Use of csf1r-targeted drugs in the preparation of a medicament for the prevention of radiation-induced intestinal injury

Abstract

The application discloses a use of a CSF1R targeting drug in preparation of a drug for preventing radiation-induced intestinal injury. Specifically, the application proves by systematic experimental data that a CSF1R inhibitor can significantly improve survival and regeneration in a specific dose / time window, directly proves the protection of the CSF1R inhibitor on a "crypt stem cell core damage chain", and provides a clearer path for clinical application and transformation.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of CSF1R-targeting drugs in the preparation of drugs for the prevention of radiation-induced intestinal injury. Background Technology

[0002] Radiation-induced intestinal injury (RIII) and gastrointestinal acute radiation syndrome (GI-ARS) are common complications after radiotherapy for pelvic and abdominal tumors. Their pathological features mainly include apoptosis of intestinal crypt stem cells, destruction of crypt structure, and damage to the mucosal barrier, which can lead to intestinal failure, infection, and even death.

[0003] Currently, clinical preventative measures for RIII or GI-ARS are extremely limited. Existing clinical drugs are mostly symptomatic supportive agents or non-specific protectants, unable to directly block the core chain of "acute apoptosis of crypt stem cells—regeneration failure," resulting in limited efficacy under high-dose irradiation. Stem cell regeneration promotion strategies are significantly dose- and timing-dependent, and their efficacy fluctuates or even has negative effects in different models, limiting their clinical application. Previous data also suggest that long-term CSF1R blockade can disrupt crypt homeostasis, and whether it damages intestinal stem cell niches remains inconclusive. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the present invention aims to provide the use of CSF1R-targeting drugs in the preparation of drugs for the prevention of radiation-induced intestinal injury. The optimal effective dose and administration time window of the CSF1R-targeting drug in radiation protection are determined to ensure stable and repeatable protective effects.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A first aspect of the present invention provides the use of a CSF1R-targeting drug in the preparation of a medicament for the prevention and / or treatment of radiation-induced intestinal injury, wherein the CSF1R-targeting drug is a CSF1R inhibitor or a pharmaceutically acceptable salt thereof.

[0006] In this invention, CSF1R encodes the colony-stimulating factor 1 receptor, also known as the giant cell colony-stimulating factor receptor, with gene ID 1436. In the context of this invention, CSF1R includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed CSF1R, as well as any form of CSF1R derived from cells and processed. The term encompasses naturally occurring variants of CSF1R (e.g., splice variants or allelic variants). The term encompasses, for example, the CSF1R gene, the CSF1R protein, the human CSF1R, and CSF1R from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats).

[0007] In this invention, "pharmaceutical-grade salt" and "pharmaceutical-acceptable salt" are used interchangeably. A pharmaceutically acceptable salt refers to any acid addition salt or base addition salt whose counterion is non-toxic to the patient at the pharmaceutical dose of the salt. The main components of pharmaceutically acceptable salts are well known in the art. If pharmaceutically acceptable salts of the compounds of this application are used in these compositions, those salts are preferably derived from inorganic acids or organic acids and bases. Such acid salts include, but are not limited to, the following: acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butylates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, diglucuronates, dodecyl sulfates, ethanesulfonates, fumarates, luchophenate, glycerophosphates, hemisulfates, heptanates, hexanoates, hydrochlorides, hydrobromide, hydroiodates, 2-hydroxyethanesulfonate, lactates, maleates, methanesulfonates, and 2-naphthalenesulfonic acid. Salts, nicotinates, oxalates, pyrates, pectates, persulfates, 3-phenyl-propionates, picrates, pentanoates, propionates, succinates, tartrates, thiocyanates, toluenesulfonates, undecanoates, hydrohalides (e.g., hydrochlorides and hydrobromates), sulfates, phosphates, nitrates, aminosulfonates, malonates, salicylates, methylene-bis-β-hydroxynaphthylcarboxylate, gentianates, hydroxyethylsulfonates, di-p-toluyl tartrate, ethanesulfonates, cyclohexylaminosulfonates, quinates, etc. Pharmaceutically acceptable base addition salts include, but are not limited to, those derived from alkali metal or alkaline earth metal bases or conventional organic bases, such as triethylamine, pyridine, piperidine, morpholine, N-methylmorpholine, ammonium salts, alkali metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts containing organic bases (such as dicyclohexylamine salts, N-methyl-D-glucosamine), and salts containing amino acids such as arginine and lysine.

[0008] Furthermore, the CSF1R inhibitor is selected from PLX5622, PLX3397, CSF1R neutralizing antibody, BLZ945, and GW2580.

[0009] In some embodiments, the CSF1R inhibitor is PLX3397.

[0010] In some embodiments, if the subject is a mouse, the effective dose of the CSF1R inhibitor includes any range.

[0011] In some embodiments, the effective dose of the CSF1R inhibitor is selected from 40-120 mg / kg. For example, it can be 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, and any dose between any two of the above.

[0012] In some embodiments, the effective dose of the CSF1R inhibitor is 120 mg / kg.

[0013] The drug can be administered preventively before radiation exposure. The preventive administration time can be within 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, or 2 hours before radiation exposure, or between any two of the above time points.

