CN116212020A

CN116212020A - Application of JAK inhibitor in medicine for preventing and treating radiation-induced bone marrow suppression and intestinal injury

Info

Publication number: CN116212020A
Application number: CN202211570683.5A
Authority: CN
Inventors: 李德冠; 岳同鹏; 李文璇; 王欣悦; 霍启东; 董辉; 赵瑜; 张俊伶; 路璐; 贺欣; 武欣
Original assignee: Institute of Radiation Medicine of CAMMS
Current assignee: Institute of Radiation Medicine of CAMMS
Priority date: 2022-12-09
Filing date: 2022-12-09
Publication date: 2023-06-06

Abstract

The invention discloses a new application method of a JAK inhibitor as a medicament for preventing or treating radiation-induced bone marrow suppression and intestinal injury. The modern pharmacological experiments and biological experiments prove that the administration of a certain dose of JAK inhibitor by mice gastric lavage has obvious protection effect on bone marrow suppression and intestinal damage induced by radiation, and the therapeutic administration is suggested to be capable of preventing and treating the damage of bone marrow and intestinal tract induced by radiation, and can be used as a medicament in the aspects of protecting living body damage caused by radioactive substance radiation, nuclear power station leakage, space ionizing radiation and tumor radiotherapy.

Description

Application of JAK inhibitor in medicine for preventing and treating radiation-induced bone marrow suppression and intestinal injury

Technical Field

The invention belongs to the technical field of medicines, and relates to application of a JAK inhibitor in the pharmaceutical field. More specifically, the application of JAK inhibitor in preparing medicines for preventing and treating radiation-induced bone marrow depression (bone marrow suppressive death) or intestinal injury.

Background

External physical, chemical and biological harmful factors can cause damage to the body through various effects, wherein ionizing radiation can generate DNA damage, apoptosis and oxidative stress, and the damage to the body is usually more serious and fatal. Bone marrow hematopoiesis and normal execution of the intestinal tract have a key role in the maintenance of life and health of the body, together with their high sensitivity to ionizing radiation damage, and are therefore important in research applications for radiation protection and treatment. With the widespread use of radioactive substances and the increasing number of people receiving radiotherapy, the development of effective radioprotections and treatments, and drugs for reducing side effects of radiotherapy, particularly drugs for reducing radiation-induced myelosuppression, is becoming increasingly urgent and important.

Radiation-induced myelosuppression manifests itself in reduced myeloproliferative capacity, reduced numbers of peripheral blood cells, particularly white blood cells, reduced adaptive immunity, and the like. Aiming at bone marrow transplantation, at present, growth factor medicines and compounds or traditional Chinese medicine preparations for enhancing nutrition state and promoting hematopoietic stem cell proliferation are mainly adopted clinically, and antioxidant injury is assisted. Radiation-induced intestinal damage is in early death, radiation enteritis, etc. The combined use of multiple drugs inevitably causes an increase in the treatment cost and the generation of multiple side effects, so that the existing treatment modes have limitations, and treatment scheme optimization measures such as drug application with better curative effects are required.

The Janus kinase (JAK) family is a family of signal molecules that link up intracellular cytokine receptors, and members are non-receptor tyrosine kinases that were found during the study of IFN signaling pathways. Mammalian JAKs comprise four total: JAK1, JAK2, JAK3 and TYK2. Four JAKs share four domains: (1) FERM domain, mediating interactions with transmembrane receptors and involved in activation of kinase activity; (2) SH2 domains, which mediate interactions with receptors; (3) a pseudo kinase domain that modulates kinase activity; (4) a kinase domain, which exerts tyrosine kinase activity. JAK is capable of recognizing receptors for more than 50 cytokines, including the transmembrane receptors for Interferons (IFNs), interleukins (ILs), colony Stimulating Factors (CSF), and various hormones, and exerting transduction and regulation effects through the JAK-STAT signaling pathway. When cytokine ligands bind to the receptor, receptor dimerization causes phosphorylation activation of JAKs, which exert tyrosine kinase activity to phosphorylate the receptor and form STAT binding sites, which in turn are released by JAK phosphorylation activation, followed by nuclear activation of gene transcription.

