EA043011B1 - CHITOSAN WITH ANIONIC CHARGE - Google Patents

Description

The present invention relates to carboxyalkyl chitosan, compositions containing it, a method for its preparation and various applications thereof, in particular in the field of therapy, rheumatology, ophthalmology, aesthetic medicine, plastic surgery, visceral surgery, dermatology or the cosmetic field.

State of the Art

Chitosan derivatives are already known, in particular disclosed in Kiomed Pharma patent applications published under the numbers WO 2016/016463 and WO 2016/016464 and related patents. These patent applications target the physical, chemical, or physico-chemical properties of chitosan derivatives. However, there remains a need to improve these compositions, in particular in the context of therapeutic treatment in such a way as to provide patients who may require the use of such compositions, an optimized therapeutic effect and, in particular, increase the benefit/risk ratio.

It is possible to obtain soluble chitosan by increasing the degree of acetylation (DA) by reacetylation of fungal chitosan. It is indeed possible to obtain formulations soluble at physiological pH by reacetylation of fungal chitosan, however, the occurrence of the following is noted:

very rapid degradation in vivo or similar conditions;

in a subject to which this chitosan is administered or implanted, for example by injection or intra-articular implantation, an immune response occurs;

chitosan is not suitable for targeted applications and therefore does not guarantee a sufficiently satisfactory therapeutic application of chitosan, in particular by injection or intra-articular implantation.

There are various publications regarding the carboxyalkylation of chitosan and, in particular, the carboxymethylation of chitosan, mainly with the aim of solubilizing chitosan. In theory, chitosan has a formula that does not contain an N-acetyl-glucosamine unit, but in practice, chitosan is obtained from chitin, which, in turn, contains N-acetyl-glucosamine units, and chitosan has a certain degree of acetylation (DA), which is the proportion of N-acetyl-glucosamine units in chitosan. The degree of acetylation (DA) of chitosan is generally low. Above, especially above 30%, it is usually reacetylated chitosan.

In addition, the Chinese patent application CN 1431229 A concerns carboxymethylated chitosan derivatives, in particular, their hydrating ability, however, said patent application is limited to this feature.

The carboxymethylation of chitin derived from animal sources, in particular crustaceans, has also been discussed in the prior art. However, crustacean-derived chitin is difficult to replace; in particular, it must be frozen and alkalized in order to be able to replace it (see, for example, Chinese patent application CN 106474569). This process is difficult to implement and, in particular, to implement on an industrial scale. On the other hand, such a substitution process based on crustacean chitin is energy-consuming, has low reproducibility, and can degrade the polymer and hydrolyze the acetyl groups of the N-acetyl-glucosamine units to a large extent, while also being difficult to control.

Objectives of the invention

The aim of the invention is to solve the technical problem associated with providing a corresponding chitosan derivative that is suitable for use in humans or animals, in particular in the field of internal medicine, rheumatology, ophthalmology, aesthetic medicine, plastic surgery, visceral surgery, dermatology or the cosmetic field.

More specifically, the aim of the invention is to solve the technical problem associated with providing a suitable chitosan derivative that is suitable for use in humans or animals in the therapeutic field and is particularly suitable as a viscosupplement, and also, in particular, can be introduced into synovial fluid or mixed with it.

In particular, the aim of the invention is to solve the technical problem associated with the provision of restored synovial fluid, i. a composition that restores the properties of a joint, for example by making it capable of lubricating cartilage surfaces.

Yet another object of the invention is to solve the technical problem associated with providing a derivative of chitosan or a composition containing it, which exhibits good properties and compatibility in admixture with synovial fluid and in particular with the synovial fluid of a human or animal subject, for example, for the treatment of articular or articular pathology, or deterioration of the synovial fluid in question.

The aim of the invention is also to solve the technical problem associated with the provision of a chitosan derivative or composition containing it, which limits the immune response of the subject and, in particular, the subject of a human or animal that receives such an introduction, for example, by injection of a chitosan derivative or composition containing it.

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Another object of the invention is also to solve the technical problem associated with providing a derivative of chitosan or composition containing it, which has characteristics that have low variability depending on pH.

It is also an object of the invention to solve the technical problem associated with providing a chitosan derivative or composition containing it, which has a suitable osmolality and pH value, which is considered suitable for its use in contact with the tissue of a human or animal subject, and is acceptable in terms of lifespan in situ, immunological response and/or reaction to a foreign body and biomechanical properties, depending on the target therapeutic indication, in particular in the context of regenerative medicine.

Description of the invention

Surprisingly, it has been found that the chitosan derivative according to the present invention solves at least one, and preferably all, of the technical problems described herein above.

Surprisingly, it has been found that a chitosan derivative exhibiting an electrostatic charge (characterized by its zeta potential) in the pH range corresponding to the pH of the medium in which it is introduced, and in particular at a pH of 7.5, i.e. below a certain value has solved at least one, and preferably all, of the technical problems described or proposed. In particular, such a chitosan derivative provides a means to limit the immune response of a subject who has been administered a chitosan derivative or composition containing it, usually by injection or implantation.

The present invention relates, according to a second aspect, to a chitosan derivative having glucosamine units, N-acetyl-glucosamine units and glucosamine units substituted with a carboxyalkyl group, said carboxyalkylchitosan having a zeta potential measured at pH 7.5, i. -10 mV or less, and preferably -15 mV or less.

In particular, it has been found that a fungal-derived chitosan derivative solves at least one and preferably all of the technical problems described or proposed. In particular, a chitosan derivative of fungal origin provides a means to limit the immune response of a subject who has been administered a chitosan derivative or composition containing it, typically by injection or implantation.

The present invention relates, according to a first aspect, to a fungal-derived carboxyalkyl chitosan having glucosamine units, N-acetylglucosamine units and glucosamine units substituted with a carboxyalkyl group, said carboxyalkyl chitosan preferably having a degree of substitution with a carboxyalkyl group that is greater than 20%, expressed as the number of moles of the substituent relative to the number of moles of all units.

Reference is also made to a chitosan derivative or a substituted chitosan derivative.

Chitosan, for example, is listed in the Chemical Abstracts Service under registration number (CAS number) 9012-76-4.

The chitosan used for the invention is predominantly of fungal origin and is preferably obtained from the mycelium of the fungus of the type Ascomycetes, and in particular from Aspergillus niger, and/or from the fungus Basidiomycetes, and in particular from Lentinula edodes (shiitake) and/or Agaricus bisporus (champignon). Preferably the chitosan is derived from Agaricus bisporus. Chitosan preferably has a high degree of purity, i. has an extremely low content of impurities due to its fungal origin or manufacturing process, and a microbiological quality that is compatible with its use as an implant or pharmaceutical composition. One way to obtain chitosan is the method described in patents WO 03/068824 (EP 1483299; US 7556946).

Typically, chitin is placed in an aqueous suspension in the presence of sodium hydroxide, after which the medium is heated to a high temperature for a variable duration, which varies depending on the desired molecular weight. The chitosan is then purified by solubilization in an acid medium and precipitated in an alkaline medium, then washed and dried.

Preferably, the chitosan is of sufficient purity to be suitable for pharmaceutical use.

The chitosan is preferably purified and then preferably dried. After purification, the process of the invention may include the step of drying the carboxyalkyl chitosan, then optionally grinding it into a powder. The carboxyalkyl chitosan can be dried, for example, by evaporating water, for example, by a spray drying process, a fluidized bed process, or by thermal drying under vacuum or atmospheric pressure, or even by lyophilization.

The carboxyalkyl chitosan can be solubilized in an aqueous solution and, for example, in pharmaceutically acceptable quality water suitable for injection or implantation in the body, and in particular in the human body.

The resulting chitosan can have various molecular weights, typically ranging from 10,000 to 500,000.

According to one embodiment, the average molecular weight is from 20,000 to 60,000.

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According to another embodiment, the average molecular weight is from 60,000 to 100,000.

According to another embodiment, the average molecular weight is from 100,000 to 120,000.

According to another embodiment, the average molecular weight is from 120,000 to 150,000.

According to another embodiment, the average molecular weight is from 150,000 to 220,000.

According to another embodiment, the average molecular weight is from 220,000 to 300,000.

According to another embodiment, the average molecular weight is from 300,000 to 500,000.

In the case where the chitosan is cross-linked, the molecular weight of the cross-linked polymer can, of course, be much higher.

It is possible to hydrolyze chitosan to reduce its molecular weight.

Preferably herein, the average molecular weight is the viscosity average molecular weight (Mv) calculated from intrinsic viscosity according to the Mark-Houwink equation. Intrinsic viscosity is measured by capillary viscometry using a capillary viscometer of the Ubbelohde type in accordance with the method described in monograph 2.2.9 of the European Pharmacopoeia. Measurement of the flow time of the solution through a suitable capillary tube (Lauda, e.g. Ubbelohde 510 01 capillary tube with a diameter of 0.53 mm) is performed using an I-Visc automatic viscometer (Lauda), first at the initial concentration of chitosan, then for several dilutions, for example, in in accordance with the recommendations in monograph 2.2.9. The reduced intrinsic viscosity is displayed for each of the concentrations.

The reduced viscosity is plotted against temperature and the value at concentration 0 is extrapolated to derive intrinsic viscosity from it. For example, it is necessary to plot the reduced viscosity (h _red in ml/g) i dilutions as a function of the concentration C i dilutions (g/ml) in accordance with formula 5.

[h red] = (ti - to) - (1 - C) Formula 2

To calculate the average viscometric mass, the Mark-Houwink equation is applied with the k and alpha constants recommended by Rinaudo et al. (in Int. J. Biol. Macromol., 15, 281, 1993), according to the degree of acetylation (DA) of chitosan according to one of the following three formulas.

Mv = ([h]/0.082) ^(1/0 ' ⁷⁶ ) for DA 2%

Mv = ([h]/0.076) ^(1/ °' ⁷⁶⁾ for DA 10% (e.g. 11.5%)

Mv = ([h]/0.074) ^(1/0 ' ⁷⁶ ) _for DA 20% (eg 21%)

Formula 3

Formula 4

Formula 5

For intermediate DA values, a linear interpolation is performed to calculate the average viscometric mass (Mv).

Preferably, the chitosan used has an average molecular weight in the range of 120,000 to 150,000, or even in the range of 150,000 to 220,000, or even more in the range of 220,000 to 300,000, or even higher than 300,000 and usually up to 500,000.

It is also possible to measure the final molecular weight of the carboxyalkylchitosan: for example, one can measure its intrinsic viscosity using capillary viscosity, deriving from its average molecular weight (Mw) (having previously determined the K and alpha parameters of the carboxyalkylchitosan), or by a chromatographic method, such as gel permeation chromatography.

As a rule, in the carboxyalkylchitosan according to the invention, the glucosamine units are D-glucosamine units (D-glucosamine units, N-acetyl-O-glucosamine units and at least one of the D-glucosamine units and N-acetyl-O- glucosamine units is substituted).

In one embodiment, the substituted chitosan has only D-glucosamine units substituted.

In accordance with another variant, the substituted chitosan has the substitution of D-glucosamine and N-acetyl-O-glucosamine units simultaneously, in which the carboxyalkyl group is covalently bonded to the amino groups of chitosan alone, in accordance with one variant, or simultaneously with the amino groups and hydroxyl groups of chitosan, in accordance with another option.

The substitution is, as a rule, only partial, not all links are necessarily substituted.

In accordance with one embodiment, the degree of substitution of D-glucosamine units, expressed as the number of moles of D-glucosamine units relative to the number of moles of all units (D-glucosamine and N-αcetyl-D-glucosamine units, substituted or unsubstituted) of the substituted chitosan, is from 30 to 250%.

In accordance with one embodiment, the degree of substitution of the carboxyalkyl group is greater than 50%, expressed as the number of moles of the substituent relative to the number of moles of all units.

In accordance with one embodiment, the degree of substitution of D-glucosamine units, expressed as the number of moles of D-glucosamine units relative to the number of moles of all units (D-glucosamine and N-αcetyl-D-glucosamine units, substituted or unsubstituted) of the substituted chitosan, is from 50 to 200% and more preferably more than 70%.

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In accordance with one embodiment, the degree of substitution of the carboxyalkyl group is less than 80%, expressed as the number of moles of the substituent relative to the number of moles of all units.

As a rule, substitution is carried out by linking with a covalent bond.

According to one embodiment, the carboxyalkyl chitosan is N,O-carboxyalkyl chitosan. The proportion of units substituted by a carboxyalkyl group in the O-position (O3 or O6 of glucosamine and/or N-acetyl-glucosamine units) and/or in the N-position (glucosamine units) varies. The degree of substitution can thus be more than 100%.

