Pharmaceutical composition and application thereof in preparation of drugs for treating mutation type myeloproliferative diseases

Abstract

The invention discloses a pharmaceutical composition and application thereof in preparation of drugs for treating mutation type myeloproliferative diseases, and relates to a drug combination method capable of being applied to targeting MPLW515L mutation type myeloproliferative diseases by taking an MPLW515L gene mutation MPN mouse model as a research object. According to the application disclosed by the invention, the STAT3-IN25 is found to have a relatively remarkable inhibition effect on p-STAT3 (Ser727), and the STAT3-IN25 and Ruxolitinib are in a synergistic effect, so that a basis is provided for combined treatment of clinical medicines of patients with myelofibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition and its application in the preparation of drugs for treating myeloproliferative disorders with mutations. Background Technology

[0002] Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are a group of malignant clonal diseases originating from hematopoietic stem cells. They are hematologic disorders characterized by abnormal proliferation of one or more myeloid cell lineages and progressive myelofibrosis, including primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocythemia (ET). The pathogenesis of MPNs primarily stems from gene mutations in the patient's own body, with mutations in Janus kinase (JAK2), thrombopoietin receptor (MPL), and calreticulin careticulin (CALR) being common pathogenic gene mutations. This disease can further transform into leukemia and is currently incurable.

[0003] Ruxolitinib (Rux), a JAK1 / JAK2 inhibitor, is the first FDA-approved targeted therapy for high-risk MPN / PMF. Ruxolitinib has shown significant efficacy in improving symptom burden, alleviating splenomegaly, and reducing cytokine storm, improving the quality of life in approximately 50% of patients. However, due to the potential mutational complexity of clonal hematopoietic progenitor cells, as a JAK2 inhibitor, ruxolitinib often leads to disease progression or intolerance in most patients within 2–3 years, and it does not reduce the burden of mutated genes. Numerous small-molecule inhibitors that can be combined with ruxolitinib, such as Aurora A kinase inhibitors, histone deacetylase inhibitors, PI3K and mTOR inhibitors, and heat shock protein 90 inhibitors, can improve remission rates, but they still cannot overcome the side effects of ruxolitinib or reverse fibrosis. Therefore, there is an urgent need to find more effective combination therapies to truly improve the efficacy of ruxolitinib in treating PMF. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0005] This invention provides a pharmaceutical composition that has a synergistic inhibitory effect on myeloproliferative disorders with the MPLW515L mutation type, wherein the pharmaceutical composition consists of STAT3-IN25 (CAS No.: 2591440-75-2) and Ruxolitinib.

[0006] In this invention, MPLW515L refers to the substitution of leucine for the 515th amino acid, tryptophan, in the thrombopoietin receptor (MPL) protein.

[0007] The composition can be used for the synergistic treatment of myeloproliferative disorders with the MPLW515L mutation, including polycythemia vera, essential thrombocythemia, and essential myelofibrosis.

[0008] Preferably, the molar ratio of STAT3-IN25 to Ruxolitinib is 20-25:1.

[0009] Preferably, the molar ratio of STAT3-IN25 to Ruxolitinib is 22.5:1.

[0010] The beneficial effects of this invention: This invention uses an MPN mouse model with an MPLW515L gene mutation as the research object to explore a combination of drugs that can be applied to myeloproliferative diseases targeting the MPLW515L mutation type. This invention found that STAT3-IN25 has a significant inhibitory effect on p-STAT3 (Ser727), and that STAT3-IN25 and Ruxolitinib have a synergistic effect, providing a basis for clinical combination drug treatment for patients with myelofibrosis. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0012] Figure 1 To detect the activation level of p-STAT3 (Ser727) in peripheral blood cells, primary hematopoietic stem cells with MPLW515L mutation, and megakaryocytes of 6133 / MPL in patients with PMF. Figure 1 A represents the expression level of p-STAT3 (Ser727) in PBMCs of PMF patients and healthy volunteers detected by Western Blot. Figure 1 B represents the expression level of p-STAT3 (Ser727) in MPLW515L mutant primary hematopoietic stem cells; Figure 1 C represents the effect of Rux treatment on the expression levels of STAT3, p-STAT3 (Ser727), and p-STAT3 (Tyr705) in 6133 / MPL megakaryocytes, and is analyzed by grayscale values.

