New application and medicine of CSF1R inhibitor

Abstract

The invention belongs to the field of medicines, discloses an application of a CSF1R inhibitor in preparation of a medicine for treating or relieving myelofibrosis, and also discloses a medicine for treating or relieving myelofibrosis. According to the application disclosed by the invention, a delta (5q) MDS mouse model constructed based on Diaph1- / -miR-146a- / -(DKO) is treated by giving a CSF1R inhibitor PLX3397, and the following positive effects are observed: PLX3397 can relieve anemia symptoms of a DKO mouse, remarkably reduce the number of peripheral blood and fibrocytes in the spleen of the DKO mouse, reduce deposition of reticular fibers and collagenous fibers in bone marrow and effectively improve myelofibrosis. In addition, PLX3397 can also reduce the clone formation ability of peripheral blood and spleen of the DKO mouse and prolong the lifetime of the DKO mouse. Flow cytometry analysis shows that PLX3397 treatment can increase the number of early erythroid progenitor cells and LSK cells in bone marrow of a DKO mouse, so that hematopoietic function defects of the DKO mouse are partially improved. Generally speaking, the invention reveals the potential curative effect of PLX3397 in treating MDS mouse disease models, and provides a new direction for treating and relieving myelofibrosis caused by clinical treatment of MDS.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and in particular relates to a new use and medicine of a CSF1R inhibitor. Background Art

[0002] Myelodysplastic syndrome (MDS) is a disease caused by abnormal hematopoietic stem cells. Its characteristics include hematopoietic dysfunction, cell dysplasia, bone marrow failure, decreased peripheral blood cell counts and the risk of transformation to acute myeloid leukemia (AML). MDS is common in the elderly, with a median age of diagnosis between 60 and 75 years old. About 10% of patients are under 50 years old. The incidence rate increases significantly with age. It is a typical senile disease associated with aging. The complexity of other factors of cell reduction makes the diagnosis of MDS challenging. Clinicians need to comprehensively consider the patient's clinical characteristics, bone marrow and peripheral blood cell morphology (such as peripheral blood smears), immunophenotype (such as immunohistochemistry), genetic testing, and other auxiliary test results, such as flow cytometry, cytogenetics, molecular genetics, etc. The genetic changes and chromosomal abnormalities of MDS include dup(1q), del(5q), -7 and del(7q), +8, del(11q), del(12p) and t(12p), del(17p) and iso(17q), del(18q), +21q, and complex karyotypes. With the rapid development of detection technologies related to chromosomal abnormalities and gene mutations, we have a new understanding of the pathogenesis and prognosis of MDS. Clonal and recurrent cytogenetic abnormalities have been observed in approximately 50% of MDS patient samples, including chromosome 8 trisomy, chromosome 12 long arm deletion (del(12q)), chromosome 7 monosomy (-7), chromosome 7 long arm deletion (del(7q)), chromosome 5 long arm deletion (del(5q)), and complex karyotypes. High-throughput technologies, such as single nucleotide polymorphism microarray (SNP-array) and next-generation sequencing (NGS), can reveal that heterozygous deletions that lead to haploid gene underexpression (e.g., CSNK1A1, DDX41, CUX1, EZH2) are involved in the pathogenesis of MDS. Whole-genome and whole-exome sequencing methods have found that about 80% to 90% of patients with MDS have more than 50 recurrently mutated genes. The most common mutated genes can be divided into the following categories: RNA splicing factors (e.g., SF3B1, SRSF2, U2AF1, and ZRSR2); DNA methylation (e.g., TET2, DNMT3A, and IDH1 / 2); histone modification (ASXL1 and EZH2); transcriptional regulation (RUNX1 and TP53); and signal transduction / kinases (FLT3 and JAK2).

[0003] The regimen for the treatment of MDS is as follows:

[0004] 1. Publication No. HK40106881A, titled: Venetoclax dosing regimen for the combined treatment of myelodysplastic syndrome with a CYP3A inhibitor and azacitidine, which discloses a regimen for the combined administration of venetoclax, azacitidine and a CYP3A inhibitor;

[0005] 2. Publication No. HK40103688A, the subject is: Iron transporter inhibitors for treating myelodysplastic syndrome (MDS), which discloses the use of iron transporter inhibitors for treating myelodysplastic syndrome.

[0006] 3. Publication No. HK40079174A, the subject is: A combination of a TIM-3 inhibitor and a hypomethylating agent for treating myelodysplastic syndrome or chronic myelomonocytic leukemia, which discloses a regimen of combining a TIM-3 inhibitor and a hypomethylating agent to treat myelodysplastic syndrome.

[0007] 4. Publication No. HK40059640A, titled: ALK5 inhibitors for treating myelodysplastic syndrome, discloses the use of ALK5 inhibitors for treating myelodysplastic syndrome.

