KR102271821B1 - Composition Comprising Orlistat and Akkermansia muciniphila EB-AMDK19 - Google Patents

Abstract

The present invention relates to a composition comprising orlistat and Akkermansia muciniphila. When the composition of the present invention is administered, it is possible to prevent or treat obesity, metabolic diseases, and inflammatory diseases, and it is possible to alleviate or prevent side effects caused by orlistat.

Description

Composition Comprising Orlistat and Akkermansia muciniphila EB-AMDK19 Strains {Composition Comprising Orlistat and Akkermansia muciniphila EB-AMDK19}

The present invention relates to a composition comprising Orlistat and the Akermansia muciniphila EB-AMDK19 strain.

It is reported that about 300 million adults worldwide are obese, and about 1.7 billion people are overweight. According to a report by the Samsung Economic Research Institute, the direct and indirect social costs caused by obesity among adults in Korea in 2011 are estimated to be close to KRW 3.4 trillion. It is said that the risk of dyslipidemia is more than doubled, increasing health care expenditure. In the United States, as of 2006, obese people paid 42% more for medical expenses than normal-weight people, and it is known that more than 80% of diabetic patients and 69% of nonalcoholic fatty liver patients are obese. . In addition, since mast cells secrete inflammatory signaling factors, obese people have a higher risk of developing inflammatory diseases than normal people.

A typical example of a treatment for obesity is orlistat, a fat absorption inhibitor. However, orlistat inhibits the absorption of fat-soluble vitamins and has side effects such as fatty stools, frequent bowel movements, fecal incontinence, and acute renal failure.

Therefore, a composition capable of suppressing or alleviating these side effects or a therapeutic agent for obesity without such side effects will be needed, and there is an urgent need to develop a therapeutic agent that can help metabolic and inflammatory diseases, rather than just preventing obesity.

Republic of Korea Patent Publication No. 10-2008-0102832 (November 26, 2008) Republic of Korea Patent Publication No. 10-2128287 (2020.06.24)

One aspect is to provide a pharmaceutical composition for preventing and treating obesity, including Orlistat (Orlistat) and Akkermansia muciniphila (Akkermansia muciniphila).

Another aspect is to provide a pharmaceutical composition for preventing and treating metabolic diseases, including Orlistat and Akkermansia muciniphila.

Another aspect is to provide a pharmaceutical composition for preventing and treating inflammatory diseases, including Orlistat and Akkermansia muciniphila.

As used herein, the term "prevention" refers to any action of suppressing or delaying the onset of obesity, metabolic disease, or inflammatory disease by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to temporarily or permanently alleviating the symptoms of obesity, metabolic disease, or inflammatory disease by administering the pharmaceutical composition according to the present invention, eliminating the cause of the symptom, or the disease or means preventing or delaying the onset of symptoms of a condition.

One aspect is to provide a pharmaceutical composition for preventing and treating obesity, comprising Orlistat and Akkermansia muciniphila.

The molecular formula of the orlistat is C ₂₉ H ₅₃ NO ₅ , and the chemical formula is the same as in Chemical Formula 1 below.

[Formula 1]

The Akermansia muciniphila strain may be monococci or dicocci with oval cells having a size of 0.5-1 μm. The Akermansia muciniphila strain may be anaerobic, non-motile, Gram-negative, non-endospore-forming, mucin-degrading bacteria.

In one embodiment, the Akermansia muciniphila may be an Akermansia muciniphila EB-AMDK19 strain. For the characteristics of the Akermansia muciniphila EB-AMDK19 strain, reference may be made to Korean Patent Publication No. 10-2128287.

The state of Akkermansia muciniphila may be a liquid state or a dry state, and the drying method may include ventilation drying, natural drying, spray drying and freeze drying, but is not limited thereto.

In one embodiment, the composition may be to alleviate the side effects of the orlistat inhibitor.

In one embodiment, alleviating the side effects may be effective in alleviating one or more symptoms of abdominal bloating, fatty stools, inflammation in the kidneys, and fibrosis in the kidneys. The alleviation may be to improve or suppress symptoms appearing as side effects.

