New Pathways in Drug Discovery for Alzheimer’s Disease Eric R. Siemers, MD, Robert A. Dean, MD, PhD, Ronald Demattos, PhD, and Patrick C. May, PhD

Corresponding author Eric R. Siemers, MD Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA. E-mail: [email protected] Current Neurology and Neuroscience Reports 2006, 6:372–378 Current Science Inc. ISSN 1528-4042 Copyright © 2006 by Current Science Inc.

Specific treatments for Alzheimer’s disease (AD) were first introduced in the 1990s using the acetyl-cholinesterase inhibitors. More recently, the N-methyl-D-aspartate (NMDA) antagonist memantine has become available. Although these treatments do provide a modest improvement in the cognitive abnormalities present in AD, their pharmacology is based on manipulation of neurotransmitter systems, and there is no compelling evidence that they interfere with the underlying pathogenic process. Pathologic and genetic data have led to the hypothesis that a peptide called amyloid β (Aβ) plays a primary role in the pathophysiology of AD. Several investigational therapies targeting Aβ are now undergoing clinical trials. This paper reviews the available data regarding Aβ -directed therapies that are in the clinic and summarizes the approach to biomarkers and clinical trial designs that can provide evidence of modification of the underlying disease process.

Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder causing progressive decline in memory and other aspects of cognition. The average duration from onset of symptoms to nursing home placement is 5 to 7 years, and the average duration from symptom onset to death is 7 to 9 years [1,2]. Based on pathologic data showing a decrease in markers for the neurotransmitter acetylcholine [3], symptomatic treatment with cholinesterase inhibitors first became available in the 1990s. More recently, the N-methyl-D-aspartate (NMDA) receptor antagonist memantine also became available. Although treatment with these drugs can lead to modest symptomatic improvement, their mechanism of action is related to

manipulation of neurotransmitter systems, and there is no compelling evidence that they slow the underlying rate of disease progression [4,5]. Although the cause of AD is unknown, evidence began to accrue in the 1980s that the pathology is related to extracellular accumulations of β-amyloid plaques and intracellular accumulations of neurofibrillary tangles [6–8]. Whether amyloid plaques or neurofibrillary tangles are the primary cause of cell death in AD has not yet been resolved; however, a number of therapies under development are based on what is now known as the “amyloid hypothesis.” A 38- to 42-amino acid peptide known as amyloid β (Aβ) may be critical to the pathogenesis of AD [8–11]. Aβ, particularly the Aβ1–42 variant, is a major constituent of amyloid plaques, and mutations in the amyloid precursor protein (APP) gene that encode amino acid substitutions near the cleavage sites for Aβ formation have been linked to AD in several families. Mutations in the genes for presenilin (PS-1 and PS-2), proteins thought to be involved in the cleavage of APP and formation of Aβ, also lead to familial AD. Additionally, a common polymorphism in the apolipoprotein (apo) E ε4 allele is a major risk factor for the development of AD, and one hypothesis for how apoE influences risk for AD is through its presumptive role in the trafficking of Aβ [12]; however, other hypotheses for the role of apoE ε4 in AD exist [13]. Neuronal degeneration around a subset of Aβ plaques is one of the pathologic hallmarks of the disease, suggesting that amyloid plaque may be toxic to neurons. Alternatively, other oligomeric species of Aβ have been suggested recently as the primary source of toxicity [14]. Because plaque, Aβ monomers, and Aβ oligomers are all likely to exist in equilibrium, treatments targeting one of these forms of Aβ may indirectly affect others. Aβ peptide is generated from APP by the sequential cleavage of APP by two proteases: β-site APP amyloid cleaving enzyme (BACE) and γ-secretase. BACE first cleaves APP and generates a large, secreted protein product known as sAPPβ and a smaller, membrane-bound fragment. This latter fragment is cleaved at several sites by γ-secretase to generate Aβ peptides of various lengths; however, the primary products of this cleavage are known as Aβ1–40 and Aβ1–42 . γ-Secretase is a complex enzyme,

