Use of cyclosporin in the treatment of radiation damage to the bowel

Abstract

The application discloses application of cyclosporin in treatment of intestinal radiation injury. The application discloses a new use of cyclosporin in prevention and treatment of intestinal radiation injury. Researches show that cyclosporin can effectively prolong the survival time of a mouse model of intestinal radiation injury, reduce apoptosis of intestinal crypt cells, and increase the number of intestinal crypt stem cells after radiation, and both intraperitoneal injection and oral administration have the prevention and treatment effects, indicating that the cyclosporin has great potential market and application prospect in prevention and / or treatment of intestinal radiation sickness and gastrointestinal radiation injury caused by tumor radiotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of cyclosporine in the treatment of intestinal radiation injury. Background Technology

[0002] Secondary nuclear disasters (nuclear power plant accidents, drilling accidents), accidental radioactive nuclear accidents, and medical irradiation targeting tumors can all lead to acute radiation sickness (ARS). ARS is a systemic disease, classified into bone marrow type, intestinal type, and cerebral type based on the primary affected organ; and into acute, subacute, and chronic types based on the duration of exposure. Intestinal type ARS is an extremely severe form of ARS caused by exposure to radiation exceeding 10 Gy in a single event or over a short period (several days), characterized by intestinal damage and manifested primarily by vomiting, diarrhea, and bloody stools. This type of ARS is extremely serious, progresses rapidly, and is so severe that no patients have survived despite aggressive comprehensive treatment.

[0003] For decades, abdominal radiotherapy has been a crucial component of treatment and palliative care for patients with intestinal or pelvic cancers, accounting for approximately 70% of all cancer patients. Radiotherapy plays a key role in treating 25% of tumors. Despite advancements in radiotherapy technology, radiation inevitably causes damage to normal intestinal tissue while killing tumor cells. Radiation enteritis refers to intestinal complications caused by radiotherapy for malignant tumors in the pelvic cavity, abdominal cavity, or retroperitoneum. Depending on the affected site, it includes radiation-induced rectal, colonic, and small enteritis. Early symptoms include erosion, ulceration, or bleeding of the intestinal mucosa, with clinical symptoms such as acute diarrhea, abdominal pain, bloody stools, and mucus in the stool. Later stages manifest as fistulas, perforations, stenosis, fibrosis, or cancer, resulting in rectal stenosis, difficulty defecating, and even intestinal obstruction. Iatrogenic radiation enteritis not only increases the economic burden on patients and severely impacts their quality of life but also limits the radiation dose that can be used in radiotherapy. Currently, there are no specific drugs or treatments for radiation enteritis; prevention is paramount.

[0004] Therefore, finding drugs that can treat radiation damage to the intestines is of great importance in this field. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention provides the application of cyclosporine in the treatment of intestinal radiation injury.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides the use of cyclosporine in the preparation of medicaments for the prevention / treatment of intestinal radiation injury.

[0008] Furthermore, the intestinal radiation injury is intestinal radiation injury caused by gamma rays.

[0009] Furthermore, the irradiation dose of the gamma rays is greater than or equal to 10 Gy.

[0010] Furthermore, the irradiation dose of the gamma rays is 12-15 Gy.

[0011] Furthermore, the prevention / treatment of intestinal radiation injury includes inhibiting crypt cell apoptosis and / or promoting the regeneration and repair of intestinal crypts.

[0012] Furthermore, the promotion of intestinal crypt regeneration and repair includes promoting the survival of intestinal stem cells.

[0013] Furthermore, the intestinal stem cells include Olfm4 cells and / or Paneth cells.

[0014] Furthermore, the cyclosporine can be administered via intraperitoneal injection, subcutaneous injection, or oral administration.

[0015] Furthermore, the cyclosporine is administered via intraperitoneal injection.

[0016] A second aspect of the invention provides the use of cyclosporine in the preparation of medicaments for the prevention / treatment of crypt injuries.

[0017] Furthermore, the intestinal radiation injury is intestinal radiation injury caused by gamma rays.

[0018] A third aspect of the invention provides a medicament for the prevention / treatment of intestinal radiation injury / crypt injury, the medicament comprising cyclosporine.

[0019] Furthermore, the drug also includes other drugs for treating intestinal radiation injury.

[0020] Furthermore, the other drugs for treating intestinal radiation injury include one or more of the following: probiotics, antibiotics, nonsteroidal anti-inflammatory drugs, and hormones.

[0021] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0022] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: diluent, binder, surfactant, humectant, adsorbent, lubricant, filler, and disintegrant.

[0023] Furthermore, the dosage form of the drug includes granules, powder, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions, or solutions.

