Identification of human p450 isoforms involved in the metabolism of the antiallergic drug, oxatomide, and its inhibitory effect on enzyme activity

Oxatomide is an antiallergic drug used for the treatment of diseases mediated by type I allergy. Recently, it has been reported that terfenadine and astemizole, which have antiallergic actions similar to those of oxatomide, show side effects on the cardiovascular system, such as QT prolongation, ventricular arrhythmia and cardiac arrest. This might be because concomitant drugs such as itraconazole inhibit cytochrome P450 3A4 (CYP3A4), the enzyme responsible for degradation of terfenadine and astemizole, and thus the blood concentrations of the drugs are abnormally increased. On the other hand, isoforms of P450 involved in the metabolism of oxatomide have not been clarified. Therefore, we attempted to identify these isoforms using microsome preparations of in vitro expression systems derived from a human lymphoblastoid cell line. Oxatomide was metabolized by CYP2D6-Val and CYP3A4, but not by CYP1A2, CYP2C9-Arg, CYP2C9-Cys or CYP2C19. We also examined whether oxatomide showed inhibitory effects on metabolic activity of individual P450 isozymes using model substrates for each isozyme. Oxatomide did not inhibit the metabolism of the model substrates for CYP1A2, CYP2C9-Arg, CYP2C9-Cys and CYP2C19, but inhibited the degradation of those for CYP2D6-Val and CYP3A4. It was found that oxatomide is metabolized by CYP2D6 and CYP3A4 in human liver microsomes, and simultaneously acts as an inhibitor for these isoforms, responsible for the metabolism of the drug itself.     

Identifying a selective substrate and inhibitor pair for the evaluation of CYP2J2 activity

CYP2J2, an arachidonic acid epoxygenase, is recognized for its role in the first-pass metabolism of astemizole and ebastine. To fully assess the role of CYP2J2 in drug metabolism, a selective substrate and potent specific chemical inhibitor are essential. In this study, we report amiodarone 4-hydoxylation as a specific CYP2J2-catalyzed reaction with no CYP3A4, or other drug-metabolizing enzyme, involvement. Amiodarone 4-hydroxylation enabled the determination of liver relative activity factor and intersystem extrapolation factor for CYP2J2. Amiodarone 4-hydroxylation correlated with astemizole O-demethylation but not with CYP2J2 protein content in a sample of human liver microsomes. To identify a specific CYP2J2 inhibitor, 138 drugs were screened using terfenadine and astemizole as probe substrates with recombinant CYP2J2. Forty-two drugs inhibited CYP2J2 activity by ≥50% at 30 μM, but inhibition was substrate-dependent. Of these, danazol was a potent inhibitor of both hydroxylation of terfenadine (IC(50) = 77 nM) and O-demethylation of astemizole (K(i) = 20 nM), and inhibition was mostly competitive. Danazol inhibited CYP2C9, CYP2C8, and CYP2D6 with IC(50) values of 1.44, 1.95, and 2.74 μM, respectively. Amiodarone or astemizole were included in a seven-probe cocktail for cytochrome P450 (P450) drug-interaction screening potential, and astemizole demonstrated a better profile because it did not appreciably interact with other P450 probes. Thus, danazol, amiodarone, and astemizole will facilitate the ability to determine the metabolic role of CYP2J2 in hepatic and extrahepatic tissues.     

Identification of the human cytochrome P450 enzymes involved in the in vitro metabolism of artemisinin

