Use of cd47 as a target in the treatment of heatstroke

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of CD47 as a target spot in treatment of heat stroke. The invention provides application of CD47 in preparation of a medicine or a medicine composition for treating heat stroke. The CD47 is used as a heat stroke treatment target for the first time, and a remarkable treatment effect is achieved. In a heat stroke mouse model, the function damage of multiple organs such as lung, kidney and the like caused by heat stroke can be relieved by injecting the CD47 monoclonal antibody into the caudal vein, the survival rate of the heat stroke mouse is increased, and the survival rate can be increased to about 80% by applying the CD47 monoclonal antibody under the dosage of 10 mg / kg. Therefore, the CD47 can be used as a therapeutic target for preparing the medicine for treating the heat stroke.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202410962191.3 (22) Application Date 2024.07.17 (71) Applicant Chengdu Sainjin Biotechnology Co., Ltd. Address 610041, Chengdu, Sichuan Province, China (Sichuan) Pilot Free Trade Zone, Chengdu High-tech Zone, 4th Floor, Building 2, Rooms 401 and 402 (72) Inventors Zhang Wei, Feng Zhongxue, Kang Yan (74) Patent Agency Beijing Zhizhong Deben Intellectual Property Agency Co., Ltd. 16083 Patent Attorney Guan Wei (51) Int.Cl. A61K 45 / 00 (2006.01) A61P 43 / 00 (2006.01) (54) Invention Title: Use of CD47 as a Target in the Treatment of Heatstroke (57) Abstract: This invention belongs to the field of pharmaceutical technology, specifically relating to the use of CD47 as a target in the treatment of heatstroke. This invention provides the use of CD47 in the preparation of drugs or pharmaceutical compositions for the treatment of heatstroke. This invention is the first to use CD47 as a therapeutic target for heatstroke, achieving significant therapeutic effects. In a mouse model of heatstroke, intravenous injection of a monoclonal antibody containing CD47 can alleviate multi-organ dysfunction caused by heatstroke, including lung and kidney damage, and improve the survival rate of mice with heatstroke. Administration of the CD47 monoclonal antibody at a dose of 10 mg / kg can increase the survival rate to approximately 80%. Therefore, CD47 can be used as a therapeutic target in the preparation of drugs for the treatment of heatstroke. Claims (2 pages), Description (21 pages), Sequence List (electronic publication), Drawings (4 pages), CN 121360229 A 2026.01.20 CN 1 21 36 02 29 A 1.CD47 Use in the preparation of a medicament or pharmaceutical composition for treating heatstroke; Preferably, the heatstroke includes classic heatstroke and exertional heatstroke; Preferably, the heatstroke also includes heatstroke complications; More preferably, the heatstroke complications include heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or combinations thereof. 2. The use according to claim 1, characterized in that the CD47 comprises CD47 nucleic acid molecules and / or CD47 protein molecules; and / or, the drug comprises a CD47 inhibitor; preferably, the CD47 inhibitor is a substance that inhibits, downregulates, weakens, antagonizes, and / or regulates the level of CD47 molecules; and / or, the CD47 inhibitor is a substance that blocks the interaction between CD47 and SIRPα; more preferably, the substance comprises nucleic acid drugs, protein drugs, small molecule inhibitors, or combinations thereof.3. The use according to claim 2, characterized in that the nucleic acid drug comprises siRNA, miRNA, ASO, shRNA, sgRNA, Aptamer or a combination thereof, preferably siRNA, more preferably the nucleotide sequence of the siRNA shown in SEQ ID NO:1-2; and / or, the protein drug comprises antibody, polypeptide, antibody-drug conjugate, polypeptide-drug conjugate, ribozyme, CAS protein or a combination thereof, preferably antibody, more preferably monoclonal antibody; and / or, the small molecule inhibitor comprises NCGC00138783, NCGC00138783TFA, NCGC00138783free base, 4-MU, 4-MUB, RS17, DMUP, NCGC00538431, RRx-001, Metformin, JQ1, PQ-912, SEN177, RS-17, Pep-20, D4-2 or a combination thereof. 4. The use according to any one of claims 1-3, characterized in that the drug or drug composition further comprises a second therapeutic agent for treating heatstroke, preferably 0.9% saline, Ringer's injection, glucose injection, norepinephrine, epinephrine, benzodiazepines, Angong Niuhuang Wan, Tanreqing injection, Xingnaojing injection, compound musk injection, biological agents containing coagulation factors, biological agents containing platelets, heparin, ulinastatin, glucocorticoids or combinations thereof. 5. Use of CD47 inhibitors in the preparation of medicaments or pharmaceutical compositions for treating heatstroke; preferably, the heatstroke includes classic heatstroke and exertional heatstroke; preferably, the heatstroke also includes heatstroke complications; more preferably, the heatstroke complications include heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or combinations thereof; preferably, the medicament or pharmaceutical composition further includes a second therapeutic agent for treating heatstroke, preferably 0.9% saline, Ringer's injection, glucose injection, norepinephrine, epinephrine, benzodiazepines, Angong Niuhuang Wan, Tanreqing injection, Xingnaojing injection, compound musk injection, biological agents containing coagulation factors, biological agents containing platelets, heparin, ulinastatin, glucocorticoids, or combinations thereof. 6. The use according to claim 5, characterized in that the CD47 inhibitor is a substance that inhibits, downregulates, weakens, antagonizes, and / or regulates the CD47 molecular level; and / or, the CD47 inhibitor is a substance that blocks the interaction between CD47 and SIRPα; preferably, the molecular level includes the nucleic acid molecular level and / or the protein molecular level; preferably, the substance includes nucleic acid drugs, protein drugs, small molecule inhibitors, or combinations thereof.Claims 1 / 2 Page 2 CN 121360229 A 7. The use according to claim 5 or 6, characterized in that the nucleic acid drug includes siRNA, miRNA, ASO, shRNA, sgRNA, Aptamer and combinations thereof, preferably siRNA, more preferably siRNA with a nucleotide sequence as shown in SEQ ID NO:1-2; and / or, the protein drug includes antibodies, peptides, antibody-drug conjugates, peptide-drug conjugates, ribozymes, CAS proteins and combinations thereof, preferably antibodies, more preferably monoclonal antibodies; and / or, the small molecule inhibitor includes NCGC00138783, NCGC00138783TFA, NCGC00138783free base, 4-MU, 4-MUB, RS17, DMUP, NCGC00538431, RRx-001, Metformin, JQ1, PQ-912, SEN177, RS-17, Pep-20, D4-2 or combinations thereof. 8. The use according to any one of claims 5-7, characterized in that the drug or pharmaceutical composition comprises 0.1-1000 mg of a CD47 inhibitor, preferably 1-1000 mg, more preferably 1-200 mg, for example: 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5, 45.0, 47.5, 50.0, 52.5, 55.0, 57.5. 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 80.0, 90.0, 92.5, 95.0, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 mg; Preferably, the drug or drug composition comprises 10 or 100 mg of CD47 inhibitor. 9. The use according to any one of claims 5-8, characterized in that the drug or pharmaceutical composition comprises 0.1-1000 mg / kg of CD47 inhibitor, preferably 1-1000 mg / kg, more preferably 1-200 mg / kg, for example: 1.0, 1.5, 2.0, 2.5, ...3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5, 45.0, 47.5, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 80.0, 90.0 92.5, 95.0, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 mg / kg; Preferably, the drug or drug composition comprises 10 or 100 mg / kg of CD47 inhibitor. 10. The use according to any one of claims 5-9, characterized in that the drug or pharmaceutical composition comprises 0.1-1000 nmol of CD47 inhibitor, preferably 1-1000 nmol, more preferably 10-1000 nmol, further, 1-100 nmol, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nmol, and further, 20 nmol. Claims 2 / 2 Page 3 CN 121360229 A Use of CD47 as a target in the treatment of heatstroke Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of CD47 as a target in the treatment of heatstroke, and particularly relating to the use of CD47 in the preparation of medicaments or pharmaceutical compositions for the treatment of heatstroke. Background Art

[0002] Heatstroke (HS) is the most serious condition among heat-related emergencies, namely severe heatstroke, caused by...Exposure to high temperature and humidity causes an imbalance in the body's regulatory functions, with heat production exceeding heat dissipation, leading to a rapid rise in core temperature exceeding 40°C. This severe and fatal illness is accompanied by burning skin, altered consciousness (e.g., delirium, convulsions, coma), and multiple organ dysfunction. It is the most serious type of heatstroke, with an extremely high mortality rate. Depending on the cause and susceptible population, heatstroke can be divided into exertional heat stroke (EHS) and classic heat stroke (CHS) (also known as non-exertional heat stroke).

