Application of JAK inhibitor in drugs for preventing and treating radiation-induced myelosuppression and intestinal injury

Abstract

The present invention discloses a new application method of JAK inhibitors as drugs for preventing or treating radiation-induced myelosuppression and intestinal injury. Through modern pharmacological experiments and biological experiments, it is proved that intragastric administration of a certain dose of JAK inhibitors to mice has an obvious protective effect on radiation-induced myelosuppression and intestinal injury, suggesting that therapeutic administration can prevent and treat radiation-induced damage to the bone marrow and intestine, and can be used as a drug for protecting living organisms from damage caused by radioactive substance radiation, nuclear power plant leakage, space ionizing radiation, and tumor radiotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to the application of JAK inhibitors in the pharmaceutical field. More specifically, it relates to the application of JAK inhibitors in the preparation of drugs for preventing and treating radiation-induced myelosuppression (myelosuppressive death) or intestinal injury. Background Art

[0002] External physical, chemical, and biological harmful factors can cause damage to the body through various effects. Among them, ionizing radiation can cause DNA damage, apoptosis, and oxidative stress, and the damage to the body is usually more severe and fatal. The normal function of bone marrow hematopoiesis and the intestine plays a key role in maintaining the life and health status of the body. In addition, due to their high sensitivity to ionizing radiation damage, they have become important contents in the research and application of radiation protection and treatment. With the wide application of radioactive substances and the increase in the number of people receiving radiotherapy, it is becoming increasingly urgent and important to develop effective radiation protection and treatment drugs, especially drugs that can reduce radiation-induced myelosuppression, to reduce the side effects of radiotherapy.

[0003] Radiation-induced myelosuppression is manifested as a decrease in bone marrow proliferation ability, a decrease in the number of peripheral blood cells, especially white blood cells, and a weakening of adaptive immunity. For bone marrow transplantation, currently, growth factor drugs and enhanced nutritional status are mostly used clinically to promote the proliferation of hematopoietic stem cells, supplemented with compounds or traditional Chinese medicine preparations that can resist oxidative damage. Radiation-induced intestinal injury includes early death, radiation enteritis, etc. The combined use of multiple drugs inevitably leads to an increase in treatment costs and the occurrence of various side effects, making the current treatment method have limitations and requiring optimization measures such as the application of drugs with better efficacy.

[0004] The Janus kinase (JAK) family is a family of signal molecules that connect intracellularly to cytokine receptors. Its members are non-receptor tyrosine kinases, which were discovered during the study of the IFN signaling pathway. Mammals have a total of four JAKs: JAK1, JAK2, JAK3, and TYK2. These four JAKs share four domains: ① the FERM domain, which mediates interactions with transmembrane receptors and participates in the activation of kinase activity; ② the SH2 domain, which mediates interactions with receptors; ③ the pseudokinase domain, which regulates kinase activity; ④ the kinase domain, which exhibits tyrosine kinase activity. JAK can recognize the receptors of more than 50 cytokines, including interferon (IFN), interleukin (IL), colony-stimulating factor (CSF), and the transmembrane receptors of various hormones, and plays a transduction and regulatory role through the JAK-STAT signaling pathway. When a cytokine ligand binds to a receptor, receptor dimerization causes the phosphorylation and activation of JAK. The activated JAK exhibits tyrosine kinase activity to phosphorylate the receptor and form a STAT binding site. The recruited STAT is then phosphorylated and activated by JAK and dissociates, and subsequently enters the nucleus to play a regulatory role in gene transcription.

[0005] The JAK-STAT signaling pathway is considered to be one of the central regulatory pathways of cell functions and plays a significant role in the regulation of the body's immune system. It is involved in processes such as the differentiation and maturation of immune cells and the function of hematopoietic stem cells. Therefore, JAK is also considered a key target for immune and inflammatory diseases, and JAK inhibitors have great potential in the treatment of hematopoietic and immune system diseases. To date, 8 JAK inhibitors have been approved for marketing globally, including 5 first-generation JAK inhibitors (broad JAK inhibitors): Ruxolitinib, Tofacitinib, Baricitinib, Peficitinib, Delgocitinib, and 4 second-generation JAK inhibitors (selective JAK inhibitors): Fedratinib, Upadacitinib, Filgotinib, Abrocitinib. Among them, Tofacitinib, Baricitinib, Ruxolitinib, Upadacitinib, and Abrocitinib have been approved in China. These JAK inhibitors are mainly used to treat rheumatoid arthritis, atopic dermatitis, ulcerative colitis, psoriatic arthritis, myelofibrosis, and graft-versus-host disease, etc.

