Use of CHP for preventing, improving or treating peritoneal fibrosis

Abstract

The present invention relates to the use of CHP for preventing, improving or treating peritoneal fibrosis, and more specifically, to a pharmaceutical composition containing CHP for preventing or treating peritoneal fibrosis, a peritoneal dialysis fluid for preventing or improving peritoneal fibrosis, a method for preventing or treating peritoneal fibrosis using CHP, a peritoneal dialysis method using CHP, the use of CHP in preparing a pharmaceutical composition for preventing or treating peritoneal fibrosis, and / or the use of CHP in preparing a peritoneal dialysis fluid.

Description

Technical Field

[0001] The present invention relates to the use of CHP for preventing, improving or treating peritoneal fibrosis, and more specifically, to a pharmaceutical composition containing CHP for preventing or treating peritoneal fibrosis, a peritoneal dialysis fluid for preventing or improving peritoneal fibrosis, a method for preventing or treating peritoneal fibrosis using CHP, a peritoneal dialysis method using CHP, the use of CHP in preparing a pharmaceutical composition for preventing or treating peritoneal fibrosis, and / or the use of CHP in preparing a peritoneal dialysis fluid. Background Art

[0002] Peritoneal dialysis is one of the representative renal replacement therapies and is a dialysis method that uses the peritoneum, which is the body's own filter. However, it has a fatal drawback that it cannot be sustained if the peritoneum, which is the only dialysis membrane, is not functional enough.

[0003] As a result, peritoneal fibrosis (PF) has become the main cause of peritoneal structural changes and ultrafiltration disorders in patients with continuous ambulatory peritoneal dialysis (CAPD). Encapsulating peritoneal sclerosis (ESP), an extreme form of peritoneal fibrosis, is a representative cause of difficulty in continuous peritoneal dialysis. There is currently no clear prevention method. Due to the characteristics of fibrosis-sclerosis, normal function cannot be restored. Therefore, once diagnosed, the course of the disease is very fatal.

[0004] As described above, attempts to suppress peritoneal fibrosis in renal patients can not only suppress progression to chronic kidney disease that causes socioeconomic losses, but is also considered to be an important issue that can determine the sustainability of renal replacement therapy, and therefore the development of therapeutic agents therefor is urgently needed.

[0005] In addition, it is known that the main characteristics of peritoneal fibrosis are the epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMC) and the peritoneal accumulation of extracellular matrix (ECM) proteins, and transforming growth factor (TGF)-β1 plays a key role in the development of peritoneal fibrosis.

[0006] This epithelial-mesenchymal transition is known to be characterized by a decrease in E-cadherin expression and accumulation of de novo α-smooth muscle actin (α-SMA) expression. Epithelial-mesenchymal transition is known to be a reversible process, so inhibition of epithelial-mesenchymal transition can be considered a therapeutic target for preserving peritoneal function.

[0007] In this context, the present inventors confirmed that CHP (Cyclo-His Pro) showed the effects of inhibiting epithelial-mesenchymal transition of the peritoneum, inhibiting cell apoptosis of the peritoneum or infiltration of immune cells into the peritoneum in an animal model of induced peritoneal fibrosis, and was effective in treating peritoneal fibrosis, thereby completing the present invention.

[0008] In addition, Korean Patent Publication No. 10-2013-0006170 discloses a composition for regulating blood sugar containing soybean hydrolyzate containing CHP (CYCLO (His-Pro)) at a high concentration, but the anti-fibrotic effect of CHP is not yet clear. Summary of the invention

[0009] Technical issues

[0010] An object of the present invention is to provide a pharmaceutical composition comprising CHP for preventing or treating peritoneal fibrosis.

[0011] Another object of the present invention is to provide a peritoneal dialysis solution containing CHP.

[0012] Still another object of the present invention is to provide a method for preventing or treating peritoneal fibrosis using CHP.

[0013] Another object of the present invention is to provide a peritoneal dialysis method using CHP.

[0014] Another object of the present invention is to provide the use of CHP in preparing a pharmaceutical composition for preventing or treating peritoneal fibrosis.

[0015] Another object of the present invention is to provide the use of CHP in preparing peritoneal dialysis fluid.

