an endocytosis only mechanism. Finally, as the bulk concentration increases the fraction detected in the cytoplasm increases suggesting the simultaneous presence of a non-endocytotic mechanism of entry. Overall, FCS and ICS demonstrate that they provide invaluable information on the cellular delivery of peptides at therapeutic levels. See reference below for additional reading 1. Lee HL, et al. J Am Chem Soc 2008;130:9364–70. 2. Kolin DL, Wiseman PW. Cell Biochem Biophys 2007;49:141–64. 3. Hebert B, et al. Biophys J 2005;88:3601–14.

doi:10.1016/j.drudis.2010.09.402

A55 Tat-LK15, a Tat-fusion peptide, to deliver therapeutic siRNA in chronic myeloid leukemic cells Yamini Arthanari ∗ , Costas Demonacos, Harmesh Aojula, Alain Pluen School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom ∗

Corresponding author. E-mail: [email protected] (Y. Arthanari). Chronic myeloid leukaemia (CML) is caused by the reciprocal translocation of chromosomes 9 and 22 resulting in the formation of the BCR-ABL fusion protein, which exhibits deregulated tyrosine kinase activity. Hence, BCR-ABL would be a key target for developing a therapy for CML. We have used the potential of RNA interference to study the silencing of this oncoprotein. siRNA has been used to target wide range of genes in various cell types using cell penetraing peptides (CPPs). In this study we have evaluated the ability of the Tat fusion peptide, Tat-LK15 [1] to study uptake of www.drugdiscoverytoday.com 1099

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Coupling of these ligands showed a highly beneficial effect for transfection efficacy to TNF-␣ activated endothelial cells compared to non-targeted SAINT-O-Somes. The intracellular delivery of anti VE-cadherin siRNA SAINT-OSomes to activated endothelial cells resulted in a specific, 70% down-regulation of VE-cadherin gene expression. In conclusion, we demonstrated that SAINT-O-Somes are stable, high capacity carriers for effective siRNA delivery into endothelial cells that present the requirements for in vivo application.

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siRNA and also silencing of the BCR-ABL protein in K562 CML cells. Tat-LK15 peptide [1], a fusion of Tat and membrane lytic peptide LK15, was used to non-covalently complex siRNA targeting the BCR-ABL mRNA (b3a2 isoform). Complexation of siRNA by Tat-LK15 was studied using fluorescence correlation spectroscopy (FCS) in the presence of the intercalating dye YOPRO-1. Cy5 labelled siRNA was used to study uptake in K562 cells using flow cytometry and confocal microscopy. The reduction in BCR-ABL protein levels was observed by Western blot. Results were compared with K562 cells transfected with lipofectamine/siRNA complexes. MTT assay was performed to study the cytotoxicity of the Tat-LK15/siRNA complexes. The YOPRO-1 competitive binding assay revealed efficient condensation of siRNA by Tat-LK15 and LipofectamineTM at charge ratios higher than 3:1 (less than 10% of YOPRO-1 labelled siRNA). Flow cytometry studies using varying amounts of siRNA showed an increase in intracellular existence of Cy5-siRNA also leading to an increase in percentage positive transfected cells. Confocal microscopy confirmed the increase in intracellular localization upon transfection with higher amount of siRNA 4 hours and 24 hours post-transfection. Finally RNAi was observed using siRNA, which resulted in 70–80% reduction in BCR-ABL protein levels at lower concentrations. However, silencing observed using siRNA did not last longer than 48 hours. Cytotoxicity studies show that TatLK15/siRNA complexes are not toxic when lower concentrations of siRNA are used. Here, we show that Tat-LK15 can be a potential vector in delivering siRNA targeting genes of clinical significance. Reference 1. Saleh AF, et al. Improved Tat-mediated plasmid DNA transfer by fusion to LK15 peptide. J Control Release 2010;143:233–42.

doi:10.1016/j.drudis.2010.09.403

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A56 Carbon nanotube-dendron series for siRNA delivery: mechanisms of cellular internalisation

Reference 1. Herrero MA, et al. J Am Chem Soc 2009;131:9843.

Chang Guo 1,∗ , Khuloud Al-Jamal 1 , Alberto Bianco 2 , Maurizio Prato 3 , Kostas Kostarelos 1,∗ 1 Nanomedicine Lab, Centre for Drug Delivery Research, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK 2 CNRS, Institut de Biologie Moleculaire et Cellulaire, Laboratoire d’Immunologie et Chimie Therapeutiques, 67000 Strasbourg, France 3 Dipartimento di Scienze Farmaceutiche, Universita di Trieste, 34127 Trieste, Italy

A57 Cellular internalisation of humanized IgG antibody changes by functionalization onto multi-walled carbon nanotubes

∗

Corresponding author. E-mails: [email protected] (C. Guo), [email protected] (K. Kostarelos). Carbon nanotubes have been attracting attention as tools for various biomedical applications. Chemical surface functionalization of multi-walled carbon nanotubes (MWNT) has shown remarkably increased aqueous solubility and debundling of nanotube aggregates that makes this material a promising candidate for biological applications. In this work, a series of dendron-MWNT derivatives were synthesized as potential vectors for siRNA delivery [1]. To elucidate the mechanism of cellular internalization characteristics of the dentron-MWNT:siRNA complexes, a fluorescence probed, non-coding siRNA sequence was used and its nanotubemediated cytoplasmic delivery was studied in comparison to that by cationic liposomes. siRNA delivered by the dendron-MWNT was found throughout the cytoplasm including the nucleus. The siRNA delivered by cationic (DOTAP:cholesterol) liposomes was co-localized with endosomal markers indicating primarily an endocytosis pathway for internalization as previously described in the literature. The cellular transport of the siRNA was significantly increased with higher dendron generations conjugated on the nanotube surface at physiological conditions (37 ◦ C) as well as under endocytosis-inhibiting conditions (4 ◦ C). This work demonstrated that clathrin-coated endocytosis is a contributing but not the major pathway for the cellular internalization of the dentron-MWNT:siRNA complexes and could offer a great advantage via direct cytoplasmic delivery of siRNA for effective gene silencing.

doi:10.1016/j.drudis.2010.09.404

Chang Guo 1,∗ , Enrica Venturelli 2 , Alberto Bianco 2 , Kostas Kostarelos 1,∗ 1 Nanomedicine Lab, Centre for Drug Delivery Research, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK 2 CNRS, Institut de Biologie Moleculaire et Cellulaire, Laboratoire d’Immunologie et Chimie Therapeutiques, 67000 Strasbourg, France ∗

Corresponding author. E-mails: [email protected] (C. Guo), [email protected] (K. Kostarelos). Antibodies have been extensively used as anti-neoplastic therapeutics clinically and preclinically as they allow for therapeutic and specific targeting to specific cell receptors. The humanized CTMO1 IgG antibody was raised against the membrane-associated antigen of human milk fat globules (HMFG) derived from the anti-HMFG mouse monoclonal antibody CTMO1, but with similar affinity to the polymorphic epithelial mucin-1 (MUC-1). Anticancer drugs derived from murine HMFG1 have been under development in phase III clinical trial [1]. Carbon nanotubes have remarkable physicochemical properties offering an array of interesting features. In the context of this study, their large surface area offered a template for conjugation with a variety of monoclonal antibodies. Multi-walled carbon nanotubes (MWNT) were chemically functionalized with humanized CTMO1 IgG. The MWNT-IgG constructs were observed to target MUC-1 positive cells, but were retained at the plasma membrane with limited internalization. In contrast, a time-dependent cell surface binding and internalization was observed for the humanized CTMO1 IgG alone. The co-localization of the fluorescently labeled IgG with markers of specific cellular compartments was also studied using confocal laser scanning microscopy, to determine its mechanism of cellular uptake and trafficking pathway. The results here indicated that the size and aggregation state of the MWNT-IgG constructs played a determinant role in their interaction with cells. The design and development of CNT-antibody con-

structs needs further optimization in order to constitute a viable novel platform for cancer treatment with the purpose of combinatory therapeutic/diagnostic functionality.

3.

Reference 1. Verheijen RH, et al. J Clin Oncol 2006;24:571.

4.

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A58 Role of cell-surface carbohydrates and plasma membrane components in the internalization of cell-penetrating peptides Chérine Bechara ∗ , Chen-Yu Jiao, Fabienne Burlina, Isabel D. Alves, Gérard Chassaing, Sandrine Sagan Universite Pierre et Marie Curie, Laboratory of Biomolecules, CNRS, ENS, Paris, France ∗ Corresponding author. E-mail: [email protected] (C. Bechara). Among cell-penetrating peptides, penetratin is widely used as a molecular device to cross membranes and transport biologically active molecules inside cells [1,2]. But, the underlying internalization mechanisms for such behaviour is still studied and discussed [3]. The idea is now well accepted that the physico-chemical properties of the cargo [4], the cell-penetrating peptide [5], and the disulfide-bridge in the conjugate [6], have an impact in the intracellular delivery pathways of the conjugate. Therefore, it is obvious that the internalization pathways and the final localization of conjugates within cells can hardly be anticipated. We have previously reported that penetratin internalizes in cells at 37 ◦ C and 4 ◦ C, thus through translocation and endocytosis pathways [7]. The translocation process occurs at low micromolar penetratin, while endocytosis is activated at higher concentrations. We have now studied the impact of cell-surface (GAG, sialic acid) and plasma membrane (cholesterol) components in the temperature-dependent cell internalization efficiency [8] and pathways [7] of penetratin and other well-studied cell-penetrating peptides. These results will be presented and discussed.

Reference 1. Davidson TJ, et al. Highly efficient small interfering RNA delivery to primary mammalian neurons induces MicroRNA-like effects before mRNA degradation. J Neurosci 2004;24:10040–6. 2. Muratovska A, Eccles MR. Conjugate for efficient delivery of short interfering RNA

5.

6.

7.

8.

(siRNA) into mammalian cells. FEBS Lett 2004;558:63–83. Alves ID, Jiao CY, Aubry S, Aussedat B, Burlina F, Chassaing G, Sagan S. Cell biology meets biophysics to unveil the different mechanisms of penetratin internalization in cells. Biochim Biophys Acta 2010;1798(12):2231–9. Maiolo JR, et al. Effects of cargo molecules on the cellular uptake of arginine-rich cellpenetrating peptides. Biochim Biophys Acta 2005;1712:161–72. Aussedat B, et al. Modifications in the chemical structure of Trojan carriers: impact on cargo delivery. Chem Commun (Camb) 2008:1398–400. Aubry S, et al. Cell-surface thiols affect cell entry of disulfide-conjugated peptides. FASEB J 2009;23:2956–67. Jiao C-Y, et al. Translocation and endocytosis for cell-penetrating peptide internalization. J Biol Chem 2009;284:33957–65. Burlina F, et al. A direct approach to quantification of the cellular uptake of cell-penetrating peptides using MALDI-TOF mass spectrometry. Nat Protoc 2006;1:200–5.

doi:10.1016/j.drudis.2010.09.406

A59 Development of a microwell device for correlative light and electron microscopy Edward Sayers 1,2,∗ , Chris Allender 1 , David Barrow 2 , Arwyn T. Jones 1 1 Welsh School of Pharmacy, Redwood Building, Cardiff University, Cardiff CF10 3NB, UK 2 Cardiff School of Engineering, Queens Buildings, The Parade, Cardiff University, CF24 3AA, UK ∗

