Neuropharmacology 79 (2014) 412e419

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Pharmacological characterization of the novel g-secretase modulator AS2715348, a potential therapy for Alzheimer’s disease, in rodents and nonhuman primates Yasuyuki Mitani a, *, Hiroki Akashiba a, Kyoko Saita a, Junko Yarimizu a, Hiroshi Uchino a, Mayuko Okabe a, Makoto Asai a, Shingo Yamasaki b, Takashi Nozawa c, Noritoshi Ishikawa d, Yoshitsugu Shitaka a, Keni Ni a, Nobuya Matsuoka a a

Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan Chemistry Research Laboratories, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan c Analysis and Pharmacokinetics Research Laboratories, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan d Drug Safety Research Laboratories, Astellas Pharma Inc., Osaka, Japan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 February 2013 Received in revised form 27 October 2013 Accepted 16 December 2013

g-Secretase is the enzyme responsible for the intramembranous proteolysis of various substrates, such as amyloid precursor protein (APP) and Notch. Amyloid-b peptide 42 (Ab42) is produced through the sequential proteolytic cleavage of APP by b- and g-secretase and causes the synaptic dysfunction associated with memory impairment in Alzheimer’s disease. Here, we identified a novel cyclohexylaminederived g-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 Ab42 without increasing levels of APP b-carboxyl terminal fragment or inhibiting Notch signaling. Additionally, the compound increased Ab38 production, suggesting a shift of the cleavage site in APP. The inhibitory potency of AS2715348 on endogenous Ab42 production was similar across human, mouse, and rat cells. Oral administration with AS2715348 at 1 mg/kg and greater significantly reduced brain Ab42 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 Ab42 levels in cynomolgus monkeys. These findings collectively show the promise for AS2715348 as a potential disease-modifying drug for Alzheimer’s disease. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Alzheimer’s disease g-Secretase Amyloid-b Cognition

1. Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disease associated with dementia. Although senile plaques composed of insoluble amyloid-b (Ab) fibrils are a pathological hallmark of AD, the soluble form Ab is more relevant to synaptic deterioration and memory impairment (Lue et al., 1999; McDonald et al., 2010; McLean et al., 1999), indicating that reduction in levels of soluble Ab may be a potential therapeutic strategy. Ab is produced through sequential proteolytic cleavage of amyloid precursor protein (APP), with g-secretase being the enzyme responsible for the final step of its production (De Strooper et al., 1998; Wolfe et al., 1999). However, while g-secretase inhibitors (GSIs) can reduce total * Corresponding author. Tel.: þ81 29 863 7169; fax: þ81 29 852 2955. E-mail address: [email protected] (Y. Mitani). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.12.013

Ab production, they also cause accumulation of APP-b-carboxylterminal fragment (b-CTF or C99) and inhibit processing of other substrates, such as Notch. We recently reported that two GSIs, semagacestat and avagacestat, cause memory impairment in mice, probably due to b-CTF accumulation (Mitani et al., 2012), which may be relevant to the fact that both GSIs worsened cognition scores in AD patients (Coric et al., 2012; Doody et al., 2013). Further, inhibition of Notch processing in animals causes severe side effects such as intestinal goblet cell hyperplasia, thymus atrophy, spleen hypertrophy, and skin carcinoma (Li et al., 2007; Milano et al., 2004; Wong et al., 2004). In addition to findings in animal studies, Notch-related side effects of gastrointestinal symptoms and skin cancer has been observed in humans treated with semagacestat (Doody et al., 2013; Fleisher et al., 2008). As such, dissociating the inhibitory effects on Notch processing is sorely needed.

