Expert Opinion on Biological Therapy

Editorial Multi-focal motor neuropathy: one treatment works but many uncertainties remain Yusuf A Rajabally Neuromuscul...

Author: EDITOR-IN-CHIEF: Michael Morse: Duke University Medical Centre | USA

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Editorial

Multi-focal motor neuropathy: one treatment works but many uncertainties remain Yusuf A Rajabally Neuromuscular Clinic, Department of Neurology, University Hospitals of Leicester, Leicester, UK

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Expert Opin. Biol. Ther. (2011) 11(3):257-259

Multifocal motor neuropathy (MMN) is a relatively newly described purely motor neuropathy [1], which is characteristically treatable, responding to intravenous immunoglobulin (IVIg) therapy in a majority of cases [2]. Identification of the condition relies typically on a clinical picture consisting of asymmetrical predominantly upper limb, distal motor weakness and wasting, occurring more frequently in middle-aged men [2]. The hallmark of MMN is the presence of conduction block (CB) on motor nerve conduction studies. In some forms of MMN, CBs are not present or cannot be detected with routinely available diagnostic techniques, and this may complicate matters as to which patients without typical electrophysiology still have to be tried on treatment. MMN is immune-mediated and may be associated with antiganglioside antibodies to GM1 of IgM type. Different series describe discrepant results regarding the frequency of such antibodies, varying widely, between only 30% and up to 70% of cases. MMN is a rare disease, with estimated prevalence of only 1 -- 2 per 100,000 [3]. This compares with approximately 5 per 100,000 for amyotrophic lateral sclerosis [4]. Nevertheless, the cost of treating MMN is very high, and the recent rapid increase in the cost of IVIg (of about 60% between 2002 and 2010 in the United Kingdom) has heightened the need to find other more cost-effective therapies for the disorder. Furthermore, potential options for immunoglobulin-unresponsive patients remain most uncertain. In the this issue, van Schaik and Eftimov provide a detailed up-to-date, clinically useful and relevant review of the treatment of MMN [5]. IVIg is a treatment of proven short-term effect although its long term efficacy has not been studied. Responder rates as high as 94% of treated patients, have been described recently [6]. As the authors rightly remind us, the literature is on the whole inconsistent on the important issue of, on one hand, the possible benefits of continuing high-dose IVIg therapy in an attempt to prevent axonal loss [7-10] the latter itself representing a marker of poor response, and, on the other, the conflicting need to reduce IVIg usage due to cost. Discrepancies in the literature regarding other prognostic markers of IVIg response are also highlighted, such as presence of CBs and of antiGM1 antibodies. The authors raise the question of side-effects with IVIg, importantly including thromboembolic events, which although possibly more likely in those with multiple vascular risk factors, worryingly, are not known to be currently preventable [11]. The subcutaneous route of administration of immunoglobulins appears promising for MMN patients in the future although, as the authors rightly point out, treatment timing and frequency as well as local adverse effects may be limiting factors. The effects of other theoretically justifiable therapies such as steroids and plasma exchanges are well discussed, and the reasons why they are no longer acceptable for treating MMN are explained. Many other anecdotally attempted agents/methods, namely methotrexate, azathioprine, cyclosporine, interferon beta 1a and autologous stem cell transplantation, are mentioned and the lack of evidence for the use of all of them, properly documented. This is most helpful to the uncertain clinician, either

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Multi-focal motor neuropathy: one treatment works but many uncertainties remain

confronted to an IVIg-unresponsive patient or, alternatively, feeling the cost-pressure and having to justify high dosage use, for an IVIg-responsive patient, ‘too’ dependent on the treatment. In the case of mycophenolate mofetil, what we do now know is that it does not work for MMN, this having been demonstrated in a properly-conducted randomized controlled trial (RCT), the only one of its kind performed for MMN besides those with IVIg [12]. In the case of rituximab, for which enthusiasm has grown in recent years for its potential role in MMN as in other immune-mediated neuropathies, available evidence for MMN, none of which is RCT-based, is conflicting. This is factually well documented in this review, which also includes the latest negative open-label prospective study of six MMN patients, published in September 2010 [13]. Recently reported cases of progressive multifocal leucoencephalopathy (PML) with Rituximab in HIV-negative patients are mentioned [14]. This is a serious concern for any treatment, even if licensed and of proven effect, which is obviously far from being the case for rituximab for MMN. Cyclophosphamide is also well discussed in its historical context in the treatment of MMN. Experience with this immunosuppressive agent is the largest available, and as explained by the authors, of the 41 published cases treated with cyclophosphamide, 28 (68%) actually improved. Side-effects are welldocumented, potentially very serious, and constitute the main concern, again in the absence of evidence from RCTs. So, what shall we do in clinical practice? It appears clear that immunoglobulin therapy is the only treatment one can justifiably start with, and that the intravenous route, administered initially in the hospital environment seems the logical initial way to proceed. Standard dose utilized remains 400 mg/kg daily over 5 days by most, although the issue of initial dosage remains of utmost economic importance, with, it could be argued, a persistent need for adequate doseranging studies in MMN as in other immune-mediated neuropathies like chronic inflammatory demyelinating polyneuropathy (CIDP). Subsequent maintenance doses described in this paper are those obtained from cohort studies due to unavailability of RCT evidence, this again representing a major drawback for such a costly therapy. Functional monitoring and the best way to perform this are beyond the scope of this review but are of obvious vital interest to all those treating patients with MMN. First, a proportion of patients, albeit small, may not need a further course, and second, a placebo effect is well-known to potentially occur, by all neuromuscular neurologists using IVIg repeatedly, and unless the use of objective, regular, reproducible measures of continued effectiveness becomes the rule, overtreatment is likely to continue. This was illustrated in the recent trial of methotrexate for CIDP [15]. On the other hand, the possibility that sustained high-dose IVIg therapy may prevent axonal deterioration and consequent clinical progression cannot be discounted [9,10]. In conclusion, we simply do not know at this time whether, what van Schaik and Eftimov, as well as most of us generally advocate for IVIg-treated neuropathies, that 258

is, regular tapering and gradual dose reduction [16], is really in the best long-term clinical interests of patients with MMN. Long-term dose-comparative studies are needed to try and answer this question. When difficulties arise with the intravenous route, subcutaneous infusion seems a promising alternative, for which the evidence of efficacy versus side-effects remains, however, limited. A trial of subcutaneous administration showed that it appears, nevertheless, a relatively safe, reasonable and reversible option to consider, in selected cases. The same is not true for addon treatment with immune therapies. Rituximab, mainly in view of the latest published negative open-label study, together with the absence so far, of truly convincing effects in other immune-mediated neuropathies, is not an agent that can be recommended at this time. Although cyclophosphamide appears to have helped a majority of MMN patients who have used it, the evidence is of class IV level and most treated cases were not on IVIg (which if used, one may assume, would have probably helped the majority). Also, side-effects are potentially very serious. Most importantly, whether or not to consider such a drug should exclusively remain the clinician’s clinical decision unprompted by financial constraints, as important as these may be in the real world. The low prevalence of MMN is in this regard an essential point to remember and convey to those managing our drug budgets. It would appear at the present time ethically unjustified to use any such agent in immunoglobulin-responsive cases exclusively to reduce dose requirements, especially in the absence of serious side-effects and if adequate functional monitoring is confirmatory of continuing effectiveness. On the other hand, in the rare immunoglobulin-resistant cases, and after reconsideration of the diagnosis, cyclophosphamide may, in specific circumstances of severe progressive functional decline, represent an option, to be attempted with extreme caution. In summary, the treatment of MMN remains very much at the end of 2010, little else other than immunoglobulins. Optimal route of administration, initial dose, long term therapeutic dosage and frequency appear to represent the main questions and challenges, still for the most part unanswered. There are few candidates for replacing immunoglobulin maintenance therapy, and even fewer alternatives in immunoglobulinunresponsive patients. At the present time, the clinical focus should therefore remain on the adequate, functionally-effective, regularly and properly monitored, safe use of immunoglobulins. Despite the understandable temptation to investigate new agents, research should for its part, not neglect the essential issues of dose-ranging and frequency of immunoglobulin administration, both for short-term action as well as longterm prevention of secondary axonal loss, utilizing, what remains so far, the only proven treatment for MMN.

Declaration of interest YA Rajabally has been the recipient of an educational grant from UCB.

Expert Opin. Biol. Ther. (2011) 11(3)

Rajabally

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2.

motor neuropathy. Neurology 2010;75:818-25

Pestronk A, Cornblath DR, Ilyas A, et al. A treatable multifocal motor neuropathy with antibodies to GM1 ganglioside. Ann Neurol 1988;24:73-8

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van Schaik IN, Bouche P, Illa I, et al. European Federation of Neurological Societies/Peripheral Nerve Society guideline on management of multifocal motor neuropathy. Eur J Neurol 2006;13:802-8

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Terenghi F, Cappellari A, Bersano A, et al. How long is IVIg effective in multifocal motor neuropathy? Neurology 2004;62:666-8

Slee M, Selvan A, Donaghy M. Multifocal motor neuropathy: the diagnostic spectrum and response to treatment. Neurology 2007;69:1680-7

3.

Nobile-Orazio E, Cappelari A, Priori A. Multifocal motor neuropathy: currents concepts and controversies. Muscle Nerve 2005;31:663-80

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Vucic S, Black KR, Chong PST, Cros D. Multifocal motor neuropathy. Decrease in conduction blocks and reinnervation with long-term IVIg. Neurology 2004;63:1264-9

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Abhinav K, Stanton B, Johnston C, et al. Amyotrophic lateral sclerosis in South-East England: a population-based study. The South-East England register for amyotrophic lateral sclerosis (SEALS Registry). Neuroepidemiology 2007;29:44-8

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Baumann A, Hess CW, Sturzenegger M. IVIg dose increase in multifocal motor neuropathy: a prospective six month follow-up. J Neurol 2009;256:608-14

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van Schaik IN, Eftimov F. Immunotherapy of multifocal motor neuropathy. Expert Opin Biol Ther 2011;11:xxx-xxx Cats EA, van der Pol WL, Piepers S, et al. Correlates of outcome and response to IVIg in 88 patients with multifocal

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Caress JB, Hobson-Webb L, Passmore LV, et al. Case-control study of thromboembolic events associated with IV immunoglobulin. J Neurol 2009;256:339-42 Piepers S, Van den Bergh-Vos R, Van der Pol WR, et al. Mycophenolate mofetil as adjunctive therapy for MMN patients: a randomized, controlled trial. Brain 2007;130:2004-10

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Chaudhry V, Cornblath DR. An open-label trial of rituximab (RituxanÒ) in multifocal motor neuropathy. J Peripher Nerv Syst 2010;15:196-201

14.

Carson KR, Evens AM, Richey EA, et al. Progressive multifocal leukoencephalopathy after rituximab therapy in HIV-negative patients: a report of 57 cases from the Research on Adverse Drug Events and Reports project. Blood 2009;113:4834-40

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RMC Trial Group. Randomised controlled trial of methotrexate for chronic inflammatory demyelinating polyradiculoneuropathy (RMC trial): a pilot, multicentre study. Lancet Neurol 2009;8:158-64

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Rajabally YA, Seow H, Wilson P. Dose of intravenous immunoglobulins in chronic inflammatory demyelinating polyneuropathy. J Peripher Nerv Syst 2006;11:325-9

Affiliation Yusuf A Rajabally Neuromuscular Clinic, Department of Neurology, University Hospitals of Leicester, Leicester, LE5 4PW, UK Tel: +44 116 258 4576; Fax: +44 116 258 4875; E-mail: [email protected]

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Future Perspective

1.

Introduction

2.

Applying the technology of L. lactis for current health issues

3.

Expert opinion and future

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perspective

Targeting diseases with genetically engineered Lactococcus lactis and its course towards medical translation Julio Villatoro-Hernandez†, Roberto Montes-de-Oca-Luna & Oscar P Kuipers †

University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Molecular Genetics Group, Haren, The Netherlands

The use of the lactic acid bacterium Lactococcus lactis, primarily used in food fermentations, as therapeutic agent is no longer speculative but an imminent reality. After the successful completion of Phase I and II clinical trials in humans for the treatment of inflammatory bowel disease, an ongoing clinical trial to alleviate oral mucositis as well as the development of a pneumococcal and a flu vaccine using genetically modified L. lactis, many exciting possibilities exist to develop novel therapeutic and prophylactic biopharmaceuticals to alleviate a wide range of diseases. Here, we discuss existing characteristics of the systems currently employed and the nature of the immune responses evoked. We also discuss the criteria that are fundamental to making the systems feasible and efficient which should ultimately translate into human therapies. Finally, we examine the prospects for L. lactis to become a commercially viable therapeutic agent. Keywords: biopharmaceuticals, human therapy, Lactococcus lactis, vaccine Expert Opin. Biol. Ther. (2011) 11(3):261-267

1.

Introduction

Recent reviews have described early and contemporary achievements in the use of the lactic acid bacterium (LAB) Lactococcus lactis as a therapeutic agent for different human diseases and for the delivery of biologically active immunomodulating proteins in vivo [1-3]. The results presented in these reports provide solid evidence supporting the appropriate use of genetically engineered L. lactis to target diseases and modulate immune responses. Two distinctions must be made in this aspect. The first is the development of a L. lactis able to produce a specific antigen, hence, regarded as a ‘vaccine’. The second is the production of a non-antigenic immunomodulatory protein by L. lactis (e.g., cytokines) to stimulate the immune system. One of the earliest reports regarding L. lactis as a vehicle to elicit immune responses describes the genetic manipulation of strain IL1403 (previously referred to as Streptococcus lactis) to express the surface protein antigen (Pac) of Streptococcus mutans. The resulting genetically modified organism (GMO) was then used to immunize mice. Analysis of salivary and serological samples revealed the presence of IgA and IgG in salivary secretions and in serum, which where specifically reactive against S. mutans [4]. These and other studies explored the suitability of L. lactis in the development of (live or dead) vaccines. Although other approaches using pathogenic microorganisms, for example. Salmonella or Listeria species [5], were

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Targeting diseases with genetically engineered Lactococcus lactis and its course towards medical translation

Article highlights. .

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Genetically modified Lactococcus lactis are currently being used to treat a variety of diseases. With the increasing number of clinical trials and applications, L. lactis has the potential to become a commercially available biopharmaceutical in the coming years. Several aspects affect the efficacy of L. lactis vaccines. The choice of antigen to be expressed by L. lactis, the natural attributes and composition of the antigen, the final cellular localization in L. lactis, as well as the route of vaccine administration are all important factors which need to be considered. The safety of administration of genetically manipulated L. lactis to humans must be thoroughly assessed. Deletion of essential genes or auxotrophic strains prevent long-lasting viable cells and undesired cell proliferation. Oral administration of genetically engineered L. lactis able to produce IL-10 is currently being employed to treat inflammatory bowel disease in humans (Crohn’s Disease). This same strain is currently in clinical trials to treat moderate ulcerative colitis and oral mucositis. Improving protein production by synthetic biology and facilitating the antigen reaching the immune system are viable options to create a better immune response of the therapeutic agent.

This box summarizes key points contained in the article.

providing promising results in the induction of immune responses, there was a much more keen interest in Lactococcus because of its natural non-pathogenic and non-colonizing properties [2]. Such an approach eliminated the drawbacks of using pathogenic or nocuous attenuated microorganisms that is the high risk and infeasible use in humans and in clinical trials. Presently, there is a vast collection of reports evidencing the safe and effective use of L. lactis, firstly to produce a wide variety of proteins and peptides efficiently, but most importantly, to deliver these biologically active molecules in vivo without disturbing their specific activity [2,3]. In 2006, the first breakthrough in the application of L. lactis in humans was reported, with Braat and colleagues revealing a clinical approach to treat Crohn’s Disease with an IL-10-secreting L. lactis. In the investigation they showed that the oral administration of IL-10-secreting L lactis in patients suffering from this disease resulted in a reduction of the intestinal lesions and a consequent alleviation of the illness [6]. As an added safeguard to ensure limited survival of the IL-10-expressing L. lactis once is released in the environment, the heterologous gene allowing for IL-10 expression substituted an essential gene, thymidylate synthase (thyA). The survival of this thyA-deficient GMO depends on the presence of thymidine or thymine in the growth medium. In this way, the microorganism can be regarded as ‘biologically contained’ [7]. This study, along with others, paved the path 262

for many other investigators into translational applications of existing strains of L lactis for the treatment of other diseases. While L. lactis possesses many characteristics such as easy genetic manipulation and a long history of safe use in food, that make it an attractive choice for further medical applications, the focus has been on fermenting LAB with the ability to produce heterologous antigens rather than the optimization of this bacterium for use as a vaccine. Cellular antigen location in L. lactis The location of the antigen is important for its immunemodulating activity. For certain proteins, such as cytokines or antibody fragments, extracellular secretion is necessary due to their activity [7,8], whereas for other antigens it is unclear whether the antigen must be produced extracellularly, retained in the cytoplasm or attached to the cell wall. Several factors influence whether intracellular or extracellular localization is desirable. These include the nature of the protein, that is its folding, accumulation and toxicity for the producing cell and unwanted processing by cellular/ membrane-located proteases. However, most important is the capability of the produced antigen to undergo the crucial antigen processing by the immune machinery to ultimately cause the desired specific (humoral and/or cellular) immune response in the host. In this regard, there is an ongoing debate among researchers to select the final location of the L. lactis expressed-antigen. In several studies, immunization with L. lactis expressing an intracellularly localized antigen caused protection in mice when they were challenged with the respective pathogen. In one such study, Hannify et al. demonstrated that intranasal immunization with a strain of L. lactis producing intracellularly-localized pneumococcal surface protein A (PspA) from Streptococcus pneumoniae, was able to cause protection in the immunized group when they were challenged with S. pneumoniae, the causal agent of pneumonia [9]. In another study, three strains of L. lactis (all derived from L. lactis NZ9000 [10]) were developed that were able to produce the E7 antigen of the human papilloma virus type 16 (HPV-16) at different cellular locations, that is intracellular, secreted and attached to the cell wall. After immunization of three groups of mice with the three strains, it was demonstrated that L. lactis with the E7 antigen attached to the cell wall displayed the highest immunogenicity as compared with the other two strains, even though equal amounts of E7 were produced by each strain [11]. There are many other examples of the effect of antigen location and immunogenicity and the reader is referred to a recent review [3]. Vaccination using different mucosal surfaces may cause a systemic production of IgA at different mucosal locations. In this regard it seems remarkable that nasal mucosal immunization not only stimulates an immune response in the respiratory tract, but can also give rise to a strong genital--vaginal mucosal immune response [12]. 1.1

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Villatoro-Hernandez, Montes-de-Oca-Luna & Kuipers

Therefore, it is highly relevant to study the nature of the antigen to be expressed by L. lactis (the candidate to create the vaccine) to determine the most suitable cellular compartment where it will be delivered and the most adequate route of administration. While antigen localization and immunization routes are important aspects in eliciting a good immune response by the host, the correct presentation of the antigen by L. lactis to the antigen-presenting cells (APCs) is crucial if an effective vaccine is to be developed. Antigen modeling to optimize MHC priming It has been calculated that from 104 degraded proteins only 1 peptide binds to the MHC class I molecule [13]. This is a very modest amount of peptide compared with the massive amounts of antigen available when L. lactis is administered and yet it is sufficient to cause the prophylactic and therapeutic effects [9,11]. Considering the ease with which genetic manipulations can be made in L. lactis, including the ability to express the antigen in different cellular locations, it may be possible to confer desirable characteristics to antigens to be produced by the bacterium, resulting in a more effective vaccine. This could involve the fusion of virus-derived or bacteria-derived proteins to the antigen to facilitate MHC I presentation. This can result in a better cellular immunity response, as in previous experiments that have demonstrated how fusing a protein from bacterial origin (e.g., a domain from a toxin) to a particular antigen, results in an increment in the immunogenicity and an augmentation in the immune response. These experiments nevertheless, were performed employing DNA vaccines to produce the chimeric antigen [14-17]. Using adenoviral vectors, we have demonstrated that the fusion of peptides commonly found in the endoplasmic reticulum (ER) (e.g., calreticulin) to an antigen, resulted in the retention of the antigen in the ER, improving its presentation to the MHC I [18]. These experiments indicate that enhanced antigen presentation to the MHC I results in a better cellular immune response [17-19]. Taking in account these results, it is possible to use the same strategy and, for example, fusing calreticulin to antigens expressed by L. lactis in order to increase the immunogenicity. Alternatively, also to increase the antigenicity and obtain a more robust immune response, molecules capable of tight binding to APCs like dendritic cells (e.g., bacterial heat shock proteins) may be fused to the antigen of interest, as has been shown [15,16]. Such approaches in the design of immunomodulatory proteins or antigens expressed by L. lactis would result in a more acute immune response since they would be tailormade to the type of immunity, when either humoral or cellular immunity is desired. Currently, the immunological responses evoked by genetically manipulated strains of L. lactis have achieved in the majority of the cases, humoral responses in animal models [3]. In these cases, the immunization, with strains of L. lactis able

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1.2

to produce a specific antigen results in the development of specific antibodies against the pathogen (the causal agent) and the alleviation of the disease [9,20]. Upon immunization, the genetically engineered L. lactis should express the antigen, retain antigen already synthesized or both. At some point the antigens (or fragments of them) are internalized by APCs and loaded into the endosomal compartment to the MHC II, where they are later transported to the cell surface. The final outcome of the above processes induce the generation of specific antibodies, which later will recognize (and neutralize) the causal agent and continue with the antibody-mediated immune response. This humoral immunity resulting is suitable for extracellular pathogens (e.g., bacteria, fungi and protozoa). But what about for other diseases where cell-mediated immunity plays an essential role, like for example in the case of virus infection and even more importantly, for tumors? Is it possible to create a strategy using L. lactis that will target cancerous cells and will act as an anti-tumor agent? In this context, it was demonstrated by BermudezHumara´n and colleagues that immunization with a combination of two strains of L lactis expressing either E7 or IL-12 resulted in both prophylactic and therapeutic effects in a cervical cancer model [21]. In this report, it was demonstrated how the E7 strain solely can confer (to a much lesser extent) antitumoral activity. Moreover, very few mice immunized with the non-expressing wild type strain of L. lactis had tumors of similar size compared with the group of mice immunized only with the strain expressing E7. This suggests that either the vaccination with L. lactis wild type alone may enhance at some level a nonspecific immune response that affected the growth of the tumors, or that some of the mice vaccinated with L. lactis-E7 did not respond to the treatment and the tumor growth was similar as in the group vaccinated with the non-expressing wild type strain. Nevertheless, in this study it was demonstrated how an antigen-expressing L. lactis is capable of inducing a cellular immune response using simultaneously two strains of L. lactis expressing the E7 antigen of the HPV and cellular-immunity activating IL-12 [21].

Applying the technology of L. lactis for current health issues

2.

In emerging diseases, like the recent epidemic caused by the H1N1 virus, the immediate availability (and distribution) of a vaccine against a pathogen would have a tremendous effect on the evolution and prevention of the epidemic. Unfortunately, currently, after identifying the causal agent of an emerging disease, the process to finally develop a safe and functional vaccine (and make it commercially available) is lengthy and tedious.

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Targeting diseases with genetically engineered Lactococcus lactis and its course towards medical translation

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Table 1. Perspectives and criteria for safe and effective vaccines using Lactococcus lactis. Criteria

Consideration

Ref.

Biological contingency of genetically manipulated LAB

Deletion of essential genes, auxotrophic strains

[7]

Expression systems employed

Constitutive expression, inducible systems (e.g., nisin-inducible system [NICE])

[10,22,28]

Augmentation of the immune response

Intracellular antigen targeting (antigen fusion with viral proteins, antigen-presenting cells binding proteins), sub-cellular antigen retention (e.g., in the rugged endoplasmic reticulum), immunomodulators (interleukins, viral or heat shock proteins)

[14-18,21,29]

Increasing protein expression

Codon optimization for Lactococcus, synthetic promoters, protease deficient strains

[30,31]

Route of administration

Dependent on the immune response most adequate for the targeted disease and the entry route of the causal agent

[3]

Antigen location in L. lactis

Cytoplasmic, extracellular or associated with the cell

[11]

For this reason, the existence of a ‘plug-in and ready-toproduce’ vaccine using L. lactis could expedite the production stage of a vaccine, and therefore, influence an ongoing epidemic. Moreover, because preparation of growth medium and cultures of L. lactis is relatively easy, the need for highly skilled personnel is minimal and makes it advantageous to proceed also in developing countries. Moreover, the route of administration, either intranasal or oral, avoids the use of infectious material, like needles and syringes commonly used to apply non-oral vaccines, decreasing the risk of consequent infections and reducing the cost of the vaccination. This becomes attractive in situations where substantial numbers of doses are needed in adverse conditions (environmental, geographical or limited human resources). In a previous case we mentioned that the immunization with a genetically modified strain of L. lactis conferred protection to mice when they were challenged with the pathogen [9]. Using L. lactis, for example to develop a pneumococcal vaccine for humans, would prevent the need to use attenuated strains of the pathogen intended to be used as a vaccine, or lengthy and costly procedures to purify recombinant antigens used for vaccination. At the present time, a patented technology called ActobioticsÔ consists of biologically contained strains of L. lactis able to secrete biological active interleukins in situ [22]. These strains of L. lactis are being employed to alleviate inflammatory bowel disorders in humans (e.g., Crohn’s disease and ulcerative colitis). Actobiotics are delivered to patients via oral administration (they are usually formulated in capsules) avoiding the use of syringes and needles. This technology is designed to selectively be delivered to receptors and cells localized in the gastrointestinal tract, reducing the exposure to non-target organs to a minimal (For detail information of Actobiotics and its characteristics the reader is referred to www.actogenix.com). 264

ActoGeniX, the company that exploits Actobiotics, has completed Phase IIA of clinical trial to treat ulcerative colitis in humans and is now in Phase I of a clinical trial to treat oral mucositis in cancer patients using Actobiotics [Data from ActoGeniX press release]. Safety is also an essential issue, for that reason, ActoGeniX has also taken measures to prevent undesired microbial endurance inside the host. This was achieved by substituting an essential gene of L. lactis (thymidylate synthase) with the gene of interest (IL-10) to prevent prevalence of the genetically manipulated L. lactis once they are released to the environment [7]. Another company focusing on the generation of vaccines using L. lactis is Mucosis, who recently developed FluGEMÔ, an intranasal vaccine to prevent influenza. Moreover, this company deals with a novel variety of a non-genetically modified strain of L. lactis, able to load antigens of viral, parasitic or bacterial origin on the cell envelope. The remarkable property of the system named MimopathÔ is that the L. lactis cells loaded with the antigen are administered dead, thus, the vaccine is regarded as a non-genetically modified non-live vaccine [23,24]. 3.

Expert opinion and future perspective

The use of L. lactis as a therapeutic agent has been documented as safe and efficient, giving good results in a variety of disease models. No side effects or undesired reactions have been detected up to now. There have been only two medical cases that reported L. lactis infection in humans, causing cardiovascular complications. The patients in these two cases suffered from previous cardiac abnormalities. In one of these patients the infection caused by L. lactis was firstly treated with antibiotics. Once the infection was cleared, the myoxoma, a possible pre-existing condition, was surgically extirpated. After the surgery, the patient did not develop any further infection with L. lactis [25]. These isolated two

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Table 2. List of clinical trials and diseases targeted with Lactococcus lactis. Targeted disease

Route of administration

Physiological system targeted

Condition of L. lactis at administration

Model protein used

Completed clinical trial

Ulcerative colitis*

Oral

Digestive

Live (biologically contained)

IL-10

Phase II

Inflammatory bowel disease (Crohn’s disease)*

Oral

Digestive

Live (biologically contained)

IL-10

Phase I

Mucositis*

Oral

Digestive

Live (biologically contained)

Human Trefoil factor 1 (hTFF1)

Ongoing Phase IB

Influenzaz

Oral or intranasal

Respiratory

Dead particles loaded with antigen

Diverse influenza antigens

Unavailable

*Obtained from ActoGeniX press release. z Obtained from Mucosis press release.

cases probably represent opportunistic infection of (in this case) L. lactis, due to the weakened general health of the patients [25,26]. To our knowledge no cases of infection by L. lactis have been reported in healthy individuals. This two incidents involving opportunistic infection by L. lactis in patients with deteriorated health should not discourage further use of L. lactis in clinical trials or in any other medically related research. The application of L. lactis as a vaccine and therapeutic element is a viable option that can be used safely, and even more importantly, that can be developed rapidly to provide functional and effective vaccines in newly emerging diseases and world wide epidemics. The use of L. lactis as an established vaccine could be achieved by the creation of a ‘ready-to-use’ reliable system that allows the rapid integration of the antigen-coding DNA in L. lactis to provide an expeditious vaccine whenever is required, for example in emerging epidemics or when large amounts of vaccines are required in very little time. Presently it is possible to optimize expression levels and overall protein production using synthetic biology for example with dual codon optimization, strong synthetic promoters or synthetic genes. This and other considerations are reviewed in Table 1, where the different present alternatives to achieve an efficient vaccine using L. lactis are also highlighted. The low cost of the overall production of the vaccine when L. lactis is employed is an advantage that would allow developing countries to have access to vaccines and therapies that usually are expensive and scarce. A few examples are the availability of vaccines for tuberculosis, against HPV or for pneumonia using L. lactis in countries where the prevalence of these diseases is high (usually in countries with a large population with low income per capita), could decrease the

number of current pandemics that have persisted for decades due to the lack of commercial available vaccines that are readily accessible for the population. In less than 5 years the use of L. lactis in clinical trials progressed from Phase I to Phase IIA (Table 2). There is also an ongoing clinical trial Phase IB to treat oral mucositis in cancer patients, using this same therapeutic agent. Other pre-clinical trials are currently being prepared to treat celiac disease, type 1 and 2 diabetes, and allergic diseases (Data from ActoGeniX press release), as well as the development of a pneumococcal vaccine (Mucosis) [27]. With this, there are precedents that encourage and support further applications of L. lactis to alleviate other diseases. In the coming years it will be important that scientists supporting the use of L. lactis as a therapeutic agent transfer their achievements to endorsed proposals for clinical trials in humans to provide more evidence for future successful applications, ultimately culminating in the use of L. lactis as a commercial available biopharmaceutical against any disease of relevance.

Acknowledgements The authors thank CE Price for critically reviewing this manuscript. J Villatoro-Hernandez would like to thank E Garza-Salinas for the constructive advice and support.

Declaration of interest J Villatoro-Hernandez and OP Kuipers are supported by the research programme of the Kluyver Centre for Genomics of Industrial Fermentation, which is part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research.

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Targeting diseases with genetically engineered Lactococcus lactis and its course towards medical translation

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Braat H, Rottiers P, Hommes DW, et al. A Phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin Gastroenterol Hepatol 2006;4:754-9 This report, to our knowledge, is the first clinical trial using a genetically modified strain of L. lactis in humans.

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Lee P, Faubert GM. Expression of the Giardia lamblia cyst wall protein 2 in Lactococcus lactis. Microbiology 2006;152:1981-90

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Yewdell JW. Not such a dismal science: the economics of protein synthesis, folding, degradation and antigen processing. Trends Cell Biol 2001;11:294-7

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Bosma T, Kanninga R, Neef J, et al. Novel surface display system for proteins on non-genetically modified Gram-positive bacteria. Appl Environ Microbiol 2006;72:880-9

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Cuesta AM, Suarez E, Larsen M, et al. Enhancement of DNA vaccine potency through linkage of antigen to filamentous bacteriophage coat protein III domain I. Immunology 2006;117:502-6

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van Roosmalen ML, Kanninga R, El Khattabi M, et al. Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria. Methods 2006;38:144-9

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Zechini B, Cipriani P, Papadopoulou S, et al. Endocarditis caused by Lactococcus lactis subsp. lactis in a patient with atrial myxoma: a case report. Diagn Microbiol Infect Dis 2006;56:325-8

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Pellizzer G, Benedetti P, Biavasco F, et al. Bacterial endocarditis due to Lactococcus lactis subsp. cremoris: case report. Clin Microbiol Infect 1996;2:230-2

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Audouy SA, van Selm S, van Roosmalen ML, et al. Development of lactococcal GEM-based pneumococcal vaccines. Vaccine 2007;25:2497-506

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Mierau I, Kleerebezem M. 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 2005;68:705-17

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Kang TH, Kim KW, Bae HC, et al. Enhancement of DNA vaccine potency by antigen linkage to IFN-gamma-inducible protein-10. Int J Cancer 2010, doi: 10.1002/ijc.25391

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de Ruyter PG, Kuipers OP, de Vos WM. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 1996;62:3662-7

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Vandenbroucke K, de Haard H, Beirnaert E, et al. Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis. Mucosal Immunol 2009;1:49-56

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Hanniffy SB, Carter AT, Hitchin E, et al. Mucosal delivery of a pneumococcal vaccine using Lactococcus lactis affords protection against respiratory infection. J Infect Dis 2007;195:185-93

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Loera-Arias MJ, Martinez-Perez AG, Barrera-Hernandez A, et al. Targeting and retention of HPV16 E7 to the endoplasmic reticulum enhances immune tumour protection. J Cell Mol Med 2010;14:890-4 Hung CF, Monie A, Alvarez RD, et al. DNA vaccines for cervical cancer: from bench to bedside. Exp Mol Med 2007;39:679-89

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Mijakovic I, Petranovic D, Jensen PR. Tunable promoters in systems biology. Curr Opin Biotechnol 2005;16:329-35

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Affiliation

Julio Villatoro-Hernandez†1,2, Roberto Montes-de-Oca-Luna3 & Oscar P Kuipers1,2 † Author for correspondence 1 University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Molecular Genetics Group, Kerklaan 30, 9751 NN, Haren, The Netherlands Tel: +31 50 363 2107; Fax: +31 50 363 2348; E-mail: [email protected] 2 Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands 3 Facultad de Medicina, Universidad Auto´noma de Nuevo Leo´n, Madero y Aguirre Pequen˜o S/N, Monterrey, Nuevo Leo´n, Me´xico 64460

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Review

Tits and bits of HIV Tat protein Manish K Johri, Ritu Mishra, Chintan Chhatbar, Salini K Unni & Sunit K Singh† Laboratory of Neurovirology and Inflammation Biology, Section of Infectious Diseases, Centre for Cellular and Molecular Biology (CCMB), Council of Scientific and Industrial Research (CSIR), Uppal Road, Hyderabad, India

1.

Introduction

2.

HIV genome

3.

Tat protein

4.

Secretion and uptake of HIV

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Tat protein 5.

Structure of HIV Tat protein

6.

Post translational modifications in Tat

7.

Tat: role in HIV pathogenesis

8.

Expert opinion

Introduction: HIV-Tat protein displays an array of functions that are essential for HIV replication. The structural flexibility of Tat protein has been regarded as one of the unique features responsible for sustaining diverse functions, from facilitated membrane-crossing ability to strong affinity for RNA binding. Areas covered: RNA binding ability and presence of multiple interacting domains in the same protein are very important properties of HIV-Tat protein. Tat protein has shown great ability to influence cellular and viral gene expression. We discuss the functions of HIV Tat protein, describing its structural significance, secretion and uptake of HIV Tat protein by immune cells, post-translational modifications and role of HIV Tat protein in HIV pathogenesis. Expert opinion: Perturbation in expression of many cytokines and chemokines by HIV-Tat protein exhibits downstream immune suppressive function as well as activation of several apoptotic genes. This explains the massive death of immune cells due to bystander effect of HIV Tat protein among HIV-infected patients. Keywords: AIDS, HIV, HIV neuropathogenesis, HIV pathogenesis, Tat, Tat protein, vaccine Expert Opin. Biol. Ther. (2011) 11(3):269-283

1.

Introduction

Since the discovery of HIV-1 in 1983 [1], HIV infection has emerged as a global health and socioeconomic problem. According to the joint report on HIV/AIDS by UNAIDS and World Health Organization (WHO) in 2009, approximately 33 million people are infected with HIV-1 worldwide [2]. High genetic variability and rapid evolution are the major factors in worldwide spread of this virus. The reverse transcriptase (RT) enzyme of HIV-1 is mainly responsible for its variability due to its high rate of mutation and recombination. High mutation rate and recombination result in genetically diverse populations of viral strains in infected individuals. These genetically diverse populations of viral strains are known as ‘quasispecies’. Host immune systems and antiviral drugs exert selection pressure on virus, which leads to the further evolution of virus population [3]. HIV-1 strains can be divided into three genetically distinct groups: Major (M), Outlier (O) and New (N). Group M causes most HIV-1 infections worldwide, while groups N and O are responsible for small numbers of infections in central Africa [3]. On the basis of their genome sequences, group M can be divided into nine clades/subtypes. These clades are designated by the letters A -- D, F -- H, J and K [4-6]. Genetic variation within a subtype is approximately 15 -- 20% while variation between subtypes can be up to 25 -- 40% [3,7,8]. The percentage of similarity/dissimilarity also depends on the HIV subtype and the genomic region considered for comparison [7]. HIV has the capacity to undergo the process of recombination in patients suffering from multiple subtype infection. These recombinant forms are known as circulating recombinant forms (CRF) [3]. Unlike some other viruses, infection with HIV does not provide immunity against additional 10.1517/14712598.2011.546339 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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Tits and bits of HIV Tat protein

Article highlights. . . . . .

HIV Tat protein allows a several-fold increase in rate of HIV transcription after binding to TAR. Extracellular Tat is actively released from the productively infected cells. HIV Tat is known to modulate the level of pro-inflammatory and anti-inflammatory cytokines. HIV Tat protein has been implicated in the development of cancers in AIDS patients HIV Tat disturbs homeostasis of uninfected cells by altering expression of several genes.

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This box summarizes key points contained in the article.

infections. Multiple infections lead to more rapid disease progression [9]. Different subtypes show their regional dominance in causing infection [10]. Over 50% of all infections are caused by subtype C globally, which is found mainly in subSaharan Africa, India and South America [3,5]. The most studied HIV clade (clade B), represents 10% of all infections, and is dominant in both Europe and America. Subtypes A and D are found in sub-Saharan Africa and account for 12 and 3% of infections respectively [3,5]. 2.

3.

HIV genome

HIV is a lentivirus and belongs to family retroviridae. The HIV genome consists of only 14 genes. There are two species of HIV known to exist: HIV-1 and HIV-2. HIV-1 is more virulent, and reported as a major HIV infection globally [11]. HIV-2 shows poor capacity of transmission and is largely confined to parts of West Africa [12]. HIV has two copies of single-stranded RNA in its genome. The transcript produced from the viral promoter is approximately 10 kb long. This transcript contains seven structured sub-domains throughout its length namely long terminal repeats (LTR), transactivating region (TAR), rev responsive element (RRE), psi elements (PE), a TTTTTT slippery site (SLIP), cis-acting repressive sequences (CRS), and inhibitory/instability RNA sequences (INS). The ends of each strand of HIV RNA contain an RNA sequence called an LTR. LTR regions are reported to contain promoter sequences. It has been reported that the synthesis of any reporter genes placed under control of the viral LTR gets stimulated more than 100-fold in HIV-1-infected cells as compared with uninfected cells. These promoters can be triggered either by HIV or by host cell proteins and act as switches to control production of new viruses [13]. TAR has a three dimensional hairpin structure, which helps it in binding with transactivator of transcription (Tat) protein [14]. The presence of two copies of positive single-stranded RNA in HIV [13] is believed to help in recombination events, which leads to more variable HIV population [15]. The HIV genome encodes nine open reading frames (ORFs) namely group specific antigen 270

(gag), polymerase (pol), envelope (env), transactivator of transcription (tat), regulator of virion protein expression (rev), negative effector (nef ), viral infectivity factor (vif ), viral protein R (vpr) and viral protein U (vpu). Broadly these genes can be categorized into two categories, structural and non structural genes. The gag, pol, and env genes are structural genes in HIV genome, which contain information to make the structural proteins for new virus particles. Gag, Pol, and Env are synthesized as polyproteins, which subsequently get proteolyzed into individual proteins [14]. Non structural genes can be further classified as accessory and regulatory proteins. Accessory proteins include Vif, Vpr, Vpu and Nef, which are unique to primate lentiviruses. Recent studies have demonstrated that these proteins are essential for efficient viral replication, dissemination, pathogenicity and disease progression [16]. Tat and Rev proteins are known as regulatory proteins and are directly involved in regulation of transcription and replication [16]. HIV-Tat protein is transcriptional transactivator for the LTR promoter and acts by binding to the TAR RNA element. Tat protein regulates early stages of the HIV life cycle. Whereas the major role of Rev is to regulate the expression of HIV proteins by controlling the export rate of mRNA from the nucleus [14].

Tat protein

HIV-1 encodes small nuclear transcriptional activator protein known as trans-activator of transcription (Tat) [17]. Unlike other transactivators, which only bind to DNA, HIV-Tat can bind HIV proviral DNA [18], host cell DNA [19] and viral RNA and affect transcription. The ability of Tat to bind viral RNA during its transcription distinguishes it from other transactivators. Tat allows several fold increase in the rate of HIV transcription after binding to TAR [20]. HIV-1 Tat is an 86 -- 101 residue regulatory protein with molecular mass ranging from 9 to 11 kDa [5]. Tat is the first protein to be produced after cDNA synthesis by reverse transcriptase [21]. It is not only required for initiation of viral transcription but indispensable for full-length RNA chain synthesis [22]. Association of HIV Tat protein with a number of the pathologies related to AIDS makes the HIV Tat protein an important target for development of therapeutics. The ORF of Tat is composed of two exons separated by a non-coding region (Figure 1) [23]. The length of Tat varies from one subtype to another, while different variants of the same strain show variability in sequence of the Tat [23]. The length of the Tat protein in HIV-1 ranges from 86 to 101 amino acids, whereas in HIV-2 it consists of 130 amino acids. Short HIV-1 Tat variants exist due to presence of stop codon in second exon of the Tat gene. These short and terminally truncated Tat variants are also able to transactivate HIV TAR [24]. Once the transcription of full-length Tat mRNA is completed, the tat transcripts undergo double splicing, which results in removal of the non-coding region between the two coding exons.

Expert Opin. Biol. Ther. (2011) 11(3)

Figure 1. Primary structure of HIV Tat C protein. Region I: Proline-rich region: 1 -- 21 residues (red). Region II: Cysteine-rich region: residues 22 -- 37 (green). Region III: Core region: residues 38 -- 48 (blue). Region IV: Basic region: residues 49 -- 59 (orange). Region V: Glutamine-rich region: residues 60 -- 72 (purple). Regions I to V are encoded by the first exon. Region VI is encoded by the second exon.

Region VI residues 73 – 101 Region V Glutamine region: residues 60 – 72 Region IV Basic region: 49 – 59 residues Region III Core region: 38 – 48 residues Region II Cysteine-rich region: 22 – 37 residues Region I Proline-rich region: 1 – 21 residues

MEPVDPNLEPWNHPGSQPKTA CNQCYCKRCSYHCLVC FQKKGLGISYG RKKRRQRRAP PSSEDHQNLISKQ PLPRTQGDPTGSEESKKKVESKTKTDPFD

First exon

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Second exon

Johri, Mishra, Chhatbar, Unni & Singh

Tat B protein (from HIV-1 clade B) has been reported to show better transactivation capacity, compared with Tat C (from HIV-1 clade C). However, significant difference in transactivation capacity has not been found when plasmid constructs of B and C have been used to study the comparative capacity of transactivation [25]. One of the possible reasons of differences might be the capacity of Tat to cross cell membranes because ability of crossing depends on flexibility of proteins. More structured proteins show less flexibility therefore may face difficulty in crossing the cell membranes. The flexibility of different regions of Tat depends on type of amino acids comprising that region and their abundance [26]. HIV Tat protein could be present either as an intracellular or extracellular form. The main function of HIV-Tat protein is LTR-mediated transcription of HIV RNA inside the cell. Several cellular factors hinder effective mRNA transcription of HIV-1 [27]. Tat binds to the TAR region of HIV-RNA [27] and recruits cellular proteins required to relieve the repression of HIV-LTR. Therefore, Tat makes viral promoter able to induce the expression of other viral genes [28]. Functions, other than transactivation are mostly performed by extracellular Tat. Extracellular Tat is actively released from productively infected cells [5]. Extracellularly secreted HIV-Tat protein is able to enter neighboring cells and can activate expression of various genes involved in inflammatory response such as TNF-b, IL-6, and TGF-b [23,29]. Tat is also known to repress cellular immune responses such as MHC-I presentation, IL-2, CD25 and anti-oxidative enzyme expression [23,30]. Tat -- TAR interaction The life cycle of HIV-1 can be broadly divided into two phases. The first phase includes viral RNA entry, reverse transcription of viral RNA, translocation of cDNA from cytoplasm to nucleus followed by its integration into the human genome. The second phase includes transcription of integrated cDNA, transport of RNA molecules from the nucleus to the cytoplasm, translation, assembly and release of new virus [31]. The rate of transcription of HIV-1 provirus varies greatly. It is very low in the early phases of HIV infection, while, it increases up to hundreds of folds during late phases of infection [32]. Sodroski et al. showed that induction or transactivation of transcription is due to a HIV-Tat protein [31]. In vitro studies have shown that, only short transcripts are synthesized in absence of Tat protein and most of the full-length transcripts are synthesized in the presence of HIV-Tat protein [33]. Tat requires minimal transcription of TAR before it can stimulate HIV transcription from the LTR. In the absence of Tat, RNA polymerase II (RNAPII) can initiate transcription but can’t undergo the elongation step. It stops soon after clearing the promoter (HIV LTR). In majority of cases, this short-term binding of RNA polymerase II to integrated HIV cDNA can yield short viral transcripts, which contain the TAR sequence required for Tat protein binding and transactivation [33]. Tat protein is 3.1

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Tits and bits of HIV Tat protein

expressed early after the virus integrates into the cellular genome, and stimulates the elongation of transcription by RNA polymerase II (RNAPII) [34]. In early phases of HIV infection, the rate of synthesis of HIV-Tat protein is very low and gets imported into the nucleus during late phases of HIV infection. It initiates additional rounds of transcription, which results in increased Tat production. Newly synthesized Tat protein again results in the burst of Tat-mediated transcription. Thus a positive feedback loop (between HIV-Tat protein and transcription) is established inside HIV infected cells [33]. Eukaryotic host-cell machinery plays major role in HIV transcription. Eukaryotic transcription is initiated by holoenzyme (RNA Pol II with associated factors). These additional factors help in proper positioning of RNA Pol II at the transcription initiation site [34]. Initially it was believed that HIV Tat protein is critical requirement for the transcription initiation [32]. Now it is clear that holoenzyme complex is itself capable of transcriptional initiation. However, the holoenzyme complex alone is not sufficient for elongation and requires the HIV Tat protein [14]. Efficient elongation is important for successful transcription, which depends on the positive transcription elongation factor b (P-TEFb). P-TEFb is a heterodimeric protein, which consists of CDK9 kinase and remains associated either with cyclin T1, cyclin T2a, cyclin T2b or cyclin K. The elongation activity of P-TEFb is dependent on its kinase activity. In vitro studies have shown the importance of phosphorylation of the RNAP II carboxy terminal domain (CTD) by p-TEFb (Figure 2) [35]. DRB-sensitivity-inducing factor (DSIF) binds to RNAP II during or shortly after transcription initiation. Negative elongation factors (NELF) interacts with DSIF and play roles in pausing of polymerase enzyme. P-TEFb relieves this pausing, by phosphorylating one of the subunits of DSIF (Figure 2) [36]. The Spt5 subunit of DSIF has been shown to be phosphorylated by P-TEFb in the presence of Tat. This demonstrates the mechanism for overcoming the negative effect of DSIF by P-TEFb with the help of Tat protein (Figure 2) [37]. Transactivation through Tat needs efficient functionality of binding domain and activation domain. The arginine rich motif of HIV Tat protein binds to the 5¢ bulge in TAR. It spans positions 49 -- 57, where a central arginine (R52) is key for this interaction. The activation domain of Tat consists of N-terminal core and cysteine-rich regions. The Cys-rich motif in Tat is capable of binding divalent cation metals such as zinc. Each monomer of Tat can coordinate two atoms of zinc [38]. Further studies have shown that Tat forms a Zn-mediated bridge with Cys 261 of cyclin T1. The activation domain of Tat acquires a well-ordered structure upon interaction with pPTEFb with two Zn ions [39]. Several upstream transcription cofactors also play important role in HIV-tat transactivation. Tat has been reported to interact with many of these factors including specific protein-1 (Sp1), TATA box binding protein (TBP), 272

cylinE/cyclin-dependent kinase 2 (cdk2), cAMP responsive element binding protein (CBP)/p300, cAMP responsive element binding protein-associated factor PCAF, transcription factor IIH (TFIIH), 60 kDa Tat-interactive protein (Tip60) and RNA Pol II [32]. 4.

Secretion and uptake of HIV Tat protein

For Tat to act as a paracrine molecule, it has to cross the cell membrane of an infected cell followed by entry into an uninfected cell. The mechanism of secretion of HIV Tat protein is largely unknown. Proteins can be secreted either through the conventional or non-conventional pathways. The conventional secretion pathway includes the endoplasmic reticulum-Golgi body (ER-Golgi) pathway. The destination of these secretory proteins depends on their signal sequence. Signal peptides may be either amino-terminal or internal [40]. Proteins synthesized on ribosomes enter the ER as nascent protein and they identify ER through their signal peptide. Mature protein is released through ER exit sites and the newly synthesized mature protein enters Golgi bodies, where they undergo processing, sorting and dispatching to their destination [40]. In non-conventional secretion pathways, proteins either follow ER-independent or both ER- and Golgi-independent routes. The latter category of unconventional proteins lack ER signal peptide [41]. Tat protein does not have a signal sequence [42]. Tat secretion is highly efficient and it gets secreted unconventionally without lysis of infected cells [43]. HIV Tat has also been reported in the serum of AIDS patients in nanomolar concentrations [44]. A recent study has shown the export of two-third of HIV-Tat protein extracellularly from an infected cell during its life span [42]. HIV Tat in circulation is implicated for viral multiplication, AIDS development and HIV-associated neurological disorders (HAND) [29,45,46]. Earlier reports could only demonstrate that Tat is secreted via the leaderless non-conventional pathway [47]. However, a recent study conducted on CD4+ T-cells clearly showed that Tat is secreted directly through plasma membrane and that no intracellular organelle is involved in this process. Secretion of Tat remained unaffected after treatment with cytoskeleton toxins (nocodazole, taxol), Na+, K+-ATPase inhibitor (ouabain) and endosome-neutralizing drugs (bafilomycin A1, monensin and nigericin) [48]. Rayne et al. reported, that Tat never gets accumulated at its export site, that is the plasma membrane, due to the high efficiency of the Tat transport system [48]. However, another study demonstrated the accumulation of Tat protein at plasma membrane of CD4+ T-cells. This accumulation was reported to be due to the high binding affinity of Tat with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) [42]. PI(4,5)P2 is a phospholipid and remains concentrated within the inner leaflet of the plasma membrane. HIV-Tat protein is known to recruit several other proteins involved in important cell activities such as endocytosis, phagocytosis, exocytosis and cell adhesion [49,50]. Tat interacts with a single PI(4,5)

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Johri, Mishra, Chhatbar, Unni & Singh

A.

Cyc T HIV-1TAR CDK9 5′

Pol ll

LTR DNA

3′ TFIIH Transactivator of transcription CTD

Cyclin-dependent kinase 9 Cyclin T1

Tat

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Negative elongation factor Transcription factor II H

Zn++

Cyc T

B.

RNA Polymerase-II DRB sensitivity inducing factor

Zn++

CDK9

Tat

Phosphate

HIV-1TAR

Zn++ Pol II

5′

LTR DNA 3′

P P

P P CTD

P

P P DSIF P NELF

Figure 2. Tat-mediated transactivartion of transcription from HIV trans-activation region. A. Promoter clearance. RNA polymerase II assembles on the HIV LTR promoter with associated factors to form pre-initiation complex. This complex remains capable enough to clear promoter successfully. TFIIH helps promoter clearance by phosphorylating the carboxyterminal domain of largest subunit of Pol II, while additional factors also play their role in proper positioning of Pol II (Not shown in figure). TFIIH-mediated phosphorylation is believed to be necessary for promoter clearance. B. Elongation. DSIF and NELF associate with the transcription complex during the early elongation stage and induce polymerase pausing. This pausing is relieved through PTEFb-mediated phosphorylation of the spt5 subunit of DSIF. Initial transcription gives rise to a functional TAR RNA. HIV-Tat protein via binding to TAR bulge brings PTEFb closer to the carboxy-terminal domain (CTD) of RNA pol II and Spt5. Proper positioning of PTEFb is necessary for DSIF phosphorylation.

P2 molecule via a specific motif present in its basic domain and this interaction gets stabilized by membrane insertion of the Tat tryptophan side chain [42]. Identification of other protein partners, which enable the HIV-Tat recruitment to the plasma membrane, would be helpful in understanding the unconventional secretion pathway. Most of the activities of extracellular Tat depend upon its rapid entry into variety of cells. HIV-Tat disturbs homeostasis of uninfected cells by altering expression of several genes [5]. Although much work has been done to explore the precise mechanism for its entry into the cell [51-53], the cellular mechanisms for Tat uptake and internalization, are still obscure [5]. No specific receptor has been reported for the uptake of HIV Tat inside cells [5]. However, several mechanisms have been reported for the uptake of the HIV-Tat protein inside the cells such as: macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid-raft-mediated endocytosis [5].

5.

Structure of HIV Tat protein

NMR-based studies have been performed to study the 3D structure of HIV Tat protein (Figure 3). Three 2D NMR studies with Tat variants having transactivation activity in a cellular assay showed that there is a conserved folding among Tat variants with localized structural variations due to mutations [54-56]. However, a 2D-NMR study of a peptide corresponding to a first exon of Tat (1 -- 72) showed that Tat was instead an intrinsically unstructured protein (IUP) and not having well defined secondary and tertiary structures [57]. Recently, the crystal structure of the Tat- P-TEFb complex has been solved, which throws light on Tat structure in complex with its natural ligands [39]. The folding of Tat in the complex with P-TEFb crystal has similarities with the data obtained by using NMR with Tat variants (having transactivation activity) [54-56].

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Tits and bits of HIV Tat protein

Figure 3. Three dimensional structure of HIV Tat protein. The structure was derived using solution NMR. (http://www.pdb.org/ pdb/explore/explore.do?structureId=1TBC. DOI:10.2210/pdb1tbc/pdb. PDB ID: 1TBC). It is evident that Tat has very little secondary structure (pink colored). Each of the gray lines in the structures corresponds to one of the several different conformations present in solution. The structure has been viewed using RasMol software.

Primary structure The major function of Tat protein is to act as a transactivator of transcription of HIV genes. Apart from this, Tat also performs several distinct functions which depend on its concentration and the type of cell. Functionally, Tat protein is divided in six regions (Figure 1). Region I is rich in proline and includes residues 1 -- 21 (Figure 1). Acidic amino acid residues present at positions 1 and 2 act as a pH sensor and play an important role in entry of Tat into cells. This region also has a conserved tryptophan at position 11 that is essential for release of Tat from infected cells [42] and its translocation into the cytosol of bystander cells [58]. Region II comprises residues 22 to 37 (Figure 1). This is a cysteine-rich region, having seven well conserved cysteines at the positions 22, 25, 27, 30, 31, 34 and 37 except for subtype C, which has a cysteine to serine mutation. Mutations in any of cysteines except at position 31 result in loss of transactivation ability of Tat [59]. Crystal structure of Tat-PTEFb complex shows that the conserved cysteines at positions 22, 25, 27, 30, 34, 37 and histidine at position 33 are involved in Zn ion binding [39]. Region II is essential for transactivational activity of Tat. Region III ranges from residues 38 to 48 (Figure 1). This region has a conserved phenylalanine at position 38 and the conserved sequence 41KGLGISYG48 at residues 41 -- 48. The amino acid residues 41 to 45 are part of the minimal activation domain together with the cysteine-rich domain [60]. Region IV consists of residues 49 -- 59 (Figure 1). This region is rich in basic amino acid residues and has a well conserved sequence 49RKKRRQRR56. This sequence is essential for binding of Tat to TAR [61] and translocation of Tat to nucleus [62]. Residues 55 -- 57 along with the acidic residue of the first region form a pH sensor, which controls the entry of Tat protein in the cytosol from endosomal membrane [58]. 5.1

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The basic domain is also required for efficient endocytosis of HIV-Tat protein [58]. Region V ranges from residue 60 to 72 (Figure 1). This region is a glutamine-rich region and involved in microtubule polymerization and Tat-mediated apoptosis of T-cells [63]. Region V shows the highest sequence variation among different isolates. Region VI, starts from residue 73 and can be either till residues 88 -- 89 or 101 (coded for by the second exon). This region has a conserved 78RGD80 motif of D and B subtype and involved in cell adhesion [64]. A 86ESKKKVE92 motif in this region is involved in NF-kB mediated transcription of HIV genes [65]. Region VI is also responsible for modifications in cell cytoskeleton structure as well as T-cell signaling and activation (Figure 1) [66]. Secondary structure The secondary structure of HIV Tat protein has been studied extensively [25,55]. Circular Dichroism (CD) spectroscopy has been used to study the secondary structures of chemically synthesized Tat proteins corresponding to different HIV isolates. There is no or very little a-helical structure and the major secondary structure is b-turns in aqueous conditions in about 30% residues [55]. Interestingly, however, in organic solvents 20 -- 50% of residues present in unstructured regions attain a-helical confirmation [55]. The CD studies of Tat protein from different HIV clades have shown that its secondary structure depends on its surrounding environment [55]. Comparative CD studies of Tat B and C has shown that Tat C is more structured compared with Tat B [25]. 5.2

Tertiary structure Bayer et al. conducted an NMR-based structural study of HIV-Tat protein having 86 residues under reducing conditions in the presence of dithiothreitol (DTT) [67]. This 5.3

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study showed that cysteine-rich and basic sequence regions were highly flexible [67]. Previously, it has been shown that under reducing conditions Tat loses its transactivation capacity due to reduction of cysteine residues [68]. Reducing conditions inhibit Zn binding of these residues and make the cysteinerich region flexible. The recently reported crystal structure of the Tat-PTEFb complex shows that cysteine-rich region is involved in formation of a compact structure (random coil, two helices, a tail and two bound Zn ions) [39]. Another NMR study of the first exon of Tat (residues 1 -- 72) combined with a histidine segment and T7 epitope under reducing conditions has been conducted [57]. However, investigators described the absence of a fixed conformation and natively unfolded characteristics in HIV Tat protein [57]. Watkins et al. conducted an NMR study of chemically synthesized biologically active Tat of 99 residues (Tat Eli) [54]. The Tat Eli model has a core region composed of a part of the N-terminus including the highly conserved Trp 11. The extra residues in the Tat Eli C-terminus protrude from a groove between the basic region and the cysteine-rich region and are well exposed to the solvent. Comparison of the Tat Eli structure model with NMR studies of the 86-residue Tat Bru [69] and the 87-residue Tat Mal [56] performed in the absence of reducing agents demonstrate that all three show a similar folding in the core region composed of a part of the N-terminus with the highly conserved Trp11. 6.

Post translational modifications in Tat

Post translational modifications (PTMs) are covalent modifications and play important roles in protein regulatory mechanisms. PTMs change the properties of the protein by proteolytic cleavage or by adding modifying groups onto one or more amino acids [70]. A number of PTMs have been identified which include phosphorylation, acetylation, methylation, ubiquitination and other modifications. Each modification occurs at specific amino acid residues and affects the structure and function of proteins. HIV Tat, protein, is subjected to various post-translational modifications by host cell proteins (Figure 4). Phosphorylation Protein phosphorylation is a reversible process and plays a major role in the signal transduction pathways in eukaryotes. Phosphorylation is the transfer of g-phosphate from ATP to specific amino acid residues in a protein. The reaction is catalysed by protein kinases. Ser, Thr and Tyr are the residues that are commonly phosphorylated in eukaryotes. The possibility of Tat being phosphorylated arose after in vitro studies showed that both HIV-1 Tat (Tat-1) and HIV-2 Tat (Tat-2) interacted with a cellular kinase CDK9/TAK [71]. However, only Tat-2 was shown to be phosphorylated by CDK9/TAK in vivo [71]. The phosphorylation sites of CDK9 were identified as Thr 85, Thr 89 and Ser 94, but these were found to be non-essential for Tat-2 6.1

transactivation activity [72]. Tat-1 has also been reported to be phosphorylated in vivo, but by different host factors. ds-RNA-dependent protein kinase (PKR) and CDK2/cyclin E were found to phosphorylate HIV-1 Tat both in vitro and in vivo [73,74]. The amino acids Ser-62, Thr-64 and Ser-68 of Tat-1 are phosphorylated by PKR, which results in increased binding of Tat with TAR RNA [73]. The possible sites of phosphorylation of CDK2/cyclin E are the Ser residues at positions 16 and 46 [74]. Acetylation Transfer of acetyl groups from acetyl coenzyme A to the e-amino group of lysine residues is known as acetylation. Acetylation of protein at the Lys residue is a reversible process which neutralizes the positive charge of Lys thereby affecting the function of the protein [75]. p300/CBP and PCAF are Tat-associated histone acetyltransferases (TAHs), which help in the activation of chromosomally integrated HIV-1 LTR. p300/CBP acetylates Tat-1 at Lys50 in the TAR RNA binding domain and PCAF acetylates Lys28 in the activating domain [76]. Lysine acetyltransferase 2A (hGCN5/KAT2A), a histone acetyltransferase, also acetylates Tat at the same residues as p300 [77]. In the initial stage of transcription Tat is acetylated by p300/CBP. Acetylation of Tat disrupts the formation of the Tat/TAR/cyclin T1 complex by promoting dissociation of Tat from TAR RNA during early transcription elongation. Acetylated Tat is then transferred to the elongating polymerase along with PCAF [78,79]. PCAF is bound directly to Tat through a conserved module called the bromodomain. This interaction requires acetylation at lysine 50 in Tat. PCAF and TAR RNA competes with each other to bind to the Tat acetylated at Lys 50 [79]. Acetylation and de-acetylation work in tandem, and bring about the different functions of HIV-Tat protein. Human sirutin (SIRT1) is a class III histone deacetylase (HDAC), which mediates de-acetylation of Tat [80]. De-acetylation occurs at the Lys residue at position 50. SIRT1 interacts both with acetylated and de-acetylated Tat, indicating that acetylation is not necessary for interaction of Tat with SIRT1. De-acetylation usually plays a negative role in transcriptional regulation. However, de-acetylation of Tat has a positive effect on the Tat-mediated transactivation of HIV LTR. Acetylation of Tat also plays an important role in modulating its interaction with various other proteins. Tat is bound by p73, a protein implicated in apoptosis and cell cycle control, which prevents Tat acetylation at lysine 28. However, it does not interrupt its interaction with PCAF or cyclin T1 [81]. Loss of interaction between Tat and p73 leads to induction of apoptosis in the host cell. HIV-1 splicing is also regulated by Tat acetylation. p32, a cofactor of the splicing factor alternative-splicing factor (ASF)/splicing factor 2 (SF-2), is bound by acetylated Tat leading to reduced splicing of the HIV-1 genome [82]. Thus it leads to the production of the full-length viral transcripts. Acetylation of HIV-Tat protein by TAHs also increases its interaction with 6.2

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P

hG

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0,

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Early transcription Viral RNA

SIRT1

KMT7 Me

Ac

Transactivator of transcription (TAT) Transactivating region (TAR) Ubiquitin

RNA polymerase II (RNAPII) Cyclin T1 Cyclin-dependent kinase-9 (CDK9)

p

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Me Ac

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p300/CBP-associating factor (PCAF) ds-RNA dependent protein kinase (PKR)

Figure 4. Post-translational modification in Tat and its role in transactivation. A schematic representation of the role of PTMs in Tat in transactivation. In the initial stage of transcription, ubiquitination of Tat by Mdm2 p53 binding protein homolog (Hdm2) and monomethylation by Lysine methyltransferase Set7/9 (KMT7) initiates the interaction between Tat, cyclinT1 and TAR RNA. CDK9 mediated phosphorylation of Tat-2 and the C-terminal domain (CTD) of RNAPII also occurs at this stage. Phosphorylation of Tat-1 is done by PKR. Acetylation by p300 and K (lysine) acetyltransferase 2A (human general control of amino acid synthesis protein 5 (hGCN5)/KAT2A) leads to dissociation of Tat (represented by dashed lines) from cyclin T1--TAR. Tat is then transferred to the RNAPII complex and recruits PCAF. In the late stage of transcription RNAPII, PCAF and Tat dissociates from the transcribed RNA (represented by dashed lines) Protein arginine methlytransferases (PRMT6) mediates dimethylation of Tat, causing its dissociation from the polymerase--PCAF complex and leads to its degradation by non-ubiquitin pathway. Recycling of Tat is initiated by Sirtuin (SIRT1) which deacetylates the protein.

CDK9/P-TEFb which is necessary for phosphorylation [76]. This could be a possible mechanism for regulating the phosphorylation of Tat. Methylation Methylation changes the charge of the amino acid that is targeted and thus alters the function of the protein. Tat is methylated by an enzyme, protein arginine methlytransferase (PRMT6), at Arg 52 and 53 [83,84]. Arginine methylation is a covalent PTM that involves the addition of one or two methyl groups to the guanidine nitrogen atoms of arginine [85]. This methylation leads to decreased interaction between Tat and TAR RNA and caused reduced formation of Tat-TARcyclin T1 complexes [84]. It also increases the stability of Tat 6.3

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in the cell by inhibiting a proteasome dependent degradation pathway [86]. Lys residues 50 and 51 can be methylated by SET domain, bifurcated 1 (SETDB1) in vitro leading to decreased viral transcription [87]. Lysine methyltransferase Set7/9 (KMT7) was found to monomethylate Tat at Lys 51. This residue is present in the RNA binding domain of Tat. Knockdown of KMT7 was found to suppress the transactivation of HIV LTR by Tat. However, it did not affect its transcriptional activity. Thus methylation of Tat at Lys 51 is essential during the early transactivation cycle [88]. Ubiquitination Ubiquitin is a small (~ 76 amino acids), highly conserved protein. Ubiquitination is the process by which ubiquitin 6.4

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covalently attaches to its target proteins. Ubiquitination of cellular proteins was found to regulate their half life by targeting them for degradation. Recent reports suggest that ubiquitination is a regulator of eukaryotic messenger RNA synthesis. It acts by activating transcription factors and regulating transcription [89]. E3 ligases mediate the interaction between ubiquitin and its substrate. Mouse double minute 2 p53 binding protein homolog (Hdm2), a proto-oncoprotein, ubiquitinates HIV-Tat, both in vitro and in vivo. Thus, Hdm2 acts an E3 ligase for Tat ubiquitination. In contrast to its normal role as a protein-degradation mechanism, ubiquitination of Tat leads to stimulation of its transcriptional activities [90]. Lys 71 has been identified as the site of ubiquitin attachment in Tat in vivo. Mutation of this residue has a minor effect on the ubiquitination efficiency in vitro. This suggests that Tat does not act as a substrate for Hdm2 in vitro [90]. Purified protein is required to study the basic biophysical and biochemical character of a protein. Thus production of HIV-Tat protein in its functional form is essential to understand its role in HIV pathogenesis. Escherichia coli cells are the most favored system for producing recombinant Tat at a large scale. PTMs have not been identified in prokaryotes [91]. However, many studies have reported that recombinant Tat expressed and purified from E. coli is functional [25]. A few studies have identified endogenous phosphorylation and glycosylation of eukaryotic proteins that are overexpressed in prokaryotes [92]. Thus it is possible that these endogenous mechanisms help to produce a functional Tat in prokaryotic expression systems. During natural HIV infection, Tat is produced in very limited amounts in the initial stages [93]. The various PTMs identified in Tat modify the protein in such a way that the few available molecules of HIV Tat protein can be cycled for different functions [78,79]. 7.

