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)

Oxidative metabolic pathways of propranolol consist of naphthalene ring-hydroxylations (at the 4-, 5-, and 7-positions) and side-chain N-desisopropylation in mammals. We characterized cytochrome P450 isozymes responsible for propranolol metabolism, especially N-desisopropylation and 5-hydroxylation, in human liver microsomes. 4-Hydroxy, 5-hydroxy-, and N-desisopropylpropranolol were detected as primary metabolites, whereas 7-hydroxypropranolol was in trace amounts. Good correlations were obtained for activities of propranolol 4- and 5-hydroxylases with immunochemically determined CYP2D6 content, whereas correlations of these activities with CYP1A2, CYP2C, or CYP3A4 content were relatively low. The activities also correlated highly with debrisoquine 4-hydroxylase, compared with other metabolic activities such as phenacetin O-deethylase, hexobarbital 3'-hydroxylase, and testosterone 6 beta-hydroxylase, which are typical reactions for CYP1A2, CYP2C, and CYP3A4, respectively. Propranolol N-desisopropylase activity in the samples highly correlated with CYP1A2 content and phenacetin O-deethylase activity, but not with the other P450 isozyme contents or metabolic activities. Quinidine, a specific inhibitor of CYP2D6, inhibited propranolol 4- and 5-hydroxylase activities selectively and in a concentration-dependent manner. alpha-Naphthoflavone, a potent inhibitor of CYP1A2, inhibited all of the propranolol oxidation activities, and the IC50 value for N-desisopropylase activity was much smaller than the values for ring-hydroxylase activities. Antibody directed to CYP2D inhibited propranolol 4- and 5-hydroxylase activities by 70% at an antibody/microsomal protein ratio of 1.0. Anti-CYP2C9 antibody did not inhibit any activity determined. These results indicate that propranolol 5-hydroxylation, as well as 4-hydroxylation, is mainly catalyzed by CYP2D6 in human liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cytochrome P450 isozymes involved in propranolol metabolism in human liver microsomes. The role of CYP2D6 as ring-hydroxylase and CYP1A2 as N-desisopropylase. 789 9

The oxidative metabolism of bunitrolol, an adrenergic beta-receptor antagonist was examined in human liver microsomes fortified with an NADPH-generating system. The microsomal fractions (n = 11) showed bunitrolol 4-hydroxylase activities, which correlated well with CYP2D6 contents (correlation coefficient, r = 0.854), debrisoquine 4-hydroxylase (r = 0.953) and imipramine 2-hydroxylase (r = 0.976) activities. On the other hand, the bunitrolol 4-hydroxylase activity showed relatively poor correlations with CYP3A4 content (r = 0.552) and testosterone 6 beta-hydroxylase activity (r = 0.668). The bunitrolol 4-hydroxylase activity was significantly inhibited by quinidine, a selective inhibitor for CYP2D6. Polyclonal antibodies raised against rat liver microsomal cytochrome P450BTL, which is thought to belong to the CYP2D subfamily, effectively inhibited bunitrolol 4-hydroxylation. In contrast, polyclonal antibodies raised against human liver microsomal CYP3A4 did not show any inhibitory effect on the activity. These results suggest that CYP2D6 is involved in the bunitrolol 4-hydroxylase activity in human liver microsomes.
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PMID:Involvement of a cytochrome P4502D subfamily in human liver microsomal bunitrolol 4-hydroxylation. 795 Nov 42

