STABILIZED HYALURONIC ACID

Abstract

Sterile hydrogel composition comprising crosslinked hyaluronan, wherein the amount of extractable hyaluronan having a molecular weight of less than 200 kDa is less than 15 wt. % relative to the total amount of hyaluronan.

Claims

1. A sterile hydrogel composition comprising crosslinked hyaluronan, wherein the amount of extractable hyaluronan having a molecular weight of less than 200 kDa is less than 15 wt. % relative to the total amount of hyaluronan.

2. The composition according to claim 1, wherein the extractable hyaluronan is the hyaluronan extractable from the sterile hydrogel composition by use of reductive extraction or conservative extraction.

3. The composition according to claim 1, wherein the crosslinked hyaluronan has structure according to formula I
HA-L-HA  (I), wherein each HA stands for hyaluronan or a salt thereof according to formula II ##STR00011## wherein n is an integer>1 and determines the number of repeats of the repetitive unit of formula II, and L is a linker, which linker is covalently bound to each HA by replacing one OH-moiety in the repetitive unit according to formula II, and wherein L is derived from a molecule LH.sub.2, wherein the molecule LH.sub.2 is a molecule occurring naturally in humans or a conjugate of molecules occurring naturally in humans.

4. The composition according to claim 3, wherein the linker L is a non-genotoxic and non-cytotoxic fragment of an endogenous molecule.

5. The composition according to claim 3, wherein the molecule LH.sub.2 is selected from the group consisting of glutathione, cysteamine, cysteine, homo-cysteine, beta-cysteine, a peptide comprising cysteine, a conjugate of cysteamine and an amino acid, and a disulfide dimer of any of the foregoing thereof.

6. The composition according to claim 1, further comprising at least one local anaesthetic.

7. The composition according to claim 5, wherein the local anaesthetic is selected from the group consisting of lidocaine, articaine, prilocaine, chloroprocaine, articaine, and combinations thereof.

8. The composition according to claim 1, further comprising a non-toxic stabilizer.

9. The composition according to claim 1, further comprising at least one inhibitor of hyaluronic acid degradation.

10. The composition according to claim 9, wherein said inhibitor is selected from the group consisting of 1,2,3,4,6-penta-O-galloylglucose, apigenin, beta-escin, caltrin, cis-Hinokiresinol (CHR), echinacin, eicosatrienoic acid (C20:3), fenoprofen, gold sodium thiomalate, gossypol, heparin, hesperidin phosphate, indomethacin, L-ascorbic acid, L-carnitine, L-aminocarnitine, myochrisine (sodium aurothiomalate), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), phosphorylated hesperidin, poly(sodium 4-styrene-sulphonate) (T-PSS), polyoestradiol phosphate, polyphloretin phosphate, PS53 (a hydroquinone/sulphonic acid/formaldehyde polymer), sodium polystyrene sulphonate (N-PSS), sulphated 2-hydroxyphenyl monolactobioside, sulphated hydroquinone digalactoside, the sulphated verbascose, planteose and neomycin oligosaccharides, tetradecyl sodium sulphate (TDSS), a C14:1 to C24:1 unsaturated fatty acid with one double bond, urinary trypsin inhibitor (UTI), urolithin B, WSG, glycyrrhetinic acid, and combinations thereof.

11. The composition according to claim 1, further comprising an unmodified polymer selected from the group of biocompatible polysaccharides.

12. The composition according to claim 1, further comprising a viscous and hydrophilic biocompatible polyalcohol.

13. A cosmetic comprising the composition according to claim 1.

14. A medicament comprising the composition according to claim 1.

15. A soft tissue filling comprising the composition according to claim 1.

16. The composition according to claim 11, wherein the unmodified polymer is unmodified hyaluronan.

17. The composition according to claim 12, wherein the viscous and hydrophilic biocompatible polyalcohol is glycerol.

18. A sterile hydrogel composition comprising crosslinked hyaluronan, wherein the crosslinked hyaluronan has structure according to formula I
HA-L-HA  (I), wherein each HA stands for hyaluronan or a salt thereof according to formula II ##STR00012## wherein n is an integer>1 and determines the number of repeats of the repetitive unit of formula II, and L is a linker, which linker is covalently bound to each HA by replacing one OH-moiety in the repetitive unit according to formula II, and wherein L is derived from a molecule LH.sub.2, wherein the molecule LH.sub.2 is a molecule occurring naturally in humans or a conjugate of molecules occurring naturally in humans.

