RU2783125C2 - Injection gel product - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention proposes a method for the production of an injection gel product, including stages of a) cross-linking of the first glycosaminoglycan (hereinafter – GAG) with the first cross-linking agent to obtain gel, in which the ratio of loading of the cross-linking agent to disaccharide links is less than 0.15; b) obtaining particles of gel from the stage a); c) mixing of particles of GAG gel from the stage b) with the second GAG to obtain a mixture; d) cross-linking of the mixture obtained at the stage c) with the second cross-linking agent to form transverse crosslink between GAGs of the second external phase to obtain gel having the first internal phase of particles of cross-linked GAG gel, embedded in gel of the second external phase containing the second GAG; and e) obtaining injection particles of gel from the stage d), wherein each such a particle contains a set of cross-linked particles of GAG gel of the first internal phase. The present invention also proposes an injection gel product, and an aqueous composition based on it.

EFFECT: obtaining an injection gel product.

49 cl, 9 ex, 6 dwg

Description

Technical field

The present invention relates to the field of polysaccharide gels. More specifically, the present invention relates to injectable gel products for use in cosmetic surgery.

Prior Art

One of the most widely used biocompatible polymers in medicine is hyaluronic acid. It is a naturally occurring polysaccharide belonging to the group of glycosaminoglycans (GAGs). Hyaluronic acid (HA) and other GAGs are negatively charged heteropolysaccharide chains that have the ability to absorb large amounts of water. Hyaluronic acid and products derived from hyaluronic acid are widely used in the biomedical and cosmetic fields, for example during viscosurgery and as a dermal filler. When used as an injectable filler, hard, tightly cross-linked materials are often considered to have a stronger clinical effect, referred to as "lifting potential". It is also contemplated that these materials may have greater durability in tissues. At the same time, it is suspected that very hard materials sometimes cause unwanted tissue reactions such as foreign body rejection, while softer materials are considered less problematic in this sense.

EP 466300 A2 discloses a two-phase gel slurry in which the first phase comprises swollen polymer gel particles dispersed in a second phase of a viscoelastic polymer solution.

US 2007/0196426 A1 discloses a cross-linked gel containing a number of highly cross-linked nodes interconnected by a gel that is less and less cross-linked, that is, a gel in which the number of cross-links is progressively reduced.

US 2016/0143943 A1 discloses core-shell HA gel particles with gradually increasing swelling capacity from center to surface.

EP 02011816 A1 discloses a co-crosslinked injectable gel having highly cross-linked gel particles associated with a weakly cross-linked gel. The aim is to provide a dual action after injection; first, immediate filling, and in the second stage, when the weakly cross-linked gel is biodegraded in the tissues, the phenomenon of fibroblast reactivation after a mild foreign body reaction to strongly cross-linked particles.

The essence of the invention

Glycosaminoglycan fillers, such as hyaluronic acid, are made up of small particles, allowing them to be administered with a fine needle. The inventors have found that simply mixing a hard and soft injectable excipient does not provide dual functionality in vivo as the particles will spread and separate from each other to some extent when injected into tissue. Thus, when using an injection material consisting of a mixture of hard and soft particles, the tissue can immediately be exposed to hard particles after injection, since the soft and hard particles are separated and end up in different places.

The objective of the present invention is to increase the lifting potential of the filler material. To solve this and other problems that will be apparent to a person skilled in the art from the following description, the present invention provides, in accordance with the first aspect, an injectable gel product containing a first, internal phase of a plurality of cross-linked glycosaminoglycan (GAG) gel particles embedded in a second , an outer phase of cross-linked glycosaminoglycan (GAG) gel; where the second, external phase is in the form of particles;

where the degree of modification (MoD - from the English Degree of Modification) of the gel of the first, internal phase is 0.15 or less, and

where the MoD of the gel of the second, outer phase is lower than the MoD of the gel of the first, inner phase, and where

MoD is the molar amount of bound crosslinking agent(s) relative to the total molar number of repeating GAG disaccharide units.

The first aspect of the invention is based on the understanding that in an injectable gel product, each particle will be a soft tissue material, regardless of the distribution of the particles in the tissue, thereby causing fewer tissue reactions. Since the softer outer phase breaks down over time, the inside phase will break down to a lesser extent. Over time, the outer phase decomposes to such an extent that it dissolves and is carried away from the injection site by tissue fluids. Since the material of the originally internal and harder phase will break down to a softer material during this time, the material remaining at the injection site will be softer than the internal phase material was originally. This allows the embedded material to provide a second period of function in the tissue, now with reduced hardness for less tissue response. In other words, by choosing the hardness of the inner gel and outer gel so that the outer gel is softer, the tissue may initially be exposed to the softer outer gel, but as this gel slowly breaks down, the tissue is eventually exposed to the initially harder inner gel, which becomes softer during the destruction of the outer gel, i.e. the inner gel is allowed to swell in vivo before the tissue comes into direct contact with the inner gel.

In addition, the injectable gel product can be formulated so that the tissue is exposed to a gel with similar properties, for example in terms of swelling or resilience, for a longer period of time. Thus, in some applications it may be desirable that the inner gel exposed to tissue contact after the outer gel is broken is as soft as the original outer gel. However, in other cases, the inner gel exposed to contact need not be as soft as the original outer gel. Since the effect of the internal gel will be significantly delayed and take place over a long period of time, the tissue response may be much weaker than with direct injection of the solid gel.

According to a second aspect, the present invention further provides a method for producing an injectable gel product, comprising the steps of:

a) cross-linking the first glycosaminoglycan (GAG) with the first cross-linking agent to obtain a gel, in which the loading ratio of the cross-linking agent to disaccharide units is less than 0.15;

b) obtaining gel particles from step a);

c) mixing the glycosaminoglycan (GAG) gel particles from step b) with a second glycosaminoglycan (GAG) to form a mixture;

d) cross-linking the mixture obtained in step c) with a second cross-linking agent to form a cross-link between the glycosaminoglycans (GAGs) of the second, external phase to obtain a gel having a first, internal phase of cross-linked glycosaminoglycan (GAG) gel particles embedded in the gel a second, outer phase containing a second glycosaminoglycan (GAG); and

e) obtaining injectable gel particles from step d), each such particle containing a plurality of cross-linked GAG gel particles of the first, internal phase.

As a third aspect of the invention, there is provided an aqueous composition comprising an injectable gel product according to the first aspect of the invention above and optionally a buffering agent.

Further, as a fourth aspect of the invention, there is provided a pre-filled syringe which is pre-filled with an injectable gel product according to the first aspect of the invention described above and then sterilized, or pre-filled with a sterilized aqueous composition according to the third aspect of the invention.

Detailed description of the invention

As a first aspect of the invention, there is provided an injectable gel product comprising a first, internal phase of a plurality of cross-linked glycosaminoglycan (GAG) gel particles embedded in a second, external phase of cross-linked glycosaminoglycan (GAG) gel;

where the second, external phase is in the form of particles;

where the degree of gel modification of the first, internal phase is 0.15 or less, and

where the MoD of the gel of the second, outer phase is lower than the MoD of the gel of the first, inner phase, and where

MoD is the molar amount of bound crosslinking agent(s) relative to the total molar number of repeating GAG disaccharide units.

An injectable gel product refers to a gel capable of flowing and which can be injected with a syringe, such as a syringe with a small diameter needle, which is usually required in cosmetic surgery. Needle can be 27 gauge. Thus, the injectable gel product may be ready to be packed into a syringe.

