Use of bicyclol and berberine in combination for preventing and treating non-alcoholic fatty liver disease

Abstract

The present invention discloses the use of bicyclol and berberine in combination for preventing and treating non-alcoholic fatty liver disease, belonging to the field of biomedicine technology. In the present invention, the bicyclol and berberine are both marketed drugs with good safety and suitable for long-term use. Experiments have shown that the combined use of bicyclol and berberine can more significantly prevent and treat non-alcoholic (or metabolic-related) fatty liver disease than the use of either drug alone, and the combined use of the two drugs has a synergistic effect. Therefore, the drug combination of bicyclol and berberine will have broad application prospects in the prevention and treatment of non-alcoholic (or metabolic-related) fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the use of bicyclol and berberine in combination for preventing and treating non-alcoholic fatty liver disease. The present invention belongs to the field of biomedicine technology. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a common metabolic stress-induced chronic liver disease. The disease spectrum includes non-alcoholic hepatic steatosis (NAFL) and non-alcoholic steatohepatitis (NASH). The former only causes lipid accumulation in liver tissue, while the latter is also accompanied by inflammation and / or fibrosis, and even cirrhosis. (Note: Since 2020, an international expert group has recommended that this type of disease be renamed metabolic-associated fatty liver disease (MAFLD)). With the prevalence of obesity and metabolic syndrome, the prevalence of NAFLD has increased year by year. Currently, the global prevalence of NAFLD is approximately 25% (14% to 32%), and the prevalence in China is as high as over 15%. It has become the world's number one chronic liver disease and the leading cause of abnormal liver biochemical indicators in health examinations. NAFLD can not only lead to worsening of liver disease and endanger the patient's life, but is also closely related to the high incidence of metabolic syndrome, type 2 diabetes, arteriosclerotic cardiovascular disease, and colorectal cancer, seriously affecting national health. Moreover, with the changes in lifestyle and pace, the occurrence of metabolic abnormalities tends to be younger, and fatty liver disease is also common among younger populations, which seriously affects my country's future social progress and economic development.

[0003] However, there is currently no specific treatment for NAFLD, and symptomatic treatments are the mainstay, but these are ineffective. Therefore, the development of drugs to prevent and treat NAFLD has become a hot topic and focus of research worldwide. Several drugs are in various stages of clinical development both domestically and internationally, and many new drugs have even been granted expedited review by the US Food and Drug Administration (FDA). However, due to inconsistent outcomes in clinical trials or lack of therapeutic benefit in randomized controlled trials, these clinical trials have failed one after another. To date, no approved drug remains for the clinical prevention and treatment of NAFLD.

[0004] The pathogenesis and progression of non-alcoholic fatty liver disease are complex. The "two-hit" theory is widely accepted, positing that the combined effects of multiple genetic and metabolic factors induce insulin resistance (IR), which increases lipolysis and free fatty acid transport to the liver. Large amounts of lipids accumulate in the liver, leading to a first hit to the liver and the formation of fatty liver. Simultaneously, the liver's sensitivity to metabolites and inflammatory factors increases, increasing the release of oxidative stress and inflammatory factors in the body, further exacerbating IR. This creates a vicious cycle, exacerbating liver cell damage and leading to liver cell degeneration, the second hit. With the deepening of research, the "multiple-hit" theory, including the neuroendocrine-immune-inflammatory network, has gradually replaced the classic "two-hit" theory. Insulin resistance, adipose tissue dysfunction, mitochondrial dysfunction, endoplasmic reticulum stress, inflammatory activation, fatty acids, intestinal flora, iron overload, dietary factors, as well as genetic and epigenetic factors, are all involved in the progression of NAFLD. Drug interventions and new drug development targeting specific aspects of these pathogenesis mechanisms are still ongoing. However, due to the complexity of NAFLD's pathophysiology, single-target drugs are unlikely to fundamentally prevent and treat NAFLD. In fact, due to an incomplete understanding of the role of the target, targeted drug treatment may even lead to safety drawbacks, which will be a huge challenge for future anti-NAFLD drug development. Therefore, the combined use of drugs with different mechanisms of action may be more beneficial. Finding combination therapy with marketed drugs with potential to prevent and treat NAFLD may shorten the drug development process and may lead to breakthrough progress in the prevention and treatment of NAFLD.

