Study On The Relationship Between CYP3A5, MDR1 And CNI's Dosage And Concentration In Renal Transplant Recipients

ObjectivesCalcineurin inhibitor (CNI) does much good to renal transplantation, but its narrow therapeutic windows and differences between individuals puzzled clinicians all the time. Research from home and aboard indicated that CNI's differences between individuals had close relationships with cytochrome P3A4 (CYP3A4), cytochrome P3A5 (CYP3A5) and Multi-drug resistance gene 1 (MDR1), the dosage of CNI for the same concentration decreased as follows: mutant homozygote, heterozygote and wild-type homozygote. MDR1 encoded P-glycoprotein (P-gp), while P-gp cooperated and adjusted with cytochrome P3A (CYP3A). As a conclusion, the detections of CYP3A and MDR1 polymorphisms in renal transplant recipients play an important role. Research from home showed that the genovariation rates of CYP3A4 were very low (0.5%～2%), in view of their limited significance, we didn't internalize CYP3A4 into our research.In light of the genic features of the Hans in China, we took pharmacogenomics and un-pharmacogenomics factors into consideration that influenced the absorption and metabolism of CNI in renal transplant recipients. We designed the primers and amplified the objective DNA by PCR in one time, then detected the gene polymorphisms of CYP3A5 and MDR1 ( 3435, 2677 and 1236 ) with DHPLC, the clinical data consisted of 274 Hans kidney recipients from 12 provinces in China, whose distributions of CYP3A5 and MDR1 ( 3435, 2677 and 1236 ) were analyzed. Combined with the different features of postoperative stages of renal transplantation, we used multiple linear regression to identify the factors responsible for the inter-individual variations in dosage and concentration of CNI (tacrolimus (TAC) and Cyclosporine A (CsA)) in renal transplant recipients.Methods1. Designed the primers and amplified the objective DNA by PCR in one time, then detected the gene polymorphisms of CYP3A5 and MDR1 ( 3435, 2677 and 1236) with DHPLC.2. Analyzed the distributions of CYP3A5 and MDR1 ( 3435,2677 and 1236 ) of 274 Hans kidney recipients from 12 provinces in China.3. In accordance with 118 recipients in morning periods after renal transplantation, whose immune suppressions were TAC, mycophenolate (MMF) and Prednisone (Pred), at 3,7,14 and 30 days after operation, we respectively recorded their gender, age, height, weight, dosage of TAC, dosage of Pred, diarrhea, blood fat, liver function, renal function, albumn and erythrocrit, and at the same time detected their concentrations of TAC and genetic polymorphisms of CYP3A5, MDR1 ( 3435, 2677 and 1236). Multiple linear regressions were used for analysis.4. In accordance with 137 recipients in stable phases (≥1year) after renal transplantation, whose immune suppressions were TAC, MMF and Pred, we respectively recorded their gender, age, height, weight, dosage of TAC, dosage of Pred, blood fat, liver function, renal function, albumn, erythrocrit, and at the same time detected their concentrations of TAC and genetic polymorphisms of CYP3A5, MDR1 (3435,2677 and 1236). Multiple linear regressions were used for analysis. 5. In accordance with 118 recipients in morning periods, 103 recipients 3 months, 75 recipients 6 months, 137 recipients in stable phases (≥1year) after renal transplantation, whose immune suppressions were TAC, MMF and Pred, we respectively recorded their gender, age, height, weight, dosage of TAC, dosage of Pred, blood fat, liver function, renal function, albumn, erythrocrit, and at the same time detected their concentrations of TAC and genetic polymorphisms of CYP3A5, MDR1 ( 3435,2677 and 1236). Multiple linear regressions were used for analysis.6. In accordance with 117 recipients in stable phases (≥1year) after renal transplantation, whose immune suppressions were CsA, MMF and Pred, we respectively recorded their gender, age, height, weight, dosage of CsA, dosage of Pred, blood fat, liver function, renal function, albumn, erythrocrit, leucocyte, neutrophilic leukocyte and lympholeukocyte, and at the same time detected their concentrations of CsA and genetic polymorphisms of CYP3A5, MDR1 ( 3435, 2677 and 1236). Multiple linear regressions were used for analysis.Results1. The genic mutations of CYP3A5*3, MDR14 3435C>T, MDR1 2677G>T/A and MDR1 1236C>T existed in Hans kidney recipients in China, whose frequencies were as follows 74.1%, 49.5%, 