Hydrogen sulfide releasing polymer compounds

Abstract

The invention provides a hydrogen sulfide releasing polymer compound having a polysaccharide backbone, wherein the compound has at least two substructures, and wherein said substructures are capable of releasing hydrogen sulfide by thiol activation as well as uses thereof. Additionally, a method of treatment and prevention of a skin condition, an ocular disease or osteoarthritis is provided.

Claims

1. A hydrogen sulfide releasing polymer compound comprising a polysaccharide backbone and at least two substructures, wherein said polysaccharide backbone is selected from the group consisting of hyaluronic acid, cellulose derivatives, salts of alginic acid, chondroitin sulfate, dermatan sulfate, chitosan, chitosan derivatives, pectin, and salts of pectin, wherein said substructures are capable of releasing hydrogen sulfide by thiol activation, and wherein each of the at least two substructures capable of releasing hydrogen sulfide by thiol activation: is covalently bound to the polysaccharide backbone, and is independently selected from the group consisting of acyl-protected perthiols, N-(acylthio)-benzamides, dithioperoxyanhydrides, arylthioamides, and diallyl disulfide or diallyl trisulfide structures derived from garlic.

2. The hydrogen sulfide releasing polymer compound according to claim 1, wherein the at least two substructures are acyl-protected perthiol substructures according to formula I ##STR00005## wherein R.sub.1 is an alkyl, an alkenyl, an alkinyl, an alkylaryl, an aralkyl or an aryl group, and R.sup.4 is an atom of a repetitive unit of the polysaccharide backbone to which the acyl-protected perthiol substructure is covalently bound.

3. The hydrogen sulfide releasing polymer compound according to claim 1, wherein the polymer compound comprises at least two substructures according to formula II ##STR00006## wherein R.sub.1 is an alkyl, an alkenyl, an alkinyl, an alkylaryl, an aralkyl or an aryl, wherein R.sub.4 is an atom of a repetitive unit of the polysaccharide backbone to which the substructure is covalently bound, and wherein L is a linker.

4. The hydrogen sulfide releasing polymer compound according to claim 3, wherein the at least two substructures are selected from the group consisting of formula III ##STR00007##

5. The hydrogen sulfide releasing polymer compound according to claim 1, wherein the polysaccharide backbone is chitosan or hyaluronic acid and the polysaccharide backbone has a mean molecular weight in the range of about 20 kDa to about 500 kDa if the polysaccharide backbone is chitosan or in the range of about 90 kDa to about 3 MDa if the polysaccharide backbone is hyaluronic acid.

6. The hydrogen sulfide releasing polymer compound according to claim 1, wherein the polysaccharide backbone is selected from hyaluronic acid and chitosan.

7. The hydrogen sulfide releasing polymer compound according to claim 2, wherein R.sub.1 is selected from phenyl and methyl.

8. The hydrogen sulfide releasing polymer compound according to claim 3, wherein R.sub.1 is selected from phenyl and methyl.

9. The hydrogen sulfide releasing polymer compound according to claim 1, wherein said substructures release essentially no hydrogen sulfide by hydrolysis.

10. The hydrogen sulfide releasing polymer compound according to claim 1, wherein each of the at least two substructures capable of releasing hydrogen sulfide by thiol activation is capable of releasing hydrogen sulfide upon contact with a thiol group bearing reaction partner, and wherein the thiol group containing reaction partner is selected from the group consisting of cysteine, cysteamine, N-acylcysteine, homocysteine, and glutathione.

11. The hydrogen sulfide releasing polymer compound according to claim 1, wherein said at least two substructures are the same.

12. The hydrogen sulfide releasing polymer compound according to claim 1, wherein the polysaccharide backbone is a sodium or calcium salt of alginic acid.

Description

(1) The invention will now be described in more detail by figures and the non-limiting examples.

(2) FIG. 1 shows exemplary schemes for hydrogen sulfide releasing polymer compounds according to the invention, wherein FIGS. 1A and 1B both show a hyaluronic acid with acyl-protected perthiol substructures and FIG. 1C shows a chitosan with acyl-protected perthiol substructures.