The injectable gel product can be considered as a two-phase product, i.e. containing a first, internal phase, including a plurality of gel particles embedded in a second, external gel phase. The internal phase may have a higher concentration of GAGs, may be harder and/or have a higher degree of cross-linking.

The injectable gel product may also be cohesive or exhibit cohesiveness, for example as defined in WO 11086458 A1.

Glycosaminoglycan (GAG) is a negatively charged heteropolysaccharide chain that has the ability to absorb large amounts of water. GAGs may, for example, be sulfated or non-sulfated glycosaminoglycans such as hyaluronan, chondroitin sulfate, heparan sulfate, heparosan, heparin, dermatan sulfate, and keratan sulfate. In some embodiments, the GAG is hyaluronic acid, chondroitin, or chondroitin sulfate.

The gel of the inner phase and/or the outer phase may be a hydrogel. That is, they can be considered as water-insoluble but substantially dilute cross-linked systems of GAG molecules when exposed to a liquid, typically an aqueous liquid.

The first internal phase gel and the second external phase gel are cross-linked. Thus, these gels contain a continuous network of GAG molecules that is held together by covalent cross-links, the physical interlacing of GAG chains, and various interactions such as hydrogen bonds, van der Waals forces, and electrostatic interactions.

The degree of modification (MoD) is a measure of the degree of crosslinking. MoD is expressed as mol/mol, a dimensionless number, or mol. %. The MoD describes the amount of crosslinker(s) that is bound to the GAG, i.e. the molar amount of bound crosslinker(s) relative to the total molar number of repeating GAG disaccharide units. Thus, the MoD reflects the extent to which the GAG is chemically modified by the crosslinker(s).

Another measure of the degree of crosslinking is the loading ratio, that is, the ratio between the amount of crosslinkers added to the reaction vessel and the total amount of disaccharide units added to the reaction vessel during the crosslinking reaction.

By way of example, the degree of crosslinking of the gel product can be such that the loading ratio is less than 0.1, preferably less than or equal to 0.02, when preparing gels of both the first, internal phase and the second, external phase.

The gel of the first internal phase and the gel of the second external phase are furthermore in the form of particles, that is to say in the form of fragments which may have any shape, regular or irregular. Dimensions may not be uniform, i.e. the gel product may contain particles of different sizes. In addition, the external phase gel particles contain a plurality of internal gel particles. In other words, the particle size of the outer gel is larger than the particle size of the inner gel.

The gel particles of the second outer phase may have a size ≥ 0.1 mm.

The first aspect of the invention is based on the understanding that embedding a tightly crosslinked and therefore hard material into a weakly crosslinked and therefore soft material can provide higher lifting potential in the tissue. However, the inventors have found that a MoD value of less than 0.15 in the internal phase gel and even a lower MoD value in the external phase gel is advantageous as it makes the combination gel ideal for injection. Thus, the gel product of the present invention is soft enough to cause minor tissue reactions upon injection.

Over time, the soft material degrades to the point where it dissolves and is carried away from the injection site by tissue fluids. Because the MoD is less than 0.15, the initially harder inner material will decompose to a softer state over this time. Thus, the material left at the injection site will be softer than the initially hard material. This allows the internally embedded material to provide a second period of function in the tissue, now with reduced hardness for less tissue response. In other words, due to the fact that the MoD value is less than 0.15 in the inner phase gel and the MoD value is even lower in the outer phase gel, the tissue is exposed to the soft gel for a long period of time.

It should be understood that the first, internal phase of the multiple cross-linked glycosaminoglycan (GAG) gel particles may itself include additional phases, such as smaller particles embedded in the first, internal phase of the multiple cross-linked glycosaminoglycan (GAG) gel particles, and such more small particles may even contain even smaller particles within them, and so on.

In embodiments of the invention, the degree of modification (MoD) of the gel of the first, internal phase is 0.10 or less, such as 0.05 or less, such as 0.02 or less.

In embodiments of the invention, the degree of modification (MoD) of the gel of the second, outer phase is 0.10 or less. Gels with such mildness may be suitable for tissue contact after injection.

However, the MoD of the second, outer phase may be higher than 0.05, for example higher than 0.05, but lower than the MoD of the gel of the inner phase. For example, the MoD of the gel of the second, outer phase may be from 0.08 to 0.095. Gels having this MoD have been found to be hard enough to be held together as a cohesive gel, yet still feel soft after injection.

A relevant physical property of a cross-linked GAG gel product is the volume of liquid that the gel can absorb and is related to the structural stability of the gel, often referred to as gel strength or hardness. Conventional expressions for characterizing liquid absorption are swelling, swelling capacity, liquid holding capacity, degree of swelling, swelling ratio, maximum liquid absorption, and maximum swelling. Throughout the text, the term swelling factor (SwF) will be used.

When the gel is subjected to non-settling conditions, its swelling ratio or, conversely, its minimum concentration (Cmin), ie the GAG concentration when the gel product is fully swollen, can be determined. Further addition of liquid will not further dilute the gel, i.e. a gel cannot be infinitely diluted like a solution of free molecules. Harder (low swelling) gels are generally expected to have longer in vivo half-life than softer (high swelling) gels.

In embodiments of the invention, the swelling factor (SwF) of the gel of the second, outer phase is greater than 3.0, for example greater than 4.0. Such a gel may be perceived as being soft to the tissues.

However, the swelling ratio of the gel of the second, outer phase can still be less than 10.0, i.e. from 3.0 to 10.0, for example from 3.0 to 5.0. A gel having such a swelling ratio may be soft enough to be perceived as soft, but be hard enough to retain a plurality of internal gel particles incorporated into the gel during manufacture, for example, after reduction in particle size (PSR - from English. Particle Size Reduction) external gel. PSR is a process in which the gel is forced through a sieve, resulting in the gel being broken up into particles whose average size corresponds to the mesh size of the sieve.

In embodiments of the invention, the gels of the first, internal phase and the second, external phase are cross-linked with each other.

Thus, gels can be stacked on top of each other to form a single cohesive gel, as opposed to a structure where internal gel particles are dispersed in an external gel. Crosslinks between the inner and outer gels can be made by crosslinking the outer gel. This may be advantageous as it may prevent separation of the inner gel particles from the outer gel before the outer gel is completely dissolved after injection.

In embodiments of the invention, the degree of modification (MoD) is substantially uniform throughout the second, outer phase and substantially uniform throughout the first, inner phase.

Thus, the degree of crosslinking may be substantially uniform in both gel phases, eg constant when viewed from the center of the gel and outward. Thus, this is different from a gel, in which the degree of crosslinking varies within the gel.

The outer gel particles may comprise at least five inner gel particles, such as at least ten inner gel particles, such as at least twenty inner gel particles.

In embodiments of the invention, the particle size of the outer gel is at least three times the size of the inner particles.

In addition, the internal particles may have an average size of less than 0.200 mm.

In embodiments of the invention, the dry weight of the cross-linked glycosaminoglycan (GAG) in the first, internal phase is at least 25% of the total dry weight of glycosaminoglycans (GAG) in the internal and external phases, for example, at least 50% of the total dry weight of glycosaminoglycans (GAG) in internal and external phases.

Such gels may be soft enough to provide initial softness in vivo, but have a high enough internal particle content to provide a second function after the external gel has dissolved.

For example, the dry weight content of cross-linked glycosaminoglycan (GAG) in the first, internal phase may be at least 60% of the total dry weight of glycosaminoglycans (GAG) in the internal and external phases.

