title: Chiral capillary electrophoretic analysis of verapamil metabolism by cytochrome P450 3A4.
abstract: Cytochrome P450 (CYP), which is one of the most important enzymes in human liver, is responsible for a large portion of the first-pass metabolism of drugs. Many studies have focused on the determination of CYP activity by substrate assays. Most of them used liquid chromatography (LC) as analytical technique, while only a few studies used capillary electrophoresis (CE) for the separation and quantitation of reaction components. In this study, the feasibility of using CE in an in vitro metabolism study with CYP was tested. Verapamil was chosen as the substrate for CYP 3A4 isozyme (Supersome). A chiral capillary electrophoretic method was developed and validated for the simultaneous determination of R,S-verapamil (VER) and their major metabolites, R,S-norverapamil (NOR). A method for CYP 3A4 activity assay was proposed with VER as a probe. At the same time, the enantioselective metabolism of VER was studied. Michaelis-Menten constants of R- and S-VER were determined. S-VER was metabolised faster and more extensively than R-VER, with K(m)=167+/-23 microM, V(max)=3,418+/-234 pmol/min/mg for S-VER, and K(m)=168+/-35 microM, V(max)=2,502+/-275 pmol/min/mg for R-VER.
authors.affiliation: Laboratory of Pharmaceutical Chemistry and Drug Analysis, Faculty of Pharmaceutical Sciences, O&N2, PB 923, Herestraat 49, K.U. B-3000 Leuven, Leuven, Belgium.
authors.forename: Pham Thi Thanh
authors.initials: PT
authors.lastname: Ha
authors.forename: Inge
authors.initials: I
authors.lastname: Sluyts
authors.forename: Sigrid
authors.initials: S
authors.lastname: Van Dyck
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Ron A H J
authors.initials: RA
authors.lastname: Gilissen
authors.forename: Jos
authors.initials: J
authors.lastname: Hoogmartens
authors.forename: Ann
authors.initials: A
authors.lastname: VanSchepdael
mesh_terms: D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D015394:Molecular Structure; D015203:Reproducibility of Results; D013237:Stereoisomerism; D014700:Verapamil / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; C016904:norverapamil; D014700:Verapamil; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A