Two cDNAs (pLM3c 4.1 and pLM3c 6.1) coding for rabbit cytochrome P450 3c were sequenced. cDNA 4.1 (1768 bp) exhibits an open reading frame from nucleotides 74 to 1576 encoding the 501 amino acid residues of the entire protein. cDNA 6.1 (189 bp) appears to encode the last 24 amino acids. Comparative amino acid sequence analysis indicated that P450 PCN1, PCN2, and HLp from rat and man, were 70, 67, and 73% homologous, respectively, to P450 3c. According to the cytochrome P450 nomenclature, the P450 3c gene is termed P450IIIA4. Comparison of the nucleotide sequences indicated that cDNA 6.1 was 100% homologous to cDNA 4.1. However, whereas a poly(A) tract started 23 nucleotides after the AATAAA consensus sequence in cDNA 6.1, cDNA 4.1 had a 3' untranslated region extending 101 bp beyond the polyadenylation signal, which lacked poly(A). This observation is consistent with the previous finding that both cDNA 4.1 and 6.1 hybridized with two distinct species of poly(A)RNA (1700 and 1850 bases) from rabbit liver. The extreme 3'-end 79-bp fragment of cDNA 4.1 therefore was isolated by subcloning in pUC12 (clone p18-Rsa I) and used to probe Northern blots of poly(A)RNA from control and rifampicin-treated rabbit liver. In contrast to cDNA 4.1 and 6.1, p18-Rsa I cDNA hybridized only with the largest (1850 bases) mRNA species. We conclude that rabbit liver contains two P450 3c mRNA species differing in the length of their 3' untranslated region.

Amino Acid Sequence↗

PubMedDate not supplied

Cyclosporin A drug interactions. Screening for inducers and inhibitors of cytochrome P-450 (cyclosporin A oxidase) in primary cultures of human hepatocytes and in liver microsomes.

In previous papers we demonstrated that cyclosporin A (CsA) was specifically oxidized in rabbit and human liver by cytochrome P-450IIIA. We therefore anticipated that any drug that is an inducer or an inhibitor of this cytochrome should lead to interaction with CsA when given in association with it. In order to confirm this hypothesis, primary cultures of human hepatocytes and human liver microsomes were used to "reproduce" in vitro clinically significant interactions observed between CsA and drugs known either as specific inducers (i.e., rifampicin) or as specific inhibitors (i.e., erythromycin) of P-450IIIA. Our results were in close agreement with the clinical reports. Human hepatocytes maintained in primary cultures for 72 hr in the presence of 50 microM rifampicin exhibited increased levels of P-450IIIA, determined by Western blot using specific antibodies, and concomitant increase in CsA oxidase activity, determined by HPLC analysis of extra and intracellular media. Conversely, these cultures exhibited erythromycin concentration-dependent decreases in CsA oxidase activity when incubated in the presence of 5, 20, and 100 microM erythromycin. In addition, a Lineweaver-Burk analysis of the erythromycin-mediated inhibition of CsA oxidase activity in human liver microsomes revealed competitive inhibition (with Ki of 75 microM) as expected, this macrolide being a specific substrate of P-450IIIA. Using this experimental approach, 59 molecules representative of 17 different therapeutic classes were screened for inducers and inhibitors of CsA oxidase activity. Our results allowed us to elucidate the molecular mechanism of previously observed, but unexplained, drug interactions involving CsA, and to detect drugs that should interfere with CsA metabolism as inducers or inhibitors. Drugs detected as potential inducers of CsA oxidase included: rifampicin, sulfadimidine, phenobarbital, phenytoin, phenylbutazone, dexamethasone, sulfinpyrazone, and carbamazepine. Drugs detected as potential competitive inhibitors included: triacetyloleandomycin, erythromycin, josamycin, midecamycin, ketoconazole, miconazole, midazolam, nifedipin, diltiazem, verapamil, nicardipine, ergotamine, dihydroergotamine, glibenclamide, bromocriptine, ethynylestradiol, progesterone, cortisol, prednisone, prednisolone, and methylprednisolone. Finally, cefoperazone, cefotaxime, ceftazidime, isoniazide, doxycycline, spiramycin, sulfamethoxazole, norfloxacin, pefloxacin, vancocin, trimethoprim, amphotericin B, valproic acid, quinidine, cimetidine, ranitidine, omeprazole, diclofenac, aspirin, paracetamol, debrisoquine, guanoxan, captopril, furosemide, acetazolamide, sparteine, gliclazide, and imipramine were found not to interfere with the hepatic metabolism of CsA.

