Pharmaceutical composition, preparation as well as preparation method and application of pharmaceutical composition

Abstract

The invention relates to a pharmaceutical composition, a preparation as well as a preparation method and application of the pharmaceutical composition, belongs to the technical field of pharmaceutical preparations, and at least solves the problem that an existing single medicine is insufficient in curative effect and / or safety in treatment of non-alcoholic fatty liver disease complicated with type 2 diabetes mellitus to a certain extent. The pharmaceutical composition comprises berberine and an FXR agonist, and the mass ratio of the berberine to the FXR agonist is (1: 5)-(5: 1). The berberine and the FXR agonist in the composition provided by the invention show an efficient synergistic effect under a proper compatibility relationship, so that the effects of reduction and synergism are realized, the medication safety and the compliance of patients are improved, the weight gain can be effectively inhibited, and the biochemical indexes and the weight of organisms are promoted to tend to a healthy state.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a pharmaceutical composition, a preparation and a preparation method and application thereof. BACKGROUND

[0002] Non-alcoholic fatty liver disease (NAFLD) affects about 25% of the global population, posing a major threat to global health. Non-alcoholic steatohepatitis (NASH) develops from non-alcoholic fatty liver (NAFL) and is accompanied by inflammation or fibrosis in addition to liver steatosis. NASH can further develop into cirrhosis and hepatocellular carcinoma. About 20% of NAFL patients will progress to NASH, and more than 40% of NASH patients will progress to liver fibrosis. In addition, NASH is often complicated with multiple metabolic diseases, and a study has reported that the incidence of NAFL in patients with Diabetes Mellitus type 2 (T2DM) in China is as high as 52.56%. The treatment of glucose and lipids is becoming an important development direction.

[0003] Farnesoid X Receptor (FXR) agonists are one of the most promising NASH treatment drugs. The new drug application of obeticholic acid for NASH treatment has progressed to clinical phase III, but due to the presence of unbearable side effects such as itching and liver toxicity, its marketing application for NASH has not been approved by the Food and Drug Administration (FDA). According to literature reports, other FXR agonists also have the above-mentioned side effects to varying degrees, so it is necessary to reduce the dosage of FXR agonists as much as possible to ensure their safety.

[0004] In view of the high incidence of NASH combined with T2DM, there is also a correlation between the two diseases, and the combined treatment of the two diseases is a new trend in drug development. There is a patent (Application Publication No. CN 116782898A) for using berberine and ursodeoxycholic acid in combination to treat NASH combined with T2DM, but the highest dose given to the subject in this patent is 2500mg, which reduces the compliance of clinical patients.

[0005] Therefore, it is a major task in the field of medical and pharmaceutical research to develop and study safe and effective drugs for treating non-alcoholic fatty liver disease combined with type 2 diabetes. SUMMARY

[0006] In view of the above analysis, the present application aims to provide a pharmaceutical composition, a preparation and a preparation method and application thereof, and aims to at least partially solve the deficiencies of the existing single drug in the treatment of non-alcoholic fatty liver disease combined with type 2 diabetes in terms of efficacy and / or safety.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] The present application provides a pharmaceutical composition comprising berberine and an FXR agonist, and the chemical structure of the FXR agonist is represented by the following formula (I):

[0009]

[0010] In the formula, the mass ratio of berberine to the FXR agonist is 1:5-5:1.

[0011] Further, the amount of each component in the pharmaceutical composition is calculated based on the oral administration dose of mice: berberine is 10-300 mg / kg, and the FXR agonist is 7.5-105 mg / kg.

[0012] Further, the amount of each component in the pharmaceutical composition is calculated based on the dose of healthy adults: berberine is 1.11-33.3 mg / kg, and the FXR agonist is 0.83-11.62 mg / kg.

[0013] Further, the amount of each component in the pharmaceutical composition is calculated based on the oral administration dose of mice: berberine is 50-100 mg / kg, and the FXR agonist is 22.5-45 mg / kg; and / or, based on the dose of healthy adults: berberine is 5.55-11.1 mg / kg, and the FXR agonist is 2.5-5.0 mg / kg.

[0014] Further, the mass ratio of berberine to the FXR agonist is 1.5:1-3.0:1.

[0015] Further, the mass ratio of berberine to the FXR agonist is 1.8:1-2.7:1.

[0016] The present application also provides a pharmaceutical preparation made of the pharmaceutical composition as described above, and the pharmaceutical preparation is in the form of a solid preparation or a liquid preparation.

[0017] Further, the pharmaceutical preparation comprises a powder, a granule, a tablet, a capsule, a pill, an injection, an oral liquid, a sustained-release preparation or a controlled-release preparation.

[0018] The application also provides a use of berberine and an FXR agonist in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease, and / or non-alcoholic steatohepatitis, and / or type 2 diabetes, and / or obesity, wherein the chemical structure of the FXR agonist is represented by the following formula (I):

[0019]

[0020] Further, in the use, the berberine and the FXR agonist are provided in a mass ratio of 1:5 to 5:1.

[0021] Compared with the prior art, the application can achieve at least one of the following beneficial effects:

[0022] (1) The berberine and the FXR agonist in the composition of the application exhibit a high-efficiency synergistic effect in a suitable compatibility relationship (for example, a mass ratio of 1:5 to 5:1), and can achieve a better effect of reducing both sugar and fat than the use of the drugs alone, even if the use amount of each component in the composition is reduced relative to the use amount of the drugs alone. In addition, a notable feature of the application is that the combination of berberine and the FXR agonist produces a unique synergistic effect on inhibiting weight gain, which is particularly suitable for obese people with NAFLD / NASH combined with type 2 diabetes, and has significant application prospects and economic value.

