Metabolism and disposition of the metabotropic glutamate receptor 5 antagonist (mGluR5) mavoglurant (AFQ056) in healthy subjects.

The disposition and biotransformation of (14)C-radiolabeled mavoglurant were investigated in four healthy male subjects after a single oral dose of 200 mg. Blood, plasma, urine, and feces collected over 7 days were analyzed for total radioactivity, mavoglurant was quantified in plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and metabolite profiles were generated in plasma and excreta by high-performance liquid chromatography (HPLC) and radioactivity detection. The chemical structures of mavoglurant metabolites were characterized by LC-MS/MS, wet-chemical and enzymatic methods, NMR spectroscopy, and comparison with reference compounds. Mavoglurant was safe and well tolerated in this study population. Mavoglurant absorption was ≥50% of dose reaching mean plasma Cmax values of 140 ng/ml (mavoglurant) and 855 ng-eq/ml (total radioactivity) at 2.5 and 3.6 hours, respectively. Thereafter, mavoglurant and total radioactivity concentrations declined with mean apparent half-lives of 12 and 18 hours, respectively. The elimination of mavoglurant occurred predominantly by oxidative metabolism involving primarily 1) oxidation of the tolyl-methyl group to a benzyl-alcohol metabolite (M7) and subsequently to a benzoic acid metabolite (M6), and 2) oxidation of the phenyl-ring leading to a hydroxylated metabolite (M3). The subjects were mainly exposed to mavoglurant and seven main metabolites, which combined accounted for 60% of (14)C-AUC0-72 h (area under the concentration-time curve from time 0 to infinity). The primary steps of mavoglurant metabolism observed in vivo could partially be reproduced in vitro in incubations with human liver microsomes and recombinant cytochrome P450 enzymes. After 7 days, the mean balance of total radioactivity excretion was almost complete (95.3% of dose) with 36.7% recovered in urine and 58.6% in feces.

Micropreparative isolation and NMR structure elucidation of metabolites of the drug candidate 1-isopropyl-4-(4-isopropylphenyl)-6-(prop-2-yn-1-yloxy) quinazolin-2(1H)-one from rat bile and urine.

LC-MS based drug metabolism studies are effective in the optimization stage of drug discovery for rapid partial structure identification of metabolites. However, these studies usually do not provide unambiguous structural characterization of all metabolites, due to the limitations of MS-based structure identification. LC-MS-SPE-NMR is a technique that allows complete structure identification, but is difficult to apply to complex in vivo samples (such as bile collected during in vivo drug metabolism studies) due to the presence, at high concentrations, of interfering endogenous components, and potentially also dosage excipient components (e.g. polyethylene glycols). Here, we describe the isolation and structure characterization of seven metabolites of the drug development candidate 1-isopropyl-4-(4-isopropylphenyl)-6-(prop-2-yn-1-yloxy) quinazolin-2(1H)-one from a routine metabolism study in a bile-duct cannulated rat by LC-MS-SPE. The metabolites were isolated from bile and urine by repeated automatic trapping of the chromatographic peak of each metabolite on separate Oasis HLB SPE columns. The micropreparative HPLC/MS was performed on an XBridge BEH130 C18 HPLC column using aqueous formic acid/acetonitrile/methanol as mobile phase for the gradient elution. Mass spectrometric detection was performed on a LTQ XL linear ion trap mass spectrometer using electrospray ionization. Desorption of each metabolite was performed after the separation sequence. NMR spectra ((1)H, (13)C, 2D ROESY, HSQC and HMBC were measured on a Bruker AVANCE III spectrometer (600 MHz proton frequency) equipped with a 1.7 mm (1)H{(13)C,(15)N} Bruker Biospin's TCI MicroCryoProbe™.