Application of combination of dimethyl sulfoxide and thrombopoiesis promoting drug in treatment of acute radiation sickness

Abstract

The invention discloses application of combination of dimethyl sulfoxide and thrombopoiesis promoting drugs in treatment of acute radiation sickness. It is found for the first time that a combined scheme of DMSO and thrombopoiesis promoting drugs can significantly improve the survival rate of animals irradiated by lethal and even super lethal dose, animal weight reduction caused by radiation is reversed, and a significant synergistic effect is shown. The invention provides a safe and reliable radiation prevention and treatment measure for patients suffering from acute high-dose radiation exposure, and has a wide clinical application scene.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to the application of dimethyl sulfoxide in combination with thrombolytic drugs in the treatment of acute radiation sickness. Background Technology

[0002] Acute radiation sickness is a systemic disease caused by exposure to a high dose (>1 Gy) of ionizing radiation over a short period of time. Clinically, it is classified into three types based on the radiation dose, clinical characteristics, and basic pathological changes: bone marrow type, intestinal type, and cerebral type. The main pathological feature of bone marrow type acute radiation sickness is a significant decrease in the number of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow, accompanied by a decrease in multiple blood cell lines, leading to life-threatening sepsis.

[0003] Dimethyl sulfoxide (DMSO) is a polar organic solvent with multiple biological functions. Currently, it is widely used in the medical field as a cryoprotectant for the cryopreservation of organs and cells. It possesses anti-inflammatory, analgesic, diuretic, and sedative pharmacological effects and is relatively safe and effective as a pharmaceutical solvent. Therefore, it is also widely used in the pharmaceutical industry and clinical drug delivery as a pharmaceutical solvent and drug carrier. In recent years, research on the radiation protection effects of DMSO has also received widespread attention, providing new insights into the pathogenesis and protection against radiation damage to multiple tissues and organs.

[0004] Thromboplastin-promoting drugs include recombinant human thrombopoietin (rhTPO) and thrombopoietin receptor agonists (TPO-RA). These drugs regulate megakaryocyte proliferation, differentiation, and maturation by specifically binding to the thrombopoietin (TPO) receptor, thereby promoting platelet production. In recent years, thrombopolastin-promoting drugs have been widely used to treat thrombocytopenia caused by various factors, effectively reducing the risk of bleeding, minimizing platelet transfusions, and avoiding adverse reactions associated with blood product transfusions. Currently, five thrombopolastin-promoting drugs have been approved for clinical use worldwide, including rhTPO, romiplostim, eltrombopag, hetrombopag, avatrombopag, and lusutrombopag.

[0005] In the clinical treatment of radiation accident patients, multi-drug combination therapy can effectively promote functional recovery after severe bone marrow suppression. However, the effects of combining dimethyl sulfoxide with thrombopoietin-promoting drugs on acute radiation sickness are currently unknown. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the present invention aims to provide a drug combination for the prevention and treatment of acute radiation sickness.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides the use of dimethyl sulfoxide combined with a platelet-promoting drug in the preparation of a medicament for the prevention and / or treatment of acute radiation sickness.

[0008] Furthermore, the thrombopoietin-promoting drugs include recombinant human thrombopoietin (rhTPO) and thrombopoietin receptor agonists.

[0009] Furthermore, the thrombopoietin receptor agonist includes one or more of roprostine, eltrombopag, hetrombopag, avatrombopag, and rutrombopag.

[0010] Furthermore, the platelet-promoting drug is selected from rhTPO or roprostine.

[0011] Furthermore, the acute radiation sickness includes bone marrow type acute radiation sickness, intestinal type acute radiation sickness, and cerebral type acute radiation sickness.

[0012] In this invention, the applicant has discovered for the first time that the combined use of DMSO with thrombopoietin-promoting drugs such as roprostine and rhTPO can significantly improve the survival rate of animals irradiated with lethal or even super-lethal doses, reverse radiation-induced weight loss, and exhibit a significant synergistic effect, with the synergistic effect being more pronounced the higher the irradiation dose received by the animal. Simultaneously, the applicant has found that the combined use of DMSO with thrombopoietin-promoting drugs can significantly increase the proportion and absolute number of hematopoietic stem and progenitor cells in the bone marrow of irradiated animals, enhance extramedullary hematopoietic activity in the spleen, and promote the recovery of hematopoietic function of the leukocyte, platelet, and erythrocyte lineages.

[0013] Drug combination therapy can produce multi-level interactions, ultimately manifesting as synergistic, additive, or antagonistic effects. Synergistic effects refer to the phenomenon where the combined action of two or more drugs results in a consistent direction and enhanced efficacy, categorized as additive or enhancing effects. Antagonistic effects, on the other hand, refer to a weakening or disappearance of the combined effect. Additive or synergistic effects mean that the combined effect of two drugs acting on disease-related targets is equal to or greater than the sum of their individual effects.

[0014] In this invention, recombinant human thrombopoietin (rhTPO) is the most important stimulating factor regulating platelet production. rhTPO is a full-length glycosylated thrombopoietin produced by recombinant gene technology from Chinese hamster ovary cells, after purification. It has similar pharmacological effects in increasing platelet count as endogenous thrombopoietin. Nplate is a second-generation long-acting thrombopoietin (TPO) receptor agonist that stimulates platelet production by mimicking the body's natural TPO. Both rhTPO and Nplate stimulate endogenous cytokines that promote megakaryocyte growth and differentiation, thus stimulating all stages of megakaryocyte development, including the proliferation of precursor cells and the development and maturation of polyploid megakaryocytes, thereby increasing platelet count. Those skilled in the art can obtain rhTPO and Nplate through conventional methods.

[0015] In this invention, the treatment and / or prevention refer to delaying disease progression, preventing disease progression, and / or reducing the severity of the symptoms that will or are expected to develop. Therefore, these terms include improving existing disease symptoms, preventing additional symptoms, improving or preventing underlying metabolic causes of symptoms, inhibiting disorders or diseases, for example, preventing the development of disorders or diseases, alleviating disorders or diseases, regressing disorders or diseases, reducing symptoms caused by diseases or disorders, or stopping the symptoms of diseases or disorders.

[0016] In some implementations, the acute radiation sickness includes, but is not limited to: acute radiation sickness caused by various types of radiation exposure, such as mild myeloid acute radiation sickness, moderate myeloid acute radiation sickness, severe myeloid acute radiation sickness, extremely severe myeloid acute radiation sickness, intestinal acute radiation sickness, cerebral acute radiation sickness, and acute skin injury.

[0017] Furthermore, the radiation irradiation includes alpha irradiation, beta irradiation, gamma irradiation, neutron irradiation, proton irradiation, heavy ion irradiation, electron irradiation, and / or X-ray irradiation.

[0018] A second aspect of the present invention provides a medicine box.

[0019] Furthermore, the medicine box includes: (I) A first formulation containing dimethyl sulfoxide; (II) A second formulation containing a platelet-producing drug.

[0020] Furthermore, the second formulation is selected from roprostine, eltrombopag, hetrombopag, avatrombopag, rutrombopag, TPO and / or rhTPO.

[0021] Furthermore, the second formulation is selected from roprostine and / or rhTPO.

[0022] Furthermore, the medicine box also includes an instruction manual.

[0023] Furthermore, the instructions state that the first and second preparations are used in combination to prevent and / or treat acute radiation sickness.

[0024] A third aspect of the present invention provides a pharmaceutical composition.

[0025] Furthermore, the pharmaceutical composition includes dimethyl sulfoxide and a platelet-producing drug.

[0026] Furthermore, the thrombopoietin-promoting drug includes recombinant human thrombopoietin and thrombopoietin receptor agonists.

[0027] Furthermore, the thrombopoietin receptor agonist includes one or more of roprostine, eltrombopag, hetrombopag, avatrombopag, and rutrombopag.

