Calcium alginate for use in the prevention and / or treatment of inflammation

Abstract

The present invention relates to a calcium alginate for use in the prevention and / or treatment of inflammation.

Description

[0001] DESCRIPTION

[0002] TITLE: CALCIUM ALGINATE FOR USE IN THE PREVENTION AND / OR TREATMENT OF INFLAMMATION

[0003] Technical field of the invention

[0004] The present invention relates to calcium alginates for their uses in the prevention and / or treatment of inflammation.

[0005] Technical background

[0006] Inflammation (or inflammatory reaction) is a natural process that usually occurs when an organism is subjected to microbial aggression or tissue damage, for example due to physical or traumatic chemical agents.

[0007] Inflammation can also result from a dysfunction of the immune system, for example in autoimmune diseases which cause the body to attack its own tissues (multiple sclerosis, rheumatoid arthritis, Crohn's disease, etc.), certain genetic diseases (epidermolysis bullosa, familial Mediterranean fever, Blau syndrome, etc.) or non-genetic diseases (dermatoses, diabetes, contact dermatitis, tendinitis, etc.) and cancer.

[0008] Inflammation comprises a series of events that activate immune cells and release inflammatory mediators such as cytokines and prostaglandins to clear the injured site and / or fight infection. This process promotes tissue repair and a return to homeostasis.

[0009] Inflammation can be acute or chronic.

[0010] Acute inflammation is a rapid defense mechanism of the body against external threats such as microbes or injuries, thanks to the action of immune cells such as neutrophils and macrophages that engulf pathogens. Once the threat is controlled, the tissue is repaired and the inflammation subsides.

[0011] Chronic inflammation is a prolonged response that persists when the insult is not eliminated. It involves continuous activation of immune cells such as macrophages and lymphocytes, leading to tissue damage and the formation of scar tissue, as in the case of chronic diseases or persistent infections.

[0012] Inflammation plays a role, for example, in the healing process for tissue repair, particularly of skin wounds, after an injury or trauma. Healing generally includes the following phases:

[0013] - Hemostasis to stop the bleeding,

[0014] - inflammation to eliminate pathogens and cellular debris,

[0015] - proliferation to regenerate tissues and close wounds, and

[0016] - the remodeling to result in the formation of the final scar.

[0017] In cases of non-pathological wound healing, the inflammatory phase resolves rapidly; this is the case with acute inflammation. In cases of pathological wound healing, the inflammatory phase is prolonged and hinders healing; this is the case with chronic inflammation found in chronic wounds (diabetic wounds, venous or arterial ulcers, pressure sores, etc.).

[0018] An initial inflammatory response is therefore beneficial and essential to ensure the effective triggering of the repair process. Conversely, prolonged, ineffective (so-called "low-grade") or excessive inflammation can become detrimental, leading to delayed healing or chronic wounds. Depending on the condition of the wound, a pro-healing treatment may require an anti-inflammatory or pro-inflammatory action to stimulate the healing process. Some treatments can promote healing while maintaining or even stimulating the inflammatory response. (21 ' 22 ' 23) .

[0019] Inflammation can be local or systemic.

[0020] Local inflammation typically occurs in a specific area of ​​the body where there is an injury or infection. Systemic inflammation generally affects the entire body and can be triggered by severe infections, extensive burns, autoimmune diseases, or chronic inflammatory responses.

[0021] The main cells involved in inflammation include cells derived from myeloid progenitors (neutrophils, macrophages, etc.) and lymphoid progenitors (natural killer cells and lymphocytes). Macrophages play a crucial role in regulating inflammation. Type I macrophages are characterized by the secretion of pro-inflammatory factors such as TNF-α and the production of reactive oxygen species (ROS), thus amplifying the inflammatory response. In contrast, type M2 macrophages secrete anti-inflammatory cytokines like IL-10, promote angiogenesis, and contribute to tissue repair. Therefore, the transition between type M1 and M2 macrophages regulates the shift from the inflammatory phase to the repair phase and the return to tissue homeostasis.

[0022] In summary, inflammation is essential for the body's protection and plays a role in many physiological situations. However, when left uncontrolled, it becomes debilitating and requires therapeutic intervention.

[0023] Treatment of inflammation

[0024] Pathological inflammation is typically treated with anti-inflammatory drugs to control excessive inflammation and / or prevent tissue damage. Among anti-inflammatory drugs, two main categories are distinguished: non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids (or steroidal anti-inflammatory drugs).

[0025] However, anti-inflammatory drugs often have undesirable side effects. 2)and it proves useful to diversify the therapeutic means of treating inflammation. This is what the Applicant has achieved by proposing a new means of preventing and / or treating inflammation which consists of calcium alginate.

[0026] Subject of the patent

[0027] The present invention relates to calcium alginate for its use in the prevention and / or treatment of inflammation.

[0028] The calcium alginate according to the invention can be prepared in the form of a wet composition (such as gel, semi-solid emulsion, membrane or foam), in the form of a liquid composition (such as suspension or lotion) or in the form of a dry composition (powder, film, fiber, etc.).

[0029] The calcium alginate according to the invention can be formulated or incorporated into a composition, in particular wet or dry, or a suitable device.

[0030] Detailed description of the invention

[0031] Calcium alginate

[0032] The present invention relates to calcium alginate for its use in the prevention and / or treatment of inflammation.

[0033] Calcium alginate is known in the prior art to be pro-inflammatory and thus induce and accentuate an inflammatory response. 4 5) .

[0034] For example, the study by Gail Chan et al. analyzed the biological effect of a calcium alginate gel and concluded that it promotes the inflammatory mechanism. Indeed, the calcium alginate gel induces the secretion of interleukin-1β (IL-10), a pro-inflammatory cytokine. The authors of this study indicate that in in vitro tests, the calcium alginate gel leads to the maturation of dendritic cells of the immune system and increases the secretion of inflammatory cytokines (such as IL-1β3). This is explained by the fact that dendritic cells require calcium for cytokine secretion. In another study, the inflammatory role of calcium is highlighted by its ability to trigger the innate immune response activated by Toll-I-like receptors. (5)Adib et al. confirm the pro-inflammatory effect of calcium alginate by showing that it activates the production of pro-inflammatory cytokines, such as TNF-α, IFN-γ factors, as well as the cytolytic activity of Natural Killer cells.

[0035] In contrast to these studies (3 ' 4 ' 5) Document CN-A1-111437435, hereinafter referred to as A1, describes a hydrogel cell structure and a method for preparing such a hydrogel cell structure that is said to have an anti-inflammatory effect. This hydrogel cell structure comprises a hydrogel made of calcium alginate and polyvinyl alcohol (PVA), optionally seeded with M2 phenotype macrophages. A1 indicates that such a hydrogel would exhibit an anti-inflammatory effect by promoting the differentiation of M1 macrophages into the M2 phenotype.

[0036] The Applicant reproduced the protocol of the second example of A1 and then tested the anti-inflammatory effect of the A1 hydrogel thus prepared. The protocol and results of these tests are detailed in Example 1. It appears that, contrary to what the authors of A1 claim, the test results (Figures 1a to 1d) demonstrate that the A1 hydrogel does not have an anti-inflammatory effect, that it is toxic to macrophages, and that it affects their viability regardless of their phenotype (Figure 1a).

[0037] Thus, calcium alginate has a pro-inflammatory effect according to the findings of studies (3 ' 4 ' 5) and according to the results of tests obtained by reproducing the A1 trials (Example 1), and would therefore not allow to control or prevent excessive inflammation, and the tissue damage and delays in healing that may result.

[0038] It is therefore very surprising that the Applicant has highlighted an anti-inflammatory action of calcium alginate and, in particular, its ability to stimulate the differentiation of pro-inflammatory M1 macrophages towards an anti-inflammatory M2 phenotype.

