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Keywords:

  • biomarker;
  • clinical trial;
  • disease-modifying therapy;
  • neurodegenerative disease;
  • outcome measure

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Accumulation of abnormal proteins
  5. Development of therapies targeting abnormal protein accumulation
  6. Key issues in therapy development for neurodegenerative diseases
  7. Summary
  8. Author contributions
  9. Acknowledgments
  10. Competing interests
  11. References

Neurodegenerative diseases are a group of disorders that affect a certain population of neurons, and are characterized by progressive impairment of cognitive and/or motor function. Although most current therapies aim at symptomatic relief, recent studies using cellular and animal models have given insight into the molecular pathogenesis of neurodegenerative diseases and the development of disease-modifying therapies that slow neurodegeneration. However, few clinical trials have confirmed the efficacy of disease-modifying therapy for neurological deficits. For successful translational research on neurodegenerative disorders, several challenges in basic and clinical studies should be overcome. Elucidation of the molecular basis for neurodegeneration and creation of animal models that faithfully replicate human pathology are fundamental to basic studies, while the efficiency of clinical trials needs to be improved by establishing sensitive outcome measures, enrichment of trial designs by identifying the target individuals who are expected to show a high response, and innovative study designs. Initiation of therapy at the presymptomatic stage is another key to the successful development of disease-modifying therapy for neurodegenerative diseases. Although accumulation of abnormal proteins is thought to be the pivotal molecular trigger of neurodegeneration, it has also been revealed that disease progression is influenced by various factors, including the neuron–glia interaction and the propagation of causative proteins. The entire mechanism of neurodegeneration appears to be more complicated than hitherto expected, and thus therapies that target multiple molecular pathways also need to be developed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Accumulation of abnormal proteins
  5. Development of therapies targeting abnormal protein accumulation
  6. Key issues in therapy development for neurodegenerative diseases
  7. Summary
  8. Author contributions
  9. Acknowledgments
  10. Competing interests
  11. References

Against the background of population aging, the development of therapies for age-related neurological diseases is becoming an increasingly urgent objective. Although most currently available therapies for neurodegenerative diseases act by symptomatic relief, these agents have no or only subtle effects on disease progression. On the basis of recent advancements in molecular biology, disease-modifying therapies that target pathogenesis-related molecules have been developed for devastating neurodegenerative diseases.[1] Although these disease-modifying therapies are expected to halt or slow the fundamental pathological processes, most candidate agents identified in animal studies did not demonstrate disease-modifying effects in clinical trials.[2, 3] Here, we review examples of disease-modifying therapies for neurodegenerative diseases, and discuss challenges and innovation of the translational research on fatal neurological disorders.

Accumulation of abnormal proteins

  1. Top of page
  2. Abstract
  3. Introduction
  4. Accumulation of abnormal proteins
  5. Development of therapies targeting abnormal protein accumulation
  6. Key issues in therapy development for neurodegenerative diseases
  7. Summary
  8. Author contributions
  9. Acknowledgments
  10. Competing interests
  11. References

Intra- and extracellular accumulation of abnormal proteins is a common histopathological feature of neurodegenerative diseases (Fig. 1). For example, the main pathological hallmarks of Alzheimer's disease (AD) are extracellular amyloid plaques and intracellular neurofibrillary tangles. Amyloid plaques are mainly composed of Aβ peptide, which is generated through the proteolytic cleavage of the transmembrane amyloid precursor protein (APP). Aβ deposition is detected even in early cases of AD and in subjects with mild cognitive impairment (MCI).[4] It has also been reported that Aβ peptide itself is capable of exerting toxicity in neurons and inducing cognitive deficits in animals.[5] In addition, basic and clinical studies have demonstrated the toxicity of a particular form of Aβ peptide (Aβ42) that has a strong propensity for self-aggregation into fibrils, and that plays a role in the molecular pathogenesis of AD. This view is strongly corroborated by the identification of gene mutations in familial cases, accounting for about 60% of early-onset AD patients. All known causative genes in hereditary AD, APP, presenilins 1 and 2, encode proteins that are involved in the Aβ generation pathway.[6]

image

Figure 1. Accumulation of abnormal protein in neurodegenerative diseases. Accumulation of abnormal protein is the histopathological hallmark of neurodegenerative disorders. The main pathological hallmarks of Alzheimer's disease (AD) are extracellular amyloid plaques and intracellular neurofibrillary tangles. Ubiquitinated cytoplasmic inclusions are characteristic of amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD), while nuclear accumulation of a polyglutamine-expanded protein is the common pathological feature shared by various CAG triplet repeat-mediated disorders.

