CYP3A4 mediates dextropropoxyphene N-demethylation to nordextropropoxyphene: human in vitro and in vivo studies and lack of CYP2D6 involvement

The individual cytochrome P450 isoforms in dextropropoxyphene N-demethylation to nordextropropoxyphene were determined and the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in cytochrome P4502D6 (CYP2D6) extensive (EM) and poor (PM) subjects were characterized. Microsomes from six CYP2D6 extensive metabolizers and one CYP2D6 poor metabolizer were used with isoform specific chemical and antibody inhibitors and expressed recombinant CYP enzymes. Groups of three CYP2D6 EM and PM subjects received a single 65-mg oral dose of dextropropoxyphene, and blood and urine were collected for 168 and 96 h, respectively. Nordextropropoxyphene formation in vitro was not different between the CYP2D6 extensive metabolizers (Km = 179 +/- 74 microM, Cl(int) = 0.41 +/- 0.26 ml mg(-1)h(-1)) and the PM subject (K = 225 microM, Cl(int) = 0.19 ml mg(-1) h(-1)) and was catalysed predominantly by CYP3A4. There was no apparent difference in the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in CYP2D6 EM and PM subjects. CYP3A4 is the major CYP enzyme catalysing the major metabolic pathway of dextropropoxyphene metabolism. Hence variability in the pharmacodynamic effects of dextropropoxyphene are likely due to intersubject variability in hepatic CYP3A4 expression and/or drug-drug interactions. Reported CYP2D6 phenocopying is not due to dextropropoxyphene being a CYP2D6 substrate.     

丹参注射液对人肝微粒体酶CYP2C9,CYP2C19,CYP2D6体外抑制作用的研究

目的 评价丹参注射液在体外对人肝微粒体酶CYP2C9、CYP2C19、CYP2D6活性的影响。方法 将丹参注射液与CYP450酶3种亚型(CYP2C9、CYP2C19、CYP2D6)的特异性探针底物甲苯磺丁脲、奥美拉唑、右美沙芬与大鼠肝微粒体进行孵育,采用LC-MS/MS法测定对应3种代谢产物4-羟基甲苯磺丁脲、5-羟基奥美拉唑、右啡烷的浓度,求算出IC₅₀。结果 丹参注射液对CYP2C9、CYP2C19和CYP2D6的IC₅₀值均>50 μg/mL。结论 丹参注射液对人肝微粒体酶CYP2C9、CYP2C19、CYP2D6没有抑制作用。     

Inhibitory effects and oxidation of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin via human CYP2A6 and its mouse and pig orthologous enzymes

1.?Information about the metabolism of compounds is essential in drug discovery and development, risk assessment of chemicals and further development of predictive methods.

2.?In vitro and in silico methods were applied to evaluate the metabolic and inhibitory properties of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin with human CYP2A6, mouse CYP2A5 and pig CYP2A19.

3.?6-Methylcoumarin was oxidized to fluorescent 7-hydroxy-6-methylcoumarin by CYP2A6 (K_m: 0.64–0.91?µM; V_max: 0.81–0.89?min^?1) and by CYP2A5 and CYP2A19. The reaction was almost completely inhibited at 10?µM 7-methylcoumarin in liver microsomes of human and mouse, but in pig only 40% inhibition was obtained with the anti-CYP2A5 antibody or with methoxsalen and pilocarpine. 7-Methylcoumarin was a mechanism-based inhibitor for CYP2A6, but not for the mouse and pig enzymes. 7-Formylcoumarin was a mechanism-based inhibitor for CYP2As of all species.

4.?Docking and molecular dynamics simulations of 6-methylcoumarin and 7-methylcoumarin in the active sites of CYP2A6 and CYP2A5 demonstrated a favorable orientation of the 7-position of 6-methylcoumarin towards the heme moiety. Several orientations of 7-methylcoumarin were possible in CYP2A6 and CYP2A5.

5.?These results indicate that the active site of CYP2A6 has unique interaction properties for ligands and differs in this respect from CYP2A5 and CYP2A19.     