19. The composition according to claim 18, wherein the molecule LH.sub.2 is selected from the group consisting of glutathione, cysteamine, cysteine, homo-cysteine, beta-cysteine, a peptide comprising cysteine, a conjugate of cysteamine and an amino acid, and a disulfide dimer of any of the foregoing.

20. A cosmetic comprising the composition according to claim 19.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0073] As discussed above a preferred embodiment the crosslinked hyaluronan is a reaction product of a modified hyaluronan, wherein the modified hyaluronan is modified with an endogenous molecule providing reactive group for crosslinking, such as thiol groups. In the following more details regarding the preparation of preparation of thiol modified hyaluronan, the crosslinked hyaluronan being an oxidation product as well as compositions comprising the same are given.

[0074] Introduction of the modification agent via formation of an ester bond or an amide bond between the carboxyl group of the glucuronic acid moiety of hyaluronan and the modification agent is preferred (s. formula III above). The modification agent may comprise thiol groups in the form of disulfide bonds or as protected thiol groups during the synthesis process.

[0075] In one preferred embodiment, the modification agent is linked to the carboxyl group of the glucuronic acid moiety in the hyaluronan via an amide bond. Accordingly, the modification agent comprises at least one amino group capable to form the amide bond with the carboxyl group of the glucuronic acid moiety in the hyaluronan and the modification agent comprises a thiol group. For example, the thiol modified hyaluronan is a hyaluronan-cysteamine conjugate, wherein cysteamine is linked to hyaluronan via an amide bond.

[0076] Similarly, other thiol group bearing modification agents may be used for the synthesis of thiol-modified hyaluronan via amide bond formation between an amino group (primary or secondary amino group, preferably primary amino group) of the modification agent and the carboxyl group of the glucuronic acid moiety in the hyaluronan. Potential modification agents include for example derivatives of cysteamine, cysteine or homocysteine, wherein the amino group of the cysteamine, cysteine or homocysteine is coupled with the carboxyl group of an amino acid. These derivatives are preferably synthesized by using N-protected amino acids.

[0077] A low molecular weight of the modification agent is preferred to conserve to the unique physico-chemical properties of hyaluronan as much as possible. Suitable low molecular weight modification agents to obtain a cross-linkable thiol-modified hyaluronan useful for a composition according to the invention preferably are further selected from the group comprising glutathione, cysteine and homocysteine.

[0078] Non-limiting examples of suitable linker L or naturally occurring molecule LH.sub.2 are: [0079] cystamine, the disulfide dimer of cysteamine, i.e L of formula V

—NH—CH.sub.2—CH.sub.2—S—S—CH.sub.2—CH.sub.2—NH—  (V) [0080] oxidized glutathione, the disulfide dimer of glutathione, i.e LH.sub.2 of formula VI

##STR00004## [0081] cystine, the disulfide dimer of cysteine, i.e. LH.sub.2 of formula VII

##STR00005## [0082] homocystine, the disulfide dimer of homocysteine, i.e. LH.sub.2 of formula VIII

##STR00006## [0083] an amino acid derivative of cystamine being a conjugate of the naturally occurring molecules cystamine and naturally occurring amino acids, i.e. LH.sub.2 of formula (IX)

##STR00007##

[0084] wherein both X may be identical or different to each other (symmetrical or asymmetrical derivatives) and the nature of X depends on the amino acid. In one embodiment both X are hydrogen atoms, i.e. symmetrical glycine derivative; [0085] urea, i.e. LH.sub.2 of formula X

##STR00008##

[0086] The crosslinked hyaluronan used in the hydrogel composition according to the present invention may be produces as follows: first the hyaluronan is modified with endogenous molecules with reactive groups for crosslinking, such as thiol groups. Cysteamine, cysteine and glutathione are examples of such non-genotoxic endogenous molecules which naturally occur in humans. For example the relevant substructures of thiol-modified hyaluronan crosslinkable by disulfide formation modified with cysteamine (HA-cysteamine) and glycinyl-cysteamine (HA-glycinyl-cysteamine), are shown in formula XI

##STR00009##

[0087] and formula XII

##STR00010##

[0088] respectively.

