Application of Akkermansia muciniphila in preparing a pharmaceutical composition for preventing and treating diabetes, the composition and its application

Abstract

The present invention relates to the application of Akkermansia muciniphila in the preparation of a pharmaceutical composition for preventing and treating diabetes, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two; wherein Akkermansia muciniphila AM06 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 22793; Akkermansia muciniphila AM02 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 22794. In the application, blood sugar can be lowered, insulin resistance can be reduced, and insulitis can be reduced, thereby effectively preventing and treating the occurrence, development and deterioration of diabetes and improving the quality of life of patients. The present invention also relates to a composition containing the Akkermansia muciniphila and the application thereof.

Description

Technical Field

[0001] The present invention relates to the technical field of diabetes prevention and treatment, in particular to the use of Akkermansia muciniphila in the preparation of a pharmaceutical composition for preventing and treating diabetes, a composition containing Akkermansia muciniphila and the use of the composition in the preparation of or as a medicine for preventing and treating diabetes, and a composition containing Akkermansia muciniphila. Background Art

[0002] Diabetes is a metabolic disease caused by the dysfunction of pancreatic B cells, which leads to metabolic disorders of fat, protein and carbohydrates in the human body, and further causes chronic damage and functional disorders to human organs. The onset of diabetes is related to environmental, genetic, autoimmune and other factors. With the development of society, the improvement of material living standards and changes in lifestyle, coupled with the sharp increase in the number of elderly people and the aggravation of the aging structure, the prevalence of diabetes in China has increased year by year, and it has become one of the chronic non-communicable diseases that pose a serious threat to people's health in addition to tumors and cardiovascular and cerebrovascular diseases.

[0003] Diabetes can be divided into four major categories based on etiology, including type 1 diabetes (T1DM), type 2 diabetes (T2DM), special types of diabetes and gestational diabetes (GDM). Among them, T1DM, T2DM and gestational diabetes are common clinical types. The etiology and pathogenesis of T1DM are not yet fully understood. Its significant pathological and pathophysiological characteristics are a significant decrease or loss of insulin secretion caused by a significant decrease or even disappearance of the number of pancreatic β cells. The etiology and pathogenesis of T2DM are also unclear at present. Its significant pathophysiological characteristics are a decrease in insulin's ability to regulate glucose metabolism (insulin resistance) accompanied by a decrease in insulin secretion (relative decrease) caused by a defect in pancreatic β cell function. Special types of diabetes are diabetes with relatively clear etiology. For patients with type 1 diabetes, lifelong insulin replacement therapy is required to maintain life due to the absolute lack of insulin secretion. For type 2 diabetes, metformin is the first-line medication for controlling hyperglycemia in patients with T2DM and the basic medication in drug combination. In addition, the drugs currently available for clinical treatment of diabetes include sulfonylureas, glinides, α-glucosidase inhibitors, thiazolidinediones (TZDs), dipeptidyl peptidase IV inhibitors (DPP-4i), α-glucosidase inhibitors and sodium-glucose co-transporter 2 inhibitors (SGLT2i), glucagon-like peptide-1 receptor agonists (GLP-1RA) and insulin, etc. However, the above-mentioned diabetes treatment drugs still cannot meet clinical needs, and it is still necessary to further develop new drugs for the prevention and treatment of diabetes. Summary of the invention

[0004] Based on this, the invention objective of the present application includes providing the use of Akkermansia muciniphila and a composition containing the Akkermansia muciniphila in the preparation of a pharmaceutical composition for preventing and treating diabetes, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two; wherein,

[0005] The Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 28, 2021, with the deposit number of CGMCC No.22793;

[0006] The Akkermansia muciniphila AM02 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No.22794.

[0007] In a first aspect of the present invention, there is provided a use of Akkermansia muciniphila in preparing a pharmaceutical composition for preventing and treating diabetes, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two; wherein,

[0008] The Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 28, 2021, with the deposit number of CGMCC No.22793;

[0009] The Akkermansia muciniphila AM02 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No.22794.

[0010] In some embodiments, the Akkermansia muciniphila AM06 and the Akkermansia muciniphila AM02 are each independently live bacteria, inactivated bacteria, or a combination of live bacteria and inactivated bacteria.

[0011] In some embodiments, the diabetes comprises at least one of type 1 diabetes, type 2 diabetes, a special type of diabetes, and gestational diabetes.

[0012] In some embodiments, the manner of improving blood glucose levels comprises at least one of inducing secretion of GLP-1 and reducing insulin resistance.

[0013] In some embodiments, the pharmaceutical composition includes the Akkermansia muciniphila and a pharmaceutically acceptable carrier.

[0014] In some embodiments, the pharmaceutical composition is a medicine, and its dosage form is tablets, capsules, granules, pills, ointments, solutions, suspensions, emulsions, creams, sprays, drops, patches or tube feeding preparations.

[0015] In a second aspect of the present invention, there is provided use of a composition containing Akkermansia muciniphila in the preparation of or as a medicament for preventing and treating diabetes, wherein the Akkermansia muciniphila is as defined in the first aspect of the present invention.

[0016] In some embodiments, the composition containing Akkermansia muciniphila further comprises a second active ingredient, wherein the second active ingredient comprises at least an α-glucosidase inhibitor; and / or,

[0017] The composition containing Akkermansia muciniphila is a composite probiotic, and the composite probiotic also includes probiotics different from the Akkermansia muciniphila.

[0018] In some embodiments, the α-glucosidase inhibitor is selected from one or more of acarbose, voglibose and miglitol.

[0019] In a third aspect of the present invention, a composition containing Akkermansia muciniphila is provided, wherein the Akkermansia muciniphila is as defined in the first aspect of the present invention; further, the composition containing Akkermansia muciniphila also includes a second active ingredient, and further, the second active ingredient includes at least an α-glucosidase inhibitor;

[0020] Furthermore, the α-glucosidase inhibitor is selected from one or more of acarbose, voglibose and miglitol.

[0021] The inventors of the present application isolated strains AM06 (deposit number CGMCC No. 22793) and AM02 (deposit number CGMCC No. 22794). According to comprehensive analyses such as 16S rRNA analysis, morphological analysis, metabolite component analysis, efficacy analysis (such as tolerance to artificial gastric juice and artificial intestinal juice, the ability to inhibit inflammatory factors from destroying tight junction proteins of intestinal cells, and the effect of inhibiting LPS-induced liver slice hepatitis), both strains belong to Akkermansia muciniphila, and are identified as new strains of Akkermansia muciniphila different from ATCC BAA-835 (standard strain) and other Akkermansia muciniphila.

[0022] The inventors found that the above-isolated Akkermansia muciniphila AM06 and / or AM02 can be used to prevent and treat diabetes (including but not limited to type 2 diabetes and type 1 diabetes), or to improve blood sugar levels. Therefore, the above-isolated Akkermansia muciniphila AM06 and / or AM02 can be used to prepare pharmaceutical compositions, and further medicines can be prepared. The above-isolated Akkermansia muciniphila AM06 and / or AM02 can lower blood sugar, reduce insulin resistance, and reduce insulitis, thereby effectively preventing and / or treating the occurrence and development of diabetes and its deterioration, and improving the quality of life of patients. After experimental analysis, the applicant believes that the above-isolated Akkermansia muciniphila AM06 and / or AM02 can enhance the intestinal barrier and induce intestinal L cells to secrete GLP-1, thereby lowering blood sugar, reducing insulin resistance, and reducing insulitis.

[0023] Furthermore, the inventors have also found that the newly isolated Akkermansia muciniphila AM06 and / or AM02 can also be used in combination with other diabetes treatment drugs (such as α-glucosidase inhibitors, such as acarbose) for preventing and treating diabetes, or for improving blood sugar levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0025] Figure 1 This is a colony characteristic diagram of Akkermansia muciniphila AM02 cultured in one embodiment of the present invention;

[0026] Figure 2 This is a colony characteristic diagram of Akkermansia muciniphila AM06 obtained in one embodiment of the present invention;

[0027] Figure 3 This is a microscopic observation picture of Akkermansia muciniphila AM02 cultured in one embodiment of the present invention after Gram staining;

[0028] Figure 4 This is a microscopic observation picture of Akkermansia muciniphila AM06 cultured in one embodiment of the present invention after Gram staining;

[0029] Figure 5 This is a PCA analysis diagram of metabolites of several Akkermansia muciniphila culture supernatants in one embodiment of the present invention;

[0030] Figure 6This is a fluorescence microscope image showing the effect of several muciniphilic Akkermansia on the reduction of tight junction protein ZO-1 expression induced by TNF-α and IFN-γ in Caco2 cells according to an embodiment of the present invention.

[0031] The Akkermansia muciniphila AM06 isolated in the present invention is classified and named Akkermansia muciniphila. It was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 22793; the strain was received and registered by the collection center on June 28, 2021, and was detected as a viable strain by the collection center on June 28, 2021.