As a further example, the dry weight content of cross-linked glycosaminoglycan (GAG) in the first, internal phase can be from 65% to 95% of the total dry weight content of glycosaminoglycans (GAG) in the internal and external phases.

In embodiments of the invention, the glycosaminoglycan (GAG) is hyaluronic acid.

Unless otherwise stated, the term "hyaluronic acid" covers all variants and combinations of variants of hyaluronic acid or hyaluronan with different chain lengths and charge states, as well as with different chemical modifications, i.e. this term also covers various salts of hyaluronic acid, such as sodium hyaluronate . The term also covers various modifications of hyaluronic acid such as oxidation, for example the oxidation of CH ₂ OH groups to COOH; periodate oxidation of vicinal hydroxyl groups, optionally followed by reduction or imine formation, etc.; recovery, such as reduction of COOH to CH ₂ OH; sulfation; deamidation, optionally followed by deamination or amide formation with new acids; esterification; substitution with various compounds, for example using a cross-linking agent or carbodiimide; including the condensation of various molecules such as proteins, peptides and drug active ingredients with hyaluronic acid; and deacetylation.

As an example, hyaluronic acid may be chemically unmodified hyaluronic acid or a salt of hyaluronic acid, preferably sodium hyaluronate, having an average molecular weight in the range of 0.1-10 MDa, preferably 0.8-5 MDa, more preferably 0.8-1, 2 MDa. Preferably, the hyaluronic acid is of non-animal origin, preferably bacterial.

In some embodiments, cross-linking of the inner and/or outer gel is performed using one or more polyfunctional cross-linking agents individually selected from the group consisting of divinyl sulfone, multi-epoxides, and diepoxides. In some embodiments, the multifunctional crosslinking agent(s) is individually selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane. In specific embodiments, the multifunctional crosslinker is 1,4-butanediol diglycidyl ether (BDDE).

Thus, in embodiments, cross-linking is the formation of ether bonds.

In embodiments of the invention, cross-linking of glycosaminoglycans is achieved by amide linkage of glycosaminoglycan molecules. Amide coupling can be carried out using a di- or multi-nucleophilic functional cross-linking agent.

Amide linkage using a di- or multi-amine functional crosslinker together with a linker is an attractive route to obtain cross-linked glycosaminoglycan molecules useful in the present invention.

Cross-linking can be achieved using a non-carbohydrate based di- or multinucleophilic crosslinker, eg hexamethylenediamine (HMDA), or a carbohydrate-based di- or multinucleophilic crosslinker, eg diaminotrehalose (DATH), together with a glycosaminoglycan.

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

A preferred group of di- or multi-nucleophilic functional cross-linking agents includes homo- or hetero-bifunctional primary amines, hydrazines, hydrazides, carbazates, semicarbazides, thiosemicarbazides, thiocarbazates, and aminooxy.

The di- or multinucleophilic functional di-, tri-, tetra- and oligosaccharides may be derived from nucleophilic functional polysaccharides such as chitobiose derived from chitin. Di- or multinucleophilic functional di-, tri-, tetra- and oligosaccharides can also be di-, tri-, tetra- and oligosaccharides which have been modified by introducing two or more nucleophilic functional groups.

Carbohydrate-based di- or polynucleophilic crosslinkers may be useful as they provide a hydrogel product based entirely on carbohydrate-type structures or derivatives thereof, which minimizes disruption of the natural crosslinking of glycosaminoglycans. The crosslinker itself can also help maintain or enhance the properties of the hydrogel, for example by crosslinking with a structure that correlates with hyaluronic acid, or by crosslinking with a structure with high water-retaining properties.

In embodiments of the invention, the di- or multinucleophilic crosslinker is at least partially deacetylated polysaccharide, i.e. an acetylated polysaccharide that has been at least partially deacetylated to form a polysaccharide having free amino groups. The at least partially deacetylated glycosaminoglycan may be cross-linked either alone or in combination with a second glycosaminoglycan, with the deacetylated glycosaminoglycan itself acting as a di- or multinucleophilic cross-linking agent.

In embodiments of the first aspect of the invention, the glycosaminoglycan molecules of the first, internal phase and/or the glycosaminoglycan molecules of the second, external phase are covalently cross-linked via cross-links containing a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides.

The crosslink typically consists of i) a spacer group and ii) linking groups formed by the reaction of functional groups of the crosslinker with carboxylic acid groups on GAG. The spacer group may, for example, include hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, a trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue.

Cross-linking with cross-linking agents containing a spacer group selected from the group consisting of di-, tri-, tetra-, and oligosaccharides results in a hydrogel product based entirely on carbohydrate-type structures or their derivatives, which minimizes the disruption of natural cross-linking. 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 particular di-, tri-, tetra- or oligosaccharide structure. Preferably the di-, tri-, tetra- or oligosaccharide is monodispersed or has a narrow molecular weight distribution. The use of well-defined crosslinkers based on di-, tri-, tetra- or oligosaccharides, together with a highly efficient condensation reaction, allows the synthesis of the product in a controlled manner. The crosslinker itself can also help maintain or enhance the properties of the hydrogel, for example when crosslinked with a structure that correlates with hyaluronic acid (eg, diaminohyaluronic acid tetrasaccharide), or when crosslinked with a highly water-retaining structure (eg, trehalose).

For example, at least 75% of the crosslinks may include a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides.

In addition, the spacer group may be a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, a trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue.

Moreover, the spacer group may be selected from the group consisting of di-, tri- and tetrasaccharides.

In embodiments of the first aspect of the invention, at least 90% of the bonds between the glycosaminoglycan molecules and the crosslinks are amide bonds.

In addition, less than 5% of the bonds between glycosaminoglycan molecules and cross-links may be ester bonds.

The first and/or second glycosaminoglycan can be further cross-linked using a peptide binding agent. Crosslinking using a peptide coupling agent is an advantage over many other common crosslinking methods (eg, BDDE crosslinking) because it allows crosslinking at neutral pH with minimal disruption of glycosaminoglycan molecules.

In some embodiments, the peptide binding agent is selected from the group consisting of triazine-based binding agents, carbodiimide-based binding agents, imidazolium-based binding agents, Ohut, and COMU.

In some embodiments, the peptide binding agent is a triazine binding agent. In some embodiments, the triazine coupling agent is selected from 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 some embodiments, the triazine binding agent is DMTMM.

In some embodiments, the peptide coupling agent is a carbodiimide coupling agent. In some embodiments, the carbodiimide coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS).

The injectable gel product may also contain a portion of the GAG molecules that are not crosslinked, ie. are not associated with a three-dimensional network of cross-linked GAG molecules. However, it is preferred that at least 50 wt. %, preferably at least 60 wt. %, more preferably at least 70 wt. % and most preferably at least 80 wt. % of the GAG molecules of the injectable gel product formed a network of cross-linked GAG molecules.

As a second aspect of the invention, a method for producing an injectable gel product is provided, comprising the steps:

a) cross-linking the first glycosaminoglycan (GAG) with the first cross-linking agent to obtain a gel in which the loading ratio of the cross-linking agent to disaccharide units is less than 0.15;

b) obtaining gel particles from step a);

c) mixing the glycosaminoglycan (GAG) gel particles from step b) with a second glycosaminoglycan (GAG) to form a mixture;

d) cross-linking the mixture obtained in step c) with a second cross-linking agent to form a cross-link between glycosaminoglycans (GAGs) of a second, outer phase, to obtain a gel having a first, inner phase of cross-linked glycosaminoglycan (GAG) gel particles embedded into the gel of the second, outer phase containing the second glycosaminoglycan (GAG), and

e) obtaining injectable gel particles from step d), each such particle containing a plurality of cross-linked GAG gel particles of the first, internal phase.

