EP1592715A1 - Water soluble and biocompatible gels of hyaluronic acid cross-linked with bi-functional l-aminoacids or l-aminoesters - Google Patents
Water soluble and biocompatible gels of hyaluronic acid cross-linked with bi-functional l-aminoacids or l-aminoestersInfo
- Publication number
- EP1592715A1 EP1592715A1 EP03815561A EP03815561A EP1592715A1 EP 1592715 A1 EP1592715 A1 EP 1592715A1 EP 03815561 A EP03815561 A EP 03815561A EP 03815561 A EP03815561 A EP 03815561A EP 1592715 A1 EP1592715 A1 EP 1592715A1
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- European Patent Office
- Prior art keywords
- process according
- hyaluronic acid
- gel
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- solution
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L2203/00—Applications
- C08L2203/02—Applications for biomedical use
Definitions
- the present invention refers to soluble, bio-compatible gels consisting of hyaluronic acid cross-linked with bi-functional L-aminoacids or L-aminoesters, or mixtures thereof, to a process for their preparation and to their use in the pharmaceutical, cosmetic surgical and medical fields.
- Hyaluronic acid is a mucopolysaccharide consisting of alternated units of D- glucuronic acid and N-acetyl-glucosamine, bound together by ⁇ 1-3 and ⁇ -4 bindings.
- Hyaluronic acid is found in nature in the synovial liquid of articular joints, in the vitreous humour of eyes, in the umbilical cordon and in the connective tissues.
- Hyaluronic acid can be obtained by extraction from animal tissues like cockscombs or umbilical cordons, or can be recovered from the fermenting broth of specific Streptococci.
- the present invention allows to overcome the above said problem by making available gels soluble in water and bio-compatible prepared by cross-linking hyaluronic acid with bi-functional L-aminoacids or L-aminoesters or their mixtures.
- the characteristics of the compounds according to the invention make them ideal products for use in various fields from pharmaceutical to medical.
- the product in the form of film or gel, can for example be used as anti-adherent material in surgery (in abdomen, spinal surgery etc); in this case the material forms a barrier separating the damaged tissues and is absorbed after a time which allows the formation of the new tissues.
- the products according to the present invention can be also used as substrate for tissue engineering (derma, epidermis tissue, bones, fat cells, etc.) and can be used in ophthalmology, dermatology, in the osteoartritis field etc.
- the compounds according to the invention can be prepared in water or in a mixture water/organic solvents as DMF or DMSO depending on their use.
- the reaction is carried out in two subsequent steps: firstly the hyaluronic acid is activated and thereafter the binding between hyaluronic acid a cross-linking agent is performed.
- the activation of hyaluronic acid takes place in the presence of a carbodiimide soluble in water according to known processes (see for example Tomihata, J. Biomed. Mater. Res., 1997, 37(2), 243-251; Danishefsky, Carbohydrate Res., 1971, 16, 199-205).
- a carbodiimide soluble in water the N-3-dimethylamino-propylethylcarbodiimide hydrochloride is particularly preferred.
- the cross-linking agents are bi-functional ⁇ L- amino acids, i.e. having a second functional group besides the amino acid group, or their esters or mixtures thereof.
- Particularly preferred are L-lysine, L-serine, L- lysine ethylester di-hydrochloride, L-lysine methylester di-hydrochloride, L-serine methylester hydrochloride, L-serine ethylester hydrochloride or mixtures thereof.
- the use of amino esters instead of amino acids allows the protection of the carboxylic functions of the amino acids in respect of their possible activation and their involvement in secondary reactions.
- the reaction is carried out in a reactor equipped with a stirring system and a temperature controller.
- the hyaluronic acid sodium salt is dissolved in water in a concentration comprised between 0.5 and 2.5% according to the desired characteristics of the final product, the preferred concentration is 1 - 1.5%.
- the reaction temperature is one of the essential condition for obtaining the products of the invention and it must be comprised between 0°C and 25°C, preferably between 0°C and 10°C.
