Glycosaminoglycan esters, processes for their preparation and their use in formulations for ophthalmic use

Abstract

Glycosaminoglycan esters, wherein at least part of the hydroxyl groups present on the N-acetylglucosamine residue are esterified with an apocarotenoid, their preparation, and their use in formulations for ophthalmic use are described.

Claims

1. Glycosaminoglycan ester, wherein at least some hydroxyl groups present on N-acetylglucosamine residues of the glycosaminoglycan ester are esterified with an apocarotenoid, wherein said apocarotenoid is selected from crocetin, bixin, or abscisic acid.

2. Ester according to claim 1, wherein said glycosaminoglycan ester comprises glycosaminoglycans selected from: hyaluronic acid, chondroitin sulfate, heparin, or heparan sulfate.

3. Ester according to claim 1, wherein the degree of esterification of apocarotenoid in the ester is from 0.1 to 5% and the molecular weight of said ester is from 350,000 to 2,000,000 Daltons.

4. A process for preparing ester according to claim 1, wherein said method comprises: preparing a hyaluronic acid tetrabutylammonium salt by salifying an ionic exchange resin with an aqueous solution of tetrabutylammonium hydroxide; washing the resin with water and passing a solution of sodium hyaluronate dissolved in water therethrough, thus collecting the eluate containing said salt and freeze-drying it; reacting the thus-obtained salt under heating with a selected apocarotenoid to form a reaction mixture, wherein carboxy functionality of said apocarotenoid is activated by reaction at room temperature with carbonyldiimidazole in dimethylformamide; stirring the reaction mixture; and precipitating the ester product by addition of sodium chloride and ethanol.

5. A method for protecting eyes of an individual from blue light comprising: administering to the eyes of the individual an effective amount of a glycosaminoglycan ester, wherein at least some hydroxyl groups present on N-acetylglucosamine residues of the glycosaminoglycan ester are esterified with an apocarotenoid, wherein said apocarotenoid is selected from crocetin, bixin, or abscisic acid.

6. The method according to claim 5, wherein said glycosaminoglycan ester comprises glycosaminoglycans selected from: hyaluronic acid, chondroitin sulfate, heparin, or heparan sulfate.

7. A formulation for ophthalmic use, comprising at least one glycosaminoglycan ester according to claim 1.

8. A formulation according to claim 7, wherein said formulation comprises a 0.1-1% glycosaminoglycan ester solution in purified water.

9. The formulation of claim 8, further comprising one or more of polyethylene glycols, benzalkonium chloride, or chlorhexidine.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention allows to overcome the aforesaid drawbacks with new glycosaminoglycan esters, wherein at least part of the hydroxyl groups present on the N-acetylglucosamine residue are esterified with an apocarotenoid.

(2) Glycosaminoglycan according to the invention refers to, for example: hyaluronic acid, chondroitin sulfate, heparin, heparan sulfate; hyaluronic acid is preferred.

(3) Among the apocarotenoids usable according to the invention we may mention: retinoic acid, crocetin, bixin, abscisic acid.

(4) Hyaluronic acid (hereinafter, HA) is a natural linear polysaccharide consisting of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine into repeating units.

(5) HA may be used in pure form as a drug for disorders arising from dry eye syndrome, such as keratoconjunctivitis sicca, as described by DeLuise (Annals of Ophthalmology 16: 823-824, 1984), Laflamme (Canadian Journal of Ophthalmology 23: 174-176, 1988) and Sand (Acta Ophthalmologica 67: 181-183, 1989).

(6) It is noted that according to the present invention, in contrast to the occurrence in other eye drops, hyaluronic acid is not used as an excipient but forms a new chemical product by reacting with the apocarotenoid.

(7) The products according to the present invention can be prepared according to processes known in art; in particular following the process described in US 20090239822.

(8) For example, a hyaluronic acid tetrabutylammonium (HA-TBA) salt is first prepared by salifying an ionic exchange resin (such as Amberlite) with an aqueous solution of tetrabutylammonium hydroxide (TBAI) which is percolated on the resin arranged in a chromatographic column.

(9) Once all the solution has passed, the resin is washed with water and then a solution of sodium hyaluronate dissolved in water is passed on the salified resin and finally the eluate is collected and freeze-dried.

(10) Normally, the aqueous tetrabutylammonium hydroxide solution has a concentration of 15-45%, preferably 30-40%, more preferably 40%; while the sodium hyaluronate solution in water normally has a concentration of 1.5-4.5 g/L, preferably 3 g/L.

(11) The salt (HA-TBA) obtained as described above is then reacted with the selected apocarotenoid under heating. The carboxyl function of the apocarotenoid is activated by reaction with carbonyldiimidazole at room temperature; the resulting compound is added slowly to the HA-TBA gel in dimethylformamide.

(12) The mixture is left under stirring between 25 and 35 C. for 12-20 h, then the product is precipitated by the addition of sodium chloride and ethanol.

(13) The degree of substitution of apocarotenoid in the esters according to the invention is of 0.1-5%, where the term degree of substitution indicates the number of moles of apocarotenoid per mole of glycosaminoglycan.

(14) The esters according to the invention normally have a molecular weight of 350,000-2,000,000 Daltons, thus indicating the average molecular weight of the glycosaminoglycan without considering the contribution of the apocarotenoid residue.

(15) The compounds according to the present invention can be formulated in the forms known in pharmacopeia as suitable for ocular administration.