AIMS: The study aimed to identify the specific human cytochrome P450 (CYP450) enzymes involved in the metabolism of artemisinin. METHODS: Microsomes from human B-lymphoblastoid cell lines transformed with individual CYP450 cDNAs were investigated for their capacity to metabolize artemisinin. The effect on artemisinin metabolism in human liver microsomes by chemical inhibitors selective for individual forms of CYP450 was investigated. The relative contribution of individual CYP450 isoenzymes to artemisinin metabolism in human liver microsomes was evaluated with a tree-based regression model of artemisinin disappearance rate and specific CYP450 activities. RESULTS: The involvement of CYP2B6 in artemisinin metabolism was demonstrated by metabolism of artemisinin by recombinant CYP2B6, inhibition of artemisinin disappearance in human liver microsomes by orphenadrine (76%) and primary inclusion of CYP2B6 in the tree-based regression model. Recombinant CYP3A4 was catalytically competent in metabolizing artemisinin, although the rate was 10% of that for recombinant CYP2B6. The tree-based regression model suggested CYP3A4 to be of importance in individuals with low CYP2B6 expression. Even though ketoconazole inhibited artemisinin metabolism in human liver microsomes by 46%, incubation with ketoconazole together with orphenadrine did not increase the inhibition of artemisinin metabolism compared to orphenadrine alone. Troleandomycin failed to inhibit artemisinin metabolism. The rate of artemisinin metabolism in recombinant CYP2A6 was 15% of that for recombinant CYP2B6. The inhibition of artemisinin metabolism in human liver microsomes by 8-methoxypsoralen (a CYP2A6 inhibitor) was 82% but CYP2A6 activity was not included in the regression tree. CONCLUSIONS: Artemisinin metabolism in human liver microsomes is mediated primarily by CYP2B6 with probable secondary contribution of CYP3A4 in individuals with low CYP2B6 expression. The contribution of CYP2A6 to artemisinin metabolism is likely of minor importance.     

Identification of human P450 isoforms involved in the metabolism of the antiallergic drug, oxatomide, and its kinetic parameters and inhibition constants

Oxatomide is an antiallergic drug used for the treatment of diseases mediated by type I allergy. Recently, terfenadine and astemizole, which have antiallergic actions similar to those of oxatomide, showed side effects on the cardiovascular system. This might be because concomitant drugs such as itraconazole inhibit cytochrome P450 3A4 (CYP3A4), the enzyme responsible for the degradation of terfenadine and astemizole, and thus the blood concentrations of the drugs are abnormally increased. In another article of this issue, we have reported that oxatomide is metabolized by CYP2D6-Val and CYP3A4, and simultaneously inhibits the metabolism of the model substrates for these enzymes. In this study, we performed the kinetic analysis of oxatomide metabolism using microsomes prepared from human liver, and found that the Km and Vmax values were 26.1 microM and 1254.4 pmol/mg protein/min, respectively. Ketoconazole, one of the representative inhibitors for CYP3A4, potently inhibited the metabolism of oxatomide, but other well-known CYP inhibitors did not show significant inhibition. These results suggest that the metabolism of oxatomide is principally catalyzed by CYP3A4. Furthermore, oxatomide inhibited the metabolism of (+/-) bufuralol and testosterone, model substrates for CYP2D6 and CYP3A4, respectively, in a dose-dependent manner with the Ki values of 57.4 and 24.3 microM, respectively. These observations, together with the finding that the putative highest concentration of oxatomide in blood was congruent with 40 ng/ml ( congruent with 93 nM) at 4 h after each dosage during consecutive 6-d administration, encouraged us to conclude that oxatomide won't inhibit CYP2D6 or CYP3A4 at clinical doses.     

Involvement of CYP3A in the metabolism of eplerenone in humans and dogs: differential metabolism by CYP3A4 and CYP3A5.

In vitro studies were conducted to identify the major metabolites of eplerenone (EP) and the cytochrome p450 (p450) isozymes involved in its primary oxidative metabolism in humans and dogs. The major in vitro metabolites were identified as 6 beta-hydroxy EP and 21-hydroxy EP in both humans and dogs. EP was metabolized by cDNA-expressed human CYP3A4 and dog CYP3A12 but only minimally by human CYP3A5. In human microsomes, inhibition of total metabolism by the CYP3A-selective inhibitors ketoconazole, troleandomycin, and 6',7'-dihydroxybergamottin, each at 10 micro M concentration, was 83 to 95%, whereas inhibition with inhibitors selective for other p450 isozymes was minimal. In dog liver microsomes, the percentages of inhibition were 53 to 76% with the CYP3A-selective inhibitors. A monoclonal anti-CYP3A4 antibody inhibited EP metabolism by 84%, whereas other monoclonal antibodies had minimal effects. The formation of 6 beta-hydroxy and 21-hydroxy metabolites in human liver microsomes was best correlated with CYP3A-selective dextromethorphan N-demethylation and testosterone 6 beta-hydroxylation activities. EP moderately inhibited only CYP3A (testosterone 6 beta-hydroxylase) activity in human liver microsomes by 23, 34 and 45% at concentrations of 30, 100, and 300 micro M, respectively. With human microsomes, the V(max) and K(m) for 6 beta-hydroxylation and 21-hydroxylation were 0.973 nmol/min/mg and 217 micro M, and 0.143 nmol/min/mg and 211 micro M, respectively. The human hepatic clearance calculated from total in vitro EP metabolism was 2.30 ml/min/kg, which agrees with in vivo data. In conclusion, 6 beta- and 21-hydroxylation of EP is primarily catalyzed by CYP3A4 in humans and CYP3A12 in dogs. Also, it is unlikely that EP would substantially inhibit the metabolism of other drugs that are metabolized by CYP3A4 or other p450 isoforms.     