[0024] A fourth aspect of the present invention provides a method for regulating intestinal stem cells, the method comprising administering cyclosporine.

[0025] Furthermore, the intestinal stem cells include Olfm4 cells and / or Paneth cells.

[0026] Furthermore, the method described is not for therapeutic purposes.

[0027] Advantages and beneficial effects of the present invention:

[0028] This invention reveals a new use of cyclosporine for the prevention and treatment of intestinal radiation injury. Studies have shown that prophylactic administration of cyclosporine can effectively prolong the survival time of a mouse model of intestinal radiation injury, reduce intestinal crypt cell apoptosis, and increase the number of intestinal crypt stem cells after irradiation. Both intraperitoneal injection and oral administration have preventive and therapeutic effects, indicating that cyclosporine has great potential market and application prospects for reducing gastrointestinal radiation injury before clinical abdominal tumor radiotherapy. Attached Figure Description

[0029] Figure 1 This is a time-effect diagram of cyclosporine administration regimens. Among them, 1A is a diagram showing the number of crypts after intraperitoneal injection of cyclosporine before irradiation, 1B is a statistical diagram showing the number of crypts after intraperitoneal injection of cyclosporine before irradiation, 1C is a diagram showing the number of crypts after gavage of cyclosporine before irradiation, and 1D is a statistical diagram showing the number of crypts after gavage of cyclosporine before irradiation.

[0030] Figure 2 This is a dose-response diagram of the cyclosporine administration regimen. Among them, 2A is a diagram of the number of crypts of intraperitoneal cyclosporine injected within 2 hours before irradiation, 2B is a statistical diagram of the number of crypts of intraperitoneal cyclosporine injected within 2 hours before irradiation, 2C is a diagram of the number of crypts of oral cyclosporine injected within 2 hours before irradiation, and 2D is a statistical diagram of the number of crypts of oral cyclosporine injected within 2 hours before irradiation.

[0031] Figure 3 This is a diagram showing the prevention of intestinal injury caused by radiotherapy using cyclosporine. Among them, 3A is the survival rate statistics of mice with an irradiation dose of 12 Gy, 3B is the body weight statistics of mice with an irradiation dose of 12 Gy, 3C is the survival rate statistics of mice with an irradiation dose of 15 Gy combined with bone marrow transplantation, and 3D is the body weight statistics of mice with an irradiation dose of 15 Gy combined with bone marrow transplantation.

[0032] Figure 4 These are images of intestinal cell apoptosis after cyclosporine radiation. Among them, 4A is the number of Cleaved-Caspase-3 positive apoptotic cells in intestinal crypts, 4B is the statistical chart of the number of Cleaved-Caspase-3 positive apoptotic cells in intestinal crypts, 4C is the number of TUNEL positive apoptotic cells in intestinal crypts, and 4D is the statistical chart of the number of TUNEL positive apoptotic cells in intestinal crypts.

[0033] Figure 5These are images showing the effects of cyclosporine radiation on intestinal crypt stem cells. 5A shows the number of Olfm4+ stem cells in the intestinal crypts, 5B shows the statistical data on the number of Olfm4+ stem cells in the intestinal crypts, 5C shows the number of Paneth cells in the intestinal crypts, and 5D shows the statistical data on the number of Paneth cells in the intestinal crypts. Detailed Implementation

[0034] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] This invention provides the use of cyclosporine in the preparation of medicaments for the prevention / treatment of intestinal radiation injury.

[0036] In one embodiment of the present invention, cyclosporine (Cyclosporine, Cyclosporine A, Ciclosporin) is also referred to as "cyclosporine" or "cyclosporin A".

[0037] In one embodiment of the invention, cyclosporine further includes pharmaceutically acceptable salts of cyclosporine. A pharmaceutically acceptable salt is any acid addition salt or base addition salt whose counterion is non-toxic to the patient at the pharmaceutical dose of said salt. The main components of pharmaceutically acceptable salts are well known in the art. If a pharmaceutically acceptable salt of the compound of this application is 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, diglucuronides, 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.

[0038] The intestinal radiation injury mentioned is intestinal radiation injury caused by gamma rays.

[0039] In one embodiment of the present invention, intestinal radiation damage caused by gamma rays includes intestinal damage caused by any dose of gamma rays, wherein the dose may 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, or not less than 15 Gy.

[0040] In a preferred embodiment of the present invention, the irradiation dose of the gamma rays is greater than or equal to 10 Gy, and the 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, or not less than 15 Gy.

[0041] In a more preferred embodiment of the present invention, the irradiation dose of the gamma rays is 12-15 Gy. For example, it can be 12 Gy, 13 Gy, 14 Gy, 15 Gy, or any two of the above-mentioned irradiation doses.

