WO2019002369A1

WO2019002369A1 - Glycosaminoglycan hydrogel with grafted dextran or cyclodextrin

Info

Publication number: WO2019002369A1
Application number: PCT/EP2018/067252
Authority: WO
Inventors: Hotan MOJARRADI; Lars Nord; Johan Olsson; Anne HELANDER KENNE; Craig Steven Harris; Laura JING JING
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 an aldehyde or hemiacetal functionalized dextran or cyclodextrin to the free amine groups of the crosslinked glycosaminoglycan.

Description

G LYCOSAM I N OG LYCAN HYDROGEL WITH GRAFTED DEXTRAN OR

CYCLODEXTRIN

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 is concerned with methods for preparing crosslinked and modified polysaccharides.

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. 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 reduced 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 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 cyclodextrins. For these and other objects that will be evident from this disclosure, the present invention provides according to a first aspect 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 an aldehyde or hemiacetal functionalized dextran or cyclodextrin to the free amine groups of the crosslinked

glycosaminoglycan. Cross-linking and/or other modifications of the hyaluronic acid molecule is necessary to improve its duration in vivo.

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 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. 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.

In some embodiments, the glycosaminoglycan molecules are selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof. In some embodiments, the glycosaminoglycan molecules are 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

variouscounter 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

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

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

The crosslinker comprising at least two nucleophilic functional groups may for example be a non-carbohydrate based di- or multinucleophilic crosslinker or a carbohydrate based di- or multinucleophilic crosslinker.

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

The carbohydrate based di- or multinucleophilic crosslinker may for example be selected from the group consisting of di- or multinucleophilic functional di-, tri-, tetra-, oligosaccharides, and polysaccharides.

In 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.

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, 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. 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 is performed by reductive amination using the free amine and an aldehyde or hemiacetal. 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 gel new properties.

As an example, hyaluronic acid is chemically altered using a deacetylation process which hydrolyses the N-acetyl group on GlcNAc, liberating a primary amine on the back-bone. The primary amine is used to chemically attach a dextran moiety via a single point attachment. Dextran, containing a hemiacetal in the reducing end, or optionally an aldehyde after chemical modification with a linker, rapidly reacts with the primary amine of the deacetylated hyaluronic acid, which results in an imine that is reduced to a stable secondary amine using a reducing agent, as illustrated in Scheme 2a (dextran with hemiacetal, no linker) and Scheme 2b (dextran with linker).

Scheme 2a

Scheme 2b

The present invention provides a method of preparing hydrogel products with grafted dextran or cyclodextrin moieties in an efficient and controlled manner. The capping process can be performed in aqueous or non-aqueous conditions and applied to crosslinked glycosaminoglycan hydrogels as well as polysaccharides.

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.

In 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.

In 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.

In 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. In some embodiments, the at least partially deacetylated glycosaminoglycan is deacetylated hyaluronic acid.

In 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 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.

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

The crosslinking 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.

The free amines can be used for mild and efficient coupling of the aldehyde or hemiacetal functionalized dextran or cyclodextrin to the crosslinked glycosaminoglycan. The coupling reaction may for example be effected by adding a reducing agent, e.g. NaCNBH3.

In some embodiments, the molar ratio of dextran or cyclodextrin of step ii and the disaccharides of the cross-linked glycosaminoglycans of step i is

0.1 -50%, preferably 1 -20% and more preferably 8-12%.

In some embodiments, the dextran or cyclodextrin of step ii forms a secondary amine bond with a free amine group of the cross-linked

glycosaminoglycans.

In some embodiments, the dextran or cyclodextrin is covalently grafted to the activated glycosaminoglycan by single end-point attachment.

In some embodiments, the dextran or cyclodextrin of step ii contains a linking group having an aldehyde group. In other words, in some

embodiments, the dextran or cyclodextrin of step ii has been chemically modified to comprise a linking group having an aldehyde group. One example of a useful modification agent is aminoacetaldehyde dimethylacetal.

In some embodiments, the linking group contains a C1 -6 alkyl. In some embodiments, the linking group contains a C1 -4 alkyl. In some embodiments, the dextran or cyclodextrin of step ii is a dextran aldehyde or dextran hemiacetal.

