WO2020130205A1 - Pharmaceutical composition and health function food comprising pepsin inhibitor - Google Patents

Abstract

A pharmaceutical composition and a health functional food according to the present invention each comprise a pepsin inhibitor and as such, can greatly prevent, alleviate, or treat tonsillar hypertrophy, particularly, tonsillar hypertrophy caused by the reflux of pepsin.

Description

Pharmaceutical composition and health functional food containing pepsin inhibitor

The present invention relates to a pharmaceutical composition and a dietary supplement comprising a pepsin inhibitor.

The tonsils are lymphatic (lymph node) epithelial tissues in the oropharynx and nasopharynx. Frequently speaking, the tonsils are often referred to as the tonsils on both sides of the uvula. The tonsils in this area are palatine tonsils, one of the tonsil tissues. In addition to the palatine tonsils visible in the mouth, there are pharyngeal tonsils called adenoids in the nasopharynx and tongue tonsils in the tongue roots, which are not visible in the mouth. A tonsil hypertrophy is a condition in which the tonsils become abnormally hypertrophic and cause problems without repetitive upper respiratory tract infections or other symptoms associated with “systemic” diseases.

Chronic tonsil hypertrophy can also cause airway obstruction in children. In general, “one way” and adenoids are very small at birth, but continue to grow until the age of four. More pathogenic bacteria reside in the tonsils and adenoids that have become enlarged tonsils and chronic inflammation. Normally, the tonsil tissue is enlarged when the balance is broken due to a recurrent acute viral or bacterial infection while maintaining the balance between the normal tonsils and the immune response.

It is an object of the present invention to provide a pharmaceutical composition and a dietary supplement comprising a pepsin inhibitor.

1. A pharmaceutical composition for preventing or treating tonsil hypertrophy comprising a pepsin inhibitor.

2. The composition according to the above 1, wherein the tonsil hypertrophy is caused by the reverse flow of pepsin.

3. The composition of 1 above, wherein the pepsin inhibitor is at least one selected from the group consisting of pepstatin, statin, 1-bis(diazoacetyl)-2-phenylethane, and sucralfate.

4. Health functional food for preventing or improving tonsil hypertrophy, including a pepsin inhibitor.

5. The functional food according to the above 4, wherein the tonsil hypertrophy is caused by reflux of pepsin.

6. In the above 4, the pepsin inhibitor is at least one selected from the group consisting of pepstatin, statin, 1-bis(diazoacetyl)-2-phenylethane and sucralfate.

The pharmaceutical composition and dietary supplement of the present invention contain a pepsin inhibitor, and thus can excellently prevent, ameliorate, or treat tonsil hypertrophy, particularly tonsil hypertrophy due to reflux of pepsin.

Figure 1 relates to the number of immune cells in the tonsils of tonsil hypertrophy and tonsillitis patients.

Figure 2 relates to the effect of pepsin on cell proliferation in patients with tonsil hypertrophy and tonsillitis.

Figure 3 relates to the effect of pepsin on the secretion of CD4 positive cells, IFN-γ and IL-10 and the anti-pepsin effect of IL-2 blockade.

4 shows the coexistence of pepsin and lymphocytes in the hypertrophic tonsil section sample.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating tonsil hypertrophy comprising a pepsin inhibitor.

The tonsil hypertrophy may be caused by various causes, but specifically, it may be caused by reflux of pepsin.

The pepsin inhibitor may be at least one selected from the group consisting of pepstatin, statin, 1-bis(diazoacetyl)-2-phenylethane, and sucralfate, but is not particularly limited as long as it can inhibit the activity of pepsin. .

The composition of the present invention may further include a pharmaceutically acceptable carrier, and may be formulated with a carrier. The term "pharmaceutically acceptable carrier" in the present invention refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. As a pharmaceutical carrier that is acceptable in a composition formulated as a liquid solution, as a sterile and biocompatible material, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

The composition of the present invention is applicable to any formulation containing a pepsin inhibitor as an active ingredient, and can be prepared in oral or parenteral formulations. The pharmaceutical formulation of the present invention is oral, rectal, nasal, topical (including cheek and sublingual), subcutaneous, vaginal or parenteral; intramuscular and subcutaneous. And forms suitable for administration by inhalation (including intravenous) or administration by inhalation or insufflation.

