Characterization of the enzymes involved in the in vitro metabolism of amrubicin hydrochloride

The in vitro metabolism of amrubicin by rat and human liver microsomes and cytosol was examined. The main metabolic routes in both species were reductive deglycosylation and carbonyl group reduction in the side-chain. In vitro metabolism of amrubicinol by rat and human liver microsomes and cytosol was also examined and the main metabolic route of this active metabolite was reductive deglycosylation. Metabolism of amrubicin in human liver microsomes was inhibited by TlCl₃ and that in human liver cytosol was inhibited by dicumarol and quercetin. Generation of amrubicinol was inhibited only by quercetin. The results indicate that metabolism of amrubicin is mediated by NADPH-cytochrome P450 reductase, NADPH:quinone oxidoreductase and carbonyl reductase. In addition, generation of amrubicinol is mediated by carbonyl reductase. Metabolism of amrubicinol in human liver microsomes was inhibited by TlCl₃ and that in human liver cytosol was inhibited by dicumarol. The results indicate that metabolism of amrubicinol is mediated by NADPH-cytochrome P450 reductase and NADPH:quinone oxidoreductase. To investigate the influence of cisplatin on the metabolism of amrubicin and amrubicinol, human liver microsomes and cytosol were pre-incubated with cisplatin. This did not change the rates of amrubicin and amrubicinol metabolism in either human liver microsomes or cytosol.     

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.     

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.     

Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of ethosuximide by human hepatic microsomal enzymes

1. The relative roles of human hepatic cytochrome P450 (CYP) subfamilies participating in ethosuximide metabolism have been studied in vitro using humanized heterologous CYP microsomal systems expressing either CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1 or CYP3A4. 2. Ethosuximide was incubated with each expression system at 37 degrees C, and its hydroxylated metabolite was quantified by HPLC assay. 3. K(m) and V(max) values for metabolite formation were estimated for CYP3A4 and CYP2E1. The K(m) values for the metabolite formed were 1.40 and 0.24 mM for CYP3A4 and CYP2E1, respectively. The V(max) values were 0.65 and 0.14 nmol mg-1 protein min(-1) for CYP3A4 and CYP2E1, respectively. 4. These parameters could not be measured for other enzymes, since metabolite concentrations formed were below the HPLC detection limits. 5. Immuno-inhibition studies using specific antibodies against CYP3A4 or CYP2E1 revealed that ethosuximide metabolite levels decreased when the amount of added CYP3A4 or CYP2E1 antibody increased, with anti-CYP3A4 antibodies yielding a greater inhibitory effect. 6. Simulations of scaled-up in vivo ethosuximide CL(hepatic) mediated by CYP3A4 and CYP2E1 based on the in vitro CL'(int) values, which were calculated from the foregoing respective V(max) and K(m) values, project that well over 90% of in vivo CL(hepatic) is due to CYP3A4. These results support an important role for CYP3A in human ethosuximide metabolism, and a minor role for CYP2E1.     

Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of ethosuximide by human hepatic microsomal enzymes

1. The relative roles of human hepatic cytochrome P450 (CYP) subfamilies participating in ethosuximide metabolism have been studied in vitro using humanized heterologous CYP microsomal systems expressing either CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1 or CYP3A4. 2. Ethosuximide was incubated with each expression system at 37 °C, and its hydroxylated metabolite was quantified by HPLC assay. 3. K m and V max values for metabolite formation were estimated for CYP3A4 and CYP2E1. The K m values for the metabolite formed were 1.40 and 0.24 mM for CYP3A4 and CYP2E1, respectively. The V max values were 0.65 and 0.14 nmol?mg ? 1 protein?min ? 1 for CYP3A4 and CYP2E1, respectively. 4. These parameters could not be measured for other enzymes, since metabolite concentrations formed were below the HPLC detection limits. 5. Immuno-inhibition studies using specific antibodies against CYP3A4 or CYP2E1 revealed that ethosuximide metabolite levels decreased when the amount of added CYP3A4 or CYP2E1 antibody increased, with anti-CYP3A4 antibodies yielding a greater inhibitory effect. 6. Simulations of scaled-up in vivo ethosuximide CL hepatic mediated by CYP3A4 and CYP2E1 based on the in vitro CL' int values, which were calculated from the foregoing respective V max and K m values, project that well over 90% of in vivo CL hepatic is due to CYP3A4. These results support an important role for CYP3A in human ethosuximide metabolism, and a minor role for CYP2E1.     

