A pharmaceutical composition, preparation and its preparation method and application

Abstract

The present application relates to a kind of pharmaceutical composition, preparation and its preparation method and application, belong to the technical field of pharmaceutical preparation, at least to a certain extent, the deficiency of the curative effect and / or safety of existing single drug in treating non-alcoholic fatty liver disease combined with type 2 diabetes is solved.A kind of pharmaceutical composition, including berberine and FXR agonist, the mass ratio of berberine and FXR agonist is 1:5~5:1.The berberine and FXR agonist in the composition provided by the present application show high-efficiency synergistic effect under suitable compatibility, realize the reduction of efficiency, improve the safety of drug use and the compliance of patient, can also effectively inhibit weight gain, promote the biochemical index and weight of organism more tend to healthy state.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to a pharmaceutical composition, formulation, preparation method thereof, and application. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) affects approximately 25% of the global population, posing a significant threat to global health. Non-alcoholic steatohepatitis (NASH) develops from NAFL and, in addition to hepatic steatosis, is accompanied by inflammation or fibrosis. NASH can further progress to cirrhosis and hepatocellular carcinoma. Approximately 20% of NAFL patients will progress to NASH, and over 40% of NASH patients will progress to liver fibrosis. Furthermore, NASH is often complicated by various metabolic diseases; studies have reported that the incidence of NAFL in Chinese patients with type 2 diabetes mellitus (T2DM) is as high as 52.56%. Cooperative treatment of both glucose and lipids is becoming an important direction for research.

[0003] Farnesoid X receptor (FXR) agonists are among the most promising treatments for NASH. Obeticholic acid's new drug application for NASH treatment has progressed to Phase III clinical trials, but its marketing application for NASH has not been approved by the Food and Drug Administration (FDA) due to intolerable side effects such as itching and liver toxicity. According to literature reports, other FXR agonists also have these side effects to varying degrees; therefore, it is necessary to minimize the dosage of FXR agonists to ensure their safety.

[0004] Given the high incidence of NASH combined with T2DM and the correlation between the two diseases, combination therapy for both is a new trend in drug development. Previously, there was a patent (application publication number, CN 116782898A) for the combined treatment of NASH and T2DM using berberine and ursodeoxycholic acid, but this patent administered doses up to 2500 mg to subjects, reducing patient compliance in clinical practice.

[0005] Therefore, developing and researching safe and effective drugs for the treatment of non-alcoholic fatty liver disease complicated with type 2 diabetes is a major topic in the field of medical research. Summary of the Invention

[0006] In view of the above analysis, the present invention aims to provide a pharmaceutical composition, formulation, preparation method and application thereof, with the purpose of at least partially addressing the shortcomings of existing single drugs in the efficacy and / or safety of treating non-alcoholic fatty liver disease complicated with type 2 diabetes.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention provides a pharmaceutical composition comprising berberine and an FXR agonist, the chemical structure of which is represented by formula (I):

[0009]

[0010] The mass ratio of berberine to FXR agonist is 1:5 to 5:1.

[0011] Furthermore, the amounts of each component in the pharmaceutical composition, calculated based on the oral dosage for mice, are as follows: berberine is 10-300 mg / kg, and the FXR agonist is 7.5-105 mg / kg.

[0012] Furthermore, the amounts of each component in the pharmaceutical composition, calculated based on a healthy adult dose, are: berberine 1.11-33.3 mg / kg, and FXR agonist 0.83-11.62 mg / kg.

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

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

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

[0016] The present invention also provides a pharmaceutical preparation made from the pharmaceutical composition described above, wherein the pharmaceutical preparation is a solid preparation or a liquid preparation.

[0017] Furthermore, the pharmaceutical preparations include powders, granules, tablets, capsules, pills, injections, oral liquids, sustained-release preparations, or controlled-release preparations.

[0018] This invention also provides the use of berberine and an FXR agonist in combination in the preparation of a medicament for the prevention and treatment of 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] Furthermore, in the application, 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 present invention can achieve at least one of the following beneficial effects:

[0022] (1) Berberine and FXR agonist in the composition of the present invention exhibit a highly efficient synergistic effect under appropriate compatibility (e.g., a mass ratio of 1:5 to 5:1). Even when the amount of each component in the composition is reduced compared to the amount of the drug alone, it can still achieve a better effect of reducing both glucose and lipids than the drug alone. In addition, a noteworthy feature of the present invention is that the combination of berberine and FXR agonist has a unique synergistic effect on inhibiting weight gain, which is especially suitable for obese individuals with NAFLD / NASH and type 2 diabetes, and has significant application prospects and economic value.