The composition of the present invention is administered in a pharmaceutically effective amount. The effective dose level depends on the type of patient's disease, severity, drug activity, sensitivity to the drug, time of administration, rate of administration and release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. Can be determined. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention can vary widely depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity, and an appropriate dosage is, for example, It may vary depending on the amount of drug accumulated in the body and/or the specific efficacy level of the pepsin inhibitor used. In general, it can be calculated based on the EC50 measured as effective in the in vivo animal model and in vitro, for example, may be 0.01 μg to 1 g per 1 kg of body weight, and in a unit period of daily, weekly, monthly or yearly, It may be administered once or several times per unit period, or may be continuously administered for a long period of time using an infusion pump. The number of repeated doses is determined taking into account the time the drug stays in the body and the concentration of the drug in the body. The composition may be administered for relapse even after being treated according to the course of disease treatment.

The composition of the present invention may further contain a compound that maintains/increases the solubility and/or absorption of one or more active ingredients exhibiting the same or similar function in relation to the treatment of tonsil hypertrophy. Also optionally, chemotherapeutic agents, anti-inflammatory agents, anti-viral agents and/or immunomodulators may be further included.

In addition, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

The present invention provides a health functional food for preventing or improving tonsil hypertrophy comprising a pepsin inhibitor.

The tonsil hypertrophy may be caused by various causes, but specifically, it may be caused by reflux of pepsin.

The pepsin inhibitor may be at least one selected from the group consisting of pepstatin, statin, 1-bis(diazoacetyl)-2-phenylethane, and sucralfate, but is not particularly limited as long as it can inhibit the activity of pepsin. .

The health functional food of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or improving tonsil hypertrophy.

The health functional food of the present invention refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act 6727 on the Health Functional Food, and the nutrients for the structure and function of the human body. This means taking it for the purpose of obtaining a useful effect for health purposes such as control or physiological action.

The health functional food of the present invention may include a conventional food additive, and whether or not it is suitable as a food additive is related to the item according to the general rules and general test methods of food additives approved by the Food and Drug Administration unless otherwise specified. Judging by standards and standards.

Examples of the food additives receivable include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamonic acid; Natural additives such as chromic pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; L-sodium glutamate formulations, mixed additives such as noodle additive alkalis, preservatives, tar colorants, and the like, but are not limited thereto.

For example, in the form of tablets, the dietary supplement is granulated by mixing a mixture of pepsin inhibitors with excipients, binders, disintegrants and other additives in a conventional manner, and then compression-molding with a lubricant or the like. It can be compression molded. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary.

Hard capsules in the form of capsules can be prepared by filling a mixture of pepsin inhibitors with additives such as excipients in conventional hard capsules, and soft capsules gelatin mixtures of pepsin inhibitors with additives such as excipients. It can be prepared by filling the same capsule base. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The health functional food in the form of a ring may be prepared by molding a mixture of a pepsin inhibitor and an excipient, a binder, a disintegrant, etc. by a conventionally known method, and may be peeled with a white sugar or other skin repellent if necessary, or starch. , You can also coat the surface with a material such as talc.

The health functional food in granular form may be prepared in a granular form by mixing a pepsin inhibitor with an excipient, a binder, a disintegrant, etc., and may contain a flavoring agent, a mating agent, and the like, if necessary.

The health functional food is beverage, meat, chocolate, food, confectionery. It may be pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, and dietary supplements.

Hereinafter, examples will be described in detail to specifically describe the present invention.

