Pharmacodynamics of selective inhibition of γ-secretase by avagacestat.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid ß-peptide (Aß), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aß 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 Aß 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) Aß 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 Aß40 and Aß42 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 Aß40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aß 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.

Determination of relative Notch1 and gamma-secretase-related gene expression in puromycin-treated microdissected rat kidneys.

Notch signaling pathways are involved in the regulation of cell differentiation and are highly conserved across species. Notch ligand binding leads to gamma-secretase-mediated proteolytic cleavage of the Notch receptor releasing the Notch intracellular domain, resulting in its subsequent translocation into the nucleus and gene expression regulation. To investigate the level of expression of Notch signaling pathway components in microanatomic regions following renal injury, kidneys from untreated, vehicle control, and puromycin aminonucleoside (PA, 150 mg/kg)-treated rats were evaluated. Frozen tissue sections from rats were microdissected using laser capture microdissection (LCM) to obtain glomeruli, cortical (proximal) tubules, and collecting ducts, and relative gene expression levels of Presenilin1, Notch1 and Hes1 were determined. In untreated rats, the Notch1 expression in glomeruli was higher than in the proximal tubules and similar to that in collecting ducts, whereas Presenilin1 and Hes1 expressions were highest in the collecting ducts, followed by cortical tubules and glomeruli. Following PA-induced renal injury, Hes1 gene expression increased significantly in the glomeruli and tubules compared to the collecting ducts where no injury was observed microscopically. Although these data present some evidence of change in Notch signaling related to injury, the expression of Presenilin1, Notch1, and Hes1 in the microanatomic regions of the kidney following PA treatment were not significantly different when compared to controls. These results demonstrate that there are differences in Notch-related gene expression in the different microanatomic regions of the kidneys in rats and suggest a minimal role for Notch in renal injury induced by PA. In addition, this work shows that LCM coupled with the RT-PCR can be used to determine the relative differences in target gene expression within regions of a complex organ.

Investigations on the Relationship Between Ovarian, Endocrine, and Renal Findings in Nonclinical Safety Studies of the γ-Secretase Inhibitor Avagacestat.

Avagacestat, a gamma (γ)-secretase inhibitor that was in development for treatment of Alzheimer's disease, produced ovarian granulosa-thecal cell tumors in rats and dogs and a glomerulopathy with profound proteinuria in female rats. This report describes the results of follow-up investigative studies, including the use of ovariectomized (OVX) rats, to further characterize these findings and determine their mechanism(s). Ovarian proliferative changes in rats likely resulted from: (1) inhibition of Notch signaling pathways regulating ovarian follicular differentiation/development, characterized microscopically as altered ovarian cyclicity and/or ovarian follicular degeneration; (2) subsequent disruption of the hypothalamic-pituitary-ovarian axis due to ovarian atrophy with decreases in serum estrogen and progesterone (as low as 0.45× and 0.21× controls, respectively); and (3) chronic gonadotropin stimulation and pituitary hypertrophy/hyperplasia in response to the absence of negative feedback. Gonadotropin stimulation in rats was confirmed by increases in serum follicle-stimulating hormone (up to 7.75× controls) and luteinizing hormone (up to 5.84×). A similar nongenotoxic mechanism was likely responsible for the ovarian findings in dogs although changes in serum hormone levels were not detected. The dose- and time-dependent glomerulopathy with progression to chronic progressive nephropathy in female rats appears to be a direct effect of avagacestat and was not ameliorated with coadministration of 17ß-estradiol or an antihypertensive (enalapril) and was not present in control OVX rats. In contrast, adrenocortical hypertrophy in female rats was considered secondary to ovarian changes based on the absence of this finding in avagacestat-treated OVX rats and no increase in adrenocorticotropic hormone staining in the pituitary.

Nonclinical Safety Assessment of the γ-Secretase Inhibitor Avagacestat.