[0039] More specifically, the Applicant demonstrated that calcium alginate, particularly in particulate gel form, reduces both the secretion of tumor necrosis factor alpha (TNF-α) and the production of reactive oxygen species (ROS). TNF-α is produced by various immune system cells (such as macrophages with a pro-inflammatory M1 phenotype). (6) ) and ROS species are known to be pro-inflammatory^.

[0040] Furthermore, the Applicant has demonstrated that calcium alginate, particularly in the form of a particulate gel, also increases the secretion of interleukin 10 (IL-10), which is produced by various cells of the immune system (such as anti-inflammatory M2 macrophages). 8 >) and have an anti-inflammatory effect (6) (example 2).

[0041] These advantageous properties allow for the stimulation of differentiation of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages, thereby strengthening the anti-inflammatory action.

[0042] Therefore, calcium alginate now appears as a promising therapeutic strategy for the prevention and / or treatment of inflammation, particularly in the context of wound healing and tissue regeneration (including skin, mucous membranes, tendons, ligaments, muscles, cartilage, viscera and / or bones).

[0043] Alginate is a polysaccharide mainly obtained from algae: kelp or fucus.

[0044] It is made up of two monomers: mannuronate or mannuronic acid (M) and guluronate or guluronic acid (G).

[0045] The general formula for alginate is as follows:

[0046] The proportion and distribution of these two monomers are crucial for a wide range of alginate's physical and chemical properties; alginate is thus characterized by its mannuronic acid / guluronic acid ratio (or M / G ratio). Its chemical composition varies depending on the different algae species, the different parts of the same plant, and is subject to seasonal changes. Nevertheless, by carefully selecting the source of raw materials, it is possible to obtain a variety of alginates with consistent characteristics.

[0047] The carboxyl groups of alginate allow it to bind with cations, forming alginate salts, notably calcium alginate.

[0048] Alginate-based products have natural intrinsic physical properties and are used in health, primarily for the treatment of gastroesophageal reflux by preventing acid reflux and for wound treatment by maintaining a moist environment and absorbing exudates.

[0049] Advantageously, within the framework of the present invention, calcium alginate can be devoid of cells, such as human, animal or plant cells.

[0050] In particular, calcium alginate may be devoid of M2 and / or M1 type macrophages.

[0051] Calcium alginate may or may not be formulated with polyvinyl alcohol. In one particular embodiment, calcium alginate is formulated without polyvinyl alcohol.

[0052] As mentioned above, alginate itself is a polysaccharide formed from two monomers: mannuronic acid and guluronic acid.

[0053] In a preferred embodiment, calcium alginate comprises mannuronic acid (M) and guluronic acid (G) in an M / G ratio greater than 0.5. Advantageously, the M / G ratio may be between 0.5 and 1.5. Preferably, the M / G ratio may be between 0.7 and 1.5. More preferably, the M / G ratio may be between 1 and 1.5. Even more preferably, the M / G ratio may be approximately 1 or 1.5. These M / G ratios, in particular, allow for improved anti-inflammatory properties.

[0054] These M / G ratios can be implemented regardless of the form of the calcium alginate of the invention, the composition, and / or the device comprising the calcium alginate of the invention. The preparation of the calcium alginate according to the invention can be carried out using methods known to those skilled in the art.

[0055] The M / G ratio can be determined by Nuclear Magnetic Resonance (NMR) spectroscopy or by the so-called gel strength method. (15 ' 16 ' 17 ' 18) .

[0056] According to the invention, calcium alginate has a molar mass between 60,000 and 400,000 g / mol -1 Preferably, the molar mass of calcium alginate is between 100,000 and 300,000 g.mol' 1 .

[0057] Calcium alginate may contain less than 15% calcium by weight. Preferably, the calcium content is between 3% and 12% by weight. Even more preferably, the calcium content is between 4% and 11% by weight. Advantageously, the calcium content is between 4% and 10% by weight.

[0058] According to a particular embodiment, calcium alginate is presented in a particulate form.

[0059] The term "particulate" refers to a calcium alginate that is present in the form of particles or an aggregate of particles.

[0060] Calcium alginate in particulate form comprises particles that may have a size between 5 pm and 2000 pm, and preferably between 5 pm and 1000 pm.

[0061] In particular, according to a preferred embodiment, at least 80% of the calcium alginate particles may have a size between 5 pm and 700 pm, and preferably between 5 pm and 450 pm.

[0062] Particle size can be measured according to ISO 13320 by dispersing the sample in a medium and directing a laser beam through this dispersion. The light scattered at different angles is collected by detectors, and the resulting data is processed by software that applies Mie theory to calculate the particle size. The particle size distribution profile is then established by analyzing the distribution of, for example, particle diameters within the sample.

[0063] This particulate form allows, in particular, for an increase in the anti-inflammatory action of calcium alginate, while maintaining a homogeneous structure (or, in other words, a uniform distribution) for application in a wide range of products (such as the various forms mentioned above, the composition and / or the device incorporating calcium alginate according to the invention). This is intended to guarantee uniform application and / or absorption of the calcium alginate, thus ensuring optimal efficacy of its anti-inflammatory action.

[0064] Various possible compositions including calcium alginate

[0065] The present invention also relates to a composition comprising calcium alginate, as defined above, for its use in the prevention and / or treatment of inflammation.

[0066] The composition may include between 0.4% and 100% by weight of calcium alginate relative to the total weight of the composition.

[0067] According to one embodiment, the composition comprising calcium alginate, as defined above, is presented in wet form for its use in the prevention and / or treatment of inflammation; this composition is referred to hereafter as the wet composition.

[0068] The term "wet" refers to a mixture or structure containing a significant amount of an aqueous medium (such as water or saline solution) or impregnated with such an aqueous medium.

[0069] According to another embodiment, the composition comprising calcium alginate, as defined above, is in liquid form for use in the prevention and / or treatment of inflammation; this composition is hereinafter referred to as the liquid composition. The term "liquid" means a 10- to 20-fold dilution of the wet composition in an acceptable solvent.

[0070] According to another embodiment, the composition comprising calcium alginate, as defined above, is presented in dry form for its use in the prevention and / or treatment of inflammation; this composition is hereinafter referred to as the dry composition.

[0071] The term "dry" refers to a mixture or substance devoid of an aqueous medium (such as water or physiological saline) or containing a minimal amount of this aqueous medium.

[0072] Wet composition:

[0073] The wet composition can be formulated as a gel, semi-solid emulsion, membrane, or foam.

[0074] The term "gel" means a wet preparation (e.g. colloidal) containing calcium alginate according to the invention which can be diluted or dispersed in an aqueous medium (such as water or physiological saline).

[0075] A "semi-solid emulsion" is defined as a composition comprising a lipid phase dispersed in an aqueous phase containing, for example, calcium alginate according to the invention, or vice versa. According to this embodiment, the composition may be a cream, optionally including a fatty substance.

[0076] The term "membrane" refers to a solid, generally porous structure containing a material, such as calcium alginate.

[0077] The term "foam" means a structure comprising gas bubbles trapped in a solid or liquid matrix containing, for example, calcium alginate according to the invention.

[0078] The wet composition may thus include calcium alginate as defined above, and an acceptable medium.

[0079] The acceptable medium may be an aqueous medium, preferably physiologically acceptable, such as water or saline solution. The wet composition may comprise between 80% and 98% by weight of acceptable medium relative to the total weight of the composition. Preferably, the wet composition comprises between 90% and 98% by weight of acceptable medium relative to the total weight of the composition.

[0080] The wet composition may contain less than 10% by weight of calcium alginate relative to the total weight of the composition. Preferably, the wet composition contains between 0.4% and 7% by weight of calcium alginate relative to the total weight of the composition. Even more preferably, the wet composition contains between 1% and 6% by weight of calcium alginate relative to the total weight of the composition.

[0081] The wet composition may comprise between 0.1% and 3% by weight of calcium relative to the total weight of the composition. Preferably, the wet composition may comprise between 0.2% and 2% by weight of calcium relative to the total weight of the composition.

