Beyond the neurotransmitter-focused approach in treating Alzheimer’s Disease: drugs targeting...

Aging Clin Exp Res, Vol. 21, No. 6 386

Key words: Aβ aggregation inhibitors, active immunotherapy, Alzheimer’s Disease, α-secretase activators, β-amyloid, β-secretase inhibitors,γ-secretase inhibitors, γ-secretase modulators, passive immunotherapy.Correspondence: Francesco Panza, MD, PhD, Department of Geriatrics, Center for Aging Brain, Memory Unit, University of Bari,Policlinico, Piazza Giulio Cesare 11, 70124, Bari, ItalyE-mail: [email protected] December 10, 2008; accepted in revised form December 22, 2008.

Beyond the neurotransmitter-focused approachin treating Alzheimer's Disease: drugs targetingββ-amyloid and tau protein

Aging Clinical and Experimental Research

Francesco Panza1, Vincenzo Solfrizzi1, Vincenza Frisardi1, Bruno P. Imbimbo2, Cristiano Capurso3,Alessia D’Introno1, Anna M. Colacicco1, Davide Seripa4, Gianluigi Vendemiale3,4, Antonio Capurso1and Alberto Pilotto5

1Department of Geriatrics, Center for Aging Brain, Memory Unit, University of Bari, Bari, 2Research andDevelopment Department, Chiesi Farmaceutici, Parma, 3Department of Geriatrics, University of Foggia, Foggia,4Internal Medicine Unit, IRCCS Casa Sollievo dalla Sofferenza, San Giovanni Rotondo, Foggia, 5Geriatric Unit and Gerontology-Geriatric Research Laboratory, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy

ABSTRACT. Drugs currently used to treat Alzheimer’sDisease (AD) have limited therapeutic value and do notaffect the main neuropathological hallmarks of the dis-ease, i.e., senile plaques and neurofibrillar tangles.Senile plaques are mainly formed of β-amyloid (Aβ), a 42-aminoacid peptide. Neurofibrillar tangles arecomposed of paired helical filaments of hyperphos-phorylated tau protein. New, potentially disease-mod-ifying, therapeutic approaches are targeting Aβ and tauprotein. Drugs directed against Aβ include active andpassive immunization, that have been found to accel-erate Aβ clearance from the brain. The most devel-opmentally advanced monoclonal antibody directlytargeting Aβ is bapineuzumab, now being studied in alarge Phase III clinical trial. Compounds that interferewith proteases regulating Aβ formation from amyloidprecursor protein (APP) are also actively pursued. Thediscovery of inhibitors of β-secretase, the enzymethat regulates the first step of the amyloidogenicmetabolism of APP, has been revealed to be particu-larly difficult due to inherent medicinal chemistryproblems, and only one compound (CTS-21166) hasreached clinical testing. Conversely, several com-pounds that inhibit γ-secretase, the pivotal enzymethat generates Aβ, have been identified, the most ad-vanced being LY-450139 (semagacestat), now in PhaseIII clinical development. Compounds that stimulate

α-secretase, the enzyme responsible for the non-amy-loidogenic metabolism of APP, are also being devel-oped, and one of them, EHT-0202, has recently en-tered Phase II testing. Potent inhibitors of Aβ aggre-gation have also been identified, and one of suchcompounds, PBT-2, has provided encouraging neu-ropsychological results in a recently completed PhaseII study. Therapeutic approaches directed against tauprotein include inhibitors of glycogen synthase ki-nase-3 (GSK-3), the enzyme responsible for tau phos-phorylation and tau protein aggregation inhibitors.NP-12, a promising GSK-3 inhibitor, is being tested ina Phase II study, and methylthioninium chloride, a tauprotein aggregation inhibitor, has given initial en-couraging results in a 50-week study. With all these ap-proaches on their way, the hope for disease-modifyingtherapy in this devastating disease may become a re-ality in the next 5 years.(Aging Clin Exp Res 2009; 21: 386-406)©2009, Editrice Kurtis

REVIEW ARTICLES

INTRODUCTIONSince population aging has become a worldwide phe-

nomenon, the burden of the age-related neurodegener-ative diseases, particularly dementia, is expected to in-crease dramatically in both developed and developingnations (1). Dementia is estimated as affecting approxi-

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mately 6% of the population aged 65 and older, theprevalence increasing exponentially with age, being 40-70% at the age of 95 years and over (2). In Western coun-tries, the most common forms of dementia areAlzheimer’s Disease (AD) and vascular dementia (VaD),with respective frequencies of 70% and 15% of all de-mentias (3). Therefore, AD currently affects more than 26million people worldwide with an expected increase tomore than 106 million by 2050 (4). While more than 100years have passed since the first description of the neu-ropathological hallmarks of AD, relatively little was knownabout the mechanisms driving the development and pro-gression of AD until recently. Without specific mechanismsto target, the development of disease-modifying therapiesshowed little progress. In the last decade, advances in un-derstanding the neurobiology of AD have been translatedinto an increase in clinical trials assessing several poten-tial AD treatments. However, only cholinesterase in-hibitors (ChEIs) and the N-methyl-D-aspartic acid (NMDA)-receptor antagonist memantine have received Food andDrug Administration (FDA) approval for symptomatictreatment of AD. However, ChEIs and memantine onlyslightly delay the inevitable symptomatic progression of thedisease, leading some to question their clinical utility (5).

In this review article, we focused on therapies based onthe two major neuropathological hallmarks of AD and onthe status of clinical trials of β-amyloid (Aβ) and tau pro-teins-targeted drugs, with particular emphasis on themost important pharmacological strategies aimed at in-terfering with the generation, deposition or clearanceof Aβ, the major culprit of the AD brain.

CLINICAL TARGETS FOR TREATING LATE-LIFE COGNITIVE DISORDERSIn recent years, in an effort to identify clinical targets of

potential therapeutic agents for AD, people with mildcognitive impairment (MCI) have been enrolled in trials withdrugs that were effective in patients with AD (6, 7). How-ever, previous studies have shown that not all MCI subjectshave predementia AD (8). The term “predementia syn-drome” identifies all conditions with age-related deficits incognitive function reported in the literature, including amild stage of cognitive impairment based on a normalitymodel and pathological conditions considered predictive ofearly stages of dementia (8, 9). Such predementia syn-dromes have been defined for AD, but have not yet beenoperationalized for other specific forms of dementia.Therefore, the term “predementia syndromes” includes dif-ferent conditions and, among them, MCI is at present themost widely used term to indicate non-demented aged per-sons with no significant disability and a mild memory orcognitive impairment which cannot be explained by anyrecognized medical or psychiatric condition (8, 9).

At present, the term Mild Cognitive Impairment and itsacronym MCI have frequently been used in studies on the

preclinical phases of dementia, although with differing andinconsistent definitions (10-13). There is now ample ev-idence that MCI is often a pathology-based conditionwith a high rate of progression to AD (8, 9). Therefore,MCI has also been identified as the predementia syndromefor AD. The more recently proposed multiple subtypes ofMCI were intended to reflect the heterogeneity of differ-ent types of dementia. Actually, the recent subclassifica-tions of MCI according to its cognitive features [dysex-ecutive MCI and amnestic-MCI (aMCI), or aMCI andnon-amnestic MCI (naMCI): single-domain aMCI andmultiple-domain aMCI or single-domain naMCI and mul-tiple-domain naMCI] (13), clinical presentation [MCI withparkinsonism, cerebrovascular disease (CVD), depres-sive symptoms, behavioral and psychological symptoms](14), or probable etiology (MCI-AD, vascular MCI, orMCI-Lewy body dementia) (15) all represent an attempt tocontrol this heterogeneity. A critical review has recentlybeen made in Stockholm and then in Montreal, in orderto define a new consensus on MCI (13). Modification ofPetersen’s criteria (12) was proposed during the confer-ence in Montreal. Lastly, the European Consortium onAlzheimer's Disease (EADC) working group on MCI hasvery recently proposed a novel diagnostic procedurewith different stages, combining neuropsychological eval-uation and family interview to detect MCI at the earliestpossible stage (16). Therefore, given that, at present,the predementia syndromes for VaD, LBD and FTD arenot well defined clinically, the clinical target for treatmentof late-life cognitive disorders may be restricted to AD atvarious stages, particularly mild to moderate, and aMCI,as the predementia syndrome of Alzheimer’s pathology.