- the pH is brought at 3 - 6, preferably 4-5, for example by adding diluted HCl.
- activating agent/hyaluronic acid sodium salt Another essential factor for obtaining the products according to the invention it is the ratio: activating agent/hyaluronic acid sodium salt; the activating agent must be added in quantities comprised between 0.05 - 0.5 equivalents for equivalent of monomer unit of hyaluronic acid, preferably between 0.1 and 0.2 equivalents. If higher quantities of activating agent are used irreversible binding between hyaluronic acid and activating agent takes place instead of the wanted binding hyaluronic acid/cross-linking agent which causes the formation of undesired products which are insoluble or partially insoluble in water and having bio- compatibility and purity characteristics which are different from those of the presently described products. Finally the cross-linking agent is added in quantities comprised between 0.1 - 1 equivalents for equivalent of monomer unit of starting hyaluronic acid.
- reaction mixture is kept at the fixed temperature and under stirring for 15 minutes - 4 hours, preferably 30 minutes - 2 hours.
- a solution of NaCl 1 M is added and a buffer solution at pH 7 - 8 (preferably 7.5) then the mixture is purified according to known techniques as dialysis and/or diafiltration on ultrafiltration membrane and/or precipitation with an organic solvent and/or evaporation under vacuum and/or freeze drying.
- the preferred methods are diafiltration on membrane having a cutoff of 30 KDa and freeze drying.
- the solid product can be solved in water or in physiologic solution in various concentrations in order to obtain viscous solutions or transparent gels, it is also possible to obtain thin films, membranes according to the wanted use.
- the product can be sterilised for example by filtration at 0.2 ⁇ .
- the reciprocal quantities hyaluronic acid/activating agent/cross-linking agent have an effect on the cross-linking grade and therefore they will be chosen according to the wanted viscoelastic properties. Obviously the final characteristics will also be a consequence of the hyaluronic acid used as starting product.
- hyaluronic acid having higher molecular weight will result in a liquid more viscous and compact compared to the one obtained with a hyaluronic acid having lower molecular weight.
- hyaluronic acid having a molecular weight comprised between 100.000 and 2.000.000 is used and the final products will have a molecular weight comprised between 200.000 and 2.500.000.
- the final products have a cross-linking degree of 2 - 40%.
- hyaluronic acid sodium salt (MW 1.550.000) (2.5 mmol) are dissolved in 100 ml of demineralised water. The temperature is maintained at 5°C and the pH value is brought to 4.5 by addition of diluted HCl. 0.048 g (0.1 eq) of N-3-dimethylamino- propylethylcarbodiimide hydrochloride and 0.31 g (0.5 eq) of L-lysine ethylesterdi- hydrochloride are added.
- hyaluronic acid sodium salt (MW 1.550.000) (2.5 mmol) are dissolved in 100 ml of demineralised water. The temperature is maintained at 5°C and the pH value is brought to 4.5 by addition of diluted HCl. 0.096 g (0.2 eq) of N-3-dimethylamino- propylethylcarbodiimide hydrochloride and 0.31 g (0.5 eq) of L-lysine ethylesterdi- hydrochloride are added.
- hyaluronic acid sodium salt (MW 750.000) (2.5 mmol) are dissolved in 100 ml of demineralised water. The temperature is maintained at 5°C and the pH value is brought to 4.5 by addition of diluted HCl. 0.048 g (0.1 eq) of N-3-dimethylamino- propylethylcarbodiimide hydrochloride and 0.20 g (0.5 eq) of L-serine methylester hydrochloride are added.
- hyaluronic acid sodium salt (MW 750.000) (2.5 mmol) are dissolved in 100 ml of demineralised water. The temperature is maintained at 5°C and the pH value is brought to 4.5 by addition of diluted HCl. 0.096 g (0.2 eq) of N-3-dimethylamino- propylethylcarbodiimide hydrochloride and 0.20 g (0.5 eq) of L-serine methylester hydrochloride are added.
