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PubMed1998

Biosynthesis of an aminopiperidino metabolite of irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine] by human hepatic microsomes.

Irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine++ +] is a water-soluble analogue of camptothecine used in the second-line treatment of advanced colon cancer. Recently, we identified, in the plasma of patients and in human liver microsomal incubations, the presence of a new metabolite of irinotecan, 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine (NPC), which is produced by cleavage of the distal piperidine ring of irinotecan. The kinetics of biotransformation of the lactone and carboxylate forms of irinotecan into NPC were studied using human liver microsomes. The formation of NPC was characterized by the following parameters: KM = 48.2 +/- 6.8 and 273 +/- 122 microM and Vmax = 74.1 +/- 4.9 and 78.6 +/- 27.7 pmol/min/mg of protein for the lactone and carboxylate forms of irinotecan, respectively. Interestingly, there was no formation of NPC from 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecine, a major metabolite of irinotecan that has an open distal piperidine ring and could be considered a possible metabolic precursor of NPC. The transformation of irinotecan into NPC was found to be catalyzed principally by cytochrome P450 (CYP) 3A, based on three key results, as follows: 1) the CYP3A-selective inhibitors ketoconazole (1 microM) and troleandomycin (100 microM) inhibited NPC formation by 99 and 100%, respectively; 2) of a series of microsomal preparations from transfected lymphoblastoid cells expressing specific CYPs, only those from CYP3A4 cDNA-transfected cells transformed irinotecan into NPC; and 3) incubations with 15 individual preparations of human liver microsomes yielded highly significant correlations between the formation of NPC and both immunoreactivity with anti-CYP3A antibodies and testosterone 6beta-hydroxylation (an activity specifically mediated by CYP3A). The effects of 11 drugs (used at 100 microM) on this metabolism were studied with irinotecan lactone (25 microM). Although ondansetron, loperamide, and racecadotril inhibited this pathway by 75, 95, and 95%, respectively, the concentrations used may not be clinically achievable. However, significant inhibition by ketoconazole and troleandomycin indicates that NPC formation in patients may be influenced by coadministration of drugs with known anti-CYP3A activities.

Anti-Bacterial Agents↗

PubMed1998

Involvement of cytochrome P450 3A in the metabolism and covalent binding of 14C-monocrotaline in rat liver microsomes.

The metabolism and covalent binding of 14C-monocrotaline in Sprague-Dawley (SD) rat liver microsomes was investigated using the inducers dexamethasone, clotrimazole, pregnenolone-16 alpha-carbonitrile, and phenobarbital. Monocrotaline is a pyrrolizidine alkaloid (PA) that causes a syndrome in rats that is a model for human primary pulmonary hypertension. It has been documented that bioactivation of PAs (dehydrogenation to reactive pyrroles) in the liver by cytochromes P450 is required for their toxicity. Covalent binding of these reactive pyrroles to tissue macromolecules has been hypothesized to correspond to PA toxicosis. We correlated metabolism and total microsomal covalent binding of 14C-monocrotaline with cytochrome P450 3A using the aforementioned inducers, troleandomycin (a cytochrome P450 3A inhibitor), erythromycin N-demethylase assay of cytochrome P450 3A activity, and Western blots employing anti-rat cytochrome P450 3A antibodies. In addition, autoradiography of membranes electroblotted from SDS-PAGE demonstrated the formation of radiolabeled adducts with specific protein(s). The most intensely radiolabeled protein bands have an apparent molecular weight of approximately 52 kDa, which was similar to the molecular weight detected by anti-rat cytochrome P450 3A antibodies in the Western blots. No radiolabeled proteins were detected in microsomes pretreated with troleandomycin.

Animals↗

PubMed2005

Effects of enzyme inducers and inhibitors on the pharmacokinetics of intravenous DA-8159, a new erectogenic, in rats.

