The amyloid-β peptide (Aβ)-in particular, the 42-amino acid form, Aβ1-42-is thought to play a key role in the pathogenesis of Alzheimer's disease (AD). Thus, several therapeutic modalities aiming to inhibit Aβ synthesis or increase the clearance of Aβ have entered clinical trials, including γ-secretase inhibitors, anti-Aβ antibodies, and amyloid-β precursor protein cleaving enzyme inhibitors. A unique class of small molecules, γ-secretase modulators (GSMs), selectively reduce Aβ1-42 production, and may also decrease Aβ1-40 while simultaneously increasing one or more shorter Aβ peptides, such as Aβ1-38 and Aβ1-37. GSMs are particularly attractive because they do not alter the total amount of Aβ peptides produced by γ-secretase activity; they spare the processing of other γ-secretase substrates, such as Notch; and they do not cause accumulation of the potentially toxic processing intermediate, β-C-terminal fragment. This report describes the translation of pharmacological activity across species for two novel GSMs, (S)-7-(4-fluorophenyl)-N2-(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481) and (S,Z)-17-(4-chloro-2-fluorophenyl)-34-(3-methyl-1H-1,2,4-triazol-1-yl)-16,17-dihydro-15H-4-oxa-2,9-diaza-1(2,4)-cyclopenta[d]pyrimidina-3(1,3)-benzenacyclononaphan-6-ene (BMS-986133). These GSMs are highly potent in vitro, exhibit dose- and time-dependent activity in vivo, and have consistent levels of pharmacological effect across rats, dogs, monkeys, and human subjects. In rats, the two GSMs exhibit similar pharmacokinetics/pharmacodynamics between the brain and cerebrospinal fluid. In all species, GSM treatment decreased Aβ1-42 and Aβ1-40 levels while increasing Aβ1-38 and Aβ1-37 by a corresponding amount. Thus, the GSM mechanism and central activity translate across preclinical species and humans, thereby validating this therapeutic modality for potential utility in AD.

The pharmacokinetics, pharmacodynamics, safety, and tolerability of BMS-932481, a γ-secretase modulator (GSM), were tested in healthy young and elderly volunteers after single and multiple doses. BMS-932481 was orally absorbed, showed dose proportionality after a single dose administration, and had approximately 3-fold accumulation after multiple dosing. High-fat/caloric meals doubled the Cmax and area under the curve and prolonged Tmax by 1.5 hours. Consistent with the preclinical pharmacology of GSMs, BMS-932481 decreased cerebrospinal fluid (CSF) Aβ39, Aβ40, and Aβ42 while increasing Aβ37 and Aβ38, thereby providing evidence of γ-secretase enzyme modulation rather than inhibition. In plasma, reductions in Aβ40 and Aβ42 were observed with no change in total Aβ; in CSF, modest decreases in total Aβ were observed at higher dose levels. Increases in liver enzymes were observed at exposures associated with greater than 70% CSF Aβ42 lowering after multiple dosing. Although further development was halted due to an insufficient safety margin to test the hypothesis for efficacy of Aβ lowering in Alzheimer's disease, this study demonstrates that γ-secretase modulation is achievable in healthy human volunteers and supports further efforts to discover well tolerated GSMs for testing in Alzheimer's disease and other indications.

AIM:To evaluate the single dose pharmacokinetics, pharmacodynamics, and preliminary tolerability of the γ-secretase inhibitor BMS-708163 (avagacestat) in young and elderly men and women.METHODS:All subjects received double-blinded administration of a single 50 mg dose of avagacestat in capsule form or matching placebo. Main evaluations included pharmacokinetics, safety, plasma amyloid-β (Aβ)(1-40) concentratios and exploration of Notch biomarkers.RESULTS:Avagacestat 50 mg capsule was well tolerated and rapidly absorbed among young and elderly subjects, with a median t(max) between 1 and 2 h post dose and an average half-life between 41 and 71 h. In general, subjects aged 75 years or more had higher AUC(0,∞) values than those aged less than 75 years. An exploratory analysis of Aβ(1-40) serum concentrations showed a pattern of decreasing concentrations over the first 4-6 h followed by a rise above baseline that was maintained until the end of the assessment period. Adverse events were generally mild, occurring more frequently in elderly subjects, with no observed difference between subjects receiving avagacestat and placebo. No dose limiting gastrointestinal effects of avagacestat were observed and exploratory biomarkers of Notch inhibition did not change significantly.CONCLUSIONS:The favourable safety profile and pharmacokinetic effects of avagacestat in this study support its continued development, especially in the target population of elderly subjects with mild cognitive impairment or Alzheimer's disease.