[0014] In some embodiments, the CSF1R inhibitor is administered at a time selected from 3 days prior to radiation exposure. 12 hours.

[0015] In some embodiments, the CSF1R inhibitor is administered at a time selected from 6 days prior to radiation exposure. 12 hours.

[0016] In some embodiments, the CSF1R inhibitor is administered 12 hours before irradiation.

[0017] In some embodiments, the CSF1R inhibitor may be administered via intraperitoneal injection, oral administration, subcutaneous injection, or local intestinal administration.

[0018] In some embodiments, the CSF1R inhibitor is administered via intraperitoneal injection.

[0019] Furthermore, the radiation-induced intestinal injury refers to radiation-induced intestinal injury and / or acute radiation syndrome of the gastrointestinal tract caused by gamma rays.

[0020] In this invention, radiation-induced intestinal injury and / or acute gastrointestinal radiation syndrome caused by gamma rays includes radiation-induced intestinal injury and / or acute gastrointestinal radiation syndrome caused by any dose of gamma rays, wherein the dose can be not less than 1 Gy, not less than 2 Gy, not less than 3 Gy, not less than 4 Gy, not less than 5 Gy, not less than 6 Gy, not less than 7 Gy, not less than 8 Gy, not less than 9 Gy, not less than 10 Gy, not less than 11 Gy, not less than 12 Gy, not less than 13 Gy, not less than 14 Gy, not less than 15 Gy, and the radiation dose between any two of these points.

[0021] In some embodiments, the gamma ray irradiation dose is greater than or equal to 10 Gy. The irradiation dose may be not less than 10 Gy, not less than 11 Gy, not less than 12 Gy, not less than 13 Gy, not less than 14 Gy, not less than 15 Gy, and any two of these points.

[0022] In some embodiments, the gamma ray irradiation dose is 14-15 Gy. For example, it can be 14 Gy, it can be 15 Gy, and the irradiation dose between any two points therein.

[0023] Furthermore, the prevention and / or treatment include reducing radiation-induced apoptosis of intestinal crypt cells and / or promoting regeneration and repair of intestinal crypt cells.

[0024] Furthermore, the promotion of intestinal crypt cell regeneration and repair includes maintaining or enhancing the survival of crypt stem cells.

[0025] In some embodiments, the crypt stem cells are Olfm4+ stem cells.

[0026] A second aspect of the present invention provides the use of a CSF1R-targeting drug in the preparation of a drug for the prevention and / or treatment of crypt injuries, wherein the CSF1R-targeting drug is a CSF1R inhibitor or a pharmaceutically acceptable salt thereof.

[0027] In some embodiments, the crypt damage is crypt damage caused by gamma rays.

[0028] A third aspect of the present invention provides a medicament for the prevention and / or treatment of radiation-induced intestinal injury and / or crypt injury, said medicament comprising a CSF1R inhibitor or a pharmaceutically acceptable salt thereof.

[0029] In this invention, the term "medicine" refers to a substance containing at least one bioactive compound. The medicines or compositions thereof described in this invention can be administered via intraperitoneal injection, subcutaneous injection, oral administration, non-gastrointestinal administration, inhalation spray, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration, or via an implanted drug delivery device. In some embodiments, intraperitoneal injection is preferred. The medicines or compositions thereof of this invention may contain any commonly used non-toxic pharmaceutically acceptable carriers, excipients, or excipients. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The medicines or compositions thereof of this invention can be delivered to the receptor via any route, provided the target tissue can be reached.

[0030] In some embodiments, the CSF1R inhibitor is selected from PLX5622, PLX3397, CSF1R neutralizing antibody, BLZ945, and GW2580.

[0031] In some embodiments, the CSF1R inhibitor is PLX3397.

[0032] Furthermore, the drug also includes other drugs for the prevention and / or treatment of radiation-induced intestinal injury and / or crypt injury.

[0033] In some embodiments, the other drugs for preventing and / or treating radiation-induced intestinal injury and / or crypt injury include one or more of antioxidants and cytokine inhibitors.

[0034] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0035] In this invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not induce the production of antibodies harmful to an individual receiving the drug or a combination thereof and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions may comprise fluids such as water, saline, glycerol, and ethanol. Such carriers may also contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc.

[0036] In some embodiments, the pharmaceutically acceptable carrier includes one or more of the following: diluent, excipient, flow aid, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and emulsifier.

[0037] Furthermore, the dosage forms of the drug include oral dosage forms, injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0038] In some embodiments, the dosage form of the drug includes granules, powders, tablets, capsules, syrups, suppositories, or injections.

[0039] In one embodiment of the invention, treatment means reducing or alleviating, improving or eradicating a disease or one or more symptoms related to the disease. In some embodiments, the term refers to minimizing the spread or worsening of the disease due to the administration of one or more preventive or therapeutic agents to a patient suffering from the disease. For the purposes of the various aspects and embodiments provided by the invention, treatment includes, but is not limited to, reducing, alleviating or improving one or more clinical manifestations or side effects of the treated disease or condition, improving one or more clinical outcomes, reducing the severity of the disease, delaying or slowing the progression of the disease, improving, alleviating or stabilizing the disease state, and other beneficial results described in the invention.