[0005] Berberine, also known as berberine, is an over-the-counter drug used clinically for the treatment of intestinal infections and diarrhea. Recent studies have found that berberine has multiple pharmacological effects, including anti-pathogenic microbial, anti-inflammatory, anti-tumor, cardioprotective, hypoglycemic, lipid metabolism regulation, and immunosuppressive effects. Its clinical application potential is constantly being explored, and it has been reported to have good effects on cardiovascular diseases, metabolic syndrome and its complications, tumors, abdominal adhesions, and Chlamydia trachomatis infection. Among them, pharmacological studies and clinical case reports on berberine for the treatment of NAFLD suggest its potential for the treatment of NAFLD. The specific mechanism is related to its direct triggering of insulin secretion, activation of AMPK, regulation of intestinal flora, and increased expression of low-density lipoprotein receptors.

[0006] Bicyclol (trade name: Bisino) is a Class I new drug launched in my country in 2004. Clinically, bicyclol tablets have been used in many countries to treat mild to moderate chronic hepatitis B and chronic hepatitis C associated with elevated serum aminotransferase levels, as well as various types of non-viral hepatitis. Preclinical pharmacology studies have also demonstrated that bicyclol exhibits potent hepatoprotective and anti-inflammatory effects in models of liver injury induced by chemical, immune, fatty, drug-induced, and surgical injury, and can inhibit liver fibrosis induced by carbon tetrachloride, bovine serum albumin, and bile duct ligation. Bicyclol's protective effects against fatty liver injury are associated with suppression of oxidative stress and overexpression of inflammatory factors, anti-fibrosis, and mitochondrial protection. Although the clinical efficacy of various hepatoprotective and anti-inflammatory drugs for the treatment of NAFLD has not been fully validated, the "Guidelines for the Prevention and Treatment of Non-Alcoholic Fatty Liver Disease" recommends hepatoprotective drugs as adjunctive therapy for patients with NASH and elevated aminotransferase levels or liver injury. The "Expert Recommendations on the Clinical Application of Bicyclol Tablets" also suggests that bicyclol tablets can be used for fatty liver disease to effectively improve liver function and liver histological inflammatory lesions in patients with NAFLD and abnormal liver function.

[0007] In research seeking combined drug therapies for the prevention and treatment of NAFLD, the present invention demonstrates that treatment with bicyclol and berberine significantly reduced cellular lipid deposition in a free fatty acid (FFA)-induced hepatocyte lipid accumulation model, particularly in the combined treatment group. In a mouse model of non-alcoholic fatty liver disease (NAFLD) that mimics human disease progression, both bicyclol and berberine exhibited modest preventive and therapeutic effects on NAFLD when administered alone, while their combined efficacy was significantly enhanced. Therefore, the present invention proposes the combined use of bicyclol and berberine for the prevention and treatment of NAFLD. To date, no literature has reported on this drug combination, either domestically or internationally. This combined drug regimen could provide a convenient medication reference for the clinical prevention and treatment of NAFLD, and also holds promise for the development of a bicyclol and berberine combination for the prevention and treatment of NAFLD. Summary of the Invention

[0008] The object of the present invention is to provide a method for preventing and treating non-alcoholic (or metabolism-related) fatty liver disease by combining bicyclol and berberine.

[0009] In order to achieve the above object, the present invention adopts the following technical means, specifically:

[0010] (1) Effect of combined use of bicyclol and berberine in preventing non-alcoholic fatty liver disease (NAFLD) cell model: HepG2 cells were induced with 0.1 mM free fatty acid FFA (OA:PA=2:1), and bicyclol (0.5, 2 μM) intervention group, berberine (0.5, 2 μM) intervention group, and bicyclol and berberine combination groups at different doses were set up. The drugs were induced and administered simultaneously for 24 hours, and the effects of the drugs on lipid droplets were detected by NileRed staining. The results showed that bicyclol and berberine alone could reduce lipid droplet accumulation, and the combined drug effect was significantly better than that of the single drug.