JAK-STAT signaling pathway is considered as one of the central regulation pathways of cellular functions, plays a great role in regulating the immune system of the organism, and is involved in the differentiation and maturation of immune cells, the exertion of hematopoietic stem cell functions and other processes. JAK is therefore also considered a key target for immune, inflammatory diseases, and JAK inhibitors have great potential in the treatment of hematopoietic, immune system diseases. To date, 8 JAK inhibitors have been marketed worldwide, including 5 generation JAK inhibitors (broad JAK inhibitors): ruxolitinib (Ruxolitinib), tofacitinib (Tofacitinib), baratinib (Baricitinib), piracetinib (Peficitinib), dyotinib (delgcitinib), and the type 4 second generation JAK inhibitors (selective JAK inhibitors): phenanthrene Zhuo Tini (Fedratinib), wu Pati Ni (Uppadacritinib), non-gotinib (Filgotinib), and Albaxitinib (Abrocitinib). Wherein tofacitinib, barytatinib, ponyinib, wu Pati ni and abb-xitinib have been batched in China. These JAK inhibitors are mainly used for the treatment of rheumatoid arthritis, atopic dermatitis, ulcerative colitis, psoriatic arthritis myelofibrosis, graft versus host disease and the like.

Although JAK inhibitors have been used as a common drug for the clinical treatment of immune system disorders, their use in radioprotection and therapy has not been reported. Related studies have found that JAK is critical for bone marrow cell homeostasis, including stem cell maintenance, hematopoiesis and immune cell development. Based on the current state of the deficiency of the existing medicaments for preventing and treating radiation injury, especially bone marrow suppression, the inventor conducts research on the aspect of protecting radiation injury by using a JAK inhibitor, discovers that the JAK inhibitor has obvious treatment effect on radiation injury in experiments for the first time, particularly in the aspects of treating radiation-induced bone marrow suppression and intestinal injury, conducts further experiments based on the discoveries and completes the invention.

Disclosure of Invention

The invention aims to find a medicine capable of preventing and treating ionizing radiation injury.

The invention aims at providing the application of a JAK inhibitor in preparing medicaments for preventing or treating radiation-induced bone marrow transplantation, and also aims at providing the application of the JAK inhibitor in radiotherapy protection, in particular to the preparation of a composition by mixing with nutrients and other medicaments such as medicaments for promoting bone marrow cell proliferation, so as to reduce toxic and side effects caused by radiotherapy.

In order to achieve the aim of the invention, the following technical scheme is designed:

use of a JAK inhibitor in the preparation of a medicament for preventing ionising radiation induced myelosuppression and intestinal damage. Such ionizing radiation-induced myelosuppression and intestinal damage include, but are not limited to: tumor patients receiving radiation therapy, radiation exposure staff, and staff exposed to accidental radiation isotopes.

The bone marrow suppression of the invention comprises peripheral blood leucopenia, myelodysplasia or aplastic anemia. Intestinal tract injury is mainly radiation enteritis.

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

The invention further discloses a composition containing the JAK inhibitor and other nutrient substances, a medicament for promoting bone marrow cell proliferation or an anti-tumor medicament, wherein the composition comprises a therapeutic medicament, a preventive medicament, a health food and the like in various clinical formulations. The JAK inhibitor is mixed with other nutrient substances and medicines for promoting bone marrow cell proliferation to prepare the composition.

Other nutrients described in the present invention include: other amino acid nutrient solution, vitamins, glucose, fat milk, ions, etc.

The medicine for promoting bone marrow cell proliferation includes: various Chinese medicinal preparations such as polysaccharides (fructus Lycii, tremella, radix Codonopsis, radix astragali, and Ginseng radix polysaccharide), alkaloids (berberine, radix Sophorae Flavescentis total alkaloids, etc.), saponins (ginsenoside, astragaloside, etc.), and other preparations such as LIUWEIDIHUANG oral liquid; human granulocyte macrophage colony stimulating factor (rHuGM-CSF), L-Bai Xin, inosine, levamisole, three oral liquid, kang Lilong, etc.

