title: Capillary electrophoresis to assess drug metabolism induced in vitro using single CYP450 enzymes (Supersomes): application to the chiral metabolism of mephenytoin and methadone.
abstract: Capillary electrophoresis (CE) with multiwavelength absorbance detection is demonstrated to be an effective tool for the assessment of in vitro drug metabolism studies using microsomes containing single human cytochrome P450 enzymes (CYPs) expressed in baculovirus-infected insect cells (Supersomes). Mephenytoin (MEPH), dextromethorphan, diclofenac, caffeine, and methadone (MET) were successfully applied as test substrates for CYP2C19, CYP2D6*1, CYP2C9*1, CYP1A2, and CYP3A4, respectively. For each system, the CE-based assay could be shown to permit the simultaneous analysis of the parent drug and its targeted metabolite. Using a chiral micellar electrokinetic capillary chromatography assay, the aromatic hydroxylation of MEPH catalyzed by CYP2C19 could thereby be confirmed to be highly stereoselective, an aspect that is in agreement with data obtained via urinary analysis after intake of racemic MEPH by extensive metabolizer phenotypes. The MET to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) conversion was investigated with a chiral zone electrophoresis assay. Incubation of racemic and nonracemic MET with CYP3A4 revealed no stereoselectivity for the transformation to EDDP, whereas no EDDP formation was observed with CYP1A2. CYP2C9 and CYP2C19 provided enhanced formation of R-EDDP and CYP2D6 incubation resulted in the preferential conversion to S-EDDP. Investigations using racemic MET and human liver microsomes revealed a modest stereoselectivity with an R/S EDDP ratio < 1 which is similar to the in vivo findings in urine.
authors.affiliation: Department of Clinical Pharmacology, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland.
authors.forename: Francine
authors.initials: F
authors.lastname: Prost
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Thormann
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008617:Mephenytoin / Q000032:analysis / Q000378:metabolism*; D008691:Methadone / Q000032:analysis / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000378:metabolism; D011759:Pyrrolidines / Q000032:analysis*; D013237:Stereoisomerism
chemical_list: D004364:Pharmaceutical Preparations; D011759:Pyrrolidines; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D008617:Mephenytoin; D008691:Methadone; C010724:2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine