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 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 isoforms involved in the metabolism of loperamide in human liver microsomes

Objective: The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Methods: Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. Results: The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent K_m and V_max values were 21.1 M and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 M and 412.0 pmol/min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (r_s=0.829; P<0.01), CYP2B6-catalzyed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (r_s=0.691; P<0.05), and CYP2C8-catalyzed paclitaxel 6-hydroxylation (r_s=0.797; P<0.05). Conclusion: CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.     

Characterization of the metabolism of fenretinide by human liver microsomes, cytochrome P450 enzymes and UDP-glucuronosyltransferases

BACKGROUND AND PURPOSE

Fenretinide (4-HPR) is a retinoic acid analogue, currently used in clinical trials in oncology. Metabolism of 4-HPR is of particular interest due to production of the active metabolite 4′-oxo 4-HPR and the clinical challenge of obtaining consistent 4-HPR plasma concentrations in patients. Here, we assessed the enzymes involved in various 4-HPR metabolic pathways.

EXPERIMENTAL APPROACH

Enzymes involved in 4-HPR metabolism were characterized using human liver microsomes (HLM), supersomes over-expressing individual human cytochrome P450s (CYPs), uridine 5′-diphospho-glucoronosyl transferases (UGTs) and CYP2C8 variants expressed in Escherichia coli. Samples were analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry assays and kinetic parameters for metabolite formation determined. Incubations were also carried out with inhibitors of CYPs and methylation enzymes.

KEY RESULTS

HLM were found to predominantly produce 4′-oxo 4-HPR, with an additional polar metabolite, 4′-hydroxy 4-HPR (4′-OH 4-HPR), produced by individual CYPs. CYPs 2C8, 3A4 and 3A5 were found to metabolize 4-HPR, with metabolite formation prevented by inhibitors of CYP3A4 and CYP2C8. Differences in metabolism to 4′-OH 4-HPR were observed with 2C8 variants, CYP2C8*4 exhibited a significantly lower V_max value compared with *1. Conversely, a significantly higher V_max value for CYP2C8*4 versus *1 was observed in terms of 4′-oxo formation. In terms of 4-HPR glucuronidation, UGTs 1A1, 1A3 and 1A6 produced the 4-HPR glucuronide metabolite.

CONCLUSIONS AND IMPLICATIONS

The enzymes involved in 4-HPR metabolism have been characterized. The CYP2C8 isoform was found to have a significant effect on oxidative metabolism and may be of clinical relevance.     

The in vitro metabolism of bupropion revisited: concentration dependent involvement of cytochrome P450 2C19

1. The involvement of cytochrome P450 2B6 (CYP2B6) to the in vitro and in vivo metabolism of bupropion has been well studied. In these investigations we performed a detailed in vitro phenotyping study to characterize isoforms other than CYP2B6.

2. A total of nine metabolites were identified (M1–M9) in the incubations with cDNA-expressed P450s (rhCYP) and human liver microsomes (HLM).

3. Incubations in rhCYP identified CYP2B6 as the isoform responsible for the formation of hydroxybupropion (M3). CYP2C19 was involved in bupropion metabolism primarily through alternate hydroxylation pathways (M4–M6) with higher activity at lower substrate concentrations, near 1 µM.

4. The results from HLM inhibition studies using CYP2B6 and CYP2C19 inhibitory antibodies indicated that CYP2B6 contributed to approximately 90% of M3 formation, and CYP2C19 contributed to approximately 70–90% of M4, M5, and M6 formation.

5. Studies using single donor HLM with varying degrees of CYP2B6 and CYP2C19 activities showed a good relationship between M3 formation and CYP2B6 activity and M4/M5 formation and CYP2C19 activity.

6. These results confirmed the principle role of CYP2B6 in hydroxybupropion formation, as a selective CYP2B6 probe. In addition, the new findings revealed that CYP2C19 also contributes to bupropion metabolism through alternate hydroxylation pathways.

     

