Application of selutinib in the preparation of drugs for treating and / or preventing neurodegenerative diseases and cell protection drugs

Abstract

The present invention relates to the use of selutinib in the preparation of drugs for treating and / or preventing neurodegenerative diseases and cell protection drugs. Selutinib significantly reduces nerve cell damage and significantly improves the learning and memory ability and cognitive function of Alzheimer's disease mice, reduces nerve cell death, and has a nerve cell protection effect.

Description

Technical Field

[0001] The present invention relates to the use of seletinib in preparing drugs for treating and / or preventing neurodegenerative diseases and cell protection drugs, and belongs to the field of biomedicine. The present invention provides new uses and administration methods of seletinib, including the effects of anti-Alzheimer's disease, reducing the damage of nerve cells in Alzheimer's disease; and the effects of cell protection drugs for protecting organs, tissues or cells from cell damage and dysfunction, expanding the scope of indications of seletinib; in addition, the present invention also covers the use of seletinib in preparing cell damage protection drugs. Background Art

[0002] Neurodegenerative diseases are a general term for diseases that are caused by the gradual loss or deterioration of nerve cells / neurons (cell bodies, nuclei, axons, terminals) and / or nerve myelin sheaths over time, resulting in dysfunction. There are many common neurodegenerative diseases in clinical practice, including Alzheimer's disease, Parkinson's disease, spinocerebellar ataxia, etc.

[0003] Alzheimer's disease (AD) is a common degenerative disease of the central nervous system characterized by progressive cognitive dysfunction and behavioral impairment. It is common in the elderly and is also known as senile dementia. Clinically, it manifests as learning and memory decline, cognitive decline, visual-spatial disorders, personality and behavioral changes, etc. In severe cases, it enters a state of complete dementia. The main pathological features of Alzheimer's disease are the accumulation of β-amyloid protein in the cell matrix to form senile plaques, the hyperphosphorylation of tau protein to form neurofibrillary tangles, and the degeneration and loss of neurons. Its nerve cell damage and degeneration also involve mechanisms such as oxidative stress, inflammatory response, and mitochondrial dysfunction. AD lacks effective therapeutic drugs. Existing drugs can only partially relieve symptoms and delay the course of the disease but cannot cure AD, and have obvious side effects. With the aging of the population, the incidence of AD will increase year by year, and it is of great significance to seek effective drugs for the treatment of AD.

[0004] Studies have shown that RIPK1 / RIPK3 / MLKL-dependent necroptosis exists in a variety of injury-related diseases, including ischemic stroke, Alzheimer's disease and other neurodegenerative diseases, and inhibiting RIPK1 / RIPK3 / MLKL-dependent necroptosis can inhibit or alleviate cell death and tissue damage caused by the above diseases.

[0005] Cerdulatinib is an oral multi-target tyrosine kinase inhibitor that can inhibit spleen tyrosine kinase (SYK) and Janus kinase (JAK), significantly reduce the cell activity of some non-Hodgkin's lymphoma (NHL) cell lines, and induce apoptosis of NHL cell lines with BCR signals. It can be used to treat peripheral T-cell lymphoma. However, whether Cerdulatinib has anti-necroptosis and cytoprotective effects, as well as anti-neurodegenerative diseases such as Alzheimer's disease, has not been reported. Summary of the invention

[0006] In view of the deficiencies of the prior art, one of the objects of the present invention is to provide the use of seletinib in the preparation of drugs for treating and / or preventing neurodegenerative diseases; a second object of the present invention is to provide the use of seletinib in the preparation of cell protection drugs.

[0007] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0008] Use of selutinib and / or its pharmaceutically acceptable salt in the preparation of a medicament for treating and / or preventing neurodegenerative diseases.

[0009] The structural formula of selenotinib is shown in Formula I, and the molecular formula is C 20 H 27 N 7 O 3 S:

[0010]

[0011] Furthermore, the neurodegenerative disease includes one or more of Alzheimer's disease, Parkinson's disease, Huntington's disease, and spinocerebellar ataxia.

[0012] Optionally, the active ingredient of the drug includes selegiline or a pharmaceutically acceptable salt thereof.

[0013] Furthermore, selutinib is used as a compound (drug) or a pharmaceutically acceptable salt, or cocrystal, or any stereoisomer, tautomer, hydrate, or solvate thereof, but not as an anti-tumor drug.

[0014] The method for treating or preventing Alzheimer's disease with seletinib comprises administering an effective amount of seletinib to a patient.

[0015] Furthermore, the administration of the drug includes one or more of oral administration, intramuscular injection, subcutaneous injection, intravenous injection, sublingual administration, intralesional or intracerebral or implanted delivery, and spray administration, preferably intramuscular, subcutaneous or intravenous injection.

[0016] Furthermore, the drug is administered orally, intramuscularly, subcutaneously or intravenously.

[0017] Furthermore, the drug can be prepared into any pharmaceutically acceptable dosage form.

[0018] Further, the dosage form includes one of a suspension or a ready-to-use injection solution or a temporary injection solution, a gel, an oil, a tablet, a suppository, a powder, a capsule, an oral liquid, a lozenge, a granule, a pill, a powder, an ointment, a pill, a suspension, an emulsion, a polymer, a nanoparticle, a microsphere, a rectal capsule, an enema, a paste, a patch, an ointment, a milky agent, a plaster, a drink, an implant, a spray, an aerosol, a drop, a patch, a pill, etc., and optionally, controlled release and / or sustained release are performed by a dosage form or a device. Wherein the preferred dosage form is a tablet, an injection, such as an injection, a capsule, a tablet, a granule, a powder, a spray, a liposome, an oral liquid, a pill.

