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)

We systematically characterized the levels and substrate specificity of P450s from humans and rats to extrapolate drug metabolism data from experimental animals to humans. Human P450s (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4) were expressed in Saccharomyces cerevisiae and purified. Rat P450s were purified from hepatic microsomes of rats. We investigated the catalytic activities of purified P450s in a reconstituted system. Human CYP2B6 and rat CYP2B1 had high lidocaine N-deethylation activity. Human and rat CYP2D forms had high debrisoquine 4-hydroxylation activity. Human CYP3A4 and rat CYP3A2 had high testosterone 2 beta- and 6 beta-hydroxylation activities in a modified reconstituted system with a lipid mixture. The hydroxylation site of testosterone by CYP2B6 (16 alpha- and 16 beta-positions) agreed with that by rat CYP2B1. Human CYP2E1 had the highest lauric acid (omega-1)-hydroxylation activity and also had catalytic properties similar to those of rat CYP2E1. Human CYP2A and 2C forms had catalytic properties in testosterone metabolism different from those of rats. Antibodies raised against purified P450s were used to measure the levels of hepatic P450s. The level of CYP3A4 was the highest in human hepatic microsomes, comprising 30-40% of the total P450. CYP2C9 comprised 10-20% of the total. The levels of CYP1A2, 2A6, 2C8, 2D6, and 2E1 were moderate (5-15% of total P450). CYP2B6 content was very low. The information of this study is useful for drug metabolism and toxicological studies.
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PMID:Multiple forms of human P450 expressed in Saccharomyces cerevisiae. Systematic characterization and comparison with those of the rat. 886 26

1,2-Dibromoethane (1,2-DBE) is mainly used as an additive in leaded gasoline and as a soil fumigant and it is a suspected carcinogen in humans. In this study, the oxidative bioactivation of 1,2-DBE to 2-bromoacetaldehyde (2-BA) was studied using heterologously expressed human cytochrome P450 (P450) isoenzymes and human liver microsomes. Out of ten heterologously expressed human P450 isoenzymes (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2C9, CYP2C18, CYP3A4 and CYP3A5), only human CYP2A6, CYP2B6 and CYP2E1 metabolized 1,2-DBE, albeit with strongly differing catalytic efficiencies. The apparent Km and Vmax values were 3.3 mM and 0.17 pmol/min per pmol P450 for CYP2A6, 9.7 mM and 3.18 pmol/min per pmol P450 for CYP2B6 and 42 microM and 1.3 pmol/min per pmol P450 for CYP2E1, respectively. In all of 21 human liver samples studied, 1,2-DBE was oxidized with activities ranging from 22.2 to 1027.6 pmol/min per mg protein, thus showing a 46-fold inter-individual variability. The kinetics of the oxidative metabolism of 1,2-DBE to 2-BA in human liver microsomes were linear, indicating the involvement of primarily one single P450 isoenzyme. There was a tendency towards a positive correlation between the oxidative metabolism of 1,2-DBE in the human liver microsomes and the 6-hydroxylation of chlorzoxazone, a selective substrate for CYP2E1. Furthermore, the oxidative metabolism of 1,2-DBE was inhibited by the specific CYP2E1 inhibitors disulfiram (DS) and diethyldithiocarbamate (DDC). In contrast, a poor correlation was found between the immunochemically quantified amount of CYP2E1 and the microsomal chlorzoxazone 6-hydroxylation or the 1,2-DBE oxidation. The results indicate that CYP2E1 is probably the major P450 isoenzyme involved in the oxidative hepatic metabolism of 1,2-DBE in humans. The inter-individual variability in the oxidative bioactivation of 1,2-DBE in humans, largely due to inter-individual variability in the catalytic activity of hepatic CYP2E1, may have important consequences for the risk assessment for human exposure to 1,2-DBE.
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PMID:Inter-individual variability in the oxidation of 1,2-dibromoethane: use of heterologously expressed human cytochrome P450 and human liver microsomes. 887 Jun 87

