A pharmaceutical composition, an organ preservation solution and its application

Abstract

A pharmaceutical composition, an organ preservation solution, and their applications are disclosed, wherein the pharmaceutical composition comprises cedutinib and edaravone. The mass ratio of cedutinib to edaravone is 1:0.0001-10000. The pharmaceutical composition of the present invention exhibits good cell protection effects, and cedutinib and edaravone demonstrate a synergistic effect, contributing to excellent therapeutic efficacy, reducing drug dosage, and improving clinical drug safety.

Description

[0001] This application is a divisional application of patent application CN202580001280.4, filed on January 24, 2025, entitled "A Pharmaceutical Composition, Organ Preservation Solution and Its Application". Like the parent application CN202580001280.4, this application claims priority to CN202410142933.8. Technical Field

[0002] This invention relates to a pharmaceutical composition for protecting cells, an organ preservation solution, and their applications. The composition can be used to protect individuals (especially at-risk individuals), organs, tissues, or cells from damage and belongs to the field of biomedicine. Background Technology

[0003] During the decline process or pathological conditions, especially those that may lead to cell death, organs, tissues or cells may undergo various pathological processes such as oxidative stress, calcium overload, energy metabolism disorders, and inflammatory responses. These processes can lead to apoptosis, necrosis, and other forms of death in organs, tissues or cells, ultimately resulting in cell death.

[0004] Cellular necrosis includes various pathways such as necroptosis, ferroptosis, and pyroptosis. Studies have shown that RIPK1 / RIPK3 / MLKL-dependent necroptosis exists in various injury-related diseases, such as ischemic stroke, myocardial infarction, hepatic and renal ischemia / reperfusion injury, autoimmune diseases, and neurodegenerative diseases. Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy, Huntington's disease, and Parkinson's plus syndrome. Inhibiting RIPK1 / RIPK3-dependent necroptosis, ferroptosis, or pyroptosis, such as the RIPK1 inhibitor necrostatin-1 (Nec-1), can reduce the degree of tissue, organ, and cell damage and decrease cell death. In cases of cerebral ischemia / reperfusion injury, the RIPK1 inhibitor necrostatin-1 can reduce cerebral ischemia-induced injury in mice and improve neurological function.

[0005] Oxidative stress (OS) is a state of imbalance between oxidation and antioxidation in the body, with a predominance of oxidation leading to inflammatory infiltration of neutrophils, increased protease secretion, and the production of large amounts of oxidative intermediates. Oxidative stress is a negative effect produced by free radicals in the body and is considered a significant factor contributing to aging and disease. In various injury-related diseases, including ischemic stroke, myocardial infarction, liver and kidney ischemia / reperfusion injury, autoimmune diseases, and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy, Huntington's disease, and Parkinson's plus syndrome, oxidative stress levels are elevated. Inhibiting oxidative stress can reduce the degree of tissue, organ, and cell damage and decrease cell death.

[0006] If, under multiple pathological conditions, such as ischemia / reperfusion injury of the heart and brain, apoptosis, necroptosis-like apoptosis, ferroptosis, or pyroptosis pathways are simultaneously inhibited, cell death can be synergistically suppressed, greatly reducing the degree of tissue, organ, and cell damage, decreasing cell death, and simultaneously reducing the dosage and adverse reactions of individual drugs. However, existing compounds that inhibit these pathways can only serve as tool drugs and cannot meet clinical needs.

[0007] Therefore, it is essential to find novel drug compositions (cell protectants) with greater clinical application potential for cell protection.

[0008] Cerdulatinib is an oral, multi-target tyrosine kinase inhibitor that inhibits spleen tyrosine kinase (SYK) and Janus kinase (JAK), significantly reducing cell viability in a subset of non-Hodgkin's lymphoma (NHL) cell lines and inducing apoptosis in NHL cell lines with BCR signaling. It may be used to treat peripheral T-cell lymphoma. However, its cytoprotective and anti-cell-damage effects, as well as its efficacy against neurological diseases, have not been reported. Edaravone has the chemical formula C... 10 H 10 N2O is primarily used as a free radical scavenger, mainly to improve neurological symptoms and functional disorders. However, it remains unclear whether the combination of the two drugs has a synergistic effect. Summary of the Invention

[0009] According to the present invention, the term "cell protection" refers to the effect of any reagent or compound (whether natural or non-natural) in protecting and / or preventing and / or treating cells against the consequences of pathological conditions (particularly those that may lead to cell death) or degenerative processes at the cellular level.

[0010] In this article, unless otherwise stated, “pathological condition” means, for example, symptoms or disease or trauma or exposure to various factors (especially factors that trigger cell death processes), and also includes events such as bleeding, accidental occlusion (infarction) and / or medical procedures (especially surgical procedures, such as organ transplantation).

[0011] According to the present invention, "protection" refers to preventing the occurrence of the consequences at the cellular level (especially for at-risk individuals) of pathological conditions or degenerative processes that may lead to cell death, or inhibiting, reducing or treating such consequences.

[0012] According to the present invention, "treatment" refers to preventive (advantageously for individuals at risk) and / or alleviating and / or curative treatment. It includes: a) inhibiting and / or eliminating the occurrence and / or development of a degenerative process or pathological condition; b) or mitigating the severity of such a degenerative process or pathological condition, for example, reducing the frequency or severity of symptoms associated with the degenerative process or pathological condition, improving the quality of life of individuals suffering from such a degenerative process or pathological condition, reducing the dosage of other medications required to treat the degenerative process or pathological condition, enhancing the effectiveness of another treatment for the degenerative process or pathological condition, or prolonging the lifespan of individuals suffering from such a degenerative process or pathological condition.

[0013] "Prevention" or "avoidance" refers to reducing the likelihood of development or preventing or delaying the occurrence of a decline process or pathological condition in an individual who has not yet developed such a condition but is at risk of developing one.

[0014] "At risk" means that an individual has one or more risk factors for a degenerative process or pathology, which are measurable parameters that can be associated with the development of the degenerative process or pathology and are known to those skilled in the art. Individuals exhibiting one or more of these risk factors are more likely to develop a degenerative process or pathology than individuals who do not exhibit these risk factors. For example, an individual planning to undergo surgery may be considered an at-risk individual. As another example, an individual with the following risk factors may be considered an individual with risk factors for stroke and cerebral ischemia: hypertension, carotid artery stenosis, transient ischemic attack, coronary artery disease, history of myocardial infarction, lack of physical activity, atrial fibrillation, left ventricular dysfunction or mitral stenosis, heart failure, hyperlipidemia, smoking, and diabetes.

[0015] The terms "organ," "tissue," or "cell" refer to one or more cells, a part of an organ, an entire organ, tissue, or group of tissues (limbs, etc.) of human or animal origin. This invention may be applicable to all organs, tissues, or cells. Examples include: solid organs such as the heart, liver, brain, lungs, kidneys, pancreas, intestines, and eyes; cells such as cells or stem cells of the aforementioned organs; and tissues such as skin, cornea, and vascularized composite tissue (VCA). Preferably, this invention targets solid organs; even more preferably, the brain, heart, intestines, lungs, liver, and kidneys are the targets of this invention.

[0016] In this invention, unless otherwise stated, celutinib refers to the celutinib compound (drug) or its pharmaceutically acceptable salt, co-crystal, any stereoisomer, tautomer, hydrate, or solvate thereof. Edaravone refers to the edaravone compound (drug) or its pharmaceutically acceptable salt or ester, or isomers thereof, or one of its semi-synthetic derivatives, or one of its salts (salt of the compound or salt of the semi-synthetic derivative), or one of its esters, or one of its ester salts (salt of the ester of the compound or salt of the ester of the semi-synthetic derivative), or its deuterated compound (deuterated edaravone) or isotopically labeled compound. Optionally, celutinib is the celutinib compound. Optionally, edaravone is the edaravone compound.

[0017] Optionally, the pharmaceutically acceptable salt is a pharmaceutically commonly used salt, and further, the salt is selected from one or more of the following: acetate, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, benzoate, fumarate, maleate, succinic acid, tartaric acid, citrate, oxalic acid, glyoxylic acid, aspartic acid, tartrate, 2,5-dihydroxybenzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, lecithin sulfonate, hydroquinone sulfonate, and p-toluenesulfonate, or salts formed from carboxylic acids (e.g., formic acid, acetic acid, or propionic acid).

[0018] Generally, the structural formula of cedutinib is shown in Formula I, and its molecular formula is C10. 20 H 27 N7O3S.

[0019] Formula I.

[0020] The structural formula of the edaravone compound is shown in Formula II, and its molecular formula is C. 10 H 10 N2O.

[0021] Formula II.

[0022] To address the shortcomings of existing technologies, one objective of this invention is to provide a pharmaceutical composition capable of protecting, preventing, and / or treating cells against processes that lead to cell death, such as drugs against pathological apoptosis and / or necrosis and / or necroptosis and / or ferroptosis and / or disulfide death and / or autophagy, or pharmaceutical compositions that resist surgical procedures that may lead to cell death. Another objective of this invention is to provide the application of the pharmaceutical composition in the preparation of drugs that protect, prevent, and / or treat cells against processes that lead to cell death.

[0023] More specifically, in view of the shortcomings of the prior art, the present invention aims to provide a pharmaceutical composition for protecting cells with superior efficacy; a second objective of the present invention is to provide the application of the above-mentioned pharmaceutical composition in the preparation of cell protection drugs; a third objective of the present invention is to provide the application of the above-mentioned pharmaceutical composition in the preparation of organ preservation solutions; and a fourth objective of the present invention is to provide an organ preservation solution.

[0024] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0025] A pharmaceutical composition for protecting cells, comprising cedutinib and edaravone.

[0026] Further, the mass ratio of cedutinib to edaravone is 1:0.0001-10000, preferably 1:0.001-1000, more preferably 1:0.01-100, even more preferably 1:0.05-25, even more preferably 1:0.1-10, even more preferably 1:0.15-1, and even more preferably 1:0.2-0.8.

[0027] Optionally, the mass ratio of cedutinib to edaravone is 1:0.5-20, further 1:1-10, even further 1:1.5-9, even further 1:2-8.5, and even further 1:2.5-8.

[0028] Further, the mass ratio of cedutinib to edaravone is 5-40:1-10, preferably 10-35:2-8, more preferably 15-30:3-6, even more preferably 18-25:4-5, and still more preferably 19:4-5, 20:4-5, 21:4-5, 22:4-5, 23:4-5, or 24:4-5. Further, the mass ratio of cedutinib to edaravone is 3-30:1-10, preferably 5-25:2-8, more preferably 7.5-15:3-6.

[0029] Optionally, the mass ratio of cedutinib to edaravone in the pharmaceutical composition is 20:7, 19:7, 18:7, 17:7, 16:7, 15:7, 14:7, 12:7, 11:7, 10:7, 9:7, 8:7, 7:7, 6:7, 5:7, 20:6, 19:6, 18:6, 17:6, 16:6, 15:6, 14... 6, 12:6, 11:6, 10:6, 9:6, 8:6, 7:6, 6:6, 5:6, 20:5, 19:5, 18:5, 17:5, 16:5, 15:5, 14:5, 12:5, 11:5, 10:5, 9:5, 8:5, 7:5, 6:5, 5:5, 20:4, 19:4, 18:4, 17:4, 1 6:4, 15:4, 14:4, 12:4, 11:4, 10:4, 9:4, 8:4, 7:4, 6:4, 5:4, 20:3, 19:3, 18:3, 17:3, 16:3, 15:3, 14:3, 12:3, 11:3, 10:3, 9:3, 8:3, 7:3, 6:3, 5:3, 20:2, 19:2 18:2, 17:2, 16:2, 15:2, 14:2, 12:2, 11:2, 10:2, 9:2, 8:2, 7:2, 6:2, 5:2, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, or 5:1.

