Use of obeticholic acid for the preparation of a medicament for the treatment of immune thrombocytopenia

Abstract

The application discloses application of obeticholic acid in preparation of a drug for treating immune thrombocytopenia. The obeticholic acid can intervene in an ITP mouse model, reduce platelet destruction in spleen and liver, repair damaged intestinal barrier, reduce FITC-dextran and LPS leakage in plasma, restore intestinal flora-bile acid metabolism balance, and reduce IL-12 secretion and inhibit Th1 cell differentiation by regulating an FXR-SHP channel.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of obeticholic acid in the preparation of drugs for treating immune thrombocytopenic purpura. Background Technology

[0002] Immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disorder, accounting for one-third of all hemorrhagic diseases, with an annual incidence of 5–10 per 100,000 adults. ITP patients present with isolated thrombocytopenia in peripheral blood, with or without bleeding symptoms. Due to immune intolerance to self-antigens, humoral and cellular immunity mediate excessive platelet destruction, leading to abnormal megakaryocyte count and quality, thereby limiting platelet production. The gut microbiota is associated with the development of ITP, including the colonization of pathogenic bacteria and changes in the composition of symbiotic flora. For example, Helicobacter pylori infection induces host-produced CagA antibodies that cross-react with platelet surface glycoproteins, accelerating platelet clearance by the reticuloendothelial system. Metagenomic sequencing analysis of fecal microbiota from 48 newly diagnosed ITP patients and 52 healthy volunteers revealed higher heterogeneity in the gut microbiota composition of ITP patients. ITP gut microbiota dysbiosis exhibits unique species characteristics and gene functions. Treatment-naïve ITP patients show significantly increased abundances of *Ruminococcus*, *Bifidobacterium longum*, and *Ackermania marcescens*, which correlate with clinical indicators such as platelet count and disease duration. Microbiota function analysis revealed reduced biosynthesis of secondary bile acids and flagellar assembly in the gut microbiota of ITP patients, which may affect the immune system and thus promote the development and progression of ITP. Given that bile acids can regulate monocyte, macrophage, or dendritic cell states and thus T cell differentiation and immune homeostasis through bile acid receptor-dependent or independent signaling pathways such as the farnesoid X receptor (FXR) or G protein-coupled receptor (TGR5), we believe that bile acid synthesis and metabolism disorders in the gut of ITP may mediate immune cell differentiation and homeostasis imbalance through bile acid receptor signaling pathways.

[0003] Obeticholic acid (OCA or 6-ECDCA), a synthetic bile acid, is an FXR agonist used to treat primary biliary cholangitis. Studies have shown that oral administration of obeticholic acid to mice with experimental autoimmune encephalomyelitis significantly alleviated disease symptoms and reduced CD4 counts. + The expression of BTLA and PD-1 on the surface of T lymphocytes and B lymphocytes can increase CD8 expression. + The expression of BTLA on the surface of T cells inhibits lymphocyte activation and the secretion of related cytokines. However, there are no reports on the application of obeticholic acid in the treatment of immune thrombocytopenic purpura. Summary of the Invention

[0004] The purpose of this invention is to provide a new pharmaceutical use for obeticholic acid.

[0005] In a first aspect, the present invention claims protection for the use of a1) obeticholic acid, a2) a pharmaceutically acceptable salt of obeticholic acid, or a3) a substance having obeticholic acid or a pharmaceutically acceptable salt of obeticholic acid as an active ingredient in the following respects:

[0006] To prepare products for the treatment of immune thrombocytopenic purpura.

[0007] Secondly, the present invention claims the use of a1) obeticholic acid, a2) a pharmaceutically acceptable salt of obeticholic acid, or a3) a substance having obeticholic acid or a pharmaceutically acceptable salt of obeticholic acid as an active ingredient in the preparation of a medicament for treating complications or comorbidities of immune thrombocytopenic purpura:

[0008] The comorbidities or complications of immune thrombocytopenic purpura include any of the following:

[0009] 1) Destruction of platelets in the spleen and liver;

[0010] 2) Damage to the intestinal barrier;

[0011] 3) Leakage of FITC-dextran and LPS in plasma;

[0012] 4) Disorders of gut microbiota-bile acid metabolism;

[0013] 5) The proportion of Th1 cells in the bone marrow increases.

[0014] Thirdly, this invention claims protection for a product.