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Histopathology of amyotrophic lateral sclerosis (ALS) is characterized by intraneuronal protein aggregates in motor neurons. Among these, hyaline and skein-like inclusions, which are usually ubiquitinated, are detected in the motor neurons of most sporadic cases of ALS. The main constituent of these cytoplasmic inclusions is the 43-kDa TAR DNA-binding protein (TDP-43), a DNA- and RNA-binding protein that regulates gene transcription, exon splicing and exon inclusion.[7] Intriguingly, several point mutations in the glycine-rich domain of the gene encoding TDP-43 have also been identified as the disease-causing mutations of familial and sporadic ALS. The histopathological hallmark of Parkinson's disease (PD) is the presence of Lewy bodies; the main component of which is phosphorylated α-synuclein. The fact that mutations or multiplications of the gene encoding α-synuclein cause early-onset, autosomal-dominant PD strongly implicates this protein in the central pathogenesis of dopaminergic neuron loss.[8] Polyglutamine diseases are hereditary neurodegenerative disorders caused by an abnormal expansion of a trinucleotide CAG repeat, which encodes a polyglutamine tract.[9] To date, nine polyglutamine diseases have been identified: Huntington's disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubral–pallidoluysian atrophy (DRPLA), and six forms of spinocerebellar ataxia (SCA). Proteins with an expanded polyglutamine tract have an altered conformation and propensity to form toxic oligomers that trigger neurodegenerative processes.

Development of therapies targeting abnormal protein accumulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Accumulation of abnormal proteins
  5. Development of therapies targeting abnormal protein accumulation
  6. Key issues in therapy development for neurodegenerative diseases
  7. Summary
  8. Author contributions
  9. Acknowledgments
  10. Competing interests
  11. References

Disease-modifying therapy for AD

The histopathological evidence of abnormal protein deposition has prompted genetic and pharmacological interventions that inhibit the accumulation of causative molecules in various neurodegenerative diseases. One of the most-studied targets is amyloid plaques in AD, because the majority of disease-modifying therapies for AD have been developed on the basis of the amyloid cascade hypothesis. For instance, active immunization with Aβ vaccine reduced amyloid plaque burden and improved behavioral phenotypes in a transgenic mouse model carrying mutant APP.[10] Similarly, passive immunization using anti-Aβ antibodies inhibits the formation of plaques and ameliorates behavior deficits.[11] Therapies targeting β- and γ-secretases that produce the Aβ peptide via cleavage of APP have also been developed, and some of them suppress the production of toxic Aβ peptides in animal models of AD.[12] In addition to Aβ oligomers, tau, the major component of neurofibrillary tangles, is also a critical target of therapy development for AD. It is commonly held that, following Aβ deposition, tau is hyperphosphorylated and forms neurofibrillary tangles in vulnerable neurons. Suppression of tau phosphorylation by lithium, an inhibitor of glycogen synthase kinase 3β, mitigates neurodegeneration and improves cognitive function in an animal model of AD.[2] Other candidates that dampen neurodegeneration in AD include microtubule stabilizers, antioxidants, mitochondrial protectors, immune response modulators and autophagy inducers.[13]