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

Northern blot and immunoblot analyses indicated that considerable levels of CYP2B, CYP2C, CYP2D, CYP2E, and CYP3A were expressed in the liver of untreated marmosets. CYP1A was also expressed but to lesser extents. CYP3A mRNA was also detectable in the small intestine of untreated marmoset; the amount was increased by treatment with polychlorinated biphenyl. From a liver cDNA library, two cDNA clones coding for CYP2D19 and CYP3A21 (clones CM2D-1 and CM3A-10, respectively) were isolated. CM2D-1 and CM3A-10 contained an entire coding region for polypeptide 497 and 503 amino acid residues, respectively. The deduced amino acid sequences of CYP2D19 and CYP3A21 showed 90% identities to human CYP2D6 and CYP3A4, respectively. The value of CYP3A21 was 3% lower than that of cynomolgus monkey CYP3A8. On the other hand, these values were 11 to 23% higher than those of the other experimental animals, including dogs, rabbits, guinea pigs, rats, mice, and hamsters. These results indicate that the marmoset stands at a midpoint between human and nonprimate experimental animals.
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PMID:Marmoset liver cytochrome P450s: study for expression and molecular cloning of their cDNAs. 905 37

We have previously found that for acetaminophen kinetic differences exist between the hepatic microsomal catalyzed protein binding and cysteine conjugation. We have also observed that the protein binding of acetaminophen is only to intralumenal proteins. Together these data suggested that two pools of the reactive metabolite, N-acetyl-p-benzoquinone imine (NABQI), are formed during the oxidative metabolism of acetaminophen: one on the cytosolic surface and the other within the lumen of the microsomes. This would indicate that some of forms of cytochrome P450 (CYP) catalyzing NABQI formation have their active site on the cytosolic surface and others on the lumenal surface. We have examined this question by comparing the rates of cysteine conjugation and protein binding of acetaminophen by microsomes from lymphoblasts transfected with the cDNAs for human CYPs. We found that CYP2D6 catalyzed only cysteine conjugation; CYP1A2 and 3A4 catalyzed only protein binding; CYP2E1 catalyzed both; and CYP1A1, CYP2A6 and CYP2B6 catalyzed neither. These data suggest that CYP2D6 has its active site only on the cytosolic surface; CYP1A2 and CYP3A4 only on the lumenal surface; and CYP2E1 has catalytic sites on both the lumenal and cytosolic surfaces of the membrane. In mouse studies we have found that ethanol administration increased acetaminophen protein binding by 265% but cysteine conjugation by only 61%. CYP2E1 and CYP2B increased, whereas CYP3A decreased and the others did not change. These data suggest that in control mice CYP2E1 catalyzes the bulk of protein binding, whereas CYP2D catalyzes slightly more cysteine conjugation than does CYP2E1.
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PMID:Catalysis of the cysteine conjugation and protein binding of acetaminophen by microsomes from a human lymphoblast line transfected with the cDNAs of various forms of human cytochrome P450. 915 86

The antihypertensive agent diltiazem (DTZ) impairs hepatic drug metabolism by inhibition of cytochrome P450 (CYP). The accumulation of DTZ metabolites in serum occurs during prolonged therapy and leads to decreased DTZ elimination. Thus, DTZ metabolites may contribute to CYP inhibition. This study assessed the role of human CYPs in microsomal DTZ oxidation and the capacity of DTZ metabolites to inhibit specific CYP activities. DTZ N-demethylation varied 10-fold in microsomal fractions from 17 livers (0.33-3.31 nmol/mg of protein/min). DTZ oxidation was correlated with testosterone 6beta-hydroxylation (r = 0.82) and, to a lesser extent, tolbutamide hydroxylation (r = 0.59) but not with activities mediated by CYP1A2 or CYP2E1. CYP3A4 in lymphoblastoid cell microsomes catalyzed DTZ N-demethylation but CYP2C8 and CYP2C9 were also active (approximately 20% and 10% of the activity supported by CYP3A4); seven other CYPs produced little or no N-desmethyl DTZ from DTZ. The CYP3A4 inhibitors ketoconazole and troleandomycin decreased microsomal DTZ oxidation, but inhibitors or substrates of CYP2C, CYP2D and CYP2E1 produced no inhibition. Some inhibition was produced by alpha-naphthoflavone, a chemical that inhibits CYP1As and also interacts with CYP3A4. In further experiments, the capacities of DTZ and three metabolites to modulate human CYP 1A2, 2E1, 2C9 and 3A4 activities were evaluated in vitro. DTZ and its N-desmethyl and N,N-didesmethyl metabolites selectively inhibited CYP3A4 activity, whereas O-desmethyl DTZ was not inhibitory. The IC50 value of DTZ against CYP3A4-mediated testosterone 6beta-hydroxylation (substrate concentration, 50 microM) was 120 microM. The N-desmethyl (IC50 = 11 microM) and N,N-didesmethyl (IC50 = 0.6 microM) metabolites were 11 and 200 times, respectively, more potent. From kinetic studies, N-desmethyl DTZ and N,N-didesmethyl DTZ were potent competitive inhibitors of CYP3A4 (Ki = approximately 2 and 0.1 microM, respectively). CYP3A4 inhibition was enhanced when DTZ and N-desmethyl DTZ underwent biotransformation in NADPH-supplemented hepatic microsomes in vitro, supporting the contention that inhibitory metabolites may be generated in situ. These findings suggest that N-demethylated metabolites of DTZ may contribute to CYP3A4 inhibition in vivo, especially under conditions in which N-desmethyl DTZ accumulates, such as during prolonged DTZ therapy.
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PMID:Role of CYP3A4 in human hepatic diltiazem N-demethylation: inhibition of CYP3A4 activity by oxidized diltiazem metabolites. 922 67