In order to find what types of hepatic microsomal cytochrome P450 (CYP) isozymes are involved in the metabolism of DA-8159 and in the formation of DA-8164 in rats, enzyme inducers, such as dexamethasone, phenobarbital, 3-methylcholanthrene and isoniazid, and enzyme inhibitors, such as troleandomycin and quinine, were pretreated in rats. After a 1 min intravenous administration of DA-8159 at a dose of 30 mg/kg to rats pretreated with dexamethasone (a main inducer of CYP3A1/2 in rats), the total areas under the plasma concentration-time curve from time zero to time infinity (AUC) values of DA-8159 (283 versus 349 microg min/ml) and DA-8164 (98.0 versus 79.8 microg min/ml) were significantly smaller and greater, respectively, than those in control rats. However, the AUC values of DA-8159 were not significantly different after pretreatment with phenobarbital, isoniazid and 3-methylcholanthrene (main inducers of CYP2B1/2, 2E1 and 1A1/2, respectively, in rats). In rats pretreated with troleandomycin (a main inhibitor of CYP3A1/2 in rats), the AUC values of DA-8159 (435 versus 370 microg min/ml) and DA-8164 (34.8 versus 76.5 microg min/ml) were significantly greater and smaller, respectively. However, in rats pretreated with quinine (a main inhibitor of CYP2D1 in rats), the AUC of DA-8159 was comparable to that in control rats. The above data indicate that DA-8159 was metabolized and DA-8164 was formed mainly via CYP3A1/2 in rats.

Alcohol Oxidoreductases↗

PubMed2006

Quantitative prediction of macrolide drug-drug interaction potential from in vitro studies using testosterone as the human cytochrome P4503A substrate.

OBJECTIVE: Macrolide antibiotics are mechanism-based inactivators of CYP3A enzymes that exhibit varying degrees of inhibitory potency. Our aim was to predict quantitatively the drug-drug interaction (DDI) potential of five macrolides from in vitro studies using testosterone as the CYP3A substrate, and to compare the predictions generated from human liver microsomal and recombinant CYP3A4 data. METHODS: The in vitro kinetic constants of CYP3A inactivation (K (I) and k (inact)) were estimated by varying the time of pre-incubation and the concentration of troleandomycin, erythromycin, clarithromycin, roxithromycin or azithromycin. CYP3A activity was determined from the measurement of testosterone 6beta-hydroxylation with human liver microsomes (HLM) and recombinant CYP3A4 as the enzyme sources. The mechanism-based pharmacokinetic model was fitted with inactivation data to predict the increase in oral area under the plasma concentration-time curve (AUC) for midazolam. RESULTS: All five macrolides inactivated testosterone 6beta-hydroxylation by HLM and recombinant CYP3A4 with k (inact) values in the range of 0.023 to 0.058 min(-1). The potency of inactivation (K (I)) was higher using recombinant CYP3A4 as the enzyme source. The oral AUCs for midazolam were predicted from HLM data to increase 16.6, 5.3, 4.6, 1.6 and 1.2-fold due to the inhibition of metabolic clearance by troleandomycin, erythromycin, clarithromycin, roxithromycin and azithromycin, respectively. These results are within the range of the AUC ratios reported for clinical DDI studies. The predicted AUC increases generated using recombinant CYP3A4 overestimated the magnitude of the DDIs. CONCLUSIONS: The DDI potential of five macrolide antibiotics was quantitatively predicted from in vitro studies using testosterone as the CYP3A substrate with HLM as the enzyme source.

Algorithms↗

PubMed2005

Single-point sampling for assessment of constitutive, induced, and inhibited cytochrome P450 3A activity with alfentanil or midazolam.

