Application of Menin inhibitor BN104 in preparation of medicine for preventing and treating graft versus host disease

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a Menin inhibitor BN104 in preparation of medicines for preventing and treating graft versus host disease. Animal experiment research finds that the Menin inhibitor BN104 has an obvious effect of inhibiting the acute graft versus host disease, and can be used in the development of drugs or drug combinations for preventing and treating the graft versus host disease after hematopoietic stem cell transplantation.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of Menin inhibitor BN104 in the preparation of drugs for preventing and treating graft-versus-host disease. Background Art

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective means for treating malignant hematological diseases today. Acute graft-versus-host-disease (aGVHD) is an important complication thereof. AGVHD is a pathological injury caused by lymphocytes mainly composed of T cells and B cells in the graft recognizing host antigens, sensitizing, proliferating, differentiating, and directly or indirectly attacking host tissues and organs. Currently, the classic regimen for clinically preventing and treating GVHD is cyclosporine (CSA) + short-course methotrexate (MTX). However, such drugs target the entire immune system, which not only increases the risk of infection, but also some patients still develop severe GVHD, seriously affecting the prognosis of the patients. Therefore, it is particularly important to find new drug regimens for effectively preventing and treating GVHD. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide the application of Menin inhibitor BN104 in the preparation of drugs for preventing and treating graft-versus-host disease.

[0004] The present invention provides the application of a Menin inhibitor in the preparation of drugs for preventing and / or treating graft-versus-host disease and / or graft-versus-leukemia of a host.

[0005] Further, the Menin inhibitor includes BN104, Revumenib (MI-503), Ziftomenib (JNJ-64609178), Bleximenib (JNJ-75276617), Icovamenib (BMF-219), BTC-86, MI-2, MI-3, MI-136, VTP-50469, BAY-155, KO-539, etc.; the present invention proves through screening that BN104 is the optimal one.

[0006] In the present invention, the host includes: human, mouse, rabbit, and / or sheep; the present invention does not make any limitation thereto; in the specific embodiments of the present invention, the host is a mouse.

[0007] Further, in the present invention, the dosage of BN104 for a mouse is 100 mg / kg.

[0008] In the present invention, the Menin inhibitor is used for preventing and / or treating graft-versus-host disease and / or graft-versus-leukemia in a host by: inhibiting T cells, increasing the number of Treg cells, inhibiting CD4+ T cells and / or inhibiting B cells.

[0009] Furthermore, the inhibition of T cells is the inhibition of the proliferation of CD3 / CD28-activated T cells;

[0010] The inhibition of B cells includes: inhibiting the number of B cells and / or inhibiting the secretion of TNF-α by B cells;

[0011] In the present invention, the inhibition of the number of B cells includes inhibiting the number of germinal center B cells, naive B cells, memory B cells, plasma cells, regulatory B cells (Bregs), marginal zone B cells, follicular helper B cells, B1 cells, B2 cells, B cell precursors, immature B cells, transitional B cells and / or atypical memory B cells, and the present invention does not limit this; in a specific embodiment of the present invention, it is germinal center B cells; specifically, the inhibition of the number of B cells includes inhibiting the number of germinal center B cells.

[0012] The present invention provides the use of a Menin inhibitor in the preparation of a pharmaceutical combination for preventing and / or treating graft-versus-host disease and / or graft-versus-leukemia.

[0013] Furthermore, the pharmaceutical combination includes a Menin inhibitor and other drugs for preventing and / or treating graft-versus-host disease and / or graft-versus-leukemia.

[0014] The other drugs for preventing and / or treating graft-versus-host disease include, but are not limited to: cyclosporine A (CsA), tacrolimus, methotrexate (MTX), sirolimus, abatacept, vedolizumab, antithymocyte globulin (ATG), alemtuzumab, ruxolitinib,

[0015] Other drugs for preventing and / or treating graft-versus-leukemia include: immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors, etc.) and / or cyclophosphamide (PtCy), etc.