1. In vitro metabolism of a rifamycin derivative, benzoxazinorifamycin KRM-1648, was studied using mouse, rat, guinea pig, dog, monkey and human liver microsomes. 30-Hydroxy-KRM-1648 (M2) was produced in mouse, dog, monkey and human microsomes. 25-Deacetyl-KRM-1648 (M1) was produced in dog and human microsomes, but not in mouse or monkey microsomes. Neither M1 nor M2 was detected in rat or guinea pig microsomes. 2. In dog and human liver microsomes the formation of M2 was dependent on NADPH, but the formation of M1 was not. 3. In vitro metabolism of the parent compound was studied in whole blood in some species. Only M1 was detected in mouse and rat blood, and not in dog and human blood. 4. These findings demonstrated that the metabolite pattern in dog resembled that in man, and suggested that the 30-hydroxylation of KRM-1648 was mediated by cytochrome P450, but that the 25-deacetylation was not. 5. Among the ten recombinant human P450 isoforms used, only the cell lysates including CYP3A3 and CYP3A4 catalysed the M2 formation from KRM-1648.
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PMID:In vitro metabolism of a rifamycin derivative by animal and human liver microsomes, whole blood and expressed human CYP3A isoform. 887 43

The microsomal metabolism of fentanyl, a synthetic opioid commonly used in anesthesia, was investigated in human liver. Incubation of fentanyl with human hepatic microsomes fortified with NADPH resulted in the formation of a single major metabolite, namely norfentanyl, as determined by GC/MS. No evidence was obtained for the formation of either desproprionylfentanyl or N-phenylpropionamide, the latter arising via N-dealkylation of the fentanyl amide nitrogen. Kinetic analysis of microsomal fentanyl oxidation revealed a single K(m) of 117 microM and a Vmax of 3.86 nmol of norfentanyl formed/min/nmol of cytochrome P450 (P450). Studies using chemical inhibitors of human P450 enzymes revealed that only agents known to inhibit CYP3A4 (e.g. ketoconazole and erythromycin) were capable of strongly inhibiting (> or = 90%) microsomal fentanyl oxidation. Marked inhibition (> 90%) of norfentanyl formation by liver microsomes was also observed with polyclonal antibodies to CYP3A4, whereas antibodies to other human P450s were without effect. Furthermore, rates of norfentanyl production by 10 individual human liver samples were highly correlated (r2 = 0.876, F = 56.46 p < 0.001) with immunochemically determined levels of CYP3A4 present in the samples but not with levels of CYP2C8, CYP2C9, CYP2C19, or CYP2E1. Our results indicate that CYP3A4 is the major catalyst involved in fentanyl oxidation to norfentanyl in human liver. Alterations in CYP3A4 levels or activity, as well as the concomitant administration of other therapeutic agents metabolized by this P450 enzyme, could lead to marked perturbations in fentanyl disposition and, hence, analgesic response.
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PMID:Metabolism of fentanyl, a synthetic opioid analgesic, by human liver microsomes. Role of CYP3A4. 888 1

In vitro methods were used to identify the cytochrome P450 (CYP) enzyme(s) involved in S-mephenytoin N-demethylation. S-Mephenytoin (200 microM) was incubated with human liver microsomes, and nirvanol formation was quantitated by reversed-phase HPLC. S-Mephenytoin N-demethylase activity in a panel of human liver microsomes ranged 35-fold from 9 to 319 pmol/min/mg protein and correlated strongly with microsomal CYP2B6 activity (r = 0.91). Additional correlations were found with microsomal CYP2A6 and CYP3A4 activity (r = 0.88 and 0.74, respectively). Microsomes prepared from human beta-lymphoblastoid cells transformed with individual P450 cDNAs were assayed for S-mephenytoin N-demethylase activity. Of 11 P450 isoforms (P450s 1A1, 1A2, 2A6, 2B6, 2E1, 2D6, 2C8, 2C9, 2C19, 3A4, and 3A5) tested, only CYP2B6 catalyzed the N-demethylation of S-mephenytoin with an apparent K(m) of 564 microM. Experiments with P450 form-selective chemical inhibitors, competitive substrates, and anti-P450 antibodies were also performed. Troleandomycin, a mechanism-based CYP3A selective inhibitor, and coumarin, a substrate for CYP2A6 and therefore a potential competitive inhibitor, failed to inhibit human liver microsomal S-mephenytoin N-demethylation. In contrast, orphenadrine, an inhibitor of CYP2B forms, produced a 51 +/- 4% decrease in S-mephenytoin N-demethylase activity in human liver microsomes and a 45% decrease in recombinant microsomes expressing CYP2B6. Also, both CYP2B6-marker 7-ethoxytrifluoromethylcoumarin O-deethylase and S-mephenytoin N-demethylase activities were inhibited by approximately 65% by 5 mg anti-CYP2B1 IgG/mg microsomal protein. Finally, polyclonal antibody inhibitory to CYP3A1 failed to inhibit S-mephenytoin N-demethylase activity. Taken together, these studies indicate that the N-demethylation of S-mephenytoin by human liver microsomes is catalyzed primarily by CYP2B6.
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PMID:Catalytic role of cytochrome P4502B6 in the N-demethylation of S-mephenytoin. 888 3

