WO2019002370A1

WO2019002370A1 - Crosslinked and functionalized glycosaminoglycans

Info

Publication number: WO2019002370A1
Application number: PCT/EP2018/067253
Authority: WO
Inventors: Craig Steven Harris; Johan Olsson
Original assignee: Nestlé Skin Health Sa
Priority date: 2017-06-28
Filing date: 2018-06-27
Publication date: 2019-01-03

Abstract

A method of preparing a hydrogel product comprising crosslinked glycosaminoglycan molecules, said method comprising: i) providing a crosslinked glycosaminoglycan, wherein said glycosaminoglycan is at least partially N-deacetylated such that the crosslinked glycosaminoglycan comprises free amine groups; and ii) covalently grafting a reactive side-chain to the free amine groups of the crosslinked glycosaminoglycan, wherein the reactive side-chain comprises a functional group capable of forming a covalent bond to the free amine groups, and a side-chain moiety selected from the group consisting of a hydrophobic moiety, a charged moiety, and a peptide moiety. A hydrogel product comprising a crosslinked glycosaminoglycan, wherein at least some of the acetyl groups of the N-acetyl glucosamine (GlcNAc) repeating units of the glycosaminoglycan have been substituted by a side chain comprising a side-chain moiety selected from the group consisting of a hydrophobic moiety, a charged moiety, and a peptide moiety.

Description

CROSSLINKED AND FUNCTIONALIZED G LYCOSAM I N OG LYCAN S

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 modified with hydrophobic or hydrophilic side-chains.

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 cross-linking of polymers to infinite networks. While native hyaluronic acid and certain cross-linked hyaluronic acid products absorb water until they are completely dissolved, cross-linked 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. Cross-linking 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. WO 01/85801 discloses a process for crosslinking polymers containing primary and secondary amine groups with activated dicarboxylic acids. An example is discussed in which hyaluronic acid is crosslinked with aspartic acid. However, the hyaluronic acid to be crosslinked is prepared in harsh conditions leading to degradation of the hyaluronic acid, and the crosslinked hyaluronic acid fragments do not form a hydrogel but instead a solution that is filtered and then "quenched" by sulphation of the residual amino groups.

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 increased or reduced swelling.

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 a crosslinked glycosaminoglycan, wherein said

glycosaminoglycan is at least partially N-deacetylated such that the

crosslinked glycosaminoglycan comprises free amine groups; and ii) covalently grafting a reactive side-chain to the free amine groups of the crosslinked glycosaminoglycan, wherein the reactive side-chain comprises a functional group capable of forming a covalent bond to the free amine groups, and a side-chain moiety selected from the group consisting of a hydrophobic moiety, a charged moiety, and a peptide moiety. 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 macronnolecule from which at least four chains emanate. A reaction of a reactive chain end of a linear macronnolecule with an internal reactive site of another linear macronnolecule 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 macronnolecule 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 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.

While native hyaluronic acid and crosslinked hyaluronic acid products generally are inert materials when injected, modifying said hyaluronic acid or crosslinked hyaluronic acid with a different moieity can result in a different effect in vivo and properties of the gel. This invention describes a way to modify glycosaminoglycan molecules by "capping" residual amines, formed by deacetylation of the glycosaminoglycan molecules. The capping can be performed using different functional side-chain and chemistries, resulting in different gel properties as well as potential in vivo effects. The general concept for hyaluronic acid is illustrated in Scheme 1 .

Scheme 1

As an example, hyaluronic acid or a hydrogel based on hyaluronic acid is subjected to a chemical deacetylation process which hydrolyzes the N- acetyl function on GlcNAc, liberating a primary amine on the back-bone. From this primary amine, new covalent bonds can be formed, giving the

polysaccharide of gel new properties, i.e. by capping the amine with an alkyl chain to achieve a more lipophilic hydrogel. The present invention provides a method of preparing hydrogel products with grafted side-chains in an efficient and controlled manner. The method may for example be used to provide hydrogel products with tailored swelling characteristics, hydrophobicity/hydrophilicity and/or solubility.

Applications for hydrogels with tailored properties may for example be for use as injectable fillers for tissue aumentation or for drug delivery.