[0016] Through animal experiment research, the present invention discovers that the Menin inhibitor BN104 has an obvious effect of inhibiting acute graft-versus-host disease and can be used in the development of drugs or drug combinations for preventing and treating graft-versus-host disease after hematopoietic stem cell transplantation. Brief Description of the Drawings

[0017] Figure 1 It is a survival curve graph of mice. Compared with the Control group (control group), the survival time of mice in the Menin inhibitor group (BN104 or experimental group) is significantly prolonged (P = 0.002);

[0018] Figure 2 It is a graph of the change in mouse body weight. Compared with the Menin inhibitor group (BN104 or experimental group), the body weight of mice in the Control group (control group) is significantly reduced (P = 0.03);

[0019] Figure 3 It is a curve graph of the change in mouse clinical score. Compared with the Menin inhibitor group (BN104 or experimental group), there is no significant statistical difference in the Control group (control group) (P = 0.38);

[0020] Figure 4 It is a graph of the inhibitory effect of different concentrations of BN104 on allogeneic T cell responses;

[0021] Figure 5 It is a graph of the cell percentages of CD4+Treg and CD8+Treg in the experimental group and the control group;

[0022] Figure 6 It is a graph of the cell percentages of CD3+T, CD4+T, and CD8+T in the experimental group and the control group;

[0023] Figure 7 It is a graph of the percentage of B cells in the experimental group and the control group;

[0024] Figure 8 In the experimental group and the control group, BN104 can inhibit B cells from secreting TNF-α. Detailed Embodiments

[0025] The present invention provides the use of the Menin inhibitor BN104 in the preparation of drugs for preventing and treating graft-versus-host disease. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The methods and uses of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes, or appropriate changes and combinations, to the methods and uses herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0026] The Menin inhibitor (BN104) has a molecular weight of 570.67. At present, for the prevention and treatment of graft-versus-host disease after hematopoietic stem cell transplantation, existing drugs have certain side effects, there is a lack of effective drug treatment, and there is no report on the prevention and treatment of aGVHD with Menin inhibitors.

[0027] Treg cells, Regulatory T cells;

[0028] GC B cells, Germinal Center B cells

[0029] BMT refers to "Bone Marrow Transplant";

[0030] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market. The following further elaborates the present invention in conjunction with the examples:

[0031] Example 1 Use of the Menin inhibitor BN104 in the preparation of drugs for preventing and treating graft-versus-host disease

[0032] I. Materials and methods

[0033] (I). Animals

[0034] 7- to 8-week-old SPF-grade BALB / c mice (H-2d), weighing 20-24 g; 6- to 8-week-old SPF-grade C57BL / 6 mice (H-2b), weighing 20-24 g, were purchased from the Shanghai Laboratory Animal Center, China. All procedures were carried out in accordance with the guidelines of the Animal Care Committee and the National Institutes of Health for the care and use of animals. All experimental mice were housed in isolation cages at the SPF (specific pathogen-free facility) level. The feed and bedding were sterilized by 60Co irradiation, and the drinking water was sterilized by high temperature and high pressure and acidified and added with gentamicin, and the mice were housed in a sterile laminar flow room at the Experimental Animal Center of Soochow University.

[0035] (II) Main Reagents and Consumables

[0036] BN104: Sigma, USA; Methylcellulose: Sigma, USA; PE-CD4 monoclonal antibody: BD Pharmingen, USA; APC-CD3 monoclonal antibody: BD Pharmingen, USA; PE-cy5.5-CD8 monoclonal antibody: BD Pharmingen, USA; FITC-CD69 monoclonal antibody: BD Pharmingen, USA; Foxp3 staining kit: ebioscience, USA; RPMI-1640 medium: Hyclone, USA; DMEM medium: Gibco, USA; Fetal bovine serum: Hyclone, USA; Penicillin and streptomycin: Shijiazhuang Baisted Pharmaceutical and Chemical Co., Ltd.; 96-well U-bottom culture plate: Becton Dickinson, USA

