A novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo

Cytochrome P450 (CYP) 2A6 is a major CYP responsible for the metabolism of nicotine and coumarin in humans. We identified a novel allele, designated CYP2A6*17 , which contains A51G (exon 1), C209T (intron 1), G1779A (exon 3), C4489T (intron 6), G5065A (V365M, exon 7), G5163A (intron 7), C5717T (exon 8), and A5825G (intron 8). We developed a genotyping method by polymerase chain reaction-restriction fragment length polymorphism for the CYP2A6*17 allele, targeting the G5065A mutation. The allele frequency in black subjects (n = 96) was 9.4% (95% confidence interval [CI], 5.3%-13.5%). The allele was not found in white subjects (95% CI, 0%-0.9%; n = 163), Japanese subjects (95% CI, 0%-1.6%; n = 92), and Korean subjects (95% CI, 0%-0.7%; n = 209). To examine the effects of the amino acid change in the CYP2A6*17 allele on the enzymatic activity, we expressed a wild-type or variant (V365M) CYP2A6 together with NADPH-CYP reductase in Escherichia coli . For coumarin 7-hydroxylation, the apparent Michaelis-Menten constant value of variant CYP2A6 (1.06 +/- 0.11 micromol/L) was significantly (P < .005) higher than that of wild type (0.60 +/- 0.05 micromol/L). The maximum velocity values of the wild-type and variant CYP2A6 were 0.61 +/- 0.06 and 0.64 +/- 0.07 pmol . min -1 . pmol -1 CYP, respectively. For nicotine C -oxidation, the apparent Michaelis-Menten constant values of the wild-type or variant CYP2A6 were 31.6 +/- 2.9 micromol/L and 31.3 +/- 3.1 micromol/L, respectively. The maximum velocity value of variant CYP2A6 (0.72 +/- 0.21 pmol . min -1 . pmol -1 CYP) was significantly (P < .05) lower than that of the wild type (1.80 +/- 0.42 pmol . min -1 . pmol -1 CYP). Thus the intrinsic clearance values for coumarin 7-hydroxylation and nicotine C -oxidation by the variant were both significantly (P < .05) decreased to 40% to 60% compared with the wild type. Furthermore, cotinine/nicotine ratios after 1 piece of nicotine gum was chewed, used as an index of in vivo nicotine metabolism, were significantly (P < .05) decreased in heterozygotes of the CYP2A6*17 allele (5.4 +/- 2.7, n = 12) compared with homozygotes of the wild type (11.5 +/- 10.5, n = 37). A subject with CYP2A6*17 / CYP2A6*17 revealed the lowest cotinine/nicotine ratio (1.8). We found a novel allele in black subjects that affects the nicotine metabolism in vitro and in vivo.     

Genetic polymorphisms of cytochrome P450 among patients with Balkan endemic nephropathy (BEN)

OBJECTIVES: The concept of multifactorial etiology of BEN anticipates that a combination of polymorphic gene variants and various environmental factors causes an increased risk for the disease. CYP enzymes play a key role in the metabolic activation of environmental chemicals and toxins. CYP3A enzymes are particularly relevant for xenobiotic metabolism because of their broad substrate specificity and abundant expression in the human liver, intestine, and kidney. Previous phenotyping analysis on CYP2D6 enzyme activity in BEN patients proposed a modifying effect of CYP2D6 gene variants on BEN risk, but it was not approved with molecular-genetic methods. The aim of the current case-control study was to compare the frequency of CYP2D6 and CYP3A5 polymorphisms, as well as one CYP3A4 promoter variant in BEN patients and controls in order to investigate a possible association between individual genetic variations in these genes and susceptibility to BEN. DESIGN AND METHODS: Ninety-six nonrelated Bulgarian BEN patients from endemic villages in the Vratza district and 112 healthy Bulgarians from nonendemic areas (controls) were genotyped. Identification of alleles was done by allele-specific PCR or by rapid-cycle amplification on the LightCycler, followed by sequence-specific detection. RESULTS: The UM, PM, and EM + IM genotype frequencies of CYP2D6 did not differ significantly between the two groups (P > 0.05). The CYP3A4*1B allele was only found in the heterozygous form, with allelic frequencies of 5.21% in the patients and 2.23% in the healthy individuals (P = 0.11). The CYP3A5*1 allele was more prevalent in BEN patients with a frequency of 9.38% compared to 5.36% in the controls and was associated with a higher risk for BEN (OR 2.41, 95% CI 1.09-5.33) (P = 0.02). CONCLUSIONS: Our results demonstrate that the CYP3A5*1 allele, previously reported as a marker for CYP3A5 expression in human kidney, is associated with increased risk for BEN, while CYP3A4*1B and CYP2D6 genotypes do not significantly modify the risk for the disease.     