Pathways in Drug Discovery for AD

appearing to consist of four proteins known as presenilin (PS-1 or PS-2), nicastrin, Aph-1, and Pen-2 [15]. Although the synthesis of Aβ is becoming better understood, relatively little is known about its clearance. Animal studies suggest that Aβ is actively transported from brain interstitial fluid into blood [16]. As noted previously, a polymorphism in the apoE ε4 allele is considered a major risk factor for the development of AD, and apoE is thought to be important in the removal of Aβ from the brain [12]. Thus, based on pathologic data showing accumulation of Aβ and amyloid plaques in the brains of AD patients, and genetic data showing the involvement of APP and Aβ in human AD, the amyloid hypothesis has attracted substantial attention in the development of new drugs that might slow disease progression.

Amyloid-based Investigational Treatments for AD γ-Secretase inhibitors

LY450139 is a “functional” inhibitor of γ-secretase that was developed based on its ability to inhibit the synthesis of both Aβ1–40 and Aβ1–42 [17]. Preclinically, this molecule has been shown to lower Aβ in the brain, cerebrospinal fluid (CSF), and plasma of both mice and dogs [18–20]. Further, in a study of a transgenic mouse model of AD, after 5 months of treatment with LY450139 once per day, mice given 30 mg/kg showed less accumulation of Aβ in cortex and hippocampus than mice given vehicle [21]. This dose of LY450139 results in an acute decrease in plasma Aβ of approximately 60% in these transgenic mice. In clinical trials using volunteers [22] or AD patients [23], doses of LY450139 up to 40 mg once daily caused a reduction in plasma Aβ of nearly 40%. Somewhat surprisingly, statistically significant reductions in CSF Aβ have not been demonstrated, perhaps in part due to marked variability in Aβ concentrations in lumbar CSF in humans. Drug concentrations in CSF were substantially above those necessary to inhibit γ-secretase in cultured cells [22]. Based in part on these results, changes in plasma rather than CSF Aβ may provide a method to optimize dose selection in phase II studies so that pivotal phase III trials can be initiated. The γ-secretase complex is now known to cleave several single-pass transmembrane proteins in addition to APP, and potential safety concerns with compounds that inhibit γ-secretase have been identified related to inhibition of cleavage of a protein known as Notch [24]. Despite these potential safety concerns, doses of LY450139 up to 40 mg once daily have been well tolerated [22,23], and clinical development of this compound continues. R-flurbiprofen is an allosteric modulator of γ-secretase that is currently in clinical testing for AD. Unlike the functional γ-secretase inhibitor LY450139, some allosteric modulators may have more selective effects on Aβ1–42 than

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on Aβ1–40, and may not inhibit cleavage of other proteins such as Notch [25]. In rodents, flurbiprofen causes a reduction of about 20% in brain Aβ1–42 in some [25], but not all [26], studies. Dosing for 3 days using 10 to 25 mg/kg/d reduced plasma Aβ1–42 by up to 50% in some but not all experiments [26]. Chronic treatment for 8 months reportedly slows deposition of brain Aβ in transgenic mice [27]. Whether decreases in plasma or CSF Aβ1–42 occur after administration of R-flurbiprofen to humans has not yet been reported.

BACE inhibitors BACE is the first and possibly only rate-limiting enzyme involved in the formation of Aβ from APP. The enzyme has been extensively characterized [28–32], including x-ray crystallography of the active site. If BACE is genetically knocked out from transgenic mouse models of AD, formation of amyloid plaques is reduced [33–35]. Further, BACE knockout mice have few phenotypic changes, suggesting that an inhibitor of this enzyme would be well tolerated [34,35]. Despite these striking advances in knowledge of the molecular biology of this enzyme, the task of finding a small molecule that would be useful as an oral BACE inhibitor has proven formidable. As yet, no BACE inhibitors are reported to have reached clinical trials despite extensive efforts [36,37]. Although BACE remains a very attractive target for new drug development, whether the associated medicinal chemistry challenges can be overcome is not clear.

