Application of ganoderic acid A in preparation of medicine for preventing and / or treating biliary atresia

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of ganoderic acid A in preparation of a medicine for preventing and / or treating biliary atresia. The new application of ganoderic acid A in preparation of drugs for preventing and / or treating biliary atresia is found for the first time. The method comprises the following steps: within 24 hours after a BALB / c newborn mouse is born, inducing the formation of BA by intraperitoneal injection of RRV, and from the second day after intraperitoneal injection of RRV, carrying out intraperitoneal injection of Ganoderic Acid A on the mouse every day until the twelfth day, it can be observed that the survival time of BA mice treated by the Ganoderma Acid A is prolonged, the weight loss is obviously improved, the jaundice symptom is relieved, the liver function index is obviously improved, the infiltration number of inflammatory cells around the intrahepatic bile duct is obviously reduced, and the liver bile duct injury is relieved. The invention provides a new potential target and a treatment strategy for treatment of BA, and has potential clinical treatment significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of ganoderic acid A in the preparation of a drug for preventing and / or treating biliary atresia. Background Art

[0002] Biliary atresia (BA) is a serious neonatal disease caused by inflammation and fibrotic occlusion of extrahepatic bile ducts, resulting in cholestasis and progressive liver failure. If not treated promptly, most children will die of end-stage liver disease within two years, which makes BA the most common cause of pediatric liver transplantation. The clinical features of this disease are simple and reproducible: pathological jaundice with direct or conjugated hyperbilirubinemia; acholic stools; hepatosplenomegaly of varying degrees; and the onset of symptoms is limited to the first few months after birth. At the time of diagnosis, the extrahepatic bile ducts are completely blocked. At the tissue level, there is segmental or global loss of the inner layer of the extrahepatic bile duct epithelium, accompanied by extensive fibrosis and occasional focal inflammation. Intrahepatic bile ducts are typically hyperplastic, embedded in portal tracts containing variable inflammation and fibrosis, and surrounded by lobules characterized by cholestasis and varying degrees of giant multinucleated hepatocytes.

[0003] The initial goal of clinical treatment is timely diagnosis so that surgical intervention can remove the remaining remnants of the atretic bile ducts and establish a Roux-en-Y intestinal loop for bile drainage, also known as Kasai portoenterostomy / hepatoportoenterostomy. However, most children will require a liver transplant operation to extend their survival due to recurrent cholangitis after the operation. Currently, there is no reported drug for treating BA and increasing survival time, and it can only be treated by portoenterostomy and liver transplant surgery, but the operation affects the long-term quality of life of children. Therefore, there is an urgent need to find drugs that may intervene in the occurrence and progression of BA disease, delay the disease progression, and extend the survival time.

[0004] Ganoderic acids are a diverse group of compounds, mainly including multiple subtypes such as A, B, C, D, F, H, K, etc. They share a similar chemical skeleton but differ in structure. Ganoderic acid A (GAA) is a natural triterpenoid. Ganoderma triterpenoids are a class of triterpenoid compounds found in Ganoderma lucidum, scientifically named total triterpenoids of Ganoderma lucidum, belonging to highly oxidized lanostane derivatives, and are one of the main chemical and pharmacodynamic components of Ganoderma lucidum. The Ganoderma triterpenoids contained in Ganoderma lucidum have particularly significant physiological activities. It has obvious anti-inflammatory effects, can inhibit the production of inflammatory factors, and reduce the inflammatory response. At the same time, it has antioxidant properties, can scavenge oxygen free radicals, and reduce the damage of oxidative stress to cells. Previous studies in nephropathy found that Ganoderic acid A can reduce the development of renal cysts in polycystic kidney disease and was identified as an effective inhibitor of renal fibrosis in vitro by inhibiting the TGF-β / Smad pathway. It can induce apoptosis of human breast cancer cells by regulating the Jak2 / STAT3 pathway, and the upregulation of Aβ protein clearance by GAA may be a potential drug for the treatment of Alzheimer's disease. Related studies have shown that GAA is a novel potential therapeutic agent that can rescue the neuroimmune imbalance and myelin regeneration in multiple sclerosis through an FXR receptor-dependent mechanism.

[0005] However, so far, there has been no report on the therapeutic effect of Ganoderic acid A on biliary atresia, and it is impossible to infer whether it has a therapeutic effect on biliary atresia based on the known properties of Ganoderic acid A. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of Ganoderic acid A or its derivatives in the preparation of drugs for preventing and / or treating biliary atresia.

[0007] In order to achieve the above object of the present invention, the technical solution adopted by the present invention is:

[0008] The present invention provides the use of Ganoderic acid A or its derivatives in the preparation of drugs for preventing and / or treating biliary atresia.