[0023] (2) In some preferred embodiments, the berberine and the FXR agonist in the composition of the application are further selected in a suitable dose range and an optimal dose range of each component under the premise of ensuring a suitable compatibility ratio of the medicament, which can more effectively exert the synergistic effect of the two components, promote the biochemical indicators and weight of the organism to be more healthy, and even if the dose of each component is significantly reduced, for example, reduced by 25%, at least the effect of the drugs alone can be achieved or the effect of reducing both sugar and fat can be achieved (for example, the results of the examples). In particular, the dose optimization strategy of the application can significantly alleviate the itching and other adverse side effects caused by the FXR agonist at a high dose, thereby improving the safety of the drugs. This innovative combination of drugs and dose optimization method has the potential to fill the gap and important value in the field of marketed drug preparations for NAFLD / NASH combined with type 2 diabetes in China.

[0024] (3) In some preferred embodiments, the mass ratio of berberine and FXR agonist is further optimized to maximize the beneficial effects of each component in vivo, achieve a synergistic effect of 1+1>2, and improve glycemic control and lipid-lowering, making biochemical indicators and body weight more healthy, using a more appropriate drug compatibility ratio, allowing the use of lower drug doses to maximize the positive effects on NAFLD / NASH combined with type 2 diabetes, not only reducing drug costs, but also reducing drug side effects, and further achieving the optimal balance between efficacy and safety.

[0025] (4) The application also provides a preparation method of a medicament containing the pharmaceutical composition of the application, which has simple preparation process, rapid therapeutic effect, and the prepared oral liquid containing the composition can fully exert the synergistic effect between the two drugs, effectively improve the bioavailability of the drug, and has the advantages of convenient administration, rapid absorption, and easy dosage adjustment.

[0026] (5) The pharmaceutical composition provided by the application exhibits better glycemic control and lipid-lowering effect through the synergistic effect of berberine and FXR agonist at a reduced dose compared to single drug treatment. This combination composition has significant efficacy for non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and type 2 diabetes (T2DM), specifically, after combination therapy, the liver function, blood glucose control, and biochemical indicators of the organism are significantly improved, and the combination therapy is more close to the healthy control group than the single drug therapy (with a higher dose). The pharmaceutical composition and medicament of the application exhibit excellent performance in reducing the amount and increasing the effect, improve the safety of drug use and the compliance of patients, and have potential application value in the preparation of drugs for preventing and treating one or more of NAFLD, NASH, T2DM, and obesity.

[0027] In the application, the above technical solutions can be combined with each other to achieve more preferred combination solutions. Other features and advantages of the application will be described in the subsequent specification, and some advantages will become apparent from the specification, or will be understood by implementing the application. The purpose and other advantages of the application can be achieved and obtained from the specific embodiments described in the specification and the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0028] The accompanying drawings are included to provide a further understanding of the application and are incorporated herein and constitute a part of the application. The drawings illustrate embodiments of the application and, together with the description, serve to explain the principles of the application.

[0029] Figure 1 The results of the intraperitoneal injection of the glucose tolerance test in the mice given normal diet (healthy group) and high-fat diet (model group) for 12 weeks in Example 1 are shown in Table 1.

[0030] Figure 2 Figure for NAS score of mouse liver under different intervention conditions in Example 1;

[0031] Figure 3 Figure for ballooning score of mouse liver under different intervention conditions in Example 1;

[0032] Figure 4 Figure for inflammation score of mouse liver under different intervention conditions in Example 1;

[0033] Figure 5 Figure for steatosis score of mouse liver under different intervention conditions in Example 1;

[0034] Figure 6 Figure for IPGTT (intraperitoneal glucose tolerance test) of mouse under different intervention conditions in Example 1;

[0035] Figure 7 Figure for ITT (insulin tolerance test) of mouse under different intervention conditions in Example 1;

[0036] Figure 8 Figure for body weight of mouse under different intervention conditions in Example 1;

[0037] Figure 9 Figure for liver weight of mouse under different intervention conditions in Example 1;

[0038] Figure 10 Figure for liver coefficient (liver weight / body weight) of mouse under different intervention conditions in Example 1;

[0039] Figure 11 Figure for serum ALT change of mouse under different intervention conditions in Example 1;

[0040] Figure 12 Figure for serum AST change of mouse under different intervention conditions in Example 1;

[0041] Figure 13 Figure for liver ALT change of mouse under different intervention conditions in Example 1;

[0042] Figure 14 Figure for liver AST change of mouse under different intervention conditions in Example 1;

[0043] Figure 15 Figure for serum TBA change of mouse under different intervention conditions in Example 1;

[0044] Figure 16 Figure for serum ALP change of mouse under different intervention conditions in Example 1;

[0045] Figure 17Figure for changes in serum triglyceride of mice under different intervention conditions in Example 1;

[0046] Figure 18 Figure for changes in serum cholesterol of mice under different intervention conditions in Example 1;

[0047] Figure 19 Figure for changes in serum APOA of mice under different intervention conditions in Example 1;

[0048] Figure 20 Figure for changes in serum APOB of mice under different intervention conditions in Example 1. DETAILED DESCRIPTION

[0049] The preferred embodiments of the present application will be described in detail below with reference to the drawings, wherein the drawings constitute a part of this application and illustrate the principles of the application together with the embodiments thereof, but are not intended to limit the scope of the application.

[0050] The inventors have previously obtained a potential FXR agonist with NASH treatment effect, which has been verified by in vitro and animal experiments to have good FXR selectivity and NASH treatment effect, but also shows side effects such as itching at a high dose in the clinical trial stage. At the same time, the FXR agonist has poor blood glucose control effect, and the treatment effect for NASH patients combined with T2DM is limited. Therefore, it is necessary to explore a drug combination scheme to reduce the dosage of the FXR agonist, while achieving the treatment of NASH and T2DM.

