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P Beaune

Publications and source records attributed to P Beaune.

At least 55 records · Page 3Linked to original sources
PubMed1998

Possible involvement of multiple human cytochrome P450 isoforms in the liver metabolism of propofol.

Previous studies of propofol (2,6-diisopropylphenol) pharmacology have shown that this widely used anaesthetic drug is extensively cleared from the body by conjugation of the parent molecule or its quinol metabolite. On the basis of potential influence of propofol on the metabolism of co-administered agents, many investigators have evaluated the effects of propofol on cytochrome P450 (CYP) activities. CYP isoforms involved in propofol metabolism are not defined. In this study, our objective was to elucidate further the CYP isoforms responsible for the hydroxylation of propofol. Using microsomes from 12 different human livers, we investigated CYP isoforms involved in propofol hydroxylase activity, using selective chemical inhibitors of CYP isoforms, correlation with immunoquantified specific CYP isoform content, immunoinhibition, and 11 functionally active human CYP isoforms expressed in a heterologous system (yeast and human B-lymphoblastoid cells). We found a low variability in the production of the hydroxylated metabolite of propofol, 2,6-diisopropyl-1,4-quinol. This activity was mediated by CYP and followed Michaelis-Menten kinetics with apparent K(M) and Vmax values of 18 microM (95% Cl 15.1-20.1) and 2.6 nmol min-1 mg-1 (95% Cl 2.45-2.68) respectively. Part of the propofol hydroxylase activity was mediated by CYP2C9 in human liver, especially at low substrate concentration. Moreover, propofol was likely to be metabolized by additional isoforms such as CYP2A6, 2C8, 2C18, 2C19 and 1A2, especially when substrate concentrations are high. This low specificity among CYP isoforms may contribute to the low interindividual variability (two-fold) and may contribute to the low level of metabolic drug interactions observed with propofol.

Anesthetics, Intravenous↗
PubMed1998

Cytochrome P450 CYP2D6 gene polymorphism and lung cancer susceptibility in Caucasians.

Many studies have been performed in an attempt to establish a link between the polymorphism of the cytochrome P450 CYP2D6 gene and the incidence of lung cancer. Nevertheless, whether or not this genetic polymorphism has a role in the development of the disease remains unclear. Recently, new advances in our knowledge of the CYP2D6 gene and its locus (CYP2D) have been achieved. In particular, CYP2D6 was found to be highly polymorphic and multiple novel mutations and allelic variants of the gene have been identified. In addition, a number of CYP2D rearrangements, including those with amplification of the gene, have been demonstrated. Taking this new information into account, we have reconsidered the potential influence of CYP2D6 polymorphism in lung cancer susceptibility by performing a comparative analysis of the overall mutational spectrum of CYP2D6 and of the rearrangements of CYP2D in 249 patients with lung cancer and in 265 control individuals matched on age, sex, hospital and residence area. For this purpose, a strategy based on SSCP analysis of the entire coding sequence of CYP2D6 and on RFLP analysis of the gene locus was carried out in DNA samples from each individual. Forty mutations occurring in various combinations on 42 alleles of the gene and 82 different genotypes were identified. No significant difference in the distribution of the mutations, alleles or genotypes was observed between the two groups, except a particular genotype (CYP2D6*1A/*2), which was more common in the sub-group of moderate smokers (< 30 pack-years) suffering from small cell carcinoma (Odds Ratio (OR) 3.6, 95% CI 1.1-11.9). When the phenotype was predicted according to genotype, only a trend toward a higher frequency of ultrarapid metabolizers in patients was obtained. In spite of a complete analysis of the CYP2D6 gene and its locus, this case-control study provides elements against an influence of the CYP2D6 polymorphism on lung cancer susceptibility.

Alleles↗
PubMed1998

Use of heterologously expressed human cytochrome P450 1A2 to predict tacrine-fluvoxamine drug interaction in man.