[0019] Optionally, selenotinib is a pharmaceutically acceptable salt thereof, and the pharmaceutically acceptable salt is a salt commonly used in pharmacy. Further, the salt is selected from one or more of 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, benzoylsulfonate, hydroquinonesulfonate and p-toluenesulfonate, or salts formed by carboxylic acids (such as formic acid, acetic acid or propionic acid).

[0020] Use of selutinib and / or its pharmaceutically acceptable salt in the preparation of cell protection drugs.

[0021] Furthermore, the cell protection drug refers to a drug that has the function of preventing, inhibiting and / or treating damage, degeneration and / or dysfunction of tissues, organs and / or cells.

[0022] Furthermore, the cytoprotective drugs include drugs for preventing, protecting and / or treating cells from damage to cells caused by pathological conditions or decay processes; preferably, the pathological conditions or decay processes include one or more of the diseases or conditions caused by pathological apoptosis, pathological necrosis, necroptosis, pyroptosis, ferroptosis, dependent cell death, disulfide death, and autophagy (anti-apoptotic drugs and / or anti-necrosis drugs and / or anti-necroptosis drugs and / or anti-pyroptosis drugs and / or anti-ferroptosis drugs and / or anti-dependent cell death drugs and / or anti-autophagy drugs).

[0023] Optionally, cytoprotective drugs include drugs for organs or cells such as the brain, lungs, heart, liver, spleen, blood vessels, intestines, kidneys, pancreas, skin, eyes, corneas, joints, etc. to resist cell death or processes that cause cell death, such as drugs for treating diseases caused by necrosis and / or pathological apoptosis and / or necroptosis and / or ferroptosis and / or pyroptosis and / or disulfide death and / or autophagy, or drugs for resisting surgical operations that may cause cell death processes.

[0024] Trauma and factors that trigger the cell death process can be of biological and / or chemical and / or physical origin.

[0025] Alternatively, biological sources include: asphyxia, ischemia / reperfusion, hypoxia or anoxia, nutritional deprivation, in situ generated free radicals or poisoning caused by reactive oxygen species, growth factor deficiency, cytotoxins or massive release of cytokines. It may also come from certain events, such as hemorrhage, accidental occlusion (infarction) and certain medical operations (such as using airbag inflation, artificial respirator, suture), as well as biological or chemical agents used as therapeutic agents in medical treatment (such as immunosuppressants, cell inhibition or cytotoxic agents, anti-inflammatory drugs).

[0026] Optionally, chemical sources include poisoning by toxicants, waste, pH changes, free radicals, reactive oxygen species, environmental toxins.

[0027] Optionally, physical sources include impact, laceration, exposure to radiation (X-radiation, gamma radiation, UV radiation, etc.), hyperthermia, hypothermia, or the presence of foreign matter or crystals in the organism.

[0028] Alternatively, surgical procedures that may lead to cell death processes may be, for example, procedures that require a brief interruption of blood circulation leading to systemic or local ischemia or hypoperfusion (such as the use of tourniquets, hemostats, etc.), for example during surgery, in particular during procedures such as angioplasty of organs or the heart or major or peripheral blood vessels or thoracic surgery that sometimes requires (bypassing) the cardiopulmonary system or stopping the heart, cardiac surgery or vascular surgery, as well as any surgery that requires voluntary occlusion of an organ or part of an artery or reduction of blood flow through an organ.

[0029] Alternatively, 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 disulfide death and / or autophagy.

[0030] Further, the diseases or conditions include one or more of nervous system diseases, circulatory system diseases, bleeding and thrombotic diseases, bone diseases, joint diseases and cartilage diseases, ischemic diseases or attacks of limbs, ophthalmic diseases, skin diseases, kidney diseases, blood diseases 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, diseases related to aging, dental diseases, auditory conduction pathway diseases, mitochondrial-related diseases, trauma and / or exposure to biological sources and / or chemical sources and / or physical sources and / or and / or medical operations and / or surgical operations, such as accidental infarction and bleeding, and / or medical operations and / or surgical operations, such as cell, tissue or organ transplantation.

[0031] Furthermore, the neurological diseases include hemorrhagic stroke, ischemia, intracranial hemorrhage, cerebral hemorrhage, prenatal cerebral hypoxia, adult or childhood cerebral hypoxia, Alzheimer's disease, Parkinson's disease, infantile spinal muscular atrophy, Huntington's disease, disease), Parkinson plus syndrome, trigeminal neuralgia, glossopharyngeal neuralgia, muscle disease, Bell's palsy, progressive bulbar palsy, spinal muscular atrophy, primary lateral sclerosis (PLS), pseudobulbar palsy, invertebrate disc syndrome, cervical spondylosis, hereditary muscular atrophy, plexus disorder, thoracic outlet disruption syndrome, porphyria, peripheral neuropathy, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, dementia with Lewy bodies, demyelinating disease, frontotemporal dementia, Guillain-Barré syndrome, Creutzfeldt-Jakob disease, progressive Charcot-Marie-Tooth disease, prion disease, fatal familial insomnia (FFI), Gerstmann-Strauss-Schaefer syndrome (GSS), bovine spongiform encephalopathy, epilepsy, Pick's disease, AIDS dementia syndrome, different types of spinocerebellar ataxia (SCA), intervertebral disc herniation, scoliosis, nerve damage caused by exposure to toxic compounds in the group consisting of industrial solvents, heavy metals, drugs and chemotherapeutic agents, nervous system damage caused by mechanical, physical or chemical trauma, lysosomal storage disease, Niemann-Pick disease, Gaucher disease, pain, one or more thereof; preferably, the muscle disease includes muscular dystrophy, and the muscular dystrophy includes one or more of Duchenne's muscular dystrophy, myotonic muscular dystrophy, myopathy and myasthenia, progressive muscular dystrophy; preferably, the pain includes one or more of neuropathic pain, inflammatory pain, and diabetic pain;