(3) FIG. 2 schematically shows the synthesis scheme of the polymer of FIG. 1C.

(4) FIG. 3 schematically shows the synthesis scheme of the polymer of FIG. 1A.

(5) FIG. 4 schematically shows an alternative synthesis scheme of the polymer of FIG. 1A.

(6) FIG. 5 schematically shows the synthesis scheme of the polymer of FIG. 1B.

(7) FIG. 6 shows the results from an H.sub.2S release assay, which was performed with an H.sub.2S releasing chitosan derivative.

(8) FIG. 7 shows the results from an H.sub.2S release assay, which was performed with an H.sub.2S releasing hyaluronic acid derivative.

(9) FIG. 8 shows results from cell compatibility assays, wherein the investigated cell lines were primary human dermal fibroblasts (dFb) (FIG. 8A), immortalized human microvascular endothelial cells (HMEC-1) (FIG. 8B), and keratinocytes from the cell line HaCat (FIG. 8C). “% GAG” indicates the investigated concentration of the investigated compounds. Significance is calculated using an unpaired Student's T-test, wherein results are indicated by stars according to * for p<0.05; ** for p<0.01; ***for p<0.005, and **** for p<0.001.

(10) FIG. 9 schematically shows the structure of a multi-layer wound dressing.

(11) FIG. 10 shows the preparation of a N-(benzoylthio)benzamide derivative and a H.sub.2S releasing hyaluronic acid derivative with covalently attached N-(benzoylthio)benzamide substructures. FIG. 10A shows a reaction scheme for the synthesis of a derivative of H.sub.2S Donor 5a modified with an amino group. FIG. 10B shows a H.sub.2S releasing hyaluronic acid derivative with covalently attached N-(benzoylthio)benzamide substructures.

(12) In the schemes, the polymer compounds are visualized by a section of the polymer and the brackets indicate that these sections are multiplied, i.e. multiplied n-times within the polymer. It will be appreciated that the schemes are of symbolic nature and the relation of unmodified and modified repetitive units does not represent an actual degree of modification.

EXAMPLES

Example 1: Generation of a H.SUB.2.S-Releasing Chitosan Derivative

(13) Chitosan-N-acetylcysteine hydrochloride (Chitosan-NAC), the starting material for a H.sub.2S releasing polymer according to FIG. 1C was synthesized according to a method described previously (WO 2015/169728 “Aqueous ophthalmic solution and method for treating dry eye syndrome”). Other routes for synthesis were published as well (Schmitz, Grabovac et al., 2008, Synthesis and characterization of a chitosan-N-acetyl cysteine conjugate, Int J Pharm (347): 79-85). The reaction scheme is depicted in FIG. 2.

(14) 2 g Chitosan-NAC (MW 150 kDa, degree of modification 185 g.Math.mol thiol moieties/g polymer) were dissolved in 200 mL water (WFI, water for injection). About 90 mg (approx. 400 μmol) 2,2′-dithiodipyridine were dissolved in 5 mL ethanol and were admixed to the polymer solution. The solution was stirred for one hour at ambient temperature. Then, 76 mg (1 mmol) of thioacetic acid were dissolved in 0.5 mL 1 M NaOH and were added to the reaction solution. The solution was stirred for further 35 min. Thereafter, the solution was neutralized with NaOH and the polymer was precipitated by addition of 2-propanol. The polymer was harvested by suction filtration. For further purification, the polymer was suspended in WFI and strongly acidified (pH˜1) with 5 M HCl. The polymer was again precipitated by addition of 2-propanol and harvested by suction filtration. The polymer was washed several times with 2-propanol and was finally dried under reduced pressure. 1.6 g polymer was obtained as a white, water-soluble powder with an estimated molecular weight of 150 kDa (based on the starting material) and a degree of modification with H.sub.2S releasing perthiol structures of 178 μM per g polymer. This corresponds to a conversion rate of 96% of the thiol moieties of the polymer used as starting material.

