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CYP3A4-Mediated Carbamazepine (CBZ) Metabolism: Formation of a Covalent CBZ-CYP3A4 Adduct and Alteration of the Enzyme Kinetic Profile

Departments of Cellular & Molecular Pharmacology, Biopharmaceutical Sciences, Pharmaceutical Chemistry and the Liver Center, University of California, San Francisco, California (P.K., M.L., M.A.C.); Pfizer Global Research & Development, San Diego, California (M.R.W.); and Section of Developmental Pharmacology and Experimental Therapeutics, the Children's Mercy Hospitals & Clinics, Kansas City, Missouri (J.S.L., R.E.P.)

Carbamazepine (CBZ) is a widely prescribed anticonvulsant whose use is often associated with idiosyncratic hypersensitivity. Sera of CBZ-hypersensitive patients often contain anti-CYP3A antibodies, including those to a CYP3A23 K-helix peptide that is also modified during peroxidative CYP3A4 heme-fragmentation. We explored the possibility that cytochromes P450 (P450s) such as CYP3A4 bioactivate CBZ to reactive metabolite(s) that irreversibly modify the P450 protein. Such CBZ-P450 adducts, if stable in vivo, could engender corresponding serum P450 autoantibodies. Incubation with CBZ not only failed to inactivate functionally reconstituted, purified recombinant CYP3A4 or CYP3A4 Supersomes in a time-dependent manner, but the inclusion of CBZ (0–1 mM) also afforded a concentration-dependent protection to CYP3A4 from inactivation by NADPH-induced oxidative uncoupling. Incubation of CYP3A4 Supersomes with ³H-CBZ resulted in its irreversible binding to CYP3A4 protein with a stoichiometry of 1.58 ± 0.15 pmol ³H-CBZ bound/pmol CYP3A4. Inclusion of glutathione (1.5 mM) in the incubation reduced this level to 1.09. Similar binding (1.0 ± 0.4 pmol ³H-CBZ bound/pmol CYP3A4) was observed after ³H-CBZ incubation with functionally reconstituted, purified recombinant CYP3A4(His)₆. The CBZ-modified CYP3A4 retained its functional activity albeit at a reduced level, but its testosterone 6β-hydroxylase kinetics were altered from sigmoidal (a characteristic profile of substrate cooperativity) to near-hyperbolic (Michaelis-Menten) type, suggesting that CBZ may have modified CYP3A4 within its active site.

This article has been cited by other articles:

				R. E. Pearce, W. Lu, Y. Wang, J. P. Uetrecht, M. A. Correia, and J. S. Leeder Pathways of Carbamazepine Bioactivation in Vitro. III. The Role of Human Cytochrome P450 Enzymes in the Formation of 2,3-Dihydroxycarbamazepine Drug Metab. Dispos., August 1, 2008; 36(8): 1637 - 1649. [Abstract] [Full Text] [PDF]