Pharmaceutical composition for preventing and / or treating non-alcoholic fatty liver disease and application thereof

Abstract

The invention discloses a pharmaceutical composition for preventing and / or treating a non-alcoholic fatty liver disease and application of the pharmaceutical composition. By means of rat model experiments and combined administration of curcumin and AKK bacteria, it is found that curcumin and AKK bacteria have the remarkable synergistic interaction advantage on prevention and treatment of the non-alcoholic fatty liver disease, and the effect of curcumin and AKK bacteria is remarkably superior to that of curcumin or AKK bacteria which are independently administered. The combined administration of curcumin and AKK bacteria can effectively improve liver fatty degeneration, mitochondrial injury and liver inflammation of rats with non-alcoholic fatty liver diseases, reduce blood fat and body weight, and provide a new path for developing drugs for preventing and treating non-alcoholic fatty liver diseases.

Description

Technical Field

[0001] This application belongs to the field of biological medicine technology, and specifically relates to a pharmaceutical composition for preventing and / or treating non-alcoholic fatty liver disease and its application. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a clinical syndrome that causes fatty degeneration of no less than 5% of hepatocytes except for liver damage caused by alcohol and drugs. Its main pathological manifestation is liver fatty degeneration, and it can progress to non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and even liver cancer as the disease develops. According to statistics, the number of people suffering from NAFLD accounts for about one-fourth of the global total population, and the prevalence rate in China has exceeded 29% and is on the rise, seriously endangering human health. Therefore, there is an urgent need to develop drugs that can effectively improve NAFLD to solve this increasingly serious social health problem. Due to the complex etiology and pathogenesis of NAFLD, there is currently no targeted drug. Clinically, statins, fibrates, antioxidants, and TNF-α antagonists are mainly used for anti-insulin resistance, antioxidant stress, and cytoprotective treatment, but the curative effect is poor and there are many adverse reactions. Therefore, the use of natural extracts and probiotics has gradually become a hot spot and new approach for the treatment of NAFLD.

[0003] Curcumin is a natural polyphenolic compound extracted from turmeric roots, and has various pharmacological effects such as anti-inflammatory, antioxidant, lipid-regulating, anti-aging, and anti-tumor effects. Curcumin has been reported as a natural inhibitor of 11β-HSD1, and by selectively inhibiting the activity of this enzyme, it can increase insulin sensitivity, thereby effectively improving insulin resistance and hepatic steatosis.

[0004] Akkermansia muciniphila (AKK) is a normal intestinal bacterium belonging to the phylum Verrucomicrobia, which feeds on mucin and participates in the intestinal metabolic process. This bacterium plays an important role in maintaining intestinal health and is negatively correlated with various diseases such as obesity, diabetes, cardiovascular diseases, and low-grade inflammation. AKK bacteria have been reported to be able to regulate the body's immunity, increase the synthesis of antimicrobial peptides, and maintain the homeostasis of the intestinal environment through short-chain fatty acids produced by the decomposition of mucin.

[0005] Currently, there has been no reported scheme for combining curcumin and AKK bacteria for preventing and / or treating non-alcoholic fatty liver disease. Summary of the Invention

[0006] In view of this, the primary object of the present application is to provide a new pharmaceutical composition, which is composed of a drug and probiotics, and the application of the pharmaceutical composition in the prevention and / or treatment of non-alcoholic fatty liver disease. The main components of the pharmaceutical composition are curcumin and Akkermansia muciniphila. By combining the two drugs, it can improve hepatic lipid deposition, mitochondrial damage and inflammatory response, reduce blood lipids and body weight, so as to achieve the effect of preventing and / or treating non-alcoholic fatty liver disease.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] One aspect of the present application provides a pharmaceutical composition, which is composed of curcumin and Akkermansia muciniphila.

[0009] Another aspect of the present application provides a drug for preventing and / or treating non-alcoholic fatty liver disease, which comprises the above-mentioned pharmaceutical composition.