[0008] 5. Publication No. HK1238548A, titled: Protein phosphatase 2A inhibitors for treating myelodysplastic syndrome, discloses the use of protein phosphatase 2A inhibitors for treating myelodysplastic syndrome.

[0009] 6. Publication No. HK1231762A, titled: JAK1 inhibitors for treating myelodysplastic syndrome, discloses the use of JAK1 inhibitors for treating myelodysplastic syndrome.

[0010] It can be found from the above literature that in the existing schemes for treating myelodysplastic syndrome, different inhibitor administration schemes are developed or selected from different perspectives.

[0011] In the prior art, there is no specific drug for the treatment and relief of myelofibrosis caused by myelodysplastic syndrome.

[0012] It can be seen that the development of MDS is related to multiple gene mutations. In the prior art, there is no technical literature research revealing whether macrophage Csf1r has an intrinsic correlation with MDS, let alone whether it has a correlation with myelofibrosis caused by MDS. Summary of the invention

[0013] Based on this, the first object of the present invention is to provide a use of a CSF1R inhibitor in a drug for treating or alleviating myelofibrosis.

[0014] At the same time, the invention also discloses a medicine for treating or alleviating myelofibrosis.

[0015] In order to achieve the above object, the present invention adopts the following technical solutions:

[0016] Use of a CSF1R inhibitor in preparing a drug for treating or alleviating myelofibrosis.

[0017] The CSF1R inhibitors can be selected from imibutumab, CSF1R-IN-5, CSF1R-IN-6, CSF1R-IN-20 (compound 7a), CSF1R-IN-19, CSF1R-IN-21 (compound 7e), PLX3397, etc.;

[0018] Particularly preferably, the CSF1R inhibitor PLX3397 is used in the preparation of a medicament for treating or alleviating myelofibrosis.

[0019] PLX3397 (also known as Pexidartinib) is a potent, orally active, selective ATP-competitive CSF1R (or M-CSFR) and c-Kit inhibitor that can cross the blood-brain barrier and is approved by the U.S. Food and Drug Administration (FDA) for tenosynovial giant cell tumors (TGCT). PLX3397 can reduce the number of TAMs by inhibiting the activity of CSF1R, thereby potentially weakening the tumor's immunosuppressive barrier and enhancing the body's immune response to the tumor. PLX3397 treatment has been extensively studied in solid tumors and hematological tumors. PLX3397 treatment can reduce TAMs and FOXP3+ regulatory T cells in the osteosarcoma microenvironment and enhance the infiltration of CD8+ T cells in the microenvironment of primary and metastatic osteosarcoma sites; PLX3397 has a strong macrophage and T cell regulatory effect, which may be transformed into cancer immunotherapy for bone and soft tissue sarcomas. PLX3397 has also been shown to cause regression of neurofibromas and block macrophage infiltration. In follicular lymphoma, in vivo experiments have demonstrated that the CSF1R inhibitor PLX3397 can lead to a decrease in M2 macrophages, promote the repolarization of M1 macrophages, and synergize with anti-CD20rituximab in anti-tumor effects. In vitro experiments have shown that PLX3397 combined with GM-CSF reprograms macrophages and destroys the pro-tumor effects of macrophages on AML cells. PLX3397 has also been studied in a phase 1 / 2 study in patients with relapsed or refractory acute myeloid leukemia (R / R AML) with FLT3-ITD mutations. The results showed that PLX3397 has good tolerability and anti-leukemic activity in the heavily pretreated R / R AML patient population with FLT3-ITD mutations. In summary, macrophage-targeted therapy or CSF1R inhibitor-targeted therapy is a new leukemia treatment strategy and one of the important development directions in the current leukemia treatment field.

[0020] To date, the CSF1R inhibitor PLX3397 has been used to treat a variety of tumors, including tenosynovial giant cell tumors, follicular lymphoma, leukemia, etc. However, there are currently no reports on the therapeutic application of the CSF1R inhibitor PLX3397 in MDS, let alone the use of the CSF1R inhibitor PLX3397 in the treatment or relief of myelofibrosis caused by myelodysplastic syndrome.

[0021] At the same time, among the relevant literature on the CSF1R inhibitor PLX3397, the literature reports involving myelodysplastic syndrome (MDS) are: Publication No. CN110621316A, the subject is: Patent application for combination therapy with EHMT2 inhibitors and Publication No. CN109069410A, the subject is: Patent application for IDH1 inhibitors for the treatment of hematological malignancies and solid tumors; the above two literature materials did not clearly reveal whether the CSF1R inhibitor PLX3397 has an intrinsic correlation with myelodysplastic syndrome, nor did they reveal whether it has a correlation with myelofibrosis caused by MDS.