[0003] High temperature and humidity, along with high-intensity physical activity, are the most significant risk factors for heatstroke. Classic heatstroke is mainly caused by high temperature and / or high humidity, and usually does not involve strenuous physical activity. Exertional heatstroke is mainly caused by an imbalance between heat production and heat dissipation due to high-intensity physical activity.

[0004] The pathophysiological processes of heatstroke include oxidative stress, inflammatory response, coagulation dysfunction, rhabdomyolysis, and intestinal flora imbalance. From the perspective of oxidative stress, heatstroke induces an increase in the amount of reactive oxygen species (ROS) in the human body. ROS are produced by mitochondria and can oxidize and damage biomolecules such as lipids, proteins, and DNA. Mitochondria are also a target of ROS, and damage to the mitochondrial membrane can induce apoptosis, leading to tissue damage. ROS synthesis is usually promoted through the following pathways: activation of protein kinase 2 by p38-MAPK, induction of dynein-associated protein 1-mediated mitochondrial fission or inhibition of mitochondrial complex I activity, and induction of reduced uncoupling protein content. From an inflammatory perspective, heatstroke induces a systemic inflammatory response, with increased secretion of inflammatory factors such as tumor necrosis factor, interleukin-1, interleukin-6, interleukin-8, matrix metalloproteinase-2, matrix metalloproteinase-9, intercellular adhesion molecule-1, transforming growth factor-β, and cyclooxygenase-2. These inflammatory factors can promote apoptosis through various pathways. From a coagulation perspective, heatstroke causes coagulation dysfunction, including thrombocytopenia, decreased fibrinogen levels, and delayed prothrombin time / activated partial thromboplastin time. Endothelial rupture caused by heatstroke leads to bleeding and thrombosis in multiple organs, ultimately resulting in necrosis. The pathogenesis of heatstroke is accompanied by severe oxidative stress, inflammatory response, coagulation dysfunction, rhabdomyolysis, and gut microbiota imbalance. These factors, along with apoptosis, autophagy, organ bleeding, and intestinal barrier disruption, collectively drive the progression of heatstroke, ultimately leading to multiple organ failure and even death.

[0005] Currently, the conventional treatment for heatstroke in China is a combination of chlorpromazine injection for sedation and various physical cooling methods.The patient's body temperature drops rapidly, blocking the inflammatory cascade response in the body and correcting the coagulation-anticoagulation imbalance. Continuous blood purification technology, adopted in recent years, has also improved the survival rate of heatstroke patients to some extent. However, because the disease is often accompanied by rhabdomyolysis, endotoxin release into the bloodstream, and organ failure, causing widespread irreversible damage throughout the body, the reported mortality rate remains around 20%-80%, with a poor prognosis. Current treatment methods are still insufficient to effectively improve the survival rate.

[0006] Currently, there is a lack of drugs specifically for the prevention and / or treatment of heatstroke. Instruction Manual 1 / 21 Page 4 CN 121360229 A Summary of the Invention

[0007] Cluster of Differentiation 47 (CD47), also known as integrin-Associated Protein (IAP), was first discovered in 1990. It consists of five transmembrane regions and one cytoplasmic domain. The cytoplasmic domain has four different splice isoforms. Type I splice isoform contains only 4 amino acids and is mainly expressed on epithelial and endothelial cells. Type II is the most common splice isoform, containing 16 amino acids and is mainly expressed on all hematopoietic cells, epithelial cells, and endothelial cells. Type IV splice isoform is mainly expressed in neurons, testes, and intestines, containing 36 amino acids, which is consistent with the expression site of Type III, which contains 23 amino acids. Both are related to memory function in rats. However, according to current research, the protein functions and mechanisms of action of the four splice isoforms are still unclear. CD47 is widely expressed in solid organs such as the brain, liver, lungs, kidneys, and bladder, as well as in bone marrow and immune regions such as the spleen, bone marrow, and lymph nodes. It participates in regulating innate immune responses, inflammatory responses, cell growth and differentiation, cell adhesion and migration, phagocytosis and apoptosis, angiogenesis, and immune cell homeostasis. Its main ligand is SIRPα (Signal Regulatory Protein α). SIRPα is selectively expressed in myeloid cells, including monocytes / macrophages and neutrophils. The binding of CD47 to SIRPα transmits a "Don't eat me" signal, preventing CD47-expressing cells from being phagocytosed by SIRPα-expressing myeloid cells. Blocking CD47 can enhance the phagocytic and killing effects of macrophages and neutrophils on tumor cells. Currently, more than 20 antibody drugs that block CD47 have entered clinical trials for immunotherapy of hematologic / solid tumors. In addition, CD47 has been reported to be closely related to the development of cardiovascular diseases, brain and neurodegeneration, metabolic diseases, and infectious diseases. However, no reports have been found regarding the relationship between CD47 and heatstroke.

[0008] This invention is the first to use CD47 as a target for the treatment of heatstroke.

[0009] To achieve the above-mentioned technical objectives, the present invention proposes the following technical solutions:

[0010] In one aspect, the present invention provides the use of CD47 in the preparation of a medicament or pharmaceutical composition for treating heatstroke.

[0011] In some embodiments, the heatstroke includes classic heatstroke and exertional heatstroke.

[0012] In some preferred embodiments, the heatstroke further includes heatstroke complications.

[0013] In some preferred embodiments, the heatstroke complications include, but are not limited to: heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or combinations thereof.

[0014] In some embodiments, the CD47 includes CD47 nucleic acid molecules and / or CD47 protein molecules.

[0015] In some embodiments, the medicament comprises a CD47 inhibitor.

[0016] In some embodiments, the CD47 inhibitor is a substance that inhibits, downregulates, weakens, antagonizes, and / or regulates the level of CD47 molecules.

[0017] In some embodiments, the substance includes nucleic acid drugs, protein drugs, small molecule inhibitors, or combinations thereof.

[0018] In some preferred embodiments, the CD47 inhibitor is a substance that blocks the interaction between CD47 and SIRPα.

[0019] In some preferred embodiments, the nucleic acid drug includes, but is not limited to: small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), short hairpin RNA (shRNA), single-guide RNA (sgRNA), nucleic acid aptamers, or combinations thereof.

[0020] In some preferred embodiments, the nucleic acid drug is siRNA.

[0021] In some more preferred embodiments, the nucleic acid drug is siRNA with a nucleotide sequence as shown in SEQ ID NO:1-2. Instructions for Use, Page 2 / 21, CN 121360229 A

[0022] In some preferred embodiments, the protein drug includes, but is not limited to: antibodies, peptides, antibody-drug conjugates, peptide-drug conjugates, ribozymes, CAS proteins, or combinations thereof.

[0023] In some preferred embodiments, the protein drug is an antibody.

[0024] In some preferred embodiments, the antibody includes monoclonal antibodies or polyclonal antibodies.

[0025] In some more preferred embodiments, the antibody is a monoclonal antibody.

[0026] In some preferred embodiments, the antibody is a bispecific antibody or a multispecific antibody.

[0027] In some preferred embodiments, the small molecule inhibitor includes small molecule compounds and / or small molecule peptides.

[0028] In some preferred embodiments, the small molecule inhibitor includes, but is not limited to: NCGC00138783, NCGC00138783 TFA, NCGC00138783 free base, 4-Methylumbelliferone (4-MU), 4-Methylumbelliferyl butyrate (4-MUB), DMUP, NCGC00538431, RRx-001, Metformin, JQ1, PQ-912, SEN177, or combinations thereof.

[0029] In some preferred embodiments, the small molecule peptide includes, but is not limited to: RS-17, Pep-20, D4-2, or combinations thereof.

[0030] In some embodiments, the medicament or pharmaceutical composition further includes a second therapeutic agent for treating heatstroke.

[0031] In some preferred embodiments, the second therapeutic agent includes, but is not limited to: 0.9% saline, Ringer's injection, glucose injection, norepinephrine, epinephrine, benzodiazepines (such as midazolam, lorazepam), Angong Niuhuang Wan, Tanreqing injection, Xingnaojing injection, compound musk injection, biological agents containing coagulation factors, biological agents containing platelets, heparin, ulinastatin, glucocorticoids, or combinations thereof.

[0032] In some embodiments, the medicament or pharmaceutical composition further includes at least one pharmaceutically acceptable excipient.

[0033] In another aspect, the use of a CD47 inhibitor in the preparation of a medicament or pharmaceutical composition for treating heatstroke.

[0034] In some embodiments, the heatstroke includes classic heatstroke and exertional heatstroke.

[0035] In some preferred embodiments, the heatstroke also includes heatstroke complications.

[0036] In some preferred embodiments, the heatstroke complications include, but are not limited to: heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or combinations thereof.

[0037] In some embodiments, the CD47 inhibitor is a substance that inhibits, downregulates, weakens, antagonizes, and / or regulates the level of CD47 molecules.