[0032] The Akkermansia muciniphila AM02 isolated in the present invention is classified and named Akkermansia muciniphila, and was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 22794; the strain was received and registered by the collection center on June 28, 2021, and was detected as a viable strain by the collection center on June 28, 2021. DETAILED DESCRIPTION

[0033] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present invention. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0035] the term

[0036] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0037] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0038] In the present invention, "plurality", "multiple", "multiple times", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0039] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0040] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0041] Herein, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. If multiple "preferred" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "preferred" item is independent.

[0042] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0043] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0044] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0045] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0046] In the present invention, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers in the numerical interval, including the two endpoint integers of the numerical range, and each integer between the two endpoints, is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows for broadly including quantitative intervals such as percentage intervals, ratio intervals, and ratio intervals.

[0047] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0048] In the present invention, the term "room temperature" generally refers to 4°C to 35°C, preferably 20°C ± 5°C. In some embodiments of the present invention, room temperature refers to 20°C to 30°C.

[0049] In the present invention, when it comes to the unit of the data range, if there is a unit only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 3-5h means that the units of the left endpoint "3" and the right endpoint "5" are both h (hours).

[0050] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0051] In the present invention, "Akkermansia muciniphila" can be a living bacterium, or it can be an Akkermansia muciniphila that has been inactivated, genetically recombined, transformed or modified, attenuated, chemically treated, or physically treated to fully or partially retain the original biological activity, or it can be a bacterial lysate, culture (such as supernatant) or a component extracted from the culture.

[0052] Current diabetes treatment drugs can treat diabetes in the following ways, but there is still an urgent need to further develop new drugs for the prevention and treatment of diabetes.

[0053] Insulin: Insulin therapy is an important means of controlling hyperglycemia. T1DM patients need to rely on insulin to maintain life, and must also use insulin to control hyperglycemia and reduce the risk of diabetic complications. Although T2DM does not need insulin to maintain life, when oral hypoglycemic drugs are ineffective or there are contraindications to oral drugs, insulin is still needed to control hyperglycemia and reduce the risk of diabetic complications. At certain times, especially when the course of the disease is long, insulin therapy may be the most important and even necessary measure to control blood sugar. At present, the most common adverse reaction of insulin in clinical practice is hypoglycemia. Excessive insulin dosage or failure to reduce insulin dosage in time after blood sugar is controlled, failure to eat on time or insufficient food after insulin injection, excessive exercise or no snack before exercise, too much long-acting insulin in the mixed insulin injection, etc., may induce hypoglycemia.

[0054] Metformin: The main pharmacological effect of biguanides is to lower blood sugar by reducing the output of glucose in the liver and improving peripheral insulin resistance. Many countries and international organizations have recommended metformin as the first-line drug for controlling hyperglycemia in patients with T2DM in the guidelines for the diagnosis and treatment of diabetes. The use of metformin alone does not increase the risk of hypoglycemia, but the risk of hypoglycemia may be increased when metformin is used in combination with insulin or insulin secretagogues. The main adverse reactions of metformin are gastrointestinal reactions. Long-term use of metformin can cause a decrease in vitamin B12 levels.

[0055] Sulfonylureas: These drugs are insulin secretagogues. Their main pharmacological action is to stimulate the pancreatic β cells to secrete insulin, increase the insulin level in the body and lower blood sugar. Improper use of sulfonylureas can lead to hypoglycemia, especially in elderly patients and those with liver and kidney dysfunction. Sulfonylureas can also lead to weight gain. Patients with mild renal insufficiency should choose gliquidone when using sulfonylureas.

[0056] Meglitinides: These drugs are non-sulfonylurea insulin secretagogues. They mainly reduce postprandial blood sugar by stimulating the early phase of insulin secretion. They also have a certain effect on reducing fasting blood sugar, and can reduce glycosylated hemoglobin HbA1c by 0.5% to 1.5%. Common adverse reactions of meglitinides are hypoglycemia and weight gain, but the risk and degree of hypoglycemia are milder than those of sulfonylureas. Meglitinides can be used in patients with renal insufficiency.

[0057] Thiazolidinediones (TZDs): They lower blood sugar primarily by increasing the sensitivity of target cells to the effects of insulin. TZDs do not increase the risk of hypoglycemia when used alone, but they can increase the risk of hypoglycemia when used in combination with insulin or insulin secretagogues. Weight gain and edema are common adverse reactions of TZDs, which are more pronounced when used in combination with insulin. The use of TZDs is associated with an increased risk of fractures and heart failure.

[0058] α-Glucosidase inhibitors: can be used in combination with biguanides, sulfonylureas, TZDs or insulin. Common adverse reactions of α-Glucosidase inhibitors are gastrointestinal reactions (such as abdominal distension, flatulence, etc.). Starting with a small dose and gradually increasing the dose is an effective way to reduce adverse reactions. Hypoglycemia usually does not occur when this type of drug is taken alone.

[0059] Dipeptidyl peptidase IV inhibitor (DPP-4i): By inhibiting dipeptidyl peptidase IV (DPP-4), it reduces the inactivation of GLP-1 (glucagon-like peptide-1) in the body, thereby increasing the level of endogenous GLP-1. GLP-1 increases insulin secretion and inhibits glucagon secretion in a glucose concentration-dependent manner.

[0060] Sodium-glucose co-transporter 2 inhibitors (SGLT2i): can inhibit the renal reabsorption of glucose, lower the renal glucose threshold, and thus promote the excretion of urine glucose. Currently, the SGLT2i listed in my country include dapagliflozin, empagliflozin, canagliflozin and etogluflozin. Common adverse reactions of SGLT2i are urinary and reproductive system infections and adverse reactions related to hypovolemia. Rare adverse reactions include diabetic ketoacidosis (DKA).

[0061] Glucagon-like peptide-1 receptor agonist (GLP-1RA): It stimulates insulin secretion and inhibits glucagon secretion in a glucose concentration-dependent manner by activating GLP-1 receptors, while increasing glucose uptake in muscle and adipose tissue, inhibiting glucose production in the liver, and exerting a hypoglycemic effect. It can also inhibit gastric emptying and appetite. The main adverse reactions of GLP-1RA are mild to moderate gastrointestinal reactions, including diarrhea, nausea, abdominal distension, vomiting, etc. These adverse reactions are more common in the early stages of treatment, and gradually alleviate with prolonged use.

[0062] Based on this, there is still an urgent need to develop more drugs with fewer adverse reactions, better ability to prevent and treat chronic complications, improve insulin resistance and delay pancreatic cell failure.

[0063] A first aspect of the present invention

[0064] In a first aspect of the present invention, there is provided a use of Akkermansia muciniphila in preparing a pharmaceutical composition for preventing and treating diabetes, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two.

[0065] The inventors of the present application isolated strains AM06 (deposit number CGMCC No. 22793) and AM02 (deposit number CGMCC No. 22794). According to comprehensive analyses such as 16S rRNA analysis, morphological analysis, metabolite component analysis, efficacy analysis (such as tolerance to artificial gastric juice and artificial intestinal juice, the ability to inhibit inflammatory factors from destroying tight junction proteins of intestinal cells, and the effect of inhibiting LPS-induced liver slice hepatitis), both strains belong to Akkermansia muciniphila, and are identified as new strains of Akkermansia muciniphila different from ATCC BAA-835 (standard strain) and other Akkermansia muciniphila.

[0066] In the present invention, "Akermansia muciniphila" is consistent with the common definition in the art, including but not limited to Akkermansia muciniphila AM06 and AM02.

[0067] Akkermansia muciniphila (A. muciniphila) belongs to the phylum Verucomicrobia, family Akkermansiaceae, genus Akkermansia. A. muciniphila is a mucin-degrading bacterium. It is an anaerobic, non-motile, non-spore-forming, oval Gram-negative bacterium that grows at 20-40°C and pH 5.5-8.0 (optimum temperature 37°C, optimum pH 6.5). It is commonly found in the human intestine. Although it is an anaerobic bacterium, it can also tolerate a certain degree of oxygen. A. muciniphila can colonize in the mucus layer and colon by degrading and utilizing mucin as an energy source. Furthermore, it can also produce short-chain fatty acids (SCFA) through mucin degradation. The production of these short-chain fatty acids (including acetate, propionate and butyrate) plays an important role in human health.

[0068] In the present invention, “Akermansia muciniphila described in the present invention” specifically refers to Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two, which can be recorded as “AM06 and / or AM02”.

[0069] Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No. 22793. It can be isolated by the method of Example 1, and the strain can also be identified by one or more methods including but not limited to Examples 2 to 4.

[0070] In some embodiments, the colony culture characteristics of Akkermansia muciniphila AM06 include: round protrusions, neat edges, opaque, white, and unevenly sized colonies, and further, the colony size is about 0.08 to 2.2 mm.

