KR20130021920A - Composition comprising extracellular vesicles derived from akkermansia muciniphila and bacteroides acidifaciens as an active ingredient for treating or preventing inflammatory disease - Google Patents

Abstract

The present invention is Akkermansia muciniphila or Bacteroides It relates to a composition for the treatment or prevention of inflammatory diseases containing acidifaciens- derived extracellular vesicles as an active ingredient. The composition may inhibit inflammatory diseases due to the extracellular vesicles contained therein, and thus may be used as pharmaceutical compositions, food compositions, and cosmetic compositions for the prevention or treatment of inflammatory diseases. In addition, the present invention is Akkermansia in the extracellular vesicles isolated from the body or secretion of a mammal muciniphila or Bacteroides The present invention relates to a method for diagnosing the development or progress of an inflammatory disease by quantifying an extracellular vesicle derived from acidifaciens .

Description

Composition comprising extracellular vesicles derived from Akkermansia muciniphila and Bactenseroides an anisotropic acid as an active ingredient

The present invention is Akkermansia muciniphila or Bacteroides It relates to a composition for the treatment or prevention of inflammatory diseases containing acidifaciens- derived extracellular vesicles as an active ingredient.

Inflammation is a local or systemic defense against damage or infection of cells and tissues. Inflammation is mainly caused by a cascade of biological reactions that occur by direct response to humoral mediators that make up the immune system, or by stimulating local or systemic effector systems. Major inflammatory diseases include digestive diseases such as gastritis and inflammatory enteritis, oral diseases such as periodontitis, asthma, chronic obstructive pulmonary disease, respiratory diseases such as rhinitis, and skin diseases such as atopic dermatitis, and other infectious rhinitis and allergic rhinitis. Rhinitis and sinusitis, such as chronic rhinitis, acute sinusitis and chronic sinusitis; Otitis media, such as acute purulent otitis media and chronic purulent otitis media; Pneumonia, such as bacterial pneumonia, bronchial pneumonia, lobar pneumonia, regoraella pneumonia and viral pneumonia; Acute or chronic gastritis; Enteritis, such as infectious enterocolitis, Crohn's disease, idiopathic ulcerative colitis, gastric colitis, and the like; Arthritis such as purulent arthritis, tuberculosis arthritis, degenerative arthritis and rheumatoid arthritis; And diabetes mellitus, arteriosclerosis, and the like. There is also research showing that persistent inflammatory diseases can cause cancer. Common compositions used to treat or prevent such inflammatory diseases are largely divided into steroidal and nonsteroidal compositions, many of which are accompanied by various side effects. Therefore, there is a need for development of a prophylactic or therapeutic agent for inflammatory diseases with excellent effects and fewer side effects.

On the other hand, prokaryotic or eukaryotic cells secrete extracellular vesicles, and recently released extracellular vesicles have several functions. Extracellular vesicles secreted from Gram-negative bacteria contain endotoxin (lipopolysaccharide, LPS) and bacteria-derived proteins, and are known to induce inflammatory diseases in the host. In addition, it has been reported that extracellular vesicles derived from various cancer cells contain proteins necessary for growth and metastasis of cancer cells. As described above, since tissue-derived extracellular vesicles reflect the state of tissues secreting vesicles, it has been reported that they can be used to diagnose diseases.

Akkermansia muciniphila is well known as a symbiotic bacterium that continuously lives in the human intestine, starting from young children to the elderly, and has been reported to have fewer bacteria in mucosal tissues in patients with inflammatory bowelitis than in normal controls. have. However, the effects of the bacterial extracellular vesicles on inflammatory diseases have not been studied. In addition, Bacteroides Acidifaciens has been known to have been isolated and cultured in the cecum of mice, but little is known about the secretion and role of extracellular vesicles.

The present invention provides Akkermansia for the treatment or prevention of inflammatory diseases. muciniphila Or Bacteroides acidifaciens It is an object to provide a composition or the like containing the derived extracellular vesicles as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention is Akkermansia muciniphila or Bacteroides Provided are compositions for the treatment or prevention of inflammatory diseases containing an acidifaciens- derived extracellular vesicles as an active ingredient.

In one embodiment of the invention, the composition is Akkermansia It is characterized by containing extracellular vesicles secreted naturally or artificially from muciniphila or Bacteroides acidifaciens .

