Site-specific oxidation of flavanone and flavone by cytochrome P450 2A6 in human liver microsomes

1. The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes.

2. CYP2A6 was highly active in oxidizing flavanone to form flavone, 2′-hydroxy-, 4′-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5.

3. Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2′-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities.

4. Molecular docking analysis showed that the C2′-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2′ and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively.

5. These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.

     

In vitro comparative inhibition profiles of major human drug metabolising cytochrome P450 isozymes (CYP2C9, CYP2D6 and CYP3A4) by HMG-CoA reductase inhibitors

Objective: The affinity of (+)-, (−)- and (±)-fluvastatin, a new synthetic HMG-CoA reductase inhibitor developed as a racemate, for specific human P450 monooxygenases in liver microsomes was compared with that of the pharmacologically active acidic forms of lovastatin, pravastatin and simvastatin. Methods: Affinity was determined as the inhibitory potency for prototype reactions for 3 major drug metabolising enzymes: diclofenac 4′-hydroxylation (CYP2C9), dextromethorphan O-demethylation (CYP2D6), and midazolam 1′-hydroxylation (CYP3A4). Results: Lovastatin acid, pravastatin and simvastatin acid displayed moderate affinity for all three P450 isozymes (estimated K_i > 50 μmol⋅l⁻¹). Racemic and (+)- and (−)-fluvastatin showed moderate affinity (estimated K_i > 50 μmol⋅l⁻¹) for CYP2D6 and CYP3A4, whereas their affinity for CYP2C9 was high (estimated K_i < 1 μmol⋅l⁻¹). Diclofenac 4′-hydroxylation was competitively and stereoselectively inhibited, with measured K_i’s of 0.06 and 0.28 μmol⋅l⁻¹ for (+)- and (−)-fluvastatin, respectively. Conclusion: Fluvastatin selectively inhibits a major drug metabolising enzyme (CYP2C9), the (+)-isomer (pharmacologically more active) showing 4–5 fold higher affinity. As already reported for lovastatin and simvastatin, in vivo drug interactions by inhibition of liver oxidation of CYP2C9 substrates (e.g. hypoglyceamic sulphonylureas and oral anticoagulants) may be expected. Received: 9 June 1995/Accepted in revised form: 7 November 1995     

Mechanism-based inhibition of human cytochrome P4503A4 by domperidone

1. Domperidone was evaluated in direct and time-dependent cytochrome P450 (CYP) 3A inhibition assays in human liver microsomes with midazolam and testosterone as probe substrates.

2. Domperidone was found to be a modest mechanism-based inhibitor of human and rat CYP3A. For human CYP3A, the inactivation constant (K_I) is 12 μM, and the maximum inactivation rate (k_inact) is 0.037?min^?1.

3. A rat interaction study was conducted between midazolam and either a single dose or five daily doses of domperidone. Although a single oral dose of 10?mg kg^?1 domperidone did not affect the pharmacokinetics of 10?mg kg^?1 oral midazolam, five daily oral doses of domperidone almost doubled the area under the plasma concentration versus time curve (AUC) of midazolam, and increased the maximum plasma concentration (C_max) of midazolam by 72%.

4. Based on the simulation and rat in vitro–in vivo extrapolation, it is predicted that co-administration of domperidone in humans could modestly increase (approximately 50%) the exposure of drugs that are primarily cleared by CYP3A.

     

In vitro modulation of naturally occurring flavonoids on cytochrome P450 2A6 (CYP2A6) activity

1. The effect of flavonoids on coumarin 7-hydroxylation, an activity marker of an important human liver cytochrome P450 isoform, cytochrome P450 2A6 (CYP2A6), was investigated in this study.

2. Coumarin 7-hydroxylase activity was measured fluorometrically in reaction mixtures containing cDNA-expressed CYP2A6, nicotinamide adenine dinucleotide phosphate generating system and 10 uM coumarin, at various concentrations of flavonoids.

3. Among the 23 compounds tested, most of the active members were from flavonol group of hydroxylated flavonoids, with myricetin being the most potent inhibitor followed by quercetin, galangin, and kaempferol.

4. Further exploration of the inhibition mechanism of these compounds revealed that myricetin, galangin, and kaempferol exhibited mixed-type of inhibition pattern while quercetin was observed to exhibit competitive mode of inhibition.

