The Effect Of Genetic Polymorphism On The Blood Concentration Of Tacrolimus In Chinese Heart Transplant Patients

BackgroundTacrolimus(FK506),one of the calcineurin inhibitors(CNIs),was first applied in clinic in 1989.Although there are differences in molecular structure among FK506 and CsA,FK506 works on the same signal transduction pathway as CsA.FK506 inhibit the phosphatase activity of calcineurin,thereby suppressing the activation of T cells,as well as the production of T cell dependent antibodies and the transcription of cytokines.FK506 is 10?100 times more potent than CsA is.FK506 can effectively prevent the occurrence of rejection after heart transplantation and has less adverse reaction.FK506 is characterized by a narrow therapeutic index and large intra-and interindividual pharmacokinetic variability.Therefore,routine therapeutic drug monitoring(TDM)is an integral part of tacrolimus regimen after organ transplantation to ensure appropriate exposure.The main methods for the detection of FK506 are immunoassay and liquid chromatography-mass spectrometry.Most commercially available methodologies for the routine monitoring of tacrolimus have been immunoassay based,utilizing an anti-FK506 monoclonal antibody.However,its sensitivity and specificity is low.In addition,these antibody-based assays have been shown to overestimate tacrolimus concentrations due to cross-reactivity with tacrolimus metabolites and thus may be misleading with respect to patient immunosuppression status.On the contrary,The HPLC-MS assay for tacrolimus is an ideal method for comparison studies as it is specific for the parent drug.Recent studies indicate that the large inter-individual variability in drug responses occurs as a result of genetic variants in proteins involved in drug disposition pathways(e.g.,metabolic enzymes and membrane transporters)or associated with FK506 pharmacodynamics pathways.Most of the studies on molecular variation have focused on the effect of gene variation on the expression and function of the above protein.Although genetic variants of metabolic enzyme(e.g.,CYP3A4 and CYP3A5)and transporter(e.g.,P-gp)affecting the blood concentration of tacrolimus has been extensively studied,the pharmacogenetics of drug target proteins and upstream regulators of metabolism enzyme and transporter is less known and understood.Objectives1.To evaluates the suitability of the high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)against chemiluminescence microparticle immunoassay(CMIA).2.To investigate the effect of genetic polymorphisms on tacrolimus application in the patients with heart transplantation.Methods and content1.A total of 253 heart transplant patients(n = 581)were enrolled.The concentration of tacrolimus in whole blood was determined by HPLC-MS/MS.CMIA was used Simultaneously,the consistency of results was compared by Bland-Altman method,and the correlation was analyzed by paried t test and Pearson correlation analysis.2.The SNPs genotypes of 200 heart transplantation patients and 100 healthy were determined by PCR-direct sequencing,PCR,MassArrayR SNP methods.The demographic parameters,dosage regimen,laboratory examinations were recorded.The differences FK506 Co were compared among all of the genotype groups.3.Correction for age,sex,liver function,and renal function,multiple linear regression was used to analyse the association between genetic polymorphisms and FK506 Co.Results1.The linear range by HPLC-MS/MS was 2-30 ng/ml,and the lower limit of quantification was 2 ng/ml.The intra-and inter-day precision(RSD%)by HPLC-MS/MS was less than 15%.2.According to the Bland-Altman method,the percentage of the limit can be obtained:95.52%LOA=(0.2,5.6).And 95.52%difference value was within the limit and the consistency of the two methods was good.Compared with the CMIA method,the regression equation was yrHPLC-MS/MS=0.7O011XCMiA-0.1668(R2=0.9024),and the correlation between the two methods was better.The measured value of CMIA is higher than the HPLC-MS/MS,the difference was statistically significant(P<0.05).3.All SNPs were in Hardy-Weinberg equilibrium apart from COMT rs165599,COMT rs6267 and PPP3R1 rs1868402(P<0.01),The frequency of patients with the TGF-?1 rs1800470 C and CYP3A5a*3 rs776746 G allele was higher than the healthy controls(P=0.03).The frequency of patients with the IL-6 rs1800796 C and CYP3A4?1G rs2242480 C allele was significantly higher compared with healthy controls(P=0.03).4.After the initial dose of FK506 in heart transplantation patients,there was a statistically significant difference in FK506 Co:1)homozygous wild type is higher than heterozygous/homozygous mutant type:CYP3A4*1G CYP3A4 rs339722392)homozygous wild type is lower than heterozygous/homozygous mutant type:IL-2 rs2069762 T>G3)homozygous/heterozygous wild type is lower than homozygous mutant type:CYP3A5*3,CYP3A4 rs4646437 A>Q TLR4 rs1927907 G>A4)homozygous wild type is lower than homozygous mutant type:PPP3R1 rs1868402G>A5.After correction for gender,age factors,multiple linear regression results showed that the final model accounted for 23.1%of total variation in FK506 Co.CYP3A5*3 and IL-2 rs2069762 T>G accounted for 38.7%and 15.6%of total variation involved in FK506 Co,respectively,while gender,age,CYP3A4*1G CYP3A4 rs33972239,CYP3A4 rs4646437,PPP3R1 rs1868402 G>A was excluded from the final model.6.After eliminating the influence of CYP3A5*3 factors,the results showed that the effects of CYP3A4*1Q CYP3A4 rs33972239 delA,CYP3A4 rs4646437 A>G and IL-2 rs2069762 T>G on FK506 Co were not statistically significant(P>0.05).Conclusion1.The method of HPLC-MS/MS for the determination of tacrolimus blood concentration shows a high specificity and sensitivity.Compared with the results of CMIA,the measured value by HPLC-MS/MS is low,but HPLC-MS/MS is highly selective for parent drug of tacrolimus.2.The HPLC-MS/MS and CMIA method have the good consistency and correlation in detecting olanzapine serum concentration,which can be used for conventional therapeutic drug monitoring.3.Genetic polymorphism of CYP3A5*3 and IL-2 rs2069762 T>G may be responsible in part for the large inter-individual variability of tacrolimus pharmacokinetics after first taking FK506 in heart transplantation patients.4.Combined genotyping with TDM may be a new approach to predict the optimal initial dosage of FK506,which can improve the effect of immunotherapy and reduce the toxicity reaction.