WO2017009335A1

WO2017009335A1 - Chitosan for mixing with a coagulable fluid

Info

Publication number: WO2017009335A1
Application number: PCT/EP2016/066537
Authority: WO
Inventors: Mickaël CHAUSSON; Pierre DOUETTE; Sandrine Gautier
Original assignee: Kiomed Pharma
Priority date: 2015-07-13
Filing date: 2016-07-12
Publication date: 2017-01-19
Also published as: FR3038837B1; FR3038837A1

Abstract

The invention relates to a composition comprising a mixture of a formulation of chitosan and a coagulable fluid, in which the chitosan formulation has a chitosan concentration of at least 2.5 wt% relative to the total weight of the chitosan formulation, said chitosan having a dynamic viscosity no greater than 60 mPa.s, measured in 1% acetic acid (v/v), said composition including at least one inorganic salt. The invention also relates to a medical device.

Description

Chitosan for mixing with a coagulable fluid

The present invention relates to the field of implantable compositions for repairing a tissue defect and a joint defect, and in particular cartilage. For example, the compositions may be implanted or injected into a cartilage lesion that has been debrided and has undergone a microfracture procedure that involves puncturing microtunnels to stimulate the bone marrow.

Chitosan is a polysaccharide well known in the prior art. It is a copolymer composed of glucosamine units, deacetylated, and N-acetyl-glucosamine (acetylated) units. The properties of chitosan vary in particular according to the structure of the polymer, and in particular its molecular weight, reflected by its dynamic viscosity, for example.

Chitosan presents formulation difficulties because it is soluble at acidic pH, less than 6, but insoluble at pH higher than this value.

It is known that the addition of organic salt such as glycerophosphate makes it possible to solubilize the higher pH chitosan by avoiding its precipitation. Glycerol-phosphate solutions mixed with a high molecular weight chitosan (> 200kDa) have a temperature-dependent sol-gel transition and can gel when changing from a temperature of about 4 ° C to a temperature of about 40 ° C.

It is known from the patent application WO 2011/060545 to prepare a chitosan / blood mixture having a concentration of chitosan described as ranging from 1 to 10% by weight and in which the blood / chitosan volume ratio ranges from 4/1 to 1 / 1 so as to form a clot in the form of a paste by a mechanism of coagulation of blood in the presence of chitosan. This patent application evokes the use of different organic or inorganic salts, and focuses (examples and embodiments) on the use of inorganic salts. This application recommends the use of a chitosan having a molecular mass greater than 200 kDa, and more specifically 232 kDa. The concentrations of chitosan used are about 2% by weight. Coagulation times are at best 5 minutes with human blood (see Example 2, and Figure 5). It can be noted by comparing Tables 3 and 6 that the clotting time on human blood can be very different from that obtained on animal blood, in this case the time of coagulation with human blood is clearly greater than the time of clotting. coagulation with rabbit blood (from one to five times). This document does not describe how to prepare other chitosan and blood compositions with rapid clotting time. This document does not describe how to prepare a clot that spreads easily when applied to the surface of a fabric that is not arranged in a horizontal plane. This document suggests that clots break up into at least two fragments (Tables 3 and 7). In addition, this document mentions (Table 8) that clots with a concentration of 2% of chitosan were not homogeneous. It is therefore not easy to obtain both rapid coagulation of the blood clot and that clot is intact, non-retracted, non-fragmented, resistant to compression and deformation.

The patent application WO 201 1/060544, of the same applicant as the application WO 201 1/060545 (Corporation of the Ecole Polytechnique de Montreal), describes the preparation of compositions based on chitosan for their use for tissue repair, and focuses on a comparison of formulations with or without glycerophosphate (abbreviated "GP", replaced in this case by a salt of sodium chloride while adjusting the formulation to pH 6.6 - see examples). In particular, the influence of molecular weight varies over a wide range (2.7 to 298 kDa). The molecular weight of chitosan does not appear to have any effect in the presence of NaCl but it seems that the molecular weight of chitosan influences the coagulation time in the presence of GP, the best results being obtained for a high molecular weight, ie a molecular weight 232kDa and 298kDa (see Example 2, Tables 9, 11, 13 and 15). For a molecular weight of chitosan below 232 kDa, the clotting time with rabbit blood is greater than 12 minutes in the presence of an organic salt, according to FIGS. 3 and 5. This would justify the molecular weight value of chitosan recommended by the application WO 2011/060545 (232kDa according to the examples). The concentrations of chitosan used in WO 2011/060544 are less than 2% (tables, examples). In all the examples of this application, the clots are evaluated with freshly collected blood in the rabbit, which does not allow to conclude on the coagulation time or the mechanical properties of a chitosan / human blood mixture. This document does not describe how to prepare other chitosan-based compositions having a rapid coagulation time, nor how to improve the coagulation time especially of a coagulable human fluid. This document does not describe how to prepare a clot that spreads easily when applied to a fabric that is not arranged in a horizontal plane. This document suggests that clots break up into at least two fragments (Tables 10, 12, 14 and 16). Moreover, this document mentions that the homogeneity of the chitosan / blood mixtures is variable. It is therefore not easy to obtain a rapid coagulation of the blood clot that is integral, non-retracted, non-fragmented, resistant to compression and deformation. The patent application WO 2011/060554, of the same applicant as the application WO 201 1/060545 (Corporation of the Ecole Polytechnique de Montreal), describes the preparation of compositions based on chitosan and blood pre-solidified for their use for tissue repair. The concentrations of chitosan used in WO 201 1/060554 are less than 2% (tables, examples). The coagulation times described in the presence of a composition comprising chitosan and an organic salt are greater, largely, at 20 minutes. Such coagulation time is not desired by a clinical practitioner, usually the surgeon when to inject such a composition into a tissue of a patient. It is therefore appropriate to limit the coagulation time of a formulation of chitosan mixed with human blood. This document does not describe how to prepare other chitosan-based compositions with rapid clotting time, or how to improve coagulation time. This document does not describe how to prepare a clot that spreads easily when applied to a fabric that is not arranged in a horizontal plane. This document aims to provide compositions at physiological pH (pH 7). This document does not indicate how to prevent a clot fragmenting and also does not describe how to obtain a clot that is not presolidified, in particular to allow the injection of blood-chitosan mixture through a needle in the case of an arthroscopic implantation procedure.

The patent application WO 201 1/060553 describes a teaching close to the aforementioned patent applications (WO 201 1/060545, WO 2011/060544, and WO 201 1/060554), but relates to compositions in two parts, one comprising chitosan, the other the organic salt (glycerol-phosphate). It describes compositions comprising up to 2.5% chitosan (on page 8). Feasibility studies include 2-part compositions comprising chitosan with a molecular weight greater than 300 kDa (Table 3) and concentrations of 1.5% chitosan in solution (Table 6). The concentrations of chitosan used in mixture with human blood are less than 2% by mass relative to the mass of the chitosan solution. This document does not describe how to prepare other chitosan-based compositions with rapid clotting time, or how to improve coagulation time. This document does not describe how to prepare a clot that spreads easily when applied to a fabric that is not arranged in a horizontal plane. This document does not indicate whether the clots are fragmenting and does not address the specific problem of providing both homogeneous mixtures prior to coagulation and clots with both rapid coagulation and integrity, not retracted, no fragmented, resistant to compression and deformation. The patent application WO 02/00272 describes a composition and a method for repairing and regenerating cartilage or other tissues. This application teaches pages 15/16 that it is known to mix a chitosan with a glycerol-phosphate, the chitosan having a concentration of 0.5 to 3% and a high molecular mass (several hundreds of kDa) in particular to avoid precipitation chitosan at pH greater than 6.2. This document also teaches (page 16) that chitosan can remain in the soluble state at physiological pH but that it requires reducing the concentration or the molecular weight and the degree of deacetylation of chitosan (0.1%, 350 kDa, 50%). %). The examples support a chitosan concentration of 1.5% for its applications mixed with blood (Example 4). The concentrations of chitosan solutions used in mixture with human blood are less than 2%. This document does not suggest how to prepare other chitosan-based compositions exhibiting rapid clotting time, or how to improve coagulation time. This document does not describe how to prepare a clot that spreads easily when applied to a fabric that is not arranged in a horizontal plane.

The patent application WO 99/07416 describes a thermo-gellable composition based on chitosan. This document does not suggest how to decrease the coagulation time of a mixture of such a composition with blood. This document does not describe how to prepare a clot that spreads easily when applied to a fabric that is not arranged in a horizontal plane.

It is known that solutions of chitosan and GP are generally kept separately and reconstituted in the operating room before mixing with the blood, because of their heat-sensitive nature, as described for example by the application WO 2011/060553.

In general, we seek the shortest possible therapeutic or surgical procedures, in particular to minimize personnel costs, to be able to perform more daily interventions, avoid the risks of infection related to prolonged exposure to external environment, etc. The mini-arthrotomy and arthroscopy procedures as described in the prior art, for example by Stanish et al. (Oper Tech Orthop 16: 271, 2006) seem relatively long. Thus, it would be advantageous to shorten it, in particular by accelerating the formation of the clot which currently takes 15 to 25 minutes according to Tey et al. (Arthroscopy Techniques 4, 29, 2015) and Stanish et al (2006).