THE NEUROTRANSMITTER-FOCUSEDAPPROACH Drugs currently used to treat cognitive impairment

and dementia have very limited therapeutic value, andmainly cover management of psychiatric and behavioralrather than cognitive symptoms. Clearly, it is necessary toidentify new strategies able to prevent and slow down theprogression of predementia and dementia syndromes.Since the first use of ChEIs, in addition to preventive ef-forts, two major pathways of pharmacological develop-ment have emerged (17, 18). One is a neurotransmitter-oriented approach, focusing on the functional signifi-cance of acetylcholine and glutamate in the central ner-vous system (CNS) of AD patients (17, 18). A second ap-proach builds on step-by-step achievements in neu-ropathology, molecular biology and genetics of the disease(18). The neurotransmitter-focused approach has result-ed in the creation of two classes of drugs, one based oninhibition of acetyl- and butyrylcholinesterases (ChEIs)and a second on stimulation of glutamate receptors (me-mantine). Both approaches reduce the severity of cogni-tive symptoms, improve quality of life, and decrease

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caregiver burden for patients with mild to severe dis-ease. The ChEIs (donepezil, galantamine, rivastigmine,tacrine) degrade acetylcholinesterase, allowing levels ofacetylcholine, a neurotransmitter critical to the neurons in-volved in cognition, to increase. The primary excitatoryneurotransmitter of the CNS is glutamate, which is usedby approximately two-thirds of the synapses in the neo-cortex and hippocampus (19). Glutamate is involved inmost, if not all, aspects of cognition and higher mentalfunctions (19). Memantine partially blocks the NMDAacid receptor and prevents excess stimulation of the glu-tamate system, which influences memory and learning.More direct approaches include ampakines, which arepositive modulators of the action of glutamate at α-amino-3-hydroxy-5-methyl-4 isoxazoleproprionic acid(AMPA) receptors (20). While there are no large-scalePhase III data on ampakines, there is significant clinical ev-idence for the positive effects of memantine on cognitiveimpairment in AD (21). Evidence from controlled clinicaltrials suggests that ChEIs in particular can stabilize patients’symptoms for periods of time ranging between 1 and 3years, but without modifying the progression of the disease(17). Despite many theoretical considerations suggestingthat ChEIs or memantine may have a disease-modifyingeffect, only the symptomatic effects of these compoundshave been proven (22). Individual ChEIs have additionalpharmacological effects besides the inhibition of acetyl-cholinesterase (23, 24). However, the clinical benefit ofthese additional effects has not been convincingly shown(25). A very recent large meta-analysis based on 59 stud-ies showed that both ChEIs and memantine had consistenteffects in the domains of cognition and global assessment,but summary estimates showed small effect sizes (26). Out-comes in the domains of behavior and quality of lifewere evaluated less frequently and showed less consistenteffects. Most studies were of short duration (6 months),which limited their ability to detect delay in onset orprogression of dementia (26). Three studies of this meta-analysis directly compared different ChEIs and found nodifferences in cognition or behavior (27-29). In the treat-ment of dementias, ChEIs and memantine can reducesymptoms, primarily in the domains of cognition andglobal functions (26). Clinically important differenceswere not consistently evaluated or demonstrated in thesetwo domains for all drugs. Direct comparisons amongthese drugs are limited and do not suggest importantdifferences (26). Lastly, another meta-analysis was con-ducted on ChEIs to determine the impact of these drugson MCI with data from three published and five unpub-lished trials which met the inclusion criteria (three ondonepezil, two on rivastigmine, and three on galan-tamine) (30). The duration of these trials ranged from 24weeks to 3 years. No significant differences emerged inthe probability of conversion from MCI to AD or de-mentia between the treated and placebo groups. The

rate of conversion ranged from 13% (over 2 years) to25% (over 3 years) among treated patients, and from 18%(over 2 years) to 28% (over 3 years) among those inthe placebo groups (30). Only for two studies it waspossible to derive point estimates of the relative risk ofconversion: 0.85 (95% confidence interval 0.64-1.12)(31), and 0.84 (0.57-1.25) (32). Statistically significant dif-ferences emerged for three secondary end-points. How-ever, when adjusting for multiple comparisons, only onedifference remained significant (i.e., the rate of atrophy inthe whole brain) (30). In conclusion, the use of ChEIs inMCI was not associated with any delay in the onset of ADor dementia. Moreover, the safety profile showed that therisks associated with ChEIs are not negligible.

WAYS OF PREVENTION: FROM EPIDEMIOLOGICAL STUDIES TO CLINICAL TRIALS Epidemiological studies, mainly with cross-sectional

data, have suggested that non-steroidal anti-inflammato-ry drugs (NSAIDs), estrogens, HMG-CoA reductase in-hibitors (statins) and tocopherol (vitamin E) may be ben-eficial in reducing the incidence of AD. However, bias incase selection and several other sources of error are in-herent in epidemiological studies, and subsequent clinicaltrials have often been disappointing.

Several components of the inflammatory system, suchas activated microglia, cytokines, complement proteinand acute phase reactants have been detected in ADautopsies (33). A significant number of epidemiologicalstudies have suggested that prolonged intake of NSAIDsis associated with a reduced incidence of AD (34). How-ever, the results of clinical trials did not confirm this evi-dence (35). At present, several hypotheses explain theseinconsistent results by taking into account the possibilitythat NSAIDs can act only after very long-term use, or thatthe choices of drugs or doses are inappropriate; they al-so raise the question whether the hypothesis itself is in-correct (18). As clinical trials with indomethacin lead tohigh withdrawal rates as a result of gastrointestinal and re-nal toxicity (36), to overcome these limitations a lecithinderivative of indomethacin has been developed very re-cently, limiting these peripheral adverse effects by in-creasing brain/plasma ratios and brain half-life time in an-imal models (37). Further trials with COX-2-selective(celecoxib and rofecoxib) or unselective (naproxen) NSAIDsor other anti-inflammatory drugs such as dapsone, hy-droxychloroquine and prednisone, have not shown ben-eficial effects (38, 39).

At present, there is no evidence from clinical trials thatestrogens reduce the incidence or modify the course ofAD (40, 41), in contrast to cross-sectional epidemiologicalfindings (42). A recent trial involving transdermal 17-β-estradiol and oral progesterone will end in 2008, and theNational Institute on Aging (NIA) in the US is sponsoring

Emerging drugs in Alzheimer’s Disease





an ongoing Phase III clinical trial called PREPARE (PRE-venting Postmenopausal memory loss and Alzheimer'swith Replacement Estrogens) with estrogen or estro-gen and progesterone (estimated study completion date:December 2010) (43). For women with AD, estrogentreatment trials have tended to be small and of short du-ration, and estrogen started after the onset of dementiasymptoms may not meaningfully improve cognition orslow disease progression (44). Hormone replacementtherapy (HRT) initiated after age 64 increased all-causedementia in the Women's Health Initiative MemoryStudy (WHIS) (40). However, starting HRT and usingconjugated equine estrogens or estrogen-progesteronecombinations may have influenced the outcome (18).One intriguing hypothesis is that hormone therapy, ini-tiated or used during an early critical window, may reducelater incidence of AD (44).

At present, epidemiological studies do not suggestthat intake of antioxidant vitamins is beneficial against de-mentia or AD (45, 46). Furthermore, despite the wide useof tocopherol in treating AD, this antioxidant compoundwas not effective in a prevention trial in MCI to reduceprogression to AD (32), nor clearly effective in patientswith AD (47, 48). Since 2002, the PREADVISE (Pre-vention of AD by Vitamin E and Selenium) trial has re-cruited patients with an expected final enrolment of10,400 people (estimated study completion date: De-cember 2012), and only participants who are takingpart in the SELECT study (examining the usefulness of vi-tamin E and selenium in preventing prostate cancer)may apply to participate in the PREADVISE study (49). Inaddition to tocopherol, several antioxidants have been sug-gested to modulate brain metal metabolism and attenuateoxidative stress in the prevention and treatment of AD(50). Lastly, the NIA has very recently ended enrolmentfor a Phase IB clinical trial to measure the effects of to-copherol, ascorbic acid (vitamin C), thioctic acid (alpha-lipoic acid) and coenzyme-Q on markers of oxidativestress in AD (51).

Circulating lipoproteins and lipids can be modified bydietary or pharmacologic intervention, and total cholesterolis an established marker of the effects of lipid-loweringtreatment (52). Some epidemiological studies also indicatethat the prevalence of AD may be decreased in patientstreated with 3-hydroxy-3-methylglutaryl-coenzyme A re-ductase (HMGCoAr) inhibitors (statins) (52). In particular,one observational study in three hospitals in the UnitedStates recently found that the prevalence of AD amongpatients taking statin drugs was 60-73% lower than in pa-tients not on statins (53). However, the Cache CountyStudy, evaluating the incidence of AD in users of statins,questioned older cross-sectional observations and sug-gested that statins had no particular benefits (54). Pre-liminary clinical trials in patients with mild to moderate ADreceiving simvastatin and atorvastatin did not show clear

positive effects (55, 56). Furthermore, secondary analy-sis of the Alzheimer's Disease Cholesterol-LoweringTreatment (ADCLT) trial (56) revealed a benefit only in pa-tients with higher baseline total cholesterol or anapolipoprotein E (APOE) ε4 genotype, who receivedatorvastatin 80 mg/day for 12 months (57). Other larg-er and longer-term studies with patients receiving thesame statins have recently been completed, but the resultshave not yet been published (58, 59). In particular, resultsfrom the Lipitor's Effect in Alzheimer's Dementia (LEADe)Study, in 640 mild to moderate AD patients receivingatorvastatin 80 mg daily and a background therapy ofdonepezil 10 mg daily, will furnish more definitive eval-uation of the potential of statins in treating AD (59).However, preliminary findings presented at the annualAmerican Academy of Neurology meeting, held in April2008 in Chicago, showed that, in patients with mild tomoderate AD, the addition of atorvastatin 80 mg todonepezil 10 mg revealed no significant differences in cog-nition or global functions compared with placebo plusdonepezil 10 mg. Not were any statistically significant dif-ferences seen on various cognitive, behavioral and func-tional secondary end-points. However, in that trial, theatorvastatin arm was not associated with greater cognitivedecline than the placebo arm. In a subset of 64 patientsfor whom MRI scans were available, patients in the ator-vastatin arm had significantly less decline in hippocampalvolume in the brain compared with those in the placeboarm. In addition, in a sub-analysis completed after the tri-al, men in the atorvastatin arm had a significantly slowerrate of decline in cognition compared with men in theplacebo arm, although no definitive conclusions can bedrawn from this post-hoc analysis (60).