5. Structure-function analyses revealed that degree of inhibition was closely related to the number and location of hydroxyl groups, glycosylation of the free hydroxyl groups, degree of saturation of the flavane nucleus as well as the presence of the alkoxylated function.

     

Time-dependent inhibition of CYP3A4 by gallic acid in human liver microsomes and recombinant systems

Abstract

1.?Gallic acid is a main polyphenol in various fruits and plants. Inhibitory characteristics of gallic acid on CYP3A4 were still unclear. The objective of this work is hence to investigate inhibitory characteristics of gallic acid on CYP3A4 using testosterone as the probe substrate in human liver microsomes (HLMs) and recombinant CYP3A4 (rCYP3A4) systems.

2.?Gallic acid caused concentration-dependent loss of CYP3A4 activity with IC₅₀ values of 615.2?μM and 669.5?μM in HLM and rCYP3A4 systems, respectively. IC₅₀-shift experiments showed that pre-incubation with gallic acid in the absence of NADPH contributed to 12- or 14-fold reduction of IC₅₀ in HLM and rCYP3A4 systems, respectively, supporting a time-dependent inhibition. In HLM, time-dependent inactivation variables K_I and K_inact were 485.8?μM and 0.05?min^–1, respectively.

3.?Compared with the presence of NADPH, pre-incubation of gallic acid in the absence of NADPH markedly increased its inhibitory effects in HLM and rCYP3A4 systems. Those results indicate that CYP3A4 inactivation by gallic acid was independent on NADPH and was mainly mediated its oxidative products.

4.?In conclusion, we showed that gallic acid weakly and time-dependently inactivated CYP3A4 via its oxidative products.     

Deactivation of anti-cancer drug letrozole to a carbinol metabolite by polymorphic cytochrome P450 2A6 in human liver microsomes

1. Cytochromes P450 (P450) involved in letrozole metabolism were investigated. Among 13 recombinant P450 forms examined, only P450 2A6 and 3A4 showed activities in transforming letrozole to its carbinol metabolite with small K_m and high V_max values yielding apparent V_max/K_m values of 0.48 and 0.24 nl min^?1 nmol^?1 P450, respectively.

2. The metabolic activities of individual human liver microsomes showed a significant correlation with coumarin 7-hydroxylase activities (P450 2A6 marker) at a letrozole concentration of 0.5 μM, while a good correlation was also seen with testosterone 6β-hydroxylase activities (P450 3A4 marker) at 5 μM substrate concentration with different inhibition by 8-methoxypsolaren.

3. Significantly low carbinol-forming activities were seen in human liver microsomes from individuals possessing CYP2A6*4/*4 (whole CYP2A6 gene deletion) at a letrozole concentration of 0.5 μM. A V_max/K_m value measured for CYP2A6.7 (amino acid substitution type) in human liver microsomes, in the presence of anti-P450 3A4 antibodies, was approximately seven-fold smaller than that for CYP2A6.1 (wild-type).

4. These results demonstrate that P450 2A6 and 3A4 catalyse the conversion of letrozole to its carbinol metabolite in vitro at low and high concentrations of letrozole. Polymorphic variation of CYP2A6 is considered to be relevant to inter-subject variation in therapeutic exposure of letrozole.

     

Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes

Nicotine C-oxidation by recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes was investigated using a convenient high-performance liquid chromatographic method. Experiments with recombinant human P450 enzymes in baculovirus systems, which co-express human nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-P450 reductase, revealed that CYP2A6 had the highest nicotine C-oxidation activities followed by CYP2B6 and CYP2D6; the K _m values by these three P450 enzymes were determined to be 11.0, 105, and 132 μM, respectively, and the V _max values to be 11.0, 8.2, and 8.6 nmol/min per nmol P450, respectively. CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed nicotine C-oxidation only at high (500 μM) substrate concentration. CYP1B1, 2C18, 3A5, and 4A11 had no measurable activities even at 500 μM nicotine. In liver microsomes of 16 human samples, nicotine C-oxidation activities were correlated with CYP2A6 contents at 10 μM substrate concentration, whereas such correlation coefficients were decreased when the substrate concentration was increased to 500 μM. Contribution of CYP2B6 (as well as CYP2A6) was demonstrated by experiments with the effects of orphenadrine (and also coumarin and anti-CYP2A6) on the nicotine C-oxidation activities by human liver microsomes at 500 μM nicotine. CYP2D6 was found to have minor roles since quinidine did not inhibit microsomal nicotine C-oxidation at both 10 and 500 μM substrate concentrations. These results support the view that CYP2A6 has major roles for nicotine C-oxidation at lower substrate concentration and both CYP2A6 and 2B6 play roles at higher substrate concentrations in human liver microsomes. Received: 27 October 1998 / Accepted: 11 January 1999     

Mechanism-based inactivation of cytochrome P450 2B6 by isopsoralen

1.?Isopsoralen (IPRN) is a major component in many traditional medicinal herbs widely used in Asian countries. The objective of the present study was to investigate the inhibitory effect of IPRN on cytochrome P450 2B6 (CYP2B6) and the mechanism involved in the enzyme inactivation.