[0022] Therefore, we believe that there is no literature disclosure regarding the pathogenesis of MDS mentioned in the present invention and the use of the CSF1R inhibitor PLX3397 in the treatment of myelofibrosis caused by MDS.

[0023] The core innovation of the present invention is:

[0024] The present invention is achieved by using Diaph1 - / - miR-146a - / - The study of del(5q)MDS mouse model with del(5q)as the core of DKO mice found that as DKO mice aged, their myeloid granulocytes differentiated abnormally, the cell volume increased and transformed into myeloid-derived suppressor cells (MDSCs), leading to anemia, thrombocytopenia and increased mortality of mice. Flow cytometry and other data showed that the proportion of macrophages in the bone marrow and spleen cells of DKO mice increased significantly; data analysis of gene expression profiles showed that the colony stimulating factor receptor-Csf1r was abnormally highly expressed in DKO macrophages.

[0025] Subsequently, by using the CSF1R small molecule inhibitor PLX3397 to block Csf1r expression or activity, the effect on the onset of the MDS mouse disease model and the evaluation of the therapeutic effect were explored. After research, it was found that PLX3397 can alleviate the anemia symptoms of DKO mice, significantly reduce the number of fibroblasts in their peripheral blood and spleen, reduce the deposition of reticular fibers and collagen fibers in the bone marrow, and effectively improve bone marrow fibrosis. In addition, PLX3397 can also reduce the cloning ability of the peripheral blood and spleen of DKO mice and prolong their survival. Flow cytometric analysis showed that PLX3397 treatment can increase the number of early erythroid progenitor cells and LSK cells in the bone marrow of DKO mice, thereby partially improving their hematopoietic function defects. In general, the present invention reveals the potential efficacy of PLX3397 in the treatment of MDS mouse disease models, providing a new direction for the clinical treatment of MDS.

[0026] At the same time, the present invention also discloses a drug for treating or alleviating myelofibrosis, wherein the active ingredient in the drug is a CSF1R inhibitor. More preferably, the CSF1R inhibitor is PLX3397.

[0027] The beneficial effects of the present invention are:

[0028] The present invention is based on Diaph1 - / - miR-146a - / - The del(5q)MDS mouse model constructed by DKO mice was treated with the CSF1R inhibitor PLX3397, and the following positive effects were observed: PLX3397 can alleviate the anemia symptoms of DKO mice, significantly reduce the number of fibroblasts in their peripheral blood and spleen, reduce the deposition of reticular fibers and collagen fibers in the bone marrow, and effectively improve bone marrow fibrosis. In addition, PLX3397 can also reduce the clonal formation ability of the peripheral blood and spleen of DKO mice and prolong their survival. Flow cytometric analysis showed that PLX3397 treatment can increase the number of early erythroid progenitor cells and LSK cells in the bone marrow of DKO mice, thereby partially improving their hematopoietic function defects. In general, the present invention reveals the potential therapeutic effect of PLX3397 in treating the MDS mouse disease model, providing a new direction for the clinical treatment of MDS. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A This is the WBC (white blood cell) count result;

[0030] Figure 1B It is the RCB (red blood cell) count result;

[0031] Figure 1C This is the PLT (platelet) count result;

[0032] Figure 1D It is the HCT (hematocrit) count result;

[0033] Figure 1E It is the result of HGB (hemoglobin) counting;

[0034] Figure 1F This is a statistical analysis chart of the survival time of three groups of mice;

[0035] Figure 2A WT, DKO, DKO-Csf1r + / - Photos of reticular fiber and collagen fiber staining in the femur and spleen of the three groups of mice;

[0036] Figure 2B To use ImageJ to analyze the WT, DKO, and DKO-Csf1r + / - Statistical graph of the results of quantitative analysis of the length of reticular fibers in the femurs of the three groups of mice;

[0037] Figure 2C To use ImageJ to analyze the WT, DKO, and DKO-Csf1r + / - Statistical graph of the results of quantitative analysis of the percentage of trabecular bone area in the femurs of the three groups of mice;

[0038] Figure 2D This is a statistical graph showing the results of quantitative analysis of the collagen volume fraction in the bone marrow of WT, DKO, and DKO-Csf1r+ / - mice using ImageJ;

[0039] Figure 2E To use ImageJ to analyze the WT, DKO, and DKO-Csf1r + / - Statistical graph of the results of quantitative analysis of the length of reticular fibers in the spleen of the three groups of mice;

[0040] Figure 3 Schematic diagram of bone marrow transplantation for spleen cells;

[0041] Figure 4 CD11b + Statistical results of Csf1r MFI in cells;

[0042] Figure 5A This is the WBC (white blood cell) count result;

[0043] Figure 5B It is the RCB (red blood cell) count result;

