(19)
(11)EP 3 397 651 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
06.05.2020 Bulletin 2020/19

(21)Application number: 16822477.2

(22)Date of filing:  28.12.2016
(51)Int. Cl.: 
C08B 37/00  (2006.01)
C08L 5/00  (2006.01)
C08B 37/08  (2006.01)
C08J 3/075  (2006.01)
(86)International application number:
PCT/EP2016/082783
(87)International publication number:
WO 2017/114867 (06.07.2017 Gazette  2017/27)

(54)

CARBOHYDRATE CROSSLINKER

KOHLENHYDRATVERNETZER

RÉTICULATION DE GLUCIDES


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 29.12.2015 EP 15202944
31.05.2016 EP 16172254
31.05.2016 EP 16172225
31.05.2016 EP 16172241

(43)Date of publication of application:
07.11.2018 Bulletin 2018/45

(60)Divisional application:
19204239.8 / 3623390

(73)Proprietor: Galderma S.A.
6330 Cham (CH)

(72)Inventors:
  • OLSSON, Johan
    167 33 Bromma (SE)
  • HARRIS, Craig Steven
    06410 Biot (FR)
  • MOJARRADI, Hotan
    754 31 Uppsala (SE)
  • GERFAUD, Thibaut
    06370 Mauans Sartoux (FR)
  • TOMAS, Loïc
    06410 Le Biot (FR)
  • BOITEAU, Jean-Guy
    06650 Opio (FR)

(74)Representative: AWA Sweden AB 
P.O. Box 45086
104 30 Stockholm
104 30 Stockholm (SE)


(56)References cited: : 
EP-A1- 2 727 597
US-A1- 2007 053 987
  
      
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description

    Technical field of the invention



    [0001] The present invention relates to the field of hydrogels containing crosslinked polysaccharides and the use of such hydrogels in medical and/or cosmetic applications. More specifically, the present invention is concerned with hydrogels made of crosslinked glycosaminoglycans, particularly crosslinked hyaluronic acid, chondroitin or chondroitin sulfate.

    Background of the invention



    [0002] Water-absorbing gels, or hydrogels, are widely used in the biomedical field. They are generally prepared by chemical crosslinking of polymers to infinite networks. While many polysaccharides absorb water until they are completely dissolved, crosslinked gels of the same polysaccharides can typically absorb a certain amount of water until they are saturated, i.e. they have a finite liquid retention capacity, or swelling degree.

    [0003] Hyaluronic acid, chondroitin and chondroitin sulfate are well-known biocompatible polymers. They are naturally occurring polysaccharides belonging to the group of glycosaminoglycans (GAGs). All GAGs are negatively charged heteropolysaccharide chains which have a capacity to absorb large amounts of water.

    [0004] Hyaluronic acid (HA) is one of the most widely used biocompatible polymers for medical and cosmetic use. HA is a naturally occurring polysaccharide belonging to the group of glycosaminoglycans (GAGs). 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.

    [0005] Chondroitin sulfate (CS) is a highly abundant GAG found in the connective tissues of mammals where it, together with other sulfated GAGs, is bound to proteins as part proteoglycans. It has previously been shown that hydrogels containing CS successfully can be used in biomedical applications due to their resemblance to the natural extra cellular matrix (Lauder, R.M., Complement Ther Med 17: 56-62, 2009). Chondroitin sulfate is also used in the treatment of osteoarthritis, e.g. as a dietary supplement.

    [0006] Crosslinking of the glycosaminoglycans prolongs the duration of the degradable polymers that make up the network, which is useful in may application. However, the crosslinking can also reduce the native properties of the glycosaminoglycans. Hence, it is typically desired to maintain a low degree of modification by efficient crosslinking to conserve the native properties and effects of the glycosaminoglycan itself.

    Summary of the invention



    [0007] It is an object of the present invention to provide a hydrogel having a glycosaminoglycan (GAG) as the swellable polymer.

    [0008] It is a further object of the present invention to provide a method for crosslinking GAG molecules with reduced effect of the native properties of the GAG molecules.

    [0009] It is also an object of the present invention to provide a method for preparing hydrogels of GAG molecules by mild and efficient routes.

    [0010] For these and other objects that will be evident from this disclosure, the present invention provides according to a first aspect a hydrogel product comprising glycosaminoglycan molecules as the swellable polymer, wherein the glycosaminoglycan molecules are covalently crosslinked via crosslinks essentially consisting of a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides, wherein the crosslinked glycosaminoglycan molecules are free, or essentially free from synthetic non-carbohydrate structures or linkers.

    [0011] With reference to the inventive processes of preparing hydrogel products described herein, the term "crosslinker" refers to a molecule having two or more functional groups, particularly nucleofile functional groups attached to a non-reactive spacer group, particularly a di-, tri-, tetra-, or oligosaccharide. Each of the two or more functional groups is capable of reacting with carboxylic acid groups on the GAG molecules to form stable covalent bonds. Preferably, the crosslinker consists of the two or more functional groups and the spacer.

    [0012] With reference to the inventive hydrogel products described herein, the term "crosslink" refers to the portion, or residue, of the crosslinker by which the GAG molecules are covalently linked after crosslinking. The crosslink typically consists of i) the spacer group and ii) the binding groups formed upon reaction of the functional groups of the crosslinker with the carboxylic acid groups on the GAG. The spacer group may for example be comprised of a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue.

    [0013] Crosslinking via crosslinkers comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides provides 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 di-, tri-, tetra-, or oligosaccharide is preferably well defined in terms of structure and molecular weight. Preferably the spacer is selected from one specific di-, tri-, tetra-, or oligosaccharide structure. Preferably, the di-, tri-, tetra-, or oligosaccharide is mono-disperse or has a narrow molecular weight distribution. Using well defined di-, tri-, tetra-, or oligosaccharide based crosslinkers together with a highly efficient condensation reaction allows the product to be assembled in a controlled fashion. 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 (e.g. diamino hyaluronic acid tetrasaccharide) or when crosslinking with a structure with high water retention properties (e.g. trehalose).

    [0014] The GAG may for example be sulfated or non-sulfated glycosaminoglycans such as hyaluronan, chondroitin sulphate, heparan sulphate, heparosan, heparin, dermatan sulphate and keratan sulphate. In some embodiments the GAG is hyaluronic acid, chondroitin or chondroitin sulfate. In a preferred embodiment the GAG is hyaluronic acid.

    [0015] In preferred embodiments, the GAG is a native GAG. The GAG used in connection with the invention is preferably a naturally occuring GAG. The GAG is preferably used in its native state. I.e., the chemical structure of the GAG has preferably not been altered or modified by addition of functional groups or the like. Using the GAG in its native state is preferred because this will afford a crosslinked structure more closely resembling the natural molecules, which conserves the native properties and effects of the GAG itself, and can minimize the immune response when the crosslinked GAG is introduced into the body.

