Multi-ion complex for preventing or treating inflammation

Abstract

The invention relates to a synergistic multi-ion complex that provides enhanced prevention and treatment in response to inflammation by modulating the pathway of immunity. The invention relates to a composition comprising mineral salts comprising at least one mineral salt of lithium, at least one mineral salt of magnesium and at least one mineral salt of potassium, characterized by the following molar ratio: lithium 1-magnesium [0.13-0.34]-potassium [1.20-2.40]. The compositions according to the invention may also comprise organic salts, other mineral salts, additives and / or excipients. It can be used as a medicament, medical device for the prevention and / or treatment of inflammation associated with acute or chronic pathologies involving cell degeneration.

Description

Technical Field

[0001] The invention falls within the field of preventing and / or treating inflammation and restoring cellular homeostasis therapeutic responses. Background Art

[0002] When cells are damaged or subjected to stress (psychological, metabolic, chemical, etc.), in the case of damage, the magnesium Mg in the cell 2+ consumption and the concentration will drop by 40% to 60% ( Neuropsychiatric Disease and Treatment 2017:13 275-302 In fact, the intracellular Mg 2+ Small fluctuations in Mg concentration can affect cellular processes. Mg is frequently observed in patients with diabetes, neurodegenerative diseases, and metabolic syndrome… 2+ The imbalance ( Magnesium is a key player in neuronal maturation and neuropathology, Int. J. Mol. Sci. 2019, 20, 3439 ).

[0003] Magnesium acts as a cofactor in more than 300 enzymatic reactions, among which it is essential for the metabolism of adenosine triphosphate (ATP), a source of energy ( Magnesium in Prevention and Therapy, Nutrients 2015, 7 ).

[0004] Magnesium has powerful antioxidant, anti-necrosis and anti-apoptosis effects. 2+ It is largely cytoprotective, cardioprotective, and neuroprotective against a wide range of insults ( Neuropsychiatric Disease and Treatment, 2017:13 275-302 ).

[0005] Magnesium (the fourth most common mineral in the body) also plays a vital role in neurotransmission and neuromuscular conduction. Low magnesium levels are associated with elevated glutamatergic neurotransmission, leading to a favorable environment against excitotoxicity, which can lead to oxidative stress and neuronal cell death. This process has been implicated in several neurological disorders, such as chronic pain ( The Role of Magnesium in Neurological Disorders, Nutrients, 2018, 10,730 ).

[0006] Magnesium deficiency negatively affects the insulin resistance index (HOMA-IR). Magnesium is also necessary for protein synthesis (including DNA and RNA synthesis). Magnesium in Prevention and Therapy, Nutrients, 2015, 7 ). It regulates the flow of potassium and calcium across the membrane.

[0007] Lithium is considered an essential nutrient for the functioning of the human body based on experiments in which animals were deprived of lithium. Unlike other biologically active ions, the concentration of lithium in the tissue fluid of multicellular animals is not strictly regulated. Its concentration can vary widely.

[0008] Although lithium is highly bioactive, it is tolerated at highly variable body fluid concentrations. The lack of lithium bioregulation appears to be due to the lack of specific lithium binding sites and selective screening. Instead, lithium exerts many of its physiological and biochemical effects by competing with other elements for relatively specific macromolecular sites of other cations (most notably sodium and magnesium). Towards a unified understanding of Lithium action, Topical Review, 2017, 586 ).

[0009] Lithium and magnesium have similar ionic radii (0.60 Å and 0.65 Å, respectively) and similar physicochemical properties, allowing them to successfully compete for binding to several magnesium-dependent enzyme sites ( Lithium: the pharmacodynamic actions of the amazing ion Ther. Adv. Psychopharmacol. (2013) 3(3) 163–176 ). These ionic radii allow lithium and magnesium to easily cross cell membranes.

[0010] Li + The ion activates survival and recovery mechanisms such as inositol monophosphatase (IMPase) / inositol polyphosphate 1-phosphatase (IPPase), glycogen synthase kinase 3 (GSK-3) inhibition. As a result, lithium produces significant protective, anti-apoptotic, anti-hypoxic, cellular plasticity and elastic responses ( A fully integrated new paradigm for lithium's mode of action – lithium utilizes latent cellular fail-safe mechanisms - Neuropsychiatric Disease and Treatment 2017:13 275–302 ).

[0011] Lithium Glycogen synthase kinase 3 (GSK-3β) is a serine / threonine kinase that plays a role in regulating cellular metabolism in mammals. It regulates neurogenesis, neuronal polarization, and axonal growth in the developing central nervous system. It is constitutively active in all tissues ( Role of glycogen synthase kinase-3b in ketamine- induced developmental neuroapoptosis in rats, British Journal of Anaesthesia 110 (S1): i3–i9 (2013) ).

[0012] GSK-3β is also an activator of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing-3 (NLRP3) inflammasome (S Arumugam et al Cell Death Differ 2022). NLRP3 is an intracellular sensor for microbial motifs as well as endogenous and environmental danger signals that induce the assembly and activation of the NLRP3 inflammasome. NLRP3 assembly and activation occurs in 2 steps. First, priming signals (pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs)) mediated by Toll-like receptors (TLRs) or tumor necrosis factor receptors (TNFRs) and by activating the transcription factor nuclear factor kappa light-chain-enhancer (NF-κB) involved in innate immunity lead to upregulation of NLRP3 and pro-IL-1β levels. The second step signals (PAMPs, DAMPs, extracellular ATP-P2X7R, ROS, intracellular calcium levels, etc.) lead to NLRP3 activation and assembly. The NLRP3 inflammasome promotes the caspase-1-dependent release of the proinflammatory cytokines IL-1β and IL-18 and pyroptosis-induced pyrocytic D-mediated cell death ( KV Swanson et al Nat Rev Immunol 2019 ). The NLRP3 inflammasome is involved in many acute and chronic inflammatory diseases, such as myocardial infarction, colitis, diabetes, steatohepatitis, Alzheimer's disease, traumatic brain injury, atherosclerosis, stroke, cancer, and many other diseases ( Y Li et al, Signal Transduction and Targeted Therapy 2021 ).

[0013] As shown in vitro and in vivo, lithium inhibits GSK-3, thereby improving the activity of BDNF (brain-derived neurotrophic factor). The role of lithium in increasing BDNF expression coupled with the role of BDNF in neuronal survival has led to the suggestion that lithium plays a role in the treatment of neurodegenerative diseases. The Wnt signaling pathway has been implicated in neurodegenerative diseases and cancer. Lithium inhibition of GSK-3 has been shown to specifically inhibit the Wnt signaling pathway ( Towards a unified understanding of Lithium action, Topical Review, 2017, 586 ).

[0014] As seen in vivo and in vitro, lithium inhibits GSK-3β and, therefore, suppresses the assembly and activation of the NLRP3 inflammasome, cell death, and the release of proinflammatory cytokines IL-1β and IL-18. By inhibiting GSK-3β, lithium treatment prevents reactive oxygen species (ROS) production, thereby preventing NLRP3 inflammasome activation in mouse models of ischemic stroke and spinal cord injury, and exerts anti-inflammatory and neuroprotective effects ( B Chen et al Commun Biol 2022; YJ Zhao et al Neural Regen Res 2022 ).

[0015] NLRP3 is an intracellular sensor. ATP-P2X7 receptor binding activates the potassium channel TWIK2, which induces potassium efflux. A decrease in intracellular potassium levels modifies the NLRP3 structure, leading to its activation. Potassium treatment or potassium efflux inhibition prevents NLRP3 inflammasome assembly and activation in mouse and human cells in vitro (β).

[0016] As described in vitro and in vivo, magnesium is also involved in NLRP3 inflammasome activation. Indeed, magnesium deficiency leads to NLRP3-induced pyroptosis via pyroporin D cleavage. On the other hand, magnesium supplementation inhibits calcium influx-dependent pyroporin D activation, leading to pyroptosis ( D Wang et al Cell Death Differ 2020; C Li et al Acta Biochimica and Biophysica Sinica 2021 ).

[0017] Prophylactic lithium treatment prevented the paclitaxel-induced increase in GSK-3β activity in mice by inhibiting GSK-3β, which simultaneously reduced AKT (protein kinase B) and mTOR (mechanistic target of rapamycin) activities, thereby preventing the development of paclitaxel-induced neuropathic pain ( Inhibition of glycogen synthase kinase 3beta activity with lithium prevents and attenuates paclitaxel-induced neuropathic pain, Neuroscience. 2013 December 19; 254 ).

[0018] Lithium has anti-inflammatory properties inducing a decrease in both pro-inflammatory cytokines and the interleukin TNF-α. On the other hand, lithium modulates the biosynthesis of different neurotransmitters and / or associated receptors, such as serotonin and glutamate. Lithium has an anti-allodynic effect as well as a stimulatory effect on the production of brain beta-endorphin, a MOR agonist with strong analgesic properties ( Lithium reverses mechanical allodynia through a μ opioid-dependent mechanism Molecular Pain 2018 Volume 14: 1–8 ).

[0019] Lithium also contributes to calcium homeostasis and blocks calcium-dependent activation of pro-apoptotic signaling pathways, confirming its cytoprotective effects ( Molecular actions and therapeutic potential of lithium in preclinical and clinical studies of CNS disorders Pharmacol Ther. 2010 November; 128(2): 281–304 ).

