Application of sodium-glucose cotransporter 2 inhibitors in the prevention and treatment of radiation-induced bone marrow suppression

Abstract

The present invention belongs to the field of medical technology, and relates to the application of sodium-glucose cotransporter 2 inhibitors in the field of pharmacy. More specifically, it relates to the application of sodium-glucose cotransporter 2 inhibitors as drugs for preventing and treating radiation-induced bone marrow suppression.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the application of sodium-glucose co-transporter 2 inhibitors in the pharmaceutical field, and more specifically to the application of sodium-glucose co-transporter 2 inhibitors as drugs for preventing and treating radiation-induced bone marrow suppression. Background of the Invention

[0002] Nuclear technology has played an important role in the development of the national economy, such as energy, aerospace, and medicine. However, nuclear terror, nuclear leakage, and loss of radioactive sources occur from time to time, which seriously threaten human life and health. There is still a lack of medical cures for radiation damage in clinical practice. The hematopoietic system is composed of bone marrow hematopoietic stem cells and hematopoietic cells at different developmental stages. It is sensitive to radiation and will be inhibited to varying degrees after being damaged, which is manifested as decreased bone marrow activity, increased reactive oxygen species, and DNA damage, which reduces the patient's quality of life and even endangers his life.

[0003] In recent decades, the research on radiation protection drugs has become a hot topic at home and abroad. The process of developing new drugs for radiation prevention and treatment is complicated, and the safety needs to be verified clinically several times. At present, the clinical treatment of bone marrow suppression is mostly to strengthen nutrition or give various growth factors to increase the proliferation of hematopoietic cells. In addition, some compounds or traditional Chinese medicine preparations with anti-radiation damage effects are used. There are many types of drugs, most of which are used in combination. The efficacy is not very certain when evaluated alone, and various side effects may occur.

[0004] Sodium-glucose co-transporter (SGLT) is a family of active glucose transporters with a molecular weight of 73kd. There are two main subtypes: SGLT-1 and SGLT-2. SGLT2 plays a major role in the reabsorption of glucose. It transports 90% of the glucose reabsorbed by the kidney, while SGLT1 transports the remaining 10%. Therefore, SGLT-2 inhibitors can block the reabsorption of glucose by the proximal tubule and excrete excess sugar through urine. The development of SGLT-2 inhibitors began with the discovery of phlorizin, a naturally occurring compound with glycosuria. Phlorizin is easily hydrolyzed in the intestine and has poor oral availability, but its analogs are easily absorbed after oral administration. Chemically, most SGLT2 inhibitors are glycosides derived from the phlorizin prototype.

[0005] At present, some SGLT-2 inhibitor drugs have been approved for marketing, including empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, togliflozin, rupagliflozin, sogliflozin and erpagliflozin. Among them, dapagliflozin, empagliflozin and canagliflozin were launched in China in 2017, and ipragliflozin, jointly developed by Merck and Pfizer, was approved for marketing in my country in 2020. In addition, there are some SGLT-2 inhibitors under development, such as triazoles, indoles, thiophenes, pyridazines, thiazoles and thiadiazoles, spirocyclics, phenyls, azules, isoquinolines, glucose derivatives and non-sugars. These SGLT-2 inhibitors are mainly used as oral hypoglycemic drugs for the treatment of type 2 diabetes, reducing blood sugar in diabetic patients without the risk of weight gain and hypoglycemia, and adverse reactions are rare. Although a variety of SGLT-2 inhibitors have been used as clinical treatments for type 2 diabetes, their application in the prevention and treatment of radiation damage has not been reported.

[0006] The present inventors firstly revealed in experiments that SGLT-2 inhibitors have obvious therapeutic effects in preventing and treating myelosuppression, especially in preventing and treating radiation-induced myelosuppression. Based on the above findings, the present invention was completed through further experiments. Summary of the invention

[0007] The technical problem to be solved by the present invention is to find a drug capable of protecting and preventing the damage of bone marrow cells and functions induced by radiation and treating the damage.

[0008] Therefore, the object of the present invention is to provide the use of SGLT-2 inhibitors in the preparation of drugs for preventing or treating radiation-induced bone marrow suppression.

[0009] To solve the above problems, the present invention provides the following technical solutions:

[0010] An application of an SGLT-2 inhibitor in the preparation of a drug for preventing radiation-induced bone marrow suppression. The implementation method is: the application of an SGLT-2 inhibitor in the preparation of a drug for preventing and treating radiation-induced bone marrow damage. The radiation-induced bone marrow damage includes: tumor patients receiving radiotherapy, radiation-exposed workers, and personnel accidentally exposed to radioactive isotopes.