Preferably, the degree of substitution (DS) and degree of acetylation (DA) of carboxyalkylchitosan are measured by solid phase carbon-13 nuclear magnetic resonance (NMR) spectroscopy using a Bruker Spectrometer (Avance III HD 400 MHz) equipped with a MAS VTN 400SB BL4 NP/H pH sensor . For example, the spectrum is recorded at ambient temperature, with a relaxation time of 1 to 8 seconds and a number of scans of 64 to 512. Areas of carbon signals are determined after deconvolution. The following carbon atoms are considered: acetyl CH3 (methyl carbon of the acetyl group of N-acetyl-glucosamine units, substituted or unsubstituted), C1 (carbon in position 1 of glucosamine and N-acetyl-glucosamine units) and C=O (carbonyl carbon of the carboxymethyl substituent and carbonyl C=O carbon of the acetyl group of N-acetyl-glucosamine units, substituted or unsubstituted). To determine the DS of a given carboxyalkylchitosan, it is also necessary to record the NMR spectrum at carbon 13 nuclei of the chitosan precursor of this carboxyalkylchitosan. Based on the spectrum of the chitosan precursor, the CSU ratio is calculated, i.e. the ratio of the signal area of the acetyl CH3 group (methyl carbon of the acetyl group of N-acetyl-glucosamine units) to the area of the C=O signal (carbonyl carbon of the acetyl group of N-acetyl-D-glucosamine units). DA of carboxyalkylchitosan is calculated by formula 1 and DS by formula 2, where I represents the signal area of the carbon in question.

_ ¹ acetyl CHS

VL — ---j---- Formula 1 % g _ ίSNZ / ^Jc=a f CsU Ratio _{Λ o}

DS — ------------------ Formula 2 h,

DA and DS can be determined using other known methods for carboxyalkylchitosans, for example, using proton NMR in an aqueous medium, using a magnetic resonance spectrometer, for example, in accordance with the method described by Liu et al. (in Carb. Polym., 137, 600, 2016), for example, by pre-hydrolyzing carboxyalkyl chitosan by adding a concentrated solution of deuterated hydrochloric acid to it before analysis.

If there is another NMR method that is more advantageous for a reliable estimate of the degree of substitution, then that alternative method should be used. The above methods must be adapted by one skilled in the art with regard to sample preparation and signals to be integrated, in particular depending on the resolution, stability and proton position of the signals used to calculate the degree of substitution.

The degree of carboxyalkylation of chitosan may preferably vary from 20 to 250%, preferably from 50 to 200% and for example from 70 to 170%, expressed as moles of carboxyalkyl relative to the moles of all units.

In accordance with one embodiment, the degree of carboxyalkylation of chitosan may preferably vary from 40 to 130% and, for example, from 70 to 130%, expressed as the number of moles of carboxyalkyl relative to the number of moles of all links.

The degree of substitution of chitosan usually correlates with the mass ratio of the initial reagents relative to chitosan at the beginning of the reaction. As carboxyalkylating agents, mention may be made of acid chlorides (or salts thereof, eg sodium monochloroacetate), for example those bearing one or more carboxymethyl, carboxyethyl, carboxypropyl, carboxybutyl groups, etc.

In accordance with one embodiment, the present invention relates to carboxyalkylchitosan, in which the alkyl part of the carboxyalkyl is a (C ₁ -C ₅ )alkyl group, linear or branched.

In accordance with one embodiment, the present invention relates to carboxymethylchitosan.

According to this variant, the substituted chitosan is an N-carboxyalkylated chitosan.

According to this variant, the substituted chitosan is O-carboxyalkylated chitosan.

According to this variant, the substituted chitosan is N-carboxyalkylated and O-carboxyalkylated chitosan.

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Preferably, the zeta potential, measured at pH 7.5, is -18 mV or less.

Preferably, the carboxyalkylchitosan has a zeta potential, measured at pH 7.5, of -22 mV or less, and preferably -24 mV or less.

In one particular embodiment, the substituted chitosan preferably has an average molecular weight of 150,000 to 220,000 and a degree of substitution in the range of 50 to 200%, with the molecular weight preferably written before substitution.

According to another specific embodiment, the substituted chitosan has an average molecular weight of 120,000 to 150,000 and a degree of substitution in the range of 70 to 200%, with the molecular weight preferably written before substitution.

In one particular embodiment, the substituted chitosan preferably has an average molecular weight of 220,000 to 300,000 and a degree of substitution in the range of 70 to 200%, with the molecular weight preferably written before substitution.

According to another specific embodiment, the substituted chitosan has an average molecular weight of 220,000 to 300,000 and a degree of substitution in the range of 50 to 200%, with the molecular weight preferably written before substitution.

According to another specific embodiment, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution in the range of 50 to 200%, with the molecular weight preferably written before substitution.

According to another specific embodiment, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution in the range of 70 to 200%, with the molecular weight preferably written before substitution.

In one particular embodiment, the substituted chitosan preferably has an average molecular weight of 120,000 to 150,000 and a degree of substitution in the range of 20 to 50%, with the molecular weight preferably written before substitution.

According to another specific embodiment, the substituted chitosan has an average molecular weight of 220,000 to 300,000 and a degree of substitution in the range of 20 to 50%, with the molecular weight preferably written before substitution.

According to another specific embodiment, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution in the range of 20 to 50%, with the molecular weight preferably written before substitution.

In one particular embodiment, the substituted chitosan has a degree of substitution in the range of 20 to 80% and preferably 40 to 60% and a degree of acetylation in the range of 20 to 80% and preferably 30 to 75%.

In one particular embodiment, the substituted chitosan has a degree of substitution in the range of 50 to 200% and preferably 70 to 200% and a degree of acetylation in the range of 20 to 80% and preferably 30 to 75%.

According to one particular embodiment, the substituted chitosan has a degree of substitution in the range of 50 to 200% and preferably 70 to 200% and a degree of acetylation in the range of 20 to 50% and preferably 20 to 40%.

In one particular embodiment, the substituted chitosan has a degree of substitution in the range of 50 to 200%, and preferably 70 to 200%, and a degree of acetylation in the range of 50 to 75%.

In another specific embodiment, the substituted chitosan has a degree of substitution in the range of 90 to 200% and preferably 90 to 150% and a degree of acetylation in the range of 20 to 80%, with the molecular weight preferably written before substitution.

In one particular embodiment, the substituted chitosan has a degree of substitution in the range of 90 to 200% and preferably 90 to 150% and a degree of acetylation in the range of 20 to 50% and preferably 20 to 40%.

In one particular embodiment, the substituted chitosan has a degree of substitution in the range of 90 to 200%, and preferably 90 to 150%, and a degree of acetylation in the range of 50 to 75%.

In one particular embodiment, the substituted chitosan preferably has an average molecular weight of 220,000 to 300,000, a degree of substitution in the range of 90 to 200% and preferably 90 to 150%, and a degree of acetylation in the range of 50 to 75%, the molecular weight being preferably written before replacement.

According to one embodiment, the carboxyalkyl chitosan is cross-linked. Thus, several chitosan chains can be cross-linked, for example by reaction with a cross-linking agent, such as, for example, cross-linking agents used to cross-link polysaccharides, such as, for example, genipin, butyl glycidyl ether, glutaraldehyde, epichlorohydrin, 1-bromo3 ,4-epoxybutane, 1-bromo-4,5-epoxypentane, 1-chloro-2,3-epithiopropane, 1-bromo-2,3-epithiopropane, 1-bromo3,4-epithiobutane, 1-bromo-4 ,5-epithiopentane, 2,3-dibromopropanol, 2,4-dibromobutanol, 2,5-dibromopentanol, 2,3-dibromopropanethiol, 2,4-dibromobutanethiol and 2,5-dibromopentanethiol epichlorohydrin, 2,3-dibromopropanol,

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1-chloro-2,3-epithiopropane, dimethylaminopropylcarbodiimide, oxidized dextran, gallic acid, epigallocatechin gallate, curcumin, tannic acid, or even diisocyanate compounds such as hexamethylene diisocyanate or toluene diisocyanate.

When cross-linked carboxyalkylchitosan is used, the molecular weight can be very high.

By substituting chitosan, it is possible to prepare a solution of carboxyalkylchitosan, which is soluble in an aqueous solution whose pH varies widely, while unsubstituted chitosan is soluble only at a pH below 5.5-6.5. Thus, carboxyalkylchitosan has the ability to solubilize at various pH values due to the presence of carboxyalkyl groups that modify its solubility profile, and in particular at physiological pH or at the pH of physiological fluids that are modified by pathology, such as inflammatory pathology. It is known that the pH of bodily fluids such as joint synovial fluid, aqueous humor, vitreous humor and tears can vary significantly from person to person due to various factors such as age, pathology, etc. Therefore, it is preferred that the carboxyalkyl chitosan can remain soluble over a wide pH range, such as 6.0 to 8.5, or even 5.0 to 8.5, or even 4.5 to 8.5.

According to one embodiment, the substituted chitosan formulation has an osmolality of 100 to 700 mOsm/kg, preferably 200 to 500 mOsm/kg.

Preferably, the osmolality of the substituted chitosan formulation is 250 to 400 mOsm/kg and preferably 275 to 325 mOsm/kg.

In one embodiment, the substituted chitosan formulation has an osmolality that is compatible with the joint.

In one embodiment, the substituted chitosan formulation has an osmolality that is compatible with the ocular or intraocular surface.

Preferably, the osmolality of the substituted chitosan formulation is 250 to 400 mOsm/kg, and more particularly 250 to 380 mOsm/kg.

The term water-soluble means that the carboxyalkyl chitosan does not exhibit any turbidity visible to the naked eye when placed in an aqueous solution. In particular, solubility can be verified, i.e. the absence of any turbidity, a solution of carboxyalkylchitosan at a concentration of, for example, 1% (wt./wt.) in water or a buffer, for example phosphate buffer, with an optical density / for an optical density of less than 0.5 and preferably less than 0.2, measured by spectrometry in the visible UV region at a wavelength of 500 nm, with reference to a reference tank containing only the aqueous solvent used for the measured sample, but in the absence of substituted chitosan. Another way is to visually inspect according to monograph 2.9.20 of the European Pharmacopoeia. When the chitosan is under-substituted, the composition is insoluble in a satisfactory pH range, for example in the range of 6.0 to 8.5, at ambient temperature.

The degree of acetylation (DA) of chitosan is determined, as for example described in patent applications WO 2017009335 and WO 2017009346, by potentiometric titration. Alternatively, the degree of acetylation (DA) can be measured by other methods known for chitosan, such as proton nuclear magnetic resonance (NMR) spectroscopy, carbon-13 solid state NMR spectroscopy, infrared spectrometry.

Preferably, the carboxyalkyl chitosan has a degree of acetylation in the range of 5 to 80%, expressed as the number of moles of N-acetyl-glucosamine units relative to the number of moles of all units. The degree of acetylation is expressed as the number of N-acetyl-D-glucosamine units relative to the amount of all N-acetyl-D-glucosamine and D-glucosamine units present.

Advantageously, the carboxyalkyl chitosan has a degree of acetylation between 40% and 80%, expressed as the number of moles of N-acetyl-glucosamine units relative to the number of moles of all units.

In accordance with one embodiment, the degree of acetylation is from 5 to 20%.

In accordance with one embodiment, the degree of acetylation is from 15 to 25%.

In accordance with one embodiment, the degree of acetylation is from 20 to 45%.

In accordance with one embodiment, the degree of acetylation is from 20 to 30%.

In accordance with one embodiment, the degree of acetylation is from 25 to 40%.

In accordance with one embodiment, the degree of acetylation is from 40 to 50%.

In accordance with one embodiment, the degree of acetylation is from 50 to 60%.

In accordance with one embodiment, the degree of acetylation is from 60 to 75%.

The degree of acetylation is determined by carbon-13 NMR spectroscopy or proton NMR according to the same method used to determine the degree of substitution (DS). The carboxyalkyl chitosan preferably has a controlled degree of acetylation. The term chitosan having a controlled degree of acetylation should be understood as referring to a product for which the degree of acetylation, i. the proportion of N-acetyl-glucosamine units can be controlled in a controlled manner, in particular through the acetylation reaction.

According to one variant, the composition according to the invention may be presented in the form of a solution and cannot be turned into a gel with a change in temperature (not thermogelable).

According to one embodiment, the rheological characteristics of the solution according to the invention may change with temperature, but without undergoing a sol-gel transition.

The rheological characteristics of the solution according to the invention can in particular be confirmed by the elastic modulus (G') and/or the loss modulus (G), or even the complex modulus (G*).

According to one embodiment, the rheological characteristics of the solution according to the invention are substantially constant regardless of temperature.

In one embodiment, the composition according to the invention may be in the form of a solution and be capable of thermogelation.

In one embodiment, the composition according to the invention may be in the form of a gel and not thermogelable.