[0016] Technical Solution

[0017] In order to solve the problems as described above, the present invention provides a pharmaceutical composition for preventing or treating peritoneal fibrosis, comprising CHP or a pharmaceutically acceptable salt thereof.

[0018] Furthermore, the present invention provides a peritoneal dialysis solution comprising CHP or a pharmaceutically acceptable salt thereof.

[0019] Furthermore, the present invention provides a method for preventing or treating peritoneal fibrosis, comprising the step of administering an effective amount of CHP to a subject in need thereof.

[0020] Furthermore, the present invention provides a peritoneal dialysis method comprising the step of administering an effective amount of CHP to a subject in need thereof.

[0021] Furthermore, the present invention provides the use of CHP in the preparation of a pharmaceutical composition for preventing or treating peritoneal fibrosis.

[0022] Furthermore, the present invention provides the use of CHP in the preparation of peritoneal dialysis fluid.

[0023] According to a preferred embodiment of the present invention, the peritoneal fibrosis may include at least one selected from the group consisting of retroperitoneal fibrosis and encapsulating peritoneal sclerosis.

[0024] According to another preferred embodiment of the present invention, CHP or a pharmaceutically acceptable salt thereof can show one or more effects selected from the group consisting of inhibiting epithelial-mesenchymal transition of peritoneum, inhibiting apoptosis of peritoneum and inhibiting infiltration of immune cells into peritoneum.

[0025] According to another preferred embodiment of the present invention, peritoneal fibrosis may occur via dialysis.

[0026] Effects of the Invention

[0027] The composition containing CHP of the present invention shows the effects of suppressing epithelial-mesenchymal transition, suppressing apoptosis, or suppressing infiltration of immune cells into the peritoneum in fibrosis-induced peritoneum, and thus can effectively prevent, improve, or treat peritoneal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The photographs show the peritoneum of the animal model of peritoneal fibrosis to which CHP (17.5 mg / kg and 35 mg / kg, respectively) was stained with periodic acid Schiff (PAS), Masson's Trichrome (MT) and Sirius red to confirm the degree of fibrosis.

[0029] Figure 2 The photographs are for observing the degree of peritoneal sclerosis by confirming the protein expression levels of intercellular adhesion molecule-1 (ICAM-1) and F4 / 80 in the peritoneum of the peritoneal fibrosis animal model administered with CHP (17.5 mg / kg and 35 mg / kg, respectively) by immunohistochemistry (IHC) staining.

[0030] Figure 3a Western blotting was used to confirm the changes in protein expression levels of collagen 1 and α-smooth muscle actin, which are markers of fibrosis, and p16 and p21, which are markers of apoptosis, in the peritoneal tissue of an animal model of peritoneal fibrosis that was administered CHP (17.5 mg / kg and 35 mg / kg, respectively). Figure 3b (Collagen 1), Figure 3c (α-smooth muscle actin), Figure 3d (p21) and Figure 3e (p16) is the result of quantifying the size of the band of each target and plotting it in a graph.

[0031] Figure 4a This is a graph showing changes in the gene expression level of collagen 1, a marker of fibrosis, in the peritoneal tissue of an animal model of peritoneal fibrosis to which CHP (17.5 mg / kg and 35 mg / kg, respectively) was administered. Figure 4b This is a graph showing changes in gene expression levels of p21, a marker for cell cycle arrest. Figure 4c This is a graph confirming changes in the gene expression level of E-cadherin, a cell synapse marker. DETAILED DESCRIPTION

[0032] Hereinafter, the present invention will be described in more detail.

[0033] As described above, attempts to inhibit peritoneal fibrosis in renal patients can not only inhibit progression to chronic kidney disease that causes socioeconomic losses, but is also considered to be an important issue that can determine the sustainability of renal replacement therapy, and therefore the development of therapeutic agents therefor is urgently needed.

[0034] Therefore, the present inventors confirmed that CHP (Cyclo-His Pro) showed the effects of inhibiting epithelial-mesenchymal transition of the peritoneum, inhibiting peritoneal cell apoptosis or immune cell infiltration into the peritoneum in an animal model of induced peritoneal fibrosis, and was effective in treating peritoneal fibrosis, thereby completing the present invention.

[0035] Thus, the present invention provides a pharmaceutical composition for preventing or treating peritoneal fibrosis, comprising CHP or a pharmaceutically acceptable salt thereof.

