Akkermansia muciniphila and its use for the prevention of non-alcoholic steatohepatitis

Abstract

The present invention provides Akkermansia muciniphila LWHK0003, which has the effect of preventing non-alcoholic steatohepatitis, as well as anti-obesity effects, prevention of GAN diet-induced NASH, maintenance of blood sugar stability, and anti-inflammatory functions.

Description

[Technical Field]

[0001] The present disclosure relates to Akkermansia muciniphila and uses thereof, and in particular to Akkermansia muciniphila and its use for the prevention of non-alcoholic steatohepatitis. [Background technology]

[0002] Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease, and if not properly treated, it can progress to non-alcoholic steatohepatitis (NASH), leading to irreversible liver cirrhosis and hepatocellular carcinoma. The progression of NASH is closely related to metabolic syndrome and intestinal microbiota imbalances caused by unhealthy diets.

[0003] Probiotics are active microorganisms that coexist with the host and, when properly ingested, can have a beneficial effect on the host's health. Traditional probiotics, such as lactobacilli and bifidobacteria, are widely used and are safe and well tolerated by the human body, but their effects on the human intestinal microorganisms are still limited, and there are significant differences in the effects and mechanisms of action between different strains.

[0004] Therefore, how to provide probiotics that can effectively prevent and treat NASH has become an area that needs improvement in the prior art. Summary of the Invention [Problem to be solved by the invention]

[0005] One embodiment of the present disclosure provides Akkermansia muciniphila LWHK0003, deposited at the German Collection of Microbial Cell Cultures DSMZ under accession number DSM 35051. [Means for solving the problem]

[0006] Another embodiment of the present disclosure provides a composition for preventing non-alcoholic steatohepatitis, comprising the Akkermansia muciniphila LWHK0003 and a pharmaceutically acceptable carrier.

[0007] In some embodiments, Akkermansia muciniphila LWHK0003 is killed.

[0008] In some embodiments, the killed bacteria are killed by pasteurization.

[0009] Another embodiment of the present disclosure provides a use of Akkermansia muciniphila LWHK0003 for the manufacture of a medicament or health food for preventing non-alcoholic steatohepatitis.

[0010] In some embodiments, the strain is live, killed, or a combination thereof.

[0011] In some embodiments, the bacterial strain is capable of preventing obesity, high fat, high liver triglyceride content, high nonalcoholic fatty liver disease activity score, high blood triglycerides, or a combination thereof associated with nonalcoholic steatohepatitis.

[0012] In some embodiments, the strains are capable of preventing high fat, including body fat or visceral fat, or a combination thereof, associated with non-alcoholic steatohepatitis.

[0013] In some embodiments, the bacterial strain is capable of preventing high non-alcoholic fatty liver disease activity scores, including hepatocellular ballooning, hepatitis, or a combination thereof, associated with non-alcoholic steatohepatitis.

[0014] In some embodiments, the drug or health food is in the form of a capsule, tablet, powder, or liquid.

[0015] In some embodiments, the drug or health food is prepared for oral administration. [Brief explanation of the drawings]

[0016] Various aspects of the present disclosure will be readily understood by reading the following detailed description in conjunction with the accompanying drawings. It should be noted that, in accordance with standard industry practice, various features may not be drawn to scale. In fact, the dimensions of various features may be arbitrarily increased or decreased for clarity of discussion. To enable a clearer understanding of the above and other objects, features, advantages, and embodiments of the present disclosure, the accompanying drawings are described as follows:

[0017] [Figure 1A] 1 shows the body weight of mice at 8 weeks according to one embodiment of the present disclosure (statistics were analyzed using t-test). [Figure 1B] 1 shows the change in mouse body weight over an 8-week period (statistics performed using t-test) according to one embodiment of the present disclosure. [Figure 2A] 1 shows the weight gain of mice at week 8 according to one embodiment of the present disclosure (statistics processed using t-test). [Figure 2B] 1 shows the change in weight gain of mice over an 8-week period (statistics performed using t-test) according to one embodiment of the present disclosure. [Figure 3] 1 shows relative adipose tissue weights of mice, including subcutaneous adipose tissue (SAT), epididymal adipose tissue (EAT), perirenal adipose tissue (PRAT), and brown adipose tissue (BAT) weights (statistics performed using t-test), according to one embodiment of the present disclosure. [Figure 4] 1 shows the content of triglycerides (TG) in the liver of a mouse according to one embodiment of the present disclosure (statistical analysis using t-test). [Figures 5A-5C] 1 shows a pathology section of mouse liver tissue according to an embodiment of the present disclosure. [Figure 6A] 1 shows the steatosis scores of the livers of mice according to one embodiment of the present disclosure (statistics using the Mann-Whitney U test). [Figure 6B] 1 shows hepatocyte ballooning scores for mice according to one embodiment of the present disclosure (statistics using the Mann-Whitney U test). [Figure 6C] 1 shows inflammation scores of mouse liver tissue according to one embodiment of the present disclosure (statistics using Mann-Whitney U test). [Figure 6D] 1 shows a pathological section of mouse liver tissue and non-alcoholic fatty liver disease activity score (NAFLD activity score; NAS) according to one embodiment of the present disclosure (statistics using the Mann-Whitney U test). [Figure 7A] 1 shows the blood triglyceride content of mice according to one embodiment of the present disclosure (t-test). [Figure 7B] 1 shows blood cholesterol content of mice according to one embodiment of the present disclosure (statistics processed using t-test). [Figure 8] 1 shows postprandial blood glucose levels of mice according to one embodiment of the present disclosure (statistics processed using t-test). [Figure 9A] 1 shows an oral glucose tolerance test (OGTT) of a mouse according to one embodiment of the present disclosure (statistics processed using t-test). [Figure 9B] 1 shows the area under the curve (AUC) of blood glucose in mice according to one embodiment of the present disclosure (statistical analysis by t-test). DETAILED DESCRIPTION OF THE INVENTION