[0044] Figure 5C This is the PLT (platelet) count result;

[0045] Figure 5D It is the HCT (hematocrit) count result;

[0046] Figure 5E It is the result of HGB (hemoglobin) counting;

[0047] Fig. 5F To statistically analyze the survival time of mice in the PBS group and the PLX3397-administered group;

[0048] Fig. 6A The femurs of mice in the PBS and PLX3397 groups were stained for reticular fibers and collagen fibers;

[0049] Figure 6B The results of quantitative analysis of the length of reticular fibers in the femurs of mice in the PBS and PLX3397 groups using ImageJ;

[0050] Figure 6CThe results of quantitative analysis of the percentage of trabecular bone area in the femurs of mice in the PBS and PLX3397 groups using ImageJ;

[0051] Fig. 7A The results of the quantified spleen / body weight ratio of mice in the PBS and PLX3397 groups are shown;

[0052] Figure 7B Lin in the bone marrow and spleen of PBS and PLX3397 mice - Statistical results of the absolute number of cells;

[0053] Figure 7C The statistical results are as follows: The absolute number of mouse bone marrow erythrocytes at different developmental stages (I-VI) in the PBS and PLX3397 groups of mice;

[0054] Fig.7D The statistical results are as follows: The absolute number of mouse bone marrow erythrocytes at different developmental stages (I-VI) in the PBS and PLX3397 groups of mice;

[0055] Fig. 7E The statistical results of the absolute number of hematopoietic stem and progenitor cells of different lineages in the mouse bone marrow of the PBS and PLX3397 groups of mice;

[0056] Figure 7F The statistical results of the absolute number of hematopoietic stem and progenitor cells of different lineages in the spleen of mice in the PBS and PLX3397 groups;

[0057] Fig. 8A The statistical results of the number of monocyte-derived fibrocytes in the peripheral blood of mice transplanted with PBS and PLX3397 in vitro;

[0058] Figure 8B The statistical results of the number of monocyte-derived fibrocytes in the spleen of mice transplanted with PBS and PLX3397 were cultured in vitro;

[0059] Fig.9A The statistical results of clone numbers in 10 μL peripheral blood of mice transplanted with PBS and PLX3397 are shown;

[0060] Fig. 9B For statistical analysis, 2.5×10 mice were transplanted with PBS and PLX3397. 5 Statistical results of the number of clones formed by spleen monocytes. DETAILED DESCRIPTION

[0061] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0062] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0063] Example 1

[0064] Diaph1 - / - miR-146a - / - Blood routine test and survival curve statistical analysis of (DKO)Csf1r haploid deletion mice

[0065] Csf1r is essential for the development and homeostasis of mouse microglia. Studies have shown that Csf1r in C57BL / 6 mice - / - It affects neural development, resulting in a smaller brain size, death 21 days after birth, and very few survive to adulthood.

[0066] In this example, through mouse genotyping, it was found that in newborn experimental mice, Csf1r was rarely obtained. - / - Therefore, Csf1r haploid null mice were used as research objects, and WT (Diaph1 + / + miR-146a + / + Csf1 + / + , purchased from Saiye Biotechnology Co., Ltd.), DKO (Diaph1 - / - miR-146a - / - , Diaph1 - / - Mice and miR-146a - / - Mice were purchased from Saiye Biotechnology Co., Ltd. + / - (Diaph1 - / - miR-146a - / - Csf1 + / - , Csf1r + / - Indicates Csf1r haploid deletion, Csf1r + / - Mice were purchased from Saiye Biotechnology Co., Ltd.) Three groups of mice were collected, and the peripheral blood CBC of the three groups of mice was measured at the 6th, 9th and 12th months, and the survival curves of the three groups of mice were tracked and counted.

[0067] The results showed that DKO-Csf1r + / - The red blood cells (RBC) and hemoglobin (HGB) of the mice in the DKO group at 12 months were significantly higher than those in the DKO group ( Figures 1A to 1E ); DKO-Csf1r + / - The survival time of mice in the DKO group was significantly longer than that in the DKO group ( Figure 1F), indicating that Csf1r haploid deficiency improves anemia in DKO mice and prolongs the survival time of DKO mice.

[0068] in, Figures 1A to 1E Peripheral blood was collected from the three groups of mice at the 6th, 9th, and 12th months for complete blood cell counts;

[0069] in, Figure 1A This is the WBC (white blood cell) count result;

[0070] Figure 1B It is the RCB (red blood cell) count result;

[0071] Figure 1C This is the PLT (platelet) count result;

[0072] Figure 1D It is the HCT (hematocrit) count result;

[0073] Figure 1E It is the result of HGB (hemoglobin) counting;

[0074] Above Figures 1A to 1E Medium, WT n=7, DKO, n=9, DKO-Csf1r + / - , n=8.

