In vitro characterization of clobazam metabolism by recombinant cytochrome P450 enzymes: importance of CYP2C19.

The specific cytochrome P450 (P450) isoforms mediating the biotransformations of clobazam (CLB) and those of its major metabolites, N-desmethylclobazam (NCLB) and 4'-hydroxyclobazam were identified using cDNA-expressed P450 and P450-specific chemical inhibitors. Among the 13 cDNA-expressed P450 isoforms tested, CLB was mainly demethylated by CYP3A4, CYP2C19, and CYP2B6 and 4'-hydroxylated by CYP2C19 and CYP2C18. CYP2C19 and CYP2C18 catalyzed the 4'-hydroxylation of NCLB. The kinetics of the major biotransformations were studied: CYP3A4, CYP2C19, and CYP2B6 mediated the formation of NCLB with Km = 29.0, 31.9, and 289 microM, Vmax = 6.20, 1.15, and 5.70 nmol/min/nmol P450, and intrinsic clearance (CLint) = 214, 36.1, and 19.7 microl/min/nmol P450, respectively. NCLB was hydroxylated to 4'-hydroxydesmethylclobazam by CYP2C19 with Km = 5.74 microM, Vmax = 0.219 nmol/min/nmol P450, and CLint = 38.2 microl/min/nmol P450 (Hill coefficient = 1.54). These findings were supported by chemical inhibition studies in human liver microsomes. Indeed, ketoconazole (1 microM) inhibited the demethylation of CLB by 70% and omeprazole (10 microM) by 19%; omeprazole inhibited the hydroxylation of NCLB by 26%. Twenty-two epileptic patients treated with CLB were genotyped for CYP2C19. The NCLB/CLB plasma metabolic ratio was significantly higher in the subjects carrying one CYP2C19*2 mutated allele than in those carrying the wild-type genotype. CYP3A4 and CYP2C19 are the main P450s involved in clobazam metabolism. Interactions with other drugs metabolized by these P450s can occur; moreover, the CYP2C19 genetic polymorphism could be responsible for interindividual variations of plasma concentrations of N-desmethylclobazam and thus for occurrence of adverse events.     

Contributions of human cytochrome P450 enzymes to glyburide metabolism

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

Genotypes for the cytochrome P450 enzymes CYP2D6 and CYP2C19 in human longevity Role of CYP2D6 and CYP2C19 in longevity

Objective: To test whether some genotypes for CYP2D6 or CYP2C19 could contribute to longevity, we genotyped 241 Danish nonagenarians and centenarians for CYP2D6 and CYP2C19. Methods: For CYP2D6 we identified the alleles CYP2D6*1, CYP2D6*3 and CYP2D6*4 with allele-specific polymerase chain reaction (PCR). The CYP2D6*5 alleles were identified with a long PCR method. For CYP2C19 we identified the alleles CYP2C19*1, CYP2C19*2 and CYP2C19*3 with an oligonucleotide ligation assay. Results: The four alleles for CYP2D6 did not occur in Hardy-Weinberg proportions. The frequency of poor metabolism was slightly higher (10.2%) than expected [7.7%; odds ratio (OR) = 1.36 (0.75–2.40)]. The genotypes for CYP2C19 occur in Hardy-Weinberg proportions. The frequency of poor metabolism (3.8%) was not significantly different from a young control group [3.1%; OR = 1.21 (0.26–5.75)]. Conclusion: CYP2D6 could play a role in human longevity due to the lack of Hardy-Weinberg proportions. If CYP2D6 only plays a role in longevity by protecting the poor metabolizers from cancer, we should expect a rise in the frequency in these genotypes in Denmark from 7.7% among young adults to 10–11% among very old people. We found a frequency of poor metabolism of 10.2% in the very old group. CYP2C19 is – due to the occurrence of Hardy-Weinberg proportions and the expected number of poor metabolizers – unlikely to contribute to human longevity.     

Electrochemical characterisation of the human cytochrome P450 CYP2C9

The electrochemistry of human cytochrome P4502C9 (CYP2C9) was characterised using purified His-tagged enzyme. The His-tagged enzyme was shown to have similar functional characteristics to native CYP2C9 heterologously expressed in Escherichia coli and to the CYP2C9 activity of human liver microsomes. Evidence was observed for a reversible one-electron transfer between the P450 heme and the electrode. Both pH and ionic strength influenced the electrochemical behaviour of CYP2C9. A range of substrates was investigated to determine the effect of the heme-substrate interaction on CYP2C9 redox potential. In the absence of oxygen, tolbutamide, diclofenac, warfarin and sulfaphenazole did not alter the redox potential of the iron heme. In contrast, torsemide, carbon monoxide and oxygen led to an anodic shift in redox potential. These results suggest alternative mechanisms by which CYP2C9 (and by inference other P450 enzymes) may alter redox potential to facilitate electron delivery from physiological donors.     