[0030] Furthermore, the molar ratio of cedutinib to edaravone is 5-75:3-65, preferably 6-70:4-60, and more preferably 7-68:5-58.

[0031] Furthermore, the active ingredients of the pharmaceutical composition may include or be cedutinib and edaravone.

[0032] Furthermore, the pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form according to known techniques. The pharmaceutical composition of the present invention can be formulated into the following forms: suspension or ready-to-use injection solution or temporary injection solution, gel, oil, tablet, suppository, powder, capsule, granule, suspension, emulsion, polymer, nanoparticle, microsphere, rectal capsule, enema, paste, ointment, cream, plaster, decoction, implant, spray, aerosol, etc., optionally with controlled release and / or sustained release via dosage form or device. Preferred dosage forms include injection, capsule, tablet, granule, powder, spray, liposome, oral liquid, and pellet.

[0033] The pharmaceutical combination of cedutinib and edaravone can be used (advantageously in individuals at risk) for the prevention and / or protection and / or treatment of humans and / or animals (particularly mammals, preferably humans).

[0034] Based on the same inventive concept, the present invention also provides the use of the pharmaceutical composition described above in the preparation of cell protection drugs.

[0035] Further, the cell-protective drug is a drug used to prevent, protect, and / or treat cell damage caused by pathological conditions and / or degeneration processes; the pathological conditions and / or degeneration processes include situations that may lead to cell death, preferably, the pathological conditions and / or degeneration processes include pathological apoptosis and / or pathological necrosis and / or necrotizing apoptosis and / or pyroptosis and / or ferroptosis and / or disulfide-dependent cell death and / or autophagy (anti-apoptotic drugs and / or anti-necrotizing drugs and / or anti-pyroptosis drugs and / or anti-ferroptosis drugs and / or anti-dependent cell death drugs and / or disulfide-dependent cell death drugs and / or anti-autophagy drugs) and / or diseases or conditions; preferably, the diseases or conditions include, but are not limited to: nervous system diseases, cardiovascular system diseases Hemorrhage and thrombotic diseases, diffuse connective tissue diseases, organ-specific or systemic inflammation or autoimmune diseases, autoimmune diseases, bone diseases, joint and cartilage diseases, ischemic diseases or attacks of limbs, ophthalmic diseases, skin diseases, kidney diseases, blood and vascular diseases, lung diseases, gastrointestinal diseases, liver diseases, metabolic diseases, muscle diseases, pancreatic diseases, severe poisoning caused by chemicals, infectious agents, toxins or drugs, age-related diseases, dental diseases, auditory conduction pathway diseases, mitochondrial-related diseases, and / or trauma and / or exposure to factors of biological and / or chemical and / or physical and / or medical and / or surgical procedures, such as accidental infarction and hemorrhage, and / or medical and / or surgical procedures, such as cell, tissue or organ transplantation.

[0036] Furthermore, the cell-protective drug refers to a drug that has the effect of preventing, inhibiting or treating damage, degeneration or dysfunction of tissues, organs and cells caused by hypoxia / reoxygenation;

[0037] Alternatively, the cell-protective drug refers to a drug that has the effect of preventing, inhibiting or treating damage, degeneration or dysfunction of tissues, organs and cells caused by necroptosis.

[0038] Furthermore, the neurological diseases mentioned include stroke, transient ischemic attack, focal ischemia, intracranial hemorrhage, prenatal hypoxia, adult or childhood hypoxia, neurodegenerative diseases, muscle diseases, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's palsy, progressive bulbar palsy, primary lateral sclerosis (PLS), pseudobulbar palsy, invertebral disc syndrome, cervical spondylosis, plexus disorder, thoracic outlet destruction syndrome, porphyria, peripheral neuropathy, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, Lewy body dementia, demyelinating diseases, frontotemporal dementia, Gullman-Barré syndrome, multiple sclerosis, Kreutzfeldt-Jacob disease, progressive peroneal muscular atrophy, prion disease, and lethal... Familial insomnia, G.S.-S. syndrome, bovine spongiform encephalopathy, epilepsy, hereditary ataxia, Friedreich ataxia, spinocerebellar ataxia, hereditary spastic paraplegia, dystonia, multiple system atrophy, lysosomal storage disease, Niemann-Pick disease, Gaucher disease, AIDS dementia syndrome, neurological damage caused by exposure to toxic compounds consisting of industrial solvents, heavy metals, drugs, and chemotherapeutic agents, and neurological damage caused by mechanical, physical, or chemical trauma; preferably, stroke includes one or more of ischemic and hemorrhagic strokes; preferably, the neurodegenerative disease includes Alzheimer's disease. Muscle diseases include one or more of the following: Parkinson's disease, amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy, Huntington's disease, and Parkinson's plus syndrome; preferably, the muscle diseases include one or more of the following: muscular atrophy, Duchenne's muscular dystrophy, ankylosing spondylitis, myopathy and myasthenia gravis, myasthenia gravis, progressive muscular atrophy, spinal muscular atrophy, and hereditary muscular atrophy;

[0039] Preferably, the cardiovascular diseases include myocardial ischemia and / or vascular ischemia, ischemic heart disease, angina pectoris, unstable angina pectoris, refractory angina pectoris, myocardial infarction, myocardial ischemia / reperfusion injury, hypoxia, low oxygen, chronic or acute heart failure, systolic heart failure and diastolic heart failure, left ventricular dysfunction, post-myocardial infarction left ventricular dysfunction, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, myocardial hypertrophy, hypertrophic cardiomyopathy, myocarditis, valvular heart disease, arrhythmia, paroxysmal tachycardia, atrial fibrillation, ventricular fibrillation, arteriosclerosis, atherosclerosis, peripheral vascular disease, aneurysm, peripheral vascular occlusive disease (preferably cerebral occlusion, pulmonary occlusion or intestinal occlusion), chronic venous insufficiency or varicose veins, hypertension, systemic hypertension, pulmonary hypertension, portal hypertension, and one or more of the cardiovascular toxic side effects caused by drug (preferably anticancer drug) treatment;

[0040] Preferably, the stroke includes ischemic stroke;

[0041] Preferably, the diffuse connective tissue disease includes one or more of the following: rheumatoid arthritis, juvenile idiopathic arthritis, lupus erythematosus, systemic lupus erythematosus, scleroderma, idiopathic inflammatory myopathy, polymyositis, dermatomyositis, vasculitis, necrotizing vasculitis, polyarteritis nodosa, granulomatous vasculitis, giant cell arteritis, Sjögren's syndrome, systemic scleroderma, allergic cutaneous vasculitis, and Behcet's disease;

[0042] Preferably, the organ-specific inflammation, systemic inflammation, or autoimmune disease includes one or more of chronic inflammatory bowel disease, bronchial asthma, chronic obstructive pulmonary disease, eosinophilic sinusitis, and systemic lupus erythematosus.

[0043] Preferably, the ophthalmic diseases or conditions include one or more of the following: diabetic retinopathy, glaucoma, retinal degeneration, retinitis pigmentosa, corneal reticular dystrophy, optic neuropathy and optic neuritis, optic drusen, ptosis, chronic progressive extraocular muscle paralysis, macular degeneration, retinal hole or retinal tear, retinal ischemia, retinal ischemia / reperfusion injury, retinal detachment, trauma-related acute retinopathy, inflammatory degeneration, postoperative complications, drug-induced retinopathy or cataract, wet or dry AMD-related photoreceptor degeneration;

[0044] Preferably, the skin disease includes one or more of the following: dermatitis, psoriasis, scarring, aging or altered healing process, eczema, and collagen diseases;

[0045] Preferably, the trauma and / or exposure to factors of biological and / or chemical and / or physical and / or medical and / or surgical origins include severe poisoning caused by infectious agents, toxins, chemicals, or drugs. More preferably, the symptoms of severe poisoning include one or more of sepsis, septic shock and its consequences, or iatrogenic diseases.

[0046] Furthermore, the cell-protective drug is one or more drugs used to prevent and / or protect and / or treat cell death in transplanted organs and / or organ donors and / or organ recipients, prevent acute transplant rejection of organs and / or increase long-term survival rates, limit primary organ dysfunction and / or limit the delay in the recovery of transplanted organ function and / or improve the functional recovery of transplanted organs, primarily preventing or treating cell death in transplanted organs.

[0047] Advantageously, the pharmaceutical composition containing cedutinib and edaravone may be used before, during, or after transplantation for living or clinically dead organ donors, tissue donors, cell donors, organ recipients, tissue recipients, or cell recipients; and / or more specifically, the organ, tissue, or cell may be not only in situ (e.g., in medicine, during surgery, or in pathological procedures) but also ex vivo (e.g., in certain procedures requiring temporary removal of organs, tissues, or cells from the body, particularly those that modify or purify them, or during the transport and preservation of organs, tissues, or cells, during transplantation, or during their reperfusion after reimplantation).

[0048] Therefore, this prophylaxis and protection can be applied in a general manner to individuals, donors or recipients, or organs, tissues or cells, in situ or ex vivo, for example during certain surgeries, or during their transport or preservation for reimplantation.

[0049] Ischemia primarily results from a reduced or interrupted blood supply to an organ, leading to decreased blood, oxygen, and energy supply to organs, tissues, and cells. This causes damage and death of tissues, organs, and cells, and is associated with, but not limited to, atherosclerotic plaques, thrombosis, arterial compression (e.g., through limb compression, tourniquet use, tumors, hematomas, or fluid leakage), artificial cessation of blood circulation (if necessary during surgery), hemorrhage, or insufficient perfusion. Ischemia or insufficient perfusion can affect or damage the function of all organs, particularly the brain, heart, liver, lungs, kidneys, intestines, or limbs. When blood circulation to an organ is restored after a period of ischemia, reperfusion can also cause damage to the organ, tissues, and cells, a condition known as ischemia / reperfusion injury. This can limit the recovery of function and may even jeopardize an individual's survival. Ischemia / reperfusion injury involves multiple types of cell death.

[0050] Unexpectedly, the inventors discovered that a pharmaceutical composition containing cedutinib and edaravone can reduce damage to human or animal cells from different types of organs that have undergone organ ischemia / reperfusion, as well as from organs that have been subjected to hypoxia / reoxygenation (oxygen-glucose deprivation / reoxygenation) and necroptosis-like apoptosis, and increase their survival rate.

[0051] Preferably, the cell-protective drug is a drug for preventing and / or protecting and / or treating pathological conditions or degenerative processes associated with ischemia / reperfusion symptoms (especially those that lead to cell death). More preferably, the pathological conditions or degenerative processes associated with ischemia / reperfusion symptoms include one or more of actual cold ischemia, warm ischemia, actual reperfusion, and ischemia / reperfusion phenomena.

[0052] Preferably, the cell-protective drug is a drug used to prevent and / or protect and / or treat organs, tissues or cells against ischemia / reperfusion injury. Optionally, the ischemia / reperfusion injury is caused by cold or warm ischemia and / or reperfusion and / or ischemia / reperfusion. Further, the ischemia / reperfusion injury includes one or more of cerebral ischemia / reperfusion injury, myocardial ischemia / reperfusion injury, hepatic ischemia / reperfusion injury, renal ischemia / reperfusion injury, pulmonary ischemia / reperfusion injury, intestinal ischemia / reperfusion injury and limb ischemia / reperfusion injury.

[0053] In a preferred form, the pharmaceutical composition containing cedutinib and edaravone can be used as a medicine for the prevention and / or protection and / or treatment of: neurological sequelae due to stroke or trauma, heart failure due to infarction, tissue damage affecting the heart, liver, intestine, lung or kidney after transplantation to a graft or surgery, or damage caused by a surgical procedure.