[0051] Based on this, in a first aspect, the present application provides a pharmaceutical composition comprising berberine and an FXR agonist, wherein the chemical structure of the FXR agonist is represented by the following formula (I):

[0052]

[0053] wherein the mass ratio of berberine to the FXR agonist is 1:5-5:1.

[0054] Berberine (Berberine, Berberine) is a quaternary ammonium alkaloid isolated from traditional Chinese medicine Huanglian, with a molecular weight of 336.36, a molecular formula of C20H18NO4+, a yellow needle-like crystal, a CAS number of 2086-83-1, and has been approved for listing. The indications are intestinal infections.

[0055] The compound represented by formula (I) is an FXR agonist disclosed in Chinese patent CN 110662743B.

[0056] In the present application, the purity of berberine and the FXR agonist is >98%.

[0057] The inventors of the present application surprisingly found that, compared with each component alone, the berberine and FXR agonist in the composition of the present application exhibit high synergistic effect in a suitable compatible relationship (such as a mass ratio of 1:5-5:1), and can achieve better effects of reducing both glycolipids than using each component alone, while the amount of each component in the pharmaceutical composition is reduced relative to the amount used alone. In addition, a notable feature of the present application is the unique synergistic effect produced by the combination of berberine and FXR agonist for inhibiting weight gain, which is particularly suitable for obese people with NAFLD / NASH combined with type 2 diabetes, and has significant application prospects and economic value.

[0058] It should be noted that the potential mechanism of the synergistic effect exhibited by the composition provided by the present application includes the following aspects: (1) synergistic effect of multiple organs: berberine promotes TGR5 activation GLP-1 secretion by improving intestinal flora and bile acid composition, further promoting insulin secretion; FXR agonist promotes the increase of IRS-2 tyrosine phosphorylation level in liver and peripheral tissues, and promotes the increase of insulin sensitivity. The combination of the two can promote glycogen synthesis in the liver, while inhibiting gluconeogenesis and improving insulin resistance symptoms. (2) synergistic effect of liver: both berberine and FXR agonist have liver targeting (liver / blood ratio in animal experiments is >10), berberine can inhibit liver inflammation and oxidative stress through the ROS pathway, while reducing lipid synthesis and inducing fatty acid beta oxidation through the AMPK pathway and PPARγ pathway to regulate liver lipid metabolism; FXR agonist can regulate lipid metabolism and inhibit inflammation through the FXR pathway.

[0059] In some preferred embodiments, the amount of each component in the pharmaceutical composition, calculated based on the oral administration dose of mice, is: berberine 10-300 mg / kg, and FXR agonist 7.5-105 mg / kg; and / or, calculated based on the dose of healthy adults, is: berberine 1.11-33.3 mg / kg, and FXR agonist 0.83-11.62 mg / kg.

[0060] Further preferably, the amount of each component in the pharmaceutical composition, calculated based on the oral administration dose of mice, is: berberine 50-100 mg / kg, and FXR agonist 22.5-45 mg / kg; and / or, calculated based on the dose of healthy adults, is: berberine 5.55-11.1 mg / kg, and FXR agonist 2.5-5.0 mg / kg.

[0061] It can be understood that the berberine and the FXR agonist in the composition of the present application further select the suitable dose range and the optimal dose range of each component under the premise of ensuring the suitable drug compatibility ratio, and the optimization can more effectively exert the synergistic effect of the two components, promote the biochemical indicators and the body weight of the organism to be more healthy, and at least achieve the effect comparable to the single drug even if the dose of each component is significantly reduced, for example, reduced by 25%, and the effect of double reduction of sugar and fat is achieved beyond the single drug (for example, the results of the examples). In particular, the dose optimization strategy of the present application has a significant alleviating effect on the itching and other adverse side effects caused by the FXR agonist at a higher dose, thereby improving the safety of the drug. The innovative drug combination and dose optimization method of the combined drug have the potential to fill the gap and important value in the field of marketed drug preparations for NAFLD / NASH combined with type 2 diabetes in China.

[0062] Exemplarily, the mass ratio of the berberine to the FXR agonist is 1:5, 1:2, 1:1, 1.2:1, 1.5:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 3.0:1, 3.2:1, 3.5:1, 3.7:1, 4.0:1, 4.2:1, 4.5:1, 4.7:1, 5.0:1.

[0063] Preferably, the mass ratio of the berberine to the FXR agonist is 1.5:1 to 3.0:1.

[0064] Further preferably, the mass ratio of the berberine to the FXR agonist is 1.8:1 to 2.7:1.

[0065] More preferably, the mass ratio of the berberine to the FXR agonist is 2.0:1 to 2.5:1.

[0066] It should be noted that by further optimizing the mass ratio of berberine and FXR agonist, each component can exert the maximum beneficial effect in the organism, achieve a synergistic effect of 1+1>2, thereby improve the reduction of sugar and fat, make the biochemical indicators and the body weight more healthy, adopt a more suitable drug compatibility ratio, allow the use of a lower drug dose to maximize the positive effect on NAFLD / NASH combined with type 2 diabetes, reduce the cost of drugs, and reduce the toxic and side effects of drugs, so as to further achieve the optimal balance of drug efficacy and safety.

[0067] In the second aspect, the present application provides a pharmaceutical preparation made of the pharmaceutical composition of the first aspect, and the pharmaceutical preparation is in the form of a solid preparation or a liquid preparation.

[0068] Optionally, the pharmaceutical preparation comprises a powder, a granule, a tablet, a capsule, a pill, an oral liquid, a sustained-release preparation or a controlled-release preparation.

[0069] In some preferred embodiments, the pharmaceutical preparation is an oral liquid preparation.

