COMPOSITIONS AND THEIR USE FOR RESTORING BOWEL PERMEABILITY AND / OR FOR THE PREVENTION OR CONTROL OF MULTIFACTORY DISEASES

Description

Technical field

[0001] The invention relates to the prevention and / or treatment of multifactorial diseases. In particular, the invention relates to compositions comprising several selected specific molecules and their use for reducing intestinal permeability and / or restoring the function of normal intestinal permeability and / or for preventing or combating various multifactorial diseases involving intestinal permeability. State of the art

[0002] Most diseases, especially chronic diseases, are multifactorial. They always involve a genetic component and immunological and toxic components linked to the "environment," i.e., bacteria, viruses, chemical agents, etc.

[0003] Among the known multifactorial chronic diseases, we can cite in particular degenerative diseases such as Parkinson's disease, Alzheimer's disease, inflammatory post-stroke effects, Charcot's disease (or amyotrophic lateral sclerosis), Huntington's disease, etc.

[0004] Degenerative diseases are pathologies that lead to a progressive degradation of one or more organs. Often of genetic origin, they can be triggered after strong and long exposure to biological and / or toxic products. Indeed, any excessive passage of bacterial constituents and / or other toxins through the mucous membranes, leads to immune activation, which, in patients with chronic conditions, always resumes the pathway of stimulating self-reactive clones attacking the target tissues (degeneration). Degenerative diseases are therefore closely linked to intestinal hyperpermeability, like many infectious diseases and particularly viral diseases.

[0005] In a healthy human intestine, small particles (< 4 Angstrom radius) can migrate through the claudin pores, which are the largest component of tight junctions, but particles up to 10-15 Angstrom (3.5LDa) can transit through the absorptive pathway of the paracellular space. The intestine normally exhibits a certain permeability, which allows nutrients to pass through the intestine, while maintaining a barrier function to prevent potentially harmful substances from leaving the intestine and migrating more widely into the body.

[0006] The intestinal flora constantly interacts with the intestinal immune system, with which it is in contact. The interaction between the considerable mass of antigens constituted by the intestinal microflora and the intestinal immune system is extremely complex, but in a balanced organism, this maintains an "immune background noise," an essential balance between the bacteria of the flora and the immune system that regulates them.

[0007] An unbalanced flora, called dysbiosis, leads to an exacerbated response of the intestinal immune system, increasing inflammation, which results in an alteration of tight junctions. The passage of fragments of walls of bacteria destroyed by the intestine, and passing into the general circulation, endotoxins, is accompanied by the significant secretion by the body of inflammatory cytokines, and if the arrival of endotoxins is massive, can be the starting point of septic shock, respiratory distress, renal ischemia or cytokine storm. The dosage of endotoxins in the blood is also a reliable marker of intestinal hyperpermeability.

[0008] The causes of intestinal hyperpermeability are extremely numerous. Any disruption of the intestinal biotope, any dysbiosis, triggers a response from the immune system, with the release of inflammatory cytokines, which results in an inflammatory state of the mucosa of the small intestine and colon, with alteration of the epithelium. The causes of this dysbiosis can include: Stress, which significantly reduces the volume and quality of all digestive juices, Emotional and psychological shocks, Significant efforts or physical shocks Antibiotic therapies, Viral, bacterial or parasitic infections, Radiation Toxic chemical agents: air, water and food pollution; pesticides and heavy metals; medications (statins, anti-inflammatories, immunosuppressants, etc.).

[0009] Intestinal hyperpermeability, in addition to contributing to the triggering of degenerative diseases in genetically predisposed patients, is the cause of inflammatory, infectious and autoimmune diseases, but also of very disabling food allergies and intolerances.

[0010] Currently, the solutions proposed to prevent or combat dysbiosis essentially consist of administering probiotics and / or prebiotics, but their effectiveness is very variable and depends on the patient's basic intestinal flora.

[0011] Furthermore, with regard to degenerative diseases specifically, these are often very burdensome for the patient. Some symptoms can be reduced, but these diseases are never completely cured and there is currently no satisfactory treatment. Many chemical agents have therapeutic properties, but either their toxicity, their lifespan, or their rapid elimination make them unsuitable for the treatment of degenerative diseases.

[0012] Indeed, while they are multifactorial, current treatments only target one of the factors causing these diseases, and do not seek to act on intestinal hyperpermeability. For example, Lou Gehrig's disease is linked to many factors, in particular oxidative stress, mitochondrial dysfunction, neuroinflammation, excitotoxicity, oligodendrocyte dysfunction and degeneration, impaired proteostasis, an impaired DNA repair system, alterations in nucleocytoplasmic RNA and in RNA linked to transport proteins, defective axonal transport, defective vesicular transport. However, currently, the recommended treatment for Lou Gehrig's disease is Riluzole ®< which acts only on the inhibition of glutamate release to combat excitotoxicity, and in addition no action on intestinal permeability is envisaged. The observation is identical for other degenerative diseases.

[0013] There is therefore a need for compositions capable of acting on different factors to prevent and / or combat the effects of intestinal hyperpermeability and associated multifactorial diseases, in particular against degenerative, autoimmune, inflammatory or infectious diseases. Summary of the invention

[0014] To meet this need, the invention proposes a composition which both: reduces the inflammatory process decreases intestinal permeability has an antioxidant effect has an anti-radical effect has an antibacterial, antiviral and antifungal effect, and preferentially rebalances neurological metabolisms causing the degeneration of neurons and motor neurons.

[0015] For this purpose, the invention relates to a composition comprising at least the following molecules: oleic acid, palmitic acid, lauric acid, linoleic acid, azelaic acid, farnesyl cysteine, palmitoleic acid, cholesterol, thioctic acid, myristic acid, orotic acid, acetic acid, butyric acid, lactic acid, propionic acid, and / or a salt and / or an ester and / or an anhydride of one or more of these molecules.

[0016] Advantageously, these molecules each act on different factors, allowing them to act in a combined manner on the inflammatory process, intestinal permeability, toxic chemical agents, free radicals and toxic microorganisms. The molecules act together to restore the function of intestinal permeability disrupted by an imbalance in the microbiota.

[0017] To increase the efficacy and bioavailability of these molecules, some are preferentially conjugated to specific polymers and others, which cannot be directly conjugated to these polymers, are preferentially in the form of co-polymers or encapsulated in micelles. The invention also relates to a method for manufacturing these compositions.

[0018] The compositions according to the invention can be used as a medicament alone or combined with the use of at least one other composition, and the invention also relates to the compositions for their use as a medicament.

[0019] Other characteristics and advantages will emerge from the detailed description of the invention, the examples and the figures which follow. Brief description of the Figures

[0020] [ Fig. 1a] represents a polymerization scheme: L-Met NCA and L-Lys NCA (trifluoroacetate) are mixed and polymerized to obtain the copolymer (Poly-(L-Lysine-co-methionine). (DMF = dimethylformamide). [ Fig. 1b ] represents a deprotection scheme: the copolymer of the Figure 1a is deprotected to obtain a Poly-(L-Lysine-co-methionine) in the form of bromide salt. [ Fig. 2a ] represents a polymerization scheme L-Cys NCA (trityl) and L-Lys NCA (trifluoroacetate) are mixed and polymerized to obtain the copolymer Poly-(L-lysine-co-L-cysteine). [ Fig2 b ] represents a deprotection scheme: the copolymer of the Figure 2a is deprotected to obtain a Poly-(L-lysine-co-L-cysteine) in the form of bromide salt. (DMF = dimethylformamide). [ Fig3] represents a scheme of micelle formations according to one embodiment of the invention: 10 = micelles; 12 = polymer (for example) poly-lysine; 14 = covalently conjugated molecules (for example fatty acids); 16 = highly hydrophobic molecules not conjugatable to polylysine (for example cholesterol, farnesyl-cysteine, co-enzyme Q10). [ Fig.4 ] is a graphical representation of a PLS-DA analysis of the comparison of the microbiota of WT and SOD1 mice [ Fig 5 ] is a graphical representation of a comparative analysis of the beta diversity of the mouse intestinal microbiota via the MDS - Jaccard method as a function of treatment. [ Fig 6 ] is a graphical representation of a comparative analysis of the beta diversity of the mouse intestinal microbiota via the MDS - Bray Curtis method as a function of treatment. [ Fig 7] is a graphical representation of a comparative analysis of the mouse gut microbiota as a function of treatment via PLS-DA analysis. [ Fig 8 ] is a graphical representation of a correlation analysis between WT or SOD1 mice based on bacterial families. [ Fig 9 ] is a graphical representation of a correlation analysis between low-dose treated and untreated SOD1 mice. Detailed description of the invention Definition

[0021] For the purposes of the invention, the term "animal" means any animal with the exception of human beings. This may include, in particular, mammals.

[0022] For the purposes of the invention, the term “amphiphilic conjugate” means a conjugate formed by a hydrophobic molecule X and a hydrophilic polymer Y, the conjugate thus having an amphiphilic characteristic.

[0023] For the purposes of the invention, the term “molecule X conjugated to a polymer Y” means a molecule X linked by a covalent bond to a polymer Y, said bond being preferably able to be an amide, urea or carbamate bond depending on the chemical nature of the molecule X and the polymer Y. Composition C1

[0024] The present invention therefore relates to a composition C1 comprising at least the following active molecules: oleic acid, palmitic acid, lauric acid, linoleic acid, azelaic acid, farnesyl cysteine, palmitoleic acid, cholesterol, thioctic acid, myristic acid, orotic acid, acetic acid, butyric acid, lactic acid, propionic acid, and / or a salt and / or an ester and / or an anhydride of one or more of these molecules.

[0025] Composition C1 may optionally also comprise pyruvic acid and / or a salt and / or an ester and / or an anhydride.

[0026] For all the active molecules present in composition C1, when they are mentioned in the present application, these may be the molecules (example butyric acid) as such and / or a salt (example: butyrate) and / or an ester and / or an anhydride of these molecules (example: butyric acid ester) and / or an anhydride. Preferably:

[0027] the quantity of butyric acid in composition C1 is greater than that of each of the other molecules taken individually, and / or the quantity of thioctic acid in composition C1 is greater than that of each of the other molecules taken individually, except butyric acid, and / or the quantity of lauric acid in composition C1 is greater than that of each of the following molecules taken individually: palmitic acid, linoleic acid, azelaic acid, farnesyl cysteine, palmitoleic acid, cholesterol, myristic acid, orotic acid, the quantity of oleic acid on the one hand and lactic acid on the other hand in composition C1 is greater than that of each of the following molecules taken individually: palmitic acid, lauric acid, linoleic acid, azelaic acid, farnesyl cysteine, palmitoleic acid, cholesterol, acid myristic, orotic acid, acetic acid.

[0028] Preferably, each of the active molecules in composition C1 represents between 0.5 E-05 M and 10 E-05 M.

[0029] According to one embodiment, to improve the solubility, efficacy and bioavailability of the molecules of composition C1, at least one of the molecules of the composition chosen from oleic acid, palmitic acid, lauric acid, linoleic acid, azelaic acid, palmitoleic acid, thioctic acid, myristic acid, orotic acid, acetic acid, butyric acid, lactic acid, propionic acid, the salts of these acids, the esters of these acids and the anhydrides of these acids, is covalently conjugated to at least one molecule of a polymer chosen from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine.

[0030] Indeed, the molecules chosen from oleic acid, palmitic acid, lauric acid, linoleic acid, azelaic acid, palmitoleic acid, thioctic acid, myristic acid, orotic acid, acetic acid, butyric acid, lactic acid, propionic acid, the salts of these acids, the esters of these acids and the anhydrides of these acids, are all covalently conjugatable with a polymer chosen from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine, in particular by an amide, urea or carbamate bond.

[0031] According to a particularly suitable variant, all the molecules chosen from oleic acid, palmitic acid, lauric acid, linoleic acid, azelaic acid, palmitoleic acid, thioctic acid, myristic acid, orotic acid, acetic acid, butyric acid, lactic acid, propionic acid, the salts of these acids and the esters of these acids, present in the composition C1 according to the invention are covalently conjugated to at least one molecule of a polymer chosen from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine.

