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PubMed2003

Atorvastatin reduces the ability of clopidogrel to inhibit platelet aggregation: a new drug-drug interaction.

BACKGROUND: We observed that the prodrug clopidogrel was less effective in inhibiting platelet aggregation with coadministration of atorvastatin during point-of-care platelet function testing. Because atorvastatin is metabolized by cytochrome P450 (CYP) 3A4, we hypothesized that clopidogrel might be activated by CYP3A4. METHODS AND RESULTS: Platelet aggregation was measured in 44 patients undergoing coronary artery stent implantation treated with clopidogrel or clopidogrel plus pravastatin or atorvastatin, and in 27 volunteers treated with clopidogrel and either erythromycin or troleandomycin, CYP3A4 inhibitors, or rifampin, a CYP3A4 inducer. Atorvastatin, but not pravastatin, attenuated the antiplatelet activity of clopidogrel in a dose-dependent manner. Percent platelet aggregation was 34+/-23, 58+/-15 (P=0.027), 74+/-10 (P=0.002), and 89+/-7 (P=0.001) in the presence of clopidogrel and 0, 10, 20, and 40 mg of atorvastatin, respectively. Erythromycin attenuated platelet aggregation inhibition (55+/-12 versus 42+/-12% platelet aggregation; P=0.002), as did troleandomycin (78+/-18 versus 45+/-18% platelet aggregation; P<0.0003), whereas rifampin enhanced platelet aggregation inhibition (33+/-18 versus 56+/-20% platelet aggregation, P=0.001). CONCLUSIONS: CYP3A4 activates clopidogrel. Atorvastatin, another CYP3A4 substrate, competitively inhibits this activation. Use of a statin not metabolized by CYP3A4 and point-of-care platelet function testing may be warranted in patients treated with clopidogrel.

Adolescent↗
PubMed1999

Cytochrome CYP sources of N-alkylprotoporphyrin IX after administration of porphyrinogenic xenobiotics to rats.

Cytochrome P-450 (CYP) 3A2 and CYP2C11 are sources of 70 and 30%, respectively, of N-vinylprotoporphyrin IX (N-vinylPP) formation after administration of 3-[(arylthio)ethyl]sydnone (TTMS) to rats. Female rats receiving TTMS were pretreated with dexamethasone, which induces CYP3A1 preferentially to CYP3A2. The resulting 12-fold increase in N-vinylPP formation showed that CYP3A1 was also a source of N-vinylPP. Phenobarbital (PB) pretreatment, which induces CYP2B1/2 and 3A1/2 in male rats, increased N-vinylPP formation after TTMS administration. Troleandomycin, a selective CYP3A inhibitor, was unable to decrease TTMS-mediated N-vinylPP formation in PB-treated male rats, indicating that CYP2B1/2 were sources of N-vinylPP. This conclusion was supported by demonstrating a 15-fold increase in TTMSinduced N-vinylPP formation in female rats after CYP2B1/2 induction with PB pretreatment. Allylispropylacetamide (AIA) inactivates rat CYP2B1/2, 2C6, 2C7, 2C11, and 3A1/2. Troleandomycin was unable to decrease N-AIA protoporphyrin IX adduct (N-AIAPP) formation, showing that CYP3A1/2 were not susceptible to AIA-mediated N-alkylation. N-AIAPP formation in females was approximately 30% of that in males, and thus we attribute 30% of N-AIAPP formation in males to the non-gender-specific isozymes (CYP2C6, 2C7, and/or 2B1/2), whereas approximately 70% originates from CYP2C11. PB treatment in female rats resulted in a 5-fold increase in N-AIAPP formation, showing that CYP2B1/2 were also susceptible to N-alkylation mediated by AIA. 1-Aminobenzotriazole elicited formation of equivalent amounts of N'N-aryl bridged protoporphyrin IX in male and female rat liver, demonstrating that nonselective mechanism-based inactivation is accompanied by nonselective conversion of the CYP heme moieties to N'N-aryl bridged protoporphyrin IX.

Allylisopropylacetamide↗
PubMed2001

Metabolism of levo-alpha-Acetylmethadol (LAAM) by human liver cytochrome P450: involvement of CYP3A4 characterized by atypical kinetics with two binding sites.