Thus, the invention allows, in one embodiment, to prepare a composition capable of thermogelation which is fluid at a temperature below the temperature of use, typically at a temperature lower than physiological temperature, for example 37° C., but which is in the form of a gel at a temperature of use, typically at physiological temperature, for example 37°C, at neutral pH (pH 7) or at physiological pH and, for example, from 7 to 8.5, with an osmolality that is suitable for the intended application. This is, for example, physiological osmolality.

According to one embodiment, the composition capable of thermogelation has a thermoreversible sol-gel transition.

Preferably, the present invention makes it possible to provide a composition with a low concentration of substituted chitosan.

Preferably, the concentration of carboxyalkylchitosan is less than 10%, eg 5% or less by weight, based on the total weight of the composition (w/w).

In accordance with one embodiment, the concentration of chitosan is less than 4%, for example equal to 3% or less, or even, for example, equal to 2% or less by weight relative to the total weight of the composition (wt./wt.).

Preferably, the composition of the invention may also contain a biopolymer other than substituted chitosan. According to one preferred embodiment, the biopolymer is a polysaccharide, whether oxidized or not, cross-linked or non-covalently cross-linked, such as hyaluronic acid or sodium hyaluronate.

Hyaluronic acid can have a molecular weight of up to 5 million Da. The molecular weight of hyaluronic acid can be determined from its inherent viscosity or its dynamic viscosity in solution. Hyaluronic acid may have a density in the range of 1 to 4 m ³ /kg and, for example, may be characterized as having low (eg, about 1 to 2 m ³ /kg) or high (eg, about 2 to 4 m ^{3 /} kg). kg) molecular weight.

Preferably, the concentration of hyaluronic acid is less than 4%, for example equal to 3% or less, or even, for example, equal to 2% or less by weight relative to the total weight of the composition (w/w).

In one specific embodiment, the concentration of hyaluronic acid is less than 1.9% (w/w), expressed on a weight basis relative to the weight of the final composition. Preferably the concentration of hyaluronic acid is from 0.5 to 1.5% (w/w), expressed by weight relative to the weight of the final composition. In one particular embodiment, the concentration of hyaluronic acid is approximately 0.9, 1.0, 1.1, 1.2, 1.3, 1.5% (w/w), expressed as a mass relative to the mass final composition.

The ratio between chitosan and hyaluronic acid can, for example, vary from 5 to 95%, for example from 10 to 90% and even, for example, from 30 to 70% of substituted chitosan, and from 5 to 95%, for example from 10% to 90% and even, for example, from 30 to 70%, respectively, of hyaluronic acid, while the percentages are expressed in relation to the dry weight of the substituted chitosan/dry weight of hyaluronic acid. According to one embodiment, this ratio between chitosan and hyaluronic acid is 1/1 (ie 50% chitosan and 50% hyaluronic acid). According to another embodiment, the ratio between chitosan and hyaluronic acid is 1.5/0.5 (ie 75% chitosan and 25% hyaluronic acid).

According to one embodiment, hyaluronic acid can be cross-linked between different hyaluronic acid chains.

The present invention also relates to a process for the production of carboxyalkylchitosan.

According to one embodiment, the process for producing carboxyalkyl chitosan according to the invention includes the production of fungal-derived chitosan, reacetylation of the chitosan, and carboxyalkylation of the reacetylated chitosan. Thus, the invention relates to reacetylated chitosan or reacetylated carboxyalkyl chitosan.

According to one embodiment, it is thus possible to dissolve the chitosan in an aqueous medium, preferably slightly acidic (eg pH 6). Acetic anhydride can be added to the chitosan solution one or more times. Then a basic agent is added, such as, for example, sodium hydroxide and/or urea.

Thereafter, an alkylating agent such as, for example, sodium monochloroacetate (ie sodium chloroacetic acid) or chloroacetic acid is added, then the substituted chitosan is purified, reduced and dried.

In accordance with one embodiment, the method of obtaining carboxyalkylchitosan in accordance with the invention includes obtaining chitosan, carboxyalkylation of chitosan, then reacetylation of carboxyalkylated chitosan. Preferably, such a method provides precise control of the degree of acetylation of the final carboxyalkylchitosan and, in particular, obtaining a high degree of acetylation, for example, above 40%. Thus, the invention relates to reacetylated and then carboxyalkylated chitosan or reacetylated carboxyalkylchitosan.

In accordance with one embodiment, the method for producing carboxyalkylchitosan in accordance with the invention includes obtaining chitin of fungal origin, carboxyalkylation of chitin and optionally reacetylation of carboxyalkylated chitin to obtain carboxyalkylchitosan in accordance with the invention.

In accordance with one embodiment, the method for obtaining carboxyalkylated chitosan in accordance with the invention includes obtaining chitin of fungal origin, deacetylation of chitin, carboxyalkylation of chitin, and optionally reacetylation of carboxyalkylated chitin to obtain carboxyalkylchitosan in accordance with the invention.

According to one embodiment, the carboxyalkylation step includes an alkalinization step to alkalinize the hydroxyl groups of the chitosan to promote high substitution both at the N-position of the glucosamine units and at the O-position of the glucosamine and N-acetyl-glucosamine units, for example.

According to one embodiment, the method of the invention includes an alkalization step comprising dispersing the chitosan in an alcohol solution such as isopropanol in the presence of an alkaline agent such as, for example, sodium hydroxide and stirring for a period of at least one hour. at a temperature of at least -32°C and preferably at a maximum of 15°C. To this suspension is then added an alkylating agent such as, for example, monochloroacetic acid. Thereafter, the substituted chitosan is purified, isolated and then dried.

In accordance with one embodiment, the method of obtaining carboxyalkylated chitosan in accordance with the invention includes obtaining chitosan, carboxyalkylation of chitosan and then reacetylation of carboxyalkylated chitosan.

According to one embodiment, the carboxyalkylation step does not include an alkalinization step.

The step of reacetylation of the carboxyalkyl chitosan may, for example, include one or more additions of acetic anhydride to the carboxyalkyl chitosan solution.

The final stages of purification, filtration and drying to collect the purified carboxyalkylchitosan are carried out in accordance with known methods in order to obtain carboxyalkylchitosan in accordance with the desired degree of purity, which generally depends on the intended application, for example, using precipitation and solubilization cycles or by dialysis.

According to one embodiment, the ratio of acetic anhydride/chitosan (v/w) varies from 0.1 to 10 and preferably from 0.1 to 2 during the reacetylation step.

According to one embodiment, the weight ratio of carboxyalkylating agent/chitosan varies from 1 to 50 and preferably from 2.5 to 25 during the carboxyalkylation step.

According to one embodiment, a weight ratio of reactants that provide a carboxyalkyl group (ie, alkylating agent) to base agent is used that is necessary and sufficient to obtain the desired degree of substitution. For example, the weight ratio of alkylating agent to base agent is greater than 1 and less than 10, and is preferably in the range of 1.4 to 3.3.

The invention also relates to a process for preparing a composition according to the invention.

According to one embodiment, the method generally comprises dissolving the substituted chitosan in an aqueous solution, preferably a buffered solution, preferably having a pH in the range of 6.2 to 8.5 and preferably 6.5 to 7.5;

optionally adjusting the pH to the desired pH, typically physiological pH for the intended use, for example by adding a buffering agent, acid or base;

optional addition of other excipients, such as, for example, reducing sugar, for example, sorbitol or mannitol;

optional regulation of the final osmolality of the composition.

Preferably, the method also includes an additional filling step for filling an injection or implantation device, such as, for example, a syringe, with carboxyalkyl chitosan or a composition containing it. Preferably, the injection device, such as, for example, a syringe, may then be subjected to a steam sterilization process. This device, for example a syringe, can then be packaged, preferably in a sterile manner. It can also be a package, vial or bottle, which serves for instillation of a solution of carboxyalkylchitosan.

It is preferable to use chitosan that is sufficiently pure for the intended use.

Preferably, the method also includes an additional filling step for filling an injection, implantation or instillation device, such as, for example, a syringe, with the composition according to the invention. Preferably, the injection device, such as, for example, a syringe, may then be subjected to a steam sterilization process. This device, such as a syringe, may then be packaged, preferably in a sterile manner.

According to one embodiment, the carboxyalkyl chitosan according to the invention is sterilized by filtration and/or steam sterilization before it is filled into an injection, implantation or instillation device, such as, for example, a syringe or vial.

It is preferable to use chitosan that is sufficiently pure for the intended use.

More specifically, the present invention relates to an injectable composition containing carboxyalkyl chitosan in accordance with the invention.

The invention also relates to a pharmaceutical composition containing at least one carboxyalkyl chitosan defined in accordance with the invention.

According to one embodiment, the carboxyalkyl chitosan, or a composition containing it, is used as a pharmaceutical composition that can be injectable, implantable, or instillable, or a medical device that is injectable, implantable, or instillable.

The invention also covers carboxyalkyl chitosan or a composition containing it in dry form, in particular in lyophilized form. In particular, it is possible to (re)disperse and preferably solubilize the lyophilized product before use.

More specifically, the present invention relates to a composition according to the invention for use in therapeutic treatment, for example, comprising the injection of said composition by subcutaneous, intradermal, intraocular or intra-articular route, for example, to restore, regenerate or fill at least one body tissue in need of repair or filling.

Preferably, in accordance with one embodiment, the carboxyalkyl chitosan according to the invention exhibits immunocompatibility of fungal-derived carboxymethyl chitosan formulations in a mouse air sac model, 24 hours after injection, and is expressed as a white blood cell count that is less than 10 x 10 ⁶ cells/ml and is preferably less than 8x10 ⁶ cells/ml.

Preferably, in accordance with one embodiment, the carboxyalkyl chitosan according to the invention exhibits immunocompatibility of fungal-derived carboxymethyl chitosan formulations in a mouse air sac model, 24 hours after injection, and is expressed by an interleukin 1 beta (IL1-beta) concentration that is less than 10 x 10 ^-9 g/ml and preferably less than 5x10 ^-9 g/ml.

Preferably, in accordance with one embodiment, the carboxyalkyl chitosan according to the invention exhibits immunocompatibility of fungal-derived carboxymethyl chitosan formulations in a mouse air sac model, 24 hours after injection, and is expressed by the concentration of chemokine ligand 1 containing the C-X-C motif (KC/CXCL1 ), which is less than 50x10 ^-9 g/ml and preferably less than 30x10 ^-9 g/ml.

Preferably, in accordance with one embodiment, the carboxyalkyl chitosan according to the invention exhibits immunocompatibility of fungal-derived carboxymethyl chitosan formulations in a mouse air sac model, 24 hours after injection, and is expressed by a tumor necrosis factor alpha (TNF-alpha) concentration that is less than 150 x 10 ^{- 9} g/ml and preferably less than 125 x 10 ^-9 g/ml.

Preferably, in accordance with one embodiment, the carboxyalkyl chitosan according to the invention exhibits immunocompatibility of fungal-derived carboxymethyl chitosan formulations in a mouse air sac model, 24 hours after injection, and is expressed by the concentration of chemokine ligand 1 containing the C-X-C motif (KC/CXCL1 ), which is less than 50x10 ^-9 g/ml and preferably less than 30x10 ^-9 g/ml.

- 9 043011

Preferably, in accordance with one embodiment, the carboxyalkyl chitosan according to the invention exhibits immunocompatibility of fungal-derived carboxymethyl chitosan formulations in a mouse air sac model, 24 hours after injection, while meeting the maximum limits specified herein above for white blood cell counts and concentrations ILl-beta, KC/CXCL1 and TNF-alpha.

Measurements of the number of white blood cells and the concentrations of SH1-beta, KC/CXCL1 and TNF-alpha are carried out in accordance with the protocols indicated in the examples.

Preferably, in accordance with one embodiment, the carboxyalkyl chitosan according to the invention has a half-life in the presence of a mixture of lysozyme and hyaluronidase enzymes at 37°C, which is more than 500 min according to the measurement protocol in example 6.

Preferably, in accordance with one embodiment, in particular for intra-articular application, the carboxyalkyl chitosan according to the invention has a coefficient of friction (COF ₀ ) less than 5 and preferably equal to 4 or less (according to the protocol in example 9).

The present invention relates in particular to a composition according to the invention for use in treatment in the fields of rheumatology, ophthalmology, aesthetic medicine, plastic surgery, visceral surgery, for example for the prevention of postoperative tissue adhesions, in cosmetic or dermatological surgery.

In one embodiment, the body tissue is selected from tissues belonging to the vocal cords, muscles, ligaments, tendons, cartilage, genitals, bones, joints, eyes, dermis, epidermis, one or more layers of skin, mesoderm, or any combination thereof, and, more specifically, belonging to the eyes, dermis and articular joints.

The present invention also relates to a composition according to the invention for use in the therapeutic treatment of dry eye syndrome, corneal injury or injury, or joint inflammation.

In addition, the present invention relates to the use of the composition according to the invention by instillation on the surface of the eye to prevent or combat corneal injury or injury, or dry eye syndrome, in particular for the purpose of lubricating or regenerating the ocular surface.

Thus, the invention also relates to an eye drop composition containing carboxyalkyl chitosan as defined in accordance with the present invention.

In one embodiment, the subject is afflicted with an inflammatory pathology (eg, osteoarthritis).

More specifically, the present invention relates to a composition according to the invention for the treatment of arthrosis (osteoarthritis), arthritis or the repair of a cartilage defect, for example by injection into a synovial cavity or by implantation at the site of a cartilage defect.

More specifically, the present invention relates to a medical device, such as a medical implant, characterized in that it contains or consists of a composition according to the invention.

According to one preferred embodiment, the invention thus relates to a medical device containing a chamber containing carboxyalkyl chitosan in dry form, in particular in lyophilized form, and optionally one or more other chambers containing one or more active products, additives or auxiliary substances.

The composition in accordance with the present invention may also contain one or more additives or excipients that allow its properties to be modulated.

The present invention also relates to a composition according to the invention for use in a method of therapeutic treatment, for example, comprising instillation or injection of said composition by subcutaneous, intradermal, ocular, intraocular or intraarticular route, for example, to repair or fill at least one body tissue requiring restoration or filling.

The present invention also relates to a composition according to the invention for use in a method for treating osteoarthritis or for repairing a defect in cartilage, for example by injection into synovial fluid or after mixing with blood and implantation into cartilage.

The present invention also relates to a composition according to the invention for use in a method of treatment or method of aesthetic care provided by filling the dermis (dermal filling). This particularly includes, for example, injection of the composition according to the invention subcutaneously or intradermally.

The present invention also relates to a composition according to the invention for use in a method of treatment for treating the surface of the skin by means of multiple intradermal injections. Such compositions can generally be used in dermatology as a treatment for aesthetic purposes. Such a method, for example, is intended to increase the volume of the skin so that it loses its wrinkled appearance (treatment of wrinkles and/or fine lines). Such treatment may be used in a subject who wishes to improve the appearance of their skin by rejuvenation.

- 10 043011

The present invention also relates to a composition according to the invention for use in a treatment method in which the composition is a viscosupplementation agent. In this case, for example, it comprises administering a composition of the invention to a site within a joint, in particular to limit friction on the cartilaginous surfaces of the joint.

The present invention also relates to a composition according to the invention for use as a cellular vector, for one or more cell types, and/or one or more active agents. These may be active agents from a pharmaceutical or biological point of view. The composition of the invention may be substantially compatible with the presence of cells, preferably living cells. Live cells of interest include, for example: chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligament fibroblasts (ligament), skin fibroblasts (skin), tenocytes (tendons), myofibroblasts (muscles), mesenchymal stem cells, red blood cells (blood) and keratinocytes (skin). The composition of the invention may also be considered as a therapeutic vector for targeted delivery and/or controlled release of at least one therapeutic agent.

According to one embodiment, the addition of blood, or plasma, or platelet lysate, or platelet-rich plasma, or any biological fluid to the composition of the invention can thereby, for example, improve the characteristics of the product.

In accordance with one embodiment, the composition in accordance with the invention is formulated in solid form (eg, film or porous foam), which is solubilized after implantation.

According to one embodiment, the composition is in the form of a nebulized (nebulized) composition.

The present invention also relates to a composition according to the invention for use in a treatment method or aesthetic care method for one or more tissues or organs that have been exposed to excessive temperature, as in the case of a burn.

The present invention also relates to a composition according to the invention for use in a method of treating cartilage to repair cartilage (eg, by implantation into a defect in cartilage to promote the regeneration of such cartilage).

The present invention also relates to a composition according to the invention for use in a method of treatment for the prevention of tissue adhesions after surgery: the product is applied to tissues at the end of an operation, such as gynecological surgery, abdominal surgery, etc.

The present invention also relates to a composition for use as an artificial synovial fluid, which contains carboxyalkylchitosan in accordance with the invention.

The composition in accordance with the present invention provides the ability to mimic healthy synovial fluid or improve healthy or defective or degenerative synovial fluid, for example, by improving its lubricity to reduce friction in the joint and/or its damping capacity (determined by the elastic modulus G'), while being easily administered, for example, for filling a syringe or for injection into a human or animal body. For illustrative purposes only, the elastic modulus G' of a healthy synovial fluid is 40 to 100 Pa and its loss modulus G is 1 to 10 Pa.

According to one embodiment, the carboxyalkyl chitosan or a composition containing it is administered in the form of a solution. Preferably, according to this embodiment, the carboxyalkyl chitosan or composition containing it is easily injected through a fine needle, for example a 21 gauge needle at room temperature. The term easy administration means that preferably the force applied to such a syringe, under which the composition according to the invention flows through a 21 gauge needle, is less than 50 Newtons, preferably the force is less than 20 Newtons.

The present invention also relates to a composition for use as artificial tears, which contains carboxyalkylchitosan in accordance with the invention.

Generally, the osmolality and pH ranges required in biomedical applications are close to the following ranges:

osmolality:

1) isosmolarity with plasma: 285-295 mOsm/kg;

2) isosmolarity with synovial fluid: 404±57 mOsml/kg according to Clin. Orthop. Relat. Res., 235, 289-95, 1988 and Biochem. Biophys. Res. Comm., 422, 455-461, 2012;

3) from 280 to 350 mOsm/kg;

pH.

Typically, the physiological pH is above 6.8, in particular above 7.0, in particular 7.4 (as in plasma or synovial fluid).

Typically, the plasma pH is 7.4. As a rule, the pH value of the synovial fluid is 7.768±0.044 according to J. Bone. joint. Surg. Br., 41-B (2), 388-400, 1959, or 7.3 according to Acta Orthop Scand 634-641, 1969, or also according to Clin. Rheumatol., 25, 886-888, 2006.

- 11 043011

It is generally believed that the pH value of synovial fluid in osteoarthritis or arthritis is lower than in healthy synovial fluid.

Thus, the present invention relates to a mixture of synovial fluid with a composition in accordance with the present invention, for example, based on the volume ratio between (i) carboxyalkylchitosan or a composition containing it; and (ii) synovial fluid that ranges from 20/80 to 80/20 (v/v) and is, for example, 50/50 (v/v).

Preferably the composition according to the present invention is sterile. Highly preferably, the composition according to the present invention is sterilized by increasing the temperature, preferably in an autoclave.

According to one embodiment, the compositions of the invention are transparent or translucent.

The term translucent means that an object can be seen when the composition is placed between the observer's eye and said object. The term transparent means that it is possible to distinguish alphanumeric characters when the composition is placed between the observer's eye and the observed characters. As a rule, this assessment is carried out on a composition with a thickness of approximately 1 cm. It is also possible to follow the method described in the monograph 2.9.20 of the European Pharmacopoeia for visual inspection. It is also possible to measure the optical density of the composition, for example, by spectrometry in the visible UV region at 500 nm, and verify that the optical density is less than 0.5, preferably 0.2, compared to the reference solvent.

In one embodiment, the compositions of the invention are not opalescent or are only slightly opalescent.

The term opalescent means that the solution results in a diffraction of light visible to the naked eye, for example, by visual inspection according to a method such as in the monograph 2.9.20 of the European Pharmacopoeia, and by comparison with reference solutions having different levels of opalescence from the European Pharmacopoeia. According to one embodiment, the composition according to the invention is colorless, ie, in particular, the observer observing with the naked eye does not attribute any particular color to the composition. In one embodiment, the opalescence is below the maximum allowable level for the intended application.

The invention relates in particular to articles or packages, preferably sterile, containing one or more instillation or injection devices pre-filled with a composition according to the invention. Typically, these are devices that allow the product to be administered in the form of drops or in pre-filled syringes.

The composition according to the invention may preferably be sterilized. Thus, the invention relates to sterilized carboxyalkyl chitosan. The carboxyalkyl chitosan is thus sterile, in particular for applications in which this is required.

According to one embodiment, the composition according to the invention is steam sterilized, for example, by raising the temperature to a temperature above 100°C and preferably above 120°C, for example, in the range from 121 to 138°C in an autoclave for a period of time sufficient for sterilization, for example, typically within 15 to 20 minutes. Alternatively, the composition may be sterilized by filtration using filters adapted for this purpose, such as filters with a porosity of less than or equal to 0.2 µm.

The advantage is that, according to one preferred embodiment, the loss of intrinsic viscosity of the carboxyalkylchitosan during the steam sterilization process is less than 40%.

The invention also covers the composition according to the invention in dry form, in particular in lyophilized form.

In particular, it is possible to (re)disperse this lyophilized composition before use.

The present invention also encompasses a method of therapeutic treatment comprising injecting a composition according to the invention.

The present invention also covers the use of a composition according to the invention for the manufacture of a pharmaceutical composition, in particular for therapeutic treatment, for example as defined more specifically by the invention.

The present invention also encompasses an aesthetic care method, in other words a non-therapeutic method, comprising injecting a composition according to the invention. This includes, for example, filling in wrinkles or filling in one or more areas of damaged visible tissue, such as after an accident or aesthetic surgery.

A tissue is a collection of cells that are similar and have the same origin, grouped together as a functional unit, i.e. contribute to the same given function. Tissues include epithelial tissue, connective tissue, muscle

- 12 043011 tissue and nervous tissue.

The term composition in accordance with the invention or equivalent terms should be understood as referring to the composition defined in the present invention, including in accordance with any of the options, specific or specific embodiments, independently or in accordance with any of their combinations, including according to preferred features.

Other objects, features and advantages of the invention will become apparent to a person skilled in the art upon reading the explanatory description, which refers to the examples given solely by way of illustration and which in no way limits the scope of the invention.

The examples form an integral part of the present invention, and any feature that appears new in comparison to any prior art document based on the description taken as a whole, including the examples, forms an integral part of the invention with respect to its function and applicability.

Thus, each example is general in scope.

On the other hand, in the examples, all percentages are by weight unless otherwise indicated, temperature is in degrees Celsius unless otherwise indicated, and pressure is atmospheric pressure unless otherwise indicated.

Examples

The precursor chitosans of the substituted chitosans according to the invention have an average molecular weight in terms of viscosity (determined by capillary viscometry) and degree of acetylation (DA, content of N-acetyl-D-glucosαminα units determined by potentiometric titration) within the ranges indicated in table 1. The molecular weight of chitosan can also be determined from the dynamic viscosity of a solution having a concentration of 1% (w/w) chitosan in acetic acid having a concentration of 1% (v/v) measured using a rotating viscometer, such as a viscometer Brookfield, as described in Table. 1.

Table 1

Characteristics of chitosan precursors

Range Average molecular weight range (capillary viscometry) Viscosity range at 1% (mPa s) DA range (mole%) "Super Low" approx. 20000 - 60000 5-20 5 - 20% "Short" approx. 60000 - 120000 20-50 15-25% "Average" approx. 120000 - 150000 50-80 20 - 30% "High" approx. 150000-220000 80 - 120 25 - 40% "Ultrahigh" approx. 220000 - 300000 120 - 200 25 - 40% "400k" approx. 300000 - 500000 200 - 600 35 - 50%

The following methods were used in the present invention unless otherwise indicated.

A method for measuring the zeta potential.

The composition to be analyzed was diluted in phosphate buffer to obtain a final polymer concentration of 0.05%, then lightly stirred until homogenization was achieved. The solution was then separated into different fractions, and the pH of each of the fractions was adjusted to the desired pH range of 4-8, either by adding 0.1N sodium hydroxide solution or by adding 0.1N hydrochloric acid solution. The zeta potential of each fraction was measured with a NanoZ instrument (Zeta-Sizer range, Malvern Instruments).

Method for measuring the solubility range of chitosan polymers.

The solubility range was established by preparing a solution of the polymer to be tested at a concentration of 1% and pH 9 by dividing it into several fractions, the pH of which was adjusted to various pH values in the range from 9 to 1. For each fraction, the polymer was tested to ensure that it dissolved, i.e. it does not form any turbidity, according to the method of visual control described in monograph 2.9.20 of the European Pharmacopoeia. The pH range in which the polymer is soluble or insoluble was noted.

Model of the subcutaneous air sac in mice.