[0089] The reactions are carried out under conditions which do not negatively affect the molecular mass of hyaluronic acid, such as temperatures of less than 40° C. and very limited if any exposure times to pH values in the range of pH 11 or higher and less than pH 4.

[0090] Aeschlimann (EP 1 115 433 B1) describes a method of functionalization of hyaluronan which does not compromise the molecular weight of hyaluronan and which further provides hyaluronan molecules that are well tolerated in vivo and are biodegradable. The method is used to generate hyaluronan with different terminal functional groups for crosslinking, such as thiol groups. These side chains are introduced into hyaluronan by carbodiimide-mediated coupling of primary (protected) thiol group containing amines or disulfide-bond containing diamino or dihydrazide ligands to the carboxyl group of the glucuronic acid moiety using an active ester intermediate. Intermediate products with disulfide bonds are then reduced and intermediate products with protected thiol groups are then deprotected by removing the protecting group.

[0091] EP 0 587 715 discloses how to synthesize water insoluble anionic polysaccharides via dissolving at least one polyanionic polysaccharide (e.g., hyaluronan), in an aqueous mixture; activating the polyanionic polysaccharide with an activating agent such as a diimide, e.g. EDC or ETC, or BOP; modifying the activated polyanionic polysaccharide with a modifying compound such as 1-hydroxybenzotriazole hydrate (HOBt) or 1-hydroxybenzotriazole monohydrate; and reacting the activated polyanionic polysaccharide with a suitable nucleophile (such as an amino thiol) to form the desired insoluble composition. The inventors state that one major advantage of the BOP activation of polyanionic polysaccharide is that the molecular weight of the polyanionic polysaccharide is not decreased upon coupling to the nucleophile.

[0092] Triazine-mediated amidation with DMT-MM for efficient and controlled functionalization of hyaluronic acid with cysteamine is described in Borke et al. In comparison to other coupling reagents (such as EDC-mediated substitution) the mild reaction conditions and the minimal degradation of the polysaccharide chain are listed as advantages of using this group of coupling agents (Borke et al., Carbohydrate Polymers 116 (2015) 42-50). Liang et al. describe the introduction of thiol groups to hyaluronan via an amidation reaction of the side carboxylates with cystamine in the presence of CDMT and NMM, followed by a reducing reaction with DTT (Liang et al. Carbohydrate Polymers 132 (2015) 472-480).

[0093] The state of the art is silent about the preservation of the molecular weight of hyaluronan during crosslinking in the course of the preparation of sterile hydrogel compositions.

[0094] The modified hyaluronan is then purified while it is still uncrosslinked, allowing for very efficient purification by different methods such as precipitation, chromatography and dialysis.

[0095] Then, the modified and purified hyaluronan is crosslinked to form a highly viscous gel. It is then not necessary to further purify the gel via dialysis, which is the only purification method routinely applied to highly viscous aqueous gels. The condition for crosslinking depend on the nature of the linker. In case, the hyaluronan is modified with endogenous molecules with reactive thiol groups, the crosslinking involves the oxidation of the thiol groups to form inter and intra molecular disulfide bonds.

[0096] Once the gels are filled into syringes, they need to be sterilized.

EXAMPLES

Example 1: Crosslinking with Urea

[0097] 8 g sodium hyaluronate is dissolved in 72 g saline. A solution is prepared separately dissolving 4 g urea in 0.2M 16 g HCl. The two solutions prepared are mixed until the final solution is homogeneous; the pH is measured which has to be in the range from 3.5 to 4. The product is thermostated at 35(+/−2) ° C. for 20-24 hours, the excess of urea is then eliminated; once purified, the pH of the product obtained was measured which was comprised from 5.5 to 7.5.

[0098] The product is then filled into syringes and sterilized with an autoclave.

Example 2: Crosslinking with Cystamine

[0099] In order to prepare a hydrogel the HA-cysteamine powder with a MW of at least about 700 kDa is dissolved in aqueous medium. Unmodified HA with a MW of at least 1000 kDa and optionally a local anesthetic such as lidocaine HCl are added to this solution. Crosslinking of HA-cysteamine via the formation of disulfide bonds takes then place under mild oxidative conditions (pH 7.4, presence of O.sub.2,) and at room temperature which results in a hydrogel suitable for a soft tissue filler. No further purification of the gel (e.g. via dialysis) is required. The hydrogels are filled into syringes and are sterilized with an autoclave.

