| β-elemene is a sesquiterpene ingredient extracted from one of the traditional Chinese herbal medicine Curcuma wenyujin Chen et C. Ling. Recent studies have shown thatβ-elemene possesses specific cytotoxicity. As a broad-spectrum anti-tumor agent,β-elemene exhibits pharmacological effects including anti-tumor, antibacterial, antivirus, improving immunity, inhibiting platelet aggregation and improving microcirculation, etc. The preparations ofβ-elemene, such as oral emulsion, injection, are extensively used in clinic to treat ascites resulting from cancer, lung cancer, hepatoma, brain cancer, etc. in China. Due to the potent efficacy ofβ-elemene in clinic to treat these diseases, the research ofβ-elemene has become a hot topic in recent years in China andβ-elemene has also become a typical example of TCM made by Hesperian and modern preparation technology.It has been reported that the oral bioavailability ofβ-elemene is very low due to its insolubility in water. Theβ-elemene is rapidly eliminated by injection and the bioavailability ofβ-elemene by i.p. is 8.49%. One of the adverse effects ofβ-elemene formulation used in clinic is the irritability to blood vessel, and the incidence rate of phlebitis is about 30%. Therefore, the purposes of this paper are to reduce irritability to blood vessel, to increase its bioavailability and to prolong its duration in the circulation after i.v. and to target to tumor and to improve efficacy. Three kinds of liposomes, including conventional liposome, PEGylated liposome, thermosensitive long-circulating liposome, were used as the means of fulfilling these purposes.The following parts are included in this paper:1.β-elemene was isolated from Rhizoma Curcumae. The physicochemical properties ofβ-elemene, including n-octanol/water partition coefficient, the solubilities in various solvent, refractive index, specific optical rotation, stability, were determined. The results indicate that:β-elemene is insoluble in water or PBS and soluble in organic solvents. Its n-octanol/water partition coefficient is 199.5, refractive index is 1.4932, specific optical rotation is -16.359°. The concentration ofβ-elemene in ethanol aqueous decreased at 40℃or 60℃for 6 or 12 months.2. The CLE,PLE were prepared by ethanol injection method. Single factor experiment and orthogonal experimental design were used to optimize the formulation of CLE. The effects of various factors on the encapsulation efficiency (EE％) were investigated. The results indicated that the ratio of PC toβ-elemene, different aqueous media and concentration of PC had larger effect on EE％than the other factors. The optimized formulation of CLE was 5％PC, 0.83％CH, 0.5％β-elemene, pH7.4 PBS (33.23mmol/L). The EE％of optimized CLE formulation was above 92％. The quantity of DSPE-PEG2000 in formulation was investigated and the formulation of PLE was optimized on the basis of CLE formulation. The EE％of PLE was 95.2％. The Tm and EE％were used to evaluate the formulation of TLLE prepared by film hydration method. The Tm of TLLE formulation was 41.8℃, which accord with the requirement of the clinical heat treatment. The EE％of TLLE formulation was 87.9％. The characteristics of CLE,PLE,TLLE were investigated. The results indicated that the liposomes were mainly unilamellar and the stability of CLE, PLE and TLLE were investigated. The results of stability indicated that CLE, PLE were stable at 4℃or 25℃for 12 months. But the stability of TLLE was not good.3. The pharmacokinetics and tissue distribution of CLE, PLE and TLLE were investigated. A simple, accurate, rapid GC method for the determination of 13-elemene in biological sample was developed. The pharmacokinetic behavior in rats via i.v. administration showed that the mean t1/2, AUC(0→t) and MRT(0→t) of PLE was 2.7 fold, 3.9 fold, 2.3 fold higher than those of EE. There was significant difference (P＜0.001). The mean t1/2, AUC(0→t) and MRT(0→t) of TLLE was 3.2, 4.3, 2.3 times higher than those of EE. Meanwhile, AUG(0→t) showed a significant difference (P＜0.05) between CLE and EE. Other PK parameters showed no significant difference (P＞0.05) between CLE and EE. The results obtained from pharmacokinetics study showed that the circulation time in vivo ofβ-elemene in TLLE and PLE was significantly prolonged (P＜0.001) and the bioavailability of TLLE, PLE, CLE was significantly improved (P＜0.001). In in vivo tissue distribution study, the dosage was 60mg/kg and the biodistribution behavior ofβ-elemene in the H22 bearing mice via i.v. was changed by encapsulated in conventional liposome, PEGylated liposome and thermosensitive long-circulating liposome. The results of biodistribution showed that: the quantity ofβ-elemene in tumor in different groups was in order that, TLLE+HT＞PLE+HT＞PLE＞CLE＞EE+HT＞EE. The ability to target to tumor of TLLE+HT was maximal and TI of it was 2.57 when it compared with EE+HT. At 37℃, PLE and CLE had greater tumor target than EE. The TI of PLE and CLE was 2.17, 1.33, respectively. The accumulation ofβ-elemene in liver and spleen in TLLE+HT group and PLE group decreased, which indicated that TLLE and PLE can decrease uptake of liver and spleen and can target to tumor. Especially, TLLE had the thermosensitive property and the ability to target to tumor rapidly increased with HT. Moreover, the accumulation ofβ-elemene encapsulated into TLLE, PLE, CLE in fat drastically decreased, which can be used to resolve the accumulation of EE in fat in clinic.4. In pharmacodynamics, we studied the anti-tumor effect of PLE, CLE, TLLE and EE for six cancer cell lines in vitro by MTr assay. The cytotoxicity of different formulations was in order that: PLE＞CLE≈TLLE＞EE. Morphologic change of CoCl cell observed by transsion electron micrograph was consistent with the result of MTT assay. In in vivo anti-tumor experiment, H22 beating mice was used as animal model and IRT and lifespan were used to evaluate the efficacy following intravenous administration at a dose of 40mg/kg. The IRT of different groups was in order that: TLLE+HT(69％)＞PLE+HT (62％)＞CLE+HT(58％)=TLLE (58％)＞PLE(56％)＞EE+HT(51％)＞CLE (48％)＞EE(40％)＞HT(11％)。The IRT of TLLE combining with HT was 69％. PLE had high IRT(56％) at normal animal heat. The survival time of different groups was in order that: PLE≈CLE＞TLLE. The survival time of PLE and CLE group increased 100％. The percent weight loss was used to evaluate the toxicity of different formulations. The percent weight loss was not above 15％, which indicated that the 40mg/kg dosage is proper and the toxicity of CLE, PLE, TLLE and EE was low. In conclusion, the anti-tumor activities of CLE, PLE and TLLE in vivo and in vitro were superior to EE.5. We also studied the pharmaceutical safety of CLE, PLE, TLLE to determine if these formulations were safe via iv administration. The results indicated that (1) no hemolysis, no irritability and no hypersensitivity were found in three formulations, (2) These formulation were tolerated for mice at maximal concentration and maximal injection volume (150mg/kg), which showed the safety of these formulations.
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