METABOLISM OF CYCLOSPORINE-A .4. PURIFICATION AND IDENTIFICATION OF THE RIFAMPICIN-INDUCIBLE HUMAN-LIVER CYTOCHROME-P-450 (CYCLOSPORINE-A OXIDASE) AS A PRODUCT OF P450IIIA GENE SUBFAMILY

  • 1 March 1989
    • journal article
    • research article
    • Vol. 17  (2) , 197-207
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 Ka(app) of 25 .mu.M, 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 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 in NF oxidase, or a very closely related gene, and is strongly inducible by rifampicin pretreatment.