Contributions of human cytochrome P450 enzymes to glyburide metabolism

Glyburide (GLB) is a widely used oral sulfonylurea for the treatment of gestational diabetes. The therapeutic use of GLB is often complicated by a substantial inter‐individual variability in the pharmacokinetics and pharmacodynamics of the drug in human populations, which might be caused by inter‐individual variations in factors such as GLB metabolism. Therefore, there has been a continued interest in identifying human cytochrome P450 (CYP) isoforms that play a major role in the metabolism of GLB. However, contrasting data are available in the present literature in this regard. The present study systematically investigated the contributions of various human CYP isoforms (CYP3A4, CYP3A5, CYP2C8, CYP2C9 and CYP2C19) to in vitro metabolism of GLB. GLB depletion and metabolite formation in human liver microsomes were most significantly inhibited by the CYP3A inhibitor ketoconazole compared with the inhibitors of other CYP isoforms. Furthermore, multiple correlation analysis between GLB depletion and individual CYP activities was performed, demonstrating a significant correlation between GLB depletion and the CYP3A probe activity in 16 individual human liver microsomal preparations, but not between GLB depletion and the CYP2C19, CYP2C8 or CYP2C9 probe activity. By using recombinant supersomes overexpressing individual human CYP isoforms, it was found that GLB could be depleted by all the enzymes tested; however, the intrinsic clearance (V_max/K_m) of CYP3A4 for GLB depletion was 4–17 times greater than that of other CYP isoforms. These results confirm that human CYP3A4 is the major enzyme involved in the in vitro metabolism of GLB. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Characterization of the cytochrome P450 isoenzymes involved in the in vitroN-dealkylation of haloperidol

Aims The present study was carried out to identify the cytochrome P450 isoenzyme(s) involved in the N-dealkylation of haloperidol (HAL). Methods In vitro studies were performed using human liver microsomes and c-DNA-expressed human P450 isoforms. N-dealkylation of HAL was assessed by measuring the formation of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP). Results There was a tenfold variation in the extent of CPHP formation amongst the nine human liver microsomal preparations. The CPHP formation rates as a function of substrate concentration, measured in three livers, followed monophasic enzyme kinetics. K_m and V_max values ranged respectively from 50 to 78 μm and from 180 to 412 pmol mg⁻¹ min⁻¹. 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 activity of dextromethorphan O-demethylase (CYP2D6), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Ketoconazole, an inhibitor of CYP3A4, inhibited competitively CPHP formation (K_i=0.1 μm ), whereas sulphaphenazole (CYP2C9), furafylline (CYP1A2) and quinidine (CYP2D6) gave only little inhibition (IC₅₀>100 μm ). CPHP formation was, moreover, enhanced by α-naphtoflavone, an effect common to CYP3A4 mediated reactions. Anti-CYP3A4 antibodies strongly inhibited CPHP formation, whereas no inhibition was observed in the presence of CYP2D6 antibodies. Among the recombinant human CYP isoforms tested, CYP3A4 exhibited the highest activity with respect to CPHP formation rate, with no detectable effect of other CYP isoforms (CYP1A2, CYP2D6 and CYP2C9). HAL inhibited dextromethorphan O-demethylase (CYP2D6) with IC₅₀ values between 2.7 and 8.5 μm, but not (IC₅₀>100 μm ) dextromethorphan N-demethylase (CYP3A4), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Conclusions These results strongly suggest that the N-dealkylation of HAL in human liver microsomal preparations is mediated by CYP3A4.     

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.     

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.     

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。     

The role of human cytochrome P450 enzymes in metabolism of acrylamide in vitro

Abstract

Objective: Acrylamide (AA), a probable human carcinogen, is present in fried and baked starch-rich food. In vivo, the substance is partly biotransformed to glycidamide (GA), which may account for carcinogenic effects. Existing data suggest an important but not exclusive contribution of CYP2E1 to GA formation. The aim of this project was to derive respective enzyme kinetic parameters for CYP2E1 and to assess a possible role of other important human CYPs for this reaction in vitro.

Methods: AA (0.2–20?mM) was incubated with human liver microsomes (HLM) and human cytochrome P450 enzymes (supersomes?). GA was quantified by a specific LC-MS/MS method. Enzyme kinetic parameters were estimated assuming a single binding site. Furthermore, inhibition experiments were performed with diethyldithiocarbamate (DDC), a potent inhibitor of CYP2E1.

Results: The mean?±?SD maximum formation rate (V_max) and Michaelis–Menten constant (K_m) for GA formation in HLM were 199?±?36?pmol GA/mg protein/min and 3.3?±?0.5?mM, respectively. In AA incubations with supersomes?, only for CYP2E1 measurable GA formation was detected in all tested AA concentrations (V_max and K_m were 5.4?nmol GA/nmol CYP2E1/min and 1.3?mM, respectively). Inhibition constant (IC₅₀) of DDC was 3.1?±?0.5?µM for HLM and 1.2?±?0.2?µM for CYP2E1 supersomes?. Therefore, relevant participation of CYPs other than CYP2E1 in the metabolism of AA to GA in humans does not seem likely.

Conclusion: Our results confirm the major role of CYP2E1 in GA formation from AA, albeit with low affinity and low capacity. Further studies are needed to identify other pathways of GA formation.     