[0054] A more preferred form is a pharmaceutical composition containing cedutinib and edaravone, which can be used as a medicine for the prevention and / or protection and / or treatment of ischemic stroke caused by actual hot or cold ischemia and / or actual reperfusion and / or ischemia / reperfusion.

[0055] A more preferred form is a pharmaceutical composition containing cedutinib and edaravone, which can be used as a medicine for the prevention and / or protection and / or treatment of myocardial infarction caused by actual hot or cold ischemia and / or actual reperfusion and / or ischemia / reperfusion.

[0056] According to the present invention, the pharmaceutical composition containing cedutinib and edaravone is used in a physiologically effective amount.

[0057] As a medicine, the pharmaceutical composition containing cedutinib and edaravone can be formulated for use in the digestive tract or external gastrointestinal tract.

[0058] Furthermore, the cell-protective drug is a drug used to prevent and / or protect and / or treat one or more of the following: heart failure caused by infarction, neurological sequelae caused by stroke or trauma, tissue damage affecting liver, intestine, heart, lung or kidney transplantation to grafts or surgery, or consequences of surgical procedures.

[0059] Alternatively, the cell-protective drug is a drug for preventing and / or protecting and / or treating one or more of nerve cells (a drug for protecting the brain), heart cells (a drug for protecting the heart), liver cells (a drug for protecting the liver), kidney cells (a drug for protecting the kidneys), intestinal cells, or lung cells. Preferably, the cell-protective drug is a drug for protecting nerve cells, vascular endothelial cells, brain endothelial cells, and / or cardiomyocytes.

[0060] Optionally, the factors that trigger the cell death process may be of biological and / or chemical and / or physical origin.

[0061] The use of the pharmaceutical composition described above in the preparation of medicaments for the prevention, protection and / or treatment of ischemia / reperfusion injury and / or neurodegenerative diseases.

[0062] Optionally, the ischemia / reperfusion injury includes one or more of the following: cerebral ischemia / reperfusion injury, myocardial ischemia / reperfusion injury, hepatic ischemia / reperfusion injury, renal ischemia / reperfusion injury, pulmonary ischemia / reperfusion injury, intestinal ischemia / reperfusion injury, and limb ischemia / reperfusion injury; preferably, cerebral ischemia / reperfusion injury includes ischemic stroke;

[0063] The neurodegenerative diseases include one or more of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy, Huntington's disease, and Parkinson's plus syndrome.

[0064] The application of the pharmaceutical composition described above in the preparation of organ preservation solutions.

[0065] Optionally, biological sources include, for example, asphyxiation, ischemia / reperfusion, hypoxia or oxygen deprivation, nutrient deprivation, poisoning from in-situ generated free radicals or reactive oxygen species, growth factor deficiency, and massive release of cytotoxins or cytokines. They may also originate from events such as hemorrhage, accidental occlusion (infarction), and certain medical procedures (e.g., cuff inflation, ventilator use, sutures), as well as biological or chemical agents used as therapeutic agents in medical treatment (e.g., immunosuppressants, cell inhibitors or cytotoxic agents, anti-inflammatory drugs).

[0066] Optionally, chemical sources include, for example, poisoning from toxins, waste, pH changes, free radicals, reactive oxygen species, and environmental toxins.

[0067] Alternatively, physical sources include, for example, impact, cuts, exposure to radiation (X-ray radiation, gamma radiation, UV radiation, etc.), hyperthermia, hypothermia, or the presence of foreign objects or crystals in the organism.

[0068] Alternatively, surgical procedures that may lead to cell death processes may include, for example, procedures that require a brief interruption of blood circulation resulting in systemic or local ischemia or hypoperfusion (such as the use of tourniquets, hemostatic forceps, etc.), which occur during, for example, surgery, particularly angioplasty of organs or the heart or main or peripheral blood vessels, or thoracic surgery, cardiac surgery or vascular surgery that sometimes requires bypassing the cardiopulmonary system or stopping the heart, and any surgery that requires voluntary obstruction of an organ or part of an artery or reduction of blood flow through an organ.

[0069] Optionally, very important diseases or symptoms that may lead to cell death processes include (but are not limited to) the following diseases or symptoms (which are often accompanied by apoptosis and / or necrosis and / or necroptosis and / or ferroptosis and / or pyroptosis and / or autophagy):

[0070] Neurological disorders include stroke, transient ischemic attack, focal ischemia, intracranial hemorrhage, prenatal hypoxia, hypoxic-ischemic encephalopathy in adults or children, neurodegenerative diseases, muscle diseases, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's palsy, progressive bulbar palsy, primary lateral sclerosis (PLS), pseudobulbar palsy, invertebral disc syndrome, cervical spondylosis, plexus disorder, thoracic outlet destruction syndrome, porphyria, peripheral neuropathy, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, Lewy body dementia, demyelinating diseases, frontotemporal dementia, Gullman-Barré syndrome, multiple sclerosis, Kreutzfeldt-Jacob disease, progressive peroneal muscular atrophy, prion disease, and fatal familial insomnia. The following are included in the list of neurodegenerative diseases: Göttingen-Schwarz syndrome, bovine spongiform encephalopathy, epilepsy, hereditary ataxia, Friedreich-type ataxia, spinocerebellar ataxia, hereditary spastic paraplegia, dystonia, multiple system atrophy, lysosomal storage diseases, Niemann-Pick disease, Gaucher disease, AIDS dementia syndrome, neurological damage caused by exposure to toxic compounds in the group consisting of industrial solvents, heavy metals, drugs and chemotherapeutic agents, and neurological damage caused by mechanical, physical or chemical trauma; preferably, stroke includes one or more of ischemic stroke and hemorrhagic stroke; preferably, the neurodegenerative disease includes Alzheimer's disease. Muscle diseases include one or more of the following: Parkinson's disease, amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy, Huntington's disease, and Parkinson's plus syndrome; preferably, the muscle diseases include one or more of the following: muscular atrophy, Duchenne's muscular dystrophy, ankylosing spondylitis, myopathy and myasthenia gravis, myasthenia gravis, progressive muscular atrophy, spinal muscular atrophy, and hereditary muscular atrophy;

[0071] Pain, such as neuropathic pain, inflammatory pain, and diabetic pain;

[0072] Cardiovascular diseases, such as myocardial ischemia and / or vascular ischemia, ischemic heart disease, angina pectoris, unstable angina pectoris, refractory angina pectoris, myocardial infarction, myocardial ischemia / reperfusion injury, hypoxia, hypoxia, chronic or acute heart failure, systolic heart failure and diastolic heart failure, left ventricular dysfunction, post-myocardial infarction left ventricular dysfunction, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, myocardial hypertrophy, hypertrophic cardiomyopathy, myocarditis, valvular heart disease, arrhythmia, paroxysmal tachycardia, atrial fibrillation, ventricular fibrillation, arteriosclerosis, atherosclerosis, peripheral vascular disease, aneurysm, peripheral vascular occlusive disease (especially cerebral occlusion, pulmonary occlusion or intestinal occlusion), chronic venous insufficiency or varicose veins, hypertension, systemic hypertension, pulmonary hypertension, portal hypertension, cardiovascular toxicity due to drug treatment (especially anticancer drugs);

[0073] Myocardial remodeling includes myocardial remodeling after myocardial ischemia / hypoxia (such as myocardial infarction), myocardial remodeling after cardiac surgery, myocardial remodeling after arterial valve disease, vascular hypertrophy (smooth muscle cell hypertrophy), and vascular remodeling.

[0074] Hemorrhage and thrombotic diseases, thromboembolic diseases and venous thrombosis, vascular permeability disorders, restenosis, acute coronary syndrome, venous embolism occurring after thrombolytic therapy or coronary angioplasty, venous embolism, pulmonary embolism caused by venous thrombosis, cerebrovascular syndromes such as embolic stroke, transient ischemic attack, and occlusive coronary thrombosis. Coagulopathy, thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, thromboangiitis obliterans, and thrombotic diseases related to heparin-induced thrombocytopenia. Thrombotic complications related to cardiopulmonary bypass, thrombotic complications related to instruments such as cardiac or other endovascular catheterization, intraaortic balloon catheterization, coronary stenting, or cardiac valves, and conditions requiring assistive devices and similar symptoms;

[0075] Diffuse connective tissue diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, lupus erythematosus, systemic lupus erythematosus, scleroderma, idiopathic inflammatory myopathy, polymyositis, dermatomyositis, vasculitis, necrotizing vasculitis, polyarteritis nodosa, granulomatous vasculitis, giant cell arteritis, Sjögren's syndrome, systemic scleroderma, allergic cutaneous vasculitis, Behcet's disease, etc.

[0076] Organ-specific inflammation or systemic inflammation or autoimmune diseases, such as chronic inflammatory bowel disease, bronchial asthma, chronic obstructive pulmonary disease, and eosinophilic sinusitis or systemic lupus erythematosus.

[0077] Autoimmune diseases or symptoms, including but not limited to Hashimoto's thyroiditis, autoimmune atrophic gastritis, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune thrombocytopenia, autoimmune alopecia, ulcerative colitis, hemolytic anemia, pernicious anemia, sympathetic ophthalmia, Graves' disease, primary biliary cirrhosis, chronic invasive hepatitis, conjunctival tetragonal lesions, and systemic lupus erythematosus;

[0078] Bone diseases, joint diseases, and cartilage diseases, such as osteoporosis, osteomyelitis, ischemic necrosis, arthritis (including osteoarthritis and psoriatic arthritis), spondyloarthritis, ankylosing spondylitis, rickets, progressive ossifying fibrous proliferative disease, and Cushing's syndrome.

[0079] Ischemic limb disease or attack;

[0080] Ophthalmic diseases or conditions such as diabetic retinopathy, glaucoma, retinal degeneration, retinitis pigmentosa, corneal reticular dystrophy, optic neuropathy and optic neuritis, optic drusen, ptosis, chronic progressive extraocular muscle paralysis, macular degeneration, retinal hole or retinal tear, retinal ischemia, retinal ischemia / reperfusion injury, retinal detachment, trauma-related acute retinopathy, inflammatory degeneration, postoperative complications, drug-induced retinopathy or cataracts, wet or dry AMD-related photoreceptor degeneration;

[0081] Skin conditions such as dermatitis, psoriasis, scarring, changes in the aging or healing process, eczema, and collagen diseases;

[0082] Kidney diseases, such as renal fibrosis, acute kidney disease, renal ischemia, renal capillary infarction, acute kidney injury, acute or chronic interstitial nephropathy, glomerulonephritis, diabetic nephritis, renal artery sclerosis, renal insufficiency, acute or chronic renal failure or dialysis side effects, and renal failure after myocardial ischemia / reperfusion.

[0083] Blood disorders, such as anemia, bleeding, angioamyloidosis, sickle cell disease, neutropenia, erythrocyte fragmentation syndrome, pancytopenia, leukopenia, myelodysplastic syndrome, thrombocytopenia, and hemophilia;

[0084] Lung diseases, such as pulmonary hypertension, acute respiratory distress syndrome, respiratory infections, chronic obstructive pulmonary disease, such as chronic bronchitis and emphysema, asthma, cystic fibrosis, and pulmonary cystic disease.

[0085] Gastrointestinal diseases, such as chronic inflammatory bowel disease, ulcers or mesenteric infarction, portal hypertension;

[0086] Liver diseases, such as autoimmune hepatitis, viral hepatitis or hepatitis caused by other infectious agents, liver fibrosis, alcoholic liver disease (ALD), alcoholic hepatitis, fulminant hepatitis, cirrhosis, and liver diseases caused by toxins or drugs.

