New uses and drugs for CSF1R inhibitors

Abstract

The present invention belongs to the field of medicine and discloses the use of a CSF1R inhibitor in preparing a drug for treating or alleviating myelofibrosis. At the same time, the present invention also discloses a drug for treating or alleviating myelofibrosis. ‑ / ‑ miR‑146a ‑ / ‑ A del(5q) MDS mouse model constructed by DKO mice was established. Treatment with the CSF1R inhibitor PLX3397 demonstrated the following positive effects: PLX3397 alleviated anemia in DKO mice, significantly reduced the number of fibrocytes in their peripheral blood and spleen, and decreased reticular and collagen fiber deposition in the bone marrow, effectively improving myelofibrosis. Furthermore, PLX3397 reduced the clonogenic capacity of DKO mice in their peripheral blood and spleen and prolonged their survival. Flow cytometric analysis demonstrated that PLX3397 treatment increased the number of early erythroid progenitor cells and LSK cells in the bone marrow of DKO mice, partially ameliorating their hematopoietic defects. Overall, this study reveals the potential efficacy of PLX3397 in treating MDS mouse models and provides a new approach for the clinical treatment and alleviation of myelofibrosis caused by MDS.

Description

Technical Field

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

[0002] Myelodysplastic syndrome (MDS) is a disorder caused by abnormalities in hematopoietic stem cells. Its characteristics include hematopoietic dysfunction, cellular dysplasia, bone marrow failure, decreased peripheral blood cell counts, and the risk of transformation to acute myeloid leukemia (AML). MDS commonly occurs in the elderly, with a median age of diagnosis between 60 and 75 years. Approximately 10% of patients are under 50 years old, and the incidence increases significantly with age, making it a typical senile disease associated with aging. The complexity of other factors contributing to cytopenia makes the diagnosis of MDS challenging. Clinicians need to comprehensively consider the patient's clinical features, bone marrow and peripheral blood cell morphology (such as peripheral blood smears), immunophenotype (such as immunohistochemistry), genetic testing, and other ancillary test results, such as flow cytometry, cytogenetics, and molecular genetics. Genetic alterations and chromosomal abnormalities in 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 gained new insights into the pathogenesis and prognosis of MDS. Clonal and recurrent cytogenetic abnormalities, 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, have been observed in approximately 50% of MDS patient samples. High-throughput technologies such as single nucleotide polymorphism microarrays (SNP-arrays) and next-generation sequencing (NGS) have revealed that heterozygous deletions leading to haploid gene underexpression (e.g., CSNK1A1, DDX41, CUX1, and EZH2) are involved in the pathogenesis of MDS. Whole-genome and whole-exome sequencing have revealed that approximately 80–90% of patients with MDS harbor over 50 recurrent mutations in these genes. The most commonly mutated genes fall 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 following are the relevant inhibitors for the treatment of MDS:

[0004] 1. Publication No. HK40106881A, titled: Venetoclax Dosage Regimen for Combination with a CYP3A Inhibitor and Azacitidine for the Treatment of Myelodysplastic Syndrome, which discloses a regimen for the combined administration of venetoclax, azacitidine, and a CYP3A inhibitor;

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

[0006] 3. Publication No. HK40079174A, titled "Combination of a TIM-3 inhibitor and a hypomethylating agent for the treatment of myelodysplastic syndrome or chronic myelomonocytic leukemia," discloses a regimen for the combined administration of a TIM-3 inhibitor and a hypomethylating agent for the treatment of 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 the treatment of myelodysplastic syndrome," discloses the use of protein phosphatase 2A inhibitors for the treatment of 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] This shows that the development of MDS is associated with multiple gene mutations. In the existing technology, there is no technical literature research revealing whether macrophage Csf1r has an intrinsic correlation with MDS, nor 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 present 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 medicament for treating or alleviating myelofibrosis.

[0017] CSF1R inhibitors include imipenem, CSF1R-IN-5, CSF1R-IN-6, CSF1R-IN-20 (compound 7a), CSF1R-IN-19, CSF1R-IN-21 (compound 7e), PLX3397, etc.