Renal-tubule metabolism of ifosfamide to the nephrotoxic chloroacetaldehyde: pharmacokinetic modeling for estimation of intracellular levels

Ifosfamide (IF) improves survival in children with solid tumors but causes a high rate of nephrotoxicity. We hypothesized that this is caused by an oxidative metabolite of IF, chloroacetaldehyde, which is produced locally by the cells of the renal tubule (RT). For this hypothesis to be viable, one must document that chloroacetaldehyde concentrations in the RT cell are consistent with levels shown to cause nephrotoxicity in experimental systems. Using pharmacokinetic modeling of experimental data, we show that the median level of chloroacetaldehyde in RT cells is 80 micromol/L, ranging from 35 to 320 micromol/L. These concentrations are consistent with levels shown experimentally to cause functional and structural RT damage and lends validity to the hypothesis that local renal production of chloroacetaldehyde causes nephrotoxicity.     

Pharmacokinetic interaction between ketoconazole and praziquantel in healthy volunteers

BACKGROUND: Praziquantel, a broad-spectrum anthelminthic, has been reported to undergo extensive first-pass metabolism by cytochrome P450 (CYP) enzymes in vivo. Ketoconazole, a potent CYP3A4 inhibitor, is known to markedly increase plasma concentrations of many co-administered drugs. However, no data are available on the potential pharmacokinetic drug interaction between ketoconazole and praziquantel in humans. OBJECTIVE: To investigate the potential pharmacokinetic interaction of ketoconazole with praziquantel in healthy adult Thai male volunteers. METHODS: In an open-label, randomized two-phase crossover study, separated by a 2-week period, 10 healthy adult Thai male volunteers ingested a single dose of 20 mg/kg praziquantel alone or with co-administration of 400-mg ketoconazole orally daily for 5 days. Venous blood samples were collected at specific times for a 24-h period. Plasma concentrations of praziquantel were determined using high-performance liquid chromatography. A non-compartmental model was applied for pharmacokinetic parameter analysis of praziquantel. RESULTS: Concurrent administration of ketoconazole with praziquantel significantly increased the mean area under the curve from time zero to infinity (AUC(0-alpha)) and maximum plasma concentration (Cmax) of praziquantel by 93% (955.94 +/- 307.74 vs. 1843.10 +/- 336.39 ng h/mL; P < 0.01) and 102% (183.38 +/- 43.90 vs. 371.31 +/- 44.63 ng/mL; P < 0.01), respectively, whereas the mean total clearance (Cl/F) of praziquantel was significantly decreased by 58% (2.65 +/- 0.64 vs. 1.11 +/- 0.35 mL/h/kg; P < 0.01). CONCLUSION: Ketoconazole co-administration alters the pharmacokinetics of praziquantel in humans, possibly through inhibition of CYP3A, particularly CYP3A4, first-pass metabolism of praziquantel. Our data suggest that when praziquantel is co-administered with ketoconazole, the dose of praziquantel could be reduced to half the standard dose of praziquantel to reduce the cost of therapy.     

