Field of the invention

The present invention relates to the field of injectable hydrogel compositions and the use of such compositions in medical and/or cosmetic applications.

Background

One of the most widely used biocompatible polymers for medical use is hyaluronic acid (HA). It is a naturally occurring polysaccharide belonging to the group of glycosaminoglycans (GAGs). Hyaluronic acid and the other GAGs are negatively charged heteropolysaccharide chains which have a capacity to absorb large amounts of water. Hyaluronic acid and products derived from hyaluronic acid are widely used in the biomedical and cosmetic fields, for instance during viscosurgery and as a dermal filler. Water-absorbing gels, or hydrogels, are widely used in the biomedical field. They are generally prepared by chemical crosslinking of polymers to infinite networks. While native hyaluronic acid and certain crosslinked hyaluronic acid products absorb water until they are completely dissolved, crosslinked hyaluronic acid gels typically absorb a certain amount of water until they are saturated, i.e. they have a finite liquid retention capacity, or swelling degree.

Since hyaluronic acid is present with identical chemical structure except for its molecular mass in most living organisms, it gives a minimum of reactions and allows for advanced medical uses. Crosslinking and/or other modifications of the hyaluronic acid molecule is necessary to improve its duration in vivo. Furthermore, such modifications affect the liquid retention capacity of the hyaluronic acid molecule. As a consequence thereof, hyaluronic acid has been the subject of many modification attempts. Hyaluronic salts with divalent cations, e.g. zinc hyaluronate and calcium hyaluronate are known in the field, e.g. through the patent family WO9010020A1 , and zinc hyaluronan is sold under the name Curiosin ^® for promoting physiological wound healing and preventing wound infection.

WO 00/27887 discloses amide cross-linked hyaluronic acids obtainable by reaction with a polyamine. Saturated complexes of the cross-linked hyaluronic acids with divalent metal ions, i.e. 0.5 eqiuv. divalent cations per HA disaccharide, are prepared by swelling lyophilized, cross-linked hyaluronic acid in concentrated solutions of the metal ions. The resulting saturated complexes of the cross-linked hyaluronic acids are suggested to exhibit good stability but are not subjected to heat sterilization.

Description of the invention

An object of the present invention is to provide improved injectable hydrogel compositions, preferably hyaluronic acid based hydrogel compositions comprising divalent cations, preferably Zn ²⁺ , for use as dermal fillers and/or for slow release of the divalent cations in a subject.

An object of the present invention is to provide improved injectable hydrogel compositions, preferably hyaluronic acid based hydrogel compositions comprising divalent cations, preferably Zn ²⁺ , which exhibit decreased degradation of the composition during autoclaving.

An object of the present invention is to provide improved injectable hydrogel compositions, preferably hyaluronic acid based hydrogel compositions comprising divalent cations, preferably Zn ²⁺ , which exhibit increased stability after autoclaving.

An object of the present invention is to provide improved injectable hydrogel compositions, preferably hyaluronic acid based hydrogel compositions comprising divalent cations, preferably Zn ²⁺ , which upon injection of the composition in a subject provide a slow and safe release of the divalent cations to the subject. Hydrogel compositions, such as hyaluronic acid based hydrogel

compositions, for use in injection need to be sterilized before use. Sterilization is generally performed by heat treatment, such as autodaving. The heat treatment generally leads to a reduction of the rigidity or viscosity of the composition.

One problem which has been observed with prior art hyaluronic acid compositions comprising zinc, is that the compositions exhibit increased degradation of the hyaluronic acid during autodaving and decreased stability of the composition after autodaving as compared to identical compositions with sodium as the counterion. Thus, although an initial stabilization (i.e. increased viscosity or G') of the composition is achieved due to electrostatic interaction between the positively charged Zn ²⁺ and negatively charged hyaluronate, this stabilizing is decreased or eliminated due to degradation of the hyaluronate during autodaving. This in turn decreases the longevity of the compositions in vivo.

According to aspects illustrated herein, there is provided a method of preparing a sterilized injectable hydrogel composition, comprising the steps: a) providing an amide crosslinked glycosaminoglycan, and

b) swelling the amide crosslinked glycosaminoglycan in a solution comprising a divalent cation to form a hydrogel composition, and

c) sterilizing the hydrogel composition by autodaving to form a sterilized injectable hydrogel composition.

The term "injectable" means that the composition is provided in a form which is suitable for parenteral injection, e.g. into soft tissue, such as skin, of a subject or patient. An injectable composition should be sterile and free from components that may cause adverse reactions when introduced into soft tissue, such as the skin, of a subject or patient. This implies that no, or only very mild, immune response occurs in the treated individual. That is, no or only very mild undesirable local or systemic effects occur in the treated individual.

According to certain embodiments, the glycosaminoglycan is selected from the group consisting of hyaluronic acid, heparosan, chondroitin and

chondroitin sulfate, and mixtures thereof. According to some embodiments, the glycosaminoglycan is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof. In a preferred embodiment, the glycosaminoglycan is hyaluronic acid.

The divalent cation is preferably selected from the group consisting of Ca ²⁺ , Cu ²⁺ , Mg ²⁺ and Zn ²⁺ . In a preferred embodiment, the divalent cation is Zn ²⁺ .

In some embodiments, the Zn ²⁺ in the solution provided by a Zn-salt selected from the group consisting of ZnC , Zn-gluconate and Zn-citrate.

The present inventors have identified that in a hyaluronic acid gel

composition, Zn ²⁺ in a concentration in the range of 0.01 to 4 mM, such as 0.05 to 4 mM, and preferably in the range of 0.01 to 2 mM, such as 0.05 to 2 mM or 0.1 to 1 mM acts to stabilize the composition, whereas higher concentrations of Zn ²⁺ instead cause degradation of the composition.

Specifically, Zn ²⁺ and other divalent cations in a non-saturating concentration in the range of 0.01 to 4 mM, such as 0.05 to 4 mM, and preferably in the range of 0.01 to 2 mM, such as 0.05 to 2 mM provide a stable composition upon heat treatment. A preferred concentration providing a useful stability to the composition is above 0.01 mM, such as 0.01 to 1 mM of Zn ²⁺ and other divalent cations. Higher concentrations of Zn ²⁺ and other divalent cations, and in particular saturating concentrations of Zn ²⁺ and other divalent cations, instead cause degradation of the composition upon heat treatment. Thus, in some embodiments, the concentration of the divalent cation in the solution is in the range of 0.01 to 4 mM, such as 0.05 to 4 mM, preferably in the range of 0.01 to 2 mM, such as 0.05 to 2 mM or 0.01 to 1 mM. It is preferred that the concentration of the divalent cation in the solution is not saturating the HA component, i.e. less than 0.5 eqiuv. divalent cations per HA disaccharide.

The viscosity and/or elastic modulus G' of the hydrogel composition may be measured according to various methods, well known to the person skilled in the art. Viscosity may for example be measured as the "Zero shear viscosity, rjo" by rotational viscometry using an Ares G2 rheometer (Measuring system cone plate or parallel plates, Gap 1 .00 mm). Other methods of measuring viscosity may also be applicable. The elastic modulus G' may for example be measured using a Ares G2 Reometer (Measure system parallel plates, Gap 1 .00 mm) by performing a strain sweep to find the linear viscoelastic region (LVR) and then measuring the viscoelastic properties within the LVR. Other methods of measuring elastic modulus G' may also be applicable. The composition may be present in an aqueous form, but it may also be present in dried or precipitated form, e.g. in ethanol.