[0075] Figure 1F For statistical analysis of the survival time of the three groups of mice, all error bars represent the SEM of the mean, WT n = 6, DKO n = 11, DKO-Csf1r + / - n = 8. Survival analysis was performed using log-rank (Mantel–Cox) tests, *P < 0.05.

[0076] Example 2

[0077] DKO-Csf1r + / - Mouse tissue section staining analysis

[0078] This example is a comparison of the WT, DKO, and DKO-Csf1r of Example 1. + / - The femurs and spleens of the three groups of mice were stained for reticular fibers, and the femurs were stained for collagen fibers ( Figure 2A ), and the length of reticular fibers, percentage of bone trabecular area, and collagen proportion in the bone marrow and spleen of the two groups of mice were calculated using software.

[0079] The results showed that compared with DKO mice, Csf1r haploid deletion significantly inhibited the accumulation of reticular fibers and collagen fibers in the bone marrow of DKO transplanted mice and effectively alleviated the bone marrow fibrosis of DKO mice, but the percentage of trabecular bone area in the bone marrow was not much different from that of the DKO group ( Figure 2B-D). Compared with the DKO group, DKO-Csf1r + / - The number of reticular fibers in the spleen of group mice was also significantly reduced ( Figure 2E ).

[0080] Figure 2A WT, DKO, DKO-Csf1r + / - Photographs of reticular and collagen fiber staining of the femurs and spleens of the three groups of mice. The scale bar in the Reticular image is 50 μm; the scale bar in the Trabecular and Masson images is 100 μm.

[0081] Figure 2B To use ImageJ to analyze the WT, DKO, and DKO-Csf1r + / - Statistical graph of the results of quantitative analysis of the length of reticular fibers in the femur of the three groups of mice, all error bars represent the SEM of the mean, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, t-test.

[0082] Figure 2C To use ImageJ to analyze the WT, DKO, and DKO-Csf1r + / - Statistical graph of the results of quantitative analysis of the percentage of trabecular area in the femur of the three groups of mice. All error bars represent the SEM of the mean, and t-test was performed.

[0083] Figure 2D This is a statistical graph showing the results of quantitative analysis of the collagen volume fraction in the bone marrow of WT, DKO, and DKO-Csf1r+ / - mice using ImageJ. Collagen proportion = the percentage of collagen-positive blue area to the total area of ​​the bone marrow cavity. All error bars represent the SEM of the mean. *P<0.05, t-test.

[0084] Figure 2E To use ImageJ to analyze the WT, DKO, and DKO-Csf1r + / - Statistical graph showing the results of quantitative analysis of the length of reticular fibers in the spleens of the three groups of mice.

[0085] Example 3

[0086] DKO mouse spleen cells were transplanted and PLX3397 was administered

[0087] DKO mice aged 12 months or older were selected and killed by cervical dislocation, and the abdominal skin was disinfected with 75% alcohol. The mouse abdominal cavity was opened and the spleen was taken out and placed in a well plate containing PBS; the spleen was transferred to a sterile mortar, 1-2 mL PBS was added and gently ground with a grinding rod (to avoid damaging the cells) to a cell suspension, which was placed on ice and filtered with a 70 μM filter into a sterile centrifuge tube; the supernatant was removed, 1 mL ACK Lysis Buffer was added, and the mixture was gently blown and mixed, and the mixture was placed on ice to lyse red blood cells for 5 min; 7 mL PBS was added to terminate the reaction, and the mixture was centrifuged at 1800 rpm and 4 °C for 8 min; the supernatant was removed and 1 mL PBS was added for resuspending; the cells were counted with a hemocytometer, and the recipient mice were lethally irradiated with 7.5 Gy using a biological irradiator RS2000, and 2 million cells / recipient mouse were injected intraorbitally; the recipient mice were fed with antibiotic water for 2 weeks; and the recipient mice were divided into a PBS group and a PLX3397 group one month after the transplantation.

[0088] PLX3397 group: Each mouse was intraperitoneally injected with PLX3397 at a dose of 10 mg / kg once a week ( Figure 3 PLX3397 powder (MCE) was dissolved in DMSO, and the mother solution was divided and frozen in a -80°C refrigerator. The mother solution was diluted before each use to prepare a solution containing 5% DMSO and 95% solvent (20% SBE-β-CD dissolved in PBS), and prepared before use.

[0089] PBS group: The transplant recipient mice in the control group were intraperitoneally injected with blank solvent (PBS solvent containing 20% ​​SBE-β-CD).

[0090] Figure 3 Schematic diagram of bone marrow transplantation for spleen cells.

