Study On Epirubicin Hydrochloride Long-circulating Thermosensitive Liposomes And Compound Liposomes

In recent years, some antineoplastic agents loaded liposomes have reached the market successfully, and especially those novel liposomes which having good targeting and therapeutic effects had been deeply studied through physical and chemical ways. All these studies bring great hope for people to overcome cancer. For instance, antineoplastic agents loaded long-circulating thermosensitive liposomes not only can prolong the action time of the drug, but also can make the drug be released mostly at the tumor site when the local site is heated, thus therapeutic effect is improved and system toxicity is reduced.This study includes two parts, and the first part is study on epirubicin hydrochloride(EPI) loaded long-circulating thermosensitive liposomes(LTSL). First, pharmaceutical characteristic of the model drug EPI was investigated systemically. Liposomes and free drug were separated through dextran G50. UV and HPLC were established to determine drug content. Fluorescence method were established to determine the in vitro release properties of LTSL. The solution properties of EPI was investigated in different media. The principles of drug loading and releasing were elucidated initially. The precipitation that EPI formulated with buffer salt after loaded in the intraliposomes can be dissolved and released when the temperature increased to 42℃and the liposomes phase transition happened. While when EPI-LTSL was stored in lower temperature, due to EPI existing in colloidal state precipitation, it was not easy to leak out from liposomes. Initial process and formulation was established by single factor evaluation taking encapsulating efficiency(EE) and accumulation drug release as index. And pH gradient method was used to load the drug. When drug/lipid ratio was 1:20, the ratio different lipids had little effect on the EE of liposome. When the theoretical drug loading concentration was 2 mg·mL^-1, EE of liposome decreased from 99% to 80% with lipid concentration decreasing from 75 mg·mL^-1 to 30mg·mL^-1. So lipid concentration had larger effect on EE of liposome. It can improve the stability of liposome and almost without any effect on the transition temperature of liposome through addition of a little DSPG. The DPPC/MSPC ratio also had large effect on drug release of EPI-LTSL, and the in vitro release amount increased with MSPC content increasing or DSPG decreasing. Therefore, Drug/lipid ratio, DPPC/MSPC ratio and the hydration medium concentration were taken as evaluation factors, and the formulation and process was optimized by orthogonal design experiments with thermosensitive drug release amount at 37℃and 42℃as evaluation index. The results demonstrated that thermosensitive drug release amount was significantly influenced by the hydration medium concentration. The optimized formulation were as follows: drug/lipid was 1:10; DPPC/MSPC was 82:8; the hydration medium was 250mM pH4.0 citrate buffer. The optimized process were as follows: liposomes were homogenated for six times at 15000psi; extruded through 100 nm membranefor 3 times.The HPLC method was established to determine the related substances of EPI-LTSL. The result demonstrated that the specificity of the method was good. Three batches of liposomes were prepared according to optimal formula and their physicochemical properties were as follows. Drug concentration was 2.4mg/mL with 2.6% of total related substances .The EE was 99%, and the mean particle diameter was not more than 100nm. The pH of liposome was 7.6⁷.7, and phase transition temperature was about 42.8℃. The in vitro release test of EPI-LTSL demonstrated that the drug release amount was not more than 5% at 37℃in 15 min, while about 90% of the total drug released at 42 or 43℃in 4 min. Stability test of EPI-LTSL demonstrated that EE and pH changed little when stored at 4℃or -20℃for 150 days. The mean particle diameter increased about 20nm and 50nm with drug content decreasing 9.6% and 2.9% respectively. The mean particle diameter and EE almost had not changed after diluted 10 times with normal saline or 5% glucose for 2h. While the mean particle diameter increase about 20nm with EE decreasing about 1.5% when EPI-LTSL was diluted by new born calf serum with equal volume and stored for 24h.As we know, keeping high EE and little changed particle diameter after re-hydration of freeze-dried liposomes is a bottle neck for water-soluble drug loaded liposomes studying. Qualified liposomes were prepared in the test after adjusting formulation, choosing cryoprotectant and freeze-drying process. By addition of proper amount of DSPG, it can be avoided that the mean particle diameter of liposome increased due to aggregation when freeze-dried liposomes were rehydrated .It was found that the cryoprotectant concentration in the external and internal phase of liposomes can have a significant effect on the characteristics, re-hydration rate. Futhermore, the cryoprotectant type can influence the stability of liposomes after rehydration. Lactose was an optimized cryoprotectant, with an optimized concentration of 9% in the intraliposomes as well as extralioposomes, and the freeze-dried liposomes had a homogeneous bright red color with porous structure. Which can be rehydrated fast by water with little drug leaking out and mean particle diameter increasing little. It was also found in the test that pre-freezing temperature and rates and freeze-drying procedure also can influence the characteristics