Characterization of human cytochrome P450 enzymes involved in the in vitro metabolism of perospirone

In vitro studies were carried out to identify the major contribution of CYP2C8, CYP2D6 and CYP3A4 to the metabolism of perospirone (cis-N-[4-[4-(1,2-benzisothiazol-3-yl)-1-piperazinyl]butyl]cyclohexane-1,2-dicarboximide monohydrochloride dehydrate), a novel antipsychotic agent, using human liver microsomes and expressed P450 isoforms. Quinidine (a specific inhibitor of CYP2D6) did not markedly affect the metabolism of perospirone, whereas quercetin (an inhibitor of CYP2C8) and ketoconazole (an inhibitor of CYP3A4) caused a decrease in the metabolism with human liver microsomes in a concentration dependent fashion. With 10 microM quercetin, the metabolism of perospirone was inhibited by 60.0% and with 1 microM ketoconazole almost complete inhibition was apparent. Anti-CYP2C8 and anti-CYP2D6 antisera did not exert marked effects, whereas anti-CYP3A4 antiserum caused almost complete inhibition. With expressed P450s, K(m) and V(max) values were 1.09 microM and 1.93 pmol/min/pmol P450 for CYP2C8, 1.38 microM and 5.73 pmol/min/pmol P450 for CYP2D6, and 0.245 microM and 61.3 pmol/min/pmol P450 for CYP3A4, respectively. These results indicated that the metabolism of perospirone in human liver was mainly catalysed by CYP3A4, and to a lesser extent CYP2C8 and CYP2D6 were responsible because kinetic data (K(m) and V(max)) of CYP2C8 and CYP2D6 suggested catalytic potential.     

Identification of cytochrome P450 isoform involved in the metabolism of YM992, a novel selective serotonin re-uptake inhibitor,in human liver microsomes

1. In vitro studies were conducted to identify the hepatic cytochrome P450 isoform involved in the metabolism of YM992, ((S)-2-[[(fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride), a novel serotonin re-uptake inhibitor, in human liver microsomes. 2. Microsomes prepared from yeast expressing CYP1A1, CYP1A2 and CYP2D6 effectively metabolized YM992. A significant correlation was observed between the rate of YM992 metabolism and 7-ethoxyresorufin O-deethylation, CYP1A1/2 specific activity, in liver microsomes from 16 individual donors (r² = 0.628, p<0.001). alpha-Naphtoflavone and isosafrole, CYP1A1}2 inhibitors, suppressed the metabolism of YM992 in human liver microsomes in a concentration-dependent manner. 3. The metabolism of YM992 in human liver microsomes was inhibited by ~95% by antibodies which recognize both CYP1A1 and CYP1A2 whereas antibodies specific for CYP1A1 did not show inhibitory effects. 4. The same major metabolites, M6 and M7, were generated from YM992 after incubation with human liver microsomes and recombinant human CYP1A2. 5. These results suggest that the metabolism of YM992 in human liver microsomes is mainly catalysed by CYP1A2, and that YM992 might increase plasma concentration of concomitant drugs metabolized by CYP1A2 due to competitive inhibition.     

Identification of CYP isozymes involved in benzbromarone metabolism in human liver microsomes