INTRODUCTION: The clearances of oral and intravenous alfentanil are in vivo probes for assessing first-pass and hepatic cytochrome P450 (CYP) 3A activity. Clearance determinations require time-consuming and expensive serial blood samples and quantitative analysis. This investigation tested the hypothesis that a single concentration measurement can accurately predict alfentanil clearance and compared limited sampling results for alfentanil and midazolam. METHODS: Data were analyzed from 2 previous studies in healthy volunteers (n = 10 in each). By use of a dose-finding protocol, subjects received oral alfentanil (23, 30, 43, or 75 microg/kg). By use of a CYP3A modulation protocol, subjects received oral or intravenous alfentanil and midazolam after pretreatment with rifampin (INN, rifampicin) (CYP3A induction), troleandomycin (CYP3A inhibition), grapefruit juice (intestinal CYP3A inhibition), or nothing (control). Alfentanil and midazolam area under the concentration-time curve (AUC) was calculated by noncompartmental analysis. Correlations between AUC and plasma concentration at each time point were evaluated by unweighted and weighted (1/y, 1/y(2)) linear regression, and the optimal weight and sampling time were determined. RESULTS: Correlations between AUC and plasma concentration were best with 1/y(2) weights. Optimal correlations were at 1.5 to 2 hours for each alfentanil dose and also in an all-dose composite analysis. With the CYP3A modulation protocol, correlations for the control, grapefruit juice, rifampin, and troleandomycin sessions were optimal at 2, 3, 1.5, and 12 hours, respectively, for oral alfentanil, at 2, 3, 0.5, and 9 hours, respectively, for intravenous alfentanil, at 5, 5, 1.5, and 13 hours, respectively, for oral midazolam, and at 3, 1.75, 1, and 13 hours, respectively, for intravenous midazolam. In a composite analysis of all treatments, correlations (oral and intravenous) were optimal at 4 to 5 hours for alfentanil and 5 to 6 hours for midazolam. AUC estimated by a single-point concentration was highly predictive of measured AUC (r(2) = 0.91 and 0.93 for oral and intravenous alfentanil, respectively, and r(2) = 0.90 and 0.87 for oral and intravenous midazolam, respectively). CYP3A induction or inhibition was detected equally well by single-point alfentanil concentrations and by formal AUC determinations. CONCLUSION: A single plasma concentration can predict alfentanil AUC and hence clearance, independent of dose, and thus appears to be an effective and efficient strategy for assessing CYP3A modulation over a broad spectrum of enzyme activity.

Administration, Oral↗

PubMed2002

Human cytochrome P450s involved in the metabolism of 9-cis- and 13-cis-retinoic acids.

The purpose of this work was to identify the principal human cytochrome P450s (CYPs) involved in the metabolism of the retinoic acid (RA) isomers, 9-cis- and 13-cis-RA, by using a combination of techniques including human liver microsomes (correlation of activity and inhibition), and lymphoblast microsomes expressing a single CYP. Concerning the 9-cis-RA, 4-OH- and 4-oxo-9-cis-RA were formed with human liver microsomes, and their formation correlated with activities linked to CYPs 3A4/5, 2B6, 2C8, 2A6, and 2C9. The use of lymphoblast microsomes expressing a single human CYP identified CYPs 2C9>2C8>3A7 as the most active in the formation of 4-OH-9-cis-RA. With regard to 13-cis-RA, specific P450 activities linked to CYPs 2B6, 2C8, 3A4/5, and 2A6 were correlated with the formation of 4-OH- and 4-oxo-13-cis-RA. Microsomes expressing a single CYP identified CYPs 3A7, 2C8, 4A11, 1B1, 2B6, 2C9, 2C19, 3A4 (decreasing activity) in the formation of 4-OH-13-cis-RA. The use of CYP-specific inhibitors in human liver microsomes disclosed that the formation of the 4-OH-9-cis-RA was best inhibited by sulfaphenazole (72%) and quercetin (66%), whereas ketoconazole and troleandomycin inhibited its formation by 55 and 38%, respectively; the formation of 4-OH-13-cis-RA was best inhibited by troleandomycin (54%) and ketoconazole (46%), whereas quercetin was a weak inhibitor (14%). In conclusion, adult human CYPs 2C9, 2C8, 3A4 have been identified as active in the 9-cis-RA metabolism, whereas CYPs 3A4 and 2C8 were active in 13-cis-RA metabolism. The fetal form CYP3A7 was also identified as very active in either 9-cis- or 13-cis-RA metabolism. The role of these human CYPs in the biological response or resistance to RA isomers remains to be determined.

Alitretinoin↗

PubMed2003

High-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry.