Blotting, Western↗

PubMedDate not supplied

Identification of the rabbit and human cytochromes P-450IIIA as the major enzymes involved in the N-demethylation of diltiazem.

Oxidative metabolism of diltiazem (DTZ), a calcium channel blocker, was investigated in rabbit and human liver microsomes as well as in primary cultures of human hepatocytes. DTZ N-demethylation, the major metabolic pathway in man, was strongly increased by treatment of animals, patients, and hepatocyte cultures with rifampicin and other inducers of the P-450IIIA subfamily. In a reconstituted system with purified forms of P-450 and NADPH cytochrome P-450 reductase, P-450IIIA7 exhibited the highest DTZ N-demethylase activity. In both rabbit and human liver microsomes, this activity was highly correlated with erythromycin demethylase, a characteristic substrate of P-450IIIA, or with an immunoquantitated level of P-450IIIA, and was specifically inhibited by anti-P-450IIIA7 polyclonal and monoclonal antibodies. Cyclosporin A, another specific substrate of P-450IIIA in rabbit and human, competitively inhibited DTZ N-demethylase in both species. In primary cultures of human hepatocytes treated with various inducers, including rifampicin, dexamethasone, phenobarbital, phenylbutazone or beta-naphthoflavone, the rate of release of N-demethyl-DTZ in the extracellular medium was highly correlated with the intracellular level of P-450IIIA, which appeared to be strongly induced by rifampicin and phenobarbital and to a lesser extent by dexamethasone and phenylbutazone. In aggregate, these results are consistent with the view that in both rabbit and human, cytochromes P-450 from the P-450IIIA subfamily are the major enzymes involved in the N-demethylation of DTZ. Accordingly, drugs which may be specific substrates or inducers of this P-450 are likely to influence both the side effects and the efficacy of this molecule.

Adult↗

PubMedDate not supplied

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.

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.

Blotting, Western↗

PubMedDate not supplied

Metabolism of cyclosporin A. I. Study in freshly isolated rabbit hepatocytes.

The metabolism of cyclosporin A (CsA), a widely used immunosuppressive agent, was evaluated in freshly isolated rabbit hepatocytes by HPLC which separated CsA from its major group of derivatives, e.g. "first generation" metabolites (monohydroxylated and N-demethylated) and "second generation" derivatives (dihydroxylated and dihydroxy-N-demethylated). After exposure of hepatocytes to radiolabeled CsA (0.5 mg/liter), CsA was rapidly accumulated inside the cells and metabolized. The dihydroxylated metabolites represent the major intracellular forms after 1 hr. CsA metabolites synthesized inside the cells are then rapidly detected in the extracellular compartment. Unchanged drug and the various metabolites are concentrated inside the cells with transmembrane chemical gradients ranging between 20:1 and 40:1. Transport and metabolic processes for CsA have been evaluated over the following CsA extracellular concentration range, 0.1-10 mg/liter. Metabolism appears to be the rate-limiting step. The apparent affinity constant of CsA for the enzyme system involved in its metabolism is approximately 15 microM. Besides the lipophilicity of the molecule, which is responsible for the retention of CsA and its metabolites in the intracellular compartment, the presence of a binding component(s) in the hepatocytes was also demonstrated. CsA and its metabolites seem to have similar affinities for this binding site. These studies demonstrate that CsA is rapidly transformed inside the hepatocytes to various metabolites which may play an important role in the pharmacological activity of the drug and/or in its clinical toxicity.