    [0016] The covalently crosslinked GAG molecules preferably consist, or essentially consist of carbohydrate type structures or derivatives thereof. This means that the crosslinked GAG molecules are preferably free, or essentially free from synthetic non-carbohydrate structures or linkers. This can be achieved by using a GAG in its native state together with a crosslinker which consist, or essentially consist of carbohydrate type structures or derivatives thereof. Functional groups of the crosslinker are then covalently bound directly to carboxyl groups of the GAG. The crosslinks of the covalently crosslinked GAG thus preferably consist, or essentially consist of di-, tri-, tetra-, and oligosaccharide spacer groups.

    [0017] The present invention provides according to a second aspect a process of preparing a hydrogel product comprising crosslinked glycosaminoglycan molecules, comprising the steps of:
    1. (a) providing a solution of glycosaminoglycan molecules;
    2. (b) activating carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated, glycosaminoglycan molecules;
    3. (c) crosslinking the activated glycosaminoglycan molecules via their activated carboxyl groups using a di- or multinucleophile functional crosslinker comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides to obtain crosslinked glycosaminoglycan molecules.


    [0018] The present invention involves crosslinking of glycosaminoglycan molecules by covalent bonds, preferably amide bonds, typically using an activating agent for the carboxyl groups on the glycosaminoglycan molecule backbone and a di- or multinucleophile functional crosslinker comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides. Crosslinking according to the inventive method can be achieved by mild and efficient routes resulting in high yields with minimal degradation of the GAG molecules.

    [0019] The di- or multinucleophile functional crosslinker contains a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides, which remains in the crosslinks between the GAG molecules. The di- or multinucleophile functional di-, tri-, tetra-, and oligo-saccharides comprise at least two nucleophile functional groups attached thereto. The at least two nucleophile functional groups are preferably separated by the spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides.

    [0020] The di- or multinucleophile functional crosslinker comprises two or more functional groups capable of reacting with functional carboxyl groups of the GAG, resulting in the formation of covalent bonds, preferably amide bonds. The nucleophile functional groups are preferably capable of reacting with carboxyl groups on the glycosaminoglycan molecule to form amide bonds. In some embodiments the nucleophile functional groups of the di-, tri-, tetra-, and oligosaccharides are selected from the group consisting of primary amine, hydrazine, hydrazide, carbazate, semi-carbazide, thiosemicarbazide, thiocarbazate and aminoxy.

    [0021] The di- or multinucleophile functional di-, tri-, tetra-, and oligo-saccharides may be derived from nucleophile functional polysaccharides, such as chitobiose derived from chitin. The di- or multinucleophile functional di-, tri-, tetra-, and oligo-saccharides may also be di-, tri-, tetra-, and oligo-saccharides which have been modified by introduction of two or more nucleophile functional groups.

    [0022] A preferred group of di- or multinucleophile functional crosslinker includes homo- or heterobifunctional primary amines, hydrazines, hydrazides, carbazates, semi-carbazides, thiosemicarbazides, thiocarbazates and aminoxy.

    [0023] In certain embodiments, the activation step (b) and the crosslinking step (c) occur simultaneously. In other embodiments, the activation step (b) occurs prior to and separately from the crosslinking step (c).

    [0024] In a preferred embodiment, step (c) further comprises providing particles of the crosslinked GAG molecule, having an average size in the range of 0.01-5 mm, preferably 0.1-0.8 mm.

    [0025] In one preferred embodiment, the coupling agent of step (b) is a peptide coupling reagent. The peptide coupling reagent may be selected from the group consisting of triazine-based coupling reagents, carbodiimide coupling reagents, imidazolium-derived coupling reagents, Oxyma and COMU. A preferred peptide coupling reagent is a triazine-based coupling reagent, including the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), preferably DMTMM. Another preferred peptide coupling reagent is a carbodiimide coupling reagent, preferably N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) combined with N-hydroxysuccinimide (NHS).

    [0026] According to a related aspect, the present disclosure also provides use of the hydrogel product as a medicament, such as in the treatment of soft tissue disorders. There is provided a method of treating a patient suffering from a soft tissue disorder by administering to the patient a therapeutically effective amount of the hydrogel product. There is also provided a method of providing corrective or aesthetic treatment to a patient by administering to the patient a therapeutically effective amount of the hydrogel product.

    [0027] Other aspects and preferred embodiments of the present invention will be evident from the following detailed disclosure of the invention and the appended claims.

    [0028] In a preferred embodiment, at least 95 % of the bonds between glycosaminoglycan molecules and crosslinks are amide bonds.

    [0029] In a preferred embodiment, less than 5 % of the bonds between glycosaminoglycan molecules and crosslinks are ester bonds.

    [0030] In a preferred embodiment, less than 1 % of the bonds between glycosaminoglycan molecules and crosslinks are ester bonds.

    [0031] In a preferred embodiment, the crosslinking of step (c) provides amide bonds between glycosaminoglycan molecules and crosslinkers.

    [0032] In a preferred embodiment, the coupling agent and the crosslinker are added to the glycosaminoglycan simultaneously.

    [0033] In a preferred embodiment, step (c) further comprises providing particles of the crosslinked glycosaminoglycan, having an average size in the range of 0.01-5 mm, preferably 0.1-0.8 mm.

    [0034] In preferred 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.

    [0035] In a preferred embodiment, the process is further comprising the step:
    (d) subjecting the crosslinked glycosaminoglycan molecules obtained in step (c) to alkaline treatment.

    Detailed description of the invention



    [0036] The present invention provides advantageous processes for preparing hydrogels made of crosslinked glycosaminoglycan (GAG) molecules, the resulting hydrogel products and uses thereof. GAGs are negatively charged heteropolysaccharide chains which have a capacity to absorb large amounts of water. In the hydrogel products according to the invention, the crosslinked GAG molecule is the swellable polymer which provides the gel properties. The preparation process described herein is mild to the GAG molecules but provides an efficient crosslinking.

    [0037] Thus, the current invention provides GAG molecule hydrogels by crosslinking in aqueous media using di- or multinucleophile functional crosslinker capable of forming covalent bonds directly with carboxylic acid groups of GAG molecules by a reaction involving the use of a coupling agent.

    [0038] The GAG according to the invention is preferably selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate. In a preferred embodiment, the GAG molecule is hyaluronic acid. Hyaluronic acid (HA) is one of the most widely used biocompatible polymers for medical and cosmetic use. HA is a naturally occurring polysaccharide belonging to the group of glycosaminoglycans (GAGs). 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.

