| The 4-aminoquinoline drug piperaquine is a long acting antimalarial drug and it is one of suitable partner drugs in artemisinin drug-based combination therapy?ACT?.Extensive studies on the pharmacokinetics of piperaquine showed that piperaquine was characterized by a low clearance(?2.1 L·h-1·kg-1),a large volume of distribution(?730 L·kg-1)and a long terminal half-life?20?30 days?.The main metabolites of piperaquine were the mon-N-oxi dated and carboxylic products.The pharmacokinetics of its mono-N-oxidated metabolite has not been studied.There was no reports on the bioanalytical methods for the determination of PQ metabolites.Piperaquine and its metabolites displayed remarkable antimalarial activity(IC50<200 nmol·L-1)in vitro.However,the antimalarial activities of PQ metabolites have not been evaluated in vivo.This study was designed to evaluate the pharmacokinetics of piperaquine and its N-oxidated metabolite in rats and healthy Chinese subjects.Additionally,the antimalarial activities of piperaquine and its metabolites were evaluated in vivo1.The pharmacokinetics of piperaquine in ratsA sensitive and efficient liquid chromatography tandem mass spectrometry?LC-MS/MS?method was developed and validated for the simultaneous determination of piperaquine?PQ?and its N-oxidated metabolite?M1?in rat plasma.The validated method was successfully applied to a pharmacokinetic study performed in rats after a single oral administration of piperaquine(60 mg·kg-1)The sample preparation,liquid chromatography and mass spectrometric conditions were optimized.The optimized conditions were as follows:40 ?L of rat plasma samples were pre-treated by a simple protein precipitation procedure.Adequate chromatographic retention was achieved on the Venusil C18 column under gradient elution with acetonitrile and 2 mmol·L-1 aqueous ammonium acetate containing 0.15%formic acid and 0.05%trifluoroacetic acid.A triple-quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode.The validated method had good specificity,and the hemolyzed plasma had no influence on the determination.The method was linear in the range of 2.0-400.0 ng·mL-1 for PQ and 1.0-50.0 ng·mL-1 for M1.The lower limits of detection?LLOD?were established at 0.4 ng·mL-1 and 0.2 ng·mL-1 for PQ and M1 respectively.The accuracy was 96.1-106.0%for PQ and 93.7-102.3%for M1,and the precision was less than 11.0%,which showed that the method was accurate and reliable.The mean extraction recoveries were 80.3-89.6%for PQ and 80.0-88.7%for M1.The matrix effect was negligible under the current conditions.PQ and Ml were stable during storage and analysis.The validated LC-MS/MS method was applied for the determination of PQ and Ml in rat plasma after a single oral administration of PQ(60 mg·kg-1).For PQ,the maximum of concentration?Cmax?was 381.77 ± 87.33 nmol·L-1,AUC0-1 was 7.96 ±3.53 h·?mol·L-1,the time to peak?Tmax?observed was 5.5?2.0,12.0?h,T1/2 was 90.4 ±22.6 h and CL/F was 17.11 ± 10.29 L·h-1·kg-1.For M1,Cmax was 43.33 ± 12.73 nmol·L-1,AUC0twas 0.80±0.36 h·?mol·L-1,Tmax observed was 5.5?3.0,8.0?h and T1/2 was 22.4 ± 6.8 h.The results indicated that both PQ and Ml were eliminated slowly in rats.2.The pharmacokinetics of piperaquine in healthy Chinese subjectsA selective and sensitive LC-MS/MS method was established for the determination of piperaquine and its N-oxidated metabolite in human plasma.The pharmacokinetics of PQ and M1 were evaluated in healthy Chinese subjects after a two-day oral administration of Artequick?QHS-PQ?or Artekin?DHA-PQ?.The sample preparation,liquid chromatography and mass spectrometric conditions were optimized.The following conditions were applied:50 ?L of human plasma samples were pre-treated by a simple