Example 2: Generation of an HS-Releasing Hyaluronic Acid Derivative

(15) An S-mercaptonicotinic acid-protected thiolated hyaluronic acid derivative was used as intermediate for the introduction of acyl-protected perthiol groups to the polymer. The schematic reaction scheme of this synthesis step is shown in FIG. 3. The protection of thiol-groups of thiolated polymers via mercaptonicotinic acid (step b) and its derivatives is described in EP 2 482 852 “Mucoadhesive polymers having vitamin B partial structures”.

Step a) Synthesis of 2-(2-aminoethyldisulfanyl)pyridine-3-carboxylic Acid

(16) 2-Mercaptonicotinic acid was dispersed in H.sub.2O, and dissolved completely after addition of 5 M NaOH until pH was 9-10. After pH adjustment to about 8.0 with HCl, a 30% solution of H.sub.2O.sub.2 was added drop wise, while keeping the pH constantly in a range of 7.5-8.5. After acidification to pH=1.0-1.5 with 5 M HCl, the dimer compound 2-(3-carboxypyridin-1-ium-2-yl)disulfanylpyridin-1-ium-3-carboxylic acid precipitated as a white solid. The product was filtered and washed with 1 M HCl and 2-propanol. Finally, it was dried under reduced pressure at 50° C.

(17) The dimer (2-[3-carboxypyridin-1-ium-2-yl]disulfanylpyridin-1-ium-3-carboxylic acid) was suspended in 96% ethanol and dissolved upon addition of triethylamine. A solution of cysteamine-HCl (1.1 equiv.) in 96% ethanol was neutralized with an equimolar amount of triethylamine. The cysteamine solution was added to the dimer solution and the mixture was allowed to react for 45 hours. The precipitate was filtered off, washed with ethanol and dried under reduced pressure at 50° C. (mp [uncorrected]=184-186° C.).

Step b) Synthesis of S-Mercaptonicotinic Acid-Protected Thiolated Hyaluronic Acid

(18) 1) 3.2 g of free hyaluronic acid (mean molecular weight (MMW) approximately 0.6 MDa) were suspended in 300 mL DMSO. 2) 900 μL of triethylamine were added. 3) The suspension was stirred at ambient temperature until complete dissolution of the hyaluronic acid-triethylamine salt. 4) The “pH value” of the solution was adjusted to 2.55 to 2.75 by addition of HCl in a suitable organic solvent. 5) 470 mg of N,N′-carbonyldiimidazole in 10 mL DMSO were added, and the solution was stirred for 30 min at ambient temperature. 6) 600 mg of 2-(2-aminoethyldisulfanyl)pyridine-3-carboxylic acid (s. step a) were added, and the solution was stirred for 20 hrs. 7) The reaction solution was acidified with 5 M HCl. 8) The S-mercaptonicotinic acid-protected thiolated hyaluronic acid was precipitated by addition of 2-propanol and harvested by centrifugation. 9) The polymer was dissolved in 300 mL H.sub.2O and re-precipitated with 2-propanol. 10) The polymer was harvested by centrifugation and dried under reduced pressure.

(19) 2.6 g of a white, water soluble powder was obtained with a degree of modification of about 250 g.Math.mol mercaptonicotinic acid bearing sidechains/g polymer.

Step c) Synthesis of H.SUB.2.S-Releasing Hyaluronic Acid Derivative Starting from the Product of Step b)

(20) 1) The polymer obtained in step 10 during the synthesis of S-mercaptonicotinic acid-protected thiolated hyaluronic acid as described above was dissolved in 300 mL H.sub.2O and the pH was adjusted to 7.3. 2) 228 mg (3 mmol) thioacetic acid was mixed with 1.50 mL 1 M NaOH and added to the polymer solution. 3) The solution was stirred for 15 min. 4) The solution was strongly acidified by addition of HCl. 5) The final polymer was precipitated by addition of 500 mL 2-propanol and harvested by centrifugation. 6) The solid polymer was suspended in 2-propanol, filtrated by suction, washed three times with 50 mL 2-propanol and twice with 50 mL ethanol, until the filtrate was colourless. 7) The polymer was dried under reduced pressure at 35-40° C.

