KR102522724B1 - Pharmaceutical composition for preventing, ameliorating or treating inflammatory bowel disease comprising 8-HVA - Google Patents

Abstract

(12E,18R,20Z)-8-hydroxyberia ablin (8-HVA) of the present invention relieves weight loss, diarrhea and bloody stool symptoms in mice induced with chronic enteritis induced by DSS (dextran sodium sulfate) and increases the length of the large intestine. and, as a result of flow cytometry analysis of immune cells infiltrating the lamina propria of the large intestine, it was confirmed that administration of the compound of the present invention relieves inflammation, so it can be usefully used for preventing or treating inflammatory bowel disease.

Description

Pharmaceutical composition for preventing, ameliorating or treating inflammatory bowel disease comprising 8-HVA as an active ingredient

It relates to a pharmaceutical composition for the prevention, improvement or treatment of inflammatory bowel disease comprising 8-HVA ((12E,18R,20Z)-8-hydroxyberia ablin) as an active ingredient.

Inflammatory bowel disease is a disease that causes inflammation in the digestive tract chronically for more than 6 months, repeating improvement and exacerbation. It belongs to the category, and in a broad sense may include infectious enteritis such as bacterial, viral, amebic, and tuberculous enteritis, and all inflammatory diseases occurring in the intestine such as ischemic enteritis, radiation enteritis, and chronic enteritis.

One of the leading causes of inflammatory bowel disease identified so far is thought to be caused by disruption of intestinal homeostasis due to excessive production of proinflammatory mediators (Singh UP et al., Chemokine and cytokine levels in inflammatory bowel disease). patients, Cytokine. 2016 Jan;77:44-9). Inflammation mediators play an important role in defense against foreign substances and reconstitution in innate immunity, but excessively secreted inflammatory mediators destroy intestinal immune homeostasis and induce invasion of immune cells into intestinal tissue, which leads to continuous inflammation. It expresses mediators and develops into cancer in severe cases.

Recently, due to the influence of westernized lifestyles, the frequency of outbreaks is increasing in Korea. In particular, patients with inflammatory bowel disease are more than six times more likely to develop colon than normal people, so timely treatment is essential. However, a treatment that can fundamentally treat inflammatory bowel disease has not yet been developed. ), 5-aminosalicylic acid (5-ASA)-type drugs that block the production of 5-ASA, for example, sulfasalazine, etc., or steroid-type immunosuppressive drugs are used, but the treatment effect is insignificant, abdominal weakness, headache , liver disease, peptic ulcer, diabetes, and side effects such as steroid-induced nephropathy have become a problem.

Accordingly, the inventors of the present invention recognized the above problems and studied, and as a result of administering a compound isolated from Ircinia sp. to an animal model in which chronic enteritis was induced, weight loss of mice, diarrhea and bloody stool symptoms The present invention was completed by confirming that the colon length was restored and the activity of macrophages infiltrated into the intestinal tissue was reduced.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory bowel disease.

Another object of the present invention is to provide a pharmaceutical composition for preventing or improving inflammatory bowel disease.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

.

.

In addition, the present invention provides a health functional food for preventing or alleviating inflammatory bowel disease, comprising a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

.

.

(12E,18R,20Z)-8-hydroxyberia ablin (8-HVA) of the present invention relieves weight loss, diarrhea and bloody stool symptoms in mice induced with chronic enteritis induced by DSS (dextran sodium sulfate) and increases the length of the large intestine. and, as a result of flow cytometry analysis of immune cells infiltrating the lamina propria of the large intestine, it was confirmed that administration of the compound of the present invention relieves inflammation, so it can be usefully used for preventing or treating inflammatory bowel disease.

1 is a diagram showing a high-resolution mass chromatogram of the compound 8-HVA of Example 1 according to the present invention isolated from spiny sponges (Ircinia sp.).
Figure 2 is a view showing the hydrogen NMR spectrum (top) and carbon NMR spectrum (bottom) of the compound 8-HVA of Example 1 according to the present invention isolated from fine filamentous sponges (Ircinia sp.).
Figure 3 is a diagram showing the body weight of each mouse group during the experimental period in which chronic enteritis is induced by feeding mice with DSS.
(NC: normal control
VC: enteritis control group
8-HVA: 8-HVA administration group)
Figure 4 is a diagram showing the disease activity (disease activity index, DAI) calculated based on the body weight and enteritis symptoms of each mouse group during the experimental period of inducing chronic enteritis by feeding DSS to mice.
5 is a diagram showing the results of one-way ANOVA performed for each mouse group by obtaining an AUC (area under the curve) value of the disease activity of each mouse group.
Figure 6 is a view showing a photograph of the large intestine extracted from each mouse group after the end of the experiment of inducing chronic enteritis by feeding DSS to mice.
Figure 7 is a graph showing the length of the large intestine extracted from each mouse group after the end of the experiment of inducing chronic enteritis by feeding DSS to mice.
8 is a diagram showing the results of flow cytometry analysis of immune cells in the lamina propria of the large intestine excised from each group of mice after the end of an experiment in which chronic enteritis was induced by feeding mice with DSS.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

.