Corresponding author. E-mail: [email protected] (E. Sayers). New innovations and techniques are constantly being developed within the field of microscopy with the aim of generating higher through-put analysis and/or gaining the maximum data out of a single sample. Correlative light electron microscopy or CLEM involves bringing together the two most common aspects of microscopy, fluorescence and electron microscopy. The weaknesses in fluorescence microscopy, low resolution, can be counteracted by the highly detailed electron microscopy images. On the other hand, the weakness of electron microscopy for live intracellular tracking, can be counteracted using fluorescence microscopy. This project involves the development of a microwell array technique to allow a user to correlatively image the same cell under both fluorescence microscopy and scanning electron microscopy (SEM). Microwell arrays were ablated into borosilicate glass and PDMS (silicone elastomer) cover slips using 193 nm and 157 nm excimer lasers

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(MetaFAB, Cardiff University). The surface of the substrate is first coated with a sacrificial layer before ablation thus providing an important step in helping to remove ablation debris during sonication. PDMS surfaces were further modified to optimise cell adhesion by oxidizing the surface using UV/ozone treatment and reacting with APTES (aminopropyltriethoxysilnae) to create an amine modified surface. Initially, for proof of concept, KG1a (acute myelogenous leukaemia) cells were allowed to settle into the microwells before being exposed to transferrin as an endocytic marker or a pro-apoptotic peptide linked to the cell penetrating peptide R8 to determine whether apoptosis can be monitored. The cells were then imaged by confocal microscopy then fixed, dehydrated, dried and splutter coated for imaging by SEM. We have successfully imaged uptake of transferrin and the effects of a proapoptotic peptide whilst cells were resting within the microwells. We have also obtained correlative images of KG1a cells imaged before fixation under light microscopy and the same cells under SEM. By comparing cell number and their position within the microwells before and after fixation we are confident of achieving correlative microscopy. For adherent cells we are able to create microwell arrays of varying sizes in both glass and PDMS. Post-ablation processing increased microwell quality whilst the auto-fluorescence in glass was reduced by various cleaning steps. However, switching to PDMS provided a much lower auto-fluorescent substrate on which to work. PDMS is naturally very hydrophobic (contact angle ∼105◦ ); using UV/ozone we were able to reduce the hydophobicity of the surface (contact angle ∼40◦ ). This formation of hydroxyl groups on the surface allowed for further modification using APTES, which improved cell adhesion. We can now obtain correlative images using confocal microscopy and SEM of the same cells and are developing further methods for TEM correlative light electron microscopy studies. doi:10.1016/j.drudis.2010.09.407

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A60 Formulation of new reducible liposomes for gene delivery Daniele Pezzoli 1,∗ , Laura Ciani 2 , Sandra Ristori 2 , Alberto Cigada 1 , Roberto Chiesa 1 , Gabriele Candiani 1 1 Politecnico di Milano, Via Mancinelli 7, 20131 Milan, Italy 2 Dipartimento di Chimica & CSGI, Università di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy ∗

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Corresponding author. E-mail: [email protected] (D. Pezzoli). Gene therapy aims to eradicate causes rather than symptoms of diseases and is believed by many to be the therapy of the future. Improved liposome formulations are a valuable alternative to viral gene delivery vectors and the rapid disulfide linkages cleavage by the intracellular reductive environment can induces fast reducible lipoplex dissociation and efficient DNA release, yielding increased gene expression. On the light of these findings, we developed four different liposome formulations based on SS14, a reducible cationic gemini-like surfactant. SS14 was previously synthesized by our group [1,2]. Helper lipids bearing different alkyl chain and/or polar head types were chosen and four formulations were investigated: DMPC/SS14:0.75/0.25; DOPC/SS14:0.75/0.25; DMPC/DMPE/SS14:0.5/0.25/0.25; DOPC/DOPE/SS14:0.5/0.25/0.25 molar ratios. SS14-containing liposomes were prepared by repeated extrusions through polycarbonate filters of 100 nm pore diameter. Three out of four liposome formulations showed a size distribution with a monodisperse population (polydispersity index, P.I. ≤ 0.3) while in DMPC/DMPE/SS14 liposomes, large aggregates (∅ > 1 ␮m) were found together with the main liposome population, possibly due to fluctuating lamellar sheets. All liposome dimensions were between 95 and 120 nm. Zeta potential, within experimental error, was the same for all the formulations, ranging from +39 ± 7 mV and +55 ± 8 mV. By monitoring the displacement of SYBR-Green I from DNA, a negative trend of fluorescence in function of CR was noticed for each formulation with a plateau reached beyond CR5. Since between the reducing intracellular space and the oxidizing extracellular environment a high redox potential difference exists (∼100–1000-fold), by agarose gel electrophoresis we demonstrated the ability of GSH to enable DNA release. Transfection activity and

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cytotoxicity of the four formulations were compared at CR5 and CR15 on U87-MG, Cos-7, HeLa and MG63 cell line using pEGFP-N1 as plasmid DNA. Firstly, liposome effectiveness was not inhibited by the presence of serum in transfection experiments. Secondly, the introduction of helper lipids bearing PE polar heads in twocomponent liposome formulations increased significantly transfection efficiency up to 7-fold (p < 0.05). This may be due to the high fusogenic properties of their phosphoethanolamine (PE) polar head. Finally, three-component formulations were more cytotoxic. In particular, DOPC/DOPE/SS14:0.5/0.25/0.25 CR5 liposomes demonstrated superior transfection efficiency (24.4 ± 2.7% by FACS analysis on U87-MG cells) and modest cytotoxicity. The mechanisms beneath intracellular reduction, transfection enhancement and increased cytotoxicity will be the subject of further investigation. Reference 1. Candiani G. ChemMedChem 2007;2:292–6. 2. Candiani G. J Gene Med 2008;10:637–45.

doi:10.1016/j.drudis.2010.09.408

A61 Strategies for microsphere-mediated delivery of oligonucleotides J.M. Cardenas-Maestre ∗ , A. Seth, R.M. Sanchez-Martın Chemical Biology Section, School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK ∗

Corresponding author. E-mail: [email protected] (J.M. Cardenas-Maestre). An efficient intracelllar delivery of oligonucleotides is a vital step for gene therapy. Many technologies have been developed to design efficient transfection agents. Many of these agents are promising tools in vitro but they fail when in vivo assays are carried out. Recently we have developed a polystyrene microspherebased system designed to efficiently deliver biological materials into a broad range of cell lines. Additionally, these particles have been successfully test in vivo. The fact that these polymer particles are easy to functionalise with high controllability over the cargo loading, showing any undesired cytotoxic effect, make them enormously attractive as delivery system. Our recent advances in the design of strategies for the delivery of oligonucleotides using microspheres as transfection system will be presented.

See reference below for additional reading 1. Sánchez-Martín RM, et al. ChemBioChem 2005;6:1341–5. 2. Sánchez-Martín RM, et al. In: Pignataro B, editor. Ideas in Chemistry and Molecular Sciences: Where Chemistry Meets Life. Wiley VCH Ed.; 2010. p. 117–40. Chapter 5. 3. Alexander LM, et al. Bioconjug Chem 2009;20:422–6. 4. Tsakiridis A, et al. Biomaterials 2009;30:5853–61.

doi:10.1016/j.drudis.2010.09.409

A62 Microsphere-mediated delivery of therapeutic peptides on neuronal cells Ana M. Pérez-López 2 , Juan Manuel Cárdenas-Maestre 1 , Sonia Panadero-Fajardo 2,∗ , José F. Domínguez-Seglar 2 , José A. Gómez-Vidal 2 , Rosario Sánchez-Martín 1 1 Chemical Biology Section, School of Chemistry & Institute of Stem Cell Research, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK 2 Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Campus de Cartuja s/n, 18071 Granada, Spain ∗

Corresponding author. E-mail: [email protected] (S. Panadero-Fajardo). Many proteins exert their biological roles as components of complexes, and the functions of proteins are often determined by their specific interactions with other proteins. The identification of inhibitory peptides and derived peptidomimetics has been developed as potent inhibitors of protein–protein interaction. More specifically protein–protein interaction domains that couples the NMDA receptor to intracellular proteins are potential targets for the development of new therapies to combat neurodegenerative diseases [1]. Different studies of the PDZ domain in nNOS inhibitors have been carried out. The peptidic nature of these compounds has obstructed their uptake into the cell. Amino cross-linked microspheres have been used previously for the delivery of therapeutic molecules [2–5]. The design, synthesis and biological evaluation of microspheres as carrier systems to facilitate the cellular uptake of these peptidic sequences on SH-SY5Y neuroblastoma cells will be presented. See reference below for additional reading 1. Pawson T, Scott JD. Science 1997;278:2075–80. 2. Sánchez-Martín RM, et al. In: Pignataro B, editor. Ideas in Chemistry and Molecular Sciences: Where Chemistry Meets Life. Wiley VCH Ed.; 2010. p. 117–40. Chapter 5.

3. Sánchez-Martín RM, et al. Angew Chem Int Ed 2006;45:5472–4. 4. Alexander LM, et al. Bioconjug Chem 2009;20:422–6. 5. Sánchez-Martín RM, et al. ChemBioChem 2009;10:1453–6.

doi:10.1016/j.drudis.2010.09.410

A63 siRNA versus pharmacological inhibition of endocytic pathways for studying cellular uptake of cell penetrating peptides and other drug delivery vectors Monerah H. Al-Soraj 1,∗ , Catherine L. Watkins 1 , Dries Vercauteren 2 , Stefaan De Smedt 2 , Kevin Braeckmans 2 , Arwyn T. Jones 1 1 Welsh School of Pharmacy, Cardiff University, Cardiff, Wales CF10 3NB, UK 2 Laboratory of General Biochemistry & Physical Pharmacy, Ghent University, Harelbekestraat 72, Ghent, Belgium ∗

Corresponding author. E-mail: [email protected] (M.H. Al-Soraj). Cell-penetrating peptides (CPPs) have the potential to deliver numerous therapeutic macromolecules into cells including peptides, proteins, and nucleic acids. Under defined conditions endocytosis is thought to be of significant importance for CPP entry but identifying the exact uptake mechanism and pathway(s) involved has been difficult. Multiple pathways have been reported to contribute to uptake, including macropinocytosis and those regulated by clathrin and cavaeolin-1. This project aims enhance the use of cell penetrating peptides as drug delivery vectors by developing new technologies to study their mechanisms of uptake. Traditionally studies investigating the uptake of these molecules, and other drug delivery vectors, have been performed using chemical inhibitors but these are often toxic and lack specificity [1]. We have developed siRNA-based assays to silence endocytic proteins that have previously been shown to regulate distinct endocytic pathways. The effect of depleting these proteins was then assessed to investigate their roles in mediating the uptake of well characterised endocytic probes and CPPs. Two cell lines were predominantly used, HeLa (cervical cancer epithelial) and A431 (human epithelial carcinoma). Endocytic proteins clathrin heavy chain, flotillin-1, dynamin II, caveolin1 and P21-activated kinase (PAK-1) were depleted using single siRNA sequences; siRNA against GFP was used as a control. In siRNA treated cells the uptake of fluorescent endo-

cytic markers including; Alexa488-transferrin (clathrin mediated endocytosis), 40 kDa FITC Dextran (fluid phase uptake and macropinocytosis), FITC conjugated anti-CD59 antibody (flotillin-1 dependent uptake) and the uptake of Alexa488 CPPs (RRRRRRRRGC-Alexa488-R8, and GRKKRRQRRRPPQ-Alexa488-HIV-TAT) were measured by flow cytometry. Protein depletion was assessed from protein lysates using SDS PAGE and Western blotting. Overall, the data shows that siRNA transfection method could effectively reduce expression of clathrin heavy chain, caveolin-1 and flotillin-1 from HeLa cells and this then allowed for us to study effects on endocytosis of various probes. PAK-1 has been shown to regulate macropinocytosis and we show that the induction of macropinocytosis and PAK-1 expression are highly cell line dependent. Paralleled with this was our findings that cationic CPPs induce an increase in fluid phase uptake of dextran and the extent of this was cell line dependent. Comparative analysis of these experiments with those performed using pharmacological inhibitors, allowed us to determine the usefulness of this approach for drug delivery research. Reference 1. Vercauteren D, et al. The use of inhibitors to study endocytic pathways of gene carriers: optimisation and pitfalls. Mol Ther 2010;18:561–9.

