Population Pharmacokinetics Of Vivdesine: The Effect Of ABCB1 Genetic Polymorphism And Epigentic Variance

Vindesine is a vinca alkaloid,which is a synthetic derivative of vinblastine,is widely used in treatment of solid malignance,as well as hematological malignance.ABCB1,an important drug transpoters,is correlated with multi-drug resistance of various malignance to chemotherapy as it effluxes drugs.It was proved that vindesine is substrate of ABCB1 in vitro.Genetic polumorphism could influence the phenotype of ABCB1,and it was reported that epi-genetic variance could influence the expression of ABCB1.However,there is no report about the effect of ABCB1 genetic polumorphism on vindesine pharmacokinetics in vivo.Until now,there is no report about the effect of epi-genetic variance on pharmacokinetics in vivo.Since classic pharmacokinetic research methods require dense data sets in each subject,leading to poor compliance,it is difficult to use it widely in pharmacokinetics research in patients,especially those with hematological disorders.Thus we performed a series of study including the following aspects:(1)To characterize the pharmacokinetics of intravenous Vindesine in Chinese Han Subjects with hematological malignant disorders by using population pharmacokinetic analysis with limited sampling strategy.(2)To compare the genetic polymorphism and haplotype distribution between Chinese Han health subjects and pateients with hematological malignance.At the same time,to investigate the difference of methylation status in MDR1 promoter region among variety of ABCB1 genetic polymorphism.(3)To evaluate the effect of genotypes and haplotypes of ABCB1 on pharmacokinetics of Vindesine in vivo.(4)To investigate the correlation between methylation status in MDR1 promoter region and vindesine pharmacokinetics in vivoIn order to performe this study,the follwing methods were used.1.To establish population pharmacokinetic model and limited sampling strategy for intravenous Vindesine in Chinese subjects with hematological malignant disorders100 unrelated Chinese Han subjects(62 Males and 38 females)with Hematological Malignant Disorders were enrolled in this study.All patients received 3 mg of Vindesine as an 1 min rapid infusion.Three random sampling times derived from 0min,5min,20min,1hour,3hours, 6hours,12hours,24hours,48hours,72hours after dosing were:made by using NONMEN software and blood samples were collected. HPLC-MS/MS methods was used to analyze Vindesine.The population pharmacokinetic parameters of mizoribine in healthy subjects were estimated using a nonlinear mixed effects model(NONMEM)program.2.Genotyping and haplotype analysis of ABCB1A polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)assay was used for the detection of C1236T, G2677T/A and C3435T polymorphism.The haplotypes were reconstructed using PHASE 2.0.3.To determine the methylation status in MDR1 promoter regionThe methylation status of CpG sites within the proximal promoter region of ABCB1 gene was assessed using the combined bisulfite restriction analysis(COBRA)method.2μg of DNA were diluted to 50μl. DNA was then denatured,and incubated with 3M sodium bisulfite at 50℃for 16 hours.This combination of bisulfite treatment and PCR amplification results in the conversion of unmethylated cytosine residues to thymine and methylated cytosine residues to cytosine.PCR was performed and PCR productes were digested with TagI endonuclease and separated in nondenaturing polyacrylamide gels.Gels were stained with ethidium bromide.The proportion of methylated vs unmethylated product was quantitated by densitometric analysis to determine the density of methylation.4.Statistical methodsGenotype frequencies for the variants SNPs were assessed for deviation from Hardy-Weinberg equilibrium using the Chi-square test. Statistical significance of LD between SNP pairs was assessed using Fisher's Exact Test.Nonparametric tests including Kruskal-Wallis method and Mann-Whitney U test were used to compare the methylation status among different genotypes.Spearman analysis were performed to investigate the correlation between Methylation Status in Promoter Region and vindesine pharmacokinetics.A p value＜0.05 was deemed to indicate a significant difference.All statistical analyses were performed using SPSS version 13.0The present series studies found that:1.The mean value of Clearance(CL),halftime(T_1/2),apparent distribution volume(V/F),and area under the curve(AUC)was estimated to be 0.381±0.108L/h,22.8±10.4h,170.12±69.29L,1 49±67μg/L·h^-1, respectively.In addition,pharmacokinetic parameters in individual 100 subjects were obtained from population estimates according to Bayesian' theorem.The Mean value of Clearance is a little higher than early reports, and the other parameters were similar.2.Among Chinese Han patients with Hematological Malignant Disorders,the frequencies of homozygote of C3435T SNP wild type(CC) was 34%,and homozygote of C3435T SNP mutant type(TT)was 22%, heterozygots of C3435T SNP mutant type(CT)was 44%.The frequencies of homozygote of C1236T SNP wild type(CC)was 10%,and homozygote of C1236T SNP mutant type(TT)was 43%,heterozygots of C1236T SNP mutant type(CT)was 48%.The frequencies of homozygote of G2677T/A SNP wild type(GG)was 18%,and homozygote of G2677T/A SNP mutant type,including TT,TA,and AA was 16%,9%, 2%,respectively;heterozygots of G2677T/A SNP mutant type,including GT and GA was 42%,13%,respectively.9 haplotypes derived from these three variants were reconstructed using PHASE method;in which four haplotypes TTT,TGC,CGC,and CAC(in order of 1236-2677-3435) composed more than 90%in Chinese Han population with Hematological Malignant Disorders.No significant genetic differences were found between Chinese Han patients with Hematological Malignant Disorders and health volunteers.3.In our study,it was revealed that significant difference of methylation status between different genotypes of C3435T and G2677T/A(P＜0.05),but there is no significant difference in between different genotypes of C1236T(P＞0.05).We also found significant difference of methylation status between Chinese Han health volunteers and patients4.In our study on the effect of ABCB1 genetic polymorphism and haplotype on pharmacokinetic of Vindesine,There is no significant difference of pharmacokinetic parameters of Vindesine among different genotypes and haplotypes(P＞0.05).And the main pharmacokinetic parameters of Vindesine showed 3.5-fold variance.5.In our study to investigate the correlation between methylation status in MDR1 promoter region and vindesine pharmacokinetics in vivo, it was found that there was significant positive correlation between CL/F (P=0.000,r_s=0.365)and ABCB 1 Methylation Status in Promoter Region, and negative correlation between Vd(P=0.001,r_s=-0.339)and AUC (P=0.000,r_s=-0.366).However,we found no correlation between ABCB1 methylation status in promoter region and T 1/2(P=0.252).The present series studies achieved the following conclusions:1.A population pharmacokinetic model and a limited sampling strategy forintravenous vindesine have been developed,indicating that the pharmacokinetics of Vindesine is well described by a three-compartment model with first-order elimination.The model can be used to clinical pharmacokinetic study and therapeutic drug monitoring.2.Genetic frequencies and distribution of ABCB1 G2677T/A, C3435T,C1236T polymorphisms,as well as haplotypes,in patients with Hematological Malignant Disorders was similar with that in Chinese health subjects.This suggested no association between G2677T/A, C3435T,C1236T polymorphisms and Hematological Malignant Disorders.An association between MDR1 genetic polymorphism C3435T and G2677T/A with methylation status of MDR1 promoter region was also found in this study.Further investigations are needed to explore the clinical significant and molecular mechanism of this correlation.3.There is no association between G2677T/A,C3435T,C1236T polymorphisms with pharmacokinetics of Vindesine in Chinese Han subjects.However,it was found that there is association in some degree between ABCB1 methylation status in promoter region and some vindesine pharmacokinetics parameters in vivo.The actual effect and mechanism need further investigation.