[0032] Circulatory system diseases include one or more of anoxia, hypoxia, chronic or acute heart failure, systolic heart failure and diastolic heart failure, left ventricular dysfunction, left ventricular dysfunction after myocardial infarction, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, myocardial hypertrophy, hypertrophic cardiomyopathy, myocarditis, valvular heart disease, arrhythmia, paroxysmal tachycardia, atrial fibrillation, ventricular fibrillation, peripheral vascular disease, aneurysm, chronic venous insufficiency or varicose veins, hypertension, systemic hypertension, pulmonary hypertension, portal hypertension, and cardiovascular toxicity caused by drug (especially anticancer drug) treatment;

[0033] Myocardial remodeling includes one or more of myocardial remodeling after myocardial ischemia / hypoxia (such as myocardial infarction), myocardial remodeling after cardiac surgery, myocardial remodeling after aortic valve (membrane) disease, vascular hypertrophy (smooth muscle cell hypertrophy), and vascular remodeling;

[0034] Bleeding and thrombotic diseases include one or more of vascular permeability disorders, bone diseases, joint diseases and cartilage diseases occurring after thrombolytic therapy or coronary angioplasty, such as osteoporosis, osteomyelitis, avascular necrosis, spondyloarthropathies, rickets, fibromatosis ossificans progressiva, and Cushing's syndrome;

[0035] Ophthalmic diseases include one or more of diabetic retinopathy, glaucoma, retinal degeneration, retinitis pigmentosa corneal reticular dystrophy, optic neuropathy and optic neuritis, optic nerve drusen, ptosis, chronic progressive ophthalmoplegia, macular degeneration, retinal hole or retinal tear, retinal ischemia, retinal ischemia / reperfusion injury, retinal detachment, acute retinopathy associated with trauma, inflammatory degeneration, postoperative complications, drug-induced retinopathy or cataract, wet or dry AMD-related photoreceptor degeneration;

[0036] Renal diseases include one or more of renal fibrosis, acute renal disease, renal ischemia, renal capillary infarction, acute renal injury, acute or chronic interstitial nephropathy, glomerulonephritis, diabetic nephropathy, renal arteriosclerosis, renal insufficiency, acute or chronic renal failure or dialysis side effects, and renal failure after myocardial ischemia / reperfusion;

[0037] Pulmonary diseases include pulmonary hypertension, acute respiratory distress syndrome, respiratory tract infection, chronic obstructive pulmonary disease such as chronic bronchitis and emphysema, cystic fibrosis, and cystic lung disease.

[0038] Gastrointestinal diseases include ulcer or mesenteric infarction, portal hypertension or one or more of them;

[0039] Liver diseases include one or more of autoimmune hepatitis, viral hepatitis or hepatitis caused by other infectious agents, liver fibrosis, alcoholic liver disease (ALD), alcoholic hepatitis, fulminant hepatitis, cirrhosis, liver disease caused by toxins or drugs, and steatosis, where steatosis is caused by liver ischemia or drug-induced exogenous poisoning, alcoholic or non-alcoholic steatohepatitis (NASH);

[0040] Metabolic diseases include one or more of diabetes, diabetic complications, diabetic nephropathy, diabetic retinopathy, diabetic foot disease, thyroiditis, Hashimoto's thyroiditis, glucose intolerance syndrome, obesity, abetalipoproteinemia, hyperlipidemia, hypothalamic-pituitary axis dysfunction, diabetes insipidus, galactosemia, glycogen disease, gout, Wilson's disease or Weber-Christian disease;

[0041] Pancreatic diseases include chronic pancreatitis or acute pancreatitis;

[0042] Severe poisoning caused by chemical agents, infectious agents, toxins or drugs including sepsis, septic shock and their consequences or iatrogenic diseases;

[0043] Aging-related diseases include accelerated aging syndrome;

[0044] Dental diseases include those that cause tissue damage, such as periodontitis;

[0045] Auditory pathway disorders include antibiotic-induced deafness and otosclerosis;

[0046] Mitochondrial-related diseases (mitochondrial pathology) include congenital muscular dystrophy with structural mitochondrial abnormalities and Friedrich's ataxia;

[0047] Trauma and / or exposure to biological and / or chemical and / or physical sources and / or medical and / or surgical procedures including unexpected bleeding, cell, tissue or organ transplantation.

[0048] Advantageously, the above-mentioned drugs are used to prevent and / or protect and / or treat nerve cells (drugs to protect brain cells), cardiomyocytes (drugs to protect the heart), liver (drugs to protect the liver), and kidneys (drugs to protect the kidneys), and are preferably used to protect nerve cells (drugs to protect the brain), cardiomyocytes (drugs to protect the heart), and liver (drugs to protect the liver), and more preferably nerve cells and cardiomyocytes.

