GRAFT COPOLYMER

Abstract

A graft copolymer comprising: a core polymer comprising a crosslinked or non-crosslinked polysaccharide, a plurality of primary graft polymers covalently grafted to the core polymer, a plurality of secondary graft polymers covalently grafted to each primary graft polymer, an injectable dermal aesthetic formulation comprising such a graft copolymer and a method of preparing such a graft copolymer.

Claims

1. A graft copolymer comprising: a core polymer comprising a crosslinked or non-crosslinked polysaccharide, a plurality of primary graft polymers covalently grafted to the core polymer, a plurality of secondary graft polymers covalently grafted to each primary graft polymer.

2. A graft copolymer according to claim 1, wherein the core polymer comprises hyaluronic acid.

3. A graft copolymer according to claim 1, wherein the core polymer has a molecular weight in the range of 50-5000 kDa, preferably in the range of 100-1000 kDa.

4. A graft copolymer according to claim 1, wherein the core polymer is crosslinked.

5. A graft copolymer according to claim 1, wherein the core polymer comprises a polysaccharide gel.

6. A graft copolymer according to claim 1, wherein the primary graft polymers comprise a water soluble polymer.

7. A graft copolymer according to claim 1, wherein the primary graft polymers are dextran.

8. A graft copolymer according to claim 1, wherein the primary graft polymers have a molecular weight in the range of 1-1000 kDa.

9. A graft copolymer according to claim 1, wherein the secondary graft polymers are chondroitin sulphate.

10. A graft copolymer according to claim 1, wherein the secondary graft polymers have a molecular weight in the range of 10-1000 kDa, preferably in the range of 20-500 kDa.

11. A graft copolymer according to claim 1, wherein the primary graft polymers are covalently grafted, directly or through a linking group, to the core polymer by single end-point attachment.

12. A graft copolymer according to claim 1, wherein the secondary graft polymers are covalently grafted, directly or through a linking group, to the primary graft polymers by single end-point attachment.

13. A graft copolymer according to claim 1, wherein the core polymer is a crosslinked hyaluronic acid, the primary graft polymers are dextran and the secondary graft polymers are chondroitin sulphate.

14. An injectable dermal aesthetic formulation comprising a graft copolymer according to claim 1.

15. A method of preparing a graft copolymer comprising the steps: a) providing a core polymer comprising a crosslinked or non-crosslinked polysaccharide comprising a plurality of pendant functional groups, b) providing a primary graft polymer comprising a terminal functional group and a plurality of pendant functional groups, c) providing a secondary graft polymer comprising a terminal functional group, d) mixing the core polymer with the primary graft polymer under conditions allowing covalent binding of the primary graft polymer exclusively to the pendant functional groups of the core polymer via the terminal functional group of the primary graft polymer, e) mixing the grafted product of step d) with the secondary graft polymer under conditions allowing covalent binding of the secondary graft polymer exclusively to the pendant functional groups of the primary graft polymer via the terminal functional group of the secondary graft polymer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1 is a schematic illustration of an embodiment of a graft copolymer according to the present disclosure. The graft copolymer comprises two main substructures, wherein the first substructure consists of a crosslinked polysaccharide network (core polymer) and the second main substructure consists of multiple polysaccharide chains grafted onto a polymer chain, which in turn is grafted onto the first main substructure (secondary and primary grafts respectively)

EXAMPLES

Example 1a. Generation of the Core Polymer

[0084] The core polymer consists of hyaluronic acid (500-1000 kDa) that has been stabilized (crosslinked) with 1,4-butanediol diglycidyl ether, using methods previously described in WO9704012A1, followed by micronization to particle sizes of 0.01-5 mm and treating with sodium borohydride (0-25° C. during 0.5-2 hours) prior purification by precipitation and drying in vacuum, to obtain a crosslinking degree of 0.1-10% as determined by nuclear magnetic resonance spectroscopy using methods previously described (Edsman, K. et al. Dermatol Surg 38:1170-1179, 2012).

Example 1b. Synthesis of a Primary Graft

[0085] A primary graft has been prepared from dextran according to the following procedure.

