WO2018164295A1

WO2018164295A1 - Pharmaceutical composition for treating colorectal disease containing cystatin a

Info

Publication number: WO2018164295A1
Application number: PCT/KR2017/002514
Authority: WO
Inventors: Myung Jun Chung
Original assignee: Cell Biotech Co., Ltd.
Priority date: 2017-03-06
Filing date: 2017-03-08
Publication date: 2018-09-13
Also published as: KR101791876B1

Abstract

The present invention relates to a pharmaceutical composition for treating colorectal disease, which contains cystatin A. The pharmaceutical composition of the present invention, which contains cystatin A, exhibits a significantly increased inhibitory effect against colorectal cancer compared to when cystatin is not used. Thus, the pharmaceutical composition of the present invention will be effectively used as a therapeutic agent against colorectal cancer in the medical field.

Description

PHARMACEUTICAL COMPOSITION FOR TREATING COLORECTAL DISEASE CONTAINING CYSTATIN A

The present invention relates to a pharmaceutical composition for treating colorectal disease, which contains cystatin A.

Colorectal diseases refer to a group of various diseases that occur in the colon which include colorectal cancer, colitis, irritable bowel syndrome, Crohn's disease and the like. The number of patients with colorectal diseases is increasing in modern times, due to frequent stress, Westernized eating habits, and drinking. In particular, colorectal cancer is the second most common cancer in cancer deaths, accounting for about 13% of all cancers. It was reported that about 50,310 people died of colorectal cancer annually and 136,830 cases of new colorectal cancer occur in the United States. In South Korea, the death rate of colorectal cancer is the fourth highest following lung, liver, and stomach cancer. The incidence of colorectal cancer is increasing which accounts for 12.9% of all cancers in 2012 compared with 5.8% in 1980, 6.1% in 1985, 8.2% in 1995, and 11.2% in 2002. And despite a decrease in the death rate of gastric cancer, liver cancer and cervical cancer in 2011 compared with 1999, the death rate of colorectal cancer has increased by 5.6%. Accordingly, many chemical anticancer drugs have been developed for the treatment of colorectal cancer, such as fluoropyrimidine-based drugs(fluorouracil, tegafur-uracil, capecitabine and the like), irinotecan, oxaliplatin, and targeted therapeutic agents(bevacizumab, cetuximab and the like). However such chemical anticancer drugs have a high side-effect risk according to high-dose and long-term administration., Therefore, it is necessary to develop a pharmaceutical composition of natural ingredients.

Meanwhile, cystatin is a substance that inhibits the activity of intracellular proteases, and is currently classified into family 1 (cystatin-alpha, and cystatin-beta) that is present in cells, secretory family 2 (cystatin C, cystatin S, egg white cystatin, and milk cystatin), family 3 (kininogen), and the like. Cystatin-alpha exists in leukocyte, epithelial tissue of epidermis, digestive tracts, vagina, and etc., and the like, and functions to protect the human body from the invasion of pathogenic microbes. Cystatin-beta is present in most cytosols, and functions to inhibit the activity of proteases to thereby prevent viral infections. Egg white cystatin also helps prevent viral infections, and a substance having the same function as that of egg white cystatin is oryzacystatin that is found in rice seeds. Cystatin C is a functional protein that is produced in certain amounts every moment by all the cells of the human body and functions to prevent the onset of diseases and maintain normal physiology, and particularly, it is known to play an important role in the defense function of the immune system. Elucidation of the secretory mechanism and function of cystatin D is still insufficient. In recent years, many studies have been conducted on the therapeutic effects of cystatin, including the effects of cystatin A on prostate cancer (US 2003-0113762 A1), the effects of cystatin C on inflammation (US 2016-0245824 A1), the effects of cystatin SN on colorectal cancer (CN 103740854 B) and the like. However, the technology for the treatment of cystatin A in colorectal cancer has not been developed sufficiently.

Therefore, the present invention is directed to a pharmaceutical composition for treating colorectal disease, which contains cystatin A. The composition of the present invention has been demonstrated to have significant effects on the inhibition of colorectal cancer growth, and thus will be effectively used as a therapeutic agent against colorectal cancer in the medical field.