[0020] Because Li + Ions inhibit the GSK-3 enzyme, which is an important enzyme at the convergence of several signaling systems, so its impact includes ionotropic glutamate signaling, multiple transcription factors, and multiple downstream targets of the integrated pathway of wingless (Wnt) / β-catenin. Wnt signaling plays a role in structural brain processes such as neuronal development, synapse formation, and neuronal plasticity ( LITHIUM IN THE TREATMENT OF BIPOLAR DISORDER: PHARMACOLOGY AND PHARMACOGENETICS, Mol Psychiatry. 2015 June; 20(6): 661–670 ).

[0021] Normal concentrations of ATP and Mg found in plasma or cytoplasm 2+In addition, high levels of ATP corresponding to biological activity or response to stress in the cytoplasm, organelles (such as mitochondria), and extracellular matrix serve as accumulated lithium ions (Li + The bimetallic phosphate complex ATP-Mg-Li was found to be a potential reservoir for 2+ The biologically active form of lithium co-bound to phosphate with a ligand or receptor cofactor or enzyme action. The ATP-Mg-Li complex allows lithium ions to act by regulating the normal function of ATP as a ligand for cell surface purinergic receptors, of which there are two subtypes: P2X (which mediates the absorption of extracellular calcium ions (Ca 2+ ) ion channels that flow into the cytoplasm) and P2Y (which binds to G receptor proteins (GPCRs) that activate inositol triphosphate second messenger channels to release calcium ions from intracellular stores) to regulate signaling in the central nervous system and periphery ( A Molecular Model for Lithium's Bioactive Form, Biophysical Journal 111, 294–300, July 26, 2016 ).

[0022] One of the major limitations of lithium therapy is its toxicity. Indeed, research on bipolar therapy has focused intensely on identifying alternatives to lithium carbonate salts that reduce toxicity and improve the therapeutic window. Therefore, one of the challenges of using lithium in the treatment of diseases with inflammatory characteristics is to broaden the currently available therapeutic window.

[0023] Potassium is also an essential element. It is the most abundant cation in intracellular fluids and plays a key role in maintaining cell function, especially in excitable cells such as muscles and nerves. Potassium deficiency is associated with glucose intolerance and diabetes. + With extracellular Na + The interaction increases the activation energy for water movement that promotes ion exchange and equilibration, especially that of lithium. Potassium Intake, Bioavailability, Hypertension, and Glucose Control, Nutrients 2016, 8, 444 ).

[0024] Dysregulation of the activity of these three ions is involved in neurogenic inflammation, also known as neuroinflammation, "low-noise inflammation" or "inflammaging" (inflammation associated with aging), including chronic inflammation, such as is involved in fibromyalgia, neuropathy, osteoarthritis, polyarthritis, metabolic syndrome, diabetes, obesity, atherosclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease (inflammatory bowel disease), multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Charcot-Marie-Tooth lateral sclerosis, migraine, asthma, pain, or inflammatory responses involving an acute component, such as is involved in stroke, systemic inflammatory response syndrome, infarction, sepsis, burns, coronavirus disease, trauma or postoperative inflammation.

[0025] These detunings have an effect both individually and in combination.

[0026] A simultaneous balanced approach is reasonable to provide a therapeutic response to inflammation and its physiological consequences, such as cellular degradation.

[0027] The present inventors have found that a composition containing a specific mixture of lithium, magnesium and potassium in specific molar ratios allows (and is alone) to be particularly effective in preventing and / or treating inflammation, in particular in oral, parenteral or ocular administration. Summary of the invention

[0028] The present invention relates to synergistic polyionic complexes that provide an enhanced biological response to chronic or acute inflammation.

[0029] The polyionic complex for oral or parenteral treatment of inflammation or prevention of inflammation is a polyionic composition comprising a salt comprising at least one mineral or organic salt of lithium, magnesium and potassium, characterized by the following molar ratios: Lithium 1-magnesium [0.13-0.34]-potassium [1.20-2.40].

[0030] Preferably, the lithium concentration in the composition is between 0.01 mmol / kg and 10 mmol / kg.

[0031] Preferably, the molar ratio of magnesium to lithium is [0.14-0.32], preferably [0.15-0.30], preferably [0.18-0.25].

[0032] Preferably, the molar ratio of potassium to lithium is [1.30-2.30], preferably [1.35-2.15], preferably [1.40-2.15], preferably [1.55-1.75], preferably [1.58-1.72], again preferably [1.61-1.70].

[0033] This polyion complex consists of a novel combination of "magnesium-lithium-potassium" according to a specific molar ratio algorithm. Depending on the therapeutic response to inflammation, a change in one of the components leads to a proportional change in the concentration of the other two elements.

[0034] This novel combination of algorithms aims (especially through oral or parenteral administration) to simultaneously break the pro-inflammatory vicious cycle that it feeds, restore the cellular homeostasis of these ions found altered in many pathologies, promote a dynamic competition for binding sites of these ions for corrective physiological responses (enzymatic, metabolic, neurotransmission...), and generate a reinforced synergistic response that will strengthen the cellular response to damage to quickly and sustainably manage inflammation. It is also demonstrated below that the combination of these three elements significantly reduces the toxicity of lithium (compared to lithium alone) and allows considering its use in the treatment of bipolar disorder.

[0035] The action of a complex of multiple ions, especially magnesium and its ionic form Mg, promotes the restoration of the homeostasis altered by the inflammatory response. 2+ and potassium and its ionic form K + (whose concentration can be significantly influenced) and contribute to the restoration of optimal cell physiological function, improving cell metabolism and restoring cytoprotective and anti-apoptotic activity at the neuronal level.

[0036] Its action promotes the rebalancing of potassium and restores its key role in maintaining cellular function, especially in excitable cells such as nerve cells. In addition, it promotes the interaction of potassium with sodium (interaction of ionized form K+ with extracellular Na+) and increases fluid movement that promotes ion exchange and balance, especially that of lithium. Lithium does have a strong biological activity, and since it does not have its own system responsible for regulating its concentration in intracellular and extracellular fluids, which can vary significantly, its external contribution must be regulated, coordinated and weighted according to therapeutic needs.

[0037] As mentioned above, the bioactive polyion complex brings together lithium, magnesium, and potassium to exert their multiple physiological and biochemical effects.

[0038] Lithium combines its important anti-inflammatory properties with a modulatory effect on the biosynthesis of neurotransmitters such as serotonin and glutamate, with a stimulatory effect on the production of pain-reducing brain beta-endorphins.

[0039] Lithium and magnesium are linked. The bimetallic phosphate complex ATP-Mg-Li was found to be the biologically active form of lithium, which binds to Mg 2+ This double metal phosphate complex binds to ATP and Mg in plasma or cytoplasm. 2+ In addition, under stress conditions that increase ATP levels in the cytoplasm and organelles such as mitochondria, the extracellular matrix will act as a reservoir for Li accumulation. + potential reservoir.

[0040] Only the specific combination of molar ratios according to the invention ensures the absence of cytotoxicity, thus ensuring compliance with tissue homeostasis (especially of the more sensitive nervous tissues) and allows obtaining a significant effectiveness in the prevention and / or treatment of neurogenic inflammation. Only the composition that meets the definition according to the invention allows in particular a protective effect of neurons, a protective and restorative effect of neurites (axons-dendrites) and myelin sheaths against the damage caused by cisplatin, which is considered an inducer of neurogenic inflammation. Without prejudging the underlying mechanism for obtaining this surprising effect, it can be considered that only this combination of lithium, magnesium and potassium in the molar ratios according to the invention makes it possible to maximize the biological activity of each of these elements in the complex environment of human and animal organisms, all involving the inhibition of the level of the pro-inflammatory vicious cycle, the restoration of the cellular homeostasis of these ions that are found to be altered (especially the restoration of the cellular homeostasis of magnesium), its ionic form Mg being essential for the optimal functioning of mitochondria. 2+ and potassium and its ionic form K + , a dynamic competitive effect at the level of ion channels and of the fixation sites of these ions, associated with an increase in intracellular and extracellular fluid movement to reduce lithium toxicity, in particular the activation of immune, enzymatic, metabolic and neurotransmission responses, a synergistic response that increases resistance to damage and intensifies cellular responses through cytoprotective effects, restoring neuronal excitatory potential and inactivating apoptotic pathways activated by extracellular ATP.

[0041] Lithium is present in the composition at a concentration between 0.01mmol / kg and 100mmol / kg. The lithium concentration in the composition is preferably between 0.1mmol / kg and 50mmol / kg, preferably between 0.2mmol / kg and 25mmol / kg, for example between 0.5mmol / kg and 15mmol / kg, or between 1mmol / kg and 6mmol / kg. Ideally, target cells cannot be effectively exposed to concentrations greater than 10mmol / kg, with the risk that toxicity outweighs the benefits of treating inflammation. However, in the presence of magnesium and potassium, the toxicity of lithium is significantly reduced compared to the toxic concentrations generally described, as shown below. Below 0.01mmol / kg of lithium, the effectiveness of the composition is compromised.