Methadone N-demethylation in human liver microsomes: lack of stereoselectivity and involvement of CYP3A4

AIMS: To investigate the kinetics of CYP-mediated N-demethylation of methadone in human liver microsomes, and examine the role of stereoselectivity and CYP isoforms involved. METHODS: The kinetics of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) formation via N-demethylation of rac-, (R)- and (S)-methadone in human liver microsomes prepared from six liver samples were determined by h.p.l.c., and inhibition of metabolic function was studied using isoform-specific chemical inhibitors and monoclonal antibodies. Microsomes containing expressed CYP3A4, CYP2D6 and CYP2C19 were also used to examine the formation of EDDP. RESULTS: The V max, Km, and CLint values for the formation of EDDP from rac-, (R)- and (S)-methadone were in the ranges of 20-77 nmol mg-1 protein h-1, 125-252 microm, and 91-494 ml h-1 g-1 protein. Km and CLint values for (R)- and (S)-methadone were not statistically significantly different (P >0.05), while V max values for (S)-methadone were 15% (P=0.045) lower than for (R)-methadone. Expressed CYP3A4 and CYP2C19 showed similar reaction rates for both (R)- and (S)-methadone, while CYP2D6 did not catalyse this reaction. Selective chemical inhibitors of CYP3A (troleandomycin, ketoconazole) and monoclonal human CYP3A4 antibodies significantly inhibited (P<0.05) the formation of EDDP in a concentration dependent manner by up to 80%. Sulphaphenazole (CYP2C9) also significantly inhibited (P<0.05) EDDP formation (range 14-25%). There were no statistically significant differences in the inhibition observed between the three substrates. Selective inhibitors of CYP1A2 (furafylline), CYP2A6 (coumarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (quinidine) and CYP2E1 (diethyldithiocarbamic acid sodium salt and monoclonal human CYP2E1 antibodies) had no significant (P >0.05) effect. CONCLUSIONS: The N-demethylation of methadone in human liver microsomes is not markedly stereoselective, and is mediated mainly by CYP3A4 with the possible involvement of CYP2C9 and CYP2C19. Thus, the large interindividual variation reported for methadone pharmacokinetics may be due to variability in the expression of these CYP isoforms, and the reported stereoselectivity in the systemic clearance of methadone in vivo is not due to stereoselectivity in N-demethylation.     

In vitro study on the involvement of CYP1A2, CYP2D6 and CYP3A4 in the metabolism of haloperidol and reduced haloperidol

Objective: To investigate in vitro which CYP isoforms (CYP1A2, CYP2D6 and CYP3A4) are involved in the biotransformation of haloperidol (HAL) and reduced haloperidol (RHAL). Methods: The biotransformation of HAL and RHAL is evaluated by measuring HAL and RHAL remaining after incubation with human liver microsomes and with supersomes from human baculovirus-infected cells expressing human P ₄₅₀ isoforms. The influence of chemical- and immuno-inhibition of specific isoforms on the disappearance of HAL and RHAL was also studied. Results: After 60-min incubation of 2 μM and 20 μM HAL or RHAL with human liver microsomes, for HAL, 58% and 64%, respectively, remained in the incubation mixture, for RHAL, 53% and 66%, respectively. Ketoconazole had the most pronounced inhibitory effect on the biotransformation of both substrates, while for quinidine and furafylline there was only a weak or no influence. Anti-CYP3A4 antibodies inhibited strongly the biotransformation of HAL and RHAL, while the influence of anti-CYP2D6 antibodies was much less pronounced. After incubation with supersomes of recombinant CYP3A4, HAL and RHAL disappeared rapidly; disappearance was slow after incubation with CYP2D6 supersomes, and negligible with CYP1A2 supersomes. Conclusion: The results show that CYP3A4 is the most important CYP isoenzyme involved in the biotransformation of HAL and RHAL, and that the metabolism by CYP2D6 is only a minor pathway; CYP1A2 has no or only a negligible influence. Received: 12 April 1999 / Accepted in revised form: 2 August 1999     

体外中国成人肝微粒体中氯胍活化为氯胍三嗪由CYP2C19和CYP3A4介导(英文)

