Multi-ion complex for preventing or treating inflammation

Abstract

The present invention relates to a synergistic multi-ion complex that enhances the preventive and therapeutic responses to inflammation by modulating the immune pathway. This is a composition containing inorganic salts, characterized in that it contains at least one inorganic salt of lithium, magnesium, and potassium, respectively, in a molar ratio of lithium 1 - magnesium [0.13 to 0.34] - potassium [1.20 to 2.40]. The composition according to the present invention may also contain organic salts, or other inorganic salts, additives, and / or excipients. The composition can be used as a drug or medical device for the prevention and / or treatment of inflammation associated with acute or chronic pathological conditions accompanied by cell degeneration.

Description

Technical Field

[0001] The present invention is included in the field of therapeutic responses for preventing and / or treating inflammation and restoring cellular homeostasis.

Background Art

[0002] When cells are damaged or stressed (due to mental, metabolic, chemical causes, etc.), subsequent depletion of intracellular magnesium Mg 2+ occurs, and in the case of damage, the concentration may decrease by 40 - 60% (Non-Patent Document 1). In fact, slight fluctuations in the intracellular concentration of Mg 2+ can affect cell processes. Abnormal regulation of Mg 2+ is frequently seen in patients suffering from diabetes, neurodegenerative diseases, and metabolic syndrome, etc. (Non-Patent Document 2).

[0003] Magnesium acts as a cofactor in more than 300 enzymatic reactions and is extremely important for the metabolism of adenosine triphosphate (ATP), an energy source (Non-Patent Document 3).

[0004] Magnesium has strong antioxidant, anti-necrosis, and anti-apoptosis effects. Mg 2+ itself is generally cytoprotective, cardioprotective, and neuroprotective against a wide range of damage (Non-Patent Document 1).

[0005] Magnesium, the fourth most abundant mineral in the body, also plays an important role in neurotransmission and neuromuscular conduction. Low magnesium concentration is associated with an increase in glutamatergic neurotransmission, creating an environment prone to excitotoxicity, which can lead to oxidative stress and neuronal death. This process is involved in some neuropathies such as chronic pain (Non-Patent Document 4).

[0006] Magnesium deficiency affects the insulin resistance index (HOMA-IR). Magnesium is also essential for protein synthesis, as well as DNA and RNA synthesis (Non-Patent Document 3). Magnesium regulates the transmembrane movement of potassium and calcium.

[0007] Based on experiments in which lithium is depleted in animals, lithium is considered an essential nutrient for human body functions. Unlike other biologically active ions, the lithium concentration in the body fluids of multicellular animals is not strictly regulated. The concentration can vary widely.

[0008] Lithium has strong biological activity, but large changes in its concentration in body fluids are also tolerated. This lack of biological regulation of lithium is thought to be due to the absence of lithium-specific binding sites and selectivity filters. Rather, lithium exerts its diverse physiological and biochemical effects by competing with other elements at relatively specific macromolecular sites for other cations, especially sodium and magnesium (Non-Patent Document 5).

[0009] Lithium and magnesium have similar ionic radii (0.60 and 0.65 angstroms, respectively) and similar physicochemical properties, and can competitively bind to some magnesium-dependent enzyme sites (Non-Patent Document 6). Due to these ionic radii, lithium and magnesium can easily pass through cell membranes.

[0010] Li + Ions activate survival and recovery mechanisms such as the inhibition of inositol monophosphatase (IMPase) / inositol polyphosphate 1-phosphatase (IPPase) and glycogen synthase kinase 3 (GSK-3). Therefore, lithium produces excellent protection, anti-apoptosis, anti-anoxia, cell plasticity, and recovery responses (Non-Patent Document 7).

[0011] The lithium target of glycogen synthase kinase 3 (GSK-3β) is a serine / threonine kinase, which plays a role in regulating mammalian cell metabolism. GSK-3β regulates neurogenesis, neuronal polarization, and axon growth in the central nervous system during development. GSK-3β is constitutively active in all tissues (Non-Patent Document 8).

[0012] GSK3β is also an activator of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing-3 (NLRP3) inflammasome (Non-Patent Document 9). NLRP3 is an intracellular sensor for microbial motifs and endogenous and environmental danger signals, and the construction and activation of the NLRP3 inflammasome are induced by these motifs and signals. The construction and activation of NLRP3 occur in two steps. First, the upregulation of NLRP3 and pro-IL-1β concentrations is caused through the activation of the transcription factor nuclear factor κ-light chain enhancer (NF-κB) involved in innate immunity by a priming signal (pathogen-associated molecular pattern (PAMP) or damage-associated molecular pattern (DAMP)) via Toll-like receptor (TLR) or tumor necrosis factor receptor (TNFR). The second-stage signal (PAMPs, DAMPs, extracellular ATP-P2X7R, ROS, intracellular calcium concentration, etc.) causes the activation and construction of NLRP3. The NLRP3 inflammasome promotes the release of caspase-1-dependent inflammatory cytokines IL-1β and IL-18 and gasdermin D-mediated cell death by pyroptosis (Non-Patent Document 10). 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, and cancer (Non-Patent Document 11).

[0013] Lithium inhibits GSK-3, thereby improving the activity of BDNF (brain-derived neurotrophic factor) as shown in vitro and in vivo. The fact that lithium plays a role in increasing the expression of BDNF and BDNF plays a role in neuron survival suggests that lithium plays a role in the treatment of neurodegenerative diseases. The Wnt signaling pathway is involved in neurodegenerative diseases and cancer. Lithium inhibition of GSK-3 has been shown to specifically inhibit the Wnt signaling pathway (Non-Patent Document 5).

[0014] Lithium inhibits GSK3β, thereby inhibiting the assembly and activation of the NLRP3 inflammasome, cell death, and the release of IL-1β and IL-18 inflammatory cytokines as seen in vivo and in vitro. Lithium treatment prevents the generation of reactive oxygen species (ROS) via inhibition of GSK3β, thereby preventing the activation of the NLRP3 inflammasome in mouse models of ischemic stroke and spinal cord injury, and exerting an anti-inflammatory effect and a neuroprotective effect (Non-Patent Document 12).

[0015] NLRP3 is an intracellular sensor. When the ATP-P2X7 receptor binds, the potassium channel TWIK2 is activated, thereby inducing potassium efflux. When the intracellular potassium concentration decreases, the structure of NLRP3 changes, leading to its activation. Performing potassium treatment or inhibiting potassium efflux prevents the assembly and activation of the NLRP3 inflammasome in mouse and human cells (β) in vitro.

[0016] It has been reported in vitro and in vivo that magnesium is also involved in the activation of the NLRP3 inflammasome. In fact, magnesium deficiency causes NLRP3-induced pyroptosis by cleavage of gasdermin D. On the other hand, magnesium supplementation inhibits calcium influx-dependent gasdermin D activity and induces pyroptosis (Non-Patent Document 13).

[0017] When prophylactic treatment is performed using lithium, inhibition of GSK3β prevents the increase in taxol-induced GSK3β activity in rats, which simultaneously reduces the activities of AKT (protein kinase B) and mTOR (mechanistic target of rapamycin), thereby preventing the development of taxol-induced neuropathic pain (Non-Patent Document 14).

[0018] Lithium has anti-inflammatory properties, resulting in a decrease in both inflammatory cytokines and interleukin TNF-α. On the other hand, lithium regulates the biosynthesis of various neurotransmitters such as serotonin and glutamate and / or related receptors. In addition to having an anti-allodynic effect, lithium also has the effect of stimulating the production of brain β-endorphin, a strong analgesic MOR agonist (Non-Patent Document 15).

[0019] Lithium also contributes to calcium homeostasis and prevents the calcium-dependent activation of the apoptosis-promoting signaling pathway. This supports the cytoprotective effect of lithium (Non-Patent Document 16).

[0020] Li + The ion inhibits the GSK-3 enzyme, an important enzyme located at the center of several signaling systems. Therefore, it affects multiple downstream targets such as ion channel-type glutamate signaling, multiple transcription factors, and the Wingless (Wnt) / β-catenin integration pathway. Wnt signaling is involved in structural brain processes such as neural development, synapse formation, and neural plasticity (Non-Patent Document 17).