[0087] Steatosis, such as liver ischemia or drug-induced exogenous poisoning, alcoholic or non-alcoholic steatohepatitis (NASH);

[0088] Metabolic diseases, such as diabetes, diabetic complications, diabetic nephropathy, diabetic retinopathy, diabetic foot, thyroiditis, Hashimoto's thyroiditis, glucose intolerance syndrome, obesity, abeta-lipoproteinemia, hyperlipidemia, hypothalamic-pituitary axis dysfunction, diabetes insipidus, galactosemia, glycogenopathies, gout, Wilson's disease, or Weber-Christian disease;

[0089] Pancreatic diseases, such as chronic pancreatitis or acute pancreatitis;

[0090] Severe poisoning caused by infectious agents, toxins, chemicals or drugs, such as sepsis, septic shock and its consequences or iatrogenic diseases;

[0091] Age-related diseases, such as accelerated aging syndrome;

[0092] Dental diseases, such as those that cause tissue damage, such as periodontitis;

[0093] Diseases of the auditory conduction pathway, such as antibiotic-induced deafness and otosclerosis;

[0094] Mitochondrial-related diseases (mitochondrial pathology), such as congenital muscular dystrophy with structural mitochondrial abnormalities and Friedrich's ataxia;

[0095] And / or trauma and / or exposure to factors of biological and / or chemical and / or physical origin and / or events, such as accidental bleeding and infarction, and / or medical procedures and / or surgical procedures, such as cell, tissue or organ transplantation.

[0096] Advantageously, the above-described pharmaceutical composition is used, for example, for the prevention and / or protection and / or treatment of nerve cells (a drug for protecting brain cells), cardiomyocytes (a drug for protecting the heart), lungs (a drug for protecting the lungs), intestines (a drug for protecting the intestines), liver (a drug for protecting the liver), and kidneys (a drug for protecting the kidneys), preferably for the protection of nerve cells (a drug for protecting the brain), cardiomyocytes (a drug for protecting the heart), and intestines (a drug for protecting the intestines), and very preferably for nerve cells and cardiomyocytes.

[0097] The pharmaceutical compositions of the present invention, comprising cedutinib and edaravone, are intended for concurrent, separate, or time-interval use as medicines, particularly for the prevention and treatment of individuals at risk and / or for individuals suffering from at least one pathological condition or at least one of the aforementioned degenerative processes. It should be understood that, for concurrent use, the compounds present in the pharmaceutical compositions comprising these compounds may be mixed together or physically separated; for separate or time-interval use, the present compounds must be physically separated.

[0098] The pharmaceutical compositions of the present invention, comprising cetuximab and edaravone, are used concurrently, separately, or at time intervals as cell-protective drugs or medicines for the prevention and / or protection and / or treatment of the consequences of the aforementioned pathological conditions or degenerative processes at the cellular level.

[0099] The pharmaceutical compositions of the present invention, comprising cedutinib and edaravone, are intended for simultaneous, separate, or time-interval use as cellular protective agents or medicines for the prevention and / or protection and / or treatment of the consequences of the aforementioned pathological conditions or degenerative processes at the cellular level and / or for the treatment of subjects suffering from one of the aforementioned pathological conditions or degenerative processes.

[0100] In a highly preferred form, the present invention relates to pharmaceutical compositions comprising cedutinib, edaravone, or a pharmaceutically acceptable salt or ester thereof, or an isomer thereof, or a semi-synthetic derivative thereof, or a salt thereof, or an ester thereof, or a deuterated compound thereof, or an isotopically labeled compound thereof, for use as a cellular protective agent or medicine for preventing and / or protecting and / or treating the consequences of the aforementioned pathological conditions or degenerative processes at the cellular level and / or treating a subject suffering from one of the aforementioned pathological conditions or degenerative processes.

[0101] Optionally, with respect to this aspect of the invention relating to the composition, consideration should also be given to the use of cedutinib and edaravone in the pharmaceutical compositions of the invention as cell-protective agents, as well as the implementation methods and definitions relating to administration and dosage.

[0102] The pharmaceutical composition of the present invention, comprising cedutinib and edaravone, is for use concurrently, separately, or at time intervals as a medicine for preventing and / or protecting and / or treating cells, tissues, and organs against ischemia / reperfusion injury (which may occur during actual cold or warm ischemia and / or actual reperfusion and / or ischemia / reperfusion).

[0103] In a highly preferred form, the present invention relates to pharmaceutical compositions as medicines for preventing and / or protecting and / or treating cells, tissues, and organs against ischemia / reperfusion injury (which may occur during actual cold or hot ischemia and / or actual reperfusion and / or ischemia / reperfusion).

[0104] In a highly preferred form, the present invention relates to pharmaceutical compositions as medicines for preventing and / or protecting and / or treating cells, tissues, and organs against degeneration and degenerative changes such as neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy, Huntington's disease, Parkinson's plus syndrome, and multiple sclerosis).

[0105] According to one embodiment of the present invention, a pharmaceutical composition of cedutinib and edaravone, wherein the amount of cedutinib can be from 0.0001 mg to 40000 mg / mg edaravone, preferably from 1 mg to 6000 mg (cedutinib) / mg edaravone. Optionally, the amount of edaravone can be from 0.0001 mg to 40000 mg / mg cedutinib, preferably from 0.01 mg to 100 mg edaravone / mg cedutinib.

[0106] According to the present invention, cedutinib and edaravone in the pharmaceutical composition may be a common dosage form. Also according to the present invention, cedutinib and edaravone in the composition may be the same dosage form or different dosage forms.

[0107] Therefore, it should be understood that, according to the present invention, when the composition of cedutinib and edaravone is a common dosage form, they can be administered simultaneously (i.e., at the same time) and via the same route of administration.

[0108] It should also be understood that, according to the present invention, when cedutinib and edaravone of the composition are in the same or different dosage forms, they can be administered simultaneously, sequentially or separately via the same or different routes of administration.

[0109] Preferably, when administered sequentially, cedutinib and edaravone are administered at intervals not exceeding about 1 hour (preferably not exceeding about 10 minutes, even more preferably not exceeding about 1 minute).

[0110] According to the present invention, cedutinib and edaravone can be mixed with one or more acceptable excipients or inert carriers (i.e., pharmaceutically inactive and non-toxic excipients) to impart a specific consistency or other specific physical or taste characteristics to the finished product, avoiding any chemical interaction between the two drugs. These include saline solutions, isotonic solutions, physiological solutions, buffer solutions, etc., which are compatible with the pharmaceutical use and known to those skilled in the art. The pharmaceutical compositions of the present invention may contain one or more reagents or media selected from the group consisting of: solubilizers, dispersants, stabilizers, sweeteners, preservatives, flavoring agents, lubricants, anti-caking agents, disintegrants, adsorbents, etc. In particular, reagents or media (liquid and / or injectable and / or solid) that can be used in formulations include: methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, polysorbate 80, cyclodextrin, gelatin, mannitol, lactose, PEG, animal oils or vegetable oils, etc. The pharmaceutical compositions of the present invention can be formulated as follows: suspensions or ready-to-use or temporary injection solutions, oils, gels, tablets, powders, suppositories, capsules, granules, emulsions, suspensions, polymers, microspheres, nanoparticles, rectal capsules, enemas, pastes, creams, ointments, plasters, implants, decoctions, sprays, aerosols, etc.; optionally, controlled release and / or sustained release can be achieved through dosage forms or devices. For this type of formulation, reagents such as carbonates, cellulose, or starch are advantageously used.

[0111] In one embodiment, the pharmaceutical composition may be formulated into a powder form and reconstituted for intravenous injection.

[0112] Administration may be performed by any method known to those skilled in the art, preferably orally or parenterally, such as by intramuscular injection, intravenous administration, subcutaneous injection, intra-arterial injection, intraperitoneal injection, intracerebral injection or intrathecal injection, oral administration, sublingual administration, delivery to the lesion or brain or implantation, or aerosol administration. Intramuscular injection, intravenous administration, subcutaneous injection or oral administration are preferred. For long-term treatment, oral, sublingual or transdermal routes of administration are preferred.

[0113] For injection, the compound may be packaged as a suspension or liquid solution and administered using a syringe or infusion set. It should be understood that those skilled in the art can adjust the amount to be administered or the volume and / or rate of injection based on pathology, route of administration, individual circumstances, etc. It should be understood that repeated administration may be performed in combination with other active ingredients and / or any pharmaceutically acceptable carrier (buffer solution, isotonic solution, saline solution, in the presence of stabilizers, etc.).

[0114] According to some aspects, the pharmaceutical compositions of the present invention can be administered before, during, or after a process that may lead to cell death, such as before surgery, during surgery requiring bypassing the heart, or when an individual is at risk of potential ischemic injury (e.g., myocardial ischemia or vascular ischemia). According to some aspects, the pharmaceutical compositions of the present invention can be administered after a process that may lead to cell death has occurred (e.g., after an infarction).

[0115] In cases of pathological conditions and / or degenerative processes and / or cell death processes caused at least in part by ischemia-reperfusion, the compositions of the present invention may be administered before and / or during and / or after ischemia and / or before and / or during and / or after reperfusion.

[0116] Whether the organ is in situ or ex vivo, contact between the organ and the composition of the present invention can be made by any known means, such as direct contact with the organ by spraying, irrigation, immersion, dipping, rinsing, etc.

[0117] Typically, the daily dose of the compound will be the minimum dose required to achieve the desired therapeutic effect. Optionally, for humans, the dose of cedutinib is typically from 0.01 mg / kg / day to 150 mg / kg / day, preferably from 0.1 mg / kg / day to 60 mg / kg / day, and even more preferably from 0.5 mg / kg / day to 10 mg / kg / day.

[0118] Alternatively, for humans, the dose of edaravone is typically from 0.001 mg / kg / day to 150 mg / kg / day, preferably from 0.01 mg / kg / day to 60 mg / kg / day, and even more preferably from 0.1 mg / kg / day to 1 mg / kg / day.

[0119] Optionally, as needed, the daily dosage may be administered as one, two, three, four, five, six or more doses per day, or as multiple sub-dose at appropriate intervals throughout the day.

[0120] Optionally, the amount selected may depend on a variety of factors, particularly the route of administration, duration of administration, time of administration, rate of elimination of the compound, different products used in combination with the composition, the individual's age, weight and physical condition, as well as the individual's medical history, the nature of the pathological condition or degenerative process he or she is facing, and any other medically known information.

[0121] Optionally, the doctor may begin with a lower dose than normally used and then gradually increase these doses to better control potential side effects. Preferably, the compositions of the present invention can be administered for a period of 1 day to 20 years, and even more preferably for a period of 1 day to 3 years.

[0122] The present invention also relates to a treatment method that may include administering a therapeutically effective amount of a pharmaceutical composition comprising cedutinib and edaravone to a person or animal in need.

[0123] The present invention also relates to a method for preparing a pharmaceutical composition comprising cedutinib and edaravone, wherein the compound of the composition of the present invention is mixed with an acceptable excipient (particularly a pharmaceutically acceptable excipient) according to methods known per se.

[0124] The present invention also relates to the use of pharmaceutical compositions containing cedutinib and edaravone in the preparation of organ preservation solutions. For example, organ preservation solutions containing pharmaceutical compositions containing cedutinib and edaravone can be prepared from any existing organ preservation solution (e.g., any solution used for infusion, storage, transport, and / or flushing of organs). Such solutions may be, for example, Belzer cryopreservation solution, preservation solutions, or mixtures thereof.

[0125] Based on the same inventive concept, the present invention also provides: an organ preservation solution comprising the pharmaceutical composition described above.

[0126] Further, in the organ preservation solution, the concentration of cedutinib is from 0.01 mg / L to 1000 mg / L, preferably from 0.1 mg / L to 100 mg / L, more preferably from 1 mg / L to 10 mg / L; and the concentration of edaravone is from 0.1 mg / L to 150 mg / L, preferably from 1 mg / L to 50 mg / L, more preferably from 1 mg / L to 10 mg / L.

