Pharmacokinetics And Pharmacodynamics Evaluation Of Choline Derivative Modified Artelinic Acid Complex Nanoliposomes

Objective:The commonality of Plasmodium-infected red blood cells and malignant tumor cells was abnormal choline metabolism.Based on this fact,artelinic acid-choline derivative?AD?was synthesized in order to target Plasmodium-infected erythrocytes and malignant tumor cells.AD liposome?ADLs?was prepared and optimized by the single factor method and its formulation properties were investigated.The preliminarily pharmacokinetics study of ADLs in rats was performed and the in vivo activity of ADLs against Plasmodium yoelii BY265?Py BY265?and the in vitro and in vivo activities of ADLs on mouse breast cancer?4T1?cells were evaluated.Methods:1.Synthesis and characterization of choline derivative modified artelinic acid.In this study,dihydroartemisinin?DHA?was used as raw material to synthesize artelinic acid?AA?through dehydration condensation and hydrolysis reaction.And AD was synthesized by esterification between AA and quaternized choline derivatives.The structure of componds was confirmed by Fourier transform ion cyclotron resonance mass spectrometry?FT-ICR MS?,¹H-NMR and ¹³C-NMR.2.Preparation and prescription optimization of choline derivative artelinic acid complex nanoliposomes.The in vitro analytical method of AD was established by high performance liquid chromatography?HPLC?.ADLs were prepared by thin-film hydration method,and the ratio of drug and lipid,hydration temperature and cholesterol dosage on the prescription of ADLs were investigated with encapsulation efficiency?EE?,drug loading efficiency?DL?,particle size?Size?and polydispersity index?PDI?as evaluation indexes to obtain the optimal formulation.The surface morphology of ADLs was observed by transmission electron microscopy?TEM?,the EE of ADLs was determined by ultra-filtration,and the release behavior of liposomes was determined by in vitro dialysis.The stability of ADLs in one month was also investigated.3.Pharmacokinetics of choline derivative modified artelinic acid complex nanoliposomes in ratsA high performance liquid chromatography-tandem mass spectrometry?HPLC-MS/MS?method was established for the determination of AD and AA in rat plasma.24 male Sprague-Dawley rats were randomly divided into 4 groups?n=6?,respectively.Rats were injected with artemisinetic acid solution?AA-SOL?,artemisinetic acid liposome?AALs?,AD solution?AD-SOL?and ADLs,respectively.And 0.5 ml of blood samples were obtained by the orbital plexus at 0.083,0.167,0.25,0.5,0.75,1,2,4,6,8,12 and 24 h,put it into heparinized tubes followed by centrifugation at 3500 r/min for 10 min.The upper plasma was stored in a-20?until analysis.The concentration of AD and AA in the blood was determined by HPLC-MS/MS.4.In vivo antimalarial effect of choline derivative modified artelinic acid complex nanoliposomes.The mice infected with PyBY265 were used as murine model.The untreated group and the blank liposome group were used as the negative control group.The dihydroartemisinin liposomes group?DHALs?,artelinic acid liposomes?AALs?,choline derivative liposomes?CDLs?and equimolar physical mixed liposome groups of AALs and CDLs?AALs-CDLs?were used as the control group.Infection rate,inhibition rate,negative conversion rate,recurrence rate and survival rate were used as evaluation indexes.The anti-malarial activity of ADLs in vivo was examined by Peter's 4-day test.5.In vitro and in vivo antitumor activities of choline derivative modified artelinic acid complex nanoliposomes.The cytotoxicity of ADLs on Balb/C mouse breast cancer cells?4T1?was evaluated by MTT assay.Tumor models were established by inoculating breast of Balb/C mice with 4T1 cells.The tumor growth volume,mouse body weight,tumor inhibition rate and survival rate were used as evaluation indexes,and the in vivo antitumor activity of ADLs was examined by four consecutive days of administration.Results:1.Synthesis and characterization of choline derivative modified artelinic acid complex.The results of the FT-ICR MS,¹H-NMR and ¹³C-NMR were consistent with the structure of the corresponding compounds,indicating that