Technical field of the invention

The present invention relates to the field of hydrogels containing cross- linked polysaccharides and the use of such hydrogels in medical and/or cosmetic applications. More specifically, the present invention deals with cross-linked hyaluronic acid hydrogels, functionalized with dextran or cyclodextrin.

Background of the invention

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. Another widely used biocompatible polymer is dextran. Dextran is a complex, branched glucan composed of chains of varying lengths (from 1 to 2000 kD). The straight chain consists of a-1 ,6 glycosidic linkages between glucose molecules, while branches begin from a-1 ,3 linkages.

Cyclodextrins (sometimes called cycloamyloses), also referred to herein as CDs, are a family of compounds made up of sugar molecules bound together in a ring (cyclic oligosaccharides). Cyclodextrins are produced from starch by means of enzymatic conversion. Typically, cyclodextrins are constituted by 6-8 glucopyranoside units, and have a structural conformation resembling toroids with the primary hydroxyl groups of the glucopyranoside units arranged along the smaller opening of the toroid and the secondary hydroxyl groups of the glucopyranoside units arranged along the larger opening of the toroid. Because of this arrangement, the interior of the toroids is considerably less hydrophilic than the aqueous environment and thus able to host other hydrophobic molecules. In contrast, the exterior is sufficiently hydrophilic to impart cyclodextrins (or their complexes) water solubility.

When a hydrophobic molecule (the guest) is contained, fully or partially, within the interior of the cyclodextrin (the host), this is referred to as an inclusion complex or guest/host complex. The formation of the guest/host complex can greatly modify the physical and chemical properties of the guest molecule, mostly in terms of water solubility. This is a reason why

cyclodextrins have attracted much interest in pharmaceutical applications: because inclusion compounds of cyclodextrins with hydrophobic molecules are able to penetrate body tissues, these can be used to release biologically active compounds under specific conditions. In most cases the mechanism of controlled degradation of such complexes is based on change of pH, leading to the cleavage of hydrogen or ionic bonds between the host and the guest molecules. Other mechanisms for the disruption of the complexes include heating or action of enzymes able to cleave a-1 ,4 linkages between glucose monomers. Summary of the invention

It is an object of the present invention to provide a cross-linked hyaluronic acid product suitable for use as a dermal filler.

It is a further object of the present invention to provide a cross-linked hyaluronic acid product suitable having improved durability in use as a dermal filler.

It is a further object of the present invention to provide a cross-linked hyaluronic acid product suitable having desired swelling.

It is a further object of the present invention to provide a cross-linked Hyaluronic acid product that treat skin aging by promoting collagen

neosynthesis in the skin.

It is a further object of the present invention to provide improved formulations for administration of pharmaceutical and/or cosmetic

substances.

In particular, it is an object of the present invention to provide a stable cross-linked hyaluronic acid gel product having a significant amount of grafted aminodextran.

An alternative object of the present invention is to provide a stable cross-linked hyaluronic acid gel product having a significant amount of grafted cyclodextrins.

It is also an object of the present invention to provide a process for preparing improved formulations for administration of pharmaceutical and/or cosmetic substances.

In particular, it is an object of the present invention to a process for providing a stable cross-linked hyaluronic acid gel product having a significant amount of grafted aminodextran.

An alternative object of the present invention is to provide a process for providing a stable cross-linked hyaluronic acid gel product having a significant amount of grafted cyclodextrins

For these and other objects that will be evident from this disclosure, the present invention provides according to a first aspect thereof, a method of preparing a hydrogel product comprising crosslinked glycosaminoglycan molecules, said method comprising:

(i) providing an amide crosslinked glycosaminoglycan;

(ii) activating carboxyl groups on the glycosaminoglycan with a coupling agent to form an activated glycosaminoglycan;

(iii) grafting an aminodextran and/or an aminocyclodextrin to the activated glycosaminoglycan via the activated carboxyl groups.

Cross-linking and/or other modifications of the hyaluronic acid molecule is necessary to improve its duration in vivo. The present invention provides a simple, mild and efficient manufacturing process for preparing crosslinked and grafted glycosaminoglycan molecules.

The term cross-linking as used herein refers to a reaction involving sites or groups on existing macronnolecules or an interaction between existing macronnolecules that results in the formation of a small region in a

macromolecule from which at least four chains emanate. A reaction of a reactive chain end of a linear macromolecule with an internal reactive site of another linear macromolecule results in the formation of a branch point or graft, but is not regarded as a cross-linking reaction.