Y. Mitani et al. / Neuropharmacology 79 (2014) 412e419

g-Secretase modulators (GSMs) were first identified from a subset of non-steroidal anti-inflammatory drugs, which selectively reduced Ab42, a toxic Ab species, without any concomitant increase in b-CTF or Notch processing inhibition (Weggen et al., 2001, 2003). However, tarenflurbil (R-flurbiprofen), the first GSM clinically tested, failed to show any beneficial effects on AD patients, likely due to its low pharmacological potency and brain penetration (Eriksen et al., 2003; Green et al., 2009; Lanz et al., 2005). Here, we identified a novel cyclohexylamine-derived GSM (AS2715348) and clarified its pharmacological profile, including cognitive improvement in Tg2576 AD model mice as well as brain Ab42 reduction not only in rodents but also in nonhuman primates. We also determined whether or not chronic treatment with AS2715348 causes Notch-related toxicity in rats. 2. Materials and methods 2.1. Ethics statement All animal experiments were performed at an American Association for Accreditation of Laboratory Animal Care (AAALAC)-approved facility. All procedures involving animals and their care were conducted in accordance with AAALAC guidelines and Astellas Pharma Inc. guidelines and were approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc. 2.2. Drug AS2715348 ({(1R*,2S*,3R*)-3-[(cyclohexylmethyl)(3,3-dimethylbutyl)amino]-2[4-(trifluoromethyl)phenyl]cyclohexyl}acetic acid) was synthesized at Astellas Pharma Inc. (Tsukuba, Japan). Although the absolute configuration is not determined, the chemical structure with the relative configuration of the compound is shown in Fig. 1. 2.3. Ab ELISA Ab1-42 and Ab1-40 ELISA kits (IBL, Gunma, Japan), and Ab x-42 and Ab x-40 ELISA kits (Wako, Osaka, Japan) were used unless otherwise specified. The Ab1-42/ 1-40 ELISA can detect full-length human/monkey Abs, while the Ab x-42/x-40 ELISA can detect both human/monkey and rat/mouse Abs including N-terminal truncated forms.

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800 g for 10 min. The supernatant was ultracentrifuged at 100,000 g for 1 h, and the ensuing membrane pellet was resuspended with buffer A and stored at 80  C. The membrane suspension was solubilized with 1% CHAPSO for 30 min and then mixed with a reaction buffer (20 mM HEPES, 2 mM EDTA, 0.1% BSA; pH 7.4, final 0.25% CHAPSO) containing Complete Protease Inhibitor Cocktail, 4 mM C100-FmH, and various concentrations of AS2715348 in 1% dimethylsulfoxide. All procedures were performed at 4  C or on ice. The mixture was incubated at 37  C for 3 h, and generated Abs were measured using the Ab x-42 and Ab x-40 ELISA kits. 2.4.2. Cell-based Ab production assay Ab production assay using APP-overexpressing H4 cells was performed as previously described (Mitani et al., 2012). Briefly, H4 cells stably overexpressing human wild-type APP695 were cultured in Dulbecco’s modified Eagle’s medium and treated with AS2715348 at various concentrations in 0.5% dimethylsulfoxide for 24 h. Levels of Ab1-43, Ab1-42, Ab1-40, and Ab1-38 in the media and b-CTF in the cell lysates were measured using separate ELISA kits (IBL). Ab production assay using SK-N-BE(2) cells was performed as previously described (Mitani et al., 2013). Briefly, cells were cultured in RPMI1640 medium in poly-D-lysine-coated 96-well plates and treated with AS2715348 at various concentrations in 0.5% dimethylsulfoxide for 24 h. Ab levels in the media were measured using the Ab1-42 and Ab1-40 ELISA kits. Neuro2A cells were cultured in Dulbecco’s modified Eagle’s medium in poly-Dlysine-coated 96-well plates and treated with AS2715348 at various concentrations in 0.5% dimethylsulfoxide for 24 h. Ab levels in the media were measured using the Ab x-42 and Ab x-40 ELISA kits. All culture media used above were supplemented with 10% fetal bovine serum and penicillin/streptomycin. Rat cortical cells were isolated from embryonic day-18e20 Wistar rats and cultured for 7 days in Nerve-Cell Culture Medium (Sumitomo Bakelite, Tokyo, Japan) in poly-D-lysine-coated 96-well plates, and then treated with AS2715348 at various concentrations in 0.1% dimethylsulfoxide for 24 h. Ab levels in the media were measured using the Ab x-42 and Ab x-40 ELISA kits. Cell viability was evaluated using the CellTiter-Glo Luminescent Cell Viability Assay in all cellular experiments (Promega, Fitchburg, WI, USA). 2.4.3. Cell-based Notch signaling assay Drug effects on cellular Notch signaling were evaluated as previously described (Mitani et al., 2012). Briefly, a human NotchDE expression vector was constructed and transiently introduced into H4 cells together with an RBP-Jk-responsive luciferase vector (SA Biosciences, Frederick, MD, USA), and then exposed to various concentrations of AS2715348 or 0.05 mM Compound E (Merck KGaA, Darmstadt, Germany) for 16 h. Notch signaling was measured based on luciferase activity in the cell lysate using the Dual-Glo Luciferase Assay System (Promega).