[0023] (2) In some preferred embodiments, the berberine and FXR agonist in the composition of the present invention, while ensuring appropriate drug compatibility, further refine the suitable and optimal dosage ranges of each component. This optimization can more effectively leverage the synergistic effect of the two components, promoting a healthier state in the organism's biochemical indicators and weight. Even with a significant reduction in the dosage of each component, such as by 25%, it can at least achieve an effect roughly equivalent to that of monotherapy, or even surpass the effect of monotherapy in reducing both glucose and lipids (e.g., the results of the examples). In particular, the dosage optimization strategy of the present invention significantly reduces adverse side effects such as itching caused by higher doses of FXR agonists, thereby improving the safety of medication. This innovative combination drug composition and dosage optimization method has the potential to fill a gap and has significant value in the field of marketed drug formulations for NAFLD / NASH combined with type 2 diabetes in my country.

[0024] (3) In some preferred embodiments, the mass ratio of berberine and FXR agonist has been further optimized so that each component can exert the maximum beneficial effect in the body and achieve a synergistic effect of 1+1>2, thereby improving blood sugar and lipid reduction, making biochemical indicators and weight more in line with a healthy state, adopting a more suitable drug compatibility ratio, allowing the use of lower drug doses to maximize the positive effect on NAFLD / NASH combined with type 2 diabetes, not only reducing drug costs, but also reducing the toxic side effects of drugs, and further achieving the optimal balance between efficacy and safety.

[0025] (4) The present invention also provides a method for preparing a pharmaceutical preparation containing the pharmaceutical composition of the present invention. The preparation process is simple and the therapeutic effect is rapid. The oral liquid containing the composition prepared can give full play to the synergistic effect between the two drugs, effectively improve the bioavailability of the drugs, and has the advantages of convenient administration, fast absorption and easy dosage adjustment.

[0026] (5) The pharmaceutical composition provided by this invention, through the synergistic effect of berberine and FXR agonists, exhibits better hypoglycemic and lipid-lowering effects compared to monotherapy with reduced dosage. This combination therapy demonstrates significant efficacy against non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and type 2 diabetes mellitus (T2DM). Specifically, after combination therapy, the liver function, blood glucose control, and biochemical indicators of the organism are significantly improved, and the results are closer to those of the healthy control group compared to monotherapy (at higher doses). The pharmaceutical composition and its formulation of this invention exhibit excellent efficacy with reduced dosage, improving medication safety and patient compliance, and have potential application value in the preparation of drugs for the prevention and treatment of one or more of NAFLD, NASH, T2DM, and obesity.

[0027] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0028] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

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

[0030] Figure 2 The graph shows the NAS score results of mouse livers under different intervention conditions in Example 1;

[0031] Figure 3 This is a graph showing the scoring results of mouse liver ballooning degeneration under different intervention conditions in Example 1;

[0032] Figure 4 This is a graph showing the results of liver inflammation scores in mice under different intervention conditions in Example 1;

[0033] Figure 5 The graph shows the results of liver steatosis scoring in mice under different intervention conditions in Example 1;

[0034] Figure 6 The graph shows the results of the IPGTT (intraperitoneal glucose tolerance test) in mice under different intervention conditions in Example 1;

[0035] Figure 7 The graph shows the results of the ITT (insulin tolerance test) in mice under different intervention conditions in Example 1;

[0036] Figure 8 Figure 1 shows the body weight of mice under different intervention conditions in Example 1;

[0037] Figure 9 The images show the liver weights of mice under different intervention conditions in Example 1.

[0038] Figure 10 This is a graph showing the liver coefficient (liver weight / body weight) of mice under different intervention conditions in Example 1;

[0039] Figure 11 This is a graph showing the changes in serum ALT in mice under different intervention conditions in Example 1;

[0040] Figure 12 This is a graph showing the changes in serum AST in mice under different intervention conditions in Example 1;

[0041] Figure 13 This is a graph showing the changes in ALT levels in the liver of mice under different intervention conditions in Example 1;

[0042] Figure 14 This is a graph showing the changes in AST in mouse livers under different intervention conditions in Example 1;

[0043] Figure 15 This is a graph showing the changes in serum TBA in mice under different intervention conditions in Example 1;

[0044] Figure 16 This is a graph showing the changes in serum ALP levels in mice under different intervention conditions in Example 1;

[0045] Figure 17This is a graph showing the changes in serum triglycerides in mice under different intervention conditions in Example 1;

[0046] Figure 18 This is a graph showing the changes in serum cholesterol in mice under different intervention conditions in Example 1;

[0047] Figure 19 This is a graph showing the changes in serum APOA in mice under different intervention conditions in Example 1;

[0048] Figure 20 This is a graph showing the changes in serum APOB in mice under different intervention conditions in Example 1. Detailed Implementation

[0049] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0050] The inventors previously developed a potential FXR agonist with therapeutic effects for NASH, which has been validated in vitro and in animal experiments as exhibiting good FXR selectivity and NASH therapeutic efficacy. However, during clinical trials, it also showed side effects such as itching at high doses. Furthermore, this FXR agonist showed poor glycemic control and limited efficacy in treating patients with both NASH and T2DM. Therefore, it is necessary to explore combination drug regimens to reduce the dosage of the FXR agonist while simultaneously achieving dual treatment for both NASH and T2DM.