Immune-mediated therapies Immunologic treatments for AD have been studied as a means to increase clearance of soluble Aβ or to remove amyloid plaque directly by Fc-mediated phagocytosis. Both active and passive immunization strategies have been investigated. Published human experience with immune treatments for AD is limited to an active immunization strategy [38]. An active immunization clinical trial was stopped when intramuscularly administered aggregated Aβ1–42 with QS-21 adjuvant (AN1792) appeared to cause meningoencephalitis in 18 of 298 patients with mild to moderate AD [39]. This event may have been T-cell mediated rather than related to antibodies directed against Aβ as plasma antibody titers did not appear to be related to the onset of meningoencephalitis [39,40], and T-lymphocyte meningoencephalitis was described pathologically. Antibodies that are generated after active immunization bind primarily to the N-terminal region of Aβ [41]. In addition to these safety concerns, positive results have also been reported from active immunization studies. Immunohistochemical staining of three brains available from patients who had participated in the AN1792 trials reportedly showed areas of cortex with much less amyloid plaque than would be expected for patients with mild to moderate AD [42–44]. Hock et al. [45] reported pre-

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liminary findings showing improved cognitive scores from patients who generated antibodies against Aβ compared with those without such a response. A later report using the complete study population showed no effect on standard cognitive scores or on activities of daily living, but did show improved cognition as assessed by a battery of neuropsychological tests in subjects who demonstrated an antibody response [46]. Concentrations of tau, a putative biomarker of AD severity [47], were also reduced in the CSF of patients developing an antibody response [46]. Volumetric magnetic resonance imaging (vMRI) showed that brain parenchymal volumes were reduced and ventricular size was increased in antibody responders [48]. This was unexpected because brain volumes are lost at a greater rate in AD patients compared with age-matched control subjects, and a disease-modifying treatment of AD would be expected to attenuate the rate of volume loss [49]. An explanation for this unexpected effect on this biomarker is not yet clear. Given the substantial safety concerns raised by the initial clinical experience with active immunization, passive immunization using monoclonal antibodies directed against Aβ has been suggested as a possible treatment for AD. Treatment of transgenic mice was first reported using antibodies that bind to the N-terminal region of Aβ [50]. The N-terminal antibodies used in these studies bind robustly to deposited Aβ; this binding is thought to be important in the degradation of plaques by Fc-mediated microglial activation. Other studies have used an antibody (m266.2) that binds to the mid-domain of Aβ and is selective for soluble rather than deposited Aβ. Treatment of transgenic mice for 5 months using this antibody also results in a reduction of plaque burden [51]. Just 24 hours following a single administration of this antibody, cognitive improvement was demonstrated in two tests in transgenic mice [52]. In contrast to the Fc fragment/microglia-based mechanism of action of the N-terminal antibodies, the m266.2 antibody is thought to alter the soluble Aβ equilibrium between the central nervous system and periphery, favoring peripheral clearance and thus resulting in decreased Aβ deposition. Although the use of monoclonal antibodies may reduce the risk of meningoencephalitis, other potential adverse events must still be considered. Antibodies binding to the N-terminal region of Aβ have caused microhemorrhage in preclinical studies [53], whereas a study using a different antibody that binds only to soluble Aβ did not show such a result [54]. The antibodies generated in the phase II AN1792 trial were primarily directed toward the N terminus of Aβ and bind to parenchymal amyloid plaque and vascular amyloid [40]. Humanized monoclonal antibodies directed against Aβ are now being studied in early phase AD clinical trials. Their safety and tolerability, as well as their effect on rate of progression of AD, will need to be evaluated carefully.