[0021] The bimetallic phosphate complex ATP-Mg-Li is formed by ATP and Mg at normal concentrations found in plasma or cytoplasm. 2+ Furthermore, high concentrations of ATP, corresponding to biological activity or responding to stress in the cytoplasm, organelles (such as mitochondria), and extracellular matrix, are lithium ions (Li+ ) functions as a potential reservoir for accumulating. The bimetallic phosphate complex ATP-Mg-Li has Mg 2+ and has been found to be a bioactive form of lithium that acts by simultaneous binding to phosphate or receptor cofactors or enzymes having ligands. The action of lithium ions in regulating the normal function of ATP as a ligand for cell surface purine receptors is made possible by the ATP-Mg-Li complex. There are two subtypes of the said receptor: P2X is an ion channel that mediates the influx of extracellular calcium (Ca 2+ ) ions into the cytoplasm; P2Y is coupled to the G receptor protein (GPCR), activates the second messenger pathway of inositol trisphosphate, releases calcium ions stored intracellularly, and regulates central nervous system and peripheral signal transduction (Non-Patent Document 18).

[0022] One of the main limitations of lithium treatment is its toxicity. In fact, research on the treatment of bipolar disorder has focused on identifying alternatives to lithium carbonate salts that are used to reduce toxicity and also improve the therapeutic concentration range. Therefore, one of the challenges in using lithium for the treatment of inflammation-based diseases is to broaden the currently available therapeutic concentration range.

[0023] Potassium is also an essential element. It is the most abundant cation in intracellular fluid and plays an important role in maintaining cell function, especially in excitable cells such as muscle and nerve. Potassium deficiency is associated with impaired glucose tolerance and diabetes. K + and extracellular Na + interaction increases the activation energy for the movement of water that promotes ion exchange and equilibrium of lithium in particular (Non-Patent Document 19).

[0024] Dysregulation of the activity of these three ions is involved in neurogenic inflammation, including chronic inflammation, also known as "low-noise inflammation" or "inflammaging" (inflammation associated with aging), and is involved in, for example, fibromyalgia, neuropathy, osteoarthritis, rheumatoid arthritis, 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, or pain, or is involved in an inflammatory response with acute elements involved in, for example, stroke, systemic inflammatory response syndrome, infarction, sepsis, burns, Covid, trauma, or postoperative inflammation.

[0025] These dysregulations have a strong impact individually or simultaneously. To provide a therapeutic response to inflammation and its physiological effects, such as cell degeneration, it is reasonable to take a balanced approach simultaneously.

[0026] The inventors have found that a composition containing a specific mixture of lithium, magnesium, and potassium in a specific molar ratio enables the prevention and / or treatment of inflammation particularly effectively, especially in oral, parenteral, or ocular administration.

Prior Art Documents

Non-Patent Documents

[0027]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Summary of the Invention

[0028] The present invention relates to a synergistic multi-ion complex that provides an enhanced biological response to chronic or acute inflammation. This is an oral or parenteral multi-ion composition for the treatment or prevention of inflammation, characterized by containing at least one inorganic or organic salt of lithium, magnesium, and potassium, respectively, in a molar ratio of lithium 1 - magnesium [0.13 - 0.34] - potassium [1.20 - 2.40].

[0029] The present invention will be better understood from the following description with reference to the drawings.

Brief Description of the Drawings

[0030]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Mode for Carrying Out the Invention

[0031] Preferably, the lithium concentration in the composition is 0.01 to 10 mmol / kg. Preferably, the molar ratio of magnesium to lithium is [0.14 to 0.32], preferably [0.15 to 0.30], preferably [0.18 to 0.25].

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

[0033] This multi-ion complex consists of a novel combination of "magnesium-lithium-potassium" according to a specific molar ratio algorithm. If one of the components is adjusted according to the therapeutic response to inflammation, the concentrations of the other two elements will be proportionally adjusted.

[0034] The combination according to this novel algorithm aims to simultaneously interrupt the self-perpetuating vicious cycle of inflammation promotion, especially by oral or parenteral administration, restore the cellular homeostasis of these ions that vary in many pathological conditions, promote the dynamic competition of the binding sites of these ions, obtain modified physiological responses (such as enzymatic reactions, metabolic reactions, and neurotransmission), and produce an enhanced synergistic reaction that strengthens the cellular response to damage and rapidly and continuously manages inflammation. It is also shown below that by combining these three elements, the toxicity of lithium is significantly reduced (compared to taking lithium alone), making its use in the treatment of bipolar disorder conceivable.

[0035] The action of the multi-ion complex promotes the restoration of the homeostasis changed by the inflammatory reaction, especially that of magnesium and its ionic form Mg 2+ and that of potassium and its ionic form K + which can assist in promoting the restoration of homeostasis, the restoration of optimal cellular physiological functions, the improvement of cellular metabolism, and the restoration of cell protection and anti-apoptotic activities at the neuronal cell level.

[0036] Its action promotes the balance of potassium and restores an important role in maintaining cell functions, especially in excitable cells such as neuronal cells. It also affects the interaction between potassium and sodium in the ionic form of K + and extracellular Na +It promotes as an interaction and increases the movement of a liquid that facilitates the exchange and equilibrium of ions, particularly lithium ions. Lithium has quite powerful biological activity. Lithium does not have its own system for regulating its concentration, and the concentration of lithium can vary significantly in intracellular and extracellular fluids. Therefore, it is necessary to adapt the external contribution according to the therapeutic need and perform adjustment and weighting.

[0037] The bioactive multi-ion complex binds lithium, magnesium, and potassium, and these exert the above-mentioned multiple physiological and biochemical effects. In addition to its important anti-inflammatory property, lithium has a regulatory effect on the biosynthesis of neurotransmitters such as serotonin and glutamate, which have the effect of stimulating the production of brain β-endorphin that reduces pain.

[0038] Lithium and magnesium are linked. The bimetallic phosphate complex ATP-Mg-Li has been found to be the bioactive form of lithium that acts by simultaneously binding to Mg 2+ to phosphate having a ligand or a receptor cofactor or an enzyme. This bimetallic phosphate complex is formed by ATP and Mg 2+ at normal concentrations found in plasma or cytoplasm. Also, in a high-stress situation that increases the ATP concentration in the cytoplasm and organelles such as mitochondria, the extracellular matrix functions as a potential reservoir for accumulating Li + .

[0039] Only specific combinations of molar ratios according to the present invention guarantee the absence of cytotoxicity and thus guarantee compatibility with tissue homeostasis, particularly with the higher sensitivity of neural tissue, enabling high effectiveness for the prevention and / or treatment of neurogenic inflammation. Only compositions meeting the definitions according to the present invention enable, in particular, the protective action on neurons and the protection and repair action on neurites (axons-dendrites) and myelin sheaths against the damage caused by cisplatin, a treatment considered an inducer of neurogenic inflammation. Without prejudging the mechanism behind the achievement of such a surprising effect, it can be thought that only this combination of lithium, magnesium, and potassium in the molar ratios according to the present invention enables the optimization of the respective biological activities of these elements in the complex environment of humans and animals. It includes both the inhibitory effect at the level of the vicious cycle of inflammation promotion, the restoration of the cell homeostasis of these ions when changes are seen in them, particularly magnesium and its ionic form Mg 2+ , and potassium and its ionic form K + when changes are seen in the cell homeostasis of these ions; and the dynamic competition effect at the level of ion channels and on the binding sites of these ions, an effect associated with an increase in the movement of intracellular and extracellular fluids for reducing the toxicity of lithium; including both, particularly, the activation of immune, enzymatic, metabolic, and neurotransmission reactions, the enhanced synergistic reaction that strengthens the resistance to damage and the cell response by the cytoprotective action, the restoration of the potential of neuronal excitability, and the inhibition of the apoptotic pathway activated by extracellular ATP.

[0040] Lithium is present in the composition at a concentration of 0.01 to 100 mmol / kg. The concentration of lithium in the composition is preferably 0.1 to 50 mmol / kg, preferably 0.2 to 25 mmol / kg, for example, 0.5 to 15 mmol / kg, or 1 to 6 mmol / kg. Ideally, the target cells are not actually exposed to a concentration exceeding 10 mmol / kg where the risk of toxicity outweighs the benefit for the treatment of inflammation. However, in the presence of magnesium and potassium, it is shown below that the toxicity of lithium is significantly reduced compared to the generally described toxic concentrations. When lithium is below 0.01 mmol / kg, the effect of the composition is impaired.

[0041] Preferably, lithium is present in the composition at a concentration of 0.01 to 100 mmol / kg. The concentration of lithium in the composition is preferably 0.1 to 50 mmol / kg, preferably 0.2 to 25 mmol / kg, for example, 0.5 to 15 mmol / kg, or 1 to 6 mmol / kg.