[0009] The molecular formula of Ganoderic acid A used in the present invention is C 30 H 44 O7, with a molecular weight of 516.67, CAS registration number: 81907-62-2, and its chemical structure is shown in formula (Ⅰ):

[0010]

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

[0012] In some embodiments of the present invention, the virus includes herpes virus, cytomegalovirus, rotavirus or reovirus.

[0013] In some embodiments of the present invention, the biliary atresia also presents as jaundice caused by biliary atresia and liver diseases caused by biliary atresia.

[0014] In some embodiments of the present invention, the liver diseases caused by biliary atresia include liver fibrosis or cirrhosis.

[0015] In some embodiments of the present invention, the biliary atresia is biliary atresia caused by viral infection in the neonatal period, infancy or childhood.

[0016] In some embodiments of the present invention, the ganoderic acid A or its derivatives include at least one of pharmaceutically acceptable salts and pharmaceutically acceptable modifications.

[0017] In some embodiments of the present invention, the pharmaceutically acceptable salts include at least one of metal salts, ammonium salts, salts formed with organic bases, and salts formed with basic amino acids.

[0018] In some embodiments of the present invention, the metal salts include alkali metal salts and alkaline earth metal salts.

[0019] In some embodiments of the present invention, the alkali metal salts include at least one of sodium salts and potassium salts.

[0020] In some embodiments of the present invention, the alkaline earth metal salts include at least one of calcium salts, magnesium salts, barium salts, and aluminum salts.

[0021] In some embodiments of the present invention, the salts formed with organic bases include salts formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0022] In some embodiments of the present invention, the salts formed with basic amino acids include salts formed with the following basic amino acids: at least one of arginine, lysine, and ornithine.

[0023] In some embodiments of the present invention, the pharmaceutically acceptable modifications include at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phosphatidylation, and halogenation. For example, the lipophilicity can be enhanced by introducing fluorine atoms (fluoroethyl) to improve the blood-brain barrier permeability. The corresponding modification methods can be selected according to the actual use of the drug.

[0024] In some embodiments of the present invention, the drug includes pharmaceutically acceptable excipients and / or any one or more other active ingredients.

[0025] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickening agents, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, carriers.

[0026] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and serve as inactive ingredients of pharmaceutical agents. Compilations of pharmaceutically acceptable excipients can be found in "Handbook of Pharmaceutical Excipients" (2nd edition, edited by A. Wade and P. J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994); "List of Names of Pharmaceutical Excipients in the Pharmacopoeia of the People's Republic of China", and other reference books.

[0027] The present invention provides a drug and a corresponding pharmaceutical dosage form using ganoderic acid A or its derivative as an active ingredient for the treatment of biliary atresia.

[0028] In some embodiments of the present invention, the pharmaceutical dosage form of the drug includes a dosage form for gastrointestinal administration or a dosage form for non-gastrointestinal administration.

[0029] In some embodiments of the present invention, the dosage forms for gastrointestinal administration include at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, chewable tablets;

[0030] In some embodiments of the present invention, the dosage forms for non-gastrointestinal administration include at least one of injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0031] In some embodiments of the present invention, the pharmaceutical dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults.

[0032] In some embodiments of the present invention, the children include neonates within 28 days after birth, infants within 1 year old, toddlers aged 1 - 3 years old, toddlers aged 3 - 6 years old, or children aged over 6 years old and under 18 years old.

[0033] In some embodiments of the present invention, the adults are adult women during pregnancy, perinatal adult women, or lactating adult women.

[0034] The carriers selected in the preparation of oral preparations can be conventional pharmaceutical excipients such as starch, dextrin, or cyclodextrin and various chemically modified cyclodextrins, sucrose, stearate, etc. The later preparation processes and equipment for each preparation belong to the conventional technologies in the pharmaceutical field, and the present invention does not limit this.

[0035] In some embodiments of the present invention, the application dose of Ganoderic Acid A in the mouse model is 15 - 25 mg / kg. In other animals or humans, the corresponding adjustment of the drug dose can be carried out, and the present invention does not limit this.