Aβ binding and antiaggregating agents The process that leads to Aβ aggregation in amyloid plaques in AD is not well understood, but a possible contributor is a physical-chemical association of glycosaminoglycans (GAGs) with Aβ that induces aggregation [55]. Studies of specific GAGs have suggested that inhibitors of GAG-induced amyloid formation could be developed [56]. Tramiprosate is a GAG mimetic that is thought to interact with the GAG-binding region of Aβ, and thus decrease fibrillization and formation of amyloid plaque. Chronic treatment of transgenic mice with tramiprosate is reported to reduce accumulation of brain Aβ [57]. When given to patients with AD, CSF concentrations of Aβ1–42 are lowered [57], although the mechanism leading to this lowering is not clear. Phase III clinical studies of tramiprosate in AD patients in North America and Europe are ongoing. Metal ions, including zinc, copper, and iron, might also play a role in Aβ aggregation [58]. Clioquinol, an antibiotic that chelates zinc and copper and passes the blood-brain barrier, has been studied in transgenic mice given 30 mg/kg/d for 9 weeks [59]. Treatment resulted in a greater than 40% reduction in total brain Aβ. In this study, soluble (not aggregated) Aβ increased by about 50%; however, the fact that soluble Aβ concentrations are relatively small compared with total concentrations should be noted. Clioquinol has also been studied in a small phase II trial of AD patients [60]. Somewhat surprisingly, cognitive scores improved in a short period of time in drug-treated compared with placebo-treated subjects. Aβ1–42 in plasma was decreased in drug-treated subjects but increased in placebo-treated subjects. Unfortunately, clioquinol has not been available for routine clinical use since the early 1970s because of a serious adverse event known as subacute myelo-optico-neuropathy. Although this adverse event might be prevented by vitamin B12 supplementation [61], this potential safety concern is likely to prevent clioquinol from being used routinely for the treatment of AD. Nevertheless, these preclinical and clinical studies show promise for this therapeutic approach.

Use of Biomarkers in AD Drug Development Numerous research tools have evolved in recent years to detect, characterize, and quantify the biochemical, morphologic, and functional aspects of AD pathology. Although initially applied to elucidation of AD pathology, many of these research tools are also now being applied to the discovery and development of candidate therapeutics. A variety of neuroimaging methods have helped define regional abnormalities and alterations in brain morphology (vMRI), function (fluoro-dexoy-glucose positron emission tomography [PET]) and amyloid plaque burden (PET imaging with Pittsburgh Compound B and single photon emission computed tomography imaging with 6-iodo-2-(4’-dimethylamino-)phenyl-imidazol

Pathways in Drug Discovery for AD

[1,2-a]pyridine). The development and application of these clinical research tools and the potential for use in clinical practice have been thoroughly reviewed elsewhere [49,62••]. A major government-academic-industry collaborative study known as the Alzheimer’s Disease Neuroimaging Initiative (ADNI) is underway to define the utility and limitations of selected clinical neuroimaging and biochemical modalities to assess predisposition, diagnosis, and progression of this disorder [62••]. Biochemical assessment of AD has focused predominantly on the constituents of amyloid plaque and neurofibrillary tangles. These histopathologic findings provide the basis for postmortem confirmation of the diagnosis. A variety of analytical methods have been described for measurement of Aβ species in brain parenchyma, CSF, and plasma. Immunoassay methods are most widely applied in both the basic science and clinical research settings. Enzyme-linked immunosorbent assay (ELISA)-based methods have proven sufficiently sensitive and specific for detection and quantification of soluble Aβ1–40, Aβ1–42 , and other Aβ variants [23,63,64]. Measurement of Aβ1–42 in CSF is of particular interest as it is presumed to reflect concentrations of soluble Aβ1–42 in interstitial fluid in brain parenchyma. Soluble Aβ1–42 is likely to be a critical intermediate in the central nervous system trafficking of Aβ and thus may be handled differently given normal clearance compared with abnormal pathway(s) resulting in plaque deposition. Although differences in calibration and reagent antibody affinities for various ELISA methods produce marked differences in measured concentrations across methods, Aβ1–42 concentrations in AD patient populations are consistently lower than those of healthy, cognitively intact, age-matched populations when measured by the same method [47,62••]. The consistency of this finding has led to the development and commercialization of CSF Aβ1–42 research-use-only and in vitro diagnostic assays for clinical application [65–67]. One such assay provides simultaneous measurement of Aβ1–42, total tau, and phospho-tau 181 in CSF [68]. Recent data based on the three measures derived from this multiplex analysis correlate well with plaque imaging methods [69]. These three measures performed in a multiplex assay format also identified those patients with mild cognitive impairment (MCI) likely to progress to AD over a 4- to 6-year period [70]. The diagnostic sensitivity and specificity of this laboratory panel for progression to AD were 95% and 83%, respectively. Apart from the robust CSF Aβ1–42 finding, analyses of other Aβ variants in routinely accessible clinical specimens such as plasma and CSF have yet to distinguish AD from healthy populations or detect risk for or characterize the progression of AD. Thus, although CSF measures of Aβ1–42, tau, and phospho-tau have not yet been established as part of the routine diagnosis of AD, studies to evaluate their use are ongoing, and they are currently available to clinicians as a research tool. There is currently no established link between AD diagnosis or progression and available measures of plasma Aβs.