[0071] In some embodiments, Akkermansia muciniphila AM06 is isolated from breast milk.

[0072] Akkermansia muciniphila AM02 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No. 22794. It can be isolated by the method of Example 1, and the strain can also be identified by one or more methods including but not limited to Examples 2 to 4.

[0073] In some embodiments, the colony culture characteristics of Akkermansia muciniphila AM02 of CGMCC No. 22794 include: round protrusions, neat edges, opaque, white, and unevenly sized colonies, and further, the colony size is about 0.08 to 2.2 mm.

[0074] In some embodiments, the applicant also conducted an artificial gastric juice and artificial intestinal juice tolerance test. Among them, in the artificial gastric juice tolerance test, the number of live bacteria in different groups of 0.9wt% NaCl solution group, pH3 artificial gastric juice group and pH2 artificial gastric juice group was statistically analyzed after anaerobic incubation at 37±2℃ for 0h, 1.5h and 3h. The results showed that the artificial gastric juice tolerance of Akkermansia muciniphila strain was AM02>AM06>standard strain ATCC BAA-835. Among them, in the artificial intestinal juice tolerance test experiment, the number of live bacteria after anaerobic incubation at 37±2℃ for 0h, 4h and 8h was statistically analyzed, and the artificial intestinal juice tolerance was better than AM06>AM02>ATCC BAA-835.

[0075] In some in vitro experiments, Akkermansia muciniphila AM06 and AM02 were superior to the standard strain BAA-835 in terms of tolerance to artificial gastric juice and artificial intestinal juice, ability to inhibit inflammatory factors from destroying tight junction proteins of intestinal cells, and effect of inhibiting LPS-induced liver slice hepatitis.

[0076] Akkermansia muciniphila AM06 and AM02 can each independently be living bacteria, or inactivated Akkermansia muciniphila (i.e. inactivated bacteria, which can be fully or partially inactivated), or a bacterial lysate, culture (such as supernatant) or a component extracted from the culture.

[0077] In some embodiments, Akkermansia muciniphila AM06 and Akkermansia muciniphila AM02 are each independently live bacteria, inactivated bacteria, or a combination of live bacteria and inactivated bacteria. The inactivated bacteria may be inactivated bacteria with complete morphological structure, inactivated bacteria with incomplete morphological structure, or a combination thereof.

[0078] In some embodiments, Akkermansia muciniphila AM06 and Akkermansia muciniphila AM02 are each independently one or more of live bacteria, inactivated bacteria with intact morphological structures, and inactivated bacteria with incomplete morphological structures.

[0079] In some embodiments, the Akkermansia muciniphila of the present invention is selected from live bacteria of Akkermansia muciniphila.

[0080] In some embodiments, the Akkermansia muciniphila of the present invention is selected from inactivated Akkermansia muciniphila bacteria.

[0081] In the present invention, "morphologically intact" may include, but is not limited to, Akkermansia muciniphila that has been completely or partially inactivated through an inactivation treatment.

[0082] In the present invention, the situation of "incomplete morphological structure" may include but is not limited to bacterial lysate, culture (such as supernatant) or components extracted from the culture.

[0083] The inventors have found through a large number of experiments that the above-isolated Akkermansia muciniphila AM06 and / or AM02 can be used to prevent and treat diabetes (including but not limited to type 2 diabetes and type 1 diabetes) or to improve blood sugar levels. Therefore, the above-isolated Akkermansia muciniphila AM06 and / or AM02 can be used to prepare pharmaceutical compositions, and further can be used to prepare medicines. The above-isolated Akkermansia muciniphila AM06 and / or AM02 can lower blood sugar, reduce insulin resistance, and reduce insulitis, thereby effectively preventing and / or treating the occurrence and development of diabetes and its deterioration, and improving the quality of life of patients. After experimental analysis, the applicant believes that the above-isolated Akkermansia muciniphila AM06 and / or AM02 can enhance the intestinal barrier and induce intestinal L cells to secrete GLP-1, thereby lowering blood sugar, reducing insulin resistance, and reducing insulitis.

[0084] For preparing pharmaceutical compositions

[0085] In the present invention, "prevention" includes prevention, treatment, adjuvant therapy and other aspects. As used herein, "prevention" means to alleviate, delay the progression, attenuate, prevent, or maintain an existing disease or condition. "Prevention" also includes curing one or more symptoms of a disease or condition, preventing its development, or alleviating it to a certain extent. In the present invention, "prevention" and "prevention and / or treatment" have the same meaning and can be used interchangeably.

[0086] In the present invention, the "composition" may be a combination of a plurality of substances, and further, may be used in combination or may be a mixture of the combination.

[0087] In the present invention, "drug" includes any agent, compound, composition or mixture that provides pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the pharmacological effects of "drug" in vivo is not particularly limited, and it can be a systemic effect or only a local effect. The activity of the "drug" is not particularly limited, and it can be an active substance that can interact with other substances, or it can be an inert substance that does not interact.

[0088] In the present invention, "pharmaceutical composition" refers to a composition that has the effect of preventing and treating diseases or symptoms and can be used as a medicine or can be used to prepare a medicine.

[0089] In the present invention, "drug" refers to a directly administrable pharmaceutical preparation, which usually has a prescribed usage and dosage.

[0090] In some embodiments, the diabetes comprises at least one of type 1 diabetes, type 2 diabetes, a special type of diabetes, and gestational diabetes.

[0091] In some embodiments, the diabetes comprises at least one of type 1 diabetes and type 2 diabetes.

[0092] In some embodiments, the diabetes comprises at least type 1 diabetes.

[0093] In some embodiments, the diabetes is type 1 diabetes.

[0094] In some embodiments, when used to prevent and treat type 1 diabetes, the Akkermansia muciniphila (AM06 and / or AM02) described in the present invention can effectively prevent and treat type 1 diabetes in mice (such as spontaneous type 1 diabetes), reduce and delay the onset of diabetes and reduce insulitis, and the therapeutic effect AM06>AM02>BAA-835.

[0095] In some embodiments, the diabetes comprises at least type 2 diabetes.

[0096] In some embodiments, the diabetes is type 2 diabetes.

[0097] In some embodiments, Akkermansia muciniphila AM06 and / or AM02 can effectively prevent and treat type 2 diabetes in animals (such as mice), reduce body weight, lower fasting blood glucose and insulin levels, reduce insulin resistance and reduce liver fat accumulation, and the therapeutic effect of AM06>AM02>BAA-835.

[0098] In some embodiments, the pharmaceutical composition comprises the Akkermansia muciniphila (AM06 and / or AM02) of the present invention and a pharmaceutically acceptable carrier.

[0099] In the present invention, "pharmaceutically acceptable" refers to those ligands, materials, compositions and / or dosage forms that are suitable for administration to a patient within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0100] In the present invention, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. As used herein, the phrase "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with drug administration. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Suitable examples include, but are not limited to: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffered saline; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0101] In the present invention, "carrier" includes but is not limited to mannitol, sorbitol, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium ethylenediaminetetraacetate (disodium EDTA), calcium sodium EDTA, carbonates, acetates, phosphates or aqueous solutions of monovalent alkali metals, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, oligofructose, dextran, glycine, starch, sucrose, dextrin (such as maltodextrin), lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, and magnesium stearate.

[0102] In some embodiments, the pharmaceutical composition may further include other active pharmaceutical ingredients, which may be pharmaceutical ingredients having a preventive and therapeutic effect on diabetes or a pharmaceutical ingredient having a preventive and therapeutic effect on other diseases.

[0103] In some embodiments, other active pharmaceutical ingredients can be appropriately selected from existing diabetes prevention and treatment drugs: sulfonylureas, glinides, α-glucosidase inhibitors, thiazolidinediones (TZDs), dipeptidyl peptidase IV inhibitors (DPP-4i), α-glucosidase inhibitors and sodium-glucose co-transporter 2 inhibitors (SGLT2i), glucagon-like peptide-1 receptor agonists (GLP-1RA) and insulin, etc.

[0104] In some embodiments, the other active pharmaceutical ingredients include at least an α-glucosidase inhibitor. The α-glucosidase inhibitor can be selected from one or more of acarbose, voglibose and miglitol. In some embodiments, the pharmaceutical composition comprises the Akkermansia muciniphila (AM06 and / or AM02) of the present invention and acarbose.

[0105] In the present invention, "improving blood sugar level" means beneficially lowering blood sugar level, and "beneficial" means bringing about beneficial effects on health.

[0106] In some embodiments, the method for improving blood sugar level is not particularly limited. For example, in some embodiments, the method for improving blood sugar level includes at least one of inducing secretion of GLP-1 and reducing insulin resistance. In other embodiments, the method for improving blood sugar level is selected from one or more of inducing secretion of GLP-1 and reducing insulin resistance.

