Application of AIM2 inhibitor in preparation of medicine for treating biliary tract diseases

Abstract

The invention discloses application of an AIM2 inhibitor in preparation of a medicine for treating biliary tract diseases. The AIM2 inhibitor, including ODN A151, has a certain treatment effect on BA, can significantly prolong the survival time of BA mice, improve the liver function of the BA mice and reduce the infiltration quantity of inflammatory cells around intrahepatic bile ducts, and provides a new potential target and a treatment strategy for treatment of BA.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to the application of AIM2 inhibitors in the preparation of drugs for treating biliary tract diseases. Background Art

[0002] Biliary atresia (BA) is a severe biliary tract disease mediated by extrahepatic bile duct obstruction, which usually occurs in the neonatal period. It is characterized by inflammation and fibrosis of the bile ducts, resulting in obstruction of bile flow and liver damage, causing pathological jaundice and liver failure, with a high mortality rate. The causative factors are complex and not yet clear. The Kasai operation (portoenterostomy) is currently the preferred method for treating biliary atresia, but most children with BA still develop cirrhosis due to progressive intrahepatic bile duct inflammatory damage and finally die of liver failure within 2 years.

[0003] Clinically, it has been found that the progression of biliary atresia is closely related to progressive inflammatory damage of the bile ducts. Repeated bile duct inflammatory damage easily leads to irreversible bile duct obstruction, ultimately resulting in cirrhosis and liver failure. However, the specific triggering factors are currently unclear, and the treatment means are limited. Most children ultimately need liver transplantation. Therefore, it is urgent to clarify the pathological mechanism of the occurrence of bile duct inflammatory damage in biliary atresia, and there is an urgent need for new strategies for specific prevention and treatment of bile duct inflammatory damage. Summary of the Invention

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

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides 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 a large amount of pro-inflammatory cytokines, including TNF-α, IL-1β, LIX, and MIP-2. Excessive secretion of TNF-α and IL-1β activates CD177 + cells, thereby regulating their ROS metabolism, leading to an increase in NETs formation, which in turn causes bile duct damage. At the same time, the PANoptosis pathway in M-MDSCs is activated. It can be observed that the expression of the key target AIM2 in its signaling pathway is significantly up-regulated. The use of AIM2 inhibitors can selectively inhibit the signal of the PANoptosis pathway, significantly improve the liver and bile duct damage of patients with biliary tract diseases, and increase body weight and survival rate.

[0008] In some embodiments of the present invention, examples of AIM2 inhibitors include oligodeoxynucleotides (ODNs) A151 (ODN A151, also known as ODN TTAGGG), a synthetic oligonucleotide that contains 4 repeats of the TTAGGG motif and has the following nucleotide sequence, where the bases are linked by phosphorothioate bonds: 5'-TTAGGGTTAGGGTTAGGGTTAGGG-3' (SEQ ID NO:1); and 5'-TTAGGGTTAGGGTTAGGGTTAGGG-3' (SEQ ID NO:2), which contains phosphodiester bonds. Additional oligonucleotide sequences include other types of modified SEQ ID NO:2, such oligonucleotide sequences having the same nucleotide sequence but having different modified backbones.

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

[0010] In some embodiments of the present invention, the biliary atresia also presents as food allergy caused by biliary atresia, jaundice caused by biliary atresia, cholangitis caused by biliary atresia, liver diseases caused by biliary atresia, and intestinal diseases caused by biliary atresia.

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

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

[0013] In some embodiments of the present invention, the jaundice includes pathologic jaundice or neonatal jaundice.

[0014] In some embodiments of the present invention, the pathologic jaundice includes pathologic jaundice caused by viruses or neonatal pathologic jaundice.

[0015] In some embodiments of the present invention, the pathologic jaundice also includes neonatal pathologic jaundice caused by viruses; or the pathologic jaundice caused by viruses includes cytomegalovirus-induced jaundice.

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

[0017] In some embodiments of the present invention, the bacterial cholangitis further includes bacterial cholangitis caused by biliary atresia and bacterial cholangitis complicated after 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 a dosage form suitable for adults; preferably, the children include neonates within 28 days after birth, infants within 1 year old, toddlers aged 1 - 6 years old, and children aged 6 - 18 years old; preferably, the adults include female adults during pregnancy, female adults during the perinatal period, and female adults during lactation.