[0036] In the present invention, "CHP (Cyclo-HisPro)" refers to a naturally occurring cyclic dipeptide composed of histidine-proline which is a metabolite of thyrotropin-releasing hormone (TRH) or a physiologically active dipeptide synthesized in vivo through the metabolic process of thyrotropin-releasing hormone and de novo, and refers to a substance widely distributed throughout the brain, spinal cord, gastrointestinal tract, etc.

[0037] In the composition of the present invention, the CHP may be synthesized or commercially available, and may be purified from substances containing CHP, such as prostate extracts and soybean hydrolyzates, for use.

[0038] The term "purified" is used to mean that CHP is in a concentrated form compared to the form that can be obtained from natural sources such as prostate extract. Purified components can be concentrated from their natural sources or obtained by chemical synthesis methods.

[0039] In the present invention, "peritoneal fibrosis" refers to a state in which the peritoneum is fibrotic, and refers to abnormal formation of fibroblasts in the peritoneum, and includes, for example, retroperitoneal fibrosis and encapsulating peritoneal sclerosis, but is not limited thereto. For example, peritoneal fibrosis can occur through dialysis.

[0040] In the composition for preventing, improving or treating peritoneal fibrosis of the present invention, CHP or a pharmaceutically acceptable salt thereof may exhibit one or more effects selected from the group consisting of inhibiting epithelial-mesenchymal transition of peritoneum, inhibiting apoptosis of peritoneum or infiltration of immune cells into peritoneum.

[0041] In the present invention, the term "epithelial-mesenchymal transition" refers to the phenotype of epithelial cells being transformed into mesenchymal cells. Specifically, epithelial-mesenchymal transition is a phenomenon in which the connection between cells is loosened and the cell skeleton changes, thereby allowing the tissue to acquire mobility, and refers to the phenomenon that cells lose their original cell phenotype and transform into the phenotype of mesenchymal cells. If epithelial-mesenchymal transition is induced, it can lead to the accumulation of fibroblasts, and the induction of epithelial-mesenchymal transition is considered to be an important mechanism of peritoneal fibrosis.

[0042] Therefore, in one embodiment of the present invention, the expression changes of epithelial-mesenchymal transition markers such as E-cadherin and α-smooth muscle actin according to the administration of CHP were confirmed in an animal model of peritoneal fibrosis, thereby evaluating the anti-fibrotic effect. Figure 3a , Figure 3c and Figure 4c It was confirmed that CHP inhibited the expression of α-smooth muscle actin, a marker of fibrosis, and increased the expression of E-cadherin, a cell synaptic marker, in peritoneal tissue. The results showed that CHP showed anti-fibrotic effects by inhibiting the epithelial-mesenchymal transition of the peritoneum.

[0043] In another specific embodiment of the present invention, in order to evaluate the apoptosis of peritoneal cells in response to administration of CHP in an animal model of induced peritoneal fibrosis, the expression changes of apoptosis markers, namely p16 and p21, were confirmed. Figure 3a , Figure 3d , Figure 3e and Figure 4b It was confirmed that the expressions of p16 and p21 in the CHP-treated group were significantly decreased, indicating that cell apoptosis was suppressed.

[0044] The term "fibrosis" refers to a condition, disease or disorder characterized by dysregulated proliferation or activity of fibroblasts, abnormal accumulation of fibronectin, and / or pathological or excessive accumulation of collagen tissue.

[0045] Therefore, in another specific embodiment of the present invention, in an animal model of induced peritoneal fibrosis, changes in the expression of collagen 1 in response to administration of CHP were confirmed, thereby evaluating the anti-fibrotic effect. Figure 1 (Sirius red staining results), Figure 3a , Figure 3b and Figure 4a It was confirmed that CHP significantly reduced the expression of collagen 1 that was increased due to fibrosis induction, which showed that it had an excellent anti-fibrotic effect.

[0046] In another specific embodiment of the present invention, in an animal model of induced peritoneal fibrosis, the infiltration degree of immune cells in response to administration of CHP is confirmed, thereby evaluating the therapeutic effect of peritoneal fibrosis. Figure 1 (Iodic acid-Schiff staining results) and Figure 2 It was confirmed that CHP concentration-dependently reduced the deposition of immune cells that increased due to fibrosis induction, reduced the expression of intercellular adhesion molecule-1 (ICAM-1) that induced the initial immune response, and reduced the expression of f4 / 80 positive cells, a marker of macrophages and dendritic cells, thereby rapidly reducing the infiltration of immune cells. Therefore, it can be seen that CHP hinders the progression of peritoneal fibrosis by reducing the infiltration of immune cells.

