Elsevier

Neurobiology of Aging

Volume 34, Issue 1, January 2013, Pages 35-61
Neurobiology of Aging

Review
Advancing neurotrophic factors as treatments for age-related neurodegenerative diseases: developing and demonstrating “clinical proof-of-concept” for AAV-neurturin (CERE-120) in Parkinson's disease

https://doi.org/10.1016/j.neurobiolaging.2012.07.018Get rights and content

Abstract

Neurotrophic factors have long shown promise as potential therapies for age-related neurodegenerative diseases. However, 20 years of largely disappointing clinical results have underscored the difficulties involved with safely and effectively delivering these proteins to targeted sites within the central nervous system. Recent progress establishes that gene transfer can now likely overcome the delivery issues plaguing the translation of neurotrophic factors. This may be best exemplified by adeno-associated virus serotype-2-neurturin (CERE-120), a viral-vector construct designed to deliver the neurotrophic factor, neurturin to degenerating nigrostriatal neurons in Parkinson's disease. Eighty Parkinson's subjects have been dosed with CERE-120 (some 7+ years ago), with long-term, targeted neurturin expression confirmed and no serious safety issues identified. A double-blind, controlled Phase 2a trial established clinical “proof-of-concept” via 19 of the 24 prescribed efficacy end points favoring CERE-120 at the 12-month protocol-prescribed time point and all but one favoring CERE-120 at the 18-month secondary time point (p = 0.007 and 0.001, respectively). Moreover, clinically meaningful benefit was seen with CERE-120 on several specific protocol-prescribed, pairwise, blinded, motor, and quality-of-life end points at 12 months, and an even greater number of end points at 18 months. Because the trial failed to meet the primary end point (Unified Parkinson's Disease Rating Scale motor-off, measured at 12 months), a revised multicenter Phase 1/2b protocol was designed to enhance the neurotrophic effects of CERE-120, using insight gained from the Phase 2a trial. This review summarizes the development of CERE-120 from its inception through establishing “clinical proof-of-concept” and beyond. The translational obstacles and issues confronted, and the strategies applied, are reviewed. This information should be informative to investigators interested in translational research and development for age-related and other neurodegenerative diseases.

Introduction

Neurotrophic factors offer one of the most compelling opportunities to significantly improve the treatment of serious age-related, neurological diseases such as Alzheimer's and Parkinson's, as well as Huntington's and amyotrophic lateral sclerosis. The therapeutic potential of neurotrophic factors to alleviate the symptoms and slow or even halt disease progression in neurodegenerative diseases, including Parkinson's disease (PD), is widely acknowledged (Apfel et al., 2000, Eriksdotter Jönhagen et al., 1998, Mufson et al., 1999, Seiger et al., 1993) and has been independently supported by research conducted by numerous laboratories around the world. A major translational advantage of neurotrophic factors is that they offer the opportunity to treat both the symptoms of a disease (thus improving clinical status) as well as its pathogenesis (thus delaying disease progression) without any prerequisite, deep insight into the etiology or specific pathogenic variables driving the disease process. An editorial written more than two decades ago, entitled “Neurotrophic factors: can the degenerating brain be induced to heal itself,” helps illustrate the enthusiasm many of us have felt for a long time, excerpted here: “When one considers the history of neurology, the idea that one might be able to treat patients so that their brain cells might either withstand deadly perturbations or regenerate to a healthier, more functional state is truly revolutionary. Never before in the history of medical science could we imagine the means to induce damaged parts of the brain to heal” (Bartus, 1989a). While it was clearly too early to know whether neurotrophic factors might eventually live up to those early expectations, it would have been even more difficult for anyone to have known that after more than two decades of animal research and many clinical trials attempting to show efficacy in humans, their ability to treat human neurodegenerative diseases would continue to remain unfulfilled this long.

Neurotrophic factors are endogenous proteins that have consistently demonstrated that under conditions of neurodegeneration they are able to activate neuronal repair genes when supra-physiological (i.e., biopharmaceutical) levels are achieved. Induction of these repair genes routinely produces morphological and functional restoration of the degenerating neurons, significantly slowing further neurodegeneration and even protecting against cell death (Hefti et al., 1989). Thus, decades of research using numerous animal models argues that neurotrophic factors provide the opportunity to substantially improve neuronal vitality and function in human neurodegenerative diseases (thus potentially improving symptoms and extending the value of current pharmacotherapies), as well as to protect against further neurodegeneration (possibly slowing, halting, or even reversing disease progression).