Several mechanisms of how statins may interfere withAD pathology have been discussed (52). Some statins suchas lovastatin, simvastatin and cerivastatin (no longer avail-able in the US or Italy) are able to cross the brain-bloodbarrier (BBB) into the CNS, whereas others such as ator-vastatin, pravastatin, rosuvastatin and fluvastatin do not(52). These statins are thought to reduce the amount ofAβ peptides by reducing cholesterol from the bloodand/or cerebrospinal fluid (CSF) (61). By reducing theamount of Aβ peptides, they may reduce the incidence orprogression of AD. Some authors believe that thosestatins which do not cross the BBB should be preferred,as those which do cross it may increase the rate of neu-ronal death by decreasing cholesterol beyond what isneeded for neuronal formation (62). Statins also havepleiotropic effects, including regulation of endothelial ni-tric oxide synthase (eNOS) activity and NO production,modulation and inflammatory processes, antioxidant ac-tivity, angiogenesis, and immunomodulation, as well as re-duction of free total cholesterol and inhibition of choles-terol ester accumulation in macrophages by inhibiting low-density lipoprotein (LDL) endocytosis and reducing meval-






der“ (64) was confirmed by subsequent observations (65).These neuropathological hallmarks of AD have stronglyinfluenced recent therapeutic approaches (18). SPs are theresult of misprocessing of the amyloid precursor pro-tein (APP), a type-1 transmembrane protein, by β- and γ-secretase, to form a toxic Aβ peptide of 40-42 aminoacids (66) (Fig. 1) which aggregates and initiates apathogenic self-perpetuating cascade, ultimately leadingto neuronal loss and dementia. Extracellular, and perhapsalso intracellular, Aβ exerts neurotoxic effects (67). Ex-tracellular Aβ peptides cluster in a β-sheet structure toform SPs. According to the “amyloid cascade hypothesis”(68), the development of SPs is thought to precede andprecipitate the formation of NFTs, as a result of cellularchanges, and the oligomeric forms of Aβ is the maincause of neuronal death in AD. APP may be metabolicallyprocessed according to two pathways (Fig. 1). In the so-called non-amyloidogenic pathway, α-secretase cleaves

onate byproducts essential for cholesterol esterification.Other potential effects of statins include reduction ofatherosclerotic plaque formation, endothelial protection,and reduction of oxidized LDL (63).

THE NEUROPATHOLOGICAL HALLMARK FOCUSED APPROACHBased on current nosology, AD is the most common

type of dementia and primary neurodegenerative disorderin the elderly (3). AD gradually leads to complete psy-chological and physical dependency, and finally to death,within one to two decades. It involves aberrant proteinprocessing and is characterized by the presence of both in-traneuronal protein clusters composed of paired helical fil-aments of hyperphosphorylated tau protein [neurofibril-lary tangles (NFTs)], and extracellular protein aggregates[senile plaques (SPs)]. Therefore, Alzheimer’s classicpathological description of AD as a “two-hallmark disor-



Fig. 1 - Overview of amyloid precursor protein (APP) processing. The upper part of the Figure shows APP region comprising trans-membrane sequence (underlined, bold) and sequences of Aβ1-40 (D1-V40) and Aβ1-42 (D1-A42) peptides. β-Secretase cleaves at D1 andY10. α-Secretase cleaves at Lys16, and the γ-secretase cleaves at Val40 and/or Ala42. Below the upper part of the Figure there is a rep-resentation of APP with residue numbers of interest in β- and γ-secretase cleavage. Panel A shows the non-amyloidogenic α-secretase path-way in which sAPPα and C83 are generated. Subsequent hydrolysis by γ-secretase produces a p3 peptide which does not form amyloiddeposits. Panel B shows the amyloidogenic pathway in which cleavage of APP by β-secretase to liberate sAPPβ and C99 is followed byγ-secretase processing to release β-amyloid peptides (Aβ1-40 and Aβ1-42) found in plaque deposits.

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APP within the Aβ sequence and releases its transmem-brane fragment, sAPPα, which appears to exert neuro-protective activity. In the amyloidogenic pathway, β-sec-retase releases APP plus a 12-kDa protein fragment(C99), which in turn is cleaved by γ-secretase giving wayto Aβ. Accumulation of toxic, aggregated forms of Aβseem to be crucial in the pathogenesis of familial forms ofAD (69). Many studies have shown a weak correlation be-tween Aβ deposits and cognitive status (70) and othersthat cognitively healthy elderly people may have a sub-stantial amyloid burden (71, 72).

THE ββ-AMYLOID TARGET:IMMUNOTHERAPY IN ALZHEIMER’SDISEASEThe idea of eliciting an immune response to exoge-

nously administered Aβ peptide in humans was originallydescribed in 1990 in a U.S. patent application by a physi-cian and an experimental immunologist (73). The patentprotected the idea of administering low amounts (up to 10g/day) of Aβ peptide by the parenteral route to slowdown the development of SPs. The inventors describedthree AD patients who were treated for 3-5 months andshowed dramatic improvements on “objective tests”. Lat-er, in 1996, Solomon et al. found that monoclonal anti-bodies raised against Aβ peptide inhibited its aggregation(74). In 1997, in another patent application, Schenk et al.(75) at Athena Neurosciences proposed the same idea asKline and McMichael (73) but with higher doses of Aβ andthe concomitant use of immune adjuvants. The inven-tors did not describe studies in humans but provided hugeamounts of data from animal experiments, including his-tological results in APP transgenic mice; some of these da-ta were published in 1999 (76). Therefore, vaccination wasthe first treatment approach to have a genuine effect onthe AD process, at least in animal models. Later, a num-ber of active and passive anti Aβ immunization procedureswere developed, all with the aim of removing Aβ aggre-gates from the brain of AD patients (77).

Active ImmunizationThe striking biological effect of vaccination in pre-

clinical testing and the apparent lack of side-effects intransgenic mice encouraged the launch of clinical trialswith AN1792, a vaccine containing preaggregatedAβ1–42 and the saponin QS21. The initial U.K. trial in80 patients with mild to moderate AD was designed to as-sess the antigenicity and toxicity of multiple-dose immu-nization (78). In Phase II of this trial, 372 patients wereenrolled, 300 receiving the aggregated Aβ1-4 (AN1792)with QS21 in the polysorbate 80 formulation.

This trial was stopped early because of symptoms ofacute meningoencephalitis in 18 patients (79). However,the autopsies of a few participants showed clearance ofparenchymal plaques, confirming the validity of this ap-

proach for amyloid clearance in human beings (80-82).Amyloid clearance in most cases was associated withmicroglia showing Aβ immunoreactivity, suggestingphagocytosis. Some patients also had a detrimental T-cellreaction surrounding some cerebral vessels, suggestive ofan excessive Th1 immune response. Immune reaction trig-gered by AN1792 seemed to be a double-edged sword:the benefits of humoral response against Aβ were over-shadowed in some individuals by a detrimental T-cell-mediated inflammatory response (82, 83).

Clinical cognitive benefits in the active vaccinationgroup compared with placebo were also very modest(84). The probable involvement of an excess cell-mediatedresponse in toxicity was supported by analysis of partic-ipants’ peripheral blood mononuclear cells. When stim-ulated in vitro with Aβ, cells from most participantswho showed a response produced interleukin 2 and in-terferon γ, indicative of a class II (CD4+) Th1-type re-sponse (85). Hence, a redesigned vaccine will need to pre-vent this by avoiding stimulation of Th1 lymphocytes, sothat the vaccine could potentially elicit a purely humoralresponse using non-toxic and non-fibrillogenic Aβ ho-mologous peptides, so that the immunogen does notproduce direct toxicity, and enhancing the peripheral-sinkeffect rather than central action (77). Another modifica-tion of vaccination in AD may involve mucosal applicationof the antigen, which stimulates predominantly IgA pro-duction with less cellular immunity. Positive results havebeen shown in transgenic animals with intranasal appli-cation of Aβ (86). Other approaches may use gene vac-cination to bypass Th1 activation (87-89).

Passive ImmunizationPassive transfer of exogenous monoclonal Aβ anti-

bodies seems to be the easiest way to provide antibodieswithout eliciting Th1-mediated autoimmunity. Transgenicmice treated this way had significant decreases in Aβconcentration and cognitive benefits (90, 91). The majorchallenges of this approach are high costs, BBB pene-tration, microhemorrhage, off-target cross-reactivity, andloss of the antibody to a peripheral sink. Nevertheless, atleast four clinical trials for passive immunization withvarious approaches are under way (77).