2.4. In vitro studies

2.5. In vivo studies

2.4.1. Cell-free g-secretase assay Cell-free Ab production assay using a recombinant substrate and solubilized gsecretase was performed as previously described with some modifications (Beher et al., 2004; Qi et al., 2003; Takahashi et al., 2003). Briefly, cDNA encoding the C terminal 100 amino acids of human APP (596e695) fused to an FLAG tag was constructed into a pTrcHis2 TOPO vector (Invitrogen, Carlsbad, CA, USA). The recombinant protein, C100-FLAG/c-Myc/His (C100-FmH), was produced in Escherichia Coli and purified by nickel-chelating affinity chromatography. Solubilized g-secretase was prepared from a membrane fraction of H4 cells. Cells were grown in Dulbecco’s modified Eagle’s medium and homogenized with buffer A (20 mM Pipes, 5 mM EGTA, 140 mM KCl, 250 mM sucrose; pH 7.0) containing Complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany), followed by centrifugation at

2.5.1. Ab reduction in rats AS2715348 was suspended in 0.5% methylcellulose and orally administered to male SpragueeDawley rats. Blood, cerebrospinal fluid (CSF), and brains were collected under isoflurane anesthesia at defined time points, and the hippocampi were isolated and stored at 80  C. Each frozen hippocampus was homogenized in an ultrasonic homogenizer with 10-fold volume of TBS (Tris 25 mM, NaCl 137 mM, KCl 2.68 mM; pH 7.4) containing Complete Protease Inhibitor Cocktail on ice followed by ultracentrifugation at 100,000 g and 4  C for 1 h. Ab levels in the supernatant as well as in plasma and CSF samples were measured using the Ab x-42 and Ab x-40 ELISA kits.

Fig. 1. Chemical structure of AS2715348. The absolute configuration is not determined.

2.5.2. Notch-related toxicity AS2715348 was dissolved in 30% (v/v) propylene glycol-solvent (propylene glycol: HCO-40: Tween 80 ¼ 4: 2: 1, v/v) aqueous solution containing 4.5% citric acid and orally administered to male and female SpragueeDawley rats at 100 mg/kg once daily for 28 consecutive days. Measurements of body weight, spleen weight, and the number of circulating leukocytes as well as anatomical and histological examinations of the gastrointestinal tract (stomach, duodenum, ileum, jejunum, ileum, cecum, colon, and, rectum), spleen, and thymus were executed. 2.5.3. Y-maze test Female 5.5-month-old Tg2576 mice expressing human APP695 with the Swedish mutation (K670N/M671L) were used (Hsiao et al., 1996). Drug effects on spatial working memory were evaluated by recording spontaneous alternation behavior in the Y-maze task as previously described (Mitani et al., 2012). Briefly, AS2715348 was suspended in 0.5% methylcellulose and orally administered once daily for 8 days. Three hours after administration on Days 1 and 8, each animal was placed at the end of one arm of the apparatus and allowed to freely explore for 8 min. The alternation rate was defined as entries into all three arms on consecutive occasions using the following formula: Alternation rate (%) ¼ Number of alternations/(Number of total arm entries  2)  100. Data were eliminated in cases where the number of total arm entries was less than 10.

