Human P450 3A4 Research Articles

Dataset for genotyping validation of cytochrome P450 2A6 whole-gene deletion (CYP2A6*4) by real-time polymerase chain reaction platforms.

This data article contains a supplementary figure and validation data relating to the research article entitled “Genotyping of wild-type cytochrome P450 2A6 and whole-gene deletion using human blood samples and a multiplex real-time polymerase chain reaction method with dual-labeled probes” (Shimizu et al., Clinica Chimica Acta 441, 71–74, 2015), which presents a multiplex real-time polymerase chain reaction method with dual-labeled probes for human P450 2A6 wild-type and whole-gene deletion. Real-time methods have dramatically improved the speed of complex genetic diagnostics compared to conventional assays based on restriction enzyme digestion. Here, we show the basic assay validation data by single and multiplex determinations in comparison with commercial TaqMan copy number assays for P450 2A6.

Substrate Selectivities and Catalytic Activities of Marmoset Liver Cytochrome P450 2A6 Differed from Those of Human P450 2A6.

The common marmoset (Callithrix jacchus), a New World primate species, is potentially a useful animal model for preclinical studies in drug development. However, cytochrome P450 (P450) enzymes have not been fully identified and characterized in marmosets. In this study, we identified P450 2A6 cDNA with the sequence highly identical (91-94%) to human P450 2A6, 2A7, and 2A13 cDNA and cynomolgus monkey P450 2A23, 2A24, and 2A26 cDNA. Among the tissue types examined, marmoset P450 2A6 mRNA was most abundantly expressed in livers where P450 2A6 protein was also detected by immunoblotting. Phylogenetic analysis showed that marmoset P450 2A6 was more closely clustered with human and cynomolgus monkey P450 2As than P450 2As of dog, rat, and mouse (the species also used in drug metabolism). Marmoset P450 2A6 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation, similar to human P450 2A6 and 2A13 and cynomolgus monkey P450 2A23, 2A24, and 2A26, but much less effectively coumarin 7-hydroxylation, showing some difference as well. Interestingly, marmoset P450 2A6 and cynomolgus monkey P450 2A23 catalyzed phenacetin O-deethylation, which is catalyzed by human P450 1A2 and 2A13, but not by P450 2A6. Marmoset P450 2A6 also exhibited catalytic activity toward testosterone by the multiple sites, but not rat P450 2A-specific testosterone 7α-hydroxylation activity. These results indicated that marmoset P450 2A6 had functional characteristics different from those of human and cynomolgus monkey P450 2As in terms of partially different substrate specificities and catalytic activities, indicating its importance of further studies for P450 2A-dependent drug metabolism in marmosets.

Kinetic Analysis of the Processivity of Human Cytochrome P450 17A1

Human cytochrome P450 (P450) 17A1 is one of several P450 monoxygenases involved in the biosynthesis of steroids. Notably, P450 17A1 is responsible for 17α‐hydroxylation of the 21‐carbon steroids pregnenolone and progesterone, which are used in glucocorticoid production. 17α‐Hydroxypregnenolone and 17α‐hydroxyprogesterone are also further oxidized by P450 17A1 in a 17α,20‐lyase reaction generating the 19‐carbon androgens dehydroepiandrosterone and androstenendione, respectively. Selective stimulation of the 17α,20‐lyase reaction is observed with human cytochrome b5 (b5). Inhibition of P450 17A1 is a goal in some cancer therapies because of its role in generating sex hormone precursors through the cleavage reaction. However, complete inhibition of the enzyme concomitantly impedes glucocorticoid synthesis. This issue poses the question of whether the 17α,20‐lyase reaction succeeds 17α‐hydroxylation in a successive or distributive manner. To date, the processivity of human P450 17A1 (with and without b5) has yet to be systematically determined. Steady‐state kinetic parameters have been measured for human P450 17A1 reactions and binding constants have been obtained via spectral titrations. Pulse‐chase assays using radio‐labeled substrates and pre‐steady‐state analyses further elucidate the rate constants of key steps in the two‐reaction mechanism for both steroids. Human P450 17A1 is partially processive with pregnenolone. The kinetic analyses yield data to further model the processivity in the presence and absence of b5 utilizing KinTek Explorer® software.