参与抗癌新药F318体外代谢的大鼠细胞色素P450酶

目的:体外研究大鼠肝微粒体中F318代谢的酶促动力学．及利用选择性细胞色素(CYP)酶抑制剂明确参与F318代谢的CYP亚型。方法:优化F318在大鼠肝微粒体中孵育的条件,并进行酶促动力学研究;探讨CYP酶的选择性抑制剂α-萘磺酮(CYP1A2),磺胺苯吡唑(CYP2C9),噻氯匹定(CYP2C19),查尼丁(CYP2D6),4-甲基吡唑(CYP2E1),酮康唑(CYP3A1)对其代谢的影响及参与其代谢的CYP亚型。结果:F318代谢的酶促动力学参数:最大反应速率(V_(max))为(1．40±0．03)μmol·min~(-1)·mg~(-1),米氏常数(K_m)为(19．36±2．30)μmol·L~(-1)。酮康唑可以显著地抑制F318代谢(抑制效果≈70％),而其他CYP特异性抑制剂对F318的代谢没有明显的影响。结论: CYP3A1主要参与了F318的代谢,CYP1A2,2C19和2D6也起到了部分代谢作用。     

Characterization of human cytochrome P450 enzymes involved in the metabolism of cyamemazine

Recombinant human liver microsomal enzymes of the cytochrome P450 family (CYP1A2, CYP2A6, CYP3A4, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1) were used to determine the metabolic fate of the antipsychotic anxiolytic agent cyamemazine. An LC/MS–MS tandem methodology was developed specifically for identifying the presence of cyamemazine and its metabolites in reaction media. All P450 enzymes investigated, with the exception of CYP2A6 and CYP2E1, degraded cyamemazine, albeit to a different extent, with CYP1A2, CYP2C8 and CYP2C19 being the most efficient (>80%). However, in microsomes prepared from native human hepatocytes, only relatively specific competitors (inhibitors and/or substrates) of CYP1A2, CYP2C8, CYP2C9 and CYP3A4 reduced notably the degradation cyamemazine. The main routes of cyamemazine biotransformation are N-mono-demethylation (CYP1A2, CYP3A4 and CYP2C8) and mono-oxidation (either S-oxidized or hydroxylated derivatives which could not be discriminated because characterized by the same mass value) by CYP1A2 and CYP2C9. Secondary metabolic routes yields N,N-di-demethylated and N-demethylated mono-oxidized products. Thus, under in vitro conditions, cyamemazine is extensively degraded by at least four distinct P450 enzymes, into two primary hydrophilic metabolites. These results suggest that cyamemazine detoxification process is unlikely to be significantly impaired by co-administration of therapeutic agents that are substrates of the CYP metabolic system.     

Characterization of the human cytochrome P450 enzymes involved in the metabolism of dihydrocodeine