CYP3A5 and CYP3A4 but not MDR1 single-nucleotide polymorphisms determine long-term tacrolimus disposition and drug-related nephrotoxicity in renal recipients

The impact of CYP3A and MDR1 gene single-nucleotide polymorphisms on long-term tacrolimus disposition and drug-related toxicity has not been assessed. A study was performed in 95 genotyped recipients by measuring (12 and 4 h) concentration-time curves on day 7; 3, 6 months; 1, 2, 3, 4, and 5 years after transplantation. In contrast to recipients carrying the CYP3A4*1/CYP3A5*1 or CYP3A4*1B/CYP3A5*1 genotypes, dose-corrected tacrolimus exposure almost doubled over 5 years in patients with the CYP3A4*1/ CYP3A5*3 genotype (AUC(0-12 h): from 41.7+/-18.7 to 80+/-39.2 ng h/ml/mg; P<0.05), whereas apparent oral steady-state clearance and dose requirements significantly decreased accordingly. The CYP3A4*1/CYP3A5*1 and CYP3A4*1B/CYP3A5*1 genotypes were significantly more frequently associated with the development of biopsy-proven tacrolimus-related nephrotoxicity than the CYP3A4*1/ CYP3A5*3 genotype (37.5 vs 11.2%; P=0.03 and 42.8 vs 11.2%; P=0.02). The lack of a time-related increase in dose-corrected tacrolimus exposure observed with the CYP3A4*1/CYP3A5*1 and CYP3A4*1B/CYP3A5*1 genotypes is associated with tacrolimus-related nephrotoxicity, possibly as a result of higher concentrations of toxic metabolites.     

Major role of human liver microsomal cytochrome P450 2C9 (CYP2C9) in the oxidative metabolism of celecoxib, a novel cyclooxygenase-II inhibitor

In vitro studies were conducted to identify the cytochromes P450 (CYP) involved in the oxidative metabolism of celecoxib. The hydroxylation of celecoxib conformed to monophasic Michaelis-Menten kinetics (mean +/- S.D., n = 4 livers, K(m) = 3.8 +/- 0.95 microM, V(max) = 0.70 +/- 0.45 nmol/min/mg protein) in the presence of human liver microsomes, although substrate inhibition was significant at higher celecoxib concentrations. The treatment of a panel of human liver microsomal samples (n = 16 subjects) with antibodies against CYP2C9 and CYP3A4 inhibited the formation of hydroxy celecoxib by 72 to 92% and 0 to 27%, respectively. The presence of both antibodies in the incubation suppressed the activity by 90 to 94%. In addition, the formation of hydroxy celecoxib significantly correlated with CYP2C9-selective tolbutamide methyl hydroxylation (r = 0.92, P <. 001) and CYP3A-selective testosterone 6beta-hydroxylation (r = 0.55, P <.02). In contrast, correlation with activities selective for other forms of CYP was weak (r 相似文献   