[0040] Advantages and benefits of the present invention: The present invention provides the use of CSF1R-targeting drugs in the preparation of drugs for the prevention of radiation-induced intestinal injury. The system experimental data demonstrates that CSF1R inhibitors can significantly improve the survival and regeneration of the cells within a specific dose / time window, overcoming and correcting the negative expectations and technical biases of the prior art. It directly proves the protection of the "core damage chain of crypt stem cells" by CSF1R inhibitors, providing a clearer path for clinical application and translation. Attached Figure Description

[0041] Figure 1 The results of CSF1R neutralizing antibody in improving intestinal crypt injury in radiation-induced intestinal injury are shown in Figure A, where A is the flow cytometry result of peripheral blood macrophage proportion at 6 h, 12 h and 24 h after mouse administration; B is the statistical graph of flow cytometry results; C is the representative graph of BrdU-labeled proliferating cells detected by immunohistochemical staining; and D is the statistical graph of BrdU-labeled proliferating cells.

[0042] Figure 2 The figures show the results of a study on the intestinal radioprotective effect of CSF1R inhibitors. In the figure, A is a representative figure of the number of positive crypts labeled with BrdU detected by immunohistochemical staining, B is a statistical figure of the number of positive crypts at different doses of PLX5622, and C is a statistical figure of the number of positive crypts at different doses of PLX3397.

[0043] Figure 3 The figure shows the results of a study on the time-series effect of CSF1R inhibitors on intestinal radioprotection. In the figure, A is a representative figure of the number of positive crypts labeled with BrdU detected by immunohistochemical staining, B is a statistical figure of the number of positive crypts after PLX5622 administration at different time intervals before irradiation, and C is a statistical figure of the number of positive crypts after PLX3397 administration at different time intervals before irradiation.

[0044] Figure 4 The graph shows the effect of CSF1R inhibitors on mice subjected to lethal doses of whole-body irradiation combined with bone marrow transplantation. In the graph, A is the weight statistics of mice after lethal doses of whole-body irradiation following bone marrow transplantation, and B is the survival rate.

[0045] Figure 5 The images show the results of CSF1R inhibitors reducing apoptosis in mouse intestinal crypt cells induced by gamma ray irradiation. In the images, A is a representative immunohistochemical image of Cleaved-Caspase-3 in intestinal crypt cells of the PLX3397 prophylactic administration group, B is a statistical graph of the number of Cleaved-Caspase-3 positive apoptotic cells, C is a representative immunohistochemical image of TUNEL in intestinal crypt cells of the PLX3397 prophylactic administration group, and D is a statistical graph of the number of TUNEL positive apoptotic cells.

[0046] Figure 6 The image shows the results of CSF1R inhibitors improving high-dose radiation-induced damage to intestinal crypt structure. In the image, A is a representative image of Olfm4 (a small intestinal stem cell-specific marker) immunofluorescence staining, and B is a statistical chart of the percentage of Olfm4+ stem cell-positive areas. Detailed Implementation

[0047] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0048] Example 1: Study on the effect of CSF1R-targeted drugs in preventing intestinal radiation injury I. Experimental Materials 1.1 Experimental animals: C57BL / 6J male mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., China).

[0049] 1.2 Reagents and materials: as shown in Table 1.

[0050] Table 1. Experimental Materials

[0051] II. Experimental Methods 2.1 Animal feeding: C57BL / 6J mice, 6 8 weeks old, weighing 20 22g male mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and housed at the Animal Center of the Academy of Military Medical Sciences (SPF grade). The housing temperature was 22±2℃, humidity 55±5%, and bedding was sterilized with gamma rays and changed twice weekly. Five mice were per cage, with 12 hours of light and 12 hours of darkness daily, fed standard gamma-ray sterilized feed and acidified water. All mice were housed in the animal facility for one week after purchase to acclimatize before experiments were conducted.

[0052] 2.2 Mouse Grouping: 1) Time-effect experiment of CSF1R neutralizing antibody (CSF1R-Ab): 18 C57BL / 6J mice were used. The CSF1R-Ab treatment group received an intraperitoneal injection of 250 μg CSF1R-Ab, and the NC group received an intraperitoneal injection of 250 μg of isotype IgG antibody as a negative control. Peripheral blood was collected at 6h, 12h, and 24h after administration to detect the proportion of macrophages. There were 3 mice in each group. 2) Intervention experiment on radiation intestinal injury caused by CSF1R-Ab prophylaxis: 6 C57BL / 6J mice were used. The CSF1R-Ab prophylaxis group received an intraperitoneal injection of 250 μg CSF1R-Ab 12h before irradiation, followed by irradiation. The irradiation control group (IR) received an intraperitoneal injection of 250 μg of isotype IgG antibody. There were 3 mice in each group. 3) CSF1R inhibitor (PLX5622 / PLX3397) dose-effect experiment: 24 C57BL / 6J mice were intraperitoneally injected with PLX5622 or PLX3397 at different concentrations (40, 80, 120 mg / kg). The irradiation control group was given the drug solvent. There were 3 mice in each group. 4) CSF1R inhibitor (PLX5622 / PLX3397) time-effect experiment: 24 C57BL / 6J mice were administered CSF1R inhibitor (PLX5622 / PLX3397) 3 h, 6 h, and 12 h before irradiation. The irradiation control group was given the drug solvent 3 h before irradiation. There were 3 mice in each group.