The aim of the present study was to evaluate the use of recombinant human cytochrome P-450 1A2 (rH-CYP1A2) in studies performed in vitro in order to predict metabolic drug-drug interactions occurring in man. In vitro metabolism of tacrine (a CYP1A2 probe) in the presence and absence of fluvoxamine, a CYP1A2 inhibitor, was investigated in human liver mircrosomes and with different rH-CYP. Vmax, Km and Ki determined with human liver microsomes were compared with those observed using rH-CYP1A2, assuming that 1 mg of liver microsomes contains, on average, 69 pmol of CYP1A2. The extent of tacrine metabolism inhibition procured by fluvoxamine with rH-CYP1A2, was compared with previous results observed in man. The Vax and Km for 1-hydroxytacrine formation rates obtained with rH-CYP1A2 were in good agreement with those observed in human liver microsomes (175+/-9 versus 140+/-60 pmol/min/mg for Vmax and 14+/-2 versus 16+/-2 microM for Km, respectively. The Ki of fluvoxamine on 1-hydroxytacrine formation rate observed with rH-CYP1A2 was similar to that observed with human liver microsome (0.35+/-0.05 versus 0.20+/-0.20 microM, respectively). Using the Km, Vmax and Ki determined with rH-CYP1A2, we calculated that fluvoxamine produced an inhibition of 1-, 2- and 4-hydroxytacrine formation rate of 91, 87 and 88%, respectively, in the range of tacrine and fluvoxamine concentrations observed in man. These percentages of inhibition calculated in vitro were in agreement with the percentage of fluvoxamine-dependent decrease in tacrine apparent oral clearance previously observed in man (83+/-13%). We conclude that human CYP1A2 expressed in yeast is a powerful tool to predict and to quantify drug-drug interactions in man.

Antidepressive Agents, Second-Generation↗
PubMed1998

Effects of the bacterial status of rats on the changes in some liver cytochrome P450 (EC 1.14.14.1) apoproteins consequent to a glucosinolate-rich diet.

The aim of the present work was to investigate the influence of the intestinal microflora on the changes in hepatic cytochrome P450 apoproteins induced by dietary glucosinolates. Ten rats harbouring a conventional digestive microflora were offered either a diet containing 390 g myrosinase-free rapeseed meal/kg (n 5) or a control diet devoid of glucosinolates (n 5). A similar trial was performed using germ-free rats. After 4 weeks of exposure to the dietary regimens, animals were slaughtered and their livers removed for preparation of microsomes and analysis of cytochrome P450 (EC 1.14.14.1). The glucosinolate-rich diet decreased the concentration of total cytochrome P450 in conventional rats only (-34%). The bacterial status did not modify the concentration of apoproteins CYP1A2 and CYP2B1/B2, but greatly decreased the concentration of the male constitutive isoform CYP2C11 (-53 and -45% respectively in conventional and germ-free rats). Germ-free rats fed on the glucosinolate-rich diet had a greater concentration of CYP3A (+139%) and a lower concentration of CYP2E1 (-32%) than their counterparts fed on the control diet. However, these differences were absent in conventional animals. On the whole, the influence of the intestinal microflora on the changes in hepatic cytochrome P450 due to the consumption of cruciferous vegetables is very complex and obviously involves different mechanisms according to the apoprotein.

Animals↗
PubMed1997

Possible involvement of multiple cytochrome P450S in fentanyl and sufentanil metabolism as opposed to alfentanil.

Fentanyl, sufentanil, and alfentanil are commonly used as opioid analgesics. Alfentanil clearance has previously been shown to exhibit an important interindividual variability, which was not observed for fentanyl or sufentanil. Differences in pharmacokinetic parameters of alfentanil have previously been associated with the wide distribution of CYP3A4, the only known hepatic cytochrome P450 monooxygenase (CYP) involved in the conversion of alfentanil to noralfentanil. Little is known about the involvement of CYP enzymes in the oxidative metabolism of fentanyl and sufentanil. Microsomes prepared from different human liver samples were compared for their abilities to metabolize fentanyl, sufentanil and alfentanil, and it was found that disappearance of the three substrates was well correlated with immunoreactive CYP3A4 contents but not with other CYPs, including CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP2E1. Specific known inhibitors of CYP enzymes gave similar results, whereas the use of recombinant human CYP enzymes expressed in yeast provided information about the possible involvement of other CYPs than CYP3A4 in the biotransformation of fentanyl and sufentanil. The possible in vivo interaction of fentanyl and sufentanil with other drugs catalyzed by CYP3A4 is also discussed.

Alfentanil↗
PubMed1997

Pregnenolone-7beta-hydroxylating activity of human cytochrome P450-1A1.