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Mice were decapitated immediately after the test on Day 8, and hippocampi were collected and stored at 80  C. The hippocampal levels of Ab x-42 and Ab x-40 were measured as described above. Aliquots of the hippocampal homogenate were solubilized with 2% SDS, and b-CTF levels were measured using an ELISA kit. 2.5.4. Ab reduction in nonhuman primates Seven male cynomolgus monkeys were used. AS2715348 was dissolved in saline containing 10% (v/v) dimethylformamide, 30% (v/v) propylene glycol and 1N HCl, and orally administered to three animals. The other four animals were administered a vehicle. Blood was sequentially collected 0.5, 1, 2, and 4 h after administration, and animals were euthanized by exsanguination under anesthesia induced by an injection of sodium pentobarbital into the cephalic vein. The brain was dissected and cut into 4-mm coronal sections on ice and stored at 80  C. Plasma samples were also stored at 80  C until use. One brain section per animal was partially thawed, and the hippocampus and cingulate cortex were isolated, followed by homogenization and centrifugation as described above. Ab levels in the supernatant as well as in plasma samples were measured using the Ab1-42 and Ab1-40 ELISA kits. Plasma samples were also subjected to the Ab x-42 ELISA kit, as the basal level of plasma Ab1-42 was found to be near the quantitation limit. 2.6. Drug concentrations in biological samples AS2715348 concentrations in plasma, CSF and the brain were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Briefly, aliquots of plasma, CSF, and brain homogenates were treated by protein precipitation with acetonitrile containing an appropriate internal standard, followed by centrifugation. The supernatant was separated on an XBridge C18 column (Waters, Milford, USA) using a gradient mixture of water and acetonitrile containing 0.1% formic acid, and the drug was detected by multiple-reaction monitoring using an LC-MS/MS system. 2.7. Statistical analysis All statistical analyses were conducted using SAS software (SAS Institute, Cary, NC, USA). Dunnett’s multiple comparison test was used to compare multiple doses in the drug-treated groups and vehicle-treated group. An unpaired Student’s t-test was used to compare the single-dose drug-treated group and vehicle-treated group, and the Tg2576 group and wild-type group. For all tests, a value of p < 0.05 was considered statistically significant.

3. Results 3.1. AS2715348 inhibits both cell-free and cellular Ab42 production but not Notch signaling To evaluate the direct effects of AS2715348 on g-secretase activity, we used a cell-free system with detergent-solubilized gsecretase from H4 cell membranes and an exogenous substrate C100-FmH. This system was validated using a transition state analog GSI, L-685,458, as it potently and equally inhibited the production of Ab42 and Ab40 (data not shown). AS2715348 potently inhibited Ab42 production but weakly inhibited Ab40 production with IC50 values of 0.450 and 7.33 mM, respectively (Fig. 2A). Next, we evaluated the drug effects on Ab production in APPoverexpressing H4 cells. AS2715348 potently inhibited Ab42 production and weakly inhibited Ab40 production with IC50 values of 0.128 and 9.44 mM, respectively (Fig. 2B). These values were in the similar range to those observed in the cell-free assay. Further, while AS2715348 also inhibited Ab43 production with an IC50 value of 0.846 mM, the compound increased Ab38 production up to 2-fold. Finally, AS2715348 did not affect b-CTF levels. Cell viability was not

Fig. 2. Cell-free and cell-based APP processing assays. A, Effects of AS2715348 on cell-free Ab production. Solubilized g-secretase from H4 cell membranes were incubated with a recombinant substrate, C100-FmH, and various concentrations of AS2715348. B, Effects of AS2715348 on Ab production in APP-overexpressing H4 cells. AS2715348 most potently reduced Ab42 production and increased Ab38 production without affecting b-CTF levels. All data are presented as mean  SEM (n ¼ 3).

affected at any of the tested concentrations (data not shown). In this assay system, semagacestat inhibited the production of all Ab isoforms with a similar potency and increased b-CTF levels (Mitani et al., 2012). The drug effects on Notch signaling were evaluated using a Notch intracellular domain reporter gene assay system in NotchDEtransfected H4 cells. In this system, AS2715348 showed no inhibition on Notch signaling even at 10 mM, while the GSI, Compound E, showed potent inhibition (Table 2). Further, AS2715348 did not show any potent inhibition in a multi-target binding screening against neurotransmitter receptors, ion channels, transporters, and neuronal enzymes (Tables S1 and S2), suggesting that AS2715348 is a pure GSM.