[0011] (2) The effect of combined use of bicyclol and berberine in preventing non-alcoholic fatty liver disease (NAFLD): Western diet was used to induce male C57BL / 6J mice. Berberine (50 or 200 mg / kg / day) intervention group, bicyclol (50 or 200 mg / kg / day) intervention group, and different doses of bicyclol and berberine combination group were set up at the same time. The induction and administration were given for 16 weeks. The results showed that the combined use of bicyclol and berberine was safe. The combined use of bicyclol and berberine at different doses could more significantly improve the yellowish appearance of gross lipid deposition in the liver than the use of either drug alone, and more significantly reduce the NAFLD activity score (NASScore) in histopathology. At the same time, the combined use of bicyclol and berberine could more significantly improve the liver function (serum ALT, AST) of NAFLD mice, and more significantly reduce the accumulation of triglycerides (TG) and cholesterol (CHO) in the liver. The overall effect was better than that of either drug alone.

[0012] (3) Effect of combined use of bicyclol and berberine in the treatment of non-alcoholic fatty liver disease (NAFLD): After 4 weeks of Western diet + carbon tetrachloride (CCl4)-induced simple fatty liver in male C57BL / 6J mice, the mice were randomly divided into a model group, a berberine (50 or 200 mg / kg / day) intervention group, a bicyclol (50 or 200 mg / kg / day) intervention group, and a bicyclol and berberine combination group at different doses. The model induction and drug administration were continued for 8 weeks. The results showed that the combined use of bicyclol and berberine was safe. The combined use of bicyclol and berberine at various doses significantly reduced NAS scores and fibrosis compared with the use of either drug alone. At the same time, the combined use of the two drugs significantly improved the liver function (serum ALT, AST) and liver lipid deposition (liver TG, CHO) levels of NAFLD mice, and the overall effect was better than that of either drug alone.

[0013] Therefore, the drug combination of bicyclol and berberine described in the present invention is expected to be used as a combined drug regimen for preventing and treating NAFLD (MAFLD) in clinic or to develop a combination drug for preventing and treating NAFLD (MAFLD).

[0014] Based on the above research, the present invention proposes the use of a pharmaceutical composition containing bicyclol and berberine in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease (NAFLD), wherein the bicyclol or berberine is in the form of its original drug, pharmaceutical salt, or pharmaceutical derivative based on the skeleton structure of bicyclol or berberine.

[0015] Among them, preferably, the non-alcoholic fatty liver disease (NAFLD) includes non-alcoholic hepatic steatosis (NAFL) and non-alcoholic steatohepatitis (NASH).

[0016] Among them, preferably, the active ingredients of the pharmaceutical composition are composed of bicyclic alcohol and berberine, wherein the bicyclic alcohol or berberine is in the form of its original drug, pharmaceutical salt or pharmaceutical derivative based on the skeleton structure of bicyclic alcohol or berberine.

[0017] Among them, preferably, the dosage ratio of bicyclol and berberine is 1:4 to 4:1, wherein the bicyclol or berberine is in the form of its original drug, pharmaceutical salt or pharmaceutical derivative based on the skeleton structure of bicyclol or berberine.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] NAFLD (MAFLD) is a chronic liver disease with complex pathogenesis and progression. Combining clinically known drugs with different mechanisms of action and suitable for long-term use may offer unexpected therapeutic benefits. Bicyclol and berberine, two traditional drugs, have not been used together to prevent and treat NAFLD (MAFLD). This study combined the two drugs and found that a dosage ratio of 1:4 to 4:1 was more effective in preventing and treating NAFLD than using either drug alone. This discovery represents a promising combination therapy with significant clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Light microscopic observation of the combined use of bicyclol and berberine to prevent lipid droplet accumulation in HepG2 cells (representative images of each group);

[0021] Figure 2 The quantitative results of the combined use of bicyclol and berberine in preventing lipid droplet accumulation in HepG2 cells are presented;