- hyaluronic acid sodium salt (MW 1.600.000) (2.5 mmol) are dissolved in 100 ml of demineralised water. The temperature is maintained at 5°C and the pH value is brought to 4.5 by addition of diluted HCl. 0.048 g (0.1 eq) of N-3-dimethylamino- propylethylcarbodiimide hydrochloride and 0.18 g (0.5 eq) of L-lysine are added.
- Example 1 The solid obtained in Example 1 is dissolved in water in a concentration of 3-4g/l and filtered, under sterile condition, at 0.2 microns.
- the filtered gel is sterile and has a content of endotoxines lower than 0.2
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Abstract
Soluble and bio-compatible gels consisting of hyaluronic acid cross-linked with bifunctional L-amino acids or L-amino esters, or mixture thereof are described; also described is a process for their preparation and their use in pharmaceutical, cosmetic and medical-surgical field.
Description
WATER SOLUBLE AND BIOCOMPATIBLE GELS OF HYALURONIC ACID CROSS-LINKED WITH BIFUNCTIONAL L-AMINOACIDS OR L-AMINOESTERS
Field of the invention
The present invention refers to soluble, bio-compatible gels consisting of hyaluronic acid cross-linked with bi-functional L-aminoacids or L-aminoesters, or mixtures thereof, to a process for their preparation and to their use in the pharmaceutical, cosmetic surgical and medical fields.
State of the art
Hyaluronic acid is a mucopolysaccharide consisting of alternated units of D- glucuronic acid and N-acetyl-glucosamine, bound together by β 1-3 and β-4 bindings.
Hyaluronic acid is found in nature in the synovial liquid of articular joints, in the vitreous humour of eyes, in the umbilical cordon and in the connective tissues.
Hyaluronic acid can be obtained by extraction from animal tissues like cockscombs or umbilical cordons, or can be recovered from the fermenting broth of specific Streptococci.
The development of biotechnology allowed the optimisation and improvement of the last described method of production of hyaluronic acid which is nowadays the one considered the most simple and rewarding. The applications of hyaluronic acid, in surgery, pharmacology and in general in biomedicine, are widely described in literature, see for example: Balazs et al.
"Hyaluronan Biomaterials: Medical Applications", Handbook of Biomaterials and
Applications, Ed. D Wise et al, 1995, 2719-2741; US - 5,559,104, 1996; Pape,
Balazs, Ophthalmology, 87, No 7, 1980; Iwata, Clin. Orthop., 289, 285-291; 1993; US 5,128,326; US - 4,500,676; US - 5,840,046; US - 5,795,584; US -
6,010,692; US - 5,658,331.
Since the important role played by hyaluronic acid in the human body is due to the peculiar viscoelastic, lubricant and hydrophilic characteristics of its aqueous solutions the searchers are studying and preparing hyaluronic acid derivatives wherein such properties are changed in order to obtain compounds for various uses.
A wide literature report various products derived from the cross-linking of
hyaluronic acid with, for example, formaldheyde (Balazs, US 4,713,448, 1987), divinyl sulphone (Balazs, US 4,582,865, 1986), aziridine, alcohols (Delia Valle, US 4,851 ,521, 1989) and mono-functional amino acids (Hamilton, US 4,937,270, 1990). In particular Miller et al., US 5,760,200, 1998 described meythods for the preparation of insoluble derivatives of hyaluronic acids, for example with L-lysine ethylester, in the presence of carbodiimide soluble in water, the reaction was performed in the present of a high excess of activating agent and amino acid in order to obtain an insoluble gel. Moreover in WO 01/58961, in the name of the present Applicant, derivatives of hyaluronic acid cross-linked with bi-functional L- amino acids and L-amino esters, and process for their preparation both in water and in organic solvents are described, however the therein described products, in spite of their quality, should be improved in respect of their purity, viscolelastic and biocompatible properties in view of their possible pharmacological, medical or surgical use.