Tat: role in HIV pathogenesis

Tat: cause of general immune suppression Immune-suppression is the main characteristic feature of AIDS, which develops in due course of time among HIV patients. HIV productively infects T helper cells and macrophages and the immune-compromised status is generated by the loss of immune cells, mainly T helper cells. HIV-Tat suppresses the immune status among HIV-infected individuals through different mechanisms. Extracellular Tat can induce the production of various cytokines like TGF-b, IL-2 and IL-6 [94-97]. However, overexpression of Tat was found to reduce the expression of IL-2 and IL-2R mRNA [98]. Tat can also inhibit the production of IL-12 from PBMCs [99]. Tat proteins from different HIV clades have been found to differentially modulate cytokine expression in human primary monocytes [100]. Tat B caused significant upregulation of pro-inflammatory cytokines like IL-6 and TNF-a while Tat C caused upregulation of anti-inflammatory molecules like IL-4 and IL-10 [100]. Since IL-10 has strong inhibitory action upon T cell, its 7.1

upregulation by Tat could suppress T cell functions [101]. HIV-Tat has been reported to suppress gp120-specific T-cell response in an IL-10-dependent manner. This was confirmed by studies showing that an IL-10-deficient mouse doesn’t show the immunosuppressive effect of Tat. The second mechanism of immunosuppression involves the alteration of secretion of interferon (IFN). HIV-Tat has been shown to upregulate IFN expression during HIV infection. The increased expression of IFN-a has been shown to suppress uninfected T cells functions [102]. The downregulation of MHC I expression on HIV infected cells by HIV-Tat protein suggests another mechanism for the development of immune evasion and immune suppression. Since MHC I presents intracellular processed antigens to CD8+T cells, the downregulation of MHC I could severely affect CD8+T cell-associated immune responsiveness [30]. HIV-Tat is known to inhibit the production of NO by host macrophages. This in turn makes the host vulnerable to infections, because NO provides the first line of defense against general opportunistic pathogens [103]. 7.2

Tat: decline in CD4+ T cells

An increased virus load and the progressive loss of CD4+ T cells is the hallmark of disease progression in HIV-infected individuals [104]. This could be either due to direct infection or due to extracellular effects of HIV Tat protein. Uninfected CD4+ T cells have been reported to undergo apoptosis due to bystander effect of HIV Tat protein [105]. Microtubules are also shown to be involved in Tat mediated apoptosis of CD4+ T cells. Tat binds to tubulin and enhances tubulin polymerization and prevents its depolymerization [63]. HIV-Tat protein binds to tubulin through a four-amino-acid subdomain of its conserved core region, leading to changes in the microtubule dynamics. Perturbation of microtubule dynamics generates a death signal, which gets transduced by B cell leukamia/lymphoma--2 (bcl-2) interacting mediator of cell death (Bim; a pro-apoptotic Bcl-2 family relative) gene and results in Tat-induced apoptosis [106]. Tat has been shown to upregulate TNF-related apoptosisinduced ligand (TRAIL), caspase-3 and caspase-8 in human monocytes [107]. HIV-Tat protein upregulates Fas, Fas ligand (FasL/CD95 L) and TNF-a in human astrocytes [108]. Modulation of the Fas/FasL system by HIV-Tat protein represents an important mechanism for regulating apoptotic cell death [109]. The mitochondria-dependent apoptotic pathway is also targeted by HIV-Tat protein, which activates caspases through the PI3K pathway. On the other hand, Bcl-2, an anti-apoptotic gene has been found to be increased following HIV-1 infection. This increase could be mimicked by the treatment with Tat [110]. Stable transfection of Tat in Jurkat cells have also been found to induce the expression of Bcl-2 [111]. Thus, tat could play a role in cell survival by upregulation of the anti-apoptotic gene Bcl-2. Thus HIV tat can play very opposite roles of being proapoptotic and antiapoptotic.

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Tat: Mimicking chemokines HIV-Tat protein is secreted outside the infected cells and can be detected in the serum of HIV-1-infected individuals during late stages of HIV infection [112]. The extracellular Tat protein binds to cell surfaces through electrostatic forces, as well as by binding to integrin and chemokine receptors [113]. Tat protein also competes with cognate ligands to bind CXCR4, CCR2 and CCR3 chemokine receptors in primary human monocytes and PBMCs. Peptides spanning the HIV Tat cysteine-rich region show the interaction with and competitive behavior for chemokine receptors. Tat is able to displace binding of b-chemokines from the b-chemokine receptors CCR2 and CCR3, but not from CCR1, CCR4 and CCR5 [114]. Tat also mimics the features of b-chemokines, which in turn may serve to locally recruit chemokine receptorexpressing monocytes and macrophages toward HIV producing cells chemotactically and facilitate their activation and infection. Extracellular Tat protein has also been found to mimic matrix molecules through its RGD (Arg-Gly-Asp) sequence [115] and its basic domain. Cross-desensitization studies have indicated that Tat shares receptors with MCP-1, MCP-3 and eotaxin [68]. It also influences the integrity of the extracellular matrix via dysregulation of matrix metalloproteinase (MMP) activity in HIV-infected individuals, which facilitate disease progression. Recently, it was shown that extracellular Tat upregulates the expression of MMP 9 via an NF-kB-dependent pathway in astrocytes [116]. Tat triggers Ca2+ mobilization in macrophages in a concentration-dependent manner through CCR2 and CCR3 [44]. The change in Ca2+ concentration is likely to affect the availability of amino acids for protein synthesis and further modifying other cellular processes [117].

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7.3

Tat: as antigen and adjuvant HIV Tat protein is produced very early during infection and secreted from infected cells [43]. This makes Tat a very important vaccine candidate for humoral immunity. A few clinical trials have also been documented with native Tat protein as a vaccine candidate. Tat is well known to induce both TH1and TH2-type specific immune responses along with humoral response [118,119]. Tat protein has been reported to enhance the proteolytic activities of the immunoproteasome in dendritic cells, which enhances processing and presentation of MHC-I displayed CTL epitopes. Gavioli et al. reported that when other HIV proteins injected with Tat protein in mice showed TH1-type response and the CTL responses were directed to a larger number of T cell epitopes compared with the administration of other proteins without HIV Tat [120]. Another study showed that the core region of Tat controls the autoadjuvant phenomenon in which dimers of Tat are formed by the oxidation of two cysteine residues at position 34 and raise antibody response similar to that observed with wild-type protein without any adjuvants [121]. These studies show that apart from being an important vaccine candidate, Tat can also be 7.4

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used as adjuvant in HIV vaccines as well as other vaccines aimed at inducing antibody response. Tat: oxidative stress HIV infection is typically linked with increased systemic oxidative stress and inflammation [122]. Reactive oxygen species are able to activate NF-kB transcription factors, particularly the p50--p65 heterodimers. It can bind to kappa B-response elements in the HIV LTR promoter, and plays a stimulatory role in the transcription of HIV-1 genes [123]. Activated NADPH oxidase is a key mediator of the apoptosis leading to depletion of the CD4+ T lymphocytes and also plays a role in the induction of AIDS related dementia [124]. Tat is known to modulate the level of pro-inflammatory and anti-inflammatory cytokines. It can induce the production of pro-inflammatory cytokines, such as TNF-a, CCL2, IL-6 and IL-10 from monocytes and macrophages [22]. HIV-infected individuals have been reported to have increased levels of IL-10 [125]. IL-10 is an anti-inflammatory cytokine produced by a wide variety of cells and has been reported to downregulate MHC-II [126]. Induction of IL-10 by Tat results into rapid progression to AIDS. Tat has been shown to induce IL-10 production in monocytes through a calcium-dependent mechanism [127]. Individuals with higher plasma levels of IL-10 have been shown with severely compromised T helper cell function followed by lower T helper cell counts [128]. 7.5

Tat: an inducer for cancer Cases of neoplasms in HIV infected individuals are well documented. HIV Tat protein has been implicated in the development of cancers in AIDS patients [129]. Tat has been shown to induce the expression of the DNA polymerase beta gene, which encodes a key protein in the DNA baseexcision repair pathway [130]. This alteration might affect the stability of DNA in the host genome. Tat has been implicated for its role in double-strand break (DSB) DNA repair. Cellular extracts containing the Tat protein have a reduced ability to re-join linearized DNA [131]. HIV Tat protein downregulates the expression of the p53 gene and might play a role in the AIDS-related cancers [132]. Tat has potential to increase protease production, and invasion, which might result in tumorigenesis and metastasis [133]. In addition to the modulation of HIV genes, Tat upregulates a number of non-HIV viral genes [134]. In the case of oncogenic viruses, such as human papillomavirus (HPV) and Kaposi Sarcoma (KS)-associated Herpes Virus (KSHV) this activation could result in neoplasia [135]. Kaposi-sarcoma-associated lymphoproliferative disorders and cervical cancers have also been strongly associated with the progression of AIDS [136]. HIV Tat protein has been reported to contribute to these incidences and act as eliciting factor towards the aggressive growth of KS [137]. The abundance of KS due to facilitated replication of KSHV in HIV-infected individuals has been attributed to 7.6

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the role of Tat [135]. HIV Tat protein can act as co-factor and activate lytic cycle replication of KSHV [138]. The HIV positive patients show an enhanced prevalence of anogenital human papillomavirus (HPV) infection. These oncogenic viruses show aggressive behavior of invasive cervical cancer in HIV-infected patients. Tat has been shown to transactivate the HPV LTR, hence stimulating their replication [139]. JC virus (JCV), a human neurotropic polyomavirus, is the etiologic infectious agent of progressive multifocal leukoencephalopathy (PML). Immunosuppressive conditions among AIDS patients tend to show significantly higher incidence of PML than in patients with other immunosuppressive conditions [45]. This suggests the possible molecular link between HIV and JCV infection. HIV Tat protein has been reported to activate the JCV enhancer/promoter [140]. Tat induces the basal activity of the JCV late promoter, and causes JCV leukoencephalopathy in glial cells. The JCV early protein, the large tumor antigen, stimulates expression of JCVL and HIV-1 LTR promoters in both glial and non-glial cells [141]. 8.

Expert opinion

helpful for understanding the alterations in transactivation and other cascades of pathogenesis. Biologically active HIV Tat protein has immunomodulatory features that make it an attractive adjuvant for other antigens. HIV Tat protein is released by acutely infected cells and affects the neighboring uninfected cells in a bystander fashion. Tat is produced very early after HIV infection, even before the virus integrates with the host cell’s genetic machinery. HIV Tat protein can function as both an antigen and a potent adjuvant.

Acknowledgements The authors are highly grateful to Ch. Mohan Rao, Director, Centre for Cellular & Molecular Biology (CCMB), Hyderabad, for providing all the necessary support during preparation of this manuscript. MK Johri, R Mishra and C Chhatbar contributed equally.

Declaration of interest

HIV Tat protein plays pivotal role in the HIV life cycle. Knowledge of the structure of HIV Tat protein with its various classes of post--translational modifications would be

The authors have received financial support from the Department of Biotechnology (DBT) (BT/PR10709/AGR/36/598/ 2008), Government of India, New Delhi.

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100. Gandhi N, Saiyed Z, Thangavel S, et al. Differential effects of HIV type 1 clade B and clade C Tat protein on expression of proinflammatory and antiinflammatory cytokines by primary monocytes. AIDS Res Hum Retroviruses 2009;25:691-9 101. Gupta S, Boppana R, Mishra GC, et al. HIV-1 Tat suppresses gp120-specific T cell response in IL-10-dependent manner. J Immunol 2008;180:79-88 102. Zagury D, Lachgar A, Chams V, et al. Interferon alpha and Tat involvement in the immunosuppression of uninfected T cells and C-C chemokine decline in AIDS. Proc Natl Acad Sci USA 1998;95:3851-6 103. Barton CH, Biggs TE, Mee TR, Mann DA. The human immunodeficiency virus type 1 regulatory protein Tat inhibits interferon-induced iNos activity in a murine macrophage cell line. J Gen Virol 1996;77:1643-7 104. Gougeon ML, Lecoeur H, Dulioust A, et al. Programmed cell death in peripheral lymphocytes from HIV-infected persons: increased susceptibility to apoptosis of CD4 and CD8 T cells correlates with lymphocyte activation and with disease progression. J Immunol 1996;156:3509-20

107. Zheng L, Yang YD, Lu GC, Salvato MS. Extracellular HIV Tat and Tat cysteine rich peptide increase CCR5 expression in monocytes. J Zhejiang Univ Sci B 2005;6:668-72 108. Chauhan A, Turchan J, Pocernich C, et al. Intracellular human immunodeficiency virus Tat expression in astrocytes promotes astrocyte survival but induces potent neurotoxicity at distant sites via axonal transport. J Biol Chem 2003;278:13512-19 109. Nakajima H, Mizuta N, Fujiwara I, et al. Blockade of the Fas/Fas ligand interaction suppresses hepatocyte apoptosis in ischemia-reperfusion rat liver. Apoptosis 2008;13:1013-21 110. Zhang M, Li X, Pang X, et al. Bcl-2 upregulation by HIV-1 Tat during infection of primary human macrophages in culture. J Biomed Sci 2002;9:133-9 111. Zauli G, Gibellini D. The human immunodeficiency virus type-1 (HIV-1) Tat protein and Bcl-2 gene expression. Leuk Lymphoma 1996;23:551-60 112. Westendorp MO, Frank R, Ochsenbauer C, et al. Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120. Nature 1995;375:497-500 113. Sarkaria JN, Busby EC, Tibbetts RS, et al. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 1999;59:4375-82 114. Albini A, Ferrini S, Benelli R, et al. HIV-1 Tat protein mimicry of chemokines. Proc Natl Acad Sci USA 1998;95:13153-8 115. Brake DA, Debouck C, Biesecker G. Identification of an Arg-Gly-Asp (RGD) cell adhesion site in human immunodeficiency virus type 1 transactivation protein, tat. J Cell Biol 1990;111:1275-81

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Affiliation Manish K Johri, Ritu Mishra, Chintan Chhatbar, Salini K Unni & Sunit K Singh† † Author for correspondence Laboratory of Neurovirology & Inflammation Biology, Section of Infectious Diseases, Centre for Cellular and Molecular Biology (CCMB), Council of Scientific and Industrial Research (CSIR), Uppal Road, Hyderabad-500007, (A.P), India Tel: +91 40 2719 2865; Fax: +91 40 27160591/311; E-mail: [email protected]

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Review

Antiviral drugs for HBV liver disease Mauro Vigano` & Pietro Lampertico†

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1.

Introduction

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Treatment indications

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End-points of therapy

4.

Therapeutic strategies

5.

Prevention of resistance

6.

Conclusions

7.

Expert opinion

“ A. M. and A. Migliavacca” Center for Liver Disease, 1st Gastroenterology Unit, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Universita` degli Studi di Milano, Milan, Italy

Introduction: Chronic hepatitis B (CHB) virus infection affects about 400 million people around the globe and is among the world’s leading causes of death. The management of CHB has evolved rapidly, several therapeutic options are now available to prevent both progression of liver disease and anticipated liver morbidity and mortality. Areas covered: Current treatment modalities for CHB patients, together with suggestions from our own experience are summarized. The most relevant works published in recent years on pegylated interferon, nucleos(t)ides analogues (NUC) and the 2009 update of the American Association for the Study of Liver Diseases Practice Guidelines and the 2009 European Association for the Study of the Liver (EASL) Clinical Practice Guidelines on the management of chronic hepatitis B are discussed. Expert opinion: Pegylated interferon and NUC have advantages and limitations, as short-term interferon treatment induces a sustained virological response in a third of patients, whereas long-term suppressive therapy by NUC rapidly inhibits HBV replication in most patients but drug resistance and safety in the long-term will remain the most important unresolved questions. Careful evaluation of patient history, staging of liver disease and virological factors should guide the start of treatment and the choice to the most appropriate individualized treatment strategy in all CHB patients. Keywords: antiviral treatment, chronic hepatitis B, entecavir, HBV, pegylated-interferon, tenofovir Expert Opin. Biol. Ther. (2011) 11(3):285-300

1.

Introduction

Chronic HBV infection affects about 400 million people around the globe and is one of the most common infectious diseases and among the world’s leading causes of death. Antiviral therapy of chronic hepatitis B (CHB) is aimed to improve quality of life and survival by preventing progression of liver damage to cirrhosis, endstage liver disease or liver cancer (hepatocellular carcinoma(HCC)) thus preventing anticipated liver-related death. This goal is achieved by suppression of HBV replication in a sustained or maintained manner either by short-term ‘curative’ treatment with pegylated interferon (PEG-IFN) or long-term ‘suppressive’ therapy with potent nucleos(t)ide analogues (NUC). Since both strategies have advantages and disadvantages the wise treatment of a patient with CHB requires careful balance between prediction of the natural history of HBV and of the potential benefit of anti HBV therapy. PEG-IFN, owing to its immunomodulatory and antiviral properties, causes a durable suppression of viral replication in a significant proportion of patients with short treatment duration. However, PEG-IFN is less tolerated, has side effects, is less applicable to patients with severe hepatitis and less frequently causes permanent hepatitis suppression compared with NUC. Recent data on the long-term efficacy, excellent tolerability and low risk of drug-resistance by such third generation NUC as entecavir or tenofovir, have tipped 10.1517/14712598.2011.546340 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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Antiviral drugs for HBV liver disease

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Chronic HBV carriers with histological, biochemical or clinical evidence of progressive liver disease, require antiviral therapy. Complete suppression of HBV replication, that is undetectable HBV DNA by real time PCR assay (< 10 -- 15 IU/ml) is the primary efficacy endpoint of antiviral therapy. A short-term PEG-IFN based strategy or long-term NUC-based suppressive therapy serve this purpose. Of patient treated with PEG-IFN, 20 to 30% achieve a sustained virological response. These results can and must be improved by careful selection of patients to start on PEG-IFN and early identification, that is at week 12 or 24, of primary non responders who can stop therapy. First- and second-generation nucleoside/ nucleotide anlogues (NUC), such as lamivudine, adefovir and telbivudine, are not recommended any more as starting therapeutic choice in NUC-naive patients. New potent and high-genetic-barrier NUC such as entecavir or tenofovir are currently recommended as first line monotherapies in NUC naive patients independently of disease severity, serology or viral load. For patients developing NUC resistance, internationally agreed rescue protocols based upon selection of a non-cross-resistant drug, early rescue and combination therapy, are available and proven to be very effective. Long-term suppression of HBV replication normalizes ALT levels, remodels liver fibrosis, prevents clinical decompensation, and reduces HCC risk.

This box summarizes key points contained in the article.

the balance towards long-term suppression therapy as the first-line option for most patients with CHB, independently on the HBeAg status. The advantages of the ‘suppressive’ strategy are the good tolerability and rapidity of action, with the disadvantages of having to continue therapy indefinitely, having to manage the emergence of drug resistance and not yet having strong evidence of long-term effectiveness and safety. Since, however, long-term NUC treatment does not translate to HBV eradication in all patients, owing to the persistence of covalently closed circular DNA (cccDNA) in the infected hepatocytes, questions arise regarding whether monotherapy with the new NUC will result in permanent suppression of viral replication in all patients. Close monitoring of serum HBV DNA levels together with the pro-active management of the patients with incomplete virological response or a virological breakthrough, by early addition of a non-cross-resistant drug, may optimize therapy. 2.

Treatment indications

Not every patient with CHB needs antiviral therapy as only patients with clinical or histological predictors of HBV progression should be prioritized to anti-HBV treatment [1-3]. HBV envelope antigen (HBeAg)-seropositive patients with 286

persistently normal alaninoaminotransferase (ALT) levels, high serum HBV DNA and no family history of HCC or cirrhosis, do not require immediate therapy. These patients should be monitored at 3 -- 6 month intervals and when ALT levels become elevated liver biopsy and antiviral treatment should be considered. Liver biopsy and treatment should also be considered in patients with persistent borderline normal or slightly elevated ALT levels, particularly if the patient is above the age of 40 [1]. HBeAg-negative, antiHBe--positive patients with normal ALT levels and HBV DNA < 2,000 IU/ml should be monitored with ALT determination every 3 months during the first year to verify that they are truly in the ‘inactive carrier state’ and then every 6 -- 12 months. Such patients do not require therapy as well as patients with mild CHB, that is slightly elevated ALT (less than 2 times the upper limit of normal) and mild histological lesions (less than grade A2 or stage F2 by METAVIR scoring). According to AASLD Guidelines, CHB patients with serum HBV DNA > 20,000 IU/ml, (> 2,000 IU/ml in HBeAg-negative cases), persistent or intermittent elevation in ALT levels and with liver biopsy showing chronic hepatitis with moderate or severe necroinflammation should be considered for treatment [1]. According to EASL guidelines [3], both HBeAg-positive and HBeAg-negative patients should be considered for treatment when HBV DNA levels exceed 2,000 IU/ml and/or the serum ALT levels are above the upper limit of normal (ULN), and/or a liver biopsy (or eventually non-invasive markers when validated in HBV patients) show moderate to severe active necro-inflammation and/or fibrosis using a standardised scoring system (at least grade A2 or stage F2 by METAVIR scoring). While indications for treatment must also take into account age, health status, and availability of anti-viral agents in individual countries, current guidelines support also the view that all patients with compensated or decompensated cirrhosis and detectable serum HBV DNA (even < 2000 IU/ml) must receive antiviral treatment regardless of ALT. The main goal of treatment in this group of severely ill patients is to completely inhibit viral replication in order to improve liver function and survival and, in case of liver transplantation, to prevent graft re-infection. 3.

End-points of therapy

The treatment paradigm of CHB is to persistently suppress serum HBV DNA to as low a level as possible. This goal can be obtained either on- or off-treatment, with subsequent biochemical remission, histological improvement, prevention of complications and improved long-term outcomes. Maintenance of undetectable levels of HBV DNA is mandatory to prevent resistance to NUC and increase the rates of HBeAg and hepatitis B surface antigen (HBsAg) seroconversion. In both HBeAg-positive and HBeAg-negative patients, the ideal end-point of therapy is sustained HBsAg loss with seroconversion to anti-HBs [4-6]. Recent studies suggest that

Expert Opin. Biol. Ther. (2011) 11(3)

Vigano` & Lampertico

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quantitative assessment of serum HBsAg levels may identify the different phases of the natural history of HBV and predict the virological response to antiviral therapy [7-9]. In HBeAg-positive patients, a sustained HBeAg seroconversion is a realistic and satisfactory end-point associated with improved prognosis [10-12]. In HBeAg-positive patients unable to seroconvert to anti-HBe and in HBeAg-negative patients, a maintained undetectable HBV DNA level on treatment with NUC or a sustained undetectable HBV DNA level after interferon-based treatment is the next most desirable end-point, as the induction of persistent biochemical and virological remission appears to be the most important therapeutic target in CHB [13-19]. 4.

Therapeutic strategies

Currently, there are seven drugs licensed for treatment of CHB: standard interferon (IFNa), pegylated IFNa (PEGIFNa), lamivudine, adefovir dipivoxil, entecavir, telbivudine and tenofovir disoproxil fumarate. Interferon couples antiviral and immunomodulatory activities and is administered subcutaneously, whereas the oral antiviral agents which are analogs of natural nucleosides (lamivudine, entecavir, telbivudine) or nucleotides (adefovir dipivoxil, tenofovir disoproxil fumarate) are administered once daily. Two different therapeutic approaches can be used: short-term potentially ‘curative’, treatment based upon PEG-IFN administration and long-term ‘suppressive’ treatment based upon NUC therapy. Pegylated interferon Interferons are a family of cytokines naturally produced by cells in response to various stimuli, including viral infections. Two types of interferon are commercially available for viral hepatitis treatment: standard IFN has been used for many decades but has been largely superseded by PEG-IFN. The attachment of a PEG molecule to IFN led to an improved pharmacokinetic profile with a significant increase in halflife and a relatively continuous drug exposure during the dosing interval, allowing weekly administration. Two PEG-IFN have been developed: a large branched 40-kDa PEG linked to IFNa-2a (PEG-IFNa-2a) and a small linear 12-kDa PEG linked to IFNa-2b (PEG-IFNa-2b). The PEG-IFNa-2a has a longer half-life (approximately 80 h), it is mainly catabolised in the liver, and has active metabolites. The smaller PEG-IFNa-2b has a shorter half-life (approximately 40 h) and may act as a pro-drug depot, slowly releasing interferon. Almost worldwide, PEG-IFNa-2a is registered for the treatment of CHB as a 48-week course given by subcutaneous injection once weekly in a dosage of 180 µg, while PEG-IFNa-2b has only been licensed for the treatment of CHB in specific Asian countries. Treatment with PEG-IFN is often complicated by the occurrence of side-effects. In particular, flu-like symptoms, cytopenia, headache, myalgia, fatigue and local reaction at the injection site occur frequently (> 30% of patients) but 4.1

often do not require treatment discontinuation. Anorexia, thyroid dysfunction, hepatitis flares and psychiatric adverse events occur less frequently. Clinical relevant neutropenia or thrombocytopenia, severe complications of bacterial infection and exacerbation of liver disease occur infrequently and have been observed in patients with more advanced stages of cirrhosis or in patients with decreased cell counts before starting therapy [20]. PEG-IFN is absolutely contraindicated in decompensated liver disease and contraindicated in patients with advanced cirrhosis because of the increased risk of hepatic decompensation due to IFN-induced hepatitis flares and severe bacterial infection. Other important contraindications of treatment with PEG-IFN are severe psychiatric co-morbidity (depression and suicidal ideation), severe cardiac disease and severe autoimmune disorders. PEG-IFN in HBeAg-positive patients Two large trials of 1-year treatment with PEG-IFNa-2a (180 µg per week) or PEG-IFNa-2b (100 µg per week), demonstrated that one third of HBeAg-positive patients achieved HBeAg seroconversion and 3 -- 5% seroconverted to anti-HBs, 6 months after stopping therapy [21,22]. Sustained viral suppression (HBV DNA below 5 log copies/ml) was observed in 27 -- 32% and ALT was normal in 32 -- 41% of patients. In both studies, the addition of lamivudine to PEG-IFN did not increase the sustained response rates compared with PEG-IFN monotherapy, although the former additional treatment was associated with more potent viral suppression during treatment [21,22]. Rates of antiHBe seroconversion and undetectable HBV DNA at one year of therapy with PEG-IFN in comparison with NUC are reported (Figure 1). After 3 years of post treatment follow-up, 37 and 11% of the patients who received 1 year of PEG-IFNa-2b became HBeAg and HBsAg seronegative, respectively. Among the initial responders (defined as HBeAg negativity at 26 weeks posttreatment), HBeAg was sustained in 81% of cases and HBsAg loss increased to 30% and overall, HBsAg was negative in 11% of patients at the last visit [23]. HBeAg seroconversion was more likely to occur in patients with high ALT (above 3 times ULN), low HBV DNA (below 7 log IU/ml) and high activity scores (‡ A2) on liver biopsy [20]. An HBV DNA decrease to less than 20,000 IU/ml at week 12 of IFN treatment is associated with a 50% chance of HBeAg seroconversion [24]. Recently, a dynamic prediction model adding HBV DNA decline during therapy provided a more accurate prediction of PEG-IFN induced sustained response than baseline factors; it is recommended that PEG-IFN treatment should be stopped by 24 weeks if HBV DNA declined less than 2 log10 [25]. Patients with genotype A have higher rates of sustained HBeAg loss, durability of HBeAg seroconversion and HBsAg loss compared with those infected with nonA genotype [21-23,26]. Despite increased risk of side effects and contraindications, a 48-week course of PEG-IFN is a recommended first-line therapy for young, highly motivated, 4.1.1

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Undetectable HBV DNA

100% 80%

Anti-HBe seroconversion

74% 67% 60%

60% 39%

40% 25%

30%

26%

22%

21%

20%

22% 21%

12%

0% IFN LAM ADV

G-

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PE

T

LD

V

ET

F

TD

IFN LAM ADV

G-

PE

T

LD

V

ET

F

TD

Figure 1. 48-week rates of virological response (HBV DNA < 300/400 copies/ml) and anti-HBe seroconversion in NUC-nay¨ve HBeAg-positive patients. Entecavir (ETV) and tenofovir disoproxil fumarate (TDF) inhibited HBV replication in most patients over 48 weeks of therapy but this did not translate into higher rates on HBeAg seroconversion compared to PEG-IFN. Collation of currently available data -- not from head-to-head studies (lower limit of quantification of HBV DNA assays: 300 -- 400 copies/ml). Adapted from [3]. ADV: Adefovir dipivoxil; LAM: Lamivudine; LDT: Telbivudine; PEG-IFN: Pegylated IFN.

non-cirrhotic HBeAg-positive patients with genotype A or B having high pre-treatment serum levels of ALT [3]. PEG-IFN in HBeAg-negative patients A large multinational trial compared the long-term virological and biochemical response after 48 weeks of PEG-IFNa-2a (180 µg/week) ± lamivudine with lamivudine monotherapy alone [27]. Combination of PEG-IFNa-2a and lamivudine leads to more profound end of treatment virological response than PEG-IFNa-2a alone (92 versus 81%), but this effect is lost 6 months after treatment cessation when rates of responders were 43 and 44%, respectively. Rates of undetectable HBV DNA at one year of therapy with PEG-IFN in comparison with NUC are reported in Figure 2. The long-term follow-up of 230 patients treated with PEG-IFN ± lamivudine and originally enrolled in the Marcellin et al. study, has showed that 5 years after the end of treatment, sustained response was higher (21%) among PEG-IFN ± lamivudine-treated compared with lamivudinetreated patients. The rates of HBsAg seroclearance among PEG-IFN treated patients gradually increased over time reaching 12% 5 years after the end of therapy with 35% of sustained responders achieving this end point (Figure 3) [27-29]. In HBeAg-negative patients, combination of PEG-IFN + lamivudine did not significantly improve the response rates compared with monotherapy, except for genotype D infected patients [30]. Likewise, the addition of adefovir dipivoxil or ribavirin to PEG-IFN did not increase the rates of sustained virologic response after the end of therapy compared to PEG-IFN alone [31,32]. To improve the suboptimal sustained virological responses to PEG-IFN in HBeAg-negative patients, different 4.1.2

288

approaches, such as a longer than 48-week treatment regimen, stratification by baseline predictors and on-treatment assessments, have been suggested. A pilot study demonstrated the superiority of 60 weeks of PEG-IFNa-2a treatment compared with 48 weeks [33] and a recent Italian multicenter study showed that the end of treatment response were similar in patients treated with PEG-IFNa-2a for 48 or 96 weeks, whereas the 1-year sustained response was significantly higher in patients treated for 96 versus 48 weeks (29 versus 12%, p = 0.02) (Figure 4). Three patients in the extended-duration group, compared with none of those treated for 48 weeks, became HBsAg negative (6 versus 0%) and another 2 patients in the former group had low levels of HBsAg at the end of the follow-up [34]. Baseline high ALT and low HBV DNA levels, female gender, younger age and HBV genotype significantly influence the response to PEG-IFN. At 1 year post-treatment, HBV genotype and ALT above 5 times the upper limit of normal were the strongest predictor of efficacy for patients treated with PEG-IFNa-2a ± lamivudine [30]. The best on-treatment predictor of response is HBsAg reduction; a decrease of 0.5 and 1 log10 IU/ml of serum HBsAg levels at weeks 12 or 24 of therapy showed negative predictive value (NPV) 90% and positive predictive value (PPV) 89% for week 12 and NPV 97%, PPV 92% for week 24 [35]. A study revealed that 35% of HBeAg-negative patients under PEGIFNa-2a ± lamivudine with HBsAg levels of 1500 IU/ml or less at week 12 of treatment, had HBsAg seroclearance by 4 years after treatment [36]. Rijckborst et al. [37] showed a very high negative predictive value of the combination of HBsAg and HBV DNA declines at week 12 of treatment from the baseline, none of the 20 patients without decrease

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Percentage of patients with HBV DNA < limit of quantification

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100% 80%

90%

91%

LDT

ETV

TDF

72% 63%

60%

51%

40% 20% 0% PEG-IFN

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88%

LAM

ADV

Figure 2. 48-week rates of virological response (HBV-DNA < 300/400 copies/ml) in NUC-nay¨ve HBeAg-negative patients. 90% of the patients treated with entecavir (ETV) or tenofovir disoproxil fumarate (TDF) achieved undetectable levels of HBV DNA within the first year of therapy. Collation of currently available data -- not from head-to-head studies (lower limit of quentification of HBV DNA assays: 300 -- 400 copies/ml). Adapted from [3]. ADV: Adefovir dipivoxil; LAM: Lamivudine; LDT: Telbivudine; PEG-IFN: Pegylated IFN.

in serum HBsAg levels and less than 2 log copies/ml HBV DNA levels decline at week 12 achieved a response. Nucleos(t)ides analogues There are currently five NUC available for the treatment of CHB: lamivudine, adefovir, telbivudine, entecavir and tenofovir. These antiviral agents affect the reverse transcription step of HBV replication resulting in different long-lasting viral suppression. Despite being well tolerated, long-term administration of NUC is hampered by the selection of drugresistant mutants, leading to loss of efficacy, that differ according to the drug. The in vitro cross resistance profile of a drug guides the selection of treatment rescue (Table 1). Nucleoside analogues such as telbivudine and entecavir are not the ideal choice for treating lamivudine resistance, since they share a similar resistance profile characterized by the mutation at position 204 plus several compensatory mutations [38]. By contrast, the resistance pattern of the nucleotide analog adefovir is characterized by changes at position 236 and 181, that is N23T and A181V, thereby offering a chance for suppressing lamivudine resistance. As a general rule, a nucleotide analog is recommended for rescue of HBV patients resistant to nucleoside analogues, whereas a nucleoside analog is recommended for a nucleotide-related resistance. Tenofovir is the preferred nucleotide for the rescue of nucleoside resistant patients because of its superior efficacy, safety and cost-effectiveness compared with adefovir (Table 2). First-generation NUC, such as lamivudine, and second-generation NUC, like adefovir and telbivudine, have been replaced by third-generation NUC, like entecavir and tenofovir, characterized by high potency and genetic barrier and low rates of resistance (Figure 5). 4.2

First and second generation NUC Lamivudine was the first nucleoside analogue for the treatment of both HBeAg-positive and HBeAg-negative patients. One 4.2.1

year of lamivudine treatment achieved HBV DNA undetectable (< 102 copies/ml) in 36 -- 44% of HBeAg-positive patients, with HBeAg seroconversion rate of approximately 20%, and in 60 -- 73% of HBeAg-negative patients [39-41]. However, longterm lamivudine monotherapy inexorably ends with the selection of specific mutations in the HBV polymerase gene, that is M204I/V as a primary mutation and L80, L180M, V173L as the most relevant secondary mutations at rates that increase from 20% after 1 year to peak at 70% after 5 years of therapy. In HBeAg-positive patients, non-Asian ethnicity, high pretreatment serum HBV DNA level, male sex, longer treatment duration and high body mass index are likely predictors of lamivudine resistance [42], whereas factors associated with the onset of lamivudine resistance in HBeAg-negative patients are poorly defined [19,43]. As a rule, patients with incomplete suppression of HBV replication at week 24 are at higher risk of generation of mutated strains [44,45]. The development of lamivudine resistance is responsible for virological rebound, ALT flares, histological worsening, progression to cirrhosis, clinical decompensation, HCC and delisting from liver transplantation [18,19,38,46]. In addition, the emergence of lamivudine resistance may decrease the efficacy of other anti-HBV agents as a result of cross-resistance [47]. Although lamivudine has proven to be well tolerated and has the most extensive safety record, it is no longer considered a first-line agent for long-term CHB monotherapy due to the exceedingly high rate of development of drug-resistant HBV and cross resistance to other drugs. However, permanent viral suppression in the few (25%) lamivudine long-term responders or in lamivudine-resistant patients rapidly rescued with ‘early add-on’ adefovir dipivoxil therapy, reduces disease progression by preventing clinical decompensation and improving portal hypertension but not the development of hepatocellular carcinoma in cirrhotic patients [48,49]. To limit the development of lamivudine resistance and increase the efficacy, the ‘de novo’ combination of lamivudine with

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Patients with HBsAg clearance (%)

20

n = 230

15 11% 10

5

12%

9% 5%

6%

0 1

2

3

4

5

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Years of post-IFN follow-up

Figure 3. In HBeAg-negative chronic hepatitis B patients treated with PEG-IFN a-2a ± lamivudine for 48 weeks, the rates of HBsAg loss progressively increased up to 5 years. Adapted from [27-29].

another nucleotides analogue has been suggested. However, results with the 1-year lamivudine and telbivudine combination therapy were similar to those obtained with telbivudine alone [50]. The combination therapy with lamivudine and adefovir in comparison with lamivudine monotherapy showed lower serum HBV DNA levels and higher rates of ALT normalization, lower rates of lamivudine resistance (19 versus 44%, respectively) but similar HBeAg seroconversion [51]. Adefovir dipivoxil was the first nucleotide analogue approved for use in patients with CHB showing significant improvements in liver histology and HBV DNA reductions compared with placebo [52,53]. Adefovir dipivoxil is less potent than lamivudine and the other more recently introduced oral agents. In 48-week registration trials, adefovir dipivoxil achieved undetectable serum HBV DNA (< 102 copies/ml) in only 13 -- 21% of HBeAg-positive, with 12% of HBeAg seroconversion and in 50 -- 65% of HBeAg-negative patients [52-54]. The advantages of adefovir dipivoxil are its limited resistance during the first 2 years of treatment and the absence of cross-resistance with lamivudine that allowed its use as rescue treatment for lamivudine resistance in CHB patients and in patients with liver decompensation associated with lamivudine resistance prior to and after liver transplantation [55-57]. Adefovir dipivoxil is the only agent with long term (5 years) data from a controlled study in naı¨ve HBeAg-negative patients. In this study adefovir dipivoxil showed maintained virological and biochemical response in nearly 70% of the patients and 5% of HBsAg seroclearance [58]. Drug withdrawal in patients with persistent undetectable HBV DNA by PCR assays, results in a universal virological relapse, although approximately 70% of the relapsers could maintain serum levels of HBV DNA below 10,000 copies/ml and ALT levels within the normal range for at least 12 -- 18 months [59]. Because of the significant rates (29% after 5 years of treatment) of genotypic resistance (rtN236T and/or rtA181V/T mutations) over long-term 290

administration and the suboptimal rates of virological response, adefovir dipivoxil monotherapy is no longer considered in HBV patients [3,58,60]. Moreover, treatment with adefovir dipivoxil may be complicated by renal toxicity. Although none of the patients treated with adefovir dipivoxil (10 mg/daily for 48 weeks) showed a ‡ 0.5 mg/dl increase of the serum creatinine [52], a significant increase in serum creatinine (> 0.5 mg/dl versus baseline) was reported in 3 -- 5% of patients treated with adefovir dipivoxil (10 mg) for up to 2 years [53,61]. Long-term safety data of adefovir dipivoxil monotherapy, for up to 5 years, showed that 9% of patients had confirmed increases of 0.5 mg/dl from baseline in serum creatinine [62]. Telbivudine was approved for use in patients with CHB infection in 2006. Telbivudine was tested against lamivudine in a large (GLOBE trial) multicenter study of HBeAgpositive and -negative patients [63]. In HBeAg-positive patients, at the end of year 1, telbivudine was superior to lamivudine in achieving undetectable HBV DNA levels (< 102 copies/ml) (60 versus 40%) but not in HBeAg seroconversion (23 versus 22%). Also in HBeAg-negative patients, telbivudine was superior to lamivudine in achieving undetectable serum HBV DNA (88 versus 71%) but not in achieving histological or biochemical improvement [63]. Second-year results of the GLOBE trial showed that rates of undetectable viremia (< 300 copies/ml) with telbivudine were significantly superior to that of lamivudine in both HBeAg-positive (55.6 versus 38.5%) and HBeAg-negative patients (82 versus 56.7%). The frequency of resistance to telbivudine at 1 year was 5% in HBeAg-positive and only 2% in HBeAg-negative patients [63]. By the end of year 2, however, resistance emerged in 25% of HBeAg-positive patients and in 11% of HBeAg-negative group [64]. Telbivudine is tolerated as well as lamivudine and appears to be quite safe, however, grade 3/4 increases in creatine kinase levels were more common in telbivudine-treated than in lamivudine-treated

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Vigano` & Lampertico

Percentage of patients with virological response

50% 43% 40% 30%

22% 20%

20% 10% 0% Marcellin* (n = 177)

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20%

Piccolo‡ (n = 30)

Rijckborst‡ (n = 69)

Lampertico‡ (n = 51)

Figure 4. The 24-week post-treatment HBV DNA suppression rates in HBeAg-negative chronic hepatitis B patients treated with a 48-week course of PEG-IFN a-2a ranged between approximately 20% in studies including mainly genotype D infected patients to 40% in studies performed in patients circulating genotype B or C patients. Based on data from [27,31,32,34]. *HBV DNA < 4000 U/ml. z HBV DNA < 2000 U/ml.

patients after 2 years of therapy (13 versus 4%, p < 0.001). Durability of HBeAg responses was similar to that achieved with the other oral agents whereas relapse was common after discontinuation of therapy in HBeAg-negative patients. A 4- year telbivudine treatment in the 213 HBeAg-positive patients showed undetectable serum HBV DNA (< 300 copies/ml) and normal serum ALT in 79 and 86% of patients, respectively and 42% of patients achieved HBeAg seroconversion. Among the 185 HBeAg-negative patients treated for 4 years with telbivudine, undetectable HBV DNA and ALT normalisation was achieved in 91 and 84% of patients, respectively [65]. Early HBV DNA response positively correlated with virologic and clinical efficacy response over 2 years of treatment in both HBeAg-positive and -negative patients: patients who had undetectable serum HBV DNA at treatment week 24 in the GLOBE trial showed the highest rates of virological primary response and the lowest rates of resistance after 2 years of treatment [64,66]. Patients treated with telbivudine in comparison with adefovir dipivoxil, achieved more frequently an undetectable viral load in both HBeAgpositive and -negative patients, however, sustained HBeAg loss and seroconversion and ALT normalization rates were similar in the treatment arms [67]. However, because of its cross-resistance with lamivudine and the significant rates of resistance, current international guidelines do not recommend telbivudine as an optimal first line therapy for patients with chronic hepatitis B [3]. Entecavir This drug is the prototype of a third generation NUC for naive patients, as it couples high potency and high genetic barriers to resistance. Entecavir was well tolerated, with similar frequencies of adverse events occurring between groups including predominantly constitutional symptoms such as headache, cough and fatigue. 4.3

Entecavir in HBeAg-positive patients Chang et al. reported that 1-year of entecavir (0.5 mg/day) led to undetectable HBV DNA by PCR assay in 67% of NUCnaı¨ve HBeAg-positive patients with normalization of ALT, improved histology and HBeAg loss in 68, 72 and 22% of patients, respectively [68]. A small randomized trial reported significant differences in undetectable HBV DNA by PCR assay among the 21 HBeAg-positive patients treated with entecavir compared with 21 treated with lamivudine (52 versus 36%), however without significant differences in terms of HBeAg seroreversion (15 versus 18%) [69]. Although entecavir showed a continuous viral decline beyond week 48, rates of HBeAg loss and seroconversion remain relatively low [70,71]. In addition, when therapy was discontinued after a 48-week treatment period, the majority of patients had recurrent viremia, as well as biochemical breakthrough, indicating a need for continued therapy and identifying the need to define optimal treatment duration [72]. Continuous entecavir use for up to 5 years [73] (0.5 mg/day the first year and then 1 mg/day) showed that among 146 patients who met criteria for enrolment in the long-term study, at year 5, 94% (88/94) of patients had undetectable serum HBV DNA, 80% (78/98) had normal ALT levels, 23% (33/141) achieved HBeAg seroconversion and 1.4% (2/145) lost HBsAg. Resistance emerges, in NUC-naive HBeAg-positive patients, at rates of 1.2% after 5 years of entecavir monotherapy [74]. In one study comparing entecavir with adefovir dipivoxil, entecavir achieved higher viral load reduction, however, ALT normalization as well as HBeAg seroconversion rates were similar in the treatment groups [75]. Despite data indicating the efficacy of entecavir in both nucleoside-naı¨ve and lamivudine-refractory patients, the potential for resistance remains a concern. When lamivudine-resistance substitutions L180M and M204V are present, HBV susceptibility to entecavir has been shown to 4.3.1

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Table 1. Cross-resistance data (in vitro). Nucleosides

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Wild type M204I L180M + M204V L180M + M204V/I ± I169T ± V173L ± M250V A181T/V N236T

Nucleotides

LAM

LDT

ETV

ADV

TDF

S R R R I S

S R R R S S

S I I R S S

S S S S R R

S S S S S I

The in vitro cross resistance profile of nucleoside analogues is significantly different from that of nucleotides, suggesting how to select the preferred rescue drug in patients developing resistance. Adapted from [3]. ADV: Adefovir dipivoxil; ETV: Entecavir; I: Intermediate susceptibility; LAM: Lamivudine; LDT: Telbivudine; R: Resistant; S: Susceptible; TDF: Tenofovir disoproxil fumarate.

be decreased by eightfold. Additional substitutions at either T184, S202, L180 or M250 further decrease susceptibility by 10- to 250-fold [76]. Therefore, entecavir displays reduced efficacy and higher incidence of entecavir-resistance when used in patients with lamivudine-resistance substitutions [77-79]. The antiviral efficacy of entecavir was not influenced by prior treatment with adefovir dipivoxil or presence of adefovir dipivoxil resistance but was less effective in patients with previous lamivudine-resistance and in patients with both lamivudine resistance and adefovir dipivoxil resistance [79-81]. Several case reports have documented in nucleoside-naı¨ve patients with CHB who experienced virologic rebound on entecavir the presence of lamivudine-resistance-associated mutations, suggesting that the patients were infected with resistant virus [82-84]. Entecavir in HBeAg-negative patients One-year of entecavir (0.5 mg/day) led to undetectable HBV DNA by PCR assay in 90% of NUC-naı¨ve HBeAg-negative patients with normalization of ALT and improved histology in 78 and 70% of patients, respectively [85]. Virological rebound and genotypically confirmed drug resistance occurred in 2 and 0% of the patients, only. After the first year of study, the vast majority of patients included in the study had a virological response, but were, per protocol, forced to withdraw from entecavir, with a viral rebound in all. The 25 patients who continued treatment for 2 years and the 16 who were treated for 3 years could maintain a virological response [86]. In 57 patients under long-term entecavir treatment with adequate baseline biopsy samples, a second liver biopsy evaluation after a median time of 6 years (range: 3 -- 7 years) showed a significant histological improvement and regression of fibrosis and cirrhosis. At the time of long-term biopsy, all patients had a serum HBV DNA level less than 300 copies/ml and histological improvement (a ‡ 2 point decrease in the Knodell necroinflammatory score and no worsening of the Knodell fibrosis score) was observed in 4.3.2

292

96% of patients, and a ‡ 1 point improvement in the Ishak fibrosis score was found in 88% of patients, including all 10 patients with advanced fibrosis or cirrhosis at baseline [87]. Entecavir in field practice A multi-center cohort study from European Vigilance Against Viral Resistance Network (VIRGIL) Surveillance Study Group in 104 naı¨ve patients showed that entecavir monotherapy, during a median follow-up of 11 (range 3 -- 23) months, induced virological response (HBV DNA < 80 IU/ml) in 79% of patients and none of them developed genotypic entecavir resitance. Moreover, the efficacy of entecavir was not influenced by prior treatment with lamivudine or by the presence of adefovir dipivoxil resistance [79]. In a field practice study in Italy of 376 NUC-naive patients (mostly HBeAg-negative), 2 years treatment with entecavir resulted in high rates of virological suppression (96% of patients with undetectable serum HBV DNA by PCR assay) with 11% of partial virological responders after 1 year of treatment, with no cases of viral resistance [88]. A US study in 153 patients treated with entecavir over a 3-year period showed higher response rates in patients who had never been treated before and in HBeAg-negative patients who displayed response rates of 90, 96 and 100% over 12, 24 and 36 months, respectively [89]. 4.3.3

Entecavir in patients with cirrhosis One-year of entecavir treatment was reported to have good efficacy and safety profile in patients with advanced fibrosis or histological cirrhosis resulting in undetectable HBV DNA (< 300 copies/ml) in > 90%, ALT normalisation in over 60% and histological improvement in > 70% of patients with compensated cirrhosis [90]. One-year of treatment with entecavir 0.5 mg/day showed similarly efficacy in both decompensated and compensated patients achieving undetectable serum HBV DNA and HBeAg loss in 89% and 48% versus 78% and 41%, respectively. Among patients with decompensated cirrhosis, 65% achieved class A Child-Pugh 4.3.4

Expert Opin. Biol. Ther. (2011) 11(3)

Vigano` & Lampertico

Table 2. Management of HBV resistance. Lamivudine resistance Telbivudine resistance Entecavir resistance Adefovir resistance

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Tenofovir resistancez

Add tenofovir Add tenofovir* Add tenofovir* Switch to tenofovir and add a second drug If N236T, add lamivudine, entecavir* or telbivudine* or switch to Truvada If A181V/T, add entecavir * or switch to Truvada Add entecavir*, telbivudine, lamivudine or switch to Truvada

In patients developing NUC resistance, EASL 2009 guidelines recommend the early add on strategy with a non-cross-resistant analogue. In patients developing resistance to adefovir, tenofovir and entecavir combination is the recommended strategy, although the long-term safety of this combination is unproven. Adapted from [3]. *The long-term safety of these combinations is unknown. z Not seen so far. Do genotyping and phenotyping in an expert lab to determine the cross-resistance profile.

score and 49% showed improvement of at least 2 points in the Child-Pugh score with a cumulative transplantation-free survival of 87% [91]. In a randomized, open-label study in 195 patients with chronic hepatitis B infection and decompensated cirrhosis (mean pre-treatment Model for End-Stage Liver Disease (MELD) score = 16, 45% HBeAg-negative and approximately one third previously exposed and resistant to lamivudine) entecavir (1 mg daily) was compared to adefovir dipivoxil (10 mg daily) [92]. At week 48, entecavir demonstrated greater viral suppression compared to adefovir dipivoxil and was associated with clinical benefits including improved survival: 57% of patients on entecavir achieved an undetectable viral load, compared with 20% of patients on adefovir dipivoxil (p < 0.0001). At week 48, MELD score declined by 2.6 points in patients treated with entecavir compared with 1.7 points for patients treated with adefovir dipivoxil. By week 48, 5% of entecavir recipients, but none of the adefovir dipivoxil recipients had lost HBsAg. The overall safety profile was comparable across the two treatments but increases in serum creatinine levels of > 0.5 mg/dl from baseline were observed in 17 and 24% of patients treated with entecavir and adefovir dipivoxil, respectively. Although the study design did not include prospective measurements of serum lactate levels, a retrospective analysis of the 22 patients with baseline MELD score ‡ 22 (15 on entecavir and 7 on adefovir dipivoxil) identified only one case of lactic acidosis in an entecavir-treated patient who spontaneously resolved on continued entecavir treatment [92]. Recently, few cases of lactic acidosis were reported in decompensated (MELD > 22) cirrhosis that resolved in most patients after drug withdrawal [93]. This warning suggests that entecavir should be applied cautiously in

patients with severe liver damage and high baseline MELD scores, particularly those in the waiting list of liver transplantation. Tenofovir Tenofovir, an acyclic nucleotide phosphonate, is the other third generation NUC. Registration trials have compared the efficacy of tenofovir to that of adefovir dipivoxil in both nucleotide-naı¨ve [54] and -experienced patients [94]. In these studies, more patients receiving tenofovir than adefovir dipivoxil achieved an undetectable viral load and improved histology. 4.4

Tenofovir in NUC-naive HBeAg-positive patients

4.4.1

In a long term ongoing trial of tenofovir treatment of HBeAgpositive CHB infection, 198 patients (74% of the initial enrolled cohort) completed 4 years of therapy [95]. In an intent-to-treat analysis, 77% of patients had sustained suppression of HBV DNA (< 400 copies/ml), 74% of patients who received tenofovir monotherapy for the entire study and 84% of those who started on adefovir dipivoxil and later switched to tenofovir. In a per-protocol analysis, 96% of patients in the ongoing tenofovir arm and 99% in the adefovir dipivoxil-to-tenofovir arm achieved undetectable HBV DNA, 41% of participants achieved HBeAg loss, 31% experienced HBeAg seroconversion with an overall cumulative rate of HBsAg loss of 10%. Creatinine levels remained stable through year 4 with only one patient experiencing a greater than 0.5 mg/dl increase in serum creatinine. Resistance analysis showed no amino acid substitutions that could be associated with tenofovir resistance. Tenofovir in NUC-naive HBeAg-negative patients

4.4.2

In a 4-year study of 315 patients, 86% of HBeAg-negative patients had full suppression of HBV DNA in an intent-totreat analysis, with similar efficacy in patients who received tenofovir monotherapy for the entire study (85%) and those who started on adefovir dipivoxil and later switched to tenofovir (87%) [96]. In a per-protocol analysis, 99% of participants in the ongoing tenofovir arm and 100% in the adefovir dipivoxil-to-tenofovir arm achieved undetectable HBV viral load. Resistance analyses showed no amino acid substitutions that could be associated with tenofovir, only two patients experienced a 0.5 mg/dl increase in creatinine. An excellent record of durable antiviral activity, safety and tolerability was also demonstrated in a 4-year study comparing tenofovir and adefovir dipivoxil in Asian patients and in high viremic patients. Among 145 Asian patients who completed the 4-year study, HBV DNA was < 400 copies/ml in 79% of patients (74% in HBeAg-positive and 84% in HBeAg-negative patients) [97]. Among the 129 CHB patients with baseline high viral load (‡ 9 log10 copies/ml), 71% of

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1st generation

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Patients developing resistance (%)

80 67

70 2nd generation

60 49 38

40

3rd generation 29 24 20

18

17

11 0

0 LAM

3 ADV

4

? LDT

0.5 1.2 0.2 1.2 1.2 0 0 0 0 ? ETV

TDF

Figure 5. The incidence of resistance in NUC-nay¨ve patients treated up to 5 years progressively increased for lamivudine, adefovir and telbivudine, but not for entecavir and tenofovir. Collation of currently available data -- not from head-to-head studies. Adapted from [3]. ADV: Adefovir dipivoxil; ETV: Entecavir; LAM: Lamivudine; LDT: Telbivudine; TDF: Tenofovir disoproxil fumarate.

patients, achieved undetectable serum HBV DNA by year 4 [98], as per intention-to-treat analysis. Tenofovir in patients with cirrhosis In a sub-analysis of 2-year tenofovir treatment, Buti et al. showed similar proportions of cirrhotic and non-cirrhotic patients achieving HBV DNA suppression (< 400 copies/ml), with also a similar safety profile [99]. Recently, a Phase II, double-blind study randomized 112 patients with CHB and decompensated liver disease to receive either tenofovir (n = 45), combination therapy with emtricitabine plus tenofovir (n = 45), or entecavir (n = 22) [100]. After 48 weeks of treatment, a similar rates of response (HBV DNA < 400 copies/ml) was observed across the three treatment arms (71, 88 and 73%). ALT normalization occurred in 57, 76 and 55% of patients in the three groups. A 2 point median MELD score reduction and a 1 point median Child Pugh score reduction were observed in all the three treatment arms. No significant renal safety difference was observed between groups, in fact the proportion of subjects with a confirmed increase in serum creatinine ‡ 0.5 mg/dl from baseline or confirmed serum phosphorus < 2.0 mg/dl were 9, 7 and 5%, respectively. 4.4.3

Tenofovir in NUC-experienced or -resistant patients

4.4.4

Van B€ommel in 2004, described the use of tenofovir in patients with genotypic evidence of lamivudine-resistance and high baseline serum HBV DNA (> 6 log10 copies/ml) showing a strong and early suppression of HBV DNA 294

compared with the HBV DNA decline in patients treated with adefovir dipivoxil [94]. In fact, after 1-year of treatment 100% of tenofovir-treated patients had HBV DNA levels < 105 copies/ml versus 44% of adefovir dipivoxiltreated patients. The efficacy of tenofovir as rescue drug for HBV-infected patients with lamivudine resistance and incomplete virological response to adefovir dipivoxil was also reported; 3.5 months after the switch from adefovir dipivoxil to tenofovir, 19 of 20 patients had undetectable serum HBV DNA (< 400 copies/ml), whereas ALT normalized in 10 of 14 patients after a median of 12 months. During the tenofovir treatment no side effects were reported [101]. A recent retrospective multicenter study evaluated the use of tenofovir monotherapy in patients with prior failure or resistance to different NUC treatments: 131 patients with previous treatment with lamivudine (n = 18), adefovir dipivoxil (n = 8), sequential lamivudine-adefovir dipivoxil (n = 73), add-on combination therapy with both drugs (n = 29), entecavir (n = 3) and evidence of genotypic lamivudine and adefovir dipivoxil resistance in 62 and 19% of patients, respectively [102]. The overall cumulative proportion of patients achieving undetectable serum HBV DNA (< 400 copies/ml) was 79% after a mean treatment duration of 23 months (range, 6 -- 60). Although lamivudine resistance did not influence the antiviral efficacy of tenofovir, the presence of adefovir dipivoxil-resistance impaired tenofovir efficacy (100 versus 52% probability of HBV DNA < 400 copies/ml, respectively). Neither a virological breakthrough nor significant adverse events were noticed during tenofovir monotherapy.

Expert Opin. Biol. Ther. (2011) 11(3)

Vigano` & Lampertico

A combination of emtricitabine and tenofovir from the start versus tenofovir as monotherapy has been evaluated for patients with incomplete response to adefovir dipivoxil. At week 48, 81% of patients initially given tenofovir or tenofovir/emtricitabine had HBV DNA levels below 400 copies/ml. The presence of baseline lamivudine- or adefovir dipivoxil-resistance-associated mutations did not affect response, however adherence to therapy appeared to be the primary factor associated with HBV DNA levels below 400 copies/ml at week 48 [103].

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4.5

6.

de-novo combination

Monotherapy with one class of antiviral would decrease treatment options as a single mutation can confer resistance across the class for nucleoside and nucleotide analogues. Similarly to HIV, the possibility of combining antivirals to more effectively treat CHB and minimize the risk of resistance development has been proposed [104,105]. However, combination therapy does not appear to affect the number of HBeAg seroconversions. One study assessed the safety and tolerability of tenofovir in comparison to combination therapy based on emtricitabine plus tenofovir and entecavir in decompensated liver disease, mostly in patients with HBeAg-negative chronic hepatitis B [106]. After 48 weeks of treatment, a similar rate of response (HBV DNA < 400 copies/ml) was observed across the three treatment arms: 71, 88 and 73%, whereas the rates of confirmed kidney impairment were 9, 7 and 5%, and no patient lost serum HBsAg. While long-term de-novo combination has the potential advantages of increased efficacy and lower resistance rates compared with monotherapy, it has the cons of increased cost, poorly known safety, and reimbursement in several countries. At present, de-novo combination of two third-generation NUC is not a recommended strategy for NUC-naive patients. 5.

NUC, evidence-based algorithms for rescuing these patients have not been generated, apart from expert opinions. De-novo combination might represent the best approach for highly viremic patients, to increase the antiviral efficacy while rescuing the risk of resistance, but there are no studies supporting this approach in HBV patients. In general, the best strategy to prevent resistance is to start with potent and high-geneticbarrier drugs and to evaluate adherence to carry a negligible risk of drug resistance in long-term clinical practice.

The management of chronic hepatitis B has evolved rapidly, several therapeutic options are now available and nowadays hepatitis B is a treatable disease, in the sense that HBV replication can now be inhibited long-term. Therapy must reduce HBV DNA to as low a level as possible to ensure a degree of virological suppression that will then lead to biochemical remission, histological improvement and prevention of disease progression. Interferon and NUC have both advantages and disadvantages, short-term treatment with PEG-IFN induces a sustained virological response in a third of patients, long-term NUC treatment inhibits HBV replication in most of the patients but drug resistance and safety in the longterm will remain the most important unresolved questions. With the anti-HBV analogues available today, HBV replication can be easily suppressed for many years in most of the patients but, as none of the drugs available today given as a monotherapy suppress viral replication in all patients forever, some patients will require a rescue therapy added to the current drug. Careful evaluation of patient history, staging of liver disease and virological factors should guide the start of treatment and the choice to the most appropriate treatment strategy. 7.

Prevention of resistance

Resistance to anti-HBV analogues can be delayed or prevented by careful selection of patients to be treated, administration of de-novo combination therapy, use of thirdgeneration NUC as first-line therapy and early adoption of antiviral therapy especially in partial responders. In the search for predictors of resistance to improve the cost-effectiveness of HBV therapy, a relationship between residual viral load at week 24 and risk of developing resistance at week 48 or 96 has been demonstrated [107]. The clinical relevance of partial virological responders (PVR) to NUC relates to the high risk these patients face of developing resistance to longterm anti-HBV treatment [3]. However, for PVR to entecavir and tenofovir, known to carry a lower risk of resistance to long-term monotherapy, the association between residual viremia at week 48 and secondary treatment failure during follow-up has not been fully established. Despite the strong rationale for adapting antiviral therapy, at least for selected

Conclusions

Expert opinion

The antiviral management of patients with chronic hepatitis B is entering in the modern era. In PEG-IFN treated patients, treatment is now personalized by using baseline and more importantly on-treatment predictors, among which HBsAg levels play a major role. Stopping rules have also been suggested, in an attempt to start therapy in a larger number of patients but to continue after 12 or 24 weeks in only a subset of responders. By the same token, long-term suppressive therapy has been revolutionized by third-generation NUC such as entecavir and tenofovir, which can inhibit HBV replication in the vast majority of patients for 3 -- 5 years as a monotherapy. Early diagnosis and effective rescue algorithms for patients developing viral resistance have been developed to rapidly keep viral replication under control. All these achievements serve the main purpose of anti-HBV therapy, that is sustained or maintained suppression of virus replication. This had and will have a tremendous clinical effect by significantly reducing

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liver necroinflammation but also fibrosis on histological grounds, preventing evolution to cirrhosis and end-stage liver disease, with a residual risk of hepatocellular carcinoma. However, some unsolved issues do still exist, such as when to start antiviral therapy, how to improve the management of high-viral-load patients, how to rescue patients with partial virological response, how to increase the rates of HBsAg seroconversion during long-term suppressive Bibliography

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Ren FY, Piao DM, Piao XX. A one-year trial of entecavir treatment in patients with HBeAg-positive chronic hepatitis B. World J Gastroenterol 2007;13:4264-7

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Gish RG, Lok AS, Chang TT, et al. Entecavir therapy for up to 96 weeks in patients with HBeAg-positive chronic hepatitis B. Gastroenterology 2007;133:1437-44

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Colonno RJ, Rose RE, Baldick CJ, et al. High barrier to resistance results in no emergence of entecavir resistance in nucleoside-naive subjects during the first two years of therapy. J Hepatology 2006;44(Suppl 2):S182

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Shouval D, Lai CL, Chang TT, et al. Relapse of hepatitis B in HBeAg negative

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73.

Chang TT, Lai CL, Yoon SK, et al. Entecavir treatment for up to 5 years in patients with hepatitis B e antigen-positive chronic hepatitis B. Hepatology 2010;51:422-30

74.

Tenney DJ, Rose RE, Baldick CJ, et al. Long-term monitoring shows hepatitis B virus resistance to entecavir in nucleoside-naive patients is rare through 5 years of therapy. Hepatology 2009;49:1503-14

75.

Leung N, Peng CY, Hann HW, et al. Early hepatitis B virus DNA reduction in hepatitis B e antigen-positive patients with chronic hepatitis B: a randomized international study of entecavir versus adefovir. Hepatology 2009;49:72-9

76.

Colonno RJ, Rose R, Baldick CJ, et al. Entecavir resistance is rare in nucleoside naive patients with hepatitis B. Hepatology 2006;44:1656-65

77.

Sherman M, Yurdaydin C, Sollano J, et al. Entecavir for treatment of lamivudine-refractory, HBeAg-positive chronic hepatitis B. Gastroenterology 2006;130:2039-49

78.

Suzuki F, Toyoda J, Katano Y, et al. Efficacy and safety of entecavir in lamivudine-refractory patients with chronic hepatitis B: randomized controlled trial in Japanese patients. J Gastrol Hepatol 2008;23:1320-6

79.

Reijnders JG, Deterding K, Petersen J. Antiviral effect of entecavir in chronic hepatitis B: influence of prior exposure to nucleos(t)ide analogues. J Hepatol 2010;52:493-500

80.

Shim JH, Suh DJ, Kim KM, et al. Efficacy of entecavir in patients with chronic hepatitis B resistant to both lamivudine and adefovir or to lamivudine alone. Hepatology 2009;30:1064-71

81.

Cho SW, Koh KH, Cheong JY, et al. Low efficacy of entecavir therapy in adefovir-refractory hepatitis B patients with prior lamivudine resistance. J Viral Hepat 2010;17:171-7

82.

Tong CW, Mullen JE, Wong T. Early hepatitis B virological rebound on entecavir through selection of

Vigano` & Lampertico

lamivudine-associated mutations. J Antimicrob Chemother 2009;64:875-7 83.

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84.

85.

86.

87.

..

88.

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Kobashi H, Fujioka S, Kawaguchi M, et al. Two cases of development of entecavir resistance during entecavir treatment for nucleoside-naive chronic hepatitis B. Hepatol Int 2009;3:403-10 Tenney DJ, Rose RE, Baldick CJ, et al. Two-year assessment of entecavir resistance in lamivudine-refractory hepatitis B virus patients reveals different clinical outcomes depending on the resistance substitutions present. J Antimicrob Chemother 2007;51:902-11 Lai CL, Shouval D, Lok AS, et al. Entecavir versus lamivudine for patients with HBeAg-negative chronic hepatitis B. N Engl J Med 2006;354:1011-20 Shouval D, Akarca US, Hatzis G, et al. Continued virologic and biochemical improvement through 96 weeks of entecavir treatment in HBeAg(-) chronic hepatitis B patients (Study ETV-027). J Hepatol 2006;44(Suppl 2):S21-2 Chang TT, Liaw Y-F, Wu SS, et al. Long-term entecavir therapy results in reversal fibrosis/cirrhosis and continued histologic improvement in chronic hepatitis B patients. Hepatology 2010;52:886-93 88% of the 57 patients with maintained undetectable serum HBV DNA under entecavir therapy, undergoing long-term liver biopsy, showed a > or =1-point improvement in the Ishak fibrosis score, including all 10 patients with advanced fibrosis or cirrhosis at baseline.

90.

