title: The effect of organic solvents on enzyme kinetic parameters of human CYP3A4 and CYP1A2 in vitro.
abstract: Abstract Enzyme kinetic parameters provide essential quantitative information about characterization of individual steps in drug metabolism. Such enzymes are located in a (partially) aqueous environment. For in vitro measurements potential lipophilic substrates regularly require organic solvents to achieve concentrations sufficient for access of the drug to the binding site of the enzyme. However, solvents may interact with the enzymes. In this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethyl sulfoxide (1% to 4%) on the assessment of km, Vmax and Clint for the metabolism of midazolam via CYP3A4 to 1-hydroxymidazolam and the metabolism of caffeine to paraxanthine via CYP1A2 using expressed enzymes in vitro. The presence of acetonitrile proved the highest apparent Vmax value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. The km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to 8-fold lower for solvent free samples compared to solvent containing samples. The present example suggests that effects of solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. These effects illustrate a difference for individual enzyme--substrate pairs, solvents, and solvent concentrations. What remains is the determination to which extent these effects compromise in vitro-in vivo extrapolations, and which solvents are most appropriate.
authors.affiliation: Department of Pharmacology, University of Cologne , Cologne , Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Rokitta
authors.forename: Kay
authors.initials: K
authors.lastname: Pfeiffer
authors.forename: Christina
authors.initials: C
authors.lastname: Streich
authors.forename: Henrik
authors.initials: H
authors.lastname: Gerwin
authors.forename: Uwe
authors.initials: U
authors.lastname: Fuhr
mesh_terms: D002110:Caffeine / Q000494:pharmacology; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008874:Midazolam / Q000494:pharmacology; D009930:Organic Chemicals / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry*
chemical_list: D009930:Organic Chemicals; D012997:Solvents; D002110:Caffeine; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D008874:Midazolam