In many human and murine tissues, both pregnenolone and dehydroepiandrosterone are hydroxylated at the 7alpha and 7beta positions by a cytochrome P450-containing microsomal complex. The 7alpha- and 7beta-hydroxysteroids produced were shown to activate an immune response in mice. Based upon identification by crystallization to constant specific activity and gas chromatography-mass spectrometry analysis, we ascertained that a yeast-expressed human cytochrome P450-1A1 was able to 7beta-hydroxylate pregnenolone (K(M) from 3.2 +/- 0.5 to 4.1 +/- 0.4 microM, turnover number from 117 +/- 15 to 135 +/- 13 pmol/min/nmol of cytochrome P450-1A1). The other human cytochromes P450 tested did not produce identifiable quantities of 7alpha- or 7beta-hydroxylated derivatives of pregnenolone or dehydroepiandrosterone. These findings indicate that cytochrome P450-1A1 involvement in the 7beta-hydroxylation of pregnenolone may contribute to the production of the 7-hydroxylated steroids necessary for activation of the immune defences.

Chromatography, Gas↗
PubMed1997

Cytochrome P450 mediated bioactivation of methyleugenol to 1'-hydroxymethyleugenol in Fischer 344 rat and human liver microsomes.

Cytochrome P450 mediated metabolism of methyleugenol to the proximate carcinogen 1'-hydroxymethyleugenol has been investigated in vitro. Kinetic studies undertaken in liver microsomes from control male Fischer 344 rats revealed that this reaction is catalyzed by high affinity (Km of 74.9 +/- 9.0 microM, Vmax of 1.42 +/- 0.17 nmol/min/nmol P450) and low affinity (apparent Km several mM) enzymic components. Studies undertaken at low substrate concentration (20 microM) with microsomes from livers of rats treated with the enzyme inducers phenobarbital, dexamethasone, isosafrole and isoniazid indicated that a number of cytochrome P450 isozymes can catalyze the high affinity component. In control rat liver microsomes, 1'-hydroxylation of methyleugenol (assayed at 20 microM substrate) was inhibited significantly (P < 0.05) by diallylsulfide (40%), p-nitrophenol (55%), tolbutamide (30%) and alpha-naphthoflavone (25%) but not by troleandomycin, furafylline, quinine or cimetidine. These results suggested that the reaction is catalyzed by CYP 2E1 and by another as yet unidentified isozyme(s) (most probably CYP 2C6), but not by CYP 3A, CYP 1A2, CYP 2D1 or CYP 2C11. Administration of methyleugenol (0-300 mg/kg/day for 5 days) to rats in vivo caused dose-dependent auto-induction of 1'-hydroxylation of methyleugenol in vitro which could be attributed to induction of various cytochrome P450 isozymes, including CYP 2B and CYP 1A2. Consequently, high dose rodent carcinogenicity studies are likely to over-estimate the risk to human health posed by methyleugenol. The rate of 1'-hydroxylation of methyleugenol in vitro in 13 human liver samples varied markedly (by 37-fold), with the highest activities being similar to the activity evident in control rat liver microsomes. This suggests that the risk posed by dietary ingestion of methyleugenol could vary markedly in the human population.

Animals↗
PubMed1997

Role of CYP2D6 in the N-hydroxylation of procainamide.

Sequential oxidations at the arylamine moiety of the procainamide molecule leading to the formation of N-hydroxyprocainamide and its nitroso derivative may be responsible for lupus erythematosus observed in patients treated with the drug. The objective of the present study was to characterize major cytochrome P450 isozyme(s) involved in the N-hydroxylation of procainamide. Firstly, incubations were performed with microsomes from either lymphoblastoid cells or yeast transfected with cDNA encoding for specific human cytochrome P450 isozymes. Experiments performed with these enzyme expression systems indicated that the highest formation rate of N-hydroxyprocainamide was observed in the presence of CYP2D6 enriched microsomes. Additional experiments demonstrated that the formation rate of N-hydroxyprocainamide by CYP2D6 enriched microsomes was decreased from 45 +/- 4% to 93 +/- 1% by quinidine at concentrations ranging from 30 nM to 100 microM (all p < 0.05 vs control) and by approximately 75% by antibodies directed against CYP2D6. Secondly, incubations were performed with microsomes prepared from 15 human liver samples. Using this approach, an excellent correlation was observed between the formation rate of N-hydroxyprocainamide and dextromethorphan O-demethylase activity (CYP2D6; r = 0.9305; p < 0.0001). In contrast, no correlation could be established between N-hydroxyprocainamide formation rate and caffeine N3-demethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), S-mephenytoin N-demethylase (CYP2B6), tolbutamide methlhydroxylase (CYP2C9), S-mephenytoin 4'-hydroxylase (CYP2C19), chlorzoxazone 6-hydroxylase (CYP2E1), dextromethorphan N-demethylase (CYP3A4), testosterone 6 beta-hydroxylase (CYP3A4/5) or lauric acid 12-hydroxylase (CYP4A11) activities. Furthermore, formation rate of N-hydroxyprocainamide was decreased in a concentration-dependent manner by quinidine (300 nM to 100 microM) and by antibodies directed against CYP2D6 but not by furafylline 20 microM (CYP1A2), ketoconazole 1 microM (CYP3A4), sulfaphenazole 10 microM (CYP2C9) or antibodies directed against CYP1A1/1A2, CYP2C, CYP2A6, CYP2E1 or CYP3A4/3A5. In conclusion, the results obtained in the present study demonstrate that CYP2D6 is the major human cytochrome P450 isozyme involved in the formation of the reactive metabolite of procainamide, namely N-hydroxyprocainamide.