[0086] 1) Derivatization of the hydroxyl groups via a spacer by reacting dextran (100-500 kDa, 5-50 mg/mL) in an alkaline solution consisting of 0.1-2 M sodium hydroxide at pH 10-14, with 6-bromohexanoic acid (1-100 molar equivalents to dextran hydroxyl groups) at 15-95° C. for 2-24 hours followed by purification by precipitation in ethanol and drying in vacuum. The carboxylated dextran (5-50 mg/mL in aqueous solution) is subsequently converted to the corresponding protected hydrazide derivative (e.g. N-Boc) by activation of the carboxylic group, using 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM) together with tert-butyl carbazate (1-100 molar equivalents of either reagent to introduced carboxylic groups on dextran during 1-120 hours at 0-50° C. and pH 4-10) followed by purification by precipitation in ethanol, drying in vacuum.

[0087] 2) Derivatization of the reducing end of the modified dextran (5-50 mg/mL) by covalent addition of with a linker consisting of hexamethylene diamine (2-100 molar equivalents to reducing ends) in an aqueous solution containing sodium cyanoborohydride (2-100 molar equivalents to reducing ends) during 2-90 hours at 0-90° C. and pH 6-12 followed by precipitation in ethanol and drying in vacuum and characterization by nuclear magnetic resonance spectroscopy.

Example 1c. Conjugation of the Primary Graft to the Core Polymer

[0088] The modified polymer (primary graft) obtained in Example 1 b is covalently linked to the core polymer of Example 1a via an amidation reaction between the primary amines on the linker (located on the reducing end of the primary graft) and a carboxyl groups of the core polymer by treating an aqueous mixture of the core polymer (5-25 mg/mL) and the primary graft (5-25 mg/mL) with DMTMM (1-100 molar equivalents to primary amines) during 1-120 hours at 0-70° C. and pH 4-10. After purification by washing with excess deionized water and filtration, the N-Boc groups of the spacers on the primary grafts are removed by treating with diluted acid (0.2-4 M HCl or trifluoroacetic acid) during 1-120 hours at 0-70° C. and pH 1-6.

Example 1d. Conjugation of the Secondary Graft to the Primary Graft

[0089] In the final stage, secondary grafts are linked by reducing ends to free hydrazide groups on the primary grafts by mixing the dextran-grafted core polymer (5-25 mg/mL) with chondroitin sulfate (10-100 kDa, 1-100 molar equivalents with regard to reducing ends towards hydrazide groups) in an aqueous solvent containing sodium cyanoborohydride (2-100 molar equivalents to reducing ends) during 2-90 hours at 0-50° C. and pH 6-10, followed by precipitation in ethanol and drying in vacuum. The final product obtained is verified by determining the ratio between the polymers that compose the core polymer and the respective grafts using nuclear magnetic resonance (NMR) spectroscopy. The product obtained has a molecular structure as schematically illustrated in FIG. 1.

Example 2. Alternative Synthesis Route

[0090] Following the preparation of a core polymer as described in example 1a, an alternative route to synthesis of a primary graft involves direct derivatization of the reducing end of Dextran (1-100 kDa) as described in step 2) of example 1b, without modification of hydroxyl groups on the Dextran back-bone. The resulting Dextran derivative is further conjugated to the core polymer as described in example 1c.

[0091] The resulting product is subjected to mild oxidation using sodium periodate (≦1 molar equivalents to Dextran monosaccharide units) in aqueous media yielding aldehyde functionalities exclusively on the back-bone of the primary graft. A secondary graft polymer is prepared by derivatizing the reducing end of chondroitin sulfate (5-200 kDa, 40 mg/mL) with an excess of hexamethylene diamine dihydrochloride (15 mg/mL) in the presence of sodium cyanoborohydride (2-100 molar equivalents to reducing ends) in borate buffer at pH 10, followed by purification by dialysis (3.5 kDa molecular weight cutoff) and lyophilization.

[0092] The final product is then prepared by mixing an excess of the secondary graft (20-40 mg/mL) with the Dextran-grafted HA-gel particles (5-25 mg/mL) and sodium cyanoborohydride (1-50 molar equivalents) in aqueous media (room temperature, 16-24 h). Purification is carried out by extensive washing using sodium chloride (0.1-0.5 M) followed by precipitation and simultaneous washing in 70% ethanol and drying in vacuum.