A method has been developed for the high-throughput inhibition screening of the major human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) using an in vitro substrate cocktail and liquid chromatography-tandem mass spectrometry (LC-MS-MS). A cocktail consisting of the selective substrates phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), bufuralol (CYP2D6), and midazolam (CYP3A4) was incubated with human liver microsomes. The metabolic reactions were terminated with methanol containing dextrorphan as an internal standard. Following centrifugation, the supernatant was analyzed by LC-MS-MS employing a fast gradient. The concentrations of the substrate metabolites-paracetamol, 4-hydroxytolbutamide, 5-hydroxyomeprazole, 1'-hydroxybufuralol, and 1'-hydroxymidazolam-in each sample were determined by LC-MS-MS in a single assay. The method was validated by incubating known CYP inhibitors (furafylline, CYP1A2; sulfaphenazole, CYP2C9; s-mephenytoin, CYP2C19; quinidine, CYP2D6; and troleandomycin, CYP3A4) with the individual substrates they were known to inhibit and with the substrate cocktail. IC50s (microM) determined using the substrate cocktail were in good agreement with those obtained with individual substrates (furafylline, 2.9 vs. 2.0; sulfaphenazole, 0.75 vs. 0.72; s-mephenytoin, 170 vs. 180; quinidine, 0.17 vs. 0.24; troleandomycin, 2.6 vs. 3.2) and with previously reported values in the literature.

Cytochrome P-450 Enzyme System↗

PubMed1999

Metabolism-dependent stimulation of reactive oxygen species and DNA synthesis by cyclosporin A in rat smooth muscle cells.

The clinical use of the immunosuppressive drug cyclosporin A (CsA) is limited by its side effects, namely hypertension and nephrotoxicity. It has been proposed that reactive oxygen species (ROS) could be involved as mediators of the toxic effects of CsA. Here, we have studied the possible interrelationship between CsA metabolism and production of ROS. Using cultures of rat aortic smooth muscle cells (RASMC), CsA (1 microM) produced a rapid (within 10 min) increase in reactive oxygen species, detected by oxidation of the fluorescent probes 2,7-dichlorofluorescin and dihydrorhodamine-123. DNA synthesis was increased in the presence of CsA as assessed by [3H]thymidine incorporation. The superoxide dismutase inhibitor diethyldithiocarbamate (1 mM) and the iron chelator desferal (5 microM), as well as ketoconazole (1 microM) and troleandomycin (10 microM), inhibitors of the cytochrome P-450 3A, were able to block both effects. High-performance liquid chromatography analysis revealed that RASMC were capable to metabolize CsA to its primary metabolites (AM1, AM9 and AM4N), and that their formation was inhibited by ketoconazole and troleandomycin. Furthermore, mRNAs encoding cytochrome P-450 3A1 and 3A2 were detected in RASMC by reverse transcriptase-polymerase chain reaction. Our data suggest that CsA is metabolized by cytochrome P-450 3A in RASMC producing reactive oxygen species, most likely superoxide and the hydroxyl radical, known to damage lipids and DNA.

Animals↗

PubMed1997

Applications of primary human hepatocytes in the evaluation of pharmacokinetic drug-drug interactions: evaluation of model drugs terfenadine and rifampin.

The utility of primary human hepatocytes in the evaluation of drug-drug interactions is being investigated in our laboratories. Our initial approach was to investigate whether drug-drug interactions observed in humans in vivo could be reproduced in vitro using human hepatocytes. Two model drugs were studied: terfenadine and rifampin, representing compounds subjected to drug-drug interactions via inhibitory and induction mechanisms, respectively. Terfenadine was found to be metabolized by human hepatocytes to C-oxidation and N-dealkylation products as observed in humans in vivo. Metabolism by human hepatocytes was found to be inhibited by drugs which are known to be inhibitory in vivo. Ki values for the various inhibitors were derived from the in vitro metabolism data, resulting in the following ranking of inhibitory potency: For the inhibition of C-oxidation, ketoconazole > itraconazole > cyclosporin approximately troleandomycin > erythromycin > naringenin. For the inhibition of N-dealkylation, itraconazole > or = ketoconazole > cyclosporin > or = naringenin > or = erythromycin > or = troleandomycin. Rifampin induction of CYP3A, a known effect of rifampin in vivo, was also reproduced in primary human hepatocytes. Induction of CYP3A4, measured as testosterone 6 beta-hydroxylation, was found to be dose-dependent, treatment duration-dependent, and reversible. The induction effect of rifampin was observed in hepatocytes isolated from all 7 human donors studied, with ages ranging from 1.7 to 78 years. To demonstrate that the rifampin-induction of testosterone 6 beta-hydroxylation could be generalized to other CYP3A4 substrates, we evaluated the metabolism of another known substrate of CYP3A4, lidocaine. Dose-dependent induction of lidocaine metabolism by rifampin is observed. Our results suggest that primary human hepatocytes may be a useful experimental system for preclinical evaluation of drug-drug interaction potential during drug development, and as a tool to evaluate the mechanism of clinically observed drug-drug interactions.