[0049] Use of selutinib and / or a pharmaceutically acceptable salt thereof in preparing an in vitro tissue and / or organ preservation solution.

[0050] Optionally, the ex vivo tissue and / or organ includes one or more of the brain, lung, heart, liver, spleen, blood vessels, intestines, kidneys, pancreas, skin, eyes, cornea, and joints.

[0051] The present invention relates to selenotinib as a cytoprotective drug, which is used for preventing and / or protecting and / or treating cell death of transplanted organs and / or organ donors and / or organ recipients, and / or increasing long-term survival rate, and / or limiting primary organ dysfunction and / or limiting delayed recovery of the function of transplanted organs and / or improving functional recovery of transplanted organs, mainly preventing or treating cell death of transplanted organs.

[0052] Advantageously, selecitinib can be used in living or clinically dead organ donors, tissue donors, cell donors, organ recipients, tissue recipients, cell recipients before, during or after transplantation; and / or more specifically, organs, tissues or cells are not only in situ organs, tissues or cells (e.g. in medicine, surgery or in pathological processes), but also ex vivo organs, tissues or cells (e.g. in certain specific surgeries that require temporary removal of organs, tissues or cells from the body, especially those for their modification or purification, or during the transportation and storage of organs, tissues or cells, during transplantation or during their reperfusion after reimplantation of organs, tissues or cells).

[0053] Thus, this prevention and protection can be carried out in a general manner on an individual, donor or recipient, or on an organ, tissue or cell in situ or ex vivo, for example during certain surgeries, or during its transport or during its storage for reimplantation.

[0054] In a preferred form, selutinib is used to prevent and / or protect and / or treat: neurological sequelae due to stroke or trauma, heart failure due to infarction, tissue damage affecting the heart, liver, intestine, lung or kidney after transplantation or surgery, or damage caused by surgical procedures.

[0055] Furthermore, the drug as described above is used in the preparation of a method for preventing or treating cardiovascular toxicity of anticancer drugs, wherein the anticancer drugs include but are not limited to anthracyclines, tyrosine kinase inhibitors, fluorouracils, VEGF signaling pathway inhibitors, immune checkpoint inhibitors, platinum antitumor drugs, and other antitumor drugs.

[0056] Furthermore, the anticancer drugs include anthracycline antibiotics including doxorubicin (i.e., adriamycin), epirubicin (i.e., epirubicin), pirarubicin, aclarubicin, idarubicin, daunorubicin, mitoxantrone, etc.; tyrosine kinase inhibitors, such as ninotinib, sunitinib, lapatinib, regorafenib, ponatinib and dasatinib; fluorouracils including fluorouracil, capecitabine, tegafur, tegafur; VEGF signaling pathway inhibitors including bevacizumab; immune checkpoint inhibitors such as trastuzumab; platinum anti-tumor drugs such as cisplatin; other anti-tumor drugs such as paclitaxel and cyclophosphamide.

[0057] Unless otherwise stated, with regard to the use involving selutinib, the embodiments and definitions used to describe the use of selutinib of the present invention as an anti-Alzheimer's disease and cytoprotective drug and related to administration and dosage should also be considered.

[0058] In a preferred embodiment, cilutinib can be used as a drug for preventing and / or protecting and / or treating neurodegenerative diseases, such as one or more of Alzheimer's disease, Parkinson's disease, and spinocerebellar ataxia.

[0059] The inventors found that seletinib has the effect of resisting hypoxia / reoxygenation and necroptosis-like apoptosis inducer TSZ (TNF-α, SM-164 and Z-VAD-FMK)-induced neuronal cell damage, inhibiting neuronal cell necroptosis, reducing neuronal cell death, and significantly improving the learning and memory ability and cognitive function of APP / PS1 transgenic mice (Alzheimer's disease mouse model), and has a neuronal cell protective effect and an anti-Alzheimer's disease effect. The inventors found that seletinib has a neuronal cell protective effect and can significantly improve the learning and memory ability and cognitive function of Alzheimer's disease mice.

[0060] The inventors unexpectedly discovered that selegiline has the effect of resisting hypoxia / reoxygenation and necroptosis-like apoptosis inducer TSZ-induced neuronal cell damage, has the protective effect of neuronal cells, can reduce the death of Alzheimer's disease neuronal cells, significantly improve the behavioral function of APP1 / PS1 transgenic Alzheimer's disease mice, and significantly improve the learning and memory ability and cognitive function of Alzheimer's disease mice. The drug of the present invention has a significant effect of improving Alzheimer's disease, and may be used to treat Alzheimer's disease, and may further be used for the protection of organs such as the heart and brain, and has good development and application prospects. The inventors found that selegiline has a neuronal cell protective effect and can significantly improve the learning and memory ability and cognitive function of Alzheimer's disease mice.

[0061] The present invention expands the indications that can be resisted or treated by seletinib, and can be applicable to cell protection and anti-Alzheimer's disease.