[0028] Furthermore, the platelet-promoting drug is selected from rhTPO or roprostine.

[0029] Furthermore, the dose ratio of dimethyl sulfoxide to the platelet-promoting drug is 100-1,000,000:1.

[0030] Preferably, the dose ratio of dimethyl sulfoxide to the platelet-promoting drug is 1000-100000:1.

[0031] Preferably, the dose ratio of dimethyl sulfoxide to the platelet-producing drug is 100,000:1.

[0032] In some embodiments, the appropriate dosage of the drug or pharmaceutical composition described in this invention can be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate, and responsiveness. Skilled physicians can usually easily determine the prescription and the desired dosage that is effective for treatment, as long as it can produce the expected therapeutic and / or preventive effect on acute radiation sickness, such dosage is within the protection scope of this invention.

[0033] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

[0034] In some embodiments, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0035] In some embodiments, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, and water. The binder includes, but is not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginate and alginates, xanthan gum, and hydroxypropyl cellulose. The surfactant includes, but is not limited to, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol. The humectant includes, but is not limited to, glycerol and starch. The adsorbent carrier includes, but is not limited to, starch, lactose, bentonite, and soap clay. The lubricant includes, but is not limited to, zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric sucrose, and magnesium lauryl sulfate. The filler includes, but is not limited to, mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, and starch. The disintegrants include, but are not limited to: crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosylcarboxymethyl cellulose sodium, soybean polysaccharides, etc.

[0036] In some embodiments, the pharmaceutical composition may also contain other drugs that can be used to treat and / or prevent acute radiation sickness, including but not limited to: sulfur-containing drugs (e.g., N-acetylcysteine, aminoethyl isothiourea, amifostine for injection, etc.), amine drugs (e.g., acetylcysteine ​​tablets, indoleamine, catecholamines, etc.), and traditional Chinese medicine drugs (e.g., anemarrhena, ganoderma, rubia, ginseng, etc.).

[0037] Furthermore, the dimethyl sulfoxide and the platelet-producing drug may have the same or different dosage forms.

[0038] Furthermore, the dosage form includes gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0039] Furthermore, the gastrointestinal dosage forms include solutions, granules, tablets, capsules, suspensions, powders, sustained-release preparations, effervescent tablets, emulsions, syrups, drops, and chewable tablets.

[0040] Furthermore, the non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0041] In some embodiments, the injectable dosage forms include, but are not limited to, various injectables such as subcutaneous injections, intravenous injections, intramuscular injections, intradermal injections, and intracavitary injections; the respiratory dosage forms include, but are not limited to, sprays, aerosols, and powder inhalers; the cavity dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pills, for use in the rectum, vagina, urethra, nasal cavity, and ear canal; the mucosal dosage forms include, but are not limited to, eye drops, nasal drops, ointments, mouthwashes, sublingual tablets, adhesive tablets, and patches; and the skin dosage forms include, but are not limited to, topical solutions, lotions, liniments, ointments, plasters, pastes, and patches.

[0042] In some embodiments, the drugs or pharmaceutical compositions provided by the present invention can be formulated in various forms, such as subcutaneous injection dosage forms, for the prevention and treatment of acute radiation sickness, particularly to reduce hematopoietic damage caused by radiation or chemotherapy drugs, increase blood cell levels, or for adjuvant cancer therapy.

[0043] Furthermore, the dimethyl sulfoxide and the platelet-producing drug are administered simultaneously or sequentially.

[0044] In some embodiments, the administration of the dimethyl sulfoxide and the thrombolytic drug in combination includes, but is not limited to, single or sequential administration within two weeks before, during, and / or within two weeks after exposure to radiation.

[0045] In some embodiments, the timing of administration of the dimethyl sulfoxide and the thrombolytic drug in combination includes, but is not limited to, single or sequential administration within 5 days before radiation exposure, during radiation exposure, and / or within 3 days (inclusive) after radiation exposure.

[0046] In some embodiments, the timing of administration of the dimethyl sulfoxide and the thrombolytic drug in combination includes, but is not limited to, single or consecutive sequential administration within 3 days before, during, and / or 3 days after radiation exposure.

[0047] In some embodiments, the timing of administration of the dimethyl sulfoxide and the thrombolytic agent in combination includes, but is not limited to, single or sequential administration within 24 hours before and / or within 2 hours after exposure to radiation.

[0048] In some implementations, the combined administration of dimethyl sulfoxide and the thrombolytic drug is performed as follows: 12 hours before irradiation, preferably 1 hour before irradiation, followed by administration of the thrombolytic drug within 30 minutes or 24 hours after irradiation.

[0049] In some embodiments, the administration time and dosage of the combined dimethyl sulfoxide and thrombolytic drug are as follows: oral administration of (0.1~10) g / kg dimethyl sulfoxide 1 hour before irradiation; subcutaneous administration of (1) g / kg dimethyl sulfoxide within 30 minutes or 24 hours after irradiation. 100) μg / kg of platelet-producing drugs.

[0050] In this invention, those skilled in the art can adjust the administration time and / or dosage of the dimethyl sulfoxide and the platelet-promoting drug according to actual conditions, and the adjusted administration time and / or dosage are also included within the protection scope of this invention.

[0051] The fourth aspect of the present invention provides any of the following applications: (1) The use of dimethyl sulfoxide and thrombolytic drugs in combination in the preparation of drugs for the prevention and / or treatment of acute radiation sickness; (2) Application of platelet-producing drugs in the preparation of drugs for improving the therapeutic effect of dimethyl sulfoxide on acute radiation sickness; (3) The application of dimethyl sulfoxide in the preparation of drugs for improving the therapeutic effect of thrombopoietin-promoting drugs on acute radiation sickness; (4) The use of dimethyl sulfoxide and thrombolytic drugs in combination in the preparation of drugs for the prevention and / or treatment of bone marrow suppression caused by radiation exposure; (5) The use of dimethyl sulfoxide and thrombolytic drugs in combination in the preparation of drugs for the prevention and / or treatment of death in animals or humans caused by radiation exposure; (6) The use of dimethyl sulfoxide and thrombolytic drugs in combination in the preparation of drugs for the prevention and / or treatment of peripheral blood pancytopenia, leukopenia, neutropenia, lymphopenia, thrombocytopenia and / or anemia caused by radiation exposure; (7) Application of dimethyl sulfoxide and platelet-producing drugs in the preparation of drugs for the prevention and / or treatment of radiation-induced damage to hematopoietic stem and / or progenitor cells.

[0052] Furthermore, the thrombopoietin-promoting drug includes recombinant human thrombopoietin and thrombopoietin receptor agonists.

[0053] Furthermore, the thrombopoietin receptor agonist includes one or more of roprostine, eltrombopag, hetrombopag, avatrombopag, and rutrombopag.

[0054] Furthermore, the platelet-promoting drug is selected from rhTPO or roprostine.