[0032] Among the set of molecules to be conjugated, 2 of them are highly hydrophobic (LogP values ​​greater than 1, preferably greater than 2 and preferably greater than 3) and chemically not suitable for covalent conjugation, either due to the absence of reactive functional groups or due to chemical incompatibilities. These are cholesterol and farnesyl-cysteine.On the other hand, considering both the hydrophilic character of certain polymers (such as poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine or poly-histidine) and the hydrophobic character of most molecules suitable for covalent conjugation (oleic acid, palmitic acid, lauric acid, linoleic acid, azelaic acid, palmitoleic acid, thioctic acid, myristic acid, orotic acid, acetic acid, butyric acid, lactic acid, propionic acid), according to the invention these particular conjugates behave as surfactants in aqueous solution. In this way, some of these conjugates (amphiphiles) act as polymeric micelles in aqueous media, generating a hydrophobic pocket, capable of encapsulating hydrophobic species, thus considerably improving their solubility and stability in aqueous solution.

[0033] Farnesyl cysteine ​​and cholesterol, and the esters, salts and anhydrides of farnesyl cysteine ​​or cholesterol cannot covalently bond, cannot be conjugated, to a polymer selected from poly-lysine, polyethylene glycol, poly-ornithine, poly-arginine and poly-histidine. Also, preferably, the farnesyl cysteine ​​and / or cholesterol, and / or their esters, present in composition C1 are encapsulated in micelles. Thus, composition C1 preferably comprises micelles in which at least farnesyl cysteine ​​and / or cholesterol and / or one of their esters and / or salts and / or anhydrides are encapsulated.According to a particularly suitable embodiment, at least one of the micelles is formed by amphiphilic conjugates each consisting of at least one hydrophobic molecule covalently conjugated to a molecule of a polymer chosen from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine. Even more preferably, as shown in the . figure 3, at least one of the micelles 10 is formed by amphiphilic conjugates each consisting of at least one hydrophobic molecule 14 preferentially chosen from the fatty acids of the composition and in particular at least one molecule chosen from oleic acid, palmitic acid, lauric acid, linoleic acid, palmitoleic acid, myristic acid, the salts of these acids, the esters of these acids and the anhydrides of these acids, covalently conjugated to a molecule of a polymer 12 chosen from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine, said micelles encapsulating farnesyl cysteine ​​and / or cholesterol and / or one of their esters and / or salts and / or anhydrides 16.Indeed, oleic acid, palmitic acid, lauric acid, linoleic acid, azelaic acid, palmitoleic acid, myristic acid, propionic acid, the salts of these acids, the esters of these acids and the anhydrides of these acids, are hydrophobic and poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine are hydrophilic polymers.

[0034] Thus, composition C1 preferably comprises: conjugates of oleic acid, palmitic acid, lauric acid, linoleic acid, azelaic acid, palmitoleic acid, thioctic acid, myristic acid, orotic acid, acetic acid, butyric acid, lactic acid, propionic acid, salts of these acids, esters of these acids and anhydrides of these acids, with poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine or poly-histidine, and micelles formed by at least one of these conjugates, said micelles encapsulating farnesyl cysteine ​​and cholesterol.

[0035] This configuration allows in particular: to increase the half-life of the active molecules of the composition in the body, to target the tissues or cells on which the molecules of the composition must act, to allow the active molecules of the composition to pass the blood-brain barrier and penetrate into the cells, in particular into neurons and motor neurons, to increase the stability and bioavailability of the active ingredient.

[0036] The effectiveness of the composition is therefore greater and it is possible to administer lower doses and reduce the acute or chronic toxicity of the active molecules contained in the composition.

[0037] Preferably, the polymer(s) conjugated to the molecules are chosen from poly-L-lysine, poly-ethylene glycol, poly-L-ornithine, poly-L-arginine and poly-L-histidine.

[0038] According to one embodiment, it is poly-lysine, preferably poly-L-lysine. The polylysine is preferably linear. In particular, the poly-lysine used is an epsilon poly-L-lysine with a molecular weight of between 12,000 and 20,000 Da.

[0039] Thus, according to one variant, composition C1 comprises at least: A. the following conjugates, each conjugate consisting of a molecule covalently linked to a poly-lysine: one or more Oleyl-Poly-L-Lysine conjugates one or more Palmitic-Poly-L-Lysine conjugates one or more Lauryl-Poly-L-Lysine conjugates one or more Azelayl-Poly-L-Lysine conjugates one or more Palmitoleyl-Poly-L-Lysine conjugates one or more Thioctyl-Poly-L-Lysine conjugates one or more Myristyl-Poly-L-Lysine conjugates one or more Orotyl-Poly-L-Lysine conjugates one or more Acetate-Poly-L-Lysine conjugates one or more Butyrate-Poly-L-Lysine conjugates one or more Lactate-Poly-L-Lysine one or more conjugates Propionate-Poly-L-Lysine one or more conjugates Linoleyl-Poly-L-Lysine, and B. farnesyl cysteine ​​and cholesterol, and / or an ester of these molecules, encapsulated in micelles, preferably in micelles formed by one or more of the conjugates listed in list A.

[0040] In this composition, poly-L-Lysine may be replaced by another poly-lysine or by polyethylene glycol, poly-L-ornithine, poly-L-arginine or poly-L-histidine.

[0041] Composition C1 according to the invention may be in liquid form or in solid form. When it is in liquid form, the composition comprises at least water and the constituents mentioned above. The solid form is preferably obtained from the liquid form, preferably by lyophilization. Thus, when composition C1 in liquid form comprises micelles and is lyophilized in solid form, the micelles reform when composition C1 in solid form is again placed in an aqueous solution.

[0042] Composition C1 also preferably comprises pharmaceutically acceptable excipients. These excipients may be chosen in particular to meet the pH and osmolarity of injectable solutions in humans or animals. These may be, for example, acids or bases to adjust the pH or NaCl to adjust the osmolarity.

[0043] Composition C1 according to the invention is intended to be administered to humans or animals and is therefore in a form suitable for such administration. When it is in liquid form, it is preferably suitable for subcutaneous or intravenous administration, in particular intravenous administration by perfusion, and it is packaged in suitable containers known to those skilled in the art for packaging this type of product. Composition C1 can also be administered in liquid form via a pump, such as insulin pumps.

[0044] When it comes in solid form, it is preferentially suitable for administration: by transcutaneous route and it is preferably formulated and packaged in the form of a patch, or by nasal route in powder form, or by sublingual route in powder or tablet form, or by transmucosal route and it is preferably formulated and packaged in the form of a mucoadhesive tablet. Manufacturing process of composition C1

[0045] Composition C1 according to the invention can be manufactured by any suitable process.

[0046] If the molecules that make up composition C1 are used as they are in a solvent, they can be mixed all together in said solvent.

[0047] If the molecules in composition C1 are conjugated to polymers for some part and in micelles for others, the manufacturing process can comprise the following steps: has. create an amphiphilic premix: mix in an aqueous solution one or more conjugates chosen from: one or more Oleyl-Poly-L-Lysine conjugates one or more Palmitic-Poly-L-Lysine conjugates one or more Lauryl-Poly-L-Lysine conjugates one or more Azelayl-Poly-L-Lysine conjugates one or more Palmitoleyl-Poly-L-Lysine conjugates one or more Thioctyl-Poly-L-Lysine conjugates one or more Myristyl-Poly-L-Lysine conjugates one or more Orotyl-Poly-L-Lysine conjugates one or more Acetate-Poly-L-Lysine conjugates one or more Butyrate-Poly-L-Lysine conjugates one or more Lactate-Poly-L-Lysine one or more Propionate-Poly-L-Lysine conjugates, one or more Linoleyl-Poly-L-Lysine conjugates, and b.adding to the amphiphilic premix at least farnesylcysteine ​​and cholesterol, and stirring so as to form micelles formed by one or more of the conjugates of the amphiphilic premix encapsulating the farnesylcysteine ​​and cholesterol.

[0048] Indeed, a variant of the invention consists in taking advantage of the amphiphilic nature of most of the individual components, to create an amphiphilic premix which allows the controlled solubilization of the hydrophobic species. The solubilization process according to a preferred embodiment consists of a controlled addition of the hydrophobic molecules to the amphiphilic premixes and allowing the necessary time for their dissolution under stirring.

[0049] Poly-L-lysine can be replaced by another poly-lysine or by polyethylene glycol, poly-L-ornithine, poly-L-arginine or poly-L-histidine.

[0050] Preferably, the stirring is carried out for at least 60 minutes, even more preferably between 5 and 20 minutes, and preferably at a stirring speed of less than or equal to 900 revolutions per minute, in particular at a stirring speed of between 50 and 800 revolutions per minute.

[0051] According to a preferred embodiment, the manufacturing process according to the invention also comprises a step c. of separation of the soluble and insoluble phases, to recover the soluble phase. In this case, the insoluble phase is eliminated and the soluble phase constitutes the composition C1 according to the invention. Indeed, a physical separation process (filtration, ultrafiltration) is preferentially carried out to ensure the isolation of the soluble fraction, containing both the amphiphilic premixture and the solubilized molecules.

[0052] Composition C1 in liquid form can then be lyophilized or dehydrated to be in solid form, preferably by slow lyophilization for example between 12 and 36 hours.

[0053] Furthermore, if active molecule-polymer conjugates of composition C1 are not integrated into the premix of step a, these can be added to the mixture after step b, i.e. after the formation of the micelles and the encapsulation of the farnesylcysteine ​​and cholesterol.

[0054] The active molecule-polymer conjugates can be manufactured by any means known to those skilled in the art for covalently conjugating a molecule to a polymer depending on their chemical nature. Thus, lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, orotic acid, azelaic acid, thioctic acid, acetic acid, palmitoleic acid, butyric acid, lactic acid, propionic acid, and oleic acid are conjugated to the polymer (preferably poly-L-Lysine) by an amide bond.

[0055] Examples of the realization of amide, urea and carbamate bonds are described for example in: Ryser HJ, Shen WC. Conjugation of methotrexate to poly (L-lysine) as a potential way to overcome drug resistance. Cancer. 1980 Mar 15;45(5 Suppl):1207-11 (liaisons amide), Zhuxian Z, Jianbin T, Qihang S, William J. A multifunctional PEG-PLL drug conjugate forming redox-responsive nanoparticles for intracellular drug delivery Issue 38, 2015. Journal of Materials Chemistry B (liaisons amide), Scheper V, Wolf M, Scholl M, Kadlecova Z, Perrier T, Klok HA, Saulnier P, Lenarz T, Stöver T. Potential novel drug carriers for inner ear treatment: hyperbranched polylysine and lipid nanocapsules. Nanomedicine (Lond). 2009 Aug;4(6):623-35 (liaisons urée), Stéphanie Gac-Breton, Jean Coudane, Mahfoud Boustta & Michel Vert (2004) Norfloxacin-Poly(l-Lysine Citramide Imide) Conjugates and Structure-dependence of the Drug Release, Journal of Drug Targeting, 12:5, 297-307, (liaisons carbamate), Elmore, W. M. (2013).Nanoparticles Stabilized with MPEG-Polylysine Carbamate: Synthesis and Characterization, (carbamate bonds), Ning-Ping Huang, Janos Vörös, Susan M. De Paul, Marcus Textor, and Nicholas D. Spencer. Biotin-Derivatized Poly(l-lysine)-g-poly(ethylene glycol): A Novel Polymeric Interface for Bioaffinity Sensing. Langmuir 2002 18 (1), 220-230 (carbamate bonds). . Use of composition C1

[0056] Composition C1 is a generic formulation particularly suitable for preventing and / or treating multifactorial diseases, including chronic multifactorial diseases, and in particular diseases involving intestinal permeability.