[0062] The present invention relates to the use of seletinib in the preparation of drugs for treating Alzheimer's disease and cell protection drugs, which is the first disclosure, and can significantly reduce nerve cell damage and significantly improve the learning and memory ability and cognitive function of Alzheimer's disease mice, reduce nerve cell death, and have a nerve cell protective effect, which is unexpected, has nothing to do with the known uses of seletinib, and there is no other existing compound to provide relevant inspiration, has outstanding substantive characteristics, has significant progress in the treatment of Alzheimer's disease and cell protection, and has good application prospects. The present invention finds that seletinib has a protective effect on nerve cells and can significantly improve the learning and memory ability and cognitive function of Alzheimer's disease mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 These are graphs showing the effect of seletinib on cell viability and LDH release of neural cells (HT-22, SH-SY5Y) treated with hypoxia / reoxygenation and necroptosis inducer TSZ in Example 1, A-graph showing the effect of seletinib on cell viability of HT-22 treated with hypoxia / reoxygenation; B-graph showing the effect of seletinib on cell viability of SH-SY5Y treated with hypoxia / reoxygenation; C-graph showing the effect of seletinib on cell viability of HT-22 treated with necroptosis inducer TSZ; D-graph showing the effect of seletinib on cell viability of SH-SY5Y treated with necroptosis inducer TSZ; E-graph showing the effect of seletinib on LDH release of SH-SY5Y treated with hypoxia / reoxygenation; F-graph showing the effect of seletinib on LDH release of SH-SY5Y treated with necroptosis inducer TSZ.

[0064] Figure 2Figure 1 is a graph showing the regulatory effect of selenotinib on the necroptosis-related molecule MLKL protein and phosphorylation of neural cells (SH-SY5Y) treated with hypoxia / reoxygenation and necroptosis inducer TSZ in Example 1. A-graph showing the regulatory effect of selenotinib on the necroptosis-related molecule MLKL protein and phosphorylation of neural cells (SH-SY5Y) treated with hypoxia / reoxygenation; B-graph showing the regulatory effect of selenotinib on the necroptosis-related molecule MLKL protein of neural cells (SH-SY5Y) treated with hypoxia / reoxygenation; C-graph showing the regulatory effect of selenotinib on the necroptosis-related molecule MLKL protein of neural cells (SH-SY5Y) treated with hypoxia / reoxygenation A-Statistical graph showing the regulatory effect of Ceruleinib on the phosphorylation of necroptosis-related molecule MLKL; D-Statistical graph showing the regulatory effect of Ceruleinib on the protein and phosphorylation of necroptosis-related molecule MLKL in nerve cells (SH-SY5Y) treated with necroptosis inducer TSZ; E-Statistical graph showing the regulatory effect of Ceruleinib on the protein and phosphorylation of necroptosis-related molecule MLKL in nerve cells (SH-SY5Y) treated with necroptosis inducer TSZ; F-Statistical graph showing the regulatory effect of Ceruleinib on the phosphorylation of necroptosis-related molecule MLKL in nerve cells (SH-SY5Y) treated with necroptosis inducer TSZ.

[0065] Figure 3 This is the effect of selenotinib on the learning and memory ability and cognitive function of APP / PS1 transgenic Alzheimer's disease model mice in Example 2 - novel object recognition experiment.

[0066] Figure 4 Figure 2 shows the effects of selenotinib on the learning and memory ability and cognitive function of APP / PS1 transgenic Alzheimer's disease model mice in Example 2 - Y maze experiment.

[0067] Figure 5 Figure 2 shows the effects of selegiline on spatial learning and memory ability and cognitive function of APP / PS1 transgenic Alzheimer's disease model mice in Example 2 - Morris water maze experiment, A-time (latency) for mice to find the original platform, B-number of times mice cross the platform. DETAILED DESCRIPTION

[0068] The present invention will be described in detail below with reference to embodiments.

[0069] The use of seletinib in the preparation of drugs for treating Alzheimer's disease and drugs for cell protection.

[0070] Materials and methods:

[0071] To prove that selecitinib has anti-Alzheimer's disease and cell protection effects, the applicant used an APP / PS1 transgenic Alzheimer's disease mouse model and treated it with selecitinib every day. The applicant used novel object recognition experiments, Y-maze experiments and Morris water maze experiments to detect neuronal cell damage and the learning and memory abilities and cognitive functions of mice.

[0072] To demonstrate the protective effect of selenotinib on cells, the applicant used a hypoxia / reoxygenation injury model and a necroptosis cell injury model induced by the necroptosis inducer TSZ (TNF-α+SM-164+Z-VAD-FMK), and treated the cells with selenotinib. Cell viability and cell damage were detected by MTS and LDH, respectively.

[0073] APP / PS1 transgenic male mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0074] The drugs used in each example: Cerutinib (the compound of Cerutinib, whose structural formula is shown in Formula I) was purchased from a reagent company, and TSZ was purchased from Beyotime (Jiangsu).

[0075] Example 1

[0076] Cell experiment: The protective effect of selenotinib on HT-22 and SH-SY5Y neural cells treated with hypoxia / reoxygenation and necroptosis inducer TSZ.

[0077] HT-22 and SH-SY5Y neural cells were purchased from Xiangya Cell Bank of Central South University, and TSZ was purchased from Beyotime (Jiangsu).

[0078] Cell culture method: According to the conventional cell culture method, the above HT-22 and SH-SY5Y neural cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum). When the cells grew to 80-90% confluence, they were digested with trypsin. When the cells shrank and became round and the intercellular gaps were obvious, the digestion was immediately terminated with culture medium. The cells were dispersed and blown into a single suspension state, and then sub-bottled and passaged. The cells were incubated at 37°C with 5% CO 2 cultured in a cell culture incubator.