[0070] In a third aspect, the present application further provides a preparation method of the oral liquid preparation, comprising the following steps:

[0071] (1) weighing the required berberine and FXR agonist according to the predetermined dosage, and then adding them into an aqueous solution containing a cosolvent to obtain a mixed solution, wherein, in terms of mouse oral dosing, the concentration of berberine ranges from 8.0 to 12.0 milligrams per milliliter (mg / mL) of the aqueous solution, and the concentration of FXR agonist ranges from 3.0 to 6.0 milligrams per milliliter (mg / mL) of the aqueous solution;

[0072] For example, in terms of mouse oral dosing, the concentration of berberine is 8.5, 9.0, 9.5, 10.0, 10.5, 11.0 or 11.5 milligrams per milliliter (mg / mL) of the aqueous solution, and the concentration of FXR agonist is 3.5, 4.0, 4.5, 5.0 or 5.5 milligrams per milliliter (mg / mL) of the aqueous solution;

[0073] Or, in terms of healthy adult oral dosing, the concentration of berberine ranges from 0.88 to 1.32 milligrams per milliliter (mg / mL) of the aqueous solution, and the concentration of FXR agonist ranges from 0.33 to 0.66 milligrams per milliliter (mg / mL) of the aqueous solution;

[0074] For example, in terms of healthy adult oral dosing, the concentration of berberine is 0.93, 0.99, 1.04, 1.10, 1.15, 1.21 or 1.26 milligrams per milliliter (mg / mL) of the aqueous solution, and the concentration of FXR agonist is 0.38, 0.44, 0.50, 0.55 or 0.60 milligrams per milliliter (mg / mL) of the aqueous solution;

[0075] (2) subjecting the obtained mixed solution to ultrasonic treatment to facilitate complete dissolution of the berberine and FXR agonist.

[0076] Optionally, the cosolvent comprises at least one of sodium carboxymethyl cellulose, Tween-80, cyclodextrin, polyethylene glycol, N-methyl pyrrolidone and propylene glycol.

[0077] Preferably, the cosolvent is sodium carboxymethyl cellulose.

[0078] Exemplarily, the "solution containing a cosolvent" refers to a 0.5% carboxymethyl cellulose sodium aqueous solution. The 0.5% carboxymethyl cellulose sodium aqueous solution is a solution with carboxymethyl cellulose sodium (CMC-Na) as solute and water as solvent. "0.5%" means mass percentage concentration, i.e. 0.5 grams of carboxymethyl cellulose sodium is contained in 100 grams of the aqueous solution.

[0079] Preferably, in the preparation method, the ultrasonic frequency of the ultrasonic treatment is 30-50 KHz, the ultrasonic power is 200-300 W, and the ultrasonic time is 8-15 min. More preferably, the ultrasonic frequency of the ultrasonic treatment is 35-45 KHz, the ultrasonic power is 240-260 W, and the ultrasonic time is 10-12 min.

[0080] Preferably, the ultrasonic treatment adopts a shaking ultrasonic mode.

[0081] In a fourth aspect, the present application further provides a use of berberine and an FXR agonist in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease, and / or non-alcoholic steatohepatitis, and / or type 2 diabetes, and / or obesity, wherein the chemical structure of the FXR agonist is represented by the following formula (I):

[0082]

[0083] In some embodiments, in the use, the berberine and the FXR agonist are provided in a mass ratio of 1:5-5:1. Exemplarily, the berberine and the FXR agonist are provided in a mass ratio of 1:5, 1:2, 1:1, 1.2:1, 1.5:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 3.0:1, 3.2:1, 3.5:1, 3.7:1, 4.0:1, 4.2:1, 4.5:1, 4.7:1, 5:1.

[0084] Preferably, in the use, the berberine and the FXR agonist are provided in a mass ratio of 1.5:1-3.0:1.

[0085] Further preferably, in the use, the berberine and the FXR agonist are provided in a mass ratio of 1.8:1-2.7:1.

[0086] More preferably, in the use, the berberine and the FXR agonist are provided in a mass ratio of 2.0:1-2.5:1.

[0087] In some preferred embodiments, in said use, said berberine and said FXR agonist are used in amounts as follows, calculated as mouse oral dosing: berberine 10-300 mg / kg, FXR agonist 7.5-105 mg / kg; and / or, calculated as healthy adult dosing: berberine 1.11-33.3 mg / kg, FXR agonist 0.83-11.62 mg / kg.

[0088] Exemplarily, calculated as mouse oral dosing: berberine 10 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, 275 mg / kg, 300 mg / kg, such as berberine 50-300 mg / kg; FXR agonist 7.5 mg / kg, 10 mg / kg, 12.5 mg / kg, 15.0 mg / kg, 17.5 mg / kg, 20.0 mg / kg, 22.5 mg / kg, 25 mg / kg, 27.5 mg / kg, 30.0 mg / kg, 32.5 mg / kg, 35.0 mg / kg, 37.5 mg / kg, 40.0 mg / kg, 42.5 mg / kg, 45.0 mg / kg, 50.0 mg / kg, 52.5 mg / kg, 75 mg / kg, 90.0 mg / kg, 105.0 mg / kg, such as FXR agonist 7.5-52.5 mg / kg.

[0089] Exemplarily, calculated as healthy adult dosing: berberine 1.11 mg / kg, 5.55 mg / kg, 11.1 mg / kg, 16.65 mg / kg, 22.2 mg / kg, 27.75 mg / kg, 33.3 mg / kg, such as berberine 5.55-33.3 mg / kg; FXR agonist 0.83 mg / kg, 1.66 mg / kg, 2.5 mg / kg, 3.32 mg / kg, 4.15 mg / kg, 5.0 mg / kg, 5.83 mg / kg, 7.47 mg / kg, 9.13 mg / kg, 11.62 mg / kg, such as FXR agonist 0.83-5.83 mg / kg.

[0090] Further preferably, in said application, the berberine and the FXR agonist are used in the following amounts, calculated as oral dose in mice: berberine 50-100 mg / kg, FXR agonist 22.5-45 mg / kg; and / or, calculated as healthy adult dose: berberine 5.55-11.1 mg / kg, FXR agonist 2.5-5.0 mg / kg.