Animals↗

PubMedDate not supplied

Metabolism of cyclosporin A. II. Implication of the macrolide antibiotic inducible cytochrome P-450 3c from rabbit liver microsomes.

The in vitro metabolism of cyclosporin A (CsA) was investigated by rabbit liver microsomes in order to identify the form(s) of cytochrome P-450 responsible for its biotransformation. Metabolites including monohydroxy-, N-demethylated, dihydroxy- and dihydroxy-N-demethylated derivatives were detected and quantified by HPLC from incubates of liver microsomes, CsA, and NADPH. Kinetic data indicated that monohydroxy- and N-demethylated derivatives were first generated and then served as substrates for production of dihydroxylated derivatives. Liver microsomes from phenobarbital-, beta-naphthoflavone-, triacetyloleandomycin-, erythromycin-, or rifampicin-treated and untreated rabbits were investigated, but only microsomes from animals treated with macrolide antibiotics (specific inducers of form P-450 3c) exhibited a type I binding spectrum upon CsA addition (Ks = 1.5 +/- 0.5 microM) and extensively metabolized the drug to all groups of derivatives (Km = 5.0 +/- 0.5 microM, Vmax = 1.0 +/- 0.2 nmol/mg/min). A linear correlation existed between CsA oxidase activity and P-450 3c specific content. Antibodies to P-450 3c strongly inhibited CsA oxidase activity of microsomes from macrolide antibiotic-induced animals, whereas antibodies to other forms, including P-450 2, 3b, 4, and 6, did not. When highly purified forms of P-450, including P-450 2, 3b, 3c, and 4, were assayed in a reconstituted system, only P-450 3c exhibited type I binding spectrum upon CsA addition (Ks = 1.4 +/- 0.5 microM) and extensively metabolized the drug to all derivatives. We conclude that the macrolide antibiotic-inducible form P-450 3c (or P-450 3c related from(s)) is responsible for the major part of CsA metabolism by rabbit liver microsomes.

Animals↗

PubMedDate not supplied

Metabolism of cyclosporin A. III. Interaction of the macrolide antibiotic, erythromycin, using rabbit hepatocytes and microsomal fractions.

The interaction between cyclosporin A (CsA) and the macrolide antibiotic, erythromycin, has been studied in freshly isolated rabbit hepatocytes and in rabbit liver microsomal fractions. In hepatocytes, CsA was rapidly accumulated inside the cells and metabolized to its different groups of derivatives (mono- and/or dihydroxylated and/or N-demethylated metabolites) [Fabre, Bertault-Peres, Fabre, Maurel, Just, and Cano: Drug Metab. Dispos. 15, 384 (1987)]. In the presence of erythromycin in the extracellular compartment, CsA metabolism was inhibited in a concentration-dependent manner. However, erythromycin did not affect intracellular CsA accumulation and binding of CsA to its intracellular protein binding site(s). Since CsA was specifically metabolized by the cytochrome P-450 LM3c isozyme [Bertault-Peres, Bonfils, Fabre, Just, Cano, and Maurel: Drug Metab. Dispos. 15, 391 (1987)], we further studied the effect of erythromycin on CsA metabolism by liver microsomal fractions. In the presence of erythromycin, CsA metabolism was also decreased. Lineweaver-Burk analysis of erythromycin-CsA interaction demonstrated that erythromycin was a competitive inhibitor (Ki = 156 microM) of CsA metabolism (Km = 0.43 microM; Vmax = 4.8 nmol/min). In agreement with these data, CsA inhibited (i) erythromycin N-demethylation to a large extent and (ii) the appearance of the erythromycin-cytochrome P-450 LM3c complex. We could conclude that the interaction between CsA and erythromycin most likely results from the fact that both drugs are extensively metabolized by the same cytochrome P-450 form: P-450 LM3c or P-450 III A4 according to the new nomenclature.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