The terms and definitions used in relation to the second aspect of the invention are discussed above in relation to the first aspect.

In embodiments of the invention, the injectable particles obtained in step e) are a gel product as disclosed in relation to any embodiment of the first aspect above.

The method according to the second aspect has the advantage that it is a convenient method for producing a combined gel, ie for incorporating a tightly crosslinked and therefore hard material into a weakly crosslinked and therefore soft material. Such a combination gel can provide higher tissue lifting potential. The inventors have found that the loading ratio in step a) is less than 0.15, i.e. when preparing "internal" GAG gel particles, and an even lower loading ratio in step d), i.e. when embedding internal GAG gel particles in an external matrix, promotes the formation of a gel product, which can increase the potential for tissue lifting.

In addition, the method according to the second aspect of the invention has the advantage that it provides an efficient method for obtaining a gel product for injection, since small amounts of a crosslinking agent can be used.

The steps of incorporating the already cross-linked particles into the gel which is subsequently cross-linked can be repeated any number of times, for example at least two or at least three times. Thus, step d) may further comprise the preparation of particles of the obtained gel, and these obtained particles can be used in the repeated step c), in which they are mixed with, for example, a third glycosaminoglycan (GAG) to obtain a mixture, which is then crosslinked in the repeated step d ) to form a gel. The particles of this gel can be mixed again with other GAGs, etc.

Thus, steps c) and d) can be repeated any number of times. The crosslinking agent used in the crosslinking steps may be as described above in the first aspect of the invention.

For example, cross-linking in step a) and/or step b) can lead to the formation of ether linkages.

As a further example, crosslinking in step a) and/or step b) can result in covalent crosslinking of GAG molecules via crosslinks containing a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides.

The crosslinking in step d) can be performed using the same crosslinking conditions as the crosslinking in step a). The loading ratio in step d) may be the same as in step a) or different.

In addition, cross-linking in step d) can be performed using the same cross-linking agent as in step a), or using a different cross-linking agent than the cross-linking agent used in step a). Therefore, the first and second crosslinkers may be the same or different.

In addition, cross-linking in steps a) and/or d) can be accomplished by first adding a cross-linking agent to improve cross-linking homogeneity in the first and/or outer gel.

The load ratio in step a) may be less than 0.10, for example less than 0.05.

In embodiments, the loading ratio of crosslinker to disaccharide units in step d) is less than 0.10, such as less than 0.05. The inventors have found that such a low loading ratio when making an external gel matrix can still provide a gel product with suitable in situ degradation characteristics.

In embodiments of the invention, the loading ratio of crosslinker to disaccharide units in step d) is less than the loading ratio in step a).

In embodiments of the invention, cross-linking in step d) further comprises making cross-links between the first, internal phase and second, external phase gels. Thus, the second crosslinking agent used in step d) may further assist in crosslinking the outer gel matrix with the incorporated gel particles and thus may assist in retaining the embedded gel particles within the gel when preparing the injectable particles in step e).

In embodiments of the invention, step c) comprises dry mixing the glycosaminoglycan (GAG) gel particles of step b) with a second glycosaminoglycan (GAG) in the dry state. The dry gel particles may, for example, be in the form of a powder or in the form of dry entangled filaments. Dry blending can be advantageous as it provides good mixing between the first and second GAGs, ie ensures uniform distribution of the first internal phase gel particles within the external phase.

However, step c) may involve first dissolving the glycosaminoglycan (GAG) gel particles of step b) and mixing this solution with a solution containing a second glycosaminoglycan (GAG).

In embodiments of the invention, the step of obtaining the particles of step b) further comprises precipitating and drying said particles obtained from the gel of step a).

Precipitation of the internal gel particles is advantageous in that it further enhances the ability to wash the gel particles of the first, internal phase, for example, to reduce the amount of by-products such as unreacted crosslinkers. The precipitation itself can act as a purification step, ie. unwanted by-products may be separated from the gel particles during the actual precipitation.

In addition, precipitation of the internal gel particles may make the internal gel particles more accessible to crosslinkers during the second crosslinking step, ie during step d). This means that smaller amounts of crosslinking agent can be used in the second crosslinking step, i.e. precipitation increases the efficiency of using crosslinkers and thus increases the efficiency of the overall process.

Moreover, the precipitated powder can be stored and therefore the second crosslinking in step d) need not be carried out immediately after precipitation. Thus, step c) can be performed at least 1 hour after precipitation, such as at least 5 hours, such as at least 10 hours, such as at least 24 hours, such as at least 48 hours after precipitation . Therefore, the step of settling and drying the particles prepared from the gel in step a) increases the flexibility of the process.

Therefore, the precipitation step is advantageous in that it allows the gel particles to be purified, the process becomes more flexible since the two crosslinking steps can be separated into more individual and separate crosslinking steps, and the overall efficiency of the process is increased due to the use of less crosslinking agent.

In embodiments of the second aspect of the invention, the mixture obtained in step c) contains at least 45% by dry weight of the GAG gel particles obtained in step b), for example at least 50% by dry weight of the GAG gel particles obtained in step b ). Thus, the dry weight content of cross-linked glycosaminoglycan (GAG) in the first, internal phase can be at least 50% of the total dry weight content of glycosaminoglycan (GAG) in step c).

For example, the mixture obtained in step c) may contain at least 60% by dry weight of the GAG gel particles obtained in step b).

As a further example, the mixture obtained in step c) may contain from 65% to 95% by dry weight of the GAG gel particles obtained in step b). Thus, the dry weight content of cross-linked glycosaminoglycan (GAG) in the first, internal phase can be from 65% to 95% of the total dry weight content of glycosaminoglycans (GAG) in the internal and external phases.

As a third aspect of the invention, there is provided an aqueous composition comprising an injectable gel product according to any embodiment of the first aspect described above and optionally a buffering agent.

The aqueous composition may be suitable for injection. The aqueous composition may typically contain a physiological saline buffer. The composition may further contain other suitable additives such as local anesthetics (eg lidocaine hydrochloride), anti-inflammatory drugs, antibiotics and other suitable supportive drugs such as growth factors or bone cells.

Administration of the injectable gel product or aqueous composition may be by any suitable means, for example by injection from standard cannulas and suitably sized needles, or by surgical insertion, for example in the case of film insertion. The introduction is carried out in those places where it is desirable to increase soft tissues, such as the chin, cheeks, or anywhere else on the face or body.

Thus, as a fourth aspect of the invention, there is provided a pre-filled syringe which is pre-filled with a sterilized injectable gel product according to any embodiment of the first aspect described above or a sterilized aqueous composition according to the third aspect described above.

The gel product may be stored in precipitated form in a syringe, which is converted to non-precipitated form before injection or in the body after injection.

The gel product can optionally be autoclaved as this is the most convenient way to sterilize the final product. This allows the preparation of a sterilized injectable gel product.

In some embodiments, the injectable gel product or aqueous composition according to the first and third aspects above is useful as a drug or medical device in a medical or surgical manner.