[0091] In particular, the obesity includes simple obesity, secondary obesity. The pharmaceutical composition or the pharmaceutical preparation of the present application is suitable for overweight people with body mass index (BMI) between 25 and 29.9, and especially suitable for obese people with degree I to III obesity with body mass index (BMI) of 30 and above.

[0092] The technical solutions of the present application are further described in detail below in combination with specific examples.

[0093] The test materials in the following examples are as follows:

[0094] (1) Berberine: produced by Bailingwei Technology Co., Ltd., batch number 275920, purity 98%;

[0095] (2) FXR agonist: produced by Shijiazhuang Zhongqi Pharmaceutical Technology Co., Ltd.

[0096] Example 1

[0097] This example describes the effect of the pharmaceutical composition of berberine and FXR agonist on the NASH combined with T2DM mouse animal model constructed after high-fat feeding induction; in this example, the chemical structure of the FXR agonist is represented by the following formula (I):

[0098]

[0099] 1. Test design: the modeling of this test includes two steps of high-fat diet feeding and mouse intervention;

[0100] (a) High-fat diet feeding, constructing NASH combined with T2DM mouse animal model;

[0101] Specifically, the initial weight of mice ranged from 17.5 to 18 g, and the model evaluation was performed after feeding with 60% high-fat feed (item number D12492i) for 12 weeks. The weight of mice ranged from 47.2 to 60.4 g after 12 weeks. The evaluation of NASH relied on the diagnostic gold standard: liver pathological section. The liver of mice was fixed by polyoxymethylene, paraffin section, and finally hematoxylin-eosin staining (HE) was performed. The HE staining results were scored by two pathologists independently, and the specific scoring criteria referred to the NAS score (Table 1 and Table 2). The NAS score of 5 points or more was judged as NASH, 3-4 points was possible NASH, and 1-2 points was non-NASH.

[0102] Table 1. Pathological score of non-alcoholic steatohepatitis (NASH)

[0103]

[0104] Table 2. Diagnostic criteria of non-alcoholic steatohepatitis (NASH)

[0105]

[0106] The model evaluation results showed that the NAS score of mice after modeling was generally 4-6, which showed the typical symptoms of NASH.

[0107] The evaluation of T2DM was performed by intraperitoneal glucose tolerance test (IPGTT), which was similar to OGTT (the gold standard for clinical diagnosis of T2DM). The blood glucose of mice was measured at different times after administration of glucose to diagnose T2DM. From the results of Figure 1 , compared with the healthy group, the mice in the model group showed obvious glucose regulation abnormalities after being fed with high-fat feed for 12 weeks.

[0108] It should be noted that Figures 1-20 , ns: no statistical difference; *: p < 0.05; **: p < 0.01; ****: p < 0.0001.

[0109] (b) Intervention of mice with NASH combined with T2DM

[0110] After the model was successfully established, the mice were randomly divided into 5 groups (6-8 mice per group), including:

[0111] (1) Healthy control group: mice without high-fat feed were given the same volume of 0.5% carboxymethylcellulose sodium aqueous solution orally;

[0112] (2) Solvent control group: mice fed with high-fat diet, and given the same volume of 0.5% sodium carboxymethyl cellulose aqueous solution orally;

[0113] (3) Single-dose FXR group: the dose of FXR agonist is 45 mg / kg, the predetermined dose of FXR agonist is weighed according to the weight of the mouse, and is added into 0.5% sodium carboxymethyl cellulose aqueous solution to prepare an oral solution for oral administration to the mouse;

[0114] (4) Single-dose berberine group: the dose of berberine is 100 mg / kg, the predetermined dose of berberine is weighed according to the weight of the mouse, and is added into 0.5% sodium carboxymethyl cellulose aqueous solution to prepare an oral solution for oral administration to the mouse;

[0115] (5) FXR and berberine combination group: the dose of FXR agonist is 33.75 mg / kg, and the dose of berberine is 75 mg / kg, the predetermined dose of FXR agonist and berberine is weighed according to the weight of the mouse, and is added into 0.5% sodium carboxymethyl cellulose aqueous solution to prepare an oral solution for oral administration to the mouse.

[0116] For groups (3)-(5), when the oral solution containing the drug is prepared, the concentration of berberine is 10 mg of berberine per 1 ml of 0.5% sodium carboxymethyl cellulose aqueous solution, and the concentration of FXR agonist is 4.5 mg of FXR agonist per 1 ml of 0.5% sodium carboxymethyl cellulose aqueous solution.

[0117] Among them, for group (5), when the FXR agonist is combined with berberine, the mass ratio of berberine to FXR agonist is 2.22:1.

[0118] For the above five groups, the blank solvent or different drugs are administered by gavage once a day, and the administration is continued for 28 days. Subsequently, the pharmacodynamic evaluation is carried out: 1. HE staining of liver pathological sections, NAS scoring; 2. intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) for evaluation of glucose tolerance and insulin resistance; 3. detection of physiological indexes, biochemical indexes and inflammatory indexes to comprehensively evaluate the efficacy and synergistic effect of single drug and two-drug combination on reducing blood sugar and blood lipid.

[0119] 2. Efficacy evaluation: the effect of the drug combination provided by the application and the medicament thereof on the improvement of NASH, the improvement of T2DM, the effect of weight loss, and the influence on biochemical indexes are comprehensively evaluated;

[0120] (1) NAS score results of mice;

[0121] Test purpose: Primary efficacy endpoint for NASH treatment. Used to evaluate the therapeutic effect of NASH after drug intervention.

