Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.13.97 (CYP3A4)
 6,365 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Polymorphisms have been detected in a variety of xenobiotic-metabolizing enzymes at both the phenotypic and genotypic level. In the case of four enzymes, the cytochrome P450 CYP2D6, glutathione S-transferase mu, N-acetyltransferase 2 and serum cholinesterase, the majority of mutations which give rise to a defective phenotype have now been identified. Another group of enzymes show definite polymorphism at the phenotypic level but the exact genetic mechanisms responsible are not yet clear. These enzymes include the cytochromes P450 CYP1A1, CYP1A2 and a CYP2C form which metabolizes mephenytoin, a flavin-linked monooxygenase (fish-odour syndrome), paraoxonase, UDP-glucuronosyltransferase (Gilbert's syndrome) and thiopurine S-methyltransferase. In the case of a further group of enzymes, there is some evidence for polymorphism at either the phenotypic or genotypic level but this has not been unambiguously demonstrated. Examples of this class include the cytochrome P450 enzymes CYP2A6, CYP2E1, CYP2C9 and CYP3A4, xanthine oxidase, an S-oxidase which metabolizes carbocysteine, epoxide hydrolase, two forms of sulphotransferase and several methyltransferases. The nature of all these polymorphisms and possible polymorphisms is discussed in detail, with particular reference to the effects of this variation on drug metabolism and susceptibility to chemically-induced diseases.
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PMID:Metabolic polymorphisms. 836 90

We have investigated: (a) the formation of N-acetyl-p-aminobenzoquinone imine (NAPQI) from acetaminophen (APAP) by reconstituted human liver CYP3A4, (b) the kinetics of NAPQI formation in microsomes prepared from four human livers varying in CYP1A2, 2E1 and 3A4 content determined by Western blot analysis, (c) the contribution of CYP3A4 to the total formation of NAPQI from 0.1 mM APAP in human liver microsomes using troleandomycin as a specific inhibitor, and (d) the relationship between the contribution of CYP3A4 to NAPQI formation and relative CYP3A4 content. The Km of CYP3A4 for APAP was found to be approximately 0.15 mM, similar to concentrations observed in humans after therapeutic doses of the drug. The kinetics of formation of NAPQI in human liver microsomes were complex; the lower Km was similar to that found for reconstituted CYP3A4. The contribution of CYP3A4 to total NAPQI formation varied from 1 to 20% among livers, and correlated with the relative CYP3A4 content, r2 = 0.88, P < 0.05. Our findings indicate that CYP3A4, the major P450 isoform in human liver and enterocytes, contributes appreciably to the formation of the cytotoxic metabolite NAPQI at therapeutically relevant concentrations of APAP and suggest that APAP may be a previously unrecognized inhibitor of this enzyme.
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PMID:Oxidation of acetaminophen to N-acetyl-p-aminobenzoquinone imine by human CYP3A4. 838 97

The activity of metabolizing enzymes determines plasma concentrations and hence effects of drugs. Identification of these enzymes may allow the prediction of both the interaction potential of drugs and the variability deriving from certain pathways. The antiarrhythmic propafenone is extensively biotransformed to the active metabolites 5-hydroxypropafenone and N-desalkylpropafenone. Whereas 5-hydroxylation is catalyzed by CYP2D6, the enzyme involved in N-dealkylation has not been identified. We, therefore, characterized the enzyme involved in the formation of N-desalkylpropafenone by using both in vitro [human liver microsomes, specific antibodies or inhibitors, and stably expressed cytochrome P450 (P450) enzymes] and in vivo (formation of N-desalkylpropafenone in patients under conditions of chronic therapy) approaches. Formation of N-desalkylpropafenone can be described by Michaelis-Menten kinetics. A strong correlation was observed between maximum rate of formation (Vmax) of N-desalkylpropafenone and the amount of CYP1A2 (r = 0.83, p < 0.001) and CYP3A (r = 0.54, p < 0.05) in the microsomal fraction of 20 human livers. In vitro intrinsic clearances (derived from Vmax/Km) indicated a wide interindividual variability in seven human livers (from 0.01 to 0.1 ml/hr/mg of protein). Antibodies directed against CYP3A and CYP1A2 inhibited formation of N-desalkylpropafenone by 54 +/- 10% and 24 +/- 16%, respectively. The CYP2D6-mediated formation of 5-hydroxypropafenone was unaffected by these antibodies. Verapamil (substrate of CYP3A4 and CYP1A2) and midazolam (substrate of CYP3A4) were competitive inhibitors of N-desalkylpropafenone formation (Ki = 70 microM and 25 microM for verapamil and midazolam, respectively). Coding sequences for CYP1A2 and CYP3A4 were inserted in a yeast expression vector and introduced into Saccharomyces cerevisiae strain W(R). Both CYP1A2 and CYP3A4 catalyzed N-dealkylation of propafenone, with specific activities of 0.32 pmol/min/pmol of P450 and 0.16 pmol/min/pmol of P450, respectively. Our data indicate that N-dealkylation of propafenone is mediated via CYP3A4 and CYP1A2. From experiments on the molecular level interactions of propafenone with other drugs that are metabolized by CYP3A4 and CYP1A2 can be predicted. Such interactions have been reported for cyclosporin, rifampicin, warfarin, and theophylline. Moreover, in vitro intrinsic clearances showed a wide interindividual variability. Therefore, variable plasma concentrations of the active metabolite N-desalkylpropafenone are expected in vivo. We tested this hypothesis in 14 patients (dose of 150 mg of propafenone three times per day) during chronic oral therapy and observed steady state plasma concentrations of N-desalkylpropafenone ranging from 4 to 293 ng/ml.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification and characterization of the cytochrome P450 enzymes involved in N-dealkylation of propafenone: molecular base for interaction potential and variable disposition of active metabolites. 842 65

