Effects Of Different Length Fatty Acids On Liposomes Of Combrestastatin A4 Prodrugs

Drug release contributes a lot to improve therapeutic efficacy and decrease systemic side effects of the therapeutic drug.Especially,the process of the parent drug released or converted from the prodrug,which play an important role in improving the efficacy of the parent drug.In this study,the novel anticancer drug combretastatin A4(CA4)was used as drug model to study how the parent drug released or converted from the prodrug influence the efficacy,pharmacokinetic and tissue distribution of the parent drug by studying a series of CA4 prodrugs,and to screen a suitable preparation for CA4 prodrug with improved anticancer efficacy.This study included five chapters:(1)Synthesis and preformulation study of acylate CA4 prodrugs,including synthesis,identification and preformulation study of acylate CA4 prodrugs;(2)Preparation and characterization of acylate CA4 prodrugs liposomes,including optimizing the liposomes prescription and studies of the in vitro drug release behavior;(3)In vitro studies of acylate CA4 prodrugs liposomes,including the stability in rat plasma and the in vitro cytotoxicity of acylate CA4 prodrugs liposomes;(4)In vivo pharmacokinetics and biodistribution studies;(5)In vivo antitumor activity studies and evaluation the safety.In the first chapter,CA4 reacted with fatty acyl chloride with different chain to synthesis the acylate CA4 prodrugs.The products were identified through H-NMR spectrometry,CNMR spectrometry and LC/MS.The partition coefficient of the acylate CA4 prodrugs were determined by shake-flask method.In the second chapter,the film dispersion method was used to prepare the acylate CA4 prodrugs liposomes.The effects of different content of DSPE-PEG2000,different ratio of lipid to drug and different ratio of lipid to cholesterol on size,Zeta potential and encapsulation efficiency of the liposomes were evaluated through the single factor experiments.The finall prescription was that the content of DSPE-PEG2000 was 0.2%,the ratio of lipid to drug was 1:9,the ratio of lipid to cholesterol was 9:1.The reverse bulk equilibrium dialysis method was used to evaluate the in vitro drug release behavior.And the result showed that the longer the modified acyl chain for CA4,the slower release in vitro.In the third chapter,the stability of CA4 P,CA4-6-L,CA4-10-L,CA4-14-L,CA4-16-L and CA4-18-L in rat plasma was investigated,which were also the percent conversion of the prodrug in rat plasma.The result indicated that the slower release in vitro,the slower conversion rate.Then the cytotoxicity of CA4-6-L,CA4-10-L,CA4-14-L,CA4-16-L,CA4-18-L,CA4 P solution and free CA4 solution was evaluated,using CCK8 assay.And the IC50 values were CA4 < CA4-6-L< CA4-10-L< CA4-14-L< CA4-16-L < CA4-18-L,which meant the slower rate of drug release and conversion,the lower cytotoxicity.In the fourth chapter,in vivo pharmacokinetics of CA4-6-L,CA4-10-L,CA4-18-L and CA4 P were valuated by HPLC.The results demonstrated that CA4-18-L with slow drug release and conversion,could prolong the circulation time in vivo while the acylate CA4 prodrugs with faster drug release and conversion were easy to eliminate from the body.when compared to other treatment groups,CA4-18-L can increase the T1/2 and AUC of CA4 significantly.S180 tumor-bearing mice were used to investigate the biodistribution of CA4-18-L,CA4-10-L,CA4 P.The results demonstrated that CA4-18-L can increase the concentration of CA4 in tumor tissue at the equal dosage due to the slow drug release.In the fifth chapter,the S180 tumor-bearing mice were established to evaluate the in vivo antitumor effects of the different CA4 prodrugs.At the equal dosage,the tumor inhibition rate of CA4 P solution,CA4-10-L and CA4-18-L were 57.87%,43.06%,and 94.44%,respectively,which proves that the rate of drug release and conversion have remarkable effect on properties in vivo and in vitro.Slow drug release could prolong the circulation time and increase the antitumor effect.Especially,CA4-18-L was found even had better antitumor efficacy than CA4 P which is undergoing phase II/III clinical trials.Furthermore,the hemolytic and vascular irritation of CA4-18-L were tested.It meant that CA4-18-L had no effect on hemolytic and vascular irritation.