Optimization and biological evaluation of imidazopyridine derivatives as a novel scaffold for γ-secretase modulators with oral efficacy against cognitive deficits in Alzheimer's disease model mice.

Gamma-secretase modulators (GSMs) selectively lower amyloid-ß42 (Aß42) and are therefore potential disease-modifying drugs for Alzheimer's disease (AD). Here, we report the discovery of imidazopyridine derivatives as GSMs with oral activity on not only Aß42 levels but also cognitive function. Structural optimization of the biphenyl group and pyridine-2-amide moiety of compound 1a greatly improved GSM activity and rat microsomal stability, respectively. 5-{8-[(3,4'-Difluoro[1,1'-biphenyl]-4-yl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-N-methylpyridine-2-carboxamide (1o) showed high in vitro potency and brain exposure, induced a robust reduction in brain Aß42 levels, and exhibited undetectable inhibition of cytochrome p450 enzymes. Moreover, compound 1o showed excellent efficacy against cognitive deficits in AD model mice. These findings suggest that compound 1o is a promising candidate for AD therapeutics.

Discovery of N-ethylpyridine-2-carboxamide derivatives as a novel scaffold for orally active γ-secretase modulators.

Gamma-secretase modulators (GSMs) are promising disease-modifying drugs for Alzheimer's disease because they can selectively decrease pathogenic amyloid-ß42 (Aß42) levels. Here we report the discovery of orally active N-ethylpyridine-2-carboxamide derivatives as GSMs. The isoindolinone moiety of 5-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-2-ethyl-2,3-dihydro-1H-isoindol-1-one hydrogen chloride (1a) was replaced with a picolinamide moiety. Optimization of the benzyl group significantly improved GSM activity and mouse microsomal stability. 5-{8-[([1,1'-Biphenyl]-4-yl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-N-ethylpyridine-2-carboxamide hydrogen chloride (1v) potently reduced Aß42 levels with an IC50 value of 0.091 µM in cultured cells without inhibiting CYP3A4. Moreover, 1v demonstrated a sustained pharmacokinetic profile and significantly reduced brain Aß42 levels in mice.

Discovery of novel scaffolds for γ-secretase modulators without an arylimidazole moiety.

Gamma-secretase modulators (GSMs) selectively inhibit the production of amyloid-ß 42 (Aß42) and may therefore be useful in the management of Alzheimer's disease. Most heterocyclic GSMs that are not derived from nonsteroidal anti-inflammatory drugs contain an arylimidazole moiety that potentially inhibits cytochrome P450 (CYP) activity. Here, we discovered imidazopyridine derivatives that represent a new class of scaffold for GSMs, which do not have a strongly basic end group such as arylimidazole. High-throughput screening identified 2-methyl-8-[(2-methylbenzyl)oxy]-3-(pyridin-4-yl)imidazo[1,2-a]pyridine (3a), which inhibited the cellular production of Aß42 (IC50 = 7.1 µM) without changing total production of Aß. Structural optimization of this series of compounds identified 5-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-2-ethylisoindolin-1-one (3m) as a potent inhibitor of Aß42 (IC50 = 0.39 µM) but not CYP3A4. Further, 3m demonstrated a sustained pharmacokinetic profile in mice and sufficiently penetrated the brain.

Intravenous transplantation of bone marrow-derived mononuclear cells prevents memory impairment in transgenic mouse models of Alzheimer's disease.

Stem cell transplantation therapy is currently in clinical trials for the treatment of ischemic stroke, and several beneficial aspects have been reported. Similarly, in Alzheimer's disease (AD), stem cell therapy is expected to provide an efficient therapeutic approach. Indeed, the intracerebral transplantation of stem cells reduced amyloid-ß (Aß) deposition and rescued memory deficits in AD model mice. Here, we show that intravenous transplantation of bone marrow-derived mononuclear cells (BMMCs) improves cognitive function in two different AD mouse models, DAL and APP mice, and prevents neurodegeneration. GFP-positive BMMCs were isolated from tibiae and femurs of 4-week-old mice and then transplanted intravenously into DAL and APP mice. Transplantation of BMMCs suppressed neuronal loss and restored memory impairment of DAL mice to almost the same level as in wild-type mice. Transplantation of BMMCs to APP mice reduced Aß deposition in the brain. APP mice treated with BMMCs performed significantly better on behavioral tests than vehicle-injected mice. Moreover, the effects were observed even with transplantation after the onset of cognitive impairment in DAL mice. Together, our results indicate that intravenous transplantation of BMMCs has preventive effects against the cognitive decline in AD model mice and suggest a potential therapeutic effect of BMMC transplantation therapy.