In an embodiment of the present invention, when the orlistat and Akermansia muciniphila EB-AMDK19 strain were co-administered to mice fed a high-fat diet, intestinal distension was alleviated compared to mice administered with orlistat, and the stool was neutral It was confirmed that the fat was reduced, and it was confirmed that inflammation and fibrosis in the kidney were reduced.

The composition may exhibit a more pronounced anti-obesity effect than the single effect of orlistat or Akermansia muciniphila EB-AMDK19 strain.

In an embodiment of the present invention, when the orlistat and Akermansia muciniphila EB-AMDK19 strain were co-administered to mice fed a high-fat diet, body weight, liver, It was confirmed that the increase rate of spleen, mesenteric fat, and subcutaneous fat decreased.

The pharmaceutical composition may be orlistat and Akkermansia muciniphila EB-AMDK19 strains separately ingested in each form, or dissolved in a solvent and ingested together, but is not limited thereto.

The pharmaceutical composition used in the present invention should be in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level may vary depending on the type and severity of the subject, age, sex, It can be determined by the type of virus infected, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including concurrent drugs, and other factors well known in the medical field. An effective amount may vary depending on the route of treatment, the use of excipients, and the potential for use with other agents, as will be appreciated by those skilled in the art.

The pharmaceutical compositions of the present invention may be formulated into pharmaceutical formulations using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. In the preparation of formulations, it is preferable to mix or dilute the active ingredient with a carrier, or to enclose the active ingredient in a carrier in the form of a container.

Accordingly, the pharmaceutical composition of the present invention may be used in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., depending on the method, and suitable carriers and excipients commonly used in the preparation of the composition or a diluent may be further included.

For example, carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, all, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it can be prepared using a diluent or excipient such as a filler, a weight agent, a binder, a wetting agent, a disintegrant, a surfactant, etc. normally used.

Another aspect is to provide a pharmaceutical composition for preventing and treating metabolic diseases comprising orlistat and Akkermansia muciniphila.

The description of orlistat, Akkermansia muciniphila and the pharmaceutical composition are the same as those described above.

In one embodiment, the Akermansia muciniphila may be an Akermansia muciniphila EB-AMDK19 strain. For the characteristics of the Akermansia muciniphila EB-AMDK19 strain, reference may be made to Korean Patent Publication No. 10-2128287.

In one embodiment, the metabolic disease may be hyperlipidemia, fatty liver, diabetes, hypertension, hypercholesterolemia.

In an embodiment of the present invention, when orlistat and Akermansia muciniphila EB-AMDK19 strain are co-administered to mice, blood insulin concentration, GTP concentration, and liver triglyceride and liver cholesterol concentration are lowered by Orlistat or Akkermansia mu It was confirmed that the Siniphila EB-AMDK19 strain was more significantly reduced than that of the single administration. In addition, when Orlistat and Acermansia muciniphila EB-AMDK19 strain were administered in combination to mice, glucose tolerance was improved as much as that of mice on a normal diet, compared to administration of Orlistat or Akkermansia muciniphila EB-AMDK19 strain alone. Confirmed.

Another aspect is to provide a pharmaceutical composition for preventing and treating inflammatory diseases comprising orlistat and Akkermansia muciniphila.

Descriptions of orlistat, Akkermansia muciniphila and the pharmaceutical composition are the same as those described above, and thus are omitted to avoid excessive duplication of the specification.

In one embodiment, the Akermansia muciniphila may be an Akermansia muciniphila EB-AMDK19 strain. For the characteristics of the Akermansia muciniphila EB-AMDK19 strain, reference may be made to Korean Patent Publication No. 10-2128287.

In one embodiment, the inflammatory disease may be colitis, nephritis, hepatitis.

In an embodiment of the present invention, when Orlistat and Akermansia muciniphila EB-AMDK19 strain were co-administered to mice, more inflammatory cytokines than mice administered with Orlistat or Akkermansia muciniphila EB-AMDK19 strain alone It was confirmed that the levels of IL-6, TLR2, and TLR4 were further significantly reduced.

In an embodiment of the present invention, when Orlistat and Akermansia muciniphila EB-AMDK19 strain were co-administered to mice, intrahepatic fibrosis compared to mice administered with Orlistat or Akermansia muciniphila EB-AMDK19 strain alone was confirmed to decrease.