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

[0052]

[0053] The mass ratio of berberine to FXR agonist is 1:5 to 5:1.

[0054] Berberine is a quaternary ammonium alkaloid isolated from the traditional Chinese medicine Coptis chinensis. It has a molecular weight of 336.36 and a molecular formula of C20H18NO4+. It is a yellow needle-like crystal with CAS number 2086-83-1. It has been approved for marketing and its indication is intestinal infection.

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

[0056] The purity of berberine and FXR agonist in this invention is >98%.

[0057] The inventors of this invention unexpectedly discovered that, compared to the individual components, berberine and the FXR agonist in the composition of this invention exhibit a highly efficient synergistic effect under suitable compatibility (e.g., a mass ratio of 1:5 to 5:1). Even when the dosage of each component in the pharmaceutical composition is reduced compared to the dosage of the individual drugs, it can still achieve better blood sugar and lipid reduction effects than the individual drugs. In addition, a noteworthy feature of this invention is the unique synergistic effect of berberine and the FXR agonist in inhibiting weight gain when used together, which is particularly suitable for obese individuals with NAFLD / NASH and type 2 diabetes, and has significant application prospects and economic value.

[0058] It should be noted that the potential mechanisms of the synergistic effect exhibited by the composition provided by the present invention include the following aspects: (1) Synergistic effect of multiple organs: Berberine promotes the activation of GLP-1 secretion by TGR5 by improving the composition of intestinal flora and bile acids, and further promotes insulin secretion; FXR agonists promote the increase of IRS-2 tyrosine phosphorylation level in the liver and peripheral tissues, and promote 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 the liver: Both berberine and FXR agonists have liver targeting (the liver / blood ratio is >10 in animal experiments). Berberine can inhibit liver inflammation and oxidative stress through the ROS pathway, and at the same time regulate liver lipid metabolism by reducing lipid synthesis and inducing fatty acid β oxidation through the AMPK and PPARγ pathways; FXR agonists can regulate lipid metabolism and inhibit inflammation through the FXR pathway.

[0059] In some preferred embodiments, the amounts of each component in the pharmaceutical composition are calculated based on oral mouse dosage: berberine 10-300 mg / kg, FXR agonist 7.5-105 mg / kg; and / or, calculated based on healthy adult dosage: berberine 1.11-33.3 mg / kg, FXR agonist 0.83-11.62 mg / kg.

[0060] More preferably, the amounts of each component in the pharmaceutical composition are calculated based on the oral dosage for mice: berberine 50-100 mg / kg, FXR agonist 22.5-45 mg / kg; and / or, calculated based on the dosage for healthy adults: berberine 5.55-11.1 mg / kg, FXR agonist 2.5-5.0 mg / kg.

[0061] It is understood that, while ensuring appropriate drug compatibility, the berberine and FXR agonist in the composition of this invention have further refined the suitable and optimal dosage ranges of each component. This optimization can more effectively leverage the synergistic effect of the two components, promoting a healthier state in the organism's biochemical indicators and weight. Even with a significant reduction in the dosage of each component, such as by 25%, it can still achieve at least an effect roughly equivalent to that of monotherapy, or even surpass the effect of monotherapy in reducing both glucose and lipids (e.g., the results of the examples). In particular, the dosage optimization strategy of this invention significantly reduces adverse side effects such as itching caused by higher doses of FXR agonists, thereby improving the safety of medication. This innovative combination drug composition and dosage optimization method has the potential to fill a gap and has significant value in the field of marketed drug formulations for NAFLD / NASH combined with type 2 diabetes in my country.

[0062] For example, the mass ratio of 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, or 5.0:1.

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

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

[0065] More preferably, the mass ratio of 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 agonists, the beneficial effects of each component can be maximized in vivo, achieving a synergistic effect of 1+1>2. This can improve blood sugar and lipid reduction, bring biochemical indicators and weight closer to a healthy state, and allow for the use of lower drug doses to maximize the positive effects on NAFLD / NASH combined with type 2 diabetes. This not only reduces drug costs but also mitigates drug toxicity and side effects, further achieving an optimal balance between efficacy and safety.

[0067] In a second aspect, the present invention provides a pharmaceutical preparation made from the pharmaceutical composition as described in the first aspect, wherein the pharmaceutical preparation is a solid or liquid preparation.

[0068] Optionally, the pharmaceutical preparation includes powders, granules, tablets, capsules, pills, oral liquids, sustained-release preparations, or controlled-release preparations.