[0011] The bone marrow suppression (bone marrow suppressive death) described in the present invention includes decreased bone marrow cell activity, increased reactive oxygen species in bone marrow cells, or bone marrow cell DNA damage.

[0012] The experimental contents of the present invention on radiation-induced bone marrow suppression mainly include the following aspects:

[0013] 1.SGLT-2 inhibitors have a protective effect on bone marrow cell damage induced by in vitro irradiation.

[0014] 2. SGLT-2 inhibitors have a protective effect on bone marrow cell function damage in mice subjected to whole-body irradiation.

[0015] Instruction Manual

[0016] Figure 1 The effect of empagliflozin on the viability of bone marrow cells;

[0017] Figure 2 The effect of empagliflozin on ROS in bone marrow cells;

[0018] Figure 3 The effect of empagliflozin on apoptosis of mouse bone marrow cells;

[0019] Figure 4 The effect of empagliflozin on peripheral blood and unilateral femoral counts;

[0020] Figure 5 The effect of empagliflozin on ROS in bone marrow cells of mice;

[0021] Figure 6 The effect of empagliflozin on Nox-4 in bone marrow cells of mice;

[0022] Figure 7 This is the effect of empagliflozin on γH2AX of bone marrow cells in mice. DETAILED DESCRIPTION

[0023] The following pharmacological experiments further illustrate the pharmacological experiments of SGLT-2 inhibitors in preventing or treating radiation-induced bone marrow suppression.

[0024] 1. Experimental Methods

[0025] Example 1 Bone marrow mononuclear cell isolation

[0026] Aseptically remove the femur of the mouse, wash the bone marrow with PBS buffer, filter and count, adjust the required cell concentration, add 1640 culture medium containing 10% FBS and 1% double antibody, and prepare mononuclear cell suspension for use.

[0027] Example 2 Effect on bone marrow cell viability

[0028] Cell viability assay: Add 100ul of mononuclear cell suspension to the designated wells of a 96-well plate, add 100ul of the treatment drug as designed, place in a 37°C carbon dioxide incubator, and culture for 18h. Take out the culture plate, place it at room temperature, add 10ul of the bioluminescent reagent cell-titer, shake and mix, and transfer to a white assay plate. TMThe luminescence detector uses Promega's own detection program Cell-titer Protocol for detection. The detection results automatically generate Excel data.

[0029] Isolate mouse bone marrow mononuclear cells and adjust the cell concentration to 1×10 6 / ml. 100ul of cell suspension and SGLT-2 inhibitor empagliflozin were added to a 96-well culture plate. After incubation for 18 hours, cell viability was determined by bioluminescence. Results ( Figure 1 ) found that the SGLT-2 inhibitor empagliflozin was non-toxic to bone marrow cells in vitro and had a significant protective effect on irradiated mouse bone marrow cells at concentrations of 500 nmol / L and 250 nmol / L.

[0030] Example 3 Effects on Reactive Oxygen Species in Mouse Bone Marrow Cells

[0031] Determination of reactive oxygen species in bone marrow cells After culturing the above bone marrow cells for 18 hours, DCFH-DA reactive oxygen species detection reagent was added and incubated at 37 degrees for 20 minutes. Flow cytometry was used to analyze the effect of empagliflozin on reactive oxygen species in mouse bone marrow cells.

[0032] Isolate mouse bone marrow mononuclear cells and adjust the cell concentration to 2.5×10 5 / ml. Take 1ml of cell suspension and SGLT-2 inhibitor empagliflozin and add them to 6-well culture plates. After incubation for 18 hours, DCFH-DA was used to measure cell reactive oxygen species. The results are shown in Figure 2 , found that the SGLT-2 inhibitor empagliflozin significantly reduced reactive oxygen species in bone marrow cells of irradiated mice.

[0033] Example 4 Effect on apoptosis of mouse bone marrow cells

[0034] Bone marrow cell apoptosis assay After 18 hours of culture, the above bone marrow cells were added with Annexin V-FITC / PI reagent and incubated at room temperature in the dark for 15 minutes. Flow cytometry was used to analyze the effect of empagliflozin on mouse bone marrow cell apoptosis.

[0035] Isolate mouse bone marrow mononuclear cells and adjust the cell concentration to 2.5×10 5 / ml. Take 1ml of cell suspension and SGLT-2 inhibitor empagliflozin and add them to 6-well culture plates. After incubation for 18 hours, Annexin V-FITC / PI was used to determine cell apoptosis. The results are shown in Figure 3 , found that the SGLT-2 inhibitor empagliflozin significantly reduced apoptosis in bone marrow cells of irradiated mice.