Oxidative stress accelerates amyloid deposition and memory impairment in a double-transgenic mouse model of Alzheimer's disease.

Oxidative stress is known to play a prominent role in the onset and early stage progression of Alzheimer's disease (AD). For example, protein oxidation and lipid peroxidation levels are increased in patients with mild cognitive impairment. Here, we created a double-transgenic mouse model of AD to explore the pathological and behavioral effects of oxidative stress. Double transgenic (APP/DAL) mice were constructed by crossing Tg2576 (APP) mice, which express a mutant form of human amyloid precursor protein (APP), with DAL mice expressing a dominant-negative mutant of mitochondrial aldehyde dehydrogenase 2 (ALDH2), in which oxidative stress is enhanced. Y-maze and object recognition tests were performed at 3 and 6 months of age to evaluate learning and memory. The accumulation of amyloid plaques, deposition of phosphorylated-tau protein, and number of astrocytes in the brain were assessed histopathologically at 3, 6, 9, and 12-15 months of age. The life span of APP/DAL mice was significantly shorter than that of APP or DAL mice. In addition, they showed accelerated amyloid deposition, tau phosphorylation, and gliosis. Furthermore, these mice showed impaired performance on Y-maze and object recognition tests at 3 months of age. These data suggest that oxidative stress accelerates cognitive dysfunction and pathological insults in the brain. APP/DAL mice could be a useful model for exploring new approaches to AD treatment.

Pharmacological characterization of the novel γ-secretase modulator AS2715348, a potential therapy for Alzheimer's disease, in rodents and nonhuman primates.

γ-Secretase is the enzyme responsible for the intramembranous proteolysis of various substrates, such as amyloid precursor protein (APP) and Notch. Amyloid-ß peptide 42 (Aß42) is produced through the sequential proteolytic cleavage of APP by ß- and γ-secretase and causes the synaptic dysfunction associated with memory impairment in Alzheimer's disease. Here, we identified a novel cyclohexylamine-derived γ-secretase modulator, {(1R*,2S*,3R*)-3-[(cyclohexylmethyl)(3,3-dimethylbutyl)amino]-2-[4-(trifluoromethyl)phenyl]cyclohexyl}acetic acid (AS2715348), that may inhibit this pathological response. AS2715348 was seen to reduce both cell-free and cellular production of Aß42 without increasing levels of APP ß-carboxyl terminal fragment or inhibiting Notch signaling. Additionally, the compound increased Aß38 production, suggesting a shift of the cleavage site in APP. The inhibitory potency of AS2715348 on endogenous Aß42 production was similar across human, mouse, and rat cells. Oral administration with AS2715348 at 1 mg/kg and greater significantly reduced brain Aß42 levels in rats, and no Notch-related toxicity was observed after 28-day treatment at 100 mg/kg. Further, AS2715348 significantly ameliorated cognitive deficits in APP-transgenic Tg2576 mice. Finally, AS2715348 significantly reduced brain Aß42 levels in cynomolgus monkeys. These findings collectively show the promise for AS2715348 as a potential disease-modifying drug for Alzheimer's disease.

Amelioration of cognitive deficits in plaque-bearing Alzheimer's disease model mice through selective reduction of nascent soluble Aß42 without affecting other Aß pools.

Given that amyloid-ß 42 (Aß42) is believed to be a culprit in Alzheimer's disease (AD), reducing Aß42 production should be a potential therapeutic approach. γ-Secretase modulators (GSMs) cause selective reduction of Aß42 or both reduction of Aß42 and Aß40 without affecting total Aß through shifting the γ-cleavage position in amyloid precursor protein. We recently reported on GSM-2, one of the second-generation GSMs, that selectively reduced brain Aß42 level and significantly ameliorated cognitive deficits in plaque-free 5.5-month-old Tg2576 AD model mice. Here, we investigated the effects of GSM-2 on 10-, 14-, and 18-month-old mice which had age-dependent increase in amyloid plaques. Eight-day treatment with GSM-2 significantly ameliorated cognitive deficits measured by Y-maze task in the mice of any age. However, GSM-2 reduced brain soluble Aß42 only in 10-month-old mice. In contrast, GSM-2 markedly reduced newly synthesized soluble Aß42 in both 10- and 18-month-old mice with similar efficacy when measured using the stable isotope-labeling technique, suggesting that nascent Aß42 plays a more significant role than plaque-associated soluble Aß42 in the cognitive deterioration of Tg2576 mice. These findings further indicate the potential utility of approach to reducing Aß42 synthesis in AD therapeutic regimens.

Differential effects between γ-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice.