    [0039] 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. That is, the term also encompasses the various hyaluronate salts of hyaluronic acid with various counter ions, such as sodium hyaluronate. 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.

    [0040] The term "chondroitin" refers to GAGs having a disaccharide repeating unit consisting of alternating non-sulfated D-glucuronic acid and N-acetyl-D-galactosamine moieties. For avoidance of doubt, the term "chondroitin" does not encompass any form of chondroitin sulfate.

    [0041] The term "chondroitin sulfate" refers to GAGs having a disaccharide repeating unit consisting of alternating D-glucuronic acid and N-acetyl-D-galactosamine moieties. The sulfate moiety can be present in various different positions. Preferred chondroitin sulfate molecules are chondroitin-4-sulfate and chondroitin-6-sulfate.

    [0042] The chondroitin molecules 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 chondroitin molecule is typically in the range of 1-500 kDa, but other molecular weights are possible.

    [0043] The crosslinked GAG comprises crosslinks between the GAG molecule chains, which creates a continuous network of GAG molecules which is held together by the covalent crosslinks.

    [0044] The GAG molecule chains are preferably crosslinked to each other via crosslinkers comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides.

    [0045] It is preferred that the crosslinkers are bound to the glycosaminoglycan molecules by amide bonds.

    [0046] The crosslinked GAG product is preferably biocompatible. This implies that no, or only very mild, immune response occurs in the treated individual. That is, no or only very mild undesirable local or systemic effects occur in the treated individual.

    [0047] The crosslinked product according to the invention is a gel, or a hydrogel. That is, it can be regarded as a water-insoluble, but substantially dilute crosslinked system of GAG molecules when subjected to a liquid, typically an aqueous liquid.

    [0048] The gel 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 GAG molecule 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. It is also useful for treatment of soft tissue disorder and for corrective or aesthetic treatment. It is preferably used as an injectable formulation.

    [0049] Crosslinking of the GAG molecule may be achieved by activation with a coupling agent, followed by reaction with a crosslinking agent. The GAG molecule concentration and the extent of crosslinking affect the mechanical properties, e.g. the elastic modulus G', and stability properties, of the gel. Crosslinked GAG molecule gels can be characterized in terms of "degree of modification". The degree of modification of GAG molecule gels generally range between 0.01 and 15 mole%. The degree of modification (mole%) describes the amount of crosslinking agent(s) that is bound to the GAG molecule, i.e. molar amount of bound crosslinking agent(s) relative to the total molar amount of repeating disaccharide units. The degree of modification reflects to what degree the GAG molecule has been chemically modified by the crosslinking agent. Reaction conditions for activation and crosslinking and suitable analytical techniques for determining the degree of modification are all well known to the person skilled in the art, who easily can adjust these and other relevant factors and thereby provide suitable conditions to obtain a desirable degree of modification and verify the resulting product characteristics with respect to the degree of modification.

    [0050] The hydrogel product may also comprise a portion of GAG molecules which are not crosslinked, i.e not bound to the three-dimensional crosslinked GAG molecule network. However, it is preferred that at least 50 % by weight, preferably at least 60 % by weight, more preferably at least 70 % by weight, and most preferably at least 80 % by weight, of the GAG molecules in a gel composition form part of the crosslinked GAG molecule network.

    [0051] The crosslinked GAG molecule is preferably present in the form of gel particles. The gel particles preferably have an average size in the range of 0.01-5 mm, preferably 0.1-0.8 mm, such as 0.2-0.5 mm or 0.5-0.8 mm.

    [0052] The hydrogel product may be present in an aqueous solution, but it may also be present in dried or precipitated form, e.g. in ethanol. The hydrogel product is preferably injectable.

    [0053] The hydrogel product may be prepared by a process comprising the steps of:
    1. (a) providing a solution of glycosaminoglycan molecules;
    2. (b) activating carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated, glycosaminoglycan molecules;
    3. (c) crosslinking the activated glycosaminoglycan molecules via their activated carboxyl groups using a di- or multinucleophile functional crosslinker comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides to obtain crosslinked glycosaminoglycan molecules.


    [0054] The GAG according to the invention is preferably selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate. In a preferred embodiment, the GAG molecule is hyaluronic acid.

    [0055] In the activation step (b), the carboxyl groups on the GAG molecules are activated with a coupling agent to form activated GAG molecules.

    [0056] In one preferred embodiment, the peptide coupling reagent is selected from the group consisting of triazine-based coupling reagents, carbodiimide coupling reagents, imidazolium-derived coupling reagents, Oxyma and COMU.

    [0057] The peptide coupling reagent is preferably a triazine-based coupling reagent, such as 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). A preferred triazine-based peptide coupling reagent is DMTMM.

    [0058] Other preferred peptide coupling reagent are carbodiimide coupling reagents, preferably N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) combined with N-hydroxysuccinimide (NHS).

    [0059] In the crosslinking step (c), crosslinking of the activated GAG molecules occurs via their carboxyl groups using a crosslinker. The crosslinker is a di- or multinucleophile functional crosslinker comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides. The crosslinker connects the GAG chains to each other via carboxyl groups on the GAG backbone. The spacer group may for example be a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue. By the term "residue" is meant here that the structure of the compound is similar but not identical to the patent compounds hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose respectively. The structure of the residue may differ from the structure of the parent compound in that it has been provided with two or more nucleofile functional groups and optionally covalently linked via said nucleofile functional groups carboxyl groups on the GAG backbone.

    [0060] The di- or multinucleophile functional crosslinker comprises two or more functional groups capable of reacting with functional carboxyl groups of the GAG, resulting in the formation of covalent bonds, preferably amide bonds.

    [0061] A preferred group of di- or multinucleophile functional crosslinker includes homo- or heterobifunctional primary amines, hydrazines, hydrazides, carbazates, semi-carbazides, thiosemicarbazides, thiocarbazates and aminoxy. Non limiting examples of such heterobifunctional crosslinkers useful in the present invention include:

    Diaminotrehalose (6,6'-diamino-6,6'-dideoxy trehalose);

    Diaminosucrose (6,6'-diamino-6,6'-dideoxy sucrose);

    Chitobiose (2,2'-diamino-2,2'-dideoxy cellobiose);

    Diaminolactose (6,6'-diamino-6,6'-dideoxy lactose);

    "Reduced N-Deacetylated hyaluronic acid tetrasaccaride" or "Reduced diamino hyaluronic acid tetrasaccharide"; and

    Diaminoraffinose (6,6"-diamino-6,6"-dideoxy raffinose).

