The Influence Of Gene Polymorphisms Of Drug Metabolism Enzyme On Tacrolimus Dosage-requirements In Patients Following Liver Transplantation

Part IThe experimental study of liver graft injury by ischemia and reperfusion secondary to retrograde reperfusionAim:The aim of this study was to evaluate the influence of retrograde reperfusion via the vena cava on ischemic/reperfusion injury (I/R-injury) in a rat liver transplantation model, under comparing different kinds of reperfusion.Methods:Fifty four orthoptic rat liver transplantations were performed on male SD rats. Three groups (n=18) were formed. Group I:Antegrade reperfusion via the portal vein. Group II:Antegrade reperfusion, simultaneously, via the portal vein and the hepatic artery. Group III:Retrograde reperfusion via the vena cava. Serum parameters were determined one,6and24h after operation. Furthermore, the liver was taken for histological assessment. The early cell apoptosis index was examined also.Results:Rats of group III showed significantly lower aspartate amino transferase and alanine aminotransferase serum levels compared with group I and group II rats. Aspartate amino transferase and alanine aminotransferase serum level were significantly lower in group I than in group II. In histology, group III livers showed significantly less leukocytic infiltrate than group I and group II livers, however, group II livers showed the most serious. The apoptosis index in the group III livers were lower than in the group I and group II livers.Conclusions:Our data suggested that the expression of I/R-injury correlates with the type of reperfusion. Furthermore, this study was able to demonstrate that in a rat model, the retrograde reperfusion leads to a lower expression of I/R-injury than the antegrade reperfusion. Objectives:According to the serum alanine transarninase, serum total bilirubin and the characteristics of the acute rejection in hepatic tissue, to investigate the significance of CYP3A mRNA expression in rat's blood concentration of tacrolimus following liver transplantation and immunosuppressive effect of tacrolimus.Methods:With Sprague-Dawley rats as donors and male Wistar rats as recipients, seventy two acute rejection models of orthotopic liver transplantation in the rat were established. Recipients were divided into4groups:blank control group, experimental group, enzyme inhibition group and enzyme induction group. Every six recipients were sacrificed respectively at day1,4,7after OLT to detect the changes of serum alanine transarninase, serum total bilirubin, tacrolimus whole-blood trough concentrations and the grafts histology. CYP3A mRNA levels in rat hepatic tissue were determined by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).Results:The success rate of acute rejection model of orthotopic liver transplantation in blank control group was88.9%(16/18). Rats of blank control group showed the highest levels of serum alanine transarninase, serum total bilirubin and the histological score of the acute rejection in hepatic tissue compared with other three groups, however, rats of enzyme inhibition group showed the lowest levels, enzyme induction group was higher than experimental group. The blood drug level of tacrolimus in rats of enzyme inhibition group was the highest compared with other three groups, however, blank control group showed the lowest, enzyme induction group was lower than experimental group. CYP3A mRNA levels in hepatic tissue of enzyme induction group showed the highest compared with other three groups, however, enzyme inhibition group showed the lowest, blank control group was higher than experimental group.Conclusions:Acute rejection experimental model of liver transplantation in rat could be stably established using SD rat as donor and Wistar rat as recipient. The expression of CYP3A mRNA levels in hepatic tissue may influence the tacrolimus whole-blood trough concentration and immunosuppressive effect of tacrolimus furthermore. Objectives:1. To summarize the clinical data in considerable interindividual differences of tacrolimus dosage and trough blood concentration during the early period after liver transplantation in patients.2. To evaluate the distribution characteristics of CYP3A5and ABCB1gene polymorphisms in Chinese liver transplantation recipients.3. To investigate the possible association of the ABCBl gene C3435T polymorphism and the CYP3A5gene A6986G polymorphism with tacrolimus trough concentration and dose requirements during the early period after liver transplantation in patients.Methods:1. The clinical data of sixty seven liver transplantation recipients were analyzed and tacrolimus whole-blood trough concentrations were measured by enzyme-linked immunospecific assay. Dose requirements and dose-adjusted trough concentrations (concentration/dose [C/D] ratios) were analyzed.2. CYP3A5and ABCBl genotyping were performed by direct DNA sequencing using the polymerase chain reaction restriction sites polymorphism-based procedure.3. Tacrolimus was administered twice daily at specified times post-transplant period according to the trough-targeting strategy. Sixty recipients with stable graft function were studied beyond1-month post transplantation. Whole blood samples were collected lw,2w and lm after both the morning and evening doses during hospitalization.Results1. Tacrolimus dose requirements and dose-adjusted trough concentrations (concentration/dose [C/D] ratios) after liver transplantation had great variation between individuals.2. The CYP3A5*1/*1was observed in15subjects (22.4%),23(34.3%) carried*l/*3, and29(43.3%) carried*3/*3. CYP3A5*3/*3variant was associated with significant lower tacrolimus dose at lw,2w and lm compared with patients who did not (CYP3A5*3/*3) after transplantation. The tacrolimus C/D ratios were obviously higher in recipients carrying CYP3A5*3/*3.3. A synonymous single nucleotide polymorphism (SNP) of ABCBl in various exon was different. Furthermore, ABCBl3435C>T polymorphisms was significantly correlated with tacrolimus dose-adjusted pre-dose concentrations at various time points. The ABCB13435C/C was observed in22subjects (32.8%), whereas40(59.7%) carried3435C/T and5(7.5%) carried3435T/T.Conclusions1. Tacrolimus dose requirement is characterized by a large interindividual variability requiring the use of therapeutic drug monitoring in daily clinical practice.2. The present study shows that genetic polymorphisms in CYP3A5and ABCB1may be responsible, in part, for the large interindividual variability of tacrolimus pharmacokinetics during the early period after liver transplantation in patients.3. Patients in CYP3A5non-expressors require a low dose of tacrolimus to reach target levels compared with expressors. Patients in ABCB1low-expressors require a low dose of tacrolimus to reach target levels compared with high-expressors.4. In the future, genotyping patients for CYP3A5and ABCB1polymorphisms may help to optimize the tacrolimus individualized immunosuppressive therapy in liver transplantation.