Application of Metformin in the Prevention and Treatment of Biliary Atresia

Abstract

The present invention discloses the application of metformin in the prevention and treatment of biliary atresia. The present invention uses a classical biliary atresia mouse model induced by RRV as the research object, and injects metformin into neonatal mice on the first day after birth; the results show that, compared with the disease group, metformin has a therapeutic effect on biliary atresia mice, can prolong their survival time, improve their liver function, reduce the degree of jaundice in mice, and reduce the number of inflammatory cell infiltrations around intrahepatic bile ducts, suggesting that metformin can be used as a potential therapeutic drug for biliary atresia; it provides a new idea for the preparation of drugs for the protection of biliary atresia and broadens the application scope of metformin.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of metformin in the prevention and treatment of biliary atresia. Background Art

[0002] Biliary atresia (BA) is a serious neonatal disease caused by progressive inflammatory destruction and fibrotic occlusion of intrahepatic and extrahepatic bile ducts, leading to cholestasis and liver failure. If not treated in time, most children will die of end-stage liver disease within two years, which makes BA the most common cause of pediatric liver transplantation. Currently, there is no reported direct and effective treatment drug, and only Kasai operation and liver transplantation can be used for treatment, but the long-term quality of life of children is affected after the operation. 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 prolong the survival time.

[0003] Metformin (1,1-Dimethylbiguanide) can inhibit the mitochondrial respiratory chain in the liver, resulting in the activation of AMPK and enhanced insulin sensitivity, and can be used in the research of type 2 diabetes. In addition, metformin can cross the blood-brain barrier and induce autophagy. Besides its hypoglycemic effect, metformin can cross the blood-brain barrier, induce autophagy, and also has anti-inflammatory, antioxidant, anti-aging, weight loss, cardiovascular protection, lipid-lowering, anti-tumor, and reversal of cognitive impairment effects, and its application in the treatment of BA disease has not been reported. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of metformin in the preparation of drugs for preventing and / or treating biliary atresia.

[0005] The technical solution adopted by the present invention is as follows:

[0006] The present invention provides the application of metformin or a pharmaceutically acceptable salt thereof in the preparation of drugs for preventing and / or treating biliary atresia.

[0007] Preferably, the biliary atresia includes biliary atresia caused by viruses.

[0008] Preferably, the viruses include herpes virus, cytomegalovirus, rotavirus or reovirus.

[0009] Preferably, the biliary atresia also presents as jaundice caused by biliary atresia and liver diseases caused by biliary atresia.

[0010] Preferably, the liver diseases caused by biliary atresia include liver fibrosis or cirrhosis.

[0011] Preferably, the biliary atresia is biliary atresia caused by viral infection in the neonatal period, infancy or childhood.

[0012] Preferably, the dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults.

[0013] Preferably, the children are 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.

[0014] Preferably, the adults are female adults during pregnancy, female adults during the perinatal period or female adults during lactation.

[0015] Preferably, the pharmaceutically acceptable salt is metformin hydrochloride.

[0016] The dosage forms of the drug include capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honeyed pills, pills, granules, honey-refined ointments, sustained-release and controlled-release preparations, oral liquid preparations, injections, chewable tablets, oral films, transdermal patches and effervescent tablets.

[0017] Preferably, the administration methods of the drug include but are not limited to oral administration and injection.

[0018] Preferably, the injection includes subcutaneous injection, intravenous injection, intraperitoneal injection, etc.

[0019] Preferably, the drug also includes pharmaceutically acceptable excipients.

[0020] Preferably, the pharmaceutical excipients include one or more of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

[0021] Preferably, the drug contains an effective dose of metformin or its pharmaceutically acceptable salt.

[0022] Preferably, the unit dose of metformin or its pharmaceutically acceptable salt in the drug is 0.1 mg - 1 g.

[0023] Preferably, the drug also includes at least one other active ingredient that can be used for preventing and / or treating biliary atresia.

[0024] In the present invention, there is also provided a method for preventing and / or treating biliary atresia, including administering the above-mentioned drug to a subject, wherein the appropriate daily dose range of metformin or its pharmaceutically acceptable salt is 1 mg - 1 g / kg body weight; the above dose can be administered in one dose unit or divided into several dose units, depending on the doctor's clinical experience and the dosing regimen including the use of other treatment means.