[0122] Specific steps: The liver tissue fixed in 4% paraformaldehyde solution for 24h was dehydrated with 70%, 80%, 90%, 95% ethanol and anhydrous ethanol gradient, and then paraffin-embedded, cut into 4μm thin sections and spread on glass slides, dried in an oven, and then subjected to subsequent staining. First, staining was performed with xylene solvent, and then dewaxing and washing were performed with 100%, 95%, 80%, ultrapure water, followed by staining with hematoxylin staining solution, distilled water washing, 1% hydrochloric acid ethanol solution immersion, distilled water washing, 0.5% ammonia water solution blueing, distilled water washing, 1% eosin staining solution re-staining, distilled water washing, and then 80%, 95%, 100% ethanol washing, and finally xylene solution cleaning. After staining, the cell nucleus was blue, and the cytoplasm, muscle fibers, collagen fibers and red blood cells were red in different shades.

[0123] Result analysis: After 4 weeks of drug intervention, from Figure 2 It can be seen that, compared with the healthy control group, the NAS score of the solvent control group was significantly increased. The single-dose FXR group, the single-dose berberine group and the combination of FXR and berberine can significantly reduce the NAS score, and it is worth noting that the combination group can more significantly reduce the NAS score than the single drug use and show more significant differences. The NAS score is composed of three phenotypes of steatosis, ballooning and inflammation. Continue to analyze the effect of different drugs and combinations on the phenotype, see Figures 3-5 , compared with FXR and berberine single drug, the combination group can more significantly reduce the level of steatosis and inflammation in the liver.

[0124] Therefore, it is shown that the berberine and FXR agonist in the composition of the present application show high efficient synergistic effect under suitable compatibility, and in the case that the dosage of each component in the composition is reduced by 25% compared with the dosage of single drug, better NASH treatment effect than single drug can still be achieved.

[0125] (2) Evaluation of the results of mouse IPGTT (intraperitoneal glucose tolerance test) and ITT (insulin tolerance test);

[0126] Test purpose: Primary efficacy endpoint for T2DM treatment. Used to evaluate the improvement of blood glucose control and insulin secretion after drug intervention.

[0127] (a) IPGTT evaluation, which includes the following specific steps:

[0128] 1. The day before the experiment, the mice were changed into clean cages and fasted for 6-8 hours, with free access to water;

[0129] 2. The weight of each mouse was measured;

[0130] 3. The mice were taken out, and the tail ends of the mice were cut off about 1-2 mm. The tail vein of the mice was squeezed to make the blood accumulate into a drop. The first drop of blood was discarded, and the second drop of blood was used to measure the fasting blood glucose with a blood glucose meter (Roche Accu-Chek Active blood glucose meter) and blood glucose test paper (Roche Accu-Chek Active blood glucose test paper), which was the blood glucose value at 0 min;

[0131] 4. A 200 mg / ml glucose solution was prepared with normal saline, and intraperitoneal injection was performed according to a proportion of 0.01 ml / g (0.01 milliliter of glucose solution was received by each gram of body weight of the mice). The time was counted from the completion of the injection, and blood glucose test paper was used for detection at different time points (15, 30, 60, 90, and 120 minutes).

[0132] (b) ITT evaluation, the specific steps of which include:

[0133] 1. The day before the experiment, the mice were changed into clean cages and fasted for 6-8 hours, with free access to water;

[0134] 2. The weight of each mouse was measured;

[0135] 3. The mice were taken out, and the tail ends of the mice were cut off about 1-2 mm. The tail vein of the mice was squeezed to make the blood accumulate into a drop. The first drop of blood was discarded, and the second drop of blood was used to measure the fasting blood glucose with a blood glucose meter (Roche Accu-Chek Active blood glucose meter) and blood glucose test paper (Roche Accu-Chek Active blood glucose test paper), which was the blood glucose value at 0 min;

[0136] 4. A 0.05 U / ml insulin solution was prepared with normal saline, and intraperitoneal injection was performed according to a proportion of 0.01 ml / g (0.01 milliliter of insulin solution was received by each gram of body weight of the mice). The time was counted from the completion of the injection, and blood glucose test paper was used for detection at different time points (15, 30, 60, 90, and 120 minutes).

[0137] Results analysis: from Figure 6 and Figure 7It can be seen that the blood glucose level of the solvent control group is significantly increased after administration of glucose or insulin compared with the healthy control group, indicating a decrease in insulin sensitivity and a decrease in insulin secretion capacity. Administration of the FXR agonist alone does not improve the blood glucose curve of the IPGTT, indicating that it cannot affect the secretion capacity of insulin, but it can reduce the blood glucose level after ITT, indicating that it can improve insulin resistance to some extent, because insulin resistance is mainly due to the impairment of insulin sensitivity of the insulin receptor in the liver and muscle, and the FXR agonist can improve the metabolic abnormalities of the liver to improve its insulin sensitivity. Berberine can have a beneficial effect on insulin secretion and insulin sensitivity, but it shows a phenomenon of secondary increase in blood glucose in the IPGTT, indicating that it is still insufficient for blood glucose control in the disease state.

[0138] In comparison, the berberine and FXR agonist in the composition of the present application exhibit a highly efficient synergistic effect in a suitable compatible relationship. When the dosage of each component in the composition is reduced by 25% compared with the single drug, a better therapeutic effect on T2DM than the single drug can still be achieved. Specifically, the combination of FXR and berberine can ensure more stable blood glucose fluctuations and has a better improvement capacity for insulin secretion and insulin resistance than the single drug.

[0139] (3) Changes in body weight and liver weight of mice

[0140] From Figures 8-10 It can be seen that after 4 weeks of drug intervention, the solvent control group has a significant increase in body weight, liver weight, and liver weight to body weight ratio compared with the healthy control group. Administration of the FXR agonist or berberine alone can reduce the body weight and / or liver weight of the NASH combined with T2DM mice to varying degrees, but the single-dose FXR agonist group does not show a significant difference, indicating that administration of the FXR agonist alone does not have a significant effect on the body weight of the mice. The combination of FXR and berberine shows a more obvious trend of body weight and liver weight reduction and is closer to the healthy control group, indicating that the combination can better improve the body weight and liver weight increase induced by high-fat diet.

