Use of ivermectin in the preparation of a medicament for treating immune thrombocytopenia

Abstract

This invention discloses the application and method of a STAT1-targeting inhibitor in the treatment of immune thrombocytopenic purpura (ITP). It employs ivermectin, a STAT1 nuclear translocation inhibitor, and fludarabine, a STAT1 activation inhibitor. By injecting either the activation inhibitor or the nuclear translocation inhibitor, the platelet count in a mouse model of ITP is increased. Fludarabine, a fluorinated nucleotide analog of vidarabine, is non-radioactive and a small-molecule phosphorylation inhibitor. Ivermectin is a small-molecule inhibitor of nuclear translocation mediated by α / β1 introgression protein. Both have high bioavailability and are widely used to treat various hematological diseases with good safety profiles. Their application in treating ITP is safe, effective, and shows high compliance.

Description

Technical Field

[0001] This invention relates to the application of a drug for treating immune thrombocytopenic purpura, belonging to the field of biomedicine, and particularly to a method for using a STAT1-targeting inhibitor in the treatment of immune thrombocytopenic purpura. Background Technology

[0002] Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disease characterized by low platelet counts and an increased risk of bleeding complications. The annual incidence of ITP is approximately 2 to 6 cases per 100,000 children and 3 cases per 100,000 adults. Importantly, ITP can also occur secondary to many other diseases, including autoimmune diseases such as systemic lupus erythematosus, cancer, and viral diseases such as hepatitis C (HCV), HIV / AIDS, and COVID-19. Recent reports also indicate that some COVID-19 vaccines may increase the risk of ITP. ITP results from the accumulation of multiple cellular mechanisms, including impaired platelet production, autoantibody-mediated macrophage phagocytosis and destruction of platelets, and direct toxicity from cytotoxic T cells. Among the complex pathological factors of ITP, the enhanced macrophage phagocytic function leading to excessive platelet destruction is one of the most important links in the pathogenesis of ITP. However, research on how macrophages regulate platelet phagocytosis in the ITP microenvironment is lacking, which hinders the development of therapeutic strategies and drugs targeting macrophage phagocytosis to ultimately cure ITP.

[0003] The current treatment goal is to stop active bleeding and reduce the risk of future bleeding. If a patient experiences active, major bleeding, emergency treatment is required. Appropriate and specific measures include discontinuing anticoagulants and antiplatelet drugs, and using platelet transfusions, glucocorticoids, intravenous immunoglobulin (IVIG), or a combination of all these drugs / methods. Drug treatment for ITP patients who do not respond initially to glucocorticoids or whose platelet counts repeatedly decline after discontinuation of glucocorticoids includes thrombopoietin receptor agonists, rituximab, and fostatinib. However, ITP treatment requires long-term medication, most patients are not sensitive to glucocorticoids, and patient compliance with injectable drugs such as IVIG is low. Clinical trials of oral drugs such as eltrombopag have shown that these treatments have long onset times, short durations of effectiveness, and a high risk of relapse, and are accompanied by significant side effects such as bone marrow suppression. Splenectomy, a non-pharmacological treatment, has no predictive value and has high patient inclusion criteria, making it a non-preferred treatment option.

[0004] Specifically, eltrombopag is a thrombopoietin receptor agonist approved by the U.S. Food and Drug Administration (FDA) and used as a daily tablet (with dietary restrictions). It takes effect within 1 to 2 weeks of administration and usually requires continued treatment to maintain its effectiveness. Only 10-30% of patients can discontinue treatment after several months or years of receiving thrombopoietin receptor agonists; most patients experience late relapse. Regarding safety, the main risk is an increased risk of venous thromboembolism; other adverse reactions include elevated liver transaminase levels.

[0005] Rituximab is the most widely used immunomodulatory agent for ITP patients, although it is not approved by the FDA for this indication. Retrospective studies show that 60% of patients experience partial remission of ITP after using rituximab, demonstrating a high response rate. The effect is onset within 1 to 2 weeks of administration, and some patients achieve complete symptom remission after 6 months of continuous administration, with a long duration of remission (more than 2 years). Although a large proportion of ITP patients relapse, most respond to rituximab retreatment. Regarding adverse reactions, rituximab may increase the risk of mild infections and, extremely rarely, progressive multifocal leukoencephalopathy.

[0006] Fuscatinib is an oral tyrosine kinase (Syk) inhibitor approved by the FDA in 2018 for patients with ITP who have previously failed treatment. Its efficacy is lower than eltrombopag and rituximab (overall response rate approximately 43%, stable response rate approximately 18%), with a median response time of 15 days. Common adverse reactions include diarrhea, hypertension, elevated transaminases, and nausea. Other immunomodulators such as mycophenolate mofetil, azathioprine, dapsone, and danazol are also used clinically in ITP patients. Data supporting their use are largely limited to retrospective observational studies, with approximately 30-60% of patients showing a drug response.