The glycosaminoglycan of the composition is preferably a hyaluronic acid. Unless otherwise provided, the term "hyaluronic acid" encompasses all variants and combinations of variants of hyaluronic acid, hyaluronate or hyaluronan, of various chain lengths and charge states, as well as with various chemical modifications, including crosslinking. That is, the term also encompasses the various hyaluronate salts of hyaluronic acid with various counter ions, such as sodium hyaluronate. Various modifications of the hyaluronic acid are also encompassed by the term, such as oxidation, e.g. oxidation of -CH2OH groups to -CHO and/or -COOH; periodate oxidation of vicinal hydroxyl groups, optionally followed by reduction, e.g. reduction of -CHO to -CH2OH or coupling with amines to form imines followed by reduction to secondary amines; sulphation; deamidation, optionally followed by deamination or amide formation with new acids; esterification; crosslinking; substitutions with various compounds, e.g. using a crosslinking agent or a carbodiimide assisted coupling; including coupling of different molecules, such as proteins, peptides and active drug components, to hyaluronic acid; and deacetylation. Other examples of modifications are isourea, hydrazide, bromocyan, monoepoxide and monosulfone couplings. In some embodiments, the glycosaminoglycan in step a) of the method is provided in the form of a salt with a monovalent cation, e.g. Na ⁺ .

The hyaluronic acid can be obtained from various sources of animal and non- animal origin. Sources of non-animal origin include yeast and preferably bacteria. The molecular weight of a single hyaluronic acid molecule is typically in the range of 0.1 -10 MDa, but other molecular weights are possible.

In certain embodiments the concentration of the glycosaminoglycan is in the range of 1 to 100 mg/ml, such as 5 to 100 mg/ml. In some embodiments the concentration of the glycosaminoglycan is in the range of 2 to 50 mg/ml, such as 5 to 50 mg/ml. In specific embodiments the concentration of the

glycosaminoglycan is in the range of 5 to 30 mg/ml or in the range of 10 to 30 mg/ml. The glycosaminoglycan is covalently crosslinked. The covalently crosslinked glycosaminoglycan may be obtained by covalently crosslinking a

glycosaminoglycan using a bi- or polyfunctional crosslinking agent, or it may be obtained by so called linker free crosslinking where a coupling agent is used to form covalent bonds directly between functional groups already present in the glycosaminoglycan, but where the coupling agent does not form part of the crosslink.

The term "amide crosslinked glycosaminoglycan" as used herein refers either to a glycosaminoglycan crosslinked directly by amide bonds formed between carboxylic and amine functions present on the glycosaminoglycan backbone, or to a glycosaminoglycan crosslinked indirectly by a di- or multiamine crosslinker, whereby amide bonds are formed between carboxylic groups present on the glycosaminoglycan backbone and amines of the crosslinker.

Crosslinking of the glycosaminoglycan can be achieved by modification with a crosslinking agent. In preferred embodiments, crosslinking of the

glycosaminoglycan is achieved by amide coupling of glycosaminoglycan molecules. Amide coupling using a using a di- or multiamine functional crosslinker together with a coupling agent is an attractive route to preparing crosslinked glycosaminoglycan molecules useful for hydrogel products.

Crosslinking can be achieved using a non-carbohydrate based di- or multinucleophilic crosslinker, for example hexamethylenediamine (HMDA), or a carbohydrate based di- or multinucleophilic crosslinker, for example diaminotrehalose (DATH) together with a glycosaminoglycan. Crosslinking can also be achieved using an at least partially deacetylated

glycosaminoglycan, either alone or in combination with a second

glycosaminoglycan, whereby the deacetylated glycosaminoglycan itself acts as the di- or multinucleophilic crosslinker.

Crosslinking of the glycosaminoglycan may for example be achieved in aqueous media using a crosslinker comprising at least two nucleophilic functional groups, for example amine groups, capable of forming covalent bonds directly with carboxylic acid groups of GAG molecules by a reaction involving the use of a coupling agent. The crosslinker comprising at least two nucleophilic functional groups may for example be a non-carbohydrate based di- or multinucleophilic crosslinker or a carbohydrate based di- or multinucleophilic crosslinker.

Carbohydrate based di- or multinucleophilic crosslinkers are preferred, since they provide a hydrogel product based entirely on carbohydrate type structures or derivatives thereof, which minimizes the disturbance of the crosslinking on the native properties of the glycosaminoglycans. The crosslinker itself can also contribute to maintained or increased properties of the hydrogel, for example when crosslinking with a structure that correlates to hyaluronic acid or when crosslinking with a structure with high water retention properties.

The carbohydrate based di- or multinucleophilic crosslinker may for example be selected from the group consisting of di- or multinucleophilic functional di-, tri-, tetra-, oligosaccharides, and polysaccharides. In a preferred embodiment, the di- or multinucleophilic crosslinker is an at least partially deacetylated glycosaminoglycan, i.e. an acetylated

glycosaminoglycan which has been at least partially deacetylated to provide a glycosaminoglycan having free amine groups. An at least partially

deacetylated glycosaminoglycan, can be crosslinked either alone or in combination with a second glycosaminoglycan, whereby the deacetylated glycosaminoglycan itself acts as the di- or multinucleophilic crosslinker.

The coupling agent may for example be selected from the group consisting of triazine-based coupling agents, carbodiimide coupling agents, imidazolium- derived coupling reagents, Oxyma and COMU. A preferred coupling agent is a triazine-based coupling agent, including the group consisting of 4-(4,6- dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and 2- chloro-4,6-dimethoxy-1 ,3,5-triazine (CDMT), preferably DMTMM. Another preferred coupling agent is a carbodiimide coupling agent, preferably N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) combined with N- hydroxysuccinimide (NHS).

In some embodiments, the covalently crosslinked GAG is obtained by preparing a mixture of a GAG molecule, such as hyaluronic acid together with a crosslinker agent, such as diamino trehalose, DATH, (0.001 - 10 molar equivalents of amine towards carboxylic acid groups, or preferably 0.001 - 1 molar equivalents) and a coupling agent such as DMTMM (0.01 - 10 molar equivalents to carboxylic acid groups, or preferably 0.05 - 1 molar

equivalents). Incubating the mixture at 5 - 50 °C, preferably 10 - 40 °C or even more preferred 20 - 35 °C, during 2 - 120 hours, preferably 4 - 48 hours, followed by alkaline treatment, neutralization, precipitation, washing and dried under vacuum, yields a crosslinked polysaccharide as a solid. The precipitate was swelled in phosphate buffer containing NaCI to form a hydrogel, the hydrogel is preferably micronized to hydrogel particles in the size of 0.01 - 5 mm, preferably 0.1 - 1 mm. Diaminotrehalose (DATH) can be synthesized as described in "Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties"; Keisuke Kurita, ^* Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, 7544-7549.

In other embodiments, the crosslinked GAG is obtained by:

1 ) crosslinking at least partially deacetylated GAG to partially deacetylated GAG in the presence ot a coupling agent using free amine and carboxylic acid groups present in the at least partially deacetylated GAGs; or

2) crosslinking at least partially deacetylated GAG to a non-deacetylated GAG in the presence ot a coupling agent using free amine groups present in the at least partially deacetylated GAG and carboxylic acid groups present in the GAG. The sterilized injectable composition formed using the inventive method is a hydrogel. That is, it can be regarded as a water-insoluble, but substantially dilute crosslinked system of glycosaminoglycan molecules when subjected to a liquid, typically an aqueous liquid. The sterilized injectable hydrogel composition contains mostly liquid by weight and can e.g. contain 90-99.9% water, but it behaves like a solid due to a three-dimensional crosslinked hyaluronic acid network within the liquid. Due to its significant liquid content, the gel is structurally flexible and similar to natural tissue, which makes it very useful as a scaffold in tissue engineering and for tissue augmentation. The hydrogel composition is preferably biocompatible. This implies that no, or only very mild, immune response occurs in the treated individual. That is, no or only very mild undesirable local or systemic effects occur in the treated individual. As mentioned, crosslinking of a glycosaminoglycan such as hyaluronic acid, to form the crosslinked glycosaminoglycan, may for example be achieved by modification with a crosslinking agent. The glycosaminoglycan concentration and the extent of crosslinking affects the mechanical properties, e.g. the elastic modulus G', and stability properties of the hydrogel. Crosslinked glycosaminoglycan gels are often characterized in terms of "degree of modification".

The degree of modification of hyaluronic acid gels generally range between 0.1 and 15 mole%.