The term grafting as used herein refers to a reaction in which one or more species are connected to the main chain of a macromolecule as side- chains having constitutional or configurational features that differ from those in the main chain.

The 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 hydrogel product 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 product 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.

According to a preferred embodiment, the cross-linked hydrogel product is in the form of gel particles having an average size in the range of 0.01 -5 mm, preferably 0.1 -0.8 mm.

Step (i) of the inventive method comprises providing an amide crosslinked glycosaminoglycan. 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 multinucleophilic crosslinker, whereby amide bonds are formed between carboxylic groups present on the glycosaminoglycan backbone and nucleophiles of the crosslinker.

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

glycosaminoglycan is 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.

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. In some embodiments the concentration of the glycosaminoglycan is in the range of 2 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.

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

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.

According to some embodiments, (i) comprises the steps:

a) providing a solution comprising an at least partially deacetylated

glycosaminoglycan and optionally a second glycosaminoglycan;

b) activating carboxyl groups on the at least partially deacetylated

glycosaminoglycan and/or the optional second glycosaminoglycan with a coupling agent, to form activated glycosaminoglycans;

c) crosslinking the activated glycosaminoglycans via their activated carboxyl groups using amino groups of the at least partially deacetylated

glycosaminoglycans to provide an amide crosslinked glycosaminoglycan.

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.

According to some embodiments, the at least partially deacetylated glycosaminoglycan is selected from the group consisting of deacetylated hyaluronic acid, deacetylated chondroitin and deacetylated chondroitin sulfate, and mixtures thereof. According to some embodiments, the at least partially deacetylated glycosaminoglycan is deacetylated hyaluronic acid. According to some embodiments, the at least partially deacetylated glycosaminoglycan has a degree of acetylation of 99% or less, preferably 98% or less, preferably 97% or less, preferably 96% or less, preferably 95% or less, preferably 94% or less, preferably 93% or less, and a weight average molecular weight of 0.1 MDa or more, preferably 0.5 MDa or more.

According to some embodiments, the at least partially deacetylated glycosaminoglycan is obtained by a method for at least partial deacetylation of a glycosaminoglycan, comprising: a1 ) providing a glycosaminoglycan comprising acetyl groups;

a2) allowing the glycosaminoglycan comprising acetyl groups to react with hydroxylamine (NH2OH) or a salt thereof at a temperature of 100 °C or less for 2-200 hours to form an at least partially deacetylated glycosaminoglycan; and

a3) recovering the at least partially deacetylated glycosaminoglycan.

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

Crosslinking of the glycosaminoglycan may also 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.

According to some embodiments, (i) comprises the steps:

(a) providing a solution of one or more glycosaminoglycans; (b) activating carboxyl groups on the glycosaminoglycans with a coupling agent to form activated glycosaminoglycans; (c) crosslinking the activated glycosaminoglycans via their activated carboxyl groups using a di- or multinucleophilic functional crosslinker comprising a spacer group selected from the group consisting of di-, tri-,

tetra-, and oligosaccharides to provide an amide crosslinked

glycosaminoglycan.

According to some embodiments, the glycosaminoglycans are selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof. According to some embodiments, the glycosaminoglycans are hyaluronic acid.

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 some embodiments, the crosslinks comprise a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides.

In some embodiments, the spacer group is a hyaluronic acid

tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue.

In some embodiments, the spacer group is selected from the group consisting of di-, tri-, and tetrasaccharides. In some embodiments, the nucleophilic groups of the crosslinker are selected from the group consisting of primary amine, hydrazine, hydrazide, carbazate, semi-carbazide, thiosemicarbazide, thiocarbazate and aminoxy. In some embodiments, the nucleophilic groups of the crosslinker are primary amine.

In some embodiments, the crosslinker is a dinucleophilic functional crosslinker.

In some embodiments, the crosslinker is selected from the group consisting of diamino hyaluronic acid tetrasaccharide, diamino hyaluronic acid hexasaccharide, diamino trehalose, diamino lactose, diamino maltose, diamino sucrose, chitobiose, or diamino raffinose.

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.

According to some embodiments, the grafting of step (iii) provides amide bonds between the glycosaminoglycan molecules and graft chains. Amide bonds are stable covalent bonds that are not easily hydrolysed.

