Tadakatsu Mandai

Efficient Drug Delivery of Paclitaxel Glycoside: A Novel Solubility Gradient Encapsulation into Liposomes Coupled with Immunoliposomes Preparation

Although the encapsulation of paclitaxel into liposomes has been extensively studied, its significant hydrophobic and uncharged character has generated substantial difficulties concerning its efficient encapsulation into the inner water core of liposomes. We found that a more hydrophilic paclitaxel molecule, 7-glucosyloxyacetylpaclitaxel, retained tubulin polymerization stabilization activity. The hydrophilic nature of 7-glucosyloxyacetylpaclitaxel allowed its efficient encapsulation into the inner water core of liposomes, which was successfully accomplished using a remote loading method with a solubility gradient between 40% ethylene glycol and Cremophor EL/ethanol in PBS. Trastuzumab was then conjugated onto the surface of liposomes as immunoliposomes to selectively target human epidermal growth factor receptor-2 (HER2)-overexpressing cancer cells. In vitro cytotoxicity assays revealed that the immunoliposomes enhanced the toxicity of 7-glucosyloxyacetylpaclitaxel in HER2-overexpressing cancer cells and showed more rapid suppression of cell growth. The immunoliposomes strongly inhibited the tumor growth of HT-29 cells xenografted in nude mice. Notably, mice survived when treated with the immunoliposomes formulation, even when administered at a lethal dose of 7-glucosyloxyacetylpaclitaxel in vivo. This data successfully demonstrates immunoliposomes as a promising candidate for the efficient delivery of paclitaxel glycoside.

A semisynthesis of paclitaxel via a 10-deacetylbaccatin III derivative bearing a β-keto ester appendage

Abstract A semisynthesis of paclitaxel has successfully been accomplished starting from a newly developed baccatin III derivative bearing a β-keto ester appendage on C-13.

Synthesis of new baccatin III derivatives through transesterification of β-keto esters with a protected 10-deacetylbaccatin III

Abstract Synthesis of a new array of baccatin III derivatives bearing a β-keto ester appendage on C-13 is successfully achieved through transesterification of a wide range of β-keto esters with a protected baccatin III.

Evaluation of glycosylated docetaxel-encapsulated liposomes prepared by remote loading under solubility gradient

Abstract Docetaxel comprises one of the most effective anti-cancer drugs despite of serious side effects. Liposomes encapsulation is practically feasible to deliver the drug. However, due to the significant hydrophobicity, docetaxel will be integrated into the lipid bilayer resulting in poor encapsulation capacity. Here, we evaluated a remote loading strategy using a solubility gradient made between the two solvents for 7-glucosyloxyacetyldocetaxel, which has enhanced water solubility of docetaxel with a coupled glucose moiety. Therefore, 7-glucosyloxyacetyldocetaxel was more effectively encapsulated into liposomes with 71.0% of encapsulation efficiency than docetaxel. While 7-glucosyloxyacetyldocetaxel exhibited 90.9% of tubulin stabilisation activity of docetaxel, 7-glucosyloxyacetyldocetaxel encapsulated in liposomes significantly inhibited the growth of tumour in vivo with side effects less than unencapsulated drug. Collectively, the encapsulation of 7-glucosyloxyacetyldocetaxel into liposomes by remote loading under the solubility gradient is considered to be a promising application to prepare practical drug delivery system.

Practical Liposomal Formulation for Taxanes with Polyethoxylated Castor Oil and Ethanol with Complete Encapsulation Efficiency and High Loading Efficiency

Taxanes including paclitaxel and docetaxel are effective anticancer agents preferably sufficient for liposomal drug delivery. However, the encapsulation of these drugs with effective amounts into conventional liposomes is difficult due to their high hydrophobicity. Therefore, an effective encapsulation strategy for liposomal taxanes has been eagerly anticipated. In this study, the mixture of polyethoxylated castor oil (Cremophor EL) and ethanol containing phosphate buffered saline termed as CEP was employed as a solvent of the inner hydrophilic core of liposomes where taxanes should be incorporated. Docetaxel-, paclitaxel-, or 7-oxacetylglycosylated paclitaxel-encapsulating liposomes were successfully prepared with almost 100% of encapsulation efficiency and 29.9, 15.4, or 29.1 mol% of loading efficiency, respectively. We then applied the docetaxel-encapsulating liposomes for targeted drug delivery. Docetaxel-encapsulating liposomes were successfully developed HER2-targeted drug delivery by coupling HER2-specific binding peptide on liposome surface. The HER2-targeting liposomes exhibited HER2-specific internalization and enhanced anticancer activity in vitro. Therefore, we propose the sophisticated preparation of liposomal taxanes using CEP as a promising formulation for effective cancer therapies.