In another embodiment of the invention, the inflammatory disease is a respiratory disease, including asthma and chronic obstructive pulmonary disease; Inflammatory diseases in the oral cavity including periodontitis; Digestive diseases including gastritis and inflammatory colitis; Skin diseases including atopic dermatitis; Atherosclerosis and its complications; It is characterized in that it is selected from the group consisting of lung cancer, stomach cancer, and colon cancer caused by inflammation.

In another embodiment of the present invention, the extracellular vesicles are characterized in that the average diameter of 20 to 800nm.

In addition, the present invention Akkermansia muciniphila or Bacteroides Provided are pharmaceutical compositions containing, as an active ingredient, extracellular vesicles derived from acidifaciens .

In addition, the present invention Akkermansia muciniphila or Bacteroides Provided are food compositions containing an acidifaciens- derived extracellular vesicles as an active ingredient.

In one embodiment of the invention, the food composition is Akkermansia An extracellular vesicle derived from fermented foods fermented using muciniphila or Bacteroides acidifaciens as an active ingredient.

In addition, the present invention Akkermansia muciniphila or Bacteroides It provides a cosmetic composition containing an acidifaciens- derived extracellular vesicles as an active ingredient.

Akkermansia of the invention muciniphila Or Bacteroides The composition containing the acidifaciens- derived extracellular vesicles as an active ingredient is expected to be used as pharmaceutical compositions, food compositions and cosmetic compositions for the treatment and / or prevention of inflammatory diseases.

Also Akkermansia muciniphila or Bacteroides Extracellular vesicles isolated from fermented foods fermented with acidifaciens can be used to suppress inflammatory diseases, and thus can be used to prevent or treat inflammatory diseases. .

In addition, Akkermansia muciniphila or Bacteroides in extracellular vesicles isolated from mammals' bodies or secretions By quantifying the acidifaciens- derived extracellular vesicles, it is expected to be applicable to the diagnosis or development of inflammatory diseases.

Figure 1 is a schematic diagram showing an experimental design for collecting stool by inducing inflammatory enteritis in C57BL / 6 mice using dextran sodium sulfate (DSS).
FIG. 2 is a transmission electron microscope (TEM) image of extracellular vesicles isolated from feces of normal mice, and extracellular vesicles isolated from feces at day 5 of inflammatory enteritis induced mice.
Figure 3 is a graph showing the results of measuring the size of the extracellular vesicles separated from the mouse stool collected by date using dynamic light scattering (DLS).
4 is a graph showing the result of separating extracellular vesicles from mouse stool collected by date, performing protein quantification, and converting the amount of extracellular vesicles per weight (gram) of stool according to date.
5 is an SDS-PAGE photograph stained using Coomassie blue after separating extracellular vesicles from mouse feces collected by date.
6 is a diagram showing the results of analyzing the composition of bacteria at the phylum level after performing metanomics (metagenomics) with bacteria and extracellular vesicles isolated from feces.
7 is a diagram showing the results of analyzing the composition of bacteria at the species level after performing metanomics (metagenomics) with bacteria and extracellular vesicles isolated from feces.

The present inventors analyzed the extracellular vesicles isolated from the stool of a mammal suffering from inflammatory enteritis, Akkermansia muciniphila or Bacteroides The present invention was completed by confirming that the acidifaciens- derived extracellular vesicles rapidly decreased to 3% or less.

The present invention is Akkermansia muciniphila or Bacteroides Provided are compositions for the treatment or prophylaxis of inflammatory diseases containing acidifacien s-derived extracellular vesicles as an active ingredient.

The compositions of the present invention can be used for the treatment or prevention of inflammatory diseases. The treatment or prevention of the inflammatory disease is meant to include alleviation, alleviation and improvement of symptoms of the inflammatory disease, and also to reduce the possibility of developing the inflammatory disease.

In the present invention, an inflammatory disease means a disease caused by an inflammatory response in a mammalian body, and examples thereof include respiratory diseases such as asthma, chronic obstructive pulmonary disease and rhinitis; Skin diseases such as atopic dermatitis; Digestive diseases such as gastritis and inflammatory enteritis; Arteriosclerosis, sepsis, inflammatory joint disease, inflammatory brain disease, and the like. In addition, the inflammatory disease is used as a meaning including cancer associated with the inflammatory response, in addition to the general inflammatory disease, and includes lung cancer, stomach cancer, colon cancer, and the like.

The large intestine contains about 10 times as many bacteria as host cells. Bacteria secrete extracellular vesicles, which are known to induce inflammatory responses when absorbed locally or systemically. However, little is known about the mechanism by which pathogenic bacteria-derived extracellular vesicles inhibit the development of inflammatory responses. The present inventors induced inflammatory enteritis in mice using DSS, collected feces before and after the disease, and then performed extracellular vesicles from feces to carry out metanomics. Meta-genomics showed that 25% of extracellular vesicles from Akkermansia muciniphila and 27% of extracellular vesicles of Bacteroides acidifaciens were found in the extracellular vesicles isolated from control rats. In the case of inflammatory enteritis induced mice, however, extracellular vesicles of Akkermansia muciniphila accounted for 0.4% and extracellular vesicles of Bacteroides acidifaciens accounted for 2.3% (see Example 5).

From the above results, the Akkermansia of the present invention The composition containing muciniphila or Bacteroides acidifaciens- derived extracellular vesicles as an active ingredient is expected to be applicable to the treatment or prevention of inflammatory diseases. Accordingly, the present invention provides a pharmaceutical composition, a food composition, a cosmetic composition and the like containing the extracellular vesicles as an active ingredient.

The composition is Akkermansia muciniphila or Bacteroides culture of acidifaciens , or Akkermansia muciniphila or Bacteroides The extracellular vesicles isolated from fermented foods fermented with acidifaciens are not particularly limited. The extracellular vesicles can be separated from the culture medium or fermented food, by centrifugation, ultracentrifugation, filtration by filter, gel filtration chromatography, pre-flow electrophoresis, capillary electrophoresis, or a combination thereof. have. In addition, the separation process may further include a process such as washing to remove impurities, concentration of the separated extracellular vesicles. The extracellular vesicles separated by the above method may be naturally secreted or artificially produced, and may have an average diameter of 20 to 800 nm, but preferably 50 to 200 nm.

The pharmaceutical composition of the present invention contains the bacteria-derived extracellular vesicles as an active ingredient, and may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes, but is not limited to, saline solution, sterile water, Ringer's solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc. And other conventional additives. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations may be preferably formulated according to each component using the methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, and external skin preparation.

Another aspect of the invention provides a method of treating an inflammatory disease by administering to a subject a pharmaceutically effective amount of a pharmaceutical composition comprising an extracellular vesicle as an active ingredient. In the present invention, an individual means a subject in need of treatment of a disease, and more specifically, a human, or a non-human primate, a mouse, a rat, a dog, a cat, a horse, a cow, and the like. Mean mammal. In addition, it will be apparent to those skilled in the art that the pharmaceutically effective amount in the present invention may be variously adjusted in the range depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease of the patient. Do.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. However, preferably, it is administered at 0.001 to 100 mg / kg body weight per day, more preferably 0.01 to 30 mg / kg body weight. Administration may be administered once a day or may be divided several times. The extracellular vesicles of the present invention may be present in an amount of 0.0001 to 10% by weight, preferably 0.001 to 1% by weight, based on the total weight of the total composition.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. The method of administration is not limited and may be administered, for example, by oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine dural, or intra cerbroventricular injection.

The food composition of the present invention contains the bacteria-derived extracellular vesicles as an active ingredient, and may include ingredients commonly added during food production. For example, it may include, but is not limited to, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. In addition, when the food composition of the present invention is prepared with a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, and juice may be further included in addition to the extracellular vesicles of the present invention. Also Akkermansia muciniphila or Bacteroides Fermented foods fermented using acidifaciens can be used.

The cosmetic composition of the present invention contains the bacteria-derived extracellular vesicles as an active ingredient, and may include components commonly used in cosmetic compositions. For example, it may include, but is not limited to, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and the like.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it may be prepared in the formulation of a nourishing cream, astringent lotion, flexible lotion, lotion, essence, nourishing gel and massage cream.

In another aspect, the present invention of the extracellular vesicles isolated from the secretions or body of a mammal, Akkermansia The present invention provides a method of quantifying extracellular vesicles derived from muciniphila or Bacteroides acidifaciens to diagnose the development or progress of inflammatory diseases.

The diagnostic method includes the steps of collecting a substance or secretion in the body of a mammal, centrifuging the collected material to remove impurities, animal cells, and bacteria, performing ultracentrifugation to separate extracellular vesicles, And quantifying the isolated extracellular vesicles. The extracellular vesicles can be isolated from the oral cavity, small intestine, and feces of a mammal.