Schiff E, Simsek H, Lee WM, et al. Efficacy and safety of entecavir in patients with chronic hepatitis B and advanced hepatic fibrosis or cirrhosis. Am J Gastroenterol 2008;103:2776-83

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Shim JH, Lee HC, Kim KM, et al. Efficacy of entecavir in treatment-naive patients with hepatitis B virus-related decompensated cirrhosis. J Hepatol 2010;52:176-82

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Liaw Y-F, Raptopoulou-Gigi M, Cheinquer H, et al. Efficacy and safety of entecavir versus adefovir in chronic hepatitis B patients with evidence of hepatic decompensation [abstract 422]. 60th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2009); October 30 -- November 1 2009, Boston

93.

Lange CM, Bojunga J, Hofmann WP, et al. Severe lactic acidosis during treatment of chronic hepatitis B with entecavir in patients with impaired liver function. Hepatology 2009;50:2001-6

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Van Bommel F, Wunsche T, Mauss S, et al. Comparison of adefovir and tenofovir in the treatment of lamivudine-resistant hepatitis B virus infection. Hepatology 2004;40:1421-5

95.

Heathcote E, Gane EJ, De Man RA, et al. Long term (4 Year) efficacy and safety of tenofovir disoproxil fumarate (TDF) treatment in HBeAg-Positive patients (HBeAg+) with chronic hepatitis B (Study 103): preliminary Analysis [abstract 477]. 61th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2010); Boston

96.

Lampertico P, Vigano M, Facchetti F, et al. Entecavir for NUC-naive chronic hepatitis B patients in clinical practice: long-term effectiveness from a large multicenter cohort study in 376 patients [abstract 220]. 60th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2009); October 30 -- November 1 2009, Boston Baqai SF, Hana Yi D, Gish RG. Profound virologic response in chronic hepatitis B (CHB) patients treated with entecavir [abstract 476]. 60th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2009); October 30 -- November 1 2009, Boston

97.

Marcellin P, Buti M, Krastev Z, et al. Continued efficacy and safety through 4 Years of tenofovir disoproxil fumarate (TDF) treatment in HBeAg-negative patients with chronic hepatitis B (Study 102): preliminary Analysis [abstract 476]. 61th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2010); Boston Gane EJ, Lee SS, Heathcote E, et al. Four years efficacy and safety of tenofovir disoproxil fumarate (TDF) in asians with HBeAg-positive and HBeAg-negative chronic hepatitis B (CHB), preliminary analysis [abstract 481]. 61th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2010); Boston

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99.

Buti M, Hadziyannis S, Mathurin P, and others. Two years safety and efficacy of tenofovir disoproxil fumarate (TDF) in patients with HBV-induced cirrhosis [abstract 21]. 44th Annual Meeting of the European Association for the Study of the Liver (EASL 2009); 22 -- 26 April 2009; Copenhagen, Denmark

100. Liaw YF, Sheen IS, Lee CM, et al. Tenofovir disoproxil fumarate (TDF), emtricitabine/TDF and entecavir in patients with decompensated chronic hepatitis B liver disease. Hepatology 2010: published online 27 October 2010, doi: 10.1002/hep.23952 101. van Bommel F, Zollner B, Sarrazin C. Tenofovir for patients with lamivudine-resistant hepatitis B virus (HBV) infection and high HBV DNA level during adefovir therapy. Hepatology 2006;44:318-25 102. van Bommel F, de Man RA, Wedemeyer H, et al. Long-term efficacy of tenofovir monotherapy for hepatitis B virus-monoinfected patients after failure of nucleoside/nucleotide analogues. Hepatology 2010;51:73-80 103. Berg T, Marcellin P, Zoulim F, et al. Tenofovir is effective alone or with emtricitabine in adefovir-treated patients with chronic hepatitis B infection. Gastroenterology 2010;139:1207-17 104. Brunell MN, Jacquard AC, Pichoud C, et al. Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir. Hepatology 2005;41:1391-8 105. Delaney WE, Yang H, Miller MD, et al. Combinations of adefovir with nucleoside analogs produce additive antiviral effects against hepatitis B virus in vitro. Antimicrob Agents Chemother 2004;48:3702-10 106. Liaw Y-F, Lee C-M, Akarca U, et al. Interim 48 week data of a phase 2, double-blind, randomized study comparing the safety of tenofovir disoproxil fumarate (TDF), emtricitabine

299

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plus TDF (Truvada, TVD) and entecavir (ETV) in subjects with decompensated chronic hepatitis B liver disease [abstract 122]. 60th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2009); October 30 -- November 1 2009; Boston 107. Keeffe EB, Zeuzem S, Koff RS, et al. Report of an international workshop: roadmap for management of patients receiving oral therapy for chronic hepatitis B. Clin Gastroenterol Hepatol 2007;5:890-7

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Affiliation

Mauro Vigano`1 & Pietro Lampertico†2 † Author for correspondence 1 Hepatology Unit, Ospedale San Giuseppe, Via San Vittore 12, Milan, Italy 2 “A. M. and A. Migliavacca” Center for Liver Disease, 1st Gastroenterology Unit, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Universita` degli Studi di Milano, Via F. Sforza 35, 20122 Milan, Italy Tel: +39 0255035432; Fax: +39 0250320410; E-mail: [email protected]

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Review

Interferon in the treatment of chronic hepatitis C: a drug caught between past and future 1.

Introduction

2.

Mechanism of action of

Elena Vezali†, Alessio Aghemo & Massimo Colombo †

interferons 3.

Interferons in treatment of hepatitis C -- current treatment

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options 4.

New IFN molecules

5.

IFN in the directly acting antivirals (DAA) era

6.

IFN in combination with therapeutic vaccines

7.

Expert opinion

A.M. Migliavacca Center for Liver Disease, First Division of Gastroenterology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Universita` degli studi di Milano, Milan, Italy; and Hygeia Diagnostic and Therapeutic Center of Athens, Athens, Greece

Introduction: Chronic hepatitis C affects 2.2 -- 3% of the world population (130 million -- 170 million). Pegylated IFN-a in combination with ribavirin, the currently approved therapy, leads to viral eradication in about 50% of treated patients, nevertheless it is associated with not negligible adverse events. Continuous efforts are being applied to improve the efficacy by means of new interferon molecules. Consensus IFN-a is currently available for use, while albumin-interferon, IFN-l and IFN-w are under investigation. Research is focussed on the directly acting antivirals, such as Telaprevir and Boceprevir, which are anticipated to enter the drug market during the next few years. Areas covered: This paper reviews the classification of interferons, mechanism of action, pegylated IFN-a molecules and their differences, current chronic hepatitis C treatment regimen and new drugs for HCV treatment. Expert opinion: Pegylated IFN-a will remain the basis of anti-HCV therapy during the next decade, since new IFN formulation are not proven to be superior or are in early stages of development and directly acting antivirals show limited antiviral activity as monotherapy. Keywords: albumin-interferon, consensus interferon, directly acting antivirals, hepatitis C, interferon-alfa, interferon-lambda, interferon-omega Expert Opin. Biol. Ther. (2011) 11(3):301-313

1.

Introduction

Chronic infection with HCV is a major health problem affecting 130 million -170 million people worldwide (2.2 -- 3% of the world population) [1]. Currently it is a principal cause of death from liver disease and the leading indication for liver transplantation in the USA [2], with some evidence that mortality related to HCV will continue to increase over the next two decades [3]. Infection is sustained by six virus genotypes (1 -- 6), varying in nucleotide sequence by 30 -- 50%, that are differently distributed in various regions of the world [4]. Genotypes 1, 2 and 3 are most common in the USA, Europe, Australia and East Asia. Genotype 4 is endemic in Central and South Africa, but it is becoming more prevalent in Europe and North America due to immigration, while genotypes 5 and 6 are of limited frequency, being found predominantly in South Africa and Southeast Asia, respectively. The prognosis of chronic HCV depends on fibrosis progression with the risk of cirrhosis ranging from 5 to 25% over periods of 25 to 30 years [5]. Persons with HCV-related cirrhosis are at risk of hepatic decompensation (30% over 10 years) as well as hepatocellular carcinoma (1 -- 8% per year) [6,7]. Viral eradication is the only therapeutic intervention that may halt the disease progression, reduce HCV-related mortality and improve the quality of life of infected patients [8,9]. The efforts to develop a therapy for chronic hepatitis C started in the mid-1980s, when interferon was empirically tested following the demonstration of its activity 10.1517/14712598.2011.552906 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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Article highlights. .

.

.

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.

IFN-a in combination with the nucleoside analogue ribavirin is the current standard of care for hepatitis C patients, but viral eradication is achieved in about 50% of patients only and therapy is associated with non-negligible adverse events. New interferon molecules, such as albumin-IFN-a2b, IFN-l, IFN-w are currently being investigated, while consensus interferon is available for non-responders to previous treatment. A triple therapy regimen based on the combination of PegIFN-a, ribavirin and a directly acting antiviral drug will soon be available, leading to improved rates of HCV clearance in genotype-1-infected patients. Future areas of research include long-term studies of pegylated IFN-a in children and in hard-to-treat patients, including those with HIV co-infection, non-responders and those with a previous treatment failure.

This box summarizes key points contained in the article.

against other hepatitis viruses [10]. Since that pioneering era, antiviral therapy for HCV infection has constantly evolved, with the current standard of care (SOC) for chronic hepatitis C being pegylated IFN-a (PegIFN-a) in combination with the guanosine analogue ribavirin [11]. Nevertheless, the rates of sustained virological response (SVR), defined as absence of detectable HCV RNA 6 months after treatment cessation, still do not top 50% for the more difficult to cure HCV genotype 1. IFN-a based therapy is also associated with many potentially serious side effects that ultimately reduce the treatment efficacy and acceptability by patients [12]. For this reason over the past decade intensive effort has been made to develop new agents for anti-HCV treatment, as well as alternative IFN preparations. This review highlights the most recent advances in new interferon formulations, concentrating on those that have already progressed to the clinical development stage. 2.

Mechanism of action of interferons

IFNs are natural glycoproteins, produced by the cells of immune system in response to infectious organisms and/or tumor cells, endowed with antiviral, antiproliferative, antitumor and immunomodulatory actions in various organs and cell lines. Based upon the type of receptor they interact with, three major classes of human IFNs are defined: types I, II and III (Table 1). Human type I IFNs is a family of innate genetically and structurally similar cytokines that include IFN-a, IFN-b and IFN-w. They bind to two receptor complexes, IFNAR-1 and IFNAR-2, both ubiquitously expressed [13], and are all secreted by leukocytes, except for IFN-b, which is also produced by fibroblasts. IFN-g represents the only type II 302

IFN and is produced exclusively by activated thymus-derived T cells and NK cells. It binds to a receptor consisting of two subunits: a-chain (IFNGR1) and b-chain (IFNGR2) [14]. Type III IFNs represent a novel class of IFNs first described in 2003 by two independent groups, that originally classified them as type II IFNs [15,16]. This type includes IFN-l1 (IL-29), IFN-l2 (IL-28A) and IFN-l3 (IL-28B) with biological activities similar to that of type I IFNs. IFN-l binds to a receptor composed of two chains, IFNLR-1 (specific for IFN-l) and IL-10R2 (shared with the IL-10, IL-22 and IL-26 receptors). IL-10R2 is ubiquitously expressed, whereas IFNLR-1 is expressed mainly in epithelial-derived cells and in B-lymphocytes [17,18]. A detailed description of the extremely complex mechanisms of action of IFN-a is beyond the scope of this review, but still a quick glance is important to understand the strengths and limits of the drug. Briefly, the subcutaneous administration of IFN-a triggers a cascade of intracellular reactions that activate numerous IFN-inducible genes [19], leading to the synthesis of IFN-induced proteins that are believed to inhibit virus multiplication within single cells. Among these is the RNA-dependent protein kinase (PKR), which inhibits translation initiation and 2¢,5¢-oligoadenylate synthetase (OAS) which in turn mediates RNA degradation [20,21]. Moreover, IFN-a also binds to immune cells triggering a complex and intricate pathway including class I MHC antigen expression, activation of effector cells and complex interactions with the cytokine cascade [22,23]. As part of its immunomodulatory activities, IFN-a also stimulates the production of TH1 cells while reducing the production of TH2 (suppressor) cells. The result of such a concerted action is the induction of a non-virus-specific antiviral state in infected cells, resulting in direct inhibition of viral replication whilst also enhancing the host’s specific antiviral immune responses through IFN-related immunomodulatory stimuli [24].

Interferons in treatment of hepatitis C -- current treatment options

3.

Efficacy Recombinant IFN-a at a dose 3 -- 6 MU trice weekly for 48 weeks was the first treatment regimen for chronic hepatitis C treatment, but only 10 -- 20% SVR rates could be achieved [25]. In fact, the efficacy of standard IFN-a was negatively affected by the pharmacokinetic properties of the recombinant formulations, such as rapid absorption with a peak in serum levels 7 -- 12 h after administration, a large tissue volume of distribution and rapid elimination through the kidneys. These properties ultimately led to wide fluctuations in serum concentration over a 1-week period, impairing viral replication suppression. Although the addition of the guanosine analogue ribavirin increased the SVR rate to 41 -- 43% [26,27] through mechanisms of action that are still poorly characterized, further improvement of the SVR rates 3.1

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Vezali, Aghemo & Colombo

Table 1. Characteristics of the main human interferons. Type I IFNs

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Gene Receptor Producing cells

Type II IFN

Type III IFNs

IFN-a

IFN-b

IFN-v

IFN-g

IFN-l (IL-29)

IFN-l2/3 (IL-28A/B)

IFNA IFNAR-1/2 Hematopoetic cells, mainly leukocytes

IFNB IFNAR-1/2 Fibroblasts and some epithelial cell types

IFNW IFNAR-1/2 Hematopoetic cells, mainly leukocytes

IFNG IFNGR-1/2 Immune cells, mainly T-cells and NK cells

IFNL IFNLR-1/IL-10R2 Hematopoetic cells, mainly monocytes

IFNL IFNLR-1/IL-10R2 Hematopoetic cells, mainly monocytes

was necessary to alter the pharmacokinetic properties of IFN-a. For this reason pegylated IFN-a (PegIFN-a) was developed and commercially registered in 2000 -- 2001. The process of modification of molecules by covalent conjugation with polyethylene glycol (PEG) [28], aimed to enhance the delivery of therapeutic molecules by modifying their pharmacokinetic and pharmacodynamic properties. PEG moieties are inert, nontoxic and water soluble, and can be manufactured in a range of size up to 5 -- 10 times larger than the original protein volume and £ 69 kDa in size. Pegylation reduces renal clearance of the active drug, permits its sustained absorption and results in long-lasting circulation in the plasma. Additional advantages of pegylation include decreased antigenicity, immunogenicity and proteolytic degradation of the active protein [29]. Ultimately two forms of PegIFN-a are available: PegIFN-a2a (Pegasys, Roche, Basel, Switzerland) and PegIFN-a2b (PegIntron, Merck & Co./Shering-Plough, Kenilworth, New Jersey, USA), from a purely pharmacokinetic point of view both compounds are significantly better than the standard IFN formulation [30]. However, the different choices of the two manufacturers in terms of size and nature of the attached PEG molecule resulted in different properties of two drugs (Table 2) [31]. Briefly, the branched 40 kDa PEG chain of PegIFN-a2a is covalently attached to lysine residues of IFN-a2a and circulates as an intact molecule. It has very restricted volume of distribution, longer half-life and reduced clearance, compared with standard IFN-a and can be administrated once weekly independently of body weight. PegIFN-a2b has a linear 12 kDa PEG chain covalently attached to histidine-34 of IFN-a2b through an unstable bond that is hydrolyzed after injection, releasing native IFN-a2b. PegIFN-a2b has a shorter half-life, compared with PegIFN-a2a, requires weight-adjusted dosing and is excreted by the kidney, while PegIFN-a2a is metabolized by both liver and kidney and does not require dose modification in patients with renal impairment. Differences between the two pegylated interferons

3.2

Although both PegIFNs-a combined with ribavirin proved to be more effective than standard IFN combined with ribavirin in prospective registration trials [32-34], the controversy regarding whether the two drugs were equally effective or if

one drug had an advantage over the other was partially solved only 10 years after their development. Indeed, three randomized head to head studies designed to compare the SVR rates achieved by the two PegIFN-a plus ribavirin showed PegIFN-a2a to achieve slightly higher SVR rates in two independent studies. In the Milan Safety Tolerability (MIST) study, SVR rates were 66% for PegIFN-a2a versus 54% for PegIFN-a2b (p = 0.02) while in the study by Ascione and colleagues, SVR rates were 69 versus 54%, respectively (p = 0.008) [35,36]. No significant difference emerged from the largest randomized controlled trial (RCT) in HCV genotype 1 patients, the Individualized Dosing Efficacy versus Flat Dosing to Assess Optimal Pegylated Interferon Therapy (IDEAL) trial. There was a 41% rate of SVR for PegIFN-a2a versus a 40% rate of SVR for standard dose PegIFN-a2b, both in combination with ribavirin (p = 0.57) [12]. A recent meta-analysis of 12 RCTs comparing the two PegIFN-a regimens confirmed PegIFN-a2a to be associated with a higher chance of an SVR versus PegIFN-a2b (47 versus 41%, risk ratio 1.11, 95% CI 1.04 -- 1.19; p = 0.004) [37]. The potential reasons that explain the different results in the three RCTs are various and have already been extensively analyzed [38-40] still it is important to note that the role of ribavirin is quite hard to assess in all three of them as a consequence of either a different initial dosing in the two PegIFN-a regimens or different dose reductions schemes. Given the crucial role of ribavirin in reducing viral breakthroughs and relapse rates especially in HCV-1 patients [41], it is clear this is no small confounder [12,42-44]. 3.3

Predictors of treatment response Pre-treatment

3.3.1

A number of host and viral factors have been associated with the chances of achieving an SVR to IFN-based therapies. The pivotal PegIFN + ribavirin studies have identified the HCV genotype and the pre-treatment viral load as major predictors of treatment success [11]. Indeed, the SVR rates are higher among patients infected with HCV genotypes 2 or 3 (60 -- 80%), compared to genotypes 1 or 4 (40 -- 50%), and in those with pre-treatment viral load below 600,000 IU/ml [11]. Other reported baseline characteristics associated with a favourable response include female gender,

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Table 2. Structural and paramcokinetics characteristics of standard and pegylated interferons. Parameter

IFN-a

PegIFN-a2a

PegIFN-a2b

Chemical parameters Molecular weight (kDa) Polyethylene glycol structure

19 --

40 Two 20 kDa chains, linked to form a branched 40 kDa chain

31 Linear

Pharmacokinetic parameter Absorbtion half-life, h* Time to Tmax, h Mean Cmax, µg/l Elimination half-life, h Volume of distribution, l Clearance, ml/h

2.3 5 -- 7 14 -- 30 6.5 -- 6.8 31 -- 73 6000

50 45 -- 78 14 -- 26 65 8 -- 12 60 -- 100

50 15 -20 -22 -31 -725

Posology and administration Route of administration Dosage

Subcutaneous 3 -- 6 MU t.i.w

Subcutaneous 180 µg qw

Subcutaneous 1.5 µg/kg qw

44 32 60 73

*Following subcutaneous dosing. IFN-a: Recombinant interferon a; MU: Million units; na: Non applicable; PegIFN-a: Pegylated interferon a; qw: Once weekly; t.i.w: Thrice weekly.

age less than 40 years, non-African-American race, body weight (< 75 kg), the absence of insulin resistance, elevated alanine aminotransferase levels, the absence of bridging fibrosis or cirrhosis on liver biopsy and a dose of ribavirin > 10.6 mg/kg [11]. Genome-wide association studies have shown that genetic variation of single nucleotide polymorphism (SNP) near the IL-28B gene (rs12979860) on chromosome 19, encoding IFN-l-3, predicts the virological response to combination treatment in HCV-1 patients [45,46]. Although the C/C genotype was found to be associated with higher chances of SVR in patients with genotype 1 HCV infection of all ancestries (European-American ancestry (Odds ratio (OR) 7.3, 95% CI 5.1 -- 10.4), African ancestry (OR 6.1, 95% CI 2.3 -- 15.9) and Hispanics (OR 5.6, 95% CI 1.4 -- 22.1), the different prevalence of this genotype among races (higher prevalence in Asian and Caucasian patients compared with African-American patients) partially explains the differences in SVR rates between these population groups [47]. The effect of rs12979860 polymorphism on treatment response was further validated in other studies [48] and for other genotypes, showing that C/C genotype is a strong predictor of SVR (OR 7.88, 95% CI 3.10 -- 20.05, p 1.48 10-5) [49]. Although the discoveries regarding IL28B can be generally considered an important breakthrough in the understanding of the influence of host factors on PegIFN-a + ribavirin efficacy, the usefulness of IL-28B determination in clinical practice is still somewhat obscure. The major drawback of IL-28B polymorphism determination is its low negative predictive value (29 -- 42%), thus the absence of favorable allele should not preclude treatment [50]. Moreover, achievement of a rapid virological response (RVR) after 4 weeks of treatment leads to high SVR rates even for the unfavorable TT genotype. Surprisingly its clinical role is easier to understand in 304

HCV-2 and HCV-3 patients where the SNP does not significantly influence SVR rates when looking at the entire treatment population, but if one focuses on those patients not achieving an RVR, IL-28B type is strongly associated with treatment outcome (SVR: 87% versus 67% versus 29% for C/C versus C/T versus T/T; OR, 4.0; 95% CI, 1.9 -- 8.5; p = 0.0002), suggesting that IL-28B polymorphism should be tested in this subgroup of patients to eventually decide on continuing or stopping treatment [51]. Additionally, the role of genetic testing in other settings, such as treatment of recurrent HCV infection after liver transplantation, HCV/HIV co-infection or in predicting treatment response to IFNs other than PegIFN-a remains to be determined. On-treatment Early on-treatment viral kinetics is also essential in the optimization of therapy duration or as an early stopping rule, thus limiting toxicity and reducing the economic cost. The absence of detectable HCV RNA in serum after week 4 of treatment is defined as RVR, while after week 12 is defined as early virological response (EVR). The RVR is predictive of SVR, independent of genotype or treatment regimen [52]. For genotypes 1 and 4 the positive predictive value (PPV) and the negative predictive value (NPV) of RVR for achieving SVR are 91 -- 95% and 50%, respectively. For genotypes 2 and 3 these numbers are 87 and 45%, respectively [51]. The utility of RVR is mainly in tailoring treatment duration as several trials proposed abbreviated treatment schemes for patients with an RVR. A total treatment duration of 12 -- 16 weeks has been shown to be highly effective for HCV genotypes 2 or 3, while 24 weeks for those infected with genotypes 1 or 4 with a low baseline HCV RNA level have been shown to achieve similar rates compared with the standard 48 week treatment schedule [53,54]. 3.3.2

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On the other hand, detectable HCV RNA after week 12 of therapy (absence of EVR) is a negative predictor of an SVR. Among genotype-1-infected patients 79 -- 100% of those who fail to achieve an EVR, also fail to clear the infection after 48 weeks of combination treatment [55]. HCV-1 subjects, clearing HCV RNA between week 12 and 24 of treatment (slow virological responders) can benefit from treatment extension from 48 to 72 weeks, although at the price of increased costs and side effects [56,57]. Whether extended treatment would increase SVR rates in patients achieving HCV RNA negativity between weeks 4 and 12 remains to be determined. Safety The effects of IFN are broad and not just limited to its antiviral activity; indeed IFN exerts its actions through receptors that are present in various tissues, resulting in systemic effects. Although some of these effects might be beneficial in other medical conditions such as melanoma or leukaemia, they ultimately can be considered to be side effects when PegIFN-abased therapies are aimed at HCV clearance. The most common adverse effects include fatigue (54%), headache (47%), pyrexia (43%), myalgia (42%), haematological abnormalities (2 -- 34%), rigors (24%) and psychiatric symptoms (22 -- 37%) [58]. Adverse effects require dose reduction or cessation of therapy in up to 32% [33-35] potentially resulting in suboptimal SVR rates. Hematological abnormalities, such as anaemia (13 -- 43%), neutropenia (2 -- 32%) and thrombocytopenia (6 -- 24%), are often seen in patients receiving PegIFN-a and ribavirin [59]. Anaemia is attributed mainly to ribavirin causing haemolysis, while neutropaenia and thrombocytopenia are considered to be the direct consequence of myelosuppression by IFN. The off-label use of haematological growth factors in the treatment of chronic hepatitis C has been introduced in recent years in order to improve adherence and compliance. Erythropoietic agents are effective in treating anaemia and preventing ribavirin dose reduction, and can improve patients’ quality of life, but a positive effect on SVR is not substantiated by studies to date [60]. Also, there is no clear cut evidence that indicating that neutropenia during PegIFN-a and ribavirin therapy correlates with infections [59]. For this reason even if granulocyte colony forming stimulation factor is an effective therapeutic option to elevate absolute neutrophil count, its use is still not universally accepted for managing this side effect during PegIFN and ribavirin therapy. Indeed, a PegIFN-dose-reduction strategy is still the standard of care measure to manage a decrease in neutrophil count during therapy. Regarding thrombocytopenia, eltrombopag, a new oral thrombopoietin mimetic, is currently under investigation. Although the final data from these studies have not been revealed yet, if proven effective this drug might radically affect the treatment of patients with HCV infection as it would allow treatment of patients with severe baseline thrombocytaemia

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3.4

at baseline as well as improving the management of IFN induced thrombocytaemia. Psychiatric symptoms (anxiety, insomnia, depression) are often the main cause of treatment discontinuation and represent at least one-third of the reported serious adverse events (SAE). For example in the IDEAL study 28% of all reported SAE were classified as psychiatric or related to the nervous system. Notwithstanding that severe depression or suicidal attempts very rarely occur during the treatment of chronic hepatitis C (less than 1% of the cases), expert management of psychiatric disorders is still warranted during IFN-a-based treatment [11,61]. Since most of these side effects are generally mild, treatment-related SAE are rather uncommon with the reported incidence in large RCTs raging from 3 to 4% [33-35]. While their incidence might be higher in clinical practice where patients are less selected and therefore more often affected by significant co-morbidities, this is not supported by scientific data as in real life studies such as MIST the incidence of SAE was 1% only. 4.

New IFN molecules

Since currently available IFNs are still imperfect in terms of effectiveness, tolerability and convenience, extensive research is being performed to develop more portent and/or better tolerated IFNs. Albumin-IFN-a Albumin-IFN-a2b (AlbIFN-a) is an 85.7 kDa molecule resulting from the genetic fusion of human albumin and IFN-a2b, which was designed to increase drug exposure and improve patients compliance through a less frequent dosing. A less frequent administration of the drug is the direct consequence of its prolonged half life, as Phase I/II studies in treatment-naive and IFN-experienced patients showed. AlbIFN-a has a half-life of approximately 6 days, twofold longer than PegIFN-a2a and fourfold longer than PegIFN-a2b, thereby supporting dosing at 2 -- 4-week intervals. AlbIFN-a induced the IFN-specific OAS1 gene, with a mechanism of action similar of that of PegIFN-a causing a rapid significant HCV RNA reduction during the first phase, followed by slower second-phase decline [62]. These studies showed that the effectiveness and tolerability of AlbIFN-a are dose-dependent providing a rationale for exploring 900 and 1200 µg dosing in future studies. Nevertheless, two recent parallel Phase III studies in genotype 1 (ACHIEVE 1) [63] and in genotype 2 or 3 (ACHIEVE 2/3) [64] failed to demonstrate better tolerability of Alb-IFNa, compared with the SOC (PegIFN-a plus ribavirin). In the ACHIEVE-1 study, 1331 patients were randomized to three open-label 48-week treatment groups: PegIFN-a2a 180 µg weekly, or AlbIFN-a 900 or 1200 µg every two weeks, with weight-based oral ribavirin 1000 -- 1200 mg/d. In the ACHIEVE-2/3 study, 933 patients 4.1

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were randomized to open-label treatment with PegIFN-a2a 180 µg weekly, or AlbIFN-a 900 or 1200 µg every two weeks for 24 weeks, each administered with ribavirin (800 mg/d). The primary end-point of noninferiority to PegIFNa2a was achieved: the SVR rates for PegIFN-a2a, AlbIFN-a 900 µg and AlbIFN-a 1200 µg being 51.0, 48.2, and 47.3% in the genotype 1 arm and 84.8, 79.8, and 80.0% in the genotype 2/3 arm, respectively. However, the expected improvement in terms of adverse events was not observed. Conversely, side effects were more common for AlbIFN-a than in PegIFNa2a groups in both studies: alopecia (43 -- 44% versus 25%), cough (38 -- 41% versus 29%), and weight loss (25 -- 28% versus 15%), whereas rash was less frequent with Alb-IFNa compared with PegIFN-a2a (14 versus 20%). On top of these findings during both studies, the Data Monitoring Committee recommended AlbIFN-a dose reduction from 1200 to 900 µg due to increased incidence of pulmonary adverse effects. In the ACHIEVE-1 study, there were more discontinuations due to adverse effects with 900 µg AlbIFN-a than with Peg-IFNa-2a (10 versus 4%), including two documented events of progressive interstitial lung disease, both in the 1200 µg AlbIFN-a group, one of which was fatal. The pulmonary complications, although known adverse events during PegIFN-a administration [12], raised safety concern in the USA and Europe to the point that European regulatory authorities requested additional data regarding the therapeutic benefit offered by AlbIFN-a dosed once every two weeks versus the relative risk of pulmonary complications. Since these data could not reasonably be generated within the timeframe allowed in the European Centralized Procedure, the applications for approval of the drug to both the US FDA and European Health Care Agency were withdrawn [65,66] IFN-lambda IFN-l1 is a type III IFN, also known as IL-29, which suppresses HCV replication in vitro through mechanisms similar to type I IFNs. Both IFN-l and IFN-a act through up-regulation of the MHC class I antigen expression while stimulating the JAK--signal transducer and activator of transcription (STAT) pathway that ultimately induces IFN-stimulated genes [67]. While exhibiting a slightly lower antiviral activity compared with IFN-a, IFN-l shows a more rapid activation of STAT and ultimately a delayed yet prolonged effect on the expression of various IFN stimulated genes; these differences coupled with the in vitro evidence that anti-HCV activity of either IFN type is enhanced by a low dose of the other, suggests that the two IFNs interact while also playing a complimentary role in the suppression of HCV [68]. Importantly, expression of IFN-l1 receptors is restricted to B-lymphocytes and epithelium-derived cells, including hepatocytes, providing the rationale for reduced adverse events, especially haematological toxicity, compared with IFN-a, which exerts its actions through widely distributed IFN-a tissue 4.2

306

receptors [69,70]. Pegylated IFN-l1(PegIFN-l1) developed as a recombinant form of human IFN-l1 conjugated with a 20-kDa linear PEG chain, has been evaluated in Phase Ib open-labelled dose-escalation study, in which PegIFN-l1 was given for 4 weeks in HCV genotype 1 treatment-naı¨ve and treatment-experienced patients, either as monotherapy or either in combination with ribavirin [71]. Antiviral activity was observed at all doses ranging from 0.5 to 3.0 µg/kg, given either every two weeks or weekly. Overall HCV RNA became undetectable after 4 weeks in 13% of patients, the best results being observed in patients receiving higher weekly doses of PegIFN-l1. Overall, the drug was well tolerated with most common adverse events being fatigue (29%), nausea (12%) and myalgia (11%), while haematological adverse were minimal following PegIFN-l1 monotherapy. These encouraging preliminary results have led to the conduct of a dose-finding, Phase II study in combination with ribavirin, from which final results are expected in 2012. Although PegIFN-l1 has been in development for some years, much of its scientific popularity derives from the recent discovery, by three independent genome-wide association studies, that single nucleotide polymorphisms located near the IL-28B encoding region on chromosome 19, are strongly associated with HCV patients’ response to PegIFN-a and ribavirin, as discussed previously. Since genome-wide association studies cannot be considered as hypothesis-driven research, these findings need to be validated clinically and biologically, but still IL-28B studies strongly suggest a primary role for the IL-28B product IFN-l3, in determining HCV clearance [72,73]. The connection between IL-28B and PegIFN-l1 is substantial, since IFN-l3 shares the same receptor and approximately 70% sequence identity with IFN-l1, suggesting a potential role for the IFN-l family in the treatment of chronic hepatitis C. IFN-omega Human IFN-w, like other IFNs, is a type of IFN that is secreted from cells in response to viral infection and exerts antiviral, anti-proliferative and immunomodulatory activities [74]. This type I IFN has 62% amino acid identity, binds to the same receptors and activates the similar pathway to IFN-a. Two recombinant IFN-w have been developed -- a non-glycosylated with similar antiviral potency to IFN-a [75], and a fully glycosylated form, which exhibits more potent antiviral activity in vitro compared either with IFN-a or the non-glycosylated IFN-w [76]. Most recent data on IFN-w are derived from a Phase II study, in which 102 genotype-1 patients were randomized to receive 25 µg IFN-w with or without ribavirin for 48 weeks, a 12 week SVR was achieved in 6% in the monotherapy group and in 36% in the combination group, with an adverse effect profile similar to that of PegIFN-a [77]. Nevertheless, since the publication of these results in abstract form, no other data have been reported. 4.3

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An obvious drawback of IFN-w, which can affect patients’ compliance and weakens its effectiveness, is the need of daily subcutaneous administration. To overcome this, a proprietary delivery system (Omega DUROS device) has been developed, that is implanted subcutaneously and provides continuous drug delivery for 48 weeks [78]. The results of a Phase Ib study, enrolling genotype-1 relapsers after a previous treatment with SOC, assessing the effectiveness and safety profile of Omega DUROS device, are currently awaited [79]. Consensus IFN-a IFN alphacon-1 or consensus IFN (CIFN) is a synthetic, recombinant type I interferon derived by aligning the most commonly observed amino acid in each position of several alpha interferon nonallelic subtypes to generate a consensus sequence [80]. CIFN initially was approved for treatment of chronic hepatitis C in 1997, before combination treatment with PegIFN-a and ribavirin became available. Currently the use of this drug is restricted to a subgroup of patients who have failed a previous course of treatment. The approval of CIFN for retreatment was based on the results of the Daily-Dose Consensus Interferon and Ribavirin: Efficacy of Combined Therapy (DIRECT) trial [81]. This Phase III, open-label, multicenter, United States-based registration study evaluating two daily dosages of CIFN (9 µg/d and 15 µg/d) plus ribavirin in 343 PegIFN-a plus ribavirin nonresponders showed SVR rates of 7 and 11% for the 9 and 15 µg/d arms, respectively. SVR rates were less (4.2%) in African American compared with Caucasian patients (11%) and in cirrhotic patients versus non-cirrhotic ones (4% only versus 8 -- 13%). The level of HCV-RNA reduction achieved during previous therapy with SOC was predictive of a response to re-treatment: 13.3% of patients receiving 9 µg CIFN per day and 30.0% of those receiving 15 µg per day with no or mild liver fibrosis who had a ‡ 2 log10 viral reduction following previous therapy, attained a SVR. The CIFN regimen is far from being ideal in terms of patients’ compliance too, since it needs daily subcutaneous administration. Efforts to develop a pegylated form of CIFN have been unsuccessful [82]. Thus CIFN may provide benefit only in highly selected, Caucasian non-cirrhotic patients, non-responders to a full course of PegIFN-a plus ribavirin, who had ‡ 2 log10 decrease of viral load during previous treatment. CIFN is approved in the USA, but not in Europe, where it is available in several countries under a named patient program only. A summary of new interferon molecules is presented in Table 3.

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4.4

IFN in the directly acting antivirals (DAA) era

5.

The important technical advances in cell culture systems and replication assays introduced in the last decade have finally

provided researchers and pharmaceutical companies with the tools to develop and study DAA drugs for the treatment of chronic hepatitis C. Indeed, most if not all steps of the HCV life cycle have been targeted for drug development [83,84] with the non-structural viral RNA-dependent RNA polymerase (NS5B) and the serine protease (NS3) with its cofactor (NS4A) being the most successful targets to date. The NS3/NS4A protease is required for viral replication, with its principal function being in post-translational processing [85]. Two NS3/4A protease inhibitors, telaprevir (Vertex Pharmaceuticals, Cambridge, MA, USA) and boceprevir (Merck & Co., Inc. Whitehouse Station, NJ, USA) are currently in the late stages of their Phase III programs, with an anticipated entry into the drug market in 2011 -- 2012. Although both telaprevir and boceprevir have a strong antiviral activity against HCV-1, causing a 4 log10 HCV RNA decline and 2 log10 HCV RNA decline following 14 days of monotherapy [86], a sustained antiviral response is negatively influenced by the rapid emergence of HCV-resistant strains in the NS3 domain, as these amino acid changes result in reduced or null antiviral activity of the drug [87,88] Since the antiviral effect induced by IFN is not weakend by these NS3-resistant strains, PegIFN-a will remain the backbone of both drugs as shown by the respective Phase II and Phase III studies. The Phase II studies of telaprevir and boceprevir have also shown quite clearly that ribavirin will still be essential with this first generation DAA, since it determines higher SVR rates, lower breakthrough and relapse rates both in treatment-naı¨ve and treatment-experienced patients [89-91]. In fact, a triple therapy with PegIFN-a plus ribavirin plus a DAA will be the new SOC for HCV-1 patients in 2012 with a practical 70 -- 75% SVR rates in naı¨ve patients, 60 -- 70% in previous relapsers and unfortunately only to 25 -- 40% in previous non responders to PegIFN-a plus ribavirin patients (Figure 1). Although these rates are significantly higher than what is currently attainable with PegIFN-a plus ribavirin therapy, 50, 25 and about 10% respectively, they will come at the price of more side effects like anaemia, rash and gastrointestinal side effects, that ultimately will result in more patients discontinuing treatment for these reasons. Not surprisingly attempts are being made to simplify the new therapeutic regimens not just by reducing the number of DAA pills or by making their assumption more user friendly [92], but also by improving their tolerability. Indeed, a proof of concept study in HCV genotype-1-infected patients has shown that IFN may become expandable in the near future, thus improving the side effect profile of DAA based therapies. In the Phase I double-blind study by Gane et al., 64 HCV-1 patients received the combination of HCV polymerase inhibitor RG7128 and NS3/4A protease inhibitor RG7227 for 14 days at various dosages without PegIFN-a or ribavirin. In the 1000 mg RG7128 and 900 mg RG7227 twice a day arms, 83% of the patients achieved HCV RNA negativity below the limit of quantification of < 42 IU/ml and 63% below the limit of

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Table 3. New interferon molecules. Consensus IFN

AlbIFN-a2b

IFN-l1

IFN-v

Study phase Route of administration Dosage

Licensed in USA Subcutaneous 15 µg daily*

Stopped after Phase III Subcutaneous 900 or 1200 µg q2w

I Subcutaneous 25 µg daily

Potential advantages over PegIFN-a

None

Longer half-life permitting every 2 or 4 week administration

Phase Ib completed Subcutaneous Under investigation. Range 0.5 -- 3.0 µg/kg qw or q2w Reduced adverse events

Improved efficacy, reduced adverse events

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*Dosage for combination with ribavirin therapy, as monotherapy (although not indicated anymore) the dosage is 9 or 15 µg every other day. AlbIFN: Albumin-interferon; PegIFN-a: Pegylated interferon a; qw: Once weekly; q2w: Every two weeks.

A.

TVR + PegIFN-α2a + Rbv PegIFN-α2a + Rbv Relapsers

Naive

Non responders

100 p = 0.004 80

p < 0.001

p < 0.001

75

76

p < 0.001 83

p < 0.001

p < 0.001

SVR (%)

69 60 46

44 38

40

29 20

20

24 9

5

0 Phase ll

Phase lll

Phase ll

Phase lll

Phase ll

Phase lll

BOC + PegIFN-α2a + Rbv

B.

PegIFN-α2a + Rbv Naive

Relapsers

100 p < 0.0001 80

p non available

75 66

66 SVR (%)

p < 0.0001

60 40

38

38 21

20 0 Phase ll

Phase lll

Phase lll

Figure 1. The sustained virological response rate on triple anti-HCV therapy in Phase II and Phase III studies. BOC: Boceprevir; PegIFN: Pegylated interferon; Rbv: Ribavirin; SVR: Sustained virological response; TVR: Telaprevir.

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detection of < 15 IU/ml at day 13. The median HCV RNA decline was 5.1 log10 IU/ml without the emergence of resistance [93]. Although these data are extremely promising, they should not yet be considered as a definite proof for future IFN-free regimens. Indeed viral suppression does not equal viral eradication, as the latter endpoint implies viral clearance from infected hepatocytes, a feature that is unlikely to be achieved by DAAs. Moreover, recent data from other DAA combination regimens given for 28 days have clearly shown that the addition of PegIFN and ribavirin to a protease and polymerase inhibitor, improve the RVR rates while reducing the risk of viral resistance in HCV-1 naı¨ve patients [94]. Given the importance of PegIFN for maximising the efficacy of the new DAAs, it seems likely that all pre-treatment factors associated with the chances of an SVR in the PegIFN and ribavirin era will still hold true in the DAA era. Early data showing that not only cirrhosis and high viral load will reduce the SVR rates to telaprevir, but also that the IL28B SNPs will still predict treatment outcome [95]. The possibility of IFN-free regimens is looking more likely for HCV-2 and 3 patients, that are known to achieve high SVR rates even with lower doses of both drugs [96]. Indeed clinical trials without PegIFN-a are already being designed, like the Phase II trial investigating the efficacy of Alisporivir, a cyclophillin A inhibitor with anti-HCV activity previously known as Deb025 [97]. Interestingly, this study assesses a combination of Alisporivir with ribavirin, administered for 24 weeks without PegIFN-a [98]. Another approach is to investigate the effectiveness of the herbal drug silibinin (Legalon Sil; Madaus, K€oln, Germany), a mixture of flavolignans extracted from the milk thistle [99]. The oral preparation of the drug has low bioavailability and low activity against HCV. Nevertheless, the preliminary results of a dose-finding study including 26 null-responders to the SOC (PegIFN-a plus ribavirin) revealed that a 7-day intravenous course of 5 -- 20 mg/kg daily silibinin led to a viral load decrease ranging from 0.55 to 3.02 log10 IU/ml. The addition PegIFN-a plus ribavirin to the ongoing silibinin therapy at day 8 resulted in a further decline in the viremia levels (log10 drop 3.69 or 4.85 in the 15 or 20 mg/kg group), resulting in six patients achieving a complete virologic response (undetectable HCV RNA) at day 15 [100]. Currently a non-randomized open-label dose comparison study of intravenous silibinin, followed by PegIFN-a2a plus ribavirin is ongoing [101] with preliminary results showing 28 of 63 (44%) patients having a HCV RNA under the limit of quantification at the end of silibinin treatment (day 15 or 22), 39.3% of them with undetectable HCV RNA at week 25 [102]. Although the exact mechanisms of action of silibinin are yet to be determined, Alisporivir has the peculiarity of acting through the inhibition of a host protein, cyclophillin A, that is implicated in HCV replication through its interaction

with the NS5B polymerase. Therefore it should have the theoretical advantage of having a higher genetic barrier that should result in lower rates of viral resistance, plus the resistant strains should be sensitive to NS3 protease inhibitors and in theory also to other NS5B polymerase inhibitors, effectively allowing for a rescue therapy in case of development of viral resistance. Although these concepts are interesting, the data on DAA are still preliminary and the role of PegIFN-a with these agents remains unknown. Although at the moment the future clinical landscape for HCV infection is still intricate and at times hardly foreseeable, it is possible that IFN will not remain the backbone therapy for the third generation of DAA.

IFN in combination with therapeutic vaccines

6.

Other novel immune-based strategies, such as combination of SOC with therapeutic vaccines, are currently under development. The immunotherapeutic vaccine GI-5005 consists of recombinant Saccharomyces cerevisiae yeast expressing an HCV NS3-core fusion protein designed to elicit antigenspecific host CD4+ and CD8+ T-cell responses. GI-5005 has demonstrated immunogenicity in preclinical in vitro and in vivo models, displayed anti-HCV efficacy and was well tolerated as monotherapy in a Phase Ib clinical trial. The Phase II trial that evaluated a triple therapy of GI-5005 in combination with PegIFNa and ribavirin is ongoing with preliminary results showing improved early virological responses [103]. Several additional vaccines are in early Phase (I and II) development including DNA vaccines, T cell vaccines, dendritic cell immunotherapy and more traditional peptide-based strategies. 7.

Expert opinion

After 25 years since its first use in the treatment of chronic hepatitis C, recombinant IFN-a is still an essential part of the SOC. Given the enormous advances made in understanding the HCV life cycle and the mechanisms of action that underlie the antiviral activity of IFN-a, it is fascinating but also somewhat disappointing that no further progress has been made in the development of new IFNs. Indeed pegylation of the old recombinant IFN-a molecule can be considered only a minor breakthrough as it just provided clinicians and patients with a slightly more effective drug. Things are not much brighter if one looks at the other IFN molecules in development, to date neither CIFN, Alb-IFN, PegIFN-l1 or IFN-w have actually shown any evidence that they can either improve efficacy or reduce side effects compared with PegIFN-a. Although Alb-IFN might have at least reduced the need for weekly injections in our patients, for safety and commercial reasons it is highly unlikely, if not impossible, that this drug will

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Interferon in the treatment of chronic hepatitis C: a drug caught between past and future

actually ever become available. PegIFN-l1 does seem to be a promising drug, but it is still in the early phases of development and ultimately might become available in an era where DAA with potent anti-HCV activity and high genetic barriers to resistance, might allow us to spare our patients from the side effects of IFN. Still what we know for certain is that PegIFN-a will remain the backbone of Telaprevirand Boceprevir-based therapies once they become available for HCV-1 patients. Hopefully, when the second or third generation of DAAs enter the market, IFN will be finally able to enjoy its golden retirement after 30 years of (almost) impeccable service.

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E Vezali has received a research/educational grant from the Hellenic Society for the Study of the Liver. She has also received travel support from Bristol-Myers Squibb, Roche and Shering-Plough. A Aghemo is on the Advisory committee for Roche, and has received travel support from BristolMyers Squibb, Roche and Glaxo-Smith-Kline. M Colombo has received grant and research support from Shering- Plough, Roche, Novartis and Bristol-Myers Squibb. He is also on the Advisory Committees for Shering-Plough, Roche, Novarits, Vertex, Gilead Sciences, Bristol-Myers Squibb and Bayer.

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Affiliation

Elena Vezali†1,2, Alessio Aghemo1 & Massimo Colombo1 † Author for correspondence 1 A.M. Migliavacca Center for Liver Disease, First Division of Gastroenterology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Universita` degli studi di Milano, Via F. Sforza 35, 20122 Milan, Italy Tel: +39 0255 035432; Fax: +39 0250320410; E-mail: [email protected] 2 Hygeia Diagnostic and Therapeutic Center of Athens, Athens, Greece

313

Review

AAV-mediated gene therapy for liver diseases: the prime candidate for clinical application? 1.

Introduction

2.

Essential properties of AAV

Luc JW van der Laan, Yigang Wang, Hugo W Tilanus, Harry LA Janssen & Qiuwei Pan† Erasmus MC-University Medical Center, Department of Gastroenterology & Hepatology, Rotterdam, The Netherlands

vectors 3.

Clinical development

4.

Liver-directed vector delivery

5.

Candidate diseases for hepatic

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gene therapy 6.

Immunological barriers

7.

Conclusions

8.

Expert opinion

Introduction: Diseases of the liver represent a major health problem. Often treatments are ineffective, prompting the need for new therapeutic strategies. From extensive preclinical studies, gene therapy in particular mediated by adeno-associated virus (AAV)-derived vectors, has now emerged as the prime candidate for clinical application. AAV-mediated gene therapy for inherited liver diseases has now become a clinical reality, in particular for the treatment of hemophilia B. Areas covered: This review provides a summary of current literature on AAVmediated gene therapies for both inherited and acquired liver diseases and outlines different strategies to overcome current clinical limitations. The unique properties of AAV over other viral vectors are highlighted as well as the current challenges which are faced for wide-ranging clinical application. Expert opinion: Despite the extensive positive results from animal models, successful application in clinical settings is hampered by immunological barriers. However, immune suppression and other strategies can be employed to overcome these limitations. Given some of their unique advantages, AAV vectors are currently the most obvious candidate for hepatic gene therapy applications, however, serotype-related issues of immune reactivity still represent a formidable barrier for clinical success. Keywords: AAV, adeno-associated virus, hemophilia, hepatic delivery, hepatic gene therapy, RNAi, serotypes, viral vector Expert Opin. Biol. Ther. (2011) 11(3):315-327

1.

Introduction

The liver is a solid organ performing essential functions in metabolism, detoxification and production of plasma proteins. A variety of factors have been reported to cause liver diseases, including inherited genetic disorders, alcohol abuse, druginduced toxicity, viral hepatitis infection, malignancy, blood or bile flow abnormalities. There are also many pediatric liver diseases including alpha-1 antitrypsin (AAT) deficiency, biliary atresia, alagille syndrome, progressive familial intrahepatic cholestasis and Langerhans cell histiocytosis. Unfortunately, very limited therapies are available to prevent or cure most of these diseases. Currently, liver transplantation is the only option for end-stage liver disease. Gene therapy represents an attractive alternative treatment option, not only for correction of genetic liver diseases but also for systemic delivery of therapeutic proteins. The success of gene therapy relies on the effective delivery of genetic material (DNA or RNA) into cells of the target organ. In contrast to non-viral vehicles, viruses possess unparalleled predominance because of their natural tropisms to host cells, thereby ensuring efficient and safe gene delivery across cell membranes. The majority of currently-used viral vectors are derived from engineered human 10.1517/14712598.2011.548799 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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AAV-mediated hepatic gene therapy

Article highlights. . .

.

.

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.

.

Liver diseases represent a major health problem and often treatments are ineffective. Extensive preclinical studies have demonstrated the potential of adeno-associated virus (AAV) vectors for gene therapy of various liver diseases. AAV-mediated gene therapy has moved into clinical use and is being tested in trials for specific inherited liver diseases. Immune reactivity against the AAV vector or cargo appears to be a major obstacle to long-term clinical success. Many strategies are being explored to overcome these immunological barriers, which include serotype substitution, immunosuppression, liver-targeted delivery and transgene expression. AAV vectors are also suitable for delivery of RNA interference and microRNA-based therapies.

This box summarizes key points contained in the article.

or primate animal viruses, including adenoviruses (Ad), adeno-associated viruses (AAV), herpes simplex viruses (HSV), baculoviruses, Epstein--Barr viruses, lentiviruses and other retroviruses. Given some of their unique properties, AAV vectors are currently considered the prime candidate vector for clinical gene therapy applications, including for the treatment of various liver diseases. In this review, we provide a background for the use of AAV in liver diseases and an update on progress made in recent pre-clinical and clinical studies. Current limitations hampering wide-scale clinical applications are discussed. 2.

Essential properties of AAV vectors

2.1

Biological features

AAV is a member of the Parvovirus family with a small single-stranded DNA genome of about 4,700 base pairs. AAV was originally described as a contaminant of Ad preparations which gave the virus its name [1]. The genome consists of two 145 nucleotide long inverted terminal repeats that can form hairpin structures. This secondary structure provides a 3¢ hydroxyl group for the initiation of viral DNA synthesis of double-stranded replication intermediates [2]. Although wild-type AAV has been shown to be able to integrate into the host genome, gene delivery by recombinant AAV vectors often results in episomal persistence [3]. A distinct merit of AAVs is that both wild-type and engineered vectors have a wide host spectrum with high infectivity for both nondividing and dividing cells and can achieve persistence in transgene expression without pathogenicity. So far, a number of serotypes of AAVs have been identified and engineered into vectors. By the definition of serotyping, AAV1 -- 5 and AAV7 -- 9 can be defined as true serotypes. The serology of AAV6 is almost identical to that of AAV1 and serological profiles of AAV10 and AAV11 are not well characterized. In 316

addition there are over 100 new isolates, termed AAV variants due to the lack of serological profiling [4]. AAV2 was the first to beused as a vector for gene delivery and was tested in several in vitro and in vivo models as well as in a series of gene therapy trials [5]. AAV8, a new member of the AAV family isolated from rhesus monkeys, is less prone to recognition by prevailing antibodies in humans [6]. It is an attractive candidate for hepatic gene transfer because of a 10- to 100-fold increased transduction efficiency in mouse liver models, compared with the AAV2-based vectors [7]. Self-complementary AAV vectors As a single-stranded DNA genome of AAV, secondstrand DNA synthesis and/or strand annealing are required to generate a double-stranded DNA template for transcription [8]. This process is a rate-limiting step in single-stranded (ss) AAVmediated transgene expression [9,10]. Self-complementary (sc) AAV vectors were invented to bypass this rate-limiting conversion step, dramatically improving the speed and efficiency of transduction. scAAV vectors were generated by packaging both coding and complementary, non-coding strands as a single molecule. Using scAAV, a significant reduction of the amount of vector required for sustained transgene expression in mouse liver was achieved [11]. One apparent downside to scAAV is the approximately 50% reduction in the cargo capability, which results a maximum 2,000 base pair transgene size. Still, this is barely sufficient for many of the protein-coding genes driven by a simple promoter. However, this does not matter at all in the context of delivery of small RNA expression cassettes and appears to be suitable for RNA interference (RNAi)- and microRNA (miRNA)-based applications [12]. 2.2

Scalable vector production Production of scalable vector with compatibilitywith good manufacturing practice (GMP) is a crucial step towards clinical application of AAV-mediated gene therapy [13]. As a result of extensive research efforts, several vector production systems have been established [14]. These include the transient transfection method [15,16], Ad-helper virus infection-dependent AAVproducer cell lines [17], the stable AAV rep/cap cell line/ Ad--AAV hybrid infection method [18], the recombinant HSV-helper infection-based AAV production method [19], and the recombinant baculovirus/insect-cell-based production system [20-22]. Transient transfection of multiple plasmids encoding the requisite genes for vector biosynthesis into cell lines, such as naive 293 cells, is the most commonly used packaging system. It provides great flexibility and speed, but is unable to be scaled up. Therefore, it is suitable for the early stages of investigational product development [14,23]. In contrast, the generation of stable transfected-cell-line-based methods is more complex and time-consuming, but ultimately provides better scalability and cost-effectiveness [14]. As pointed out by Wright [13], the development of an AAV gene therapeutic 2.3

Expert Opin. Biol. Ther. (2011) 11(3)

van der Laan, Wang, Tilanus, Janssen & Pan

candidate from laboratory to clinical use, to eventually be licensed and commercialized as a new drug, ideally requires parallel vector production systems initially providing speed and flexibility, but eventually providing optimal quality, scalability and cost-effectiveness.

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3.

Clinical development

Since the first successful clinical trial started in 1990 for the treatment of a four-year-old girl suffering from severe combined immunodeficiency (SCID) [24], a genetic disease caused by mutation in adenosine deaminase gene in T cells, gene therapy has gained particular attention worldwide. However, a tragic setback occurred during the end of the last century and the beginning of this century. It started with a tragic victim, an 18 years old boy undergoing gene therapy of congenital Ornithine Carbamoyltransferase deficiency in the USA in 1999 [25], followed by a French patient having developed leukemia with gene therapy for SCID, leaving the field in turmoil [26]. After this setback, clinical gene therapy was temporary halted, but now many hundreds of clinical studies have been performed which have slowly but surely given credibility to safety and efficacy of newer gene therapy vectors. The field of gene therapy has now become a clinical reality, although still a very young one: still full of conceptual and technical challenges [27,28]. The three recently-reported clinical studies of AAVmediated gene therapy for inherited retinal diseases are a landmark for the development of ocular gene therapy. Subretinal injections of AAV carrying a normal retinal pigment epithelium, 65 kDa (RPE65) gene have been found to be safe and led to moderate improvement of retinal function in seven of nine patients [29-31]. A follow-up study [32] by Maguire et al. in which they treated both child and adult patients with higher doses of AAV-RPE65 showed sustained improvement of light sensitivity in all 12 partially blind patients. Most importantly, the greatest improvement was found in children, all of whom gained ambulatory vision. These encouraging results paved the way for wide clinical application of AAV vectors, including for gene therapy of liver diseases. Up to summer 2010, 75 gene therapy trials using AAV vectors have been registered at The Journal of Gene Medicine Clinical Trial site [33]. These trials were approved for treating various diseases, including cancer, monogenic diseases, neurological diseases, ocular diseases, cardiovascular diseases and infectious diseases (Figure 1). There is a single liver-directed Phase I trial in patients with severe hemophilia B using AAV to deliver the normal human Factor IX (FIX) gene. This study achieved only transient therapeutic gene expression, probably due to the preexisting immunity to the AAV2 serotype [34]. Two additional trials of liverdirected AAV gene therapy for hemophilia B are currently recruiting participants [35], and will be further discussed in the following section.

4.

Liver-directed vector delivery

The endothelium of hepatic sinusoids displays fenestrations that allow macromolecules and viral particles to cross the vascular barrier and directly encounter hepatocytes [36,37]. Hepatic artery and portal vein are the best studied routes for intra-hepatic delivery of vectors. This local delivery, in theory, reduces the amount of vector required as compared with systemic vector administration [38]. In rodent models, hydrodynamic injection has been shown to be an efficient means of targeting the liver but induces considerable tissue injury [39]. A minimally invasive method to mimic hydrodynamic injection was reported to achieve preferential hepatic delivery of viral vector by using balloon occlusion catheters, which could be applied in larger animals and has potential for clinical application [40]. Since the first gene therapy trial, treating patients with Familial hypercholesterolemia (FH) by retrovirus ex vivo engineering hepatocytes [41], this approach has gained great interest owing to the rapid development of integrating lentiviral vectors and stem cell therapy. Although AAV is not ideal for ex vivo tranducing hepatocytes/stem cells because of its nonintegrating property, it is applicable for ex vivo delivery to the whole liver graft. Early studies have demonstrated that ex vivo perfusion of cold-preserved rat liver graft with Ad vectors in the organ preservation solution, resulted in uptake and expression of transgenes and induction of transplant tolerance [42,43]. It is a particular attractive approach in the context of liver transplantation that donor grafts are often ex vivo perfused and stored for transportation for a few hours before transplantation into a receipt. This setting could represent a unique opportunity for delivery of AAV vectors expressing therapeutic genes or small RNAs, for treatment of ischemic injury, to induce tolerance or to prevent recurrent diseases as such hepatitis C virus (HCV) reinfection [12,44].

Candidate diseases for hepatic gene therapy

5.

5.1

Inherited diseases Hemophilia

5.1.1

Hemophilia is a recessive X-linked disorder that affects blood clotting and is characterized by potentially life threatening bleeding episodes in soft tissues, joints or muscles. There are two main types of hemophilia, hemophilia A and B, caused by missing or low levels of clotting factors VIII (FVIII) and FIX, respectively. Given the inherited feature of hemophilia A and B, both diseases are theoretically curable by gene therapy. Several approaches have been reported to achieve muscle or liverdirected gene transfer of FVIII or FIX using different types of vectors, including Ad vectors, AAV, retroviral and lentiviral vectors [45-47]. Following the excellent results obtained in animal models of hemophilia [48], several Phase I/II studies of somatic-cell gene therapy were conducted in patients with

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Others, 4 Infectious diseases, 2

Monogenic diseases, 29

Phase III, 8

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Phase I/II, 11

Trials approved/initiated by year

C. 12 10 Trial records

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Cancer, 17

16

Figure 1. Clinical trials of AAV-mediated gene therapy registered at the Journal of Gene Medicine Clinical Trial site [33]. A. In total, there are 75 registered AAV trials, including 29 for monogenic diseases, 17 for cancers, 6 for ocular diseases, 3 for cardiovascular diseases, 2 for infectious diseases and 4 for other indications. B. 45 trials are at Phase I, 11 at Phase I/II, 11 at Phase II and 8 at Phase III. C. Distribution of trials according to the year of approval/initiation. Two trials lacking the information regarding the year they were approved/initiated and two from 2010 are not listed in the graph.

FVIII or FIX deficiency. So far these studies resulted in a measurable levels of FVIII or FIX, however, they only resulted in modest and transient effects on blood clotting parameters [49-51]. Since both FVIII and FIX are primarily produced in the liver, liver-directed gene delivery using AAV vector is considered a favorable option. Indeed this has been demonstrated in various animal models including nonhuman primates [52-59]. Unexpectedly, in the first clinical trial of AAV2-mediated hepatic delivery of FIX in hemophilia B patients, transgene expression was detectable but only short lived, persisting for approximately 4 weeks and then gradually declining 4 -- 6 weeks after infusion. Evidence was found that this decline was due to the reactions of the host immune system to the viral vectors [34] (further discussed in Section 6). New clinical trials with optimized protocols are now underway. A Phase I/II dose escalation trial of liver-directed scAAV8 gene transfer is currently recruiting for treatment of hemophilia B, sponsored by the St. Jude Children’s Research Hospital. Meanwhile, another trial, sponsored by The Children’s Hospital of Philadelphia, is recruiting hemophilia B patients and using AAV2 vector, which will be delivered via hepatic artery and in combination of transient immunosuppression [60]. 318

Familial hypercholesterolemia FH is a hereditary disorder of lipoprotein metabolism, characterized by extreme elevations of total cholesterol and low density lipoprotein (LDL), accelerated atherosclerosis and premature death from myocardial infarction [61]. It is caused by a defect in the LDL receptor gene resulting in dysfunction of the receptor on the cell surface. Although LDL receptors are expressed ubiquitously, the hepatic LDL receptor has the greatest effect on controlling plasma LDL levels. As FH homozygous patients are highly resistant to the current drug therapy, gene therapy appears to be an ideal alternative and a straightforward strategy by delivery of a normal copy of LDL receptor gene to hepatocytes. Homozygous FH was among the earliest diseases tested for clinical gene therapy. The trial reported in the mid 1990s used an ex vivo approach by transducing hepatocytes with retroviral vector expressing normal LDL receptor. The genetically modified cells were then transplanted into the patient’s liver. It was safe and transgene expression persisted for months after gene therapy. However, the patients experienced only a suboptimal decrease in total and LDL cholesterol, which could be explained by the limited number of cells engrafted and their poor repopulation capability after engraftment [41,62]. 5.1.2

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A more extensively investigated approach is, rather than cells, direct delivery of vector to the liver. Different vectors, such as first-generation Ad, helper-dependent Ad, lentiviral and AAV vectors, have been tested in mouse or rabbit models of FH [63-66]. In LDL receptor knockout mice, AAV-mediated expression of LDL receptor driven by a ubiquitous promoter only resulted in transient reduction of plasma cholesterol due to immune reactivity against the transgene [64]. Despite this general problem of immune responses against allogenic proteins, AAV remains the best candidate vector for hepatic gene therapy of FH [64,67]. Other inherited disorders Crigler--Najjar disease is caused by mutations in the UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) gene, which encodes the enzyme required for the conjugation of bilirubin. The enzyme is primarily expressed in the endoplasmic reticulum of hepatocytes. Defects in this enzyme lead to jaundice (yellow discoloration of skin and eyes) and a high risk of brain damage. In an animal model for Crigler-Najjar, the UGT1A1-deficient Gunn rat, liverdirected delivery of the UGT1A1 gene using different viral and nonviral vectors has been shown to be effective in normalizing serum bilirubin [68-72]. Based on the promising results in animal models and clinical experience of using AAV vector, now the first clinical trials of AAV-mediated gene therapy for Crigler--Najjar are in preparation (Dr P. Bosma, University of Amsterdam, pers. commun.). Other indications, such as ornithine carbamoyltransferase deficiency, Wilson’s disease and AAT deficiency, could also fall under the category of AAV-directed hepatic gene therapy.

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5.1.3

5.2

Viral hepatitis Chronic hepatitis B

5.2.1

Chronic HBV infection occurs in approximately 6% of the world’s population. The wide application of vaccination accounted for the landmark of controlling the infection, the already infected individuals however are only partially responsive to the current therapy, IFN-a or nucleotide analogues, and at high risk of developing cirrhosis and hepatocellular carcinoma (HCC). Among the new technologies, RNA interference (RNAi) is one of the most promising avenues currently being explored as novel anti-viral stratagies. RNAi is a sequence-specific inhibition of gene expression at posttranscriptional level. It is triggered by siRNAs of 21 nucleotides, which can be introduced into cells directly as synthetic siRNAs or indirectly as precursor long double-stranded RNAs or short-hairpin RNAs (shRNAs). These siRNAs are assembled into a multicomponent complex, known as the RNA-induced silencing complex, which unwinds the siRNA and uses its single-stranded form as a guide to target homologous mRNA sequences, resulting in translational repression and mRNA degradation [73,74]. The functional process of exogenous RNAi delivery shares the endogenous machinery of microRNA (miRNA) biogenesis.

Mature miRNAs are approximately 22 nucleotide non-coding RNAs that can downregulate gene products by inducing either cleavage or a reduction in the translational efficiency of the target mRNA [75]. Therefore, structural mimics of miRNAs are designed as a second-generation of shRNA to induce RNAi. Indeed, potent and specific gene silencing technology has greatly fuelled the study of biology of viral infections and led to the discovery of new antiviral targets for therapy [73,74]. The HBV genome is a partially double-stranded DNA of approximately 3200 bases in length with four open reading frames that encode viral proteins. RNAi targeted to all four major HBV transcripts has shown to limit viral replication in vitro and in vivo [76-78]. Several studies have demonstrated the feasibility of using AAV vectors to deliver antiHBV RNAi [79-82]. Hepatotropic AAV8-mediated delivery of anti-HBV shRNA into the liver of the transgenic mice resulted in potent and persistent inhibition of viral replication. The suppression declined after the first 21 days due to the host immune response but persisted for up to 120 days [79]. Notably, AAV-mediated expression of shRNA was shown to evoke liver toxicity in mice [83]. This toxicity was due to the saturation of endogenous miRNA processing machinery by overexpressed shRNA [84]. However, it was suggested that AAV expressing shRNA driven by a liverspecific promoter represented a much safer and more effective approach for anti-HBV RNAi delivery [82]. Chronic hepatitis C HCV attacks approximately 170 million people with 3 to 4 million de novo infections each year. There is no effective vaccine or antibodies available for prevention of the infection. A range of 55 -- 85% of those infected individuals fail to clear the virus and progress to a chronic infection state which is associated with a high risk of developing cirrhosis, liver failure and HCC [85]. The current standard therapy for HCV, pegylated IFN-a in combination with ribavirin, has achieved substantial success however with overall half of the patients developing a sustained virological response [86]. Liver transplantation is the only treatment for the patients who eventually developed end-stage liver disease or HCC. However, the course of re-infection is more aggravated after transplantation and often resistant to the antiviral therapy [87]. Gene therapy, in particular smallRNA-based technologies, is one of the most promising avenues currently being explored as novel anti-HCV therapy [88]. The hepatic-abundant miRNA, miR-122, has been shown to be a crucial positive regulator of HCV replication [89]. Recently a so-called locked nucleic acid (LNA) antisense probe has been developed to effectively antagonize the function of miR-122 [90]. Treatment of chronically infected chimpanzees with a LNA-anti-miR-122 probe led to long-term suppression of HCV viremia without evidence of viral resistance or toxicity [91]. Hopefully, the upcoming clinical trials will soon be revealing the safety and efficacy in HCV patients. 5.2.2

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AAV-mediated hepatic gene therapy

Since the HCV genome is a single-stranded RNA that functions as both a template for transcription and a template for a negative strand RNA replication intermediate, it is a prime target for RNAi. Numerous studies have demonstrated the feasibility of targeting different regions of the viral genome by RNAi [92-95]. However, due to the lack of proofreading abilities of the NS5B RNA-dependent RNA-polymerase, HCV has a high mutation rate, resulting in a high risk of resistance development under the pressure of genome-targeted RNAi silencing [96-98]. Host cellular factors involved in the HCV life cycle, such as entry receptors CD81, SR-B1, claudin-1 and occludin, represent another group of targets, which are thought to be less prone to mutation [99]. However, RNAi silencing of host factors probably disturbs the cellular functions of the genes and does not prevent compensatory mutations by the virus to escape treatment. A combinatorial strategy of simultaneously targeting both viral and host factors by multiple shRNAs was proposed to be more effective to prevent mutational escape and suppress virus infection [99,100]. Few studies have reported the application of AAV for delivery of RNAi in the context of HCV. The biotech company, Tacere therapeutics, has pioneered the development of an AAV-based anti-HCV RNAi regimen, termed ‘TT-033’. It contains three shRNA cassettes with one targeting the internal ribosome entry sites (IRES) and two targeting NS5b regions and these targets are conserved across multiple viral genotypes. TT-033 was shown to inhibit three separate regions of the HCV genome simultaneously in mice for more than two months after a single administration [101]. Clinical trials are the next step to evaluate its safety and efficacy in HCV patients. Hepatic malignancies Hepatic malignancies consist of primary and secondary liver cancers. Human primary liver cancer, HCC, accounts for the most common primary liver tumors in adults and colorectal liver metastases is the most common secondary liver tumor. Surgical resection or liver transplantation are applied as potentially curative treatments. However, only a highly selected patient population can benefit from these therapies [102]. Gene therapy of liver cancer covers a variety of strategies, including gene directed enzyme/prodrug therapy, inhibition of oncogenes and restoration of tumorsuppressor genes, immunotherapy, anti-angiogenesis and virotherapy [103]. In particular, RNAi-based approaches [104,105] and miRNA-targeted strategies [106,107] have been recently explored as new anti-cancer avenues. Some of these strategies have reached early clinical development with limited success [108-110]. More importantly, gene therapy could be considered as a potential adjuvant of other therapies and a number of preclinical [111-113] and clinical studies [114-116] have provided the proof of principle. 5.3

320

From the early excitement of gene therapy in experimental liver cancer models, researchers in this field soon realized its limitations and are now systematically addressing fundamental issues to solve them. Despite 17 trials using AAV vector for gene therapy of cancer registered at The Journal of Gene Medicine Clinical Trial site [33], none of these target liver cancer. Efficient transfer of genes/small RNAs to the majority of cancer cells is still unrealistic for solid tumors, even with the best vectors available to date [103]. 6.