[0055] Advantages and beneficial effects of the present invention: This invention is the first to discover that a multi-drug combination regimen, administering DMSO for prophylaxis before irradiation and rhTPO or roprostine for treatment after irradiation, can significantly improve the survival rate of animals exposed to lethal and even super-lethal doses of radiation, reverse radiation-induced weight loss, and exhibit a significant synergistic effect. Compared with monotherapy, the combination therapy can significantly increase the proportion and absolute number of hematopoietic stem and progenitor cells in the bone marrow, enhance extramedullary hematopoietic activity in the spleen, and promote the recovery of hematopoietic function of the leukocyte, platelet, and erythrocyte lineages. This discovery provides a safe and reliable radiation protection measure for patients suffering from acute high-dose radiation exposure, applicable to the prevention and treatment of large-scale irradiated populations during sudden radiation and nuclear events. Attached Figure Description

[0056] Figure 1 The study showed the effects of combined administration of DMSO and rhTPO on the survival and body weight of mice subjected to different doses of lethal irradiation. Figure 1 A, Figure 1 C and Figure 1 E shows the animal survival rate after different doses of irradiation; Figure 1 B Figure 1 D and Figure 1 F shows the changes in animal body weight after different doses of irradiation; Figure 2 The effects of combined administration of DMSO and rhTPO on survival and peripheral blood cell recovery in mice irradiated with 9.5 Gy were shown; among which Figure 2 A shows the animal survival rate after photography. Figure 2 B shows the white blood cell count; Figure 2 C shows the number of neutrophils; Figure 2 D shows the platelet count; Figure 2 E indicates the number of red blood cells; Figure 2 F indicates the hemoglobin level; Figure 3 The effect of combined administration of DMSO and rhTPO on the recovery of peripheral blood counts in mice irradiated with 6.5 Gy was shown; among which Figure 3 A shows the white blood cell count. Figure 3 B shows the number of neutrophils. Figure 3 C indicates the number of lymphocytes. Figure 3 D shows the platelet count. Figure 3 E indicates the number of red blood cells; Figure 3 F indicates the hemoglobin level; Figure 4 The study showed the effects of combined administration of DMSO and rhTPO on bone marrow histopathology and nucleated cell count in mice 10 days after 6.5 Gy irradiation; among which, Figure 4 A and B show histopathological images of bone marrow tissue from healthy mice and mice irradiated with 6.5 Gy, respectively. Figure 4C, D, and E show the bone marrow histopathological images of mice in the DMSO intervention group, rhTPO treatment group, and combined drug administration group, respectively. Figure 4 F shows the number of nucleated cells in the mouse femur; Figure 5 This study demonstrated the effect of combined administration of DMSO and rhTPO on the recovery of bone marrow hematopoietic stem and progenitor cells in mice 10 days after 6.5 Gy irradiation; among which... Figure 5 A shows a schematic diagram of flow cytometry analysis. Figure 5 B shows the proportion and number of bone marrow hematopoietic progenitor cells (LK). Figure 5 C shows the proportion and number of bone marrow hematopoietic stem and progenitor cells (LSK). Figure 5 D shows the proportion and number of MPP4 pluripotent progenitor cells in the bone marrow. Figure 5 E displays the MPP3 ratio and quantity. Figure 5 F displays the MPP2 ratio and quantity. Figure 5 G indicates the proportion and number of short-term hematopoietic stem cells (ST-HSC). Figure 5 H represents the proportion and number of long-term hematopoietic stem cells (LT-HSC). Figure 5 I shows the proportion of MPPs with different differentiation biases in the LSK population; Figure 6 This study demonstrated the effect of combined administration of DMSO and rhTPO on the recovery of bone marrow hematopoietic stem and progenitor cells 14 days after 6.5 Gy irradiation in mice; among which... Figure 6 A shows the number of nucleated cells in the femoral bone marrow. Figure 6 B shows the proportion and quantity of LK in the bone marrow. Figure 6 C shows the proportion and number of LSK in the bone marrow. Figure 6 D shows the proportion and quantity of MPP4 in the bone marrow. Figure 6 E shows the proportion and quantity of MPP3 in the bone marrow. Figure 6 F shows the proportion and quantity of MPP2 in the bone marrow. Figure 6 G indicates the proportion and number of ST-HSCs in the bone marrow. Figure 6 H shows the proportion and number of LT-HSCs in the bone marrow. Figure 6 I shows the proportion of MPPs with different differentiation biases in the bone marrow LSK population; Figure 7 The study showed the effects of combined administration of DMSO and rhTPO on spleen organ coefficients, nucleated cell counts, and histopathological changes in mice 10 days after 6.5 Gy irradiation; among which... Figure 7 A, B, and C show the size of the mouse spleen, the spleen coefficient, and the number of nucleated cells in the mouse spleen, respectively. Figure 7 D, E, F, G, and H show HE staining of the spleens of healthy mice, irradiated mice, DMSO-treated mice, rhTPO-treated mice, and combined drug-treated mice, respectively. Figure 8 This study demonstrated the effect of combined administration of DMSO and rhTPO on the recovery of spleen hematopoietic stem and progenitor cells in mice 10 days after 6.5 Gy irradiation; among which... Figure 8 A shows a schematic diagram of flow cytometry analysis. Figure 8 B and C show the proportion and number of LK cells and LSK cells in the spleen, respectively. Figure 8 D, E, and F show the proportion and quantity of MPP4, MPP3, and MPP2 in the spleen, respectively. Figure 8 G, H, and I show the proportion and number of ST-HSCs, LT-HSCs, and the proportion of MPPs with different differentiation biases in the LSK population, respectively. Figure 9 The study showed the effects of combined administration of DMSO and rhTPO on spleen organ coefficients and nucleated cell counts in mice 14 days after 6.5 Gy irradiation; among which, Figure 9 A shows the size of the mouse spleen. Figure 9 B shows the spleen coefficient in mice. Figure 9 C shows the number of nucleated cells in the mouse spleen; Figure 10 This study demonstrated the effect of combined administration of DMSO and rhTPO on the recovery of spleen hematopoietic stem and progenitor cells 14 days after 6.5 Gy irradiation in mice; among which... Figure 10 A and B show the proportion and number of LK cells and LSK cells in the spleen, respectively. Figure 10 C, D, and E show the proportion and quantity of MPP4, MPP3, and MPP2 in the spleen, respectively. Figure 10 F shows the proportion and number of ST-HSCs in the spleen. Figure 10 G shows the proportion and number of LT-HSCs in the spleen. Figure 10 H shows the proportion of MPPs with different differentiation biases in the spleen LSK population; Figure 11 The study showed the effects of combined administration of DMSO and roprolastine on the survival and body weight of mice subjected to 10.5 Gy lethal irradiation; among which, Figure 11 A shows the survival rate of mice irradiated with 10.5 Gy. Figure 11 B shows the body weight of mice irradiated with 10.5 Gy; Figure 12 The study showed the effects of combined administration of DMSO and roprolastine on the survival and peripheral blood cell recovery of mice subjected to 9.0 Gy lethal irradiation; among which, Figure 12 A shows the 30-day survival rate. Figure 12 B, C, D, E, and F show the numbers of white blood cells, platelets, lymphocytes, red blood cells, and hemoglobin, respectively; * indicates comparison with the irradiation group, and # indicates comparison with the combined drug administration group; Figure 13This study showed the effect of combined administration of DMSO and roprolastine on bone marrow hematopoietic recovery in mice 10 days after 6.5 Gy irradiation; among which... Figure 13 A shows the bone marrow nucleated cell count. Figure 13 BH shows the absolute values ​​of each cell population and their proportion of the total number of nucleated cells in the bone marrow. Figure 13 I shows the proportion of cell populations with different differentiation biases in the bone marrow LSK population; * indicates comparison with the irradiation group, # indicates comparison with the combined drug administration group; Figure 14 This study demonstrated the effect of combined administration of DMSO and roprolastine on extramedullary hematopoiesis in the spleen of mice 10 days after 6.5 Gy irradiation; among which... Figure 14 AC displays spleen size, spleen coefficient, and number of nucleated cells, respectively; Figure 15 This study demonstrated the effect of combined administration of DMSO and roprolastine on extramedullary hematopoiesis in the spleen of mice 10 days after 6.5 Gy irradiation; among which... Figure 15 A displays the door-drawing strategy. Figure 15 BH shows the absolute values ​​of each cell population and their proportion of the total number of nucleated cells in the spleen. '* indicates comparison with the irradiated group, and # indicates comparison with the combined drug administration group. Detailed Implementation

[0057] The present invention will be further described below with reference to specific embodiments. However, the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0058] Unless otherwise specified in the following examples, the conditions should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0059] The experimental materials and methods used in specific embodiments of the present invention are as follows: 1. Laboratory animals Male C57BL / 6J mice aged 6-8 weeks were purchased from Beijing Jicui Yaokang Biotechnology Co., Ltd. Mice were randomly assigned to cages, with no more than 5 mice per cage, and housed in an SPF (specific pathogen-free) barrier environment at the Experimental Animal Center of the Academy of Military Medical Sciences. During the experiment, the animals had free access to maintenance pelleted feed (60Co sterilized) and acidified water (pH 2.5-3.0). The pelleted feed was purchased from Beijing Huafukang Biotechnology Co., Ltd. The housing environment was maintained with a 12-hour light / 12-hour dark cycle, a temperature of 23±2°C, and a relative humidity of 50±20%. Mice were acclimatized to the environment for at least 7 days before the experiment. All animal experiments were approved by the Experimental Animal Welfare and Ethics Committee of the Academy of Military Medical Sciences and strictly followed the institution's guidelines for laboratory animal operations. The "Regulations on the Management of Laboratory Animals in Beijing" were strictly followed, and mice were euthanized according to the "Guidelines for Ethical Review of Laboratory Animal Welfare" designated by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. The mice underwent cervical dislocation after CO2 inhalation.