[0053] 2.3 Irradiation Protocol: The boxes containing the mice were placed 2.5 meters away from the gamma-ray radiation source for irradiation. The total irradiation dose was 14 Gy. After irradiation, the mice were allowed free movement and feeding in cages with an SPF barrier environment. 3.5 days later, the intestines were dissected and used for pathological observation.

[0054] 2.4 Dosing Regimen: 1) CSF1R neutralizing antibody (CSF1R-Ab) and isotype IgG antibody were purchased from BioXcell (USA). After dilution with PBS, they were administered to mice via intraperitoneal injection at the given drug dose. 2) PLX5622 hydrochloride and PLX3397 hydrochloride are salts of PLX5622 and PLX3397, respectively, to increase the water solubility of the drugs. The drugs were prepared with 5% DMSO, 30% PEG400, 5% Tween-80, and 60% physiological saline, and sonicated to obtain a clear and transparent physiological saline solution, which was then administered to mice via intraperitoneal injection at the given drug dose.

[0055] 2.5 Flow Cytometry Detection of Mouse Peripheral Blood Macrophages: 50 μL of tail vein blood was collected from mice. Red blood cells were lysed using pre-chilled ammonium chloride lysis buffer, and peripheral blood mononuclear cells were obtained after centrifugation and washing. Cells were surface-stained with APC-labeled anti-F4 / 80 antibody and PE-labeled anti-CD11b antibody, incubated at 4°C in the dark for 30 minutes, and then washed and resuspended. Data were acquired using flow cytometry. Mononuclear cell populations were delineated by forward and side scattering. After excluding adherent cells, the F4 / 80 and CD11b double-positive cell populations were analyzed to characterize the macrophage content in peripheral blood.

[0056] 2.6 BrdU Immunohistochemistry: 1) BrdU incorporation is required before BrdU immunohistochemistry: BrdU is injected intraperitoneally into mice 2 hours before sacrifice, at a dose of 120 mg / kg.

[0057] 2) Sample collection: Mice were euthanized by cervical vertebrae dislocation. After soaking in alcohol, the abdominal wall was cut open in the lower abdomen to expose the abdominal cavity, and the target intestinal segment was taken. The intestinal cavity was rinsed with pre-cooled PBS until clean. The small intestinal tissue could be cut to an appropriate length and placed in a plastic embedding cassette.

[0058] 3) Fixation: Place the tissue from the previous step in 10% formalin or 4% paraformaldehyde and fix at room temperature for 8-24 hours.

[0059] 4) Dehydration, clearing, and paraffin impregnation: Rinse with running water to remove excess fixative. Trim the fixed intestinal tissue appropriately to remove surrounding tissues such as the mesentery. Then, cut the small intestine tissue into segments of approximately 5 mm. The cut tissue is then dehydrated sequentially through different concentrations of ethanol solution: 70% → 80% → 90% → 95% → 95% → 100% → 100%, each concentration for 40-60 minutes. Next, clear the tissue in two xylene baths for 10-20 minutes each. Finally, apply paraffin wax in three baths at 65-70℃ for 0.5-1 hour each.

[0060] 5) Embedding: Turn on the tissue embedding machine in advance, fill the metal base with liquid paraffin, place the intestinal segment vertically at the bottom of the base, place it on the freezing table to cool and solidify, and then separate the paraffin block from the metal base for later use.

[0061] 6) Sectioning, spreading, and baking: Turn on the slide spreader and baking machine in advance. Fill the slide spreader with water and set the temperature to 40℃, and the baking machine to 60℃. After trimming the paraffin block in the previous step, fix it on the microtome and cut it into thin sections, generally 3-5 μm. Use tweezers to spread the cut sections into 40℃ water. After the tissue in the sections has fully expanded, remove them with a glass slide and place them on the 60℃ baking machine to dry for 3-6 hours.

[0062] 7) Dewaxing: Dissolve paraffin in xylene in 3 tanks for 6 minutes each, then add alcohol to water in a gradient: 100% 6 min → 100% 6 min → 95% 4 min → 95% 4 min → 90% 4 min → 80% 4 min → 75% 4 min, then place in distilled water for later use.

[0063] 8) Antigen retrieval: Autoclave at pH 6.0 with 10 mM sodium citrate solution for 4 min, then allow to cool naturally to room temperature. Wash three times with PBS, 3 min each time.

[0064] 9) Endogenous peroxidase blockade: Draw hydrophobic zones around the tissue and block with an endogenous peroxidase inhibitor at room temperature for 20 min. Wash three times with PBS, 3 min each time.

[0065] For BrdU immunohistochemistry, steps 10) and 11) are performed; for labeling other antibodies, step 12) is performed directly.

[0066] 10) Acidification: Treat with 2 M HCl at room temperature for 19 min.

[0067] 11) Protease digestion: Digest tissue with pepsin digestion solution at room temperature for 3 min. Wash with PBS 3 times, 3 min each time.

