Use of HDAC6 specific inhibitor ACY-1215 in anti-radiation damage

Abstract

The present invention discloses a new pharmaceutical use of ACY-1215. The new use is its application in the following aspects: 1) application in the preparation of cell radiation protectors; 2) application in the preparation of drugs for preventing and / or resisting radiation damage; 3) application in the preparation of products that promote cell proliferation after radiation. The pharmacodynamics experiments of the present invention show that ACY-1215 improves the survival rate and proliferation ability of cells after irradiation; improves the survival rate of humans or mammals after radiation; increases the number of white blood cells in humans or mammals after radiation; and increases the spleen index and thymus index of humans or mammals after radiation. It provides a new idea for the preparation of drugs for preventing and / or resisting radiation damage.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a use of a Histone Deacetylase 6 (HDAC6) specific inhibitor 2-(diphenylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]-5-pyrimidinecarboxamide (ACY-1215) in resisting radiation damage. Background Art

[0002] Ionizing radiation has appeared in all walks of life in human production and life, and has been widely used in the fields of industry, agriculture, national defense, science and technology, and medicine and health. While enjoying the huge benefits brought by it, the danger of human exposure to radiation environment is also increasing. Ionizing radiation is a kind of energy that can be transmitted without the participation of any medium. It can cause considerable damage to organisms and can be divided into direct damage and indirect damage. Direct damage refers to the direct effect of radiation on biological macromolecules such as DNA and protein to change their molecular structure and biological characteristics; indirect damage refers to the effect of radiation on water molecules in the body, causing them to ionize and produce a large number of free radicals, thereby causing a variety of related damaging diseases. The anti-radiation damage drugs currently under research and development mainly include aminothiol, cytokine, hormone, nitroxide free radical and natural medicine, but the comprehensive efficacy of these drugs is not ideal, the therapeutic effect of some drugs has not been recognized, and some drugs have serious side effects after use, which limits the clinical application of anti-radiation damage drugs. Therefore, the development of high-efficiency, low-toxic or non-toxic anti-radiation damage drugs has great application prospects and social benefits.

[0003] In mammalian cells, the HDAC family is very large and is divided into four main categories based on sequence homology: Class I (HDAC1-3 and HDAC8) is mainly distributed in the nucleus and has homology with yeast Rpd3; Class IIa (HDAC4, 5, 7 and 9) is expressed in both the cytoplasm and the nucleus and has a common catalytic site with HDAC1, while Class IIb (HDAC6 and 10) is mainly distributed in the cytoplasm and has two catalytic sites; Class III (SIRT1-7) has homology with yeast scSir2; Class IV only contains HDAC11. Histone deacetylase 6 (HDAC6) is a special member of the HDACs family, belonging to type IIb HDACs, mainly located in the cytoplasm, containing two N-terminal deacetylase domains and a C-terminal ubiquitin binding domain, and plays an important role in regulating gene expression, cell proliferation, apoptosis, migration, immune response and protein degradation.

[0004] ACY-1215 is a specific HDAC6 inhibitor that inhibits the activity of HDAC6 protein. Studies have shown that it can inhibit the proliferation of tumor cells and promote cell apoptosis.

[0005] Currently, there is no report on the application of HDAC6 inhibitor ACY-1215 in anti-radiation damage. Summary of the invention

[0006] The purpose of the present invention is to provide an application of ACY-1215, a HDAC6 specific inhibitor.

[0007] The use of ACY-1215 provided by the present invention is its application in the following aspects:

[0008] 1) Application in the preparation of cell radiation protectants;

[0009] 2) Application in the preparation of drugs for preventing and / or resisting radiation damage;

[0010] 3) Application in the preparation of products that promote cell proliferation after radiation.

[0011] In the present invention, the cell may be a normal cell of a eukaryotic organism.

[0012] The eukaryotic organism may be a human or a mammal; the normal cell is a cell derived from a normal tissue; the cell includes a liver cell, a lung cell, an umbilical vein endothelial cell, an intestinal epithelial cell, and a gastric epithelial cell.

[0013] The liver cells may be L-02; the lung cells may be MRC-5; the umbilical vein endothelial cells may be HUVEC; the small intestinal epithelial cells may be HIEC; and the gastric epithelial cells may be GES-1.

[0014] The cell radiation protectant specifically improves the survival rate of cells after radiation.

[0015] The prevention and / or resistance to radiation damage is specifically embodied in at least one of the following aspects:

[0016] 1) Improved survival rate of humans or mammals after radiation;

[0017] 2) Increased white blood cell count in humans or mammals after radiation;

[0018] 3) Increased spleen index in humans or mammals after radiation;

[0019] 4) Increased thymus index in humans or mammals after radiation;

[0020] 5) Protect human or mammalian organs (such as spleen, thymus, etc.) after radiation.