[0057] It is capable of acting in a combined manner: as an immunomodulator and regulator of autoimmunity, in particular due to the presence of oleic acid, palmitic acid, palmitoleic acid, as an anti-inflammatory, in particular due to the presence of palmitic acid, azelaic acid, farnesyl cysteine, palmitoleic acid, myristic acid, as an antibacterial, in particular due to the presence of lauric acid, azelaic acid, as an antifungal, in particular due to the presence of lauric acid, as an anti-radical, in particular due to the presence of farnesyl cysteine, as an antagonist of isoprenylated proteins, in particular due to the presence of farnesyl cysteine, on the stabilization of cell membranes, in particular due to the presence of cholesterol, as a scavenger of free radicals and metals, in particular due to the presence of thioctic acid.as an anti-viral, in particular thanks to the presence of myristic acid, as a cellular synergistic substrate, in particular thanks to the presence of butyrate, to promote the development of intestinal flora, in particular thanks to the presence of orotic acid, to regulate the mucous membranes and intestinal flora, in particular thanks to the presence of acetate, lactate, propionate and Butyrate.

[0058] The different molecules present act in synergy and in particular help to restore the function of intestinal permeability.

[0059] Thus, composition C1 can be used to restore the function of the intestinal membrane and / or to maintain intestinal permeability and / or prevent or combat hyperpermeability of the intestinal mucosa.

[0060] According to one variant, the invention relates to the use of a composition C1 as a food supplement in a healthy person or a healthy animal, in particular to reduce the intestinal permeability of the person or animal for whom it is intended.

[0061] The intestine therefore plays a key role as an initiator and regulator of the chronicity of diseases. Thus, composition C1 can be administered to humans or animals to prevent or combat diseases in which the intestine plays an important role, including chronic diseases, and in particular chronic degenerative diseases, such as neurodegenerative diseases such as, for example, Lou Gehrig's disease. Composition C1 can also be used as an activator of the immune system to prevent or combat autoimmune and infectious diseases.

[0062] The composition can be used in particular to reduce the chronicity of chronic diseases.

[0063] Preferably, composition C1 is used for prevention or treatment from the first symptoms of intestinal hyperpermeability or a chronic disease, so as to prevent the development of the chronicity of said disease and thus avoid the inflammatory storm which results from prolonged intestinal hyperpermeability.

[0064] Specifically, composition C1 is particularly useful for at least: reduce the inflammatory process, and / or reduce the autoimmune process, and / or reduce intestinal permeability, and / or an antioxidant effect, and / or an anti-radical effect, and / or an antibacterial and / or antiviral / or antifungal effect.

[0065] The invention therefore also relates to a composition C1 according to the invention for its use as a medicament, in particular in humans or animals, that is to say for a human or animal subject.

[0066] In particular, the subject of the invention is a composition C1 for its use in the prevention and / or treatment of a multifactorial pathology, in particular a chronic multifactorial pathology, in particular a pathology chosen from inflammatory diseases, neurodegenerative diseases, bacterial diseases, viral diseases, autoimmune diseases, food allergies and intolerances.

[0067] According to a particular embodiment, the invention relates to a composition C1 for its use in the prevention and / or treatment of a pathology chosen from eczema, psoriasis, chronic inflammatory intestinal diseases (in particular Crohn's disease and Celiac disease), Alzheimer's disease, Parkinson's disease, multiple sclerosis, Charcot's disease.

[0068] Composition C1 can be used alone or with one or more other compositions.

[0069] According to one embodiment, composition C1 is preferentially used with a composition C2. Composition C2

[0070] The present invention therefore also relates to a composition C2 comprising at least the following active molecules: Taurine, Cysteine, Methionine Spermine Retinoic Acid, Pantothenic Acid, Biotin, Co-enzyme Q10, Glutathione, Ascorbic Acid, GABA, Alpha-Tocopherol, and / or a salt and / or an ester and / or an anhydride of one or more of these molecules.

[0071] For all the active molecules present in composition C2, when they are mentioned in the present application, these may be the molecules (example ascorbic acid) as such and / or a salt (example: ascorbate) and / or an ester of these molecules (example: ascorbic acid ester) and / or an anhydride (example: ascorbic acid anhydride). Preferably:

[0072] the amount of retinoic acid in composition C2 is greater than that of each of the other molecules taken individually, and / or the amount of taurine, the amount of methionine and the amount of GABA are equivalent in composition C2 and are each greater than that of each of the other molecules taken individually, except retinoic acid and coenzyme Q10.

[0073] Preferably, each of the active molecules in composition C2 represents between 6 E-05 M and 18 E-05 M.

[0074] According to one embodiment, to improve the solubility, efficacy and bioavailability of the molecules of composition C2, at least one of the molecules of the composition chosen from taurine, spermine, pantothenic acid, biotin, glutathione, ascorbic acid, GABA, and alpha-tocopherol, the salts of these molecules, the esters of these molecules and the anhydrides of these molecules, is covalently conjugated to at least one molecule of a polymer chosen from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine.

[0075] Indeed, the molecules chosen from taurine, spermine, pantothenic acid, biotin, retinoic acid, glutathione, ascorbic acid, GABA, and alpha-tocopherol, the salts of these molecules, the esters of these molecules and the anhydrides of these molecules, are all covalently conjugatable with a polymer chosen from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine, in particular by an amide, urea or carbamate bond.

[0076] According to a particularly suitable variant, all the molecules chosen from taurine, spermine, retinoic acid, pantothenic acid, biotin, glutathione, ascorbic acid, GABA, and alpha-tocopherol, the salts of these molecules, the esters of these molecules and the anhydrides of these molecules, present in the composition C1 according to the invention are covalently conjugated to at least one molecule of a polymer chosen from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine.

[0077] Cysteine ​​and methionine, and esters, salts and anhydrides of cysteine ​​or methionine cannot covalently bond, cannot be conjugated, to a polymer selected from poly-lysine, polyethylene glycol, poly-ornithine, poly-arginine and poly-histidine. Also, preferably, cysteine ​​and / or methionine, and / or their esters and / or salts, present in composition C2 are bonded to said polymer by copolymerization to form a copolymer. Preferably, cysteine ​​and / or methionine, and / or their esters and / or salts and / or anhydrides are included in the polypeptide backbone of the polymer to form a copolymer.

[0078] The copolymerization can be carried out in particular by implementing a polymerization step (for example L-Met NCA or L-Cys NCA and L-Lys NCA are mixed and polymerized to obtain a copolymer) then a step of deprotection of the copolymer obtained after polymerization. Examples are presented on the Figures 1a, 1b (Poly-(L-Lysine-co-methionine), 2a and 2b (Poly-(L-lysine-co-L-cysteine): 1a: Polymerization: L-Met NCA and L-Lys NCA (trifluoroacetate) are mixed and polymerized in the appropriate ratio to obtain the appropriate random copolymer, 1b: Deprotection of PLys-TFA: The previous copolymer is deprotected to obtain PLys in the form of bromide salt. 2a: Polymerization: L-Cys NCA (trityl) and L-Lys NCA (trifluoroacetate) are mixed in the appropriate ratio to obtain the appropriate random copolymer. 2b: Deprotection of Lys-TFA: The previous copolymer is deprotected to obtain L-Lys in the form of bromide salt and L-Cys in its deprotected form.

[0079] Thus, in composition C2, cysteine ​​and methionine are each conjugated to a molecule of a polymer selected from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine, by polymerization, said molecule and said polymer forming a copolymer. Preferably, the copolymers have between 5 and 20% cysteine ​​or methionine and between 80 and 95% polymer (selected from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine). According to a preferred variant, the copolymers have 10% cysteine ​​or methionine and 90% polymer (ratio of 1 to 10).

[0080] Among all the molecules to be conjugated, one of them is highly hydrophobic (LogP values ​​greater than 1, preferably greater than 2 and preferably greater than 3) and chemically not suitable for covalent conjugation, due to the absence of reactive functional groups and due to chemical incompatibilities. This is coenzyme Q10. On the other hand, considering both the hydrophilic character of certain polymers (such as poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine or poly-histidine) and the hydrophobic character of certain molecules suitable for covalent conjugation (alpha-tocopherol, retinoic acid), according to the invention these particular conjugates behave as surfactants in aqueous solution.In this way, some of these conjugates (amphiphiles) act as polymeric micelles in aqueous media, generating a hydrophobic pocket, capable of encapsulating the hydrophobic coenzyme Q10, thus considerably improving their solubility and stability in aqueous solution.

[0081] Co-enzyme Q10, and the esters, salts and anhydrides of co-enzyme Q10 cannot in fact bind covalently, cannot be conjugated, to a polymer chosen from poly-lysine, polyethylene glycol, poly-ornithine, poly-arginine and poly-histidine. Also, preferably co-enzyme Q10 and / or the esters and salts of co-enzyme Q10 are encapsulated in micelles. Thus, composition C2 preferably comprises micelles in which at least co-enzyme Q10 and / or esters and / or salts and / or anhydrides of co-enzyme Q10 are encapsulated.

[0082] According to a particularly suitable embodiment, at least one of the micelles is formed by amphiphilic conjugates each consisting of at least one hydrophobic molecule covalently conjugated to a molecule of a polymer chosen from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine. Even more preferably, as shown in the figure 3, at least one of the micelles 10 is formed by amphiphilic conjugates each consisting of at least one molecule 14 chosen from taurine, spermine, pantothenic acid, biotin, glutathione, ascorbic acid, GABA, and alpha-tocopherol, the salts of these molecules, the esters of these molecules and the anhydrides of these molecules, covalently conjugated to a molecule of a polymer 12 chosen from poly-lysine, poly-ethylene glycol, poly-ornithine, poly-arginine and poly-histidine, encapsulating co-enzyme Q10 and / or esters and / or salts and / or anhydrides of co-enzyme Q10 16.Indeed, taurine, spermine, pantothenic acid, biotin, glutathione, ascorbic acid, GABA, and alpha-tocopherol, the salts of these molecules, the esters of these molecules and the anhydrides of these molecules, are hydrophobic and poly-L-lysine, poly-ethylene-glycol, poly-ornithine, poly-arginine or poly-histidine are hydrophilic polymers.

[0083] Thus, composition C2 preferably comprises: conjugates of taurine, retinoic acid, spermine, pantothenic acid, biotin, glutathione, ascorbic acid, GABA, and alpha-tocopherol, salts of these molecules, esters of these molecules and / or anhydrides of these molecules, with poly-lysine, polyethylene glycol, poly-ornithine, poly-arginine or poly-histidine, and micelles formed by at least one of these conjugates, said micelles encapsulating co-enzyme Q10, and copolymers of methionine and poly-lysine or polyethylene glycol or poly-ornithine or poly-arginine or poly-histidine, and / or copolymers of cysteine ​​and poly-lysine or polyethylene glycol or poly-ornithine or poly-arginine or polyhistidine.

[0084] This configuration allows in particular: to increase the half-life of the active molecules of the composition in the body, to target the tissues or cells on which the molecules of the composition must act, to allow the active molecules of the composition to pass the blood-brain barrier and penetrate into the cells to increase the stability and bioavailability of the active ingredient.

[0085] The effectiveness of composition C2 is therefore greater and it is possible to administer lower doses and reduce the acute or chronic toxicity of the active molecules contained in the composition.

[0086] Preferably, the polymer(s) conjugated to the molecules or with which the molecules are copolymerized are chosen from poly-L-lysine, polyethylene glycol, poly-L-ornithine, poly-L-arginine and poly-L-histidine.

[0087] According to one embodiment, it is poly-lysine, preferably poly-L-lysine. Preferably, the poly-lysine used is a poly-L-lysine with a molecular weight of between 12,000 and 20,000 Da.

[0088] Thus, according to one variant, composition C2 comprises at least: A. the following conjugates, each conjugate consisting of a molecule covalently linked to a poly-lysine: one or more Taurine-Poly-L-Lysine conjugates one or more Spermine-Poly-L-Lysine conjugates one or more Pantothenyl-Poly-L-Lysine conjugates one or more Alpha tocopherol-Poly-L-Lysine conjugates one or more Biotinyl-Poly-L-Lysine conjugates one or more Glutathione-Poly-L-Lysine conjugates one or more Ascorbyl-Poly-L-Lysine conjugates one or more GABA-Poly-L-Lysine conjugates, one or more Retinoyl-Poly-L-Lysine conjugates, and

[0089] B. Co-enzyme Q10, and / or an ester and / or a salt of Co-enzyme Q10, encapsulated in micelles, preferably in micelles formed by one or more of the conjugates listed in point A, and C. methionine-Poly-L-Lysine copolymers and cysteine-Poly-L-Lysine copolymers, the methionine and / or the cysteine ​​being able to be replaced by an ester or a salt of these molecules.

