Application of pyroptosis inhibitor in preparation of medicine for treating biliary atresia

Abstract

The invention discloses an application of a pyroptosis inhibitor in preparation of a product for treating biliary atresia, the pyroptosis inhibitor 2-bromohexadecanoic acid has a certain treatment effect on biliary atresia mice, can significantly prolong the survival time of the mice, improve jaundice, improve the liver function of the mice, reduce the infiltration quantity of inflammatory cells around intrahepatic bile ducts, and can be used for treating the biliary atresia. A new potential target and a treatment strategy are provided for treatment of biliary atresia.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of a pyroptosis inhibitor in the preparation of a drug for treating biliary atresia. Background Art

[0002] Biliary atresia (BA) is a severe bile duct disease mediated by extrahepatic bile duct obstruction, usually occurring 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 fatality rate. The pathogenic factors are complex and not yet clear. The Kasai operation (hepatic 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 ultimately die of liver failure within 2 years.

[0003] Clinical findings have shown 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 methods 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 urgently need new strategies for specific prevention and treatment of bile duct inflammatory damage. Summary of the Invention

[0004] The present invention provides the application of a pyroptosis inhibitor in the preparation of a product for treating biliary atresia.

[0005] In some embodiments, the pyroptosis inhibitor includes 2-bromohexadecanoic acid, its geometric isomers, or its pharmaceutically acceptable salts.

[0006] In some embodiments, the treatment of biliary atresia includes at least one of the following:

[0007] Improving jaundice in a subject with biliary atresia;

[0008] Improving liver function indexes in a subject with biliary atresia;

[0009] Reducing the infiltration of inflammatory cells around the intrahepatic bile ducts in a subject with biliary atresia;

[0010] Reducing bile duct damage in a subject with biliary atresia.

[0011] In some embodiments, the improvement of liver function indexes in a subject with biliary atresia includes reducing the content of at least one of the following in the blood of a subject with biliary atresia: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, total bilirubin, direct bilirubin, total bile acid.

[0012] In some embodiments, the product is a drug.

[0013] In some embodiments, the drug further comprises other drugs for treating biliary atresia.

[0014] In some embodiments, the other drugs for treating biliary atresia include at least one of bile acid metabolism regulating drugs and anti-fibrotic drugs.

[0015] In some embodiments, the bile acid metabolism regulating drugs include at least one of drugs that inhibit BAs synthesis (such as obeticholic acid (OCA)), drugs that inhibit the enterohepatic circulation of BAs (such as ASBT inhibitors (such as A4250), NTCP inhibitors (such as Myrcludex-B)).

[0016] In some embodiments, the anti-fibrotic drugs include at least one of drugs that inhibit macrophage activation (such as cenicriviroc (CVC)), drugs that inhibit inflammatory factors (such as pentoxifylline (PTX)), monoclonal antibodies (such as CTGF monoclonal antibody FG-3019).

[0017] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0018] In some embodiments, the pharmaceutically acceptable carrier and / or excipient includes at least one of the following: pH regulator, binder, lubricant, diluent, stabilizer, buffer, emulsifier, viscosity regulator, surfactant, preservative, flavoring agent, coloring agent.

[0019] In some embodiments, the dosage form of the drug is an injection or an oral preparation.

[0020] In some embodiments, the administration method of the drug includes any one of subcutaneous injection, intravenous injection, intraperitoneal injection, and oral administration.

[0021] Term Definition

[0022] "Pyroptosis" is a form of cell death. Usually, after cleavage of the GSDM (gasdermin) family proteins, the N-terminal free peptide segments are formed. These peptide segments will induce the formation of pores in the cells and cause cell rupture, thereby releasing cytoplasmic components. "Pyroptosis inhibitor" refers to a compound that can inhibit the process of pyroptosis.

[0023] "Treatment" refers to a regimen used to obtain a beneficial or desired result, including a clinical result. Beneficial or desired clinical results can include, but are not limited to: alleviation or improvement of one or more symptoms or conditions, reduction in the severity of a disease, stabilization (i.e., non-worsening) of a disease state, prevention of the spread of a disease, delay or slowing of disease progression, improvement or alleviation of a disease state, and remission (whether partial or complete), whether detectable or not. The term can also refer to an extension of survival compared to what would be expected without treatment.