The present invention has been made in order to solve the above-described problems occurring in the prior art, and is directed to a pharmaceutical composition for treating colorectal disease, which contains cystatin A.

However, the technical object to be achieved by the present invention is not limited to the above technical object, and other objects that are not mentioned above can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to figures. In the following description, numerous specific details are set forth, such as specific configurations, compositions, and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in combination with other known methods and configurations. In other instances, known processes and preparation techniques have not been described in particular detail in order to not unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

Unless otherwise specified in the specification, all the scientific and technical terms used in the specification have the same meanings as commonly understood by those skilled in the technical field to which the present invention pertains.

In one embodiment of the present invention, the term “cystatin” refers to a substance that inhibits the activity of intracellular proteases. Cystatin is currently classified into family 1 (cystatin-alpha, and cystatin-beta) that is present in cells, secretory family 2 (cystatin C, cystatin D, cystatin S, egg white cystatin, and milk cystatin), family 3 (kininogen), and the like. Cystatin-alpha exists in leukocytes, the epithelial tissue of epidermis, digestive tracts, vagina, and etc., and the like, and functions to protect the human body from the invasion of pathogenic microbes. Cystatin-beta is present in most cytosols, and functions to inhibit the activity of proteases to thereby prevent viral infections. Egg white cystatin also helps prevent viral infections, and a substance having the same function as that of egg white cystatin is oryzacystatin that is found in rice seeds. Cystatin C is a functional protein that is produced in certain amounts every moment by all the cells of the human body and functions to prevent the onset of diseases and maintain normal physiology, and particularly, it plays an important role in the defense function of the immune system. However, it was reported that blood cystatin C levels increase in various pathologies. If blood cystatin C levels are 1 mg/L or higher, the incidence rate of heart attack, stroke, sudden cardiac death or chronic renal failure increases as the value increases. Elucidation of the secretory mechanism and function of cystatin D is still insufficient.

In one embodiment of the present invention, the term “colorectal disease” refers to a group of various diseases that occur in the colon. The colorectal disease is preferably colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease, but is not limited thereto. More preferably, the colorectal disease is colorectal cancer.

In one embodiment of the present invention, “pharmaceutical composition” refers to a composition to be administered for a specific purpose. For the purpose of the present invention, the pharmaceutical composition of the present invention contains, as active ingredients, cystatin A. The pharmaceutical composition of the present invention may contain protein and a pharmaceutically acceptable carrier, excipient or diluent, which are involved therein. The "pharmaceutically acceptable" carrier or excipient means one approved by a regulatory agency of the Federal or a state government, or one listed in the pharmacopoeia or other generally recognized pharmacopoeia for use in vertebral animals, and more particularly in humans.

For parenteral administration, the pharmaceutical composition of the present invention can be in the form of suspensions, solutions, or emulsions, in oily or aqueous vehicles, and can be in the form of solid or semi-solid, preferably liquid. Furthermore, the pharmaceutical composition of the present invention can contain formulatory agents such as suspending, stabilizing, solubilizing, and/or dispersing agents, and can be sterilized. The pharmaceutical composition can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. Alternatively, the pharmaceutical composition of the present invention can be in sterile powder form for reconstitution with a suitable vehicle before use. The pharmaceutical composition can be presented in unit dose form, in microneedle patch, in ampoules, or other unit-dose containers, or in multi-dose containers. Alternatively, the pharmaceutical composition can be stored in a freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example, water for injection immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules or tablets.

In some non-limiting embodiments, the pharmaceutical composition of the present invention may be formulated as liquid or contained as microspheres in liquids. In some non-limiting embodiments, excipients that are suitable for the pharmaceutical composition of the present invention may include preservatives, suspending agents, stabilizers, dyes, buffers, antibacterial agents, antifungal agents, and isotonic agents, for example, sugars or sodium chloride. As used herein, the term "stabilizer" refers to a compound optionally used in the pharmaceutical composition of the present invention in order to increase storage life. In non-limiting embodiments, additional stabilizers may be sugars, amino acids or polymers. In addition, the pharmaceutical composition of the present invention can comprise one or more pharmaceutically acceptable carriers. The carrier can be a solvent or dispersion medium. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), oils, and suitable mixtures thereof. Non-limiting examples of sterilization techniques that are applied to the pharmaceutical composition of the present invention include filtration through a bacterial-restraining filter, terminal sterilization, incorporation of sterilizing agents, irradiation, sterilization gas irradiation, heating, vacuum drying, and freeze drying.

