DMD #048264 1 Discovery and Characterization of Novel, Potent ...

DMD #048264
role of CYP2J2 in drug metabolism and its potential involvement in drug-drug interaction
in a clinical setting.
Materials and Methods
Materials. CYP substrates, inhibitors, metabolite standards, and all other
materials were obtained from the following sources: all compounds from Table 1, except
olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome
(HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol,
omeprazole, 4'-hydroxytolbutamide, 1’-hydroxybufuralol, 6β-hydroxytestosterone,
acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH)
were purchased from Sigma-Aldrich (St. Louis, MO, USA); testosterone was purchased
from Acros Organics (Morris Plains, NJ, USA); 5’-hydroxyomeprazole was purchased
from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-
desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA); potassium phosphate (monobasic and dibasic) and magnesium chloride
hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs
and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA, USA);
high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO),
methanol, and formic acid used for liquid chromatography-tandem mass spectrometry
(LC-MS/MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA, USA).
CYP2J2 Activity Study. Astemizole O-demethylation, a well-known reaction
catalyzed by CYP2J2, was measured and characterized in all studies to evaluate CYP2J2
activity, and hereafter it will be the functional assay used for CYP2J2 activity. The
substrate, astemizole, was diluted sequentially by DMSO to yield the final required
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