Analysis Of Drug Metabolism Enzyme Gene Expression In Peripheral Blood After Liver Transplantation

Objective:We had systematically and simultaneously evaluated the expression profile of drug-metabolizing enzymes in peripheral blood of liver transplant. To explore the application of drug metabolic enzyme gene expression in liver transplantation.Methods:Sixty-four patients were included in the study, who had undergone liver transplant in Tianjin First Center Hospital during 2013 to 2014. Peripheral blood samples were collected before the morning dose was administered at the third week after transplantation. The samples were stored at-80℃ until required for analysis.We analyzed the clinical data by NONMEM, and found that clearance rate of tacrolimus were mainly affected by TBIL, Hemoglobin, Post-operative day and BUN,moreover TP and albumin were included. By these factors, we chose 8 samples at third week after transplantation, which were divided into 2 groups according to concentration of tacrolimus, low group and high group. We had systematically and simultaneously evaluated the expression profile of the two groups using the Drug Metabolism RT2 Profile PCR Array. And we used custom-made RT2 Profiler? PCR Array to validate the above results with an ABI 7900 instrument through 64 samples from the 3 week after transplantation. The relationship of drug-metabolizing enzymes and immunosuppressive agents were validated by recombinase assay. The Genotype of drug-metabolizing enzyme was analyzed by i MLDR.Results:(1) Clinical data at third week after transplantation was analyzed. We found that the level of ALT, GGT, TBIL was abnormal. It showed that the liver was injured.Correlation analysis were done among these factors, and it was found that the plasma levels of tacrolimus were inversely related to level of TBIL(r=-0.300,P<0.05) and statistically significant.(2) Every sample of the two groups was performed using the 3 different human drug metabolisms RT2 Profiler? PCR Array. Gene expression analysis showed that m RNAs expression of 29 gene were significantly higher in low tac group compared with high tac group(fold>1.5, P<0.05).(3) These differently expressed genes belonged to cytochrome P450, Aldehyde Dehydrogenase, Alcohol Dehydrogenase, Esterase, Prostaglandin-endoperoxide Synthase, Xanthine Dehydrogenase, Decarboxylases, Glutathione Transferases,N-Acetyltransferases, Sulfotransferases. Twelve genes belonged to cytochrome P450.(4) To validate the result from RT2Profiler? PCR Array, we used custom-made RT2Profiler? PCR Array in 64 liver transplantation patients. The result showed that6 genes were meaningful(P<0.05), which were CYP3A5, CYP2B6, CYP4A11,CYP1A1, CYP19A1, CYP17A1. Correlational analyses were conducted, the plasma levels of tacrolimus were inversely related to expression level of CYP3A5(r= ﹣ 0.381, P<0.05), CYP4A11(r= ﹣ 0.331, P<0.05), CYP2B6(r= ﹣0.339, P<0.05).(5) The mix of the three immunosuppressant drugs(mycophenolate mofetil,tacrolimus, methyl prednisolone) were incubated with recombinant human CYP17A1, CYP1A1, CYP4A11, CYP2B6, CYP3A5, and we used HPLC-MS to detect the decreased amount of the three immunosuppressant drugs. The result showed that tacrolimus was not a substrate of CYP17A1, CYP1A1, CYP4A11,CYP2B6, tacrolimus was a substrate of CYP3A5, and mycophenolate mofetil was a substrate of CYP17A1.(6) Amultiplex PCR-ligase detection reaction method was used for genotyping six target SNPs of CYP17A1. We found that expression of CYP17A1 with G/G genotype(rs17115149) was higher than G/T genotype(P<0.05). No significant association was found for other SNPs(P>0.05).Conclusion:The plasma level of tacrolimus was related to expression level of CYP17A1,CYP1A1, CYP4A11, CYP2B6, CYP19A1 in peripheral blood at the third week after liver transplant. The gene expression level of drug-metabolizing enzymes in peripheral blood can reflect liver injury. Tacrolimus was a substrate of CYP3A5, and mycophenolate mofetil was a substrate of CYP17A1. The Genotype(rs17115149)may affected the expression of CYP17A1.