Benzbromarone (BBR) is metabolized to 1′‐hydroxy BBR and 6‐hydroxy BBR in the liver. 6‐Hydroxy BBR is further metabolized to 5,6‐dihydroxy BBR. The aim of this study was to identify the CYP isozymes involved in the metabolism of BBR to 1′‐hydroxy BBR and 6‐hydroxy BBR and in the metabolism of 6‐hydroxy BBR to 5,6‐dihydroxy BBR in human liver microsomes. Among 11 recombinant P450 isozymes examined, CYP3A4 showed the highest formation rate of 1′‐hydroxy BBR. The formation rate of 1′‐hydroxy BBR significantly correlated with testosterone 6β‐hydroxylation activity in a panel of 12 human liver microsomes. The formation of 1′‐hydroxy BBR was completely inhibited by ketoconazole in pooled human liver microsomes. On the other hand, the highest formation rate of 6‐hydroxy BBR was found in recombinant CYP2C9. The highest correlation was observed between the formation rate of 6‐hydroxy BBR and diclofenac 4′‐hydroxylation activity in 12 human liver microsomes. The formation of 6‐hydroxy BBR was inhibited by tienilic acid in pooled human liver microsomes. The formation of 5,6‐dihydroxy BBR from 6‐hydroxy BBR was catalysed by recombinant CYP2C9 and CYP1A2. The formation rate of 5,6‐dihydroxy BBR was significantly correlated with diclofenac 4′‐hydroxylation activity and phenacetin O‐deethylation activity in 12 human liver microsomes. The formation of 5,6‐dihydroxy BBR was inhibited with either tienilic acid or α‐naphthoflavone in human liver microsomes. These results suggest that (i) the formation of 1′‐hydroxy BBR and 6‐hydroxy BBR is mainly catalysed by CYP3A4 and CYP2C9, respectively, and (ii) the formation of 5,6‐dihydroxy BBR is catalysed by CYP2C9 and CYP1A2 in human liver microsomes. Copyright © 2012 John Wiley & Sons, Ltd.     

Different in vitro metabolism of paclitaxel and docetaxel in humans, rats, pigs, and minipigs.

We investigated cytochrome P450 (P450)-catalyzed metabolism of the important cancer drugs paclitaxel and docetaxel in rat, pig, minipig, and human liver microsomes and cDNA-expressed P450 enzymes. In rat microsomes, paclitaxel was metabolized mainly to C3'-hydroxypaclitaxel (C3'-OHP) and to a lesser extent to C2-hydroxypaclitaxel (C2-OHP), di-hydroxypaclitaxel (di-OHP), and another unknown monohydroxylated paclitaxel. In pig and minipig microsomes, this unknown hydroxypaclitaxel was the main metabolite, whereas C3'-OHP was a minor product. In minipigs, C2-OHP was the next minor product. In human liver microsomes, 6 alpha-hydroxypaclitaxel (6 alpha-OHP) was the main metabolite, followed by C3'-OHP and C2-OHP. Among different cDNA-expressed human P450 enzymes (CYP1A2, 1B1, 2A6, 2C9, 2E1, and 3A4), only CYP3A4 enzyme formed C3'-OHP and C2-OHP. Docetaxel was metabolized in pig, minipig, rat, and human liver microsomes mainly to hydroxydocetaxel (OHDTX), whereas CYP3A-induced rat microsomes produced primarily diastereomeric hydroxyoxazolidinones. Human liver microsomes from 10 different individuals formed OHDTX at different rates correlated with CYP3A4 content. Troleandomycin as a selective inhibitor of CYP3A inhibited the formation of C3'-OHP, C2-OHP, and di-OHP, as well as the unknown OHP produced in rat, minipig, and pig microsomes. In human liver microsomes, troleandomycin inhibited C3'-OHP and C2-OHP formation, and a suitable inhibitor of human CYP2C8, fisetin, strongly inhibited the formation of 6 alpha-OHP, known to be catalyzed by human CYP2C8. In conclusion, the metabolism of docetaxel is the same in all four species, but metabolism of paclitaxel is different, and 6 alpha-OHP remains a uniquely human metabolite. Pigs and minipigs compared with each other formed the same metabolites of paclitaxel.     

Terfenadone is a strong inhibitor of CYP2J2 present in the human liver and intestinal microsomes

Cytochrome P450 2J2 (CYP2J2) is involved in the metabolism of drugs, including albendazole, astemizole, ebastine, and endogenous substrates. In a previous study, we used recombinant CYP2J2 and determined whether danazol, hydroxyebastine, telmisartan, and terfenadone inhibited CYP2J2 by using four representative CYP2J2 substrates, namely albendazole, astemizole, ebastine, and terfenadine. In this study, we evaluated the inhibitory potential of these four chemicals on human liver and intestinal microsomes, which are commonly used in a reaction phenotyping study. Among the four CYP2J2 inhibitors tested, terfenadone was strongest inhibitor of CYP2J2-mediated metabolism of albendazole, astemizole, and terfenadine with IC₅₀ values of 0.31, 0.15, and 2.11 μM, respectively, in human liver microsomes (HLMs). In addition, terfenadone had strong inhibitory effect on the metabolism of the abovementioned drugs in human intestinal microsomes (HIMs), with IC₅₀ values of 0.43, 0.08 and 1.07 μM, respectively. Danazol, weakly inhibited CYP2J2-mediated metabolism of albendazole and astemizole with IC₅₀ values of 13.8 and 18.3 μM, respectively in HLMs, whereas it strongly inhibited the CYP2J2-mediated ebastine hydroxylase activity in HLMs and HIMs (IC₅₀ = 1.93–1.95 μM). Our data suggest that terfenadone may be used as a general CYP2J2 inhibitor in reaction phenotyping study using HLMs and HIMs regardless of the substrate used.     