[0022] Figure 3 The effect of bicyclol and berberine combined for the prevention of NAFLD on food intake and body weight in mice;

[0023] Among them, A is the change of mouse food intake, and B is the change of mouse body weight;

[0024] Figure 4A and 4BThe gross observation of the liver and the qualitative observation of liver pathology under light microscopy in the combination of bicyclol and berberine for the prevention of NAFLD (representative figures of each group);

[0025] Figure 5 The quantitative results of NAS Score of the combined use of bicyclol and berberine in preventing NAFLD liver pathology;

[0026] Figure 6 To investigate the effect of bicyclol and berberine combined for the prevention of NAFLD on liver damage indicators and liver lipid content indicators.

[0027] Figure 7 The effect of bicyclol and berberine combined in the treatment of NAFLD on the body weight of mice;

[0028] Figure 8A and 8B The gross observation of the liver and the qualitative observation of liver pathology under light microscopy in the treatment of NAFLD with the combination of bicyclol and berberine (representative figures in each group);

[0029] Figure 9 The quantitative results of NAS Score and fibrosis of liver pathology of NAFLD treated with the combination of bicyclol and berberine are presented;

[0030] Figure 10 To investigate the effects of bicyclol and berberine combined in the treatment of NAFLD on liver damage indicators and liver lipid content indicators. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to specific examples. These examples are for illustrative purposes only and do not in any way limit the scope of the present invention.

[0032] OA and PA used in the cell experiments were saponified with sodium hydroxide and then mixed with equal volumes of 25% BSA to obtain OA and PA stock solutions. Cell culture medium containing 0.1 mM FFA was prepared at a ratio of OA:PA = 2:1. Bicyclol and berberine were dissolved in DMSO and diluted to 0.5 and 2 μM with 0.1 mM FFA solution. OA (Cat. No. O1008), PA (Cat. No. P0500), BSA (Cat. No. B2064), and DMSO (Cat. No. D8418) for the model, control, and treatment groups were purchased from Sigma. Bicyclol (Bic) and berberine (BBR) used in the animal experiments were mixed with high-fat diet (equivalent to an oral dose of 50 mg / kg / day or 200 mg / kg / day body weight). High-fat and high-cholesterol Western diet (catalog number TP26300122) and control low-fat diet (catalog number LAD0011) were purchased from Nantong Trophi Feed Technology Co., Ltd.; the concentration of D-fructose (Sigma, F0127) in high-sugar drinking water was 23.1 g / L, and the concentration of D-glucose (Sigma, G8270) was 18.9 g / L; carbon tetrachloride (CCl4) was purchased from Tianjin Fuchen Chemical Reagent Factory.

[0033] The SPF-grade male C57BL / 6J mice used were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0034] C57BL / 6J mice were housed under the following conditions: ambient temperature of 22 ± 0.5°C, with 12 h / 12 h light / dark cycles.

[0035] Experimental data are presented as mean ± standard deviation and representative figures. One-way ANOVA was used to compare the mean values of the bicyclol and berberine groups alone with the model group, and the combination group with either the individual bicyclol or berberine groups or the model group. Nonparametric analyses were performed using the Kruskal-Wallis H test and the Mann-Whitney U test. *P < 0.05 or **P < 0.01 indicated statistically significant differences.

[0036] Example 1 Cell experiment

[0037] The cell-specific slide was placed in a 24-well plate, and the logarithmic phase HepG2 cells were plated at 10 5Cells were plated at a density of 100 cells / well in 24-well plates. After overnight attachment, the model group was induced with 0.1 mM FFA. The drug-treated groups were treated with FFA containing 0.5 μM and 2 μM berberine (BBR) and bicyclol (Bic) for 24 h. The corresponding control groups were treated with solvent. The experiment was divided into 10 groups, labeled as: Control, FFA (0.1 mM), FFA + BBR (0.5 μM), FFA + BBR (2 μM), FFA + Bic (0.5 μM), FFA + Bic (2 μM), FFA + BBR (0.5 μM) + Bic (0.5 μM), FFA + BBR (0.5 μM) + Bic (2 μM), FFA + BBR (2 μM) + Bic (0.5 μM), and FFA + BBR (2 μM) + Bic (2 μM). At the end of the experiment, Nile Red staining was performed and photos were taken under a fluorescence microscope (630×). Three representative fields of view were selected from each group and the lipid droplet area was quantified using Image-Pro Plus 6.0 software to evaluate the effect of drugs on lipid droplets.