[0007] Aside from medication, splenectomy remains the most effective treatment for ITP, resulting in durable remission in 60-70% of patients. However, due to the availability of effective medications, the potential complications of splenectomy, and the unpredictability of which patients will respond, consideration for splenectomy is typically limited to patients who do not respond to or cannot tolerate standard medication and who have been diagnosed for at least one year (allowing for remission). Short-term risks of splenectomy include surgical and postoperative complications, while long-term risks include venous thromboembolism and sepsis. Patients undergoing splenectomy have an increased risk of bacterial infection and require repeated vaccinations. Other vascular complications such as coronary artery disease, stroke, and chronic thromboembolic pulmonary hypertension have also been occasionally reported.

[0008] In summary, current treatments and drugs for ITP are relatively limited. However, drugs or treatment strategies developed targeting the physiological process of macrophages phagocytizing platelets can fundamentally increase platelet count by inhibiting platelet clearance from the body. This represents a significant expansion and supplement to current clinical approaches. Furthermore, both of these drugs are repurposed existing drugs with high safety, few side effects, and broad application prospects. Summary of the Invention

[0009] The main technical problem addressed by this invention is how to provide a targeted STAT1 inhibitor, developed for the physiological process of macrophage phagocytosis of platelets, for the treatment of immune thrombocytopenic purpura and its application method.

[0010] To solve the above-mentioned technical problems, one technical solution adopted by the present invention is to provide an application of a STAT1-targeting inhibitor in the treatment of immune thrombocytopenic purpura.

[0011] In a preferred embodiment, the application is to activate the inhibitor to increase the number of platelets.

[0012] In a preferred embodiment, the inhibitor is fludarabine.

[0013] In a preferred embodiment, the application is a nuclear injection inhibitor that increases platelet count.

[0014] In a preferred embodiment, the inhibitor is ivermectin.

[0015] A method for using a STAT1-targeting inhibitor, specifically fludarabine, in the treatment of immune thrombocytopenic purpura includes the following steps:

[0016] S1: Weigh out fludarabine, a STAT1 activation inhibitor;

[0017] S2: Dissolve the STAT1 activation inhibitor fludarabine weighed in step S1 in DMSO;

[0018] S3: Dilute the DMSO in step S2 with physiological saline.

[0019] The final volume content of DMSO in the diluted solution is 0-5%.

[0020] A method for using a STAT1-targeting inhibitor in the treatment of immune thrombocytopenic purpura, wherein the inhibitor is ivermectin, includes the following steps:

[0021] SS1: Weigh out ivermectin, a STAT1 nuclear entry inhibitor;

[0022] SS2: Dissolve the STAT1 nuclear inhibitor ivermectin weighed in step S1 in DMSO;

[0023] SS3: Dilute the DMSO in step SS2 with physiological saline or medical corn oil.

[0024] The final volume content of DMSO in the diluted solution ranges from 0% to 5%.

[0025] In a preferred embodiment, during the dilution process in step SS3, physiological saline is used for dilution when the DMSO in step SS2 is at a low concentration.

[0026] In a preferred embodiment, during the dilution process in step SS3, when the DMSO in step SS2 is at a high concentration, corn oil for injection is used for dilution.

[0027] The beneficial effects of this invention are as follows: Fludarabine in this invention is a fluorinated nucleotide analog of vidarabine, which is non-radioactive and is a small molecule phosphorylation inhibitor. At the same time, ivermectin is a small molecule inhibitor of nuclear translocation mediated by protein α / β1. Both have high bioavailability and are widely used to treat various hematologic diseases with good safety. They are also used to treat immune thrombocytopenic purpura, which is safe, effective and has high compliance. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort, wherein:

[0029] Figure 1 This invention relates to the application and method of targeting STAT1 inhibitors in the treatment of immune thrombocytopenic purpura and platelet counts in ITP (Adap- / -) mice after treatment with fludarabine (50 mg / kg) or saline for 4 days.

[0030] Figure 2 This invention relates to the application and method of targeting STAT1 inhibitors in the treatment of immune thrombocytopenic purpura and platelet counts in ITP (Adap- / -) mice after 5 days of treatment with ivermectin (1 mg / kg and 3 mg / kg).

[0031] Figure 3 This invention relates to the application and method of targeting STAT1 inhibitors in the treatment of immune thrombocytopenic purpura and platelet counts in ITP (Adap- / -) mice after 5 days of treatment with saline.