Anti-Arrhythmia Agents↗
PubMed1997

Expression and localization of CYP3A4 and CYP3A5 in human lung.

Expression in the lung of procarcinogen-metabolizing P450 enzymes in the CYP3A subfamily may contribute to the initiation of pulmonary carcinogenesis by agents that require metabolic activation, such as tobacco-derived polycyclic aromatic hydrocarbons. Expression and localization of CYP3A4 and CYP3A5 proteins in human lung were determined by immunohistochemistry with three antibodies, one specific for members of the CYP3A subfamily and two antipeptide antibodies specific for CYP3A4 and CYP3A5, respectively. Positive immunostaining in one or several cell types of the lung was observed in all patients with anti-CYP3A4 and anti-CYP3A5 antibodies. With the anti-CYP3A4 antibody epithelial staining was observed in five cases and staining of alveolar macrophages in 12 of 27 cases. To determine which CYP3A genes are transcribed in lung tissue, analysis by reverse-transcriptase-polymerase chain reaction with gene-specific primers for CYP3A4, CYP3A5, and CYP3A7 was performed. CYP3A5 mRNA was detected in all eight samples studied, CYP3A4 mRNA in one sample, and CYP3A7 mRNA in none of the samples. CYP3A5 was localized by immunohistochemistry in the ciliated and mucous cells of the bronchial wall, bronchial glands, bronchiolar columnar and terminal cuboidal epithelium, type I and type II alveolar epithelium, vascular and capillary endothelium, and alveolar macrophages, whereas CYP3A4 was found in bronchial glands, bronchiolar columnar and terminal epithelium, type II alveolar epithelium, and alveolar macrophages. These data establish that CYP3A5 is the predominant CYP3A form in human lung, that CYP3A4 is expressed in about 20% of individuals, and considerable variation of pulmonary expression occurs in both CYPs between individuals.

Bronchi↗
PubMed1997

Inhibition by omeprazole of proguanil metabolism: mechanism of the interaction in vitro and prediction of in vivo results from the in vitro experiments.

Both the antimalarial prodrug proguanil and the gastric proton pump inhibitor omeprazole are substrates for cytochrome P450 (CYP)2C19 and CYP3A. However, the relative contribution of each enzyme to proguanil bioactivation to cycloguanil and to the metabolism of omeprazole, as well as their potential to interact, remains to be examined. The bioactivation of proguanil to its active metabolite cycloguanil was studied in vitro in human liver microsomes and in vivo in 12 healthy subjects, in the absence and in the presence of omeprazole. The formation of cycloguanil from proguanil exhibited biphasic kinetic behavior in four of six human livers, indicating that at least two enzymes are responsible for this metabolic step. Cycloguanil formation activity did not correlate with immunoreactive CYP3A4 content or with CYP3A4 activity, as measured by testosterone 6beta-hydroxylation, suggesting that CYP3A4 plays a limited role in cycloguanil formation. Furthermore, troleandomycin (10 microM) inhibited only 10 to 17% of cycloguanil formation at proguanil concentrations of 100 and 500 microM. At a proguanil concentration of 20 microM, omeprazole at 10 microM inhibited cycloguanil formation in vitro by 47 +/- 59%. These in vitro results were consistent with the results of our in vivo study in healthy subjects, which showed a 32 +/- 11% decrease in proguanil apparent oral clearance and a 65 +/- 8% decrease in proguanil partial metabolic clearance to cycloguanil in the presence of omeprazole (both P < .001). We conclude that in vitro studies of proguanil metabolism and interactions are predictive of in vivo situations, that CYP2C19 is the main enzyme responsible for proguanil bioactivation to cycloguanil and that omeprazole inhibits this biotransformation in vitro and in vivo by inhibiting this enzyme.