[0038] In some embodiments, the molecular level includes the nucleic acid molecular level and / or the protein molecular level.

[0039] In some embodiments, the CD47 inhibitor is a substance that blocks the interaction between CD47 and SIRPα.

[0040] In some embodiments, the substance includes nucleic acid drugs, protein drugs, small molecule inhibitors, or combinations thereof.

[0041] In some preferred embodiments, the nucleic acid drug includes, but is not limited to: small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), short hairpin RNA (shRNA), single-guide RNA (sgRNA), nucleic acid aptamers, or combinations thereof.

[0042] In some preferred embodiments, the nucleic acid drug is siRNA.

[0043] In some more preferred embodiments, the nucleic acid drug is siRNA with a nucleotide sequence as shown in SEQ ID NO:1-2. Specification 3 / 21 page 6 CN 121360229 A

[0044] In some preferred embodiments, the protein drug includes, but is not limited to: antibodies, peptides, antibody-drug conjugates, peptide-drug conjugates, ribozymes, CAS proteins, or combinations thereof.

[0045] In some preferred embodiments, the protein drug is an antibody.

[0046] In some preferred embodiments, the antibody includes monoclonal antibodies or polyclonal antibodies.

[0047] In some more preferred embodiments, the antibody is a monoclonal antibody.

[0048] In some preferred embodiments, the antibody is a bispecific antibody or a multispecific antibody.

[0049] In some preferred embodiments, the small molecule inhibitor comprises a small molecule compound and a small molecule peptide.

[0050] In some preferred embodiments, the small molecule compound includes, but is not limited to: NCGC00138783, NCGC00138783 TFA, NCGC00138783 free base, 4-Methylumbelliferone (4-MU), 4-Methylumbelliferyl butyrate (4-MUB), Gefitinib, MUP, NCGC00538431, RRx-001, Metformin, JQ1, PQ-912, SEN177, or combinations thereof.

[0051] In some preferred embodiments, the small molecule peptide includes, but is not limited to: RS17, Pep-20, D4-2, or combinations thereof.

[0052] In some embodiments, the medicament or pharmaceutical composition comprises 0.1-1000 mg of a CD47 inhibitor.

[0053] In some embodiments, the medicament or pharmaceutical composition comprises 1-1000 mg of a CD47 inhibitor.

[0054] In some embodiments, the medicament or pharmaceutical composition comprises 10-1000 mg of a CD47 inhibitor, for example: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg.

[0055] In some embodiments, the drug or pharmaceutical composition comprises 10-300 mg of CD47 inhibitor.

[0056] In some embodiments, the drug or pharmaceutical composition comprises 10-200 mg of CD47 inhibitor.

[0057] In some embodiments, the drug or pharmaceutical composition comprises 1-300 mg of CD47 inhibitor.

[0058] In some embodiments, the drug or pharmaceutical composition comprises 1-200 mg of a CD47 inhibitor, for example: 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5, 45.0, 47.5. 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 80.0, 90.0, 92.5, 95.0, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200mg.

[0059] In some preferred embodiments, the drug or drug composition comprises 0.1-20 mg of a CD47 inhibitor, for example: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0mg. Instructions for Use, Page 4 / 21, CN 121360229 A

[0060] In some preferred embodiments, the drug or pharmaceutical composition comprises 10 mg of CD47 inhibitor.

[0061] In some preferred embodiments, the drug or pharmaceutical composition comprises 100 mg of CD47 inhibitor.

[0062] In some embodiments, the drug or pharmaceutical composition comprises 0.1-1000 mg / kg of CD47 inhibitor.

[0063] In some embodiments, the drug or pharmaceutical composition comprises 1-1000 mg / kg of CD47 inhibitor.

[0064] In some embodiments, the drug or pharmaceutical composition comprises 10-1000 mg / kg of CD47 inhibitor, for example: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520530, 540, 550, 560, 570, 580, 590, 600, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg / kg.

[0065] In some embodiments, the drug or pharmaceutical composition comprises 10-300 mg / kg of CD47 inhibitor.

[0066] In some embodiments, the drug or pharmaceutical composition comprises 10-200 mg / kg of CD47 inhibitor.

[0067] In some embodiments, the drug or pharmaceutical composition comprises a CD47 inhibitor of 1-300 mg / kg.

[0068] In some embodiments, the drug or pharmaceutical composition comprises a CD47 inhibitor of 1-200 mg / kg, for example: 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5, 45.0, 47.5, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 80.0, 90.0, 92.5, 95.0, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200mg / kg.

[0069] In some preferred embodiments, the drug or drug composition comprises 0.1-20 mg / kg of CD47 inhibitor, for example: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.55.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0 mg / kg.

[0070] In some preferred embodiments, the drug or pharmaceutical composition comprises 10 mg / kg of CD47 inhibitor.

[0071] In some preferred embodiments, the drug or pharmaceutical composition comprises 100 mg / kg of CD47 inhibitor.

[0072] In some embodiments, the drug or pharmaceutical composition comprises 0.1-1000 nmol of CD47 inhibitor.

[0073] In some embodiments, the drug or pharmaceutical composition comprises 1-1000 nmol of CD47 inhibitor.

[0074] In some embodiments, the drug or pharmaceutical composition comprises 10-1000 nmol of CD47 inhibitor.

[0075] In some preferred embodiments, the drug or pharmaceutical composition comprises 1-100 nmol of a CD47 inhibitor, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nmol.

[0076] In some preferred embodiments, the drug or pharmaceutical composition comprises 20 nmol of CD47 inhibitor. Specification 5 / 21 pages 8 CN 121360229 A

[0077] In some embodiments, the medicament or pharmaceutical composition further includes a second therapeutic agent for treating or adjunctive treatment of heatstroke.

[0078] In some preferred embodiments, the second therapeutic agent includes, but is not limited to: 0.9% saline, Ringer's injection, glucose injection, norepinephrine, epinephrine, benzodiazepines (such as midazolam, lorazepam), Angong Niuhuang Wan, Tanreqing injection, Xingnaojing injection, compound musk injection, biological agents containing coagulation factors, biological agents containing platelets, heparin, ulinastatin, glucocorticoids, or combinations thereof.

[0079] In some embodiments, the medicament or pharmaceutical composition further includes at least one pharmaceutically acceptable excipient.

[0080] In a third aspect, the present invention provides a method for treating heatstroke, the method comprising:

[0081] administering a therapeutically effective amount of any of the aforementioned CD47 inhibitors to a subject in need.

[0082] In some embodiments, the heatstroke includes classic heatstroke and exertional heatstroke.

[0083] In some embodiments, the subject has been exposed to a high temperature and high humidity environment.

[0084] In some preferred embodiments, the heatstroke also includes heatstroke complications.

[0085] In some preferred embodiments, the heatstroke complications include, but are not limited to: heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or a combination thereof.

[0086] In some preferred embodiments, the subject has an imbalance in thermoregulation.

[0087] In some preferred embodiments, the subject's lung tissue is damaged.

[0088] In some more preferred embodiments, the subject has enlarged alveolar septa.

[0089] In some preferred embodiments, the subject's liver tissue is damaged.

[0090] In some more preferred embodiments, the subject has swollen liver lobules, disordered hepatic cords, reduced hepatic sinusoids, increased red blood cells in the hepatic sinusoids, or a combination thereof.

[0091] In some embodiments, the subject has a coagulation disorder or severe coagulation disorder.

[0092] In some more preferred embodiments, the subject's serum fibrinogen level is significantly elevated, where "significant" means that the fibrinogen level is significantly different from that in a normal physiological state, p < 0.05, preferably p < 0.01, more preferably p < 0.001.

[0093] In a fourth aspect, the present invention provides a method for alleviating or reducing lung tissue damage in a subject, the method comprising:

[0094] administering a therapeutically effective amount of any of the aforementioned CD47 inhibitors to a subject in need.

[0095] In some embodiments, the subject is suspected of or diagnosed with heatstroke.

[0096] In some preferred embodiments, the heatstroke includes classic heatstroke and exertional heatstroke.

[0097] In some preferred embodiments, the heatstroke also includes heatstroke complications.

[0098] In some preferred embodiments, the heatstroke complications include, but are not limited to: heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or a combination thereof.

[0099] In some embodiments, the subject has been exposed to a high temperature and high humidity environment.

[0100] In some preferred embodiments, the subject has an imbalance in thermoregulation.

[0101] In some preferred embodiments, the subject has enlarged alveolar septa.

[0102] In some preferred embodiments, the subject's liver tissue is damaged. Instructions for Use, Page 6 / 21, CN 121360229 A

[0103] In some more preferred embodiments, the subject has swollen liver lobules, disordered hepatic cords, reduced hepatic sinusoids, increased red blood cells in the hepatic sinusoids, or a combination thereof.

