Use of synephrine in the preparation of medicaments for the treatment of cholestatic liver disease

Abstract

This invention relates to a novel use of synephrine in the preparation of drugs for treating cholestatic liver disease, belonging to the field of pharmaceutical technology. Cholestatic liver disease is characterized by jaundice and conjugated bilirubin and / or total bilirubin hyperconjugation caused by impaired bilirubin excretion due to various reasons. Its causes include common bile duct stones, pancreatic duct cancer, common bile duct malignant tumors, pancreatic cancer, biliary parasitic diseases, viral hepatitis cirrhosis, alcoholic liver disease, fatty liver disease, drug-induced liver injury, primary biliary cirrhosis, intrahepatic sclerosing cholangitis, and certain congenital diseases such as Dubin-Johnson syndrome and Rotor syndrome. This invention establishes a mouse cholestasis model using bile duct ligation and administers synephrine by gavage to evaluate its protective effect against liver injury. The results showed that synephrine significantly improved jaundice caused by cholestasis, reduced hepatocellular necrosis and inflammatory infiltration caused by cholestasis, alleviated bile duct dilation, decreased serum total bilirubin (TBIL) and direct bilirubin (DBIL), and reduced the levels of total bile acids (BA) in liver tissue and plasma. Furthermore, the above-mentioned effects of synephrine in improving cholestasis showed a clear dose-dependent effect. This invention reveals for the first time the application of synephrine in cholestatic liver disease, demonstrating good safety and promising clinical development prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a novel use of synephrine, a natural bioactive ingredient, in the preparation of drugs for treating cholestasis-related liver injury. More specifically, this invention reveals the significant ameliorative effect of synephrine on liver tissue damage, bile metabolism disorders, and bile duct dilation caused by bile acid stasis, making it a potential drug for treating cholestatic liver disease. Background Technology

[0002] Cholestatic liver disease is a group of clinical conditions caused by impaired bile production, secretion, or excretion, leading to the accumulation of bile acids in the liver and subsequently causing hepatocellular damage, cholangitis, and even cirrhosis. Common causes include primary biliary cholangitis, primary sclerosing cholangitis, cholestasis of pregnancy, and drug-induced liver injury. Currently, the main drugs used clinically to treat cholestatic diseases include ursodeoxycholic acid (UDCA) and obeticholic acid (OCA), which alleviate the stasis by promoting bile secretion and bile acid transport. However, these treatments have limitations, including limited efficacy, narrow indications, poor long-term tolerability, and significant side effects, leaving clinical needs unmet. Therefore, there is an urgent need to develop new natural or small-molecule drugs to alleviate tissue damage caused by cholestasis. Synephrine is a natural alkaloid extracted from citrus plants and has been widely used in research on metabolic regulation and anti-inflammation. However, there is currently no publicly available literature or patent disclosure on the therapeutic effects of synephrine in the regulation of bile acid metabolism and cholestatic liver disease.

[0003] Current technologies for the treatment of cholestasis still have the following main shortcomings:

[0004] 1. Most drugs have a single mechanism of action and are difficult to comprehensively alleviate hepatocellular damage caused by the disruption of bile acid homeostasis;

[0005] 2. Some medications have potential hepatotoxicity, and long-term use poses safety risks;

[0006] 3. The treatment effect is limited for complex pathological processes such as elevated bilirubin, inflammation, and oxidative stress.

[0007] Therefore, developing a novel treatment strategy with multiple mechanisms of action, high safety, and natural origin has become an urgent need in the field of cholestasis treatment. Summary of the Invention

[0008] In view of the problems of limited efficacy, insufficient safety and lack of new natural active ingredients in existing treatments for cholestatic liver disease, this invention provides the application of synephrine, a phenethylamine alkaloid from a natural source, in the preparation of drugs for treating cholestasis-related diseases.