[0042] Preferably, lithium is present in the composition at a concentration between 0.01 mmol / kg and 100 mmol / kg. The lithium concentration in the composition is preferably between 0.1 mmol / kg and 50 mmol / kg, preferably between 0.2 mmol / kg and 25 mmol / kg, for example between 0.5 mmol / kg and 15 mmol / kg, or between 1 mmol / kg and 6 mmol / kg.

[0043] In the context of oral or parenteral use / administration, the composition of the invention is intended to restore these ions (particularly Mg 2+ and potassium and its ionic form K + ) of the cell to promote dynamic competition for binding sites and generate an enhanced synergistic response.

[0044] The composition of the invention may also be administered by the ocular route, for example, in the form of eye drops.

[0045] The composition of the invention is used for preventing and / or treating inflammation (including chronic inflammation or acute inflammation).

[0046] Neurogenic inflammation, also known as neuroinflammation, “low-noise inflammation” or “inflammage” (age-related inflammation), includes chronic inflammation (as implicated in, for example, fibromyalgia, neuropathy, osteoarthritis, polyarthritis, metabolic syndrome, diabetes, obesity, atherosclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease (inflammatory bowel disease), multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, migraine, asthma, pain) or involves inflammatory responses with an acute component (as implicated in, for example, stroke, systemic inflammatory response syndrome, infarction, sepsis, burns, coronavirus disease, trauma or postoperative inflammation).

[0047] The release of inflammatory markers (such as TNF-α, IL-1β, IL-18 and / or NFκB) or the activation of enzymes (such as caspases) are recurrent in all these pathologies, underpinning the cause of inflammation. The composition of the invention makes it possible to inhibit the release of these markers or the activation of these enzymes both in a preventive context and in a therapeutic context.

[0048] The composition of the invention can be used for oral or parenteral administration to prevent and / or treat inflammation.

[0049] Preferably, the composition of the invention is used for treating and / or preventing Alzheimer's disease, Huntington's disease, Parkinson's disease, Charcot-Marie-Tooth disease by oral or parenteral administration.

[0050] Preferably, the composition of the invention is used for oral or parenteral treatment and / or prevention of stroke.

[0051] Preferably, the composition of the invention is used for oral or parenteral treatment of bipolar disorder.

[0052] At least one mineral salt or organic salt of lithium, magnesium and potassium means that these substances are present in ionic form. + Mg 2+ and K + Combined with one or more anions, ie they may be from a mixed salt and / or each from a different salt.

[0053] Advantageously, the lithium is introduced into the composition in the form of lithium chloride, lithium hydroxide, lithium carbonate, lithium citrate, lithium gluconate, lithium orotate and / or any other pharmaceutically acceptable salt.

[0054] Preferably, for oral or parenteral use, the composition includes lithium chloride.

[0055] Advantageously, magnesium is introduced into the composition in the form of magnesium chloride, magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium sulfate, magnesium silicate, magnesium bisglycinate, magnesium malate, magnesium glycerophosphate, magnesium stearate, magnesium ascorbate, magnesium taurate, magnesium citrate, magnesium gluconate, magnesium taurate and / or any other pharmaceutically acceptable mineral or organic salt.

[0056] Preferably, for oral or parenteral use, the composition comprises magnesium chloride, optionally hydrated magnesium chloride.

[0057] Advantageously, potassium is introduced into the composition in the form of potassium chloride, potassium bromide, potassium iodide, potassium phosphate, potassium carbonate, potassium hydroxide, potassium silicate, potassium gluconate, potassium citrate, potassium malate, potassium glycerophosphate, potassium lactate, potassium pidolate, potassium aspartate and / or any other pharmaceutically acceptable mineral or organic salt. Preferably, for oral or parenteral use, the composition comprises potassium chloride.

[0058] The compositions of the invention may include other mineral salts or organic salts.

[0059] The composition may, for example, comprise at least one silicon salt, preferably a silicate, such as sodium silicate (Na2SiO3) (optionally hydrated sodium silicate), potassium silicate, orthosilicic acid, monomethylsilanetriol and / or any other pharmaceutically acceptable mineral or organic salt, in particular for oral or parenteral use.

[0060] Silicon is a cofactor of prolyl hydroxylase, which is involved in the stimulation of fibroblasts. It reduces the permeability of capillaries and has anti-edematous, soothing and refreshing effects.

[0061] It plays a role in the regulation of the cell cycle of lymphocytes, which ultimately affects immune and inflammatory responses.

[0062] Silicon reduced the expression levels of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), nuclear factor of activated B-cell light chain (NF-κB), various cytokines (TNF-α and IL-1β) in the inflamed areas.

[0063] Depending on the assumed pathological basis of the inflammation, the proportion of silicon relative to the other salts is adjusted.

[0064] The composition may, for example, include at least one manganese salt, such as manganese chloride, manganese sulfate, manganese carbonate, manganese citrate, manganese malate, manganese glycerophosphate, manganese lactate, manganese pidolate, manganese aspartate, manganese gluconate, manganese alginate, manganese picolinate and / or any other pharmaceutically acceptable mineral or organic manganese salt, in particular for oral or parenteral use.

[0065] Manganese is involved in tissue physiology and biology through its role as a cofactor in many enzymatic processes. Manganese plays an important role in the regulation of glucose tolerance by acting synergistically with magnesium, which specifically allows for the reduction of neural and epidermal metabolic stress.

[0066] The composition may, for example, include at least one mineral or organic salt of zinc, such as zinc citrate, zinc orotate, zinc sulfate, zinc citrate, zinc malate, zinc glycerophosphate, zinc lactate, zinc pidolate, zinc aspartate, zinc gluconate, which plays an important role in many important enzymatic processes (such as DNA and protein synthesis, wound healing, insulin metabolism, development and normal function of the nervous system...).

[0067] The composition may for example comprise at least one mineral or organic salt of copper, such as copper carbonate, copper citrate, copper malate, copper glycerophosphate, copper lactate, copper pidolate, copper aspartate, copper gluconate. Copper is a component of several enzymes that are involved in the metabolism of carbohydrates, lipids and iron. It has an antioxidant effect.

[0068] An oral or orally administrable composition is a composition intended to be ingested. Preferably, the composition is in the form of a tablet, a pill, a capsule, a solution, a syrup, a lyophilisate, ie it may contain the excipients necessary for the desired formulation.

[0069] Parenteral compositions or parenteral administration refer to compositions that are injectable beside the digestive tract, intravenously (injected via a hypodermic needle, catheter or catheter port), subcutaneously, intramuscularly or intraperitoneally. In the case of parenteral compositions, they are in liquid form.

[0070] In the case of parenteral compositions, the mineral salts or organic salts of the composition of the invention are dissolved in water, that is, the main solvent of the composition is preferably water. Water accounts for at least 50% by weight of the composition, preferably at least 75% by weight, at least 80% by weight, preferably at least 90% by weight or 95% by weight. The composition may include other liquid ingredients that a person skilled in the art considers useful, in particular to promote stability and biocompatibility (pH, plasma osmotic pressure, sodium blood).

[0071] The composition of the invention may also include other molecules, additives or excipients, such as preservatives, antibiotics, stabilizers, thickeners, antioxidants, emulsifiers or gelling agents. These additives may be used to preserve the composition of the invention.

[0072] The composition may, for example, include one or more vitamins, such as vitamin E. Vitamin E is a potent antioxidant and modulates immune function.

[0073] The invention relates to the use of the composition as a human or veterinary medicine.

[0074] Neurogenic inflammation, also known as neuroinflammation, “low-noise inflammation” or “inflammage” (age-related inflammation), includes chronic inflammation (as implicated in, for example, fibromyalgia, neuropathy, autonomic neuropathy, osteoarthritis, polyarthritis, metabolic syndrome, diabetes, obesity, atherosclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease (inflammatory bowel disease), multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, migraine, asthma, pain) or involves an inflammatory response with an acute component (as implicated in, for example, stroke, systemic inflammatory response syndrome, infarction, sepsis, burns, coronavirus disease, trauma or postoperative inflammation). BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The invention will be better understood using the following description with reference to the accompanying drawings, which show: Figure 1 : Effect of the composition of the invention on the survival of cortical neurons after glutamate injury; Figure 2 : The effect of the composition of the invention on the release of TNF-α after cisplatin injury; Figure 3 : The effect of the composition of the invention on the release of IL-1β after cisplatin injury; Figure 4 : Effects of the composition of the invention on the level of caspase-3 in Schwann cells injured by cisplatin; Figure 5 : The effect of the composition of the invention on the survival of dopaminergic neurons after MPP injury; Figure 6 : Effects of the composition of the invention on the survival of cortical neurons after oxygen and glucose deprivation; Figure 7 : The anti-inflammatory effect of the composition of the invention on human mononuclear blood cells stimulated by LPS in vitro; Figure 8 :Anti-inflammatory effects of single ions on LPS-stimulated human mononuclear blood cells in vitro; Figure 9 : Anti-inflammatory effects of three variants of the composition of the invention on human mononuclear blood cells stimulated by LPS in vitro; Figure 10 : Anti-inflammatory properties of the inventive composition in an in vivo model of DSS-induced ulcerative colitis. DETAILED DESCRIPTION

[0076] The algorithm combination was tested in vitro and in vivo on various models of diseases known to be prominent inflammatory diseases as described above.