[0104] In some embodiments, the subject has coagulation dysfunction or severe coagulation disorder.

[0105] In some more preferred embodiments, the subject has significantly elevated levels of fibrinogen in serum, where "significant" means that there is a significant difference in fibrinogen levels compared to normal physiological conditions, p < 0.05, preferably p < 0.01, more preferably p < 0.001.

[0106] In a fifth aspect, the present invention provides a method for alleviating or reducing liver tissue damage in a subject, the method comprising:

[0107] administering a therapeutically effective amount of any of the aforementioned CD47 inhibitors to a subject in need.

[0108] In some embodiments, the subject is suspected of or diagnosed with heatstroke.

[0109] In some preferred embodiments, the heatstroke includes classic heatstroke and exertional heatstroke.

[0110] In some preferred embodiments, the heatstroke also includes heatstroke complications.

[0111] In some preferred embodiments, the heatstroke complications include, but are not limited to: heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or a combination thereof.

[0112] In some embodiments, the subject has been exposed to a high temperature and high humidity environment.

[0113] In some preferred embodiments, the subject's thermoregulation is imbalanced.

[0114] In some preferred embodiments, the subject has swollen liver lobules, disordered hepatic cords, reduced hepatic sinusoids, increased red blood cells in the hepatic sinusoids, or a combination thereof.

[0115] In some preferred embodiments, the subject's lung tissue is damaged.

[0116] In some more preferred embodiments, the subject's alveolar septa are thickened.

[0117] In some embodiments, the subject has coagulation dysfunction or severe coagulation disorder.

[0118] In some more preferred embodiments, the subject's serum fibrinogen level is significantly elevated, where "significant" means that there is a significant difference in fibrinogen level compared with normal physiological state, p < 0.05, preferably p < 0.01, more preferably p < 0.001.

[0119] In a sixth aspect, the present invention provides a method for alleviating or reducing coagulation dysfunction in a subject, the method comprising:

[0120] administering a therapeutically effective amount of any of the aforementioned CD47 inhibitors to a subject in need.

[0121] In some embodiments, the subject is suspected of or diagnosed with heatstroke.

[0122] In some preferred embodiments, the heatstroke includes classic heatstroke and exertional heatstroke.

[0123] In some preferred embodiments, the heatstroke also includes heatstroke complications.

[0124] In some preferred embodiments, the heatstroke complications include, but are not limited to: heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or a combination thereof.

[0125] In some embodiments, the subject has been exposed to a high temperature and high humidity environment.

[0126] In some preferred embodiments, the subject's thermoregulation function is imbalanced.

[0127] In some preferred embodiments, the subject's serum fibrinogen level is significantly elevated, where "significant" means that there is a significant difference in fibrinogen level compared with the normal physiological state, p < 0.05, preferably p < 0.01, more preferably p < 0.001. Instructions 7 / 21 pages 10 CN 121360229 A

[0128] In some preferred embodiments, the alveolar septa of the subject are thickened.

[0129] In some preferred embodiments, the liver tissue of the subject is damaged.

[0130] In some more preferred embodiments, the subject has swollen liver lobules, disordered hepatic cords, reduced hepatic sinusoids, increased red blood cells in the hepatic sinusoids, or a combination thereof.

[0131] This invention is the first to use CD47 as a therapeutic target for heatstroke, achieving significant therapeutic effects. In a mouse model of heatstroke, intravenous injection of a monoclonal antibody against CD47 can alleviate multi-organ dysfunction caused by heatstroke and improve the function of lungs, kidneys, etc.The survival rate of mice with heatstroke can be increased to approximately 80% by administering CD47 monoclonal antibodies at a dose of 10 mg / kg. Therefore, CD47 can be used as a therapeutic target in the preparation of drugs for treating heatstroke, and monoclonal antibodies / small molecule inhibitors / siRNAs targeting CD47 can also be a preferred treatment strategy for heatstroke. Figure Descriptions

[0132] Figure 1 shows the H&E staining of lung tissue from mice with heatstroke in different treatment groups.

[0133] Figure 2 shows the H&E staining of liver tissue from mice with heatstroke in different treatment groups.

[0134] Figure 3 shows the changes in survival rate of mice with heatstroke in different treatment groups.

[0135] Figure 4 shows the changes in serum physiological parameters of mice with heatstroke in different treatment groups.

[0136] Figure 5 shows the changes in the secretion of fibrinogen, a coagulation marker, in mice with heatstroke in different treatment groups. In the above figures, * represents a significant difference between the two experimental groups, p≤0.05; ** represents a significant difference between the two experimental groups, p≤0.01; *** represents a significant difference between the two experimental groups, p≤0.001; **** represents a significant difference between the two experimental groups, p≤0.0001. Detailed Implementation

[0137] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, not all of them. Based on the described embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0138] This invention can be implemented in other specific forms without departing from the basic attributes of this invention. It should be understood that, without conflict, any and all embodiments of this invention can be combined with the technical features of any other embodiment or multiple other embodiments to obtain other embodiments. This invention includes other embodiments obtained by such combinations.

[0139] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. If any use or terminology used in any publication or patent incorporated by reference conflicts with that used in this disclosure, the use and terminology of this disclosure shall prevail.

[0140] Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter.

[0141] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate...When a term is used in the singular form, it will also include the plural form, and vice versa.

[0142] Unless the context clearly indicates otherwise, the expressions “a” and “an” as used herein include plural references. For example, reference to “a cell” includes a plurality of such cells and equivalents known to those skilled in the art, etc. Specification 8 / 21 pages 11 CN 121360229 A

[0143] As shown in this disclosure, the term “about” indicates a range of ±20% of the following numerical value. In some embodiments, the term “about” indicates a range of ±10% of the following numerical value. In some embodiments, the term “about” indicates a range of ±5% of the following numerical value.

[0144] The numerical ranges used in this disclosure should be understood to mean that all numbers within that range have been enumerated. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0145] As shown in this disclosure, the terms “comprises” or “comprising” mean “including, but not limited to”. The term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term “comprising” includes the more restrictive terms “consisting of” and “substantially consisting of”. In one embodiment, the term “comprising” used throughout the disclosure, particularly in the claims, may be replaced by the term “consisting of”.

[0146] As shown in this disclosure, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or circumstance described below may, but does not necessarily, occur, and the description includes the occasion in which the event or circumstance occurs or does not occur. In this invention, "an" and "a" are used to refer to one or more grammatical objects.

[0147] As disclosed herein, "and / or" should be understood to mean any one of the options or any combination of two or more of the options.

[0148] Heatstroke

[0149] As disclosed herein, the term "heatstroke" refers to severe heatstroke, which is a serious acute heat-induced illness caused by exposure to a hot and humid environment, resulting in an imbalance in the body's thermoregulation function, with heat production exceeding heat dissipation, leading to a rapid increase in core temperature exceeding 40°C, accompanied by burning skin, altered consciousness (such as delirium, convulsions, coma), and multiple organ dysfunction. It is the most severe type of heatstroke. Heatstroke is divided into classic heatstroke (also known as non-exertional heatstroke, CHS) and exertional heatstroke.stroke, EHS). For specific diagnostic criteria for "heatstroke", please refer to: Expert Group on Prevention and Treatment of Heatstroke of the PLA, Expert Consensus Group on Emergency Diagnosis and Treatment of Heatstroke. Expert Consensus on Emergency Diagnosis and Treatment of Heatstroke (2021 Edition). Chinese Journal of Emergency Medicine, 2021, 30(11):1290-1299.

[0150] As shown in this disclosure, the terms "classic heatstroke", "classic type heatstroke" or "non-exertional heatstroke" refer to heatstroke mainly caused by high temperature and / or high humidity environmental factors, usually without strenuous physical activity. Classic type heatstroke is more common in people with insufficient thermoregulation ability (such as the elderly and weak, children), those with underlying diseases (such as mental disorders, sequelae of cerebral hemorrhage, etc.) and those who have been in a high temperature environment for a long time.

[0151] As shown in this disclosure, the term "exertional heatstroke" refers to heatstroke mainly caused by an imbalance between heat production and heat dissipation caused by high-intensity physical activity. Exertional heatstroke is more common in previously healthy young adults.

[0152] In some instances, the heatstroke also includes heatstroke complications.

[0153] In some preferred instances, the heatstroke complications include, but are not limited to: heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or combinations thereof.

[0154] CD47

[0155] As disclosed herein, the terms “nucleic acid,” “nucleotide,” and “polynucleotide” are used interchangeably and refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and polymers thereof in single-stranded, double-stranded, or multi-stranded forms. The term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and / or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic, or derived nucleotide bases. In some examples of specification 9 / 21, page 12, CN 121360229 A, nucleic acids can include mixtures of DNA, RNA, and analogues. The term also covers nucleic acids containing known analogues of natural nucleotides, having similar binding properties to the reference nucleic acid, and being metabolized in a manner similar to naturally occurring nucleotides. “Nucleic acid” is used interchangeably with “gene,” “DNA,” and “mRNA” encoded by a gene.