Background: gamma-Secretase inhibitors (GSIs) are being investigated for their potential to modify the progression of Alzheimer disease based on their ability to regulate amyloid-beta (A beta) accumulation. BMS-708163 (avagacestat) is an oral GSI designed for selective inhibition of A beta synthesis currently in development for the treatment of mild to moderate and predementia AD. In addition to the desired effect on A beta synthesis, GSIs affect Notch processing, which is thought to mediate some toxic adverse effects reported with this drug class. Avagacestat produced up to 190-fold greater selectivity for A beta synthesis than Notch processing in preclinical studies and may therefore produce less toxic adverse events than other less selective compounds. Presented here are the results of the first inhuman study for this new GSI compound.Objective: The goal of this study was to assess the tolerability profile, pharmacokinetic properties, and effects on pharmacodynamic markers (A beta, trefoil factor family 3 protein, dual specificity phosphatase 6, and hairy and enhancer of split-1) of single, oral doses of avagacestat in healthy, young, male volunteers.Methods: This was a multicenter, randomized, double-blind, placebo-controlled, single-ascending dose study in 8 healthy young men (age, 18-45 years) per dosing panel. Each study participant was randomized to receive a single dose of placebo (n = 2) or avagacestat (n = 6 for each dose) as an oral solution in 1 of 9 sequential dose panels (0.3, 1.5, 5, 15, 50, 100, 200, 400, and 800 mg). For determination of avagacestat, blood samples were obtained before dosing and for up to 144 hours after dosing. For participants in the 800-mg avagacestat dose panel, additional samples were obtained at 216, 312, and 648 hours. For 40 amino acid isoform of A beta (A beta(1-40)) assessment, plasma samples were collected before avagacestat administration and up to 72 hours after dosing.Results: Avagacestat concentrations peaked quickly after oral administration and then had a biphasic decrease in concentrations with a prolonged terminal phase. Exposures were proportional with doses up to 200 mg. Avagacestat was well tolerated at single oral doses up to 800 mg, with a biphasic effect on plasma A beta(1-40). Adverse events were predominately mild to moderate in severity with no evidence of dose dependence up to 200 mg.Conclusions: Results from this single-ascending dose study suggest that avagacestat was tolerated at a single-dose range of 0.3 to 800 mg and suitable for further clinical development. ClinicalTrials.gov identifier: NCT0145 4115. (Clin Ther. 2012;34:654-667) (C) 2012 Elsevier HS Journals, Inc. All rights reserved.

Charles F. Albright, Randy C. Dockens, Jere E. Meredith Jr., Richard E. Olson, Randy Slemmon, Kimberley A. Lentz, Jun-Sheng Wang, R. Rex Denton, Gary Pilcher, Paul W. Rhyne, Joseph J. Raybon, Donna M. Barten, Catherine Burton, Jeremy H. Toyn, Sethu Sankaranarayanan, Craig Polson, Valerie Guss, Randy White, Frank Simutis, Thomas Sanderson, Kevin W. Gillman, John E. Starrett, Jr., Joanne Bronson, Oleksandr Sverdlov, Shu-Pang Huang, Lorna Castaneda, Howard Feldman, Vlad Coric, Robert Zaczek, John E. Macor, John Houston, Robert M. Berman, and Gary Tong

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid β-peptide (Aβ), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aβ and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aβ and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aβ levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aβ40 and Aβ42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aβ40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aβ levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

Avagacestat is an orally active γ-secretase inhibitor that selectively inhibits amyloid β (Aβ) synthesis in cell culture and animal models. The objective of the current study was to assess the pharmacokinetics, pharmacodynamics, safety and tolerability of multiple doses of avagacestat over 28 days in healthy young men and elderly men and women in a placebo-controlled, sequential-panel, ascending multiple-dose study.