[0090] In this composition, poly-L-Lysine may be replaced by another poly-lysine or by polyethylene glycol, poly-L-ornithine, poly-L-arginine or poly-L-histidine.

[0091] Composition C2 according to the invention may be in liquid form or in solid form. When it is in liquid form, the composition comprises at least water and the constituents mentioned above. The solid form is preferably obtained from the liquid form, preferably by lyophilization. Thus, when composition C2 in liquid form comprises micelles and is lyophilized in solid form, the micelles reform when composition C2 in solid form is again placed in an aqueous solution.

[0092] Composition C2 also preferably comprises pharmaceutically acceptable excipients. These excipients may be chosen in particular to meet the pH and osmolarity of injectable solutions in humans or animals. These may be, for example, acids or bases to adjust the pH or NaCl to adjust the osmolarity.

[0093] Composition C2 according to the invention is intended to be administered to humans or animals and is therefore in a form suitable for such administration. When it is in liquid form, it is preferably suitable for subcutaneous or intravenous administration, in particular intravenous administration by perfusion, and it is packaged in suitable containers known to those skilled in the art for packaging this type of product. Composition C2 can also be administered in liquid form via a pump, such as insulin pumps.

[0094] When it comes in solid form, it is preferentially suitable for administration: by transcutaneous route and it is preferably formulated and packaged in the form of a patch, or by nasal route in powder form, or by sublingual route in powder or tablet form, or by transmucosal route and it is preferably formulated and packaged in the form of a mucoadhesive tablet. Manufacturing process of composition C2

[0095] Composition C2 according to the invention can be manufactured by any suitable process.

[0096] If the molecules that make up composition C2 are used as they are in a solvent, they can be mixed all together in said solvent.

[0097] If the molecules in composition C2 are conjugated to polymers for some, copolymerized for others and in micelles for others, the manufacturing process can comprise the following steps: a. create an amphiphilic premix: mix in an aqueous solution one or more conjugates chosen from: one or more Taurine-Poly-L-Lysine conjugates one or more Spermine-Poly-L-Lysine conjugates one or more Pantothenyl-Poly-L-Lysine conjugates one or more Alpha tocopherol-Poly-L-Lysine conjugates one or more Biotinyl-Poly-L-Lysine conjugates one or more Glutathione-Poly-L-Lysine conjugates one or more Ascorbyl-Poly-L-Lysine conjugates one or more GABA-Poly-L-Lysine conjugates, one or more Retinoyl-Poly-L-Lysine conjugates, and b. adding to the amphiphilic premix at least co-enzyme Q10, and stirring so as to form micelles formed by one or more of the conjugates of the amphiphilic premix encapsulating co-enzyme Q10, adding methionine-Poly-L-Lysine co-polymers and cysteine-Poly-L-Lysine co-polymers to the mixture.

[0098] Indeed, a variant of the invention consists in taking advantage of the amphiphilic nature of most of the individual components, to create an amphiphilic premix which allows the controlled solubilization of the hydrophobic co-enzyme Q10. The solubilization process according to a preferred embodiment consists of a controlled addition of the hydrophobic molecules to the amphiphilic premixes and allowing the necessary time for their dissolution under stirring.

[0099] Poly-L-lysine can be replaced by another poly-lysine or by polyethylene glycol, poly-L-ornithine, poly-L-arginine or poly-L-histidine.

[0100] Preferably, the stirring is carried out for at least 5 minutes, even more preferably between 5 and 20 minutes, and preferably at a stirring speed of less than or equal to 900 revolutions per minute, in particular at a stirring speed of between 50 and 800 revolutions per minute.

[0101] According to a preferred embodiment, the manufacturing process according to the invention also comprises a step d. of separating the soluble and insoluble phases, to recover the soluble phase. In this case the insoluble phase is eliminated and the soluble phase constitutes the composition C2 according to the invention. Indeed, a physical separation process (filtration, ultrafiltration) is preferentially carried out to ensure the isolation of the soluble fraction, containing both the amphiphilic premix, the copolymers and the solubilized molecules.

[0102] Composition C2 in liquid form can then be lyophilized or dehydrated to be in solid form, preferably by slow lyophilization for example between 12 and 36 hours.

[0103] Furthermore, if active molecule-polymer conjugates and / or co-polymers of composition C2 are not integrated into the premix of step a or c, these can be added to the mixture after step b, i.e. after the formation of the micelles and the encapsulation of the co-enzyme Q10.

[0104] Active molecule-polymer conjugates can be manufactured by any means known to those skilled in the art for covalently conjugating a molecule to a polymer depending on their chemical nature. Thus: Ascorbic acid is conjugated to a polymer by a carbamate bond, spermine and taurine are conjugated to a polymer by a urea bond. Glutathione, pantothenic acid, aminobutyric acid, biotin, alpha tocopherol and GABA, retinoic acid are conjugated to a polymer by an amide bond.

[0105] Examples of the production of urea and carbamate bonds are described for example in: Zhuxian Z, Jianbin T, Qihang S, William J. A multifunctional PEG-PLL drug conjugate forming redox-responsive nanoparticles for intracellular drug delivery Issue 38, 2015. Journal of Materials Chemistry B (liaisons amide), Scheper V, Wolf M, Scholl M, Kadlecova Z, Perrier T, Klok HA, Saulnier P, Lenarz T, Stöver T. Potential novel drug carriers for inner ear treatment: hyperbranched polylysine and lipid nanocapsules. Nanomedicine (Lond). 2009 Aug;4(6):623-35 (liaisons urée), Stéphanie Gac-Breton, Jean Coudane, Mahfoud Boustta & Michel Vert (2004) Norfloxacin-Poly(l-Lysine Citramide Imide) Conjugates and Structure-dependence of the Drug Release, Journal of Drug Targeting, 12:5, 297-307, (liaisons carbamate), Elmore, W. M. (2013). Nanoparticles Stabilized with MPEG-Polylysine Carbamate: Synthesis and Characterization, (liaisons carbamate), Ning-Ping Huang, Janos Vörös, Susan M. De Paul, Marcus Textor, and Nicholas D. Spencer.Biotin-Derivatized Poly(l-lysine)-g-poly(ethylene glycol): A Novel Polymeric Interface for Bioaffinity Sensing. Langmuir 2002 18 (1), 220-230 (carbamate bonds). .

[0106] The copolymers can be manufactured by any means known to those skilled in the art. The methionine or cysteine ​​molecules are incorporated into the structure of the polymer preferably in the presence of an anhydrous solvent and according to one embodiment in a ratio of 1 molecule of cysteine ​​or methionine for 10 monomers of the polymer. Using composition C2

[0107] Composition C2 is a formulation particularly suitable for preventing and / or treating multifactorial diseases, including chronic multifactorial diseases. Composition C2 is particularly useful for the prevention and treatment of neurodegenerative, autoimmune, infectious diseases or cancers. In particular, it is highly effective in the prevention and treatment of Charcot's disease. Composition C2 acts on classic and known processes to reduce the chronicity of diseases. Its action is particularly suitable for neurodegenerative, autoimmune, cancer and infectious diseases.

[0108] In particular, it is capable of acting in a combined manner: as an immunomodulator and regulator of autoimmunity, in particular thanks to the presence of retinoic acid and GABA, as a neuroprotector, in particular thanks to the presence of taurine, co-enzyme Q10, GABA as an anti-inflammatory, in particular thanks to the presence of spermine, as a cell protector, in particular thanks to the presence of pantothenic acid, co-enzyme Q10 (protection in particular at the level of the mitochondrial chain) as a protector of cell membranes, in particular thanks to the presence of alpha-tocopherol, as an antioxidant, in particular thanks to the presence of taurine, cysteine, methionine, spermine, pantothenic acid, alpha-tocopherol, co-enzyme Q10, glutathione, ascorbic acid, as a regulator of cellular metabolism, scavenging oxygenated derivatives, in particular thanks to ascorbic acid, as a precursor of glutathione, in particular thanks to the presence of cysteine, on the stabilization of membranes cellular,as a scavenger of free radicals and metals, in particular thanks to the presence of taurine, cysteine, methionine, spermine, pantothenic acid, co-enzyme Q10, glutathione.

[0109] The different molecules present act in synergy and in particular help to prevent or combat the factors causing chronic diseases and in particular Charcot's disease.

[0110] Thus, composition C2 can be used to prevent and / or combat chronic diseases, particularly to act on the specific metabolisms of the pathology. Composition C2 can be used in particular to reduce the chronicity of chronic diseases.

[0111] Preferably, composition C2 is used for prevention or treatment from the first symptoms of a chronic disease, in particular a neurodegenerative disease and specifically Charcot's disease.

[0112] The invention therefore also relates to a composition C2 according to the invention for its use as a medicament, in particular in humans or animals, that is to say for a human or animal subject.

[0113] In particular, the invention relates to a composition C2 for its use in the prevention and / or treatment of a multifactorial pathology, in particular a chronic multifactorial pathology, in particular a pathology chosen from inflammatory diseases, neurodegenerative diseases, bacterial diseases, viral diseases, autoimmune diseases, food allergies and intolerances, and in particular Charcot's disease.

[0114] According to a particular embodiment, the invention relates to a composition C1 for its use in the prevention and / or treatment of a pathology chosen from eczema, psoriasis, chronic inflammatory intestinal diseases (in particular Crohn's disease and Celiac disease), Alzheimer's disease, Parkinson's disease, multiple sclerosis, Charcot's disease.

[0115] Composition C2 is very preferentially used with composition C1.

[0116] A particular subject of the invention is therefore composition C1 for its use with composition C2 as a medicament, in particular in the prevention and / or treatment of a multifactorial pathology, in particular a chronic multifactorial pathology, in particular a pathology chosen from inflammatory diseases, neurodegenerative diseases, bacterial diseases, viral diseases, autoimmune diseases, food allergies and intolerances, and in particular in the prevention and / or treatment of a pathology chosen from eczema, psoriasis, Crohn's disease, celiac disease, chronic inflammatory bowel diseases, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Charcot's disease.

[0117] This use as a medicinal product of composition C1 and composition C2 is simultaneous or sequenced in time. In particular, it is preferable to administer composition C1 first, then composition C2, the two administrations being preferably separated by at least 1 hour, even more preferably by at least 4 hours, ideally one in the morning and one in the evening.

[0118] Compositions C1 and C2 can also be used to treat or prevent diseases associated with pathological dysbiosis of the intestinal microbiota. In particular, compositions C1 and C2 can be used to prevent or treat: a neurodegenerative disease associated with pathological dysbiosis of the intestinal microbiota, for example a disease selected from amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, and Alzheimer's disease an intestinal disease associated with pathological dysbiosis of the intestinal microbiota, for example a disease selected from Crohn's disease, chronic inflammatory bowel diseases, ulcerative colitis, irritable bowel syndrome, ulcerative colitis, rheumatoid arthritis and food intolerance, in particular gluten intolerance.

[0119] The invention is now described by examples and test results. Examples Example 1: composition C1

[0120] An example of a C1 composition suitable for testing on mice is presented below.

[0121] The following amounts of conjugates were weighed according to Table 1 to prepare a 300 mL stock of 10x composition C1. All conjugates were weighed with an analytical balance in a laminar flow hood. [Table 1] PLL conjugate (PLL = Poly-L-Lysine) Quantity (mg) Oléyl-PLL 201 Palmitic-PLL 78 Lauryl-PLL 189 Lynoleyl-PLL 78 Azelayl-PLL 75 Plamitoleyl-PLL 156 Thioctyl-PLL 225 Myristyl-PLL 78 Orothyl-PLL 72 Acetate-PLL 174 Butyrate-PLL 285 Lactate-PLL 177 Propionate-PLL 177

[0122] Each conjugate was weighed into a sterile 50 mL centrifuge tube and dissolved in 30 mL of water using a vortex mixer. Each conjugate was stirred for 10 to 20 minutes depending on the solubility of the conjugate.