doi:10.1016/j.drudis.2010.09.411

A64 SAINTargs, a novel lipid-based targeting device for siRNA delivery Niek G.J. Leus 1,∗ , Piotr S. Kowalski 1 , Sigridur A. Ásgeirsdóttir 1 , Eduard G. Talman 2 , Marcel H.J. Ruiters 2 , Grietje Molema 1 , Jan A.A.M. Kamps 1 1 University Medical Center Groningen, Department of Pathology & Medical Biology, Laboratory of Endothelial Medicine & Vascular Drug Targeting, Hanzeplein 1, 9713 GZ Groningen, The Netherlands 2 Synvolux Therapeutics, L.J. Zielstraweg 1, 9713 GX Groningen, The Netherlands ∗

Corresponding author. E-mail: [email protected] (N.G.J. Leus). The endothelium represents an important therapeutic target because its pivotal role in many diseases such as chronic inflammation and cancer and its accessibility for systemic administration. RNA interference by small interfering RNA (siRNA) has become in the last decade a very powerful tool in basic research, and has huge potential to

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become an important new class of therapeutics for humans. However, due to their size and charge, siRNAs have no bioavailability to enter unperturbed cells. To overcome this problem, our laboratory developed a non-viral lipid-based targeting device which efficiently and specifically delivers siRNA into endothelial cells. Molecular determinants expressed on the surface of inflammationactivated endothelial cells, like certain adhesion molecules and receptors involved in endocytosis, are excellent candidates to increase carrier-mediated siRNA uptake. Therefore we conjugated monoclonal anti-E-selectin antibodies to the cationic amphiphilic lipid, 1-methyl-4-(cis-9-dioleyl)methyl-pyridiniumchloride (SAINT-18) which was complexed in a 1:2000 molar ratio with the transfection agent SAINT-MIX (SAINT:DOPE, 1:1), and siRNA, resulting in a siRNA containing lipoplex called anti-E-selectin-SAINTarg [1]. Our findings demonstrate that anti-E-selectin-SAINTargs maintained the antigen recognition capacity of the parental antibody and showed increased siRNA uptake in otherwise difficult-to-transfect primary human umbilical vein endothelial cells (HUVEC) as compared to non-targeted SAINT-MIX. Moreover, anti-E-selectin-SAINTargs superior binding and uptake efficiency was corroborated by improved silencing of both geneand protein expression of VE-cadherin in activated HUVEC. The VE-cadherin gene expression could be silenced up to 95% by VE-cadherin specific siRNA, at low siRNA concentrations (30 pmol/ml). Furthermore, no non-specific silencing by scrambled or VE-cadherin specific siRNA was observed. To optimize siRNA delivery into activated endothelial cells we also synthesized anti-VCAM-1-SAINTargs which were as efficient in VE-cadherin silencing as anti-E-selectin-SAINTargs. Because of the heterogeneous expression of adhesion molecules on inflammation-activated endothelial cells in vivo, a combination of these two SAINTargs may result in enhanced siRNA effects. Taken together, SAINTargs demonstrate specific and efficient targeting to inflammation-activated difficult-to-transfect primary endothelial cells and results in strong siRNA specific gene silencing at low siRNA concentrations. Reference 1. Asgeirsdottir SA, Talman EG, de GI, Kamps JA, Satchell SC, Mathieson PW, Ruiters MH, Molema G. Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro - A quantitative study. J Control Release 2010 Jan 25;141(2):241–51.

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Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

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A65 Cationic star homo- and co-polymers for gene delivery Theoni K. Georgiou 1,∗ , Mark A. Ward 1 , Phillip Knight 1 , Maria D. Rikkou 2 , Maria Vamvakaki 3,4 , Edna N. Yamasaki 5 , Leonidas A. Phylactou 6 , Costas S. Patrickios 2 1 Department of Chemistry, The University of Hull, HU6 7RX, Hull, UK 2 Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus 3 Department of Materials Science and Technology, University of Crete, P.O. Box 2208, 710 03 Heraklion, Crete, Greece 4 Foundation for Research and Technology, Institute of Electronic Structure and Laser, P.O. Box 1527, 711 10, Heraklion, Crete, Greece 5 Department of Life and Health Sciences, School of Sciences, University of Nicosia, 46 Makedonitissas Ave, 1700 Nicosia, Cyprus 6 Cyprus Institute of Neurology and Genetics, P.O. Box 23462, 1683 Nicosia, Cyprus ∗

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Corresponding author. E-mail: [email protected] (T.K. Georgiou). Several groups of star polymers were synthesized and evaluated as gene delivery vehicles. All polymers were synthesized by group transfer polymerisation and were based on 2-(dimethylamino)ethyl methacrylate (DMAEMA). In particular, one group of DMAEMA star homo-polymers of different molecular weights and three groups of star copolymers of different architectures were prepared. The three groups of copolymers were based on the DMAEMA monomer and a second hydrophilic monomer comprising either poly(ethylene glycol) methacrylate, methacrylic acid or glycerol methacrylate. All series of star polymers were characterized by gel permeation chromatography and nuclear magnetic resonance spectroscopy. Aqueous solutions of the star polymers were studied by turbidimetry, hydrogen ion titration, and dynamic light scattering. All but the most recent star polymers were evaluated for their ability to transfect cells. The transfection efficiency was affected by the molecular weight of the star polymer, the star architecture and the nature of the second co-monomer. doi:10.1016/j.drudis.2010.09.413

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A66 Gene electrotransfer: comparison between 2D cultured cells and multicellular tumor spheroid model L. Chopinet ∗ , L. Wasungu, M.P. Rols Institut de Pharmacologie et de Bilogie Structurale, 205 route de narbonne, 31077 Toulouse Cedex, France ∗

Corresponding author. E-mail: [email protected] (L. Chopinet). Electroporation is a physical method to deliver molecules into cells and tissues. Clinical applications have been successfully developed for antitumoral drug delivery and clinical trials for gene electrotransfer are underway [1]. However, little is known about the mechanisms involved in these processes. The main difficulties stem from the lack of cell models which reliably replicate the complex in vivo environment. To increase our understanding of the DNA electrotransfer mechanisms, we recently exploited multicellular tumor spheroids (MCTS) as an ex vivo model of tumor [2]. This 3-dimensional model can replicate the in vivo in complex environment and therefore enables us to develop new strategies for studying mechanisms of molecules delivery by electric field pulses. In the present study, we observed cells response to electric field pulses for propidium iodide and plasmid DNA delivery. HCT116 cells were pulsed either in suspension (2D culture) or in MCTS (3D culture) and 10 pulses lasting 5 ms were applied at different voltages. Confocal and biphotonic microscopy allowed us to visualize the repartition of permeabilized and transfected cells in MCTS subjected to electric pulses. Flow cytometery analysis was used to obtain quantitative analysis both on cells pulsed in suspension or on cells pulsed in MCTS (in that case, cells were dissociated by an enzymatic treatment). Results show differences in electric field sensitivity between cell in suspension and MCTS. Permeabilization process (revealed by propidium iodide uptake) is affected only the first cell layers of MCTS. A maximum of 30% of cells being permeabilized was obtained at 400 V cm−1 . Increasing the field strength above that value did not further increase the number of permeabilized cell. On the contrary, in the case of cells pulsed in suspension, up to 90% of cells were shown to be permeabilized at 700 V cm−1 . DNA delivery process (revealed by GFP expression) showed that less than 5% cells were transfected when present in the spheroid model while, under the same conditions, about 25% of them were

transfected when pulsed in suspension. These results point out the difficulty DNA has to cross the multicellular barrier and give an explanation for the different of responses of cells in vitro and in vivo [3]. Taken together, these results are in agreement with the ones obtained in tumors and indicate that the spheroid model is more relevant to an in vivo situation than cells cultured as monolayers. They validate the spheroid model as a way to study electromediated gene delivery processes. Reference 1. Daud AI, et al. Phase I trial of interleukin12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 2008;26:5896–903. 2. Wasungu L, et al. A 3D in vitro spheroid model as a way to study the mechanisms of electroporation. Int J Pharm 2007;379:278–84. 3. Rols MP, et al. In vivo electrically mediated protein and gene transfer in murine melanoma. Nat Biotechnol 1998;16:168–71.

doi:10.1016/j.drudis.2010.09.414

A68 Combination of a triblock copolymer L64 with electrotransfer increases gene delivery in vitro Luc Wasungu 1,∗ , Anne-Laure Marty 2,3 , Michel Francis Bureau 2,3 , Michel Bessodes 2,3 , Justin Teissie 1 , Daniel Scherman 2 , Marie-Pierre Rols 1 , Nathalie Mignet 2,3 1 CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077, Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France 2 Unité de Pharmacologie Chimique et Génétique; CNRS, UMR 8151, Inserm U 640, Université Paris Descartes, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, F-75270 Cedex, France 3 ENSCP, Paris, F-75231 Cedex, France ∗

Corresponding author. E-mail: [email protected] (L. Wasungu). Gene transfer into muscle cells is a key issue in biomedical research. Indeed, it is important for the development of new therapy for many genetic disorders affecting this tissue and for the use of muscle tissue as a secretion platform of therapeutic proteins. Electrotransfer is a promising method to achieve gene expression in muscles. However, this method can lead to some tissue damage especially on pathologic muscles. Therefore there is a need for the development of new and less deleterious methods. Triblock copolymers as pluronic L64 are starting to be used to improve gene transfer mediated by several agents into muscle tissue.