[0100] Preparation of TH-260417-1, TH-270217-2, TH-220317-2 and TH-070217-2

[0101] For the preparation of the sterile hydrogel composition TH-260417-1 3580 mg HA-cysteamine (MW 730 kDa), 600 mg lidocaine HCl and 1160 mg NaCl were dissolved in 185 g water for injection under mechanical stirring at room temperature for about 3 hours. 1000 mg sodium hyaluronate (MW 2400 kDa) were then added to the solution under continued stirring at room temperature for about another 3 hours. Phosphate buffer pH 11 was then added to a final amount of 200 g formulation. The solution was homogenized for about 15 min. After incubation overnight at room temperature the now crosslinked hydrogel was filled into 1 ml glass syringes and sterilized via autoclavation. The sterile hydrogel had a pH of about 7.7 and an osmolality in the range of 270-330 mOsmol/kg.

[0102] TH-270217-2 and TH-070217-2 were produced according to the same method but in a smaller batch size (50 g). The sterile hydrogel composition TH-220317-2 was produced according to the same method as sterile hydrogel compositions TH-270217-2 and TH-070217-2 but with HA-cysteamine raw material with a MW of about 900 kDa.

[0103] Preparation of TH-260417-2

[0104] For the preparation of the sterile hydrogel composition TH-260417-2 3580 mg HA-cysteamine (MW 730 kDa), 600 mg lidocaine HCl and 1160 mg NaCl were dissolved in 185 g water for injection under mechanical stirring at room temperature for about 3 hours. 1000 mg sodium hyaluronate (MW 1300 kDa) were then added to the solution under continued stirring at room temperature for about another 3 hours. Phosphate buffer pH 11 was then added to a final amount of 200 g formulation. The solution was homogenized for about 15 min. After incubation over night at room temperature the now crosslinked hydrogel was filled into 1 ml glass syringes and sterilized via autoclavation. The sterile hydrogel had a pH of about 7.7 and an osmolality in the range of 270-330 mOsmol/kg.

[0105] Preparation of TH-260917_200

[0106] For the preparation of the sterile hydrogel composition TH-260917_200 3580 mg HA-cysteamine sodium salt (MW 730 kDa, degree of modification 151 μmol/g polymer), 600 mg lidocaine HCl and 1160 mg NaCl were dissolved in 185 g water for injection under mechanical stirring at room temperature for about 3 hours. 1000 mg sodium hyaluronate (MW 2400 kDa) were then added to the solution under continued stirring at room temperature for about another 3 hours. Phosphate buffer pH 11 was then added to a final amount of 200 g formulation. The solution was homogenized for about 15 min. After incubation over night at room temperature the now highly viscous gel was pressed through a filter plate with a mesh size of 200 μm. The hydrogel was then filled into 1 ml glass syringes and sterilized via autoclavation (121° C./15 min). The sterile hydrogel had a pH of about 7.5 and an osmolality in the range of 270-330 mOsmol/kg.

[0107] Preparation of TH-250417-1

[0108] For the preparation of the sterile hydrogel composition TH-250417-1 2685 mg HA-cysteamine (MW 730 kDa), 450 mg lidocaine HCl and 870 mg NaCl were dissolved in 85.5 g phosphate buffer pH 3 under mechanical stirring over night at room temperature. The pH of the solution comprising HA-cysteamine was then adjusted to pH 7.6 via addition of an alkaline phosphate buffer to obtain a solution comprising 2.7% (m/m) HA-cysteamine. After 10 min of homogenisation the solution was incubated over night without stirring at room temperature. Next, the crosslinked gel was pressed through a filter plate with a mesh size of 200 μm. A solution containing 1.5% (m/m) sodium hyaluronate with a MW of 2400 kDa in a 10 mM phosphate buffer pH 6.7 was prepared. 1 part solution containing 1.5% (m/m) sodium hyaluronate with a MW of 2400 kDa in a 10 mM phosphate buffer pH 6.7 was then added to two parts sieved and crosslinked hydrogel. After mechanical mixing for 10 min the finished product was then filled into 1 ml glass syringes and sterilized via autoclavation. The sterile hydrogels had a pH of about 7.4 and an osmolality in the range of 270-330 mOsmol/kg.