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

[0070] Thirdly, the present invention also provides a method for preparing the oral liquid formulation, comprising the following steps:

[0071] (1) Weigh the required berberine and FXR agonist according to the predetermined dosage, and add them to an aqueous solution containing a solubilizer to obtain a mixed solution. The concentration range of berberine for oral administration to mice is 8.0-12.0 mg / mL per milliliter of aqueous solution, and the concentration range of FXR agonist is 3.0-6.0 mg / mL per milliliter of aqueous solution.

[0072] For example, in oral doses for mice: the concentration of berberine is 8.5, 9.0, 9.5, 10.0, 10.5, 11.0 or 11.5 mg of berberine per milliliter of aqueous solution, and the concentration of FXR agonist is 3.5, 4.0, 4.5, 5.0 or 5.5 mg of FXR agonist per milliliter of aqueous solution;

[0073] Alternatively, for oral dosage in healthy adults: the concentration range of berberine is 0.88–1.32 mg / mL in aqueous solution, and the concentration range of FXR agonists is 0.33–0.66 mg / mL in aqueous solution;

[0074] For example, the oral dosage for healthy adults is: berberine concentration of 0.93, 0.99, 1.04, 1.10, 1.15, 1.21 or 1.26 mg per milliliter of aqueous solution, and FXR agonist concentration of 0.38, 0.44, 0.50, 0.55 or 0.60 mg per milliliter of aqueous solution;

[0075] (2) The resulting mixed solution is subjected to ultrasonic treatment to promote the complete dissolution of the berberine and FXR agonist.

[0076] Optionally, the co-solvent includes at least one of sodium carboxymethyl cellulose, Tween-80, cyclodextrin, polyethylene glycol, N-methylpyrrolidone, and propylene glycol.

[0077] Preferably, the co-solvent is sodium carboxymethyl cellulose.

[0078] For example, the "solution containing a cosolvent" refers to a 0.5% aqueous solution of sodium carboxymethyl cellulose. The 0.5% sodium carboxymethyl cellulose aqueous solution is a solution with sodium carboxymethyl cellulose (CMC-Na) as the solute and water as the solvent. "0.5%" indicates a mass percentage concentration, meaning that 0.5 grams of sodium carboxymethyl cellulose are contained in every 100 grams of the aqueous solution.

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

[0080] Preferably, the ultrasonic treatment is performed using a shaking ultrasonic method.

[0081] Fourthly, the present invention also provides the use of berberine and an FXR agonist in combination in the preparation of a medicament for the prevention and treatment of 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, the berberine and the FXR agonist are provided in a mass ratio of 1:5 to 5:1 in the application. Exemplarily, the berberine and the FXR agonist are provided in mass ratios 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, and 5:1.

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

[0085] More preferably, in the application, the berberine and the FXR agonist are provided in a mass ratio of 1.8:1 to 2.7:1.

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

[0087] In some preferred embodiments, the dosages of berberine and the FXR agonist in the application are as follows, calculated based on oral mouse dosage: berberine 10-300 mg / kg, FXR agonist 7.5-105 mg / kg; and / or, calculated based on healthy adult dosage: berberine 1.11-33.3 mg / kg, FXR agonist 0.83-11.62 mg / kg.

[0088] For example, based on oral administration doses in mice: berberine at doses of 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, and 300 mg / kg, or berberine at 50-300 mg / kg; and FXR agonists at doses of 7.5 mg / kg, 10 mg / kg, and 12.5 mg / kg. g / kg, 15.0mg / kg, 17.5mg / kg, 20.0mg / kg, 22.5mg / kg, 25mg / kg, 27.5mg / kg, 30.0mg / kg, 32.5mg / kg, 35.0mg / kg, 37.5mg / kg , 40.0mg / kg, 42.5mg / kg, 45.0mg / kg, 50.0mg / kg, 52.5mg / kg, 75mg / kg, 90.0mg / kg, 105.0mg / kg, such as FXR agonist is 7.5-52.5mg / kg.

[0089] For example, based on the dosage for healthy adults: berberine is 1.11 mg / kg, 5.55 mg / kg, 11.1 mg / kg, 16.65 mg / kg, 22.2 mg / kg, 27.75 mg / kg, and 33.3 mg / kg, such as berberine ranging from 5.55 to 33.3 mg / kg; FXR agonists are 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, and 11.62 mg / kg, such as FXR agonists ranging from 0.83 to 5.83 mg / kg.

[0090] More preferably, in the application, the dosages of berberine and the FXR agonist are as follows, calculated based on oral administration doses in mice: berberine 50-100 mg / kg, FXR agonist 22.5-45 mg / kg; and / or, calculated based on healthy adult doses: berberine 5.55-11.1 mg / kg, FXR agonist 2.5-5.0 mg / kg.

[0091] Specifically, the obesity includes simple obesity and secondary obesity. The pharmaceutical composition or formulation of the present invention is suitable for overweight individuals with a body mass index (BMI) between 25 and 29.9, and is particularly suitable for individuals with grade I to III obesity with a BMI of 30 or higher.