The capping process can be performed in aqueous or non-aqueous conditions and applied to crosslinked glycosaminoglycan hydrogels as well as polysaccharides. The capping process can be performed using different types of chemistries resulting in different functional groups, typical amides, ureas, thioureas, carbamates etc. Scheme 2 illustrates the concept of capping by formation of more lipophilic amides from a amide crosslinked linker-free gel with residual amines.

Scheme 2

Glycosaminoglycans in their native form are N-acetylated. The present invention is based on the realization that free amine groups formed by deacetylation of glycosaminoglycans can advantageously be used as graft points for attachment of functional side-chains to the glycosaminoglycan backbone. The present invention has been made possible through the recent inventive realization that hydroxylamine (NH2OH) and salts thereof can advantageously be used for deacetylation of glycosaminoglycans, e.g.

hyaluronic acid, comprising N-acetyl groups under mild reaction conditions. Using hydroxylamine or a salt thereof for deacetylation has been found to allow for N-deacetylation under mild conditions resulting in only minor degradation of the polymeric backbone of sensitive polysaccharides such as HA. Using hydroxylamine or a salt thereof for deacetylation thus allows for production of deacetylated HA with retained high molecular weight. This is in contrast to previously known methods, such as deacetylation using hydrazine or NaOH as the deacetylating agent, where high degrees of deacetylation have been inevitably accompanied by severe degradation of the polymeric backbone.

Deacetylation by hydroxylaminolysis allows not only for deacetylation of native glycosaminoglycans, but also deacetylation of crosslinked

glycosaminoglycans and glycosaminoglycan hydrogels. This means that amine functionalized hydrogels can be achieved under controlled conditions, with minimal degradation of the glycosaminoglycan network. Functional side- chains can then be grafted to the free amine groups using mild and efficient coupling techniques.

By the term "at least partial deacetylation" as used herein as used herein with reference to the glycosaminoglycan, we mean that at least some of the N-acetyl groups of a glycosaminoglycan comprising N-acetyl groups are cleaved off, resulting in the formation of free amine groups in the glycosaminoglycan. By the term "at least partial deacetylation" as used herein, we mean that a significant portion of the N-acetyl groups of the glycosaminoglycan, particularly at least 1 %, preferably at least 2 %, at least 3 %, at least 4 %, or at least 5 %, of the N-acetyl groups of the

glycosaminoglycan are converted to free amine groups. More preferably, at least 3 % of the N-acetyl groups of the glycosaminoglycan are converted to free amine groups.

By the term "at least partially deacetylated" as used herein with reference to the glycosaminoglycan, we mean a glycosaminoglycan comprising N-acetyl groups in which at least some of the N-acetyl groups have been cleaved off, resulting in the formation of free amine groups in the glycosaminoglycan. By "at least partially deacetylated" as used herein, we mean that a significant portion of the N-acetyl groups of the

glycosaminoglycan, particularly at least 1 %, preferably at least 2 %, at least 3 %, at least 4 %, at least 5 %, of the N-acetyl groups of the glycosaminoglycan have been converted to free amine groups. More preferably, at least 3 % of the N-acetyl groups of the glycosaminoglycan have been converted to free amine groups. The free amines can be used for mild and efficient coupling of various reactive side-chains to the glycosaminoglycan. Preferably, the reactive side- chain comprises a functional group capable of forming a covalent bond to the free amine, and a side-chain moiety.

The functional group preferably comprises a carboxylic acid or aldehyde functional group. Carboxylic acid and aldehyde groups are preferred because they can readily be used, together with a suitable coupling agent, to form stable amide or secondary amine bonds together with free amine under mild reaction conditions. In other words, step ii comprises covalently grafting aldehyde or carboxylic acid containing side chains to the free amine groups of the crosslinked glycosaminoglycan.

In some embodiments, the side-chain moiety is selected from a hydrophobic moiety and a peptide moiety.

According to some embodiments, the reactive side-chain comprises a hydrophobic moiety. Thus, the side-chain moiety may comprise a

hydrophobic moiety.

According to some embodiments, the hydrophobic moiety comprises a linear or branched, saturated or unsaturated, C2-C22 alkyl group, preferably a linear or branched, saturated or unsaturated, C2-C12 alkyl group, preferably a linear or branched, saturated or unsaturated, C2-C6 alkyl group.

