1. The effects of tangeretin, green tea flavonoids, and other flavonoids on 7-ethoxyresorufin-O-deethylase (EROD; 450 1A), 7-pentoxyresorufin-O-dealkylase (PROD; P450 2B), p-nitrophenol hydroxylase (PNPH, P450 2E1), and erythromycin-N-demethylase (ERDM; P450 3A) were examined in induced rat liver microsomes. EROD, PNPH, ERDM, and nifedipine oxidase (NIFO; P450 3A4) were examined in human liver microsomes. 2. All flavonoids tested inhibited EROD activity at higher concentrations in liver microsomes. Flavone and tangeretin were potent inhibitors of EROD, with IC50's of 0.7 and 0.8 microM respectively in rat liver microsomes and 0.15 and 16 microM respectively in human liver microsomes. The green tea flavonoid (-)-epicatechin-3-gallate (ECG) was the most potent inhibitor of EROD in human liver microsomes (IC50 = 75 microM). The effect of the green tea flavonoids on EROD was complex; in addition to inhibition at high concentrations of flavonoid, moderate activation was seen at lower concentrations. 3. 450 2B-, 2E1- and 3A-dependent activities in rat and human liver microsomes were only moderately inhibited by any of the flavonoids tested, and, in general, ECG was the most potent inhibitor for these activities with IC50's ranging from 75 to 300 microM. 4. Tangeretin inhibited EROD activity (P450 1A2) in human liver microsomes in a competitive manner with a Ki = 68 nM. Tangeretin inhibited NIFO activity (P450 3A4) in human liver microsomes in an uncompetitive manner with Ki = 72 microM.

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A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid b-peptide (Ab), generated by g-secretasemediated cleavage of the amyloid precursor protein (APP). Therefore, g-secretase inhibitors (GSIs) may lower brain Ab and offer a potential new approach to treat AD. As g-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 Ab 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) Ab 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 Notchregulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Ab40 and Ab42 levels similarly. Chronic administration in rats and dogs, and 28-day, single-and multipleascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Ab40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Ab 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.

BMS-986094, a 2'-C-methylguanosine prodrug that was in development for treatment of chronic hepatitis C infection was withdrawn from Phase 2 clinical trials because of unexpected cardiac and renal adverse events. Investigative nonclinical studies were conducted to extend the understanding of these findings using more comprehensive endpoints. BMS-986094 was given orally to female CD-1 mice (25 and 150 mg/kg/d) for 2 weeks (53/group) and to cynomolgus monkeys (15 and 30 mg/kg/d) for up to 6 weeks (2-3/sex/group for cardiovascular safety, and 5/sex/group for toxicology). Endpoints included toxicokinetics; echocardiography, telemetric hemodynamics and electrocardiography, and tissue injury biomarkers (monkey); and light and ultrastructural pathology of heart, kidney, and skeletal muscle (mouse/monkey). Dose-related and time-dependent findings included: severe toxicity in mice at 150 mg/kg/d and monkeys at 30 mg/kg/d; decreased left ventricular (LV) ejection fraction, fractional shortening, stroke volume, and dP/dt; LV dilatation, increased QTc interval, and T-wave flattening/inversion (monkeys at ≥ 15 mg/kg/d); cardiomyocyte degeneration (mice at 150 mg/kg/d and monkeys at ≥ 15 mg/kg/d) with myofilament lysis/myofbril disassembly; time-dependent proteinuria and increased urine β-2 microglobulin, calbindin, clusterin; kidney pallor macroscopically; and tubular dilatation (monkeys); tubular regeneration (mice 150 mg/kg/d); and acute proximal tubule degeneration ultrastructurally (mice/monkeys); and skeletal muscle degeneration with increased urine myoglobin and serum sTnI. These studies identified changes not described previously in studies of BMS-986094 including premonitory cardiovascular functional changes as well as additional biomarkers for muscle and renal toxicities. Although the mechanism of potential toxicities observed in BMS-986094 studies was not established, there was no evidence for direct mitochondrial toxicity.

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