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.

     

甘草次酸在人细胞色素CYP450中体外代谢研究(英)

甘草根是中医临床常用解毒草药, 其活性成分甘草次酸主要是通过肝脏代谢。本文研究了人肝微粒体以及人源性CYP450s对甘草次酸的体外代谢影响, 以及甘草次酸对几种CYP450酶活性的影响。实验结果表明, 甘草次酸体外主要代谢酶为CYP3A4。体外药代动力学参数K_m, V_max和CL_int分别为18.6 μmol·L⁻¹, 4.4 nmol·mg⁻¹(protein)·min⁻¹和0.237 mL·mg⁻¹(protein)·min⁻¹。体外抑制试验显示, 50 μmol·L⁻¹甘草次酸可以抑制CYP2C19、CYP2C9、CYP3A4酶的活性, 其抑制率可高达50%以上。     

Benzydamine N-oxidation as an index reaction reflecting FMO activity in human liver microsomes and impact of FMO3 polymorphisms on enzyme activity

AIMS: The role of flavin containing monooxygenases (FMO) on the disposition of many drugs has been insufficiently explored. In vitro and in vivo tests are required to study FMO activity in humans. Benzydamine (BZD) N-oxidation was evaluated as an index reaction for FMO as was the impact of genetic polymorphisms of FMO3 on activity. METHODS: BZD was incubated with human liver microsomes (HLM) and recombinant enzymes. Human liver samples were genotyped using PCR-RFLP. RESULTS: BZD N-oxide formation rates in HLM followed Michaelis-Menten kinetics (mean Km = 64.0 microM, mean Vmax = 6.9 nmol mg-1 protein min-1; n = 35). N-benzylimidazole, a nonspecific CYP inhibitor, and various CYP isoform selective inhibitors did not affect BZD N-oxidation. In contrast, formation of BZD N-oxide was almost abolished by heat treatment of microsomes in the absence of NADPH and strongly inhibited by methimazole, a competitive FMO inhibitor. Recombinant FMO3 and FMO1 (which is not expressed in human liver), but not FMO5, showed BZD N-oxidase activity. Respective Km values for FMO3 and FMO1 were 40.4 microM and 23.6 microM, and respective Vmax values for FMO3 and FMO1 were 29.1 and 40.8 nmol mg-1 protein min-1. Human liver samples (n = 35) were analysed for six known FMO3 polymorphisms. The variants I66M, P135L and E305X were not detected. Samples homozygous for the K158 variant showed significantly reduced Vmax values (median 2.7 nmol mg-1 protein min-1) compared to the carriers of at least one wild type allele (median 6.2 nmol mg-1 protein min-1) (P < 0.05, Mann-Whitney-U-test). The V257M and E308G substitutions had no effect on enzyme activity. CONCLUSIONS: BZD N-oxidation in human liver is mainly catalysed by FMO3 and enzyme activity is affected by FMO3 genotype. BZD may be used as a model substrate for human liver FMO3 activity in vitro and may be further developed as an in vivo probe reflecting FMO3 activity.     

Identification of the human cytochrome P450 isoforms mediating in vitro N-dealkylation of perphenazine