In an example of the present invention, when orlistat and Akermansia muciniphila EB-AMDK19 strain were co-administered to mice, it was confirmed that inflammation was reduced in nephritis caused by orlistat.

Another aspect is to provide a method for treating obesity comprising administering to a subject a composition comprising orlistat and Akermansia muciniphila.

Another aspect is to provide a method of treating a metabolic disease comprising administering a composition to a subject comprising orlistat and Akermansia muciniphila.

In one embodiment, the metabolic disease may be hyperlipidemia, fatty liver, diabetes, hypertension, hypercholesterolemia.

Another aspect is to provide a method for treating an inflammatory disease comprising administering to a subject a composition comprising orlistat and Akermansia muciniphila.

In one embodiment, the inflammatory disease may be colitis, nephritis, hepatitis.

When the composition of the present invention is administered, there is an effect of preventing or treating obesity, metabolic diseases and inflammatory diseases. In addition, administration of the composition of the present invention can alleviate or prevent side effects caused by orlistat.

In the figure, Normal is normal diet group, HFD is obesity induction group, Orlistat is orlistat administered group, EB-AMDK19 is Akermansia muciniphila EB-AMDK19 strain administered group, ORL+EB-AMDK19 is orlistat and Akkermansia. It refers to the group administered with the Muciniphila EB-AMDK19 strain.
1A is a graph showing the change in weight of each group, and B is a graph showing the weight increase rate of each group after 12 weeks.
2A is data showing the amount of food intake of each group, and B is data showing the calories consumed by each group.
3 is data comparing the weights of livers of each group.
4 is data comparing the weight of the spleen of each group.
5 is data showing the difference in weight of mesenteric fat in A and subcutaneous fat in each group.
6 is a graph comparing the glucose tolerance of each group.
7 is a graph comparing the glucose tolerance in each group, where A is the measurement of blood glucose immediately before glucose administration and B is 30 minutes after glucose administration.
8 is data comparing the concentration of insulin in each group.
9 is data comparing GPT concentrations in each group.
10 is data obtained by analyzing lipid biochemical indicators in the liver in each group, where A is liver triglyceride and B is data comparing total cholesterol in the liver.
11 is data confirming the expression concentration of LPL, an adipocyte-related factor, in the liver of each group.
12 is data confirmed by staining the degree of fat accumulation in the liver.
13 is data confirmed by staining the degree of fibrosis in the liver.
14 is data comparing the expression of IL-6 in the intestinal mucosa of each group.
15 is data comparing the expression of TLR2 in the intestinal mucosa of each group.
16 is data comparing the expression of TLR4 in the intestinal mucosa of each group.
17 is a photograph comparing the shapes of the small intestine and large intestine of each group.
18 is a graph comparing the amount of triglycerides in the feces of each group
19A is the data obtained by staining whether or not the kidneys in each group are inflammatory, and B is data comparing the inflammation level through the F4/80 antibody in the immunochemical staining graph.
20A is data obtained by staining the degree of fibrosis of each group's kidneys, and B is data comparing the numerical values in a graph.

Hereinafter, one or more specific embodiments will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1. Strain isolation and sample preparation

Acermansia muciniphila EB-AMDK19 (KCTC 13761BP) was isolated from the feces of a healthy Korean (female, 35 years old, BMI 23.3). The isolated Akermansia muciniphila EB-AMDK19 (hereinafter 'EB-AMDK19') was prepared at a concentration of ^{1×10 8 CFU per 150 μl of PBS containing 25% glycerol and 0.05% cysteine.} Orlistat (Xenical, Orlistat 120 mg, Roche, USA) was purchased and used commercially.

Example 2. Preparation of mice for animal experiments and model production

Animal experiments were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC). Experimental animals purchased an 8-week-old male mouse C57BL and had an adaptation period in an environment of 22°C, 40-60% relative humidity, and 12 hours of light-dark cycle for one week. After that, the experiment was conducted for 12 weeks. Experimental animals were randomly divided into 5 groups as shown in Table 1, with 9 animals as one group.