    [0062] Reaction schemes 1a-1h schematically illustrate examples of coupling by heterobifunctional primary amine (1a), aminoxy (1b), carbazate (1c), semi-carbazide (1d), thiosemicarbazide (1e), thiocarbazate (1f), hydrazine (1g) and hydrazide (1h)

















    [0063] The di- or multinucleophile functional crosslinker contains a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides, which remains in the crosslinks between the GAG molecules.

    [0064] The process may be performed in a one-pot approach in aqueous media, involving the covalent coupling of di- or multinucleophile functional crosslinkers directly to inherent carboxylic acid groups on the native GAGs using a suitable coupling agent. In a preferred embodiment, the activation step (b) and the crosslinking step (c) occur simultaneously.

    [0065] In another embodiment, the activation step (b) occurs prior to and separately from the crosslinking step (c).

    [0066] The process for generating the crosslinked hydrogel typically involves preparing a mixture of a GAG molecule, such as hyaluronic acid together with a crosslinker agent, such as diamino trehalose, DATH, (0.001 - 10 molar equivalents of amine towards carboxylic acid groups, or preferably 0.001 - 1 molar equivalents) and a coupling agent such as DMTMM (0.01 - 10 molar equivalents to carboxylic acid groups, or preferably 0.05 - 1 molar equivalents). Incubating the mixture at 5 - 50 °C, preferably 10 - 40 °C or even more preferred 20 - 35 °C, during 2 - 120 hours, preferably 4 - 48 hours, followed by alkaline treatment, neutralization, precipitation, washing and dried under vacuum, yields a crosslinked polysaccharide as a solid. The precipitate was swelled in phosphate buffer containing NaCl to form a hydrogel, the hydrogel is preferably micronized to hydrogel particles in the size of 0.01 - 5 mm, preferably 0.1 - 1 mm.

    [0067] A typical application of the resulting hydrogel product involves the preparation of injectable formulations for treatment of soft tissue disorders, including, but not limited to, corrective and aesthetic treatments.

    [0068] In one more specific embodiment, crosslinking of chondroitin sulfate with DATH may be achieved as follows:
    Diaminotrehalose (DATH) is 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.

    [0069] Chondroitin Sulfate (CS) (10 - 200 kDa) is weighed in a Falcon tube. A stock solution of diaminotrehalose (DATH) is prepared by dissolving DATH in phosphate buffer pH 7.4. DMTMM is weighed in a vessel and the DATH-solution is added to the DMTMM. The pH of the DMTMM-DATH solution is adjusted to approx. 7 by addition of 1.2 M HCI or 0.25 M NaOH, and the mixture is subsequently added to CS. The contents are thoroughly homogenized and then incubated at 15-55 °C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh two times and swelled in NaOH. The gel is neutralized with 1.2 M HCI to pH 7 and precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The obtained solid is dried at 25 °C under vacuum. The precipitate is swelled in 0.7 % NaCl phosphate buffer pH 7.4 and pressed through a filter mesh three times. The crosslinked CS-gel is filled on syringes and sterilized.

    [0070] In another more specific embodiment, crosslinking of HA with diaminosucrose may be achieved as follows:
    Diaminosucrose is prepared as described in "Library of mild and economic protocols for the selective derivatization of sucrose under microwave irradiation"; M. Teresa Barros, Krasimira T. Petrova, Paula Correia-da-Silva and Taterao M. Potewar; Green Chem., 2011, 13, 1897-1906.

    [0071] Hyaluronic acid (HA) (10 - 1 000 kDa) is weighed in a vessel. A stock solution of diaminosucrose is prepared by dissolving diaminosucrose in phosphate buffer pH 7.4. DMTMM is weighed in a vessel and the diaminosucrose-solution is added to the DMTMM. The pH of the DMTMM-diaminosucrose solution is adjusted to approx. 7 by addition of 1.2 M HCI or 0.25 M NaOH, and the mixture is subsequent added to HA. The contents are thoroughly homogenized and then incubated at 15-55 °C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh two times and swelled in NaOH. The gel is neutralized with 1.2 M HCI to pH 7 and precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The obtained solid is dried at 25 °C under vacuum. The precipitate is swelled in 0.7 % NaCl phosphate buffer pH 7.4 and pressed through a filter mesh three times. The crosslinked HA-gel is filled on syringes and sterilized.

    [0072] In another more specific embodiment, crosslinking of HA with chitobiose may be achieved as follows:
    Hyaluronic acid (HA) (10 - 1 000 kDa) is weighed in a vessel. A stock solution of chitiobiose (purchased from Carbosynth Ltd. UK) is prepared by dissolving chitobiose in phosphate buffer pH 7.4. DMTMM is weighed in a vessel and the chitobiose-solution is added to the DMTMM. The pH of the DMTMM-chitobiose solution is adjusted to approx. 7 by addition of 1.2 M HCI or 0.25 M NaOH, and the mixture is subsequent added to HA. The contents are thoroughly homogenized and then incubated at 15-55 °C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh two times and swelled in NaOH. The gel is neutralized with 1.2 M HCI to pH 7 and then precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The obtained solid is dried at 25 °C under vacuum. The precipitate is swelled in 0.7 % NaCl phosphate buffer pH 7.4 and pressed through a filter mesh three times. The crosslinked HA-gel is filled on syringes and sterilized.

    [0073] In another more specific embodiment, crosslinking of HA with a reduced diamino HA-tetrasaccharide may be achieved as follows:
    Hyaluronic acid (HA) (10 - 1 000 kDa) is weighed in a vessel. A stock solution of a reduced diamino HA-tetrasaccharide is prepared by dissolving reduced diamino HA-tetrasaccharide in phosphate buffer pH 7.4. DMTMM is weighed in a vessel and the reduced diamino HA-tetrasaccharide solution is added to the DMTMM. The pH of the DMTMM and reduced diamino HA-tetrasaccharide solution is adjusted to approx. 7 by addition of 1.2 M HCI or 0.25 M NaOH, and the mixture is subsequent added to HA. The contents are thoroughly homogenized and incubated at 15-55 °C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh two times and swelled in NaOH. The gel is neutralized with 1.2 M HCI to pH 7 and precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The obtained solid is dried at 25 °C under vacuum. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and pressed through a filter mesh three times. The crosslinked HA-gel is filled on syringes and sterilized.