[0141] Therefore, the berberine and FXR agonist in the composition of the present application exhibit a highly efficient synergistic effect in a suitable compatible relationship. When the dosage of each component is reduced by 25% compared with the single drug, not only can a better therapeutic effect on non-alcoholic steatohepatitis (NASH) and type 2 diabetes (T2DM) be achieved, but an additional benefit of controlling body weight gain is also unexpectedly exhibited. For overweight or obese people with NASH combined with type 2 diabetes, the pharmaceutical composition and medicament provided by the present application exhibit a positive application prospect.

[0142] (4) Evaluation of mouse biochemical indicators

[0143] Objective: T2DM and NASH diseases can cause changes in physiological and biochemical parameters, and with the remission of T2DM and NASH symptoms, some physiological or biochemical parameters also have varying degrees of improvement. Therefore, monitoring these biochemical parameters is crucial for evaluating the effects of drug intervention and discovering possible side effects.

[0144] Specific steps: ALT, AST and cholesterol of liver were detected by ELISA kit (ALT kit brand Solarbio, item number BC1555; AST kit brand Solarbio, item number BC1565; Cholesterol kit brand AAT Bioquest, item number 40006), liver tissue was added to PBS homogenate solution at a ratio of 1:9, and then tissue homogenate was carried out at 4°C. Then take 3500g tissue homogenate, centrifuge at 4°C for 10 min, take the supernatant and determine according to the kit instructions. The parameters of serum were detected by blood biochemical analyzer.

[0145] By respectively determining the ALT and AST levels of liver, and detecting the alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acid (TBA), total cholesterol (TC), triglyceride (TG), apolipoprotein A (APOA), apolipoprotein B (APOB), cholesterol in serum, the results of Figures 11-20 were obtained.

[0146] Result analysis:

[0147] a) ALT, AST and ALP are enzymes secreted by the liver, mainly used for the diagnosis and differential diagnosis of skeletal and hepatobiliary system diseases, and their abnormal increase represents inflammation or necrosis of the liver, especially the increase of AST in blood represents severe necrosis, and mitochondria in cells enter the blood. From Figure 11 and Figure 12It can be seen that in serum, ALT and AST of the solvent control group were significantly higher than those of the healthy control group, indicating that the liver cells of mice were severely damaged under the intervention of high-fat diet, while the single-dose FXR group, the single-dose berberine group and the FXR+berberine combined group could reduce the levels of ALT and AST to different extents, among which the combined group was close to the healthy control group. Similarly, from Figure 16 It can be seen that in serum, ALP of the solvent control group was significantly higher than that of the healthy control group, and was significantly improved after drug intervention, and the FXR+berberine combined group tended to be close to the healthy control group.

[0148] b) from Figure 13 and Figure 14 It can be seen that in the liver, ALT and AST of the FXR+berberine combined group were close to those of the healthy control group.

[0149] c) Bile acids are produced by the liver and excreted by bile, and after enterohepatic circulation, they are distributed in blood and tissues and organs. Abnormal increase of bile acids can cause a series of diseases such as inflammation, thereby promoting the progression of NASH. From Figure 15 In serum, TBA of the solvent control group was significantly higher than that of the healthy control group, and was significantly improved after drug intervention, and the FXR+berberine combined group had more obvious improvement effect.

[0150] d) TC and TG can reflect the distribution of cholesterol and triglyceride in serum, from Figure 17 and Figure 18 It can be seen that both of them were significantly increased in the solvent control group, while berberine showed the effect of reducing triglyceride, and FXR agonist showed the effect of reducing cholesterol, and the combined use of the two drugs could achieve the simultaneous improvement of cholesterol and triglyceride.

[0151] e) APOA and APOB are mainly used for binding and transporting blood lipids to tissues of the body for metabolism and utilization, and their function is very important for maintaining blood lipid balance. From Figure 19 and Figure 20 It can be seen that APOA and APOB were significantly increased in the solvent control group, FXR agonist could appropriately reduce the level of APOA and significantly reduce the level of APOB, and berberine had no improvement effect on APOA; the combination of the two drugs could produce certain beneficial effects and reduce the levels of APOA and APOB.

[0152] Thus, FXR alone significantly improved some indicators, such as cholesterol and APOA, but had limited effects on triglycerides. In contrast, berberine alone significantly improved triglycerides, but had no significant effect on cholesterol, APOA, and other indicators. The combination of FXR and berberine, when used in an appropriate combination, exhibited a highly effective synergistic effect. When the dosage of each component was reduced by 25% relative to the single-dose dosage, they synergistically improved abnormal biochemical indicators of NASH and T2DM, enhancing the therapeutic effect. Furthermore, biochemical indicators tended toward those of the healthy control group, demonstrating that the combination therapy did not produce significant side effects. Thus, compared to the liver toxicity and other side effects of existing NASH drugs (e.g., obeticholic acid), the drug combination provided by the present invention, based on the highly effective synergistic effect of FXR and berberine in an appropriate ratio, can simultaneously effectively improve NASH and T2DM with reduced dosage. Furthermore, for overweight or obese individuals, it can also achieve the additional beneficial effect of weight loss while maintaining safety.

[0153] Example 2

[0154] The difference between this example and Example 1 is that in the mouse intervention step after model establishment, the FXR and berberine combination group: the dose of FXR agonist is 35.0 mg / kg, the dose of berberine is 63 mg / kg, and the mass ratio of berberine to FXR agonist is 1.8:1; the remaining steps and parameters are similar to Example 1.