[0006] Although JAK inhibitors have been commonly used as clinical drugs for treating immune system disorders, their application in radiation protection and treatment has not been reported. Related studies have found that JAK is crucial for the homeostasis of bone marrow cells, including the maintenance of stem cells, hematopoiesis, and the development of immune cells. Based on the current insufficient status of drugs for preventing and treating radiation injuries, especially for bone marrow suppression, the inventor of the present invention has carried out research on the radiation injury protection of JAK inhibitors and for the first time found in experiments that JAK inhibitors have an obvious therapeutic effect on radiation injuries, especially in the treatment of radiation-induced bone marrow suppression and intestinal injuries. Further experiments were conducted based on the above findings to complete the present invention. Summary of the Invention

[0007] The present invention aims to find drugs that can prevent and treat ionizing radiation injuries.

[0008] An object of the present invention is, on the one hand, to provide the use of JAK inhibitors in the preparation of drugs for preventing or treating radiation-induced bone marrow transplantation, and on the other hand, to provide the use of JAK inhibitors in radiotherapy protection, specifically in combination with nutrients and other drugs such as drugs that promote bone marrow cell proliferation to form a composition, reducing the toxic and side effects brought by radiotherapy.

[0009] To achieve the above invention objectives, the following technical solutions are designed:

[0010] The use of a JAK inhibitor in the preparation of drugs for preventing ionizing radiation-induced bone marrow suppression and intestinal injuries. The ionizing radiation-induced bone marrow suppression and intestinal injuries include, but are not limited to: cancer patients receiving radiotherapy, radiation-exposed workers, and personnel accidentally exposed to radioactive isotopes.

[0011] The bone marrow suppression described in the present invention includes a decrease in peripheral blood white blood cells, myelodysplasia, or aplastic anemia. Intestinal injury is mainly radiation enteritis.

[0012] The present invention further discloses a pharmaceutical composition containing a JAK inhibitor, which is prepared by mixing a therapeutically effective amount of the JAK inhibitor with a pharmaceutically acceptable pharmaceutical excipient.

[0013] The present invention further discloses a composition containing a JAK inhibitor and other nutrients, drugs that promote bone marrow cell proliferation, or anti-tumor drugs, including therapeutic drugs, preventive drugs, and health foods in various clinical dosage forms, etc. It is prepared by mixing the JAK inhibitor with other nutrients and drugs that promote bone marrow cell proliferation.

[0014] The other nutrients described in the present invention include: other amino acid nutrient solutions, vitamins, glucose, fat emulsion, ions, etc.

[0015] The drugs for promoting the proliferation of bone marrow cells according to the present invention include: various traditional Chinese medicine preparations such as polysaccharides (wolfberry, tremella, codonopsis pilosula, astragalus membranaceus, ginseng polysaccharide, etc.), alkaloids (berberine, total alkaloids of sophora flavescens, etc.), saponins (ginsenoside, astragaloside, etc.), and other traditional Chinese medicine preparations for increasing white blood cells such as Liuwei Dihuang Oral Liquid; human granulocyte macrophage colony-stimulating factor (rHuGM-CSF), Shengbaixīn, inosine, levamisole, Sanzhu Oral Liquid, stanozolol, etc.

[0016] The JAK inhibitors according to the present invention are mixed with pharmaceutically acceptable pharmaceutical excipients to form various pharmaceutical compositions, including: tablets, capsules, granules, pills, dripping pills, pre-emulsions, microemulsions, suspensions, syrups, various enteric preparations or injection preparations, etc. Each preparation can be prepared according to conventional processes. Pharmaceutically acceptable excipients can be added when preparing into medicaments. The pharmaceutically acceptable excipients include conventional diluents, fillers (such as mannitol, lactose, polyethylene glycol) in the preparation, binders (starch, microcrystalline cellulose), disintegrants (such as carboxymethyl cellulose, low-substituted hydroxypropyl cellulose), lubricants (such as talc powder, magnesium stearate), wetting agents (such as propylene glycol, ethanol), stabilizers (EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, ethanolamine, sodium bicarbonate), etc.