[0082] The wet composition may include other components, such as salts, preservatives, stabilizers, active ingredients, copolymers, etc. The wet composition may include less than 10% by weight of these other components relative to the total weight of this composition.

[0083] Preferably, calcium alginate in the wet composition is in particulate form.

[0084] Calcium alginate in particulate form comprises particles that may have a size between 5 pm and 2000 pm, and preferably between 10 pm and 1000 pm.

[0085] In particular, according to a preferred embodiment, at least 80% of the calcium alginate particles may have a size between 10 pm and 700 pm, preferably between 40 pm and 450 pm.

[0086] The wet composition as defined above can be in the form of a calcium alginate gel. For example, the preparation of the calcium alginate gel according to the invention can be carried out by mixing or dispersing a solution containing alginate and calcium in an acceptable medium. In particular, the preparation of calcium alginate gel in particulate form is well known to those skilled in the art. Calcium alginate particles can be obtained by gelling droplets of an aqueous phase containing sodium alginate in contact with a phase containing divalent cations (such as calcium), called the gelation phase. Thus, the use of calcium as a divalent cation allows for the production of calcium alginate particles.

[0087] Particle size is typically defined by the droplet size before contact with the phase containing divalent cations. For example, for calcium alginate particles, the aqueous alginate phase can be contained in a syringe fitted with a needle. Droplets are obtained by expelling the aqueous phase through the needle. The droplet size will be determined by the needle's exit diameter, the viscosity of the aqueous phase, and the height between the needle's exit point and the surface of the phase containing the calcium ions. Removing the gelling phase by filtration allows the formation of a gel composed of calcium alginate particles.

[0088] A non-limiting example of the preparation of the calcium alginate gel according to the invention is described in Example 2. This calcium alginate gel according to the invention provides an anti-inflammatory effect.

[0089] Advantageously, the preparation of the calcium alginate gel according to the invention yields a smooth, viscoelastic texture that allows for easy and homogeneous application, ensuring good adhesion, conformability, and uniform coverage of the treated area. The wet composition according to the invention results in a creamy and soft formulation, providing a feeling of comfort and hydration.

[0090] The calcium alginate gel according to the invention can serve as a basis for the formulation of other wet composition(s), such as emulsions, foams and / or membranes.

[0091] The liquid composition can be formulated by diluting the wet composition 10 to 20 times in a suitable solvent. The liquid composition can be formulated as a suspension or a lotion.

[0092] The acceptable solvent can be an aqueous solvent or a solvent miscible with water and compatible with alginate.

[0093] The term "suspension" refers to a heterogeneous mixture comprising fine solid particles, containing, for example, calcium alginate according to the invention, dispersed in a liquid without being dissolved. For example, the suspension comprising calcium alginate may have a water content different from that of the calcium alginate gel.

[0094] The term "lotion" refers to an oil-in-water emulsion, particularly one with a higher aqueous phase content than a cream. For example, a lotion containing calcium alginate may also contain an oily substance in addition to the calcium alginate suspension. The liquid composition may contain between 98% and 99.95% by weight of an acceptable solvent relative to the total weight of the composition.

[0095] The liquid composition may contain less than 1% by weight of calcium alginate relative to the total weight of the composition. Preferably, the liquid composition comprises between 0.02% and 0.7% by weight of calcium alginate relative to the total weight of the composition. Even more preferably, the liquid composition comprises between 0.5% and 0.6% by weight of calcium alginate relative to the total weight of the composition.

[0096] The liquid composition may contain between 0.005% and 0.3% by weight of calcium relative to the total weight of the composition. Preferably, the wet composition may contain between 0.01% and 0.2% by weight of calcium relative to the total weight of the composition.

[0097] The liquid composition may include other components, such as salts, preservatives, stabilizers, active ingredients, copolymers, etc. The liquid composition may contain less than 1% by weight of these other components relative to the total weight of the composition.

[0098] Dry composition:

[0099] The dry composition can be formulated as a film, fiber, or powder. The term "film" refers to a generally thin and compact solid structure comprising a material (namely, calcium alginate). The film is prepared by drying a wet calcium alginate composition according to the invention.

[0100] The term "fiber" refers to a filamentous structure (containing, for example, calcium alginate according to the invention) that is generally elongated and thin. The preparation of calcium alginate fiber is well known to those skilled in the art by extruding a sodium alginate solution through a die in a calcium chloride bath. The calcium alginate fiber according to the invention can be used for the manufacture of yarns, compresses, and wicks.

[0101] The term "powder" means a structure comprising solid particles (containing, for example, calcium alginate according to the invention) dispersed in the form of grains or fragments.

[0102] The dry composition thus comprises calcium alginate as defined above. The dry composition may optionally include less than 20% by weight of aqueous medium relative to the total weight of the composition. Preferably, the dry composition may include between 2% and 20% by weight of aqueous medium relative to the total weight of the composition.

[0103] The dry composition may comprise more than 10% by weight of calcium alginate relative to the total weight of the composition. Preferably, the dry composition comprises between 10% and 100% by weight of calcium alginate relative to the total weight of the composition. Even more preferably, the dry composition comprises between 30% and 90% by weight of calcium alginate relative to the total weight of the composition.

[0104] The dry composition may contain less than 15% by weight of calcium relative to the total weight of the composition. Preferably, the dry composition contains between 2% and 12% by weight of calcium relative to the total weight of the composition. Even more preferably, the dry composition contains between 2.5% and 10% by weight of calcium relative to the total weight of the composition.

[0105] Preferably, calcium alginate in the dry composition is in a particulate form whose characteristics are detailed below.

[0106] Calcium alginate in particulate form comprises particles that may have a size between 5 pm and 2000 pm, and preferably between 5 pm and 1000 pm.

[0107] For example, the dry composition in powder form may be in the form of a plurality of granular particles. The particles advantageously have a size less than 200 pm, preferably less than 75 pm, which is defined by sieving or by laser diffraction measurement.

[0108] The powder in the form of a plurality of granular particles in the dry composition according to the invention can be prepared as described in patent FR-B3-2823979 filed by the Applicant.

[0109] According to one embodiment, the powder also takes the form of a plurality of cylindrical particles obtained from fibers.

[0110] Cylindrical particles can have a length between 20 pm and 2000 pm and a diameter between 5 pm and 50 pm.

[0111] The powder in the form of a plurality of cylindrical particles in the dry composition according to the invention can be prepared as described in patent EP-B1-2836243 filed by the Applicant.

[0112] Adding ingredients

[0113] The composition according to the invention, whether in its dry or wet form, can advantageously be combined with one or more anti-inflammatory agents. For example, the anti-inflammatory agent can be chosen from a steroidal anti-inflammatory agent (such as cortisone), a non-steroidal anti-inflammatory agent (such as ibuprofen), a natural active ingredient (such as curcumin), and / or ions (such as zinc). The composition comprising calcium alginate can be combined with an ion, in particular zinc.

[0114] Zinc is a physiological trace element found in the extracellular matrix (ECM) of the dermis and epidermis. Thanks to its widely studied and recognized pharmacological properties, zinc is used for its anti-inflammatory, antioxidant, and immunoregulatory effects: it reduces pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1), and tumor necrosis factor 30 (TNF-30). Zinc contributes to maintaining skin tissue homeostasis and to skin repair and regeneration processes. (9) .

[0115] Combining zinc with calcium alginate enhances the anti-inflammatory action.

[0116] The composition may contain less than 1% by weight of zinc relative to the total weight of the composition. Preferably, the composition contains between 0.02% and 0.05% by weight of zinc relative to the total weight of the composition. Even more preferably, the composition contains between 0.027% and 0.035% by weight of zinc relative to the total weight of the composition.

[0117] The composition may also include one or more ingredients selected from a buffer, a stabilizing agent (such as pectins, cellulose derivatives, xanthan gum, etc.), a preservative (such as benzalkonium chloride, potassium sorbate, sodium benzoate, tocopherol, etc.), a surfactant, a vitamin, a mineral, one or more ions (such as zinc), a peptide, proteins, amino acids, nucleic acids, oligosaccharides, lipids, hormones, any element of an extracellular matrix, a pH adjuster, an antibiotic, cells, bacteriophages, viruses, an antimicrobial and an antibiofilm.Various possible packagings including calcium alginate The composition including calcium alginate, as defined above, can be incorporated into different types of devices such as threads, dressings, wicks, compresses, aerosols, sprays, liquid (oral or injectable) or semi-solid forms, suppositories, oral solid forms.

[0118] For example, dressings, gauze, and compresses can be chosen from highly absorbent fibers, hydrofibers, alginates, hydrocellular materials, hydrocolloids, hydrogels, petrolatum-based dressings, interface materials, activated charcoal dressings, silver dressings, and hyaluronic acid-based dressings. These devices are generally used for hemostasis and wound healing.

[0119] > Various possible applications

[0120] In general, the present invention relates to calcium alginate, the composition and / or the device comprising calcium alginate according to the invention, for use in the prevention and / or treatment of any pathology having an inflammatory component.

[0121] Inflammation is generally manifested by pain, redness, edema or swelling, and heat.

[0122] For example, inflammation can result from or be associated with various factors, including but not limited to:

[0123] - an infection (viral, bacterial, fungal or parasitic),

[0124] - trauma (e.g., physical injuries such as burns, (post-)surgical wounds, sutured wounds, etc.),

[0125] - an abnormal immune reaction (such as an allergy),

[0126] - chemical irritants (such as toxins, pollutants, certain industrial chemicals, etc.),

[0127] - chronic conditions (such as chronic diseases, chronic wounds, oxidative stress, etc.),

[0128] - cancers, and - lifestyle (such as after sports activity, smoking, alcoholism and / or stress).

[0129] The inflammation that the present invention aims to treat can occur in various organs, and the method of administration and formulation of calcium alginate will be chosen according to the target organ to be treated.

[0130] In particular, the location of the inflammation may be cutaneous or extracutaneous (such as cartilaginous, mucosal, muscular, tendinous, ligamentous, visceral and / or osseous).

[0131] By way of illustration and without limitation, inflammatory skin disorders may include eczema, atopic dermatitis, urticaria, acne, seborrheic dermatitis, contact dermatitis, couperose, erythrosis, psoriasis, etc.

[0132] Inflammatory skin disorders can also be genetic and / or rare diseases, such as epidermolysis bullosa.

[0133] Extracutaneous inflammatory disorders can include, but are not limited to, rheumatoid arthritis, inflammatory bowel diseases (such as Crohn's disease), chronic diseases (such as asthma), autoimmune diseases (such as systemic lupus erythematosus), etc.

[0134] Inflammatory cartilage disorders can include, but are not limited to, osteoarthritis.

[0135] Mucosal inflammatory disorders can include, but are not limited to, bronchitis, gastritis, stomatitis, conjunctivitis, cystitis, etc.

[0136] Muscle inflammatory disorders can include, but are not limited to, various myositis (such as infectious, ossifying, etc.), fibromyalgia, etc.

[0137] Inflammatory tendon disorders can include, but are not limited to, various forms of tendinitis (such as shoulder, wrist, etc.).

[0138] Ligament inflammatory disorders can include, but are not limited to, ligamentitis, ligament sprains, etc. According to specific embodiments, calcium alginate, the composition, and / or the device comprising calcium alginate according to the invention are used for the prevention and / or treatment of inflammation of joints, muscles, tendons, and ligaments, which can occur, for example, during sports activities. The present invention further relates to calcium alginate, the composition, and / or the device comprising calcium alginate according to the invention, for their use in the prevention and / or treatment of inflammation of wounds or tissue lesions to promote their healing. Tissue wounds can be cutaneous, extracutaneous, cartilaginous, mucosal, muscular, tendinous, ligamentous, visceral, and / or osseous lesions.

[0139] It is also known that aging can be accompanied and exacerbated by chronic, low-level systemic inflammation. (19)This latent inflammation, although not pathological, increases the risk of developing diseases, and it is advantageous to prevent or limit its development. Thus, the present invention also relates to a method for the non-therapeutic prevention and / or treatment of the low-level, non-pathological, chronic, and systemic inflammatory component of aging, comprising the administration of calcium alginate according to the invention, the composition, and / or the device comprising the calcium alginate according to the invention. According to this embodiment, the present invention may more particularly relate to a cosmetic treatment method for skin aging, comprising the administration of calcium alginate according to the invention, the composition, and / or the device comprising the calcium alginate according to the invention.

[0140] The term "low level" in the context of the low-level, non-pathological, chronic systemic inflammatory component of aging is a well-established concept in the scientific literature on physiological aging – see reference (19) of this application. Furthermore, the term "low level" does not refer to an arbitrary value, but rather to measurable biological thresholds that can be defined by comparison with reference values ​​in young or healthy individuals. > Routes of administration

[0141] Calcium alginate, the composition and / or the device may be administered by any route, including topically, orally (or buccally), nasally, rectally or by injection.

[0142] The topical route can allow the composition according to the invention to be administered locally to the skin or mucous membranes, for example, to treat inflammatory skin conditions or to control inflammation during the healing process.

[0143] The topical route can also allow the composition according to the invention to be administered locally to certain tissues, for example, to treat inflammatory tissue conditions other than those of the skin, such as tendons, ligaments, muscles and / or bones.

[0144] The oral route can be used to treat inflammatory conditions, including inflammatory bowel diseases such as Crohn's disease, as well as other systemic inflammatory conditions such as rheumatoid arthritis.

[0145] The nasal route can be used to administer the composition according to the invention to treat inflammatory conditions of the nasal mucosa and sinuses, such as allergic rhinitis, chronic sinusitis and other inflammatory nasal conditions.

[0146] The rectal route can allow the composition according to the invention to be administered to treat inflammatory conditions affecting the rectum, colon or anus, such as ulcerative colitis, hemorrhoids and other inflammatory bowel diseases.

[0147] The injectable route allows the composition according to the invention to be administered by transcutaneous, intramuscular, or intravenous injection to treat inflammatory tissue conditions other than those of the skin, such as tendons, ligaments, muscles, viscera, and / or bones. This is intended to prevent and / or rapidly treat inflammations, such as flare-ups of rheumatoid arthritis, exacerbations of inflammatory bowel disease, or other viscera and / or bone diseases.

[0148] For example, gel, cream, lotion, aerosol and / or foam can be applied directly to wounds for local absorption and targeted action on the inflamed area.

[0149] The tablets can be administered orally.

[0150] The oral formulation can be administered when the composition is liquid and as an alternative to tablets.

[0151] As mentioned above, the injectable formulation (such as in gel form) can be administered intramuscularly, intravenously, or intra-articularly to rapidly treat inflammation.

