negatively charged chitosan

Abstract

The present invention relates to a carboxyalkyl chitosan of fungal origin, having glucosamine units, N-acetyl-glucosamine units and glucosamine units substituted with carboxyalkyl groups, said carboxyalkyl chitosan having a degree of substitution of carboxyalkyl groups greater than 50% expressed in moles of substituents relative to the moles of total units. The present invention further relates to compositions comprising this carboxyalkyl chitosan, to methods for its preparation and to various uses thereof, in particular in the fields of therapy, rheumatology, ophthalmology, aesthetic medicine, plastic surgery, internal and external surgery, dermatology or cosmetics.

Description

[0001] This application is a divisional application of application No. 202210266925.5, filed on November 09, 2018, with the title “Negatively charged chitosan”. TECHNICAL FIELD

[0002] The present application relates to carboxyalkyl chitosans, compositions comprising the carboxyalkyl chitosans, methods for their preparation and various uses thereof, in particular in the fields of therapy, rheumatology, ophthalmology, aesthetic medicine, plastic surgery, internal surgery, dermatology or cosmetics. BACKGROUND

[0003] Chitosan derivatives are known, in particular disclosed in the patent applications and corresponding patents published by Kiomed Pharma under the publication numbers WO 2016 / 016463 and WO 2016 / 016464. These patent applications focus on the physical, chemical or physico-chemical properties of chitosan derivatives. However, there is still a need to improve these compositions, in particular in the case of therapeutic treatments, in order to provide the best therapeutic benefit to patients who can have to use them, and in particular to increase the benefit / risk ratio.

[0004] By increasing the degree of acetylation (DA) through reacetylation of fungal chitosan, it is possible to obtain soluble chitosan. By reacetylation of fungal chitosan, it is in fact possible to obtain a preparation that is soluble at physiological pH, but it is noted that the following occurs:

[0005] Degradation too fast in vivo or similar conditions;

[0006] Immune reaction in the subject into which the chitosan is injected or implanted, for example by injection or intra-articular implantation;

[0007] The chitosan is not suitable for the target application, so that the chitosan suitable for use, in particular by injection or intra-articular implantation, does not guarantee a sufficiently satisfactory therapeutic use.

[0008] There are many publications on the carboxyalkylation, and in particular on the carboxymethylation, of chitosan, mainly to solubilize chitosan. The molecular formula of chitosan does not theoretically contain N-acetyl-glucosamine units, but in fact chitosan is derived from chitin, which itself contains N-acetyl-glucosamine units, and therefore chitosan has a certain degree of acetylation (DA), i.e. the proportion of N-acetyl-glucosamine units in chitosan. The DA of chitosan is generally low. In particular, chitosan, usually reacetylated chitosan, is more than 30%.

[0009] Furthermore, Chinese patent application CN1431229A relates to derivatives of carboxymethylated chitosan, more specifically to their hydrating capacity, which is however still limited to this feature.

[0010] Carboxymethylation of chitin of animal origin, in particular of crustacean origin, has also been envisaged in the prior art. However, chitin of crustacean origin is difficult to access; in particular, it must be frozen and alkalized in order to be able to be substituted (see for example Chinese patent application CN106474569). This method is difficult to implement and in particular to implement on an industrial scale. On the other hand, this substitution method based on chitin of crustacean origin is expensive in terms of energy, has low reproducibility and the polymer is prone to degradation and the acetyl group of the N-acetyl-glucosamine units is prone to a high degree of hydrolysis, which is also difficult to control. SUMMARY

[0011] The object of the present application is to solve the technical problem of providing a suitable chitosan derivative, which is suitable for use in humans or animals, in particular in the fields of therapy, rheumatology, ophthalmology, aesthetic medicine, plastic surgery, internal and external surgery, dermatology or the cosmetics field.

[0012] More particularly, the object of the present application is to solve the technical problem of providing a suitable chitosan derivative, which is suitable for use in humans or animals in the field of therapy and which can in particular be used as a viscosifying agent and which in particular has the ability to be injected into or mixed with synovial fluid.

[0013] The object of the present application is in particular to solve the technical problem of providing a reconstituted synovial fluid, i.e. of providing a composition which restores the properties of a joint, for example the ability to lubricate the surface of the cartilage.

[0014] Another object of the present application is to solve the technical problem of providing a chitosan derivative or a composition comprising it, which exhibits good properties and compatibility in a mixture with synovial fluid, and in particular with synovial fluid of a human or animal subject, for example for treating a joint or joint pathology or synovial fluid deterioration in question.

[0015] The object of the present application is also to solve the technical problem of providing a chitosan derivative or a composition comprising it, which limits the immune response in a subject, in particular a human or animal subject, receiving the chitosan derivative or the composition comprising it, for example by injection.

[0016] Another object of the present application is also to solve the technical problem of providing a chitosan derivative or a composition comprising it, which exhibits a feature which varies little with pH.

[0017] The present invention also aims to solve the technical problem of providing chitosan derivatives or compositions comprising thereof, which, particularly in the context of regenerative medicine, have suitable osmolality and pH value according to the indication for the target treatment, are considered suitable for tissue contact with human or animal subjects, and are acceptable in terms of lifespan in situ, immune response and / or foreign body response, and biomechanical properties. Detailed Implementation

[0018] Surprisingly, it has been found that the chitosan derivative according to the present invention can solve at least one of the above-mentioned technical problems, and preferably all of the above-mentioned technical problems.

[0019] Surprisingly, it has been found that chitosan derivatives exhibiting a static charge (expressed as zeta potential) below a certain value can solve at least one, and preferably all, of the technical problems described or mentioned above, within a pH range approximately equal to the pH of the medium in which they are applied, and particularly at a pH equal to 7.5. In particular, such chitosan derivatives provide a means of limiting the immune response in subjects who have typically been administered chitosan derivatives or compositions containing chitosan derivatives via injection or implantation.

[0020] According to a second aspect, the present invention relates to chitosan derivatives having glucosamine units, N-acetyl-glucosamine units and carboxyl-substituted glucosamine units, said carboxyl-substituted chitosan having a zeta potential less than or equal to -10 mV, preferably less than or equal to -15 mV, as measured at pH 7.5.

[0021] In particular, fungal-derived chitosan derivatives have been found to solve at least one, and preferably all, of the technical problems described or mentioned above. Specifically, fungal-derived chitosan derivatives provide a means of limiting the immune response in subjects who have typically been administered chitosan derivatives or compositions containing chitosan derivatives via injection or implantation.

[0022] According to a first aspect, the present invention relates to a carboxyalkyl chitosan of fungal origin, having glucosamine units, N-acetylglucosamine units and carboxyalkyl-substituted glucosamine units; expressed as the number of moles of substituents relative to the total number of units, the carboxyalkyl chitosan preferably has a degree of carboxyalkyl substitution greater than 20%.

[0023] It also mentions chitosan derivatives or substituted chitosan derivatives.

[0024] Chitosan can be, for example, the substance referred to in Chemical Abstracts Service registration number (CAS number) 9012-76-4.

[0025] The chitosan used in this invention is advantageously derived from fungi, and preferably from ascomycetes. Ascomycetes Fungal mycelium, and especially derived from Aspergillus niger. Aspergillus niger ; and / or derived from basidiomycetes Basidiomycetes Fungi, especially shiitake mushrooms Lentinula edodes (Shiitake mushroom) and / or button mushroom Agaricus bisporus (Oyster mushroom). Preferably, the chitosan is derived from button mushrooms. Agaricus bisporus Chitosan is preferably very pure, meaning it is derived from fungal sources or has extremely low levels of impurities from the production process, and possesses a microbiological grade appropriate for its use as an implant or pharmaceutical composition. One method for preparing chitosan is described in patent WO 03 / 068824 (EP 1483299; US 7556946).

[0026] Generally, chitosan is placed in an aqueous suspension in the presence of sodium hydroxide, and the medium is then heated to a high temperature for a duration varying depending on the desired molecular weight. Purification is then performed by dissolving the chitosan in an acidic medium, followed by precipitation in an alkaline medium, and then washing and drying.

[0027] Preferably, the chitosan has a sufficient purity grade to be suitable for pharmaceutical use.

[0028] Advantageously, the chitosan is purified and then preferably dried. After purification, the method of the present invention may include the step of drying the carboxyl chitosan, followed optionally by grinding to obtain a powder. The carboxyl chitosan can be dried, for example, by evaporation of water, such as by spray drying, fluidized bed drying, or by heating under vacuum or atmospheric pressure, or even by freeze drying.

[0029] Carboxyalkyl chitosan can be dissolved in aqueous solutions, such as water of pharmaceutically acceptable quality suitable for injection or implantation, particularly for the human body.

[0030] The prepared chitosan can have a variety of different molecular weights, typically ranging from 10,000 to 500,000.

[0031] According to one variant, the average molecular weight is between 20,000 and 60,000.

[0032] According to another variant, the average molecular weight is 60,000 to 100,000.

[0033] According to another variant, the average molecular weight is between 100,000 and 120,000.

[0034] According to another variant, the average molecular weight is 120,000 to 150,000.

[0035] According to another variant, the average molecular weight is between 150,000 and 220,000.

[0036] According to another variant, the average molecular weight is 220,000 to 300,000.

[0037] According to another variant, the average molecular weight is between 300,000 and 500,000.

[0038] When chitosan is cross-linked, the molecular weight of the cross-linked polymer may actually be higher.

[0039] Chitosan can be hydrolyzed to reduce its molecular weight.

[0040] In this document, preferably, the average molecular weight is the viscosity-average molecular weight (Mv), calculated from the intrinsic viscosity according to the Mark-Houwink equation. This intrinsic viscosity is measured using a Ubbelohde capillary viscometer according to the method in European Pharmacopoeia Monograph 2.2.9. The flow time of the solution through a suitable capillary (Lauda, ​​e.g., a Ubbelohde 510 01 capillary with a diameter of 0.53 mm) is measured using an automatic viscometer I-Visc (Lauda), for example, according to the specification of Monograph 2.2.9, first at the initial chitosan concentration and then subsequently at multiple dilutions. The reduced intrinsic viscosity is inferred from each concentration. This reduced viscosity is plotted against temperature, and the value at a concentration of 0 is extrapolated to infer the intrinsic viscosity. For example, according to Equation 5, the reduced viscosity (h) of the i-th dilution... 降低 The concentration C (g / mL) of the i-diluted solution is plotted relative to the concentration C (g / mL), expressed in mL / g.

[0041] Equation 2: [h 降低 ] = (t1- t0) - (1 - C).

[0042] To calculate the average viscosity mass, the Mark-Houwink equation is used, where, as suggested by Rinaudo et al., constants k and α are used (see Int. J. Biol. Macromol, 15, 281, 1993), and one of the following three formulas is chosen based on the DA of chitosan:

[0043] Equation 3: Mv = ([h] / 0.082) (1 / 0.76) For 2% DA;

[0044] Equation 4: Mv = ([h] / 0.076) (1 / 0.76) For 10% DA (e.g., 11.5%).

[0045] Equation 5: Mv = ([h] / 0.074) (1 / 0.76) For 20% DA (e.g., 21%).

[0046] For intermediate DA values, linear interpolation is performed to calculate the average viscosity mass (Mv).

[0047] Preferably, the average molecular weight of the chitosan used is 120,000 to 150,000, or even 150,000 to 220,000, or even 220,000 to 300,000, or even greater than 300,000, and typically as high as 500,000.

[0048] The final molecular weight of carboxyalkyl chitosan can also be measured: for example, by measuring the intrinsic viscosity using capillary viscosity method, thereby inferring its average molecular weight (Mw) (by pre-determining the parameters K and α of the carboxyalkyl chitosan), or by chromatographic methods, such as gel permeation chromatography.

[0049] Typically, in the carboxyalkyl chitosan according to the invention, the glucosamine unit is a D-glucosamine unit (D-glucosamine unit, N-acetyl-D-glucosamine unit, and at least one of the D-glucosamine unit and N-acetyl-D-glucosamine unit is substituted).

[0050] According to one variant, the substituted chitosan has only the substitution of D-glucosamine units.

[0051] According to another variant, the substituted chitosan has substitutions for both D-glucosamine and N-acetyl-D-glucosamine units, wherein, according to one variant, the carboxyl group is covalently bonded only to the amino group of chitosan, or according to another variant, the carboxyl group is covalently bonded to both the amino and hydroxyl groups of chitosan.

[0052] Replacement is usually only partial; not all units need to be replaced.

[0053] According to one embodiment, the molar number of D-glucosamine units is expressed relative to the molar number of all units of the substituted chitosan (substituted or unsubstituted D-glucosamine and substituted or unsubstituted N-acetyl-D-glucosamine units), wherein the degree of substitution of the D-glucosamine units is from 30% to 250%.

[0054] According to one embodiment, the degree of substitution of the carboxyl alkyl group is greater than 50%, expressed as the number of moles of substituents relative to the total number of units.

[0055] According to one embodiment, the molar number of D-glucosamine units is expressed relative to the molar number of all units of the substituted chitosan (substituted or unsubstituted D-glucosamine and substituted or unsubstituted N-acetyl-D-glucosamine units), and the degree of substitution of the D-glucosamine units ranges from 50% to 200%, and more preferably greater than 70%.

[0056] According to one embodiment, the degree of substitution of the carboxyl alkyl group is less than 80%, expressed as the number of moles of substituents relative to the total number of units.

[0057] Typically, substitution is achieved through covalent bonding.

[0058] According to one variant, carboxyl chitosan is N,O-carboxyl chitosan. The proportion of units at the O-position (O3 or O6 of glucosamine and / or N-acetyl-glucosamine units) and / or the N-position (N-position of glucosamine units) substituted with carboxyl groups varies. Therefore, the degree of substitution can be greater than 100%.

[0059] Advantageously, the degree of substitution (DS) and degree of acetylation (DA) of carboxyl chitosans were measured by solid-state carbon-13 nuclear magnetic resonance (NMR) spectroscopy using a Bruker spectrometer (AvanceIII HD 400 MHz) equipped with a PH MAS VTN 400SB BL4 NP / H probe. For example, spectra were recorded at ambient temperature with scan numbers ranging from 64 to 256 and relaxation times from 1 to 8 seconds. The area of ​​the carbon signal was determined after deconvolution. The carbons considered were: “acetyl CH3” (the methyl carbon of the acetyl group in substituted or unsubstituted N-acetyl-glucosamine units), “C1” (the carbon at position 1 of glucosamine and N-acetyl-glucosamine units), and “C=O” (the carbonyl carbon of the carboxymethyl substituent and the carbonyl carbon of the acetyl group in substituted or unsubstituted N-acetyl-glucosamine units). To determine the degree of substitution of a given carboxyl chitosan, the carbon-13 NMR spectrum of the carboxyl chitosan precursor chitosan also needed to be recorded. Based on the spectrum of the precursor chitosan, the "CSU ratio" was calculated, which is the ratio of the signal area of ​​the "acetyl CH3" group (the methyl carbon of the acetyl group in the N-acetyl-D-glucosamine unit) to the signal area of ​​the "C = O" group (the carbonyl carbon of the acetyl group in the N-acetyl-D-glucosamine unit). The DA of carboxyalkyl chitosan was calculated according to Equation 1, and the DS was calculated according to Equation 2, where I represents the signal area of ​​the carbon atoms considered.

[0060] Formula 1:

[0061]

[0062] Formula 2:

[0063]

[0064] The DA and DS of carboxyl chitosan can be determined by other known methods, such as by proton NMR in an aqueous medium using magnetic resonance spectroscopy, or by the method described by Liu et al. (see: Carb Polym 137, 600, 2016), for example by pre-hydrolyzing the carboxyl chitosan by adding a concentrated solution of deuterated hydrochloric acid prior to analysis.

[0065] If an alternative NMR method exists that is more advantageous for reliably estimating the degree of substitution, that alternative method should be used. The above method will be adapted by those skilled in the art with respect to sample preparation and the signal to be integrated, particularly based on the resolution, robustness, and proton position of the signal used to calculate the degree of substitution.

[0066] The degree of carboxylation of chitosan can advantageously be 20% to 250%, preferably 50% to 200%, for example 70% to 170%, expressed as the number of moles of carboxyl groups relative to the total number of units.

[0067] According to one variation, expressed in terms of the number of moles of carboxyl groups relative to the total number of units, the degree of carboxylation of chitosan can advantageously be from 40% to 130%, for example from 70% to 130%.

[0068] The degree of substitution of chitosan is generally related to the mass ratio of the starting reagent to chitosan at the start of the reaction. For carboxylating agents, acyl chlorides (or their salts, such as sodium monochloroacetate) can be mentioned, such as carboxylating agents containing one or more carboxymethyl, carboxyethyl, carboxypropyl, carboxybutyl, or other carboxylating groups.

[0069] According to one variation, the present invention relates to carboxyl chitosan, wherein the alkyl portion of the carboxyl group is a straight-chain or branched C1-C5 alkyl group.

[0070] According to one variation, the present invention relates to carboxymethyl chitosan.

[0071] According to this variant, the substituted chitosan is N-carboxyalkylated chitosan.

[0072] According to this variant, the substituted chitosan is O-carboxyalkylated chitosan.

[0073] According to this variant, the substituted chitosan is an N-carboxyalkylated and O-carboxyalkylated chitosan.

[0074] Advantageously, the zeta potential measured at pH 7.5 is less than or equal to -18 mV.

[0075] Advantageously, carboxyl chitosan has a zeta potential of less than or equal to -22 mV, preferably less than or equal to -24 mV, when measured at pH 7.5.

[0076] According to a specific variant, the substituted chitosan preferably has an average molecular weight of 150,000 to 220,000 and a degree of substitution of 50% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0077] According to another specific variant, the substituted chitosan has an average molecular weight of 120,000 to 150,000 and a degree of substitution of 70% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0078] According to a specific variant, the substituted chitosan preferably has an average molecular weight of 220,000 to 300,000 and a degree of substitution of 70% to 200%, where the molecular weight preferably represents the molecular weight before substitution.

[0079] According to another specific variant, the substituted chitosan has an average molecular weight of 220,000 to 300,000 and a degree of substitution of 50% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0080] According to another specific variant, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution of 50% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0081] According to another specific variant, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution of 70% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0082] According to a specific variant, the substituted chitosan preferably has an average molecular weight of 120,000 to 150,000 and a degree of substitution of 20% to 50%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0083] According to another specific variant, the substituted chitosan has an average molecular weight of 220,000 to 300,000 and a degree of substitution of 20% to 50%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0084] According to another specific variant, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution of 20% to 50%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0085] According to a specific variant, the substituted chitosan has a degree of substitution of 20% to 80%, preferably 40% to 60%, and a degree of acetylation of 20% to 80%, preferably 30% to 75%.

[0086] According to a specific variant, the substituted chitosan has a degree of substitution of 50% to 200%, preferably 70% to 200%, and a degree of acetylation of 20% to 80%, preferably 30% to 75%.

[0087] According to a specific variant, the substituted chitosan has a degree of substitution of 50% to 200%, preferably 70% to 200%, and a degree of acetylation of 20% to 50%, preferably 20% to 40%.

[0088] Depending on a specific variant, the substituted chitosan has a degree of substitution of 50% to 200%, preferably 70% to 200%, and a degree of acetylation of 50% to 75%.

[0089] According to another specific variant, the substituted chitosan has a degree of substitution of 90% to 200%, preferably 90% to 150%, and a degree of acetylation of 20% to 80%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0090] According to a specific variant, the substituted chitosan has a degree of substitution of 90% to 200%, preferably 90% to 150%, and a degree of acetylation of 20% to 50%, preferably 20% to 40%.

[0091] Depending on a specific variant, the substituted chitosan has a degree of substitution of 90% to 200%, preferably 90% to 150%, and a degree of acetylation of 50% to 75%.

[0092] According to a specific variant, the substituted chitosan preferably has an average molecular weight of 220,000 to 300,000, a degree of substitution of 90% to 200%, preferably 90% to 150%, and a degree of acetylation of 50% to 75%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0093] According to one variant, carboxyl chitosan is cross-linked. Therefore, several chitosan chains can be cross-linked, for example, by reacting with a cross-linking agent, such as those used for cross-linking polysaccharides, including genipin, butyl diglycidyl ether, glutaraldehyde, epichlorohydrin, 1-bromo-3,4-epoxybutane, 1-bromo-4,5-epoxypentane, 1-chloro-2,3-cyclothiopropane, 1-bromo-2,3-cyclothiopropane, 1-bromo-3,4-cyclothiobutane, 1-bromo-4,5-cyclothiopentane, 2,3- Dibromopropanol, 2,4-dibromobutanol, 2,5-dibromopentanol, 2,3-dibromopropanethiol, 2,4-dibromobutanethiol and 2,5-dibromopentane-thiol epichlorohydrin, 2,3-dibromopropanol, 1-chloro-2,3-cyclothiopropane, dimethylaminopropylcarbodiimide, oxidized dextran, gallic acid, epigallocatechin gallate, curcumin, tannic acid or even diisocyanate compounds, such as hexamethylene diisocyanate or toluene diisocyanate.

[0094] Cross-linked carboxyl chitosan can have very high molecular weights.

[0095] By substituting chitosan, carboxyl-based chitosan solutions can be prepared that dissolve in aqueous solutions with a wide pH range, whereas unsubstituted chitosan is only soluble at pH values ​​below 5.5 to 6.5. Therefore, due to the altered solubility profile caused by the presence of carboxyl groups, carboxyl-based chitosans possess the ability to dissolve at a variety of pH values, particularly at physiological pH values ​​or at pH values ​​of physiological fluids altered by pathological factors (e.g., inflammatory pathology). It is known that the pH of physiological fluids such as synovial fluid, aqueous humor, vitreous fluid, and tears can vary significantly between individuals due to various factors such as age and pathology. Therefore, it is advantageous for carboxyl-based chitosans to remain soluble over a wide pH range (e.g., 6.0 to 8.5, or even 5.0 to 8.5, or even 4.5 to 8.5).

[0096] According to one embodiment, the substituted chitosan formulation has an osmolality of 100 to 700 mosm / kg, preferably 200 to 500 mosm / kg.

[0097] Advantageously, the permeability of the substituted chitosan formulation is 250 to 400 mosm / kg, preferably 275 to 325 mosm / kg.

[0098] According to one variant, the substituted chitosan formulation has joint-compatible permeability.

[0099] According to one variant, the substituted chitosan formulation has permeability compatible with the eye or the inner surface of the eye.

[0100] Preferably, the permeability of the substituted chitosan formulation is 250 to 400 mosm / kg, and more specifically 250 to 380 mosm / kg.

[0101] The term "water-soluble" should be understood to mean that no visible turbidity is observed when carboxyl chitosan is placed in an aqueous solution. More specifically, the solubility (i.e., the absence of any turbidity) of an aqueous solution or buffer solution (e.g., phosphate buffer) of carboxyl chitosan at a concentration of, for example, 1% (m / m) is confirmed by UV-Vis spectroscopy at a wavelength of 500 nm with a density less than 0.5, preferably less than 0.2, relative to a reference cell containing only the aqueous solvent used for the measurement sample and without substituted chitosan. Another method is visual inspection according to European Pharmacopoeia 2.9.20. When the chitosan is not sufficiently substituted, the composition does not remain soluble within a satisfactory pH range (e.g., pH 6.0 to 8.5) at ambient temperature.

[0102] The degree of acetylation (DA) of chitosan is determined by potentiometric titration, as described, for example, in patent applications WO2017009335 and WO 2017009346. Alternatively, the DA of chitosan can be measured by other known methods, such as proton nuclear magnetic resonance (NMR) spectroscopy, solid-state carbon-13 NMR, and infrared spectroscopy.

[0103] Advantageously, the carboxyl chitosan has a degree of acetylation of 5% to 80%, which is expressed as the molar number of N-acetyl-glucosamine units relative to the total number of units. The degree of acetylation is also expressed as the number of N-acetyl-D-glucosamine units relative to the total number of N-acetyl-D-glucosamine and D-glucosamine units present.

[0104] Advantageously, carboxyalkyl chitosan has a degree of acetylation of 40% to 80% relative to the total number of moles of N-acetyl-glucosamine units.