Immunological barriers

Immunity to hepatic gene transfer: a continued challenge?

6.1

The liver is located in the peritoneal cavity and receives a blood supply not only from the hepatic artery, but also the venous drainage of the intestines, pancreas and spleen via the portal vein. Approximately 30% of the total blood volume of the human body passes through a network of hepatic sinusoids every minute. The liver is comprised of parenchymal cells, the hepatocytes, and a variety of non-parenchymal cell types, like biliary epithelial cells (cholangiocytes), sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells [117]. Various types of leukocytes cells are enriched in the liver, like NK cells, NKT cells, T and B lymphocytes, which all have distinct phenotype and functions from their counterparts residing in other organs [118-120]. The presence of suppressive cytokines and intrahepatic lymphocytes with regulatory activity, such as CD4+CD25+FoxP3+ regulatory T cells, may contribute to the known immunological tolerance assocated with the liver [121]. The tolerogenic property of liver has been demonstrated, most convincingly, in the setting of allogenic organ transplantation, showing spontaneous acceptance of liver grafts in many species without immunosuppression [122], as well as inducing acceptance of other co-transplanted organs [123]. The nonpathogenic nature of AAV does not present itself as a danger signal to the host. It therefore causes only a minimum level of vector-related toxicity and immune responses in AAVbased gene transfers [124]. In contrast to most other viruses and vectors, AAV vectors do not appear to engage pattern recognition receptors such as Toll-like receptors [125] and induce type I IFN responses [124]. The inability of AAV vectors to efficiently transduce or activate antigen-presenting cells probably contributes to their decreased immunogenicity [126]. Several studies have demonstrated induction of immune tolerance to hepatocyte-derived transgenic proteins, which possibly involves the induction of regulatory T cells [127-129]. Despite the long-term transgene expression in experimental animals, in the first trial of AAV2-mediated hepatic delivery of FIX in hemophilia B patients, transgene expression was short lived [34]. Although no T cell response against the transgene was detectable, AAV2 capsid-specific cytotoxic T cells were detectable following vector administration and their frequency subsequently expanded, which resulted in killing

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and clearance of transduced hepatocytes. Conceivably, these patients harbor a population of capsid-specific memory cytotoxic T cells, formed during childhood infection with the wild-type virus, probably AAV2, which were reactivated when the subject were exposed to AAV capsid via vector infusion [130]. Consistently, a recent study showed that antigen presentation by hepatocytes, although modest, was sufficient to flag transduced cells for cytotoxic-T-cell-mediated lysis in vitro [131]. Therefore, the host cytotoxic T cell response to vector remains a potential challenge to successful hepatic gene therapy. 6.2

Strategies to overcome immunological barriers Serotype substitution and capsid modification

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6.2.1

Preexisting immunity due to a previous exposure from a natural infection can significantly limit the efficacy of AAVmediated gene transfer via neutralization of the incoming vector particles in a serotype-specific manner. The frequencies of antibodies to AAV1 and AAV2 have been reported as ranging from 30 to 80% among humans [132-134]. Since AAV8 was isolated from non-human primates, neutralizing factors against this virus were reported to be rather low compared with those for other serotypes [135,136]. Moreover, AAV8 capsid cannot effectively activate cytotoxic T cells in mice or non-human primates, presumably due to an attenuated capacity of interaction with dendritic cells [137]. AAV8 is also more efficient than AAV2 for hepatic gene transfer in mice. This may be contributed by the induction of tolerance to the transgene product [138]. However, whether the substitution of AAV2 with AAV8 could successfully evade the pre-existing naturalizing antibody in humans remain questionable. Because AAV8 neutralizing antibodies have been detected in the human population, although with much lower frequency and lower neutralizing activity than AAV2 [6]. Hopefully, the ongoing trial using scAAV8 for treatment of hemophilia B will provide more insight in the near future. Modification of capsid represents another strategy to avoid neutralization. It starts with generating mutations in the capsid by site-directed mutagenesis or high-throughput approaches, then selecting the variants with the ability to evade neutralizing antibodies in the presence of anti-AAV serum [139-141]. The main challenge of this approach is to retain the tissue tropism and the high infectivity of the re-engineered vector. Applying adjuvant immune suppression Immunosuppressants are widely used in organ transplantation to prevent allograft rejection. There are various types of agents with distinct immunosuppressive mechanism used in clinical settings, including steroids (dexamethasone and prednisolone), calcineurin inhibitors (cyclosporine A and tacrolimus), mycophenolate mofetil (MMF), rapamycin, anti-IL-2 receptor antibodies and rabbit anti-thymocyte globulin (rATG). In the context of AAV-mediated gene therapy, several studies have provided proof of concept for using immunosuppressants as 6.2.2

adjuvant. Combination of MMF and tacrolimus with AAV8 expressing FIX neither induced toxicity nor impaired liverdirected vector transduction or transgene expression in rhesus macaque, a natural host of AAV8 [142]. A recent study supported the importance of transient immunosuppression for preventing immune responses to the FIX transgene product [143]. Capsid or other vector-proteins of AAV2 or AAV6 often induce immune responses in outbred dogs [144]. However, transient immunosuppression with a combination of rATG, cyclosporine A and MMF was shown to be sufficient to prevent immune reactivity and permit long-term and effective transgene expression in skeletal muscles [145]. In a similar model of AAV2-mediated gene transfer, treatment with cyclosporine A and MMF partially improved the vector transduction and gene expression [146]. Furthermore, this combination of immunosuppressive drugs was shown to abrogate immune responses to AAV6 vectors in AAT-deficient dogs [147]. However, therapeutic effects slowly declined over time indicating that this immunosuppressive regimen maybe still suboptimal to completely block all immune reactivity. Still more studies are required to optimize immunosuppressive drug regimens for AAV clinical trials. Clearly, just combining more compounds may not be beneficial, as illustrated by a study in non-human primates. In this study it was found that combining the anti-IL-2 receptor antibody (daclizumab) with a regimen of MMF and rapamycin actually increased humoral immune response to AAV2 vector [129]. In this setting, the regimen of MMF and rapamycin alone was fully effective in preventing B cell responses. Therefore, further immunesuppression protocols need to be carefully tested in order to identify the optimal regimen for future clinical trials. Hepatocytes-restricted transgene expression Transcriptional targeting can be achieved by using hepatocyte-specific promoters, which, in principle, limits gene expression to hepatocytes. Many promoters are considered liver specific, including the albumin, AAT, apolipoprotein E, HBV or combinational chimeric promoters [148,149]. Promoters that drive restricted expression patterns are challenging to reconstitute, therefore, the specificity varies extensively among these promoters. Given the nature of suppressing target gene expression by cellular miRNA, tissue-specific miRNA offers an innovative tool to regulate the tropisms of transgene expression [150]. By incorporating target sites for a specific miRNA into a transgene, expression becomes susceptible to regulation in cells that express that particular miRNA [151]. For instance, miR-142-3p is highly expressed in antigen-presenting dendritic cells but not in hepatocytes. By incorporating a fully complementary miR-142-3p target sequence into a lentiviral vector genome, long-lasting transgene (FIX) expression was established due to the diminished induction of immune reactivity by dendritic cells [152]. A combination of a liverspecific promoter driving the expression of UGT1A1 with four copies of the miR-142-3p target sequences resulted in 6.2.3

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significantly prolonged normalization of bilirubin levels without elimination of transduced cells by the immune system [70]. Conceivably, exploitation of miRNA targeting strategies will be of great benefit for AAV-mediated hepatic gene therapy by increasing the persistence of the vector and the evasion of host immunity.

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7.

Conclusions

This review summarizes the potential application of AAVmediated gene therapy for both inherited and acquired liver diseases. With the rapid development of vector engineering and scalable production, gene therapy using AAV has now become a clinical reality. Several trials have attempted to cure recessive liver disease, in particular hemophilia. Although it remains at a preclinical stage for many of the acquired liver diseases, the compelling results from various large-animal models continuously encourage researchers in the field to move ahead. From laboratory to clinic, researchers also learned the challenges of the field, in particular the induction of immunity against hepatic gene transfer. Therefore, several strategies have been presented for optimization, including serotype substitution, capsid modification, immunosuppressant adjuvants, liver-targeted vector delivery and transcriptional regulation. 8.

Expert opinion

Acknowledgements

Despite the conceptual simplicity of restoring a single mutated gene, the reality has taught us the potential complexity in patients, even for a well-investigated disease such as hemophilia. Two ongoing trials intend to improve the efficacy by switching to scAAV8 or by combining AAV with immunosuppressants. Transient immunosuppression has emerged as one of the most favorable strategies to overcome host immune reactivity. However, proper design of the immunosuppressive protocol is of vital importance to ensure adequate protection against vector immunity [153,154]. The calcineurin inhibitors are effective but often cause nephrotoxicity and, particularly

322

higher doses of cyclosporine A, have been reported to be associated with a higher de novo cancer risk [155]. Both cyclosporine A and MMF have been shown to be antiviral against a spectrum of viruses, prompting further studies to address whether there is any effect on AAV vector delivery. The fact that cyclosporine A [156] and MMF [157] also have potent antiHCV activity suggests that both regimens might be beneficial in the setting of treating hepatitis C. Immunosuppressive drugs are usually used life-long after liver transplantation, which could potentially facilitate the application of AAV-mediated gene therapy after transplantation, such as to induce tolerance, stimulate liver regeneration, treat tissue injury or prevent recurrence diseases. With accumulating experience from gene therapy trials and existing knowledge from organ transplantation, more sophisticated immunosuppressive protocols will probably be developed specifically tailored for application in AAV-mediated gene therapy. To achieve the final success in clinical settings, probably requires a combination of multiple approaches to optimize AAV-mediated hepatic gene transfer and it is more important to implant and incorporate new technologies into the field. After all, a cycle between laboratory and clinical investigations is crucial to drive the field forward, and create a brighter future for AAV-mediated gene therapy of liver diseases.

We would like to thank HJ Metselaar and J Kwekkeboom of the Erasmus MC, Rotterdam for their general support.

Declaration of interest This study has been supported financially by National Natural Science Foundation of China (No. 30800093), Natural Science Foundation of Zhejiang Province in China (No. Y2090935), the Erasmus Medical Center Translational Research Fund and the Liver Research Foundation (SLO) Rotterdam.

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Affiliation Luc JW van der Laan1*, Yigang Wang2*, Hugo W Tilanus1, Harry LA Janssen3 & Qiuwei Pan†3 *These authors contributed equally to this work † Author for correspondence 1 Erasmus MC-University Medical Center, Department of Surgery and Laboratory of Experimental Transplantation and Intestinal Surgery, Rotterdam, The Netherlands 2 Zhejiang Sci-Tech University, Xin Yuan Institute of Medicine and Biotechnology, School of Life Sciences, Hangzhou, Zhejiang, China 3 Erasmus MC-University Medical Center, Department of Gastroenterology & Hepatology, 3000 CA Rotterdam, The Netherlands E-mail: [email protected]

327

Review

Immunotherapy of multifocal motor neuropathy Filip Eftimov† & Ivo N Van Schaik

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University of Amsterdam, Neurology, Academic Medical Center, Amsterdam, Netherlands 1.

Introduction

2.

Treatment of MMN

3.

Expert opinion and conclusions

Introduction: Multifocal motor neuropathy (MMN) is an immune-mediated disease, which usually runs a chronic course. Current treatments are aiming at modulation of the immune response to maintain functional status. Although patients can remain stable with prolonged maintenance intravenous immunoglobuin (IVIg) treatment, most patients slowly deteriorate over many years despite increasing IVIg doses. Areas covered: The paper identifies studies since 1985 for patients with MMN. Randomised and non-randomized studies examining the effects of any therapeutic agent were selected, to give a full overview of the different treatments for MMN. The review provides a treatment algorithm together with our views on current treatments, ongoing trials and possible directions for further research. Expert opinion: IVIg remains the only proven treatment for MMN, although questions around dosages and duration of treatment remain. The presence of an effective therapy for MMN should not discourage but stimulate further research into alternative treatments as ongoing axonal damage results in increasing impairment in patients with MMN. Keywords: focal neuropathy, immunotherapy, inflammatory neuropathy, MMN, multifocal motor neuropathy, treatment Expert Opin. Biol. Ther. (2011) 11(3):329-342

1.

Introduction

Multifocal motor neuropathy (MMN) is a rare immune-mediated disorder characterised by slowly progressive, asymmetric, predominantly distal weakness of one or more limbs without sensory loss [1]. The arms are more involved than the legs, a feature that distinguishes this neuropathy from most other neuropathies in which the legs are affected predominantly. The neuropathy leads to loss of dexterity resulting in disability for tasks such as writing, getting dressed and manipulating keys. In one study, one-fifth of patients reported severe disability of the arms despite treatment [2]. Affected motor nerves in the legs can lead to walking impairment. Muscle atrophy may develop early in the disease corresponding with the innervation of the affected motor nerves. Minor vibration sense disturbances at neurological examination are present in about 22% of MMN patients and do not preclude a diagnosis of MMN [2] Fatigue is reported in more than a half of the patients. As the disease progresses, the clinical picture can resemble a symmetrical distal motor polyneuropathy. The prevalence is estimated to be 1 -- 2 in 100,000 [3]. The mean age of onset of initial symptoms is around 40, with a considerable age range [2-4]. Possibly, the age of onset in men is slightly earlier than in women [2]. Men are more frequently affected than women. At initial presentation, muscle weakness and atrophy can closely resemble lower motor neuron disease, especially progressive spinal muscular atrophy and segmental spinal muscular atrophy. In a minority of patients brisk reflexes can be found corresponding with the weakened muscles making the differential diagnosis even more challenging [2]. The hallmark of MMN is the electrophysiological finding of 10.1517/14712598.2011.548798 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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Immunotherapy of multifocal motor neuropathy

Article highlights. .

.

.

.

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.

.

Multifocal motor neuropathy (MMN) is an immune-mediated disease presenting with asymmetric weakness and conduction blocks on electrophysiological testing. European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) guidelines for diagnostic criteria have ended a long during debate about the proper criteria for MMN. Intravenous immunoglobulin (IVIg) remains the only successful treatment with evidence from randomized controlled trials. Subcutaneous immunoglobulin treatment (SCIg) is a possible alternative for chronic treatment. Alternative treatments are needed in cases of insufficient response to IVIg or intolerable adverse events and because of the very high costs of chronic IVIg treatment. Several immunosuppressive therapies have been tried, mostly in small open-label studies, without convincing evidence of efficacy. A treatment algorithm is proposed for a newly diagnosed MMN patient.

This box summarizes key points contained in the article.

conduction blocks at locations outside usual nerve entrapment sites, possibly caused by focal demyelination [5]. A conduction block is a reduction in the amplitude or area of the compound muscle action potential obtained by proximal versus distal stimulation of motor nerves. Extensive electrophysiological studies, especially of the motor nerves in both arms, are needed to identify these conduction blocks. The first reports recognizing the motor neuropathy and the presence of conduction blocks appeared in 1985 while the term multifocal motor neuropathy was introduced by Pestronk et al. in 1988 [6-9]. For several years, there has been no consensus on the electrophysiological criteria for the number and the extent of conduction blocks required for the diagnosis MMN. The European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) Task Force published a set of diagnostic criteria for MMN based on clinical and strict electrophysiological assessment that is now considered to be the reference standard [1]. Some patients with a typical picture of MMN do not have conduction blocks or do not fulfil the criteria for conduction block on electrophysiological testing but do respond to treatment with intravenous immunoglobulin (IVIg). In the EFNS criteria, response to treatment is considered a supportive criterion for the diagnosis MMN [1]. Apart from clinical improvement after IVIg treatment, the presence of elevated IgM anti-ganglioside GM1 antibodies and MRI of the brachial plexuses showing gadolinium contrast enhancement and/or hypertrophy are also considered supportive criteria for the diagnosis [1]. Other tests such as cerebrospinal fluid (CSF) protein, nerve biopsies and laboratory testing can be useful to detect alternative 330

conditions but are not considered standard investigations. The presence of anti IgM anti-ganglioside GM1 antibodies and their diagnostic value has been a matter of debate ever since the identification of these antibodies, especially because they are also found in other related diseases such as the Guillain-Barre´ syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and lower motor neuron disease. There are no accepted standardised ELISA protocols for anti-GM1 antibody detection, making it difficult to compare various studies [10]. Currently, the diagnostic value of these antibodies is being investigated in a Cochrane diagnostic test accuracy meta-analysis [11]. Inflammation of nerves and plexuses can sometimes be seen with MRI but the value of routine MRI testing has never been studied [12]. CSF analysis can be performed if an infectious cause is suspected (elevated white cell count) or when the clinical picture resembles motor CIDP. CSF protein in MMN is normal or only slightly elevated (up to 1 g/l), whereas CSF protein in CIDP is often higher than 1g/l. Nerve biopsy can be performed if there is suspicion of a vasculitic neuropathy. The association of MMN with the presence of anti-GM1 antibodies and the response to IVIg both suggest an inflammatory pathogenesis. However, high serum concentration of anti-GM1 antibodies does not necessarily indicate a more serious immune response and more severe disease. It is not completely understood yet if antibodies targeting gangliosides are the initial culprit and if so, how these antibodies lead to focal demyelination and conduction blocks. GM1 is mostly present on axons and myelin of motor nerves but can also be found in sensory nerves. There is no adequate explanation for the selectivity of anti-GM1 antibodies for motor nerves, as sensory nerves are spared except for some cases with longstanding disease [13]. Some studies have described IgM depositions in demyelinated nerves of MMN patients [14-16]. Furthermore, in vivo and in vitro studies have shown that sera containing high levels of anti-GM1 antibodies can induce demyelination and conduction abnormalities [17,18]. However, in a rather large proportion of patients with MMN, anti-GM1 antibodies cannot be identified. A more dynamic complexity of the pathogenic role of anti-GM1 antibody has recently been emphasized by studies suggesting that formation of anti-ganglioside complexes can negatively influence anti-GM1 binding to GM1 in addition to the fact that there can be variable hiding of the reactive antigens such as GM1 [19,20]. Finally, the inflammatory path induced by the antigen--antibody interaction is not clear although there is growing evidence that complement activation plays a significant role [20]. MMN is a chronic disease. A minority of patients achieve prolonged remissions upon treatment, but most patients need treatment for years, which may lead to adverse events. Also, these therapies are usually accompanied by a considerable social and financial burden. Therapies aim to reduce demyelination blocks, to slow down ongoing axonal degeneration or even to promote reinnervation and

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remyelination. However, despite treatment, MMN often runs a slowly progressive course resulting in decreased dexterity of hands, atrophy and disability. New treatments are needed to prevent irreversible axonal damage. In this review we give an overview of the evidence on different treatment options for MMN, with emphasis on the evidence from randomised controlled trials. Finally, we provide our views on current treatments, ongoing trials and possible directions for future studies. 2.

Intravenous immunoglobulin The mode by which IVIg exerts its immunomodulatory effect is complex. Several mechanisms affecting different immune system pathways have been suggested, although none has conclusively been identified to be the dominant pathway [21]. IVIg neutralises pathogenic antibodies and superantigens, inhibits antibody production by B-cells and accelerates catabolism of antibodies, suppresses pro-inflammatory mediators produced by T cells, inhibits complement-mediated inflammation and damage, induces blockade of Fc-receptors on macrophages and regulates proliferation and adhesion of T cells [22-25]. Finally, modulation of dendritic cell maturation and function and a possible effect on remyelinisation has been suggested [26,27]. Response to IVIg treatment in patients with MMN was first described in 1992, after the publication of several studies on successful IVIg treatment in CIDP [28-32]. Several casereports and case series followed. There are four small double-blind placebo-controlled randomised studies, all with a cross-over design, including a total of 45 patients [33-36]. The electrophysiological criteria for the diagnosis MMN differed between the studies. Van den Berg et al. [34] included patients (n = 6) who previously had responded to IVIg, while Leger et al. [36] (n = 19) included both patients who previously had responded to IVIg treatment (n = 9) and patients who were treatment naı¨ve (n = 10). The other two studies included only treatment-naı¨ve patients [33,35] One study used 2.5 g IVIg/kg during 5 consecutive days [36], while the other studies used 2.0 g/kg during 5 days. Different outcome scales were used. A Cochrane meta-analysis using data on 34 patients from these 4 studies concluded that there was improvement of strength in 78% of patients treated with IVIg and in 4% of placebo-treated patients [37]. The improvement with IVIg treatment compared with placebo corresponded with a number need to treat of 1.4. Disability improved in 39% of IVIg treated patients compared with 11% of the placebo-treated patients. A summary of case reports and case series up to 2004 reported improvement in muscle strength in 81% (373 patients) and improvement in disability in 74% (123 patients) [37]. More recently, three relatively large cohort studies reported similar rates of improvement [4,38,39]. The largest study so far reported improvement in muscle strength in even 94% of 84 IVIg-treated patients [2]. 2.1

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Treatment of MMN

The European Federation of Neurological Societies (EFNS) and the Peripheral Nerve Society currently considers in their guideline IVIg as the first line treatment for MMN as there is considerable evidence (Level A) of short-term improvement [1]. Optimal treatment should prevent secondary axonal damage which is mostly irreversible [2,40]. However, the long-term efficacy of IVIg is less clear. Evidence for IVIg as maintenance therapy is only provided by a few cohort studies. Different IVIg regimens have been used, making comparison between studies difficult [38]. Some studies highlighted the progression of the disease during longterm follow-up resulting in increased atrophy and disability, despite increasing dosages [2,41,42]. Another study reported sustained improvement in muscle strength and even reinnervation with higher maintenance dosages (mean 1.63 g/kg/ month) during a mean of a 7-year follow-up [43]. Recently, Baumann et al. increased IVIg maintenance dosages in nine patients with a persistent paresis resulting in further improvement in six patients [44]. Alternatively, lack of response is associated with more axon loss and longer disease duration before start with IVIg treatment [2]. Some studies have tried to identify patient characteristics that can predict IVIg treatment response. Shorter disease duration before first IVIg treatment, a greater number of conduction blocks and absence of axonal involvement have been proposed to have a positive predictive value for a good response to IVIg but this has not been confirmed by others [2,4,40,45]. High anti-GM1 antibody titres were found in 11 of 12 patients with significant improvement whereas 6 patients not responding to IVIg therapy were all antiGM1-antibody-negative [46]. However, a more recent study found no difference in the presence of anti-GM1 antibodies between responders and non-responders [4]. An equally important question is whether patients need prolonged IVIg maintenance therapy. In contrast to CIDP, where remissions are frequently seen, most MMN patients are believed to require ongoing maintenance treatment [41,46]. In one retrospective study including 40 patients, 8 (20%) were able to stop IVIg treatment without subsequent clinical deterioration [38]. In another study in 12 (15%) of the 79 IVIg-responsive patients, IVIg maintenance treatment could be discontinued [2]. Adverse reactions to IVIg are usually mild and transient and include headache, rigors, myalgia, chest discomfort, high blood pressure, fatigue, fever and nausea. Aseptic meningitis, haemolysis and skin reactions are less frequent. Serious adverse reactions are rare and include thromboembolic events such as stroke and myocardial infarction due to increased serum viscosity, renal tubule necrosis and anaphylactic reactions [23]. The incidence of adverse reactions varies throughout the literature. In one series the incidence of headache was 54% [47]. A large long-term observational study reported adverse reactions in 7% of 1093 patients with autoimmune diseases treated with IVIg [48]. Of thsee adverse reactions, 90% were mild-to-moderate. Serious adverse reactions

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Immunotherapy of multifocal motor neuropathy

occurred in 0.3% of the patients. In this study no relationship was found between the frequency of adverse reactions and elevated infusion rates or high dosages. Precautionary measures can be taken to avoid serious adverse events. Risk factors for thromboembolic events are immobilisation, low cardiac output, atherosclerotic stenosis, high age, recent deep vein thrombosis and pre-existent hyperviscosity syndromes such as cryoglobulinemaemia, monoclonal gammopathies and high lipoprotein levels [23,49]. There is no evidence for the effectiveness of measures such as achieving a good hydration status and prophylactic use of low-dose heparin or antiplatelet agents to avoid thromboembolic events. Recently, in a retrospective case-control study including IVIg-treated patients with and without thromboembolic events there was no difference in the percentage of patients with ongoing use of platelet inhibitors or anticoagulants [50]. Very low infusion rates have been suggested by several authors for patients with risk factors [23,49]. Severe anaphylactic reactions can occur in patients with selective IgA deficiency and anti-IgA antibodies. Renal tubular necrosis can be avoided by screening for pre-existent renal disease and achieving a good hydration status. Other drawbacks of IVIg treatment are possible difficulties with venous access and the needfor regular treatments, which must be given in a clinical or home-care setting under supervision of specialised nurses. Furthermore, IVIg is very expensive and in some countries there is a significant shortage of IVIg [51]. These drawbacks have led to large trials in patients with immunodeficiency syndromes investigating Ig maintenance therapy by subcutaneous administration. In these studies, weekly subcutaneous immunoglobulin treatment (SCIg) proved to be a good alternative to IVIg in terms of treatment effect and patients’ satisfaction due to the possibility of self-infusion. Two case reports reported sustained improvement of muscle strength after a switch from IVIg to SCIg treatment in three patients with CIDP and two patients with MMN [52,53]. A small randomised single-blinded crossover study including nine patients showed no difference in muscle strength between short-term SCIg and IVIg treatment [54]. Recently, the same authors reported 2-year followup data on five of these patients and one newly-diagnosed patient [55]. All patients had stable muscle strength and treatment was well-tolerated. In another open study, including 10 patients with MMN switching from IVIg to two different SCIg regimens, all 4 patients starting with SCIg equivalent to 50% of the previous monthly IVIg dose deteriorated in muscle strength despite a later increase of the SCIg dose while in 4 of 5 patients starting with an equivalent of the IVIg dose muscle strength remained stable over 6 months after baseline measurement [56]. One patient withdrew due to local pain after the first SCIg administration. A possible advantage of SCIg is a more stable day-by-day immunoglobulin concentration, which would avoid some IVIg-related adverse events such as headache and rigors that are attributed to the high peak dose concentration 332

of immunoglobulins. In earlier studies in patients with immunodeficiency, SCIg self-infusions led to a higher level of independence from caregivers when compared with hospital-based IVIg infusions, less emotional distress and better therapy convenience [57]. The drawbacks of SCIg treatment are largely related to the volume of the immunoglobulin solution that has to be administered. The treatment is more time consuming than IVIg, even in case of simultaneous administration at multiple injections sites, and has to be administered once or twice a week. At the start of treatment most patients experience some local adverse effects such as tenderness, swelling and redness which usually subside after several treatments. Finally, similar to IVIg treatment, SCIg treatment is also very expensive. Corticosteroids Corticosteroids inhibit many events of the inflammatory process and immune response by various modes which are divided into genomic and non-genomic mechanisms [58]. There are no randomised controlled trials investigating corticosteroids in MMN. The general opinion that corticosteroids should not be administered to patients with MMN is based on early case-reports and small case-series in which patients failed to respond to treatment [8,32,59-62] In the largest of these case-series there was no improvement in all 13 patients after corticosteroid treatment [60]. Rapid deterioration within a few weeks after start of treatment was reported in a small number of patients by different authors [62-64]. More recently, Slee et al. reported marked deterioration in six out of nine patients treated with corticosteroids who subsequently stabilized upon steroid withdrawal [4]. In contrast, there are a few reports suggesting a good response to corticosteroids; these have been summarised by Umapathi et al. [65]. In the EFNS guidelines corticosteroid treatment is not recommended for treatment of MMN [1]. 2.2

Plasma exchange Plasma exchange (PE) or plasmapheresis is a method by which blood from patients is removed and separated into plasma and blood cells by means of filtration and centrifugation. Blood cells and substitute plasma are then re-administered to the patient. PE is primarily used to extract pathogenic antibodies in a variety of autoimmune diseases. The fact that antiGM1 antibodies are frequently found in MMN and are considered to be pathogenic by some provides the rationale for treatment with PE. There are no randomised controlled trials with PE. One retrospective study included seven patients who did not respond to IVIg and/or cyclophosphamide, three of these patients had elevated anti-GM1 antibodies [66]. Four patients received IVIg or cyclophosphamide directly after the last PE. Two patients (both had elevated anti-GM1 antibodies) improved on MRC sum score and on two of the three functional scales used. Treatment led to a transient decrease of anti-GM1 antibodies in both patients, while 2.3

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electrophysiological data did not change. Two patients slightly deteriorated and in three patients PE had no effect. Only three other patients are reported to have improved after PE [59,67,68], while in six patients PE did not have any effect [8,32,59]. Two other patients worsened after PE [69,70] One patient with MMN was treated by immunoadsorption without a response [71]. PE is a complicated treatment which has to be administered in hospital by specially trained staff. Complications and adverse events such as paraesthesia, hypotension, urticaria and nausea occur in about 4% of the PE sessions [72,73]. Severe side effects such as hypotension warranting treatment discontinuation and arrhythmia occur in about 1%. Other immunosuppresive and immunomodulating agents 2.4.1 Cyclophosphamide 2.4

Cyclophosphamide is an alkylating agent, which inhibits cell replication and is primarily used in haematological malignancies and some autoimmune disorders. Following the recognition of MMN as a separate entity, Pestronk et al. reported successful cyclophosphamide treatment in two patients [8]. Several small case series followed, the largest including nine patients treated with cyclophosphamide, of which eight patients improved. We identified 41 patients treated with different cyclophosphamide regimens. Some or good improvement was reported in 28 patients (68%), Table 1 [2,8,28,32,42,46,59-63,74-78]. Only nine of these patients were previously treated with IVIg, all with insufficient response. The initial enthusiasm for cyclophosphamide in MMN diminished when IVIg treatment appeared to be effective. Nowadays, cyclophosphamide is usually only administered when other treatments have failed. In three studies including a total of 15 patients, adding cyclophosphamide to regular IVIg treatment resulted in a reduced frequency of IVIg infusions in 10 patients (Table 1). However, one of these studies using a longterm follow up of 5 to 12 years reported gradual clinical worsening despite increasing IVIg doses, regardless of oral cyclophosphamide administration. Cyclophosphamide is an agent asociated with potentially serious adverse events. Gastrointestinal symptoms and alopecia often occur. Serious adverse events such as prolonged bone marrow suppression, haemorrhagic cystitis and neoplasia are relatively frequent. Mycophenolate mofetil Mycophenolate mofetil (MMF) inhibits more specifically the DNA synthesis and proliferation in lymphocytes. It is used primarily after organ transplantation to prevent rejection and in some immune-mediated diseases. There is one high quality randomised double-blind placebo-controlled trial including 28 MMN patients treated with IVIg in which oral MMF (1000 mg twice daily) was studied as add-on therapy [79]. Primary outcome was defined 2.4.2

as a reduction of the IVIg dose by 50% or more after 1 year of treatment. Only one patient using MMF reached this primary endpoint. There was no significant difference in mean IVIg dose reduction between patients using MMF or placebo. Earlier, Benedetti et al. treated four patients with MMN with MMF (oral, 1000 mg twice daily) [80]. Three patients achieved a 50% reduction of the IVIg dose, two of these patients were even able to discontinue IVIg treatment. The last patient achieved a reduction of 25% of IVIg dose but experienced a relapse 4 months later. Two patients also used other immunosuppressive therapy which could be discontinued after start of MMF treatment. Umapathi and Hughes reported one MMN and four CIDP therapyresistant patients in which MMF did not result in a clinically significant benefit [81]. Gastrointestinal side effects such as nausea, loss of appetite and abdominal pain are common. Serious side effects such as myelotoxicity and liver toxicity are believed to be less frequent than with other immunosuppressive agents. Rituximab Rituximab is a monoclonal antibody that targets the CD-20 antigen on B lymphocytes. Pestronk et al. treated 21 patients with different neuropathies and IgM antibodies [82]. Of these patients, 14 had an asymmetrical motor neuropathy, 9 had anti-GM1 antibodies and 11 had conduction blocks found on electrophysiological testing. A control group of 13 untreated patients, including 8 motor neuropathies were also included. Rituximab was administered by a weekly infusion of 375 mg/m2 for 4 weeks. Patients who improved but later experienced an increase in muscle weakness were treated with new infusions. After 2 years, there was an increase in muscle strength in most treated patients. The mean change in muscle strength between the treated patients and the control group was 23%. In the same year, others reported a patient with MMN previously treated with IVIg who deteriorated 2 months after starting rituximab treatment [83]. More recently, three patients with MMN were treated with rituximab because of declining efficacy of IVIg [84]. After discontinuing IVIg, rituximab monotherapy was administered by two infusion of 375 mg/m2 1 week apart, followed by more infusions if peripheral blood B cells increased during follow-up. All three patients experienced a sustained improvement in muscle strength for over 2 years of follow-up. Rituximab has also been tried as add-on therapy in nine patients. Ru¨egg et al. treated one patient who became increasingly unresponsive to IVIg treatment despite cyclophosphamide and IFN-b1a therapy [85]. Yearly cycles of rituximab infusions during a 4-week period were administered and stabilised the increasing need for IVIg infusions. In a small open-label prospective study including different inflammatory neuropathies on maintenance IVIg treatment, Gorson et al. treated two MMN patients with weekly rituximab infusion of 375 mg/m2 for 4 weeks [86]. In one patient the IVIg doses 2.4.3

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Treatment regimen

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[2]

Orally 1.5 -- 3 mg/kg/day depending on white cell count (3000 -- 3500/mm2). Treatment after first or second IVIg infusion, mean duration 32 months Orally, dose not stated

Two patients had stable improvement during 5 -- 12 year follow-up

Seven

Six

Two, reduction in IVIg infusion frequency IVIg frequency reduced after CTX, relapse in three patients that discontinued both therapies

Two

None

Three

*,†: Indicate fatalities. BCC: Blood cell count; CTX: Cyclophosphamide; i.v.: Intravenous.

[42]

[78]

Add-on therapy [62] Orally 1.5 -- 3 mg/kg/day

None

One

One

High dose followed by autologous stem cell transplantation Unknown

One remission

Three

[77]

[76]

[75]

One Five

Orally 2 mg/kg/day Three patients i.v. 1 g/m2 every month Two orally 1 -- 1.5 mg/kg/day i.v. followed by orally 100 mg/day for 6 months i.v. 200 mg/kg divided over 4 days

[74] [46]

Two (one also after stopping treatment) One One improved (i.v.) One IVIg frequency reduction (oral) Three

Two

Orally 100 mg/day

[63]

Three One Eight

Eight (three patients worsened upon stopping treatment) Three None Five

Nine

Not stated Orally 100 mg/day 1 month i.v. 3 g/m2 divided over 5 alternate days + orally 2 mg/kg after 1 month or BCC > 3 109/l over 6 months

Two One

Number of improved patients

Two Two

Number of treated patients

[32] [28] [61]

Monotherapy [8] i.v. 3 g/m2 + orally [59] One patient i.v. 3 g/m2 + orally 50 -- 100 mg/day; one patient only orally 150 mg/day [60] i.v. 3 g/m2 + orally 2 mg/kg/day

Ref.

Table 1. Cyclophosphamide treatment in MMN.

Seven

Six patients, all responded to IVIg therapy

Five

Not given

All patients unresponsive to other treatments Unresponsive to other treatments Frequent IVIg needed

One responded to IVIg after stopping CTX Not given All patients unresponsive to IVIg

Not given Later Not given

Not given

Not given Not given

IVIg response

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Two patients developed hemorrhagic cystitis, resolving after stopping treatment. One patient developed persistent amenorrhea Not mentioned

Not mentioned

Transient alopecia, neutropenic fever, supraventricular tachycardia Clinical worsening with respiratory insufficiency and ventilation Unknown

Unknown

One leukemia Not mentioned In total 43 patients treated with MMN and different motor neuron disease: two respiratory failure*, one pulmonary embolus*, five hematuria; one severe leukopenia and one pneumonia with chronic diarrhea One patient developed Listeria meningitis and abdominal sepsis† Not mentioned All three i.v. treated patient developed leucopenia rendering dose reduction

Mild leucopenia and alopecia

Two leucopenia Not mentioned

Adverse events

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could be reduced by 43%, in the other patient the IVIg dose had to be increased to maintain muscle strength. Recently, an open-label prospective study reported six patients with MMN and absent anti-GM1 antibodies in which two infusions of rituximab (1000 mg) did not result in reduction of the IVIg dose or improvement in strength [87]. Treatment with rituximab has been regarded as relatively safe. Most common adverse events are chills and hypotension during infusions [82]. However, concern has risen after reports of progressive multifocal leucoencephalopathy in patients with hematological malignancies and systemic lupus erythematosus treated with rituximab [88]. Methotrexate Methotrexate inhibits DNA synthesis in proliferating cells and is used in a variety of immune-mediated disorders. Recently, Nobile-Orazio et al. treated 8 MMN patients on maintenance IVIg treatment with add-on methotrexate (MTX) in an increasing dose to 15 mg/week for a mean of 12.6 months [89]. Importantly, in the previous 6 months IVIg dose was reduced in all patients, resulting in a clinical worsening indicating IVIg-dependency. MTX was beneficial and well tolerated in two patients; one was able to stop IVIg treatment while muscle strength improved and the other’s monthly IVIg dose could be reduced by 30%. Another patient only experienced subjective improvement. The remaining five patients stopped MTX treatment; three due to adverse events and two because of the modest decrease in IVIg dose (10% and 25%). Common serious side effects are gastrointestinal symptoms, myelotoxicity and liver toxicity.

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2.4.4

Azathioprine Azathioprine is a widely used immunosuppressive agent that inhibits DNA synthesis in proliferating cells. However, in MMN experience with the use of azathioprine is limited. Only two patients have been reported to respond to azathioprine monotherapy, one did not respond to corticosteroids previously and the other failed to respond to IVIg [90,91]. Krarup et al. treated one patient with 200 mg per day for 8 months with no improvement [59]. Azathioprine (150 mg daily) was used as add-on therapy in one patient with MMN and Hashimoto’s thyroiditis allowing a prolongation of the IVIg interval from 3 to weeks 4 weeks [92]. Dose was later reduced to 100 mg due to increased level of transaminases. In another study three out of five patients had a good response to a combination of IVIg, prednisone and azathioprine [93]. Possible serious adverse events due to azathioprine are bone marrow toxicity, acute pancreatitis and liver toxicity. Screening for low thyopurine methyltransferase (TPMT) enzyme activity has been propagated as this activity is associated with increased risk for myelotoxicity. 2.4.5

Cyclosporine A Cyclosporine A inhibits specifically the proliferation of T-lymphocytes. There is only one published account 2.4.6

reporting two patients improving in muscle strength and disability 2 months after starting cyclosporine A (5 mg/kg/day) monotherapy [94]. Interferon-beta1a IFN-b1a is a natural cytokine, which downregulates the inflammatory response. In multiple sclerosis, interferon reduces the frequency of exacerbations [95]. There are only three open label studies reporting results from IFN-b1a treatment. Martina et al. treated three patients refractory to other treatments such as IVIg, prednisone, cyclophosphamide and PE with IFN-b1a, 6 million IU three times a week for at least 6 months [96]. Clinical improvement was found in all three patients after 2 -- 4 weeks with a maximal improvement at 3 months follow-up. However, disability improved only in one patient. Van den Berg-Vos et al. treated nine patients, all of them with prior good response to IVIg treatment [97]. Five patients received maintenance IVIg treatment that was stopped after starting treatment with IFN-b1a, 6 million IU three times a week. Three patients had prolonged improvement in muscle strength lasting 6 -- 12 months after stopping treatment. Six patients did not improve after treatment. Four of these patients, all on prior maintenance IVIg treatment, deteriorated, rendering reinitiating IVIg treatment necessary. In three of these patients IVIg maintenance dose could be reduced during IFN-b1a treatment when compared with the dose prior to the start of IFN-b1a treatment. Recently, the possible role of IFN-b1a (44 µl three times a week) as adjunctive therapy was studied in three patients using maintenance IVIg treatment [98]. There was no change in muscle strength or disability. Two patients were able to increase the IVIg interval with 2 weeks, in one patient this effect was persistent after discontinuing IFN-b1a treatment. Both patients chose not to continue IFN-b1a treatment due to side-effects. Adverse events of interferons consist of flu-like symptoms and mild leucocytopenia and alteration of liver function. Another drawback of interferon treatment is the high cost. Immunomodulating drugs can also be hazardous, as illustrated by several reports of development of demyelinating neuropathies including MMN after treatment of different autoimmune diseases with TNF-a blockers such as infliximab and etanercept [99-101]. Usually, this neuropathy requires IVIg treatment although cases of a self-limiting neuropathy have also been reported [100,102,103]. 2.4.7

Hematopoietic autologous stem cell transplantation

2.5

Autologous stem cell transplantation is a high-risk procedure in which CD34+ stem cells are harvested from peripheral blood after mobilisation with high dose cyclophosphamide and G-CSF. Patients are afterwards treated with myeloablative chemotherapy followed by stem cell reinfusion. Axelson et al. reported a patient with MMN who did not respond to this treatment [77].

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3.

Expert opinion and conclusions

Initial management Most patients with MMN improve after initial IVIg treatment, which is currently considered to be the only evidencebased first-line treatment for MMN. The IVIg treatment should be started as early as possible as there is some evidence from prospective cohort studies that early treatment can possibly prevent or limit secondary axonal damage [2,43]. Patients with very mild and stable symptoms and signs which do not or only slightly interfere with activities of daily living can be monitored without treatment. In these cases the chance of IVIg-related adverse events does not outweigh the possible small amelioration of impairment and disability. The standard starting IVIg dose is 2 g/kg given as 0.4 g/kg on 5 consecutive days or 1.0 g/kg on two consecutive days (Figure 1). IVIg treatment should be initiated in a hospital setting because of the very small but possible risk of an anaphylactic shock. Precautionary measures such as a low infusion rate to start with and good hydration status will help to avoid possible adverse events. Treatment effect can usually be noticed within the first weeks after start of treatment [5]. This fast improvement suggests that IVIg not only influences the immune-mediated response but probably also reduces reversible conduction blocks [5]. A minority of patients do not respond to IVIg treatment. This could be due to settled, irreversible axonal damage. If there is only limited axonal damage in such a patient, one should reconsider the diagnosis of MMN before trying alternative treatment options. One should consider another course of IVIg (2 g/kg) as there are no good therapeutic alternatives for the initial management of MMN patients. The number of courses needed before the conclusion may be drawn that IVIg is ineffective is not known. Our opinion is that a third course would be futile if there is no improvement whatsoever after a second course of IVIg. Switching to another IVIg brand has no additional value, this is supported by the findings of Kuitwaard et al. who found no difference in treatment effect between different IVIg brands in patients with CIDP [104]. In cases of insufficient improvement IVIg maintenance treatment can be combined with immunosuppressive treatment. Alternatively, in cases of no improvement after IVIg, immunosuppressive treatment should be given as monotherapy. Corticosteroids and plasmapheresis do not seem to be effective and may even be harmful. Several immunosuppressive and immunomodulating drugs have been tried (Table 2). Cyclophosphamide is probably the most studied drug, in which effectiveness as monotherapy was reported in 74% of the 38 patients. Most studies are prior to the introduction of IVIg treatment and selection bias could play a role. Due to relatively frequent serious adverse events we use cyclophosphamide only when other therapies have failed. Treatment with other immunosuppressive and immunomodulating drugs has only been reported in very small groups of patients, and often with conflicting results making recommendations difficult.

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3.1

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Long term management Long term data is solely provided by retrospective cohort studies and one nation-wide cross-sectional descriptive study using retrospective data (class IV evidence). Patients who improve after initial IVIg treatment should be observed closely. Only a minority of patients do not need further treatment [2]. Responders who subsequently deteriorate should receive maintenance treatment with IVIg. Maintenance therapy can usually be given in a single day. If high doses are necessary to maintain functional status (> 60 g) or adverse events occur IVIg can be divided over two days. If possible, home-based treatment is preferred as it increases patients’ comfort, which is an important aspect in light of the need for prolonged treatment. The amount per course and interval between courses need to be titrated according to the individual response. Median dose of maintenance treatment differs and may range from 12.5 to 22.5 g/week [2,4]. Repeat courses are usually needed every 2 -- 6 weeks. Treatment intervals should be chosen such to avoid deterioration, which is sometimes seen just before the next IVIg course. IVIg effectiveness tends to decrease over time making increase of the maintenance dose necessary [2,4]. Remissions are less frequent compared with CIDP. Approximately 85% from patients responding to IVIg needs prolonged maintenance IVIg treatment [2]. To avoid treatment with higher dose than necessary one should try to taper IVIg dose gradually on a regular basis [105]. It seems sensible to make a first attempt to taper therapy after one year of treatment with at least six months of stable muscle strength and functional performance. Maintenance IVIg dose should be lowered first before one decides to broaden the IVIg intervals [37]. These patients have to be monitored closely as the majority will experience deterioration, in which case increasing the dose is necessary. SCIg has been studied recently and may be considered as a possible alternative to IVIg maintenance treatment in terms of treatment effect and patients’ satisfaction due to the possibility of self-infusion [54-56]. However, only a small number of patients have been studied so far. SCIg can be tried in patients who are stable and do not need large amounts of immunoglobulin. Large volumes will usually result in troublesome local side-effects at the infusion site. The dose of SCIg should be at least equivalent to the dose of IVIg measured over the same time-period. SCIg is given in weekly infusions. In cases of clear deterioration shortly after starting SCIg, we advise switching back immediately to IVIg treatment. If patients deteriorate after a longer period of being stable on SCIg, a loading dose of IVIg is recommended to bring a patient back to their base-line level while a subsequent increase of the SCIg dose can lead to a new stable situation. Add-on therapy with immunosuppressive or immunomodulating drugs should be considered in case of insufficient effect of IVIg, when large amounts of IVIg are needed, in case of IVIg-related adverse events or difficulties with venous access. Add-on therapy is sometimes also started to 3.2

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Sufficient impairment or disability to warrant treatment

Very mild impariment or disability

Initial treatment Course of IVIg (2 g/kg) over 2 to 5 days

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Insufficient improvement or deterioration

Sufficient improvement

Reconsider diagnosis and second course of IVIg (2 g/kg) over 2 to 5 days

Sufficient improvement

Maintenance IVIg treatment (Find lowest possible dose with longest possible intervals to maintain patient’s functional status. Usually 0.4 g/kg once every 2 to 4 weeks)

Insufficient improvement or deterioration

No improvement

Wait and see monitor without treatment Deterioration

Maintenance IVIg treatment and add second-line treatment see table 2 (Find lowest possible dose with longest possible intervals to maintain patient’s functional status. Usually 0.4 g/kg once every 2 to 4 weeks)

No deterioration No treatment

Reconsider diagnosis. Consider starting immunosuppressive therapy

Figure 1. Treatment algorithm for immunotherapy of multifocal motor neuropathy.

reduce the high costs associated with IVIg treatment. Unfortunately, there is insufficient evidence on efficacy of these drugs to be able to make recommendation on first choice of add-on treatment. Cyclophosphamide is the most frequently reported immunosuppressive treatment for MMN (class IV evidence). However, most reports are prior to the acceptance of IVIg as first-choice treatment for MMN and the evidence as add-on treatment complementary to IVIg is limited. The relatively frequent serious adverse events deter many physicians from using this agent as first choice immunosuppressant.

Ongoing trials Effectiveness of a 10%-solution of IVIg is being studied in a randomised, placebo controlled, cross-over study [106] with an expected number of 40 patients. Earlier, equal clinical efficacy was demonstrated of this liquid IVIg preparation and freezedried preparations for maintenance treatment of CIDP. Subcutaneous immunoglobulin is currently being studied in a third small open label study [107] with a 24-week followup period. No other therapeutic studies currently registered in the international trial registers (Current Controlled Trials and Clinical trials.gov) were found. 3.3

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Table 2. Treatments in MMN. First-line treatment*

Second-line treatment‡

Treatment proven to be ineffective

Intravenous immunoglobulin (IVIg)

Rituximab Cyclophosphamide Methotrexate IFN b-1a (intramuscular) Subcutaneous immunoglobulin (Azathioprine) (Cyclosporine A)

Mycophenolate mofetil

*Sufficient evidence from randomized controlled trials. z Efficacy based on case reports and/or case series.

Future trials All but one of the randomised studies in MMN have focussed on the short term effect of treatment, particularly IVIg. The most recent cohort studies and one large casecontrol study illustrates the fact that MMN should be regarded as a chronic disease in which most patients require treatment for many years. Future trials should take this into account, especially as the disease tends to progress over years despite increasing IVIg doses. Initial IVIg treatment (2 g/kg) is very effective within the first weeks in the majority of patients. This effect will probably be unlikely to be equalled by any other immunosuppressive or immunomodulating drugs. Trials should therefore focus on add-on therapies which reduce IVIg dose and interval. Ideally, these therapies would also limit the secondary axonal degeneration. There are currently no real candidates for substitution of IVIg maintenance treatment. SCIg could be 3.4

338

an alternative with regards to increasing treatment satisfaction due to self-infusion, but this treatment is associated also with very high costs. For both treatment options, comparison in a long-term follow-up study will be needed. If IVIg reduction is chosen as primary outcome in a trial studying an add-on therapy, dependence on IVIg treatment has to be proven by decreasing IVIg dose in the months before starting the study treatment so that the lowest possible IVIg maintenance dose can be found [105]. There is no consensus what an appropriate treatment effect should be, although 30 -- 50% reduction of the IVIg dose was considered a significant effect in earlier studies [79,105]. Possible candidates for add-on therapy trials are rituximab, methotrexate, cyclophosphamide and azathioprine. Short-term and especially long-term adverse events should also be taken into account when choosing the proper candidate.

Declaration of interest IN Van Schaik serves on scientific advisory boards for and has received honoraria for lecturing and consultancy from CSL-Behring. He has also received research support and honoraria for lecturing and consultancy from Actelion Pharmaceuticals Ltd. He has received research support from the Netherlands Organisation for Scientific Research [940-33-024 (PI) and 903-51-201 (PI)], and from the Prinses Beatrix Fonds [MAR-01-023(PI)]. All consulting fees for IN Van Schaik were donated to the Stitching Klinische Neurologie, a local foundation that supports research in the field of neurological disorders. F Eftimov declares no conflict of interest.

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Affiliation

Filip Eftimov† & Ivo N Van Schaik † Author for correspondence University of Amsterdam, Neurology, Academic Medical Center, PO Box 22700 Amsterdam, Netherlands E-mail: [email protected]

342

Expert Opin. Biol. Ther. (2011) 11(3)

Review

Antibody-based therapy in Alzheimer’s disease Refik Pul, Richard Dodel & Martin Stangel† †

Hannover Medical School, Department of Neurology, Hannover, Germany

1.

General introduction

2.

Rationale for immunotherapy in Alzheimer’s disease

3.

Putative mode of action of monoclonal and polyclonal antibodies

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4.

Clinical studies with monoclonal antibodies in Alzheimer’s disease

5.

Clinical studies with polyclonal antibodies in Alzheimer’s disease

6.

Antibody-based therapies for other neurodegenerative disorders

7.

Conclusion

8.

Expert opinion

Introduction: Alzheimer’s disease (AD) is a progressive, age-dependent, neurodegenerative disorder being the most common cause of dementia. The pathological hallmarks are extracellular amyloid-beta (Ab) deposition and intracellular neurofibrillary tangles containing hyperphosphorylated tau. Stunning results in vaccination trials in transgenic animals led to an antibody-based approach to develop immunotherapeutics for AD. Thus, several monoclonal antibodies raised against Ab as well as polyclonal antibodies are currently in clinical testing and some of them already entered Phase III clinical trials. New upcoming experimental approaches like antibodies recognizing specific conformational epitopes, single-chain variable fragment antibodies, or intrabodies give hope of further drug development for this incurable disease. Areas covered: The rationale and putative mode of action of antibodybased immunotherapy in AD and delineates correlations for other neurodegenerative diseases. Current clinical trials and recent results of therapeutic antibodies in AD are presented. Expert opinion: Antibodies against Ab will not only broaden the therapeutic repertoire but also our knowledge about the pathology in AD. Further results of the clinical trials and new antibody-based approaches will shed light on the importance of Ab and its removal in AD. Keywords: Alzheimer’s disease, anti-amyloid antibodies, immunotherapy, IVIg, monoclonal antibodies, neurodegenerative disease, passive immunization Expert Opin. Biol. Ther. (2011) 11(3):343-357

1.

General introduction

Alzheimer’s disease (AD) is the most common neurodegenerative disorder with a prevalence of 9.7% in subjects older than 70 years [1]. It first and foremost affects the regions of the brain responsible for higher cognitive functions such as learning and memory. Cortical atrophy, neuronal loss, region-specific amyloid deposition, congophilic amyloid angiopathy, neuritic plaques and neurofibrillary tangles (NFTs) make up the key neuropathological features in the AD brain. NFTs consist of intracellularly formed hyperphosphorylated tau protein. Tau is a highly soluble microtubule-associated protein and its main function is to modulate the stability of axonal microtubules [2]. Hyperphosphorylation of the tau protein, however, can result in the self-assembly of tangles of paired helical filaments and straight filaments, and consecutively to the disintegration and structural dysfunction of the protein. Furthermore, the formation of insoluble b-amyloid (Ab) peptides may lead to extracellular plaque deposition in the brain. Ab, a peptide of 38 -- 43 amino acids, arises from a larger precursor protein, the amyloid precursor protein (APP), through enzymatic cleavage and is deposited as plaques in the diseased brain [3]. Emerging experimental evidence identified the acidic early endosomes as the principal generation site for Ab peptides [3,4], and low molecular weight aggregates of Ab have recently been shown to be the crucial toxic species [5-7]. It was further suggested that small Ab oligomers form intracellularly before being released into the 10.1517/14712598.2011.552884 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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Antibody-based therapy in Alzheimer’s disease

Article highlights. .

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Alzheimer’s disease is a progressive dementia with cerebral deposits of extracellular amyloid-beta (Ab) and intracellular tangles containing hyperphosphorylated tau. Immunotherapies in animal models of Alzheimer’s disease led to stunning results. Antibodies against Ab can lead to clearance of cerbral Ab deposits. Polyclonal intravenous immunglobulin (IVIg) preparations contain antibodies against Ab. Several monoclonal antibodies against Ab are currently in clinical testing. A clinical Phase III trial is investigating the efficacy of IVIg in Alzheimer’s disease.

This box summarizes key points contained in the article.

extracellular medium where they may interfere with synaptic activity or act as seeds to accelerate fibril formation [8,9]. Thus, preventing or reversing the formation of amyloid would appear to be a promising strategy for AD treatment. Based on the ‘amyloid cascade hypothesis’ a large number of therapeutic approaches are currently under consideration, including active/passive immunization against Ab [10]. In transgenic APP-expressing mice, immunization against Ab peptides has been shown to be effective in clearing plaques and also in affecting behaviour [11]. Active immunization in transgenic mice impairs fibril formation, promotes clearance of Ab plaques and also may interfere with phosphorylation of tau [12,13]. Similarly, passive immunization with antibodies that recognize the amino-terminal and the mid-terminal domain of Ab peptide were also effective [14]. Based on these data, several clinical trials using passive or active immunization approaches have been initiated (Tables 1 -- 3). The aim of this review is to collect current knowledge from basic research and clinical trials, in order to provide a comprehensive overview of antibody-based therapy for AD.

Rationale for immunotherapy in Alzheimer’s disease 2.

In the last decade a great deal of research has been devoted to immune-mediated mechanisms in AD initiated by the observation that anti-Ab antibodies could inhibit Ab peptide fibrillization/oligomerization and prevent cell-culture-based neurotoxicity [15,16]. In 1999 Schenk et al. administered peripheral injections of fibrillized Ab1 -- 42 emulsified in complete Freund’s adjuvant, originally to exacerbate AD-like pathology in the platelet-derived growth factor promoter expressing amyloid precursor protein (PDAPP) mouse model of AD. Unexpectedly, they found that the treated mice had brains virtually devoid of b-amyloid plaques [12]. High titres of antibodies directed against Ab and co-localization of MHC II-positive microglia with Ab deposits suggested that the Ab vaccine promoted antibody-mediated clearance of 344

b-amyloid plaques [12]. This led to vaccination of AD transgenic mice with Ab1 -- 42 coinjected with Freund’s adjuvant confirming distinct reductions in Ab deposition and cognitive improvement [13,17]. Another vaccine, K6A-A1-30, a fragment of the Ab peptide containing the two major immunogenic sites, also reduced plaques in the amyloid precursor protein (APP) transgenic mice whereas a nonfibrillogenic Ab derivative just ameliorated cognitive impairments [18-20]. Interestingly, cognitive improvements upon immunotherapy seemed to be without a relationship to Ab load [13,17,21]. A reduction of the amyloid burden in the brain and the reverse of memory deficits were also attainable by passive transfer of anti-Ab antibodies from vaccinated AD transgenic mice, indicating that the therapeutic effect of the vaccine may only be mediated by an antibody response [14,22,23]. All monoclonal IgG antibodies (10D5, 3D6, m266) as well as a polyclonal immunoglobulin (PabAb1 -- 42) were administered intraperitoneally. While 10D5 and PabAb1 -- 42 significantly reduced plaque burden, 3D6 trimmed down diffuse amyloid deposits and small aggregates of Ab [14,22]. Brain sections of mice treated either with 10D5 or 3D6 showed plaques that were strongly decorated with antibody, suggesting that monoclonal antibodies can enter the CNS and may directly trigger amyloid clearance [22]. However, in some studies, little or no influence on Ab burden but learning and memory improvements were reported [24,25]. Antibodies specifically directed against amino acids 4 -- 10 of Ab recognize monomeric and fibrillar Ab, but not diffuse Ab deposits, which may explain the different results [26]. Moreover, polyclonal anti-Ab antibodies were detected infrequently in cell culture supernatants derived from Ebstein-Barr virus (EBV)-transformed lymphocytes from AD patients as well as healthy individuals [27,28]. Intravenous or direct administration into brain of L11.3, a human IgM antibody engendered by a lymphoblastoid cell line derived from EBV-immortalized peripheral blood lymphocytes, improved cognition in aged SAMP8 mice, a model of AD [29]. Furthermore, experiments in a triple transgenic mouse model applying antibodies against Ab also showed that tau associated changes could be attained in the presence of those antibodies but not with antibodies against tau [30]. More recently, polyclonal antibodies against Ab were also detected in human IgG fractions and they were able to reduce Ab toxicity and to inhibit Ab fibrillation in vitro [31,32]. Due to the promising results in animal models Phase I/II clinical trials of aggregated Ab vaccination, designated as AN1792, and the TH1 response-activating QS21 as adjuvant were initiated. During the Phase I clinical trial (80 patients) no safety concerns arose and patients who responded to this treatment showed slower decline of cognitive functions as compared with non-responders [33,34]. The Phase IIa clinical trial, however, had to be terminated prematurely due to meningoencephalitis in 6% (18 patients) of the patients. However, in more than 30% of the patients with an antibody response approximately 20% did not respond to the active

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Table 1. Different approaches for antibody-based therapy in Alzheimer’s disease. Passive immunisation

Epitopes

Active immunization

Humanized monoclonal antibodies Human ‘monoclonal’ antibodies Parts of Ab-recognizing antibodies Nanobodies Polyclonals

Ab: Oligomers/Fibrills/Plaques

Adapted antigens Adjuvants Phages

Peripheral/central effect

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Table 2. Current clinical trials with active immunization//passive immunization for Alzheimer’s disease. Substance Active immunization ACC-001 + QS-21 (NCT00960531) Affitope AD01 (NCT00711139) CAD-106 (NCT01097096) Passive immunization (monoclonal antibodies) Bapineuzumab (AAB-001) Solenazumab (LY2062430) PF-04360365/RN-1219 Gantenerumab/R1450/RO4909832 V950 GSK933776A MABT5102A

Epitope

Phase

Company

N-terminus N-terminus N-terminus

II I II

Wyeth & Elan Pharmaceuticals Affiris Novartis

N-terminus Mid-section C-terminus N-terminus and internal epitope N-terminus Not published Not published

III III II I

Wyeth & Elan Pharmaceuticals Eli Lilly Pfizer & Rinat Neuroscience Hoffmann-LaRoche & MorphoSys

I I I

Merck GlaxoSmithKline Genentech

Data adapted from www.clinicaltrials.org.

immunization [35]. Autopsy results indicated aseptic meningoencephalitis, presumably induced by T-cell-mediated and/or Fc-mediated immune responses [35-38], and studies in animals revealed convincingly the cause of those side-effects [39]. Although, there was a striking effect on Ab clearance of parenchymal plaques in the brains of autopsy cases, N-terminally directed antibodies did not seem to modify the cognitive decline in those patients [40]. Despite the disappointing effects obtained in the active vaccination trial [41], new vaccination strategies are currently being tested in clinical trials (Table 2). In order to circumvent unwanted T-cell responses direct administration of human/humanized monoclonal/polyclonal antibodies against Ab is moving more and more into the spotlight.

Putative mode of action of monoclonal and polyclonal antibodies

3.