[0127] The present invention also relates to a solution comprising a pharmaceutical composition of cedutinib and edaravone, intended for use in organ preservation (preservation solution). For example, the preservation solution of the present invention can be used to inject an organ in situ prior to retrieval from a donor, optionally to cool it, and / or after retrieval for, for example, static flushing and / or storage and / or transport of the organ, or by, for example, infusion via an infusion machine (with or without oxygen supply) at different temperatures ranging from hypothermia to normal body temperature.

[0128] According to the present invention, the preservation solution can contain an amount of the pharmaceutical composition of cedutinib and edaravone sufficient to prevent / mitigate / limit lesions caused by processes that can lead to cell death (particularly lesions caused by ischemia-reperfusion). For example, the concentration of cedutinib in the preservation solution used in the pharmaceutical composition containing cedutinib and edaravone can be from 0.01 mg / L to 1000 mg / L, preferably from 0.1 mg / L to 100 mg / L, and even more preferably from 1 mg / L to 10 mg / L. The concentration of edaravone in the preservation solution can be from 0.1 mg / L to 150 mg / L, preferably from 1 mg / L to 50 mg / L, and even more preferably from 1 mg / L to 10 mg / L.

[0129] Advantageously, the organ preservation solution of the present invention can be used for infusion and / or storage and / or transportation and / or flushing of organs, such as the liver, lungs, heart, kidneys, or pancreas, preferably the liver. Optionally, the pharmaceutical composition comprising cedutinib and edaravone of the present invention may be added to the solution several hours to minutes before using the organ preservation solution for infusion and / or storage and / or transportation and / or flushing of the organ.

[0130] Furthermore, a pharmaceutical composition comprising cedutinib and edaravone may be added to the preservation solution when the organ may already be present in the preservation solution. For example, the pharmaceutical composition comprising cedutinib and edaravone may be added to the preservation solution at any time during warm ischemia, cold ischemia, or reperfusion.

[0131] Optionally, according to the present invention, when cedutinib and edaravone in the pharmaceutical composition are of the same dosage form, they may be added simultaneously to the preservation solution. According to the present invention, when cedutinib and edaravone in the pharmaceutical composition are of the same dosage form or different dosage forms, they may be added simultaneously or sequentially to the preservation solution.

[0132] Another object of the present invention relates to a method for temporarily preparing an organ preservation solution comprising cedutinib and edaravone, the method comprising the step of mixing cedutinib and edaravone. According to the present invention, when cedutinib and edaravone are used to prepare a transplant organ preservation solution, the composition can be formulated into a form compatible with such use.

[0133] The present invention also relates to a method for preventing and / or protecting and / or treating organs, tissues or cells against ischemia-reperfusion injury (which may occur during actual warm or cold ischemia and / or actual reperfusion and / or ischemia-reperfusion), the method comprising contacting said organ, tissue or cells with an organ preservation solution of the composition of cedutinib and edaravone of the present invention.

[0134] Another object of the present invention relates to the use of the composition of cedutinib and edaravone of the present invention, the medicament, or the preservation solution of the present invention in the prevention and / or protection and / or treatment of organs, tissues, or cells against ischemia-reperfusion injury (which may occur during actual warm or cold ischemia and / or actual reperfusion and / or ischemia-reperfusion).

[0135] Another object of the present invention relates to the use of the cedutinib and edaravone compositions, pharmaceuticals, compositions or preservative solutions of the present invention in the prevention and / or protection and / or treatment of lesions in which organs, tissues or cells may resist during ischemia-reperfusion.

[0136] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0137] The pharmaceutical composition of the present invention has excellent cell protection effect. The combined use of cedutinib and edaravone shows a synergistic effect, which helps to achieve excellent efficacy, reduce drug dosage, improve clinical drug safety, and has good clinical application prospects.

[0138] The pharmaceutical compositions of the present invention can more effectively prevent and / or treat ischemia / reperfusion injury, especially nerve cell damage, and have excellent protective effects against ischemic stroke, significantly reducing cerebral ischemia / reperfusion injury. Attached Figure Description

[0139] Figure 1 Figure A shows the TTC staining and infarct volume measurement of mouse brain tissue from different groups in Example 1.1.

[0140] Figure 1 B is a graph showing the neurological function scores of mice in different groups in Example 1.1.

[0141] Figure 2 Figure A shows the infarct volume measurement of different groups of mouse brain tissue in Example 1.2.

[0142] Figure 2 B is a graph showing the neurological function scores of mice in different groups in Example 1.2.

[0143] Figure 3 Figure A shows the measurement of brain tissue and infarct volume in different groups of mice in Example 1.3.

[0144] Figure 3 B is a graph showing the neurological function scores of mice in different groups in Example 1.3.

[0145] Figure 4 A is a graph showing the infarct area of ​​myocardial tissue in different groups of mice in Example 2.

[0146] Figure 4 B is a graph showing the CK activity of different groups in Example 2.

[0147] Figure 5 A is a diagram showing the escape incubation period of different groups in Example 3.

[0148] Figure 5 B is a graph showing the ratio of target quadrant dwell time for different groups in Example 3.

[0149] Figure 6 A is a diagram showing the incubation period of different groups in Example 4.

[0150] Figure 6 B is a diagram showing the gripping force of different groups in Example 4. Detailed Implementation

[0151] The present invention will be described in detail below with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0152] The present invention will be described in detail below with reference to embodiments.

[0153] Application of cedutinib and edaravone in the preparation of cell protection drugs.

[0154] Materials and methods:

[0155] To demonstrate the role of edaravone and edaravone in cell protection, the applicant used various animal models, such as ischemic stroke mouse model, myocardial ischemia / reperfusion injury mouse model, Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Parkinson's disease, and administered cedutinib and edaravone at different time points. Cell damage and the protective effect of the drugs were detected according to the corresponding detection methods for each disease model.

[0156] Experimental reagents: Certutinib (compound) and Edaravone (compound) were purchased from a reagent company and dissolved and prepared according to the company's reagent instructions.

[0157] Example 1

[0158] Investigating the anti-ischemic stroke effect of the combination of cedutinib and edaravone.

[0159] Animal experiments: Using a mouse model of ischemic stroke, the effects of the combination of cedutinib and edaravone on ischemic stroke were investigated.

[0160] Experimental animals: 7-week-old male C57BL / 6J mice. All experimental animals were housed in an SPF-grade housing for one week at a temperature of 22℃±2℃, relative humidity of 45%±15%, with free access to water and following a 12-hour light / dark cycle. Then, they were administered drugs according to the requirements of each experimental group.

[0161] Methods for establishing a mouse model of ischemic stroke: A mouse model of cerebral ischemia / reperfusion was established using the middle cerebral artery occlusion (MCAO) method. The steps are as follows: Eight-week-old male C57BL / 6J mice were anesthetized by intraperitoneal injection of 0.3% (w / v) sodium pentobarbital (20 mL / kg). The left common carotid artery (CCA) was isolated, and the left external carotid artery (ECA) and internal carotid artery (ICA) were isolated superiorly. The ECA and ICA were temporarily clamped with ophthalmic forceps, and the proximal end of the CCA was ligated. Then, a knotted spare suture was placed at the distal end of the CCA, and a small slit was cut at the lower end of the suture. The suture was inserted into the internal carotid artery, the arterial clamps on the ECA and ICA were released, and the suture was advanced into the cranium along the ICA. When resistance was encountered, the suture was tightened and fixed. After 1 hour of ischemia, the suture was removed, the skin was sutured, and the animals were treated after 24 hours of reperfusion.

[0162] The Longa 5-point scale was used to score the neurological deficits in the mouse model of cerebral ischemia / reperfusion injury (neurological function score). 0 points: no neurological deficit symptoms; 1 point: right forelimb cannot be fully extended; 2 points: rotation to the right; 3 points: walking towards the right and falling; 4 points: inability to walk spontaneously, loss of consciousness. Scores of 1-4 indicate a valid model.

[0163] TTC staining and infarct volume determination in mouse brains. After anesthetizing mice, the brains were quickly removed, the olfactory bulb and hindbrain were discarded, and 3-5 coronal slices were taken starting from the frontal pole. These slices were immediately placed in 1% (w / v) TTC solution and incubated at 37°C in the dark for 30 min. Then, they were fixed by soaking in 10% (w / v) paraformaldehyde solution. Infarcted areas appeared white, and non-infarcted areas appeared red. Each group of brain slices was neatly arranged and scanned. ImageJ was used to measure the infarct area of ​​each slice, and the corresponding volume was calculated using the formula: Infarct volume (%) = (Volume of brain tissue contralateral to the infarct - Volume of brain tissue in the non-infarcted area on the infarcted side) / Volume of brain tissue contralateral to the infarct × 100%.

[0164] Depending on the administration method and dosage, the study was divided into three parts: detecting the neurological function score of mice and measuring the cerebral infarction volume to evaluate the drug's efficacy.

[0165] Statistical analysis: GraphPad Prism 9.0 software was used for statistical analysis. All data are expressed as mean ± standard error (±SEM). Differences among multiple groups were analyzed using one-way ANOVA or two-way ANOVA, followed by Tukey's test to compare whether there were significant differences between groups. Neurological function scores were analyzed using Kruskal-Wallis and Wilcoxon tests. P < 0.05 was considered statistically significant (the same applies below).

[0166] Example 1.1

[0167] Experimental grouping and administration method: The experimental animals were randomly divided into the following 7 groups. All relevant drugs were dissolved in the solvent (10% DMSO + 30% PEG400 + 60% physiological saline), i.e.:

[0168] Sham group: The internal and external carotid arteries were separated without inserting sutures into the arteries.

[0169] Cerebral ischemia / reperfusion group (I / R group): cerebral ischemia for 1 hour (hour, h), reperfusion for 24 hours.

[0170] Cerdulatinib + I / R: During the above-mentioned reperfusion period, cerebral ischemia / reperfusion group (Cerdulatinib + I / R) was administered intramuscularly 1 hour after reperfusion.

[0171] Edaravone + I / R group: During the above reperfusion period, edaravone (6 mg / kg) was administered intramuscularly 1 hour after reperfusion.

[0172] Cerdulatinib + Edaravon + I / R: During the above reperfusion period, 1 hour after reperfusion, cerebral ischemia / reperfusion group was given intramuscular injection of cerebral ischemia / reperfusion group (Cerdulatinib + Edaravon + I / R): Cerdulatinib (15 mg / kg) and edaravon (3 mg / kg) were administered intramuscularly.

[0173] Cerdulatinib + Edaravon + I / R: During the above reperfusion period, 1 hour after reperfusion, cerebral ischemia / reperfusion group was given intramuscular injection of cerebral ischemia / reperfusion group (Cerdulatinib + Edaravon + I / R): Cerdulatinib (7.5 mg / kg) and edaravon (6 mg / kg) were administered intramuscularly.

[0174] The solvent + cerebral ischemia / reperfusion group (Vehicle + I / R) was given an intramuscular injection of solvent 1 hour after reperfusion during the above reperfusion period.

[0175] The solvent consists of 10% DMSO, 30% PEG400, and 60% physiological saline (v / v, the same below); cedutinib and / or edaravone are dissolved in the solvent and then injected (the same below).

[0176] The neurological function scores of mice and the volume of cerebral infarction were measured to evaluate the drug's efficacy.