Table 1 Summary of IC50 values of AS2715348 on cell-free and cellular Ab production in various cell types. IC50 (mM)

Ab40 Ab42 Ab43

Cell-free

APP-H4

SK-N-BE(2)

Neuro2A

Rat primary

7.33  1.39 0.450  0.128 NA

9.44  2.01 0.128  0.008 0.846  0.078

>3 0.062  0.012 NA

>3 0.156  0.55 NA

>3 0.121  0.019 NA

Values are shown as mean  SEM for three independent experiments. NA: not assessed.

Y. Mitani et al. / Neuropharmacology 79 (2014) 412e419 Table 2 Drug effects on Notch signaling in NotchDE-transfected H4 cells.

Control AS2715348

Compound E

Concentration (mM)

Reporter activity (%)

e 0.1 1 10 0.05

100.0 109.6 111.7 109.8 8.3

    

2.6 5.3 2.3 2.5 0.1

Values are shown as mean  SEM; n ¼ 3.

3.2. AS2715348 inhibits endogenous Ab42 production in human and rodent cells We also evaluated drug effects on endogenous Ab production in human neuroblastoma SK-N-BE(2) cells. AS2715348 selectively inhibited endogenous Ab42 production with an IC50 value of 0.062 mM, a potency that was similar to that observed in APPoverexpressing H4 cells (Fig. 3 and Table 1). In this assay system, while L-685,458 showed potent non-selective reductions in Ab42 and Ab40, tarenflurbil showed a less potent but selective Ab42 reduction with an IC50 value of 280 mM and no reduction in Ab40 up to 1000 mM (data not shown), potencies that were in line with the previous literature (Eriksen et al., 2003). To determine whether or not the drug efficacy differed between human and rodent cells, we evaluated the drug effects on endogenous Ab production also in mouse neuroblastoma Neuro2a cells and rat cortical primary neurons. AS2715348 selectively inhibited endogenous Ab42 production in these cells with IC50 values of 0.156 and 0.121 mM, respectively, potencies that were similar to those observed in human cells, indicating no apparent difference in the drug efficacy between humans and rodents as well as neuroblastoma cells and primary neurons (Fig. 3 and Table 1). Cell viabilities were not affected at any of the tested concentrations (data not shown). 3.3. AS2715348 reduces endogenous Ab42 in the brain, plasma, and CSF in rats without causing Notch-related toxicity In vivo Ab-lowering effects of AS2715348 and pharmacokineticsepharmacodynamics (PKePD) relationships in the brain, plasma, and CSF were evaluated using rats. PK time courses and parameters after oral administration at 5 mg/kg are shown in Fig. 4A and Table 3. AS2715348 penetrated into the brain and achieved a brain/plasma AUC ratio of 0.22. The drug treatment reduced Ab42 levels in all compartments, effects that were