MPTP-induced model of Parkinson's disease in cytochrome P450 2E1knockout mice

Evidence for involvement of cytochrome P450 2E1 in the MPTP-induced mouse model of PD has been reported [Vaglini, F., Pardini, C., Viaggi, C., Bartoli, C., Dinucci, D., Corsini, G.U., 2004. Involvement of cytochrome P450 2E1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease. J. Neurochem. 91, 285–298]. We studied the sensitivity of Cyp2e1(−/−) mice to the acute administration of MPTP in comparison with their wild-type counterparts. In Cyp2e1(−/−) mice, the reduction of striatal DA content was less pronounced 7 days after MPTP treatment compared to treated wild-type mice. Similarly, TH immunoreactivity analysis of the substantia nigra of Cyp2e1(−/−) mice did not show any neuronal lesions after MPTP treatment. In contrast to this, wild-type animals showed a minimal but significant lesioning by the toxin as evaluated also by means of non-stereologic computerized assisted analysis of this brain area. Striatal levels of DA metabolites after 7 days were variably affected by the toxin, but consistent differences between the two animal strains were not observed.We evaluated short-term changes in the levels of striatal DA and its metabolites, and we monitored striatal MPP⁺ levels. Striatal MPP⁺ was cleared more rapidly in Cyp2e1(−/−) mice than in wild-type animals and, consistently, striatal DA content decreased faster in Cyp2e1(−/−) mice than in wild-type animals, and 3-methoxytyramine and HVA levels showed an early and sharp rise. Our findings suggest that Cyp2e1(−/−) mice are weakly sensitive to MPTP-induced brain lesions, markedly in contrast with a protective role of the enzyme as suggested previously. The differences observed between the knockout mice and their wild-type counterparts are modest and may be due to an efficient compensatory mechanism or genetic drift in the colonies.     

Unexpected contribution of cytochrome P450 enzymes CYP11B2 and CYP21, as well as CYP3A4 in xenobiotic androgen elimination - Insights from metandienone metabolism

The metabolism of a variety of anabolic steroids frequently misused for doping purposes has been investigated in the last years. This research mainly focused on main and long-term metabolites suitable for detection, but detailed clearance mechanisms have rarely been elucidated. Recent studies on metandienone focused on the identification of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20βOH-NorMD) as long-term metabolite, however, the metabolic pathway of its generation remained unclear. Metandienone and its Wagner-Meerwein rearrangement product 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one (NorMD) were hydroxylated by different human cytochrome P450 enzymes (CYPs). Some of their hydroxylation products were chemically synthesized and characterized by mass spectrometry to allow for their trace detection in urine samples. Following oral administration of metandienone or NorMD in one human volunteer each the post administration urines were checked for the presence of those hydroxylated metabolites using GC-MS/MS analysis. The human mitochondrial steroid hydroxylating enzymes CYP11B1 and CYP11B2 were capable to metabolize metandienone leading to the formation of 11β-hydroxymetandienone and 18-hydroxymetandienone. Following Wagner-Meerwein rearrangement, the resulting products could be assigned to 20βOH-NorMD and 11βOH-NorMD. The contribution of CYP11B1 and CYP11B2 in human metabolism of metandienone was confirmed by analysis of post-administration samples of metandienone and NorMD. Combined with the results from a previous study, enzymatic pathways were identified that involve CYP21 and CYP3A4 in the hydroxylation of NorMD, while CYP21, CYP3A4 and CYP11B2 take part in 20βOH-NorMD generation from MD. The current study represents a valuable contribution to the elucidation of clearance mechanisms of anabolic steroids and also indicates that mainly non-liver CYPs seem to be involved in these processes.     

