Abstract 4461: Anti-cancer activity of immunoliposomes encapsulated effective amount of glycosylated paclitaxel with novel loading strategy
Authors: Shigehiro Tsukasa , Kasai Tomonari , Mizutani Akifumi , Murakami Hiroshi , Mikuni Katsuhiko , Mandai Tadakatsu , Hamada Hiroki , Seno Masaharu 
Published: May 2015

Targeted liposomal drug delivery is considered to enhance the therapeutic effects of anti-cancer agents because high amount of drug could be administered with least side effects. However, significant hydrophobic compounds such as paclitaxel (PTX) are not suitable for efficient encapsulating. We conferred water-solubility of PTX by coupling glucose at 7-OH to produce glycosylated PTX (gPTX). gPTX appeared to have a 10-fold higher solubility than PTX in 40% ethylene glycol (EG) and in Cremophor® EL/Ethanol/PBS (12:12:76 volume%) (CEP). The maximal solubility of gPTX in these solvents was 1.1 and 20.0 mg/ml, respectively. An anti-cancer activity of PTX is due to inhibit tubulin depolymerization. We confirmed tubulin polymerization activity of gPTX in vitro. The activity of gPTX to stabilize tubulin polymerization was lower than that of PTX by only 10%. We successfully prepared gPTX-encapsulated liposome (gPTX-L) with the remote loading method by solubility gradient between 40% EG and CEP. The encapsulation efficiency and loading efficiency were 70.8 % and 8.0 mol% (drug mol / initial lipid mol). Then, trastuzumab was conjugated on the surface of gPTX-L to prepare immunoliposome (gPTX-IL) for targeting HER2 positive cancer cells. The particle size of gPTX-IL was around 150 nm and Z potential was -8.1 mV. We evaluated cellular cytotoxicity of PTX, gPTX, gPTX-L and gPTX-IL in HER2 positive cancer cell lines, SK-BR-3 and HT-29, and HER2 negative cancer cell line, MDA-MB-231, by MTT assay. The IC50 values after 72 hours drug-exposure showed gPTX-L exhibited lower IC50 values than free-gPTX in all cell line, which indicated gPTX-L internalized with liposomal formulation contained high concentration of gPTX. Furthermore, gPTX-IL exhibited lower IC50 values than gPTX-L in HER2 positive cancer cell lines but no significant statically difference in this assay condition. We then evaluated the time required to inhibit the growth at the half maximal level (IT50) at the minimum concentration to show maximal cytotoxicity after 72 hours drug-exposure. gPTX-IL showed the shortest IT50 values of 6.4 and 4.7 h in SK-BR-3 and HT-29 cells, respectively. On the other hand, gPTX-IL showed the same IT50 value of gPTX-L in MDA-MB-231 cells. We then evaluated the anti-cancer efficacy of the formula in vivo using tumor bearing ICR-nu/nu mice. The mice were intravenously injected at the dose of 150 mg/kg of gPTX in a day, which was the lethal dose of free-gPTX. While gPTX-L and free-trastuzumab did not inhibit tumor growth, gPTX-IL significantly decreased tumor volume with low side effects. We further evaluated dose dependency of gPTX-IL using tumor bearing BALB/c-nu/nu mice. The doses of over 100 mg/kg decreased tumor volume and the dose of 150 mg/kg showed the most efficient anti-cancer activity. Collectively, the gPTX-IL with novel encapsulation strategy should be a candidate of highly efficient targeting drug delivery system.

Citation Format: Tsukasa Shigehiro, Tomonari Kasai, Akifumi Mizutani, Hiroshi Murakami, Katsuhiko Mikuni, Tadakatsu Mandai, Hiroki Hamada, Masaharu Seno. Anti-cancer activity of immunoliposomes encapsulated effective amount of glycosylated paclitaxel with novel loading strategy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4461. doi:10.1158/1538-7445.AM2014-4461