and rehydration of the lyophilized liposomes. Middle speed pre-freezing (-65℃) and first-drying slowly may be beneficial to obtain good characteristics of liposomes having smooth surface with no collapse.The pharmacokinetics study was carried out with SD rats. The LC-MS method was established to determine EPI content in vivo. This method was simple, quick, sensitive and the lower limit of quantitation is 10 ng/ml. The pharmacokinetics results showed that EPI solution ,EPI-LIP and EPI-LTSL was fast distributed and slowly eliminated When the same dosage (12mg/kg) of EPI was administered in rats by i.v, which was fit for the three compartment model. The t1/2α, t1/2β, t1/2γ, AUC, and MRT of EPI-LTSL group calculated by DAS software were 7.8, 1.3, 12.6, 15.1, 6.2 times those of EPI solution group; and 1.6, 1.4,12.3, 3.0, 3.6 times those of EPI-LIP group. Moreover, the CL of the latter two groups was about 14 times of the former EPI-LTSL group. It's obvious that EPI-LTSL can significantly prolong the circulation time of EPI in vivo.Tumor-bearing mouse model was founded by subcutaneous injection of Lewis cancer cell. Non thermosensitive long circulating liposomes(NTSL) group and EPI injection group were taken as control group, and normal saline group as blank group. The drug solution or normal saline were administered three times i.v. in all the groups. The tumor growth and weight diversity curve were drawn. The tumor inhibitory rate was calculated taken tumor weight as index. The results indicated that the tumor weight of EPI-LTSL, NTSL and injection groups all had extremely significant difference (P<0.01) compared with normal saline group. And the tumor inhibitory rate of EPI-LTSL had significant difference (P<0.05) compared with that of NTSL or injection group, whose mean tumor inhibitory rate was about 61.1% while the other two only 39.6% and 43.1%. But there was no significant (P>0.05) difference between NTSL and EPI injection group. The mice weight of EPI-LTSL and NTSL group decreased the least, while EPI solution group decreased more, which indicated that EPI loaded liposome had less systemic toxic effect in mice. The results of histopathologic examination of tumors in each group indicated that tumors of EPI-LTSL group had more extensive necrosis than that of the other two control group. And there wasn't any change founded in the cardiac muscle of the mice in all the groups. This may be due to relatively smaller administration dose of EPI.The second part is the study of the compound liposomes loaded with EPI and paclitaxel (TAX). First, the in vitro cell growth inhibitory effect was evaluated when EPI and TAX were used EPI and TAX separately or together. The joint action effect was calculated by King's equation. The cell toxicity test indicated that the human breast cancer cell of MCF-7,ZR75^-1 are more sensitive to EPI than TAX. When the two drugs used separately, the cell inhibitory effect of them was time-dependent. When used together at high concentration(>1μg·mL^-1), there was addition effect to inhibit both MCF-7,and ZR75^-1. In the test, two anticancer drugs with large difference in their physiochemical properties were loaded in one liposome(which we called compound liposome). The formulation and process of the compound liposome was optimized. HPLC method was established which can determine the content of EPI and TAI at the same time. The mean particle diameter of the compound liposome prepared by the optimized process and formulation was about 100 nm; the EE of TAX and EPI was 95% and 99%; the content was 0.83 mg·mL^-1 and 0.27mg·mL^-1 respectively. Two dose (Low and high) pharmacodynamics tests were carried out. High dose test(TAX 30mg·kg^-1,EPI 10mg·kg^-1) results demonstrated that the compound liposomes group had addition tumor growth inhibitory effect when compared with single drug loaded liposome.But there weren't any significant difference (p>0.05) among compound liposomes group, physical blended liposomes group and physical blended solution group whether with high or Low dose(TAX 15mg·kg^-1,EPI 5mg·kg^-1).The EPI-LTSL prepared in the first part of this study had stable preparation process and desirable thermosensitive drug release behavior in vitro. EPI-LTSL not only prolong the circulation time of EPI, but also could make the drug enriched in local tumor site when heating. Therefore, Therapeutic effect was improved and systemic toxic effect was reduced in lewis bearing mice. Although EPI loaded conventional and long-circulation liposomes were reported in some literatures, but the pharmacokinetics and pharmacodynamics in vivo of EPI loaded long-circulation thermosensitive liposomes haven't been reported. This study indicated EPI-LTSL had better lewis tumor inhibitory effect than conventional lipsomes. In the second part of this study, the compound liposomes loaded with EPI and TAX with high EE was initially studied. The HPLC method was first established which can determine the content of EPI and TAI at the same time. The EPI-TAX compound liposomes haven't been reported .The in vitro cytotoxicity test with breast cancer cell MCF-7 and ZR75^-1 using EPI and TAX alone or together have been done ,and the combination effect has been calculated. The formulation of EPI-LTSL was systemically evaluated in this study, and in vitro and in vivo evaluation was also carried out in EPI-LTSL and EPI and TAX loaded compound liposomes. It had done meaningful exploration for new dosage form of EPI and provided reference for clinical development and application of the new preparations.