Cells, Cultured↗

PubMed2003

Structural insight into the role of the ribosomal tunnel in cellular regulation.

Nascent proteins emerge out of ribosomes through an exit tunnel, which was assumed to be a firmly built passive path. Recent biochemical results, however, indicate that the tunnel plays an active role in sequence-specific gating of nascent chains and in responding to cellular signals. Consistently, modulation of the tunnel shape, caused by the binding of the semi-synthetic macrolide troleandomycin to the large ribosomal subunit from Deinococcus radiodurans, was revealed crystallographically. The results provide insights into the tunnel dynamics at high resolution. Here we show that, in addition to the typical steric blockage of the ribosomal tunnel by macrolides, troleandomycin induces a conformational rearrangement in a wall constituent, protein L22, flipping the tip of its highly conserved beta-hairpin across the tunnel. On the basis of mutations that alleviate elongation arrest, the tunnel motion could be correlated with sequence discrimination and gating, suggesting that specific arrest motifs within nascent chain sequences may induce a similar gating mechanism.

Amino Acid Sequence↗

PubMed1999

Comparison of (S)-mephenytoin and proguanil oxidation in vitro: contribution of several CYP isoforms.

AIMS: To compare the oxidative metabolism of (S)-mephenytoin and proguanil in vitro and to determine the involvement of various cytochrome P450 isoforms. METHODS: The kinetics of the formation of 4'-hydroxymephenytoin and cycloguanil in human liver microsomes from 10 liver samples were determined, and inhibition of formation was studied using specific chemical inhibitors and monoclonal antibodies directed towards specific CYP450 isoforms. Expressed CYP450 enzymes were used to characterize further CYP isoform contribution in vitro. Livers were genotyped for CYP2C19 using PCR amplification of genomic DNA followed by restriction endonuclease digestion. RESULTS: All livers were wildtype with respect to CYP2C19, except HLS#5 whose genotype was CYP2C19*1/CYP2C19*2. The Km, Vmax and CLint values for the formation of 4'-hydroxymephenytoin from (S)-mephenytoin and the formation of cycloguanil from proguanil ranged from 50.8 to 51.6 and 43-380 microm, 1.0-13.9 and 0.5-2.5 nmol mg-1 h-1, and 20.2-273.8 and 2.7-38.9 microl h-1 mg-1, respectively. There was a significant association between the Vmax values of cycloguanil and 4'-hydroxymephenytoin formation (rs=0.95, P=0.0004). Cycloguanil formation was inhibited significantly by omeprazole (CYP2C19/3A), troleandomycin (CYP3A), diethyldithiocarbamate (CYP2E1/3A), furafylline (CYP1A2), and (S)-mephenytoin. 4'-Hydroxymephenytoin formation was inhibited significantly by omeprazole, diethyldithiocarbamate, proguanil, furafylline, diazepam, troleandomycin, and sulphaphenazole (CYP2C9). Human CYP2E1 and CYP3A4 monoclonal antibodies did not inhibit the formation of cycloguanil or 4'-hydroxymephenytoin, and cycloguanil was formed by expressed CYP3A4 and CYP2C19 supersomes. However, only expressed CYP2C19 and CYP2C19 supersomes formed 4'-hydroxymephenytoin. CONCLUSIONS: The oxidative metabolism of (S)-mephenytoin and proguanil in vitro is catalysed by CYPs 2C19 and 1A2, with the significant association between Vmax values suggesting that the predominant enzymes involved in both reactions are similar. However the degree of selectively of both drugs for CYP isoforms needs further investigation, particularly the involvement of CYP3A4 in the metabolism of proguanil. We assert that proguanil may not be a suitable alternative to (S)-mephenytoin as a probe drug for the CYP2C19 genetic polymorphism.