[0013] Figure 2 The STAT3 inhibitor IN25, in combination with ruxolitinib, synergistically inhibits the proliferation of 6133 / MPL megakaryocytes. Figure 2A represents the Western Blot analysis of the protein expression levels of STAT3, p-STAT3 (Ser727), and p-STAT3 (Tyr705) in 6133 / MPL megakaryocytes after treatment with different concentrations of STAT3-IN25; Figure 2 B is the effect of different concentrations of STAT3-IN25 on the proliferation of 6133 / MPL megakaryocytes detected by CCK-8 proliferation assay. Figure 2 C is a statistical analysis chart of the joint index (Cl) of STAT3-IN25 and Ruxolitinib.

[0014] Figure 3 The effect of STAT3-IN25 combined with ruxolitinib on peripheral blood counts in MPLW515L transplanted mice. Figure 3 A, Figure 3 B, Figure 3 C, Figure 3 D represents the changes in white blood cells, red blood cells, hematocrit, and platelets in the peripheral blood of MPLW515L transplanted mice before and after the end of gavage treatment.

[0015] Figure 4 The combination of STAT3-IN25 and ruxolitinib improved blood glucose levels and splenomegaly in MPLW515L transplanted mice. Figure 4 A represents the fasting blood glucose level in PMF-transplanted mice after gavage treatment. Figure 4 B represents the spleen size and weight of PMF mice in each treatment group after treatment. Figure 4 C is the calculated spleen index.

[0016] Figure 5 The combination of STAT3-IN25 and ruxolitinib reduced the protein expression levels of STAT3, p-STAT3 (Ser727), and p-STAT3 (Tyr705) in MPLW515L transplanted mice.

[0017] Figure 6 To reduce GFP in the spleen and bone marrow of MPLW515L transplanted mice by combining STAT3-IN25 with ruxolitinib + The ratio of tumor cells to megakaryocyte lineage cells. Figure 6 A and B are GFP in the spleen + Flow cytometry analysis of the proportion of tumor cells and megakaryocyte lineage cells; Figure 6 C and D are GFP in bone marrow + Flow cytometry analysis of the proportion of tumor cells and megakaryocyte lineage cells.

[0018] Figure 7 The infiltration of STAT3-IN25 combined with ruxolitinib into the spleen, bone marrow, and sternum of MPLW515L transplanted mice was investigated.

[0019] Figure 8 The synergistic inhibitory effect of STAT3-IN20 combined with ruxolitinib on MPLW515L mutant cells. Detailed Implementation

[0020] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0021] Materials used in this invention:

[0022] Cells: 6133 / MPL megakaryocytes are megakaryocytes derived from OTT-MAL mice carrying the MPLW515L mutation. (Platinum-E cells) are retroviral packaging cell lines.

[0023] Experimental methods:

[0024] 1. Western blot analysis of p-STAT3 (Ser727) expression levels in peripheral blood mononuclear cells (PBMCs) of PMF patients: Peripheral blood samples were collected from PMF patients and healthy volunteers. The samples were collected and lysed by centrifugation and red blood cell lysis. Cell proteins were separated on a 12% SDS gel, transferred to a nylon (NC) membrane, blocked, incubated with primary antibody, incubated with secondary antibody, and detected by ECL substrate colorimetric analysis. Grayscale values ​​were analyzed using ImageJ.

[0025] 2. Detection of p-STAT3 (Ser727) protein levels in primary cells of MPLW515L mutant mice:

[0026] (1) Packaging retroviruses carrying the MPLW515L mutant gene: Plat-E cells were packaged at 1.5 × 10⁻⁶ cells per cell line. 6 Plating the cells at the specified density in each well, gently mixing the transfection stimulating reagent, adding serum-free OPTI-MEM and 2 μg of the target plasmid to an EP tube, and adding 6 μl of the transfection stimulating reagent at a ratio of 1:3, mixing well, and incubating at room temperature for 15 min, then dropping the transfection complex into a Plat-E cell culture dish, and collecting the viral supernatant at 48 h and 72 h.