[0107] Formulation type

[0108] In some embodiments, the pharmaceutical composition is a drug.

[0109] In the present invention, "drug" refers to a pharmaceutical preparation that can be directly administered, which usually has a prescribed usage and dosage, consistent with the above definition.

[0110] In some embodiments, the pharmaceutical preparation can be a liquid preparation or a solid preparation. Liquid preparations refer to preparations containing a liquid phase, non-limiting examples of which include solutions, suspensions, emulsions, etc. Non-limiting examples of solid preparations include tablets, capsules, granules, pills, etc.

[0111] In some embodiments, depending on the different modes of administration, the pharmaceutical preparation can be an oral preparation, an injection, a drop, a patch, a tube feeding preparation, etc.

[0112] In some embodiments, the dosage form of the drug is a tablet, capsule, granule, pill, ointment, solution, suspension, emulsion, cream, spray, drop, patch or tube feeding preparation.

[0113] It should be understood that the medicine contains a therapeutically effective amount of Akkermansia muciniphila (AM06 and / or AM02) of the present invention.

[0114] In the present invention, "therapeutically effective amount" refers to the amount of the active pharmaceutical ingredient that will cause a biological or medical response in an individual for a disease, disorder and / or symptom, for example, the amount of Akkermansia muciniphila of the present invention that brings a pharmacologically positive effect to an individual, wherein the pharmacologically positive effect includes but is not limited to reducing or inhibiting the activity of an enzyme or protein or improving symptoms, alleviating symptoms, slowing down or delaying the progression of a disease, or preventing a disease.

[0115] In the present invention, "subject" refers to a patient who takes the pharmaceutical composition.

[0116] In the present invention, "subject" is an animal, preferably a mammal, more preferably a human, and the subject includes but is not limited to a patient with a disease, illness and / or symptom. The subject in the present invention is preferably a mammal. The term "mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, humans, etc., preferably primates, more preferably humans.

[0117] In some embodiments, the subject is a mammal.

[0118] In some embodiments, the pharmaceutical composition is suitable for humans or other mammals. In the present invention, "other mammals" are not humans, and non-limiting examples of "other mammals" include cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, etc., and further include mice or rats; non-limiting examples of "other mammals" include primates.

[0119] In some embodiments, the subject is a human, mouse, or rat.

[0120] In some embodiments, the subject is a human or a mouse.

[0121] In the present invention, "patient" refers to an animal, preferably a mammal, such as a human, a mouse, or a rat.

[0122] Different subjects may result in different selection ranges of other ingredients in the aforementioned pharmaceutical composition.

[0123] Second aspect of the present invention

[0124] In a second aspect of the present invention, a composition containing Akkermansia muciniphila is provided for use in the preparation or as a drug for preventing and treating diabetes. The "composition containing Akkermansia muciniphila" herein refers to a composition containing "Ackermansia muciniphila (AM06 and / or AM02) of the present invention". The definition of "Ackermansia muciniphila of the present invention" can refer to the first aspect of the present invention.

[0125] Furthermore, the Akkermansia muciniphila described in the present invention is Akkermansia muciniphila AM06 (with a deposit number of CGMCC No. 22793), Akkermansia muciniphila AM02 (with a deposit number of CGMCC No. 22794), or a combination of the two.

[0126] In some embodiments, the composition containing Akkermansia muciniphila is a pharmaceutical composition.

[0127] In the second aspect of the present invention, the definitions of "composition", "prevention and treatment", "diabetes", "drug", "improvement of blood sugar level", and "pharmaceutical composition" can refer to the first aspect of the present invention. The definition of "drug" can also refer to the fourth aspect of the present invention.

[0128] When the composition containing Akkermansia muciniphila is used to prepare medicines, it is a pharmaceutical composition, in which the active pharmaceutical ingredients include at least one of Akkermansia muciniphila AM06 and Akkermansia muciniphila AM02.

[0129] The Akkermansia muciniphila described in the present invention may be a living bacterium, or an inactivated Akkermansia muciniphila (i.e., an inactivated bacterium, which may be fully or partially inactivated), or a lysate of the bacterium, a culture (such as a supernatant), or a component extracted from the culture.

[0130] In some embodiments, the composition comprising Akkermansia muciniphila further comprises a second active ingredient.

[0131] In the present invention, the "second active ingredient" refers to other active ingredients that have the effect of improving blood sugar levels, in addition to the Akkermansia muciniphila (AM06 and / or AM02) described in the present invention.

[0132] In some embodiments, the second active ingredient can be a modified bacterium of the muciniphilic Akkermansia (AM06 and / or AM02) described in the present invention. The modification methods include, but are not limited to, gene editing, chemical treatment, physical treatment, and the like. The modified muciniphilic Akkermansia forms a new strain different from AM06 and AM02. The modified bacteria may still have the function of preventing and treating diabetes or improving blood sugar levels, and may also be endowed with other new pharmacological and / or physiological functions.

[0133] In some embodiments, the composition comprising Akkermansia muciniphila further comprises a second pharmaceutically active ingredient.

[0134] In the present invention, "second active pharmaceutical ingredient" refers to other active pharmaceutical ingredients having the effect of preventing and treating diabetes, in addition to Akkermansia muciniphila (AM06 and / or AM02) described in the present invention. "Second active pharmaceutical ingredient" is within the scope of "second active ingredient".

[0135] In some embodiments, non-limiting examples of the second active pharmaceutical ingredient include: insulin, metformin, sulfonylureas, glinides, α-glucosidase inhibitors, thiazolidinediones (TZDs), dipeptidyl peptidase IV inhibitors (DPP-4i), α-glucosidase inhibitors, sodium-glucose co-transporter 2 inhibitors (SGLT2i), glucagon-like peptide-1 receptor agonists (GLP-1RA), and one or more thereof.

[0136] In some embodiments, the second pharmaceutically active ingredient includes at least an α-glucosidase inhibitor.

[0137] In some embodiments, the α-glucosidase inhibitor may be selected from one or more of acarbose, voglibose, and miglitol.

[0138] In some embodiments, the second pharmaceutically active ingredient is acarbose.

[0139] In some embodiments, the composition containing Akkermansia muciniphila is a composite probiotic. In this case, the composition contains, in addition to the Akkermansia muciniphila (AM06 and / or AM02) described in the present invention, other probiotics different from the Akkermansia muciniphila described in the present invention.

[0140] In the present invention, the "composite probiotics" contain at least two probiotics. It should be understood that the "composite probiotics containing Akkermansia muciniphila" includes the Akkermansia muciniphila described in the present invention, and also includes other probiotics different from the Akkermansia muciniphila described in the present invention.

[0141] In some embodiments, other probiotics (different from the muciniphilic Akkermansia described in the present invention) may include modified bacteria of the muciniphilic Akkermansia described in the present invention and probiotics of other species. Probiotics of other species are probiotics different from muciniphilic Akkermansia, and may include but are not limited to Bacteroides fragilis, Christensen, Saccharomyces boulardii, Lactobacillus rhamnosus, Lactobacillus bifidum, Enterococcus hilarii, etc.

[0142] In some embodiments, the complex probiotic includes multiple species of Akkermansia muciniphila.

[0143] In some embodiments, the composite probiotic consists of at least one of Akkermansia muciniphila AM06 and AM02, and at least one of the modified bacteria of AM06 and the modified bacteria of AM02.

[0144] In some embodiments, the composite probiotics include the Akkermansia muciniphila described in the present invention, and also include probiotics of other species. The probiotics of other species are defined as above. It further includes modified bacteria of the Akkermansia muciniphila described in the present invention.

[0145] In the present invention, each component in the "composition containing Akkermansia muciniphila" can appear at any appropriate time. For example, it can be directly formulated into a mixture and opened for use, or it can be packaged separately and formulated into a mixture when used, or it can be administered to a subject separately so that it appears simultaneously at a local location in the body and exerts a combined effect. When the "composition containing Akkermansia muciniphila" also contains other active pharmaceutical ingredients, the combination of Akkermansia muciniphila (AM06 and / or AM02) and other active pharmaceutical ingredients described in the present invention can also appear at any appropriate time; the definition of "other active pharmaceutical ingredients" can refer to the first aspect of the present invention. The "composition containing Akkermansia muciniphila" that appears at any appropriate time is within the scope of protection of the present invention.

[0146] The third aspect of the present invention

[0147] In a third aspect of the present invention, a composition comprising Akkermansia muciniphila is provided;

[0148] The definition of "composition containing Akkermansia muciniphila" may refer to the second aspect of the present invention.

[0149] In some embodiments, the composition containing Akkermansia muciniphila of the present invention further comprises a second active ingredient. The definition of "second active ingredient" can refer to the second aspect of the present invention.

[0150] In some embodiments, the second active ingredient includes at least an α-glucosidase inhibitor.