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sustained at 8 h and returned toward baseline levels at 24 h, time courses that were similar to those of PK (Fig. 4B). Maximum reductions in the brain, plasma, and CSF were 33%, 56%, and 32%, respectively. In contrast, Ab40 levels in all compartments showed no obvious changes. The dose dependency of Ab42-lowering effects at 4.5 h after administration is shown in Fig. 5. AS2715348 significantly reduced Ab42 levels in the brain and plasma by 20%e37% and 15%e49%, respectively, at 1e10 mg/kg in a dose-dependent manner. To determine whether or not administering AS2715348 could avoid Notch-related toxicity in vivo, the compound was orally administered to rats at 100 mg/kg/day for 28 days, and a histological study of the gastrointestinal tract and immune organs along with a hematological study were conducted. In the previous reports on nicastrin hetero-knockout mice (Li et al., 2007), and GSI-treated mice (Wong et al., 2004) and rats (Milano et al., 2004), intestinal goblet cell hyperplasia, spleen hypertrophy, thymus atrophy, a decrease in numbers of circulating lymphocytes, and an increase in numbers of circulating neutrophils were considered findings for Notch-related toxicity. In the present study, AS2715348-treated rats did not show any gross or microscopic changes in the gastrointestinal tract, spleen, or thymus, nor any changes in the number of circulating lymphocytes or neutrophils (Table 4). Other leukocytes such as eosinophils, basophils, and monocytes were also unaffected (data not shown). Plasma drug exposure (AUC0e24h) at 100 mg/kg on Day 1 was 75-fold higher than that at the pharmacological effective dose of 1 mg/kg (Table 3). Apparent differences were not observed in PK parameters at 100 mg/kg on Days 1 and 28 (data not shown). These findings confirmed that AS2715348 did not cause Notch-related toxicity. 3.4. AS2715348 reduces brain Ab42 and ameliorates cognitive deficits in Tg2576 mice To investigate the functional consequence of reducing Ab42 levels via AS2715348, effects on cognitive function were evaluated in Tg2576 mice. Mice aged 5.5 months were orally administered with AS2715348 or a vehicle and then subjected to the Y-maze task 3 h later. Vehicle-treated Tg2576 mice showed significantly lower spontaneous alternation rates than their wild-type littermates, suggesting deficits in spatial working memory. These memory deficits were significantly ameliorated by treatment with AS2715348 at 0.3e3 mg/kg (Day 1 in Fig. 6A). Given that we previously reported that GSIs failed to ameliorate cognitive deficits in Tg2576 mice after 8-day dosing despite showing improvement after acute dosing (Mitani et al., 2012), the mice were further administered AS2715348 for a subsequent 7 days and again subjected to the Y-maze task. The

Fig. 3. Effects of AS2715348 on Ab production in various cell types. Human neuroblastoma SK-N-BE(2), mouse neuroblastoma Neuro2A, and rat cortical primary neurons were exposed to various concentrations of AS2715348, and levels of endogenous Ab secreted in the media were measured. AS2715348 showed selective Ab42 reduction with dosee response curves that were similar for the three cell types. All data are presented as mean  SEM (n ¼ 3).

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Fig. 5. PD doseeresponse study in rats. Hippocampal and plasma Ab42 levels 4.5 h after oral administration with AS2715348 were measured. Both levels were significantly reduced by drug treatment in a dose-dependent manner. *, p < 0.05; **, p < 0.01 compared with vehicle group by Dunnett’s test. All data are presented as mean  SEM (n ¼ 4).

The effects of AS2715348 on the brain levels of Ab and b-CTF were evaluated on the hippocampus isolated immediately after the second Y-maze test. AS2715348 reduced hippocampal Ab42 levels at 0.3e3 mg/kg by 4%e31% in a dose-dependent manner, while the compound did not affect the levels of Ab40 or b-CTF (Fig. 6B). 3.5. AS2715348 reduces endogenous Ab42 in the brain and plasma in nonhuman primates The Ab-lowering effects of AS2715348 were also tested in nonhuman primates. Drug (10 mg/kg) or vehicle was orally administered to cynomolgus monkeys (n ¼ 3e4), and blood was collected over time. Animals were euthanized 4 h after administration, and the brain was dissected. Plasma Cmax was 1321 ng/mL (Fig. 7A), and the brain drug level at 4 h was 390 ng/g. Plasma Ab42 levels were reduced after administration (34% reduction at 4 h compared to vehicle group), while Ab40 levels were unaffected (Fig. 7B). Brain Ab42 levels were also significantly reduced by 21% and 16% in the hippocampus and cortex, respectively, while Ab40 levels were unchanged (Fig. 7C). 4. Discussion Fig. 4. PK-PD study in rats. A, Time course of AS2715348 concentrations in the brain, plasma (left axis), and CSF (right axis) after oral administration at 5 mg/kg. The Cmax and AUC0e24 calculated from these data are described in Table 3. B, Time course of Ab levels in the brain, plasma, and CSF. A sustained reduction in Ab42 levels after dosing was observed in all compartments, while Ab40 levels were unchanged. All data are presented as mean  SEM (n ¼ 4).

memory deficits were significantly ameliorated also after multiple dosing at the same dosage as with acute dosing (Day 8 in Fig. 6A). No significant changes in the number of total arm entries were found with drug treatment (data not shown). Table 3 Pharmacokinetic parameters of AS2715348 in rats after oral administration. Pharmacokinetic parameters of AS2715348 in rats after oral administration.