[0027] (2) Obtain CD117 +Mouse bone marrow cells: Mice were euthanized by cervical dislocation. The abdominal skin was disinfected, cut open, and the femur, tibia, and ilium were bluntly dissected and placed in pre-chilled Facs-buffer. The separated bone was ground in a sterile tissue homogenizer, and an appropriate amount of Facs-buffer was aspirated to flush out the bone marrow cells. The mixture was then filtered through a 100 μm cell filter. This process was repeated 3-4 times, and all cells were collected in centrifuge tubes. The cells were centrifuged at 1300 rpm for 5 min to obtain a single-cell suspension. After centrifugation, the suspension was incubated with CD117-positive magnetic beads in the dark for 10-15 min. Unbound magnetic beads were washed away, and the sorting column was placed in the magnetic field of a separator for sorting to obtain magnetically labeled bone marrow cells.

[0028] (3) Retroviral transfection with c-kit + Cells: Collect the virus suspension and transfect c-kit by adding 500 μL of original virus solution at a 1:1 ratio under conditions of 32℃ and 2500 rpm. + Cells, and repeated infections.

[0029] (4) After 24 hours, cells were collected to obtain cell precipitate for Western blot (WB) analysis.

[0030] 3. Western blotting analysis of the expression levels of STAT3, p-STAT3 (Ser727), and p-STAT3 (Tyr705) in 6133 / MPL megakaryocytes treated with Ruxolitinib: 6133 / MPL megakaryocytes in good growth condition were used, and Ruxolitinib was added at a concentration of 1.5 × 10⁻⁶ cells / mL. 6 / well, prepare the master tube to take the corresponding cell suspension and add it to the 6-well plate; add the drug solution to the corresponding cell well according to the corresponding concentration to make the final volume 3ml, mix gently, incubate in a cell culture incubator at 37℃ for 12h, centrifuge to collect the cell pellet, and perform WB analysis and gray value analysis.

[0031] 4. Cell proliferation assay to detect the synergistic effect of STAT3-IN25 combined with Ruxolitinib on 6133 / MPL megakaryocytes: 6133 / MPL megakaryocytes in good growth condition were used, and the cells were proliferated at a rate of 5 × 10⁻⁶ cells / mL. 3 For each well, prepare a master tube to take the corresponding cell suspension, add fresh culture medium at a volume of 50 μL per well, and add it to the 96-well plate; prepare the corresponding master tube according to different drug concentrations and replicates, add drug solution to the corresponding cell wells to make the final volume 100 μL, gently shake to mix, and incubate in a 37℃ cell culture incubator for 48 h, measure the absorbance value at 450 nm, and use ComPusyn software to perform co-index (Cl) analysis.

[0032] 5. Obtain c-Kit from C57BL / 6 mice transfected with MigR1-MPLW515L retrovirus. + Cells (using the same steps as above) were used for bone marrow transplantation via tail vein injection. Two weeks post-transplantation, complete blood cell counts (CBCs) were measured in mice, and the GFP positivity rate in peripheral blood was detected by flow cytometry to confirm the successful establishment of the MPLW515L mutant PMF mouse model.

[0033] Mice were randomly divided into four groups and administered drugs 5 days a week. The treatment consisted of gavage with Captisol (15%), STAT3-IN25 (10 mg / kg), ruxolitinib (30 mg / kg), or STAT3-IN25 (10 mg / kg) combined with ruxolitinib (30 mg / kg). The treatment was administered 5 days a week for 2 weeks.

[0034] Whole blood cell analysis of tail vein blood: Mice were fixed in a tail restraint device, disinfected with alcohol swabs, and 30–100 μL of blood was collected in an EDTA anticoagulant tube. Hemostasis was achieved by applying pressure with a cotton ball. White blood cell count, platelet count, and other parameters were monitored using a whole blood cell analyzer.

[0035] 6. Measure the weight and size of the spleen in each treatment group of mice after treatment, and measure the fasting blood glucose level of PMF mice after treatment using blood glucose test strips: Fast the mice in each treatment group for 6–8 hours, collect 5–10 μL of tail vein blood, contact the blood droplet with the absorbent end of the test strip to ensure sufficient blood absorption, read the blood glucose value and record it. Two weeks after gavage treatment, sacrifice the mice, remove the spleen tissue, weigh the spleen using an electronic balance, and record the weight.

[0036] 7. The infiltration of tumor cells and the degree of bone marrow fibrosis in the spleen and bone marrow were detected by flow cytometry, histochemical staining (HE), and silver staining of sternal reticular protein.