[0036] The beneficial effects of the present invention are:

[0037] The present invention first discovers that Ganoderic Acid A can be used to prepare a new use for preventing and / or treating biliary atresia. BA is induced by intraperitoneal injection of RRV within 24 hours after the birth of BALB / c neonatal mice, and starting from the second day after intraperitoneal injection of RRV, Ganoderic Acid A is intraperitoneally injected into the mice every day until the 12th day. It can be observed that the survival time of BA mice treated with Ganoderic Acid A is prolonged, the weight loss is significantly improved, the jaundice symptoms are alleviated, the liver function indicators are significantly improved, the number of inflammatory cell infiltrations around the intrahepatic bile ducts is significantly reduced, and the liver bile duct damage is alleviated. The present invention provides a new potential target and treatment strategy for the treatment of BA, and has potential clinical treatment significance. Description of the Drawings

[0038] The following further describes the present invention in conjunction with the drawings and embodiments, where:

[0039] Figure 1 Shows the survival status of each group of mice, where A is the appearance diagram of each group of mice on the 12th day; B is the fluorescence angiography of the extrahepatic bile duct; C shows the phenotypic results of each group of mice, including survival rate, body weight, and jaundice rate.

[0040] Figure 2 Shows the liver function indicators of each group of mice on the 12th day, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), total bilirubin (TBIL), and direct bilirubin (DBIL); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

[0041] Figure 3H&E staining results of the liver and CK19 immunohistochemical staining results of intrahepatic bile ducts of mice in each group on the 12th day; ****p<0.0001.

[0042] Figure 4 Multi-factor results of in vitro culture of liver monocytes of BA children and Ganoderic acid A Detailed implementation manners

[0043] The following will clearly and completely describe the concept and technical effects of the present invention in combination with embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0044] Example 1 Screening of Ganoderic acid A

[0045] Referring to relevant literature on metabolomics (DOI: 10.1038 / s41598-024-66893-2, DOI: 10.1016 / j.jhepr.2024.101208), in this example, non-target metabolomics research was carried out on the livers of children with biliary atresia to screen key metabolites. It was found that Ganoderic acid A had the highest VIP value among the differential metabolites. Subsequently, preliminary experiments showed that by intraperitoneally injecting Ganoderic acid A into BA mice, the expression of monocytes in BA mice was inhibited, the formation of bile duct atresia in BA mice was effectively alleviated, the jaundice rate was reduced, and the body weight and survival rate were increased. After the preliminary experiment, ethical approval for the formal experiment and informed consent from patients were applied for, and the formal experiment was verified.

[0046] Example 2 Verification process of the therapeutic effect of Ganoderic acid A on BA

[0047] The relevant experimental materials and methods of the present invention are as follows:

[0048] 1. Experimental materials

[0049] Experimental animals: BALB / c WT neonatal mice within 24 hours after birth.

[0050] Experimental cells: Monocytes derived from the livers of children with biliary atresia, and informed consent from patients was obtained for relevant experiments (Approval No.

[2023] No. 130B01 of the Ethics Committee of Guangzhou Women and Children's Medical Center).

[0051] Main reagents and antibodies: (1) Rhesus rotavirus (RRV), (American Type Culture Collection (ATCC), VR-1739), with a titer of 1.5×10 6PFU / mL, dose 20 μL.

[0052] (2) Ganoderic Acid A (MCE, HY-N1447).

[0053] 2. Experimental methods

[0054] 1) Animal grouping: Newborn BALB / c mice were randomly divided into the following three groups:

[0055] ① Normal saline group;

[0056] ② Experimental group;

[0057] ③ Drug treatment group.

[0058] 2) Injection method: Treatment of the disease model group (experimental group): Within 24 hours after the birth of newborn BALB / c mice, 20 μL of RRV (titer: 1.5×10 6 PFU / mL) was intraperitoneally injected using a disposable sterile insulin syringe to induce the formation of biliary atresia; Treatment of the normal control group (normal saline group): Within 24 hours after the birth of mice, 20 μL of normal saline was intraperitoneally injected using a disposable sterile insulin syringe; Treatment of the intervention group (drug treatment group): After injecting RRV into mice within 24 hours after birth, starting from the second day, Ganoderic acid A (dose: 20 mg / kg) was intraperitoneally injected using a disposable sterile insulin syringe once a day until the 12th day.

[0059] 3) Observe and record the survival status, survival body weight, and skin jaundice of mice in each group every day, and collect blood samples and liver tissue samples on the 12th day.

[0060] 4) Fluorescent cholangiography of extrahepatic bile ducts: Newborn mice in each group on the 12th day 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 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 the microscope and photographed.

[0061] 5) Blood collection and biochemical detection: On the 12th day after the birth of neonatal mice, cardiac blood collection was performed. 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 notch was cut on the left side of the diaphragm with scissors to expose the heart. An insulin syringe was used to pierce the apex of the mouse heart and slowly draw blood following the heart rhythm (after inserting the needle into the apex, there is an obvious sense of breakthrough, which means entering the left ventricle) until no more blood can be drawn. The drawn blood was transferred into an anticoagulation tube, marked, and centrifuged at 3000 rpm for 10 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 future detection. The serum to be tested was taken to the hospital laboratory for liver function detection using a biochemical detection instrument.