[0042] In the context of oral or parenteral use / administration, the composition of the present invention aims to simultaneously restore the cellular homeostasis of these ions, particularly Mg 2+ , as well as potassium and its ionic form K + , promote dynamic competition for binding sites, and produce an enhanced synergistic reaction. The composition of the present invention can also be administered via the ocular route, for example, in the form of eye drops.

[0043] The composition of the present invention is useful for the prevention and / or treatment of inflammation, including chronic or acute inflammation. Neurogenic inflammation, including chronic inflammation, also known as "low-grade inflammation" or "inflammaging" (inflammation associated with aging), is involved in, for example, fibromyalgia, neuropathy, osteoarthritis, rheumatoid arthritis, 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, or pain, or is involved in an inflammatory response with acute elements related to, for example, stroke, systemic inflammatory response syndrome, infarction, sepsis, burns, COVID, trauma, or postoperative inflammation.

[0044] The release of inflammatory markers such as TNF-α, IL-1β, IL-18, and / or NFκB, or the activation of enzymes such as caspases, is repeated in all these pathologies and forms the basis of the inflammatory cause. With the composition of the present invention, it is possible to inhibit the release of these markers or the activation of these enzymes from a preventive and a therapeutic perspective.

[0045] The composition of the present invention can be used orally or parenterally for the prevention and / or treatment of inflammation. Preferably, the composition of the present invention is for the treatment and / or prevention of Alzheimer's disease, Huntington's disease, Parkinson's disease, Charcot-Marie-Tooth disease by oral or parenteral administration.

[0046] Preferably, the composition of the present invention is for treating and / or preventing stroke orally or parenterally. Preferably, the composition of the present invention is for treating bipolar disorder orally or parenterally.

[0047] At least one inorganic or organic salt of lithium, magnesium, and potassium each means that these species exist in ionic form. The cations Li + , Mg 2+ , and K +is associated with one or more anions. That is, they can be from a mixture of salts and / or each can be from a different salt.

[0048] Advantageously, 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.

[0049] Preferably, for oral or parenteral use, the composition contains lithium chloride. 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 taurinate, and / or any other pharmaceutically acceptable inorganic or organic salt.

[0050] Preferably, for oral or parenteral use, the composition contains magnesium chloride, which may be in a hydrated form. 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 inorganic or organic salt. Preferably, for oral or parenteral use, the composition contains potassium chloride.

[0051] The composition of the present invention may contain other inorganic or organic salts. The composition may contain, for example, at least one silicon salt, preferably a silicate, especially for oral or parenteral use. Examples thereof include sodium silicate (Na2SiO3) which may be a hydrate, potassium orthosilicate, monomethylsilanetriol, and / or any other pharmaceutically acceptable pharmaceutical or organic salt, etc.

[0052] Silicon is a cofactor of prolyl hydroxylase involved in the stimulation of fibroblasts. Silicon reduces the permeability of capillaries and has an anti-edema effect, a sedative effect, and a refreshing effect.

[0053] Silicon plays a role in regulating the cell cycle of lymphocytes, which ultimately affects the immune and inflammatory responses. Silicon reduces the expression levels of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), kappa-activated B cell light chain nuclear factor (NF-κB), and various cytokines (TNF-α and IL-1β) at the inflammatory site.

[0054] Adapt the ratio of silicon to other salts according to the inflammation underlying the assumed pathological condition. The composition may contain, for example, at least one manganese salt, especially for oral or parenteral use. Examples thereof include 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 inorganic or organic magnesium salt, etc.

[0055] Manganese is involved in tissue physiology and biology through its role as a cofactor in many enzymatic processes. Manganese plays an important role in regulating glucose tolerance by acting synergistically with magnesium, thereby particularly enabling a reduction in metabolic stress in the nerves and epidermis.

[0056] The composition may contain at least one inorganic 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, etc. These play important roles in many essential enzymatic processes, such as DNA and protein synthesis, wound healing, insulin metabolism, nervous system development, and proper functioning.

[0057] The composition may contain at least one inorganic or organic salt of copper, such as copper carbonate, copper citrate, copper malate, copper glycerophosphate, copper lactate, copper pidolate, copper aspartate, copper gluconate, etc. Copper is a component of several enzymes involved in the metabolism of carbohydrates, lipids, and iron. Copper has antioxidant effects.

[0058] An oral composition, or an orally administered composition, is a composition intended for ingestion. Preferably, the composition is in the form of tablets, pills, capsules, liquids, syrups, lyophilized agents. That is, the composition may contain excipients necessary for the desired formulation.

[0059] A parenteral composition, or a parenterally administered composition, means a composition that can be injected adjacent to the digestive tract, intravenously (injection via a subcutaneous injection needle, catheter, or cath port), subcutaneously, intramuscularly, or intraperitoneally. In the case of a parenteral composition, it is liquid.

[0060] In the case of a parenteral composition, the inorganic or organic salts of the composition of the present invention are dissolved in water. That is, the main solvent of the composition is preferably water. Water corresponds to at least 50% by weight, preferably at least 75% by weight, at least 80%, and preferably at least 90% or 95% of the composition. The composition may contain other liquid components that those skilled in the art consider useful, particularly to promote stability and biocompatibility (pH, plasma osmotic pressure, natremia).

[0061] The composition of the present invention may also contain other molecules, additives, or excipients, such as preservatives, antibiotics, stabilizers, thickeners, antioxidants, etc. These additives are useful for the preservation of the composition of the present invention.

[0062] The composition may contain one or more vitamins, such as vitamin E. Vitamin E is a powerful antioxidant and regulates immune function. The present invention relates to the use of the composition as a pharmaceutical for human or veterinary use.

[0063] Neurogenic inflammation, including chronic inflammation, also known as "low-grade inflammation" or "inflammaging" (inflammation associated with aging), is involved in, for example, fibromyalgia, neuropathy, osteoarthritis, rheumatoid arthritis, 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, or pain, or is involved in an inflammatory reaction with an acute component related to, for example, stroke, systemic inflammatory response syndrome, infarction, sepsis, burns, COVID, trauma, or postoperative inflammation.

[0064] Combinations of algorithms were tested in vitro and in vivo in each of the above models known as major inflammatory diseases. Example 1 - Preparation of solutions of various concentrations for in vitro testing A solution containing lithium chloride, magnesium chloride, and potassium chloride was prepared with reverse osmosis water.

[0065] They are shown in Table 1 below.

[0066]

Table 1

[0067] Example 2 - Effect of the composition of the present invention on the Alzheimer's disease model of glutamate injury in cortical neurons Rat cortical neurons are cultured according to the procedure described by Singer (1999).

[0068] Female rats on the 15th day of pregnancy were sacrificed by cervical dislocation (Wistar rats; January Lab), and the fetuses were removed from the uterus. The embryonic cortex was collected and placed in ice-cold Leibovitz 15 (L15; PanBiotech, product number: P04-27055, batch: 3870221) medium containing 2% penicillin-streptomycin (PS; PanBiotech, product number: P06-07100, batch: 5870421) and 1% bovine serum albumin (BSA; PanBiotech, product number: P06-1391100, lot: H210207). The cortex was separated by trypsin treatment at 37°C for 20 minutes (trypsin EDTA 1×; PanBiotech, product number: P10-023100, batch: 3030221). The reaction was stopped by the addition of Dulbecco's modified Eagle's medium (DMEM; PanBiotech, product number: P04-03600, batch: 1130721) containing DNase I grade II (0.1 mg / mL; PanBiotech, product number: P60-37780100, batch: H181015) and 10% fetal bovine serum (FCS; Invitrogen, product number: 10270106, lot: 2275115). Then, the cells were mechanically separated by passing a 10 mL pipette through them three times. Subsequently, the cells were centrifuged at 515×g at +4°C for 10 minutes. The supernatant was discarded, and the cell pellet was resuspended in a synthetic medium consisting of Neurobasal (Invitrogen, product number: 11570556, batch: 2327396) containing 2% B27 (Invitrogen, product number: 11530536, batch: 2337321), L-glutamine (2 mM; PanBiotech, product number: P04-80100, batch: 2770620), 2% PS, and 10 ng / mL BDNF (Peprotech, product number: 450-02, batch: 102061). Next, viable cells were counted with a Neubauer cytometer using the trypan blue exclusion test.Cells were seeded at a density of 30,000 cells / well in 96-well plates (pre-coated with poly-D-lysine; Greiner, product number: 655940, lot: E200933A) and grown at 37°C in a humidified air (95%) / CO2 (5%) atmosphere. The central half was exchanged with cooled medium every two days.