[0151] In some embodiments, the α-glucosidase inhibitor may be selected from one or more of acarbose, voglibose and miglitol.

[0152] A fourth aspect of the present invention

[0153] In a fourth aspect of the present invention, a method for preventing and treating diabetes is provided, comprising administering a therapeutically effective amount of Akkermansia muciniphila of the present invention to a subject (ie, administering a therapeutically effective amount of Akkermansia muciniphila of the present invention to a subject).

[0154] In some embodiments, a method for preventing and treating diabetes is provided, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition containing the Akkermansia muciniphila of the present invention.

[0155] In some embodiments, a method for preventing and treating diabetes is provided, comprising administering to a subject a therapeutically effective amount of a medicament containing the Akkermansia muciniphila of the present invention.

[0156] In the fourth aspect of the present invention, the definitions of "Akkermansia muciniphila", "Akkermansia muciniphila described in the present invention", "composition", "pharmaceutical composition", "medicine", "prevention and treatment", "diabetes", "therapeutically effective amount" and "improvement of blood sugar level" can refer to the first aspect or the second aspect of the present invention.

[0157] In a fourth aspect of the present invention, the Akkermansia muciniphila of the present invention can be used in combination with other active pharmaceutical ingredients to prevent and treat diabetes or improve blood sugar levels. The definition of "other active pharmaceutical ingredients" can refer to the first aspect of the present invention. For example, other active pharmaceutical ingredients can be pharmaceutical ingredients with diabetes prevention and treatment effects, or can be pharmaceutical ingredients with other disease prevention and treatment effects. In some embodiments of the present invention, the other active pharmaceutical ingredient is the second active ingredient defined in the second aspect of the present invention.

[0158] In some embodiments, the Akkermansia muciniphila described herein is used in combination with an α-glucosidase inhibitor.

[0159] In some embodiments, the Akkermansia muciniphila of the present invention is used in combination with acarbose. Each may be administered in a suitable unit. For example, acarbose may be provided in an amount of compound per unit body weight, and the Akkermansia muciniphila of the present invention may be provided in an amount of bacteria per person or per animal. For example, in Example 8, "30 mg / kg of acarbose + 10 9 CFU / piece AM06" method.

[0160] In the present invention, the Akkermansia muciniphila of the present invention, the pharmaceutical composition containing the Akkermansia muciniphila of the present invention, and the medicine containing the Akkermansia muciniphila of the present invention all belong to the category of "medicine" defined in the present invention.

[0161] In some embodiments, the administration of the drug includes, but is not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection, and topical administration, inhalation.

[0162] Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffer. Solid dosage forms such as tablets, sugar pills, capsules, pills and granules may be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active ingredient in such compositions may be delayed in a certain part of the digestive tract. Examples of embedding components that may be used are polymeric substances and waxes. If necessary, the active ingredient may be formed into microcapsules with one or more of the above-mentioned excipients.

[0163] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, specifically, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices. For example, the suspension may contain a suspending agent, specifically, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitan esters, microcrystalline cellulose, aluminum methylate and agar or mixtures of these substances.

[0164] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0165] Dosage forms for topical administration include ointments, powders, patches, sprays and inhalants, which are prepared by mixing the active ingredient with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required under sterile conditions.

[0166] The "active ingredient" in the above-mentioned pharmaceutical preparation of the present invention refers to the ingredient in the pharmaceutical composition that can play the role of "drug".

[0167] It is to be understood that the drugs of the embodiments of the present invention can be added with different pharmaceutically acceptable carriers to prepare suitable clinical dosage forms, which include but are not limited to the dosage forms described above.

[0168] Some specific embodiments are provided below.

[0169] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0170] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0171] In the following examples, 10-fold gradient dilution means that each gradient is diluted 10 times. The unit "day" can be expressed as "d".

[0172] Example 1. Isolation and identification of Akkermansia muciniphila

[0173] 1.1. Isolation and identification of strain AM02

[0174] Use a sterile sampling spoon to take soybean-sized feces (samples are from healthy adult men) into a 10mL centrifuge tube. After sampling, transfer the sample immediately to a 37℃ anaerobic workstation (85% N2, 10% H2, 5% CO2, volume percentage) and dilute the sample to 10 by 10-fold gradient dilution. -9 , take 1 mL of each dilution solution and inoculate it into 9 mL of basal medium with mucin as the sole carbon source, and culture it anaerobically for 7 days. -4 Dilution: 1 mL of culture medium was inoculated and diluted to 10 in a 1:10 gradient dilution. -6, 100 μL of each dilution was spread on mucin agar medium, cultured anaerobically for 7 days, and a single colony was picked and inoculated into 2 mL of BHI broth (containing N-acetyl-D-glucosamine medium). The cultured bacterial solution was sequenced by 16s rRNA, and the 16S rRNA sequence was compared on the NCBI database. The isolated strain was identified as Akkermansia muciniphila, and the 16S rRNA sequencing results are as follows:

[0175]

[0176] The sequence was compared with the 16S rRNA sequence of ATCC BAA-835 at NCBI and the Per.Ident value was 99.43%.

[0177] 1.2. Isolation and identification of strain AM06

[0178] Freshly collected breast milk samples (from healthy adult women) were immediately injected into 5 mL anaerobic vials for storage. The samples were then transferred to a 37°C anaerobic workstation (85% N2, 10% H2, 5% CO2, volume percentage) and diluted to 10 by 10-fold gradient dilution. -6 , take 1 mL of each dilution solution and inoculate it into 9 mL of basal medium with mucin as the sole carbon source, and culture it anaerobically for about 1 month. -1 ~10 -4 Dilution: 1 mL of culture medium inoculated with 1:10 dilution to 10 -6 , 100 μL of each dilution was spread on mucin agar medium, cultured anaerobically for 7 days, and a single colony was picked and inoculated into 2 mL of BHI broth (containing N-acetyl-D-glucosamine medium). The cultured bacterial solution was sequenced and identified by 16S rRNA. The 16S rRNA sequence was compared with the NCBI database, and the result was identified as Akkermansia muciniphila. The sequencing results of 16S rRNA are as follows:

[0179]

[0180] The sequence was compared with the 16S rRNA sequence of ATCC BAA-835 at NCBI and the Per.Ident value was 99.22%.

[0181] 1.3. Strain deposit information

[0182] Akkermansia muciniphila AM02, whose classification name is Akkermansia muciniphila, was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.22794; the strain was received and registered by the collection center on June 28, 2021, and was tested as a viable strain by the collection center on June 28, 2021.

[0183] Akkermansia muciniphila AM06, whose classification name is Akkermansia muciniphila, was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.22793; the strain was received and registered by the collection center on June 28, 2021, and was tested as a viable strain by the collection center on June 28, 2021.

[0184] Example 2. Cultivation of Akkermansia muciniphila and preparation of inactivated bacteria

[0185] 2.1. Streak the Akkermansia muciniphila onto BHA plates and culture anaerobically for 3 days. Observe the colony morphology, staining properties, size, club shape, and distribution.

[0186] Colony characteristics: After 3 days of culture on the above medium, Akkermansia muciniphila AM02 and AM06 showed round, convex, neatly edged, opaque, white, and unevenly sized colonies, with a size of about 0.08 to 2.2 mm. Figure 1 (Akkermansia muciniphila AM02) and Figure 2 (Akermansia muciniphila AM06).

[0187] Microscopic morphology: Gram staining of Akkermansia muciniphila AM02 and AM06 showed that they were Gram-negative bacteria, oval in shape, arranged singly or in chains, as shown in Figure 3 (Akkermansia muciniphila AM02) and Figure 4 (Akermansia muciniphila AM06).

[0188] The colony characteristics of the standard strain ATCC BAA-835 are: round protrusions, neat edges, opaque, white, and unevenly sized colonies. Under the microscope, they are gram-negative bacteria, oval, single or in chain arrangement.

[0189] 2.2. A single colony was selected and inoculated into BHI broth for 48 hours (temperature was 37°C), and the obtained bacterial solution was centrifuged at a speed of 16000×g for 30 minutes, and the supernatant was removed and the precipitate was collected to obtain the Akkermansia muciniphila sludge. AM02, AM06, and ATCC BAA-835 Akkermansia muciniphila were obtained by culture.

[0190] 2.3. Preparation of inactivated bacteria: Take an appropriate amount of bacterial mud, add physiological saline at a ratio of bacterial mud: physiological saline (m:v) = 1:10, use a stirrer to stir at 1000rpm for 5 to 20min to evenly disperse the bacteria. Take 100mL of the dispersed bacterial solution and place it in a sterile three-necked round-bottom flask (try to ensure that the bacterial solution does not adhere to the inner wall of the flask). Place the three-necked round-bottom flask on the heating plate of the magnetic stirrer, put in a sterile stirring bar, and insert a temperature electrode, set the speed to 300 to 500rpm, the temperature to 70℃, and heat for 30min to obtain inactivated bacteria of Akkermansia muciniphila.