Aims Using human liver microsomes from donors of the CYP2D6 poor and extensive metabolizer genotypes, the role of individual cytochromes P-450 in the oxidative metabolism of dihydrocodeine was investigated.
Methods The kinetics of formation of N- and O -demethylated metabolites, nordihydrocodeine and dihydromorphine, were determined using microsomes from six extensive and one poor metabolizer and the effects of chemical inhibitors selective for individual P-450 enzymes of the 1A, 2A, 2C, 2D, 2E and 3A families and of LKM1 (anti-CYP2D6) antibodies were studied.
Results Nordihydrocodeine was the major metabolite in both poor and extensive metabolizers. Kinetic constants for N -demethylation derived from the single enzyme Michaelis-Menten model did not differ between the two groups. Troleandomycin and erythromycin selectively inhibited N -demethylation in both extensive and poor metabolizers. The CYP3A inducer, α-naphthoflavone, increased N -demethylation rates. The kinetics of formation of dihydromorphine in both groups were best described by a single enzyme Michaelis-Menten model although inhibition studies in extensive metabolizers suggested involvement of two enzymes with similar K _m values. The kinetic constants for O -demethylation were significantly different in extensive and poor metabolizers. The extensive metabolizers had a mean intrinsic clearance to dihydromorphine more than ten times greater than the poor metabolizer. The CYP2D6 chemical inhibitors, quinidine and quinine, and LKM1 antibodies inhibited O -demethylation in extensive metabolizers; no effect was observed in microsomes from a poor metabolizer.
Conclusions CYP2D6 is the major enzyme mediating O -demethylation of dihydrocodeine to dihydromorphine. In contrast, nordihydrocodeine formation is predominantly catalysed by CYP3A.     

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.     

Characterization of human cytochrome p450 enzymes involved in the metabolism of cilostazol.

Cilostazol (OPC-13013; 6-[4-(1-cyclohexl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone) is widely used as an antiplatelet vasodilator agent. In vitro, the hydroxylation of the quinone moiety of cilostazol to OPC-13326 [6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-4-hydroxy-2(1H)-quinolinone], is the predominant route, and the hydroxylation of the hexane moiety to OPC-13217 is the second most predominant route. This study was carried out to identify and kinetically characterize the human cytochrome P450 (P450) isozymes responsible for the formation of the two major metabolites of cilostazol, namely, OPC-13326 and OPC-13217 [3,4-dihydro-6-[4-[1-(cis-4-hydroxycyclohexyl)-1H-tetrazol-5-yl)butoxy]-2(1H)-quinolinone)]. In in vitro studies using 14 recombinant human P450 isozymes, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11, cilostazol was metabolized to OPC-13326 mainly by CYP3A4 (K(m) = 5.26 muM, intrinsic clearance (CL(int)) = 0.34 microl/pmol P450/min), CYP1B1 (K(m) = 11.2 microM, CL(int) = 0.03 microl/pmol P450/min), and CYP3A5 (K(m) = 2.89 microM, CL(int) = 0.05 microl/pmol P450/min) and to OPC-13217 mainly by CYP3A5 (K(m) = 1.60 microM, CL(int) = 0.57 microl/pmol P450/min), CYP2C19 (K(m) = 5.95 microM, CL(int) = 0.16 microl/pmol P450/min), CYP3A4 (K(m) = 5.35 microM, CL(int) = 0.10 microl/pmol P450/min), and CYP2C8 (K(m) = 33.8 microM, CL(int) = 0.009 microl/pmol P450/min). The present study showed that the two major metabolites of cilostazol in vitro, namely, OPC-13326 and OPC-13217, are mainly catalyzed by CYP3A4 and CYP3A5, respectively.     

Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation.     

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 the rat liver cytochrome P450 enzymes involved in the metabolism of the calcium channel blocker dipfluzine hydrochloride

This study aimed to identify the specific cytochrome P450 (CYP450) enzymes involved in the metabolism of dipfluzine hydrochloride using the combination of a chemical inhibition study, a correlation analysis and a panel of recombinant rat CYP450 enzymes. The incubation of Dip with rat liver microsomes yielded four metabolites, which were identified by liquid chromatography-coupled tandem mass spectrometry (LC/MS/MS). The results from the assays involving eight selective inhibitors indicated that CYP3A and CYP2A1 contributed most to the metabolism of Dip, followed by CYP2C11, CYP2E1 and CYP1A2; however, CYP2B1, CYP2C6 and CYP2D1 did not contribute to the formation of the metabolites. The results of the correlation analysis and the assays involving the recombinant CYP450 enzymes further confirmed the above results and concluded that CYP3A2 contributed more than CYP3A1. The results will be valuable in understanding drug–drug interactions when Dip is coadministered with other drugs.     