[0010] Another aspect of the present application provides the application of curcumin combined with Akkermansia muciniphila, or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating non-alcoholic fatty liver disease.

[0011] The curcumin mentioned herein refers to curcumin and its derivatives. The curcumin refers to dimethoxycurcumin. The curcumin derivatives refer to substances obtained by modifying the molecular structure of curcumin in various known ways. For example, it can be Curc-OEG, biphenyldifluoroketone EF24, etc., but is not limited thereto. In some specific embodiments of the present application, the curcumin is dimethoxycurcumin, and its specific chemical structural formula is as follows:

[0012]

[0013] The Akkermansia muciniphila mentioned herein refers to Akkermansia muciniphila, which is an oval-shaped Gram-negative bacterium isolated from the human intestine. In some specific embodiments of the present application, the Akkermansia muciniphila is an anaerobic bacterium, and its specific structure is as Figure 1 shown therein.

[0014] In the present application, it has been determined through experiments that curcumin and Akkermansia muciniphila have a synergistic effect. It can improve the therapeutic effect on non-alcoholic fatty liver disease while reducing the drug dosage, thereby reducing side effects. Moreover, the combined agent can effectively delay or prevent the generation of drug resistance by affecting multiple targets simultaneously. In addition, the combined agent can act on multiple targets and signaling pathways simultaneously, thus more comprehensively intervening in the occurrence and development of the disease. And curcumin has anti-inflammatory and antioxidant effects, regulates lipid metabolism, improves insulin resistance, and regulates the intestinal flora; Akkermansia muciniphila has functions such as regulating the intestinal flora, enhancing the intestinal barrier, reducing inflammatory responses, and improving metabolism. The two acting synergistically also have the effect of preventing NAFLD.

[0015] As used herein, "combination" and "combined use" mean that two or more active substances can be administered to a subject simultaneously as separate single-agent preparations, or as separate single-agent preparations in any order sequentially or at intervals, or formulated into a single combined preparation and administered to the subject.

[0016] Among them, the combination of single-agent preparations is the combination of single-agent preparations containing curcumin and single-agent preparations containing Akkermansia muciniphila. The combined preparation is a combined preparation containing curcumin and Akkermansia muciniphila.

[0017] There is no particular limitation on the ratio of the two active substances curcumin and Akkermansia muciniphila in this article. Those skilled in the art can determine it through experiments known in the art. In some specific embodiments of the present application, in the combined drug composition composed of the two active substances, the ratio of curcumin to Akkermansia muciniphila is (1-2 mg):(1×10 8 ~2×10 8 live bacteria).

[0018] The drugs for preventing and / or treating non-alcoholic fatty liver disease described herein include the above drug composition, and also include any pharmaceutically acceptable excipients, excipients or carriers.

[0019] Specifically, the selection of excipients, fillers or carriers is based on the different dosage forms of the drug, and the specific dosage form of the drug is adjusted according to the different administration routes. The pharmaceutical composition or drug in this article can be prepared into corresponding dosage forms by administration routes such as oral administration and injection, preferably oral dosage forms. Specifically mentioned dosage forms can include: powders, tablets (including various coated tablets, sustained-release or controlled-release tablets), capsules (including soft capsules and hard capsules), granules, pills, dispersible powders, aqueous or oily suspensions, aqueous or oily solutions, emulsions, elixirs, syrups, etc., but are not limited thereto. According to the different dosage forms, their pharmaceutically acceptable carriers, excipients or fillers include but are not limited to fillers (or diluents), binders, disintegrants, lubricants, wetting agents, auxiliary lipids, glidants, sweeteners, flavoring agents, solvents, solubilizing aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, coloring agents, foaming agents, etc. Those skilled in the art can select the above carriers, excipients or fillers according to actual needs. No specific elaboration will be made here.

[0020] In some preferred examples of the present application, the dosage form of the drug is a suspension or a capsule. More preferably, the dosage form of the drug is a capsule.

[0021] Another aspect of the present application discloses a kit of medicines, which includes the above-mentioned pharmaceutical composition or the above-mentioned drug.