With the aim to assess the involvement of distinct forms of cytochrome P450 enzymes in the activation of procarcinogens, we have developed by means of retroviral infection a series of NIH/3T3 cell lines stably expressing human CYP1A1, CYP1A2, CYP2C10, CYP2D6, and CYP2E1 cDNA. The levels of cytochrome P450 enzyme activities were determined using specific substrates. An increase in specific catalytic activity could be observed in all cell lines compared to background activity in vector-infected cells. Furthermore, we developed a test system in which we are able to combine P450-expressing cells with a shuttle vector containing the lacZ' gene, which serves as a reporter gene for mutations. Using this system, we investigated the cytotoxicity and mutagenicity of the mycotoxin ochratoxin A. Natural occurrence of ochratoxin A in food commodities has been linked to an increased incidence of urinary tract tumors in certain geographic regions. Although biotransformation seems to play a crucial role in ochratoxin A toxicity, the possible contribution of metabolites to genotoxicity and carcinogenicity remained unelucidated. We have demonstrated that the mutation frequency of ochratoxin A was increased dependent upon concentration in NIH/3T3 cell lines, stably expressing human CYP1A1, CYP1A2, CYP2C10, and CYP3A4. In contrast, neither in vector-infected NIH/3T3 cells nor in CYP2D6- and CYP2E1-expressing cells was an increase of mutation frequency observed.
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PMID:Development of human cytochrome P450-expressing cell lines: application in mutagenicity testing of ochratoxin A. 854 84

Cytochromes P450 (CYP) constitute a superfamily of enzymes involved in the metabolism of xenobiotics. Within the same subfamily, the isoforms present strong similarities, making them difficult to characterize and differentiate. Using heterologous expression in bacteria, five pure human CYP (1A1, 1A2, 2C9, 2E1, 3A4) were easily obtained and used as antigens to raise specific antibodies. These antibodies were characterized for their specificity and sensitivity by immunoblots; anti-CYP3A4 was immunoinhibitor. These antibodies could be used in association with other means to identify the CYPs responsible for production of a given metabolite. The use of our human recombinant CYP1A2 as antigen and the corresponding specific antibody enabled us to quantify the CYP1A2 content in 43 human livers. The average level was 69 pmol of CYP1A2/mg of microsomal proteins. Finally, these antibodies were also used to evaluate the level of heme incorporation in human microsomal CYP expressed in yeasts.
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PMID:Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies. 857 93