levo-alpha-Acetylmethadol (LAAM) is a long-acting opioid agonist prodrug used for preventing opiate withdrawal. LAAM undergoes bioactivation via sequential N-demethylation to nor-LAAM and dinor-LAAM, which are more potent and longer-acting than LAAM. This study examined LAAM and nor-LAAM metabolism using human liver microsomes, cDNA-expressed CYP, CYP isoform-selective chemical inhibitors, and monoclonal antibody to determine kinetic parameters for predicting in vivo drug interactions, involvement of constitutive CYP isoforms, and mechanistic aspects of sequential N-demethylation. N-Demethylation of LAAM and nor-LAAM by human liver microsomes exhibited biphasic Eadie-Hofstee plots. Using a dual-enzyme Michaelis-Menten model, K(m) values were 19 and 600 microM for nor-LAAM and 4 and 450 microM for dinor-LAAM formation, respectively. LAAM and nor-LAAM metabolism was inhibited by the CYP3A4-selective inhibitors troleandomycin, erythromycin, ketoconazole, and midazolam. Of the cDNA-expressed isoforms examined, CYP2B6 and 3A4 had the highest activity toward LAAM and nor-LAAM at both low (2 microM) and high (250 microM) substrate concentrations. N-Demethylation of LAAM and nor-LAAM by expressed CYP3A4 was unusual, with hyperbolic velocity curves and Eadie-Hofstee plots and without evidence of positive cooperativity. Using a two-site model, K(m) values were 6 and 0.2 microM, 1250 and 530 microM, respectively. Monoclonal antibody against CYP2B6 inhibited CYP2B6-catalyzed but not microsomal LAAM or nor-LAAM metabolism, whereas troleandomycin inhibited metabolism in all microsomes studied. The ratio [dinor-LAAM/(nor-LAAM plus dinor-LAAM)] with microsomes and CYP3A4 decreased with increasing LAAM concentration, suggesting most dinor-LAAM is formed from released nor-LAAM that subsequently reassociates with CYP3A4. Based on these results, we conclude that LAAM and nor-LAAM are predominantly metabolized by CYP3A4 in human liver microsomes, and CYP3A4 exhibits unusual multisite kinetics.

Antibodies, Monoclonal↗
PubMed2001

Metabolism of methadone and levo-alpha-acetylmethadol (LAAM) by human intestinal cytochrome P450 3A4 (CYP3A4): potential contribution of intestinal metabolism to presystemic clearance and bioactivation.

Methadone and levo-alpha-acetylmethadol (LAAM) are opioid agonists used for analgesia and preventing opiate withdrawal. Methadone is sequentially N-demethylated to the inactive metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP). LAAM is essentially a prodrug that undergoes bioactivation via sequential N-demethylation to levo-alpha-acetyl-N-normethadol (nor-LAAM) and levo-alpha-acetyl-N,N-dinormethadol (dinor-LAAM). Methadone and LAAM are metabolized by CYP3A4 in human liver. Since they are administered orally, and CYP3A4 is expressed in human intestine, we tested the hypotheses that human intestine can metabolize methadone and LAAM, and evaluated the participation of CYP3A4. Intestinal microsomal methadone N-demethylation exhibited hyperbolic noncooperative kinetics and biphasic Eadie-Hofstee plots. Using a dual-enzyme Michaelis-Menten model, K(m) values were 11 and 1200 microM for EDDP and 23 and 930 microM for EMDP formation, respectively. CYP3A4 inhibitors (troleandomycin and ketoconazole) inhibited EDDP and EMDP formation by >70%. Methadone N-demethylation by CYP3A4 showed biphasic Eadie-Hofstee plots without evidence of positive cooperativity; K(m) values were 10 and 1100 microM for EDDP and 20 and 1000 microM for EMDP formation. Intestinal microsomal LAAM and nor-LAAM N-demethylation also exhibited hyperbolic kinetics and biphasic Eadie-Hofstee plots. K(m) values were 21 and 980 microM for nor-LAAM from LAAM and 18 and 1200 microM for dinor-LAAM from nor-LAAM. Troleandomycin and ketoconazole inhibited N-demethylation by >70%. LAAM and nor-LAAM metabolism by CYP3A4 showed biphasic Eadie-Hofstee plots without evidence of positive cooperativity; K(m) values were 8 and 1300 microM, 6 and 950 microM, respectively. Predicted in vivo intestinal extraction of methadone and LAAM is 21 and 33%, respectively. We conclude that methadone, LAAM, and nor-LAAM are metabolized by human intestinal microsomes; CYP3A4 is the predominant cytochrome P450 isoform; CYP3A4-catalyzed methadone, LAAM, and nor-LAAM metabolism is characterized by noncooperative, multisite kinetics; and intestinal metabolism may contribute to presystemic methadone inactivation and LAAM bioactivation.

Algorithms↗
PubMed2004

Relationship between content and activity of cytochrome P450 and induction of heterocyclic amine DNA adducts in human liver samples in vivo and in vitro.