目的:鉴定中国成人肝微粒体中介导氯胍(PG)活化为氯胍三嗪(CG)的细胞色素P450(CYP450).方法:分析中国成人(n=6)肝微粒体中PG活化为CG的酶促动力学,各种CYP450抑制剂对该代谢的作用及其与S-美芬妥英4′-羟化的关系.结果:6个标本中,除一个外(两酶米氏模型),PG活化为CG的酶促动力学符合米氏一酶模型;CYP3A4和CYP2E1的选择性抑制剂醋竹桃霉素(81.1％)和二乙二硫基苯甲酸(47.23％)可抑制CG生成,其它抑制剂没有明显作用;在低PG浓度时,PG的环化与S-美芬妥英4′-羟化显著相关(r=0.805,P<0.05),高浓度时相关性明显减小.结论:在中国成人肝微粒体中CYP2C19和CYP3A4参与了PG活化为CG.     

Genetic Polymorphism of Drug Metabolizing Enzymes: New Mutations in CYP2D6 and CYP2A6 Genes in Japanese

Genetic polymorphism of drug metabolizing enzymes, particularly cytochrome P450 (CYP), is an important cause of adverse drug reactions. Multiple gene mutations in CYP have been shown to be phenotype. The occurrence of genetic polymorphism has been seen in genes for CYP1A1, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A5. This review discusses the molecular mechanism of two genetic polymorphisms, debrisoquine/sparteine (CYP2D6) coumarin (CYP2A6) polymorphisms. In addition, elucidation of gene mutations of CYP2D6 and CYP2A6 in Japanese will be discussed.     

丹参注射液对家兔体内CYP1A2、CYP2D6和CYP3A4活性的影响

目的：用Cocktail探针药物法研究丹参注射液对家兔细胞色素P450亚型酶活性的影响。方法：将家兔随机分成四组,耳缘静脉注射给予丹参注射液,分低、中、高剂量三个实验组,空白对照组给予生理盐水。用HPLC法测定各组Cock-tail探针药物（咖啡因、美托洛尔和氨苯砜）的血药浓度,比较药时曲线及药代动力学参数的变化,评价各组细胞色素P450亚型酶的活性。结果：丹参注射液低、中、高剂量组CYP2D6的活性与对照组相比降低,具有统计学意义（P〈0.05或P〈0.01）;CYP1A2和CYP3A4的活性与对照组相比差异均无统计学意义。结论：丹参注射液对家兔CYP2D6有抑制作用,对CYP1A2和CYP3A4的影响不显著。提示临床应用丹参注射液时,应关注其对CYP2D6的抑制作用,避免联合用药引发CYP2D6介导的药物相互作用,促进临床安全合理用药。     

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.     

The role of CYP2C and CYP3A in the disposition of 3-keto-desogestrel after administration of desogestrel

AIMS: Our objective was to study in vivo the role of CYP2C and CYP3A4 in the disposition of 3-keto-desogestrel after administration of desogestrel, by using the selective inhibitors fluconazole (CYP2C) and itraconazole (CYP3A4). METHODS: This study had a three-way crossover design and included 12 healthy females, the data from 11 of whom were analyzed. In the first (control) phase all subjects received a single 150 microg oral dose of desogestrel alone. In the second and third phases subjects received a 4 day pretreatment with either 200 mg fluconazole or 200 mg itraconazole once daily in a randomized balanced order. Desogestrel was given 1 h after the last dose of the CYP inhibitor. Plasma 3-keto-desogestrel concentrations were determined for up to 72 h post dose. RESULTS: Pretreatment with itraconazole for 4 days significantly increased the area under the plasma concentration-time curve (AUC) of 3-keto-desogestrel by 72.4% (95% confidence interval on the difference 12%, 133%; P = 0.024) compared with the control phase, whereas fluconazole pretreatment had no significant effect (95% CI on the difference -42%, 34%). Neither enzyme inhibitor affected significantly the maximum concentration (95% CI on the difference 14%, 124% for itraconazole and -23%, 40% for fluconazole) or elimination half-life (95% CI on the difference -42%, 120% for itraconazole and -24%, 61% for fluconazole) of 3-keto-desogestrel. CONCLUSIONS: According to the present study, the biotransformation of desogestrel to 3-keto-desogestrel did not appear to be mediated by CYP2C9 and CYP2C19 as suggested earlier. However, the further metabolism of 3-keto-desogestrel seems to be catalyzed by CYP3A4.     