[3H]Loratadine was incubated with human liver microsomes to determine which cytochrome P450 (CYP) enzymes are responsible for its oxidative metabolism. Specific enzymes were identified by correlation analysis, by inhibition studies (chemical and immunoinhibition), and by incubation with various cDNA-expressed human P450 enzymes. Descarboethoxyloratadine (DCL) was the major metabolite of loratadine detected following incubation with pooled human liver microsomes. Although DCL can theoretically form by hydrolysis, the conversion of loratadine to DCL by human liver microsomes was not inhibited by the esterase inhibitor phenylmethylsulfonyl fluoride (PMSF), and was dependent on NADPH. A high correlation (r2 = 0.96, N = 10) was noted between the rate of formation of DCL and testosterone 6 beta-hydroxylation, a CYP3A-mediated reaction. With the addition of ketoconazole (CYP3A4 inhibitor) to the incubation mixtures, the residual rate of formation of DCL correlated (r2 = 0.81) with that for dextromethorphan O-demethylation, a CYP2D6 reaction. Rabbit polyclonal antibodies raised against the rat CYP3A1 enzyme (5 mg IgG/nmol P450) and troleandomycin (0.5 microM), a specific inhibitor of CYP3A4, decreased the formation of DCL by 53 and 75%, respectively, when added to 1.42 microM loratadine microsomal incubations. Quinidine (5 microm), a CYP2D6 inhibitor, inhibited the formation of DCL approximately 20% when added to microsomal incubations of loratadine at concentrations of 7-35 microM. Incubation of loratadine with cDNA-expressed CYP3A4 and CYP2D6 microsomes catalysed the formation of DCL with formation rates of 135 and 633 pmol/min/nmol P450, respectively. The results indicated that loratadine was metabolized to DCL primarily by the CYP3A4 and CYP2D6 enzymes in human liver microsomes. In the presence of a CYP3A4 inhibitor, loratadine was metabolized to DCL by the CYP2D6 enzyme. Conformational and electrostatic analysis of loratadine indicated that its structure is consistent with substrate models for the CYP2D6 enzyme.
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PMID:Identification of human liver cytochrome P450 enzymes that metabolize the nonsedating antihistamine loratadine. Formation of descarboethoxyloratadine by CYP3A4 and CYP2D6. 861 85

CYP3A4 represents the most important form of human cytochrome P450 active in drug metabolism. Reconstitution of this enzyme has in the past been a major problem. Using purified cDNA-expressed CYP3A4 incorporated into membranous vesicles made from microsomal phospholipids, rates of nifedipine and testosterone oxidation of about 60 nmol/nmol P450/min were achieved, whereas similar reconstitution into dilauroyl-phosphatidylcholine micelles was unsuccessful. A higher Vmax for nifedipine oxidation was obtained in negatively charged vesicles as compared to neutral membranes, whereas the membrane charge did not influence the Km. It is concluded that the native function of CYP3A4 requires a negatively charged microsomal membrane.
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PMID:High rates of substrate hydroxylation by human cytochrome P450 3A4 in reconstituted membranous vesicles: influence of membrane charge. 861 53

The aim of this study was to identify which human P450 enzymes are involved in the metabolism of lansoprazole. In the presence of NADPH and oxygen, human liver microsomes converted lansoprazole to lansoprazole sulfide, lansoprazole sulfone and 5-hydroxylansoprazole. Formation of lansoprazole sulfide occurred nonenzymatically. The formation of lansoprazole sulfone appeared to be catalyzed by a single, low-affinity enzyme (apparent Km approximately 100 microM). In contrast, lansoprazole 5-hydroxylation appeared to be catalyzed by two kinetically distinct enzymes (apparent Km approximately 100 microM and approximately 15 microM). When human liver microsomes (n = 16) were incubated with 100 microM lansoprazole, both the 5-hydroxylation and sulfoxidation of lansoprazole appeared to be catalyzed by CYP3A4/5 (based on correlation analyses). Antibodies against rat CYP3A enzymes inhibited the rate of both 5-hydroxylation (approximately 55%) and sulfoxidation (approximately 70%) and cDNA-expressed CYP3A4 catalyzed both the 5-hydroxylation and sulfoxidation of lansoprazole (apparent Km approximately 100 microM). However, at the pharmacologically relevant substrate concentration of 1 microM, lansoprazole sulfoxidation was still highly correlated with CYP3A4/5 activity (r2 = .905), but lansoprazole 5-hydroxylation appeared to be catalyzed by CYP2C19 (r2 = .875) rather than CYP3A4/5 (r2 = .113). Antibodies and chemical inhibitors of CYP2C enzymes preferentially inhibited the 5-hydroxylation of lansoprazole, whereas lansoprazole sulfoxidation was preferentially inhibited by antibodies and chemical inhibitors of CYP3A4/5. The cDNA expressed enzymes CYP2C8, CYP2C9 and CYP2C19 catalyzed varying rates of lansoprazole 5-hydroxylation at a substrate concentration of 50 microM, but only CYPC19 catalyzed this reaction at 1 microM. These results suggest that at pharmacologically relevant concentrations, the 5-hydroxylation of lansoprazole is primarily catalyzed by CYP2C19, whereas the sulfoxidation of lansoprazole is primarily catalyzed by CYP3A4/5. It is possible that individuals lacking CYP2C19 will be poor metabolizers of lansoprazole.
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PMID:Identification of the human P450 enzymes involved in lansoprazole metabolism. 862 62