Three types of anti-amyloid antibodies binding to linear epitopes within the Ab sequence have been described: antibodies against the N-terminal epitope (amino acids ~ 1 -- 10), the central region (amino acids ~ 17 -- 32) and the C-terminal region of Ab (amino acids ~ 32 -- 42) [42,43]. Antibodies directed to the N-terminus of Ab have been shown to act within the CNS by binding to Ab aggregates in plaques,

triggering microglial phagocytic clearance of amyloid plaques via an Fc receptor-mediated mechanism [22], or inhibiting aggregation or neurotoxicity of Ab [26,44]. F(ab¢)2 fragments that lack the Fc region of the antibody may also be effective [45-47]. The anti-Ab mAb m266 raised against the central domain of the Ab peptide has a high affinity for soluble Ab but lesser affinity to aggregated b-amyloid [14]. Its intraperitoneal administration caused a rapid increase in the level of plasma Ab while chronic treatment with m266 even affected Ab deposition, suggesting that it changed the equilibrium of Ab levels between brain interstitial fluids and blood, thereby accelerating the Ab efflux [14,24]. In other words, the site of antibody action is in the periphery, where soluble forms of Ab are sequestrated in the peripheral circulation and thus drive an efflux of Ab from the brain to the blood plasma, providing a ‘peripheral sink’ for Ab clearance [14,48]. The idea of a draining ‘peripheral sink’ is supported by observations which show that Ab binding reagents, like gelsolin or soluble lipoprotein receptors, also increased the Ab efflux [49,50]. In contrast, Seubert et al. attributed the increased plasma Ab levels to a reduced clearance rate of Ab complexed to antibody as they did not observe any amyloid reduction with m266 [51]. Another observation showed that despite an increase in plasma Ab anti-Ab antibodies did not change the levels of total Ab [52]. Yamada et al. demonstrated that peripheral

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Table 3. Clinical trials in Alzheimer’s disease patients with IVIg. IVIg (passive immunization, polyclonal antibodies)

Patients

Duration

Cognitive outcome

Octagam + Piracetam [129] Octagam [127]

? Mild to moderate MMSE (n = 5) Mild to moderate MMSE (n = 8)

12 months 6 months

? ADAS-cog: MI:3.7 pts MMSE: MI: 1.6 pts MMSE: MI: 2.5 points

Gammagard Liquid; Gammagard S/D

Mild to moderate MMSE (n = 24*)

Gammagard

Mild to moderate (n ~ 360)

6 months; washout 3 months; 1 g/kg/2 weeks for 3 months followed by 0.4 g/kg/2 weeks for 6 months 6 months followed by 12 months open-label treatment 70 weeks

Octagam

Mild to moderate (n = 56)

24 weeks

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Gammagard S/D [128]

ADAS-cog MI: 5.27 pts NPI" Primary outcome: ADAS-cog; CGCI Primary outcome: change of Ab in serum/CSF

*16 patients received Gammagard Liquid; 8 patients received Gammagard S/D. ADAS-cog: Alzheimer disease assessment scale - cognitive; CSF: Cerebrospinal fluid; MI: Myocardial infarction; MMSE: Mini-Mental state examination; NPI: Neuropsychiatric inventory.

administration of m266 actually retards the efflux transport of Ab from brain to bloodstream and increases the level of the soluble monomer form of Ab in the brain. Thus, they suggested that the beneficial effect is mediated by inhibition of Ab forming into oligomers and amyloid fibrils [53]. Ab oligomers are recognized as the major toxic form that impairs neuronal function by altering the expression of memory-related receptors [54], inducing aberrant neuronal responses to electrical stimulation [55] and neuronal death [56,57]. mAb 22C4, directed against the C-terminus of Ab, reduced brain Ab levels and protected hippocampal neurons against oxidative stress [58]. Upon intra-ventricular administration 22C4 did not show any effect on central or peripheral Ab but when applied intravenously it lowered brain Ab levels and reduced neurotoxic effects [58,59]. mAb 1A10 selectively altered plasma Ab levels whereas mAb 2286 mediated Ab clearance both by central and peripheral mechanisms of action [60,61]. However, in a subsequent study with mAb 2286 cerebral microhemorrhages were observed [62]. The same authors suggested the use of deglycosylated antibodies as unmodified IgGs probably promote the redistribution of disaggregated Ab from brain parenchyma to cerebral vasculature which may result in increased congophilic amyloid angiopathy causing a ‘weakening’ of the blood vessel wall [63]. Cerebral microhemorrhages also occurred following administration of mAb directed to the N-terminus of Ab, but were not observed with the anticentral domain antibody m266 [48,64,65]. New epitope targets may avoid such adverse effects. In this regard, a new format of antibodies (Anti-Trx (Ab15)4, NAB61) was developed recognizing specific conformational epitopes and binding to aggregated Ab such as protofibrils, oligomers or amyloid plaques, but not to monomeric Ab or Ab precursor protein [25,66-69]. Accordingly, emphasis 346

has shifted to soluble oligomers and protofibrils as pathological species of Ab42 because their level correlates positively with the severity of dementia [5,6,70,71]. In vitro and ex vivo experiments revealed that conformation-dependent anti-Ab antibodies of the IgG isotype prevented cytotoxicity mediated by these small soluble Ab aggregrates [72-74]. In vivo, they have been shown to prevent/reverse disruption of hippocampal long-term potentiation mediated by Ab oligomers [6,25,75]. Interestingly, repeated immunization of aged canines with aggregated Ab1 -- 42 (AN1792) has been shown to drift toward the elevation of antibodies that recognize conformational epitopes [76]. Lindhagen-Persson et al. even reported that endogenous anti-Ab IgM auto-antibodies from a group of healthy individuals displayed a preference for oligomeric Ab [77]. Intriguingly, Goni et al. used a polymerized British amyloidosis (ABri) related peptide in a predominantly b-sheet oligomeric form. This peptide is involved in a rare highly amyloidogenic form of familial human amyloidosis and has no sequence homology to Ab or other human proteins. It induced an antibody response through conformational mimicry to the toxic Ab in APP/PS1 AD transgenic mice but also to paired helical filaments that are core constituents of the NFTs [78]. These mice had a cognitive benefit associated with a reduction in the amyloid burden, Ab40/42 levels, as well as reduced Ab oligomer levels [78]. In recent studies using synthetic Ab oligomers the cellular prion protein (PrPc) has been shown to be a high-affinity specific binding site for Ab oligomers [79,80]. Accordingly, PrPc seems to be important for acute Ab-oligomer-mediated suppression of synaptic plasticity in hippocampal slices while in another study memory impairment induced by acute injection of Ab oligomers did not depend on PrPc [79,80]. Nevertheless, PrPc expression has recently been shown to be necessary

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for memory impairment in an AD transgenic mouse model [81]. A monoclonal anti-PrP antibody 6D11 has been demonstrated to block Ab oligomer toxicity in hippocampal slices [80]. Distinctive cognitive improvements without any change in the amyloid burden or Ab peptide levels in the APP/PS1 transgenic mouse model were reported to occur just after two weeks of treatment with 6D11 [82]. A further interesting approach is the use of single-chain variable fragment antibodies (ScFv) which even if they vary from conventional mAbs exhibit similar antigen binding properties with the advantage of lacking of the Fc part. Their small size facilitates tissue penetration, but also results in a rapid clearance from circulation [83]. They inhibit/amyloid fibril formation and Ab-mediated cytotoxicity in vitro [57,84-86] as well as reduce Ab deposits in vivo [87]. As ScFv are still considered to be large for crossing the blood--brain barrier (BBB), where transport is restricted when the size of the molecule exceeds 400 Da, new strategies like fusion of ScFv to a mAb against the human insulin receptor are proposed to enable BBB penetration [88]. ScFv can also be expressed intracellularly as an intrabody, which can be directed to intracellular as well as to extracellular epitopes. Intrabodies directed to nicastrin, an essential component of the g-secretase complex, or to a peptide adjacent to the b-secretase cleavage site led to suppression of the g-secretase activity or inhibited generation of Ab, respectively [89,90]. ScFv directed to Ab can be delivered to the brain by recombinant adeno-associated virus (rAAV) [91]. A very elegant approach was demonstrated by Sudol et al. who transfected transgenic AD mice with rAAV vectors expressing engineered intrabodies directed to intracellular Ab. They observed a partial clearance of Ab deposits [92]. Other engineered antibody fragments included rFabs and V-domains preventing the formation of mature amyloid fibrils by stabilizing Ab protofibrils [93,94]. Certain antibodies can catalyze the hydrolysis of peptide bonds, leading to permanent inactivation of polypeptide antigens [95,96]. A monoclonal IgM antibody from a patient with Waldenstr€om’s macroglobulinemia as well as polyclonal IgM antibodies purified from the sera of patients with AD were shown to hydrolyze Ab40 at the Lys-28 -- Gly-29 bond and Lys-16 -- Ala-17 bonds [97]. Recently, a ScFv that has a-secretase-like activity has also been shown to hydrolyze Ab in vitro [98]. Furthermore, the receptor for advanced glycation end products (RAGE)-mediated influx of circulating Ab is one of the crucial regulators of the BBB transport of Ab [99]. In AD as well as in related animal models upregulation of RAGE has been observed and supposed to result in cerebral Ab retention [100,101]. Although direct evidence is lacking, sequestration of Ab in the circulation is assumed to reduce RAGE-mediated Ab influx across the BBB [102]. Antibodies raised against Ab were detected in the blood as well as in the cerebrospinal fluid of non-immunized subjects [103]. Intravenous immunoglobulins (IVIg) as a fractionated product consisting of pooled polyclonal

immunoglobulins is assumed to contain a wide range of variable regions of antibodies present in normal serum [43]. Polyclonal IgG antibodies against Ab were also detected in IVIg which reduced Ab toxicity and inhibited Ab fibrillation in vitro [32]. Furthermore, IVIg dissolved preformed fibrils, increased cellular tolerance to Ab, enhanced microglial migration towards Ab deposits and mediated their phagocytosis [104]. Immunotherapy against Tau The occurrence of neurofibrillary tangles along with plaque deposition are the neuropathological changes defining AD. Hyperphosphorylation of the microtubule associated protein tau leads to self-assembly into abnormal fibers (‘paired helical filaments’) and can form higher order aggregates. Although Ab is the primary trigger of AD according to the amyloid cascade hypothesis, neurofibrillary degeneration of abnormally hyperphosphorylated tau is apparently required for the clinical expression of this disease. Recently, reports on active immunization against tau in animal models have been published [105,106], although tau is an intracellularly located protein. Asuni and coworkers vaccinated P301L mice, that develop neurofibrillary tangles in several brain regions and the spinal cord, with a tau fragment peptide (amino acids 379 -- 408) phosphorylated at Ser396 and Ser404 (with aluminum as adjuvant) starting at 2 months for a three month period [107]. Vaccination reduced aggregated tau in the brain and slowed progression of the tangle-related behavioral phenotype compared with control animals. Antibodies against this vaccine were found to cross the blood--brain barrier, bind to phosphorylated tau and exert their function in the brain. In contrast, Boimel et al. applied Tau195 -- 213[P202/205], Tau207 -- 220[P212/214] and Tau224 -- 238[P231] peptides, which contain the AD/tauopathy-related phosphorylation epitopes. The epitope seem to play an important role for vaccination as the same group has previously reported that wild-type mice vaccinated with full-length tau (emulsified in complete Freund adjuvant/pertussis toxin) resulted in severe side effects including NFT-like structures, axonal damage and motor phenotypes as well as triggering an autoimmune response. These data reveal the potential hazards of using tau as an immunogen, and stress the importance of the appropriate conformation and target epitope for active and passive immunization approaches [108,109]. In the experimental setting Boimel et al. treated either double-mutant (DM) tau [K257T/P301S] transgenic mice or those mice pretreated with myelin oligodendrocyte glycoprotein starting at 4 months of age [110]. They found a significant decrease of about 40% in NFT burden accompanied by an increase in microglial burden. Antibodies were detected in serum and blood vessels in the CNS, while no encephalitogenicity was detectable. Recently, circulating naturally occurring antibodies against tau (IgM and IgG) have been detected in the blood and with much lower titers in the cerebrospinal fluid 3.1

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(CSF) [111]. In the sera of 9 AD and 8 healthy individuals and in 20 AD patients and 22 subjects suffering from other neurological disorders, Rosenmann and coworkers found antibodies against unphosphorylated as well as phosphorylated tau. Similar to naturally occurring Abs against Ab naturally occurring tau antibodies decrease with age. Unfortunately, no further studies regarding passive immunization against tau are currently available.

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4. Clinical studies with monoclonal antibodies in Alzheimer’s disease

Currently, a large number of mAbs are in clinical trials at various stages of development (Table 2). The development of bapineuzumab is the most advanced. It was evaluated in a small placebo-controlled single-ascending-dose trial comprising 30 patients. Infusions were administered in different doses (0.5, 1.5 or 5 mg/kg body weight, or placebo) and elevated plasma Ab levels were observed with a peak concentration 24 h after infusion. However, with the highest dosage of this substance three patients developed a reversible vasogenic brain edema [112]. In the subsequent Phase II trial 234 patients were enrolled [113]. As bapineuzumab has a half-life of 21 -- 26 days, 13-week intervals were chosen for dosing for a total period of 78 weeks. In contrast to the Phase I trial the highest dose was limited to 2 mg/kg body weight (randomized to 0.15, 0.5, 1 and 2 mg/kg body weight, or placebo) [113]. The study was completed by 65% of the patients in the treatment group and 71% in the placebo group. Primary clinical outcomes were assessed in the Alzheimer’s Disease Assessment Scale -- Cognitive sub-scale (ADAS-Cog) and the Disability Assessment for Dementia (DAD). Additionally, the Neuropsychological Test Battery (NTB) 17, the Mini-Mental State Examination (MMSE), and the Clinical Dementia Rating-Sum of Boxes (CDRSB) were measured [113]. Essential treatment differences were reported in the completers population on the parameters ADAS-Cog (p = 0.003), NTB (p = 0.045) and DAD (p = 0.041). However, when patients who received at least one dose of study drug were included no differences were seen except trends on the ADAS-Cog (p = 0.078) and NTB (p = 0.068) [113]. Unfortunately, 12 of the treated subjects developed vasogenic edema during the protocol which occurred after the initial or second dose of study drug. One patient had to be treated with corticosteroids. The occurrence of vasogenic edema seems to increase at higher doses of bapineuzumab. Moreover, apolipoprotein E e4 (APOE e4) allele frequency was higher in this group than in the general study population [113]. Analysis of data of APOE e4 non-carriers revealed treatment differences in the ADAS-Cog (p = 0.026), NTB (p = 0.006), MMSE (p = 0.043) and CDR-SB (p = 0.04) whereas no treatment differences were seen in the APOE e4 carriers. Biomarker analyses revealed a trend towards lowered CSF tau, a marker of neuronal loss, in the treated versus placebo group in a 348

subset of patients (p = 0.056) [113]. Overall, no differences were noted in the MRI volumetric measurements. Subgroup analyses of APOE e4 non-carriers showed that non-carriers have less brain volume loss (10.7 ml) as compared with the placebo group (p = 0.004) [113]. In another Phase II trial (28 patients) results of the carbon-11-labelled Pittsburgh compound B (11C-PiB) positron emission tomography was the primary outcome [114]. 11 C-PiB is a thioflavin that binds fibrillar oligomeric proteins such as Ab and serves as an in vivo marker of cortical amyloid [115]. Patients were randomly assigned to three different doses (0.5, 1.0 or 2.0 mg/kg body weight) and they received up to 6 infusions at 13-week intervals. Comparison of the placebo group versus the total treatment group (pooled data for all three doses) revealed significantly lower 11C-PiB signals in the treatment cohort, suggesting that bapineuzumab reduced the level of cerebral Ab [114]. Solenazumab (LY2062430), the humanized version of the mAb m266, was examined in a small clinical trial (19 patients) to assess safety and biomarker outcomes after a single dose [116]. According to the different doses administered (0.5, 1.5, 4.0 and 10.0 mg/kg) patients were stratified into four treatment groups. Except for infusion reactions at higher dosages no adverse events attributed to the treatment were seen over the short course of the trial. A dose-dependent increase of Ab in plasma and CSF Ab was observed, although changes in cognitive scores were not noted [116]. In another Phase II trial with solenazumab in which 52 patients over a 12 week study period were enrolled, four treatment groups were assigned. Doses of solenazumab varied based on dose (100 versus 400 mg) and frequency of administration (weekly versus every 4 weeks) and a fifth group served as the control [117]. Even after 1 year of follow up no adverse effects were reported, especially no vasogenic edema. After injection of solenazumab distinct plasma elevations of Ab and an increase of Ab42 and a decrease of Ab40 in the CSF were reported [117]. A large multicenter trial with solenazumab is currently conducted worldwide. The humanized IgG2 mAb PF-04360365 (Pfizer; NCT00722046) [118] targets the C-terminal end of the Ab1--40 peptide, particularly Ab33--40. Although two Phase I studies have been completed, only preliminary data are available which were presented at the Alzheimer’s Association International Conference of Alzheimer Disease (AAICA) 2009. Singledose data on 37 patients were obtained and indicate that the substance is well-tolerated over a 0.1 -- 10 mg/kg body weight dose range. In MRI examinations vasogenic edema did not occur. A mild central antibody penetration over the BBB is supposed as two out of eight patients in the 10 mg/kg body weight cohort had measurable concentrations in CSF (~ 0.5% of plasma values). However, no remarkable changes in cognition (ADAScog, MMSE) have been noted yet (AAICA 2009, abstract of the presentation O4-04-03). Gantenerumab (R1450 or RO4909832; Hoffmann-La Roche; NCT00531804 [119]), V950 (Merck; NCT00464334

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[120]),

MABT5102A (Genentech; NCT00736775 [121]) and GSK933776A (GlaxoSmithKline; NCT00459550 [122]) are further mAb currently evaluated in Phase I/II trials. Detailed information on these trials is currently not available.

Clinical studies with polyclonal antibodies in Alzheimer’s disease

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5.

Studies with polyclonal antibodies or oligoclonal combinations of antibodies are scarce as high regulatory hurdles exist for approval of such drugs. Currently, only data on commercially available IVIg preparations, which have been used in clinical trials in AD patients, are available. IVIg is a fractionated blood product that is used in the treatment of a variety of health conditions including a number of indications in neurological disorders [123]. The rationale for their use in Alzheimer’s disease stems from data that commercially available IVIg contain naturally occurring autoantibodies against Ab (nAbs-Ab) [31]. nAbsAb were detected in the blood and CSF of healthy and diseased persons. Although there are contrasting data, the current evidence indicates that their concentration is reduced in patients with AD [103,124,125]. Naturally occurring antibodies make up to two thirds of the human antibody pool, however, their function is diverse and far from being understood [126]. The nAbs-Ab seem to have an important physiological role in the clearance of Ab in healthy but also in diseased states by inhibiting the propensity to fibrilize, blocking its toxicity and interfering with its metabolism and hydrolysis [97,127,128]. However, several issues have not been addressed and our knowledge, especially regarding the interaction with Ab and its clearance, their role in plaque deposition in vivo and their effect on behaviour, has not been investigated thoroughly. However, based on the hypothesis that a reduced concentration of those nAbs-Ab exists in AD patients, their supplementation would be a therapeutic option. Currently, several pilot studies including a total of 37 patients have been performed using different preparations of IVIg and a large Phase III study has been initiated in the USA (Table 3). The first study investigated 16 patients with AD: 8 patients received 140 g piracetam for one year only and 8 patients received an additional monthly dose 0.2 g/kg IVIg [129]. Unfortunately, the study results are only available in abstract form and, therefore, a detailed consideration is not feasible. The authors, however, concluded that ‘the treatment demonstrated a significant improvement in the group of patients with the additional IVIg treatment’. An independently started small pilot study included five patients with AD who were given monthly infusions (a total of 1.2 g/kg body weight over a three day period) for a duration of six months. The primary outcome was the shift of Ab from the central to the peripheral pool [127]. A significant decrease was detected in the Ab1--40 CSF concentration and an increase was also found in the serum after 6 months of

IVIg treatment. Similar findings were reported by an US pilot trial that included eight patients with mild to moderate AD receiving IVIg for a total period of 18 months (6 months treatment/3 months washout/9 months observation) at different concentrations and infusion intervals [128]. Although, the small number of patients and the lack of a control group in both studies precluded a meaningful analysis of cognitive changes, none of the patients deteriorated during the study period and in the US study an improvement of MMSE scores by a mean of 2.5 points was reported. In a randomized and placebo-controlled dose finding study, the same group investigated IVIg in 24 mild to moderate AD patients for a period of 6 months. Four different dosing regimens and two different application times (0.2 g/kg/2 weeks (n = 4); 0.4 g/kg/2 weeks (n = 4); 0.4 g/kg/4 weeks (n = 4); 0.8 g/kg/4 weeks (n = 4); placebo (n = 8)) were applied. The ADAS-cog difference between the control group and verum was 5.27 at 6 months, however, this was not significant. CGIC and NPI improved significantly in the combined group as compared with the placebo group. Plasma Ab increased in a similar fashion as in the previous study. In the FDG-PET in a subgroup of the study, the glucose metabolism in mildly hypometabolic brain regions and in severely hypometabolic brain regions was preserved. Based on these promising results a large Phase III clinical trial using Kiovig in patients with AD has been initiated recently (Baxter; NCT00818662 [130]). This placebo controlled randomized trial, which follows current international recommendations for such trials, will recruit over 360 patients with mild to moderate AD and will be randomized into three treatment arms: i) IVIg 0.4 g/kg every 2 weeks for 70 weeks, ii) IVIg 0.2 g/kg every 2 weeks for 70 weeks, and iii) placebo. Primary outcome measures include cognition and global function. Data from this trial will be available in 2013. Recently, another clinical trial using IVIg (Octagam 10%) was initiated as a dose-finding study with centers in the USA and Germany (Octapharma; NCT00812565 [131]). Furthermore, Shankle et al. reported long-duration data of an open study using IVIg in patients with AD (n = 10) and Lewy body dementia (n = 4) [132]. They were treated for a mean duration of 21.4 ± 15.3 months (7 -- 46 months) and 14.3 ± 14.5 (7 -- 36 months) with IVIg, respectively. No major side effects occurred and with respect to their cognitive testing no deterioration was found over the investigated time period in both treatment groups. Further evidence for an effect of antibody-based therapy and nAbs-Ab stems from a study in which a retrospective case-control analysis to evaluate the incidence of AD and related disorders in an IVIg treated (with various diagnoses as indication for IVIg use, mainly humoral immunodeficiency syndromes) versus an untreated population was performed [133]. A total of 847 IVIg treated patients were matched (1:100) to 84,700 untreated controls. The proportion of patients diagnosed with dementia was 2.0%

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Antibody-based therapy in Alzheimer’s disease

among treated cases and 4.2% among untreated controls (p < 0.002). The relative risk reported was 0.58 for treated patients versus controls (p = 0.024) ‘indicating a 42 percent lower incidence rate of dementia in patients treated with IVIg’. Although evidence has accumulated that IVIg and treatment based on nAbs-Ab may have a positive effect on cognition in Alzheimer’s disease, it is too early to recommend IVIg as a treatment outside of clinical trials. Many more issues have to be addressed including clinical aspects (e.g., dosing, treatment intervals, etc.) but also basic aspects (e.g., physiological role of nAbs-Ab, long-term results of reducing Ab, etc.). Indeed, the results of the large clinical Phase III trial will show whether IVIg may provide the urgently expected next step to the development of an appropriate treatment for this devastating disorder. 6. Antibody-based therapies for other neurodegenerative disorders

Neurodegenerative disorders actually have no cure and most therapies are oriented towards symptomatic relief. One common feature of several neurodegenerative diseases is the occurrence of misfolded proteins. Although the mechanisms by which these altered proteins lead to neurodegeneration remains unknown much effort has been devoted to preventing the formation of misfolded conformers or promoting their removal. For instance, in Parkinson’s disease the presynaptic protein a-synuclein has been implicated as a contributor to the pathogenesis [134]. Recently, vaccination strategies and application of antibodies have been found to exert a beneficial effect in animal models [38,135,136]. Several ScFv that bind to specific regions of a-synuclein have been reported [137-140]. Human ScFv as well as anti-a-synuclein intrabodies led to inhibition of a-synuclein oligomers, amelioration of cell morphological abnormalities, reduction of intracellular a-synuclein aggregation and toxicity [135,137,138,140]. These promising results were supported by results of active immunization of human a-synuclein transgenic mice with recombinant human a-synuclein [141]. The treated animals produced antibodies against the C-terminal region of human a-synuclein and exhibited a reduction in a-synuclein deposits in the histopathological examinations and in Western Blot analyses. The decrease in the deposits did not only affect the Lewy body-like inclusions but also the a-synuclein aggregates in the presynaptic terminals, as shown by laser scanning microscopy [141]. Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a cellular prion protein (PrPC) into a misfolded toxic protein (PrPSc). The human variants include very rare diseases such as Kuru, Creutzfeldt--Jakob disease, Gerstmann--Straussler--Scheinker disease, fatal familial insomnia, and sporadic fatal insomnia [142]. An antibody-mediated approach was first investigated in scrapie susceptible neuroblastoma cells [143-146]. This approach 350

became more popular after the investigation of White et al. showing that anti-prion antibodies are effective in treating animal prion disease [147]. Other researchers confirmed the effectiveness of antibody-mediated therapy in delaying the onset of disease in vivo [148-152]. Studies involving transgenic mice with a skewed anti-PrPC B-cell repertoire (6H4µ) showed that PrPC-specific B cells are produced, even if endogenous PrPC is expressed, providing a basis for vaccination [153]. Thus, this was followed by vaccinating scrapie-infected mice with recombinant PrP [154] and PrP peptides [155]. An alternative way to achieve a primarily humoral response is mucosal vaccination, which has been shown to prevent oral prion infection. This type of response produces primarily secretory IgA antibodies, but when the antigen is coadministrated with adjuvants such as a live attenuated strain of Salmonella typhimurium expressing the mouse PrP gene substantial serum IgG titres can be achieved [156,157]. The immunotherapeutic strategies directed against the native cellular form of the prion protein have recently been extended to scFv, which cleared PrPSc in cultured cells [158]. Anti-PrPC scFvs delivered by rAAV type 2 were shown to reduce the PrPSc burden and to delay the onset of prion disease in animals [149]. However, sequestration of free cholesterol in cell membranes, significant overexpression of apolipoprotein E (apoE), and activation of cytoplasmic phospholipase A2 as well as production of prostaglandin E2 as toxic effects have been observed in vitro [159]. Another progressive neurodegenerative disease is Huntington’s disease caused by an expansion of a polyglutamine sequence in mutant huntingtin that results in the formation of nuclear and cytoplasmic neuronal inclusion bodies. Accordingly, anti-N-terminal huntingtin intrabodies (C4 sFv) reduced aggregation of huntingtin and cellular toxicity in vitro [160-162]. In a Drosophila model of Huntington’s disease these intrabodies slowed down the aggregation of huntingtin and increased the proportion of flies surviving the adulthood from 23 to 100% [163]. Recently, in an animal model of amyotrophic lateral sclerosis intracerebroventricular administration of a monoclonal antibody raised against toxic misfolded forms of superoxide dismutase 1 (mSOD1) led to reduction of mSOD1 by 23% in the spinal cord and prolonged the lifespan of transgenic mice [164]. In a small open-label trial (seven patients) IVIg treatment with additional administration of oral cyclophosphamid of patients did not improve clinical outcomes in patients with amyotrophic lateral sclerosis [165]. 7.

Conclusion

Antibody-based therapies have led to a novel concept for the treatment of chronic neurodegenerative disorders in the last decade. Several different concepts including passive and active immunization have been investigated and some of them have been already translated into clinical trials (Tables 1 and 2) whose results are expected to be available in the next two

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years. This will elucidate whether antibody-based therapies will result in a clinical benefit for those devastating disorders. However, as a recent review has outlined, a long journey is still ahead of us: looking at ‘the timelines in the field of immunotherapeutic development for neoplastic disorders approximately 20 years elapsed from when the results of first trials of monoclonal antibodies in animal models were reported in 1977 until the first FDA approval of a monoclonal antibody-based therapeutic for a human cancer in 1997’ [11]. Thus, we are on a long journey but fascinated to elucidate the fundamental link between immunological and neurodegenerative pathways and hopefully elaborate an effective cure for our patients. 8.

Expert opinion

Promising results of vaccination in transgenic animals led promptly to clinical studies in AD patients. In the largerscale Phase II trial of AN1792 the development of aseptic meningitis of immunized patients led to cessation of this study [33]. A reassessment was enabled in a subset of cases by neuropathological examinations which revealed perivascular T cells and mononuclear cells. Indeed, the impressive loss of Ab noticed in these examinations confirmed that the principle was correct [35-38]. However, Ab should be attacked but without activating the immune system. Thus, the immunotherapeutic approach turned to monoclonal/polyclonal anti-Ab antibodies which in animal models have been shown to be effective as well [14,22,23]. The mechanism of passive immunization still remains elusive. Passively administered antibodies have been shown to enter the CNS either to decorate plaques and induce clearance of preexisting amyloid or to prevent plaque formation [22,26,44]. A therapeutic benefit may be provided by both, acting within the CNS and/or by acting in the periphery as a peripheral sink [166]. The ‘peripheral sink’ hypothesis of Holtzman and Paul is supported by Bell et al. who measured an efflux of b-amyloid from brain to blood in mice [167]. Accordingly, elevated plasma Ab levels were reported in the Phase I clinical trials of the mAb bapineuzumab and solenazumab, favouring a peripheral mode of action [112,116]. Moreover, in the smaller Phase II trial of bapineuzumab the treatment group had less 11 C-PiB signals than the untreated cohort as assessed by positron emission tomography, suggesting a decrease of the cerebral Ab [114]. However, if all participating subjects are taken into account the results of the bigger Phase II trial revealed

no differences in the primary outcomes of the study, which was actually disappointing [113]. Similar to AN1792 bapineuzumab is burdened with an adverse effect. Vasogenic brain edema occurred preferentially at higher doses and seems also to be associated with APOE e4 carriers [113]. In a population of Alzheimer’s patients 65% in case of sporadic AD and 80% in case of familial AD carry at least one APOE e4 allele [168]. If this association can be confirmed, then bapineuzumab will be available for only a small group of non-carriers. However, critical adverse effects were not reported in the Phase I/II trial of solenazumab and PF-04360365. IVIg is well known in clinical practice and considered to be a relatively safe treatment [123]. In all completed studies an increase of plasma and a decrease of CSF Ab1 -- 40 was detected [43,127,128]. Despite small sample sizes cognitive improvements or stability were noted which, consequently, motivated for further Phase III trials [43]. As expected, no serious adverse effects were reported in the completed trials. The antibody-based approach in AD is strongly focused on Ab peptide and recently to its smaller soluble intermediates. A novel approach is antibodies raised against PrPc which block downstream interactions of Ab-mediated oligomer toxicity in transgenic animals rather than just removing abnormal proteins [80,82]. Another important target in AD is the NFTs and their precursors, composed primarily of hyperphosphorylated tau proteins. Follow-up analyses of AD patients in the AN1792 trial revealed a reduced tau load in neuronal processes but not in the neuronal cell bodies [169]. Less data about antitau antibodies is available [105,110]. Further experiments will show whether antibodies raised against tau protein will have any influence on neurodegeneration. Other neurodegenerative diseases sharing the common feature of misfolded proteins provide scope for similar antibody-based approaches. Antibodies directed against toxic prion protein are the most advanced development but still too far away from clinical use [149].

Declaration of interest R Pul declares no conflict of interest. M Stangel has received honoraria for lectures and travel expenses for scientific meetings from Baxter, CSL-Behring and Talecris. R Dodel has received honoraria from Baxter, CSL-Behring and Octapharma. He holds two patents related to the subject of this review.

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Affiliation

Refik Pul1, Richard Dodel2 & Martin Stangel†1 † Author for correspondence 1 Hannover Medical School, Department of Neurology, Carl-Neuberg-Str. 1, 30625 Hannover, Germany Tel: +49 511 532 6676; Fax: +49 511 3115; E-mail: [email protected] 2 Philipps-University, Department of Neurology, Marburg, Germany

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Review

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Introduction

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Identifying a transplantable

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A transplantation method

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The survival of the graft

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Expert opinion

Intramuscular cell transplantation as a potential treatment of myopathies: clinical and preclinical relevant data Daniel Skuk† & Jacques P Tremblay †

CHUQ Research Center -- CHUL, Neurosciences Division -- Human Genetics, Quebec, Quebec, Canada

Introduction: Myopathies produce deficits in skeletal muscle function and, in some cases, literally progressive loss of skeletal muscles. The transplantation of cells able to differentiate into myofibers is an experimental strategy for the potential treatment of some of these diseases. Areas covered: Among the two routes used to deliver cells to skeletal muscles, that is intramuscular and intravascular, this paper focuses on the intramuscular route due to our expertise and because it is the most used in animal experiments and the only tested so far in humans. Given the absence of recent reviews about clinical observations and the profusion based on mouse results, this review prioritizes observations made in humans and non-human primates. The review provides a vision of cell transplantation in myology centered on what can be learned from clinical trials and from preclinical studies in non-human primates and leading mouse studies. Expert opinion: Experiments on myogenic cell transplantation in mice are essential to quickly identify potential treatments, but studies showing the possibility to scale up the methods in large mammals are indispensable to determine their applicability in humans and to design clinical protocols. Keywords: cell transplantation, clinical trials, myopathies, non-human primates, skeletal muscle Expert Opin. Biol. Ther. (2011) 11(3):359-374

1.

Introduction

Transplantation of myogenic cells (given the different possible meanings of the term ‘myogenic’, in the present review this is defined as corresponding to ‘giving rise to or forming muscular tissue’) is an experimental approach under study for the potential treatment of diseases of the skeletal muscle. Since myoblasts were the only myogenic cells identified for several decades (the earliest references to myoblast cultures date from at least 1915 [1]), they were the first cells to be proposed for this therapeutic use [2]. The term myoblasts defines the proliferating mononuclear myogenic cells that fuse among themselves to form myotubes, elongated and narrow syncytia that under appropriate conditions mature to form myofibers, the contractile multinucleated cells of the skeletal muscle parenchyma. This term applies to every instance in which this phenomenon takes place, that is, during histogenesis in utero, in postnatal myofiber regeneration and in in vitro culture of muscle cells. Partridge, Grounds and Sloper set the starting point of this approach in 1978, suggesting that ‘in subjects suffering from inherited recessive myopathies, muscle function might be restored if normal myoblasts could be made to fuse with defective muscle fibers’ [2]. Among all myopathies, Duchenne muscular dystrophy (DMD) has being the major target of myogenic cell transplantation. The reason is the relative frequency of this disease (a prevalence of 50 cases per million in the male 10.1517/14712598.2011.548800 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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The intramuscular transplantation of cells able to differentiate into myofibers is a potential therapeutic tool for the treatment of myopathies. For clinical purposes in myology, transplantable cells may accomplish at least one of the following: direct fusion with recipient myofibers to induce gene complementation, neoformation of myofibers and differentiation into satellite cells. The protocol of cell delivery to the target tissue must be defined in animal models allowing proper extrapolation to humans. The survival of the graft is mainly threatened by acute rejection, which is present when the grafted cells are allogeneic or express foreign epitopes. Current preclinical work may solve the main problems in the field, that is, restricted topographical participation of the grafted cells and acute rejection, by developing protocols that proven to be potentially useful in humans with an adequate balance between potential benefits and risks.

This box summarizes key points contained in the article.

population) and its severity: skeletal-muscle degeneration in the limbs and trunk, progressing during infancy and puberty, leading to severe to complete muscle loss, respiratory insufficiency and ultimately death from respiratory or cardiac complications. The early saga of clinical myoblast transplantation in myology is a good example of the relevance of having an appropriate preclinical basis to design clinical tests of innovative therapeutic procedures. The clinical trials conducted in the 1990s were undertaken after a few experiments in rodents, whose results were overstated and, in addition, gave no clues about the transplantation parameters to be used in humans (for a table summarizing these clinical trials see [3]). Using improvised transplantation protocols, these clinical trials reported scarce and limited results at the molecular level. With present knowledge, it is evident that these premature trials were designed with exaggerated expectancies about the properties of the transplanted cells. It was obviously believed that arbitrarily chosen amounts of myoblasts injected in a few sites of a skeletal muscle would diffuse spontaneously throughout the muscle to fuse with so many myofibers that a therapeutic effect would be achieved. Later research showed that this was optimistic speculation. Hence, a main lesson of that frustrated clinical saga is that it is crucial to know the actual behavior of the grafted cells under appropriate experimental conditions before planning clinical protocols able to give relevant information. This review aims to cover some information potentially relevant for clinical development of intramuscular cell transplantation in myology. For this reason, priority will be given to data obtained in humans and non-human primates. The profuse research in mice helped to define several properties 360

of myogenic-cell transplantation, but also yields much material without clear clinical significance. Conversely, preclinical studies in non-human primates helped to define technical and pharmacological parameters to improve the clinical protocols. 2.

Identifying a transplantable cell

Cell transplantation implies the implantation into the body of either differentiated cells that form the parenchyma and eventually the stroma of the organs, or precursor cells with the capacity to differentiate into the former. With regard to skeletal muscle, it seems technically difficult to transplant myofibers -- the differentiated elements of the parenchyma -- in order to reconstruct a tissue. Therefore, cell transplantation in myology was directed toward the second possibility: a precursor mononuclear cell. The task was facilitated by the fact that the skeletal muscle has specific committed stem cells, the satellite cells [4]. The niche of satellite cells is between the sarcolemma and the myofiber’s basal lamina, where they remain dormant until an event such as focal or total necrosis of a nearby myofiber is produced. Myofiber necrosis elicits the removal of myofiber debris by macrophages and the activation of satellite cells, which differentiate into proliferating myoblasts [5]. Satellite cells can completely regenerate myofibers without needing other sources of myogenic cells [6]. However, it is worth noting that other cells with the capacity to give rise to myoblasts were reported in mice and/or human muscles. These include ‘side-population cells’, ‘muscle-derived stem cells’, myoendothelial cells, mesoangioblasts/pericytes and CD133+ cells (for a review about these cells see [7]) and, more recently, a population of cells identified in human muscles that show aldehyde dehydrogenase activity [8]. It could be convenient to use the adjective ‘adult’ for myoblasts responsible of myofiber regeneration in postnatal life [9]. This would establish a difference from embryonic and fetal myoblasts that form skeletal muscles during histogenesis. This distinction is important because adult cells do not necessarily display the same gene expression profiles as embryonic or fetal counterparts [10]. One example of the different molecular dependence of myoblasts in the embryonic, fetal, neonatal and adult ages is the different relevance of myogenic determinants such as paired box proteins Pax3 and Pax7 [11]. Notably, adult myoblasts have different behavior and morphology from embryonic and fetal myoblasts in cell culture [12]. Satellite cells can be released from skeletal muscle fragments by standard enzymatic procedures of cell culture and can be easily sub-cultured as myoblasts, able to fuse to form myotubes that will eventually differentiate into myofibers [13]. The simplicity of obtaining and expanding these cells in vitro, facilitated the use of myoblasts for cell transplantation approaches not only in myology but, later, in cell-based therapies for heart pathologies [14].

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Skuk & Tremblay

Properties needed in a cell to be candidate for transplantation in myology

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2.1

Lipton and Schultz reported in 1979 two of the three main properties that cell transplantation needs to be useful in myology [15]. They reported in rats and quails that intramuscularly grafted myoblasts fused with the recipient’s myofibers and, in a minor proportion, fused among themselves to form new small myofibers. Fusion of the grafted cells with the recipient’s myofibers gives rise to a phenomenon named gene complementation, meaning that the myofiber’s syncytia express proteins encoded by exogenous and endogenous nuclei [16]. Myofibers expressing exogenous and endogenous proteins are called mosaic or hybrid [16,17]. Gene complementation allows grafted myogenic cells to be vehicles of therapeutic genes. The typical example is the introduction of normal genomes in the genetically abnormal myofibers of patients with recessive genetic myopathies. The second property reported by Lipton and Schultz [15], the fusion of the grafted cells among themselves to form new myofibers, encourages research trying to restore functional contractile parenchyma in muscles in which too many myofibers were lost due to a degenerative or traumatic pathological condition. A third property was first reported by Yao and Kurachi in 1993 [18]: the possibility of myogenic cell transplantation creating a new pool of muscle precursor cells in the recipient muscle. Gene complementation The first report of gene complementation restoring a genetically missing protein in a myopathic animal was that of Partridge et al. in 1989 [19]. They transplanted normal mouse myoblasts in skeletal muscles of mdx mice, a mouse strain that develops a myopathy caused by the lack of dystrophin, the protein whose deficiency triggers DMD. They detected dystrophin in several myofibers after follow-ups of at least 3 weeks. Similar results were reported thereafter by other researchers [20,21] and this has become routine in myogeniccell transplantation research, including restoration of other proteins such as merosin in dy/dy mice (a model of merosindeficient congenital muscle dystrophy) [22] and dysferlin in SJL mice (a model of limb-girdle muscular dystrophy 2B) [23]. In humans, sporadic observations of enhanced dystrophin expression in DMD patients following normal-myoblast allotransplantation were reported during the clinical trials conducted in the 1990s (see [3] for a table summarizing these results). Nevertheless, most of the patients at that time gave negative results and only one study confirmed that the dystrophin detected after transplantation was of donor origin [24]. It was not until later clinical trials, using a protocol of cell injection and immunosuppression set up in non-human primates, which was shown that donor-derived dystrophin can be reliably detected in muscles of DMD patients allotransplanted with myoblasts from normal donors (Figure 1) [25-27]. 2.2

A key factor that makes myogenic-cell implantation difficult is the fact that intracellular proteins in myofibers remain near the nucleus of origin, in a region named the nuclear domain [28]. This is both because the mRNA diffuses only 100 µm from the nucleus [29] and because the proteins have also a limited spread [30]. Consequently, proteins of donor origin remain expressed in the myofiber regions in which the transplanted cells fused. The nuclear domain’s length varies for each protein, depending on factors such as if they remain soluble or anchored to stationary cellular components [30]. This factor conditions the analysis of transplantation results and a striking example was the comparison between green fluorescent protein and dystrophin in one study: following transplantation of cells expressing dystrophin and green fluorescent protein in mdx mice, dystrophin was detected through about 116 µm in myofibers, while green fluorescent protein diffused up to 1500 µm [31]. It is especially important to consider this factor, together with the differences in sensitivity among detection methods, when the results for a transgenic label in a transplantation experiment are extrapolated to a therapeutic protein, such as dystrophin in DMD. Dystrophin restoration in limited myofiber regions was remarked upon in some clinical trials of normal myoblast allotransplantation in DMD patients. Gussoni et al. [32] reported dystrophin expression ranging from 300 to more than 450 µm (the largest size of their analysis) in the myofibers, although without confirming whether this was actually donor-derived dystrophin. In another DMD patient in which donor-derived dystrophin was effectively detected, myofiber regions expressing donor-derived dystrophin were from at least 700 µm to more than 2 mm in size [26]. Restoration of myofibers In DMD, as in other myopathies, worsening of muscle weakness is produced by the progressive loss of myofibers. The optimal treatment for the advanced phases of these diseases must involve not only molecular correction but also restitution of functional myofibers. This second factor becomes even more important as the disease progresses. In mouse experiments, myoblast transplantation showed the capacity to completely or almost completely regenerate muscles destroyed by different acute treatments, recovering muscle mass and strength [33-36]. However, these acute experimental conditions are different from a chronic degenerative myopathy. In these mouse experiments, the damage was massive and acute, preserving the endomysial scaffold, in which the basal lamina seems important for an orderly muscle regeneration as observed in rats and rabbits [37]. In contrast, myofiber necrosis is insidious in chronic degenerative myopathies and the muscle is progressively replaced by adipose and/or fibrous tissue, losing the endomysial support. Mouse experiments showed that, under some conditions, myoblast transplantation could form new myofibers independent of a previous endomysial scaffold. This was the case of the fusion of the implanted myoblasts among themselves in 2.3

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A.

B.

C.

Figure 1. Allotransplantation of normal myoblasts from an adult related donor in 1 cm3 of the Tibialis anterior of a DMD patient under tacrolimus immunosuppression, in a Phase IA clinical trial done by the authors. A. Several parallel intramuscular cell-injections were done with a 100-µl Hamilton precision syringe. The cells were injected homogeneously during the needle extraction, and the number and distribution of injections was monitored with the help of a sterile transparent dressing with a 5-mm grid. B. The whole cross-section of one of the biopsies done 1 month posttransplantation in a cell-grafted site is shown stained for fluorescent immunodetection of dystrophin. C. A schematic representation of B helps to illustrate the distribution of the dystrophin+ myofibers, represented in black on the gray background of the tissue.

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mdx mice in which muscle irradiation was done prior to transplantation [38]. Exogenous myoblasts were also able to neoform ectopical muscles after subcutaneous transplantation in mice [39]. Otherwise, whether myogenic-cell transplantation could form functional myofibers in skeletal muscles that degenerated to fibrosis and/or fat substitution remains insufficiently studied. One study showed that myotubes and regenerating myofibers developed across implants of adipose tissue inserted in regenerating skeletal muscles in mice, suggesting that perhaps adipose tissue in dystrophic muscle would not be an impediment for the neoformation of myofibers [40]. The presence of fat tissue in the muscle, however, could induce the transdifferentiation of some human muscle precursor cells into adipocytes, depending on whether they express or not CD34 [41]. Cell sorting could be useful to separate CD34+ human muscle-derived cells, which adipogenic potential after transplantation in immunodeficient mice, from CD34-negative cells, which did not transdifferentiate into fat cells [41]. Moving from these few observations in mice towards a useful procedure of muscle restitution in humans is still a challenge, among other factors because mice have inherently a better muscle regeneration competence than large animals [42]. Nevertheless, some clinical observations could be encouraging: in DMD patients allotransplanted with normal myoblasts, clusters of small dystrophin+ myofibers were interpreted as neo-formed by the fusion of the implanted myoblasts among themselves [25]. Formation of donor-derived satellite cells If some of the grafted cells remain in the muscle as mononuclear muscle-precursor cells, and especially as satellite cells, this would imply that the effect of transplantation may not be limited to the early fusion of the implanted cells but that it would provide also a source of normal muscle-committed stem cells to participate later in muscle hypertrophy and regeneration. Potentially, this could suggest that the amount of myofibers expressing dystrophin in DMD patients receiving transplants of normal myoblasts may increase over time, proven that a phenomenon similar to that reported in mdx mice is produced, that is, the expansion of clusters of myofibers expressing revertant dystrophin [43]. This property was extensively reported after transplanting mouse and human myoblasts in mice. The pioneer paper of Yao and Kurachi in 1993 reported that up to 2% of myoblast colonies obtained in culture from muscles grafted with mouse myoblasts were of donor origin, even after 5 months, and that these myoblasts were able to fuse with myofibers after a new transplantation, giving rise again to mononuclear cell precursors [18]. Gross and Morgan grafted mouse myoblasts into irradiated muscles and produced muscle necrosis four times at 3-week intervals using notexin, a substance that damages myofibers but not satellite cells [44]. Since there were regenerating donor-derived 2.4

myofibers after each muscle injury, this proved that some grafted myoblasts remained as muscle precursor cells competent to contribute to muscle regeneration after repeated muscle damages. Similar results were reported following the fate of fluorescent-labeled mouse myoblasts by in vivo fluorescence imaging [45]. Irintchev et al. were the first to report that some intramuscularly-transplanted myoblasts became satellite cells in the recipient [36,39]. They partially reconstituted destroyed muscles by mouse myoblast transplantation and they identified donor-derived mononuclear cells expressing M-cadherin in the border with myofibers, a characteristic of satellite cells [36]. In a second experiment, they formed ectopic muscles by subcutaneous myoblast implantation, and these neomuscles showed M-cadherin+ cells adjacent to myofibers, indenting the sarcolemma under the basal lamina in a typical satellite-cell position [39]. Heslop et al. [46] and Xu et al. [45] further identified donor-derived satellite cells in muscles that received transplants of mouse myoblasts, respectively by immunodetection of CD34 and Pax7. Similar results were reported with human myoblasts transplanted into immunodeficient mice. Abundant donorderived satellite cells were detected in mouse muscles one month after transplantation of myoblasts from adult humans (Figure 2) [47]. These human-derived satellite cells explain the presence of human donor-derived muscle-precursor cells evidenced in experiments of culture, re-transplantation and regeneration in immunodeficient mice [47] and confirmed preliminary observations suggesting that some human nuclei in isolated human/mouse hybrid myofibers were satellite cells [48]. Human fetal myoblasts also gave rise to mononucleated muscle-precursor cells following transplantation in immunodeficient mice [49]. Clinical observations suggest that formation of donorderived satellite cells could also occur following myoblast allotransplantation in DMD patients. Donor-derived mononuclear cells were detected in the muscles of DMD patients that received myoblast allotransplantations, and some of the donor-derived nuclei were in an anatomical position susceptible to correspond to satellite cells [25,47]. Other human-derived cells that were reported to form donor-derived satellite cells following transplantation in immunodeficient mice were muscle-derived CD133+ cells [50], and probably muscle-derived aldehyde-dehydrogenase+ cells [8]. 3.

A transplantation method

Identifying cells suitable for transplantation is the first step in developing a cell-based therapy. A crucial second stage is to deliver these cells to the target tissue in a way to reach a therapeutic result. Two major routes were studied in order to deliver myogenic cells to skeletal muscles: intramuscular injection and intravascular infusion. Since the subject of the present review

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Figure 2. Donor-derived satellite cells evidenced by fluorescent immunodetection in cross-sections of skeletal muscles of immunodeficient mice transplanted with myoblasts from an adult human donor 4 weeks before. A. A region of a mouse muscle composed almost entirely by human-derived myofibers, identified by a monoclonal antibody that reacts with human dystrophin but not with mouse dystrophin (red fluorescence). In a serial double-stained section, it can be seen that the region shown in A is filled with human nuclei (B., green fluorescence using an anti-human lamin A/C monoclonal antibody) and contains several nuclei expressing Pax7, a marker of satellite cells (C, red fluorescence). D to G show the region in the white rectangle in C, illustrating the co-detection of Pax7, human nuclei (human lamin A/C+) and basal lamina (laminin, blue fluorescence). Arrows indicate several human donor-derived nuclei that express Pax7 (a marker of satellite cells) and are placed in the periphery of myofibers and inside the basal lamina (i.e., in the anatomical position of satellite cells). Arrowheads indicate human Pax7+ nuclei outside the basal lamina. H to K show a single myofiber with the same triple co-detection, but with a higher magnification and in images taken with confocal microscopy. Confocal microscopy allows better discrimination of the intra-nuclear typical staining of Pax7, the peripheral nuclear labeling of lamin A/C and the anatomical position of the two donor-derived satellite cells in the periphery of the myofiber under the basal lamina. The lower magnification images taken by standard fluorescence microscopy aim to illustrate the abundance of donor-derived satellite cells in these conditions. Scale bars = 100 µm (A -- C) and 10 µm (D -- K).

is the intramuscular route, we will only mention that, up to now, a specific type of cells called mesoangioblasts was the only one that seems to be sufficiently efficient by intraarterial infusion in animal models of muscular dystrophies, including dogs with dystrophinopathy [51,52], although some positive results were also reported with CD133+ cells in mice [53]. Other cell types more recently reported as able to differentiate in muscle precursor cells following transplantation in mouse muscles require direct intramuscular injection (for a recent review about these cells see reference [54]). In the event that these cells prove to be useful for intramuscular transplantation in the clinics, they will be probably concerned by the same considerations made below. 364

Cell injection strategy: a crucial factor The major restriction of the intramuscular route is that the grafted cells fuse essentially with the myofibers near the injection trajectories. In non-human primates in which myoblasts are injected into normal muscles without pre-treatments, each myoblast-injection leads a strip of hybrid myofibers in muscle cross sections (Figures 3 and 4), as if the injected cells fused just with the myofibers damaged by the injection [55-57]. The pattern of donor-dystrophin expression in DMD patients receiving allotransplants of normal myoblasts was sometimes similar [24,25,27], but in other cases it was less defined (Figure 1) [25-27]. The local fusion of the implanted myoblasts would not be a problem if the donor-derived proteins were capable of 3.1

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Grafted myogenic cell Recipient’s myoblast Macrophage

Figure 3. Schematic representation of the mechanism allowing the incorporation of the grafted myoblasts in the recipient’s myofibers of normal skeletal muscles. The point of departure of the illustration is the cross section of a macaque skeletal muscle that was treated, one month before, with an injection of autologous myoblasts genetically modified ex vivo to express a micro-dystrophin under a MCK promoter and coupled to a peptide tag (I, peroxidase immunodetection of the peptide tag). A shows a needle traversing a muscle fascicle in this muscle and injecting the cells homogeneously during the needle extraction (orange arrow). B to G illustrate the process of injected-cell uptake in a myofiber isolated from this fascicle. This myofiber (B) is damaged by the needle and undergoes segmental necrosis (C). The necrotic region is invaded by macrophages (D) with two main functions: phagocytosis of the necrotic debris and release of factors helping myofiber regeneration. Myofiber regeneration is produced by the activation of the recipient’s satellite cells, which proliferate as myoblasts that fuse together (E). This regenerative process recruits some of the injected myogenic cells (E). The nuclei of the transplanted cells that participated in this regeneration are integrated in the myotubes that fill the gap lead by segmental necrosis (F). Later, these donor-derived nuclei will produce donor-derived proteins throughout a restricted length of the myofiber (G), leading to restricted regions of donor-protein expression in the fascicle (H). In histological cross sections of the cell-grafted muscle, this restricted fusion is observed as ‘strips’ of donor-derived protein expression (I).

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Figure 4. Intramuscular myogenic-cell transplantation in non-human primates using different injection devices. A. A precision Hamilton syringe operated manually. B. A repeating PB600-1 dispenser with a Hamilton syringe. C. A specific prototype for repetitive intramuscular cell injections. As in DMD patients, the density of cell injections is controlled with a sterile transparent dressing with a 5-mm grid. After at least one month, the fusion of the injected cells in the recipient’s myofibers is analyzed in cross sections of muscle biopsies using histological techniques to detect the transgenic proteins in the transplanted cells (D to F). The images correspond to cross sections of macaque muscles grafted with myoblasts labeled either with b-galactosidase (D, E) or a micro-dystrophin coupled to a peptide tag (F). Myofibers expressing donor-derived proteins are respectively detected by histochemical detection of b-galactosidase (D, E, dark staining) or fluorescent immunohistological detection of the peptide tag (F). The distribution of the myofibers expressing donor-derived proteins reproduces the pattern of the original cell-injection trajectories (indicated by the arrows). The density of b-galactosidase+ myofibers is higher in E than in D, because the density of cell injections was higher: 25 per cm2 in D and 100 per cm2 in E. Scale bars: 500 µm (D -- F).

diffusing throughout each myofiber. However, given that proteins are restricted to nuclear domains, which are very short in the case of dystrophin, myogenic-cell transplantation must insure a diffuse fusion of the implanted myoblasts throughout the muscle. If myoblasts are injected in a saline solution and without muscle pre-treatments, the only possibility to achieve a significant homogeneous expression of donor-derived proteins is to perform cell injections very close to each other and through the full muscle thickness [55]. With this technique, the amount of muscle expressing a donor-derived protein depends on the number of injections per muscle volume (Figure 4), that is, the more injections done, the better result is achieved in terms of donorprotein expression in the muscle, such as b-galactosidase or 366

dystrophin in monkeys [56,57] or dystrophin in DMD patients [25-27]. Looking to make a difference from the earlier clinical trials in which myoblast transplantation was performed by few injections away from each other, this strategy was called a high-density injections protocol [3], meaning by ‘density’ the amount of injections per volume of muscle. Is it possible to increase the efficiency of myoblast injections?

3.2

It is obvious that reducing the number of cell injections per volume of muscle will facilitate transplantation. To achieve that, the amount of muscle expressing a therapeutic protein (e.g., dystrophin in DMD) per cell injection must be increased.

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The factors reducing gene complementation to the vicinity of cell injections seems to be only two: i) the grafted cells fuse only with myofibers around the injection trajectory, and ii) proteins remain in nuclear domains. Therefore, the possibilities to reduce the number of injections are also two: i) to make grafted cells fuse more diffusely, and ii) to increase the nuclear domain of the therapeutic protein. Two factors may explain why the injected cells fuse mostly in the thin regions traversed by the cell injections: i) the transplanted cells lack the ability to move in the recipient muscled and/or ii) a muscle regeneration process is obligatory to draw the grafted cells into fusing with the recipient myofibers and, at least in a normal muscle, this occurs only in the regions damaged by the injections. In the belief that the first factor was the key one, several studies attempted to promote the intramuscular migration of the grafted myoblasts by inducing in them the secretion of enzymes that degrade the extracellular matrix and are crucial for the intra-tissular motion of cells [58-62]. Some of these studies reported an improved migration of myoblasts under particular experimental conditions in vitro and in vivo in mice, and even an improved transplantation success. Nonetheless, tests in non-human primates exposing myoblasts to motogenic factors (basic fibroblast growth factor and IGF-1) produced an enhanced migration in vitro and in some in vivo conditions in immunodeficient mice, but did not increase the success of allotransplantation in macaques [62]. Indeed, recent experiments in macaques indicate that transplanted myoblasts have by themselves the ability to migrate into the muscle, but that they migrate basically when there is a muscle damage and to fuse with regenerating myofibers [63]. More experiments addressed the second factor, with better transplantation results. They increased the uptake of the engrafted cells in the recipient muscle by increasing the number of regenerating myofibers. Intramuscular injection of myotoxins, like phospholipases derived from snake venoms [21,64] and local anesthetics [65,66], were efficient for myoblast transplantation in mice. Snake venom phospholipases like notexin and cardiotoxin are regularly used for studies of myogenic-cell transplantation in mice, and were used in other animal models such as rabbits [67]. Looking for an easier method applicable in humans, a study in mice increased myofiber regeneration in mdx mice by intense muscular exercise, almost doubling the success of myoblast transplantation [68]. In mice, hindering the recipient’s satellite cell proliferation favors the participation of the transplanted cells in muscle regeneration. The most frequent method to inhibit the proliferation of the recipient’s satellite cells is to submit skeletal muscles to high doses of ionizing radiation before transplantation [21,33,34,64]. Freezing the recipient muscles (cryoinjury) combines myofiber necrosis with killing the recipient’s satellite cells, and was used in mice as a pretreatment to increase the engraftment of the implanted myogenic cells [35,36,48,50].

Cryoinjury, which necroses also vessels and nerves, is appropriate for experiments in small muscles of mice but seems unthinkable for clinical use. Up to now, only the co-injection of myoblasts with notexin improved the success of cell transplantation in nonhuman primates, although this was observed only when the injections were highly concentrated in small volumes of muscle [55,69]. The possibility of increasing the nuclear domain of donorderived proteins was poorly studied. There is only one study in mdx mice, reporting a threefold expansion of dystrophin’s nuclear domain after transplanting transgenic myoblasts overexpressing dystrophin 50-fold [70]. Tools for intramuscular cell transplantation In clinical conditions, a protocol of myogenic cell transplantation involving many intramuscular injections makes the manual use of single precision syringes (Figures 1A and 4A) acceptable only for very small volumes of muscle [25,27]. Done in that way, the procedure is extremely slow and very demanding in terms of permanently ensuring cell delivery at the right depth. In a first attempt to partially improve the technique, some dispensers used for repetitive delivery of small volumes of liquid in serological analysis were tested [71]. The monosyringe dispenser PB600-1 from Hamilton was used for myoblast transplantations in monkeys (Figure 4B) [57,71], rabbits [72] and even an adult DMD patient [26]. Nevertheless, its clinical use seems restricted to small muscles or muscle regions and, yet again, the accuracy required to deliver cells specifically in the muscle through a thick skin could be a challenge. For this reason, it could be important to develop tools specialized for the percutaneous intra-tissular injection of cells, focusing on clinical use. As a first step, a semimanual prototype was developed that delivers very small volumes of cell suspension homogeneously through the intramuscular trajectory via several needles at the same time, avoiding as much as possible the delivery of cells into the skin and hypodermis (Figure 4C) [73]. 3.3

4.

The survival of the graft

Producing sufficient amounts of an optimal transplantable cell and delivering them adequately to the recipient must be completed by the survival of the graft. This survival should be analyzed at two periods: early and long-term. Early cell death: a poorly understood but not critical phenomenon

4.1

Several studies indicate that grafted myoblasts undergo a considerable mortality mostly within the first three days after transplantation. This was inferred from the post-transplantation loss of different markers used to label myoblasts in mice [74-79] and pigs [80]. Additionally, morphological evidence of apoptosis (by immunodetection of active caspase 3) and necrosis (by

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histochemical detection of intracellular calcium deposition) was reported among the implanted myoblasts during this early post-transplantation period [79]. The importance of this early cell death is frequently overstated, that is it is fallaciously referred as a factor ‘limiting’ myoblast transplantation. In fact, this phenomenon does not affect the level of cell engraftment, which is actually dependent on the technique of cell implantation. Indeed, not all grafted myoblasts die [78-80] and, in mouse experiments, the proliferation of the surviving cells compensates partially [78] or totally [79] for the cell death. This process of cell death and proliferation is still not well understood. Some studies are contradictory, and, in some cases, this is due to methodological differences and even mistakes [79]. Different factors were blamed of this death, but experiments trying to control each of them never prevented the whole cell death and only minimal enhancements of survival were reported (understanding as ‘survival’ the extrapolation of quantification in the recipient muscle of a label present in the grafted cells). An exception was the administration of an anti- lymphocyte function associated antigen 1 (LFA1) antibody in specific experimental conditions in mice [76]. Initial studies in mice accused acute inflammation of killing the grafted myoblasts [76,81]. A specific study, however, did not find evidence that neutrophils, macrophages or NK cells deplete the grafted myoblast population [82]. Other causes suggested to induce apoptosis of the grafted myoblasts were hypoxia [68] and anoikis [83]. A research group raised the hypothesis that the survival of the myoblast graft is due to a minor subpopulation of cells with specific capacities to evade the early cell death and to proliferate to a great extent [78,84]. This hypothesis leaves unanswered the cause of the cell death but could explain the curious fact that, even if one or more factors could indiscriminately kill the grafted cells, the whole grafted-cell population is never eliminated. Considering that the early cell death never eliminates the cell graft and that the surviving cells proliferate to ensure graft success, the sole possible advantage of controlling this cell death would be, in theory, a reduction of the amount of cells needed for transplantation. A clear type of cell death observed after muscle-cell transplantation in non-human primates is the ischemic necrosis of the inner region of the intramuscular accumulations of injected cells (Figure 5) [85]. Since the implanted cells form avascular collections whose survival depends on the limited diffusion of oxygen and nutrients from the surrounding tissue, only a peripheral layer of around 100 -- 200 µm survives the first hours post-transplantation. Given that the extent of ischemic necrosis depends on the size of the cell graft, a massive ischemic death can be prevented or reduced by transplantation strategies in which formation of too large cell accumulations is avoided, ensuring that most cells stay within 100 -- 200 µm of the surrounding tissue [85]. 368

Long-term survival Immune rejection seems the sole factor menacing the longterm survival of myogenic-cell transplantation. The first description of lymphocyte infiltration with lost of the myoblast graft after allogeneic transplantation was done in mice as early as in 1979 [86]. Posterior reports identified CD8+ and CD4+ cells in these infiltrates [87-89]. Focal infiltrations of CD8+ lymphocytes and lymphocyte invasion of myofibers were observed in macaques that received myoblast allotransplantation with low immunosuppression [56,69,90]. In contrast with early cell death, acute rejection, if not controlled, prevents the success of cell transplantation. Nevertheless, this factor is well defined and there are strategies to control it, essentially pharmacological immunosuppression. Special attention should be given, however, to the choice of immunosuppressive drugs, since some of them can be detrimental for the transplanted cells (for a table about immunosuppressive drugs and myogenic cell transplantation see [3]). Several tests supported the usefulness of tacrolimus for myoblast allotransplantation in mice [21,22] and non-human primates [55,56,69,71,90,91]. For this reason, the most recent clinical trials of myoblast allotransplantation were carried out under tacrolimus monotherapy [25-27]. The first of them, involving nine DMD patients, had a follow-up of just one month [25,27], but in a DMD patient in which the follow-up was of 18 months, donor-derived dystrophin was preserved throughout this period: 27.5% of the myofibers were donor-dystrophin-positive one month post-transplantation and 34.5% at 18 months [26]. Considering the adverse effects of immunosuppression, a major goal in clinical transplantation is specific immune tolerance, that is long-term unresponsiveness to the graft in the recipient, preserving immune responses against pathogens or cancer. Transient immunosuppression allowed immune tolerance to myoblast grafts in some mouse strains [92], although not in other mouse strains [35,92] nor in non-human primates [55]. For myoblast allotransplantation in mice, central tolerance via mixed chimerism [93,94] was superior to anti-CD154 administration and donor-specific transfusion [95]. Nevertheless, central tolerance in the context of myogenic-cell allotransplantation would not include neoantigens in the hybrid myofibers which would need also peripheral tolerance [96]. A different experimental strategy to elude immunosuppression is the autotransplantation of cells genetically corrected ex vivo (for a recent review detailing this topic see [54]). Results in mice support a potential development of this strategy, and the viability of this approach was verified in non-human primates albeit for only one month post-transplantation [57]. This strategy avoids incompatibility of the MHC among the recipient and the transplanted mononuclear cells, but leaves the possibility of eliciting a specific immune response if the genetically-modified cells express epitopes not shared with the recipient [97]. 4.2

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Figure 5. Ischemic necrosis in a collection of grafted myoblasts one day after a unique injection of 20 106 myoblasts in the skeletal muscle of a macaque. The whole section of a muscle biopsy is shown (the epimysium is observed in the top of the biopsy), stained for histochemical detection of the oxidative enzyme nicotinamide adenine dinucleotide reduced diaphorase. The cell-suspension infiltrated the muscle in a polymorphic pattern, giving rise to cell accumulations of different sizes (a -- e). The arrowheads circumscribe these collections of transplanted myoblasts. Two regions are clearly delimitated in the largest accumulations of implanted cells (a -- c): a peripheral ring strongly stained (living cells with oxidative activity) and a central region almost devoid of oxidative reaction (necrosed cells without oxidative activity). The small accumulations of implanted cells (d, e) do not exhibit an ischemic central region. Scale bar: 500 µm.

5.

Expert opinion

Paraphrasing Brazelton and Morris [98], we realize that several ‘promising’ transplantable cells were not included in this review. These authors, in a review of pharmacology, made a distinction between a clinical drug and a molecule, stressing that ‘few molecules become drugs’ and specifying that they called molecule ‘a substance that, when injected into a rodent, results in abstracts, publications, grant funds, academic promotions and public stock offerings’ [98]. Since sometimes a similar concern could be pertinent in the context of cell transplantation, we kept some distance from reports claiming the discovery in mice of cells with huge myogenic potential (on the other hand, there are several reviews dealing with that topic). This does not imply a blind reluctance but rather a wary eye on that topic: there were already shooting stars in the sky of cell-based therapies in myology, such as the overvaluation of mouse observations about the potential of bone marrow transplantation to provide circulating myogenic progenitors able to restore dystrophin in DMD skeletal

muscles, which when analyzed in a human patient or tested experimentally in dystrophic dogs with dystrophinopathy showed to be nil or irrelevant [99,100]. It is therefore important to assess in the best animal model as possible the extent to which a ‘promising’ cell type has actually some clinical potential or, in contrast, to what extent the properties reported are only curiosities observed in specific experimental conditions and/or unique to the mouse biology. We also realize that the basis of the present review were studies in which the transplanted cells were the mononuclear precursor cells whose function is to fuse among themselves in order to form myotubes giving rise to myofibers, that is, myoblasts. In fact, regarding clinical transplantation, adult myoblasts were the only myogenic cells properly tested in myology, restoring dystrophin in variable amounts of myofibers in DMD patients and forming putative new myofibers. This was possible by using a method of intramuscular implantation previously developed in non-human primates and a quite suitable control of acute rejection with an immunosuppressant also tested in non-human primates. If other myogenic cells are finally tested clinically in the future for intramuscular transplantation, it is possible that they will be affected by the same challenges as adult myoblasts, that is, the restricted integration to the skeletal muscle only in the regions around the injection trajectories and the need to control their acute rejection with immunosuppression. These are thus the two important challenges in order to facilitate the clinical applicability of intramuscular cell transplantation in clinical settings with any benefit to myopathic patients: i) to expand the cell engraftment while, at the same time, reducing as much as possible the complications inherent to a protocol of cell delivery using a high density of cell injections, and ii) to reduce the toxicity of the approaches to control acute rejection. The last issue could be attained by refinements of the immunosuppression protocols or, ideally, through efficient strategies of immune tolerance. These are major goals in the global field of transplantation, and myogenic cell transplantation will be subsidiary to the progress in this research. Otherwise, cell transplantation potentially offers a specific alternative to avoid acute rejection or, at least, to control it with lower nuisances to the recipient, through the in vitro manipulation of cells. Regarding the promises of gene therapy or the intravascular delivery of myogenic cells, the difficulties associated with the intramuscular transplantation of cells seems to relegate this approach or to deny the difficulties through the quest for a ‘magic bullet’. However, as the treatment of cancer needs frequently the combination of different approaches such as surgery, radiotherapy and chemotherapy, the treatment of myopathies such as DMD could also need the combination of several approaches. It is logical to predict that effectively correcting the molecular problem in a disease such as DMD will stop its progression, but it also should be clear that this does not restore the lost muscle. Restoring muscle in patients in whom too many myofibers were lost, will inevitably need

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strategies overlapping cell transplantation and tissue engineering. Thus, a third important challenge in the field of cell transplantation in myology is to address some research to clarify the factors that could contribute to forming new skeletal muscle in muscles in which myofibers were lost and replaced by fibrosis and fat tissue. Another weakness in the field of cell transplantation is that, when moving to clinical trials, some researchers seem to disregard the actual behavior of the transplanted cells. As in the early clinical trials of myoblast transplantation, there is a risk with proceeding as if the simple act of injecting cells into a tissue, no matter how, is ‘cell transplantation’. Something similar to putting a kidney from a donor into a recipient without performing vascular and ureterovesical anastomoses being called ‘kidney transplantation’: the term is semantically correct but the procedure, done that way, is a medical aberration. This obvious statement, however, does not seem so evident in the field of cell transplantation: often arbitrarily chosen amounts of cells are injected, no matter how, with no more rationale that the hypothetical way the cells are expected to act, and no more experimental basis than mouse experiments not designed to clarify the transplantation parameters which could be needed in the larger organs of a human. Therefore, a good knowledge of the behavior of the transplanted cells in the recipient is crucial for designing Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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adequate clinical strategies for cell transplantation, avoiding exaggerated expectations and rapid frustration. Ideally, the ultimate goal of any treatment for a myopathic patient would be a complete cure, no matter what is the state of evolution of the disease. Whatever the experimental therapeutic approach under study, this objective appears, in the short- and medium-term, unattainable. A more realistic goal for the short- or medium-term, regarding myogenic cell transplantation, would be to at least mitigate the worst consequences of the disease, and, whenever possible, improving the function of muscle groups to ensure a better quality of life. For a patient with a severe handicap, every little improvement will be an achievement.