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doi:10.1016/j.drudis.2010.09.415

A69 A receptor-mediated gene delivery system using CXCR4 ligand-conjugated cross-linking peptides A.A. Egorova, A.V. Kiselev ∗ , M.S. Bogacheva, V.S. Baranov Ott’s Institute of Obstetrics and Gynecology, 199034 Saint-Petersburg, Mendeleevskaya line, 3, Russian Federation ∗

Corresponding author. E-mail: [email protected] (A.V. Kiselev). Application of DNA as therapeutics requires efficient cell and tissue-specific targeting which can be achieved by modification of vehicles with a ligand for certain receptor. CXCR4 is a receptor of chemokine SDF-1 and is expressed on some types of cancer and stem cells. Cystein-flanked peptides which are capable of forming small and stable DNA condensates because of cross-linking are considered to be a perspective group of non-viral vehicles. The aim of this project is to characterize a CXCR4 ligand-conjugated cross-linking peptides as a receptor-mediated gene delivery system. We studied four types of DNA/peptide complexes with different ratio between cystein-flanked arginine-rich peptide modified with N-terminal sequence of the chemokine SDF-1 (residues 1–17) and peptide (CHRRRRRRHC) – 100%, 50%, 10% and 0% (ligand-free control). The peptides modification with histidine residues facilitates the escape of DNA from endosomes. Template polymerization of cross-linking peptides was used to form DNA/peptide complexes. EtBr

exclusion and DNA retardation assays proved peptides ability to condense DNA. Transfection activity was studied in CXCR4(+),(A172 and HeLa) and CXCR4(−) (CHO) cell lines with lacZ as a reporter gene. Transfection efficacy of ligand-conjugated vehicles in CXCR4(+) HeLa and A172 cells was 10-times higher compared to control peptide. The level of transgene expression with ligand-conjugated peptides in low N/P ratios was comparable with the efficacy of control PEI. Otherwise transfection efficacy of ligand-conjugated peptides on CXCR4(−) CHO cells was lower than in control PEI. Thus these results demonstrate that ligand-conjugated peptide-based vehicles reported can be a perspective approach for effective gene delivery to CXCR4 expressing cells. Acknowlegements This work was supported by Carl Zeiss fellowship and RFBR grant 10-04-01236-a. doi:10.1016/j.drudis.2010.09.416

A70 Antibody targeting of lipid nanocapsules for directed drug delivery: physicochemical characterization and in vitro study P. Sánchez-Moreno, H. Boulaiz, J.A. Marchal, J.L. Ortega-Vinuesa, J.M. Peula García, A. Martín-Rodríguez ∗ Biocolloids and Fluids Physics Group, Applied Physics Department, Faculty of Sciences, University of Granada, Granada 18071, Spain ∗

Corresponding author. E-mail: [email protected] (A. Martín-Rodríguez). Lipid nanocapsules are recently developed as nanocarriers for lipophilic drugs delivery. The surface characteristics of these colloidal particles are determinant in order to provide a controlled and directed delivery on target tissues with specific markers. We report the development of immuno-nanocapsules, in which antibodies are conjugated to nanocapsules offering the promise of selective drug delivery to specific cells. Several nanocapsule systems were prepared by the solvent displacement technique obtaining an oily core surrounded by a functional shell with surface carboxylic groups. Antibodies were conjugated with nanoparticles by the carbodiimide method that allows it the covalent immobilization of protein molecules through these carboxylic surface groups. A complete physico-chemical characterization of the immuno-nanocapsules was developed confirming the immobilization of protein molecules on the colloidal

nanoparticles via electrokinetic and colloidal stability experiments. The immunoreactivity of the protein–nanocapsules complexes was studied following the changes in the turbidity after addition of specific antigens, showing an adequate surface disposition of the covalent bound antibodies in order to a specific immunological recognize. Finally, nanocapsules were conjugated to a specific antibody to HER2 oncoprotein. In this case, in addition to the colloidal characterization, an ‘in vitro’ study was developed using this surface modified system with different lipophilic anti-cancer drugs entrapped in their oily core. Flow cytometry experiments were used in order to evaluate the cytotoxicity (IC50) of our modified nanocapsules with wild-type and HER2 over expressing tumoral, cell lines. The obtained results have shown the capacity of the immuno-nanocapsules to increase their uptake in tumoral cells, suggesting their ability to a selective deliver drugs. doi:10.1016/j.drudis.2010.09.417

A71 Characterization of polymer-coated nanoparticles based on DNA condensation via spermine A. Rata-Aguilar ∗ , A.B. Jódar-Reyes, J.M. Peula-García, M.J. Galvez, J.L. Ortega-Vinuesa, A. Martín-Rodríguez Biocolloids and Fluids Physics Group, Applied Physics Department, Faculty of Sciences, University of Granada, Granada 18071, Spain ∗

Corresponding author. E-mail: [email protected] (A. Rata-Aguilar). The combination of the complete human genome sequence and the understanding of molecular pathways of some diseases including cancer, could lead to develop several interesting new treatments, such as gene therapy. But one of the major obstacles preventing this therapy from being used is the lack of specific and efficient delivery systems. The uptake of vectors by living cells depends on the degree of DNA condensation, thus we used a demonstrated condensing agent of nucleic acids: spermine. Nanoparticles based on DNA condensation by this natural polyamine were synthesized. In order to protect DNA against DNase degradation, these nanoparticles were coated with the positive charged polymers chitosan or polyethyleneimine (PEI). Folic acid was covalently bound to chitosan with the aim of enhance nanoparticle endocytosis via folate receptor, which is over-expressed in cancer

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Their mechanism of action is still under investigation. The combination of electrotransfer and triblock copolymers, in allowing softening electric field conditions leading to efficient DNA transfection, could potentially represent a milder and more secure transfection method. In the present study, we address the possible synergy that could be obtained by combining the copolymer triblock L64 and electroporation. The synthesis of fluorescent probes L64-rhodamine and DNA-rhodamine is presented here. These probes allowed us to gain some insights into the mechanism of transfection of the combined physical and chemical methods. We have found that a pretreatment of cells with L64 could improve the transfection efficiency. Neither interaction of DNA with the cell membrane, nor L64 membrane interaction seemed to be related to the gain obtained in these transfecting conditions.

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cells. Nanoparticles were characterized and some preliminary in vitro studies were done, showing that nucleic acids are efficiently condensed with this system, which appears to have a potential use in cancer gene therapy. doi:10.1016/j.drudis.2010.09.418

A72 Reduced transgene persistence and trafficking to nuclear periphery are barriers to transfection in lipid substituted nonviral cationic polymer Charlie Hsu ∗ , Hasan Uludag 808 Chemical and Materials Engineering Building, University of Alberta, Edmonton, Alberta T6G 2G6, UK ∗

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Corresponding author. E-mail: [email protected] (C. Hsu). Background: Polyethylenimine (PEI) is one of the most sought after cationic polymer for nonviral gene delivery owing to its ability to transfect a variety of cell types efficiently. The amine groups found on the polymer renders high density of cationic charges, which facilitates efficient binding to DNA, while allowing polymer to be derivatized conveniently. Recently, our lab has derived a novel amphiphilic polymer by grafting linoleic acid (LA) to a low molecular weight PEI (2 kDa). The resulting polymer, PEI2k-LA, displayed significant improvement in transfection efficiency in HEK 293T cells over the ineffective, unmodified 2 kDa PEI. However, when PEI2k-LA was used to transfect rat bone marrow stromal cell (rBMCS), low transfection was observed despite 80% of the cells showing polyplex uptake. We aim to further improve PEI2k-LA transfection efficiency in primary cell line by gaining better understanding of its intracellular kinetics in transfection. In this study, we compared the efficiency of polyplexes trafficking to the nuclear periphery with respect to cellular uptake and transgene expression. Polyplexes routing to the nuclear periphery may facilitate passive nuclear uptake of transgene DNA following mitosis, which may increase the probability of transgene expression. Methods: A mammalian expression vector encoding the green fluorescent protein is covalently labeled with Cy5 (Mirus Bio Label IT® Tracker). Plasmid DNA labelled using this method maintains transcriptional activity, permitting simultaneous tracking of DNA and transgene expression. Labelled DNAs are complexed with PEI2k-LA or 25 kDa branched PEI (bPEI25k) to transfect tissue cultured rBMSC; cells and nuclei

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are processed for analysis by flow cytometry at 0.16, 1, 4, and 7 days to assess for DNA uptake and transgene expression. Results and discussion: GFP-expression was detected in bPEI25k transfected cells, but not with PEI2k-LA treated cells. PEI2k-LA was able to deliver DNA with similar efficiency as bPEI25k; both carriers delivered DNA to >90% of cells by Day 1. However, the percent of cells with DNA uptake reduced to <50% at an earlier time point with PEI2k-LA than with bPEI25k (∼1.7-fold difference between carriers by Day 4). There were significantly fewer nuclei with plasmid DNA associated from PEI2k-LA treated cells than bPEI25k (6% versus 43%, Day 7). Further, the nuclei from PEI2k-LA treated cells had, on average, fewer amounts of DNA associated (∼11-fold lower). Taken together, these data suggest that the lack of transfection in rBMSC by PEI2k-LA may be attributed to reduced transgene trafficking to the nuclear periphery and reduced intracellular retention of transgene DNA. Carrier efficiency in transfection may be improved by concurrently enhancing its DNA protective ability and nuclear routing capability. doi:10.1016/j.drudis.2010.09.419

A74 Utilising the fluorescent properties of Laurdan to study plasma membrane fluidity in cells treated with the cell penetrating peptide R8 Catherine Watkins ∗ , Onyebuchi Ochonogor, Arwyn T. Jones Welsh School of Pharmacy, Cardiff University, Cardiff, CF10 3NB, Wales, UK ∗

Corresponding author. E-mail: [email protected] (C. Watkins). Despite a large body of research, the mechanism of CPP translocation across biological barriers remains unclear. CPP interactions with membrane lipids have been studied by numerous groups and are hypothesised to be critical determinants for internalisation into cells. The possibility exists that cationic CPPs such as octaarginine (R8) and HIV-TAT, at certain concentrations, affect the phase behaviour of the membrane bilayer [1,2]. This phenomenon may explain our earlier studies with leukaemia cells; R8 freely crosses the plasma membrane at concentrations >5 ␮M, in cells depleted of cholesterol and also at low temperatures [3]. We therefore determined what effects different temperatures, and cholesterol manipulations had on the fluidity and phase behaviour of the plasma membrane

of leukemic KG1a and K562 cells and then compared the data with that obtained from experiments in cells incubated with R8. Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) is a fluorescent membrane probe that possesses different spectral properties depending on the phospholipid phase state of the membrane. Upon passing from the gel phase to the liquid crystalline phase a shift of the emission maxima is observed, from 440 nm to 490 nm and the emission/excitation values obtained can be used to determine membrane fluidity. The results confirm that for both cell lines, over the temperature range of 4–37 ◦ C, the plasma membrane fluidity increased with increasing temperature. Extraction of plasma membrane cholesterol results in an influx of R8-Alexa488 into the cytosol of cells incubated at 37 ◦ C with 2 ␮M peptide but this effect can be reversed by adding back cholesterol to cholesterol depleted cells. M␤CD treatment caused an increase in plasma membrane fluidity but this was unchanged in cells in which had been incubated with M␤CD:Chol. Direct plasma membrane translocation of R8-Alexa488 was previously seen in the majority of both KG1a and K562 cells within 10 min of peptide addition (10 ␮M) while the peptide was restricted to intracellular vesicles at 2 ␮M thus raising the possibility that translocation at high concentration was the result of peptide induced effects on membrane fluidity. This was however not the case as no effects on membrane fluidity were observed when similar Laurdan measurements were performed in R8 treated cells. Overall the data show that under conditions where direct translocation of R8 is observed, the fluidity of the plasma membrane is unperturbed. Reference 1. Herce H, Garcia A. Molecular dynamics simulations suggest a mechanism for translocation of the HIV-1 TAT peptide across lipid membranes. Proc Natl Acad Sci USA 2007;104:20805–10. 2. Herce H, et al. Arginine-rich peptides destabilize the plasma membrane, consistent with a pore formation translocation mechanism of cell-penetrating peptides. Biophys J 2009;97:1917–25. 3. Watkins C, et al. Low concentration thresholds of plasma membranes for rapid energyindependent translocation of a cell-penetrating peptide. Biochem J 2009;420:179–89.

doi:10.1016/j.drudis.2010.09.420

Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

Éva Molnár ∗ , Eugen Barbu, Chun-Fu Lien, Dariusz C. Górecki, John Tsibouklis School of Pharmacy and Biomedical Sciences, University of Portsmouth, St Michael’s Building, White Swan Road, Portsmouth PO1 2DT, UK ∗

