Identification Of A Novel Cytochrome P450 2C9 Allele And Its Impact On Drug Metabolism

Variation in drug toxicity and efficacy is consistently observed across patient populations. It contributes to enviromental and genetic factors. Enviromental factors contain irrational administrating, nutritional intake, smoking and drinking, etc. Genetic factors refer to a number of inherited sequence variants of genes encoding drug-metabolising enzymes and drug receptors, which are the major cause of interindividual and ethnic variability.Cytochrome P450 2C9 (CYP2C9), a member of the CYP2C enzyme subfamily, ranks amongst the most important drug metabolizing enzymes in human. It makes up about 20% of the total Cytochrome P-450 protein in liver microsomes, and hydroxylates about 16% of drugs in current clinical use.The CYP2C9 gene spans approximately 55-kb with nine exons and encodes a protein of 490 amino acid residues. Human CYP2C9 has been shown to exhibit geneticly polymorphic. In addition to the wild-type protein CYP2C9*1, at least eleven single nucleotide polymorphisms (SNPs) have been reported within the coding region of the CYP2C9 gene producing the variant allozymes,CYP2C9*2-CYP2C9*12. Most of the variant alleles are associated with altered enzyme activity. CYP2C9*3 (Ile359Leu) is the most widely studied allele, The frequency of CYP2C9 alleles among Caucasian, African, and Asian populations is 7%, 3% and 2%, respectively. In Chinese populations, the most important variant allele is CYP2C9*3 with a gene frequency of about 3.3%, while the other CYP2C9 alleles are rare or absent.Discovery of CYP2C9 alleles has stimulated numerous in vitro and in vivo investigations designed to evaluate their catalytic activities. The COS cell system is among the most widely used for the transient expressions of P450 enzymes because of its simplicity and have endogenous P450 reductase and cytochrome bs to support enzyme activities. However, due to the low levels of expression, different spectroscopy cannot easily and accurately be used to determine absolute P450 holoenzyme contents, and COS cell system is fit for the initial determination of catalytic activities.A number of investigations have demonstrated significantly lower intrinsic clearance of certain CYP2C9 substrates in cells expressing the CYP2C9*3 allele compared with wild-type. It contributes to the different extent of increase of Km and decrease of Fmax. In addition, human studies have shown that, in homozygous carriers of the *3 allele, mean clearances of S-warfarin, tolbutamide, glipizide, celecoxid, and fluvastatin were 25% below that of the wild type. In the more common heterozygous carriers of the *3 allele, the clearances were between 40% and 75% of wild type. Therefore, CYP2C9*3 allele is often used to be the positive control for catalytic activities.CYP2C9 has been shown as the primary enzyme responsible for thebiotransformation of the NSAID lornoxicam to its major metabolites, 5'-hydroxylornoxicam in human liver microsomes. Previously Laboratory of Drug Metabolism and Pharmacokinetics in Shenyang Pharmaceutical University studied the lornoxicam pharmacokinetics in 18 subjects and found a subject with lornoxicam half-life of about 105 hours was markedly longer than that of other subjects. In the present study, we performed genetic analysis of the CYP2C9 gene in this Chinese poor metabolizer and found identified a new variant CYP2C9 allele involving a T269C transversion in exon 2 that leads to a Leu90Pro substitution in the encoded protein (CYP2C9*13). Frequency analysis indicated approximately 2% of the Chinese population carry the allele. Then, the pharmacokinetics of tolbutamide in the PM subjects was investigated. The results showed that he was a phenotypically poor metabolizer of tolbutamide as well. Since tolbutamide is widely accepted as a probe substrate for the assessment of hepatic CYP2C9 activity in vivo, this study confirms that the study subject has severely impaired CYP2C9 catalytic efficiency. The result provides further evidence that lornoxicam pharmacokinetics is a reliable indicator of the genetic polymorphism in the CYP2C9 gene. The CYP2C9 genotype