[0109] Preparation of TH-250417-2

[0110] For the preparation of the sterile hydrogel composition TH-250417-2 2685 mg HA-cysteamine (MW 730 kDa), 450 mg lidocaine HCl and 870 mg NaCl were dissolved in 103.5 g phosphate buffer pH 3 under mechanical stirring overnight at room temperature. The pH of the solution comprising HA-cysteamine was then adjusted to pH 7.6 via addition of an alkaline phosphate buffer to obtain a solution comprising 2.2% (m/m) HA-cysteamine. After 10 min of homogenisation the solution was incubated over night without stirring at room temperature. Next, the crosslinked gel was pressed through a filter plate with a mesh size of 200 μm. A solution containing 2.5% (m/m) sodium hyaluronate with a MW of 1300 kDa in a 10 mM phosphate buffer pH 6.7 was prepared. 1 part solution containing 2.5% (m/m) sodium hyaluronate with a MW of 1300 kDa in a 10 mM phosphate buffer pH 6.7 was then added to three parts sieved and crosslinked hydrogel. After mechanical mixing for 10 min the finished product was then filled into 1 ml glass syringes and sterilized via autoclavation. The sterile hydrogels had a pH of about 7.6 and an osmolality in the range of 270-330 mOsmol/kg.

[0111] Preparation of THM-040717-1-53

[0112] For the preparation of the sterile hydrogel composition THM-040717-1-53 750 mg HA-cysteamine sodium salt (MW 730 kDa, degree of modification 151 μmol/g polymer), 450 mg sodium hyaluronate (MW 2400 kDa), 450 mg lidocaine HCl and 795 mg NaCl were dissolved in 132 g 0.01 M HCl under mechanical stirring at room temperature for about 21 hours. Phosphate buffer pH 12.5 was then added to a final amount of 150 g formulation. The solution was homogenized for about 15 min. After incubation overnight at room temperature the now highly viscous hydrogel was filled into 1 ml glass syringes and sterilized via autoclavation. The sterile hydrogel had a pH of 7.3 and an osmolality of 267 mOsmol/kg.

Example 3 Measurement of MW of Extractable HA in Sterile Hydrogel Compositions Comprising Disulfide Crosslinked HA

[0113] Sample preparation (reductive extraction) After sterilization of hydrogels comprising disulfide crosslinked HA and free HA a reducing agent was added to the hydrogels to quantitatively break disulfide bonds. The MW distribution of modified HA in its reduced (uncrosslinked) form and free HA was then determined simultaneously. About 900 mg of the hydrogel was diluted with 1500 mg water for injection followed by addition of a reducing agent (2500 mg TCEP.HCl (Tris(2-carboxyethyl)phosphine hydrochloride (2.5 mg/ml water for injection) to cleave disulfide bridges. After a reducing time of 3 hours 400 mg of the reaction solution were acidified with 50 μl 5 N HCl. Free HA and the modified hyaluronan were precipitated with ethanol. The precipitate was recovered by centrifugation followed by solubilization in 4 ml of an aqueous solution containing a capping agent for free thiol moieties (2-(2-aminoethyldisulfanyl)pyridine-3-carboxylic acid) in a concentration of 2 mg/ml. After 3 h incubation at room temperature the sample was further diluted with PBS.

[0114] Molecular Weight Determination

[0115] For size exclusion chromatography (SEC) analysis, sample solutions were diluted with SEC eluent resulting in a final HA concentration of 0.1 mg/ml. A Viscotek TDAmax temperature controlled, multi-detector SEC system comprising high sensitivity detectors in series—Photodiode Array UV, Light Scattering (both RALS and LALS), Refractive Index and Viscometer was used for the measurements. The refractive index detector recorded the concentration of the sample resulting in the respective distribution curve. In combination with the light scattering detectors, the molecular weight MW was determined.

[0116] Results

[0117] It was found that the low MW fraction (MW<200 kDa) of HA in sterile hydrogel compositions prepared according to example 2 was in the range of 8% to 15% after storing the samples at room temperature for the indicated number of days (Table 1).