Corresponding author. E-mail: [email protected] (É. Molnár). Targeting therapeutic compounds to the central nervous system (CNS) via systemic administration requires crossing the blood–brain barrier (BBB). This is currently one of the most challenging problems in CNS drug development. A series of alkylglyceryl chitosans with systematically varied degrees of grafting were prepared through synthetic steps that involved the protection of amino moieties via the formation of phthaloyl chitosan. These alkylglyceryl-modified chitosans were formulated into nanoparticles via a standard ionic gelation technique using sodium tripolyphosphate; the stability and size distribution profiles of nanoformulations were determined using dynamic light scattering. The mean diameter of the particles was found to range between 200 and 350 nm, with the zeta potential between +37 and +41 mV. The stability of nanoformulations was investigated under physiological conditions: it was found that an increase in pH from 4 to 7.4 resulted in a raised hydrodynamic diameter of particles and in a corresponding decrease of their zeta potential. A further chemical modification involving a partial quaternisation of the alkylglyceryl-modified chitosan improved the stability of the formulation at neutral pH, as shown by the changes in the zeta potential and particle size. Preliminary in vitro tests using mouse-brain endothelial cells demonstrated no toxicity and an efficient uptake and indicated that butylglyceryl chitosan and butylglyceryl N,N,N-trimethyl chitosan nanoparticles are promising formulations for BBB targeting. doi:10.1016/j.drudis.2010.09.421

A76 A study of the interaction of novel, coated microparticles with alveolar macrophages and their application in tuberculosis treatment via inhalation C. Lawlor 1,2,∗ , M. O’Sullivan 2 , S. O’Leary 2 , R. Bowie 2 , J. Keane 2 , P.J. Gallagher 1 , S.A. Cryan 1 1 School of Pharmacy, RCSI, Dublin, UK 2 Institute of Molecular Medicine, Trinity College Health Science Building, St. James’ Hospital, UK ∗

Corresponding author. E-mail: [email protected] (C. Lawlor). Introduction: Mycobacterium tuberculosis (MTb) is a pathogenic mycobacterium and the main causative agent of tuberculosis infection in humans. Current treatment involves a multi-dose drug regimen for a minimum of 6–9 months. Approximately 80% of all MTb cases affect the pulmonary region. Despite this fact therapy is currently based on oral and parenteral formulations [1]. Aerosol delivery of anti-tubercular agents aims to reduce the systemic toxicity associated with conventional therapy, to maximise local concentrations of therapeutics in the alveolar region and target alveolar macrophages (AM), the niche environment of the MTb bacilli. We have bioengineered novel, inhalable microparticles designed to efficiently target drugs intracellularly to alveolar macrophages using opsonic coatings. The aims of this study were: (i) to determine the effect of the coatings on the uptake and intracellular trafficking of the microparticles in AMs and (ii) to assess the effect of coated and uncoated microparticles on macrophage activation. Materials and methods: Poly-lactide-co-glycolide (PLGA) microparticles were manufactured using a solvent evaporation method and coated with a number of opsonic proteins. THP-1 cells were differentiated using phorbol 12-myristate13-acetate (PMA) into a macrophage-like cell and where necessary infected with MTb. Noninfected or infected cells were treated with fluorescently labelled microparticles, fixed and counterstained using LAMP-1 and DAMP. Their uptake and intracellular trafficking was visualised using confocal laser scanning microscopy (CLSM). THP-1 blue cells were used to assess the effect of the microparticles on AM activation. This cell line produces a reporter protein when NFkB is activated. These cells were also differentiated using PMA and subsequently treated with microparticles. Results: The coated microparticles were efficiently internalised by infected THP-1 cells and showed some degree of co-localisation with MTb after 1 h.

Microparticle-treatment led to significant activation of NFkB. The degree of activation was found to be microparticle size and coating dependent. Conclusion: Opsonic coating of inhalable microparticles significantly increases their uptake into TB-infected AMs and facilitates co-localisation with the mycobaterium. Previous work by us and others has shown that empty microparticle treatment of MTb infected cells can decrease mycobacterial viability. The increase in NFkB expression associated with microparticle treatment may explain this phenomenon via induction of pro-inflammatory cytokines important for mycobacterium control. Overall this work suggests that microparticles may have immunopotentiator applications in MTb control. Reference 1. Muttil P, et al. Pharm Res 2009;26:2401–16.

doi:10.1016/j.drudis.2010.09.422

A77 Development of a high throughput method for screening of novel nanotechnologies for siRNA transfection of airway cells using high content screening (HCS) A. Hibbitts ∗ , C. Kelly, J. Barlow, C. Jefferies, F. O’Brien, S.A. Cryan School of Pharmacy, Royal College of Surgeons in Ireland, York House, York St, Dublin 2, Ireland ∗

Corresponding author. E-mail: [email protected] (A. Hibbitts). Introduction: RNA interference (RNAi) is an endogenous system in eukaryotic cells whereby sequence-specific RNAs are able to bind and degrade their complementary mRNA. Properly applied, this system could potentially be used to control and treat a wide range of respiratory diseases including cystic fibrosis, lung cancer and inflammatory lung disease. However, siRNA delivery problems encountered in the lungs include poor airway mucus penetration, insufficient cell uptake, poor cell-type specific targeting and rapid clearance. To overcome these problems, we have developed a range of novel nanotechnologies for transfection of airway epithelial cells and alveolar macrophages. The aim of this study was to develop a high throughput method for screening novel nanotechnologies for siRNA transfection of airway cells using high content screening (HCS). Materials and methods: A range of polyethyleneimine-polyethyleneglycol (PEI-PEG) polymers was synthesised and complexed with fluorescent siRNA (fl-siRNA) and

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A75 Alkylglyceryl chitosan nanoparticles for drug delivery across the blood–brain barrier

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used to transfect an airway epithelial cell line, Calu-3 cells. A range of targeted, mannosylated liposomes were also synthesized, fl-siRNA was encapsulated therein and these complexes were used to transfect an alveolar macrophagelike cell line, THP-1. Each of the systems was characterised for size, zeta-potential and encapsulation efficiency prior to transfection. To determine the efficiency of fl-siRNA transfection facilitated by these nanoparticles a protocol was specifically designed to qualitatively and quantitatively monitor siRNA uptake using InCell 1000 high content screening. Results: A number of the PEI-PEG nanoparticles significantly increased siRNA uptake into Calu-3 cells and a number of the mannosylated liposomes were capable of efficiently transfecting alveolar macrophages, a particularly difficult to transfect cell type. Conclusion: HCS facilitated the screening of a large number of novel nanoparticles rapidly and comprehensively for siRNA delivery efficiency, providing both high quality cell images and quantitative data on siRNA uptake, thereby avoiding the need for separate microscopy and quantification studies. doi:10.1016/j.drudis.2010.09.423

A78 Endosomal DNA release studies using giant unilamellar vesicles as model endosomal membranes Laila Kudsiova ∗ , Jasmine Jian-min Goh Pharmacy Department, King’s College London, 150 Stamford Street, London SE1 9NH, UK ∗

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Corresponding author. E-mail: [email protected] (L. Kudsiova). Endosomal DNA release is one of the main barriers to successful non-viral gene delivery, since the inability of DNA to escape from the endosome at an early stage leads to its degradation through trafficking to the lysosomal compartment. It is therefore essential to understand the interactions between commonly used gene delivery vectors and endosomal membranes. While membrane interactions are often studied using small unilamellar vesicles (SUVs) as model bilayers, it is proposed that giant unilamellar vesicles (GUVs) present more realistic models due to their larger size, their superior lipid packing due to reduced surface curvature and the ability to visualise them using light or confocal microscopy. GUVs composed of a mixture of neutral or neutral and negatively charged lipids, representing early or late stage endosomal membranes respectively were prepared by electroforma-

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tion in calcein, followed by the addition of cobalt chloride to quench background fluorescence. GUVs were then observed by confocal fluorescence microscopy before and after the addition of lipid:DNA complexes composed of equimolar mixture of dimethyldioctadecylammonium bromide (DDAB) with the helper lipid dioleoylphosphatidyl-ethanolamine (DOPE) incorporating a 10 mol% rhodamine-labelled DOPE at a 4:1 lipid:DNA charge ratio. Furthermore, in order to visualise the DNA in relation to the encapsulated calcein (green) and the lipid (red), 4 ,6-diamidino-2-phenylindole (DAPI) was added to highlight the DNA blue. Both endosomal models formed spherical GUVs approximately 10–90 ␮m in diameter and were visible as green calcein-encapsulating vesicles. Upon the addition of lipid:DNA complexes to the early endosomal model, a large number of GUVs were shown to lose fluorescence due to calcein leakage, which was concentration dependent first order kinetics. This was also associated with visible alignment of the lipid (red) and the DNA (blue) around the GUV with possible pore formation and DNA translocation across the endosomal membrane. When lipid:DNA complexes were added to the late endosomal membrane model (which incorporated a small percentage of anionic lipid), calcein leakage and pore formation on the surface of the GUV membranes were clearly visible. Additionally, and exclusively to this model, however, a high number of GUVs were shown to deform after the addition of the complexes with or without calcein leakage. This was thought to be due to electrostatic interactions between the cationic DDAB and the anionic lipid domains of the endosomal membrane. In conclusion, it is thought that DDAB-DOPE:DNA complexes interact with both early and late endosomal membranes, causing pore formation and DNA translocation across the membrane, however the nature of the interaction changes as the endosomes traffic from early to late stages. doi:10.1016/j.drudis.2010.09.424

A79 Characterisation of a cytosolic shuttle based upon ricin toxin S.C.W. Richardson ∗ , A.K. Kotha School of Science, University of Greenwich, Kent, UK ∗

Corresponding author. E-mail: [email protected] (S.C.W. Richardson). We have cloned and codon optimised both modified ricin B chain (containing N-terminal 6 His and V5 motifs) and disarmed ricin A chain (containing either a deletion (deleted amino acids 177–183) or mutation (amino acids 177–183 mutated to Gly) within the active site). These molecules were expressed in Escherichia coli BL21*DE3 and affinity purified from E. coli lysate using Talon affinity resin. Following an initial round of characterisation by SDS PAGE and Coomassie brilliant blue staining, Western blotting (using commercially available anti-ricin A or B chain antibodies as well as antibodies specific for N- and C-terminal epitopes) was successfully used to confirm the production of both species of molecule. Both ricin A and B chains were tested for toxicity against a panel of cell lines either individually, after mixing the A and the B chains, or after re-folding using published protocols. Having ascertained that, relative to wild-type ricin A chain, minimal toxicity was displayed by the disarmed A chain analogues, further controls were undertaken to investigate the character of the recombinant B chain. These studies are reported here and show that the recombinant B chain demonstrates both lectinic activity and the ability to translocate to the Golgi, being localised to GM130 positive structures as depicted by epifluorescence microscopy. Further, crude subcellular fractionation and Western blotting of Vero cells exposed to refolded ricin toxin containing disarmed A chain show the disarmed A chain in the cytosol and the differential sedimentation of the B chain within membrane delimited structures. This data suggests the potential of these materials as cyotosolic delivery vehicles suitable for use with gene medicines such as antisense oligonucleotides or RNAi agents. doi:10.1016/j.drudis.2010.09.425

Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

Xiaona Jing 1 , Anders Husted Simonsen 1 , Marina Kasimova 1 , Henrik Franzyk 2 , Camilla Foged 1 , Hanne Morck Nielsen 1,∗ 1 Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark 2 Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark ∗