In some embodiments the hyaluronic acid gel has a degree of modification of 12 mole% or less, such as 12 mole% or less, such as 10 mole% or less, for example in the range of 0.1 to 12 mole%, such as in the range of 0.2 to 10 mole%, such as in the range of 0.3 to 8 mole%.

In some embodiments the hyaluronic acid gel has a degree of modification of 12 mole% or less, such as 12 mole% or less, such as 10 mole% or less, for example in the range of 2 to 12 mole%, such as in the range of 3 to 10 mole%, such as in the range of 4 to 8 mole%.

In some embodiments, the hyaluronic acid gel has a degree of modification of 2 mole% or less, such as 1 .5 mole% or less, such as 1 .25 mole% or less, for example in the range of 0.1 to 2 mole%, such as in the range of 0.2 to 1 .5 mole%, such as in the range of 0.3 to 1 .25 mole%.

The degree of modification (mole%) describes the amount of crosslinking agent(s) that is bound to glycosaminoglycan, i.e. molar amount of bound crosslinking agent(s) relative to the total molar amount of repeating

glycosaminoglycan disaccharide units. The degree of modification reflects to what degree the glycosaminoglycan has been chemically modified by the crosslinking agent. Reaction conditions for crosslinking and suitable analytical techniques for determining the degree of modification are all well known to the person skilled in the art, who easily can adjust these and other relevant factors and thereby provide suitable conditions to obtain a degree of modification in the range of 0.1 -15% and verify the resulting product characteristics with respect to the degree of modification. A BDDE (1 ,4- butandiol diglycidylether) crosslinked hyaluronic acid gel may for example be prepared according to the method described in Examples 1 and 2 of published international patent application WO 9704012.

In a preferred embodiment the crosslinked glycosaminoglycan of the composition is present in the form of a crosslinked hyaluronic acid crosslinked by a crosslinking agent, wherein the concentration of said hyaluronic acid is in the range of 2 to 50 mg/ml and the degree of modification with said

crosslinking agent is in the range of 0.1 to 2 mole%. In some embodiments, the solution further comprises a buffering compound. The purpose of the buffering compound is to maintain the pH of the

composition at a suitable value, typically in the range of 5 to 8, preferably in the range of 6 to 7.5. Thus, the pH value of the solution is in the range of 5 to 8, preferably in the range of 6 to 7.5.

The injectable hydrogel composition may further comprise a therapeutically relevant concentration of a local anesthetic. A local anesthetic is a drug that causes reversible local anesthesia and a loss of nociception. When it is used on specific nerve pathways (nerve block), effects such as analgesia (loss of pain sensation) and paralysis (loss of muscle power) can be achieved. The local anesthetic may be added to the composition to reduce pain or discomfort experienced by the patient due to the injection procedure.

According to certain embodiments the local anesthetic is selected from the group consisting of amide and ester type local anesthetics, for example bupivacaine, butanilicaine, carticaine, cinchocaine (dibucaine), clibucaine, ethyl parapiperidinoacetylaminobenzoate, etidocaine, lignocaine (lidocaine), mepivacaine, oxethazaine, prilocaine, ropivacaine, tolycaine, trimecaine, vadocaine, articaine, levobupivacaine, amylocaine, cocaine, propanocaine, clormecaine, cyclomethycaine, proxymetacaine, amethocaine (tetracaine), benzocaine, butacaine, butoxycaine, butyl aminobenzoate, chloroprocaine, dimethocaine (larocaine), oxybuprocaine, piperocaine, parethoxycaine, procaine (novocaine), propoxycaine, tricaine or a combination thereof.

According to some preferred embodiments the local anesthetic is lidocaine.

According to specific embodiments the local anesthetic is lidocaine. Lidocaine is a well-known substance, which has been used extensively as a local anesthetic in injectable formulations, such as hyaluronic acid compositions. The concentration of the amide or ester local anesthetic may be selected by the skilled person within the therapeutically relevant concentration ranges of each specific local anesthetic or a combination thereof.

In some embodiments the concentration of said local anesthetic is in the range of 0.1 to 30 mg/ml. In certain embodiments the concentration of said local anesthetic is in the range of 0.5 to 10 mg/ml. When lidocaine is used as the local anesthetic, the lidocaine may preferably be present in a concentration in the range of 1 to 5 mg/ml, more preferably in the range of 2 to 4 mg/ml, such as in a concentration of about 3 mg/ml. The method described herein involves sterilization of the composition by autoclaving, i.e sterilization using saturated steam. The autoclaving may be performed at an Fo-value > 4. The autoclaving may preferably be performed at an Fo-value in the range of 10 to 50, such as in the range of 20 to 30.The Fo value of a saturated steam sterilisation process is the lethality expressed in terms of the equivalent time in minutes at a temperature of 121 °C delivered by the process to the product in its final container with reference to

microorganisms posessing a Z-value of 10.

In a preferred embodiment, the glycosaminoglycan is hyaluronic acid at a concentration in the range of 2-50 mg/ml, such as 5-50 mg/ml, the divalent cation is Zn ²⁺ at a concentration in the range of 0.01 to 4 mM, such as 0.05 to 4 mM, preferably in the range of 0.01 to 2 mM, such as 0.05 to 2 mM or 0.01 to 1 mM, and sterilization is performed by autoclaving at an Fo-value > 4.

Experimental data provided by the inventors show that the sterilized injectable hydrogel compositions formed according to the inventive method exhibit increased stability compared to identical compositions without the divalent cation.

The term stability, as used herein, is used to denote the ability of the sterilized injectable hydrogel composition to resist degradation during storage and handling prior to use. It is known that the addition of constituents to a glycosaminoglycan, such as hyaluronic acid or hyaluronic acid gel, may affect the stability of said glycosaminoglycan. Stability of a hydrogel composition comprising a glycosaminoglycan can be determined by a range of different methods. Methods for determining stability include, but are not limited to, assessing homogeneity, color, clarity, pH, gel content and rheological properties of the composition. Stability of a hydrogel composition comprising a glycosaminoglycan is often determined by observing or measuring one or more of said parameters over time. Stability may for example be determined by measuring the viscosity and/or elastic modulus G' of the composition over time. Viscosity may for example be measured as the "Zero shear viscosity, rjo" by rotational viscometry using a Bohlin VOR rheometer (Measuring system C14 or PP 30, Gap 1 .00 mm). Other methods of measuring viscosity may also be applicable. The elastic modulus G' may for example be measured using a Bohlin VOR Reometer (Measure system PP 30, Gap 1 .00 mm) by performing a strain sweep to find the linear viscoelastic region (LVR) and then measuring the viscoelastic properties within the LVR. Other methods of measuring elastic modulus G' may also be applicable.

According to aspects illustrated herein, there is provided a sterilized injectable hydrogel composition obtainable by the method described above.

According to aspects illustrated herein, there is provided a sterilized injectable hydrogel composition comprising

i) an amide crosslinked glycosaminoglycan, and

ii) a divalent cation.

The crosslinked glycosaminoglycan the composition may be further defined as set out herein above with reference to the inventive method of preparing a sterilized injectable hydrogel composition.

In certain embodiments, the glycosaminoglycan is selected from the group consisting of hyaluronic acid, heparosan, chondroitin and chondroitin sulfate, and mixtures thereof. In some embodiments, the glycosaminoglycan is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof. In a preferred embodiment, the glycosaminoglycan is a hyaluronic acid. The glycosaminoglycan is covalently crosslinked. The covalently crosslinked glycosaminoglycan may be obtained by covalently crosslinking a

glycosaminoglycan using a bi- or polyfunctional crosslinking agent, or it may be obtained by so called linker free crosslinking where a coupling agent is used to form covalent bonds directly between functional groups already present in the glycosaminoglycan, but where the coupling agent does not form part of the crosslink.

In some embodiments, crosslinking of the glycosaminoglycan is achieved by modification with a crosslinking agent. In preferred embodiments, crosslinking of the glycosaminoglycan is achieved by amide coupling of

glycosaminoglycan molecules using a di- or multiamine functional crosslinker together with a coupling agent.