中国汉族和回族药物代谢酶细胞色素P450(CYP)3A4、CYP2C9、CYP2C19及CYP2D6基因多态性分析

目的对中国汉族、回族健康人群细胞色素P450(CYP)3A4、CYP2C9、CYP2C19及CYP2D6进行基因多态性分析,比较汉族和回族健康人群基因表型和基因频率分布。方法多聚酶链反应-限制性片段长度多态性(PCR-RFLP)法,对300名志愿者的几种基因进行分型。结果汉族、回族CYP3A4*5等位基因频率均为0,CYP3A4*18等位基因频率分别为0.18,0.19;汉族、回族CYP2C9*2等位基因频率分别为0.01,0.05;CYP2C9*13等位基因频率均为0;汉族、回族CYP2C19*2等位基因频率分别为0.39,0.50;CYP2C19*3等位基因频率分别为0.05,0.05;汉族、回族CYP2D6*10等位基因频率分别为0.57,0.39。结论汉族、回族健康人群的CYP3A4*18、CYP2C9*2、CYP2C19*2、CYP2C19*3均没有显著性差异;在汉族、回族健康人群中未发现CYP3A4*5和CYP2C9*13突变;汉族、回族CYP2D6*10等位基因频率有显著性差异(P<0.01);回族人群CYP2D6中速代谢型(*10/*10)频率为13.4%,明显低于汉族的33.1%(P<0.01)。     

Stereoselective hydroxylation by CYP2C19 and oxidation by ADH4 in the in vitro metabolism of tivantinib

1.?In prior studies, it has been shown that tivantinib is extensively metabolized in humans to many oxidative metabolites and glucuronides. In order to identify the responsible enzymes, we investigated the in vitro metabolism of tivantinib and its four major circulating metabolites.

2.?The primary isoforms involved in the elimination of tivantinib were CYP2C19 and CYP3A4/5. CYP2C19 showed catalytic activity for the formation of M5 (hydroxylated metabolite), but not for M4 (a stereoisomer of M5), whereas CYP3A4/5 catalyzed the formation of both metabolites. For the elimination of M4, M5 and M8 (keto-metabolite), CYP3A4/5 was the major cytochrome P450 isoform and UGT1A9 was mainly involved in the glucuronidation of M4 and M5.

3.?ADH4 was identified as one of the major alcohol dehydrogenase isoforms contributing to the formation of M6 (sequential keto-metabolite of M4 and M5) and M8. The substrate preference of ADH for M4, and not M5, was observed in the formation of M6.

4.?In conclusion, CYP2C19, CYP3A4/5, UGT1A9 and ADH4 were the primary drug metabolizing enzymes involved in the in vitro metabolism of tivantinib and its metabolites. The stereoselective hydroxylation by CYP2C19 and substrate stereoselectivity of ADH4-catalyzed oxidation in the in vitro metabolism of tivantinib was discovered.     

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.     

Effects of notoginsenoside R1 on CYP1A2, CYP2C11, CYP2D1, and CYP3A1/2 activities in rats by cocktail probe drugs

Context: Notoginsenoside R1 (NGR1) is the main component with cardiovascular activity in Panax notoginseng (Burk.) F. H. Chen, an herbal medicine that is widely used to enhance blood circulation and dissipate blood stasis.

Objective: The objective of this study is to investigate NGR1's effects on CYP1A2, CYP2C11, CYP2D1, and CYP3A1/2 activities in rats in vivo through the use of the Cytochrome P450 (CYP450) probe drugs.

Materials and methods: After pretreatment with NGR1 or physiological saline, the rats were administered intraperitoneally with a mixture solution of cocktail probe drugs containing caffeine (10?mg/kg), tolbutamide (15?mg/kg), metoprolol (20?mg/kg), and dapsone (10?mg/kg). The bloods were then collected at a set of time-points for the ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) analysis.