According to a further aspect, the use of an injectable gel product according to the first aspect described above or an aqueous composition according to the third aspect described above is provided in cosmetic or medical surgery. In other words, what is provided is an injectable gel product or aqueous composition for use in cosmetic or medical surgery.

In some embodiments of the invention, dermal filling and body contouring are chosen in cosmetic surgery. In some other embodiments, the invention is used as a medicament in the treatment and/or medical surgery selected from dermal filling, body contouring, anti-adhesion, channeling, incontinence and orthopedic applications.

In one aspect, there is provided a method of treating a subject undergoing cosmetic or medical surgery, comprising administering an injectable gel product according to the first aspect described above, or an aqueous composition according to the third aspect described above, to a subject in need thereof.

In some embodiments, the subject is undergoing cosmetic surgery selected from dermal filling and body contouring. In some other embodiments, the subject is undergoing medical surgery or medical treatment for a condition selected from dermal filling, body contouring, tissue adhesion prevention, channeling, incontinence treatment, and orthopedic applications.

The glycosaminoglycan itself can act as a cross-linking agent in a cross-linked gel, for example when deacetylated glycosaminoglycan is used. In such cases, the degree of modification (MoD), which is an important property when using cross-linking agents other than the glycosaminoglycan itself, may correspond to the degree of deacetylation of the glycosaminoglycan. Thus, all of the embodiments and examples of MoDs discussed in connection with the previous aspects above may also be relevant when the glycosaminoglycan itself acts as a crosslinking agent, and also as the degree of deacetylation of the glycosaminoglycan.

Thus, as a general aspect of the present invention, there is provided an injectable gel product comprising a first, internal phase of a plurality of cross-linked glycosaminoglycan (GAG) gel particles embedded in a second, external phase of a cross-linked glycosaminoglycan (GAG) gel;

where the second, external phase is in the form of particles;

where the maximum number of crosslinks in relation to the total molar number of repeating disaccharide GAG units in the gel of the first, internal phase is 0.15 or less, and

where the maximum number of crosslinks in relation to the total molar number of recurring GAG disaccharide units in the gel of the second, external phase is lower than the maximum number of crosslinks in relation to the total molar number of recurring GAG disaccharide units in the gel of the first, internal phase.

The maximum number of crosslinks relative to the total molar number of repeating disaccharide units of the GAG may be, for example, MoD if a separate crosslinker other than the GAG itself is used for crosslinking, or the degree of deacetylation if the GAG itself acts as a crosslinker. In addition, when the glycosaminoglycan itself acts as a cross-linking agent in the cross-linked gel, alternative aspects discussed below may become relevant. The terms and definitions used in relation to these alternative aspects are the same as discussed above in relation to the previous aspects.

Thus, as an alternative aspect of the present invention, there is provided an injectable gel product comprising a first, internal phase of a plurality of cross-linked glycosaminoglycan (GAG) gel particles embedded in a second, external phase of a cross-linked glycosaminoglycan (GAG) gel;

where the second, outer phase is in the form of particles and

where glycosaminoglycan (GAG) of the first, internal phase and/or glycosaminoglycan (GAG) of the second, external phase contain cross-links, in which the glycosaminoglycan itself acts as a di- or multinucleophilic cross-linking agent.

In embodiments of the invention, the degree of deacetylation of the gel of the first, internal phase is 0.15 or less, and the degree of deacetylation of the gel of the second, external phase is lower than the degree of deacetylation of the gel of the first, internal phase.

In one of the embodiments of the invention, only the glycosaminoglycan (GAG) of the first, internal phase contains cross-links, in which the glycosaminoglycan itself acts as a di- or multinucleophilic cross-linking agent.

In one embodiment of the invention, only the glycosaminoglycan (GAG) of the second, outer phase contains cross-links, in which the glycosaminoglycan itself acts as a di- or multinucleophilic cross-linking agent.

In one of the embodiments of the invention glycosaminoglycan (GAG) of the first, internal phase and glycosaminoglycan (GAG) of the second, external phase contain cross-links, in which the glycosaminoglycan itself acts as a di- or multinucleophilic cross-linking agent.

For example, the deacetylated glycosaminoglycan itself can act as a di- or multinucleophilic crosslinker.

Thus, the glycosaminoglycan itself, acting as a di- or multinucleophilic cross-linking agent, may be at least partially deacetylated glycosaminoglycan, that is, an acetylated glycosaminoglycan that is at least partially deacetylated to form a glycosaminoglycan having free amino groups. The at least partially deacetylated glycosaminoglycan may be cross-linked either alone or in combination with a second glycosaminoglycan, with the deacetylated glycosaminoglycan itself acting as a di- or multinucleophilic cross-linking agent.

As another alternative aspect of the present invention, a method for preparing an injectable gel product is provided, comprising the steps of:

a) crosslinking the first glycosaminoglycan (GAG) to obtain a gel,

b) obtaining gel particles from step a);

c) mixing the glycosaminoglycan (GAG) gel particles from step b) with a second glycosaminoglycan (GAG) to form a mixture;

d) cross-linking the mixture obtained in step c) to form a cross-link between the glycosaminoglycans (GAGs) of the second, external phase to obtain a gel having a first, internal phase of cross-linked glycosaminoglycan (GAG) gel particles embedded in the gel of the second, external a phase containing a second glycosaminoglycan (GAG); and

e) obtaining injectable gel particles from step d), each such particle containing a plurality of cross-linked GAG gel particles of the first, internal phase.

Crosslinking in step a) and/or step d) can be achieved using at least partially deacetylated glycosaminoglycan, either alone or in combination with a second glycosaminoglycan, with the deacetylated glycosaminoglycan itself acting as a di- or multinucleophilic crosslinking agent.

The degree of deacetylation of the first glycosaminoglycan in step a) may be less than 0.15. The degree of deacetylation of the second glycosaminoglycan in step d) may be less than the degree of deacetylation of the first glycosaminoglycan.

The term "at least partially deacetylated", as used herein in relation to glycosaminoglycan, refers to glycosaminoglycan containing N-acetyl groups, in which at least some of the N-acetyl groups have been cleaved, which leads to the formation of free amino groups in the glycosaminoglycan. By the term "at least partially deacetylated" in the context of the present description, we mean that a significant part of the N-acetyl groups of the glycosaminoglycan, in particular at least 1%, preferably at least 2%, at least 3%, at least 4 %, at least 5% of the N-acetyl groups of glycosaminoglycan are converted into free amino groups. More preferably, at least 3% of the N-acetyl groups of the glycosaminoglycan are converted to free amino groups.

In some embodiments, the at least partially deacetylated glycosaminoglycan may have an acetylation degree of less than 99%, preferably less than 98%, less than 97%, less than 97%, less than 96%, less than 95%, less than more than 94%, or less than 93%.

Crosslinking of the deacetylated glycosaminoglycan can be carried out with a coupling agent, such as a peptide coupling agent.

In some embodiments, the peptide binding agent is selected from the group consisting of triazine-based binding agents, carbodiimide-based binding agents, imidazolium-based binding agents, Ohut, and COMU.

In some embodiments, the peptide binding agent is a triazine binding agent. In some embodiments, the triazine coupling agent is selected from 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 some embodiments, the triazine binding agent is DMTMM.

In some embodiments, the peptide coupling agent is a carbodiimide coupling agent. In some embodiments, the carbodiimide coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS).