In some embodiments, the covalently crosslinked glycosaminoglycan has a degree of modification of 2 mole% or less, such as 1 .5 mole% or less, such as 1 .25 mole% or less.

In some embodiments, the covalently crosslinked glycosaminoglycan has a degree of modification in the range of 0.1 to 2 mole%, such as in the range of 0.2 to 1 .5 mole%, such as in the range of 0.3 to 1 .25 mole%. In some embodiments, the concentration of the glycosaminoglycan in the composition is in the range of 1 to 100 mg/ml, such as 5 to 100 mg/ml.

In some embodiments, the concentration of the glycosaminoglycan in the composition is in the range of 2 to 50 mg/ml, such as 5 to 50 mg/ml.

In some embodiments, the concentration of the glycosaminoglycan in the composition is in the range of 5 to 30 mg/ml, such as 10 to 30 mg/ml.

In some embodiments, the divalent cation is selected from the group consisting of Ca ²⁺ , Cu ²⁺ , Mg ²⁺ and Zn ²⁺ . In a preferred embodiment, the divalent cation is Zn ²⁺ . The concentration of the divalent cation in the composition is preferably in the range of 0.01 to 4 mM, such as 0.05 to 4 mM, more preferably in the range of 0.01 to 2 mM, such as 0.05 to 2 mM or 0.01 to 1 mM. It is preferred that the concentration of the divalent cation in the composition is not saturating the HA component, i.e. less than 0.5 eqiuv. divalent cations per HA disaccharide.

In some embodiments, the composition further comprises a buffering compound. The purpose of the buffering compound is to maintain the pH of the composition at a suitable value, typically in the range of 5 to 8, preferably in the range of 6 to 7.5. Thus, the pH value of the solution is in the range of 5 to 8, preferably in the range of 6 to 7.5.

In some embodiments, the composition further comprises a therapeutically relevant concentration of a local anesthetic. The local anesthetic is preferably lidocaine.

The compositions described herein have preferably been subjected to sterilization by autoclaving, i.e sterilization using saturated steam. The autoclaving, may be performed at an Fo-value > 4. The Fo value of a saturated steam sterilisation process is the lethality expressed in terms of the equivalent time in minutes at a temperature of 121 °C delivered by the process to the product in its final container with reference to microorganisms posessing a Z-value of 10. The inventive composition preferably exhibits increased stability compared to an identical composition without the divalent cation.

The components, features, effects and advantages of the composition may be further defined as described above with reference to the method of preparing the sterilized injectable hydrogel composition.

The sterilized injectable hydrogel compositions according to the invention may be provided in the form of a pre-filled syringe, i.e. a syringe that is pre- filled with the injectable hydrogel composition and autoclaved.

The sterilized injectable hydrogel compositions as described herein may advantageously be used for the transport or administration and slow or controlled release of various parmaceutical or cosmetic substances.

The sterilized injectable hydrogel compositions described herein may be employed in medical as well as non-medical, e.g. purely cosmetic, procedures by injection of the composition into soft tissues of a patient or subject. The compositions have been found useful in, e.g., soft tissue augmentation, for example filling of wrinkles, by hyaluronic acid gel injection. The compositions have also been found useful in a cosmetic treatment, referred to herein as skin revitalization, whereby small quantities of the hyaluronic acid composition are injected into the dermis at a number of injection sites distributed over an area of the skin to be treated, resulting in improved skin tone and skin elasticity. Skin revitalization is a simple procedure and health risks associated with the procedure are very low. The composition is useful, for example in the treatment of various

dermatological conditions. Particularly, there is provided an injectable hyaluronic acid composition as described above for use in a dermatological treatment selected from the group consisting of wound healing, treatment of dry skin conditions or sun-damaged skin, treatment of hyper pigmentation disorders, treatment and prevention of hair loss, and treatment of conditions that have inflammation as a component of the disease process, such as psoriasis and asteototic eczema. In other words, there is provided an injectable hyaluronic acid composition as described above for use in the manufacture of a medicament for use in a dermatological treatment selected from the group consisting of wound healing, treatment of dry skin conditions or sun-damaged skin, treatment of hyper pigmentation disorders, treatment and prevention of hair loss, and treatment of conditions that have inflammation as a component of the disease process, such as psoriasis and asteototic eczema.

According to other aspects illustrated herein, there is provided the use of an injectable hyaluronic acid composition as described above for cosmetic, nonmedical, treatment of a subject by injection of the composition into the skin of the subject. A purpose of the cosmetic, non-medical, treatment may be for improving the appearance of the skin, preventing and/or treating hair loss, filling wrinkles or contouring the face or body of a subject. The cosmetic, nonmedical, use does not involve treatment of any form of disease or medical condition. Examples of improving the appearance of the skin include, but are not limited to, treatment of sun-damaged or aged skin, skin revitalization, skin whitening and treatment of hyper pigmentation disorders such as senile freckles, melasma and ephelides.

According to aspects illustrated herein, there is provided the sterilized injectable hydrogel composition as described herein for use as a medicament.

According to aspects illustrated herein, there is provided the sterilized injectable hydrogel composition as described herein for use in the treatment of a condition susceptible to treatment with the divalent cation.

In some embodiments, the sterilized injectable hydrogel composition is injected into the skin of a subject. The composition preferably provides a slow release of the divalent cation in the skin of the subject upon injection. The release of the divaalent cation in the skin of the subject should be well below the toxic dose of the divalent cation. As an example, for a compostion with Zn ²⁺ the release in the skin of the subject upon injection is preferably below 0.1 mmol/day. According to aspects illustrated herein, there is provided the manufacture of a medicament for treatment of a condition susceptible to treatment with the divalent cation. According to aspects illustrated herein, there is provided a method of treating a patient suffering from a condition susceptible to treatment with a divalent cation by administering to the patient a therapeutically effective amount of the sterilized injectable hydrogel composition according as described herein. According to aspects illustrated herein, there is provided a method of cosmetically treating skin, which comprises administering to the skin a sterilized injectable hydrogel composition as described herein.

The inventive methods and compositions are described herein particularly with reference to a preferred embodiment, wherein the glycosaminoglycan is hyaluronic acid and the divalent cation is Zn ²⁺ . However, the invention is not restricted to this particular embodiment. Other glycosaminoglycans and other divalent cations are also contemplated within the scope of the present. The person skilled in the art realizes that the present invention by no means is limited to the preferred embodiments described herein. On the contrary, many modifications and variations are possible within the scope of the appended claims. Additionally, variations to the disclosed embodiments can be understood and effected by the skilled person in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Itemized listing of embodiments

The following is a non-limiting and itemized listing of embodiments of the present disclosure, presented for the purpose of describing various features and combinations provided by the invention in certain of its aspects.

1 . A method of preparing a sterilized injectable hydrogel composition, comprising the steps:

a) providing an amide crosslinked glycosaminoglycan, and

b) swelling the amide crosslinked glycosaminoglycan in a solution comprising a divalent cation to form a hydrogel composition, and

c) sterilizing the hydrogel composition by autoclaving to form a sterilized injectable hydrogel composition.

2. The method according to item 1 , wherein the glycosaminoglycan is selected from the group consisting of hyaluronic acid, heparosan, chondroitin and chondroitin sulfate, and mixtures thereof, such as wherein the

glycosaminoglycan is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof. 3. The method according to item 2, wherein the glycosaminoglycan is a hyaluronic acid.

4. The method according to any one of the preceding items, wherein the glycosaminoglycan in step a) is provided in the form of a salt with a monovalent cation, e.g. Na ⁺ .

5. The method according to any one of the preceding items, wherein the amide crosslinked glycosaminoglycan is provided by covalently crosslinking a glycosaminoglycan using a bi- or polyfunctional crosslinking agent.

6. The method according to any one of the preceding items, wherein the amide crosslinked glycosaminoglycan is provided by amide coupling of glycosaminoglycan molecules using a di- or multinucleophilic functional crosslinker together with a coupling agent.

7. The method according to item 6, wherein the di- or multinucleophilic functional crosslinker is a carbohydrate based di- or multinucleophilic crosslinker.

