Use of the bach1 inhibitor asp-8731 in the preparation of a medicament for the prevention and / or treatment of biliary atresia

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of BACH1 inhibitor ASP-8731 in preparation of a medicine for preventing and / or treating biliary atresia. The application verifies the treatment potential of BACH1 inhibitor ASP-8731 on biliary atresia for the first time. The experimental results show that, compared with a control group, ASP-8731 treatment can significantly improve the survival rate of model mice, make the body weight growth trend close to a normal level, and effectively reduce the incidence of jaundice, indicating that BACH1 inhibitor ASP-8731 can improve the survival state of a diseased individual as a whole. In terms of liver function protection, ASP-8731 administration significantly improves the liver function index of mice. Histopathological examination further confirms that ASP-8731 can greatly reduce liver inflammatory cell infiltration. The finding is expected to make up for the current clinical short board that there is still a lack of medicines for effectively controlling inflammation and fibrosis after Kasai operation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the BACH1 inhibitor ASP-8731 in the preparation of drugs for the prevention and / or treatment of biliary atresia. Background Technology

[0002] Biliary atresia is a congenital hepatobiliary disease occurring in infants and young children, characterized by progressive inflammation and fibrotic obstruction of the intrahepatic and extrahepatic bile ducts, leading to cholestasis, liver fibrosis, and cirrhosis. It is the leading cause of liver transplantation in children, and without treatment, affected children typically die within 1-2 years from liver failure. Currently, the standard treatment for biliary atresia is the Kasai procedure (hepatic-jejunostomy). This procedure aims to reconstruct the bile drainage pathway. However, the success rate of the Kasai procedure is limited, and even when successful, most children will still develop progressive liver fibrosis and portal hypertension due to the ongoing inflammation and fibrosis, ultimately requiring liver transplantation. Therefore, the treatment of biliary atresia faces a significant unmet clinical need: there is an urgent need for effective drug therapies that can inhibit or reverse the underlying pathological processes of biliary atresia—namely, biliary reactivity, inflammation, and liver fibrosis.

[0003] The exact etiology of biliary atresia is not fully understood, but it is generally believed to be related to autoimmune responses triggered by viral infections (such as rotavirus and reovirus) and abnormal damage to bile duct epithelial cells. BACH1 (BTB and CNC homology 1) is a key transcription factor that has been found to play a central role in regulating oxidative stress, inflammation, and fibrosis in recent years. However, there are currently no studies on the therapeutic effect of inhibiting BACH1 on biliary atresia. Summary of the Invention

[0004] The applicant's previous research found that the BACH1 inhibitor (ASP-8731) has a therapeutic effect on a mouse model of RRV biliary atresia, significantly improving intrahepatic peribiliary inflammation. Therefore, the primary objective of this invention is to provide a pharmaceutical use for the BACH1 inhibitor (ASP-8731) in the treatment of biliary atresia. To achieve this objective, the technical solution adopted by this invention is as follows: This invention provides the use of BACH1 inhibitors in the preparation of medicaments for the prevention and / or treatment of biliary atresia.

[0005] In some embodiments of the present invention, the BACH1 inhibitor comprises at least one of the following: (a1) Substances that inhibit the activity of BACH1 protein; (a2) Substances that reduce the content of BACH1 protein; (a3) Substances that knock out the BACH1 gene; (a4) Substances that inhibit the expression of the BACH1 gene.

[0006] In some embodiments of the present invention, the BACH1 inhibitor includes nucleic acid molecules, protein molecules, and small molecule compounds; In some embodiments of the present invention, the nucleic acid molecule is microRNA, siRNA, shRNA, dsRNA, sgRNA, nuclease and / or antisense oligonucleotide.

[0007] In some embodiments of the present invention, the protein molecule is a BACH1-specific antibody or a PROTAC molecule that degrades the BACH1 protein.

[0008] In some embodiments of the present invention, the small molecule compound is a small molecule compound that inhibits BACH1.

[0009] In some embodiments of the present invention, the small molecule compound that inhibits BACH1 is ASP-8731.

[0010] In some embodiments of the present invention, the structural formula of the ASP-8731 is shown in formula (I): Formula (I).

[0011] In some embodiments of the present invention, the biliary atresia includes biliary atresia caused by a virus or biliary atresia caused by bile stasis.

[0012] In some embodiments of the present invention, the biliary atresia is caused by viral infection during the neonatal, infancy, or childhood period.

[0013] In some embodiments of the present invention, the virus includes herpesvirus, cytomegalovirus, rotavirus, or reovirus.

[0014] In some embodiments of the invention, the ASP-8731 comprises a pharmaceutically acceptable salt.

[0015] In some embodiments of the present invention, the pharmaceutically acceptable salt includes acid addition salts and base addition salts.

[0016] "Pharmaceutically acceptable acid addition salts" refer to salts that retain the biological effectiveness and properties of the free base, are not undesirable in biological or other respects, and are formed from an inorganic acid and an organic acid, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and such as, but not limited to, acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor 10 sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfate, ethane 1,2 disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, fumaric acid, galactopyric acid, gentian acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, etc.

