Genotype-phenotype Association Analysis Of The Polymorphic Drug-metabolizing Enzymes With The Metabolism Of Anti-cancer Drugs In Human Liver Microsomes

Background:Cancer chemotherapy is characterized by a broad range of efficacy and toxicity among patients, which is closely related to the wide interindividual variability in pharmacokinetics and narrow therapeutic windows of most anticancer drugs. Since drug metabolism is often an essential determinant of interindividual variability in pharmacokinetics, and pharmacogenomic studies of drug-metabolizing enzymes are of great importance for predicting the efficacy and toxicity of the anti-cancer drugs, as well as guidance of the personalized cancer chemotherapy.Tegafur-based chemotherapy has been widely used in the treatment of gastrointestinal cancers and other tumors. It is a pro-drug, primarily converted to 5-fluorouracil (5-FU) by cytochrome P450 2A6 (CYP2A6) in human liver to exert its antitumor effect. CYP2A6 is highly polymorphic, and shows large interindividual and interethnic variations in its expression levels and conversion activities. Studies have demonstrated that CYP2A6 genotype is associated with the differences in pharmacokinetics and efficacy of tegafur-based chemotherapy.Irinotecan, an anticancer prodrug, is currently used as the first-line chemotherapy for metastatic colorecta cancer. It is activated by carboxylesterases to SN-38 (7-ethyl-10-hydroxycamptothecin), which is then further metabolized in the liver by uridine diphosphateglucuronosyltransferases (UGTs) to an inactive metabolite, SN-38 glucuronide (SN-38G). Among the UGT isoforms, UGT1A1 and UGT1A9 were identified as the main hepatic enzymes involved in process of SN-38 glucuronidation, they are encoded by a single gene by exon sharing of individual exons 1 with common exons 2 to 5 in UGT1A gene locus. Numerous studies have demonstrated that the genetic polymorphisms of UGT1A are associated with the differences in pharmacokineitics of SN-38 and the efficacy and toxicity of irinotecan-base chemotherapy.Aim:To comprehensively investigate the distribution of genetic polymorphisms of CYP2A6, UGT1A1, and UGT1A9 in Chinese population, and analyze their effects on the metabolism of tegafur and SN-38 in human liver microsomes, respectively, thus to reveal the molecular mechanism underlying the variability of tegafur and SN-38 metabolism in Chinese livers.Methods:A panel of 53 livers was genotyped mainly by sequencing, including the promotors, all the exons and adjacent introns, and 3'untranslated region (3'UTR) of CYP2A6, UGT1A1, and UGT1A9. By using real-time PCR, immunoblotting, and high performance liquid chromatography, the mRNA and protein levels of detected genes, as well as the metabolites formation from tegafur and SN-38 in over 45 human livers were detected, respectively. Finally, using statistical methods, the effects of identified genetic polymorphisms and haplotypes on measured phenotypes were analyzed.Results;1. Association analysis of CYP2A6 genotypes and haplotypes with 5-fluorouracil formation from tegafur in human liver microsomes1) A 6-fold variation in 5-FU formation rates from tegafur was detected across 45 human liver microsome samples, and it was significantly correlated with CYP2A6 mRNA and protein expression levels. 2) In the present study,20 different variants and 20 haplotypes of CYP2A6 were identified in a panel of 53 Chinese livers; the variation pattern in this Chinese population was generally comparable with other Asian populations but markedly different from whites and blacks.3) Livers carrying CYP2A6*4 showed significantly lower 5-FU formation rates and CYP2A6 mRNA/protein levels compared with CYP2A6*4 non-carriers. Haplotype14, a novel CYP2A6*1B allele, was associated with increased 5-FU formation rates and CYP2A6 expression levels; this may be attributed to the combined effects of three single variants (g.22C>T, g.1620T>C, and a gene conversion in the 3'UTR) included in this haplotype.2. Association analysis of UGT1A genotypes and haoplotypes with SN-38 glucuronidation activity in human liver microsomes1) A 20-fold variation in SN-38 glucuronidation rates was detected across 45 human liver microsome samples, and it was highly correlated with UGT1A1 mRNA and protein expression levels, but poorly correlated with UGT1A9 mRNA or protein levels.2) A total of 36 variants were identified in UGT1A1 and UGT1A9. According to the linkage disequilibrium analysis, UGT1A can be divided into three blocks:promoter regions and exon 1 of UGT1A9 and UGT1A1 in blockl and block2, respectively, and the common exons 2 to 5 in block3. Analysis of the estimated UGT1A haplotypes showed that linkage of the functional polymorphic sites or haplotypes was not only found within the blocks but also existed between the blocks. 3) Genotype-phenotype association analysis showed that UGTlA9*lb was associated with increased SN-38G formation rates and UGT1A9 mRNA/protein levels. However, a further analysis revealed that the increased effect of UGT1A9*lb are probably caused by its close linkage with high enzyme activity allele UGT1A1*1. UGT1A1*60 was associated with decreased SN-38G formation rates and UGT1A1 mRNA/protein levels, when it combined with UGT1A1*6 or*1B haplotype, would make its decreased effect more intensified. The decreased SN-38G formation rates and UGT1A mRNA/protein levels were associated with the number of UGT1A1 reduced-function alleles (UGT1A1*60, UGT1A1*28, UGT1A1*6, or * 1B) the livers carried, indicated that UGT1A1 genetic polymorphisms may play an important role in the interindividual variability in SN-38 glucuronidation activity.Conclusions:This is the first report to systematically investigate the distribution of genetic polymorphisms and haplotypes of CYP2A6, UGT1A1, and UGT1A9 in Chinese population, as well as the molecular mechanisms underlying the variability in the metabolism of tegafur and SN-38 in human liver microsomes. We concluded that CYP2A6*4 and the novel CYP2A6*1B variant were the major genetic determinants of interindividual variability in 5-FU formation from tegafur in Chinese livers, and haplotypes containing more UGT1A1 reduced-function alleles were mainly responsible for the decreased SN-38 glucuronidation activities in Chinese livers. Our findings should facilitate the in vivo pharmacogenomics study of tegafur and irinotecan in Asian populations, and provide valuable information for the implementation of personalized medicine of tegafur-based and irinotecan-based chemotherapy.