8. The method according to item 6, wherein the di- or multinucleophilic crosslinker comprises a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides.

9. The method according to item 6, wherein the di- or multinucleophilic crosslinker is an at least partially deacetylated glycosaminoglycan. 10. The method according to any one of the preceding items, wherein the covalently crosslinked glycosaminoglycan has a degree of modification of 2 mole% or less, such as 1 .5 mole% or less, such as 1 .25 mole% or less and/or wherein the covalently crosslinked glycosaminoglycan has a degree of modification in the range of 0.1 to 2 mole%, such as in the range of 0.2 to 1 .5 mole%, such as in the range of 0.3 to 1 .25 mole%.

1 1 . The method according to any one of the preceding items, wherein the concentration of said glycosaminoglycan is in the range of 1 to 100 mg/ml, such as 5 to 100 mg/ml.

12. The method according to item 1 1 , wherein the concentration of said glycosaminoglycan is in the range of 2 to 50 mg/ml, such as 5 to 50 mg/ml.

13. The method according to item 12, wherein the concentration of said glycosaminoglycan is in the range of 5 to 30 mg/ml, such as 10 to 30 mg/ml. 14. The method according to any one of the preceding items, wherein the divalent cation is selected from the group consisting of Ca ²⁺ , Cu ²⁺ , Mg ²⁺ and Zn ²⁺ , and preferably wherein the divalent cation is Zn ²⁺ . 15. The method according to item 14, wherein the Zn ²⁺ in the solution is provided by a Zn-salt selected from the group consisting of ZnC , Zn- gluconate and Zn-citrate.

16. The method according to any one of the preceding items, wherein the concentration of the divalent cation in the solution is in the range of 0.01 to 4 mM or 0.05 to 4 mM, preferably in the range of 0.01 to 2 mM or 0.05 to 2 mM; or in the range of 0.01 to 1 mM.

17. The method according to any one of the preceding items, wherein the solution is comprising less than 0.5 eqiuv. of the divalent cation per HA disaccharide.

18. The method according to any one of the preceding items, wherein the solution further comprises a buffering compound.

19. The method according to any one of the preceding items, wherein the pH value of the solution is in the range of 5 to 8, preferably in the range of 6 to 7.5. 20. The method according to any one of the preceding items, wherein the solution further comprises a therapeutically relevant concentration of a local anesthetic.

21 . The method according to item 20, wherein the local anesthetic is lidocaine. 22. The method according to any one of the preceding items, wherein the autoclaving is performed at an Fo-value > 4.

23. The method according to any one of the preceding items, wherein the formed sterilized injectable hydrogel composition exhibits increased stability compared to an identical composition without the divalent cation.

24. A sterilized injectable hydrogel composition obtainable by the method according to any one of items 1 -23.

25. A sterilized injectable hydrogel composition comprising

i) an amide crosslinked glycosaminoglycan, and

ii) a divalent cation. 26. The composition according to item 25, wherein the glycosaminoglycan is selected from the group consisting of hyaluronic acid, heparosan, chondroitin and chondroitin sulfate, and mixtures thereof, such as wherein the

glycosaminoglycan is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof.

27. The composition according to item 26, wherein the glycosaminoglycan is a hyaluronic acid.

28. The composition according to any one of items 25-27, wherein the covalently crosslinked glycosaminoglycan has a degree of modification of 2 mole% or less, such as 1 .5 mole% or less, such as 1 .25 mole% or less.

29. The composition according to any one of items 25-27, wherein the covalently crosslinked glycosaminoglycan has a degree of modification in the range of 0.1 to 2 mole%, such as in the range of 0.2 to 1 .5 mole%, such as in the range of 0.3 to 1 .25 mole%. 30. The composition according to any one of items 25-29, wherein the concentration of said glycosaminoglycan is in the range of 1 to 100 mg/ml, such as 5 to 100 mg/ml. 31 . The composition according to item 30, wherein the concentration of said glycosaminoglycan is in the range of 2 to 50 mg/ml, such as 5 to 50 mg/ml.

32. The composition according to item 31 , wherein the concentration of said glycosaminoglycan is in the range of5 to 30 mg/ml, such as 10 to 30 mg/ml.

33. The composition according to any one of items 25-32, wherein the divalent cation is selected from the group consisting of Ca ²⁺ , Cu ²⁺ , Mg ²⁺ and Zn ²⁺ , and preferably wherein the divalent cation is Zn ²⁺ . 34. The composition according to any one of items 25-33, wherein the concentration of the divalent cation in the composition is in the range of 0.01 to 4 mM or 0.05 to 4 mM, preferably in the range of 0.01 to 2 mM or 0.05 to 2 mM; or in the range of 0.01 to 1 mM. 35. The composition according to any one of items 25-34, comprising less than 0.5 eqiuv. of the divalent cation per HA disaccharide.

36. The composition according to any one of items 25-35, further comprising a buffering compound.

37. The composition according to any one of items 25-36, wherein the pH value of the composition is in the range of 5 to 8, preferably in the range of 6 to 7.5. 38. The composition according to any one of items 25-37, further comprising a therapeutically relevant concentration of a local anesthetic. 39. The composition according to item 38, wherein the local anesthetic is lidocaine.

40. The composition according to any one of items 25-39, wherein the composition has been subjected to sterilization by autoclaving.

41 . The composition according to any one of items 25-40, wherein the composition has been subjected to sterilization by autoclaving at an Fo-value > 4.

42. The composition according to any one of items 25-41 , wherein the composition exhibits increased stability compared to an identical composition without the divalent cation. 43. A sterilized injectable hydrogel composition according to any one of items 24-42 for use as a medicament.

44. A sterilized injectable hydrogel composition according to any one of items 24-42 for use in the treatment of a condition susceptible to treatment with the divalent cation.

45. The sterilized injectable hydrogel composition for use according to item 43 or 44, wherein the composition is injected into the skin of a subject. 46. The sterilized injectable hydrogel composition for use according to item 45, wherein the composition provides a slow release of the divalent cation in the skin of the subject upon injection.

47. The sterilized injectable hydrogel composition for use according to item 45 or 46, wherein the release of the divalent cation in the skin of the subject upon injection is below 0.1 mmol/day. 48. Use of a sterilized injectable hydrogel composition according to any one of items 24-42 for the manufacture of a medicament for treatment of a condition susceptible to treatment with the divalent cation. 49. A method of treating a patient suffering from a condition susceptible to treatment with a divalent cation by administering to the patient a

therapeutically effective amount of the sterilized injectable hydrogel composition according to any one of items 24-42. 50. A method of cosmetically treating skin, which comprises administering to the skin a sterilized injectable hydrogel composition according to any one of items 24-42.

Brief description of the drawings

The invention is further illustrated by figures 1 -9. Figures 1 -9 represent exemplary embodiments only.

Figure 1 a is a diagram showing the stabilizing effect of Zn on a hyaluronic acid gel with a degree of modification of 1 mole%.

Figure 1 b is a diagram showing the stabilizing effect of Zn on a hyaluronic acid gel with a degree of modification of 5 mole%. Figure 2a and 2b are diagrams showing that the stabilizing effect of Zn is also present when lidocaine is added to the composition.

Figure 3 is a diagram showing that the stabilizing effect of Zn is also present when tris is used as the buffer.

Figure 4 is a diagram showing that different Zn salts have a similar stabilizing effect. Figure 5a is a diagram showing the concentration dependence of the stabilizing effect of Zn. Figure 5b is a diagrams showing the effect of Zn on the storage stability of the composition.

Figure 6 is a diagram showing the fraction of Zn released from the gel containing 1 mM zinc chloride as a function of time.

Figure 7 is a diagram showing corrected swelling degree (SwCC) of gels crosslinked with DATH in different buffers.

Figure 8 is a diagram showing the gel part (GelP) of gels crosslinked with DATH in different buffers.

Figure 9 is a diagram showing how much gel part of gels crosslinked with DATH in different buffers that is left after day 14 at 60 °C compared to at day 0.