[0091] Flow cytometry detection of Csf1r MFI in peripheral blood of transplanted mice

[0092] On the first day after PLX3397 administration in the PLX3397 group of Example 3, in this example, mice in the PBS and PLX3397 groups were fixed on a fixer, exposing their tails; 1-2 mm of the tail tip was cut with scissors, and massaged from the base of the tail to the tip of the tail with both hands, and the blood flowing out was collected with an anticoagulant tube containing EDTA, 3-5 drops were enough; after blood collection, cotton balls were used to press and stop bleeding; 10 μL of mouse peripheral blood was drawn and placed in a clean EP tube, and 200 μL of RBC Lysis Buffer was added to the tube, shaken and mixed, and lysed on ice for five minutes; 8000rpm, 4°C, centrifuged for 5min; discarded the supernatant, added 1mL PBS, 8000rpm, 4°C, centrifuged for 5min; discarded the supernatant, retained the cell pellet, added PBS containing 1:200 diluted antibodies (CD11b, CSF1R) to resuspend the cells, 50 μL / tube was stained, and stained on ice for 30min away from light; after staining, 1mL was added to each tube PBS was terminated, centrifuged at 8000rpm, 4℃ for 5min; the supernatant was discarded, and 200μL PBS (containing 1mg / mL PI) was added to each sample to resuspend the cell pellet. The Csf1r MFI in the peripheral blood of the two groups of mice was detected using a CytoFLEX high-performance flow cytometer. The experimental results showed that compared with the control group, the CSF1R inhibitor PLX3397 could significantly reduce the peripheral blood CD11b of transplanted mice. + Expression of Csf1r in cells ( Figure 4 ).

[0093] Figure 4 CD11b + Statistical results of Csf1r MFI in cells.

[0094] Blood routine test and statistical analysis of survival curves of transplanted mice

[0095] The mice were fixed on a fixture, with their tails exposed. The tail tip was cut with scissors to 1-2 mm, and the bleeding was massaged from the base of the tail to the tip with both hands. The blood flowed from the mouth of the anticoagulant tube containing EDTA to the bottom of the tube. 20-30 μL of blood was collected. After the blood was collected, cotton balls were used to press and stop the bleeding. The mixture was flicked and tested with an automatic blood cell analyzer (Hemo 3600V). The results showed that compared with the control group, the CSF1R inhibitor PLX3397 could alleviate anemia in transplanted mice ( Figure 5A -E), and significantly prolonged the survival of DKO mice ( Fig. 5F ).

[0096] FIG. 5A to FIG. 5E The results are from the peripheral blood test of mice after intraperitoneal administration of PLX3397;

[0097] in, Figure 5A This is the WBC (white blood cell) count result;

[0098] Figure 5B It is the RCB (red blood cell) count result;

[0099] Figure 5C This is the PLT (platelet) count result;

[0100] Figure 5D It is the HCT (hematocrit) count result;

[0101] Figure 5E It is the result of HGB (hemoglobin) counting;

[0102] Fig. 5F To statistically analyze the survival time of mice in the PBS group and the PLX3397-treated group, log-rank (Mantel–Cox) tests were used for the significance of survival analysis, *P<0.05.

[0103] Staining analysis of tissue sections from transplanted mice

[0104] The dying transplanted mice were weighed, the body weight was recorded, and the mice were killed by cervical dislocation. Use scissors to cut open the mouse's abdominal cavity, cut off the sternum, remove the ribs, and cut off excess tissue. Separate the spleen, remove the connective tissue on the spleen, collect one lobe of the lung, liver and kidney to obtain complete organs, and fix all the organs collected above in a 10% formalin solution in the dark. In this example, the femurs of the mice in the treatment group and the control group were stained with reticular fibers (Wuhan Pinofi Biotechnology Co., Ltd.), and the femurs were stained with collagen fibers Masson (Wuhan Bo Derivative Reagent Business Department) ( Fig. 6A ), the length of reticular fibers and percentage of trabecular area of ​​the two groups of transplanted mice were counted, and the results showed that compared with the PBS group, the CSF1R inhibitor PLX3397 treatment significantly reduced the accumulation of reticular fibers and collagen fibers in the bone marrow of DKO transplanted mice, and could effectively alleviate the bone marrow fibrosis of DKO mice ( Figure 6B -C);

[0105] Fig. 6A The femurs of the PBS and PLX3397 groups of mice were stained for reticular fibers and collagen fibers. The scale bar in the reticular image is 50 μm; the scale bar in the trabecular and masson images is 100 μm.

[0106] Figure 6B The results of quantitative analysis of the length of reticular fibers in the femurs of mice in the PBS and PLX3397 groups using ImageJ, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, t-test.

[0107] Figure 6CThe results of quantitative analysis of the percentage of trabecular area in the femurs of the PBS and PLX3397 groups of mice were analyzed using ImageJ, and t-test was performed.