[0017] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acid and are not undesirable in biological or other respects. These salts are prepared by the addition of an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, thiazolinone, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0018] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients.

[0019] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

[0020] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0021] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0022] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0023] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0024] In some embodiments of the present invention, the administration method of the drug includes oral, subcutaneous, intravenous, or intraperitoneal injection.

[0025] In some embodiments of the present invention, the drug is suitable for mammals.

[0026] In some embodiments of the present invention, the drug is suitable for children or adults.

[0027] In some embodiments of the present invention, the child is a newborn within 28 days of birth, an infant within 1 year of age, a toddler aged 1-3 years, a toddler aged 3-6 years, or a child aged 6 to 18 years.

[0028] In some embodiments of the present invention, the adult is a pregnant female adult, a perinatal female adult, or a lactating female adult.

[0029] In some embodiments of the invention, the medicament includes other active ingredients for treating biliary atresia.

[0030] The beneficial effects of this invention are: This invention, through the construction of a rotavirus-induced biliary atresia model in newborn mice, for the first time validated the therapeutic potential of the BACH1 inhibitor ASP-8731 for this disease, providing a novel approach to clinical drug intervention. Experimental results showed that, compared with the control group, ASP-8731 treatment significantly improved the survival rate of model mice, brought weight gain closer to normal levels, and effectively reduced the incidence of jaundice, indicating that the BACH1 inhibitor ASP-8731 can improve the overall survival status of affected individuals. Regarding liver function protection, after ASP-8731 administration, key indicators in mouse serum, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, direct bilirubin, alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and total bile acids, all decreased significantly, indicating that this compound can simultaneously alleviate hepatocellular damage, restore bile excretion function, and alleviate bile duct damage and cholestasis. Histopathological examination further confirmed that ASP-8731 can significantly reduce inflammatory cell infiltration in the liver. This discovery is expected to fill the current clinical gap of lacking effective drugs for controlling inflammation and fibrosis after Kasai surgery. Attached Figure Description

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results show the survival curves of mice in each group according to this invention.

[0032] Figure 2 The results show the changes in body weight of mice in each group according to this invention.

[0033] Figure 3 The results show the changes in jaundice rate over time in mice of each group in this invention.

[0034] Figure 4 The results of liver function indicators in mice of each group on day 6 of this invention.

[0035] Figure 5 The results of HE staining of the livers of mice in each group on days 6 and 12 of this invention are shown in the scale bar (100 μm). Detailed Implementation

[0036] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0037] Terminology Explanation: Biliary atresia (BA) is a condition in which the intrahepatic and extrahepatic bile ducts are partially or completely blocked. It is a bile duct disease that can eventually lead to liver failure and seriously endanger the patient's life. It is a neonatal disease of unknown etiology.

[0038] BACH1 (BTB and CNC Homology 1): BACH1 is the name of a gene and the protein it encodes. It is a transcriptional regulator that plays a central role in cellular responses to oxidative stress and heme homeostasis.

[0039] ASP-8731 is a selective inhibitor of BACH1. ASP-8731 can inhibit inflammation and vascular occlusion, and induce elevated fetal hemoglobin levels in sickle cell disease.

[0040] Example 1. Experimental materials Laboratory animals: BALB / c WT suckling mice within 24 hours of birth.

[0041] Main reagents and antibodies: (1) Rhesus monkey MMU18006 rotavirus (RRV), titer 1.5 × 10⁻⁶ 6 PFU / mL, dose 20μL; (2) DMSO; (3)ASP-8731 (MCE, HY-167874).

[0042] 2. Experimental methods: 2.1 Animal grouping: Newborn BALB / c mice were randomly divided into the following 3 groups: 1) Control group (CONT group); 2) RRV+DMSO group; 3) RRV+ASP-8731 group.

[0043] 2.2 Injection Method: Normal control group (CONT group): Within 24 hours of birth, mice were intraperitoneally injected with 20 μL of physiological saline using a disposable sterile insulin syringe. Negative control group (RRV+DMSO group): Within 24 hours of birth, mice were intraperitoneally injected with 20 μL of RRV (titer: 1.5 × 10⁻⁶) using a disposable sterile insulin syringe. 6DMSO was induced to form BA using PFU / mL. Starting from day 2, DMSO was injected intraperitoneally using a disposable sterile insulin syringe every other day until day 6 and day 12. In the drug group (RRV+ASP-8731 group), mice were injected with RRV within 24 hours after birth. Starting from day 2, ASP-8731 20 mg / kg was injected intraperitoneally using a disposable sterile insulin syringe every other day until day 6 and day 12.

[0044] 2.3 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 6 and day 12, respectively.