This study was designed to estimate a correlation between metabolic activation phenotypes and formation of DNA adducts by heterocyclic amines (HCA) in 15 liver samples from healthy donors. The correlation between the amount of endogenous DNA adducts and the content of cytochrome P450 in human liver samples in vivo was statistically significant at r(2) = 0.71 and P < 0.005. Furthermore, the isolated human liver microsomes were treated in vitro with two HCAs, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-9H-pyrido[2,3-b]indole (A alpha C), which have been recognized to induce two DNA adducts: 3',5'-diphosphate-N-(2'-deoxyguanosin-8-yl)-PhIP (3',5'-pdGp-C8-PhIP) and 3',5'-diphosphate-N-(2'-deoxyguanosin-8-yl)-A alpha C (3',5'-pdGp-C8-A alpha C). The correlations between the amount of DNA adducts induced by both compounds in vitro and the content of cytochrome P450 in human microsomes are statistically significant at r(2) = 0.69 and r(2) = 0.62 (P < 0.001), respectively. Furthermore, the level of DNA adducts after treatment with PhIP and A alpha C correlated with the activities of three isozymes of cytochrome P450: CYP1A1, CYP1A2, and CYP3A4. Therefore, three chemical inhibitors were used in the experiments: ellipticine against CYP1A1, furafylline against CYP1A2, and troleandomycin against CYP3A4. The highest inhibition levels in the formation of 3',5'-pdGp-C8-PhIP and 3',5'-pdGp-C8-A alpha C adducts were estimated to occur in the presence of furafylline at 56% and 69%, respectively. Ellipticine was involved in the inhibition of 40% of 3',5'-pdG-C8-PhIP adducts and in only 18% of the inhibition of 3',5'-pdGp-C8-A alpha C adducts. Troleandomycin did not significantly inhibit the formation of 3',5'-pdGp-C8-PhIP adducts under these conditions, but it inhibited the formation of 31% of the 3',5'-pdGpC8-A alpha C adducts. We conclude that the formation of DNA adducts can be used as a relevant marker of interindividual variability in the metabolic activation of HCAs in humans.

Adult↗
PubMedDate not supplied

Terfenadine metabolism in human liver. In vitro inhibition by macrolide antibiotics and azole antifungals.

To determine whether the clinical adverse interactions of terfenadine with azole antifungals and macrolide antibiotics may be related to inhibition of terfenadine biotransformation, an in vitro system was developed to follow the metabolism of terfenadine by rat liver S9 or human liver microsomes. When test compounds were coincubated with terfenadine, the metabolites formed and unchanged terfenadine was quantitatively analyzed by HPLC. Five metabolites of terfenadine were formed by rat liver S9: predominantly alcohol metabolite (III), with four minor metabolites--azacyclonol (I), acid metabolite (II), an unidentified metabolite (IV), and a new ketone metabolite (V). By human liver microsomes, two major metabolites were formed: azacyclonol (I) and alcohol metabolite (III). Ketoconazole, fluconazole, itraconazole, erythromycin, clarithromycin, and troleandomycin potently inhibited terfenadine metabolism by human liver (IC50 = 4-10 microM), but inhibition by rat liver was weaker (IC50 = 87-218 microM) and 18% maximally for troleandomycin. Other CYP3A substrates (cyclosporin A, naringenin, and midazolam) also demonstrated potent inhibition of terfenadine biotransformation in human liver microsomes (IC50 = 17-24 microM). Substrates of other P450 families [sparteine (CYP2D6), caffeine (CYP1A), and diclofenac (CYP2C)] only very weakly inhibited terfenadine metabolism. Dixon plot analyses for human liver revealed competitive/reversible inhibition by the azole antifungals and macrolide antibiotics of azacyclonol and alcohol metabolite formations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Metabolic activation of the nitroaromatic antiandrogen flutamide by rat and human cytochromes P-450, including forms belonging to the 3A and 1A subfamilies.

The in vitro metabolic activation of flutamide, a nitroaromatic antiandrogen which produces hepatitis in a few recipients, was first studied with male rat liver microsomes. There was no electron spin resonance evidence for the reduction of flutamide by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase into a nitro anion free radical. In contrast, flutamide was oxidatively transformed by cytochrome P-450 into reactive metabolite(s) that covalently bound to microsomal proteins. Covalent binding required oxygen and NADPH, and was decreased by the nucleophile glutathione and by the cytochrome P-450 inhibitors SKF 525-A, piperonyl butoxide and troleandomycin (an inhibitor of the cytochrome P-450 3A subfamily). Covalent binding was increased markedly by pretreatment with dexamethasone (an inducer of the cytochrome P-450 3A subfamily) and moderately by pretreatment with beta-naphthoflavone (an inducer of the 1A family). Covalent binding was immunoinhibited markedly by anticytochrome P-450 3A immunoglobulin G and moderately by anticytochrome P-450 1A immunoglobulin G. Covalent binding was much lower with liver microsomes from female rats (not expressing P-450 3A2). Covalent binding of flutamide also occurred with human liver microsomes (where it was inhibited by troleandomycin), and with yeast microsomes expressing human liver cytochromes P-450 1A1, 1A2 or 3A4. We concluded that flutamide was oxidatively transformed into chemically reactive metabolite(s) by rat and human cytochromes P-450, including forms belonging to the 3A and 1A subfamilies.