AIMS: To identify the human cytochrome P450 (CYP) isoforms mediating the N-dealkylation of the antipsychotic drug perphenazine in vitro and estimate the relative contributions of the CYP isoforms involved. METHODS: cDNA-expressed CYP isoforms were used to identify the isoforms that are able to mediate the N-dealkylation of perphenazine, which is considered a major metabolic pathway for the drug. Using human liver microsomal preparations (HLM), inhibition studies were carried out to establish the relative contributions of the CYP isoforms involved in the N-dealkylation reaction. RESULTS: CYP isoforms 1A2, 3A4, 2C8, 2C9, 2C18, 2C19 and 2D6 were able to mediate the N-dealkylation of perphenazine. Reaction velocities and their relative abundance in HLM suggested that CYP1A2, 3A4, 2C19 and 2D6 were the most important contributors to N-dealkylation. Apparent Km values of CYP1A2 and CYP2D6 were in the range 1-2 microM, and Km values of CYP2C19 and CYP3A4 were 14 microM and 7.9 microM, respectively. Ketoconazole inhibition of N-dealkylation mediated by a mixed HLM indicated that CYP3A4 accounted for about 40% of perphenazine N-dealkylation at therapeutically relevant concentrations.The contribution of the CYP isoforms 1A2, 2C19 and 2D6 amounted to 20-25% each as measured by the percentage inhibition obtained by addition of furafylline, fluvoxamine or quinidine, respectively. HLM-mediated N-dealkylation of perphenazine accounted for 57% of the total amount of substrate consumed during incubation. CONCLUSIONS: The present in vitro study suggests that CYP isoforms 1A2, 3A4, 2C19 and 2CD6 are primarily involved in the N-dealkylation of perphenazine. The relatively modest role of CYP2D6 is at variance with in vivo studies, which indicate a greater contribution of this isoform. Alternative metabolic pathways, corresponding to 43% of the HLM-mediated metabolism of the drug, may depend more strongly on CYP2D6.     

肝细胞微粒体的制备和细胞色素P450氧化酶活性测定

目的：为测定人肝细胞微粒体细胞色素Ｐ４５０氧化酶的活性。方法：用差速离心法制备３例人肝细胞微粒体。结果：细胞色素Ｐ４５０的含量为０．５２３±０．００５ｎｍｏｌ·ｍｇ－１；细胞色素ｂ５为０．２８５±０．０２５ｎｍｏｌ·ｍｇ－１；氨基比林Ｎ－脱甲基酶的活力为０．５±０．６ｎｍｏｌ·ｍｇ－１；乙基吗啡Ｎ－脱甲基酶活力为０．９８±０．０８ｎｍｏｌ·ｍｇ－１。结论：Ｐ４５０酶活性影响因素较多，个体差异大。临床用药时应考虑患者的个体情况。     

豆腐果苷对人肝微粒体CYP450酶体外抑制作用研究

目的:通过评价豆腐果苷在体外对人肝微粒体CYP450酶的7种亚型酶活性的影响,预测服用豆腐果苷可能出现的食物-药物及药物-药物代谢的影响。方法:将豆腐果苷与CYP450酶7种亚型的特异性探针底物咖啡因(CYP1A2)、右美沙芬(CYP2D6)、甲苯磺丁脲(CYP2C9)、S-美芬妥因(CYP2C19)、氯唑沙宗(CYP2E1)、香豆素(CYP2A6)及咪达唑仑(CYP3A4)与人肝微粒体进行孵育反应,采用HPLC和LC-MS/MS法测定对应的7种代谢产物(1,7-二甲基黄嘌呤、去甲右美沙芬、4-羟基甲苯磺丁脲、4-羟基美芬妥因、6-羟基氯唑沙宗、7-羟基香豆素和1-羟基咪达唑仑)的浓度,与对照组比较,确定豆腐果苷对以上7种亚酶活性的影响。结果:豆腐果苷在1～100μmol.L-1时对7种酶的抑制作用均无明显统计学意义(P>0.05)。结论:豆腐果苷可能不会引起有临床意义的CYP450酶抑制现象的发生。     

Assay of caffeine metabolism in vitro by human liver microsomes using radio-high-performance liquid chromatography

The low turnover of caffeine in vitro by human liver microsomes makes the study of the metabolic pathways of this compound difficult. Analytical methods with high sensitivity and specificity are needed for the detection of its metabolic products. A method based on the on-line radiometric determination of [8C-³H]caffeine and its principal metabolite (paraxanthine) in man has been developed using reversed-phase high-performance liquid chromatography. The method has been successfully employed in preliminary studies of the kinetics of this reaction.     

Drug metabolism by flavin-containing monooxygenases of human and mouse

Introduction: Flavin-containing monooxygenases (FMOs) play an important role in drug metabolism.