In some embodiments, the reactive side-chain is a carboxylic acid selected from the group consisting of propanoic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, and icosanoic acid. In preferred embodiments, the reactive side-chain is a carboxylic acid selected from the group consisting of propanoic acid, butanoic acid, pentanoic acid, hexanoic acid.

According to some embodiments, the reactive side-chain is a charged moiety, comprising one or more positively or negatively charged groups.

Examples of positively charged groups include, but are not limited to, amino acids such as lysine, proline and serine. Examples of negatively charged groups include, but are not limited to, diacids such as malonic acid, succinic acid, adipic acid and aspartic acid.

According to some embodiments, the reactive side-chain is a peptide moiety. Examples of peptide moieties include, but are not limited to, amino acids such as serine, cysteine, lysine, aspartic acid and leucine.

According to some embodiments, the reactive side-chain is a hydrophobic peptide moiety.

In some embodiments, the molar ratio of the reactive side-chain to the disaccharides of the glycosaminoglycan is 0.1 -50%, preferably 1 -20% and more preferably 8-12%.

According to some embodiments, the crosslinked glycosaminoglycan provided in step i) is crosslinked by amide bonds.

According to some embodiments, the crosslinked glycosaminoglycan provided in step i) is:

- a crosslinked glycosaminoglycan formed by crosslinking an at least partially N-deacetylated glycosaminoglycan by amide bonds between carboxyl groups and free amine groups on the glycosaminoglycan backbone, wherein the crosslinked glycosaminoglycan comprises residual free amine groups; or

- a crosslinked glycosaminoglycan formed by subjecting an already

crosslinked glycosaminoglycan to at least partial N-deacetylation.

According to some embodiments, the crosslinked glycosaminoglycan provided in step i) is crosslinked by ether bonds. As an example, the glycosaminoglycan provided in step i) may have been crosslinked by a crosslinking agent selected from the group consisting of divinyl sulfone, multiepoxides and diepoxides, such as selected from the group consisting of 1 ,4-butanediol diglycidyl ether (BDDE), 1 ,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane.

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 glycosaminoglycans crosslinked by amide bonds.

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.

In some embodiments, the crosslinked glycosaminoglycan provided in step i) has a Degree of Acetylation of at least 75%, such as at least 80%, such as at least 85%, such as at least 90%.

In some embodiments, the crosslinked glycosaminoglycan provided in step i) has a Degree of Acetylation between 75-99%, such as between 80- 98 %.

In some embodiments, the crosslinked glycosaminoglycan provided in step ii) forms an insoluble gel network. Thus, the crosslinked

glycosaminoglycan may be in the form of a hydrogel and not be in solution.

The crosslinked glycosaminoglycan provided in step ii) may form an insoluble network before and/or after grafting of the reactive side-chain to the free amine groups of the crosslinked glycosaminoglycan. As an example, the crosslinked glycosaminoglycan provided in step ii) may form an insoluble gel network after heat sterilization.

As an example, the glycosaminoglycan used for crosslinking in step i) may have a M_w of above 300 kDa, such as above 400 kDa, such as above 500 kDa, such as above 700 kDa.

In some embodiments, the glycosaminoglycan used for crosslinking in step i) has a viscosity of above 9.0 dl/g. This may correspond to the glycosaminoglycan having a M_w of about 465 kDa.

In some embodiments, the crosslinked glycosaminoglycan provided in step i) forms an insoluble gel network. The insoluble gel network may have a Swelling capacity in saline (SwC) above 70 mL/g.

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.

In embodiments, the reactive side-chain is grafted to the free amine groups by amide bonds.

Thus, in some embodiments, the grafting of step (ii) provides amide bonds between glycosaminoglycan molecules the reactive side-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.

In some embodiments, at least 90 % of the bonds between glycosaminoglycan molecules and the reactive side-chains are amide bonds.

In some embodiments, less than 5 % of the bonds between glycosaminoglycan molecules and the reactive side-chains are ester bonds.