protein precipitation procedure Adequate chromatographic retention was achieved on the Venusil C18 column under gradient elution with acetonitrile and 2 mmol·L-1 aqueous ammonium acetate containing 0.15%formic acid and 0.05%trifluoroacetic acid.A triple-quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode.No endogenous interfering peak derived from either biological matrices or co-administrated drug was observed.The validated method was linear in the range of 2.0-400.0 ng·mL-1 for PQ and 1.0-200.0 ng·mL-1 for M1.The lower limits of quantification?LLOQ?were established at 2.0 ng·mL-1 and 1.0 ng·mL-1 for PQ and Ml respectively.The accuracy was 95.1-109.9%for PQ and 92.0-104.1%for M1,and the precision was less than 9.0%,which indicated that the method was accurate and reliable.More than 82.0%of the extraction recoveries were obtained.No matrix effect was found in the bioanalysis process.The results to evaluate stability showed that PQ and Ml were stable during storage and analysisThe validated LC-MS/MS was applied for the determination of PQ and M1 in plasma of healthy subjects recieving a two-day regimen of DHA-PQ.The pharmacokinetic parameters of PQ and M1 were obtained using the TOPFIT software For PQ,Cmax was 902.15 ± 202.88 nmol·L-1,AUC0-t was 47.54 ± 11.25 h·?mol·L-1,Tmax was 34.5?32.0,36.0?h,T1/2 was 248.8 ± 25.6 h and CL/F was 0.87 ± 0.26 L·h-1·kg-1.For M1,Cmax was 270.09 ± 83.53 nmol·L-1,AUC0-t was 27.01±8.27 h·?mol·L-1,Tmax was 35.5?34.0,36.0?h and T1/2 was 205.0 ± 24.4 h.The method was also applied for the bioanalysis of PQ and M1 in plasma of healthy subjects recieving a two-day regimen of QHS-PQ,and the pharmacokinetic parameters of PQ and M1 were obtained.For PQ,Cmax was 424.51 ± 345.10 nmol·L01,AUC0-t was 25.67 ± 14.47 h·?mol·L-1,Tmax was 26.5?25.25,36.0?h,T1/2 was 277.0 ± 58.6 h and CL/F was 1.84± 0.71 L·h-1·kg-1.For M1,Cmax was 143.13 ± 77.21 nmol·L-1,AUC0-t was 1 8.93 ± 6.54 h·?mol·L-1,Tmax was 46.3?28.0,96.0?h and T1/2 was 231.7±38.0 h.The results showed that PQ and M1 were eliminated slowly with a low elimination clearence and a long terminal half-life.After repeated administrations of piperaquine to humans,accumulation could happen for piperaquine and its mono-N-oxidated metabolite.3.The evaluation of antimalarial activity of piperaquine and its metabolites using rodent P.yoelii-infected miceA rodent model for the evaluation of antimalarial activity was established,and chloroquine was selected as the positive drug.The model was then applied to evaluate the antimalarial activity of piperaquine and its metabolites.The ICR male mice were injected intraperitoneally with Plasmodium yoelii BY 265?1 × 107?strain to establish rodent plasmodium-infected model.At 24 h after infection,cloroquine was administrated by gavage to the mice in the positive control group,and the vehicle solution was given in the negative control group.Several doses of piperaquine or its metabolites were administrated in the experimental groups for three consecutive days.The microscopic method was applied to evaluate the parasitaemia in each group.The results showed that chloroquine showed obvious antimalarial activity with ED90 of 1.91 mg·kg-1,which was in correspondence with previous reports(2.04 mg·kg-1).Piperaquine and its two N-oxidated metabolites?M1 and M2?had the potent antimalarial effect against Plasmodium yoelii with ED90 of 1.54 mg·kg-1?piperaquine?,1.31 mg·kg-1?Ml?and 3.22 mg·kg-1?M2?,respectively.The antimalarial activity of the carboxylic metabolite?M3?was not obvious under the designed doses(?32.0 mg·kg-1);The dealylated metabolites?M4 and M5?had good antimalarial activity(ED90 of 14.90 mg·kg-1 for M4 and 20.33 mg·kg-1 for M5). |
PDF Full Text Request