Acknowledgements This work was supported by a grant of the Jesse’s Journey Foundation for Gene and Cell Therapy of Canada to Daniel Skuk.

Declaration of interest JP Tremblay has shares in CellGene Inc., a biotechnological company created to accelerate the development of cell therapies. D Skuk declares no conflict of interest.

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Lipton BH, Schultz E. Developmental fate of skeletal muscle satellite cells. Science 1979;205:1292-4 This pioneer paper was one of the first two publications on myogenic-cell transplantation, and described for the first time, to our knowledge, that intramuscularly implanted myoblasts either fuse with the recipient’s myofibers to be incorporated in large myofibers or fuse between themselves to form new small myofibers.

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Watt DJ, Lambert K, Morgan JE, et al. Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse. J Neurol Sci 1982;57:319-31 Another pioneer paper on myogenic-cell transplantation, demonstrating that the donor-derived and recipient-derived proteins coexist in skeletal muscles of mice following myoblast transplantation.

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Kikuchi T, Doerr L, Ashmore CR. A possible mechanism of phenotypic expression of normal and dystrophic genomes on succinic dehydrogenase activity and fiber size within a single myofiber of muscle transplants. J Neurol Sci 1980;45:273-86

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Yao SN, Kurachi K. Implanted myoblasts not only fuse with myofibers but also survive as muscle precursor cells. J Cell Sci 1993;105:957-63 This study has the merit of being the first demonstration, to our knowledge, that some transplanted myoblasts remain as mononuclear muscle-precursor cells in the recipient’s muscles.

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Partridge TA, Morgan JE, Coulton GR, et al. Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature 1989;337:176-9 A seminal paper that institutes the principle that cell transplantation could be a way to introduce donor-derived dystrophin into dystrophin-deficient dystrophic muscle. This paper also states some of the main problems of myoblast transplantation.

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Karpati G, Pouliot Y, Zubrzycka-Gaarn E, et al. Dystrophin is expressed in mdx skeletal muscle fibers after normal myoblast implantation. Am J Pathol 1989;135:27-32

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Leriche-Guerin K, Anderson LV, Wrogemann K, et al. Dysferlin expression after normal myoblast transplantation in SCID and in SJL mice. Neuromuscul Disord 2002;12:167-73 Mendell JR, Kissel JT, Amato AA, et al. Myoblast transfer in the treatment of Duchenne’s muscular dystrophy. N Engl J Med 1995;333:832-8 This study was the first, to our knowledge, to confirm that donor-derived dystrophin can be detected in myofibers of a DMD patient following normal myoblast allotransplantation under cyclosporine immunosuppression. Skuk D, Goulet M, Roy B, et al. Dystrophin expression in muscles of Duchenne muscular dystrophy patients after high-density injections of normal myogenic cells. J Neuropathol Exp Neurol 2006;65:371-86 This clinical trial is the first demonstration, to our knowledge, that donor-derived dystrophin can be systematically observed in myofibers of DMD patients receiving normal myoblast allotransplantations by combining a method of intramuscular implantation of ‘high-density injections’ with a tacrolimus immunosuppression. The paper describes several other new clinical observations, such as the putative neoformation of small myofibers exclusively from donor origin and the putative presence of donor-derived satellite cells, and analyzes the immune responses in this context. Skuk D, Goulet M, Roy B, et al. First test of a “high-density injection” protocol for myogenic cell transplantation throughout large volumes of muscles in a Duchenne muscular dystrophy patient: eighteen months follow-up. Neuromuscul Disord 2007;17:38-46 This clinical paper is the first demonstration, to our knowledge, that donor-derived dystrophin can be maintained as long as desired (18 months in this case) in a DMD patient that received normal myoblast allotransplantation, as long as tacrolimus immunosuppression is maintained. This was also the first time that a protocol of ‘high-density cell injections’ was tested in several

Expert Opin. Biol. Ther. (2011) 11(3)

muscle regions of a DMD patient, showing the feasibility of the method. 27.

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Skuk D, Roy B, Goulet M, et al. Dystrophin expression in myofibers of Duchenne muscular dystrophy patients following intramuscular injections of normal myogenic cells. Mol Ther 2004;9:475-82 A preliminary report of the first clinical trial, to our knowledge, demonstrating that donor-derived dystrophin can be systematically observed in myofibers of DMD patients receiving normal myoblast allotransplantations by combining a method of intramuscular implantation of ‘high-density injections’ with tacrolimus immunosuppression.

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Gussoni E, Blau HM, Kunkel LM. The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat Med 1997;3:970-7 An interesting analysis of the relationship between donor-derived nuclei and dystrophin expression in biopsies of DMD patients receiving allotransplantations of normal myoblasts

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regarding the regenerative capacity of skeletal muscles. This article highlights the importance of performing experiments in large mammals to test the observations involving muscle regeneration in mice.

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Irintchev A, Langer M, Zweyer M, et al. Functional improvement of damaged adult mouse muscle by implantation of primary myoblasts. J Physiol (Lond) 1997;500:775-85 This paper demonstrated for the first time, to our knowledge, in mice that some of the intramuscularly implanted myoblasts remain specifically as satellite cells in the recipient’s muscles.

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Kinoshita I, Vilquin JT, Tremblay JP. Mechanism of increasing dystrophin-positive myofibers by myoblast transplantation: study using mdx/beta-galactosidase transgenic mice. Acta Neuropathol 1996;91:489-93

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Ehrhardt J, Brimah K, Adkin C, et al. Human muscle precursor cells give rise to functional satellite cells in vivo. Neuromuscul Disord 2007;17:631-8 This study was the first, to our knowledge, to show that human myoblasts (of fetal origin) remain as mononuclear muscle-precursor cells in the recipient’s muscles in immunodeficient mice.

Irintchev A, Rosenblatt JD, Cullen MJ, et al. Ectopic skeletal muscles derived from myoblasts implanted under the skin. J Cell Sci 1998;111:3287-97 This is the only study, to our knowledge, showing the capacity of myoblasts in mice to form neomuscles after ectopic transplantation.

Borisov AB. Regeneration of skeletal and cardiac muscle in mammals: do nonprimate models resemble human pathology? Wound Repair Regen 1999;7:26-35 An enlightening and well-documented analysis of the differences between small rodents and large mammals

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Sampaolesi M, Blot S, D’Antona G, et al. Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature 2006;444:574-9 This study was the first, to our knowledge, to report that some intramuscularly transplanted myoblasts and CD133+ cells of postnatal human origin form satellite cells in muscles of immunodeficient mice.

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Sampaolesi M, Torrente Y, Innocenzi A, et al. Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 2003;301:487-92

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Skuk D, Goulet M, Roy B, Tremblay JP. Myoblast transplantation in whole muscle of nonhuman primates. J Neuropathol Exp Neurol 2000;59:197-206

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Skuk D, Goulet M, Roy B, Tremblay JP. Efficacy of myoblast transplantation in nonhuman primates following simple intramuscular cell injections: toward defining strategies applicable to humans. Exp Neurol 2002;175:112-26

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El Fahime E, Mills P, Lafreniere JF, et al. The urokinase plasminogen activator: an interesting way to improve myoblast migration following their transplantation. Exp Cell Res 2002;280:169-78

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Lafreniere JF, Mills P, Bouchentouf M, Tremblay JP. Interleukin-4 improves the migration of human myogenic precursor cells in vitro and in vivo. Exp Cell Res 2006;312:1127-41

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Mills P, Dominique JC, Lafreniere JF, et al. A synthetic mechano growth factor E Peptide enhances myogenic precursor cell transplantation

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Lafreniere JF, Caron MC, Skuk D, et al. Growth factor coinjection improves the migration potential of monkey myogenic precursors without affecting cell transplantation success. Cell Transplant 2009;18:719-30

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Skuk D, Goulet M, Paradis M, Tremblay JP. Intramuscular migration capacity of transplanted myoblasts after subcutaneous implantation: a study in nonhuman primates. Tissue Eng 2009;15:713

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Cesar M, Roussanne-Domergue S, Coulet B, et al. Transplantation of adult myoblasts or adipose tissue precursor cells by high-density injection failed to improve reinnervated skeletal muscles. Muscle Nerve 2008;37:219-30 Richard PL, Gosselin C, Laliberte T, et al. A first semimanual device for clinical intramuscular repetitive cell injections. Cell Transplant 2010;19:67-78 A first attempt at developing a specific device for the intramuscular injection of cells. Beauchamp JR, Morgan JE, Pagel CN, Partridge TA. Quantitative studies of efficacy of myoblast transplantation. Muscle Nerve 1994;(suppl 1):S261

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Guerette B, Skuk D, Celestin F, et al. Prevention by anti-LFA-1 of acute myoblast death following transplantation. J Immunol 1997;159:2522-31

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Skuk D, Roy B, Goulet M, Tremblay JP. Successful myoblast transplantation in primates depends on appropriate cell delivery and induction of regeneration in the host muscle. Exp Neurol 1999;155:22-30 Kinoshita I, Vilquin JT, Asselin I, et al. Transplantation of myoblasts from a transgenic mouse overexpressing dystrophin produced only a relatively small increase of dystrophin-positive

Skuk D, Goulet M, Tremblay JP. Use of repeating dispensers to increase the efficiency of the intramuscular myogenic cell injection procedure. Cell Transplant 2006;15:659-63

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Beauchamp JR, Morgan JE, Pagel CN, Partridge TA. Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J Cell Biol 1999;144:1113-22 This paper described the most valuable method to analyze the early donor-cell death versus the donor-cell proliferation following myogenic cell transplantation. It was the first observation, to our knowledge, that both phenomena arise simultaneously during the first post-transplantation days in such a way that cell proliferation compensates for cell death. Skuk D, Caron NJ, Goulet M, et al. Resetting the problem of cell death following muscle-derived cell Expert Opin. Biol. Ther. (2011) 11(3)

transplantation: detection, dynamics and mechanisms. J Neuropathol Exp Neurol 2003;62:951-67 80.

Holzer N, Hogendoorn S, Zurcher L, et al. Autologous transplantation of porcine myogenic precursor cells in skeletal muscle. Neuromuscul Disord 2005;15:237-44

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Guerette B, Asselin I, Skuk D, et al. Control of inflammatory damage by anti-LFA-1: increase success of myoblast transplantation. Cell Transplant 1997;6:101-7

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Sammels LM, Bosio E, Fragall CT, et al. Innate inflammatory cells are not responsible for early death of donor myoblasts after myoblast transfer therapy. Transplantation 2004;77:1790-7

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Bouchentouf M, Benabdallah BF, Rousseau J, et al. Induction of Anoikis following myoblast transplantation into SCID mouse muscles requires the Bit1 and FADD pathways. Am J Transplant 2007;7:1491-505

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Cousins JC, Woodward KJ, Gross JG, et al. Regeneration of skeletal muscle from transplanted immortalised myoblasts is oligoclonal. J Cell Sci 2004;117:3259-69

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Irintchev A, Zweyer M, Wernig A. Cellular and molecular reactions in mouse muscles after myoblast implantation. J Neurocytol 1995;24:319-31 One of the first studies describing the immune responses in mice following myogenic-cell transplantation.

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Kinoshita I, Vilquin JT, Gravel C, et al. Myoblast allotransplantation in primates. Muscle Nerve 1995;18:1217-18 This was, to our knowledge, the first complete approach to the study of the acute rejection response following myogenic-cell transplantation in non-human primates.

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transplantation at the age of 1 year, showing that 13 years thereafter less than 1% of the myofibers showed nuclei from donor origin. 100. Dell’Agnola C, Wang Z, Storb R, et al. Hematopoietic stem cell transplantation does not restore dystrophin expression in Duchenne muscular dystrophy dogs. Blood 2004;104:4311-18 . A long-term study in seven dogs with dystrophinopathy demonstrating that successful transplantation of bone marrow from normal littermates does not produce detectable contribution of bone-marrow-derived cells to skeletal muscle.

Affiliation

Daniel Skuk†1 & Jacques P Tremblay2 † Author for correspondence 1 CHUQ Research Center -- CHUL, Neurosciences Division -- Human Genetics, 2705 Boulevard Laurier, Quebec, Quebec G1V 4G2, Canada E-mail: [email protected] 2 Centre de Recherche du Centre Hospitalier Universitaire de Que´bec, Sainte-Foy, Quebec, Canada

Drug Evaluation

Platelet-derived growth factor applications in periodontal and peri-implant bone regeneration 1.

Introduction

2.

Osseous regeneration

3.

PDGF for bone regeneration in periodontal and peri-implant sites

Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by HINARI on 02/10/11 For personal use only.

4.

Expert opinion

Darnell Kaigler, Gustavo Avila, Leslie Wisner-Lynch, Marc L Nevins, Myron Nevins, Giulio Rasperini, Samuel E Lynch & William V Giannobile† †

University of Michigan School of Dentistry, Department of Periodontics and Oral Medicine and Michigan Center for Oral Health Research, Ann Arbor, MI, USA

Introduction: Achieving successful tissue regeneration following traditional therapeutic protocols, combining bone grafts and barrier membranes, may be challenging in certain clinical scenarios. A deeper understanding of periodontal and peri-implant wound healing and recent advances in the field of tissue engineering have provided clinicians with novel means to obtain predictable clinical outcomes. The use of growth factors such as recombinant human platelet-derived growth factor-BB (rhPDGF) with biocompatible matrices to promote tissue regeneration represents a promising approach in the disciplines of periodontology and implantology. Areas covered: This review covers the basic principles of bone and periodontal regeneration, and provides an overview of the biology of PDGF and its potential to predictably and reproducibly promote bone regeneration in regular clinical practice. The results of preclinical and clinical human studies evaluating the effectiveness of growth-factor-enhanced matrices are analyzed and discussed. Expert opinion: Current available evidence supports the use of rhPDGF-enhanced matrices to promote periodontal and peri-implant bone regeneration. Keywords: dental implants, periodontal regeneration/periodontal diseases, platelet-derived growth factor, regenerative medicine Expert Opin. Biol. Ther. (2011) 11(3):375-385

1.

Introduction

Clinicians are frequently faced with the challenge of treating patients with significant alveolar bone loss due to periodontal disease, congenital abnormalities, tumors, traumatic injury, or resorption secondary to tooth loss. Conventional treatment procedures may be ineffective in achieving bone regeneration, leaving both the clinician and the patient dissatisfied with the outcome. Growth factors (GFs) have long been believed to have the potential to accelerate the healing process and, therefore, enhance tissue regeneration in challenging clinical scenarios. GFs are natural biological mediators that regulate key cellular events that are part of the process of tissue repair and regeneration. After binding of GFs to specific cell membrane receptors of target cells, intracellular signaling pathways are induced, which typically results in the activation of genes that may ultimately change cellular activity and phenotype. However, the effect of each GF is regulated through a complex system of feedback loops, which involve other GFs, enzymes and binding proteins. Recent advances in the areas of cellular and molecular biology allowed better understanding of the functions of GFs and their participation in the different phases of wound healing. In vitro and in vivo studies have confirmed that GFs can enhance

10.1517/14712598.2011.554814 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

375

rhPDGF-BB

Article highlights. . .

.

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.

.

Polypeptide growth factors play an important role in the natural oral wound healing process. Delivery of platelet-derived growth factor (PDGF) can offer an approach to repair tooth-supporting (periodontal) and oral implant osseous defects. New intraoral applications using growth factor delivery are demonstrating indications and potential to repair edentulous ridge defects including extraction socket defects and in horizontal and vertical ridge repair. Novel biomaterials are being developed that can control the release of growth factors using nanospheres and other drug delivery systems to sustain the production of PDGF at local bone defects. Future work will also be applying new drug delivery systems exploiting both gene transfer of PDGF DNA as well as the targeting of PDGF-producing progenitor cells to local oral osseous defects.

This box summarizes key points contained in the article.

the proximal tibia [3]. However, the use of autogenous bone grafts may also involve a series of disadvantages, such as limited availability and increased morbidity associated with a second surgical site. When autogenous bone is not readily available, alternative bone sources can be utilized. Tissue matrices, including allogenic, xenogenic and synthetic graft materials are available for use. These bone substitutes function primarily by passively guiding or ‘conducting’ cell migration through the matrix, eventually leading to repair of the defect. These materials may be used alone, in combination with autogenous grafts or in combination with titanium meshes, barrier membranes, or other passive materials designed to act as a physical guide or cell-occlusive element [4]. While these options are useful for maintaining space and a framework for tissue deposition during the process of bone regeneration, results obtained with passive therapeutic matrices have been variable, depending upon their inherent physical and chemical properties as well as the patient’s individual healing response [5,6]. Tissue engineering approaches for bone regeneration

2.2

the capacity of tissues to regenerate by regulating cell chemoattraction, differentiation and proliferation [1,2]. One of the most extensively studied GFs is plateletderived growth factor (PDGF). This growth factor has been well characterized and has broad wound healing activities in both hard (bone) and soft (skin, gingival) tissue. Recently, a combination of purified recombinant human PDGF (rhPDGF) with synthetic ceramic matrices [betatricalcium phosphate (b-TCP)] has been proposed as a periodontal regenerative agent (Box 1). The efficacy of growth-factor-enhanced matrices in periodontal therapies has been rigorously examined in a variety of preclinical animal models and long-term human clinical studies. The results of these studies are discussed in this review, along with the potential for PDGF-enhanced matrices to predictably and reproducibly promote bone regeneration in clinical practice. 2.

Osseous regeneration

Bone grafting A variety of biologic and synthetic materials to augment existing bone and enhance bone regeneration are available to clinical practitioners. Autogenous bone grafting has been traditionally considered the gold standard for treating bone defects or deficiencies. The clinical benefits derived from the use of autogenous bone grafts are primarily due to i) the presence of osteoblasts and osteoprogenitor cells within the graft; ii) release of naturally occurring GFs and other biochemical mediators; and iii) the osteoconductive nature of the substrate itself. Autogenous grafts may be obtained from intraoral sites such as the mandibular symphysis or the ascending ramus, or may be harvested from extraoral sites such as the iliac crest or 2.1

376

Novel tissue engineering techniques have been recently developed to regenerate bone in challenging defect sites, where spontaneous repair is not achievable [7]. On a fundamental level, bone tissue engineering involves supplying four basic elements required for bone formation to the defect site: adequate blood supply, bone-forming cells, scaffolds or matrices, and signaling molecules, such as GFs (Figure 1). Osteoblasts or bone-precursor cells may be provided by direct scaffold seeding (in vitro tissue engineering) or they may be induced by biochemical mediators to migrate into the scaffold from the marginal host bone (in vivo tissue engineering). Scaffolds provide a foundation to support cell attachment and proliferation at the defect site, and facilitate blood clot stabilization, which additionally prevents tissue collapse, all essential events in the initial stages of healing regenerative processes [8]. Some scaffold materials used in bone regeneration are collagen, autogenous or allograft bone, resorbable polymers and porous calcium phosphate ceramics. GFs serve to stimulate native cell migration into the defect site and increase proliferation of these cells to populate the scaffold through specific chemotactic and mitogenic signals. Different scaffold materials (naturallyderived and synthetic) are available that are biocompatible with GFs and through their use enable the delivery of GFs to different sites. With newer bioengineered materials, GFs can be incorporated directly into the scaffold and their release from these materials controlled over a predetermined period of time [9]. This mechanism of action contrasts with that of bone morphogenetic proteins (BMPs), which primarily exert their effect through osteoinduction (stimulating differentiation of mesenchymal cells into bone-forming cells) [10,11].

Expert Opin. Biol. Ther. (2011) 11(3)

Kaigler, Avila, Wisner-Lynch, Nevins, Nevins, Rasperini, Lynch & Giannobile

Box 1. Drug summary. Drug name Phase Indication Pharmacology description Route of administration Pivotal trial(s)

rhPDGF-BB III Bone regeneration Platelet-derived growth factor alpha and beta receptor agonist Local implantation NCT00583375, NCT01008891 [44]

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Pharmaprojects -- copyright to Citeline Drug Intelligence (an Informa business). Readers are referred to Pipeline (http://informa-pipeline. citeline.com) and Citeline (http://informa.citeline.com).

Cells

Blood supply Scaffolds and matrices

Molecular mediators

Figure 1. Four basic elements are required for periodontal repair and regeneration: adequate blood supply and wound stability, a source of bone and ligament forming cells, a supporting scaffold or matrix, and growth factors to regulate cell migration, proliferation, synthesis and angiogenesis for revascularization of the site.

PDGF for bone regeneration in periodontal and peri-implant sites

3.

GFs and BMPs have been the focus of considerable attention in recent years by dental and orthopedic researchers [12,13]. These biological molecules offer the possibility of using biomaterials that are readily available for the clinician to attempt bone regeneration in a controlled, predictable manner. While a considerable number of bone GFs and morphogens have been identified, one of the most extensively characterized growth factors for clinical applications is PDGF. In the late 1980s, Lynch and co-workers first discovered in an animal study that PDGF promotes regeneration of periodontal tissues including bone, cementum and periodontal ligament [14]. Since then, numerous studies have been published providing a deeper understanding of the mechanism of action and therapeutic potential of this molecular mediator. PDGF is a naturally occurring protein that is found abundantly in the bone matrix in at least three different

forms: PDGF-AA, PDGF-AB and PDGF-BB [15]. This GF is locally released by blood platelets during clotting following soft- or hard-tissue injury [16]. Once it is released from the platelets, PDGF binds to specific cell-surface receptors promoting rapid cell migration (chemotaxis), and proliferation (mitogenesis), in the area of injury [17]. In vitro and in vivo studies have demonstrated that PDGF is a potent chemotactic and mitogenic factor for gingival and periodontal ligament fibroblasts, cementoblasts and osteoblasts [14,18-20]. While growth factor proteins have been shown to be potent stimulators of wound repair, the ability to utilize concentrated forms of these proteins contained within blood platelets for routine oral surgical treatment was not introduced until 1998, when Marx and co-workers proposed the use of autologous platelet concentrates [21]. The preparation of platelet concentrates consists of isolating the platelets naturally present in whole autologous blood by a selective process of centrifugation, and subsequently activating them to release their growth factor content, including superphysiologic concentrations of PDGF, TGF-b and IGF-I, among others [22]. These factors are directly applied to the treatment site in order to promote tissue regeneration or repair. Thrombin/calcium preparations initiate clotting, including the conversion of fibrinogen to fibrin, resulting in a clinically useful platelet-rich plasma (PRP) gel that can additionally improve the handling and efficacy of particulate autografts and bone substitutes. A variety of protocols to produce platelet concentrates have been described to date. However, studies evaluating the effect of platelet gel concentrates alone, or in combination with osteoconductive matrices, on graft maturity, bone density and new bone formation in a number of different clinical applications have demonstrated somewhat variable outcomes ranging from excellent results in some studies to no apparent benefit in others [23]. The differences in outcomes are thought to be a result of variability in platelet concentration as well as individual patient healing responses. In summary, platelet concentrates provide good handling characteristics alone or in combination with a variety of matrices, however with the primary disadvantage of the technique being the need to obtain blood from the patient and lack of a predictable response following treatment according to current evidence [23]. To overcome some of these limitations other therapeutic approaches have been developed. Advances in recombinant technology have lead to the synthesis of proteins in a controlled manner, which in turn enables production of concentrated and purified molecules in large quantities. This has led to the development and commercialization of recombinant growth factor/matrix combination products. Combination products represent an emerging new trend in regenerative therapeutics and have gained increasing attention as a strategy to optimize tissue regeneration. These products synergize tissue-specific matrices with highly concentrated bioactive proteins to actively recruit progenitor cells for the treatment of tissue deficiencies. The ability to

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combine highly concentrated forms of signaling proteins with scaffolds has enabled clinical researchers to develop improved regenerative products taking advantage of the physical and chemical characteristics required for specific cell attachment, growth and differentiation. Recombinant human platelet-derived growth factor (rhPDGF) was the first recombinant protein to be approved by the US FDA for treatment of chronic foot ulcers in diabetic patients (Regranex, Ethicon, Inc. Somerville, NJ) [24,25]. Widespread use in this application has established the safety and effectiveness of PDGF for soft tissue regeneration [26]. Additionally, rhPDGF for bone regeneration has been rigorously tested in preclinical studies, which indicate that PDGF has the potential to be used to direct and control bone regeneration in humans. For example, a tibial osteotomy study using PDGF in rabbits demonstrated that this protein substantially increased the rate of fracture repair as compared with untreated control sites [27]. In addition, the biomechanical strength of the repair tissue in rhPDGF-treated animals was not significantly different from un-operated normal intact bone. Furthermore, when PDGF was injected subperiosteally in long bones, it induced intramembranous bone formation [28]. In a detailed study in osteoporotic animals involving dual-energy X-ray absorptiometry (DEXA) bone density scans, quantitative computed tomography scans, biomechanical testing and histological analyses, periodic systemic injection of rhPDGF substantially increased bone density in the long bones and in the spine [29]. Preclinical studies using PDGF for periodontal and peri-implant regeneration

3.1

In order to evaluate the potential and safety of this therapy, extensive in vivo preclinical studies have been performed using PDGF alone or in combination with other GFs such as IGF to treat periodontal and peri-implant defects. In a previously mentioned study, Lynch and co-workers first published evidence of the regenerative potential of PDGF-BB when used to treat naturally occurring periodontal defects in dogs [14]. Most notably, this study showed increased cellular activity after treatment with PDGF-BB, leading to increased bone, cementum and periodontal ligament regeneration. In a related study examining its use around dental implants, direct application of a rhPDGF/IGF mixture into implant sites in dogs produced a two- to three-times increase in the number of peri-implant spaces filled with bone at early time points [30]. Promising results were also seen in immediate extraction socket implants treated with polytetrafluoroethylene (PTFE) membranes and PDGF/IGF. Bone density and bone-toimplant contact were increased twofold for the growthfactor-treated sites, as compared with the membrane alone or membranes combined with bone grafts [31]. These early studies were instrumental in establishing preclinical evidence for the potential of PDGF treatment in not only periodontal, but also peri-implant sites. 378

The adjunctive effect of rhPDGF therapy for implant site development indications has also been investigated in animal models. Simion and co-workers conducted an animal study to investigate the outcomes of vertical bone augmentation following the application of rhPDGFBB [32]. Bilateral severe bone defects were surgically created in the mandibles of foxhound dogs and three months later, each of the defects was treated following one of these three augmentation protocols: i) anorganic bovine bone blocks covered with a collagen membrane, ii) anorganic bovine bone blocks infused with rhPDGF-BB, or iii) anorganic bovine bone blocks infused with rhPDGFBB covered with a collagen membrane. In all cases, anorganic bovine bone blocks were secured using two titanium dental implants. At four months the results were more favorable for the group that included the blocks with rhPDGF-BB, illustrating the importance of the periosteal blood supply in the outcomes of GF therapy. This study demonstrated that the rhPDGF-BB-infused matrix significantly enhanced bone formation and gingival healing in large, critical-size alveolar bone defects. Radiographic and histological analysis indicated that the greatest bone regeneration occurred for the rhPDGF-BB-infused graft block without the collagen membrane (Figure 2). Unlike traditional guided bone regeneration procedures, the membrane appears to block the migration of boneforming cells into the scaffold. Histological analysis indicated that bone formation occurred from both the coronal and apical surfaces of the rhPDGF-BB-treated graft, suggesting that osteoblasts and other bone-forming precursor cells were recruited into the graft from both the superior (coronal, periosteal surface) and the inferior (apical, marrow spaces) boundaries. Additionally, bone growth appeared more robust from the periosteal surface when compared with the bone formation observed at the original osseous crest. According to these results, rhPDGF-BB may have exerted a potent chemotactic effect on osteogenic cells present in the periostium, when a barrier membrane was not placed. In addition to applications of PDGF in vertical ridge augmentation procedures, preclinical studies by Schwarz and coworkers recently evaluated early healing outcomes following horizontal ridge augmentation [33]. Bilateral mandibular surgically created defects were treated with either beta-tricalcium phosphate (b-TCP) covered with a collagen membrane (CM) or a combination of b-TCP and CM plus rhPDGF-BB, following a splitmouth design. The results of this study revealed that the group that received rhPDGF-BB showed better results in terms of mineralized tissue and total augmented area at 3 weeks. Taken together, the promising preclinical evidence of PDGF therapy in periodontal, peri-implant, and bone augmentation indications established the foundation for therapeutic evaluation of PDGF in clinical applications.

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Figure 2. rhPDGF is associated with increased bone formation in a canine alveolar ridge augmentation model. The baseline surgically created defect (A, B), was treated using guided bone regeneration (GBR) with equine bone block infused with rhPDGF-BB (C). Excellent bone formation is seen at four months in the treated site (D). E and F show the histological outcomes, with special emphasis on the remarkable bone-to-implant contact achieved. Reprinted with permission from Quintessence Publishing Co., Inc. [48].

Clinical studies using PDGF for periodontal and peri-implant regeneration

3.2

The use of rhPDGF for dental implant site development (i.e., sinus augmentation) [34], horizontal bone augmentation [35] and ridge preservation [36] has been investigated in human studies, however the primary focus of clinical studies using this agent has been in periodontal and peri-implant regenerative indications [37]. An early human clinical trial to evaluate the effect of rhPDGF/IGF treatment applied to osseous periodontal defects was reported by Howell and co-workers [38]. The experimental sites received direct application of the GFs contained in a methylcellulose matrix to improve retention. A statistically significant increase in alveolar bone formation was seen in the growth-factor-treated sites at nine months post-operatively, as compared with untreated control sites. Average bone height for the PDGF/IGF group was 2.08 mm and 43.2% osseous defect fill was achieved, as compared with 0.75 mm new bone height and 18.5% fill for the control sites. Based on the principles of tissue engineering, the use of a growth-factor-enhanced matrix for periodontal regeneration consisting of rhPDGF-BB in combination with an osteoconductive scaffold (i.e., autograft, allograft, xenograft or a synthetic matrix, such as beta-TCP) was proposed [39]. The rationale underlying this approach is that PDGF stimulates angiogenesis, promotes cell migration into the bone defect

from the surrounding tissue margins and upregulates cell proliferation [40]. The matrix, in addition to its role as a growth factor delivery vehicle, provides mechanical support for migrating cells and contributes to the formation of new bone, cementum and/or periodontal ligament. Early human clinical studies utilized rhPDGF-BB combined with bone allograft. In one of these studies the effectiveness of rhPDGF-BB treatment for extensive interproximal intrabony defects and Class II furcation lesions associated with advanced periodontitis was evaluated [41,42]. Surgical procedures involved full-thickness flap reflection, thorough debridement of the defect site, and subsequent filling of the defect with demineralized freeze-dried bone allograft (DFDBA) presoaked in a solution containing rhPDGFBB at three different concentrations (0.5, 1.0 or 5.0 mg/ml). Control defect sites were filled with commercially available anorganic bovine bone in collagen (Figure 3). Clinical probing depths and attachment levels were assessed at periodic intervals up to nine months post-operatively, at which time the treated teeth were removed together for histological analysis. The study results indicated that substantial improvements in vertical and horizontal probing depths over baseline levels were achieved for all sites treated with rhPDGF-BB. Histological evaluation revealed robust periodontal regeneration in the rhPDGF-BB sites, including new bone, cementum and periodontal ligament formation (Figure 3). Statistical analysis

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D. TR

NC

NC NB PDL

NB B. TR NB

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Figure 3. A growth factor-enhanced matrix (GEM) can consist of allograft bone saturated with rhPDGF-BB. The matrix is saturated with pure, recombinant growth factor solution prior to being packed into the defect site. This case illustrates the use of rhPDGF and allograft to treat a class II furcation with a mesial wrap-around defect. A) debrided defect; B) grafted site; C and D) histology showing periodontal regeneration coronal to a reference notch placed at the base of calculus prior to treatment. NB: New bone; NC: New cementum; PDL: Periodontal ligament; TR: Tooth roots. Reproduced with permission from The American Academy of Periodontology [41].

indicated that rhPDGF-BB combined with allograft bone produced robust regeneration and improved gingival attachment in interproximal bone defects and Class II furcation defects, compared with baseline. Comparisons among doses indicated that there were no adverse reactions, even at the highest dose, indicating that rhPDGF-BB was well tolerated in periodontal defect sites. Important to note is that this was the first study, to our knowledge reporting clear histological evidence of periodontal regeneration for human Class II furcation defects using this approach [42]. The regenerative effects of rhPDGF in combination with mineralized freeze dried bone allograft have been further documented clinically in a case study reported by Nevins and co-workers [43]. Two patients presenting with extremely severe bone loss (teeth with a poor to hopeless prognosis) and requiring surgical bone grafting were treated with rhPDGF-BB-enhanced mineralized allografts. Defect sites were treated with freeze-dried bone allograft saturated with rhPDGF-BB at a concentration of 0.3 (Case 1) or 1.0 mg/ ml (Case 2). The growth-factor-enhanced matrix was packed into the defect and an absorbable barrier membrane was placed over the defect prior to soft tissue closure. Patients were followed for six months, at which time a surgical reentry procedure was performed to evaluate the healing response within these previously severe defects. Both patients in this limited study exhibited excellent soft tissue healing. At six months, probing depths for both patients were 3 mm 380

and gingival recession was 0 and 3 mm for Case 1 and Case 2, respectively. The gains in clinical attachment level relative to baseline were 7 and 2 mm for Case 1 and Case 2, respectively. No adverse effects associated with either rhPDGF-BB dose were observed. Radiographic findings of excellent bone fill at six months for both patients were confirmed upon surgical re-entry of the treated sites. These results demonstrate that rhPDGF-BB combined with freezedried bone allograft provides an effective treatment for severe periodontal bone loss. An alternative to an allograft is the use of a completely synthetic growth-factor-enhanced matrix system. rhPDGFBB has been combined with b-tricalcium phosphate (b-TCP), a well-established resorbable ceramic biomaterial commonly used in oral reconstructive surgery. The results of a large, multicenter clinical trial evaluating the effectiveness of rhPDGF-BB combined with a porous b-TCP matrix have been recently reported [44]. This study included 180 participants with one interproximal periodontal defect 4 mm or deeper after debridement. Other noteworthy inclusion criteria included a baseline probing depth of 7 mm or greater, sufficient keratinized gingiva to allow complete coverage of the defect, radiographic defect base at least 3 mm coronal to the apex of the tooth, and no evidence of localized aggressive periodontitis. Grade I and Grade II furcation defects were acceptable for inclusion in the study. Smokers who consumed up to one pack per day were also included in

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the study. Three treatment groups were evaluated: i) b-TCP plus 0.3 mg/ml rhPDGF-BB (Group I), ii) b-TCP plus 1.0 mg/ml rhPDGF-BB (Group II), and iii) b-TCP plus buffer alone (Group III). Defects were classified as 1-wall, 2-wall or 3-wall/circumferential, indicating the extent of involvement and severity. At the time of surgery, b-TCP granules were saturated with rhPDGF-BB before the graft was placed in the defect site. Patients were followed for a period of six months and outcome measures included evaluation of soft tissue changes and assessment of bone growth. Safety was monitored throughout the trial by assessing the frequency and severity of clinical and/or radiographic adverse events. Excellent healing was observed for all defects treated with rhPDGF-BB. The study results demonstrated that there was a significantly greater clinical attachment level gain at three months for the 0.3 mg/ml rhPDGFBB (Group I), as compared with the b-TCP controls (Group III), indicating an early benefit of rhPDGFBB treatment. At six months, the clinical attachment level gain for the lower rhPDGF-BB concentration group continued to be greater than that for the control group, although statistical significance was not achieved. Additionally, rhPDGF-BB treatment resulted in significantly less gingival recession at three months, as compared with the untreated control group. This difference was no longer apparent at six months, however, as the control group exhibited a slight gain in gingival height over time. Increasing the rhPDGF-BB concentration appeared to reduce the effectiveness of the growth-factor-enhanced matrix. No statistically significant differences were observed in clinical attachment level or gingival recession for the higher rhPDGF-BB concentration (Group II), as compared with the b-TCP controls. Radiographic assessment revealed that bone fill was significantly increased at six months for the lower rhPDGF-BB concentration, as compared with both the higher rhPDGF-BB concentration and the control group (Figure 4). A subgroup analysis further indicated that rhPDGF-BB treatment improved bone fill in smokers and for all defect types (1, 2 and 3 wall and circumferential) (Figure 5). Similarly, linear bone growth was also significantly greater for Group I, as compared with Groups II and III. No significant differences were observed in the number or severity of adverse events among the three groups, indicating that both rhPDGF-BB and the b-TCP matrix were safe and adequately tolerated at the defect site. The results of this study demonstrate that the use of rhPDGF-BB in combination with a synthetic b-TCP matrix accelerates the rate of bone regeneration and improves bone fill and clinical attachment level in surgically treated periodontal defects. These positive outcomes were maintained over time as reported in a case series [45]. Four patients, selected from centers participating in the original pivotal clinical trial, exhibited significantly enhanced results for sites treated with 0.3 mg/ml

rhPDGF and b-TCP in a long-term (24 month) evaluation. These results remained significantly improved over those observed for the b-TCP control group (Figure 6). Placing these clinical results in perspective, the use of 0.3 mg/ml rhPDGF and b-TCP for the treatment of periodontal defects compares very favorably with existing FDAapproved treatments in terms of clinical attachment level gain and bone fill. This therapeutic approach significantly improved both clinical soft tissue and radiographic measures, as compared with the control group. The superior results for the lower dose suggest that there may be an optimum level of rhPDGF required to effectively stimulate a cellular response that leads to regeneration in periodontal defects. This observation emphasizes the need for rigorous clinical studies of new growth factor therapies, including systematic examination of dose effects. Though delivery of rhPDGF has shown clinical efficacy in the treatment of intrabony defects, it has also been examined for the treatment of soft tissue recession defects. In a case series study conducted by McGuire and colleagues, [46] seven subjects presenting contralateral > 3 mm-deep recession defects (Miller Class I and II) received two different types of treatment in a split-mouth design. Test therapy consisted of a combination of rhPDGF/b-TCP and a collagen membrane. The control treatment applied was the gold standard for root coverage: the subepithelial connective tissue graft (CTG). Healing was evaluated at 8, 16 and 24 weeks following the interventions. Primary outcome measure was recession depth. Results indicated a favorable tissue response to the test therapy, with similar clinical outcomes to CTG in terms of root coverage and keratinized tissue width. In a later publication, McGuire and co-workers reported histologic and microtomographic findings following analysis of human en-bloc samples of teeth that received either CTG or a combination of rhPDGF/b-TCP and a wound healing dressing [47]. Two patients requiring the extraction of a total of six premolars as part of orthodontic therapy were included in the study. Gingival recession defects were surgically created in the buccal aspect of these teeth and when necessary, alveolar bone was resected to position the crest at 2 -- 3 mm from the newly created gingival margin. In all teeth reference notches were created at the level of the new gingival margin and at the bone crest. Defects were left untouched for two months prior to the performance of root coverage procedures and a total of two CTG and four rhPDGF/b-TCP procedures were performed. After a nine-month healing period biopsies were obtained and defects were grafted. Clinically, 100% root coverage was achieved in all surgical sites. However, histological and microtomographic results were distinct for the two treatment groups. While, none of the CTGtreated sites showed signs of periodontal regeneration (healing was characterized by a long juctional epithelium and parallel connective tissue fibers, with minimal new cementum formation), all four rhPDGF/b-TCP-treated

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p < 0.001 70

p = 0.002

60 p = 0.019

50 56

40 30

34

20

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80

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0

1.0 mg/ml rhPDGF 0.3 mg/ml rhPDGF

Figure 4. rhPDGF-BB in combination with b-TCP significantly improved percentage bone fill of all bone defects compared with b-TCP plus buffer [44]. Reproduced with permission from The American Academy of Periodontology [44].

One-or two-walled periodontal defects

Three-walled and circumferental periodontal defects

p < 0.001

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80

70

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65

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34

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10

0

0

21

n = 14 TCP + buffer

0.3 mg/ml rhPDGF

n = 12

n = 14

1.0 mg/ml rhPDGF 0.3 mg/ml rhPDGF

Figure 5. rhPDGF-BB treatment resulted in significantly improved bone fill in 1-, 2-, and 3-wall defects (p < 0.001); there was no significant difference in bone fill between 1- and 2- wall defects compared with 3-wall and circumferential defects (p = 0.40 in the 0.3 mg/ml group) [43]. Reproduced with permission from The American Academy of Periodontology [44].

sites exhibited periodontal regeneration. This was evidenced by the presence of periodontal ligament interposed between newly formed cementum and alveolar bone, situated above the reference apical notch. Preliminary evidence provided by these studies suggests the need for further investigation consisting of the performance of controlled clinical trials of greater magnitude, designed to evaluate the application of GF-based therapy as a more 382

favorable option than CTG for patients requiring gingival recession coverage, since the need for a second surgical site could be eliminated. 4.

Expert opinion

A combination of rhPDGF with a tissue-specific scaffold, such as those comprised of allograft materials or synthetic

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Figure 6. Representative case from pivotal clinical trial treated with 0.3 mg/ml rhPDGF. A., B. and C. baseline; D. 6 months post-surgically; E. 18 months post-surgically. Note progressive increase in radio-opacity and trabecular bone pattern in the area of the original defect. Reproduced with permission from Quintessence Publishing Co., Inc. [45].

bioresorbable ceramics, has the potential to fulfill the clinical need for a biomaterial that predictably leads to periodontal or peri-implant regeneration. The results of a large, multi-center clinical trial aimed at evaluating the clinical safety and effectiveness of rhPDGF-BB incorporated in a b-TCP matrix demonstrate that this product is safe for long-term use. This treatment approach is supported by extensive and rigorous in vitro, preclinical and clinical studies, which provide strong evidence for the mechanism of action of PDGF in periodontal and periimplant healing and regeneration. Growth-factor-enhanced matrices provide clinicians with a new, highly effective treatment option for challenging periodontal lesions, which

are difficult to treat using conventional methods. Promising clinical results for rhPDGF in combination with osteoconductive matrices in a diverse array of periodontal and periimplant sites suggest that growth-factor-enhanced matrices incorporating rhPDGF have the potential to become routine, a standard-of-care modality.

Declaration of interest L Wisner-Lynch and S Lynch are employees of Biomimetic Therapeutics, Inc. Financial. The other authors declare no conflict of interest. This work has been supported by Biomimetic Therapeutics and NIH/NCRR UL 1RR-02496.

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Schwarz F, Sager M, Ferrari D, et al. Influence of recombinant human platelet-derived growth factor on lateral ridge augmentation using biphasic calcium phosphate and guided bone regeneration: a histomorphometric study in dogs. J Periodontol 2009;80:1315-23 Nevins M, Garber D, Hanratty JJ, et al. Human histologic evaluation of anorganic bovine bone mineral combined with recombinant human platelet-derived growth factor BB in maxillary sinus augmentation: case series study. Int J Periodontics Restorative Dent 2009;29:583-91 Simion M, Rocchietta I, Dellavia C. Three-dimensional ridge augmentation with xenograft and recombinant human platelet-derived growth factor-BB in humans: report of two cases. Int J Periodontics Restorative Dent 2007;27:109-15 Nevins ML, Camelo M, Schupbach P, et al. Human histologic evaluation of mineralized collagen bone substitute and recombinant platelet-derived growth factor-BB to create bone for implant placement in extraction socket defects at 4 and 6 months: a case series. Int J Periodontics Restorative Dent 2009;29:129-39 McAllister BS, Haghighat K, Prasad HS, Rohrer MD. Histologic evaluation of extraction sockets grafted with platelet-derived growth factor. Int J Periodontics Restorative Dent 2010;30:365-73 Howell TH, Fiorellini JP, Paquette DW, et al. A phase I/II clinical trial to evaluate a combination of recombinant human platelet-derived growth factor-BB and recombinant human insulin-like growth factor-I in patients with periodontal disease. J Periodontol 1997;68:1186-93

39.

Stephan EB, Renjen R, Lynch SE, Dziak R. Platelet-derived growth factor enhancement of a mineral-collagen bone substitute. J Periodontol 2000;71:1887-92

40.

Hollinger JO, Hart CE, Hirsch SN, et al. Recombinant human platelet-derived growth factor: biology and clinical applications. J Bone Joint Surg Am 2008;90(Suppl 1):48-54

41.

Nevins M, Camelo M, Nevins ML, et al. Periodontal regeneration in humans using recombinant human platelet-derived growth factor-BB (rhPDGF-BB) and allogenic bone. J Periodontol 2003;74:1282-92

42.

Camelo M, Nevins ML, Schenk RK, et al. Periodontal regeneration in human Class II furcations using purified recombinant human platelet-derived growth factor-BB (rhPDGF-BB) with bone allograft. Int J Periodontics Restorative Dent 2003;23:213-25

43.

Nevins M, Hanratty J, Lynch SE. Clinical results using recombinant human platelet-derived growth factor and mineralized freeze-dried bone allograft in periodontal defects. Int J Periodontics Restorative Dent 2007;27:421-7

44.

Nevins M, Giannobile WV, McGuire MK, et al. Platelet-derived growth factor stimulates bone fill and rate of attachment level gain: results of a large multicenter randomized controlled trial. J Periodontol 2005;76:2205-15

45.

McGuire MK, Kao RT, Nevins M, Lynch SE. rhPDGF-BB promotes healing of periodontal defects: 24-month clinical and radiographic observations. Int J Periodontics Restorative Dent 2006;26:223-31

46.

McGuire MK, Scheyer ET. Comparison of recombinant human platelet-derived growth factor-BB plus beta tricalcium phosphate and a collagen membrane to subepithelial connective tissue grafting

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for the treatment of recession defects: a case series. Int J Periodontics Restorative Dent 2006;26:127-33 47.

McGuire MK, Scheyer T, Nevins M, Schupbach P. Evaluation of human recession defects treated with coronally advanced flaps and either purified recombinant human platelet-derived growth factor-BB with beta tricalcium phosphate or connective tissue: a histologic and microcomputed tomographic examination. Int J Periodontics Restorative Dent 2009;29:7-21

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Simion M, Nevins M, Rocchietta I, et al. Vertical ridge augmentation using an equine block infused with recombinant human platelet-derived growth factor-BB: a histologic study in a canine model. Int J Periodontics Restorative Dent 2009;29:245-55

Affiliation Darnell Kaigler1,2, Gustavo Avila1, Leslie Wisner-Lynch3, Marc L Nevins4, Myron Nevins4, Giulio Rasperini5, Samuel E Lynch3 & William V Giannobile†1,2 † Author for correspondence 1 University of Michigan School of Dentistry, Department of Periodontics and Oral Medicine and Michigan Center for Oral Health Research, 1011 North University Avenue, Ann Arbor, MI 48109 1078, USA E-mail: [email protected] 2 University of Michigan, College of Engineering, Department of Biomedical Engineering, Ann Arbor, MI, USA 3 Biomimetic Therapeutics, Inc., Franklin, TN, USA 4 Harvard School of Dental Medicine, Division of Periodontology, Department of Oral Medicine, Infection and Immunity, Boston, MA, USA 5 University of Milan, Unit of Periodontology, Department of Surgical, Regenerative and Diagnostic Science, Foundation IRCCS Ca` Granda Policlinico, Milan Italy

385

Drug Evaluation

ETC-216 for coronary artery disease Stephen J Nicholls†, Kiyoko Uno, Yu Kataoka & Steven E Nissen †

Heart & Vascular Institute, Cleveland Clinic, Department of Cardiovascular Medicine, Cleveland, OH, USA

1.

Introduction

2.

Rationale for targeting HDL functionality

3.

Current therapeutic approaches for HDL

4.

Rationale for developing AIM

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as a therapeutic strategy 5.

Early clinical experience of ETC-216

6.

Additional HDL targeted therapies

7.

Emergence of functionality as a target

8.

Future directions for ETC-216

9.

Conclusion

10.

Expert opinion

Introduction: Increasing attention has focused on the role of high-density lipoprotein function as a target for cardiprotection. Apolipoprotein A-IMilano (AIM) involves a single amino-acid mutation of the major wild-type protein carried on high-density lipoprotein (HDL) particles. Early evidence of beneficial activities of AIM has stimulated support in its development as a potential therapy to reduce cardiovascular risk. Areas covered: The importance of HDL as a target and early data supporting the beneficial effects of AIM are reviewed. All clinical studies of AIM found in PubMed are reviewed. Expert opinion: ETC-216 represents a lipid-deplete form of HDL containing recombinant AIM. While early evidence suggests that administration of ETC-216 promotes rapid regression of coronary atherosclerosis, bringing this compound to clinical practice will require further trials that evaluate its impact on cardiovascular events. Keywords: apoA-IMilano, atherosclerosis, high-density lipoprotein, risk factors Expert Opin. Biol. Ther. (2011) 11(3):387-394

1.

Introduction

Over the course of the last 2 decades, large randomized controlled trials have consistently demonstrated that lowering levels of low-density lipoprotein (LDL) cholesterol reduce cardiovascular event rates across the full spectrum of risk [1-6]. Accordingly, LDL cholesterol lowering has become the cornerstone of treatment guidelines for the prevention of cardiovascular disease. However, despite these advances many patients continue to experience clinical events. This residual risk suggests that additional therapies will be required to achieve more effective reductions in cardiovascular risk. 2.

Rationale for targeting HDL functionality

High-density lipoproteins (HDLs) have presented an attractive target for the development of new anti-atherosclerotic therapies. Population studies have consistently observed an inverse relationship between systemic levels of HDL cholesterol and prospective risk [7-9]. These findings were independent of levels of atherogenic lipid parameters. Animal studies subsequently demonstrated that promoting functionality of HDL via direct infusions or transgenic expression of its major protein, apolipoprotein (apo) A-I, has a beneficial impact on both the size and composition of atherosclerotic lesions [10-16]. HDL possesses a number of biological activities that are likely to contribute to their beneficial influence on the artery wall. The predominant functional property of HDL appears to be its central role in the facilitation of reverse cholesterol transport. Circulating HDL particles are the primary acceptor of cholesterol that is effluxed from cells in peripheral tissues. On the particle surface, cholesterol is esterified by the factor lecithin cholesterol acyltransferase (LCAT) and stored within 10.1517/14712598.2011.557061 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

387

ETC-216

Box 1. Drug summary.

biological activities is likely to have a favorable impact on both the extent and composition of atherosclerotic plaque.

Drug Name Phase

3.

Indication

Pharmacology

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Route of administration Pivotal trial(s)

ETC-216 Phase I studies with new formulation. Recently licensed by the Medicines Co. (MDCO-216) Probably patients with acute coronary syndromes, but could be expanded to others with atherosclerotic disease Mutant form of apoA-I combined with phospholipid Intravenous Proof-of-concept IVUS study demonstrated regression

apoA-I: Apolipoprotein A-I; IVUS: Intravascular ultrasound.

the core particle core. This results in the generation of larger, lipid-rich particles and maintains a relatively low concentration of cholesterol on the surface, which tends to favor ongoing efflux from peripheral tissues [17]. Increasing evidence has implicated a number of transmembranous pathways that facilitate movement of lipid from the cell to HDL. Particles of different size and degree of lipid content appear to act as the predominant acceptor for cholesterol effluxed via each of these different pathways. While the transporter, ATP-binding cassette A1 (ABCA1), is the pivotal pathway for cholesterol efflux to small, lipid-deplete forms of HDL, larger, lipid laden particles more preferentially accept cholesterol that leaves cells via ABCG1 and the scavenger receptor SR-BI mediated pathways. This relative redundancy of cholesterol efflux pathways enables ongoing movement of lipid to a wide range of circulating HDL particles [18]. Ultimately, cholesterol from HDL is taken up by the liver in a process that is facilitated by the scavenger receptor SR-BI. While cholesteryl ester transfer protein (CETP) has been reported to facilitate an alternative pathway, whereby esterified cholesterol is transferred from HDL to both LDL particles, the majority of this lipid is also likely to be delivered to the liver via the LDL receptor pathway [17]. A number of additional biological properties have been ascribed to HDL particles. Laboratory studies have demonstrated that HDL imparts a beneficial effect on a range of inflammatory, oxidative, thrombotic, apoptotic and proliferative pathways [19]. The observation that HDL increases the bioavailability of NO may contribute to a number of these potentially beneficial properties [20]. It remains to be determined to what degree these activities reflect the consequence of promoting removal of excess cholesterol from cells or whether they represent specific non-lipid transporting functions. Reports that infusing HDL has rapid beneficial effects on endothelial-dependent vascular reactivity in humans [21,22] and on models of acute vascular inflammation in animals [23] suggest that non-lipid transporting effects of HDL are observed in vivo. It is likely that the combination of these 388

Current therapeutic approaches for HDL

Use of lipid-modifying approaches currently used in clinical practice is typically associated with relatively modest increases in levels of HDL cholesterol. Mild alcohol consumption and lifestyle changes, particularly when involving intensive levels of exercise or resulting in weight loss, are accompanied by increases in HDL cholesterol by up to 10% [24]. While statins are primarily used for LDL cholesterol lowering, these agents raise HDL cholesterol by 3 -- 15%. A number of groups have reported that these modest elevations do contribute to the ability of statins to slow plaque progression and to reduce cardiovascular event rates [25-27]. Fibric acid derivatives (fibrates) elevate HDL cholesterol by 5 -- 20%, with varying effects on cardiovascular event rates in large clinical trials, depending on the individual agent studied [28]. In the early studies that demonstrated clinical efficacy of gemfibrozil, modest elevations in HDL cholesterol by ~ 6% were found to be the strongest independent predictor of clinical benefit [29]. Further lipoprotein analysis using NMR revealed that this benefit appeared to be associated with a greater elevation in the smaller HDL particles [30]. The consistent finding from all fibrate studies of benefit in patients with hypertriglyceridemia or low levels of HDL cholesterol at baseline identifies a patient cohort who is more likely to benefit with treatment with these therapies [28]. Niacin is currently the most effective HDL cholesterol raising agent in clinical practice. While studies have demonstrated beneficial effects of niacin on both disease burden [31-33] and clinical events [34], patient intolerance largely due to flushing presents a formidable challenge to its widespread and effective use. Novel formulations that aim to reduce flushing via either extended release or in combination with an epidermal prostanoid receptor antagonist are currently being evaluated in large clinical trials.

Rationale for developing AIM as a therapeutic strategy

4.

ApoA-IMilano (AIM) was first identified in a cohort of individuals in the northern Italian hamlet of Limone sul Garda, who demonstrated protection from vascular disease, despite the presence of low levels of HDL cholesterol [35]. These individuals with hypertriglyceridemia and low levels of both HDL cholesterol and apoA-I were found to be heterozygous carriers of a point mutation of apoA-I, involving an arginine to cysteine substitution at position 173 [35]. This mutation enables the formation of AIM homodimers and has been demonstrated to possess potent cholesterol efflux, anti-inflammatory and anti-thrombotic activities in cellular studies [36]. These early observations stimulated interest in the development of AIM as a potential therapeutic strategy for patients with atherosclerotic disease.

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Recombinant forms of AIM can be generated in relatively large quantities using an Escherichia coli-based expression system, although the ability to produce sufficient quantities for larger scale clinical trials has always been a challenge. When combined with phospholipid, AIM can be incorporated into lipid-deplete forms of HDL that can be used as an infusional therapy. In early studies involving arterial wall intervention in animal models, infusions were reported to have a beneficial effect. In a study of cholesterol-fed rabbits undergoing balloon injury within the femoral and iliac arteries, alternate daily infusion of AIM--phospholipid from 5 days prior to 5 days following balloon injury was associated with a reduction in intimal thickening and macrophage content [37]. A similar benefit was observed when AIM--phospholipid complexes were infused into cholesterol-fed rabbits prior to implantation of a non-occlusive collar around the carotid artery. When initiation of infusional therapy was delayed until the day of surgery, a beneficial effect on neointimal formation was not observed. Similarly, no benefit was demonstrated with animals that received phospholipid liposomes, suggesting that the favorable effects were either due to the AIM or the administration of lipid-deplete forms of HDL [38]. The subsequent finding that a single infiltration of a commercial preparation of AIM--phospholipid (ETC-216; Box 1) directly into the artery wall immediately prior to stent deployment in a pig model was associated with less intimal hyperplasia further suggested that AIM had a favorable effect on restenosis [39]. Beneficial effects of infusing AIM--phospholipid complexes have also been observed in animal models of atherosclerotic plaque. Intravenous infusions administered on alternate days from weeks 20 to 25 in apoE knockout mice fed a highcholesterol diet resulted in no progression of established atherosclerotic plaque and a reduction in both lipid and macrophage content [40]. Similar rapid changes in plaque composition 48 h following a single intravenous infusion of AIM--phospholipid in high cholesterol-fed apoE knockout mice with established atherosclerosis suggest that this strategy could potentially stabilize plaque [16]. The finding that treatment of apoE knockout mice restores endothelial-dependent vasodilation in association with reductions in both aortic cholesterol content and platelet aggregation [41] provides additional mechanistic benefits that may contribute to a favorable clinical outcome in patients with acute ischemic syndromes. The impact of infusing AIM--phospholipid complexes on atherosclerotic plaque has been further characterized using novel imaging modalities that directly visualize atherosclerotic plaque in the artery wall. In a novel study, lipid-rich plaques were generated in the carotid arteries of cholesterolfed rabbits by perivascular electric injury. AIM--phospholipid infused directly into the carotid artery was demonstrated to promote rapid regression of disease when evaluated by intravascular ultrasound (IVUS) over a 90 min period [42]. In a subsequent study, infusions administered every 4 days over a 20 day period in rabbits with established carotid atherosclerotic plaque demonstrated a beneficial impact on

disease progression, with evidence of regression at the highest doses, on serial imaging with both IVUS and MRI [43]. These findings have also been demonstrated with shorter treatment periods [44], in correlation with findings on multidetector CT [45] and associate with molecular and protein findings that are consistent with upregulation of pivotal factors involved in reverse cholesterol transport and modulating both inflammatory and oxidative pathways [44]. The potential cardiovascular benefit of AIM has been further explored with regard to its effect on the degree of ischemia/reperfusion injury sustained by end organ tissue. In early ex vivo studies using an isolated rat heart model, administration of AIM--phospholipid either immediately prior to the induction of ischemia or during the post-ischemic phase resulted in improvements in left ventricular and coronary perfusion pressures and a reduction in the amount of creatine kinase released into the coronary effluent [46]. A reduction in levels of lipid hydroperoxides was observed in myocardial tissue suggesting that a reduction in oxidative stress may have contributed to the observed benefit [46]. In the in vivo setting, administration of ETC-216 was associated with the development of smaller myocardial infarct sizes in a left anterior descending artery ligation model of ischemia and reperfusion [47]. Furthermore, the effects of AIM on lipid mobilization and inflammatory pathways are likely to underlie the recent observation that AIM infusions reversed aortic valve stenosis in an experimental rabbit model [48]. 5.

Early clinical experience of ETC-216

Given the large body of preclinical data that suggest that AIM when administered in a complex with phospholipid has beneficial effects on the artery wall, interest in its potential as a therapeutic continues to increase. In a pilot study of 57 patients with an acute coronary syndrome within the last 2 weeks, the impact of AIM in the ETC-216 preparation on atherosclerotic plaque was evaluated. Patients underwent IVUS imaging within a coronary artery and were then randomized to receive weekly intravenous infusions of either saline or ETC-216 containing either 15 or 45 mg/kg AIM for 5 weeks. Two weeks following the final infusion, patients returned for repeat IVUS imaging in the same artery. In the combined active treatment group, significant reductions in percent atheroma volume by 1.06% (p = 0.02) and total atheroma volume by 14.3 mm3 (p < 0.001) were observed, consistent with plaque regression. No differences in efficacy were observed between the two active dosing groups. These findings were observed in the absence of any increase in steadystate levels of HDL cholesterol. In general, ETC-216 was well tolerated. Two patients in the high dose group were withdrawn from the study, one for a symptomatic transaminase elevation greater than three times the upper limit of normal and the other for the development of diaphoresis, nausea and a mild rash during the administration of the infusion which was deemed to be study drug related [49].

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The findings were noteworthy for a number of reasons. While the degree of regression may appear to be relatively modest, in terms of the rapidity of the changes this is likely to be clinically meaningful. This is supported by the observation that in much longer studies a clinically meaningful therapeutic effect on IVUS appears to be demonstrated with changes in disease burden that are about 50% of the changes observed in this pilot study [50]. Regression of coronary atherosclerosis was observed in a matter of weeks, a time course that had not been achieved with any other therapeutic intervention previously. The lack of difference in efficacy in the low and high dose AIM groups suggested that there may be a lower dose that may still result in regression. To what degree the current findings support the concept that the low dose of AIM saturated efflux via the ABCA1 pathway remains to be determined. Greater regression was observed in the 10 mm segments that contained the most amount of plaque at baseline, suggesting the potential for a greater effect in the regions that are likely to contain the most amount of lipid [49,51]. Furthermore, the changes in plaque burden were demonstrated in association with reduction in vessel wall, but no changes in lumen volumes. This finding suggested that plaque regression is accompanied by a reversal of the remodeling pattern that is typically encountered with plaque accumulation. Moreover, the lack of changes in lumen dimensions suggests that evaluation of ETC-216 with conventional angiographic techniques, which assess the lumen and not the artery wall, would have completely missed the therapeutic effect [51]. The major message from this early step reflects the potential beneficial effect at the level of the artery wall by infusing a form of HDL in humans. Considerable debate continues to focus on the relative degree of benefit attributable to AIM. The development of AIM-based strategies has focused on the potential for enhanced biological activities with AIM, compared with the wild-type form of apoA-I. While some investigators have reported differences in favor of AIM, this has not been demonstrated by others [52]. Two subsequent IVUS studies have demonstrated beneficial effects of infusing different forms of HDL that do not contain AIM. In a similar study, the Effect of rHDL on Atherosclerosis-Safety and Efficacy (ERASE) investigators infused HDL complexes containing wild-type apoA-I and phospholipid. Serial IVUS evaluation revealed evidence of a trend towards regression and an improvement in a calculated plaque characterization index that the authors considered a surrogate for plaque composition [53]. Abnormal liver function tests were observed in association with administration of the highest dose of CSL-111 in this study. In a more recent study, autologous infusions of delipidated HDL similarly resulted in a trend towards plaque regression on serial IVUS imaging [54]. While the relative merits of mutated versus wild-type forms of apoA-I remain to be completely defined, these studies in general support the concept that infusions of lipid-deplete forms of HDL, which are avid acceptors of cholesterol effluxed via ABCA1, have beneficial effects on plaque burden. These findings continue 390

to stimulate interest that infusional HDL therapy might be of benefit for patients with atherosclerotic plaque, particularly in the acute phase of their disease. However, this remains to be determined in large, randomized, controlled trials that will evaluate the impact of these approaches on clinical events. 6.

Additional HDL targeted therapies

The preliminary findings of the benefits of ETC-216 in humans continued to support interest in the development of therapeutic strategies that promote HDL functionality. Clinical development programs are currently working on a number of novel agents that target various aspects of HDL generation, metabolism and function. In addition to infusion of apoA-I-based formulations, there is considerable interest in apoA-I as a therapeutic target. The ability to enhance endogenous synthesis of apoA-I presents an interesting approach, via its ability to simply generate circulating HDL particles that can perform its range of biological activities. While this concept of ‘turning on the system’ has been appealing, it has remained a challenge to identify compounds that selectively upregulate apoA-I expression. The first compound to achieve this effect, RVX-208, has been reported in early studies to raise levels of the lipid-deplete form of HDL and enhance efflux capacity in ex vivo cell assays [55]. The impact of this compound is currently being evaluated in patients with coronary artery disease. Early reports of a study in statintreated patients with stable coronary artery disease revealed a dose-dependent increase in levels of apoA-I, HDL cholesterol and large HDL particles, in association with elevations in liver transaminases [56]. An alternative strategy is to mimic the functional activity, via the generation of short peptides, which possess no amino-acid homology to the apoA-I sequence, but retain the capacity to form an amphipathic helix. This helical structure appears to be a critical determinant of the ability of both apoA-I and these mimetic peptides to carry out lipid transporting and anti-inflammatory activities. The generation of these peptides with D-type amino acids, which are resistant to gastric hydrolysis, permits the potential for oral administration. These mimetic peptides are starting to reach more advanced stages of clinical development [57]. HDL circulates in the plasma as a heterogeneous group of particles, varying in terms of their size, shape and composition of lipid and protein. This heterogeneity results from the constant remodeling of particles in response to a number of factors [58]. Considerable interest has focused on the potential of modulating these remodeling factors in order to elevate HDL cholesterol levels. Inhibition of CETP has received the most interest on the basis of observations that low CETP activity is associated with markedly elevated HDL cholesterol levels and has been reported by some, but not all, investigators to be associated with atheroprotection in both animal models and in population studies [59]. Early interest in CETP inhibition has been tempered by the finding that the pharmacological inhibitor, torcetrapib, resulted in excess

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mortality [60] and had no effect on disease progression [61-63]. The finding that torcetrapib has off-target toxicities provides some ongoing enthusiasm that another inhibitor might be beneficial from a cardiovascular perspective [64]. Accordingly, several CETP inhibitors are currently being evaluated in Phase II and III studies. The recent finding that anacetrapib has profound effects on levels of LDL and HDL cholesterol, in addition to statin therapy, has further stimulated interest in the potential effect of this therapeutic class on the arterial wall [65]. Endothelial lipase is involved in HDL remodeling. While observations suggest that low levels of endothelial lipase are associated with higher HDL cholesterol levels and less disease burden on imaging [66], no pharmacological inhibitor has moved forward in clinical development. Similarly, while LCAT plays a critical role in the formation of mature HDL particles and the facilitation of reverse cholesterol transport, it remains to be determined whether LCAT modulation represents a strategy worth further pursuit. Modulation of nuclear transcription of critical factors involved in reverse cholesterol transport provides an alternative therapeutic approach. While early generation activators of PPARs appear to upregulate expression of a range of factors involved in HDL particle formation and reverse cholesterol transport, the development of newer generation agents that are either more potent or activate multiple PPAR subtypes has proved challenging due to either lack of incremental benefit or toxicity [67]. Similarly, the development of liver X receptor agonists has been limited by the finding that early generation agents promote formation of hepatic steatosis [68]. 7.