An extremely important point for translational purposes is that neurotrophic factors appear to provide functional and morphological benefit to their responsive neurons, no matter how the neurons are damaged or impaired. Investigators have consistently shown benefit of neurotrophic factors against cutting and/or crushing axons, exposure to neurotoxins, free radical donors, inflammatory agents and other cytotoxic agents, genetic mutations, protein processing defects, and the effects of age. Thus, neurotrophic factors seem to represent a final common therapeutic pathway to achieve neuronal restoration and protection, likely providing potential benefit independent of which of many possible pathogenic cascade(s) are truly responsible for the disease and thus free of theoretical insight, assumptions, or uncertainties surrounding those issues. The potential therapeutic effects of neurotrophic factors seem to be “pathogenic neutral,” which offers a major translational advantage, given the apparent complexity of most chronic neurodegenerative diseases as well as the uncertainty and controversy regarding which pathogenic variables are most important. Therefore, if one is able to identify a neuronal population whose degeneration and/or loss of function has been linked to the symptoms or pathogenesis of a disease, then the appropriate neurotrophic factor can likely provide restorative effects independent of a clear understanding of the pathogenesis involved. This rather unique characteristic of neurotrophic factors provides a significant, perhaps unprecedented opportunity to reduce risk in the development of “first in class” therapeutics for serious, unmet needs. This approach to treat neurodegenerative diseases leverages decades of cross-disciplinary research that collectively establishes “nonclinical proof-of-concept” for the potential benefit of neurotrophic factors when degeneration of a specific neuronal population is known to represent a key feature of a disease.

This scenario makes neurotrophic factors a compelling target for translational research and development (R&D). Moreover, the complex but powerful biology of neurotrophic factors suggests that if a significant reduction in clinical symptoms can be achieved, then a slowing of disease progression should also occur, simply because the same repair genes activated by the neurotrophic factor to improve symptoms should also produce healthier neurons that are better able to withstand the pathogenic variables responsible for disease progression. As many have noted in the past, this possibility of reversing and slowing disease progression represents the “Holy Grail” for neurological diseases and neurotrophic factors arguably provide the best opportunity to accomplish this in the foreseeable future.

The major reason neurotrophic factors have not lived up to their early promise centers around the long-standing translational obstacles that impeded safe and effective delivery. While an editorial written decades ago titled “Delivery to the brain: the problem lurking behind the problem” forewarned that a major translational stumbling block for neurotrophic factors might involve successful delivery to the brain (Bartus, 1989b), that problem has proven to be far more difficult than we had reason to believe at the time. Similarly, while no one can be certain that solving delivery issues will necessarily produce the anticipated clinical benefit, it has become increasingly accepted that unless the delivery problems are solved, reliable and meaningful clinical benefit will likely not be achieved.

Numerous clinical trials, testing many different neurotrophic factors in several different neurodegenerative diseases, have been conducted over the past 20 years (Apfel, 2002, Apfel et al., 1998, Apfel et al., 2000, Eriksdotter Jönhagen et al., 1998, Gill et al., 2003, Lang et al., 2006, Marks et al., 2008, Miller et al., 1996, Nutt et al., 2003, Penn et al., 1997, Slevin et al., 2005, Tuszynski et al., 2005, Wellmer et al., 2001), with mixed results. Three relatively recent, open label studies, in particular, fueled further enthusiasm for the possible therapeutic benefits of neurotrophic factors. The first involved infusion of recombinant glial cell line-derived neurotrophic factor (GDNF) in 5 PD subjects (Gill et al., 2003); the next involved ex vivo gene transfer of nerve growth factor (NGF) in 8 Alzheimer's disease subjects, 6 of whom were evaluable (Tuszynski et al., 2005); the third involved in vivo gene transfer of NRTN (neurturin) in 12 PD subjects, distinguished also by using clear, predefined primary and secondary efficacy end points (Marks et al., 2008). While all 3 studies reported preliminary evidence of clinical benefit, the uncontrolled, open-label nature of the studies rendered all these results preliminary and merely suggestive (e.g., see Alterman et al., 2011). In fact, later studies failed to repeat the motor improvements reported for GDNF (Lang et al., 2006), the cognitive improvements reported for NGF (Arvanitakis et al., 2006), or the very early emergence (i.e., within initial postdosing months) of motor improvements seen with NRTN (Marks et al., 2010). More importantly, no controlled trial has yet established that neurotrophic factors can dramatically improve the clinical symptoms in any neurodegenerative disease, let alone delay disease progression.