[0069] The glutamic acid solution (Sigma, product number: G1501, batch: SLBD7305V) was made to 10 mM in the medium. The control solution was prepared under the same conditions. After 11 days of culture, primary cortical neurons were pretreated with the composition of Example 1 or the reference compound (BDNF, 50 ng / mL) for 1 hour, and then poisoned by incubating with glutamic acid at a concentration of 40 μM for 20 minutes.

[0070] The following conditions were tested. · Control medium · Control + Glutamic acid (40 μM, 20 minutes) · Composition TA6496 + Glutamic acid (40 μM, 20 minutes) · Composition TA6440 + Glutamic acid (40 μM, 20 minutes) · Composition TA6415 + Glutamic acid (40 μM, 20 minutes) · BDNF (50 ng / mL) + Glutamic acid (40 μM, 20 minutes) The culture was performed using 6 wells for each condition.

[0071] Main evaluation item: Measurement of the total number of cortical neurons. After 24 hours of incubation, the cells were fixed with 4% paraformaldehyde solution (AlphaAesar; product number: J19943, batch: 206909) at room temperature for 15 minutes. Next, the cells were permeabilized with phosphate-buffered saline (PBS; PanBiotech; product number: P04-36500, batch: 3410921) containing 0.1% Triton X-100 (Sigma; product number: T9284, batch: 118K01602) at room temperature for 30 minutes. Nonspecific sites were saturated with phosphate-buffered saline containing 3% BSA and 3% FCS for 1 hour. Then, the cells were incubated with chicken polyclonal anti-MAP-2 antibody (1 / 2000, Abcam; product number: ab5392, batch: GR3405939-2) in saturating buffer at 4°C for 12 hours.

[0072] These antibodies were visualized with Alexa Fluor 568 goat anti-chicken IgG (1 / 400, Molecular Probe, product number: A110041, lot: 1963088) in saturating buffer at room temperature for 1 hour. Cell nuclei were labeled with a fluorescent marker (Hoechst solution, Sigma; product number: B1155, lot: 046M4048V) in the same solution.

[0073] For each condition, 20 images per well were taken at 20× magnification using an InCell Analyzer® 2200 (GE Healthcare). Images of each cropwell were taken under the same conditions. Automatic analysis of the number of cortical neurons was performed using Developer software (GE Healthcare). A total of 6 data were provided for each experimental condition.

[0074] Statistics: Data were represented as mean ± standard error (6 data per condition, 1 culture). Overall analysis of the data was performed using one-way analysis of variance (ANOVA), followed by Dunnett's test. The significance level was set at p < 0.05.

[0075] The results are shown in Figure 1. Composition TA6415 significantly protected cortical neurons from apoptosis after glutamate injury, and the level slightly exceeded that of the BDNF control. Example 3 - Anti-inflammatory effect of the composition of the present invention on the release of TNF-α and IL-1β in sensory neurons Sensory neurons are part of the peripheral nervous system. Their cell bodies are located in the dorsal root ganglia (D.R.G.) along the spinal cord and extend to the distal ends of the extremities. One of the characteristics of sensory neurons is the ability to regenerate the extended part after nerve transection. This property is related to the presence of Schwann cells, trophic cells, and myelinating cells in the peripheral nervous system that release specific growth factors for axonal growth.

[0076] 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 miniaturized model in 96-well plates enables the analysis of the effects of various molecules on the development of sensory neurons and the myelination of axons by Schwann cells.

[0077] The purpose of this study is to examine the effects of six different concentrations of TA64 compositions on the release of TNF-α and IL-1β by sensory neurons after cisplatin injury. Protocol for co-culture of sensory neurons and Schwann cells: Female rats on the 15th day of pregnancy were sacrificed by cervical dislocation (Wistar rats; January Lab), and the fetuses were removed from the uterus. Embryonic DRGs were collected and placed in ice-cold Leibovitz 15 (L15; PanBiotech, product number: P04-27055, batch: 5331021) medium containing 2% penicillin-streptomycin (PS; PanBiotech, product number: P06-07100, batch: 9300621) and 1% bovine serum albumin (BSA; PanBiotech, product number: P06-1391100, lot: H210207). The DRGs were separated by trypsin treatment at 37°C for 20 minutes (trypsin EDTA 1×; PanBiotech, product number: P10-023100, batch: 8600621). The reaction was stopped by the addition of Dulbecco's modified Eagle's medium (DMEM; PanBiotech, product number: P04-03600, batch: 1130721) containing DNase I grade II (0.1 mg / mL; PanBiotech, product number: P60-37780100, batch: H181015) and 10% fetal bovine serum (FCS; Invitrogen, product number: 10270106, lot: 2319479). Then, the cells were mechanically separated by passing a 10 mL pipette through them three times. Next, the cells were centrifuged at 180×g for 10 minutes at +4°C on top of a layer of BSA (3.5%) in L15 medium. The supernatant was discarded, and the cell pellet was resuspended in a synthetic medium consisting of Neurobasal (Invitrogen, product number: 21103049, batch: 2348949) containing 2% B27 (Invitrogen, product number: 17504-044, batch: 2336991), L-glutamine (2 mM; PanBiotech, product number: P04-80100, batch: 2770620), 2% PS, and 50 ng / mL NGF (Sigma, product number: N1408, lot: SLCG2596). Subsequently, viable cells were counted using a Neubauer hemocytometer with the trypan blue exclusion test.Cells were seeded at a density of 12,000 cells / well in a 96-well plate (pre-coated with poly-D-lysine; Greiner, product number: 655940, lot: E200933A) and grown at 37°C in a humidified air (95%) / CO2 (5%) atmosphere. The central half was exchanged with fresh medium every two days. The cells were maintained for 7 days to grow Schwann cells and sensory neurons. On the 8th day, 50 μg / mL of ascorbic acid (AA; Sigma, product number: 092902, batch: 05316HJ-438) was added to the medium to initiate the differentiation of basal Schwann cells into myelinating Schwann cells.

[0078] After culturing for 5 days with AA under this condition, myelin sheaths are detected with an anti-MAG (myelin antigen glycoprotein) antibody against an early marker of myelin. Cisplatin preparation, exposure, and drug treatment: Treatment protocol Cis-diammineplatinum(II) dichloride (cisplatin; Sigma, product number: P4394, lot: MKCN8054) was made to 24 mM in DMSO (stock solution). A control environment was prepared under the same conditions. After culturing for 12 days with AA, primary sensory neurons were pretreated with the composition of Example 1, or a reference compound (NGF, 50 ng / ML) for 2 hours, and then poisoned with cisplatin. A cisplatin preparation diluted to a final concentration of 12 μM in the control medium was used for the neurons, and incubated for 24 hours.

[0079] The following conditions were tested. · Control medium · Control + cisplatin (12 μM, 24 hours) · Composition TA6426 + cisplatin (12 μM, 24 hours) · Composition TA6422 + cisplatin (12 μM, 24 hours) · Composition TA6419 + cisplatin (12 μM, 24 hours) · Composition TA6415 + cisplatin (12 μM, 24 hours) · Composition TA6412 + Cisplatin (12 μM, 24 hours) · Composition TA6409 + Cisplatin (12 μM, 24 hours) · Control + NGF (50 ng / mL) + Cisplatin (12 μM, 24 hours) Cultures were performed using six wells for each condition. After incubating for 6 hours in the presence of cisplatin, the conditioned medium was collected and stored at -80 °C. Measurement of TNF-α in the thawed medium was performed by flow cytometry according to the instructions (BD Bioscience, product number 561516, batch: 0171683).

[0080] The results are shown in Figure 2. After treatment with cisplatin for 6 hours, the conditioned medium was removed and stored at -80 °C. Measurement of rat IL-1β in the thawed samples was performed by ELISA according to the instructions (Bio-techne, product number DY501, lot P296813).

[0081] The results are shown in Figure 3. Data were represented as mean ± standard error (6 data per condition, 1 culture). Overall analysis of the data was performed using one-way analysis of variance (ANOVA), followed by Dunnett's test. The significance level was set at p < 0.05.

[0082] Results: Cisplatin increases the release of TNF-α and IL-1β at 12 μM. All TA64 compositions tested significantly inhibit the release of TNF-α at control levels. Compositions of TA6426, TA6419, and TA6415 significantly inhibit the release of IL-1β, although this trend is generally seen in all compositions tested.

[0083] Example 4 - Anti-apoptotic effect of the compositions of the present invention by analysis of caspase activation in sensory neurons (Alzheimer's disease model) The co-culture of sensory neurons and Schwann cells, as well as the protocol for cisplatin preparation and exposure, are the same as those detailed in Example 3.