A mouse subcutaneous air sac model was used to assess foreign body response and immunocompatibility of several different polymers derived from fungal chitosan. In this model, the air sac cavity was created by repeated subcutaneous injections of sterile air into the back of the mouse. The sac created by inflation mimics the synovial cavity, thus providing a localized environment in which to study the stimulation of an inflammatory response in the fluid flowing from the air sac and surrounding tissues, as described by Segwick et al. (in J. Pathol., 141, 483, 1983) and Sin et al. (in Ann. Rheum. Dis., 45, 873, 1986). WHO cavity

- 13 043011 suffocating bag allows you to enter up to 1 ml of the product to an animal weighing about 30g. According to the protocol described by Dawson et al. (in Int. J. Tiss. Reac., 8, 171, 1991), an air sac was placed in unrelated male CD-1 Swiss albino mice (pathogen-free, admission weight 25-35 g) according to the schedule on day 0 and day 4 by a second injection of 5 ml followed by 3 ml of sterile air. On day 7, a single subcutaneous injection of 1 ml of the product was injected directly into the cavity of the air sac. Injections of 1 ml saline and 1% carrageenan solution served as negative and positive controls, respectively. Animals were observed regularly for several hours after injection for possible clinical signs of systemic or local toxicity, and body weight gain and temperature were measured at the time of injection and sacrifice. Animals were sacrificed after 24 hours of contact with the product (3 animals per product). Immunological evaluations of the lavage fluid removed from the bag included cytological analysis (count and distribution of white and red blood cells) and quantification of major inflammatory mediators (IL-1-beta, TNF-alpha and KC/CXCL1) using ELISA kits (AbCam) carried out in accordance with the instructions. Macroscopic analysis and histopathological analysis were performed by hematoxylin/eosin staining of surrounding skin tissues. Particular attention was paid to the localization of the product on the surrounding tissues, as well as its resorption in the liquid.

A model for evaluating local tolerability of intra-articular injection in a sheep model.

According to the literature and recommendations of the Osteoarthritis Research Society International (OARSI), sheep are recognized as a well-characterized model for evaluating the effects of intra-articular injection of innovative treatments (Little et al., in Osteoarthritis Cartilage, 18, S80, 2010; Edouard et al., in Phys. Ther. Sport., 14, 116, 2013; McCoy et al., in Vet. Pathol., 52, 803, 2015). The sheep model provides the means for injection by intra-articular route of a volume formulation that mimics the clinical use of viscosupplementation therapy in humans (Fraser et al., in Semin Arthritis Rheum, 22, 9, 1993). Measurements of local tolerance include clinical signs of synovial inflammation (effusion, lameness, comfort) by using the validated semi-quantitative clinical scores described by Shafford et al. (in Vet. Anaesth. Analg., 31, 20, 2004) and Kaler et al. (in Vet. J., 180, 189, 2009), as well as cytological examination of the synovial fluid. In some cases, these analyzes may be supplemented by anatomical and histological evaluation of the injected limb. A volume of 2 ml of the composition was injected into the joint (knee) of a healthy sheep (age 2 to 6 years; weight 60 to 80 kg). After injection, clinical signs in animals were recorded daily for 15 days based on a semi-quantitative scale from 0 to 4 for effusion (by palpation of the knee joint) and lameness. A number of features were found for each of the assessments over the entire observation period. In order to evaluate the effects of a second injection of the same given formulation, a second injection into the joint of the same animals was given 1 month later and the animals were observed according to the same protocol as for the first injection. In addition, synovial fluid puncture was performed on day 15, and macroscopic (color, viscosity), cytological (number and distribution of white and red blood cells) parameters, and the amount of total serum proteins were determined according to conventional methods. Synovial fluid was considered normal if it had a clear, viscous and slightly yellowish appearance, and cytological analysis showed that the number of white blood cells was less than 1x105 cells/ml and that the concentration of total proteins was approximately 25 mg/ml.

Example 1 N-succinyl-chitosans.

N-succinyl-chitosans having different molecular weight, molecular structure (degree of acetylation (DA) and degree of succinyl group substitution (DS)) were prepared and characterized according to the general method described in patent application WO 2016/016463 or WO 2016 /016464 (patent EP 3016663). The first acetylation step by adding acetic anhydride was performed to increase the degree of acetylation, except for the CSS-1 polymer (Table 1a).

Formulations were prepared with these N-succinyl-chitosans (CSS) at a concentration of 2% at physiological pH and osmolarity and packaged in glass syringes. The syringes were sterilized in an autoclave using a standard cycle (15 min at 121°C). The formulations were checked to ensure that they did not exhibit the presence of any insoluble matter or turbidity over a wide pH range near physiological pH (pH 6.5 to 7.5) by visual inspection according to the method described in monograph 2.9.20 European Pharmacopoeia. It was verified that the level of bacterial endotoxins in the formulations was less than 20 EU/ml according to the method described in monograph 2.6.14 (D) of the European Pharmacopoeia, and that the microbiological load was less than 100 CFU/g according to the method described in monograph 2.6.12 European Pharmacopoeia, before assessing their immunocompatibility in vivo.

- 14 043011

Table 1a

Characteristics of N-succinyl-chitosans

No. Molecular weight range DA* (mole%) DS* (mole%) CSS-1 Medium 12 62 CSS-6 Low 50 34 CSS-8 Low 49 32 CSS-10 high 61 37

* Molecular weight of chitosan precursor according to Table. 1.

** DA and DS were measured by proton NMR according to the method described in EP 3016663.

The immunocompatibility of the CSS formulations was assessed using a mouse subcutaneous air sac model (Table 1b). The observed response was compared to that observed after injection of saline (negative control), highly reactive positive control (1% carrageenan solution) and a commercially available product based on Hylan GF-20, partially cross-linked hyaluronic acid (Synvisc®, Sanofi).

Table 1b

Immunocompatibility of N-succinyl-chitosan formulations using an air-pouch model in mice 24 h after injection*

No. Number of white blood cells (10 ⁶ cells / ml) ILl-beta concentration (10 ^-9 g/ml) “ Concentration of KC/CXCL1 (10' ⁹ g/ml) TNF-alpha concentration (10' ⁹ g/ml) “ CSS-1 1.5 / / / CSS-6 0.7 <3 137 < 125 CSS-8 3.7 / / / CSS-10 2.1 <3 >700 < 125 controls Negative control (saline) 0.2 17 23 272 Positive control (1% carrageenan) 33.2 28 700 767 Synvisc® (Hylan GF-20) 0.7 4 46 < 125

* Average value for 3 animals.

** Limit of detection: 3.10 ^-9 g/ml for IL1-beta and 125.10 ^-9 g/ml for TNF-alpha.

In general, these CSS-based formulations have been found to elicit an immunological foreign body type reaction, as evidenced by the number of white blood cells in the cellular infiltrate, which is higher than the negative control, but not as high as the positive control. (1% carrageenan). The formulation-induced response is characterized by neutrophil activation, as evidenced by the higher concentration of the KC/CXCL1 marker compared to the negative control and the Synvisc® product. As for the markers IL1-beta and TNF-alpha, their levels are low.

Modification of DA and/or DS CSS, or in fact modification of its molecular weight does not lead to any difference in the immunological response. Although the CSS formulations do not have any deleterious effect on tissues locally, it is necessary to be able to obtain a higher level of immunocompatibility in the case of therapeutic applications that target tissues that are weakened by the corresponding pathology, such as dry eye syndrome, corneal injury or inflammation, for example. associated with joint pathologies.

Example 2 Carboxymethylchitosans of animal origin.

It is possible to obtain two carboxymethylchitosans of animal origin for pharmaceutical or medical use, which are implantable or injectable (CC-1 and CC-2 in Table 2a).

Two other carboxymethylchitosan were tested:

one CC obtained by carboxymethylation of chitin of animal origin (CC-3);

- 15 043011 one CC obtained by carboxymethylation of chitosan of animal origin, and chitosan was obtained by deacetylation of chitin of animal origin (CC-4).

Carboxymethylchitosans (CC) were characterized by carbon-13 NMR to confirm their identity and to determine the DA (degree of acetylation) and DS (degree of carboxymethyl substitution) values with an error estimated at approximately ±10% (Table 2a). The electrostatic charge of the polymer was characterized by measuring its zeta potential at a concentration of 0.05% (wt./wt.) in the pH range from 8 to 4 and recorded the value of the zeta potential at pH 7.5 (Table 2a). The zeta potential value at pH 7.5 was observed to correlate relatively well with the DS value determined by carbon-13 NMR. For example, polymer CC-3 is substituted to a greater extent than polymer CC-4 by an anionic carboxymethyl group (in carboxylate form at pH 7.5) and has a similar DA, so its overall negative charge is higher (-28 mV vs. -11 mV). mV at pH 7.5).

It is observed that polymers CC-1 and CC-2 are not very soluble in the physiological pH range (pH 6.5-7.5). It is not possible to assess their immunocompatibility using the mouse subcutaneous air sac model. Polymers CC-3 and CC-4 are virtually soluble over a wide range of pH 1-9.

Table 2a Characteristics of carboxymethylchitosans of animal origin and their compositions

No. (concentration) DA* (mol.%) DS* (mole%) Polymer solubility range Sterilizability of the composition in an autoclave Zetapotential at pH 7.5 (mV) SS-1 (2%) 8 124 Insoluble in the pH range 3.6-8.1 / / SS-2 (2%) / / Insoluble at pH below 6.6 / / SS-3 (2%) 71 149 Soluble at all pH values No -28 SS-4 (1.5%) 69 24 Soluble at all pH values Yes - AND

* DA and DS values for carboxymethylchitosan polymers were determined using solid phase nuclear magnetic resonance (NMR) spectroscopy on carbon-13 nuclei.

Formulations CC-3 (2%) and CC-4 (1.5%) were then prepared in the same manner as in Example 1, packaged in glass syringes and then autoclaved according to a standard cycle of 15 minutes at 121°C (Table 2a). The 2% CC-3 polymer formulation was found not to withstand final autoclave sterilization, as evidenced by a loss of approximately 60% of its intrinsic viscosity, indicating hydrolysis of the polymer, as well as a 98% loss of its storage modulus G'. As an alternative to final sterilization in an autoclave, the CC-3 formulation was then filtered before being packaged in a syringe to obtain a formulation that can be evaluated using a mouse air sac model.

Formulations CC-3 (2%, filtered) and CC-4 (1.5%, autoclaved) were tested to ensure endotoxin levels and microbiological load were below 20 EU/mL and 100 CFU/g and then evaluated. their immunocompatibility using a subcutaneous air sac model in mice (Table 2b).

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Table 2b

Immunocompatibility of animal-derived carboxymethylchitosan formulations using a subcutaneous air sac model in mice 24 h post-injection*

No. Number of white blood cells (x10 ⁶ cells / ml) concentr. ILl-beta (x10 ⁹ g/ml) * * concentr. KC/CXCL1 (x10 ⁹ g/ml) concentr. TNF-alpha (x10' ⁹ g/ml) ** ss-z 0.7 <3 49 < 125 4 1.0 5 115 184 controls Negative control (saline) 0.2 17 23 272 Positive control (1% carrageenan) 33.2 28 >700 767 Synvisc® (Hylan GF20) 0.7 4 46 < 125

* Average value for 3 subjects.

** Limit of detection: 3x10 ^-9 g/ml for IL1-beta and 125.10 ^-9 g/ml for TNF-alpha.

The composition of CC-4 caused a significant reaction, characterized by a high concentration in the infiltrate of two markers simultaneously, KC/CXCL1 (neutrophil activation) and TNF-alpha. The TNF-alpha marker was not detected after injection of the CSS formulations described in Example 2 or Synvisc® (Hylan GF-20). However, it is undesirable to elicit a response characterized by the TNF-alpha marker. Because it is desirable to have good immunocompatibility in therapeutic applications where the composition is expected to be in contact with tissues weakened by the underlying pathology such as, for example, dry eye syndrome, corneal injury or injury, or joint inflammation, and to avoid neutrophil activation and TNF-alpha, animal carboxymethylchitosan CC-4 is unsuitable.

The CC-3 formulation induces a lower response characterized by some white blood cells and a moderate concentration of the KC/CXCL1 marker, similar to the levels from the Synvisc® reference product, but above the levels of the negative control (saline). Thus, formulation CC-3 appears to be less immunoreactive than formulations CSS and formulation CC-4. However, the CC-3 formulation has the disadvantage that it has a concentration of the KC/CXCL1 marker which may not be suitable for a therapeutic treatment method where very good immunocompatibility is required in the subject, and that it undergoes very significant hydrolysis of the macromolecular chains by heat. , which thus makes it impossible to sterilize it in an autoclave at the final stage at the end of the production process, i.e. after filling the syringe with the composition.

Example 3 Carboxymethylchitosans of fungal origin.

To get carboxymethylchitosan from table. 3a and 3b, starting with chitin or chitosan of fungal origin, the following reactions are carried out:

reacetylation followed by carboxymethylation of ultrahigh molecular weight fungal chitosan (CC-5 in Table 3a);

carboxymethylation followed by reacetylation of ultra-high molecular weight fungal chitosan (CC-5 to CC-9);

carboxymethylation of fungal ultra-high molecular weight chitosan (CC-11);

carboxymethylation of fungal chitin (CC-10).

For example, SS-8 was obtained as follows:

g of ultra-high molecular weight chitosan was dispersed in 220 ml of isopropanol and 68 ml of 40% sodium hydroxide (w/v). 45 g of the alkylating agent monochloroacetic acid (MCA) was dissolved in 45 ml of isopropanol and added to the chitosan suspension. The reaction was allowed to proceed for 16 h at 25°C. The polymer was isolated by ethanol precipitation, then purified by solubilization/ethanol precipitation cycles. The precipitate was dried in a ventilated oven. A mass of 15 g of the precipitate was dispersed in water and 3.75 ml of acetic anhydride was added thereto. After 30 minutes of stirring at room temperature, the pH of the medium was adjusted to 6 and 3.75 ml of acid anhydride (AC) was added thereto. After 30 min stirring at room temperature, the medium was neutralized and precipitated in ethanol. The precipitate thus obtained was redissolved and then precipitated again. The final precipitate of carboxymethylchitosan (CC-8) was dried in a ventilated oven.

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The synthesis parameters used to obtain other SS are described in table. 3a and 3b.

Table 3 a

Parameters for the synthesis of carboxyalkyl chitosan starting from fungal chitosan

Options SS-5 SS-6 SS-7 SS-8 SS-9 SS-11 Carboxymethylation step Alkylating agent (MCA or SCA)* SCA MCA MCA MCA MCA MCA NaOH/chitosan (w/w) AND 2.7 2.7 2.7 2.7 2.7 Urea Yes No No No No No Alkylating agent/chitosan (w/w) ID 9 9 4.5 4.5 6.8 Isopropanol/alkylating agent (% w/w) 0% 80% 80% 80% 80% 80% Temperature (°C) 15°C 15°C 25°С 25°С 35°С 60°С Duration(h) 22 h 16 h 16 h 16 h 4h 1 hour Reacetylation step Before and after carboxymethylation BEFORE after after after after / AC/chitosan (volume/mass) - first addition - second addition OD o, s 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 / duration and reaction temperature 0.5 h at 25 °C 0.5 h at 25 °C 0.5 h at 25°C 0.5 h at 25°C 0.5 h at 25°C /

* MCA: monochloroacetic acid; SCA: monochloroacetic acid sodium salt.

Table 3b

Parameters for the synthesis of carboxyalkylchitosan starting from fungal chitin

Options SS-10 Chitin/soda/water (w/w) 1/11.4/28.5 Duration and temperature of contact with soda 16 h at 4 °C Alkylating agent (MCA or SCA) MCA Chitin/isopropanol/alkylating agent (w/w/w) 1/28.5/4.5 Duration and reaction temperature 16 hours at 25°C

The resulting carboxymethylchitosans (CC) of fungal origin were characterized by carbon-13 NMR to confirm their structure and determine the DA and DS values (Table 3c). Taking into account the standard deviation inherent in the method of NMR spectrometry on carbon-13 nuclei (approximately ±10%), it was observed that the value of the zeta potential at pH 7.5 correlated with the molecular structure, in particular with DS.

Table 3c

Characteristics of carboxymethylchitosans of fungal origin and their compositions

No. (concentration) DA* % DS* % Solubility of the polymer Sterilizable in autoclave ** Zetapotential at pH 7.5 (mV) SS-5 (1.5%) 67 18 Soluble at any pH Yes -12.5 SS-6 (2%) 71 42 Soluble at any pH Yes -15

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SS-7 (2%) 57 70 Soluble at any pH Yes -18 SS-8 (2%) 50 80 Soluble at any pH Yes -26 SS-9 (2%) 41 101 Soluble at pH> 3.1 Yes -22 SS-10 (2%) 46 167 Soluble at any pH Yes -24.5 SS-11 (2%) 23 130 Soluble at pH > 3.5 Yes -26

* DA and DS as determined by 13C-NMR.

** Considered sterilizable if the intrinsic viscosity loss before/after sterilization is less than 40%.

All obtained SS are soluble in a wide pH range, in particular in the pH range of 4.0-8.5, without opalescence.

Formulations were prepared at 1.5% or 2% CC, packaged in glass syringes, then autoclaved according to a standard 15 min cycle at 121°C. To assess whether formulations can be autoclaved sterilized, the intrinsic viscosity of the polymers was measured before and after autoclaving by capillary viscometry after diluting 25-fold with phosphate buffer. The loss of intrinsic viscosity of the CC was less than 40%, indicating acceptable stability, in contrast to the CC-3 formulation described in Example 2 (which lost approximately 60% of its intrinsic viscosity).

Visual inspection of the sterilized compositions included in the table. 3c showed that they are transparent and non-opalescent. They were then tested to ensure that endotoxin levels and microbiological load were less than 20 EU/mL and 100 CFU/g, and then their immunocompatibility was assessed using a mouse subcutaneous air sac model.

table 3d

Immunocompatibility of fungal-derived carboxymethylchitosan formulations using a subcutaneous air sac model in mice 24 h after injection*

No. Number of white blood cells (x10 ⁶ cells/ml) Conc. ILl-beta (x10 ⁹ g/ml) Conc. KC/CXCL1 (xlO ⁹ g/ml) ** Conc. TNF-alpha (x10' ⁹ g/ml) ** SS-5 1.9 6 28 391 SS-6 17 12 20 < 125 SS-7 9.0 12 20 < 125 SS-8 2.6 <3 20 < 125 SS-9 6.5 7 20 < 125 SS-10 2.4 <3 16 < 125 controls negative control (physiological solution) 0.2 17 23 272 positive control (1% carrageenan) 33.2 28 >700 767 Synvisc® (Hylan GF20) 0.7 4 46 < 125

* Average value for 3 objects.

** Limit of detection: 3x10 g/ml for IL1-beta and 125x10 ^-9 g/ml for TNF-alpha.

In general, there is a good immunocompatibility of the fungal-derived SS formulations with a weakening or even suppression of the immune response compared to the N-succinylchitosan formulations described in example 1, and to the animal-derived carboxymethylchitosan formulations described in example 2.

A low concentration of the KC/CXCL1 marker is noted to be induced by the fungal-derived SS formulations, at the same level as the negative control (saline) and lower than the Synvisc® product. This indicates little or even no neutrophil activation, in contrast to the level reported after administration of the CSS formulations (Example 1) and

SS of animal origin (example 2).

In addition, it is noted that for some formulations of fungal-derived SS (namely SS-8 and SS-10), four parameters of the immune response are simultaneously attenuated or suppressed, ie. total white blood cells, IL1-beta marker, KC-CXCL1 marker, and TNF-alpha marker. This did not happen with CSS formulations or animal based CC formulations.

It appears that for fungal-derived CC formulations, the formulations that exhibit this simultaneous attenuation of four parameters are those with the highest negative charge (e.g. -26 mV for CC-8 and -24.5 mV for CC-10 at pH 7.5).

It is observed that the animal-derived CC-4 formulations described in Example 2 and the fungal-derived CC-5 lightly substituted with a carboxyalkyl group cause a certain activation of the TNF-alpha marker and that, on the other hand, only the animal-derived CC-4 formulation leads to significant secretion of the KC/CXCL1 marker. It was also observed that the CC-10 formulation (fungal origin) did not result in neutrophil activation in contrast to the animal origin CC-3 formulation described in Example 2.

Example 4 Evaluation of Local Tolerability of Intra-Articular Administration of Fungal Origin Carboxymethylchitosan Compounds Using a Sheep Model.

Formulations with 2% of the two fungal-derived carboxymethyl chitosans (CC-8 and CC-10) described in Example 3 were prepared to evaluate their possible use by intra-articular injection in a sheep model (sheep). A 2 ml volume of the two formulations was administered to a sheep and the local response induced by the intra-articular injection was evaluated for 2 weeks. The effects of a second injection of formulation CC-8 in the same joint 1 month after the date of the first injection were also evaluated. The Synvisc® product was administered in the same manner in a volume of 1 or 2 ml.

Clinical signs were monitored daily by palpation of the joint and observation of the onset of lameness for 15 days and scored semi-quantitatively from 0 (no signal) to a maximum score of 3 for effusion and 5 for lameness. The overall clinical outcome over a period of 15 days was presented as a frequency of occurrence in points (Table 4), as well as a characteristic of the synovial fluid produced on day 15.

Table 4

Assessment of local tolerance of fungal-derived carboxymethylchitosan and Synvisc® formulations using a sheep model by intra-articular injection

Composition No. Volume (N: number of animals) Incidence of effusions at 15 days (score from 0 to 3) Incidence of lameness in 15 days (score from 0 to 5) Characterization of synovial fluid taken by puncture on day 15 SS-8 First injection 2 ml (N = 4) Score 0: 100% Points 1-3: 0% Score 0: 100% Points 1-5: 0% Normal SS-8 Second injection 2 ml (N = 2) Score 0: 100% Points 1-3: 0% Score 0: 100% Points 1-5: 0% Normal SS-10 One injection 2 ml (n=4) Score 0: 100% Points 1-3: 0% Score 0: 100% Points 1-5: 0% Normal Synvisc® (Hylan GF20) Ν = 8, of which: - one injection of 1 ml (N = 4), - one injection of 2 ml (N = 4) Score 0: 62.5% Score 1: 37.5% Points 2-3: 0% Score 0: 62.5% Score 1: 37.5% Points 2-5: 0% Normal

Example 5 Evaluation of local tolerability of fungal-derived carboxymethylchitosan formulations after intradermal injection in rabbits.

The compositions of three carboxymethylchitosans of fungal origin at physiological pH and osmolality were prepared to evaluate the possibility of their intradermal administration in a rabbit model (Table 5a). CC-12, CC-13, CC-14 and CC-15 of fungal origin were prepared by carboxymethylation followed by reacetylation according to the general method described in Example 3 for CC-8, while modulating the synthesis parameters in such a way as to cause variation in DA and DS (Table 5a). CC-13 and CC-14 showed a low DS value of 41% and 51%, respectively, and a high DA value of 74% and 69%, respectively.

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The formulations were packaged in a 1 ml glass syringe and then autoclaved according to the standard cycle (15 min at 121°C). Three formulations were resistant to autoclave sterilization with less than 40% intrinsic viscosity loss. Finally, the formulations were tested to ensure that the level of bacterial endotoxins was less than

EU/ml and microbiological load is less than 100 cfu/ml.

The composition in a volume of 200 μl was administered to rabbits by intradermal injection through a 27 gauge needle (very small diameter needle) according to a protocol that complies with ISO 10993 (part 10) to assess primary irritation caused by an intradermal implant. Each of the six rabbits received three injections of each formulation. A commercially available cross-linked hyaluronic acid dermal filler product, Juvederm® Voluma (Allergan), was also injected through a 27 gauge needle. To determine the primary irritation score by assessing the possible occurrence of erythema, ulcer, edema and induration on a semi-quantitative scale based on scores from 0 (no signal) to 4 (maximum signal), macroscopic skin irritation signals were recorded after 12, 24 and 48 hours for all injected animals and for all sites. The primary irritation score for each product was determined as follows: the mean erythema scores for the 18 sites injected at 24, 48 and 72 hours were added together. The sum of the mean edema scores was calculated in a similar manner. The 2 sums (erythema and edema) were added and then divided by 54 to give the average primary irritation score. The same methodological approach was applied to the reference product. Points of irritation are presented in table. 5b. The CC formulations were observed to show no signs of edema and few signs of erythema, with a score lower than that of rabbits induced with Juvederm® Voluma in all cases. It can be concluded that the formulations are non-irritating and less irritating than Juvederm® Voluma.

Table 5a

Characteristics of carboxymethylchitosans of fungal origin and their compositions

No. (concentration) DA* (mole%) DS* (mole%) Solubility of the polymer Sterilizable in autoclave ** Zetapotential at pH 7.5 (mV) SS-12(3%) 41 50 Insoluble in the pH range 3.1-6.6 Yes -17 SS-13 (3%) 74 45 Soluble at any pH Yes -20 SS-14(3%) 69 51 Soluble above pH 3.6 Yes *** SS-15 (2%) 55 100 Soluble above pH 3.1 Yes -27.5

* As determined by 13C-NMR.

** Considered sterilizable if IV loss before/after autoclaving is 40% or less.

*** Value estimated from polynomial regression of the zetapotential curve at pH 7.5 as a function of DS (±20%).

Table 5b

Evaluation of the primary irritation score of carboxymethylchitosan and Juvederm® Voluma formulations after intradermal injection in rabbits

No. (concentration) Initial irritation score at 72 h Total score (and mean score) SS-12 (3%) 0.11 (0.002) SS-13 (3%) 0.22 (0.004) SS-13 (3%) 0.17(0.003) SS-14 (2%) 0(0) Juvederm® Voluma 2.67 (0.049)

The observation period was then extended to 2 weeks post-injection, with clinical signals assessed on days 5, 7, 9, 11, and 14. For possible histological analysis, 2 animals were sacrificed on day 3, day 7, and day 14 post-injection. General estimates are presented in table. 5s. The four formulations were observed to show excellent local tolerability over a period of 2 weeks following intradermal injection, with scores lower than those observed for the Juvederm® Voluma product for all general or macroscopic signs and no signs of ulceration or edema. Formulations CC-12 and CC-13 showed some signs of erythema with a low score (maximum 1), which disappeared on day 11. Formulation CC-13 showed some signs of erythema with a maximum score of 2, with 33% of cases observed on day 9, which also disappeared on day 11 and had no harmful effect on the tissue.