Areas covered: We focus on the role of FMOs in the metabolism of drugs in human and mouse. We describe FMO genes and proteins of human and mouse; the catalytic mechanism of FMOs and their significance for drug metabolism; differences between FMOs and CYPs; factors contributing to potential underestimation of the contribution of FMOs to drug metabolism; the developmental and tissue-specific expression of FMO genes and differences between human and mouse; and factors that induce or inhibit FMOs. We discuss the contribution of FMOs of human and mouse to the metabolism of drugs and how genetic variation of FMOs affects drug metabolism. Finally, we discuss the utility of animal models for FMO-mediated drug metabolism in humans.

Expert opinion: The contribution of FMOs to drug metabolism may be underestimated. As FMOs are not readily induced or inhibited and their reactions are generally detoxifications, the design of drugs that are metabolized predominantly by FMOs offers clinical advantages. Fmo1^(-/-),Fmo2^(-/-),Fmo4^(-/-) mice provide a good animal model for FMO-mediated drug metabolism in humans. Identification of roles for FMO1 and FMO5 in endogenous metabolism has implications for drug therapy and initiates an exciting area of research.     

Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes

Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results for 96 samples were obtained with the plate reader in less than 10 min as opposed to 15-35 min/sample required by conventional HPLC. The fluorescent substrates used to measure CYP activities were as follows: 3-cyano-7-ethoxycoumarin (CEC) for CYP1A1, CYP1A2, CYP2C9 and CYP2C19; 7-ethoxyresorufin (7-ER) for CYP1A1, CYP1A2 and CYP1B1; 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6; dibenzylfluorescein (DBF) for CYP3A4, CYP3A5 and CYP2C8; 7-methoxy-4-trifluoromethylcoumarin (7-MFC) for CYP2E1, CYP2B6 and CYP2C18; and coumarin for CYP2A6. The chemical inhibition and correlation data indicated that the following substrates can be used as specific functional probes for individual cytochrome P450 present in human liver microsomes: coumarin for CYP2A6 (r=0.82), AMMC for CYP2D6 (r=0.83) and DBF for CYP3A4 (r=0.92). The fluorescent plate reader was found to be useful for the rapid assessment of CYP activities (positive control) in both intact cells and subcellular fractions.     

Characterization of human cytochrome P450 isoforms involved in the metabolism of 7-epi-paclitaxel

1. The C-7 chiral centre in paclitaxel is subject to epimerization under physiological conditions, thus making 7-epi-paclitaxel as the principal degradant. This study was designed to characterize the cytochrome P450 (CYP) enzymes involved in 7-epi-paclitaxel metabolism, and to examine possible metabolic interactions that this C-7 epimer may have with paclitaxel.

2. In human liver microsomes, 7-epi-paclitaxel was oxidized to two monohydroxylated metabolites while the metabolic sites occurred at the C-13 side-chain for M-1 and taxane core ring for M-2. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that M-1 was generated predominantly by CYP3A4 and M-2 by CYP2C8. Co-incubation of 7-epi-paclitaxel with paclitaxel in human liver microsomes resulted in potent inhibition of 6α-hydroxypaclitaxel formation (IC₅₀?=?2.1?±?0.2 μM), thus decreasing the metabolic elimination of paclitaxel.

3. In conclusion, both CYP3A4 and CYP2C8 play a major role in biotransformation of 7-epi-paclitaxel in human liver microsomes. The existence of epimeric interactions between paclitaxel and its degradant might be a noteworthy factor resulting in the complex pharmacokinetic profile of paclitaxel.

     

Regioselective oxidation of phospho-NSAIDs by human cytochrome P450 and flavin monooxygenase isoforms: implications for their pharmacokinetic properties and safety

BACKGROUND AND PURPOSE

Phospho-ibuprofen (MDC-917) and phospho-sulindac (OXT-328) are highly effective in cancer and arthritis treatment in preclinical models. Here, we investigated their metabolism by major human cytochrome P450s (CYPs) and flavin monooxygenases (FMOs).

EXPERIMENTAL APPROACH

The CYP/FMO-catalysed metabolism of phospho-ibuprofen and phospho-sulindac was studied by using in silico prediction modelling and a direct experimental approach.