The most advanced trial is that of bapineuzumab.Elan/Wyeth recently initiated a Phase III trial and re-leased preliminary analysis of Phase II results (92). ThePhase II trial was a randomized, double-blind, placebo-con-trolled trial testing three doses of a humanized Aβ antibodyin 240 participants. In each of the escalating doses of theantibody, about 32 patients received active agent and 28received placebo. Although the study did not attain sta-tistical significance on the primary efficacy end-points inthe whole study population over the 18-month trial period,in the subgroup of participants who did not have theAPOE ε4 allele, clinically significant benefits were record-






ed on several scales, including the Mini-Mental State Ex-amination (MMSE) and the Alzheimer’s Disease assess-ment scale (ADAS). These findings suggest that this formof therapy may be effective. However, some patients inthe treatment group, but not in the control group, had va-sogenic edema, a serious adverse event (Table 1). In an-other study, intravenous immunoglobulin containing an-tibodies against Aβ affected Aβ plasma concentrations inpatients (93), and this approach is undergoing furtherstudy. Alternative approaches for passive immunizationless likely to be associated with toxicity include the use ofFv fragments or mimetics of the active antibody-bindingsite (94). Numerous studies in animal models of AD sug-gest that vaccination can prevent the devastating effectsof this prevalent disorder. However, a balance must beachieved between effective prevention and clearance ofamyloid deposits and the induction of autoimmunity.

THE ββ-AMYLOID TARGET: αα-SECRETASE ACTIVATORSThe shifting of APP metabolism to the non-amyloido-

genic pathway is an alternative way of decreasing thebrain Aβ burden in AD patients. α-Secretase cleaves APPat the Lys16-Leu17 bond within the Aβ sequence, thus pre-cluding the formation and deposition of Aβ (Fig. 1). α-Sec-retase cleavage generates an extracellular N-terminal frag-ment, termed sAPPα (soluble APPα-cleaved), and a C-ter-minal transmembrane fragment, called C83. sAPPα haspotent neuroprotective (95) and memory-enhancing effects(96). C83 undergoes further cleavage by γ-secretase, withthe generation of a small peptide, called p3, which seemsto be of no toxicological significance, since it is not foundin SPs. The identity of α-secretase has not been completelyelucidated. It is known that the enzyme is a member of theA Disintegrin And Metalloprotease (ADAM) family ofproteases and may be ADAM10, ADAM17/TACE (Tu-mour necrosis factor-Alpha-Converting Enzyme) (97), oreven ADAM9 (98). Studies on transgenic animals have re-cently identified ADAM10 as the putative α-secretase(99). Activation of α-secretase has regained interest as atherapeutic drug target in AD (100). Overexpression ofADAM10 in transgenic animals leads to a decrease in amy-loid pathology, whereas the transgenic expression of a cat-alytic inactive form of the enzyme resulted in an increasein amyloid pathology (99). The activation of α-secretasemay have the double advantage of not only precluding theformation of the neurotoxic Aβ peptide but also generat-ing the putatively neuroprotective sAPPα. Although neu-rons have a certain level of basal α-secretase activity,proteolysis by this enzyme can be increased pharmaco-logically with a variety of approaches (101).

One strategy may be to upregulate ADAM10 orADAM17/TACE α-secretase gene expression. Anotherapproach may be to use statins, retinoids or neuropeptidessuch as pituitary adenylate cyclase-activating polypep-

tide (PACAP), to stimulate α-secretase or protein kinaseC (PKC) activities (18). The mechanisms underlying thisapparent risk reduction of AD in people taking statins, asseen above, are poorly understood, but one hypothesis re-gards the ability of statins to increase sAPPα levels via α-secretase activation. Indeed, in vitro studies have shownthat lovastatin (102), atorvastatin (103), simvastatin (104)and rosuvastatin (105) stimulated sAPPα shedding fromhuman cell lines. The stimulating effects of simvastatin onsAPPα have been observed in the CSF of AD patients af-ter 1-year treatment with a dose of 20 mg/day but, un-fortunately, the study was uncontrolled (106). Whetherstatins act by altering the distribution of APP and the var-ious secretases between raft and non-raft regions of thecell membranes, or by altering the fluidity of the mem-brane, is not clear. Alternatively, the expression of APPand secretases may be modified by statins (107). It has al-so been proposed recently that the stimulating effects ofstatins on sAPPα is mediated, at least partly, by modu-lation of the isoprenoid pathway and Rho-associatedprotein kinase 1 (ROCK1) (104).

The activation of α-secretase is also controlled by theprotein phosphorylation signal transduction pathway ofPKC. Thus, enhancement of α-secretase activity can be ob-tained by direct stimulation of PKC or by activation of re-ceptors that work through PKC. Phorbol esters are directPKC activators and have been shown to significantly en-hance the secretion of sAPPα in vitro (108), reduce Aβ invitro (109) and in vivo (110) and prevent Aβ toxicity in rathippocampal neurons (111). Unfortunately, phorbol estersare tumor promoters and therefore novel PKC activa-tors may offer an alternative, but their safety for human useremains to be demonstrated. Benzolactam derivativeshave been shown to bind to PKC (112) and 8-(1-de-cynyl)benzolactam-enhanced sAPPα secretion in fibroblastsfrom AD patients (113). A 17-week intraperitoneal treat-ment of APP transgenic mice with 1 mg/kg of this com-pound produced a significant increase in brain sAPPαconcentrations and a decrease in brain Aβ40 levels (114).Bryostatin 1, a macrolide lactone isolated from Bugula ner-itina with promising anticancer activity, exhibited sub-nanomolar affinity for PKC (115) and, at subnanomolarconcentrations, dramatically enhanced sAPPα in fibroblastsfrom AD patients (114). Prolonged intraperitoneal treat-ment with bryostatin 1 (40 g/kg three times per week) inAPP+PS1 double transgenic mice significantly reducedbrain Aβ42 levels (114). The Blanchette Rockefeller Neu-rosciences Institute in the U.S. is sponsoring a Phase IIclinical trial not yet open for participant recruitment of asingle dose of bryostatin 1 in patients with mild to mod-erate AD (single one-hour intravenous infusion of 10 or15 µg/m2 of bryostatin 1) (116) (Table 1). A number ofneurotransmitters (glutamate, serotonin) and growth fac-tors (epidermal growth factor) also appear to stimulate α-secretase via PKC activation (107).






A physiological alternative could be to stimulate α-sec-retase activity via muscarinic receptors with selectiveM1 muscarinic agonists or ChEIs, which have clearlydemonstrated this capacity both in vitro and in vivo(117). The lowering effects of talsaclidine, a selectiveM1 agonist, on both Aβ42 and Aβ40 concentrations inthe CSF of AD patients were documented in two 4-

week, double-blind, placebo-controlled studies involving 24and 40 subjects, respectively (118, 119). The lowering ef-fects on total Aβ levels in the CSF of AD patients were al-so described for selective M1 muscarinic receptor agonistAF102B (120). Unfortunately, M1 selective agonistshave significant cholinergic-mediated side-effects, themost serious being syncope (121), which limit their clin-



Classes of drugs Compound Mechanisms of action Side-effects Status(company/institution)

Aβ: amyloid-β; APOE: apolipoprotein E; sAPPα: soluble amyloid precursor protein α-cleaved; GABA: γ-aminobutyric acid; CSF: cerebrospinal fluid; NSAID: non-steroidal anti-inflammatory drug; MPAC: metal-protein attenuating compound.

Table 1 - Potentially disease-modifying drugs in clinical testing for treatment of Alzheimer’s disease. Compounds aimed at interferingwith generation, deposition or clearance of β-amyloid (Aβ).

Passive immunotherapy

αα-Secretase Activators

ββ-Secretase Inhibitors

γγ-Secretase Inhibitors

Aββ AggregationInhibitors

Bapineuzumab (Elan/Wyeth)

Bryostatin 1(Blanchette RockefellerNeurosciences Institute)

EHT-0202(ExonHit)

CTS-21166 (ASP-1702)(CoMentis/Astellas)

LY450139Semagacestat(Eli-Lilly)

PBT-2(Prana Biotechnology Ltd)

Scyllo-cyclohexanehexol(AZD-10, ELND005)(Transition Therapeutics/Elan)

Prevention of Aβdeposition and promotionof Aβ clearance basedprimarily on eliciting ofhumoral response by several mechanisms, not mutually exclusive

Increase in brain sAPPαconcentrations anddecrease in brain Aβ40levels

Regulates GABA-A re-ceptors inducing amyloidprecursor protein produc-tion and reducing Aβplaque formation

Reduction ofAβ concentrations

Reduction of Aβ concentrations

MPAC; inhibition of Aβ-metal binding, i.e.precipitation of Aβ byzinc and its radicalizationby copper; reduction ofCSF Aβ levels; significantimprovement above base-line in performance onexecutive tests

Reduces accumulation of Aβ and amyloid betaplaques in the brain, aswell as reduce or elimi-nate learning deficits in aleading transgenic mousemodel of AD