[0009] The technical solution of this invention is:

[0010] 1. The use of synephrine or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a pharmaceutical formulation for treating cholestatic liver disease and its clinical manifestations caused by multiple factors, wherein the synephrine has the following structural formula:

[0011]

[0012] 2. The application according to claim 1, characterized by cholestatic liver disease, is characterized by jaundice and hyperconjugated bilirubin and / or total bilirubin due to bilirubin excretion disorders caused by various factors. The causes include common bile duct stones, pancreatic duct cancer, malignant tumors of the common bile duct, pancreatic cancer, biliary parasitic diseases, viral hepatitis cirrhosis, alcoholic liver disease, fatty liver disease, drug-induced liver injury, primary biliary cirrhosis, intrahepatic sclerosing cholangitis, and certain congenital diseases such as Dubin-Johnson syndrome and Rotor syndrome.

[0013] 3. According to the application described in claim 1, the clinical symptoms caused by cholestasis include itching, fatigue, steatorrhea, xanthomas, hepatic osteomalacia, jaundice, etc.

[0014] 4. The application according to claim 1, the treatment, characterized in that its mechanism of action is by reducing bilirubin levels, including direct bilirubin and total bilirubin levels.

[0015] 5. The application and treatment according to claim 1, characterized in that it reduces hepatocyte necrosis.

[0016] The application and treatment according to claim 1 are characterized by reducing the infiltration of inflammatory cells in liver tissue.

[0017] 6. The application and treatment according to claim 1, characterized in that it reduces the inner diameter of the bile duct, relieves bile duct dilation, and improves bile excretion.

[0018] 7. The application and treatment according to claim 1, characterized in that it reduces the level of total bile acids in the liver and serum, improves bile metabolism disorders, and maintains bile acid homeostasis.

[0019] 8. The application according to claim 1, wherein the pharmaceutical formulation comprises a sustained-release formulation administered orally or via a parenteral route.

[0020] To verify the above applications, this invention used a mouse cholestasis model constructed by bile duct ligation (BDL) and administered different doses of synephrine via gavage, observing its effects on liver tissue structure and biochemical indicators. The results showed that synephrine significantly alleviated liver tissue structural damage, inflammatory cell infiltration, and fibrosis induced by BDL, and reduced serum TBIL, DBIL, and BA levels in a dose-dependent manner.

[0021] HE staining results further showed that, compared with the model group, synephrine treatment improved liver tissue cell structure and reduced necrosis and inflammatory infiltration, suggesting that it has a significant tissue protective effect.

[0022] This invention reveals for the first time the potential value of synephrine in the prevention and treatment of cholestatic liver disease, filling a gap in its application in bile acid metabolism-related diseases. This ingredient is naturally derived, has good biosafety, and shows promise as a novel bile metabolism regulating drug, possessing significant theoretical and practical value. Attached image description:

[0023] Figure 1 This is a flowchart of the invention, illustrating the process of establishing a BDL model and subsequent synephrine intervention;

[0024] Figure 2 This invention presents the HE staining results of liver tissue after synephrine intervention. Liver tissues from mice in the blank group, model group, and each dose treatment group were collected, paraffin sections were prepared, and HE staining was performed. Photos were taken from any selected field of view at a scale bar of 100 μm. The results showed that hepatocyte necrosis and inflammatory infiltration were significantly alleviated in the synephrine group, and the tissue structure tended to be intact.

[0025] Figure 3 This invention relates to the effect of synephrine intervention on the diameter of intrahepatic bile ducts. The diameter of bile ducts in transverse sections of liver tissue was measured using ImageJ software. Five different fields of view were randomly selected from each mouse for measurement, and the average value was taken. The average bile duct diameter of each group was normalized relative to the average value of the normal group. The results showed that the bile duct diameter was significantly enlarged in the model group, while the degree of bile duct dilation was significantly reduced in the medium- and high-dose synephrine intervention groups, with a smaller diameter than the model group, which was statistically significant (*P<0.05, **P<0.01, ***P<0.001). This suggests that synephrine can effectively alleviate bile duct dilation caused by cholestasis and help improve bile excretion function. Figure 4 This study investigated the effects of synephrine on serum ALT and AST levels, using a kit to detect serum ALT and AST activities. Compared with the model group, synephrine significantly inhibited the elevation of both liver enzyme levels, which was statistically significant (*P<0.05, **P<0.01, ***P<0.001), suggesting that it has the function of alleviating liver damage.