[0045] 2.4 Blood Collection and Biochemical Detection: Cardiac blood was collected from newborn mice on days 6 and 12 after birth. Mice were anesthetized with isoflurane inhalation before blood collection. The abdominal skin was held with forceps on a clean microsurgical table, 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 clear 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 samples were taken to a hospital laboratory for liver function testing using a biochemical analyzer.

[0046] 2.5 HE staining: Fresh mouse liver tissue from days 6 and 12 of each group 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] 3. Observation indicators and detection methods: 3.1 General observation of mice: The survival status, weight, jaundice, and color of urine and feces of each group of mice were observed and recorded daily.

[0048] 3.2 Mouse dissection and sample collection: On days 6 and 12, mice were euthanized and dissected to observe the appearance of the liver and bile ducts.

[0049] 3.3 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.

[0050] 3.4 Liver histopathological examination: Liver tissue was fixed, embedded, sectioned, and stained with hematoxylin and eosin (HE). HE staining can be used to observe the infiltration of inflammatory cells around the intrahepatic bile ducts.

[0051] 4. Experimental Results 1) ASP-8731 treatment improved the survival status of mice. The survival status of mice in each group is as follows: Figures 1-3 As shown.

[0052] Compared with the disease control group, the survival rate of mice treated with ASP-8731 was significantly improved. Figure 1 By day 14 of the experiment, the survival rate of the disease control group was 0%, while the survival rate of the treatment group was 82.35%.

[0053] On day 12, the average weight of mice in the normal group reached 9.313 g, while the average weight of mice in the disease control group was only 3.863 g. The weight of mice in the treatment group reached 6.007 g on day 12, showing a trend towards weight gain similar to the normal group. Figure 2 This indicates that ASP-8731 treatment effectively promoted normal growth in mice.

[0054] The jaundice rate in the treatment group mice was significantly lower than that in the disease control group. Figure 3 By day 8 of the experiment, the jaundice rate in the disease control group was 100%, while it decreased to 58.82% in the treatment group, a reduction of approximately 41.15%, suggesting that ASP-8731 can alleviate jaundice symptoms.

[0055] 2) ASP-8731 treatment improves liver function indicators in mice Liver function indicators of mice in the RRV group and the RRV+ASP-8731 treatment group on day 6 were as follows: Figure 4 As shown.

[0056] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are key enzymatic indicators reflecting the degree of hepatocellular damage; total bilirubin (TBIL) and direct bilirubin (DBIL) are important parameters for assessing bile excretion function; alkaline phosphatase (ALP) and gamma-glutamyl transferase (γ-GT) are closely related to bile duct injury and cholestasis. The results showed that, compared with the RRV group, the serum levels of ALT, AST, TBIL, DBIL, ALP, γ-GT, and total bile acids (TBA) in mice treated with RRV+ASP-8731 were significantly reduced, indicating that ASP-8731 can effectively alleviate hepatocellular damage, improve bile excretion function, and reduce bile duct injury and cholestasis.

[0057] 3) ASP-8731 treatment reduced liver inflammation in mice. The liver HE staining results of mice in the RRV group and the RRV+ASP-8731 treatment group on days 6 and 12 are as follows: Figure 5 As shown.

[0058] In the RRV group, mice showed significant pathological changes in their liver tissue, including inflammatory cell infiltration, bile duct hyperplasia, and focal necrosis, with the pathological damage worsening as the disease progressed. In contrast, the RRV+ASP-8731 treatment group showed a significant reduction in inflammatory cell infiltration in their liver tissue, relatively intact hepatocyte structure, and a marked reduction in bile duct hyperplasia. The pathological damage was significantly improved at all time points compared to the RRV group, indicating that ASP-8731 can effectively inhibit the inflammatory response in the liver.

[0059] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The use of BACH1 inhibitors in the preparation of drugs for the prevention and / or treatment of biliary atresia; The BACH1 inhibitor includes at least one of the following: (a1) Substances that inhibit the activity of BACH1 protein; (a2) Substances that reduce the content of BACH1 protein; (a3) Substances that knock out the BACH1 gene; (a4) Substances that inhibit BACH1 gene expression; The BACH1 inhibitors include nucleic acid molecules, protein molecules, and small molecule compounds.

2. The application according to claim 1, characterized in that: The small molecule compound includes ASP-8731; The structural formula of the ASP-8731 is shown in formula (I): Equation (I).

3. The application according to claim 1, characterized in that: The biliary atresia includes biliary atresia caused by viruses or biliary atresia caused by bile stasis.

4. The application according to claim 3, characterized in that: The viruses include herpesviruses, cytomegaloviruses, rotaviruses, or reoviruses.

5. The application according to claim 2, characterized in that: The ASP-8731 comprises a pharmaceutically acceptable salt.

6. The application according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients.

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

8. The application according to claim 6, characterized in that: The drug can be administered orally, subcutaneously, intravenously, or intraperitoneally.

9. The application according to claim 6, characterized in that: The drug is suitable for mammals.

10. The application according to any one of claims 6 to 9, characterized in that: The drug includes other active ingredients for treating biliary atresia.

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