[0155] In the NAS score results and the mouse IPGTT (intraperitoneal glucose tolerance test) and ITT (insulin tolerance test) and body weight / liver weight results, the hypoglycemic and lipid-lowering effects and weight loss effects of this example were better than those of the single drug, but were slightly lower than the corresponding effects of the combination drug group in Example 1.

[0156] Example 3

[0157] The difference between this example and Example 1 is that in the mouse intervention step after model establishment, the FXR and berberine combination group: the dose of FXR agonist is 28.51 mg / kg, the dose of berberine is 77.0 mg / kg, and the mass ratio of berberine to FXR agonist is 2.7:1; the remaining steps and parameters are similar to those in Example 1.

[0158] In the NAS score results and the mouse IPGTT (intraperitoneal glucose tolerance test) and ITT (insulin tolerance test) and body weight / liver weight results, the hypoglycemic and lipid-lowering effects and weight loss effects of this example were better than those of the single drug, but were slightly lower than the corresponding effects of the combination drug group in Example 1.

[0159] Example 4

[0160] The difference between this example and Example 1 is that in the mouse intervention step after model establishment, the FXR and berberine combination treatment group: the dose of FXR agonist was 40.0 mg / kg, the dose of berberine was 60.0 mg / kg, and the mass ratio of berberine to FXR agonist was 1.5:1; the remaining steps and parameters were similar to those in Example 1.

[0161] In the NAS score results and the mouse IPGTT (intraperitoneal glucose tolerance test) and ITT (insulin tolerance test) and body weight / liver weight results, the hypoglycemic and lipid-lowering effects and weight loss effects of this example are better than those of the single drug, but are slightly lower than the corresponding effects of the combination drug group in Example 2.

[0162] Example 5

[0163] The difference between this example and Example 1 is that in the mouse intervention step after model establishment, the FXR and berberine combination group: the dose of FXR agonist was 26.67 mg / kg, the dose of berberine was 80.0 mg / kg, and the mass ratio of berberine to FXR agonist was 3.0:1; the remaining steps and parameters were similar to those in Example 1.

[0164] In the NAS score results and the mouse IPGTT (intraperitoneal glucose tolerance test) and ITT (insulin tolerance test) and body weight / liver weight results, the hypoglycemic and lipid-lowering effects and weight loss effects of this example were better than those of the single drug, but were slightly lower than the corresponding effects of the combination drug group in Example 3.

[0165] Example 6

[0166] The difference between this example and Example 1 is that in the mouse intervention step after model establishment, the FXR and berberine combination treatment group: the dose of FXR agonist was 90 mg / kg, the dose of berberine was 18 mg / kg, and the mass ratio of berberine to FXR agonist was 1:5; the remaining steps and parameters were similar to those in Example 1.

[0167] In the NAS score results and the mouse IPGTT (intraperitoneal glucose tolerance test) and ITT (insulin tolerance test) and body weight / liver weight results, the hypoglycemic and lipid-lowering effects and weight loss effects of this example are better than those of the single drug, but are slightly lower than the corresponding effects of the combination drug group in Example 4.

[0168] Example 7

[0169] The difference between this example and Example 1 is that in the mouse intervention step after model establishment, the FXR and berberine combination treatment group: the dose of FXR agonist was 18 mg / kg, the dose of berberine was 90.0 mg / kg, and the mass ratio of berberine to FXR agonist was 5.0:1; the remaining steps and parameters were similar to those in Example 1.

[0170] In the results of NAS score, mouse IPGTT (intraperitoneal glucose tolerance test) and ITT (insulin tolerance test), the hypoglycemic, hypolipidemic and weight loss effects of the combination of the present embodiment are all superior to the single drug, but slightly lower than the corresponding effects of the combination of the present embodiment in Example 5.

[0171] The above merely describes the preferred embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises berberine and an FXR agonist, wherein the chemical structure of the FXR agonist is represented by the following formula (I): The mass ratio of berberine to FXR agonist is 1:5 to 5:

1.

2. The pharmaceutical composition according to claim 1, characterized in that The amount of each component in the pharmaceutical composition is calculated based on the oral dosage for mice: berberine is 10-300 mg / kg, and the FXR agonist is 7.5-105 mg / kg.

3. The pharmaceutical composition according to claim 1, characterized in that The amount of each component in the pharmaceutical composition is calculated based on the dosage for a healthy adult: berberine is 1.11-33.3 mg / kg, and the FXR agonist is 0.83-11.62 mg / kg.

4. The pharmaceutical composition according to claim 3, characterized in that The amounts of the components in the pharmaceutical composition are calculated based on the oral dosage for mice: berberine is 50-100 mg / kg, and the FXR agonist is 22.5-45 mg / kg; and / or, calculated based on the dosage for healthy adults: berberine is 5.55-11.1 mg / kg, and the FXR agonist is 2.5-5.0 mg / kg.

5. The pharmaceutical composition according to claim 1, characterized in that The mass ratio of the berberine to the FXR agonist is 1.5:1 to 3.0:

1.

6. The pharmaceutical composition according to claim 5, characterized in that The mass ratio of the berberine to the FXR agonist is 1.8:1 to 2.7:

1.

7. A pharmaceutical preparation, characterized in that The pharmaceutical preparation is prepared from the pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical preparation is a solid preparation or a liquid preparation.

8. The pharmaceutical preparation according to claim 7, characterized in that: The pharmaceutical preparations include powders, granules, tablets, capsules, pills, injections, oral solutions, sustained-release preparations or controlled-release preparations.

9. Use of berberine and an FXR agonist in combination for the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease, and / or non-alcoholic steatohepatitis, and / or type 2 diabetes, and / or obesity, wherein the chemical structure of the FXR agonist is represented by the following formula (I):

10. The use according to claim 9, characterized in that The berberine and the FXR agonist are provided in a mass ratio of 1:5 to 5:1.

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