[0025] In the present invention, when medicating animals, the dosage of metformin or its pharmaceutically acceptable salt per administration is 50 mg - 200 mg / kg of mice, and / or the administration time is 1 - 2 times a day, and / or the total cycle is 10 - 25 days.

[0026] Preferably, the animals include but are not limited to humans, rats, mice, cattle, sheep, etc.

[0027] The beneficial effects of the present invention are as follows:

[0028] The present invention provides the application of metformin in the preparation of drugs for treating biliary atresia. The present invention takes the classical BA mouse model induced by RRV as the research object, and injects metformin into neonatal mice on the first day after birth; the results show that, compared with the disease group, metformin has a therapeutic effect on BA mice, can extend their survival time, improve their liver function, reduce the degree of jaundice in mice, and reduce the number of inflammatory cell infiltrations around the intrahepatic bile ducts, suggesting that metformin can be used as a potential drug for treating BA; it provides a new idea for the preparation of drugs for protecting against biliary atresia and broadens the application scope of metformin. Description of the Drawings

[0029] Figure 1 : A: Appearance pictures of mice in each group on the 12th day; B: Survival curves of mice in each group; C: Body weight curves of mice in each group; D: Jaundice rate graphs of mice in each group.

[0030] Figure 2 : A: Representative pictures of fluorescence angiography of extrahepatic bile ducts of mice in each group; B: Atresia rates of mice in each group on the 12th day.

[0031] Figure 3 : A: Representative pictures of the liver appearance of mice in each group on the 12th day; B: Liver function indexes of mice in each group on the 12th day, where: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyl transpeptidase. *p < 0.05, **p < 0.01, ***p < 0.001.

[0032] Figure 4 : A: Representative results of H&E staining of the liver of mice in each group on the 12th day; B: Quantitative graph of liver inflammation infiltration of mice in each group on the 12th day; C: Representative results of immunohistochemical staining of CK19 of intrahepatic bile ducts of mice in each group on the 12th day; D: Quantitative graph of the number of intrahepatic bile ducts of mice in each group on the 12th day. Detailed Embodiments

[0033] The concept of the present invention and the resulting technical effects will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0034] To better understand the essence of the present invention, the application of metformin in the treatment of biliary atresia mice will be described below in conjunction with experiments and results.

[0035] The structural formula of metformin (Metformin, 1,1 - Dimethylbiguanide) is as follows:

[0036]

[0037] Purchased from MCE Company;

[0038] Classical BA mice induced by Rhesus rotavirus (RRV) can better simulate the pathogenesis and clinical manifestations of BA. Studies in BA mice are helpful for understanding the disease and finding better treatment methods.

[0039] Example 1

[0040] 1) Newborn BALB / c mice were randomly divided into four groups, namely:

[0041] ① Normal control group: Saline group;

[0042] ② Disease model group: RRV group;

[0043] ③ Drug treatment group: RRV + Metformin group.

[0044] Treatment of the disease model group (RRV 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 with a disposable sterile insulin syringe to induce the formation of BA;

[0045] Treatment of the normal control group (Saline group): Within 24 hours after the birth of the mice, 20 μL of normal saline was intraperitoneally injected with a disposable sterile insulin syringe;

[0046] Treatment of the drug treatment group (RRV + Metformin group): Within 24 hours after the mice were born, they were injected with RRV. From the second day, metformin (100 mg / kg) was intraperitoneally injected using a disposable sterile insulin syringe once a day until the 12th day.

[0047] Treatment of the drug control group (Metformin group): On mice not treated with RRV, referring to the drug treatment group, at the same time points, the same volume of 20 μL of metformin (100 mg / kg) was intraperitoneally injected using a disposable sterile insulin syringe once a day until the 12th day after birth.

[0048] 2) Observe and record the survival status, survival body weight, and skin jaundice of the mice in each group every day.