39.9% / 13.7% and 72.1% , and their distributions between different provinces had no statistical distinctions.2. The fitting degrees of stepwise regression equations in recipients with TAC in morning periods after renal transplantation were low, at 3, 7, 14 and 30 days after operation, the adjusted R~2 was 0.284, 0.267, 0.417 and 0.324 respectively. From the aspect of pharmacogenomics, the main factors included MDR1 2677, MDR1 1236 and MDR1 3435, which varied intensively. Age, albumn, renal function, blood fat and liver function were important factors too.3. The fitting degrees of stepwise regression equations in recipients with TAC in stable phases after renal transplantation were high, adjusted R~2 = 0.739. The influence of the 6 independent variables on the dependent variable decreased as follows: MDR1 3435 (1), MDR1 1236 (1), MDR1 2677 (1), MDR1 1236 (2), age and AST. In the solitude analysis, MDR1 3435 (1), MDR1 1236 (1), age, MDR1 1236 (2) and MDR1 2677 (1) showed statistical significance.4. Factors responsible for inter-individual variations in dosage and concentration of TAC in renal transplant recipients: 1) Patients in early stage following renal transplantation showed rather poor fitting of the stepwise regression model, which increased obviously 3 months after the operation and further increased till reaching a stable level at 6 months. Multiple factors were found to affect TAC dosage and concentration in the early postoperative stage, during which period these factors underwent drastic variations and became stable 3 months later. 2) In terms of pharmacogenomics, the major factors affecting TAC dosage and concentration included MDR1 3435, MDR1 2677 and MDR1 1236 polymorphisms, which vastly varied between the patients early after the operation. Of these polymorphic sites, CYP3A5 produced only minor effects on TAC dosage and concentration, and was not included as an active factor until the stable phases ( over 1 year ) following the transplantation; MDR1 3435 was found to be the predominant factor affecting TAC metabolism in the stable phase. 3) Age, liver function, albumin and hematocrit were found to be positively correlated to the independent variable TAC concentration / (dosage * body surface area), and identified as important factors responsible for the intra-individual variation of TAC dosage and concentration.5. The fitting degrees of stepwise regression equations in recipients with CsA in stable phases after renal transplantation were high, adjusted R~2 = 0.824. The influence of the independent variables on the dependent variable decreased as follows: MDR1 3435 (1), MDR1 1236 (1) , CYP3A5 (2), LDL, DBIL, Age, TC, HDL and L. In the solitude analysis, MDR1 3435 (1), MDR1 1236(1), CYP3A5 (2), DBIL, Age, LDL and HDL showed statistical significance.Conclusions1. The genic mutations of CYP3A5*3, MDR1 3435C>T, MDR1 2677G>T/A and MDR1 1236OT generally exist in Hans kidney recipients in China, and their distributions have no differences between different provinces.2. The main reasons for the differences of TAC dosage and concentration between individuals in morning periods after renal transplantation are medicines and changes of internal environment after operation. The genetic polymorphisms of MDR1, age, albumn, renal function, blood fat and liver function are important factors too.3. The main reasons for the differences of TAC dosage and concentration between individuals in stable phases after renal transplantation are MDR1 3435, MDR1 1236, age, MDR1 2677 and liver function, and the extents of their influences decrease in order.4. The variations in the factors affecting TAC dosage and concentration after renal transplantation are consistent with the clinical features of the renal transplantation, and these factors vary with the postoperative stages. Pharmacogenomic factors produce the most conspicuous effect on TAC dosage and concentration differences, and agent that may interfere with TAC absorption and metabolism should be avoided after the operation. Age, liver function, albumin and hematocrit are also important factors responsible for the inter-individual variations in dosage and concentration of TAC in renal transplant recipients.5. The main reasons for the differences of CsA dosage and concentration between individuals in stable phases after renal transplantation are MDR1 3435, MDR1 1236, CYP3A5, DBIL, age and blood fat, and their influences decrease in order.