Aryl Hydrocarbon Hydroxylases↗
PubMed1996

Stable expression of human cytochrome P450 3A4 in conjunction with human NADPH-cytochrome P450 oxidoreductase in V79 Chinese hamster cells.

V79 Chinese hamster cells were constructed for stable expression of human cytochrome P450 3A4 with and without coexpression of human NADPH-cytochrome P450 oxidoreductase. Expression of the cDNAs was shown by Northern and Western analyses. Activity was tested by 6 beta-hydroxylation of testosterone for cytochrome P450 3A4 and by cytochrome c reduction for NADPH-cytochrome P450 reductase. Five V79 cell lines were obtained expressing cytochrome P450 3A4, human NADPH-cytochrome P450 oxidoreductase, and both. Cytochrome P450 3A4 activity depended highly on cytochrome P450 reductase activity, with lowest activity when only the parental Chinese hamster cytochrome P450 reductase was present, 5- and 10-fold higher when coexpressed with the human NADPH-cytochrome P450 reductase. Correspondingly, cytotoxic and genotoxic potency of aflatoxin B1 was increased by orders of magnitudes when human cytochrome P450 3A4 was coexpressed with the human NADPH-cytochrome P450 reductase. The effect of NADPH-cytochrome P450 reductase coexpression on cytochrome P450 3A4 activity was also tested by nifedipine oxidation and midazolam hydroxylation. Nifedipine oxidation was increased about 10-fold, 1-hydroxylation of midazolam and 4-hydroxylation of midazolam were increased 15-fold.

Aflatoxin B1↗
PubMed1996

Expression of cytochrome P 450 3A enzymes in human lung: a combined RT-PCR and immunohistochemical analysis of normal tissue and lung tumours.

We have previously demonstrated expression of cytochrome P 450 3A (CYP3A) protein in pulmonary carcinomas and surrounding normal tissue, using immunohistochemistry. These results suggested that different CYP3A enzymes may be expressed in normal and tumour tissue. Therefore, the aim of the present study was to identify specific CYP3A enzymes expressed in normal human lung and lung tumours. Both normal lung tissue and tumour tissue from eight patients was analyzed for CYP3A4, CYP3A5 and CYP3A7 mRNA using a specific RT-PCR (reverse transcriptase-polymerase chain reaction) method. Identical samples were subjected to immunohistochemical analysis of CYP3A protein. CYP3A5 was the major enzyme of the CYP3A subfamily present at the mRNA level in both normal human lung and lung tumours. CYP3A5 mRNA was detected in normal lung tissue in all eight cases and in tumour tissue in four cases. CYP3A7 mRNA was detected in five cases in normal tissue and in one tumour. Notably, no CYP3A4 mRNA was found in any of the samples. Immunohistochemical staining for CYP3A protein was found in normal lung tissue in each case. Interestingly, all pulmonary carcinomas showed immunostaining for CYP3A, while mRNA for CYP3A enzymes was found in only four cases. In summary, our study indicates a specific expression pattern of the members of the CYP3A subfamily in normal human lung and lung tumours. These findings have potential clinical significance, since it has been recently shown that CYP3A5 catalyzes the activation of the anticancer pro-drugs cyclophosphamide and ifosfamide. Thus, local activation of these agents may take place in pulmonary carcinomas and surrounding normal tissues.

Aged↗
PubMed1996

Interindividual variability in catalytic activity and immunoreactivity of three major human liver cytochrome P450 isozymes.