[0105] According to one variant, the degree of acetylation ranges from 5% to 20%.

[0106] According to one variant, the degree of acetylation ranges from 15% to 25%.

[0107] According to one variant, the degree of acetylation ranges from 20% to 45%.

[0108] According to one variant, the degree of acetylation is 20% to 30%.

[0109] According to one variant, the degree of acetylation ranges from 25% to 40%.

[0110] According to one variant, the degree of acetylation is 40% to 50%.

[0111] According to one variant, the degree of acetylation is 50% to 60%.

[0112] According to one variant, the degree of acetylation is 60% to 75%.

[0113] The degree of acetylation is determined by 13-carbon NMR or proton NMR using the same method as for measuring DS. Carboxyalkyl chitosans advantageously possess a controlled degree of acetylation. The term "chitosan with a controlled degree of acetylation" is understood to refer specifically to a product whose degree of acetylation, i.e., the ratio of N-acetylglucosamine units, can be controlled in a controlled manner through the acetylation reaction.

[0114] According to one variation, the composition according to the invention can be in the form of a solution and does not gel due to temperature changes (it cannot gel thermally).

[0115] According to one variation, the rheological properties of the solution according to the invention can vary with temperature, but do not undergo a sol-gel transition. The rheological properties of the solution according to the invention can be characterized in particular by the elastic modulus (G′) and / or loss modulus (G″) or even the complex modulus G*.

[0116] According to one variation, the rheological properties of the solution according to the invention are substantially constant regardless of temperature.

[0117] According to one variation, the composition according to the invention can be in solution form and is capable of thermogelation.

[0118] According to one variation, the composition according to the invention can be in gel form and cannot be thermally gelled.

[0119] Therefore, according to one variation, the present invention can prepare compositions capable of thermal gelation, which are fluid at temperatures below the operating temperature (typically below physiological temperatures, e.g., below 37°C), but in gel form at the operating temperature (typically at physiological temperatures, e.g., 37°C), and have a permeability suitable for the intended use at neutral pH (pH 7) or physiological pH (e.g., between 7 and 8.5), for example, it is physiological permeability.

[0120] According to one variant, compositions capable of thermogelling exhibit a thermally reversible sol-gel transition.

[0121] Advantageously, the present invention enables the provision of compositions having a low concentration of substituted chitosan.

[0122] Advantageously, the concentration (m / m) of carboxyalkyl chitosan relative to the total mass of the composition is less than 10% by mass, for example less than or equal to 5% by mass.

[0123] According to one variant, the concentration (m / m) of chitosan relative to the total mass of the composition is less than 4% by mass, for example less than or equal to 3% by mass, or even less than or equal to 2% by mass.

[0124] Advantageously, the compositions of the present invention may also comprise a non-substituted chitosan biopolymer. According to an advantageous variant, the biopolymer is an oxidized or unoxidized, covalently cross-linked or uncross-linked polysaccharide, such as hyaluronic acid or sodium hyaluronate.

[0125] Hyaluronic acid can have molecular weights up to 5 million Da. The molecular weight of hyaluronic acid can be reflected by its intrinsic viscosity or its dynamic viscosity in solution. Hyaluronic acid can have a molecular weight of 1 m³. / kg to 4 m³ / The density is measured in kg, and can be characterized, for example, as having a low density (e.g., about 1 m³). / kg to 2 m³ / kg) or high (e.g., about 2 m³) / kg to 4 m³ / The molecular weight (kg).

[0126] Advantageously, the concentration (m / m) of hyaluronic acid relative to the total mass of the composition is less than 4% by mass, for example less than or equal to 3% by mass, or even less than or equal to 2% by mass.

[0127] According to a specific variation, the concentration of hyaluronic acid, expressed relative to the mass of the final composition, is less than 1.9% by mass (m / m). Advantageously, the concentration of hyaluronic acid, expressed relative to the mass of the final composition, is from 0.5% by mass to 1.5% by mass (m / m). According to a specific variation, the concentration of hyaluronic acid, expressed relative to the mass of the final composition, is approximately 0.9% by mass, 1.0% by mass, 1.1% by mass, 1.2% by mass, 1.3% by mass, and 1.5% by mass (m / m).

[0128] The ratio of chitosan to hyaluronic acid can be 5% to 95%, for example 10% to 90%, or even 30% to 70% of substituted chitosan, and 5% to 95%, for example 10% to 90%, or even 30% to 70% of hyaluronic acid, respectively, with the percentages expressed based on the dry weight of the substituted chitosan / the dry weight of the hyaluronic acid. According to one variation, the ratio between chitosan and hyaluronic acid is 1 / 1 (i.e., 50% chitosan and 50% hyaluronic acid). According to another variation, the ratio between chitosan and hyaluronic acid is 1.5 / 0.5 (i.e., 75% chitosan and 25% hyaluronic acid).

[0129] According to one variant, hyaluronic acid can be cross-linked between different chains of hyaluronic acid.

[0130] The present invention also relates to a method for preparing carboxyalkyl chitosan.

[0131] According to one variation, the method for preparing carboxyalkyl chitosan according to the present invention includes the preparation of fungal-derived chitosan, reacetylation of chitosan, and carboxylation of the reacetylated chitosan. Therefore, the present invention relates to a reacetylated chitosan or a reacetylated carboxyalkyl chitosan.

[0132] Therefore, according to one embodiment, chitosan can be dissolved in an aqueous medium, preferably in a slightly acidic (e.g., pH 6) aqueous medium. Acetic anhydride can be added to the chitosan solution once or multiple times. A basic reagent, such as sodium hydroxide and / or urea, is then added. An alkylating agent, such as sodium monochloroacetate (i.e., sodium chloroacetate salt) or chloroacetic acid, is then added. Subsequently, the substituted chitosan is purified, recovered, and dried.

[0133] According to one variation, the method for preparing carboxyalkyl chitosan according to the invention comprises the preparation of chitosan, the carboxylation of the chitosan, and then the reacetylation of the carboxyalkylated chitosan. Advantageously, this method allows for precise control of the final degree of acetylation of the carboxyalkyl chitosan, particularly achieving a high degree of acetylation, for example, above 40%. Therefore, the present invention relates to chitosan that is first reacetylated and then carboxylated, or to reacetylated carboxyalkyl chitosan.

[0134] According to one variation, a method for preparing carboxyalkyl chitosan according to the invention comprises the preparation of fungal-derived chitin, carboxylation of the chitin, and optional reacetylation of the carboxylated chitin to obtain carboxyalkyl chitosan according to the invention.

[0135] According to one variation, the method for preparing carboxyalkylated chitosan according to the invention comprises the preparation of fungal-derived chitin, deacetylation of chitin, carboxylation of chitin, and optional reacetylation of carboxyalkylated chitin to obtain carboxyalkylated chitosan according to the invention.

[0136] According to one embodiment, the carboxylation step includes an alkalinization step of alkalizing the hydroxyl groups of chitosan to promote, for example, high substitution at the N position of the glucosamine unit and at the O positions of the glucosamine and N-acetyl-glucosamine units.

[0137] According to one embodiment, the method of the present invention includes an alkalization step, which comprises dispersing chitosan in an alcoholic solution (such as isopropanol) in the presence of an alkaline reagent (such as sodium hydroxide) and stirring at a temperature of at least -32°C and preferably at a maximum of 15°C for at least one hour. An alkylating agent (such as monochloroacetic acid) is then added to the suspension. Subsequently, the substituted chitosan is purified, recovered, and then dried.

[0138] According to one variation, the method for preparing carboxyalkylated chitosan according to the present invention includes the preparation of chitosan, the carboxylation of the chitosan, and the subsequent reacetylation of the carboxyalkylated chitosan.

[0139] According to one embodiment, the carboxylation step does not include an alkalization step.

[0140] The reacetylation step of carboxyalkyl chitosan may, for example, include adding acetic anhydride to the carboxyalkyl chitosan solution once or multiple times.

[0141] To collect purified carboxyl chitosan, the final steps of purification, filtration, and drying are performed according to known methods, such as by cycling the precipitation and dissolution or by dialysis, with the aim of obtaining carboxyl chitosan with the desired purity, which typically depends on the intended application.

[0142] According to one variation, in the reacetylation step, the ratio (volume / mass) of acetic anhydride to chitosan is 0.1 to 10, preferably 0.1 to 2.

[0143] According to one variation, in the carboxylation step, the mass ratio of carboxylating agent to chitosan is 1 to 50, preferably 2.5 to 25.

[0144] According to one variation, the mass ratio of the reagent (i.e., the alkylating agent) to the basic reagent is used to obtain the required degree of substitution, which is necessary and sufficient for the formation of carboxyl groups. For example, the mass ratio of the alkylating agent to the basic reagent is greater than 1 and less than 10, preferably from 1.4 to 3.3.

[0145] The present invention also relates to a method for preparing the composition according to the present invention.

[0146] Depending on a variant, the method typically includes:

[0147] The substituted chitosan is dissolved in an aqueous solution, preferably in a buffer solution, and more preferably in a buffer solution with a pH of 6.2 to 8.5, and more preferably 6.5 to 7.5.

[0148] Optionally, the pH may be adjusted to the desired pH, typically the physiological pH, by adding a buffer, acid, or base, for the target application.

[0149] Optional addition of other excipients, such as reducing sugars, such as sorbitol or mannitol;

[0150] The final permeability of the composition may be adjusted optionally.

[0151] Advantageously, the method further includes a further filling step, filling the injection or implantation device (e.g., a syringe) with carboxymethyl chitosan or a composition containing therein. Advantageously, the injection device (e.g., the syringe) may subsequently undergo a steam sterilization process. The device (e.g., the syringe) is then preferably packaged aseptically. It may also be a bag, vial, or bottle for instilling a carboxymethyl chitosan solution.

[0152] Advantageously, chitosan with sufficient purity for the intended application is used.

[0153] Advantageously, the method further includes a further filling step, filling the injection, implantation, or infusion device (e.g., a syringe) with the composition according to the invention. Advantageously, the injection device (e.g., the syringe) may subsequently undergo a steam sterilization process. The device (e.g., the syringe) is then preferably packaged aseptically.

[0154] According to one variation, the carboxyalkyl chitosan according to the invention is sterilized by filtration and / or steam sterilization before being filled into an injection, implantation, or infusion device (such as a syringe or vial).

[0155] Advantageously, chitosan with sufficient purity for the intended application is used.

[0156] More specifically, this invention relates to injectable compositions comprising carboxyl chitosan according to the invention.

[0157] The present invention also relates to pharmaceutical compositions comprising at least one carboxyl chitosan as defined in the present invention.

[0158] According to one variant, carboxyl chitosan or a composition containing carboxyl chitosan is used as an injectable, implantable or infusion-suitable pharmaceutical composition, or an injectable, implantable or infusion-suitable medical device.

[0159] The present invention also covers carboxyl chitosan or compositions comprising it in a dry form, particularly a freeze-dried form. The freeze-dried product can be (re)dispersed, and preferably dissolved, before use.

[0160] More specifically, the present invention relates to the use of compositions according to the invention for therapeutic treatment, such as by injecting the compositions via subcutaneous, intradermal, intraocular, or intra-articular routes, for example for repairing, regenerating, or filling at least one body tissue requiring repair or filling.

[0161] Advantageously, according to one implementation, a mouse air sac model is used. -pouch model), 24 hours after injection, the carboxyl chitosan according to the invention exhibits immunocompatibility with fungal-derived carboxymethyl chitosan formulations, expressed as a white blood cell count of less than 10 × 10⁻⁶. 6 Cells / mL, preferably less than 8 × 10⁻⁶ 6 Cells / mL.

[0162] Advantageously, according to one embodiment, using a mouse airbag model, 24 hours after injection, the carboxyalkyl chitosan according to the invention exhibited immunocompatibility with fungal-derived carboxymethyl chitosan formulations, and was expressed as a concentration of interleukin 1β (IL1-β) below 10 × 10⁻⁶. -9 g / mL, preferably below 5×10 g / mL -9 g / mL.