[0021] The radiation is ionizing radiation, including α-ray radiation, γ-ray radiation, X-ray radiation, neutron ray radiation, and the like. 60 Co γ-ray radiation. 60The radiation dose of Co gamma irradiation can be 6 Gy – 12 Gy.

[0022] The radiation damage may be organ damage caused by radiation, such as spleen damage, thymus damage, gonad damage, pancreas damage, lung damage, etc.

[0023] The present invention also protects an in vitro method for promoting cell proliferation after irradiation.

[0024] The method comprises treating irradiated cells with ACY-1215.

[0025] The concentration of ACY-1215 in the treatment system may be 0.5 μM - 5 μM.

[0026] The cells include liver cells, lung cells, umbilical vein endothelial cells, small intestinal epithelial cells, and gastric epithelial cells.

[0027] The liver cells may be L-02; the lung cells may be MRC-5; the umbilical vein endothelial cells may be HUVEC; the small intestinal epithelial cells may be HIEC; and the gastric epithelial cells may be GES-1.

[0028] The radiation protector for normal cells of eukaryotic organisms or the drug for preventing and / or resisting radiation damage prepared with ACY-1215 as the active ingredient also falls within the protection scope of the present invention.

[0029] The eukaryotic normal cell radiation protector or drug for preventing and / or resisting radiation damage can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation methods; or introduced into the body after being mixed or wrapped with other substances.

[0030] When necessary, one or more pharmaceutically acceptable carriers may be added to the above-mentioned drugs, including conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0031] The eukaryotic normal cell radiation protector or drug for preventing and / or resisting radiation damage prepared with ACY-1215 as the active ingredient can be prepared in various forms such as injection, tablet, powder, granule, capsule, oral solution, ointment, cream, etc. The above-mentioned drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 In Example 1, whether ACY-1215 was administered and whether 60 Changes in the viability of HUVEC cells treated with Co γ-irradiation.

[0033] Figure 2 In Example 2, whether ACY-1215 was administered and whether 60 Proliferation changes of HUVEC cells treated with Co γ-irradiation.

[0034] Figure 3 In Example 3, whether ACY-1215 was administered and whether 60 Survival of mice after Co γ-irradiation for 30 days.

[0035] Figure 4 In Example 4, whether ACY-1215 was administered and whether 60 Changes in the number of leukocytes in mice 7 days after Co γ-irradiation.

[0036] Figure 5 In Example 4, whether ACY-1215 was administered and whether 60 Changes in spleen index of mice 7 days after Co γ-irradiation.

[0037] Figure 6 In Example 4, whether ACY-1215 was administered and whether 60 Changes in thymic index of mice 7 days after Co γ-irradiation. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0041] The quantitative experiments involved in the following examples were all repeated at least 3 times, and the results were averaged.

[0042] The immortalized human umbilical vein endothelial cells (HUVEC) used in the following examples were obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0043] Example 1: Detection of in vitro cell viability after radiation

[0044] Materials and Methods

[0045] 1. HUVEC Cell Culture

[0046] HUVEC cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. When the cells were in the logarithmic growth phase, they were trypsinized, the cell density was calculated using a cell counter, and 5,000 cells were seeded per well in a 96-well plate and incubated in the incubator for 24 hours. The drug administration experiment was performed after the cells attached to the wall and grew stably. The cells were incubated for 24 hours and the drug administration was performed when the cell density reached 70-80%. 60 Co γ-ray irradiation treatment.

[0047] 2.ACY-1215 pair 60 Detection of the effect of Co γ-rays on the loss of HUVEC cell viability

[0048] HUVEC cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. When the cells were in the logarithmic growth phase, trypsin was digested, the cell density was calculated using a cell counter, and 5,000 cells were seeded in a 96-well plate per well and incubated in the incubator for 24 hours. After the cells attached to the wall and grew stably, ACY-1215 was administered at a final concentration of 1 μM and incubated for 24 hours. 60 Co γ-ray irradiation of 8 Gy, dose rate 1 Gy / min (+IR+ ACY-1215), untreated control group (-IR+vehicle), simple drug group (-IR+ ACY-1215), simple irradiation group (+IR+vehicle) and cell-free blank group were set up at the same time, and cultured in a 37°C, 5% CO2 incubator. After 72 hours, the test was performed, CCK-8 solution was added to each well, incubated at 37°C in the dark for 1 hour, and the absorbance value D was measured at 450 nm using a microplate reader.

