RT Journal Article
SR Electronic
T1 Methanol Solvent May Cause Increased Apparent Metabolic Instability in in Vitro Assays
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 185
OP 193
VO 29
IS 2
A1 Hequn Yin
A1 Phi Tran
A1 Gerald E. Greenberg
A1 Volker Fischer
YR 2001
UL http://dmd.aspetjournals.org/content/29/2/185.abstract
AB 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 apparentKm of 35 mM and aVmax of 7.9 nmol/min/mg of protein. The concentration of formaldehyde reached as high as 600 μM 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. The American Society for Pharmacology and Experimental Therapeutics