γ-Secretase inhibitors (GSIs) reduce amyloid-ß (Aß) peptides but inevitably increase the ß-C-terminal fragment (ß-CTF) of amyloid precursor protein (APP), potentially having undesirable effects on synapses. In contrast, γ-secretase modulators (GSMs) reduce Aß42 without increasing ß-CTF. Although the Aß-lowering effects of these compounds have been extensively studied, little effort has been made to investigate their effects on cognition. Here, we compared the effects of two GSIs--(2S)-2-hydroxy-3-methyl-N-[(2S)-1-{[(1S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-3-benzazepin-1-yl]amino}-1-oxopropan-2-yl]butanamide (LY450139, semagacestat) and (2R)-2-[[(4-chlorophenyl)sulfonyl][[2-fluoro-4-(1,2,4-oxazol-3-yl)phenyl]methyl]amino-5,5,5-trifluoropentanamide (BMS-708163)--and a second-generation GSM [{(2S,4R)-1-[(4R)-1,1,1-trifluoro-7-methyloctan-4-yl]-2-[4-(trifluoromethyl)phenyl]piperidin-4-yl}acetic acid (GSM-2)] on spatial working memory in APP-transgenic (Tg2576) and nontransgenic mice using the Y-maze task. While acute dosing with either GSI ameliorated memory deficits in 5.5-month-old Tg2576 mice, these effects disappeared after 8 d subchronic dosing. Subchronic dosing with either GSI rather impaired normal cognition in 3-month-old Tg2576 mice, with no inhibition on the processing of other γ-secretase substrates, such as Notch, N-cadherin, or EphA4, in the brain. LY450139 also impaired normal cognition in wild-type mice; however, the potency was 10-fold lower than that in Tg2576 mice, indicating an APP-dependent mechanism likely with ß-CTF accumulation. Immunofluorescence studies revealed that the ß-CTF accumulation was localized in the presynaptic terminals of the hippocampal stratum lucidum and dentate hilus, implying an effect on presynaptic function in the mossy fibers. In contrast, both acute and subchronic dosing with GSM-2 significantly ameliorated memory deficits in Tg2576 mice and did not affect normal cognition in wild-type mice. We demonstrated a clear difference between GSI and GSM in effects on functional consequences, providing new insights into strategies for developing these drugs against Alzheimer's disease.

Differential involvement of cell cycle reactivation between striatal and cortical neurons in cell death induced by 3-nitropropionic acid.

Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G(1) phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca(2+) and activation of calpain, a Ca(2+)-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca(2+) and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease.

Ablation of p27 enhance kainate-induced seizure and hippocampal degeneration.

p27 is a cyclin-dependent kinase inhibitor which arrests cell cycle at G1-S phase. Using RNA interference method, we previously showed that reduction of endogenous p27 expression induces cell death through cell cycle progression in cultured cortical neurons. In this study, we investigated responses to kainate treatment using p27 knockout mice. Injection of kainic acid induced p27 downregulation and retinoblastoma protein phosphorylation in wild-type mouse hippocampus. No change was observed in hippocampal cell viability in untreated adult p27 heterozygous and homozygous mice compared with wild type (+/+). p27 homozygous mice, however, displayed enhanced seizure and hippocampal degeneration after kainic acid treatment. This study first suggests that ablation of p27 enhance kainate-induced seizure and hippocampal cell death in vivo.

Calpain activation is required for glutamate-induced p27 down-regulation in cultured cortical neurons.

Recent evidence suggests that cell cycle-related molecules play pivotal roles in multiple forms of cell death in post-mitotic neurons. Nevertheless, it remains unclear what molecular mechanisms are involved in the regulation of expression levels and activities of these molecules. We showed previously that treatment with extracellular glutamate decreases cyclin-dependent kinase inhibitor p27 before neuronal cell death. In this study, we demonstrate that reductions of both p27 and neuronal viability were dependent on activity of calpain, a Ca(2+)-dependent protease, but not on activity of caspase 3. Interestingly, the glutamate-induced reduction of p27 was not dependent on the ubiquitin-proteasome system. In fact, p27 was present only in the neuronal nucleus, whereas calpain 1, a ubiquitous calpain, was observed both in the neuronal nucleus and cytoplasm in control cultures. Glutamate treatment did not change the localization patterns of p27 and calpain 1. It reduced p27 expression level in the nucleus in a calpain-dependent manner. In vitro experiments using neuronal cell lysate and p27 recombinant protein revealed that p27 was degraded as a substrate of activated calpain 1. These results suggest that calpain(s), activated by glutamate treatment, degrade(s) p27 in the nucleus of neurons, which might promote aberrant cell cycle progression.