Preparation Of Dacarbazine Long-circulating Liposomes And Its Preliminary Evaluation Of Pharmacokinetics And Pharmacodynamics In Vivo

Malignant melanoma?MM?is a malignant tumor derived from melanocytes in the skin and mucous membranes.In recent years,it has become one of the fastest growing tumors with an annual growth rate of 3%-5%.The proportion of deaths and new cases of melanoma in China is significantly higher than that in the global average.The treatment of melanoma is difficult and its mortality rate is high.Melanoma has become a disease that seriously threatens human health.At present,early melanoma can achieve better results by surgical resection,but the prognosis of middle and advanced melanoma is poor.There is no effective treatment,and the main method is to provide systemic adjuvant chemotherapy after the operation.Dacarbazine?DTIC?is the first-line chemotherapy drug for malignant melanoma.However,dacarbazine has significant defects,such as only single dosage form,poor stability,and complicated preparation before using,poor compliance,low clinical effective rate,severe adverse reactions,and ineffective for brain metastases.In order to expand its chemotherapy regimen of malignant melanoma,improve the anti-tumor effect of dacarbazine and reduce the toxic and side effects,this paper has carried out a preliminary exploration in improving the dosage form of dacarbazine and A long circulating liposomes were prepared for loading and delivering dacarbazine as targeted drug delivery systems.And its preliminary evaluation of pharmacokinetics and pharmacodynamics in vivo was carried out.This main studies were as the following sections:In the first part,The reverse vaporization method and ammonium sulfate gradient method were both used to decide better preparation method for liposome containing dacarbazine by selecting the soybean lecithin and cholesterol as the material and the encapsulation efficiency as the index.The factors affecting the encapsulation efficiency of dacarbazine liposomes were investigated with univariate analysis to screen best prescriptions and processes.The results showed that the ammonium sulfate gradient method was more suitable for the preparation of dacarbazine liposome,and the liposome entrapment efficiency was 63.67%.The univariate study showed that factors such as the phospholipid-cholesterol ratio,the ratio of drug to lipid,the concentration of ammonium sulfate solution,the type of dialysate,dialysis time had a great influence on the entrapment efficiency of dacarbazine liposome,while the incubation temperature and incubation time had little effect on it.In the second part,on the basis of general liposomes of dacarbazine,the functionalized phospholipid of Distearoyl Phosphatidylethanola mine-Polyethylene Glycol 2000(DSPE-PEG₂₀₀₀)was added to prepare dacarbazine long circulating liposomes modified with polyethylene glycol?DTIC-PEG-LP?.And the encapsulation efficiency,drug loading rate,morphology,particle size distribution,Zeta potential,stability,and drug release behavior in vitro of DTIC-PEG-LP were evaluated and compared with Dacabazine normal liposomes.The prepared DTIC-PEG-LPs were showed round shape and smooth surface,uniform particle size,with an average particle size of 100-200 nm,a PDI value of about 0.2,and a Zeta potential of about-50 mV.The encapsulation efficiency was greater than 60%and the drug loading rate was greater than 5%.The stability experiment showed that the prepared DTIC-PEG-LPs were rather stable in 4?refrigerator for 7 days,and the appearance and entrapment efficiency of the liposome suspension were not significantly changed.The prepared DTIC-PEG-LPs releasing experiments in vitro showed a certain slow release function in PBS6.5 and PBS7.4 release media.In the third part,High Performance Liquid Chromatography tandem Mass Spectrometry?HPLC-MS/MS?was established to determine the concentration of dacarbazine in rat plasma.Methodology such as specificity,standard curve and lower limit of quantitation,precision and accuracy,matrix effect and stability were validated.The plasma concentration of dacarbazine was determined by HPLC-MS/MS at each time point after the intravenous administration of DTIC-PEG-LPs via rat tail vein to investigate the pharmacokinetics of dacarbazine,draw the harmacokinetic curves,and calculate the pharmacokinetic parameters.Dacarbazine solution was compared as a control.Methodology validation showed that the established HPLC-MS/MS method is sensitive and reliable.Pharmacokinetics experiments showed that the AUC_0-t and C_maxax of DTIC-PEG-LPs in rats were greater than that of dacarbazine solutions.Compared with dacarbazine solutions,DTIC-PEG-LPs could increase the plasma concentration of dacarbazine,enhancing the anti-tumor efficacy of dacarbazine.In the fourth part,a melanoma tumor-bearing mouse model was established by subcutaneous injection of B16-F10 cell suspensions into the dorsal skin of C57BL6mice.The body weights,tumor sizes,and survival time of mice were investigated as the anti-tumor effect of dacarbazine long-circulating liposomes with saline group,blank liposome group,and dacarbazine solution group as controls.The results showed that the average tumor volumes of DTIC-PEG-LPs mice group?2056.78mm3?was significantly smaller than that of dacarbazine solution group?5128.93mm3?,and the median survival time of DTIC-PEG-LPs mice group?31 days?.Significantly longer than the dacarbazine solution group?26 days?.In melanoma tumor-bearing mouse models,the antitumor effect of dacarbazine long-circulating liposomes was better than that of dacarbazine solution.