Effect of eurycomanone on cytochrome P450 isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 in vitro

Eurycomanone, an active constituent isolated from Eurycoma longifolia Jack, was examined for modulatory effects on cytochrome P450 (CYP) isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 using in vitro assays. The IC₅₀ value was determined to assess the potencies of modulation for each CYP isoform. Our results indicated that eurycomanone did not potently inhibit any of the CYP isoforms investigated, with IC₅₀ values greater than 250 μg/ml. Hence there appears to be little likelihood of drug–herb interaction between eurycomanone or herbal products with high content of this compound and CYP drug substrates via CYP inhibition.     

Genetic polymorphisms of cytochrome P450 enzymes and antidepressant metabolism

INTRODUCTION: The cytochrome P450 (CYP) enzymes are the major enzymes responsible for Phase I reactions in the metabolism of several substances, including antidepressant medications. Thus, it has been hypothesized that variants in the CYP network may influence antidepressant efficacy and safety. Nonetheless, data on this field are still contradictory. The authors aim to give an overview of the published studies analyzing the influence of CYP highly polymorphic loci on antidepressant treatment in order to translate the acquired knowledge to a clinical level. AREAS COVERED: The authors collected and compared experimental works and reviews published from the 1980s to the present and included in the Medline database. The included studies pertain to the effects of CYP gene polymorphisms on antidepressant pharmacokinetic parameters and clinical outcomes (response and drug-related adverse effects), with a focus on applications in clinical practice. The authors focused mainly on in vivo studies in humans (patients or healthy volunteers). EXPERT OPINION: Great variability in antidepressant metabolism among individuals has been demonstrated. Thus, with the current interest in individualized medicine, several genetic tests to detect CYP variants have been produced. They provide a potentially useful way to anticipate some clinical outcomes of antidepressant treatment, although they will only be extensively used in clinical practice if precise and specific treatment options and guidelines based on genetic tests can be provided.     

Phenytoin metabolism by human cytochrome P450: involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation.

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.     

Inhibition and induction of human cytochrome P450 (CYP) enzymes

1. The present authors have previously developed a transgenic rat carrying a chimeric gene of the mouse whey acidic protein promoter and the structural portion of human growth hormone (GH) gene. Among this (hGH-TG) rat, a line (low GH rat) missing a male-specific pulsatile GH secretary pattern due to suppression of endogenous GH secretion and having a continuous low GH (hGH and rat GH) level in the peripheral circulation was identified. The latter rat was also characterized as having severe obesity with age. This strain (low Gh rat) was used to correlate the sex-specific secretory pattern of GH with the sex-specific expression of cytochrome P450 (CYP) in rat. 2. Comparisons were made between the low GH rat and the non-transgenic rat as to the expression of liver microsomal CYP isozymes. The following enzyme activities were assessed: testosterone (T) hydroxylation and oxidation; ethoxyresorufin O-dealkylation (EROD); bunitrolol (BTL) 4-hydroxylation and T5 alpha-reduction. Protein expression of CYP1A, CYP2C11, CYP2D, CYP2E1, CYP3A2 and CYP4A1 were also assessed by Western blot analysis. 3. Enzyme activities and protein expression of CYP2C11 (T16 alpha and 2alpha-hydroxylase and 17-oxidase activities) and CYP3A2 (T6beta and 2beta-hydroxylase activities) levels, which are known to be higher in the male than in the female rat, were significantly lower in the adult male low GH rat than in the control male rat. In contrast, CYP2A1 (T7 alpha hydroxylase) and T5-alpha-reductase activities, which are known to be specifically elevated in the female, were significantly higher in the adult male low GH rat than in the control male rat. Thus, the loss of male-specific secretory pattern of GH results in feminization of the pattern of expression of CYP and T5 alpha-reductase activity in the liver. 4. In contrast to other GH-deficient models so far studied, an increase in CYP4A1 and a decrease in CYP2E1 protein expression were observed in the low GH rat. These trends are consistent with the characteristic phenotype of obesity in the transgenic rat because CYP4A1 and CYP2E1 enhance fatty acid excretion and glyconeogenesis from fatty acids respectively.     