Biotransformation of 6-methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole (BPR0L075), a novel antimicrotubule agent, by mouse, rat, dog, and human liver microsomes.

6-Methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole (BPR0L075) is a novel synthetic indole compound with microtubule binding activity. Incubation of BPR0L075 with mouse, rat, dog, and human liver microsomes in the presence of NADPH resulted in the formation of six metabolites. Liquid chromatography-tandem mass spectrometry and comparison with the synthetic reference standards identified two metabolites (M1 and M5) as the products derived from hydroxylation on the indole moiety of the molecule. M3 was also identified as a product derived from hydroxylation, but the structure of this metabolite was not identified because of the lack of a reference standard. M2, M4, and M6 were identified as the products derived from O-demethylation. M2, 6-desmethyl-BPR0L075, was the major metabolite formed by the liver microsomes of the four species. No qualitative species difference in the metabolism of BPR0L075 was observed. There was quantitative species difference in the metabolism of BPR0L075 among the four species. Whereas mouse and rat liver microsomes metabolized BPR0L075 predominantly via O-demethylation, dog liver microsomes metabolized BPR0L075 by O-demethylation and hydroxylation to about the same extent. The rank order of intrinsic clearance rates for the conversion of BPR0L075 to 6-desmethyl-BPR0L075 was mouse > rat > human > dog. Incubation of BPR0L075 with baculovirus-insect cell-expressed human cytochrome P450 (P450) isozymes showed that CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the O-demethylation and hydroxylation of BPR0L075 but to a different degree. Among the six P450 isozymes tested, CYP1A2 and 2D6 were most active on catalyzing the metabolism of BPR0L075. CYP1A2 catalyzed mainly the formation of M1, M2, and M3. M2 was the predominant metabolite formed by CYP2D6.     

Drug-drug interactions of Z-338, a novel gastroprokinetic agent, with terfenadine, comparison with cisapride, and involvement of UGT1A9 and 1A8 in the human metabolism of Z-338

In the present study, the inhibitory properties of N-[2-(diisopropylamino)ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl)amino]-1,3-thiazole-4-carboxamide monohydrochloride trihydrate (Z-338), a novel gastroprokinetic agent, were investigated and compared with those of cisapride to establish its potential for drug-drug interactions. There was no notable inhibition of terfenadine metabolism or of any of the isoforms of cytochrome P450 (CYP1A1/2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) by Z-338 in in vitro studies using human liver microsomes. Z-338 was mainly metabolized to its glucuronide by UGT1A9 (UDP glucoronosyltransferase 1 family, polypeptide A9) and UGT1A8, and did not show marked inhibition of P-glycoprotein activity. On the other hand, cisapride strongly inhibited CYP3A4 and markedly inhibited CYP2C9. Furthermore, we used the whole-cell patch-clamp technique to investigate the effects of Z-338 and cisapride on potassium currents in human embryonic kidney (HEK) 293 cells transfected with the human ether-a-go-go-related gene (hERG). Z-338 had no significant effect on hERG-related current at the relatively high concentration of 10 microM. In contrast, the inhibition by Z-338 was very small compared with that of cisapride at 10 nM, which was a thousand-fold lower concentration. In the prediction method for the drug interaction between terfenadine and cisapride based on the K(i) and PK parameters, we suggest the possibility that terfenadine mainly affect the QT interval, since its plasma concentration would be markedly increased, but cisapride may not be changed. Thus, in contrast with cisapride, Z-338 did not inhibit CYP and the hERG channel, and is predominantly metabolized by glucuronide conjugation, Z-338 is considered unlikely to cause significant drug-drug interactions when coadministered with CYP substrates at clinically effective doses.     