The grafting of the reactive side-chains to the free amine groups of the glycosaminoglycan molecules is preferably effected by means of a suitable coupling agent. According to some embodiments, the coupling agent is a peptide coupling agent. The coupling agent may for example be selected from the group consisting of triazine-based coupling agents, carbodiimide coupling agents, imidazolium-derived coupling agents, 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 DMTMIW disaccharide repeating unit ratio is in the range of 4-6.

The hydrogel product is further provided with 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 embodiments the preferred 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.

According to some embodiments, the method further comprises providing particles of the hydrogel product, having an average size in the range of 0.01 -5 mm, preferably 0.1 -0.8 mm.

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

According to a further aspect of the invention, there is provided a hydrogel product comprising a crosslinked glycosaminoglycan, wherein at least some of the acetyl groups of the N-acetyl glucosamine (GlcNAc) repeating units of the glycosaminoglycan have been substituted by a side- chain comprising a side-chain moiety selected from the group consisting of a hydrophobic moiety, a charged moiety, and a peptide moiety.

According to some embodiments, the side-chain comprises a hydrophobic moiety.

According to some embodiments, the side-chain is a charged moiety, comprising one or more positively or negatively charged groups.

According to some embodiments, the side-chain is a peptide moiety, preferably a hydrophobic peptide moiety. In some embodiments, the side-chain is attached to the glucosamine repeating unit by an amide bond.

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 herein 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 herein.

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.

Brief description of the drawings

Figure 1 shows the experimental results summarized from Example 3.

EXAMPLES

Terms and 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).

SWCCPS - Corrected swelling degree, total liquid uptake of one gram PS, corrected for GelP (mL/g).

SwF

SwC C _PS

Ceil' * [HA]

[PS] - The polysaccharide concentration (mg/g).

GelP - Gel part is a description of the percentage of PS that is a part of the gel network. A number of 90% means that 10% of the polysaccharide is not a part of the gel network.

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

-amide crosslinks

amide

nHA disaccharides

CrD_amide

∑(Area amide crosslinked HA fragments)

* (100

∑(Area 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 (%) = Integral acetylgroup * 100

+ Integral C2-C2

MoDsc - Degree of modification (%) by side-chains, analyzed by NMR and defined as:

^linked side chains

MoD side chains (%) =

^HA disaccharides

Example 1 - Deacetylation of Hyaluronic Acid by Hvdroxylaminolvsis

20 g of HA (Mw 2 500 kDa, DoA 100%) was solubilised in hydroxylamine (Sigma-Aldrich 50 vol% solution). The solution was stirred in darkness and under inert atmosphere at 40 °C. After 72 hrs, the mixture was precipitated by adding ethanol under stirring. The obtained precipitate was filtered, washed with ethanol and then re-dissolved in water. The precipitation procedure was repeated two more times and the obtained solid was subsequently dried under vacuum to obtain partly deacetylated HA (Mw 1700 kDa, DoA 95%) as a white powder Example 2 - Crosslinking of Deacetylated Hyaluronic Acid

The coupling agent DMTMM was dissolved in Na-phosphate buffer (pH 7.4), and pH was controlled and adjusted if necessary. The DMTMM solution was subsequently added to deacetylated HA. 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 for 24 hours. The reaction was stopped by removal from the incubator and the gel was pressed through a 1 mm steel mesh two times followed by addition of 0.25 M NaOH to the resulting material (pH >13) and mixed for 60 minutes and subsequently neutralized with 1 .2 M HCI. After neutralization, the gels particle-size reduced through a fine filter mesh (315 μιτι) three times and subsequently precipitated in EtOH and washed with 70 w/w% EtOH and EtOH. The resulting material was dried under vacuum over night. The experiments and results are summarized in Table 1 .

Table 1 .

Example 3 - Capping of a Hvdroqel by Activated Carboxylate

The carboxylate side-chain (1 equiv./HA disaccharide), was dissolved in acetonitrile. Then, /V-hydroxysulfosuccinimide sodium salt (1 equiv./HA disaccharide) dissolved in water was added dropwise under stirring at 23 °C, followed by dropwise addition of A/-(3-dimethylaminopropyl)- A/'(ethylcarbodiimide hydrochloride solution in water. The reaction mixture was stirred for 15 hours and subsequently neutralized with 0.25 M NaOH. Thereafter, the precipitated gel powder was added in the reaction mixture and continued to stir at 23 °C for 15 hours. The gel suspension pH was controlled and adjusted to 7.0-7.5. After neutralization, the gel was precipitated in EtOH and washed with 70 w/w% EtOH + 100 mM NaCI (1x), 70 w/w% EtOH (1 x) followed by EtOH (2x) and dried in vacuum over night. The experiments and results are summarized in Table 2 and Figure 1 .