[0092] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0093] The test materials in the following examples were sourced from the following sources:

[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., a subsidiary of CSPC Pharmaceutical Group.

[0096] Example 1:

[0097] This embodiment describes the effect of a pharmaceutical composition of berberine and an FXR agonist on a mouse model of NASH combined with T2DM induced by a high-fat diet; in this embodiment, the chemical structure of the FXR agonist is represented by the following formula (I):

[0098]

[0099] 1. Experimental Design: The modeling process in this experiment included two steps: feeding mice with a high-fat diet and intervention in mice.

[0100] (a) A mouse model of NASH combined with T2DM was established by feeding the mouse with a high-fat diet;

[0101] Specifically, mice with an initial body weight ranged from 17.5 to 18 g were fed a 60% high-fat diet (catalog number D12492i) for 12 weeks before model evaluation. After 12 weeks, their body weight ranged from 47.2 to 60.4 g. NASH evaluation relied on the gold standard for diagnosis: liver pathological sections. Mouse livers were fixed with paraformaldehyde, sectioned in paraffin, and finally stained with hematoxylin-eosin (HE). Two pathologists independently scored the HE staining results, using the NAS scoring criteria (Tables 1 and 2). A NAS score of 5 or higher indicated NASH, 3-4 might indicate NASH, and 1-2 indicated no NASH.

[0102] Table 1. Pathological scores for non-alcoholic steatohepatitis (NASH)

[0103]

[0104] Table 2. Diagnostic criteria for nonalcoholic fatty liver disease (NASH)

[0105]

[0106] The model evaluation results showed that the NAS scores of mice after modeling were generally between 4 and 6, exhibiting typical symptoms of NASH.

[0107] The evaluation of type 2 diabetes mellitus (T2DM) was performed using the intraperitoneal glucose tolerance test (IPGTT), which is similar to the oral glucose tolerance test (OGTT) (the gold standard for clinical T2DM diagnosis). T2DM is diagnosed by measuring changes in blood glucose levels in mice at different time points after glucose administration. Figure 1 The results showed that, compared with the healthy group, the mice in the model group who were fed a high-fat diet for 12 weeks exhibited obvious abnormalities in glucose regulation.

[0108] It needs to be explained that, Figures 1-20 In the mean squares, ns: no statistical difference; *: p<0.05; **: p<0.01; ****: p<0.0001.

[0109] (b) Mouse intervention with NASH combined with T2DM

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

[0111] (1) Healthy control group: Mice that were not fed a high-fat diet were given an equal volume of 0.5% sodium carboxymethyl cellulose aqueous solution orally;

[0112] (2) Solvent control group: Mice fed a high-fat diet were given an equal volume of 0.5% sodium carboxymethyl cellulose aqueous solution orally;

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

[0114] (4) Single dose berberine group: The dose of berberine was 100 mg / kg. The predetermined dose of berberine was weighed according to the weight of the mice and added to 0.5% sodium carboxymethyl cellulose aqueous solution to prepare an oral solution for oral administration to the mice.

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

[0116] For groups (3)-(5), when preparing oral solutions containing the drugs, 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] In group (5), when FXR agonists and berberine were used in combination, the mass ratio of berberine to FXR agonists was 2.22:1.

[0118] For the five groups mentioned above, blank solvents or different drugs were administered by gavage once daily for 28 consecutive days. Pharmacodynamic evaluation was then performed: 1. Liver pathological sections were stained with hematoxylin and eosin (HE) and NAS scores were calculated; 2. Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed to evaluate glucose tolerance and insulin resistance; 3. Physiological, biochemical, and inflammatory indicators were detected to comprehensively evaluate the efficacy and synergistic effects of single-drug and two-drug combinations in lowering blood sugar and lipids.

[0119] 2. Efficacy evaluation: The efficacy of the drug composition and its agents provided by this invention will be comprehensively evaluated from four aspects: its effect on improving NASH, its effect on improving T2DM, its effect on weight loss, and its effect on biochemical indicators.

[0120] (1) Mouse NAS score results;

[0121] Purpose of the test: To assess the primary efficacy endpoint of NASH treatment and to evaluate the therapeutic effect of drug intervention on NASH.

[0122] Specific steps: Liver tissue fixed in 4% paraformaldehyde solution for 24 hours was dehydrated in a gradient of 70%, 80%, 90%, 95% ethanol and anhydrous ethanol, then embedded in paraffin. After paraffin embedding, it was cut into 4μm thin sections, spread on glass slides, and dried in an oven before subsequent staining. First, it was stained with xylene solvent, then dewaxed and washed with 100%, 95%, 80%, and ultrapure water, then stained with hematoxylin staining solution, washed with distilled water, immersed in 1% hydrochloric acid ethanol solution, washed with distilled water, blued with 0.5% ammonia solution, washed with distilled water, counterstained with 1% eosin staining solution, washed with distilled water, then washed with 80%, 95%, and 100% ethanol in sequence, and finally washed with xylene solution. After staining, it was immediately mounted with neutral resin, air-dried for several days, and then observed, scanned, and processed under a 40× microscope using a digital pathology slide scanner. After staining, the cell nuclei appear blue, while the cytoplasm, muscle fibers, collagen fibers, and erythrocytes appear in varying shades of red.