[0036] Example 5 Effects on the Peripheral Blood and Unilateral Femoral Nucleated Cell Counts in Radiation-Exposed Mice

[0037] After irradiation, the mice were given SGLT-2 inhibitors for 7 days. The mice were killed 10 days later, and peripheral blood was collected from the mice and the nucleated cells of the unilateral femur were washed and counted using a blood cell counter.

[0038] Mice were divided into 0 and 4 Gy according to the radiation dose, 5 mice / group. After irradiation, normal saline and SGLT-2 inhibitor empagliflozin 20 mg / kg / d were given by gavage for 7 days, 5 mice in each treatment group. After 10 days of irradiation, peripheral blood and unilateral femoral bone marrow cells were collected for counting. The results showed that ( Figure 4 ), 4Gy irradiation had a significant inhibitory effect on the nucleated cell counts in the peripheral blood and unilateral femur of mice, while empagliflozin had a significant protective effect on the nucleated cell counts in the peripheral blood and unilateral femur of mice irradiated with 4Gy, compared with the control group, P < 0.05.

[0039] Example 6 Effects on the Peripheral Blood and Unilateral Femoral Nucleated Cell Counts in Radiation-Exposed Mice

[0040] Determination of reactive oxygen species in hematopoietic stem and progenitor cells Mouse bone marrow cells were separated, and the cell concentration was adjusted by counting cells with a blood cell counter. The corresponding surface antibodies were added and incubated in the dark. Then, ROS dye was added and incubated at 37 degrees for 20 minutes. Flow cytometry was used to analyze the effect of SGLT-2 inhibition on the reactive oxygen species of mouse hematopoietic stem and progenitor cells.

[0041] Mice were divided into 0 and 4 Gy according to the radiation dose, 5 mice / group. After irradiation, normal saline and SGLT-2 inhibitor empagliflozin 20 mg / kg were given by gavage for 7 days, 5 mice in each treatment group. After 10 days of irradiation, bone marrow cells were obtained, cell staining was performed, and ROS levels were detected. The results showed that ( Figure 5 ), empagliflozin can significantly reduce the ROS level in irradiated mouse cells.

[0042] Example 7 Effects on Nox-4 Levels in Radiation-Exposed Mouse Cells

[0043] Determination of Nox-4 and γH2AX in hematopoietic stem cells Mouse bone marrow cells were separated, and the cells were counted by a blood cell counter to adjust the cell concentration. The corresponding surface antibodies were added and incubated in the dark. The membrane was fixed with a fixative for 24 hours, and then the corresponding antibodies were added and incubated at 4 degrees for 30 minutes. The effect of SGLT-2 inhibition on Nox-4 and γH2AX in mouse hematopoietic stem cells was analyzed by flow cytometry.

[0044] Mice were divided into 0 and 4 Gy according to the radiation dose, 5 mice / group. After irradiation, normal saline and SGLT-2 inhibitor empagliflozin 20 mg / kg were given by gavage for 7 days, 5 mice in each treatment group. Ten days after irradiation, bone marrow cells were obtained, cell staining was performed, and Nox-4 levels were detected. The results showed that ( Figure 6 ),

[0045] Example 8 Effects on γH2AX Levels in Radiation-Exposed Mouse Cells

[0046] Mice were divided into 0 and 4 Gy according to the radiation dose, 5 mice / group. After irradiation, normal saline and SGLT-2 inhibitor empagliflozin 20 mg / kg were given by gavage for 7 days, 5 mice in each treatment group. Ten days after irradiation, bone marrow cells were obtained, cell staining was performed, and γH2AX levels were detected. The results showed that ( Figure 7 ), empagliflozin could significantly reduce the level of γH2AX in cells from irradiated mice.

[0047] According to the results of in vitro and in vivo experiments, the SGLT-2 inhibitor empagliflozin has a certain protective effect on radiation-induced bone marrow cell function damage, suggesting that preventive or therapeutic administration can prevent and treat radiation-induced bone marrow damage.

Claims

1. Application of empagliflozin in the preparation of drugs to protect against radiation-induced bone marrow suppression.

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 to 2, characterized in that A therapeutically effective amount of empagliflozin is mixed with pharmaceutically acceptable excipients to prepare a composition.

4. The use according to claim 3, characterized in that The composition is prepared into tablets, capsules, granules, pills, dripping pills, pre-emulsions, suspensions, syrups, various enteric-coated preparations and injections.

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