Chemo-Enzymatic Synthesis of Glycolyl-Ester-Linked Taxol-Monosaccharide Conjugate and Its Drug Delivery System Using Hepatitis B Virus Envelope L Bio-Nanocapsules

Chemo-enzymatic synthesis of glycolyl-ester-linked taxol-glucose conjugate, ie, 7-glycolyltaxol 2′-O-α-D-glucoside, was achieved by using α-glucosidase as a biocatalyst. The water-solubility of 7-glycolyltaxol 2′-O-α-D-glucoside (21 μM) was 53 fold higher than that of taxol. The hepatitis B virus envelope L particles (bio-nanocapsules) are effective for delivering 7-glycolyltaxol 2′-O-α-D-glucoside to human hepatocellular carcinoma NuE cells.

Abstract 4461: Anti-cancer activity of immunoliposomes encapsulated effective amount of glycosylated paclitaxel with novel loading strategy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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

Abstract LB-8: A novel remote loading method with solubility gradient to encapsulate effevtive amount of taxanes into liposomes.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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, water-insoluble hydrophobic and non-weak base chemical 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 7-alpha-glucosyloxyacetylpaxcitacel (gPTX). gPTX showed enhanced water-solubility with the 40 % ethylene glycol (EG) in PBS and with the CEP buffer (Cremophor EL and Ethanol in PBS). The maximal solubility of gPTX in these solvents was 1 and 20 mg/ml, respectively. We prepared gPTX encapsulated liposome (gPTX-L) with remote loading method by solubility gradient between 40 % EG and CEP buffer. gPTX efficiently encapsulated into liposomes. The encapsulation efficiency and loading efficiency were 72.9 % and 8.5×10−2 drug mol / initial lipid mol. Then, trastuzumab was conjugated on the surface of gPTX-L to prepare immunoliposome (gPTX-IL) for targeting HER2 overexpressing cancer cells. The particle size of gPTX-IL was under 150 nm and Z potential was -3.07 mV. We evaluated IC50 values by MTT assay after 72 hours exposure. The IC50 values of PTX, gPTX, gPTX-L and gPTX-IL for SK-BR-3 cells were 5.5, 18.9, 6.6 and 5.3 nM, respectively. gPTX-IL exhibited the highest toxicity in these paclitaxel formulations. Similarly, the IC50 values for HT-29 cells were 1.3, 11.0, 7.6 and 6.8 nM, respectively. We then evaluated the time required for each formula to inhibit the growth at the half maximal level (IT50) at the minimum concentration to show maximal cytotoxicity (IC100) after 72 hours exposure. gPTX-IL showed the shortest IT50 values of 1.6 and 1.1 h in SK-BR-3 and HT-29 cells respectively. We then evaluated in vivo distribution of liposome and immunoliposome encapsulating HSA conjugated Cy5.5 in ICR-nu/nu mice bearing HT-29 cell tumors. Both liposome and immunoliposome accumulated at the tumor site, but immunoliposome exhibited less accumulation at liver than liposome. Based on these results, we then evaluated the anti-cancer efficacy of the formula in vivo using tumor bearing ICR-nu/nu mice. While gPTX-L and free trastuzumab did not inhibit tumor growth, gPTX-IL decreased tumor volume and inhibited tumor growth with low side effects at the dose of 150 mg/kg gPTX, which was the lethal dose of naked gPTX exhibiting 25% survival rate of mice one week after the injection of gPTX. Collectively, the gPTX-encapsulated immunoliposome with novel encapsulation strategy should be an excellent candidate of highly efficient drug delivery system of cancer treatment targeting HER2. Citation Format: Tsukasa Shigehiro, Tomonari Kasai, Akifumi Mizutani, Hiroshi Murakami, Katsuhiko Mikuni, Tadakatsu Mandai, Hiroki Hamada, Masaharu Seno. A novel remote loading method with solubility gradient to encapsulate effevtive amount of taxanes into liposomes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-8. doi:10.1158/1538-7445.AM2013-LB-8