Distribution of CYP2C9*13 allele in the Chinese Han and the long-range haplotype containing CYP2C9*13 and CYP2C19*2

Cytochrome P450 2C9 (CYP2C9) and CYP2C19, located in tandem on chromosome 10q23–24, are known as genetically polymorphic. CYP2C9*13 is an important CYP2C9 variant in Asian populations, and is correlated with the reduced plasma clearance of some clinically important drugs. In this research, the allele frequency of CYP2C9*13 was determined to be 0.42% (95% CI of 0.17% to 0.86%) in 839 Chinese Han, male subjects. All detected subjects with CYP2C9*13 carry the CYP2C19*2 allele, too. Sequencing results infer the CYP2C9*13 haplotype, which contains eight linked SNPs, originates from the CYP2C9*1B haplotype group. CYP2C9*1B has been reported to be linked with CYP2C19*2. These indicate a long‐range haplotype containing the CYP2C9*13 and CYP2C19*2 mutation, which means most CYP2C9*13 carriers will carry the CYP2C19*2 allele and the six SNPs of the CYP2C9*1B haplotype group, and may have more reduced intrinsic clearance of drugs such as phenytoin, tolbutamide and chlorpropamide that are metabolized by both CYP2C9 and CYP2C19. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of a null allele of CYP2C9 in an African-American exhibiting toxicity to phenytoin.

Cytochrome P450 (CYP) 2C9 is the principal enzyme responsible for the metabolism of numerous clinically important drugs. Two polymorphic alleles CYP2C9*2 and CYP2C9*3 have been documented which affect the metabolism and clinical toxicity of drugs such as phenytoin, warfarin, glipizide, and tolbutamide. The present study reports the first example of a null polymorphism in CYP2C9. This mutation dramatically affects the half-life and clinical toxicity of phenytoin. The study subject was a female African-American presented to the emergency department with phenytoin toxicity evidenced by mental confusion, slurred speech, memory loss and the inability to stand. She exhibited extremely poor clearance of phenytoin with an elimination half-life of approximately 13 days. Genotyping studies demonstrated that the patient did not possess any known variant CYP2C9 alleles. Phenytoin is metabolized to a minor extent by the polymorphic CYP2C19, but this individual did not possess any variant CYP2C19 alleles. Sequencing studies revealed that the individual was homozygous for a new CYP2C9 allele (CYP2C9*6) with the deletion of an adenine at base pair 818 of the cDNA. The clearance of phenytoin in this individual is estimated to be approximately 17% of that observed in normal patients. The frequency of this allele was 0.6% (95% confidence limits of 0.1 to 3.5%) in 79 African-Americans and 0% (95% confidence limits of 0 to 1.1%) in 172 Caucasians. The study also demonstrates the severe clinical consequences to patients with a null mutation in CYP2C9 after treatment with normal doses of phenytoin.     

Characterization of the novel defective CYP2C9*24 allele.

CYP2C9 is one of the major drug-metabolizing enzymes, and it is involved in the oxidative metabolism of approximately 10% of clinically important drugs, among which some, such as the anticoagulant warfarin, have a narrow therapeutic index. The human CYP2C9 gene is highly polymorphic. We found a new sequence variation in exon 7 of the CYP2C9 gene (1060G>A) resulting in a substitution of acidic amino acid glutamate to basic lysine (E354K) when translated. The allele, designated CYP2C9(*)24, was present in heterozygous state in one warfarin-treated patient. To characterize the CYP2C9(*)24 allele, we expressed the wild-type and CYP2C9.24 protein in a recombinant yeast expression system and a human embryonic kidney (HEK)-293 cell system. Carbon monoxide difference spectra were recorded on dithionite-reduced microsomes, and protein was determined by Western blotting. Transfection with CYP2C9.1 cDNA resulted in detectable CYP2C9 protein in yeast or HEK-293 cells, whereas only small amounts of the protein were detected in yeast transfected with CYP2C9.24 cDNA. A strong differential absorption peak at 450 nm was observed with microsomes of yeast transfected with CYP2C9.1 cDNA, whereas no peak was detected with microsomes of yeast transfected with CYP2C9.24 cDNA or empty pYeDP60 plasmid. These results suggest that CYP2C9.24 may be improperly folded, both in yeast and mammalian cells, resulting in improper heme incorporation and rapid intracellular degradation. The data obtained in the expression systems are consistent with our findings in vivo. In conclusion, we have identified a novel defective CYP2C9 variant allele of potential importance for drug metabolism in vivo.     

Catalytic roles of CYP2C9 and its variants (CYP2C9*2 and CYP2C9*3) in lornoxicam 5'-hydroxylation.