[0017] When the JAK inhibitor is mixed with pharmaceutically acceptable pharmaceutical excipients to form a pharmaceutical composition, the amount of the active ingredient JAK inhibitor contained in the composition can be specifically applied according to the patient's condition and the doctor's diagnosis. The amount or concentration of the JAK inhibitor used is adjusted within a relatively wide range. Generally, the amount range of the JAK inhibitor is 0.5% - 90% (by weight) of the composition. Another preferred range is 0.5% - 70%. Still another preferred range is 3% - 50%.

[0018] The JAK inhibitors according to the present invention are mixed with other nutrients and drugs for promoting the proliferation of bone marrow cells to form a pharmaceutical composition, or the JAK inhibitor is used as a supporting drug in combination with anti-tumor drugs. Generally, conventional techniques are used, combined with pharmaceutically acceptable solid or liquid carriers, and optionally combined with pharmaceutically acceptable adjuvants and excipients to prepare into microparticles or microspheres. Solid dosage forms include tablets, dispersible granules, capsules, sustained-release tablets, sustained-release pellets, etc. The solid carrier can be at least one substance, which can act as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, disintegrant, and coating agent. Inert solid carriers include magnesium phosphate, magnesium stearate, powdered sugar, lactose, pectin, propylene glycol, polysorbate 80, dextrin, starch, gelatin, cellulose substances such as methyl cellulose, microcrystalline cellulose, low-melting paraffin, polyethylene glycol, mannitol, cocoa butter, etc. Liquid dosage forms include solvents, suspensions such as injections, lyophilized powder for injections, etc.

[0019] The experimental contents of the present invention on radiation-induced bone marrow suppression mainly include the following aspects:

[0020] 1.JAK inhibitors have no toxic effect on bone marrow cells in normal state.

[0021] 2.JAK inhibitors can improve the survival rate of mice receiving whole-body irradiation.

[0022] 3.JAK inhibitors reduced ROS levels in bone marrow cells of radiation-exposed mice.

[0023] 4.JAK inhibitors reduced the level of apoptosis in bone marrow cells of radiation-exposed mice.

[0024] The present invention uses the first-generation pan-JAK inhibitor tofacitinib for relevant research. The following biological experiments further illustrate the experimental results of JAK inhibitors in preventing or treating radiation- and chemical-induced bone marrow suppression.

[0025] Specific experimental methods

[0026] 1. Determination of the survival rate of mice after whole-body irradiation

[0027] C57BL / 6 mice were randomly divided into groups, 10 mice / group. Tofacitinib was administered by oral gavage (20 mg / kg / d) 1 h before irradiation. After whole-body irradiation of 7.2 Gy, the mice were weighed regularly, and the mice were administered by oral gavage daily and the death of the mice was observed.

[0028] 2. Bone marrow mononuclear cell isolation

[0029] The femur of the mouse was aseptically removed, and the bone marrow was flushed with Hunks solution containing 2% FCS to prepare a mononuclear cell suspension, which was then washed, counted, and adjusted to the required cell concentration for later use.

[0030] 3. Cell Viability Assay

[0031] Mouse bone marrow cells were isolated and cultured in a 96-well plate. 100 μl of mononuclear cell suspension was added to each well. 100 μl of treatment drugs were added as designed. After irradiation with 1 Gy, the plates were incubated in a 37°C incubator for 18 h. The plates were removed and placed at room temperature. 20 μl of the bioluminescent reagent cell-titer was added. After oscillation and mixing, the plates were transferred to a black assay plate. GloMax TM The luminescence detector uses Promega's own detection program Cell-titer Protocol for detection. The detection results automatically generate Excel data.

[0032] 4. Determination of Cellular Reactive Oxygen Species

[0033] Isolate mouse bone marrow cells and culture them in 96-well plates. Add 100 μl of mononuclear cell suspension to each well, add 100 μl of the treatment drug according to the design, irradiate with 1 Gy, and incubate in a 37 °C incubator for 18 h. Take out the culture plates, place them at room temperature, and add the prepared DFCH reagent (500 μl / sample). After washing with PBS, resuspend the cells and detect the intracellular reactive oxygen species level by flow cytometry.