Food-drug interactions via human cytochrome P450 3A (CYP3A)

Food-drug interactions have been reported to occur in various systems in the body. The causes of these interactions are mainly divided into pharmacodynamic and pharmacokinetic processes. Among these processes, drug metabolism plays a crucial role in drug interactions. Metabolic food-drug interactions occur when a certain food alters the activity of a drug-metabolizing enzyme, leading to a modulation of the pharmacokinetics of drugs metabolized by the enzyme. A variety of interactions have been documented so far. Foods consisting of complex chemical mixtures, such as fruits, alcoholic beverages, teas, and herbs, possess the ability to inhibit or induce the activity of drug-metabolizing enzymes. According to results obtained thus far, cytochrome P450 3A4 (CYP3A4) appears to be a key enzyme in food-drug interactions. For example, interactions of grapefruit juice with felodipine and cyclosporine, red wine with cyclosporine, and St John's wort with various medicines including cyclosporine, have been demonstrated. The results indicate the requirement of dosage adjustment to maintain drug concentrations within their therapeutic windows. The CYP3A4-related interaction by food components may be related to the high level of expression of CYP3A4 in the small intestine, as well as its broad substrate specificity, as CYP3A4 is responsible for the metabolism of more than 50% of clinical pharmaceuticals. This review article summarizes the findings obtained to date concerning food-drug interactions and their clinical implications. It seems likely that more information regarding such interactions will accumulate in the future, and awareness is necessary for achieving optimal drug therapy.     

Coumarin metabolism by rat esophageal microsomes and cytochrome P450 2A3

The rat esophagus is strikingly sensitive to tumor induction by nitrosamines, and it has been hypothesized that this tissue contains cytochrome P450 enzymes (P450s) which catalyze the metabolic activation of these carcinogens. The metabolic capacity of the esophagus is not well characterized. In the study described here, the products of 14C-coumarin metabolism by rat esophageal microsomes were identified and quantified. Metabolite characterization was by LC/MS/MS and GC/MS and comparison to standards, quantification was by radioflow HPLC. The coumarin metabolites formed by rat esophageal microsomes were compared to those formed by P450 2A3. The major metabolites formed by esophageal microsomes were 8-hydroxycoumarin, o-hydroxyphenylacetaldehyde (o-HPA), and o-hydroxyphenylacetic acid (o-HPAA). A smaller amount of 5-hydroxycoumarin, about one-third the 8-hydroxycoumarin, was also formed. o-HPA and o-HPAA are products of coumarin 3,4-epoxidation. The relative rates of coumarin 8-hydroxylation and 3,4-epoxidation were similar. Coumarin 8-hydroxylation has not previously been reported as a major pathway in any tissue, and no P450s have yet been reported to catalyze this reaction. P450 2A3 catalyzed both the 7-hydroxylation and 3,4-epoxidation of coumarin. P450 2A3 was previously characterized as a coumarin 7-hydroxylase, however, in this study, we report that it catalyzes the formation of o-HPA more efficiently. The Km and Vmax were 1.3 +/- 0.35 microM and 0.65 +/- 0.06 nmol/min/nmol P450 for coumarin 7-hydroxylation and 1.4 +/- 0.58 microM and 3.1 +/- 0.46 nmol/min/nmol P450 for o-HPA formation.     

Coleus forskohlii extract induces hepatic cytochrome P450 enzymes in mice

Coleus forskohlii root extract (CFE) is popular for use as a weight loss dietary supplement. In this study, the influence of standardized CFE containing 10% active component forskolin on the hepatic drug metabolizing system was investigated to evaluate the safety through its drug interaction potential. Male ICR mice were fed AIN93G-based diets containing 0-5% CFE or 0.05% pure forskolin for 2-3 weeks. Intake of two different sources of 0.5% CFE significantly increased the relative liver weight, total content of hepatic cytochrome P450 (CYP) and induced CYPs (especially 2B, 2C, 3A types) and glutathione S-transferase (GST) activities. CFE significantly increased mRNA expression of CYPs and GST with dose related responses. However, unlike the CFE, intake of 0.05% pure forskolin was found to be associated with only weak induction in CYP3A and GST activities with no significant increases in relative liver weight, total hepatic content or other CYPs activities. The inductions of CYPs and GST by CFE were observed at 1 week of feeding and rapidly recovered by discontinuation of CFE. These results indicated the induction potential of CFE on CYPs, and that this effect was predominantly due to other, as yet unidentified constituents, and not forskolin contained in CFE.     