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2

Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity.

Flexible docking-based molecular dynamics/steered molecular dynamics calculations of protein-protein contacts in a complex of cytochrome P450 1A2 with cytochrome b5.

Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2-cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2-cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450-cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.

Thalidomide Increases Human Hepatic Cytochrome P450 3A Enzymes by Direct Activation of the Pregnane X Receptor

Heterotropic cooperativity of human cytochrome P450 (P450) 3A4/3A5 by the teratogen thalidomide was recently demonstrated by H. Yamazaki et al. ( ( 2013 ) Chem. Res. Toxicol. 26 , 486 - 489 ) using the model substrate midazolam in various in vitro and in vivo models. Chimeric mice with humanized liver also displayed enhanced midazolam clearance upon pretreatment with orally administered thalidomide, presumably because of human P450 3A induction. In the current study, we further investigated the regulation of human hepatic drug metabolizing enzymes. Thalidomide enhanced levels of P450 3A4 and 2B6 mRNA, protein expression, and/or oxidation activity in human hepatocytes, indirectly suggesting the activation of upstream transcription factors involved in detoxication, e.g., the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). A key event after ligand binding is an alteration of nuclear receptor conformation and recruitment of coregulator proteins that alter chromatin accessibility of target genes. To investigate direct engagement and functional alteration of PXR and CAR by thalidomide, we utilized a peptide microarray with 154 coregulator-derived nuclear receptor-interaction motifs and coregulator and nuclear receptor boxes, which serves as a sensor for nuclear receptor conformation and activity status as a function of ligand. Thalidomide and its human proximate metabolite 5-hydroxythalidomide displayed significant modulation of coregulator interaction with PXR and CAR ligand-binding domains, similar to established agonists for these receptors. These results collectively suggest that thalidomide acts as a ligand for PXR and CAR and causes enzyme induction leading to increased P450 enzyme activity. The possibilities of drug interactions during thalidomide therapy in humans require further evaluation.

Identification of Key Licorice Constituents Which Interact with Cytochrome P450: Evaluation by LC/MS/MS Cocktail Assay and Metabolic Profiling

Licorice has been shown to affect the activities of several cytochrome P450 enzymes. This study aims to identify the key constituents in licorice which may affect these activities. Bioactivity assay was combined with metabolic profiling to identify these compounds in several complex licorice extracts. Firstly, the inhibition potencies of 40 pure licorice compounds were tested using an liquid chromatography/tandem mass spectrometry cocktail method. Significant inhibitors of human P450 isozymes 1A2, 2C9, 2C19, 2D6, and 3A4 were then selected for examination of their structural features by molecular docking to determine their molecular interaction with several P450 isozymes. Based on the present in vitro inhibition findings, along with our previous in vivo metabolic studies and the prevalence of individual compounds in licorice extract, we identified several licorice constituents, viz., liquiritigenin, isoliquiritigenin, together with seven isoprenylated flavonoids and arylcoumarins, which could be key components responsible for the herb-drug interaction between cytochrome P450 and licorice. In addition, hydrophilic flavonoid glycosides and saponins may be converted into these P450 inhibitors in vivo. These studies represent a comprehensive examination of the potential effects of licorice components on the metabolic activities of P450 enzymes.

Drug interactions of diclofenac and its oxidative metabolite with human liver microsomal cytochrome P450 1A2-dependent drug oxidation