Animals↗
PubMed1995

Metabolism of the immunosuppressant tacrolimus in the small intestine: cytochrome P450, drug interactions, and interindividual variability.

The small intestinal metabolism of tacrolimus, which is used as an immunosuppressant in transplantation medicine, was investigated in this study. Tacrolimus was metabolized in vitro by isolated human, pig, and rat small intestinal microsomes. The metabolites generated were identified by HPLC/MS. Tacrolimus and its metabolites were quantified using HPLC or HPLC/MS. The cytochrome P450 (CYP) enzymes responsible for tacrolimus metabolism in small intestine were identified using specific CYP antibodies and inhibitors. For characterization of the interindividual variability, microsomes were isolated from small intestinal samples of patients who had undergone resection for various reasons. In an in vitro model using pig small intestinal microsomes, 32 drugs were analyzed for their interactions with tacrolimus metabolism. After incubation with human, rat, and pig small intestinal microsomes, the metabolites 13-O-demethyl and 13,15-O-demethyl tacrolimus were identified. The metabolism of tacrolimus by human small intestine was inhibited by anti-CYP3A, troleandomycin, and erythromycin, indicating that, as in the liver, CYP3A enzymes are the major enzymes for tacrolimus metabolism in the human small intestine. Metabolism of tacrolimus by small intestinal microsomes isolated from 14 different patients varied between 24 and 110 pmol/13-O-demethyl tacrolimus/min/mg microsomal protein, with a mean +/- SD of 54.2 +/- 29.2 pmol/min/mg. Of 32 drugs tested, 15 were found to inhibit small intestinal tacrolimus metabolism: bromocryptine, corticosterone, cyclosporine, dexamethasone, ergotamine, erythromycin, ethinyl estradiol, josamycin, ketoconazole, nifedipine, omeprazole, progesterone, rapamycin, troleandomycin, and verapamil. All of these drugs inhibited tacrolimus metabolism by human liver microsomes as well. It is concluded that tacrolimus is metabolized by cytochrome CYP3A enzymes in the small intestine. The rate of the CYP3A enzymatic activities varies about 5 times from patient to patient, and drugs that interfere with the in vitro metabolism of tacrolimus in the liver also inhibit its small intestinal metabolism.

Animals↗
PubMed1995

Microsomal metabolism of the 5-lipoxygenase inhibitors L-746,530 and L-739,010 to reactive intermediates that covalently bind to protein: the role of the 6,8-dioxabicyclo[3.2.1]octanyl moiety.

Hepatic microsomes from different species were used to study the oxidative metabolism of L-746,530 and L-739,010, two potent and specific 5-lipoxygenase inhibitors. HPLC analysis of the incubates obtained from the microsomal incubations of L-739,010 and L-746,530 showed only traces of metabolites. However, recovery of the starting material in the supernatant was less than quantitative in all of the species studied (approximately 90% in rat, approximately 70% in the dexamethasone-induced rat, approximately 70-90% in humans, and approximately 60% in the rhesus monkey for both compounds). The recovery of the starting material was found to be time- and NADPH-dependent, suggesting that metabolite(s) were formed and reacting with the microsomal proteins. Evidence that the cytochrome P4503A (CYP3A) contributed to the formation of the reactive metabolite(s) was shown by the low recovery of material that was observed upon incubation with microsomes obtained from dexamethasone-treated rats (a CYP3A inducer), compared with microsomes obtained from untreated rats. Also, the recovery of material was improved when troleandomycin, a CYP3A inhibitor, was added to rhesus monkey microsomal incubations (25% more parent compound detected in the supernatant with 100 microM of troleandomycin). Using radiolabeled L-746,530 and gel electrophoresis analysis, it was confirmed that radiolabeled material was covalently bound to the microsomal protein. Incubations of L-739,010 and L-746,530 in the presence of semicarbazide resulted, in both cases, in the formation of two adducts. Using a combination of NMR, liquid secondary-ion MS, and UV techniques, these adducts were identified as isomers of an oxidized metabolite that had been trapped by semicarbazide. The site of oxidation was determined to be on the dioxabicyclo moiety. The importance of this moiety in the formation of reactive metabolite(s) was verified by incubating analogs of the 5-lipoxygenase inhibitors that contained blocking methyl groups at the proposed site of oxidation on the bicyclo moiety. Incubations of these gemdimethyl analogs of L-746,530 and L-739,010 with microsomes from different species resulted in significantly improved recovery of the starting material (approximately 94% in the rat, 85% in the dexamethasone-induced rat, 95% in humans, and 85% in the rhesus monkey for both compounds) and significantly less radioactive binding to the microsomal protein.