[0068] 12) Blocking: Block the tissue with sheep serum at room temperature for 1 h.

[0069] 13) Labeling primary antibody: Dilute the antibody to the recommended concentration and incubate the tissue, then place it in a humidified chamber at 4°C overnight.

[0070] 14) Washing with primary antibody: After removing the tissue sections from the 4℃ refrigerator, warm them again and wash them with PBS 3 times, 3 min each time.

[0071] 15) Labeling secondary antibody: Tissue samples were incubated with enzyme-labeled goat anti-mouse / rabbit IgG polymer at room temperature for 1 h, and washed 3 times with PBS for 3 min each time. 16) DAB color development: After preparing the DAB color development solution according to the reagent instructions, add the color development solution to the tissue sample and observe the color development effect under a microscope, and terminate the color development reaction in time.

[0072] 17) Counterstaining, dehydration, and mounting: Counterstain in hematoxylin for 5 min → rinse with running water for 1 min → differentiate with 1% hydrochloric acid alcohol for 1-3 s → rinse with running water for 10 min → dehydrate with gradient alcohol (75% 1 min, 85% 1 min, 95% 1 min, 95% 2 min, 100% 4 min) → xylene (3 min * 2) → mount with neutral resin.

[0073] III. Experimental Results 1) CSF1R neutralizing antibody (CSF1R-Ab) improves intestinal crypt damage in radiation-induced intestinal injury: CSF1R neutralizing antibody is the most specific targeting mechanism of CSF1R. In this study, peripheral blood was collected from mice at 6 h, 12 h, and 24 h after intraperitoneal injection of 250 μg of CSF1R-Ab. Macrophages in the peripheral blood of mice were labeled with a combination of CD11b and F4 / 80 antibodies, and the proportion of macrophages in the peripheral blood of mice was detected by flow cytometry to evaluate the onset time of CSF1R-Ab in mice. The results are as follows: Figure 1 As shown in A and B, the proportion of macrophages in the peripheral blood of mice was significantly reduced 12 hours after CSF1R-Ab administration. Therefore, 250 μg / mouse of CSF1R-Ab was injected intraperitoneally 12 hours before irradiation to study its protective effect against intestinal radiation damage.

[0074] When intestinal tissue is damaged, intestinal stem cells located at the base of the crypts are significantly activated, accelerating mucosal regeneration and functional reconstruction through accelerated division. To investigate the mechanism by which CSF1R inhibitors regulate radiation-induced intestinal injury repair, this study selected BrdU (5-bromodeoxyuracil) as a DNA synthesis marker. This compound can incorporate newly synthesized DNA strands into cells during the S phase by mimicking the structure of thymine, thereby achieving specific tracking of proliferating cells. Mice were subjected to... 60 After 3.5 days of Coγ-ray irradiation (14 Gy), animals were intraperitoneally injected with BrdU (100 mg / kg). One hour later, the animals were sacrificed to obtain intestinal samples. After paraformaldehyde fixation and antigen retrieval, immunohistochemical staining was used to detect BrdU-labeled proliferating cells (BrdU+ cells > 5 were considered 1 positive crypt). The differences in intestinal crypt cell proliferation among the groups of mice were compared. Quantitative analysis results showed ( Figure 1 In the C and D groups, compared with the control group, the number of intestinal BrdU-positive crypts in the CSF1R-Ab 250 μg / mouse prophylactic administration group was significantly increased (P<0.01).

[0075] 2) CSF1R inhibitors PLX5622 and PLX3397 improve intestinal crypt damage in radiation-induced intestinal injury: PLX5622 and PLX3397 are both small-molecule CSF1R inhibitors commonly used in neuroscience research to target microglia. They differ in chemical structure and application. To further evaluate the effects of CSF1R-targeting drugs on the repair function of radiation-induced intestinal injury, this study used CSF1R inhibitors PLX5622 and PLX3397 to investigate their intestinal radioprotective effects. The experimental design employed a two-factor analysis strategy using dose gradient and time window. The specific procedure was as follows: Mice were subjected to... 60 After 3.5 days of Co-γ ray irradiation (14 Gy), the number of BrdU-labeled positive crypts was detected using immunohistochemical staining. Quantitative analysis results showed ( Figure 2 In the AC study, compared with the control group, the BrdU-positive crypts in the crypt region of the PLX5622 and PLX3397 treatment groups showed a significant increasing trend (P<0.01), with the 120 mg / kg dose group reaching the peak proliferation response, suggesting that this dose has the optimal therapeutic effect. In the time-series effect study ( Figure 3 In the case of AC), pre-treatment with 120 mg / kg of the drug 3-12 hours before irradiation significantly enhanced the regenerative capacity of intestinal stem cells after injury. Among them, there was no statistically significant difference in proliferation promotion effect between the group treated 6 hours before irradiation and the group treated 3 hours before irradiation, indicating that the drug intervention window period covers at least 6 hours before irradiation.

[0076] Example 2: Effect of CSF1R inhibitors on survival in mice with radiation-induced intestinal injury I. Experimental Materials 1.1 Experimental animals: C57BL / 6J male mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., China).