Nevertheless, none of the agents thus far mentioned has demonstrated beneficial effects in clinical trials, even though they ameliorate histopathological and behavioral findings in AD model animals. Aβ vaccine AN1792, which contains a synthetic form of the Aβ42 peptide, was the first immunotherapy tested in clinical trials. Although anti-Aβ antibodies were produced in a subset of AD patients treated, the phase 2a study of the vaccine was discontinued due to serious adverse events, including meningoencephalitis and leukoencephalopathy.[14] Intriguingly, AN1792 reduces the amyloid plaque burden in the brains of AD patients compared with those of unvaccinated cases, suggesting that the active immunization ameliorates AD pathology in humans.[15] However, the effects of this therapy on cognitive function or survival were not clearly shown in the phase 2a trial or in a long-term follow-up of a phase 1 study.[14, 16] Passive immunization with anti-Aβ monoclonal antibodies, bapineuzumab and solanezumab, has also been tested in humans. Primary endpoints, both cognitive and functional, were not met in the phase 3 trials of either drug, although solanezumab showed small, but statistically significant effects on some cognitive functions and independent activity of daily living in patients with mild AD.[17]

Moreover, even with passive immunization, there can be vasogenic edema and microhemorrhage that depend, in part, on ApoE allele status, a genetic risk factor of AD.[18] Neither other Aβ aggregation inhibitors nor γ-secretase modulators showed benefits over placebo in clinical trials.[19]

Although various factors appear to underlie the discrepancies between the histopathological and functional outcomes in clinical trials of Aβ vaccine, several studies strongly indicate the possibility that inhibition of Aβ aggregation, the upstream event in the pathological process, is not sufficient for suppressing disease progression at a symptomatic stage of AD.[12] This view is supported by the observation that Aβ aggregation as detected with positron emission tomography using Pittsburgh Compound B and the decreased Aβ42 in cerebrospinal fluid antedated the onset of cognitive deficits in AD patients.[20] Hippocampal atrophy is also detectable in magnetic resonance imaging (MRI) before the manifestation of clinical symptoms in familial cases of AD, suggesting that molecular changes are already extensive when the disease is diagnosed.[21] In addition, it is supposed that immunotherapies are most effective when they are initiated at early stages of the disease in animal models of AD.[6] Together, these observations suggest that current disease-modifying therapies for AD have limited effects on disease progression in symptomatic patients, in whom various molecular events downstream to Aβ aggregation overwhelm the neuroprotection by the interventions.

Disease-modifying therapy for polyglutamine diseases

In view of their monogenic natures, polyglutamine diseases are thought to be a pragmatic model in the development of disease-modifying therapies for neurodegenerative disorders. Genetic modulation inhibiting the nuclear localization of polyglutamine-expanded protein results in marked suppression of disease manifestation in mouse models of polyglutamine diseases, suggesting that inhibition of abnormal protein accumulation is a fundamental strategy to slow pathophysiological progression in polyglutamine diseases.[22] This hypothesis has been rigorously tested in SBMA, a polyglutamine disease characterized by progressive loss of lower motor neurons within the spinal anterior horn and brainstem. SBMA is caused by the expansion of a CAG repeat in the gene coding androgen receptor (AR), and exclusively affects males. Leuprorelin, a potent luteinizing hormone-releasing hormone (LHRH) analog, has been shown to inhibit the testosterone-dependent nuclear accumulation of the polyglutamine-expanded AR, resulting in a marked improvement of neuromuscular phenotypes seen in the AR-97Q mouse, a transgenic animal model of SBMA.[23] In a phase 2 clinical trial, 12 months treatment with leuprorelin significantly diminished the serum level of creatine kinase, and suppressed the nuclear accumulation of the polyglutamine-expanded AR in the scrotal skin. Of note is the observation that the frequency of neurons bearing pathogenic AR in the anterior horn and brainstem of an autopsied patient, who received leuprorelin for 2 years, was less than in non-treated SBMA patients.[24] Nevertheless, no definite effects on motor functional scores were observed in a 48-week randomized placebo-controlled multicenter clinical trial, although there was improvement of swallowing function, measured as the pharyngeal barium residue in videofluorography in a subgroup of patients whose disease duration was <10 years, as well as a decrease in AR accumulation in scrotal skin biopsies and serum levels of creatine kinase.[25] Also, a phase 2 clinical trial of dutasteride, a 5-α-reductase inhibitor that blocks the conversion of testosterone to dihydrotestosterone, did not demonstrate effectiveness.[26] Although the results of these studies are inconclusive, their findings do not exclude the possibility that ligand-targeted therapies slow the progression of SBMA. Given the strong evidence shown in basic studies, this hypothesis needs to be further verified in clinical trials with a rigorous and efficient design.