It is therefore evident, in view of the above said, the importance of making available new compounds capable of widening and improving the use of hyaluronic acid in the known or in new fields. Detailed description of the invention The present invention allows to overcome the above said problem by making available gels soluble in water and bio-compatible prepared by cross-linking hyaluronic acid with bi-functional L-aminoacids or L-aminoesters or their mixtures. The characteristics of the compounds according to the invention make them ideal products for use in various fields from pharmaceutical to medical. The product, in the form of film or gel, can for example be used as anti-adherent material in surgery (in abdomen, spinal surgery etc); in this case the material forms a barrier separating the damaged tissues and is absorbed after a time which allows the formation of the new tissues. The products according to the present invention can be also used as substrate for tissue engineering (derma, epidermis tissue, bones, fat cells, etc.) and can be used in ophthalmology, dermatology, in the osteoartritis field etc. The compounds according to the invention can be prepared in water or in a
mixture water/organic solvents as DMF or DMSO depending on their use. In both cases the reaction is carried out in two subsequent steps: firstly the hyaluronic acid is activated and thereafter the binding between hyaluronic acid a cross-linking agent is performed. The activation of hyaluronic acid takes place in the presence of a carbodiimide soluble in water according to known processes (see for example Tomihata, J. Biomed. Mater. Res., 1997, 37(2), 243-251; Danishefsky, Carbohydrate Res., 1971, 16, 199-205). Among the various carbodiimides soluble in water the N-3-dimethylamino-propylethylcarbodiimide hydrochloride is particularly preferred. According to the present invention the cross-linking agents are bi-functional α L- amino acids, i.e. having a second functional group besides the amino acid group, or their esters or mixtures thereof. Particularly preferred are L-lysine, L-serine, L- lysine ethylester di-hydrochloride, L-lysine methylester di-hydrochloride, L-serine methylester hydrochloride, L-serine ethylester hydrochloride or mixtures thereof. The use of amino esters instead of amino acids allows the protection of the carboxylic functions of the amino acids in respect of their possible activation and their involvement in secondary reactions.
The reaction is carried out in a reactor equipped with a stirring system and a temperature controller. The hyaluronic acid sodium salt is dissolved in water in a concentration comprised between 0.5 and 2.5% according to the desired characteristics of the final product, the preferred concentration is 1 - 1.5%. The reaction temperature is one of the essential condition for obtaining the products of the invention and it must be comprised between 0°C and 25°C, preferably between 0°C and 10°C. The pH is brought at 3 - 6, preferably 4-5, for example by adding diluted HCl.
Another essential factor for obtaining the products according to the invention it is the ratio: activating agent/hyaluronic acid sodium salt; the activating agent must be added in quantities comprised between 0.05 - 0.5 equivalents for equivalent of monomer unit of hyaluronic acid, preferably between 0.1 and 0.2 equivalents. If higher quantities of activating agent are used irreversible binding between hyaluronic acid and activating agent takes place instead of the wanted binding hyaluronic acid/cross-linking agent which causes the formation of undesired
products which are insoluble or partially insoluble in water and having bio- compatibility and purity characteristics which are different from those of the presently described products. Finally the cross-linking agent is added in quantities comprised between 0.1 - 1 equivalents for equivalent of monomer unit of starting hyaluronic acid.
After the above said the reaction mixture is kept at the fixed temperature and under stirring for 15 minutes - 4 hours, preferably 30 minutes - 2 hours. A solution of NaCl 1 M is added and a buffer solution at pH 7 - 8 (preferably 7.5) then the mixture is purified according to known techniques as dialysis and/or diafiltration on ultrafiltration membrane and/or precipitation with an organic solvent and/or evaporation under vacuum and/or freeze drying. The preferred methods are diafiltration on membrane having a cutoff of 30 KDa and freeze drying. The solid product can be solved in water or in physiologic solution in various concentrations in order to obtain viscous solutions or transparent gels, it is also possible to obtain thin films, membranes according to the wanted use. The product can be sterilised for example by filtration at 0.2 μ. The reciprocal quantities hyaluronic acid/activating agent/cross-linking agent have an effect on the cross-linking grade and therefore they will be chosen according to the wanted viscoelastic properties. Obviously the final characteristics will also be a consequence of the hyaluronic acid used as starting product.