Vasogenic edema, partic-ularly in carriers of APOEε4 allele

Myalgia, fatigue, nausea,headache, vomiting,anorexia, anemia andlymphopenia are com-monly reported side-effects

Well tolerated

No severe side-effectsreported

Gastrointestinal prob-lems; goblet cell hyper-plasia in intestinal epithe-lium; changes in immunesystem with decrease inlymphocytes in spleenand thymus; hair colorchanges; skin rashes;transient period of Aβreduction may be followed by elevated AβHeadache; dizziness; somnolence

Well tolerated

Exogenous monoclonalAβ antibody; completedPhase II clinical trial;Phase III in progress with800 patients (2007-2010)

Phase II clinical trialin progress

Phase II clinical trialin progress

Completed Phase Iclinical trial in 2008;Phase II clinical trialis planned

Completed Phase II clinicaltrial in 2007; Phase IIIclinical trial in progress

Completed Phase II clinicaltrial in 2007; Phase IIIclinical trial in progress

Phase II clinicaltrials in progress




ical use. Overexpression of the α-secretase ADAM10 oractivation of its promoter with retinoic acid may reach thesame objective (122). None of these alternatives havereached the clinic with the exception of muscarinic agonistswhich have already been extensively tested in AD pa-tients. Non-selectivity of these muscarinic agonists and se-vere side-effects due to non-selectivity have so far precludedfurther clinical development of these drugs. Other agentswhich can also increase the non-amyloidogenic cleavageof APP include 17-estradiol, bradykinin, copper, testos-terone, insulin, calmodulin, and Ginkgo biloba extracts(107). Indeed, at present, most of the available α-secretaseactivators are drugs intended for other pharmacological ac-tions, and this lack of specificity is their major limitation.

Nonetheless, promising preclinical results have re-cently been disclosed for EHT-0202. This agent is anovel α-secretase stimulator which specifically regulates γ-aminobutyric acid (GABA)-A receptors, inducing the pro-duction of APP and reducing Aβ plaque formation. EHT-0202 has demonstrated GABA-A-dependent neuropro-tective and procognitive effects in several in vitro and invivo AD and aging-related pharmacological models, in-cluding Aβ intoxication, scopolamine-induced amnesia andBarnes tests. In Phase I clinical trials, the compoundwas shown to be orally bioavailable, well tolerated, notsedative or emetic, and devoid of adverse effects. APhase II study of EHT-0202 in AD patients is currentlyongoing (123). Regulatory approval to initiate a Phase IIclinical trial of EHT-0202 in patients with AD has recentlybeen granted. The multicenter, double-blind trial will ran-domize 135 patients and has the primary end-point ofsafety and tolerability of EHT-0202 when administeredorally for a 3-month period in conjunction with an acetyl-cholinesterase inhibitor. The trial will also provide pre-liminary data on clinical efficacy, particularly regarding itseffects on cognition and behavior (Table 1).

THE ββ-AMYLOID TARGET: ββ-SECRETASE INHIBITORSIn 1999, β-Amyloid Cleaving Enzyme-1 (BACE-1,

memapsin-2, Asp-2) was identified as a protease with β-secretase activity (124). BACE-1 is a 501-amino acidtype-1 integral membrane protein, with aspartic residuesat positions 93 and 289, which are thought to be re-sponsible for proteolytic activity (125). The first step in theprocess of cleavage of membrane-bound APP is due toBACE-1, which forms soluble APP (sAPPα) and a 12-kDapeptide, C99 (Fig. 1) (125). In knockout animals, lack ofBACE-1 abolishes Aβ generation in the absence of furtherabnormalities, suggesting that blockade of BACE-1 mayreduce progression of amyloid pathology without majoradverse effects (126). For these reasons, BACE-1 and itsclosely related homolog, BACE-2 (memapsin-1, Asp-1),which has 79% sequence identity with BACE-1 and alsocleaves APP at the β-secretase site, have become impor-

tant drug targets (127, 128). The biological functions ofBACE-2 have yet to be clarified, although it is known thatit also cleaves APP within the Aβ domain (127). β-Secre-tase inhibitors are not easy to find, because BACE-1 hasa large catalytic side that may not avidly bind smallmolecules (129). The search for β-secretase inhibitorsshould be greatly facilitated by its resemblance to other as-partic protease targets, particularly renin and HIV protease,and the ability to use structure-based design (107).

Several peptide-based β-secretase inhibitors havebeen described to date (130). They have centered onpeptide-derived structures, which act as transition-statemimetics based on amino acid sequences at the cleavagesite of APP by BACE-1 (131-133). Descriptions of non-peptidic β-secretase inhibitors not derived from the“transition-state mimetic” approach are scarce and aredisclosed primarily in patent applications (107). Therehave also been sporadic reports of naturally occurringnon-competitive inhibitors, such as hispidin (134) orcatechins (135), but their micromolar potency and poorspecificity versus other proteases limit their pharmaco-logical development as such.

Although the first-generation β-secretase inhibitorsOM99-1 and OM99-2 were very potent, they were es-sentially peptidic in nature, lacking drug-like characteristics(131). Modification of OM99-2 resulted in the identifica-tion of smaller inhibitors (molecular weight in the range of700 Da) with maintained nanomolar potency (132). Oth-er modifications of OM99-2 recently led to functionalized15- or 16-residue cycloamide-urethane derivatives (136).Recently, the crystal structure of a potent 13-residue in-hibitor called OM03-4, bound to β-secretase, revealedthe existence of three other subsites (S5–S7) and the in-teraction of a tryptophan with the S6 subsite of the enzyme(137). A β-secretase inhibitor designed to penetrate theBBB has been shown in proof-of-concept experiments toreduce both Aβ40 and Aβ42 in the brain of transgenic ADmice Tg2576 (138). Lastly, reduction of brain Aβ in ADmice as a result of BACE-1 immunization, in which anti-bodies of BACE-1 are the inhibitors, has been shown toimprove cognitive performance in the animals (139), thussubstantiating the therapeutic target.

In conclusion, crystal structure-based design cycleshave allowed much progress to be made in the develop-ment of drug-like β-secretase inhibitors (129). The currentgeneration of inhibitors is sufficiently small to penetrate cellmembranes and the BBB, yet they retain potency for theinhibition of Aβ production in vitro and in vivo. Re-cently developed GRL-8234 has shown both excellent en-zyme inhibitory activity and cellular inhibitory potency inChinese hamster ovary cells (140). It also has very im-pressive in vivo properties. Intraperitoneal administrationof GRL-8234 to Tg2576 mice resulted in a 65% re-duction of Aβ40 production at 3 h, after a single dose of8 mg/kg (140). Therefore, β-secretase inhibition ap-






pears to be another viable target (141). The possibility thatsome β-secretase inhibitors may be drug candidates is en-couraged by the recent announcement that the U.S.biotech company CoMentis has been reported to havecompleted a Phase I study of its orally bioavailable smallmolecule CTS-21166 (ASP-1702), described by Co-Mentis as a highly potent, highly selective and effica-cious brain-penetrating β-secretase inhibitor (142). ThePhase I trial in healthy volunteers was designed as adose escalation study to assess the safety, tolerabilityand pharmacokinetics and pharmacodynamics of CTS-21166 following intravenous administration. Forty-eightsubjects received one of six different doses or placebo. Thestudy measured levels of CTS-21166 and plasma Aβ. Sin-gle-dose administration of CTS-21166 produced a greaterthan 60% reduction in plasma Aβ, measured either byarea-under-curve (AUC) over 24 hours or as maximal re-duction relative to predose levels. The top doses of CTS-21166 further demonstrated a sustained reduction inAUC, which exceeded 40% over 72 hours. Because ofthe urgent need for AD treatment, Phase II studies forCTS-21166 are planned for AD patients in 2008 (143)(Table 1). In view of the significant overall progress in thisfield, it is likely that many other β-inhibitors will bebrought to clinical trials soon.

THE ββ-AMYLOID TARGET: γγ-SECRETASEINHIBITORS AND MODULATORSγ-Secretase InhibitorsThe last metabolic step that generates Aβ involves

enzymatic intramembrane cleavage of APP by a highmolecular weight complex called γ-secretase (Fig. 1). γ-Secretase is formed by at least four proteins: presenilin(PS), nicastrin, anterior pharynx (Aph-1) and presenilin en-hancer 2 (Pen-2) (108). Presenilins are of exceptionalpathophysiological importance, since more than 150autosomal dominant point mutations are known in theseproteins, all of which cause aggressive early-onset AD.These mutations result in increased production of Aβ1-42,the highly self-aggregating and neurotoxic form of Aβ.Thus, inhibition of the catalytic unit (presenilin) of the γ-secretase enzymatic complex appears to be a logicalstrategy to contrast Aβ accumulation in the brain of ADpatients (144).