[0049] 3. Processing and statistics of ACY-1215 results

[0050] The absorbance values ​​of different groups are used to show the viability of cells, reflecting the changes in the viability of cells after irradiation, as well as the effect of ACY-1215 on the loss of viability of cells after irradiation. The cell viability is the ratio of the absorbance value of the experimental group to the absorbance value of the drug-free, non-irradiated group (i.e., the untreated control group), expressed as a percentage:

[0051] Survival rate = (D 450 Experimental Group-D 450 Blank group) / (D 450 Control group-D 450 Blank group)*100%

[0052] The results were statistically analyzed using GraphPad Prism 7.04 software. Compared with the irradiation group, * P <0.05.

[0053] result:

[0054] The experiment was repeated three times independently, and the CCK-8 test results are shown in Figure 1 ACY-1215 can inhibit 60 Co γ-rays cause loss of HUVEC cell viability and increase cell survival rate after irradiation.

[0055] Example 2: Detection of in vitro cell proliferation after radiation

[0056] Materials and Methods

[0057] 1. HUVEC Cell Culture

[0058] Same as Example 1.

[0059] 2. Effect of ACY-1215 on HUVEC cells 60 Detection of proliferation after Co γ-ray irradiation

[0060] HUVEC cells were used for cloning experiments under irradiation and drug administration conditions. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The cells were trypsinized when they were in the logarithmic growth phase, and the cell density was calculated using a cell counter. 2000 cells were seeded in a 6-well plate per well and incubated in the incubator for 24 hours. After the cells attached to the wall and grew stably, ACY-1215 was administered at a final concentration of 1 μM and incubated for 24 hours. 60 Co γ-ray irradiation of 8 Gy, dose rate 1 Gy / min, untreated control group, simple drug group and simple irradiation group were set up at the same time, and culture was continued at 37 ° C, 5% CO2 incubator. The medium was changed every 3 days, the old medium was discarded, and fresh medium was slowly added. The cell state was observed and cultured for 14 days. After cloning was completed, PBS was washed once, 1 mL of 4% paraformaldehyde was added to each well and fixed at room temperature for 30 minutes, and PBS was washed once. 1 mL of crystal violet stain was added to each well, stained for 15 minutes, and washed 3 times with PBS. Dry, take pictures, and use ImageJ 1.48v software to count the number of clones formed N.

[0061] 3. Processing and statistics of ACY-1215 results

[0062] The number of clones formed in different groups is used to show the proliferation of cells, reflecting the changes in the proliferation ability of cells after irradiation, as well as the effect of ACY-1215 on the proliferation ability of cells after irradiation. The clone survival rate is the ratio of the number of clones formed in the experimental group to the number of clones formed in the drug-free and non-irradiated group (i.e., the untreated control group), expressed as a percentage:

[0063] Survival rate = N experimental group / N control group * 100%

[0064] The results were statistically analyzed using GraphPad Prism 7.04 software. Compared with the irradiation group, * P <0.05.

[0065] result

[0066] The experiment was repeated three times independently, and the results of cell clone formation are shown in Figure 2 ACY-1215 can inhibit 60 Co γ-rays weakened the proliferation ability of HUVEC cells, while Co γ-rays increased the proliferation ability of HUVEC cells after irradiation.

[0067] Example 3: Experiment on the survival rate of mice after 30 days of radiation

[0068] Materials and Methods

[0069] 1. C57BL / 6J mouse breeding

[0070] Forty male C57BL / 6J mice weighing 18 to 22 g were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The mice were kept in an environment with a temperature of 22°C ± 2°C and a relative humidity of 50% to 60%.

[0071] 2.ACY-1215 pair 60 Effect of single whole-body irradiation with Co γ-rays at 8 Gy on the 30-day survival rate of C57BL / 6J mice

[0072] Drug preparation: Accurately weigh 5 mg of ACY-1215 powder, slowly add 50 μL DMSO, 400 μL PEG, 50 μL Tween 80 and 500 μL water in sequence, vortex mix until the solution is clear, and prepare a 5 mg / mL solution.

[0073] Animal grouping and treatment: 40 C57BL / 6J mice were randomly divided into 4 groups, 10 mice in each group, including untreated control group, simple drug administration group, simple irradiation group and drug administration irradiation group. The mice in the untreated group were intraperitoneally injected with an equal volume of the above solvent (5% DMSO + 40% PEG + 5% Tween 80 + 50% water), and the mice in the ACY-1215 administration group were intraperitoneally injected once at a dose of 50 mg / kg 24 hours and 1 hour before irradiation.