The effects of allelic variants of CYP2C9 (CYP2C9*2 and CYP2C9*3) on lornoxicam 5'-hydroxylation were studied using the corresponding variant protein expressed in baculovirus-infected insect cells and human liver microsomes of known genotypes of CYP2C9. The results of the baculovirus expression system showed that CYP2C9.3 gives higher K(m) and lower V(max) values for lornoxicam 5'-hydroxylation than does CYP2C9.1. In contrast, K(m) and V(max) values of CYP2C9.1 and CYP2C9.2 for the reaction were comparable. Lornoxicam 5'-hydroxylation was also determined in liver microsomes of 12 humans genotyped for the CYP2C9 gene (*1/*1, n = 7; *1/*2, n = 2; *1/*3, n = 2; *3/*3, n = 1). A sample genotyped as *3/*3 exhibited 8- to 50-fold lower intrinsic clearance for lornoxicam 5'-hydroxylation than did samples genotyped as *1/*1. However, the values for intrinsic clearance for *1/*3 were within the range of values exhibited by samples of *1/*1. In addition, no appreciable differences were observed in kinetic parameters for lornoxicam 5'-hydroxylation between *1/*1 and *1/*2. In conclusion, this study showed that lornoxicam 5'-hydroxylation via CYP2C9 was markedly decreased by the substitution of Ile359Leu (CYP2C9.3), whereas the effect of the substitution of Arg144Cys (CYP2C9.2) was nonexistent or negligible. Additional in vivo studies are required to confirm that individuals with homologous CYP2C9*3 allele exhibit impaired lornoxicam clearance.     

Role of CYP2C9 and its variants (CYP2C9*3 and CYP2C9*13) in the metabolism of lornoxicam in humans.

CYP2C9 is an important member of the cytochrome P450 enzyme superfamily with some 12 CYP2C9 alleles (*1-*12) being previously reported. Recently, we identified a new CYP2C9 allele with a Leu90Pro mutation in a Chinese poor metabolizer of lornoxicam [Si D, Guo Y, Zhang Y, Yang L, Zhou H, and Zhong D (2004) Pharmacogenetics 14:465-469]. The new allele, designated CYP2C9*13, was found to occur in approximately 2% of the Chinese population. To examine enzymatic activity of the CYP2C9*13 allele, kinetic parameters for lornoxicam 5'-hydroxylation were determined in COS-7 cells transiently transfected with pcDNA3.1 plasmids carrying wild-type CYP2C9*1, variant CYP2C9*3, and CYP2C9*13 cDNA. The protein levels of cDNA-expressed CYP2C9*3 and *13 in postmitochondrial supernatant (S9) from transfected cells were lower than those from wild-type CYP2C9*1. Mean values of Km and Vmax for CYP2C9*1, *3, and *13 were 1.24, 1.61, and 2.79 microM and 0.83, 0.28, and 0.22 pmol/min/pmol, respectively. Intrinsic clearance values (Vmax/Km) for variant CYP2C9*3 and CYP2C9*13 on the basis of CYP2C9 protein levels were separately decreased to 28% and 12% compared with wild type. In a subsequent clinical study, the AUC of lornoxicam was increased by 1.9-fold and its oral clearance (CL/F) decreased by 44% in three CYP2C9*1/*13 subjects, compared with CYP2C9*1/*1 individuals. This suggests that the CYP2C9*13 allele is associated with decreased enzymatic activity both in vitro and in vivo.     

Lack of differences in diclofenac (a substrate for CYP2C9) pharmacokinetics in healthy volunteers with respect to the single CYP2C9 * 3 allele

Objectives: Evidence exists to suggest that diclofenac is metabolised by CYP2C9. The present study was undertaken in order to evaluate the effect of the single CYP2C9*3 variant on drug metabolism using diclofenac as a probe drug. Methods: A single dose of diclofenac was administered orally to 12 healthy subjects in whom the genotype of CYP2C9 had been determined previously. The disposition kinetics of diclofenac were compared between homozygotes for the wild type (CYP2C9*1/*1, n=6) and heterozygotes for the Leu359 variant (CYP2C9*1/*3, n=6). Results: For diclofenac, the following kinetic parameters were observed in the CYP2C9*1/*1 and CYP2C9*1/*3 subjects, respectively (mean ± SD): apparent oral clearance (ml/kg/h) 355.8 ± 56.9 and 484.4 ± 155.3; area under plasma concentration–time curve (μg h/ml) 2.7 ± 0.7 and 1.9 ± 0.6. The formation clearance of 4′-hydroxydiclofenac (ml/kg/h) was 63.6 ± 19.1 in the CYP2C9*1/*1 subjects compared with 75.9 ± 27.6 in the CYP2C9*1/*3 subjects. There were no significant differences in any of the kinetic parameters for either diclofenac disposition or formation clearance of 4′-hydroxydiclofenac between the two genotype groups. Conclusion: Since the disposition kinetics of diclofenac does not change in subjects with the single CYP2C9*3 mutant allele, it is suggested that the effects of CYP2C9 polymorphisms on the drug metabolism tend to be substrate specific. Received: 4 October 1999 / Accepted in revised form: 12 January 2000     