[0060] 2. The sources of experimental reagents and antibodies are shown in Table 1.

[0061] Table 1. Sources of experimental reagents and antibodies

[0062] 3. Experimental Methods (1) Drug preparation and administration Preparation and injection of rhTPO: rhTPO was provided in liquid form at a concentration of 50 μg / ml and stored at 4°C protected from light. The experimental dose of rhTPO was 100 μg / kg, administered in a volume of 0.2 ml, diluted with sterile physiological saline and subcutaneously 30 minutes after irradiation. Mice in the solvent control group received a subcutaneous injection of 0.2 mL of 0.9% NaCl solution.

[0063] Preparation and subcutaneous administration of roprostine: Roprostine is provided in solid powder form. Before use, add 0.72 mL of physiological saline to each vial of solid powder, gently shake to completely dissolve into a colorless liquid with a concentration of 500 μg / mL, aspirate with a syringe and place in a 1.5 mL centrifuge tube, aliquot, and store at -80℃ protected from light. When using, thaw and centrifuge before use, avoiding repeated freeze-thaw cycles. Gently shake to mix, and administer subcutaneously at a concentration of 100 μg / kg. The solvent is sterile physiological saline, and the dosage is 0.2 mL per mouse.

[0064] Preparation and administration of DMSO: DMSO is a colorless liquid with a concentration of 1.1 g / mL, stored at room temperature in a sealed container protected from light. The dosage of DMSO used in the experiment was 10 g / kg, which was diluted 1:1 with 0.9% NaCl and administered orally by gavage one hour before irradiation. Mice in the solvent control group received an equal volume of 0.9% NaCl by gavage.

[0065] (2) Animal irradiation All irradiation experiments were conducted between 9:00 AM and 11:00 AM. Before irradiation, unanesthetized mice were placed in well-ventilated restraint devices divided into 20 independent compartments by plexiglass partitions. They were then subjected to whole-body irradiation at the Cobalt-60 gamma-ray irradiation facility of the Radiation Medicine Institute, Academy of Military Medical Sciences. The source dose rate was measured using a dosimeter (PTW UNIDOS) connected to a finger-shaped ionization chamber (30013 0.6cc). The animal dose was determined by considering the decay of the cobalt source over time and the attenuation caused by air and tissue during irradiation. The whole-body irradiation doses in this study were 6.5 Gy, 9.5 Gy, 10.5 Gy, and 11.5 Gy, with a dose rate of approximately 40.56 Gy / min.

[0066] (3) Study on the survival of irradiated animals To evaluate the radiation protection effect of the combined use of DMSO and rhTPO, a 30-day survival experiment was conducted on mice. Mice received whole-body irradiation at doses of 9.5 Gy, 10.5 Gy, and 11.5 Gy. Before the experiment, mice with a body weight deviating from the population mean ± 10% were excluded and randomly assigned to groups. At each dose, mice were divided into four groups (n = 10 per group), identified by their tail tattoo numbers: Group 1: Vehicle; Group 2: DMSO; Group 3: rhTPO; Group 4: DMSO + rhTPO. Mice survival was observed after irradiation using the above method, and survival data were expressed as Kaplan-Meier curves using GraphPadPrism 10.1.2 software. Mice were euthanized after the experiment using the above method.

[0067] (4) Evaluation of drug synergistic effects This patent uses the King's equation to calculate the Q value to evaluate the synergistic effect of combined drug therapy. The equation is: Q = E(A + B) / (EA + EB - EA × EB), where EA and EB represent the survival rate of the single drug, and E(A + B) represents the survival rate of the combined drug therapy. The criteria for determining the Q value are: when Q < 0.85, it indicates an antagonistic effect; when 0.85 ≤ Q < 1.15, it indicates an additive effect; and when Q ≥ 1.15, it indicates a synergistic effect.

[0068] (5) Hematological studies of irradiated animals 9.5 Gy irradiated mice were irradiated for 7, 10, 14, and 18 days post-irradiation with blood samples collected via the tail vein (10 μl). Mice were returned to their original cages after blood collection, provided no abnormal bleeding was observed at the collection site. The blood samples were mixed with 2 ml of hematology analyzer diluent (MEK-640) and then used in pre-dilution mode on a Celltac ES fully automated hematology analyzer (NIHON KOHDEN) for complete blood cell count and white blood cell differential, counting red blood cells, platelets, white blood cells, lymphocytes, monocytes, and neutrophils.

[0069] (6) Flow cytometry analysis of bone marrow and spleen hematopoietic stem and progenitor cells (HSPCs) Mice irradiated with 6.5 Gy were sacrificed on day 10 post-irradiation. The mice were dissected, and the femur and spleen were separated. Bone marrow and spleen were extracted. Bone marrow hematopoietic cells were flushed from the femur using RPMI-1640. The spleen was ground to prepare a single-cell suspension. The prepared bone marrow and spleen single cells (6 × 10⁶ cells) were first added to an anti-mouse Fc receptor blocker (TruStainFcX™ (anti-mouse CD16 / 32)) to block the non-specific binding of antibody Fc fragments to the cells. Antibodies including Lineage cocktailPerCP-Cy5.5, Sca-1 PE-Cy7, C-kit APC-H7, CD150 PE, CD48 FITC, and CD135 APC were added at a 1:100 ratio to DPBS (2% FBS) to prepare an antibody mixture. The antibody sources are as described in the aforementioned table. Cells were incubated with antibody mixture at 4°C for 30 minutes, followed by the addition of cell lysis buffer. After 5 minutes, the cells were washed with DPBS (2% FBS), and finally, a certain amount of DPBS (2% FBS) was added. The mixture was then filtered through a 40 μm sieve into flow cytometry tubes containing 7-AAD. Data were then analyzed using a BD FACS Verse™ flow cytometer and BD FACSuite software. The cell populations analyzed included: lymphoid progenitor cells (LK: Lineage-Sca-1-C-Kit+), hematopoietic stem / progenitor cells (LSK: Lineage-Sca-1+C-Kit+), pluripotent progenitor cells 4 (MPP4: Lineage-Sca-1+C-Kit+CD135+), pluripotent progenitor cells 3 (MPP3: Lineage-Sca-1-C-Kit+CD135-CD48+CD150-), pluripotent progenitor cells 2 (MPP2: Lineage-Sca-1-C-Kit+CD135-CD48+CD150+), long-term hematopoietic stem cells (LT-HSC: Lineage-Sca-1-C-Kit+CD135-CD48-CD150+), and short-term hematopoietic stem cells (ST-HSC: Lineage-Sca-1-C-Kit+CD135-CD48-CD150-).