[0047] In the composition for preventing, improving or treating peritoneal fibrosis of the present invention, the terms "prevention", "improvement" and / or "treatment" refer to all actions that inhibit or delay a disease or condition, all actions that improve or beneficially change the state of a disease or condition, and all actions that delay, stop or reverse the progression of a disease or condition.

[0048] In the present application, the term "pharmaceutically acceptable" means that it is physiologically acceptable and generally does not cause allergic reactions or similar reactions when administered to the human body. Preferably, the above salt is an acid addition salt formed by a pharmaceutically acceptable free acid.

[0049] The above-mentioned pharmaceutically acceptable salt can be an acid addition salt formed by an organic acid or an inorganic acid, and the above-mentioned organic acid includes formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or methylsulfonic acid. Inorganic acid includes for example hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid or boric acid. The acid addition salt is preferably a chlorate or acetate form, more preferably a chlorate form.

[0050] Besides these, other examples of acceptable salts include γ-aminobutyric acid (GABA) salts, gabapentin salts, pregabalin salts, nicotinate salts, adipate salts, hemi-malonate salts, cysteine ​​salts, acetylcysteine ​​salts, methionine salts, arginine salts, lysine salts, ornithine salts or aspartate salts and the like.

[0051] In addition, the pharmaceutical composition of the present invention may also include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration include water, appropriate oils, saline solutions, aqueous glucose solutions, ethylene glycol, etc. In addition, the above-mentioned carrier may also include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl parahydroxybenzoate or propyl parahydroxybenzoate and chlorobutanol. Other pharmaceutically acceptable carriers may refer to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0052] The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. The method of parenteral administration can be intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, and rectal administration, but is not limited thereto.

[0053] The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the administration route as described above. In the case of formulation, it can be prepared by using more than one buffer (e.g., saline solution or phosphate buffered saline (PBS)), carbohydrates (e.g., glucose, mannose, sucrose or dextran, etc.), antioxidants, antibacterial agents, chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA) or glutathione), fillers, extenders, adhesives, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrating agents, surfactants, diluents or excipients.

[0054] Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions or capsules, etc. Such solid preparations can be prepared by mixing at least one excipient with the pharmaceutical composition of the present invention, and examples of the above excipients include starch (including corn starch, wheat starch, rice starch and potato starch), calcium carbonate, sucrose, lactose, glucose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose or gelatin, etc. For example, after the active ingredient is mixed with the solid excipient and crushed, a suitable auxiliary agent is added thereto and processed into a granular mixture, thereby obtaining a tablet or a sugar-coated tablet.

[0055] In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration correspond to suspensions, internal liquids, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water or liquid paraffin, they can also include several excipients such as wetting agents, sweeteners, aromatics or preservatives.

[0056] Furthermore, depending on the situation, cross-linked polyvinyl pyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrator, and anticoagulants, lubricants, wetting agents, flavors, emulsifiers and preservatives may also be added.

[0057] In the case of parenteral administration, the pharmaceutical composition of the present invention can be formulated into an injection form, a transdermal agent and an intranasal inhalation agent together with a suitable carrier for parenteral administration by methods known in the art. The above-mentioned injection must be sterilized and needs to be protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections may include water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), mixtures thereof and / or solvents or dispersants including vegetable oils, but are not limited thereto. More preferably, Hanks solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, or isotonic solution (such as 10% ethanol, 40% propylene glycol or 5% glucose) can be used as a suitable carrier. In order to protect the above-mentioned injection from microbial contamination, the injection may also contain various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and thimerosal, etc. Also, in most cases, the above injections may also contain isotonic agents such as sugars or sodium chloride.

[0058] Forms of transdermal preparations include ointments, creams, lotions, gels, external solutions, plasters, coatings and aerosols, etc. The above-mentioned "transdermal administration" refers to the topical administration of a pharmaceutical composition to the skin to deliver an effective amount of active ingredients contained in the pharmaceutical composition into the skin.

[0059] In the case of inhalants, the compound used according to the present invention can be conveniently delivered from a pressurized package or atomizer using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas in the form of an aerosol spray. In the case of a pressurized aerosol, the dosing unit can be determined by providing a valve that delivers the measured amount. For example, gelatin capsules and cartridges used in inhalers or insufflators can be formulated into a powder mixture containing the compound and a suitable powder material such as lactose or starch. The dosage form for parenteral administration is described in a formulary (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour) that is generally known in pharmaceutical chemistry.

[0060] When the pharmaceutical composition of the present invention includes an effective amount of CHP, a preferred effect of preventing, improving or treating peritoneal fibrosis can be provided. In the present application, the term "effective amount" refers to an amount showing more responses compared to a control group, preferably an amount sufficient to prevent, improve or treat peritoneal fibrosis. The pharmaceutical composition of the present invention may include 0.01% to 99.9% CHP, with the remainder being a pharmaceutically acceptable carrier. The effective amount of CHP included in the pharmaceutical composition of the present invention may vary depending on the form in which the composition is commercialized.

[0061] The total effective amount of the pharmaceutical composition of the present invention can be administered to the patient in a single dose, or in multiple doses by a fractionated treatment protocol for long-term administration. The pharmaceutical composition of the present invention can change the content of the active ingredient according to the degree of the disease. For example, based on CHP, 0.001 mg to 100 mg is administered per 1 kg of body weight per day, once or more per day, more preferably, 0.01 mg to 10 mg is administered per 1 kg of body weight per day, once or more per day. However, the dosage of the above-mentioned CHP considers various factors such as the route of administration and the number of treatments of the pharmaceutical composition, the patient's age, weight, health status, gender, severity of the disease, diet and excretion rate to determine the effective dose for the patient. Therefore, in view of this, as long as the ordinary technicians in the field can determine the above-mentioned CHP for the prevention, treatment or improvement of peritoneal fibrosis The appropriate effective dose for a specific purpose. As long as the pharmaceutical composition of the present invention shows the effect of the present invention, its dosage form, route of administration and method of administration are not particularly limited.

[0062] The pharmaceutical composition for preventing or treating peritoneal fibrosis of the present invention can be used alone or in combination with a method using surgery, radiotherapy, hormone therapy, chemotherapy or a biological response controller.

[0063] Furthermore, the present invention provides a peritoneal dialysis solution comprising CHP or a pharmaceutically acceptable salt thereof. The above-mentioned CHP and a pharmaceutically acceptable salt thereof are as described above.

[0064] The CHP or a pharmaceutically acceptable salt thereof of the present invention inhibits epithelial-mesenchymal transition and apoptosis of peritoneal cells, and thus can be included in peritoneal dialysis fluid to improve, delay, prevent or treat peritoneal fibrosis.

[0065] The peritoneal dialysis solution comprising CHP or a pharmaceutically acceptable salt thereof of the present invention may comprise one or more selected from the group consisting of an osmotic agent, a buffer, an electrolyte and a combination thereof. For example, an osmotic agent comprises glucose, a glucose polymer (e.g., maltodextrin, icodextrin), a glucose polymer derivative, a cyclodextrin, a modified starch, hydroxyethyl starch, a polyol, fructose, an amino acid, a peptide, a protein, an amino sugar, glycerol, N-acetylglucosamine (NAG) or a combination thereof. The buffer may comprise bicarbonate, lactate, pyruvate, acetate, citrate, tris (i.e., trishydroxymethylaminomethane), an amino acid, a peptide or a combination thereof. The electrolyte may comprise sodium, potassium, magnesium, calcium and chloride.

[0066] The peritoneal dialysis fluid of the present invention may contain one or more dialysis components (factors or components of the dialysis fluid) and a therapeutically effective amount of an effective substance, and may contain CHP or a pharmaceutically acceptable salt thereof as a component for improving, alleviating, preventing or treating peritoneal fibrosis. The peritoneal dialysis fluid may be a dialysis concentrate, and the dialysis fluid may contain about 0.1 μM to about 1000 μM CHP or a pharmaceutically acceptable salt thereof.

[0067] The peritoneal dialysis fluid can be used as a single dialysis fluid in a single container or a dialysis portion of a container containing a single or multiple chambers, and can be administered to a patient simultaneously with a currently used dialysis fluid or at intervals during peritoneal dialysis.