The first report of in vivo inhibition of brain Aβ withDAPT, a peptidomimetic γ-secretase inhibitor, was pub-lished in 2001 (145). Several other non-peptidic, orallyavailable, γ-secretase inhibitors have been synthesized(146). Historically, the first γ-secretase inhibitor thatreached the clinic was a compound synthesized by Bristol-Myers Squibb (BMS-299897) (144). Human testing ofBMS-299897 started in 2001, but the clinical data havenever been fully described. The long-lasting lack of infor-mation on its clinical development may indicate that it hasbeen abandoned. At least six other γ-secretase inhibitors

reached the clinic: LY-450139 (semagacestat) (147), MK-0752 (148), E2012 (144), BMS-708163 (149), PF-3084014 (150) and GSI-953 (begacestat) (151).

LY-450139 is the best documented γ-secretase in-hibitor that has reached clinical testing and for which clin-ical studies have been fully published (Table 1). Most of theinformation on the other compounds derives fromcongress communications. LY-450139 is a γ-secretase in-hibitor in development at Eli-Lilly. LY-450139 is only 3-fold selective in inhibiting APP and Notch cleavage (APPIC50 = 15 nM, Notch EC50 = 49 nM) (149). In experi-mental animals, the effects of LY-450139 on Aβ levels inbrain, CSF and plasma were well characterized in trans-genic mice (152), non-transgenic mice (153), guineapigs (154), and dogs (155). However, the drug failed toshow a statistically significant effect on brain plaque de-position in chronic studies in transgenic mice expressingmutated human APPV717F (PDAPP mice) (156). Moreimportantly, no data are available on the cognitive or be-havioral effects of the drug in animal models of AD.

In man, the first Phase I study of this drug evaluated thesafety, tolerability and biomarker responses to singleoral doses of 60, 100 or 140 mg in 31 healthy male andfemale volunteers (40 years and older) (157). No clinicallysignificant adverse events or laboratory changes wereobserved. A dose-dependent decrease in plasma Aβ1-40 levels was also demonstrated, with maximum inhibition(-73%) 6 h after administration of the 140-mg dose. A re-bound effect on plasma Aβ1-40 levels was observed at 8-12 hours after administration and lasted for at least 24 h.CSF concentrations of Aβ were unchanged 4 hours afteradministration. In a second Phase I study, LY-450139 wasadministered to healthy men and women (45 years andolder) for up to 14 days at doses of 5, 20, 40 and 50 mgonce daily (158). The 50-mg dose caused a maximal40% reduction in total plasma Aβ, which returned to base-line within 8 hours. After returning to baseline, plasma Aβlevels increased to about 300% of baseline values at 15hours before slowly declining again. At lower doses,smaller and shorter decreases in plasma Aβ were ob-served, although the subsequent plasma Aβ increaseswere similar. No significant changes in CSF Aβ levels weredetected (158). The extent to which Aβ reduction inplasma or CSF needs to be achieved with a γ-secretase in-hibitor in order to modify the rate of AD progression re-mains largely unknown. In this Phase I study, it is unlikelythat the extent of this effect on peripheral Aβ and the ab-sence of a measurable inhibition in CSF Aβ will be able toreveal a beneficial cognitive effect in patients. Two subjectsin the 50-mg dose group developed possibly drug-relatedadverse events and discontinued treatment. The first sub-ject had significant increases in serum amylase and lipase,and complained of moderate abdominal pain. The othersubject reported diarrhea which was positive for occultblood. A potential cause of toxicity of γ-secretase in-






hibitors is their inhibition of Notch cleavage, which hasbeen associated with goblet cell hyperplasia in intestinalepithelium and changes in the immune system, with de-creased lymphocytes in the spleen and thymus (144).Much more data must be collected from human studies todistinguish between well-tolerated and toxic doses of γ-sec-retase inhibitors in chronic treatment of AD patients.Recently, hair color changes in animals have been linkedto inhibition of Notch processing (159). The use of aNotch-related toxicity biomarker such as adipsin may beuseful for early detection of potential toxicity (160).

LY-450139 has also been evaluated in AD patients. Inthe first randomized, placebo-controlled trial, 70 patientsreceived the drug for 6 weeks (30 mg once a day for 1week, followed by 40 mg once a day for 5 weeks) (161).Six patients taking LY-450139 reported diarrhea. A 76-year-old man on LY-450139 had gastrointestinal bleedingassociated with a Barrett esophagus, a clinical conditioncharacterized by the conversion of normal squamous cellsinto abnormal specialized columnar cells. Approximately4 months after discontinuing treatment, the patient de-veloped endocarditis and approximately 1 month there-after, died. In the LY-450139-treated group, circulatingCD69, T lymphocytes, eosinophils and serum concen-trations of potassium and inorganic phosphorus showedstatistically significant changes, although these findingswere reported as “clinically irrelevant”. The plasma Aβ1-40 concentrations of patients taking the drug decreased sig-nificantly by 38% compared with baseline values. Aβ1-40concentrations in CSF did not decrease significantly. An-other study evaluated the safety, tolerability, and Aβ re-sponse to LY-450139 in 51 AD patients treated for 14weeks (162). Patients were randomized to receive placebo(n=15) or LY-450139 (n=36). Patients on LY-450139 re-ceived 60 mg/day for 2 weeks, then 100 mg/day for 6weeks, and then either 100 or 140 mg/day for 6 moreweeks. Forty-three patients completed the study. Therewere 7 cases of skin rashes and 3 reports of hair colorchange in the treated groups. There were 3 adverseevent-related discontinuations, including 1 transient bow-el obstruction. Compared to placebo, Aβ1-40 plasmaconcentrations were reduced by 58% in the 100-mggroup and 65% in the 140-mg group. No significant re-duction was seen in CSF Aβ levels. No differences wereseen in cognitive or functional measures between placeboand LY-450139-treated patients (162). Therefore, nodata have been reported on the effects of LY-450139 onAβ1-42 levels. This is a clear limitation, since Aβ1-42 is theneurotoxic Aβ species that has major pathological rele-vance. Recently, the results of a study on the effects of LY-450139 on Aβ synthesis and clearance in CSF of AD pa-tients were reported at a conference (163). CSF was col-lected hourly with a lumbar catheter. Fractional synthesisand clearance rates of CSF Aβ showed significant effectson newly synthesized Aβ with LY-450139 doses of 140

and 280 mg. A maximum decrease in Aβ concentrationwas seen 9 hours after administration (163).

In April 2008, Eli Lilly initiated its first Phase III clini-cal trial of LY-450139 in mild to moderate AD patients.LY-450139 is being tested to determine if it can slow dis-ease progression. The randomized, double-blind, placebo-controlled trial, called IDENTITY will be conducted in theU.S. and 21 other countries and will evaluate 1500 pa-tients for 21 months. An open-label extension will beavailable to all participants completing the study. Pa-tients who are taking currently available symptomaticdrugs for AD will be permitted to continue such treatmentsfor the duration of the study. The study incorporates a“randomized delayed start” design with subjects initially as-signed to the placebo arm and later treated with LY-450139 to evaluate whether the drug has effects ondisease progression. Both study subjects and investigatorswill be blinded to the exact timing of this delayed start drugadministration (144).

γ-Secretase ModulatorsAs seen above, although γ-secretase has in many ways

been an attractive target for AD therapeutics, interferencewith Notch processing and signaling may lead to toxicitythat precludes clinical use of the inhibitors of this protease(164). However, compounds that can modulate the en-zyme to alter or block Aβ production with little or no effecton Notch would bypass this potential block to therapeutics.Recent studies suggest that the protease complex containsallosteric binding sites that can alter substrate selectivity andthe sites of substrate proteolysis. A subset of NSAIDs(e.g., ibuprofen, indomethacin and sulindac sulfide) can re-duce the production of the highly aggregation-proneAβ42 peptide by indirectly modulating γ-secretase activi-ty (shifting Aβ1-42 to Aβ1-38 production) and binding toAPP (165), a pharmacological property independent of in-hibition of cyclooxygenase (166). In transgenic mousemodels of CNS amyloid deposition, treatment with theseagents significantly reduced amyloid accumulation (167).But in human beings, the toxicity of high doses of suchNSAIDs limits the feasibility of this approach.

However, among γ-secretase modulators, in a mousemodel of AD, tarenflurbil (MPC-7869; former non-pro-prietary name R-flurbiprofen, FlurizanTM) reduced brainconcentrations of Aβ1-42, and chronic dosing in thismodel prevented defects in learning and memory (167,168). The concentrations of tarenflurbil required to produceAβ1-42-lowering effects in vitro and in vivo can beachieved in human beings at doses that have been well tol-erated (169). A Phase II trial of tarenflurbil was designed toshow slowing of cognitive and functional decline in mild tomoderate AD, with enrolment of 210 patients in a 1-yeartrial (170). The primary analysis failed: there was nooverall effect on primary outcomes. But planned analysesdid suggest that the drug had an influence on outcome






measures: there was a significant interactive effect oftreatment and baseline cognitive function on change in out-comes (171). A separate analysis with patients grouped ac-cording to whether they had mild or moderate cognitiveimpairment at baseline showed the favorable effect oftreatment on activities of daily living (Alzheimer’s Dis-ease Cooperative Study activities of daily living scale,ADCS-ADL) and global functions (clinical dementia ratingsum of boxes, CDR-SB) in the subgroup of patients withmild AD. The results of a 12-month blinded extension tothe trial suggested the long-term tolerability, and perhapsefficacy, of 800 mg tarenflurbil twice per day in patientswith mild AD (170). However, after these encouraging re-sults, a large 18-month Phase III trial with 1,684 mild ADpatients turned out to be completely negative (172). Thereasons for this major failure are unclear, but preclinicalstudies have shown that tarenflurbil has low potency in in-hibiting the secretion of Aβ1-42 in vitro as well as apoor CNS penetration in rodents with inadequate brainconcentrations achieved in vivo (173). The promisingpharmacological results obtained with tarenflurbil by theMayo Clinic group (owner of the patent) after short-termadministration in transgenic mice (167) have never beenconfirmed by other groups (174). The neuropathologicaland behavioral effects of tarenflurbil after chronic admin-istration in transgenic mice are inconsistent and ques-tionable from a methodological point of view (168).