[0034] 5. Detection of cell apoptosis

[0035] Isolate mouse bone marrow cells and culture them in 96-well plates. Add 100 μl of mononuclear cell suspension to each well, add 100 μl of the treatment drug according to the design, irradiate with 1 Gy, and incubate in a 37 °C incubator for 18 h. Take out the culture plates, place them at room temperature, add Annexin V / PI apoptosis detection kit binding buffer (200 μl / tube), mix well, then add 1 μl of Annexin V-FITC and 2 μl of PI to each well in sequence, and incubate at room temperature in the dark for 15 min. Subsequently, detect the cell apoptosis level by flow cytometry. Description of the drawings

[0036] Figure 1 The JAK inhibitor tofacitinib has no toxic effect on bone marrow cells in the normal state;

[0037] Figure 2 The JAK inhibitor tofacitinib increases the survival rate of mice irradiated with 7.2 Gy of total body irradiation;

[0038] Figure 3 The JAK inhibitor tofacitinib reduces the ROS level in bone marrow cells of irradiated mice;

[0039] Figure 4 The JAK inhibitor tofacitinib reduces the apoptosis level of bone marrow cells in irradiated mice. Detailed implementation manners

[0040] The following further describes the present invention in conjunction with embodiments. These embodiments are only typical descriptions of the present invention, but the present invention is not limited thereto. Detailed implementation manners

[0042] To more fully explain the implementation of the present invention, the following formulation embodiments are provided. These embodiments are only for explanation and do not limit the scope of the present invention.

[0043] Example 1

[0044] 100 mg of JAK inhibitor, 50 mg of lactose, 80 mg of microcrystalline cellulose, 50 mg of starch, 40 mg of hydroxypropyl methylcellulose, and 5 mg of magnesium stearate. The active ingredient, lactose, starch, and microcrystalline cellulose are passed through a 100-mesh sieve and thoroughly mixed. An aqueous solution of 2% hydroxypropyl methylcellulose is added to the above mixed powder and mixed, and then passed through a 20-mesh sieve to make soft materials. The obtained wet granules are dried at 45 - 55 °C. Sodium carboxymethyl starch and magnesium stearate are added to the above dried granules and pressed into tablets.

[0045] Example 2

[0046] 100 mg of JAK inhibitor, 500 mg of leucogen, 4 g (lactose - microcrystalline cellulose 5:1), 1% of magnesium stearate. Granulate with 70% ethanol and press into tablets to obtain.

[0047] Example 3

[0048] 10 g of JAK inhibitor, 60 g of mannitol, dissolved in 1000 ml of injection water, make up to 2000 ml with injection water, add appropriate amount of activated carbon to remove pyrogen, filter through a 0.2 - μm microporous membrane, fill, and freeze-dry to obtain 2000 vials of freeze-dried powder injections. Specification: 70 mg / vial, for intravenous injection.

[0049] Example 4

[0050] 10 g of JAK inhibitor, add 10 g of dextrin and 30 g of lactose, granulate with 60% ethanol, dry, and fill into capsules to obtain 1000 capsules. Specification: 50 mg / capsule.

[0051] In summary, the content of the present invention is not limited to the embodiments. Those with knowledge in the same field can easily propose other embodiments within the guiding ideology of the technical solution of the present invention, but such embodiments are all included within the scope of the present invention.

Claims

1. Use of tofacitinib in the preparation of a medicament for treating or preventing radiation-induced myelosuppression.

2. The use according to claim 1, wherein the radiation includes radiation in radiotherapy, radiation exposure in the working environment, and accidental exposure to radioactive isotopes.

3. The use according to any one of claims 1-2, characterized in that the myelosuppression includes a decrease in peripheral blood white blood cells, myelodysplasia, and aplastic anemia.

4. The use according to any one of claims 1-2, characterized in that a therapeutically effective amount of tofacitinib is mixed with a pharmaceutically acceptable excipient to form a composition.

5. The use according to any one of claims 1-2, characterized in that tofacitinib is mixed with nutrients and drugs that promote the proliferation of bone marrow cells to form a composition, or tofacitinib is used as a supportive drug in combination with anti-tumor drugs.

6. The use according to claim 4, characterized in that the composition is made into tablets, capsules, granules, pills, suspensions, syrups, various enteric preparations, and injections.

7. The use according to claim 6, characterized in that the pills include dripping pills.

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