2.?Pre-incubation of CYP2B6 with IPRN resulted in a time- and concentration-dependent enzyme activity loss. The values of K_I and k_inact were found to be 7.89?μM and 0.067?min^?1, respectively. Ticlopidine exhibited protective effect on the IPRN-induced enzyme inactivation. The estimated partition ratio of the inactivation was 122. The GSH trapping experiments indicate that an epoxide and/or γ-ketoenal intermediate were/was generated in IPRN-fortified microsomal incubations. The synthetic work verified the formation of the reactive intermediate(s). Additionally, CYPs2E1, 2C19, 2B6 and 1A2 were found to be the major enzymes participating in the bioactivation of IPRN.

3.?IPRN was characterized as a mechanism-based inactivator of CYP2B6. An IPRN-derived furanoepoxide and/or γ-ketoenal intermediate(s) were/was generated and may be responsible for the inactivation of CYP2B6.     

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.     

Inhibition of cytochrome P450 by ethambutol in human liver microsomes

Although cytochrome P450 inhibition is the major drug–drug interaction (DDI) mechanism in clinical pharmacotherapy, DDI of a number of well-established drugs have not been investigated. Rifampicin, isoniazid, pyrazinamide and ethambutol combination therapy inhibits clearance of theophylline in patients with tuberculosis. We determined the inhibitory effects of ethambutol on the activities of nine CYP isoforms including CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 in pooled human liver microsomes (HLM). As measured by liquid chromatography–electrospray ionization tandem mass spectrometry, ethambutol exhibited strong inhibitory potential against CYP1A2 and CYP2E1, moderate against CYP2C19 and CYP2D6 and weak against CYP2A6, CYP2C9 and CYP3A4, based on the IC₅₀ values. The K_i value of ethambutol for CYP1A2 was 1.4 μM and for CYP2E1 was 2.9 μM. Inhibition of CYP1A2 and CYP2E1 was not increased by preincubation with ethambutol and β-nicotinamideadenine dinucleotide phosphate (NADPH), suggesting that the ethambutol-induced CYP inhibition may not be metabolism-dependent. Kinetic analysis showed that the inhibition of CYP1A2 and CYP2E1 by ethambutol was best fit to a competitive inhibition model. Formation of 1-methylxanthene and 1,3-dimethyluric acid from theophylline in HLM was decreased to 47% and 36%, respectively, by 3.0 μM ethambutol, which is comparable to its IC₅₀ value against CYP1A2. Considering its maximal plasma concentrations of ∼10 μM and long half-life of ∼22 h, our findings raise the possibility that ethambutol causes significant DDIs in clinical situations with drugs with narrow therapeutic index, such as theophylline, in clinical situations.     

Comparison of mechanism-based inhibition of human cytochrome P450 2C19 by ticlopidine,clopidogrel, and prasugrel

1. Mechanism-based inhibition of CYP2C19 in human liver microsomes by the thienopyridine antiplatelet agents clopidogrel, prasugrel and their thiolactone metabolites was investigated by determining the time- and concentration-dependent inhibition of the activity of S-mephenytoin 4′-hydroxylase as typical CYP2C19 activity and compared with ticlopidine and its metabolite.

2. Clopidogrel was shown to be a mechanism-based inhibitor of CYP2C19 with the inactivation kinetic parameters, k_inact and K_I, equal to 0.0557?min^?1 and 14.3?μM, respectively, as well as ticlopidine (0.0739?min^?1 and 3.32?μM, respectively). The thiolactone metabolite of ticlopidine and clopidogrel inhibited CYP2C19 only in a concentration-dependent manner. In contrast, neither prasugrel nor its thiolactone metabolite inhibited CYP2C19 at concentrations up to 100?μM.