[0079] 1.1 Establishment of Hypoxia / Reoxygenation (H / R) Injury Model of HT-22 and SH-SY5Y Neuronal Cells and Drug Treatment

[0080] When the fusion rate of cultured HT-22 or SH-SY5Y cells reaches 80% to 90%, the plates can be inoculated with DMEM high-glucose medium containing 10% fetal bovine serum and incubated in an atmosphere of 95% air, 5% CO 2The cells were cultured in a cell culture incubator at 37°C. When the fusion rate reached about 60%, they were synchronized for 12 hours using DMEM high-glucose medium containing 1% fetal bovine serum. Then, sugar-free DMEM was replaced with 95% N 2 , 5% CO 2 The cells were cultured in a cell culture incubator at 37°C for 8 h. After the hypoxia ended, the cells were replaced with DMEM high-glucose culture medium containing 10% fetal bovine serum and 95% air and 5% CO. 2 , and continue culturing in a cell culture incubator at 37°C for 24 h.

[0081] The experimental groups are as follows:

[0082] Normal control group (Control group): cultured under normoxic conditions for 36 h;

[0083] Hypoxia / reoxygenation group (H / R group): hypoxia (1% O 2 )8h, reoxygenation 24h;

[0084] Hypoxia / reoxygenation + Cerdulatinib group [+ Cerdulatinib group]: 5-4000 nM Cerdulatinib was added to the cell culture medium 1 hour before hypoxia and during reoxygenation, and hypoxia was carried out for 8 hours and reoxygenation for 24 hours. Cerdulatinib was first dissolved in DMSO and then diluted to the working concentration;

[0085] Hypoxia / reoxygenation + solvent group (DMSO): During hypoxia / reoxygenation, an equal volume of solvent (DMSO) as that of the selegiline group was added to the culture medium.

[0086] MTS was used to detect cell viability and LDH release rate was used to detect cell damage.

[0087] 1.2 Establishment of necroptosis model of HT-22 and SH-SY5Y neural cells induced by TSZ and drug treatment:

[0088] When the fusion rate of cultured HT-22 or SH-SY5Y cells reaches 80% to 90%, the plates can be inoculated with high-glucose DMEM containing 10% fetal bovine serum and incubated in an atmosphere of 95% air, 5% CO 2 , and cultured in a cell culture incubator at 37°C. When the fusion rate reached about 60%, the cells were synchronized with high-glucose DMEM medium containing 1% fetal bovine serum for 12 h, and then replaced with high-glucose DMEM medium containing TSZ (dilution ratio of 1:250) for 24 h.

[0089] The experimental groups are as follows:

[0090] Normal control group (Control group): cultured under normal conditions;

[0091] TSZ group: TSZ treatment for 24 h;

[0092] TSZ+Cerdulatinib group: 5-10000 nM Cerdulatinib was added to the cell culture medium during TSZ treatment;

[0093] TSZ+vehicle group (DMSO): During TSZ treatment, an equal volume of vehicle (DMSO) as that of the selegiline group was added to the culture medium.

[0094] MTS was used to detect cell viability and LDH release rate was used to detect cell damage.

[0095] 1.3 MTS cell viability assay

[0096] The assay was performed according to the MTS kit [Promega (Beijing) Biotech Co., Ltd) operating instructions, and the groups were designed according to the experimental requirements, and a background blank control group (no cells) was set up. When the cell fusion rate reached 80-90%, it was inoculated into a 96-well plate. When the cell fusion rate was about 60%, the subsequent experiment was started. After the experimental treatment was completed, MTS was added to the 96-well plate in the dark at 10 μl / well, and incubated in the dark for about 1 hour. The absorbance at 490nm was measured by an enzyme marker (the absorbance was controlled at 0.7-1.2). The size of the absorbance value reflects the relative size of the cell viability. Calculation: Cell viability (%) = absorbance of the drug group / absorbance of the control group × 100%. When calculating the absorbance value of each group, the absorbance value of the background blank control group was first subtracted.

[0097] 1.4 Determination of lactate dehydrogenase (LDH) release rate

[0098] The assay was performed according to the operating instructions of the lactate dehydrogenase kit (Biyuntian, Jiangsu). The groups were designed according to the experimental requirements, and a background blank control group (no cells) and a maximum enzyme release group were set up. The LDH release reagent with a volume of 1 / 10 of the original culture medium was added to the sample maximum enzyme activity well group, and the culture was continued for 1 hour. 120 μL of supernatant was aspirated from each well and added to a new 96-well plate. 60 μL of LDH detection working solution was added to each well, mixed, incubated at room temperature for 30 minutes (avoiding light), and the sample absorbance was measured at 490 nm. Calculation: Cell LDH release rate (%) = absorbance of each group (including drug treatment group) / absorbance of maximum enzyme release group × 100%. When calculating the absorbance value of each group, the absorbance value of the background blank control group was subtracted first.