Examples

Without desiring to be limited thereto, the present invention will in the following be illustrated by way of examples. DEFINITIONS

Mw - The mass average molecular mass.

SwF - Swelling factor analysis in saline, volume for a 1 g gel that has swelled to its maximum (mL/g).

SwC - Swelling capacity in saline, total liquid uptake per gram PS (mL/g). SwCC - Corrected swelling degree, total liquid uptake of one gram PS, corrected for GelP (mL/g).

SwF

SwCC

Ceil' * [PS]

[PS] - The polysaccharide concentration (mg/g), measured with LC-SEC-UV or NIR.

GelP - Gel part is a description of the percentage of polysaccharide that is a part of the gel network. A number of 90% means that only 10% of

polysaccharide is not a part of the network. The amount of free

polysaccharide in the gel was measured with LC-SEC-UV.

CrDamide - Degree of amide cross-linking (%) was analyzed with SEC-MS and defined as:

^L HA disaccharides

Y(Area amide crosslinked HA fraqments)

Q _r D = — '- _ - * (100 amide ∑( j _rea amide crosslinked HA fragments + Area HA amine fragments)

- DoA)

DoA - Degree of Acetylation. The degree of acetylation (DoA) is the molar ratio of acetyl groups compared to hyaluronic acid disaccharides. DoA can be calculated from NMR spectra by comparing the integral of the acetyl signal of the hyaluronan disaccharide residues to the integral of the C2-H signal of the deacetylated glucosamine residues according to the equation.

Integral acetylgroup

DoA (%) ( jnteqral acetylqroup _τ

^ — + Integral , C„2„-,H, ) * l^O CrR - Effective crosslinking ratio was analyzed with LC-SEC-MS and defined as: mol crosslinked crosslinker with amide bonds

mol linked crosslinker with amide bonds

A CrR of 1 .0 means that all of the crosslinker has crosslinked.

GENERAL PROCEDURES

A. General Procedure for manufacture of diaminotrehalose (DATH)

Diaminotrehalose (DATH) was synthesized as described in "Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties"; Keisuke Kurita ^* Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, 7544-7549.

B. General procedure for alkaline hydrolysis

The crosslinked material was swelled in 0.25 M NaOH (1 g material : 9 g 0.25 M NaOH resulting in pH 13) for at least 1 h at room temperature. The gel was neutralized with 1 .2 M HCI to pH 7 and then precipitated with ethanol. The resulting precipitate was washed with 100 mM NaCI in ethanol (70% w/w) to remove excess reagents and then with ethanol (70% w/w) to remove salts and finally with ethanol to remove water.

C. General procedure for heat hydrolysis

The crosslinked material was swelled in 0.7% NaCI 8 mM phosphate buffer pH 7.4 at room temperature. The pH was adjusted to 7.2-7.5 if needed. The gel was left at 70 °C for 20-24 h and then precipitated with ethanol. The resulting precipitate was washed with 100 mM NaCI in ethanol (70% w/w) to remove excess reagents and then with ethanol (70% w/w) to remove salts and finally with ethanol to remove water. Example 1 - Hyaluronic acid gel with MoD 1 %

A BDDE (1 ,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole% and a hyaluronic acid content of 20 mg/mL was prepared by first transferring hyaluronic acid (Mw 1 MDa) to a plastic jar. A solution of 1 % NaOH and 0.3% BDDE was added and the mixture was homogenized. The jar was transferred to an incubator to perform the crosslinking step. The resulting gel was divided in two parts. The first part was allowed to swell to a HA concentration of about 20 mg/mL in a buffer solution containing sodium phosphate, HCI and NaCI, at a pH about 7.4 (Reference). The second part was allowed to swell to a HA concentration of about 20 mg/mL in a buffer solution containing sodium phosphate, HCI, NaCI and 1 mM ZnC . The pH of the formulations was adjusted to 7.4 and the

formulations were filled in glass syringes and autoclaved in a Getinge 6610 ERC-1 autoclave (Fo ~19).

The rheological properties of the formulations were analyzed using an Ahres G2 reometer (measure system PP 40, gap 1 .00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the

viscoelastic properties were measured within the LVR.

Fig. 1 a shows the difference in G' between the HA gel sample swelled in buffer with ZnCb and the Reference sample swelled in buffer only.

A stabilizing effect on the gel can be seen when 1 mM ZnCb is used in the formulation.

Example 2 - Hyaluronic acid gel with MoD 5%

A BDDE (1 ,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a of modification of 5 mole% was prepared by mixing 3 g of hyaluronate (Mw 2 MDa) with a mixture of BDDE (1 ,4-butandiol diglycidylether) in 1 % NaOH. The BDDE/NaOH solution was prepared by diluting BDDE in 1 % NaOH to get to give a basic BDDE solution diluted to 1/100. 19 g of the prepared BDDE solution was then added to hyaluronate and homogenized. The mixture was placed in a water bath at 50 °C for 2 h.

The resulting crosslinked hyaluronic acid was swelled in phosphate buffer saline (1 mM PBS) and pH was adjusted to 7. The gel was then purified by dialysis (MWCO 15000) for 2 days. The gel was then divided in two parts, to one of the gels 1 M ZnC was added to get a concentration of 1 mM ZnC in the final gel, PBS buffer was added to get a final hyaluronic acid

concentration of 20 mg/mL. To the second part of the gel PBS buffer was added to get a final hyaluronic acid concentration of 20 mg/mL. The pH of the formulations was adjusted to 7.4 and the formulations were filled in glass syringes and autoclaved in a Getinge 6610 ERC-1 autoclave (Fo -19).

The rheological properties of the formulations were analyzed using an Ahres G2 reometer (measure system PP 40, gap 1 .00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the

viscoelastic properties were measured within the LVR.

Fig. 1 b shows the difference in G' between the HA gel sample swelled in buffer with ZnC and the Reference sample swelled in buffer only.

A stabilizing effect on the gel can be seen when 1 mM ZnC is used in the formulation. The stabilizing effect on the gel from the addition of ZnC is thus present both in the case of a gel with 1 % and 5% degree of modification. Example 3 - Hyaluronic acid gels with different ZnC concentrations

A BDDE (1 ,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole% and a hyaluronic acid content of 20 mg/mL was prepared according to Example 1 , with the difference that the gel was divided into 8 parts after the crosslinking step. The gels parts were swelled to about 20 mg/mL with PBS buffer containing 0, 0.01 , 0.05, 0.1 , 0.5, 1 , 2, 4 and 8 mM ZnC , respectively. The pH of the formulations was adjusted to 7.4 and the formulations were filled in glass syringes and autoclaved in a Getinge 6610 ERC-1 autoclave (F ₀ ~19).

Figure 5a shows the effect on the rheological properties following autoclaving of the gel at different concentrations of ZnCb.

A stability study was also performed on the gels. Autoclaved syringes were put in an incubator for 1 and 2 weeks and the rheological properties were then analyzed.

Figure 5b shows the rheological properties of the gel at different

concentrations of ZnCb as a function of time when the gels is subjected to 60 °C for 1 and 2 weeks. A stabilizing effect in the gel was seen with 0.01 - 2 mM ZnCb and 0.05 - 2 mM ZnC while degradation of the gel was observed with concentration at 4 mM ZnCb and in particular above 4 mM ZnCb. A preferred concentration range providing a useful stability to the composition is 0.01 to 1 mM

ZnCb.The degradation rate of the gel was higher when a higher concentration of ZnCb was used in heat sterilization.

Example 4 - Hyaluronic acid gel with different Zn salts

A BDDE (1 ,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole% and a hyaluronic acid content of 20 mg/mL was prepared according to Example 1 , with the difference that the gel was divided into 4 parts after the crosslinking step. As shown in Table 1 , one part was swelled in PBS buffer (1 mM phosphate), one part was swelled in PBS buffer with 1 mM ZnCb, one part was swelled in PBS buffer with 1 mM Zn- gluconate and one part was swelled with PBS buffer with 1 mM Zn-citrate. All gels were swelled to a HA concentration of about 20 mg/mL. The pH of the formulations was adjusted to 7.4 and the formulations were filled in glass syringes and autoclaved in a Getinge 6610 ERC-1 autoclave (Fo -19). The Theological properties of the formulations were analyzed using an Ahres G2 reometer (measure system PP 40, gap 1 .00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the

viscoelastic properties were measured within the LVR.