[0042] This invention provides a drug for the prevention / treatment of intestinal radiation injury / crypt injury, the drug comprising cyclosporine.

[0043] The medication also includes other medications for treating radiation-induced intestinal injury.

[0044] In one embodiment of the present invention, the other drugs for treating intestinal radiation injury include one or more of probiotics, antibiotics, nonsteroidal anti-inflammatory drugs, and hormones.

[0045] The probiotics include one or more of lactobacillus, bifidobacteria, enterococci, or lactic acid bacteria.

[0046] The antibiotics include one or more of mesalazine, betamethasone, metronidazole, and ciprofloxacin.

[0047] The nonsteroidal anti-inflammatory drugs include one or more of sulfasalazine, balsalazine, mesalazine, and olsalazine.

[0048] The hormones mentioned include glucocorticoids.

[0049] The drug also includes a pharmaceutically acceptable carrier.

[0050] In one embodiment of the invention, a pharmaceutically acceptable carrier refers to a material that is compatible with the recipient, preferably a mammal, more preferably a human, and is suitable for delivering the active agent to the target site without terminating the activity of the agent. The toxicity or side effects (if present) associated with the pharmaceutically acceptable carrier are preferably proportionate to a reasonable risk / benefit ratio for the intended use of the active agent.

[0051] Pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants. These pharmaceutically acceptable carriers may be used as needed to aid in the stability of the formulation or to contribute to its activity or bioavailability, or to produce an acceptable taste or odor when taken orally. The formulation may be used in this drug in the form of the original compound itself or optionally in the form of a pharmaceutically acceptable salt thereof. The drug thus formulated may be administered as needed by any appropriate method known to those skilled in the art.

[0052] The diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water.

[0053] Adhesives include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropylcellulose and hydroxypropylmethylcellulose.

[0054] Surfactants include, but are not limited to, polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol.

[0055] Humectants include, but are not limited to, glycerin.

[0056] Adsorption carriers include, but are not limited to, bentonite, silica gel, kaolin and soap clay.

[0057] Lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium dodecyl sulfate.

[0058] Fillers include, but are not limited to, mannitol (granular or powdered), xylitol, sorbitol, maltose, erythritol, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, and sodium bicarbonate.

[0059] Disintegrants include, but are not limited to, crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropylmethyl, crosylcarboxymethyl cellulose sodium, and soybean polysaccharides.

[0060] In one embodiment of the invention, the drug can be manufactured using methods well-known in the art, such as conventional granulation, mixing, dissolving, encapsulation, lyophilization, or emulsification. The drug can be formulated into various forms, including granules, precipitates or microparticles, powders (including lyophilized powders, rotary-dried powders, spray-dried powders, and amorphous powders), tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions.

[0061] In one embodiment of the invention, the drug is formulated for administration to mammals. The drug can be administered via various routes, including intravenous injection, intraperitoneal injection, subcutaneous injection, intramuscular injection, oral administration, transmucosal administration, rectal administration, transdermal administration, or inhalation.

[0062] In a preferred embodiment of the present invention, cyclosporine is administered via intraperitoneal injection, subcutaneous injection, or oral administration.

[0063] In a specific embodiment of the present invention, the administration method of cyclosporine is selected from intraperitoneal injection and oral administration.

[0064] Injectable formulations can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques, such as sterile injectable aqueous or oily suspensions. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable mediators and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids, such as oleic acid, are used in injectable formulations. Injectable formulations can be sterilized, for example, by filtering through a bacterial-retaining filter, or by adding a sterilizing agent in the form of a sterile solid composition soluble in sterile water or other sterile injectable media before use. Drugs formulated for parenteral administration can be administered by rapid injection or by timed bolus injection, or by continuous infusion.

[0065] Oral liquid dosage forms include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the compounds of this application, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, cyclodextrin, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

[0066] Solid dosage forms for oral administration include capsules, tablets, powders, and granules. In these solid dosage forms, the cyclosporine of this application or a pharmaceutically acceptable salt thereof is mixed with at least one inert, pharmaceutically acceptable excipient, such as sodium citrate or dicalcium hydrogen phosphate, and / or the following: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerin; d) disintegrants, such as agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) dissolution delayers, such as paraffin; f) absorption enhancers, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glyceryl monostearate; h) adsorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, lauryl, sodium sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer, such as a phosphate or carbonate.

[0067] Examples of encapsulation compositions that can be used include lactose or toffee and high molecular weight polyethylene glycol as excipients, employing similar types of solid compositions as fillers in soft and hard-filled gelatin capsules. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells, such as enteric coatings and other coatings well known in the art. They may optionally contain light-blocking agents and may also be compositions that optionally release only or preferentially the active ingredient in a portion of the intestine in a delayed manner. Examples of encapsulation compositions that can be used include polymeric substances and waxes. Examples of similar types of solid compositions that can be used include lactose or toffee and high molecular weight polyethylene glycol as excipients, employing similar types of solid compositions as fillers in soft and hard-filled gelatin capsules.

[0068] In one embodiment of the invention, the dosage regimen of the drug will vary based on known factors, such as the pharmacodynamic characteristics of the particular drug and its administration mode and route; the recipient's age, sex, health, medical condition, and weight; the nature and severity of symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration; and the desired effect. In some embodiments, the drug of the invention may be administered in a single daily dose, or the total daily dose may be administered in divided doses two, three, or four times daily.

[0069] In one embodiment of the present invention, if the subject is a mouse, the dosage of cyclosporine can be any range, for example, 1 mg / kg, 3 mg / kg, 5 mg / kg, 7 mg / kg, 10 mg / kg, 12 mg / kg, 13 mg / kg, 15 mg / kg, 17 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 23 mg / kg, 24 mg / kg, 30 mg / kg, and any two of the above.

[0070] 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.

[0071] In one embodiment of the invention, administration refers to the method of giving a dose / effective amount of a drug or compound or pharmaceutical composition to a subject (e.g., a patient). Administration can be by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and intralesional administration if intended for local treatment. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending on whether the administration is transient or long-term, administration can be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are covered in this application, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0072] In one embodiment of the invention, an effective amount or therapeutically effective amount refers to an amount of compound sufficient to provide therapeutic or preventive benefit in the treatment or prevention of a disease, or sufficient to delay or minimize disease-related symptoms. Furthermore, a therapeutically effective amount of a compound used in the invention refers to an amount of the compound, alone or in combination with other therapies that provide therapeutic benefit in the treatment or prevention of a disease. When used in conjunction with compounds used in the invention, this term may include amounts that improve overall treatment, reduce or avoid symptoms or causes of a disease, enhance the therapeutic efficacy of another therapeutic agent, or synergize with another therapeutic agent. The full therapeutic effect does not necessarily occur with the administration of a single dose (or a single administration), but may occur only after a series of doses. Therefore, an effective amount may be administered in one or more administrations.

[0073] The subject refers to any animal, including both human and non-human animals. Non-human animals include all vertebrates, such as mammals, including non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; as well as non-mammals, such as chickens, amphibians, reptiles, etc.

[0074] 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.

[0075] The present invention provides a method for regulating intestinal stem cells, the method comprising administering cyclosporine.

[0076] In one embodiment of the present invention, the regulation of intestinal stem cells is to promote the survival of intestinal stem cells.

[0077] 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.

[0078] Example 1: Study on the administration regimen of cyclosporine to reduce radiation damage in the intestinal crypts of mice.

[0079] 1. Experimental Materials

[0080] 1.1 Laboratory Animals

[0081] C57BL / 6J male mice, 20-22 g, 6-8 weeks old, Beijing Huafukang Biotechnology Co., Ltd., China

[0082] 1.2 Reagents and Materials

[0083] Cyclosporine Injection (Sandimin) 5ml: 250mg Novartis Pharmaceuticals Switzerland, Physiological Saline Shijiazhuang No.4 Pharmaceutical China, BrdU Selleck Chem USA, Pepsin Digestion Solution Beijing Zhongshan Jinqiao China, Endogenous Peroxidase Inhibitor Beijing Zhongshan Jinqiao China, Blocking Goat Serum (Working Solution) Beijing Zhongshan Jinqiao China, pH 6.0 Citrate Repair Solution Beijing Zhongshan Jinqiao China, BrdU Antibody Reagent (Immunohistochemistry) Beijing Zhongshan Jinqiao China, Enzyme-Labeled Goat Anti-Mouse / Rabbit IgG Polymer Beijing Zhongshan Jinqiao China, DAB Colorimetric Reagent Kit Beijing Zhongshan Jinqiao China, Hydrophobic Pen Beijing Zhongshan Jinqiao China, Concentrated HCl Sinopharm Chemical Reagent China, Hematoxylin Staining Solution Leica Biosystems USA, Paraffin Leica Biosystems USA, Xylene Sinopharm Chemical Reagent China, Anhydrous Ethanol Sinopharm Chemical Reagent China, Paraformaldehyde Sinopharm Chemical Reagent China, Neutral Resin Wuxi Jiangyuan Industrial China, Adhesive Slides Jiangsu Shitai Laboratory Equipment China.

[0084] 2. Experimental Methods

[0085] 2.1 Animal husbandry

[0086] C57BL / 6J mice, 6-8 weeks old, weighing 20-22g, male, were purchased from SPF (Beijing) 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.