[0084] The following conditions were tested. · Control medium · Control + cisplatin (12 μM, 24 hours) · Control + composition TA6426 + cisplatin (12 μM, 24 hours) · Control + composition TA6422 + cisplatin (12 μM, 24 hours) · Control + composition TA6419 + cisplatin (12 μM, 24 hours) · Control + composition TA6415 + cisplatin (12 μM, 24 hours) · Control + composition TA6412 + cisplatin (12 μM, 24 hours) · Control + composition TA6409 + cisplatin (12 μM, 24 hours) Cultivation was performed using six wells for each condition.

[0085] After 24-hour treatment in the presence of cisplatin, cells were fixed at -20°C for 5 minutes with a solution of 5% acetic acid (Sigma; product number: 33209; lot: SHBJ9236) and 95% ethanol (VWR; product number: 83813.360; lot: 20G074013), and control conditions were set up in the same procedure. Next, the cells were permeabilized and non-specific sites were saturated with phosphate-buffered saline (PBS; PanBiotech; product number: P04-36500, batch: 5371021) containing 0.1% newt X-100 (Sigma; product number: T9284, batch: 118K01602) at room temperature for 30 minutes. Non-specific sites were saturated with a PBS solution containing 3% BSA and 3% FCS for 1 hour. Then, the cells were incubated with mouse anti-β-tubulin monoclonal antibody (1 / 2000, Sigma; product number: T8660, batch: 034M4790V) and rabbit cleaved anti-caspase-3 polyclonal antibody (1 / 500, Abcam, product number: ab13847, batch: GR3286166-1) in the saturation buffer at +4°C for 12 hours. The antibodies were visualized at room temperature for 1 hour using goat anti-mouse IgG Alexa Fluor 647 (1 / 400; Molecular Probe; product number: A212235; lot: 1922319) and goat anti-rabbit IgG Alexa Fluor 568 (1 / 400; Molecular Probe; product number: A11011; batch: 2192277) in the saturation buffer. The cell nuclei were labeled with a fluorescent marker (Hoechst solution, Sigma; product number: B1155, batch: 046M4048V) under the same conditions.

[0086] For each condition, 20 images per well were taken at 20x magnification using an InCell Analyzer® 2200 (GE Healthcare). Images of each crop well were taken under the same conditions. The number of sensory neurons and caspase-3 concentration were automatically evaluated using Developer software (GE Healthcare). A total of 6 data were provided for each experimental condition.

[0087] Data are presented as mean ± standard error (6 data per condition, 1 culture). Overall analysis of the data was performed using one-way analysis of variance (ANOVA), followed by Dunnett's test. The significance level was set at p < 0.05.

[0088] The results are shown in Figure 4. Compositions TA6426, TA6422, TA6419, and TA6415 significantly reduce caspase-3 release in primary Schwann cells after cisplatin injury.

[0089] Example 5 - Effect of the composition of the present invention in an in vitro model of Parkinson's disease with MPP+ (1-methyl-4-phenylpyridinium+) lesions of midbrain neurons Dopaminergic neurons of rats were cultured as described by Schinelli et al. (1988). Briefly, female rats on the 15th day of pregnancy were sacrificed by cervical dislocation (Wistar rats; January Lab), and the fetuses were removed from the uterus. The embryonic midbrain was collected and placed in ice-cold Leibovitz 15 (L15; PanBiotech, product number: P04-27055, batch: 3870221) medium containing 2% penicillin-streptomycin (PS; PanBiotech, product number: P06-07100, batch: 1201220) and 1% bovine serum albumin (BSA; PanBiotech, product number: P06-1391100, lot: H200403). Only the ventral part of the midbrain flexure was used for cell preparation, as this region of the developing brain is rich in dopaminergic neurons. The midbrain was separated by trypsin treatment at 37 °C for 20 minutes (trypsin EDTA 1×; PanBiotech, product number: P10-023100, batch: 9821220). The reaction was stopped by the addition of Dulbecco's modified Eagle's medium (DMEM; PanBiotech, product number: P04-03600, batch: 3090221) containing DNase I grade II (0.1 mg / mL; PanBiotech, product number: P60-37780100, batch: H181015) and 10% fetal bovine serum (FCS; Invitrogen, product number: 10270106, lot: 2275115). Subsequently, the cells were mechanically separated by passing a 10 mL pipette through them three times. Next, the cells were centrifuged at 180 × g for 10 minutes at +4 °C on top of a layer of BSA (3.5%) in L15 medium.The supernatant was discarded, and the cell pellet was resuspended in a synthetic medium consisting of Neurobasal (Gibco, product number: 21103049, batch: 2242402) containing 2% B27 (Gibco, product number: 17504044, batch: 2295367), L-glutamine (2 mM; PanBiotech, product number: P04-80100, batch: 3301019), 2% PS, 10 ng / mL BDNF (Peprotech, product number: 450-02, batch: 071961), and 10 ng / mL glial cell line-derived neurotrophic factor (GDNF; PanBiotech, product number: 450-10, lot: 0606B64). Subsequently, viable cells were counted with a Neubauer hemocytometer using the trypan blue exclusion test. The cells were seeded into 96-well plates (pre-coated with poly-D-lysine; Greiner, product number: 655940, batch: E20093UL) at a density of 40,000 cells / well and grown at 37 °C in a humidified atmosphere (95%) / CO2 (5%). The central half was replaced with fresh medium every two days. Under these conditions, astrocytes were present in the culture after 5 days of culture, releasing growth factors to enable neuronal differentiation. Under these conditions, 2-5% of the neurons were dopaminergic neurons.

[0090] MPP+ (1-methyl-4-phenylpyridinium) (Sigma, product number: D048, lot 0000047439) was made up to 10 mM (stock solution) in the synthetic medium. A control environment was prepared under the same conditions. After 7 days of culture, primary midbrain neurons were pretreated with the test compound or reference compound (BDNF, 50 ng / mL) for 1 hour and then poisoned by incubation with MPP+ at a final concentration of 8 μM for 48 hours, inducing approximately 45% ± 5% neuronal death.

[0091] The following conditions were tested. · Control medium · Control + MPP+ (16 μM, 48 hours) · TA6496 + MPP+ (16 μM, 48 hours) · TA6440 + MPP+ (16 μM, 48 hours) ·TA6415 + MPP+(16 μM, 48 hours) ·BDNF(50 ng / mL) + MPP+(16 μM, 48 hours) After 48 hours of intoxication in the presence or absence of the test compound, the cells were fixed with a 4% paraformaldehyde solution (Alpha Aesar, product number J19943, batch: 202585) for 20 minutes at room temperature, and fixation was also performed for the control conditions following the same procedure. Next, the cells were permeabilized, and non-specific sites were saturated with phosphate-buffered saline (PBS; PanBiotech; product number P04-36500, batch: 6300421) containing 0.1% saponin (Sigma; product number: S7900, lot: BCBL8667V) and 1% FCS for 15 minutes at room temperature. The cells were incubated with an anti-tyrosine hydroxylase mouse monoclonal antibody (TH, 1 / 10,000, Sigma; product number: T1299, lot: 014M4835) in PBS solution at 4°C for 12 hours. The antibody against TH labeled dopaminergic neurons.

[0092] Alexa Fluor 488 goat anti-mouse IgG (1 / 400, Molecular Probe, product number: A11001, batch: 2247988) in PBS containing 1% FCS and 0.1% saponin was added, and visualization of the staining was performed for 1 hour at room temperature. The cell nuclei were labeled with a fluorescent marker (Hoechst, Sigma; product number: B1155, batch: 046M4048V) in the same solution.

[0093] For each condition, 20 images per well were taken at 20x magnification using an InCell Analyzer® 2200 (GE Healthcare). Images of each cropped well were taken under the same conditions. Analysis of the cell bodies of positive TH neurons was performed using Developer software (GE Healthcare). A total of 6 data were provided for each experimental condition.

[0094] Data were expressed as mean ± standard error (6 data per condition, 1 culture). Overall analysis of the data was performed using one-way analysis of variance (ANOVA), followed by Dunnett's test. The significance level was set at p < 0.05.

[0095] The results are shown in Figure 5. The TA6415 composition significantly protects dopaminergic neurons from apoptosis after MPP+ injury.

[0096] Example 6 - Effect of the composition of the present invention in a stroke model using oxygen deprivation and glucose Here, we study the survival of neurons in primary cortical cultures of rats injured by oxygen / glucose deprivation (OGD).