Omeprazole (OP) is a potent antiulcer drug that is metabolized by liver cytochrome P450 (P450) enzymes. However, the identities of the P450 isoforms responsible for its metabolism have been controversial. 5-Hydroxyomeprazole (5OH-OP) formation cosegregates with the polymorphism of (S)-mephenytoin 4'-hydroxylation in humans, which is now known to be mediated by CYP2C19. Previous in vitro studies have indicated that liver microsomal 50H-OP formation correlates with both (S)-mephenytoin 4'-hydroxylase and CYP3A content. Inhibitor and CYP2C antibody studies also suggested that both enzymes may be involved in the 5-hydroxylation of OP, whereas CYP3A appears to be the predominant enzyme involved in OP sulfone (OP-S) formation. The present studies assessed the contribution of various CYP2C and CYP3A4 enzymes to OP metabolism by using recombinant human enzymes. CYP2C19, CYP2C8, CYP2C18, and CYP2C9 formed a single metabolite with an HPLC retention time identical to that of 5OH-OP. The turnover number for CYP2C19 was 13.4 +/- 1.4 nmol/min/nmol of P450, whereas those for CYP2C8, CYP2C18, and CYP2C9 were 2.2 +/- 0.1, 1.5 +/- 0.1, and approximately equal to 0.5 nmol/min/nmol of P450, respectively. Recombinant human CYP3A4 formed 5OH-OP and OP-S with turnover numbers of 5.7 +/- 1.1 and 7.4 +/- 0.9 nmol/min/nmol of P450, respectively, and formed a minor unidentified metabolite. CYP2C19 had a substantially lower KM for 5OH-OP formation than did CYP3A4, CYP2C8, or CYP2C18. Antibody to CYP2C proteins inhibited approximately equal to 70% of OP 5-hydroxylation at low substrate concentrations, comparable to those that may be encountered at therapeutically relevant doses, whereas antibody to CYP3A4 inhibited approximately equal to 30% of the activity. At high substrate concentrations, the contributions of the two enzymes to OP hydroxylation were roughly comparable (40-50%). In contrast, OP-S formation was completely inhibited by antibody to CYP3A4 proteins. The present study provides the first direct confirmation, using human recombinant P450 enzymes and selective antibody inhibition, that CYP2C19 is a major high affinity OP 5-hydroxylase and CYP3A4 is a low affinity OP-hydroxylating enzyme. The current work also shows, for the first time, that other CYP2C enzymes (CYP2C8, CYP2C9, and CYP2C18) may contribute to OP hydroxylation at high substrate concentrations. In contrast, OP-S was formed principally by CYP3A4.
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PMID:Human CYP2C19 is a major omeprazole 5-hydroxylase, as demonstrated with recombinant cytochrome P450 enzymes. 889 8

The fluoroquinolone antibacterial agents have gained widespread use in the treatment of a broad range of bacterial infections. We recently described a possible interaction concerning the concomitant use of cyclosporine A and norfloxacin in pediatric renal transplant patients. We examined the effect of two common fluoroquinolone antibiotics on cytochrome P450-mediated drug biotransformations in human and rat liver microsomes. Rats were pretreated with inducers, which increased the levels of the P450 isozymes CYP3A2, CYP1A, CYP2E1, and CYP4A1. Ciprofloxacin and norfloxacin significantly depressed the N-demethylation of erythromycin by CYP3A4 in human microsomes and by CYP3A2 in rat microsomes. The inhibition was determined to be competitive in nature in rat microsomes, with ciprofloxacin and norfloxacin both exhibiting similar Ki values of 2.0 and 2.3 mM, respectively. Ciprofloxacin and norfloxacin also inhibited ethoxyresorufin-O-dealkylase (CYP1A). In contrast, ciprofloxacin and norfloxacin did not inhibit the metabolism of substrates that are specific for the P450 isozymes CYP2E1 and CYP4A1. Rats treated chronically with norfloxacin revealed no alterations in hepatic CYP3A2 protein levels or activity. These studies in hepatic microsomes demonstrate that fluoroquinolones can decrease CYP3A- and CYP1A-mediated biotransformation by competitive inhibition and that they have the potential to cause drug interactions with agents metabolized by these enzymes.
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PMID:Fluoroquinolone antibiotics inhibit cytochrome P450-mediated microsomal drug metabolism in rat and human. 889 16