.

The inflammatory bowel disease may be ulcerative colitis, Crohn's disease, Behcet's disease or enteritis.

The present invention confirmed that as a result of administering the pharmaceutical composition to an animal model in which chronic enteritis was induced, weight loss was significantly recovered compared to the enteritis control group (FIGS. 3 to 5)

In addition, the present inventors confirmed that, as a result of administering the pharmaceutical composition to an animal model in which chronic enteritis was induced, there was an effect of reducing diarrhea and bloody stool symptoms (FIGS. 4 and 5).

In addition, the present inventors confirmed that, as a result of administering the pharmaceutical composition to an animal model in which chronic enteritis was induced, the colon length was significantly recovered compared to the enteritis control group (FIGS. 6 and 7).

In addition, the present inventors confirmed that inflammation was alleviated as a result of flow cytometry of immune cells in the lamina propria of the colon after administering the pharmaceutical composition to an animal model in which chronic enteritis was induced (FIG. 8).

Therefore, the pharmaceutical composition according to the present invention alleviates clinical symptoms such as weight loss, diarrhea and bloody stool in chronic enteritis-induced mice, and exhibits signs of relieving inflammation in the lamina propria of the large intestine, specifically reducing IL17A (interleukin-17A) and can reduce the degree of colonic infiltration of inflammatory cells, so it can be usefully used for the treatment of inflammatory bowel disease.

The pharmaceutical composition of the present invention may further include at least one active ingredient exhibiting the same or similar function in addition to the above active ingredient.

The pharmaceutical composition of the present invention may include carriers, diluents, excipients, or mixtures thereof commonly used in biological preparations. Any pharmaceutically acceptable carrier may be used as long as it is suitable for delivering the composition in vivo. Specifically, the carrier is described in Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol or mixtures thereof. In addition, conventional additives such as antioxidants, buffers, bacteriostatic agents and the like may be added as needed.

When formulating the composition, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.

The composition of the present invention may be formulated as an oral preparation or a parenteral preparation. Oral preparations may include solid preparations and liquid preparations. The solid preparation may be a tablet, pill, powder, granule, capsule or troche, and such a solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or mixtures thereof. In addition, the solid preparation may include a lubricant, examples of which include magnesium stearate and talc. Meanwhile, the liquid formulation may be a suspension, internal solution, emulsion or syrup. In this case, the liquid formulation may include excipients such as wetting agents, sweeteners, fragrances, and preservatives.

The parenteral formulation may include injections, suppositories, powders for respiratory inhalation, aerosols for sprays, powders and creams, and the like. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, and the like. At this time, vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, or injectable esters such as ethyl oleate may be used as non-aqueous solvents or suspending solvents.

The composition of the present invention may be administered orally or parenterally, depending on the desired method. Parenteral administration may include intraperitoneal, intrarectal, subcutaneous, intravenous, intramuscular or intrathoracic injection modes.

The composition may be administered in a pharmaceutically effective amount. This may vary depending on the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the administration time, the route of administration, the treatment period, and the drugs used simultaneously. However, for desirable effects, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.0001 to 100 mg/kg, specifically 0.001 to 70 mg/kg. The administration may be once or several times a day.

The compositions of the present invention may be administered alone or in combination with other therapeutic agents. When administered in combination, administration can be sequential or simultaneous.

In addition, the present invention provides a health functional food for preventing or alleviating inflammatory bowel disease, comprising a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

.

.

The inflammatory bowel disease may be ulcerative colitis, Crohn's disease, Behcet's disease or enteritis.

The composition of the present invention may be added to food as it is or used together with other foods or food ingredients. At this time, the content of the active ingredient added may be determined according to the purpose, and may generally be 0.01 to 90 parts by weight of the total food weight.

The form and type of health functional food is not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids and pills. The health functional food may include various flavors, sweeteners, or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include thaumatin and stevia extract. On the other hand, examples of synthetic sweeteners include saccharin and aspartame. In addition, the natural carbohydrates may be monosaccharides, disaccharides, polysaccharides, oligosaccharides and sugar alcohols.

