Use of aim2 inhibitors in the manufacture of a medicament for the treatment of biliary tract disease

Abstract

This invention discloses the application of AIM2 inhibitors in the preparation of drugs for treating biliary tract diseases. AIM2 inhibitors, including ODN A151, have a certain therapeutic effect on biliary tract infections (BA), significantly prolonging the survival time of BA mice, improving their liver function, and reducing the number of inflammatory cells infiltrating the perihepatic bile ducts, thus providing a new potential target and treatment strategy for BA.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of AIM2 inhibitors in the preparation of drugs for treating biliary tract diseases. Background Technology

[0002] Biliary atresia (BA) is a serious biliary disease mediated by extrahepatic bile duct obstruction, usually occurring in the neonatal period. It is characterized by inflammation and fibrosis of the bile ducts, leading to obstruction of bile flow and liver damage, causing pathological jaundice and liver failure. It has a high mortality rate, and the causative factors are complex and remain unclear. The Kasai procedure (hilar intestinal anastomosis) is currently the preferred treatment for biliary atresia; however, most children with BA still develop cirrhosis due to progressive intrahepatic bile duct inflammation and damage, ultimately dying from liver failure before the age of two.

[0003] Clinical findings indicate that the progression of biliary atresia is closely related to progressive inflammatory damage to the bile ducts. Repeated inflammatory damage to the bile ducts easily leads to irreversible bile duct obstruction, ultimately resulting in cirrhosis and liver failure. However, the specific precipitating factors are currently unclear, and treatment options are limited, with most affected children ultimately requiring liver transplantation. Therefore, it is urgent to elucidate the pathological mechanisms of biliary atresia and inflammatory bile duct damage, and new prevention and treatment strategies specifically targeting inflammatory bile duct damage are urgently needed. Summary of the Invention

[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide the use of AIM2 inhibitors in the preparation of drugs for treating biliary tract diseases.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention proposes the application of AIM2 inhibitors or their derivatives in the preparation of drugs for treating biliary tract diseases.

[0007] Myeloid-derived suppressor cells (M-MDSCs) derived from biliary tract diseases secrete large amounts of pro-inflammatory cytokines, including TNF-α, IL-1β, LIX, and MIP-2. Excessive secretion of TNF-α and IL-1β can activate CD177. + This process modulates the ROS metabolism of cells, leading to increased NET formation and consequently bile duct damage. Simultaneously, the panapoptosis pathway in M-MDSCs is activated. Significant upregulation of AIM2, a key target in this signaling pathway, was observed. AIM2 inhibitors selectively inhibited panapoptosis signaling, significantly improving liver and bile duct damage in patients with biliary tract diseases, increasing body weight, and improving survival rates.

[0008] In some embodiments of the invention, examples of AIM2 inhibitors include oligonucleotides (ODN) A151 (ODN A151, also known as ODN TTAGGG), a synthetic oligonucleotide containing four repeats of the TTAGGG motif, having the following nucleotide sequences, wherein the bases are linked by phosphothioester bonds: 5'-TTAGGGTTAGGGTTAGGGTTAGGG-3' (SEQ ID NO:1); and 5'-TTAGGGTTAGGGTTAGGGTTAGGG-3' (SEQ ID NO:2), which contain phosphodiester bonds. Additional oligonucleotide sequences include SEQ ID NO:2 with other types of modifications, having the same nucleotide sequence but with different modified backbones.

[0009] In some embodiments of the present invention, the biliary tract diseases include biliary atresia, cholangitis, cholecystitis, gallstones, biliary parasitic diseases, and jaundice.

[0010] In some embodiments of the present invention, the biliary atresia may also manifest as food allergies caused by biliary atresia, jaundice caused by biliary atresia, cholangitis caused by biliary atresia, liver disease caused by biliary atresia, and intestinal disease caused by biliary atresia.

[0011] In some embodiments of the present invention, the liver disease caused by biliary atresia includes liver function impairment caused by biliary atresia and / or inflammatory liver disease caused by biliary atresia.

[0012] In some embodiments of the present invention, the intestinal diseases caused by biliary atresia include inflammatory intestinal diseases caused by biliary atresia.

[0013] In some embodiments of the present invention, the jaundice includes pathological jaundice or infantile jaundice.

[0014] In some embodiments of the present invention, the pathological jaundice includes viral pathological jaundice or infantile pathological jaundice.

[0015] In some embodiments of the present invention, the pathological jaundice also includes viral-induced infantile pathological jaundice; or the viral-induced pathological jaundice includes cytomegalovirus jaundice.