[0077] Example 1 - Preparation of solutions at various concentrations for in vitro testing A solution containing lithium chloride, magnesium chloride and potassium chloride was prepared in reverse osmosis water.

[0078] They are shown in Table 1 below.

[0079]

[0080]

[0081] Table 1.

[0082] Example 2 - Effect of the composition of the invention on glutamate damage of cortical neurons (cortical neurons) in Alzheimer's model The influence of type Rat cortical neurons were cultured according to the procedures described by Singer (1999).

[0083] Female mice (Wistar mice; January Lab) at 15 days of gestation were killed by cervical dislocation, and fetuses were removed from the uterus. Embryonic cortices were collected and placed in ice-cold Leibovitz 15 (L15; PanBiotech, No.: P04-27055, Lot: 3870221) medium containing 2% penicillin-streptomycin (PS; PanBiotech, No.: P06-07100, Lot: 5870421) and 1% bovine serum albumin (BSA; PanBiotech, No.: P06-1391100, Lot: H210207). Cortices were dissociated by trypsinization at 37°C for 20 minutes (min) (Trypsin EDTA 1X; PanBiotech, No.: P10-023100, Lot: 3030221). The reaction was stopped by adding Dulbecco's modified Eagle's medium (DMEM; PanBiotech, No. P04-03600, Lot: 1130721) containing grade II DNase I (0.1 mg / mL; PanBiotech, No. P60-37780100, Lot: H181015) and 10% fetal calf serum (FCS; Invitrogen, No. 10270106, Lot: 2275115). The cells were then mechanically dissociated 3 times by a 10 mL pipette. The cells were then centrifuged at 515 x g for 10 min at +4°C. The supernatant was discarded and the cell pellet was resuspended in defined medium consisting of Neurobasal medium (Invitrogen, No. 11570556, Lot: 2327396) supplemented with 2% B27 (Invitrogen, No. 11530536, Lot: 2337321), L-glutamate (2 mM; PanBiotech, No. P04-80100, Lot: 2770620), 2% PS and 10 ng / mL BDNF (Peprotech, No. 450-02, Lot: 102061). Viable cells were then counted in a Neubauer cell counter using a trypan blue incompatibility test. Cells were seeded in 96-well plates (pre-coated with poly-D-lysine; Greiner, No. 655940, Lot No. E200933A) at a density of 30,000 cells / well and grown at 37°C in a humidified air (95%) / CO2 (5%) atmosphere. Half of the medium was replaced with cold medium every 2 days.

[0084] Glutamate solution (Sigma, No. G1501, Lot No. SLBD7305V) was reconstituted in the culture medium at 10 mM. A control solution was prepared under the same conditions.

[0085] After 11 days of culture, primary cortical neurons were pretreated with the composition of Example 1 or a reference compound (BDNF, 50 ng / mL) for 1 hour, and then incubated with glutamate intoxication at a concentration of 40 μM for 20 minutes.

[0086] Test the following conditions: Control medium Control medium + glutamate (40 μM, 20 min) Combination TA6496 + glutamate (40 μM, 20 min) Combination TA6440 + glutamate (40 μM, 20 min) Combination TA6415 + glutamate (40 μM, 20 min) BDNF (50 ng / mL) + glutamate (40 μM, 20 min) Culture was performed using 6 wells for each condition.

[0087] Primary endpoint: Measurement of the total number of cortical neurons.

[0088] After 24 h of incubation, cells were fixed with 4% paraformaldehyde solution (Alpha Aesar; No. J19943, Lot No. 206909) at room temperature for 15 min. Cells were then permeabilized with saline phosphate buffer (PBS; PanBiotech; No. P04-36500, Lot No. 3410921) containing 0.1% triton X-100 (Sigma; No. T9284, Lot No. 118K01602) at room temperature for 30 min. Nonspecific sites were saturated with saline phosphate buffer solution containing 3% BSA and 3% FCS for 1 h. Cells were then incubated with chicken polyclonal anti-MAP-2 antibody (1 / 2000, Abcam; No. ab5392, Lot No. GR3405939-2) in saturation buffer at 4 °C for 12 h.

[0089] These antibodies were revealed with Alexa Fluor 568 goat anti-chicken IgG (1 / 400, Molecular Probes, No. A110041, Batch: 1963088) in saturation buffer for 1 hour at room temperature. Nuclei were labeled with a fluorescent marker (Hoechst solution, Sigma; No. B1155, Batch: 046M4048V) in the same solution.

[0090] For each condition, 20 pictures per well were taken at 20x magnification using the InCell AnalyzerTM 2200 (GE Healthcare). Images of each crop well were taken under the same conditions. Automated analysis of the number of cortical neurons was performed using the Developer software (GE Healthcare). A total of 6 data were provided for each experimental condition.

[0091] statistics: Data (6 data sets per condition, 1 culture) are presented as mean ± SE. Overall analysis of data was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. The significance level was set at p < 0.05.

[0092] The results are shown in Figure 1 The composition TA6415 significantly protected cortical neurons from apoptosis after glutamate insult at a level slightly higher than the BDNF control.

[0093] Example 3 - Anti-inflammatory effect of the composition of the invention on the release of TNF-α and IL-1β in sensory neurons .

[0094] Sensory neurons are part of the peripheral nervous system. Their cell bodies are located in the dorsal root ganglia (DRG) along the spinal cord and send out extensions to the most distal limbs. One of the properties of sensory neurons is their ability to regenerate their extensions after the nerve is cut. This property is related to the presence of Schwann cells, which are trophic and myelin-forming cells of the peripheral nervous system that release specific growth factors for axonal growth.

[0095] The in vitro model used is a culture of sensory neurons myelinated by Schwann cells (Callizot et al., 2011, Exp Cell Res 317: 2374-2383). This model, miniaturized in 96 wells, enables the analysis of the effects of molecules on the development of sensory neurons and the myelination of axons by Schwann cells.

[0096] The aim of this study was to investigate the effects of TA64 compositions at 6 different concentrations on TNF-α and IL-1β release from sensory neurons following cisplatin injury.

[0097] Protocol for co-culture of sensory neurons and Schwann cells: Female mice (Wistar mice; January Lab) at 15 days of gestation were killed by cervical dislocation, and fetuses were removed from the uterus. Embryonic DRGs were collected and placed in ice-cold medium containing 2% penicillin-streptomycin (PS; PanBiotech, No.: P06-07100, Lot: 9300621) and 1% bovine serum albumin (BSA; PanBiotech, No.: P06-1391100, Lot: H210207) in Leibovitz 15 (L15; PanBiotech, No.: P04-27055, Lot: 5331021). DRGs were dissociated by trypsinization at 37°C for 20 minutes (min) (Trypsin EDTA 1X; PanBiotech, No.: P10-023100, Lot: 8600621). The reaction was terminated by adding Dulbecco's modified Eagle's medium (DMEM; PanBiotech, No. P04-03600, Lot No. 1130721) containing grade II DNase I (0.1 mg / mL; PanBiotech, No. P60-37780100, Lot No. H181015) and 10% fetal calf serum (FCS; Invitrogen, No. 10270106, Lot No. 2319479). The cells were then mechanically dissociated 3 times by a 10 mL pipette. The cells were then centrifuged at 180 x g for 10 min at +4°C on a layer of BSA (3.5%) in medium L15. The supernatant was discarded and the cell pellet was resuspended in defined medium consisting of Neurobasal medium (Invitrogen, No. 21103049, Lot: 2348949) supplemented with 2% B27 (Invitrogen, No. 17504-044, Lot: 2336991), L-glutamate (2 mM; PanBiotech, No. P04-80100, Lot: 2770620), 2% PS and 50 ng / mL NGF (Sigma, No. N1408, Lot: SLCG2596). Viable cells were then counted in a Neubauer cell counter using a trypan blue incompatibility test. Cells were seeded in 96-well plates (pre-coated with poly-D-lysine; Greiner, No. 655940, Batch: E200933A) to a density of 12,000 cells / well and grown at 37°C in a humidified air (95%) / CO2 (5%) atmosphere. Half of the medium was replaced with fresh medium every 2 days. Cells were maintained for 7 days to allow proliferation of Schwann cells and sensory neurons. On day 8, the medium was supplemented with 50 μg / mL ascorbic acid (AA; Sigma, No. 092902, Batch: 05316HJ-438) to initiate differentiation of basal Schwann cells into myelinating Schwann cells.

[0098] Under these conditions, after 5 days of incubation with AA, myelin sheaths were detected using anti-MAG (myelin antigen glycoprotein) antibody, an early marker of myelin.

[0099] Cisplatin preparation, exposure, and drug treatment: treatment regimen Cis-diaminedichloroplatinum(II) (Cisplatin; Sigma, No.: P4394, Batch: MKCN8054) was reconstituted in DMSO at 24 mM (stock solution). A control environment was prepared under the same conditions. After 12 days of culture with AA, primary sensory neurons were pretreated with the composition of Example 1 or with a reference compound (NGF, 50 ng / ML) for 2 hours and then intoxicated with cisplatin. A cisplatin preparation diluted in control medium at a final concentration of 12 μM was used for incubation of neurons for 24 hours.