[0015] The product claimed in this invention has obeticholic acid or a pharmaceutically acceptable salt of obeticholic acid as its active ingredient; the product has any of the following uses:

[0016] 1) Treatment of immune thrombocytopenic purpura;

[0017] 2) Treatment of complications or comorbidities of immune thrombocytopenic purpura;

[0018] The complications or comorbidities of immune thrombocytopenic purpura include any of the following:

[0019] 1) Destruction of platelets in the spleen and liver;

[0020] 2) Damage to the intestinal barrier;

[0021] 3) Leakage of FITC-dextran and LPS in plasma;

[0022] 4) Disorders of gut microbiota-bile acid metabolism;

[0023] 5) The proportion of Th1 cells in the bone marrow increases.

[0024] The product described in this invention may specifically be a pharmaceutical product.

[0025] When necessary, one or more pharmaceutically acceptable carriers may be added to the above-mentioned drugs; the carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbents, lubricants, etc., which are commonly used in the pharmaceutical field.

[0026] The above-mentioned drugs can be formulated into various forms such as injections, tablets, powders, granules, capsules, oral liquids, ointments, and creams; all of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0027] The aforementioned drugs can be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, through injection, spray, nasal drops, eye drops, osmosis, absorption, or physical or chemical mediated methods; or they can be introduced into the body after being mixed with or encapsulated by other substances.

[0028] Fourthly, the present invention claims protection for a medicament for treating immune thrombocytopenic purpura.

[0029] The medicament for treating immune thrombocytopenic purpura claimed in this invention has an active ingredient of obeticholic acid or a pharmaceutically acceptable salt of obeticholic acid.

[0030] Fifthly, the present invention claims protection for a medicament for treating complications or comorbidities of immune thrombocytopenic purpura.

[0031] The medicament claimed in this invention for treating complications or comorbidities of immune thrombocytopenic purpura has an active ingredient of obeticholic acid or a pharmaceutically acceptable salt of obeticholic acid.

[0032] In all of the above aspects, the products described can be pharmaceuticals.

[0033] Obeticholic acid is a safe, oral FXR agonist used to treat primary biliary cholangitis. Many clinical trials (Phase I-III) have begun using obeticholic acid to treat non-alcoholic steatohepatitis, type 2 diabetes, and other conditions. Combined with research in various autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, disruption of the intestinal barrier may play a significant role in the persistent chronic immune imbalance in autoimmune diseases. Coupled with the resulting disturbances in gut microbiota and bile acid metabolism, obeticholic acid can specifically correct this gut microbiota-bile acid metabolic imbalance, providing a new approach to the treatment of autoimmune diseases. Attached Figure Description

[0034] Figure 1 A indicates that Z-GS significantly inhibits FXR activation in mouse bone marrow-derived dendritic cells, while OCA significantly promotes FXR activation in mouse bone marrow-derived dendritic cells.

[0035] Figure 1 B indicates that when mouse bone marrow-derived dendritic cells interact with CD4... + When T cells are co-cultured, Z-GS pretreatment significantly increases dendritic cell-induced Th1 cell differentiation, while OCA and GW4064 reduce Th1 cell differentiation induced by Z-GS pretreatment of dendritic cells.

[0036] Figure 1 C indicates that Z-GS significantly increases IL-12 secretion from mouse bone marrow-derived dendritic cells, while OCA significantly reduces IL-12 secretion from mouse bone marrow-derived dendritic cells.

[0037] Figure 2 The results indicate that Z-GS can significantly increase the expression of p38, p-p38, and p-JNK in mouse bone marrow-derived dendritic cells, while OCA and GW4064 can reduce the p38 expression induced by Z-GS pretreatment of dendritic cells.

[0038] Figure 3 A indicates that OCA can significantly increase platelet count in ITP mice.

[0039] Figure 3 B indicates that OCA can significantly reduce platelet retention and destruction in the liver and spleen of ITP mice.

[0040] Figure 4 A indicates that OCA can significantly reduce the leakage of FITC-dextran in the plasma of ITP mice and repair the intestinal barrier.

[0041] Figure 4 B indicates that OCA can significantly reduce LPS leakage in the plasma of ITP mice and repair the intestinal barrier.

[0042] Figure 4 C indicates that OCA can significantly reduce the proportion of Th1 cell differentiation in the bone marrow of ITP mice.

[0043] Figure 4 D indicates that OCA can significantly reduce the plasma IL-12 cytokine secretion concentration in ITP mice.