Animals↗
PubMed1996

Role of cytochrome P450 in oxazaphosphorine metabolism. Deactivation via N-dechloroethylation and activation via 4-hydroxylation catalyzed by distinct subsets of rat liver cytochromes P450.

The roles of individual liver cytochrome P450 (P450) enzymes in N-dechloroethylation leading to deactivation and neurotoxification of the isomeric alkylating agent prodrugs ifosfamide (IF) and cyclophosphamide (CPA) were investigated using an in vitro rat liver model. Rats were pretreated with a panel of drugs, including phenobarbital (a strong inducer of liver P450 2B1/2B2) and dexamethasone (a strong inducer of P450 3A enzymes), to examine the effects of these P450-inducing agents on IF and CPA N-dechloroethylation catalyzed by rat hepatic microsomes. The P450 3A-specific inhibitor troleandomycin and inhibitory monoclonal antibodies reactive with P450 2B and 2C enzymes were used to identify the individual P450 subfamilies involved in microsomal N-dechloroethylation of IF and CPA. It was found that dexamethasone pretreatment preferentially elevated microsomal CPA N-dechloroethylation activity (12-fold increase) and that P450 3A enzymes catalyzed up to > 95% of this reaction in both uninduced and drug-induced liver. In contrast, IF N-dechloroethylation activity was stimulated (approximately 8-fold increase) in liver microsomes by phenobarbital pretreatment, and P450 2B1/2B2 were responsible for the majority of this activity. In addition, P450 2C11 catalyzed approximately 50% of IF N-dechloroethylation in uninduced male rat liver microsomes. inducers of P450 1A and 4A enzymes had no effect on N-dechloroethylation of IF or CPA. These P450 enzyme patterns for the N-dechloroethylation reaction are distinct from those previously determined for IF and CPA activation via 4-hydroxylation. In accord with this observation, the balance between oxazaphosphorine activation (4-hydroxylation pathway) and deactivation/neurotoxication (N-dechloroethylation pathway) could be modulated by P450 form-selective inducers and inhibitors. Thus, dexamethasone pretreatment substantially decreased the extent of IF N-dechloroethylation, from 47% to 24% of total metabolism, whereas it increased CPA N-dechloroethylation from 29% to 84% of total metabolism. Moreover, troleandomycin selectively inhibited CPA N-dechloroethylation, thereby increasing net metabolism of the drug via the therapeutically productive 4-hydroxylation pathway. Oxazaphosphorine activation and deactivation/neurotoxication are thus catalyzed by distinct subsets of liver P450 enzymes, in a manner that may allow for improvements in therapeutic indices for this class of drugs by using P450 form-selective modulators.

Animals↗
PubMed1997

Fentanyl metabolism by human hepatic and intestinal cytochrome P450 3A4: implications for interindividual variability in disposition, efficacy, and drug interactions.

The synthetic opioid fentanyl undergoes extensive metabolism in humans. Systemic elimination occurs primarily by hepatic metabolism. When administered as a lozenge for oral transmucosal absorption, swallowed fentanyl is subject to first pass metabolism in the liver and possibly small intestine. Little is known, however, about the identity and formation of human fentanyl metabolites. This investigation identified routes of human liver microsomal fentanyl metabolism and their relative importance, tested the hypothesis that fentanyl is metabolized by human duodenal microsomes, and identified the predominantly responsible cytochrome P450 isoforms. A GC/MS assay using deuterated internal standards was developed for fentanyl metabolites. Piperidine N-dealkylation to norfentanyl was the predominant pathway of liver microsomal metabolism. Amide hydrolysis to despropionylfentanyl and alkyl hydroxylation to hydroxyfentanyl were comparatively minor pathways. Hydroxynorfentanyl was identified as a minor, secondary metabolite arising from N-dealkylation of hydroxyfentanyl. Liver microsomal norfentanyl formation was significantly inhibited by the mechanism-based P450 3A4 inhibitor troleandomycin and the P450 3A4 substrate and competitive inhibitor midazolam, and was significantly correlated with P450 3A4 protein content and catalytic activity. Of six expressed human P450 isoforms (P450s 1A2, 2B6, 2C9, 2D6, 2E1, and 3A4), only P450 3A4 exhibited significant fentanyl dealkylation to norfentanyl. These results indicate the predominant role of P450 3A4 in the primary route of hepatic fentanyl metabolism. Human duodenal microsomes also catalyzed fentanyl metabolism to norfentanyl; the average rate was approximately half that of hepatic metabolism. Rates of duodenal norfentanyl formation were diminished by troleandomycin and midazolam, and were significantly correlated with P450 3A4 activity, suggesting a prominent role for P450 3A4. These results demonstrate that human intestinal as well as liver microsomes catalyze fentanyl metabolism, and N-dealkylation by P450 3A4 is the predominant route in both organs. The fraction of fentanyl lozenge that is swallowed likely undergoes significant intestinal, as well as hepatic, first-pass metabolism. Intestinal and hepatic first-pass metabolism, as well as systemic metabolism, may be subject to individual variability in P450 3A4 expression and to drug interactions involving P450 3A4.