[0018] To make the description of the present disclosure more detailed and complete, the following provides illustrative descriptions of embodiments and specific examples of the present disclosure, but these are not the only ways to implement or use the specific examples of the present disclosure. The examples disclosed below may be combined with or substituted for each other when beneficial, and one example may be added to another example without further description or explanation. In the following description, numerous specific details are described in detail to enable readers to fully understand the following examples. However, the examples of the present disclosure can be practiced without these specific details.

[0019] As used herein, "a," "an," and "the" generally refer to one or more, unless the context otherwise limits the use of the articles. It will be further understood that, as used herein, "comprises," "includes," "having," and similar terms refer to the features, regions, integers, steps, operations, elements, and / or assemblies described therein, but do not exclude the above or additional one or more other features, regions, integers, steps, operations, elements, assemblies, and / or combinations thereof.

[0020] The Akkermansia muciniphila LWHK0003 strain of the present disclosure has been deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute Foundation under accession number BCRC911209. The Akkermansia muciniphila LWHK0003 strain of the present disclosure has also been deposited at the German Collection of Microbial Cell Cultures DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) under accession number DSM 35051, deposited on June 14, 2024.

[0021] The presently disclosed Akkermansia muciniphila LWHK0003 is a highly anaerobic bacterium isolated from the feces of a healthy human. The strain is oval in shape, approximately 0.4 to 0.6 microns in diameter, and approximately 0.6 to 1 micron in length. Live Akkermansia muciniphila bacteria of the present disclosure are cultured in brain heart infusion broth (BHI broth) in an anaerobic environment at 37°C.

[0022] In some specific embodiments of the present disclosure, the strain is administered to an individual orally or parenterally, hi some specific embodiments of the present disclosure, the strain is administered to an individual in an oral dosage form selected from the group consisting of a solution, a suspension, an emulsion, a powder, a tablet, a pill, a syrup, a lozenge, a tablet, a chewing gum, and a capsule.

[0023] In some embodiments, pharmaceutically acceptable carriers include water, alcohols, glycols, preserving agents, antioxidants, solvents, emulsifiers, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption enhancers, active agents, humectants, odor absorbers, fragrances, pH adjusting agents, occlusive agents, emollients, thickeners, solubilizing agents, and the like. These include, but are not limited to, agents, penetration enhancers, anti-irritants, colorants, propellants, surfactants, and other similar or compatible carriers of the present invention.

[0024] As used herein, the term "GAN diet" refers to Gubra-Amylin NASH. Previously, a high-fat diet known as the American lifestyle-induced obesity syndrome (ALIOS) diet was shown to promote experimental NASH. Hansen et al. modified it, naming it the Amylin liver NASH (AMLN) diet, which reliably induces NASH-related metabolic biochemistry and liver pathology in C57BL / 6J mice (AMLN diet-induced NASH; AMLN DIO NASH) and ob / ob mice lacking the receptor (AMLN ob / ob-NASH). However, in 2018, the FDA banned the use of trans fatty acids as a food additive, leading to the development of the Gubra-Amylin NASH (GAN) diet, which is similar to the AMLN diet but lacks trans fatty acids. Specifically, the GAN diet is a diet #D09100310i (Source: Research Diets, INC.) that contains 40 kcal of fat, 20 kcal of fructose, and 2% cholesterol.

[0025] The following provides some examples and experimental examples to more clearly explain the Akkermansia muciniphila of the present disclosure and its use for preventing non-alcoholic steatohepatitis, but these are merely illustrative and are not intended to limit the present invention. The scope of protection of the present disclosure should be determined by the following appended claims.