(21) 2.4 g of a white, water soluble powder was obtained with an estimated degree of modification of about 250 μmol S-acetyl moieties and with an estimated molecular weight of 0.6 MDa (based on the starting material). The absence of aromatic signals in .sup.1H-NMR spectra showed complete conversion of S-mercaptonicotinic acid-protected thiolated hyaluronic acid to the desired product.

(22) A similar synthesis was also applicable for generation of another H.sub.2S-releasing hyaluronic acid derivative with thiobenzoic acid in step c) 2) as schematically shown in FIG. 5.

Example 3: Generation of an H.SUB.2.S-Releasing Hyaluronic Acid Derivative-Alternative Synthesis Route

(23) Alternatively, H.sub.2S releasing hyaluronic acid derivatives were synthesized according to the reaction scheme depicted in FIG. 4 using a thiolated hyaluronic acid as starting material.

(24) 1 g of hyaluronic acid-cysteamine conjugate (degree of modification ˜150 μmol thiol moieties per g polymer, MW 270 kDa) was dissolved in 100 mL WFI. About 45 mg (approx. 195 μmol) 2,2′-dithiodipyridine were dissolved in 1.5 mL ethanol and were admixed to the polymer solution. The solution was stirred for 2 hours at ambient temperature.

(25) About 38 mg (approx. 490 μmol) thioacetic acid were dissolved in 245 μL 1 M NaOH and the resulting solution was added to the polymer solution. After two hours stirring at ambient temperature, the polymer solution was neutralized by the addition of NaOH. Upon addition of 2-propanol, the polymer precipitated. The product was harvested by suction filtration and purified by multiple washings with ethanol. Finally, the polymer was dried under reduced pressure.

(26) 850 mg polymer was obtained as a white, fibrous product with an estimated molecular weight of 270 kDa (based on the starting material) and a degree of modification with H.sub.2S releasing perthiol structures of 147 μM per g polymer. This corresponds to a conversion rate of 98% of the thiol moieties of the polymer used as starting material. The polymer was insoluble in water, but dissolved, after being suspended in WFI in presence of 10-fold excess L-cysteine, to a clear viscous solution while H.sub.2S was released. Emerging H.sub.2S was detected by embrowning Pb-acetate paper.

Example 4: Release of Hydrogen Sulfide

(27) The investigated polymer compounds according to the invention (as shown in FIGS. 1A and 1C) were analyzed for their hydrogen sulfide release properties in presence of a thiol containing component with an amperometric detection device (H.sub.2S Micro-Sensor with H.sub.2S permeable membrane; AMT Analysenmesstechnik GmbH). The H.sub.2S release was triggered by the addition of L-cysteine.

(28) FIG. 6 depicts the results of a H.sub.2S release assay, wherein 50 mL of a 0.1% (m/m) solution of the H.sub.2S releasing chitosan derivative, which was synthesized as described in example 1, was prepared with 50 mM acetate buffer (pH 5.5). To the resulting solution, which had a pH of 5.3, 1.5 mL of a 100 mM solution of L-cysteine were added. The resulting release of H.sub.2S was monitored at room temperature.

(29) FIG. 7 depicts the results of a H.sub.2S release assay, wherein 0.1% (m/m) of a H.sub.2S releasing hyaluronic acid derivative of example 2 (FIG. 1A) were incubated in DMEM (Dulbecco's modified eagle medium) in the presence of 3 mM L-cysteine (at room temperature and pH 7.6-7.8). After a short lag time, the H.sub.2S release continued for more than 15 hours at the investigated conditions.

