Studies On Pharmacokinetics And Bioequivalence Of Dihydroartemisinin And Piperaquine Phosphate Tablets With Parallel Design

Objectives:1. To establish an electro-spray ionization high performance liquid chromatography-mass spectrometry (HPLC-MS) method for the determination of dihydroartemisinin and piperaquine in human plasma.2. To study the pharmacokinetics and bioequivalence of dihydroartemisinin and piperaquine phosphate tablets with parallel design in healthy Chinese male volunteers, and then evaluate the absorption, distribution, metabolism, elimination characteristics of them.3. To investigate the effect of terminal elimination phase and two pharmacokinetic softwares on studying pharmacokinetics and bioequivalence of dihydroartemisinin and piperaquine phosphate tablets by using different statistically analytical methods.Methods:1. The dihydroartemisinin and piperaquine in plasma were all determined by HPLC-MS. The MS conditions of dihydroartemisinin were as follows: positive ion mode; capillary voltage4000V; source temperature350℃, drying gas flow12L·min-1; nebulizer gas45psi; the pseudo-molecular ions [M+H]+for dihydroartemisinin and artemisinin (internal standard) were m/z221.2and m/z283.2, which were selected as the target ions for quantification in the selected ion monitoring (SIM) mode, and fragment electric voltage for them were80V and70V, respectively. The separation was performed on Inertsil ODS-3column (4.6×150mm,5μm), and eluted by the mobile phase of lOmmol ammonium acetate (with0.1%formic acid)-methanol-acetonitrile (37:43:20, V/V/V) at a flow rate of0.8mL·min-1. The column temperature was maintained at30℃. The MS conditions of piperaquine were as follows:positive ion mode; capillary voltage4000V; source temperature350℃, drying gas flow11L·min-1; nebulizer gas50psi; the pseudo-molecular ions [M+H]+for piperaquine and lidocaine (internal standard) were m/z535.2and m/z235.1, which were selected as the target ions for quantification in the selected ion monitoring (SIM) mode, and fragment electric voltage for them were120V and90V, respectively. The separation was performed on Gemini C18column (4.6×250mm,5μm), and eluted by the mobile phase of2.5mmol ammonium acetate (adjust pH value to10.5by ammonia water)-acetonitrile (37:63, V/V) at a flow rate of0.8mL·min-1. The column temperature was maintained at25℃.2. The study used a single-center, randomized, parallel and controlled trial design.72healthy Chinese male subjects were randomly assigned to two groups and received dihydroartemisinin and piperaquine phosphate tablets test or reference. Blood samples (6mL) were collected into sodium heparin (0.4%)-containing tubes before and0.25,0.5,0.75,1,1.5,2,3,4,5,6,8,10,12,24,36h and3,5,7,10,14,21,28d after administration. Samples were immediately centrifuged at6000rpm for5min, plasma separated and transported into EP tubes, labeled and stored at-20℃for pending analysis. The dihydroartemisinin and piperaquine levels in plasma were determined by established HPLC-MS.According to the measured plasma concentration-time data of dihydroartemisinin and piperaquine, the following pharmacokinetic parameters were obtained for test or reference:Cmax, Tmax, t1/2, AUC0-t (from0to last measurable time), AUC0-∞(from0to infinity), CL (clearance) and Vd (apparent volume of distribution), using a software WinNonLin6.2.1. The ratios and90%confidence interval analysis (CIs) for the Cmax and AUC0-t of dihydroartemisinin and piperaquine were calculated for both test and reference formulations, which were used to evaluate bioequivalence of them.3. According to the measured plasma concentration-time data during0-72h of piperaquine, the AUC0-72was obtained from the software WinNonLin6.2.1. The ratios