(16) For example, they can be formulated as a 0.1-1% solution of compound in purified water, with the possible addition of polyethylene glycols to increase viscosity and benzalkonium chloride or chlorhexidine as preservatives (alternatively, single-dose packaging may be considered).

(17) The examples below illustrate what is claimed in higher detail.

Example 1

(18) Preparation of the Hyaluronic Acid Tetrabutylammonium (HA-TBA) Salt

(19) ##STR00001##

(20) where n is between 250 and 5000.

(21) An ion-exchange resin (Amberlite in acid form) is used, having a capacity of 1.9 eq/L.

(22) 1 L of resin is loaded into a chromatographic column and is then washed with demineralized water and then an aqueous solution of 40% tetrabutylammonium hydroxide (TBAI) is percolated on the resin from above by means of a peristaltic pump.

(23) Once all the solution has passed, the resin is washed with demineralized water until the eluate has a constant pH of 9.5-10.

(24) One liter of resin thus salified allows to salify about 75-80 g of sodium hyaluronate. The sodium hyaluronate is dissolved in water (concentration of about 3 g/L) and the resulting gel is passed in a column containing the previously prepared resin. The eluate is collected and freeze-dried.

Example 2

(25) Preparation of a Hyaluronic Acid Ester with Crocetin

(26) ##STR00002##

(27) where n is between 250 and 5000

(28) 1.5 g HA-TBA is dissolved in 500 mL DMF in a reactor with thermostat and is placed under stirring at 30 C.

(29) 0.8 g crocetin is dissolved in 400 mL DMF in a reactor protected against moisture and 1 eq N,N-carbonyldiimidazole is added to the mixture.

(30) After 1 h, the solution is slowly added to the HA-TBA gel and the mixture thus obtained is allowed to stir at 30 C. for 16 h.

(31) The reaction is stopped by the addition of 90 mL of saturated solution of sodium chloride. The product is precipitated by the addition of a 96% volume of ethanol; the supernatant is discarded and the filtered residue is washed several times with ethanol in different concentrations and then dried under vacuum.

(32) The resulting product has a degree of substitution of 0.5%.

Example 3

(33) Preparation of a Hyaluronic Acid Ester with Crocetin

(34) 1.5 g HA-TBA is dissolved in 500 mL DMF in a reactor with thermostat and is placed under stirring at 30 C.

(35) 0.4 g crocetin is dissolved in 200 mL DMF in a reactor protected against moisture and 1 eq N,N-carbonyldiimidazole is added to the mixture.

(36) After 1 h, the solution is slowly added to the HA-TBA gel and the mixture thus obtained is allowed to stir at 30 C. for 16 h.

(37) The reaction is stopped by the addition of 70 mL of saturated solution of sodium chloride. The product is precipitated by the addition of a 96% volume of ethanol; the supernatant is discarded and the filtered residue is washed several times with ethanol in different concentrations and then dried under vacuum.

(38) The resulting product has a degree of substitution of 0.25%.

Example 4

(39) Preparation of a Hyaluronic Acid Ester with Bixin

(40) ##STR00003##

(41) where n is between 250 and 5000

(42) 1.5 g HA-TBA is dissolved in 500 mL DMF in a reactor with thermostat and is placed under stirring at 30 C.

(43) 0.95 g bixin is dissolved in 450 mL DMF in a reactor protected against moisture and 1 eq N, N-carbonyldiimidazole is added to the mixture.

(44) After 1 h, the solution is slowly added to the HA-TBA gel and the mixture thus obtained is allowed to stir at 30 C. for 16 h.

(45) The reaction is stopped by the addition of 90 mL of saturated solution of sodium chloride. The product is precipitated by the addition of a 96% volume of ethanol; the supernatant is discarded and the filtered residue is washed several times with ethanol in different concentrations and then dried under vacuum.

(46) The resulting product has a degree of substitution of 0.4%.

Example 5

(47) Preparation of a Hyaluronic Acid Ester with Bixin

(48) 1.5 g HA-TBA is dissolved in 500 mL DMF in a reactor with thermostat and is placed under stirring at 30 C.

(49) 0.5 g bixin is dissolved in 250 mL DMF in a reactor protected against moisture and 1 eq N, N-carbonyldiimidazole is added to the mixture.

(50) After 1 h, the solution is slowly added to the HA-TBA gel and the mixture thus obtained is allowed to stir at 30 C. for 16 h.

(51) The reaction is stopped by the addition of 80 mL of saturated solution of sodium chloride. The product is precipitated by the addition of a 96% volume of ethanol; the supernatant is discarded and the filtered residue is washed several times with ethanol in different concentrations and then dried under vacuum.

(52) The resulting product has a degree of substitution of 0.15%.

Example 6

(53) Preparation of a Hyaluronic Acid Ester with Retinoic Acid

(54) ##STR00004##

(55) where n is between 250 and 5000.

(56) 1.5 g HA-TBA is dissolved in 500 mL DMF in a reactor with thermostat and is placed under stirring at 30 C.

(57) 0.7 g retinoic acid is dissolved in 350 mL DMF in a reactor protected against moisture and 1 eq N,N-carbonyldiimidazole is added to the mixture.

(58) After 1 h, the solution is slowly added to the HA-TBA gel and the mixture thus obtained is allowed to stir at 30 C. for 16 h.

(59) The reaction is stopped by the addition of 90 mL of saturated solution of sodium chloride. The product is precipitated by the addition of a 96% volume of ethanol; the supernatant is discarded and the filtered residue is washed several times with ethanol in different concentrations and then dried under vacuum.

(60) The resulting product has a degree of substitution of 0.2%.