[0070] (7) Statistical analysis All experimental data were processed using GraphPad Prism 10.0 software (GraphPad Software Inc., USA), and data are expressed as mean ± standard deviation (Mean ± SD). Differences between groups were analyzed using a two-tailed Student's t-test; survival analysis was performed using the Kaplan-Meier method to plot survival curves. Statistical significance was defined as follows: compared with the irradiation group: *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001; compared with the combined treatment group: #P<0.05, ##P<0.01, ###P<0.001, and ####P<0.0001.

[0071] Example 1: Combined administration of DMSO and rhTPO synergistically protects mice from lethal doses of gamma radiation. To investigate whether pre-irradiation administration of DMSO for prophylaxis and post-irradiation administration of a high dose of rhTPO for rescue would produce additive or synergistic protective effects, we first tested this in a mouse model of total body irradiation (TBI) at a lethal dose of 9.5 Gy. In the experiment, C57BL / 6 mice were divided into a solvent control group (receiving only saline solution before and after irradiation), a DMSO prophylaxis group (receiving a single dose of DMSO 10 g / kg by gavage 1 hour before irradiation), an rhTPO rescue group (receiving a single dose of rhTPO 100 µg / kg subcutaneously 30 minutes after irradiation), and a DMSO+rhTPO combined administration group (receiving a single dose of DMSO 10 g / kg by gavage 1 hour before irradiation and a single dose of rhTPO 100 µg / kg subcutaneously 30 minutes after irradiation). Figure 1 As shown, all total body irradiation (TBI) mice exhibited a similar degree of moderate weight loss during the first 9 days post-irradiation. Subsequently, the solvent control group mice experienced sustained weight loss and all died within 14 days of irradiation. The rhTPO and DMSO groups showed transient weight loss at 20 days post-irradiation, while the DMSO+rhTPO group showed almost no weight loss within 30 days. Consistent with the post-irradiation weight loss, the 30-day survival rates of mice in the rhTPO, DMSO, and DMSO+rhTPO groups were 70%, 40%, and 100%, respectively. Based on the King's Law, we found that the combined use of DMSO and rhTPO resulted in a Q value of 1.22 (greater than 1.15), demonstrating a significant synergistic effect.

[0072] Subsequently, we further evaluated the synergistic effect of combined use of DMSO and rhTPO in a superlethal dose irradiation mouse model. When the whole-body irradiation dose was increased to 10.5 Gy, all mice in the solvent control group died within 14 days after irradiation. Mice in the rhTPO and DMSO groups showed significant weight loss from days 13 to 28 after irradiation, with 30-day survival rates of 10% and 80%, respectively. However, no significant weight loss was observed in the DMSO+rhTPO group within 30 days after irradiation, and the 30-day survival rate was 100%. Based on the King's Law, the Q value for the combined use of DMSO and rhTPO was 1.22, indicating a synergistic effect. When the whole-body irradiation dose was increased to 11.5 Gy, mice in the solvent control and rhTPO groups showed continuous weight loss after irradiation and all died within 10 days after irradiation. Mice in the DMSO group showed continuous weight loss starting from day 13 after irradiation and all died within 18 days after irradiation. It was found that the weight loss in the DMSO+rhTPO group was not significant within 30 days after irradiation, and the 30-day survival rate was 100%. Based on the King's formula, the Q value of DMSO and rhTPO used together is infinite, showing a significant synergistic effect.

[0073] The above results indicate that the combined administration of DMSO and rhTPO can significantly improve the survival rate of lethal and superlethal irradiated mice, exhibiting a synergistic effect, and the synergistic effect is more prominent with a higher irradiation dose.

[0074] Example 2: Combined administration of DMSO and rhTPO significantly promoted the recovery of multilineage hematopoiesis in irradiated mice. To evaluate the effect of combined administration of DMSO and rhTPO on hematological recovery in irradiated mice, we compared the effects of DMSO and rhTPO, alone or in combination, on hematopoietic recovery on days 7, 10, 14, and 18 after 9.5 Gy whole-body irradiation in a mouse model. Figure 2As shown, the peripheral blood counts of the irradiated control mice decreased significantly as expected, reaching their lowest point 10 days after irradiation, and all mice died within 12 days. In the DMSO monotherapy group, the levels of white blood cells, platelets, red blood cells, and hemoglobin reached their lowest points on days 7 or 10, respectively, and then began to recover. The levels of white blood cells and neutrophils on day 7, platelets on days 7 and 10, and hemoglobin on day 10 were significantly higher than those in the solvent control group. During the observation period, 2 out of 10 DMSO-treated mice died. In the rhTPO monotherapy group, the white blood cells and platelets reached their lowest points on day 10 after irradiation, and then recovered somewhat, but the red blood cells and hemoglobin showed a continuous downward trend. On day 7, the white blood cell and lymphocyte levels in the rhTPO group were significantly higher than those in the solvent control group; only 1 mouse survived during the observation period. In mice in the DMSO+rhTPO combination group, the levels of white blood cells, neutrophils, lymphocytes, platelets, erythrocytes, and hemoglobin were significantly higher than those in the solvent control group and the DMSO and rhTPO single-drug groups at multiple observation time points. Notably, the platelet count in the combination group recovered to pre-irradiation levels by day 10 after irradiation, with absolute values ​​being 15.3 times, 3.3 times, and 6.7 times higher than those in the solvent control group, DMSO group, and rhTPO group, respectively. Furthermore, the erythrocyte and hemoglobin levels in the combination group mice did not show a significant decrease throughout the observation period, and all 10 mice survived.

[0075] To further evaluate the promoting effect of combined administration of DMSO and rhTPO on hematological recovery, we used a 6.5 Gy sublethal dose irradiation model and ensured that all animals survived for 30 days after irradiation. Figure 3 As shown, on day 10 post-irradiation, the number of white blood cells, neutrophils, lymphocytes, platelets, and red blood cells, as well as hemoglobin levels, were successively higher in the DMSO group, rhTPO group, and DMSO+rhTPO group than in the solvent control group. Specifically, the platelet count and hemoglobin levels in the DMSO group, and the neutrophils, platelets, red blood cells, and hemoglobin levels in the rhTPO group were significantly better than those in the solvent control group. The DMSO+rhTPO combination group showed significantly better results than the DMSO and rhTPO single-drug groups in multiple indicators, including white blood cells, neutrophils, lymphocytes, and platelets. By day 14 post-irradiation, all three groups were higher than the solvent control group in all hematological indicators, and the DMSO+rhTPO combination group still showed better recovery of white blood cells, neutrophils, and lymphocytes than the single-drug groups.

[0076] In conclusion, the combined administration of DMSO and rhTPO significantly promoted the recovery of multilineage hematopoiesis in lethal and sublethal irradiated mice, with efficacy significantly superior to DMSO or rhTPO alone. Furthermore, the higher the irradiation dose, the more significant the therapeutic advantage of the combined administration.

[0077] Example 3: Combined administration of DMSO and rhTPO significantly promoted the regeneration of hematopoietic stem and progenitor cells in the bone marrow of irradiated mice. Total body irradiation (TBI)-induced bone marrow suppression is a key factor leading to hematopoietic system damage, primarily manifested as a significant reduction in bone marrow cell count. Based on this, we compared the effects of DMSO and rhTPO, alone or in combination, on bone marrow hematopoietic recovery in mice subjected to 6.5 Gy total body irradiation on day 10 post-irradiation. Figure 4 As shown, longitudinal sections of the femur from mice in the solvent control group irradiated with 6.5 Gy revealed a significant decrease in cell density, a reduction in the number of megakaryocytes, and the presence of adipocytes filling the bone marrow cavity, indicating severe impairment of bone marrow hematopoietic function. In contrast, femur sections from mice receiving DMSO and rhTPO, alone or in combination, showed more hematopoietic cell regeneration microfoci, increased cell density, and a greater number of megakaryocytes, indicating a significant recovery of bone marrow hematopoietic function. The DMSO and rhTPO combination group showed better bone marrow recovery on day 10 post-irradiation than the single-drug groups. Consistent with histological observations, the number of bone marrow nucleated cells (BMNCs) in the DMSO, rhTPO, and DMSO+rhTPO groups was significantly higher than that in the solvent control group, being 1.6 times, 1.7 times, and 3.1 times higher, respectively.