Analgesics, Opioid↗
PubMed1997

In vitro comparison of cytochrome P450-mediated metabolic activities in human, dog, cat, and horse.

As domestic animals such as cat, horse, and dog increasingly become the clinical targets for drug discovery programs, the need to understand how these animals metabolize xenobiotics becomes more important. In the present study, substrates and inhibitors that were reported to be selective for particular P450 isozymes were used as probes to study in vitro metabolism in horse, dog, cat, and human liver microsomes. Seven selective catalytic activity markers for cytochrome P450-mediated reactions were measured: phenacetin O-deethylase (P4501A1/2), coumarin 7-hydroxylase (P4502A6), tolbutamide hydroxylase (P4502C8/9), S-mephenytoin 4'-hydroxylase (P4502C19), dextromethorphan O-demethylase (P4502D6), chlorzoxazone 6-hydroxylase (P4502E1), and testosterone 6beta-hydroxylase (P4503A4). Metabolic activity was found in every species with each substrate. Under the conditions of this study, it was observed that no one species was more active for any given substrate. However, rather large interspecies differences were observed. There was no marked sex difference in the way the various species metabolized the different substrates. The effect of selective P450 inhibitors on the various activities was tested with furafylline (P4501A2), mouse monoclonal antibody inhibitory to CYP2A6, sulfaphenazole (P4502C9), tranylcypromine (P4502C19), quinidine (P4502D6), diethyldithiocarbamate (P4502E1), and troleandomycin (P4503A4). In most cases, these inhibitors were effective to varying degrees against the activity seen in horse, dog, and cat liver microsomes. However, even at high concentrations, furafylline did not inhibit phenacetin O-deethylase activity in cat and troleandomycin did not affect testosterone 6beta-hydroxylase activity in horse. Sulfaphenazole was not tested in dog and cat because of the low tolbutamide hydroxylase activity. Overall, these results show that there are also large interspecies differences in the way the selective P450 inhibitors affect the in vitro metabolism of the various substrates in horse, dog, and cat liver microsomes.

Adolescent↗
PubMed1998

Identification of the human P-450 enzymes responsible for the sulfoxidation and thiono-oxidation of diethyldithiocarbamate methyl ester: role of P-450 enzymes in disulfiram bioactivation.

Diethyldithiocarbamate methyl ester (DDTC-Me) is a precursorto the formation of S-methyl-N,N-diethylthiolcarbamate sulfoxide, the active metabolite proposed to be responsible for the alcohol deterrent effects of disulfiram. The present study investigated the role of human cytochrome P-450 (CYP) enzymes in sulfoxidation and thiono-oxidation of DDTC-Me, intermediary steps in the activation of disulfiram. Several approaches were used in an attempt to delineate the particular P-450 enzyme(s) involved in the sulfoxidation and thiono-oxidation of DDTC-Me. These approaches included the use of cDNA-expressed human P-450 enzymes, correlation analysis with sample-to-sample variation in human P-450 enzymes in a bank of human liver microsomes, and chemical and antibody inhibition studies. Multiple human P-450 enzymes (CYP3A4, CYP1A2, CYP2A6, and CYP2D6) catalyzed the sulfoxidation of DDTC-Me, as determined with cDNA-expressed enzymes. Several lines of evidence suggest that the sulfoxidation of DDTC-Me by human liver microsomes is primarily catalyzed by CYP3A4/5, including (1) a high correlation between DDTC-Me sulfoxidation and testosterone 6beta-hydroxylation; (2) increased DDTC-Me sulfoxidation in the presence of alpha-naphthoflavone, an activator of CYP3A enzymes; (3) inhibition of this reaction by inhibitors of CYP3A4/5 enzymes, such as troleandomycin and ketoconazole; and (4) inhibition of DDTC-Me sulfoxidation by antibodies against CYP3A enzymes. On the other hand, several lines of evidence suggested that the thiono-oxidation of DDTC-Me by human liver microsomes is catalyzed in part by CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5, including (1) these human P450 enzymes among others have the capacity to catalyze this reaction, as determined with cDNA-expressed enzymes; (2) a high correlation between DDTC-Me thiono-oxidation and testosterone 6beta-hydroxylation, weak inhibition by ketoconazole, troleandomycin, and anti-CYP3A antibodies suggested a minor role for CYP3A4; (3) a high correlation with immunoreactive CYP2B6 suggested involvement of this enzyme; (4) weak inhibition of DDTC-Me thiono-oxidation by furafylline and anti-CYP1A antibody suggested involvement of CYP1A2; and (5) inhibition of DDTC-Me thiono-oxidation by DDTC and anti-CYP2E antibodies suggested a role for CYP2E1. Collectively, these data suggested CYP3A4/5 enzymes are the major contributors to the sulfoxidation of DDTC-Me by human liver microsomes, and CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5 contribute toward DDTC-Me thiono-oxidation by human liver microsomes. This study, in conjunction with others (Madan et al., Drug Metab. Dispos. 23:1153-1162, 1995), may help explain the variability in disulfiram's effectiveness as an alcohol deterrent.