[0152] Brief description of the figures

[0153] Other features and advantages of the invention will become apparent upon reading the detailed description that follows, for an understanding of which reference should be made to the attached figures in which:

[0154] Figure 1a represents a histogram illustrating the effect on the viability of M1 macrophages of the hydrogel formed from calcium alginate and polyvinyl alcohol from document A1, this hydrogel being obtained from the protocol of example 2 of A1;

[0155] Figure 1b represents a histogram illustrating the percentage of cells positive for the CD206 marker by control M1 macrophages, M1 in contact with the A1 hydrogel, and by M2 macrophages;

[0156] Figure 1c represents a histogram illustrating the co-expression of CD163 and CD206 as a percentage of control M1 macrophages, M1 in contact with the hydrogel of A1 and M2;

[0157] Figure 1d represents a histogram illustrating the secretion of the anti-inflammatory interleukin IL-10 by control M1 macrophages, M1 in contact with the hydrogel of A1 and M2; Figure 2a represents a histogram illustrating the effect on the viability of M1 macrophages of the calcium alginate gel in particulate form according to the invention;

[0158] Figure 2b represents a histogram illustrating the co-expression of CD163 and CD206 as a percentage of control M1 macrophages, M1 in contact with the calcium alginate gel in particulate form according to the invention and M2;

[0159] Figure 2c represents a histogram illustrating the secretion of the anti-inflammatory interleukin IL-10 by control macrophages M1, M1 in contact with calcium alginate gel in particulate form according to the invention and M2;

[0160] Figure 2d represents a histogram illustrating the ratio of secretion of the pro-inflammatory factor TNF-a to the secretion of the anti-inflammatory interleukin IL-10 by control macrophages M1, M1 in contact with the calcium alginate gel in particulate form according to the invention and M2;

[0161] Figure 2e represents a histogram illustrating the production of pro-inflammatory ROS by control M1 macrophages, M1 in contact with calcium alginate gel in particulate form according to the invention and M2;

[0162] Figure 3a represents a histogram illustrating the effects on the viability of M1 macrophages of two calcium alginate gels according to the invention at different M / G ratios (G1 and G2);

[0163] Figure 3b represents a histogram illustrating the co-expression of CD163 and CD206 as a percentage of M1 macrophages in contact with two calcium alginate gels according to the invention at different M / G ratios (G1 and G2) compared to control M1 and M2 macrophages;

[0164] Figure 3c represents a histogram illustrating the secretion of the anti-inflammatory interleukin IL-10 by M1 macrophages in contact with two calcium alginate gels according to the invention at different M / G ratios (G1 and G2) compared to control M1 and M2 macrophages;

[0165] Figure 3d shows a histogram illustrating the TNF-α secretion ratio to the interleukin IL-10 secretion ratio by M1 macrophages in contact with two calcium alginate gels according to the invention at different M / G ratios (G1 and G2) compared to control M1 and M2 macrophages; Figure 4 shows a histogram illustrating the TNF-α secretion ratio to the interleukin IL-10 secretion ratio by M1 macrophages in contact with calcium alginate gels (G) and calcium alginate associated with zinc (G.Zn). 2+ ) compared to control M1 and M2 macrophages;

[0166] Figure 5 shows a line graph illustrating the effect of treatment with calcium alginate (G) and zinc-associated calcium alginate (G.Zn) gels. 2+ ) on the severity of psoriasis in mice.

[0167] Example 1 - Calcium alginate according to the prior art

[0168] Preparation of the hvdroqel of document A1

[0169] The hydrogel formed from calcium alginate and polyvinyl alcohol described in A1 was prepared from the protocol of example 2 of A1.

[0170] This protocol is briefly summarized below:

[0171] - add 100 mg of sodium alginate (marketed by FMC, which is the English acronym for "Food Machinery Corporation" (renamed "International N&H Manufacturing UK Limited") and has an M / G ratio of approximately 1; referenced as SA in A1) into 12.5 ml of deionized water, and stir magnetically until the sodium alginate dissolves completely;

[0172] - add a quantity of 500 mg of PVA into a volume of 12.5 ml of demineralized water, and stir at 80°C until the PVA dissolves completely;

[0173] - mix the two solutions of sodium alginate and PVA evenly;

[0174] - add an aqueous solution of calcium chloride of 74 pl (having a concentration of 5 mg / ml);

[0175] - adjust the pH: a drop of 1 M sodium hydroxide (NaOH) (VWR) was added to obtain a pH of 6.5 and form an intermediate hydrogel which was crosslinked with calcium ion (referenced as Ca-SA / PVA hydrogel in A1) with a degree of crosslinking of 1%; - perform a primary freeze-thaw cycle: the Ca-SA / PVA hydrogel was placed in a glass Petri dish, and frozen for 22h at -20°C then thawed for 2h at 25°C then a lyophilization cycle was performed to obtain a lyophilized gel;

[0176] - add 500 pl of phosphate buffer (PBS) solution at pH 7.4 into the pores of the lyophilized gel so that the lyophilized gel absorbs the PBS solution and thus obtain a final hydrogel referenced (Ca-SA / PVA)1 FT+FD in A1.

[0177] Preparation of pro- and anti-inflammatory macrophages M1 and M2

[0178] PBMC cells (acronym for peripheral blood mononuclear cells) from a healthy individual (i.e., without inflammatory disorders) are isolated by centrifugation in a Ficoll solution.

[0179] Monocytes with the classic CD14+ / CD16- markers are purified.

[0180] These purified monocytes are placed in a 24-well plate (containing, for example, 400,000 cells / mL / well) and differentiated as follows: M1 macrophages:

[0181] - monocytes are incubated with a culture medium containing 10% human serum in RPMI medium (English acronym for "Roswell Park Mondial Institut Medium") with 1% of a Penicillin and Streptomycin (PS) based antibiotic, 1% LG (acronym for "L-Glutamine") and 50 ng / mL of GM-CSF (English acronym for "Granulocyte Macrophage Colony Stimulating Factor");

[0182] - the plate in an incubator for 6 days;

[0183] - from the sixth day of culture, half of the medium is replaced by another culture medium containing: 10% FBS (acronym for fetal bovine serum) in an RPMI medium with 1% PS, 1% LG, 50 ng / mL of GM-CSF factor and 50 ng / mL of LPS / IFN-γ activators (acronym for Lipopolysaccharide and Interferon-γ), and incubated for 24h for differentiation into pro-inflammatory M1 macrophages.

[0184] M2 Macrophages:

[0185] - monocytes are incubated with a culture medium containing 10% human serum in an RPMI medium with 1% PS, 1% LG and 50 ng / ml of an M-CSF factor (English acronym for "Monocyte colony-stimulating factor") to differentiate monocytes into anti-inflammatory M2 macrophages;

[0186] - a plate is placed in an incubator for 6 days;

[0187] - from the sixth day of culture, half of the medium is replaced with another culture medium comprising: 10% SVF in an RPMI medium, 1% PS, 1% LG, 50 ng / mL of the M-CSF factor, 20 ng / mL of the cytokines IL-4 (acronym for interleukin 4) and IL-13 (acronym for interleukin 13), and incubated for 24h for differentiation into anti-inflammatory M2 macrophages.

[0188] Results: Effects of the hydrogel in document A1

[0189] The anti-inflammatory activity of A1 hydrogel was tested on pro-inflammatory M1 macrophages.

[0190] Figures 1a to 1d illustrate the effects of A1 hydrogel on M1 macrophages (designated "A1").

[0191] In particular, Figure 1a illustrates the viability, as a percentage of live cells, of M1 macrophages in contact with A1 hydrogel compared to pro-inflammatory control M1 macrophages. In this figure, the viability of M1 macrophages in contact with A1 hydrogel is significantly reduced compared to that of control M1 macrophages.

[0192] Thus, A1 hydrogel has a toxic effect on cells, particularly on pro-inflammatory M1 macrophages.

[0193] A1 describes a single marker CD206 to assess the anti-inflammatory profile of macrophages.

[0194] Figure 1b illustrates the percentage (%) of macrophages expressing the CD206 marker among M1 macrophages in contact with the A1 hydrogel and that of macrophages in control condition (M1 and M2).

[0195] In Figure 1b, the percentage of cells positive for the CD206 marker by control macrophages M1 and M2 is almost identical; it can be deduced that the observation of the percentage of CD206-positive cells is not correlated with a pro- or anti-inflammatory phenotype and does not allow differentiation between an M1 or M2 profile. Some studies (10 ' 11) They mention that the CD206 marker is not sufficient to indicate an anti-inflammatory state. Therefore, it is not a relevant marker for evaluating an anti-inflammatory effect. (10 ' 11 ' 12 ' 13 ' 14) .