KEY RESULTS

The CYP isoforms catalyse the oxidation of non-steroidal anti-inflammatory drugs (NSAIDs) and phospho-NSAIDs, with distinct activity and regioselectivity. CYP1A2, 2C19, 2D6 and 3A4 oxidize phospho-ibuprofen, but not ibuprofen; whereas CYP2C9 oxidizes ibuprofen, but not phospho-ibuprofen. All CYPs tested oxidize phospho-sulindac, but not sulindac. Among the five CYPs evaluated, CYP3A4 and 2D6 are the most active in the oxidation of phospho-ibuprofen and phospho-sulindac respectively. FMOs oxidized phospho-sulindac and sulindac, but not phospho-ibuprofen or ibuprofen. FMOs were more active towards phospho-sulindac than sulindac, indicating that phospho-sulindac is a preferred substrate of FMOs. The susceptibility of phospho-NSAIDs to CYP/FMO-mediated metabolism was also reflected in their rapid oxidation by human and mouse liver microsomes, which contain a full complement of CYPs and FMOs. Compared with conventional NSAIDs, the higher activity of CYPs towards phospho-ibuprofen and phospho-sulindac may be due to their greater lipophilicity, a key parameter for CYP binding.

CONCLUSIONS AND IMPLICATIONS

CYPs and FMOs play an important role in the metabolism of phospho-NSAIDs, resulting in differential pharmacokinetic profiles between phospho-NSAIDs and NSAIDs in vivo. The consequently more rapid detoxification of phospho-NSAIDs is likely to contribute to their greater safety.     

In vitro metabolism of an estrogen‐related receptor γ modulator,GSK5182, by human liver microsomes and recombinant cytochrome P450s

GSK5182 (4‐[(Z)‐1‐[4‐(2‐dimethylaminoethyloxy)phenyl]‐hydroxy‐2‐phenylpent‐1‐enyl]phenol) is a specific inverse agonist for estrogen‐related receptor γ, a member of the orphan nuclear receptor family that has important functions in development and homeostasis. This study was performed to elucidate the metabolites of GSK5182 and to characterize the enzymes involved in its metabolism. Incubation of human liver microsomes with GSK5182 in the presence of NADPH resulted in the formation of three metabolites, M1, M2 and M3. M1 and M3 were identified as N‐desmethyl‐GSK5182 and GSK5182 N‐oxide, respectively, on the basis of liquid chromatography‐tandem mass spectrometric (LC‐MS/MS) analysis. M2 was suggested to be hydroxy‐GSK5182 through interpretation of its MS/MS fragmentation pattern. In addition, the specific cytochrome P450 (P450) and flavin‐containing monooxygenase (FMO) isoforms responsible for GSK5182 oxidation to the three metabolites were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 and FMO isoforms. GSK5182 N‐demethylation and hydroxylation is mainly mediated by CYP3A4, whereas FMO1 and FMO3 contribute to the formation of GSK5182 N‐oxide from GSK5182. The present data will be useful for understanding the pharmacokinetics and drug interactions of GSK5182 in vivo. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of diethyldithiocarbamate (DDC) and ticlopidine on CYP1A2 activity and caffeine metabolism: an in vitro comparative study with human cDNA-expressed CYP1A2 and liver microsomes

The aim of the present study was to test the effect of diethyldithiocarbamate (DDC), which is regarded as a cytochrome P450 (CYP) CYP2A6 and CYP2E1 inhibitor, and ticlopidine, an efficient CYP2B6, CYP2C19 and CYP2D6 inhibitor, on the activity of human CYP1A2 and the metabolism of caffeine (1-N-, 3-N- and 7-N-demethylation, and C-8-hydroxylation). The experiment was carried out in vitro using human cDNA-expressed CYP1A2 (Supersomes) and human pooled liver microsomes. The effects of DDC and ticlopidine were compared to those of furafylline (a strong CYP1A2 inhibitor). A comparative in vitro study provides clear evidence that ticlopidine and DDC, applied at concentrations that inhibit the above-mentioned CYP isoforms, potently (as compared to furafylline) inhibit human CYP1A2 and caffeine metabolism, in particular 1-N- and 3-N-demethylation.     