[0177] result:

[0178] Effects of the combination of cedutinib and edaravone on cerebral infarction volume and neurological function in mice

[0179] like Figure 1 As shown in the figure, the I / R group had obvious white infarct foci, while the cedutinib, edaravone monotherapy group and the combination therapy group reduced the infarct volume in mice. Figure 1 A) and improving neurological function ( Figure 1 B), and the effect of combination therapy was significantly better than that of monotherapy. (Data are expressed as mean ± standard error, n=6, **P<0.01 vs sham surgery group; # P<0.05 vs I / R group; + P<0.05 vs I / R + drug alone group).

[0180] Conclusion: The combination of cedutinib and edaravone is more effective than the single drug, showing a synergistic effect. It can significantly reduce neuronal cell death, alleviate cerebral ischemia / reperfusion injury, and has a neuroprotective effect. It can be used to prepare drugs to alleviate cerebral ischemia / reperfusion injury for the treatment of ischemic stroke.

[0181] Example 1.2

[0182] Experimental grouping and administration: The experimental animals were randomly divided into the following 6 groups. All relevant drugs were dissolved in the solvent (10% DMSO + 30% PEG400 + 60% physiological saline), i.e.:

[0183] Sham group: The internal and external carotid arteries were separated without inserting sutures into the arteries.

[0184] Cerebral ischemia / reperfusion group (I / R group): cerebral ischemia for 1 hour, reperfusion for 24 hours.

[0185] Cerdulatinib + I / R group: During the above-mentioned reperfusion period, cerebral ischemia / reperfusion group (Cerdulatinib + I / R) was administered intramuscularly 1 hour after reperfusion.

[0186] Edaravone + I / R group: During the above reperfusion period, edaravone (3 mg / kg) was administered intramuscularly 1 hour after reperfusion.

[0187] Cerdulatinib + Edaravon + I / R: During the above reperfusion period, 1 hour after reperfusion, cerebral ischemia / reperfusion group was given intramuscular injection of cerebral ischemia / reperfusion group (Cerdulatinib + Edaravon + I / R): Cerdulatinib (7.5 mg / kg) and edaravon (3 mg / kg) were administered intramuscularly.

[0188] The solvent + cerebral ischemia / reperfusion group (Vehicle + I / R) was given an intramuscular injection of solvent 1 hour after reperfusion during the above reperfusion period.

[0189] The solvent consists of 10% DMSO, 30% PEG400 and 60% physiological saline; cedutinib and / or edaravone are dissolved in the solvent and then injected (the same applies below).

[0190] The neurological function scores of mice and the volume of cerebral infarction were measured to evaluate the drug's efficacy.

[0191] result:

[0192] Effects of the combination of cedutinib and edaravone on cerebral infarction volume and neurological function in mice

[0193] The I / R group had obvious white infarct foci, such as Figure 2 As shown, cedutinib, edaravone monotherapy, and combination therapy reduced the infarct volume in mice. Figure 2 A) and improving neurological function ( Figure 2 B), and the effect of combination therapy was significantly better than that of monotherapy. (Data are expressed as mean ± standard error, n=6, **P<0.01 vs sham surgery group; # P<0.05 vs I / R group; + P<0.05 vs I / R + drug alone group).

[0194] Conclusion: The combination of cedutinib and edaravone is more effective than the single drug, showing a synergistic effect. It can significantly reduce neuronal cell death, alleviate cerebral ischemia / reperfusion injury, and has a neuroprotective effect. It can be used to prepare drugs to alleviate cerebral ischemia / reperfusion injury for the treatment of ischemic stroke.

[0195] Example 1.3

[0196] Experimental grouping and administration method: The experimental animals were randomly divided into the following 6 groups. All relevant drugs were dissolved in the solvent (10% DMSO + 30% PEG400 + 60% physiological saline), i.e.:

[0197] Sham group: The internal and external carotid arteries were separated without inserting sutures into the arteries.

[0198] Cerebral ischemia / reperfusion group (I / R group): cerebral ischemia for 1 hour, reperfusion for 24 hours.

[0199] Cerdulatinib + I / R: During the above-mentioned reperfusion period, cerebral ischemia / reperfusion group (1.5 mg / kg) was administered intraperitoneally 1 h and 5 h after reperfusion.

[0200] Edaravone + I / R group: During the above reperfusion period, edaravone (dose: 4 mg / kg) was administered intraperitoneally 1 h and 5 h after reperfusion.

[0201] Cerdulatinib + Edaravon + I / R: During the reperfusion period, cerdulatinib (dose: 1.5 mg / kg per administration) and edaravon (dose: 4 mg / kg per administration) were administered intraperitoneally 1 h and 5 h after reperfusion, respectively. Cerdulatinib and edaravon were administered simultaneously at each administration.

[0202] The solvent + cerebral ischemia / reperfusion group (Vehicle + I / R): During the above reperfusion period, the solvent was administered intraperitoneally 1 hour and 5 hours after reperfusion.

[0203] The solvent consists of 10% DMSO, 30% PEG400, and 60% physiological saline; cedutinib and / or edaravone are dissolved in the solvent and then injected.

[0204] The neurological function scores of mice and the volume of cerebral infarction were measured to evaluate the drug's efficacy.

[0205] result:

[0206] Effects of the combination of cedutinib and edaravone on cerebral infarction volume and neurological function in mice

[0207] The I / R group had obvious white infarct foci, such as Figure 3 As shown, cedutinib, edaravone monotherapy, and combination therapy (cedutinib + edaravone + cerebral ischemia / reperfusion group) reduced the infarct volume in mice. Figure 3 A) and improving neurological function ( Figure 3 B), and the effect of combination therapy was significantly better than that of monotherapy. (Data are expressed as mean ± standard error, n=6, **P<0.01 vs sham surgery group; # P<0.05 vs I / R group ## P<0.01 vs I / R group; ++ P<0.01 vs I / R + drug alone group).

[0208] Conclusion: The combination of cedutinib and edaravone is more effective than the single drug, showing a synergistic effect. It can significantly reduce neuronal cell death, alleviate cerebral ischemia / reperfusion injury, and has a neuroprotective effect. It can be used to prepare drugs to alleviate cerebral ischemia / reperfusion injury for the treatment of ischemic stroke.

[0209] Example 2

[0210] Investigating the effects of the combination of cedutinib and edaravone on myocardial ischemia / reperfusion injury.

[0211] Animal experiments: Using a mouse model of myocardial ischemia / reperfusion injury, the effects of the combination of cedutinib and edaravone on myocardial ischemia / reperfusion injury were investigated.

[0212] Experimental animals: 7-week-old male C57BL / 6J mice. All experimental animals were housed in an SPF-grade housing for one week at a temperature of 22℃±2℃, relative humidity of 45%±15%, with free access to water and following a 12-hour light / dark cycle. Then, they were administered drugs according to the requirements of each experimental group.

[0213] Methods for establishing a mouse model of myocardial ischemia / reperfusion: Eight-week-old male C57BL / 6J mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital (20 mL / kg), followed by endotracheal intubation, with the endotracheal tube fixed using pressure-sensitive adhesive. Ventilator parameters were set as follows: tidal volume 3.2 mL / kg, frequency 110 breaths per minute. The left anterior descending artery (LAD) was ligated using 8-0 sutures after thoracotomy. Myocardial infarction was confirmed by whitening at the apex of the heart, and the ligation was maintained for 1 hour. After 1 hour of ischemia, the LAD suture knot was loosened to restore cardiac perfusion. The thoracic cavity was sutured layer by layer, and the ventilator was removed; the mice regained spontaneous breathing. Twenty-four hours after cardiac perfusion was restored, the mice were anesthetized again with 0.3% sodium pentobarbital (20 mL / kg), and the LAD was ligated again in situ after thoracotomy. The abdominal cavity was opened, and 0.2 mL of 2% (w / v) Evans Blue solution was injected via the inferior vena cava. When the lower lip of the mouse turned blue, blood was collected from the apex of the heart, and the heart was removed. The heart was frozen at -20°C for 1 hour, then sliced ​​into 1 mm thick sections. The sections were then incubated with 1% TTC staining solution (prepared in PBS) at 37°C for 15 minutes. After staining, the staining solution was removed, the heart was washed once with PBS, and then fixed with 4% (w / v) paraformaldehyde for 24 hours. The heart sections were then removed, the staining was observed, and photographs were taken. The areas of ischemic and infarcted regions were determined using ImageJ software.

[0214] The blue area in the heart slice represents normal tissue; the area outside the blue area is the ischemic area (also known as the risk area, AOR); the white area is the infarct area (AOI). The percentage of infarct area (the ratio of the infarct area AOI to the ischemic area AOR) in each slice is calculated, and the ratio of the infarct area is compared between the drug group and the model group.

[0215] Serum creatine kinase (CK) activity assay

[0216] After ischemia / reperfusion surgery, approximately 150 μL of whole blood was collected from the mouse orbital cavity. The blood was centrifuged at 3000 rpm, 4 ºC for 10 min, and the supernatant was collected and stored at -40 ºC. Serum CK activity was measured according to the instructions of a commercially available kit as follows: 10 mL of R2 was dissolved in one vial of R1 to prepare the working solution. 4 μL of serum was added to 200 μL of the CK kit working solution and incubated at 37 ºC for 2 min. The wavelength of the microplate reader was set to 340 nm. Absorbance readings (A0, A1, A2, A3) were taken at 0, 1, 2, and 3 min, respectively. The change in average absorbance per minute (ΔA) was calculated, and the concentration of CK in the serum (U / L) was calculated.

[0217] Experimental grouping: Experimental animals were randomly divided into groups of 6. Based on preliminary experimental results, drug concentrations and administration times were set. The drug groups were treated with different concentrations of cedutinib, edaravone, and a combination of both drugs, respectively.

[0218] Sham group: The mice underwent heart surgery but did not have their blood vessels ligated.

[0219] Model group (myocardial ischemia / reperfusion group): left anterior descending coronary artery was ligated for 1 hour for ischemia treatment, the suture was cut, and reperfusion was performed for 24 hours;

[0220] Cerdulatinib + I / R: Cerdulatinib (dose: 1.5 mg / kg) was administered intraperitoneally to mice 30 min after ischemia and 5 h after reperfusion.

[0221] Edaravone + myocardial ischemia / reperfusion group (Edaravon + I / R): Mice were given intraperitoneal injection of edaravon (dose: 4 mg / kg each time) 30 min after ischemia and 5 h after reperfusion.

[0222] Cerdulatinib + Edaravon + I / R: Mice were administered cerdulatinib (dose: 1.5 mg / kg per dose) and edaravon (dose: 4 mg / kg per dose) intraperitoneally 30 min after ischemia and 5 h after reperfusion, respectively. Cerdulatinib and edaravon were administered simultaneously at each dose.

[0223] The solvent + myocardial ischemia / reperfusion group (Vehicle + I / R) was treated with solvent after mice were ischemic for 30 min.

[0224] The solvent consists of 10% DMSO, 30% PEG400, and 60% physiological saline.

[0225] Blood and myocardial tissue were collected and relevant indicators were measured: myocardial infarction area was measured in mice and serum creatine kinase activity (CK activity) was detected.

[0226] result:

[0227] like Figure 4 As shown, cedutinib, edaravone monotherapy, and the combination of the two drugs have anti-myocardial ischemia / reperfusion injury effects in mice, and can reduce the infarct area of ​​myocardial tissue in mice. Figure 4 A), reduces serum creatine kinase ( Figure 4 B), and the effect of combination therapy was significantly better than that of monotherapy. (Data are expressed as mean ± standard error, n=6, **P<0.01 vs sham surgery group; # P<0.05 vs I / R group; + P<0.05 ++ P<0.01 vs I / R + drug alone group).

[0228] Conclusion: The combination of cedutinib and edaravone is more effective than single-drug therapy, exhibiting a synergistic effect. It can significantly reduce cardiomyocyte death, alleviate myocardial ischemia / reperfusion injury, and has a cardioprotective effect. It can be used to prepare drugs to alleviate myocardial ischemia / reperfusion injury for the treatment of myocardial infarction.