Background The concentration of amyloid b (Ab) peptides in cerebrospinal fluid (CSF) is a biomarker for Alzheimer’s disease (AD) pathology, and has been used to evaluate the effectiveness of c-secretase inhibition. Avagacestat is a selective c-secretase inhibitor in development for the treatment of AD. The primary objective of this study was to assess the effects of single oral doses of avagacestat on the CSF Ab concentrations in healthy male subjects. Secondary objectives included single-dose pharmacokinetics in CSF and plasma, safety and tolerability. Methods This was a double-blind, placebo-controlled, randomized, single-dose study. Healthy male subjects were assigned to one of three sequential avagacestat dose panels (50, 200 and 400 mg) or placebo as single oral doses. Results 34 subjects were enrolled. Administration of a single dose of 200 or 400 mg of avagacestat resulted in a marked decrease in CSF Ab1‐38 ,A b1‐40 and Ab1‐42 concentrations vs placebo; with smaller decreases observed in the 50 mg dose group. Avagacestat was quickly absorbed into the systemic circulation, with a mean time to reach maximum plasma concentration (tmax) of approximately 1‐2 h, and a CSF tmax of approximately 3 h. Adverse events were uncommon and occurred with similar frequency in the placebo and avagacestat groups. Conclusion Avagacestat was safe, well tolerated, and resulted in a notable decrease in CSF Ab concentrations, suggestive of c-secretase inhibition. The results warrant further clinical study in patients with AD.

A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

BMS-708163 is an oral γ-secretase inhibitor designed for selective inhibition of amyloid beta synthesis in late phase development for the treatment of Alzheimer's disease. The primary objective of this randomized, double-blind, 4-period, 4-treatment crossover study was to determine the effect of BMS-708163 on the QTc interval (QT interval corrected for heart rate). Each subject (N = 62) was exposed to 4 single-dose treatments, each separated by a washout period of ≥ 14 days. Treatment doses included BMS-708163 (200 mg oral solution), BMS-708163 (800 mg oral solution), moxifloxacin (400 mg, open-label), and matching placebo. Single doses of BMS-708163 were chosen to match (200 mg), or exceed (800 mg), peak concentrations achieved at steady state in Phase 2 studies following 125 mg QD. Two distinct sets of ECG data were collected during the study: serial triplicate ECG measurements and single 12-lead safety ECG measurements. Primary outcome measures were the change from baseline in QTc (▵QTc) and the time-matched difference between the mean ▵QTc after administration of BMS-708163 or moxifloxacin, and the mean ▵QTc after administration of placebo (▵▵QTc). Absence of a clinically relevant effect of BMS-708163 on QTc would be confirmed if the upper 90% confidence interval (CI) for ▵▵QTc was ≤ 10 msec at every time point. Fifty-five subjects completed the study. QTcF (Fridericia's correction) was the most appropriate formula to correct QT interval for heart rate. Assay sensitivity was confirmed with moxifloxacin (lower 90% CI for ▵▵QTc > 10 msec at each of the 2-, 3-, and 4-hour post-dose). Peak exposures comparable to 125 mg QD and peak exposures 3-times of that did not have any clinically relevant effect on QTc interval in healthy subjects (upper 90% CI for ▵▵QTc < 2 msec at each time point post-dose). Although a mean increase in heart rate (< 10 bpm) was observed in the 800 mg group, there was no apparent difference between any of the treatment groups with respect to mean QRS, ▵QRS, PR, or ▵PR profiles. Both single doses of BMS-708163 were well-tolerated and safe in healthy subjects. BMS-708163 (200 mg and 800 mg) had no clinically relevant effect on the QTc interval.

BMS-708163, in Phase II development for Alzheimer's Disease, selectively inhibits Aβ synthesis relative to Notch, and is a potent and orally active γ-secretase inhibitor. The primary objective of this double-blind, placebo-controlled, randomized, single-dose study was to assess the effects of single oral BMS-708163 doses on CSF Aβ. Healthy non-Japanese (n = 8) and Japanese (n = 3) males aged 20-45y were assigned to each of 3 sequential panels (50 mg, 200 mg, 400 mg) to receive BMS-708163 or placebo as a single oral dose. CSF samples were obtained prior to dosing and hourly until 39h post-dose. Plasma samples were collected until 144h post-dose. Thirty-four subjects were randomized; 33 completed the study. One subject in the placebo group discontinued from the study on Day 7 due to difficulties with the spinal fluid collection procedure. Median CSF Aβ1-40 concentrations 12h post-dose were 111%, 88%, 63%, and 60% of baseline values after placebo, 50-mg, 200-mg, and 400-mg doses of BMS-708163, respectively. Corresponding values for CSF Aβ1-38 were 111%, 90%, 60%, and 58%, respectively; and for CSF Aβ1-42 were 112%, 97%, 68%, and 66%, respectively. BMS-708163 was quickly absorbed (Tmax, 1-2h); Cmax and AUC at 200 mg and 400 mg were similar. Plasma profiles were biphasic, with a terminal half-life of ˜40h. Exposure for BMS-708163 in CSF was much lower (<1%) than in plasma. AEs occurred with similar frequency between drug and placebo; the most common AEs were headache (n = 12), post-lumbar puncture syndrome (n = 8), and back pain (n = 7). CSF Aβ reduction responses were similar in Japanese and non-Japanese; Japanese had higher mean plasma AUC0-t and AUCinf (17% and 39%, respectively) and higher mean CSF AUC0-t and AUCinf (5% and 69%, respectively) versus non-Japanese subjects, but the actual values were within the range of values for non-Japanese subjects. Single 200-mg or 400-mg BMS-708163 doses resulted in marked decreases in CSF Aβ; single 50-mg doses resulted in smaller and less-sustained decreases. Evidence of saturation in exposure was present at doses >200 mg. CSF exposure correlated with plasma exposure but was of lower magnitude. BMS-708163 was safe and well tolerated. CSF Aβ reduction responses were similar in Japanese and non-Japanese.