[0087] 2.2 Mouse grouping

[0088] A mouse model of intestinal radiation injury caused by 12 Gy whole-body irradiation was used to evaluate the protective effect of oral or intraperitoneal administration of cyclosporine against radiation damage to intestinal crypts. ① Time-effect study of different administration routes of cyclosporine: Thirty-six C57BL / 6J mice were administered cyclosporine 30 mg / kg orally or intraperitoneally. Each administration route was further divided into an irradiation control group, a group administered 24 h before irradiation, a group administered 12 h before irradiation, a group administered 6 h before irradiation, a group administered 2 h before irradiation, and a group administered 0.5 h before irradiation, with three mice in each group. ② Time-effect study of different administration routes of cyclosporine: Thirty-six C57BL / 6J mice were administered cyclosporine 2 hours before irradiation, either orally or intraperitoneally. Each administration route was further divided into an irradiation control group, a group administered cyclosporine 1 mg / kg, a group administered cyclosporine 5 mg / kg, a group administered cyclosporine 10 mg / kg, a group administered cyclosporine 20 mg / kg, and a group administered cyclosporine 30 mg / kg, with three mice in each group.

[0089] 2.3 Irradiation scheme

[0090] The mice were placed in boxes 2.5 meters away from the gamma-ray source and irradiated with a total dose of 12 Gy. After irradiation, the mice were allowed free movement and feeding in cages with an SPF barrier environment. The intestines were dissected and used for pathological observation 3.5 days later.

[0091] 2.4 Dosing regimen

[0092] Cyclosporine injection (Sandimin) was purchased from Novartis AG, Switzerland. After being diluted with physiological saline, mice were administered the drug at different time points before treatment by gavage or intraperitoneal injection at the given drug dose. The control group mice were given the same volume of physiological saline.

[0093] 2.5 Brdu Immunohistochemistry

[0094] (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.

[0095] (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 box.

[0096] (3) Fixation: The tissue from the previous step was placed in 10% formalin or 4% paraformaldehyde and fixed at room temperature for 8~24 h.

[0097] (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, and then cut the small intestinal tissue into segments of about 5 mm. The cut tissue is then dehydrated step by step in ethanol solutions of different concentrations: 70%→80%→90%→95%→95%→100%→100%, with each concentration taking 40~60 min. Then, it is cleared in xylene in two tanks for 10~20 min each, and finally in paraffin in three tanks at a temperature of 65~70℃ for 0.5~1 h each.

[0098] (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.

[0099] (6) Sectioning, spreading, and baking: Turn on the slicer and baking machine in advance. After filling the slicer with water, set the temperature to 40℃ and the baking machine to 60℃. After trimming the paraffin block in the previous step, fix it on the slicer and cut it into thin sections, generally 3~5 μm. Use tweezers to spread the cut thin sections in 40℃ water. After the tissue in the thin sections is fully expanded, take them out with a glass slide and place them on the baking machine at 60℃ to dry for 3~6 hours.

[0100] (7) Dewaxing: Dissolve paraffin in xylene in 3 tanks for 6 min each, then add alcohol to water in a gradient, specifically 100% 6 min → 100% 6 min → 95% 4 min → 95% 4 min → 90% 4 min → 80% 4 min → 75% 4 min, and then place in distilled water for later use.

[0101] (8) Antigen retrieval: pH 6.0 10 mM sodium citrate retrieval solution was used for high temperature and high pressure retrieval for 4 min, followed by natural cooling to room temperature. The antigen was washed 3 times with PBS for 3 min each time.

[0102] (9) Endogenous peroxidase blockade: Draw hydrophobic zones around the tissue and block with endogenous peroxidase inhibitor at room temperature for 20 min. Wash with PBS 3 times, 3 min each time.

[0103] When performing BrdU immunohistochemistry, steps (10) and (11) are performed, while when labeling other antibodies, step (12) is performed directly.

[0104] (10) Acidification: 2 M HCl was applied at room temperature for 19 min.

[0105] (11) Protease digestion: Digest tissue with pepsin digestion solution at room temperature for 3 min. Wash with PBS 3 times, 3 min each time.

[0106] (12) Blocking: The tissue was blocked with sheep serum at room temperature for 1 h.

[0107] (13) Labeling primary antibody: Dilute the antibody according to the recommended concentration and incubate the tissue. Place it in a humidified box and freeze at 4°C overnight.

[0108] (14) Washing with primary antibody: After taking the tissue sections out of the 4℃ refrigerator, warm them again and wash them with PBS 3 times, 3 min each time.