[0097] Stroke induces brain damage through the combined action of several mechanisms, including excitotoxicity, free radical generation, inflammation, and apoptosis. Excitotoxicity results from the excessive release of the excitatory neurotransmitter glutamate from dead or dying neurons, accompanied by inappropriate stimulation of the N-methyl-D-aspartic acid (NMDA-R) receptor. The initial attack associated with a large influx of Ca 2+ Neighboring neurons that survive the initial attack associated with a large influx of Ca cannot survive such massive activation when the supply of glucose and oxygen is limited, and are killed by high levels of glutamate (Verleye et al., 2016).

[0098] The aim of this study was to examine the protective effects of three different concentrations of TA64 compounds on neuron survival in primary cortical cultures of rats injured by oxygen / glucose deprivation (OGD), an in vitro stroke model.

[0099] Cortical cells were cultured as described in Example 2. After 11 days of culture, primary cortical neurons were pretreated with TA64 or a reference compound (BDNF, 50 ng / mL) for 1 hour. Then, the medium was removed and fresh glucose-free medium was added. This medium consisted of glucose-free DMEM (PanBiotech, product number: P04-01549, batch: 5471021) containing 2% B27, 0.2 mM L-glutamine, 1% PS solution, and 10 ng / mL BDNF. The cells were transferred to an anaerobic incubator at 37 °C with 95% N2 and 5% CO2. After 2 hours, 25 mM D-glucose (Sigma, product number: G8644, batch: RNBJ8362) with or without compound TA64 was added to the medium, and the cells were transferred to a normal incubator at 37 °C with 95% air / 5% CO2 for 24 hours.

[0100] The following conditions were tested. · 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, the cells were fixed at -20°C for 5 minutes using a solution of 5% acetic acid (Sigma, product number: 33209, batch: SHBJ9236) and 95% ethanol (VWR, product number: 83813.360, batch: 20G074013), and fixation was also carried out for the control conditions following the same procedure. Next, the cells were permeabilized, and non-specific sites were saturated at room temperature for 15 minutes with phosphate-buffered saline (PBS; PanBiotech; product number: P04-36500, batch: 5371021) containing 0.1% saponin (Sigma; product number: S7900, lot: BCBL8667V) and 1% FCS. The cells were incubated with an anti-MAP-2 mouse monoclonal antibody (1 / 5,000, Sigma; product number: M4403, lot: 035M4780V) in PBS solution at 4°C for 12 hours. Dopaminergic neurons were labeled with an anti-TH antibody.

[0101] Goat anti-mouse IgG Alexa Fluor 568 (1 / 400, Molecular Probe, product number: A11004, batch: 2332536) in PBS containing 1% FCS and 0.1% saponin was added, and visualization of the staining was carried out at room temperature for 1 hour. Cell nuclei were labeled with a fluorescent marker (Hoechst, Sigma; product number: B1155, batch: 046M4048V) in the same solution.

[0102] For each condition, 20 images per well were taken at a magnification of 20× using an InCell Analyzer® 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 Developer software (GE Healthcare). A total of 6 data were provided for each experimental condition.

[0103] The data were represented as mean ± standard error (6 data per condition, 1 culture). Global analysis of the data was performed using one-way analysis of variance (ANOVA), followed by Dunnett's test. The significance level was set at p < 0.05.

[0104] The results are shown in Fig. 6. The TA6415 composition significantly protects primary rat cortical neurons from apoptosis better than the 50 ng / mL BDNF control.

[0105] Conclusion The markers evaluated in Examples 2, 3, and 4 are representative of Alzheimer's disease. The markers evaluated in Examples 3, 4, and 5 are representative of Parkinson's disease. The markers evaluated in Examples 3, 4, and 6 are also involved in stroke.

[0106] Thus, the composition according to the present invention has been shown to have a preventive and therapeutic effect on these models in vitro, suggesting a significant effect in vivo. The release levels of these markers, particularly TNF-α, IL-1β, and caspase-3, are typical of inflammatory pathologies in general. Therefore, it is reasonable to consider that the composition of the present invention has a positive effect on all inflammatory pathologies.

[0107] In addition to the anti-inflammatory activity of the composition of the present invention, the intrinsic toxicity of lithium, magnesium, and potassium ions individually or in combination was also evaluated. This is repeated in the following examples.

[0108] Example 7 - Effect of the multi-ion complex on the neurite length of sensory neurons Treatment of healthy cells with the multi-ion compositions TA6496, TA6440, and TA6415 for 24 hours induces a slight increase in the neurite length of sensory neurons, but the increase is not significant (31%, 34%, and 54% respectively compared to the control).

[0109] 12 μM cisplatin induces a significant decrease in the neurite length of sensory neurons (65% loss of neurites (p < 0.01)). Treatment with the multi-ion compositions TA6440 and TA6415 according to the present invention can effectively protect the neurite length of sensory neurons from cisplatin injury for 24 hours (neurite loss was 19% (p<0.05) and 11% (p<0.05), respectively).

[0110] However, treatment with the multi-ion composition TA6496 can partially protect the neurite length of sensory neurons from cisplatin injury for 24 hours, but the protection is not significant (neurite loss was 28%).

[0111] In parallel, the effects of individual ions or combinations of two ions on the survival and neurite length of sensory neurons were also evaluated under the same conditions. All solutions were prepared from the chlorides of the test ions.

[0112] The results are shown in Table 2.

[0113]

Table 2-1

[0114]

Table 2-2

[0115] Rows 11 to 14 of the table show that lithium is highly toxic at 40 mg / kg when combined with only one of magnesium or potassium ions, but lithium is no longer toxic at the same concentration in the presence of the other two ions and in the variant of the algorithm in row 14. Therefore, the combination of the three ions lithium, magnesium, and potassium actually has a synergistic effect.

[0116] The ability of individual ions to protect the survival and neurite length of sensory neurons was also tested in parallel. The results are shown in Table 3.

[0117]

Table 3

[0118] Combinations of multi-ions significantly protect sensory neurons from cisplatin-induced apoptosis, but none of the individual ions significantly protect sensory neurons when tested at the same concentration. The same is true for the combination of potassium and magnesium, which does not significantly improve the survival of sensory neurons. This difference is probably because the combination of multi-ions has lower toxicity compared to individual ions, and thus the protective effect is dominant over toxicity.

[0119] Manganese was also tested in the concentration range of 0.15 mg / kg to 1.2 mg / kg under the same conditions, but it did not show a significant effect on neuron survival or neurite length. Other combinations of lithium-magnesium-potassium according to the present invention were also tested. They are shown in Table 4.

[0120]

Table 4

[0121] Compositions C12 - C16 are outside the range of molar ratios according to the present invention, and the other compositions, C1 - C11, represent the algorithm of the present invention. The effects of ion combinations on the survival of sensory neurons and neurite length were evaluated under the same conditions as above. All solutions were prepared from chlorides of the test ions.

[0122] The results are shown in Table 5.

[0123]

Table 5

[0124] The above results indicate that the composition of the present invention significantly reduces the apoptosis of sensory neurons induced by cisplatin in all beaches as claimed. When potassium and / or magnesium are below and / or above the range of molar ratios according to the present invention (C12 - C16), the combination no longer functions significantly.

[0125] Example 8 - Neuroprotective effects of three different concentrations of multi-ion complexes on primary myelinated rat sensory neurons, neurites, and myelin sheaths after cisplatin injury Example 8a - Effect of multi-ion complex on the length of myelin sheaths of sensory neurons (treatment protocol) Treatment of healthy cells with three different concentrations of multi-ion complexes TA6496, TA6440, and TA6415 for 24 hours did not regulate the length of the myelin sheaths of sensory neurons (89%, 97%, and 89% respectively compared to the control).

[0126] 12 μM cisplatin induces a significant decrease in the length of the myelin sheaths of sensory neurons (myelin loss is 78%, p < 0.001). Treatment with two different concentrations of multi-ion complexes TA6440 and TA6415 can significantly protect the length of the myelin sheaths of sensory neurons from cisplatin injury for 24 hours (myelin loss is 46% (p < 0.05) and 34% (p < 0.01) respectively).

[0127] However, treatment with the TA6496 multi-ion complex induces a slight increase in the length of the myelin sheaths after 24 hours of cisplatin injury, but the increase is not significant (myelin loss is 66%).