    [0074] In another more specific embodiment, crosslinking of HA with dicarbazate trehalose may be achieved as follows:
    α,α-D-Trehalose (1 equiv.) (anhydrous) (Carbosynth Ltd. UK) is dissolved in dry dimethylformamid (DMF), and triethylamine (2-6 equiv.) is added subsequently. The flask is cooled to 0 °C (ice/water) and under N2-atmosphere. 4-Nitrophenyl chloroformate (2-6 equiv.) is added into the flask dropwise. The resulting mixture is allowed to stir at room temperature for 2-48 h and then concentrated, purified by FC and dried under vacuum. The product is dissolved in DMF, and hydrazine monohydrate (2-20 equiv.) is added to the solution and stirred at 0 - 50 °C for 4 - 48 h. The reaction is then concentrated, purified by FC and dried under vacuum to obtain α,α-D-6,6'-dideoxy-6,6'-dicarbazate trehalose (dicarbazate trehalose, DCT).

    [0075] Hyaluronic acid (HA) (10 - 1 000 kDa) is weighed in a vessel. A stock solution of dicarbazate trehalose (DCT) is prepared by dissolving DCT in phosphate buffer pH 7.4. DMTMM is weighed in a vessel and the DCT-solution is added to the DMTMM. The pH of the DMTMM-DCT solution is adjusted to approx. 7 by addition of 1.2 M HCI or 0.25 M NaOH, and the mixture is subsequent added to HA. The contents are thoroughly homogenized and incubated at 15-55 °C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh two times and swelled in NaOH. The gel is neutralized with 1.2 M HCI to pH 7 and precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The obtained solid is dried at 25 °C under vacuum. The precipitate is swelled in 0.7 % NaCl phosphate buffer pH 7.4 and then pressed through a filter mesh three times. The crosslinked HA-gel is filled on syringes and sterilized.

    [0076] In another more specific embodiment, crosslinking of HA with diaminoxytrehalose may be achieved as follows:
    To a stirred suspension of α,α-D-Trehalose (1 equiv.) (anhydrous) (Carbosynth Ltd. UK) in anhydrous THF, N-hydroxyphthalimide (2-10 equiv.) and triphenylphosphine (2-10 equiv.) is added, and the mixture is stirred for 5-60 min. Diisopropyl azodicarboxylate (DIAD, 2-10 equiv.) is then added dropwise at 0 - 40 °C and the mixture is stirred for 2-48 h at 0 - 40 °C. The solvent is removed in vacuo and the crude product is purified by FC and dried under vacuum. A suspension of the product in a mixture of MeOH and CH2Cl2 is treated with hydrazine monohydrate (2-20 qeuiv.), and the mixture is stirred at 0 - 40 °C for 2-24 h followed by concentration, purification by FC and drying under vacuum to obtain the diaminoxytrehalose.

    [0077] Hyaluronic acid (HA) (10 - 1 000 kDa) is weighed in a vessel. A stock solution of diaminoxytrehalose (DAOT) is prepared by dissolving DAOT in phosphate buffer pH 7.4. DMTMM is weighed in a vessel and the DAOT-solution is added to the DMTMM. The pH of the DMTMM-DAOT solution is adjusted to approx. 7 by addition of 1.2 M HCI or 0.25 M NaOH, and the mixture is subsequent added to HA. The contents are thoroughly homogenized and incubated at 15-55 °C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh two times and swelled in NaOH. The gel is neutralized with 1.2 M HCI to pH 7 and precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The obtained solid is dried at 25 °C under vacuum. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and then pressed through a filter mesh three times. The crosslinked HA-gel is filled on syringes and sterilized.

    EXAMPLES



    [0078] Without desiring to be limited thereto, the present invention will in the following be illustrated by way of examples.

    Definitions and Analysis



    [0079] 

    SwF - Swelling factor analysis was done in saline.

    [PS] - Polysaccharide concentration, e.g. HA concentration. The PS concentration was measured with LC-SEC-UV or NIR.

    GelP - Gel part (also sometimes referred to as gel content or GeIC) is a description of the percentage of polysaccharide that is a part of the gel network. A number of 90% means that only 10% of polysaccharide is not a part of the network. The amount of free polysaccharide in the gel was measured with LC-SEC-UV.

    SwC - swelling capacity is the total liquid uptake of one gram polysaccharide, not corrected for gel part.

    SwCC - Corrected swelling capacity (also sometimes referred to as SwDC) is the total liquid uptake of one gram polysaccharide, corrected for gel part.

    CrR - Effective crosslinking ratio was analyzed with LC-SEC-MS and defined as:



    [0080] A CrR of 1.0 means that all of the crosslinker has crosslinked.

    Alkaline or heat hydrolysis



    [0081] In some of the examples below, the product was subjected to alkaline or heat hydrolysis in order to hydrolyze ester bonds formed during the crosslinking process. The alkaline/heat hydrolysis results in only amide crosslink bonds in the end product. The alkaline/heat hydrolysis was performed as follows:

    Alkaline hydrolysis



    [0082] The material was swelled in 0.25 M NaOH (1 g material : 9 g 0.25 M NaOH resulting in pH 13) for at least 1 h at room temperature. The gel was neutralized with 1.2 M HCI to pH 7 and then precipitated with ethanol. The resulting precipitate was washed with 100 mM NaCl in 70% ethanol to remove excess reagents and then with 70% ethanol to remove salts and finally with ethanol to remove water. Ethanol was removed in a vacuum dryer overnight.

    [0083] The precipitate was swelled in 0.7% NaCl phosphate buffer pH 7.4 and then pressed through a fine filter mesh three times. The gel was filled on syringes and sterilized. In some cases a couple of the syringes were not sterilized to see the effect of sterilization.

    Heat hydrolysis



    [0084] The material was swelled in 0.7% NaCl phosphate buffer pH 7.4 at room temperature. The pH was adjusted to 7.2-7.5 if needed. The gel was left at 70 °C for 20-24 h and then particle-size reduced through a fine filter mesh three times. The gel was filled on syringes and sterilized. In some cases a couple of the syringes were not sterilized to see the effect of sterilization.

    Synthesis of hyaluronic diaminotrehalose



    [0085] Diaminotrehalose (DATH) was synthesized as described in "Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties"; Keisuke Kurita,* Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, 7544-7549.

    Example 1 - Crosslinking of Hyaluronic Acid with diaminotrehalose (DATH)



    [0086] A series of experiments (Examples 1-1 to 1-5) were performed which involved crosslinking of hyaluronic acid (HA) of different molecular weights with various molar ratios of DATH using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling agent. Ratios between HA, DATH and DMTMM are set out in Table 1 below.

    [0087] The hyaluronan (molecular weight (Mw) from about 100 kDa to about 1 000 kDa) was weighed in a Falcon tube. A stock solution of diaminotrehalose (DATH) was prepared by dissolving DATH (0.001 - 0.005 equiv.) in phosphate buffer pH 7.4. DMTMM (0.05 equiv.) 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 or 0.25 M NaOH and then added to the HA. The contents were thoroughly homogenized and then incubated at 35 °C for 24 h.