In fact, the other condition being unvaried, the use of a hyaluronic acid having higher molecular weight will result in a liquid more viscous and compact compared to the one obtained with a hyaluronic acid having lower molecular weight. Preferably according to the invention hyaluronic acid having a molecular weight comprised between 100.000 and 2.000.000 is used and the final products will have a molecular weight comprised between 200.000 and 2.500.000. The final products have a cross-linking degree of 2 - 40%. The invention will be better understood in view of the following examples. EXAMPLE 1
1 g of hyaluronic acid sodium salt (MW 1.550.000) (2.5 mmol) are dissolved in 100 ml of demineralised water. The temperature is maintained at 5°C and the pH value
is brought to 4.5 by addition of diluted HCl. 0.048 g (0.1 eq) of N-3-dimethylamino- propylethylcarbodiimide hydrochloride and 0.31 g (0.5 eq) of L-lysine ethylesterdi- hydrochloride are added. After 1 hours 10 ml of NaCl solution 1 M and 10 ml of buffer solution at pH 7.5 are added, the gel is diafiltered with 10 volumes of demineralised water on a membrane with cutoff 30 KDa and finally freeze-dried. EXAMPLE 2
1 g of hyaluronic acid sodium salt (MW 1.550.000) (2.5 mmol) are dissolved in 100 ml of demineralised water. The temperature is maintained at 5°C and the pH value is brought to 4.5 by addition of diluted HCl. 0.096 g (0.2 eq) of N-3-dimethylamino- propylethylcarbodiimide hydrochloride and 0.31 g (0.5 eq) of L-lysine ethylesterdi- hydrochloride are added.
After 1 hours 10 ml of NaCl solution 1 M and 10 ml of buffer solution at pH 7.5 are added; the gel is diafiltered with 10 volumes of demineralised water on a membrane with cutoff 30 KDa and finally freeze-dried. EXAMPLE 3
1 g of hyaluronic acid sodium salt (MW 750.000) (2.5 mmol) are dissolved in 100 ml of demineralised water. The temperature is maintained at 5°C and the pH value is brought to 4.5 by addition of diluted HCl. 0.048 g (0.1 eq) of N-3-dimethylamino- propylethylcarbodiimide hydrochloride and 0.20 g (0.5 eq) of L-serine methylester hydrochloride are added.
After 1 hours 10 ml of NaCl solution 1 M and 10 ml of buffer solution at pH 7.5 are added, the gel is diafiltered with 10 volumes of demineralised water on a membrane with cutoff 30 KDa and finally freeze-dried. EXAMPLE 4
1 g of hyaluronic acid sodium salt (MW 750.000) (2.5 mmol) are dissolved in 100 ml of demineralised water. The temperature is maintained at 5°C and the pH value is brought to 4.5 by addition of diluted HCl. 0.096 g (0.2 eq) of N-3-dimethylamino- propylethylcarbodiimide hydrochloride and 0.20 g (0.5 eq) of L-serine methylester hydrochloride are added.
After 1 hours 10 ml of NaCl solution 1 M and 10 ml of buffer solution at pH 7.5 are added, the gel is diafiltered with 10 volumes of demineralised water on a
membrane with cutoff 30 KDa and finally freeze-dried.
EXAMPLE 5
1 g of hyaluronic acid sodium salt (MW 1.600.000) (2.5 mmol) are dissolved in 100 ml of demineralised water. The temperature is maintained at 5°C and the pH value is brought to 4.5 by addition of diluted HCl. 0.048 g (0.1 eq) of N-3-dimethylamino- propylethylcarbodiimide hydrochloride and 0.18 g (0.5 eq) of L-lysine are added.