[0037] (III) Experimental Methods

[0038] 1. Preparation of mouse splenocyte and hematopoietic stem cell suspensions

[0039] (1) Preparation of mouse splenocyte suspension

[0040] The mouse was sacrificed by cervical dislocation. The spleen of the mouse was aseptically removed in a laminar flow hood. The spleen was gently ground on a glass slide until most of the splenocytes were ground out. The cell suspension obtained was filtered through a nylon mesh and centrifuged at 1200 rpm at 4°C for 5 min. The supernatant was discarded. 2 mL of 1× red blood cell lysate was added, mixed well, and left at room temperature for 5 min. 5 mL of RPMI-1640 medium was added, and the mixture was centrifuged at 1200 rpm at 4°C for 5 min. The supernatant was discarded. The cells were resuspended in 1 mL of 1× PBS and counted. They were placed at 4°C for later use.

[0041] (2) Preparation of mouse hematopoietic stem cell suspension

[0042] The mouse was sacrificed by cervical dislocation. The spine, femur, and tibia of the mouse were aseptically removed in a laminar flow hood and gently squeezed and ground in a mortar. This operation was repeated until most of the cells were ground out. The cell suspension obtained was filtered through a nylon mesh and centrifuged at 1200 rpm at 4°C for 5 min. The supernatant was discarded. 3 mL of 1× red blood cell lysate was added, mixed well, and left at room temperature for 5 min. 5 mL of RPMI-1640 medium was added, and the mixture was centrifuged at 1200 rpm at 4°C for 5 min. The supernatant was discarded. The cells were resuspended in 1 mL of 1× PBS and counted. They were placed at 4°C for later use.

[0043] 2. Establishment of acute graft-versus-host disease model

[0044] One week before the experiment, SPF-grade BALB / C mice were fed with gentamicin aqueous solution to prevent intestinal infection. The mice were divided into an experimental group (5 mice) and a control group (5 mice). Subsequently, the mice were given a whole-body irradiation of 8.5 Gy; 4 hours after irradiation, hematopoietic stem cells (1×10 7 ), and splenocytes (5×10 6 ) of the donor mice (C56BL / 6 mice) were injected via the tail vein to induce an acute graft-versus-host disease model; the date of injecting hematopoietic stem cells and splenocytes of the donor mice (C56BL / 6 mice) via the tail vein was recorded as day 0. The experimental group was given BN104 (100 mg / kg, 100 μL per mouse, daily from -3d to +3d) by gavage, and the control group was given an equal amount of placebo (0.8% methylcellulose) (100 μL per mouse, daily from -3d to +3d) by gavage.

[0045] 3. Observation of clinical manifestations of aGVHD

[0046] The mice were observed once a day after transplantation, and their body weights were measured once every 2 days. Clinical aGVHD indicators such as weight loss, postural changes, activity level, hair coat changes, and skin integrity were observed. Table 1 is the clinical scoring standard table for aGVHD in mice after allogeneic hematopoietic stem cell transplantation.

[0047] Table 1

[0048]

[0049] 4. Obtaining splenic lymphocytes of mice after transplantation

[0050] The mice were sacrificed by cervical dislocation. Under sterile conditions in a laminar flow hood, the spleens of the mice were removed. The spleens were gently ground on a glass slide until most of the splenocytes were ground out. The resulting cell suspension was filtered through a nylon mesh, centrifuged at 1200 rpm at 4°C for 5 min, the supernatant was discarded, 2 mL of 1× erythrocyte lysate was added, mixed well, and left at room temperature for 5 min. Then 5 mL of RPMI-1640 medium was added, centrifuged at 1200 rpm at 4°C for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of RPMI-1640 medium. 10 μL was taken for counting. The obtained sterile splenocytes can be used for mixed lymphocyte reaction and flow cytometry detection.