[0196] It is known from the state of the art that the co-expression of the CD163 and CD206 markers allows differentiation of macrophages of profile M1 (low co-expression) from profile M2 (high co-expression / 14 ' 15) .

[0197] Figure 1c thus illustrates this CD163 / CD206 co-expression for M1 macrophages in contact with A1 hydrogel and for pro- and anti-inflammatory macrophages, M1 and M2, in control condition.

[0198] In Figure 1c, the percentage of CD163 and CD206 co-expression in control M2 macrophages is higher than that observed in control M1 macrophages. This confirms that this CD163 and CD206 co-expression allows for differentiation between the phenotype of pro-inflammatory M1 and anti-inflammatory M2 macrophages.

[0199] Furthermore, in this figure 1c, the M1 macrophages in contact with the A1 hydrogel have a low co-expression of CD163 and CD206 compared to the M2 macrophages in the control condition, but also compared to the M1 macrophages in the control condition.

[0200] Thus, A1 hydrogel does not induce the differentiation of M1 macrophages into M2 macrophages. Since the co-expression of the CD163 and CD206 markers obtained in the presence of A1 hydrogel was lower than that measured for control M1 macrophages, A1 hydrogel did not activate M1 macrophages into M2 macrophages. This confirms that A1 hydrogel does not have an anti-inflammatory effect.

[0201] Finally, Figure 1d illustrates the secretion of the anti-inflammatory interleukin IL-10 by M1 macrophages in contact with the A1 hydrogel and by pro- and anti-inflammatory macrophages, M1 and M2, in control conditions.

[0202] In Figure 1d, the secretion of interleukin IL-10 by anti-inflammatory M2 macrophages is, as expected, much higher than that of pro-inflammatory M1 macrophages. The secretion of interleukin IL-10 by M1 macrophages in contact with the A1 hydrogel is lower than that of control M1 and M2 macrophages.

[0203] Thus, A1 hydrogel decreased the secretion of interleukin IL-10, even compared to control M1 macrophages. A1 hydrogel did not activate M1 macrophages into M2 macrophages. This also confirms that A1 hydrogel does not exhibit an anti-inflammatory effect.

[0204] Contrary to what A1 claims, the results obtained and presented in figures 1a to 1d demonstrate that the hydrogel of A1 does not induce an M2 phenotype and does not have anti-inflammatory properties.

[0205] Example 2 - Preparation and evaluation of the effect of calcium alginate according to the invention

[0206] The anti-inflammatory effect of calcium alginate according to the invention in gel form was tested on macrophages.

[0207] Preparation of a calcium alginate gel for testing

[0208] A first calcium ion solution is prepared in 2 liters (L). This first solution contains 1% by weight of calcium chloride (CaCl2), 0.6% by weight of sodium chloride (NaCl), and 98.4% by weight of water, preferably demineralized. Approximately 0.4% by weight of zinc gluconate is added to this first solution to obtain a zinc-enriched gel (ZnG). 2+ ).

[0209] A second sodium alginate (AlgNa) solution, commercially available from FMC, is prepared in 2 liters (L). This second solution contains 2% by weight of sodium alginate, 0.9% by weight of NaCl and 97.1% by weight of water, preferably demineralized.

[0210] The second solution is sprayed through a nozzle into the first solution contained in a tank. The entire assembly is housed in a suitable enclosure, and the tank is continuously agitated using one or more magnetic stirrers. This spraying is carried out with the following parameters: - pressure in the tank of approximately 1.8 bar,

[0211] - casing pressure of approximately 2.5 bar, and

[0212] - spraying duration of approximately 90 minutes.

[0213] The powdered solution is then centrifuged several times, for example three times, at a speed of approximately 2500 rpm for about 2 minutes. After each centrifugation, the pellet is resuspended in a quantity of vacuum-filtered demineralized water at approximately 0.1 µm to reach a mass of 300 g. The pellet from the last centrifugation is sterilized by passing it through a UV reactor to obtain the calcium alginate gel at the outlet of the UV reactor. This gel is in particulate form.

[0214] Several calcium alginate gels according to the invention have been prepared from the preparation described above, such as:

[0215] - a G gel with an M / G ratio of approximately 1.5;

[0216] - a G.Zn gel 2+ with an M / G ratio of approximately 1.5;

[0217] - a G1 gel with an M / G ratio of approximately 0.7; and

[0218] - a G2 gel with an M / G ratio of approximately 1.

[0219] Gel G or Gel G.Zn alginate 2+ has a molar mass of 400,000 g.mol' 1

[0220] The alginate in gel G1 has a molar mass of 160,000 g.mol' 1 .

[0221] The alginate in gel G2 has a molar mass of 125,000 g.mol' 1 .

[0222] The calcium level in the G gel or the G.Zn gel 2+ is between 4 and 10% relative to the total weight of the dry gel.

[0223] The particle size distribution of each of the gels G, G.Zn 2 and G1 was measured. For this, a particle size analysis of calcium alginate was performed by laser light diffraction of G, G.Zn gels. 2+ and G1, was carried out according to the ISO 13320:2020 standard for particle size analysis (European Pharmacopoeia 2.9.31).

[0224] Particle size distribution was measured using a Malvern Instruments Mastersizer 3000 laser particle size analyzer capable of determining particle size distributions ranging from 0.5 µm to 3 mm. This equipment features a Hydro LV liquid analysis module and an Aero S dry analysis module.

[0225] The particle size distribution of a sample can be characterized by various parameters that represent the shape of the distribution. The parameters that will be used are:

[0226] - Total particle distribution interval,

[0227] - Dv10: 10% of the particles in the analyzed sample are smaller than x pm,

[0228] - Dv50: 50% of the particles in the analyzed sample are smaller than x pm,

[0229] - Dv90: 90% of the particles in the analyzed sample are smaller than x pm,

[0230] - Dv[4:3] : average particle size relative to volume.

[0231] It should be noted that approximately 80% of the particles can be found between Dv10 and Dv90.

[0232] For each sample, six measurements are taken to verify the repeatability of the results according to the criteria of the ISO 13320:2020 standard.

[0233] The results of the particle size distribution of calcium alginate for G, G.Zn gels 2+ and G1 are summarized in Table 1 below:

[0234] Evaluation of the effect of calcium alginate gel on pro-inflammatory M1 macrophages

[0235] Preparation of pro- and anti-inflammatory macrophages M1 and M2

[0236] PBMCs (peripheral blood mononuclear cells) from a healthy individual (i.e., without inflammatory disorders) are isolated by Ficoll. Monocytes with the classic markers CD14 + / CD16' are purified. These purified monocytes are placed in a 24-well plate (containing, for example, 400,000 cells / mL / well) and differentiated as follows: M1 Macrophages:

[0237] - monocytes are incubated with a culture medium containing 10% human serum in RPMI medium with 1% of a Penicillin and Streptomycin-based antibiotic, 1% LG and 50 ng / ml of a GM-CSF factor;

[0238] - the plate is placed in an incubator for 6 days;

[0239] - from the sixth day of culture, half of the medium is replaced by another culture medium containing: 10% SVF in an RPMI medium with 1% PS, 1% L-G, 50 ng / mL of GM-CSF factor and 50 ng / mL of LPS / IFN-y activators (acronym for Lipopolysaccharide and Interferon-y) and incubated for 24h for differentiation into pro-inflammatory M1 macrophages.

[0240] M2 Macrophages:

[0241] - monocytes are incubated with a culture medium containing 10% human serum in RPMI medium with 1% PS, 1% LG and 50 ng / mL of an M-CSF factor to differentiate monocytes into anti-inflammatory M2 macrophages;

[0242] - the plate is placed in an incubator for 6 days;

[0243] - from the sixth day of culture, half of the medium is replaced with another culture medium comprising: 10% SVF in RPMI medium, 1% PS, 1% LG, 50 ng / mL of M-CSF factor, 20 ng / mL of IL-4 cytokines (acronym for interleukin 4) and IL-13 cytokines (acronym for interleukin 13) and incubated for 24h for differentiation into anti-inflammatory M2 macrophages.