    [0088] The resulting material was pressed through a 1 mm steel mesh two times and then treated with a NaOH solution. The gel was neutralized with 1.2 M HCI to pH 7 and then precipitated with ethanol. The resulting precipitate was washed with 100 mM NaCl in 70% ethanol to remove excess reagents and then with 70% ethanol to remove salts and finally with ethanol to remove water. Ethanol was removed in a vacuum dryer over night.

    [0089] The precipitate was swelled in 0.7% NaCl phosphate buffer pH 7.4 and then pressed through a filter mesh three times. The gel was filled on syringes and sterilized.
    Table 1.
    Example<Mw> (MDa)Eq DMTMMEq DATHMonophasicat [HA] 50mg/mLCrRGelC (%)SwCC (ml/g)
    1-1 1.06 0.05 0.003 No 0.99 NA NA
    1-2 1.06 0.05 0.001 Yes 0.99 74 101
    1-3 0.64 0.05 0.005 Yes NA 93 38
    1-4 0.31 0.05 0.005 Yes NA 85 49
    1-5 0.11 0.05 0.005 Yes NA 84 123
    NA - not available since the analysis was not done

    Example 2 - Crosslinking of Hyaluronic Acid with diaminotrehalose (DATH)



    [0090] A series of experiments (Examples 2-1 to 2-11) were performed which involved crosslinking of hyaluronic acid (HA) of different molecular weights with various molar ratios of DATH using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling agent. Ratios between HA, DATH and DMTMM are set out in Table 2 below.

    [0091] Hyaluronic acid was weighed in a reaction vessel. A stock solution of the crosslinker (DATH) was prepared by dissolving it in phosphate buffer pH 7.4. DMTMM was weighed in a PTFE-container and the crosslinker-solution was added to the DMTMM to dissolve it. The pH of the DMTMM-crosslinker solution was adjusted to 6-7 with 1.2 M HCI or 0.25 M NaOH and 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 treated with either heat or alkaline. The results are displayed in Table 2.
    Table 2. Summary crosslinking Hyaluronic Acid with (DATH)
    Example<Mw> (MDa)DMTMM/HA (mol%)DATH/HA (mol%)DMTMM/ DATHHydrolysisCrRGelP after sterilization (%)SwCC (ml/g)
    2-1 1 5.0 0.08 61 Alkaline 0.99 74 101
    2-2 1 0.7 0.06 12 Heat 0.51 45 309
    2-3 1 2.4 0.03 74 Heat 0.94 55 259
    2-4 (n = 4) 0.6 1.2 0.32 3.6 Heat 0.45 72±4 132±23
    2-5 (n = 4) 0.6 1.2 0.32 3.6 Alkaline 0.43 63±1 218+13
    2-6 0.3 5.0 0.25 20 Alkaline   77 79
    2-7 0.2 4.9 0.57 8.7 Heat 0.86 77 106
    2-8 0.2 4.0 0.57 7.0 Alkaline 0.63 52 445
    2-9 0.1 4.5 0.65 7.0 Heat 0.55 50 337
    2-10 0.1 6.9 0.79 8.8 Heat 0.89 91 91
    2-11 0.1 5.5 0.65 8.5 Heat 0.75 87 146
    Empty cells - no analysis done.

    Example 3 - Crosslinking of Hyaluronic Acid with Chitobiose (CB)



    [0092] Hyaluronic acid was weighed in a reaction vessel. A stock solution of the crosslinker (chitobiose) was prepared by dissolving it in phosphate buffer pH 7.4. DMTMM was weighed in a PTFE-container and the crosslinker-solution was added to the DMTMM to dissolve it. The pH of the DMTMM-crosslinker solution was adjusted to 6-7 with 1.2 M HCI and then added to the HA. The contents 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 treated with either heat or alkaline according to the general procedures. Ratios between HA, chitobiose and DMTMM are set out below in Table 3 (Examples 3-1 to 3-2).

    Example 4 - Crosslinking of Hyaluronic Acid with diaminotetra-HA (DA-4HA)



    [0093] Diaminotetra-HA (DA-4HA) was synthesized according to below scheme:


    Step 1



    [0094] A solution of HA-4 (500 mg, 0.61 mmol) in water (5 ml) at room temperature was treated with sodium borohyride (23.05 mg, 0.61 mmol) and the resulting solution was stirred for 3 h, concentrated to dryness to afford the reduced product 1 (532 mg, assumed 100%) as a white foam.
    LCMS (tr = 0.28 min., ES+ = 779.4 (M-2 Na + 2H)

    Step 2



    [0095] The reduced product 1 (532 mg) was dissolved in aqueous NH2OH (5 ml, 50% v/v/) and solid NH4l (100 mg) was added. The resulting suspension was heated at 70 °C for 48 h, cooled to room temperature and concentrated to dryness to afford a residue. The residue was precipitated in neat EtOH and the resulting precipitate was collected by filtration and dried to a constant weight to afford the a 1:1 mixture of diamine 2 and mono-amine 3 in quantitative yield. The crude reaction product was used without further purification.
    2 : LCMS (tr = 0.16 min., ES+ = 695.36 (M-2 Na + 2H)
    3: LCMS (tr = 0.19 min., ES+ = 737.47 (M-2 Na + 2H)

    [0096] Hyaluronic acid was weighed in a reaction vessel. A stock solution of the crosslinker (diaminotetra-HA), synthesized as described above, and DMTMM respectively were prepared by dissolving it in phosphate buffer pH 7.4. The pH of the solutions were adjusted to 7 and then added to the HA. The contents were thoroughly homogenized and then incubated at 23 °C for 24 h. The resulting material was pressed through a 1 mm steel mesh two times and then treated with heat according to the general procedures. Ratio between HA, diaminotetra-HA and DMTMM are set out below in Table 3 (Example 4).

    Example 5 - Crosslinking of Heparosan (HEP) with diaminotrehalose (DATH)



    [0097] The coupling agent DMTMM and the crosslinker DATH were weighed in separate reaction vessels and dissolved in phosphate buffer (pH 7.4). The solutions pH was adjusted to pH 7-7.5 with 1.2 M HCI or 0.25 M NaOH. Thereafter, DMTMM- and DATH-solutions were successively added to the heparosan weighed in a reaction vessel. 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 treated with heat according to the general procedures. Ratios between heparosan, DATH and DMTMM are set out in Table 3 below (Examples 5-1 to 5-2).