TABLE-US-00001 TABLE 1 Molecular weight (MW) results for various hydrogels according to the invention MW <200 kDa Days of MW since HA fraction of Measurement pro- Test material [kDa] HA [%] date duction TH-270217-2 566 15 10 Mar. 2017 11 TH-070217-2 581 13 10 Mar. 2017 31 TH-220317-2 757 8 24 Mar. 2017 2 TH-250417-1 670 9 6 May 017 11 TH-250417-2 710 9 6 May 2017 11 TH-260417-1 790 8 6 May 2017 10 TH-260417-2 700 8 6 May 2017 10 TH-260917_200 632 13 16 Oct. 2017 20 THM-040717-1-53 n.d. 13 27 Feb. 2018 238

Example 4 (Comparative) Decrease of the Molecular Weight of Hyaluronic Acid Under Reaction Conditions which are Necessary for Crosslinking with TMP (Trimetaphosphate) as Described in WO 2016/005785A1

[0118] Hydration Step

[0119] Sodium hyaluronate (HA) with an initial molecular weight of 2.4 MDa was hydrated in 0.01 M NaOH in a final concentration of 90 mg/ml for 2.5 h with mechanical homogenization (pH 11).

[0120] Simulated Crosslinking Step

[0121] One sample of the mixture obtained during the hydration step was incubated for 48 h at 70° C. Degradation of the hyaluronan backbone was stopped by neutralization using phosphate buffer pH 7.0.

[0122] Molecular Weight Determination

[0123] Molecular weight was determined of the sodium hyaluronate used as starting material, of sodium hyaluronate after the hydration step and of all three samples obtained during mimicking of the crosslinking step.

[0124] For size exclusion chromatography (SEC) analysis, sample solutions were diluted with SEC eluent resulting in a final HA concentration of 0.1 mg/ml. A Viscotek TDAmax temperature controlled, multi-detector SEC system comprising high sensitivity detectors in series—Photodiode Array UV, Light Scattering (both RALS and LALS), Refractive Index and Viscometer was used for the measurements. The refractive index detector recorded the concentration of the sample resulting in the respective distribution curve. In combination with the light scattering detectors, the molecular weight Mw of HA was determined.

[0125] Results

[0126] It was found that exposure to high temperatures and high pH values increases the low MW fraction of HA. After 48 h at pH 11 and 70° C. the low MW fraction was 30%. It is to be expected that the low MW fraction will be further increased during the sterilization step which is necessary to produce the final soft tissue filler formulations.

TABLE-US-00002 TABLE 2 Results from molecular weight analysis MW <200 kDa MW of HA fraction of HA Test material [kDa] [%] HA starting material 2414 0 HA incubated for 48 h at 349 30 pH11 and 70° C.

Example 5 In Vivo Residence Time of Sterile Hydrogel Compositions Comprising Disulfide Crosslinked HA

[0127] The degradation dynamics of sterile hydrogel compositions TH-250417-1 and TH-260417-1 (see example 2 and 3) was determined over a period of 2 months after intradermal injection into the back skin of female Sprague Dawley rats with a total of 12 applications per formulation. The volume of the applied filler depots was monitored via MRT scans. Mean depot volumes relative to the depot volumes at starting point were calculated. At 108 days post implantation the mean relative depot volume was 115% for TH-260417-1 and 106% for TH-250417-1 indicating a high resistance of both sterile hydrogel compositions comprising disulfide crosslinked HA towards degradation.

Example 6 Comparison Between MW Distribution of HA in Compositions Comprising Disulfide Crosslinked HA and State of the Art BDDE-Crosslinked HA

[0128] Investigated composition comprising disulfide crosslinked HA (cystamine crosslinked HA) A thiol-modified hyaluronan with a degree of modification of 147 μmol thiol groups per g polymer (MW 730 kDa) was used to produce a composition comprising 17.9 mg/mL crosslinked HA-cysteamine sodium salt, 3 mg/mL lidocaine HCl and 5 mg/mL unmodified sodium hyaluronate (MW 1.94 MDa). For adjustment of the pH and the osmolality to physiologically acceptable values, the hydrogel further comprised 10 mM phosphate buffer and 95 mM NaCl. In brief, HA-cysteamine sodium salt, sodium hyaluronate, lidocaine HCl and sodium chloride were dissolved in 001 M HCl via stirring at room temperature for 8 hours. Crosslinking was initiated by addition of 1 part of 100 mM phosphate buffer pH 12.1 to 9 parts of the solution for adjustment of the pH to 7.4, followed by addition of a diluted hydrogen peroxide solution so that the molar ratio of free thiol groups of thiol-modified hyaluronan to hydrogen peroxide was 2:1. After 48 hours of crosslinking at room temperature, the hydrogel was sieved, filled into 1 ml glass syringes and sterilized via autoclavation. Mean reduced post-sterilisation molecular weight (MRPMW) of crosslinked polymer in the composition was 610 kDa. The sterile hydrogel had a pH of 7.5 and an osmolality of 296 mOsmol/kg.