1. The purpose of this study was to investigate the inhibitory effects of diclofenac on human cytochrome P450 1A2-, 2C19- and 3A4-mediated drug oxidations and to evaluate the drug interaction potential of diclofenac and 4′-hydroxydiclofenac.2. Diclofenac was converted to 4′-hydroxydiclofenac by recombinantly expressed human P450 1A2 with Km and Vmax values of 33 µM and 0.20 min−1, respectively. Diclofenac and 4′-hydroxydiclofenac suppressed flurbiprofen 4′-hydroxylation by P450 2C9 strongly and moderately, respectively; however, they did not affect P450 2C19-dependent S-mephenytoin hydroxylation or P450 3A4-dependent midazolam hydroxylation.3. Although the caffeine 3-N-demethylation activity of liver microsomal P450 1A2 was inhibited by simultaneous incubation with diclofenac, the riluzole N-hydroxylation activities of recombinant P450 1A2 and human liver microsomes were inhibited after preincubation with diclofenac or 4′-hydroxydiclofenac for 20 min in the presence of NADPH. Using the inhibition constant (37 µM) of diclofenac on caffeine 3-N-demethylation and the reported 95th percentiles of maximum plasma concentration (10.5 µM) after an oral dose of diclofenac, the in vivo estimated increase in area under the plasma concentration–time curve was 29%.4. These results suggest that diclofenac could inhibit drug clearance to a clinically important degree that depends on P450 1A2. Clinically relevant drug interactions in vivo with diclofenac are likely to be invoked via human P450 1A2 function in addition to those caused by the effect of diclofenac on P450 2C9.

Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline

1. Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline.2. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes.3. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4′-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation.4. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys.5. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

In Vivo Drug Interactions of the Teratogen Thalidomide with Midazolam: Heterotropic Cooperativity of Human Cytochrome P450 in Humanized TK-NOG Mice

In vivo drug interactions of the teratogen thalidomide with the model cytochrome P450 (P450) 3A substrate midazolam were investigated in mice with humanized livers. The clearance of midazolam (administered intravenously, 10 mg kg(-1)) in chimeric mice was enhanced by orally co-administered thalidomide (100 mg kg(-1)). A larger area under the curve of the major metabolite 1'-hydroxymidazolam (1.7-fold) was obtained with thalidomide because of the heterotropic cooperativity of human P450 3A enzymes. A larger area under the curve of the minor metabolite 4-hydroxymidazolam (3.5-fold) was seen with daily pretreatment with thalidomide for 3 days, presumably because of human P450 3A induction. These results demonstrate that livers of humanized mice mediate drug interactions of thalidomide and suggest interactions of therapeutic agents during therapies with thalidomide.

Expression of active human P450 3A4 on the cell surface of Escherichia coli by Autodisplay

The cytochrome P450 enzyme system comprises a large group of enzymes catalyzing a broad diversity of reactions and an extensive substrate specificity, which makes them the most versatile known catalysts. CYP3A4 is one of the important human P450 enzymes and involved in the oxidation of a large range of substrates including toxins and pharmaceuticals. Bottlenecks in studying this enzyme include the difficulty in expressing it in a bacterial host, its need for membrane surroundings and the limited substrate accessibility of enzymes expressed within the cell. To circumvent these difficulties, human CYP3A4 was expressed on the outer membrane of Escherichia coli using Autodisplay. Transport of CYP3A4 to the cell surface was monitored by SDS-PAGE and Western blot analysis of outer membrane proteins. Localization on the cell envelope was determined by flow cytometry after immunolabeling, a whole cell ELISA and a protease accessibility assay. A HPLC assay confirmed the catalytic activity of displayed CYP3A4, using testosterone as a substrate. This activity required the external addition of electron supplying enzymes, however surprisingly, we found that the external addition of a heme group was not necessary. Our results indicate that human CYP3A4 can be recombinantly expressed by surface display in a gram-negative bacterium.

Functional Expression and Characterization of Recombinant NADPHP450 Reductase from Malassezia globosa

Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.

Simultaneous pharmacokinetics assessment of caffeine, warfarin, omeprazole, metoprolol, and midazolam intravenously or orally administered to Microminipigs