[0077] 1.2 Reagents and materials: as shown in Table 2.

[0078] Table 2. Experimental Materials

[0079] II. Experimental Methods 2.1 Animal feeding: C57BL / 6J mice, 6 8 weeks old, weighing 20 22g male mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and housed at the Animal Center of the Academy of Military Medical Sciences (SPF grade). The housing temperature was 22±2℃, humidity 55±5%, and bedding was sterilized with gamma rays and changed twice weekly. Five mice were per cage, with 12 hours of light and 12 hours of darkness daily, fed standard gamma-ray sterilized feed and acidified water. All mice were housed in the animal facility for one week after purchase to acclimatize before experiments were conducted.

[0080] 2.2 Mouse grouping: A mouse model of 14 Gy whole-body irradiation combined with bone marrow transplantation was established. A total of 30 mice were used, with 10 mice in each of the PLX5622 and PLX3397 drug administration groups and 10 mice in the irradiation control group.

[0081] 2.3 Irradiation Protocol: The boxes containing the mice were placed 2.5 meters away from the gamma-ray source for irradiation. The total irradiation dose was 14 Gy. Six hours after irradiation, all mice received 1×10⁻⁶ gamma rays. 6 A mouse model of whole-body irradiation combined with bone marrow transplantation was established by injecting mouse bone marrow cells via the tail vein. After irradiation, the mice were allowed free movement and feeding in cages with an SPF barrier environment.

[0082] 2.4 Dosage Regimen: PLX5622 hydrochloride and PLX3397 hydrochloride were prepared using 5% DMSO, 30% PEG400, 5% Tween-80, and 60% physiological saline. The solutions were sonicated to obtain a clear, transparent saline solution, which was administered intraperitoneally to mice at a dose of 120 mg / kg 3 hours before irradiation. Control mice received the same volume of solution. Mice survival and body weight were observed for 30 days after irradiation.

[0083] III. Experimental Results To eliminate the interference of hematopoietic system damage caused by whole-body irradiation on mouse survival, the inventors observed the effect of CSF1R inhibitors on the survival of mice subjected to a lethal dose of 15 Gy gamma rays combined with bone marrow transplantation. Figure 4 As shown in Figures A and B, all mice in the control group experienced rapid weight loss after irradiation and died within 7 days, with a median survival time of 4 days post-irradiation. Mice in the PLX5622-treated group showed significantly prolonged survival, with all dying within 17 days and a median survival time of 13 days. Mice in the PLX3397-treated group died only one on day 7 and one on day 8 post-irradiation, with a 30-day survival rate of 80%, and surviving mice recovered to their pre-irradiation weight. This indicates that the CSF1R inhibitors PLX5622 and PLX3397 can prolong the survival time of mice with radiation-induced intestinal injury. At the same dosage, PLX3397 is significantly more effective than PLX5622 in preventing radiation-induced intestinal injury. Therefore, PLX3397 will be selected for further research on the mechanism of CSF1R inhibitors in preventing radiation-induced intestinal injury.

[0084] Example 3: CSF1R inhibitors reduce gamma-ray irradiation-induced apoptosis of mouse intestinal crypt cells. I. Experimental Materials 1.1 Experimental animals: C57BL / 6J male mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., China).

[0085] 1.2 Reagents and materials: as shown in Table 3.

[0086] Table 3. Experimental Materials

[0087] II. Experimental Methods 2.1 Animal feeding: C57BL / 6J mice, 6 8 weeks old, weighing 20 22g male mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and housed at the Animal Center of the Academy of Military Medical Sciences (SPF grade). The housing temperature was 22±2℃, humidity 55±5%, and bedding was sterilized with gamma rays and changed twice weekly. Five mice were per cage, with 12 hours of light and 12 hours of darkness daily, fed standard gamma-ray sterilized feed and acidified water. All mice were housed in the animal facility for one week after purchase to acclimatize before experiments were conducted.

[0088] 2.2 Mouse Grouping: A mouse model of intestinal radiation injury induced by whole-body irradiation with 14 Gy was used to evaluate the protective effect of intraperitoneal injection of the CSF1R inhibitor PLX3397 120 mg / kg 3 h before irradiation against apoptosis of intestinal crypt cells induced by γ-ray irradiation. Twelve C57BL / 6J mice were divided into an irradiation control group and a PLX3397 administration group. The intestines of the mice were dissected and pathologically examined at 6 h and 24 h after irradiation, with 3 mice in each group.

[0089] 2.3 Dosage regimen: PLX3397 hydrochloride was prepared using 5% DMSO, 30% PEG400, 5% Tween-80, and 60% physiological saline, and dissolved by sonication to obtain a clear and transparent saline solution. Mice were intraperitoneally injected with 120 mg / kg 3 h before irradiation. Control mice received the same volume of solvent as a control.

[0090] 2.4 The Cleaved-caspase 3 immunohistochemical method is the same as described in Example 1.

[0091] 2.5 TUNEL assay for intestinal cell apoptosis: 1) The steps of sampling, fixation, dehydration, embedding, sectioning, and dewaxing are as described above.