In one embodiment of the present invention, “food composition” can be used in various foods, for example, beverages, gums, teas, vitamin complexes, health supplement foods or the like, and may be used as pills, powders, granules, infusions, tablets, capsules or beverages, and the content (wt%) of the food composition in foods is not limited.

The food composition of the present invention may contain conventional food additives known in the art, for example, flavorings, colorants, fillers, stabilizers and the like. The food composition according to the present invention is not particularly limited, as long as it is a composition containing, as essential ingredients, cystatin A. The food composition of the present invention may additionally contain various flavorings or natural carbohydrates as in conventional beverages. Examples of the natural carbohydrates include conventional sugars, such as monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), polysaccharides (e.g., dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, erythritol or the like. Examples of flavorings that may be used in the present invention include natural flavorings (thaumatin, stevia extracts, such as rebaudioside A, glycyrrhizin, etc.) and synthetic flavorings (saccharin, aspartame, etc.). The natural carbohydrate is used in an amount of about 1-20 g, preferably about 5-12 g, based on 100 mL of the composition of the present invention.

In addition, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, colorants, extenders (cheese, chocolate, etc.), pectic acid and its salt, alginic acid and its salt, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonizing agents as used in carbonated beverages, etc. Such components may be used individually or in combination. Although the percentage of such additives is not of great importance, it is generally selected in a range of 0 to about 20 parts by weight based on 100 parts by weight of the composition of the present invention.

In one embodiment of the present invention, “administration” means introducing the composition of the present invention into a patient by any suitable method. The composition of the present invention may be administered by any general route, as long as it can reach a target tissue. Specifically, the composition of the present invention may be administered orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, intrapulmonarily, intrarectally, intracavitally or intrathecally. However, the pharmaceutical composition of the present invention, which contains, as active ingredients, cystatin A, is administered intravenously as a liquid formulation or administered as orally as a powder, tablet, capsule or liquid formulation, but is not limited thereto.

A method for treating colorectal disease according to the present invention may comprise administering a pharmaceutically effective amount of the pharmaceutical composition. In the present invention, the effective amount can be determined depending on various factors, including the kind of disease, the severity of the disease, the kinds and contents of active ingredient and other ingredients in the composition, the kind of formulation, the patient’s age, body weight, general health condition, sex and diet, the time of administration, the route of administration, the secretion rate of the composition, the period of treatment, and other drugs that are concurrently used.

In one embodiment, the present invention provides a method of treating colorectal disease, the method comprising administering to a patient in need thereof a pharmaceutical composition containing an effective amount of cystatin A. In the method, the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease. Preferably, the colorectal disease is colorectal cancer.

In another embodiment of the present invention, the present invention provides a use of cystatin A thereof, for preparation of a medicament for treatment of colorectal disease. In the use, the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease. Preferably, the colorectal disease is colorectal cancer.

In another embodiment of the present invention, the present invention provides a pharmaceutical composition for treating colorectal disease, which contains, as active ingredients, cystatin A. In the pharmaceutical composition, the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease. Preferably, the colorectal disease is colorectal cancer.

In still another embodiment of the present invention, the present invention provides a food composition for alleviating colorectal disease, which contains, as active ingredients, cystatin A. In the food composition, the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease. Preferably, the colorectal disease is colorectal cancer.

Hereinafter, each step of the present invention will be described in detail.

The number of patients with colorectal diseases is increasing in modern times, due to frequent stress, Westernized eating habits, and drinking. In particular, colorectal cancer is the second most common cancer in cancer deaths. The pharmaceutical composition of the present invention, which contains cystatin A, exhibits a significantly increased inhibitory effect against colorectal cancer compared to when cystatin is not used. Thus, the pharmaceutical composition of the present invention will be effectively used as a therapeutic agent against colorectal cancer in the medical field.