Experimental group I, a normal diet group, was allowed to freely consume 10% fat feed, and other experimental groups other than experimental group I were allowed to consume high-fat feed (60 kcal fat; Research Diets Inc. NJ, USA), and drinking water was freely consumed by all experimental groups. made to do Experimental group III was orally administered with 10 mg/kg (150 μl) of orlistat daily. Experimental group IV received 1×10 ⁸ CFU of live EB-AMDK19, and experimental group V received 1×10 ⁸ CFU of 1×10 8 CFU of live EB-AMDK19 orally, followed by oral administration of 150 μl of orlistat 10 mg/kg and 150 μl of orlistat daily. . In addition, in order to exclude the effect of stress, etc. due to administration, the same amount of phosphate buffered saline (25% glycerol, 0.05% cysteine/PBS) was orally administered to experimental groups I and II daily.

Experimental group I normal diet group Experimental group Ⅱ Experimental diet fed obesity induction group Experimental group Ⅲ Experimental diet fed and orlistat (10 mg/kg, 150 μl) administration group Experimental group IV Experimental diet and EB-AMDK19 (live cells 1×10 ⁸ CFU) administration group Experimental group Ⅴ Experimental diet and EB-AMDK19 (live cells 1×10 ⁸ CFU), orlistat (10 mg/kg, 150 μl) administration group

Example 3. Confirmation of changes in body weight and food intake

The weight and body fat of each experimental group were measured every week while dieting and administration as in Example 2 for 12 weeks, and the degree of body fat increase was measured after 12 weeks.

As a result, as shown in FIG. 1 , compared to the obesity-inducing group that consumed only high-fat feed, body weight was significantly reduced in the group administered with orlistat and EB-AMDK19 strain, the group administered with the EB-AMDK19 strain, and the group administered with orlistat. In addition, in the group administered with orlistat and EB-AMDK19 strain, the weight increase rate after 12 weeks was significantly decreased compared to the weight increase rate of the group administered with orlistat or EB-AMDK19 strain alone.

On the other hand, as shown in Figure 2, the food intake of each group was relatively higher in the normal diet group than in the other groups, but there was no significant difference. In addition, the calorie intake was relatively higher in the other group consuming a high-fat diet compared to the normal diet group, but there was no significant difference.

Therefore, it was confirmed that when orlistat and EB-AMDK19 strain were administered in combination, the rate of increase in weight compared to food intake or calories intake was reduced.

Example 4. Changes in tissue and body fat weight

After 12 weeks, at the end of the experiment, the liver, spleen, mesenteric fat, and subcutaneous fat were extracted under anesthesia with _{CO 2 , washed with physiological saline, water was removed, and the weight was measured.}

As shown in FIG. 3 , it was confirmed that the liver tissue weight of the obesity induction group was significantly increased compared to the liver tissue weight of the normal diet group, and although a high fat diet was consumed as in the obesity induction group, Orlistat, EB-AMDK19 strain, Orly It was confirmed that the liver tissue weight was reduced in the group administered with the start and EB-AMKD19 strains compared to the obesity induction group.

In addition, as shown in FIG. 4 , it was confirmed that the weight of the spleen tissue of the obesity induction group was significantly increased compared to the weight of the spleen tissue of the normal diet group. It was confirmed that the group that consumed a high-fat diet and administered orlistat slightly decreased compared to the obesity-inducing group, but it was not significantly decreased. On the other hand, it was confirmed that the spleen tissue weight was significantly reduced in the group that consumed a high-fat diet as in the obesity-inducing group but administered the EB-AMDK19 strain, orlistat and EB-AMKD19 strains compared to the obesity-inducing group.

In addition, as shown in FIG. 5 , it was confirmed that the mesenteric fat and subcutaneous fat weights of the obesity-inducing group were significantly increased compared to the mesenteric fat and subcutaneous fat weights of the normal diet group. It was confirmed that the group that consumed a high-fat diet and administered orlistat slightly decreased compared to the obesity-inducing group, but it was not significantly decreased. On the other hand, it was confirmed that the mesenteric fat and subcutaneous fat weights were significantly reduced in the group administered with the EB-AMDK19 strain, orlistat and EB-AMKD19 strains despite the high fat diet as in the obesity induction group compared to the obesity induction group. In addition, it was confirmed that the weight of mesenteric fat and subcutaneous fat was significantly reduced in the group administered with orlistat and the EB-AMKD19 strain than the group administered with the EB-AMDK19 strain alone.

