Studies On Liposomal Formulations For Irinotecan Hydrochloride

Irinotecan hydrochloride(CPT-11) is a water-soluble derivate of camptochecin. It has been studied extensively recently because of its better anti-cancer activity and lower toxicity in comparison with other camptothecins. CPT-11 was demonstrated to specifically inhibit the function of the topoisomerase I which is a ubiquitous enzyme involved in DNA replication and RNA transcription. Preclinical studies showed that CPT-11 has demonstrated anti-tumor activity against abroad range of rumor cell. It has been approved for therapy of metastatic colorectal cancer which resist to 5-flurouracil by FDA. But like other camptothecins, CPT-11 mainly has two defects, its lactone structure can be easily hydrolyzed to inactive carboxylate form at physiology condition and its has acute toxicity, mainly including neutropenia and diarrhea. To improve the therapeutic effect of CPT-11 and reduce its adverse reacts, we prepared CPT-11 conventional liposomes by ammonium sulfate gradient method, an active loading procedure. In order to provide a sustained-release effect and reduce the frequency of injection administration, we prepared CPT-11 multivesicular liposome, a unique lipid-based depot-delivery system.We developed the RP-HPLC method to determine the drug concentration. The solubilities of CPT-11 in PBS with different pH were determined by HPLC. The HPLC method for the content of CPT-11 in liposomes was established. The entrapment efficiency of CPT-11 liposome was determined by microcolumn centrifuge-HPLC method.The CPT-11 conventional liposomes were prepared by the traditional methods, for example thin-film dispersion, ethanol injection, reverse-phase evaporation and active remote loading -ammonium sulfate gradient method. The results showed that a higher encapsulation efficiency were obtained by ammonium sulfate gradient method. Through optimize the formulation by single and orthogonal design, we prepared liposomal CPT-11 by ammonium sulfate gradient with hydrogenated soybean phosphatidylcholine (HSPC) and soybean phosphatidylcholine(SPC).The entrapment efficiency of CPT-11 liposome was above 80%.The physicochemical properties of CPT-11 conventional liposomes were studied. The CPT-11 liposomes were spheroidal and uniform, the volume diameters of CPT-11 conventional liposomes prepared with HSPC(HSPC-L) and SPC(SPC-L) were 142±7.8nm and 198±10.4 nm, respectively. The pH of them were 7.06 and 7.39, respectively. The entrapment efficiency of them were 88.4±1.30 and 90.5±0.95, respectively. Studies on the release of CPT-11 conventional liposomes in vitro showed that the CPT-11 leakage from HSPC-L was slower than that from SPC-L. The results of stability tests indicated that the CPT-11 conventional liposomes were sensitive to high temperature and high light, but they had a good stability at 4℃. The CPT-11 conventional liposome composed with HSPC was more stable than that of SPC. The biological safety test indicated that the CPT-11 conventional liposomes was non-irritant, the SPC-L was non-hematolytic, but the HSPC-L showed hemolysis after 3 hours. Therefore, the HSPC-L formulation should be optimized further.The CPT-11 multivesicular liposome was prepared to increase the action duration of CPT-11. The entrapment efficiency method of CPT-11 multivesicular liposome was established. Through optimize the formulation of CPT-11 multivesicular liposome by single design, we prepared the CPT-11 multivesicular liposome, which had a good appearance. The entrapment efficiency of CPT-11 multivesicular liposome was 30.13±2.00%. The volume diameter of the CPT-11 multivesicular liposome was 33.11±13.82μm. Compared with the CPT-11 conventional liposomes, the CPT-11 multivesicular liposomes had about 10～100 fold larger particle sizes. The release time of CPT-11 from multivesicular liposomes can last 48 h, indicating that the liposomes have good sustained-release property.