From a clinical point of view, accurate examination ofthe results of the Phase II study (170) reveals that the ap-parently positive effects of the drug in mildly affected pa-tients at a dose of 800 mg twice a day on the ADCS-ADLand CDR-SB scales were probably due to an anoma-lous deterioration rate observed in patients treated withplacebo, rather than to the drug itself (173). Interest-ingly, for the cognitive measure ADAS-Cog (cognitive sub-scale) which showed consistent mean decays in mild andmoderate placebo patients, no significant effects of taren-flurbil were observed. Accordingly, there were no ef-fects at all of tarenflurbil on MMSE, another measure ofcognition. Lastly, moderately affected patients who re-ceived the same dose of tarenflurbil (800 mg twice aday) had highly significant greater clinical deteriorationcompared to placebo on the CDR-SB scale. The detri-mental effects of tarenflurbil on the global clinical status(CDR-SB) of the AD patients were again observed inthe large Phase III study carried out in mild patients, inwhich patients receiving 800 mg twice a day had a sig-nificantly higher deterioration than placebo on the CDR-SB scale at the end of the 18-month treatment period(p=0.004) (172). One explanation for the surprisingly neg-ative effects of tarenflurbil on global status of the AD pa-tients is that the compound, although it is the R enan-tiomer of flurbiprofen, still maintains significant anti-COX activity at the high doses used in the study. The oc-currence of several gastro-intestinal adverse events in

the AD patients treated with tarenflurbil during the PhaseIII study, including 8 cases of peptic ulcer (vs only one inthe placebo group) confirms this hypothesis. Similardetrimental effects compared with placebo have alreadybeen observed in the past in other large, long-term, con-trolled studies with anti-inflammatory drugs, includingprednisone in AD patients (175), rofecoxib in patients withMCI (176), celecoxib and naproxen in elderly subjects atrisk of developing AD (177). Thus, it is possible thatthe negative results obtained by tarenflurbil in mild AD pa-tients in the recently completed Phase III study are due toits poor pharmacological profile and its residual anti-in-flammatory activity (173).

THE ββ-AMYLOID TARGET: Aββ AGGREGATION INHIBITORSIn the last few years, a large number of compounds in-

terfering with Aβ aggregation (Aβ aggregation inhibitors)have been discovered (107). Their common theoretical ad-vantage is that they do not interfere with physiologicalAPP metabolism. In vivo animal testing of a limited sub-set of these compounds has produced encouraging results(107). The Metals Hypothesis of AD, based upon obser-vations of the precipitation of Aβ by zinc and its radical-ization by copper, both metals markedly enriched in SPs(178, 179), led to the discovery of small-molecule chela-tors that perturb Aβ-metal binding. Interestingly, clio-quinol (PBT-1, Prana Biotechnology Ltd), an antibiotic,provided the first evidence that a compound interferingwith Aβ aggregation may produce measurable biologicaleffects in AD patients. Clioquinol partially dissolves SPs invitro and prevents plaque deposition in transgenic mice(180). Nonetheless, clinical trials were halted due to an im-purity, but some evidence was provided of reduced plas-ma Aβ and improved cognition (181, 182). To differen-tiate these compounds from high-affinity, medicinal met-al chelators, they were termed metal-protein attenuatingcompounds (MPACs). The second-generation 8-OHquinoline derivative of clioquinol, PBT-2, which hasgreater BBB penetration, recently completed its firstdouble-blind, placebo-controlled, 12-week Phase II clini-cal trial in 78 subjects for the treatment of early AD(Table 1). Results revealed that the drug was safe and welltolerated at 50 mg and 250 mg daily doses, CSF Aβ lev-els were significantly lowered by the 250 mg dose, andthere was a significant improvement above baseline in per-formance on executive tests of the NeuropsychologicalTest Battery (NTB) at 12 weeks (183). These results ap-pear to be the basis for proceeding with further Phase IIbor Phase III testing of what may be a disease-modifyingdrug based upon the Metal Hypothesis (178). Finally, an-other promising Aβ aggregation inhibitor, scyllo-cyclo-hexanehexol (AZD-10, ELND005, scyllo-inositol) wasidentified after the observation that phosphatidylinositolstimulates Aβ aggregation (184). Scyllo-cyclohexane-






hexol binds an Aβ oligomer to inhibit further aggregationand toxicity (185) and reduces plaque deposition andcognitive deficits in a transgenic mouse model (186). Atpresent, scyllo-inositol is in Phase II clinical trials (estimatedenrollment: 340 patients; estimated study completiondate: May 2010) (Transition Therapeutics/Elan) (187). Avariety of other molecules that prevent Aβ aggregationhave also been identified (188). One potential problem foraggregation inhibitors is a shift in the equilibrium be-tween the less toxic aggregated forms to more toxic sol-uble intermediates, such as protofibrils (189) (Table 1).

Agents that mimic glycosaminoglycan structure or in-terfere with binding of heparan sulfate to Aβ have beenstudied with the aim of inhibiting Aβ deposition or toxicity.Tramiprosate (3-aminopropanesulfonic acid, NC-531,AlzhemedTM) has been claimed to mimic the anionicproperties of glycosaminoglycans and to inhibit Aβ fibrilformation and deposition in vitro significantly (190). Intransgenic mice expressing the human APP Swedishmutation (TgCRND8), chronic administration oftramiprosate (100 mg/kg for 8 weeks) induced a 61% re-duction in Aβ plasma levels and a 30% reduction in thenumber and size of brain Aβ plaques (191). The safetyand tolerability of tramiprosate in AD patients have beenevaluated in a double-blind, placebo-controlled study in-volving 58 subjects (192). Again the most frequent adverseevents were nausea and vomiting, and the drug was de-tected in the CSF of patients. A large Phase III study last-ing 18 months was completely negative (193).

THE TAU PROTEIN TARGET: GLYCOGENSYNTHASE KINASE 3 (GSK-3) INHIBITORSThe microtubule-associated protein tau is the principal

component of the paired helical filaments (PHF) thatcomprise NFTs, the other pathological hallmark of AD,and also of a number of neurodegenerative disorders, suchas FTD. Tau is a 50- to 75-kDa protein with six differentsplice variants (194). Tau binds and stabilizes micro-tubules, while hyperphosphorylated tau from AD brain dis-rupts microtubule structure (195). This process severelyimpairs axonal transport. The presence of NFTs in ADand their correlation with cognitive status suggests that animportant role is played by abnormal tau in dementia(194). Hyperphosphorylation of tau presents a second andprobably critical target for AD therapy (18).The twomost characteristic changes of tau in AD are phospho-rylation and aggregation. A number of tau-focused targetsfor treatment of tauopathies in general and in AD areslowly emerging, following the recent development oftransgenic animal models expressing tau abnormalities andtangles (18). Tau-based therapeutics include kinase in-hibitors, microtubule stabilizers and catabolism inducers(184). Recent evidence implicates the protein kinaseglycogen synthase kinase 3 (GSK-3) in the regulation ofboth tau and amyloid pathological processes (196, 197).

GSK-3 activity may also promote cell death, conversely,inhibition of GSK-3 has been associated with increased cellsurvival in a variety of cytotoxic conditions (198). GSK-3is also involved in the mechanisms underlying learning andmemory (199) and in local cerebral inflammatory re-sponses (200). Although direct evidence that GSK-3plays a central role in AD (201) is still limited, it is knownthat GSK-3 expression is upregulated in the hippocampusof AD patients (202) and in circulating peripheral lym-phocytes (203). All these data, taken together, clearlypoint GSK-3 as one of the most promising targets for de-veloping innovative drugs to attack AD pathogenesis onmultiple fronts simultaneously (204), thus providing anovel avenue for therapeutic intervention in this cognitivedisorder (205).