Plasma

Brain CSF

Dose (mg/kg)

Cmax (ng/mL or g)

AUC0e24h (ng h/mL or g)

1 5 100 1 5 5

368 1878 18,970 169 379 13

4575 23,562 342,040 2310 5162 165

PK parameters were calculated from the mean drug concentrations of four animals. All four rats were male for doses at 1 and 5 mg/kg; two male and two female rats for the dose at 100 mg/kg. No apparent gender difference was observed.

Here, we clarified the pharmacological profiles of a novel cyclohexylamine-derived GSM, AS2715348. The compound selectively reduced brain Ab42 levels in rats without causing Notchrelated side effects and significantly ameliorated cognitive deficits in Tg2576 mice. AS2715348 also reduced brain Ab42 levels in nonhuman primates, suggesting potential efficacy in humans. The Ab production assay using APP-overexpressing H4 cells revealed that AS2715348 reduced Ab42 production 74-fold more potently than Ab40 production, increased Ab38 production, and resulted in no accumulation of b-CTF (Fig. 2B and Table 1), effects that were similar to structurally related piperidine-derived GSMs, one of the second generation GSMs (Hall et al., 2010; Mitani et al., 2012). Further, AS2715348 reduced the production of Ab43, another possibly toxic peptide (Saito et al., 2011), although the potency of this reduction was 6.6 times lower than that against Ab42. While the molecular mechanism of modulating Ab production by AS2715348 remains to be addressed, the results of our cell-free study nevertheless indicate the direct modulation of g-secretase (Fig. 2A), an action that may be similar to the piperidine-derived GSM, which binds to presenilin 1, a core component of g-secretase, thereby inducing a conformational change that results in a shift of the cleavage position in APP (Ohki et al., 2011).

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Table 4 Absence of Notch-related toxicity in rats after oral administration with AS2715348 at 100 mg/kg for 28 days. Sex [n]

Body weight (g)

Spleen weight (g)

Hematology (103/mL) Lymphocyte

Control AS2715348

M [5] F [5] M [5] F [4]

354 241 355 231

   

5 7 12 5

0.59 0.45 0.64 0.44

   

0.03 0.03 0.07 0.05

9.9 4.9 8.7 5.0

   

0.7 0.7 0.5 0.6

Histology Neutrophil 1.5 1.2 1.4 0.74

   

0.3 0.2 0.3 0.08

GI/Spleen/Thymus No abnormal finding

Values are shown as mean  SEM. No statistical significance was detected between the groups of control and AS2715348 by Student’s t-test. GI: gastrointestinal tract (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum).

AS2715348 also showed selective Ab42 reduction in SK-N-BE(2) human neuroblastoma with an IC50 value similar to that in APPoverexpressing H4 cells (Fig. 3 and Table 1), even though the production of Ab1-42 and Ab1-40 at a basal level was two magnitudes lower. This feature is quite different from GSIs, which show a potency shift that is dependent on the substrate quantity and an enhancement of Ab production at low drug concentrations (Burton et al., 2008). We also found that the IC50 values of semagacestat and avagacestat on Ab production were 83 and 70 times lower, respectively, in APP-overexpressing H4 cells than in SK-N-BE(2) cells, where low concentrations of either GSI enhanced Ab production (unpublished data), suggesting a significant distinction between GSM and GSI. Additionally, AS2715348 showed selective Ab42 reduction in Neuro2A mouse neuroblastoma cells and rat cortical primary neurons with IC50 values similar to that in human cells (Fig. 3 and Table 1). This finding warranted the use of rats for our subsequent in vivo studies that showed that AS2715348