The toxicity of avagacestat, a sulfonamide-based gamma (γ)-secretase inhibitor that was in development as a treatment for Alzheimer's disease, was evaluated in a comprehensive nonclinical toxicology program that included 6-month and 1-year repeat-dose toxicity studies in rats and dogs, respectively. There was a spectrum of mechanism-based changes attributed to inhibition of Notch signaling that regulates the differentiation and proliferation of cells throughout development and in adult tissues. In both rats and dogs, ovarian follicular degeneration and atrophy and a low incidence of granulosa cell hyperplasia and benign granulosa-thecal cell tumors were observed. Gastrointestinal (GI) findings, including goblet cell metaplasia, dilatation of intestinal crypts/glands, mucosal epithelial necrosis and regeneration, and villous atrophy, were limited to dogs that had clinical evidence of GI toxicity. Other avagacestat-related findings attributed to interference with Notch signaling included decreases in peripheral lymphocytes (T and/or B cells) and lymphoid depletion in lymph nodes and the spleen in both species, as well as epiphyseal cartilage and trabecular bone changes in rats. Pharmacologically mediated decreases in brain and cerebrospinal fluid levels of ß-amyloid (Aß) peptides Aß40 and Aß42 and decreased expression of white blood cell mRNA levels of the Notch-regulated gene hairy and enhancer of split-1 confirmed target engagement at all doses. Reductions in brain Aß peptide levels (22 to 34%) in dogs after 1 year at exposures up to the no-observed-effect level for GI toxicity of 1.1× the human plasma exposure, and reversible GI changes at a 3.2× multiple, indicated that a sustained pharmacodynamic effect was attained at exposures without dose-limiting toxicity.

Targeting Prodromal Alzheimer Disease With Avagacestat: A Randomized Clinical Trial.

IMPORTANCE: Early identification of Alzheimer disease (AD) is important for clinical management and affords the opportunity to assess potential disease-modifying agents in clinical trials. To our knowledge, this is the first report of a randomized trial to prospectively enrich a study population with prodromal AD (PDAD) defined by cerebrospinal fluid (CSF) biomarker criteria and mild cognitive impairment (MCI) symptoms. OBJECTIVES: To assess the safety of the γ-secretase inhibitor avagacestat in PDAD and to determine whether CSF biomarkers can identify this patient population prior to clinical diagnosis of dementia. DESIGN, SETTING, AND PARTICIPANTS: A randomized, placebo-controlled phase 2 clinical trial with a parallel, untreated, nonrandomized observational cohort of CSF biomarker-negative participants was conducted May 26, 2009, to July 9, 2013, in a multicenter global population. Of 1358 outpatients screened, 263 met MCI and CSF biomarker criteria for randomization into the treatment phase. One hundred two observational cohort participants who met MCI criteria but were CSF biomarker-negative were observed during the same study period to evaluate biomarker assay sensitivity. INTERVENTIONS: Oral avagacestat or placebo daily. MAIN OUTCOMES AND MEASURE: Safety and tolerability of avagacestat. RESULTS: Of the 263 participants in the treatment phase, 132 were randomized to avagacestat and 131 to placebo; an additional 102 participants were observed in an untreated observational cohort. Avagacestat was relatively well tolerated with low discontinuation rates (19.6%) at a dose of 50 mg/d, whereas the dose of 125 mg/d had higher discontinuation rates (43%), primarily attributable to gastrointestinal tract adverse events. Increases in nonmelanoma skin cancer and nonprogressive, reversible renal tubule effects were observed with avagacestat. Serious adverse event rates were higher with avagacestat (49 participants [37.1%]) vs placebo (31 [23.7%]), attributable to the higher incidence of nonmelanoma skin cancer. At 2 years, progression to dementia was more frequent in the PDAD cohort (30.7%) vs the observational cohort (6.5%). Brain atrophy rate in PDAD participants was approximately double that of the observational cohort. Concordance between abnormal amyloid burden on positron emission tomography and pathologic CSF was approximately 87% (κ = 0.68; 95% CI, 0.48-0.87). No significant treatment differences were observed in the avagacestat vs placebo arm in key clinical outcome measures. CONCLUSIONS AND RELEVANCE: Avagacestat did not demonstrate efficacy and was associated with adverse dose-limiting effects. This PDAD population receiving avagacestat or placebo had higher rates of clinical progression to dementia and greater brain atrophy compared with CSF biomarker-negative participants. The CSF biomarkers and amyloid positron emission tomography imaging were correlated, suggesting that either modality could be used to confirm the presence of cerebral amyloidopathy and identify PDAD. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00890890.