[0022] It can be understood that the kit of medicines also includes an instruction manual, which states that the dosage of the drug is an effective amount. Specifically, the effective amount refers to the dosage sufficient to achieve the expected therapeutic effect (such as inhibiting disease progression, relieving symptoms, preventing diseases, etc.) in the target subject (such as humans or animals), and varies according to the subject, dosage form of the drug, etc. There is no special limitation, and those skilled in the art can determine it based on known experimental methods. In some examples, taking rats weighing 210 - 240 g as an example, the dosage of the drug is: curcumin 10 mg / mL, 100 - 200 μl / d, Akkermansia muciniphila (1×10 9 CFU) 100 - 200 μl / d; preferably, the dosage of the drug is: curcumin 10 mg / mL, 150 μl / d, Akkermansia muciniphila 1×10 9 CFU, 150 μl / d.

[0023] The dosage of the drug in the present application is an effective amount and can be determined by known experimental methods, so there is no special limitation. The effective amount here refers to the amount containing enough to prevent or treat the symptoms or diseases of a medical condition. After being used for a specific patient or medical subject, the following changes can occur: the medical condition to be treated and the overall health of the patient / subject are improved.

[0024] Beneficial effects of the present application:

[0025] The present application provides a new use of the combination of curcumin and Akkermansia muciniphila in the prevention and treatment of non-alcoholic fatty liver disease. By co-administering curcumin and Akkermansia muciniphila, it is found that the two drugs have a synergistic effect in the prevention and treatment of NAFLD, and the combined effect of the two drugs is significantly better than that of administering curcumin or Akkermansia muciniphila alone.

[0026] In the present application, co-administering curcumin and Akkermansia muciniphila can effectively improve hepatic steatosis, mitochondrial damage, and liver inflammation in NAFLD rats, reduce blood lipids and body weight, providing a new option for the preparation of drugs for the prevention and treatment of NAFLD, and having important application value in new drug research and development. Description of the drawings

[0027] Figure 1 It is the microscopic structure of Akkermansia muciniphila.

[0028] Figure 2 It is the experimental result of the body weight of rats in the example, * indicates P < 0.05, ** indicates P < 0.01, and **** indicates P < 0.0001.

[0029] Figure 3 It is the experimental result of the liver index of rats in the example, indicates P < 0.001 compared with the control group, ** indicates P < 0.01 compared with the model group, and *** indicates P < 0.001.

[0030] Figure 4 It is the experimental result of the appearance of the liver of rats in the example.

[0031] Figure 5 It is the hematoxylin-eosin staining result of the liver of rats in the example.

[0032] Figure 6 It is the oil red O staining result of the liver of rats in the example.

[0033] Figure 7 It is the transmission electron microscopy photograph result of the liver of rats in the example.

[0034] Figure 8 It is the hematoxylin-eosin staining result of the rectum of rats in the example.

[0035] Figure 9 It is the detection result of the biochemical indexes in the serum of rats in the example. Among them, Figure 9 a is the experimental result of the serum triglyceride content, Figure 9 b is the experimental result of the serum total cholesterol content, Figure 9 c is the experimental result of the serum low-density lipoprotein content, Figure 9 d is the experimental result of the serum alanine aminotransferase content, Figure 9e is the experimental result of the content of serum glutamic-oxaloacetic transaminase, Figure 9 f is the experimental result of the content of serum high-density lipoprotein. # indicates P < 0.0001 compared with the control group; * indicates P < 0.05 compared with the model group, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. Specific implementation manners

[0036] The embodiments of the present application will be described in detail below. The following described embodiments are exemplary and are only used to explain the present application, and should not be construed as a limitation to the present application.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application in this specification are only for the purpose of describing specific implementation manners and are not intended to limit this application. Additionally, unless otherwise specified, the methods without specific conditions or steps recorded are all conventional methods, and the reagents and materials used can be obtained from commercial channels.