[0068] Furthermore, the present invention relates to a method for preventing or treating peritoneal fibrosis comprising the step of administering an effective amount of CHP to an individual in need thereof.

[0069] Furthermore, the present invention relates to a peritoneal dialysis method comprising the step of administering an effective amount of CHP to a subject in need thereof.

[0070] In the methods of the present invention, the term "subject" includes any animal (e.g., humans, horses, pigs, rabbits, dogs, sheep, goats, non-human primates, cattle, cats, guinea pigs, or rodents), but is not limited thereto. This term does not indicate a particular age or gender. Thus, female / female, male / male, adult / adult and newborn subjects and embryos are included. Patient refers to a subject suffering from a disease or disorder. The term "patient" includes humans and veterinary subjects.

[0071] In the method for preventing or treating peritoneal fibrosis of the present invention, the effect of CHP and the description of its administration route, administration frequency, dosage, etc. are the same as those described above, and therefore, description thereof will be omitted.

[0072] The peritoneal dialysis method of the present invention can be performed on all patients whose kidneys are damaged and who are unable to excrete water and wastes on their own. For example, the above-mentioned patients may be patients with chronic renal failure, but are not limited thereto.

[0073] The peritoneal dialysis method of the present invention can be performed by methods known in the art, for example, by the following method, but not limited thereto: a catheter leading to the abdominal cavity is inserted into the lower abdomen, dialysate is allowed to enter the body, waste and water in the body escape into the dialysate through diffusion and osmosis, thereby removing water and waste.

[0074] Furthermore, the present invention provides the use of CHP in the preparation of a pharmaceutical composition for preventing or treating peritoneal fibrosis.

[0075] Furthermore, the present invention provides the use of CHP in the preparation of peritoneal dialysis fluid.

[0076] In the use of the present invention, the effect of CHP, the pharmaceutical composition for preventing or treating peritoneal fibrosis, and the description of the peritoneal dialysis solution are the same as those described above, and thus the description thereof will be omitted.

[0077] Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention may be implemented in various variations and may have various embodiments. The specific embodiments and descriptions described below are only to help understand the present invention, and the present invention is not limited to the specific disclosed embodiments. It should be understood that the scope of the present invention includes all variations, equivalent technical solutions or alternative technical solutions within the scope of the present invention's ideas and techniques.

[0078] Embodiments of the present invention

[0079] Preparation Example

[0080] CHP used in the following examples was purchased from BACHEM, Switzerland, and used.

[0081] Example 1

[0082] Confirmation of the protective effect of CHP administration against peritoneal sclerosis in an animal model of peritoneal fibrosis

[0083] 1-1. Administration of CHP to the peritoneal fibrosis animal model and negative control group

[0084] In order to prepare an animal model of peritoneal fibrosis, 7-week-old C57BL / 6 male mice were purchased from Koatech Technology Co., Ltd. and raised under specified conditions (temperature: 22±2°C, relative humidity: 55±10%, daily cycle: 12 hours). Each group of 5 mice was divided into a group, and water and food were freely available in the cage. They were acclimated one week before the experiment and used for the experiment. After the adaptation period, 0.1% chlorohexidine was administered by intraperitoneal injection every 3 days for 30 days to establish an animal model of peritoneal fibrosis. The animals were divided into 4 groups, and the first group of experimental animals was set as a control group (Vehicle) that was administered with phosphate buffer and did not develop peritoneal fibrosis, and the second group was set as a control group (CG-PD) that was administered with phosphate buffer and developed peritoneal fibrosis. The third and fourth groups were the groups with peritoneal fibrosis. In the third group, CHP at a concentration of 17.5 mg / kg (CG-PD+CHP(17.5)) was orally administered via a 1 ml syringe for 30 days. In the fourth group, CHP at a concentration of 35 mg / kg (CG-PD+CHP(35)) was orally administered via a 1 ml syringe for 30 days.