Corresponding author. E-mail: [email protected] (H.M. Nielsen). Biomacromolecules as proteins and nucleic acids are promising drug candidates. However, one problem with biomacromolecules is that they usually have to pass the cell membrane to exert their effect. Utilization of cell penetrating peptides (CPPs) might be a way to transport biomacromolecules across the cell membrane. It is becoming increasingly evident that CPP uptake pathways may vary depending on the physico-chemical properties of the CPP and the cargo they deliver, the specific cell types and the specific experimental conditions. Nevertheless, the interaction between CPPs and membrane is the very first step of the internalization. Analysis of the CPPs interaction with liposomes is expected to provide information about the CPPs interaction with the cell membrane. We have performed a thermodynamic characterization and spectroscopic of the binding between a series of novel CPPs and anionic liposomes. Recently, we described a new class of CPPs, which seem to show superior biological effect compared to the well described CPPs. The molecular design of these alpha-peptide-beta-peptoid chimeras is based on alternating repeats of (-amino acids and (-peptoid residues. The rationale was to benefit from the structure-promoting effects and lipophilicity from the unnatural chiral (peptoid residues, and the (-amino acid residues providing cationic properties and hydrogen bonding possibilities. The chimeras are very stable toward proteolysis, non-hemolytic, possess antibacterial activity and promising cell-penetrating potential. Interpretation of the data obtained in ITC-experiments showed that an increased number of basic residues in

the novel CPPs sequence resulted in a more favorable interaction with the anionic liposomes. Additional experiments revealed that a hydrophobic interaction was a part of the binding. From CD spectra it was concluded, that no major structural changes occurred in the novel CPPs when they were in the presence of anionic liposomes. The initial electrostatic attraction in CPPs internalization mechanism was confirmed by comparing Gibbs free energy ((G) with the number of basic residues. Furthermore, it is proposed that the hydrophobic interaction registered could be between hydrophobic groups on the novel CPP and the hydrophobic region of the liposome. Another possibility could be simultaneously increased lipid-lipid interaction in the hydrophobic region of the liposome. In conclusion, when comparing the novel CPPs with results obtained for the well described CPP penetratin it seems, that the binding to anionic liposomes is more favorable for all novel CPPs investigated. doi:10.1016/j.drudis.2010.09.426

A81 Studies towards improved cellpenetrating peptide-promoted macromolecular drug delivery Jacob A.D. Clausen 1,2,∗ , Lars Linderoth 3 , Rikke Bjerring Andersen 3 , Henrik Franzyk 1 , Hanne M. Nielsen 2 1 Department of Medicinal Chemistry, University of Copenhagen, Denmark 2 Department of Pharmaceutics and Analytical Chemistry, Universitetsparken 2, DK-2100 Copenhagen, Denmark 3 Novo Nordisk, Novo Nordisk Park, DK-2760 Måløv, Denmark ∗

Corresponding author. E-mail: [email protected] (J.A.D. Clausen). The general concept of drug delivery facilitated by cell-penetrating peptides (CPPs) is well-known; however its practical utility for delivery of biopharmaceuticals necessitates further development concerning in vivo stability and efficiency of these peptidic carriers. In the present project, the aim is to increase the stability towards enzymatic degradation as well as to improve membrane translocation properties by incorporating novel unnatural amino acids into the naturally occurring CPP penetratin. The CPP efficiency of these penetratin analogues will be tested upon conjugation to a therapeutic biomacromolecule. Nine novel and unique amino acid building blocks have been synthesized from enantiopure aziridines to form

amino acids with additional cationic charges as compared to natural amino acids. An increased number of cationic charges in CPPs have been shown to improve the interaction between CPPs and the cell membrane. The novel amino acids will be incorporated into penetratin to increase its cationic charge and to generate more efficient and stable CPPs. The enzymatic stability of penetratin is estimated by testing its resistance towards degradation by intestinal juice from rats. The metabolites are analyzed by an Orbitrap MS to identify the initial sites of cleavage and the largest non-degradable fragment as well. Thereby the optimal sites for incorporation of the novel amino acids may be revealed. The modified penetratin molecules will be tested for stability and CPP efficiency. doi:10.1016/j.drudis.2010.09.427

A82 New configuration of an in vitro blood–brain barrier model Eduard Urich ∗ , Per-Ola Freskgard CNS Research, F. Hoffmann – La Roche, Building 93/5.38, CH-4070 Basel, Switzerland ∗

Corresponding author. E-mail: [email protected] (E. Urich). It is an undeniable fact that neuroscience has an urgent need for a reliable and translatable in vitro model to investigate the human blood–brain barrier (B3). The use of human primary cerebral capillary endothelial cells is considered to provide such a model. The aim of the present study was to compare a B3-model based on two novel immortalized human primary brain endothelial cell (hBEC) lines. The human cerebral cortex microvascular endothelial cell (hCMEC-D3) and the human brain capillary endothelial cell line (NKIM-6) were used. These cell lines were used to investigate the potential transport of large molecules across the cell monolayer. The B3 is unique in that it consists of highly selective endothelial cell interface that create tight junctions around the capillaries separating the bloodstream from the brain parenchyma. Brain endothelial cells in association with astrocytes display complex tight junctions, polarized expression of enzymes, transporters and receptors. In order to take advantage of the influence associated with astrocytes we established an in vitro coculture model of hBECs with primary human astrocytes. The co-culture was performed either by growing the cells on either side of a permeable membrane or growth in direct contact. Using a cell-based kinetic profiling approach

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A80 Microcalorimetric and spectroscopic studies on the mechanism of interaction between novel peptoids and lipid bilayers - effect of length, charge and N-terminal end group

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we studied the optimal conditions for attachment and proliferation of the astrocytes and hBECs. Furthermore, we monitored the effect of hBEC growing directly on the surface of an adherent astrocytic monolayer. The tight junctions between the brain endothelial cells forms a diffusion barrier that is responsible for the high paracellular resistance which is a crucial characteristic for any B3-model. In order to test the integrity of this barrier in the B3-model and simultaneously measure the transcellular transport we combined fluorescent compounds and dye labelled large molecules to test the permeability across the barrier. This strategy allows for the discrimination between transcellular and paracellular transport. doi:10.1016/j.drudis.2010.09.428

A83 Solid lipid nanoparticles for gene delivery into prostate cancer cells Marcelo Bispo de Jesus 1,2 , Carmen Veríssima Ferreira 2 , Eneida de Paula 2 , Dick Hoekstra 1 , Inge S. Zuhorn 1,∗ 1 University of Groningen, University Medical Center Groningen, Dept. of Cell Biology/Membrane Cell Biology, Groningen, The Netherlands 2 State University of Campinas, Dept. of Biochemistry, Institute of Biology, São Paulo, Brazil ∗

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Corresponding author. E-mail: [email protected] (I.S. Zuhorn). Prostate adenocarcinoma is the most common cancer occurring in male. The aim of this study is to develop a gene delivery system based on solid lipid nanoparticles (SLNs) for the transfer of tumor suppressor genes that are able to induce death into prostate cancer cells. Formulations of cationic SLNs, consisting of stearic acid/DOTAP/pluronic, were produced. Additionally, formulations with and without 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in various molar ratios were tested. The SLNs produced were approximately 100 nm in size and showed a positive surface charge (+40 mV) in water. The SLNs showed excellent stability, as evidenced by size, zeta potential, transfection efficiency over 140 days, and possibility of lyophilization and/or sterilization without loss of efficiency. The SLNs were able to protect genetic material against DNase digestion and showed a transfection capacity comparable to that of Lipofectamine 2000® , a commercially available gene carrier. Interestingly, we found that the transfection efficiency of SLNs in prostate cancer PC3 cells was signifi-

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cantly higher when compared to that in normal human prostate PNT2-C2 cells. Further examination revealed that this is due to enhanced endosomal escape rather than enhanced internalization of SLNs in prostate cancer cells. These results indicate that cationic SLNs are a promising tool for gene delivery into prostate cancer cells.

A85 Peptide-based nano-particle for in vivo delivery of siRNA A. Rydstrom ∗ , S. Deshayes, K. Konate, L. Crombez, G. Aldrian, G. Divita CRBM-CNRS-UMR5237, Dept. Molecular Biophysics & Therapeutics, 1919 route de Mende, Montpellier, France

doi:10.1016/j.drudis.2010.09.429 ∗

A84 Kinases in cationic lipid/polymermediated gene delivery Zia ur Rehman, Dick Hoekstra, Inge S. Zuhorn ∗ Department of Cell Biology/Section Membrane Cell Biology, University Medical Center Groningen, University of Groningen, The Netherlands ∗

Corresponding author. E-mail: [email protected] (I.S. Zuhorn). Cationic lipids/polymers, complexed with DNA (also called lipo/polyplexes), are promising tools for gene delivery or transfection. Lipo/polyplexes have low toxicity, a relative low immunological response and can be synthesized on large scale. Lipo/polyplexes are internalized by cells via endocytosis. The endocytotic pathway that is used by lipo/polyplexes depends on the cell type and the type of lipo/polyplexes, and likely contributes to transfection efficiency. We have recently shown that adhesion receptors are involved in binding and endocytosis of lipoplexes. Cell receptors also have been described for the endocytosis of polyplexes. Receptor occupation can initiate signaling cascades, commonly mediated by kinases, which in turn tightly regulate endocytosis and endocytotic processing. The elucidation of cellular signaling signatures, initiated by lipo/polyplexes and/or those that allow or preclude gene delivery, will be instrumental in understanding the interaction between lipo/polyplexes and cells at the molecular level and contribute to the design of protocols with improved gene delivery efficiency. In this study we have performed a screen with a wide range of validated pharmacological kinase inhibitors, and evaluated their effects on lipo/polyplex transfection efficiency. In this screen a kinase is identified that specifically influences the transfection efficiency of a polyplex. It is further demonstrated that, as a part of the underlying mechanism, this kinase regulates the endocytotic processing of the polyplex and, as a consequence, controls its endosomal escape. doi:10.1016/j.drudis.2010.09.430

Corresponding author. E-mail: [email protected] (A. Rydstrom). The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible of patholological disorders. However their clinical application remains limited by their poor cellular uptake, low bioavailability, and insufficient capability to reach targets in vivo. We have designed a novel approach, based on short amphipathic peptides ‘CADY’ that promotes efficient delivery of siRNA into wide variety of mammalian cell lines and in vivo upon systemic and topical administrations. This carrier consisting of a balance between hydrophobic and hydrophilic domains and forms stable discrete ‘nanoparticles’ with siRNA, through non-covalent interactions. Cellular uptake mechanism of CADY/siRNA nanoparticles is dependent on the size of the particle and involves membrane potential and dynamic, which enables a rapid release of the siRNA into the cytoplasm and promotes a robust down-regulation of target mRNA. CADY-carriers were applied to the delivery of siRNA targeting the cell cycle regulatory protein Cyclin B1 into cancer cells. We demonstrated that when associated with CADY, sub-nanomolar concentrations of siRNA Cyclin B1 significantly knocked down Cyclin B1 protein levels resulting in cell cycle arrest in G2 arrest and blocked cancer cell proliferation. The surface of CADY particles can be functionalized and addition of cholesterolmoiety significantly improves siRNA stability in vivo, thereby enhancing the efficiency of this technology for systemic administration following intravenous injection. We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in various mouse tumour models and demonstrate that CADY-mediated delivery of cyclin B1 siRNA prevents tumour growth in vivo following systemic intravenous injection. Moreover, we showed that functionalization of CADY particles with other chemical groups or biological moieties can be applied to generate formulations to target specific cell types or tissues which can

Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

non-toxic reagents for the delivery of DNA into mouse lung. These reagents, contrary to the most of chemical carriers commercially available, might offer a viable chemical alternative to viral transfection.

doi:10.1016/j.drudis.2010.09.431

Reference 1. Unciti-Broceta A, et al. Tripod-like cationic lipids as novel gene carriers. J Med Chem 2008;51:4076–84.