[0128] Example 8b - Neuroprotective effects of three different concentrations of multi-ion complexes on primary myelinated rat sensory neurons, neurites, and myelin sheaths after cisplatin injury: Protection protocol The main evaluation items were the measurement of the survival of sensory neurons after cisplatin intoxication and in the absence of intoxication, as well as the evaluation of the neurite length and myelin sheath length of sensory neurons.

[0129] Cis-diammineplatinum(II) dichloride (cisplatin; Sigma, product number: P4394, lot: MKCL0026) was made into 10 mg / mL in the medium (stock solution). A control environment was prepared under the same conditions. After 12 days of culture using AA, primary sensory neurons were pretreated with multi-ion complexes at three concentrations for 2 hours, and then intoxication with cisplatin was carried out. A cisplatin preparation diluted to a final concentration of 12 μM in the control medium was used for the neurons, and incubation was carried out for 24 hours.

[0130] The concentrations of the multi-ion complexes were as follows: 1. TA6496: Li 10.37 mmol / kg, Mg 2.27 mmol / kg, K 16.88 mmol / kg 2. TA6440: Li 5.76 mmol / kg, Mg 1.26 mmol / kg, K 9.38 mmol / kg 3. TA6415: Li 1.73 mmol / kg, Mg 0.38 mmol / kg, K 2.81 mmol / kg The following conditions were evaluated: · Control medium · Control + cisplatin (12 μM, 24 hours) · Three different concentrations of multi-ion complexes TA6496, TA6440, or TA6415 + cisplatin (12 μM, 24 hours) · Three different concentrations of multi-ion complexes TA6496, TA6440, or TA6415.

[0131] This study was carried out using six wells for each condition and was performed twice for result validation. Analysis was carried out including the combined data of the results of the two studies. After 24-hour treatment with or without cisplatin, the cells were fixed at -20°C for 5 minutes in a solution of acetic acid / ethanol (5 / 95), and the control conditions were fixed following the same procedure. Next, the cells were permeabilized, and non-specific sites were saturated with phosphate-buffered saline (PBS; PanBiotech; product number: P04-36500, batch: 2620121) containing 0.1% saponin (Sigma; product number: S7900, batch: BCBL8667V) and 1% FCS at room temperature for 15 minutes. Next, the cells were incubated with polyclonal anti-rabbit neurofilament antibody (NF; 1 / 500, Sigma; product number: N0142, batch: 083M4833) and anti-MAG mouse monoclonal antibody (1 / 400, Sigma; product number: MAB1567, batch: 3227322) in a PBS solution containing 1% FCS and 0.1% saponin at 4°C for 12 hours.

[0132] These antibodies were visualized at room temperature for 1 hour using goat anti-mouse Alexa Fluor 488 (1 / 400, Molecular Probe, product number: A11001, 2247988) and goat anti-rabbit IgG Alexa Fluor 633 (1 / 400, Molecular Probe, product number: A21070, lot: 1700326) in PBS containing 1% FCS and 0.1% saponin. The cell nuclei were labeled with a fluorescent marker (Hoechst solution, Sigma; product number: H-33258, lot: 046M4048V) in the same solution.

[0133] For each condition, 20 images per well were taken at 20x magnification using an InCell Analyzer® 2200 (GE Healthcare). Images of each cropwell were taken under the same conditions. Analysis was performed using Developer software (GE Healthcare). For each of the two studies above, a total of 6 data were provided per experimental condition.

[0134] Data are presented as mean ± e.g. (6 data per condition per crop for 2 crops). Overall analysis of the data was performed using one-way analysis of variance (ANOVA), followed by Dunnett's test. The significance level was set at p < 0.05.

[0135] Effect of multi-ion complex on the survival of sensory neurons Treatment of healthy cells with three different concentrations of multi-ion complexes TA6496, TA6440, and TA6415 for 24 hours did not regulate cell survival (91%, 89%, and 95% respectively compared to the control).

[0136] 12 μM cisplatin induces a significant decrease in neuron survival (64% cell death, p < 0.0001). Pretreatment with three different concentrations of multi-ion complexes TA6496, TA6440, and TA6415, 2 hours before cisplatin injury and during 24 hours of cisplatin injury, partially and significantly protected neurons from cell death (35% (p < 0.0001), 30% (p < 0.0001), and 30% (p < 0.0001) cell death respectively).

[0137] Effect of multi-ion complex on the neurite length of sensory neurons Treatment with the multi-ion complex at concentration TA6496 for 24 hours did not affect the neurite length of sensory neurons (111% compared to the control).

[0138] Treatment with the multi-ion complex at concentration TA6440 for 24 hours induced a slight increase in neurite length, but the increase was not significant (129% of the control). Treatment with the multi-ion complex at concentration TA6415 for 24 hours significantly increased the neurite length of sensory neurons (140% (p < 0.01) compared to the control).

[0139] 12 μM cisplatin induces a significant decrease in the neurite length of sensory neurons (neurite loss is 51% (p < 0.0001)). Pretreatment for 2 hours with three different concentrations of the multi-ion complexes TA6496, TA6440, and TA6415 can effectively maintain the neurite length of sensory neurons damaged by cisplatin over 24 hours (83% (p < 0.01), 100% (p < 0.0001), and 102% (p < 0.0001) relative to the control, respectively).

[0140] Effect of multi-ion complexes on the length of the myelin sheath of sensory neurons Treatment of healthy cells with three different concentrations of the multi-ion complexes TA6496 and TA6415 for 24 hours induces a slight increase in the length of the myelin sheath of sensory neurons, but the increase is not significant (138% and 129% relative to the control, respectively). Treatment of healthy cells with the multi-ion complex at concentration TA6440 for 24 hours also has no effect on the length of the myelin sheath (111% relative to the control).

[0141] 12 μM cisplatin induces a significant decrease in the length of the myelin sheath of the extended part of sensory neurons (69% myelin loss, p < 0.0001). Pretreatment for 2 hours with three different concentrations of the multi-ion complexes TA6496, TA6440, and TA6415 significantly protects the length of the myelin sheath of sensory neurons from cisplatin damage over 24 hours (29% (p < 0.001), 37% (p < 0.01), and 29% (p < 0.001) myelin loss, respectively).

[0142] On the other hand, at the concentrations used for TA6496, TA6440, and TA6415, none of the individual ions showed a significant protective effect on the length of the myelin sheath. Manganese was also tested under the same conditions in the concentration range of 0.15 mg / kg to 1.2 mg / kg, but it showed no significant effect on the length of the myelin sheath.

[0143] Example 9 - In Vitro Anti-Inflammatory Effect on Human Monocytes The following compositions were tested. · TA6496 & TA64128: Ion complex (Mg 0.2 / Li 1 / K 1.6) · Li alone (chloride) · Mg alone (chloride) · K alone (chloride) · C17 - C18 multi-ion complex (chloride): (Mg 0.22 / Li 1 / K 1.3) · C19 - C20 multi-ion complex (chloride) (Mg 0.14 / Li 1 / K1.6) · C21 - C22 multi-ion complex (chloride) (Mg 0.22 / Li 1 / K 1.76) Method: Monocytes are isolated from the blood of healthy donors by Ficoll density gradient (Ficoll-Paque PLUS; GE Healthcare; 11778538) using a density gradient centrifugation tube (SepMate; StemCell Technologies; 85450). Monocytes are deposited in 96-well plates (200,000 cells per well), pretreated for 2 hours with vehicle (0.179% PBS), or the compositions according to the invention, or individual ions (see Table 6), and then stimulated at 37°C, 5% CO2 for 6 hours in RPMI medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, either without lipopolysaccharide (vehicle 0.002% H2O) or with (100 ng / mL LPS; Invivogen, tlrl-b5lps). After 6 hours of incubation, the culture supernatant is collected and stored at -20°C until cytokine analysis. The concentrations of cytokines (IL-1β and IL-18) in the culture supernatant are quantified by ELISA according to the instructions (R&D Systems; DY201, DY318-05). Table 6 summarizes the ratios of the measured IL-1β and IL-18 cytokines for each condition to the vehicle (ns: not significant).

[0144]

Table 6-1

[0145]

Table 6-2

[0146] Effects of TA6496 and TA64128 compounds When monocytes were stimulated with LPS (100 ng / mL), a significant increase in the concentrations of IL-1β and IL-18 in the culture supernatant was induced. TA6496 and TA64128 significantly decreased the IL-1β and IL-18 concentrations in a concentration-dependent manner (Figure 7).