[0018] Particularly preferred is the use of the CSF1R inhibitor PLX3397 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 crosses the blood-brain barrier and is approved by the US Food and Drug Administration (FDA) for tenosynovial giant cell tumors (TGCT). By inhibiting CSF1R activity, PLX3397 reduces the number of TAMs, potentially weakening the tumor's immunosuppressive barrier and enhancing the body's anti-tumor immune response. PLX3397 therapy has been extensively studied in both solid and hematologic tumors. PLX3397 treatment reduced TAMs and FOXP3+ regulatory T cells in the osteosarcoma microenvironment and enhanced CD8+ T cell infiltration in the microenvironment of primary and metastatic osteosarcoma sites. PLX3397 exhibits potent macrophage and T cell regulatory effects, potentially translating into cancer immunotherapy for bone and soft tissue sarcomas. PLX3397 has also been shown to induce neurofibroma regression and block macrophage infiltration. In follicular lymphoma, in vivo studies have demonstrated that the CSF1R inhibitor PLX3397 can reduce M2 macrophages, promote M1 macrophage repolarization, and synergize with anti-CD20 rituximab in its anti-tumor effects. In vitro studies have shown that PLX3397, combined with GM-CSF, reprograms macrophages and disrupts their tumor-promoting effects 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) harboring FLT3-ITD mutations. Results showed that PLX3397 was well tolerated and exhibited anti-leukemic activity in heavily pretreated R / R AML patients harboring FLT3-ITD mutations. In summary, macrophage-targeted therapy or CSF1R inhibitor-targeted therapy represents a novel leukemia treatment strategy and represents an important development direction in the current field of leukemia treatment.

[0020] To date, the CSF1R inhibitor PLX3397 has been used to treat various tumors, including tendon sheath giant cell tumors, follicular lymphoma, and leukemia. 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 treating or alleviating 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; neither of the above two documents clearly discloses whether the CSF1R inhibitor PLX3397 has an intrinsic correlation with myelodysplastic syndrome, nor does it disclose 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 and the use of the CSF1R inhibitor PLX3397 in the treatment of myelofibrosis caused by MDS mentioned in the present invention.

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

[0024] The present invention is achieved by using Diaph1 - / - miR-146a - / - Studies using a del(5q) MDS mouse model with a de novo knockout knockout (DKO) mouse model have revealed that as DKO mice age, myeloid granulocytes undergo abnormal differentiation, increasing in size and transforming into myeloid-derived suppressor cells (MDSCs), leading to anemia, thrombocytopenia, and increased mortality. Flow cytometry and other data indicate a significant increase in the proportion of macrophages in the bone marrow and spleen of DKO mice. Gene expression profiling analysis reveals abnormally high expression of the colony-stimulating factor receptor, Csf1r, in DKO macrophages.

[0025] Subsequent studies using the small molecule CSF1R inhibitor PLX3397 to block Csf1r expression or activity were conducted to investigate the effects of treatment on the pathogenesis of MDS in a mouse model and evaluate the therapeutic efficacy. These studies revealed that PLX3397 alleviated anemia in DKO mice, significantly reduced the number of fibrocytes in the peripheral blood and spleen, and decreased reticular and collagen fiber deposition in the bone marrow, effectively improving myelofibrosis. Furthermore, PLX3397 reduced the clonogenic capacity of DKO mice in the peripheral blood and spleen and prolonged their survival. Flow cytometric analysis demonstrated that PLX3397 treatment increased the number of early erythroid progenitors and LSK cells in the bone marrow of DKO mice, partially ameliorating their hematopoietic defects. Overall, this study reveals the potential efficacy of PLX3397 in treating MDS in a mouse model and provides a new approach for the clinical treatment of MDS.

[0026] The present invention also discloses a drug for treating or alleviating myelofibrosis, wherein the active ingredient of 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 - / - A del(5q) MDS mouse model constructed by DKO mice was established. Treatment with the CSF1R inhibitor PLX3397 demonstrated the following positive effects: PLX3397 alleviated anemia in DKO mice, significantly reduced the number of fibrocytes in their peripheral blood and spleen, and decreased reticular and collagen fiber deposition in the bone marrow, effectively improving myelofibrosis. Furthermore, PLX3397 reduced the clonogenic capacity of DKO mice in their peripheral blood and spleen and prolonged their survival. Flow cytometric analysis demonstrated that PLX3397 treatment increased the number of early erythroid progenitor cells and LSK cells in the bone marrow of DKO mice, partially ameliorating their hematopoietic defects. Overall, this study reveals the potential efficacy of PLX3397 in treating MDS mouse models and provides 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 This 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 This is the HGB (hemoglobin) count result;