Example 5: Cell Compatibility of H.SUB.2.S-Releasing Polymer

(30) In order to study cell compatibility, especially the effect of proliferation, the H.sub.2S releasing hyaluronic acid derivative according to FIG. 1A (s. above,) was investigated in comparison with unmodified HA (sodium salt of hyaluronic acid, “NA HA”) and an S-mercaptonicotinic acid-protected thiolated hyaluronic acid (“Thiomer CAPPY”). As further control, the well established commercially available H.sub.2S donor N-(benzoylthio)benzamide (Donor 5a is NSHD1, a small molecule with a thiol activation mechanism) was included in the experiments.

(31) Three types of skin cells were used as models: primary human dermal fibroblasts (dFb), the immortalized line of human microvascular endothelial cells (HMEC-1) and the keratinocyte cell line HaCat. The cells were cultured in their appropriate medium. Synthetic medium Fibrolife with supplements and gentamycin was used for dFb, EBM+supplements was used for HMEC-1 and DMEM with 10% FCS and 1% CellShield was used for HaCat cells. Furthermore, 3 mM cysteine was added to all conditions investigated. The addition of 3 mM cysteine to all media did not affect cell growth as tested in pilot experiments. Cells were seeded and grown at least overnight before medium was changed and the HA components were added according to the agreed protocol. Two concentrations (0.05% and 0.1%) of HA samples were applied. The exposition was performed for 48 h and 96 h. During 48 h of experiment, one additional medium exchange was performed after 24 h of initial exposition. During 96 h of experiment, two additional medium exchanges were performed after 24 h and 72 h of initial exposition. Cell proliferation was measured indicated by using a WST-1-based colorimetric detection reagent according to manufacturers' protocol with fresh medium without addition of any HA-derivative. All experiments have been repeated 3-6 times each with 5 parallel samples of each condition. After each medium exchange the successful generation of H.sub.2S from was verified with Pb-acetate paper.

(32) The study demonstrated that H.sub.2S releasing hyaluronic acid derivative is not toxic to the cell types analyzed here in the concentrations and culture settings used. The experimental outcome seems to be valid since known effects of induced fibroblast and endothelial cell proliferation by non-modified HA (0.1% conc.) could be observed. Endothelial cells might be most sensitive to H.sub.2S as found for “H.sub.2S CAPPY” and the control Donor 5a.

Example 6: Wound Healing Preparation—Sponge

(33) An aqueous hydrogel solution comprising 1.5% (m/m) H.sub.2S releasing chitosan derivative with a degree of modification of about 185 μmol S-acetyl moieties according to the invention is prepared. The solution is cast into 12 well cell culture plates—in an amount of 0.9 g per well) and freeze dried. The resulting lyophilisate is a sponge with a round shape and has a diameter of about 2 cm. The sponges are sealed individually in pouches. The pouches and secondary packaging material are then sterilized via gamma irradiation.

(34) For the treatment of wounds the sterile sponges are applied on the skin wound. A few drops of a sterile buffered solution of N-acetylcysteine in a concentration of 5 mg/mL may be applied on the sponge to immediately start the release of H.sub.2S. The wound area is then covered with a secondary wound dressing with low gas permeability, such as a film consisting of polyethylene, polypropylene or polytetrafluoroethylene or a fabric or fleece that has reduced gas permeability, for example, a polypropylene fleece with an almost gas-tight layer such as polyurethane.

Example 7: Skin Treatment—Topical Hydrogel

(35) An aqueous hydrogel solution comprising 1% (m/m) H.sub.2S releasing hyaluronic acid derivative with a degree of modification of about 250 g.Math.mol S-acetyl moieties according to the invention is prepared in a phosphate buffer with a physiologically acceptable pH value. The solution is then filled into single use containers (e.g. single dose polypropylene containers or glass syringes with a filling volume of about 3 mL) and sterilized with moist heat. A second composition comprising N-acetylcysteine in a physiologic buffer solution in a concentration of 10 mg/mL is also provided in a single use container. The resulting hydrogel with a low viscosity is applied on intact skin or inflamed skin areas, immediately followed by the application of approximately the same amount of the composition comprising N-acetylcysteine to the same skin area.

(36) The treatment with the hydrogel may be preferred in case of cosmetic skin conditions or skin disease such as for treatment of scars and closed wounds or inflammatory diseases of the skin.