[0100] Test the following conditions: Control medium Control medium + cisplatin (12 μM, 24 h) Combination TA6426 + cisplatin (12 μM, 24 h) Combination TA6422 + cisplatin (12 μM, 24 h) Combination TA6419 + cisplatin (12 μM, 24 h) Combination TA6415 + cisplatin (12 μM, 24 h) Combination TA6412 + cisplatin (12 μM, 24 h) Combination TA6409 + cisplatin (12 μM, 24 h) Control medium + NGF (50 ng / mL) + cisplatin (12 μM, 24 h) Six wells were used for each condition. After 6 h of incubation in the presence of cisplatin, the conditioned medium was collected and stored at -80 °C. TNF-α was measured by flow cytometry on the thawed medium according to the manufacturer's instructions (BD Bioscience, No. 561516, Lot No. 0171683).

[0101] The results are shown in Figure 2 middle.

[0102] After 6 hours of treatment with cisplatin, the conditioned medium was removed and stored at -80° C. Murine IL-1β was measured by ELISA on thawed samples according to the manufacturer's instructions (Bio-techne, No. DY501, batch P296813).

[0103] The results are shown in Figure 3 middle.

[0104] Data (6 data sets per condition, 1 culture) are presented as mean ± SE. Overall analysis of data was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. The significance level was set at p < 0.05.

[0105] result: Cisplatin at 12 μM induced an increase in the release of TNF-α and IL-1β. All TA64 compositions tested significantly inhibited the release of TNF-α at control levels. Compositions TA6426, TA6419 and TA6415 significantly inhibited the release of IL-1β, while a general trend could be observed for all tested compositions.

[0106] Example 4 - Inventive Combination by Analyzing Caspase Activation in Sensory Neurons (Alzheimer's Model) Anti-apoptotic effect of substances.

[0107] The protocol used for co-culture of sensory neurons and Schwann cells and preparation and exposure to cisplatin was the same as described in Example 3.

[0108] Test the following conditions: Control medium Control medium + cisplatin (12 μM, 24 h) Control medium + combination TA6426 + cisplatin (12 μM, 24 h) Control medium + combination TA6422 + cisplatin (12 μM, 24 h) Control medium + combination TA6419 + cisplatin (12 μM, 24 h) Control medium + combination TA6415 + cisplatin (12 μM, 24 h) Control medium + combination TA6412 + cisplatin (12 μM, 24 h) Control medium + combination TA6409 + cisplatin (12 μM, 24 h) Six wells were used for each condition.

[0109] After treatment in the presence of cisplatin for 24 hours, cells were fixed with a solution of 5% acetic acid (Sigma; No.: 33209; Lot: SHBJ9236) and 95% ethanol (VWR; No.: 83813.360; Lot: 20G074013) at -20°C for 5 min, and control conditions were set up in the same manner. Cells were then permeabilized and nonspecific sites were saturated with saline phosphate buffer (PBS; PanBiotech; No.: P04-36500, Lot: 5371021) containing 0.1% newt X-100 (Sigma; No.: T9284, Lot: 118K01602) for 30 min at room temperature. Nonspecific sites were saturated with a PBS solution containing 3% BSA and 3% FCS for 1 hour. The cells were then incubated with mouse anti-β-tubulin monoclonal antibody (1 / 2000, Sigma; No.: T8660, Lot: 034M4790V) and rabbit cleaved anti-caspase-3 polyclonal antibody (1 / 500, Abcam, No.: ab13847, Lot: GR3286166-1) in saturation buffer at +4°C for 12 hours. Antibodies were revealed with goat anti-mouse IgG AlexaFluor 647 (1 / 400; Molecular Probes; No.: A212235; Lot: 1922319) and goat anti-rabbit IgG Alexa Fluor 568 (1 / 400; Molecular Probes; No.: A11011; Lot: 2192277) in saturation buffer at room temperature for 1 hour. Nuclei were labeled with fluorescent markers (Hoeschst solution, Sigma, No.: B1155, Lot: 046M4048V) under the same conditions.

[0110] For each condition, 20 pictures per well were taken at 20x magnification using the InCell AnalyzerTM 2200 (GE Healthcare). Images of each crop well were taken under the same conditions. The number of sensory neurons and caspase-3 levels were automatically assessed using the Developer software (GE Healthcare). A total of 6 data were provided for each experimental condition.

[0111] Data (6 data sets per condition, 1 culture) are presented as mean ± SE. Overall analysis of data was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. The significance level was set at p < 0.05.

[0112] The results are shown in Figure 4 middle.

[0113] The compositions TA6426, TA6422, TA6419 and TA6415 significantly reduced the release of caspase-3 in primary Schwann cells after cisplatin injury.

[0114] Example 5 - Effect of the composition of the invention on the MPP (1-methyl-4-phenylpyridinium +) injury of midbrain neurons in Parvovirus in an in vitro model of Kinson's disease.

[0115] Rat dopaminergic neurons were cultured as described by Schinelli et al., 1988. Briefly, female rats (Wistar rats; January Lab) at 15 days of gestation were killed by cervical dislocation and the fetuses were removed from the uterus. The embryonic midbrains were collected and placed in ice-cold medium containing 2% penicillin-streptomycin (PS; PanBiotech, No.: P06-07100, Lot: 1201220) and 1% bovine serum albumin (BSA; PanBiotech, No.: P06-1391100, Lot: H200403) in Leibovitz 15 (L15; PanBiotech, No.: P04-27055, Lot: 3870221). Only the ventral part of the midbrain flexure was used for cell preparation because this region of the developing midbrain is rich in dopaminergic neurons. The midbrain was dissociated by trypsinization for 20 min at 37°C (Trypsin EDTA 1X; PanBiotech, No.: P10-023100, Lot: 9821220). The reaction was stopped by adding Dulbecco's modified Eagle's medium (DMEM; PanBiotech, No.: P04-03600, Lot: 3090221) containing grade II deoxyribonuclease I (0.1 mg / mL; PanBiotech, No.: P60-37780100, Lot: H181015) and 10% fetal calf serum (FCS; Invitrogen, No.: 10270106, Lot: 2275115). The cells were then mechanically dissociated 3 times by a 10 mL pipette. The cells were then centrifuged at 180 x g for 10 min at +4°C on a layer of BSA (3.5%) in medium L15. The supernatant was discarded and the cell pellet was resuspended in a defined medium consisting of Neurobasal medium (Gibco, No.: 21103049, Lot: 2242402) with 2% B27 (Gibco, No.: 17504044, Lot: 2295367), L-glutamate (2 mM; PanBiotech, No.: P04-80100, Lot: 3301019), 2% PS, 10 ng / mL BDNF (PeproTech, No.: 450-02, Lot: 071961), and 10 ng / mL glial-derived neurotrophic factor (GDNF; PanBiotech, No.: 450-10, Lot: 0606B64). Viable cells were then counted in a Neubauer cell counter using a trypan blue incompatibility test. Cells were seeded in 96-well plates (pre-coated with poly-D-lysine; Greiner, No.: 655940, Batch: E20093UL) to a density of 40,000 cells / well and grown at 37°C in a humidified air (95%) / CO2 (5%) atmosphere.Half of the culture medium was replaced with fresh medium every 2 days. Under these conditions, after 5 days of culture, astrocytes were present in the culture and released growth factors, allowing neuronal differentiation. Under these conditions, 2% to 5% of the neurons were dopaminergic neurons.

[0116] MPP+ (1-methyl-4-phenylpyridinium+) (Sigma, No.: D048, Batch 0000047439) was reconstituted at 10 mM (stock solution) in defined medium. Control environments were prepared under the same conditions. After 7 days in culture, primary midbrain neurons were pretreated with test compounds or reference compounds (BDNF, 50 ng / mL) for 1 hour and then incubated with MPP+ intoxication at a final concentration of 8 μM for 48 hours to induce approximately 45% + / - 5% neuronal cell death.

[0117] Test the following conditions: Control medium Control medium + MPP+ (16μM, 48h) ·TA6496+MPP+ (16μM, 48h) ·TA6440+MPP+ (16μM, 48h) ·TA6415+MPP+ (16μM, 48h) ·BDNF (50ng / mL)+MPP+ (16μM, 48h) After 48 hours of intoxication in the presence or absence of the test compound, the cells were fixed with 4% paraformaldehyde solution (Alpha Aesar, No.: J19943, Lot: 202585) at room temperature for 20 minutes, and the control condition was fixed according to the same procedure. The cells were then permeabilized and nonspecific sites were saturated with phosphate-buffered saline (PBS; PanBiotech; No.: P04-36500, Lot: 6300421) containing 0.1% saponin (Sigma; No.: S7900, Lot: BCBL8667V) and 1% FCS for 15 minutes at room temperature. The cells were incubated with anti-tyrosine hydroxylase mouse monoclonal antibody (TH, 1 / 10000, Sigma; No.: T1299, Lot: 014M4835) in PBS solution at 4°C for 12 hours. The anti-TH antibody labels dopaminergic neurons.