Alcohol Deterrents↗
PubMed1999

Comparison of cytochrome P-450-dependent metabolism and drug interactions of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors lovastatin and pravastatin in the liver.

In an in vitro study, the cytochrome P-450 3A (CYP3A)-dependent metabolism and drug interactions of the 3-hydroxy-3-methylglutaryl-Co A reductase inhibitors lovastatin and pravastatin were compared. Lovastatin was metabolized by human liver microsomes to two major metabolites: 6'beta-hydroxy [Michaelis-Menten constant (Km): 7.8 +/- 2.7 microM] and 6'-exomethylene lovastatin (Km,10.3 +/- 2.6 microM). 6'beta-Hydroxylovastatin formation in the liver was inhibited by the specific CYP3A inhibitors cyclosporine (Ki, 7.6 +/- 2.3 microM), ketoconazole (Ki, 0.25 +/- 0.2 microM), and troleandomycin (Ki, 26.6 +/- 18.5 microM). Incubation of pravastatin with human liver microsomes resulted in the generation of 3'alpha,5'beta, 6'beta-trihydroxy pravastatin (Km, 4,887 +/- 2,185 microM) and hydroxy pravastatin (Km, 20,987 +/- 9,389 microM). The formation rates of 3'alpha,5'beta,6'beta-trihydroxy pravastatin by reconstituted CYP3A enzymes were (1,000 microM pravastatin) 1.9 +/- 0.6 pmol.min-1.pmol CYP3A4 and 0.06 +/- 0.04 pmol.min-1.pmol CYP3A5, and the formation rates of hydroxy pravastatin were 0.12 +/- 0.02 pmol.min-1.pmol CYP3A4 and 0.02 +/- 0.004 pmol.min-1.pmol CYP3A5. The specific CYP3A inhibitors cyclosporine, ketoconazole, and troleandomycin significantly inhibited hydroxy pravastatin formation by human liver microsomes, but only ketoconazole inhibited 3'alpha, 5'beta,6'beta-trihydroxy pravastatin formation, suggesting that other CYP enzymes are involved in its formation. It is concluded that, compared with lovastatin [CLint formation 6'beta-hydroxylovastatin (microl.min-1.mg-1): 199 +/- 248, 6'-exomethylene lovastatin: 138 +/- 104)], CYP3A-dependent metabolism of pravastatin [CLint formation 3'alpha,5'beta, 6'beta-trihydroxy pravastatin (microl.min-1.mg-1): 0.03 +/- 0.03 and hydroxy pravastatin: 0.02 +/- 0.02] is a minor elimination pathway. In contrast to lovastatin, drug interactions with pravastatin CYP3A-catalyzed metabolism cannot be expected to have a clinically significant effect on its pharmacokinetics.

Biotransformation↗
PubMed2004

Pharmacokinetics of clarithromycin in rats with acute renal failure induced by uranyl nitrate.

In rats pretreated with dexamethasone (an inducer of CYP3A1/2 in rats) and troleandomycin (an inhibitor of CYP3A1/2 in rats), the area under the plasma concentration-time curve from time zero to time infinity (AUC) values of clarithromycin were significantly smaller (365 compared with 600 micro g min/ml) and greater (1410 compared with 581 micro g min/ml), respectively, than those in control rats. This indicated that clarithromycin was metabolized via CYP3A1/2 in rats. The expression of CYP3A1(23) increased in rats with acute renal failure induced by uranyl nitrate (rats with U-ARF). Hence, it could be expected that AUC of clarithromycin could be smaller in rats with U-ARF. However, after intravenous administration of clarithromycin at a dose of 20mg/kg, the AUC and time-averaged total body (Cl) and nonrenal (Cl(nr)) clearance values were comparable between the two groups of rats. The 9000 x g supernatant fraction of liver homogenates in rats with U-ARF had comparable metabolic activities for clarithromycin compared with those in control rats, suggesting that the CYP3A isozyme responsible for metabolism of clarithromycin seemed not to be expressed considerably in the rats. This could explain the comparable AUC, Cl and Cl(nr) values of clarithromycin between the two groups of rats.