[0032] Figure 4This invention relates to the application and method of targeting STAT1 inhibitors in the treatment of immune thrombocytopenic purpura and fluorescence spectra of Fc receptor expression on the surface of splenic macrophages in ITP (Adap- / -) mice after 5 days of treatment with ivermectin (1 mg / kg and 3 mg / kg).

[0033] Figure 5 This is a fluorescence spectrum of the expression of Fc receptor on the surface of splenic macrophages after ITP (Adap- / -) mice were treated with saline for 5 days, and the application and method of the STAT1 inhibitor targeted by this invention in the treatment of immune thrombocytopenic purpura.

[0034] Figure 6 This is a diagram showing the average fluorescence intensity of Fc receptor expression on the surface of splenic macrophages after ITP (Adap- / -) mice were treated with ivermectin (1 mg / kg and 3 mg / kg) for 5 days, in comparison with the application and method of the STAT1 inhibitor in the treatment of immune thrombocytopenic purpura.

[0035] Figure 7 This is a diagram showing the average fluorescence intensity of Fc receptor expression on the surface of splenic macrophages after ITP (Adap- / -) mice were treated with saline for 5 days, comparing the application and method of the STAT1-targeting inhibitor in the treatment of immune thrombocytopenic purpura.

[0036] Figure 8 This is a schematic flowchart of the method for preparing fludarabine for the treatment of immune thrombocytopenic purpura using STAT1 according to the present invention.

[0037] Figure 9 This is a schematic flowchart of the method for preparing ivermectin for treating immune thrombocytopenic purpura using STAT1 according to the present invention. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] The application of a STAT1 nuclear translocation / activation inhibitor in the treatment of immune thrombocytopenic purpura, wherein the application is that the STAT1 activation inhibitor increases the number of platelets, and the inhibitor is fludarabine.

[0041] Example 2

[0042] The application of a STAT1 nuclear translocation / activation inhibitor in the treatment of immune thrombocytopenic purpura, wherein the application is that the STAT1 nuclear translocation inhibitor increases the platelet count, and the inhibitor is ivermectin.

[0043] Example 3

[0044] A method for preparing a fludarabine solution for treating immune thrombocytopenic purpura using a STAT1 activation inhibitor includes the following steps:

[0045] S1: Weigh out fludarabine, a STAT1 activation inhibitor;

[0046] S2: Dissolve the STAT1 activation inhibitor fludarabine weighed in step S1 in DMSO;

[0047] S3: Dilute the DMSO in step S2 with physiological saline.

[0048] The final volume content of DMSO in the diluted solution is 0-5%.

[0049] Example 4

[0050] A method for preparing ivermectin solution for treating immune thrombocytopenic purpura using a STAT1 nuclear translocation inhibitor includes the following steps:

[0051] SS1: Weigh out ivermectin, a STAT1 nuclear entry inhibitor;

[0052] SS2: Dissolve the STAT1 nuclear inhibitor ivermectin weighed in step S1 in DMSO;

[0053] SS3: Dilute the DMSO in step SS2 with physiological saline or medical corn oil.

[0054] The final volume content of DMSO in the diluted solution ranges from 0% to 5%.

[0055] During the dilution process in step SS3, when the DMSO in step SS2 is at a low concentration (0-5%), it is diluted with physiological saline.

[0056] During the dilution process in step SS3, when the DMSO in step SS2 is at a high concentration (>5%), corn oil for injection is used for dilution.

[0057] Example 5

[0058] A method for treating immune thrombocytopenic purpura by using the STAT1 activation inhibitor fludarabine to increase platelet count includes the following steps:

[0059] 1. Weigh an appropriate amount of the STAT1 activation inhibitor fludarabine, dissolve it in DMSO, and then dilute it with physiological saline so that the final volume content of DMSO does not exceed 5%. Use it immediately after dilution.

[0060] 2. The administration method is intravenous injection, with a dose of 50 mg / kg, for one week.

[0061] Example 6

[0062] A method for treating immune thrombocytopenic purpura by using the STAT1 nuclear entry inhibitor ivermectin to increase platelet count includes the following steps:

[0063] 1. Weigh an appropriate amount of ivermectin, a STAT1 nuclear inhibitor, and dissolve it in DMSO. For low concentrations, dilute with physiological saline; for high concentrations, dilute with corn oil for injection to ensure the final DMSO volume content does not exceed 5%. Use immediately after dilution.

[0064] 2. The administration method is intraperitoneal injection, with a dose of 1 mg / kg or 3 mg / kg, for one week.

[0065] In the specific experimental process, we have provided specific experimental data reference graphs, specifically in the graphs:

[0066] Figure 1 Platelet counts were measured in control and ITP (Adap- / -) mice after 4 days of treatment with fludarabine (50 mg / kg) or saline. Control mice had a WT value of n=4, and ITP mice had an Adap- / - value of n=4.