At present, several chemically diverse families haveemerged as GSK-3 inhibitors (206), including peptides(207), metal ions (208) and diverse small heterocycles(209). Lithium, a drug used in acute bipolar depression, isa small monovalent cation discovered in 1996 as the firstdirect, reversible GSK-3 inhibitor. Lithium reduces GSK-3 activity in two ways: both directly and by increasing theinhibitory phosphorylation of GSK-3 (210). As a direct in-hibitor, lithium competes with magnesium ions for bind-ing to GSK-3 but not to substrate or ATP (211). Indirectly,this inhibitor of GSK-3 increases the inhibitory N-terminalserine phosphorylation of GSK-3 which inactivates en-zyme function. This dual mechanism explains the dis-crepancies between the administration of lithium and di-rect inhibition of GSK-3. The effects of lithium requirechronic treatment (212) whereas the direct inhibition ofGSK-3 is a fast reaction. The first group of GSK-3 in-hibitors, paullones 1 (213), indirubines 2 (214) and bisin-dol-maleimides 3 (215), were discovered in screeningprograms designed to find inhibitory activity in GSK-3 incompounds previously reported to have different bio-logical properties. The disadvantages of the first group ofGSK-3 inhibitors were their lack of selectivity and com-petition with ATP for its binding site. Apart from GSK-3inhibition, these compounds can inhibit other kinasessuch as CDK1 or CDK2. Two kinds of molecules, thia-diazolidinones (TDZD) and halomethylketones (HMK),were described as the first non-ATP competitive in-hibitors for GSK-3 (216). They do not inhibit different ki-nases like PKA, PKC, CK-2 and CDK1/cyclin B, andstudies in primary culture neuronal cells showed a decreaseof tau phosphorylation in the presence of these inhibitors(217, 218). In the conditional tet/GSK-3 model on dou-ble transgenic mice that over-expressed GSK-3 specificallyin the hippocampus (219), it was demonstrated thatchronic oral treatment with TDZDs decreases tau hy-perphosphorylation in a dose- and time-dependent way(220). In addition, in a double transgenic model thatover-expressed hAPP (with London mutation) and PS-1,chronic oral treatment for 3 weeks decreased brain-soluble






Aβ1-40 and Aβ1-42 levels in a dose-dependent wayand oral treatment for two months reduced the amyloidplaque load in the cortex and hippocampus (221). Theseresults, together with some other in vivo neuroprotectiveproperties found in this family of compounds (222), con-firm the potential of TDZDs as new therapeutic agents toinhibit GSK-3 and tau phosphorylation in AD. Various re-views of the discovery of GSK-3 inhibitor leads have re-cently been published (223, 224) focusing both on thechemical structures of the new drugs and on potential ther-apeutic benefits in several diseases mediated by GSK-3,with special attention to their potential application inAD. Two GSK-3 inhibitors, AZD1080 and NP031112,were in clinical trials for AD (225), but on January 2008the halt of AZD1080 development was announced, leav-ing only NP031112 (NP-12), a drug which belongs to theTDZD family from Neuropharma, a Spanish biophar-maceutical company now named Noscira.

Noscira has started a Phase II clinical study on NP-12 inpatients with AD and other tauopathies (226). In three pre-vious Phase I studies involving more than 160 healthy sub-jects of young and old age, oral administration of NP-12was generally well tolerated (226). In animal models of AD,it was shown that NP-12 improves cognitive performanceand reduces amyloid deposits, hyperphosphorylation andtau aggregation, neuroinflammation and, most impor-tantly, neuron loss. Specifically, the effects of chronicadministration of NP-12 have been studied in three mod-els of transgenic mice that replicate the typical patholog-ical features of AD: a double transgenic conditionalTet/GSK-3 that over-expresses GSK-3 and produces hy-perphosphorylated tau protein deposits in the brain, a dou-ble transgenic APPV717I x PS1A246E, producing Aβ andbrain SPs, and a double-transgenic APPK670N-M671L xTauVLW, producing fibrillary deposits of hyperphospho-rylated tau, SPs, gliosis and neuron loss. Chronic oraltreatment with NP-12 reduced reactive inflammatory glio-sis and tau phosphorylation in Tet/GSK-3β transgenicmice, brain amyloid burden and plaque volume in AP-PV717IxPS1A246E transgenic mice and amyloid de-posit, tau phosphorylation, reactive gliosis and cell deathin APPK670N-M671LxTauVLW transgenic mice. Cog-nitive improvement was also observed in AP-PV717IxPS1A246E and APPK670NM671LxTauVLWmice. NP-12 improves all major neurodegenerative pa-rameters of AD in animal models, particularly neuronloss, which is the ultimate cause of the clinical profile ofprogressive and widespread deterioration (226).

Recently, at the International Conference onAlzheimer’s Disease (ICAD) 2008, the results of a PhaseII study on methylthioninium chloride (RemberTM, TauRxTherapeutics) were released (227). This drug dissolvesPHF isolated from AD brain, and prevents tau aggregationin cell models, showing efficacy in tau transgenic animalmodels, reversing cognitive and other behavioral defects,

and reversing tau pathology in the brain. In this trial, 321mild-to-moderate AD patients received 30, 60 or 100 mg(three times per day) or placebo for 24 weeks. Participantswere not taking other AD drugs. At the end of 24 weeks,patients with moderate AD in the 60-mg group declined5.4 points less on the ADAS-cognitive subscale (ADAS-Cog) than those with moderate AD taking the placebo. Inpatients taking the 100-mg dose, there was no effect andthis was ascribed to an interaction between the drug atthat concentration and the capsule preventing the drugfrom being absorbed. Results from the 100-mg groupwere then added to those of the placebo group. At 24weeks, there were no differences in the ADAS-Cogscores between those treated with RemberTM and thosegiven placebo in the group with mild AD (227). Thiswas ascribed to the fact that the test scores of the place-bo group did not decline in 24 weeks. The study was ex-tended to 50 weeks, at which point results were significantin both mild and moderate groups at the 60-mg dose(227). Looking at the mild and moderate groups togeth-er, those taking the treatment did not show a signifi-cant decline in their performance on the ADAS-Cog,whereas those on placebo declined by 6.8 points. A fol-low-up of those on the 60-mg dose at 84 weeks showedno significant decline in their ADAS-Cog performance.Because there was no placebo group after 50 weeks, it isunclear how this would compare with no treatment (227).Some of the participants underwent brain imaging(SPECT) during and/or after the trial and reduced bloodflow in certain brain regions associated with AD wasobserved in the placebo group, but not in those receivingtreatment (228). SPECT tests were confirmed by FDG-PET tests measuring reduced glucose use in the brain(229). The safety of RemberTM appeared good, but someparticipants experienced diarrhea. TauRx plans to startPhase III trials of the drug in 2009.

CONCLUSIONSIn the last 10 years, the neurotransmitter-focused ap-

proach has resulted in two classes of drugs, ChEIs and me-mantine, which reduce the severity of cognitive symptoms,improve functionality and attenuate behavior disturbancesin patients with mild to severe AD, but which do not showreal disease-modifying effects. Today, the neuropatho-logical hallmarks of the disease (SPs and NFTs) are driv-ing the efforts for identifying new therapeutic approach-es able to modify the natural history of the disease.Among Aβ-based therapies, the most revolutionary ap-proach is represented by active and passive vaccines,which have been shown to accelerate Aβ clearance fromthe brain of AD patients and are now under extensive clin-ical testing with bapineuzumab monoclonal antibodies.The most biologically attractive Aβ-based approach isinhibition of β-secretase, but identification of orally ab-sorbed and brain penetrant inhibitors is technically difficult.






Another target of Aβ-based therapy is γ-secretase whichalthough an unusual proteolytic complex turns out to bedrugable, and several potent inhibitors have been identi-fied. The most advanced γ-secretase inhibitor is LY-450139 (semagacestat), under investigation in two largelong-term Phase III studies. Unfortunately, after encour-aging Phase II findings, tarenflurbil, a compound be-lieved to modulate the activity of γ-secretase, producednegative results in the largest ever study in AD patients.Aβ aggregation inhibitors are also being actively pur-sued, and PBT-2 has recently completed a Phase II study,showing lowering effects on Aβ levels in CSF and im-proved performance on executive tests. Another inter-esting Aβ aggregation inhibitor, ELND005 (AZD-103),which has received fast-track designation from the FDA,has recently completed the enrolment of a 18-monthPhase II study. Protein tau-based therapeutics are main-ly represented by GSK-3 inhibitors and tau aggregation in-hibitors. A recent Phase II study on methylthioninium chlo-ride has raised many hopes. Thus, several new potentiallydisease-modifying therapeutic approaches have beenidentified and are being tested in AD patients. However,the way forward appears to be a long process. Thecholinergic hypothesis was generated in 1976 (230). Itsinitial clinical validation was achieved 17 years later withthe cholinesterase inhibitor tacrine (231), and that of asafe and effective cholinergic agent, donepezil, was ob-tained only after 20 years (232). When we recall that theamyloid cascade hypothesis was proposed in 1991 (68),its initial clinical validation, with these promising Aβ-based therapeutics, has again been reached in 17 years,i.e., in 2008. We may need to wait until 2011 for an ef-fective and safe agent able to slow down or to arrest thedevastating disease that is AD.

ACKNOWLEDGEMENTThis work was supported by the Italian Longitudinal Study on Aging

(ILSA) (Italian Research Council - CNR-Targeted Project on Ageing -Grants 9400419PF40 and 95973PF40) and Ministero della Salute, IR-CCS Research Program 2006-2008, Line 2: “Malattie di rilevanza so-ciale”. Conflicts of interest: No Disclosures to Report

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Beyond the neurotransmitter-focused approach in treating Alzheimer’s Disease: drugs targeting...

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