After 1 hours 10 ml of NaCl solution 1M and 10 ml of buffer solution at pH 7.5 are added; the gel is diafiltered with 10 volumes of demineralised water on a membrane with cutoff 30 KDa and finally freeze-dried. EXAMPLE 6
The solid obtained in Example 1 is dissolved in water in a concentration of 3-4g/l and filtered, under sterile condition, at 0.2 microns.
The filtered gel is sterile and has a content of endotoxines lower than 0.2
EU/mg(ss). The physico-chemical characterisation showed a cross-linking degree of 10% a loss by drying of 18-20% and the following 1 H-NMR signals (solvent D20):
1.2 ppm (t, 3H, J = 10.6 Hz, CH3-CH2O-Lys)
1.4 ppm (m, 2H, Ch δ Lys)
1.6 ppm (m, 2H, CH? γ Lys) 1.7-1.9 ppm (m, 2H, CH2 β Lys)
1.9-2.0 ppm (m, 3H, CH3-CONH hyaluronic acid)
2.9 ppm (t, 2H, J = 11.2 Hz, CH2 ε Lys)
3.0-3.9 ppm (m, CHOH hyaluronic acid)
4.0 ppm (t, 1 H, J = 9.6 Hz, CH α Lys) 4.2 ppm (q, 2H, J = 10.6 Hz, CHs-ChbO-Lys)
Claims
1. A process for the preparation of soluble, biocompatible gels consisting of hyaluronic acid cross-linked with bi-functional cross-linking agents wherein:
- the hyaluronic acid is solved in water at a concentration of 0.5 2.5% (w/w) and at a temperature of 0° - 25°C and the solution pH is brought at 3-6; the solution is added with bi-functional cross-linking agents, in a quantity comprised between 0.1 - 1 equivalent for each equivalent of monomer unit of hyaluronic acid, and an activating agent, in a quantity comprised between 0.05 - 0.5 equivalents for each equivalent of monomer unit of hyaluronic acid; - the solution is stirred for 15 minutes - 4 hours;
- the solution is added with a solution of NaCl 1 M and a buffer solution at pH 7 - 8;
- the formed gel is collected and purified according to known techniques.
2. Process according to claim 1 wherein the bi-functional cross-linking agents are L-amino acids and/or L-amino esters or mixtures thereof.
3. Process according to claim 2 wherein the bi-functional cross-linking agents are: L-lysine, L-serine, L-lysine ethylester dihydrochloride, L-lysine methylester dihydrochloride, L-serine ethylester hydrochloride, L-serine methylester hydrochloride or mixtures thereof.
4. Process according to Claims 1 - 3 wherein the reaction temperature is comprised between 0 - 10°C.
5. Process according to claim 4 wherein the reaction pH is comprised between 3 - 6.
6. Process according to claim 5, wherein the reaction pH is comprised between 4 - 5.
7. Process according to claims 1-6, wherein the activating agent is a carbodiimide soluble in water.
8. Process according to claim 7, wherein the activating agent is N-3- dimethylamino-propylethylcarbodiimide hydrochloride.
9. Process according to claims 1-8, wherein the activating agent is added in a quantity comprised between 0.1 - 0.2 equivalents for each equivalent of monomer unit of hyaluronic acid.
10. Process according to claims 1-9, wherein the reaction time is comprised between 30 minutes and 2 hours.
11. Process according to claims 1-10, wherein the finally obtained gel is added with a solution of NaCl 1 M and a buffer solution at pH 7.5
12. Process according to claims 1-11 , wherein the gel is purified by dialysis and/or diafiltration on ultrafiltering membrane and/or precipitation with an organic solvent and/or evaporation under vacuum and/or freeze-drying.
13. Process according to claim 12, wherein the gel is purified by diafiltration on a membrane with cutoff 30 KDa and freeze-dried.
