title: CYP3A4-Mediated carbamazepine (CBZ) metabolism: formation of a covalent CBZ-CYP3A4 adduct and alteration of the enzyme kinetic profile.
abstract: 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 (3)H-CBZ resulted in its irreversible binding to CYP3A4 protein with a stoichiometry of 1.58 +/- 0.15 pmol (3)H-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 (3)H-CBZ bound/pmol CYP3A4) was observed after (3)H-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 6beta-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.
authors: Department of Cellular & Molecular Pharmacology, Liver Center, University of California, San Francisco, 600 16th Street N572F, San Francisco, CA 94158-2280, USA.
authors: Ping
authors: P
authors: Kang
authors: Mingxiang
authors: M
authors: Liao
authors: Michael R
authors: MR
authors: Wester
authors: J Steven
authors: JS
authors: Leeder
authors: Robin E
authors: RE
authors: Pearce
authors: Maria Almira
authors: MA
authors: Correia
mesh_terms: D002220:Carbamazepine / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D018360:Crystallography, X-Ray; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004917:Erythromycin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000378:metabolism; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002220:Carbamazepine; D013739:Testosterone; D004917:Erythromycin; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D051544:Cytochrome P-450 CYP3A; C022657:steroid hormone 6-beta-hydroxylase; C510163:CYP3A4 protein, human