[0191] Example 3. Non-targeted metabolic differential analysis of Akkermansia muciniphila culture supernatants

[0192] Sample preparation

[0193] After the culture of each Akkermansia muciniphila (AM02, AM06, ATCC BAA-835) in Example 2 was completed, 1 mL of the culture solution was centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was taken as a sample to be tested for non-targeted metabolomics analysis. Five parallel samples to be tested were prepared for each strain.

[0194] 3.2 Experimental results

[0195] PCA is a data dimensionality reduction method, which is to reduce multiple variables to a set of new comprehensive variables, and then select the first few principal components that reflect the original variable information as much as possible, so as to achieve the purpose of dimensionality reduction. The PCA graph reflects the true distribution of samples, and is mainly used to observe the separation trend between sample groups and whether there are abnormal points. At the same time, it reflects the variability between and within groups from the original data.

[0196] The experimental results can be found in Figure 5 . Figure 5The PCA analysis of quality control samples (QC samples) and all samples was included. The QC samples were clustered together in the two principal component analysis diagrams, indicating that the instrument was stable during the test and the data collection had good repeatability. At the same time, the results also showed that the metabolites in the culture supernatant of AM06 were close to those in the culture supernatant of BAA-835, and the metabolites in the culture supernatants of AM02 and BAA-835 were quite different.

[0197] The results of the comparison of the number of differential metabolites between the strains can be seen in Table 1. It can be seen that compared with the standard strain BAA-835, the number of differential metabolites detected by positive ion (pos) mode and negative ion (neg) mode of AM02 was 205 and 135, respectively, and compared with the standard strain BAA-835, the number of differential metabolites detected by positive ion (pos) mode and negative ion (neg) mode of AM06 was 111 and 62, respectively.

[0198] Table 1. Statistics of differential metabolites

[0199]

[0200] Example 4. Efficacy verification of Akkermansia muciniphila

[0201] 4.1. Tolerance of Akkermansia muciniphila to artificial gastric fluid

[0202] 4.1.1. Experimental methods and grouping

[0203] Table 2. Experimental groups

[0204]

[0205] Table 3. Experimental methods

[0206]

[0207] “+” indicates that testing is required.

[0208] 4.1.1.1. Take one tube of Akkermansia muciniphila, remove the label, wipe and disinfect the outer surface of the glycerol cryopreservation tube with 75% (v / v) alcohol, vortex and oscillate to mix, and open. Pipette 100-500 μL of bacterial solution and inoculate into 10 mL / tube of BHI broth, shake well, prepare 3 tubes in total, and do not inoculate bacteria as a negative control, place at 37°C for anaerobic culture for 2-4 days to obtain the first-level seed solution.

[0209] Gram staining of the primary seed solution should show Gram-positive bacteria without spores or other bacteria.

[0210] 4.1.1.2. Take 10 mL of the above-mentioned first-level seed solution, centrifuge at 12000×g and 4°C for 10 min, discard the supernatant, add 1 mL of 0.9wt% NaCl solution and resuspend to prepare bacterial suspension for use.

[0211] 4.1.1.3. According to Table 2 and Table 3, add the bacterial suspension of AM06, AM02 and standard strains to 0.9wt% NaCl, pH3.0 and pH2.0 artificial gastric juice, mix well, dispense into 5mL / tube, place in an anaerobic glove box and incubate at 37℃ for 0h, 1.5h and 3h, then take out and test the bacterial concentration of each sample. Each experimental group is carried out in parallel 3 times.

[0212] 4.1.1.4. Determination of viable count:

[0213] Take the experimental sample and dilute it in a 10-fold series to different dilutions. Take 100 μL of the dilution and inoculate it on the BHA plate. Spread it evenly. Make 2 plates for each dilution. Generally, make 2 to 3 dilutions. At the same time, take 100 μL of the dilution on the BHA plate as a negative control. Place all the coated plates upright and culture them under anaerobic conditions for about 3 to 5 days. Observe the growth of the colonies on the plates and count them.

[0214] The number of viable bacteria was calculated based on the sum of the colony counts on the two plates using the following formula:

[0215] Viable bacteria count (CFU / mL) = sum of colony counts on 2 plates / 2 × 10 × final dilution

[0216] Survival rate calculation:

[0217]

[0218] 4.1.2 Experimental results

[0219] The experimental results can be seen in Table 4. The artificial gastric juice tolerance of different mucinotropic Akkermansia strains is AM02>AM06>standard strain BAA-835 from high to low.

[0220] Table 4. Statistical table of survival rate of Akkermansia muciniphila in artificial gastric fluid tolerance

[0221]

[0222] 4.2. Tolerance of Akkermansia muciniphila to artificial intestinal fluid

[0223] 4.2.1. Experimental methods and grouping

[0224] Table 5. Experimental groups

[0225]

[0226] 4.2.1.1. Preparation of primary seed solution

[0227] Take one tube of Akkermansia muciniphila and remove the label. Use 75% (v / v) alcohol to wipe and disinfect the outer surface of the glycerol cryopreservation tube. Vortex and oscillate to mix well and open. Pipette 100 μL of bacterial solution and inoculate into 10 mL / tube of BHI broth. Shake well and prepare 3 tubes in total. At the same time, do not inoculate bacteria as a negative control and place at 37°C for anaerobic culture for 2 to 4 days to obtain the first-level seed solution.

[0228] Gram staining of the primary seed solution should show Gram-positive bacteria without spores or other bacteria.

[0229] 4.2.1.2. Preparation of bacterial sludge

[0230] The above-mentioned first-level seed liquid was divided into 1.5 mL / tube, centrifuged at 12000×g for 10 min, and the supernatant was discarded to obtain bacterial sludge. 3 tubes of bacterial sludge were prepared for each of AM06, AM02 and standard strains.

[0231] 4.2.1.3. Evaluation of strain tolerance to artificial intestinal fluid

[0232] As shown in Tables 5 and 6, 1.5 mL of artificial intestinal fluid was added to each tube of the prepared bacterial sludge, mixed, and then each tube of solution was divided into 3 tubes at 0.5 mL / tube, and incubated anaerobically at 37°C for 0, 4, and 8 hours, respectively, and samples were taken to detect the number of viable bacteria. Three parallels were made for each group.

[0233] Table 6. Experimental methods

[0234]

[0235] “+” indicates that testing is required.

[0236] 4.2.1.4. Determination of viable count

[0237] Take the incubated samples separately, dilute them in 10-fold gradient series, take 100 μL of the dilution and inoculate them on the BHA plate, spread it evenly, make 2 plates for each dilution, usually make 2 to 3 dilutions, and take 100 μL of the dilution on the BHA plate as a negative control. Place all the coated plates upright and culture them under anaerobic conditions for about 3 to 5 days. Observe the growth of the colonies on the plates and count them.

[0238] Viable bacteria count (CFU / mL) = sum of colony counts on 2 plates / 2 × 10 × final dilution

[0239] Survival rate calculation:

[0240] Survival rate = number of viable bacteria at each time point / corresponding number of viable bacteria at 0h × 100%

[0241] 4.2.2 Experimental results

[0242] The results can be seen in Table 7. ATCC BAA-835, AM02, and AM06 strains have good tolerance to artificial intestinal fluid, and the tolerance is in the order of AM02>AM06>standard strain BAA-835.

[0243] Table 7. Survival rate statistics

[0244]

[0245] Effects of Akkermansia muciniphila on the expression of tight junction protein ZO-1 in Caco2 cells induced by TNF-α and IFN-γ

[0246] TNF-α: tumor necrosis factor-α; IFN-γ: interferon-γ.

[0247] 4.3.1. Experimental methods and grouping

[0248] Caco2 cells were inoculated into 96-well plates and cultured until the confluence was 80% to 90%. After inducing Caco2 cells with 100 ng / mL TNF-α+100 ng / mL IFN-γ for 24 h, AM02, AM06, and BAA-835 were added to continue incubating with the cells for 24 h. The experimental grouping can be seen in Table 8, and 5 replicates were made for each group. Immunofluorescence was used to observe the effects of BAA-835, AM06, and AM02 on the expression of tight junction protein ZO-1 in Caco2 cells induced by TNF-α and IFN-γ.

[0249] Table 8. Experimental groups

[0250]

[0251] 4.3.2 Experimental results

[0252] The fluorescence intensity of the captured images is counted, and the results can be seen in Figure 6 and Table 9. Compared with the blank control group, the fluorescence intensity of the intercellular space in the inflammatory model group was significantly weakened after TNF-α and IFN-γ induction for 48 hours, indicating that the expression of ZO-1 protein was reduced and the tight junctions between cells were destroyed. Compared with the inflammatory model group, the fluorescence intensity of the treated group after intervention with AM02, AM06, and BAA-835 increased significantly (p<0.05), and the ability of AM02 and AM06 to inhibit the reduction of ZO-1 protein in Caco2 cells induced by inflammatory factors was significantly better than that of BAA-825 (p<0.05).

