MXPA06007323A

MXPA06007323A - Compositions of semi-interpenetrating polymer network

Info

Publication number: MXPA06007323A
Application number: MXPA/A/2006/007323A
Authority: MX
Inventors: James White Barry; Isabella Rodden Gillina
Original assignee: Innomed Ltd
Priority date: 2003-12-23
Filing date: 2006-06-23
Publication date: 2007-04-20

Abstract

Novel compositions consisting of semi-interpenetrating network of cross-linked water soluble derivatives of basic polysaccharides and a non-cross-linked component, which is an anionic polysaccharide are provided. Methods for the production of such compositions are also disclosed. Preferably the basic polysaccharide is chitosan or a derivative thereof and the anionic polysaccharide is hyaluronic acid. The compositions can be formed into gels or films, for example, and thus find use in a wide range of medical applications in the fields of dermatology, plastic surgery, urology and orthopaedics.

Description

connective tissue that are influenced in their development, the migration and proliferation in the medium in which they are exposed to a polymeric network of hyaluronic acid that is not normally found there. There is growing evidence in the scientific literature that natural hyaluronic acid administered exogenously stimulates the synthesis of endogenous hyaluronic acid and therefore it can be postulated that a biological material that has a biopolymer network whose in vivo residence time could be modified and at the same time time can produce exogenous hyalorunic acid in its natural structure not chemically modified for an extended period of time would have potential benefits on hyaluronic acid in several biomedical applications. It can further be postulated that such a biological material could have applications as a mimic of an extracellular matrix if other polysaccharide components of the extracellular natural matrix such as chondroitin sulfates, dermatan and qeuratin are incorporated into the polymer network. Chitosan, an amino group containing basic polysaccharides, a derivative of biopolymer chitin, has been reported in the scientific literature because it has excellent biocompatibility and is used in several biomedical applications. The North American patent no. No. 5,977,330 discloses substituted N-crosslinked chitosan derivatives in which the substitution is carried out by means of hydroxyacyl compounds carrying carboxylic acids subsequently crosslinked using polyepoxides. No attempt has been made to define semi-I PN using those cross-linked derivatives. The North American patent no. No. 6,379,702 discloses a mixture of chitosan and a hydrophilic poly (N-vinyl lactam). This document does not disclose any cross-linking of the chitosan or the formation of semi-IPN. US Patent 6,224,893 discloses compositions for forming a semi-interpenetrating or interpenetrating polymer network for drug delivery and tissue engineering wherein the semi IPN is prepared from synthetic and / or natural polymers with a photoinitiator wherein the crosslinking is initiates by means of the generation of free radicals by means of electromagnetic radiation. The North American patent no. No. 5,644,049 describes a biological material comprising an interpenetrating polymer network in which one of the components, an acidic polysaccharide, is crosslinked to a second component, a synthetic chemical polymer to create an infinite network. There are no descriptions of the cross-linking of acidic polysaccharides with basic polysaccharides. The North American patent no. 5,620,706 discloses a biological material comprising a polyanic complex of xanthan and chitosan for the encapsulation and controlled release of biologically active substances. There is no description of basic polysaccharides crosslinked covalently with acidic polysaccharides. Berger et al. European Journal of Pharmaceutics and Biopharmaceutics, 57 (2004), 1 9-34, discusses various structures of crosslinked chitosan hydrogels, including semi-IPN structures. Therefore we have developed a new range of biological materials, which are based on the formation of a semi-IPN with cationic polysaccharide derivatives that are crosslinked in the presence of anionic polysaccharides under conditions which prevent the formation of ionic complexes between the two polymers and which allow the subsequent release of anionic polysaccharides from the crosslinked network. DESCRIPTION OF THE INVENTION Thus in a first aspect the present invention provides a composition consisting of an interpenetrating polymer network consisting of at least one water-soluble crosslinked derivative of the basic polysaccharide, having primary and / or secondary amine groups, wherein the Anionic polysaccharide resides within the semi-interpenetrating polymer network. An interpenetrating polymer network is a combination of at least two polymers formed by covalently crosslinking at least one of the polymers in the presence but not with the other polymers, and having at least one of the polymers in the network a linear or crosslinked crosslinked polymer. In the context of the present invention, a basic cationic polysaccharide is a polysaccharide containing at least one functional group that is capable of undergoing ionization to form a cation, for example a protonated amino group, while an acidic anionic polysaccharide is a polysaccharide which it contains at least one functional group that is capable of undergoing ionization to form an anion, for example a carboxylate or sulfate ion. The compositions of the present invention have use as biological materials, which can be formulated for example as hydrogels, which in turn can be placed in soft tissues as a mimic of the extracellular matrix. In one embodiment of the present invention, the water-soluble derivative of a basic polysaccharide is a derivative of chitosan, in particular N-carboxy methyl chitosan, O-carboxy methyl chitosan or O-hydroxy ethyl chitosan or an N-acetylated chitosan. Partially N-acetylated chitosan can be produced by partially deacetylating chitin or by re-acetylating chitosan. In any case, in one embodiment the partially N-acetylated chitosan has a degree of acetylation in the range of 45 to 55%. In another preferred embodiment, the non-crosslinked component is hyaluronic acid. In addition, the other anionic polysaccharide components of the extra cellular matrix can be included. The crosslinking component of the composition can be crosslinked using crosslinking agents such as diglycidyl ethers, diisocyanates or aldehydes. In particular, 1,4-butanedioldiglycidyl (BDDE) ethers can be used. The reaction between the epoxide rings at either end of the BDDE molecule and the amino groups on the chitosan chains occur through nucleophilic attack by reactive amino groups with subsequent epoxide ring opening as described in "Chitin in. Nature and Technology ", RA , Muzarelli, C. Jeuniaux and G.W. Godday, Plenum Press, New York, 1986, p. 303. The compositions of the present invention can be in the form of films, sponges, hydrogels, threads or nonwoven matrices.