[0078] Hematopoietic stem cells (HSCs) and pluripotent progenitor cells (MPPs) play a central role in hematopoietic regeneration. Radiation exposure can significantly reduce the number of hematopoietic stem and progenitor cells, thereby impairing bone marrow regeneration capacity. Given the potential of combined administration of DMSO and rhTPO to improve bone marrow function and enhance post-radiation survival, we further investigated its effects on the regeneration of hematopoietic stem and progenitor cells in the bone marrow of irradiated mice. Based on existing literature, we used multiparameter flow cytometry to analyze bone marrow cells from mice subjected to 6.5 Gy whole-body irradiation with different treatments. In mice, hematopoietic stem cells (HSCs) are mainly found in the Lin... The Sca-1+ c-Kit+ (LSK) fraction contains a hematopoietic stem / progenitor cell (HSPC) population. The HSPC population can be further subdivided into long-term (LT) HSCs (LSK+ CD135). CD48 CD150+) and short-term (ST) HSC (CD135) CD48 CD150 LSK). Hematopoietic stem cells can differentiate into multiple lineage-biased pluripotent progenitor cell (MPP) subsets, among which MPP2 (CD135) is the most common. CD48+ CD150+ LSK) and MPP3 (CD135) CD48+ CD150 LSK tends to differentiate into myeloid cells, MPP2 is mainly involved in megakaryocyte / erythrocyte differentiation, and MPP3 is biased towards granulocyte / monocyte differentiation; while MPP4 (CD135+ CD150) is more inclined towards myeloid differentiation. LSK (Lymphoid Strain) is mainly involved in lymphoid differentiation.

[0079] like Figure 5 As shown, on day 10 after irradiation, the percentage and total number of LK cells in the bone marrow of mice in the DMSO group did not change significantly compared with the solvent control group, while the percentage of LSK cells increased slightly, and the total number of LSK cells was significantly higher than that in the control group. The percentage and total number of LK and LSK cells in mice in the rhTPO group and the DMSO+rhTPO group were significantly higher than those in the solvent control group, with the DMSO+rhTPO combination group showing the most significant effect, with the total number of LSK cells being 3.3 times, 2.2 times, and 11.0 times higher than that in the DMSO group, rhTPO group, and solvent control group, respectively. Compared with the solvent control group, DMSO and rhTPO, alone or in combination, significantly reduced the proportion of HSCST cells in the LSK compartment, but had no significant effect on HSCLT cells. Specifically, DMSO-treated mice showed only an increase in the proportion of MPP2 and a decrease in the proportion of HSCST cells; while rhTPO-treated mice showed a significant increase in the proportions of MPP2, MPP3, and MPP4, with a more significant decrease in the proportion of HSCST cells. In mice treated with the combined DMSO and rhTPO administration, the proportion of MPP4 increased further, while the proportion of MPP2 decreased significantly, and the proportion of HSCST remained unchanged. Consistent with the changes in the proportions of HSCs and MPPs in the LSK compartment, the frequencies and absolute numbers of MPP2, MPP3, and HSCST in the bone marrow nucleated cells of mice in all treatment groups were significantly increased. Among them, the frequency and absolute number of MPP4 in the rhTPO group were significantly higher than those in the control group, while the frequency of HSCST decreased accordingly. The frequencies and absolute numbers of MPP3 and MPP4 in the bone marrow of mice treated with the combined DMSO and rhTPO administration were significantly higher than those in the single administration groups. The absolute number of MPP4 was 73.8 times, 4.1 times, and 254.9 times that of the DMSO group, rhTPO group, and solvent control group, respectively.

[0080] Consistent with the recovery trend in peripheral blood counts, bone marrow hematopoietic function in all groups of mice was significantly improved on day 14 after irradiation compared to day 10. Figure 6As shown, the number of bone marrow nucleated cells (BMNCs) in mice treated with DMSO, rhTPO alone, and in combination was significantly higher than that in the solvent control group, with the number of BMNCs in the combination treatment group being significantly higher than that in the DMSO group. The percentages and total number of LK and LSK cells in the bone marrow of mice in each treatment group were significantly higher than those in the control group, but there were no significant differences between the treatment groups. In the LSK compartment, the trends of MPP2 and MPP4 ratios on day 14 were basically consistent with those on day 10, but the MPP3 ratio decreased in all treatment groups. The proportion of HSCLTs in the LSK compartment of mice in the DMSO group was significantly higher than that in the solvent control group. Consistent with the trends of HSCs and MPPs in the LSK compartment, the frequencies and absolute numbers of MPP4 and HSCLTs, as well as the absolute number of MPP2, in the bone marrow of mice in each treatment group were significantly higher than those in the control group. Specifically, the frequency and absolute number of MPP4 in the DMSO and rhTPO combination treatment group were significantly higher than those in the single treatment group, and the HSC... LT The absolute numbers were also significantly higher than those in the rhTPO group. Surprisingly, the MPP2 and HSC levels in the bone marrow of mice in the DMSO group were also significantly higher. LT The frequency and absolute number of the disease were significantly higher than those of the control group and the other two treatment groups.

[0081] In summary, DMSO primarily alleviates bone marrow hematopoietic injury by enhancing the reconstitution capacity of hematopoietic stem cells, while rhTPO mainly reduces radiation-induced bone marrow injury by promoting multi-lineage differentiation of hematopoietic stem cells. The combined administration of DMSO and rhTPO achieves a good balance between promoting hematopoietic stem cell reconstitution and differentiation, thus exhibiting a stronger bone marrow hematopoietic recovery effect.

[0082] Example 4: Combined administration of DMSO and rhTPO significantly enhanced extramedullary hematopoietic function of the spleen in irradiated mice. Hematopoietic stress can induce hematopoietic stem cells (HSCs) in the bone marrow to migrate to the spleen, thereby triggering extramedullary hematopoiesis (EMH). Extramedullary hematopoiesis in the spleen plays a crucial role in hematopoietic system remodeling after radiation injury. For example... Figure 7As shown, on day 10 post-irradiation, mice subjected to 6.5 Gy total body irradiation (TBI) exhibited a significant reduction in spleen volume, with spleen coefficient and nucleated cell count decreasing by 2.3 times and 7.2 times, respectively, compared to unirradiated mice. Prophylactic administration of DMSO significantly alleviated radiation-induced reductions in spleen coefficient and nucleated cell count, while salvage administration of rhTPO had a weaker effect. Notably, combined administration of DMSO and rhTPO significantly improved splenic hematopoietic status, resulting in a significant increase in spleen volume on day 10 post-irradiation. The spleen coefficient was 1.3 times and 1.7 times higher in the DMSO group and rhTPO group, respectively, and the splenic nucleated cell count was also significantly higher than in the irradiated control group and the rhTPO monotherapy group. Histopathological analysis revealed that after irradiation, the white and red pulp structures of the spleen in mice were disordered and poorly defined, splenic corpuscles were atrophied and sparsely arranged, the red pulp splenic sinuses were dilated and congested, and the number of hematopoietic cells was reduced. After receiving DMSO and rhTPO alone or in combination, the red marrow region expanded significantly due to the infiltration of a large number of hematopoietic cells, and megakaryocytes were visible, indicating active extramedullary hematopoiesis in the spleen, with the combined administration group showing particularly obvious results.