Small minipigs (Microminipig, registered as a novel variety of pig in Japan) were developed for use in non-clinical pharmacological/toxicological studies for new drug development. To assess the pharmacokinetics of selective substrates of human cytochrome P450s in Microminipigs, caffeine (human P450 1A2), warfarin (P450 2C9), omeprazole (P450 2C19), metoprolol (P450 2D6), and midazolam (P450 3A) were administered in combination, intravenously (0.20 mg kg(-1))( )or orally (1.0 mg kg(-1)). Plasma samples obtained, up to 24 hr after dosing, from four male and four female Microminipigs were analyzed by liquid chromatography tandem mass spectrometry to estimate typical pharmacokinetic parameters for each analyte. Bioavailabilities were approximately 80% for caffeine and warfarin, but less than 10% for omeprazole, metoprolol, and midazolam. No significant differences were noted, for the five probes, in area under the plasma concentration-time curve and peak plasma concentration values obtained from male and female Microminipigs. Clearance of caffeine, warfarin, omeprazole or midazolam in vivo, mediated mainly by cytochrome P450s 1A, 2C or 3A in Microminipigs, was similar to data reported for human. However, metoprolol metabolism, mediated by P450 2D enzymes in Microminipigs, was faster than reported for in vivo human kinetic parameters and in vitro in a human liver microsomal system. The results of this study suggest that the Microminipig is a suitable animal model for use in biological experiments for comparisons of pharmacokinetics of drugs in humans. The five-probes in combination used in this study demonstrate the disposition of typical P450 drugs in Microminipigs in vivo, with the aim of use in non-clinical pharmacological/toxicological studies.

Cytochrome P450-mediated metabolism and DNA binding of 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline and its carcinogenic isomer 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in mice.

2-Amino-1,7-dimethylimidazo[4,5-g]quinoxaline (MeIgQx) is a recently discovered heterocyclic aromatic amine (HAA) that is formed during the cooking of meats. MeIgQx is an isomer of 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), a rodent carcinogen and possible human carcinogen that also occurs in cooked meats. MeIgQx is a bacterial mutagen, but knowledge about its metabolism and carcinogenic potential is lacking. Metabolism studies on MeIgQx and MeIQx were conducted with human and mouse liver microsomes, and recombinant human P450s. DNA binding studies were also investigated in mice to ascertain the genotoxic potential of MeIgQx in comparison to MeIQx. Both HAAs underwent comparable rates of N-oxidation to form genotoxic N-hydroxylated metabolites with mouse liver microsomes (0.2-0.3 nmol/min/mg protein). The rate of N-oxidation of MeIQx was 4-fold greater than the rate of N-oxidation of MeIgQx with human liver microsomes (1.7 vs 0.4 nmol/min/mg protein). The rate of N-oxidation, by recombinant human P450 1A2, was comparable for both substrates (6 pmol/min/pmol P450 1A2). MeIgQx also underwent N-oxidation by human P450s 1A1 and 1B1 at appreciable rates, whereas MeIQx was poorly metabolized by these P450s. The potential of MeIgQx and MeIQx to form DNA adducts was assessed in female C57BL/6 mice given [(14)C]-MeIgQx (10 μCi, 9.68 mg/kg body wt) or [(14)C]-MeIQx (10 μCi, 2.13 mg/kg body wt). DNA adduct formation in the liver, pancreas, and colorectum was measured by accelerator mass spectrometry at 4, 24, or 48 h post-treatment. Variable levels of adducts were detected in all organs. The adduct levels were similar for both HAAs, when adjusted for dose, and ranged from 1 to 600 adducts per 10(7) nucleotides per mg/kg dose. Thus, MeIgQx undergoes metabolic activation and binds to DNA at levels that are comparable to MeIQx. Given the high amounts of MeIgQx formed in cooked meats, further investigations are warranted to assess the carcinogenic potential of this HAA.

Different Metabolites of Human Hepatotoxic Pyrazolopyrimidine Derivative 5‐n‐Butyl‐Pyrazolo[1,5‐a]Pyrimidine Produced by Human, Rat and Monkey Cytochrome P450 1A2 and Liver Microsomes

Different Metabolites of Human Hepatotoxic Pyrazolopyrimidine Derivative 5‐<i>n</i>‐Butyl‐Pyrazolo[1,5‐<i>a</i>]Pyrimidine Produced by Human, Rat and Monkey Cytochrome P450 1A2 and Liver Microsomes