The metabolism of clopidogrel is catalyzed by human cytochrome P450 3A and is inhibited by atorvastatin.

The prodrug clopidogrel (Plavix) is activated by cytochrome p450 (p450) to a metabolite that inhibits ADP-induced platelet aggregation. Clopidogrel is frequently administered to patients in conjunction with the CYP3A4 substrate atorvastatin (Lipitor). Since clinical studies indicate that atorvastatin inhibits the antiplatelet activity of clopidogrel, we investigated whether CYP3A4 metabolized clopidogrel in vitro. Microsomes prepared from dexamethasone-pretreated rats metabolized clopidogrel at a rate of 3.8 nmol min(-1) nmol of p450(-1), which is 65 and 1270% faster than the rate of metabolism by microsomes from control and beta-napthoflavone-treated rats, respectively. To identify the human p450s responsible for clopidogrel oxidation, genetically engineered microsomes containing a single human p450 isozyme were tested for their ability to oxidize clopidogrel. CYP3A4 and 3A5 metabolized clopidogrel at a significantly higher rate than eight other p450 isozymes, suggesting that CYP3A4 and 3A5 are primarily responsible for in vivo clopidogrel metabolism. Clopidogrel interacts with human CYP3A4 with a spectral dissociation constant (K(s)), K(m), and V(max) of 12 microM, 14 +/- 1 microM and 6.7 +/- 1 nmol min(-1) nmol p450(-1), respectively. Atorvastatin lactone, the physiologically relevant substrate, inhibits clopidogrel with a K(i) of 6 microM. When clopidogrel and atorvastatin are present at equimolar concentrations, clopidogrel metabolism is inhibited by greater than 90%. Since CYP3A4 and 3A5 metabolize clopidogrel faster than other human p450 isozymes and are the most abundant p450s in human liver, they are predicted to be predominantly responsible for the activation of clopidogrel in vivo.     

Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of rosiglitazone

AIMS: To identify the human cytochrome P450 enzyme(s) involved in the in vitro metabolism of rosiglitazone, a potential oral antidiabetic agent for the treatment of type 2 diabetes-mellitus. Method The specific P450 enzymes involved in the metabolism of rosiglitazone were determined by a combination of three approaches; multiple regression analysis of the rates of metabolism of rosiglitazone in human liver microsomes against selective P450 substrates, the effect of selective chemical inhibitors on rosiglitazone metabolism and the capability of expressed P450 enzymes to mediate the major metabolic routes of rosiglitazone metabolism. Result The major products of metabolism following incubation of rosiglitazone with human liver microsomes were para-hydroxy and N-desmethyl rosiglitazone. The rate of formation varied over 38-fold in the 47 human livers investigated and correlated with paclitaxel 6alpha-hydroxylation (P<0.001). Formation of these metabolites was inhibited significantly (>50%) by 13-cis retinoic acid, a CYP2C8 inhibitor, but not by furafylline, quinidine or ketoconazole. In addition, both metabolites were produced by microsomes derived from a cell line transfected with human CYP2C8 cDNA. There was some evidence for CYP2C9 playing a minor role in the metabolism of rosiglitazone. Sulphaphenazole caused limited inhibition (<30%) of both pathways in human liver microsomes and microsomes from cells transfected with CYP2C9 cDNA were able to mediate the metabolism of rosiglitazone, in particular the N-demethylation pathway, albeit at a much slower rate than CYP2C8. Rosiglitazone caused moderate inhibition of paclitaxel 6alpha-hydroxylase activity (CYP2C8; IC50=18 microm ), weak inhibition of tolbutamide hydroxylase activity (CYP2C9; IC50=50 microm ), but caused no marked inhibition of the other cytochrome P450 activities investigated (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, 3A and 4A). Conclusion CYP2C8 is primarily responsible for the hydroxylation and N-demethylation of rosiglitazone in human liver; with minor contributions from CYP2C9.     

Terfenadine t-butyl hydroxylation catalyzed by human and marmoset cytochrome P450 3A and 4F enzymes in livers and small intestines

1.?Roles of human cytochrome P450 (P450) 3A4 in oxidation of an antihistaminic drug terfenadine have been previously investigated in association with terfenadine–ketoconazole interaction. Several antihistamine drugs have been recently identified as substrates for multiple P450 enzymes. In this study, overall roles of P450 3A4, 2J2, and 4F12 enzymes in terfenadine t-butyl hydroxylation were investigated in small intestines and livers from humans, marmosets, and/or cynomolgus monkeys.