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 some embodiments, the dextran aldehyde or dextran hemiacetal has an average molecular weight of less than 10 kDa. In some embodiments, the dextran aldehyde or dextran hemiacetal has an average molecular weight of less than 5 kDa.

In some embodiments the glycosaminoglycan is hyaluronic acid and the graft chain is a dextran. 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 the skin.

In some embodiments the glycosaminoglycan is hyaluronic acid and the graft chain is a cyclodextrin moiety. 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. This is achieved with the present invention by formation of stable secondary amine bonds between an aldehyde group of the cyclodextrin (or guest/host complex) and the free amine group of the deacetylated glycosaminoglycan.

In some embodiments, the dextran or cyclodextrin of step ii is a cyclodextrin aldehyde. In some embodiments, the cyclodextrin aldehyde is constituted by 5-32 glucopyranoside units. In some embodiments, the cyclodextrin aldehyde is constituted by 6-8 glucopyranoside units. In some embodiments, the cyclodextrin aldehyde is constituted by 6 glucopyranoside units (a-cyclodextrin). In some embodiments, the cyclodextrin aldehyde is constituted by 7 glucopyranoside units (β-cyclodextrin). In some

embodiments, the cyclodextrin aldehyde is constituted by 8 glucopyranoside units (γ-cyclodextrin).

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.

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.

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.

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

Particularly, according to a further aspect 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 dextran or a cyclodextrin.

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 according 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. EXAMPLES

Characterization

Reductive amination on dextran

The aldehyde-modified dextran was analyzed with 1 H NMR.

Deacetylated material

Degree of acetylation (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.

a ₃ + integral C2— H The molecular weight was analyzed using SEC-MALLS on hyaluronan materials that were not hydrogels.

Hydrogels

SwF - Swelling factor was analyzed by fully dispersing 0.5 g of gel material in an excess of saline. The gel material is allowed to sediment in a 10 mL graded cylinder and the gel volume is read after 24 h.

GelP - Gel part is a description of the percentage of hyaluronan that is a part of the gel network. A number 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 (PS), corrected for gel part, according to the equation below. SwF

~ [PS] * GelP

MODDEX/CHHA - Degree of modification, which is the amount of dextran grafted to hyaluronan, was analyzed with 1 H NMR. This is done by by comparing the anomeric protons of dextran (4.94 ppm) with the N-acetyl protons of HA (2.02 ppm). Since the number average molecular weight (Mn) of dextran used is 849 Da each dextran polysaccharide is approximately 4.1 glucose units. The anomeric signal is therefore divided by this number according to

( integral of anomeric proton dextran

4 1

* 100 integral of N - acetyl

3

Materials

Hydroxylamine,NaCNBH3, 4-aminobutyraldehyde diethylacetal,

Aminoacetaldehyde dimethylacetal, and 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)- 4-methyl-morpholinium chloride (DMTMM) were purchsed from Sigma Aldrich.

Example 1 - Deacetylation of hyaluronan

20 g of HA (Mw 2500 k, 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. The results are presented in Table 1 . Table 1 .

Example 2 - Crosslinking of deacetylated hvaluronan using DMTMM

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. The results are presented in Table 2.

Table 2.

Example Mw (k) DoA (%) DMTMM SWCCPS GelP

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

2 1000 89 3.0 96 81 Example 3 - Crosslinking of hyaluronan using BDDE

Ether-crosslinked

hyaluronic acid

BDDE and 1 % NaOH was mixed and added to hyaluronan. 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. The results for the resulting powder is presented in Table 3. Table 3.

Example Mw (k) BDDE SWCCPS GelP

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

3 1000 1 .8 136 87

Example 4 - Crosslinking of hyaluronan using diaminotrehalose (PATH)

Amide-crosslinked hyaluronic acid

Hyaluronan was weighed in a Falcon tube. A stock solution of DATH was prepared by dissolving DATH in phosphate buffer pH 7.4. DMTMM was weighed in a PTFE container and the DATH-solution was added to DMTMM to dissolve it. The pH of the DMTMM-DATH solution was adjusted to 6-7 with 1 .2 M HCI then added to the HA. The contents were thoroughly homogenized and then incubated at 35 °C for 24 h. The resulting material was pressed through a 1 mm steel mesh two times and then swelled in water. The pH was adjusted to 7 and then gel was incubated at 70 °C for 24 h. 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. The results are presented in Table 4.