Dydrogesterone metabolism in human liver by aldo-keto reductases and cytochrome P450 enzymes

1.?The metabolism of dydrogesterone was investigated in human liver cytosol (HLC) and human liver microsomes (HLM). Enzymes involved in dydrogesterone metabolism were identified and their relative contributions were estimated.

2.?Dydrogesterone clearance was clearly higher in HLC compared to HLM. The major active metabolite 20α-dihydrodydrogesterone (20α-DHD) was only produced in HLC.

3.?The formation of 20α-DHD by cytosolic aldo-keto reductase 1C (AKR1C) was confirmed with isoenzyme-specific AKR inhibitors.

4.?Using recombinantly expressed human cytochrome P450 (CYP) isoenzymes, dydrogesterone was shown to be metabolically transformed by CYP3A4 and CYP2C19.

5.?A clear contribution of CYP3A4 to microsomal metabolism of dydrogesterone was demonstrated with HLM and isoenzyme-specific CYP inhibitors, and confirmed by a significant correlation between dydrogesterone clearance and CYP3A4 activity.

6.?Contribution of CYP2C19 was shown to be clearly less than CYP3A4 and restricted to a small group of human individuals with very high CYP2C19 activity. Therefore, it is expected that CYP2C19 genetic variations will not affect dydrogesterone pharmacokinetics in man.

7.?In conclusion, dydrogesterone metabolism in the liver is dominated primarily by cytosolic enzymes (particularly AKR1C) and secondarily by CYP3A4, with the former exclusively responsible for 20α-DHD formation.     

In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, is extensively metabolized to eupatilin glucuronide, 4-O-desmethyleupatilin and 4-O-desmethyleupatilin glucuronide in human liver microsomes. This study characterized the human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of eupatilin. The specific CYPs responsible for O-demethylation of eupatilin to the major metabolite, 4-O-desmethyleupatilin were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by human cDNA-expressed CYP enzymes. UGT enzymes involved in the eupatilin glucuronidation were identified using pooled human liver microsomes and human cDNA-expressed UGT enzymes. Eupatilin was predominantly metabolized by CYP1A2 and, to a lesser extent, CYP2C8 mediated O-demethylation of eupatilin to 4-O-desmethyleupatilin. Eupatilin glucuronidation was catalysed by UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10.     

CYP2C8 and CYP3A4 are the principal enzymes involved in the human in vitro biotransformation of the insulin secretagogue repaglinide

AIMS: To identify the principal human cytochrome P450 (CYP) enzyme(s) responsible for the human in vitro biotransformation of repaglinide. Previous experiments have identified CYP3A4 as being mainly responsible for the in vitro metabolism of repaglinide, but the results of clinical investigations have suggested that more than one enzyme may be involved in repaglinide biotransformation. METHODS: [14C]-Repaglinide was incubated with recombinant CYP and with human liver microsomes (HLM) from individual donors in the presence of inhibitory antibodies specific for individual CYP enzymes. Metabolites, measured by high-performance liquid chromatography (HPLC) with on-line radiochemical detection, were identified by liquid chromatography-mass spectrophotometry (LC-MS) and LC-MS coupled on-line to a nuclear magnetic resonance spectrometer (LC-MS-NMR). RESULTS: CYP3A4 and CYP2C8 were found to be responsible for the conversion of repaglinide into its two primary metabolites, M4 (resulting from hydroxylation on the piperidine ring system) and M1 (an aromatic amine). Specific inhibitory monoclonal antibodies against CYP3A4 and CYP2C8 significantly inhibited (> 71%) formation of M4 and M1 in HLM. In a panel of HLM from 12 individual donors formation of M4 and M1 varied from approximately 160-880 pmol min-1 mg-1 protein and from 100-1110 pmol min-1 mg-1 protein, respectively. The major metabolite generated by CYP2C8 was found to be M4. The rate of formation of this metabolite in HLM correlated significantly with paclitaxel 6alpha-hydroxylation (rs = 0.80; P = 0.0029). Two other minor metabolites were also detected. One of them was M1 and the other was repaglinide hydroxylated on the isopropyl moiety (M0-OH). The rate of formation of M4 in CYP2C8 Supersomes was 2.5 pmol min-1 pmol-1 CYP enzyme and only about 0.1 pmol min-1 pmol-1 CYP enzyme in CYP3A4 Supersomes. The major metabolite generated by CYP3A4 was M1. The rate of formation of this metabolite in HLM correlated significantly with testosterone 6beta-hydroxylation (rs = 0.90; P = 0.0002). Three other metabolites were identified, namely, M0-OH, M2 (a dicarboxylic acid formed by oxidative opening of the piperidine ring) and M5. The rate of M1 formation in CYP3A4 Supersomes was 1.6 pmol min-1 pmol-1 CYP enzyme but in CYP2C8 Supersomes it was only approximately 0.4 pmol min-1 pmol-1 CYP enzyme. CONCLUSIONS: The results confirm an important role for both CYP3A4 and CYP2C8 in the human in vitro biotransformation of repaglinide. This dual CYP biotransformation may have consequences for the clinical pharmacokinetics and drug-drug interactions involving repaglinide if one CYP pathway has sufficient capacity to compensate if the other is inhibited.     