[0038] Example 1

[0039] In this example, a NAFLD rat model was constructed by feeding rats with a high-fat diet. The effects of curcumin and Akkermansia muciniphila (AKK) alone and in combination on the treatment of non-alcoholic fatty liver disease induced by a high-fat diet were studied. The combination of curcumin and AKK can effectively improve liver steatosis, liver inflammation, and mitochondrial damage in NAFLD rats, and reduce blood lipid and body weight in rats, showing an obvious synergistic promoting effect.

[0040] The specific experimental methods are as follows:

[0041] 1. Preparation of experimental materials:

[0042] Curcumin: Catalog number S31628, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0043] Freeze-dried powder of Akkermansia muciniphila (AKK): Catalog number A.muciniphila BAA-835, purchased from the American Type Culture Collection.

[0044] Curcumin suspension: Weigh 0.4 g of curcumin, add 39.6 ml of sterile 0.5% CMC, and prepare a 40 ml curcumin suspension with a final concentration of 10 mg / ml.

[0045] Akkermansia muciniphila suspension: The freeze-dried powder of A. muciniphila BAA-835 was inoculated into Brain Heart Infusion Broth (BHI) (Meilun, MMT040-1) supplemented with 0.05% type II mucin and cultured at 37 °C for 24 hours under anaerobic conditions consisting of 10% H2, 10% CO2, and 80% N2 in an AW500SG anaerobic workstation. After culturing, the samples were collected and centrifuged at 4500 revolutions per minute for 10 minutes, washed twice with pre-cooled sterile phosphate buffer saline, and then resuspended in sterile PBS containing 20% glycerol to reach a concentration of 1×10 9 colony-forming units (CFU) per milliliter.

[0046] Male Sprague-Dawley rats, 6 weeks old, weighing 210 - 240 g, were purchased from the Nanjing Branch of Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0047] Normal diet, product number: SWS9102, energy contribution ratio: 20.6% protein, 12% fat, 67.4% carbohydrates, was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.

[0048] High-fat diet, product number: XTHF45-1, energy contribution ratio: 20% protein, 45% fat, 35% carbohydrates, was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.

[0049] 2. Experimental grouping:

[0050] After 1 week of adaptive feeding with normal diet, 6-week-old male Sprague-Dawley rats were weighed and randomly divided into 5 experimental groups, with 5 rats in each group. The specific grouping is as follows:

[0051] (1) Control group: Fed with normal diet for 8 weeks; from the 3rd week, gavaged with glycerol (300 μl) at the same time every day for 5 weeks, and the body weight of the rats was recorded weekly.

[0052] (2) Model group: Fed with high-fat diet for 8 weeks; from the 3rd week, gavaged with glycerol (300 μl) at the same time every day for 5 weeks, and the body weight of the rats was recorded weekly.

[0053] (3) Curcumin group: Fed with high-fat diet for 8 weeks; from the 3rd week, gavaged with curcumin suspension (10 mg / ml, 300 μl) at the same time every day for 5 weeks, and the body weight of the rats was recorded weekly.

[0054] (4) Akkermansia muciniphila group: Fed with high-fat diet for 8 weeks; from the 3rd week, gavaged with Akkermansia muciniphila suspension (1×10 9 CFU, 300 μl) at the same time every day for 5 weeks, and the body weight of the rats was recorded weekly.

[0055] (5) Combined treatment group (curcumin + Akkermansia muciniphila): Fed with high-fat diet for 8 weeks; From the 3rd week, gavaged with a suspension of combined administration of curcumin (10 mg / ml, 150 μl) and a suspension of Akkermansia muciniphila (1×10 9 CFU, 150 μl) at the same time every day for 5 weeks, and the body weight of the rats was recorded weekly.

[0056] It should be noted that for the rats in the above 5 experimental groups, in the week before gavage, an antibiotic mixture (ampicillin 1 g / L, neomycin 1 g / L, metronidazole 1 g / L, and vancomycin 0.5 g / L) was used to clear the intestinal microbiota for one week.