The JAK inhibitor is mixed with pharmaceutically acceptable pharmaceutical excipients to prepare various pharmaceutical compositions, which comprise: tablets, capsules, granules, pills, drop pills, pre-emulsions, microemulsions, suspensions, syrups, various enteric or injectable formulations and the like. Each formulation may be prepared according to conventional techniques. Pharmaceutically acceptable excipients can be added in the preparation of the medicament, wherein the pharmaceutically acceptable excipients comprise conventional diluents, fillers (such as mannitol, lactose and polyethylene glycol), binders (starch and microcrystalline cellulose), disintegrants (such as carboxymethyl cellulose and low-substituted hydroxypropyl cellulose), lubricants (such as talcum powder and magnesium stearate), wetting agents (such as propylene glycol and ethanol), stabilizers (EDTA-2 Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, ethanolamine and sodium bicarbonate) and the like.

When the JAK inhibitor is mixed with pharmaceutically acceptable pharmaceutical excipients to form a pharmaceutical composition, the amount of the JAK inhibitor as an active ingredient contained in the composition can be specifically applied according to the illness state of a patient and the diagnosis situation of a doctor, the amount or concentration of the JAK inhibitor is regulated within a wider range, and the amount of the JAK inhibitor is usually in the range of 0.5-90% by weight of the composition. Another preferred range is 0.5% -70%. Yet another preferred range is 3% -50%.

The JAK inhibitor is mixed with other nutrient substances and medicines for promoting bone marrow cell proliferation to prepare a pharmaceutical composition or the JAK inhibitor is used as a supporting medicine to be matched with an anti-tumor medicine. Microparticles or microspheres are typically prepared using conventional techniques, in combination with a pharmaceutically acceptable solid or liquid carrier, and optionally in combination with pharmaceutically acceptable adjuvants and excipients. Solid dosage forms include tablets, dispersible granules, capsules, sustained release tablets, sustained release pellets, and the like. The solid carrier may be at least one substance which may act as a diluent, a flavoring agent, a solubilizer, a lubricant, a suspending agent, a binder, a disintegrant, and a coating agent. Inert solid carriers include magnesium phosphate, magnesium stearate, powdered sugar, lactose, pectin, propylene glycol, polysorbate 80, dextrin, starch, gelatin, cellulosic materials such as methylcellulose, microcrystalline cellulose, low melting point waxes, polyethylene glycol, mannitol, cocoa butter, and the like. Liquid dosage forms include solvents, suspensions such as injections, powder injections, and the like.

The test content of the invention for radiation induced bone marrow suppression mainly comprises the following aspects:

jak inhibitor normal state bone marrow cells are nontoxic.

Jak inhibitors can increase survival in mice receiving systemic exposure.

Jak inhibitors reduce ROS levels in bone marrow cells of radiation-exposed mice.

Jak inhibitors reduced the level of bone marrow apoptosis in radiation-exposed mice.

The invention selects a generation of JAK inhibitor tofacitinib for relevant research, and the experimental condition of the JAK inhibitor in preventing or treating the effect of radiation and chemical poison induced bone marrow suppression is further described through biological experiments.

Specific experimental method

1. Whole body irradiated mice survival assay

C57BL/6 mice were randomly grouped, 10 mice/group, tofacitinib was given by gavage (20 mg/kg/d) 1h before the mice were irradiated, and after the whole body was irradiated with 7.2Gy, the mice were weighed periodically, given by gavage daily and observed for death.

2. Bone marrow mononuclear cell isolation

The femur of the mouse is aseptically taken, bone marrow is flushed with Hunks solution containing 2% FCS, mononuclear cell suspension is prepared, washed, and the required cell concentration is adjusted by counting for later use.

3. Cell viability assay

The mouse bone marrow cells were isolated and cultured in 96-well plates, 100. Mu.l of mononuclear cell suspension was added to each well, 100ul of treatment drug was added as designed, and incubated in an incubator at 37℃for 18 hours after 1Gy irradiation. Taking out the culture plate, standing at room temperature, adding bioluminescence reagent cell-titer 20ul, shaking, mixing, transferring into black assay plate, and GloMax ^TM The luminescence detector uses Promega self-contained detection program Cell-timer Protocol for detection. And automatically generating Excel data according to the detection result.

4. Cellular active oxygen assay

The mouse bone marrow cells were isolated and cultured in 96-well plates, 100. Mu.l of mononuclear cell suspension was added to each well, 100ul of treatment drug was added as designed, and incubated in an incubator at 37℃for 18 hours after 1Gy irradiation. The plate was removed, allowed to stand at room temperature, and the prepared DFCH reagent (500. Mu.l/sample) was added. Cells were resuspended after washing with PBS and cellular reactive oxygen levels were detected by flow cytometry.