Example 8: Wound Healing Preparation—Hydrogel

(37) An aqueous hydrogel comprising 2% (m/m) H.sub.2S releasing hyaluronic acid derivative with a degree of modification of about 250 g.Math.mol S-acetyl moieties according to the invention is prepared in a phosphate buffer with a physiologically acceptable pH. The gel is then filled into single use containers (such as syringes) and sterilized with moist heat.

(38) The resulting hydrogel is applied on the skin wound. A small volume of a sterile buffered solution of N-acetylcysteine in a concentration of 100 mg/mL may be sprayed on the gel to immediately start the release of H.sub.2S. The wound area is then covered with a secondary wound dressing with low gas permeability, such as a film consisting of polyethylene, polypropylene or polytetrafluoroethylene or a fabric or fleece that has reduced gas permeability, for example, a polypropylene fleece with an almost gas-tight layer such as polyurethane.

Example 9: Injectable Dermal Filler Formulation

(39) Hyaluronic acid is stabilized via crosslinking with 1,4-butanediol diglycidyl ether (BDDE). After purification via dialysis the crosslinked hyaluronic acid is mixed with a buffered solution (phosphate buffer, pH 7.4 comprising a H.sub.2S releasing hyaluronic acid derivative according to the invention, so that the final concentration of H.sub.2S releasing hyaluronic acid derivative in the formulation is 5 mg/mL. The formulation is then filled into syringes and sterilized with moist heat.

(40) The thiol-group containing reaction partner may be an endogenous reaction partner which is present in the skin, such as reduced glutathione.

Example 10: Wound Healing Preparation—Multi-Layer Wound Dressing

(41) The H.sub.2S releasing polysaccharide polymer according to the invention may be provided in a multi-layer wound dressing (or dermal patch) as shown in FIG. 9. A reservoir of dry H.sub.2S releasing polymer powder 1 is used as the absorbent layer of the dressing. Optionally, this layer may additionally comprise a thiol-group donor such as L-cysteine, homocysteine, N-acetylcysteine or reduced glutathione. Alternatively, the thiol-group reaction partner is not applied but provided endogenously e.g. in the wound exudate. The absorbent layer is covered by an outer protective membrane with low gas permeability. The outer protective layer 2 further comprises a layer of adhesive, which is used to adhere the wound dressing to the skin. The outer layer 2 extends beyond the edges of the other layers (i.e., the absorbent layer 1 and the wound contact layer 4) to form an adhesive rim 4. The polymer reservoir is further covered by an inner non-adherent and porous membrane, for example, a woven nylon fabric. This wound contact layer 4 is permeable to fluids (wound exudate), hypoallergenic and non-irritant.

(42) The wound contact layer 4 and the exposed part of the adhesive layer 2 are protected prior to use by a release-coated protective membrane. The protective membrane is formed from paper release-coated with a silicone.

(43) The multilayered wound dressing is packaged in a hermetically sealed pouch and sterilised by gamma-irradiation.

Example 11: Injectable Formulation for the Treatment of Acne Scars

(44) An aqueous hydrogel comprising 2% (m/m) H.sub.2S releasing hyaluronic acid derivative according to the invention with a degree of modification of about 250 μmol S-acetyl moieties is prepared in a phosphate buffer with a physiologically acceptable pH. A second composition comprising N-acetylcysteine in a physiologically acceptable buffer solution in a concentration of 0.5% (m/m) is prepared. The gel and the N-acetylcysteine solution are then filled into single use dual chamber syringes and sterilized with moist heat.

Example 12: Injectable Formulation for Intraarticular Viscosupplementation

(45) An aqueous hydrogel comprising 1.0% (m/m) H.sub.2S releasing hyaluronic acid derivative according to the invention is prepared in a phosphate buffer with a physiologically acceptable osmolarity and pH value. The gel is filled into syringes and sterilized with moist heat.

(46) The thiol-group containing reaction partner may be an endogenous reaction partner which is present in the synovial fluid, such as reduced glutathione.