[0037] Two weeks after gavage treatment, the mice were sacrificed, and their spleens and bone marrow were ground, filtered through a cell filter to obtain a single-cell suspension, lysed with red blood cell lysis buffer, and the cells were collected, washed with PBS, and placed in centrifuge tubes for later use.

[0038] Megakaryocytic lineage cell (CD41-PE-Cy7) detection: 50 μL of cell suspension and 50 μL of PBS were added to a flow cytometry tube. 1 μL of CD41-PE-Cy7 antibody was added at a ratio of 1:100. The cells were incubated at 4°C in the dark for 30 min. After washing once with PBS, the cells were centrifuged at 1300 rpm for 5 min. The cells were resuspended in 200 μL of PBS and detected by flow cytometry. Data were analyzed using FlowJo software.

[0039] The spleen, femur, and tibia of mice were dissected and separated, placed in embedding cassettes, and fixed with 4% formaldehyde fixative. After routine dehydration, paraffin embedding, and embedding, the infiltration of tumor cells was observed under a microscope and analyzed by HE staining. Sternal bone tissue was dissected, fixed in 4% formaldehyde fixative, routinely dehydrated, embedded, and then silver-stained for reticular protein.

[0040] The above experimental methods are applicable to the following embodiments.

[0041] Example 1:

[0042] Reference Appendix Figure 1 The expression level of p-STAT3 (Ser727) in PBMCs of healthy volunteers and PMF patients was detected, and it was found that the expression level of p-STAT3 (Ser727) was increased in PMF patients and primary hematopoietic stem cells with MPLW515L mutation. Figure 1 A, B). Subsequently, 6133 / MPL megakaryocytes were treated with the JAK2 inhibitor ruxolitinib (Rux) for 12 h, and the protein expression levels of STAT3, p-STAT3 (Ser727), and p-STAT3 (Tyr705) in the 6133 / MPL megakaryocytes were detected. The results showed that at a treatment concentration of 0.5 μM ruxolitinib, the expression of p-STAT3 (Ser727) protein was hardly affected. Figure 1 C), and even at a treatment concentration of 3 μM, p-STAT3 (Ser727) protein was not completely inhibited, indicating that blocking the JAK2 signaling pathway cannot completely inhibit the activation of p-STAT3 (Ser727) in PMF megakaryocytes. This suggests that the activation of p-STAT3 (Ser727) plays an important role in PMF progression and may be a potential escape signaling pathway for JAK2 inhibitors.

[0043] Example 2:

[0044] Reference Appendix Figure 2 The STAT3 inhibitor STAT3-IN25 used in this study is a STAT3 diphosphorylation site inhibitor. This invention found that it has a significant inhibitory effect on p-STAT3 (Ser727), and at low doses, it can be considered a specific inhibitor of p-STAT3 (Ser727). Treatment of 6133 / MPL megakaryocytes with different concentrations of STAT3-IN25 showed that cell proliferation was significantly inhibited with increasing inhibitor concentration. After treatment with 0.6 μM STAT3-IN25, cell viability was 67.9%, significantly inhibiting the phosphorylation level of p-STAT3 (Ser727) protein, while having no significant effect on the phosphorylation level of p-STAT3 (Tyr705). Figure 2 A, B).

[0045] Subsequently, 6133 / MPL megakaryocytes were treated with STAT3-IN25 at the same concentration as those that inhibited p-STAT3 (Ser727) but not p-STAT3 (Tyr705), in combination with ruxolitinib. ComPusyn software was used for combination index (Cl) analysis. The Cl value of STAT3-IN25 combined with ruxolitinib was less than 1, indicating that STAT3-IN25 combined with ruxolitinib synergistically inhibited 6133 / MPL megakaryocytes (…). Figure 2 C, Table 1). Table 1 is a statistical analysis table of the combined index (Cl) of STAT3-IN25 and Ruxolitinib. This indicates that simultaneous targeting of the p-STAT3 (Ser727) and JAK pathways has a significant synergistic inhibitory effect on abnormal megakaryocytes.