[0129] Investigated Composition Comprising BDDE Crosslinked HA

[0130] A sterile hydrogel comprising BDDE crosslinked HA (MW 2.7 MDa) in a concentration of 23 mg/ml was obtained. The hydrogel had pH 7 of and an osmolality of 298 mOsmol/kg.

[0131] Sample preparation for MW determination by conservative extraction Both hydrogel compositions were investigated as follows. About 200 mg of the hydrogel were diluted with 1800 mg PBS. After physical (“conservative”) extraction of free HA for four hours, the dispersion was centrifuged followed by recovering of the supernatant.

[0132] For size exclusion chromatography (SEC) analysis, the supernatant was diluted with SEC eluent resulting in a final HA concentration of about 0.1 mg/ml. A Viscotek TDAmax temperature controlled, multi-detector SEC system comprising high sensitivity detectors in series—Photodiode Array UV, Light Scattering (both RALS and LALS), Refractive Index and Viscometer was used for the measurements. The refractive index detector recorded the concentration of the sample resulting in the respective distribution curve. In combination with the light scattering detectors, the molecular weight MW was determined.

[0133] Results

TABLE-US-00003 TABLE 3 Results from molecular weight analysis Total HA MW of Concentration <200 KDa <200 KDa Hydrogel concentration extracted HA of extracted MW fraction MW fraction composition (mg/mL) (MDa) HA (mg/mL) per peak (%) of total HA (%) Cystamine 22.9 0.84 5.2 <5 <1 crosslinked HA BDDE 23 0.89* 1.3 <5 23 crosslinked 0.11* 6.2 85 HA *bimodal MW distribution

[0134] The concentration of extracted HA (5.2 mg/mL) of the sterile hydrogel comprising disulfide crosslinked HA corresponded very well to the concentration of unmodified HA (5 mg/mL), which was added to the composition during hydrogel preparation. This indicates that the crosslinking process was both very efficient and mild, since no significant amount of modified HA remained uncrosslinked during preparation of the hydrogel or was set free upon degradation of hyaluronan chains. The first measurements (Table 3) were performed within one month after hydrogel production. Repeated measurements 8 months later did not indicate an increase in the concentration of extracted HA or the <200 KDa MW fraction. The amount of extractable hyaluronan having a molecular weight of less than 200 kDa was less than 1 wt. % relative to the total amount of hyaluronan (including modified and unmodified HA).

[0135] In contrast, the total concentration of extracted HA was 7.5 mg/ml in the hydrogel comprising BDDE crosslinked HA, which means that about one third of the HA used for hydrogel production remained uncrosslinked or was set free upon degradation of hyaluronan chains during crosslinking, sterilization and storage. The concentration of free HA which was actively added during production of the product was not specified by the manufacturer. The amount of extractable hyaluronan having a molecular weight of less than 200 kDa was 23 wt. % relative to the total amount of hyaluronan.