Evaluation of xenobiotic N- and S-oxidation by variant flavin-containing monooxygenase 1 (FMO1) enzymes.

The flavin-containing monooxygenase gene family (FMO1-6) in humans encodes five functional isoforms that catalyze the monooxygenation of numerous N-, P- and S-containing drugs and toxicants. A previous single nucleotide polymorphism (SNP) analysis of FMO1 in African-Americans identified seven novel SNPs. To determine the functional relevance of the coding FMO1 variants (H97Q, I303V, I303T, R502X), they were heterologously expressed using a baculovirus system. Catalytic efficiency and stereoselectivity of N- and S-oxygenation was determined in the FMO1 variants using several substrates. The I303V variant showed catalytic constants equal to wild-type FMO1 for methimazole and methyl p-tolyl sulfide. Catalytic efficiency (V(max)/K(m)) of methyl p-tolyl sulfide oxidation by R502X was unaltered. In contrast, methimazole oxidation by R502X was not detected. Both H97Q and I303T had elevated catalytic efficiency with regards to methyl p-tolyl sulfide (162% and 212%, respectively), but slightly reduced efficiency with regards to methimazole (81% and 78%). All the variants demonstrated the same stereoselectivity for methyl p-tolyl sulfide oxidation as wild-type FMO1. FMO1 also metabolized the commonly used insecticide fenthion to its (+)-sulfoxide, with relatively high catalytic efficiency. FMO3 metabolized fenthion to its sulfoxide at a lower catalytic efficiency than FMO1 (27%) and with less stereoselectivity (74% (+)-sulfoxide). Racemic fenthion sulfoxide was a weaker inhibitor of acetylcholinesterase than its parent compound (IC(50) 0.26 and 0.015 mM, respectively). The (+)- and (-)-sulfoxides were equally potent inhibitors of acetylcholinesterase. These data indicate that all the currently known FMO1 variants are catalytically active, but alterations in kinetic parameters were observed.     

Metabolism and effect of para-toluene-sulfonamide on rat liver microsomal cytochrome P450 from in vivo and in vitro studies

AIM: To study the in vivo and in vitro metabolism and the effect of para-toluene-sulfonamide (PTS) on cytochrome P450 enzymes (CYP450). METHODS: Total CYP450 and microsome protein content were determined after iv pretreatment of rats with PTS. CYP-specific substrates were incubated with rat liver microsomes. Specific CYP isoform activities were determined by using HPLC. CYP chemical inhibitors added to the incubation mixture were used to investigate the principal CYP isoforms involved in PTS metabolism. The effect of PTS on CYP isoforms was investigated by incubating PTS with specific substrates. RESULTS: The groups treated with 33 and 99 mg/kg per d PTS, respectively, had a total CYP content of 0.66+/-0.17 and 0.60+/-0.12 nmol/mg. The K(m) and V(max) were 92.2 micromol/L and 0.0137 nmol/min per mg protein. CYP2C7, CYP2D1 and CYP3A2 might contribute to PTS metabolism in the rat liver. The inhibitory effects of sulfaphenazole and ketoconazole on PTS metabolism were shown to have a mixed mechanism, whereas PTS metabolism was inhibited noncompetitively by quinidine. PTS had little effect on the activities of the selected CYP isoforms. CONCLUSION: Generally speaking, it is relatively safe for PTS to be co-administered with other drugs. However, care should be taken when administering PTS with CYP inhibitors and the substrates of CYP2C, CYP2D and CYP3A.     

In vitro identification of human cytochrome P450 isoforms involved in the metabolism of Geissoschizine methyl ether,an active component of the traditional Japanese medicine Yokukansan

1.?Yokukansan (YKS) is a traditional Japanese medicine also called kampo, which has been used to treat neurosis, insomnia, and night crying and peevishness in children. Geissoschizine methyl ether (GM), a major indole alkaloid found in Uncaria hook, has been identified as a major active component of YKS with psychotropic effects. Recently, GM was reported to have a partial agonistic effect on serotonin 5-HT_1A receptors. However, there is little published information on GM metabolism in humans, although several studies reported the blood kinetics of GM in rats and humans. In this study, we investigated the GM metabolic pathways and metabolizing enzymes in humans.