Pharmacogenetic relevance of the CYP2C9*3 allele in a tenoxicam bioequivalence study performed on Spaniards

We performed a study to quantify CYP2C9 and CYP2C8 alleles influence on the variability observed in tenoxicam pharmacokinetic (PK) and implication in a bioequivalence study design performed on Spaniards. Eighteen healthy volunteers were included in an open, randomized, crossover, phase I bioequivalence study. Significant increases were found in CYP2C9*3 alleles vs. *1 and *2 in AUC_0–∞ (median (min–max)): 256 (230–516) vs. 150 (100–268) and 169 (124–197) μg h/mL (p < 0.01) and half-life time (t1/2) 102 (79–36) vs. 56 (45–94) and 64 (60–80) h (p < 0.01). Non-significant differences were observed in C_max 1.9 (1.8–2.9) vs. 2.4 (1.7–3.4), 2.5 (1.6–2.9) μg/mL or in according to CYP2C8 alleles presence. CYP2C9*3 allele is associated to a longer elimination time of tenoxicam. PK parameters calculated in bioequivalence studies (AUC_0–∞, t1/2) may be influenced by the presence of CYP2C9*3 allele resulting in a high variability. Thus, bioequivalence studies of tenoxicam formulations should be designed considering genotype profile.     

Linkage disequilibrium between the CYP2C19*17 allele and wildtype CYP2C8 and CYP2C9 alleles: identification of CYP2C haplotypes in healthy Nordic populations

Purpose

To determine the distribution of clinically important CYP2C genotypes and allele frequencies in healthy Nordic populations with special focus on linkage disequilibrium.

Methods

A total of 896 healthy subjects from three Nordic populations (Danish, Faroese, and Norwegian) were genotyped for five frequent and clinically important CYP2C allelic variants: the defective CYP2C8*3, CYP2C9*2, CYP2C9*3, and CYP2C19*2 alleles, and the CYP2C19*17 allele that causes rapid drug metabolism. Linkage disequilibrium was evaluated and CYP2C haplotypes were inferred in the entire population.

Results

Ten CYP2C haplotypes were inferred, the most frequent of which (49%) was the CYP2C wildtype haplotype carrying CYP2C8*1, CYP2C9*1, and CYP2C19*1. The second most frequent haplotype (19%) is composed of CYP2C19*17, CYP2C8*1, and CYP2C9*1. This predicted haplotype accounts for 99.7% of the CYP2C19*17 alleles found in the 896 subjects.

Conclusion

CYP2C19*17 is a frequent genetic variant in Nordic populations that exists in strong linkage disequilibrium with wildtype CYP2C8*1 and CYP2C9*1 alleles, which effectively makes it a determinant for a haplotype exhibiting an efficient CYP2C substrate metabolism.     

Effect of the single CYP2C9*3 allele on pharmacokinetics and pharmacodynamics of losartan in healthy Japanese subjects

Objective Losartan is metabolized to the active carboxylic acid metabolite EXP3174 by CYP2C9. In this study, we determined the effects of the single CYP2C9*3 variant on the pharmacokinetics and pharmacodynamics of losartan.Methods Seven healthy Japanese subjects (CYP2C9*1/*1, n=4 and CYP2C9*1/*3, n=3) were phenotyped with a single dose of losartan (25 mg). Blood and urine samples were collected and assayed for losartan and EXP3174. Blood pressure and pulse rate were also measured using a sphygmomanometer.Results The maximum plasma concentration of EXP3174 was significantly (P<0.05) lower in the CYP2C9*1/*3 (n=3) group than in the CYP2C9*1/*1 (n=4) group. Diastolic blood pressure in the CYP2C9*1/*1 group, but not that in the CYP2C9*1/*3 group except for at 6 h and 8 h, was reduced from 1.5 h to 12 h compared with the baseline level. Systolic blood pressure in the CYP2C9*1/*1 group, but not that in the CYP2C9*1/*3 group, was reduced from 1 h to 12 h compared with the baseline level. The metabolic ratio (MR) of EXP3174 concentration to the losartan concentration in plasma at 6 h post-dosing and the 4-h to 8-h urinary EXP3174/losartan MR were significantly lower in the CYP2C9*1/*3 group than in the CYP2C9*1/*1 group. The plasma 6-h MR and the 4-h to 8-h urinary MR were significantly (P<0.05) correlated with the plasma AUC ratio (AUC_EXP3174/AUC_losartan), with Spearman rank correlation coefficients of 0.75 and 0.89, respectively.Conclusion The single CYP2C9*3 variant reduces the metabolism of losartan and its hypotensive effect. Plasma MR, as well as urine MR, may be useful for phenotyping assays of CYP2C9 activity.     