Table 2.

SEC Ms

DoA (%) SwC

MoDsc (Mw)

Gel

[delta- powder Carboxylate (%)

Example Precipitated after hexa- from for capping (mL/g) gel before capping deacHA- example

capping X]

After capping

3-1 2-1 95 Butyric acid 2 1 164.358 89

Hexanoic

3-2 2-1 96 2 1 192.39 97 acid

3-3 2-2 95 Butyric acid 2 1 164.358 71

Hexanoic

3-4 2-2 95 2 1 192.39 68 acid

Example 4 - Crosslinking of Hvaluronan using BDDE

Ether-crosslinked

hyaluronic acid

BDDE (1 .8 mol%/HA) and 1 % NaOH was mixed and added to hyaluronan (1 000 kDa). The sample was mixed and incubated in 23 °C for 24 h. The resulting material was cut in to pieces and swelled in diluted acid to obtain neutral pH. The gel was particle size reduced through a 125 m mesh, precipitated in ethanol, washed with ethanol (70 w/w%) and dried in vacuum overnight to obtain a white powder (GelP 87%; SwCC 36 mL/g)

Example 5 - Deacetylation of Hvaluronan gel by Hvdroxylaminolvsis

1 g of gel powder was swelled in hydroxylamine (Sigma-Aldrich 50 vol% solution). The sample was incubated in darkness and under argon at 40 or 55 °C for 72 hours. After incubation, the gel was precipitated by ethanol. The obtained precipitate was filtered, washed with ethanol and then swelled in water. The gel powder was purified by ultrafiltration and subsequently lyophilized to obtain the deacetylated gel as a white solid. The results are presented in Table 5.

Table 5.

Example 6 - Capping of a Ether-linked Hvdroqel by Activated Carboxylate

The carboxylate side-chain (1 equiv./HA disaccharide), is dissolved in acetonitrile. Then, /V-hydroxysulfosuccinimide sodium salt (1 equiv./HA disaccharide) dissolved in water is added dropwise under stirring at 23 °C, followed by dropwise addition of A/-(3-dimethylaminopropyl)- A/'(ethylcarbodiimide hydrochloride solution in water. The reaction mixture is stirred for 15 hours and subsequently neutralized with 0.25 M NaOH. Thereafter, the precipitated gel powder (from example 5) is added to the reaction mixture and continued to stir at 23 °C for 15 hours. The gel suspension pH is controlled and adjusted to 7.0-7.5 if necessary. After neutralization, the gel is precipitated in EtOH and washed with 70 w/w% EtOH + 100 mM NaCI (1x), 70 w/w% EtOH (1 x) followed by EtOH (2x) and dried in vacuum over night. The experimental setup is presented in Table 6. Table 6.

DoA (%)

Gel

powder Carboxylate

Example Precipitated

from for capping gel before

example

capping

Hexanoic

6-1 5-1 95

acid

Hexanoic

6-2 5-2 82

acid

Claims

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

glycosaminoglycan is at least partially N-deacetylated such that the

crosslinked glycosaminoglycan comprises free amine groups; and ii) covalently grafting a reactive side-chain to the free amine groups of the crosslinked glycosaminoglycan, wherein the reactive side-chain comprises a functional group capable of forming a covalent bond to the free amine groups, and a side-chain moiety selected from the group consisting of a hydrophobic moiety, a charged moiety, and a peptide moiety.

2. The method according to claim 1 , wherein the functional group capable of forming a covalent bond to the free amine groups is a carboxylic acid or aldehyde functional group.

3. The method according to claim 1 , wherein the side chain moiety is selected from a hydrophobic moiety and a peptide moiety.