[0024] "Biliary atresia (BA)" is characterized by the progressive inflammation and fibrotic obliteration of the intrahepatic and extrahepatic biliary tract system, resulting in severe obstructive cholestasis, and ultimately leading to cholestatic cirrhosis, portal hypertension, and liver failure, which is the main cause of pediatric liver transplantation currently.

[0025] "2-Bromohexadecanoic acid (2-BP; CAS No.: 18263-25-7)" is a palmitoylation inhibitor that targets the DHHC (Asp-His-His-Cys) protein palmitoyltransferase. 2-BP inhibits the palmitoylation of GSDME-C and suppresses pyroptosis mediated by the BAK / BAX-Caspase 3-GSDME pathway. 2-BP inhibits the DHHC protein palmitoyltransferase: 2-BP blocks the catalytic action by irreversibly binding to the active center (Cys residue) of the DHHC family proteins, thereby inhibiting protein palmitoylation modification. Palmitoylation is a lipid modification that regulates protein membrane localization, stability, and function, and is widely involved in signal transduction, cell death, and metabolic processes. 2-BP blocks the palmitoylation of GSDME-C: GSDME (Gasdermin E) is a key effector molecule of pyroptosis, and the palmitoylation of its C-terminus is crucial for its membrane localization and pore-forming activity. 2-BP inhibits the palmitoylation of GSDME-C, weakens its activity on the membrane, and thus prevents the occurrence of pyroptosis. 2-BP inhibits the BAK / BAX-Caspase 3-GSDME pathway: In the pyroptosis pathway, the mitochondrial apoptosis proteins BAK / BAX trigger the change in mitochondrial membrane permeability and activate Caspase-3. Caspase-3 cleaves GSDME, releasing its N-terminal effector domain (GSDME-N), inducing membrane pore formation and pyroptosis. 2-BP inhibits the activity of this pathway by preventing the palmitoylation modification of key proteins, reducing pyroptosis and related inflammatory responses.

[0026] "Subject" refers to a mammal, including but not limited to, humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc. In certain embodiments, the subject is a human. "Subject with biliary atresia" refers to a subject suffering from biliary atresia.

[0027] "Pharmaceutically acceptable" means recognized in the pharmaceutical field for use in animals, especially for use in humans. "Pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is compatible with the subject and the active ingredient pharmacologically and / or physiologically, which is well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention provides the application of a pyroptosis inhibitor in the preparation of a product for treating biliary atresia. The pyroptosis inhibitor 2-bromopalmitic acid has a certain therapeutic effect on mice with biliary atresia, can significantly prolong their survival time, improve jaundice, improve their liver function, and reduce the number of inflammatory cell infiltrations around the intrahepatic bile ducts, providing a new potential target and therapeutic strategy for the treatment of biliary atresia. Brief Description of the Drawings

[0030] Figure 1 Survival status of mice in each group, where (A) external appearance map of mice in each group on the 12th day, (B) cholangiography of extrahepatic bile ducts of mice in each group, (C) body weight curve of mice in each group, (D) jaundice rate map of mice in each group, (E) survival curve of mice in each group.

[0031] Figure 2 Liver function indexes of mice in each group on the 12th day. Among them, *p < 0.05, **p < 0.01, ***p < 0.001.

[0032] Figure 3 HE staining result map of the liver and CK19 immunohistochemical staining result map of intrahepatic bile ducts of mice in each group on the 12th day, ***p < 0.001, ****p < 0.0001. Detailed Description of the Embodiments

[0033] 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 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.

[0034] Example 1 Therapeutic Effect of 2-BP on BA Mice

[0035] This example aims to explore the therapeutic effect of 2-BP on BA mice. BA was induced by intraperitoneal injection of RRV within 24 hours after the birth of BALB / c neonatal mice. Starting from the second day after intraperitoneal injection of RRV, the mice were intraperitoneally injected with 2-BP (dose: 5 mg / kg) every day until the 12th day. It was observed that the survival time of BA mice treated with 2-BP was prolonged, the jaundice symptoms were alleviated, the liver function indexes were significantly improved, and the number of inflammatory cell infiltrations around the intrahepatic bile ducts was significantly reduced. The specific experimental methods and results are as follows:

[0036] (I) Experimental Materials:

[0037] Experimental animals: BALB / c wild-type neonatal mice within 24 hours after birth.

[0038] Main reagents and antibodies:

[0039] (1) Rhesus rotavirus (RRV) MMU18006, titer 1.5×10 6 PFU / mL, dose 20 μL.