[0026] Figure 5 This study investigated the effects of synephrine on serum TBIL and DBIL levels. Serum total bilirubin and direct bilirubin levels were measured in mice in the blank group, BDL model group, and treatment group. Data are expressed as mean ± SD. The results showed that both indicators in the synephrine group were significantly lower than those in the model group, which was statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

[0027] Figure 6 The study investigated the effects of synephrine on total bile acid (BA) levels in plasma and liver tissue. The results showed that synephrine significantly reduced the BA content in plasma and liver tissue induced by bile acid metabolism (BDL), with statistical significance (*P<0.05, **P<0.01, ***P<0.001), suggesting its potential to regulate bile acid metabolism homeostasis. Detailed implementation method:

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0029] Unless otherwise specified, the equipment and materials used in this embodiment are commercially available. The sources of the reagents, experimental animals, and materials used in this embodiment are described for the purpose of more detailed description of the invention, but this is not intended to limit the scope of this application. Synephrine was purchased from Shanghai Maclean Biotechnology Co., Ltd.; male C57BL / 6J mice (20±2g, 7 weeks old, SPF grade) were purchased from Nanjing Jicui Pharmaceutical Co., Ltd.; total bilirubin (TBIL) kit, direct bilirubin (DBIL) kit, total bile acid (BA) kit, aspartate aminotransferase (AST / GOT) test kit, and alanine aminotransferase (ALT / GPT) test kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0030] Example 1: Establishment of the BDL mouse model

[0031] Animal preparation: 6-8 week old male C57BL / 6 mice were selected. They were fasted for 12 hours before the operation, but water was provided as usual.

[0032] Anesthesia: Administer 1% sodium pentobarbital intraperitoneally at a rate of 100 μL / 20 g body weight. Once the animal is fully anesthetized, begin the surgical procedure.

[0033] Surgical area treatment and fixation: Spray 75% alcohol to disinfect the abdominal skin, shave the abdominal hair from the manubrium of the sternum to the perineum, and fix the animal to a sterilized foam board.

[0034] Surgical procedure: An incision is made along the midline of the abdomen, and the skin and abdominal wall tissues are dissected layer by layer to expose the hilar structures. The bile ducts accompanying the portal vein are identified, and the tissues surrounding the bile ducts are carefully dissected. Two silk sutures are used to ligate the bile ducts above and below, and two knots are tied before the middle segment of the bile duct is cut to create a complete bile duct occlusion model. Postoperatively, the muscle layer and skin are sutured sequentially, an antibacterial ointment is applied to the surface, and the patient is transferred to a warm and dry environment for recovery.

[0035] Model confirmation: The mice showed normal activity and no obvious wound infection within 48 hours after surgery, indicating that the model was successfully constructed and can be used for subsequent experiments.

[0036] Example 2: Synephrine Gavage Administration Experiment

[0037] After the model was established, the animals were randomly divided into groups of 8 each. They were given different doses of synephrine (20 mg / kg, 40 mg / kg, and 80 mg / kg) by gavage according to their body weight, once a day for 7 consecutive days. The blank control group was given an equal volume of physiological saline.

[0038] During the administration period, the general condition and weight changes of the mice were observed. The mice were fasted for 12 hours after the last administration before sample collection began.

[0039] Example 3: HE staining analysis

[0040] HE staining: Mouse liver tissue was collected, fixed in 10% neutral formalin for 24 hours, routinely embedded in paraffin, and sectioned to a thickness of 4 μm. Hematoxylin-eosin staining was used to observe changes in hepatocyte structure, degree of necrosis, and inflammatory cell infiltration.

[0041] Example 4: Measurement of Intrahepatic Bile Duct Diameter

[0042] To evaluate the effect of synephrine on bile duct dilation in cholestatic mice, paraffin sections of liver tissue (same as HE-stained sections) were taken. Five different fields of view were randomly selected under a microscope, and the transverse diameter of the intrahepatic bile ducts was measured using ImageJ software. Five intrahepatic bile duct measurements were taken from each mouse, and the average value was calculated. The results were normalized relative to the bile duct diameter of the normal group.