In vitro identification of the human cytochrome p450 enzymes involved in the oxidative metabolism of loxapine

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) enzymes responsible for the oxidative metabolism of loxapine to 8-hydroxyloxapine, 7-hydroxyloxapine, N-desmethylloxapine (amoxapine) and loxapine N-oxide. These studies included use of cDNA-expressed enzymes, correlation analysis with 12 phenotyped human liver microsomal samples, and use of selective inhibitors of cytochrome P450s. The resultant data indicated that loxapine was mainly metabolized by human liver microsomes to (i) 8-hydroxyloxapine by CYP1A2, (ii) 7-hydroxyloxapine by CYP2D6, (iii) N-desmethyloxapine by CYP3A4 and (iv) loxapine N-oxide by CYP3A4. The involvement of flavin-containing monooxygenase (FMO) in the formation of loxapine N-oxide was also observed.     

Characterization of the cytochrome P450 enzymes involved in the metabolism of a new cardioprotective agent KR-33028

KR-33028 (N-[4-cyano-benzo[b]thiophene-2-carbonyl]guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. This study was performed to characterize the cytochrome P450 (CYP) enzymes that are involved in the metabolism of KR-33028. Hydroxylation (5-hydroxy- and 7-hydroxy-KR-33028) is major pathways for the metabolism of KR-33028 in human liver microsomes. Among the nine c-DNA expressed CYP isoforms tested, KR-33028 was 5-hydroxylated by CYP3A4 and 7-hydroxylated by CYP1A2, CYP3A4, and CYP2C19. These findings were supported by the combination of chemical inhibition studies in human liver microsomes and correlation analysis. Furafylline and ketoconazole potently inhibited hydroxylation of KR-33028 in human liver microsomes. Correlation analysis between the known CYP enzyme activities and the rates of the formation of 5-hydroxy- and 7-hydroxy-KR-33028 in the 16 human liver microsomes has showed significant correlations with CYP3A4-mediated midazolam 1'-hydroxylation and CYP1A2-mediated phenacetin O-deethylation, respectively. A 7-hydroxy-KR-33028 formation is also weakly correlated with CYP3A4-mediated midazolam 1'-hydroxylation. The kinetics of the major biotransformation of KR-33028 were studied: CYP3A4 mediated the formation of 5-hydroxy-KR-33028 from KR-33028 with Cl(int)=0.22microl/min/pmol CYP. The intrinsic clearance for 7-hydroxy-KR-33028 formation by CYP1A2, CYP2C19, and CYP3A4 were 0.26, 0.19, and 0.03microl/min/pmol CYP, respectively. Taken together, these results provide evidence that CYP3A4 and CYP1A2 are the major isoforms responsible for the hydroxy metabolites formation from KR-33028.     

Characterization of human cytochrome p450 enzymes involved in the metabolism of the piperidine-type phenothiazine neuroleptic thioridazine.

The aim of the present study was to identify human cytochrome P450 enzymes (P450s) involved in mono-2-, di-2-, and 5-sulfoxidation, and N-demethylation of the piperidine-type phenothiazine neuroleptic thioridazine in the human liver. The experiments were performed in vitro using cDNA-expressed human P450s (Supersomes 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4), liver microsomes from different donors, and P450-selective inhibitors. The results indicate that CYP1A2 and CYP3A4 are the main enzymes responsible for 5-sulfoxidation and N-demethylation (34-52%), whereas CYP2D6 is the basic enzyme that catalyzes mono-2- and di-2-sulfoxidation of thioridazine in human liver (49 and 64%, respectively). Besides CYP2D6, CYP3A4 contributes to a noticeable degree to thioridazine mono-2-sulfoxidation (22%). Therefore, the sulforidazine/mesoridazine ratio may be an additional and more specific marker than the mesoridazine/thioridazine ratio for assessing the activity of CYP2D6. In contrast to promazine and perazine, CYP2C19 insignificantly contributes to the N-demethylation of thioridazine. Considering serious side-effects of thioridazine and its 5-sulfoxide (cardiotoxicity), as well as strong dopaminergic D2 and noradrenergic alpha1 receptor-blocking properties of mono-2- and di-2-sulfoxides, the obtained results are of pharmacological and clinical importance, in particular, in a combined therapy. Knowledge of the catalysis of thioridazine metabolism helps to choose optimum conditions (a proper coadministered drug and dosage) to avoid undesirable drug interactions.     

