The Study Of Pharmacokinetics And Pharmacodynamics Of Four Dihydroartemisinin Trimers Self-assembled Nanoparticles

Objective:In order to solve the shortcomings of dihydroartemisinin?DHA?in the body with high reignition rate and short half-life and improve antimalarial activity,four dihydroartemisinin trimers[?DHA-CC?₃,?DHA-SS?₃,?DHA-SCS?₃ and?DHA-S?₃]were synthesized,by using carbon-carbon bond,disulfide bond,spacer sulfur bond and single sulfur bond as linking arm and glycerol as skeleton,respectively.Four trimers were made into self-assembled nanoparticles[?DHA-CC?₃NPs,?DHA-SS?₃NPs,?DHA-SCS?₃NPs and?DHA-S?₃NPs]by nano-precipitation method.The prescriptions were optimized.The pharmacokinetics and antimalarial pharmacodynamics of four DHA trimers self-assembled nanoparticles were evaluated in rats and Plasmodium yoelii BY265?PyBY265?mice respectively,which will provide basis and guidance for the treatment of malaria for trimers self-assembled nanoparticles.Methods:1.Synthesis of four dihydroartemisinin trimmersCarbon-carbon bonds DHA trimer was synthesized by esterification reaction using AS and glycerol.Disulfide bonds,spacer sulfur bonds and monosulfide bonds DHA trimers were formed needing two steps.The first step was that 3,3-dithiodipropionic acid,methylenebis?thioglycolic acid?and 2,2-thiodiacetic acid with DHA was taken esterification reaction,respectively.The second step was the esterification reaction between the reaction product of the first step and glycerol.Finally,the products were confirmed by high resolution mass spectrometry?HRMS?and nuclear magnetic resonance spectrum?¹H^-NMR?.2.Preparation of four dihydroartemisinin trimers self-assembled nanoparticles and investigation of the physicochemical propertiesSelf-assembled nanoparticles were prepared by nano-precipitation method.The single-factor optimization method was used to screen out better prescriptions.The physicochemical properties,including size,Zeta potential,polydispersity index?PDI?,entrapment efficiency?EE?,drug-loading efficiency?DL?,stability and drug release behavior,were investigated.The classical release method in vitro was used for four kinds of self-assembled nanoparticles in 30%ethanol aqueous solution containing 10 mM DTT or 10 mM H₂O₂,respectively.3.Pharmacokinetics of four dihydroartemisinin trimers self-assembled nanoparticlesForty-two male SD rats were randomly divided into six groups?each n=7?.Rats were injected intravenously with DHA solution,AS injection and four trimers self-assembled nanoparticles at a corresponding equivalent dose of 6.3 DHA mg?kg^-1,respectively.The pharmacokinetic characteristics of all groups were measured by HPLC-MS/MS.Pharmacokinetic parameters were calculated by non-compartment method.4.Antimalarial activity of four dihydroartemisinin trimers self-assembled nanoparticlesThe ICR mice were infected with 0.2 mL of infected blood by intraperitoneal injection.After 2 h,the infected mice were treated with saline,DHA solution,AS injection,?DHA-CC?₃NPs,?DHA-SS?₃NPs,?DHA-SCS?₃NPs and?DHA-S?₃NPs,respectively.All treatment group expect saline received five different doses[3.52,8.8,17.6,26.4,35.2?mol??kg·day?^-1,each n=6].All groups were treated once a day for four consecutive days by intravenous injection?Peters 4-day suppression test?.Thin blood smears were made from mouse-tail blood and stained on the 1st days,7th days and 28th days after drug withdrawal.The inhibition ratio,infection ratio,ED₅₀,ED₉₀,negative conversion fraction,average survival days,cure ratio and recurrence ratio were calculated.The body weight of all mice was measured from 1st day to 28th day.Results:1.Synthesis of four dihydroartemisinin trimmersThe four DHA trimers were confirmed by HRMS and ¹H-NMR.The yields of the?DHA-CC?₃,?DHA-SS?₃,?DHA-SCS?₃ and?DHA-S?₃ were 80%,39%,53%and 62%,respectively.2.Preparation of four dihydroartemisinin trimers self-assembled nanoparticles and investigation of the physicochemical propertiesThe optimized prescription was 7.5 mg?ml^-1 of injection drug concentration,860r?min^-1 of stirring speed,12 d?min^-1 of injection speed,25?of temperature and 20%TPGS.The four DHA trimers self-assembled nanoparticles were regularly spherical-shaped with a uniform size distribution.The size was less than 140 nm.The PDI was less than 0.2.The absolute value of Zeta potential was greater than 10.The EE and DL was more than 98%and 78%,respectively.The PDI,Zeta potential and EE of four DHA trimers self-assembled nanoparticles had no significant changes when stored at 4?for 28days.The 30%ethanol aqueous solution containing hydrogen peroxide?H₂O₂,10 mM?or dithiothreitol?DTT,10 mM?was used as the release medium.In 30%ethanol aqueous solution,30%ethanol aqueous solution containing 10 mM DTT and 30%ethanol aqueous solution containing 10 mM H₂O₂,the release of DHA from the?DHA-CC?