BMS-708163, in Phase II development for Alzheimer's Disease, selectively inhibits Aβ synthesis relative to Notch, and is a potent and orally active γ-secretase inhibitor. We evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of single ascending doses (SAD) and multiple ascending doses (MAD) of BMS-708163 in healthy young and elderly Japanese subjects in 2 randomized, double-blind, placebo-controlled studies. SAD: Healthy young Japanese men (n = 24) were randomized to single doses of BMS-708163 50, 100, or 200 mg, or placebo (sequential panels); MAD: Young Japanese men (n = 16) and elderly Japanese healthy volunteers (n = 16) were randomized to BMS-708163 50 or 100 mg or placebo for 14 days. SAD: There were no gastrointestinal tract-related adverse events (AEs) or clinically significant abnormalities in vital signs, electrocardiograms, clinical laboratory tests, fecal occult blood tests, stool frequency and consistency evaluations, urinary and salivary cortisol, thyroid function tests, or lymphocyte immunophenotyping. BMS-708163 was quickly absorbed (mean Tmax 1.0-1.3h); plasma profiles were biphasic; mean terminal T1/2 was ˜30h; plasma AUC was dose-proportional in the range 50-200 mg. BMS-708163 decreased plasma Aβ1-40 dose-dependently after dosing. MAD: The most common AE was positive fecal occult blood test (n = 7, all mild). BMS-708163 was quickly absorbed (median Tmax 1.0-1.5h); the exposure at 100 mg was 2- to 3-fold higher than that at 50 mg; mean terminal T1/2 was ˜32h (young) and ˜44-55h (elderly). BMS-708163 reduced plasma Aβ1-40 in a dose-dependent fashion. BMS-708163 was safe and well tolerated at single doses of up to 200 mg (young Japanese) and multiple doses up to 100 mg (young and elderly Japanese), with no evidence of Notch-related dose-limiting toxicities. In both studies, BMS-708163 demonstrated rapid absorption and dose-proportional exposure. T1/2 was longer in elderly patients vs. younger patients in the multiple-dose study. BMS-708163 produced dose-dependent marked decreases in plasma Aβ1-40 levels following single- and multiple-dose administration.

The ATP-binding cassette (ABC) transporter protein subfamily B1 line (ABCB1) transporter P-glycoprotein (P-gp) plays an important role in the blood-brain barrier limiting a broad spectrum of substrates from entering the central nervous system. In the present study, the transport activity of P-gp for sertraline, desmethylsertraline, bupropion, and the major metabolites of bupropion, threo-amino alcohol (TB), erythro-amino alcohol (EB), and hydroxy metabolite (HB) was studied using an ATPase assay in expressed human P-gp membranes by measuring concentrations of inorganic P(i) in expressed human P-gp membranes. Verapamil was included as a positive control. The Michaelis-Menten equation was used for characterizing the kinetic data. Sertraline and desmethylsertraline showed high affinity for P-gp. The V(max)/K(m) values of sertraline (1.6 min(-1) x 10(-3)) and desmethylsertraline (1.4 min(-1) x 10(-3)) were comparable with that of verapamil (1.7 min(-1) x 10(-3)). Bupropion and its three metabolites showed very weak affinity for P-gp, with V(max)/K(m) values lower than 0.01 min(-1) x 10(-3). The results of the present study indicate that sertraline and desmethylsertraline have high affinity for P-gp, whereas bupropion and its three major metabolites TB, EB, and HB have very weak affinity for P-gp. These findings may help to explain observed drug-drug interactions among antidepressants.