[0109] (15) Labeling secondary antibody: Enzyme-labeled goat anti-mouse / rabbit IgG polymer was incubated at room temperature for 1 h, and the tissue samples were washed 3 times with PBS for 3 min each time.

[0110] (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.

[0111] (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) → Neutral resin sealing.

[0112] 3. Experimental Results

[0113] To demonstrate the protective effect of cyclosporine against intestinal radiation injury, a BrdU incorporation assay was conducted in a mouse model of intestinal radiation injury induced by whole-body irradiation with 12 Gy gamma rays. BrdU is a thymidine analogue that, like thymidine, can be incorporated into DNA synthesized by cells. By detecting the expression level of BrdU in mice receiving BrdU infusion, the cell proliferation status can be reflected.

[0114] Time-effect study of cyclosporine administration regimens: C57BL6 / J mice were divided into an irradiation control group and cyclosporine administration groups at different time points. The cyclosporine administration groups received intraperitoneal injections or oral administration of cyclosporine 30 mg / kg at 24 h, 12 h, 6 h, 2 h, and 0.5 h before (-) whole-body irradiation with 12 Gy γ-rays. The irradiation control group received an equal volume of physiological saline 2 h before irradiation. Mice were sacrificed 3.5 days after irradiation, and small intestine tissue sections were collected for pathological examination. One hour before sacrifice, all mice received 120 mg / kg of BrdU intraperitoneally. Immunohistochemistry was used to detect BrdU expression in mouse small intestinal crypt cells, and the proliferation differences of intestinal crypt cells among the groups were compared by counting the number of BrdU-positive crypt cells (BrdU+ cells > 5 were considered 1 crypt). Results are as follows: Figure 1As shown in A and 1B, intraperitoneal injection of cyclosporine 30 mg / kg before irradiation can increase the number of proliferative intestinal crypts; the optimal therapeutic effect is achieved with intraperitoneal injection 2 to 6 hours before irradiation. Figure 1 As shown in C and 1D, pre-irradiation administration of cyclosporine at 30 mg / kg via gavage also increased the number of proliferative intestinal crypts. Administration via gavage 24 to 12 hours before irradiation showed good efficacy. These experiments demonstrate that both intraperitoneal injection and gavage administration of cyclosporine are effective against radiation damage to intestinal crypts, and offer the advantage of a longer administration window.

[0115] Dose-response study of cyclosporine administration regimens: C57BL6 / J mice were divided into an irradiation control group and cyclosporine administration groups with different doses. The cyclosporine administration groups received intraperitoneal or oral administration of cyclosporine at doses of 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, and 30 mg / kg, respectively, 2 hours before total body irradiation with 12 Gy γ-rays. The irradiation control group received an equal volume of physiological saline 2 hours before irradiation. Mice were sacrificed 3.5 days after irradiation, and small intestine tissue sections were collected for pathological examination. One hour before sacrifice, all mice received an intraperitoneal BrdU dose of 120 mg / kg. Immunohistochemistry was used to detect BrdU expression in the small intestinal crypt cells of mice, and the results are as follows: Figure 2 As shown, intraperitoneal injection within 2 hours before irradiation ( Figure 2 A, 2B) or oral administration ( Figure 2 (C, 2D) Cyclosporine at doses of 1 to 30 mg / kg significantly increased the number of intestinal crypts during the proliferative phase. The efficacy of intraperitoneal injection of cyclosporine at 20 mg / kg was comparable to that at 30 mg / kg. Intraperitoneal injection of cyclosporine provided better protection against radiation damage to crypts than oral administration.

[0116] Example 2: Effect of a single injection of cyclosporine on the survival of mice with intestinal radiation injury

[0117] 1. Experimental Materials

[0118] 1.1 Laboratory Animals

[0119] C57BL / 6J male mice, 20-22 g, 6-8 weeks old, Beijing Huafukang Biotechnology Co., Ltd., China.

[0120] 1.2 Reagents and Materials

[0121] Cyclosporine Injection (Sandimin) 5ml: 250mg Novartis Pharmaceuticals Switzerland, saline solution Shijiazhuang No.4 Pharmaceutical Co., Ltd. China.

[0122] 2. Experimental Methods

[0123] 2.1 Animal husbandry

[0124] C57BL / 6J mice, 6-8 weeks old, weighing 20-22g, male, were purchased from SPF (Beijing) 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.

[0125] 2.2 Mouse grouping

[0126] Two intestinal radiation injury models were used to evaluate the efficacy of cyclosporine: ① a mouse model of survival after 12 Gy whole-body irradiation and ② a mouse model of survival after 15 Gy whole-body irradiation combined with bone marrow transplantation. Each model used 20 mice, which were divided into two groups of 10 mice each (control group and experimental group).