[0092] 2) Proteinase K permeabilization treatment: Draw a hydrophobic zone, add an appropriate amount of 20 μg / ml DNase-free proteinase K, incubate at room temperature for 20 min, and wash the sections with PBS for 3 min × 3 times.

[0093] 3) Endogenous peroxidase blockade: Add an appropriate amount of endogenous peroxidase inhibitor, incubate at room temperature for 20 min, and wash the sections with PBS for 3 min × 3 times.

[0094] 4) Biotin labeling: Prepare biotin labeling solution according to the instructions in the TUNEL apoptosis detection kit. Add 50 μl of biotin labeling solution to each sample, incubate at 37°C in the dark for 1 h, and wash the sections once with PBS.

[0095] 5) Termination of labeling reaction: Add labeling reaction termination solution, incubate at room temperature for 10 min, and wash the slides with PBS for 3 min × 3 times.

[0096] 6) HRP labeling: Prepare Streptavidin-HRP working solution according to the instructions in the TUNEL apoptosis detection kit. Add 50 μl of Streptavidin-HRP working solution to each sample, incubate at room temperature for 30 min, and wash the sections with PBS for 3 min × 3 times.

[0097] 7) Color development: Prepare DAB color development solution according to the instructions in the TUNEL apoptosis detection kit, add an appropriate amount to cover the tissue, observe the color development reaction under a microscope, and stop the process in time to avoid over-development.

[0098] 8) Counterstaining and mounting are the same as before.

[0099] III. Experimental Results High-dose ionizing radiation induces rapid and widespread apoptosis in the intestine, particularly in intestinal stem and progenitor cells, directly impacting the intestine's proliferative and regenerative capacity. To determine the effect of prophylactic administration of the CSF1R inhibitor PLX3397 on intestinal cell apoptosis after high-dose radiation, we performed Cleaved-Caspase-3 immunohistochemical staining and TUNEL assays. Results showed that after 14 Gy whole-body irradiation, intestinal crypt cells in the PLX3397 prophylactic administration group were Cleaved-Caspase-3 positive (…). Figure 5 The number of apoptotic cells (A and B) and the number of TUNEL-positive apoptotic cells ( Figure 5 In the C and D groups, the apoptosis rates at 6 h and 24 h post-irradiation were significantly lower than those in the irradiation-only group. These results suggest that prophylactic administration of the CSF1R inhibitor PLX3397 significantly inhibited apoptosis of intestinal crypt cells after irradiation.

[0100] Example 4: Verification of the radiation-protective effect of CSF1R inhibitors on small intestinal crypt stem cells. I. Experimental Materials 1.1 Experimental animals: C57BL / 6J male mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., China).

[0101] 1.2 Reagents and materials: as shown in Table 4.

[0102] Table 4. Experimental Materials

[0103] II. Experimental Methods 2.1 Animal feeding: C57BL / 6J mice, 6 8 weeks old, weighing 20 22g male mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and housed at the Animal Center of the Academy of Military Medical Sciences (SPF grade). The housing temperature was 22±2℃, humidity 55±5%, and bedding was sterilized with gamma rays and changed twice weekly. Five mice were per cage, with 12 hours of light and 12 hours of darkness daily, fed standard gamma-ray sterilized feed and acidified water. All mice were housed in the animal facility for one week after purchase to acclimatize before experiments were conducted.

[0104] 2.2 Mouse Grouping: A mouse model of intestinal radiation injury caused by whole-body irradiation with 14 Gy was used to evaluate the radiation-protective effect of intraperitoneal injection of the CSF1R inhibitor PLX3397 hydrochloride 120 mg / kg 3 h before irradiation on small intestinal crypt stem cells. Six C57BL / 6J mice were divided into an irradiation control group and a PLX3397 administration group. The intestines of the mice were dissected and pathologically examined 3.5 days after irradiation, with 3 mice in each group.

[0105] 2.3 Dosing regimen: PLX3397 hydrochloride was purchased from Hubei Chengfeng Chemical Co., Ltd. It was prepared using 5% DMSO, 30% PEG400, 5% Tween-80, and 60% physiological saline, and dissolved by sonication to obtain a clear and transparent saline solution. Mice were intraperitoneally injected with 120 mg / kg 3 hours before irradiation. Control mice received the same volume of solvent as a control.

[0106] 2.4 Olfm4 Immunofluorescence: Mouse intestinal tissue was fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned (4 μm thick). Immunofluorescence staining was performed: after antigen retrieval, the tissue was blocked with 5% BSA at room temperature for 1 hour; rabbit anti-Olfm4 primary antibody (concentration 1:200) was added and incubated overnight at 4°C; after washing with PBS, Alexa Fluor 594-labeled goat anti-rabbit IgG secondary antibody (red fluorescence) was added and incubated at room temperature in the dark for 1 hour; cell nuclei were counterstained with DAPI. After mounting, images were acquired using a laser confocal microscope, with uniform exposure parameters.

[0107] Image analysis was performed using ImageJ software: images were converted to 8-bit grayscale, a uniform threshold was set to distinguish specific signals from the background, and the "Analyze Particles" function was used to quantify the area of ​​fluorescent positive regions. At least three mice were analyzed in each group, and at least three non-overlapping images were taken from each mouse. Data are expressed as mean ± standard deviation.