analysis in the family of the poor metabolizer was then performed and showed the new exon 2 change and CYP2C9*3 occurred on different alleles. Thus the PM status of this subject could be attributed to his being heterozygous for the CYP2C9* 13 allele together with the CYP2C9*3, suggesting that this novel CYP2C9 allele was correlated with reduced plasma clearance of both lornoxicam and tolbutamide.To further characterize the catalytic activity of CYP2C9*13 in vitro, we separately expressed CYP2C9*1, CYP2C9*3 and CYP2C9*13 protein in COS-7cells and compared their functional differences toward three CYP2C9 substrates one of which was luminescent. The expression levels of CYP2C9 protein were quantified by immunoblotting with microsomes from insect cells expressing human CYP2C9 as standard. The protein levels of cDNA-expressed CYP2C9*3 and * 13 in postmitochondrial supernatant (S9) from COS-7 cells were lower than in wild-type CYP2C9*1. Our studies clearly showed that relative to the wild type, CYP2C9*13 had lower intrinsic clearance of all investigated substrates. This contributes to the different extent of increase in Km and decrease or no change in Fmax. The reduction of intrinsic clearance altered with the substrates, indicating the metabolic impact of the CYP2C9*13 depends on the substrate being metabolized. CYP2C9*13 had lower intrinsic clearance than CYP2C*3 due to more increase in Km and more decrease in Fmax. This suggests that C YP2C9* 13 had lower affinity and catalytic efficiency towards all three substrate relative to CYP2C9*3. The Ile359Leu (*3) is located in a region coding for substrate recongnition site 5 of the C YP2C9 protein, accounting for the lower binding capacity and intrinsic clearance observed relative to wild-type. However, the Leu90Pro (*13) having lower catalytic activity compared with CYP2C*3 is located in non-heme-binding region and far from the binding pocket of substrate according to a crystal structure of CYP2C9 published by Williams et al. and Wester et al. Thus the mechanism which explains the CYP2C9*13-mediated reduction of drug metabolism is not very clear. Homology modeling based on the crystal structure of human CYP2C9 is ongoing in our laboratory.Compared with wild type CYP2C9*1, the CYP2C9*13 variant also has lower intrinsic clearance for lornoxicam 5'-hydroxylation due to a 2.3-fold increase inKm and 73% decrease in Fmax on the basis of CYP2C9 protein level. The results are consistent with our in vivo observation that individuals with CYP2C9*1/*13 genotype have an impaired clearance of lornoxicam compared to individuals with CYP2C9*1/*1 genotype. Interestingly, in our study, individuals with CYP2C9*l/*3 and CYP2C9*1/*13 genotypes reveal the same extent of reduction in oral clearance of lornoxicam despite the fact that, in vitro, CYP2C9*I3 is associated with a lower intrinsic clearance of lornoxicam than CYP2C9*3.Given the small number of CYP2C9*!/*13 subjects studied (n=3), and in the absence of any individuals homozygous for the CYP2C9*13 allele, we recognize that further in-vivo studies are required in order to draw firm conclusions about the role of the CYP2C9*13 allele.In conclusion, relative to the wild type, the Leu90Pro substitution of CYP2C9*15 markedly decreases the intrinsic clearance of all studied substrates in vitro or in vivo. Thus individuals with CYP2C9*13 allele may also face the safe question of clinical drugs administration as do individuals with CYP2C9*3 allele. It was reported that individuals with CYP2C9*3 allele had a 66%-90% decrease of intrinsic clearance of S -warfarin compared with wild-type and were more at risk of bleeding. When administrating to tolbutamide, an antidiabetic agents, individuals with CYP2C9*3 allele were more liable for the occurrence of adverse drug effects. An individual with CYP2C9*3 allele caused the central nerve toxicity after orally routine dose of phenytoin. Therefore CYP2C9 polymorphisms are particularly relevant to the metabolism of drugs with narrow therapeutic indices such as warfarin, tolbutamide and phenytoin. Our in-vitro results showed that the magnitude of CYP2C9*13-reduced drug metabolism is larger than that of...