In a second aspect, the present invention provides a method for the preparation of a composition of the invention which consists in crosslinking at least one derivative of a basic polysaccharide containing primary and / or secondary amine groups, in the presence of at least one anionic polysaccharide, under the conditions that prevent protonation of the primary and secondary amine groups in the basic polysaccharide and which also prevents the reaction of other functional groups in the water-soluble anionic polysaccharide. As already discussed, the present invention may be in the form of different biological materials for use in medical applications. For example to produce an injectable hydrogel: An aqueous solution of a water-soluble derivative of a basic polysaccharide containing primary and / or secondary amine groups is formed, to which a water-soluble anionic polysaccharide is added. The crosslinking of the basic polysaccharide is then initiated in the presence of a polyfunctional crosslinking agent, under essentially neutral conditions that only crosslink with the primary or substituted amines leaving the anionic polysaccharide entrapped within the crosslinked polymer network. To produce a water-insoluble film: An aqueous solution of a water-soluble derivative of a basic polysaccharide containing primary and / or secondary amine groups, to which a water-soluble anionic polysaccharide is added. A polyfunctional crosslinking agent is then added and the mixture allowed to evaporate until dry to allow the crosslinking reaction to take place. Chitosan becomes soluble in aqueous solutions only when protonated with acid. The polymer thus formed is positively charged and thus will interact with negatively charged species such as hyaluronic acid and other polyanions. These anionic complexes should be avoided in order to form smei IPN, which is the object of the present invention. Thus the chitosan must be solubilized either as an anionic polyelectrolyte or as an ionic polymer in a medium either neutral or slightly alkaline. As already described, suitable derivatives include N-carboxy methyl chitosan, O-carboxy methyl chitosan, O-hydroxy ethyl chitosan or partially N-acetylated chitosan. In a preferred embodiment, approximately 50% of the re-acetylated chitosan is used since it can be solubilized in neutral medium without the protonation of the amine groups. In another preferred embodiment, the re-acetylated chitosan has a degree of deacetylation in the range of 45% to 55% in order to obtain solubility properties in water. The crosslinking reaction in the presence of the polyfunctional crosslinking agent is generally carried out under neutral or slightly alkaline conditions, in a pH range of 7 to 8, which ensures that essentially only the primary or secondary amine groups of the basic polysaccharide can react with the agent crosslinker. Thus, the crosslinking of the anionic polysaccharide or indeed the crosslinker between the acidic and basic polymers is avoided. The degree of crosslinking can be controlled by varying the molar feed ratio of the basic polysaccharide to the crosslinking agent. In this way the release profile of the trapped anionic polysaccharide can be altered / modified to suit the biomedical application in which it is to be used. Generally the crosslinking reaction will be carried out at a pH of about 7, preferably between a pH of 6.8 and 8. In a third aspect the present invention provides a biological material containing a composition of the invention. In a fourth aspect of the present invention, the use in medicine of a composition or a biological material of the invention is provided. In a fifth aspect of the present invention the use of a composition of the preparation of a biological material is provided. In particular the biological material to be used in dermatology, plastic surgery, urology and in the field of orthopedics. These biological materials can take the form of films, sponges, hydrogels, threads and non-woven matrices. Preferred aspects of each aspect of the invention are mutatis mutandis for each of the other aspects. The invention will now be described with reference to the following examples illustrating the invention that should not be construed as limiting. EXAMPLES With respect to the following examples a control experiment was performed using HA and BDDE under the same conditions as for the preparation of all the gels, only that chitosan was not used.