[0083] To further evaluate the promoting effect of combined administration of DMSO and rhTPO on the expansion of splenic hematopoietic stem / progenitor cells (HPSCs), we used flow cytometry to analyze the changes in various HPSC subsets in the mouse spleen on day 10 after irradiation. Figure 8 As shown, the frequency and number of LSK cells in the spleen of mice were significantly reduced after irradiation, and LK cells were almost undetectable. DMSO and rhTPO administration alone could increase the frequency or number of LK and LSK cells to varying degrees, but the increase was limited; while combined administration significantly increased the frequency and number of LK and LSK cells, with the absolute number of LSK cells being 18.4 times and 18.1 times that of the DMSO group and rhTPO group, respectively. DMSO and rhTPO administration alone could increase the number of long-term hematopoietic stem cells (HSCs) in the LSK compartment. LT ), short-term hematopoietic stem cell (HSC) ST The effects on the proportions of MPP2 and MPP4 were relatively small, while combination therapy significantly increased the proportion of MPP2. Notably, rhTPO monotherapy primarily increased the frequency and quantity of MPP2 and MPP4 in the spleen, while DMSO monotherapy primarily increased HSCs. ST The frequency and number of HSCs in the spleen were significantly increased by combination therapy. LT HSC ST And the frequency and number of each lineage biased towards the MPP subgroup.

[0084] On day 14 post-irradiation, extramedullary hematopoietic function of the spleen in 6.5 Gy TBI mice had recovered somewhat, with LSK cell frequency and number significantly increasing compared to day 10. Although there were no significant differences in spleen index and nucleated cell count between mice treated with DMSO and rhTPO alone or in combination and unirradiated mice, both LK and LSK cell frequencies and numbers were significantly increased, with the combination treatment group showing the most significant effect. In the LSK compartment, the DMSO and rhTPO single-drug groups showed a significant increase in HSCs. LT HSC ST The proportions of each MPP subset were not significantly affected, while the proportion of MPP4 was significantly increased in the combination therapy group. Consistent with the trends of HSCs and MPPs in the LSK compartment, rhTPO monotherapy significantly increased the frequency and number of MPP2, MPP3, and MPP4 in the spleen, while DMSO monotherapy had little effect on MPPs, and the proportion of MPP3, MPP4, and HSCs in the spleen was significantly increased in the combination therapy group. ST The frequency and number of HSCs were significantly higher than those in the single-drug group. LT There were no significant differences among the groups. Figure 9 and Figure 10 ).

[0085] In conclusion, the combined administration of DMSO and rhTPO significantly enhances the extramedullary hematopoietic activity of the spleen in mice irradiated with 6.5 Gy and significantly advances the initiation time of extramedullary hematopoiesis, with better effects than DMSO or rhTPO alone.

[0086] Example 5: Combined administration of DMSO and roprostine synergistically protects mice from superlethal doses of gamma radiation. Thromboplastin-promoting drugs include recombinant human thrombopoietin (rhTPO) and thrombopoietin receptor agonists (TPO-RA). Romiplostim, a second-generation thrombopoietin (TPO) receptor agonist developed by Kyowa Kirin, is used to treat chronic primary immune thrombocytopenic purpura (ITP) in adults. This drug utilizes a peptide-like structure to achieve cross-species TPO receptor activation, avoiding immunogenic cross-reactivity. To verify the synergistic effect of DMSO and thrombopoietin-promoting drugs in preventing acute radiation sickness, we further observed the effects of DMSO and romiplostim, alone and in combination, on the 30-day survival rate of mice irradiated with a superlethal dose of 10.5 Gy. In the experiment, C57BL / 6 mice were divided into a solvent control group (receiving only saline solution before and after irradiation), a DMSO prevention group (receiving a single dose of DMSO 10 g / kg by gavage 1 hour before irradiation), a roprostine treatment group (receiving a single dose of roprostine 100 µg / kg subcutaneously 30 minutes after irradiation), and a DMSO + roprostine combined administration group (receiving a single dose of DMSO 10 g / kg by gavage 1 hour before irradiation, and a single dose of roprostine 100 µg / kg subcutaneously 30 minutes after irradiation). Figure 11 As shown, mice in the solvent control group and the roprostine group experienced a continuous decrease in body weight after irradiation, and all died within 11 and 15 days, respectively. Mice in the DMSO group began to experience a continuous decrease in body weight from day 13 after irradiation, with only one mouse remaining after day 16. It was found that the DMSO + roprostine group showed no significant decrease in body weight within 30 days after irradiation, and the 30-day survival rate was 100%. Based on the King's Law, the Q-value of the combined use of DMSO and roprostine was 10, demonstrating a significant synergistic effect.

[0087] Example 6: Combined administration of DMSO and roprostine significantly promoted the recovery of multilineage hematopoiesis in lethally irradiated mice. To evaluate the effect of combined administration of DMSO and roprostine on hematological recovery in irradiated mice, we compared the effects of DMSO and roprostine alone and in combination on survival and peripheral blood cell recovery in mice irradiated with 9.5 Gy in a lethal irradiation mouse model. Figure 12As shown, all mice in the solvent control group died within 15 days after irradiation, two mice in the roprostine treatment group died on days 10 and 30 after irradiation, respectively, while all mice in the DMSO prevention group and the DMSO + roprostine combined administration group survived. On day 7 after irradiation, although there was no significant difference in white blood cell and lymphocyte counts between the DMSO and roprostine single administration groups and the control group, the red blood cell count and hemoglobin content were significantly higher in the DMSO group and the control group. The platelet count in the DMSO group was significantly higher than that in the control group, while it was significantly lower in the roprostine group. The white blood cell, lymphocyte, platelet, and red blood cell counts and hemoglobin content in the DMSO + roprostine combined administration group were significantly higher than those in the control group and the single administration groups, suggesting that the combined administration of DMSO and roprostine has a synergistic effect. On days 14 and 18 after irradiation, the control group mice were nearly or completely dead. There was no significant difference in the recovery of peripheral blood counts between the irradiated mice and the DMSO and roprostine single-drug groups. It was found that the DMSO + roprostine combined-drug group was significantly faster than the single-drug group, which further illustrates that the combined administration of the two has a significant synergistic effect.

[0088] Example 7: Combined administration of DMSO and roprostine significantly promoted the regeneration of hematopoietic stem and progenitor cells in the bone marrow of irradiated mice. Given the potential of synergistic improvement in post-irradiation animal survival rates through the combined administration of DMSO and roprostine, we further compared the effects of DMSO and roprostine, alone or in combination, on bone marrow hematopoietic stem and progenitor cell regeneration on day 10 after 6.5 Gy whole-body irradiation in mice. Figure 13 As shown in Figure A, administration of roprostine after irradiation significantly increased the number of bone marrow nucleated cells (BMNCs) in mice 10 days after irradiation, while prophylactic administration of DMSO had no significant effect on the number of BMNCs. It was found that the number of bone marrow BMNCs in the DMSO + roprostine combined administration group was significantly higher than that in the respective single administration groups and the solvent control group.

[0089] Multiparameter flow cytometry was used to analyze bone marrow cells from mice that underwent 6.5 Gy whole-body irradiation with different treatments. Figure 13As shown in the BI, on day 10 post-irradiation, the percentage and total number of LK cells and the total number of LSK cells in the bone marrow of mice in the DMSO group showed no significant changes compared to the solvent control group, although the percentage of LSK cells was slightly increased. The percentage and total number of LK and LSK cells in mice in the roprostine group and the DMSO + roprostine group were significantly higher than those in the solvent control group, with the DMSO + roprostine combination group showing the most significant effect, with the total number of LSK cells being 5.24 times, 1.99 times, and 15.22 times higher than those in the DMSO group, roprostine group, and solvent control group, respectively. Whole-body gamma irradiation with 6.5 Gy significantly increased the proportion of HSCST cells in the LSK region of mouse bone marrow on day 10 post-irradiation, while the proportion of MPP4 cells decreased sharply. Compared with the solvent control group, roprostine administration after irradiation significantly reduced the proportion of HSCST cells in the LSK compartment and increased the proportions of MPP4, MPP3, and MPP2. However, DMSO prophylactic administration did not increase the proportion of MPP4. The effects on other cell populations in the LSK compartment were similar to those of roprostine. It was found that the combined administration of DMSO and roprostine significantly increased the proportion of MPP4 and significantly decreased the proportion of MPP2 compared with the administration of either drug alone. Compared with the solvent control group, the frequencies and absolute numbers of MPP4, MPP3, MPP2, and HSCLT in the bone marrow nucleated cells of mice in the roprostine group and the DMSO+roprostine combination group were significantly increased, while the frequency of HSCST decreased accordingly. In the DMSO group, only the frequencies of MPP2 and HSCLT and the absolute number of MPP2 were increased. It was found that the frequencies and absolute numbers of MPP4, MPP3, and HSCLT in the bone marrow of mice in the DMSO+roprostine combination group were significantly higher than those in their respective single-drug groups. The absolute number of MPP4 was 225.85 times, 3.17 times, and positive infinity, respectively, compared with the DMSO group, roprostine group, and solvent control group. The absolute number of HSCLT was 5.80 times, 4.35 times, and 61.78 times, respectively, compared with the DMSO group, roprostine group, and solvent control group.