[0123] Results analysis: After 4 weeks of drug intervention, from Figure 2 As can be seen, the NAS score of the solvent control group was significantly higher than that of the healthy control group. The single-dose FXR group, the single-dose berberine group, and the combination of FXR and berberine all significantly reduced the NAS score. Notably, the combination therapy group showed a more significant reduction in NAS score compared to single-drug use, exhibiting a more statistically significant difference. The NAS score consists of three phenotypes: fatty degeneration, ballooning degeneration, and inflammation. Further analysis of the effects of different drugs and combinations on the phenotypes will be conducted; please refer to [link to relevant documentation]. Figures 3-5 Except for ballooning degeneration (which has a low incidence), the combination therapy group significantly reduced the levels of steatosis and inflammation in the liver compared to FXR and berberine monotherapy.

[0124] This demonstrates that berberine and the FXR agonist in the composition of the present invention exhibit a highly efficient synergistic effect under appropriate compatibility. Even when the dosage of each component in the composition is reduced by 25% compared to the dosage of the single drug, it can still achieve better therapeutic effects for NASH than the single drug.

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

[0126] Purpose of the test: To assess the primary efficacy endpoint of T2DM treatment, specifically its ability to improve glycemic control and insulin secretion after drug intervention.

[0127] (a) IPGTT evaluation, the specific steps of which include:

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

[0129] 2. Weigh each mouse;

[0130] 3. Remove the mouse and cut off about 1-2 mm from the end of the mouse's tail. Squeeze the tail vein to collect a drop of blood. Discard the first drop of blood. Use a blood glucose meter (Roche Gold Glucose Meter) and blood glucose test strips (Roche Gold Glucose Test Strips) to measure the fasting blood glucose of the second drop of blood. This is the blood glucose value at 0 min.

[0131] 4. Prepare a 200 mg / ml glucose solution using physiological saline and administer it intraperitoneally at a ratio of 0.01 ml / g (calculated based on the mouse's body weight, with each gram of mouse body weight receiving 0.01 ml of glucose solution). Start timing immediately after injection and use blood glucose test strips to detect blood glucose levels 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 placed in clean cages and fasted for 6-8 hours, with free access to water;

[0134] 2. Weigh each mouse;

[0135] 3. Remove the mouse and cut off about 1-2 mm from the end of the mouse's tail. Squeeze the tail vein to collect a drop of blood. Discard the first drop of blood. Use a blood glucose meter (Roche Gold Glucose Meter) and blood glucose test strips (Roche Gold Glucose Test Strips) to measure the fasting blood glucose of the second drop of blood. This is the blood glucose value at 0 min.

[0136] 4. Prepare a 0.05 U / ml insulin solution using physiological saline and administer it intraperitoneally at a ratio of 0.01 ml / g (calculated based on the mouse's body weight, with each gram of mouse body weight receiving 0.01 ml of insulin solution). Timing begins immediately after injection, and blood glucose levels are measured using blood glucose test strips at different time points (15, 30, 60, 90, and 120 minutes).

[0137] Results analysis: From Figure 6 and Figure 7As can be seen, compared to the healthy control group, the blood glucose level in the solvent control group was significantly higher after administration of glucose or insulin, indicating a decrease in insulin sensitivity, accompanied by a decline in insulin secretion. Administration of FXR agonists alone did not improve the blood glucose curve of the IPGTT, indicating that it could not affect insulin secretion, but it could lower the blood glucose level after the ITT, suggesting that it could improve insulin resistance to some extent. This is because insulin resistance is mainly due to impaired insulin receptor sensitivity in the liver and muscles, and FXR agonists can improve insulin sensitivity by improving hepatic metabolic abnormalities. Berberine has a beneficial effect on both insulin secretion and insulin sensitivity, but it showed a secondary increase in blood glucose in the IPGTT, indicating that its ability to control blood glucose in disease states is still insufficient.

[0138] In comparison, the berberine and FXR agonist in the composition of the present invention exhibit a highly efficient synergistic effect when properly matched. Even when the dosage of each component in the composition is reduced by 25% compared to the dosage of the single drug, it can still achieve better therapeutic effects for T2DM than the single drug. Specifically, the combined use of FXR and berberine can ensure more stable blood glucose fluctuations and has a better ability to improve insulin secretion and insulin resistance compared to the single drug.