The health functional food of the present invention, in addition to the above-mentioned additional ingredients, nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectanes and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, and preservatives , glycerin, alcohol, and the like may be further included. These components may be used independently or in combination. The ratio of the additives may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, examples and experimental examples of the present invention will be specifically illustrated and described. However, the Examples and Experimental Examples to be described below are merely illustrative of a part of the present invention, and the present invention is not limited thereto.

< Example 1> compound 8- HVAs Preparation of pharmaceutical composition for prevention and treatment of inflammatory bowel disease through isolation

(12E,18R,20Z)-8-hydroxyvariabilin (8-HVA)

IUPAC name: (Z)-5-((2R,E)-13-(furan-3-yl)-10-hydroxy-2,6,10-trimethyltridec-6-en-1-ylidene)-4-hydroxy -3-methylfuran-2(5H)-one

A pharmaceutical composition containing the compound 8-HVA (C ₂₅ H ₃₆ O ₅ ) (Fig. 1, Fig. 2) obtained by isolating from Ircinia sp. (Spiny sponge) collected in Vietnam in 2018 was prepared.

Example 1 NMR spectrum data of compound (8-HVA) (CD ₃ OD NMR solvent, 500 MHz for ¹ H) number carbon chemical shift value hydrogen chemistry movement value One 143.9, CH 7.38, dd(1.7, 1.5) 2 111.9, CH 6.29, brs 3 126.4, C. 4 140.1, CH 7.26, brs 5 26.2 CH ₂ 2.40,t(7.1) 6 25.6 CH ₂ 1.59, qint(7.1) 7 42.2 CH ₂ 1.46 m 8 73.2, C. 9 26.9 CH ₃ 1.12, s 10 42.6 CH ₂ 1.43 m 11 23.5 CH ₂ 1.98 m 12 126.0, CH 5.11,t(6.1) 13 135.8, C 14 15.8 CH ₃ 1.56, s 15 40.4 CH ₂ 1.95 m 16 26.8 CH ₂ 1.37 m 17 37.6 CH ₂ 1.30 m / 1.38 m 18 31.8, CH 2.73 m 19 21.1, CH ₃ 1.04, d(6.6) 20 115.3, CH 5.25,d(10.3) 21 145.6, C. 22 165.6, C 23 97.6, C 24 174.2, C. 25 6.1, CH3 1.72, s

< Experimental Example 1> Production of chronic enteritis animal model and 8- HVAs administration

Female C57BL/6 mice purchased from Japan SLC were given an acclimatization period of more than 10 days to allow sufficient weight gain, and when the weight reached an average of 19±2 g, they were randomly divided into 3 groups of 6 mice each and placed in individual wire cages. was put in and the experiment was carried out. The three groups are a normal control (NC), a vehicle control (VC), and an 8-HVA administration group (8-HVA), respectively. In order to induce human inflammatory bowel disease (IBD) in mice in the enteritis control group and 8-HVA-administered group, a method of administering DDS (dextran sodium sulfate) through drinking water was used. Specifically, the inducer was fed 1.5% DSS (colitis grade DSS purchased and used from MP bioscience) as negative water for 7 days, and then fed with sterile breeding water for 7 days, repeating 3 times for a total of 42 days ( Table 2). To the mice in the 8-HVA administration group, the test substance 8-HVA was orally administered once daily at a dose of 10 mg/kg.

1 cycle
2 cycles 3 cycles acclimatization period 1.5% DSS
(7 days) water
(7 days) 1.5% DSS
(7 days) water
(7 days) 1.5% DSS
(7 days) water
(7 days) End of experiment Clinical symptom observation and weight measurement

< Experimental example 2> Observation of clinical symptoms and weight change

In order to confirm whether 8-HVA affects the clinical symptoms and body weight of the chronic enteritis model, the chronic enteritis symptoms and body weight changes of each mouse group were measured during the experimental period. Specifically, body weight was measured between 9 and 10 am once daily during the experiment period, and enteritis symptoms were observed once a day with white paper placed under the cage to record the presence or absence of diarrhea and bloody stool. Based on the measured body weight and enteritis symptoms, the disease activity index (DAI) was calculated according to Table 3 below. The disease activity (DAI score) was modified based on the existing reference literature, measured for 42 days, and the degree of change was graphed. For the disease activity analysis, the AUC (area under the curve) value was obtained and one-way ANOVA was performed for each group. conducted. The results of recording weight change are shown in FIG. 3, and the results of disease activity analysis are shown in FIG.

weight loss ① Average weight on the day of the enteritis control group (VC) - Weight on the day of all enteritis induction groups
(However, negative values are evaluated as 0) diarrhea
(loose stool) ② 0 = no symptoms (negative), 1 = moderate (moderate), 2 = severe (severe) bloody stool
(blood in the stool) ③ 0 = no symptoms (negative), 2 = symptoms (positive)
(Evaluate with weight on blood stool) DAI ①+②+③

As a result, the normal control group (NC) gradually gained weight, but the enteritis control group (VC) and the 8-HVA-administered group (8-HVA) showed a decrease in body weight every DSS cycle, with the largest increase in the 3rd cycle. Weight loss was observed. In the 8-HVA-administered group, it was confirmed that the range of body weight fluctuation was smaller than that of the enteritis control group (FIG. 3). In addition, it was confirmed that the DAI AUC value in the 8-HVA-administered group was significantly reduced to an average of 41.17 ± 18.58 compared to 57.74 ± 19.29 in the enteritis control group (FIGS. 4 and 5).

< Experimental Example 3> Measurement of colon length

In order to confirm whether 8-HVA affects the length of the large intestine in an animal model with chronic enteritis, after the end of the experiment, each mouse was euthanized, and the large intestine was removed by autopsy. Photographs were taken, and then the colon tissue was longitudinally incised to empty the lumen and washed with cold PBS. The results are shown in Figures 6 and 7.

As a result, the colon length of the control group with enteritis was observed to be 5.52 ± 0.54 cm on average, whereas the colon length of the 8-HVA-administered group was 6.24 ± 0.29 on average, and recovery was observed (FIGS. 6 and 7).

< Experimental Example 4> lamina propria of my immune cells flow cell analyze

Flow cytometry was performed using FACScelesta ^TM to analyze CD4+ T cells and CD11b+ mononuclear cells in immune cells infiltrating the lamina propria of the colon. Specifically, in order to isolate cells of the lamina propria of each mouse colon, epithelial cells were removed with EDAT-added PBS solution, and single cells were obtained through collagenase VIII treatment. Then, only immune cells were extracted using a percoll gradient solution. The obtained cells were stained with fixible viability dye and surface antigens of CD4 and CD11b before fixation. Thereafter, intracellular transcription factors (RORγT, foxp3) and cytokine staining were performed through cell fixation and permeabilization. The results are shown in FIG. 8 .

As a result, the absolute numbers (# events) of CD4+ T cells and CD11b+ mononuclear cells decreased in the 8-HVA-administered group, but the ratio (FVD, %) of total immune cells increased in CD4+ T cells and decreased in CD11b+ cells. Therefore, the degree of colonic infiltration of inflammatory cells was reduced, and among them, the decrease in the population of macrophages and mononuclear cells expressing CD11b+ is considered to be a finding of alleviation of enteritis. In additional CD4+ T cell analysis, a decrease in Th17 (T helper 17), a pro-inflammatory cell, and IL17A (interleukin-17A) secreted by the cell were observed, and Foxp3+ (forkhead, known as an inflammatory regulator) box P3) It was observed that both the absolute number of cells and the percentage of total immune cells increased. Therefore, it is judged that inflammation was alleviated even in CD4+ T cells (FIG. 8).

Through the above experiments, as a result of administering compound 8-HVA to mice with chronic enteritis induced by DSS, it was confirmed that clinical symptoms such as weight loss and diarrhea were alleviated, and enteritis was alleviated due to reduced infiltration of inflammatory cells in the intestinal tissue, and Th17 was reduced and an increase in Foxp3 T cells. Therefore, 8-HVA can be usefully used for the treatment of chronic enteritis.

In the above, the present invention has been described in detail through preferred manufacturing examples, examples and experimental examples, but the scope of the present invention is not limited to specific examples, and should be interpreted by the appended claims. In addition, those skilled in the art should understand that many modifications and variations are possible without departing from the scope of the present invention.

Claims (7)

A pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

.
According to claim 1,
The inflammatory bowel disease is a pharmaceutical composition for preventing or treating inflammatory bowel disease, characterized in that ulcerative colitis or Crohn's disease.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating inflammatory bowel disease, characterized in that it reduces symptoms of diarrhea or bloody stool.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating inflammatory bowel disease, characterized in that it reduces the degree of colonic infiltration of inflammatory cells.
According to claim 4,
The pharmaceutical composition is characterized by reducing interleukin-17A (IL17A), a pharmaceutical composition for preventing or treating inflammatory bowel disease.
A functional food for preventing or improving inflammatory bowel disease comprising a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

.
According to claim 6,
The inflammatory bowel disease is a health functional food for preventing or improving inflammatory bowel disease, characterized in that the ulcerative colitis (ulcerative colitis) Crohn's disease (Crohn's disease).

Images