Synthesis, Biological Evaluation and Low-Toxic Formulation Development of Glycosylated Paclitaxel Prodrugs

Abstract

Paclitaxel (PTX) is a famous anti-cancer drug with poor aqueous solubility. In clinical practices, Cremophor EL (polyethoxylated castor oil), a toxic surfactant, is used for dissolution of PTX, which accounts for serious side effects. In the present study, a single glucose-conjugated PTX prodrug (SG-PTX) and a double glucose-conjugated PTX prodrug (DG-PTX) were synthesized with a glycosylated strategy via succinate linkers. Both of the two prodrugs presented significant solubility improvement and drug-like lipophilicities. Compared to DG-PTX, SG-PTX manifested more promising release of the parent drug in serum. A high percentage of PTX released from SG-PTX could be detected after enzymatic hydrolysis of β-glucuronidase. Besides, both of the two prodrugs exhibited effective cytotoxicity against breast cancer cells and ovarian cancer cells, but presented reduced cytotoxicity against normal breast cells. Moreover, SG-PTX manifested impressive solubility in a low toxic formulation (without ethanol) with a different percentage of Cremophor EL. These results indicated that glycosylation is a promising strategy for PTX modification and SG-PTX may be a feasible and potential type of PTX prodrug. In addition, ethanol-free formulation with a low percentage of Cremophor EL might have the potential to develop a safer formulation for further studies of glycosylated PTX prodrugs.

Keywords: anti-cancer; paclitaxel; prodrug; solubility; toxic surfactant.

Figure 1

Chemical structure of paclitaxel.

Scheme 1

Synthesis of single glucose-conjugated paclitaxel (SG-PTX). Reagents and conditions: (a)DMAP, DCM, 16 h, 99%; (b) PTX, DMAP, DCC, THF, 24 h, 57%; (c) H₂, Pd/C, MeOH, AcOH, 24 h, 75%. “DMAP” stands for 4-dimethylaminopyridine, “DCM” for dichloromethane, “PTX” for paclitaxel, “DCC” for dicyclohexylcarbodiimide and “THF” for tetrahydrofuran.

Scheme 2

Synthesis of double glucose-conjugated paclitaxel (DG-PTX). Reagents and conditions: (a) TESCl, imidazole, 4 h, 66%; (b) compound 3, DMAP, DCC, THF, 24 h, 53%; (c) TBAF, THF, 6 h, 66%; (d) compound 3, DMAP, DCC, THF, 24 h, 55%; (e) H₂, Pd/C, AcOH, 24 h, 85%. “TESCl” stands for chlorotriethyl silane and “TBAF” for tetrabutylammonium fluoride.

Figure 2

HPLC chromatography of PTX, SG-PTX, and DG-PTX.

Figure 3

Free PTX released from the prodrugs in serum. (a) PTX released from SG-PTX and DG-PTX in FBS, (b) PTX released from SG-PTX and DG-PTX in RS. All values are expressed as mean ± SD (n = 5). “FBS” stands for fetal bovine serum and “RS” for rabbit serum.

Figure 4

Enzymatic hydrolysis of SG-PTX and DG-PTX. All values are expressed as mean ± SD (n = 5). Compared to SG-PTX group, ** p < 0.01.

Figure 5

Cytotoxicity of PTX and PTX prodrugs against cancer cells and normal cells. (a) breast cancer cells MDA-MB-231, (b) ovarian cancer cells A2780, (c) normal breast cells Hs578Bst. All values are expressed as mean ± SD (n = 6). Compared to PTX group, * p < 0.05, ** p < 0.01; compared to SG-PTX group, ^# p < 0.05 ^## p < 0.01.

Figure 6

Cell apoptosis inducing effect of PTX and PTX prodrugs on MDA-MB-231 cells. (a) Apoptosis of MDA-MB-231, (b) Quantification of apoptosis data. All values are expressed as mean ± SD (n = 3). Compared to control group, * p <0.05, ** p < 0.01; compared to PTX group, ^# p < 0.05; compared to SG-PTX group, ⁺ p < 0.05.

Figure 7

Solubility of SG-PTX at different pH conditions in three common media. All values are expressed as mean ± SD (n = 4).

Figure 8

Solubility of SG-PTX in various formulations with different proportion of surfactants (Cremophor EL, Tween-80, and PEG-400). All values are expressed as mean ± SD (n = 4).