Therefore, step a) may include sub-steps:

i) obtaining a solution containing at least partially deacetylated glycosaminoglycan and possibly additional glycosaminoglycan;

ii) activating the carboxyl groups of the at least partially deacetylated glycosaminoglycan and/or possibly additional glycosaminoglycan with a binding agent to form activated glycosaminoglycans;

iii) crosslinking the activated glycosaminoglycans via their activated carboxyl groups using the amino groups of the at least partially deacetylated glycosaminoglycans to form an amide crosslinked glycosaminoglycan gel.

As a further example, the second glycosaminoglycan may be at least partially deacetylated glycosaminoglycan, and step d) may include substeps:

i) activating the carboxyl groups of the at least partially deacetylated second glycosaminoglycan with a coupling agent to form activated glycosaminoglycans;

ii) cross-linking the activated glycosaminoglycans via their activated carboxyl groups using the amino groups of the at least partially deacetylated glycosaminoglycans to form a second, external phase comprising a second glycosaminoglycan (GAG) cross-linked with amide bonds.

In other embodiments, the cross-linked GAG in step a) and/or step d) is produced by:

1) cross-linking at least partially deacetylated GAG with partially deacetylated GAG in the presence of a binding agent using free amine and carboxyl groups present in at least partially deacetylated GAGs; or

2) cross-linking at least partially deacetylated GAG with non-deacetylated GAG in the presence of a coupling agent using free amino groups present in the at least partially deacetylated GAG and carboxyl groups present in the GAG.

In embodiments of the invention, the at least partially deacetylated glycosaminoglycan has an acetylation degree 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 produced by a method for at least partially deacetylated glycosaminoglycan, comprising:

a1) obtaining glycosaminoglycan containing acetyl groups;

a2) the implementation of the interaction of glycosaminoglycan containing acetyl groups, with hydroxylamine (NH ₂ OH) or its salt at a temperature of 100°C or lower for 2-200 hours with the formation of at least partially deacetylated glycosaminoglycan; and

a3) recovering the at least partially deacetylated glycosaminoglycan.

Brief description of graphic materials

In FIG. 1 is a block diagram of embodiments of the method of the present invention. PSR=particle size reduction

In FIG. 2 shows the results of the fracture tests.

In FIG. 3 shows the results of the fracture tests.

In FIG. 4 shows gel particles with gel particles incorporated therein at 10x magnification.

In FIG. 5 shows a microscopic image of the particles of the gel obtained in Example 6 at 10x magnification.

In FIG. 6 shows a microscope image of the particles of the gel obtained in Example 9.

Experimental examples

The following non-limiting examples further illustrate the present invention. In these examples, the GAG used is hyaluronic acid, designated HA and referred to as sodium hyaluronate.

Analytical test methods

Gel content (GelC)

GelC describes in % the proportion of total HA that is bound in gel form. The gel content is defined as the amount of HA in the sample that does not pass through the 0.22 µm filter. GelC is calculated from the amount of HA collected in the filtrate and expressed as a percentage of the total amount of HA in the gel sample.

MoD (degree of modification)

MoD describes the molar amount of bound crosslinker(s) relative to the total number of repeating disaccharide units in the HA. This measure does not distinguish between mono-linked and actually cross-linked cross-linking agent(s), i.e., all cross-linking agent(s) that is linked to the HA via at least one covalent bond is included. For example, a MoD of 1% for a BDDE cross-linked HA gel means that there is 1 linked (mono-linked or cross-linked) BDDE molecule for every 100 disaccharide units in the HA gel.

MoD can be determined by NMR spectroscopy of the enzymatically degraded gel product. Before breaking the gel, soluble HA, residual (unbound) crosslinking agent(s) and its derivatives are washed out by filtration on a 0.22 µm filter. The gel product is destroyed at 37° C. by enzymatic treatment using chondroitinase AC from Arthrobacter aurescens. The decomposed gel product is subjected to NMR spectroscopy by recording one-dimensional ¹ H NMR spectra on a 400 MHz spectrometer equipped with a standard 5 mm probe.

NMR spectra can be estimated by integrating the signal at δ _H 1.6 ppm, which comes from four protons in the bound BDDE molecule, and the signal at δ _H 2.0 ppm, which comes from three protons in the CH groups ₃ on N-acetylglucosamine residues of HA disaccharides. The ratio between the integrals for these two signals is proportional to the ratio between the molar amount of bound BDDE and disaccharides after adjusting for the number of protons responsible for each signal, which gives MoD.

Load ratio

Another measure of the degree of crosslinking is the loading ratio, that is, the ratio between the amount of crosslinkers added to the reaction vessel and the total amount of disaccharide units added to the reaction vessel during the crosslinking reaction. The loading ratio may differ from the MoD since the crosslinking reaction may be incomplete, i.e. all crosslinkers may not react to form crosslinks.

Swelling factor (SwF)

The strength or network density of the gel after crosslinking can be assessed/determined, for example by allowing the gel to absorb water or saline until equilibrium. As a result of chain breaks during gel degradation, the network becomes weaker and less dense, which can be determined from the increasing swelling factor (SwF). SwF is measured according to the following protocol: about 1 g of the gel product is weighed into a beaker. Saline solution is added, the gel is thoroughly dispersed and allowed to absorb water until equilibrium. After precipitation, the volume of saturated gel is measured. The ratio of the final volume to the initial volume is referred to as the swelling factor (SwF).

Example 1 - General scheme of the method for obtaining a combined gel

An embodiment of the process for preparing an injectable gel product cross-linked with BDDE is schematically represented in FIG. 1. In this method, the mass is precipitated and dried after the first stage of particle size reduction (PSR). The dry cross-linked gel powder is then mixed with HA, BDDE and NaOH for a second cross-linking in which the previously cross-linked gel particles are incorporated into the second gel material. The second material is then subjected to the same processing steps as in the conventional process. The particles from the first PSR stage are smaller than the particles from the second PSR stage to allow room for the inner gel in the outer gel.

The first and second crosslinking is usually carried out at a temperature of 10-75 degrees Celsius, such as 10-40 degrees Celsius, such as 10-35 degrees Celsius or 10-30 degrees Celsius, but preferably this step is performed at 15-35 degrees Celsius, such as 15 -30 degrees Celsius, and especially at room temperature, such as 20-25 degrees Celsius. Preferred temperature ranges are 10-50 degrees Celsius, such as 18-40 degrees Celsius.

The reaction time is usually in the range of 2-40 hours, such as 4-36 hours. Longer reaction times than 2 hours are beneficial for reproducibility, especially on large scales. A longer reaction time than 40 hours results in a lower strength gel or may even break the gel completely. The reaction time is preferably in the range of 8-30 hours, such as 12-24 hours, such as 16-24 hours.

For example, the crosslinking step can be carried out at 15-35 degrees Celsius for 2-40 hours, eg at room temperature for 16-24 hours.

Fig. 4 shows a microscopic image at 10x magnification of gel particles produced by the method of the present invention, with smaller particles embedded in larger particles.

Example 2 - Preparation of a one-component gel

A one-component gel was prepared as follows: 100 g of HA (Mw 1MDa) were mixed and allowed to interact with 200 g of NaOH 3% by weight and 1.8 g of BDDE, resulting in a loading ratio of 0.036. After crosslinking, the gel was neutralized and heat treated to inactivate any residual crosslinker. Particle size reduction (PSR) was performed using a sieve with a mesh size of 80 μm. The gel was precipitated in EtOH to give a gel powder which was washed and dried.