There is no evidence of a gel formed after the HA was incubated with BDDE at 50 ° C for 3 hours. Therefore, it can be concluded that under the conditions used to form the semi-IPN, the HA does not contribute to gel formation and remains as a non-crosslinked linear polymer that is trapped in a cross-linked chitosan matrix. The water absorption capacity (Q) of the gels and the films prepared in the following examples was calculated using the following equation: Q% = (total wet mass of the polymer - total dry mass of the polymer) x 100 dry mass of the cross-linked polymer EXAMPLE 1 - GEL Re-acetylated chitosan (2g, DDA% = 54%, Mv = 680,000 g / mol) prepared from squid ink chitosan, hydrated in deionized water to give a solution having a final concentration of 5%. % by weight of polymer. HA (2g, prepared by means of fermentation, Hyaltech Ltd) was dissolved in water to give a solution having a final concentration of 5% by weight of polymer. The two solutions were cooled overnight to aid in the dissolution of the polymers. The solutions of the two polymers were then mixed together in a high tear mixer and a 1,4-bitanodio diglycidyl ether (2.5 g, Sigma) was added and stirred in the polymer mixture using a mechanical stirrer. The solution is then reticulated with gentle agitation in a water bath at 50 ° C for 3 hours. The gel formed was then immersed in deionized water and allowed to swell until it reached a constant weight, during which time the water was replaced 4-5 times to remove residual unreacted crosslinkers. The water absorption capacity of the gel was 9654% and it has a concentration of 10mg / ml of each polymer. The sample was homogenized in the high tear mixer to allow the gel to be injected from a syringe through a 30G needle. The average particle size (D4.3) was 302 μm. The sample has a value of modulus of elasticity G 'of 500 to 600 Pa measured in an oscillatory tear in a frequency range of 0.01 -10 Hz. An in vitro test was performed to monitor the release of HA from the gel over a period of time. extended time. The same experiment was performed in the presence of lysozymes. The results are shown below.

EXAMPLE 2- GEL Re-acetylated chitosan (2g, DDA% = 54%, Mv = 680,000 g / mol) prepared from squid ink chitosan, hydrated in deionized water to give a solution having a final concentration of 5%. % by weight of polymer. HA (1 g, prepared by means of fermentation, Hyaltech Ltd) was dissolved in water to give a solution having a final concentration of 5% by weight of polymer. The two solutions were cooled overnight to aid in the dissolution of the polymers. The solutions of the two polymers were then mixed together in a high tear mixer and a 1,4-bitanodiol diglycidyl ether (2.5 g, Sigma) was added and stirred into the polymer mixture using a mechanical stirrer. The solution is then reticulated with gentle agitation in a water bath at 50 ° C for 3 hours. The gel formed was then immersed in deionized water and allowed to swell until it reached a constant weight, during which time the water was replaced 4-5 times to remove residual unreacted crosslinkers. The water absorption capacity of the gel was 4551% and it has a concentration of 22 mg / ml of reacetylated chitosan and 12 mg / ml of HA. The sample was homogenized in the high tear mixer to allow the gel to be injected from a syringe through a 30G needle. The average particle size (D4.3) was 225 μm. The sample has a value of modulus of elasticity G 'of 2000 to 3000 Pa measured in an oscillatory tear in a frequency range of 0.01 -10 Hz. An in vitro test was performed to monitor the release of HA from the gel over a period of time. extended time. The same experiment was performed in the presence of lysozymes. The results are shown below.