In vitro identification of metabolic pathways and cytochrome P450 enzymes involved in the metabolism of etoperidone

1. In vitro studies have been carried out to investigate the metabolic pathways and identify the hepatic cytochrome P450 (CYP) enzymes involved in etoperidone (Et) metabolism. 2. Ten in vitro metabolites were profiled, quantified and tentatively identified after incubation with human hepatic S9 fractions. Et was metabolized via three metabolic pathways: (A) alkyl hydroxylation to form OH-ethyl-Et (M1); (B) phenyl hydroxylation to form OH-phenyl-Et (M2); and (C) N-dealkylation to form 1-m-chlorophenylpiperazine (mCPP, M8) and triazole propyl aldehyde (M6). Six additional metabolites were formed by further metabolism of M1, M2, M6 and M8. 3. Kinetic studies revealed that all metabolic pathways were monophasic, and the pathway leading to the formation of OH-ethyl-Et was the most efficient at eliminating the drug. On incubation with microsomes expressing individual recombinant CYPs, formation rates of M1-3 and M8 were 10-100-fold greater for CYP3A4 than that for other CYP forms. The formation of these metabolites was markedly inhibited by the CYP3A4-specific inhibitor ketoconazole, whereas other CYP-specific inhibitors did not show significant effects. In addition, the production of M1-3 and M8 was strongly correlated with CYP3A4-mediated testosterone 6 β -hydroxylase activities in 13 different human liver microsome samples. 4. Dealkylation of the major metabolite M1 to form mCPP (M8) was also investigated using microsomes containing recombinant CYP enzymes. The rate of conversion of M1 to mCPP by CYP3A4 was 503.0 ± 3.1 pmole nmole^?1 min^?1. Metabolism of M1 to M8 by other CYP enzymes was insignificant. In addition, this metabolism in human liver microsomes was extensively inhibited by the CYP3A4 inhibitor ketoconazole, but not by other CYP-specific inhibitors. In addition, conversion of M1 to M8 was highly correlated with CYP3A4-mediated testosterone 6 β -hydroxylase activity. 5. The results strongly suggest that CYP3A4 is the predominant enzyme-metabolizing Et in humans.     

In vitro identification of metabolic pathways and cytochrome P450 enzymes involved in the metabolism of etoperidone

1. In vitro studies have been carried out to investigate the metabolic pathways and identify the hepatic cytochrome P450 (CYP) enzymes involved in etoperidone (Et) metabolism. 2. Ten in vitro metabolites were profiled, quantified and tentatively identified after incubation with human hepatic S9 fractions. Et was metabolized via three metabolic pathways: (A) alkyl hydroxylation to form OH-ethyl-Et (M1); (B) phenyl hydroxylation to form OH-phenyl-Et (M2); and (C) N-dealkylation to form 1-m-chlorophenylpiperazine (mCPP, M8) and triazole propyl aldehyde (M6). Six additional metabolites were formed by further metabolism of M1, M2, M6 and M8. 3. Kinetic studies revealed that all metabolic pathways were monophasic, and the pathway leading to the formation of OH-ethyl-Et was the most efficient at eliminating the drug. On incubation with microsomes expressing individual recombinant CYPs, formation rates of M1-3 and M8 were 10-100-fold greater for CYP3A4 than that for other CYP forms. The formation of these metabolites was markedly inhibited by the CYP3A4-specific inhibitor ketoconazole, whereas other CYP-specific inhibitors did not show significant effects. In addition, the production of M1-3 and M8 was strongly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activities in 13 different human liver microsome samples. 4. Dealkylation of the major metabolite M1 to form mCPP (M8) was also investigated using microsomes containing recombinant CYP enzymes. The rate of conversion of M1 to mCPP by CYP3A4 was 503.0 +/- 3.1 pmole nmole(-1) min(-1). Metabolism of M1 to M8 by other CYP enzymes was insignificant. In addition, this metabolism in human liver microsomes was extensively inhibited by the CYP3A4 inhibitor ketoconazole, but not by other CYP-specific inhibitors. In addition, conversion of M1 to M8 was highly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activity. 5. The results strongly suggest that CYP3A4 is the predominant enzyme-metabolizing Et in humans.     