[0163] Advantageously, according to one embodiment, using a mouse airbag model, 24 hours after injection, the carboxyalkyl chitosan according to the invention exhibited immunocompatibility with a fungal-derived carboxymethyl chitosan formulation, and the concentration expressed as chemokine CXC motif ligand 1 (KC / CXCL1) was less than 50 × 10⁻⁶. -9 g / mL, preferably less than 30 × 10 g / mL -9 g / mL.

[0164] Advantageously, according to one embodiment, using a mouse airbag model, 24 hours after injection, the carboxyalkyl chitosan according to the invention exhibited immunocompatibility with a fungal-derived carboxymethyl chitosan formulation, and the concentration of tumor necrosis factor α (TNF-α) was less than 150 × 10⁻⁶. -9 g / mL, preferably less than 125 × 10 g / mL -9 g / mL.

[0165] Advantageously, according to one embodiment, using a mouse airbag model, 24 hours after injection, the carboxyalkyl chitosan according to the invention exhibited immunocompatibility with a fungal-derived carboxymethyl chitosan formulation, and the concentration expressed as chemokine CXC motif ligand 1 (KC / CXCL1) was less than 50 × 10⁻⁶. -9 g / mL, preferably less than 30 × 10 g / mL -9 g / mL.

[0166] Advantageously, according to one embodiment, using a mouse airbag model, 24 hours after injection, the carboxyalkyl chitosan according to the invention exhibits immunocompatibility with a fungal-derived carboxymethyl chitosan formulation, while meeting the maximum limits shown above in terms of leukocyte count and concentrations of IL1-β, KC / CXCL1 and TNF-α.

[0167] The white blood cell count and the concentrations of IL1-β, KC / CXCL1, and TNF-α were measured according to the protocol shown in the examples.

[0168] Advantageously, according to one embodiment, and according to the measurement scheme in Example 6, the carboxyl chitosan according to the invention has a half-life of more than 500 minutes at 37°C in the presence of a mixture of lysozyme and hyaluronidase.

[0169] Advantageously, according to one embodiment, particularly in intra-articular applications, the carboxyalkyl chitosan according to the invention has a coefficient of friction (COF0) of less than 5, preferably less than or equal to 4 (according to the scheme in Example 9).

[0170] The present invention particularly relates to compositions according to the invention for treatment in the fields of rheumatology, ophthalmology, cosmetic medicine, plastic surgery, internal medicine and surgery, for example for preventing postoperative tissue adhesions in cosmetic surgery or dermatological surgery.

[0171] According to one variant, the body tissue is selected from one or more layers of tissue belonging to the vocal cords, muscles, ligaments, tendons, cartilage, sex organs, bones, joints, eyes, dermis, epidermis, skin, mesoderm, or any combination thereof; more specifically, the body tissue is selected from tissue belonging to the eyes, dermis, and joints.

[0172] The present invention also relates to compositions according to the invention for therapeutic treatment of dry eye, corneal injury or damage, or joint inflammation.

[0173] Furthermore, the present invention also relates to the application of the compositions according to the invention by instillation onto the ocular surface to prevent or resist corneal damage or injury, or dry eye syndrome, particularly for lubrication or regeneration of the ocular surface.

[0174] Therefore, the present invention also relates to eye drop compositions comprising carboxyalkyl chitosan as defined in the present invention.

[0175] According to one variant, the subject had inflammation (such as osteoarthritis).

[0176] The present invention relates more particularly to compositions according to the invention for treating arthropathy (osteoarthritis), arthritis, or repairing cartilage defects, for example by injection into the synovial cavity or by implantation into a cartilage defect site.

[0177] More specifically, the present invention relates to a medical device, such as a medical implant, characterized in that it comprises or is composed of a composition according to the invention.

[0178] Therefore, according to a preferred variant, the present invention relates to a medical device comprising a cavity containing carboxyalkyl chitosan in a dried form, particularly a freeze-dried form, and one or more other cavities optionally containing one or more active products, additives or excipients.

[0179] The compositions according to the invention may further contain one or more additives or excipients capable of adjusting the properties of the composition.

[0180] The present invention also relates to the use of the compositions according to the invention in therapeutic treatments, such as instilling or injecting the compositions via subcutaneous, intradermal, ocular, intraocular, or intra-articular routes, for example, for repairing or filling at least one body tissue requiring repair or filling.

[0181] The present invention also relates to the use of the compositions according to the invention in a treatment method for osteoarthritis, or in the repair of cartilage defects, for example by injection into synovial fluid or by mixing with blood and implanting into cartilage.

[0182] The present invention also relates to the use of compositions according to the invention for therapeutic or cosmetic body care methods provided by filling the dermis (“dermal filling”). This particularly includes, for example, subcutaneous or intradermal injection of compositions according to the invention.

[0183] This invention also relates to a treatment method using the compositions according to the invention for topical treatment of the skin via multiple injections through an intradermal route. Such compositions are generally used in dermatology to achieve treatments for cosmetic and body shaping purposes. For example, the aim of this method is to plump up the skin, thereby eliminating the appearance of wrinkles (treatment of wrinkles and / or fine lines). This treatment can be performed on subjects who wish to revitalize their skin.

[0184] The present invention also relates to the use of compositions according to the invention in therapeutic applications, wherein the composition is a thickening agent. For example, in this case, it relates to injecting the compositions of the invention into intra-articular sites, particularly to limit friction on the cartilage surface at the joint.

[0185] This invention also relates to the use of compositions according to the invention as cell carriers for one or more cell types, and / or one or more active agents. These may be active agents from a pharmaceutical or biological perspective. The compositions of the invention are practically compatible with the presence of cells, preferably living cells. Among the living cells of interest, examples may be mentioned such as: chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligament fibroblasts (ligaments), skin fibroblasts (skin), tendon cells (tendons), myofibroblasts (muscle), mesenchymal stem cells, erythrocytes (blood), and keratinocytes (skin). The compositions of the invention can also be contemplated as therapeutic carriers for targeted delivery and / or controlled release of at least one therapeutic agent.

[0186] According to one variation, blood, plasma, platelet lysate, platelet-rich plasma, or any biological fluid may be added together with the composition of the present invention, thereby, for example, improving the performance of the product.

[0187] According to one variation, the composition according to the invention is formulated in a solid form (e.g., a membrane or porous foam) that dissolves once implanted.

[0188] According to one variant, the composition is formulated into an atomizable (sprayable) composition.

[0189] The present invention also relates to the use of the compositions according to the invention in treatment or cosmetic care methods for one or more tissues or organs that have been affected by high temperatures, such as in cases of burns.

[0190] The present invention also relates to a cartilage treatment method for repairing cartilage using compositions according to the invention (e.g., by implanting cartilage defects to promote the regeneration of such cartilage).

[0191] The present invention also relates to a treatment method for preventing postoperative tissue adhesions using the composition according to the invention: the product is applied to the tissue at the end of surgery (e.g., gynecological surgery, abdominal surgery, etc.).

[0192] The present invention also relates to compositions for use as artificial synovial fluids, comprising carboxyalkyl chitosan according to the present invention.

[0193] The compositions according to the invention, by maximizing their lubricating capacity to reduce friction in joints and / or their shock-absorbing properties (identifiable by their elastic modulus G'), while being readily injectable into a syringe or injected into the human or animal body, offer the possibility of mimicking healthy synovial fluid or improving healthy or defective synovial fluid. 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 100 Pa.

[0194] According to one variation, carboxyl chitosan or a composition containing carboxyl chitosan is injected in solution form. Advantageously, according to this variation, carboxyl chitosan or a composition containing carboxyl chitosan is readily injected at ambient temperature through a fine needle (e.g., a needle with a diameter of 21). The term "readily" should be understood to mean that a force preferably applied to such a syringe to make the composition according to the invention flow through a 21-gauge needle is less than 50 Newtons, preferably less than 20 Newtons.

[0195] The present invention also relates to compositions for use as artificial tears, comprising carboxyl chitosan according to the invention.

[0196] In biomedical applications, the required ranges for permeability and pH are typically as follows:

[0197] Osmotic degree:

[0198] Isotonic with plasma: 285 mosm / kg to 295 mosm / kg;

[0199] Isotonic 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";

[0200] 280 mosm / kg to 350 mosm / kg.

[0201] pH:

[0202] Physiological pH is usually above 6.8, especially above 7.0, and particularly 7.4 (such as plasma or synovial fluid).

[0203] The pH of blood plasma is typically 7.4. Generally, according to "J Bone Joint Surg Br, 41-B (2), 388-400, 1959", the pH of synovial fluid is typically 7.768 + / - 0.044; or according to "Acta Orthop Scand 634–641, 1969", or further according to "Clin Rheumatol 25, 886–888, 2006", the pH of synovial fluid is typically 7.3.

[0204] It is generally believed that in cases of osteoarthritis or arthritis, the pH of the synovial fluid is lower than that of healthy synovial fluid.

[0205] Therefore, the present invention relates to a mixture of synovial fluid and a composition according to the invention, wherein, for example, (i) carboxyl chitosan or a composition comprising therein and (ii) synovial fluid, the volume ratio is 20 / 80 to 80 / 20 (v / v), for example, 50 / 50 (v / v).

[0206] Advantageously, the compositions according to the invention are sterile. Most advantageously, the compositions according to the invention are sterilized by increasing the temperature, preferably in an autoclave.

[0207] According to one variation, the composition of the present invention is transparent or translucent.

[0208] The term "semi-transparent" is understood to mean that the object is recognizable when the composition is placed between the observer's eye and the object. The term "transparent" is understood to mean that the alphanumeric symbol is recognizable when the composition is placed between the observer's eye and the observed alphanumeric character. Generally, this evaluation is performed with a composition thickness of approximately 1 cm. Visual inspection can also be performed according to the method in European Pharmacopoeia Monograph 2.9.20. The optical density of the composition can also be measured, for example, by UV-Vis spectroscopy at 500 nm, ensuring that the optical density is less than 0.5, preferably 0.2, compared to the reference solvent.

[0209] According to one variation, the compositions of the present invention are not milky white or are only very slightly opalescent.

[0210] The term "milky white" is understood to mean that the solution produces visible light diffraction, for example by visual inspection according to methods such as those in European Pharmacopoeia Monograph 2.9.20, and by comparison with reference solutions in the European Pharmacopoeia that have different degrees of milky whiteness. According to one variation, the compositions of the present invention are colorless, i.e., in particular, the observer does not assign any particular color to the composition when observing it with the naked eye. According to another variation, the milky whiteness is less than the maximum permissible level for the intended application.

[0211] This invention relates particularly to articles or packages, preferably sterile articles or packages, comprising one or more drip or injection devices prefilled with compositions according to the invention. These are typically devices capable of dispensing products, in the form of drops or prefilled syringes.

[0212] Advantageously, the compositions of the present invention can be sterile. Therefore, the present invention relates to a sterile carboxyl chitosan. Thus, the carboxyl chitosan is sterile, particularly for applications requiring it.

[0213] According to one variation, the compositions of the present invention are sterilized by steam sterilization, for example by raising the temperature to above 100°C, preferably above 120°C, such as 121°C to 138°C, in an autoclave for a sufficient sterilization time, typically 15 to 20 minutes. According to another variation, the compositions can be sterilized by filtration using a filter suitable for this purpose, such as a filter with pore sizes less than or equal to 0.2 μm.

[0214] Advantageously, according to a preferred embodiment, the intrinsic viscosity loss of carboxyalkyl chitosan is less than 40% during steam sterilization.

[0215] This invention also covers the compositions of the invention in dried form, particularly the compositions of the invention in freeze-dried form.

[0216] The freeze-dried composition can be (re)dispersed, in particular, before use.

[0217] This invention also covers therapeutic treatment methods, including injecting compositions according to the invention.

[0218] The present invention also covers the use of the compositions according to the invention in the preparation of pharmaceutical compositions, particularly in pharmaceutical compositions for therapeutic purposes, as more specifically defined in the present invention.

[0219] This invention also covers methods for cosmetic and body care, in other words, non-therapeutic methods, including injecting compositions according to the invention. This relates to, for example, filling wrinkles or, for example, filling one or more damaged visible tissue areas after an accident or surgical intervention, for cosmetic purposes.

[0220] Tissues are collections of similar cells from the same source that aggregate into a functional group, that is, perform the same function. Among tissues, epithelial tissue, connective tissue, muscle tissue, and nerve tissue are worth mentioning.

