Pharmacogentic Study On Cyclosporine In Chinese Renal Transplant Patients

PART I Association of MDR1, CYP3A4*1G and CYP3A5*3genetic polymorphisms with the pharmacokinetics of cyclosporine A in Chinese renal transplant recipientsBackground and ObjectiveRenal transplantation is the preferred treatment of choice for most patients with end stage renal failure. The recent advent of novel immunosuppressive agents, especially cyclosporine A (CsA), has significantly improved the clinical outcome of renal transplant recipients. CsA is characterized by a narrow therapeutic index and high interindividual pharmacokinetic variations. Monitoring CsA blood levels is therefore highly recommended to improve the efficacy and reduce the toxicity of CsA treatment. CsA is a substrate of both P-glycoprotein (P-gp) and Cytochromes P4503A (CYP3A). The CYP3A and MDR1gene, encoding the P-gp, are polymorphic and we speculate these polymorphisms may contribute to CsA interindividual variance. Therefore, this study is to retrospectively evaluate the effects of MDR1, CYP3A4*1G and CYP3A5*3genetic polymorphisms on CsA pharmacokinetics in Chinese renal transplant patients during different post-transplant periods.MethodsA total of132Chinese renal transplant patients of Han ethnicity, receiving CsA, mycophenolate mofetil and prednisolone as immunosuppression regimen, were enrolled. The genotypes of MDR1C1236T, G2677T/A and C3435T were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP); and the genotypes of CYP3A4*1G and CYP3A5*3were determined using polymerase chain reaction-ligase detection reaction (PCR-LDR). The predose and two hour postdose concentrations of CsA (Co and C2, respectively) were determined by fluorescence polarization immunoassay. Dose-adjusted Co and C2were calculated by dividing the Co and C2by the corresponding24-hour dose on a mg/kg basis. The relationships of dose-adjusted Co and C2with corresponding genotypes and haplotypes in different post-transplant periods (day0-7, day8-15, day16-30, Month2to12and after one year) were investigated using ANOVA (or Kruskal-Wallis H test) and multiple linear regression. The influence of investigated polymorphisms and other clinical variables, such as age, gender, hematocrit (HCT), serum creatinine (Scr), Alanine transaminase (ALT) and serum albumin (ALB), was determined by multiple linear regression.Results1. Single nucleotide polymorphisms (SNPs) among transplant patientsThe frequencies of MDR11236T,2677T,2677A, C3435T, CYP3A4*1G and CYP3A5*3in132renal transplant patients were64.8%,39.4%,15.5%,37.5%,22.3%and74.2%, respectively. There was a strong linkage disequilibrium between CYP3A4*1G and CYP3A5*3(D’=0.83) and low linkage disequilibrium between other SNPs. Construction of haplotypes (CYP3A4*1G-CYP3A5*3) via expectation maximization resulted in four haplotypes and the frequencies of the CYP3A4*1G-CYP3A5*3haplotypes were71.5%for GG,19.5%for AA,6.2%for GA, and2.8%for AG, respectively.2. Analysis of each SNPsUnivariate analysis showed that MDR1C1236T was associated with the Co of day8to15and affected the Co and C2after one year post-transplant. MDR1G2677T/A was associated with the Co and C2after one year post-transplant. MDR1C3435T was associated with the C2during day16to30and the Co and C2after one year post-transplant. Both CYP3A4*1G and CYP3A5*3were associated with the Co and C2after one month post-transplant.We further investigated the association between each SNP and CsA dose-adjusted concentrations using multiple linear regression, when including clinical covariates. The results suggested that MDR1C1236T was associated with the Co during day1to15and C2after one year post-transplant. MDR1G2677T/A was associated with the Co during day16to one year and with C2after one year post-transplant. MDR1C3435T was related to the Co during day16to30and after one year, and also affected the C2during day8to30. CYP3A4*1G had an impact on the Co after one year and C2after8days post-transplant, while CYP3A5*3was only related to Co after one year. The results of multiple linear regression were summarized in Table1. Table1Association between each SNP and CsA dose-adjusted concentrations (multiple linear regression)3. Analysis of combined CYP3A4*1G and CYP3A5*3Combined CYP3A4*1G and CYP3A5*3genotype was associated with C2post-transplant and Co after one year post-transplant. The results were summarized in Table2. Patients with AA-AA genotype have a lower dose-adjusted concentration compared to those with GG-GG genotype, while the resulting model explained only <20%of