[0156] As shown in this disclosure, the term “mRNA” refers to messenger RNA. mRNA is a single-stranded RNA molecule that plays a role in transmitting genetic information from the nucleus to protein synthesis sites in the cytoplasm. This process is a key step in gene expression, which refers to the process by which genetic information in DNA is transcribed into mRNA, and then mRNA is translated into protein.

[0157] As described in this disclosure, the term "CD47 protein" refers to a cell surface receptor expressed on healthy cells and typically overexpressed on cancer cells, which protects cancer cells from phagocytosis by interacting with SIRPα. CD belongs to the immunoglobulin superfamily and has two isoforms: one containing N-glycosylation modification, and the other containing glycosaminoglycan modification at serine residues 64 and 79. The CD47 structure comprises three parts: an N-terminal extracellular immunoglobulin variable (IgV) domain, a highly hydrophobic five-transmembrane segment, and a short variable C-terminal intracellular segment.

[0158] The CD47 described in this disclosure includes the CD47 gene and / or the CD47 protein. In some preferred embodiments, the CD47 is the human CD47 gene and / or the human CD47 protein.

[0159] The Gene ID of the human CD47 gene described in this disclosure is 961 (https: / / www.ncbi.nlm.nih.gov / gene / 961), and the Uniprot ID of the human CD47 protein is Q08722 (https: / / www.uniprot.org / uniprotkb / Q08722 / entry).

[0160] In some instances, the CD47 or CD47 molecule comprises a CD47 nucleic acid molecule and / or a CD47 protein molecule.

[0161] In some preferred instances, the CD47 or CD47 molecule is CD47 mRNA.

[0162] In some preferred instances, the CD47 or CD47 molecule is the CD47 gene.

[0163] In some preferred instances, the CD47 or CD47 molecule is cDNA of CD47 mRNA.

[0164] In some preferred instances, the CD47 or CD47 molecule is a protein.

[0165] CD47 Inhibitor

[0166] As disclosed herein, the term "antibody" refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which has the ability to specifically bind an antigen. The antibody is an immunoglobulin with an Fc domain and an antigen-binding region. Antibodies typically contain two CH2-CH3 regions and a linker region, such as a hinge region, such as at least an Fc domain. Variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with the antigen. The constant region or "Fc" region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as the first component C1q in the classical activation pathway of complement. The Fc region is typically in a dimerized form, for example, via a disulfide bridge connecting two hinge regions and / or a non-covalent interaction between two CH3 regions.It can be a homodimer (where the amino acid sequences of the two Fc region monomers are identical) or a heterodimer (where the amino acid sequences of the two Fc region monomers differ in one or more amino acids). The antigen-binding region refers to the region that specifically recognizes the antigen, namely the CD47 protein.

[0167] As shown in this disclosure, the term "siRNA (small interfering RNA)" generally refers to a class of double-stranded RNA molecules that can mediate the cleavage, degradation, or silencing of their complementary target RNA (e.g., mRNA, tRNA, and viral RNA). siRNA is usually double-stranded, including an antisense strand complementary to the target RNA and a sense strand complementary to the antisense strand. Methods for preparing siRNA are well known to those skilled in the art, such as chemical synthesis, in vitro transcription, in vitro preparation of long dsRNAs via RNase III (or Dicer) degradation, and preparation by transfection of host cells with an siRNA expression vector. Specification 10 / 21 pages 13 CN 121360229 A

[0168] As disclosed herein, the term “SIRPα” refers to signal regulatory protein α, a key inhibitory regulator expressed on the surface of myeloid cells, the second identified endogenous CD47 ligand, and expressed on all myeloid cells. SIRPα is a transmembrane glycoprotein whose extracellular N-terminal domain includes three immunoglobulin-like domains, and its cytoplasmic domain contains four tyrosine phosphorylation sites and two immunoreceptor tyrosine-based inhibitory motifs (ITIM) (Barclay, AN. and Brown, M.H. (2006) The SIRP Family of Receptors and Immune Regulation. Nature Reviews Immunology, 6, 457-464). CD47 expressed on normal cells can activate various protein tyrosine phosphatases (such as SHP1 and SHP2) by binding to the N-terminal domain of SIRPα expressed on macrophages and inducing ITIM phosphorylation. This activates multiple intracellular molecular pathways, including the dephosphorylation of immune receptor tyrosine-based activation motifs (ITAMs) (Barclay, A.N. and Van den Berg, T.K. (2014) The Interaction between Signal Regulatory Protein Alpha (SIRPα) andCD47: Structure, Function, and Therapeutic Target. Nature Reviews Immunology, 32, 25-50. The interaction between CD47 and SIRPα also mediates the adhesion of B lymphocytes to unactivated endothelial cells, regulates B cell aggregation, and participates in B lymphocyte regeneration, playing an important role in the regulation of the immune system (Yoshida, H., Tomiyama, Y., Oritani, K., et al. (2002) Interaction between Src Homology 2 Domain Bearing Protein Tyrosine Phosphatase Substrate-1 and CD47 Mediates the Adhesion of Human B Lymphocytes to Nonactivated Endothelial Cells. Journal of Immunology, 168, 3213-3220).

[0169] As disclosed herein, "CD47 inhibitor" means a substance that inhibits, weakens, prevents, blocks, and / or antagonizes the normal physiological function of CD47 molecules.

[0170] In some instances, the CD47 inhibitor is a substance that inhibits, downregulates, weakens, prevents, or blocks the expression of CD47 molecules on the cell surface.

[0171] In some instances, the CD47 inhibitor is a substance that inhibits, downregulates, weakens, antagonizes, and / or regulates the level of CD47 molecules.

[0172] In some instances, the CD47 inhibitor is a substance that inhibits CD47 gene replication, transcription, translation, post-transcriptional modification, and / or post-translational modification.

[0173] In some instances, the CD47 inhibitor is a substance that inhibits the content, activity, and / or function of CD47 protein.

[0174] In some instances, the CD47 inhibitor refers to a substance that blocks CD47-SIRPα interaction.

[0175] In some instances, the CD47 inhibitor includes, but is not limited to, nucleic acid drugs, protein drugs, and small molecule inhibitors.

[0176] In some instances, the CD47 inhibitor is a nucleic acid drug.

[0177] In some preferred embodiments, the nucleic acid drug includes, but is not limited to: small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), aptamers, or combinations thereof.

[0178] In some preferred embodiments, the CD47 inhibitor is siRNA.

[0179] In some preferred embodiments, the target gene has a sequence complementary to the antisense strand of the siRNA that inhibits the expression of the target gene. More specifically, the siRNA in this invention is an RNA comprising a double-stranded RNA portion consisting of a sense RNA strand and an antisense RNA strand, wherein the sense RNA strand consists of a sequence homologous to a continuous RNA sequence in the mRNA encoding the CD47 protein, and the antisense RNA strand consists of a sequence complementary to the sense RNA sequence.

[0180] The design and manufacture of such siRNAs and mutant siRNAs discussed below fall within the skill of those skilled in the art. Those skilled in the art can suitably select any continuous RNA region of mRNA, which is a transcript of the CD47 gene sequence, to prepare a double-stranded RNA corresponding to that region within a normal procedure. Those skilled in the art can also suitably select siRNA sequences with more effective RNAi effects from mRNA sequences, which are transcripts of those sequences, using known methods.

[0181] In some preferred embodiments, the siRNA sequence targeting CD47 is as shown in mCD47-1031-s: GCAGAACUACUUGGAUUAGUUTT (SEQ ID NO:1); mCD47-1031-a: AACUAAUCCAAGUAGUUCUGCTT (SEQ ID NO:2).

[0182] In some embodiments, the protein drug includes, but is not limited to: antibodies, peptides, antibody-drug conjugates, peptide-drug conjugates, ribozymes, CAS proteins, or combinations thereof.

[0183] In some embodiments, the CD47 inhibitor is an antibody.

[0184] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0185] In some preferred embodiments, the antibody is a monoclonal antibody.

[0186] In some preferred embodiments, the antibody includes, but is not limited to: Urabrelimab, CC-90002, Ligufalimab, SRF-231, IBI188, magrolimab, lemzoparlimab, AO-176, AK117, etc.

[0187] In some embodiments, the antibody is a bispecific antibody or a multispecific antibody.

[0188] In some preferred embodiments, the antibody is a bispecific antibody, such as: CD20 / CD47 bispecific antibody, CD19 / CD47 bispecific antibody, GPC3 / CD47 bispecific antibody, PD-L1 / CD47 bispecific antibody, etc.