Adult↗

PubMed2005

Metabolism of territrem B and C in liver microsomes from 14-wk-old Wistar rats is catalyzed by cytochrome P-450 3A.

The metabolism of territrem B (TRB) and territrem C (TRC) in liver microsomes of 14-wk-old male and female Wistar rats was investigated. Metabolism of TRB to 4beta-hydroxylmethyl-4beta-demethylterritrem B (MB2), O-demethylation of the methoxy group of the aromatic moiety of TRB to form MB4 (same structure as TRC), and metabolism of TRC to 4beta-hydroxylmethyl-4beta-demethylterritrem C (MC) were observed in both genders. However, the amounts of MB2, MB4, and MC formed in females were much lower than in males. To investigate which cytochrome P-450 (CYP450) isoforms were involved in each step, four CYP450 isotype-specific inhibitors (furafylline, orphenadrine, cimetidine, and troleandomycin) and antibodies against CYP1A1, CYP2B1, CYP2C11, or CYP3A2 were used. Formation of MB2, MB4, and MC was markedly inhibited by cimetidine and troleandomycin, but less by furafylline and orphenadrine. Anti-CYP3A2 antibody completely inhibited MB, MB, and MC formation, while antibodies against CYP1A1, CYP2B1, or CYP2C11 produced no marked effect. Of the seven tested supersomes from baculovirus-transformed insect cells expressing rat CYP450 isoforms (1Al, 1A2, 2B1, 2C11, 2C12, 3A1, and 3A2), only those expressing CYP3A1 and CYP3A2 metabolized TRB and TRC. The amounts of MB2, MB4, and MC formed by male and female rat liver microsome preparations were related to the testosterone 6beta-hydroxylase activity and CYP3A1/2 protein content of the preparation. Immunoblotting showed that CYP3A1 was expressed in both genders, but at different levels, while CYP3A2 was only expressed in males. These results suggest that the formation of MB2, MB4, and MC in liver microsomes from 14-wk-old rats of either gender is mediated by CYP3A1 and CYP3A2.

Animals↗

PubMed2005

Pharmacogenetic determinants of human liver microsomal alfentanil metabolism and the role of cytochrome P450 3A5.

BACKGROUND: There is considerable unexplained interindividual variability in the clearance of alfentanil. Alfentanil undergoes extensive metabolism by cytochrome P4503A4 (CYP3A4). CYP3A5 is structurally similar to CYP3A4 and metabolizes most CYP3A4 substrates but is polymorphically expressed. Livers with the CYP3A5*1 allele contain higher amounts of the native CYP3A5 protein than livers homozygous for the mutant CYP3A5*3 allele. This investigation tested the hypothesis that alfentanil is a substrate for CYP3A5 and that CYP3A5 pharmacogenetic variability influences human liver alfentanil metabolism. METHODS: Alfentanil metabolism to noralfentanil and N-phenylpropionamide was determined in microsomes from two groups of human livers, characterized for CYP3A4 and CYP3A5 protein content: low CYP3A5 (2.0-5.2% of total CYP3A, n = 10) and high CYP3A5 (46-76% of total CYP3A, n = 10). Mean CYP3A4 content was the same in both groups. The effects of the CYP3A inhibitors troleandomycin and ketoconazole, the latter being more potent toward CYP3A4, on alfentanil metabolism were also determined. RESULTS: In the low versus high CYP3A5 livers, respectively, noralfentanil formation was 77 +/- 31 versus 255 +/- 170 pmol . min . mg, N-phenylpropionamide formation was 8.0 +/- 3.1 versus 20.5 +/- 14.0 pmol . min . mg, and the metabolite ratio was 9.5 +/- 0.4 versus 12.7 +/- 1.4 (P < 0.05 for all). There was a poor correlation between alfentanil metabolism and CYP3A4 content but an excellent correlation when CYP3A5 (i.e., total CYP3A content) was considered (r = 0.81, P < 0.0001). Troleandomycin inhibited alfentanil metabolism similarly in the low and high CYP3A5 livers; ketoconazole inhibition was less in the high CYP3A5 livers. CONCLUSION: In microsomes from human livers expressing the CYP3A5*1 allele and containing higher amounts of CYP3A5 protein, compared with those with the CYP3A5*3 allele and little CYP3A5, there was greater alfentanil metabolism, metabolite ratios more closely resembled those for expressed CYP3A5, and inhibitors with differing CYP3A4 and CYP3A5 selectivities had effects resembling those for expressed CYP3A5. Therefore, alfentanil is metabolized by human liver microsomal CYP3A5 in addition to CYP3A4, and pharmacogenetic variability in CYP3A5 expression significantly influences human liver alfentanil metabolism in vitro. Further investigation is warranted to assess whether the CYP3A5 polymorphism is a factor in the interindividual variability of alfentanil metabolism and clearance in vivo.