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Table 5c

Evaluation of local tolerability of carboxymethylchitosan formulations after intradermal injection in rabbits (2 weeks) (expressed as frequency of occurrence in points)

No. (concentration) Time period (number of plots)* Day 3 (18) Day 5 (12) Day 7 (12) Day 9 (6) Day 11 (6) Day 14 (6) Erythema score SS-12 (3%) Score 0-1 Score 2-4 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% SS-13 (3%) Score 0-1 Score 2-4 100% 0% 100% 0% 83% 17% 67% 33% 100% 0% 100% 0% SS-14 (3%) Score 0-1 Score 2-4 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% SS-15 (2%) Score 0-1 Score 2-4 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% Juvederm® Voluma Score 0-1 Score 2-4 6% 94% 17% 83% 17% 83% 17% 83% 33% 67% 33% 67% Ulcer and edema scores SS-12 (3%) Score 0-1 Score 2-4 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% SS-13 (3%) Score 0-1 Score 2-4 100% 0% 100% 0% 83% 17% 67% 33% 100% 0% 100% 0% SS-14 (3%) Score 0-1 Score 2-4 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% SS-15 (2%) Score 0-1 Score 2-4 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% Juvederm® Voluma Score 0-1 Score 2-4 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%

* Number of sites assessed macroscopically in each time period after injection.

In addition, four compositions (CC-12, CC-13, CC-14 and CC-15) can be easily administered through a fine 27 gauge needle suitable for intradermal administration.

Example 6 Susceptibility to enzymatic degradation in vitro.

In this example, the rate at which the formulation is degraded in the presence of a mixture of two enzymes, lysozyme and hyaluronidase, present in biological fluids (eg, synovial fluid, tears, or interstitial fluid of connective tissues) was compared. The enzyme lysozyme is generally known for its ability to hydrolyze chitosan-based biomaterials.

Fungal carboxymethylchitosan CC-16 was prepared in the same manner as CC-8 in Example 3 by modulating the synthesis parameters to control DA and DS. 2% formulations CC-16, CC-8 (example 3) and CC-10 (example 3) were prepared in phosphate buffer with 3.5% sorbitol. A 2% composition of CC-3 of animal origin was also evaluated (Example 2) and two commercially available viscosupplementation products based on non-crosslinked hyaluronic acid (HA-1 and HA-2).

The composition was diluted 25 times in phosphate buffer. The solution was then stirred for 12 hours and a mixture of lysozyme and hyaluronidase enzymes was added to the diluted solution at doses of 184 and 2.6 units/ml, respectively. Intrinsic viscosity measurements were made at regular intervals using an I-Visc automatic capillary viscometer (Lauda) equipped with an Ubbelohde type capillary (model 0a). The half-life of each formulation was then calculated, i.e. the time required for the intrinsic viscosity of the polymer to reach half of its initial value (Table 6).

Table 6

Half-life of formulations in the presence of a mixture of lysozyme/hyaluronidase enzymes at 37°C

SS-8 (2%) SS-10 (2%) SS-16 (2%) ss-z (2%) ON-1 ON 2 DA/DS (mole%)* 50/80 46/167 67/115 71/149 / / half life (min) 1200 min 958 min 612 min 88 min 73 min and min

*Determined using solid phase nuclear magnetic resonance (NMR) spectroscopy on carbon-13 nuclei.

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Based on the results obtained, it becomes clear that carboxymethylchitosan formulations are hydrolyzed by a mixture of lysozyme/hyaluronidase and that hydrolysis kinetics can be modulated by the molecular structure of carboxymethylchitosan. This allows you to adjust the residence time of the product in accordance with the intended therapeutic application. It also follows that fungal-derived carboxymethylchitosans degrade less rapidly than animal-derived carboxymethylchitosan (CC-3) and two commercially available hyaluronic acid products.

Example 7. Exposure to heat.

In this example, the syringes containing the formulations were placed in an oven at a temperature maintained at 60° C. for a period of 11 days, which makes it possible to evaluate their heat resistance at a temperature that is higher than the usual storage temperature. At each time interval, the syringe was withdrawn and the intrinsic viscosity of the polymer was measured according to the method described in Example 6. The ratio between the viscosity at the given time interval and the initial viscosity (in % of the initial viscosity) is shown.

Two fungal-derived carboxymethylchitosan, CC-17 and CC-18, were prepared in the same manner as CC-8 in Example 3 by modulating the synthesis parameters to control DA and DS and characterized by carbon-13 NMR ( Table 7). They were formulated at a concentration of 2% in the presence of a reducing sugar, sorbitol or mannitol, respectively. Also evaluated were 2% of the CC-3 composition of animal origin indicated in example 2 (with 3.5% sorbitol), as well as a commercially available viscosupplementation product based on non-crosslinked hyaluronic acid indicated in example 6 (HA-2).

Table 7

Change in intrinsic viscosity of formulations,

stored at 60°C (in % of initial viscosity) No. (concentration, reducing sugar) DA/DS (mole %) * Day 3 Day 7 Day 11 SS-17 (2%, 3.5% sorbitol) 71/49 96% 95% 91% SS-18 (2%, 3.5% mannitol) 71/49 100% 92% 91% SS-3 (2%, 3.5% sorbitol) 71/149 84% 80% 70% ON 2 / 94% 92% 81%

* As determined by solid state nuclear magnetic resonance (NMR) spectroscopy on carbon-13 nuclei.

It can be concluded that the fungal-derived carboxymethylchitosan formulations are heat stable and less sensitive than the animal-derived formulation (CC-3) and the commercially available hyaluronic acid product.

Example 8. Methods for reducing the microbiological load of carboxymethylchitosan.

Fungal-derived carboxymethyl chitosan, CC-19, was prepared according to the method used to prepare CC-8 in Example 3, starting with ultra-high molecular weight chitosan. It has the following characteristics: DA 67% and DS 115% (according to NMR on carbon-13 nuclei), soluble at any pH, transparent, non-opaque. The zeta potential of the formulation at pH 7.5 is estimated to be -27 mV (from polynomial regression of the zeta potential versus DS curve). A 2% formulation of CC-19 in phosphate buffer with 3.5% sorbitol was prepared as described in Example 3. It has the following characteristics: pH 7.3 and osmolality 279 mOsm/kg. To test their applicability and effect on final formulation performance, two known methods were effectively applied to reduce the microbiological load of aqueous formulations, and the intrinsic viscosity of the formulation (diluted 25 times) was compared using an I-Visc capillary viscometer (Lauda, capillary 0a), and its refractive index was measured with a HI88713 refractometer (Hanna) before and after the process (Table 8). These two methods were as follows:

autoclave: the formulation was placed in a glass syringe and then the syringe was autoclaved according to the standard process (15 min at 121°C);

filtration: a volume of 300 ml of the composition was filtered using a 0.2 μm porous filter (Preflow capsule filter, Pall).

The filtration process on the 0.2 µm filter was suitably carried out at a constant pressure of 2 bar and a constant flow rate of approximately 6 l/h.

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Table 8

Influence of filtration and autoclaving methods on the composition of 2% CC-19

Index Filtration method (0.2 µm) Autoclaving method (15 min 12GS) Intrinsic Viscosity Difference (%) 14% reduction in initial viscosity 20% reduction in initial viscosity Refractive index difference No difference No difference

It can be concluded that both methods have little effect on material reduction (refractive index) and polymer degradation (intrinsic viscosity). Therefore, they are applicable in industry for the production of medical devices and pharmaceutical products based on carboxymethylchitosan formulations.

Example 9 Lubricity of fungal-derived carboxymethylchitosan formulations in vitro.

The purpose of this example is to illustrate the lubricity of the formulations of two fungal-derived carboxymethyl chitosans, CC-8 in Example 3 (2 and 1%) and CC-19 in Example 8 (2%), with respect to their possible use as an intra-articular viscosupplement or as ophthalmic drops for the surface of the eye. The lubricity was evaluated using an in vitro model that measures the reduction in friction between two surfaces. Also characterized the composition of CC-3 (2%) of animal origin, specified in example 2, and commercially available products based on hyaluronic acid:

intra-articular viscosupplements: Synvisc® (Sanofi) and two viscosupplements based on non-crosslinked hyaluronic acid, HA-2, indicated in examples 6 and 7, and HA-3;

eye drops: two products based on non-crosslinked hyaluronic acid (HA-4 and HA-5).

In addition, a synovial fluid sample was taken from the knee of a patient suffering from osteoarthritis prior to a surgical procedure to install a knee prosthesis. The liquid was stored at -20°C, then brought to 25°C before analysis of the coefficient of friction.

The coefficient of friction was measured by the following method. Two discs made from a polyacrylate-type biomaterial used for the manufacture of hydrophobic intraocular lenses (as described in EP 1830898) having a diameter of 16.15 mm were pre-hydrated by immersion in water at 60° C. for a period of approximately 2 hours, then fixed to the upper and lower geometries of the Discovery Hybrid Rheometer-2 (DHR-2) (TA Instruments). The test liquid in a volume of approximately 100 μl was placed on the lower disk, then the upper geometry was lowered until contact was reached between the two disks, to an applied normal force of 5 newtons. Friction coefficient measurements were made at 25°C for 150 s at a constant normal force (5 N), an oscillation frequency of 1.256 rad/s, and a strain angle of approximately 0.05 radians according to a protocol adapted from the protocol described by Waller et al. (in J. Rheumatol., 39, 7, 1473, 2012). The option to correspond to the zero point of the beginning of the oscillatory movement is activated. At each measurement point, the torque value was recorded and then the coefficient of friction (COF) was calculated using the formula

COF=torque/(1/3xdisc diameternormal force)

For each composition, the measurement was repeated 5 times. The friction coefficient value was obtained by extrapolating the intercept at the beginning of each COF vs. time curve (COF0, Table 7).

Table 9

Lubricity of carboxymethylchitosan formulations of fungal origin

No. (concentration) coefficient( friction factor (COFo) Dimension 1 Dimension 2 Dimension 3 Synovial fluid from a patient with arthritis 20 40 12 SS-8 (2%) 1.2 1.7 1.3 SS-8 (1%) 2.4 3.7 3.9 SS-19 (2%) 4.0 3.2 h, o SS-3 (2%) 6.2 6.1 6.0 Synvisc® 3.2 3.1 3.8 ON 2 6.7 6.3 6.6 ON-3 5.3 5.6 4.5 ON-4 6.8 6.7 6.0 ON-5 29.4 13.3 26.1

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In the presence of formulations of 2% and 1% carboxymethylchitosan, the coefficient of friction is low, of the same order of magnitude as commercially available products for intra-articular and ophthalmic use, or even lower and significantly lower than arthritic synovial fluid under measurement conditions. It follows that carboxymethylchitosan-based formulations have the potential to act as a lubricant by reducing friction between two surfaces, for example cartilage surfaces of a joint after intra-articular injection or ocular surfaces after instillation in the form of drops. Mushroom-derived CC formulations are more effective in reducing friction than animal-derived CC-3 formulations.

Example 10 Use of fine needles to administer a fungal-derived carboxymethylchitosan formulation intradermally.

The purpose of this example is to show that the 2% fungal-derived carboxymethylchitosan formulation can be easily injected into the dermis, in particular by using very fine needles designed to be injected into the superficial layers of the dermis. The test consists of measuring the force required to expel a product from a syringe fitted with a needle at a given injection rate on a compression test stand. In an empirical sense, it is believed that the introduction of the product is easy and comfortable for the doctor and the patient when the force of expulsion, measured using this test, is less than 50 newtons, and the introduction occurs in a uniform and smooth manner. It is useful to administer the product using needles having a diameter of less than 0.3 mm (30G) to minimize pain and bleeding upon injection and the subsequent risk of bruising and skin redness.