Table 1 .

Fig 4. shows the difference in G' between an the samples swelled in PBS with 1 mM ZnC , 1 mM Zn-gluconate and Zn-citrate and the reference sample swelled in PBS only. A stabilizing effect can be seen when Zn is added either as ZnCb, Zn-gluconate or Zn-citrate.

Example 5 - Hyaluronic acid gel with ZnCb and different buffers

A BDDE (1 ,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole% and a hyaluronic acid content of 20 mg/mL was prepared and analyzed according to Example 1 , with the difference that Tris buffer (50 mM) in saline was used instead of PBS during the swelling of the gel.

As shown in Fig. 3, the stabilizing effect of 1 mM ZnCb after autoclaving is present also when Tris buffer (50 mM) in saline is used in the formulation.

Example 6 - HA gel with Zn and local anesthetics

A BDDE (1 ,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole% and a hyaluronic acid content of 20 mg/mL was prepared according to Example 1 , with the difference that the gel was divided into 8 parts after the crosslinking step. The gel parts were swelled in 10 mM PBS buffer with or without 1 mM ZnC , according to Table 2. Stock solutions of lidocaine hydrochloride monohydrate and ropivacaine hydrochloride monohydrate, respectively, were prepared by dissolving lidocaine and ropivacaine in water. The stock solutions were added to the hyaluronic acid gel with 1 mM ZnC and 10 mM phosphate buffer saline to a final concentration of 1 mg/mL for ropivacaine and 3 mg/mL for lidocaine. The gels were homogenized by stirring and pH of the formulations was adjusted to 6.5 or 7.3 according to Table 2 and the formulations were filled in glass syringes and autoclaved in a Getinge 6610 ERC-1 autoclave (Fo -19).

The rheological properties of the formulations were analyzed using an Ahres G2 reometer (measure system PP 40, gap 1 .00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the

viscoelastic properties were measured within the LVR.

Table 2. Formulations and rheological data for gels prepared according to Example 6.

Formulation HA Gel ZnCI ₂ Lidocaine Ropivacaine PH G' at

(mg/mL) (mM) (mg/mL) (mg/mL) 0.1 Hz

(kPa)

6a 20 0 0 0 7.3 0.71

6b 20 0 3 0 7.3 0.74

6c 20 1 0 0 7.3 0.83

6d 20 1 3 0 7.3 0.85

6e 20 0 0 0 6.6 0.80

6f 20 0 0 1 6.6 0.75

6g 20 1 0 0 6.6 0.84

6h 20 1 0 1 6.5 0.86 As shown in Table 2 and Figs. 2a and 2b the stabilizing effect of the 1 mM ZnC on the gel is seen also when a caine is present in the formulation.

Example 7 - Release of Zn from a gel containing ZnCb

The zinc release from the gels was measured by the USP-paddle apparatus using special gel containers in which the gel was placed. A gel containing 1 mM ZnCb was manufactured according to Example 1 . The gel was filled into 7 gel containers with a fixed volume of 1 ml. The geometry of the filled gel was a cylinder 1 cm in diameter and 3 mm deep. The gels were covered by a mesh-size plastic net (PA80 m = 200 mesh, AB Derma) and a coarse stainless steel net, care was taken to assure that no air was trapped between the gel and the plastic net.

Each gel container was immersed in 600 ml thermostated release medium, stirred at 30 rpm and maintained at 37°C using a Distek Evolution 6100

(North Brunswick, New Jersey). At predetermined time points, gel containers were removed and the Zn content in the container was determined using ICP.

The results are shown in Fig. 6. The release of Zn from the gel is very slow. In this experimental setup, a small molecule freely diffusing in a similar gel without any interactions between the gel and the molecule is normally fully released after 6 hours.

After 8 days, less than 50% of the Zn in the gel has been released. This gives a release of ~ 0.06 μηηοΙ/day. These results indicate that the gel could be safely used in vivo to effect a slow release of Zn to a subject.

Example 8 - Release of Zn from gels containing different Zinc salts

Gels prepared according to example 4 containing zinc chloride (1 mM), zinc gluconate (1 mM) and zinc citrate (1 mM) respectively were analyzed as described in Example 7. Table 3 shows the relative release rate for the different zinc salts Table 3. Time for 25% release zinc for the different salts

A larger fraction of dissociated zinc (free Zn ²⁺ ) results in a slower release, probably as a result of the interaction between Zn and the hyaluronan in the gel. The zinc that is complexed with its counterion will not interact with the gel, and the larger the fraction of zinc that is complexed with its counterion, the more rapid is the release. Example 9 - Manufacturing of amide crosslinked hyaluronan gels

Amide-crosslinked hyaluronic acid

Crosslinking of hyaluronan using diaminotrehalose (DATH) as a crosslinker and 4-(4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as condensating agent was done as follows.

Hyaluronic acid was weighed in a reaction vessel. A stock solution of the crosslinker (DATH) was prepared by dissolving it in buffer pH 7. DMTMM was weighed in a PTFE-container and the crosslinker-solution was added to the DMTMM to dissolve it. The pH of the DMTMM-crosslinker solution was adjusted to 6-7 with 1 .2 M HCI and then added to the HA. The contents was thoroughly homogenized and then incubated at 23 °C for 24 h. The resulting material was pressed through a 1 mm steel mesh two times, swelled in 0.9% NaCI and the pH adjusted to 7.3-7.5 with diluted acid/base. The gel was subjected to heat (70 °C, 24 h) in order to hydrolyze any potential ester bonds. The gel was particle size reduced through a 125 m mesh followed by precipitation with ethanol and the precipitate was washed with 100 mM NaCI in ethanol (70% w/w) to remove excess reagents and then with ethanol (70% w/w) to remove salts and finally with ethanol to remove water. The precipitate was then dried in vacuum over night.

Three different gels were manufactured using different molecular weight of HA and different amounts of DATH and DMTMM. A summary of the reaction conditions is provided in Table 4.

Table 4.

Example 10 - Crosslinking of deacetylated hvaluronan using DMTMM

Deacetylation of hyaluronan was done as follows. HA (Mw 2500 kDa, degree of acetylation DoA 100%) was solubilised in hydroxylamine (Sigma-Aldrich 50 vol% solution). The solution was incubated in darkness and under argon at 55 °C for 72 hours. After incubation, the mixture was precipitated by ethanol. The obtained precipitate was filtered, washed with ethanol and then re-dissolved in water. The solution was purified by ultrafiltration and subsequently lyophilized to obtain the deacetylated HA as a white solid. Degree of acetylation of the material was determined to 89% and the molecular weight to 1000 kDa.

The coupling agent DMTMM was dissolved in Na-phosphate buffer (pH 7.4). The reaction mixture was homogenized by shaking for 3.5 minutes and mixing with a spatula. The reaction mixture was placed in a water bath at 23 °C for 24 hours. The reaction was stopped by removal from the water bath and the gel was cut in to small pieces with a spatula. The reaction mixture was adjusted to pH >13 with 0.25 M NaOH, stirred for approx. 60 minutes and subsequently neutralized with 1 .2 M HCI. After neutralization, the gels were particle size reduced through a 125 m mesh, precipitated in ethanol, washed with ethanol (70 w/w%) and dried in vacuum overnight. A summary of the gel properties is provided in Table 5. Table 5.

Example 1 1 - Crosslinkinq of deacetylated hvaluronan

The coupling agent DMTMM was dissolved in Na-phosphate buffer (pH 7.4). if needed pH was adjusted on the DMTMM mixture and the solution was subsequently added to deacetylated HA (DeAcHA). The reaction mixture was homogenized by shaking for 3.5 minutes, by mixing with a spatula or by pressing the mixture though a filter. The reaction mixture was incubated at 23 or 35 °C. After 24 hours, the reaction was stopped by cutting the gel in to small pieces with a spatula or pressing the gel through a filter, the resulting material was subjected to alkaline treatment and subsequent dried in vacuum over night. A summary of the gel properties is provided in Table 6. Table 6.