Emergence of functionality as a target

The search to identify new therapeutic strategies to promote the biological activity of HDL has been based on the premise that substantially raising levels of HDL cholesterol should be beneficial. However, a number of observations suggest that the relative functionality of HDL, as opposed to quantity of HDL cholesterol, may be more important. HDL cholesterol simply measures the quantity of cholesterol carried in the HDL pool. It is a suboptimal marker of HDL particles and reverse cholesterol transport and provides no information with regard to potentially important, non-lipid-transporting activities. A number of investigators have reported that relatively modest increases in HDL cholesterol correlate with the clinical benefit of currently available lipid-modifying therapies [25-27,29]. Furthermore, some individuals develop coronary disease despite the presence of very high levels of HDL cholesterol. Many of these individuals are subsequently found to have HDL particles that promote, rather than inhibit, inflammatory pathways [69]. This concept of dysfunctional HDL particles has been further stimulated with the finding that torcetrapib, an agent that substantially raises HDL cholesterol, was found to have an adverse effect on

cardiovascular events [60]. While there has never been any evidence that torcetrapib does generate dysfunctional HDL particles, the finding of an adverse effect on clinical outcome has provided further impetus to define the factors that might potentially modulate HDL functionality. In combination, these observations suggest that the relative functionality of HDL may be an important factor to consider in the development of new therapies. Recent insights have elucidated the role of inflammatory and pro-oxidant mediators in the formation of apparent dysfunctional HDL [70-72]. Accordingly, these insights provide the potential to develop either additional markers of functionality or might by themselves provide additional targets for the development of new therapies. Finally, proteomic analyses of HDL continue to broaden the scope of proteins that are carried on HDL particles [73]. Many of these proteins regulate a range of inflammatory, immune and oxidative cascades and should be considered with regard to their therapeutic potential [74]. Accordingly, when the early benefits of ETC-216 on atherosclerotic plaque in humans are further explored, in the absence of any increase in HDL cholesterol levels, it will be important to consider their overall impact on HDL functionality. 8.

Future directions for ETC-216

Despite the wave of enthusiasm generated by the early findings on IVUS, clinical development of ETC-216 has not progressed. This is likely to reflect the difficulty of generating sufficient quantities of a pharmacological-grade agent that can be used effectively in larger scale clinical trials. This compound has been recently licensed by the Medicines Co. (MDCO-216) and will now proceed in clinical development. It will be important to observe to what degree emerging insights into the relative functionality of HDL are reflected in the clinical development of the compound. 9.

Conclusion

Promoting the biological activity of HDL remains an attractive target for the development of new cardioprotective therapies. Early evidence forming lipid-deplete HDL complexes with AIM and phospholipid suggest that this approach can have a rapid, beneficial effect at the level of the artery wall. The ultimate determinant of its effects will be resolved in the completion of large clinical trials that will evaluate its impact on cardiovascular events. 10.

Expert opinion

The residual cardiovascular risk observed despite use of LDL cholesterol lowering therapies highlights the need to develop additional therapeutic strategies to prevent clinical events. Widespread evidence from population and animal studies suggest that HDL represents an attractive target.

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ETC-216

This is further supported by findings that even modest changes in HDL cholesterol levels contribute to the cardiovascular benefit observed with current lipid-modifying therapies. However, the failure of torectrapib has placed considerable uncertainty, not only on the development of CETP inhibitors, but also on the premise that HDL should be a useful target. At the same time, the relative functionality of HDL has received increasing attention. AIM continues to represent an intriguing target for the development of new therapies, given the promising data from both preclinical studies and in a pivotal proof-ofconcept study using coronary IVUS in humans. As the development of this intravenous approach evolves it will be essential to demonstrate that it reduces cardiovascular morbidity and mortality in patients with established atherosclerotic disease.

Declaration of interest SE Nissen reports that the Cleveland Clinic Center for Clinical Research has received funding perform clinical trials from Pfizer, AstraZeneca, Novartis, Novo Nordisk, Roche, Daiichi-Sankyo, Karo Bio, Takeda, Sanofi-Aventis, Resverlogix, and Eli Lilly. SE Nissen is involved in these clinical trials, but receives no personal remuneration for his participation. SE Nissen consults for many pharmaceutical companies, but requires them to donate all honoraria or consulting fees directly to charity so that he receives neither income nor a tax deduction. SJ Nicholls has received funding from Pfizer, AstraZeneca, Takeda, Merck, Roche, LipoScience, Inc., Anthera Pharmaceuticals, Novo Nordisk, Novartis, Resverlogix Corp. and Eli Lilly. K Uno and Y Kataoka declare no conflict interest.

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Jun M, Foote C, Lv J, et al. Effects of fibrates on cardiovascular outcomes: a systematic review and meta-analysis. Lancet 2010;375:1875-84

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Robins SJ, Collins D, Wittes JT, et al. Relation of gemfibrozil treatment and lipid levels with major coronary events: VA-HIT: a randomized controlled trial. JAMA 2001;285:1585-91

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Sirtori CR, Calabresi L, Franceschini G, et al. Cardiovascular status of carriers of the apolipoprotein A-I(Milano) mutant: the Limone sul Garda study. Circulation 2001;103:1949-54

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Calabresi L, Sirtori CR, Paoletti R, Franceschini G. Recombinant apolipoprotein A-IMilano for the treatment of cardiovascular diseases. Curr Atheroscler Rep 2006;8:163-7

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Ameli S, Hultgardh-Nilsson A, Cercek B, et al. Recombinant apolipoprotein A-I Milano reduces intimal thickening after balloon injury in hypercholesterolemic rabbits. Circulation 1994;90:1935-41

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Soma MR, Donetti E, Parolini C, et al. Recombinant apolipoprotein A-IMilano dimer inhibits carotid intimal thickening induced by perivascular manipulation in rabbits. Circ Res 1995;76:405-11

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Kaul S, Rukshin V, Santos R, et al. Intramural delivery of recombinant apolipoprotein A-IMilano/phospholipid complex (ETC-216) inhibits in-stent stenosis in porcine coronary arteries. Circulation 2003;107:2551-4 Shah PK, Nilsson J, Kaul S, et al. Effects of recombinant apolipoprotein A-I(Milano) on aortic atherosclerosis in apolipoprotein E-deficient mice. Circulation 1998;97:780-5 Kaul S, Coin B, Hedayiti A, et al. Rapid reversal of endothelial dysfunction in hypercholesterolemic apolipoprotein

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E-null mice by recombinant apolipoprotein A-I(Milano)-phospholipid complex. J Am Coll Cardiol 2004;44:1311-19 42.

Chiesa G, Monteggia E, Marchesi M, et al. Recombinant Apolipoprotein A-IMilano infusion into rabbit carotid artery rapidly removes lipid from fatty streaks. Circ Res 2002;90:974-80

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Parolini C, Marchesi M, Lorenzon P, et al. Dose-related effects of repeated ETC-216 (recombinant apolipoprotein A-I Milano/1-palmitoyl-2-oleoyl phosphatidylcholine complexes) administrations on rabbit lipid-rich soft plaques: in vivo assessment by intravascular ultrasound and magnetic resonance imaging. J Am Coll Cardiol 2008;51:1098-103

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Ibanez B, Vilahur G, Cimmino G, et al. Rapid change in plaque size, composition, and molecular footprint after recombinant apolipoprotein A-I Milano (ETC-216) administration: magnetic resonance imaging study in an experimental model of atherosclerosis. J Am Coll Cardiol 2008;51:1104-9

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Ibanez B, Cimmino G, Benezet-Mazuecos J, et al. Quantification of serial changes in plaque burden using multi-detector computed tomography in experimental atherosclerosis. Atherosclerosis 2009;202:185-91

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Marchesi M, Booth EA, Rossoni G, et al. Apolipoprotein A-IMilano/POPC complex attenuates post-ischemic ventricular dysfunction in the isolated rabbit heart. Atherosclerosis 2008;197:572-8

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Marchesi M, Booth EA, Davis T, et al. Apolipoprotein A-IMilano and 1-palmitoyl-2-oleoyl phosphatidylcholine complex (ETC-216) protects the in vivo rabbit heart from regional ischemia-reperfusion injury. J Pharmacol Exp Ther 2004;311:1023-31

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Speidl WS, Cimmino G, Ibanez B, et al. Recombinant apolipoprotein A-I Milano rapidly reverses aortic valve stenosis and decreases leaflet inflammation in an experimental rabbit model. Eur Heart J 2010;31:2049-57

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Nissen SE, Tsunoda T, Tuzcu EM, et al. Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial. JAMA 2003;290:2292-300

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Nicholls SJ, Hsu A, Wolski K, et al. Intravascular ultrasound-derived measures of coronary atherosclerotic plaque burden and clinical outcome. J Am Coll Cardiol 2010;55:2399-2407 Nicholls SJ, Tuzcu EM, Sipahi I, et al. Relationship between atheroma regression and change in lumen size after infusion of apolipoprotein A-I Milano. J Am Coll Cardiol 2006;47:992-7 Weibel GL, Alexander ET, Joshi MR, et al. Wild-type ApoA-I and the Milano variant have similar abilities to stimulate cellular lipid mobilization and efflux. Arterioscler Thromb Vasc Biol 2007;27:2022-9 Tardif JC, Gregoire J, L’Allier PL, et al. Effects of reconstituted high-density lipoprotein infusions on coronary atherosclerosis: a randomized controlled trial. JAMA 2007;297:1675-82 Waksman R, Torguson R, Kent KM, et al. A first-in-man, randomized, placebo-controlled study to evaluate the safety and feasibility of autologous delipidated high-density lipoprotein plasma infusions in patients with acute coronary syndrome. J Am Coll Cardiol 2010;55:2727-35 Bailey D, Jahagirdar R, Gordon A, et al. RVX-208: a small molecule that increases apolipoprotein A-I and high-density lipoprotein cholesterol in vitro and in vivo. J Am Coll Cardiol 2010;55:2580-89

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cholesterol levels and are favorably affected by simvastatin treatment. Circulation 2003;108:2751-6 70.

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Affiliation

Stephen J Nicholls†1,2,3, Kiyoko Uno1, Yu Kataoka1 & Steven E Nissen1 † Author for correspondence 1 Heart & Vascular Institute, Cleveland Clinic, Department of Cardiovascular Medicine, Mail Code JJ-65, 9500 Euclid Avenue, Cleveland, OH 44195, USA Tel: +1 216 444 2678; Fax: +1 216 445 5497; E-mail: [email protected] 2 Cleveland Clinic, Department of Cell Biology, Cleveland, OH, USA 3 Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic, Cleveland, OH, USA

Drug Evaluation

Abagovomab for ovarian cancer Jacobus Pfisterer†, Philipp Harter, Cecilia Simonelli, Martin Peters, Jonathan Berek, Paul Sabbatini & Andreas du Bois †

Klinik fu¨r Gyna¨kologie und Geburtshilfe Sta¨dtisches Klinikum Solingen, Gotenstr. 1, 42653 Solingen, Germany

1.

Introduction

2.

Anti-idiotypic network and

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abagovomab generation 3.

Abagovomab pre-clinical characterization

4.

Abagovomab clinical studies

5.

Conclusions

6.

Expert opinion

Introduction: Ovarian cancer (OC) is the fifth most common cancer in women. Unfortunately, more than 70% of cases are detected at an advanced stage with a risk of recurrence, after front line therapy, of over 75%. The need for new therapeutic strategies is extremely high. Areas covered: The development status and the possible role of specific immunotherapy of abagovomab are discussed in the context of the possible therapeutic options for maintenance therapy in advanced OC. An overview of abagovomab, generation and mechanism of action, Phase I/II results and the status of the Phase II/III ongoing trial is given. Expert opinion: Abagovomab stimulates the humoral immune response and the cell-mediated immune response in the studies conducted to date. In the proof of concept (POC) study abagovomab prolonged overall survival in those OC recurrent patients who showed an immune response. Abagovomab has an excellent safety and tolerability profile. These characteristics make abagovomab an optimal candidate for a maintenance treatment for OC patients after frontline therapy. The final results of the Phase II/III pivotal study evaluating abagovomab in this setting will be available in the first half of 2011. Keywords: abagovamab, maintenance therapy, ovarian cancer Expert Opin. Biol. Ther. (2011) 11(3):395-403

1.

Introduction

Ovarian cancer is the fifth most common cancer in women [1,2] accounting for 66,430 new cases every year all over Europe [3]. More than 21,000 new cases will be diagnosed in 2010 in the USA [4]. In both the EU and USA, ovarian cancer is the leading cause of gynecological cancer deaths. Approximately 70% of the ovarian cancer cases are detected at advanced stages (Federation Internationale de Gynecologie et d’ Obstetrique (FIGO) stages III and IV) and the overall 5-year survival rate for patients with advanced stage disease is about 20 -- 30% [5]. Standard primary treatment for advanced disease consists of cytoreductive (debulking) surgery plus platinum--taxane based chemotherapy regimens [6]. Due to the chemosensitivity of the tumour, most patients have a clinical complete response at the end of primary treatment. Nevertheless, the majority of patients will have a recurrence and therefore additional good tolerable therapies able to ‘maintain’ the complete response after frontline therapy with minimal toxicity represent a strong medical need [7]. Indeed, the optimal management of patients who achieve a clinical complete response (CR) after first-line chemotherapy remains to be determined [8]. Until now, neither consolidation therapy nor maintenance therapy have been shown to extend survival in patients with advanced ovarian cancer. The Gynecologic Oncology Group (GOG) 178 Trial which tested the efficacy of prolonged paclitaxel administration (12 additional cycles versus 3 additional cycles) showed a clinically longer progression-free survival (PFS) in the long-paclitaxel-treated arm patients, but no improvement in overall survival (OS) was observed [9,10];

10.1517/14712598.2011.553598 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

395

Abagovomab

Drug name Phase Indication

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Pharmacology description Route of administration Pivotal trial

Abagovomab Phase II/III Maintenance therapy in patients with advanced ovarian cancer in complete remission after frontline therapy Murine anti-idiotypic antibody mimics CA125 Subcutaneous Ongoing; results available first half 2011

in addition an Italian study demonstrated that consolidation treatment with six cycle of paclitaxel did not prolong either PFS or OS [11]. The first positive results of a Phase III clinical trial with a targeted therapy given concurrently with chemotherapy and in the maintenance setting were presented a few months ago. Bevacizumab (15 mg/kg intravenously every 21days) added to the first-line chemotherapy and continued for 15 subsequent cycles is able to prolong PFS by 3.8 months in comparison with 6 cycles of chemotherapy alone or with only 6 cycles of chemotherapy plus concomitant bevacizumab only [12]. In addition, positive results have been reported for a second Phase III trial testing bevacizumab in a similar setting but with lower dose and shorter treatment duration [13]. However, for these two trials OS results are still pending since follow-up is too short and there is a lack of events. In this described scenario, the maintenance immunotherapy could be one alternative potential treatment leading to possible improvement in PFS and OS, with a limited number of side effects and consequently without affecting patients’ quality of life (QOL). Cancer antigen 125 (CA125) is one of the known tumor-associated antigens (TAA) for ovarian cancer and is highly expressed on epithelial cancer cells. CA125 is detected with a high frequency in sera of more than 80% of patients with epithelial ovarian cancer, and the sero-prevalence and serum titers of this TAA become elevated with cancer progression [14,15]. CA125 is a validated tumor marker used to evaluate therapeutic response as well as to predict disease recurrence. CA125 is, however, poorly immunogenic. Therefore, specific immunotherapy targeting CA125 might enhance the host immune response and lead to clinical benefit. The past experience demonstrated that immunotherapy could elicit a specific CA125 response. Unfortunately, Oregovomab, a murine monoclonal antibody, able to directly recognize and bind CA125 failed to demonstrate a prolongation of PFS in P Phase III clinical trial [16]. Consequently, the research is now focusing on alternative regimens that might improve the specific response [17] or on more immunogenic agents: abagovomab might represent one of these (Box 1). 396

Anti-idiotypic network and abagovomab generation

2.

Box 1. Drug summary.

The ‘immune network hypothesis’ formulated by Niels Jerne [18] affirmed that the variable regions of an antibody (Ab1) contain a structure which is recognizable by the variable region of another antibody, and such determinant is called an idiotope that is immunogenic and able to induce antibodies. A collection of idiotopes is called an idiotype of an antibody, and the antibody (Ab1) induced by a specific antigen induces anti-idiotypic antibodies (Ab2) which in turn induces antianti-idiotypic antibodies (Ab3). Within this network, a subset of the anti-idiotypic antibodies (Ab2) has a structure closely resembling the epitope of the original antigen and induces an antibody-response similar to that induced by the original antigen (Figure 1); likewise, a proportion of the anti-anti-idiotypic antibodies (Ab3) has the same structure (epitope) as Ab1. Abagovomab is a murine-monoclonal antibody generated by the immunization of mice with the OC125 antibody against CA125. Based on the Neils Jerne hypothesis, abagovomab (anti-idiotypic antibodies Ab2) administration could induce the production of Ab3 antibodies in patients, with a proportion of them having the same structure (epitope) of Ab1 antibodies directed against the original CA125 antigen (Figure 2). 3.

Abagovomab pre-clinical characterization

The binding of 125I-labeled abagovomab to the anti-CA125 antibody OC125 coated on costar strips demonstrated that the affinity constant, calculated by Scatchard analysis, is 2.3 109 l/mol, which is similar to that for CA125 to OC125; in addition, binding of abagovomab to OC125 is completely inhibited by the nominal antigen. Immunization of rats with the F(ab¢)2 leads to anti-CA125 IgG and IgM antibodies (Ab3) that bind both abagovomab and CA125. Furthermore, the induction of a non-MHC-restricted cell-mediated lysis of CA125-expressing tumor cells has been demonstrated, as well as complement-dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) [19]. All these characteristics make abagovomab a good candidate for an effective immunotherapy to elicit an immune response against CA125 in vivo with the potential of being of clinical benefit. 4.

Abagovomab clinical studies

The clinical development of abagovomab includes three completed Phase I/II clinical studies [20-23] wherein abagovomab was administered to a total of 197 patients, along with an ongoing Phase II/III pivotal trial. The main characteristics and status of the studies are reported in Table 1. Phase I/II studies In all the three completed studies, the production of antibody (Ab3) to abagovomab was confirmed in 68.1 to 100% of treated subjects. 4.1

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Pfisterer, Harter, Simonelli, Peters, Berek, Sabbatini & du Bois

CA 125

Tumour cell

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(OC125) Ab 1

Abagovomab (Ab2)

Figure 1. The injection of a tumor associated antigen (TAA) binding antibody (Ab1) leads to an immune response containing antibodies (Ab2) mimicking the structure of the original antigen. Antibody (Ab1) that binds to cancer antigen 125 (CA125) induces antibodies (Ab2) which have a structure closely resembling that of the antibody-binding region of the original antigen CA125, thereby inducing an immune response. OC125 is a mouse monoclonal antibody that binds to CA125, and it is thought that there exists an OC125-binding epitope in the tandemly repeated domain among the three domains constituting CA125 (i.e., the N-terminal domain, tandemly repeated domain and C-terminal domain) [37,38].

CA125

Tumour cell

Abagovomab (Ab2)

Ab3/AB1

Figure 2. Vaccination with a cancer antigen 125 (CA125)-mimicking antibody (Ab2) leads to an immune response against the original antigen. The antibody (Ab2) possessing a structure closely resembling CA125, induces immune responses to the original antigen CA125. Ab2: Anti-idiotypic antibody; Ab3: Anti-anti-idiotypic antibody.

The first proof-of-concept (POC) study, a Phase Ib/II trial, was conducted in 119 patients with recurrent epithelial ovarian cancer or CA125-positive malignant tumors, who had previously received debulking surgery and platinum based chemotherapy. Results of the study showed a significantly longer OS for Ab3-positive patients than for Ab3-negative patients (23.4 versus 4.9 months, p < 0.0001), and suggested an association between the specific anti-tumor immune response and the prolongation of patients’ survival [21]. Furthermore, a proportion of treated patients, almost 40%,

showed the presence of Ab1 (anti CA125)-specific antibodies; additionally, the formation of Ab1 immunocomplexes were seen in 42 patients and a total of 60 patients (50%) developed either Ab1 or Ab1 immunocomplexes. Finally, ADCC activity could be demonstrated in the sera of 32 treated patients. The simultaneous detection of Ab3 and ADCC did not lead to further improvement of survival compared with Ab3 response without measurable ADCC. Therefore, the influence of ADCC in Ab3-positive patients on survival time is unclear and ADCC reactivity might be regarded as

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397

398

Proof of concept study

Phase Ib/II (July 1994 -- June 2002) [21]

Safety Tolerability Immunological response

Recurrence-free survival Overall survival Safety, Tolerability Immunological response

AGO-OVAR 2.8

ABA-01/MIMOSA/ AGO-OVAR 10

Phase I/II (July 2003 -- April 2004) [23]

Phase II/III (December 2006 -- ongoing)

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FIGO: Federation Internationale de Gynecologie et d’ Obstetrique.

[24]

[22]

Safety Immunological subsequently 2 times every 4 weeks, total 6 times response Dose/Route

Immunological Safety

Objectives

ACA125-01

Phase I/II (January 2003 -- April 2004)

Trial code

Trial

Table 1. Abogovomab trials.

Double blind Randomization Placebo-controlled

Non-blind

Non-blind Randomization

Non-blind

Design

Injection, 2 mg, subcuatneous (2 groups), 4 times every 2 weeks, subsequently every 4 weeks

Injection, 2 mg, subcuatneous (2 groups), 4 times every 2 weeks, subsequently 2 times or 5 times every 4 weeks, total 6 or 9 times

Injection, 0.2 mg/2 mg, intramuscular/subcutaneous (total 4 groups), 4 times every 2 weeks, subsequently 2 times every 4 weeks, total 6 times

Injection, 2 mg, intramuscular (1group), 4 times every 2 weeks, subsequently every 4 weeks

Abagovomab dose/ administration route

888

36

42

119

Number of patients

14 weeks/ 26 weeks

Until 94 weeks after the final subject enrolled (maximum of 48 months or to recurrence)

FIGO III -- IV epithelial ovarian carcinoma in clinical complete response after first line standard treatment, (surgery + chemotherapy)

14 weeks

Relapsed FIGO II -- IV epithelial ovarian carcinoma in clinical complete response after chemotherapy

Relapsed FIGO II -- IV epithelial ovarian carcinoma in clinical complete response after chemotherapy

Until disease progression

Duration

Relapsed ovarian carcinoma, (FIGO I -- IV) or with CA125-positive malignant tumor

Target population

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Abagovomab

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Pfisterer, Harter, Simonelli, Peters, Berek, Sabbatini & du Bois

only one of several potent effector mechanisms induced by abagovomab [21]. Overall, this observation clearly proved that abagovomab could elicit a specific anti-CA125 response after repeated administration, which is associated with prolonged OS. Two additional Phase I/II studies were performed in order to evaluate the effect of different doses, routes of administration and regimen durations on Abagovomab immunogenicity measured as Ab3 response (Study Code: ACA125-01, Study Code: AGO-Ovar 2.8 study). In the ACA125-01 study, a Phase I/II trial was conducted in 42 patients with asymptomatic residual disease or in CR patients with FIGO stage II -- IV recurrent epithelial ovarian cancer who had received debulking surgery and platinum based chemotherapy. This study did not show a statistical significant difference in Ab3 titer between routes of administration (intramuscular or subcutaneous), thus recommending progression with the subcutaneous route which is mostly preferred by the patients as compared to the commonly more painful intramuscular route. In addition, it did not show a statistical significant difference between two dose groups (0.2 or 2 mg), although Ab3 titer was higher in the ‘2 mg’ treatment groups than in the ‘0.2 mg’ treatment groups [22]. Based on these data, 2 mg SC was judged to be the optimal dosage and administration. The AGO-Ovar 2.8 study, a Phase I/II trial, was conducted in 36 patients with relapsed epithelial ovarian cancer. The patients had a history of debulking surgery and platinum-based first-line chemotherapy and had completed chemotherapy for recurrent disease with a condition of clinical complete response or being asymptomatic even with a measurable residual disease. This study showed that positive Ab3 was obtained in all of the 33 patients eligible for evaluation of immune reactions. Ab3 positive were indicated regardless of the administration period (the shortperiod administration group; 14 weeks/6 injections, and the long-period administration group; 26 weeks/9 injections), and a greater amount of increased Ab3 titers was observed in the long-period administration group with 9 injections [23]. In both studies, ACA125-01 and AGO-Ovar 2.8, the specific cellular immune response was evaluated in a subset of treated patients as the frequency of CA125 specific T-cell expressing gamma-interferon measured by spot ELISA (ELISpot) and cytokine flow cytometry, respectively. In both studies it was possible to identify patients who showed an increase in CA125 T-cell population expressing gamma-interferon during abagovomab administration. These data confirmed the ability of abagovomab to induce in vivo a specific cellular response, even if obtained from a limited number of treated patients [22,23]. Phase I/II safety results Overall, abagovomab treatment was safe and well tolerated. The most frequent adverse events (AE) related to the 4.2

abagovomab application were injection-site reactions. These reactions can be explained by the injection itself and by the inflammatory response due to the immunogenicity of abagovomab. The vast majority of adverse reactions were transient and mild (grade 1 or 2 according to the common toxicity criteria). There were no critical findings regarding laboratory parameters, vital signs, or signs and symptoms at the physical examinations, nor was there any characteristic trends that would suggest toxicity of the drug. No dose-limiting toxicity was observed. No deaths due to toxicity were reported [20-23]. Phase II/III pivotal study On the basis of the very promising data of the POC study and according to the concept that immunotherapy might be more active when the disease burden is minimal, a Phase II/III pivotal study, aiming to evaluate abagovomab as a maintenance therapy in ovarian cancer patients with no residual disease after frontline therapy, was initiated in 2006. In detail the AGO OVAR 10 trial, known also as the Monoclonal antibody Immunotherapy for Malignancies of Ovary by Subcutaneous Abagovomab (MIMOSA) trial, evaluates repeated abagovomab administrations versus placebo in patients with serum CA125-positive epithelial ovarian cancer (FIGO stage III/IV) who had achieved CR following debulking surgery and standard platinum/taxane first-line chemotherapy; the randomization is 2:1 in favor of abagovomab which is given at the 2 mg dose subcutaneously every 2 weeks for the first four administrations (induction phase), then monthly (maintenance phase). All patients enrolled are administered the study drug until disease recurrence or until completion of the doubleblind observation period (21 months after the last patient randomized is enrolled). At the end of the double-blind period, analysis of the benefit of abagovomab versus placebo on the efficacy (recurrence-free survival) and safety assessments will be done, and patients will be continue to be followed-up for an additional 5 years in order to evaluate the benefit of abagovomab versus placebo in terms of OS. The study is being conducted at approximately 150 sites in the USA and eight European countries (Germany, Italy, Belgium, Spain, Poland, the Czech Republic, Hungary and France). Enrollment was completed, as planned, in December 2008. Preliminary data reporting the blinded results on safety and immunogenicity were presented at the 2010 ASCO meeting [24]. Briefly, 888 patients were enrolled. The mean age of study population was 56.8 years, the majority of patients (82%) showed a serous/ papillary histology with a G3-G4 grade of differentiation (61.7%), and more than 90% of patients had an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 1 or less. Regarding main prognostic factors, it is noteworthy that almost 60% of patients had the best combination of the three favourable prognostic factors. Distribution of baseline characteristics in the AGO-OVAR 10/MIMOSA trial reflects epidemiological data reported in stage III/IV ovarian cancer in Western populations. As of April 2010, after a mean 4.3

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Abagovomab

Table 2. Blinded human anti-mouse antibodies (HAMA) (A) and antibodies against abagovomab (Ab3) measurements (B) at weeks 0, 10, 22 and 58. A. Total number of patients

Visit (week)

HAMA- Number of patients (%)

HAMA+ Number of patients (%)

Median value (ng/ml)

863 802 692 319

V1 V2 V3 V6

822 379 226 104

41(4.8%) 423 (52.7%) 466 (67.3%) 215 (67.4%)

113 515 6415 17,000

Baseline week 10 week 22 week 58

(95.2%) (47.2%) (32.7%) (32.6%)

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B. Total number of patients

Visit (week)

Ab3- Number of patients (%)

Ab3+ Number of patients (%)

Median value (ng/ml)

859 790 677 291

V1 V2 V3 V6

785 (91.4%) 238 (30.1%) 193 (28.5%) 88 (30.2%)

74 (8.6%) 552 (69.9%) 484 (71.5%) 203 (69.8%)

2890 62,050 316,500 716,000

Baseline week 10 week 22 week 58

Modified from [24].

exposure of 12.8 months to study treatment, only 19 potentially treatment-related serious adverse events (SAE) (placebo/ abagovomab) were reported. Overall, 14.3% of patients discontinued study treatment due to AEs, but only 2.1% because of at least potentially treatment related AEs. Among the non-serious AEs, most were mild and moderate and the most frequent were injection site reactions and fatigue. The ongoing Data Safety Monitoring Board review has found no safety issues and recommended continuing the study until its completion as planned. The overall immune response was evaluated measuring the blinded human anti-mouse antibodies (HAMA) and Ab3 (anti-abagovomab) at week 0, 10, 22 and 58, and the median values at each different time points are reported in Table 2. At baseline, 4.8% patients were positive for the presence HAMA and 8.6% of patients were positive for Ab3 (Table 2A and B, respectively). These baseline percentages are consistent with data observed in previous Phase I/II ABA studies, however it is worth noting that these are blinded data referred to the overall (abagovomab/placebo) population. At week 10, 22 and 58, about 70% of overall treated patients (abagovomab/placebo) were positive for Ab3 (Table 2B). Again, taking into consideration the randomization 2:1 ratio in favour of abagovomab treatment, data obtained at week 10 and 22 are also consistent with the results observed in previous Phase I/II studies (Figure 3) in which patients were treated with four abagovomab doses for induction and up to a maximum of subsequent five monthly maintenance administrations [22-24]. 5.

Conclusions

Abagovomab has been demonstrated to stimulate the humoral immune response and the cell-mediated immune response in 400

the clinical studies conducted to date. Furthermore, in the first POC study abagovomab prolonged OS in patients with recurrent OC who showed an immune response to its repeated administration (Ab3 responders). In addition, the overall studies and in the ongoing Phase II/III trial so far showed that abagovomab has an excellent safety and tolerability profile. 6.

Expert opinion

It is well known that OC, even if highly chemosensitive, is associated with a risk of recurrence greater than 75% [5,25]. Maintenance therapy after frontline therapy represents a possible strategy to improve clinical outcome. In the past, early clinical experiences in this setting, mainly based on single-agent chemotherapy [26], extended cycles of combination chemotherapy [27-29], or intra-peritoneal chemotherapy, failed to significantly improve the clinical outcome. Only the prolonged administration of paclitaxel after frontline therapy gave a clinically significant longer PFS in the GOG178 study but, again, the trial did not demonstrate any benefit in OS [9-11]. The improvement in PFS still is the main goal of recent clinical research in a maintenance setting. However, the major concerns deriving from early experiences are the toxicity and the consequent effect of maintenance regimens on the patients’ quality-of-life. Therefore, recent clinical research has been mainly focused on the development of new antiblastic drugs/regimens with limited toxicity such as polyglutammate paclitaxel [30], or regimens including agents targeting alternative pathways, for exampleVEGF, such as AGO-OVAR 12 and AGOOVAR 16 testing BIBF 1120 and pazopanib respectively [31,32], EGFR [33], thymidine kinase (TK) [34], farnesyl transferases [35], mammalian target of rapamycin (mTOR)

Expert Opin. Biol. Ther. (2011) 11(3)

Pfisterer, Harter, Simonelli, Peters, Berek, Sabbatini & du Bois

Study ACA125-01

90000

Study AGO-OVAR 2.8 360000

Ab3 values (ng/ml)

Ab3 values (ng/ml)

300000

60000

30000

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MIMOSA

ACA125-01

0

240000

AGO-OVAR 2.8 AGO-OVAR 2.8 long

180000

120000

MIMOSA

60000 0

2 mg sc

2 mg sc

2 mg im

0.2 mg sc 0.2 mg im

2 mg sc

Week 10

Week 10

Week 22

Figure 3. Median Ab3 values from previous abagovomab Phase I/II studies (ACA125-01 and AGO-OVAR 2.8) and median Ab3 values for Ab3-positive patients in the ongoing MIMOSA trial. Patients were treated with four Abagovomab doses for induction and up to a maximum of five subsequent monthly maintenance administrations.

inhibitors [36], and on boosting the host’s immune response. Immunotherapy is usually well tolerated and it is supposed to be particularly active when the disease burden is very limited. Therefore, immunotherapy might represent an excellent weapon in the context of maintenance therapy when the cancer burden is minimal and the treatments, which should be administrated over a long period, require an optimal tolerability and patient acceptance. So far, only the prolonged administration of paclitaxel (up to 12 cycles) and more recently the combination of bevacizumab plus chemotherapy followed by long maintenance cycles of 12 -- 15 administrations of bevacizumab at different doses (7.5 -- 15 mg/kg) gave a statistically significant PFS prolongation of 6 and 3.8 months, respectively [9,10]. However, as mentioned above, the additional paclitaxel [9,10] cycles did not have any effect on OS, while the OS data are still pending for bevacizumab trials. Both approaches imply a long treatment duration that might affect the patients’ quality of life. Moreover, the overall treatment costs for bevacizumab regimens might be an additional concern and the optimal dose and schedules might require further debate. Therefore, even if great improvement in the context of

maintenance therapy in OC has been registered recently, the need of more efficacious and feasible treatments still remains high. In this context, immunotherapy, as previously discussed, might be the most suitable therapeutic approach. Among such approaches abagovomab is the most advanced product with demonstrated immunogenicity. Furthermore, its optimal safety and tolerability profile along with its convenient route/schedule of administration make it a very promising candidate for maintenance therapy in advanced OC patients. The results of the AGO-OVAR 10/MIMOSA trial, expected for the first half of 2011, will certainly clarify the possible role of abagovomab in this setting.

Declaration of interest J Pfisterer is a member of the AGO Study Group and has received speaker’s honoraria and research funding for the MIMOSA study from Menarini. C Simonelli is an employee of Menarini. J Berek and P Sabbatini are members of the COGI Study Group and have received speaker’s honoraria and research funding for the MIMOSA study from Menarini. P Harter, M Peters and A du Bois declare no conflict of interest.

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Affiliation

Jacobus Pfisterer†1, Philipp Harter2 MD, Cecilia Simonelli3 MD, Martin Peters1 MD, Jonathan Berek4 MD, Paul Sabbatini5 MD & Andreas du Bois2 † Author for corresponcence 1 Professor, Klinik fu¨r Gyna¨kologie und Geburtshilfe Sta¨dtisches Klinikum Solingen, Gotenstr. 1, 42653 Solingen, Germany E-mail: [email protected] 2 Klinikum Essen Mitte, Department of Gynecologic Oncology, Henricistrasse 92, Essen, Germany 3 Menarini Ricerche, via Tito Speri 10, Pomezia, Rome, Italy 4 Chief of Obstetrics and Gynecology, Stanford Universtiy School of Medicine, Stanford University Hospital and Clinics, Stanford, CA 94305-5317, USA 5 Memorial Sloan-Kettering Cancer Center, 1275 York Ave, H906, New York, USA

403

Drug Evaluation

The role of bevacizumab in colorectal cancer: understanding its benefits and limitations 1.

Introduction

2.

Cytotoxic chemotherapy

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for CRC 3.

Tumor angiogenesis

4.

Bevacizumab

5.

Limitations and resistance mechanisms to the use of bevacizumab

6.

Expert opinion

Karen Mulder†, Andrew Scarfe, Neil Chua & Jennifer Spratlin †

Cross Cancer Institute, Department of Medical Oncology, 11560 University Avenue, Edmonton, Alberta, Canada

Introduction: Angiogenesis is a key factor in the development of aberrant blood vessels required for malignant growth, invasion and progression. Inhibiting VEGF is by far the most clinically advanced anti-angiogenic target. Bevacizumab (BV), the only humanized mAb directed against VEGF, is approved for use in multiple tumor types after successful clinical trial results demonstrated benefits in progression-free survival and/or overall survival when combined with common cytotoxic chemotherapies. Areas covered: The review focuses on the use of BV in colorectal cancer, discusses the clinical trial data supporting its increasing use and explores its limitations. Readers will gain a succinct description of the trial data demonstrating a modest survival benefit in metastatic colorectal cancer (mCRC) and the lack of benefit of BV when utilized in the adjuvant setting. A review of common BV toxicities and a discussion about possible BV resistance mechanisms are also provided. Expert opinion: Although BV has demonstrated efficacy in mCRC, there is an urgent need to improve the understanding of its mechanism of action and the development of BV resistance. Furthermore, there is a need for delineating predictive markers of BV efficacy and toxicity. Keywords: angiogenesis, bevacizumab, colorectal cancer, VEGF, VEGFRs Expert Opin. Biol. Ther. (2011) 11(3):405-413

1.

Introduction

Colorectal cancer (CRC) is the fourth most common cancer in men and third most common cancer in women worldwide [1,2]. CRC incidence rates vary markedly with increased rates in newly economically developed countries, whereas these rates have stabilized or decreased in longstanding economically developed countries [3]. The adoption of a Western lifestyle including obesity, decreased activity and smoking in economically transitioning countries is probably contributing to the growing international CRC burden. Increasing CRC mortality is still occurring in countries with limited resources; however, mortality rates have declined in longstanding and newly economically developed countries attributed mostly to introduction of early detection programs and improved therapies [4]. Angiogenesis is a critical step in the propagation of malignant tumor growth and blood supply. Bevacizumab (BV) is a humanized mAb against vascular endothelial growth factor (VEGF), which is the major mediator of angiogenesis. BV is the first drug developed as an inhibitor of angiogenesis to be approved by the FDA based on the survival benefit seen in a landmark trial for the first-line treatment of metastatic colorectal cancer (mCRC) when combined with conventional chemotherapy (Box 1) [5]. In this review, we review cytotoxic management of CRC while focusing on VEGF biology, the development of BV in the treatment of CRC, and both the limitations and possible mechanisms of resistance of BV. 10.1517/14712598.2011.557657 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

405

Bevacizumab

Box 1. Drug summary.

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Drug name Phase Indication Pharmacology description Route of administration Pivotal trial(s)

Bevacizumab Launched mCRC VEGFR antagonist Angiogenesis inhibitor Parenteral 1. AVF2107g [5]: Landmark trial which showed efficacy of bevacizumab in combination with irinotecan, fluorouracil and leucovorin for first-line treatment of mCRC 2. E3200 [42]: Second-line mCRC study showing efficacy of bevacizumab in combination with oxaliplatin, fluorouracil and leucovorin

mCRC: Metastatic colorectal cancer; VEGFR: Vascular endothelial growth factor receptor.

2.

Cytotoxic chemotherapy for CRC

3.

The benefit of 5-fluorouracil (FU)-based chemotherapy was first established in patients with mCRC with median survival in several clinical trials reaching 11 -- 13 months [6]. Two subsequent large randomized trials using FU/leucovorin (LV) or capecitabine without effective salvage therapy confirmed median overall survival (OS) of 12 -- 13 months [7,8]. Irinotecan, a topoisomerase I inhibitor, was initially studied as a second-line therapy in FU/LV refractory patients demonstrating an OS benefit [9,10]. Combining irinotecan with FU/LV (IFL) either as bolus or continuous infusion (Douillard regimen) emerged as the new standard therapy as an OS benefit was achieved over FU/LV [11,12]. Effective second-line treatment was established when the combination of oxaliplatin + infusional FU/LV (FOLFOX) improved outcomes after IFL failure or intolerance as compared to bolus or infusional FU/LV or single agent oxaliplatin [13]. The first-line Intergroup N9741 trial demonstrated superiority of FOLFOX over IFL in terms of efficacy and safety [14]. However, since then, IFL has fallen out of favor due to improved tolerance and efficacy of FOLFIRI (modified infusion of FU and irinotecan) when compared first-line [15,16]. There does not appear to be an optimal sequencing to FOLFIRI and FOLFOX as either FOLFIRI followed by FOLFOX or FOLFOX followed by FOLFIRI have the same median OS [17]. Importantly, this study reported the longest median OS in advanced CRC highlighting the importance of using all three chemotherapeutic agents (FU, irinotecan and oxaliplatin). The benefit of FU-based adjuvant chemotherapy in reducing recurrence and improving OS for stage III, but not equivocally for stage II colon cancer, is well established. The first large scale study demonstrating a significant reduction in the risk of death with adjuvant FU plus levamisole in patients with stage III colon cancer was the Intergroup trial 406

INT-0035 [18]. Subsequently, FU/LV became the standard of care in this setting [19-21]. Increased antitumor activity and modest survival benefit of IFL in metastatic disease led to the Phase III trial comparing IFL to FU/LV in patients with stage III colon cancer [22]. No improvement in disease-free survival (DFS) or OS was observed, though an increase in both lethal and nonlethal toxicity was reported in the IFL arm. Furthermore, two randomized Phase III trials, ACCORD02 and PETACC-3, combining irinotecan with infusional FU/LV, did not improve survival confirming that the benefits of irinotecan-based chemotherapy in mCRC do not translate into advances in the adjuvant setting [23,24]. In contrast, the MOSAIC trial, which compared infusional FU/LV to FOLFOX showed a significant benefit in 3- and 5-year DFS and 6-year OS for patients receiving FOLFOX [25,26]. The clinical benefit reached statistical significance only in patients with stage III colon cancer thus making FOLFOX the standard of care adjuvant regimen; the role of adjuvant therapy remains controversial in stage II patients.

Tumor angiogenesis

Angiogenesis, the formation of new blood vessels from preexisting vasculature, is a tightly regulated process in normal adults. Under physiologic conditions, the growth of new capillaries is tightly controlled by interplay of regulatory proteins that either act to stimulate or inhibit blood vessel growth with the balance between these forces favoring inhibition and growth restraint [27]. Pathologic angiogenesis occurs in diseases such as cancer, retinopathy and endometriosis. Tumor angiogenesis has been well studied and is known to differ from physiologic angiogenesis. Blood supply is considered essential for tumor growth beyond a diameter of 1 -- 2 mm and requires a process called ‘angiogenic switch’ [28]. Currently, six ligands have been identified in the VEGF family. VEGF-A (also called VEGF) is the major mediator of tumor angiogenesis and acts on vascular endothelial cells together with VEGF-B, VEGF-E and placental growth factor (PlGF). VEGF-C and VEGF-D act on lymphatic vessel endothelial cells. VEGF signals mainly through VEGFR-2 which is overexpressed by endothelial cells engaged in angiogenesis and by circulating bone marrow-derived endothelial progenitor cells [29]. The binding of VEGF to VEGFR-2 leads to a cascade of different signaling pathways that upregulate genes involved in proliferation, migration and survival [30]. The role of VEGFR-1 is unknown with respect to VEGFmediated angiogenesis. Despite the 10-fold greater binding affinity of VEGF to VEGFR-1 compared to VEGFR-2, it has significantly weaker signal-transducing properties [31]. Overexpression of VEGF has been detected in almost all human cancers including CRC. Preclinical work on colon cancer specimens showed a correlation between increased VEGF expression and proliferative activity in tumors [32]. Furthermore, overexpression of VEGF correlates with disease stage and development of distant metastasis. In vivo,

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Mulder, Scarfe, Chua & Spratlin

murine antihuman mAbs targeted against VEGF inhibited growth of human tumor xenografts [33]. Based on these findings, inhibition of VEGF is considered a rational strategy to treat CRC.

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4.

Bevacizumab

In order to reduce the antigenecity triggered by the human immune system with the introduction of mouse antibodies, BV was developed by genetically engineered ‘humanization’ technology applied to the mouse antibody, A4.6.1 [34]. The monoclonal mouse antibody A4.6.1 was developed by immunizing mice with human VEGF165. The isolated antibody was shown to act as a neutralizing antibody against human VEGF and it exhibited antitumor activity in mouse xenograft models engrafted with different human cancer cell lines [35]. In studies using nude mice transplanted with various human tumor types, administration of A46.1 antibody or BV suppressed tumor growth by 25 -- 95% compared to the control group [36]. Combining chemotherapy with A4.6.1 antibody or BV gave a more pronounced tumor effect than chemotherapy alone. 4.1

Bevacizumab in the treatment of mCRC Phase I trials

4.1.1

In humans, the original Phase I dose escalation trial enrolled 25 patients with multiple tumor types who were refractory to standard therapies [37]. BV, at doses up to 10 mg/kg, was safely administered with no grade 3 or 4 adverse events definitely related to the antibody. Common grade 1 and 2 toxicities were asthenia, headache, fever, rash, oral symptoms, nausea, cough and dyspnea. Three episodes of tumor-related bleeding occurred. Pharmacokinetic data appeared linear with a half-life of ~ 21 days, consistent with other mAbs. Maximum drug concentration increased with dose and drug did not accumulate. Serum VEGF levels increased postBV administration, explained as either a change in synthesis and distribution of VEGF or decrease in VEGF clearance. There were no complete or partial responses to BV, though 12 patients achieved stable disease (SD). The Phase I study combining BV with various chemotherapeutic agents, including FU/LV, established a favorable safety and tolerability profile [38]. Dose limiting toxicities included diarrhea, thrombocytopenia and leucopenia which were probably caused by the cytotoxic drugs; the combinations were otherwise well tolerated. No pharmacologic interactions between BV and the chemotherapeutic regimens were noted. One mCRC patient received 40 doses of BV with FU/LV and obtained SD. Phase II trials AVF0780g, randomizing 104 patients with previously untreated mCRC to bolus FU/LV alone versus FU/LV + BV 5 mg/kg versus FU/LV + BV 10 mg/kg, demonstrated statistically significant improvement in response rate (RR) 4.1.2

and time to progression favoring the BV 5 mg/kg arm (Table 1) [39]. The AVF 2192 g trial included 209 mCRC patients who were not felt to be ideal candidates for firstline irinotecan-based chemotherapy. Patients were randomized to FU/LV + placebo or FU/LV + BV 5 mg/kg every 2 weeks [40]. A statistically significant benefit in progressionfree survival (PFS) and duration of response favored the BV containing arm; however, it did not meet its primary end point of improving OS (Table 1). Phase III trials The landmark Phase III trial by Hurwitz et al., AVF2107g, which compared IFL + BV to IFL + placebo in mCRC had led to clinical approval of BV [5]. Median OS was 20.3 versus 15.6 months in favor of the BV containing arm. Statistically significant improvements in RR and PFS were also demonstrated (Table 2). However, at the time of publication, the IFL regimen had fallen out of favor due to the improved tolerance and efficacy of FOLFIRI as compared to IFL [15]. The BICC-C trial was modified in April 2004 to assess the efficacy of BV to both arms resulting in the FOLFIRI + BV arm having improved PFS and median OS compared to IFL + BV (Table 2). The NO16966 study was designed to establish whether capecitabine and oxaliplatin (XELOX) were non-inferior to FOLFOX as first-line therapy in mCRC but was then amended to a 2 2 factorial design with a secondary randomization to BV or placebo following the release of the AVF2107g data [41]. The addition of BV to the pooled XELOX plus FOLFOX group improved the primary outcome of PFS. However, in the subgroup analysis, the addition of BV to FOLFOX did not result in clinically or statistically significant improved PFS. BV did not improve secondary outcomes of RR or OS (Table 2). It is hypothesized that failure to improve median OS was due to early discontinuation of capecitabine or FU and BV when oxaliplatin-induced neurotoxicity occurred, thereby diminishing the impact of BV. In the second-line setting, there is a single Phase III study, E3200, comparing FOLFOX to FOLFOX + BV [42]. The addition of BV provided a modest OS benefit (12.9 vs 10.8 months, p = 0.001) and improved PFS and RR (Table 2). These results lead to the approval of BV in combination with FU-based chemotherapy in the second-line treatment of mCRC. 4.1.3

Bevacizumab in the treatment of resected colon cancer

4.2

The clinical efficacy of BV in mCRC led to two pivotal Phase III clinical trials in patients with resected stage III or high risk stage II colon cancer. NSABP-C08 reported the addition of 1 year of BV to FOLFOX did not improve 3-year DFS [43]. Similarly, the AVANT trial, a randomized, adjuvant three-arm, Phase III study of 3451 patients evaluating BV in combination with XELOX or FOLFOX versus FOLFOX alone also failed to meet its primary end point of

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Bevacizumab

Table 1. Phase II clinical trials combining bevacizumab with cytotoxic chemotherapy. Study

Regimen

AVF780g [39]

FU/LV* FU/LV* + BV 5 mg/kg FU/LV* + BV 10 mg/kg

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AVF2192g [40]

FU/LV* FU/LV* + BV 5 mg/kg

Efficacy Response rate 17% 40% (vs FU/LV, p = 0.029) 24% (vs FU/LV, p = 0.434)

Time to progression 5.2 months 9 months (vs FU/LV, p = 0.005) 7.2 months (vs FU/LV, p = 0.217)

Overall survival 13.8 months 21.5 months (vs FU/LV, p = 0.137) 16.1 months (vs FU/LV, p = 0.582)

Response rate 15.2% 26% (p = 0.055)

Progression-free survival 5.5 months 9.2 months (p = 0.0002)

Overall survival 12.9 months 16.6 months (p = 0.16)

*Roswell Park regimen: LV 500 mg/m2 over 2 h and FU 500 mg/m2 as a bolus midway through the LV infusion administered weekly for the first 6 weeks of each 8-week cycle. BV: Bevacizumab; FU: 5-Fluorouracil; LV: Leucovorin.

Table 2. Phase III clinical trials combining bevacizumab with cytotoxic chemotherapy. Study

Regimen

Efficacy Overall response rate

Progression-free survival

Overall survival 15. 6 months 20.3 months (p < 0.001) 17.6 months 23.1 months (vs mIFL, NSD) 18.9 months (vs mIFL, NSD) 19.2 months 28 months (p = 0.037) 19.9 months 21.3 months (p = 0.0769) 10.8 months 12.9 months (p = 0.0011)

AVF2107g [5]

IFL + placebo IFL + BV 5 mg/kg

34.8% 44.8% (p = 0.004)

6.2 months 10.6 months (p < 0.001)

BICC-C [15,16] Period 1

mIFL FOLFIRI

43.3% 47.2% (vs mIFL, NSD)

5.9 months 7.6 months (vs mIFL, p = 0.004)

CapeIRI

38.6% (vs mIFL, NSD)

5.8 months (vs mIFL, NSD)

BICC-C [15,16] Period 2

mIFL + BV 5 mg/kg FOLFIRI + BV 5 mg/kg

53.3% 57.9% (NSD)

8.3 months 11.2 months (NSD)

NO16966 [41]

FOLFOX/XELOX + placebo FOLFOX/XELOX + BV 5 mg/kg FOLFOX FOLFOX + BV 10 mg/kg

38% 38% (p = 0.99)

8 months 9.4 months (p = 0.0023)

8.6% 22.7% (p < 0.0001)

4.7 months 7.2 months (p < 0.0001)

E3200 [42]

BV: Bevacizumab; capeIRI: Capecitabine + irinotecan; IFL: Irinotecan, fluorouracil and leucovorin; mIFL: Modified IFL; NSD: No statistical difference.

improving DFS [44]. The conclusion from these studies is that the addition of BV to adjuvant chemotherapy fails to add to the benefit provided by cytotoxics in eradicating micrometastatic tumor cells. Safety and tolerability Clinical trials utilizing BV routinely excluded patients with clinically significant cardiovascular, cerebrovascular and/or peripheral vascular disease; uncontrolled hypertension (HTN); non-healing wounds or major surgery within the preceding 28 days; bleeding diatheses; known brain metastases; regular use of acetylsalicylic acid > 325 mg/day (or the equivalent non-steroidal inflammatory agent); or therapeutic anticoagulation. There are limited data for the safe use of BV in patients with these co-morbidities. 4.3

408

Thromboembolic events A meta-analysis of 12,617 patients in 20 randomized trials with a variety of solid tumors evaluated arterial thromboembolic (ATE) events [45]. The highest incidence (6.1%) of all-grade ATE occurred in CRC patients treated with BV. In CRC patients, BV significantly increased the risk of allgrade ATE compared to controls (relative risk 2.79; p = 0.001). In mCRC patients, the incidence of highgrade ATE was 1.9% which conferred a relative risk of 2.18 (95% CI 0.99 -- 4.80). The influence of BV on ATE was independent of dose. A meta-analysis of 7956 patients in 15 randomized trials with a variety of solid tumors evaluated the risk of venous thromboembolic events (VTE) [46]. Among the BV treated patients, the incidence of all- and high-grade VTE was 4.3.1

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Mulder, Scarfe, Chua & Spratlin

11.9 and 6.3%, respectively. Compared to the control group, BV resulted in a relative risk of 1.29 and 1.38 for all- and high-grade VTE, respectively, independent of dose. In CRC patients, the incidence of all- and high-grade VTE was 19.1 and 7.3%, respectively. Hypertension and renal toxicity A meta-analysis of 1850 patients with a variety of solid tumors in seven randomized trials evaluated HTN and proteinuria with the use of BV [47]. The incidence of all-grade HTN ranged from 2.7 to 36% for the low dose (£ 7.5 mg/kg/ dose) group and 17.6 -- 32% for the high dose (‡ 10 mg/ kg/dose) group. Grade 3 HTN (requiring more than one anti-hypertensive agent or more intensive therapy than previously required) incidence was 8.7, 16 and 1.7% for the low dose, high dose and control groups, respectively. Compared to the control group, low and high dose BV conferred a relative risk for HTN of 3 and 7.5, respectively. Proteinuria incidence ranged from 21 -- 41% for low dose to 22 -- 63% for high dose BV therapy. Grade 3 proteinuria (4 + dipstick or > 3.5 g protein on a 24 h urine collection) incidence was 1% for low versus 1.8% for high dose BV. As compared to controls, low and high dose BV conferred a relative risk of proteinuria of 1.4 and 2.2, respectively.

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4.3.2

Wound healing complications BV has been associated with multiple wound healing complications, such as dehiscence, ecchymosis, surgical site bleeding and wound infection. In NSABP C-08, the addition of BV with FOLFOX postoperatively increased the incidence of grade 3 or higher wound complications from 0.3 to 1.7% (p = 0.01) [48]. For patients undergoing metastectomy following BV-containing chemotherapy, an observational study reported 4.9% all-grade and 1.8% high-grade wound healing complications [49]. Another observational cohort study reported a 4.4% serious wound healing complication rate [50]. In this study, for patients undergoing surgery within 2 weeks, 2 to < 4 weeks, 4 to < 6 weeks and 6 to < 8 weeks of their last BV dose, the incidence of wound healing complications was 9.7, 3.2, 3 and 5.9%, respectively. In patients for whom surgery occurred > 8 weeks from their last BV dose, the incidence dropped to 2.2%. The optimal timing of surgery in patients receiving neoadjuvant BV has not been confirmed in prospective studies. 4.3.3

Gastrointestinal perforations A meta-analysis of 12,294 patients with a variety of solid tumors in 17 randomized trials evaluated the risk of gastrointestinal (GI) perforation when using BV [51]. The incidence of GI perforation was 0.9%. Compared to controls, BV conferred a relative risk of 2.14. There was a dose-dependent trend with the relative risk of perforations of 1.61 and 2.67 for low and high dose BV, respectively. For the CRC subset of patients treated with BV, the relative risk of perforation was 3.10, increasing to 3.68 if metastatic, Most 4.3.4

perforations occurred within the first 6 months of treatment and were associated with death in 21.7% of patients treated with BV as compared to 15.7% of patients in the control arm; BV conferred a relative risk of death of 2.7 in this population. It was not possible to ascertain whether this risk is modified by factors such as an unresected primary tumor, presence of gastric ulcer or diverticular disease, chemotherapy or radiation therapy-induced bowel injury, peritoneal carcinomatosis, previous surgery or recent endoscopy.

Limitations and resistance mechanisms to the use of bevacizumab

5.

In mCRC, addition of BV to current standard chemotherapy leads to a modest improvement in survival. There are multiple postulated mechanisms for resistance to anti-angiogenic therapy which are not mutually exclusive [52,53]. Anti-angiogenic therapy may lead to selection of tumor cells that can survive in hypoxic conditions [29]. These agents also induce the upregulation of other pro-angiogenic factors including those in the VEGF pathway such as PlGF as well as those extrinsic to it including fibroblast growth factor and d-like ligand 4 [53]. These effects are not limited to cancer cells as tumorassociated fibroblasts secrete platelet-derived growth factor C to stimulate tumor angiogenesis [54]. Furthermore, bone marrow-derived cells also secrete pro-angiogenic factors to stimulate angiogenesis in addition to providing endothelial and pericyte progenitor cells. This may lead to rapid vascular remodeling with increased pericyte coverage obviating the need for VEGF by the blood vessels. Tumors may also co-opt normal vasculature avoiding the need for revascularization [52]. Concerns have been raised that by withdrawing antiangiogenic therapy, after induction of these mechanisms of resistance, induction of rapid re-growth of the tumor may occur [55]. However, the relapse pattern and survival outcome in a meta-analysis of Phase III trials studying BV in different tumor types do not support this hypothesis [56]. To date, preclinical and clinical studies showing efficacy of anti-angiogenic therapy have mostly been on localized tumors or established metastatic disease, but it is largely unknown how effective it is at blocking the early stages of metastatic development [57]. Some preclinical studies mimicking adjuvant therapy have raised the possibility that anti-angiogenic therapy may facilitate tumor growth and metastasis. In a mouse model of breast cancer, decreasing the level of myeloid cell-derived VEGF led to normalization of tumor vasculature and higher tumor burden [58]. The sensitivity to chemotherapeutic agents was also increased. Similarly, short-term therapy (7 days) in mice with sunitinib, a tyrosine kinase inhibitor, after injection of tumor cells or after removal of primary tumor resulted in higher metastatic tumor burden and shorter life than in controls [57]. Furthermore, in a pancreatic neuroendocrine tumor mouse model, treatment with an antibody against VEGFR-2 (DC101) resulted in a more invasive phenotype and increased metastasis [59]. In the NSABP

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Bevacizumab

C-08 adjuvant trial, the addition of BV to FOLFOX did not appear to increase development of metastasis nor shorten survival [43]. This may account for the initial separation of the curve favoring the BV arm during the delivery of FOLFOX chemotherapy which may be explained by the hypothesis that BV may work by normalizing tumor vasculature thereby improving the delivery of cytotoxic chemotherapy to the tumor resulting in higher cell kill [60]. By extending the BV monotherapy an additional 6 months, one may hypothesize that given the above preclinical data this may negate the benefits obtained from the chemotherapy with BV. Early attempts which used traditional methods of looking at clinical or tumor-related variables in identifying the optimal subgroup of patients to receive BV have been unsuccessful. Tumor microvessel density, tumor VEGF and thrombospondin-2 expression, and mutations in k-ras and p53 do not predict for benefit from BV in CRC [61,62]. Early data in other tumor types suggest that VEGF single nucleotide polymorphisms may serve as biomarkers to predict subgroups of patients who will experience the optimal outcome and as well as those who will be at higher risk of BV-induced toxicity [63,64]. 6.

Expert opinion

While angiogenesis is a critical step in malignant tumor growth, anti-angiogenic therapy has not been universally effective across all tumor groups. In CRC, a modest survival benefit has been demonstrated in patients with metastatic disease with the addition of BV to cytotoxic chemotherapy though this does not translate to the adjuvant setting. A critical look at the available data suggests the strongest evidence for BV use in mCRC is second-line in combination with FOLFOX. Though the addition to BV to irinotecanbased regimens is widely accepted, the benefits of first-line FOLFIRI + BV are limited to nonrandomized Phase III

410

comparisons and observational cohort data. The benefits of FOLFOX/XELOX + BV were disappointing after the gains noted with IFL + BV and question the risk:benefit ratio of its use in this setting. Furthermore, there is a lack of evidence for BV combined with FOLFIRI in second-line questioning its use if FOLFOX is used in first-line therapy. The value of continuing treatment with BV beyond progression remains unanswered outside of observational cohort data [65]. However, this has become the practice in many centers in the US, prompting the need for a randomized controlled trial to establish a prospective reference. It is also important to note that the addition of BV to modern cytotoxic chemotherapy in mCRC does not appear to improve RR. Hence, its role in improving resectability in the borderline resectable patients with liver metastases is unknown. Randomized controlled trials are needed to determine if there is benefit in this patient population. In contrast, emerging data with upfront use of EGFR inhibition in combination with cytotoxics in mCRC with wild-type KRAS, particularly irinotecan-based therapies, are encouraging with regard to improving RR. It is not yet clear whether this will translate into improved resectability or survival advantages. In the immediate future, there is an urgent need to better understand the mechanism of action of BV as well as the development of tumor resistance to this agent. The monetary cost and toxicity of this agent heighten the need for more effective implementation highlighting the importance of further delineating predictive markers for both efficacy and toxicity. If this cannot be achieved, it is plausible that other targeted agents will take over the role BV currently has in the treatment of mCRC.

Declaration of interest The authors declare no conflict of interest and have received no payment in preparation of this manuscript.

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Mulder, Scarfe, Chua & Spratlin

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Miles D, Ellis LM, Reardon DA. Is there really a yin and yang to VEGF-targeted therapies? Lancet Oncol 2010;11:809-11

57.

Ebos JML, Lee CR, Cruz-Munoz W, et al. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell 2009;15:232-9

58.

Stockmann C, Doedens A, Weidemann A, et al. Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis. Nature 2008;456:814-18

Mulder, Scarfe, Chua & Spratlin

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Paez-Ribes M, Allen E, Hudock J, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 2009;15:220-31

60.

Jain R. Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy. Nat Med 2001;7:987-9

61.

Ince WL, Jubb AM, Holden SN, et al. Association of k-ras, b-raf, and p53 status with the treatment effect of bevacizumab. J Natl Cancer Inst 2005;97:981-9

62.

Jubb AM, Hurwitz HI, Bai W, et al. Impact of vascular endothelial growth factor-A expression thrombospondin-2 expression, and microvessel density on the treatment effect of bevacizumab in metastatic colorectal cancer. J Clin Oncol 2006;24:217-27

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Schneider BP, Wang M, Radovich M, et al. Association of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 genetic polymorphisms with outcome in a trial of paclitaxel compared with paclitaxel plus bevacizumab in advanced breast cancer: ECOG 2100. J Clin Oncol 2008;26:4672-8

64.

Schultheis AM, Lurje G, Rhodes KE, et al. Polymorphisms and clinical outcome in recurrent ovarian cancer treated with cyclophosphamide and bevacizumab. Clin Cancer Res 2008;14:7554-63

65.

Grothey A, Sugrue MM, Purdie DM, et al. Bevacizumab beyond first progression is associated with prolonged overall survival in metastatic colorectal cancer: results from a large observational cohort study. J Clin Oncol 2008;26:5326-34

Expert Opin. Biol. Ther. (2011) 11(3)

Affiliation

Karen Mulder†1,2 BSc MSc MD FRCPC, Andrew Scarfe1,2, Neil Chua1,3 & Jennifer Spratlin1,2 † Author for correspondence 1 Cross Cancer Institute, Department of Medical Oncology, 11560 University Avenue, Edmonton, Alberta, Canada, T6G 1Z2 Tel: +780 432 8248; Fax: +780 432 8888; E-mail: [email protected] 2 Assistant Professor, Faculty of Medicine & Dentistry, University of Alberta, Alberta, T6G 1Z2 Canada 3 Assistant Clinical Professor, Faculty of Medicine & Dentistry, University of Alberta, Alberta, T6G 1Z2 Canada

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Drug Evaluation

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Intradermal influenza vaccine and new devices: a promising chance for vaccine improvement 1.

Introduction

Filippo Ansaldi, Paolo Durando & Giancarlo Icardi†

2.

Body of review

University of Genoa, Department of Health Sciences, Genoa, Italy

3.

Conclusion

4.

Expert opinion

Introduction: The main public health strategy for reducing the influenza burden is annual vaccination. However, because the immune response in the elderly and frail subjects is known to be less vigorous than in younger adults, novel strategies have been explored in the last decade to augment immune response after immunization. These have included an increased dosage, multiple dose vaccinations, use of vaccine adjuvants and more efficient routes of vaccine delivery. Areas covered: This review discusses recent advances in intradermally administered influenza vaccines with particular attention to immunogenicity and safety data from clinical trials performed using appropriate injection systems which, combining consistent injection depth and volume, safety, simplicity and ease-of-use, allowed large scale evaluation. Expert opinion: Substantial experience has been accumulated with Intanza 9 and Intanza 15 µg/IDflu 15 µg, administered using BD’s Soluvia device, and are the first intradermal trivalent inactivated split-virion influenza vaccines to be licensed for use in 18 -- 59 year adults and in elderly people, respectively. Clinical data showed that they are safe and immunogenic and can offer significant advantages in terms of higher acceptability, higher immunogenicity in the elderly and dose-sparing in adults younger than 60 years when compared with intramuscularly administered vaccine. Keywords: device, immunogenicity, influenza vaccine, intradermal, safety Expert Opin. Biol. Ther. (2011) 11(3):415-427

1.

Introduction

Seasonal influenza represents a considerable public health burden for its contribution to determining out-patient visits, hospitalizations and deaths in the community. The impact of influenza may vary widely according to the epidemiological picture observed in different seasons and in different countries, age groups, case definitions and detection methods used for surveillance. Age-specific influenza attack rates, based on surveillance studies, ranged from 6.6% (range: 2.6 -- 15.5%) for working-age adults to 20.3% (range: 7.5 -- 25.8%) for children under 5 years old. [1-3]. Rates of influenza-attributable hospitalizations and deaths, based on excess seasonal rates, showed that the highest impact is on adults aged > 65 years: in this age group, hospitalization or death rates for influenza/pneumonia were 10- and 130-fold higher than that observed among young adults, respectively [4]. The main public health strategy for reducing influenza burden is annual vaccination. Traditionally, annual vaccination has been targeted to people classified as being at high risk, particularly the elderly, but in recent years, there has been a broadening of vaccination recommendations to target larger numbers of people. This has recently culminated in the recommendation that annual vaccination be administered to all people aged ‡ 6 months for the 2010 -- 11 influenza season in the US [5]. Universal vaccination is being debated in other countries in Western 10.1517/14712598.2011.557658 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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Intanza

Box 1. Drug summary. Drug name

Indication

Intradermally administered split-virion trivalent inactivated influenza vaccine (Intanza/IDflu) Prophylaxis of influenza in 18 -- 59 and ‡ 60 year adults

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Pharmacology description/ mechanism of action Route of administration Intradermal route The recommended site of administration is the region of the deltoid [71,72,75,77,78,81] Pivotal studies

Europe, where vaccination is actually recommended for adults aged > 65 years, healthcare workers and high-risk individuals, including people from the age of 6 months with chronic diseases and pregnant women [6]. However, because the immune response in the elderly and in frail subjects is known to be less vigorous than in younger adults, research in the last decade has been focused on improving the immune response to vaccination and increasing the protection of aged and high-risk populations [7,8]. Novel strategies have been explored to augment immune response after immunization in the elderly, including an increased dosage, multiple dose vaccinations, use of vaccine adjuvants and more efficient routes of vaccine delivery. This review discusses recent advances in intradermally administered influenza vaccines with particular attention to immunogenicity and the safety data of clinical trials performed using appropriate injection systems which, combining consistent injection depth and volume, safety, simplicity and ease-of-use, allowed large scale evaluation. 2.