[0108] Analysis of hematopoietic phenotypes of bone marrow and spleen in transplanted mice

[0109] The transplanted mice in both groups were killed by dislocating the neck, and one of the hind legs of the mice was stripped. The stripped leg bones were soaked in PBS solution, and a certain volume of PBS solution was drawn with a 5mL syringe to flush out all the bone marrow cells in the leg bones. The volume was adjusted to 5mL with PBS, and filtered with a 70μm filter to obtain a bone marrow single cell suspension. The mouse spleen was separated, and the connective tissue or fat on the spleen tissue was removed. Soaked in pre-cooled PBS to keep the spleen moist and prevent internal cell necrosis, and the surface moisture was absorbed with non-chip paper and weighed. After weighing, the ratio of the spleen weight of each mouse to its body weight was calculated. About 50mg was cut from each mouse and placed in a mortar, and 1mL PBS was added to grind (gently and slowly) to avoid damaging the cells. After grinding, filter with a 70um filter, rinse the mortar with 1-2mL PBS and collect the rinse solution to filter and collect the cells, and the volume was adjusted to 5mL with PBS to obtain a single cell suspension. Take an appropriate amount of bone marrow or spleen cells (about 1×10 6 ) in a 1.5mL EP tube, add 50μL of staining solution containing fluorescent labeled antibodies (1:200) to each tube, mix well by pipetting, and place on ice to avoid light for 30 minutes. After the staining, add 1mL PBS to each tube to terminate the reaction, centrifuge to remove the supernatant, add an appropriate amount of buffer to resuspend, use flow cytometer to collect fluorescence signals, and count the cells. When several different fluorescent labeled antibodies are added for staining at the same time, compensation should be adjusted for each fluorescent antibody separately. The flow cytometry antibodies used include: PE-Cy7-CD117, FITC-CD34, APC-TER119, APC-135, PE-CD44, Pacific Blue-Sca1, BV510-CD16 / CD32, PE-CD127, etc. The experimental results showed that compared with the PBS group, PLX3397 treatment improved splenomegaly in DKO transplanted mice ( Fig. 7A ), increase Lin in bone marrow - Cell number ( Figure 7B ), while there was little difference in the spleen. Compared with the control group, PLX3397 treatment significantly restored the generation of early erythroid progenitor cells II and III and the number of LSK cells in the bone marrow of DKO transplanted mice ( Figure 7C -E), the number of LS cells in the spleen increased ( Figure 7F ).

[0110] Fig. 7A The results of the quantified spleen / body weight ratio of mice in the PBS and PLX3397 groups are shown;

[0111] Figure 7B Lin in the bone marrow and spleen of PBS and PLX3397 mice - Statistical results of the absolute number of cells;

[0112] Figure 7C The statistical results are as follows: The absolute number of mouse bone marrow erythrocytes at different developmental stages (I-VI) in the PBS and PLX3397 groups of mice;

[0113] Fig.7D The statistical results are as follows: The absolute number of mouse bone marrow erythrocytes at different developmental stages (I-VI) in the PBS and PLX3397 groups of mice;

[0114] Fig. 7E The statistical results of the absolute number of hematopoietic stem and progenitor cells of different lineages in the mouse bone marrow of the PBS and PLX3397 groups of mice;

[0115] Figure 7F The statistical results of the absolute number of hematopoietic stem and progenitor cells of different lineages in the spleen of mice in the PBS and PLX3397 groups.

[0116] Transplantation of mouse peripheral blood and spleen cells to culture fibroblasts in vitro

[0117] Steps for extracting monocytes and culturing fibroblasts: Take 500 μL of peripheral blood from two groups of mice in an anticoagulant tube, and mix the peripheral blood with 1 mL of PBS buffer by inversion. Grind and filter the mouse spleen to 2 mL of single cell suspension; preheat the Ficoll-Paque density gradient medium to 20°C before use and invert several times to mix thoroughly; under sterile conditions, draw an equal volume of Ficoll-Paque medium to the sample and place it in a centrifuge tube; carefully layer the diluted peripheral blood sample or spleen single cell suspension on the Ficoll-Paque medium solution (do not mix the Ficoll-Paque medium and the sample); centrifuge at 20°C 400x g for 35 minutes; use a sterile pipette to extract the upper layer containing plasma and platelets, leaving the mononuclear cell layer undisturbed at the interface; use a sterile pipette to transfer the mononuclear cell layer to a sterile centrifuge tube; estimate the volume of mononuclear cells transferred. Add at least 3 volumes of PBS buffer to the mononuclear cells in the centrifuge tube; gently pipette up and down to suspend the cells; centrifuge at 20°C 400 x g for 15 min; remove the supernatant; add 6-8 mL PBS buffer to resuspend the mononuclear cells; centrifuge at 20°C 400 x g for 10 min; remove the supernatant; add Fibrocytes medium (Lifeline Cell Technology, LM-0001) to resuspend and count; add 2.5×10 5 ~5×10 5The cells were cultured in a 24-well plate with 500 μL / well of culture medium. The cells were gently patted and placed in a 37°C incubator. On the 5th day, the cell induction was observed and the number of fibroblasts was counted. The results showed that compared with the PBS group, PLX3397 treatment significantly reduced the number of monocyte-derived fibroblasts in the peripheral blood and spleen of DKO transplanted mice, inhibited fibroblast differentiation, and alleviated myelofibrosis ( Fig. 8A -B).