Example 1. Experimental method

1. Subject

A total of 71 patients were enrolled in the study from January 2015 to December 2016. They visited the hospital for tonsillectomy surgery due to tonsillectomy or sleep apnea/snoring due to chronic inflammation. Criteria for inclusion are: When diagnosed with chronic tonsillitis due to tonsil hypertrophy or physical examination or medical history. The criteria for inclusion are as follows: preoperative examination of suspected abscess, head and neck trauma, malignant tumor, radiation therapy, systemic disease and other clinical problems. After general anesthesia, tonsillectomy was performed, the lower part of the tonsil was selected for tissue sampling, and whole blood was selected for blood sampling. There were no complications after surgery.

2. Pepsin

The main human enzyme is pepsin 3, called pepsin, and is similar to the porcine pepsin used in this experiment.

3. Isolation and phenotypic analysis of tonsil-derived cells

All tonsils were treated within 20 minutes after surgery. In the aseptic condition, the tonsils are washed with PBS and cut into small pieces 1-2 mm ^{2 in} size using a sterile razor blade. The chopped tissue was sequentially passed through a 70 μm nylon cell filter (BD Falcon, MA, USA) to obtain a single cell suspension, and centrifuged at 1500 rpm for 10 minutes at 4°C. The separated cells were treated with RBC lysis buffer (BioLegend, CA, USA) for 5 minutes at room temperature and washed with PBS. For phenotypic analysis of cells derived from tonsils, 1 x 10 ⁶ cells were fluorescein bound CD4 (PE conjugation, Miltenyi Biotec, Bergisch Gladbach, Germany), CD14 (PerCP conjugation, Miltenyi Biotec) and CD19 (PE conjugation, Miltenyi Biotec) antibody was labeled on ice for 15 minutes and analyzed by flow cytometry (FC500, Beckman Coulter, CA, USA).

4. Cell selection and in vitro culture

Cells were isolated using a magnetically activated cell sorting (MACS, Miltenyi Biotec) system. To perform MACS separation, cells derived from 5 x 10 ⁷ tonsils were resuspended in 80 μl of MACS buffer (PBS with 0.5% BSA and 2 mM EDTA) and 20 μl of CD4, CD14 and CD19 microbeads ( MiltenyBiotec) was added, followed by incubation at 4°C for 15 minutes. Cells were washed by adding 5 ml of MACS buffer and centrifuged at 1500 rpm for 10 minutes. The supernatant was aspirated completely, 500 μl of MACS buffer was resuspended and placed on ice. The MS column (MiltenyBiotec) was placed in the magnetic field of a MACS separator (MiltenyBiotec) and the cell suspension was passed through an MS column. Unlabeled cells were collected with passage buffer, and the column was washed 3 times with 500 μl MACS buffer. The column was removed from the separator, and a plunger was pushed into the column to immediately flush out magnetically labeled cells into a new tube. Each isolated cell was divided into 4 experimental groups: basal medium (Con); Basal medium containing 1 μg/ml acidic pepsin (1P); Basal medium with 1 μg/ml acidic pepsin and 0.5 μg/ml pepstatin A (1P + 0.5 PS); Base medium containing 1 μg/ml acidic pepsin and 1 μg/ml pepstatin A (1P + 1 PS). In this experiment, even if all cells were treated with pepsin, they were cultured and incubated in a normal pH environment (not acidic). However, the pepsin used in the experiment (pepsin from the mucous membrane of the pig, Sigma, #P7012) was used in 1% (10 mg/ml) of deionized water and 0.4% (4 mg/ml) of hydrochloric acid solution according to the manufacturer's instructions. Was used. The solution at pH 4.4 was stable at -20°C and maintained at -20°C. Pepsin was treated on day 0 and additionally on day 3. Peptstatin A (pepstatin A, Sigma, #P7012 from a microbial source) was dissolved in a mixed solution of methanol and acetic acid (methanol: acetic acid = 9: 1). The experimental concentration (0.5 to 1.0 μg/ml) was followed by the manufacturer's instructions. All media were pre-incubated at 37°C for 30 minutes, and the cells were maintained at 37°C in a humidified environment of 5% CO ₂ . To investigate the effect of pepsin on the proliferation of CD4, CD14 and CD19 positive cells, all cells were cultured for 7 days and cell counted.