    Example 6 - Crosslinking of Chondroitin Sulfate (CS) with diaminotrehalose (DATH)



    [0098] The coupling agent DMTMM and the crosslinker DATH were weighed in separate reaction vessels and dissolved in phosphate buffer (pH 7.4). The solutions pH was adjusted to pH 7-7.5 with 1.2 M HCI or 0.25 M NaOH. Thereafter, DMTMM- and DATH-solutions were successively added to the chondroitin sulfate weighed in a reaction vessel. 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 treated with heat according to the general procedures. Ratios between chondroitin sulfate, DATH and DMTMM are set out below in Table 3 (Examples 6-1 to 6-2).
    Table 3. Summary crosslinking examples 3-6
    ExamplePS MwCrosslinkerDMTMM/PS (mol%)Crosslinker/PS (mol%)DMTMM/crosslinkerHydrolysisGelP (%)SwCC (mL/g)SwC (mL/g)G' 0.1 Hz (kPa)
    3-1 HA CB 2.4 0.13 18.5 Heat 60 224    
    1 MDa
    3-2 HA CB 2.4 0.13 18.5 Alkaline 45 335    
    1 MDa
    4 HA 0.2 MDa DA-4HA 24 1.0 24 Heat     47  
     
    5-1 HEP DATH 7 1.0 7 Heat 36 175    
    140 kDa
    5-2 HEP DATH 10.5 1.5 7 Heat 80 101    
    140 kDa
    6-1 CS DATH 35 5.0 7.0 Heat     48 1.6
    30 kDA
    6-2 CS DATH 35 5.0 7.0 Heat     44 1.5
    30 kDa
    PS = polysaccharide, HA = hyaluronan, HEP = heparosan, CS = chondroitin sulfate, DA-4HA = diaminotetra-HA, CB = chitobiose
    Empty cells - no analysis done.



    Claims

    1. A hydrogel product comprising glycosaminoglycan molecules as the swellable polymer, wherein the glycosaminoglycan molecules are covalently crosslinked via crosslinks essentially consisting of a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides, wherein the crosslinked glycosaminoglycan molecules are free, or essentially free from synthetic non-carbohydrate structures or linkers.
     
    2. A hydrogel product according to claim 1, wherein the glycosaminoglycan molecules are hyaluronic acid.
     
    3. A hydrogel product according to any one of the preceding claims, wherein the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue.
     
    4. A hydrogel product according to any one of the preceding claims, wherein the spacer group is selected from the group consisting of di-, tri-, and tetrasaccharides.
     
    5. A hydrogel product according to any one of the preceding claims, wherein at least 90 % of the bonds between glycosaminoglycan molecules and crosslinks are amide bonds.
     
    6. A hydrogel product according to any one of the preceding claims, wherein less than 5 % of the bonds between glycosaminoglycan molecules and crosslinks are ester bonds.
     
    7. A hydrogel product according to any one of the preceding claims, in the form of an injectable formulation.
     
    8. A process of preparing a hydrogel product comprising crosslinked glycosaminoglycan molecules, comprising the steps of:

    (a) providing a solution of glycosaminoglycan molecules;

    (b) activating carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated, glycosaminoglycan molecules;

    (c) crosslinking the activated glycosaminoglycan molecules via their activated carboxyl groups using a di- or multinucleophile functional crosslinker comprising a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides to obtain crosslinked glycosaminoglycan molecules, wherein the crosslinked glycosaminoglycan molecules are free, or essentially free from synthetic non-carbohydrate structures or linkers.


     
    9. A process according to claim 8, wherein the glycosaminoglycan molecules are hyaluronic acid.
     
    10. A process according to any one of claims 8-9, wherein the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue.
     
    11. A process according to any one of claims 8-10, wherein the spacer group is selected from the group consisting of di-, tri-, and tetrasaccharides.
     
    12. A process according to any one of claims 8-11, wherein the nucleophilic groups of the crosslinker are selected from the group consisting of primary amine, hydrazine, hydrazide, carbazate, semi-carbazide, thiosemicarbazide, thiocarbazate and aminoxy.
     
    13. A process according to any one of claims 8-12, wherein the crosslinking of step (c) provides amide bonds between glycosaminoglycan molecules and crosslinkers.
     
    14. A process according to any one of claims 8-13, wherein the coupling agent is a triazine-based coupling reagent, preferably DMTMM.
     
    15. A method of cosmetically treating skin, which comprises administering to the skin a hydrogel product according to any one of claims 1-7 or a hydrogel product obtainable by a method according to any one of claims 8-14.
     


    Ansprüche

    1. Hydrogelprodukt, umfassend Glykosaminoglykanmoleküle als quellbares Polymer, wobei die Glykosaminoglykanmoleküle über im Wesentlichen aus einer aus der aus Di-, Tri-, Tetra- und Oligosacchariden bestehenden Gruppe ausgewählten Spacer-Gruppe bestehenden Quervernetzungen kovalent quervernetzt sind, wobei die quervernetzten Glykosaminoglykanmoleküle frei oder im Wesentlichen frei von synthetischen Nichtkohlenhydratstrukturen oder -linkern sind.
     
    2. Hydrogelprodukt nach Anspruch 1, wobei es sich bei den Glykosaminoglykanmolekülen um Hyaluronsäure handelt.
     
    3. Hydrogelprodukt nach einem der vorhergehenden Ansprüche, wobei es sich bei der Spacer-Gruppe um einen Hyaluronsäuretetrasaccharid-, Hyaluronsäurehexasaccharid-, Trehalose-, Lactose-, Maltose-, Saccharose-, Cellobiose- oder Raffinoserest handelt.
     
    4. Hydrogelprodukt nach einem der vorhergehenden Ansprüche, wobei die Spacer-Gruppe aus der aus Di-, Tri- und Tetrasacchariden bestehenden Gruppe ausgewählt ist.
     
    5. Hydrogelprodukt nach einem der vorhergehenden Ansprüche, wobei es sich bei mindestens 90% der Bindungen zwischen Glykosaminoglykanmolekülen und Quervernetzungen um Amidbindungen handelt.
     
    6. Hydrogelprodukt nach einem der vorhergehenden Ansprüche, wobei es sich bei weniger als 5% der Bindungen zwischen Glykosaminoglykanmolekülen und Quervernetzungen um Esterbindungen handelt.
     
    7. Hydrogelprodukt nach einem der vorhergehenden Ansprüche in Form einer Injektionsformulierung.
     
    8. Verfahren zur Herstellung eines quervernetzte Glykosaminoglykanmoleküle umfassenden Hydrogelprodukts, welches die folgenden Schritte umfasst:

    (a) die Bereitstellung einer Lösung von Glykosaminoglykanmolekülen,

    (b) die Aktivierung von Carboxylgruppen an den Glykosaminoglykanmolekülen mit einem Kupplungsmittel unter Bildung aktivierter Glykosaminoglykanmoleküle,

    (c) das Quervernetzen der aktivierten Glykosaminoglykanmoleküle über ihre aktivierten Carboxylgruppen unter Verwendung eines di- oder multinukleophil funktionalen Quervernetzers, welcher eine Spacer-Gruppe ausgewählt aus der aus Di-, Tri-, Tetra- und Oligosacchariden bestehenden Gruppe umfasst, unter Erhalt von quervernetzten Glykosaminoglykanmolekülen, wobei die quervernetzten Glykosaminoglykanmoleküle frei oder im Wesentlichen frei von synthetischen Nichtkohlenhydratstrukturen oder -linkern sind.


