Injectable Homogeneous Aqueous Solution of Chitosan Having a pH Close to the Physiological pH

Abstract

The present invention relates to an injectable homogeneous aqueous solution of chitosan containing, in a physiologically acceptable medium, between 0.1 and 4.5% by weight of a chitosan having a degree of acetylation less than 20% and a weight average molecular mass of between 100,000 and 1,500,000 g/mol, said solution having a pH greater than or equal to 6.2, and advantageously between 6.2 and 7.2, said solution not containing any chitosan having a degree of acetylation greater than 20%, said solution being liquid and homogeneous at ambient temperature. The invention also relates to an aqueous solution such as previously described, characterised in that it can be prepared by a method comprising at least the following steps: dissolving the chitosan in water by adding acid, such as a weak acid, said weak acid being advantageously chosen from the group consisting of acetic acid, glycolic acid, lactic acid, glutamic acid, and the mixtures of same, and readjusting the pH by dialysis, preferably at ambient temperature, in order to obtain an aqueous solution having a pH greater than or equal to 6.2, advantageously between 6.2 and 7.2, and preferably between 6.25 and 7.1.

Claims

1. Injectable homogeneous aqueous solution of chitosan containing, in a physiologically acceptable medium, between 0.1 and 4.5% by weight of a chitosan having a degree of acetylation less than 20% and a weight average molecular mass of between 100,000 and 1,500,000 g/mol, said solution having a pH greater than or equal to 6.2, and advantageously between 6.2 and 7.2, said solution not containing any chitosan having a degree of acetylation greater than 20%, said solution being liquid and homogeneous at ambient temperature.

2. The aqueous solution according to claim 1, characterized in that the chitosan has a degree of acetylation less than 19%, preferably less than 17%, more advantageously less than or equal to 15%, and most preferably less than 10%.

3. The aqueous solution according to claim 1, characterized in that it contains between 1 and 3.5% by weight of chitosan, in particular between 2 and 3.5% by weight of chitosan, with respect to the total weight of the aqueous solution.

4. The aqueous solution according to claim 1, characterized in that the chitosan has a weight average molecular mass between 200,000 and 1,000,000 g/mol, more advantageously between 250,000 and 800,000 g/mol, and still more advantageously between 300,000 and 600,000 g/mol.

5. The aqueous solution according to claim 1, characterized in that it has a pH between 6.25 and 7.1, preferably between 6.3 and 7.0.

6. The aqueous solution according to claim 1, characterized in that it is able to be prepared by a method comprising the following steps: dissolving the chitosan in water by adding acid, such as a weak acid, in order to obtain a homogeneous aqueous solution of chitosan containing, in a physiologically acceptable medium, between 0.1 and 4.5% by weight of a chitosan having a degree of acetylation less than 20% and a weight average molecular mass of between 100,000 and 1,500,000 g/mol, said solution having a pH less than 6.2, said acid being advantageously chosen from the group consisting of acetic acid, glycolic acid, lactic acid, glutamic acid, and the mixtures of same, and readjusting the pH by dialysis, preferably at ambient temperature, in order to obtain an aqueous solution having a pH greater than or equal to 6.2, advantageously between 6.2 and 7.2, and preferably between 6.25 and 7.1.

7. The aqueous solution according to claim 6, characterized in that during the dissolving step, the acid is added in a strictly necessary quantity for dissolving the chitosan.

8. The aqueous solution according to claim 6, characterized in that it is dialyzed against a buffer solution having a pH greater than 6.2, advantageously between 6.2 and 7.2, preferably between 6.25 and 7.1.

9. The aqueous solution according to claim 8, characterized in that the buffer solution is a phosphate-buffered saline solution comprising an acid salt NaH.sub.2PO.sub.4, a basic salt Na.sub.2HPO.sub.4 and NaCl.