[0099] 1.5 Western blot (WB) detection of MLKL and phosphorylated MLKL (p-MLKL) expression levels

[0100] According to the conventional method, the cells were collected, washed with PBS, and lysed with 5-7 times the cell pressure. The cells were lysed in an ice bath for 1 hour, and vortexed every 10 minutes. After lysis, the cells were centrifuged at 4°C and 12000rpm for 15 minutes, and the supernatant was taken to determine the protein concentration by BCA method. 20-30μg protein samples were separated by 8% SDS-PAGE gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking, they were incubated with MLKL and p-MLKL (Abcam, Cambridge, UK) antibodies and β-actin (Beyotime, Jiangsu) antibodies at 4°C overnight, washed, and incubated with horseradish peroxidase (HRP)-conjugated corresponding secondary antibodies (Beyotime, Jiangsu), and developed by Molecular Imager ChemiDoc XRS System (Bio-Rad, Philadelphia, PA), with β-actin as an internal reference. The protein gray value was determined by Image J software to detect the protein expression level.

[0101] 1.4 Experimental results:

[0102] 1.4.1 Selenotinib has the ability to resist hypoxia / reoxygenation and necroptosis inducer TSZ-induced HT-22 and SH-SY5Y neuronal cell damage, reduce hypoxia / reoxygenation and TSZ-induced HT-22 and SH-SY5Y neuronal cell death (manifested by increased cell viability and decreased LDH release rate), and has a neuronal cell protective effect.

[0103] Figure 1 The figure shows the effect of seletinib on the viability and necrosis (LDH release rate) of HT-22 and SH-SY5Y cells treated with hypoxia / reoxygenation (H / R) and TSZ. As can be seen from the figure, seletinib administration can significantly inhibit hypoxia / reoxygenation ( Figure 1 A, B) and TSZ-induced decrease in the viability of HT-22 and SH-SY5Y neurons ( Figure 1 C, D) and increased LDH release rate ( Figure 1 E, F), indicating that selenotinib has an anti-hypoxia / reoxygenation and TSZ-induced neuronal cell damage effect; data are expressed as mean ± standard error, n = 3, **P < 0.01 vs Control, # P<0.05, ## P<0.01vs H / R or TSZ group.

[0104] 1.4.2 Cerutinib has an anti-hypoxia / reoxygenation and TSZ-induced necroptosis in SH-SY5Y neurons

[0105] like Figure 2As shown, the necroptosis-related molecule MLKL and phosphorylation level p-MLKL of SH-SY5Y neurons induced by hypoxia / reoxygenation (Figure A, B, C) and TSZ (Figure D, E, F) were upregulated, and selenotinib could significantly inhibit the upregulation of p-MLKL, indicating that selenotinib has an anti-hypoxia / reoxygenation and TSZ-induced necroptosis of neurons; data are expressed as mean ± standard error, n = 3, **P < 0.01 vs Control, ## P<0.01vs H / R or TSZ group.

[0106] The above examples further confirm that selenotinib can reduce neuronal cell death and necroptosis induced by hypoxia / reoxygenation and TSZ, has a neuronal cell protective effect, can reduce neuronal cell damage, and can be used to prepare drugs for treating neuronal cell protection.

[0107] However, the present invention is not limited to nerve cells. Other cell injuries and other diseases involve similar injury mechanisms, so the drug is also suitable for treating other cell injuries and related diseases.

[0108] Example 2

[0109] Animal experiments: Anti-Alzheimer's disease effects of selenotinib.

[0110] Experimental animals: APP / PS1 transgenic mice (animal model of Alzheimer's dementia, mouse background is C57BL / 6J mice), male, 5 months old, weighing 24-30 g, and C57BL / 6J mice of the same age (male, 5 months old, weighing 26-30 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. All experimental animals were kept in an SPF-level breeding room with a temperature of 22°C ± 2°C, a relative humidity of 45% ± 15%, free drinking water, and a 12-hour light / dark cycle.

[0111] The experimental animals were randomly divided into 6 groups, with 6 animals in each group, namely:

[0112] Normal control group: C57BL / 6J mice + intramuscular injection of solvent

[0113] APP / PS1+vehicle group: APP / PS1 mice + intramuscular injection of vehicle (i.e. 10% DMSO + 30% PEG400 + 60% saline, continuous administration for 1 month)

[0114] APP / PS1+cerdulatinib group: APP / PS1 mice+cerdulatinib (5 mg / kg cerdulatinib was injected intramuscularly daily starting from 5 months of age for 1 month; cerdulatinib was dissolved in 10% DMSO+30% PEG400+60% normal saline).

[0115] The novel object recognition test, Y-maze and Morris water maze tests were used to detect the learning and memory ability and cognitive function of mice.

[0116] Testing methods and results:

[0117] (1) Effects of cilutinib on non-spatial learning and memory ability and cognitive function of Alzheimer's disease model mice - novel object recognition experiment.

[0118] The experiment was conducted according to the requirements of the new object recognition experiment. The experiment included three stages: adaptation period, training period and test period. One week before the new object experiment, the experimental mice were stroked for 2 to 3 minutes every day to reduce their tension. The experiment was conducted in an open field test box of 40×40 cm. The experimental mice were placed in the experimental room to adapt to the environment for 20 to 30 minutes before the experiment. After the experimental mice adapted to the environment, the new object training experiment began. Two objects with the same color, shape and material (respectively marked as A and B) were placed in the open field test box, and the mice were placed in it for training for 10 minutes. After the training period, a new object test experiment was conducted 24 hours after the end of the training period to evaluate the short-term non-spatial learning and memory of the experimental mice. One of the two objects was replaced by another object with a different shape and color (recorded as C), and the time the experimental mice explored the two different objects within 10 minutes was recorded. Cognitive index = new object exploration time / (new object exploration time + old object exploration time) × 100%.