[0118] Fig. 8A The statistical results of the number of monocyte-derived fibrocytes in the peripheral blood of mice transplanted with PBS and PLX3397 in vitro;

[0119] Figure 8B The statistical results of the number of monocyte-derived fibrocytes in the spleen of mice transplanted with PBS and PLX3397 were cultured in vitro. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, t-test.

[0120] Analysis of the clone-forming ability of peripheral blood and spleen cells in transplanted mice

[0121] Take 10 μL of EDTA whole blood from two groups of mice in EDTA anticoagulant tubes, add 200 μL of ACK Lysis buffer to each tube, mix thoroughly, and lyse red blood cells on ice for 5 minutes; add 1 mL of PBS to terminate the reaction, centrifuge at 4°C, 1800 rpm for 5 minutes; discard the supernatant, add 1 mL of PBS to resuspend the cells, centrifuge at 4°C, 1800 rpm for 5 minutes; repeat the washing 2 to 3 times. Use a pipette to slowly aspirate the culture medium (MethoCult TM M3234) were placed in a 24-well cell culture plate, and the cell suspension containing cytokines (10 ng / mL IL3, 10 ng / mL IL6, 50 ng / mL SCF, 1% Penicillin-Streptomycin) was slowly added to the culture medium (MethoCult TM M3234), gently swirl the cell culture plate to mix the cells and the culture medium thoroughly; after thorough mixing, add sterile PBS to the gaps in the cell culture plate, maintain humidity, gently place it in a cell culture incubator, culture for 7 to 14 days, and count the number of clones. Repeat the above operation with spleen mononuclear cells extracted with Ficoll-Paque medium. The results showed that compared with the PBS group, PLX3397 treatment reduced the number of clones in the peripheral blood and spleen of DKO transplanted mice ( Fig.9A -B).

[0122] Fig.9A The statistical results of clone numbers in 10 μL peripheral blood of mice transplanted with PBS and PLX3397 are shown;

[0123] Fig. 9B For statistical analysis, 2.5×10 mice were transplanted with PBS and PLX3397. 5 Statistical results of the number of clones formed by spleen monocytes.

[0124] Comprehensive analysis:

[0125] 1. Through the experiments of Examples 1 and 2 above, it can be clearly seen that Csf1r haploid deficiency improves anemia in DKO mice and prolongs the survival time of DKO mice. Csf1r haploid deficiency significantly inhibits the accumulation of reticular fibers and collagen fibers in the bone marrow of DKO transplanted mice and effectively alleviates bone marrow fibrosis in DKO mice. + / - The number of reticular fibers in the spleen of the mice in the control group was also significantly reduced; this indicates that the role of macrophage Csf1r in MDS is closely related to the occurrence and development of the disease;

[0126] 2. Through the experiment of Example 3, it can be clearly seen that PLX3397 can alleviate the anemia symptoms of DKO mice, significantly reduce the number of fibroblasts in their peripheral blood and spleen, reduce the deposition of reticular fibers and collagen fibers in the bone marrow, and effectively improve bone marrow fibrosis. In addition, PLX3397 can also reduce the clonal formation ability of the peripheral blood and spleen of DKO mice and prolong their survival. Flow cytometry analysis showed that PLX3397 treatment can increase the number of early erythroid progenitor cells and LSK cells in the bone marrow of DKO mice, thereby partially improving their hematopoietic function defects.

[0127] In general, the present invention reveals the potential therapeutic effect of PLX3397 in treating MDS mouse disease models, providing a new direction for the clinical treatment of MDS.

Claims

1. Use of a CSF1R inhibitor in the preparation of a medicament for treating or alleviating myelofibrosis.

2. The use according to claim 1, characterized in that The CSF1R inhibitor is PLX3397.

3. The use according to claim 1, characterized in that The myelofibrosis refers to myelofibrosis caused by myelodysplastic syndrome.

4. A drug for treating or alleviating myelofibrosis, characterized in that: The active ingredient in the drug is a CSF1R inhibitor.

5. The drug for treating or alleviating myelofibrosis according to claim 4, characterized in that: The CSF1R inhibitor is PLX3397.

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