₃NPs was 28.75±0.85%,26.75±0.17%and 29.75±0.54%,respectively;the release of DHA from the?DHA-SS?₃NPs was 15.79±2.59%,36.93±0.44%and 18.14±0.12%;the release of DHA from the?DHA-SCS?₃NPs was 21.49±1.04%,27.85±1.64%and 46.13±0.90%;the release of DHA from the?DHA-S?₃NPs was 58.48±3.43%,68.84±0.97%and 74.86±0.98%,respectively.3.Pharmacokinetics of four dihydroartemisinin trimers self-assembled nanoparticlesFor the area under the plasma concentration-time curve(AUC_0-t)of the trimer and active metabolites DHA in DHA solution,AS injection and the four DHA trimers self-assembled nanoparticles were compared.The order of AUC_?0-t?of DHA was:?DHA-S?₃NPs group??DHA-CC?₃NPs group>DHA solution group?AS injection group>?DHA-SCS?₃NPs group>?DHA-SS?₃NPs group;The order of AUC_?0-t?of trimers was:?DHA-SCS?₃NPs group>?DHA-S?₃NPs group>?DHA-SS?₃NPs group>?DHA-CC?₃NPs group;The AS of?DHA-CC?₃NPs group and AS injection group could be measured.The MRT of DHA in DHA solution,AS injection,?DHA-CC?₃NPs,?DHA-SS?₃NPs,?DHA-SCS?₃NPs and?DHA-S?₃NPs group were 0.29±0.027^b,0.23±0.068^b,0.33±0.11^b,0.40±0.19^b,0.28±0.06^b and 0.75±0.33 h,respectively[^bP<0.05 vs?DHA-S?₃NPs group].The MRT of DHA trimers in?DHA-CC?₃NPs,?DHA-SS?₃NPs?DHA-SCS?₃NPs,and?DHA-S?₃NPs group were 0.042±0.02^b,0.21±0.08^b,0.36±0.25^b and 0.97±0.38 h,respectively[^bP<0.05 vs?DHA-S?₃NPs group].The total drug concentrations?the sum of 3 times of the molar concentration of the trimers,DHA and AS molar plasma concentrations?(nmol?ml^-1)were used to characterize the concentration-time curve and calculate pharmacokinetic parameters of different groups.The AUC_0-t of DHA solution,AS injection,?DHA-CC?₃NPs,?DHA-SS?₃NPs,?DHA-SCS?₃NPs and?DHA-S?₃NPs were 2.59±0.34,3.09±0.49,5.87±1.58^*#,3.96±1.11^*,18.85±5.02^*#and 14.49±1.49^*#h?nmol?ml^-1 in rat plasma,respectively?^*P<0.05vs DHA solution group;^#P<0.05 vs AS injection group?;The MRT were 0.29±0.027^b,0.20±0.055^b,0.34±0.095^b,0.18±0.09^b,0.18±0.07^b and 0.75±0.12 h,respectively[^bP<0.05 vs?DHA-S?₃NPs group].4.Antimalarial activity of four dihydroartemisinin trimers self-assembled nanoparticlesThe infection ratio and inhibition ratio were calculated at 1st days.The five doses of six administration groups had different degrees of inhibition compared to saline group.And all groups shows dose dependence at 35.2,17.6,and 3.52?mol??kg?day?^-1.The ED₉₀of DHA solution,AS injection,?DHA-CC?₃NPs,?DHA-SS?₃NPs and?DHA-SCS?₃NPs,?DHA-S?₃NPs were 14.68±0.98,14.34±1.96,7.82±1.16^*#,6.39±0.65^*#,5.13±1.17^*#and 3.40±0.88^*#?mol??kg?day?^-1,respectively?^*P<0.05 vs DHA solution group;^#P<0.05 vs AS injection group?.The infection ratio was calculated at 7th day.The infection ratio of five different doses were increased in the DHA solution group and the AS injection group compared to1th days.The?DHA-CC?₃NPs group in 35.2 and 26.4?mol??kg?day?^-1 dose,the?DHA-SS?₃NPs group in 35.2,26.4,17.6?mol??kg?day?^-1 dose,the?DHA-SCS?₃NPs group in 35.2,26.4,17.6?mol??kg?day?^-1 dose,the?DHA-S?₃NPs group in 35.2,26.4,17.6,8.80?mol??kg?day?^-1 dose were no statistically significant difference compared to 1th days.On the 28th days,the average survival days of the trimers self-assembled nanoparticle were longer than those of the DHA solution and the AS injection group at the same dose.The average survival days of the four trimers self-assembled nanoparticle groups at the same dose were[17.6,8.8,3.52?mol??kg?day?^-1]:?DHA-S?₃NPs>?DHA-SCS?₃NPs>?DHA-SS?₃NPs>?DHA-CC?₃NPs.The order of negative conversion fraction and cure ratio were the same as the average survival days.Conclusion:1.Synthesis of four dihydroartemisinin trimmers and Preparation of four dihydroartemisinin trimers self-assembled nanoparticlesFour dihydroartemisinin trimers were successfully synthesized.The four DHA trimers self-assembled nanoparticles were prepared.The nanoparticles were round and spherical,with smaller particle size,higher EE and DL.They were stable at 4?for 28 day's shelf life.?DHA-SS?₃NPs had reduction sensitivity,?DHA-SCS?₃NPs had redox sensitivity and?DHA-S?₃NPs had redox and reduction sensitivity.2 Pharmacokinetics of four dihydroartemisinin trimers self-assembled nanoparticlesThe four trimers self-assembled nanoparticles increased the total drug concentrations and the?DHA-S?₃NPs group prolonged the MRT.3.Antimalarial activity of four dihydroartemisinin trimers self-assembled nanoparticlesBy comparing different evaluation indexes,the antimalarial activity of the four trimers self-assembled nanoparticles were stronger than that of DHA solution and AS injection.The order of antimalarial activity was:?DHA-S?₃NPs>?DHA-SCS?₃NPs>?DHA-SS?₃NPs>?DHA-CC?₃NPs.