Example 13: Injectable Dermal Filler

(47) A hyaluronic acid-cysteamine derivative is stabilized via crosslinking with disulfide bonds (formation of cystamine bridges). The crosslinked hyaluronic acid is then mixed with a buffered solution (phosphate buffer, pH 7.4) comprising a H.sub.2S releasing hyaluronic acid derivative according to the invention, so that the final concentration of H.sub.2S releasing hyaluronic acid derivative in the formulation is 5 mg/mL. The formulation is then filled into syringes and sterilized with moist heat.

(48) The thiol-group containing reaction partner may be an endogenous reaction partner, which is present in the skin, such as reduced glutathione.

Example 14: Injectable Dermal Filler

(49) An aqueous hydrogel comprising 2.5% (m/m) H.sub.2S releasing hyaluronic acid derivative according to the invention is prepared in a phosphate buffer with a physiologically acceptable osmolarity and pH value. The gel is filled into syringes and sterilized with moist heat.

(50) The thiol-group containing reaction partner may be an endogenous reaction partner which is present in the skin, such as reduced glutathione.

Example 15: Injectable Formulation for Intraarticular Viscosupplementation

(51) An aqueous hydrogel comprising 0.5% (m/m) hyaluronic acid-cysteamine derivative and another aqueous hydrogel comprising 0.5% (m/m) H.sub.2S releasing hyaluronic acid derivative according to the invention are prepared in a phosphate buffer with a physiologically acceptable osmolarity and pH value. The gels are individually filled into dual chamber syringes and sterilized with moist heat.

Example 16: Eye Drop Formulation

(52) An aqueous ophthalmic solution comprising 0.1% (m/m) H.sub.2S releasing chitosan derivative according to the invention is prepared in a borate buffer with a physiologically acceptable osmolarity and pH value. The solution is sterilized via filtration and filled into single use ophthalmic dosage units.

(53) The thiol-group containing reaction partner may be an endogenous reaction partner, which is present on the ocular surface such as reduced glutathione.

Example 17: Intraocular Implant

(54) An aqueous hydrogel comprising 4.0% (m/m) H.sub.2S releasing hyaluronic acid derivative according to the invention is prepared in a phosphate buffer with a physiologically acceptable osmolarity and pH value. The gel is filled into syringes and sterilized with moist heat.

(55) The thiol-group containing reaction partner may be an endogenous reaction partner, such as reduced glutathione.

Example 18: Vitreous Body Substitute

(56) A hyaluronic acid-cysteamine derivative is stabilized via crosslinking with disulfide bonds (formation of cystamine bridges). The crosslinked hyaluronic acid is then mixed with a buffered solution (phosphate buffer, pH 7.4) comprising a H.sub.2S releasing hyaluronic acid derivative according to the invention, so that the final concentration of H.sub.2S releasing hyaluronic acid derivative in the formulation is 3 mg/mL The formulation is then filled into syringes and sterilized with moist heat.

(57) The thiol-group containing reaction partner may be an endogenous reaction partner, such as reduced glutathione.

Example 19: Solid Intraocular Implant

(58) Tablets with a weight of approximately 2 mg comprising 1 mg of a H.sub.2S releasing hyaluronic derivative with a degree of modification of about 250 μmol S-acetyl moieties according to the invention and 0.2 mg N-acetylcysteine are prepared. The tablets are sealed individually in pouches. The pouches and secondary packaging material are then sterilized via gamma irradiation.

Example 20: Preparation of 4-Amino-N-(Benzoylthio)Benzamide

(59) 4-Amino-N-(benzoylthio)benzamide is a NH.sub.2-modified derivative of the H.sub.2S Donor 5a comprising a benzoylthiobenzamide substructure. The schematic reaction scheme of this synthesis is shown in FIG. 10A.