14. Process according to claims 1- 13 wherein the purified gel is sterilised by filtration at 0.2 microns.
15. Process according to claims 1-14 wherein the hyaluronic acid has a MW comprised between 100.000 and 2.000.000.
16. Soluble and bio-compatible gels consisting of hyaluronic acid cross-linked with bi-functional cross-linkants, obtained through a process according to claims 1 - 14.
17. Gel according to claim 16 having a cross-linking degree of 2-40%.
18. Gel according to claims 16 and 17 having a MW of 200.000 - 2.500.000.
19. Use of a gel according to claims 16-18 as anti-adherent material in surgical field.
20. Use of a gel according to claims 16 - 18 as a substrate for tissue engineering (derma, epidermis tissue, bones, fat cells).
21. Use of a gel according to claims 16 - 18 as a product for applications in ophthalmology, dermatology, or in the field of osteoartritis.
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PCT/IB2003/000321 WO2004067575A1 (en) | 2003-01-31 | 2003-01-31 | Water soluble and biocompatible gels of hyaluronic acid cross-linked with bi-functional l-aminoacids or l-aminoesters |
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PT1817347T (en) | 2004-11-24 | 2017-07-21 | Albumedix As | Method of cross-linking hyaluronic acid with divinylsulfone |
JP5523338B2 (en) | 2007-12-19 | 2014-06-18 | エヴォニク ゴールドシュミット ゲーエムベーハー | Cross-linked hyaluronic acid in emulsion |
US9228027B2 (en) | 2008-09-02 | 2016-01-05 | Allergan Holdings France S.A.S. | Threads of Hyaluronic acid and/or derivatives thereof, methods of making thereof and uses thereof |
US20110172180A1 (en) | 2010-01-13 | 2011-07-14 | Allergan Industrie. Sas | Heat stable hyaluronic acid compositions for dermatological use |
DK3078388T3 (en) | 2010-03-22 | 2019-05-20 | Allergan Inc | CROSS-BREAKED HYDROGEN WAVES |
WO2011148116A2 (en) * | 2010-05-27 | 2011-12-01 | Laboratoire Idenov | Modified hyaluronic acid, method for manufacturing same and uses thereof |
US9408797B2 (en) * | 2011-06-03 | 2016-08-09 | Allergan, Inc. | Dermal filler compositions for fine line treatment |
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RU2626513C2 (en) * | 2011-09-14 | 2017-07-28 | Аллерган, Инк. | Dermal filler composition for small wrinkles treating |
US20140315828A1 (en) | 2013-04-22 | 2014-10-23 | Allergan, Inc. | Cross-linked silk-hyaluronic acid compositions |
ES2761558T3 (en) | 2014-09-30 | 2020-05-20 | Allergan Ind Sas | Stable hydrogel compositions including additives |
US10300169B2 (en) | 2016-08-24 | 2019-05-28 | Allergan, Inc. | Co-crosslinked hyaluronic acid-silk fibroin hydrogels for improving tissue graft viability and for soft tissue augmentation |
US20230190997A1 (en) | 2017-06-26 | 2023-06-22 | Evolved By Nature, Inc. | Silk-hyaluronic acid based tissue filers and methods of using the same |
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US6174999B1 (en) * | 1987-09-18 | 2001-01-16 | Genzyme Corporation | Water insoluble derivatives of polyanionic polysaccharides |
IT1317091B1 (en) * | 2000-02-08 | 2003-05-26 | S F I R Societa Fondaria Ind R | CROSS-LINKED HYALURONIC ACID GEL WITH B-FUNCTIONAL L-AMINO ACIDS. |
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- 2003-01-31 WO PCT/IB2003/000321 patent/WO2004067575A1/en not_active Application Discontinuation
- 2003-01-31 AU AU2003303820A patent/AU2003303820A1/en not_active Abandoned
- 2003-01-31 EP EP03815561A patent/EP1592715A1/en not_active Withdrawn
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