Effect of antifungal drugs on cytochrome P450 (CYP) 2C9, CYP2C19, and CYP3A4 activities in human liver microsomes

The effects of five antifungal drugs, fluconazole, itraconazole, micafungin, miconazole, and voriconazole, on cytochrome P450 (CYP) 2C9-mediated tolbutamide hydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, and CYP3A4-mediated nifedipine oxidation activities in human liver microsomes were compared. In addition, the effects of preincubation were estimated to investigate the mechanism-based inhibition. The IC50 value against tolbutamide hydroxylation was the lowest for miconazole (2.0 microM), followed by voriconazole (8.4 microM) and fluconazole (30.3 microM). Similarly, the IC50 value against S-mephenytoin 4'-hydroxylation was the lowest for miconazole (0.33 microM), followed by voriconazole (8.7 microM) and fluconazole (12.3 microM). On the other hand, micafungin at a concentration of 10 or 25 microM neither inhibited nor stimulated tolbutamide hydroxylation and S-mephenytoin 4'-hydroxylation, and the IC50 values for itraconazole against these were greater than 10 microM. These results suggest that miconazole is the strongest inhibitor of CYP2C9 and CYP2C19, followed by voriconazole and fluconazole, whereas micafungin would not cause clinically significant interactions with other drugs that are metabolized by CYP2C9 or CYP2C19 via the inhibition of metabolism. The IC50 value of voriconazole against nifedipine oxidation was comparable with that of fluconazole and micafungin and higher than that of itraconazole and miconazole. The stimulation of the inhibition of CYP2C9-, CYP2C19-, or CYP3A4-mediated reactions by 15-min preincubation was not observed for any of the antifungal drugs, suggesting that these drugs are not mechanism-based inhibitors.     

Drug metabolism by CYP2C8.3 is determined by substrate dependent interactions with cytochrome P450 reductase and cytochrome b5

Genetic polymorphisms in CYP2C8 can influence the metabolism of important therapeutic agents and cause interindividual variation in drug response and toxicity. The significance of the variant CYP2C8*3 has been controversial with reports of higher in vivo but lower in vitro activity compared to CYP2C8*1. In this study, the contribution of the redox partners cytochrome P450 reductase (CPR) and cytochrome b5 to the substrate dependent activity of CYP2C8.3 (R139K, K399R) was investigated in human liver microsomes (HLMs) and Escherichia coli expressed recombinant CYP2C8 proteins using amodiaquine, paclitaxel, rosiglitazone and cerivastatin as probe substrates. For recombinant CYP2C8.3, clearance values were two- to five-fold higher compared to CYP2C8.1. CYP2C8.3's higher k_cat seems to be dominated by a higher, but substrate specific affinity, towards cytochrome b5 and CPR (K_D and K_m,red) which resulted in increased reaction coupling. A stronger binding affinity of ligands to CYP2C8.3, based on a two site binding model, in conjunction with a five fold increase in amplitude of heme spin change during binding of ligands and redox partners could potentially contribute to a higher k_cat. In HLMs, carriers of the CYP2C8*1/*3 genotype were as active as CYP2C8*1/*1 towards the CYP2C8 specific reaction amodiaquine N-deethylation. Large excess of cytochrome b5 compared to CYP2C8 in recombinant systems and HLMs inhibited metabolic clearance, diminishing the difference in k_cat between the two enzymes, and may provide an explanation for the discrepancy to in vivo data. In silico studies illustrate the genetic differences between wild type and variant on the molecular level.     

Stereoselective metabolism of omeprazole by human cytochrome P450 enzymes.

This study demonstrates the stereoselective metabolism of the optical isomers of omeprazole in human liver microsomes. The intrinsic clearance (CL(int)) of the formation of the hydroxy metabolite from S-omeprazole was 10-fold lower than that from R-omeprazole. However, the CL(int) value for the sulfone and 5-O-desmethyl metabolites from S-omeprazole was higher than that from R-omeprazole. The sum of the CL(int) of the formation of all three metabolites was 14.6 and 42.5 microl/min/mg protein for S- and R-omeprazole, respectively. This indicates that S-omeprazole is cleared more slowly than R-omeprazole in vivo. The stereoselective metabolism of the optical isomers is mediated primarily by cytochrome P450 (CYP) 2C19, as indicated by studies using cDNA-expressed enzymes. This is the result of a considerably higher CL(int) of the 5-hydroxy metabolite formation for R- than for S-omeprazole. For S-omeprazole, CYP2C19 is more important for 5-O-desmethyl formation than for 5-hydroxylation. Predictions of the CL(int) using data from cDNA-expressed enzymes suggest that CYP2C19 is responsible for 40 and 87% of the total CL(int) of S- and R-omeprazole, respectively, in human liver microsomes. According to experiments using cDNA-expressed enzymes, the sulfoxidation of both optical isomers is metabolized by a single isoform, CYP3A4. The CL(int) of the sulfone formation by CYP3A4 is 10-fold higher for S-omeprazole than for R-omeprazole, which may contribute to their stereoselective disposition. The results of this study show that both CYP2C19 and CYP3A4 exhibit a stereoselective metabolism of omeprazole. CYP2C19 favors 5-hydroxylation of the pyridine group of R-omeprazole, whereas the same enzyme mainly 5-O-demethylates S-omeprazole in the benzimidazole group. Sulfoxidation mediated by CYP3A4 highly favors the S-form.     