4. The method according to claim 3, wherein the reactive side-chain comprises a hydrophobic moiety.

5. The method according to claim 4, wherein the hydrophobic moiety comprises a linear or branched, saturated or unsaturated, C2-C22 alkyl group, preferably a linear or branched, saturated or unsaturated, C2-C12 alkyl group, preferably a linear or branched, saturated or unsaturated, C2-C6 alkyl group.

6. The method according to any one of claims 1 -2, wherein the reactive side- chain is a charged moiety, comprising one or more positively or negatively charged groups.

7. The method according to any one of claims 1 -2, wherein the reactive side- chain is a peptide moiety, preferably a hydrophobic peptide moiety.

8. The method according to any one of claims 1 -7, wherein the reactive side- chain is grafted to the free amine groups using a coupling agent.

9. The method according to any previous claim, wherein the reactive side- chain is grafted to the free amine groups by amide bonds.

10. The method according to any previous claim, wherein the crosslinked glycosaminoglycan provided in step i) has a Degree of Acetylation of at least 75%, such as at least 80%, such as at least 85%, such as at least 90%.

1 1 . The method according to any previous claim, wherein the crosslinked glycosaminoglycan provided in step ii) forms an insoluble gel network.

12. The method according to claim 1 1 , wherein the crosslinked

glycosaminoglycan provided in step ii) forms an insoluble gel network after heat sterilization.

13. The method according to any previous claim, wherein the

glycosaminoglycan used for crosslinking in step i) has a viscosity of above 9.0 dl/g.

14. The method according to any previous claim, wherein the crosslinked glycosaminoglycan provided in step i) forms an insoluble gel network.

15. The method according to claim 14, wherein the insoluble gel network has a Swelling capacity in saline (SwC) above 70 mL/g.

16. The method according to claim 1 , wherein the crosslinked

glycosaminoglycan provided in step i) is:

- a crosslinked glycosaminoglycan formed by subjecting an already crosslinked glycosaminoglycan to at least partial N-deacetylation.

17. The method according to claim 1 , wherein the crosslinked

glycosaminoglycan provided in step i) is crosslinked by ether bonds.

18. The method according to claim 1 , wherein 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

glycosaminoglycans to provide glycosaminoglycans crosslinked by amide bonds.

19. The method according to claim 18, wherein 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, preferably deacetylated hyaluronic acid.

20. The method according to any one of claims 18-19, wherein 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.

21 . The method according to any one of claims 18-20, wherein 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.

22. The method according to any one of claims 18-21 , wherein the second glycosaminoglycan is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof, preferably hyaluronic acid.

23. The method according to any one of claims 8-22, wherein the coupling agent is a peptide coupling agent.

24. The method according to claim 23, wherein the coupling agent is

DMTMM.

25. The method according to claim 24, wherein the DMTMM/ disaccharide repeating unit ratio is in the range of 1 -3, preferably in the range of 1 -1 .5.

26. The method according to claim 24, wherein the DMTMM/ disaccharide repeating unit ratio is in the range of 3-8, preferably in the range of 4-6.

27. A hydrogel product obtainable by the method according to any one of claims 1 -26.

28. A hydrogel product comprising a crosslinked glycosaminoglycan, wherein at least some of the acetyl groups of the N-acetyl glucosamine (GlcNAc) repeating units of the glycosaminoglycan have been substituted by a side chain comprising a side-chain moiety selected from the group consisting of a hydrophobic moiety, a charged moiety, and a peptide moiety.

29. The hydrogel product according to claim 28, wherein the side-chain comprises a hydrophobic moiety.

30. The hydrogel product according to claim 29, wherein the hydrophobic moiety comprises a linear or branched, saturated or unsaturated, C2-C22 alkyi group, preferably a linear or branched, saturated or unsaturated, C2-C12 alkyi group, preferably a linear or branched, saturated or unsaturated, C2-C6 alkyi group.

31 . The hydrogel product according to claim 28, wherein the reactive side- chain is a charged moiety, comprising one or more positively or negatively charged groups.

32. The hydrogel product according to claim 28, wherein the reactive side- chain is a peptide moiety, preferably a hydrophobic peptide moiety.

33. A hydrogel product according to any one of claims 27-32 for use as a medicament.

34. A method of cosmetically treating skin, which comprises administering to the skin a hydrogel product according to any one of claims 27-33.