Accordingly, hydrogel products according to the invention, where both crosslinking and grafting is effected by amide bonds will be less sensitive to degradation than similar products where crosslinking or grafting is effected by a weaker bond. Using the same types of bond in crosslinking and grafting may also provide a more predictable degradation behaviour of the product.

According to some embodiments, the molar ratio of aminodextran and/or aminocyclodextrin of step (iii) and the disaccharides of the cross-linked glycosaminoglycans of step (i) is 0.1 -50%, preferably 1 -20% and more preferably 8-12%.

According to some embodiments, the aminodextran and/or

aminocyclodextrin of step (iii) contain a linking group having an amino group, and wherein the linking group of the aminodextran and/or aminocyclodextrin forms an amide bond with a carboxyl group of the cross-linked glycosaminoglycans. According to some embodiments, the linking group contains a C1 -6 alkyl. According to some embodiments, the linking group contains a C1 -4 alkyl.

According to some embodiments, the aminodextran and/or

aminocyclodextrin of step (iii) is an aminodextran. According to some embodiments, the aminodextran has an average molecular weight of less than 10 kDa. According to some embodiments, the aminodextran has an average molecular weight of less than 5 kDa. According to some

embodiments, the aminodextran in step (iii) is covalently grafted to the activated glycosaminoglycan by single end-point attachment. According to some embodiments, the aminodextran is functional ized at the reducing end with a diamine. Preferably, the functionalization of dextran is only done on the reducing end such that grafting of the modified dextran to hyaluronic acid can only be done via single-point attachment.

Unless otherwise provided, the term "dextran" encompasses all variants and combinations of variants of dextran, of various chain lengths and charge states, as well as with various chemical modifications. Dextran is a complex, branched glucan composed of chains of varying lengths (from 1 to 2000 kD). The straight chain consists of a-1 ,6 glycosidic linkages between glucose molecules, while branches begin from a-1 ,3 linkages. Dextran is a bacterial polysaccharide and may be synthesized from sucrose by certain lactic-acid bacteria, for example, Leuconostoc mesenteroides and

Streptococcus mutans. Dextran is a nontoxic polysaccharide, for which biocompatibility has been well documented. Dextran has been extensively explored in biomedical and pharmaceutical applications. Dextrans are commonly used to decrease vascular thrombosis, reduce inflammatory response and prevent ischemia reperfusion injury in organ transplantation, in which dextran acts as a mild reactive oxygen species scavenger and reduces excess platelet activation.

In a preferred embodiment, a hydrogel comprising amide crosslinked hyaluronic acid is grafted with dextran chemically modified with a primary amine at the reducing end. The modified dextran is grafted on the amide- crosslinked hyaluronic acid gel via single-point attachement using a condensating agent as illustrated in Scheme 1 .

hyaluronic acid hydrogel

Dextran Dextran modified with

nucleophile

Scheme 1

In some embodiments, the aminodextran and/or aminocydodextrin is an aminocydodextrin. In some embodiments, the aminocydodextrin is constituted by 5-32 glucopyranoside units. In some embodiments, the aminocydodextrin is constituted by 6-8 glucopyranoside units. In some embodiments, the aminocydodextrin is constituted by 6 glucopyranoside units (a-cyclodextrin). In some embodiments, the aminocydodextrin is constituted by 7 glucopyranoside units (β-cyclodextrin). In some embodiments, the aminocydodextrin is constituted by 8 glucopyranoside units (γ-cyclodextrin).

In some embodiments, the aminocydodextrin is selected from the group consisting of 2-aminocyclodextrin, 3-aminocyclodextrin and 6- aminocyclodextrin. In some embodiments, the aminocydodextrin is selected from the group consisting of 3-aminocyclodextrin and 6-aminocyclodextrin. In some embodiments, the aminocydodextrin is 6-aminocyclodextrin.

According to some embodiments, the hydrogel product is further comprising a guest molecule capable of forming a guest-host complex with the cyclodextrin molecule acting as a host. The guest molecule may be selected from drugs and/or biologically active substances used in the treatment of disorders in the field of dermatology, aesthetics, ophthalmology, gynaecology, oncology, angiology, neurology, orthopaedics, rheumatology or aesthetic dermatology.