the total variation. Table2Effect of combined CYP3A4*1G and CYP3A5*3genotype on CsA dose-adjusted concentrations4. Combined analysis of CYP3A4/5and MDR1To investigate the potential interaction of CYP3A5, CYP3A4and MDR1, multiple linear regression was applied. CYP3A4/5combined genotype and MDR1combined genotype were added into the same regression model to investigate their effect on CsA concentrations. The results presented here showed that CYP3A4/5combined genotype affected CsA dose-adjusted Co and C2after1month post-transplant and Co during the first month post-transplant. MDR1combined genotype was only associated with dose-adjusted Co after1year post-transplant. The results were summarized in Table3. Combined genetic variation of CYP3A4/5and MDR1could explained36.3%of the total variance of CsA Co and12.9%of CsA C2after one year post-transplant.Table3Effect of combined CYP3A4/5genotype and combined MDR1genotype on CsA dose-adjusted concentrations5. The effect of non-genetic factors on CsA concentrationsAge, HCT, ALT and Scr were found to related to CsA dose-adjusted Co and C2in different post-transplant periods. ALB and gender had a relatively minor effect on the concentration of CsA.ConclusionThe data suggested that the CYP3A4*1G-CYP3A5*3combined genotype affected CsA dose-adjusted trough and peak concentration after1month post-transplant and CsA dose-adjusted trough concentration during the first month post-transplant. Patients with CYP3A4*1G-CYP3A5*3AA-AA genotypes may require higher doses of CsA to reach the target levels. MDR1C1236T-G2677T/A-C3435T combined genotype was associated with steady-state CsA dose-adjusted trough concentration after1year post-transplant. Although MDR1and CYP3A4/5could not explain the individual variance of CsA concentrations alone, combined genetic variation of CYP3A4/5and MDR1could explain36.3%of the total variance of CsA Co and12.9%of CsA C2after one year post-transplant. Interindividual differences in CsA concentrations in Chinese transplant patients were not only related to metabolism-associated genetic variation, but also affected by non-genetic factors, such as age, HCT, liver and kidney function. The results of this study could explain the individual differences of CsA concentration of the renal transplant recipients to a certain extent. PART Ⅱ Association of MDR1, CYP3A4*1G, CYP3A5*3and FOXP3genetic polymorphisms with the pharmacodynamics cyclosporine A in Chinese renal transplant recipientsBackgroud and ObjectiveCsA is characterized by high interindividual variations, not only in pharmacokinetics but also in pharmacodynamics, which means that the same plasma concentration may lead to different drug effects. At present, CsA is monitored mainly by determining drug blood levels and exploring the effects of the polymorphisms of drug transporters and metabolic enzyme on the CsA pharmacokinetics, while pharmacodynamic monitoring of CsA was less conducted. Therefore, our study further explored the polymorphisms of CYP3A4*1G, CYP3A5*3, MDR1C1236T, G2677T/A and C3435T on the immunosuppressive effect of CsA on the basis of the first part of study.CsA not only inhibit the activation of T helper cells, by inhibiting IL-2production, but also decrease CD4+CD25+Foxp3+regulatory T cell (Treg) proliferation. FOXP3gene is known to be important for the development and function of Treg, and is associated with the rejection of human kidney transplants. FOXP3gene is polymorphic and three loci in the promoter region:-3499A/G (rs3761547),-3279A/C (rs3761548) and-924A/G (rs2232365) have a relatively higher mutation rate in eastern Asians. It has been reported that some genetic variants in the FOXP3gene may contribute to the genetic susceptibility of some autoimmune diseases. However, the effect of FOXP3gene polymorphisms on immunosuppressive effect of CsA in renal transplant patients has not been reported.This part of study was therefore conducted to retrospectively evaluate the association of CYP3A4*1G, CYP3A5*3, MDR1(C1236T, G2677T/A and C3435T) and FOXP3(rs3761547, rs3761548and rs2232365) genetic polymorphisms with the immunosuppressive effects (preventing rejection) of CsA in Chinese renal transplant patients.MethodsA total of131Chinese renal transplant patients of Han ethnicity, receiving CsA, mycophenolate mofetil and prednisolone as immunosuppressive regimen, were categorized into either the Rejection group (58patients) or No rejection group (73 patients). Rejection, confirmed by biopsy, was classified into acute antibody-mediated rejection, acute