Induction of P450 3A1/2 and 2C6 by gemfibrozil in Sprague-Dawley rats

Fibrates are a group of peroxisome proliferator-activated receptor α agonists used in the treatment of dyslipidemia; however, they have been reported to cause species-related hepatocarcinogenesis and clinical myotoxicity. Gemfibrozil is one of the most commonly used fibrates, and it shows the highest risk for myotoxicity among the fibrates. The inhibitory drug-drug interaction mechanism associated with gemfibrozil has been explored recently, and the induction of human P450 3A4 and 2C8 has been reported. In this study, in vivo induction of rat P450 by gemfibrozil was studied in Sprague-Dawley rats. After the rats were dosed with gemfibrozil by oral gavage, microsomes were prepared. The metabolic activities of P450 3A1/2,2C6, and 2D2 were assayed using probe substrates, and the systemic concentration of gemfibrozil during its administration was determined. P450 3A1/2 and 2C6 activities were induced 32-77% in the rats by gemfibrozil when the exposure concentration was in the clinical range. These data indicate that the inducibility of homologous P450 isoforms by gemfibrozil is similar in Sprague-Dawley rats and in humans. Inductive drug-drug interactions and inhibitory actions are involved in the co-administration of gemfibrozil with other drugs, which suggests the relevance for a fibrate-toxicology investigation.

Cloning, Expression and Functional Characterization of Cytochrome P450 3A37 from Turkey Liver with High Aflatoxin B1 Epoxidation Activity

Cytochrome P450s (P450) play an important role in the formation of carcinogenic and mutagenic electrophilic intermediates from a wide range of xenobiotics, including naturally occurring dietary compounds. The pathogenesis of hepatotoxic and hepatocarcinogenic action of the mycotoxin aflatoxin B(1) (AFB(1)) involves initial bioactivation by P450s to a reactive and electrophilic intermediate exo-aflatoxin B(1)-8,9-epoxide (exo-AFBO). Poultry, especially turkeys are extremely sensitive to AFB(1), a condition due, in part, to efficient epoxidation by P450 1A and 3A enzymes. We previously reported the discovery of P450 1A5 from turkey liver, which like its human homologue, 1A2, bioactivated AFB(1) to exo-AFBO and aflatoxin M(1) (AFM(1)). Here, we describe P450 3A37, the 3A4 homologue from turkey liver. This gene has an open reading frame (ORF) of 1512 bp, and the protein is predicted to be 504 amino acids with 97% identity to chicken P450 3A37. A truncated construct of the turkey P450 3A37 gene with 11 amino acids deleted from the hydrophobic N-terminal region was heterologously expressed in Escherichia coli, the protein from which exhibited a CO difference spectrum typical of P450s. Like human P450 3A4, 3A37 biotransformed AFB(1) to exo-AFBO and aflatoxin Q(1) (AFQ(1)) and possessed nifedipine oxidation activity, both of which were inhibited by the P450 3A4 inhibitor 17alpha-ethynylestradiol. Oxidation of AFB(1) to exo-AFBO and AFQ(1) by P450 3A37 followed sigmoidal Hill kinetics, suggestive of an allosteric interaction between the enzyme and AFB(1). The Hill coefficient (n) value was 1.9 for exo-AFBO and 1.6 for AFQ(1), indicative of positive cooperativity. The calculated K(m) and V(max) values for the formation of exo-AFBO were 287 +/- 21 muM and 1.45 +/- 0.07 nmol/min/nmol P450, respectively, whereas those of AFQ(1) formation were 302 +/- 51 muM and 7.86 +/- 0.75 nmol/min/nmol P450, respectively. These data strongly suggest that P450 3A37, along with P450 1A5, plays an important role in AFB(1) epoxidation in turkey liver.