Alfentanil↗

PubMed1998

In-vitro characterization of the cytochrome P450 isoenzymes involved in the back oxidation and N-dealkylation of reduced haloperidol.

In-vitro studies were performed using human liver microsomes and c-DNA-expressed human P450 isoforms to identify the cytochrome P450 isoenzyme(s) involved in the back oxidation and N-dealkylation of reduced haloperidol. Back oxidation and N-dealkylation of reduced haloperidol were assessed by measuring the formation of haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), respectively. The haloperidol and CPHP formation rates as a function of substrate concentration, measured in three livers, followed monophasic enzyme kinetics. For haloperidol formation Km values ranged from 51-59 microM, and Vmax values from 190-334 pmol mg(-1) min(-1); for CPHP formation Km values were 44-49 microM, and Vmax values 74-110 pmol mg(-1) min(-1). Haloperidol and CPHP formation rates in the nine liver preparations were significantly correlated with dextromethorphan N-demethylase activity (a marker of CYP3A4 activity), but not with the CYP2D6, CYP1A2 and CYP2C9 activity. Ketoconazole and troleandomycin, inhibitors of CYP3A4, inhibited competitively both haloperidol and CPHP formation, with a Ki value lower than 0.2 microM for ketoconazole and lower than 0.3 microM for troleandomycin. Sulphaphenazole (CYP2C9), furafylline (CYP1A2) and quinidine and paroxetine (CYP2D6) gave only little inhibition (IC50 > 60 microM). CPHP and haloperidol formation were, moreover, enhanced by alpha-naphthoflavone, an effect known for CYP3A4 mediated reactions. Anti-CYP3A4 antibodies strongly inhibited haloperidol and CPHP formation, whereas CYP2D6 antibodies did not. Among the recombinant human CYP isoforms tested, CYP3A4 exhibited the highest activity with respect to haloperidol and CPHP formation rates, with no detectable effect of CYP1A2, CYP2D6 and CYP2C9. These results strongly suggest that back oxidation and N-dealkylation of reduced haloperidol in human liver microsomal preparations are mediated by CYP3A4.

Adolescent↗

PubMed1994

In vitro proguanil activation to cycloguanil by human liver microsomes is mediated by CYP3A isoforms as well as by S-mephenytoin hydroxylase.

1. The activation of proguanil to cycloguanil by human liver microsomes was studied to define the cytochrome P450 (CYP) isoforms involved in this reaction. 2. Apparent Km values for proguanil ranged from 35 microM to 183 microM with microsomes from four human livers. 3. There was a 6.3-fold range of activity with microsomes from seventeen human livers. Rates of proguanil activation correlated significantly with CYP3A activities (benzo[a]pyrene metabolism, caffeine 8-oxidation and omeprazole sulphone formation) and CYP3A immunoreactive content. There was also a highly significant correlation with rates of hydroxyomeprazole formation. Correlations with activities selective for CYP1A2, CYP2C9/10 and CYP2E1, and with immunoreactive CYP1A2 content were not significant. 4. Proguanil activation was inhibited by R,S-mephenytoin, troleandomycin and by inhibitory anti-CYP3A antiserum and anti-CYP2C IgG and was activated by alpha-naphthoflavone. Inhibitors selective for CYP1A2, CYP2E1, CYP2A6 or CYP2C9/10 had little or no effect on proguanil activation. The extents of inhibition by R,S-mephenytoin, troleandomycin and the two antibodies varied with the immunoreactive CYP3A content of the microsomes used. 5. It is concluded that proguanil activation to cycloguanil by human liver microsomes is mediated both by S-mephenytoin hydroxylase and isoforms of the CYP3A subfamily. This has implications for the use of proguanil as an in vivo probe for the S-mephenytoin poor metaboliser phenotype.