[0108] III. Experimental Results Based on the core role of small intestinal crypt stem cells (ISCs) in the repair of intestinal radiation damage, this invention focuses on the effect of the CSF1R inhibitor PLX3397 on the survival of ISCs after ionizing radiation. The experiment used Olfm4 (a specific marker for small intestinal stem cells) immunofluorescence staining to quantitatively analyze the changes in the number of Olfm4+ stem cells in the intestinal crypts of mice 3.5 days after 14 Gy whole-body irradiation. Semi-quantitative analysis of the immunofluorescence signal (percentage of positive area) was performed using ImageJ software. The results showed that the percentage of Olfm4+ stem cell-positive areas in the intestinal crypts of the irradiation-only group (IR group) was 3.8% ± 1.2%, while the PLX3397 pretreatment group (120 mg / kg, administered 12 h before irradiation) significantly inhibited this damage effect, resulting in an Olfm4+ cell-positive area percentage of 6.5% ± 1.8% (P < 0.01). Figure 5 (A and B in the original text). This finding indicates that the CSF1R inhibitor PLX3397 can effectively improve the destruction of intestinal crypt structure induced by high-dose radiation by regulating the survival rate of intestinal stem cells, providing new experimental evidence for stem cell protection strategies against radiation-induced intestinal injury. Figure 6 (A and B in the text).

[0109] Existing technologies suggest that long-term or deep CSF1R blockade may disrupt crypt homeostasis and affect Paneth cells and Lgr5+ stem cell niches, thus creating safety concerns regarding the use of CSF1R inhibitors for intestinal injury. This invention, through a prophylactic, short-term, window-period dosing strategy, demonstrates through systematic experimental data that within a specific dose / time window, not only does it not damage crypt stem cells, but it also significantly improves their survival and regeneration, overcoming and correcting the negative expectations and technical biases of existing technologies. A multi-level, strong causal endpoint system demonstrates the technical efficacy: early inhibition of crypt apoptosis (decreased Cleaved-Caspase-3 and TUNEL); promotion of crypt proliferation and regeneration (significant increase in BrdU-positive crypts); direct protection of ISCs (increased proportion of Olfm4+ stem cell area); and conversion into survival benefits (prolonged median survival and improved 30-day survival rate). This closed-loop evidence of "mechanism-structure-function-survival" makes the protective effect more explicit and reliable, directly demonstrating the protection of the "core damage chain of crypt stem cells" by CSF1R inhibitors, providing a clearer path for clinical application and translation.

[0110] 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. The use of CSF1R-targeting drugs in the preparation of drugs for the prevention and / or treatment of radiation-induced intestinal injury, characterized in that, The CSF1R-targeting drug is a CSF1R inhibitor or a pharmaceutically acceptable salt thereof; The CSF1R inhibitors are selected from PLX5622 and PLX3397.

2. The application according to claim 1, characterized in that, The effective dose of the CSF1R inhibitor is selected from 40-120 mg / kg.

3. The application according to claim 2, characterized in that, The effective dose of the CSF1R inhibitor is 120 mg / kg.

4. The application according to claim 1, characterized in that, The CSF1R inhibitor was administered 3-12 hours before radiation exposure.

5. The application according to claim 4, characterized in that, The CSF1R inhibitor was administered 6-12 hours before radiation exposure.

6. The application according to claim 5, characterized in that, The CSF1R inhibitor was administered 12 hours before irradiation.

7. The application according to claim 1, characterized in that, The CSF1R inhibitors can be administered via intraperitoneal injection, oral administration, subcutaneous injection, or local intestinal administration.

8. The application according to claim 7, characterized in that, The CSF1R inhibitor is administered via intraperitoneal injection.

9. The application according to claim 1, characterized in that, The radiation-induced intestinal injury refers to radiation-induced intestinal injury caused by gamma rays and / or acute radiation syndrome of the gastrointestinal tract.

10. The application according to claim 9, characterized in that, The radiation-induced intestinal damage caused by gamma rays includes crypt damage.

11. The application according to claim 9, characterized in that, The irradiation dose of the gamma rays is greater than or equal to 10 Gy.

12. The application according to claim 11, characterized in that, The irradiation dose of the gamma rays is 14-15 Gy.

13. The application according to claim 1, characterized in that, The prevention and / or treatment include reducing radiation-induced apoptosis of intestinal crypt cells and / or promoting regeneration and repair of intestinal crypt cells.

14. The application according to claim 13, characterized in that, The promotion of intestinal crypt cell regeneration and repair includes maintaining or enhancing the survival of crypt stem cells.

15. The application according to claim 14, characterized in that, The crypt stem cells are Olfm4+ stem cells.

16. The application according to claim 1, characterized in that, The therapeutic agents also include pharmaceutically acceptable carriers.

17. The application according to claim 16, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: diluents, excipients, glidants, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, and emulsifiers.

18. The application according to claim 1, characterized in that, The dosage forms of the therapeutic drugs include oral dosage forms, injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

19. The application according to claim 1, characterized in that, The dosage forms of the therapeutic drugs include granules, powders, tablets, capsules, syrups, suppositories, or injections.

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