5. Effect of IL-2 blocking on pepsin-induced CD4 proliferation

To confirm that pepsin is involved in IL-2 mediated CD4 cell proliferation, IL-2 was blocked with an anti-IL-2 antibody. CD4 cells were divided into 8 experimental groups: basal medium (CTL), basal medium with 1 μg/ml acidic pepsin (1 pep), basal medium with 1 μg/ml pepstatin A (1PS), 1 μg/ It was divided into basic medium containing ml acidic pepsin and 1 μg/ml pepstatin A (1 pep + 1 PS), and additionally 20 ng/ml of anti-IL-2 antibody (human IL-2 antibody, MAB202, R & D systems) were treated individually in each experimental group. All media were pre-incubated at 37° C. for 30 minutes, and all cell cultures were maintained at 37° C. in a humidified environment of 5% CO ₂ . To measure the effect of anti-IL-2 antibody on the proliferation of pepsin-induced CD4+ cells, each group was cultured for 7 days, and 3 days using an automatic cell counter (EVETM, NanoEnTek, Seoul, Korea) And cells were counted on day 7. To confirm that pepsin is involved in the proliferation of IL-2 mediated CD4 cells, IL-2 was blocked with an anti-IL-2 antibody (#MAB202, R&D system).

6. Cytokine Analysis

To identify cytokines for proliferation of CD4+ cells with or without pepsin, ELISA for IL-2, IFN-γ and IL-10 was performed. After incubation for 7 days, culture medium was collected, and cells and cell debris were precipitated by centrifugation at 1500 rpm for 10 minutes. The supernatant was collected and stored in a deep freezer at -80°C. Levels of IL-2 and IFN-γ were measured using a specific ELISA kit (Quantikine ELISA kit, R & D Systems, MN, USA) and performed according to the manufacturer's instructions.

7. Double immunofluorescence staining

To characterize pepsin-A positive cells, double immunofluorescence was performed in tonsil tissue, and deparaffinization and antigen recovery were performed. Non-specific antibody binding was blocked for 45 minutes with PBS containing 0.1% normal donkey serum (Vector Laboratories) and 0.3% Triton X-100 (Sigma). The sections were then incubated with anti-pepsin-A antibody (1:100; sc-99081, Santa Cruz) diluted overnight at 4° C. in PBS containing 0.1% bovine serum albumin (Sigma). After rinsing, the donkey Cy3-bound anti-rabbit IgG secondary antibody (1: 100; EMD Millipore, Billerica MA, USA) was applied at room temperature for 1 hour. For double labeling, after blocking with PBS containing 10% normal goat serum and 0.3% Triton X-100, sections were incubated overnight at 4° C. with anti-CD4, CD19 and CD68 (1: 100; Santa Cruz). Did. Alexa488-bound anti-mouse IgG secondary antibody (1: 100, Invitrogen, Carlsbad CA, USA) was applied at room temperature for 1 hour. Sections were mounted with an anti-fading solution containing 4',6-diamidino-2 phenylindole (DAPI, Vector Laboratories), and fluorescence microscopy ( Carl Zeiss Microscopy GmbH, Jena, Germany).

8. Statistical analysis

All data are expressed as mean±S.E.M. Comparisons between groups were analyzed by two-tailed tests. Probability values (P) <0.05 were considered significant.

Example 2. Characteristics of patients

All patients underwent physical examination to confirm the diagnosis of tonsil hypertrophy or chronic tonsillitis. The average tonsil size was 2.5 in the tonsil hypertrophy group and 1.0 in the chronic tonsillitis group. Prior to surgical treatment, tonsil tissue was obtained from 49 children (29 men, 20 women, average age 8 years) and 22 adults (9 men, 13 women, average age 17 years).

