Preclinical Pharmacokinetics Study Of A Novel Anti-HIV Drug Combination Using HPLC-MS/MS Method

The novel anti-HIV drug combination（TDF 200 mg+3TC 300 mg+EFV 400 mg,qd）in our study was firstly proposed by Chin-Ming Ho of UCLA that applied the feedback system control（FSC）approach to rapidly screen drug combinations from various antiviral drugs used clinically in China to determine the optimal drug combinations and doses with high efficacy and low toxicity for the treatment of AIDS.For a better understanding of the pharmacokinetic processes including absorption,distribution,metabolism and excretion in animals,we developed and validated a series of sensitive and selective LC-MS/MS methods for the preclinical pharmacokinetic studies,which provided the experimental datas and references for the declaration of new drugs and further clinical trials.一、Development and validation of analytical methods for tenofovir,lamivudine and efavirenz in biological samplesIn our study,we developed rapid,sensitive and reliable liquid chromatography-tandem mass spectrometry（LC-MS/MS）methods for the determination of TEF,3TC and EFV in biological samples using ACV,FTC or HCT as the internal standard.Protein precipitation with methanol or acetonitrile（0.1%formic acid）was performed to extract the analytes.The analytes and IS were chromatographed on an Agilent ZORBAX SB-Aq（150×4.6 mm,5μm）column or a Ultimate XB-C18（50×4.6 mm,5μm）in a gradient elution with a mobile phase composed of methanol and water with 25.0 mM ammonium acetate in0.1%formic acid or acetonitrile and water in 0.1%formic acid.An API 4000 electrospray tandem mass spectrometer equipped with an electrospray ionization sourse was operated in multiple reaction monitoring（MRM）with the precursor-to-product ion transitions m/z288.3→176.4（TEF）、m/z 230.3→111.9（3TC）、m/z 316.0→244.0（EFV）、m/z 226.1→152.2（ACV,IS）、m/z 247.8→129.9/101.1（FTC,IS）in positive mode and m/z 314.0→243.9（EFV）、m/z 296.0→204.8（HCT,IS）in negative mode.The methods for the quantification of TEF,3TC and EFV in different biological matrix were fully validated according to the requirements of FDA and CFDA and were successfully applied to the preclinical pharmacokinetics of the novel anti-HIV drug combination.二、The potential pharmacokinetic interaction study of the novel anti-HIV drug combination in Beagle dogsA self-controlled,randomized crossover trial was conducted to evaluate the potential pharmacokinetic interaction of a novel anti-HIV drug combination in Beagle dogs.From the plasma concentration-time curves and pharmacokinetic parameters after single dose of individual and combinational drugs,we found that there were significant differences among different Beagle dogs,while the main pharmacokinetic parameters of TEF,3TC and EFV showed no significant differences（p>0.05）.It was suggested that the result of no interaction among TEF,3TC and EFV was possibly caused by the significant individual differences in Beagle dogs,which required a further investigation.三、The pharmacokinetic study of the novel anti-HIV drug combination in Beagle dogsThe validated LC-MS/MS method was used to investigate the pharmacokinetic features of the novel anti-HIV drug combination in Beagle dogs.After a linear regression processing of the pharmacokinetic parameters(C_max and AUC_0-τ)to the corresponding doses,the correlation coefficients of C_max and AUC_0-τfor TEF were 0.4214 and 0.6849,for3TC were 0.7889 and 0.8286,for EFV were 0.2289 and 0.3047,indicating that the estimated pharmacokinetic parameters AUC_0-τof TEF and 3TC were positive correlated with the doses（low,middle and high dose）of the novel anti-HIV drug combination orally administrated by Beagle dogs.On the fourth day of multiple-dose test,the plasma concentrations of TEF,3TC and EFV in Beagle dogs had reached to the steady state.The mean AUC cumulative ratios of TEF,3TC and EFV after single-and multiple-dose administration of combinational drug were 1.89,0.95 and 1.17,respectively.Taken the accepted criteria of bioequivalence as the reference（the upper limit was defined as 1.25）,we found that there was no obvious accumulation of 3TC and EFV in Beagle dogs after seven days’administration,while TEF had a little accumulation.四、The pharmacokinetic study of the novel anti-HIV drug combination in SD ratsIn order to better understand the pharmacokinetic features of the novel anti-HIV drug combination,further pharmacokinetic and drug-drug interaction studies were carried out in SD rats using the validated LC-MS/MS method.After a linear regression processing of the pharmacokinetic parameters(C_max and AUC_0-τ)to the corresponding doses,the correlation coefficients of C_max and AUC_0-τfor TEF were 0.3035 and 0.7803,for 3TC were 0.6134 and0.8646,for EFV were 0.6886 and 0.5314,which indicated that the AUC_0-τof TEF,3TC and EFV were positive correlated with the doses,respectively.Compared with the plasma concentrations-time curves and corresponding pharmacokinetic parameters of TEF,3TC and EFV after single dose of individual and combinational drugs,there were no significant differences in the parameters of TEF and 3TC（p>0.05）,while it was found that significant differences occurred among the individuals and the parameters of EFV changed greatly with the significant differences in C_max and MRT（p<0.05）.It indicated that there was no significant effect on the pharmacokinetics of TEF and 3TC in SD rats after oral administration of the drug combination,while EFV was influenced on the pharmacokinetic process of absorption and elimination.Whether there was a potential drug-drug interaction among TEF,3TC and EFV remained to be further investigated.五、Study on tissue distribution and excretion of the novel anti-HIV drug combination in SD ratsTissue distribution and excretion of the novel anti-HIV drug combination in SD rats were studied by the validated LC-MS/MS methods.The results of anti-HIV drug combination distributed in different tissues showed that TDF was rapidly metabolized to TEF and then distributed to kidney quickly.The concentration of TEF was highest in kidney and followed by liver.For TEF was not easily accessible through the blood-brain barrier,it was less distributed in brain tissue.Two hours after oral administration,3TC reached the peak concentration in kidney and maintained a higher concentration at 24 h.Also 3TC reached a higher concentration level in rich blood-perfusion tissues（heart,liver,spleen and lung）and had a lower concentration in brain tissue.The concentration of EFV in each tissue was in the order of liver>heart,spleen,kidney,brain>lung.EFV was absorbed by the gastrointestinal tract and had the highest concentration in liver.It was easy for EFV to enter the brain tissue through the blood-brain barrier and therefore,it was widely distributed in the brain tissue and had a higher concentration level.The results of excretion experiment showed that TDF was mainly excreted in the form of TEF from urine,feces and bile after oral administration of anti-HIV drug combination.The mean accumulated excretion rates of TDF in urine,feces and bile were12.50%、28.84%and 0.285%,which was converted from TEF.3TC mainly excreted in prototype form and the mean accumulated excretion rates of 3TC in urine,feces and bile were 21.37%、27.61%和1.293%,respectively.The mean accumulated excretion rates of EFV in urine,feces and bile were 0.005%、5.86%and 0.014%,respectively.The total excretion rate of EFV as unchanged was 5.879%,indicating that EFV was mainly eliminated by metabolism and it was necessary to do the further study on the metabolites of EFV in vivo.