Metabolism of cyclosporin A. IV. Purification and identification of the rifampicin-inducible human liver cytochrome P-450 (cyclosporin A oxidase) as a product of P450IIIA gene subfamily

Abstract

A cytochrome P-450 involved in the metabolism of cyclosporin A (CsA) was isolated and purified to electrophoretic homogeneity from human liver microsomes of renal transplant donors. This cytochrome, designated P-450(CsA), exhibited a type I binding spectrum in the presence of CsA with a Ks(app) of 25 microM, a molecular weight of 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a maximal absorbance at 449 nm when reduced in the presence of carbon monoxide. The N-terminal sequence of P-450(CsA), determined by Edman degradation reaction, was 63% homologous with that of the rabbit liver CsA oxidase P-450 3c and 100% homologous with that of the human liver isozyme P-450(HLp/NF), recently identified as the human nifedipine (NF) oxidase. Polyclonal and monoclonal antibodies directed against P-450 3c and P-450(HLp/NF), respectively, recognized native microsomal and highly purified P450(CsA). As observed in the rabbit, human liver microsomes were shown to generate mono- and dihydroxy, as well as dihydroxy and/or monohydroxy N-demethylated, derivatives of CsA. Production of these metabolites was shown to be specifically inhibited by anti-P-450 3c polyclonal antibodies. CsA oxidase, NF oxidase, and erythromycin demethylase were shown to be closely correlated with the level of P-450(CsA) determined from Western blot or enzyme-linked immunosorbent assay. Moreover, these monoxygenase activities and the hepatic level of P-450(CsA) were simultaneously increased in the liver of patients treated for 4 days with 600 mg of rifampicin per day. Finally, NF was shown to be a competitive inhibitor of CsA oxidation and vice versa. We conclude that P-450(CsA) is responsible for most (80%) of CsA oxidase activity in human liver, is encoded by gene P450IIIA3, as is NF oxidase, or a very closely related gene, and is strongly inducible by rifampicin pretreatment.