[0067] Figure 2 Platelet counts were measured in control and ITP (Adap- / -) mice after 5 days of treatment with ivermectin (1 mg / kg and 3 mg / kg). Control mice had a WT of n=4; ITP mice had an Adap- / - of n=4.

[0068] Figure 3 Platelet counts were measured in control and ITP (Adap- / -) mice after 5 days of treatment with saline. Control mice had a WT value of n=4; ITP mice had an Adap- / - value of n=4.

[0069] Figure 4 The figure shows the expression of Fc receptors on the surface of splenic macrophages in control and ITP (Adap- / -) mice after 5 days of treatment with ivermectin (1 mg / kg and 3 mg / kg). The fluorescence spectra of FcγRI and FcγRIV were measured by flow cytometry (FACS).

[0070] Figure 5The figure shows the expression of Fc receptors on the surface of splenic macrophages in control and ITP (Adap- / -) mice after 5 days of treatment with saline. The fluorescence spectra of FcγRI and FcγRIV were measured by flow cytometry (FACS).

[0071] Figure 6 The figure shows the expression of Fc receptors on the surface of spleen macrophages in control and ITP (Adap- / -) mice after 5 days of treatment with ivermectin (1 mg / kg and 3 mg / kg). The histogram shown in the figure represents the average fluorescence intensity.

[0072] Figure 7 The figure shows the expression of Fc receptors on the surface of spleen macrophages in control and ITP (Adap- / -) mice after 5 days of treatment with saline. The histogram shown in the figure represents the average fluorescence intensity.

[0073] Through the above embodiments, we have found that,

[0074] 1) From the perspective of drug action mechanism, we found a correlation between low ADAP gene levels in the spleen of ITP patients and mice and low platelet counts. Molecular mechanism studies have shown that ADAP interacts with STAT1 and competes for binding to STAT1's nuclear shuttle receptor, importin 5. In the tissue microenvironment of ITP, the absence or reduction of ADAP levels increases the assembly of the STAT1-importin 5 complex, promotes STAT1 nuclear entry, and leads to enhanced transcriptional selectivity of the phagocytic receptor FcγRI / IV in macrophages. Therefore, inhibiting STAT1 or targeting the interaction of STAT1-importin 5 can alleviate thrombocytopenia.

[0075] 2) We used ADAP knockout mice as ITP model mice to screen for potential therapeutic drugs. In this ITP mouse model, daily treatment with a STAT1 activity inhibitor (fludarabine) at a dose of 50 mg / kg significantly increased peripheral blood platelet counts, reaching levels comparable to control mice after 6 days. A STAT1 nuclear translocation inhibitor (ivermectin), after treatment with 1 mg / kg or 3 mg / kg, induced an increase in platelet counts in both control and ITP mice on day 3. Furthermore, treatment with 3 mg / kg significantly increased platelet counts in ITP mice, reaching levels comparable to control mice. In addition, ivermectin treatment reduced the expression of phagocytic FcγRI and FcγRIV in splenic macrophages, decreased macrophage activation, inhibited platelet clearance, and thus increased platelet counts.

[0076] Therefore, the present invention has the following advantages:

[0077] 1. Fludarabine is a fluorinated nucleotide analog of vidarabine. It is non-radioactive, a small molecule phosphorylation inhibitor with high bioavailability. It is widely used to treat various hematologic diseases, has good safety profile, and its application in the treatment of immune thrombocytopenic purpura expands its applicability. It is safe, effective, and has high compliance.

[0078] 2. Ivermectin is a small molecule inhibitor of nuclear translocation mediated by introgression protein α / β1. It has strong antiviral activity by interfering with the recognition of viral proteins such as HIV, DENV, and SARS-CoV2 by NLS. It has good safety and its application in the treatment of immune thrombocytopenic purpura expands its scope of application. It is safe, effective, and has high compliance.

[0079] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. The application of a STAT1-targeting inhibitor in the preparation of a drug for treating immune thrombocytopenic purpura, wherein the application is a nuclear-entry inhibitor that increases platelet count, and the inhibitor is ivermectin.

2. Use of a STAT1 inhibitor, which is ivermectin, for the preparation of a medicament for the treatment of immune thrombocytopenia, characterized in that, Includes the following steps: SS1: Weigh out ivermectin, a STAT1 nuclear entry inhibitor; SS2: Dissolve the STAT1 nuclear inhibitor ivermectin weighed in step S1 in DMSO; SS3: Dilute the DMSO in step SS2 with physiological saline or medical corn oil; The final volume content of DMSO in the diluted solution ranges from 0-5%.

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