Today, there is a general tendency to practice endoscopic surgical procedures, to minimize the invasiveness, the risk of infection and to promote tissue repair. For example, Stanish et al. (Oper Tech Orthop 16, 271, 2006; J Bone Joint Surg Am. 95, 1640, 2013) indicate that implantation of a clot and blood and chitosan can be achieved by arthroscopy, if the lesion is fully visible in the field of view. Steinwachs et al. (Arthroscopy Techniques 3, 399, 2014) and Tey et al. described it in the case of knee and hip respectively. In this case, it is recommended that the mixture is injected before clot formation on the lesion comprising the microtunnels, and wait for the clot to form before removing the instruments. Tey et al. used a 18 gauge needle to inject the clot (inside diameter of 800 to 880μπι).

Tey et al (Arthroscopy Techniques 4, 29, 2015) have also recently shown that the clot formed by the mixing of blood and an ultra-high molecular weight chitosan solution could be used on joints other than the knee, despite the effect of gravity because the hip, for example, is in a vertical position during the operation of the patient. Until now, it was advisable to keep the patient in a position that places the site to be treated in a horizontal position, as described for example by Stanish et al (2013). and Steinwachs et al (2014), which is easily possible only with the knee. The need for repair of cartilage in the hip and shoulder is important. This approach has been proposed by Tey et al. to be able to use the blood / chitosan clot even in vertical position, by a procedure of arthroscopy in two stages, in particular because it was noticed that the clot was relatively adherent on the surface. However, it is necessary to improve the product to generalize its easy use for non-horizontal positions, including playing on its adhesion to the lesion and facilitating its administration by arthroscopy.

The prior art does not suggest how to improve this cartilage repair technology.

Thus, it emerges from the previous applications that, while certain technical approaches have been envisaged for preparing chitosan compositions having a low coagulation time mixed with human blood, the solution proposed by the present invention was not suggested. On the contrary, it appears that a person skilled in the art had focused on a concentration of chitosan less than or equal to 2% for its applications in mixture with the blood, and more specifically focused on chitosans having a molecular mass greater than 200kDa. Compositions having a high chitosan concentration have the technical disadvantage, in the presence of an organic sugar or polyol salt, of gelling at room temperature. This therefore poses a problem of stability of the chitosan formulation in the presence of such a salt organic in time, at room temperature. The skilled person was therefore encouraged to use a chitosan concentration of less than 2%.

In the case of the use of such a composition for the regeneration of cartilage, for example, the patient must be operated so that the composition is implanted at the level of the defective cartilage. During this operation, the clinical practitioner must wait for the composition containing chitosan mixed with the blood of the patient to coagulate, as described for example by Stanish et al. (Oper Tech Orthop 16: 271, 2006) and Tey et al. (Arthroscopy Techniques 4, 29, 2015), from 15 to 25 minutes. The longer the time, the more the patient is immobilized in the operating room. However, in general, the shortest possible surgical procedures are sought, in particular to minimize personnel costs, to be able to carry out more daily interventions, to avoid the risks of infection related to prolonged exposure to the patient. external environment, etc. Thus, it would be advantageous to shorten them, in particular by accelerating the formation of the clot. Furthermore, when the operation relates to joints other than the knee, for example the shoulder, or any other joint that does not appear in a horizontal plane when the patient is lying on the operating table, the composition may present a texture, and in particular a viscosity that is not adapted. In such a case, the composition, when brought into contact with the cartilage, may not remain in position or even flow. There is therefore the need to provide compositions having good integrity, good homogeneity of the constituents, good mechanical properties, good adhesion to the surface of the fabric to allow optimal implantation on a fabric that is not arranged in the plane horizontal when the patient is lying on an operating table.

The present invention aims to solve one or preferably many of the technical problems set forth above.

The object of the present invention is in particular to solve the technical problem of providing a composition based on chitosan and a coagulable fluid that can be used for tissue regeneration and that has a short coagulation time in the presence of a coagulable fluid, and in particular whole blood.

The present invention also aims to provide a composition based on chitosan which is homogeneous in the presence of a coagulable fluid, and in particular blood.

Another object of the present invention is to improve the mechanical properties of the coagulated chitosan / coagulable fluid mixture, and in particular of the chitosan / blood mixture. More specifically, the present invention aims to provide a composition having both rapid coagulation time, good homogeneity, and good mechanical properties, particularly to facilitate implantation on a non-horizontal plane.

The present invention also aims to provide such a composition so that it is easily injectable, in particular through a needle, and more precisely through a needle provided for an arthroscopy procedure.

In order for the arthroscopic procedure to become generalized, it is desirable for the mixture between the blood and the chitosan solution to be homogeneous, otherwise it would compromise the success of the repair over the entire lesion and lead to a non-homogeneous neo-cartilage. ; The injection mixture must also be low viscosity, to pass through a needle easily. It must finally be coagulated quickly so that it does not flow beyond the lesion to be treated.

It is also an object of the present invention to provide a chitosan-based composition that meets the maximum of technical criteria to provide an integral, non-shrunk, unfragmented, compressive and deformable clot.

The present invention also aims to provide stable compositions over time.

The present invention also aims to provide storage systems and administration of suitable compositions for their manufacture and preservation, and easy to use for the surgeon.

The present invention aims in particular to solve these technical problems reliably, industrial and inexpensive.

Blood coagulation is a complex phenomenon (primary and secondary hemostasis). Different parameters may positively or negatively affect the coagulation process during clot formation, especially when forming a blood clot.

it has been surprisingly discovered by the present inventors that it is possible to solve one or more of the aforementioned technical problems by preparing a composition comprising a mixture of a chitosan formulation and a coagulable fluid, wherein the formulation of chitosan has a concentration of at least 2.5% of mass chitosan relative to the total mass of chitosan formulation, said chitosan having a dynamic viscosity of less than or equal to 60 mPa.s, measured in acetic acid at 1 % (v / v), said composition comprising at least one inorganic salt. The coagulation rate is particularly optimized when the concentration of chitosan is greater than or equal to 3% by weight, relative to the mass of the chitosan formulation.

The composition according to the invention covers when it refers to the mixture of chitosan and fluid coagulable both to the fluid composition prior to coagulation of the coagulable fluid in the presence of chitosan, and the coagulated mixture. According to a variant of the composition of the invention relates to the fluid mixture. According to another variant, the composition of the invention relates to the coagulated mixture.

The term "chitosan formulation" means the solution comprising chitosan, the inorganic salt and any other ingredients of the formulation, just before mixing with the coagulable fluid. This is not the "chitosan solution" which refers to the chitosan solution before it comes into contact with the inorganic salt. Any other meaning is specified specifically, where appropriate, or the context makes this clarification unnecessary.

The term "coagulable fluid" means a body fluid (or biological) of a mammal, preferably a human being, which can be isolated from the body, for example by means of a syringe of samples, and which coagulates, that is, it ultimately forms a clot, in other words a thrombus. Among the coagulable fluids that may be mentioned in particular: a blood, a plasma, a platelet lysate, a platelet-rich plasma, a plasma rich in platelets without erythrocytes, and mixtures thereof. Blood refers to both whole blood and processed blood (that is, having undergone a technical operation), venous blood, arterial blood, bone blood, spinal cord blood, umbilical cord blood, placental blood.

The coagulable fluid can be enriched by amplifying the presence of one or more of its components. A component of the fluid may for example be erythrocytes, leucocytes, lymphocytes, polymorphonuclear cells, monocytes, platelets, fibrinogens, stem cells, thrombin hemoglobin, neutrophils, eosinophils, basophils, a coagulation factor.

According to one variant, human embryonic stem cells are excluded from stem cells.

The coagulable fluid may be supplemented with an exogenous agent, for example one or more coagulation factors, to obtain an optimal coagulation time, and / or the desired mechanical properties, when the coagulable fluid is coagulated, for example a calcium salt. . The coagulable fluid may also be supplemented with agents therapeutic. Among the therapeutic substances that may be mentioned are polysaccharides, polypeptides, liposomes, DNAs, antibodies, RNAs, proteins, peptides, extracellular matrix fragments, growth factors, chemotactic factors, cytokines, the factors angiogenic, living cells, for example chondrocytes, osteoblasts, stem cells.

it has surprisingly been found that increasing the concentration of chitosan in the chitosan formulation decreases the clotting time of the coagulable fluid when mixing this chitosan formulation with a coagulable fluid, and especially blood. Such a formulation of chitosan makes it possible to coagulate the coagulable fluid, in particular the blood, rapidly. Such a chitosan formulation makes it possible to form a three-dimensional structure with the coagulable fluid, forming an interpenetrating network with the components of the fluid and the chitosan in solution.

The chitosan formulation according to the invention makes it possible to obtain satisfactory results in terms of retractions of the coagulable fluid clot, in particular blood clots.

The chitosan formulation according to the invention makes it possible to obtain satisfactory mechanical properties, especially in terms of spreading and adhesion to the tissue to be repaired, in particular when it is not arranged in a horizontal plane.

The inventors have in particular discovered that when the concentration of chitosan is greater than 2%, the coagulation is not satisfactory if the dynamic viscosity of the chitosan solution is not in accordance with the present invention.

It has also been identified by the present inventors that compositions according to the prior art, comprising a mixture of chitosan with a molecular mass of greater than 200 kDa, in the presence of an inorganic salt, do not exhibit optimum viscosity and could lead to manipulation of the pre-coagulation composition and the blood clot. Indeed, it is difficult to easily prepare a blood / chitosan solution mixture prior to coagulation which is homogeneous, that is to say for which the two parts of the composition are intimately mixed and form an interpenetrating network. However, it is important that the pre-coagulation composition is homogeneous, since the entire surface of the lesion as well as the microtunnels that are formed in the cartilage lesion must be treated with the same amount of chitosan and blood, to promote formation of a new cartilage of good quality spread over the newly formed volume. The compositions of the invention have a dynamic viscosity allowing a good spreading of the composition of chitosan mixed with a coagulable fluid when it is coagulated.

The coagulated compositions of the invention also exhibit good adhesion at the site of application, for example on cartilage, and more generally on bone tissue or other tissue.

A tissue is a set of similar cells of the same origin, grouped into a functional whole, that is to say, contributing to the same function.

Chitosan is for example referenced under the CAS number 90 2-76-4.

The chitosan used for the invention is advantageously of fungal origin, and preferably derived from the mycelium of a mushroom of the Ascomycete type, and in particular of Aspergillus niger, and / or of a Basidiomycete fungus, and in particular Lentinula edodes. (shiitake) and / or Agaricus bisporus (Paris mushroom). Preferably, the chitosan is derived from Agaricus bisporus. The chitosan is preferably very pure, that is to say containing few impurities from its fungal origin, and a microbiological quality compatible with its use as an implant or pharmaceutical composition. A method for preparing chitosan is that described, for example, in patents of the family of WO 03/068824 (EP 1483299, US 7,556,946, etc.).

The average molecular weight of chitosan can be estimated by the dynamic viscosity value of the 1% (w / w) solution of chitosan in 1% (v / v) acetic acid. The "1% (m / m) solution of chitosan" refers to a distilled water solution comprising 1% by weight of chitosan relative to the total mass of the chitosan solution in distilled water. To this 1% (w / w) solution of chitosan is added acetic acid to obtain a 1% (w / w) solution of chitosan and 1% (v / v) of acetic acid. The chitosan powder can also be added in an acetic acid solution to reach 1%. Acetic acid 1% (v / v) means a concentration of 1% by volume of acetic acid relative to the volume of the final distilled water solution.

The dynamic apparent viscosity is measured using a rotating mobile viscometer, for example a rotational mobile viscometer of the Brookfield brand, for example equipped with an S 34 type spindle at an adjusted speed. depending on the viscosity by the man of the art of 0.3 to 100tr / min according to the recommendations related to the viscometer, and for example 5 rev / min, and at a temperature of 25 ° C.

Advantageously, the chitosan used according to the invention has, according to the aforementioned measurement method, a dynamic viscosity of between 5 and 60 mPa.s, for example from 5 to 55mPa.s when in solution at the concentration of 1% (m / m) in 1% (v / v) acetic acid.

According to one variant, the chitosan used according to the invention has a dynamic viscosity of between 5 and 25mPa.s, for example from 8 to 20mPa.s, when it is in solution at a concentration of 1% (m / m). in acetic acid 1% (v / v).

According to one variant, the chitosan used according to the invention has a dynamic viscosity of between 26 and 50mPa.s, for example from 30 to 45mPa.s, when it is in solution at a concentration of 1% (m / m). in acetic acid 1% (v / v).

The chitosan can be of different molecular weights, in the molecular weight range according to the present invention.

In particular, the invention relates to a chitosan having a molecular weight Mv ranging from 20,000 (20k) to 145,000 (145k).

According to one variant, the average viscosity (Mv) molecular weight of chitosan is between 20,000 (20k) and 120,000 (120k).

According to one variant, the average viscosity (Mv) molecular weight of chitosan is between 20,000 (20k) and 60,000 (60k).

According to another variant, the average molecular weight in viscosity (Mv) of chitosan is between 60,000 (60k) and 100,000 (100k).

It is possible to hydrolyze chitosan to reduce its molecular weight. Preferably, the average molecular weight is the viscosity average molecular weight (Mv), calculated from the intrinsic viscosity according to the Mark-Houwink equation. The intrinsic viscosity is measured by capillary viscosimetry, with a Ubbelohde capillary viscometer, according to the method of the European Pharmacopoeia monograph EP2.2.9. The flow time of the solution is measured through a suitable capillary tube (Lauda, for example the Ubbelohde 510 01 tube of 0.53 mm diameter) with the aid of a Lauda Vise automatic viscometer, first at the initial concentration of chitosan, then for several dilutions, for example according to the recommendations of method EP2.2.9. The reduced intrinsic viscosity is deduced for each of the concentrations. The reduced viscosity is plotted as a function of temperature, and the value is extrapolated to the concentration 0 to deduce the intrinsic viscosity. For example, the reduced viscosity (r | _red in ml / g) of the dilutions as a function of the concentration C of the dilutions (g / ml) according to formula 2 should be used.

Formula 2. [¾] = (t, - 1 ₀ ) - (1 - C).

To calculate the average viscometric mass, we apply the Mark-Houwink equation with the k and alpha constants recommended by Rinaudo et al. (J. Biol. Macromol, 1993, 15, 281-285), according to the degree of acetylation (DA) of chitosan, according to one of the following three formulas.

Formula 3. Mv = ([η] / 0.082) ^(1/0 - ⁷⁶⁾ , for a DA of 2%;

Formula 4. Mv = ([η] / 0.076) ⁽ 100% ⁾ , for a DA of 10% (for example 11.5%);

Formula 5. Mv = ([η] / 0.074) ^(1/76) , for a DA of 20% (for example 21%).

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

Advantageously, chitosan has a degree of acetylation of between 5 and 50%. The degree of acetylation is expressed as the number of moles of N-acetyl-D-glucosamine units relative to the number of moles of total N-acetyl-D-glucosamine and D-glucosamine units present.

According to one variant, the degree of acetylation is between 25 and 40%.

According to one variant, the degree of acetylation is between 20 and 30%.

According to one variant, the degree of acetylation is between 15 and 25%.

According to a preferred variant, the degree of acetylation is between 5 and 20%.

According to a preferred variant, the degree of acetylation is from 5 to 20%, and more preferably from 5 to 15%.

The degree of acetylation is determined by potentiometric titration. Chitosan is dissolved in a solution of hydrochloric acid. The excess of unreacted hydrochloric acid with the amino functions of chitosan is determined by a standard solution of sodium hydroxide. This gives the number of moles of D-glucosamine unit present in the chitosan and thus by subtraction the degree of acetylation.

Chitosan advantageously has a controlled degree of acetylation. By the term "chitosan having a controlled degree of acetylation" is meant a product whose degree of acetylation, i.e. the proportion of N-acetyl-glucosamine units, can be adjusted in a controlled manner.

According to a specific variant, the invention uses a chitosan having a degree of acetylation of between 5 and 20%, a dynamic viscosity (measured as described above, with a 1% (m / m) solution of chitosan in acetic acid 1% (v / v)) of 5 and 60mPa.s, for example from 5 to 55mPa.s.

According to a very advantageous variant, the invention uses a chitosan having a degree of acetylation of between 5 and 20%, and a dynamic viscosity (measured as described above, with a 1% solution (m / m) of chitosan in acetic acid 1% (v / v)) of 5 and 25mPa.s, for example from 8 to 20mPa.s. According to a very advantageous variant, the invention uses a chitosan having a degree of acetylation of between 5 and 15%, and a dynamic viscosity (measured as described above, with a 1% solution (m / m)). of chitosan in acetic acid 1% (v / v)) of 5 and 25mPa.s, for example from 7 to 20mPa.s.

The chitosan is advantageously purified and then preferably dried. After purification, the process of the invention may comprise a step of drying the substituted chitosan, and optionally grinding it to obtain a powder. The chitosan can be dried, for example by evaporation of water, for example by a spray-drying (atomization), fluidized bed, vacuum drying or atmospheric pressure, or by freeze-drying.

The chitosan may be solubilized in an aqueous solution, and for example in a pharmaceutical grade water acceptable for injection or implantation into a body, and in particular a human body.

Advantageously, the dissolution of chitosan in an aqueous solution is carried out by adjusting the pH to a sufficiently acidic pH by the addition of an acid. Hydrochloric acid, and preferably an organic acid, and more preferably acetic acid, can be used advantageously. Advantageously, the aim is a pH of between 4.5 and 8.5, and preferably between 5.5 and 6.5.

Thus according to one variant, the chitosan solution or formulation comprises an acid. The acid may be present at a concentration of 0.001 to 0.1N, for example about 0.01 N.

A composition according to the invention having a higher concentration of chitosan than the compositions of the prior art is interesting because of the intrinsic properties of chitosan which are beneficial for the repair of cartilage, as described by Hoemann et al. (Osteoarthritis & Cartilage 15, 78, 2007), Chen et al. (Cartilage 2, 173, 2011), or Stanish et al. (J Bone Joint Surg 95, 1640, 2013) in combination with a microfracture bone marrow stimulation procedure, or as described by Oprenyeszk et al. (Osteoarthritis Cart 21, 1099, 2013; Plos One 10, 2015).

In particular, reference may be made to application WO 2011/060553 for the various complement activation pathways.

According to one variant, the chitosan formulation has a chitosan concentration of greater than 2.5% and less than 10% by weight relative to the final formulation mass. Advantageously, the concentration of chitosan is greater than or equal to 2.8%, for example greater than or equal to 3%, or even greater than or equal to 3.2% by weight relative to the total weight of the formulation of chitosan (m / m).

According to a specific variant, the concentration of chitosan ranges from 2.9 to 3.5% (w / w), expressed in mass relative to the total weight of the chitosan formulation.

According to a specific variant, the concentration of chitosan is greater than 3.5% (m / m), expressed in mass relative to the total mass of the chitosan formulation.

According to a specific variant, the concentration of chitosan is less than 6% (w / w), expressed in mass relative to the total mass of the chitosan formulation.

According to a specific variant, the concentration of chitosan ranges from 3.6 to 4.5% (w / w), expressed in mass relative to the total weight of the chitosan formulation.

According to a specific variant, the concentration of chitosan ranges from 4.6 to 5.5% (w / w), expressed in mass relative to the total weight of the chitosan formulation.

According to a particular variant, the concentration of chitosan is approximately 3.0%

(m / m), expressed in mass relative to the mass of the chitosan formulation.

According to a particular variant, the concentration of chitosan is approximately 4.0% (w / w), expressed in mass relative to the mass of the chitosan formulation.

According to one particular variant, the concentration of chitosan is approximately 5.0% (w / w), expressed in mass relative to the mass of the chitosan formulation.

According to a very advantageous variant, the invention relates to a chitosan formulation and a chitosan solution for preparing said chitosan formulation, said chitosan, having a degree of acetylation of between 5 and 20%, and a dynamic viscosity (measured as as previously described, with a 1% (m / m) solution of chitosan in 1% (v / v) acetic acid) of 5 and 25mPa.s, for example from 8 to 20mPa.s, said chitosan formulation having a chitosan concentration greater than or equal to 3%, or even greater than or equal to 3.2% by weight relative to the total weight of the chitosan formulation (m / m).

According to a very advantageous variant, the invention uses a chitosan formulation and a chitosan solution to prepare this chitosan formulation, said chitosan having a degree of acetylation ranging from 5 to 15% and a dynamic viscosity (measured as as previously described, with a 1% (w / w) solution of chitosan in 1% (v / v) acetic acid) of 5 and 25mPa.s, for example 7 to 20mPa.s., said formulation of chitosan having a chitosan concentration greater than or equal to 2.8%, for example greater than or equal to 3%, or even greater than or equal to or 3.2% by weight relative to the total mass of the chitosan formulation (m / m).

The invention advantageously avoids the technical problems related to the presence of a sol-gel transition at room temperature and are particularly stable at room temperature or refrigerated.

The pH of the chitosan solution can be adjusted by the addition of an acid, preferably a weak organic acid, preferably acetic acid, which has the advantage of providing a composition whose pH is easily modulated. and precisely.

Advantageously, the pH of the chitosan solution is greater than the pKa of the amine groups of chitosan in aqueous solution and less than 6.5. Advantageously, the pH of the chitosan solution is greater than 5.5. According to a specific variant, the pH of the chitosan solution is about 6.0.

According to one variant, the pH ranges from 5.0 to 6.5, and preferably from 5.5 to 6.5, and more preferably from 5.8 to 6.2.

According to a preferred variant, the pH is less than 7.0.

According to a specific variant, the pH of the chitosan formulation is 6.2 +/-

0.2.

According to another specific variant, the pH is 6.1 +/- 0.2.

According to another specific variant, the pH is 6.0 +/- 0.2.

According to another specific variant, the pH is 5.9 +/- 0.2.

The pH is measured on the chitosan formulation. The pH is determined by following the method described in the European Pharmacopoeia (EP 2.2.3). The pH meter used is a pH meter from the Sartorius range with a combined glass electrode (PY-P1 1). PH measurements are made between 20 and 25 ° C.

According to one embodiment, the chitosan formulation has an osmolality of 100 to 700mosm / kg, preferably 200 to 50mosm / kg.

Advantageously, the osmolality of the chitosan formulation is between 250 and 400 mosm / kg, and preferably from 275 to 325 mosm / kg.

According to one variant, the chitosan formulation exhibits an osmolality that is compatible with a joint.

It is preferable that the osmolality of the chitosan formulation be between 250 and 400, and more specifically between 250 and 380mosm / kg.

The determination of the osmolality of the solutions is carried out with an automatic micro osmometer (Osmometer Type 15M of the brand Lôser Messtechnik). The equipment is calibrated beforehand with a solution of 300mosm / kg. The sample is placed in a container provided for this purpose, and is put to the standard temperature of the measurement.

Advantageously, the inorganic salt is an agent adjusting the osmolarity of the chitosan solution.

According to one variant, the composition comprises a buffer, for example an acetate buffer.

Buffering agents are known to those skilled in the art.

At least one polyol and in particular a reducing sugar may be used in the composition of the invention. Such polyols may for example be selected from the group consisting of: isopropanol, sorbitol, mannitol, alkylene glycol such as propylene glycol, poly (alkyl glycol), for example poly (ethylene glycol), fructose, galactose, ribose, glucose, xylose, rhamnulose, sorbose, erythrulose, deoxyribose, ketose, mannose, arabinose, fuculose, fructopyranose, ketoglucose, sedoheptulose, trehalose, tagatose, sucrose, allose, threose, xylulose, hexose, methylthio-ribose, methylthio-deoxy-ribulose, and any of their mixtures.

Preferably, the composition comprises at least one reducing sugar, for example mannitol.

According to one variant, the chitosan formulation may also comprise one or more other inorganic salts.

Among the inorganic salts (main salt or additional salt, designated as other inorganic salt), mention may be made, for example, of a sodium salt, a salt of chloride, of potassium, of calcium, of magnesium, of phosphate, of sulphate, or of salt any of their mixtures. According to a particular variant, the inorganic salt may be potassium chloride, sodium chloride, cesium chloride, calcium chloride, cesium fluoride, potassium perchlorate, sodium nitrate, calcium sulphate, or any of their mixtures.

According to an advantageous variant, the chitosan formulation comprises a chloride salt or a sodium salt.

According to a specific variant, the chitosan formulation comprises a salt of sodium chloride.

According to one variant, the chitosan formulation comprises a calcium salt as another inorganic salt (secondary or additional inorganic salt).

According to one variant, the present invention relates to a chitosan formulation comprising at least 2% by weight of chitosan, and preferably from 1 to 10% by weight. inorganic salt, for example sodium chloride, and optionally from 0.1 to 20% reducing sugar. Advantageously, the chitosan formulation comprises 2.8 to 6.5% by weight of substituted chitosan and of 0.1 and 5%, preferably about 1%, by weight of inorganic salt, for example sodium chloride, and optionally from 0.1 to 15% by weight of reducing sugar, relative to the total mass of the composition.

The chitosan formulation and solution of the invention does not include an organic salt which would render the resulting mixture gellable (presence of a sol-gel transition, particularly under the conditions of use). In other words, the chitosan formulation according to the invention advantageously does not comprise any agent which would cause the gelation or the thermo-gelable character of the chitosan solution.

Advantageously, the chitosan formulation is not gellable, that is to say does not undergo a sol-gel transition at a temperature above 10 ° C and preferably between 10 and 50 ° C. Thus, the invention advantageously relates to a non-thermogelifiable, and preferably non-gellable, chitosan formulation.

In particular, the formulation and the chitosan solution of the invention do not comprise a glycerol phosphate salt.

In general, from a safety point of view, it is preferred to limit the number and the dose of the components of any composition for therapeutic or medical use, in particular to limit the composition to the compounds which are necessary for its composition. clinical effect and / or its mode of action, especially in the case of an implantable or injectable composition to be in contact with the cartilage and the bone for a prolonged period. The compositions without thermogelifiable agent or without organic polyol salt or sugar are therefore advantageous. Another advantage is that the chitosan formulation can be packaged and stored in a single package without the need for reconstitution of the formulation prior to mixing with the blood. Another advantage is that it is stable over time because it is not likely to gel.

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

This process typically comprises:

dissolving a chitosan in an aqueous solution, a dilute acid, preferably water and a dilute acid, preferably a weak acid, preferably an organic acid, optionally a buffered solution of an acid and a weak base, preferably at a pH of between 4.5 and 8.5, and preferably between 4.5 and 7.0, more preferably between 5.5 and 6.5;

the possible adjustment of the pH for example to the pH of 6.0; the mixture with an inorganic salt solution, for example a salt of sodium chloride;

the possible adjustment of the osmolality of the solution;

the possible adjustment of the viscosity of the solution.

Chitosan formulations in the form of a solution with at least one inorganic salt can be prepared as follows:

the dispersion of chitosan in an aqueous solution, such as for example water,

the addition of an acidic solution, preferably a weak organic acid, preferably acetic acid, for solubilizing chitosan and adjusting the pH, for example at a pH of between 4.5 and 6.9, and preferably between 5.0 and 6.9, and for example preferably at pH 6.0 to solubilize chitosan in the form of a solution,

the addition of at least one inorganic salt and optionally of formulation excipients,

sterilization, preferably by steam autoclave,

- possibly packaging for the preservation of the solution.

Chitosan formulations in the form of a solution with at least one inorganic salt can also be prepared as follows:

the dispersion of chitosan in an aqueous solution, such as for example water,

the addition of an acidic solution for adjusting the pH, for example at a pH of between 4.5 and 7, and preferably between 5 and 6.9, and for example at a pH of 6, preferably for solubilizing chitosan under solution form,

sterilization, preferably by steam autoclave,

- Optionally the packaging for the preservation of the chitosan solution, for example a bottle or a syringe.

optionally drying or lyophilization of the chitosan solution in the packaging,

contacting the chitosan with at least one inorganic salt and optionally excipients, preferably in the form of an inorganic salt solution, preferably sterilized, preferably by filtration or by autoclave,

- optionally sterilization, preferably with steam,

- possibly packaging for the preservation of the solution. Chitosan formulations in the form of a solution with at least one inorganic salt can be further prepared in the following manner:

- the supply of a chitosan in dry or freeze-dried form,

optionally dispersing the chitosan in an aqueous solution, such as for example water,

- sterilization, preferably by steam,

optionally drying or lyophilizing the chitosan solution,

optionally packaging for the preservation of chitosan, optionally in dry or freeze-dried form.

Contacting the chitosan with at least one inorganic salt, an acid for adjusting the pH, preferably at a pH of 5.5 to 6.5, and for example at pH 6, preferably for solubilizing chitosan in the form of solution in the presence of the inorganic salt, and optionally excipients, preferably to provide an acidic solution of chitosan and inorganic salt,

- optionally sterilization, preferably with steam,

- possibly packaging for the preservation of the solution.

The inorganic salt (s) may be contacted with the chitosan solution as one or more solutions of one or more inorganic salts. Inorganic salt solutions are commercially available.

The inorganic salt can be added to the chitosan solution after sterilization thereof, for example.

According to one variant, the dissolution of chitosan is carried out in water comprising a reducing sugar, such as, for example, mannitol.

According to one variant, the buffering agent is an acetate buffer.

Advantageously, the method also comprises a subsequent step of filling an injection device, such as for example a syringe, with the chitosan formulation or the chitosan solution. Advantageously, the injection device, such as for example a syringe, can then undergo steam sterilization. This device, for example a syringe, can then be packaged, preferably in a sterile manner.

It is advantageous to use a chitosan having a degree of purity sufficient for the intended application.

The invention also covers a chitosan or a formulation of chitosan in a dry form, especially in a freeze-dried form. In particular, the lyophilized product can be (re) dispersed and preferably solubilized before use.

According to one variant, the chitosan may be in dry form, in particular in a freeze-dried form, before it is put in contact with the inorganic salt, which may be in the form of an aqueous solution.

Thus, the invention relates to a chitosan in dry form, in particular in powder form, for example in freeze-dried form, suitable and intended to be dissolved in a solution comprising at least one inorganic salt, such as those mentioned above, the resulting formulation being suitable and intended to be placed in contact with a coagulable fluid.

According to a preferred variant, the invention therefore relates to a medical device comprising a chamber containing a chitosan in a dry form, in particular in a freeze-dried form, and a chamber containing a solution of at least one inorganic salt.

According to another variant, the chitosan may be formulated with at least one inorganic salt, then dried, and in particular freeze-dried. The resulting freeze-dried formulation can be dispersed, and preferably solubilized as an aqueous solution, before mixing with the coagulable fluid.

Thus, the invention relates to a medical device comprising or consisting of a chitosan formulation at a concentration of at least 2.5% of mass chitosan with respect to the volume of chitosan formulation, said chitosan having a dynamic viscosity less than or equal to 60mPa.s, measured in 1% (v / v) acetic acid, to be mixed with a coagulable fluid, and at least one inorganic salt.

Advantageously, the medical device comprises at least one, and preferably only one, container containing a formulation of chitosan and at least one inorganic salt in the form of a solution, said chitosan having a dynamic viscosity of less than or equal to 60 mPa.s. , measured in 1% (v / v) acetic acid, said formulation having a concentration greater than or equal to 2.5% of chitosan by mass relative to the weight of chitosan formulation.

The invention further relates to a medical device comprising a plurality of containers, said containers being possibly physically independent ("kit-of-part"), characterized in that the medical device comprises at least (i) a container containing a chitosan in dry form or in solution, said chitosan having a dynamic viscosity less than or equal to 60mPa.s, measured in 1% (v / v) acetic acid, and (ii) a container containing a solution of at least one inorganic salt, in wherein the chitosan and the solution of at least one inorganic salt have a concentration capable of being greater than or equal to 2.5% of mass chitosan relative to the weight of chitosan formulation, the chitosan formulation being formed by the in contact with chitosan, in dry form or in solution, and said solution of at least one inorganic salt.

By way of example, mention may be made of double-chamber syringe type devices in series or in parallel, for example described in patent EP 051 1402, EP 0728492, US 3749084, US 4538920, US 5971953, US 20120265171, or else the different double-chamber syringes marketed by Vetter-Pharma (Vetter Lyo-Ject® dual-chamber syringe), Unilife (EZMix Genesis ™), Tyfill (double chamber syringe), VDW (2Seal easymiX®).

The invention also relates to a medical device comprising a composition, a solution or a dry form (powder for example) of chitosan according to the invention.

Advantageously, the chitosan formulation comprising at least one inorganic salt can therefore be disposed in a chamber of a medical device comprising a single chamber comprising the chitosan formulation comprising at least one inorganic salt. According to a preferred variant, the invention therefore relates to a medical device comprising a chamber, and for example a single chamber, containing a chitosan formulation mixed with at least one inorganic salt.

The invention therefore relates in particular to one or more articles or packaging, preferably sterile, comprising one or more pre-filled medical injection devices, said medical devices being as defined in the invention. These are typically syringes or bottles for biological use pre-filled with a chitosan formulation, a chitosan solution or a chitosan powder.

Such formulations of chitosan (comprising or not the inorganic salt mixed with chitosan) or such medical devices may for example be stored at ambient temperature (20 ° C.), which is very advantageous. Advantageously, these chitosan solutions are stable for several months at an ambient or refrigerated temperature (in general 4 ° C. to 8 ° C.), and in particular at 4 ° C.

For mixing with the coagulable fluid, the chitosan formulation or the chitosan solution is contacted with the coagulable fluid, and then the assembly is mixed.

Alternatively, the initial chitosan solution is contacted with the inorganic salt, and then the resulting chitosan formulation is mixed with the coagulable fluid, and in particular the blood. According to one variant, the initial chitosan solution is mixed with the coagulable fluid, and in particular the blood, and then the inorganic salt solution is added to the mixture.

Mixing with the coagulable fluid can be accomplished in the medical device containing the chitosan formulation or solution (with or without inorganic salt) via a system designed to make this mixture, or in a separate device. According to a variant which concerns an implantation procedure, the clinical practitioner takes the coagulable fluid by a syringe and then injects this fluid into a bottle containing the formulation or the chitosan solution, and then shakes this bottle. The mixture is left to stand or stirred until a satisfactory coagulation. Then, the clinical practitioner takes the coagulated mixture with a spatula to arrange or implant the clot at the desired location. According to another variant which relates for example to an arthroscopic procedure, the clinical practitioner takes the coagulable fluid by a syringe and then injects this fluid into a bottle containing the formulation or the chitosan solution, and then agitates this bottle. The present invention relates to a composition comprising or consisting of a mixture of chitosan and coagulable fluid in a coagulable fluid / chitosan volume ratio ranging from 1: 1 to 4: 1, and for example from 2: 1 to 3: 1, this ratio being expressed as a coagulable fluid volume with respect to the volume of chitosan formulation (v / v).

According to one variant, the coagulable fluid / chitosan volume ratio is 3: 1.

It is indeed not desired to reduce the proportion of blood in the mixture, for example in order to promote the homogeneity of the mixture or to promote faster clot formation, since it is desired to maintain a concentration of active components of the mixture. blood, these components being beneficial for cartilage repair. This ratio of volume 3: 1 has been shown to be favorable for cartilage repair (Stanish et al., 2006).

The present invention more particularly relates to an injectable or implantable composition comprising or consisting of a composition according to the invention.

According to one variant, the composition according to the invention is used as a pharmaceutical composition or an injectable or implantable medical device.

The present invention allows sterilization, especially sterilization by steam autoclaving chitosan solutions or formulations.

Advantageously, the chitosan solution or formulation according to the present invention is sterile. Very advantageously, the formulation and / or solution of chitosan according to the present invention are sterilized by temperature rise, preferably under autoclave.

According to one variant, the formulation or solution of chitosan is sterilized by steam, for example by raising the temperature to a temperature greater than 100 ° C., and preferably greater than 120 ° C., for example between 121 and 138 ° C. under an autoclave, for a sufficient period of sterilization, for example in general from 15 to 25 minutes.

According to one variant, the chitosan formulations of the invention are transparent or translucent.

By "translucent" we mean that we can distinguish an object when placing the formulation of chitosan between the eye of the observer and the object. By "transparent" it is meant that alphanumeric characters can be distinguished when placing the chitosan formulation between the observer's eye and the observed characters. In general, this evaluation is carried out with a thickness of about 1 cm.

According to one variant, the chitosan formulation of the invention is colorless, that is to say in particular that an observer with the naked eye does not assign a specific color to the chitosan formulation.

In particular, the present invention relates to a composition, formulation, solution or chitosan powder according to the invention, possibly included in a medical device, for its use in a therapeutic treatment method.

The invention relates to a composition, formulation, solution or chitosan powder according to the invention, optionally included in a medical device, for its use in a method of therapeutic treatment of an articular disease.

The present invention relates more particularly to a composition or a medical device according to the invention for its use in a method of therapeutic treatment, for example comprising the injection or implantation subcutaneously, intradermally, intraocularly, intraosseous or intra- joint, of said composition, for example for the repair or filling of at least one body tissue requiring repair or filling.

According to one variant, the body tissue is chosen from the tissues belonging to the vocal cords, muscles, ligaments, tendons, cartilages, sexual organs, bones, joints, eyes (cornea), skin (dermis), a periodontal tissue, a maxillary tissue. facial tissue, temporomandibular tissue, abscess, resected tumor, ulcer, or any of their combinations, and more particularly at joint joints.

The invention notably allows the treatment of a meniscus. The present invention relates more particularly to a composition according to the invention for its use in a method of treating osteoarthritis, or the repair of a cartilage defect, for example by injection or after mixing with the coagulable fluid and implantation in the cartilage.

The present invention relates more particularly to a medical device, for example a medical implant, characterized in that it comprises or consists of a composition according to the invention and optionally a system for mixing the components of the composition and a system of administration of the composition.

The present invention may also include one or more additives or excipients for modulating its properties. According to a specific variant, the composition of the invention comprises a suspension of particles, for example solid or gel particles.

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

According to the invention, the chitosan formulation is particularly intended to be mixed with blood or plasma, or a platelet lysate, or platelet-rich plasma, or any coagulable fluid in the presence of the formulation of the invention allowing, for example, 'increase product performance.

The present invention thus relates to a method of therapeutic treatment implementing the injection or implantation of a composition according to the present invention. Therapeutic treatment methods cover any use of the composition such as for example those mentioned above.

The present invention particularly covers an in situ treatment method and the preparation of the chitosan composition in situ. So mixing with a fluid coagulable can be achieved at the implantation site or injection, the coagulable fluid is available at this site.

The invention also relates to the use of a composition according to the invention for the preparation of a pharmaceutical composition for a therapeutic treatment.

As stated above, the invention relates in particular to a method of therapeutic treatment of a joint, such as the meniscus.

According to one variant, the chitosan formulation is applied directly at the injection or implantation site for coagulation with a coagulable fluid available at the injection or implantation site.

The methods of treatment according to the invention can be implemented according to the methods known to those skilled in the art.

The term "composition according to the invention", "chitosan formulation according to the invention", "chitosan solution according to the invention", or equivalent terms, a composition, a chitosan formulation, or a chitosan solution, respectively, as defined in the present invention, including any of the variants, particular, specific or advantageous embodiments, independently or in any of their combinations, including according to the preferred features.

By "between" a numerical value and another numerical value is meant a numerical range including terminals. These terminals can however be excluded.

The present invention also covers a method of aesthetic care, in other words non-therapeutic, comprising the injection or implantation of a composition according to the invention. This is for example the filling of wrinkles or the filling of one or more visible tissue areas damaged, for example following an accident or surgery, for aesthetic purposes.

Other objects, features and advantages of the invention will become apparent to those skilled in the art following the reading of the explanatory description which refers to examples which are given by way of illustration only and which can not in in no way limit the scope of the invention.

The examples are an integral part of the present invention and any features appearing novel from any prior art from the description taken as a whole, including the examples, form an integral part of the invention in its function and its generality.

Thus, each example has a general scope. On the other hand, in the examples, all percentages are given by weight, unless otherwise indicated, and the temperature is expressed in degrees Celsius unless otherwise indicated, and the pressure is atmospheric pressure unless otherwise indicated. Examples

Example 1 Characteristics of Chitosan of Different Molecular Weights

Chitosans of different molecular masses are prepared in the following manner: the chitin is suspended in the presence of aqueous sodium hydroxide, then the medium is heated at high temperature for a variable duration depending on the desired molecular weight. The chitosan is then purified by solubilization in acid medium and precipitated in an alkaline medium, washed and dried. The dynamic viscosity of solutions of different fungal and crustacean chitosan of concentration 1% (w / w) in 1% (v / v) acetic acid was determined using a Brookfield rotating mobile viscometer equipped with of an S-type spindle 34. The viscosimetric average molecular weight (Mv) is also determined by capillary viscometry, and the degree of acetylation by potentiometric titration.

Table 1- Characteristics of original chitosan and variable molecular mass

Viscosity

DDA

Ref Dynamic range range at 1% Mv

(Mol%)

(MPa.s)

Ultra-

CS1 A.bisporus 10 55000 86

low (UL)

Ultra-

CS2 A.bisporus 15 75000 85

low (UL)

CS3 Low (L) A.bisporus 33 100000 81

CS4 Low (L) A.bisporus 40 115000 78

Medium

CS5 A.bisporus 50 130000 75

(M)

CS6 High (H) A.bisporus 154 220000 72 Ultra-

CS7 high Crustacean 489 ND 83

(UH)

Ultra-

CS8 A.bisporus 12 ND 87

low (UL)

CS9 Low (L) A.bisporus 32 100300 81

Medium

CS10 A.bisporus 63 150400 77

(M)

Medium

CS11 A.bisporus 78 ND 75

(M)

CS12 High (H) A.bisporus 101 190500 73

ND - Not determined

It can be seen that the average molecular weight of chitosans, determined by capillary viscometry, and the dynamic viscosity of their solutions at a concentration of 1% (w / w) in 1% (v / v) acetic acid are correlated. Chitosan are classified by molecular weight range, from 'ultra-low' to 'ultra-high'.

Thus, the viscosimetric average molecular weight of a chitosan can be estimated from the dynamic viscosity of the 1% (w / v) solution in 1% (v / v) acetic acid. For example, the crustacean CS7 reference chitosan has an estimated viscosimetric average molecular weight greater than 400,000, given its solution viscosity of 489mPa.s.

Thus, the 1% dynamic viscosity value can be used to estimate the molecular weight of a chitosan.

Example 2 - Dynamic viscosity of solutions of chitosan in acetic acid 1% (v / v) as a function of the chitosan concentration (%, m / m)

The chitosans of different molecular masses prepared according to Example 1 are dissolved in 1% (v / v) acetic acid at concentrations of 0.5% to 5%. The dynamic viscosity is measured using a Brookfield rotating mobile viscometer equipped with an S 34 type spindle. Table 2 - Dynamic viscosity (mPa.s) as a function of chitosan concentration

Concentration

0.5% 1.0% 1, 5% 2.0% 2.5% 3.0% 4.0% 5.0%

(%, m / m)

Reference

Dynamic Viscosity Range (mPa.s)

chitosan

UL CS1 10 39

UL CS2 15 360

L CS3 33 130

M CS5 50 250

H CS6 154 700

UH CS7 489 1540

UL CS8 12 22 38 64 105 244

L CS9 32 79 162 319 594 1905 5712

M CS10 62 171 451 996 2060 7700 38000

M CS11 78 237 579 1194 2790 11900

H CS12 27 101 307 828 2105 4210 24940

It is understood from Table 2 that the viscosity of the chitosan solutions increases with the concentration of chitosan, especially as the molecular weight is high. The relation between the logarithm of the viscosity and the concentration of chitosan is linear. The slope of the curve of the log of the viscosity as a function of the concentration is all the higher as the molecular weight of the chitosan is high. The inventors have surprisingly put forward that the chitosans of the invention are particularly well suited for preparing blood clots in an easy manner and which are homogeneous, because for high molecular weight chitosans, the chains are too long and may have reduced mobility and result in inhomogeneous mixtures of chitosan with the blood before the clot forms.

Chitosan of lower molecular weight, for example in the 'low' and 'ultra-low' range, make it possible to increase the concentration without compromising the homogeneity of the mixture with the blood.

Example 3 - rapidity of clot formation with solutions of chitosan with pH and variable compositions

In this example, mixtures of chitosan solutions of molecular weight (expressed in terms of viscosity at 1%) and fresh blood taken from human volunteers are formed in order to obtain as quickly as possible less than 15 minutes, a clot that best satisfies the following properties preferably simultaneously: homogeneous, firm, non-retracted, unfragmented, resistant to manual compression and to a deformation such as passage in a trocar.

The clinical objective is that this clot can be easily handled by a surgeon, to affix it on an osteochondral surface, for example a lesion of the cartilage having been previously pierced by microfractures - in the form of microtunnels. The procedure can be done by implantation, or by atheroscopy, that is to say through a small diameter needle, under endoscopy, as described by Stanish et al. (J Bone Joint Surg Am. 95, 1640, 2013) and Steinwachs et al. (Arthroscopy Techniques 3, 399, 2014). In this case, it is advantageous for the clot to be flexible, non-fragmented and resistant so that it can be injected through the needle and positioned homogeneously at the entrance of the microtunnels, in order to fill them and stimulate the regeneration of the cartilage in an equivalent way over the entire lesion.

It is desired that the mixture of the fresh blood of the patient and the chitosan solution is homogeneous before the formation of the clot; indeed the repair must be done homogeneously over the entire surface of the lesion, that is to say, it is intended that the filling microtunnels is equivalent to a microtunnel to another and that the amount of the composition (of chitosan mixed with the blood) in contact with the osteochondral surface should be homogeneously distributed. In addition, the clot must not break and must be deformable to spread properly on the cartilage surface and not be unhooked during the procedure.