[0221] The term "composition according to the invention" or equivalent terms are to be understood as referring to the composition as defined in this invention, including any composition independently or in any combination thereof according to any variation, specific or particular embodiment, and including compositions according to preferred features.

[0222] Other objects, features, and advantages of the invention will become apparent to those skilled in the art after reading the exemplary description of embodiments given by way of illustration only and not by way of limiting the scope of the invention.

[0223] These embodiments form part of the present invention, and any feature that is novel relative to any prior art is part of the present invention in terms of its function and integrity, when the specification (including the embodiments) is considered as a whole.

[0224] Therefore, each embodiment is general in scope.

[0225] On the other hand, in the embodiments, unless otherwise stated, all percentages are expressed in mass, and unless otherwise stated, temperatures are expressed in degrees Celsius; and unless otherwise stated, pressures are in atmospheres.

[0226] Example

[0227] The substituted chitosan precursor according to the invention has a viscosity-average molecular weight (determined by capillary viscometer) and degree of acetylation (DA, the proportion of N-acetyl-D-glucosamine units determined by potentiometric titration) within the ranges shown in Table 1. The molecular weight of the chitosan can also be defined as the dynamic viscosity of a 1% (m / m) chitosan solution in 1% (v / v) acetic acid, measured by a rotor viscometer, such as a Brookfield viscometer, as described in Table 1.

[0228]

[0229] Unless otherwise stated, the present invention employs the following method:

[0230] Method of measurement of zeta potential

[0231] The formulation to be analyzed was diluted in phosphate buffer to obtain a final polymer concentration of 0.05%, and then gently stirred until homogenized. The solution was then divided into aliquots, and the pH of each aliquot was adjusted to the desired value (pH 4 to pH 8) by adding 0.1 N sodium hydroxide or 0.1 N hydrochloric acid. The zeta potential of each aliquot was measured using a Nano-Z device (Zeta-Sizer series, Malvern Instruments).

[0232] Method of measurement of solubility range of chitosan polymers

[0233] The solubility range was determined by preparing a 1% solution of the polymer at pH 9, including dividing it into several portions and adjusting the pH of each portion to a different value within the range of 9 to 1. For each portion, it was examined to determine its solubility—that is, the absence of any turbidity—according to the visual inspection described in European Pharmacopoeia Special Issue 2.9.20. The pH range at which the polymer was soluble or insoluble was recorded.

[0234] Mouse subcutaneous air pouch model

[0235] A mouse subcutaneous air sac model was used to evaluate the foreign body response and immunocompatibility of various polymers of chitosan derived from fungi. In this model, air sac cavities were created by repeated subcutaneous injections of sterile air into the back of mice. The sac cavities created by the inflatation of air mimic synovial cavities, thus providing a local environment capable of studying the stimulation of inflammatory responses when fluid is aspirated from the air sac cavity and surrounding tissues, as described by Segwick et al. (see: J Pathol 141, 483, 1983) and Sin et al. (see: Ann Rheum Dis 45, 873, 1986). The cavity of the air sac provides a method for injecting up to 1 mL of product from the air sac cavity into animals weighing approximately 30 g. Following the protocol described by Dawson et al. (see: Int J Tiss Reac 8, 171, 1991), air sacs were established in non-inbred male CD-1 Swiss albino mice (pathogen-free, weighing 25–35 g at reception) on days 0 and 4 of the timeline by repeated injections of 5 mL followed by 3 mL of sterile air. On day 7, 1 mL of the product was injected directly into the air sac cavity via a single subcutaneous injection. Injections of 1 mL of saline and 1% carrageenan solution served as negative and positive controls, respectively. Animals were monitored regularly for several hours post-injection to detect possible clinical signs of systemic or local toxicity, as well as increases in body weight and temperature at injection and euthanasia. Animals were euthanized 24 hours after exposure to the product (3 animals per product). Immunological evaluation was performed on the cleansing fluid recovered from the cyst cavity, including cytological analysis (counting and distribution of white blood cells and red blood cells) and quantitative analysis of major inflammatory mediators (IL-1-β, TNF-α, and KC / CXCL1) using the "ELISA" kit (AbCam) according to the manufacturer's instructions. Macroscopic and histopathological analysis was performed on the surrounding skin tissue by staining with hematoxylin / eosin. Special attention was paid to the product's localization in the surrounding tissues and its reabsorption in the fluid.

[0236] Model for evaluation of local tolerability of intra-articular administration in sheep model

[0237] Based on literature and recommendations from the Osteoarthritis Research Society International (OARSI), sheep are considered a good model for evaluating the efficacy of innovative intra-articular injection therapies (Little et al., see: Osteoarthritis Cartilage 18, S80, 2010; Edouard et al., see: PhysTher Sport 14, 116, 2013; McCoy et al., see: Vet Pathol 52, 803, 2015). The sheep model provides a method for injecting a specific amount of the formulation via the intra-articular route, mimicking the clinical application of viscoelasticity supplementation therapy in humans (Fraser et al., see: Semin Arthritis Rheum 22, 9, 1993). Measurements of local tolerance included assessing the clinical signs of synovitis (effusion, lameness, comfort) using empirical semi-quantitative clinical scoring scales described by Shafford et al. (see: Vet Anaesth Analg 31, 20, 2004) and Kaler et al. (see: Vet J 180, 189, 2009) and cytological evaluation of synovial fluid. In some cases, these analyses could be supplemented by anatomical and histological evaluation of the injected limb. Two mL of the formulation was injected into the joint (hind knee) of healthy sheep (2 to 6 years old; weighing 60 to 80 kg). Clinical signs of the animals were recorded daily over 15 days based on a semi-quantitative score of effusion (by palpation of the hind knee) and lameness from 0 to 4. The number of symptoms encountered for each score was reported throughout the observation period. To assess the effect of repeated injections of the same given formulation, a second injection was given at the same joint of the same animal one month later, and the animal was monitored according to the same protocol as the first injection. In addition, synovial fluid was aspirated on day 15, and macroscopic parameters (color, viscosity), cytological parameters (white blood cell and red blood cell counts and distribution), and total serum protein levels were determined using standard methods. If the synovial fluid appeared clear, viscous, and slightly yellow, and cytological analysis showed a white blood cell count less than 1 × 10⁻⁶, then the synovial fluid was considered acceptable. 5 If the cell / mL and total protein concentration is approximately 25 mg / mL, the synovial fluid is considered normal.

[0238] Example 1 N-succinyl-chitosan

[0239] N-succinyl chitosans with different molecular weights and molecular structures (degree of acetylation (DA) and degree of succinyl substitution (DS)) are prepared and characterized according to the general methods described in patent applications WO 2016 / 016463 or WO 2016 / 016464 (patent EP 3016663). In addition to polymer CSS-1 (Table 1a), a first acetylation step is performed by adding acetic anhydride to increase the degree of acetylation.

[0240] Formulations were prepared using these N-succinyl chitosans (CSS) at a concentration of 2% at physiological pH and osmotic pressure and packaged in glass syringes. The syringes were sterilized by autoclaving at a standard cycle (121°C for 15 minutes). The formulations were visually inspected according to method EP2.9.20 to ensure they were free of insoluble matter or turbidity over a wide pH range (approximately pH 6.5 to 7.5). The bacterial endotoxin levels in the formulations were examined to ensure they were below 20 EU / mL according to method 2.6.14(D) of the European Pharmacopoeia, and the microbial load was examined to ensure it was below 100 cfu / g according to method 2.6.12 of the European Pharmacopoeia before assessing their in vivo immunocompatibility.

[0241]

[0242] * Molecular weight of the precursor chitosan according to Table 1; **DA and DS were measured by proton NMR according to the method described in EP 3016663 patent.

[0243] Immunocompatibility of the CSS formulation was evaluated using a mouse subcutaneous air sac model (Table 1b). Observed responses were compared with responses observed after injection of saline solution (negative control), a highly reactive positive control (1% carrageenan solution), and a commercially available product based on Hylan GF-20 (Synvisc®, Sanofi) containing partially cross-linked hyaluronic acid.

[0244]

[0245] *Average value of 3 animals; **Limit of detection: 3.10 for IL1-β. -9 g / mL for TNF-α is 125.10 -9 g / mL

[0246] Generally, these CSS-based formulations appear to induce a foreign body-related immune response, as evidenced by a higher number of leukocytes in cell infiltration compared to 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 higher concentrations of the marker KC / CXCL1 compared to the negative control and the marker concentrations in Synvisc® products. Levels of the markers IL1-β and TNF-α are lower.

[0247] Changing the DA and / or DS of CSS, or actually altering its molecular weight, does not result in a difference in immune response. Although CSS formulations do not cause any harmful effects on tissues locally, high immunocompatibility must be achieved for therapeutic applications where the target tissue is impaired by underlying pathology, such as dry eye, corneal damage, or inflammation associated with joint disease.

[0248] Example 2 - Animal-derived carboxymethyl chitosan

[0249] Two animal-derived carboxymethyl chitosans (CC-1 and CC-2 in Table 2a) are available for implantable or injectable pharmaceutical or medical use.

[0250] Two other carboxymethyl chitosans were tested:

[0251] A type of carboxymethyl group (CC-3) obtained by carboxylation of animal-derived chitin.

[0252] A CC (CC-4) is obtained by carboxylation of animal-derived chitosan, wherein the chitosan is prepared by deacetylation of animal-derived chitin.

[0253] Carboxymethyl chitosan (CC) was characterized by carbon-13 NMR to identify it, and the values ​​of DA (degree of acetylation) and DS (degree of carboxymethyl substitution) were measured with an estimated error range of approximately ±10% (Table 2a). The electrostatic charge of the polymer was characterized by measuring the zeta potential at a concentration of 0.05% (m / m) in the pH range from pH 8 to pH 4, and the zeta potential value at pH 7.5 was recorded (Table 2a). It was observed that the zeta potential value at pH 7.5 correlated relatively well with the DS value determined by carbon-13 NMR. For example, polymer CC-3 had a higher degree of substitution of anionic carboxymethyl groups (in the carboxylate form at pH 7.5) than polymer CC-4 and had a similar DA, thus its total negative charge was greater (-28 mV vs. -11 mV at pH 7.5).

[0254] Polymers CC-1 and CC-2 were observed to be poorly soluble within the physiological pH range (pH 6.5 to 7.5). Their immunocompatibility could not be assessed using a mouse subcutaneous air sac model. Polymers CC-3 and CC-4 were practically soluble over a wide pH range from 1 to 9.

[0255]

[0256] *The DA and DS values ​​of the carboxymethyl chitosan polymer were measured by solid-state carbon-13 nuclear magnetic resonance (NMR) spectroscopy.

[0257] The CC-3 (2%) and CC-4 (1.5%) formulations were then prepared using the same method as in Example 1 and packaged in glass syringes, followed by autoclaving at 121°C for 15 minutes in a standard cycle (Table 2a). It was found that the 2% formulation of polymer CC-3 could not withstand final autoclaving, as evidenced by a loss of approximately 60% in intrinsic viscosity (mapped for polymer hydrolysis) and a 98% loss in its storage modulus G'. As an alternative to final autoclaving, the CC-3 formulation was filtered and subsequently packaged in syringes to produce a formulation that could be evaluated using a mouse air sac model.

[0258] The formulations of CC-3 (2%, filtered) and CC-4 (1.5%, autoclaved) were examined to determine endotoxin levels and microbial loads below 20 EU / mL and 100 cfu / g, respectively, and their immunocompatibility was then assessed using a mouse subcutaneous air sac model (Table 2b).

[0259]

[0260] *Average value of 3 subjects; **Limit of detection: 3.10 for IL1-β. -9 g / mL, and for TNF-α it was 125.10. -9 g / mL.

[0261] The formulation of CC-4 elicited a significant response, characterized by high concentrations of two markers, KC / CXCL1 (neutrophil activation) and TNF-α, in the infiltrate. The marker TNF-α was not detected after injection of the CSS formulation described in Example 2 or Synvisc® (Hylan GF-20). However, inducing a response characterized by the marker TNF-α is undesirable. Animal-derived carboxymethyl chitosan CC-4 is unsuitable because good immunocompatibility is required for therapeutic applications, and it is desirable for the formulation to come into contact with potentially pathologically compromised tissues (e.g., dry eye, corneal injury or damage, or joint inflammation) and to avoid neutrophil activation and TNF-α.