In some embodiments, at least 90 % of the bonds between

glycosaminoglycan molecules and crosslinks and between

glycosaminoglycan molecules and graft chains are amide bonds.

In some embodiments, less than 5 % of the bonds between

glycosaminoglycan molecules and crosslinks and between

glycosaminoglycan molecules and graft chains are ester bonds.

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). In preferred embodiments, the coupling agent is DMTMM.

When a hyaluronic acid gel is grafted with dextran, it has unexpectedly been found that the molar ratio between DMTMM and the disaccharide repeating units of the hyaluronic acid affects the swelling of the grafted gel. Specifically, gels with the same concentration of grafted dextran will exhibit lower swelling when the grafting has been done with a higher

DMTMM/disaccharide repeating unit ratio. Accordingly, the swelling of the gel product can be controlled by variation of the amount of DMTMM used.

In some embodiments, the DMTMM/disaccharide repeating unit ratio is in the range of 1 -10. In some embodiments, the DMTMM/ disaccharide repeating unit ratio is in the range of 1 -3. In some embodiments, the DMTMM/ disaccharide repeating unit ratio is in the range of 1 -1 .5. In some

embodiments, the DMTMM/ disaccharide repeating unit ratio is in the range of 3-10. In some embodiments, the DMTMM/ disaccharide repeating unit ratio is in the range of 3-8. In some embodiments, the DMTMM/ disaccharide repeating unit ratio is in the range of 4-6.

In some embodiments the glycosaminoglycan is hyaluronic acid and the graft chain is an aminodextran. Hyaluronic acid products obtained according to the present invention, comprising a cross-linked hyaluronic acid with one or more dextran molecules grafted, display several advantageous and surprising properties, e.g. including improved stability to thermal, hydrolytic, radical and enzymatic degradation resulting in improved durability in use as a dermal filler, and decreased swelling capacity. Reduced swelling capacity means that harder gels can be produced, without increasing the amount of cross-linker in the hyaluronic acid.

The cross-linked hyaluronic acid products obtained according to the invention can be used, e.g., as injectable compositions for cosmetic or medical surgery, like dermal filling and body contouring. The cross-linked hyaluronic acid products obtained according to the invention, combining hyaluronic acid with dextran, exhibit decreased swelling compared to hyaluronic acid products without dextran. This is useful, since it means that harder gels can be produced, without increasing the amount of cross-linker in the hyaluronic acid. The cross-linked hyaluronic acid products obtained according to the invention have also been found to have a better thermal stability as well as better stability to radical and enzymatic degradation as compared hyaluronic acid products without dextran. A possible explanation is that the hyaluronic acid backbone is protected by the dextran. This leads to an improved of durability in vivo of the cross-linked hyaluronic acid products according to the invention as compared hyaluronic acid products without dextran. In addition to improved stability, the cross-linked hyaluronic acid products obtained according to the invention combining hyaluronic acid with dextran, have been shown to promote collagen neosynthesis.

In some embodiments the glycosaminoglycan is hyaluronic acid and the graft chain is amino functionalized cyclodextrin. This allows for a significant modification of the cross-linked hyaluronic acid with cyclodextrins without inducing depolymerisation of the cross-linked polymer mixture.

The cyclodextrin molecules are useful as carriers (hosts) for a pharmaceutical agent (guest). When a pharmaceutical agent (the guest) is contained, fully or partially, within the interior of the cyclodextrin (the host), this is referred to as an inclusion complex or guest/host complex. The cyclodextrin may then release the pharmaceutical agent under specific conditions, e.g. due to change in pH leading to the cleavage of hydrogen or ionic bonds between the host and the guest molecules.

The cyclodextrin molecules are attached to the cross-linked polymer mixture, preferably to the hyaluronic acid component, in order to reduce migration of the cyclodextrin (or guest/host complex) from the site of administration, e.g. injection. In this way, the site of release of the

pharmaceutical agent from the cyclodextrin can be controlled.

Also, in order to increase temporal control of the release of the pharmaceutical agent, it has been found that the influence of cleavage of the bonds between the cyclodextrin (or guest/host complex) and the cross-linked polymer mixture should be minimized. In other words, it is desired that the release of the pharmaceutical agent is, as far as possible dependent on the physical release from the cyclodextrin rather than on chemical degradation.

