Inhibition kinetics of monoclonal antibodies against cytochromes P450.
Monoclonal antibodies (MAbs) inhibitory to individual cytochromes P450 (P450s) are of tremendous utility in identification of P450s responsible for the metabolism of a given drug or drug candidate in pharmaceuticals. In the present study, two inhibitory MAbs against CYP2D6 (MAb(2D6-50,) IgG(2b) and MAb(2D6-184), IgG(2a)) were developed by hybridoma technology to exhibit their high specificity and potency. The MAbs were further employed to assess the quantitative role (47-93%) of CYP2D6 to the metabolism of bufuralol in human liver microsomes from seven donors. Together with the MAb inhibitory to CYP3A4 as previously reported (Mei et al., 1999), the MAbs were used to study the inhibition kinetics of dextromethorphan O-demethylation (CYP2D6), testosterone 6beta-hydroxylation (CYP3A4) and aflatoxin B1 3-hydroxylation (CYP3A4), respectively, with an adequate size of sample measurement. A kinetic model was proposed to fit the experimental observations with three-dimensional nonlinear regression, thereby resulting in a solution of kinetic parameters, i.e., K(I), K(S), V(max), alpha, and beta (changes in K(I) or K(S) and V(max) in the presence of the MAb). As a result, dissociation constants (K(I)) of the MAbs for the enzymes and the maximal inhibition (beta) values for the P450-catalyzed reactions were predicted to have 0.04 to 0.25 microM and > or =94%, respectively. The results have demonstrated that the model can accurately predict the kinetic parameters and provide some insights into the understanding of the mechanism of MAb interaction with P450 enzyme in nature and the applications of the MAbs in qualitative and quantitative identification of P450s involved in drug metabolism.
Mei
Qin
Q
Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Tang
Cuyue
C
Lin
Yuh
Y
Rushmore
Thomas H
TH
Shou
Magang
M
D000348:Aflatoxins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000918:Antibody Specificity; D019389:Cytochrome P-450 CYP2D6 / Q000276:immunology / Q000378:metabolism; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism; D003915:Dextromethorphan / Q000378:metabolism; D004983:Ethanolamines / Q000378:metabolism; D006801:Humans; D006825:Hybridomas; D006900:Hydroxylation; D007074:Immunoglobulin G / Q000494:pharmacology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008253:Macaca mulatta; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D008954:Models, Biological; D051381:Rats; D013045:Species Specificity; D013739:Testosterone / Q000378:metabolism
D000348:Aflatoxins; D000911:Antibodies, Monoclonal; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004983:Ethanolamines; D007074:Immunoglobulin G; D007527:Isoenzymes; D013739:Testosterone; D003915:Dextromethorphan; C010831:bufuralol; D003577:Cytochrome P-450 Enzyme System; D019389:Cytochrome P-450 CYP2D6