[0262] The CC-3 formulation induced a weaker response, characterized by moderate white blood cell counts and KC / CXCL1 marker concentrations, similar to the control product Synvisc® but higher than the negative control (saline). Therefore, the CC-3 formulation appears to be less immunoreactive than the CSS and CC-4 formulations. However, a drawback of the CC-3 formulation is that the KC / CXCL1 marker concentration may prove unsuitable for treatments requiring very high immunocompatibility in subjects, and the macromolecular chains undergo significant hydrolysis under heat, making autoclaving impossible during the final step of the manufacturing process, after filling the syringe with the formulation.

[0263] Example 3 – Carboxymethyl chitosan of fungal origin

[0264] To obtain the carboxymethyl chitosans listed in Tables 3a and 3b, starting with chitin or chitosan of fungal origin, the following reaction is performed:

[0265] The "ultra-high" molecular weight fungal chitosan was reacetylated and then carboxymethylated (CC-5 in Table 3a).

[0266] The fungal chitosan with "ultra-high" molecular weight was carboxymethylated and then reacetylated (CC-5 to CC-9).

[0267] Carboxymethylation of "ultra-high" molecular weight fungal chitosan (CC-11).

[0268] Carboxymethylation of fungal lamina (CC-10).

[0269] For example, CC-8 can be prepared in the following way:

[0270] 10 g of “ultra-high” chitosan was dispersed in 220 ml of isopropanol and 68 ml of 40% sodium hydroxide (m / 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 carried out at 25°C for 16 hours. The polymer was recovered by precipitation in ethanol, followed by purification through a cycle of dissolution / precipitation in ethanol. The precipitate was dried in a ventilated oven. 15 g of the precipitate was dispersed in water, and 3.75 mL of acetic anhydride was added. After stirring at ambient temperature for 30 minutes, the pH of the medium was adjusted to 6, and 3.75 mL of acetic anhydride (AC) was added. After stirring at ambient temperature for 30 minutes, the medium was neutralized and precipitation was carried out in ethanol. The precipitate thus obtained was redissolved and then precipitated again. The final precipitate of carboxymethyl chitosan (CC-8) was dried in a ventilated oven.

[0271] Tables 3a and 3b show the synthesis parameters used to prepare other CCs.

[0272]

[0273] *MCA: Monochloroacetic acid; SCA: Sodium monochloroacetate.

[0274]

[0275] The fungal-derived carboxymethyl chitosan (CC) was characterized by carbon-13 NMR to confirm its structure and determine the values ​​of DA and DS (Table 3c). Considering the inherent standard deviation of carbon-13 NMR spectroscopy (approximately ±10%), a correlation was observed between the zeta potential at pH 7.5 and the molecular structure, particularly with DS.

[0276]

[0277] *DA and DS determined by 13C-NMR; **If the loss of intrinsic viscosity before / after sterilization is less than 40%, it is considered sterilizable.

[0278] The obtained CC is soluble over a wide pH range, especially in the pH range of 4.0 to 8.5, where no milky white color was observed.

[0279] Formulations with CC concentrations of 1.5% or 2% were prepared and packaged in glass syringes, then sterilized in an autoclave at 121°C for 15-minute cycles according to standard procedures. To assess whether the formulations could be autoclaved, the intrinsic viscosity of the polymer was measured before and after autoclaving using a capillary viscometer after dilution 25-fold with phosphate buffer. Unlike the CC-3 formulation described in Example 2 (which had an intrinsic viscosity loss of approximately 60%), the intrinsic viscosity loss of CC was less than 40%, indicating acceptable resistance.

[0280] Visual examination of the sterilized formulations shown in Table 3c indicated that they were clear, not milky white. They were then examined to determine endotoxin levels and microbial loads below 20 EU / mL and 100 cfu / g, respectively, and their immunocompatibility was evaluated using a mouse subcutaneous air sac model.

[0281]

[0282] *Average value of 3 subjects; **Limit of detection: 3.10 for IL1-β. -9 g / mL, and for TNF-α it was 125.10. -9 g / mL.

[0283] Generally, it is noted that the fungal-derived CC formulation exhibits good immunocompatibility and weakens or even inhibits the immune response compared to the N-succinyl chitosan formulation described in Example 1 and the animal-derived carboxymethyl chitosan formulation described in Example 2.

[0284] It was noted that the fungal-derived CC formulation induced low concentrations of the biomarker KC / CXCL1, the same as the negative control (saline) and lower than the product Synvisc®. This reflects negligible or even absent neutrophil activation, contrary to the levels reported after injection of CSS (Example 1) and animal-derived CC (Example 2) formulations.

[0285] Furthermore, it was noted that for certain fungal-derived CC formulations (i.e., CC-8 and CC-10), four parameters of the immune response—total white blood cell count, the marker IL-1β, the marker KC-CXCL1, and the marker TNF-α—were simultaneously weakened or inhibited. This did not occur with CSS formulations or animal-derived CC formulations.

[0286] It appears that, for fungal-derived CC, the formulations that exhibit a simultaneous decrease in all 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).

[0287] The formulations of animal-derived CC-4 and fungal-derived CC-5 with low carboxyl substitution, as described in Example 2, were observed to induce some activation of the biomarker TNF-α. However, only the animal-derived CC-4 formulation resulted in significant secretion of the biomarker KC / CXCL1. It was also observed that, unlike the animal-derived CC-3 formulation described in Example 2, the (fungal-derived) CC-10 formulation did not induce neutrophil activation.

[0288] Example 4 - Evaluation of local tolerance to intra-articular administration of fungal-derived carboxymethyl chitosan formulations using a sheep model

[0289] Formulations containing 2% of the two fungal-derived carboxymethyl chitosans (CC-8 and CC-10) described in Example 3 were prepared to evaluate their potential for intra-articular injection in a sheep model. Two mL volumes of both formulations were administered to sheep, and local responses to intra-articular injection were assessed over a 2-week period. The effect of a second injection of the CC-8 formulation into the same joint one month after the first injection was also evaluated. Synvisc® was administered in the same manner in either 1 mL or 2 mL volumes.

[0290] Clinical symptoms were monitored daily over a 15-day period by palpating the joints and observing limping, and were assessed using semi-quantitative scores ranging from 0 (asymptomatic) to a maximum score of 3 for effusion and a maximum score of 3 for limping. Overall clinical outcomes over the 15-day period were recorded based on the incidence of scores and the characteristics of the synovial fluid produced on day 15 (Table 4).

[0291]

[0292] Example 5 – Evaluation of local tolerance in rabbits following intradermal injection of a fungal-derived carboxymethyl chitosan formulation

[0293] Formulations of three fungal-derived carboxymethyl chitosans were prepared at physiological pH and osmotic conditions to evaluate their potential for intradermal administration using a rabbit model (Table 5a). Fungal-derived CC-12, CC-13, CC-14, and CC-15 were prepared by carboxymethylation followed by reacetylation according to the general method described in Example 3 for CC-8, while adjusting synthetic parameters to alter DA and DS (Table 5a). CC-13 and CC-14 exhibited lower DS of 41% and 51%, and higher DA of 74% and 69%, respectively.

[0294] The formulations were packaged in 1 mL glass syringes and then sterilized in an autoclave according to a standard cycle (121°C for 15 minutes). All three formulations were resistant to autoclaving, with a loss of intrinsic viscosity of less than 40%. Finally, the formulations were examined to ensure bacterial endotoxin levels were below 40 EU / mL and microbial loads were below 100 cfu / mL.

[0295] Following the protocol of ISO 10993 (Part 10), 200 µL of the formulation was administered intradermally to rabbits using a 27-gauge needle (a very small needle) to evaluate primary irritation caused by intradermal implants. Six rabbits received three injections of each formulation. Juvéderm® Voluma (Allergan), a commercially available dermal filler based on cross-linked hyaluronic acid, was also injected using a 27-gauge needle. Macroscopic signals of skin irritation at 12, 24, and 48 hours were recorded using a semi-quantitative scoring scale, ranging from 0 (asymptomatic) to 4 (maximum symptom), to determine the primary irritation score. The primary irritation score for each product was determined by summing the average erythema scores at 24, 48, and 72 hours for the 18 injection sites. The average edema scores were summed in the same manner. The two sums (erythema and edema) were added together and then divided by 54 to obtain the average score for primary irritation. The same procedure was followed for comparative products. Table 5b records the irritation scores. It was observed that the CC formulations did not produce edema symptoms and only a small amount of erythema symptoms, and in all cases, the scores were lower than those obtained by Juvéderm® Voluma. It was concluded that these formulations are non-irritating and low-irritant compared to Juvéderm® Voluma.

[0296]

[0297] *Determined by 13C-NMR;**A product is considered sterilizable if the loss of intrinsic viscosity before / after autoclaving is less than or equal to 40%;***Value estimated (±20%) based on polynomial regression of the zeta potential curve as a function of DS at pH 7.5.

[0298]

[0299] The observation period was then extended to two weeks post-injection, with clinical signs assessed on days 5, 7, 9, 11, and 14. Two animals were euthanized on days 3, 7, and 14 post-injection for possible histological analysis.

[0300] The total scores are shown in Table 5c. Excellent local tolerability was observed in all four formulations within 2 weeks of intradermal injection, with lower scores for all gross or visible symptoms compared to those observed with Juvéderm® Voluma, and no eschar or edema. Formulations CC-12 and CC-13 showed some low-score erythema symptoms (maximum score 1), which resolved by day 11. Formulation CC-13 showed some erythema symptoms with a maximum score of 2, occurring in 33% of cases by day 9; these symptoms also resolved by day 11 without adverse tissue effects.

[0301]

[0302] *Number of sites assessed macroscopically within each timeframe after injection.

[0303] In addition, the four compositions (CC-12, CC-13, CC-14 and CC-15) can be easily injected via a 27-gauge fine needle suitable for intradermal administration.

[0304] Example 6 - Sensitivity to Enzyme Degradation in Vitro

[0305] In this embodiment, the degradation rate of the formulation is compared when a mixture of two lysozymes and hyaluronidases is present in biological fluids (e.g., synovial fluid, tears, or interstitial fluid of connective tissue). Lysozymes are generally considered to have the ability to hydrolyze chitosan-based biomaterials.

[0306] Fungal carboxymethyl chitosan CC-16 was prepared by adjusting the DA and DS parameters in the same manner as the preparation of CC-8 shown in Example 3. Formulations of 2% CC-16, CC-8 (Example 3), and CC-10 (Example 3) were prepared in phosphate buffer containing 3.5% sorbitol. A 2% animal-derived CC-3 formulation (Example 2) and two commercially available viscoelastic supplements based on non-crosslinked hyaluronic acid (HA-1 and HA-2) were also evaluated.

[0307] The formulations were diluted 25-fold in phosphate buffer. The solution was then stirred for 12 hours, and a mixture of lysozyme and hyaluronidase was added to the diluted solution at doses of 184 units / mL and 2.6 units / mL, respectively. The intrinsic viscosity was measured periodically using an automated capillary viscometer I-Visc (Lauda) equipped with a Ubbelohde type capillary (model 0a). The half-life of each formulation, i.e., the time required for the polymer intrinsic viscosity to reach half its initial value, was then calculated (Table 6).

[0308]

[0309] *Determined by solid carbon-13 nuclear magnetic resonance (NMR) spectroscopy.

[0310] The results indicate that carboxymethyl chitosan formulations are hydrolyzed by a mixture of lysozyme and hyaluronidase, and the hydrolysis kinetics can be modulated by the molecular structure of carboxymethyl chitosan. This allows for adjustment of the product's residence time based on the target therapeutic application. Furthermore, it can be inferred that fungal-derived carboxymethyl chitosan degrades more slowly than animal-derived carboxymethyl chitosan (CC-3) and two commercially available hyaluronic acid-based products.