Key issues in therapy development for neurodegenerative diseases

  1. Top of page
  2. Abstract
  3. Introduction
  4. Accumulation of abnormal proteins
  5. Development of therapies targeting abnormal protein accumulation
  6. Key issues in therapy development for neurodegenerative diseases
  7. Summary
  8. Author contributions
  9. Acknowledgments
  10. Competing interests
  11. References

Elucidation of molecular pathogenesis

The disappointing results of clinical trials underscore the need to overcome challenges in the translational research on disease-modifying therapies for neurodegenerative diseases (Fig. 2). Although abnormal protein deposition is the salient pathological hallmark of neurodegenerative diseases, several studies have indicated that the insoluble aggregates are likely formed as a result of cellular defense reactions coping with the pathogenic protein.[27] This view is clearly exemplified in familial AD cases with the E693Δ mutation in the APP gene, which produces the mutant Aβ peptides showing the unique aggregation property of enhanced oligomerization but no fibrillization.[28] Soluble oligomers of polyglutamine-expanded proteins also play an important role in the initiation of neurodegeneration.[29, 30] These findings suggest the need for the identification of toxic species of the pathogenic proteins and development of agents that selectively target such species. In addition, although the accumulation of abnormal proteins appears be the primary target of therapy at pre-onset or early stages of disease, multiple molecular events may need to be treated at later stages (Fig. 3).

image

Figure 2. Challenges in development of disease-modifying therapies The lack of positive results in clinical trials of disease-modifying therapies for neurodegenerative diseases indicate several issues to be resolved. Conventional approaches are not necessarily appropriate for the development of disease-modifying therapies for neurodegenerative diseases; therefore, both basic and clinical studies are now subjected to conceptual innovation.

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image

Figure 3. Therapeutic targets of neurodegeneration Although the accumulation of abnormal proteins appears be the primary target of therapy at pre-onset or early stages of disease, multiple molecular events may need to be treated at later stages. In particular, recent studies strongly indicate the roles of the neuron–glia interaction in the progression of the neurodegenerative process, providing novel therapeutic targets. Propagation of pathogenic proteins also gains attention as the molecular mechanism of disease progression as well as the target of therapy development. In addition to protein toxicity, the RNA transcript of causative genes appears to play a causative role in neurodegenerative processes.

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Animal models

Previous attempts to elucidate the molecular pathogenesis of neurodegenerative diseases relied largely on the findings of animal models carrying mutant human genes identified in familial cases. These studies may clarify certain pathological pathways, but the insights obtained from them are not necessarily applicable to the sporadic forms of the diseases, which are usually more numerous. Therefore, novel animal models that recapitulate the pathological processes in sporadic cases are needed to improve the preclinical evaluation of promising compounds. How to evaluate the efficacy of compounds in preclinical trials is another critical issue in the development of disease-modifying therapies. Although clinical trials are usually designed on the basis of data obtained from a single animal study, other laboratories seldom replicate these preclinical research results. Thus, the reproduction of positive results from animal experiments that analyze efficacy of compounds at symptomatic stages might improve the credibility of preclinical studies. The outcome measures used in pre-clinical studies, including rotarod task and cumulative survival, are not necessarily relevant to those in clinical trials, such as functional scores. Therefore, as suggested in cancer research, utilization of species-independent biomarkers that are applicable to humans is another key to bridging the gap between basic and clinical research. For example, an integrated proteomic approach identified common biomarkers, such as insulin-like growth factor binding protein 2, in human cancer cells and plasma from tumor-bearing mice.[31] These issues are now included in guidelines of preclinical studies, the implementation of which will hopefully increase the chances of success in translational research on disease-modifying therapies.[32]