Results: NGR1 was shown to exhibit an inhibitory effect on CYP1A2 by increased caffeine C_max (43.13%, p?<?0.01) and AUC_0???∞ (40.57%, p?<?0.01), and decreased CL/F (62.16%, p?<?0.01) in the NGR1-treated group compared with those of the control group, but no significant changes in pharmacokinetic parameters of tolbutamide, metoprolol, and dapsone were observed between the two groups, indicating that NGR1 had no effects on rat CYP2C11, CYP2D1, and CYP3A1/2.

Discussion and conclusion: When NGR1 is co-administered with drugs that are metabolized by CYP1A2, the pertinent potential herb–drug interactions should be monitored.     

Growth retardation of fetal rats exposed to nicotine in utero: possible involvement of CYP1A1, CYP2E1, and P-glycoprotein

To elucidate the possible metabolic mechanism of intrauterine growth retardation induced by nicotine, this study determines the effects of prenatal nicotine exposure on fetal development and cytochrome P4501A1 (CYP1A1), CYP2E1, and P-glycoprotein (Pgp) expression in maternal liver and placenta. Pregnant rats were given 1.0 mg/kg nicotine subcutaneously twice a day from gestational day (GD) 8 to GD 15, 18, or 21. In nicotine-treated groups, fetal developmental parameters including body weight were significantly lower. The activities of CYP1A1 and CYP2E1 in maternal liver microsomes in nicotine-treated groups increased significantly with progressing gestation when compared with the corresponding control, but returned to the level similar to the control in late pregnancy. Nicotine-treated groups induced pathological changes and increased malondialdehyde (MDA) content in the placenta when compared with the control. The gene expressions of CYP1A1 and CYP2E1 in the placenta increased significantly in nicotine-treated groups on GD 15 and GD 18, but returned to the level similar to the corresponding control on GD 21. In nicotine group, there was a decrease of mdr1a expression on GD 15, GD 18, and GD 21, with the most significant decrease on GD 15. In contrast, no significant difference was found in mdr1b mRNA expression between the nicotine-treated animals and the corresponding control. In comparison with the corresponding control, the placental Pgp protein significantly decreased on GD 15 and GD 18. Our results showed that prenatal nicotine exposure resulted in inhibition of fetal growth significantly. The induction of CYP2E1 and CYP1A1 gene expression by nicotine in the maternal liver and placenta may be involved with the observed increase in oxidative stress and lipid peroxidation. The inhibition of the placental Pgp expression by nicotine may also contribute to an increased susceptibility of the fetus to environmental toxins.     

Evidence for involvement of polymorphic CYP2C19 and 2C9 in the N-demethylation of sertraline in human liver microsomes

AIMS: The present study was designed to define the kinetic behaviour of sertraline N-demethylation in human liver microsomes and to identify the isoforms of cytochrome P450 involved in this metabolic pathway. METHODS: The kinetics of the formation of N-demethylsertraline were determined in human liver microsomes from six genotyped CYP2C19 extensive (EM) and three poor metabolisers (PM). Selective inhibitors of and specific monoclonal antibodies to various cytochrome P450 isoforms were also employed. RESULTS: The kinetics of N-demethylsertraline formation in all EM liver microsomes were fitted by a two-enzyme Michaelis-Menten equation, whereas the kinetics in all PM liver microsomes were best described by a single-enzyme Michaelis-Menten equation similar to the low-affinity component found in EM microsomes. Mean apparent Km values for the high-and low-affinity components were 1.9 and 88 microm and V max values were 33 and 554 pmol min-1 mg-1 protein, respectively, in the EM liver microsomes. Omeprazole (a CYP2C19 substrate) at high concentrations and sulphaphenazole (a selective inhibitor of CYP2C9) substantially inhibited N-demethylsertraline formation. Of five monoclonal antibodies to various cytochrome P450 forms tested, only anti-CYP2C8/9/19 had any inhibitory effect on this reaction. The inhibition of sertraline N-demethylation by anti-CYP2C8/9/19 was greater in EM livers than in PM livers at both low and high substrate concentrations. However, anti-CYP2C8/9/19 did not abolish the formation of N-demethylsertraline in the microsomes from any of the livers. CONCLUSIONS: The polymorphic enzyme CYP2C19 catalyses the high-affinity N-demethylation of sertraline, while CYP2C9 is one of the low-affinity components of this metabolic pathway.     