[0044] Figure 4 E indicates that OCA can induce FXR expression in bone marrow cells of ITP mice and activate the FXR pathway.

[0045] Figure 5This indicates that OCA can repair the disordered bile acid pool in the intestine of ITP mice and significantly reduce the concentrations of FXR-inhibiting bile acids such as UDCA, α-MCA, and β-MCA.

[0046] Figure 6 This indicates that OCA can regulate the gut microbiota of ITP mice and promote the recovery of bacterial communities such as Lachnospiraceae and Eubacterium. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] Example 1, In vitro experiment

[0050] In vitro culture of mouse bone marrow-derived dendritic cells

[0051] (1) Isolation of mouse bone marrow mononuclear cells: Four bones from the femur and tibia of each mouse were used. Ice-cold PBS was drawn into a syringe to flush the cells from the bone marrow into a 50 mL centrifuge tube. The cells were gently pipetted with a sterile dropper and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in 4 mL PBS. 3 mL Ficoll separation solution was added to a new 15 mL centrifuge tube. The cell suspension was slowly added to the upper layer of the separation solution and centrifuged at 450 g for 20 minutes at 22°C. The white membrane layer was slowly aspirated and 10 mL PBS was added. The cells were gently pipetted and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded. 2 mL of erythromycin lysate was added and placed on ice for 5 minutes. 2 mL of ice-cold PBS was added and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the cells were washed once with PBS and resuspended in complete RPMI 1640 medium.

[0052] (2) Dendritic cell induction culture: IL-4 (10 ng / mL) and GM-CSF (20 ng / mL) were added to the culture system and the cells were cultured in a 37°C cell culture incubator. The medium was changed every other day, and cytokines were added to the system at the same time to observe changes in cell morphology. On day 7, LPS was added to induce dendritic cell maturation.

[0053] Drug intervention:

[0054] On day 8 of culture, after dendritic cell maturation was induced, z-guggulsterone (50 μmol / L), OCA (50 μmol / L) and GW4064 (5 μmol / L) were added to the system, and the cells were incubated at 37°C for 48 hours. Cells were then collected for co-culture or corresponding detection.

[0055] detection indicators

[0056] The activation or inhibition of FXR by different drugs was detected by luciferase reporter gene transfection. The ability of drugs to induce Th1 cell differentiation in DC cells was detected by cell co-culture and flow cytometry. The concentration of IL-12, a cytokine that induces Th1 cell differentiation, secreted by DC cells in the cell culture supernatant after drug treatment was detected by ELISA. Western blot was used to detect the expression of key molecules in the MAPK signaling pathway, such as p38, JNK, and ERK1 / 2, and the expression of phosphorylated products in DC cells after drug treatment.

[0057] result

[0058] Figure 1 A indicates that Z-GS significantly inhibits FXR activation in mouse bone marrow-derived dendritic cells, while OCA significantly promotes FXR activation in mouse bone marrow-derived dendritic cells.

[0059] Figure 1 B indicates that when mouse bone marrow-derived dendritic cells interact with CD4... + When T cells are co-cultured, Z-GS pretreatment significantly increases dendritic cell-induced Th1 cell differentiation, while OCA and GW4064 reduce Th1 cell differentiation induced by Z-GS pretreatment of dendritic cells.

[0060] Figure 1 C indicates that Z-GS significantly increases IL-12 secretion from mouse bone marrow-derived dendritic cells, while OCA significantly reduces IL-12 secretion from mouse bone marrow-derived dendritic cells.

[0061] Figure 2 The results indicate that Z-GS can significantly increase the expression of p38, p-p38, and p-JNK in mouse bone marrow-derived dendritic cells, while OCA and GW4064 can reduce the p38 expression induced by Z-GS pretreatment of dendritic cells.

[0062] Example 2, In vivo experiment

[0063] ITP mouse model construction:

[0064] (1) Preparation of platelet suspension: 8-10 week old C57BL / 6 male mice were anesthetized with isoflurane. Blood was collected from the eyeballs into centrifuge tubes containing 10% CPDA (prepared with CPDA and PBS). The tubes were immediately and gently inverted to mix, ensuring no hair or clots appeared under light. The tubes were centrifuged at 180g for 18 minutes at room temperature. The supernatant (rich in platelets) was carefully transferred to another clean centrifuge tube in a laminar flow hood and centrifuged at 450g for 18 minutes at room temperature. The supernatant was discarded. 1 mL of anticoagulant was added for resuspending. After gentle mixing, platelets from multiple mice were combined, inverted to mix, and centrifuged at 450g for 18 minutes at room temperature. The white precipitate at the bottom of the centrifuge tube was the platelets. The tubes were resuspended in PBS to a concentration of 1×10⁻⁶. 9 / mL.