[0127] 2.3 Irradiation scheme

[0128] ① A mouse-containing box was placed 2.5 meters away from a gamma-ray source for irradiation, with a total dose of 12 Gy, to establish a mouse survival model of whole-body irradiation with 12 Gy; ② A mouse-containing box was placed 2.5 meters away from a gamma-ray source for irradiation, with a total dose of 15 Gy. Six hours after irradiation, all mice received 1×10 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.

[0129] 2.4 Dosing regimen

[0130] Cyclosporine injection (Sandimin) was purchased from Novartis Pharmaceuticals Ltd. in Switzerland. After being diluted with physiological saline, mice were intraperitoneally injected at a dose of 20 mg / kg 2 hours before the treatment. The control group mice were given the same volume of physiological saline.

[0131] 2.5 Mouse survival status

[0132] The survival and weight of mice were observed 30 days after irradiation.

[0133] 3. Experimental Results

[0134] The protective effect of prophylactic administration of cyclosporine was observed in a radiotherapy-induced intestinal injury model induced by whole-body irradiation of C57BL / 6J mice with 12 Gy gamma rays. Healthy C57BL / 6J mice were intraperitoneally injected with cyclosporine 20 mg / kg or the same volume of physiological saline 2 hours before irradiation. Ten mice were in each group. Survival and body weight were recorded 30 days after irradiation. Results are as follows: Figure 3As shown in Figure A, the saline-treated group experienced rapid weight loss after treatment, and all mice died within 7 days. The cyclosporine prophylactic treatment group showed slower weight loss. Figure 3 (B) Mice irradiated with a total dose of 12 Gy showed a statistically significant difference in average body weight compared to the control group 3 to 6 days post-irradiation, and all died by day 11. Survival analysis indicated that cyclosporine effectively prolonged the survival time of mice irradiated with a total dose of 12 Gy. To eliminate the interference of hematopoietic system damage in mice irradiated with a total dose of 15 Gy gamma rays on mouse survival after bone marrow transplantation, the effect of cyclosporine on the survival of mice irradiated with a lethal dose of 15 Gy gamma rays was observed. Figure 3 As shown in C, all control group mice died rapidly within 9 days after irradiation, while the survival rate of mice in the cyclosporine group (20 mg / kg intraperitoneally injected 2 hours before irradiation) was 80% 30 days after irradiation, and the surviving mice recovered to their pre-irradiation weight. Figure 3 (D) indicates that cyclosporine significantly promoted the survival of mice under 15 Gy whole-body irradiation combined with bone marrow transplantation.

[0135] Example 3: Prevention of apoptosis of intestinal crypt cells in mice induced by intraperitoneal injection of cyclosporine

[0136] 1. Experimental Materials

[0137] 1.1 Laboratory Animals

[0138] C57BL / 6J male mice, 20-22 g, 6-8 weeks old, Beijing Huafukang Biotechnology Co., Ltd., China.

[0139] 1.2 Reagents and Materials

[0140] Anti-Cleavd-Caspase-3 antibody, Cell Signal Technology (USA); TUNEL apoptosis detection kit, Beyotime Biotechnology (China).

[0141] The rest is the same as in Examples 1 and 2.

[0142] 2. Experimental Methods

[0143] 2.1 Animal husbandry

[0144] C57BL / 6J mice, 6-8 weeks old, weighing 20-22g, male, were purchased from SPF (Beijing) 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.

[0145] 2.2 Mouse grouping

[0146] A mouse model of intestinal radiation injury induced by whole-body irradiation with 12 Gy was used to evaluate the protective effect of intraperitoneal injection of cyclosporine 20 mg / kg 2 hours before irradiation against apoptosis of intestinal crypt cells induced by gamma ray irradiation. Twelve C57BL / 6J mice were divided into an irradiation control group and a cyclosporine 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.

[0147] 2.3 Dosing regimen

[0148] Cyclosporine injection (Sandimin) was purchased from Novartis Pharmaceuticals Ltd. in Switzerland. After being diluted with physiological saline, mice were injected intraperitoneally at a dose of 20 mg / kg 2 hours before treatment. The control group mice were given the same volume of physiological saline.

[0149] 2.4 Cleaved-caspase 3 Immunohistochemical Method

[0150] Same as Example 1

[0151] 2.5 TUNEL assay for intestinal cell apoptosis

[0152] (1) The steps of material collection, fixation, dehydration, embedding, sectioning, and dewaxing are as described above.

[0153] (2) Proteinase K permeabilization treatment: Draw a hydrophobic zone, add an appropriate amount of 20 μg / ml proteinase K without DNase, incubate at room temperature for 20 min, and wash the slices with PBS for 3 min × 3 times.