A86 High-efficient transfection using cationic lipids with programmed biodegradability 1,∗

1

Asier Unciti-Broceta , Loredana Moggio , Kevin Dhaliwal 2 , Laura Pidgeon 1 , Keith Finlayson 1 , Chris Haslett 2 , Mark Bradley 1 1 School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK 2 MRC Centre for Inflammation Research, University of Edinburgh, 47 Little France Crescent, EH9 3JJ Edinburgh, UK ∗

Corresponding author. E-mail: [email protected] (A. Unciti-Broceta). Delivery of nucleic acids into cells has an ever-increasing number of applications with outstanding advances in both gene therapy and biotechnology, highlighting the induction of pluripotency in somatic cells. While the use of viral vectors is currently the most efficient transfection method, their antigenicity along with the risk of potential mutagenesis, among other inconvenients, are important limitations that hinder its application in medicine. Non-viral delivery systems (cationic lipids and polymers) represent an attractive alternative, particularly because of their low-cost, tuneable design and procedural simplicity. However, the in vivo efficacy of these carriers needs to be increased for both research purposes and clinical application. As repetitive dosing would be required in any gene therapy treatment, the cytotoxicity due to the use of these chemicals needs to be reduced, ideally by regulating their metabolic fate. To address these issues, a tripodal cationic lipid [1] was specifically designed to undergo complete intracellular metabolisation into naturally occuring compounds aiming to minimise the toxicity associated with its cytoplasmatic residence. Besides the toxicity issue, the incorporation of hydrolysisprone linkages was addressed to enhance the cationic lipid-DNA dissociation once the lipoplexes have entered the cell by endocytosis. The novel compounds showed remarkable transfection efficiency along with reduced toxicity in a variety of immortalized cells and stem cells. Moreover, preliminary in vivo studies underlined the potential applicability of these

doi:10.1016/j.drudis.2010.09.432

A87 Immune stimulation following microneedle delivery of influenza virus-like particle (VLP) vaccines to human skin Marc Pearton 1 , Sang-Moo Kang 2 , Jae-Min Song 2 , Yeu-Chun Kim 3 , Fu-Shi Quan 2 , Matthew Ivory 1 , Mark R. Prausnitz 3 , Richard W. Compans 2 , James C. Birchall 1,∗ 1 Welsh School of Pharmacy, Cardiff University, UK 2 Department of Microbiology and Immunology, Emory University, UK 3 School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, UK ∗ Corresponding author. E-mail: [email protected] (J.C. Birchall). Virus-like particles (VLPs) possess a number of features that make them attractive vaccine candidates for immunization against infectious disease. Efficient intra-epidermal delivery of VLP vaccines would exploit the abundance of Langerhans cells (LCs) that reside within the skin epidermis to generate an efficient host immune response. Microneedles (MNs) are currently being developed for the convenient and pain-free delivery of drugs and vaccines across the skin barrier layer. Whilst MN-based vaccines have demonstrated proof-of-concept in mice, it would be extremely valuable to understand how MN targeting of influenza VLP vaccines to the skin epidermis affects activation and migration of LCs in the real human skin environment. MNs with lengths of 700 ␮m were laser-etched from stainless steel sheets and surface-coated with either influenza H1 (A/PR/8/34) or H5 (A/Viet Nam/1203/04) VLPs. The coated MNs easily and reproducibly penetrated freshly excised human skin, depositing approximately 80% of the vaccine load within 60 s. Experiments conducted in cultured human skin showed that H1 and H5 VLPs, delivered via MNs, stimulated LCs causing morphological changes and a significant decline in total LCs number in epidermal sheets at 24–48 hours compared to untreated skin at the same time

points. Histological sections showed that LCs in VLP treated samples were more dispersed throughout the epidermis with substantial numbers in the vicinity of the basement membrane. The response made by LCs was more manifest in human skin treated with H1 VLPs, compared with H5 VLPs. These findings corroborate observations in mouse studies, where H1 VLPs were shown to be significantly more immunogenic than H5 VLPs. Our data provide strong evidence that MN-facilitated delivery of influenza VLP vaccines initiates a stimulatory response in LCs in human skin epidermis. The results complement and support data gained from animal models, suggesting dendritic cells (DCs), including LCs, targeted through intraepidermal or intra-dermal deposition of the vaccine generates immune response. This study also emphasizes the value of cultured human skin alongside animal studies for informative preclinical testing of intra-dermal vaccines. doi:10.1016/j.drudis.2010.09.433

A88 Electrically based transdermal techniques for delivery of therapeutic macromolecules Rakesh Kumar Tiwari 1,∗ , Ritesh Kumar 2 1 University of Wales Instituture Cardiff, UK 2 Ravishankar College of Pharmacy, Bhopal 462010, Madhya Pradesh, India ∗

Corresponding author. Advances in molecular biology have given us a wide range of protein and peptide based drugs that are unsuitable for oral delivery because of their high degree of first-pass metabolism. Though parenteral delivery is successful for developed and commercially available protein and peptide based drugs, chronic and self administration formulations are not the ideal choice through this route. Transdermal delivery is emerging as the biggest application target for these agents, however, the skin is extremely efficient at keeping out such large molecular weight compounds and therapeutic levels are never going to be realistically achieved by passive absorption. Therefore novel transdermal drug delivery systems have been developed with the aim to achieve the objective of systemic medication through topical application to the intact skin surface with benefits of deliver therapeutic macromolecules in desire therapeutic doses to overcome the difficulties associated with the oral route, namely poor bioavailability of drug and the tendency to produce rapid blood

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be of a major interest for future development. Given the biological response yielded through this approach, we propose that non-covalent, peptide-based delivery technologies hold a strong promise for therapeutic administration of siRNA.

DELEGATE ABSTRACTS

Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

DELEGATE ABSTRACTS

level. Some newly active rate controlled electrically based transdermal techniques including: iontophoresis, electroporation, ultrasound and photomechanical waves have been developed and commercialized for the delivery of troublesome therapeutic protein and peptide based macromolecular drugs. This study covers the development of different electrically based transdermal techniques for delivery of therapeutic protein and peptide based macromolecular drugs, current status and assesses the pros and cons of each technique and summarises the evidence-base of their drug delivery capabilities. doi:10.1016/j.drudis.2010.09.434

A89 Molecularly imprinted polymers: macromolecule recognition Marc Kelly ∗ , Jenna Bowen, Mark Gumbleton, Chris Allender Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3NB, UK ∗

Delegate abstracts•MONITOR

Corresponding author. E-mail: [email protected] (M. Kelly). Molecular imprinting is a technique used to engineer synthetic antibody mimics by the polymerisation of so-called functional monomers and cross-linkers around a target (template) species. Following removal of the template from the polymer matrix, cavities remain which display both chemical and steric selectivity for the imprinted molecule. To date the imprinting of biologically relevant macromolecules has been somewhat unsuccessful due to the inherent complexity of imprinting such moieties in aqueous media. Unlike small, organic molecules that are typically employed as templates, macromolecular structures such as peptides and proteins can exist in a multitude of conformations which leads to the development of heterogeneous binding sites as opposed to the well defined cavities formed during the regular imprinting process. The proteins will denature in traditional imprinting environments due to the presence of organic solvents and elevated temperatures. Additionally, the size of these biomolecules means that removal from the polymer matrix and subsequent re-binding is often inefficient. As a consequence, molecular imprinting has yet to achieve its true potential as efficacious, robust, reliable and cost-effective alternatives to the currently used antibody-based recognition systems. Projects currently underway within

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our laboratories aim to utilise target-selective peptides, derived from a phage display library, as a high affinity ‘functional monomers’ in a hybrid peptide-polymer molecularly selective system. Targets include lipopolysaccharide (LPS), the major pathogenic determinant of Gram negative bacteria and prion protein which is believed to be the causative agent of a group of invariably fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). Work to date has focused on optimisation of surface chemistries. Bifunctionalised polystyrene resin and glass surfaces have been synthesised to facilitate the independent immobilisation of peptide moiety and an initiator species. Polymer growth from the surface has been monitored by Fourier transform infra-red spectroscopy and atomic force microscopy. Future work will involve optimising a number of polymerisation variables and incorporating the phage-display derived peptide into the system to fully evaluate its potential as an antibody mimic. doi:10.1016/j.drudis.2010.09.435

A90 Interference of mycobacterium tuberculosis with the endocytic pathways on macrophages and dendritic cells from healthy donors: role of cathepsins D. Pires 1 , P. Bettencourt 1 , N. Carmo 1 , T. Bergant 2 , L. Jordao 1 , E. Anes 1,∗ 1 Centro de Patogénese Molecular, Fac. Farmácia, Univ. Lisboa e Instituto de Medicina Molecular, Lisboa, Portugal 2 Department of Biochemistry, Molecular and Structural Biology, Joˇzef Stefan Institute, Ljubljana, Slovenia ∗

Corresponding author. E-mail: [email protected] (E. Anes). Antigen-presenting cells (APC) such as macrophages and dendritic cells (DCs) play a pivotal role in tuberculosis pathogenesis. Macrophages are also key effector cells in mycobacteria killing. In order to survive inside the host immune cells mycobacteria developed different strategies. Among them blocking of phagosome-lysosome fusion and consequential reduced phagosome acidification assumes a crucial role allowing mycobacteria to escape acidic pH and destruction by proteolytic enzymes present in phagolysosomes. Since phagosome acidification varies between macrophages and DCs this may allow different kinetics of acquisition and activity for the enzymes involved. The aim of the present

study was to compare the distribution of two key cathepsins: the exopeptidase cathepsin B and the endopeptidase cathepsin S inside human monocyte derived macrophages and DCs infected with Mycobacterium spp. Infected immune cells were collected after 3 hours and 1 day post-infection and prepared either for immunofluorescence confocal microscopy or for immunogold electron microscopy on ultrathin cryo sections. In macrophages we did not observe significant co-localization between either BCG or Mycobacterium tuberculosis and cathepsins B or S indicating that phagosome-lysosome fusion was strongly hindered. Similar results were observed for Mycobacterium tuberculosis after infection of DCs. In DCs the acquisition of cathepsin B into the phagosomes containing BCG was different from the acquisition of cathepsin S. Cathepsin S content was decreased by 30% after 1 day of infection whereas cathepsin B content inside BCG-positive phagosomes was increased. Our data indicate that cathepsins might be involved in differential mycobacterial persistence in macrophages compared to dendritic cells. Acknowlegements Financial support of FCT project PIC/82859/2007 is gratefully acknowledged. doi:10.1016/j.drudis.2010.09.436

A91 Role of mycobacterium tuberculosis outermembrane porins in bacterial survival within macrophages D. Pires, P. Bettencourt, N. Carmo, M. Niederweis, E. Anes ∗ Centro de Patogénese Molecular, Fac. Farmácia, Univ. Lisboa e Instituto de Medicina Molecular, Lisboa, Portugal ∗

Corresponding author. E-mail: [email protected] (E. Anes). Mycobacterium tuberculosis (Mtb) is the etiologic agent of tuberculosis a major worldwide health concern. One important feature in Mtb virulence is the ability to withstand the detrimental conditions of the phagosome within macrophages. Most of the virulence factors of Mtb are PAMPS from the outer membrane of the bacilli. Outer membrane porins participate in the inflow of hydrophilic compounds and we have shown that they are important for mycobacteria intracellular survival. Several porins have already been described as a means for nutrient acquisition but also as a possible pathway for antibiotic inflow. Previous studies showed that mutant

Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

Acknowlegements Financial support of FCT project PIC/82859/2007 is gratefully acknowledged. doi:10.1016/j.drudis.2010.09.437

A92 In vivo phage display to identify peptides that target the brain Mathew W. Smith ∗ , Mark Gumbleton Welsh School of Pharmacy, Cardiff CF10 3XF, UK ∗