[0043] Example 5: Detection of biochemical indicators in serum, plasma and liver tissue

[0044] On day 7 of the experiment, blood was collected by enucleation to prevent hemolysis. Approximately 500 μL of blood was collected from each mouse, and after standing at room temperature, the serum and plasma were separated by centrifugation.

[0045] Bilirubin detection: Use a bilirubin detection kit to detect the levels of TBIL and DBIL in serum, following the instructions.

[0046] Liver function test: The levels of ALT and AST in serum are measured to reflect the degree of liver damage.

[0047] Bile acid content detection: Further detection of total BA levels in plasma and liver tissue to assess bile metabolism.

[0048] Analysis of Experimental Results

[0049] 1. HE staining results showed that the liver tissue of mice in the BDL model group showed obvious hepatocyte necrosis and inflammatory cell infiltration, while the liver tissue damage in the synephrine treatment group was significantly reduced and the structure was more intact, showing a dose-dependent improvement trend.

[0050] 2. Measurements of intrahepatic bile duct diameter showed that the bile duct diameter in the BDL model group was significantly higher than that in the normal group, indicating significant bile duct dilation (P < 0.001). The bile duct diameter in the medium- and high-dose synephrine intervention groups was significantly lower than that in the model group (P < 0.05 or P < 0.01), indicating that synephrine can effectively alleviate bile duct dilation caused by cholestasis.

[0051] 3. Serum tests showed that synephrine significantly reduced the levels of TBIL and DBIL in the serum of BDL model mice (P < 0.05 or P < 0.01), suggesting its ability to improve bilirubin metabolism disorders. AST and ALT levels also decreased significantly due to synephrine intervention (P < 0.05 or P < 0.01), reflecting its protective effect on hepatocytes and improvement of liver function.

[0052] 4. Synephrine significantly reduced the total BA content in liver tissue and plasma (P < 0.05 or P < 0.01), indicating that it has a good regulatory ability on bile acid metabolism.

[0053] in conclusion

[0054] The above results indicate that synephrine significantly improves liver damage, bile duct dilation, cell necrosis, and inflammatory infiltration caused by bile acid ligation in a mouse model of bile duct ligation, and effectively regulates bilirubin and bile acid levels. This invention reveals for the first time the potential application value of synephrine in the treatment of cholestatic liver disease.

[0055] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various modifications or improvements without departing from the spirit and principles of the present invention, and all such modifications or improvements should be considered to fall within the protection scope of the present invention.

Claims

1. The use of synephrine or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a pharmaceutical formulation for treating cholestatic liver disease and its clinical manifestations caused by multiple factors, wherein the synephrine has the following structural formula:

2. The application according to claim 1, characterized by cholestatic liver disease, is characterized by jaundice and hyperconjugated bilirubin and / or total bilirubin due to bilirubin excretion disorders caused by various factors. The causes include common bile duct stones, pancreatic duct cancer, malignant tumors of the common bile duct, pancreatic cancer, biliary parasitic diseases, viral hepatitis cirrhosis, alcoholic liver disease, fatty liver disease, drug-induced liver injury, primary biliary cirrhosis, intrahepatic sclerosing cholangitis, and certain congenital diseases such as Dubin-Johnson syndrome and Rotor syndrome.

3. According to the application described in claim 1, the clinical symptoms caused by cholestasis include itching, fatigue, steatorrhea, xanthomas, hepatic osteomalacia, jaundice, etc.

4. The application according to claim 1, the treatment, characterized in that its mechanism of action is by reducing bilirubin levels, including direct bilirubin and total bilirubin levels.

5. The application and treatment according to claim 1, characterized in that it reduces hepatocyte necrosis.

6. The application according to claim 1, wherein the treatment is characterized by reducing inflammatory cell infiltration in liver tissue.

7. The application and treatment according to claim 1, characterized in that it reduces the inner diameter of the bile duct, relieves bile duct dilation, and improves bile excretion.

8. The application and treatment according to claim 1, characterized in that it reduces the level of total bile acids in the liver and serum, improves bile metabolism disorders, and maintains bile acid homeostasis.

9. The application according to claim 1, wherein the pharmaceutical formulation comprises a sustained-release formulation administered orally or via a parenteral route.

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