Table 4.

Example Mw (k) HA DATH DMTMM SWCCPS GelP

(w/w%) (mol%) (mol%) (mL/g) (%)

4 170 7.5 1 .8 0.7 215 78 Example 5 - Deacetylation of hyaluronan gels

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 - Modification of dextran with aminoacetaldehyde dimethylacetal

5.0 g dextran with a number average molecular weight (Mn) of 849 Da, 6.2 g aminoacetaldehyde dimethylacetal and 3.7 g NaCNBhb were weighed in a 50 ml_ glass bottle. The reagents were dissolved in 15 g water. The pH was adjusted to pH 10.0 with 1 .5 g 1 M NaOH. The bottle was placed in a water bath at 40 °C for 6 h.

The reaction crude was neutralized to pH 7 with 1 .2 M HCI and approximately 5 g of NaCI was added to facilitate the following precipitation. The reaction crude was precipitated by slowly adding it to a beaker containing ethanol while stirring with an over-head stirrer to the final ethanol concentration of 90%. The precipitate was washed with 90% ethanol numerous times to completely remove non-bonded reaction chemicals and dried in a vacuum dryer. The results are presented in Table 6. Table 6.

Example 7 - acidic hydrolysis of aminoacetaldehyde dimethtylacetal attached to dextran producing DEX-ALD

DEX DEX-ALD

The dialkyl acetal on the dextran moitey was removed by acidic hydrolysis. The sample from Example 6 was dissolved in 0.1 M HCI to obtain pH 1 and left for 24 h. Afterwards, the solution was neutralized with 1 M NaOH to pH 7 and lyophilized. 1 H NMR can be used to confirm hyrolysis of the dimethyl acetal.

Example 8 - Grafting of aldehyde functionalized dextran to deacetylated hyaluronan

Deactylated hyaluronan from Example 1 is dissolved in water over night to a concentration of 1 mg/mL hyaluronan. DEX-ALD from Example 7 is added together with NaCNBH3. The pH is adjusted to 5.5 with diluted HCI and is left to react at 50 °C for 24 h. Afterwards, the sample is purified using diafiltration (Mw cut-off 30 k) and lyophilized..

Example 9 - Grafting of non-modified dextran to deactylated hyaluronan

The hemiacetal of dextran is used to graft non-modified dextran to

deacetylated hyaluronan. A solution of deacetylated hyaluronan from

Example 1 is dissolved in water to a concentration of 1 mg/mL hyaluronan. Dextran is added to the solution of deacetylated hyaluronan. The pH is increased to 10 with diluted NaOH and NaCNBhb is added. The reaction vessel is incubated at 60 °C for 4 h, after which it is neutralized to pH 7 with diluted acid. Afterwards, the sample is purified using diafiltration (Mw cut-off 30 k) and lyophilized. The coupling between hyaluronan and dextran can be shown by using LC-MS and 1 H NMR. The planned experimental setup is presented in Table 7.

Table 7.

Example 10 - Grafting of aldehyde functionalized dextran to deacetylated hyaluronan gels

DEX-ALD from Example 7 and NaCNBhb is dissolved in water. The pH is adjusted to approx. 5.5 with diluted HCI. Hyaluronan gel with residual amines from Example 2 or 5 is added to the mixture resulting in a concentration of 20 mg/mL HA. The pH is controlled and adjusted if necessary to pH 5.5 and mixture is left at 50 °C for 24 h. Afterwards, the pH is increased to 7 and the gel is purified by precipitation with ethanol and washing with 70% ethanol followed by drying of the obtained precipitate under vacuum. The gel powder will analyzed and the coupling between hyaluronan and dextran is shown using LC-MS and 1 H NMR. The planned experimental setup is presented in Table 8.

Table 8.