[0026] Example

[0027] Experimental group

[0028] C57BL / 6 male mice were divided into three groups and all were administered the GAN diet. The positive control group (hereafter referred to as BAA-835) received 1 × 10 9The experimental group (hereafter referred to as LWHK03) was administered 1 × 10 pasteurized Akkermansia muciniphila (ATCC BAA-835) in 200 μL of PBS containing 2.5% glycerol at a dose of CFU / 200 μL. In addition to the GAN diet, the experimental group (hereafter referred to as LWHK03) was also administered 1 × 10 pasteurized Akkermansia muciniphila (ATCC BAA-835) in a tube per mouse daily. 9 Mice were administered pasteurized Akkermansia muciniphila (LWHK0003) at a concentration of CFU / 200 μL (PBS containing 2.5% glycerol). The control group (Con) received 200 μL of PBS containing 2.5% glycerol via tube daily in addition to the GAN diet. The volume was the same as that of the positive control and experimental groups. Body weight, food intake, and water intake were recorded weekly. An oral glucose tolerance test (OGTT) was performed at week 7, and mice were sacrificed at week 8 to collect blood, multiple fat samples, and liver samples.

[0029] Example 1 Weight Gain

[0030] The experimental groups were as described above, and body weights were recorded from week 0 to week 8. The results are shown in Figures 1A and 1B. At week 8, the average body weight of the control group mice was 31.17 grams, while the average body weight of the LWHK03 group mice with GAN diet-induced NASH alleviation was 29.36 grams, demonstrating superior efficacy compared to the BAA-835 group, which had an average body weight of 30.16 grams.

[0031] The results are shown in Figures 2A and 2B. At week 8, the average weight gain of mice in the LWHK03 group with GAN diet-induced NASH suppression was 6.48 grams, a superior effect to the average weight gain of the BAA-835 group (7.32 grams), compared with the average weight gain of mice in the control group (8.64 grams).

[0032] Example 2 Fat Weight

[0033] The experimental groups were as described above, and mice were sacrificed at week 8 to collect fat samples from multiple sites. The results are shown in Figure 3. At week 8, the average weight of subcutaneous fat in the control group was 1.34 grams, while the average weight of subcutaneous fat in the LWHK03 group was significantly reduced to 1.07 grams, a more effective treatment than the average weight of 1.1 grams in the BAA-835 group. Furthermore, the average weight of perirenal fat in the control group was 0.54 grams, while the average weight of perirenal fat in the LWHK03 group was significantly reduced to 0.41 grams, a more effective treatment than the average weight of 0.45 grams in the BAA-835 group.

[0034] Example 3 Liver triglyceride content

[0035] The experimental groups were as described above. At week 8, mice were sacrificed and liver tissue was collected. The triglyceride / liver weight ratio was calculated by dividing the liver triglyceride levels by the liver weight. The results are shown in Figure 4. After 8 weeks of GAN diet administration, the mean triglyceride / liver weight ratio in the control group was 81.39 μg / mg, while the mean triglyceride / liver weight ratio in the LWHK03 group was significantly reduced to 66.6 μg / mg, a significantly better effect than the mean value of 68.57 μg / mg in the BAA-835 group.

[0036] Example 4 Liver pathology sections and liver pathology scores

[0037] The experimental groups were as described above. At week 8, mice were sacrificed and liver tissue samples were collected. For section staining (Figures 5A-5C) and interpretation of the results, a veterinary pathologist calculated scores based on the severity of pathological features, such as fatty degeneration of the liver, hepatocellular ballooning, and liver tissue inflammation, based on the 2005 paper by Kleiner et al. (Figures 6A-6C). Based on the 2005 paper by Kleiner et al., scores for each NAS indicator and a total score were calculated (Figure 6D). A maximum score of 8 was reached, and a total score of ≥ 5 was considered a diagnosis of NASH. The higher the score, the more severe the NASH condition.

[0038] As can be seen from the results, there was no significant difference in steatosis between the groups after 8 weeks of GAN diet administration (Figure 6A). Regarding the hepatocellular ballooning index (Figure 6B), of the 8 mice in the control group, 1 received a score of 1 and 7 received a score of 2. Of the 12 mice in the LWHK03 group, 2 received a score of 0, 5 received a score of 1, and 5 received a score of 2. Compared to the control group, the degree of hepatocellular ballooning in the LWHK03 group was significantly lower, and the effect was superior to that of the 12 mice in the BAA-835 group, where 3 received a score of 0 and 9 received a score of 2. Regarding the inflammatory response index (Figure 6C), of the 8 mice in the control group, 3 received a score of 0 and 5 received a score of 1. Of the 12 mice in the LWHK03 group, 6 received a score of 0 and 6 received a score of 1. Compared with the control group, the degree of inflammatory response in the LWHK03 group was significantly lower, and the effect was superior to that of the 12 mice in the BAA-835 group, where 1 mouse scored 0, 9 mice scored 1, and 2 mice scored 2. Regarding the NAS index (Figure 6D), of the 8 mice in the control group, 4 mice scored a total score of 5 and 4 mice scored a total score of 6. Of the 12 mice in the LWHK03 group, 1 mouse scored a total score of 3, 3 mice scored a total score of 4, 6 mice scored a total score of 5, and 2 mice scored a total score of 6. Compared with the control group, the NAS index in the LWHK03 group was significantly lower, and the effect was superior to that of the 12 mice in the BAA-835 group, where 1 mouse scored a total score of 2, 1 mouse scored a total score of 3, 1 mouse scored a total score of 4, 7 mice scored a total score of 6, and 2 mice scored a total score of 7.