[0085] 1-2. Tissue extraction and staining of peritoneal fibrosis animal model mice

[0086] In the experimental animals designed in Example 1-1, a wide peritoneum was obtained from the xyphoid process to the upper part of the pelvic cavity of the mice in the group that was given the drug and 0.1% chlorhexidine for 30 days, and then sacrificed by whole blood sampling. From the skin to the parietal peritoneal layer, the tissue on the left side of the navel was removed in a size of 0.5cm×1.0cm, and a part was fixed in 10% formalin and embedded in paraffin, thereby immunostaining. The tissue sections were cut into 4μm thickness, and in order to confirm the degree of peritoneal fibrosis, periodic acid Schiff staining, Masson's trichrome staining, and Sirius red staining were performed for evaluation. Photos were taken after observation under an optical microscope (Olympus BX-50, Olympus Optical, Tokyo, Japan).

[0087] 1-3. Confirmation of antifibrotic effects in an animal model of peritoneal fibrosis

[0088] Experimental results, such as Figure 1As shown, the deposition of immune cells was confirmed to increase in the fibrosis control group (CG-PD) compared with the negative control group (vehicle) by iodic acid Schiff staining, and the deposition of immune cells was confirmed to be significantly reduced in a concentration-dependent manner by administering CHP. In addition, the peritoneal layer was confirmed to be thickened in the fibrosis control group (CG-PD) compared with the negative control group (vehicle) by Masson's trichrome staining, and it was confirmed that the thickened peritoneal layer in the 35mg / kg CHP administration group was significantly reduced compared with the 17.5mg / kg administration group. The accumulation of collagen (Collagen) as an extracellular matrix was confirmed to be significantly reduced in a concentration-dependent manner according to the administration of CHP by Sirius red staining.

[0089] The above results confirmed the excellent therapeutic effect of CHP on peritoneal fibrosis.

[0090] Example 2

[0091] Confirmation of the effect of preventing peritoneal sclerosis in an animal model of peritoneal fibrosis

[0092] Peritoneal tissue was removed from mice of each group of Example 1-1, fixed in 10% formalin and embedded in paraffin, and immunostained. The expression was confirmed by coloration with diaminobenzidine tetrahydrochloride (DAB) after reacting with anti-intercellular adhesion molecule 1 antibody and anti-F40 / 80 antibody at 4°C using the streptavidin biotin peroxidase (Vector Laboratories, USA) method. The expression levels were observed under an optical microscope and photographed (Olympus BX-50, Olympus Optical, Tokyo, Japan).

[0093] Experimental results, such as Figure 2 As shown, it was confirmed that the expression level of intercellular adhesion molecule-1, which induces the initial immune response, increased in the fibrosis control group (CG-PD) compared with the negative control group (vehicle), and it was confirmed that the expression level of intercellular adhesion molecule-1 decreased with the decrease in the severity of peritoneal sclerosis by administration of CHP. In addition, it was confirmed that in the CG-PD group, the expression level of f4 / 80 positive cells, which are known as macrophage and dendritic cell markers among immune cells, increased with the increase in peritoneal sclerosis, and then the infiltration of immune cells was rapidly reduced by administration of CHP.

[0094] The above results confirmed that the infiltration of immune cells was suppressed by administration of CHP, thereby inhibiting the progression of peritoneal sclerosis.

[0095] Example 3

[0096] To confirm the protein expression of collagen 1 and α-smooth muscle actin, which are fibrosis markers, and p16 and p21, which are apoptosis markers, in an animal model of peritoneal fibrosis induced by CHP administration

[0097] Isoflurane required for anesthesia and dissection of mice in each group of Example 1-1 was purchased from Hana Pharm, Korea, and the RC2 Rodent CircuitController Anesthesia System of VetEquip Inc., USA was prepared. Phosphate buffer was purchased from Hyclone Laboratories Inc., USA. In order to separate the peritoneal tissue of the mouse, the mouse was anesthetized by respiratory anesthesia using 3% to 3.5% isoflurane. After blood was collected from the heart of the anesthetized mouse, the peritoneal tissue was immediately removed, washed with phosphate buffer, 50 mg of peritoneal tissue was cut off and placed in 500 ul of RIPA buffer supplemented with protease and phosphatase inhibitors, and crushed using a T10 homogenizer from IKA, Germany. After placing on ice for 15 minutes, centrifuge at 15,000 rpm at 4°C. The supernatant was recovered and the protein concentration was determined by the BCA quantitative method, and the protein concentration was determined by Bolt TM Proteins of the same amount of samples were separated by a protein gel electrophoresis system and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk for 1 hour at room temperature and then reacted with primary antibodies of collagen 1, α-smooth muscle actin, NRF2, p16, p21, and GAPDH at 4°C overnight. The membrane was washed with TBST for 3 times, 10 minutes each time, and then reacted with secondary antibodies for 1 hour at room temperature. The membrane was washed with TBST for 3 minutes, 10 minutes each time, and then reacted with ECL to determine the expression level. The size of the bands shown was quantified using the ImageJ program, and the size of each band was divided by the size of the GAPDH band for correction. The statistical significance was analyzed by the Student's t-test statistical method compared with the peritoneal fibrosis control group (CG-PD) (*p<0.05, **p<0.05, ***p<0.0005).