[0189] In some embodiments, the CD47 inhibitor is a small molecule inhibitor.

[0190] In some instances, the small molecule inhibitor comprises small molecule compounds and small molecule peptides.

[0191] In some preferred instances, the small molecule compound comprises, but is not limited to: NCGC00138783, NCGC00138783 TFA, NCGC00138783 free base, 4-Methylumbelliferone (4-MU), 4-Methylumbelliferyl butyrate (4-MUB), Gefitinib, MUP, NCGC00538431, RRx-001, Metformin, JQ1, PQ-912, SEN177, or combinations thereof.

[0192] The chemical structural formula of “NCGC00138783” disclosed herein is as follows:

[0193]

[0194] The chemical structural formula of “NCGC00138783 TFA” disclosed herein is as follows: Specification 12 / 21 Page 15 CN 121360229 A

[0195]

[0196] The chemical structural formula of “NCGC00138783free base” disclosed herein is as follows:

[0197]

[0198] The chemical structural formula of “4-Methylum belliferone (4-MU)” disclosed herein is as follows:

[0199] The chemical structural formula of “4-Methylumbelliferyl butyrate (4-MUB)” disclosed herein is as follows:

[0200] The chemical structural formula of “Gefitinib” disclosed herein is as follows:

[0201]

[0202] The chemical structural formula of “MUP (4-Methylumbelliferyl phosphate)” disclosed herein is as follows:

[0203] The chemical structure of “NCGC00538431” disclosed herein is shown below: Specification 13 / 21 Page 16 CN 121360229 A

[0204]

[0205] The chemical structure of “RRx-001” disclosed herein is shown below:

[0206]

[0207] The chemical structure of “Metformin” disclosed herein is shown below:

[0208]

[0209] The chemical structure of “JQ1” disclosed herein is shown below:

[0210]

[0211] The chemical structure of “PQ-912” disclosed herein is shown below:

[0212]

[0213] The chemical structure of “SEN177” disclosed herein is shown below: Specification 14 / 21 Page 17 CN 121360229 A

[0214]

[0215] In some examples, the small molecule peptides include, but are not limited to: RS17, Pep-20 or D4-2.

[0216] Drug / Drug Composition

[0217] As disclosed herein, the term “pharmaceuticalally acceptable carrier, excipient, and / or excipient” means a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delay agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and the like. Absorption delay agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as being able to stabilize the desired activity of the active ingredient in a pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0218] As disclosed herein, the term “pharmaceuticalally acceptable” means those compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues within the limits of reasonable medical judgment without excessive toxicity, irritation, allergic response, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0219] The “pharmaceutical / pharmaceutical composition” described in this disclosure comprises a therapeutically effective amount of a CD47 inhibitor.

[0220] In some instances, the pharmaceutical or pharmaceutical composition comprises 0.1-1000 mg of a CD47 inhibitor.

[0221] In some instances, the pharmaceutical or pharmaceutical composition comprises 1-1000 mg of a CD47 inhibitor.

[0222] In some instances, the pharmaceutical or pharmaceutical composition comprises 10-1000 mg of a CD47 inhibitor, for example: 10, 15, ...20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540 550, 560, 570, 580, 590, 600, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg.

[0223] In some examples, the drug or drug composition contains 10-300 mg of CD47 inhibitor.

[0224] In some examples, the drug or drug composition contains 10-200 mg of CD47 inhibitor.

[0225] In some examples, the drug or drug composition contains 1-300 mg of CD47 inhibitor. Instructions for Use, pages 15 / 21, CN 121360229 A

[0226] In some instances, the drug or drug composition comprises 1-200 mg of CD47 inhibitor, for example: 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5, 45.0, 47.5, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 80.0, 90.0, 92.5, 95.0, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 mg.

[0227] In some preferred embodiments, the drug or drug composition comprises 0.1-20 mg of a CD47 inhibitor, for example:0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0 mg.

[0228] In some preferred embodiments, the drug or drug composition comprises 10 mg of CD47 inhibitor.

[0229] In some preferred embodiments, the drug or drug composition comprises 100 mg of CD47 inhibitor.

[0230] In some embodiments, the drug or drug composition comprises 0.1-1000 mg / kg of CD47 inhibitor.

[0231] In some embodiments, the drug or drug composition comprises 1-1000 mg / kg of CD47 inhibitor.

[0232] In some instances, the drug or pharmaceutical composition comprises 10-1000 mg / kg of CD47 inhibitor, for example: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530540, 550, 560, 570, 580, 590, 600, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg / kg.

[0233] In some examples, the drug or pharmaceutical composition contains 10-300 mg / kg of CD47 inhibitor.

[0234] In some examples, the drug or pharmaceutical composition contains 10-200 mg / kg of CD47 inhibitor.

[0235] In some examples, the drug or pharmaceutical composition contains 1-300 mg / kg of CD47 inhibitor.

[0236] In some instances, the drug or pharmaceutical composition comprises a CD47 inhibitor of 1-200 mg / kg, for example: 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5, 45.0, 47.5, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 80.0, 90.0, 92.5, 95.0, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200mg / kg.

[0237] In some preferred embodiments, the drug or pharmaceutical composition comprises 0.1-20 mg / kg of CD47 inhibitor, for example: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.65.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, Instruction Manual 16 / 21 pages 19 CN 121360229 A 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0 mg / kg.

[0238] In some preferred embodiments, the drug or pharmaceutical composition comprises 10 mg / kg of CD47 inhibitor.

[0239] In some preferred embodiments, the drug or pharmaceutical composition comprises 100 mg / kg of CD47 inhibitor.

[0240] In some embodiments, the drug or pharmaceutical composition comprises 0.1-1000 nmol of CD47 inhibitor.

[0241] In some instances, the drug or pharmaceutical composition comprises 1-1000 nmol of CD47 inhibitor.

[0242] In some instances, the drug or pharmaceutical composition comprises 10-1000 nmol of CD47 inhibitor.

[0243] In some preferred instances, the drug or pharmaceutical composition comprises 1-100 nmol of CD47 inhibitor, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nmol.

[0244] In some preferred embodiments, the medicament or pharmaceutical composition comprises 20 nmol of CD47 inhibitor.

[0245] In some embodiments, the medicament / pharmaceutical composition comprises a second therapeutic agent for treating or adjunctive treatment of sepsis-related diseases.

[0246] In some instances, the second therapeutic agent may be administered separately, sequentially, or simultaneously with the CD47 inhibitor.

[0247] In some instances, the second therapeutic agent may be prepared separately from the CD47 inhibitor as different formulations, or may be prepared as the same formulation.

[0248] In some instances, the second therapeutic agent includes, but is not limited to: 0.9% saline, Ringer's injection, glucose injection, norepinephrine, epinephrine, benzodiazepines (such as midazolam, lorazepam), Angong Niuhuang Wan, Tanreqing injection, Xingnaojing injection, compound musk injection, biological agents containing coagulation factors, biological agents containing platelets, heparin, ulinastatin, glucocorticoids, or combinations thereof.

[0249] In some instances, the drug / drug composition further includes at least one pharmaceutically acceptable excipient.

[0250] Treatment Methods

[0251] As disclosed herein, the term "subject" includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc.

[0252] As disclosed herein, the term “effective amount” or “therapeutic effective amount” refers to the amount that, alone or in combination with other doses, achieves the desired response or desired effect. In the case of treating a particular disease or condition, the desired response preferably involves inhibiting the disease process. This includes slowing the progression of the disease, particularly interrupting or reversing the progression of the disease. The desired response in the treatment of a disease or condition may also be delaying the onset of said disease or condition, or preventing the onset of said disease or condition. The effective amount of the drug or pharmaceutical composition described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physical condition, size and weight, duration of treatment, type of concomitant treatment (if present), specific route of administration, and similar factors. Therefore, the dosage of the drug or pharmaceutical composition described herein may depend on several such parameters. In cases where the response in the patient is insufficient at the initial dose, a higher dose (or an effective higher dose achieved through a different, more localized route of administration) may be used.

[0253] This disclosure also provides a method for treating heatstroke, the method comprising:

[0254] administering a therapeutically effective amount of any of the aforementioned CD47 inhibitors to a subject in need. Specification 17 / 21 pages 20 CN 121360229 A

[0255] In some instances, the heatstroke includes classic heatstroke and exertional heatstroke.

[0256] In some instances, the subject has been exposed to a high-temperature and high-humidity environment.

[0257] In some preferred instances, the heatstroke also includes heatstroke complications.

[0258] In some preferred instances, the heatstroke complications include, but are not limited to: heatstroke-induced shock, heatstroke...Coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or a combination thereof.