Example 3. Tonsil cell types and numbers in children with hypertrophy

On the day of surgery, CD4, CD14, CD68 and CD19 positive cells were identified using flow cytometry from tonsil cells. As shown in Figure 1, the proportion of CD4 positive cells was significantly lower in children than adults, and the proportion of CD68 and CD19 positive cells was significantly higher in children than adults. The proportion of CD14 positive cells did not differ between the two groups. Interestingly, there was a difference between tonsil tissue and peripheral blood of the hypertrophic tonsils. The pediatric peripheral blood samples showed lymphocytes, CD14 and CD206 positive cells, and the lymphocytes were significantly higher than the adult samples. These results suggest that lymphocytes may play an important role in tonsil hypertrophy and tonsillitis.

Example 4. Reaction of tonsil CD4 positive cells to pepsin

To identify a cell population that responds to pepsin in the hypertrophic tonsil, CD4-, CD14- and CD19-positive cells were purified by magnetically activated cell sorting (MACS) and cultured in the presence of pepsin or pepstatin to inhibit pepsin. Purification of tonsil macrophages was intentionally excluded due to the low level of CD68 in tissues as shown in FIG. 1A. In children's tonsil tissue, the number of CD4 positive cells increased significantly when treated with pepsin, compared to CD4 positive control cells not treated with pepsin, but pepstatin (1P + 0.5 PS and 1P + 1PS; Figure 2A) It decreased with addition. However, there was no statistically significant difference in the number of CD14 and CD19 positive cells (FIG. 2A ). In addition, there was no statistically significant difference between tonsil cell populations in tonsil tissue (FIG. 2B ). These results suggest that CD4 cells respond to pepsin and that the reaction is inhibited by pepstatin. Therefore, it may be related to CD4 cell proliferation, and children are more likely to be exposed to pepsin than adults.

Example 5. Mediating pepsin-stimulated CD4 cell proliferation of IL-2

To reveal possible mechanisms of pepsin-mediated CD4 cell proliferation, levels of IL-2 and IFN-γ capable of stimulating lymphocyte proliferation were measured in culture medium by ELISA. IL-2 and IFN-γ levels increased significantly in response to pepsin treatment, but decreased when pepsin was inhibited by pepstatin. The level of cytokine IL-10, known as a representative anti-inflammatory agent, decreased in CD4 cells treated with pepsin and recovered by pepstatin (FIG. 3A ). IL-2 may be more sensitive to pepsin than IFN-γ and L-10. To confirm that pepsin is involved in IL-2 mediated CD4 cell proliferation, IL-2 was blocked with an anti-IL-2 antibody (FIG. 3B ). Blocking of IL-2 reduced the increased CD4 cell number on day 3 by pepsin (1 pep). However, on the 3rd day, no augmentation effect or synergistic effect of IL-2 blocking and pepstatin addition could be found. No effect was found on the 7th day.

Example 6. Expression of pepsin and lymphocytes in hypertrophic tonsils

Immunohistochemical staining was performed to investigate the role of pepsin in lymphocyte infiltration and hyperplasia in the hypertrophic tonsil. Although few pepsin-positive cells co-localized with CD4 cells, pepsin-positive cells surrounding CD4-positive hypertrophic tonsil cells were found (Figure 4A). B cells reflected by CD19 were also like lymphocytes (FIG. 4B ). Pepsin positive cells coexisted with CD68 positive cells (Figure 4C).

Claims (6)

1. A pharmaceutical composition for preventing or treating tonsil hypertrophy comprising a pepsin inhibitor.
2. The composition of claim 1, wherein the tonsil hypertrophy is due to reflux of pepsin.
3. The composition of claim 1, wherein the pepsin inhibitor is at least one selected from the group consisting of pepstatin, statin, 1-bis(diazoacetyl)-2-phenylethane, and sucralfate.
4. Health functional food for preventing or improving tonsil hypertrophy, including a pepsin inhibitor.
5. The method according to claim 4, wherein the tonsil hypertrophy is due to the reflux of pepsin health functional food.
6. The method according to claim 4, wherein the pepsin inhibitor is at least one selected from the group consisting of pepstatin, statin, 1-bis(diazoacetyl)-2-phenylethane and sucralfate.

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