2.?Human liver microsomes and liver and small intestine microsomes from marmosets and cynomolgus monkeys effectively mediated terfenadine t-butyl hydroxylation. Ketoconazole and N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine (a P450 4A/F inhibitor) almost completely and moderately inhibited these activities, respectively, in human liver microsomes; however, these chemicals did not show substantially suppression in marmoset liver. Anti-human P450 3A and 4F antibodies showed the roughly supportive inhibitory effects.

3.?Recombinant P450 3A4/90 and 4F12 showed high terfenadine t-butyl hydroxylation activities with substrate inhibition constants of 84–144?μM (under 26–76?μM of K_m values), in similar manners to liver and intestine microsomes.

4.?These results suggest that human and marmoset P450 3A4/90 and 4F12 in livers or small intestines played important roles in terfenadine t-butyl hydroxylation. Marmosets could be a model for humans during first pass extraction of terfenadine and related substrates.     

In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Yondelis®, Trabectidin®), a novel marine anti-cancer drug

Summary ET-743 is a potent marine anti-cancer drug and is currently being investigated in phase I and II clinical trials, e.g. in combination with other anti-cancer agents. To assess the biotransformation and CYP reaction phenotype and their potential implications for human pharmacology and toxicology, the in vitro metabolism of ET-743 was characterized using incubations with human liver preparations, cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) supersomes. CYP supersomes and liver microsomes showed that ET-743 was metabolized mainly by CYP3A4, but also by CYP2C9, 2C19, 2D6, and 2E1. ET-743 showed the highest affinity for CYP3A4 and the highest maximal metabolic rate for CYP2D6 among the CYPs shown to metabolize ET-743. In addition, the K_m value of ET-743 in female microsomes was significantly lower compared to male microsomes, while the V_max values did not differ. ET-743 glucuronidation, catalyzed by UGT2B15, was observed in microsomes and S9 fraction. In addition, conjugation by glutathione-S-transferase and no sulphation was observed for ET-743 in cytosol and S9 fraction. ET-743 was more extensively metabolized when CYP activity was combined with phase II enzymes UGT and glutathione-S-transferase (GST), indicating that CYP, UGT, and GST simultaneously metabolize ET-743 in the S9 fraction. These results provide evidence that CYP3A4 has a major role in the metabolism of ET-743 in vitro with additional involvement of CYP2C9, 2C19, 2D6, and 2E1. Furthermore, ET-743 is conjugated by UGT and GST. This information could be important for interpretation of the pharmacokinetic data of clinical trials and prediction of drug-drug interactions.     

Identification of cytochrome P450 enzymes involved in the metabolism of FK228, a potent histone deacetylase inhibitor, in human liver microsomes

FK228 (FR901228, depsipeptide) is a potent histone deacetylase inhibitor currently in phase II clinical trials for cancer treatment. In the present study, the cytochrome P450 (P450) enzymes responsible for FK228 metabolism in human liver microsomes were investigated. Incubation with human liver microsomes in the presence of an NADPH-generating system revealed that FK228 is metabolized to at least 10 different metabolites. Km and Vmax values for FK228 disappearance were 20.3 microM and 561.9 pmol/min/mg protein, respectively. Further studies were performed at a substrate concentration of 10 microM (half the Km value for FK228 disappearance). FK228 disappearance activities in human liver microsomes from 12 individuals strongly correlated (r2=0.957) with testosterone 6beta-hydroxylase activities, a marker enzyme activity of CYP3A4/5, but not with other P450 enzyme-specific activities (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 4A). Among 14 recombinant heterologously expressed human P450s examined, CYP3A4 exhibited the highest activity of FK228 disappearance. CYP3A5, 1A1, 2B6, and 2C19 showed 16.8%, 5.2%, 1.6%, and 1.3% of the activity of CYP3A4, respectively. Other P450s showed no significant metabolic activity toward FK228. In addition, FK228 disappearance in human liver microsomes was markedly inhibited by ketoconazole, a potent CYP3A4 inhibitor, and an anti-CYP3A4 antibody. These results indicate that the metabolism of FK228 in human liver microsomes is catalyzed mainly by CYP3A enzymes, particularly CYP3A4.     

In vitro human metabolism and interactions of repellent N,N-diethyl-m-toluamide.