[0147] Therefore, the composition of the present invention exhibits significant anti-inflammatory effects on human immune cells in a situation where inflammation is progressing. Effects of Mg, Li, and K ions alone When monocytes were stimulated with LPS, a significant increase in the concentrations of IL-1β and IL-18 in the culture supernatant was induced. None of magnesium, lithium, and potassium alone showed a significant decrease in the IL-1β and IL-18 concentrations (Figure 8).

[0148] Effects of C17~C22 chloride-based multi-ion complexes: When monocytes were stimulated with LPS, a significant increase in the concentrations of IL-1β and IL-18 in the culture supernatant was induced. All of the C17~C22 ion complexes showed a significant decrease in the IL-18 concentration, and the C18 complex with 96 mg / L lithium showed a significant decrease in the IL-1β concentration. Therefore, the C1~C22 ion complexes exhibit significant anti-inflammatory effects on human immune cells in a situation where inflammation is progressing (Figure 9).

[0149] Example 10 - Effects of the multi-ion complex of the present invention administered orally on an in vivo inflammation model A dextran sulfate sodium (DSS)-induced acute ulcerative colitis model was used. TNF-α and IFNγ markers were monitored.

[0150] TAR64 is an oral dosage form of TA64. Induction: Dextran sulfate sodium (DSS) is administered for 7 days starting from day 1, followed by a 5-day washout (without DSS) to induce ulcerative colitis. According to the standard procedure for inducing acute ulcerative colitis, an appropriate concentration of DSS (4%) is added to the drinking water and made available for the animals to consume freely, and this is replaced with fresh one every two days.

[0151] Buprenorphine (0.5 mg / kg) is administered to reduce the discomfort of the animals. Animals: The experiments were conducted using female syngeneic mice of the Balb / c strain. The mice are housed in groups of 4 animals per study group. Each mouse is uniquely identified. The animals are housed in a ventilated cage (type II (16×19×35 cm, floor area = 500 cm 2 )) under the following conditions: · Room temperature (22 ± 2 °C). · Humidity measurement (55 ± 10%). · Photoperiod (12:12 hour light-dark cycle (7 am: 7 pm)). · Food and water are provided ad libitum.

[0152] The mice were acclimatized to the environment for 7 days before the start of the experiment. If necessary according to the procedure to be performed, the mice are anesthetized by isoflurane inhalation. Analgesic treatment is carried out on the mice: During the induction of ulcerative colitis, buprenorphine (0.5 mg / kg) is administered to reduce the discomfort of the mice.

[0153] Groups and treatments: All treatments were started on day 1 and administered to each group according to Table 7 below until the end of the test (day 14).

[0154]

Table 7

[0155] The daily dosage by element is shown in Table 8 below:

[0156]

Table 8

[0157] On the 14th day, approximately 200 μL of blood is collected from the retro-orbital region of all animals under anesthesia. The blood is transferred to an EDTA-K3 tube and gently (manually) inverted and stirred 8 - 10 times. The blood is centrifuged at 800 g for 5 minutes at 2 - 4°C. The plasma is separated and transferred to a labeled polypropylene tube and centrifuged at 10,000 g for 5 minutes at 2 - 4°C. After separating the collected plasma, it is transferred to a labeled polypropylene tube and immediately frozen on dry ice or in a -80°C freezer and stored until cytokine bead array (CBA) analysis.

[0158] CBA analysis: CBA analysis of the plasma is performed using a CBA FLAX mouse kit according to the instructions. (TNF-α; BN; 558299)(IFNγ; BD; 558296) Results: DSS-induced ulcerative colitis induced a significant increase in the plasma concentrations of TNF-α and IFNγ on the 7th and 14th days. Treatment with TAR64200 significantly decreased TNF-α on the 7th day and IFNγ on the 14th day. Therefore, TAR64200 exhibits anti-inflammatory effects in vivo in the ulcerative colitis model (Figure 10).

[0159] Example 11 - In vivo potassium toxicity test Objective: In vivo toxicity test. TAV64 is an injectable form of compound TA64.

[0160] The TAV64 composition consists of lithium, potassium, and magnesium, and all three elements are in the form of chlorides: LiCl, KCl, and MgCl2 in a ratio of 0.2 - 1 - 1.6 (similar to the TA64 composition shown in Table 1). Regarding the toxicity of these three salts contained in the TAV64 composition, KCl has the lowest 24-hour intravenous median lethal dose (LD50) in rats, with LD50 = 142 mg / kg (Chemical Economics Handbook (CEH) Marketing Research Reports (1999). SRI International, p. 764.1000 I.), and thus may have the most important significance in the intravenous administration of TAV64.

[0161] To investigate whether the combination of the above three inorganic chlorides affects the 24-hour LD50 of KCl, TAV64 was injected into male Wistar rats under the following conditions. A first group consisting of 3 male Wistar rats was intravenously injected with a TAV64 composition containing 127 mg / kg of KCl in a total dose over 24 hours, that is, 90% of the LD50 of KCl, and all rats survived.

[0162] A second group consisting of 3 male Wistar rats was intravenously injected with TAV64 containing 288 mg / kg of KCl in a total dose over 24 hours, that is, 203% of the LD50 of KCl, and all rats survived.

[0163] A third group consisting of 8 male Wistar rats was intraperitoneally injected with lipopolysaccharide (LPS) to induce systemic inflammation. Then, a TAV64 composition containing 240 mg / kg of KCl in a total dose over 24 hours, that is, an amount 69% more than the LD50 of KCl, was intravenously injected, and 5 out of 8 rats survived.

[0164] In conclusion, TAV64 showed high tolerance and non-toxicity in vivo in Wistar rats. The composition based on the three chlorides of Li, Mg, and K enabled administration up to 203% compared to the LD50 of single KCl administration in healthy rats, and was also able to increase the LD50 by 69% when KCl was injected into diseased rats in a sepsis-induced model. From these results, it was revealed that the composition of TAV64 had a favorable effect on the LD50 of KCl, and that TAV64 had a large safety margin in its use by intravenous injection.

Claims

1. At least one of each of lithium, magnesium, and potassium salts in the following molar ratios: Lithium-1-Magnesium [0.13-0.34]-Potassium [1.20-2.40] Includes inorganic and / or organic salts contained in A multi-ionic composition for oral or parenteral treatment or prevention of inflammation.

2. The composition according to claim 1, wherein lithium is present at a concentration of 0.01 to 100 mmol / kg.

3. The composition according to any one of claims 1 and 2, wherein the lithium salt is an inorganic salt such as lithium chloride, lithium hydroxide, or lithium carbonate.

4. The composition according to any one of claims 1 and 2, wherein the lithium salt is an organic salt such as lithium citrate, lithium gluconate, or lithium orotate.

5. The composition according to any one of claims 1 and 2, wherein the magnesium salt is an inorganic salt such as a chloride, hydroxide, sulfate, oxide, silicate, or carbonate.

6. The composition according to any one of claims 1 and 2, wherein the magnesium salt is an organic salt such as magnesium bisglycinate, magnesium malate, magnesium glycerophosphate, magnesium stearate, magnesium ascorbate, magnesium taurate, magnesium citrate, magnesium gluconate, or magnesium taurinate.

7. The composition according to any one of claims 1 and 2, wherein the potassium salt is an inorganic salt such as potassium chloride, potassium bromide, potassium iodide, potassium phosphate, potassium carbonate, potassium hydroxide, or potassium silicate.

8. The composition according to any one of claims 1 and 2, wherein the potassium salt is an organic salt such as potassium gluconate, potassium citrate, potassium malate, potassium glycerophosphate, potassium lactate, potassium pyrulate, potassium aspartate, or potassium gluconate.

9. A composition according to any one of claims 1 and 2, for the prevention and / or treatment of chronic or acute inflammation.

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

11. The composition according to claim 9 for use in a method for treating or preventing a condition selected from chronic neuroinflammation.

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

13. The composition according to claim 9 for use in a method of treating or preventing a condition selected from stroke, infarction, sepsis, burns, systemic inflammatory response syndrome, COVID, trauma, or postoperative inflammation.

14. A composition according to any one of claims 1 and 2 for reducing the release of TNF-α and / or IL-1β and / or IL-18.

15. A composition according to any one of claims 1 and 2 for reducing caspase activation.

16. A composition according to any one of claims 1 and 2, for use in a method of oral treatment or prophylaxis of a condition selected from bipolar disorder.

17. The composition according to any one of claims 1 and 2, further comprising at least one additive selected from stabilizers, emulsifiers, preservatives, antioxidants, and / or gelling agents.

18. The composition according to any one of claims 1 and 2, further comprising one or more other inorganic or organic salts, such as salts of zinc, manganese, copper, and / or silicon.

Images