[0062] 6) H&E staining: 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.

[0063] 7) CK19 immunohistochemical staining: 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 an appropriate secondary antibody for 30 minutes at room temperature. 3,3'-Diaminobenzidine (DAB) was used as a chromogenic agent to visualize the immunohistochemical staining. The sections were observed under a microscope, images were obtained, and analyzed as needed.

[0064] 8) Detection of multiple inflammatory factors: Monocytes (CD14-positive cells) were sorted from the livers of BA patients using magnetic bead sorting method. And Ganoderic Acid A (0.5 mmol / L) was added for in vitro culture for 48 hours. Then the supernatant was collected for the determination of human multiple inflammatory factors based on the principle of Elisa.

[0065] 3. Observation indicators and detection methods:

[0066] 1) Observation of general condition of mice: The survival status, body weight, skin jaundice condition, and the colors of urine and feces of mice in each group were observed and recorded every day.

[0067] 2) Mouse dissection and sample collection: On the 12th day, 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.

[0068] 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.

[0069] 4) Histopathological examination of liver tissue: The liver tissue was fixed, embedded, sectioned, and stained with H&E and CK19 immunohistochemistry. H&E staining can observe the infiltration of inflammatory cells around the intrahepatic bile ducts, and CK19 immunohistochemistry can observe the damage of the intrahepatic bile ducts.

[0070] Example 3 Effect of Ganoderic Acid A on the survival status of BA mice

[0071] The experimental results are as Figure 1 shown. In the RRV group mice treated with Ganoderic Acid A, the skin jaundice of BA symptoms was significantly improved ( Figure 1 A in), the extrahepatic bile ducts were completely unobstructed ( Figure 1 B in), the survival time was significantly prolonged, the decrease in average survival body weight was significantly slowed down, and the jaundice rate decreased ( Figure 1 C in).

[0072] Example 4 Effect of Ganoderic Acid A on liver function of BA mice

[0073] The results are as Figure 2 shown. Compared with the RRV group, alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (γ-GT), total bilirubin (TBIL), direct bilirubin (DBIL), and total bile acid (TBA) all decreased, showing significant differences. The liver function of BA mice treated with Ganoderic Acid A was significantly improved.

[0074] Example 5 Effect of Ganoderic Acid A on periportal inflammation infiltration and intrahepatic bile ducts of BA mice

[0075] The experimental results are as Figure 3 shown. On the 12th day, the infiltration of inflammatory cells around the intrahepatic bile ducts in the RRV group mice increased significantly, and the intrahepatic bile ducts were blocked. Compared with the RRV group mice, the infiltration of inflammatory cells around the intrahepatic bile ducts in the RRV mice treated with Ganoderic Acid A decreased significantly, there was a normal intrahepatic bile duct structure, and the degree of bile duct damage was significantly reduced.

[0076] Example 6: In vitro experiments prove that ganoderic acid A downregulates the expression of inflammatory factors in liver monocytes of patients with biliary atresia

[0077] The experimental results are as Figure 4 shown. After sorting monocytes in the liver of BA children and culturing them in vitro with ganoderic acid A (0.5 mmol / L) for 48 hours, the supernatant was collected for detection of multiple inflammatory factors. The results showed that the expression of inflammatory factors in liver monocytes of biliary atresia children treated with ganoderic acid A was significantly downregulated.

[0078] 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. Various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of ganoderic acid A or its derivatives in the preparation of drugs for preventing and / or treating biliary atresia.

2. The use according to claim 1, characterized in that: The biliary atresia includes biliary atresia caused by viruses or biliary atresia caused by cholestasis.

3. The use according to claim 2, characterized in that: The biliary atresia is biliary atresia caused by viral infection in the neonatal period, infancy or childhood.

4. The use according to claim 1, characterized in that: The ganoderic acid A or its derivatives include at least one of pharmaceutically acceptable salts and pharmaceutically acceptable modifications.

5. The use according to claim 4, characterized in that: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an organic base, and a salt formed with a basic amino acid.

6. The use according to claim 4, characterized in that: The pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

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

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

9. The use according to claim 8, characterized in that: The dosage form of the drug includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration; Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

10. The use according to claim 9, 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 child is a newborn within 28 days of birth, an infant under 1 year old, a toddler between 1 and 3 years old, a toddler between 3 and 6 years old, or a child between 6 and 18 years old; Preferably, the adult is a female adult during pregnancy, a female adult during the perinatal period, or a female adult during lactation.

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