Example 3 - Preparation of a one-component gel

Performed as in Example 2 but using 30 g HA, 73.5 g NaOH 2.5% by weight and 0.22 g BDDE, giving a loading ratio of 0.015.

Example 4 - Preparation of a two-component gel

A two-component gel was prepared as follows: 5 g of the gel powder from Example 2 was mixed and allowed to react with 5 g of HA (Mw 1MDa), 45.5 g of NaOH 1.33% w/w and 90 mg of BDDE, resulting in a loading ratio of 0.036 . The PSR of Example 2 was repeated but using a 315 µm sieve. The two-component gel powder was hydrated to a final concentration of 20 mg/ml in phosphate buffer and autoclaved. The gel was exposed to a temperature of 90° C. and the gel content was measured at 16 hour intervals (see FIG. 2). Thus, in Example 4, the loading ratio in both the first and second crosslinking reactions was 0.036.

Example 5 - Preparation of a two-component gel

Performed as in Example 4, but using 7 g of the gel powder from Example 2 and 3 g of HA, resulting in a loading ratio of 0.043. The gel was exposed to a temperature of 90° C. and the gel content was measured at 16 hour intervals (FIG. 2). Restylane Lyft (from Galderma) was used as a standard. Thus, in Example 5, the loading ratio was 0.036 in the first crosslinking reaction and 0.043 in the second crosslinking reaction.

The gels of Examples 4 and 5 had the following initial characteristics:

As seen in FIG. 2, the 70% combined gel decomposes until the outer gel disappears, and then flattens out with about 70% gel remaining, which decomposes at a slower rate. The same type of behavior was observed with the 50% combination gel, but this gel flattened out at 50%. A standard gel containing only one type of gel decomposes at a more constant rate. These results clearly demonstrate the concept of a combination gel, ie a combination gel having an outer gel that first decomposes before the inner gel begins to degrade. Similar behavior is expected in vivo.

Example 6 - Preparation of a two-component gel.

Performed as in Example 4 but with 2.5 g of the gel powder from Example 3 and 2.5 g of HA (Mw 1MDa), 15 g of NAOH 2% by weight and 35 mg of BDDE, resulting in a loading ratio of 0.022. The gel was exposed to a temperature of 90° C. and the swelling ratio was measured at 16 hour intervals (FIG. 3). For comparison, the gel powder from Example 3 was crosslinked a second time to match the inner gel from Examples 6 and 7. FIG. 5 shows a microscopic image of the gel particles obtained in Example 6 at 10x magnification.

Thus, in Example 6, the loading ratio was 0.015 in the first crosslinking reaction and 0.022 in the second crosslinking reaction.

Example 7 - Preparation of a two-component gel.

Performed as Example 6, but with 3.5 g of the gel powder from Example 3 and 1.5 g of HA, resulting in a load ratio of 0.025. The gel was exposed to a temperature of 90° C. and the swelling ratio was measured at 16 hour intervals (FIG. 3). Thus, in Example 7, the loading ratio was 0.015 in the first crosslinking reaction and 0.025 in the second crosslinking reaction.

The gels of Examples 6 and 7 had the following initial characteristics:

The 50% combo gel increases swelling more than other gels because this gel includes most of the softer outer gel, which is the main contributor to swelling. After a certain period of time (about 40 hours for a 50% combined gel) the outer gel disappears and only the harder inner gel can contribute to the swelling. This causes the measured SwF to drop, which then slowly rises again due to the swelling of the internal gel. The combined 70% gel shows a similar but less distinct behavior as it contains less external gel. A gel having only an inner gel (one-component gel) exhibits a more consistent increase in swelling characteristics. Thus, the inner gel, after decomposition of the outer gel, will exhibit a swelling ratio similar to that of the original combined gel.

The table below shows the loading ratios used to prepare the two-component gels in Experimental Examples 4-7 above:

In addition, conducting the first and second crosslinking reactions with loading ratios according to the above table resulted in the following degree of modification (MoD) for the inner and outer gels in the prepared two-component gels in Experimental Examples 4-7:

Thus, the MoD of the inner gels in all examples is higher than the MoD of the outer gels, which is due to the fact that the crosslinkers diffuse into the inner gel during the second crosslinking reaction to further crosslink this gel, thereby increasing the MoD of the inner gel during the second stitching steps.

Example 8 - Preparation of one-component gels cross-linked with amide bonds.

Gels were also prepared using amide linkage in which hyaluronic acid (HA), DATH (diaminotrehalose), and DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) are mixed and allowed to react. DMTMM acts as a binding agent while DATH acts as a crosslinking agent.

A one-component gel was prepared as follows: HA was mixed and allowed to react with appropriate amounts of DATH and DMTMM dissolved in water. After crosslinking, the gel was diluted to 20 mg/g with saline and heated to 70° C. for about 20 hours. Particle size reduction (PSR) was performed using a 125 µm sieve. The gel was precipitated in EtOH to give a gel powder which was washed and dried.

Example 9 - Preparation of two-component gels cross-linked with amide bonds

The dry powder of the amide crosslinked HA gel prepared as in Example 8 was then mixed and allowed to react with the appropriate amounts of HA, DATH and DMTMM dissolved in water for a second crosslinking in which the previously crosslinked gel particles were incorporated into the second gel material.

After the second crosslinking, the gel was diluted to 20 mg/g with saline and heated to 70° C. for about 20 hours. Particle size reduction (PSR) was performed using a 315 µm sieve. The gel was precipitated in EtOH to give a gel powder which was washed and dried. The two-component gel powder was hydrated to a final concentration of 20 mg/ml in phosphate buffer and autoclaved.

A micrograph of the prepared two-component gel from Example 9 is shown in FIG. 6.

The gel characteristics of gels cross-linked with amide bonds are shown in the table below:

The person skilled in the art understands that the present invention is in no way limited to the preferred embodiments described above. On the contrary, many modifications and variations are possible within the scope of the appended claims. In addition, variations of the disclosed embodiments may be understood and practiced by one skilled in the art of the claimed invention based on examination of the drawings, the description, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The fact that certain measures are set forth in mutually distinct dependent claims does not mean that a combination of these measures cannot be used to advantage.

Claims (60)