Example 5. Measurement of changes in glucose tolerance

In order to confirm the effect of the administration of the Acermacia municipilla EB-AMDK19 strain on glucose tolerance, 12 weeks after the experiment was performed, the mice were fasted for 18 hours and glucose (Glucose 2 g/kg) was orally administered. Blood was collected from the tail vein immediately before glucose administration and 30, 60, 90 and 120 minutes after administration, and blood glucose was measured with a glucometer.

As shown in FIGS. 6 and 7 , the amount and level of increase in blood glucose level was lower in all administration groups than in the obesity induction group 30 minutes after glucose administration, and among them, in the group administered with orlistat and the group administered with orlistat and EB-AMKD19 strain, It was confirmed that the blood glucose level was significantly lower, similar to that of the normal group.

Example 6. Analysis of blood lipid biochemical indicators

After fasting for 18 hours in Example 5, blood was collected from each experimental animal and serum was separated. Insulin levels and glutamic pyruvic transaminase (GPT), an indicator of liver function, were measured with the isolated plasma. Insulin concentration was measured using an insulin ELISA kit (Morinaga, Japan), and GPT concentration was quantified using an individual measurement kit purchased from Asan Pharmaceutical.

As shown in FIG. 8 , it was confirmed that the insulin level was increased in the obesity induction group compared to the normal group, and the insulin level of the other experimental groups, orlistat, EB-AMDK19 strain, orlistat and EB-AMKD19 strain, was administered to the group. It was confirmed that the values of the normal group were significantly reduced.

In addition, as shown in FIG. 9 , it was confirmed that the GPT level was increased in the obesity induction group compared to the normal group. On the other hand, it was confirmed that the group administered with orlistat, EB-AMDK19 strain, orlistat and EB-AMKD19 strain decreased to a lower level than that of the normal group.

Example 7. Analysis of lipid biochemical indicators in the liver

After all experiments were completed, the liver was removed from the mouse, and the levels of triglyceride (TG) and total cholesterol (TC) in the liver were analyzed. 120 μl of PBS was added to 30 mg of liver tissue, and after grinding using a homogenizer, 320 μl of chloroform and 160 μl of methanol were added to make a mixture. The mixture was mixed in a shaking incubator at room temperature for one day, centrifuged at 2000 rpm, and only the supernatant was separated and the solvent was evaporated. After that, the supernatant obtained by evaporating the solvent was dissolved in 1 ml of isopropanol, and then confirmed using a TG and TC measuring kit (Asan Pharmaceutical, Korea).

As shown in FIG. 10 , it was confirmed that triglyceride and total cholesterol levels were increased in the obesity induction group compared to the normal group. On the other hand, it was confirmed that the group administered with orlistat, EB-AMDK19 strain, orlistat and EB-AMKD19 strain decreased significantly compared to the obesity induction group. In addition, it was confirmed that the concentrations of total cholesterol and triglycerides in the liver were the lowest in the group administered with orlistat and EB-AMKD19 strains. Therefore, it was confirmed that administration of orlistat and EB-AMKD19 strain helps to reduce hepatocellular damage and improve glucose and lipid metabolism for obesity treatment.

Example 8. Analysis of anti-obesity and inflammatory markers in the liver

In order to evaluate the effect on the expression of steatosis-related proteins in the liver, real-time PCR was performed with the primers in Table 2 below to analyze the mRNA expression of LPL, a fatty liver-related mediator. RNA was extracted from the cell monolayer using TRI reagent (Sigma, USA) according to the manufacturer's instructions, and cDNA was synthesized from 1 μg of total RNA using the M-MLV cDNA synthesis kit (Enzynomics, Korea). PCR is a Quant Studio 3 real time PCR system (Applied Biosystems, USA), and pre-incubation (for UDG) is 4 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 15 seconds at 95°C, 1 at 60°C. min. Afterwards, the data were analyzed by delta CT method using the built-in program of QuantStudio Design & Analysis Software v1.4.3.