[0016] In some embodiments of the present invention, the cholangitis includes bacterial cholangitis, viral cholangitis, cholangitis caused by biliary atresia, and cholangitis complicated by biliary atresia surgery.

[0017] In some embodiments of the present invention, the bacterial cholangitis also includes bacterial cholangitis caused by biliary atresia and bacterial cholangitis following biliary atresia surgery.

[0018] In some embodiments of the present invention, the dosage form of the drug is a dosage form suitable for children or an adult; preferably, the children include newborns within 28 days of birth, infants within 1 year of age, toddlers aged 1 to 6 years, and children aged 6 to 18 years; preferably, the adults include pregnant adult women, perinatal adult women, and lactating adult women.

[0019] In some embodiments of the invention, the medicament further includes other active ingredients comprising at least one known biliary tract disease treatment agent or compound, such as antibiotics, folic acid, ursodeoxycholic acid, phenobarbital, cholestyramine, or a PDE inhibitor (e.g., dipyridamole).

[0020] In some embodiments of the present invention, the dosage form of the drug includes capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, granules, honey-infused pastes, sustained-release preparations, controlled-release preparations, oral liquid preparations, injections, chewable tablets, oral tablets, transdermal patches, and effervescent tablets; or, the dosage form of the drug includes gastrointestinal dosage forms or non-gastrointestinal dosage forms.

[0021] In some embodiments of the present invention, the gastrointestinal dosage form includes powders, tablets, granules, capsules, sustained-release preparations, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, etc.

[0022] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage forms include injectable dosage forms (e.g., injections, including various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections); respiratory dosage forms (e.g., sprays, aerosols, powder inhalers, etc.); skin dosage forms (e.g., topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal dosage forms (e.g., eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive tablets, films, etc.); and cavity dosage forms (e.g., suppositories, aerosols, effervescent tablets, drops, pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.).

[0023] In some embodiments of the present invention, the unit dose of the AIM2 inhibitor or its derivative in the drug is 0.1 mg to 1 g; preferably, the unit dose of the AIM2 inhibitor or its derivative in the drug is 0.1 mg, 0.5 mg, 1.0 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, or 10.0 mg; preferably, the unit dose of the AIM2 inhibitor or its derivative in the drug is 1.0 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, or 5.0 mg.

[0024] The beneficial effects of this invention are:

[0025] AIM2 inhibitors, including ODN A151, have certain therapeutic effects on BA, significantly prolonging the survival time of BA mice, improving their liver function, and reducing the number of inflammatory cells infiltrating the perihepatic bile ducts, providing a new potential target and treatment strategy for BA. Attached Figure Description

[0026] Figure 1 This invention illustrates the effect of ODN A151 on the survival of BA mice. A shows the appearance of mice in each group on day 12; B shows extrahepatic bile duct fluorescence imaging of mice in each group on day 12.

[0027] Figure 2 The figures show the survival curves of mice in each group in the embodiments of the present invention.

[0028] Figure 3 The figures show the body weight curves of mice in each group in the embodiments of the present invention.

[0029] Figure 4 The image shows the jaundice rate of mice in each group according to the embodiments of the present invention.

[0030] Figure 5 This invention relates to the effect of ODN A151 on liver function in BA mice, where A represents alanine aminotransferase (ALT); B represents aspartate aminotransferase (AST); C represents alkaline phosphatase (ALP); D represents gamma-glutamyl transferase (γ-GT); E represents total bilirubin (TBIL); F represents direct bilirubin (DBIL); and G represents total bile acids (TBA).

[0031] Figure 6The following are the results of the effects of ODN A151 on the intrahepatic bile ducts and perihepatic inflammatory infiltration in BA mice according to the embodiments of the present invention. Among them, A is the liver H&E staining results of mice in each group on day 12; B is the area of ​​inflammatory cell infiltration in the intrahepatic bile ducts of mice in each group on day 12 by CK19 immunohistochemical staining; C is the area of ​​bile duct epithelial cells in the intrahepatic bile ducts of mice in each group on day 12 by CK19 immunohistochemical staining. Detailed Implementation

[0032] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0033] Example

[0034] This embodiment tests the therapeutic effect of ODN A151 on BA mice. The specific process is as follows:

[0035] (I) Experimental Materials:

[0036] Laboratory animals: BALB / c WT suckling mice within 24 hours of birth.

[0037] Main reagents and antibodies:

[0038] (1) Rhesus monkey MMU18006 rotavirus (RRV), titer 1.5 × 10⁻⁶ 6 PFU / mL, dose 20μL.