[0065] (2) Immunization of CD61 KO mice: 6-10 week old CD61 KO mice were selected and injected weekly via tail vein with 1×10⁻⁶ ozontally. 8 Platelet suspensions were continuously diluted for 4-6 weeks, and the titer of IgG antiplatelet antibodies in the plasma of CD61 KO mice was determined by flow cytometry gradient dilution.

[0066] (3) Preparation and injection of spleen cell suspension: CD61 KO mice with high platelet antibody titers were selected, and the spleens were removed after sacrifice. The spleens were gently ground into PBS through a 70 μm filter. After lysing red blood cells, cell counting was performed. The PBS resuspending concentration was (2.5-10)×10⁻⁶. 7 / mL, the specific concentration is determined according to the antibody titer, and each mouse is given an intraperitoneal injection of 200μL of spleen cell suspension.

[0067] (4) Platelet Count Detection and Sample Collection: On days 3, 5, 7, 10, 12, and 15 after spleen cell injection, 20 μL of blood was collected from the tail tip. The platelet count level of mice was measured using the pre-dilution method, and subcutaneous hemorrhage was observed. Before sacrifice, 3-5 pellets of fresh feces weighing approximately 50-200 mg were collected from mice in sterile cryopreservation tubes, flash-frozen in liquid nitrogen, and stored at -80℃ for 16S amplicon sequencing and bile acid targeted metabolomics detection. On day 15 after spleen cell injection, mice were anesthetized with isoflurane, and 1.0-1.5 mL of blood was collected from the eyeball (anticoagulated with heparin). After cervical dislocation and sacrifice, the liver, spleen, tibia / femur, and entire colon of the mice were collected and processed according to the experimental procedure of pathological specimen preparation, RNA extraction, protein extraction, and cell suspension preparation.

[0068] Obeticholic acid in vivo intervention

[0069] ITP mice were randomly divided into a blank control group (CTRL), a model control group (ITP-vector), and an obeticholic acid treatment group (ITP-OCA) in a 1:1:1 ratio. OCA was purchased from AbMole, USA. It was dissolved in DMSO to a concentration of 100 g / L as a stock solution, diluted with PBS, and then mixed with methylcellulose to form a homogeneous suspension, which was administered to mice via gavage. Starting 7 days after spleen cell injection, mice in the treatment group were administered the drug at a dose of 30 mg / kg / day via gavage once daily. The blank control group and the model control group were administered an equal volume of DMSO+PBS+methylcellulose suspension via gavage. Platelet monitoring was performed as before.

[0070] Before euthanasia, collect 3-5 fresh fecal pellets from a sterile cryopreservation tube and retrieve an appropriate amount of contents from the cecum, weighing approximately 50-200 mg. Flash freeze in liquid nitrogen and store at -80°C for bile acid-targeted metabolomics analysis. After anesthetizing mice with isoflurane, collect 1.0-1.5 mL of blood from the eye (anticoagulated with heparin), centrifuge, and retain the plasma. After euthanasia by cervical dislocation, collect the tibia / femur and flush out bone marrow mononuclear cells.

[0071] Intestinal barrier testing

[0072] On day 14 of ITP mouse model establishment, after a 12-hour fast, mice were administered FITC-dextran (molecular weight 3000–5000 kD) by gavage at a dose of 0.6 mg / g body weight, with a gavage volume not exceeding 200 μL per mouse. Four hours after gavage, mice were anesthetized with isoflurane, and 1.0–1.5 mL of blood was collected from the eyeballs (anticoagulated with heparin). The blood was centrifuged at 4°C and 12000 rpm for 10 minutes, and 100 μL of the supernatant plasma was transferred to a 96-well plate. The FITC fluorescence intensity of the plasma in both groups was then measured.