In preferred hydrogel products, the cyclodextrin molecules are attached to the hyaluronic acid by amide bonds. The use of amide bonds in the cyclodextrin-hyaluronic acid linkage (graft) has been found to be

advantageous compared to e.g. ester bonds, since the amide bond is more stable to degradation in vivo. The use of a less stable bond between the hyaluronic acid and cyclodextrin molecules could lead to premature loss of cyclodextrin (or guest/host complex) from the site of injection.

According to a further aspect there is provided a hydrogel product obtainable by the method described above.

According to a further aspect there is provided a hydrogel product comprising an amide crosslinked glycosaminoglycan, grafted with a dextran and/or a cyclodextrin, wherein said dextran and/or cyclodextrin is covalently bound to the glycosaminoglycan by amide bonds.

The hydrogel product 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 may further involve sterilization of the hydrogel product by autoclaving, i.e sterilization using saturated steam.

Accordingly, in some embodiments the hydrogel product has been subjected to sterilization by autoclaving. 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. 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 components, features, effects and advantages of the hydrogel product may be further defined as described above with reference to the method of preparing the hydrogel product.

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

The hydrogel product 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 hydrogel product may be useful, for example in the treatment of various dermatological conditions. Particularly, there is provided an hydrogel product 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 hydrogel product 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 a hydrogel product as described above for cosmetic, non-medical, 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 a further aspect there is provided a hydrogel product as described above for use as a medicament.

According to a further aspect there is provided a method of

cosmetically treating skin, which comprises administering to the skin a hydrogel product as described above.

Other aspects and preferred embodiments of the present invention will be evident from the following detailed disclosure of the invention and the appended claims. Without desiring to be limited thereto, the present invention will in the following be illustrated by way of examples. EXAMPLES

Characterization

Reductive amination on dextran

The total DEX-HMDA (including mono- and cross-linked HMDA to dextran and free dextran) was determimed by ¹ H NMR by comparing the integral of the signals from the anomeric proton of dextran (4.94 ppm),

hexamethylenediamine signal (1 .33 ppm) and KHP (7.62 ppm, internal standard).

LC-QToF-MS analysis was made to evaluate the residual

hexamethylenediamine (HMDA) in the powder after reductive amination.

Amide crosslinked hyaluronan hydrogels and hydrogels grafted with dextran The hydrogels were degraded with chondroitinase ABC or HCI prior to the analysis. The amount of grafted dextran on hyaluronan was analyzed with ¹ H NMR.

All gels were swelled in saline. GelP - "Gel part" is a description of the percentage of hyaluronan that is a part of the gel network. A GelP of 90% means that only 10% of hyaluronan is not a part the network. The amount of free hyaluronan in the gel was measured with LC-SEC-UV. SwCC - "Corrected swelling capacity" is the total liquid uptake of one gram polysaccharide, corrected for gel part.

MODDEX/CHHA - "degree of modification of dextran on hyaluronan" is measured with ¹ H NMR. The integral of the anomeric proton of dextran is compared to the integral of the N-acetyl group of hyaluronan. The average number of glucose units in the dextran and thus anomeric protons is 3.7, which the integral is divided with according to the equation below. ' Integral of anomeric proton of dextran ">

MoD _D Ex/diHA (%)— I integral of N -acetyl of hyaluronan ) * 100

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

Integral acetylgroup , , _„

- — —+ Intearal C2-

Example 1 - Reductive amination of dextran

Dextran (100.0 g) with a number average molecular weight (M _n ) of 849 Da, hexamethylenediamine dihydrochloride (HMDA, 193.3 g) and NaCNBH ₃ (64.8 g ) were added to a reaction vessel. The reagents were dissolved in water (500 g) and the pH was adjusted to pH 10.0 by adding 1 M NaOH. The reaction mixture was incubated at 60 °C.

After 4 hours, the reaction was neutralized to pH 7 by adding HCI (aq. 1 .2 M) and NaCI (approx. 10 g) was added to facilitate precipitation. The crude reaction was precipitated by slowly addition of the mixture to ethanol under agitation by an over-head stirrer until the final ethanol concentration was 90%. The precipitate was washed with ethanol (90% ) numerous times to

completely remove residual chemicals and subsequently dried in under vacuum. The dry powder was dissolved in D2O and analyzed by ¹ H NMR. The total HMDA modified dextran (DEX-HMDA) content was 75 w/w%. The residual HMDA in the powder was 0.03 w/w%.