[0253] Table 9. Fluorescence intensity statistics

[0254] serial number Group Fluorescence intensity A Blank control group 36.2647±5.8977** B Inflammation model group 25.6588±4.5303 C AM02 Group 41.7059±6.5664**aa D AM06 Group 40.2949±4.5437**aa E BAA-835 group 33.6688±4.13336**

[0255] Note: ** indicates that compared with the model group, the difference is extremely significant p < 0.01; aa indicates that compared with the BAA-835 group, the difference is extremely significant p < 0.01.

[0256] Example 5. Akkermansia muciniphila induces GLP-1 secretion in NCI-H716 cells

[0257] 5.1. Experimental methods and grouping

[0258] Take NCI-H716 cells in the logarithmic growth phase, centrifuge and discard the supernatant, add 0.02g / L BSA solution to mix the cells, and press 3×10 6 The cells were inoculated with an inoculum of 100 cells / well into a 6-well plate. After culturing for 2 h, appropriate amounts of 0.02 g / L bovine serum albumin (BSA) solution, AM02, AM06, and BAA-835 were added to the cells according to the experimental groups in Table 10 and the cells were incubated for 36 h. The supernatant was collected and the GLP-1 content in the supernatant was detected by ELISA.

[0259] Table 10. Experimental groups

[0260] serial number Group Test substance A Blank control group / B AM02 Group <![CDATA[1×10 8 CFU / mL AM02]]> C AM06 Group <![CDATA[1×10 8 CFU / mL AM06]]> D BAA-835 group <![CDATA[1×10 8 CFU / mL BAA-835]]>

[0261] 5.2 Experimental results

[0262] The experimental results can be seen in Table 11. After adding AM02, AM06, and BAA-835, NCI-H716 cells were significantly induced to secrete a large amount of GLP-1 (p<0.01), and compared with BAA-835, the content of GLP-1 induced by AM02 and AM06 was significantly increased (p<0.05).

[0263] Table 11. GLP-1 content (pg / mL)

[0264] serial number Group GLP-1 content (pg / mL) A Blank control group 134.5±28.34 B AM02 Group 536.56±17.35**a C AM06 Group 573.45±23.21**a D BAA-835 group 475.46±34.14**

[0265] Note: ** indicates that compared with the model group, the difference is extremely significant p<0.01; a indicates that compared with the BAA-835 group, the difference is significant p<0.05.

[0266] Example 6 Experimental study on the efficacy of Akkermansia muciniphila in preventing and treating spontaneous type 2 diabetes mouse model

[0267] Experimental design and methods

[0268] Experimental animals: SPF grade db / db mice, male, 4-5 weeks old, 100; SPF grade C57BL / KsJ mice, male, 4-5 weeks old, 10. Breeding place during the experiment: SPF grade mouse room.

[0269] Experimental groups: The blank group consisted of C57BL / KsJ mice, 10 animals. The other experimental groups consisted of db / db mice, divided into: model group, positive drug group (semaglutide, 0.36 mg / kg, subcutaneous injection), AM06 low-dose group (10 6 CFU / mL), AM06 medium dose group (10 8 CFU / mL), AM06 high-dose group (10 10 CFU / mL), AM02 medium dose group (10 8 CFU / mL), ATCC BAA-835 medium dose group (10 8 CFU / mL), and the inactivated bacteria groups of AM06, AM02 and BAA-835 (the dosage of each group was 10 8 / mL), 10 animals per group. After a one-week adaptation period, each group began to be given the corresponding drug. From the first day to the 42nd day of the experiment, each group of mice was given a normal diet and each group was given the drug once a day. On the 43rd day, the mice in each group were sampled.

[0270] Experimental design test:

[0271] On the 43rd day of the experiment, the mice were fasted for 12 hours and then euthanized with CO2. Blood samples were collected by cardiac puncture for the detection of blood glucose and insulin levels. The terminal body weight and liver weight were recorded. The liver tissue of the mice was taken, fixed with 4% paraformaldehyde, embedded in paraffin, sliced ​​at 5 μm, stained with HE, and the pathological changes of the liver were observed under a light microscope and the fatty degeneration was scored. The fatty degeneration scoring standard: 0 points means that the fat droplets in the hepatocytes are scattered, rare, and basically normal; 1 point means that the hepatocytes containing fat droplets do not exceed 1 / 4; 2 points means that the hepatocytes containing fat droplets do not exceed 1 / 2; 3 points means that the hepatocytes containing fat droplets do not exceed 3 / 4; 4 points means that the liver tissue is almost replaced by fat droplets.

[0272] 6.2 Experimental results

[0273] The results of each test index can be found in Table 12 and Table 13.

[0274] Table 12. Statistics of various test indicators (mean ± SD, n = 10)

[0275]

[0276] Table 13.

[0277]

[0278] In Table 12 and Table 13, Note 1: * indicates that compared with the model group, the difference is significant p<0.05, ** indicates that compared with the model group, the difference is extremely significant p<0.01; Note 2: a indicates that compared with the BAA-835 medium-dose group, the difference is significant p<0.05, aa indicates that compared with the BAA-835 medium-dose group, the difference is extremely significant p<0.01;

[0279] Note 3: b means that compared with the medium-dose group of BAA-835, the difference is significant p<0.05, bb means that compared with the medium-dose group of BAA-835, the difference is extremely significant p<0.01.

[0280] 6.2.1. Mouse body weight

[0281] As shown in Table 12, compared with the blank group, the body weight of the mice in the model group increased significantly (p<0.01). Compared with the model group, the AM06 medium- and high-dose groups, the AM02 medium-dose group, the AM06 inactivated bacteria group and the AM02 inactivated bacteria group could significantly reduce body weight (p<0.05), and the other drug-administered groups had a trend of reducing the body weight of mice, but there was no significant difference. The body weight of the AM06 medium-dose group was significantly lower than that of the BAA-835 medium-dose group (p<0.05), and the body weight of the AM02 medium-dose group had a trend lower than that of the BAA-835 medium-dose group. The body weight of the AM02 and AM06 inactivated bacteria groups was significantly lower than that of the BAA-835 inactivated bacteria group. This shows that the live bacteria and inactivated bacteria of AM02 and AM06 are more effective than the live bacteria and inactivated bacteria of BAA-835 in reducing the weight gain of diabetic mice.

[0282] 6.2.2. Blood glucose, insulin and insulin resistance index in mice

[0283] As shown in Tables 12 and 13, compared with the blank group, the blood glucose level, insulin level and insulin resistance index of the mice in the model group were significantly increased (p<0.01), indicating that the mice in the model group had hyperglycemia and hyperinsulinemia, i.e., insulin resistance. Compared with the model group, each administration group can reduce the blood glucose and insulin levels of animals to varying degrees, and significantly reduce the insulin resistance index (p<0.01). And at the same dose level, the insulin resistance index of the live or inactivated bacteria of AM06 and AM02 is significantly better than that of the live or inactivated bacteria of BAA-835. This shows that the live and inactivated bacteria of AM02 and AM06 are better than the live and inactivated bacteria of BAA-835 in reducing blood sugar and reducing insulin resistance.

[0284] 6.2.3. Mouse liver pathology scoring

[0285] The liver pathological HE staining of mice in each group was performed, and the score was scored according to the degree of fatty degeneration of the sections. The results can be seen in Table 13. Compared with the blank control group, the liver fatty degeneration score of the model group increased significantly (p<0.01), indicating that the animals had fatty liver. Compared with the model group, each drug-treated group could reduce the liver fatty degeneration score to varying degrees, and the low-dose groups of AM06 and AM02 could significantly reduce the liver fatty degeneration score (p<0.05). At the same dose level, the liver fatty degeneration score of the live or inactivated bacteria of AM02 and AM06 tended to be lower than that of the live or inactivated bacteria of BAA-835, indicating that the live and inactivated bacteria of AM02 and AM06 were slightly better than the live and inactivated bacteria of BAA-835 in reducing liver fat accumulation.

[0286] In summary, Akkermansia muciniphila AM02 and AM06 can effectively prevent and treat spontaneous type 2 diabetes in mice, reduce body weight, lower fasting blood glucose and insulin levels, reduce insulin resistance and reduce liver fat accumulation, and the therapeutic effects of each strain are AM06>AM02>BAA-835.

[0287] Example 7. Experimental study on the efficacy of Akkermansia muciniphila in preventing and treating type 1 diabetes in mice

[0288] 7.1. Experimental animals: 80 SPF NOD mice, female, 4-5 weeks old; 10 SPF BALB / c mice, female, 4-5 weeks old. Breeding place during the experiment: SPF mouse room.