[0039] (2) ODN A151(MCE)

[0040] (II) Experimental Methods and Procedures:

[0041] 1. Grouping: BALB / c WT suckling mice within 24 hours of birth were randomly divided into 3 groups: normal control group (Saline group, saline group), disease model group (RRV group, experimental group), and intervention group (RRV+ODN A151 group, drug treatment group).

[0042] 2. Injection method: RRV group treatment: Within 24 hours after birth, BALB / c newborn mice were intraperitoneally injected with 20 μL of RRV (titer: 1.5 × 10⁻⁶) using a disposable sterile insulin syringe. 6PFU / mL) to induce BA formation; Saline group treatment: Within 24 hours after birth, mice were intraperitoneally injected with 20uL of saline using a disposable sterile insulin syringe; RRV+ODN A151 group treatment: Within 24 hours after birth, mice were injected with RRV, and from the second day, they were intraperitoneally injected with RRV+ODN A151 (dose: 50mg / kg) using a disposable sterile insulin syringe, once every other day until the 12th day.

[0043] 3. Observe and record the survival status, weight, and jaundice of mice in each group every day, and collect blood and liver tissue samples on day 12; continue to observe the parallel groups.

[0044] 4. Extrahepatic bile duct fluorescence contrast imaging: Mice in each group were anesthetized and analgesic on the 12th day after birth using 2% sodium pentobarbital (40 mg / kg) and buprenorphine (0.05 mg / kg). On a clean microsurgical table, the liver, gallbladder and extrahepatic bile ducts of the mice were dissected with sterile forceps and scissors to fully expose the mice. An insulin syringe containing fluorescent contrast agent solution was inserted into the gallbladder cavity and the contrast agent was slowly injected. The contrast agent was observed under a microscope to see if it reached the jejunum through the extrahepatic bile duct and photographed.

[0045] 5. Blood Collection and Biochemical Testing: Cardiac blood was collected from newborn mice on day 12. Mice were anesthetized with isoflurane inhalation before blood collection. On a clean microsurgical table, the abdominal skin was held by forceps, and the abdomen and chest were cut open to fully expose the diaphragm. A notch was cut on the left side of the diaphragm to expose the heart. Using an insulin syringe, blood was slowly drawn from the apex of the heart, following the heartbeat rhythm (a noticeable breakthrough sensation indicates entry into the left ventricle after needle insertion from the apex) until no more blood could be drawn. The drawn blood was transferred to an anticoagulant tube, labeled, and centrifuged at 3000 rpm for 5 minutes at room temperature to separate the serum. The separated serum was transferred to a new EP tube. Serum volumes less than 120 μL were diluted to 120 μL with PBS, and the dilution factor was recorded. The serum was then stored at -30°C for testing. The serum was then taken to a hospital laboratory for liver function testing using a biochemical analyzer.

[0046] 6. HE staining: Fresh mouse liver tissue from each group on day 12 was fixed in 10% formalin and incubated overnight, then embedded in paraffin and sectioned. The sections were then dewaxed, hydrated, stained with hematoxylin, differentiated with 1% hydrochloric acid alcohol, and stained with eosin. Finally, the pathological changes in the liver tissue were observed under a microscope.

[0047] 7. CK19 Immunohistochemical Staining: Liver tissue sections were dewaxed and hydrated. The sections were then immersed in Tris-EDTA buffer (pH 9.0) and heated in a microwave oven at 95°C for 10 minutes for antigen retrieval. The sections were then exposed to 3% hydrogen peroxide solution for 10 minutes to remove endogenous peroxidase. The sections were treated with 5% goat serum to block nonspecific binding. Rabbit-mouse CK19 primary antibody (1:200 dilution) was added to the sections and incubated overnight at 4°C. The sections were then incubated with an appropriate secondary antibody at room temperature for 30 minutes. Immunohistochemical staining was visualized using 3,3'-diaminobenzidine (DAB) as a chromogenic agent. The sections were observed under a microscope, images were acquired, and analysis was performed as needed.

[0048] Observation indicators and detection methods:

[0049] 1. General observation of mice: Observe and record the survival status, weight, jaundice, and color of urine and feces of each group of mice every day.

[0050] 2. Mouse dissection and sample collection: On day 12, the mice were euthanized and dissected to observe the appearance of the liver and bile ducts, and fluorescent contrast agent was used to perform fluorescent contrast imaging of the extrahepatic bile ducts.

[0051] 3. Liver function index detection: The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), alkaline phosphatase (ALP), gamma-glutamyl transferase (γ-GT), and total bile acids (TBA) in mouse serum were detected using a biochemical analyzer.