To clarify the mechanism of the species difference in the metabolism of bisoprolol enantiomers, in vitro metabolic studies were performed using dog liver microsomes and human cytochrome P450 (CYP) isoforms. The O-deisopropylation of bisoprolol enantiomers showed biphasic kinetics in dog liver microsomes. The intrinsic clearance (Vmax/Km) for O-deisopropylation of R(+)-bisoprolol was higher than S(-)-isomer in both high-affinity and low-affinity components. The R/S ratio of the intrinsic clearance in high- and low-affinity components was 1.34 and 1.65, respectively. The inhibition studies in dog liver microsomes using CYP isoform-selective inhibitors indicated that the O-deisopropylation of both bisoprolol enantiomers was mediated via the CYP2D and CYP3A subfamily, and suggested that high-affinity oxidation was dependent on CYP2D. The kinds of CYP subfamilies in dogs, which contribute to the metabolism of bisoprolol enantiomers, were the same as those in humans. The intrinsic clearance for O-deisopropylation of R(+)bisoprolol by human recombinant CYP2D6 was also different from that of S(-)-enantiomers (R/S:1.50). However, unlike the dog microsomes, the intrinsic clearance by the human recombinant CYP3A4 did not show a stereoselective difference. Therefore, the species difference in the R/S ratio of metabolic clearance for the oxidation of bisoprolol enantiomers (dog > human) is mainly due to the species difference in the stereoselectivity of one of the cytochrome P450 subfamilies (CYP3A).
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PMID:Stereoselective metabolism of bisoprolol enantiomers in dogs and humans. 976 5

1. Cytochrome P450 (P450) isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21) have been identified in rat and man. 2. Anti-CYP2C11 antibody inhibited the N-deethylation of S- and R-NS-21 in rat hepatic microsomes by 84 and 66% respectively, indicating that CYP2C11 is mainly responsible for these activities in male rats. 3. Of several human recombinant P450 isoforms, CYP3A4 had the activities for the N-deethylation of S- and R-NS-21. In addition, triacetyloleandomycin (TAO), an inhibitor of the CYP3A subfamily, significantly inhibited the N-deethylation of S- and R-NS-21 in human hepatic microsomes by 67 and 69%, respectively. CYP3A4 therefore contributes to it in man. 4. Quinine, an inhibitor of the rat CYP2D subfamily, significantly inhibited the cyclohexane-4-cis-hydroxylation of S-NS-21 by 48% in rat hepatic microsomes. In contrast, this inhibitor had little effect on the cyclohexane-4-trans-hydroxylation of S-NS-21, and the cyclohexane-4-cis- and trans-hydroxylation of R-NS-21. 5. Human recombinant CYP3A4 catalysed the cyclohexane-4-trans-hydroxylation of S-NS-21, and CYP2D6 supported the cyclohexane-4-cis- and trans-hydroxylation of S-NS-21. Quinidine, an inhibitor of human CYP2D6, had little effect on these latter activities in human hepatic microsomes. TAO significantly inhibited the cyclohexane-4-trans-hydroxylation of S-NS-21 by 75%, indicating that CYP3A4 catalyses this reaction.
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PMID:Cytochrome P450 isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of NS-21. 1021 65