[0019] In some embodiments of the present invention, the drug further includes other active ingredients, and the other active ingredients include at least one therapeutic preparation or compound for biliary tract diseases known in the art, such as at least one of antibiotics, folic acid, ursodeoxycholic acid, phenobarbital, cholestyramine, and PDE inhibitors (such as dipyridamole).

[0020] In some embodiments of the present invention, the dosage form of the drug includes capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honeyed pills, pills, granules, honeyed refined extracts, sustained - release and controlled - release preparations, oral liquid preparations, injections, chewable tablets, oral tablets, transdermal patches, and effervescent tablets; or, the dosage form of the drug includes dosage forms for administration via the gastrointestinal tract or dosage forms for non - gastrointestinal administration.

[0021] In some embodiments of the present invention, the dosage forms for administration via the gastrointestinal tract include powders, tablets, granules, capsules, sustained - release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, etc.

[0022] In some embodiments of the present invention, the dosage forms for non - gastrointestinal administration include injection dosage forms (such as injections, including various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections); respiratory dosage forms (such as sprays, aerosols, powder aerosols, etc.); skin dosage forms (such as topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal dosage forms (such as eye drops, nasal drops, ophthalmic ointments, gargles, sublingual tablets, adhesive tablets, film - forming agents, etc.); and cavity - route dosage forms (such as suppositories, aerosols, effervescent tablets, drops, dripping 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 the present invention are as follows:

[0025] The AIM2 inhibitor, including ODN A151, has a certain therapeutic effect on BA, can significantly prolong the survival time of BA mice, improve their liver function, and reduce the number of inflammatory cell infiltrations around the intrahepatic bile ducts, providing a new potential target and treatment strategy for the treatment of BA. Description of the Drawings

[0026] Figure 1 It shows the influence of ODN A151 of the embodiment of the present invention on the survival status of BA mice; among them, A shows the external appearance map of each group of mice on the 12th day; B is the fluorescence angiogram of the extrahepatic bile ducts of each group of mice on the 12th day.

[0027] Figure 2 It is the survival curve of each group of mice in the embodiment of the present invention.

[0028] Figure 3 It is the body weight curve of each group of mice in the embodiment of the present invention.

[0029] Figure 4 It is the jaundice rate map of each group of mice in the embodiment of the present invention.

[0030] Figure 5 It shows the influence of ODN A151 of the embodiment of the present invention on the liver function of BA mice. Among them, A is alanine aminotransferase (ALT); B is aspartate aminotransferase (AST); C is alkaline phosphatase (ALP); D is γ-glutamyl transpeptidase (γ-GT); E is total bilirubin (TBIL); F is direct bilirubin (DBIL); G is total bile acid (TBA).

[0031] Figure 6This is the result of the effect of ODN A151 in the embodiment of the present invention on the intrahepatic bile ducts and periductal inflammatory infiltration in BA mice. Among them, A is the H&E staining result diagram of the liver of each group of mice on the 12th day; B is the inflammatory cell infiltration area diagram of CK19 immunohistochemical staining of intrahepatic bile ducts of each group of mice on the 12th day; C is the bile duct epithelial cell area diagram of CK19 immunohistochemical staining of intrahepatic bile ducts of each group of mice on the 12th day. Detailed implementation manners

[0032] The content of the present invention will be further described in detail through specific embodiments below. The raw materials, reagents or devices used in the embodiments and comparative examples can be obtained from conventional commercial channels or can be obtained by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are all conventional methods in the art.

[0033] Embodiment

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

[0035] (1) Experimental materials:

[0036] Experimental animals: BALB / c WT neonatal mice within 24 hours after birth.

[0037] Main reagents and antibodies:

[0038] (1) Rhesus rotavirus (RRV) MMU18006, with a titer of 1.5×10 6 PFU / mL, and the dose is 20 μL.