3. The oxidation of the thiophene moiety of clopidogrel to form their respective thiolactones was found to be the critical reaction that produces the chemically reactive metabolites which cause the mechanism-based inhibition of CYP2C19.

4. Estimation of in vivo drug–drug interaction using in vitro parameters predicted clinically observed data. For clopidogrel, there was no increase in the area under the curve (AUC) at its clinical dose level as predicted by the in vitro parameters, and for ticlopidine the prediction agreed with the clinically observed AUC increase.

5. In conclusion, clopidogrel is potent mechanism-based inhibitors of CYP2C19 as well as ticlopidine, whereas prasugrel did not inactivate CYP2C19. Administration of prasugrel would not cause a clinically relevant interaction with CYP2C19.

     

Metabolism of bilirubin by human cytochrome P450 2A6

The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14-22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K_i of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme.     

CYP2D6 and CYP3A4 involvement in the primary oxidative metabolism of hydrocodone by human liver microsomes

AIM: To determine the Michaelis-Menten kinetics of hydrocodone metabolism to its O- and N-demethylated products, hydromorphone and norhydrocodone, to determine the individual cytochrome p450 enzymes involved, and to predict the in vivo hepatic intrinsic clearance of hydrocodone via these pathways. METHODS: Liver microsomes from six CYP2D6 extensive metabolizers (EM) and one CYP2D6 poor metabolizer (PM) were used to determine the kinetics of hydromorphone and norhydrocodone formation. Chemical and antibody inhibitors were used to identify the cytochrome p450 isoforms catalyzing these pathways. Expressed recombinant cytochrome p450 enzymes were used to characterize further the metabolism of hydrocodone. RESULTS: Hydromorphone formation in liver microsomes from CYP2D6 EMs was dependent on a high affinity enzyme (Km = 26 microm) contributing 95%, and to a lesser degree a low affinity enzyme (Km = 3.4 mm). In contrast, only a low affinity enzyme (Km = 8.5 mm) formed this metabolite in the liver from the CYP2D6 PM, with significantly decreased hydromorphone formation compared with the livers from the EMs. Norhydrocodone was formed by a single low affinity enzyme (Km = 5.1 mm) in livers from both CYP2D6 EM and PM. Recombinant CYP2D6 and CYP3A4 formed only hydromorphone and only norhydrocodone, respectively. Hydromorphone formation was inhibited by quinidine (a selective inhibitor of CYP2D6 activity), and monoclonal antibodies specific to CYP2D6. Troleandomycin, ketoconazole (both CYP3A4 inhibitors) and monoclonal antibodies specific for CYP3A4 inhibited norhydrocodone formation. Extrapolation of in vitro to in vivo data resulted in a predicted total hepatic clearance of 227 ml x h-1 x kg-1 and 124 ml x h-1 x kg-1 for CYP2D6 EM and PM, respectively. CONCLUSIONS: The O-demethylation of hydrocodone is predominantly catalyzed by CYP2D6 and to a lesser extent by an unknown low affinity cytochrome p450 enzyme. Norhydrocodone formation was attributed to CYP3A4. Comparison of recalculated published clearance data for hydrocodone, with those predicted in the present work, indicate that about 40% of the clearance of hydrocodone is via non-CYP pathways. Our data also suggest that the genetic polymorphisms of CYP2D6 may influence hydrocodone metabolism and its therapeutic efficacy.     

细胞色素P-450 CYP2D6基因分型与表型的吻合率

目的:研究细胞色素P-450 2D6基因分型测定方法及其与表型的吻合率。方法:利用等位基因特异扩增法基本原理,对CYP2D6酶缺陷等位基因CYP2D6*3,*4,*6和*7进行测定。结果:通过168例基因分型,并将结果与表型对照,发现同时测定CYP2D6*3,*4,*6和*7等位基因时,125例快代谢者和43例慢代谢者的基因分型结果与表型结果的吻合率为100％。快代谢者至少有一个野生型CYP2D6等位基因,基因型为*1/*1,*1/*3和*1/*4。发现慢代谢者是CYP2D6突变型纯合子,基因型为*3/*4,*4/*4,*3/*6,*4/*7,*4/*6和*6/*6。结论:对CYP2D6*3,*4,*6和*7等位基因的测定能够准确预测其表型。     

Dose-dependent inhibition of CYP1A2, CYP2C19 and CYP2D6 by citalopram,fluoxetine, fluvoxamine and paroxetine