[0090] In conclusion, the combination of DMSO and roprostine, compared to DMSO alone, may enhance post-irradiation bone marrow HSC. LT Reconstruction and MPP4 differentiation showed stronger efficacy in promoting hematopoietic recovery.

[0091] Example 8: Combined administration of DMSO and roprostine significantly enhanced extramedullary hematopoietic function of the spleen in irradiated mice. Extramedullary hematopoiesis in the spleen plays a crucial role in the reconstitution of the hematopoietic system after radiation injury. For example... Figure 14 As shown, the spleen volume of mice subjected to 6.5 Gy total body irradiation (TBI) was significantly reduced on day 10 after irradiation. Post-irradiation administration of roprostine significantly alleviated the radiation-induced reduction in spleen coefficient and nucleated cells, while the prophylactic administration of DMSO had a weaker effect. The combined administration of DMSO and roprostine significantly increased the spleen coefficient and the number of nucleated cells in the spleen, but there was no statistically significant difference compared with the roprostine group.

[0092] Flow cytometry was used to analyze the changes in various subsets of HPSCs in the spleen of mice on day 10 after irradiation. Figure 15 As shown, the frequency and number of LSK cells in the spleen of mice were significantly reduced after irradiation, and LK cells were almost undetectable. Compared with the solvent control group, the frequency and number of LK and LSK cells in the spleen of mice in the roprostine group and the DMSO + roprostine combination group were significantly increased, while there was no statistically significant difference between the DMSO group and the solvent control group. Consistent with the changes in spleen LSK, the frequency and number of MPP4, MPP3, MPP2, and HSCLT cells in the spleen of the roprostine group and the DMSO + roprostine combination group were significantly higher than those in the solvent control group, while there was no statistically significant difference in any cell population in the DMSO group compared with the solvent control group; compared with the single administration, the combined administration of DMSO and roprostine significantly increased spleen HSC. ST and MPP4 frequency and HSC ST quantity.

[0093] In conclusion, the combined administration of DMSO and roprostine significantly enhanced the extramedullary hematopoietic activity of the spleen in mice irradiated with 6.5 Gy, with some indicators showing significantly better efficacy than DMSO or roprostine alone.

[0094] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Use of dimethyl sulfoxide in combination with a thrombopoietic agent in the preparation of a medicament for preventing and / or treating acute radiation sickness.

2. Use according to claim 1, characterized in that, The thrombopoietic agent comprises recombinant human thrombopoietin and thrombopoietin receptor agonist; Preferably, the thrombopoietin receptor agonist comprises one or more of romiplostim, eltrombopag, heptaplostim, avatrombopag and lusfusopag; Preferably, the thrombopoietic agent is selected from rhTPO or romiplostim.

3. Use according to claim 1, characterized in that, The acute radiation sickness comprises bone marrow acute radiation sickness, intestinal acute radiation sickness and brain acute radiation sickness.

4. A kit characterized in that, The kit comprises: (I) a first preparation comprising dimethyl sulfoxide; (II) a second preparation comprising a thrombopoietic agent; Preferably, the second preparation is selected from romiplostim, eltrombopag, heptaplostim, avatrombopag, lusfusopag, TPO and / or rhTPO; Preferably, the second preparation is selected from romiplostim and / or rhTPO; Preferably, the kit further comprises an instruction; Preferably, the instruction indicates that the first preparation and the second preparation are used in combination to prevent and / or treat acute radiation sickness.

5. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises dimethyl sulfoxide and a thrombopoietic agent.

6. The pharmaceutical composition of claim 5, wherein, The thrombopoietic agent comprises recombinant human thrombopoietin and thrombopoietin receptor agonist; Preferably, the thrombopoietin receptor agonist comprises one or more of romiplostim, eltrombopag, heptaplostim, avatrombopag and lusfusopag; Preferably, the thrombopoietic agent is selected from rhTPO or romiplostim.

7. The pharmaceutical composition of claim 5, wherein, The dose ratio of the dimethyl sulfoxide to the thrombopoietic agent is 100-1000000:1; Preferably, the dose ratio of the dimethyl sulfoxide to the thrombopoietic agent is 1000-100000:1; Preferably, the dose ratio of the dimethyl sulfoxide to the thrombopoietic agent is 100000:1; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

8. The pharmaceutical composition of claim 5, wherein, The dimethyl sulfoxide and the thrombopoietic agent are in the same or different dosage forms; Preferably, the dosage forms comprise enteral administration dosage forms, parenteral administration dosage forms; Preferably, the enteral administration dosage forms comprise solutions, granules, tablets, capsules, suspensions, powders, sustained-release preparations, effervescent preparations, emulsions, syrups, drops, chewable preparations; Preferably, the parenteral administration dosage forms comprise injection administration dosage forms, respiratory administration dosage forms, cavity administration dosage forms, mucosal administration dosage forms, skin administration dosage forms; Preferably, the dimethyl sulfoxide and the thrombopoietic agent are administered simultaneously or sequentially; Preferably, the dimethyl sulfoxide and the thrombopoietic agent are administered sequentially before and after irradiation.

9. Use of any one of: (1) dimethyl sulfoxide in combination with a thrombopoietic agent in the preparation of a medicament for preventing and / or treating acute radiation sickness; (2) a thrombopoietic agent in the preparation of a medicament for improving the therapeutic effect of dimethyl sulfoxide on acute radiation sickness; (3) dimethyl sulfoxide in the preparation of a medicament for improving the therapeutic effect of a thrombopoietic agent on acute radiation sickness; (4) Use of dimethyl sulfoxide and a thrombopoietic agent in combination in the preparation of a medicament for preventing and / or treating myelosuppression caused by radiation exposure; (5) Use of dimethyl sulfoxide and a thrombopoietic agent in combination in the preparation of a medicament for preventing and / or treating death in an animal or human caused by radiation exposure; (6) Use of dimethyl sulfoxide and a thrombopoietic agent in combination in the preparation of a medicament for preventing and / or treating peripheral cytopenia, leukopenia, neutropenia, lymphopenia, thrombocytopenia, and / or anemia caused by radiation exposure; (7) Use of dimethyl sulfoxide and a thrombopoietic agent in combination in the preparation of a medicament for preventing and / or treating damage to hematopoietic stem and / or progenitor cells caused by radiation exposure.

10. Use according to claim 9, characterized in that, The thrombopoietic agent includes recombinant human thrombopoietin and a thrombopoietin receptor agonist; Preferably, the thrombopoietin receptor agonist includes one or more of romiplostim, eltrombopag, heptaplostim, avatrombopag, and luspatercept; Preferably, the thrombopoietic agent is selected from rhTPO or romiplostim; Preferably, the radiation exposure includes alpha radiation exposure, beta radiation exposure, gamma radiation exposure, neutron exposure, proton exposure, heavy ion exposure, electron exposure, and / or X-ray exposure.

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