[0311] Example 7 – Thermal Effects

[0312] In this embodiment, syringes containing the formulation were placed in an oven at a temperature controlled at 60°C for 11 days to assess their heat resistance at temperatures above conventional storage temperatures. At each time interval, the syringes were removed, and the intrinsic viscosity of the polymer was measured according to the method described in Example 6. The ratio of the viscosity within a given time interval to the initial viscosity (expressed as a percentage of the initial viscosity) was recorded.

[0313] Two fungal-derived carboxymethyl chitosans, CC-17 and CC-18, were prepared by adjusting the synthesis parameters to regulate DA and DS, in the same manner as CC-8 in Example 3, and characterized by carbon-13 NMR (Table 7). They were formulated at a concentration of 2% in the presence of reducing sugar, sorbitol, or mannitol, respectively. A 2% formulation of the animal-derived CC-3 shown in Example 2 (containing 3.5% sorbitol) and a commercially available viscoelastic supplement product (HA-2) based on non-crosslinked hyaluronic acid, shown in Example 6, were also evaluated.

[0314]

[0315] *Determined by solid-state carbon-13 nuclear magnetic resonance (NMR) spectroscopy

[0316] The conclusion is that fungal-derived carboxymethyl chitosan formulations are heat-resistant and less sensitive than animal-derived formulations (CC-3) and commercially available hyaluronic acid-based products.

[0317] Example 8 - Method for reducing the microbial load of carboxymethyl chitosan

[0318] Following the method used to prepare CC-8 in Example 3, fungal-derived carboxymethyl chitosan CC-19 was prepared starting with "ultra-high" chitosan. Its characteristics are as follows: 67% DA and 115% DS (measured by carbon-13 NMR); soluble at any pH; transparent; not milky white. The zeta potential of this formulation at pH 7.5 is estimated to be -27 mV (based on polynomial regression of the zeta potential curve as a function of DS). As described in Example 3, a 2% formulation of CC-19 was prepared in phosphate buffer containing 3.5% sorbitol. Its characteristics are as follows: pH 7.3, permeability 279 mOsm / kg. To test their feasibility and impact on the final properties of the formulation, two known methods were effectively implemented to reduce the microbial load of the aqueous formulation. The intrinsic viscosity of the formulation (25-fold dilution) was compared using an I-Visc capillary viscometer (Lauda, ​​capillary 0a), and the refractive index was compared before and after treatment using a HI88713 refractometer (Hanna) (Table 8). The two methods are as follows:

[0319] Autoclaving: Place the preparation in a glass syringe and then autoclave the syringe according to the standard procedure (121°C for 15 minutes);

[0320] Filtration: Filter 300 mL of the formulation using a 0.2 µm porous filter (Preflow capsule filtre, Pall).

[0321] Filtration was performed using a 0.2 µm filter at a constant pressure of 2 bar and a constant flow rate of approximately 6 liters per hour.

[0322]

[0323] Our conclusion is that both methods have minimal impact on reducing the material's refractive index and degrading the polymer's intrinsic viscosity. Therefore, they are industrially applicable for obtaining medical devices and pharmaceutical products based on carboxymethyl chitosan formulations.

[0324] Example 9: In vitro lubrication ability of fungal-derived carboxymethyl chitosan formulation

[0325] This example illustrates the lubricating properties of two fungal-derived carboxymethyl chitosan formulations: CC-8 (2% and 1%) shown in Example 3 and CC-19 (2%) shown in Example 8, to demonstrate their suitability for use as intra-articular tackifiers or as ocular drops. The lubricating properties were evaluated using an in vitro model, allowing for assessment of the reduction in friction between the two surfaces. The animal-derived CC-3 formulation (2%) shown in Example 2 and a commercially available hyaluronic acid-based product were also characterized.

[0326] Intra-articular tackifiers: Synvisc® (Sanofi) and two tackifiers based on non-crosslinked hyaluronic acid (HA-2 as shown in Examples 6 and 7) and HA-3.

[0327] Eye drops: Two products based on non-crosslinked hyaluronic acid (HA-4 and HA-5).

[0328] In addition, prior to surgery to place a knee prosthesis, synovial fluid samples are extracted from the knees of patients with osteoarthritis. This fluid is stored at -20°C, then heated to 25°C, and the coefficient of friction is analyzed.

[0329] The coefficient of friction was measured using the following method. Two 16.15 mm diameter discs (as described in patent EP 1830898), made of a polyacrylate-type biomaterial used to manufacture hydrophobic intraocular lenses, were pre-hydrated by immersing them in water at 60°C for approximately 2 hours. These discs were then fixed to the upper and lower geometries of a Discovery Hybrid Rheometer-2 (DHR-2) (TAInstruments). Approximately 100 µL of the test fluid was placed on the lower disc, and the upper geometry was lowered until contact was achieved between the two discs, up to a normal force of 5 Newtons. The coefficient of friction was measured for 150 seconds at 25°C, under a constant normal force (5 Newtons), an oscillation frequency of 1.256 radians / second, and a deformation angle of approximately 0.05 radians, following a scheme adjusted according to that described by Waller et al. (see: JRheumatol 39, 7, 1473, 2012). Activate the "Zero point of oscillating motion start" option. At each measurement point, record the torque value, and then calculate the coefficient of friction (COF) using the following formula: COF = Torque / (1 / 3 × disk diameter × normal force). Repeat the measurement 5 times for each formulation. The value of the coefficient of friction is recorded by extrapolating the intercept at the start of each COF curve as a function of time (COF0, Table 7).

[0330]

[0331] In the presence of 2% and 1% carboxymethyl chitosan formulations, the coefficients of friction were low under the measured conditions, comparable to or even lower than those of commercially available products for intra-articular and ocular use, and significantly lower than the coefficient of friction of synovial fluid in arthritis. This suggests that carboxymethyl chitosan formulations have the potential to act as lubricants by reducing friction between two surfaces, such as the articular cartilage surface after intra-articular injection or the ocular surface after drop instillation. In terms of reducing friction, the fungal-derived formulation CC was more effective than the animal-derived formulation CC-3.

[0332] Example 10 - Intradermal administration of fungal-derived carboxymethyl chitosan formulation using a fine needle

[0333] This embodiment demonstrates that a 2% fungal-derived carboxymethyl chitosan formulation can be readily injected into the dermis, particularly using very fine needles designed for injection into the superficial layers of the dermis. The test measures the force required to eject the product from a syringe equipped with a needle at a given jet rate using a compression test bench. Empirically, when the jet force measured by this test is less than 50 Newtons, injection of the product is easy and comfortable for both physician and patient, and the jetting is regular and smooth. It is advantageous to use needles with a diameter less than 0.3 mm (30 G) to administer the product in order to minimize pain and bleeding during injection, as well as the subsequent risk of hematoma and skin redness.

[0334] According to the general method for CC-8 described in Example 3, a fungal reference carboxymethyl chitosan CC-20 was prepared starting from "ultra-high" type chitosan, with the following variations: for 10 g of chitosan, 228 ml of isopropanol, 57 ml of 40% sodium hydroxide, and 47 g of MCA were used. The reaction was carried out at 35°C for 23 hours. For 15 g of intermediate carboxymethyl chitosan, 7.5 ml of acetic anhydride was added each time, and three purification cycles were performed before drying and recovering the final carboxymethyl chitosan. The properties of CC-20 are as follows: DA 53% and DS 85% (determined by carbon-13 NMR), soluble in water at any pH (according to the method described above), forming a clear and non-emulsifying solution, and an estimated zeta potential of -24 mV at pH 7.5 (based on polynomial regression of the zeta potential curve as a function of DS).

[0335] As described in Example 3, a 2% (m / m) formulation of CC-20 was prepared. This formulation was packaged in a 1 mL glass syringe (BD-Medical) containing a needle. The force required to spray the product was measured using a MultiTest 2.5-i compression test bench (Mecmesin) equipped with a 100 N compression unit, by applying a constant spray rate of 80 mm / min. The maximum force the device withstood was approximately 70 Newtons. The following needles were tested: 30G, 32G, 33G, and an Invisible Needle. TM (TSK Laboratory).

[0336] For comparative purposes, commercially available products based on non-crosslinked hyaluronic acid (controls HA-6 and HA-7) and commercially available products based on crosslinked hyaluronic acid (HA-8) for skin rejuvenation via the intradermal route were evaluated in their original syringes using the same method.

[0337] Example 11 - Low-concentration formulation of fungal-derived carboxymethyl chitosan as eye drops

[0338] This example demonstrates that a low-concentration solution of fungal-derived carboxymethyl chitosan can be used to prepare eye drops for relieving symptoms of ocular surface diseases or protecting the ocular surface. In addition to physiological properties, i.e., a pH of approximately 7.2 and an osmotic pressure of approximately 200 mOsm / kg, the eye drops should preferably possess the following physicochemical properties: a low refractive index (close to 1.33) and the ability to minimize friction between the ocular surface and the conjunctiva and eyelids (lubricating ability). Finally, its rheological properties should ensure that the product is not so fluid as to fail to deposit on the eye, but rather spreads uniformly and is not overly sticky. Its rheological properties should also facilitate eyelid fluttering or blinking without accumulation, i.e., low viscosity at high shear rates. In particular, Orobia et al. found that during ocular movements, the viscosity (excluding eyelid fluttering) is advantageously greater than 10 mPa·s, for example, approximately 20 mPa·s (see: Clinical Ophthalmology 12, 453, 2018). It should be noted that viscosity values ​​can vary depending on the measurement method, particularly the equipment, measurement mode and parameters, temperature, and the shear rate applied to the product being measured.

[0339] Reference carboxymethyl chitosan CC-21 of fungal origin was prepared using the same method as CC-20 shown in Example 10. The molecular structures of CC-20 and the acidic form of CC-21 were compared using Fourier transform infrared spectroscopy on a Nicolet iS5 spectrometer equipped with an ID7-ATR, as described by Chen et al. (Carbohydrate Polymers 53, 355, 2003). It was determined that their molecular structures are in the range of 1200 to 1800 cm⁻¹. -1 They have 99% similarity within the wavelength range, therefore their DA and DS are similar.

[0340] Two carboxymethyl chitosan CC-21 formulations at concentrations of 0.7% and 0.4% (m / m) were prepared in glycerol-containing phosphate buffer. The pH was adjusted to 7.2 ± 0.2, and the permeability to 200 ± 20 mOsm / kg. The formulations were then filtered, and their lubricating properties and viscosity characteristics were characterized according to the methods described below. Eye drops with different compositions based on hyaluronic acid (HA) (HA-5 to HA-9) were characterized using the same method. The results are presented in Table 11b.

[0341] Method of measurement of coefficient of friction (applicable to eye drops)

[0342] Lubrication capability, i.e., the ability to reduce friction between two contacting surfaces, was evaluated by measuring the coefficient of friction between two disks made of the same polyacrylate biomaterial as shown in Example 9. The two disks were fixed to the upper and lower geometry of a DHR-2 rheometer (TA Instruments). Approximately 100 μL of the test product was placed on the lower disk, and the upper geometry was then lowered until contact was achieved between the two disks, up to the application of a normal force of 5 Newtons. The coefficient of friction was measured for 60 seconds at 25°C, with a constant normal force (5 N), an oscillation frequency of 6 radians / second, and a deformation angle of approximately 1.71 radians, according to a scheme adjusted according to Waller et al. (J Rheumatol 39, 7, 1473, 2012) and parameters simulating blinking kinematics conditions described by Kwon et al. (J Royal Society Interface 10, 2, 2013). The "Zero point matching the start of oscillating motion" option was activated. At each measurement point, the torque value is recorded, and the coefficient of friction (COF) is calculated using the following formula: COF = Torque / (1 / 3 × disk diameter × normal force). The average COF is determined between 0 and 60 seconds. The average coefficient of friction and standard deviation of five consecutive measurements are then calculated. The minimum COF is 52 when the two surfaces are not in contact. The upper limit of COF corresponds to the condition where the two disks are no longer moving relative to each other.

[0343] Since COF values ​​vary between different series, it is necessary to use the same disk in a random order to characterize the products to be compared within the same given series. Then, according to the criteria in Table 11a, the products tested within the same series are classified using a relative rating scale, from most effective (score 1) to least effective (score 3). Limit value "COF" A "and COF B "The series was defined by the COF of two commercially available eye drops, which served as references for each series: eye drop A (composed of 0.15% hyaluronic acid and 0.25% carbomer) and eye drop B (0.15% hyaluronic acid). According to this rating scale, for lubricating drops used to lubricate the ocular surface, a friction reduction score of 1 or 2 is advantageous, and preferably 1. For products with a score of 3, the lubrication ability is unsatisfactory, and the measured values ​​vary considerably."