Example 7 Preparation of bis(glycyl)-cystamine dihydrochloride

[0136] To a mixture of cystamine dihydrochloride (1 g, 4.44 mmol) and N-(tert-butoxycarbonyl)-glycine (1.59 g, 9.10 mmol) in dry dichloromethane:THF=1:1 (20 mL) first triethylamine (1270 μL, 9.16 mmol) was added, followed by addition of a solution of EDC*HCl (1.75 g, 9.10 mmol) in dichloromethane. The reaction solution was stirred for 5 h at ambient temperature, then volatiles were evaporated under reduced pressure. The residue was taken up in ethyl acetate (250 mL) and washed with 1 n HCl (2×50 mL), half saturated NaHCO.sub.3 (50 mL) and water (50 mL). The organic layer was dried over Na.sub.2SO.sub.4, volatiles were evaporated under reduced pressure to give the N-Boc protected bis(glycyl)-cystamine as a colorless oil. Yield: 1.575 g (88%). 1H NMR (400 MHz, CDCl3) δ 6.97 (s, 1H, NH), 5.53 (s, 1H, NH), 3.81 (d, J=5.8 Hz, 2H, α-CH.sub.2), 3.58 (aq, J=6.3 Hz, 2H, —CH.sub.2—NH—), 2.82 (t, 2H, —CH.sub.2—S—), 1.45 (s, 9H, —CH.sub.3 t-Bu); m/z=467.1 [M+H].sup.+, 489.1 [M+Na].sup.+.

[0137] To a solution of the N-Boc protected bis(glycyl)-cystamine (300 mg, 0.64 mmol) in MeOH (5 mL) was added acetyl chloride (300 μL, 4.20 mmol). After the exothermic reaction had ceased, the mixture was stirred in a sealed flask for 5 h at ambient temperature, then toluene (2 mL) was added and volatiles were evaporated until the product precipitated. The white solid was isolated via suction filtration and washed with n-pentane (2×5 mL). Yield: 146 mg (67%). m.p.=184° C. (decomp.); 1H NMR (400 MHz, D.sub.2O) δ 3.81 (s, 2H, α-CH.sub.2), 3.59 (at, J=6.3 Hz, 2H, —CH.sub.2—NH), 2.88 (at, 2H, —CH.sub.2—S—); m/z=266.9 [M+H]+, 288.9 [M+Na]+.

[0138] Bis(glycyl)-cystamine dihydrochloride is a modification agent that allows to prepare the hyaluronan-glycyl-cysteamine sodium salt (HA-GLYC). This modified hyaluronan forms the crosslinked hyaluronan HA-L-HA, wherein the linker LH.sub.2 is formally derived from the conjugate of the amino acid glycine and cystamine (i.e. LH.sub.2 of formula (IX), wherein R is H).

Example 8 Formulation and Characterisation of a Hydrogel Composition Comprising Crosslinked Hyaluronan-Glycyl-Cysteamine

[0139] A sterile hydrogel composition comprising 17.9 mg/mL crosslinked hyaluronan-glycyl-cysteamine sodium salt (HA-GLYC) and 5 mg/mL unmodified sodium hyaluronate was produced. In brief, 537 mg HA-GLYC (dry weight, MMW 610 kDa, degree of modification 162 μmol/g polymer) and 150 mg sodium hyaluronate (dry weight, MMW 2.4 MDa) were dissolved in 26 g 0.01 M HCl (comprising NaCl) under mechanical stirring at room temperature for about 5 hours. To 19.02 g of this solution, were added 2.115 mL of 100 mM phosphate buffer pH 11.85, which resulted in an adjustment of the pH to about pH 7.4. Then 273 μL of a 0.3% H.sub.2O.sub.2 solution was added and the mixture was homogenized for 15 min at ambient temperature and then left over night for crosslinking. The crosslinked hydrogel was filled into 1 mL glass syringes and sterilized via autoclavation. The sterile hydrogel had a pH of about 7.2.

Example 9 Formulation and Characterisation of a Hydrogel Composition Comprising Crosslinked Hyaluronan-Homocysteine

[0140] A sterile hydrogel composition comprising 17.9 mg/mL crosslinked hyaluronan-homocysteine sodium salt (HA-HCYS) and 5 mg/mL unmodified sodium hyaluronate was produced. In brief, 537 mg HA-HCYS (dry weight, MMW 610 kDa, degree of modification 136 μmol/g polymer) and 150 mg sodium hyaluronate (dry weight, MMW 2.4 MDa) were dissolved in 26 g 0.01 M HCl (comprising NaCl) under mechanical stirring at room temperature for about 5 hours followed by 1 hour resting time to remove air bubbles. To 23.68 g of the solution, 2.63 ml 100 mM phosphate buffer pH 12.04 was added, which resulted in an adjustment of the pH of the solution to about pH 7.2. The mixture was left for 48 h at room temperature for crosslinking, then the crosslinked hydrogel was filled into 1 mL glass syringes and sterilized via autoclavation. The sterile hydrogel had a pH of about 7.0.