The separation method of the extracellular vesicles can be separated in the same manner as the method of separating the extracellular vesicles from the body or secretion of the normal mammals described above, and if the extracellular vesicles can be purely separated, the separation method is particularly It is not limited.

Quantifying the isolated endoplasmic reticulum is Akkermansia in the isolated extracellular vesicles measuring muciniphila or Bacteroides acidifaciens specific genotypes; Or Akkermansia muciniphila or Bacteroides measuring acidifaciens specific proteins; Or Akkermansia muciniphila or Bacteroides The method may include measuring an immune response to an acidifaciens- derived vesicle itself or a protein in the endoplasmic reticulum.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example  1. In the feces of normal and inflammatory enteritis mice Extracellular  Vesicle Separation

Six-week-old mice (C57BL / 6) were collected from stool daily for five days after treatment with dextran sodium sulfate (DSS), which causes inflammatory enteritis. The weight (grams) of the collected mouse feces was measured and then dispersed in PBS. In order to remove the foreign matter, centrifugation was performed continuously for 5 minutes at 500 × g, 5 minutes at 3,000 × g, and 5 minutes at 4,350 rpm. After the supernatant was separated by centrifugation, ultrafast centrifugation was performed at 10,000 × g for 20 minutes to remove bacteria contained in the feces. The separated supernatant was filtered using a 0.45 μm filter, followed by protein quantification. After making 0.8M and 2.5M sucrose cushion, ultra-high centrifugation was repeated twice for 2 hours at 4 ° C. at 28,500 rpm. Finally, ultrafast centrifugation was performed for 2 hours at 40,200 rpm at 4 ° C. to obtain fecal-derived extracellular vesicles, and the obtained extracellular vesicles were dispersed and stored in PBS.

Example  2. Isolation from Feces of Normal and Inflammatory Enteritis Mice Extracellular  Structural analysis of endoplasmic reticulum

In order to confirm the presence and size of the extracellular vesicles collected from the feces collected by date, the extracellular vesicles were dispersed at a concentration of 50 μg / mL, and then observed by transmission electron microscopy (TEM). The observed result is shown in FIG.

As a result of the TEM observation, it was confirmed that the extracellular vesicles were separated from the feces before and after the DSS treatment, and also had spherical particles.

The exact size of the extracellular vesicles isolated from the feces collected by date was confirmed using dynamic light scattering (DLS). In order to use the DLS method, the extracellular vesicles were dispersed in PBS at a concentration of 5 μg / ml, and then placed in a size measuring device (Zetasizer nano ZS) to measure the size thereof. The results are shown in Fig.

Stool-derived extracellular vesicles were shown to have a size of 100-800 nm regardless of the date collected.

Example  3. Isolation from Feces of Normal and Inflammatory Enteritis Mice Extracellular  Protein pattern analysis of endoplasmic reticulum

The secretion amount of the extracellular vesicles was calculated by comparing the extracellular vesicles collected from the stool collected by date with the weight (gram) of the stool. The converted result is shown in FIG.

As shown in Figure 4, 5 days after the induction of inflammatory enteritis, it was confirmed that the amount of extracellular vesicles isolated from the stool was the highest.

In addition, in order to analyze the protein pattern, the same amount of extracellular vesicles derived from stool (10μg) was isolated using SDS-PAGE (10% resolving gel). The separated protein was stained with Coomassie blue staining solution for 8 hours, and then destained for 4 hours in a destaining solution, and then analyzed for protein pattern. The results are shown in Fig.

In the stool-derived extracellular vesicles of normal mice, 7 to 8 protein bands were observed, but as inflammatory enteritis progressed by DSS, the number of protein bands decreased, and only 3 to 4 protein bands were observed on day 5. As a result, as the inflammatory enteritis progressed, it was confirmed that the total number of protein bands decreased.

Example  4. Metagenenomics of Bacteria in Feces of Normal and Inflammatory Enteritis Mice metagenomics )

Metagenenomics was performed to identify bacteria present in the feces of normal mice and inflammatory enteritis mice. To do this, in order to check whether bacteria exist in the feces, PCR was performed using 16s rRNA primers (27F: 5'-AGAGTTTGATCMTGGCTCAG-3 ', 1492R: 3'-GGTTACCTTGTTACGACTT-5'). It was confirmed to exist. Subsequently, meta-nomics were performed in the order of barcode sorting, prescreen by quality, trimming primer sequences, removing non-target sequences, and taxonomic assignment to analyze bacteria present in feces. The results are shown in FIGS. 6 and 7.

6 shows the bacteria present in the stool of normal and inflammatory enteritis mice at the phylum level. Actinobacteria , Bacteroidetes , and Firmicutes were the main bacteria in the stool, and there was no significant difference in the percentage of bacteria in the stool of normal and inflammatory enteritis mice.

The stool sample of FIG. 7 shows Akkermansia in feces at the species level. muciniphila and Bacteroides This shows how much acidifaciens is present. As shown in Figure 7, Akkermansia in the stool of normal mice The proportion of muciniphila and Bacteroides acidifaciens accounted for 0.33% and 0.21% of the total bacteria, respectively, and 0.87% and 0.43%, respectively, in the stool of inflammatory diseased mice. there was.

Example  5. Isolation from Feces of Normal and Inflammatory Enteritis Mice Extracellular  Metagenetics for the endoplasmic reticulum ( metagenomics )

Metagenenomics was performed to analyze the extracellular vesicles isolated from the feces of normal and inflammatory enteritis mice. To this end, extracellular vesicles were isolated from feces of normal and inflammatory enteritis mice in the same manner as in Example 1, and metanomics were performed for the extracellular vesicles isolated in the same manner as in Example 4. The results are shown in FIGS. 6 and 7.

As shown in FIG. 6, when compared at the phylum level, the extracellular vesicles of Bacteroidetes and Verrucomicrobia accounted for 48% and 26.1% of total bacteria, respectively, in stool of normal mice. Stool was significantly reduced to 22.6% and 0.5%, respectively.

Analysis of the meta Genomics the extracellular vesicles, as shown in Figure 7 in the longitudinal (species) level is in the feces of normal mice Akkermansia muciniphila and Bacteroides The proportion of acidifaciens accounted for 25.07 % and 27.68% of the total bacteria, respectively, compared to 0.4% and 2.3% in stool of inflammatory diseased mice, respectively.

The above examples are the first to identify the change of bacteria secreting the extracellular vesicles present in normal and diseased state by performing meta-genomics on the extracellular vesicles isolated from the stool of normal and inflammatory enteritis mice. In other words, Akkermansia in steady state feces muciniphila , Bacteroides While the proportion of acidifaciens accounted for 0.33% and 0.21% of the total bacteria, the extracellular vesicles derived from these bacteria accounted for 25.07% and 27.68%, respectively. In contrast, Akkermansia in the stool if the disease state muciniphila , Bacteroides The proportion of acidifaciens was 0.87% and 0.43% of the total bacteria, respectively, and the proportion of these bacteria was increased compared to the normal state, whereas the extracellular vesicles were 0.4% and 2.31%, respectively. The ratio of was significantly reduced. Which is Akkermansia muciniphila or Bacteroides This means that extracellular vesicles derived from acidifaciens play a crucial role in immune homeostasis, which suppresses disease and maintains it in a normal state.

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (8)

Akkermansia muciniphila or Bacteroides A composition for the treatment or prevention of inflammatory diseases containing acidifaciens- derived extracellular vesicles as an active ingredient.
The method of claim 1,
The extracellular vesicles are Akkermansia muciniphila or Bacteroides A composition, characterized in that it is secreted naturally or artificially in acidifaciens .
The method of claim 1,
The inflammatory diseases include respiratory diseases, including asthma and chronic obstructive pulmonary disease; Inflammatory diseases in the oral cavity including periodontitis; Digestive diseases including gastritis and inflammatory colitis; Skin diseases including atopic dermatitis; Atherosclerosis and its complications; The composition, characterized in that selected from the group consisting of lung cancer, stomach cancer, and colon cancer caused by inflammation.
The method of claim 1,
The extracellular vesicles are characterized in that the average diameter of 20 to 800nm.
The method according to any one of claims 1 to 4,
The composition is characterized in that the pharmaceutical composition.
The method according to any one of claims 1 to 4,
Wherein said composition is a food composition.
The method according to claim 6,
The food composition is Akkermansia muciniphila or Bacteroides A composition comprising the extracellular vesicles derived from fermented foods fermented with acidifaciens as an active ingredient.
The method according to any one of claims 1 to 4,
The composition is characterized in that the cosmetic composition, the composition.

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