[0052] 4. Liver histopathological examination: Liver tissue was fixed, embedded, and sectioned, and subjected to H&E staining and CK19 staining. H&E staining can be used to observe the infiltration of inflammatory cells around the intrahepatic bile ducts, while CK19 immunohistochemical staining can be used to observe the destruction or occlusion of the intrahepatic bile ducts.

[0053] Figures 1-4 The effect of ODN A151 on the survival status of BA mice. Figure 1 Image A shows the appearance of mice in each group on day 12; Figure 1 Image B shows the extrahepatic bile duct fluorescence contrast imaging of mice in each group on day 12; Figure 2 Survival curves for each group of mice; Figure 3 The body weight curves for each group of mice; Figure 4 The graph shows the jaundice rate in each group of mice.

[0054] It can be seen that the jaundice rate in the RRV group mice is greater than 90%, indicating that the aforementioned RRV treatment can successfully establish the model; the jaundice of the BA symptoms in the RRV group mice treated with ODN A151 was significantly improved. Figure 1In the middle A group, the jaundice rate decreased, with 0% in the saline group, 100% in the experimental group, and 58.82% in the drug treatment group. Figure 4 The extrahepatic bile ducts are completely patent. Figure 1 In group B, the average weight loss during survival was significantly slower, with the following values: saline group 8.82±0.52g, experimental group 3.68±0.21g, and drug treatment group 5.67±2.42g. Figure 3 The survival time of the mice was significantly extended. In the experimental group, all mice died on day 17, while nearly half of the mice in the drug treatment group survived for more than 20 days. Figure 2 ).

[0055] Figure 5 This study demonstrates the effects of ODN A151 on liver function in BA mice, where A represents alanine aminotransferase (ALT); B represents aspartate aminotransferase (AST); C represents alkaline phosphatase (ALP); D represents gamma-glutamyl transferase (γ-GT); E represents total bilirubin (TBIL); F represents direct bilirubin (DBIL); and G represents total bile acids (TBA). *P<0.05, **P<0.01, ***P<0.001.

[0056] It can be seen that, compared with the RRV group, the liver function of BA mice treated with ODN A151 was significantly improved.

[0057] Figure 6 The results show the effects of ODN A151 on inflammatory infiltration in the intrahepatic bile ducts and perihepatic bile ducts of BA mice. In this image, A shows the liver H&E staining results of mice in each group on day 12; B shows the area of ​​inflammatory cell infiltration in the intrahepatic bile ducts of mice in each group on day 12, stained by CK19 immunohistochemical staining; and C shows the area of ​​bile duct epithelial cells in the intrahepatic bile ducts of mice in each group on day 12, stained by CK19 immunohistochemical staining.

[0058] It can be seen that on day 12, the infiltration of inflammatory cells around the intrahepatic bile ducts in the RRV group mice increased significantly, and the intrahepatic bile ducts became closed. Compared with the RRV group mice, the infiltration of inflammatory cells around the intrahepatic bile ducts in the RRV mice treated with ODN A151 was significantly reduced, and the intrahepatic bile duct structure was normal, and the degree of bile duct damage was significantly reduced.

[0059] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of oligonucleotide A151 in the preparation of drugs for treating biliary atresia.

2. The application according to claim 1, characterized in that: The biliary atresia can also manifest as food allergies, jaundice, liver disease, and intestinal diseases caused by biliary atresia.

3. The application according to claim 2, characterized in that: The liver diseases caused by biliary atresia include cholangitis caused by biliary atresia.

4. The application according to claim 1, characterized in that: The drug also includes other active ingredients, which include at least one of antibiotics, folic acid, ursodeoxycholic acid, phenobarbital, cholestyramine, and PDE inhibitors.

5. The application according to claim 1, characterized in that: The dosage forms of the drugs include capsules, tablets, microcapsules, injections, suppositories, sprays, powders, pills, granules, honey-based ointments, sustained-release preparations, oral liquid preparations, and transdermal patches.

6. The application according to claim 1, characterized in that: The dosage forms of the drug include those administered via the gastrointestinal tract or those administered outside the gastrointestinal tract.

7. The application according to claim 5, characterized in that: The tablets include chewable tablets, oral tablets, and effervescent tablets.

8. The application according to claim 5, characterized in that: The capsules include soft capsules; the pills include drop pills and honey pills.

9. The application according to claim 1, characterized in that: The oligonucleotide A151 is present in a unit dose of 0.1 mg to 1 g in the drug.

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