1. A method for producing an injectable gel product, comprising the following steps: a) cross-linking the first glycosaminoglycan (GAG) with the first cross-linking agent to obtain a gel, in which the loading ratio of the cross-linking agent to disaccharide units is less than 0.15; b) obtaining gel particles from step a); c) mixing the GAG gel particles from step b) with the second GAG to form a mixture; d) cross-linking the mixture from step c) with a second cross-linking agent to form a gel having a first, internal, phase of cross-linked GAG gel particles embedded in a second, external, phase containing a second GAG; and e) obtaining injectable gel particles from step d), each such particle containing a plurality of cross-linked GAG gel particles of the first, internal, phase; where each of the first GAG and the second GAG is hyaluronic acid and where each of the first crosslinking agent and the second crosslinking agent is independently selected from (i) a polyfunctional crosslinking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane, and (ii) a crosslinking agent containing a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides. 2. The method of claim 1, wherein the loading ratio of crosslinker to disaccharide units in step d) is less than the loading ratio in step a). 3. The method according to claim 1 or 2, wherein the loading ratio of crosslinker to disaccharide units in step d) is less than 0.05. 4. The method according to claim 1 or 2, wherein the crosslinking in step d) further comprises achieving crosslinking between the first, internal, phase and second, external, phase gels. 5. The method according to claim 1 or 2, wherein step c) comprises dry mixing the glycosaminoglycan (GAG) gel particles from step b) with a second glycosaminoglycan (GAG) in a dry state. 6. The method according to claim 1 or 2, wherein the step of obtaining the particles of step b) further comprises precipitating and drying said particles obtained from the gel of step a). 7. The method according to claim 1 or 2, wherein the mixture obtained in step c) contains at least 50% of the GAG gel particles obtained in step b), based on dry weight. 8. The method according to claim 1 or 2, wherein the cross-linking in step a) and/or step b) results in the formation of ether bonds. 9. The method according to claim 1 or 2, wherein the cross-linking in step a) and/or step b) results in the GAG molecules being covalently cross-linked, comprising a spacer group selected from the group consisting of di-, tri-, tetra- and oligsaccharides. 10. The method of claim 9 wherein at least 75% of the crosslinks comprise a spacer group selected from the group consisting of di-, tri-, tetra- and oligsaccharides. 11. The method according to claim 9 or 10, where the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, a trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue. 12. The method of claim 9 or 10 wherein the spacer group is selected from the group consisting of di-, tri- and tetrasaccharides. 13. The method according to claim 9 or 10, wherein at least 90% of the bonds between the glycosaminoglycan molecules and the crosslinks are amide bonds. 14. The method according to claim 9 or 10, wherein less than 5% of the bonds between the glycosaminoglycan molecules and the crosslinks are ester bonds. 15. The method according to claim 1 or 2, wherein both the first GAG and the second GAG are covalently cross-linked with a spacer group selected from the group consisting of di-, tri-, tetra- and oligsaccharides. 16. The method of claim 1 or 2, wherein both the first GAG and the second GAG are covalently cross-linked with a polyfunctional crosslinking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane. 17. The method of claim 1 or 2, wherein the first GAG is covalently crosslinked with a polyfunctional crosslinker selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane. 18. The method of claim 17 wherein the second GAG is covalently cross-linked with a spacer group selected from the group consisting of di-, tri-, tetra- and oligsaccharides. 19. The method according to claim 18, where the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, a trehalose, lactose, maltose, sucrose, cellobiose, or raffinose residue. 20. The method of claim 1 or 2, wherein the second GAG is covalently crosslinked with a polyfunctional crosslinker selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane. 21. The method of claim 20 wherein the first GAG is covalently cross-linked with a spacer group selected from the group consisting of di-, tri-, tetra- and oligsaccharides. 22. The method according to claim 21, wherein the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, a trehalose, lactose, maltose, sucrose, cellobiose, or raffinose residue. 23. An injectable soft tissue gel product for cosmetic or medical surgery, comprising a first, internal, phase of a plurality of cross-linked glycosaminoglycan (GAG) gel particles embedded in a second, external, phase of a cross-linked GAG gel; where the second, external, phase is in the form of particles; where the degree of modification (MoD) of the gel of the first, internal, phase is 0.15 or less, and where the MoD of the gel of the second, outer, phase is lower than the MoD of the gel of the first, inner, phase, and where MoD is the molar amount of bound cross-linking agent(s) relative to the total molar number of repeating GAG disaccharide units; where the GAG of the first, internal, phase and the second, external, phase is hyaluronic acid, and where each of the first, internal, phase and second, external, phase is independently cross-linked with a cross-linking agent selected from (i) a multifunctional cross-linking agent selected from 1 ,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane, and (ii) a crosslinking agent containing a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides. 24. An injectable gel product according to claim 23, wherein the degree of modification (MoD) of the second phase gel is 0.10 or less. 25. An injectable gel product according to claim 23 or 24, wherein the swelling factor (SwF) of the second phase gel is greater than 3.0. 26. An injectable gel product according to claim 23 or 24, wherein the gels of the first, internal, phase and the second, external, phase are cross-linked with each other. 27. An injectable gel product according to claim 23 or 24, wherein the degree of modification (MoD) is substantially uniform throughout the second, outer phase and substantially uniform throughout the first, inner phase. 28. An injectable gel product according to claim 23 or 24, wherein the particle size of the outer phase gel is at least three times the particle size of the inner phase. 29. An injectable gel product according to claim 23 or 24, wherein the dry weight of the cross-linked glycosaminoglycan (GAG) in the first, internal, phase is at least 50% of the total dry weight of glycosaminoglycans (GAG) in the internal and external phases. 30. An injectable gel product according to claim 23 or 24, wherein the glycosaminoglycan (GAG) is hyaluronic acid. 31. An injectable gel product according to claim 23 or 24, wherein the cross-links are ether bonds. 32. An injectable gel product according to claim 23 or 24, wherein the glycosaminoglycan molecules of the first, internal, phase and/or the glycosaminoglycan molecules of the second, external, phase are covalently cross-linked by means of cross-links containing a spacer group selected from the group consisting of di-, tri -, tetra- and oligosaccharides. 33. The injectable gel product of claim 32, wherein at least 75% of the crosslinks include a spacer group selected from the group consisting of di-, tri-, tetra-, and oligsaccharides. 34. An injectable gel product according to claim 32 or 33, wherein the spacer group is hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, a trehalose, lactose, maltose, sucrose, cellobiose, or raffinose residue. 35. An injectable gel product according to claim 32 or 33, wherein the spacer group is selected from the group consisting of di-, tri-, and tetrasaccharides. 36. An injectable gel product according to claim 32 or 33, wherein at least 90% of the bonds between the glycosaminoglycan molecules and the crosslinks are amide bonds. 37. An injectable gel product according to claim 32 or 33, wherein less than 5% of the bonds between the glycosaminoglycan molecules and the crosslinks are ester bonds. 38. An injectable gel product according to claim 23 or 24, wherein both the first, internal, phase and the second, external, phase are covalently cross-linked with a polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2- ethanediol diglycidyl ether (EDDE) and diepoxyoctane. 39. An injectable gel product according to claim 23 or 24, wherein the first, internal, phase is covalently cross-linked with a polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane. 40. An injectable gel product according to claim 39, wherein the second, outer, phase is covalently cross-linked with a spacer group selected from the group consisting of di-, tri-, tetra-, and oligsaccharides. 41. An injectable gel product according to claim 40, wherein the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, a trehalose, lactose, maltose, sucrose, cellobiose, or raffinose residue. 42. An injectable gel product according to claim 23 or 24, wherein the second, outer, phase is covalently cross-linked with a polyfunctional cross-linking agent selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane. 43. An injectable gel product according to claim 42, wherein the first, internal, phase is covalently cross-linked with a spacer group selected from the group consisting of di-, tri-, tetra-, and oligsaccharides. 44. An injectable gel product according to claim 43, wherein the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, a trehalose, lactose, maltose, sucrose, cellobiose, or raffinose residue. 45. An injectable gel product according to claim 23 or 24, wherein the first, internal phase and the second, external phase are covalently cross-linked with a spacer group selected from the group consisting of di-, tri-, tetra-, and oligsaccharides. 46. An injectable gel product according to claim 45, wherein the spacer group is a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, a trehalose, lactose, maltose, sucrose, cellobiose, or raffinose residue. 47. An injectable gel product according to claim 23 or 24, wherein said injectable gel product is in a syringe and then sterilized. 48. An aqueous composition for cosmetic or medical surgery containing an injectable gel product according to claim 23 or 24 and optionally a buffering agent. 49. An aqueous composition according to claim 48, wherein said aqueous composition is in a syringe.

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