[0051] 5. Cell surface staining and intracellular staining

[0052] The splenic lymphocytes of the mice obtained in "Obtaining splenic lymphocytes of mice after transplantation" in method 4 were taken respectively, and plated according to the cell surface marker (1 - 5)×10 5 / well and the Treg cell marker (1 - 5)×10 6 / well.

[0053] Specific steps for cell surface staining: Resuspend the cells with 100 μL of PBS and place them in a 96-well plate. First, block FcR with CD16 / CD32 FcR blocking antibody at 4°C for 20 min. Centrifuge the cells to precipitate, resuspend with 200 μL of PBS, and then add cell surface marker antibodies. Combine them according to different fluorescences (CD3, CD4, CD8, CD69 / well) (CD3, CD4, CD8 / well), incubate in the dark at 4°C for 30 min, centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend and wash once with 200 μL of PBS, centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend with 200 μL of PBS, and then detect with a BD FACS Calibur flow cytometer. Single-stained tubes and negative tubes of four fluorescences should be prepared simultaneously for compensation adjustment, and analyze the data with CellQuest software.

[0054] Specific steps for intracellular staining: Take (1 - 5)×10 5 / well of splenic lymphocytes, add 1 mL of RPMI-1640 medium containing 10% FBS to a 24-well plate and mix well. Add PMA (500 ng / mL) + Ionomycin (500 ng / mL) + BFA (Brefeldin A, a protein transport inhibitor that inhibits the transport of proteins from the endoplasmic reticulum to the Golgi apparatus, thus inhibiting their extracellular secretion), stimulate in a 37°C, 5% CO2 incubator for 4 hours, collect the cells and wash them thoroughly with FACS Buffer. Resuspend with 100 μL of PBS and place in a 96-well plate. First, block FcR with CD16 / CD32 FcR blocking antibody at 4°C for 20 min. Centrifuge the cells to precipitate, resuspend with 200 μL of PBS, and then add cell surface markers CD4 and CD8 antibodies, incubate in the dark at 4°C for 30 min, centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend and wash once with 200 μL of PBS, centrifuge at 1200 rpm for 5 min, discard the supernatant, then fix the cells with 100 μL of 4% paraformaldehyde for 40 min, centrifuge at 1200 rpm for 5 min, discard the supernatant, add 1% saponin to permeabilize the membrane for 20 min, centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend and wash once with 200 μL of PBS, centrifuge at 1200 rpm for 5 min, discard the supernatant, add 1 μL each of APC-IFN-γ monoclonal antibody and PE-TNF-α monoclonal antibody respectively, incubate in the dark for 30 min, centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend and wash once with 200 μL of PBS, centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend with 200 μL of PBS and detect with a BD flow cytometer. Single-stained tubes and negative tubes of four fluorescences should be prepared simultaneously for compensation adjustment, and analyze the data with CellQuest software.

[0055] 6. Statistical analysis

[0056] The Student-Newman-Keuls a, b test was used to analyze the data, and statistical analysis and plotting were performed using GraphPad Prism 10 software (GraphPad Software Inc). The Kaplan-Meier method was used to plot the survival curve of murine leukemia, and the log-rank test was used to analyze the survival period. A P-value less than 0.05 was defined as statistically significant.

[0057] II. Experimental Results

[0058] 1. BN104 has the effect of inhibiting acute graft-versus-host disease (aGVHD) after allogeneic transplantation

[0059] After TBI pretreatment, all mice showed radiation reactions, reduced food and water intake, and began to lose weight about one week after transplantation. In the placebo (0.8% methylcellulose) control group, mice showed moderate to severe aGVHD manifestations such as lethargy, anorexia, hunchback, hair loss, diarrhea, etc. 6 days after transplantation, and also showed GVHD-related cachectic weight loss and death. Deaths occurred 11 days after transplantation, and all mice died 27 days after transplantation. In the BN104 group (experimental group), the manifestations of aGVHD in mice were mild, and deaths began to occur 20 days after transplantation. Three mice survived for more than 60 days. The survival curve, clinical score change curve, and body weight change curve of the mice are shown in Figure 1 、 Figure 2 、 Figure 3 。