Investigation of allele and genotype frequencies of CYP2C9, CYP2C19 and VKORC1 in Iran

Research has shown that there are significant ethnic variations in the frequency of highly functional mutations in genes coding for metabolic enzymes. However, few studies have examined the frequency distribution of major allelic variations within the population of Iran. The present study focused on the genotype profile of southern Iranians in order to compare the allelic frequencies of CYP2C9, CYP2C19, and VKORC1 -1639G>A (all of which have been shown to have significant roles in the metabolism of warfarin) with those of other populations. Therefore, genotyping was carried out on 150 subjects (50 healthy volunteers and 100 outpatient subjects) by polymerase chain reaction- restriction length polymorphism (PCR-RFLP). Findings indicated both similarities and differences in the distribution of polymorphic alleles of CYP2C9, CYP2C19 and VKORC1 between southern and northern Iranians. For example, the frequency of CYP2C9*3 among southern Iranians (9.8%) was found to be similar to the frequency found among Caucasians (in this case, Italians) (9.7%) but was higher than the frequency found among Africans (1%), Japanese (2.3%), and northern Iranians (0%). These findings confirmed significant inter-ethnic differences in CYP2C9 frequencies between southern and northern Iranians The reported frequency of CYP2C9*2 in our subjects (25.3%) was different from the frequencies seen in Caucasian (10-13%), African (2%) and Asian (0%) populations. The CYP2C19*2 and CYP2C19*3 allelic frequencies were similar to the Caucasian population. For VKORC1, the allelic frequency of -1639A (55.6%) was in accordance with Caucasian, but different from Chinese (96%) and African-American populations (13%). The findings confirmed some important interethnic differences in the metabolic capacity for drug clearance. Because the population of Iran consists of several ethnicities, this type of analysis can help explain the genetic diversity between the populations of northern and southern Iran. In addition, the results of this study will be useful for understanding clinical pharmacokinetics and drug dosage recommendations for Iranians.     

Polymorphic variants (CYP2C9*3 and CYP2C9*5) and the F114L active site mutation of CYP2C9: effect on atypical kinetic metabolism profiles.

CYP2C9 wild-type protein has been shown to exhibit atypical kinetic profiles of metabolism that may affect in vitro-in vivo predictions made during the drug development process. Previous work suggests a substrate-dependent effect of polymorphic variants of CYP2C9 on the rate of metabolism; however, it is hypothesized that these active site amino acid changes will affect the kinetic profile of a drug's metabolism as well. To this end, the kinetic profiles of three model CYP2C9 substrates (flurbiprofen, naproxen, and piroxicam) were studied using purified CYP2C9*1 (wild-type) and variants involving active site amino acid changes, including the naturally occurring variants CYP2C9*3 (Leu359) and CYP2C9*5 (Glu360) and the man-made mutant CYP2C9 F114L. CYP2C9*1 (wild-type) metabolized each of the three compounds with a distinctive profile reflective of typical hyperbolic (flurbiprofen), biphasic (naproxen), and substrate inhibition (piroxicam) kinetics. CYP2C9*3 metabolism was again hyperbolic for flurbiprofen, of a linear form for naproxen (no saturation noted), and exhibited substrate inhibition with piroxicam. CYP2C9*5-mediated metabolism was hyperbolic for flurbiprofen and piroxicam but linear with respect to naproxen turnover. The F114L mutant exhibited a hyperbolic kinetic profile for flurbiprofen metabolism, a linear profile for naproxen metabolism, and a substrate inhibition kinetic profile for piroxicam metabolism. In all cases except F114L-mediated piroxicam metabolism, turnover decreased and the K(m) generally increased for each allelic variant compared with wild-type enzyme. It seems that the kinetic profile of CYP2C9-mediated metabolism is dependent on both substrate and the CYP2C9 allelic variant, thus having potential ramifications on drug disposition predictions made during the development process.     