[0123] In parallel, cholesterol and farnesylcysteine ​​molecules were dissolved separately at an approximate concentration of 50 mg / ml in absolute ethanol and filtered through a 0.22 µm filter. The solvent was evaporated by a rotary evaporator. The required quantities of dry solids were weighed in a laminar flow hood, as shown in Table 2. [Table 2] Molecule Quantity (mg) Cholesterol 12 Farnesylcysteine 12

[0124] In a sterile 500 ml flask equipped with a magnetic stir bar, 23.8 ml of each previously prepared conjugate solution was added under magnetic stirring (700 rpm).

[0125] The corresponding amounts of salts (previously lyophilized) at 300 of PBS were added to the conjugate solution under magnetic stirring (700 rpm).

[0126] Cholesterol and farnesylcysteine ​​were added and the mixture was allowed to stir for 25 hours (700 rpm). 30 ml of 10x C1 formulation was withdrawn from a laminar flow hood and added to a new sterile 500 ml bottle. 270 ml of sterile PBS was added to obtain 1x C1 formulation. The formulation was stirred for 30 minutes.

[0127] The vials were prepared according to the following plan using a sterile, calibrated 5 ml pipette: C1 1X = 83 vials, 2 ml each. C1 10X = 83 vials, 2 ml each.

[0128] The 166 vials were placed in a steel freeze-drying tank and a one-day freeze-drying cycle was started. Example 2: composition C2

[0129] An example of a C2 composition suitable for mouse testing is presented below.

[0130] The following amounts of conjugates were weighed according to Table 3 to prepare a 300 mL stock of 10x C2 composition. All conjugates were weighed with an analytical balance in a laminar flow hood. [Table 3] PLL conjugate (PLL = Poly-L-Lysine) Quantity (mg) Alpha-tocopherol-PLL 210 Ascorbyl-PLL 189 Biotinyl-PLL 204 GABA-PLL 378 Glutathione-PLL 222 Pantothenic Acid-PLL 204 Retynoil-PLL 606 Spermine-PLL 198 Taurine-PLL 330 PLL / Cysteine ​​(copolymer) 180 PLL / Methionine (copolymer) 303

[0131] Each conjugate was weighed into a sterile 50 mL centrifuge tube and stored in a freezer at -20°C until use.

[0132] In parallel, coenzymeQ10 was dissolved at an approximate concentration of 50 mg / ml in absolute ethanol and filtered through a 0.22 µm filter. The solvent was evaporated by a rotary evaporator. The required quantities of dry solids were weighed in a laminar flow hood, as shown in Table 4. [Table 4] Molecule Quantity (mg) Coenzyme Q10 156

[0133] The solid was stored in a sterile centrifuge tube at -20°C until use.

[0134] The previously prepared 50 ml centrifuge tubes with the conjugates and copolymers were taken and allowed to warm to room temperature. After this time, 30 ml of water was added to each centrifuge tube.

[0135] The conjugates and copolymers were dissolved using a vortex mixer, stirring for between 10 and 20 minutes, depending on the solubility of the conjugates and copolymers.

[0136] The centrifuge tube containing coenzyme Q10 was also removed from the freezer and allowed to warm to room temperature.

[0137] A sterile 500 ml flask was taken and fitted with a magnetic stir bar. 27.3 ml of each previously prepared conjugate solution was added to the flask under magnetic stirring (700 rpm).

[0138] The corresponding amounts of salts (previously lyophilized) at 300 of PBS were added to the conjugate solution under magnetic stirring (700 rpm).

[0139] CoenzymeQ10 was added and the mixture was left to stir for 2 hours (700 rpm).

[0140] After 2 hours, 30 ml of 10x C2 formulation was withdrawn in a laminar flow hood and added to a new sterile 500 ml bottle.

[0141] 270 ml of sterile PBS was added to obtain formulation C2 x1. The formulation was stirred for 30 minutes.

[0142] The vials were prepared according to the following plan using a sterile, calibrated 5 ml pipette: DP2 1X = 83 vials, 2 ml each. DP2 10X = 83 vials, 2 ml each.

[0143] The 166 vials were placed in the steel freeze-drying tank and a one-day freeze-drying cycle was started. Example 3: composition C1

[0144] An example of a C1 composition suitable for testing on rats is presented below.

[0145] The manufacturing process of the composition is similar to that presented in Example 1. The concentrations of each of the constituents are different and presented in Table 5. [Table 5] Compounds C1 Concentration 10X (mg / mL) C1 1X Concentration (mg / mL) Acetate PLL 1,16 0,12 Azelayl-PLL 0,50 0,05 Butyrate-PLL 1,88 0,19 Lauryl-PLL 1,26 0,13 Lactate-PLL 1,18 0,12 Linoleyl-PLL 0,52 0,05 Myristyl-PLL 0,51 0,05 Oléyl-PLL 1,33 0,13 Orotyl-PLL 0,48 0,05 Palmitic-PLL 0,52 0,05 Palmitoleyl-PLL 1,04 0,10 Propionate-PLL 1,17 0,12 Thioctyl-PLL 1,49 0,15 Cholesterol 0,09 0,01 FarCys 0,07 0,01

[0146] The sum of the concentrations of PLL + cholesterol + Farnesylcysteine ​​conjugates for C1 10X is 13.2 mg / mL.

[0147] The sum of the concentrations of PLL + cholesterol + Farnesylcysteine ​​conjugates for C1 1X is 1.32 mg / ml. Example 4: composition C1

[0148] An example of a C1 composition suitable for humans is presented below.

[0149] The manufacturing process of the composition is similar to that presented in Example 1. The concentrations of each of the constituents are different and presented in Table 6. [Table 6] Compounds C1 Concentration 10X (mg / mL) C1 1X Concentration (mg / mL) Acetate PLL 7,192 0,744 Azelayl-PLL 3,1 0,31 Butyrate-PLL 11,656 1,178 Lauryl-PLL 7,812 0,806 Lactate-PLL 7,316 0,744 Linoleyl-PLL 3,224 0,31 Myristyl-PLL 3,162 0,31 Oléyl-PLL 8,246 0,806 Orotyl-PLL 2,976 0,31 Palmitic-PLL 3,224 0,31 Palmitoleyl-PLL 6,448 0,62 Propionate-PLL 7,254 0,744 Thioctyl-PLL 9,238 0,93 Cholesterol 0,558 0,062 FarCys 7,192 0,744 Example 5: composition C2

[0150] An example of a C2 composition suitable for humans is presented below.

[0151] The manufacturing process of the composition is similar to that presented in Example 2. The concentrations of each of the constituents are different and presented in Table 7. [Table 7] Compounds C1 Concentration 10X (mg / mL) C1 1X Concentration (mg / mL) Alpha-tocopherol-PLL 8,61 0,861 Ascorbyl-PLL 7,749 0,7749 Biotinyl-PLL 8,364 0,8364 GABA-PLL 15,498 1,5498 Glutathione-PLL 9,102 0,9102 Pantothenic Acid-PLL 8,364 0,8364 Retynoil-PLL 24,846 2,4846 Spermine-PLL 8,118 0,8118 Taurine-PLL 13,53 1,353 PLL / Cysteine ​​(copolymer) 7,38 0,738 PLL / Methionine (copolymer) 12,423 1,2423 Coenzyme Q10 6,396 0,6396 Test results Study of the effect of composition C1 according to the invention to restore intestinal permeability

[0152] The aim of this study is to evaluate the efficacy of an injectable formulation according to the invention on the restoration of intestinal permeability following the induction of intestinal inflammation induced by dextran sodium sulfate (DSS) in rats.

[0153] The intestinal epithelium is a dynamic barrier that limits the access of bacteria, parasites, viruses and also certain chemical molecules to the host tissues. It is composed of different types of cells, each with a well-defined role, such as enterocytes responsible for the function of nutrient absorption, goblet cells secreting mucus whose thickness keeps bacteria and other microorganisms away from enterocytes, Paneth cells, located at the bottom of the crypts of the small intestine, participating in the maintenance of homeostasis and the role of defense of the intestinal mucosa via the secretion of defensin-type antimicrobial agents and microfold cells called M cells, specialized in the endocytosis of antigens, molecules or even microorganisms present in the intestinal lumen.They act as antigen-presenting cells, presenting endocytosed antigens to the underlying immune system. The epithelial physical barrier provided by enterocytes is reinforced by a thick glycocalyx and mucus layer (thinner layer at the level of M cells for easier access to the intestinal lumen microbiome) and IgA secretion by enterocyte transcytosis produced by the underlying plasma cells. Epithelial cells form a monolayer of polarized cells resting on the lamina propria via their basolateral pole and tightly bound together by tight junctions. In this lamina propria, Peyer's patches are located, consisting of lymphocyte-rich follicles, the initiating site of pro- or anti-inflammatory responses.Many pathologies are associated with altered intestinal permeability, such as inflammatory bowel disease, obesity, multiple sclerosis, Lou Gehrig's disease, and Alzheimer's disease. This alteration leads to increased intestinal permeability, most often resulting in inflammation of the intestinal wall.

[0154] The composition tested in this study is composition C1 of Example 3.

[0155] The intestinal epithelium is a dynamic barrier that limits the access of bacteria, parasites, viruses, and certain chemical molecules to host tissues. It is composed of different cell types, each with a well-defined role, such as enterocytes responsible for the absorptive function of the intestine, goblet cells secreting mucus, M cells responsible for the passage of macromolecules and antigens from the intestinal lumen (Lamina Propria) to the underlying immune cells, and neuroendocrine cells. The physical epithelial barrier provided by the enterocytes is reinforced by a thick glycocalyx and mucus layer. The epithelial cells form a monolayer of polarized cells resting on the lamina propria via their basolateral pole and tightly bound together by tight junctions.Many pathologies are associated with altered intestinal permeability, such as inflammatory bowel disease, obesity, multiple sclerosis, Lou Gehrig's disease, and Alzheimer's disease. This alteration leads to increased intestinal permeability, most often resulting in inflammation of the intestinal wall.

[0156] The main objective of the study is to demonstrate the effectiveness of the invention in remediating intestinal permeability following the induction of chronic intestinal inflammation.

[0157] To test these hypotheses, rats received different doses of composition C1 of Example 3, administered by subcutaneous injection after induction of intestinal inflammation.

[0158] The operating protocol is described below. 1- Implementation of the chronic inflammation model

[0159] Upon arrival, the animals (Wistar rats, 280-300g) were kept in 900cm2 cages (3 animals per cage). During this acclimatization period, the animals had free access to water and food. After this period, the animals (n = 9) received ad libitum food and a 2% (w / v) solution of Dextran Sodium Sulfate (DSS) in drinking water. The induction of intestinal inflammation was monitored 3, 5, and 7 days after the start of DSS addition. A control group (n = 3) received food and water ad libitum. If signs of pain were observed, the affected animal was isolated and paracetamol (200 mg / kg) was administered by gavage twice daily. These animals were removed from the experimental design. If significant weight loss was observed, the animals were sacrificed when they had reached 80% of their initial weight. Finally, the animals were sacrificed if they displayed an unusual attitude indicating discomfort.

[0160] The accommodation conditions were as follows: temperature 20-24°C, humidity 60 plus or minus 10%, 12h / 12h cycle.