[0040] (2) 2-BP (MCE, HY-111770).

[0041] (II) Experimental Methods:

[0042] Animal grouping: BALB / c neonatal mice were randomly divided into the following three groups:

[0043] 1. Normal saline group;

[0044] 2. Experimental group;

[0045] 3. Drug treatment group.

[0046] Experimental steps:

[0047] Grouping: BALB / c WT neonatal mice within 24 hours after birth were randomly divided into 3 groups: normal saline group, experimental group, and drug treatment group.

[0048] 1. Injection method:

[0049] Treatment of the experimental group: Within 24 hours after the birth of BALB / c neonatal mice, 20 μL of RRV (titer: 1.5×10 6 PFU / mL) was intraperitoneally injected with a disposable sterile insulin syringe to induce the formation of BA.

[0050] Treatment of the normal saline group: Within 24 hours after the birth of the mice, 20 μL of normal saline with the same volume was intraperitoneally injected with a disposable sterile insulin syringe.

[0051] Treatment of the drug - treated group: After injecting RRV into mice within 24 hours after birth, starting from the second day, 2 - BP (dose: 5 mg / kg) was intraperitoneally injected using a disposable sterile insulin syringe every other day until the 12th day.

[0052] From day 0 to day 20, the survival status, survival body weight, and skin jaundice of mice in each group were observed and recorded daily, and blood was collected and liver tissue samples were taken on the 12th day.

[0053] 2. Fluorescent cholangiography of extrahepatic bile ducts:

[0054] On the 12th day after birth, mice in each group were anesthetized and analgesized with 2% sodium pentobarbital (40 mg / kg) and buprenorphine (0.05 mg / kg). On a clean microscopic operating table, dissection was performed using sterile forceps and scissors to fully expose the mouse liver, gallbladder, and extrahepatic bile ducts. An insulin syringe filled with the fluorescent contrast agent solution was inserted into the gallbladder cavity, and the contrast agent was slowly injected. Whether the contrast agent passed through the extrahepatic bile ducts to the jejunum was observed under a microscope and photographed.

[0055] 3. Blood collection and biochemical detection:

[0056] On the 12th day after birth of neonatal mice, blood was collected from the heart. When collecting blood, the mice were anesthetized by inhaling isoflurane. On a clean microscopic operating table, the abdominal skin was picked up with forceps, and the abdomen and chest were cut open with scissors to fully expose the mouse diaphragm. 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 heartbeat rhythm (after inserting the needle into the apex, when there was an obvious breakthrough feeling, it entered the left ventricle) until no more blood could be drawn. The drawn blood was transferred into an anticoagulation 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 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 using a biochemical detection instrument.

[0057] 4. HE staining:

[0058] The fresh mouse liver tissues on the 12th day of each group were 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 tissues were observed under a microscope.

[0059] 5. CK19 immunohistochemical staining:

[0060] The liver tissue sections were dewaxed and hydrated. The sections were 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 exposed to 3% hydrogen peroxide solution for 10 minutes to remove endogenous peroxidase. The sections were treated with 5% goat serum to block non-specific binding. Rabbit-mouse CK19 primary antibody (diluted 1:200) was added to the sections and incubated overnight at 4 °C. The sections were incubated with the appropriate secondary antibody for 30 minutes at room temperature. 3,3'-Diaminobenzidine (DAB) was used as a chromogen to visualize immunohistochemical staining. The sections were observed under a microscope, images were obtained, and analyzed as needed.

[0061] Observation indicators and detection methods:

[0062] 1. Observation of general condition of mice: From day 0 to day 20, the survival status, body weight, skin jaundice, and the color of urine and feces of mice in each group were observed and recorded daily.

[0063] 2. Mouse dissection and sample collection: On day 12, the mice were euthanized and dissected, the appearance of the liver and bile ducts was observed, and fluorescence cholangiography of the extrahepatic bile ducts was performed using a fluorescent contrast agent.

[0064] 3. Detection of liver function indicators: A biochemical analyzer was used 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.

[0065] 4. Histopathological examination of liver tissue: The liver tissue was fixed, embedded, sectioned, and stained with HE and CK19. HE 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.