[0038] The experimental results are as follows:

[0039] 0.1mM FFA and the doses of berberine and bicyclol had no obvious toxicity to cells. Figure 1 ) and lipid droplet quantification results ( Figure 2 ) showed that under the action of FFA, intracellular lipid droplets increased significantly, and after drug intervention, lipid droplets decreased significantly. The lipid droplet-lowering effect of bicyclol and berberine was more obvious than that of single drug.

[0040] Example 2 Animal Experiment 1

[0041] 9- to 10-week-old male C57BL / 6J mice (25-27 g) were acclimated for 3 days before the experiment. Ten groups of five mice were assigned to each group. The control group was fed a low-fat control diet with normal drinking water. The model group was induced with a Western diet (high-fat, high-cholesterol diet plus high-sugar drinking water) (i.e., WD diet). The treatment groups were fed a Western diet supplemented with low (L) or high (H) doses of berberine (BBR) or bicyclol (Bic) (equivalent to an oral dose of 50 mg / kg / day or 200 mg / kg / day body weight) for 16 weeks. The groups were labeled as follows: Control, WD, WD+BBR(L), WD+BBR(H), WD+Bic(L), WD+Bic(H), WD+BBR(H)+Bic(L), WD+BBR(L)+Bic(H), WD+BBR(H)+Bic(H), and WD+BBR(L)+Bic(L). During the experiment, the mice's food intake, body weight, and mortality were recorded. At the end of the experiment, the mice were weighed, blood was collected, and serum was separated for liver injury markers (AST, AST). Portions of the right lobe of the liver were frozen in liquid nitrogen for measurement of intrahepatic lipid content markers (TG, CHO). Portions of liver tissue from the right lobe were also fixed with formaldehyde and stained with hematoxylin and eosin (HE) and oil red for histopathological evaluation of hepatic steatosis, ballooning, and inflammation, and for quantitative assessment of the NAFLD activity score (NAS Score).

[0042] The experimental results are as follows:

[0043] No mice died during the experiment, and there was no significant difference in the food intake between the drug-treated group and the model group ( Figure 3 A). At the end of the experiment, the body weight of mice in the drug-treated group decreased compared with the model group, suggesting that drug-treated mice can reduce the weight gain induced by WD diet ( Figure 3 B). Therefore, bicyclol and berberine are safe when used alone or in combination. Gross liver observation showed that the liver of the control group was bright red, while the model group showed khaki fatty liver-like changes. The khaki color of the bicyclol and berberine alone group was reduced, and the improvement was more obvious in the combined dosage groups ( Figure 4A 、 4B ). Pathological microscopic observation of liver tissue showed that the liver of the model group mice showed severe fatty degeneration, ballooning, and inflammatory necrosis pathological features. After single drug treatment, the above lesions improved to a certain extent, and the improvement was more obvious in the combined drug dosage groups ( Figure 4A 、 4B The quantitative results of NAS scores of liver tissue pathology further proved that the effects of each combined drug dosage group were significantly better than those of the single drug group ( Figure 5 Quantitative results of serum ALT, AST and liver TG, CHO also showed that the overall improvement effect of each combined drug dosage combination was more significant than that of the single drug group ( Figure 6The above results show that the combination of bicyclol and berberine has good safety and better effect than either drug alone in preventing non-alcoholic fatty liver disease, which is a discovery with drug development and clinical application value.