[0049] The experimental results are as Figure 1 shown. The results show that the survival time of the RRV group mice treated with metformin was significantly prolonged ( Figure 1 B), the decrease in the average survival body weight was significantly slowed down ( Figure 1 C), the degree of skin jaundice was reduced ( Figure 1 A), and the jaundice rate decreased ( Figure 1 D).

[0050] Example 2

[0051] 1) Anesthetize and relieve pain in the mice on the 12th day after birth in each group with 2% sodium pentobarbital (40 mg / kg) and buprenorphine (0.05 mg / kg). On a clean microscope operating table, dissect with sterile forceps and scissors to fully expose the mouse liver, gallbladder, and extrahepatic bile duct. Insert an insulin syringe filled with the fluorescence contrast agent solution into the gallbladder cavity, slowly inject the contrast agent, and observe under the microscope whether the contrast agent reaches the jejunum through the extrahepatic bile duct and take pictures.

[0052] The experimental results are as Figure 2 shown. The results show that the extrahepatic bile duct of the RRV group mice on the 12th day was completely blocked, and the blockage rate reached 91%. While the blockage rate of the extrahepatic bile duct of the RRV mice after being treated with metformin on the 12th day was 32%.

[0053] Example 3

[0054] 1) Based on Example 2, fully expose the liver, and observe the infiltration of inflammatory foci on the surface of the mouse liver under a microscope and take pictures.

[0055] The experimental results are as Figure 3 shown in A. The results show that compared with the RRV group mice, the inflammatory infiltration foci on the surface of the liver of the BA mice after being treated with metformin were significantly reduced.

[0056] 2) 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 microscope stage, 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 notch was cut on the left side of the diaphragm with scissors to expose the heart. An insulin syringe was used to pierce into the apex of the mouse heart and slowly draw blood following the heart rhythm (after inserting the needle into the apex, there was an obvious sense of breakthrough, indicating entry into 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.

[0057] 3) Liver function test: The separated serum was transferred into 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 testing. The serum to be tested was taken to the hospital laboratory for liver function testing using a biochemical analyzer.

[0058] The experimental results are as Figure 3 shown in Figure B. It can be seen from the experimental results that compared with the RRV group, the liver function of BA mice treated with metformin was significantly improved.

[0059] Example 4

[0060] 1) H&E staining: Based on Example 3, the fresh mouse liver tissues of each group on the 12th day 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.

[0061] 2) 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 at room temperature for 30 minutes. 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.

[0062] The experimental results are as Figure 4As shown, on the 12th day, the infiltration of inflammatory cells around the intrahepatic bile ducts in the RRV group of mice increased significantly, and the intrahepatic bile ducts were blocked. Compared with the mice in the RRV group, the infiltration of inflammatory cells around the intrahepatic bile ducts in the RRV mice treated with metformin decreased significantly, with normal intrahepatic bile duct structure and significantly reduced degree of bile duct injury. However, the intrahepatic bile duct structure of normal mice was not affected after treatment with metformin.

[0063] The above specific embodiments have described the present invention in detail. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the gist of the present invention. 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 metformin or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a medicament 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 virus is herpes virus, cytomegalovirus, rotavirus or reovirus.

4. The use according to claim 1, characterized in that: The biliary atresia is biliary atresia caused by viral infection in the neonatal period.

5. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt is metformin hydrochloride.

6. The use according to any one of claims 1 to 5, characterized in that: The dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults.

7. The use according to claim 6, characterized in that: The child is a newborn within 28 days of birth, an infant under 1 year old, a toddler between 1-3 years old, a toddler between 3-6 years old, or a child aged between 6 and 18 years old.

8. The use according to claim 6, characterized in that: The adult is a female adult during pregnancy, a female adult during the perinatal period, or a female adult during lactation.

9. The use according to any one of claims 1 to 5, characterized in that: The unit dosage of metformin or its pharmaceutically acceptable salt in the medicine is 0.1 mg-1 g.

10. The use according to any one of claims 1 to 5, characterized in that: The drug is administered orally, by subcutaneous injection, intravenous injection or intraperitoneal injection.

11. The use according to any one of claims 1 to 5, characterized in that: The drug also includes pharmaceutically acceptable excipients.

12. The use according to claim 11, characterized in that: The pharmaceutically acceptable excipients include one or more of diluents, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

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