OBJECTIVE: Interindividual variations in immunoreactivity and function of three major human drug metabolising P450 monooxygenases has been investigated in liver microsomes from 42 Caucasians (kidney donors or liver biopsies). METHODS: Diclofenac 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation, measured by HPLC in incubates, were used as probes to determine CYP2C9, CYP2D6 and CYP3A4 function kinetics, respectively. Immunoquantification of the three isoforms was achieved by Western blotting, using rabbit polyclonal antibodies raised against human CYP2C9 and human CYP3A4, and mouse monoclonal antibody raised against human CYP2D6. RESULTS: Diclofenac 4'-hydroxylation exhibited Michaelis-Menten kinetics with kM = 3.4 mumol.l-1 and Vmax = 45 nmole.mg-1 P.h-1. Relative immunoreactivity of CYP2C9 was correlated with Vmax and CL(int). Dextromethorphan O-demethylation in EM (extensive metabolisers) liver microsomes also showed Michaelis-Menten kinetics, with kM = 4.4 mumol.l-1 and Vmax = 5.0 nmol.mg-1 P.h-1. Relative immunoreactivity of CYP2D6 was correlated with Vmax and CL(int). Midazolam 1'-hydroxylation also exhibited Michaelis-Menten kinetics with kM = 3.3 mumol.l-1 and Vmax = 35 nmol.mg-1 P.h-1. Relative immunoreactivity of CYP3A4 was correlated with Vmax and CL(int). Immunoreactivity and function were correlated for each isozyme, but there was no cross correlation between isozymes. CONCLUSION: The velocity of metabolite formation (Vmax) by the three major human drug metabolising P450 monoxygenases is correlated with their immunoreactivity in liver microsomes. Interindividual variation was much larger for Vmax than kM. Interindividual variability was more pronounced for CYP2D6, probably due to the presence of several different functional alleles in the population of extensive metabolisers.

Adult↗
PubMed1996

Simulation of human benzo[a]pyrene metabolism deduced from the analysis of individual kinetic steps in recombinant yeast.

Human cytochrome P450 1A1 (1A1) and microsomal epoxide hydrolase (mEH)-dependent metabolic activation of benzo[a]pyrene (BP) have been reconstituted with microsomes from yeast cells expressing the two enzymes. The formation of the postulated ultimate mutagen 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP, the so-called diol epoxide-2 (DE2) derived from the reoxidation of BP-7,8-dihydrodiol by 1A1, was estimated by HPLC measurement of its hydrolysis product 7 beta, 8 alpha, 9 alpha, 10 beta-tetrahydrotetrol-BP (T2-tetrol). The 1A1/mEH coupled system was analyzed by varying the incubation time, initial substrate concentration, and molar ratio of the two enzymes. A minimum kinetic model of BP metabolism by 1A1 and mEH was constructed on the basis of the overall kinetic parameters (Vmax, Km) for a number of individual steps determined with human 1A1 and mEH expressed in yeast. The model was converted into a set of differential equations including 30 independent kinetic constants, 15 chemical species, and 8 enzymes and enzyme/substrate complexes. Numerical simulation of the model enabled us to satisfactorily reproduce the experimental kinetics of formation of BP-phenols, -dihydrodiols, and -tetrols for all tested conditions. Such a validated model was used to investigate the kinetics of unstable genotoxic species such as BP-epoxides and diol epoxides, which were not directly measurable. Based on numerical simulation, BP-7,8-oxide and -9,10-oxide appear to accumulate rapidly to reach a plateau after 2 min, while maximal accumulation of DE2 occurs after about a half-hour and declines during the following 2 h. A contribution of BP-9,10-dihydrodiol metabolism to T2-tetrol formation via the formation of a BP-7,8-oxide-9,10-dihydrodiol is predicted to be detectable after 2 hours due to the preferential accumulation of BP-9,10-dihydrodiol following 1A1-dependent consumption of initially accumulated BP-7,8-dihydrodiol.

Benzo(a)pyrene↗
PubMed1996

Involvement of cytochrome P450 3A4 enzyme in the N-demethylation of methadone in human liver microsomes.