[0118] Staining was revealed by adding Alexa Fluor 488 goat anti-mouse IgG (1 / 400, Molecular Probes, No.: A11001, Lot: 2247988) in PBS with 1% FCS and 0.1% saponin for 1 hour at room temperature. Nuclei were labeled with a fluorescent marker (Hoechst, Sigma; No.: B1155, Lot: 046M4048V) in the same solution.

[0119] For each condition, 20 pictures per well were taken at 20x magnification using the InCell AnalyzerTM 2200 (GE Healthcare). Images of each crop well were taken under the same conditions. Analysis of the cell bodies of positive TH neurons was performed using the Developer software (GE Healthcare). A total of 6 data were provided for each experimental condition.

[0120] Data (6 data sets per condition, 1 culture) are presented as mean ± SE. Overall analysis of data was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. The significance level was set at p < 0.05.

[0121] The results are shown in Figure 5 middle.

[0122] The TA6415 composition significantly protected dopaminergic neurons from apoptosis following MPP+ injury.

[0123] Example 6 - Effect of the composition of the invention in a stroke model with hypoxia and glucose.

[0124] Here, the survival of neurons in primary cortical cultures from mice injured by oxygen / glucose deprivation (OGD) was studied.

[0125] Stroke induces brain damage through a combination of several mechanisms: excitotoxicity, free radical production, inflammation, and apoptosis. Excitotoxicity is caused by excessive release of glutamate, an excitatory neurotransmitter, from dying or apoptotic neurons with inappropriate stimulation of N-methyl-D-aspartate (NMDA-R) receptors. High levels of glutamate trigger the death of neighboring neurons, which in turn are damaged by the inappropriate stimulation of the NMDA-R receptors. 2+ Cells in the wild can survive the initial invasion associated with a massive cell influx but cannot survive this massive activation under limited glucose and oxygen supply (Verleye et al., 2016).

[0126] The aim of this study was to investigate the protective effects of TA64 compositions at 3 different concentrations on neuronal survival in rat primary cortical cultures injured by oxygen / glucose deprivation (OGD) in an in vitro stroke model.

[0127] Cortical cells were cultured as described in Example 2.

[0128] After 11 days in culture, primary cortical neurons were pretreated with TA64 or reference compound (BDNF, 50 ng / mL) for 1 hour. The medium was then removed and fresh glucose-free medium was added. The medium consisted of glucose-free DMEM (PanBiotech, No.: P04-01549, Batch No.: 5471021) supplemented with 2% B27, 0.2 mM L-glutamine, 1% PS solution, and 10 ng / mL BDNF. The cells were transferred to an anaerobic incubator with 95% N2 and 5% CO2 at 37°C. After 2 hours, 25 mM D-glucose (Sigma, No.: G8644, Batch No.: RNBJ8362) with or without compound TA64 was added to the medium, and the cells were transferred to a regular incubator with 95% air / 5% CO2 at 37°C for 24 hours.

[0129] Test the following conditions: Control (0.1% DMSO) Control + oxygen and glucose deprivation (2 hours) and oxygen and glucose reperfusion (24 hours) Compound + oxygen and glucose deprivation (2 hours) and oxygen and glucose reperfusion (24 hours) 50 ng / mL BDNF + oxygen and glucose deprivation (2 hours) and oxygen and glucose reperfusion (24 hours) After 24 hours of intoxication, cells were fixed with a solution of acetic acid 5% (Sigma, No.: 33209, Lot: SHBJ9236) and ethanol 95% (VWR, No.: 83813.360, Lot: 20G074013) at -20°C for 5 min, and control conditions were set up according to the same procedure. The cells were then permeabilized and nonspecific sites were saturated with saline phosphate buffer solution (PBS; PanBiotech; No.: P04-36500, Lot: 5371021) containing 0.1% saponin (Sigma; No.: S7900, Lot: BCBL8667V) and 1% FCS for 15 min at room temperature. Cells were incubated with anti-MAP-2 mouse monoclonal antibody (1 / 5000, Sigma; No.: M4403, Lot: 035M4780V) in PBS solution at 4°C for 12 hours. Dopaminergic neurons were labeled with anti-TH antibody.

[0130] Staining was revealed by adding goat anti-mouse IgG Alexa Fluor 568 (1 / 400, Molecular Probes, No.: A11004, Lot: 2332536) in PBS with 1% FCS and 0.1% saponin for 1 hour at room temperature. Nuclei were labeled with a fluorescent marker (Hoechst, Sigma; No.: B1155, Lot: 046M4048V) in the same solution.

[0131] For each condition, 20 pictures per well were taken at 20x magnification using the InCell AnalyzerTM 2200 (GE Healthcare). Images of each culture well were taken under the same conditions. Analysis of the cell bodies of MAP-2 positive neurons was performed using the Developer software (GE Healthcare). A total of 6 data were provided for each experimental condition.

[0132] Data (6 data sets per condition, 1 culture) are presented as mean ± SE. Overall analysis of data was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. The significance level was set at p < 0.05.

[0133] The results are shown in Figure 6 middle.

[0134] The TA6415 composition significantly protected mouse primary cortical neurons from apoptosis, even better than the BDNF control at 50 ng / mL.

[0135] in conclusion The markers evaluated in Example 2, Example 3 and Example 4 represent Alzheimer's disease. The markers evaluated in Example 3, Example 4 and Example 5 represent Parkinson's disease. The markers evaluated in Example 3, Example 4 and Example 6 also relate to stroke.

[0136] Thus, it has been shown that the composition according to the invention has a preventive and therapeutic effect on these models in vitro, indicating a significant effect in vivo.

[0137] The release levels of these markers, in particular TNF-α, IL-1β and caspase-3, are generally representative of inflammatory pathologies. Therefore, it is reasonable to consider the positive effects of the inventive composition on all inflammatory pathologies.

[0138] In addition to the anti-inflammatory activity of the inventive composition, the intrinsic toxicity of lithium, magnesium and potassium ions alone or in combination was evaluated. This is repeated in the following example.

[0139] Example 7 - Effects of polyionic complexes on the length of neurites of sensory neurons Treatment of healthy cells with the multi-ionic compositions TA6496, TA6440 and TA6415 for 24 hours induced a slight but non-significant increase in the length of neurites of sensory neurons (31%, 34% and 54% of the control, respectively).

[0140] Cisplatin at 12 μM induced a significant reduction in the length of neurites of sensory neurons (65% neurite loss and p<0.01).

[0141] Treatment with the polyionic compositions TA6440 and TA6415 according to the invention was able to effectively preserve the neurite length of sensory neurons injured by cisplatin for 24 hours (19% neurite loss and p<0.05 and 11% neurite loss and p<0.05, respectively).

[0142] However, treatment with the multi-ionic composition TA6496 was able to partially, but not significantly, preserve the neurite length of sensory neurons injured by cisplatin for 24 hours (28% neurite loss).

[0143] In parallel, the effects of individual or pairwise combinations of ions on the survival of sensory neurons and the length of neurites were evaluated under the same conditions. Solutions were prepared with chlorides of the tested ions.

[0144] The results are shown in Table 2.

[0145]

[0146] Table 2.

[0147] Rows 11 to 14 of the table show that lithium combined with only one of magnesium or potassium ions is highly toxic at 40 mg / kg, but is no longer toxic in the presence of the same concentrations of the other two ions and in the variant of the algorithm in row 14. Therefore, there is indeed a synergistic effect in the combination of the three lithium, magnesium and potassium ions.

[0148] In parallel, the ability of individual ion pairs to maintain sensory neuron survival and neurite length was tested. The results are shown in Table 3.

[0149]

[0150] Table 3.

[0151] None of the individual ions significantly protected sensory neurons from cisplatin-induced apoptosis, whereas the multi-ion combination tested at similar concentrations protected sensory neurons from cisplatin-induced apoptosis. The same was true for the combination of potassium and magnesium, which did not significantly improve the survival of sensory neurons. This difference may be due to the reduced toxicity of the multi-ion combination compared to the individual ions, which makes the protection superior to the toxicity.

[0152] Manganese was also tested under the same conditions at concentrations ranging from 0.15 mg / kg to 1.2 mg / kg without demonstrating any significant effects on neuronal survival or neurite length.

[0153] Other combinations of lithium-magnesium-potassium according to the invention were also tested. They are shown in Table 4.

[0154]

[0155] Table 4.

[0156] Compositions C12 to C16 are outside the range of molar ratios according to the invention, whereas the other compositions C1 to C11 represent the inventive algorithm.

[0157] The effects of ion combinations on sensory neuron survival and neurite length were evaluated under the same conditions as above. Solutions were all prepared with chlorides of the tested ions.

[0158] The results are shown in Table 5.

[0159]

[0160] Table 5.

[0161] The above results show that, for all claimed ranges, the composition of the invention significantly reduces apoptosis of sensory neurons induced by cisplatin. When potassium and / or magnesium are below and / or above the range of molar ratios according to the invention (C12 to C16), the combination no longer has a significant effect.

[0162] Example 8 - Effects of three different concentrations of polyionic complexes on primary myelinated rat sensory neurons after cisplatin injury Neuroprotection of neurons, neurites, and myelin Example 8a - Effect of polyionic complexes on the length of myelin sheaths of sensory neurons (treatment regimen) Treatment of healthy cells with three different concentrations of the polyionic complexes TA6496, TA6440 and TA6415 for 24 h did not modulate the length of myelin sheaths of sensory neurons (89%, 97% and 89% of control, respectively).

