Methanol solvent may cause increased apparent metabolic instability in in vitro assays.

Methanol was widely used as a substrate-delivering solvent in in vitro metabolic stability screenings. Its interaction with enzyme activities, particularly those of cytochrome P450s, has been investigated extensively in the past. Little was known about the interaction of methanol, whether direct or indirect, with substrates. The present study provided data for the first time to show that use of methanol may result in the formation of artifacts, which could mislead the metabolic stability information. The disappearance of LAQ094, metaraminol, and (-)-isoproterenol following 1-h incubation with human liver microsomes was 73, 85, and 66%, respectively, in the presence of 1% methanol, but was only 3, 15, and 24%, respectively, in the absence of organic solvent. The dramatically increased instability in the presence of methanol of these three compounds, each with 1,2-diamino or 1,2-amino hydroxy functional groups, was due to the formation of [M + 12] products resulting from condensation reaction of the substrates with formaldehyde. Formaldehyde was formed from methanol by human liver microsomal enzymes with an apparent K(m) of 35 mM and a V(max) of 7.9 nmol/min/mg of protein. The concentration of formaldehyde reached as high as 600 microM following a 60-min incubation. The [M + 12] products were characterized as five-membered heterocycles by liquid chromatography and tandem mass spectrometry analysis. Inclusion of 10 mM glutathione prevented the formation of such artifacts and is therefore suggested for future in vitro screenings. Our study also documented the novel finding of enzyme-dependent conversion of NADPH to nicotinamide in microsomal incubations.

Automated-extraction, high-performance liquid chromatographic method and pharmacokinetics in rats of a highly A2-selective adenosine agonist, CGS 21680.

CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine, I) is a highly A2-selective (A2/A1 = 140), high-affinity adenosine agonist. A method has been devised to extract the compound from biological matrices with automated solid-phase extraction using C18 bonded silica columns. This is followed by reversed-phase, paired-ion chromatography on a Supelco LC-18-S column with fluorescence detection. The limit of quantitation is 5 ng/ml, but 1 ng/ml (five times the signal-to-noise ratio) can readily be detected. Tritium-labeled compound was used to study the pharmacokinetics in rats. After an intravenous dose of 0.3 mg/kg, biphasic elimination kinetics were observed for parent I, characterized by half-lives of 1.8 min (distribution) and 15 min (elimination). The volume of distribution in the terminal phase (V beta) was low (0.27 l/kg) and plasma clearance was moderate (0.83 l/kg/h). Although the compound was rapidly absorbed (mean Tmax = 13 min), low concentrations (mean Cmax = 94 ng/ml) were observed after an oral dose of 3.0 mg/kg, and bioavailability was only approximately 1.4%. Radioactivity persisted in plasma longer than parent compound after either dose, but levels were too low for isolation and structure identification of drug-derived compounds.