Xenobiotics frequently induce proteins involved in their detoxification. Because many drugs that are metabolized by human cytochromes P450 (CYP) 3A4 and 3A5 are also transported by the drug efflux pump P-glycoprotein, we determined whether expression of these proteins was altered by a variety of drugs in a cell line derived from a human colon adenocarcinoma, LS180/WT, and its adriamycin-resistant subline, LS180/AD50. P-glycoprotein and CYP3A4 were constitutively expressed in both LS180/AD50 and LS180/WT cells, and both proteins were up-regulated after treatment with many drugs, including rifampicin, phenobarbital, clotrimazole, reserpine, and isosafrole. However, there were some exceptions because P-glycoprotein was up-regulated by midazolam and nifedipine, whereas CYP3A4 was not. CYP3A5, which is also constitutively expressed in these cells, remained unchanged with most drug treatments but was up-regulated by reserpine and clotrimazole. The apparent coordinated coexpression of the CYP3A gene family and P-glycoprotein in the LS180 cells suggests that for common orally administered drugs, P-glycoprotein may play an important role in net drug absorption and drug/drug interactions of shared CYP3A4/P-glycoprotein substrates.
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PMID:Modulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up-regulate these proteins in human colon carcinoma cells. 863 64

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) forms involved in the oxidative metabolism of [14C]zileuton (ABT-077) and its N-dehydroxylated metabolite, [14C]Abbott-66193, by human liver microsomes. The two compounds were metabolized by parallel pathways to form the corresponding ring-hydroxylated and diastereomer sulfoxide metabolites. Results suggested that whereas the metabolism of zileuton and Abbott-66193 were mediated by the same CYP forms, the CYP forms responsible for hydroxylation (CYP1A2 and CYP2C9/10) were distinct from those involved in sulfoxidation (CYP3A > CYP2C9/10). Sulfoxidation (zileuton, Km = 0.82 +/- 0.40 mM, Vmax = 39.1 +/- 21.8 pmol/min/mg; Abbott-66193, Km = 0.23 +/- 0.06 mM, Vmax = 507 +/- 215 pmol/min/mg; mean +/- SD, N=3) was highly correlated with the CYP3A-specific erythromycin N-demethylase activity (r=0794-0.856; p<0.01, N=11) in human microsomes and was inhibited (32-67%) by ketoconazole and troleandomycin. In addition, purified recombinant human CYP3A4/rat NADPH-P450 reductase fusion protein catalyzed only the sulfoxidation of zileuton and Abbott-66193; no hydroxylated metabolites were detected. On the other hand, hydroxylation of the two compounds (zileuton, Km = 0.34 +/- 0.25 mM, Vmax = 17.8 +/- 5.58 pmol/min/mg; Abbott-66193,Km = 0.39 +/- 0.14 mM, Vmax = 1061 +/- 220 pmol/min/mg) was significantly correlated with 7-ethoxyresorufin O-deethylase (CYP1A2; r=0.652-0.762; p<0.01, N=11) and tolbutamide methyl hydroxylase (CYP2C9/10; r=0.863-0.935; p<0.01, N=10) activity in human liver microsome, and was inhibited (26-51%) by well-known CYP1A2 inhibitors (furafylline and alpha-naphthoflavone). Furthermore, microsomes from human B-lymphoblastoid cells expressing CYP1A2 catalyzed only the hydroxylation of zileuton and Abbott-66193; sulfoxide were not formed. Abbott-66193 was a better substrate for CYP2C9/10, when compared with zileuton: 1) the effect of sulfaphenazole on hydroxylation in human liver microsomes was more pronounced for Abbott-66193 than zileuton (56% vs. 9% inhibition); and 2) the rate of Abbott-66193 hydroxylation by purified CYP2C9 was almost 30-fold greater than that of zilueton.
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PMID:Identification of the human liver cytochrome P450 enzymes involved in the metabolism of zileuton (ABT-077) and its N-dehydroxylated metabolite, Abbott-66193. 865 6


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