Human CYP2C-mediated stereoselective phenytoin hydroxylation in Japanese: difference in chiral preference of CYP2C9 and CYP2C19

Regio- and stereoselective hydroxylation of phenytoin was determined in liver microsomes of nine extensive (EM) and three poor metabolizers (PM) of mephenytoin. Hydroxyphenytoins (HPPH) were isolated and quantified after separation into four regio- and stereoisomers. The total rates of microsomal phenytoin 4'- hydroxylation were approximately 3-fold higher than those of 3'-hydroxylation, and not significantly different in EM and PM. Formation of 4'-(R)-HPPH was 4.4-fold higher in EM than in PM, whereas no clear differences between EM and PM were detected in the formation of 4'-(S)-, 3'-(R)-, and 3'-(S)-HPPH. Cytochrome P450 (CYP)2C9, expressed in a fission yeast, Schizosaccharomyces pombe, catalyzed the formation of 4'-(R)- and 4'-(S)-HPPH stereoselectively, as observed with EM, in which predominantly 4'-(S)-HPPH was formed. Recombinant CYP2C19 was more stereoselective for 4'-(R)-HPPH formation. These results, in addition to inhibition experiments with anti-human CYP2C antibody, indicate that phenytoin hydroxylation is mainly catalyzed by CYP2C9. Furthermore, CYP2C19 showed limited contribution to phenytoin 4'-hydroxylation with a different chiral preference from CYP2C9.     

Dosage recommendation of phenytoin for patients with epilepsy with different CYP2C9/CYP2C19 polymorphisms

To search for the optimal dosage of phenytoin in patients with epilepsy based on the metabolic activities of CYP2C9 and CYP2C19 polymorphisms, a total of 169 patients receiving phenytoin treatment for more than 1 month were recruited. Phenytoin concentration, serum albumin, liver function tests, and renal function tests were measured. CYP2C9 and CYP2C19 polymorphisms were genotyped by PCR-RFLP analysis, and NONMEM models were built to evaluate factors that would affect phenytoin metabolism. Patients were divided into 5 groups according to genotyping results (G1 to G5). Compared with extensive metabolizers in both CYP2C9 and CYP2C19 (G1), the Vmax (mg/kg/d) was 8.29% and 36.96% lower in CYP2C19 poor metabolizers (G3) and CYP2C9 poor metabolizers (G4), respectively. For the patient who was identified as a poor metabolizer in both CYP2C19 and CYP2C9 (G5), the Vmax was 45.75% lower than that of G1. In respect to Km (mg/L), it was 15.09% higher in G3 and 27.36% higher in G4 compared with that in G1. The Km of G5 was 91.71% higher than that of G1. The results revealed that the CYP2C9 and CYP2C19 polymorphisms have dramatic effects on the population pharmacokinetic parameters of phenytoin, especially for CYP2C9. Based on the Vm and Km values obtained in this study, the recommended dose ranges for G1, G2, G3, G4, and G5 patients would be 5.5-7, 5-7, 5-6, 3-4, and 2-3 mg/kg/d, respectively.     

The effects of CYP2C9 and CYP2C19 genetic polymorphisms on the pharmacokinetics and pharmacodynamics of glipizide in Chinese subjects

Objective  

To study the effects of CYP2C9 and CYP2C19 genetic polymorphisms on the pharmacokinetics and pharmacodynamics of glipizide.     

细胞色素P450 CYP2C9*3对格列本脲和氯诺昔康中国人体药代动力学的影响

目的研究人体内细胞色素P450 2C9酶突变等位基因CYP2C9*3对格列本脲和氯诺昔康药代动力学的影响。方法采用PCR-RFLP方法对83名无血源关系的受试者进行CYP2C9*3等位基因的筛查，基因型为CYP2C9*1/*3(n=7)和*1/*1(n=11)的受试者分别参加了格列本脲和氯诺昔康的人体药代动力学试验。采用LC/MS/MS法分别测定受试者口服格列本脲(2.5 mg)和氯诺昔康(8 mg)后不同时刻血浆中格列本脲和氯诺昔康的浓度。结果两组受试者口服格列本脲后，CYP2C9*1/*3组AUC_0-∞显著增加，为CYP2C9*1/*1组的1.5倍，CL/F降低了40%；两组受试者口服氯诺昔康后，CYP2C9*1/*3组AUC_0-∞亦显著增加，为CYP2C9*1/*1组的2.2倍，CL/F降低了55%。结论CYP2C9酶的突变等位基因CYP2C9*3对格列本脲和氯诺昔康的药代动力学有显著性影响。