Metabolism And The Effect Of Gender On Pharmacokinetics And Activity Of Naphthoquine

The 4-aminoquinoline drug naphthoquine(NQ)is a long acting antimalarial drug and it is one of suitable partner drugs in artemisinin drug-based combination therapy(ACT).Pharmacokinetics studies of naphthoquine showed that the absorption of NQ was fast(tmax 2-4 h),and the elimination rate was slow(t1/2>255 h),and the clearance rate was low(4.49 L·h-1·kg-1).The metabolites and metabolic pathways of NQ are not yet reported.Although the pharmacokinetics of NQ is reported,the male individuals are generally chosen,and there were no reports about the differences between male and female individuals.The good antimalarial activity(IC50 4.2 nmol-L-1)was shown by NQ,but there were no reports about the antimalarial activity difference of NQ between male and female ind:ividuals.In this paper,the products of biotransformation of NQ were identified,and 5 metabolites of NQ were obtained,and the main metabolic enzymes and main metabolic pathways involved in NQ metabolism were found.At the same time,a quantitative method was established for the quantitative analysis of the biological samples of NQ LC-MS/MS with a lower limit of quantitative lower limit,and the difference of pharmacokinetics between male and female individuals was obtained.In addition,the activity of NQ in the female and male rats was evaluated.1.Research on naphthoquine biotransformationIn this chapter,a high-resolution liquid chromatography-mass spectrometry technology was applied for rapid detection of the metabolites of naphthoquine in vitro and in vivo.The multiple data acquisition and data post-processing methods were used in this study.The MS/MS fragmentation characteristics of naphthoquine and its metabolites were obtained.The main metabolites of naphtholine were obtained by the classification and quantity of the metabolites produced by seven recombinant CYP enzymes.A total of five phase I metabolites were detected(M1-M5),and no phase II metabolite was found.Four hydroxylated products(Ml,M2,M3 and M5)and one N-oxidation product were found for the first time.The main metabolic pathways including hydroxylation and N-oxidation were newly found for NQ.And the main metabolic enzyme involved in the metabolism of NQ is CYP2D6 enzyme.2.Gender effect on the pharmacokinetic of naphthoquineA selective and sensitive LC-MS/MS method was established for the determination of naphthoquine in rat plasma.The validated method was successfully applied to a pharmacokinetic study performed in rats after a single oral administration of naphthoquine(40 mg·kg-1).The sample preparation,liquid chromatography and mass spectrometric conditions were optimized.The optimized conditions were as follows:20 ?L of rat plasma samples were pre-treated by a simple protein precipitation procedure.Adequate chromatographic retention was achieved on the Poroshell 120 SB C18 column under gradient elution with acetonitrile and water containing 0.2%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.The method was linear in the range of 1.0-400.0 ng·mL-1.The lower limits of detection(LLOD)were established at 0.2 ng-mL-1.The validated LC-MS/MS method was applied for the determination of NQ in rat plasma after a single oral administration of NQ(40 mg·kg-1).For NQ in male,the maxinrmum of concentration(Cmax)was 548.83 ± 185.62 ng·mL-1,AUC0-t was 38-89 ±9.36 ?h·g·mL-1,the time to peak(Tmax)observed was 5.8 h(2.0,12,0),T1/2was194.3±44.7.For NQ in female,Cmax was 429.50 ±122.92 ng·mL-1,AUCo-t was 30.52 ±5.99 h,?g·mL-1,Tmax observed was 3.2 h(2.0,6.0h)and T1/2 was 138.3±25.1 h.The results showed that the male rats had higher AUC values and NQ was eliminated slowly in male rats.3.The evaluation of antimalarial activity of naphthoquine using male and female 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 dose of naphthoquine in the experimental groups for one day.The microscopic method was applied to evaluate the parasitaemia in each group.The results showed that chloroquine showed obvious antimalarial activity with ED90 of 2.18 mg·kg-1,which was in correspondence with previous reports(2.04 mg·kg-1).Naphthoquine had the potent antimalarial effect against Plasmodium yoelii.The 50%effective dose of ED50 in male and female malaria rats is 0.94 mg·kg-1 and 0.71 mg·kg-1 respectively,the 90%effective dose ED90 is 1.67 mg·kg-1 and 1.10 mg·kg-1 respectively.The experimental results showed that the activity of naphthoquine in male mice was higher than that of female mice.