Oxidative metabolism of the insect repellent N,N-diethyl-m-toluamide (DEET) by pooled human liver microsomes (HLM), rat liver microsomes (RLM), and mouse liver microsomes (MLM) was investigated. DEET is metabolized by cytochromes P450 (P450s) leading to the production of a ring methyl oxidation product, N,N-diethyl-m-hydroxymethylbenzamide (BALC), and an N-deethylated product, N-ethyl-m-toluamide (ET). Both the affinities and intrinsic clearance of HLM for ring hydroxylation are greater than those for N-deethylation. Pooled HLM show significantly lower affinities (K(m)) than RLM for metabolism of DEET to either of the primary metabolites (BALC and ET). Among 15 cDNA-expressed P450 enzymes examined, CYP1A2, 2B6, 2D6*1 (Val(374)), and 2E1 metabolized DEET to the BALC metabolite, whereas CYP3A4, 3A5, 2A6, and 2C19 produced the ET metabolite. CYP2B6 is the principal cytochrome P450 involved in the metabolism of DEET to its major BALC metabolite, whereas CYP2C19 had the greatest activity for the formation of the ET metabolite. Use of phenotyped HLMs demonstrated that individuals with high levels of CYP2B6, 3A4, 2C19, and 2A6 have the greatest potential to metabolize DEET. Mice treated with DEET demonstrated induced levels of the CYP2B family, increased hydroxylation, and a 2.4-fold increase in the metabolism of chlorpyrifos to chlorpyrifos-oxon, a potent anticholinesterase. Preincubation of human CYP2B6 with chlorpyrifos completely inhibited the metabolism of DEET. Preincubation of human or rodent microsomes with chlorpyrifos, permethrin, and pyridostigmine bromide alone or in combination can lead to either stimulation or inhibition of DEET metabolism.     

体外研究人细胞色素P450在雌二醇代谢中的作用(英文)

目的:研究雌二醇在cDNA表达的P450和人肝微粒体中的代谢机制,为在体内研究细胞色素P450活性与肿瘤发生的关系提供依据。方法:用HPLC-ECD法测定雌二醇的代谢产物。通过雌二醇在不同cDNA表达的P450中代谢,13例人肝微粒体中相关性研究,抑制剂对代谢的影响以及微粒体中17β-羟基脱氢化和2-羟基化代谢的催化动力学的研究来推断雌二醇的代谢机理。结果:在cDNA表达的P450中,催化2-羟基化代谢的P450按活性排列依次为CYP1A2、CYP3A4、CYP2C9。CYP2C9、CYP2C19和CYP2C8均具有较高的催化17β-羟基脱氢化活性。抑制CYP1A2与抑制CYP3A4对2-羟基化代谢产物生成的影响相似,可认为CYP1A2和CYP3A4在人肝微粒体中催化2-羟基化代谢的作用相近。雌二醇代谢的途径与底物浓度有关,低浓度时(1,10μmol/L)17β-羟基脱氢化为主要代谢途径;高浓度时(100μmol/L),2-羟基化成为主要代谢途径。结论:高底物浓度时,雌二醇主要由CYP1A2和CYP3A4催化代谢为2-羟基化产物。低底物浓度时,主要由CYP2C9、CYP2C19和CYP2C8催化生成17β-羟基去氢化产物。     

Characterization of human liver cytochrome P450 enzymes involved in the metabolism of rutaecarpine

Rutaecarpine has recently been characterized to have an anti-inflammatory activity through cyclooxygenase-2 inhibition. The incubation of rutaecarpine with human liver microsomes in the presence of NADPH generated six isobaric mono-hydroxylated metabolites. The specific cytochrome P450 (CYP) isozymes responsible for rutaecarpine metabolites were identified using the combination of chemical inhibition, immuno-inhibition and metabolism by cDNA expressed CYP enzymes. The results suggested that CYP3A4 might play major roles in the metabolism of rutaecarpine in human liver microsomes. The production of M1, M2, M3, M4 and M6 formed in human liver microsomes was inhibited by ketoconazole, a selective CYP3A4 inhibitor, and anti-CYP3A4 antibody. CYP1A2 and CYP2C9 played minor roles in the metabolism of rutaecarpine. These results were confirmed in microsomes derived from cDNA expressed lymphoblastoid cells. CYP3A4 microsome clearly formed M1, M2, M3 and M6. CYP1A2 and CYP2C9 microsomes comparably formed M5.