[0039] (2) ODN A151 (MCE)

[0040] (2) Experimental methods and steps:

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

[0042] 2. Injection method: Treatment of the RRV group: Within 24 hours after the birth of BALB / c neonatal mice, 20 μL of RRV (titer: 1.5×10 6PFU / mL) induced BA formation; Treatment of the Saline group: Within 24 hours after the mice were born, 20 μL of saline of the same volume was intraperitoneally injected with a disposable sterile insulin syringe; Treatment of the RRV+ODN A151 group: After injecting RRV into the mice within 24 hours after birth, starting from the second day, RRV+ODN A151 (dose: 50 mg / kg) was intraperitoneally injected with a disposable sterile insulin syringe every other day until the 12th day.

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

[0044] 4. Fluorescent cholangiography of extrahepatic bile ducts: The mice on the 12th day after birth in each group were anesthetized and analgesized with 2% pentobarbital sodium (40 mg / kg) and buprenorphine (0.05 mg / kg). On a clean microscopic operating table, dissection was performed with sterile forceps and scissors to fully expose the liver, gallbladder, and extrahepatic bile ducts of the mice. An insulin syringe filled with the fluorescent contrast agent solution was inserted into the gallbladder cavity, and the contrast agent was slowly injected. Observe whether the contrast agent reaches the jejunum through the extrahepatic bile ducts under the microscope and take pictures.

[0045] 5. Blood collection and biochemical detection: Cardiac blood collection was performed on the 12th day after the neonatal mice were born. When collecting blood, the mice were anesthetized by inhaling isoflurane. On a clean microscopic operating table, the abdominal skin of the mice was picked up with forceps, and the abdomen and chest were cut open with scissors to fully expose the diaphragm of the mice. A small incision was made on the left side of the diaphragm with scissors to expose the heart. An insulin syringe was inserted into the apex of the mouse heart and blood was slowly drawn following the heart rhythm (after inserting the needle into the apex, a distinct breakthrough feeling indicates entering the left ventricle) until no more blood could be drawn. The drawn blood was transferred into an anticoagulation tube, marked, 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 with a volume less than 120 μL was diluted to 120 μL with PBS, and the dilution factor was recorded. Subsequently, the serum was stored in a -30°C refrigerator for testing. The serum to be tested was taken to the hospital laboratory for liver function testing with a biochemical detection instrument.

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

[0047] 7. CK19 immunohistochemical staining: Dewax and hydrate liver tissue sections. Immerse the sections in Tris-EDTA buffer (pH 9.0) and heat them in a microwave oven at 95 °C for 10 minutes for antigen retrieval. Expose the sections to 3% hydrogen peroxide solution for 10 minutes to remove endogenous peroxidase. Treat the sections with 5% goat serum to block non-specific binding. Add rabbit-mouse CK19 primary antibody (diluted 1:200) to the sections and incubate overnight at 4 °C. Incubate the sections with the appropriate secondary antibody at room temperature for 30 minutes. Use 3,3'-diaminobenzidine (DAB) as the chromogen to visualize the immunohistochemical staining. Observe the sections under a microscope, acquire images, and analyze as needed.

[0048] Observation indicators and detection methods:

[0049] 1. Observation of general conditions of mice: Observe and record the survival status, body weight, skin jaundice, and the color of urine and feces of mice in each group every day.

[0050] 2. Dissection of mice and sample collection: On the 12th day, euthanize and dissect the mice, observe the appearance of the liver and bile ducts, and perform fluorescence angiography of the extrahepatic bile ducts using a fluorescent contrast agent.

[0051] 3. Detection of liver function indicators: Use a biochemical analyzer to detect liver function indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), and total bile acid (TBA) in the serum of mice.

[0052] 4. Histopathological examination of liver tissue: Fix, embed, and section the liver tissue, and perform H&E staining and CK19 staining. H&E staining can observe the infiltration of inflammatory cells around the intrahepatic bile ducts, and CK19 immunohistochemical staining can observe the destruction or occlusion of the intrahepatic bile ducts.

[0053] Figures 1 to 4 Effect of ODN A151 on the survival status of BA mice. Among them, Figure 1 A shows the appearance of mice in each group on the 12th day; Figure 1 B is the fluorescence angiogram of the extrahepatic bile ducts of mice in each group on the 12th day; Figure 2 is the survival curve of mice in each group; Figure 3 is the body weight curve of mice in each group; Figure 4 is the jaundice rate chart of mice in each group.