     
    9. Verfahren nach Anspruch 8, wobei es sich bei den Glykosaminoglykanmolekülen um Hyaluronsäure handelt.
     
    10. Verfahren nach einem der Ansprüche 8-9, wobei es sich bei der Spacer-Gruppe um einen Hyaluronsäuretetrasaccharid-, Hyaluronsäurehexasaccharid-, Trehalose-, Lactose-, Maltose-, Saccharose-, Cellobiose- oder Raffinoserest handelt.
     
    11. Verfahren nach einem der Ansprüche 8-10, wobei die Spacer-Gruppe aus der aus Di-, Tri- und Tetrasacchariden bestehenden Gruppe ausgewählt ist.
     
    12. Verfahren nach einem der Ansprüche 8-11, wobei die nukleophilen Gruppen des Quervernetzers aus der aus primärem Amin, Hydrazin, Hydrazid, Carbazat, Semicarbazid, Thiosemicarbazid, Thiocarbazat und Aminooxy bestehenden Gruppe ausgewählt sind.
     
    13. Verfahren nach einem der Ansprüche 8-12, wobei bei dem Quervernetzen von Schritt (c) Amidbindungen zwischen Glykosaminoglykanmolekülen und Quervernetzern ausgebildet werden.
     
    14. Verfahren nach einem der Ansprüche 8-13, wobei es sich bei dem Kupplungsmittel um ein Kupplungsmittel auf Triazinbasis, vorzugsweise DMTMM, handelt.
     
    15. Verfahren zur kosmetischen Behandlung von Haut, bei dem man ein Hydrogelprodukt nach einem der Ansprüche 1-7 oder ein durch ein Verfahren nach einem der Ansprüche 8-14 erhältliches Hydrogelprodukt auf die Haut verabreicht.
     


    Revendications

    1. Produit d'hydrogel comprenant des molécules de glycosaminoglycane en tant que polymère expansible, dans lequel les molécules de glycosaminoglycane sont réticulées de façon covalente via des liaisons de réticulation essentiellement constituées d'un groupement espaceur choisi dans le groupe constitué par les di-, tri-, tétra- et oligosaccharides, dans lequel les molécules de glycosaminoglycane réticulées sont exemptes, ou essentiellement exemptes, de structures ou de ponts synthétiques autres qu'hydrates de carbone.
     
    2. Produit d'hydrogel selon la revendication 1, dans lequel les molécules de glycosaminoglycane sont l'acide hyaluronique.
     
    3. Produit d'hydrogel selon l'une quelconque des revendications précédentes, dans lequel le groupement espaceur est un résidu de tétrasaccharide d'acide hyaluronique, d'hexasaccharide d'acide hyaluronique, de tréhalose, de lactose, de maltose, de sucrose, de cellobiose ou de raffinose.
     
    4. Produit d'hydrogel selon l'une quelconque des revendications précédentes, dans lequel le groupement espaceur est choisi dans le groupe constitué par les di- , tri- et tétrasaccharides.
     
    5. Produit d'hydrogel selon l'une quelconque des revendications précédentes, dans lequel au moins 90 % des liaisons entre les molécules de glycosaminoglycane et les liaisons de réticulation sont des liaisons amide.
     
    6. Produit d'hydrogel selon l'une quelconque des revendications précédentes, dans lequel moins de 5 % des liaisons entre les molécules de glycosaminoglycane et les liaisons de réticulation sont des liaisons ester.
     
    7. Produit d'hydrogel selon l'une quelconque des revendications précédentes, sous la forme d'une formule pour injection.
     
    8. Procédé de préparation d'un produit d'hydrogel comprenant des molécules de glycosaminoglycane réticulées, comprenant les étapes consistant à :

    (a) se munir d'une solution de molécules de glycosaminoglycane ;

    (b) activer des groupements carboxyle sur les molécules de glycosaminoglycane avec un agent de couplage pour former des molécules de glycosaminoglycane activées ;

    (c) réticuler les molécules de glycosaminoglycane activées via leurs groupements carboxyle activés en utilisant un agent de réticulation fonctionnel de type di- ou multinucléophile comprenant un groupement espaceur choisi dans le groupe constitué par les di-, tri-, tétra- et oligosaccharides pour obtenir des molécules de glycosaminoglycane réticulées, dans lequel les molécules de glycosaminoglycane réticulées sont exemptes, ou essentiellement exemptes, de structures ou de ponts synthétiques autres qu'hydrates de carbone.


     
    9. Procédé selon la revendication 8, dans lequel les molécules de glycosaminoglycane sont l'acide hyaluronique.
     
    10. Procédé selon l'une quelconque des revendications 8 et 9, dans lequel le groupement espaceur est un résidu de tétrasaccharide d'acide hyaluronique, d'hexasaccharide d'acide hyaluronique, de tréhalose, de lactose, de maltose, de sucrose, de cellobiose ou de raffinose.
     
    11. Procédé selon l'une quelconque des revendications 8 à 10, dans lequel le groupement espaceur est choisi dans le groupe constitué par les di-, tri- et tétrasaccharides.
     
    12. Procédé selon l'une quelconque des revendications 8 à 11, dans lequel les groupements nucléophiles de l'agent de réticulation sont choisis dans le groupe constitué par amine primaire, hydrazine, hydrazide, carbazate, semi-carbazide, thiosemicarbazide, thiocarbazate et aminoxy.
     
    13. Procédé selon l'une quelconque des revendications 8 à 12, dans lequel la réticulation de l'étape (c) génère des liaisons amide entre les molécules de glycosaminoglycane et les agents de réticulation.
     
    14. Procédé selon l'une quelconque des revendications 8 à 13, dans lequel l'agent de couplage est un réactif de couplage à base de triazine, préférentiellement le DMTMM.
     
    15. Méthode de traitement cosmétique de la peau, qui comprend l'administration à la peau d'un produit d'hydrogel selon l'une quelconque des revendications 1 à 7 ou d'un produit d'hydrogel pouvant être obtenu par un procédé selon l'une quelconque des revendications 8 à 14.
     




    REFERENCES CITED IN THE DESCRIPTION



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    Non-patent literature cited in the description