EXAMPLE 3- GEL Re-acetylated chitosan (2g, DDA% = 54%, Mv * 750,000 g / mol) prepared from commercial shrimp chitosan, hydrated in deionized water to give a solution having a final concentration of 5% in weight of polymer. HA (2g, prepared by means of fermentation, Hyaltech Ltd) was dissolved in water to give a solution having a final concentration of 5% by weight of polymer. The two solutions were cooled overnight to aid in the dissolution of the polymers. The solutions of the two polymers were then mixed together in a high tear blender and a 1,4-bitanodiol diglycidyl ether (1.7 g, Fluka) was added and stirred in the polymer mixture using a mechanical stirrer. The solution is then reticulated with gentle agitation in a water bath at 50 ° C for 3 hours. The gel formed was then immersed in deionized water and allowed to swell until it reached a constant weight, during which time the water was replaced 4-5 times to remove residual unreacted crosslinkers. The water absorption capacity of the gel was 12652% and it has a concentration of 7.9mg / ml of re-acetylated chitosan and 7.5mg / ml of HA. When the gel was swollen in phosphate buffered saline (PBS) the final concentration of RAC and HA was 13.54mg / m and 12.75mg / ml respectively. The gel sample that swelled in water was homogenized in the high tear mixer to allow the gel to be injected from a syringe through a 30G needle. The average particle size (D4.3) was 451 μm. The sample has a modulus of elasticity G 'of 1 000 Pa measured in an oscillatory tear in a frequency range of 0.01 -10 Hz. An in vitro test was performed to monitor the HA release of the gel over a period of time dragged on. The same experiment was performed in the presence of lysozymes. The results are shown below.

EXAMPLE 4- GEL O-hydroxyethyl chitosan (1 g, Sigma) was hydrated in deionized water to give a solution having a final concentration of 5% by weight of polymer. HA (1 g, prepared by means of fermentation, Hyaltech Ltd) was dissolved in water to give a solution having a final concentration of 5% by weight of polymer. The two solutions were cooled overnight to aid in the dissolution of the polymers.

The solutions of the two polymers were then mixed together in a high tear mixer and a 1,4-bitanodio diglycidyl ether (1.5 g, Fluka) was added and stirred into the polymer mixture using a mechanical stirrer. The solution is then reticulated with gentle agitation in a water bath at 50 ° C for 3 hours. The gel formed was then immersed in deionized water and allowed to swell until it reached a constant weight, during which time the water was replaced 4-5 times to remove residual unreacted crosslinkers. The water absorption capacity of the gel was 8525% and it has a concentration of 1.7 mg / ml of re-acetylated chitosan and 12.7 mg / ml of HA. The sample was homogenized in the high tear mixer to allow the gel to be injected from a syringe through a 30G needle. The average particle size (D4.3) was 205 μm. The sample has a value of modulus of elasticity G 'of 1000 to 2000 Pa measured in an oscillatory tear in a frequency range of 0.01 -10 Hz. EXAMPLE 5 N-carboxymethyl chitosan (0.6 g, DDA% = 85%, Heppe Ltd ), was hydrated in deionized water to give a solution having a final concentration of 5% by weight of polymer. HA (0.6g, prepared by means of fermentation, Hyaltech Ltd) was dissolved in water to give a solution having a final concentration of 5% by weight of polymer. The two solutions were cooled overnight to aid in the dissolution of the polymers. The solutions of the two polymers were then mixed together in a high tear blender and an ether of 1 was added., 4-bitanodiol diglycidyl (0.96 g, Fluka) and stirred in the polymer mixture using a mechanical stirrer. The solution is then reticulated with gentle agitation in a water bath at 50 ° C for 8 hours. The gel formed was then immersed in deionized water and allowed to swell until it reached a constant weight, during which time the water was replaced 4-5 times to remove residual unreacted crosslinkers. The water absorption capacity of the gel was 9464% and it has a concentration of 11 mg / ml of both polymers. The sample was homogenized in the high tear mixer to allow the gel to be injected from a syringe through a 30G needle. The average particle size (D4.3) was 21 8 μm. The sample has a modulus of elasticity G 'of 600 to 900 Pa measured in an oscillatory tear in a frequency range of 0.01 -10 Hz. When the sample was swollen in phosphate buffered saline the concentration of N-carboxymethyl chitosan and HA was 38 mg / ml and 39 mg / ml respectively. EXAMPLE 6 - GEL Re-acetylated chitosan (1.9g, DDA% = 54%, v ~ 680,000 g / mol) prepared from squid ink chitosan, hydrated in deionized water to give a solution having a final concentration of 5% by weight of polymer. HA (1.9g, prepared by means of fermentation, Hyaltech Ltd) was dissolved in water to give a solution having a final concentration of 5% by weight of polymer. The two solutions were cooled overnight to aid in the dissolution of the polymers. The solutions of the two polymers were then mixed together in a high tear mixer. The solution is then reticulated with gentle agitation in a water bath at 50 ° C for 7 hours. The gel formed was then immersed in deionized water and allowed to swell for a period of 2-3 days until it reached a constant weight, during which time the water was replaced 4-5 times to remove residual unreacted crosslinkers. The water absorption capacity of the gel was 7995% and it has a concentration of 12.5 mg / ml of each polymer. The gel sample was homogenized in the high tear mixer to allow the gel to be injected from a syringe through a 30G needle. The average particle size (D4.3) was 403μm. The sample has a value of modulus of elasticity G 'of 500 to 800 Pa measured in an oscillatory tear in a frequency range of 0.01 -1 0 Hz. EXAMPLE 7- FILM O-hydroxy ethyl chitosan (0.2 g) was hydrated in water Deionized (15 ml). HA (0.1 g) was added to the O-hydroxy ethyl chitosan solution and stirred until the HA had dissolved, 1,4-butanediol diglycidyl ether (0.2 g, Sigma) was added and stirred into the polymer mixture. The solution was then transferred to a Petri dish and allowed to evaporate for 18 hours during that time a cross-linked film formed. The film was subsequently immersed in the deionized water and allowed to swell. The water absorption capacity of the film was 151% and gave a concentration of 660 mg / ml of O-hydroxy ethyl chitosan and 388 mg / ml for HA. The swelling water was examined for [HA] after 48 hours and resulted in 9.38% HA being released. After leaving the film in the swelling water for another 96 hours, no further HA release was detected. EXAMPLE 8- FILM Re-acetylated chitosan (0.5g) was hydrated in deionized water with a concentration of 2%. HA (0.5g, prepared by means of fermentation, Hyaltech Ltd) was dissolved in water to give a solution having a final concentration of 2% and the two solutions were refrigerated overnight. The two solutions were mixed together and BDDE (0.3 g Fluka). The polymer mixture was transferred to a Petri dish and allowed to evaporate slowly overnight at room temperature to form a crosslinked film. The film was subsequently immersed in the deionized water and allowed to swell. The water absorption capacity of the film was 258% corresponding to a concentration of 383 mg / ml for HA and 387 mg / ml for re-acetylated chitosan. After swelling, 0.45 & of HA was released from the movie. After leaving the film in the swelling water for another 4 days, no further release of HA was detected.