Corresponding author. E-mail: [email protected] (M.W. Smith). The delivery of novel macromolecular therapeutics into brain parenchyma to treat central nervous system disorders (CNS) is hindered by the blood–brain barrier (BBB). The BBB is comprised of microvascular endothelial cells that line the capillaries traversing the brain. The existence of highly restrictive tight junctions and the relatively low abundance of morphologically evident endocytic vesicles restricts both paracellular and transcellular access to the brain of therapeutic proteins, peptides and nano-medicines [1]. As part of an ongoing programme to identify novel ligands that mediate endocytotic and transcytotic events within the BBB we report here the use of a Phage Display library to identify small cyclic

peptides (-7mer) that traverse the in vivo rat BBB. A Phage Display Library (Ph.D.-C7CTM New England Biolabs) representing 1.2 × 109 unique genotypes encoding random-7mer disulphide constrained peptides genomically fused to the pIII coat protein of the filamentous phage M13 was utilised in all studies. A synchronous selection strategy [2] was employed to select for peptides homing to a range of organs before undertaking a final selection for peptides that home to brain grey matter. In this final selection the library was injected i.v. into a rat and circulated for 15 minutes before perfusion with saline to remove freely circulating phage and then glycine buffer (pH 2.2) to strip the vasculature of binding phage. The brain was removed and the white matter and capillaries depleted before the grey matter (brain parenchyma) was homogenised and phages recovered. The recovered phages were gene sequenced to determine the corresponding peptide library sequence displayed. From the sequenced population a conserved motif AC-SXTSSTX-CGGGS was identified at a frequency of 25%; secondary phage studies and bioinformatic analysis of a large population of sequenced clones (>500) corroborated this sequence. In vivo biodistribution studies of a clone displaying the conserved motif (AC-SYTSSTM-CGGGS) revealed a selective homing to brain grey matter as demonstrated by a 4-fold increase in AUC0-∞ and 3.5-fold increase in Cmax in brain grey matter compared to insertless phage (no displayed phage). Studies are addressing the molecular pathways of entry of this peptide phage into the CNS. Reference 1. Smith MW, Gumbleton M. Endocytosis at the blood–brain barrier: from basic understanding to drug delivery strategies. J Drug Target 2006;14:191–214. 2. Kolonin MG, et al. Synchronous selection of homing peptides for multiple tissues by in vivo phage display. FASEB J 2006;20:979–81.

doi:10.1016/j.drudis.2010.09.438

A93 Phage display identification of a lung transduction peptide that affords enhanced macromolecule transport across the intact lung epithelium Christopher J. Morris 1,∗ , Peter Griffiths 2 , Neil McKeown 2 , Mark Gumbleton 1 1 Welsh School of Pharmacy, Cardiff University, Cardiff, UK 2 School of Chemistry, Cardiff University, Cardiff, UK ∗

Corresponding author. E-mail: [email protected] (C.J. Morris). Evolutionary technologies based upon the screening of combinatorial libraries, for example, phage display, are used to survey the molecular diversity of target cell surfaces with the aim of identifying peptide motifs that promote target cell binding or internalisation [1]. Here, an M13 phage peptide library displaying cyclic 7-mer peptides was biopanned against the luminal surface of primary cultures of rat lung alveolar epithelial cells. ‘Cell associated’ phage were isolated after 4 rounds of biopanning, with the peptide library repertoire contracting from 1.2 × 109 clones to a maxium of 2 × 103 clones. DNA sequencing of ‘cell associated’ phage clones indicated peptide sequences to be largely composed of hydrophillic amino acids with isoelectric points approximating neutrality. The most frequent phage clone bore the peptide sequence C-TSGTHPR-C (termed LTP-1) and displayed enhanced (>1000-fold) transport (versus phage control vector) across restrictive in vitro alveolar epithelial monolayers [2]. When the LTP-1 phage clone (LTP-1) was administered as a coarse aerosol into the airways of an isolated perfused rat lung IPRL preparation [3] the extent of phage absorption across the pulmonary epithelium was 8.6% by 120 min, some 1500-fold greater than either the insertless vector control or a library clone that displaying a control peptide sequence (C-PLLAPGI-C, termed NB-3) that was isolated from the first biopanning round. When LTP-1 phage was coadministered with a 100-fold molar excess of the synthetic LTP-1 peptide sequence (syn-LTP1) the extent of LTP-1 phage was competitively inhibited (LTP-1 phage absorption reduced to 0.1% by 120 min, p < 0.05). In contrast, the synthetic NB-3 peptide (syn-NB-3) displayed no inhibitory effect (7.6% LTP-1 phage absorbed dose absorbed by 120 min, p > 0.05). The synLTP-1 peptide sequence was grafted onto the surface of an anionic PAMAM G5.5 dendrimer

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Mycobacterium smegmatis lacking the MspA porin grow defectively due to the lack of glucose uptake but display increased resistance to several antibiotics and also to nitric oxide. Nitric oxide burst is a well described bactericidal mechanism in mouse macrophages and the inducible nitric oxide synthase is the enzyme responsible for NO release. In this study, we describe a novel putative outer membrane protein conserved between M. tuberculosis and Mycobacterium bovis BCG. We show that the absence of this protein limits bacterial growth in vitro but results in increased BCG survival within macrophages. We also demonstrate that although interferon-gamma stimulation of macrophages induces ten times increased killing of BCG, bacteria lacking this protein remain unsusceptible to this stimulation. Furthermore, quantification of iNOS and IL-1beta expression through qRT-PCR revealed that those genes were less upregulated during infection with the mutant bacteria compared to the WT strain suggesting that the increased survival of the mutants is due to lower macrophage activation and release of nitric oxide. We conclude that MtpA from Mtb complex is important to release virulence factors required for macrophage activation.

DELEGATE ABSTRACTS

DELEGATE ABSTRACTS

at a 1:1 stoichiometry to test the lung transduction functionality of the peptide using the 53 kDa dendrimer as a model macromolecular cargo. Phage peptide-dendrimer conjugates were labelled with a fluorophore and characterised by 1 H NMR and quantitative amino acid analysis prior to administration into the airways of the IPRL model. The extent of absorption of PAMAM G5.5 alone equalled 17 ± 6% of lung deposited dose absorbed by 90 min. G5.5 dendrimers displaying one syn-LTP-1 peptide per polymer (termed G5.5-syn-LTP-1) displayed a 1.8-fold greater extent of absorption (p < 0.05) cf. G5.5 alone; G5.5 dendrimer displaying one equivalent of the syn-NB3 peptide showed no evidence of enhanced absorption (p > 0.05). The enhanced absorption of G5.5-syn-LTP-1 absorption was competitively inhibited by co-administration of 100-fold molar excess of syn-LTP-1 peptide (p < 0.05) but not by syn-NB-3 peptide (p > 0.05), an observation consistent with the participation of a specific receptormediated transport mechanism. As such the LTP-1 peptide motif may serve as a platform for enhancing macromolecule absorption from the airways. Reference 1. Sergeeva, et al. Adv Drug Deliv Rev 2006;58:1622–54. 2. Campbell, et al. BBRC 1999;262:744–51. 3. Sakami, et al. Pharm Res 2006;23:270–9.

doi:10.1016/j.drudis.2010.09.439

A94 Differential transport of anionic PAMAM dendrimers across in vitro biological barriers Ghaith Aljayyoussi 1,∗ , Will Ford 1 , Neil McKeown 2 , Mark Gumbleton 1 1 Welsh School of Pharmacy, Cardiff University, Cardiff, UK 2 School of Chemistry, Cardiff University, Cardiff, UK ∗

Delegate abstracts•MONITOR

Corresponding author. E-mail: [email protected] (G. Aljayyoussi). Polyamidoamine (PAMAM) dendrimers are a class of branched polymers that have the potential to serve as drug carriers. This is primarily due to their extremely low polydispersity index, the ability to precisely control their size and charge, and the multiple functional groups that they bear on their surfaces giving the ability to conjugate a wide range of therapeutic molecules. The transport across in vitro biological barriers of cationic PAMAMs has been widely 1114

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Drug Discovery Today • Volume 15, Numbers 23/24 • December 2010

studied with reports often indicating high barrier permeability, although interpretation of such data in the context of cation-induced barrier toxicity is often omitted. We are investigating the intrinsic biological activity of intact stable anionic dendrimer-drug conjugates where the dendrimer moiety not only confers a backbone for attachment of multiple phamacological ligands but also offers a means to physically modulate in vivo tissue disposition, for example, affording access to intestinal submucosa but excluding BBB penetration. In this abstract we report the differential in vitro barrier permeability of a molecular weight series of anionic PAMAM dendrimers, that is, G1.5, 2935 Da; G3.5, 12,931 Da; G5.5, 52,907 Da which has supported our ongoing in vivo investigations. Dendrimers were fluorescently labelled and added to the apical surface of epithelial cell monolayers grown on a semi-permeable inserts (Transwell). Permeability coefficients (ρ) were determined for transport in the apical to basal direction. The epithelial models included the highly restrictive MDCK-I (TEER 5000  cm2 ), the moderately restrictive Caco2 (TEER 600  cm2 ) and the low restrictive MDCKII (TEER 200  cm2 ). For CACO-2 and MDCKII an inverse relationship was evident between dendrimer transepithelial transport and dendrimer molecular size, with dendrimer ρ decreasing approx. 5-fold G1.5 ⇒ G3.5, and decreasing approx. 10-fold G1.5 ⇒ G5.5. The permeability of the cell models to dendrimer transport declined as the paracellular restrictiveness of the monolayers increased. Indeed, for MDCKI monolayers dendrimer concentrations in the basal chamber remained at all times below the limit level of detection, but could be readily enhanced by briefly adding EDTA to the media. Nevertheless, predicted (based upon LLQ) ρ for dendrimer transport across MDCKI were at least ×10–15-fold lower than in the other cell models. Significantly, even for the smallest dendrimer, that is, G1.5, the maximum predicted (based on LLQ) ρ across MDCKI was no greater than 15% of the ρ obtained for the paracellular marker F-Na. Whereas ρ for G1.5 was 51% and 56% of that for F–Na in CACO-2 and MDCKII models, respectively. Biocompatibility studies show no affect of the anionic dendrimers upon overall barrier properties. The paracellular route is the major pathway of dendrimer transport across biological barriers. Stable pharmacologically active conjugates of dendrimer – drug are an interesting experimental therapeutic with potential to provide

differential tissue distribution/exclusion based upon physical characteristics. doi:10.1016/j.drudis.2010.09.440

A95 Non-toxic, highly efficient delivery of nucleic acids into challenging cells using safectin transfection reagent Steve Howell 1,∗ , Mark Bradley 1,2 , Asier Unciti-Broceta 1,2 1 Deliverics Ltd., 2 Blysthwood Square, Glasgow G2 4AD, UK 2 School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK ∗

Corresponding author. E-mail: [email protected] (S. Howell). Deliverics Ltd. has developed a novel cationic lipid-mediated transfection reagent for DNA and siRNA delivery into both easy and challenging to transfect eukaryotic cells: SAFEctin Transfection Reagent. This reagent is a water-based formulation of cationic and neutral lipids with programmed biodegradability. SAFEctin allows for the highest transfection efficiency of nucleic acids into many cell types (e.g. immortilized cells, mESC, hMSC) with the simplest-to-use and fastest procedure in the market: (i) mix SAFEctin and the nucleic acid (ii) followed by direct addition to cells, either in the presence or absence of serum and antibiotics. The formulation has been developed to have very low toxicity to cells and as such it is not necessary to remove or change culture medium following transfection. Combination of the highest/safest transfection rates on the market with the simplest to use protocol ensures optimal performance and fast results. The SAFEctin Transfection Reagent is a universal system that outperforms competitor’s products in each of the three defining features any researcher seeks in this kind of product: efficacy, safety and ease of use. doi:10.1016/j.drudis.2010.09.441

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