Reference carboxymethyl chitosan of fungal origin CC-20 was prepared according to the general method CC-8 specified in Example 3, starting from ultra-high type chitosan, with the following modifications: for 10 g of chitosan, 228 ml of isopropanol, 57 ml of 40% sodium hydroxide and 47 g ISA. The reaction was carried out at 35° C. for 23 hours. For 15 g of the intermediate carboxymethyl chitosan, 7.5 ml of acetic anhydride was used with each addition, and 3 additional purification cycles were applied before drying and recovering the final carboxymethyl chitosan. CC-20 has the following characteristics: DA 53% and DS 85% (determined by carbon-13 NMR), soluble in water at any pH (according to the method described herein above), forms a clear and non-opaque solution, with a zeta potential at pH 7.5 of -24 mV (from a polynomial regression of the zeta potential versus DS curve).

A formulation of 2% (w/w) CC-20 was prepared as described in Example 3. The formulation was packaged in a 1 ml glass syringe (BD-Medical) fitted with a needle. On a MultiTest 2.5-i compression test bench (Mecmesin) equipped with a 100 N compression chamber, the force required to push the product was measured by applying a constant push speed of 80 mm/min. The maximum force allowed by the equipment is approximately 70 Newtons. The following needles were tested: 30G, 32G, 33G and Invisible Needle™ (TSK Laboratory).

For comparison, commercially available products based on non-crosslinked hyaluronic acid (comparison products HA-6, HA-7) and crosslinked hyaluronic acid (HA-8) for skin rejuvenation by intradermal administration were evaluated in the same way in the original syringe.

Example 11 Evaluation of Low Concentration Fungal Origin Carboxymethylchitosan Formulations for Use as Eye Drops.

The purpose of this example is to show that a low concentration solution of fungal-derived carboxymethylchitosan can be used to make eye drops for use in reducing or protecting the symptoms of ocular surface disorders. In addition to physiological characteristics, i.e. pH of about 7.2 and osmolality of about 200 mOsm/kg, ophthalmic drops should preferably have the following physicochemical properties: a low refractive index (close to 1.33) and the ability to minimize friction between the surface of the eye and the conjunctiva and eyelids ( lubricity). Finally, its rheological profile is such that the product is not too liquid to be applied to the eye, but rather spreads evenly and is not sticky. Its rheological profile should also be such that fluttering or blinking of the eyelids is slight, without accumulation, i.e. low viscosity at high shear rate. In particular, Orobia et al. it is considered that the viscosity during eye movement (excluding eyelid flutter) preferably exceeds 10 mPa s, for example about 20 mPa s (in Clinical Ophthalmology, 12, 453, 2018). It should be noted here that the viscosity value may vary depending on the measurement method and, in particular, the equipment, measurement mode and parameters, temperature and shear rate applied to the test product.

Comparative carboxymethylchitosan CC-21 of fungal origin was obtained in the same way as CC-20 indicated in example 10. The molecular structures of CC-20 and CC-21 in acid form were compared using FT-IR spectroscopy using a Nicolet iS5 spectrometer, equipped with ID7-ATR (Thermo Scientifics), according to the method described by Chen et al. (Carbohydrate Polymers, 53, 355, 2003). It has been established that their molecular structures are identical on

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99% in the wavelength range 1200-1800 cm ^-1 and, as a result, are similar to their DA and DS.

Prepared two compositions of carboxymethylchitosan CC-21 with a concentration of 0.7 and 0.4% (wt./wt.) in phosphate buffer with glycerol. The pH value was adjusted to 7.2 ± 0.2 and the osmolality to 200 ± 20 mOsm/kg, and then the compositions were filtered, after which their lubricity and viscosity profile were characterized in accordance with the methods described below. Variable composition ophthalmic drops based on hyaluronic acid (HA) were characterized according to the same methods (from HA-5 to HA-9). The results are presented in table. 11b.

Friction coefficient measurement method (suitable for eye drops) Lubricity, i.e. to reduce friction between two contact surfaces was evaluated by measuring the coefficient of friction between two disks made from polyacrylate biomaterial, which is identical to the materials indicated in example 9. Two disks were fixed to the upper and lower geometries of the DHR-2 rheometer (TA Instruments), the test product in a volume of approximately 100 μl was placed on the lower disk, then the upper geometry was lowered until the two disks were brought into contact, to an applied normal force of 5 newtons. The coefficient of friction was measured at 25°C for 60 s at a constant normal force (5 N), an oscillation frequency of 6 rad/s, and a strain angle of approximately 1.71 radians according to a protocol adapted from Waller et al. (J. Rheumatol., 39, 7, 1473, 2012), and parameters simulating the kinematic conditions of eyelid blinking described by Kwon et al. (J. Royal Society Interface, 10, 2, 2013). The option to correspond to the zero point of the beginning of the oscillatory movement is activated. At each measurement point, the torque value was recorded, and then the coefficient of friction (CO) was calculated by the formula COF=torque/(1/3xdisk diameterxnormal force). The average COF was determined to be between 0 and 60 s. Thus, the average coefficient of friction was calculated, as well as the standard deviation of 5 consecutive measurements. The minimum COF value is 52 when two surfaces do not touch. The upper COF limit corresponds to the case when the two discs are no longer in motion relative to each other.

Given that the COF value varies from one series to another, it is necessary to characterize the products to be compared in the same series, using the same disks and in random order. A comparative score scale was then used to classify products tested in the same batch from most effective (score 1) to least effective (score 3) according to the criteria presented in Table 1. 11a. The COF _A and COFB limits are determined by the COF of two commercially available eye drops taken as a comparison for each lot: drops A (consisting of 0.15% HA and 0.25% carbomer) and drops B (0.15% HA). According to this scale, the friction reduction score is preferably 1 or 2, and preferably 1 for use as a lubricating drop to lubricate the ocular surface. For products with a score of 3, the lubricity is not satisfactory and the measurement deviations are significant.

Table 11a

Lubrication rating scale (friction reduction)

score COF limits 1 < 1.1xCOFa 2 >1.1xCOFah <1.1xCOF _b 3 > 1.1xCOFb

Methods for measuring viscosity as a function of shear.

Simmons et al. determined that the shear rate of open eye movements is about 10 s ^-1 and the shear rate of eyelid flutter is about 10,000 s ^-1 (in Clinical Ophthalmology, 11, 1637, 2017). The method of rheometric measurements was chosen depending on the range of shear rates to be studied and taking into account that the products are liquid solutions with low viscosity.

The shift range corresponding to eye movement. Viscosity was measured in rotational mode by a flow sweep test using a DHR-2 stress-controlled rheometer (TA Instruments) equipped with a Peltier plate and a 60 mm diameter cone geometry with a 2° truncation angle. The product was equilibrated within 1 min to 37°C, the temperature was controlled using a Peltier element. To avoid evaporation, the system is equipped with a solvent trap and a metal cover. The flow sweep test was then run and the viscosity was measured as a function of shear rate from 0.001 to 100 s ^-1 . Viscosity value at 10 s ^-1 is shown.

Range of shift corresponding to flutter of the eyelids. Viscosity was measured in rotational mode with a flow sweep test using a DHR-2 voltage controlled rheometer (TA Instruments) equipped with a double Couette gap (cylindrical) geometry and a concentric Peltier cylinder, equipped with a solvent trap and a metal cap. The product is equilibrated for 1 min to 37° C. and then a variable shear rate is applied ranging from 0.001 to 10,000 s ^-1 . The viscosity value at 10,000 s ^-1 was thus then compared with the value at 10 s ^-1 .

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Table 11b

Characteristics of formulations with a low concentration of carboxymethylchitosan of hybrid origin and eye drops based on hyaluronic acid

Comparison Product Refractive index Viscosity Friction reduction score at 10 s ¹ (mPa-s) at 10000 s ¹ ( mPa-s) Phosphate buffer with glycerin 1.3348 no data no data not measurable SS-21 0.7% 1.3358 27 Lower than viscosity at 10 s ¹ 1 SS-21 0.4% 1.3353 10 1 ON-5 1.3343 6 Lower than viscosity at 10 s ¹ 2 ON 10 1.3386 3 2 ON-I 1.3350 6 2 ON-12 1.3346 50 2

The results confirm that the two formulations of mushroom carboxymethylchitosan CC-21 meet the specifications of ophthalmic drops: they have a low refractive index, a satisfactory coefficient of friction (score 1, i.e. as effective as most lubricating commercially available drops), a viscosity of 10-30 mPa s under eye movement conditions and lower viscosity under eye flutter conditions. Without carboxymethylchitosan, the phosphate buffer supplemented with glycerol did not meet the technical specifications of ophthalmic drops. Finally, it should be noted that the viscosity can be reduced by lowering the concentration of carboxymethylchitosan (for example, from 0.7 to 0.4%) without adversely changing the ability to reduce friction (score 1).

Claims (22)

1. Carboxyalkylchitosan having glucosamine units, N-acetyl-glucosamine units and glucosamine units substituted with a carboxyalkyl group, wherein said carboxyalkylchitosan has a zeta potential, measured at pH 7.5, which is less than or equal to -18 mV, wherein said carboxyalkylchitosan has the degree acetylation, which is from 40 to 80%, expressed as the number of moles of N-acetyl-glucosamine units in relation to the number of moles of all units, and the specified carboxyalkylchitosan has a degree of substitution of the carboxyalkyl group, which is more than 20%, expressed as the number of moles of the substituent in relation to the number of moles of all links. 2. Carboxyalkylchitosan according to claim 1, characterized in that said carboxyalkylchitosan has a zeta potential, measured at pH 7.5, which is less than or equal to -20 mV. 3. Carboxyalkylchitosan according to claim 1 or 2, wherein said carboxyalkylchitosan has a degree of substitution with a carboxyl group, expressed as the number of moles of a substituent relative to the number of moles of all units, greater than 50%. 4. Carboxyalkylchitosan according to any one of claims 1 to 3, characterized in that said carboxyalkylchitosan has a degree of substitution with a carboxyalkyl group that is more than 70%, expressed as the number of moles of the substituent relative to the number of moles of all units. 5. Carboxyalkylchitosan according to any one of claims 1 to 4, characterized in that the chitosan is obtained from the mycelium of the Ascomycetes type fungus and, in particular, Aspergillus niger, and/or from the Basidiomycetes fungus and, in particular, Lentinula edodes (shiitake), and/ or Agaricus bisporus (champignon). 6. Carboxyalkylchitosan according to any one of claims 1 to 5, characterized in that the carboxyalkylchitosan is N-carboxyalkylated or O-carboxyalkylated. 7. Carboxyalkylchitosan according to any one of claims 1 to 6, characterized in that the carboxyalkylchitosan is reacetylated. 8. Carboxyalkylchitosan according to any one of claims 1 to 7, characterized in that it is sterilized. 9. An immunocompatible composition, characterized in that it contains at least one carboxyalkyl chitosan as defined in any one of claims 1 to 8. 10. The composition according to claim 9, characterized in that it also contains hyaluronic acid or sodium hyaluronate, cross-linked or not cross-linked by covalent bonds. 11. An injectable composition, characterized in that it contains at least one carboxyalkyl chitosan as defined in any one of claims 1 to 8. 12. Pharmaceutical composition, characterized in that it contains at least one carboxyalkylchitosan, defined according to any one of claims 1-8. 13. Composition according to claim 11 or 12, characterized in that the composition also contains hyaluronic acid or sodium hyaluronate, cross-linked or not cross-linked by covalent bonds. 14. The composition according to any one of claims 11-13, characterized in that the composition is an injectable pharmaceutical composition, an implantable pharmaceutical composition, - 27 043011 a pharmaceutical composition suitable for instillation, or an injectable medical device, an implantable medical device or a medical device suitable for instillation. 15. Use of the composition according to claim 14 in a method of therapeutic treatment. 16. The use according to claim 15, characterized in that the composition is administered by instillation or administered subcutaneously, intradermally, ocularly, intraocularly, or intra-articularly to repair or fill at least one body tissue requiring repair or filling. 17. The use according to claim 16, characterized in that the tissue of the body is selected from tissues belonging to the vocal cords, muscles, ligaments, tendons, cartilage, genitals, bones, joints, eyes, dermis, or any of their combinations and, more specifically, joints, wherein the body tissue is selected from tissues belonging to the vocal cords, muscles, ligaments, tendons, cartilage, genitals, bones, joints, eyes, dermis, or any combination thereof, and more specifically, belonging to the articular joints. 18. Use according to claim 14 or 15, wherein the method of therapeutic treatment is a method of treating osteoarthritis or repairing a cartilage defect, optionally by injection into synovial fluid or after mixing with blood and implantation into cartilage. 19. A composition according to any one of claims 11 to 14 containing a reducing sugar. 20. Medical device, characterized in that it contains or consists of a composition according to any one of claims 9-19. 21. Medical device according to claim 20, characterized in that it is a medical implant. 22. The method of obtaining a composition containing carboxyalkylchitosan, defined by any one of paragraphs.9-19, and this method includes dissolving the carboxyalkylchitosan, defined by any one of paragraphs.1-9, in an aqueous solution, optionally buffered; adjusting the pH to the desired pH; addition of other excipients; regulation of the final osmolality of the composition.