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

[0140] from Figures 8-10 It can be seen that after 4 weeks of drug intervention, the solvent control group showed significant increases in body weight, liver weight, and liver weight-to-body weight ratio compared to the healthy control group. Both FXR agonist and berberine alone reduced body weight and / or liver weight in NASH-T2DM mice to varying degrees, but the single-dose FXR agonist group did not show a significant difference, indicating that FXR agonist alone does not have a significant effect on mouse body weight. The combination of FXR and berberine showed a more significant trend of decreasing body weight and liver weight, and was closer to that of the healthy control group, suggesting that the combination therapy can better improve the increase in body weight and liver weight induced by a high-fat diet.

[0141] This demonstrates that berberine and the FXR agonist in the composition of the present invention exhibit a highly efficient synergistic effect under suitable compatibility. When the dosage of each component is reduced by 25% relative to the single-drug dosage, not only is a superior therapeutic effect achieved for non-alcoholic steatohepatitis (NASH) and type 2 diabetes mellitus (T2DM), but it also unexpectedly shows the additional benefit of controlling weight gain. The pharmaceutical composition and its formulation provided by the present invention show promising application prospects for overweight or obese individuals with NASH and type 2 diabetes.

[0142] (4) Evaluation of biochemical indicators in mice

[0143] Objective: Type 2 diabetes mellitus (T2DM) and NASH can cause changes in physiological and biochemical parameters. Along with the relief of T2DM and NASH symptoms, some physiological or biochemical parameters also improve to varying degrees. Therefore, monitoring these biochemical parameters is crucial for evaluating the effectiveness of drug interventions and identifying potential side effects.

[0144] Specific steps: Liver ALT, AST, and cholesterol were measured using ELISA kits (ALT kit brand: Solarbio, catalog number BC1555; AST kit brand: Solarbio, catalog number BC1565; cholesterol kit brand: AAT Bioquest, catalog number 40006). Liver tissue was added to PBS homogenate at a ratio of 1:9 and homogenized at 4°C. 3500g of the homogenate was then centrifuged at 4°C for 10 minutes, and the supernatant was collected for analysis according to the kit instructions. Serum parameters were measured using a blood biochemistry analyzer.

[0145] The levels of ALT and AST in the liver were measured separately, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acid (TBA), total cholesterol (TC), triglycerides (TG), apolipoprotein A (APOA), apolipoprotein B (APOB), and cholesterol in serum were detected. Figures 11-20 The result.

[0146] Results analysis:

[0147] a) ALT, AST, and ALP are enzymes secreted by the liver, mainly used for the diagnosis and differential diagnosis of diseases of the skeletal and hepatobiliary systems. Abnormally elevated levels indicate liver inflammation or necrosis, especially elevated AST levels, which indicate severe necrosis and the entry of intracellular mitochondria into the bloodstream. Figure 11 and Figure 12It can be seen that, compared with the healthy control group, the solvent control group showed significantly elevated ALT and AST levels in serum, indicating that mouse liver cells were severely damaged under high-fat diet intervention. The single-dose FXR group, the single-dose berberine group, and the FXR + berberine combination group all reduced ALT and AST levels to varying degrees, with the combination group approaching the levels of the healthy control group. Similarly, from... Figure 16 It can be seen that, in serum, compared with the healthy control group, the ALP level in the solvent control group was significantly increased, and there was a significant improvement after drug intervention. At the same time, the FXR + berberine combination group tended to be similar to the healthy control group.

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

[0149] c) Bile acids are produced by the liver and secreted by bile. They are distributed in the blood and tissues through enterohepatic circulation. Abnormally elevated levels can cause inflammation and a range of other diseases, thus promoting the NASH process. Figure 15 In serum, compared with the healthy control group, the solvent control group had significantly higher TBA levels, which significantly improved after drug intervention. Meanwhile, the FXR + berberine combination group had a more significant improvement effect.

[0150] d) TC and TG can reflect the distribution of cholesterol and triglycerides in serum, from Figure 17 and Figure 18 It can be seen that both were significantly elevated in the solvent control group. Berberine showed a triglyceride-lowering effect, while the FXR agonist showed a cholesterol-lowering effect. The combined use of the two drugs can achieve simultaneous improvement in cholesterol and triglycerides.

[0151] e) APOA and APOB are mainly used to bind and transport blood lipids to various tissues in the body for metabolism and utilization; their function is very important for maintaining blood lipid balance. Figure 19 and Figure 20 It can be seen that APOA and APOB were significantly elevated in the solvent control group. FXR agonists can appropriately reduce the level of APOA and significantly reduce the level of APOB, while berberine has no effect on improving APOA. The combination of the two drugs can produce certain beneficial effects and reduce the levels of APOA and APOB at the same time.