AD has been successfully synthesized.The yield of choline derivative?CD?produced by quaternization reaction can reach 65.65%?AA and AD were purified by column chromatography?yield of 31.71%and 63.31%,respectively?.2.Preparation and prescription optimization of choline derivative modified artelinic acid complex nanoliposomes.The HPLC methods were used to determinate the concentration of AD in vitro.After verificated by analytical methods,the methods can be used for the determination of content of AD and its preparations in vitro.The prescription for liposomes optimized by the single factor method was consisted of drug/lipid ratio of 1:10,the hydration temperature of 40?,and cholesterol/phospholipid ratio of 1:25.The ADLs showed translucent appearance,and the TEM presented that the ADLs was round or nearly circular.The average particle size of the ADLs was?164.4±1.8?nm,the Zeta potential was?40.87±0.86?mV.The entrapment efficiency of ADLs was?95.74±0.10?%and drug loading efficiency was?12.60±0.05?%.The in vitro release assay suggested that ADLs could release 48.93%at 12 h.3.Pharmacokinetics of choline derivative modified artelinic acid complex nanoliposomes in ratsThe established HPLC-MS/MS method followed up with the methodological requirements and can be used to determine the concentration of AD and AA in rat plasma.The pharmacokinetic results exhibited that the AUC,t_1/2,CLz,and Vz of ADLs were4.54,2.09,0.23,and 0.55 folds of AD-SOL,respectively.Those of AALs were 2.57,1.94,0.38 and 0.68 times of AA-SOL,respectively.This result indicated that the liposomal formulation can increase the AUC of the drug,prolong the biological half life,and reduce the clearance and volume of distribution,compared with the solution of AA and AD.AD showed a significant increase in AUC and t_1/2,and a significant decrease in CLz and Vz,compared with AA.The results demonstrated that the AA-SOL showed the fastest elimination and the lowest blood concentrations,followed by AALs,AD-SOL and ADLs.4.In vivo antimalarial effect of choline derivative modified artelinic acid complex nanoliposomes.There was no significant difference?P>0.05?between the blank liposome group and the untreated group,indicating that the blank liposome group had no inhibitory effect on Plasmodium.The infection rate,inhibition rate,negative conversion rate,recurrence rate and survival rate of each preparation group showed that the antimalarial order was ADLs>DHALs>AALs>AALs-CDLs>CDLs.5.In vitro and in vivo antitumor activities of choline derivative modified artelinic acid complex nanoliposomes.MTT assay demonstrated that the cytotoxicity of ADLs on Balb/C mice breast cancer cells?4T1?was time and concentration dependent.ADLs group showed the most obvious antitumor effect in vivo on inhibiting tumor growth,and the survival rate of mice,followed by DHA-SOL and AA-SOLConclusions:1.Choline derivative modified artelinic acid was successfully synthesized and its structure was confirmed by FT-ICR MS,¹H-NMR and ¹³C-NMR.2.ADLs were prepared by thin-film hydration method.The morphology,particle size and distribution,EE,DL,stability and release profile of the ADLs met the expected requirements.3.Pharmacokinetic experiments had shown that both preparing liposomes and binding to choline derivative could improve their pharmacokinetic characteristics of AD or AA including prolonging their biological elimination half-life,reducing their degradation in blood and increasing accumulation in target cells,thereby improving drug efficacy.4.In vivo antimalarial pharmacodynamic of ADLs was dose-dependent,and their antimalarial effects were superior to DHALs,AALs,AALs-CDLs and CDLs,and the survival rate was highest among these administrations.5.MTT assay showed that AA-SOL,ADLs and DHA-SOL inhibited the growth of4T1 cells in time and concentration dependent manner.DHA-SOL had the highest cytotoxicity to 4T1 cells,followed by ADLs and AA-SOL.The anti-tumor pharmacodynamics results in vivo suggested that compared with AA-SOL and DHA-SOL,ADLs had obvious better anti-breast cancer effect,which could significantly inhibit tumor volume and tumor weight,and improve survival rate of Balb/C mice.