[0046] Table 1. Statistical Analysis of the Combined Index (Cl) of STAT3-IN25 and Ruxolitinib

[0047]

[0048] Example 3:

[0049] Reference Appendix Figure 3 The study measured white blood cell (WBC), red blood cell (RBC), hematocrit (HCT), and platelet (PLT) counts in transplanted mice before and after treatment. Results showed that WBC levels were similar across groups before drug treatment. After gavage treatment, the white blood cell count in the control group decreased from 4 × 10⁻⁶ to 4 × 10⁻⁶. 9 / L gradually increased to 54×10 9 Compared with the control group, the increase in white blood cell count in MPLW515L transplanted mice treated with 30 mg / kg ruxolitinib or 10 mg / kg STAT3-IN25 was slowed, and the combination of the two drugs further suppressed the increase in white blood cell count than the single drug, maintaining the average white blood cell count at 4.56 × 10⁹ / L. 9 / L( Figure 3 A). Whether PMF mice were treated with ruxolitinib at 30 mg / kg or STAT3-IN25 monotherapy at 10 mg / kg, HCT levels did not change significantly, but red blood cell counts remained relatively stable. In contrast, the combined treatment group showed a significant decrease in HCT compared to the control group, maintaining an average of 64% ( Figure 3 B, C). Regarding platelet counts, all treatment groups slowed the rate of increase in platelet count, and compared to STAT3-IN25 alone, combination therapy further reduced platelet counts. Figure 3 D).

[0050] Example 4:

[0051] Reference Appendix Figure 4After gavage treatment, compared with the solvent control group, the blood glucose levels in the STAT3-IN25 group and the ruxolitinib group did not recover, but the hypoglycemia level improved after combined administration. Figure 4 A). Spleen dissection in mice revealed that neither the ruxolitinib monotherapy group nor the STAT3-IN25 monotherapy group achieved spleen reduction; the average spleen weight was 0.26g and 0.3g, respectively, greater than that of wild-type mice. However, after combination therapy, the spleen weight of mice was reduced compared to the control group, with an average spleen weight of 0.21g, alleviating the splenomegaly symptoms in transplanted mice. Figure 4 B). Compared with the single-drug group, the combination therapy group significantly reduced the spleen index (4C).

[0052] Example 5:

[0053] Reference Appendix Figure 5 Bone marrow cells were extracted from PMF mice in each treatment group, cells were lysed, and protein samples were prepared. Western blotting was used to detect the protein expression levels of STAT3, p-STAT3 (Ser727), and p-STAT3 (Tyr705). Results showed that compared to the vehicle group, the total STAT3 protein level in each treatment group remained essentially unchanged, while p-STAT3 (Tyr705) levels decreased to varying degrees, with the most significant decrease observed in the combination therapy group. The p-STAT3 (Ser727) protein level in the ruxolitinib group did not decrease significantly. However, compared to the monotherapy group, the p-STAT3 (Ser727) level was further reduced in mice treated with STAT3-IN25 combined with ruxolitinib, essentially inhibited. Figure 5 ).

[0054] Example 6:

[0055] Reference Appendix Figure 6 To further evaluate the efficacy of the combination of the STAT3 inhibitor IN25 and ruxolitinib, spleen and bone marrow samples from mice were collected for flow cytometry analysis after treatment. Results showed that in spleen tissue, compared with the control group, the GFP levels in each treatment group were significantly higher. + The proportions of tumor cells and megakaryocyte lineage cells both decreased, and the spleen GFP in the combined group mice was [increased / reduced / increased]. + Tumor cells and megakaryocyte lineage cells were further reduced, to 55% and 9.6%, respectively, significantly lower than those in the ruxolitinib and STAT3-IN25 monotherapy groups. Figure 6 A, B).

[0056] Unlike spleen tissue, compared with the control group, GFP in the bone marrow of the STAT3-IN25 or ruxolitinib monotherapy group was higher. + The ratio of tumor cells to megakaryocyte lineage cells did not change significantly, but the combination of the two resulted in GFP... +The proportions of tumor cells and abnormal megakaryocytic lineage cells were significantly lower than those in the ruxolitinib and STAT3-IN25 monotherapy groups, decreasing to 49% and 5.02%, respectively. Figure 6 C, D).

[0057] Example 7:

[0058] Reference Appendix Figure 7 The spleen, bone marrow, and sternum were pathologically evaluated to determine the effect of STAT3-IN25 combined with the JAK2 inhibitor ruxolitinib on the tissue disease status of PMF mice.