[0152] Therefore, FXR alone shows significant improvement in some indicators such as cholesterol and APOA, but has limited effect on triglycerides. Conversely, berberine alone shows significant improvement in triglycerides, but has little effect on cholesterol and APOA. The combined use of these two drugs, under appropriate compatibility, exhibits a highly efficient synergistic effect. When the dosage of each component is reduced by 25% compared to the single-drug dosage, they synergistically improve the abnormal biochemical indicators of NASH and T2DM, enhancing treatment efficacy. Simultaneously, the biochemical indicators tend towards those of the healthy control group, further indicating that the combined medication did not produce significant side effects. Thus, compared to the liver toxicity and other side effects of existing NASH drugs (such as obeticholic acid), the drug combination provided by this invention, based on the highly efficient synergy of FXR and berberine in an appropriate ratio, can effectively improve both NASH and T2DM simultaneously with reduced dosage. Furthermore, for overweight or obese individuals, it can achieve the additional beneficial effect of weight loss safely.

[0153] Example 2

[0154] The difference between this embodiment and Example 1 is that in the mouse intervention steps after model establishment, the FXR and berberine combined treatment group had an FXR agonist dose of 35.0 mg / kg and a berberine dose of 63 mg / kg, with a berberine to FXR agonist mass ratio of 1.8:1; the remaining steps and parameters were similar to those in Example 1.

[0155] In the NAS score results and 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 embodiment were better than those of single drug use, but slightly lower than the corresponding effects of the combination drug group in Example 1.

[0156] Example 3

[0157] The difference between this embodiment and Example 1 is that in the mouse intervention steps after model establishment, the FXR and berberine combined treatment group had an FXR agonist dose of 28.51 mg / kg and a berberine dose of 77.0 mg / kg, with a berberine to FXR agonist mass ratio of 2.7:1; the remaining steps and parameters were similar to those in Example 1.

[0158] In the NAS score results and 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 embodiment were better than those of single drug use, but slightly lower than the corresponding effects of the combination drug group in Example 1.

[0159] Example 4

[0160] The difference between this embodiment and Example 1 is that in the mouse intervention steps after model establishment, the FXR and berberine combined treatment group had the following dosage: FXR agonist dose of 40.0 mg / kg, berberine dose of 60.0 mg / kg, and mass ratio of berberine to FXR agonist = 1.5:1; the remaining steps and parameters are similar to those in Example 1.

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

[0162] Example 5

[0163] The difference between this embodiment and Example 1 is that in the mouse intervention steps after model establishment, the FXR and berberine combined treatment group had the following dosage: FXR agonist dose of 26.67 mg / kg, berberine dose of 80.0 mg / kg, and mass ratio of berberine to FXR agonist = 3.0:1; the remaining steps and parameters are similar to those in Example 1.

[0164] In the NAS score results and 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 embodiment were better than those of single-drug therapy, but slightly lower than the corresponding effects of the combination therapy group in Example 3.

[0165] Example 6

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

[0167] In the NAS score results and 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 embodiment were better than those of monotherapy, but slightly lower than the corresponding effects of the combination therapy group in Example 4.

[0168] Example 7

[0169] The difference between this embodiment and Example 1 is that in the mouse intervention steps after model establishment, the FXR and berberine combined treatment group had the following dosage: FXR agonist dose of 18 mg / kg, berberine dose of 90.0 mg / kg, and mass ratio of berberine to FXR agonist = 5.0:1; the remaining steps and parameters are similar to those in Example 1.

[0170] Based on the NAS score results and the results of the mouse IPGTT (intraperitoneal glucose tolerance test) and ITT (insulin tolerance test), the hypoglycemic, lipid-lowering and weight-loss effects of this embodiment are better than those of monotherapy, but slightly lower than the corresponding effects of the combination therapy group in Example 5.

[0171] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

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

1.

2. The pharmaceutical composition according to claim 1, wherein The mass ratio of berberine to the FXR agonist is 1.5:1 to 3.0:

1.

3. The pharmaceutical composition according to claim 2, characterized in that, The mass ratio of berberine to the FXR agonist is 1.8:1 to 2.7:

1.

4. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation is made from the pharmaceutical composition according to any one of claims 1-3, and the pharmaceutical preparation is in the form of a solid preparation or a liquid preparation.

5. The pharmaceutical preparation according to claim 4, characterized in that: The pharmaceutical preparations include powders, granules, tablets, capsules, pills, injections, oral liquids, sustained-release preparations, or controlled-release preparations.

6. The use of berberine and an FXR agonist in the preparation of a medicament for the prevention and treatment of 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): （I）； The berberine and the FXR agonist are provided in a mass ratio of 1:5 to 5:

1.

7. The application according to claim 6, characterized in that, The berberine and the FXR agonist are provided in a mass ratio of 1.5:1 to 3.0:

1.

8. The application according to claim 6, characterized in that, The berberine and the FXR agonist are provided in a mass ratio of 1.8:1 to 2.7:1.

Images