Clinical trial at pre-onset or early stage

Enrichment of trial designs by stratifying the target patient population is a key to the success of clinical trials, given the considerable heterogeneity in clinical features of patients with neurodegenerative diseases. In particular, the duration of disease is a critical factor which has an unequivocal influence on the outcome of a trial. The effects of disease-modifying therapies are likely limited at symptomatic stages, due to the progression of neuropathological changes during presymptomatic periods. The lack of success in clinical trials of immunotherapy for AD suggests the need for initiation of disease-modifying therapies for AD in an early or asymptomatic, prodromal phase of the disease. Consistent with this notion is the observation that, in a phase 3 trial of leuprorelin for SBMA, favorable results were seen in a subgroup of patients whose disease duration was <10 years, but not in the entire tested population.[25] Based on these views, trials of presymptomatic treatments for AD are currently being prepared.[33]

The urgent need for testing interventions before the onset of neurological symptoms or at an early stage of disease is now being recognized in the translational research on neurodegenerative diseases. Although this strategy faces practical problems, such as feasibility and ethics, the most important issue is the presymptomatic diagnosis and evaluation of therapeutic effects before the manifestation of disease phenotypes. The efficacy of tested agents is not likely to be detected by conventional outcome measures such as functional scales; therefore, much effort has been made to develop clinical and biological markers for detecting presymptomatic defects in neuronal functions, and for evaluating the clinical efficacy of disease-modifying therapies for neurodegenerative diseases. For instance, a large cohort of HD patients has clearly demonstrated that rigorous neurophysiological tests are capable of detecting functional deficits before the full manifestation of disease in the carriers of mutant huntingtin.[34] This observation underscores the importance of longitudinal studies on natural history of biological and functional markers at pre- and early symptomatic stages of disease.

Outcome measures and biomarkers

The effects of therapy are evaluated by clinical endpoints that rate motor/cognitive functions or activities of daily life in patients in most clinical trials of neurodegenerative diseases. These clinical scales are confounded by multifarious factors, including genotypic and phenotypic heterogeneities. Patient-reported outcomes are a direct method to assess how patients feel, but it may be liable to within-patient fluctuation that is irrelevant to therapy effects. It is also noteworthy that patients with neurodegenerative diseases are susceptible to placebo effects that mask the efficacy of drugs on clinical outcome measures.[35] The reliance on vulnerable rating scales decreases the success rates of clinical trials by blurring treatment efficacy. In view of the seemingly modest effects of disease-modifying therapies in humans, clinical outcome measures need to be resistant to variability, subjectivity and placebo effects. This will be achieved by refining rating scales and exploiting reliable functional tests.

Biomarkers are objectively measurable variables that indicate pathogenic processes, or pharmacological responses to a therapeutic intervention. In exploratory phase clinical trials, biomarkers may be used for evaluating pharmacological and toxicological features of tested agents. This provides valuable information on bioavailability and safety, leading to rational choices of agents to be further investigated in confirmatory-phase clinical trials. Biomarkers are also utilized for assessing the effects on pathological processes of disease and discriminating symptomatic and disease-modifying effects of tested agents. Moreover, the use of biological markers may facilitate stratification of the patients who will most likely benefit from the therapy, and enable the enrichment of subsequent trials by identifying the patient population to be included.[12, 36] Pre-onset diagnosis could also be improved by combining several biomarkers. Although the potential of biomarkers as surrogate endpoints in clinical trials have been heavily discussed, the use of them as primary outcome measures in pivotal trials for regulatory approval has not been accepted. Therefore, studies should be pursued to qualify and validate biological markers for future development of disease-modifying therapies. Moreover, since it will require a tremendous time and effort to clarify whether the effects of tested agents on biomarkers appropriately predict the efficacy in clinical trials, a paradigm shift from medical, regulatory and scientific points of view may be needed for facilitating drug development using biomarkers.