[0034] Figure 1F Statistical analysis of the survival time of the three groups of mice;

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

[0036] Figure 2B 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 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 showing the results of quantitative analysis of the length of reticular fibers in the spleens of the three groups of mice;

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

[0041] Figure 4 CD11b in peripheral blood of transplanted mice + Statistical results of Csf1r MFI in cells;

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

[0043] Figure 5B This 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 This is the HGB (hemoglobin) count result;

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

[0048] Figure 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 were obtained using ImageJ.

[0051] Figure 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 groups of mice - Statistical results of the absolute number of cells;

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

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

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

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

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

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

[0059] Figure 9A The statistical results of the number of clones in 10 μL peripheral blood of PBS and PLX3397 transplanted mice are shown;

[0060] Figure 9B For statistical analysis, 2.5×10 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 way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely 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 this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0063] Example 1

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

[0065] Csf1r is required for the development and homeostasis of mouse microglia. Studies have shown that Csf1r is required for the development and homeostasis of mouse microglia. - / - It affects neurodevelopment, 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 newborn experimental mice rarely acquired Csf1r - / - Therefore, Csf1r haploid null mice were used as research objects, and WT (Diaph1 + / + miR-146a + / + Csf1r + / + , 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 - / - Csf1r + / - , 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 deletion improved anemia in DKO mice and prolonged the survival time of DKO mice.

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

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

[0070] Figure 1B This 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 This is the HGB (hemoglobin) count result;

[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 based on the results of Example 1 for WT, DKO, and DKO-Csf1r + / - The femurs and spleens of the three groups of mice were stained for reticular fibers, and the femurs were stained for collagen fibers by Masson staining ( 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 images 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 reticular fiber length 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 quantitative analysis of the percentage of trabecular bone area in the femurs of the three groups of mice. All error bars represent the SEM of the mean, t-test analysis.

[0083] Figure 2D Figure 2: Statistical graph of the quantitative analysis of collagen volume fraction in the bone marrow of WT, DKO, and DKO-Csf1r+ / - mice using ImageJ. Collagen percentage = percentage of collagen-positive blue area to the total bone marrow cavity area. 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 three groups of mice.

[0085] Example 3

[0086] DKO mouse spleen cells were transplanted and treated with PLX3397

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

[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 aliquoted and frozen in a -80°C refrigerator. Before each use, the mother solution was diluted to prepare a solution containing 5% DMSO and 95% solvent (20% SBE-β-CD dissolved in PBS), and the solution was 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 was used to detect Csf1r MFI in peripheral blood of transplanted mice

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

[0093] Figure 4 CD11b in peripheral blood of transplanted mice + Statistical results of Csf1r MFI in cells.

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

[0095] The mice were fixed in a fixture, with their tails exposed. 1-2 mm of the tail tip was cut with scissors. The blood was massaged from the base of the tail to the tip with both hands to induce bleeding. 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, it was pressed with a cotton ball to stop the bleeding. The mixture was gently flicked and tested using an automatic hematology 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 ( Figure 5F ).

[0096] Figures 5A to 5E The results of peripheral blood testing of mice after intraperitoneal administration of PLX3397 are shown;

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

[0098] Figure 5B This 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 This is the HGB (hemoglobin) count result;

[0102] Figure 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 survival analysis, *P<0.05.

[0103] Staining analysis of tissue sections from transplanted mice

[0104] The dying transplanted mice were weighed, their body weights were recorded, and the mice were killed by cervical dislocation. The abdominal cavity of the mice was cut open with scissors, the sternum was cut off, the ribs were removed, and the excess tissue was cut off. The spleen was separated, the connective tissue on the spleen was removed, and one lobe of lung, liver and kidney were collected to obtain complete organs. All the organs collected above were fixed in 10% formalin solution in the dark. In this example, the femurs of the mice in the drug administration group and the control group were stained for reticular fibers (Wuhan Pinuofei Biotechnology Co., Ltd.), and the femurs were stained for collagen fibers (Wuhan Bo Derivative Reagent Business Department) ( Figure 6A ), the length of reticular fibers and percentage of trabecular area of ​​the two groups of transplanted mice were counted. 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] Figure 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 images 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 bone area in the femurs of mice in the PBS and PLX3397 groups were analyzed using ImageJ, and t-test was performed.