[0074] 3. Processing and statistics of the anti-radiation effect of ACY-1215

[0075] The activity status of mice in each group was observed every day, and the survival status of mice was recorded within 30 days. The results were statistically analyzed using GraphPadPrism 7.04 software to reflect the effect of ACY-1215 preventive administration on 60 Effect of whole-body irradiation with Co γ-rays 8 Gy on the 30-day survival rate of C57BL / 6J mice. Compared with the simple irradiation group, * P < 0.05.

[0076] result

[0077] ACY-1215 was administered at a dose of 50 mg / kg for prevention. 60 The results of 30-day survival rate of C57BL / 6J mice irradiated with 8 Gy of Co γ-rays are shown in Figure 3 The mice in the untreated control group and the drug-only group did not die, and the survival rate was 100%, so the curves in the figure overlapped. 60 Survival rate of C57BL / 6J mice irradiated with 8 Gy of Co γ-rays.

[0078] Example 4: Analysis of indicators after radiation in mice

[0079] Materials and Methods

[0080] 1. C57BL / 6J mouse breeding

[0081] Same as Example 3.

[0082] 2. ACY-1215 pair 60 Analysis of various parameters in C57BL / 6J mice after whole-body irradiation with Co γ-rays 8 Gy

[0083] Animal grouping and treatment: 20 C57BL / 6J mice were randomly divided into 4 groups, 5 mice in each group, including untreated control group, simple drug administration group, simple irradiation group and drug administration irradiation group. The mice in the untreated group were intraperitoneally injected with an equal volume of the above solvent (5% DMSO + 40% PEG + 5% Tween 80 + 50% water), and the mice in the ACY-1215 administration group were intraperitoneally injected once at a dose of 50 mg / kg 24 hours and 1 hour before irradiation.

[0084] Analysis of leukocyte count: 200 μL of blood was collected from the mouse eyeball on the 7th day after irradiation, added into 100 μL of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) anticoagulant solution, and the leukocyte count was detected using an automatic blood cell analyzer.

[0085] Spleen and thymus index analysis: On the 7th day after irradiation, the mice were killed by cervical dislocation, the spleen and thymus of the mice were dissected out, the blood was rinsed with PBS, the weight was measured on an analytical balance, and the results were recorded.

[0086] 3. Processing and statistics of the results of ACY-1215 anti-radiation effect

[0087] The number of white blood cells in mice of different groups was recorded, and the abnormal changes in the number of white blood cells were analyzed to reflect the effect of ACY-1215 on the anti-radiation of mice.

[0088] The spleen and thymus masses of mice in different groups, as well as the weight of mice before dissection, were recorded, and the spleen index and thymus index of mice in each group were analyzed to reflect the effect of ACY-1215 in the anti-radiation of mice.

[0089] Organ index = organ mass (mg) / body weight (g).

[0090] The results were statistically analyzed using GraphPad Prism 7.04 software. Compared with the irradiation group, * P <0.05.

[0091] result

[0092] The results of the leukocyte counts of irradiated mice treated with ACY-1215 prophylactic administration are shown in Figure 4 Compared with the simple irradiation group, ACY-1215 can increase the number of white blood cells in mice after irradiation, and has an anti-radiation damage effect to a certain extent.

[0093] The results of spleen index and thymus index of irradiated mice treated with ACY-1215 prophylactic administration are shown in Figure 5 and Figure 6 Compared with the simple irradiation group, ACY-1215 can improve the spleen index and thymus index of irradiated mice, and has a protective effect on the organs of irradiated mice to a certain extent.

Claims

1. Use of ACY-1215 in the preparation of drugs for preventing and / or resisting radiation damage; the radiation is ionizing radiation, including alpha-ray radiation, gamma-ray radiation, X-ray radiation, and neutron ray radiation; the radiation damage is manifested in at least one of the following aspects: 1) Improve the survival rate of humans or mammals after radiation; 2) Increase the number of white blood cells in humans or mammals after radiation; 3) Increase the spleen index of humans or mammals after radiation; 4) Improve the thymus index of humans or mammals after radiation.

2. Application of ACY-1215 in the preparation of products for promoting cell proliferation after radiation; the radiation is ionizing radiation, including alpha-ray radiation, gamma-ray radiation, X-ray radiation, and neutron-ray radiation; the cells are umbilical vein endothelial cells.

3. The use according to claim 2, characterized in that: The umbilical vein endothelial cells are HUVEC cells.

4. The use according to any one of claims 1 to 3, characterized in that: The radiation is 60 Co gamma radiation; 60 The radiation dose of Co γ irradiation is 6Gy-12Gy.

5. A method for promoting cell proliferation after radiation in vitro, comprising treating the radiation-treated cells with ACY-1215, wherein the cells are umbilical vein endothelial cells.

6. The method according to claim 5, characterized in that The concentration of ACY-1215 in the treatment system is 0.5M-5M.

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