[0259] In some preferred embodiments, the subject has an imbalance in thermoregulation.

[0260] In some preferred embodiments, the subject's lung tissue is damaged.

[0261] In some more preferred embodiments, the subject has enlarged alveolar septa.

[0262] In some preferred embodiments, the subject's liver tissue is damaged.

[0263] In some more preferred embodiments, the subject has swollen liver lobules, disordered hepatic cords, reduced hepatic sinusoids, increased red blood cells in the hepatic sinusoids, or a combination thereof.

[0264] In some embodiments, the subject has coagulation dysfunction or severe coagulation disorder.

[0265] In some more preferred embodiments, the fibrinogen level in the serum of the subject is significantly elevated, where "significant" means that there is a significant difference in fibrinogen level compared with the normal physiological state, p < 0.05, preferably p < 0.01, more preferably p < 0.001.

[0266] This disclosure also provides a method for alleviating or reducing lung tissue damage in a subject, the method comprising:

[0267] administering a therapeutically effective amount of any of the aforementioned CD47 inhibitors to a subject in need.

[0268] In some embodiments, the subject is suspected of or diagnosed with heatstroke.

[0269] In some preferred embodiments, the heatstroke includes classic heatstroke and exertional heatstroke.

[0270] In some preferred embodiments, the heatstroke also includes heatstroke complications.

[0271] In some preferred embodiments, the heatstroke complications include, but are not limited to: heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or a combination thereof.

[0272] In some embodiments, the subject has been exposed to a high temperature and humidity environment.

[0273] In some preferred embodiments, the subject has an imbalance in thermoregulation.

[0274] In some preferred embodiments, the subject has enlarged alveolar septa.

[0275] In some preferred embodiments, the subject's liver tissue is damaged.

[0276] In some more preferred embodiments, the subject has swollen liver lobules, disordered hepatic cords, reduced hepatic sinusoids, increased red blood cells in the hepatic sinusoids, or a combination thereof.

[0277] In some embodiments, the subject has coagulation dysfunction or severe coagulation disorder.

[0278] In some more preferred embodiments, the fibrinogen level in the serum of the subject was significantly increased,Significant means that the fibrinogen content is significantly different from that in the normal physiological state, p<0.05, preferably p<0.01, more preferably p<0.001.

[0279] This disclosure also provides a method for alleviating or reducing liver tissue damage in a subject, the method comprising:

[0280] administering a therapeutically effective amount of any of the aforementioned CD47 inhibitors to a subject in need.

[0281] In some preferred embodiments, the heatstroke includes classic heatstroke and exertional heatstroke.

[0282] In some preferred embodiments, the heatstroke also includes heatstroke complications.

[0283] In some preferred embodiments, the complications of heatstroke include, but are not limited to: heatstroke-induced shock, heatstroke-related coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or a combination thereof.

[0284] In some embodiments, the subject has been exposed to a high temperature and high humidity environment.

[0285] In some preferred embodiments, the subject has an imbalance in thermoregulation.

[0286] In some preferred embodiments, the subject has hepatic lobule swelling, disordered hepatic cord arrangement, reduced hepatic sinusoids, increased red blood cells in the hepatic sinusoids, or a combination thereof.

[0287] In some preferred embodiments, the subject's lung tissue is damaged.

[0288] In some more preferred embodiments, the subject has thickened alveolar septa.

[0289] In some instances, the subject has coagulation dysfunction or severe coagulation disorder.

[0290] In some more preferred instances, the subject's serum fibrinogen level is significantly elevated, meaning that the fibrinogen level is significantly different from that in a normal physiological state, p < 0.05, preferably p < 0.01, more preferably p < 0.001.

[0291] This disclosure also provides a method for alleviating or reducing coagulation dysfunction in a subject, the method comprising:

[0292] administering a therapeutically effective amount of any of the aforementioned CD47 inhibitors to a subject in need.

[0293] In some instances, the subject is suspected of or diagnosed with heatstroke.

[0294] In some preferred instances, the heatstroke includes classic heatstroke and exertional heatstroke.

[0295] In some preferred instances, the heatstroke also includes heatstroke complications.

[0296] In some preferred embodiments, the complications of heatstroke include, but are not limited to: heatstroke shock, heatstroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, respiratory dysfunction, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple organ failure, or combinations thereof.

[0297] In some instances, the subject was exposed to a high temperature and high humidity environment.

[0298] In some preferred instances, the subject's thermoregulation function was imbalanced.

[0299] In some preferred instances, the subject's serum fibrinogen content was significantly elevated, meaning that the fibrinogen content was significantly different from that in the normal physiological state, p<0.05, preferably p<0.01, more preferably p<0.001.

[0300] In some preferred instances, the subject's alveolar septa were thickened.

[0301] In some preferred instances, the subject's liver tissue was damaged.

[0302] In some more preferred instances, the subject had swollen liver lobules, disordered hepatic cords, reduced hepatic sinusoids, increased red blood cells in the hepatic sinusoids, or a combination thereof.

[0303] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without a specified manufacturer are commercially available conventional products. Numerous specific details are given in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in numerous publications.

[0304] The method for constructing a heatstroke mouse model in this invention is as follows:

[0305] Construction of a heatstroke mouse model: The temperature and humidity of the animal constant temperature incubator were pre-adjusted: temperature 36.5℃, humidity 60%. Mice were placed in the incubator to simulate heatstroke caused by simple heat stress. Instructions for Use, Pages 19 / 21, 22, CN 121360229 A

[0306] Example 1: Effect of CD47 monoclonal antibody treatment on lung tissue damage caused by heatstroke

[0307] C57 mice were placed in a constant temperature incubator at 36.5℃ / 60% humidity. Lung tissues of the mice were taken out at 6h, 12h, 24h, and 40h, respectively, and were sectioned and observed by H&E staining.

[0308] The results showed that the alveolar septa were significantly thickened, with the most obvious thickening at 12h. After 1h of heat stress, 10mg / kg anti-CD47 monoclonal antibody (brand Bioxcell, catalog number #BE0270, clone number MIAP301) was used. After 11h of treatment, HE staining significantly alleviated the thickening of the alveolar septa (Figure 1). This indicates that anti-CD47 monoclonal antibody treatment can significantly reduce lung damage.

[0309] Example 2: Effect of CD47 monoclonal antibody treatment on liver tissue damage caused by heatstroke

[0310] C57 mice were placed in a constant temperature incubator at 36.5℃ / 60% humidity. Lung tissues of the mice were taken out at 6h, 12h, 24h and 40h, respectively, and H&E staining was performed after sectioning.

[0311] The results showed that heat stress could cause swelling of liver lobules, disordered arrangement of hepatic cords, shrinkage of hepatic sinusoids and increase of red blood cells in hepatic sinusoids, which was most obvious at 12h-24h (Figure 2). Treatment with 10mg / kg anti-CD47 monoclonal antibody could significantly alleviate the destruction of liver lobule structure and reduce liver damage (Figure 2).

[0312] Example 3: Application of CD47 small molecule inhibitors and CD47 siRNA in the treatment of heatstroke

[0313] The siRNAs used in this example include: mCD47-1031-s:GCAGAACUACUUGGAUUAGUUTT (SEQ ID NO:1); mCD47-1031-a:AACUAAUCCAAGUAGUUCUGCTT (SEQ ID NO:2).

[0314] C57 mice were placed in a constant temperature incubator at 36.5℃ / 60% humidity. After heat stimulation for 1 hour, they were given CD47 monoclonal antibody (10mg / kg) / 4-Methylumbelliferone (4-MU, 100mg / kg) / CD47-siRNA (20nmol / mouse).

[0315] The experimental groups are as follows:

[0316] Treatment experiment. Mice were divided into 5 groups: Sham (n=4); CLP+Solvent Control (intraperitoneal injection, single injection 1 hour after CLP, n=16); CLP+Anti-CD47 (10 mg / kg intravenous injection, single injection 1 hour after CLP, n=16); CLP+4-Methylumbelliferone (100 mg / kg, intraperitoneal injection, single injection 1 hour after CLP, n=16); CLP+CD47-siRNA (20 nmol, intravenous injection, first injection 1 hour after CLP, second injection 24 hours after CLP, n=16). Survival benefits were assessed by Kaplan-Meier survival analysis, and significance was assessed by log-rank test.

[0317] Kaplan-Meier survival curve analysis showed that administration of CD47 monoclonal antibody, 4-MU and CD47-siRNA significantly improved the survival rate of mice with heatstroke (Figure 3).

[0318] Example 4: Effect of CD47 monoclonal antibody treatment on serum biochemical indicators in heatstroke model mice

[0319] C57 mice were placed in a constant temperature incubator at 36.5℃ / 60% humidity. After heat stimulation for 1 hour, CD47 monoclonal antibody (10mg / kg) was administered via the tail vein. After 11 hours of treatment, mouse serum was collected for blood biochemical related tests.