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As noted previously, however, the functional γ-secretase inhibitor LY450139 lowered Aβ species in brain, CSF, and plasma of mice and dogs as measured by ELISA [18–20]. Similarly, LY450139 administration to volunteers [22] or AD patients [23] dose-dependently lowers plasma Aβ. Curiously, administration to volunteers and AD patients did not produce statistically significant reductions in CSF Aβ. Nevertheless, the extent to which the levels or change in levels of plasma Aβ reflects levels or change in levels of soluble or other forms of brain Aβ has yet to be defined. Present attempts to compare Aβ concentrations in plasma and CSF are confounded by marked variability of Aβ concentrations in CSF obtained by lumbar puncture [22,23]. For the moment, measures of plasma Aβ have provided a means to compare and translate between humans and other species the pharmacologic effects of candidate therapeutics designed to alter amyloid production, clearance, and deposition.

Pivotal Trial Design for Amyloid-based Therapies Inherent in the development of drugs that target Aβ and amyloid is the idea that these treatments would result in effects on the underlying disease process, rather than the symptomatic effects that are seen with currently available medications. By modifying the underlying disease process, the hope is that greater long-term benefits will result for patients. The design of clinical trials for disease-modifying (sometimes termed neuroprotective) drugs is likely to be different than those of symptomatic therapies. At present, there is no general agreement among investigators about how these disease-modifying therapies should be tested; however, recent symposia have begun to address this issue in detail [71]. A distinction between symptomatic and disease-modifying effects of investigational drugs could be established easily if perfect clinical measures of cognition were available that could be obtained during trials of indefinite length [72]. Even in the absence of such ideal measures, study designs that establish disease-modification using clinical outcomes alone have been suggested. Imaging and biochemical biomarkers provide another means of demonstrating disease-modifying characteristics of a drug [62••]. The trials of AD drugs now in progress, and those likely to follow, will be evaluated closely to assess what clinical measures and what biomarkers appear to be most related to disease modification in AD.

Conclusions Investigational treatments for AD that are related to the amyloid hypothesis have moved from early concepts regarding Aβ as a potential drug target, through preclinical testing, and, in several cases, are now in clinical development (Table 1). Although each of these investigational treatments carries uncertainties with regard to potential efficacy and tolerability, successful development

N/A

~ 40% reduction Notch related

Human plasma Aβ change

Potential safety concerns

Status

Phase III

Aβ—amyloid β; SMON—subacute myelo-optico-neuropathy; Tg—triglyceride.

Phase II

Yes

Yes N/A

Yes

N/A

AN1792

Stopped

Meningo-encephalitis Specificity for Aβ versus Notch

≤ 50% reduction

60% reduction

Efficacy in Tg mouse chronic studies

Preclinical change in plasma Aβ

Flurbiprofen

LY450139

Table 1. Amyloid-related therapies in clinical or preclinical development

Phase II

Microhemorrhage / Fc mediated

N/A

Yes

N/A

N-terminal antibody

Phase II

Fc mediated

N/A

Yes

Marked increase

Mid-domain antibody

Phase III

NA

NA

Yes

N/A

Tramiprosate

Stopped

Risk of SMON

Possible decrease

Yes

N/A

Clioquinol

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of one or more of these drugs would be a major advance in the treatment of AD. As the success, or lack thereof, of each of these therapies is determined, the strategy for translational studies of AD therapies can be assessed. Whether change in plasma Aβ is a meaningful biomarker to assess drug effect will become better understood, and which imaging or biochemical biomarker best reflects disease severity and is the best indirect measure of disease-modification will be similarly determined. These understandings can be applied subsequently to additional treatments for AD and to the development of diseasemodifying therapies for other neurodegenerative diseases.

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