[0161] Animals were observed daily for signs of stress or pain. Daily weighing of animals, food, and water were performed. The onset of clinical signs of inflammation was based on body weight loss, stool consistency (normal-0, loose-2, diarrhea-4), and rectal bleeding (0-4). The results allowed for the determination of an inflammation severity index that represents the average of the scores obtained for stool consistency and rectal bleeding. Weight loss represents the percentage difference in weight of rats from D1 (DSS addition) to D7. 2- Determination of the “effective” dose

[0162] Upon arrival, the animals (Wistar rats, 280-300g) were raised in 900cm 2 cages (5 animals per cage). During this acclimatization period, the animals had free access to water and food. After this period, the animals (n = 30) were divided into 4 groups (Control - n = 5, DSS + composition C1 10X - n = 10). The "Control" group received food and water ad libitum throughout the experiment. The rats received food and a 2% DSS solution in drinking water ad libitum for 3, 5 or 7 days (duration determined during the first experiment). After induction of intestinal inflammation, each rat received an intraperitoneal injection of 1mL of composition C1. Administrations of composition C1 were carried out once a day. If signs of pain were observed, the affected animal was isolated and paracetamol (200 mg / kg) was administered by gavage twice daily.These animals were removed from the experimental design. If significant weight loss was observed, the animals were sacrificed when they reached 80% of their initial weight. Finally, the animals were sacrificed if they displayed an unusual attitude indicating discomfort.

[0163] The accommodation conditions were as follows: temperature 20-24°C, humidity 60 plus or minus 10%, 12h / 12h cycle.

[0164] The animals were observed daily for signs of stress or pain. Daily weighings of the animals, food, and water were performed.

[0165] The appearance of clinical signs of inflammation is based on body weight loss, stool consistency (normal-0, loose-2, diarrhea-4) and rectal bleeding (0 - 4).

[0166] The results allow us to determine an index of inflammation severity which represents the average of the scores obtained for stool consistency and rectal bleeding. Weight loss represents the percentage difference in the weight of the rats ((DX x D0) / D0)*100). At the end of the experiment, the animals were anesthetized (see organ sampling) and various samples were taken: blood, small intestine, cecum, colon, liver, spleen and several parameters evaluated: size of the cecum, weight of the liver, spleen, and cecum. Permeability study

[0167] A permeability study was performed on an isolated organ (jejunum or colon) to determine the effectiveness of the dose of composition C1 on the restoration of intestinal permeability. Collection of digestive fragments

[0168] This ex vivo approach uses a fragment of jejunum or colon from the rat digestive tract. The rats were anesthetized by inhalation of isoflurane (1000 mg / g) with an induction at 3% and a maintenance during the operation of 1.5%. After laparotomy and ligation of the celiac artery adjacent to the esophagus, a 10 cm fragment of jejunum or colon was removed. The sample was weighed. The rats were then sacrificed by injection of sodium pentobarbital (12 mg / 100 g). Preparation of the everted organ and removal

[0169] The serous and mucous compartments of the organ were quickly rinsed with physiological saline (37°C) and the organ was everted. A ligation was performed at one end. The second end allows the introduction of 1 mL of Krebs-Henseleit medium. This end was then ligated, the organ was weighed and then placed in a tank containing 10 mL of Krebs-Henseleit survival medium supplemented with 250 mg of FITC-dextran of 4 Kda (mucous compartment). Throughout the experiment, the everted organs were maintained at 37°C and oxygenated with an O2 / CO2 mixture (95% / 5%). Every 30 minutes, the medium of the mucous compartment was agitated by performing 3 suction-discharge cycles using a 1 mL micropipette. After 2 hours of incubation, a 1 mL sample was taken from the mucosal side.

[0170] The organs were removed from the glass tank and one end was cut. The serous medium was collected in a previously weighed tube. After collection, the tube was reweighed to assess the transfer of survival medium during the experiment. After weighing, FITC-Dextran was measured by fluorometry in duplicate (excitation, 490 nm; emission, 530 nm).

[0171] After weighing the organ, a longitudinal section was made and it was quickly frozen in liquid nitrogen. The frozen organ was then placed in a zip-lock bag and frozen in liquid nitrogen and stored at -80°C. Study of inflammation parameters

[0172] The study was conducted based on the behavior of treated animals. In fact, inflammation causes increased agitation in the animals. Results

[0173] The results of the observation of inflammation symptoms are presented in Tables 8 to 10 (before injection of composition C1) and in Tables 11 to 15 (after injection of composition C1). In these tables: The percentage change in weight is calculated as follows: ((JX x J0) / J0)*100) Stool consistency is assessed as follows: 0 = normal; 2 = loose; 3 = diarrhea Rectal bleeding is assessed as follows: 0 = nothing; 1 = light; 2 = moderate; 3= heavy; 4 = heavy. [Table 8] Days J3 J4 Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Witness Rat 1 10,7 0 0 12,14 0 0 Rat 2 11,1 0 0 13,02 0 0 Rat 3 9,5 0 0 16,07 0 0 Average 10,4 0 0 13,7 0 0 DSS 2% Rat 4 5,2 0 0 8,52 0 0 Rat 5 8,7 0 0 11,00 0 0 Rat 6 4,8 0 0 9,52 0 0 Average 6,2 0 0 9,7 0 0 DSS4% Rat 7 7,1 2 0 7,12 2 0 Rat 8 3,6 2 0 5,57 2 0 Rat 9 6,2 2 0 6,49 2 0 Average 5,6 2 0 6,4 2 0 [Table 9] Days J5 J6 Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Witness Rat 1 11,07 0 0 14,04 0 0 Rat 2 13,65 0 0 16,51 0 0 Rat 3 14,75 0 0 17,74 0 0 Average 13,2 0 0 16,1 0 0 DSS 2% Rat 4 8,52 0 0 10,03 0 0 Rat 5 10,33 0 0 15,07 0 0 Rat 6 7,94 0 0 10,54 0 0 Average 8,9 0 0 11,9 0 0 DSS4% Rat 7 6,44 2 0 8,14 2 0 Rat 8 8,20 2 0 9,64 2 0 Rat 9 5,84 2 0 7,40 2 0 Average 6,8 2 0 8,4 2 0 [Table 10] Days J7 Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Witness Rat 1 7,25 0 0 Rat 2 9,46 0 0 Rat 3 9,31 0 0 Average 8,67 0 0 DSS 2% Rat 4 4,59 2 0 Rat 5 6,50 2 0 Rat 6 4,06 2 0 Average 5,05 2 0 DSS4% Rat 7 5,08 2 0 Rat 8 4,62 2 0 Rat 9 3,90 2 2 Average 4,53 2 1 [Table 11] Days J8 J9 Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Witness Rat 1 Average 9,75 0 0 10,64 0 0 6,53 2 0 3,13 2 0 DSS 2% Rat 5 4,22 2 0 2,48 2 0 Rat 6 5,38 2 0 2,80 2 0 Average 6,92 4 0 3,67 4 0 DSS4% Rat 8 1,36 4 2 -1,10 4 4 Rat 9 4,14 4 1 1,28 4 2 Average 9,75 0 0 10,64 0 0 [Table 12] Days J10 J11 Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Witness Rat 1 Average 18,64 0 0 20,11 0 0 3,17 2 0 5,87 2 0 DSS 2% Rat 5 5,21 2 0 3,27 2 0 Rat 6 4,19 2 0 4,57 2 0 Average 4,00 4 0 4,43 4 0 DSS4% Rat 8 -0,88 4 4 -0,36 4 0 Rat 9 1,56 4 2 2,03 4 0 Average 18,64 0 0 20,11 0 0 [Table 13] Days J12 J13 Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Witness Rat 1 Average 17,50 0 0 20,18 0 0 3,73 2 0 3,63 2 0 DSS 2% Rat 5 0,00 2 0 -2,35 2 0 Rat 6 1,87 2 0 0,64 2 0 Average 4,26 4 0 3,44 4 0 DSS4% Rat 8 -1,30 4 0 -2,18 4 0 Rat 9 1,48 4 0 0,63 4 0 Average 17,50 0 0 20,18 0 0 [Table 14] Days J14 J15 Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Witness Rat 1 Average 23,79 0 0 22,89 0 0 6,53 0 0 2,87 0 0 DSS 2% Rat 5 4,22 0 0 -2,41 0 0 Rat 6 5,38 0 0 0,23 0 0 Average 6,92 4 0 2,66 4 2 DSS4% Rat 8 1,36 4 2 -2,31 4 2 Rat 9 4,14 4 1 0,18 4 2 Average 23,79 0 0 22,89 0 0 [Table 15] Days J16 J17 Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Weight variation (%) Stool consistency (0 / 2 / 4) Bleeding (0 - 4) Witness Rat 1 Average 24,57 0 0 19,64 0 0 3,20 0 0 0,93 0 0 DSS 2% Rat 5 -3,02 0 0 -5,02 0 0 Rat 6 0,09 0 0 -2,04 0 0 Average 2,46 2 2 -0,75 2 2 DSS4% Rat 8 -3,31 2 2 -2,95 2 2 Rat 9 -0,43 2 2 -1,85 2 2 Average 24,57 0 0 19,64 0 0

[0174] It was observed that the weight variation was stabilized at D12 for rats with DSS 2% and DSS4%.

[0175] Concerning the consistency of the stools, it becomes normal after 6 days of treatment with the composition according to the invention for the 2% DSS rats and after 8 days of treatment with the composition according to the invention for the 4% DSS rats.

[0176] Regarding rectal bleeding, it is absent in DSS 2% rats, which means that there has been a recovery of intestinal function.

[0177] Regarding the analysis of intestinal membrane permeability, the results of in vivo tests 10 days after injection of composition C1 into DSS 2% rats, by measurement of FITC-dextran, are presented in Table 16. [Table 16] Control (FITC) - Rat 1 DSS 2% FITC (Rat 5) DSS 2% FITC (Rat 6) Plasma 0,6519 0,6024 0,7431

[0178] It is noted that the dosage of FITC Dextran 4KDa in the plasma makes it possible to clearly discriminate the impact of composition C1 according to the invention on rats having received 2% of DSS, the fluorescence found in the blood is identical to that of the control.

[0179] Regarding the analysis of the permeability of the intestinal membrane, the results of the ex vivo tests 10 days after the injection of composition C1 into DSS 2% rats, on isolated organs (the permeability on isolated organs was carried out on a fragment of distal jejunum (5 cm) and on the ileum (5 cm) by measurement of FITC-dextran, are presented in Table 17. [Table 17] Isolated organs 30 min 60 min 90 min 120 min 150 min Control (FITC) - Rat 1 34,4 103,6 254,6 526,9 861,5 DSS 2% FITC (Rat 5) 30,7 137,9 457,2 841,4 1101,0 DSS 2% FITC (Rat 6) 27,4 85,3 293,0 659,6 1030,0

[0180] It is noted that for rats treated with composition C1 according to the invention having received 2% DSS, the values ​​are close to those observed in the control. Molecular Analysis of the Fecal Microbiota of Mice (Wild - WT, SOD1, SOD1 + composition according to the invention)

[0181] The SOD1 mouse is an animal model of axonal degeneration consisting of transgenic mice expressing the mutated form of the human superoxide dismutase gene. It is the reference in vivo model for studying amyotrophic lateral sclerosis. Preparation of the compositions:

[0182] The following amounts of conjugates were weighed to prepare a 300 mL stock of composition C1. All conjugates were weighed with an analytical balance in a laminar flow hood. [Table 18] PLL conjugate (PLL = Poly-L-Lysine) Concentration 1X (mg / mL) Concentration 10X (mg / mL) Oléyl-PLL 0,063 0,63 Palmitic-PLL 0,024 0,24 Lauryl-PLL 0,059 0,59 Linoleyl-PLL 0,025 0,25 Azelayl-PLL 0,024 0,24 Palmitoleyl-PLL 0,049 0,49 Thioctyl-PLL 0,07 0,7 Myristyl-PLL 0,024 0,24 Orothyl-PLL 0,023 0,23 Acetate-PLL 0,055 0,55 Butyrate-PLL 0,089 0,89 Lactate-PLL 0,056 0,56 Propionate-PLL 0,055 0,55

[0183] Each conjugate was weighed into a sterile 50 mL centrifuge tube and dissolved in 30 mL of water using a vortex mixer. Each conjugate was stirred for 10 to 20 minutes depending on the solubility of the conjugate.