Example 1 1 - Grafting of non-modified dextran to deacetylated hyaluronan gels The hemiacetal of dextran was used to graft non-modified dextran to hyaluronan gels containing amines. Dextran with a number average

molecular weight (Mn) of 849 Da and NaCNBhb were dissolved in water and pH was adjusted to 10 with diluted HCI. Powder of precipitated hyaluronan gel from Example 5 was added to the reaction mixture, reulsting in a

concentration of 20 mg/mL HA. The sample was incubated at 60 °C for 4 h. Afterwards, the pH was decreased to 7 with diluted acid and the gel was purified by precipitation with ethanol and washing with 70% ethanol followed by drying of the precipitate under vacuum. The gel powder was analyzed and the coupling between hyaluronan and dextran was shown using 1 H NMR. The results are presented in Table 9. Table 9.

Example Gel DoA Molar excess Molar excess MODDEX/CHHA from (%) DEX todiHA NaCNBHstodiHA (%) example

11-2 5-1 95 10 10 0.5

11-3 5-2 82 10 10 2.9

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 an aldehyde or hemiacetal functionalized dextran or cyclodextrin to the free amine groups of the crosslinked glycosaminoglycan.

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

glycosaminoglycan provided in step i) is:

3. 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.

4. The method according to any one of claims 2-3, 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.

5. The method according to claim 4, wherein the at least partially

deacetylated glycosaminoglycan is deacetylated hyaluronic acid.

6. The method according to any one of claims 2-5, 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.

7. The method according to any one of claims 2-6, 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;

a3) recovering the at least partially deacetylated glycosaminoglycan.

8. The method according to any one of claims 3-7, wherein the second glycosaminoglycan is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof.

9. The method according to any one of claims 3-8, wherein the second glycosaminoglycan is hyaluronic acid.

10. The method according to any one of claims 3-9, wherein the coupling agent of step b) is a peptide coupling agent.

1 1 . The method according to claim 10, wherein the coupling agent is

DMTMM.

12. The method according to any one of the preceding claims, wherein the molar ratio of dextran or cyclodextrin of step ii and the disaccharides of the cross-linked glycosaminoglycans of step i is 0.1 -50%, preferably 1 -20% and more preferably 8-12%.

13. The method according to any one of the preceding claims, wherein the dextran or cyclodextrin of step ii forms a secondary amine bond with a free amine group of the cross-linked glycosaminoglycans.

14. The method according to any one of the preceding claims, wherein the dextran or cyclodextrin is covalently grafted to the activated

glycosaminoglycan by single end-point attachment.

15. The method according to any one of the preceding claims, wherein the dextran or cyclodextrin of step ii contains a linking group having an aldehyde group.

16. The method according to claim 15, wherein the linking group contains a C1 -6 alkyl.

17. The method according to claim 16, wherein the linking group contains a C1 -4 alkyl.

18. The method according to any one of the preceding claims, wherein the dextran or cyclodextrin of step ii is a dextran aldehyde or dextran hemiacetal.

19. The method according to claim 18, wherein the dextran aldehyde or dextran hemiacetal has an average molecular weight of less than 10 kDa.

20. The method according to claim 19, wherein the dextran aldehyde or dextran hemiacetal has an average molecular weight of less than 5 kDa.

21 . The method according to any one of the preceding claims, wherein the dextran or cydodextrin of step ii is a cydodextrin aldehyde.

22. The method according to claim 21 , wherein the cydodextrin aldehyde is constituted by 5-32 glucopyranoside units.

23. The method according to claim 22, wherein the cydodextrin aldehyde is constituted by 6-8 glucopyranoside units.

24. The method according to claim 23, wherein the cydodextrin aldehyde is constituted by 6 glucopyranoside units (a-cyclodextrin).

25. The method according to claim 23, wherein the cydodextrin aldehyde is constituted by 7 glucopyranoside units (β-cyclodextrin).

26. The method according to claim 23, wherein the cydodextrin aldehyde is constituted by 8 glucopyranoside units (γ-cyclodextrin).

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 dextran or a cydodextrin.

A hydrogel product according to claim 27 or 28 for use as a medicament.

30. A method of cosmetically treating skin, which comprises administering to the skin a hydrogel product according to claim 27 or 28.