The majority of the recent effort with neurotrophic factors has been focused on PD, primarily testing GDNF. Nonclinical studies supported the possible use of GDNF, infusing the protein into the brain (Gash et al., 1995, Gash et al., 1996, Kirik et al., 2000a, Maswood et al., 2002), as well as delivering it via gene therapy (Choi-Lundberg et al., 1998, Kirik et al., 2000b, Kordower et al., 2000). While several subsequent clinical studies tested GDNF in moderately advanced PD patients, all of them have infused the protein using a chronically indwelling pump into an intracerebral cannula, thus providing a single “point source” of protein in the targeted areas of the large human brain. Collectively, the results of these efforts have been mixed at best (Gill et al., 2003, Lang et al., 2006, Slevin et al., 2005), with a single, controlled study showing no evidence for any real benefit (Lang et al., 2006). Many investigators have agreed that the point source of delivery employed produced poor distribution of the protein throughout the putamen, contributing to the negative results (Morrison et al., 2007, Salvatore et al., 2006, Sherer et al., 2006).

The cumulative insight gained in the past two decades of clinical trials attempting to translate the therapeutic potential of neurotrophic factors provided crucial information about the delivery requirements that must be met for neurotrophic factors to succeed as human therapeutics. For example, to treat chronic neurodegenerative diseases, adequate levels of neurotrophic factors must be maintained for very long periods of time (i.e., years), for when the proteins return to basal levels, their benefit is typically lost (Fischer et al., 1987, Hefti et al., 1989, Snider and Johnson, 1989, Sofroniew et al., 2001). Similarly, it is important that an appreciable proportion of the degenerating cell population be exposed to the neurotrophic factor in order to produce sufficient restoration of neuronal function and thus achieve measurable clinical improvement (though the exact proportion required for therapeutic benefit is currently not clear, and may differ between diseases). Because serious side effects have been observed when delivery of neurotrophic factors has inadvertently resulted in exposure to nontargeted brain sites (e.g., periventricular tissue) the importance of accurately predicting, controlling, and restricting protein delivery specifically to the intended target has become apparent (Day-Lollini et al., 1997, Eriksdotter Jönhagen et al., 1998, Kordower et al., 1999, Nauta et al., 1999, Nutt et al., 2003, Penn et al., 1997). Further complicating the translation of neurotrophic factors is the fact that because they are proteins, chronic delivery can be notoriously difficult because of aggregation, misfolding, and development of neutralizing antibodies. Moreover, as proteins, they cannot be taken orally, do not cross the blood-brain barrier (BBB) naturally and cannot typically be administered systemically, even if linked to a BBB carrier, because of side effects often induced by systemic exposure to organs and tissues (McMahon, 1996, Pezet and McMahon, 2006). These issues, individually and collectively, render safe and effective delivery of neurotrophic factors extremely challenging. It is for this reason that a consensus opinion has emerged among investigators that the successful translation of neurotrophic factors to the human clinic will first require that these crucial delivery issues be solved (Bartus, 1989a, Bartus et al., 2007, Kordower et al., 1999, Lang et al., 2006, Nutt et al., 2003, Salvatore et al., 2006, Sherer et al., 2006).