Contribution of CYP3A5 to the in vitro hepatic clearance of tacrolimus

BACKGROUND: Tacrolimus is metabolized predominantly to 13-O-demethyltacrolimus in the liver and intestine by cytochrome P450 3A (CYP3A). Patients with high concentrations of CYP3A5, a CYP3A isoenzyme polymorphically produced in these organs, require higher doses of tacrolimus, but the exact mechanism of this association is unknown. METHODS: cDNA-expressed CYP3A enzymes and a bank of human liver microsomes with known CYP3A4 and CYP3A5 content were used to investigate the contribution of CYP3A5 to the metabolism of tacrolimus to 13-O-demethyltacrolimus as quantified by liquid chromatography-tandem mass spectrometry. RESULTS: Demethylation of tacrolimus to 13-O-demethyltacrolimus was the predominant clearance reaction. Calculated K(m) and V(max) values for CYP3A4, CYP3A5, and CYP3A7 cDNA-expressed microsomes were 1.5 micromol/L and 0.72 pmol x (pmol P450)(-1) x min(-1), 1.4 micromol/L and 1.1 pmol x (pmol P450)(-1) x min(-1), and 6 micromol/L and 0.084 pmol x (pmol P450)(-1) x min(-1), respectively. Recombinant CYP3A5 metabolized tacrolimus with a catalytic efficiency (V(max)/K(m)) that was 64% higher than that of CYP3A4. The contribution of CYP3A5 to 13-O-demethylation of tacrolimus in human liver microsomes varied from 1.5% to 40% (median, 18.8%). There was an inverse association between the contribution of CYP3A5 to 13-O-demethylation and the amount of 3A4 protein (r = 0.90; P <0.0001). Mean 13-O-demethylation clearances in CYP3A5 high and low expressers, estimated by the parallel-tube liver model, were 8.6 and 3.57 mL x min(-1) x (kg of body weight)(-1), respectively (P = 0.0088). CONCLUSIONS: CYP3A5 affects metabolism of tacrolimus, thus explaining the association between CYP3A5 genotype and tacrolimus dosage. The importance of CYP3A5 status for tacrolimus clearance is also dependent on the concomitant CYP3A4 activity.     

Stereoselective metabolism of cisapride and enantiomer-enantiomer interaction in human cytochrome P450 enzymes: major role of CYP3A.

Cisapride is a chiral molecule that is marketed as a racemate consisting of two optical isomers, but little is known about its stereoselective metabolism. Studies with (-)-, (+)-, and (+/-)-cisapride were undertaken in human liver microsomes (HLMs) and recombinant cytochrome P450s (P450s) to determine the stereoselective metabolism and enantiomer-enantiomer interaction. Each enantiomer and racemic cisapride were N-dealkylated to norcisapride (NORCIS) and hydroxylated to 3-fluoro-4-hydroxycisapride (3-F-4-OHCIS) and 4-fluoro-2-hydroxycisapride (4-F-2-OHCIS). The kinetics for the formation of NORCIS from (-)-cisapride (Km = 11.9 +/- 4.8 microM; Vmax = 203 +/- 167 pmol/min/mg of protein) or (+)-cisapride (Km = 18.5 +/- 4.7 microM; Vmax = 364 +/- 284 pmol/min/mg of protein) in HLMs exhibited simple Michaelis-Menten kinetics, while a sigmoidal model characterized those of 3-F-4-OHCIS and 4-F-2-OHCIS. In vitro, NORCIS appears to be the major metabolite of both enantiomers. NORCIS and 3-F-4-OHCIS were preferentially formed from (+)-cisapride rather than (-)-cisapride, but that of 4-F-2-OHCIS was the reverse, suggesting regio- and stereoselective metabolism. The formation rate of each metabolite from each enantiomer (20 microM) in 18 HLMs was highly variable (e.g., NORCIS, >35-fold) and correlated with the activity of CYP3A (r = 0.6-0.85; p < 0.05). Coincubation of troleandomycin (50 microM) with cisapride enantiomers (15 microM) in HLMs resulted in potent inhibition of NORCIS formation (by 75-80%), while other inhibitors showed negligible effect. Of 10 recombinant human P450s tested, CYP3A4 catalyzed the formation of NORCIS, 3-F-4-OHCIS, and 4-F-2-OHCIS from each enantiomer and racemic cisapride (15 microM) with the highest specific activity (Km values close to those in HLMs). We noted that the rate of racemic cisapride metabolism by HLMs and recombinant human CYP3A4 is slower compared with equimolar concentrations of each enantiomer. When incubated simultaneously in HLMs, the enantiomers inhibit each other's metabolism. In conclusion, our data demonstrate for the first time the stereoselective metabolism and enantiomer-enantiomer interaction of cisapride. Provided that the potency or the response of the enantiomers differ, understanding the factors that control their disposition as opposed to that of racemic cisapride may better predict adverse drug interactions and the resulting prokinetic efficacy and cardiac safety of cisapride.