[0044] Example 3 Animal Experiment 2

[0045] 9- to 10-week-old male C57BL / 6J mice (25-27 g) were acclimated for 3 days before the experiment. Ten groups were divided into the control group, which was fed a low-fat control diet and received normal drinking water. The remaining groups served as modeling groups, which were induced with a Western diet (high-fat, high-cholesterol diet + high-sugar drinking water) and intraperitoneal injection of CCl4 (0.2 mL / kg body weight, once weekly) (i.e., WD / CCl4). Four weeks after the modeling established simple fatty liver, drug administration was performed. The modeling mice were randomly divided into a model group and a drug administration group. The model group continued the WD / CCl4 induction, while the drug administration group was fed a Western diet containing low (L) or high (H) doses of berberine (BBR) or bicyclol (Bic) (equivalent to an oral dose of 50 mg / kg / day or 200 mg / kg / day body weight) for 8 weeks and received an equal amount of CCl4 via intraperitoneal injection. The groups were labeled as follows: Control, WD / CCl4, WD / CCl4+BBR(L), WD / CCl4+BBR(H), WD / CCl4+Bic(L), WD / CCl4+Bic(H), WD / CCl4+BBR(H)+Bic(L), WD / CCl4+BBR(L)+Bic(H), WD / CCl4+BBR(H)+Bic(H), and WD / CCl4+BBR(L)+Bic(L). The n = 5 for the control and drug combination groups, and n = 8-9 for the other groups. Mouse body weight and mortality were monitored throughout the experiment. At the end of the experiment, mice were weighed, blood was collected, and serum was separated for liver injury markers (AST, AST). Partial tissue from the right lobe of the liver was cryopreserved in liquid nitrogen for measurement of hepatic lipid content (TG, CHO). Part of the right lobe of the liver was fixed in formalin and used for histopathological staining with HE, Oil Red, and Masson's staining to evaluate hepatic steatosis, ballooning, inflammation, and fibrosis. The NAFLD activity score (NAS Score) was calculated, and the percentage of fibrosis area was quantified in six fields of view of the Masson's stained sections of each mouse using Image J software.

[0046] The experimental results are as follows:

[0047] No mice died during the experiment, and due to the introduction of CCl4, there was no significant difference in the body weight of mice in the model group and each drug-treated group compared with the control group ( Figure 7), this example further demonstrates the good safety of the combination of bicyclol and berberine. Similar to Example 1, the liver macroscopically shows that the improvement of the liver yellowish fatty degeneration in each combination group is more obvious than that in the single drug group (Figure 8). Light microscopic observation of liver tissue pathology shows that the livers of mice in the model group have more severe fatty degeneration, ballooning, inflammatory necrosis and mild liver fibrosis pathological features. After treatment with single drugs, the above lesions have improved to a certain extent, and the improvement in each combination drug dosage group is more obvious (Figure 8). The NAS score of liver tissue pathology and the quantitative results of the percentage of liver fibrosis area further prove that the effect of combined drug treatment in the treatment of NAFLD is significantly better than that of single drug treatment ( Figure 9 ). The quantitative results of serum liver injury indicators (ALT, AST) and liver lipid content indicators (TG, CHO) also showed that the overall improvement effect of each combined drug dosage combination was more significant than that of single drug use ( Figure 10 The above results indicate that the combined use of bicyclol and berberine is more effective in treating non-alcoholic fatty liver disease than either alone, and is a discovery with value in drug development and clinical application.

[0048] The above description of the specific implementation of the present invention is merely illustrative and not restrictive of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and all such modifications or replacements will fall within the scope of protection of the present invention.

Claims

1. Use of a pharmaceutical composition containing bicyclol and berberine in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease (NAFLD), wherein: The bicyclic alcohol or berberine is its original drug or pharmaceutical salt; The dosage ratio of the bicyclol and berberine is 1:4 to 4:

1.

2. The use according to claim 1, characterized in that The non-alcoholic fatty liver disease (NAFLD) includes non-alcoholic hepatic steatosis (NAFL) and non-alcoholic steatohepatitis (NASH).

3. The use according to claim 1, characterized in that The active ingredients of the pharmaceutical composition are composed of bicyclol and berberine, wherein the bicyclol or berberine is the original drug or pharmaceutically acceptable salt thereof.

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