Example 2- Manufacturing of amide crosslinked hvaluronan gels using DATH as crosslinker.

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.

Four different gels were manufactured using different molecular weight of HA and different amounts of DATH and DMTMM. The gels were swelled in phosphate saline buffer to 20 mg/nnL HA, filled on syringes and sterilized at Fo 20 conditions. A summary of the gel properties is provided Table 1 .

Table 1 .

Example 3a - Crosslinking of deacetylated hvaluronan using DMTMM

Deacetylation of hyaluronan was done as follows. 20 g of 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 (DoA) 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 2.

Table 2.

Example 3b - Reacetylation (capping) of linker-free gel

The gel powder (300 mg), prodcuded according to example 3a, was added to water and stirred. After stirring 2 hrs, triethanol amine (175 mg) and subsequently acetic anhydride (72 mg) were added. After stirring for 60 min, the reaction mixture was adjusted to pH >13 with 0.25 M NaOH, stirred for additional 40 minutes and subsequently neutralized (pH approx. 7) with 1 .2 M HCI. The gel was precipitated by adding ethanol and the precipitae was subsequently washed with ethanol (70 w/w%) with NaCI (100 mM), ethanol (70 w/w%), ethanol (x3) and dried in vacuum overnight. The degree of acetylation (DoA) of the obtianed gel power was determined to 100% by NMR spectrscopy.

Example 4 - Grafting of functionalized dextran to an amide crosslinked hyaluronan gel

Dextran-linker Amide-crosslinked

hyaluronan gel Dextran grafted to amide-crosslinked hyaluronan gel

Dextran functionalized with hexamethylenediamine according to Example 1 was grafted to hyaluronic acid hydrogels from Example 2-1 to 2-4 (trehalose crosslinked gels) and 3b (linker-free gels). A general manufacturing

procedure is described below.

Dextran modified at the reducing end with hexamethylenediamine and

DMTMM were weighed in a glass or PTFE bottle. 1 mM phosphate buffer pH 7.4 was added to dissolve the reagents. Precipitated hyaluronan hydrogel was added to the reaction solution while stirring so that a concentration of 20 mg/mL hyaluronan was obtained. The sample was placed in a water bath 50 °C or stirred at ambient temperature for approx. 24 hrs. The reaction was stopped by increasing the pH to 13.0 with 0.25 M NaOH, and after stirring for 60-90 min the mixture was neutralized with diluted HCI to neutral pH. The gel was purified by continuously washing with 0.9% NaCI and then precipitated by slowly adding ethanol up to 70% ethanol or only

precipitated by slowly adding ethanol up to 70% ethanol. The precipitate was washed with 70% ethanol numerous times. The precipitated gel powder was subsequently washed with ethanol and dried under vacuum. The gel powder was swelled in phosphate saline buffer to 20 mg/nnL HA and filled on syringes and sterilized at Fo 20 conditions .

The reaction conditions and results are shown in Table 3. The difference in SwCC before and after grafting is calculated as shown below:

10

Table 3.

Eq = mol(X)/mol(Y), PS = polysaccharide (DEX + HA)

15 Example 5 - Grafting of functionalized cyclodextrin to an amide crosslinked hyaluronan gel

Functionalized cyclodextrin hyaluronan gel Cyclodextrin grafted to amide-crosslinked hyaluronan gel

Amino-functionlized γ-cyclodextrin (3A-Amino-3A-deoxy-(2AS,3AS)-Y- cyclodextrin Hydrate; purchased from TCI Europe) was grafted to hyaluronic acid hydrogels from example 2-1 to 2-3. A general manufacturing procedure is described below. The reaction conditions and results are shown in Table 4.

Amino-functionlized cyclodextrin and DMTMM were weighed in a PTFE vessel followed by addition of 1 mM phosphate buffer pH 7.4, the pH was adjusted if needed. Precipitated hyaluronan hydrogel was added to the reaction solution and the mixtures were incubated at 50 °C for 2 hrs, followed by 22 hrs at ambient temperature.