FIG. 1 is a schematic view showing culture of cancer cell line and cystatin treatment processes of the present invention.

FIG. 2 is a graph showing that the growth of colorectal cancer cells was inhibited by cystatin treatment to the colorectal cancer cell line HCT116 of the present invention.

FIG. 3 is a graph showing that the growth of colorectal cancer cells was inhibited by cystatin treatment to the colorectal cancer cell line DLD-1 of the present invention.

FIG. 4 is a graph showing that the growth of colorectal cancer cells was inhibited by cystatin treatment to the breast cancer cell line MCF7 of the present invention.

When the cystatin A or cystatin D was administered, the growth of the cancer cells (HCT116 and DLD-1 cells) was significantly inhibited compared to when the cystatin SN or cystatin SA was administered. However, in the case of breast cancer cell line (MCF7), every types of cystatin used in experiments had no great effect on the inhibition of the growth of cancer cells. From these results, it was found that cystatin A specifically inhibited the growth of colorectal cancer cells.

Hereinafter, the present invention will be described in further detail. It will be obvious to those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example: Analysis of the Effects of Cystatin A against Cancer Cells

Each of colorectal cancer cell lines (HCT116 and DLD-1) and breast cancer cell line (MCF7) was seeded in a 12-well plate at a density of 5x10⁴ cells/well, and then cultured for 16 hours. Experimental examples were classified as shown in Table 1 below according to treatment type of cystatin.

After completion of the cystatin treatment for 24 hours, each well was treated with 200 μl of a reagent (MTT formazan, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide) for cell survival rate measurement and incubated for 2 hours. The absorbance of each well was measured using a microplate reader at 570 nm (Amersham, Biorad, USA, Japan), and the cell survival rate was calculated based on the measured value. More specifically, FIG. 1 is schematic views showing the culture of cancer cell line and cystatin treatment processes.

Experimental Example	Definition
mock	Not treated (Negative control)
CSTA	Treated with cystatin A (novoprotein, #C087, 1 μg/mL)
CSTD	Treated with cystatin D (novoprotein, #C397, 1 μg/mL)
CSTSN	Treated with cystatin SN (novoprotein, #C462, 1 μg/mL)
CSTSA	Treated with cystatin SA (novoprotein, #C336, 1 μg/mL)

As a results are shown in FIGS. 2 to 4, when the cystatin A or cystatin D was administered, the growth of the cancer cells (HCT116 and DLD-1 cells) was significantly inhibited compared to when the cystatin SN or cystatin SA was administered. However, in the case of breast cancer cell line (MCF7), every types of cystatin used in experiments had no great effect on the inhibition of the growth of cancer cells. From these results, it was found that cystatin A or cystatin D specifically inhibited the growth of colorectal cancer cells.

Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

The present invention is directed to a pharmaceutical composition for treating colorectal disease, which contains cystatin A. The composition of the present invention has been demonstrated to have significant effects on the inhibition of colorectal cancer growth, and thus will be effectively used as a therapeutic agent against colorectal cancer in the medical field.

Claims

A method of treating colorectal disease, which comprising administering to a subject in need thereof a pharmaceutical composition containing a therapeutically effective amount of cystatin A.
The method of claim 1, wherein the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, and Crohn's disease.
The method of claim 2, wherein the colorectal disease is colorectal cancer.
Use of cystatin A thereof, for preparation of a medicament for treatment of colorectal disease.
The use of claim 4, wherein the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, and Crohn's disease.
The use of claim 5, wherein the colorectal disease is colorectal cancer.
A pharmaceutical composition for treating colorectal disease, which contains, as active ingredients, cystatin A.
The pharmaceutical composition of claim 7, wherein the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, and Crohn's disease.
The pharmaceutical composition of claim 8, wherein the colorectal disease is colorectal cancer.
A food composition for alleviating colorectal disease, which contains, as active ingredients, cystatin A.
The food composition of claim 10, wherein the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, and Crohn's disease.
The food composition of claim 11, wherein the colorectal disease is colorectal cancer.