Methadone has become one of the most widely used drugs for opiate dependency treatment. This drug is extensively metabolized by the cytochrome P450 hepatic enzyme family in man, yielding an N-demethylated metabolite that cyclizes spontaneously into 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. The specific forms of cytochrome P450 involved in this oxidative N-demethylation were examined in a panel of 20 human liver microsomal preparations previously characterized with respect to their P450 enzyme contents. Methadone was demethylated with an apparent Km of 545 +/- 258 microM (n = 3). The metabolic rates were 745 +/- 574 pmol/(min.mg of protein). This metabolic pathway was strongly correlated with estradiol 2-hydroxylation, testosterone 6 beta-hydroxylation, nifedipine oxidation, erythromycin N-demethylation, and toremifene N-demethylation, all of these monooxygenase activities being supported by P450 3A4. Furthermore, the total P450 3A content of liver microsomal samples, determined by immuno-quantification using a monoclonal anti-human P450 3A4 antibody, was correlated with methadone demethylation (r = 0.72; p < 0.003). Methadone metabolism was 60-72% inhibited either by three mechanism-based inhibitors of P450 3A4 (gestodene, TAO, and erythralosamine) or by four reversible inhibitors of P450 3A (ketoconazole, dihydroergotamine, quercetin, and diazepam with an apparent Ki of 50 microM) and by two nonspecific inhibitors (metyrapone and SKF-525A). Conversely, quinidine (inhibitor of P450 2D6), 7,8-benzoflavone (inhibitor of P450 1A), or sulfaphenazole (inhibitor of P450 2C) did not significantly inhibit, and may even have activated, methadone metabolism. Four heterologously expressed P450 proteins were able to catalyze the N-demethylation of methadone, namely, P450 2C8, P450 2C18, P450 2D6, and P450 3A4. However, referring to their relative liver content, it can be asserted that P450 3A4 is the major enzyme involved in the N-demethylation of methadone on average. Accordingly, caution should be advised in the clinical use of methadone when other drugs are also administered that induce or inhibit P450 3A4, such as rifampicin or diazepam, respectively.

Cytochrome P-450 CYP3A↗
PubMed1996

Hepatic cytochrome P450 and UDP-glucuronosyl transferase are affected by five sources of dietary fiber in germ-free rats.

The influence of dietary fiber on xenobiotic-metabolizing enzymes (XME) was assessed using germ-free rats fed inulin and other sources of fiber (wheat bran, carrot, cocoa and oat). The consumption of cocoa fiber greatly modified the hepatic cytochrome P450 isoenzymatic profile, causing a strong enhancement of 1A2 and 2B1/B2 forms, concomitant with a significant decrease of the constitutive form 2C11, compared with all of the other types of fiber. Moreover, rats fed the cocoa fiber diet had a higher specific activity of hepatic UDP-glucuronosyl transferase than their carrot fiber- and wheat bran-fed counterparts. Intestinal UDP-glucuronosyl transferase was unaffected by the type of ingested fiber. Diet composition also did not alter the specific activity of glutathione-S-transferase in the liver, small intestine, or colon. Using earlier results obtained in heteroxenic rats, we show that intestinal microflora plays a key role in some of the effects of fiber on XME, although this is not a necessary prerequisite for all of the liver alterations.

Analysis of Variance↗
PubMed1996

Evaluation of caffeine as a test drug for CYP1A2, NAT2 and CYP2E1 phenotyping in man by in vivo versus in vitro correlations.

Caffeine is used to phenotype subjects in vivo for the cytochrome P450 isoforms CYP1A2 and CYP2E1, and for N-acetyltransferase type 2 (NAT2). However, how much of the variation in phenotyping parameters may be attributed to variations in CYP1A2 and CYP2E1 activities has not been determined. Therefore, this study intraindividually compared enzyme activities and/or content in liver samples with pharmacokinetic parameters of caffeine in vivo after administration of a test dose in 25 patients undergoing hepatectomy. Parameters measured in vitro were the high affinity components of caffeine 3-demethylation and phenacetin 0-deethylation, microsomal CYP1A2 and CYP2E1 immunoreactivity, and cytosolic sulfamethazine N-acetylation. Caffeine parameters in vivo included caffeine clearance from plasma and/or saliva, paraxanthine/caffeine ratios in plasma and saliva, plasma theophylline/caffeine ratio, and several metabolite ratios from spot urine sampled 6 h postdose. Correlations between parameters were determined using weighted linear regression analyses. Caffeine clearance and paraxanthine/caffeine ratios correlated most highly to intrinsic clearance of caffeine 3-demethylation and to CYP1A2 immunoreactivity (r= 0.584-0.82), whereas urinary CYP1A2 ratios correlated less strongly with CYP1A2 parameters in vitro. Assignment of acetylator phenotype by urinary NAT2 ratios was concordant with sulfamethazine-N-acetylation in vitro. In contrast to CYP1A2 parameters in vitro, CYP2E1 immunoreactivity was not related to the theophylline/caffeine plasma ratio. CYP1A2 activity, thus, is the major determinant of caffeine clearance and the paraxanthine/caffeine ratios in vivo, of which the saliva ratio 6 h postdose appears as the most advantageous parameter. The results confirm that phenotyping using caffeine provides valid estimates of CYP1A2 and NAT2 activity.

Aged↗