In vitro metabolism of nobiletin,a polymethoxy-flavonoid,by human liver microsomes and cytochrome P450

1. Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs.

2. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4′-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4′-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL.

3. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4′-OH-NBL.

4. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

     

Characterization of cytochrome P450s mediating ipriflavone metabolism in human liver microsomes

Ipriflavone, a synthetic flavonoid for the prevention and treatment of osteoporosis, has been reported to be extensively metabolized in man to seven metabolites (M1–M7). This study was performed to characterize the human liver cytochrome P450s (CYP) responsible for the metabolism of ipriflavone. Hydroxylation at the β-ring to M3, O-dealkylation to M1 and oxidation at isopropyl group to M4 and M5 are major pathways for ipriflavone metabolism in three different human liver microsome preparations. The specific CYPs responsible for ipriflavone oxidation to the active metabolites, M1, M3, M4 and M5 were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by expressed recombinant CYP enzymes. The inhibitory potencies of ipriflavone and its five metabolites, M1–M5 on seven clinically important CYPs were investigated in human liver microsomes. Our results demonstrate that CYP3A4 plays the major role in O-dealkylation of ipriflavone to M1 and CYP1A2 plays a dominant role in the formation of M3, M4 and M5. Ipriflavone and/or its five metabolites were found to inhibit potently the metabolism of CYPs 1A2, 2C8, 2C9 and 2C19 substrates.     

Identification of human liver cytochrome P450 enzymes involved in the metabolism of SCH 351125, a CCR5 antagonist

The identification and relative contribution of human cytochrome P450 enzyme(s) involved in the metabolism of SCH 351125 were investigated. In human liver microsomes, O-deethylation was the major metabolic pathway, whereas aromatization of a piperidine ring to pyridine and the reduction of the N-oxide moiety were minor routes. Recombinant human CYP3A4 and CYP2C9 both exhibited catalytic activity with respect to the formation of rotameric O-deethylated metabolites (M12, M13), the metabolites resulting from aromatization (M22/M24) and N-oxide reduction (M31). Using the relative activity factor (RAF) approach, the relative contributions of CYP3A4 and CYP2C9 to M13 formation were estimated to be 76 and 24%, respectively. There was a high correlation (r?>?0.96) between the rate of formation of M12 and M13 and 6β-hydroxylation of testosterone catalysed by CYP3A4/5. Ketoconazole (2?µM) and CYP3A4/5-specific inhibitory monoclonal antibody inhibited the formation of M12 and M13 from human liver microsomes by approximately 60 and 71%, respectively. The results demonstrate that the in vitro metabolism of SCH 351125 is mediated primarily via CYP3A4 and that CYP2C9 plays a minor role. Clinical study designs should encompass these enzymology data to address any potential drug interactions.     

Inhibitory effects of curculigoside on human liver cytochrome P450 enzymes

1.?Curculigoside possesses numerous pharmacological activities, and however, little data available for the effects of curculigoside on the activity of human liver cytochrome P450 (CYP) enzymes.

2.?This study investigates the inhibitory effects of curculigoside on the main human liver CYP isoforms. In this study, the inhibitory effects of curculigoside on the eight human liver CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C9, 2C19, 2C8, and 3A4 were investigated in human liver microsomes.

3.?The results indicated that curculigoside could inhibit the activity of CYP1A2, CYP2C8, and CYP3A4, with IC₅₀ values of 15.26, 11.93, and 9.47?μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that curculigoside was not only a noncompetitive inhibitor of CYP1A2, but also a competitive inhibitor of CYP2C8 and CYP3A4, with Ki values of 5.43, 3.54, and 3.35?μM, respectively. In addition, curculigoside is a time-dependent inhibitor for CYP1A2, with kinact/K_I values of 0.056/6.15?μM^?1?min^?1.

4.?The in vitro studies of curculigoside with CYP isoforms suggest that curculigoside has the potential to cause pharmacokinetic drug interactions with other coadministered drugs metabolized by CYP1A2, CYP2C8, and CYP3A4. Further in vivo studies are needed in order to evaluate the significance of this interaction.