[0244] To test the activity of the prepared gels G, G.Zn 2+ G1 and G2, a 10% dilution of each of these gels in culture medium (i.e., 100 µl in 1 mL) is brought into contact with pro-inflammatory M1 macrophages. Characterization of the macrophage phenotype

[0245] After a 3-day culture in the presence of the different gels, the macrophages are recovered from the plate. After centrifugation and washing, extracellular staining is performed with the following antibodies: CD163-BV421, CD206-APC, and 7-aminoactinomycin D (7-AAD). The macrophages are then analyzed by flow cytometry. The co-expression of CD163 and CD206 allows for the identification of anti-inflammatory macrophages, and 7-AAD is used to assess cell viability. Cytokine secretion is also measured.

[0246] After a 2-day culture in contact with G, G.Zn gels 2+ In wells G1 and G2, the culture media are centrifuged to remove the different gels. After adding 50 ng / mL of LPS, these culture media are redistributed into their corresponding wells.

[0247] After an additional 24-hour culture, TNF-α and IL-10 cytokine secretions are measured in the supernatants using MACSPIex cytokine kits (Miltenyi). ROS species production is also measured.

[0248] After a 3-day culture in contact with G, G.Zn gels 2+ , G1 and G2, the cells are recovered, centrifuged and washed.

[0249] According to the instructions for kits ab113851 (abeam) and M7514 (Thermofisher), ROS production normalized to mitochondrial mass is measured on macrophages in contact with the different gels.

[0250] Results: Anti-inflammatory effects of calcium alginate gel on pro-inflammatory M1 macrophages

[0251] The anti-inflammatory activity of each of the G, G.Zn gels 2+ G1 and G2 are tested on pro-inflammatory M1 macrophages by measuring the following parameters:

[0252] - co-expression of the markers CD163 and CD206 (figures 2b and 3b),

[0253] - secretion of interleukin IL-10 (figures 2c and 3c),

[0254] - ratio of TNF-a / IL-10 factor secretion (figure 2d, 3d, 4),

[0255] - production of ROS species (figures 2e).

[0256] These assays demonstrate the reduction of pro-inflammatory components (TNF-α, ROS) and / or the increase of anti-inflammatory components (CD163 / CD206, IL-10) by M1 macrophages in the presence of each of the G, G.Zn gels. 2+ , G1 and G2. Effect of gel G on pro-inflammatory macrophages M1

[0257] Figure 2a evaluates the toxicity of gel G on M1 macrophages compared with control pro-inflammatory M1 macrophages.

[0258] In Figure 2a, the percentage of M1 macrophages alive in contact with gel G is identical to that of control M1 macrophages.

[0259] Thus, the calcium alginate in gel G does not affect macrophage survival.

[0260] Figure 2b illustrates the co-expression of CD163 and CD206 of M1 macrophages in contact with gel G compared to pro- and anti-inflammatory control macrophages, M1 and M2.

[0261] In Figure 2b, the percentage of M2 macrophages co-expressing CD163 and CD206 is, as expected, significantly increased compared to M1 macrophages. The percentage of M1 macrophages co-expressing CD163 and CD206 in the presence of gel G is higher than that of control M1 macrophages.

[0262] Gel G therefore increases the co-expression of CD163 and CD206. This co-expression of CD163 and CD206 being a marker of anti-inflammatory M2 macrophages, gel G enabled the activation of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages.

[0263] Figure 2c illustrates the secretion of the anti-inflammatory interleukin IL-10 by M1 macrophages in contact with gel G and that of pro- and anti-inflammatory macrophages, M1 and M2, under control conditions.

[0264] In Figure 2c, interleukin IL-10 secretion is, as expected, significantly increased by M2 macrophages compared to control M1 macrophages. Interleukin IL-10 secretion by M1 macrophages in the presence of gel G is greater than that of pro-inflammatory control M1 macrophages. Since interleukin IL-10 is an anti-inflammatory protein, its increase upon contact with gel G demonstrates the stimulation of M2-type anti-inflammatory activity by initially pro-inflammatory M1 macrophages. Figure 2d illustrates the ratio of TNF-α secretion to interleukin IL-10 secretion (TNF-α / IL-10) in M1 macrophages in contact with gel G and in pro- and anti-inflammatory macrophages, M1 and M2, under control conditions. This TNF-a / IL-10 ratio is, as expected, greatly reduced for M2 macrophages compared to control M1 macrophages.This TNF-a / IL-10 ratio of M1 in the presence of G gel is significantly decreased compared to the M1 control, further demonstrating an anti-inflammatory effect of G gel.

[0265] Figure 2e illustrates the production of ROS species by M1 macrophages in contact with gel G and that of pro- and anti-inflammatory macrophages, M1 and M2, under control conditions.

[0266] In Figure 2e, ROS production is, as expected, significantly reduced in M2 macrophages compared to control M1 macrophages. ROS production by M1 macrophages in the presence of gel G is also lower than that of pro-inflammatory M1 macrophages. Since ROS species are pro-inflammatory, the decrease in their production in the presence of gel G corresponds to a reduction in the pro-inflammatory activity of M1 macrophages by this gel G.

[0267] These results confirm that the calcium alginate in gel G exhibits anti-inflammatory activity. Effect of gels G1 and G2 with different M / G ratios on pro-inflammatory macrophages M1

[0268] Figure 3a evaluates the toxicity of G1 and G2 gels on M1 macrophages compared with control pro-inflammatory M1 macrophages.

[0269] In Figure 3a, the percentage of M1 macrophages in contact with each of the G1 and G2 gels is identical to that of the pro-inflammatory M1 control macrophages.

[0270] Thus, the calcium alginate in gels G1 and G2 does not affect macrophage survival. Figure 3b illustrates the co-expression of CD163 and CD206 by M1 macrophages in contact with gels G1 and G2 compared to pro- and anti-inflammatory macrophages, M1 and M2, controls.

[0271] In this figure 3b, the co-expression of CD163 and CD206 in the presence of gels G1 and G2 is greater than that of the control M1.

[0272] G1 and G2 gels have the effect of increasing the co-expression of CD163 and CD206, and have allowed the differentiation of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages.

[0273] Figure 3c illustrates the secretion of interleukin IL-10 by M1 macrophages in contact with G1 and G2 gels and that of pro- and anti-inflammatory M1 and M2 macrophages, in control condition.

[0274] In Figure 3c, the secretion of interleukin IL-10 by M1 macrophages in the presence of gel G2 is greater than that of pro-inflammatory control M1 macrophages. The increased secretion of interleukin IL-10 upon contact with gel G2 induces anti-inflammatory activity in pro-inflammatory M1 macrophages.

[0275] Thus, calcium alginate in gel form (especially G2 gel) exhibits anti-inflammatory activity.

[0276] Figure 3d illustrates the ratio of TNF-α secretion to interleukin IL-10 secretion (TNF-α / IL-10) in M1 macrophages in contact with gels G1 and G2, and in pro- and anti-inflammatory macrophages, M1 and M2, under control conditions. As expected, this TNF-α / IL-10 ratio is significantly reduced in M2 macrophages compared to control M1 macrophages. The TNF-α / IL-10 ratio in M1 macrophages in the presence of gel G2 is also reduced compared to control M1, further demonstrating the anti-inflammatory effect of gel G2.

