Chemo-enzymatic Synthesis of Ester-Linked Docetaxel-Glycoside Conjugate and its Drug Delivery System Using Hybrid-Bio-Nanocapsules Targeted With Trastuzumab and Cetuximab

Abstract

Synthesis of ester-linked glucoside conjugate of docetaxel, 7-propionyldocetaxel 3''-O-α-D-glucopyranoside, was carried out by chemo-enzymatic procedures. The EE and LE values for hybrid-bio-nanocapsules of 7-propionyldocetaxel 3''-O-α-D-glucopyranoside were much improved rather than those of docetaxel. The hybrid-bio-nanocapsules targeted with trastuzumab and cetuximab, which contained 7-propionyldocetaxel 3''-O-α-D-glucopyranoside, showed high in vivo anti-cancer activity, ie, effective suppression of tumor growth, respectively.

Docetaxel is a taxane diterpenoid, which shows inhibitory action against a variety of tumors. ¹ It has been recognized as one of the most effective and widely used drugs for the treatment of ovarian, breast, and lung cancers. In spite of its effective pharmacological activities, docetaxel has shortcomings such as low solubility in water.

Glycosylation has attracted pharmacological attention, because the glycosylation of bioactive compounds can enhance their water-solubility, physicochemical stability, and biological half-life. ^{2 -7} Particularly, glycosides are useful as prodrugs, which are hydrolyzed in living cells to release drugs. In plant cells, glycosylation reaction has diverse functions such as activation of biosynthetic intermediates and detoxification of toxic compounds generated from environment. Many of secondary metabolites such as saponins and anthocyanins, which are accumulated in the form of glycosides in plants, have specific physiological activities and have been widely used in folk medicines. Plant glycosyltransferases and glycosidases can be used as biocatalysts in organic synthesis to produce glycosides of medicines as prodrugs.

We report herein the synthesis of highly water-soluble ester-linked glycoside conjugate of docetaxel, ie, 7-propionyldocetaxel 3″-O-α-D-glucopyranoside, and its application to drug delivery system using hybrid-bio-nanocapsules targeted with trastuzumab and cetuximab.

Transglycosylation using α-glucosidase ⁸ as a biocatalyst to synthesis carboxyethyl α-D-glucopyranoside (1) was achieved by reacting phenyl α-D-glucopyranoside, and hydroxypropionic acid. 2′-TES ester of docetaxel was then reacted with 2 to provide 3 and subsequent desilylation afforded 7-propionyldocetaxel 3″-O-α-D-glucopyranoside (4) (Figure 1).

Figure 1

Chemo-enzymatic synthesis of 7-propionyldocetaxel 3″-O-α-D-glucopyranoside (4). Reagents and conditions: (i) TESCl, DMF; (ii) α-glucosidase; (iii) BnBr, NaH, DMF; (iv) KOH; (v) EDCI, DMAP, CH₂Cl₂; (vi) H₂, 10% Pd/C, HOAc/H₂O.

The encapsulation efficiency (EE) and loading efficiency (LE) values of docetaxel itself for trastuzumab-targeting hybrid-bio-nanocapsules were 0.3 and 0.05, respectively. On the other hand, those of 7-propionyldocetaxel 3″-O-α-D-glucopyranoside for trastuzumab-targeting hybrid-bio-nanocapsules were 12 and 1.6, respectively. The EE and LE values of docetaxel were much improved by modification with glucosyl ester group.

Next, 7-propionyldocetaxel 3″-O-α-D-glucopyranoside was applied to drug delivery system using hybrid-bio-nanocapsules targeted with trastuzumab and cetuximab. Although the HT-29 tumor weight at day 30 of phosphate buffered saline (PBS)-injected mouse (control) was 1220 mg, that of the hybrid-bio-nanocapsules targeted with trastuzumab encapsulating 7-propionyldocetaxel 3″-O-α-D-glucopyranoside-injected mouse was 320 mg. The gene expression omnibus (GEO) tumor weight at day 30 of control mouse was 1380 mg, whereas that of the hybrid-bio-nanocapsules targeted with cetuximab containing 7-propionyldocetaxel 3″-O-α-D-glucopyranoside-injected mouse was 270 mg.