10. The aqueous solution according to claim 8, characterized in that the dialysis is dynamic, implementing in particular the following steps: i. preparing, in a first reservoir A, a first solution comprising a weak acid, referred to as the weak acid solution, ii. preparing, in a second reservoir B, a second solution comprising the conjugate base or a base stronger than the conjugate base of the weak acid used in the weak acid solution, iii. circulating the weak acid solution through one or more dialysis pockets consisting of a dialysis membrane enclosing an aqueous solution of chitosan containing, in a physiologically acceptable medium, between 0.1 and 4.5% by weight of a chitosan having a degree of acetylation less than 20% and a weight average molecular mass of between 100,000 and 1,500,000 g/mol, said solution having a pH less than 6.2, iv. introducing said weak acid solution recovered at the end of the dialysis of step iii) into the second reservoir B, the composition of said second reservoir B being thus modified to a base+? weak acid mixture, ? representing a very small minority quantity, v. introducing the base+? weak acid mixture of reservoir B into reservoir A in order to obtain a weak acid+? base mixture in said reservoir A, vi. repeating steps iii, iv, and v until the acid-base equilibrium is attained in reservoirs A and B, thus forming, in each reservoir, a buffer solution having determined pH.

11. The aqueous solution according to claim 7, characterized in that the weak acid used in reservoir A is a mixture of dihydrogen phosphate (H.sub.2PO.sub.4.sup.?) and NaCl and the base used in reservoir B is hydrogen phosphate (HPO.sub.4.sup.2?).

12. The aqueous solution according to claim 6, characterizing that the dialysis membrane is made of a regenerated cellulose polymer (natural cellulose transformed by a series of chemical and physical operations) or a cellulose ester (for example cellulose acetate) with an exclusion threshold between 5 kDa and 30 kDa.

13. A composition comprising an aqueous solution according to claim 1, and optionally a compound or an acceptable excipient.

14. The composition according to claim 13, comprising at least one active compound such as an analgesic compound, local anaesthetic, such as lidocaine, mepivacaine, bupivacaine or ropivacaine, an angiogenic compound, a vaccine, a hormone, or even an active compound such as a growth factor or bioactive oligosaccharide, for example an hyaluronic acid oligosaccharide or chitosan oligosaccharide having a degree of polymerisation less than 20, or even a nucleic acid, a protein or an anti-cancer agent.

15. The composition according to claim 13, characterized in that it is formulated to be administered or is used by intradermic or subcutaneous, intramuscular, intra-articular or intraocular injection, and more generally into the hard or soft tissue of the body, for example the organs (digestive system or urogenital system) or the adipose tissues, mucous membranes, gums, cartilage or bones.

16. The composition according to claim 13, for use in the repair or reconstruction of tissues, in particular in the creation or substitution of biological tissues, for example as implants, or for the filling of biological tissues, for example filling of cutaneous depressions, injection into bone cartilage or into the joints.

17. The composition according to claim 13, for use as dermatological or cosmetic composition, or for use as a medical device, advantageously as a bioresorbable implant.

18. The composition according to claim 17, for use in surgery, medicine or aesthetic surgery, in urology, rheumatology, ophthalmology, odontology, orthopaedics, or angiology.

19. The composition according to claim 17, for use as a vector for active substances, for example as a vehicle for vaccines, anti-cancer agents or hormones.

Description

EXAMPLE 1: PREPARATION OF AN AQUEOUS SOLUTION OF CHITOSAN

[0158] Acid Solution of Chitosan:

[0159] An aqueous solution of chitosan was prepared by dissolving solid chitosan in water acidified by acetic acid.

[0160] The chitosan used is a chitosan having molecular mass Mw between 400,000 and 1,500,000 g.Math.mol.sup.?1 and degree of acetylation=5%.

[0161] The viscosity, measured at a concentration of 1% in aqueous solution, with 1% acetic acid at 25? C., is 9.578 Pa.Math.s

[0162] 1.5 g of chitosan were dissolved in 500 ?L of glacial acetic acid and 49.5 mL of PBS buffer (phosphate buffered saline with pH=6.5) comprising NaCl, an acid salt NaH.sub.2PO.sub.4 and a basic salt Na.sub.2HPO.sub.4.

[0163] The pH of the solution was 5.45.

[0164] Dialysis Equipment:

[0165] The dialysis was carried out using SpectraPor 4 regenerated cellulose membranes with a segregation of 12-14,000 Da.