In vitro liver metabolism of aclidinium bromide in preclinical animal species and humans: identification of the human enzymes involved in its oxidative metabolism

The metabolism of aclidinium bromide, a novel long-acting antimuscarinic drug for the maintenance treatment of chronic obstructive pulmonary disorder, has been investigated in liver microsomes and hepatocytes of mice, rats, rabbits, dogs, and humans. Due to the rapid hydrolysis of this ester compound, two distinct radiolabeled forms of aclidinium were studied. The main biotransformation route of aclidinium was the hydrolytic cleavage of the ester moiety, resulting in the formation of the alcohol metabolite (M2, LAS34823) and carboxylic acid metabolite (m3, LAS34850), which mainly occurred non-enzymatically. By comparison, the oxidative metabolism was substantially lower and the metabolite profiles were similar across all five species examined. Aclidinium was metabolized oxidatively to four minor primary metabolites that were identified as monohydroxylated derivatives of aclidinium at the phenyl (M4) and glycolyl (m6 and m7) moieties of the molecule. The NADPH-dependent metabolite m4 involved the loss of one of the thiophene rings of aclidinium. Incubations with human recombinant P450 isoforms and inhibition studies with selective chemical inhibitors and antibodies of human P450 enzymes demonstrated that the oxidative metabolism of aclidinium is primarily mediated by CYP3A4 and CYP2D6. Additionally, up to eight secondary metabolites were also characterized, involving further hydrolysis, oxidation, or glucuronidation of the primary metabolites. Also, the liver oxidative metabolism of the alcohol metabolite (LAS34823) resulted in the production of one hydroxylated metabolite (M1) mediated by human CYP2D6, whereas the acid metabolite (LAS34850) was not metabolized enzymatically, although a minor non-enzymatic and NADPH-dependent reduction was observed.     

Identification of the cytochrome P450 enzymes involved in the metabolism of cisapride: in vitro studies of potential co-medication interactions

Cisapride is a prokinetic drug that is widely used to facilitate gastrointestinal tract motility. Structurally, cisapride is a substituted piperidinyl benzamide that interacts with 5-hydroxytryptamine-4 receptors and which is largely without central depressant or antidopaminergic side-effects. The aims of this study were to investigate the metabolism of cisapride in human liver microsomes and to determine which cytochrome P-450 (CYP) isoenzyme(s) are involved in cisapride biotransformation. Additionally, the effects of various drugs on the metabolism of cisapride were investigated. The major in vitro metabolite of cisapride was formed by oxidative N-dealkylation at the piperidine nitrogen, leading to the production of norcisapride. By using competitive inhibition data, correlation studies and heterologous expression systems, it was demonstrated that CYP3A4 was the major CYP involved. CYP2A6 also contributed to the metabolism of cisapride, albeit to a much lesser extent. The mean apparent K(m) against cisapride was 8.6+/-3.5 microM (n = 3). The peak plasma levels of cisapride under normal clinical practice are approximately 0.17 microM; therefore it is unlikely that cisapride would inhibit the metabolism of co-administered drugs. In this in vitro study the inhibitory effects of 44 drugs were tested for any effect on cisapride biotransformation. In conclusion, 34 of the drugs are unlikely to have a clinically relevant interaction; however, the antidepressant nefazodone, the macrolide antibiotic troleandomycin, the HIV-1 protease inhibitors ritonavir and indinavir and the calcium channel blocker mibefradil inhibited the metabolism of cisapride and these interactions are likely to be of clinical relevance. Furthermore, the antimycotics ketoconazole, miconazole, hydroxy-itraconazole, itraconazole and fluconazole, when administered orally or intravenously, would inhibit cisapride metabolism.