Detection Of CYP3A4/5and MDR1Genepolymorphisms By High Resolution Melting And The Relationship With Pharmacokinetics Of Sirolimus In Healthy Chinese Volunteers

PurposeThis study explored the effects of MDR1, CYP3A4*1G and CYP3A5*3genetic polymorphisms on sirolimus pharmacokinetics in healthy male Chineses, the relationship between pharmacokinetics and gene polymorphisms might play some advice or guidance on individual medicine in clinical application.MethodsThe pharmacokinetic part of this study were obtained from sirolimus bioequivalence tests with a single oral dose of5mg (n=67). The experiment established a LC-MS/MS method to determine sirolimus concentration in human whole blood.32-desmethoxysirolimus was selected as the internal standard, blood samples were extracted through liquid-liquid extraction. On LC, a reversed-phase analytical column of Phenomenx C18(50×2.0mm,5μm) was adopted with gradient elution. On MS, positive electro-spray ionization and multiply reaction monitoring (MRM) mode was employed. The transition of m/z was931.3→864.3for sirolimus and901.3→816.6for32-desmethoxysirolimus. Blood samples were collected at predose,0.5,1,2,3,4,6,8,12,16,24,48,72,96,120and144hour after administration to obtain a complete concentration-time curve. MDR1C1236T, G2677T/A, C3435T, CYP3A4*1G and CYP3A5*3genotypes were determined by polymerase chain reaction-high resolution melting or direct sequencing method. ANOVA and Kruskal-Wallis Test were performed in the analysis of pharmacokinetic parameters (Cmax, AUC0-∞, and Tmax) and MDR1C1236T, G2677T/A, C3435T, CYP3A4*1G and CYP3A5*3genotypes. Multiple comparisons were used the least significant difference method (LSD), P<0.05was considered significant difference.Results1. Sirolimus concentration in whole blood of67healthy subjects were determined by LC-MS/MS method and the pharmacokinetic parameters showed a high individual differences.2. The allele frequencies and genotype frequencies of MDR1C1236T, G2677T/A and C3435T, CYP3A4*1G and CYP3A5*3conformed to Hardy-Weinberg equilibrium, and were consistent with the Chinese ethnic frequencies in reported literatures.3. Significant differences were observed in Cmax or AUC0-∞of sirolimus with the genotypes of CYP3A5*3, CYP3A4*1G and MDR1C3435T, but there was no relationship with MDR1C1236T or G2677T/A genotypes. Well, all above sites’ genotypes did not have any relationship with Tmax.1) On MDR1C3435T site, there was a significant difference in Cmax (P=0.007) between the subjects with CC genotype (n=31) and CT genotype (n=29). The Cmax of Wide homozygous CC genotype carriers was1.4-fold higher than mutate heterozygous CT carriers.2) For CYP3A4*1G site, significant difference was observed only in AUC0-∞(P=0.021) between the subjects with the CYP3A4*1G*1G genotype (n=9) and CYP3A4*1*1G(n=19), subjects with the CYP3A4*1G*1G genotype had a1.7-fold sirolimus exposure higher compared with the value seen in those subjects with CYP3A4*1*1G. But there was no statistic significance in Cmax.3) As to CYP3A5*3SNP, different genotypes significantly effected sirolimus pharmacokinetics. On Cmax, CYP3A5*3*3genotype carriers (n=37) was1.4-fold higher than those subjects with CYP3A5*1*1genotypes with a significance (P=0.036), but the AUC0-∞between the two genotypes did not reach statistic significance.The difference on AUC0-∞between Wide homozygous CYP3A5*3*3carriers (n=37) and the subjects with mutate combined CYP3A5*3*1genotype (n=24) had been statistically significant (P=0.007), the sirolimus exposure of CYP3A5*3*3carriers could be1.4times compared to CYP3A5*3*1carriers.ConclusionsThe study established a LC-MS/MS method to determine sirolimus concentration in human whole blood and this method was specific, rapid, sensitive, accurate, simple and in line with the SFDA requirements. Also, this test showed high resolution melting was an accurate and simple method to classify kinds of SNPs’genotypes with less time and effort. There was a significant difference in Cmax or AUC0-∞of sirolimus with the genotypes of CYP3A5*3, CYP3A4*1G and MDR1C3435T, but not MDR1C1236T or G2677T/A genotypes. These results suggest that the gene information of CYP3A4*1G and CYP3A5*3may help the individual application of sirolimus. CYP3A4*1G*1G genotype and CYP3A5*3*3genotype carriers require special attention. And if necessary, we should consider the adjustments of SRL doses to achieve individual administration.