[0166] The pH values were measured using a portable FiveGo FG2 pH meter (Mettler Toledo).

[0167] A PBS phosphate buffer (phosphate buffered saline with pH=6.5) was used.

[0168] The dialysis was performed against 3 L of regularly renewed buffer. The dialysis was stopped at the first signs of gelling.

[0169] The pH of the chitosan solution was measured regularly and the change over time is illustrated in FIG. 2.

[0170] The pH of the chitosan solution was thus able to be increased up to a value of 6.46, without any sign of gelling of the solution.

EXAMPLE 2: COMPARISON

[0171] The applicant attempted to reproduce the homogeneous solution of chitosan described in example 1 of patent application WO03/042250.

[0172] In order to do this, a chitosan having a degree of deacetylation of 84% was used.

[0173] 2.34 g of chitosan (deacetylated to 84%) was dissolved under agitation in 100 ml of a HCl solution (0.1 M). After 30 minutes agitation, a homogeneous solution of chitosan was obtained.

[0174] The chitosan solution was cooled in a refrigerator to 4? C. and held in an ice bath.

[0175] The pH was adjusted to 6.8 by addition, always at 4? C., of an ??-glycerophosphate disodium salt. 1.0 g of ??-glycerophosphate disodium salt was sufficient to attain the desired pH of 6.8.

[0176] However, the ??-glycerophosphate salts dispersed very poorly in the chitosan solutions, despite manual and mechanical agitation. Gel inclusions appeared very rapidly around the least soluble crystals of glycerophosphate.

[0177] Hence it was not possible to obtain homogeneous aqueous solutions of chitosan having a pH of 6.8 by reproducing example 1 of document WO03/042250.

[0178] In order to resolve this problem, the applicant reproduced this test by replacing the ??-glycerophosphate disodium salt with a ?-glycerophosphate disodium salt, for which the improved chitosan solubility properties are well-known.

[0179] For this new test, the same chitosan having a degree of deacetylation of 84% was used.

[0180] 2.34 g of chitosan (deacetylated to 84%) was dissolved under agitation in 100 ml of a HCl solution (0.1 M).

[0181] The chitosan solution was cooled in a refrigerator to 4? C. and held in an ice bath.

[0182] The pH was adjusted to 6.8 by gradual addition, always at 4? C., of 2.84 g of a ?-glycerophosphate disodium salt dispersed by manual agitation.

[0183] A homogeneous solution of chitosan at 3.4? C., having a pH of 6.8, was thus obtained. The transmittance of the solution before and after addition of ?-glycerophosphate was also measured by light diffusion, at a wavelength equal to 500 nm, through a sample of thickness 1 cm.

[0184] The solution was then heated in a bain marie until a temperature of 25? C. (ambient temperature) was attained, and was held at this temperature for 17 hours. The composition obtained was gelled and had a pronounced whitish appearance. The transmittance could no longer be measured due to the compact nature of the gel obtained.

[0185] The results are presented in the table below:

TABLE-US-00001 Without addition of ?-GP With addition of ?-GP 4? C. T? = 3.4? C. T? = 3.1? C. (T = 0) pH = 6.02 pH = 6.81 T % = 100% T % = 77% Liquid Liquid 25? C. T? = 24.5? C. T? = 24.2 (17 hours) pH = 5.60 pH = 6.81 T % = 100% T % = impossible, gel too compact. Liquid Compact gel

[0186] Hence, the solution obtained using example 1 of document WO03/042250, modified by the use of a ?-glycerophosphate disodium salt in place of an ??-glycerophosphate disodium salt, is indeed liquid and rather homogeneous (transmittance 77%) at low temperature (4? C.).

[0187] However, increasing the temperature to attain 25? C. (ambient temperature) leads to a gelling of the composition. In WO03/042250, chemical grafting of the chitosan makes it possible to obtain an injectable solution at 25? C. Without this grafting, the compositions described in WO03/042250 are in the form of a gel at 25? C. and can only be injectable at 4? C.

[0188] Conversely, the homogeneous aqueous solutions according to the present invention are in the form of an injectable liquid solution even at ambient temperature (20-25? C.), and only gel when the pH increases in situ.