[0057] 3. After 8 weeks of feeding in each experimental group, the rats were anesthetized and sacrificed, and serum and liver samples were collected. The specific operations are as follows:

[0058] All rats were fasted for 12 hours, anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg / kg), blood was taken from the inferior vena cava, left to stand at room temperature for 2 hours, then centrifuged at 4000 rpm for 10 minutes at 4°C to collect serum samples, and stored at -80°C. The liver was quickly taken out and weighed. At the same part of the right lobe of the liver, two small pieces of liver tissue were taken, rinsed with 4°C normal saline, blotted dry with filter paper, and then fixed with 4% paraformaldehyde solution and 4°C pre-cooled 2.5% glutaraldehyde solution respectively. The remaining part was stored at -80°C. The colon tissue samples of the experimental rats in each group were precisely dissected, fixed with 4% paraformaldehyde, embedded in paraffin, and made into thin sections.

[0059] 4. The pathological morphological characteristics, lipid deposition, and mitochondrial damage of the livers of rats in each group were detected by hematoxylin-eosin staining, oil red O staining, and transmission electron microscopy photography, and the pathological conditions of the colon tissues of rats in each group were detected by hematoxylin-eosin staining. The contents of serum alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglyceride, low-density lipoprotein, and high-density lipoprotein were detected by a biochemical analyzer.

[0060] Results and Analysis

[0061] 1. Compared with the control group rats, the body weight and liver index of the NAFLD model group rats were significantly increased. The combined administration of curcumin and Akkermansia muciniphila could significantly reduce the increase in body weight and liver index of rats caused by high-fat diet, and the effect was better than that of the group administered curcumin alone or the group administered Akkermansia muciniphila alone ( Figure 2 and Figure 3 ).

[0062] 2. The appearance of liver tissues in each group showed that the liver tissues of rats in the control group were normal in size, ruddy and smooth in color, and sharp in edge; the livers of rats in the model group were earthy yellow in color, with deformed and adhered liver lobes, blunt edges, and greasy cut surfaces; the livers of rats in the curcumin + AKK group were dark red, smooth in surface, and less greasy on the cut surface, and the effect was better than that of the curcumin group or the AKK group alone ( Figure 4 ).

[0063] 3. The results of hematoxylin-eosin staining, oil red staining and transmission electron microscopy of liver tissue showed that the hepatocytes of rats in the model group showed obvious fatty degeneration, inflammatory response and mitochondrial damage, while the fatty degeneration, inflammatory response and mitochondrial damage of hepatocytes of rats in the curcumin + AKK group were significantly improved, and the effect was better than that of the curcumin group or the AKK group alone ( Figure 5 , Figure 6 and Figure 7 ).

[0064] 4. The results of hematoxylin-eosin staining of colon tissue showed that the colon wall mucosa of the rats in the control group was intact, the microvilli were neatly arranged, the glands in the lamina propria were not atrophied, and the submucosal layer did not show obvious changes; the mucosal layer of the colon of the rats in the model group was atrophied and thinned, with defects and shedding, the glands in the lamina propria were atrophied, the gaps were widened, the submucosal layer was sparse, and a large number of lymphocytes infiltrated; the mucosal layer of the curcumin + AKK group was intact, the mucosal microvilli were neatly arranged, and there was a small amount of inflammatory cell infiltration, which was significantly improved compared with the model group, and the effect was better than the group treated with curcumin alone or the group treated with AKK bacteria alone ( Figure 8 ).

[0065] High-fat diet led to a significant increase in serum total cholesterol, triglycerides, and low-density lipoprotein levels, while the combined administration of curcumin and AKK bacteria significantly reversed the accumulation of these lipids in serum, and the effect was better than that of the curcumin group or the AKK bacteria group alone ( Figure 9 a~ Figure 9 c).

[0066] The increase in serum liver damage indicators (alanine aminotransferase and aspartate aminotransferase) and the decrease in high-density lipoprotein levels caused by a high-fat diet were also significantly reversed by the combined administration of curcumin and AKK bacteria, and the effect was also better than that of the curcumin group or the AKK bacteria group alone ( Figure 9 d~ Figure 9 f).