Step 1 Synthesis of S-benzoylthiohydroxylamine

(60) To a stirred solution of 5.6 g KOH (100 mmol) in 150 ml H.sub.2O and 5.65 g (50 mmol) Hydroxylamine-O-sulfonic acid 7.25 g Thiobenzoic acid (52.5 mmol) were added. After stirring at ambient temperature for 1 hour, the precipitate was collected by suction filtration and dried under reduced pressure.

Step 2 Synthesis of N-(benzoylthio)-4-nitrobenzamide

(61) 7.12 g (46 mmol) of the crude S-benzoylthiohydroxylamine were dissolved in 250 ml CH.sub.2Cl.sub.2 and 3.6 g (46 mmol) of pyridine were added. 8.53 g (46 mmol) p-NO.sub.2-benzoylchloride were suspended in 300 ml CH.sub.2Cl.sub.2 and added dropwise via dropping funnel. The reaction progress was monitored by TLC (hexane/diethylether=1:1.5), after 2 hours the solution was filtrated over a pad of Celite® and volatiles were removed under reduced pressure. The crude product was purified via silica gel column chromatography using hexane/diethylether=1:1.5 to give 3.52 g of a white solid (23% over 2 steps).

Step 3 Synthesis of 4-amino-N-(benzoylthio)benzamide

(62) 1.7 g (5.6 mmol) N-(benzoylthio)-4-nitrobenzamide were dissolved in a mixture of 270 ml THF and 180 ml H.sub.2O and cooled to 0° C. 2.42 g (14.07 mmol) of Na.sub.2S.sub.2O.sub.4 were added, whereupon the solution turned yellow. The conversion was monitored via TLC (diethylether, R.sub.f product˜0.6). After 30 minutes, THF was removed quantitatively under reduced pressure and the aqueous solution was extracted with ethyl acetate. The organic layer was dried over Na.sub.2SO.sub.4 and volatiles were evaporated under reduced pressure to give 640 mg of a yellowish powder (45%). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H, NH), 7.91 (d, J=7.3 Hz, 2H), 7.77-7.68 (m, 3H), 7.61 (d, J=7.7 Hz, 2H), 6.59 (d, J=8.6 Hz, 2H), 5.85 (s, 2H, NH.sub.2)

(63) Corresponding hydroxylated, phosphorylated or sulfonated derivatives can be prepared in analogy to the described protocol using various protecting group strategies.

(64) A first olfactory test performed with 4-amino-N-(benzoylthio)benzamide dissolved in acetone/phosphate buffer indicated H.sub.2S liberation after addition of an excess amount of L-cysteine. This finding was later confirmed by detecting H.sub.2S release after addition of excess amounts of L-cysteine or cysteamine to 4-amino-N-(benzoylthio)benzamide via embrowning Pb-acetate paper.

Example 21: Synthesis of a H.SUB.2.S Releasing Hyaluronic Acid Derivative

(65) Coupling of 4-amino-N-(benzoylthio)benzamide to hyaluronic acid is performed by dissolving hyaluronic acid in DMSO and triethylamine and adding a reagent which mediates amide coupling such as carbonyldiimidazole (CDI), carbodiimides such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), phosphonium or aminium/uronium-umonium type reagents such as bromo-tripyrrolidino-phosphonium hexafluorophosphate (PyBrOP®) or 2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethylammonium tetrafluoroborate/hexafluorophosphate (TBTU, HBTU). After precipitation, the H.sub.2S releasing hyaluronic acid derivative is isolated via filtration, washed and dried under vacuum.

(66) Coupling of hydroxylated, sulfonated or phosphorylated 4-amino-N-(benzoylthio)benzamide derivatives to hyaluronic acid is performed under aqueous conditions by dissolving sodium hyaluronate in a phosphate buffer and adding a reagent which mediates amide coupling such as a water soluble carbodiimide (e.g. N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide HCl, EDC.HCl) or a triazine derivative (e.g. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, DMT-MM). After precipitation the H.sub.2S releasing hyaluronic acid derivative is isolated via filtration, washed and dried under vacuum.

(67) A schematic structure of the resulting H.sub.2S releasing hyaluronic acid derivative is shown in FIG. 10B.