[0184] In parallel, cholesterol and farnesylcysteine ​​molecules were dissolved separately at a concentration of approximately 50 mg / ml in absolute ethanol and filtered through a 0.22 µm filter. The solvent was evaporated by a rotary evaporator. The required quantities of dry solids were weighed in a laminar flow hood. [Table 19] Molecule Concentration 1X (mg / mL) Concentration 10X (mg / mL) Cholesterol-PLL 0,004 0,04 Farnesylcysteine-PLL 0,004 0,04

[0185] In a sterile 500 ml flask equipped with a magnetic stir bar, 23.8 ml of each previously prepared conjugate solution was added under magnetic stirring (700 rpm). The corresponding amounts of salts (previously lyophilized) at 300 of PBS were added to the conjugate solution under magnetic stirring (700 rpm).

[0186] Cholesterol and farnesylcysteine ​​were added and the mixture was allowed to stir for 25 hours (700 rpm). 30 ml of 10x C1 formulation was withdrawn from a laminar flow hood and added to a new sterile 500 ml bottle. 270 ml of sterile PBS was added to obtain 1x C1 formulation. The formulation was stirred for 30 minutes.

[0187] The vials were prepared according to the following plan using a sterile, calibrated 5 ml pipette: C1 1X = 83 vials, 2 ml each. C1 10X = 83 vials, 2 ml each.

[0188] The 166 vials were placed in a steel freeze-drying tank and a one-day freeze-drying cycle was started.

[0189] Table 20 describes the composition of composition C2 used in this test. [Table 20] PLL conjugate (PLL = Poly-L-Lysine) Concentration 1X (mg / mL) Concentration 10X (mg / mL) Alpha-tocopherol-PLL 0,070 0,70 Ascorbyl-PLL 0,063 0,63 Biotinyl-PLL 0,068 0,68 GABA-PLL 0,126 1,26 Glutathione-PLL 0,074 0,74 Pantothenic Acid-PLL 0,068 0,68 Retynoil-PLL 0,202 2,02 Spermine-PLL 0,066 0,66 Taurine-PLL 0,110 1,10 PLL / Cysteine ​​(copolymer) 0,06 0,6 PLL / Methionine (copolymer) 0,101 1,01

[0190] In this test, compositions C1 (described in Table 18 and 19) and C2 (described in Table 20) were injected one after the other with a 1-hour interval starting with composition C1.

[0191] Feces from five female mice from each group (WT, SOD1, SOD1-PL-Low dose and SOD1-PL high dose), collected from the cages, were analyzed at T0 (=T6), T3 weeks (T9) and 10 weeks (T16) for a total of 55 samples.

[0192] C1 and C2 together are designated “PL” for the remainder of this test and results.

[0193] In a first step, total genomic DNA was extracted from each fecal sample using the Godon method (Godon et al., Appl Envion. Microbiol, 1997). The quality of the extracted DNA was assessed after migration of the samples on agarose gel. The DNA concentration was then determined using Nanodrop technology.

[0194] The obtained gDNA samples were then sequenced. The approach used is high-throughput sequencing of the gene coding for 16S ribosomal RNA (V3-V4 region) using Illumina technology (read length: 150 bases, read depth: 1 million reads).

[0195] The taxonomic affiliation of each obtained bacterial sequence (or OTU) was performed using the silva_nr99_v138.1 (https: / / www.arb-silva.de / ) and GTDB v202 (https: / / gtdb.ecogenomic.org / ) databases.

[0196] The ranomaly pipeline (Theil S and Rifa E. rANOMALY: AmplicoN wOrkflow for Microbial community AnaLYsis [version 1; peer review: 2 approved]. F1000Research 2021, 10:7 https: / / doi.org / 10.12688 / f1000research.27268.1 ), based on the dada2 program, was used to process all the sequences, whether for estimating the abundance of bacterial phyla / families or for performing statistical analyses (composition, diversity analyses, differential abundance analyses).

[0197] The comparison of bacterial diversity and richness between microbiota from treated and untreated mice was performed by calculating the Shanon index. Differences in population structure (β diversity) were highlighted by two multivariate analysis methods (Anova and Nonmetric Multidimensional Scaling =Method MDS) and a PLS-DA (partial least squares discriminant regression analysis) from the mixOmics packages.

[0198] Differential OTU abundance analyses were performed using the normalization and estimation tools of the DESeq2 package [Love, MI, Huber, W., Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Genome Biology 15(12):550; 2014]. Correlation Microbiota - Clinical Parameters

[0199] The potential correlation between each of the clinical and physiological parameters measured and the molecular composition of the microbiota was studied for each of the female mice whose microbiota was analyzed (5 mice per group).

[0200] Several analysis methods were tested, based on rank correlation (spearman).

[0201] The method used is the canonical correlation analysis (rcca) proposed by the mixOmics package (Rohart F, Gautier B, Singh A, and Le Cao KA,2017: mixOmics: An R package for 'omics feature selection and multiple data integration. PLoS computational biology 13(11):e1005752). This method allows cross-validation of the results of analyses of correlations between the abundance of OTUs and the different clinical parameters measured. The results obtained are presented in the form of a heat map showing the significant correlations, positive or negative, between the abundance of OTUs and the clinical parameters.

[0202] Results : Comparison of the composition of the fecal microbiota of “SOD1” KO mice with that of “Wild Type” (WT) mice

[0203] Analysis of the relative abundance of bacterial phyla, orders, families and even genera present in SDO1 mice compared to WT mice shows that the composition of the SOD1 microbiota differs from that of WT: SOD1 mice have fewer Firmicutes and more Bacteroidetes than WT Among Bacteroidetes, SOD1 mice harbor more Bacteroidales, especially Muribaculaceae, and more Porphyromonaceae, including Muribaculum, than WT Among Firmicutes, the relative abundance of Lachnospiraceae (Shaedlerella and Acetifractor) and Erysipelotrichales is decreased, while that of Lactobacillales such as Limolactobacillus and Oscillospiraceae, as well as that of Eggerthelaceae, is increased compared to WT mice.

[0204] Analysis of the diversity (α diversity) and richness (β diversity) of the fecal microbiota of the SOD1 mouse compared to that of the WT mouse made it possible to determine that there is: No significant difference in bacterial diversity (indices: Chao1, Observed, Shannon and Inv Simpson) between SOD1 and WT (α diversity). A significant difference (Jaccard: p= 0.01 and Bray Curtis: p=0.01) in bacterial species richness (β diversity). No effect of time (T0, T6 and T16) was found.

[0205] The Jaccard and Bray Curtis index or distance are two well-known metrics for measuring similarity, dissimilarity, and diversity between multiple samples.

[0206] Finally, PLS-DA analysis shows that the fecal microbiotas of WT and SOD1 mice differentiate ( Figure 4 ).

[0207] In conclusion, SOD1 mice have a disturbed intestinal microbiota compared to WT wild-type mice, characterized by lower bacterial richness, a higher proportion of Gram- bacteria (Muribaculaceae and Muribaculum) as well as Gram- bacteria Lactobacillales and Eggerthelaceae but a lower proportion of Lachnospiraceae and Erysipelotrichales. - Effect of high-dose or low-dose PL administration in SOD1 mice A - Administration of a high dose 10X

[0208] Administration of the composition at a high dose for 3 weeks results in: An increase in the relative abundance of: Bacteroidales in particular Muribaculaceae Oscillospiraceae COE1 A decrease in the abundance of: Lactobacillaceae (Lactobacillus genus) Lachnospiraceae Administration of a high dose of the composition for 3 weeks appears to accentuate the disturbances observed in SOD1 mice. B - Administration of a low dose 1X

[0209] Administration of a low dose of PL to SOD1 mice also causes changes in the relative abundance of certain microbial groups. Thus, we observe: An increase in the relative abundance of: Lachnospiraceae Oscillospirales GAC-485 A decrease in the relative abundance of: Lactobacillale

[0210] The low dose appears to partially rebalance the intestinal microbiota of SOD1 mice to bring it closer to the composition of the microbiota of wild-type (WT) mice, notably by causing an increase in Lachnospiraceae and a decrease in Lactobacillales. This effect appears to be progressive as it is more marked after 10 weeks of treatment than after 3 weeks. Comparison of bacterial diversity and richness in treated and untreated SOD1 mice.

[0211] Analysis of the α diversity of fecal microbiota did not reveal any significant effect of PL administration, at low and high doses, on bacterial diversity in SOD1 mice. Only a significant difference (Shannon index, p=0.02) was observed between the fecal microbiota of mice treated with the low dose and those treated with the high dose of PL. ( Figure 5 And 6 ).

[0212] Analysis of β diversity showed significant differences between untreated SOD1 mice and treated SOD1 mice. Controls versus high dose PL (p=0.02 Jaccard and p= 0.01 Bray Curtis) Low dose PL versus high dose PL (p = 0.02 Jaccard and p = 0.01 Bray Curtis) No significant difference between untreated control and low dose PL. Comparison between wild-type mice and SOD1 mice treated or not with different doses of PL

[0213] PLS-DA analysis of the composition of all the microbiotas studied showed that these microbiotas could be separated into 4 distinct groups, the microbiota of mice treated with the low dose of PL approaching, in the same cluster, the microbiota of wild mice ( Figure 7 ). - Conclusion

[0214] The study showed that the microbiota composition of SOD1 mice differs from that of wild-type mice, with these alterations affecting the richness of the microbiota more than its diversity. The microbiota of SOD1 mice appears disrupted since the relative abundance of certain major bacterial groups is altered. It is characterized, in particular, by a higher abundance of Muribaculaceae and Lactobacillales (Oscillospiraceae) to the detriment of Lachnospiraceae and Erysipelotrichales. PLS-DA analysis further demonstrated that the microbiotas of wild-type and SOD1 mice were separate.

[0215] Administration of a high dose of PL to SOD1 mice for 3 weeks appears to amplify the differences observed between wild-type mice and SOD1 mice (increase in Muribaculaceae and Oscillospiraceae and decrease in Lachnospiraceae). The composition of the microbiota of SOD1 mice treated with the high dose of PL is very clearly different from that of SOD1 controls but also from wild-type mice, this group of mice treated with the high dose being largely different from the others (most disturbed microbiota).

[0216] Administration of a low dose of PL to SOD1 mice for 3 to 10 weeks appears to partially rebalance the microbiota disturbances observed in control SOD1 mice. Indeed, an increase in Lachnopiraceae and a decrease in Lactobacillales, microbial groups affected in SOD1 mice compared to wild-type mice, was observed. The microbiota composition of SOD1 mice treated with a low dose of PL thus approaches that of wild-type mice, with the microbiotas of these two groups being grouped in the same cluster.

[0217] These preliminary results are of interest with regard to the administration of the lowest dose in the SOD1 mouse model. At this dose, PLs indeed appear to have a beneficial effect on the disturbed microbiota of SOD1 mice. Correlation study between symptoms and molecular composition of the microbiota in SOD1 mice after administration of a low dose of Poly-Lysine (PL).

[0218] The objective of the study was to analyze potential correlations between the molecular composition of the intestinal microbiota and clinical parameters measured in SOD1 mice versus wild-type mice and in SOD1 mice treated or not with a low dose of PL for 10 weeks.

[0219] This study was carried out in female mice whose molecular analysis of the fecal microbiota had been previously carried out (five females from each group [WT, SOD1, SOD1-Low dose), collected in the cages, at T0 (=T6), T3 weeks (T9) and 10 weeks (T16)].

[0220] Tests were performed at least 1 h after the first daily dose of test compound or vehicle.

[0221] A one-day session included a 5-min training trial at 4 RPM on the rotating apparatus (AccuScan Instruments, Columbus, USA). One hour later, animals were tested for 3 consecutive 6-min acceleration trials with the speed increasing from 0 to 40 RPM over 360 s and an inter-trial interval of at least 30 min. The latency to fall off the rod was recorded

[0222] Correlation study in SOD1 and Wild Type mice.