Aryl Hydrocarbon Hydroxylases↗

PubMed2005

Cytochrome P450 3A4 is the major enzyme responsible for the metabolism of laquinimod, a novel immunomodulator.

In the present study, the involvement of cytochrome P450 enzyme(s) in the primary metabolism of laquinimod, a new orally active immunomodulator, has been investigated in human liver microsomes. Hydroxylated and dealkylated metabolites were formed. The metabolite formation exhibited single enzyme Michaelis-Menten kinetics with apparent KM in the range of 0.09 to 1.9 mM and Vmax from 22 to 120 pmol/mg/min. A strong correlation between the formation rate of metabolites and 6beta-hydroxylation of testosterone was obtained within a panel of liver microsomes from 15 individuals (r2 = 0.6 to 0.94). Moreover, ketoconazole and troleandomycin, specific inhibitors of CYP3A4 metabolism, demonstrated a significant inhibition of laquinimod metabolism. Furthermore, in incubations with recombinant CYP3A4, all the primary metabolites were formed. In vitro interaction studies with CYP3A4 substrates and possible concomitant medication demonstrated that laquinimod inhibits the metabolism of ethinyl estradiol with an IC50 value of about 150 microM, which is high above the plasma level of laquinimod after clinically relevant doses. Ketoconazole, troleandomycin, erythromycin, prednisolone, and ethinyl estradiol inhibited the metabolism of laquinimod, and IC50 values of 0.2, 11, 24, 87, and 235 microM, respectively, were calculated. In conclusion, the present study demonstrates that laquinimod is a low affinity substrate for CYP3A4 in human liver microsomes. The likelihood for in vivo effects of laquinimod on the metabolism of other CYP3A4 substrates is minor. However, inhibitory effects on the metabolism of laquinimod by potent and specific inhibitors of CYP3A4, such as ketoconazole, are anticipated and should be considered in the continued clinical program for laquinimod.

Cytochrome P-450 CYP3A↗

PubMed2002

Enzyme kinetics for the formation of 3-hydroxyquinine and three new metabolites of quinine in vitro; 3-hydroxylation by CYP3A4 is indeed the major metabolic pathway.

The formation kinetics of 3-hydroxyquinine, 2'-quininone, (10S)-11-dihydroxydihydroquinine, and (10R)-11-dihydroxydihydroquinine were investigated in human liver microsomes and in human recombinant-expressed CYP3A4. The inhibition profile was studied by the use of different concentrations of ketoconazole, troleandomycin, and fluvoxamine. In addition, formation rates of the metabolites were correlated to different enzyme probe activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in microsomes from 20 human livers. Formation of 3-hydroxyquinine had the highest intrinsic clearance in human liver microsomes (mean +/- S.D.) of 11.0 +/- 4.6 micro l/min/mg. A markedly lower intrinsic clearance, 1.4 +/- 0.7, 0.5 +/- 0.1, and 1.1 +/- 0.2 micro l/min/mg was measured for 2'-quininone, (10R)-11-dihydroxydihydroquinine and (10S)-11-dihydroxydihydroquinine, respectively. Incubation with human recombinant CYP3A4 resulted in a 20-fold higher intrinsic clearance for 3-hydroxyquinine compared with 2'-quininone formation whereas no other metabolites were detected. The formation rate of 3-hydroxyquinine was completely inhibited by ketoconazole (1 micro M) and troleandomycin (80 micro M). Strong inhibition was observed on the formation of 2'-quininone whereas the formation of (10S)-11-dihydroxydihydroquinine was partly inhibited by these two inhibitors. No inhibition on the formation of (10R)-11-dihydroxydihydroquinine was observed. There was a significant correlation between the formation rates of quinine metabolites and activities of the CYP3A4 selected marker probes. This in vitro study demonstrates that 3-hydroxyquinine is the principal metabolite of quinine and CYP3A4 is the major enzyme involved in this metabolic pathway.

Cytochrome P-450 CYP3A↗

PubMed2002

CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes.

Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6beta-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.

Adult↗