1. The in vitro metabolism of indinavir (CRIXIVAN, MK-0639, L-735,524), an HIV protease inhibitor, was evaluated using liver microsomes from cynomolgus monkey, rhesus monkey, chimpanzee and human. Indinavir exhibited marked species differences in metabolism. The overall rate of indinavir metabolism varied > 4-fold among primates (84 pmol/min/mg protein in cynomolgus monkey versus 20.4 pmol/min/mg protein in human) and followed the rank order: cynomolgus monkey > rhesus monkey > chimpanzee > human. 2. The cis-(indan)hydroxylated metabolite of indinavir was formed only in cynomolgus and rhesus monkey livers, whereas trans-(indan)hydroxylation and N-dealkylation were observed as the major metabolites in all primates tested. Inhibition studies with P450-selective inhibitors (ketoconazole, quinine, quinidine) and monoclonal antibodies (against CYP2D6 or CYP3A4) indicated that a cytochrome P450 isoform of the CYP2D subfamily is involved in the formation of the unique cis-(indan) hydroxylated metabolite in monkey, whereas all other oxidative metabolites, including the trans-(indan)hydroxylated metabolite, are formed by CYP3A isoform(s). 3. The present study has demonstrated that monkeys were unique in their abilities to form the stereoselective metabolite and were not appropriate surrogates for the qualitative prediction of indinavir metabolism in human.
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PMID:Comparative in vitro metabolism of indinavir in primates--a unique stereoselective hydroxylation in monkey. 1071 20

The pig is increasingly being used in pharmacological and toxicological studies, and is the species of choice for future research into xenotransplantation, extracorporeal liver support and hepatocyte-based bioartificial liver. However, relatively little is known about xenobiotic-metabolizing enzymes in this species. In the present study, immunoblotting with polyclonal anti-rat and anti-human cytochrome P450 (CYP) antibodies revealed the presence of proteins in pig liver which cross-reacted with anti-human CYP1A2, CYP2D6 and CYP3A4, and with anti-rat CYP2E1 antibodies. Northern blot analysis demonstrated the presence of mRNA which hybridized to cDNA probes for human CYP2D6, CYP2E1 and CYP3A4, and to an oligonucleotide probe for pig CYP3A29. As there is a lack of a good animal model for CYP2D6, the presence of a CYP2D6-related protein in pig liver was of particular interest. Pig hepatocytes also demonstrated CYP2D6 immunoreactive protein, and mRNA hybridizable to a CYP2D6 cDNA probe. We investigated the ability of pig liver microsomes to catalyse dextromethorphan O-demethylation, a widely-used marker enzyme activity for CYP2D6. This enzyme activity demonstrated biphasic kinetics, with a high affinity apparent K(m1)=6.9+/-3.6 microM and V(max1)=10.5+/-6.1nmol/min/nmol CYP. The reaction was sensitive to inhibition by the CYP2D6-selective inhibitors quinidine, quinine, lobeline and norfluoxetine, whereas chemical inhibitors selective for other CYP isoforms failed to affect the reaction. We conclude that dextromethorphan O-demethylation is catalysed by a CYP2D enzyme which is remarkably similar to human CYP2D6, suggesting potential value of the pig as a model for predicting human metabolism of xenobiotics which undergo CYP2D6-dependent biotransformation.
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PMID:Evidence for the catalysis of dextromethorphan O-demethylation by a CYP2D6-like enzyme in pig liver. 1080 76


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