2.?Using recombinant human cytochrome P450 (CYP) isoforms and polyclonal antibodies to CYP isoforms, we found that GM was metabolized into hydroxylated, dehydrogenated, hydroxylated+dehydrogenated, demethylated and water adduct forms by some CYP isoforms.

3.?The relative activity factors in human liver microsomes were calculated to determine the relative contributions of individual CYP isoforms to GM metabolism in human liver microsomes (HLMs). We identified CYP3A4 as the CYP isoform primarily responsible for GM metabolism in human liver microsomes.

4.?These findings provide an important basis for understanding the pharmacokinetics and pharmacodynamics of GM and YKS.     

Effect of single‐walled carbon nanotubes on cytochrome P450 activity in human liver microsomes in vitro

Single‐walled carbon nanotubes (SWCNTs) are made from a rolled single sheet of graphene with a diameter in the nanometer range. SWCNTs are potential carriers for drug delivery systems because antibodies or drugs can be loaded on their surface; however, their effect on the activities of cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the effect of two kinds of SWCNTs with different lengths (FH‐P‐ and SO‐SWCNTs) on human CYP activity. In addition, other nano‐sized carbon materials, such as carbon black, fullerene‐C₆₀, and fullerene‐C₇₀ were also evaluated to compare their effects on CYP activities. Ten CYP substrates (phenacetin, coumarin, bupropion, paclitaxel, tolbutamide, S‐mephenytoin, dextromethorphan, chlorzoxazone, midazolam, and testosterone) were used. Testosterone 6β‐hydroxylation and midazolam 1′‐hydroxylation, which are catalysed by both CYP3A4 and CYP3A5 in liver microsomes, were decreased by 25% and 45%, respectively, in the presence of 0.1 mg/ml SO‐SWCNT. Dextromethorphan O‐demethylation, which is catalysed mainly by CYP2D6, was decreased by 40% in the presence of SO‐SWCNT. Other CYP activities, however, were not attenuated by SO‐SWCNT. FH‐P‐SWCNT, carbon black, fullerene‐C₆₀, and fullerene‐C₇₀ at 0.1 mg/ml had no effect on CYP activities. The K_i values for testosterone 6β‐hydroxylation, midazolam 1′‐hydroxylation, and dextromethorphan O‐demethylation in liver microsomes were 136, 34, and 56 μg/ml, respectively. SO‐SWCNT was determined to be a competitive inhibitor of CYP3A4, CYP3A5, and CYP2D6. These results suggest that the effect of SO‐SWCNT differs among CYP isoforms, and that the inhibition potency depends on the physicochemical properties of the nanocarbons.     

In vitro metabolism of mirtazapine enantiomers by human cytochrome P450 enzymes

The metabolism of mirtazapine enantiomers was investigated in vitro using human lymphoblast microsomes transfected with human cDNA to overexpress either CYP1A2, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 and assayed for mirtazapine enantiomers using a validated chiral method of high-performance liquid chromatography. (+)-Mirtazapine was extensively metabolised by CYP2D6 (K(m) = 9.3 +/- 3.3 &mgr;mol/l, V(max) = 40.9 +/- 7.9 &mgr;mol/h/mg, intrinsic clearance = 4.41 l/h/mg). CYP1A2 and CYP3A4 showed low metabolic activity towards (+)-mirtazapine and (-)-mirtazapine respectively. Neither CYP2C9 nor CYP2C19 appeared to be involved in the metabolism of the enantiomers of mirtazapine. Copyright 2001 John Wiley & Sons, Ltd.     

Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes

AIM

The most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin.

METHODS

The metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4′-hydroxylation (S)-mephenytoin 4′-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1′-hydroxylation/testosterone 6β-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences.

RESULTS

The samples of the two ethnic groups were not significantly different in cytochrome-b₅ concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A.

CONCLUSIONS

These data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins.     

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.