[0229] Example 3

[0230] Investigating the effects of the combination of cedutinib and edaravone on Alzheimer's disease

[0231] Animal experiments: Using the APP / PS1 transgenic mouse model of Alzheimer's disease, the effects of the combination of cedutinib and edaravone on Alzheimer's disease were investigated.

[0232] Laboratory animals: APP / PS1 transgenic mice (Alzheimer's disease animal model, with C57BL / 6J mice as the background), male, 5 months old, weighing 24–30 g, and age-matched C57BL / 6J mice (male, 5 months old, weighing 26–30 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. All laboratory animals were housed in an SPF-grade enclosure with a temperature of 22℃±2℃, relative humidity of 45%±15%, free access to water, and following a 12-hour light / dark cycle.

[0233] Experimental grouping: Five-month-old APP / PS1 transgenic mice were randomly divided into groups of 6. Based on the preliminary experimental results, the drug concentration and administration time were set. The drug groups were treated with different concentrations of cedutinib, edaravone, and a combination of the two drugs, respectively. The drugs were administered intraperitoneally starting from 5 months of age, once daily for 2 consecutive months. The normal control group and the APP / PS1 mouse control group were given the same volume of solvent.

[0234] Normal control group: APP / PS1 transgenic background mice - C57BL / 6J mice + intraperitoneal injection of solvent.

[0235] APP / PS1 (AD) + solvent group: APP / PS1 transgenic mice + intraperitoneal injection of solvent.

[0236] APP / PS1 (AD) + Cerdulatinib group: APP / PS1 transgenic mice were treated with intraperitoneal injection of cerdulatinib (0.75 mg / kg).

[0237] APP / PS1 (AD) + Edaravone group: APP / PS1 transgenic mice were treated with intraperitoneal injection of edaravone (4 mg / kg).

[0238] APP / PS1 (AD) + Cerdulatinib + Edaravone: APP / PS1 transgenic mice were treated with Cerdulatinib (0.75 mg / kg) and Edaravone (4 mg / kg).

[0239] The solvent consists of 10% DMSO, 30% PEG400, and 60% physiological saline; cedutinib and / or edaravone are dissolved in the solvent and then injected.

[0240] The learning and memory abilities and cognitive functions of mice were tested using experiments such as the new object recognition test and the Morris water maze test.

[0241] Data statistics: Statistical analysis was performed using GraphPad Prism 9.0 software. All data are expressed as mean ± standard error (±SEM). Differences among multiple groups were analyzed using one-way ANOVA or two-way ANOVA, followed by Tukey's test to compare whether there were significant differences between groups.

[0242] Detection methods and results:

[0243] (1) Effects of the combination of cedutinib and edaravone on non-spatial learning and memory ability and cognitive function in Alzheimer's disease model mice - new object recognition experiment.

[0244] The experiment was conducted according to the requirements for new object recognition, and included three phases: adaptation, training, and testing. For one week prior to the experiment, the mice were petted for 2-3 minutes daily to reduce stress. A 40×40cm open-field test chamber was used. Before the experiment, the mice were placed in the test chamber to acclimatize for 20-30 minutes. After acclimatization, the new object training experiment began. Two objects (A and B) of identical color, shape, and material were placed in the test chamber, and the mice were placed in it for 10 minutes of training. Twenty-four hours after the training period, a new object testing experiment was conducted to assess the mice's short-term non-spatial learning memory. One of the two objects was replaced with another object of different shape and color (C), and the time taken for the mice to explore the two different objects within 10 minutes was recorded. Cognitive index = (New object exploration time / (New object exploration time + Old object exploration time)) × 100%.

[0245] Experimental results: Compared with normal control mice, the cognitive index of APP / PS1 + solvent group mice was significantly reduced. The non-spatial learning and memory ability and cognitive function of APP / PS1 transgenic mice were weakened. After administration of cedutinib, edaravone monotherapy and the combination of the two drugs, the cognitive index of APP / PS1 transgenic mice was significantly increased. The effect of the combination of the two drugs was significantly better than that of the monotherapy.

[0246] Conclusion: The combination of cedutinib and edaravone significantly improved non-spatial learning and memory abilities and cognitive function in Alzheimer's disease mice.

[0247] (2) Effects of the combination of cedutinib and edaravone on spatial learning and memory ability and cognitive function in Alzheimer's disease model mice—Morris water maze test.

[0248] Following the requirements of the water maze experiment, the Morris water maze was divided into four quadrants. A platform with a diameter of 12cm and a height of 35cm was placed in one quadrant (the target quadrant). Water was added to submerge the platform by 1-2cm, and the water was dyed black with carbon ink. After five days of orientation and navigation experiments, the platform was removed, and a spatial exploration experiment was conducted. The time it took for the mice to reach the original platform location (the latency period) and the time spent in the target quadrant (the time spent in the quadrant where the original hidden platform was located) were recorded. The percentage of time spent in the target quadrant relative to the total exploration time was analyzed. The animals' movement trajectories and behaviors were recorded using the Smart 3.0 small animal analysis software system, and the relevant data were analyzed.

[0249] Experimental results are as follows Figure 5 As shown in the figure, compared with the normal control group mice, the APP / PS1 + solvent group mice had a significantly increased time to find the original platform location (escape latency), and a significantly decreased time spent in the target quadrant and the ratio of time spent in the target quadrant. The APP / PS1 mice given cedutinib, edaravone monotherapy, or a combination of both drugs had a significantly reduced time to find the original platform (escape latency). Figure 5 A) The time spent in the target quadrant and the percentage of time spent in the target quadrant increased significantly. Figure 5 B) In summary, the combined use of cedutinib and edaravone was significantly more effective than any single-drug group. (Data are expressed as mean ± standard error, n=6, **P<0.01 vs normal control group) ## P < 0.01 vs APP / PS1 + solvent group + P < 0.05 (vs APP / PS1 + single-drug group). The results indicate that the combination of cedutinib and edaravone significantly improved learning and memory abilities and cognitive function in Alzheimer's disease mice. Conclusion: The combination of cedutinib and edaravone significantly improved learning and memory abilities and cognitive function in Alzheimer's disease mice, exhibiting a synergistic effect.

[0250] Example 4

[0251] Investigating the effects of the combination of cedutinib and edaravone on amyotrophic lateral sclerosis (ALS).

[0252] 4.1 Animal experiments: One of the ALS animal models, B6SJL.SOD1, was used. G93A (abbreviated as SOD1) G93A A mouse model was used to investigate the effect of the combination of cedutinib and edaravone on anti-amyotrophic lateral sclerosis.

[0253] Experimental animals: ALS model was B6SJL.SOD1 G93A Mouse model, known SOD1G93A Mice with SOD1 gene mutations reproduced the pathogenesis of human ALS very well. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively affects upper and lower motor neurons.

[0254] Male SOD1 G93A The transgenic ALS mouse model was purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. All experimental animals were housed in an SPF-grade housing with a temperature of 22℃±2℃, a relative humidity of 45%±15%, free access to water, and following a 12-hour light / dark cycle.

[0255] Experimental grouping and administration method: SOD1 G93A Mice were randomly divided into groups of six at 60 days of age based on their motor function. Drug concentrations and administration times were determined according to preliminary experimental results. The drug groups received different concentrations of cedutinib, edaravone, or a combination of both drugs. Intraperitoneal injections were administered starting at 71 days of age (11 weeks) once daily until death. The normal control group and SOD1... G93A The mouse control group was given the same volume of solvent.

[0256] Normal control group: C57BL / 6J mice of the corresponding age + intraperitoneal injection of solvent

[0257] ALS+ solvent group (solvent group): SOD1 G93A Mouse + solvent intraperitoneal injection

[0258] ALS + Cerdulatinib group: SOD1 G93A Mice were treated with intraperitoneal injection of cedutinib (0.75 mg / kg).

[0259] ALS + Edaravon combination (+Edaravon): SOD1 G93A Mice treated with intraperitoneal injection of edaravone (6 mg / kg)

[0260] ALS + Cerdulatinib + Edaravon: SOD1 G93A Mice were treated with cedutinib (0.75 mg / kg) and edaravone (6 mg / kg).

[0261] The solvent consists of 10% DMSO, 30% PEG400, and 60% physiological saline; cedutinib and / or edaravone are dissolved in the solvent and then injected.

[0262] The motor function and survival rate of mice were assessed using indicators such as the rotarod test, limb grip strength test, and survival time measurement.

[0263] Data statistics: Statistical analysis was performed using GraphPad Prism 9.0 software. All data are expressed as mean ± standard error (±SEM). Differences among multiple groups were analyzed using one-way ANOVA or two-way ANOVA, followed by Tukey's test to compare whether there were significant differences between groups.

[0264] (1) Rotarod experiment

[0265] Three days before the test, the animals were allowed to acclimatize to the test environment for 30 minutes. Then, the mice were placed on a YLS-4C rotundus fatigue tester, starting at 5 rpm and increasing to 30 rpm within 180 seconds. Specific steps: Mice were first trained for 5 minutes with a rotation speed of 30 rpm, twice daily for three days. At the start of the test, three consecutive tests were conducted (180 seconds per test, with a 40-minute interval between each test). The experiment lasted 180 seconds, and the time it took for any animal to fall off the platform was recorded as the latency period, or until the end of the experiment. The mouse's time on the rotundus was recorded each time, and the longest time spent on the rotundus across the three tests was taken as the mouse's motor function. If the time exceeded 180 seconds, the test was stopped, and 180 seconds was used as the baseline. SOD1 G93A Mice were tested for motor function using the aforementioned rotarod fatigue tester starting at 60 days of age, and then observed and tested once a week until the mice died.

[0266] (2) Grip strength test

[0267] The basic method is as follows: the strength of the mouse's limb muscles is directly assessed using a mouse grip strength tester. The mouse is gently placed on the central platform of the grip strength tester, and its tail is gently pulled to encourage it to grasp the grip plate. As the mouse grips the plate, a further pull is applied to force it to release its grip. The maximum grip strength is recorded at this point. After the mouse completely releases the grip plate, the instrument automatically records the maximum grip strength value. This value is recorded. The grip strength of each mouse's forelimb and hindlimb is measured three times, and the average value is taken as the grip strength value of its forelimb or hindlimb.

[0268] (3) Survival determination: For mice that underwent behavioral evaluation, the survival rate after administration of cedutinib and edaravone was evaluated using Kaplan-Meier curves.

[0269] 4.2 Results:

[0270] 4.2.1. Rotarod test, behavioral test of ALS model mice, such as... Figure 6 As shown in A, SOD1 G93A Compared with the normal control group, the mice in the control group showed significantly weakened muscle strength and a significantly shorter time to fall off the rotisserie. Treatment with cedutinib, edaravone monotherapy, and the combination of both drugs delayed SOD1 activation. G93A The mice exhibited decreased muscle strength; among them, treatment with a combination of cedutinib and edaravone resulted in reduced SOD1 levels at 84 days of age. G93A The latency period (latency) of mice on the rotarod was significantly longer than that of the ALS solvent control group, the cedutinib group, and the edaravone monotherapy group, with statistically significant differences (P<0.05 or 0.01), which improved the motor function of mice. (Data are expressed as mean ± standard error, n=6, **P<0.01 vs normal control group;) ## P<0.01 vs ALS+ solvent group; + P<0.05 (vs ALS + drug alone)

[0271] 4.2.2. Grip test, such as Figure 6 As shown in B, SOD1 G93A Compared with the normal control group, the hind limb grip strength of the mice in the mouse group was significantly weakened. Compared with ALS model mice, treatment with cedutinib, edaravone monotherapy, and the combination of the two drugs improved SOD1 levels. G93A The hind limb grip strength of mice was reduced, while the combination of cedutinib and edaravone treatment resulted in lower SOD1 levels at 91 days of age. G93A The hindlimb grip strength of mice was significantly higher than that of the ALS solvent control group, the cedutinib group, and the edaravone monotherapy group, with statistically significant differences (P<0.05 or 0.01). (Data are expressed as mean ± standard error, n=6, **P<0.01 vs normal control group;) ## P<0.01 vs ALS+ solvent group; + P<0.05 (vs ALS + drug alone)

[0272] Compared with the normal control group, the survival time of ALS model mice was significantly shortened. The survival time of mice treated with cedutinib, edaravone monotherapy, and the combination of the two drugs was significantly longer than that of the ALS model group, while the survival time of mice in the combination therapy group was significantly better than that of mice treated with monotherapy.

