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 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.
Footnotes
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This work was supported by National Institutes of Health (NIH) Grant GM58883 to J.S.L. and M.A.C., NIH Grants DK26506 and GM44037 to M.A.C., and NIH Grant P30DK26743.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.016501.
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ABBREVIATIONS: CBZ, carbamazepine; P450, cytochrome P450; GSH, glutathione; EtOAc, ethyl acetate; b5, cytochrome b5; PAGE, polyacrylamide gel electrophoresis; MDZ, midazolam; P450 reductase, NADPH-cytochrome P450 reductase; Ni2+-NTA, nickel-nitrilotriacetic acid.
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↵1 Current affiliation: Pfizer Global Research & Development, San Diego, CA.
- Received May 3, 2007.
- Accepted December 14, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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