Unfortunately, the issues described preclude employing most traditional pharmaceutical formulations and delivery approaches as viable options for administering neurotrophic factors to patients' brains. A number of more innovative methods have therefore been devised in an attempt to effectively deliver these proteins to the central nervous system (CNS), but all have suffered serious limitations. For example, various efforts to transport neurotrophic factors across the BBB following systemic administration have been tested. One method that showed promise exploited endogenous transport receptor-mediated systems located on the abluminal surface of cerebral capillaries (e.g., transferrin transport receptors). While early nonclinical conceptual success was achieved (Bäckman et al., 1996, Bäckman et al., 1997, Bartus, 1999, Charles et al., 1996), the approach ultimately proved impractical because of serious peripheral side effects induced via exposing nontargeted tissue outside the brain to the neurotrophic factor following intravenous injections (McMahon, 1996, Pezet and McMahon, 2006), coupled with only modest benefit, compared with infusion of proteins directly into the brain. Infusions of neurotrophic factor proteins directly into the ventricles of the brain, which required a more invasive, neurosurgical approach, showed early promise in animal studies of neurodegeneration. However, significant side effects in humans occurred in the periventricular tissue exposed to high concentrations of the neurotrophic factor (Eriksdotter Jönhagen et al., 1998, Kordower et al., 1999, Nauta et al., 1999, Nutt et al., 2003, Penn et al., 1997). While subsequent infusions of the proteins directly into the degenerating parenchyma using chronically indwelling pumps and cannula reduced some, but not all safety issues (Hovland et al., 2007, Lang et al., 2006), poor diffusion of the protein from a single point source severely limited exposure of the protein to the larger area of degenerating brain tissue, thus likely limiting clinical benefit (Lang et al., 2006, Salvatore et al., 2006, Sherer et al., 2006). Similarly, complications involving indwelling hardware posed serious safety risks (e.g., the formation of neutralizing antibodies to GDNF and degeneration of distant cerebellar neurons due to protein leaking uncontrollably along paths of least resistance). In other words, while the scientific foundation for neurotrophic factors is considered well-established, attempts to translate the therapeutic potential to the clinical arena have been largely disappointing because the technology required to deliver these complex proteins in a safe, controlled, and sustained fashion to specific, targeted areas of the brain has been grossly inadequate.

Section snippets

CERE-120 (AAV2-NRTN), PD, and the use of gene transfer to solve the delivery obstacles posed by neurotrophic factors

Parkinson's disease is a chronic, debilitating disease, whose major symptoms involve loss of motor ability, including bradykinesia, tremors, and problems with gait and balance. It is widely recognized that these major symptoms result from the progressive loss of function and eventual death of the nigrostriatal dopamine neurons, linked at least in part to the accumulation of misfolded α-synuclein aggregates (McNaught and Olanow, 2006). While available pharmaceutical agents for PD are generally

Creating a paradigm for selecting initial CERE-120 doses to test in PD subjects

As reviewed in prior sections of this article, we had successfully addressed all the conventional translational issues, as well as many unique and sometimes unexpected issues required to move a novel treatment into human testing. However, an important issue that still remained to be tackled before we could actually proceed with testing in humans involved selection of the CERE-120 doses to be tested. This question presented a particularly onerous challenge because CERE-120 is a far different

Initial open-label, Phase 1 trial

Following the successful execution of the nonclinical program (previous sections), an initial Phase 1 safety trial in moderately advanced PD patients was launched. Two dose levels of CERE-120 were tested (1.3 × 1011 vg, or copies of the NRTN gene and 5.4 × 1011 vg, total) for both hemispheres. Six subjects were enrolled into each cohort with doses delivered bilaterally into the terminal fields of the degenerating nigrostriatal neurons in the putamen. Formal safety (and preliminary efficacy)

Back to the laboratory as a prerequisite to enhance the dosing strategy by targeting the SN with CERE-120

Before we would seriously consider implementing the novel dosing approach of targeting both the SN and putamen, we felt it prudent to carefully evaluate its potential risks, relative to the intended benefits. The SN is a relatively small structure that lies deep within the midbrain and therefore it presents a potentially more challenging target for stereotactic surgery. Additionally, because it lies in close proximity to other anatomical structures that mediate a diverse number of functions,

Launching a revised Phase 1/2b protocol to optimize the targeting and neurotrophic effects of CERE-120

Using the collective information and insight gained from all our research with CERE-120, but especially the results of the controlled Phase 2a trial and the autopsy cases, we designed and implemented a new “target and dose-optimized” Phase 1/2b protocol. A number of significant protocol improvements were incorporated in an attempt to increase the rate and magnitude of the neurotrophic response to CERE-120, while also improving our ability to measure the clinical benefit from that response in a

Synopsis and conclusions

Following a long and rich history of research into the powerful biological effects of neurotrophic factors and several unsuccessful attempts to translate their therapeutic potential to treat age-related neurodegenerative diseases, a significant translational breakthrough appears to have occurred. The emergence and further development of gene transfer has provided the technological means to solve the multiple delivery issues inherent with the need to selectively target these complex proteins to

Acknowledgements

The body of work reviewed in this article constitutes 10 years of strategic planning and research activity involving the assistance and input of scores of individuals who are not authors and we therefore gratefully acknowledge their invaluable contributions to the development of CERE-120. First, the input, assistance and collaborative research of Jeffrey Kordower was particularly valuable, as was the guidance and input from Warren Olanow, Eugene Johnson, and the rest of the Ceregene Scientific

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