SEQ ID NO: target primer sequence SEQ ID NO: 1 LPL F: 5'-CTC TGT ATG GCA CAG TGG CT-3' SEQ ID NO: 2 LPL R: 5'-TCC ACC TCC GTG TAA ATC AA-3'

As shown in FIG. 11 , it was confirmed that LPL, a factor related to adipocyte differentiation, was highly expressed in the obesity-induced group compared to the normal group. In all experimental groups except for the obesity induction group, it was confirmed that the expression of adipocyte differentiation-related factors decreased as much as in the normal group. In addition, it was confirmed that the group administered with orlistat and the EB-AMDK19 strain further reduced LPL expression compared to the other groups.

In order to examine the degree of fat accumulation and fibrosis in the liver, the excised liver was made into a tissue section of about 5 μm, embedded in paraffin, and the morphological change was detected by staining with hematoxtlin & eosin (H&E) or Sirius Red. The difference was observed.

As shown in FIG. 12 , it was confirmed that the number of fat cells was significantly increased in the obesity induction group compared to the normal group, and the size of the fat cells was also varied. In addition, in the orlistat-administered group, the number of adipocytes decreased, but the size of adipocytes was larger than that of the normal group. In the case of the group administered with the EB-AMDK19 strain, the size or number of adipocytes decreased compared to the obesity-inducing group, but larger adipocytes were observed compared to the normal diet group. On the other hand, it was confirmed that the number or size of adipocytes decreased in the group administered with orlistat and the EB-AMDK19 strain, similar to the normal diet group.

As shown in FIG. 13 , while fibrosis was increased in the obesity induction group, the degree of fibrosis was decreased in the orlistat-administered group and the EB-AMDK19-administered group compared to the obesity-inducing group. In addition, in the case of the group administered with orlistat and the EB-AMDK19 strain, it was confirmed that fibrosis was significantly reduced compared to the group administered with orlistat or EB-AMDK19 alone.

Example 9. Analysis of anti-inflammatory indicators in the colon

In order to evaluate the effect on the expression of inflammation-related proteins in the colon, using the primers in Table 3 below, real-time PCR was performed. Inflammation-related mediators of the colon were identified IL-6, TLR2, and TLR4.

SEQ ID NO: target primer sequence SEQ ID NO: 3 IL-6 F: 5'-TCC TAC CCC AAT TTC CAA TGC-3' SEQ ID NO: 4 IL-6 R: 5'-CAT AAC GCA CTA GGT TTG CCG-3' SEQ ID NO: 5 TLR2 F: 5'-AAG GAG GTG CGG ACT GTT TC-3' SEQ ID NO: 6 TLR2 R: 5'-GAG CCA AAG AGC TCG TAG C-3' SEQ ID NO: 7 TLR4 F: 5'-CCT CTG CCT TCA CTA CAG AGA CTT T-3' SEQ ID NO: 8 TLR4 R: 5'-TGT GGA AGC CTT CCT GGA TG-3'

As shown in FIGS. 14 to 16 , it was confirmed that the expression of IL-6, TLR2, and TLR4, which are inflammatory factors, increased in the obesity-inducing group compared to the normal group, and in all other experimental groups, the expression of the inflammatory factor compared to the obesity-inducing group It was confirmed that this decreased. In particular, it was confirmed that the group administered with orlistat and EB-AMDK19 strain was further reduced than the group administered with orlistat or EB-AMDK19 strain.

Example 10. Confirmation of orlistat drug side effects prevention effect

Example 10-1. Check changes in the shape of the large intestine and small intestine

In order to confirm the relief of abdominal bloating, oily stool, stool urge, stool incontinence, and intestinal bleeding among the side effects that may occur when taking orlistat, the colon and small intestine tissues of mice of each experimental group were separated and the shape of the intestine was observed.