[0054] It can be seen that the jaundice rate of mice in the RRV group is greater than 90%, indicating that the above-mentioned RRV treatment can successfully establish a model; the BA symptoms of skin jaundice in the RRV group of mice treated with ODN A151 were significantly improved ( Figure 1In A), the jaundice rate decreased, with 0% in the normal saline group, 100% in the experimental group, and 58.82% in the drug treatment group ( Figure 4 ), and the extrahepatic bile duct was completely unobstructed ( Figure 1 In B), the decrease in the average survival body weight slowed down significantly. Among them, the normal saline group was 8.82 ± 0.52 g, the experimental group was 3.68 ± 0.21 g, and the drug treatment group was 5.67 ± 2.42 g ( Figure 3 ), and the survival time was significantly prolonged. Among them, all the mice in the experimental group died on the 17th day, while nearly half of the mice in the drug treatment group survived for more than 20 days. ( Figure 2 ).

[0055] Figure 5 Show the effects of ODN A151 on the liver function of BA mice. Among them, A is alanine aminotransferase (ALT); B is aspartate aminotransferase (AST); C is alkaline phosphatase (ALP); D is γ-glutamyl transpeptidase (γ-GT); E is total bilirubin (TBIL); F is direct bilirubin (DBIL); G is total bile acid (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 Are the results of the effects of ODN A151 on the intrahepatic bile ducts and periductal inflammatory infiltration in BA mice. Among them, A is the H&E staining result diagram of the liver of each group of mice on the 12th day; B is the inflammatory cell infiltration area diagram of CK19 immunohistochemical staining of intrahepatic bile ducts of each group of mice on the 12th day; C is the bile duct epithelial cell area diagram of CK19 immunohistochemical staining of intrahepatic bile ducts of each group of mice on the 12th day.

[0058] It can be seen that on the 12th day, the inflammatory cell infiltration around the intrahepatic bile ducts in the RRV group of mice increased significantly, and the intrahepatic bile ducts were blocked; compared with the RRV group of mice, the inflammatory cell infiltration around the intrahepatic bile ducts in the RRV mice treated with ODN A151 decreased significantly, there was a normal intrahepatic bile duct structure, and the degree of bile duct injury was significantly reduced.

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

Claims

1. Use of AIM2 inhibitors or their derivatives in the preparation of drugs for the treatment of biliary diseases.

2. The use according to claim 1, characterized in that: The AIM2 inhibitor includes oligonucleotide A151.

3. The use according to claim 1, characterized in that: The biliary diseases include biliary atresia, cholangitis, cholecystitis, cholelithiasis, biliary parasitic diseases, and jaundice.

4. The use according to claim 1, characterized in that: The biliary atresia may also manifest as food allergies caused by biliary atresia, jaundice caused by biliary atresia, cholangitis caused by biliary atresia, liver diseases caused by biliary atresia, and intestinal diseases caused by biliary atresia.

5. The use according to claim 1, characterized in that: The jaundice includes pathological jaundice or infantile jaundice.

6. The use according to claim 1, characterized in that: The cholangitis includes bacterial cholangitis, viral cholangitis, cholangitis caused by biliary atresia, and cholangitis complicated by biliary atresia surgery.

7. The use according to claim 1, characterized in that: The dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults; preferably, the children include newborns within 28 days of birth, infants under 1 year old, toddlers aged 1 to 6 years old, and children aged 6 to 18 years old; preferably, the adults include female adults in pregnancy, female adults in the perinatal period, and female adults in lactation.

8. The use according to claim 1, characterized in that: The medicine further comprises other active ingredients, and the other active ingredients include at least one of antibiotics, folic acid, ursodeoxycholic acid, phenobarbital, cholestyramine, and a PDE inhibitor.

9. The use according to claim 1, characterized in that: The dosage forms of the drug include capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, injections, chewable tablets, buccal tablets, transdermal patches and effervescent tablets; or, the dosage forms of the drug include dosage forms for gastrointestinal administration or non-gastrointestinal administration.

10. The use according to claim 1, characterized in that: The unit dosage of the AIM2 inhibitor or its derivative in the medicine is 0.1 mg to 1 g.

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