[0066] (3) Experimental results:

[0067] 1. Effect of 2-BP on the survival status of BA mice

[0068] The survival status of mice in each group was as Figure 1 shown, where (A) external appearance of mice in each group on day 12; (B) extrahepatic bile duct angiography of mice in each group; (C) body weight curve of mice in each group; (D) jaundice rate chart of mice in each group; (E) survival curve of mice in each group.

[0069] Results showed that: In the experimental group of mice treated with 2-BP, the skin jaundice of BA symptoms was significantly improved ( Figure 1 as shown in A), and the jaundice rate decreased (saline group 0; experimental group 100%; drug treatment group 53.85%) ( Figure 1In (D), the extrahepatic bile duct was completely unobstructed. Figure 1 In (B), the significant slowdown in the decrease of the average survival body weight was obvious (saline group: 9.04 ± 0.63 g; experimental group: 4.74 ± 1.01 g; drug treatment group: 8.85 ± 1.52 g). Figure 1 In (C), all the mice in the experimental group died on the 16th day, and nearly half of the mice in the drug treatment group survived for more than 20 days, and the survival time was significantly prolonged. Figure 1 In (E).

[0070] 2.2 - BP's effect on the liver function of BA mice

[0071] The liver function indexes of each group of mice on the 12th day are as Figure 2 shown. Among them, (A) the content of alanine aminotransferase (ALT) in the blood, (B) the content of aspartate aminotransferase (AST) in the blood, (C) the content of alkaline phosphatase (ALP) in the blood, (D) the content of γ - glutamyl transpeptidase (γ - GT) in the blood, (E) the content of total bilirubin (TBIL) in the blood, (F) the content of direct bilirubin (DBIL) in the blood, (F) the content of total bile acid (TBA) in the blood.

[0072] The results show that: compared with the experimental group, the liver function of BA mice treated with 2 - BP was significantly improved.

[0073] 3.2 - BP's effect on the intrahepatic bile duct and periductal inflammatory infiltration of BA mice

[0074] The HE staining results of the livers of each group of mice on the 12th day are as Figure 3 shown in (A) in the figure, and the immunohistochemical staining results of CK19 in the intrahepatic bile duct are as Figure 3 shown in (B) in the figure.

[0075] The results show that: on the 12th day, the infiltration of inflammatory cells around the intrahepatic bile ducts of the mice in the experimental group increased significantly, and the intrahepatic bile ducts were blocked; compared with the mice in the experimental group, the infiltration of inflammatory cells around the intrahepatic bile ducts of the RRV mice treated with 2 - BP decreased significantly, there was a normal intrahepatic bile duct structure, and the degree of bile duct injury was significantly reduced.

[0076] The above results indicate that 2 - BP has a certain therapeutic effect on BA mice, can significantly prolong their survival time, improve their liver function, and reduce the number of inflammatory cell infiltrations around the intrahepatic bile ducts.

[0077] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by 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. Application of pyroptosis inhibitors in the preparation of products for the treatment of biliary atresia.

2. The use according to claim 1, characterized in that: The cell pyroptosis inhibitor includes 2-bromohexadecanoic acid, a geometric isomer thereof or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1, characterized in that: The treatment of biliary atresia comprises at least one of the following: Improve jaundice in subjects with biliary atresia; Improve liver function indicators in subjects with biliary atresia; Reduce inflammatory cell infiltration around the intrahepatic bile ducts in subjects with biliary atresia; Alleviate bile duct damage in subjects with biliary atresia.

4. The use according to claim 1, characterized in that: The improving of liver function indexes of subjects with biliary atresia comprises reducing the content of at least one of the following in the blood of subjects with biliary atresia: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, total bilirubin, direct bilirubin, and total bile acid.

5. The use according to claim 1, characterized in that: The product described is a drug.

6. The use according to claim 5, characterized in that: The medicine also includes other medicines for treating biliary atresia.

7. The use according to claim 5, characterized in that: The medicine also includes a pharmaceutically acceptable carrier and / or excipient.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable carrier and / or excipient includes at least one of the following: a pH regulator, a binder, a lubricant, a diluent, a stabilizer, a buffer, an emulsifier, a viscosity regulator, a surfactant, a preservative, a flavoring agent, and a coloring agent.

9. The use according to claim 5, characterized in that: The dosage form of the drug is injection or oral preparation.

10. The use according to claim 9, characterized in that: The administration method of the drug includes any one of subcutaneous injection, intravenous injection, intraperitoneal injection and oral administration.

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