T-cellular-mediated rejection, chronic active antibody-mediated rejection and chronic active T-cellular-mediated rejection according to the Banff05working classification criteria. A TaqMan probe technique was performed to genotype rs3761547, rs3761548and rs2232365variant alleles in the FOXP3gene. Binary logistic regression was employed to compare the rejection rates among the patients with different genotypes in the1year,2years,3years and5years post-transplant. Time-to-event analysis was performed using Kaplan-Meier estimates and log-rank tests. The first rejection was defined as an event and time of occurrence as the life time. Multivariate Cox regression analysis was further performed to calculate the hazards associated with different polymorphisms controlling potential confounders. The impact of genetic variants on acute T-cellular-mediated rejection was also analyzed using Kaplan-Meier analysis.Results1. Clinical characteristics and CsA concentrationsNo significant difference was observed in age, gender, weight, height, cold ischemia, induction antibody therapy, graft source (deceased donor vs. living donor) and CsA concentrations of1month,1year,2years,3years and5years post-transplant between Rejection group and No rejection group.2. SNPs among transplant patientsOf the131Chinese Han renal transplant recipients, the frequencies of FOXP3rs3761547G, rs3761548C and rs2232365G were13.4%,83.6%and24.8%, respectively. Regarding the LD of rs3761547-rs3761548, rs3761547-rs2232365and rs3761548-rs2232365. D" was0.99,0.96and0.57, respectively. Construction of haplotypes via expectation maximization resulted in five haplotypes. The frequencies of the rs3761547-rs3761548-rs2232365haplotypes were70.2%for ACA,13.0%for GCG,11.8%for AAG,4.6%for AAA and0.4%for GCA.3. Binary logistic regression analysisThere are41,52,54and55patients with rejection during the1,2,3and5years after kidney transplantation, respectively, and three patients experienced rejection episodes beyond the five-year observation period. There was no significant difference in the genotype frequencies of CYP3A4*1G, CYP3A5*3, MDR1C1236T, G2677T/A, C3435T, FOXP3rs3761547, rs2232365variants between patients with and without rejection history in our patient population (P>0.05). The rs3761548genotype was significantly associated with the rejection rates. The risk for allograft rejection in the rs3761548AA genotype patients was about3.79fold (95%CI1.11-12.95, P=0.034) greater than that in the CC genotype patients during the first year post-transplant. The odds ratio was5.82(95%CI1.48-15.93, P=0.012) during the two years post-transplant,5.33(95%CI1.36-14.97, P=0.017) during the three years post-transplant and5.11(95%CI1.30-14.07, P=0.019) during the five years post-transplant.4. Kaplan-Meier and Cox regression analysisKaplan-Meier survival analysis showed that the rs3761548genotype was significantly associated with allograft rejection; and that patients with the rs3761548AA genotype had a shorter mean time to first rejection episode than that of the CC genotype (35.71±14.50vs73.18±6.00months, Log rank=6.952, P=0.031). The Cox regression analysis demonstrated that the rs3761548AA genotype was associated with higher allograft rejection rate compared with the CC genotype carriers (Hazard Ratio2.70,95%CI1.22-5.96, P=0.014) after controlling potential confounders. Combined rs3761547-3761548AA-AA genotype carriers had up to about a threefold (Hazard Ratio2.83,95%CI1.22-6.58, p=0.016) higher risk of allograft rejection than those with AG-CC genotype. Other investigated polymorphisms (CYP3A4, CYP3A5, MDR1and FOXP3rs3761547, rs2232365) and clinical variables were not found to influence allograft rejection.Kaplan-Meier survival analysis of acute T-cellular-mediated rejection associated with polymorphisms suggested rs3761548polymorphism was associated to acute T-cellular-mediated rejection (Log Rank=7.853, P=0.020), while no association was found for other polymorphisms (CYP3A4*1G, CYP3A5*3, MDR1and FOXP3rs3761547,rs2232365).ConclusionOur study suggested an association between FOXP3rs3761548polymorphisms and allograft rejection in Chinese renal transplantation patients receiving CsA. AA genotype carriers had a higher risk of rejection than those CC genotype patients. Patients with the AA genotype may require higher dose of CsA than those CC genotype carriers to prevent rejection. Other investigated polymorphisms of CYP3A4*1G, CYP3A5*3, MDR1(C1236T, G2677T/A and C3435T) and FOXP3(rs3761547and rs2232365) were not found to be related to allograft rejection.