[0273] Conclusion: Treatment with cedutinib, edaravone monotherapy, and the combination of the two drugs prolonged the survival time and improved the motor function of ALS mice. The combination of cedutinib and edaravone was more effective than the monotherapy, showing a synergistic effect. The combination of cedutinib and edaravone can be used to treat amyotrophic lateral sclerosis.

[0274] Example 5

[0275] Investigating the anti-Parkinson's effect of the combination of cedutinib and edaravone

[0276] Animal experiments: PD animal models were prepared using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to investigate the anti-Parkinson's effect of the combination of cedutinib and edaravone.

[0277] Establishment of the MPTP animal model: A PD animal model was established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Male C57BL / 6 mice, 10-12 weeks old and weighing 25-30g, were given three days of pre-acclimatization training, and mice with poor motor coordination were removed. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was injected intraperitoneally at a dose of 30-40 mg / kg body weight for 5-7 consecutive days. Temporary symptoms such as trunk shaking, piloerection, tail hyperextension, reduced movement, and difficulty climbing poles appeared after the 5th-7th injection, indicating successful establishment of the Parkinson's disease (PD) mouse model.

[0278] The Parkinson's disease score is as follows: 0 points, similar to normal mice, no symptoms; 1 point, piloerection, arched back, intermittent fine tremors, but still able to move freely; 2 points, frequent swallowing, frequent tremors, hind limbs outstretched, tail tremor, and gradually restricted movement; 3 points, drooling, persistent tremors, limb stiffness, and restricted movement; 4 points, death due to general paralysis.

[0279] Experimental grouping: Experimental animals were randomly divided into groups of 6-10. Based on preliminary experimental results, drug concentrations and administration times were set. The drug groups were treated with different concentrations of cedutinib, edaravone, and a combination of both drugs, respectively.

[0280] Normal control group (Control group): C57BL / 6 mice

[0281] MPTP model group: MPTP intraperitoneal injection for 5-7 days.

[0282] Cerdulatinib + MPTP model group: Cerdulatinib was administered after modeling.

[0283] Edaravon + MPTP model group: Edaravon is applied after modeling.

[0284] Cerdulatinib + Edaravon + MPTP model group: Cerdulatinib and edaravon were administered during model creation.

[0285] Testing methods: The muscle strength and motor balance of the limbs of mice were tested using methods such as the pole climbing test and stiffness score.

[0286] (1) Pole test: The basic method is as follows: Place a rough wooden ball with a diameter of 9 mm on the top of a rough wooden stick with a circular cross-section and a length of 75 cm. Place the bottom of the stick in a mouse cage. Place the mouse on the ball and train it to climb to the bottom of the stick with its head down one day in advance. On the day of the experiment, use a stopwatch to record the time it takes for the mouse to climb to the top of the stick as A, the time it takes to climb to the bottom of the stick as B, and the time it takes for the mouse to climb the entire stick as C. C=AB. Each mouse is tested 3 to 5 times, and the average time of the 3 to 5 climbing tests is used as the statistical index.

[0287] (2) Rigidity scoring: The basic method is as follows: The mouse is held in a fixed position, with its forelimbs placed on a wooden stick 4 cm above the horizontal plane and 1 cm wide. A stopwatch is used to record the length of time the animal maintains this posture. The timer is stopped when both forelimbs are removed from the stick or the animal moves its head in an exploratory manner. The cutoff time is 300 seconds. Each mouse is tested 3 times, with an interval of 1 minute between each test. If the mouse maintains this posture for more than 30 seconds, it is considered rigid. The score is based on the time: 0 seconds = 0 points, 1 second to 150 seconds = 1 point, and 151 seconds to 300 seconds = 2 points.

[0288] Experimental results:

[0289] Compared with the control group, the time required for mice to climb the pole in the MPTP model group was significantly increased and the rigidity score was significantly elevated. Treatment with cedutinib, edaravone monotherapy, and the combination of the two drugs could significantly shorten the pole climbing time and reduce the rigidity score. The combination of cedutinib and edaravone was more effective than the monotherapy.

[0290] Conclusion: The combined use of cedutinib and edaravone is more effective than the single drug, showing a synergistic effect. It can significantly improve motor function and rigidity symptoms in Parkinson's mice, reduce neuronal death, and has a neuroprotective effect. It can be used to prepare anti-Parkinson's drugs.

[0291] Example 6

[0292] Investigating the anti-multiple sclerosis effect of the combination of cedutinib and edaravone.

[0293] Animal experiments: The effects of the combination of cedutinib and edaravone against multiple sclerosis were investigated using an experimental autoimmune encephalomyelitis (EAE) mouse model.

[0294] Experimental animals: 6-8 week old female C57BL / 6J mice, weighing 18-25 grams. All experimental animals were housed in an SPF-grade housing for one week at a temperature of 22℃±2℃, relative humidity of 45%±15%, with free access to water and following a 12-hour light / dark cycle. Then, modeling and drug administration were carried out according to the requirements of each experimental group.

[0295] Establishment of an experimental autoimmune encephalomyelitis (EAE) mouse model:

[0296] The basic experimental steps are as follows: First, prepare MOG. 35～55 The peptide was diluted with PBS to a concentration of 3 mg / ml (MOG); Mycobacterium tuberculosis was added to Freund's incomplete adjuvant (CFA) to prepare a concentration of 5 mg / ml Freund's complete adjuvant; MOG and CFA were mixed in a 1:1 ratio to prepare an antigen adjuvant emulsion. After anesthetizing mice, 0.2 ml / mouse was injected subcutaneously into the neck and back, divided into four injection points (randomly selected four points along the spine), 50 μl at each point. Subsequently, 0.1 ml (3 μg / ml, i.e., 300 ng) of pertussis toxin was injected intraperitoneally. 48 h later, 0.1 ml of pertussis toxin was injected intraperitoneally again. After immunization with MOG, mice were routinely fed and their general condition was observed. The first day of immunization was recorded as Day 0. Then, at the same time every day, two experimenters scored the neurological dysfunction of each group of mice until 28 days after MOG immunization. The modified Kono 5-point scoring scale was used for scoring, and the specific scoring details are as follows:

[0297] 0 points (no clinical symptoms); 0.5 points (decreased tail tension, with drooping tip); 1 point (tail dragging on the ground); 1.5 points (incomplete paralysis of one hind limb); 2 points (complete paralysis of one hind limb, able to roll over independently); 2.5 points (complete paralysis of one hind limb, accompanied by incomplete paralysis of the other hind limb); 3 points (complete paralysis of both hind limbs, unable to roll over independently, but can move on the ground after stimulation); 3.5 points (complete paralysis of both hind limbs, accompanied by paralysis of one forelimb); 4 points (paralysis of all four limbs or accompanied by urinary and fecal incontinence); 5 points (dying state or death).

[0298] The mice's food intake, water intake, activity, weight, and fur were recorded. A score of 0.5 to 5 was used to determine if a mouse was diseased. The time from the establishment of the model to the onset of the disease was recorded as the incubation period, and the time from the onset of the disease to the peak of the disease (no increase in the neurological dysfunction score for 3 consecutive days) was recorded as the progression period. Diseased mice were sacrificed at the peak of the disease, while unaffected mice and normal control mice were observed until 28 days after the establishment of the model and then sacrificed.

[0299] Experimental grouping: Experimental animals were randomly divided into groups of 6-10. Based on preliminary experimental results, drug concentrations and administration times were set. The drug groups were treated with different concentrations of cedutinib, edaravone, and a combination of both drugs, respectively.

[0300] Normal control group (Control group): Unmodeled C57BL / 6J mice + solvent

[0301] EAE model group (EAE group): model group + solvent.

[0302] Cerdulatinib + EAE model group: Cerdulatinib was administered after model creation.

[0303] Edaravon + EAE model group: Edaravon is applied after modeling.

[0304] Cerdulatinib + Edaravon + EAE model group: Cerdulatinib and edaravon were administered after model creation.

[0305] Detection methods: The modified Kono 5 score was used to detect the neurological function of mice and to detect changes in body weight.

[0306] result:

[0307] Effects of cedutinib, edaravone monotherapy and combination therapy on neurological function and body weight in mice.

[0308] Compared with the control group, the EAE model group showed clinical symptoms such as decreased tail tension and increased neurological function scores 7-11 days after MOG immunization. Treatment with cedutinib, edaravone monotherapy and the combination of the two drugs can reduce the neurological function scores of mice and improve their neurological function. The combination of cedutinib and edaravone is more effective than monotherapy.

[0309] Conclusion: The combination of cedutinib and edaravone is more effective than the single drug, showing a synergistic effect. It can significantly improve the neurological function of EAE mice, reduce neuronal death, and has a neuroprotective effect. It can be used to prepare drugs against multiple sclerosis for the treatment of multiple sclerosis.

[0310] The above embodiments show that the drug composition containing cedutinib and edaravone can be used to treat cerebral ischemia / reperfusion injury (including ischemic stroke), myocardial ischemia / reperfusion injury (myocardial infarction), Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and multiple sclerosis. The combination therapy is more effective than monotherapy, showing a synergistic effect, which helps to ensure efficacy and reduce drug dosage, thereby improving the safety of clinical drug use while ensuring clinical treatment effect.

[0311] However, this invention is not limited to the above-mentioned diseases; the drug is also applicable to the treatment of other diseases with similar pathogenesis.

[0312] The above embodiments should be understood as being used only to illustrate the present invention more clearly, and not to limit the scope of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims.

Claims

1. A pharmaceutical composition, characterized in that, It contains cedutinib and edaravone, wherein the mass ratio of cedutinib to edaravone is 3:10 to 1:

20.

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of cedutinib to edaravone is 3:10 to 1:

10.

3. The pharmaceutical composition according to claim 2, characterized in that, The mass ratio of cedutinib to edaravone is 0.75:4 to 1:

9.

4. The pharmaceutical composition according to claim 3, characterized in that, The mass ratio of cedutinib to edaravone is 0.75:4 to 1:8.

5.

5. The pharmaceutical composition according to claim 4, characterized in that, The mass ratio of cedutinib to edaravone is 0.75:4 to 1:

8.

6. The pharmaceutical composition according to claim 5, characterized in that, The mass ratio of cedutinib to edaravone is 0.75:4 to 0.75:

6.

7. The use of the pharmaceutical composition according to any one of claims 1-6 in the preparation of a cell-protective drug, characterized in that, The cell-protective drug is a drug used to prevent, protect against, and / or treat cell damage caused by neurodegenerative diseases.

8. The application according to claim 7, characterized in that, The neurodegenerative diseases include one or more of Alzheimer's disease and amyotrophic lateral sclerosis.

9. Use of the pharmaceutical composition according to any one of claims 1-6 in the preparation of a medicament for the prevention, protection and / or treatment of neurodegenerative diseases.

10. The application according to claim 9, characterized in that, The neurodegenerative diseases include one or more of Alzheimer's disease and amyotrophic lateral sclerosis.

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