Objectives: The purpose of this pharmacokinetic study was to investigate the dose-dependent inhibition of model substrates for CYP2D6, CYP2C19 and CYP1A2 by four marketed selective serotonin reuptake inhibitors (SSRIs): citalopram, fluoxetine, fluvoxamine and paroxetine. Methods: The study was carried out as an in vivo single-dose study including 24 young, healthy men. All volunteers had been identified as sparteine- and mephenytoin-extensive metabolisers. The volunteers received in randomised order, at weekly intervals, increasing single oral doses of one of the four SSRIs, followed 3 h later by sparteine (CYP2D6), mephenytoin (CYP2C19) and caffeine (CYP1A2) tests. Fluoxetine was given at 3-week intervals because of the long half-life of fluoxetine and its metabolite norfluoxetine. Citalopram, fluoxetine and paroxetine were given in doses of 10, 20, 40 and 80 mg and fluvoxamine was given in doses of 25, 50, 100 and 200 mg. Results: With increasing doses, there was a statistically significant increase in the sparteine metabolic ratio (MR) (P < 0.01, Page’s test for trend) for all four SSRIs. The increase was modest after intake of citalopram and fluvoxamine, while the increase was more pronounced after fluoxetine intake, although no volunteers changed phenotype from extensive metabolisers to poor metabolisers. Three of the six volunteers changed phenotype from extensive metabolisers to poor metabolisers after intake of 40 or 80 mg paroxetine. There was a statistically significant increase in the mephenytoin S/R ratio (P < 0.01, Page’s test for trend) with increasing doses of fluoxetine and fluvoxamine, but not after citalopram and paroxetine. However, no volunteers changed phenotype from extensive to poor metabolisers of S-mephenytoin. After intake of fluvoxamine, the urinary excretion of the metabolites related to N3 demethylation of caffeine were below the limit of quantification, whereas there were no significant changes in the urinary caffeine metabolic ratios after intake of the other three SSRIs. Conclusion: This investigation confirms that paroxetine and fluoxetine are potent inhibitors of CYP2D6, that fluvoxamine and fluoxetine are moderate inhibitors of CYP2C19 and that fluvoxamine is a potent inhibitor of CYP1A2 in humans in vivo. The clinical prediction of interaction from single-dose experiments may have to take the degree of accumulation during steady-state after multiple doses into account. Received: 11 December 1995/Accepted in revised form: 29 February 1996     

己烯雌酚对人肝微粒体内CYP3A4和CYP2C9酶的抑制作用

目的:体外实验考察己烯雌酚(DES)对细胞色素P450 3A4(CYP3A4)和细胞色素P450 2C9(CYP2C9)活性的抑制作用,以评佑DES通过抑制这两个重要的细胞色素P450(CYP)亚型而引发药物-药物相互作用的可能性.方法:混合人肝微粒体与不同浓度的DES(或阳性抑制剂),CYP3A4或CYP2C9的探针...     

Oxidation of 5-methoxy-N,N-diisopropyltryptamine in rat liver microsomes and recombinant cytochrome P450 enzymes

The oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), a tryptamine-type designer drug, was studied using rat liver microsomal fractions and recombinant cytochrome P450 (CYP) enzymes. 5-MeO-DIPT was biotransformed mainly into a side-chain N-deisopropylated metabolite and partially into an aromatic ring O-demethylated metabolite in liver microsomal fractions from untreated rats of both sexes. This metabolic profile is different from our previous findings in human liver microsomal fractions, in which the aromatic ring O-demethylation was the major pathway whereas the side-chain N-deisopropylation was minor [Narimatsu S, Yonemoto R, Saito K, Takaya K, Kumamoto T, Ishikawa T, et al. Oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes. Biochem Pharmacol 2006;71:1377-85]. Kinetic and inhibition studies indicated that the side-chain N-dealkylation is mediated by CYP2C11 and CYP3A2, whereas the aromatic ring O-demethylation is mediated by CYP2D2 and CYP2C6 in untreated male rats. Pretreatment of male rats with beta-naphthoflavone (BNF) produced an aromatic ring 6-hydroxylated metabolite. Recombinant rat and human CYP1A1 efficiently catalyzed 5-MeO-DIPT 6-hydroxylation under the conditions used. These results provide valuable information on the metabolic fate of 5-MeO-DIPT in rats that can be used in the toxicological study of this designer drug.     

Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes

The metabolism of (+)-fenchol was investigated in vitro using liver microsomes of rats and humans and recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human/rat P450 and NADPH-P450 reductase cDNAs had been introduced. The biotransformation of (+)-fenchol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on GC. Several lines of evidence suggested that CYP2A6 is a major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes. (+)-Fenchol oxidation activities by liver microsomes were very significantly inhibited by (+)-menthofuran, a CYP2A6 inhibitor, and anti-CYP2A6. There was a good correlation between CYP2A6 contents and (+)-fenchol oxidation activities in liver microsomes of ten human samples. Kinetic analysis showed that the V_max/K_m values for (+)-fenchol catalysed by liver microsomes of human sample HG03 were 7.25?nM^?1?min^?1. Human recombinant CYP2A6-catalyzed (+)-fenchol oxidation with a V_max value of 6.96?nmol?min^?1?nmol^?1 P450 and apparent K_m value of 0.09?mM. In contrast, rat CYP2A1 did not catalyse (+)-fenchol oxidation. In the rat (+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. Recombinant rat CYP2B1 catalysed (+)-fenchol oxidation. Kinetic analysis showed that the K_m values for the formation of fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03?mM, and V_max values were 2.94, 6.1 and 13.8?nmol?min^?1?nmol^?1 P450, respectively. Taken collectively, the results suggest that human CYP2A6 and rat CYP2B1 are the major enzymes involved in the metabolism of (+)-fenchol by liver microsomes and that there are species-related differences in the human and rat CYP2A enzymes.     

Variable contribution of CYP2D6 to the N-Dealkylation of S-(-)-propranolol by human liver microsomes

Recombinant cDNA expression systems for CYP2D6 have been shown to have significant catalytic activity with respect to the N -dealkylation of propranolol. However, the involvement of CYP2D6 in this reaction in human liver is inconclusive. We have re-evaluated the role of CYP2D6 in the dealkylation of S-(-)-propranolol using a bank of 10 human livers characterized for their specific CYP2D6 and CYP1A2 activities, the latter enzyme being known to be involved substantially in the formation of N -desisopropylpropranolol. Using quinidine (1  μm) or LKM-1 antibodies as selective inhibitors of CYP2D6, the contribution of this enzyme to net N -desisopropylation of S-(-)-propranolol (10  μm) by microsomes from the range of livers was found to vary from nil (poor metabolizer genotype) to 60%. N -desisopropylpropranolol formation inhibitable by quinidine was highly correlated with specific CYP2D6 activity, as measured by the α-hydroxylation of metoprolol ( r _s=0.90; P <0.001). The two livers with the highest proportion of CYP2D6-mediated N -dealkylation had relatively high ratios of specific CYP2D6 to CYP1A2 activity. These findings emphasize that data obtained using microsomes from single human livers or pooled microsomes from several livers may be misleading inasmuch as the relative contribution of different isoenzymes to the same metabolic reaction may show considerable between-subject variation.     

In vivo inhibition of CYP2C19 but not CYP2D6 by fluvoxamine

Studies were performed in eight healthy extensive metabolizers of mephenytoin and debrisoquine to determine the effect of fluvoxamine on the activities of S-mephenytoin 4'-hydroxylase (CYP2C19) and metoprolol α-hydroxylase (CYP2D6). Therapeutic dosing with fluvoxamine (100  mg day⁻¹) for 2 weeks caused a significant increase in the 0–8  h urinary S/R ratio of mephenytoin from 0.16 to 0.55 (95% confidence interval for difference between means: 0.28–0.50; P <0.01), accompanied by a 54% reduction in the 0–8  h urinary recovery of 4'-hydroxymephenytoin (95% confidence interval for difference between means: 3.64–16.24  mg; P <0.05). However, this did not alter the assigned phenotype of any of the subjects based on the established antimode of 0.95 (S/R-mephenytoin ratio). Two weeks after fluvoxamine was discontinued, both metabolic indices returned to their pre-study values. By contrast, fluvoxamine had no effect on either 0–8  h urinary metoprolol/α-hydroxymetoprolol ratio (95% confidence interval for difference between means: −0.38–0.46; P >0.05) or the 0–8  h urinary recovery of α-hydroxymetoprolol (95% confidence interval for difference between means: −0.61–0.70  mg; P >0.05). These results indicated fluvoxamine has a modest inhibitory effect on the activity of CYP2C19, but no effect on that of CYP2D6 in vivo .