[0320] The reagent kit for blood biochemical related tests in this example is shown below:

[0321] Albumin (ALB) assay kit (Sangon Biotech, CAT#:D721206); Alanine aminotransferase (ALT) assay kit (Sangon Biotech, CAT#:D799580); Aspartate aminotransferase (AST) assay kit (Biosharp, CAT#:BL1410B); Creatine kinase (CK) assay kit (Biosharp, CAT#:BL889B); Urea (UREAL) assay kit (Biosharp, CAT#:BL1484B); Creatinine (CREA) assay kit (Biosharp, CAT#:BL890B)

[0322] Blood biochemistry tests showed that CD47 monoclonal antibody treatment significantly reduced the levels of serum liver function-related indicators, including albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), urea (UREAL), and creatinine (CREA), in heatstroke mice (Figure 4).

[0323] Example 5: Effect of CD47 monoclonal antibody treatment on coagulation disorders in heatstroke model mice

[0324] C57 mice were placed in a constant temperature incubator at 36.5℃ and 60% humidity. After heat stimulation for 1 hour, CD47 monoclonal antibody (10mg / kg) was administered. After 11 hours of treatment, serum was collected and the coagulation parameters of the mice were measured. Instructions for use 20 / 21 pages 23 CN 121360229 A

[0325] ELISA detection showed that CD47 monoclonal antibody treatment could significantly alleviate coagulation dysfunction in heatstroke mice (Figures 3 and 5).

[0326] CD47 gene knockout heterozygous mice were prepared by Jicui Pharmaceutical Biotechnology Co., Ltd. (Jiangsu) using Crispr-Cas9 technology, and then CD47 knockout (CD47 KO) homozygous mice were bred by ourselves.

[0327] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention. Specification 21 / 21 pages 24 CN 121360229 A Figure 1 Specification Figure 1 / 4 pages 25 CN 121360229 A Figure 2 Specification Figure 2 / 4 pages 26 CN 121360229 A Figure 3 Figure 4 Specification Figure 3 / 4 pages 27 CN 121360229 A Figure 5 Specification Figure 4 / 4 pages 28 CN 121360229 A Abstract The present invention belongs to the technical field of medicines andspecifically relates to the use of CD47 as a target in the treatment of heatstroke. The present invention provides the use of CD47 in preparing a medicine or a pharmaceutical composition for treating heatstroke. The present invention for the first time takes CD47 as a target for heatstroke treatment and achieves remarkable therapeutic effects. In a mouse model of heatstroke, tail vein injection of CD47 monoclonal antibody can alleviate heatstroke-caused functional injury of multiple organs including lung and kidney and improve the survival rate of mice with heatstroke; administration of the CD47 monoclonal antibody at a dosage of 10 mg / kg can raise the survival rate to about 80%. Therefore, CD47 can be used as a therapeutic target to prepare medicines for treating heatstroke.

Claims

1. Use of CD47 in the preparation of a medicament or pharmaceutical composition for treating heat stroke; Preferably, the heat stroke comprises classic heat stroke and exertional heat stroke; Preferably, the heat stroke further comprises heat stroke complications; More preferably, the heat stroke complications comprise heat stroke shock, heat stroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, impaired respiratory function, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple system organ failure, or a combination thereof.

2. Use according to claim 1, characterized in that, The CD47 comprises a CD47 nucleic acid molecule and / or a CD47 protein molecule; and / or, the medicament comprises a CD47 inhibitor; Preferably, the CD47 inhibitor is a substance that inhibits, down-regulates, attenuates, antagonizes, and / or modulates the level of CD47 molecules; and / or, the CD47 inhibitor is a substance that blocks the interaction of CD47 with SIRPa; More preferably, the substance comprises a nucleic acid drug, a protein drug, a small molecule inhibitor, or a combination thereof.

3. Use according to claim 2, characterized in that, The nucleic acid drug comprises siRNA, miRNA, ASO, shRNA, sgRNA, Aptamer, or a combination thereof, preferably siRNA, more preferably siRNA sequences with nucleotide sequences shown in SEQ ID NO: 1-2; and / or, the protein drug comprises an antibody, a polypeptide, an antibody drug conjugate, a polypeptide drug conjugate, a ribozyme, a CAS protein, or a combination thereof, preferably an antibody, more preferably a monoclonal antibody; and / or, the small molecule inhibitor comprises NCGC00138783, NCGC00138783TFA, NCGC00138783freebase, 4-MU, 4-MUB, RS17, DMUP, NCGC00538431, RRx-001, Metformin, JQ1, PQ-912, SEN177, RS-17, Pep-20, D4-2, or a combination thereof.

4. Use according to any one of claims 1 to 3, characterized in that, The medicament or pharmaceutical composition also includes a second therapeutic agent for treating heat stroke, preferably 0.9% normal saline, Ringer's injection, glucose injection, norepinephrine, epinephrine, benzodiazepine a non-drug, An Gong Niuhuang Wan, Tanreqing injection, Xingnaojing injection, compound musk injection, biological agents containing blood coagulation factors, biological agents containing platelets, heparin, ulinastatin, glucocorticoids, or a combination thereof.

5. Use of a CD47 inhibitor in the preparation of a medicament or pharmaceutical composition for treating heat stroke; Preferably, the heat stroke comprises classic heat stroke and exertional heat stroke; Preferably, the heat stroke further comprises heat stroke complications; More preferably, the heat stroke complications comprise heat stroke shock, heat stroke coagulopathy, acute kidney injury, acute liver injury, coagulation dysfunction, central nervous system injury, impaired respiratory function, gastrointestinal dysfunction, rhabdomyolysis, disseminated intravascular coagulation, multiple system organ failure, or a combination thereof. Preferably, the medicament or pharmaceutical composition further comprises a second therapeutic agent for treating heat stroke, preferably 0.9% normal saline, Ringer's injection, glucose injection, norepinephrine, epinephrine, benzodiazepine a non-drug, An Gong Niuhuang Wan, Tanreqing injection, Xingnaojing injection, Fufangmusha injection, a biological agent containing blood coagulation factors, a biological agent containing platelets, heparin, ulinastatin, a glucocorticoid, or a combination thereof.

6. Use according to claim 5, characterized in that, The CD47 inhibitor is a substance that inhibits, down-regulates, attenuates, antagonizes, and / or modulates the level of CD47 molecules; and / or, the CD47 inhibitor is a substance that blocks the interaction of CD47 with SIRPa; Preferably, the molecular level comprises a nucleic acid molecule level and / or a protein molecule level; Preferably, the substance comprises a nucleic acid drug, a protein drug, a small molecule inhibitor, or a combination thereof.

7. Use according to claim 5 or 6, characterized in that, The nucleic acid drugs include siRNA, miRNA, ASO, shRNA, sgRNA, Aptamer and combinations thereof, preferably siRNA, more preferably siRNA with nucleotide sequence as shown in SEQ ID NO: 1-2; And / or, the protein drugs include antibodies, polypeptides, antibody drug conjugates, polypeptide drug conjugates, ribozymes, CAS proteins and combinations thereof, preferably antibodies, more preferably monoclonal antibodies; And / or, the small molecule inhibitors include NCGC00138783, NCGC00138783TFA, NCGC00138783freebase, 4-MU, 4-MUB, RS17, DMUP, NCGC00538431, RRx-001, Metformin, JQ1, PQ-912, SEN177, RS-17, Pep-20, D4-2 or combinations thereof.

8. Use according to any one of claims 5 to 7, characterized in that, The drug or pharmaceutical composition comprises 0.1-1000 mg of CD47 inhibitor, preferably 1-1000 mg, more preferably 1-200 mg, for example: 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5, 45.0, 47.5, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 80.0, 90.0, 92.5, 95.0, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 mg; Preferably, the drug or pharmaceutical composition comprises 10 or 100 mg of CD47 inhibitor.

9. Use according to any one of claims 5 to 8, characterized in that, The pharmaceutical or pharmaceutical composition comprises 0.1-1000 mg / kg of the CD47 inhibitor, preferably 1-1000 mg / kg, more preferably 1-200 mg / kg, for example: 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5, 45.0, 47.5, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 80.0, 90.0, 92.5, 95.0, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 mg / kg; Preferably, the pharmaceutical or pharmaceutical composition comprises 10 or 100 mg / kg of the CD47 inhibitor.

10. Use according to any one of claims 5 to 9, characterized in that, The pharmaceutical or pharmaceutical composition comprises 0.1-1000 nmol of the CD47 inhibitor, preferably 1-1000 nmol, more preferably 10-1000 nmol, further 1-100 nmol, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nmol, further 20 nmol.

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