and90%confidence interval analysis (CIs) for the Cmax and AUC0-72of piperaquine were calculated for both test and reference formulations, which were used to evaluate bioequivalence of them again, and then compare the bioequivalent results by AUC0-72and AUCo-672.The softwares WinNonLin6.2.1and PKsolver3.0were used to study pharmacokinetics and bioequivalence of dihydroartemisinin and piperaquine phosphate tablets, with which the following pharmacokinetic parameters were obtained for test or reference:t1/2, AUC0-t, AUC0-∞, CL and Vd and were analyzed statistically by paired t test. Then the differences between the two softwares were observed, the bioequivalent results obtained from them were also compared.Results:1. Using the established HPLC-MS, the matrix effects and recovery of dihydroartemisinin and piperaquine were96%-105%,48%-62.5%and91%～109.3%,91%～108%, respectively, meeting the determination requirements. Linearity of dihydroartemisinin was achieved over a concentration range from5to500ng-mL (r2>0.99); piperaquine was achieved over a concentration range from5to400ng-mL"1(r2>0.99). Lower limit of quantification (LLOQ) for both of them were5ng-mL-1, with accuracy of93%to107%and100%to110%, respectively, which were all within±20%and precision no more than5%. The accuracy of dihydroartemisinin and piperaquine in stability tests were ranging from86%to113%and88%to113%, respectively, and the precision of them were all less than11%, indicating that both of them were stability.2. The major pharmacokinetic parameters of dihydroartemisinin obtained from WinNonLin6.2.1for the test and reference formulations were as follows:11/2were (1.03±.17) h and (1.11±0.28) h, Tmax were (1.72±0.96) h and (1.53±0.92) h, Cmax were (236.243±91.087) ng·mL-1and (245.184±120.789) ng·mL-1, AUC0-t, were (561.613±189.513) ng·mL-1·h and (552.821±240.838) ng·mL-1·h, AUC0-∞, were (574.784±190.812) ng·mL-1·h and (565.195±241.822) ng·mL-1·h, CL were (236.0±88.9) L-h-1and (252.4±118.2) L·h-1, Vd were (347.4±141.5) L and (393.3±162.4) L, respectively. The relative bioavailability (F) for test preparations with AUC0-t of dihydroartemisinin was101.6%. The major pharmacokinetic parameters of piperaquine obtained from WinNonLin6.2.1for the test and reference formulations were as follows:t1/2were (289.08±171.11) h and (269.07±238.16) h, Tmax were (3.32±1.89) h and (3.13±0.91) h, Cmax were (223.815±110.931) ng-mL-and (225.494±105.222) ng-mL-1, AUC0-t were (8969.881±4632.410) ng-mL"’-h and (8817.516±4232.909) ng·mL-1·h, AUC0-∞were (11906.837±5792.982) ng·mL-1·h and (11461.232±5755.807) ng·mL-1·h, CL were (103.5±58.1) L·h-1and (109.6±63.4) L·h-1, Vd were (35076.5±15303.4) L and (32034.7±8566.3) L, respectively. The relative bioavailability (F) for test preparations with AUC0-t of piperaquine was101.7%.The90%CIs for the mean ratios (test:reference) with AUCo-t and Cmax of dihydroartemisinin were89.1%～121.8%and83.6%-119.3%, the90%CIs for the mean ratios (test:reference) with AUCo-t and Cmax of piperaquine were82.9%～123.8%and80.7%-120.9%, all ranging in80%～125%and75%～133%respectively.3. The relative bioavailability (F) for test preparations with AUC0-72of piperaquine was98.0%. The90%CIs for the mean ratios (test:reference) with AUC0-72of piperaquine was83.5%～117.5%, ranging in80%～125%as well.The major pharmacokinetic parameters of dihydroartemisinin obtained from PKsolver3.0for the test and reference formulations were as follows:t1/2were (1.02±0.17) h and (1.11±0.27) h, Tmax were (1.72±0.96) h and (1.53±0.92) h, Cmax were (236.243±91.087) ng·mL-1and (245.184±120.789) ng·mL-1, AUC0-t were (575.108±194.250) ng·mL-1·h and (565.444±246.383) ng·mL-1·h, AUC0-∞were (588.198±195.521) ng·mL-1·h and (577.819±247.258) ng·mL-1·h, CL were (230.7±87.2)L·h-1and (246.9±115.5) L·h-1, Vd were (338.7±140.6) L and (382.4±156.8) L, respectively. The relative bioavailability (F) for test preparations with AUC0-t of dihydroartemisinin was101.7%. The major pharmacokinetic parameters of piperaquine obtained from PKsolver3.0for the test and reference formulations were as follows:t1/2were (288.87±171.31) h and (269.07±238.16) h, Tmax were (3.32±1.89) h and (3.13±0.91) h, Cmax were (223.815±110.931) ng·mL and (225.494±105.222) ng·mL-1, AUC0-t were (9102.841±4723.946) ng·mL-1·h and (8958.835±4306.120) ng·mL-1·h, AUC0-∞were (12035.729±5875.097) ng·mL-1·h and (11602.551±5814.924) ng·mL-1·h, CL were (102.4±57.2) L·h-1and (108.3±62.9) L·h-1, Vd were (34712.0±5249.5) L and (31715.6±18516.7) L, respectively. The relative bioavailability (F) for test preparations with AUCo-t of piperaquine was101.6%. The90%CIs for the mean ratios (test:reference) with AUC0-t and Cmax of dihydroartemisinin were89.2%～121.9%and83.6%-119.3%, the90%CIs for the mean ratios (test:reference) with AUCo-t and Cmax of piperaquine were82.8%～123.7%and80.7%-120.9%, all ranging in80%-125%and75%～133%respectively.Conclusions:1. A simple, sensitive and accurate HPLC-MS method was established in the study, which was applied for the determination of dihydroartemisinin or piperaquine level in human plasma.2. Exposure of dihydroartemisinin and piperaquine were highly variable between individuals after administration of dihydroartemisinin and piperaquine phosphate tablets test or reference. Piperaquine had high apparent volume of distribution and low clearance rate, causing its long half-life. There was more than one peak on the plasma concentration-time curve, confirming that piperaquine were undergoing enterohepatic circulation in vivo.According to the pharmacokinetic characteristics of dihydroartemisinin and piperaquine, the compound preparations were not only synergetic in antimalarial mechanism, but also complementary on pharmacokinetics and pharmacodynamics.The90%CIs for the Cmax and AUC0-t of dihydroartemisinin and piperaquine showed that there were no significant differences between the two formulations and it met the State Food And Drug Administration (SFDA) criterion for bioequivalence, indicating that the test preparation of dihydroartemisinin and piperaquine phosphate tablets was bioequivalent to the established reference preparations of dihydroartemisinin and piperaquine phosphate tablets.The whole study processed successfully. There were minor adverse events happened in three subjects after administration of test or reference dihydroartemisinin and piperaquine phosphate tablets, however, these adverse events were disappeared in a short time with no relevant treatments being taken. One subject’s liver was functioning slightly abnormal after the study, which switched back within7d with no drug therapy. There were no clinically significant differences between other subjects in the laboratory examinations.3. The bioequivalent results that evaluated by AUC0-72and AUC0-672of piperaquine were similar, indicating that it was sufficiency to take blood samples up to72h to fulfill the bioequivalent research of piperaquine with long half-life.The bioequivalent results evaluated by PKsolver3.0were similar to WinNonLin6.2.1. There were small differences between the major pharmacokinetic parameters obtained from the two softwares in individuals, however, the general trend in AUCo-t and AUC0-∞of PKsolver3.0were higher than WinNonLin6.2.1, the general trend in CL and Vd of PKsolver3.0were lower than WinNonLin6.2.1, which were considered to have statistical differences. Above all, PKsolver3.0could be used for common pharmacokinetic data analysis and evaluation of bioequivalence referentially.