Simultaneously with the homogeneity of the blood / chitosan mixture prior to clot formation, a significantly faster clot formation time is sought than that of the chitosan / blood compositions described in the literature or commercially available. For example, faster clot formation is sought, depending on our method of determining clot formation time, than that of the 2.05% chitosan solution CS7 used as a control in this example, or that of chitosan solution. and beta-glycerol phosphate at pH 7 known from the prior art.

In particular, it seeks a clot formation duration of less than 10 minutes, ideally less than 5 minutes, while having a clot in one part (without fragment, maintaining its integrity), which does not retract and which resists manual compression. In this example, we therefore only characterize clots whose formation time is less than 15 min, that is to say less than or equal to 10 minutes.

In this example, the volume ratio of blood to chitosan solution of the mixture is set to 3/1 (v / v). A low molecular weight chitosan is used, which ensures good homogeneity of the blood / chitosan solution mixture prior to clot formation, and rapid clot formation.

Chitosan solutions of 2%, 3% and 4% concentration are prepared at pH 7.0 or pH 6.0.

Preparation of chitosan solutions at pH 7.0

A solution of beta-glycerophosphate (GP) is prepared by dissolving sodium betaglycerol-phosphate, pharmaceutical grade water for injection and 1N hydrochloric acid. This solution is then filtered through 0.2μιτι. The chitosan is dispersed in this solution, then a volume of glacial acetic acid variable as a function of the chitosan concentration is added to reach a pH of 7.0 ± 0.2. This solution is then sterilized by autoclave. The final GP concentration is 2% (w / w), the pH is 7.0 ± 0.2 and the osmolality is 300 ± 25 mOsmol / kg. Preparation of chitosan solutions at pH 6.0 ± 0.2

The chitosan is dispersed in pharmaceutical grade water for injection, then a volume of glacial acetic acid is added to obtain a pH of 6.0 ± 0.2. osmolarity of 275 to 325mOsm / kg This solution is then sterilized by autoclave The final sodium chloride concentration is 1% (m / m) in the final formulation, the pH is 6.0 ± 0.2 and the osmolality is 300 ± 25 mOsmol / kg.

Blood sampling and preparation of the blood / chitosan solution mixture:

Whole blood samples are taken by qualified personnel from adult human volunteers. Each blood sample is analyzed for possible abnormalities in coagulation. If an anomaly is detected, the test is not continued for this sample.

Freshly collected whole blood, ie less than 5 minutes before use, is mixed with the solution of chitosan / beta-glycerophosphate (GP) at a volume ratio of 3: 1, by mixing 1.5 ml of Chitosan / GP solution and 4.5 ml of blood. The blood / chitosan mixture is then slightly agitated manually for 10 seconds.

The clots are formed as follows:

- 0.3 ml of the chitosan / blood mixture is poured into a glass tube;

- The tube is then placed in a bath thermostated at 37 ° C without stirring. After 5, 10 and 15 minutes, the appearance of the clots is checked by gently rocking the tube. It is determined visually whether the clot is formed in the center (complete clot), or if only the edges of the clot are solidified, or if the blood is still flowing. The time necessary for the formation of the complete clot is noted.

For comparison, the clot is made with fresh blood without the addition of the chitosan / GP formulation.

Clot retraction rate:

The clots are allowed to incubate for 1 hour in a thermostated bath at 37 ° C., without stirring. After 1 hour of incubation, the clot has more or less retracted, which causes an expulsion of a certain volume of blood. To determine the rate of retraction, the tube and the clot are weighed together at the outlet of the bath. The liquid expelled from the clot is then removed using a glass syringe with a needle. The tube and the clot are again weighed together. The retraction rate is determined according to the following calculation:

% retraction

m (tube + clot exiting the bath) - m (tube + clot after withdrawal exceeding liquid)

= - r-- - ⁱ - 2 * - * 100 m (tube + · clot out of the bath)

For comparison, the clot with fresh blood shrinks by about 40%. Compression test:

Once the retraction test is done, the clots are placed in a petri dish. Compression force is manually applied to the clot to determine its strength, characterized by visual observation of the effect of compression: release of blood after compression and / or clot fragmentation. The test is always performed by the same person with a substantially identical compressive force.

Deformation test:

Then, it is evaluated visually if the clot is able to be deformed without disintegrating, for example to pass through a large needle type 18 Gauge internal diameter 800 to 880μιτΊ in the form of large drops.

Spread / adhesion test:

Next, the ability of the clot to be extended over a surface to be treated without slipping too easily is evaluated by tilting the surface vertically at an angle of about 60 ° to the horizontal axis. Homogeneity of the mixture

The homogeneity of the blood / chitosan mixture prior to coagulation is simulated and evaluated visually: the chitosan solution is prepared at pH 7.0 or pH 6.0, and a phosphate buffer is added to mimic the addition of blood, in a volume ratio of 1/3 and in a glass tube. It is stirred manually for 10 seconds, and the mixture is observed. If the chitosan formulation is evenly distributed, the mixture is considered homogeneous. If it is observed that the mixture forms some opaque zones and of 'gelatinous' consistency, it is considered that the mixture is not homogeneous. If it is observed that the mixture forms many opaque areas of gelatinous consistency, it is considered that the mixture is non-homogeneous.

Table 3 - Fresh blood clot formation in the presence of a solution of chitosan CS2 (1% viscosity of 15mPa.s) at pH 7 and pH 6 +/- 0.2 and osmolality between 275 and 325mOsm / kg.

retention

Homogeneity Aspect of

Clot Retraction Time on

Concentration of the clot mixture and

surface formation and in chitosan before resistance

clot clot ability to

% (m / m) training at the

Complete (%) ** Clot compression filling

of cavity

Freshly collected blood

No

> 15 min 40% Fragmented NS

homogeneous

Solution of chi .osa ne CS2 to pH 6.0

Very

2.0% 15 min 0.2% Segments NS

homogeneous

Integrates, no

fragmented Very good

Very adherence and

3.0% 5 min 0.2%

Homogeneous Resistant to good spreading compression, on the deformable surface

Integrates, no

fragmented Very good

Very adherence and

4.0% 5 min 0.2%

Homogeneous Resistant to good spreading compression, on the deformable surface

CS2 tosane chi solution at pH 7.0

Very

2.0% 15 min 0.1% NS NS

homogeneous

Very

3.0% 15 min 0.2% NS NS

homogeneous

Integrates, no

fragmented Good

Very adherence and

4.0% 10 min 0.2%

Homogeneous Resistant to good spreading compression, on the deformable surface

Control (2.0% CS7 chitosan solution and pH 7.0)

No

2.0% 15 min 0.2% NS NS

homogeneous

NS: Clot unsatisfactory for the assessed endpoint

These results surprisingly show that the only chitosan solutions that make it possible to provide a homogeneous mixture with fresh blood are those with the chitosans according to the invention, the viscosity at 1% in acetic acid is less than 60mPa.s.

It is further apparent from this example that clot formation which is intact, non-retracted and not fragmented can be surprisingly significantly faster with the chitosan solution at pH 6.0 and in the absence of beta-glycerophosphate. of sodium that the solution controls chitosan CS7 at 2.0%, while retaining good properties, such as homogeneity of the blood / chitosan mixture before clot formation, very low clot retraction, good compressive strength, clot remaining deformable, good ability to spread.

In addition, it is found that for the same low molecular weight chitosan (CS2), very surprisingly, a suitable clot can be formed much more rapidly at a lower chitosan concentration with a chitosan formulation at pH 6.0 in the presence of an inorganic salt (without organic salt of sugar or of polyol, and in particular without sodium glycerophosphate), than at pH 7.0 with a chitosan formulation in the presence of an organic sugar or polyol salt, and in particular of sodium glycerophosphate.

In addition, it is measured that the viscosity of the chitosan solution is low at the concentration of 4% (about 360mPa.s), and that a homogeneous mixture is formed with the clot. Maintaining a viscosity of the low chitosan solution allows easy industrial manufacturing, which is not the case for higher molecular weight chitosans as described in Example 2.

Finally, the clots formed with the 3% and 4% concentration solutions at pH 6 have a higher adhesion capacity than the clot formed with the 4% concentration solution at pH 7, when they are positioned on a mimicking surface. the vertical position, for example at an angle of 60 °.

Example 4 - Speed of formation of a clot of fresh blood in the presence of chitosan solution of different molecular masses In this example, mixtures are formed between solutions of chitosan of molecular mass (expressed in terms of viscosity at 1%) and of fresh blood taken from human volunteers, with the aim of obtaining a homogeneous mixture and a firm, supple, non-retracted and non-fragmented clot. The clinical objective is that this clot can be easily handled by a surgeon, in order to affix it to an osteo- chondral, for example a cartilage lesion having previously been pierced by microfractures - in the form of microtunnels.

The clots and mixtures are formed and evaluated according to the procedure of Example 3.