Clinical trial design

Large, randomized, placebo-controlled clinical trials are costly and call for large numbers of patients and long observation periods; therefore, promising agents need to be thoroughly screened in exploratory phase 2 studies. To effect this proof-of-concept process, several new designs of phase 2 clinical trials are being developed (Fig. 4).[37] For example, the futility study judges the necessity of further development of the candidate agents, by assessing whether the efficacy of the tested compounds reaches a prespecified threshold that is calculated from the data of previous trials and natural history. Selection design is another method to select the drug to be tested in a large-scale placebo-controlled trial by comparing the efficacy of candidate agents without a concurrent placebo group. Alternative attempts to improve the efficiency of proof-of-concept include N-of-1 (single patient), lead-in and adaptive designs. N-of-1 design is another method explored for PD that enables a rapid evaluation of agents using multiple crossovers within the same subject. Although this design is useful for symptomatic therapies, carry-over may affect the results of clinical trials that examine disease-modifying effects of molecular-targeted therapies.[38] The observation of natural disease course prior to initiation of intervention may enable stratification of trials. The lead-in design measures changes within patients, and is expected to reduce the sample size.[39] Adaptive design is an innovative method that is gaining popularity in the field of clinical trials of any kind. This type of study allows the adjustment of trial design according to the results of interim analysis, may lead to termination of trials of futile compounds and save financial and patient resources. By adopting multi-stage adaptive design, the results of early-stage studies can be utilized for designing subsequent trials. However, substantial attention has to be paid to integrity of evidence and mathematical validity.[40] In addition, several attempts have been made to utilize the historical placebo controls in phase 2 trials to improve the efficiency of drug screening.[41]

image

Figure 4. Novel designs of clinical trial The futility study judges the necessity of further development of the candidate agents, by assessing whether the efficacy of the tested compounds reaches a prespecified threshold. Unlike conventional trials, these studies test the null hypothesis, that the agents have promise and will therefore show effects that exceed a clinically meaningful threshold. If this hypothesis is rejected, further development of the compounds is considered futile. Selection design, an alternative attempt to improve the efficiency of proof-of-concept, is particularly useful when several candidate agents need to be tested in a relatively short period. This method allows the comparison of multiple compounds or combination regimens, as well as different dosages of a single compound. If the efficacy of an agent is superior to others, it will be selected for further examination in a large placebo-controlled trial. In the trials using selective design, the agents or regimens can be compared without a concurrent placebo group.

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Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Accumulation of abnormal proteins
  5. Development of therapies targeting abnormal protein accumulation
  6. Key issues in therapy development for neurodegenerative diseases
  7. Summary
  8. Author contributions
  9. Acknowledgments
  10. Competing interests
  11. References

Both basic and clinical researches are being conceptually innovated to develop and translate effective disease-modifying therapies for neurodegenerative disorders. The thorough elucidation of molecular pathways in neurodegeneration using animal models that faithfully replicate human pathology may lead to successful translation of disease-modifying therapies. Development of therapies that target multiple molecular pathways is also necessary to combat the manifold pathogenesis of neurodegeneration. Elaborate designs would increase the chance of success in clinical trials. The refinement of research frameworks that involve academic, industrial and regulatory organizations is another key issue in the translational research on neurodegenerative diseases. Systems for patient registry and clinical trial networking are also mandatory to accelerate the development of disease-modifying therapies.[37] Integration of these approaches should help to identify therapies that slow progression of neurodegeneration.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Accumulation of abnormal proteins
  5. Development of therapies targeting abnormal protein accumulation
  6. Key issues in therapy development for neurodegenerative diseases
  7. Summary
  8. Author contributions
  9. Acknowledgments
  10. Competing interests
  11. References

This work was supported by a Center-of-Excellence grant, a Grant-in-Aid for Scientific Research on Innovated Areas (No. 22110005), and KAKENHI grants from MEXT/JSPS, Japan (No. 21229011, 23390230); grants from the Ministry of Health, Labor, and Welfare (MHLW), Japan; and Core Research for Evolutional Science and Technology (CREST) from Japan Science and Technology Agency (JST).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Accumulation of abnormal proteins
  5. Development of therapies targeting abnormal protein accumulation
  6. Key issues in therapy development for neurodegenerative diseases
  7. Summary
  8. Author contributions
  9. Acknowledgments
  10. Competing interests
  11. References