Preclinical pharmacokinetics and in vitro metabolism of BMS-605339: a novel HCV NS3 protease inhibitor.

BMS-605339 is a potent HCV NS3 protease inhibitor that suppresses hepatitis C virus replication and was under investigation as an oral agent for the treatment of this disease. In vitro and in vivo studies were conducted in mouse, rat, dog, and monkey to characterize the pharmacokinetics and metabolism of this compound. BMS-605339 was predicted to be a moderate clearance compound in the human, based on human microsomal and hepatocyte data. Nearly all metabolism of BMS-605339 was oxidative; CYP3A4 is likely to play a key role in the metabolic clearance of this compound. Moderate to high Caco-2 permeability was observed for this compound, with the potential for P-glycoprotein involvement. The oral bioavailability of BMS-605339 was variable and dose dependent, suggesting low absorption, possibly because of transporter involvement. BMS-605339 possesses low intrinsic aqueous solubility and, in both rat and dog, administration of an aqueous suspension suggested that BMS-605339 absorption is likely solubility limited. Liver exposure of BMS-605339 was consistently higher than plasma exposure in all species tested (mouse, rat, and dog), indicating the potential for active uptake into hepatocytes. The overall preclinical pharmacokinetic profile supported the selection and development of BMS-605339 as a clinical candidate.

Safety and tolerability of the γ-secretase inhibitor avagacestat in a phase 2 study of mild to moderate Alzheimer disease.

OBJECTIVE: To assess the safety, tolerability, and pharmacokinetic and pharmacodynamic effects of the -secretase inhibitor avagacestat in patients with mild to moderate Alzheimer disease (AD). DESIGN: Randomized, double-blind, placebo-controlled,24-week phase 2 study. SETTING: Global, multicenter trial. PATIENTS: A total of 209 outpatients with mild to moderate AD were randomized into the double-blind treatment phase. The median age of the patients was 75 years,58.9% were APOE ε4 carriers, and baseline measures of disease severity were similar among groups. INTERVENTION: Avagacestat, 25, 50, 100, or 125 mg daily,or placebo administered orally daily. MAIN OUTCOME MEASURES: Safety and tolerability of avagacestat. RESULTS: Discontinuation rates for the 25-mg and 50-mg doses of avagacestat were comparable with placebo but were higher in the 100-mg and 125-mg dose groups.Trends for worsening cognition, as measured by change from baseline Alzheimer Disease Assessment Scale cognitive subscale score, were observed in the 100-mg and125-mg dose groups. Treatment-emergent serious adverse events were similar across placebo and treatment groups. The most common reason for discontinuation was adverse events, predominantly gastrointestinal anddermatologic. Other adverse events occurring more frequentlyin patients undergoing treatment included reversibleglycosuria (without associated serum glucose changes), nonmelanoma skin cancer, and asymptomaticmagnetic resonance imaging findings. Exploratory cerebrospinal fluid amyloid isoforms and tau biomarker analysis demonstrated dose-dependent but not statistically significant reductions in a small subset of patients. CONCLUSIONS: Avagacestat dosed at 25 and 50 mg daily was relatively well tolerated and had low discontinuation rates. The 100-mg and 125-mg dose arms were poorly tolerated with trends for cognitive worsening. Exploratory cerebrospinal fluid biomarker substudies provide preliminary support for -secretase target engagement,but additional studies are warranted to better characterize pharmacodynamic effects at the 25- and 50-mg doses.This study establishes an acceptable safety and tolerability dose range for future avagacestat studies in AD. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00810147