[0119] The experimental results are as follows Figure 3 As shown in the figure, it can be concluded that compared with the normal control group mice, the cognitive index of the APP / PS1+solvent group mice was significantly reduced, the non-spatial learning and memory ability and cognitive function of the APP / PS1 mice were weakened, and the cognitive index of the APP / PS1 mice after administration of seletinib (APP / PS1+seletinib group) was significantly increased (data are expressed as mean ± standard error, n = 6, **P < 0.01 vs normal control group, ## P<0.01vs APP / PS1+solvent group), the results showed that selegiline had a significant improvement on the non-spatial learning and memory ability and cognitive function of Alzheimer's disease mice.

[0120] (2) Y-maze test

[0121] This experiment was conducted according to reference 1 to test the spatial learning and memory ability of mice. The Y-shaped maze used in the experiment was made of gray organic plastic and consisted of three arms. The angle between adjacent arms was 120°. Each arm was 8 cm × 30 cm × 15 cm (width × length × height). The computer could automatically track and record the exploration process of the mouse based on the camera above the center of the maze. Three identical arms were randomly designated: (1) the starting arm, where the mouse began to explore (always open); (2) the novel arm (referred to as the "new arm"), which was blocked during the first trial but opened during the second trial, and (3) the other arms (always open). The novel arm was closed, and the mouse was placed in the starting arm with its back to the center of the maze, and allowed to explore the starting arm and other arms for 3 minutes. After the directional exploration, the mouse was removed from the Y maze and allowed to rest for 2 minutes. During this period, the maze was wiped with alcohol to remove the influence of the mouse's odor. After 2 minutes, the novel arm was opened, and the mouse was placed in the starting arm with its back to the center of the maze, and allowed to explore freely in the three arms for 1 minute. The movement trajectory and behavior of the animals were recorded by the smart3.0 small animal analysis software system, the time the mice stayed in the “novel arm” (i.e., the “new arm”) was recorded, and the percentage of the time the mice stayed in the new arm to the total free exploration time (i.e., the time spent in the new arm ratio) was analyzed.

[0122] The experimental results are as follows Figure 4 As shown in the figure, it can be concluded that compared with the normal control group mice, the APP / PS1+solvent group mice spent significantly less time in the "novel arm", indicating that the spatial learning and memory ability and cognitive function of the APP / PS1 mice were weakened. After administration of selegiline (APP / PS1+selegiline group), the APP / PS1 mice spent significantly more time in the "novel arm", and the time spent in the novel arm was significantly increased (data are expressed as mean ± standard error, n = 6, **P < 0.01 vs normal control group, ## P<0.01vs APP / PS1+solvent group), the results showed that selegiline had an improving effect on the spatial learning and memory ability and cognitive function of Alzheimer's disease mice.

[0123] (3) Effects of selegiline on spatial learning and memory ability and cognitive function of Alzheimer's disease model mice—Morris water maze test.

[0124] According to the requirements of the water maze experiment, the Morris water maze is divided into 4 quadrants. A platform with a diameter of 12 cm and a height of 35 cm (i.e., the target quadrant) is placed in one of the quadrants. Water is added to cover the platform by 1 to 2 cm, and the pool water is dyed black with carbon ink. After the positioning navigation experiment lasted for 5 days, the platform was removed for a spatial exploration experiment. The time for the mice to reach the original platform position (i.e., the incubation period) and the time they stayed in the target quadrant (the time they stayed in the quadrant where the original hidden platform was located) were recorded, and the percentage of the target quadrant stay time in the total exploration time was analyzed. The movement trajectory and behavior of the animals were recorded by the smart3.0 small animal analysis software system, and the relevant data were analyzed.

[0125] The experimental results are as follows Figure 5 As shown in the figure, it can be seen that compared with the normal control group mice, the time for the APP / PS1+solvent group mice to find the original platform position (escape latency) was significantly increased, and the number of times they crossed the platform was significantly reduced. After the administration of selegiline (APP / PS1+selegiline group), the time for the APP / PS1 mice to find the original platform (escape latency) was significantly reduced ( Figure 5 A), the residence time and residence time ratio in the target quadrant increased significantly ( Figure 5 B) (Data are expressed as mean ± standard error, n = 6, **P < 0.01 vs normal control group, ## P<0.01vs APP / PS1+solvent group). The results showed that selegiline had a significant improvement effect on the learning and memory ability and cognitive function of Alzheimer's disease mice.

[0126] The above results show that the seletinib described in the present invention has a protective effect on nerve cells, has a significant improvement effect on the learning and memory ability and cognitive function of Alzheimer's disease, can be used for the preparation of drugs for treating or improving the learning and memory and cognitive function of Alzheimer's disease, and broadens the indications of seletinib.

[0127] References:

[0128] 【1】Zou C, Mifflin L, Hu Z, Zhang T, Shan B, Wang H, Xing X, Zhu H, AdiconisX, Levin JZ, Li F, Liu CF, Liu JS, Yuan J. Reduction of mNAT1 / hNAT2 Contributes to Cerebral Endothelial Necroptosis and AβAccumulation in Alzheimer'sDisease.Cell Rep.2020;33(10):108447.

[0129] The contents explained in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

Claims

1. Use of selutinib and / or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a medicament for treating and / or preventing a neurodegenerative disease, characterized in that: The neurodegenerative disease is Alzheimer's disease.

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