[0073] Cell membrane fluorescent probe DIR in vivo imaging

[0074] On day 14 of ITP mouse modeling, untreated wild-type C57BL / 6 mice were anesthetized with isoflurane, and 1.0-1.5 mL of blood was collected from the eyeballs and added to PBS solution containing 10% CPDA. The mixture was centrifuged at 180g for 18 minutes at room temperature, and the supernatant platelet-rich plasma was slowly aspirated and centrifuged at 450g for 18 minutes at room temperature. The supernatant was discarded, and the resulting precipitate was platelets. The platelets were resuspended in PBS solution containing 10% CPDA and the concentration was adjusted to 10. 9 / mL, add DIR to a final concentration of 12.5ug / mL, incubate at 37℃ in the dark for 45 minutes, centrifuge at 5000rpm for 10 minutes at room temperature, discard the supernatant, and resuspend the DIR-labeled platelets in PBS to a final concentration of 10. 9 / mL, at room temperature, protected from light. ITP mice were anesthetized with isoflurane, and 200 μL / mouse of DIR-labeled platelets were injected via the tail vein to introduce them into the bloodstream. Sixty minutes after injection, the mice were anesthetized with isoflurane, and the upper abdomen was shaved with depilatory cream. Excess cream was wiped off with a damp cotton ball, and platelet retention in the spleen and liver of each group of mice was assessed using an in vivo imaging system (IVIS Spectrum, PerkinElmer, US). Mice were euthanized, and their spleen and liver were removed. Platelet retention in the spleen and liver of both groups of mice was assessed again using imaging.

[0075] detection indicators

[0076] The effects of drugs on platelet destruction in an ITP mouse model were investigated using cell membrane fluorescent probe DIR in vivo imaging. Flow cytometry was used to detect the effects of drugs on bone marrow Th1 cell differentiation. ELISA was used to detect the concentrations of IL-12, a cytokine that induces Th1 cell differentiation, and LPS, a bacterial-associated endotoxin product, in mouse plasma after drug treatment. Real-time quantitative PCR was used to detect the activation of the FXR pathway in mouse bone marrow cells. Bile acid-targeted metabolomics was used to study the changes in the composition of intestinal bile acid metabolites in the ITP mouse model. Bacterial 16S rDNA sequencing was used to identify the species and abundance of bacterial flora in the ITP mouse model.

[0077] Test results

[0078] Figure 3 A indicates that OCA can significantly increase platelet count in ITP mice.

[0079] Figure 3 B indicates that OCA can significantly reduce platelet retention and destruction in the liver and spleen of ITP mice.

[0080] Figure 4 A indicates that OCA can significantly reduce the leakage of FITC-dextran in the plasma of ITP mice and repair the intestinal barrier.

[0081] Figure 4 B indicates that OCA can significantly reduce LPS leakage in the plasma of ITP mice and repair the intestinal barrier.

[0082] Figure 4 C indicates that OCA can significantly reduce the proportion of Th1 cell differentiation in the bone marrow of ITP mice.

[0083] Figure 4 D indicates that OCA can significantly reduce the plasma IL-12 cytokine secretion concentration in ITP mice.

[0084] Figure 4 E indicates that OCA can induce FXR expression in bone marrow cells of ITP mice and activate the FXR pathway.

[0085] Figure 5 This indicates that OCA can repair the disordered bile acid pool in the intestine of ITP mice and significantly reduce the concentrations of FXR-inhibiting bile acids such as UDCA, α-MCA, and β-MCA.

[0086] Figure 6 This indicates that OCA can regulate the gut microbiota of ITP mice and promote the recovery of bacterial communities such as Lachnospiraceae and Eubacterium.

[0087] Obeticholic acid intervention in an ITP mouse model reduced platelet destruction in the spleen and liver, repaired the damaged intestinal barrier, reduced FITC-dextran and LPS leakage in plasma, restored the balance of gut microbiota and bile acid metabolism, and inhibited Th1 cell differentiation by regulating the FXR-SHP pathway and reducing IL-12 secretion. The abundance of bile acid metabolism-related bacteria such as Lachnospiraceae and Eubacterium in the gut microbiota of ITP mice was reduced, and the overall fecal bile acid level was increased, with primary bile acids α-MCA and β-MCA predominating, and a significant increase in FXR-inhibiting bile acids. Obeticholic acid intervention can regulate the gut microbiota of ITP mice and repair the disordered intestinal bile acid pool in ITP mice.

[0088] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of obeticholic acid or a pharmaceutically acceptable salt thereof in the preparation of products for the treatment of immune thrombocytopenic purpura.

2. Use according to claim 1, characterized in that: The product in question is a pharmaceutical product.

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