The oxidative metabolism of dimemorfan by human cytochrome P450 enzymes

To characterize the human cytochrome P450 (P450) forms involved in dimemorfan oxidation (DFO), human liver microsomes, and recombinant P450s were investigated. Liquid chromatography-mass spectral analysis suggested that metabolite (M)1 ([M + H]⁺ m/z at 272.200) and M2 ([M + H]⁺ m/z at 242.190) were d-3-hydroxymethyl-N-methylmorphinan and d-3-methylmorphinan, respectively. Kinetic analyses of microsomal DFO showed that the substrate concentration showing a half-maximal velocity (S₅₀) of M1 formation was less than that of M2. Microsomal M1 and M2 formation activities correlated significantly with the CYP2D6 marker, dextromethorphan O-demethylation activity. The M2 formation activity was also correlated with the CYP3A4 marker, nifedipine oxidation activity. Microsomal M1 and M2 formation was most sensitive to the inhibition by a CYP2D6 inhibitor, paroxetine and a CYP3A4 inhibitor, ketoconazole, respectively. The immunoinhibition-defined P450 contributions indicated the participation of CYP2C9, CYP2C19, and CYP2D6 in the M1 formation and CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in the M2 formation. Among recombinant P450s, CYP2D6 had the highest intrinsic clearance with a K_m value of 0.02 mM in forming M1. CYP2B6, CYP2C9, and CYP2C19 had the K_m or S₅₀ values smaller than those (1 mM) of CYP2D6 and CYP3A4 in forming M2. These results indicated the participation of multiple P450 forms in DFO. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1063–1077, 2010     

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)。     

Characterization of ebastine, hydroxyebastine, and carebastine metabolism by human liver microsomes and expressed cytochrome P450 enzymes: major roles for CYP2J2 and CYP3A.

Ebastine undergoes extensive metabolism to form desalkylebastine and hydroxyebastine. Hydroxyebastine is subsequently metabolized to carebastine. Although CYP3A4 and CYP2J2 have been implicated in ebastine N-dealkylation and hydroxylation, the enzyme catalyzing the subsequent metabolic steps (conversion of hydroxyebastine to desalkylebastine and carebastine) have not been identified. Therefore, we used human liver microsomes (HLMs) and expressed cytochromes P450 (P450s) to characterize the metabolism of ebastine and that of its metabolites, hydroxyebastine and carebastine. In HLMs, ebastine was metabolized to desalkyl-, hydroxy-, and carebastine; hydroxyebastine to desalkyl- and carebastine; and carebastine to desalkylebastine. Of the 11 cDNA-expressed P450s, CYP3A4 was the main enzyme catalyzing the N-dealkylation of ebastine, hydroxyebastine, and carebastine to desalkylebastine [intrinsic clearance (CL(int)) = 0.44, 1.05, and 0.16 microl/min/pmol P450, respectively]. Ebastine and hydroxyebastine were also dealkylated to desalkylebastine to some extent by CYP3A5. Ebastine hydroxylation to hydroxyebastine is mainly mediated by CYP2J2 (0.45 microl/min/pmol P450; 22.5- and 7.5-fold higher than that for CYP3A4 and CYP3A5, respectively), whereas CYP2J2 and CYP3A4 contributed to the formation of carebastine from hydroxyebastine. These findings were supported by chemical inhibition and kinetic analysis studies in human liver microsomes. The CL(int) of hydroxyebastine was much higher than that of ebastine and carebastine, and carebastine was metabolically more stable than ebastine and hydroxyebastine. In conclusion, our data for the first time, to our knowledge, suggest that both CYP2J2 and CYP3A play important roles in ebastine sequential metabolism: dealkylation of ebastine and its metabolites is mainly catalyzed by CYP3A4, whereas the hydroxylation reactions are preferentially catalyzed by CYP2J2. The present data will be very useful to understand the pharmacokinetics and drug interaction of ebastine in vivo.