[0223] RCCA (Regularized Canonical Correlation Analysis) correlation analysis mainly showed in WT and SOD1 mice the existence of a significant negative correlation between the clinical score, the distance traveled and the recovery time after Rotarod and the Desulfovibrionaceae family as well as a weaker negative correlation with the Prevotellaceae family. Thus, the higher the abundance of these bacterial families, the lower the clinical score, as well as the distance traveled and the recovery time ( Figure 8 ).

[0224] Although the relative abundance of the Desulfovibrionaceae family (like that of Prevotellaceae) is low compared to other bacterial families, we were nevertheless able to find the presence of OTUs of this family in SOD1 mice only (no detection in Wild Type mice). This could suggest a potential role of Desulfovibrionaceae in the pathophysiology in SOD1 mice. Correlation study in SOD1 mice treated or not treated with the low dose of PL for 10 weeks

[0225] RCCA correlation analysis performed in control SOD1 mice and mice treated with the low dose of PL revealed a strong positive correlation between the Lachnospiraceae family and the distance traveled by the mice. The more abundant this family is, the greater the distance traveled by the mice. Knowing that one of the main effects of PL on the intestinal microbiota of SOD1 mice is to increase the abundance of Lachnospiraceae species, the observed correlation suggests a role for this bacterial family in restoring the ability of SOD1 mice to travel a distance close to that traveled by Wild Type mice ( Figure 6 ).

[0226] The evidence of a negative correlation between Desulfovibrionaceae and the severity of symptoms in SOD1 mice suggests that species of this family, which produce sulfides, may be involved in the pathophysiology, as has been observed in other pathologies such as irritable bowel syndrome or chronic inflammatory bowel diseases (Crohn's, ulcerative colitis, etc.). At the same time, the lower abundance of species of the Lachnospiraceae family in SOD1 mice could result in a decrease in intracolonic butyrate concentration, as a large proportion of Lachnospiraceae species produce this metabolite with proven health effects.

[0227] Administration of low-dose PL for 10 weeks is accompanied by changes in the composition of the microbiota of the SOD1 mouse, resulting, in particular, in an increase in the abundance of Lachnospiraceae and the disappearance of Desulfovibrionaceae. This correlates with the significant improvement in the ability of the mice to travel a distance equivalent to that of Wild Type mice. The administration of PL would therefore make it possible to both partially rebalance the altered microbiota of the SOD1 mouse, generating metabolic modifications and to reduce the severity of certain symptoms in the SOD1 mouse or even to restore a normal phenotype. Comparative study of histopathological analysis of SOD1, SOD1 + low dose 1X, SOD1 + high dose 10X mice.

[0228] SOD1 mice were administered the PL compound described in Example 7 at low (1X) or high (10X) dose or vehicle control (n=14 / dose group) at 7 to 21 weeks of age.

[0229] Additionally, 14 non-carrier wild-type (WT) littermates received a control dose of vehicle.

[0230] Animals were tested for open-field behavior (at 6, 9, 13, and 15 weeks of age), Rotarod performance (at 6, 9, 13, and 15 weeks of age), and grip strength (at 6, 9, 13, 15, and 17 weeks of age). Ophthalmoscopic analysis was performed at 9, 13, and 17 weeks of age. Body weight, clinical symptoms, time to disease onset, and survival were monitored throughout the study. Blood, urine, and fecal samples were collected at 6, 9, and 16 weeks of age. Terminal sample collection was performed when an animal met a humane endpoint criterion or reached 150 days of age.

[0231] Histopathological analysis of 30 different tissue types was performed. 1. Spinal cord SOD1 Mouse Mouse SOD1 + 1X Mouse SOD1 + 10X Minimal to moderate diffuse vacuolization in males, slight to marked in females. Minimal to marked diffuse vacuolation in men, minimal to moderate in women Minimal diffuse degeneration of spinal nerve axons in both sexes 2. Brain SOD1 Mouse Mouse SOD1 + 1X Mouse SOD1 + 10X Multifocal vacuolization: mild or moderate in Mild or moderate vacuolization in both sexes Minimal multifocal vacuolization in females males, moderate in females. 3. Sciatic nerve SOD1 Mouse Mouse SOD1 + 1X Mouse SOD1 + 10X Mild or moderate axon degeneration in both sexes. Mild or moderate axon degeneration in both sexes. Minimal focal axon degeneration 4. Gastrocnemius Muscle SOD1 Mouse Mouse SOD1 + 1X Mouse SOD1 + 10X Minimal to moderate atrophy in both sexes Mild or moderate atrophy in males, minimal to moderate in females Minimal atrophy in males and females 5. Brown adipose tissue, aorta and / or kidneys SOD1 Mouse Mouse SOD1 + 1X Mouse SOD1 + 10X Mild to marked atrophy in both sexes Mild to marked atrophy in both sexes Mild diffuse atrophy in men and women 6. Rate SOD1 Mouse Mouse SOD1 + 1X Mouse SOD1 + 10X Lymphocyte necrosis of the white pulp: minimal in men, moderate in women. Minimal or mild necrosis of lymphocytes in the white pulp in females - 7. Thymus SOD1 Mouse Mouse SOD1 + 1X Mouse SOD1 + 10X Moderate decrease in lymphocyte cellularity in males. Mild or moderate lymphocytic necrosis in males, moderate in females. Moderate decrease in lymphocyte cellularity in males and females. Minimal to marked lymphocyte necrosis in females. -

[0232] Histopathological analysis shows that administration of the compound according to the invention improves the condition of the tissues. More specifically, the spinal cord, brain and gastrocnemius muscle are the tissues that present a degraded state in SOD1 mice. It is clearly observed that the composition has an effect on these tissues depending on the dose used.

[0233] Thus, the composition shows its dose-dependent effect on SOD1 mice, thus showing its promising therapeutic effect.

Claims

1. A composition comprising at least the following molecules: - oleic acid, - palmitic acid, - lauric acid, - linoleic acid, - azelaic acid, - farnesyl cysteine, - palmitoleic acid, - cholesterol, - thioctic acid, - myristic acid, - orotic acid, - acetic acid, - butyric acid, - lactic acid, - propionic acid, and / or a salt and / or an ester and / or an anhydride of one or more of these molecules.

2. The composition according to the preceding claim, characterized in that at least one of the molecules of the composition selected from among oleic acid, palmitic acid, lauric acid, linoleic acid, azelaic acid, palmitoleic acid, thioctic acid, myristic acid, orotic acid, acetic acid, butyric acid, lactic acid, propionic acid, salts of these acids, esters of these acids and anhydrides of these acids, is covalently conjugated to at least one molecule of a polymer selected from among polylysine, polyethylene glycol, polyornithine, polyarginine and polyhistidine.

3. The composition according to either of the preceding claims, characterized in that it comprises micelles in which are encapsulated at least farnesyl cysteine and / or cholesterol and / or an ester of these molecules, preferentially at least one of the micelles is formed by amphiphilic conjugates each consisting of at least one hydrophobic molecule covalently conjugated to a molecule of a polymer selected from among polylysine, polyethylene glycol, polyornithine, polyarginine and polyhistidine.

4. The composition according to the preceding claim, characterized in that at least one micelle is formed by amphiphilic conjugates each consisting of at least one molecule selected from among oleic acid, palmitic acid, lauric acid, linoleic acid, palmitoleic acid, myristic acid, salts, esters and anhydrides of these fatty acids, covalently conjugated to a molecule of a polymer selected from among polylysine, polyethylene glycol, polyornithine, polyarginine and polyhistidine.

5. The composition according to any of claims 2 to 4, characterized in that the polylysine is a poly-L-lysine with a molecular weight of between 12000 and 20000 Da.

6. The composition according to any of the preceding claims, characterized in that it comprises at least: - A. the following conjugates, each conjugate consisting of a molecule covalently bonded to a polylysine: - one or more oleyl-poly-L-lysine conjugates - one or more palmitic-poly-L-lysine conjugates - one or more lauryl-poly-L-lysine conjugates - one or more azelayl-poly-L-lysine conjugates - one or more palmitoleyl-poly-L-lysine conjugates - one or more thioctyl-poly-L-lysine conjugates - one or more myristyl-poly-L-lysine conjugates - one or more orotyl-poly-L-lysine conjugates - one or more acetate-poly-L-lysine conjugates - one or more buyrate-poly-L-lysine conjugates - one or more lactate-poly-L-lysine conjugates - one or more propionate-poly-L-lysine conjugates, - one or more linoleyl-poly-L-lysine conjugates, and - B. farnesyl cysteine and cholesterol, and / or an ester of these molecules, encapsulated in micelles, preferentially the micelles being formed by one or more of the conjugates in list A.

7. The composition according to claim 6, wherein the poly-L-lysine is replaced with another polylysine or with polyethylene glycol, a poly-L-ornithine, a poly-L-arginine or a poly-L-histidine.

8. A method for producing a composition according to any of claims 1 to 7, characterized in that it comprises the following steps: - a. creating an amphiphilic premix: mixing, in an aqueous solution, one or more conjugates selected from among: - one or more oleyl-poly-L-lysine conjugates - one or more palmitic-poly-L-lysine conjugates - one or more lauryl-poly-L-lysine conjugates - one or more azelayl-poly-L-lysine conjugates - one or more palmitoleyl-poly-L-lysine conjugates - one or more thioctyl-poly-L-lysine conjugates - one or more myristyl-poly-L-lysine conjugates - one or more orotyl-poly-L-lysine conjugates - one or more acetate-poly-L-lysine conjugates - one or more buyrate-poly-L-lysine conjugates - one or more lactate-poly-L-lysine conjugates - one or more propionate-poly-L-lysine conjugates, - one or more linoleyl-poly-L-lysine conjugates, and - b. adding at least farnesyl cysteine and cholesterol to the amphiphilic premix and stirring so as to form micelles formed by one or more of the conjugates of the amphiphilic premix encapsulating the farnesyl cysteine and cholesterol.

9. The composition according to any of claims 1 to 7, for use thereof as a drug.

10. The composition for use thereof according to claim 9, wherein the composition is associated with the use of a second composition comprising the following molecules: - taurine, - cysteine, - methionine - spermine - retinoic acid, - pantothenic acid, - biotin, - coenzyme Q10, - glutathione, - ascorbic acid, - GABA, - alpha-tocopherol and / or a salt and / or an ester and / or an anhydride of one or more of these molecules.

11. The composition for use thereof according to the preceding claim, characterized in that, in the second composition, at least one of the molecules of the composition selected from among taurine, spermine, pantothenic acid, retinoic acid, biotin, glutathione, ascorbic acid, GABA, and alpha-tocopherol, salts of these molecules, esters of these molecules, and anhydrides of these molecules is covalently conjugated to a molecule of a polymer selected from among polylysine, polyethylene glycol, polyornithine, polyarginine and polyhistidine.

12. The composition for use thereof according to any of claims 10 or 11, characterized in that, in the second composition, cysteine and methionine are each conjugated to a molecule of a polymer selected from among polylysine, polyethylene glycol, polyornithine, polyarginine and polyhistidine by polymerization, the molecule and the polymer forming a copolymer.

13. The composition for use thereof according to any of claims 10 to 12, characterized in that, in the second composition, coenzyme Q10 is encapsulated in micelles.

14. The composition for use thereof according to the preceding claim, characterized in that at least one micelle of the second composition is formed by amphiphilic conjugates each consisting of at least one molecule covalently conjugated to a molecule of a polymer selected from among polylysine, polyethylene glycol, polyornithine, polyarginine and polyhistidine, preferentially the amphiphilic conjugates each consisting of at least one molecule selected from among taurine, spermine, pantothenic acid, biotin, glutathione, ascorbic acid, GABA and alpha-tocopherol, the salts of these molecules, the esters of these molecules and the anhydrides of these molecules.

15. The composition for use thereof according to any of claims 10 to 14, in the prevention and / or treatment of pathological dysbiosis of the gut microbiota or an intestinal disease associated with pathological dysbiosis of the gut microbiota, wherein preferentially the dysbiosis is selected from among Crohn's disease, chronic inflammatory bowel diseases, hemorrhagic rectocolitis, irritable bowel syndrome, ulcerative colitis, rheumatoid arthritis and gluten intolerance.