Body of review

Overview of the market The first commercial influenza vaccines were approved for human use in the US in 1945 based on efficacy studies performed on military recruits and college students using whole-virus inactivated vaccines. Although whole-virus vaccines are still in use in some countries and are highly effective, most vaccines manufactured since the 1970s have been subvirion preparations, that is, split-virion and subunit vaccines, which present a better tolerability profile. When vaccine and circulating viruses are antigenically similar, subunit and split-virion vaccines prevent influenza illness among ~ 70 -- 90% of healthy adults < 65 years of age [9,10]. However, vaccine efficacy varies among certain populations according to the antigenic matching between vaccine and circulating strains. Subunit and split vaccines showed a reduced immunogenicity and effectiveness in infants, young children, older adults and frail subjects, which present a higher risk of influenza-related complications, hospitalizations and deaths [4]. The reduced immune response observed in the elderly is 2.1

416

particularly evident when it is evaluated against drifted circulating strains [11-14]. Therefore, there is an unmet need for improved influenza vaccines in these vulnerable populations. For these reasons, adjuvants have been developed and, in particular for influenza vaccination, a squalene-based oil-in-water emulsion, MF59, and a virosome-based carrier system were approved for human use more than a decade ago. MF59adjuvanted seasonal influenza vaccine (Fluad, Novartis) is now commercially available in 23 countries worldwide including 12 member countries of the EU and approved for adults aged > 65 years [15]. MF59 is able to increase immunogenicity of seasonal, pre-pandemic (Aflunov, Novartis) and pandemic subunit vaccines (Focetria, Novartis), maintaining acceptable safety and tolerability profiles [14-18]. Virosome-based vaccine (Inflexal V, Berna Biotech) presents an optimal tolerability profile identical to that shown by subunit and split vaccines, but controversy exists about the improvement offered by this carrier system in terms of immunogenicity [19-22]. More recently, AS03, another oil-in-water emulsion, was approved as a component of pre-pandemic H5N1 vaccine (Prepandrix, GSK) and a component of pandemic H1N1 2009 vaccine (Pamdemrix, GSK) [23-25]. A number of studies reported antigen dose--response effect showing a stronger response following vaccination with a higher dose of antigen compared with that observed using the 15 µg of hemagglutinin of each strain usually administered. In December 2009, the FDA licensed an injectable inactivated trivalent influenza vaccine (Fluzone High-Dose, Sanofi Pasteur) for people > 65 years that contains a total of 60 µg of influenza virus antigen per strain. Studies investigating the use of 60 µg antigen dose vaccine in older adults demonstrated higher GMT, seroprotection and seroconversion rates compared with that observed in subjects immunized with standard-dose vaccine (15 µg of each strain), but the incidence of adverse events, generally mild or moderate, was higher in subjects immunized with high-dose preparations [26-28]. The need to improve immunogenicity of current influenza vaccines and the lack of patient acceptance of traditional intramuscular vaccination have led to the development of new routes of vaccination delivery, including intranasal, oral, transcutaneous and intradermal. In 2003, a live cold-adapted intranasally administered influenza vaccine (Flumist, Medimmune) was approved in the US and, nowadays, it is recommended for children older than 2 years, teenagers and adults younger than 49 years. Studies comparing the efficacy of inactivated to that of live cold-adapted intranasally administered vaccine have been conducted in a variety of settings and populations using several different protocols: the difference in efficacy between the two vaccines was not statistically significant in numerous studies, but recent meta-analysis highlighted that the live formulation is more effective in pediatric populations [29-32]. Since the 2010 -- 11 season, a new microinjection system for intradermal administration called Soluvia and produced

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Ansaldi, Durando & Icardi

by Becton Dickinson (BD) has become available and used for the approved split influenza vaccine, Intanza (Sanofi Pasteur MSD, Lyon, France) (Box 1). Intradermally administered influenza vaccine: from classical intradermal administration to new devices

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2.2

The intradermal route of vaccine administration is not new and about 80 years ago Tuft provided evidence that a small dose of intradermally administered typhoid fever vaccine was better tolerated and equally immunogenic as the subcutaneous delivery of a larger dose [33]. The first experiments using whole influenza vaccine were carried out only a few years after the marketing of the first influenza intramuscular vaccine in the US, and > 20 clinical trials were performed in the following 60 years to evaluate the safety, tolerability and immunogenicity of intradermally administered influenza vaccine using conventional needles [34-55]. Historically, the use of intradermal immunization has been limited by the lack of an appropriate, reliable, easy-to-use, disposable and safe intradermal injection system and technique. The current renewed interest in intradermally administered vaccine has been largely driven by the perception that it offers a number of advantages compared with intramuscular or subcutaneous routes regarding acceptability, immunological response and logistical aspects. The potential advantages offered by intradermally administered influenza vaccine include both the improved immunogenicity in high-risk and low-responder groups and the antigen dose reduction. The dose reducing potential arouses great interest for a number of reasons including seasonal vaccine shortage due to the broadening of vaccination recommendations to target larger numbers of people or to manufacturing difficulties, such as those encountered in 1973 in the US, or contamination at a single production plant in 2004, when about 50% of the vaccine supply in the US was declared unfit for use [56,57]. Furthermore, there are concerns regarding the global under-capacity for rapid manufacture of pandemic influenza vaccines. The classical intradermal administration method using a standard or tuberculin needle and syringe requires trained and skilled personnel to inject the vaccine correctly. More importantly, injection depth and volume are not always consistent using the standard Mantoux technique, which can affect immunogenicity results, and the injection could be painful for the patient, influencing the tolerability profile and acceptability [58]. To overcome these limitations, new devices for intradermal vaccine administration have been developed and experimented with in the second half of 2000. Intradermal devices that have been extensively evaluated for influenza vaccination include the MicroJet microneedle device (NanoPass) and the intradermal microinjection system (Soluvia, BD). The first device consists of an array of four silicon microneedles, each 0.45 mm in length, which is bonded to the tip of a plastic adapter that can be mounted on any standard syringe. The microinjection system has a prefilled syringe ready for use, the volume of which can

vary from 100 to 200 µl, with an integrated minineedle ~ 1.5 mm in length able to ensure proper vaccine penetration in the dermis. Thanks to the small size of both devices, administration via microneedle penetrates the skin to a depth of a few hundred micrometers, making it almost imperceptible to the patient and, unlike deep intramuscular injection, eliminating the potential risk of injury to nerves or blood vessels. Furthermore, the elimination of the risk of injury to blood vessels offers a further advantage of reducing the risk of cross-contamination by microneedle. Taking into account the significant amelioration offered by new microinjection systems in terms of ease of use, acceptability and consistency of administered volume, this review focuses on the clinical data that have emerged from the use of MicroJet microneedle device and microinjection system in recent years. Clinical data which have emerged from studies performed in the previous decades in which a tuberculin syringe was used to administer the influenza vaccine intradermally were recently reviewed by Vankerckhoven and van Damme [59]. Furthermore, recent preclinical studies performed using different microneedle vaccines, that is, microneedles coated with influenza virus-like particles or inactivated virus, dissolving microneedle patches and microneedle patches with trehalose-stabilized influenza vaccine are likely to translate into clinical studies in the near future [60-63]. Immunological basis for potential benefits of intradermally administered influenza vaccine

2.3

The extreme richness in various resident and recruited types of dendritic cells, a professional antigen-presenting cell capable of stimulating both innate and adaptive (i.e., antigenspecific) immune responses, makes skin an attractive and efficient site for vaccination. In particular, the human dermis, although lacking cells possessing the classic subcellular characteristics of Langerhans cells that are present in the epidermis, has its own population of specialized dendritic cells [64,65]. Like Langerhans cells and macrophages recruited from circulating blood and infiltrating dermis tissue, these dermal dendritic cells (DDCs) express high levels of class II MHC and CD1 molecules and are extremely efficient in antigen presentation [66]. Dermal dendritic cells pick up the antigen, process it and re-express part of it as peptide--MHC complex on the surface; this process is profoundly affected by danger signals or danger signal-induced cytokine. Activation of Langerhans cells and DDCs includes the increased expression of MHC antigens, co-stimulatory molecules, and cytokines such as IL-1, IL-6, IL-12, as well as the enhanced emigration of these cells from the skin to the paracortical area of draining lymph nodes. Intradermal vaccination induces T-cell activation by the activation and migration of dermal dendritic cells and by lymphatic drainage of free antigen and subsequent capture by lymphonode-resident dendritic cells [67]. The intradermal approach offers potential advantages compared with intramuscular vaccination, delivering antigens directly to the

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Intanza

skin immune system. On the contrary, muscle does not possess important antigen-presenting cells and T-cell activation following intramuscular vaccination is due to transient antigen-presenting cells or antigen migration to the draining lymph node. The great richness in various resident and recruited types of antigen-presenting cells in the dermis justifies the potentially greater immunogenicity of intradermal compared with intramuscular vaccination, as confirmed by results obtained with three currently licensed vaccines: BCG, rabies, locally approved for this route in some countries, and smallpox (vaccinia), with the recently approved influenza vaccine, described in the following section, and with hepatitis B [68-70]. A recent review by Nicolas and Guy deepened the unique immunological properties of the skin and clinical experience with cutaneous immunization against infectious diseases, with a special focus on intradermal immunization [71]. Immunogenicity The first clinical trial that evaluated innovative techniques for intradermal delivery of influenza vaccine able to guarantee consistency of injection depth and volume -- not always uniform using standard Mantoux technique -- was performed by Belshe et al. during the 2001 -- 02 season. This study used a tuberculin syringe fitted with a plastic disc to limit the depth of needle penetration, essentially a forerunner of BD’s Soluvia device [72]. In Figure 1 -- 3, the difference in geometric mean titer ratio (GMTR, the ratio of the geometric mean titer before vaccination to the titer after vaccination), in seroconversion and seroprotection rates between intradermally administered, using the new delivery devices and intramuscularly administered influenza vaccines are reported. In the figures, we show only the immunogenicity parameters reported in the articles. As compared with an intramuscular injection of full-dose influenza vaccine, an intradermal injection of a reduced dose, 40% of the usual dose (6 µg for each strain), resulted in similarly vigorous antibody responses among people 18 -- 60 years of age. In subjects older than 60 years, intradermal vaccination with 6 µg elicited a strong immune response, reaching a seroprotection rate (> 60%) higher than that recommended by the European Committee for Medicinal Products for Human Use (CHMP) for this age group, but there was a trend towards a better response in the intramuscular route that was significant only for H3N2 strain (Table 1). Later studies by Belshe et al., Kenney et al. and Auewarakul et al., using a standard needle and tuberculin syringe, gave contrasting results on the immune response evoked by administration of a fifth of the standard intramuscular dose (3 µg/strain) in young adults [46,47,73]. Using an intermediate version of BD’s Soluvia device, Beran et al. did not confirm the ability of intradermal vaccine containing £ 6 µg of hemagglutinin for each antigen to elicit an immune response similar to that of intramuscular vaccination: in adults younger than 58 years, the 3 and 6 µg 2.4

418

intradermal formulations were less immunogenic than intramuscular 15 µg and no CHMP criteria for B strain were reached by vaccine with 20 and 40% of the usual dose (Table 1) [74]. In adults younger than 60 years, intradermal vaccine containing 9 µg of hemagglutinin for each antigen administered using BD’s Soluvia device met CHMP requirements for GMTR (> 2.5), seroconversion (> 40%) and seroprotection rate (> 70%) against the three vaccine strains in three studies performed by Leroux-Roels et al., Beran et al. and Arnou et al. Globally, despite the lower antigen content, the intradermal 9 µg vaccine was comparably immunogenic to the reference intramuscular vaccine, although LerouxRoels et al. showed superior responses against both A strains (H1N1, H3N2) compared with the control. Intradermal induced also antibody persistence up to a year after vaccination that was comparable with the intradermal and intramuscular vaccines [74-76]. In elderly people, two large clinical studies showed that intradermal vaccine containing 15 µg of hemagglutinin for each antigen administered using BD’s Soluvia device is more immunogenic than the standard intramuscular vaccine [77,78]. The study by Holland et al. evaluated the immunogenicity of intradermal inactivated influenza vaccine containing 15 or 21 µg of hemagglutinin per strain in volunteers > 60 years of age. For each strain and for each intradermally administered vaccine, the seroprotection rate, the seroconversion rate and the GMTR were significantly higher than those elicited by the intramuscularly administered vaccine, except for the rate of seroprotection against A(H1N1) in the group given the 15 µg intradermally administered vaccine, which did not reach significance [77]. The 3-year, randomized, controlled, open-label Phase III trial enrolling > 3700 adults aged ‡ 60 years by Arnou confirmed that the intradermal influenza vaccine containing 15 µg of hemagglutinin per strain is superior to that of a conventional intramuscular vaccine. After the first, second and third vaccinations, seroprotection rates were consistently higher with the intradermal vaccine compared with intramuscular vaccine [78]. Interestingly, a study by van Damme et al. compared the intradermal vaccine with that of the MF59-adjuvanted vaccine in the elderly: the post hoc analysis of covariance to adjust for baseline antibody titres demonstrated the non-inferiority of the intradermal vaccine by hemagglutination inhibition and single radial hemolysis assays for all three strains [79]. Only one study has investigated the effect of intradermal influenza vaccine administered using new devices on immunocompromised and high-risk patients. Sixty-two renal transplant patients with low or no hemagglutination inhibiting antibody response to an influenza vaccine were enrolled and randomly assigned to receive 15 µg intradermal or intramuscular influenza vaccines. For each strain, the intradermally administered vaccine met CHMP criteria, except for the GMTR for B strain, showing a higher seroprotection rate, seroconversion rate and GMTR for the three

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H1N1 H3N2 Soluvia ID microinjection system (Becton-Dickinson)

B

18 – 60 years, 6 µg ID vs 15 µg IM (Belshe 2004) 18 – 57 years, 9 µg ID vs 15 µg IM (Leroux 2008) 18 – 57 years, 3 µg ID vs 15 µg IM (Beran 2009) 18 – 57 years, 6 µg ID vs 15 µg IM (Beran 2009)

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19 – 58 years, 9 µg ID vs 15 µg IM (Beran 2009) 20 – 59 years, 9 µg ID vs 15 µg IM (Beran 2009) 18 – 60 years, 9 µg ID vs 15 µg IM (Arnou 2010) > 60 years, 6 µg ID vs 15 µg IM (Belshe 2004) > 60 years, 15 µg ID vs 15 µg IM (Holland 2008) > 60 years, 21 µg ID vs 15 µg IM (Holland 2008) > 60 years, 15 µg ID-ID vs 15 µg IM-IM(Arnou 2009) > 60 years, 15 µg ID-ID-ID vs 15 µg IM-IM-IM (Arnou 2009) > 65 years, 15 µg ID vs 15 µg MF59 IM (Van Damme 2010) 18 – 60 years, N.R.R.T., 15 µg ID vs 15 µg IM (Morel 2010)

MicroJet microneedle device (Nanopass) 18 – 40 years, 3 µg ID vs 15 µg IM (Van Damme 2009) 18 – 40 years, 6 µg ID vs 15 µg IM (Van Damme 2009) -12

-9

-6

-3

0

3

6

9

Figure 1. Difference in GMTR between intradermally administered, using new delivery devices, and intramuscularly administered influenza vaccines. GMTR: Geometric mean titer ratio.

components of vaccine composition than those elicited by the intramuscularly administered vaccine [80]. Also, intradermal vaccine administered by NanoPass MicroJet device showed a good immunogenicity although the experience is limited: antibody response in healthy young

(18 -- 40 years) adults elicited by a low and medium dose intradermal injection (3 and 6 µg hemagglutinin per strain, respectively) was similar to those elicited by the fulldose intramuscular vaccination. The GMTR, seroconversion and seroprotection CHMP criteria for licensure of seasonal

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Intanza

H1N1 H3N2

Soluvia ID microinjection system (Becton-Dickinson)

B

18 – 60 years, 6 µg ID vs 15 µg IM (Belshe 2004) 18 – 57 years, 9 µg ID vs 15 µg IM (Leroux 2008) 18 – 57 years, 3 µg ID vs 15 µg IM (Beran 2009)

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18 – 57 years, 6 µg ID vs 15 µg IM (Beran 2009) 19 – 58 years, 9 µg ID vs 15 µg IM (Beran 2009) 20 – 59 years, 9 µg ID vs 15 µg IM (Beran 2009) 18 – 60 years, 9 µg ID vs 15 µg IM (Arnou 2010) > 60 years, 6 µg ID vs 15 µg IM (Belshe 2004) > 60 years, 15 µg ID vs 15 µg IM (Arnou 2009) > 60 years, 15 µg ID-ID vs 15 µg IM-IM (Arnou 2009) > 60 years, 15 µg ID-ID-ID vs 15 µg IM-IM-IM (Arnou 2009) 18 – 60 years, N.R.R.T., 15 µg ID vs 15 µg IM (Morel 2010)

MicroJet microneedle device (Nanopass) 18 – 40 years, 3 µg ID vs 15 µg IM (Van Damme 2009) 18 – 40 years, 6 µg ID vs 15 µg IM (Van Damme 2009) -30

-15

0

15

30

Percentage (%)

Figure 2. Difference in seroconversion rates between intradermally administered, using new delivery devices, and intramuscularly administered influenza vaccines.

influenza vaccines were reached for both intradermal vaccines [81]. Safety and tolerability Clinical trials conducted using conventional intradermal injection techniques showed a higher incidence of local, 2.5

420

injection-site reactogenicity, but not systemic events, following intradermal versus intramuscular or subcutaneous immunization, although reactions were generally mild and transient [42,43,45,47-55]. In a recent clinical trial, 500 young adults were randomly assigned to receive an intradermal injection of 0.1 ml dose of inactivated split-virion influenza

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H1N1 H3N2 Soluvia ID microinjection system (Becton-Dickinson)

B

18 – 60 years, 6 µg ID vs 15 µg IM (Belshe 2004) 18 – 57 years, 9 µg ID vs 15 µg IM (Leroux 2008) 18 – 57 years, 3 µg ID vs 15 µg IM (Beran 2009)

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18 – 57 years, 6 µg ID vs 15 µg IM (Beran 2009) 19 – 58 years, 9 µg ID vs 15 µg IM (Beran 2009) 20 – 59 years, 9 µg ID vs 15 µg IM (Beran 2009) 18 – 60 years, 9 µg ID vs 15 µg IM (Arnou 2010) > 60 years, 6 µg ID vs 15 µg IM (Belshe 2004) > 60 years, 15 µg ID vs 15 µg IM (Arnou 2009) > 60 years, 15 µg ID-ID vs 15 µg IM-IM (Arnou 2009) > 60 years, 15 µg ID-ID-ID vs 15 µg IM-IM-IM (Arnou 2009) 18 – 60 years, N.R.R.T., 15 µg ID vs 15 µg IM (Morel 2010)

MicroJet microneedle device (Nanopass) 18 – 40 years, 3 µg ID vs 15 µg IM (Van Damme 2009) 18 – 40 years, 6 µg ID vs 15 µg IM (Van Damme 2009) -30

-20

-10

0

10

20

Percentage (%)

Figure 3. Difference in seroprotection rates between intradermally administered, using new delivery devices, and intramuscularly administered influenza vaccines.

vaccine or an intramuscular injection of 0.5 ml dose: 92 and 68% of subjects vaccinated with the intradermal preparation reported erythema and induration versus < 4% in the intramuscular group [47]. A higher incidence of erythema and induration among intradermal vaccinees using conventional

techniques was confirmed by a number of recent studies [48,51]. The safety profile of the intradermal vaccine administered with the new BD’s Soluvia device was similar to that of the reference vaccine in terms of both solicited systemic reactions

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Intanza

Table 1. Fulfillment of criteria recommended by the European CHMP according to age group and antigen dose.

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Device, author, year

Population (age class)

ID antigen dose (mg)

Vaccine strains reaching CHMP criteria GMT ratio

Soluvia ID microinjection system (Becton Dickinson) Belshe 2004 18 -- 60 years 6 µg Leroux 2008 18 -- 57 years 9 µg Beran 2009 18 -- 57 years 3 µg 18 -- 57 years 6 µg 19 -- 58 years 9 µg 20 -- 59 years 9 µg Arnou 2010 18 -- 60 years 9 µg Belshe 2004 > 60 years 6 µg Holland 2008 > 60 years 15 µg > 60 years 21 µg Arnou 2009 > 60 years 15 µg, 1 year > 60 years 15 µg, 2 year > 60 years 15 µg, 3 year Van Damme 2010 > 65 years 15 µg Morelon 2010 18 -- 60 year, 15 µg NRRT MicroJet microneedle device (NanoPass) Van Damme 18 -- 40 years 3 µg 2009 6 µg

Seroconversion rate

Seroprotection rate

No strains All strains A(H1N1) A(H1N1) and A(H3N2) All strains A(H3N2) All strains No strains All strains All strains All strains A(H1N1) and A(H3N2) All strains A(H1N1) and A(H3N2) None

All strains All strains A(H1N1) and A(H1N1) and All strains All strains All strains All strains All strains All strains A(H1N1) and A(H1N1) and All strains A(H1N1) and A(H1N1) and

All strains

All strains

All strains

All strains

All strains

All strains

A(H1N1) and All strains A(H1N1) and All strains All strains A(H3N2) All strains A(H3N2) and All strains All strains All strains A(H1N1) and All strains All strains A(H1N1) and

A(H3N2) A(H3N2)

B

A(H3N2)

A(H3N2)

A(H3N2) A(H3N2)

A(H3N2) A(H3N2) A(H3N2) B

CHMP: Committee for Medicinal Products for Human Use; GMT: Geometric mean titer; NRRT: Non-responder renal transplant.

and the number and type of spontaneously reported adverse events. In Figure 4, the difference in incidence of reactions included in the CHMP guideline between intradermally administered, using new delivery devices, and intramuscularly administered influenza vaccines is reported. In terms of safety and reactogenicity, CHMP guidelines state that within 3 days of immunization, the following events need to be assessed: injection site induration > 5 cm, injection site ecchymosis, body temperature > 38 C for ‡ 24 h, malaise and shivering. A significantly higher incidence of injection site ecchymosis in the intradermal group receiving 15 µg dose emerged from the trial by Holland et al. with a difference among groups of about 4% [77]. In all other studies, the differences in incidence of reactions included in the CHMP guideline between intradermally BD’s Soluvia and intramuscularly administered influenza vaccines are not significant and < 7%. As far as other adverse events are concerned, different studies confirmed that intradermal vaccination causes more visible injection site reactions than the intramuscular vaccine. These reactions were transient and not associated with an increase in injection site pain. When it was registered, pain occurred at comparable rates among the intradermal and intramuscular groups [76-78]. Also, intradermal vaccine administered with the MicroJet device showed that local mild and transient reactions were significantly more frequent following intradermal vaccination, but a larger study population is needed to collect more definitive data [81]. 422

Regulatory affairs Intanza 9 and Intanza 15 µg (also known in some countries as IDflu 15 µg) are the first intradermal trivalent inactivated split-virion influenza vaccines to be licensed for use in adults younger than 59 years and in elderly people (> 60 years of age), respectively. Both formulations were administered using BD’s Soluvia device. They received marketing authorization in the EU in February 2009 and in Canada in September 2010. 2.6

3.

Conclusion

The potential of intradermal vaccination is well known, but the standard Mantoux intradermal technique is difficult to perform correctly and requires highly skilled personnel. The availability of an appropriate injection system and a technique combining simplicity, safety and ease-of-use allowed large scale evaluation of the tolerability, safety and immunogenicity of intradermal influenza vaccines. Although the number of studies is low and some publications did not contain all the necessary information to make a complete comparison in terms of safety and immunogenicity, substantial experience has been accumulated with Intanza 9 and Intanza 15 µg/IDflu 15 µg, administered using BD’s Soluvia device, which are the first intradermal trivalent inactivated split-virion influenza vaccines to be licensed for use in adults younger than 59 years and in elderly people.

Expert Opin. Biol. Ther. (2011) 11(3)

Ansaldi, Durando & Icardi

Shivering Malaise Fever > 38.0ºC Injection site ecchymosis

Soluvia ID microinjection system (Becton-Dickinson)

Injection site induration 18 – 57 years, 9 µg ID vs 15 µg IM (Leroux 2008)

18 – 57 years, 6 µg ID vs 15 µg IM (Beran 2009)

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19 – 58 years, 9 µg ID vs 15 µg IM (Beran 2009)

20 – 59 years, 9 µg ID vs 15 µg IM (Beran 2009)

18 – 60 years, 9 µg ID vs 15 µg IM (Arnou 2010) > 60 years, 15 µg ID vs 15 µg IM (Holland 2008)

> 60 years, 21 µg ID vs 15 µg IM (Holland 2008) > 60 years, 15 µg ID vs 15 µg IM (Arnou 2009)

> 65 years, 15 µg ID vs 15 µg MF59 IM (Van Damme 2010) 18 – 60 years, N.R.R.T., 15 µg ID vs 15 µg IM (Morel 2010)

MicroJet microneedle device (Nanopass)

18 – 40 years, 3 µg ID vs 15 µg IM (Van Damme 2009)

18 – 40 years, 6 µg ID vs 15 µg IM (Van Damme 2009) -12

-9

-6

-3 0 3 Percentage (%)

6

9

12

Figure 4. Difference in cumulative incidence of CHMP reactions between intradermally administered, using new delivery devices, and intramuscularly administered influenza vaccines. CHMP: Committee for Medicinal Products for Human Use.

Although the incidence of injection site reactions -- mainly erythema, swelling and induration and mild pruritus -- have been reported as being higher than with intramuscular vaccine in some studies, compared with the standard intramuscular vaccine, the safety and tolerability profile of these preparation is good. In elderly people and in low responder subjects, such

as renal transplant patients, vaccine containing 15 µg of hemagglutinin for each antigen administered using BD’s Soluvia device is more immunogenic than the standard intramuscular injection. These recently emerged data open up the possibility of using the intradermal route for the routine prevention of influenza, offering a better chance of protective response in

Expert Opin. Biol. Ther. (2011) 11(3)

423

Intanza

frail subjects and a better compliance in subjects who dislike needles. Promising results were obtained from the first clinical study performed using the MicroJet device.

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4.

Expert opinion

The availability of easy-to-use devices able to guarantee a more accurate and consistent delivery of antigen and a higher vaccine acceptability than those provided by standard or tuberculin needle and syringe offers a new promising chance to bridge the gap affecting plain influenza vaccine in terms of unsatisfactory immunogenicity in the elderly and in patients with underlying chronic diseases and low compliance. The perception that the intradermal route might offer a number of clinical, including vaccine acceptability, immunological and safety advantages compared with intramuscular delivery has been largely confirmed by studies performed in recent years. Improved immunogenicity in low-responder and high-risk groups, such as adults aged > 60 years or renal transplant recipients, using 15 µg/strain formulation has been confirmed by several studies. Less clear is the potential advantage in terms of a higher immune response offered by a 9 µg formulation in younger adults that showed an immunogenicity profile similar to that of an intramuscularly administered vaccine. Reduced dose intradermal vaccines could be useful in the case of vaccine shortage and in pandemic situations, but no data are accumulated on the priming effect in naive subjects. The improved immunogenicity of intradermally administered vaccine might avoid the need to develop or incorporate adjuvants in some vaccines reducing potentially higher costs and the perception of safety concerns. The safety and tolerability profile of vaccines administered using BD’s Soluvia device is acceptable and comparable to that of intramuscularly administered vaccine, contrary to the observations made in a number of studies performed in the second half of last century using the Mantoux technique. The shortness of the needle of the MicroJet microneedle device Soluvia microinjection system reduces

424

the risk of needle-stick injuries among healthcare workers and could contribute to an increase in vaccination coverage in subjects with needle-phobia. On the other hand, substantial experience accumulated with Intanza 9 and Intanza 15 µg/IDflu 15 µg is limited to healthy adults and elderly people without unstable chronic illness. There is a clear need for studies that evaluate the immunogenicity and safety profile in other populations, that is, children or in high-risk individuals. Furthermore, the advantage offered by the intradermal route in terms of immune response should be better defined and its ability to elicit cross-protection against drifted strains, determine a prime effect against potentially pandemic strains and elicit cellular immune response should be extensively explored. So far, only one clinical study has evaluated the immunogenicity and the safety of intradermal vaccine administered using MicroJet microneedle. In conclusion, clinical data demonstrate that actually, licensed intradermal vaccine is safe and immunogenic and can offer significant advantages in terms higher acceptability, higher immunogenicity in the elderly and dose-sparing in adults younger than 60 years. These results need to be confirmed in other populations and other aspects regarding immune response should be explored. Nowadays, it can be considered a valid alternative to intramuscular and subcutaneous vaccination.

Declaration of interest F Ansaldi has previously participated at speaker’s bureaus and advisory board meetings sponsored by Novartis Vaccines and Sanofi Pasteur and has received research funding from Novartis Vaccines and Sanofi Pasteur. G Icardi has previously participated at speaker’s bureaus and adivisory board meetings sponsored by Sanofi Pasteur and has received research funding from Crucell Berna, GSK and Sanofi Pasteur. P Durando has received research funding as co-investigator from Sanofi Pasteur.

Expert Opin. Biol. Ther. (2011) 11(3)

Ansaldi, Durando & Icardi

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Affiliation

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Expert Opin. Biol. Ther. (2011) 11(3)

Filippo Ansaldi, Paolo Durando & Giancarlo Icardi† † Author for correspondence Professor, University of Genoa, Department of Health Sciences, Via Pastore 1, Genoa, 16132 Italy E-mail: [email protected]

427

Drug Evaluation

Clinical gene therapy for the treatment of RPE65-associated Leber congenital amaurosis Linda Stein, Kamolika Roy, Lei Lei & Shalesh Kaushal†

1.

Introduction

2.

The retinal system

3.

Role of RPE65 and AAV vectors

†

University of Massachusetts Medical School, Department of Ophthalmology, Worcester, MA, USA

in LCA gene therapy 4.

LCA RPE65 gene therapy

5.

A pharmacological approach:

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LCA retinoid treatment 6.

Conclusion

7.

Expert opinion

Introduction: The positive results of pioneering clinical trials using gene therapy as treatment for patients with Leber congenital amaurosis (LCA) have ushered in a new era of molecular retinal therapeutics for LCA, other blinding retinal disorders and gene therapy applications. Areas covered: This review describes the role of retinal pigment epitheliumspecific 65 kDa protein (RPE65) in the visual cycle and how RPE65 deficiency results in LCA; the extensive preclinical studies with recombinant adenoassociated virus (rAAV)-RPE65 gene vectors; and the human rAAV-RPE65 and related gene therapy clinical trials and studies. The literature search included a review of primary sources (e.g., journal articles) that reported study data results and key secondary sources such as meta-reviews available through PubMed, as well as reviews of clinical trial descriptions and results as reported in clinicaltrials.gov, conference publications and news releases. Expert opinion: LCA-RPE65 gene therapy is an example of successful, innovative, translational research. Further research is needed regarding how retinal gene therapy can be improved. Keywords: LCA, rAAV, RPE65 mutation, vision function Expert Opin. Biol. Ther. (2011) 11(3):429-439

1.

Introduction

Recent research has advanced the possibility of using gene therapy for a variety of heritable and non-heritable ocular diseases, including corneal diseases, optic neuropathies, retinal diseases, age-related macular degeneration and colorblindness. One of the most significant advances has occurred in gene therapy treatment for the retinal disease Leber congenital amaurosis (LCA), which is one of the most severe forms of inherited retinal degeneration. LCA is a key cause of blindness in children and young adults, with an estimated prevalence of 1 in 81,000 [1]. No treatment or cure was available for LCA patients prior to gene therapy. Mutations in 1 of at least 12 different genes can result in LCA. Approximately 6% of LCA cases may be attributed to a mutation in one of these genes, retinal pigment epithelium-specific 65 kDa protein (RPE65), which plays a key role in the visual cycle (Box 1) [2]. Extensive investigations of LCA-RPE65 in animal models and humans coupled with advancements in gene therapy led to several clinical trials. The results thus far of these trials indicate that gene therapy for LCARPE65 was safe and effective in restoring vision function in many, but not all, patients enrolled in the trials. Because the importance of RPE65 in the visual cycle is a key to understanding LCA gene therapy, an overview of the retinal system is provided. Results of significant murine, canine and non-human primate studies

10.1517/14712598.2011.557358 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

429

RPE65

Box 1. Drug summary. Drug name Phase Indication Mechanism Administration route Vector construct

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Pivotal trials

LCA-RPE65 gene therapy I--II--III Autosomally recessive inheritance of RPE65-deficiency associated with LCA2 Transducing rAAV vector encoding hRPE65 upregulates WT RPE65 gene expression in RPE65-deficient eyes allowing for efficient recycling of chromophore Subretinal or intravitreal injection Exon1 β-actin CMV ie enhancer ITR Intron

hRPE65 SV40 poly(A) Exon2

ITR

CHOP, Phase I Univ. of PA/Univ. of FL, Phase I Moorfields Eye Hospital, Phase I--II

Image source: Jacobson SG, et al. Safety of recombinant adeno-associated virus type 2-RPE65 vector delivered by ocular subretinal injection. Mol Ther 2006;13:1074-84 [40]. CHOP: Children’s Hospital of Philadelphia; ITR: Inverted terminal repeat; LCA: Leber congenital amaurosis; rAAV: Recombinant adeno-associated virus; RPE65: Retinal pigment epithelial-specific 65 kDa protein; WT: Wild-type.

investigating RPE65 gene therapy are presented, followed by a discussion of safety and efficacy results of human clinical trials. Non-gene therapy, pharmacological studies and a related clinical trial are briefly described as a potential LCA treatment that may be an alternative to gene therapy. Advances in molecular biology and retinal surgery have made these gene therapy clinical trials possible and pave the way for future research and trials to enhance LCA-RPE65 gene therapy and for investigating gene therapy as a treatment for other retinal disorders. 2.

The retinal system

Once light passes through the cornea, anterior chamber, lens and vitreous of the eye, the light interacts with the retina, which can be thought of as the photographic film of the eye. The retina is a diaphanous structure attached to the inner wall of the back of the eye and is generally no thicker than tissue paper. At least nine identifiable layers/components of the retina can be observed histologically (Figure 1). Clinically, the laminar structure of the retina can be seen non-invasively by optical coherence tomography, which also allows the retina specialist to quantitate the various retinal layers, especially the outer nuclear layer (ONL) where the photoreceptors (light-detecting cells) reside. Retinal health can be monitored over time to determine disease progression and also the effect of potential therapies. Light traverses the entire thickness of the retina before it interacts with photopigment proteins within photoreceptor cells. The two classes of photoreceptors are rods and cones. Rod cells contain the photopigment rhodopsin and mediate dim light and peripheral vision in humans. Cone cells contain 430

one of three pigment proteins identified in humans and are important for central and color vision. In rod-cone dystrophies, such as LCA, first rod cell death occurs, followed by subsequent cone loss. Photopigment proteins consist canonically of an apoprotein, opsin, which is a classic heptahelical GPCR that resides in the lipid bilayer of the photoreceptor cell. Opsin is covalently linked via a Schiff base to the vitamin A analog, 11-cis retinal. Phototransduction is initiated by the photoisomerization of 11-cis-retinal bound to opsin and ultimately results in a change in the release of neurotransmitters by photoreceptor cells. On light absorption, 11-cis-retinal isomerizes to all-trans retinal, which is then reduced to all-trans retinol by retinol dehydrogenase. All-trans retinol is transported out of the rod outer segment to the neighboring retinal pigment epithelium (RPE) cells, and the regeneration of 11-cis retinal occurs in the RPE through three enzymatic steps. First, lecithin retinol acyl transferase (LRAT), the first visual cycle enzyme, links alltrans retinol to phosphatidyl choline in the cell membrane to generate all-trans retinyl esters. The next step of the visual cycle involves the simultaneous hydrolysis and isomerization of all-trans retinyl esters to yield 11-cis retinol; this process is facilitated by a single enzyme, RPE65. Third, 11-cis retinol is then transferred to 11-cis retinol dehydrogenase for oxidization from 11-cis retinol to 11-cis retinal. The newly generated 11-cis retinal crosses the sub-retinal space and re-enters the outer segment of the photoreceptor. Opsin, which is synthesized in the inner segments, is also transported to the outer segments of rods and cones. Once back in the outer segment, 11-cis-retinal and opsin combine to form the photosensitive visual proteins (Figure 2).These biochemical events lead to the conversion of light into an electrical signal. This signal is

Expert Opin. Biol. Ther. (2011) 11(3)

Stein, Roy, Lei & Kaushal

A.

B. Sclera

Trabecular Anterior meshwork chamber

Retinal pigment epithelium and choroid

Pigment epithelium Rods Cones

Retina

Cornea Zonule fibers

Müller cells LIGHT

Pupil

Horizontal cells Bipolar cells Amacrine cells

Lens

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Ganglion cells

Optic nerve

Iris Ciliary body

Nerve fiber layer

Vitreous body

Figure 1. Structure and function of the eye. A. Vertical sagittal section of the adult human eye, and light micrograph of a vertical section through human peripheral retina, B. schematic enlargement of retinal cells. Source: modified from Webvision, http://webvision.med.utah.edu.

Photoreceptor

Light

Rhodopsin (opsin + 11-cis retinal)

All-trans retinal

11-cis-retinal

communicated to bipolar cells (second order neurons) and retinal ganglion cells (third order neurons), eventually leading to visual perception in the occipital cortex. Among the visual cycle’s important enzymes, RPE65 plays an essential role. The RPE65 gene encodes an abundant and evolutionarily conserved 61 kDa microsomal protein expressed almost exclusively in the RPE. The protein is necessary for the synthesis of the 11-cis chromophore of photoreceptor cell visual pigments. Loss of RPE65 function disrupts the visual cycle: all-trans retinyl will not be synthesized into 11-cis retinol without the assistance of RPE65. For a subset of LCA patients, mutations in RPE65 have been shown to involve such a disruption, with the mutated RPE65 failing to perform its usual role in the synthesis of the 11-cis chromophore. Predictably, these patients have significantly reduced light sensitivity of both rods and cones, as indicated both subjectively and by psychophysical testing. These clinical observations are the basis for the development of LCA-RPE65 gene therapy.

Role of RPE65 and AAV vectors in LCA gene therapy

3.

RPE65 RPE cell

Figure 2. The visual cycle (simplified). Absorption of light by rhodopsin pigment initiates isomerization of 11-cis-retinal to all-trans-retinal, which is transported into the RPE cell. The isomerase RPE65 uses a form of all-trans-retinal as substrate to form the chromophore 11-cis-retinal in the RPE cell. Mutations in the RPE65 gene cause LCA. 11-cis-retinal is released by the RPE cell and taken up by the photoreceptor, where it recombines with opsin to form rhodopsin. Source: [25]. LCA: Leber congenital amaurosis; RPE: Retinal pigment epithelial; RPE65: RPE-specific 65 kDa protein.

Visual function is highly regulated by a biochemical cascade involving a multitude of enzymes, including RPE65, as described in Section 2. Mutations in the RPE65 gene were the second type found to be associated with LCA; thus, LCA caused by mutations in RPE65 is distinguished as LCA2. Since the discovery of the disease-causing role of RPE65 mutations in LCA2, great strides have been made in a relatively short period of time to introduce the gene into the eyes of human LCA2 populations. The nonpathogenic adeno-associated virus (AAV) has proven to be a safe and effective vector for gene delivery.

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RPE65

Moreover, the recombinant adeno-associated virus (rAAV) vector has demonstrated increased specificity and efficiency in ocular AAV-mediated gene therapy interventions [3]. At present, at least nine AAV serotypes have been evaluated for ocular use. AAV2 is the most common serotype in humans and thus has garnered the most interest, also because of its use as a successful gene transfer vector in a variety of rodent and animal models. Recombinant AAV2 (rAAV2) vectors used for gene therapy are derived from the wild-type virus by deleting the entire viral coding region (rep and cap) and replacing them with the reporter or therapeutic transgene. Specifically, AAV2-derived vectors are produced by packaging rAAV genomes that are devoid of all viral open reading frames but harbor the minigene cassettes of a transgene, flanked by 5¢ and 3¢ inverted terminal repeats (ITRs), with rep/cap and adenovirus helper functions provided in trans. Combined AAV serotypes have also been developed. This diversity of serotypes may lead to a more specific and more efficient transduction. However, successful and efficient transfection of particular cell types in the eye still depends on other factors, such as the AAV titer, the site of injection, the amount of passenger DNA and the specific gene promoters. The promoter region of DNA, located upstream of the particular gene coding for the protein being produced, is important because it is where transcription initiation takes place. Generally, AAV2 refers to serotype 2 AAV, with both capsid and ITR from AAV2. However, there are different arrangements, according to the different capsid and ITR chosen. For example, AAV2/2 is a serotype virus with both capsid and ITR from AAV2, while AAV2/1 is a serotype 2 virus with capsid from AAV1 and ITR from AAV2. The extensive preclinical research that allowed for later human clinical trials encompassed murine, canine and non-human primate studies. Retinal degeneration and visual function loss in these genetically engineered and naturally occurring animal models were extensively characterized by a variety of methods (including histopathology, electroretinography [ERG], which measures retinal electrical response to light stimulation, or photoresponses, and transient pupillary light reflex which measures increased sensitivity to light), prior to investigations of gene therapy intervention with rAAV-vectored RPE65. 4.

LCA RPE65 gene therapy

4.1

Preclinical studies Murine RPE65 gene therapy studies

4.1.1

rAAV-mediated gene therapy was investigated in a variety of murine models of RPE65 deficiency. Studies conducted by Dejneka et al. (2004) indicated that ~ 70% of fetal (E14) eyes and 80% of young adult (1 -- 2.5 month old) eyes in RPE65-/- mice subretinally injected with AAV1-vectored human RPE65 cDNA exhibited positive treatment outcomes as measured by ERG [4]. ERG responses to light stimulation also improved in 24-month-old RPE65-/- mice that exhibited 432

a more severe retinal degeneration and were injected with the same AAV1 vector, but in a smaller percentage of vectortreated eyes. Conversely, treatment with the AAV2-vector encoding the same RPE65 cDNA in the same type of RPE65-/- mice yielded limited transduction and short-lived functional rescue [5]. A third vector, AAV5, using human RPE65 cDNA with a chicken b-actin (CBA) promoter, was injected into 2-week old RPE65rd12 mice by subretinal delivery. Efficient RPE65 transduction indicators, including rhodopsin biosynthesis, decreasing all-trans retinyl ester accumulation, improved visually guided behavior and restored retinal function as measured by ERG, were detected up to 6.5 months following the injection in the naturally occurring RPE65-deficient mice [6]. To further investigate visual pathway function beyond the retina, cortical function in AAV5 vector-treated RPE65rd12 mice injected at P18 and 6 months was investigated using visually evoked potentials and found to be relatively intact [5]. Also, 2 -- 5 week-old RPE65rd12 mice subretinally injected with rAAV2-CB(SB)-hRPE65 vector underwent extensive functional assessment by ERG at 2 and 4 months of age. Results of this study indicated an association between therapeutic efficacy and increasing vector dose and were also used to develop an in vivo bioassay for use in human gene therapy patients [5]. The biodistribution of various rAAV2/2-RPE65 doses was investigated in normal rats [7]. Specifically, vector doses of 1 1010 vg/µl rAAV2/2-RPE65 delivered in 150 µl by subretinal injection and of 3 1010 vg/µl rAAV2/2-RPE65 delivered in 150 µl by intravitreal injection were assessed at both short (10 -- 17 days) and long (7 -- 8 weeks) periods following vector administrations. At 10 -- 17 days post-injection, vector sequences were detected in all 13 subretinally injected rats; however, only four out of the six intravitreally injected rats demonstrated ocular inclusion of the vector sequences. Analysis at 7 -- 8 weeks post-injection in rats injected both subretinally and intravitreally revealed that rAAV2/2-RPE65 remained relatively contained to the injection area [7]. Canine RPE65 gene therapy studies The results of LCA studies in Swedish Briard dogs provided important impetus for the advancement of the therapeutic vector into human gene therapy trials. In the first of many proof-of-concept studies conducted in the spontaneously occurring RPE65 null canines, three dogs were injected either subretinally or intravitreally with AAV2-vectored wild-type dog RPE65 cDNA with a CMV/CBA promoter at 4 months of age [8]. At 3 months post-injection, ERG responses in eyes injected subretinally with rAAV-RPE65 revealed improved photoreceptor function, while intravitreally injected eyes showed no difference compared to untreated eyes on all ERG measures [8]. As the first-of-its-kind study to demonstrate restored visual function by multiple assessment measures in a large-animal model, this experiment served as the foundation for several follow-up experiments. 4.1.2

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Narfstr€om et al. investigated four RPE65-deficient dogs aged 4 -- 30 months subretinally injected with rAAV-RPE65 in one eye and rAAV-GFP in the other. Significant improvements in both visual behavior and ERG responses could be seen 4 weeks post-injection in the rAAV-RPE65-treated eyes and improvement in ERG responses persisted over time. Long-term results revealed the unusual finding of improved photopic ERG responses in the control rAAV-GFP-treated eyes in both young and older dogs [9,10]. Acland et al. augmented their 2001 findings in a largescale study in 2005 that included various vector pseudotypes and promoters using subretinal injection of RPE65 cDNA in 26 eyes compared to 45 untreated RPE65-mutant control eyes. With 23 of the 26 treated eyes indicating improvement in visual function, this experiment was the first to demonstrate restoration of near-normal sensitivity in both rod and cone photoreceptors in the RPE-mutant dog [11]. Jacobson et al. investigated the toxicology and biodistribution of subretinal rAAV-RPE65 gene therapy in 15 RPE65mutant dogs during a 3-month study. The dogs ranged in age from 2.7 to 7.4 months and received bilateral injections of either vehicle control (2 dogs) or rAAV-2/2.RPE65 (AAV2-CBO-hRPE65) at different dose levels (13 dogs). There was no observable increase in humoral immune response to the AAV2 capsid at 3 months post-injection. Transient abnormalities included: i) mild to moderate conjunctival reaction in 15 of the 30 vehicle- and vectortreated eyes (diminished by 3 months post-injection in most animals), ii) vitreous cellularity in 9 of the 30 vehicle- and vector-treated eyes that persisted in five eyes until 3 months post-injection and iii) lens opacities in 6 out of the 30 dogs. A pigmented lesion associated with the injection site within the tapetal retina was found at 3 months post-injection in 25 of the 30 treated eyes. Several additional lesions were discovered by ocular histopathology; these lesions were found to be either artifacts of vector administration during retinal surgery or possibly attributable to existing disease pathology. Inflammatory lesions were also noted. ONL thinning was found in a few eyes treated by subretinal injection at higher vector doses (1 and 3). Biodistribution studies of rAAV2/2-RPE65 in the ocular tissues of all 15 RPE65-mutant dogs revealed no detectable vector sequences. Analysis of other organ tissues revealed some unreplicated vector sequences in the heart, diaphragm and mandibular node of 3 of 30 dogs that were treated with either 0.0001 or 1 vector doses [7]. Jacobson et al. also evaluated efficacy in an additional cohort of three dogs injected with the rAAV2/2-RPE65 vector containing a shorter promoter (AAV2-CBSB-hRPE65). Unlike the robust ERG response evoked in normal dark-adapted dogs with increasing light stimuli, no ERG response was found in vehicle-injected RPE65-mutant dogs exposed to the same light stimuli. However, dependence of ERG photoresponses on vector dose was clearly discernible when response amplitudes from subretinal, vector-treated RPE65-mutant dogs were compared with normal dogs, untreated RPE65-mutant dogs

and vehicle-treated RPE-mutant dogs. Of the vector-treated eyes injected with ‡ 0.1 vector dose, 72% (13 of 18 eyes) responded to a flicker stimulus when light-adapted and 94% (17 of 18 eyes) responded to a white flash stimulus when dark-adapted when measured by ERG [7]. Follow-up assessments of animals from earlier proof-ofconcept studies constitute what little is known about longterm improvement in retinal function in dogs due to gene therapy intervention. ERG responses remained intact at the 3-year follow-up of two of the animals injected at 4 months of age in the Acland et al. 2001 studies [11]. More recently, Le Meur et al. investigated the specificity of the rAAV4 vector containing a human RPE65 promoter for the RPE; at 1-year post-injection, ERG responses were stable in two dogs injected subretinally at 8 months [12]. The limitations of measuring treatment efficacy in vectortreated RPE65-mutant dogs by commonly used behavioral assessments were overcome by recording of visual cortical potentials in subretinally vector-treated eyes. Additionally, cortical activation within the lateral gyrus, where the canine visual cortex resides, was evaluated by blood oxygen leveldependent functional MRI both before and after vector administration in five RPE65-mutant dogs compared with controls. Weak responses in the lateral gyrus were elicited before treatment, with much more activation observed in the lateral gyrus following vector treatment [13]. Non-human primate gene therapy studies The gene vector rAAV was also tested in monkeys, which have very similar ocular anatomy to humans. Researchers investigated the safety, efficiency and cellular specificity of subretinal injection of rAAV carrying encoding green fluorescent protein (EGFP) in four monkeys; stable and efficient EGFP transgene expression was localized to retinal cells and lasted for > 1 year, with no long-term toxicity [14]. A study of macaques and dogs found successful transgene expression, with no inflammatory response, at 2 months in seven macaques that received vitrectomies prior to subretinal injection of rAAV-GFP. Transgene expression was maintained in one macaque at 18 months with no abnormal retinal effects. Transgene expression did not occur in two other macaques that did not receive vitrectomies before subretinal injection [15]. Researchers also evaluated the safety/toxicity and upper dose limit of rAAV-RPE65 gene therapy via subretinal or intravitreal injection in monkeys at 1 week (6 monkeys) and 3 months (11 monkeys) [16]. At 3 months, generally no or minimal ocular or systemic toxicity was found; foveal anatomy was abnormal in some of the treated eyes; and no vector sequences were observed outside the retina. The no observed adverse effect level was reported to be 4.5 1012 vg. Histopathological results differed from some of the RPE65 dog studies; this primate study indicated that relatively high doses of rAAV-RPE65 via subretinal injection were not detrimental to retinal integrity. The researchers suggested that further 4.1.3

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RPE65

development of the gene delivery procedure to the central retina may be warranted to minimize risk of foveal injury from retinal detachment and thus maximize the beneficial effects of treatment [16]. LCA-RPE65 gene therapy clinical trials Original three LCA-RPE65 gene therapy clinical trials

intraoperative complication, with the following exceptions. A macular hole occurred in one patient in the CHOP trial [22]. The UPenn/UFL trial reported thinning of the fovea, the small central retinal region important for good detail and color vision, in one patient [17].

4.2

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4.2.1

The first three clinical trials investigating gene therapy treatment for LCA due to RPE65 gene mutations were initiated in 2007. The number of subjects ranged from 9 to 12 per trial with ages ranging from 5 to 44. In each trial, patients were divided into three dose cohorts (low, medium and high doses). The primary outcome measures for these trials were ocular and systemic safety, tolerability and intraocular inflammation. The secondary outcome measure was efficacy as visual function, indicated by measurements of retinal sensitivity, pupillary response, visual field, visual acuity, nystagmus, mobility, and/or response to light stimulation (as indicated by ERG measurements). All three trials used a single subretinal injection of a rAAV2 vector to deliver a human RPE65 cDNA gene to target RPE cells. Differences among the three trials included surgical protocols, DNA promoter types, patient characteristics, some of the vision testing methods used, and vector preparation and volumes [3,5]. For the low-dose cohorts in the three trials, vector doses were 1.5 1010, 5.96 1010 or 1.0 1011 particles/treated eye; vector volume delivered was 0.15 ml in two of the trials and 1 ml in the third trial [17]. For one study, the medium cohort vector dose was 4.8 1010 and the high cohort dose was 1.5 1011 [18]. These clinical trials were conducted by University College and Moorfields Eye Hospital (Moorfields) in London (www.clinicaltrials.gov, NCT00643747), Children’s Hospital of Philadelphia (CHOP) and Second University of Napoli (NCT00516477), and the University of Pennsylvania and University of Florida (UPenn/UFL) (NCT00481546) (Figure 3). Short-term results (up to 6 months) were initially reported on a total of 9 patients (3 patients per trial) in the low-dose cohorts [17,19,20], with longer-term (1 -- 1.5 year) results reported later on the same patients and/or additional patients; results for 18 patients have been reported to date [5,21,22]. Generally, statistically significant results or those reported as ‘substantial improvement’ are reported here (except where indicated) for the treated eye compared to one of the following: the control eye (same patient) at the same time period, the treated eye at baseline/pretreatment, untreated retinal regions in the treated eye, or matched control subjects. Safety results Gene therapy for patients with LCA due to RPE65 mutations appears to be safe via subretinal injection (Figure 4) at the tested doses, with no serious adverse events, no systemic toxicity, and no or minimal immune response [5,19,21,22]. Injection was achieved for the most part without 4.2.1.1

434

Efficacy results Results available thus far indicate improved visual function for many but not all of 18 patients included in the initial three LCA-RPE65 gene therapy clinical trials for several end points assessed (Table 1). Improvement often began soon after treatment (e.g., several days or weeks) and remained at 1 or 1.5 years after treatment. Initial results were reported first for the low-dose cohorts in each trial, which included patients aged 17 -- 26. Some researchers correlated visual function improvements with age, with younger patients achieving more improvement than older patients [18]. Other researchers stated that studies do not indicate that there is greater improvement in younger patients [5]; for example, decreased visual acuity in LCA-RPE65-deficient patients was not correlated with age until after the third decade of life, and outer retinal thickness did not differ by age in such patients aged 6 -- 17 [23,24]. Additional LCA-RPE65 clinical trials with more subjects and longer patient follow-up could provide further information regarding reproducibility and persistence of results, the role of rods and/or cones in treatment efficacy, the effect of age, the reason why some patients have not responded to treatment and the optimal dose for LCA-RPE65 gene therapy treatment. Efficacy results are discussed in more detail below. 4.2.1.2

Improved retinal sensitivity All three original clinical trials reported improved retinal sensitivity (increased sensitivity to light, localized to the treated retinal regions) in many patients in both the short- and/or longer-term. Improvement in dim light vision was reported by all 12 subjects in the CHOP trial, beginning in some patients as early as 2 weeks after surgery and remaining at the 1.5-year follow-up [17,18,22]. In the UPenn/UFL study, all three patients in the low-dose cohort reported increased vision in dim light conditions in the study eye; quantitative testing confirmed this improvement in visual sensitivity at 1, 2 and 3 months, which persisted at 12 months [17,21,25]. The Moorfields study reported measurable improvement in retinal sensitivity in daylight and dark-adapted retinal sensitivity in one of three patients in the low-dose cohort [19,26]. 4.2.1.2.1

Improved pupillary response Of 11 patients tested for pupillary response of the retina to light in the CHOP trial, all had improved, measurable responses in the treated eye. One 8-year-old had almost the same level of light sensitivity as that of age-matched normalsighted individuals [18]. Of the initial three patients treated in this same study, improved pupillary response persisted at the 1.5-year follow-up [22]. The UPenn/UFL study found 4.2.1.2.2

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Original 3 LCA clinical trials (initiated 2007) Phase I clinical trial of gene vector to patients with retinal disease due to RPE65 mutations (LCA) (NCT00481546) UPenn/UFL 9 subjects (3 cohorts), ages 8+ Primary outcome: safety Secondary outcome: visual field

Phase I safety study in subjects with LCA (LCARPE) (NCT00516477) Children’s Hospital of PA/2nd UNapoli 12 subjects (3 cohorts), ages 8+ Primary outcome: safety, tolerability Secondary outcome: visual field

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New clinical trials (initiated 2008 – 2010) Phase I/II safety and efficacy study of AAVRPE65 vector to treat LCA (NCT00749957) Applied Genetic Technologies, Inc. 12 subjects (4 arms), ages 8 – 17 Primary outcome: adverse events Secondary outcome: visual field, BCVA

Phase I clinical trial of gene therapy for LCA caused by RPE65 mutations (NCT00821340) Hadassah Medical Org. 10 subjects (3 cohorts) ages 8+ Primary outcome: safety Secondary outcome: visual field

Phase III safety and efficacy study in subjects with LCA (NCT00999609) Children’s Hospital of PA/UIowa,12 subjects, ages 3+ Primary outcome: PLR Secondary outcome: more visual/retinal function tests and ocular motility

Phase I/II safety study of RPE65 gene therapy to treat LCA (NCT00643747) University College, Moorfields Eye Hospital, London 12 subjects (3 doses), ages 5 – 30 Primary outcome: intraocular inflammation Secondary outcome: visual field

Phase I safety/proof of concept study to evaluate the effects of oral QLT091001 in Subjects with LCA due to RPE65 or LRAT mutations (NCT01014052), QLT Inc., 8 subjects, ages 5 – 75 Primary outcome: BCVA, visual acuity, color vision, visual field, ERG, FST Secondary outcome: safety

Phase Ib safety/proof of concept study to evaluate the effects of oral QLT091001 in subjects with Leber Congenital Amaurosis (LCA) or Retinitis Pigmentosa (RP) due to RPE 65 or LRAT mutations, ages 5 – 17 Primary outcome: BCVA, color vision, visual field, ERG Secondary outcome: safety

Genetic study of patients suffering from LCA or an early severe retinal dystrophy (NCT00422721) Nantes University Hospital, France 360 subjects Interventions include genotyping and various visual tests

Figure 3. Overview of LCA-RPE65 clinical trials. BCVA: Best corrected visual acuity; ERG: Electroretinography; FST: Full-field sensitivity testing; LCA: Leber congenital amaurosis; PLR: Pupillary light reflex; RPE65: Retinal pigment epithelial-specific 65 kDa protein.

that two of three patients tested had an improved pupillary light reflex at 1 month after treatment [5,25].

no statistically significant changes in visual acuity in a total of six patients followed for up to 1 year [5,17,19].

4.2.1.2.3

Improved visual field The CHOP study found measurable improvement in visual field (area that can be seen with the eye, including peripheral vision) of all 12 study patients: at 1 month for 10 of the patients; at 4.75 months for the 11th patient and at 2.75 months for the 12th patient. These improvements remained at the 1.5-year follow-up [18,22]. The UPenn/UFL study found increased visual fields at 1 month for all three patients in the low-dose cohort, which remained at 1 year. The Moorfields study found no clinically significant change in visual fields for the three patients in the low-dose cohort [19].

4.2.1.2.5

Improved visual acuity in one study The CHOP clinical trial reported significant measurable improvement in visual acuity (sharpness of central vision, including fine-detail vision) in all three patients in the lowdose cohort; in three of six patients in the middle dose cohort and in one of three patients in the high-dose cohort. This improvement continued or remained stable up to the 1.5-year follow-up [18,20,22]. The two other clinical trials found

4.2.1.2.6

4.2.1.2.4

Development of pseudo-fovea In the UPenn/UFL clinical trial, one patient reported better vision at the 1-year follow-up, even though testing showed no measurable changes in visual acuity or visual sensitivity. Researchers quantified the fixation of this patient’s gaze to dim light targets as well as fixation dwell time. Results suggested that this patient’s slow visual gain was accompanied in the treated eye by a shift in foveal fixation into the treated area of the retina, which created a pseudo-fovea. Development of a pseudo-fovea may reflect an experience-dependent plasticity of the visual system [27]. Reduced nystagmus Researchers found a measurable reduction in nystagmus (rapid involuntary eye movements) in the initial three patients tested after receiving LCA-RPE65 gene therapy treatment in the CHOP study [18], and this improvement remained at the 1.5-year follow-up [22]. No patients in the UPenn/UFL or Moorfields studies showed a reduction in nystagmus/ fixational eye movements [25].

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Figure 4. Schematic of injection of AAV2-RPE65 vector into the subretinal space. Source: [25]. AAV2-RPE65: Adeno-associated vector 2-RPE-specific 65 kDa protein; RPE: Retinal pigment epithelium.

larger volumes of the rAAV-RPE65 vector, evaluating higher doses and including younger patients than some of the initial 2007 trials. With a total of 12 subjects, the primary, safety outcome measure of this Phase I--II trial is the number and proportion of participants experiencing ocular or non-ocular adverse events. Secondary, efficacy outcome measures include changes in visual fields and best corrected visual acuity, which measures how well a person can see after corrective treatment. The subjects are assigned to four cohorts by age and dose. A second recent LCA-RPE65 trial, initiated in 2010, is being conducted by Hadassah Medical Organization in Israel (NCT00821340). The primary outcome measure is ocular and systemic safety, and the secondary outcome measure is visual function. The study is enrolling a total of 10 subjects divided into three cohorts by age and dose. A third recent LCA-RPE65 clinical trial, scheduled to begin in late 2010 and conducted at Children’s Hospital of Philadelphia and the University of Iowa (CHOP 2) (NCT00999609), will include 12 subjects aged 3 and older. The primary outcome measure of this Phase III trial will be efficacy assessed by pupillary light reflex. Secondary efficacy outcome measures will include additional visual/retinal function tests and ocular motility measurements and ophthalmic and systemic safety measures. This study expands on the previous CHOP clinical trial discussed above by including 12 additional subjects, some at an earlier age. LCA genetic testing clinical trial and study: potential impact on future LCA treatment

4.2.3 4.2.1.2.7

Improved mobility

Four patients in the CHOP study were better able to navigate an obstacle course after LCA-RPE65 gene therapy treatment using their treated eye, compared to their untreated eye and compared to their abilities at baseline prior to treatment [18]. One patient in the Moorfields study showed improved mobility after treatment at the 5- and 6-month follow-up visits [19]. ERG response generally not improved Except for improvement in one patient for whom multifocal ERG results were reported [5], full-field ERG testing did not detect improved ERG responses in patients in the three clinical trials [5,18,19]. Full-field ERG test responses from the entire retina and focal improvements may not have been able to be determined from these LCA patients, and the treated area may have been too small to see a response. 4.2.1.2.8

Additional LCA-RPE65 gene therapy clinical trials

Only patients with a mutation in the RPE65 gene may be responsive to LCA-RPE65 gene therapy treatment investigated in the clinical trials discussed thus far. Patients with other gene mutations that also result in LCA will not be responsive to LCA-RPE65 gene therapy. A clinical trial in France (NCT00422721) is recruiting and testing ~ 360 LCA patients to identify their phenotypes and genotypes so that future clinical trials in that country could target the correct population of LCA patients (Figure 3). Similarly, Project 3000 in the US, administered by the University of Iowa, is seeking to genotype all ~ 3000 LCA patients in the country, which will assist in targeting appropriate populations for future LCA clinical trials in the US (https://www.carverlab.org/project3000).

A pharmacological approach: LCA retinoid treatment

5.

4.2.2

Three more recent LCA-RPE65 gene therapy clinical trials were initiated between 2008 and 2010 that seek to replicate and build on the results of the initial three clinical trials discussed above, also using subretinal injection of an AAV vector as the route of administration (Figure 3). One of these clinical trials, initiated in 2008 and conducted at the University of Massachusetts Medical School and Oregon Health & Science University (NCT00749957), is delivering 436

Another current clinical trial, initiated in 2008, involves a pharmacological rather than a gene-therapy approach to LCA treatment, using a synthetic retinoid administered orally to target genetic defects that inhibit 11-cis-retinal regeneration [28]. Previous related animal studies included several murine studies, some of which indicated that a more chemically stable compound, 9-cis-retinyl acetate, a synthetic retinoid, improved vision function in LRAT or RPE65 knockout mice [29-33]. Cis-retinoid treatment in RPE65-deficient dogs has indicated

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Table 1. Summary of improved vision function for efficacy end points in three original LCA-RPE65 clinical trials. Vision function test

Retinal sensitivity

Pupillary response

Visual field

Visual acuity

Nystagmus

Mobility

ERG

Pseudo-fovea

Improved vision function (number of tested patients who improved) Results Clinical trial CHOP clinical trial

12 of 12 (self-reported)

11 of 11

12 of 12

7 of 12

3 of 3

4 of 12

1 of 1

0

UPenn/UFL clinical trial [5,17,21,27]

3 of 3

2 of 3

3 of 3

0 of 3

0 of 3

NA*

NA*

1 of 1

Moorfields clinical trial [5,19,26]

1 of 3

NA*

0 of 3

0 of 3

0 of 1

1 of 3

0 of 3

0

Total number of patients who improved

16 of 18

13 of 14

15 of 18

7 of 18

3 of 7

5 of 15

1 of 4

1 of 1

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[5,18,20,22]

*Not applicable: function not measured. CHOP: Children’s Hospital of Philadelphia; ERG: Electroretinography; LCA: Leber congenital amaurosis; Moorfields: University College and Moorfields Eye Hospital; RPE65: Retinal pigment epithelial-specific 65 kDa protein; UPenn/UFL: University of Pennsylvania/University of Florida.

that intravitreal injection with a proprietary synthetic retinoid improved ERG responses and visual behavior with no adverse effects observed [34] and resulted in better and faster navigation of obstacle courses in dim light [35]. A Phase Ia clinical trial with 20 healthy human adult subjects aged 18 -- 60 receiving an oral dose of a proprietary synthetic retinoid was completed in 2009 (NCT00765427). Results indicated that the retinoid was safe and well tolerated by the subjects [36]. Patients in the highest dose cohort experienced mild, transient adverse events including facial flushing and headache [37]. A Phase Ib trial with eight LCA patients aged 5 -- 75, initiated at Montreal Children’s Hospital in late 2009, involves the same retinoid and dosing regimen. Thus far, the trial drug has been administered to LCA patients with the LRAT mutation only; LCA patients with the RPE65 mutation will be included in the future (NCT01014052). Interim results for the Phase Ib study for the first three subjects enrolled indicated improvement in one or more measured visual function parameters and subjective patient reports of improved visual performance. The drug was generally well-tolerated, with mild to moderate adverse events including transient headache, photophobia and increased triglyceride levels [36,38,39]. 6.

Conclusion

The confluence of several factors has led to exciting proof-ofconcept retinal gene therapy trials. The self-containment of the eye makes it an excellent organ for scientific investigation, and significant advances in molecular biology have led to new studies of the retina. In addition, the availability of mouse and dog models of LCA-RPE65 mutations, as well as the development of surgical retina instruments and techniques, has greatly contributed to advances in the field. The positive results of pioneering gene therapy trials for LCA-RPE65 are forging the modern era of molecular retinal therapeutics.

These trials have made possible the discussion and planning of additional, potentially meaningful trials for otherwise blinding retinal disorders. 7.

Expert opinion

It is clear that LCA-RPE65 gene therapy can help restore, to a degree, the visual function of both rods and cones. In itself, this is a wonderful accomplishment and serves as a paradigm of translational research that is a testament to the work of many basic scientists and clinicians. As is often the case with innovative scientific research, many important questions remain unanswered that now deserve attention and further research. For example, how can retinal gene therapy be further enhanced or improved? The current published results reflect patients for whom subretinal injections resulted in partial retinal detachments and treatment. In the future, it may be desirable to detach the whole retina, thereby potentially treating the entire retina and macula. Similarly, evidence exists that identifies oxidative stress, inflammatory cytokines (presumably secreted by the RPE) and inflammatory cells (including circulating macrophages and activated microglial cells) in the subretinal space of animal models of RP as well as in humans. Gene therapy could be significantly advanced by first reducing these pathological factors by treating patients with drugs that limit inflammation (e.g., steroids or other small molecules like them) prior to subretinal surgery or intravitreal injection. Such combinatorial approaches can now be considered in the design of new, larger clinical trials.

Declaration of interest S Kaushal is a co-Principal Investigator for a current LCA gene therapy trial for RPE65 sponsored by Applied Genetic Technologies. He has also been supported by the NEI (National Eye Institute) for a previous LCA study trial.

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Linda Stein MS, Kamolika Roy BS, Lei Lei BS & Shalesh Kaushal† MD PhD † Author for correspondence University of Massachusetts Medical School, UMass Memorial Eye Center, Department of Ophthalmology, 281 Lincoln St., Worcester, MA 01605, USA Tel: +508 334 9072; Fax: +508 334 4655; E-mail: [email protected]

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