[0163] Cisplatin at 12 μM induced a significant decrease in the length of myelin sheaths of sensory neurons (78% myelin loss and p<0.001).

[0164] Treatment with 2 different concentrations of the polyionic complexes TA6440 and TA6415 significantly preserved the myelin length of sensory neurons from cisplatin insult for 24 hours (46% myelin loss and p<0.05 and 34% myelin loss and p<0.01, respectively).

[0165] However, treatment with TA6496 polyionic complexes induced a slight but non-significant increase in myelin length 24 h after cisplatin insult (66% myelin loss).

[0166] Example 8b - Effects of three different concentrations of polyionic complexes on primary myelinated rat sensory neurons after cisplatin injury, Neuroprotection of neurites and myelin: protection strategies The primary endpoints were measurement of sensory neuron survival, assessment of neurite length and myelin length of sensory neurons with and without cisplatin intoxication.

[0167] Cis-dichlorodiamine platinum (II) (Cisplatin, Sigma, No. P4394, Batch: MKCL0026) was reconstituted in culture medium at 10 mg / mL (stock solution). Control environments were prepared under the same conditions. After 12 days of culture with AA, primary sensory neurons were pretreated with 3 concentrations of the multi-ion complex for 2 hours and then intoxicated with cisplatin. Cisplatin preparations were diluted in control culture medium at a final concentration of 12 μM and used for incubation on neurons for 24 hours.

[0168] The concentration of the polyionic complex is: 1. TA6496: Li 10.37mmol / kg, Mg 2.27mmol / kg, K 16.88mmol / kg 2. TA6440: Li 5.76mmol / kg, Mg 1.26mmol / kg, K 9.38mmol / kg 3. TA6415: Li 1.73mmol / kg, Mg 0.38mmol / kg, K 2.81mmol / kg Evaluate the following conditions: Control medium Control medium + cisplatin (12 μM, 24 h) · 3 different concentrations of multi-ion complexes TA6496, TA6440 or TA6415 + cisplatin (12 μM, 24 h) · 3 different concentrations of multi-ion complexes TA6496, TA6440 or TA6415.

[0169] This study was performed with 6 wells per condition and replicated to validate the results. The data included and analyzed were the combined results of the 2 studies.

[0170] After treatment in the presence or absence of cisplatin for 24 h, cells were fixed with a solution of acetic acid / ethanol (5 / 95) at -20°C for 5 min, and control conditions were fixed in the same manner. The cells were then permeabilized and nonspecific sites were saturated with phosphate buffered saline (PBS; PanBiotech; No.: P04-36500, Lot: 2620121) containing 0.1% saponin (Sigma; No.: S7900, Lot: BCBL8667V) and 1% FCS for 15 min at room temperature. The cells were then incubated with anti-rabbit neurofilament polyclonal antibody (NF; 1 / 500, Sigma; No.: N0142, Lot: 083M4833) and anti-MAG mouse monoclonal antibody (1 / 400, Sigma; No.: MAB1567, Lot: 3227322) in a PBS solution containing 1% FCS, 0.1% saponin at 4°C for 12 h.

[0171] These antibodies were revealed with goat anti-mouse Alexa Fluor 488 (1 / 400, Molecular Probes, No.: A11001, Lot: 2247988) and goat anti-rabbit IgG Alexa Fluor 633 (1 / 400, Molecular Probes, No.: A21070, Lot: 1700326) in PBS containing 1% FCS, 0.1% saponin for 1 hour at room temperature. Cell nuclei were labeled with fluorescent markers (Hoechst solution, Sigma; No.: H-33258, Lot: 046M4048V) in the same buffer solution.

[0172] For each condition, the InCell Analyzer TM 20 images per well were taken at 20x magnification using a 2200 (GE Healthcare). Images of each crop well were taken under the same conditions. Analysis was performed using the development software (GE Healthcare). A total of 6 data sets per experimental condition were provided for each of the 2 studies.

[0173] Data (based on 6 data per crop per condition for 2 crops) are presented as mean ± SE. Overall analysis of data was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. The significance level was set at p < 0.05.

[0174] Effects of polyion complexes on the survival of sensory neurons Treatment of healthy cells with three different concentrations of the multiionic complexes TA6496, TA6440 and TA6415 for 24 h did not modulate cell survival (91%, 89% and 95% of the control, respectively).

[0175] Cisplatin at 12 μM induced a significant decrease in neuronal survival (64% cell death and p < 0.0001).

[0176] Pretreatment with three different concentrations of the multi-ionic complexes TA6496, TA6440, and TA6415 two hours before cisplatin insult partially and significantly protected neurons from cell death during 24 hours of cisplatin insult (35% cell death and p<0.0001, 30% cell death and p<0.0001, and 30% cell death and p<0.0001, respectively).

[0177] Effects of polyion complexes on the length of neurites of sensory neurons Treatment with a certain concentration of the multi-ion complex TA6496 for 24 hours had no effect on the length of neurites of sensory neurons (111% of the control).

[0178] Treatment with a certain concentration of the multiionic complex TA6440 for 24 h induced a slight but non-significant increase in neurite length (129% of control).

[0179] Treatment with a certain concentration of the multi-ion complex TA6415 for 24 hours significantly increased the length of neurites of sensory neurons (140% of the control and p < 0.01).

[0180] Cisplatin at 12 μM induced a significant decrease in neurite length of sensory neurons (51% neurite loss and p < 0.0001).

[0181] Pretreatment for two hours with three different concentrations of the multi-ion complexes TA6496, TA6440, and TA6415 effectively protected the neurite length of sensory neurons injured by cisplatin for 24 hours (83% of control and p < 0.01, 100% of control and p < 0.0001, and 102% of control and p < 0.0001, respectively).

[0182] Effects of polyionic complexes on the length of myelin sheaths of sensory neurons Treatment of healthy cells with 3 different concentrations of the polyionic complexes TA6496 and TA6415 for 24 hours induced a slight but insignificant increase in the length of the myelin sheaths of sensory neurons (138% and 129% of the control, respectively). Treatment of healthy cells with a certain concentration of the polyionic complex TA6440 for 24 hours had no effect on the length of the myelin sheaths (111% of the control).

[0183] Cisplatin at 12 μM induced a significant decrease in the length of myelin sheaths extended by sensory neurons (69% myelin loss and p<0.0001).

[0184] Pretreatment for two hours with three different concentrations of the polyionic complexes TA6496, TA6440, and TA6415 significantly protected the length of myelin sheaths in sensory neurons injured 24 hours after cisplatin (29% myelin loss and p<0.001, 37% myelin loss and p<0.01, and 29% myelin loss and p<0.001, respectively).

[0185] In contrast, none of the individual ions at the concentrations used in TA6496, TA6440, and TA6415 showed significant protective effects on myelin length.

[0186] Manganese was also tested at a concentration range of 0.15 mg / kg to 1.2 mg / kg under the same conditions without demonstrating any significant effect on myelin length.

[0187] Example 9 - In vitro anti-inflammatory effect on human mononuclear blood cells: The following compositions were tested: TA6496 and TA64128: Ionic complex (Mg 0.2 / Li 1 / K 1.6) Li (chloride) alone Mg (chloride) alone K (chloride) alone C17-C18 polyion complex (chloride): (Mg 0.22 / Li 1 / K 1.3) C19-C20 polyion complex (chloride): (Mg 0.14 / Li 1 / K 1.6) C21-C22 polyion complex (chloride): (Mg 0.22 / Li 1 / K 1.76) Methods: Mononuclear blood cells were isolated from the blood of healthy donors by density gradient centrifugation tubes (SepMate; StemCell Technologies; 85450) through a polysucrose density gradient (Ficoll-Paque PLUS; GE Healthcare; 11778538). Mononuclear blood cells were deposited in 96-well plates (200,000 cells per well), pretreated for 2 hours with vehicle (0.179% PBS) or with the composition according to the invention or ions alone (see Table 6), and then stimulated for 6 hours at 37°C, 5% CO2 in RPMI medium containing 10% fetal bovine serum and 1% penicillin / streptomycin without (vehicle 0.002% H2O) or with lipopolysaccharide (100 ng / mL of LPS; Invivogen, tlrl-b5lps). After incubation for 6 hours, the culture supernatant was collected and stored at -20°C until cytokine analysis. The levels of cytokines (IL-1β and IL-18) were quantified in the culture supernatants by ELISA according to the manufacturer's instructions (R&D Systems; DY201, DY318-05). Table 6 summarizes the percentages of IL-1β and IL-18 cytokines measured for each condition relative to vehicle (ns: not significant).

[0188]

[0189]

[0190] Table 6.

[0191] Effects of TA6496 Composition and TA64128 Composition Stimulation of mononuclear blood cells with LPS (100 ng / mL) induced a significant increase in IL-1β and IL-18 levels in the culture supernatant. TA6496 and TA64128 significantly reduced IL-1β and IL-18 levels in a concentration-dependent manner ( Figure 7 ).