As shown in FIG. 17 , no intestinal bleeding or intestinal distension was observed in all groups except the group administered with orlistat, but it was confirmed that loose stools and intestinal distension occurred in the group administered with orlistat. Side effects of orlistat when administered in combination with orlistat and EB-AMDK19 strain in that the group administered with the EB-AMDK19 strain did not affect intestinal changes, and loose stools and intestinal bloating were not observed in the group administered with the orlistat and EB-AMDK19 strains. was confirmed to alleviate the

Example 10-2. Measurement of triglyceride content in feces

Orlistat inhibits the action of lipase so that triglycerides are not absorbed and are excreted in feces. Therefore, in order to confirm the relief of steatorrhea seen in feces, which is one of the side effects of orlistat, the concentration of triglycerides in feces observed for 24 hours in mice of each experimental group was observed using an ELISA kit (Asan Pharmaceutical, Korea). .

As shown in FIG. 18 , it was confirmed that triglycerides in feces were increased in the orlistat-administered group, and triglycerides in feces were decreased in all other groups. This confirmed that the EB-AMDK19 strain had no effect on the reduction of fecal triglycerides and that the group administered with orlistat and EB-AMDK19 had an effect of reducing the increased triglycerides in feces, a side effect of orlistat.

Example 10-3. Analysis of anti-inflammatory and fibrotic indicators in the kidney

In order to confirm the degree of alleviation of the side effects of orlistat on the kidneys, nephritis and fibrosis were observed by performing immunohistochemistry (IHC) and Sirius red staining of the kidneys. The kidney tissue isolated from the mouse was embedded in paraffin, and the difference in morphological change was observed through each staining after making about 5 μm tissue sections. The degree of kidney damage through each staining was expressed in terms of percentage (%) through the Image J program.

As shown in FIG. 19 , it was confirmed that inflammation appeared at a high level in the orlistat-administered group, while decreased levels were observed in all other groups compared to the orlistat-administered group. This indicates that a high-fat diet and administration of the EB-AMDK19 strain did not affect the occurrence of kidney inflammation, and that the combined administration of orlistat and the EB-AMDK19 strain had an effect of alleviating the degree of inflammation in the kidney, a side effect of orlistat.

In addition, as shown in FIG. 20 , it was confirmed that kidney fibrosis was small in all groups except for the group administered with orlistat. In the group administered with orlistat, kidney fibrosis was widely observed, and it was confirmed that kidney fibrosis appeared in a small area in the group administered with the EB-AMDK19 strain and the group administered with the orlistat and EB-AMDK19 strains. Renal fibrosis is a characteristic of chronic renal failure, and it is known that calcium oxalate (CaOx) accumulates in the kidneys, damages the glomeruli and progresses fibrosis. Through this, it was confirmed that renal fibrosis, one of the side effects of taking orlistat, could be suppressed by concurrent administration of orlistat and the EB-AMDK19 strain.

<110> Enterobiome Co., Ltd. <120> Composition Comprising Orlistat and Akkermansia muciniphila EB-AMDK19 <130> PN200398 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LPL primer (Forward) <400> 1 ctctgtatgg cacagtggct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LPL primer (Backward) <400> 2 tccacctccg tgtaaatcaa 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-6 primer (Forward) <400> 3 tcctacccca atttccaatg c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-6 primer (Backward) <400> 4 cataacgcac taggtttgcc g 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TLR2 primer (Forward) <400> 5 aaggaggtgc ggactgtttc 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TLR2 primer (Backward) <400> 6 gagccaaaga gctcgtagc 19 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TLR4 primer (Forward) <400> 7 cctctgcctt cactacagag acttt 25 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TLR4 primer (Backward) <400> 8 tgtggaagcc ttcctggatg 20

Claims (10)

Orlistat (Orlistat) and Akkermansia muciniphila ( Akkermansia muciniphila ) As a pharmaceutical composition for preventing and treating obesity, including,
The composition is to alleviate the side effects of fat absorption inhibitors,
A pharmaceutical composition for preventing and treating obesity.
The method of claim 1,
The Akkermansia muciniphila ( Akkermansia muciniphila ) Akkermansia muciniphila ( Akkermansia muciniphila ) EB-AMDK19 strain,
A pharmaceutical composition for preventing and treating obesity.
delete The method of claim 1,
Alleviating the side effects is to have the effect of alleviating one or more of abdominal bloating, fatty stools, inflammation in the kidneys, and fibrosis in the kidneys,
A pharmaceutical composition for preventing and treating obesity.
delete delete delete delete delete delete

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