1. A structural model of CYP3A4 is reported on the basis of a novel amino acid sequence alignment between the CYP3 family and CYP102, a bacterial P450 of known crystal structure. 2. Construction of the CYP3A4 model from CYP102 is facilitated by the relatively high sequence homology between the two protein (52% homology; 27% identity) with many conservative amino acid changes, yielding a structure of low internal energy. 3. A considerable number of specific substrates, and some specific inhibitors, are shown to occupy the putative CYP3A4 active site via interactions with the same amino acid residues in almost all cases investigated. 4. The CYP3A4 model rationalizes the known positions of metabolism for many substrates of this major human P450 such that the route of metabolism in novel development compounds can be predicted.
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PMID:Molecular modelling of CYP3A4 from an alignment with CYP102: identification of key interactions between putative active site residues and CYP3A-specific chemicals. 890 20

1. We have examined the metabolism of diazepam by ten human cytochrome P450 forms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) expressed in HepG2 cells using a recombinant vaccinia virus system. 2. Among the P450 forms tested, diazepam was significantly demethylated by CYP2B6, 2C9, 2C19, 3A4 and 3A5, with 2C19 exhibiting the highest rate at concentrations < 0.1 mM, and hydroxylated only by the latter three enzymes, with 3A5 being the most active. The N-demethylation activity of diazepam by 2C19 at a concentration of 20 microM was six times of that by 3A4. However, that by 2C9 was detected at only a trace level. 3. CYP2C19, 3A4 and 3A5 of the ten human P450s catalysed the 3-hydroxylation of nordiazepam, and 2B6, the 2C subfamily and the 3A subfamily catalysed the N-demethylation of temazepam. CYP3A4 exhibited the highest activity of nordiazepam 3-hydroxylation and temazepam N-demethylation. 4. Diazepam N-demethylation by human liver microsomes correlated with diazepam 3-hydroxylation, but not S-mephenytoin 4'-hydroxylation. 5. Our results suggest that in the human liver, the metabolism of diazepam to nordiazepam is mediated by CYP3A4, which has been reported as the most abundant P450 form in human liver as well as 2C19, which has been reported as a polymorphic enzyme.
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PMID:Human liver microsomal diazepam metabolism using cDNA-expressed cytochrome P450s: role of CYP2B6, 2C19 and the 3A subfamily. 894 91

Because YM17E (1,3-bis[[1-cycloheptyl-3-(p-dimethylaminophenyl) ureido]methyl]benzene dihydrochloride) inhibits acyl coenzyme A:cholesterol acyltransferase (ACAT) it has potential application in the treatment of hypercholesterolaemia. In man and animals YM17E is extensively metabolized, via N-demethylation, to five active metabolites (M1, M2-a, M2-b, M3 and M4). The main objectives of this study were to examine inhibition of YM17E metabolism by the products and identify the cytochrome P450 isoforms in liver microsomes which catalyse in-vitro YM17E metabolism in man. In microsomes in man N-demethylation of YM17E to M1 occurred enzymatically; for up to 45 s the rate was linearly proportional to the microsomal protein concentration. This reaction was inhibited by metabolites M2-a, M2-b, M3 and M4. Further, N-demethylation of [14C]-YM17E was also inhibited by its product, M1. These results showed that primary metabolism of YM17E was inhibited by its products, and supported the finding that the non-linear increase in plasma concentration of the parent drug and metabolites observed in an in-vivo study was due to inhibition by these products. Metabolic activity in microsomes from ten individual human livers demonstrated that YM17E N-demethylase activity correlated closely with testosterone 6 beta-hydroxylase activity. When cytochrome P450 isozyme-specific substrates and chemical inhibitors were used to inhibit YM17E N-demethylase activity, CYP3A-specific substrate and inhibitors such as nifedipine, ketoconazole and triacetyloleandomycin strongly inhibited this activity, whereas CYP1A-specific substrate or inhibitor, ethoxyresorufin and alpha-naphthoflavone, inhibited weakly. Other CYP inhibitors, in contrast, had few or no effects. An inhibition study using anti-rat CYP1A1, CYP2B1, CYP2C11, CYP2E1 and CYP3A2 antibodies demonstrated that only anti-rat CYP3A2 antibody inhibited YM17E metabolism, to 40% of control level, with no other antibodies showing an inhibitory effect. Of seven cDNA-expressed P450 isoforms in man (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1 and CYP3A4), CYP3A4, CYP2D6 and CYP1A2 isozyme exhibited substantial catalytic activity of N-demethylation of YM17E. These results indicate the predominant role of CYP3A4 in liver metabolism of YM17E in man.
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PMID:In-vitro metabolism of YM17E, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, by liver microsomes in man. 895 7


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