[0289] Experimental groups: The blank group consisted of BALB / c mice, 10 animals. The other experimental groups consisted of NOD mice, divided into: model group, positive drug group (glargine insulin, 50 IU / kg, subcutaneous injection), AM06 group (10 8 CFU / mL), AM02 group (10 8 CFU / mL), ATCCBAA-835 group (10 8 CFU / mL, AM06 inactivated bacteria group (10 8 / mL), AM02 inactivated bacteria group (10 8 / mL), ATCC BAA-835 inactivated bacteria group (10 8 / mL), 10 animals in each group. After a one-week adaptation period, each group began to receive the corresponding drug, once a day for 24 weeks. On the 43rd day, samples were collected from mice in each group.

[0290] Experimental design

[0291] One drop of tail vein blood was collected every week, and random blood sugar was measured with a rapid blood glucose meter. If it was equal to or greater than 12.0 mmol / L in two consecutive readings, it was considered to be diabetes.

[0292] After the experiment, the pancreatic tissue of the mice was obtained, fixed with paraformaldehyde, embedded in paraffin, sliced, stained with HE, and observed under a light microscope to score the insulitis. The severity of islet lymphocyte infiltration was scored according to the following standards: 0 points: no lymphocyte infiltration around the islets; 1 point: local lymphocyte infiltration around the islets; 2 points: partial lymphocyte infiltration in the islets, ranging from 1 / 3; 3 points: more lymphocyte infiltration in the islets, ranging from 1 / 3 to 1 / 2; 4 points: a large number of lymphocyte infiltration in the islets, ranging from >1 / 2, but not reaching 100%; 5 points: pan-insulitis.

[0293] 7.2 Experimental results

[0294] The test results can be seen in Table 14.

[0295] Table 14. Test results of each group of animal experiments

[0296]

[0297] Note 1: * indicates that the difference is significant compared with the model group, p<0.05, ** indicates that the difference is extremely significant compared with the model group, p<0.01; Note 2: a indicates that the difference is significant compared with the BAA-835 medium-dose group, p<0.05, aa indicates that the difference is extremely significant compared with the BAA-835 medium-dose group, p<0.01;

[0298] Note 3: b means that compared with the medium-dose group of BAA-835, the difference is significant p<0.05, bb means that compared with the medium-dose group of BAA-835, the difference is extremely significant p<0.01.

[0299] 7.2.1. Prevalence of diabetes

[0300] As shown in Table 14, compared with the blank group, as the age of the mice increased, the incidence of diabetes in the model group mice gradually increased, and the incidence rate could reach 90% by the end of the experiment. Compared with the model group, each administration group reduced and delayed the occurrence of diabetes to varying degrees. The live bacteria and inactivated bacteria of AM02 and AM06 were slightly better than the live bacteria and inactivated bacteria of BAA-835 in reducing the incidence of diabetes.

[0301] 7.2.2. Pancreatic islet inflammatory score

[0302] As shown in Table 14, compared with the blank group, the pancreatic islet inflammatory score of the mice in the model group increased significantly (p<0.01), indicating that the mice in the model group had insulitis. Compared with the model group, each drug-treated group can reduce the pancreatic islet inflammatory score of the animals to varying degrees, and the positive drug group, AM06 group and AM02 group significantly reduced the pancreatic islet inflammatory score (p<0.05). Among them, the pancreatic islet inflammatory score of the AM06 group and the AM02 group was significantly lower than that of the BAA-835 group, and the pancreatic islet inflammatory score of the AM06 and AM02 inactivated bacteria groups tended to be lower than that of the BAA-835 inactivated bacteria group. This shows that the live bacteria and inactivated bacteria of AM02 and AM06 are better than the live bacteria and inactivated bacteria of BAA-835 in reducing the pancreatic islet inflammatory score.

[0303] In summary, Akkermansia muciniphila AM02 and AM06 can effectively prevent and treat spontaneous type 1 diabetes in mice, reduce and delay the onset of diabetes and reduce insulitis, and the therapeutic effects of each strain are AM06>AM02>BAA-835.

[0304] Example 8. Combination of Akkermansia muciniphila and acarbose reduces postprandial blood glucose in mice

[0305] Experimental methods

[0306] Experimental animals: SPF-grade C57 / BL mice, male, 4-5 weeks old, 70 mice; breeding place during the experiment: SPF-grade mouse room.

[0307] Experimental grouping and modeling: 10 animals were selected as the blank group, which were fed with ordinary feed and no STZ (streptozotocin) injection during the experiment. After the remaining animals were fed with high-fat feed for 4 weeks, STZ 150mg / kg was injected intraperitoneally, and then the animals with random blood sugar greater than 16.7mmol / L were selected and randomly divided into the model group, acarbose group (30mg / kg), AM06 group (10 9 CFU / mouse), acarbose+AM06 group (30 mg / kg acarbose+10 9 CFU / Animal AM06), 10 animals per group. After grouping, except for the blank group, the animals continued to be fed with high-fat diet, and each group began to be given the corresponding drug, once a day, for 3 weeks.

[0308] Blood sugar test: At the end of the experiment, all animals were fasted for 16 hours and their fasting blood sugar was tested. Then, the animals were given starch solution by gavage at a dose of 2g / kg and their blood sugar levels were tested 1 hour and 2 hours after the meal.

[0309] 8.2 Experimental results

[0310] The experimental results can be seen in Table 15. Compared with the blank group, the fasting blood glucose, blood glucose levels at 1h and 2h after meal in the model group were significantly increased (p<0.01). Compared with the model group, the fasting blood glucose, blood glucose levels at 1h and 2h after meal in the acarbose group, AM06 group, and acarbose+AM06 group were reduced to varying degrees (p<0.05 or p<0.01), and the acarbose+AM06 group was more able to reduce fasting blood glucose and postprandial blood glucose than the acarbose group (p<0.05).

[0311] Table 15. Blood glucose test results of each group of animals

[0312]

[0313] Note 1: * indicates that compared with the model group, the difference is significant p<0.05, ** indicates that compared with the model group, the difference is extremely significant p<0.01; Note 2: a indicates that compared with the acarbose group, the difference is significant p<0.05, aa indicates that compared with the acarbose group, the difference is extremely significant p<0.01.

[0314] The technical features of the above-mentioned embodiments and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0315] The above embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solution provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Use of Akkermansia muciniphila in the preparation of a pharmaceutical composition for preventing and treating diabetes, It is characterized in that The pharmaceutical composition contains a therapeutically effective amount of the Akkermansia muciniphila; The Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two; wherein, The Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 28, 2021, with the deposit number of CGMCC No.22793; The Akkermansia muciniphila AM02 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No.22794.

2. The use according to claim 1, It is characterized in that The Akkermansia muciniphila AM06 and the Akkermansia muciniphila AM02 are each independently a live bacterium, an inactivated bacterium, or a combination of live and inactivated bacteria.

3. The use according to claim 1, It is characterized in that The diabetes mellitus includes at least one of type 1 diabetes mellitus, type 2 diabetes mellitus and gestational diabetes mellitus.

4. The use according to claim 1, It is characterized in that The Akkermansia muciniphila can lower blood sugar, reduce insulin resistance, and reduce insulitis.

5. The use according to claim 1, It is characterized in that The pharmaceutical composition comprises the Akkermansia muciniphila and a pharmaceutically acceptable carrier.

6. The use according to claim 5, It is characterized in that The pharmaceutical composition is a medicine, and its dosage form is tablets, capsules, granules, pills, ointments, solutions, suspensions, emulsions, creams, sprays, drops, patches or tube feeding preparations.

7. Use of a composition containing Akkermansia muciniphila in the preparation of a medicine for preventing and treating diabetes, It is characterized in that The Akkermansia muciniphila is as defined in claim 1 or 2; wherein the medicament contains a therapeutically effective amount of the Akkermansia muciniphila.

8. The use according to claim 7, It is characterized in that The drug for preventing and treating diabetes further comprises a second active ingredient, and the second active ingredient at least comprises an α-glucosidase inhibitor.

9. The use according to claim 8, It is characterized in that The α-glucosidase inhibitor is selected from one or more of acarbose, voglibose and miglitol.

10. The use according to claim 7, It is characterized in that The composition containing Akkermansia muciniphila is a composite probiotic, and the composite probiotic also includes probiotics different from the Akkermansia muciniphila.

11. A composition comprising Akkermansia muciniphila, It is characterized in that The Akkermansia muciniphila is as defined in claim 1 or 2; The composition containing Akkermansia muciniphila is a pharmaceutical composition, which further comprises a second pharmaceutical active ingredient, wherein the second pharmaceutical active ingredient at least comprises an α-glucosidase inhibitor.

12. The composition according to claim 11, It is characterized in that The α-glucosidase inhibitor is selected from one or more of acarbose, voglibose and miglitol.

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