[0344]

[0345] Method of measurement of viscosity as a function of shear force

[0346] Simmons et al. have determined that the shear rate during eye-opening movements is approximately 10s. -1The shear rate during eyelid tremor is approximately 10,000 s. -1 (See: Clinical Ophthalmology 11, 1637, 2017). Based on the shear rate range to be studied and considering that the product is a low-viscosity fluid solution, select the rheological measurement method:

[0347] Corresponding to the shear range of eye movement. Viscosity was measured by flow sweep testing in rotational mode using a stress-controlled DHR-2 rheometer (TA Instruments), which is equipped with a Peltier plate and a "conical" geometry with a diameter of 60 mm and a cutoff angle of 2°. The product was equilibrated at 37°C for 1 minute, with the temperature controlled by Peltier. To prevent evaporation, the system was equipped with a solvent trap and a metal cap. Then, the flow sweep test was started, and the viscosity was measured based on the shear rate (0.001 s⁻¹). -1 up to 100s -1 Viscosity was measured using [a specific method / method]. The viscosity was recorded in 10 s. -1 The viscosity value at that time.

[0348] Corresponding to the shear range of eyelid tremor. Viscosity was measured by flow scanning test in rotational mode using a stress-controlled DHR-2 rheometer (TA Instruments), which is equipped with a "double gap couette" geometry and Peltier concentric columns, along with a solvent trap and metal cap. The product was allowed to equilibrate at 37°C for 1 minute, then subjected to a 0.001 s... -1 Up to 10000 s -1 The variable shear rate. Then, from this, 10000 s -1 Viscosity value at 10 s -1 The values ​​at that time are compared.

[0349]

[0350] The results confirmed that both formulations of fungal carboxymethyl chitosan CC-21 met the technical specifications for eye drops: they exhibited low refractive index, a satisfactory coefficient of friction (fraction 1, as effective as the most lubricating drops commercially available), a viscosity of 10 mPa·s to 30 mPa·s under ocular motion conditions, and low viscosity under eyelid tremor conditions. In the absence of carboxymethyl chitosan, phosphate buffer supplemented with glycerol did not meet the technical specifications for eye drops. Finally, it was noted that viscosity could be reduced by decreasing the concentration of carboxymethyl chitosan (e.g., from 0.7% to 0.4%) without adversely affecting the ability to reduce friction (fraction 1).

Claims

1. Use of carboxyalkyl chitosan in the preparation of compositions for injection, implantation, or infusion into a human or animal body, said carboxyalkyl chitosan having glucosamine units, N-acetyl-glucosamine units, and carboxyl-substituted glucosamine units, said carboxyalkyl chitosan having a zeta potential less than or equal to -18 mV as measured at pH 7.5; said carboxyalkyl chitosan having a degree of carboxyl substitution greater than 50%, expressed as the number of moles of substituents relative to the total number of units; and said carboxyalkyl chitosan having a degree of acetylation from 40% to 80%, expressed as the number of moles of N-acetyl-glucosamine units relative to the total number of units.

2. The use according to claim 1, characterized in that, The carboxyl chitosan has a degree of carboxyl substitution greater than 70%, expressed as the number of moles of substituents relative to the total number of units.

3. The use according to claim 1 or 2, characterized in that, The carboxyl chitosan has a degree of carboxyl substitution greater than 70% and less than 200%, expressed as the number of moles of substituents relative to the total number of units.

4. The use according to claim 1 or 2, characterized in that, The carboxyalkyl chitosan is of fungal origin.

5. The use according to claim 1 or 2, characterized in that, The chitosan is derived from Ascomycota. Ascomycetes Fungal mycelium.

6. The use according to claim 1 or 2, characterized in that, The chitosan is derived from fungi. Basidiomycetes and / or derived from Aspergillus niger The mycelium.

7. The use according to claim 1 or 2, characterized in that, The chitosan is derived from button mushrooms. Agaricus bisporus (Oyster mushroom) and / or shiitake mushroom Lentinula edodes The mycelium of (Shitak mushroom).

8. The use according to claim 1 or 2, characterized in that, The carboxyalkyl chitosan is an N-carboxyalkylated and O-carboxyalkylated chitosan.

9. The use according to claim 1 or 2, characterized in that, The concentration of the carboxyalkyl chitosan is less than 10% by mass relative to the total mass of the composition.

10. The use according to claim 1 or 2, characterized in that, The composition further comprises hyaluronic acid or sodium hyaluronate.

11. The use according to claim 1 or 2, characterized in that, The composition is for injection.

12. The use according to claim 1 or 2, characterized in that, The composition is a pharmaceutical composition.

13. The use according to claim 1 or 2, characterized in that, The composition also includes hyaluronic acid or sodium hyaluronate that are covalently crosslinked.

14. The use according to claim 1 or 2, characterized in that, The composition further comprises uncrosslinked hyaluronic acid or sodium hyaluronate.

15. The use according to claim 1 or 2, characterized in that, The composition is in solution form.

16. The use according to claim 1 or 2, characterized in that, The composition can be injected at ambient temperature through a needle with a diameter of 21 gauge, wherein the force applied to the syringe to cause the composition to flow through the needle is less than 50 Newtons.

17. The use according to claim 1 or 2, characterized in that, The composition is sterile.

18. The use according to claim 1 or 2, characterized in that, The composition is transparent or translucent.

19. The use according to claim 1 or 2, characterized in that, The composition is an injectable composition, an implantable pharmaceutical composition, a pharmaceutical composition suitable for infusion, or an injectable medical device, an implantable medical device, or a medical device suitable for infusion.

20. The use according to claim 1 or 2, characterized in that, The composition is used in therapeutic treatment methods.

21. The use according to claim 1 or 2, characterized in that, The composition is administered via subcutaneous, intradermal, ocular, or intra-articular routes by instillation or injection for the repair or filling of at least one body tissue requiring repair or filling.

22. The use according to claim 1 or 2, characterized in that, The composition is injected via an intraocular route for repairing or filling at least one body tissue that requires repair or filling.

23. The use according to claim 21, characterized in that, The body tissues are selected from the vocal cords, muscles, ligaments, tendons, cartilage, sex organs, bones, joints, eyes, dermis, epidermis, or any combination thereof.

24. The use according to claim 1 or 2, characterized in that, The composition is used in treatments for osteoarthritis or in the repair of cartilage defects, optionally by injection into synovial fluid or by mixing with blood and then implanting it into the cartilage.

25. The use according to claim 1 or 2, comprising injecting the composition in a cosmetic or body care method.

26. The use according to claim 25, characterized in that, The cosmetic and body care methods include filling wrinkles or filling one or more damaged, visible tissue areas.

27. The use according to claim 1 or 2, characterized in that, The preparation includes: The carboxyl chitosan according to claim 1 or 2 is dissolved in an aqueous solution, wherein the aqueous solution is optionally a buffer solution; Optionally, adjust the pH to the desired pH; Optionally, other excipients may be added; Optionally, the final permeability of the composition can be adjusted.

28. The use according to claim 27, characterized in that, The composition is steam sterilized.

29. The use according to claim 27, characterized in that, The composition is vacuum sterilized by raising the temperature to 121°C to 138°C and continuing the sterilization process in an autoclave for a sufficient time.

30. Use of carboxyalkyl chitosan in the preparation of medical devices for injection, implantation, or infusion into a human or animal body, said carboxyalkyl chitosan having glucosamine units, N-acetyl-glucosamine units, and carboxyl-substituted glucosamine units, said carboxyalkyl chitosan having a zeta potential less than or equal to -18 mV as measured at pH 7.5; said carboxyalkyl chitosan having a degree of carboxyl substitution greater than 50%, expressed as the number of moles of substituents relative to the total number of units; and said carboxyalkyl chitosan having a degree of acetylation of 40% to 80%, expressed as the number of moles of N-acetyl-glucosamine units relative to the total number of units.

31. The use according to claim 30, characterized in that, The medical device includes: a cavity containing carboxyalkyl chitosan in a dried form, and one or more other cavities optionally containing one or more active products, additives, or excipients.

32. An article or package comprising one or more drip or injection devices prefilled with a composition according to any one of claims 1 to 29.

33. A carboxyl chitosan comprising a glucosamine unit, an N-acetyl-glucosamine unit, and a carboxyl-substituted glucosamine unit, wherein the carboxyl chitosan has: The zeta potential is less than or equal to -18 mV and measured at pH 7.

5. The degree of acetylation is expressed as the number of moles of N-acetylglucosamine units relative to the total number of units, ranging from 40% to 80%. A degree of carboxyl alkyl substitution greater than 70%, expressed as the molar number of substituents relative to the total molar number of units; and The carboxyalkyl chitosan has an average molecular weight range of 20,000 to 500,000, where the molecular weight is expressed as the chitosan prior to the substitution.

34. The carboxyalkyl chitosan of claim 33, wherein the average molecular weight is between 20,000 and 60,000, and the molecular weight is expressed as the chitosan before the substitution.

35. The carboxyalkyl chitosan according to claim 33, wherein the average molecular weight is between 60,000 and 100,000, and the molecular weight is expressed as the chitosan before the substitution.

36. The carboxyalkyl chitosan of claim 33, wherein the average molecular weight is between 100,000 and 120,000, and the molecular weight is expressed as the chitosan before the substitution.

37. The carboxyalkyl chitosan according to claim 33, wherein the average molecular weight is between 120,000 and 150,000, and the molecular weight is expressed as the chitosan before substitution.

38. The carboxyalkyl chitosan according to claim 33, wherein the average molecular weight is between 150,000 and 220,000, and the molecular weight is expressed as the chitosan before substitution.

39. The carboxyalkyl chitosan according to claim 33, wherein the average molecular weight is between 220,000 and 300,000, and the molecular weight is expressed as the chitosan before substitution.

40. The carboxyl chitosan of claim 33, wherein the chitosan is derived from ascomycetes. Ascomycete Fungal mycelium and / or derived from fungi Basidiomycetes .

41. A composition comprising at least one carboxyl chitosan of claim 33.

42. An injectable composition comprising at least one carboxyl chitosan of claim 33.

43. A pharmaceutical composition comprising at least one carboxyl chitosan of claim 33.

44. An eye drop composition comprising at least one carboxyalkyl chitosan of claim 33.

45. A viscosity supplement comprising at least one carboxyalkyl chitosan of claim 33.

46. ​​A method for preparing the composition of claim 41, the method comprising: The carboxyl chitosan of claim 33 is dissolved in an aqueous solution, optionally in a buffer solution; Adjust the pH of the aqueous solution containing the carboxyl chitosan to the desired pH; Optionally, other excipients may be added to the aqueous solution; and Optionally, the permeability of the aqueous solution containing the carboxyl chitosan and optional excipients is adjusted; This forms a composition.

47. Use of carboxyalkyl chitosan in the preparation of compositions for injection, implantation, or infusion into a human or animal, said carboxyalkyl chitosan as defined in claim 45, said compositions for therapeutic treatment comprising administering the carboxyalkyl chitosan of claim 33 to a subject in need of such treatment.

48. The use according to claim 47, wherein the therapeutic treatment is for osteoarthritis or repair of cartilage defects, and wherein the carboxyalkyl chitosan is administered via injection into synovial fluid, optionally after mixing with blood, or via implantation into cartilage.

49. The use according to claim 47, wherein the carboxyalkyl chitosan is administered via subcutaneous, intradermal, ocular, or intra-articular instillation or injection.

50. The use according to claim 47, wherein the carboxyalkyl chitosan is administered via an intraocular route.

51. Use of carboxyalkyl chitosan in the preparation of compositions for injection into humans or animals, said carboxyalkyl chitosan as defined in claim 33, said compositions for cosmetic or body care, said cosmetic or body care comprising applying the carboxyalkyl chitosan of claim 45 to a subject in need of it.

52. The use according to claim 51, wherein the composition is used for skin rejuvenation.

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