[0067] Example 2

[0068] The relevant experiments were carried out according to the experimental method in Example 1, except that in the combined medication group (curcumin + AKK bacteria), from the third week onwards, curcumin suspension (10 mg / ml, 100 μl) and AKK bacteria suspension (1×109 CFU, 200 μl). Other steps and conditions were the same as those in Example 1.

[0069] Example 3

[0070] Refer to the experimental method in Example 1 for relevant experiments. The only difference was that in the combined drug group (curcumin + Akkermansia muciniphila), starting from the 3rd week, at the same time every day, intragastric administration of a combined suspension of curcumin (10 mg / ml, 200 μl) and a suspension of Akkermansia muciniphila (1×10 9 CFU, 100 μl) was performed. Other steps and conditions were the same as those in Example 1.

[0071] The results showed that the combined drug group effectively improved hepatic steatosis, liver inflammation, and mitochondrial damage in NAFLD rats, reduced blood lipid and body weight, and the effect was significantly better than that of the single-drug group.

[0072] The above research results indicate that a high-fat diet causes hepatic steatosis, induces inflammation and mitochondrial damage, increases blood lipid and body weight, while combined administration of curcumin and Akkermansia muciniphila can effectively improve hepatic steatosis, liver inflammation, and mitochondrial damage in NAFLD rats, reduce blood lipid and body weight, and the combined effect of the two drugs shows a synergistic effect, which is better than administering curcumin alone or Akkermansia muciniphila alone. The combination of curcumin and Akkermansia muciniphila has a synergistic effect in the prevention and treatment of non-alcoholic fatty liver disease, providing a new path for the development of new drugs for the prevention and treatment of non-alcoholic fatty liver disease and a new strategy for the clinical treatment of non-alcoholic fatty liver disease.

[0073] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0074] The above-described embodiments only represent several implementation manners of the present application. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition consists of curcumin and Akkermansia muciniphila.

2. The pharmaceutical composition according to claim 1, wherein In the said pharmaceutical composition, the ratio of the curcumin to the Akk bacteria is (1 - 2 mg) : (1×10 8 - 2×10 8 live bacteria).

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The pharmaceutical composition is a single compound preparation or a combination of two separate single-agent preparations; wherein, the compound preparation is a compound preparation containing curcumin and Akkermansia muciniphila; the combination of single-agent preparations is a combination of a single-agent preparation containing curcumin and a single-agent preparation containing Akkermansia muciniphila.

4. A drug for preventing and / or treating non-alcoholic fatty liver disease, characterized in that, The drug includes the pharmaceutical composition according to any one of claims 1-3.

5. The drug for preventing and / or treating non-alcoholic fatty liver disease according to claim 4, characterized in that, The drug further includes at least one pharmaceutically acceptable excipient and / or carrier.

6. The medicament for preventing and / or treating non-alcoholic fatty liver disease according to claim 4 or 5, characterized in that, The dosage form of the drug is a suspension or a capsule.

7. A kit of medicine boxes, characterized in that, The kit includes the pharmaceutical composition according to any one of claims 1-3, or the drug according to any one of claims 4-6.

8. The kit of medicine boxes according to claim 7, characterized in that, The kit further includes an instruction manual, and the instruction manual states that the dosage of the drug is: Curcumin: 10 mg / mL, 100 - 200 μl / d; Akkermansia muciniphila: 1×10 9 CFU, 100 - 200 μl / d.

9. The application of curcumin combined with Akkermansia muciniphila, or the pharmaceutical composition according to any one of claims 1-3, in the preparation of a drug for preventing and / or treating non-alcoholic fatty liver disease.

10. The application according to claim 9, characterized in that, The drug has at least one of the following effects: (1) Preventing and / or treating non-alcoholic fatty liver disease; (2) Reducing blood lipid and body weight; (3) Improving hepatic steatosis, liver inflammation and mitochondrial damage.

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