5. Apoptosis detection

The mouse bone marrow cells were isolated and cultured in 96-well plates, 100. Mu.l of mononuclear cell suspension was added to each well, 100ul of treatment drug was added as designed, and incubated in an incubator at 37℃for 18 hours after 1Gy irradiation. Taking out the culture plate, standing at room temperature, adding an Annexin V/PI apoptosis detection kit binding buffer (200 ul/tube), uniformly mixing, sequentially adding 1ul of Annexin V-FITC and 2ul of PI per well, and incubating at room temperature in a dark place for 15min. Flow cytometry was then used to detect apoptosis levels.

Drawings

FIG. 1A shows the nontoxic effect of the JAK inhibitor Tofacitinib on normal bone marrow cells;

FIG. 2 shows that the JAK inhibitor Tofacitinib increases survival in 7.2Gy whole body irradiated mice;

FIG. 3 the JAK inhibitor Tofacitinib reduces ROS levels in bone marrow cells of radiation-exposed mice;

figure 4 JAK inhibitor Tofacitinib reduced the level of bone marrow apoptosis in radiation-exposed mice.

Detailed Description

The invention is further described below with reference to examples. These examples are merely exemplary of the present invention, but the present invention is not limited thereto.

Detailed Description

To more fully explain the practice of the invention, the following formulation examples are provided. These examples are merely illustrative and are not intended to limit the scope of the invention.

Example 1

JAK inhibitor 100mg, lactose 50mg, microcrystalline cellulose 80mg, starch 50mg, hydroxymethyl cellulose 40mg, magnesium stearate 5mg. Sieving active ingredients, lactose, starch and microcrystalline cellulose with 100 mesh sieve, mixing, adding 2% aqueous solution of hydroxymethyl cellulose into the above mixed powder, mixing, sieving with 20 mesh sieve to obtain soft material, drying at 45-55deg.C, adding carboxymethyl starch sodium and magnesium stearate into the above dried granule, and tabletting.

Example 2

100mg of JAK inhibitor, 500mg of levant white, 4g (lactose-microcrystalline cellulose 5:1) and 1% of magnesium stearate are added, and the mixture is granulated with 70% ethanol and tableted.

Example 3

10g of JAK inhibitor, 60g of mannitol, adding 1000ml of injection water for dissolution, supplementing the injection water to 2000ml, adding a proper amount of active carbon for removing a heat source, filtering with a 0.2um microporous filter membrane, filling, and freeze-drying to prepare 2000 freeze-dried powder injection. Specification of: 70 mg/branch for intravenous injection.

Example 4

The JAK inhibitor 10g, dextrin 10g and lactose 30g are added, granulated, dried and encapsulated by 60% ethanol to prepare 1000 capsules. Specification of: 50 mg/grain.

In view of the foregoing, the present invention is not limited to the embodiments, and other embodiments can be easily proposed by those skilled in the art within the scope of the technical teaching of the present invention, but such embodiments are included in the scope of the present invention.

Claims

Use of a jak inhibitor for the preparation of a medicament for the treatment or prevention of radiation-induced myelosuppression, intestinal suppression.
2. The use according to claim 1, characterized in that the intestinal inhibition comprises intestinal damage.
3. The use of claim 1, wherein the radiation comprises radiation in radiotherapy, work environment radiation exposure, accidental radiation radioisotope exposure.
4. The JAK inhibitor of claim 1 comprising tofacitinib.
5. The use according to any one of claims 1 to 4, wherein said myelosuppression comprises peripheral blood leucopenia, myelodysplastic failure, aplastic anemia.
6. The use according to any one of claims 1 to 4, characterized in that a therapeutically effective amount of the JAK inhibitor is admixed with pharmaceutically acceptable pharmaceutical excipients to form a composition.
7. The use according to any one of claims 1 to 4, characterized in that it is a composition comprising a JAK inhibitor in combination with other nutrients, a drug for promoting bone marrow cell proliferation or a JAK inhibitor as a supporting drug in combination with an antitumor drug.
8. The use according to any one of claims 6 and 7, wherein the composition is formulated into tablets, capsules, granules, pills, drop pills, pre-emulsions, suspensions, syrups, various enteric preparations, injections.