[0108] Hematopoietic phenotype analysis of bone marrow and spleen transplanted mice

[0109] The transplanted mice in both groups were killed by cervical dislocation. One of the hind legs of the mouse was stripped off and the leg bones were soaked in PBS solution. A certain volume of PBS solution was drawn up 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. The surface moisture was absorbed with lint-free paper and weighed. After weighing, the ratio of the spleen weight to the body weight of each mouse was calculated. About 50mg of each mouse was cut and placed in a mortar. 1mL of 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 of PBS and collect the rinse solution. Filter the collected cells and adjust the volume 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 in the dark for 30 minutes. After 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 cytometry 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 the splenomegaly of DKO transplanted mice ( Figure 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] Figure 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 groups of mice - Statistical results of the absolute number of cells;

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

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

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

[0115] Figure 7F The statistical results show 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] To isolate monocytes and culture fibroblasts, collect 500 μL of peripheral blood from both groups of mice in an anticoagulant tube and mix the blood with 1 mL of PBS buffer by inversion. Grind and filter the mouse spleen to a 2 mL single-cell suspension. Preheat Ficoll-Paque density gradient medium to 20°C and mix thoroughly by inversion several times before use. Aseptically pipette a volume of Ficoll-Paque medium equal to the sample volume into a centrifuge tube. Carefully layer the diluted peripheral blood sample or spleen single-cell suspension on top of the Ficoll-Paque medium solution (do not mix the Ficoll-Paque medium and sample). Centrifuge at 400 x g for 35 minutes at 20°C. Use a sterile pipette to aspirate 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 400 x g at 20°C for 15 min; remove the supernatant; add 6-8 mL of PBS buffer to resuspend the mononuclear cells; centrifuge at 400 x g at 20°C for 10 min; remove the supernatant; resuspend in Fibrocytes medium (Lifeline Cell Technology, LM-0001) and count; add 2.5 × 10 cells per well. 5 ~5×10 5The cells were cultured in 24-well plates with 500 μL of culture medium per well. The cells were gently patted to mix well and placed in a 37°C incubator. On the fifth 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 ( Figure 8A -B).

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

[0119] Figure 8B Figure 3. Statistical results of the number of monocyte-derived fibrocytes cultured in the spleens of mice transplanted with PBS or PLX3397. *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 and place it in an EDTA anticoagulant tube. Add 200 μL of ACK Lysis buffer to each tube and mix thoroughly by inversion. Lyse red blood cells on ice for 5 minutes. Add 1 mL of PBS to terminate the reaction and centrifuge at 1800 rpm at 4°C for 5 minutes. Discard the supernatant and resuspend the cells in 1 mL of PBS. Centrifuge at 1800 rpm at 4°C for 5 minutes. Repeat the washing process 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 dropwise to the culture medium (MethoCult TM M3234), gently swirl the cell culture plate to mix the cells and 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 ( Figure 9A -B).

[0122] Figure 9A The statistical results of the number of clones in 10 μL peripheral blood of PBS and PLX3397 transplanted mice are shown;

[0123] Figure 9B For statistical analysis, 2.5×10 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 is clear 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 myelofibrosis in DKO mice. + / - The number of reticular fibers in the spleen of the mice in the control group was also significantly reduced, which indicates that macrophage Csf1r plays a role in MDS and is closely related to the occurrence and development of the disease.

[0126] 2. The experiments in Example 3 demonstrate that PLX3397 alleviates anemia in DKO mice, significantly reduces the number of fibrocytes in their peripheral blood and spleen, and decreases reticular and collagen fiber deposition in the bone marrow, effectively improving myelofibrosis. Furthermore, PLX3397 reduces the clonogenic capacity of DKO mice in their peripheral blood and spleen, prolonging their survival. Flow cytometry analysis demonstrates that PLX3397 treatment increases the number of early erythroid progenitor cells and LSK cells in the bone marrow of DKO mice, partially ameliorating their hematopoietic defects.

[0127] Overall, 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; the CSF1R inhibitor is PLX3397; the myelofibrosis refers to myelofibrosis caused by myelodysplastic syndrome.

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