Abstract
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 (MAb2D6–50, IgG2b and MAb2D6–184, IgG2a) 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 6β-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.,KI , KS ,Vmax, α, and β (changes inKI or KS andVmax in the presence of the MAb). As a result, dissociation constants (KI ) of the MAbs for the enzymes and the maximal inhibition (β) values for the P450-catalyzed reactions were predicted to have 0.04 to 0.25 μM and ≥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.
Footnotes
- Abbreviations used are::
- P450
- cytochrome P450
- OR
- cytochrome P450 oxidoreductase
- MAb
- monoclonal antibody
- KPi
- potassium phosphate buffer
- ELISA
- enzyme-linked immunosorbent assay
- HPLC
- high-performance liquid chromatography
- LC-MS
- liquid chromatography-mass spectrometry
- RSS
- residual sum of square
- Received December 21, 2001.
- Accepted March 5, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|