The reaction was stopped by increasing the pH to 13.0 with 0.25 M NaOH. After 60-80 min, the mixtures were neautralized with diluted HCI to neutral pH. The gel was precipitated by slowly addition of ethanol and the precipitate was subsequently washed with ethanol (70 w/w%) with NaCI (100 mM), ethanol (70 w/w%), ethanol (x3) and dried in vacuum overnight. The gels were swelled in phosphate saline buffer to 30-50 mg/mL PS and filled on syringes. Reaction conditions Results

Example Gel Eq CDx- Eq MoD GelP [PS] SwCC used amine/ DMTMIW (CDx diHA) (%) (mg/mL) (mL/g) from diHA diHA (%)

example

5-1 2-1 0.3 1 .25 8 92 42 63

5-2 2-3 0.3 5.0 5 85 34 120

5-3 2-3 0.3 1 .25 6 87 41 1 12

Table 4.

Eq = mol(X)/mol(Y), PS = polysaccharide (CDx + HA)

ITEMIZED LISTING OF EMBODIMENTS

1 . A method of preparing a hydrogel product comprising crosslinked glycosaminoglycan molecules, said method comprising:

(i) providing an amide crosslinked glycosaminoglycan;

(ii) activating carboxyl groups on the glycosaminoglycan with a coupling agent to form an activated glycosaminoglycan;

(iii) grafting an aminodextran and/or an aminocyclodextrin to the activated glycosaminoglycan via the activated carboxyl groups. 2. The method according to item 1 , wherein the glycosaminoglycan is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof.

3. The process according to any one of the preceding items, wherein the grafting of step (iii) provides amide bonds.

4. The method according to any one of the preceding items, wherein the coupling agent of step (ii) is a peptide coupling reagent. 5. The method according to any one of the preceding items, wherein the coupling agent of step (ii) is DMTMM.

6. The method according to item 5, wherein the DMTMM/ disaccharide repeating unit ratio is in the range of 1 -3, preferably in the range of 1 -1 .5.

7. The method according to item 5, wherein the DMTMM/ disaccharide repeating unit ratio is in the range of 3-8, preferably in the range of 4-6. 8. The method according to any one of the preceding items, wherein the molar ratio of aminodextran and/or aminocyclodextrin of step (iii) to the disacchahdes of the cross-linked glycosaminoglycans of step (i) is 0.1 -50%, preferably 1 -20% and more preferably 8-12%.

9. The method according to any one of the preceding items, wherein the aminodextran and/or aminocyclodextrin of step (iii) contain a linking group having an amino group, and wherein the linking group of the aminodextran and/or aminocyclodextrin forms an amide bond with a carboxyl group of the cross-linked glycosaminoglycans.

10. The method according to item 9, wherein the linking group contains a C1 -6 alkyl, preferably a C1 -4 alkyl.

1 1 . The method according to any one of the preceding items, wherein the aminodextran and/or aminocyclodextrin of step (iii) is an aminodextran. 12. The method according to item 1 1 , wherein the aminodextran has an average molecular weight of less than 10 kDa, preferably less than 5 kDa.

13. The method according to any one of items 1 1 -12, wherein the aminodextran in step (iii) is covalently grafted to the activated

glycosaminoglycan by single end-point attachment.

14. The method according to any one of items 1 1 -13, wherein the aminodextran is functionalized at the reducing end with a diamine. 15. The method according to any one of the preceding items, wherein the aminodextran and/or aminocyclodextrin of step (iii) is an aminocyclodextrin. 16. The method according to item 15, wherein the aminocyclodextrin is constituted by 6 glucopyranoside units (a-cyclodextrin).

17. The method according to item 15, wherein the aminocyclodextrin is constituted by 7 glucopyranoside units (β-cyclodextrin).

18. The method according to item 15, wherein the aminocyclodextrin is constituted by 8 glucopyranoside units (γ-cyclodextrin). 19. The method according to any one of items 15-18, wherein the

aminocyclodextrin is selected from the group consisting of 2- aminocyclodextrin, 3-aminocyclodextrin and 6-aminocyclodextrin, preferably from the group consisting of 3-aminocyclodextrin and

6-aminocyclodextrin, more preferably 6-aminocyclodextrin.

20. A hydrogel product obtainable by the method according to any one of items 1 -19.

21 . A hydrogel product comprising an amide crosslinked glycosaminoglycan, grafted with a dextran and/or a cyclodextrin, wherein said dextran and/or cyclodextrin is covalently bound to the glycosaminoglycan by amide bonds.

22. A hydrogel product according to any one of items 20-21 for use as a medicament.

23. A method of cosmetically treating skin, which comprises administering to the skin a hydrogel product according to any one of items 20-21 .