[0039] Example 5 Blood lipid analysis

[0040] The experimental groups were as described above, and mice were sacrificed at week 8 for blood collection. The results are shown in Figure 7A. After 8 weeks of GAN diet administration, the mean blood triglyceride level in the control group was 126.98 mg / dL, while the mean blood triglyceride level in the LWHK03 group was 94.92 mg / dL, a significant decrease compared with the mean blood triglyceride level of the BAA-835 group (131.17 mg / dL). There was no significant difference in blood cholesterol levels among the three groups (Figure 7B).

[0041] Example 6 Blood Glucose Analysis

[0042] The experimental groups were as described above, and blood samples were taken after a 6-hour fast on week 7 of the experiment. The results are shown in Figure 8, and there was no significant difference in blood glucose levels after fasting among the three groups.

[0043] Example 7 Oral Glucose Tolerance Test (OGTT)

[0044] The experimental groups were as described above, and an oral glucose tolerance test (OGTT) was performed at week 7. The results are shown in Figure 9A, which shows that there were no significant differences in blood glucose levels among the three groups at different time points. Furthermore, analysis of the area under the curve (AUC) of blood glucose (Figure 9B) revealed that the control group had an average AUC of 28,418, while the LWHK03 group (with an average AUC of 30,390) improved blood glucose control, which was superior to the BAA-835 average AUC of 30,577.

[0045] The Akkermansia muciniphila LWHK0003 strain of the present disclosure has the functions of preventing GAN diet-induced obesity, reducing weight gain, reducing fat accumulation (e.g., subcutaneous fat, epididymal fat, and perirenal fat), reducing liver triglyceride content, preventing GAN diet-induced NASH (e.g., reducing hepatocellular balloon degeneration and hepatic inflammatory responses), reducing blood triglycerides, and maintaining constant blood glucose. Furthermore, compared to the pasteurized strain of ATCC BAA-835, the Akkermansia muciniphila LWHK0003 strain (pasteurized) of the present disclosure has superior efficacy.

[0046] Although the present disclosure has been disclosed in the above embodiments, the present disclosure is not limited thereto, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure should be determined by the appended claims.

[0047] [Deposit of biological materials]

[0048] Akkermansia muciniphila LWHK0003 of the present disclosure is deposited with the German Collection of Microbial Cell Cultures DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) at Inhoffenstrasse 7B, 38124 Braunschweig, Germany) under the accession number DSM 35051 on the date of deposit June 14, 2024.

Claims

1. Akkermansia muciniphila LWHK0003, deposited at the German Collection of Microbial Cell Cultures DSMZ under accession number DSM 35051.

2. A composition for preventing non-alcoholic steatohepatitis, comprising: Akkermansia muciniphila LWHK0003 according to claim 1; a pharmaceutically acceptable carrier; A composition comprising:

3. The composition according to claim 2, wherein the Akkermansia muciniphila LWHK0003 is a killed bacterium.

4. The composition of claim 3, wherein the killed bacteria are killed by pasteurization.

5. Use of Akkermansia muciniphila LWHK0003 according to claim 1 for the manufacture of a drug or health food for preventing non-alcoholic steatohepatitis.

6. The use according to claim 5, wherein the bacterial strain is live, killed, or a combination thereof.

7. 6. The use of claim 5, wherein the strain is capable of preventing obesity, high fat, high liver triglycerides, high nonalcoholic fatty liver disease activity score, high blood triglycerides, or a combination thereof associated with nonalcoholic steatohepatitis.

8. 8. The use according to claim 7, wherein the strain is capable of preventing hyperlipidemia, including body fat or visceral fat or a combination thereof, associated with non-alcoholic steatohepatitis.

9. The use of claim 7, wherein the strain is capable of preventing high activity scores of non-alcoholic fatty liver disease, including hepatocellular ballooning, hepatic inflammatory response, or a combination thereof, associated with non-alcoholic steatohepatitis.

10. The use according to claim 5, wherein the drug or health food is in the form of a capsule, tablet, powder, or liquid.

11. The use according to claim 5, wherein the drug or health food is prepared for oral administration.

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