(A) Swelling of gels in phosphate buffer

The dried gels were swelled in phosphate buffer (8 mM) in 0.7% NaCI for at least two hours to a concentration of 20-35 mg/nnL The pH was controlled and adjusted if necessary to 7.4. The gel particles were reduced in size with a fine filter. The gel was filled in syringes and the syringes were sterilized by autoclavation (Fo 23). A summary of the gel properties is provided in Table 7.

Table 7.

(B) Swelling of gels in Bis-Tris buffer

The dried gels were swelled in Bis-Tris buffer (1 mM) in 0.7% NaCI for at least two hours to a concentration of approx. 20 mg/mL. The pH was controlled and adjusted if necessary to 7.4. The gel particles were reduced in size with fine filter. The gel was filled in syringes and the syringes were sterilized by autoclavation (Fo 23). A summary of the gel properties is provided in Table 8. Table 8.

(C) Swelling of gels in Bis-Tris buffer with Zn

The dried gels were swelled in Bis-Tris buffer (1 mM) in 0.7% NaCI with 1 mM ZnC for at least two hours to a concentration of approx 20 mg/mL. The pH was controlled and adjusted if necessary to 7.4. The gel particles were reduced in size with fine filter. The gel was filled in syringes and the syringes were sterilized by autoclavation (Fo 23). A summary of the gel properties is provided in Table 9.

Table 9.

Example 12 - Swelling of amide crosslinked hvaluronan gels

The gels produced in Example 9 by cross-linking of hyaluronan with diaminotrehalose (DATH) were swelled in various buffers, and the properties of the resulting gels were analyzed.

(A) Swelling of gels in phosphate buffer and lidocaine

The dried gels were swelled in phosphate buffer (1 mM) in 0.9% NaCI with 3 mg/mL lidocaine for at least two hours to a concentration of 20-35 mg/mL. The pH was controlled and adjusted if necessary to 7.4. The gel particles were reduced in size with a fine filter. The gel was filled in syringes and the syringes were sterilized by autoclavation (Fo 23). A summary of the gel properties is provided in Table 10. Table 10.

(B) Swelling of gels in Bis-Tris buffer and lidocaine

The dried gels were swelled in Bis-Tris buffer (1 mM) in 0.9% NaCI with 3 mg/mL lidocaine for at least two hours to a concentration of 20-35 mg/mL. The pH was controlled and adjusted if necessary to 7.4. The gel particles were reduced in size with fine filter. The gel was filled in syringes and the syringes were sterilized by autoclavation (Fo 23). A summary of the gel properties is provided in Table 1 1 .

Table 1 1 .

(C) Swelling of gels in Bis-Tris buffer with Zn and lidocaine

The dried gels were swelled in Bis-Tris buffer (1 mM) in 0.9% NaCI with 1 mM ZnC and 3 mg/mL lidocaine for at least two hours to a concentration of 20- 35 mg/mL. The pH was controlled and adjusted if necessary to 7.4. The gel particles were reduced in size with fine filter. The gel was filled in syringes and the syringes were sterilized by autoclavation (Fo 23). A summary of the gel properties is provided in Table 12. Table 12.

The corrected swelling degree (SwCC) of gels crosslinked with DATH in different buffers is presented in Fig. 7. The gel part (GelP) of gels crosslinked with DATH in different buffers is presented in Fig. 8. The graphs show that the hydrogels are stable after the process, also in the presence of Zn.

Example 13 - Accelerated heat stability study of DATH crosslinked HA

Gel examples 2-4 to 2-12 manufactured in Example 12 (A)-(C) were further subjected to an accelerated stability study at 60 °C for 7 and 14 days. The gel part was analyzed for each of the batches and compared to the gel part at the beginning of the study (day 0). The equation used to compare how much of the gel part that is left after day 14 compared to day 0 is shown below.

GelPCday 14)

100 * — ⁽ %)

GelP '(day 0) ^ ^J

The results are presented in Fig. 9, which shows how much gel part is left after day 14 at 60 °C compared to at day 0. The DATH crosslinked HA gels exhibit good stability at 60 °C. Example 14 - Crosslinking of heparosan with diaminotrehalose (PATH)

DMTMM (10.5 mol% DMTMM/heparosan) and the crosslinker (DATH), 1 .5 mol% DATH/Heparosan) were weighed in Falcon tubes and subsequently dissolved in phosphate buffer (1 mM, pH 7.4). The pH of the solutions was adjusted to pH 7-7.5 with 1 .2 M HCI. The DMTMM and DATH solutions were successively added to heparosan (Mw 140 kDa) weighed in a reaction vessel. The suspension was homogenized by shaking for 3.5 minutes and mixing with a spatula. The reaction mixture was placed in an incubator at 23 °C. After 24 hours the reaction was stopped by removal from incubator and the resulting material was pressed through a 1 mm steel mesh two times. The bulk material was split in two factions. The first fraction was swelled in a buffer containing 0.9% NaCI 10 mM bis-tris (pH 7.3) at room temperature to a concentration of 50 mg/nnL and pH was adjusted to 7.2-7.5. The material was left at 70 °C for 24 hours and then particle-size reduced through a fine filter mesh three times. The gel was filled on syringes and sterilized by autoclavation.

The second fraction was swelled in buffer containing 1 mM ZnC , 10 mM bis- tris and 0.9% NaCI (pH 7.3) at room temperature to a concentration of 50 mg/nnL and pH was adjusted to 7.2-7.5. The material was left at 70 °C for 24 hours and then particle-size reduced through a fine filter mesh three times. The gel was filled on syringes and sterilized by autoclavation. A summary of the gel properties is provided in Table 13.

Table 13.

Example 15 - Crosslinking of chondroitin sulfate (CS) with diaminotrehalose (PATH)

DMTMM and the crossl inker DATH were weighed in separate Falcon tubes and dissolved in phosphate buffer (1 mM, pH 7.4) and the pH of the solutions were adjusted to pH 7-7.5 with 1 .2 M HCI. DMTMM and DATH solutions were successively added to the chondroitin sulfate (Mw 30 kDA) weighed in a reaction vessel. The suspension was homogenized by shaking for 3.5 minutes and mixing with a spatula. The reaction mixture was incubated at 23 °C. After 24 hours the reaction was stopped by removal from incubator and the resulting material was pressed through a 1 mm steel mesh two times.

The bulk material was split in two factions. The first fraction was swelled in a buffer containing 0.9% NaCI 10 mM bis-tris (pH 7.3) at room temperature to a concentration of 50 mg/mL and pH was adjusted to 7.2-7.5. The material was left at 70 °C for 24 hours and then particle-size reduced through a fine filter mesh three times. The gel was filled on syringes and sterilized by

autoclavation.

The second fraction was swelled in buffer containing 1 mM ZnC , 10 mM bis-tris and 0.9% NaCI (pH 7.3) at room temperature to a concentration of 50 mg/mL and pH was adjusted to 7.2-7.5. The material was left at 70 °C for 24 hours and then particle-size reduced through a fine filter mesh three times. The gel was filled on syringes and sterilized by autoclavation. A summary of the gel properties is provided in Table 14.

Table 14.

DATH/diCS Buffer for

DMTMM/diCS SwF G at

Example (mol%) swelling 0.1 Hz

(mol%) (mL/g) (Pa)

5.0 10 mM bis-tris

4-1 35.0 2.1 1290 and 0.9% NaCI

5.0 1 mM ZnCI ₂ ,

4-2 35.0 10 mM bis-tris 2.3 890 and 0.9% NaCI

4.0 10 mM bis-tris

4-3 28.0 2.7 470 and 0.9% NaCI

4.0 1 mM ZnCI ₂ ,

4-4 28.0 10 mM bis-tris 3.1 360 and 0.9% NaCI