[0277] Effects of G and G.Zn gels 2+ Figure 4 illustrates the ratio of TNF-α secretion to interleukin IL-10 secretion (TNF-α / IL-10) in M1 macrophages in contact with G and G.Zn gels. 2+and that of pro- and anti-inflammatory macrophages, M1 and M2, under control conditions. This TNF-α / IL-10 ratio is, as expected, significantly decreased for M2 macrophages compared to control M1 macrophages. This TNF-α / IL-10 ratio of M1 in the presence of G.Zn gel 2+ is decreased compared to that of gel G. Thus, the combination of zinc with calcium alginate increases the differentiation of pro-inflammatory macrophages M1 into anti-inflammatory macrophages M2 in this G.Zn gel. 2+ .

[0278] Example 3 - Evaluation of the effect of treatment with calcium alginate gels according to the invention on the severity of psoriasis in mice

[0279] The anti-inflammatory effect of calcium alginate according to the invention in gel form was tested on mice to evaluate the treatment on the severity of psoriasis.

[0280] Preparation and treatment of mice with calcium alginate gels to be tested

[0281] Thirty female Balb / c mice (aged between 6 and 7 weeks, and weighing between 16 and 18 g) are used in a murine model of psoriasis induced by daily topical application of 65 mg of Aldara™ 5% cream (imiquimod) to the dorsal skin, from day 1 to day 14.

[0282] A control group of mice, labeled "IMI / Placebo" in Figure 5, received a neutral cream (i.e., a glycerol, petrolatum, and paraffin-based cream from BIOGARAN®) according to the same protocol. Another control group of mice, labeled "Placebo / Placebo" in Figure 5, was not induced to develop psoriasis and received the same cream.

[0283] G and G.Zn gels 2+ are tested on two groups of mice, referenced "IMI / Gel (G) and IMI / Gel (G.Zn 2+) » on Figure 5, and compared to the control group (IMI / Placebo) and to a group of mice that had been treated with a reference anti-inflammatory cream based on clobetasol (Dermoval® 0.05%) which is referenced “IMI / Clobetasol” on Figure 5.

[0284] Treatments are administered by topical application of 100 mg / day of the tested gels / creams from day 8 (T0) to day 14, four hours after psoriasis induction, under standardized conditions. The PASI score (20) is the reference measure used in clinical trials and the most validated objective measure of psoriasis severity currently available.

[0285] The severity of psoriasis is assessed daily from day 8 (T0: before treatment) to day 15 (T7) using the PASI score. Three clinical criteria are noted on the induced dorsal area of ​​the mice: erythema, desquamation (dryness or peeling), and skin thickening. Each criterion is scored from 0 (absence) to 4 (severe), for a maximum overall score of 12. The sum of the scores (0-12) gives the overall PASI score.

[0286] Photographs of the treated areas are taken on days 8, 11 and 15 in order to monitor the evolution of the lesions.

[0287] The entire study is conducted in compliance with European Directive 2010 / 63 / EU on animal experimentation, with authorization APAFIS#51288.

[0288] The PASI score results for day 8 (T0) and day 15 (T7) are summarized in Table 2 below:

[0289] [Table 2]

[0290] Placebo IMS 1MI IMI IMI

[0291] PAS) KW

[0292] For statistical analysis:

[0293] - LQ: lower quartile;

[0294] - UQ: upper quartile;

[0295] - KW: Kruskal-Wallis test; - #P < 0.05 vs Placebo / Placebo D8;

[0296] - * P < 0.05 vs IMI / Placebo D15 (Steel-Dwass test).

[0297] Results: Anti-inflammatory effects of calcium alginate gel on mice with psoriasis

[0298] Referring to Figure 5 and Table 2, we observe that the PASI score of the placebo group (IMI / Placebo) is high and stable; the placebo shows no anti-psoriatic activity. The PASI score of the reference treatment (IMI / Clobetasol) decreases progressively from T0 to T7, demonstrating a significant anti-inflammatory effect. In the mouse groups treated with G gel (IMI / Gel (G)) and G.Zn gel, the PASI score was also high and stable. 2+ (IMI / Gel (G.Zn 2+The PASI score gradually decreases between T0 and T7. This demonstrates the anti-psoriatic activity of both gels. The addition of zinc to the G.Zn gel 2+ significantly improves anti-psoriatic activity, with an accelerated and greater reduction in PASI score compared to zinc-free G gel.

[0299] These results confirm that calcium alginate, alone and also in combination with zinc, exhibits anti-psoriatic activity and therefore demonstrates in vivo anti-inflammatory activity of G and G.Zn gels 2+ for the treatment of psoriasis. It is also noted that calcium alginate combined with zinc exhibits an improvement in the in vivo anti-inflammatory effect of the gel for the treatment of psoriasis.

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Claims

1. DEMANDS 1. Calcium alginate for its use in the prevention and / or treatment of inflammation, characterized in that it comprises mannuronic acid (M) and guluronic acid (G) in an M / G ratio greater than 0.5, advantageously the M / G ratio is between 0.5 and 1.5, preferably the M / G ratio is between 0.7 and 1.5, more preferably the M / G ratio is between 1 and 1.5, even more preferably the M / G ratio is about 1 or 1.

5.

2. Composition for its use in the prevention and / or treatment of inflammation, characterized in that it comprises calcium alginate according to claim 1.

3. Composition according to claim 2, characterized in that the calcium alginate is in particulate form.

4. Composition according to claim 2 or claim 3, characterized in that it further comprises one or more ingredients selected from buffers, stabilizing agents, a preservative, surfactants, a vitamin, a mineral, one or more ions, a peptide, proteins, amino acids, nucleic acids, oligosaccharides, lipids, hormones, any element of an extracellular matrix, a pH adjuster, an antibiotic, cells, bacteriophages, viruses, a pH adjuster, an antibiotic, an antimicrobial, an antibiofilm and another anti-inflammatory.

5. Composition according to any one of claims 2 to 4, characterized in that it is incorporated into various devices such as threads, dressings, wicks, compresses, aerosols, sprays, oral or injectable liquid forms, suppositories or solid forms.

6. Composition according to any one of claims 2 to 5, characterized in that it is formulated in wet form such as in the form of a gel, semi-solid emulsion, membrane or foam.

7. Composition according to claim 6, characterized in that at least 80% of the calcium alginate particles have a size between 10 pm and 700 pm, and preferably between 40 pm and 450 pm.

8. Composition according to claim 6 or claim 7, characterized in that it comprises less than 10% by weight of calcium alginate relative to the total weight of the composition, preferably the composition comprises between 0.4% and 7% by weight of calcium alginate relative to the total weight of the composition, and even more preferably the composition comprises between 1% and 6% by weight of calcium alginate relative to the total weight of the composition.

9. Liquid composition characterized in that it is obtained from a 10- to 20-fold dilution of the wet composition according to any one of claims 6 to 8 in an acceptable solvent.

10. Composition according to any one of claims 2 to 5, characterized in that it is formulated in dry form such as in the form of film, fiber or powder.

11. Composition according to claim 10, characterized in that the particle size is less than 200 pm, preferably a size less than 75 pm.

12. Composition according to claim 10 or claim 11, characterized in that it comprises an amount greater than 10% by weight of calcium alginate relative to the total weight of the composition, preferably between 10% and 100% by weight of calcium alginate relative to the total weight of the composition, and even more preferably between 30% and 90% by weight of calcium alginate relative to the total weight of the composition.

13. Calcium alginate according to claim 1, composition and / or device comprising calcium alginate according to claim 1, for use in the prevention and / or treatment of tissue inflammation, in particular of the skin, mucous membranes, tendons, ligaments, muscles, cartilage and / or bones.

14. Calcium alginate according to claim 1, composition and / or device comprising calcium alginate according to claim 1, for use in tissue healing, in particular wounds of the skin and / or mucous membranes, skin lesions, extracutaneous lesions, cartilage lesions, muscle injuries, bone injuries, ligament injuries, tendon injuries and / or visceral injuries.

15. Method for the prevention and / or non-therapeutic treatment of the chronic and low-level systemic non-pathological inflammatory component of aging comprising the administration of calcium alginate according to claim 1, of the composition and / or device comprising calcium alginate according to claim 1.

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