[0060] 2. BN104 directly inhibits T cell activation

[0061] (1). BN104 inhibits the proliferation of CD3 / CD28-activated T cells

[0062] Donor T cells were taken and stimulated with CD3 / CD28 antibodies to proliferate T cells. By comparing with the control group, the inhibitory effect of the BN104 group on the response of donor allogeneic T cells was analyzed. The data analysis is shown in Figure 4 , and the same in vitro experiment was repeated 3 times. The results showed that the proliferation of donor T cells was significantly inhibited after BN104 treatment, and its inhibitory effect was positively correlated with the BN104 administration concentration (* indicates P < 0.05, ** indicates P < 0.01).

[0063] 3. BN104 can increase the number of Treg cells and inhibit CD4+ T and B cells

[0064] Select 10 days after transplantation as the research time point, collect spleen cells from the two groups of mice, and use flow cytometry to detect Treg, CD3+, CD4+, CD8+T, and B220+ cells in the spleen lymphocytes of the transplanted mice. Analyze the effects of BN104 on mouse Treg cells, CD4+ T cells, and GC B cells from the cell percentages. Data analysis is shown in Figure 5 , 6 , 7. Analyze and compare the data of the BN104 group and the Control group. The results show that BN104 can increase the number of Treg cells in the mouse spleen, while inhibiting the responses of CD4+ T cells and GC B cells. The differences are statistically significant (* indicates P < 0.05, ** indicates P < 0.01). The same experiment was repeated 3 times.

[0065] 4. BN104 can inhibit B cells from secreting TNF-α

[0066] Select 10 days after transplantation as the research time point, collect spleen cells from the two groups of mice, and use flow cytometry to detect the changes in the levels of the cytokine TNF-α expressed by B cells. Data analysis is shown in Figure 8 , and the level of the cytokine TNF-α in the BN104 group of cells was significantly lower than that in the control group.

[0067] Conclusion: In summary, the present invention evaluates the role of BN104 in GVHD in the GVHD model. We found that BN104 has the effect of inhibiting acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation; it can directly inhibit the responses of activated T cells after transplantation; it may be achieved by increasing the number of Treg cells, and BN104 can reduce the aGVHD effect.

[0068] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a Menin inhibitor in the preparation of a medicament for preventing and / or treating graft-versus-host disease and / or graft-versus-leukemia in a host.

2. The application according to claim 1, characterized in that, The Menin inhibitor includes BN104.

3. The application according to claim 2, characterized in that, The host includes: human, mouse, rabbit and / or sheep.

4. The application according to claim 3, characterized in that, The dosage of BN104 for mice is 100 mg / kg.

5. The application according to claim 1, wherein The prevention and / or treatment of graft-versus-host disease and / or graft-versus-leukemia in the host is achieved by: inhibiting T cells, increasing the number of Treg cells, inhibiting CD4+ T cells and / or inhibiting B cells.

6. The application according to claim 5, characterized in that, The inhibition of T cells is the inhibition of the proliferation of T cells activated by CD3 / CD28.

7. The application according to claim 5, characterized in that The inhibition of B cells includes: inhibiting the number of B cells and / or inhibiting the secretion of TNF-α by B cells.

8. The application according to claim 7, characterized in that, The inhibition of the number of B cells includes inhibiting the number of germinal center B cells.

9. Use of a Menin inhibitor in the preparation of a pharmaceutical combination for preventing and / or treating graft-versus-host disease and / or graft-versus-leukemia.

10. The application according to claim 9, characterized in that, The pharmaceutical combination includes a Menin inhibitor and other medicaments for preventing and / or graft-versus-host disease and / or graft-versus-leukemia.

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