[0059] HE staining of WT mice revealed distinct red and white pulp divisions in the spleen, with intact structures. No abnormal megakaryocyte proliferation was observed in the bone marrow or spleen. In MPLW515L transplanted mice with 15% Captisol, atypical megakaryocyte proliferation and an increase in immature megakaryocytes were observed in the spleen and bone marrow, with scattered distribution. Megakaryocyte proliferation was inhibited in the ruxolitinib and STAT3-IN25 monotherapy groups, and the combination therapy group showed a significant reduction in immature megakaryocytes and improved spleen structural damage. Figure 7 ).

[0060] The bone marrow of WT mice contained almost no stainable reticular fiber tissue, while the control group mice showed obvious diffuse reticular fibers. The single-drug group mice showed significant fiber staining in some areas, while the combination treatment group showed a significant reduction in silver-stained fibrosis. Figure 7 ).

[0061] Example 8:

[0062] Reference Appendix Figure 8 STAT3-IN20, a STAT3 inhibitor targeting the STAT3 (Tyr705) site, was selected and used to treat 6133 / MPL megakaryocytes. Cell proliferation was inhibited with increasing inhibitor concentration. After treatment with 1.2 μM STAT3-IN20, cell viability was 67.9%, significantly inhibiting the phosphorylation level of p-STAT3 (Tyr705) protein, but without significantly affecting the phosphorylation level of p-STAT3 (Ser727). Figure 2 A, B).

[0063] Subsequently, 6133 / MPL megakaryocytes were treated with different concentrations of STAT3-IN20 in combination with ruxolitinib. ComPusyn software was used for combination index (Cl) analysis. A Cl value greater than 1 for STAT3-IN20 combined with ruxolitinib indicated that STAT3-IN20 combined with ruxolitinib did not synergistically inhibit the proliferation of 6133 / MPL megakaryocytes. Figure 2 C, Table 1).

[0064] This invention primarily demonstrates that JAK pathway inhibition with ruxolitinib can cover the classical JAK-STAT3 pathway, namely the STAT3 (Tyr705) pathway, but cannot completely block the non-classical STAT3 (Ser727) pathway. Combined inhibition of JAK and STAT3 (Tyr705) does not have a synergistic inhibitory effect on 6133 / MPL megakaryocytes; only combined inhibition of the JAK and STAT3 (Ser727) pathways shows a significant synergistic inhibitory effect on 6133 / MPL megakaryocytes and even MPLW515L transplanted mice. This study delves into a novel approach to treating MPLW515L mutant myeloproliferative disorders by combining STAT3 (Ser727) with ruxolitinib, providing important insights into the previously ineffective treatment of myeloproliferative disorders by simply combining STAT3 inhibitors with ruxolitinib.

[0065] Table 2 shows that STAT3-IN20 combined with ruxolitinib did not have a synergistic inhibitory effect on MPLW515L mutant cells.

[0066] Table 2 Statistical Analysis of the Combined Index (Cl) of STAT3-IN25 and Ruxolitinib

[0067]

[0068] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A pharmaceutical composition, characterized in that: The pharmaceutical composition consists of STAT3-IN25 and Ruxolitinib.

2. The pharmaceutical composition according to claim 1, characterized in that: The molar ratio of STAT3-IN25 to Ruxolitinib is 20–25:

1.

3. The pharmaceutical composition according to claim 2, characterized in that: The molar ratio of STAT3-IN25 to Ruxolitinib is 22.5:

1.

4. The use of the pharmaceutical composition according to claim 1 in the preparation of a drug for treating myeloproliferative disorders with mutations.

5. The application according to claim 4, characterized in that: The mutation types of myeloproliferative disorders include gene mutations in the thrombopoietin receptor protein.

6. The application according to claim 5, characterized in that: The mutation types of myeloproliferative disorders include myeloproliferative disorders with the M PLW515L mutation.

7. The application according to claim 4 or 5, characterized in that: The myeloproliferative disorders mentioned include polycythemia vera, essential thrombocythemia, or essential myelofibrosis.

8. The application according to claim 4 or 5, characterized in that: The STAT3-IN25 can inhibit the expression of STAT3 protein phosphorylated at serine position 727.

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