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Biomedical subjects

G Fabre

Publications and source records attributed to G Fabre.

83 records · Page 5Linked to original sources
PubMedDate not supplied

Involvement of the cytochrome P-450IID subfamily in minaprine 4-hydroxylation by human hepatic microsomes.

4-Hydroxylation of minaprine was measured on microsomal fractions prepared from 25 different human liver samples. In vitro formation of 4-hydroxyminaprine exhibited a large interindividual variability. Indeed, minaprine 4-hydroxylase activity ranged between 0.033 and 0.421 nmol/min/mg microsomal protein. Two samples presented a particularly low enzyme activity. Minaprine 4-hydroxylation followed Michaelis-Menten kinetics with KM and Vmax values of 5.26 microM and 0.478 nmol/min/mg microsomal protein, respectively, for one particular representative sample. The effects of various compounds (substrates or inhibitors of cytochrome P-450 isoforms) on 4-hydroxyminaprine formation were investigated. Selective substrates for P-450IA [benzo(a)pyrene, theophylline, and phenacetin], IIC (hexobarbital), IIE (aniline), and IIIA (erythromycin, nifedipine, and troleandomycin) cytochrome subfamilies did not inhibit 4-hydroxyminaprine formation. The nonspecific cytochrome P-450 inhibitor, cimetidine, slightly inhibited minaprine 4-hydroxylation. The classical substrates of the P-450IID cytochrome subfamily (debrisoquine, propranolol, and sparteine) inhibited minaprine 4-hydroxylation, as did the known P-450IID specific inhibitor, quinidine. These compounds inhibited minaprine 4-hydroxylase with Ki values of 16.5 (debrisoquine), 14.4 (propranolol), 61.9 (sparteine), and 0.146 microM (quinidine). 4-Hydroxyminaprine formation rate was shown not to be correlated with the activity of both erythromycin N-demethylase (r = 0.29, non-significant) and aniline hydroxylase (r = -0.15, NS). In contrast, minaprine 4-hydroxylase was well correlated with both debrisoquine 4-hydroxylase activity (r = 0.501, p less than 0.05) and immunoquantified cytochrome P-450IID6 (r = 0.579, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P-450 Enzyme Inhibitors↗
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

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↗
PubMedDate not supplied

Evidence for CYP3A-mediated N-deethylation of amiodarone in human liver microsomal fractions.

Metabolism of amiodarone to its N-deethylated derivative was investigated on a bank of human hepatic microsomal fractions, two of them lacking the CYP2D6 isozyme. Michaelis-Menten constants for amiodarone N-deethylation were 0.33 +/- 0.11 microM and 2.38 +/- 0.74 nmol/min/mg for KM and Vmax. The specific involvement of CYP3A gene subfamily in amiodarone N-deethylation was provided by the following observations: 1) metabolism of amiodarone is inhibited in a concentration-dependent manner by ketoconazole, a specific CYP3A inhibitor, and by nifedipine, a specific substrate for CYP3A gene subfamily, with IC50 of 0.3 and 25 microM, respectively; 2) nifedipine competitively inhibits amiodarone metabolism with a Ki of 38 microM; 3) amiodarone N-deethylation is increased following incubation with hepatic microsomal fractions prepared from CYP3A-inducers such as rifampycin and triacetyloleandomycin, but also following the in vitro disruption of the "cytochrome P-450-Fe-(II)-triacetyloleandomycin nitroso derivative" complex; 4) antibodies raised against either rabbit or baboon monkey CYP3A gene subfamily inhibit amiodarone N-deethylation; and 5) microsomal fractions that specifically express CYP3A4 biotransform amiodarone to its N-deethylated derivative. These studies indicate that CYP3A isozyme(s) mainly metabolize amiodarone to its N-deethylated derivative in human hepatic microsomal fractions.

Adult↗