Thus, ester-linked glycoside conjugate of docetaxel, 7-propionyl-docetaxel 3″-O-α-D-glucopyranoside, was synthesized by chemo-enzymatic method using α-glucosidase as a biocatalyst. The EE and LE values for hybrid-bio-nanocapsules of 7-propionyl-docetaxel 3″-O-α-D-glucopyranoside were much higher than those of docetaxel. Although the IC₅₀ values of docetaxel itself and docetaxel contained in trastuzumab-targeting immunoliposomes against HT-29 cells were 15 and 27 nM, those of 7-propionyldocetaxel 3″-O-α-D-gluco-pyranoside contained in trastuzumab-targeting immunoliposomes and 7-propionyldocetaxel 3″-O-α-D-glucopyranoside encapsulated in trastuzumab-targeting hybrid-bio-nanocapsules were 3.1 and 1.2 nM. Additionally, the hybrid-bio-nanocapsules targeted with trastuzumab and cetuximab containing 7-propionyl-docetaxel 3″-O-α-D-glucopyranoside showed effective suppression of tumor growth in tumor-bearing mouse that had been transplanted with HT-29 and GEO cells, respectively.

Experimental

General

HPLC analyses were carried out with Puresil C18 column (Waters) using MeOH:H₂O (1:3, v/v) as a solvent (detect, UV₂₈₀; flow rate, 1 mL min⁻¹)

Carboxyethyl α-D-glucopyranoside (1)

To a solution of phenyl α-D-glucopyranoside, and hydroxypropionic acid in 0.1 M phosphate buffer (pH 7) was added α-glucosidase (200 U). ⁸ The mixture was stirred for 12 hours at 35°C and then was extracted with n-butanol. The organic layer was concentrated and purified by column chromatography on silica gel to afford carboxyethyl α-D-glucopyranoside (1).

7-propionyldocetaxel 3″-O-α-D-glucopyranoside (4)

To a solution of BnBr/NaH in 5 mL of DMF was added carboxyethyl α-D-glucopyranoside (1). The mixture was stirred at rt for 12 hours, followed by stirring with aqueous KOH (1.5 equiv.). The reaction mixture was quenched with saturated aqueous NaHCO₃ and extracted with ethyl acetate (20 mL ×3). The ethyl acetate layer was concentrated in vacuo and purified by silica gel column chromatography to give carboxyethyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranoside (2). To a solution of docetaxel and imidazole in dry DMF (4 mL) was added chlorotriethylsilane dropwise at rt. The reaction mixture was stirred at rt for 2 hours and diluted with ethyl acetate. The mixture was washed with water and brine, dried over MgSO₄, and concentrated in vacuo. Column chromatography of the residue on silica gel gave the 2′-TES ester of docetaxel. To a mixture of the 2′-TES ester of docetaxel in the presence of EDCI/DMAP in CH₂Cl₂ (10 mL) was added 2 (1.2 equiv.). The mixture was stirred at rt for 12 hours. The reaction mixture was extracted with ethyl acetate. The organic layer was concentrated in vacuo and purified by column chromatography on silica gel to give 3. To a solution of Pd black in HOAc-H₂O (9:1, v/v) was added 3. The suspension was stirred at room temperature for 24 hours. Extraction of the reaction mixture with n-butanol followed by column chromatography on silica gel yielded 7-propionyldocetaxel 3″-O-α-D-glucopyranoside (4).

In Vivo Anti-Tumor Effects of Hybrid-Bio-Nanocapsules Containing 7-Propionyldocetaxel 3″-O-α-D-Glucopyranoside

Docetaxel prodrug, 7-propionyldocetaxel 3″-O-α-D-glucopyranoside, was encapsulated in the hybrid-bio-nanocapsules by electroporation to give hybrid-bio-nanocapsules containing docetaxel prodrug. To prepare tumor-bearing mice, HT-29 cells or GEO cells were subcutaneously injected into 5-week-old female BALB/c-nu/nu mice (Charles River, Kanagawa, Japan). Mice were fed with sterilized food and water. When HT-29 tumor volume reached 50 to 100 mm³, mice were randomly divided into several groups, with 3 or 4 mice in each group. Hybrid-bio-nanocapsules targeted with trastuzumab encapsulating docetaxel prodrug, at a dose of 150 mg/kg, or PBS buffer (control) was injected via the tail vein twice in 3 hours. Tumor volumes were measured at 3-day intervals. After 30 days, the tumor weight of PBS-injected mouse (control) and that of the hybrid-bio-nanocapsules targeted with trastuzumab containing 7-propionyl-docetaxel 3″-O-α-D-glucopyranoside-injected mouse were weighed. When the GEO tumor volume reached 50 to 100 mm³, 7-propionyl-docetaxel 3″-O-α-D-glucopyranoside, which is encapsulated in hybrid-bio-nanocapsules targeted with cetuximab, at a dose of 150 mg/kg was injected. The tumor weight at day 30 of PBS-injected mouse (control) and that of the hybrid-bio-nanocapsules targeted with cetuximab containing 7-propionyldocetaxel 3″-O-α-D-glucopyranoside-injected mouse were weighed.