Acute Kidney Injury↗
PubMed2006

Negligible pharmacokinetic interaction between oral DA-8159, a new erectogenic, and amlodipine in rats.

A pharmacokinetic interaction between oral DA-8159 and amlodipine was evaluated in male Sprague-Dawley rats. In rats pretreated with troleandomycin (a main inhibitor of CYP3A1/2 in rats), the AUC(0-6 h) of amlodipine was significantly greater than the controls (34.5+/-6.01 compared with 28.0+/-4.70 microg min/ml), indicating that amlodipine is metabolized via CYP3A1/2 in rats. It was reported that the metabolism of DA-8159 and the formation of DA-8164 (a metabolite of DA-8159) were mainly mediated via CYP3A1/2 in rats, and amlodipine significantly inhibited the CYP3A2 in rats. Therefore, a pharmacokinetic interaction between the two drugs could be expected. However, after oral administration of DA-8159 at a dose of 30 mg/kg with or without oral amlodipine at a dose of 5 mg/kg to rats, the pharmacokinetic parameters of DA-8159 and DA-8164 were not significantly different between the two groups of rats. Similar results were also obtained from amlodipine between with and without DA-8159. The above data indicated that the pharmacokinetic interaction between oral DA-8159 and amlodipine was almost negligible in rats.

Administration, Oral↗
PubMed1996

Analysis of macrolide antibiotics by capillary electrophoresis.

Capillary electrophoresis was utilized in the study of the macrolide antibiotics (i.e. pharmaceutical glycoconjugates) clarithromycin, erythromycin, oleandomycin, troleandomycin, and spiramycin. In order to assist in analyte solubilization, two buffer systems using acetonitrile were developed. The first system involved 30 mM sodium cholate and 20% acetonitrile in 80 mM sodium phosphate, pH 6. This buffer permitted the baseline resolution of all five glycoconjugated antibiotics. In addition, erythromycin was separated from its derivatives estolate and ethylsuccinate. In the absence of surfactants, a higher acetonitrile quantity, 65%, was used in the second buffer system, with 35 mM sodium phosphate, pH 6. Selectivity between oleandomycin and clarithromycin was reversed in this system compared to the cholate buffer, indicating solute interaction with the cholate micelles in the previous system. Calibration linearity and detection sensitivity were improved in the high acetonitrile buffer, due to decreased background absorbance. It was demonstrated that both buffer systems can be utilized for the visualization of minor components that may be present in bulk pharmaceuticals.

Acetonitriles↗
PubMed1996

Hepatotoxicity of the herbal medicine germander: metabolic activation of its furano diterpenoids by cytochrome P450 3A Depletes cytoskeleton-associated protein thiols and forms plasma membrane blebs in rat hepatocytes.

Several herbal remedies have produced hepatitis in humans. The medicinal plant, germander, was recalled after its use as an adjuvant to slimming diets resulted in an epidemic of hepatitis in France. We studied the hepatotoxicity of germander in isolated rat hepatocytes. A crude fraction containing the diverse furano diterpenoids of germander, or the purified main constituents of this fraction, teucrin A and teuchamaedryn A, were hepatotoxic (correction for hepatototoxic), but not fractions containing more polar or lipophilic constituents. [3H]Teucrin A covalently bound to hepatocyte proteins. The furano diterpenoid fraction decreased cell glutathione and cytoskeleton-associated protein thiols, and led to formation of plasma membrane blebs and cell demise. Pretreatment of male rats with troleandomycin, an inhibitor of cytochrome P450 3A (CYP3A), slowed the depletion of glutathione and decreased toxicity, whereas dexamethasone, an inducer of CYP3A, had opposite effects. Female rat hepatocytes, which poorly express CYP3A, exhibited little toxicity, unless the animals were treated with dexamethasone. Feeding male rats with a sulfur amino acid-deficient diet decreased cell glutathione and enhanced toxicity, whereas supplementation of the standard diet with cystine had opposite effects. We conclude that the furano diterpenoids of germander are activated by CYP3A into electrophilic metabolites that deplete glutathione and cytoskeleton-associated protein thiols and form plasma membrane blebs. We suggest that studies in isolated hepatocytes be included in the preclinical assessment of herbal remedies.

Acetylcysteine↗