Table 4 - Fresh blood clot formation in the presence of a chitosan solution (pH 7 +/- 0.2, osmolality between 275 and 325mOsm / kg)

retention

Ref Aspect of

Clot Retraction Time on

Homogeneity of chitosan clot and

surface formation and PBS / mixture and resistance range

clot clot capacity of chitosan (viscosity at 1%, at the

complete (%) ** filling mPa.s) compression

of cavity

Freshly collected blood

> 15 min 40% NS NS

Chitosan concentration 2.0% (m / m) *

Homogeneous CS1 UL (10) 15 min 0.2% NS NS

Homogeneous CS5 M (50) 15 min 0.5% NS NS

Not very homogeneous CS 10 M (63) 15 min 0.2% NS NS

Not very homogeneous CS6 H (154) 15 min 0.2% NS NS

Integrated

Blood Control is

Uncommon 15 min 0.2% released under OK

CS7 UH (489) compression,

little deformable

Chitosan concentration 3.0% (m / m) *

Homogeneous CS1 UL (10) 15 min 0.2% NS NS

Homogeneous CS2 UL (15) 15 min 0.2% NS NS

Homogeneous CS5 M (50) 15 min 0.1% NS NS

Not very homogeneous CS10 M (63) 15min 0.2% NS NS

Non-homogeneous CS6 H (154) 15min 0.2% NS NS

Non-homogeneous CS7 UH (489) 15min 0.2% NS NS

Chitosan concentration 4.0% (m / m) *

Not fragmented

Homogeneous CS1 UL (10) 5min 0.5% Resistant, OK

deformable

Not fragmented

Homogeneous CS2 UL (15) 10 min 0.2% Resistant, OK

deformable

Slightly

fragmented

Homogeneous CS4 L (40) 10 min 0.2% OK

Resistant,

deformable Slightly

fragmented

Homogeneous CS5 M (50) 10 min 0.2% OK

Resistant,

deformable

Not very homogeneous CS10 M (63) 15min 0.2% NS NS

Non-homogeneous CS6 H (154) 15min 0.2% NS NS

Non-homogeneous CS7 UH (489) 15min 0.2% NS NS

Concentration of chitosan in the chitosan / glycerophosphate solution before mixing with the blood; ** After 1 hour at 37 ° C.

NS: Clot unsatisfactory for the evaluated criterion It follows from these results that the only solutions of chitosan that make it possible to provide a homogeneous mixture with the fresh blood are those carried out with the reference chitosan CS1, CS2, CS4 and CS5 whose viscosity with 1% is 10 to SOmPa.s. From the viscosity at 1% of 63mPa.s (CS10), the mixture is not homogeneous, which promotes the fragmentation of the clot formed.

It is further apparent from these results that the increase in chitosan concentration surprisingly accelerates clot formation significantly compared to the 2.0% CS7 chitosan control solution. Below a concentration of 4% chitosan, the clot formation time is at least 15 minutes for all chitosan tested, which is not optimal.

Clot formation time significantly lower, that is to say less than or equal to 10 minutes, is obtained for 4% chitosan solutions whose molecular weight is low (references CS1, CS2, CS4 and CS5, 1% viscosity of 10 to 50 ppm).

It is also apparent from this example that with a chitosan solution of 4% concentration for a chitosan of 1% viscosity of 10mPa.s (reference CS1), it is possible both to provide a homogeneous mixture between the blood and a solution of chitosan and glycerophosphate pH 7, and to form a clot in less than 5 minutes, this clot is very satisfactory and meets all the technical criteria (integrated, non-retracted, non-fragmented, resistant to compression and deformation) ). Example 5 Influence of the Presence of a Calcium Salt on Clot Formation with a Low Molecular Weight Chitosan Formulation

In this example, the influence of the presence of calcium chloride (CaCl ₂ ) in variable concentration on clot formation prepared with solutions of low molecular weight chitosan (CS2 reference, 1% viscosity of 15mPa was investigated. s) concentrations of 3 and 4%.

The calcium salt, in chloride form (Calcium chloride dehydrate, Merck (ref: 1.02465.1000)) is added to the chitosan solution to obtain the desired concentration, and the glycerophosphate is added.

This formulation is then mixed with fresh blood the previously described protocol.

Table 5 - Clot formation of fresh blood in the presence of a solution of chitosan CS2 (1% viscosity of 15mPa.s), sodium beta-glycerophosphate and calcium chloride (pH 7 +/- 0.2)

retention

Concentration Duration of Retraction Aspect of clot on CaCl ₂ clot formation and surface and

clot clot resistance ability to

(mg / l) full (%) ** to compression cavity filling

Chitosan concentration 3% (m / m)

Integrates, resistant to

0 15 min 0.2% NS

compression, deformable

Integrates, very resistant to

25 15 min 0.2% NS

compression, deformable

Integrates, resistant to

50 15 min 0.1% NS

compression, deformable

Integrates, resistant to

100 15 min 0.2% NS

compression, deformable

Concentration of chitosan 4% (m / m)

Integrates, resistant to good adhesion

0 5 min 0.2%

compression, deformability and good spreading

Integrates, resistant to good adhesion

25 5 min 0.2%

compression, deformability and good spreading

Very good

Integrates, very resistant to

50 5 min 0.2% membership

compression, deformable

and good spreading

Very good

Integrates, very resistant to

100 5 min 0.2% adherence

compression, deformable

and good spreading It follows from this example that the presence of calcium chloride in the chitosan solution, at equal pH and osmolarity, does not accelerate clot formation. On the other hand, the presence of calcium chloride improves its cohesion, its capacity to remain intact under compression and deformation and not to allow a part of the blood to be released under compression, and its adherent character.

This is advantageous for placing the clot on the surface to be treated, in particular by arthroscopy.

It is also advantageous to have these cohesive and reinforced adhesion properties to be able to position the clot durably on the joint to be treated, even if it is in a non-horizontal position, without the clot sliding under the effect of gravity.

Claims

A composition comprising a mixture of a chitosan formulation and a coagulable fluid, wherein the chitosan formulation has a concentration of at least 2.5% chitosan by mass based on the total weight of the formulation of chitosan, said chitosan having a dynamic viscosity of less than or equal to 60mPa.s, measured in 1% (v / v) acetic acid, said composition comprising at least one inorganic salt.

2. Composition according to claim 1, characterized in that the chitosan has a molecular weight Mv ranging from 20,000 to 145,000.

3. -Composition according to claim 1 or 2, characterized in that the chitosan formulation has a chitosan concentration greater than 2.5% and less than 10% by weight relative to the final formulation mass.

4. -Composition according to any one of claims 1 to 3, characterized in that the chitosan is a chitosan of fungal origin.

5. Composition according to any one of claims 1 to 4, characterized in that the inorganic salt is an agent adjusting the osmolarity of the chitosan solution.

6. Composition according to any one of claims 1 to 5, characterized in that the inorganic salt is chosen from a sodium salt, a salt of chloride, potassium, calcium, magnesium, phosphate, sulfate. or any of their mixtures, and more particularly of potassium chloride, sodium chloride, cesium chloride, calcium chloride, cesium fluoride, potassium perchlorate, sodium nitrate, calcium sulphate, or any of their mixtures.

7. -Composition according to any one of claims 1 to 6, characterized in that the volume ratio (v / v) between the coagulable fluid and chitosan is 1/1 to 4/1.

8. Composition, according to any one of claims 1 to 7, characterized in that the osmolality of the chitosan formulation is between 250 and 400 mosm / kg, and preferably from 275 to 325 mosm / kg.

9. Composition according to any one of claims 1 to 8, characterized in that the chitosan formulation comprises one or more other inorganic salts, and preferably a calcium salt.

10. Composition according to any one of claims 1 to 9, characterized in that the pH is from 5.0 to 6.5, and preferably from 5.5 to 6.5, and more preferably from

5.8 to 6.2.

1 1. - Composition according to any one of claims 1 to 10, characterized in that the composition comprises living cells, for example osteoblasts.

A medical device comprising or consisting of a chitosan formulation at a concentration of at least 2.5% chitosan by mass based on the volume of chitosan formulation, said chitosan having a dynamic viscosity of less than or equal to 60 mPa. s, measured in 1% (v / v) acetic acid, intended to be mixed with a coagulable fluid, and at least one inorganic salt.

13. - medical device according to claim 12, characterized in that it comprises at least one, and preferably only one container containing a formulation of chitosan and at least one inorganic salt in the form of a solution, said chitosan having a dynamic viscosity less than or equal to 60mPa.s, measured in acetic acid at 1% (v / v), said formulation having a concentration greater than or equal to 2.5% of chitosan by mass relative to the mass of chitosan formulation.

14. - medical device comprising a plurality of containers, said containers being optionally physically independent ("kit-of-part"), characterized in that the medical device comprises at least (i) a container containing a chitosan in dry form or in solution, said chitosan having a dynamic viscosity of less than or equal to 60mPa.s, measured in 1% (v / v) acetic acid, and (ii) a container containing a solution of at least one inorganic salt, wherein chitosan and the solution of at least one inorganic salt have a concentration suitable for greater than or equal to 2.5% of mass chitosan with respect to the mass of chitosan formulation, the chitosan formulation being formed by contacting the chitosan, in dry form or in solution, and said solution of at least an inorganic salt.

15. - Medical device comprising a composition according to any one of claims 1 to 1 1.

16. - Composition as defined in one of claims 1 to 1 1, optionally included in a medical device, for use in a method of therapeutic treatment.

17. - Composition as defined in one of claims 1 to 1 1, optionally included in a medical device, for use in a method of therapeutic treatment of an articular disease.