CLAIMS 1. A composition consisting of a semi-interpenetrating network consisting of at least one water-soluble crosslinked derivative of a basic polysaccharide, having primary and / or secondary amine groups, and a cross-linked component, having at least one anionic polysaccharide, in where the anionic polysaccharide resides within the semi-interpenetrating polymer network. 2. A composition according to claim 1 wherein the soluble basic polysaccharide is chitosan or a derivative thereof. 3. A composition according to claim 2 wherein the basic polysaccharide is deacetylated chitin, re-acetylated chitosan, N-carboxy methyl chitosan, O-carboxy methyl chitosan or O-hydroxy ethyl chitosan. 4. A composition according to claim 3 wherein the N-acetylated chitosan has a degree of acetylation in the range of 45% to 55%. 5. A composition according to one of claims 1 to 4 wherein the non-crosslinked component is hyaluronic acid. 6. A composition according to one of claims 1 to 5 wherein the composition also includes one or other anionic polysaccharide components of the extra-cellular matrix. 7. A method for the preparation of a composition as defined in one of claims 1 to 6, which consists of crosslinking

Claims

at least one soluble derivative of a basic polysaccharide containing primary and / or secondary amine groups in the presence of at least one anionic polysaccharide, under conditions which prevent the protonation of the primary and secondary amine groups and which also prevent the reaction of the hydroxyl groups or any other functional group in the anionic polysaccharide. The method according to claim 6 in which the reaction is carried out under neutral or slightly alkaline conditions, at a pH of 7 to 8. 9. A method according to claim 8 in which the crosslinking reaction is performed at a pH of about 7. 10. A biological material containing a composition as defined in any of claims 1 to 6. 1 1. The use in medicine of a composition according to any of claims 1 to 6 or a biological material as defined in claim 10. 12. The use of a composition according to any one of any of claims 1 to 6. in the preparation of biological material. The use according to claim 12 in which the biological material in dermatology, plastic surgery, urology and in the field of orthopedics. The use according to claim 13, wherein the biological material is in the form of a thin film, sponge, hydrogel, yarn or non-woven matrix.