[0192] Thus, the composition of the invention exhibits significant anti-inflammatory properties on human immune cells in a pro-inflammatory context.

[0193] Effects of individual Mg, Li, and K ions Stimulation of mononuclear blood cells by LPS induced a significant increase in IL-1β and IL-18 levels in the culture supernatant. Magnesium, lithium, and potassium alone did not show a significant decrease in IL-1β and IL-18 levels ( Figure 8 ).

[0194] Effects of C17 chloride to C22 chloride based polyionic complexes: LPS stimulation of mononuclear blood cells induced a significant increase in IL-1β and IL-18 levels in the culture supernatant. The C17 to C22 ion complexes all showed a significant decrease in IL-18 levels, and the C18 ion complex at 96 mg / L lithium showed a significant decrease in IL-1β. Thus, the C1 to C22 ion complexes exhibited significant anti-inflammatory properties on human immune cells in a pro-inflammatory context ( Fig. 9 ).

[0195] Example 10 - Effect of oral administration of the polyionic complex of the invention on an in vivo inflammatory model Dextran sodium sulfate (DSS)-induced acute ulcerative colitis model was used. TNF-α and IFNγ markers were monitored.

[0196] TAR64 is an oral form of TA64.

[0197] Induction: Dextran sulfate sodium (DSS) was administered for 7 days, starting on day 1, followed by a 5-day washout (no DSS) to induce ulcerative colitis. According to standard procedures for inducing acute ulcerative colitis, an appropriate concentration of DSS (4%) was added to the drinking water available to the animals ad libitum and was renewed every 2 days.

[0198] Buprenorphine (0.5 mg / kg) was administered to relieve the discomfort of the animals.

[0199] Animals: Experiments were performed on female syngeneic mice of the Balb / c strain. Mice were housed in groups of four according to the study group. Each mouse was uniquely identified. Animals were housed in ventilated cages (type II, 16 × 19 × 35 cm, floor area = 500 cm) under the following conditions. 2 )middle: Room temperature (22±2℃).

[0200] Humidity measurement (55±10%).

[0201] Photoperiod (12:12-hour day-night cycle 7am:7pm).

[0202] Unlimited food and water available.

[0203] Mice were allowed to acclimate to their environment for 7 days before the start of the experiments. Mice were anesthetized by isoflurane inhalation if required by the procedure to be performed. Mice received analgesic treatment: buprenorphine (0.5 mg / kg) was administered to reduce the discomfort of the animals during the induction of ulcerative colitis.

[0204] Groups and Treatments: All treatments started on Day 1 and were dosed according to group and Table 7 below until the end of the study (14 days).

[0205]

[0206] Table 7.

[0207] The daily doses administered by element are listed in Table 8 below:

[0208] Table 8.

[0209] On day 14, under anesthesia, 200 μL of blood was collected from the retro-orbital region of all animals. The blood was transferred to EDTA-K3 tubes and gently mixed by inversion (by hand) 8-10 times. The blood was centrifuged at 800 g for 5 min at 2-4 °C. The plasma was separated, transferred to a labeled polypropylene tube and centrifuged at 10000 g for 5 min at 2-4 °C. The recovered plasma was separated, transferred to a labeled polypropylene tube and immediately frozen on dry ice or in a -80 °C freezer and stored until flow bead array (CBA) analysis.

[0210] CBA analysis: Plasma was subjected to CBA analysis using the CBA flank mouse kit according to the manufacturer's instructions (TNF-α; BN; 558299) (IFN; BD; 558296).

[0211] Results: DSS-induced ulcerative colitis induced a significant increase in plasma TNF-α and IFNY levels on days 7 and 14. TAR64200 treatment significantly reduced TNF-α on day 7 and IFNY on day 14. Thus, TAR64200 demonstrated its anti-inflammatory properties in the in vivo ulcerative colitis model ( Fig.10 ).

[0212] Example 11 - In vivo potassium toxicity test Purpose: In vivo toxicology study.

[0213] TAV64 is an injectable form of the TA64 composition.

[0214] The TAV64 composition consists of lithium, potassium, and magnesium, with all three elements in the form of chlorides: LiCl, KCl, and MgCl2, in ratios of 0.2-1-1.6 (TA64 composition as shown in Table 1). Regarding the toxicity of the three salts that enter the TAV64 composition, KCl has the lowest intravenous lethal dose 50 (LD50) in rats within 24 hours, LD50=142 mg / kg (Chemical Economics Handbook (CEH) Marketing Research Reports (1999). SRI International, p. 764.1000 I.), and therefore may be the most critical in the intravenous administration of TAV64.

[0215] To determine whether the combination of the 3 mineral chlorides had an effect on the 24-hour LD50 of KCl, TAV64 was injected into Wistar male mice under the following conditions: The first group of 8 male Wistar rats received a total dose of a TAV64 composition containing 127 mg / kg of KCl, ie, 90% of the LD50 of KCl, via intravenous injection over 24 hours, and all rats survived.

[0216] A second group of 8 male Wistar rats received a total dose of a TAV64 composition comprising 288 mg / kg of KCl, ie, 203% of the LD50 of KCl, via intravenous injection over 24 hours, and all rats survived.

[0217] A third group of 8 male Wistar rats were treated with lipopolysaccharide (LPS) by intraperitoneal injection to induce systemic inflammation, and received intravenous injection of a total dose of a TAV64 composition containing 240 mg / kg of KCl within 24 hours, which is 69% higher than the LD50 of KCl, and 5 of the 8 rats survived.

[0218] In conclusion, TAV64 showed high tolerability and non-toxicity in vivo in Wistar rats. The composition of 3 chlorides based on Li, Mg and K allowed administration of the LD50 of KCl alone in healthy rats up to 203% and increased the LD50 of KCl injected into sick rats by 69% in a sepsis-induced model, confirming that the composition of TAV64 positively affects the LD50 of KCl and that TAV64 has a large safety margin for its use by intravenous injection.

Claims

1. A polyion composition for oral or parenteral treatment of inflammation or prevention of inflammation, the polyion composition comprising mineral salts and / or organic salts, the mineral salts and / or organic salts comprising at least one lithium salt, at least one magnesium salt and at least one potassium salt, characterized by the following molar ratios: Lithium 1-magnesium [0.13-0.34]-potassium [1.20-2.40].

2. The multi-ion composition according to claim 1, wherein Lithium is present in a concentration between 0.01 mmol / kg and 100 mmol / kg.

3. The multi-ion composition according to claim 1 , wherein: The lithium salt is a mineral salt, for example, lithium chloride, lithium hydroxide or lithium carbonate.

4. The multi-ion composition according to claim 1, wherein: The lithium salt is an organic salt, for example, lithium citrate, lithium gluconate or lithium orotate.

5. The multi-ion composition according to claim 1, wherein The magnesium salt is a mineral salt, for example, magnesium chloride, magnesium hydroxide, magnesium sulfate, magnesium oxide, magnesium silicate or magnesium carbonate.

6. The multi-ion composition according to claim 1, wherein The magnesium salt is an organic salt, for example, magnesium bisglycinate, magnesium malate, magnesium glycerophosphate, magnesium stearate, magnesium ascorbate, magnesium taurate, magnesium citrate, magnesium gluconate or magnesium taurate.

7. The multi-ion composition according to claim 1, wherein: The potassium salt is a mineral salt, for example, potassium chloride, potassium bromide, potassium iodide, potassium phosphate, potassium carbonate, potassium hydroxide, potassium silicate.

8. The multi-ion composition according to claim 1, wherein The potassium salt is an organic salt, for example, potassium gluconate, potassium citrate, potassium malate, potassium glycerophosphate, potassium lactate, potassium pidolate or potassium aspartate.

9. The polyionic composition according to one of claims 1 to 8 for use in preventing and / or treating chronic inflammation or acute inflammation.

10. The polyion composition of claim 9, for use in a method of treating or preventing a disease selected from fibromyalgia, pain, autonomic neuropathy, polyarthritis, diabetes, obesity, osteoarthritis, atherosclerosis, ulcerative colitis, Crohn's disease or irritable bowel syndrome.

11. The multi-ion composition according to claim 9, for use in a method for treating or preventing a disease selected from chronic neuroinflammation.

12. The multi-ion composition according to claim 9 or 11, which is used in a method for treating or preventing a disease selected from multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Charcot-Marie-Tooth disease or migraine.

13. The multi-ion composition of claim 9, for use in a method for treating or preventing a disease selected from stroke, infarction, sepsis, burns, systemic inflammatory response syndrome, coronavirus disease, trauma or postoperative inflammation.

14. The polyionic composition according to one of claims 1 to 13, for use in reducing the release of TNF-α and / or IL-1β and / or IL-18.

15. The polyionic composition according to one of claims 1 to 14 for use in reducing the activation of caspases.

16. The polyionic composition according to one of claims 1 to 15, for use in a method for oral treatment or prevention of a disease selected from bipolar disorder.

17. The polyionic composition according to one of claims 1 to 16, further comprising at least one additive selected from stabilizers, emulsifiers, preservatives, antioxidants and gelling agents.

18. The polyionic composition according to one of claims 1 to 17, further comprising one or more other mineral or organic salts, such as zinc salts, manganese salts, copper salts and / or silicon salts.

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