In vitro metabolism of alectinib,a novel potent ALK inhibitor,in human: contribution of CYP3A enzymes

1.?The in vitro metabolism of alectinib, a potent and highly selective oral anaplastic lymphoma kinase inhibitor, was investigated.

2.?The main metabolite (M4) in primary human hepatocytes was identified, which is produced by deethylation at the morpholine ring. Three minor metabolites (M6, M1a, and M1b) were also identified, and a minor peak of hydroxylated alectinib (M5) was detected as a possible precursor of M4, M1a, and M1b.

3.?M4, an important active major metabolite, was produced and further metabolized to M6 by CYP3A, indicating that CYP3A enzymes were the principal contributors to this route. M5 is possibly produced by CYP3A and other isoforms as the primary step in metabolism, followed by oxidation to M4 mainly by CYP3A. Alternatively, M5 could be oxidized to M1a and M1b via an NAD-dependent process. None of the non-CYP3A-mediated metabolism appeared to be major.

4.?In conclusion, this study suggests that involvement of multiple enzymes in the metabolism of alectinib reduces its potential for drug–drug interactions.     

Metabolism of Dextromethorphan in vitro: Involvement of Cytochromes P450 2D6 AND 3A3/4, with a Possible Role of 2E1

Dextromethorphan (DMO), a cough suppressing synthetic analog of codeine, undergoes parallel O-demethylation to dextrorphan (DOP), and N-demethylation to 3-methoxy-morphinan (MEM), in humans. 3-hydroxymorphinan, a didemethylated metabolite, is formed secondarily. O-demethylation activity is well established as an index reaction for CYP2D6. However, this pathway appears to be mediated by at least two different enzymes in vitro. N-demethylation activity has recently been proposed to reflect CYP3A3/4 activity. We investigated both pathways in vitro with microsomal preparations from three human livers to assess the value of DMO as a probe drug for CYP2D6 and CYP3A3/4. DMO O-demethylation displayed a biphasic pattern with a high-affinity site reflecting CYP2D6 activity (mean K_i for quinidine, 0·1 ± 0·13 μM). Kinetic parameters for the two O-demethylation mediating enzymes predict an average relative intrinsic clearance (V_max/K_m ratio) of 96% of total O-demethylation mediated via the high-affinity enzyme. Thus, in vitro data confirms the usefulness of DMO O-demethylation as an index reaction to monitor CYP2D6 activity. The Eadie–Hofstee plot of DMO N-demethylation was consistent with single-enzyme Michaelis–Menten kinetics (V_max varying from 3·3 to 6·8 nmol mg⁻¹ min⁻¹, K_m from 231 to 322 μM). However, ketoconazole, a CYP3A3/4 inhibitor, reduced N-demethylation only by 60% and had a mean K_i an order of magnitude higher (0·37 μM) compared to other pure CYP3A3/4 mediated reactions. Troleandomycin, a mechanism based CYP3A3/4 inhibitor, inhibited MEM formation by an average maximum of 46%, with an IC₅₀ varying from 1 to 2·6 μM. A polyclonal rat liver CYP3A1 antibody inhibited MEM formation only by approximately 50%. Diethyldithiocarbamate (DDC), a mechanism based CYP2E1 inhibitor, reduced MEM formation at concentrations up to 150 μM between 33 and 43%. Chemical inhibitors of CYP2D6 (quinidine), CYP1A1/2 (α-naphthoflavone), and CYP2C9 (sulfaphenazole), as well as a goat rat liver CYP2C11 polyclonal antibody (inhibitory against human CYP2C9 and CYP2C19), had minimal effect on MEM formation rate, thus excluding an involvement of any of these enzymes. DMO N-demethylation is only partly mediated by CYP3A3/4, and therefore is not a reliable index reaction for CYP3A3/4 activity either in vitro or probably in vivo. © 1997 John Wiley & Sons, Ltd.