significantly reduced brain Ab42 levels at 1 mg/kg and above in a dose-dependent manner (Fig. 5). Notch-related side effects in the gastrointestinal tract, spleen, thymus, and skin are the most important issue for GSIs. Although some GSIs have been identified as “in vitro Notch-sparing” such as GSI-953 and avagacestat, chronic treatment with even these two have shown the side effects in the gastrointestinal tract and skin of animals or humans (Coric et al., 2012; Martone et al., 2009). The “in vitro Notch-sparing” feature has also been reported in various GSMs (Hall et al., 2010; Kounnas et al., 2010; Van Broeck et al., 2011), however, whether or not this holds true for chronic treatment, which would make it unlike GSIs, is unknown. In the present study, we confirmed that 28-day treatment with AS2715348 did not cause any pathological changes in the gastrointestinal tract, spleen, or thymus of rats at drug exposures 75 times higher than pharmacologically effective exposure, proving the concept of GSM (Table 4).

Fig. 6. Acute and subchronic effects of AS2715346 on cognitive deficits in Tg2576 mice. A, Spontaneous alternation rate in Y-maze tests with 5.5-month-old Tg2576 mice. AS2715348 was orally administered for 8 days, and tests were conducted on Days 1 and 8. Alternation behavior deficits were significantly improved by drug treatment on both Days 1 and 8. WT: wild-type, Dotted lines: chance level of alternation behavior. B, Effects of AS2715348 on levels of hippocampal Ab and b-CTF on Day 8. Ab42 was significantly reduced by drug treatment, while Ab40 and b-CTF levels were unchanged. ##, p < 0.01 by Student’s t-test. *, p < 0.05; **, p < 0.01 compared with vehicle group by Dunnett’s test. All data are presented as mean  SEM (n ¼ 10e11).

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Fig. 7. PK-PD study in nonhuman primates. A, Time course of AS2715348 concentrations in plasma after oral administration at 10 mg/kg. B, Time course of Ab levels in plasma. Plasma Ab42 levels were reduced after dosing, while Ab40 levels were unchanged. C, Hippocampal and Cortical Ab levels 4 h after dosing. Ab42 levels in both sites were significantly reduced by drug treatment, while Ab40 levels were unaffected. *, p < 0.05 compared with vehicle group by Student’s t-test. All data are presented as mean  SEM (n ¼ 3e4).

Another discouraging feature of GSIs is that both semagacestat and avagacestat worsened the cognition score of AD patients (Coric et al., 2012; Doody et al., 2013). This fact may be associated with our previous finding that subchronic treatment with either GSI fails to ameliorate cognitive deficits in Tg2576 mice, rather impairs cognitive function probably due to b-CTF accumulation (Mitani et al., 2012). Although behavioral studies are useful for evaluating the functional consequences of a drug, such studies for GSMs are few, particularly on second generation GSMs. In the present study, we confirmed that both acute and subchronic treatment with AS2715348 ameliorate cognitive deficits in Tg2576 mice (Fig. 6A). Although rodents are a useful model for evaluating drug effects on Ab levels, nonhuman primates are preferred due to their analogy to humans, especially with regards to Ab amino acid sequence and its turnover rate (Cook et al., 2010). We confirmed that AS2715348 reduces brain Ab levels in cynomolgus monkeys in a manner similar to those observed in rats based on the drug exposure level (Fig. 7). In conclusion, we identified AS2715348, a structurally novel compound that modulates g-secretase activity and leads to significant reduction in brain Ab42 levels and memory improvement without Notch-related toxicity in animals. These findings strongly confirm that GSM is a promising approach to inhibit brain Ab42 production without undesirable effects and compounds such as AS2715348 therefore have potential as new agents for AD. Further investigation of the compound should be conducted for the future therapeutic application. Acknowledgments We thank Drs. Shinya Takami, Akira Nagakura, Akio Kuroda, Koichi Yonezawa, Eriko Honjo, Emiko Furukawa, Nozomu Hamakawa, and Mitsuru Hosaka for their great assistance.

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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. Am...
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