[0154] (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.

[0155] (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.

[0156] (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.

[0157] (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 slides with PBS for 3 min × 3 times.

[0158] (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-color development.

[0159] (8) Counterstaining and mounting are the same as before.

[0160] 3. Experimental Results

[0161] High-dose ionizing radiation induces rapid and widespread apoptosis in the intestine, especially in intestinal stem and progenitor cells, directly affecting the intestinal proliferative and regenerative capacity. To determine the effect of cyclosporine prophylaxis on intestinal cell apoptosis after high-dose radiation, Cleaved-Caspase-3 immunohistochemical staining and TUNEL apoptosis detection were performed. The results showed that after 12 Gy whole-body irradiation, the number of Cleaved-Caspase-3 positive and TUNEL positive apoptotic cells in the intestinal crypts of the cyclosporine prophylaxis group was significantly lower than that of the irradiation-only group at 6 h and 24 h post-irradiation. Figure 4 These results suggest that prophylactic administration of cyclosporine significantly inhibited apoptosis of intestinal crypt cells after radiation exposure.

[0162] Example 4: Verification of the radiation-protective effect of cyclosporine on small intestinal crypt stem cells.

[0163] 1. Experimental Materials

[0164] 1.1 Laboratory Animals

[0165] C57BL / 6J male mice, 20-22 g, 6-8 weeks old, Beijing Huafukang Biotechnology Co., Ltd., China.

[0166] 1.2 Reagents and Materials

[0167] Anti-Olfm4 antibody Cell Signal Technology, USA; anti-Lysozyme antibody Abcam, USA.

[0168] The rest is the same as in Example 1.

[0169] 2. Experimental Methods

[0170] 2.1 Animal husbandry

[0171] C57BL / 6J mice, 6-8 weeks old, weighing 20-22g, male, were purchased from SPF (Beijing) 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.

[0172] 2.2 Mouse grouping

[0173] A mouse model of intestinal radiation injury caused by 12 Gy whole-body irradiation was used to evaluate the protective effect of intraperitoneal injection of cyclosporine 20 mg / kg 2 hours before irradiation on radiation-damaged small intestinal crypt stem cells. Six C57BL / 6J mice were divided into an irradiation control group and a cyclosporine administration group. The intestines of the mice were dissected and pathologically examined 3.5 days after irradiation, with 3 mice in each group.

[0174] 2.3 Dosing regimen

[0175] Cyclosporine injection (Sandimin) was purchased from Novartis Pharmaceuticals Ltd. in Switzerland. After being diluted with physiological saline, mice were injected intraperitoneally at a dose of 20 mg / kg 2 hours before treatment. The control group mice were given the same volume of physiological saline.

[0176] 2.4 Olfm4 and Lysozyme Immunohistochemical Methods

[0177] Same as Example 1

[0178] 3. Experimental Results

[0179] Given the crucial role of small intestinal crypt stem cells in the regeneration and repair of the intestine after radiation injury, this study investigated the effects of cyclosporine on small intestinal crypt stem cells after radiation. Firstly, using Olfm4 immunohistochemistry, it was found that mice 3.5 days after total body irradiation with 12 Gy experienced a significant loss of Olfm4+ stem cells in the intestinal crypts, while the loss of Olfm4+ cells was significantly reduced in the cyclosporine-treated group. Figure 5 A, 5B). Secondly, Lysozyme immunohistochemistry was used to observe Paneth cells, supporting cells of the small intestinal crypt stem cells. It was found that Paneth cells were also severely damaged after radiation, while cyclosporine administration significantly inhibited the loss of Paneth cells. Figure 5 (C, 5D). The above results suggest that prophylactic administration of cyclosporine can effectively improve the survival of ISCs after high-dose ionizing radiation, thereby promoting the regeneration and repair of intestinal crypts.

[0180] 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 cyclosporin in the preparation of a medicament for preventing / treating crypt cell apoptosis caused by intestinal acute radiation sickness.

2. Use according to claim 1, characterized in that, The administration of the cyclosporin includes intraperitoneal injection, subcutaneous injection or oral administration.

3. Use according to claim 2, characterized in that, The administration of the cyclosporin is selected from intraperitoneal injection.

4. Use according to claim 1, characterized in that, The medicament further comprises a pharmaceutically acceptable carrier.

5. Use according to claim 4, characterized in that, The pharmaceutically acceptable carrier includes one or more of diluents, binders, surface active agents, wetting agents, adsorptive carriers, lubricants, fillers, disintegrants.

6. Use according to claim 1, characterized in that, The dosage form of the medicament includes granules, powders, tablets, capsules, syrups, suppositories, injections, elixirs.

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