Comparison of Cytochrome P450 3A Enzymes in Cynomolgus Monkeys and Humans

Drug metabolizing activities of cytochromes P450 (P450s, or CYPs) 3A4 and 3A5 in liver microsomes from the cynomolgus monkey [Macaca fascicularis (mf)] were investigated and compared with those of human P450 3A enzymes. Low activities for dealkylation of ethoxyresorufin and pentoxyresorufin were seen in recombinant monkey mfCYP3A4 and mfCYP3A5 and in recombinant human CYP3A4 and CYP3A5 expressed in bacterial membranes. Hydroxylation activities of mfCYP3A4 and mfCYP3A5 toward coumarin, paclitaxel, diclofenac, flurbiprofen, and S-mephenytoin were below detectable levels, as was also true for CYP3A4 and CYP3A5. Monkey mfCYP3A5 and mfCYP3A4 were highly active in bufuralol 1'-hydroxylation. mfCYP3A5 was efficient at dextromethorphan O-demethylation, although human CYP3A5 was unable to catalyze this reaction. Apparent bufuralol 1'-hydroxylation and dextromethorphan O-demethylation activities of monkey liver microsomes were higher than those of human liver microsomes, possibly because of contributions of mfCYP3A5 to these P450 2D-dependent drug oxidations. mfCYP3A5 and CYP3A5 catalyzed midazolam 1'-hydroxylation at a low substrate concentration more efficiently than the corresponding CYP3A4. mfCYP3A5 had higher testosterone 6beta-hydroxylase activity than mfCYP3A4, but the reverse relationship was observed in oxidation of nifedipine and hydroxylation of dexamethasone. These results demonstrate that monkey P450 3A enzymes have similar substrate selectivity to that of human P450 3A enzymes, but exhibit wider substrate selectivity toward P450 2D substrates.

Bioactivation of Lamotrigine in Vivo in Rat and in Vitro in Human Liver Microsomes, Hepatocytes, and Epidermal Keratinocytes: Characterization of Thioether Conjugates by Liquid Chromatography/Mass Spectrometry and High Field Nuclear Magnetic Resonance Spectroscopy

Previous studies suggested that lamotrigene (LTG) underwent bioactivation to a reactive aryl epoxide intermediate in rats. Nevertheless, definitive structures of these thioether conjugates, which are often needed to substantiate the mechanism of bioactivation and identity of reactive intermediate(s), were not fully established. In the present study, GSH, cysteinylglycine, and N-acetyl cysteine conjugates of LTG were isolated from bile of rats orally dosed with LTG (100 mg/kg), and their structures were fully elucidated by LC/MS and NMR. The definitive structural characterization of these metabolites provided evidence for the existence of a reactive aryl epoxide that was trapped as a GSH adduct. In vitro studies using various hepatic cellular and subcellular fractions obtained from human and rat were performed to demonstrate that LTG underwent bioactivation to form a GSH conjugate that was identical to the one initially characterized from in vivo studies. Human P450 2A6 and rat P450 2C11 appeared to be the primary enzymes activating LTG in human and rat liver microsomes, respectively. Interindividual variation in the bioactivation of LTG was demonstrated with 20 individual human liver microsomes. Furthermore, it was shown that human epidermal keratinocytes were capable of forming the same GSH conjugate, suggesting that LTG could be bioactivated in skin cells. The results from these studies suggest that LTG has the potential to undergo hepatic and nonhepatic bioactivation, leading to a reactive aryl epoxide intermediate in human. The bioactivation of LTG in epidermal cells provides a possible explanation for the idiosyncratic cutaneous reactions associated with LTG therapy.

Effects of Histidine-Tag on Recombinant Human Cytochrome P450 3A5 Catalytic Activity in Reconstitution Systems

Cytochrome P450 (P450) plays an important role for oxidation of a variety of chemicals. Human P450 3A5 is recognized as the secondly most important enzymes among P450 3A subfamily, with some differences in substrate specificity, catalytic activity, and region-selectivity of P450 3A4. Because addition of histidine-tag to microsomal P450s is universally used for recombinant protein purifications, purified P450 3A4 protein is commercially available, but P450 3A5 is not at present. In the present study, although catalytic activity of purified P450 3A5 expressed using the original plasmid from bacterial membranes was observed in reconstitution systems containing NADPH-P450 reductase, cytochrome b(5), and phospholipids, a histidine-tagged P450 3A5 protein did not show any detectable midazolam hydroxylation activities in the same reconstitution systems. From the docking simulation of midazolam into a P450 3A5 homology model based on the published P450 3A4 structure, it was supported that midazolam could not easily access to the center of the heme of histidine-tagged P450 3A5, with the high interaction energy compared with the original P450 3A5. In conclusion, these results suggest that the histidine-tagged system would be non-applicable for P450 3A5 function.