[0098] Experimental results, such as Figure 3a , Figure 3b and Figure 3cAs shown in Figure 2, a significant decrease in collagen, an extracellular matrix, and α-smooth muscle actin, a marker of fibroblasts, was observed in the CHP-administered group, thereby confirming that the progression of peritoneal sclerosis was suppressed. Figure 3a , Figure 3d and Figure 3e As shown, it was confirmed that the p16 and p21 proteins that induce cell cycle arrest and eventually cause apoptosis were significantly reduced by CHP administration.

[0099] This result means that CHP inhibits apoptosis and improves peritoneal fibrosis, which is confirmed as evidence that it can be used to treat peritoneal fibrosis.

[0100] Example 4

[0101] Confirmation of gene expression of collagen 1, a fibrosis marker, p21, a cell cycle arrest marker, and E-cadherin, a cell synapse marker, in an animal model of peritoneal fibrosis induced by CHP administration

[0102]

[111] Isoflurane required for anesthesia and dissection of mice in each group of Example 1-1 was purchased from Hana Pharm, Korea, and the RC2 Rodent Circuit Controller Anesthesia System of Vitrap, USA was prepared. Phosphate buffer was purchased from Hyclone, USA. In order to isolate the peritoneal tissue of mice, the mice were anesthetized by respiratory anesthesia using 3% to 3.5% isoflurane. After blood was collected from the heart of the anesthetized mice, the peritoneal tissue was immediately removed, 50 mg of peritoneum was cut and placed in 500 μL of NucleoZOL, and it was crushed using the T10 homogenizer of Aika, Germany. Then, RNA was extracted according to the total RNA isolation protocol of NucleoZOL, and 1 μg of RNA was synthesized into cDNA by reverse transcription polymerase chain reaction (Reverse Transcription Polymerase Chain Reaction) using the iScript cDNA synthesis kit. The synthesized cDNA was analyzed by real-time polymerase chain reaction using iQ SYBR Green Supermix using the forward / reverse primer sets corresponding to each gene. The expression value of each gene was corrected by dividing it by the expression value of GAPDH, which is a housekeeping gene. The statistical significance was analyzed by Student's t-test statistical method compared with the peritoneal fibrosis control group (CG-PD) (*p<0.05, **p<0.05, ***p<0.0005).

[0103] Experimental results, such as Figure 4a As shown in Figure 2, it was confirmed that the mRNA expression of collagen 1, which is an extracellular matrix, was reduced in the CHP-administered group. Figure 4bAs shown in Figure 2, it was confirmed that the p21 gene, which induces cell cycle arrest and ultimately causes cell apoptosis, was significantly reduced by administration of CHP. Figure 4c As shown, it was confirmed that the expression level of E-cadherin, a cell synapse marker, was significantly increased in the CHP-administered group.

[0104] This result shows that CHP prevents apoptosis and inhibits the progression of peritoneal fibrosis.

Claims

1. Use of CHP or a pharmaceutically acceptable salt thereof in preparing a pharmaceutical composition for preventing or treating peritoneal fibrosis.

2. The use according to claim 1, characterized in that The peritoneal fibrosis is one or more selected from the group consisting of retroperitoneal fibrosis and encapsulating peritoneal sclerosis.

3. The use according to claim 1, characterized in that CHP or a pharmaceutically acceptable salt thereof shows one or more effects selected from the group consisting of inhibition of epithelial-mesenchymal transition of peritoneum, inhibition of apoptosis of peritoneal cells, and inhibition of infiltration of immune cells into peritoneum.

4. The use according to claim 1, characterized in that The peritoneal fibrosis occurs through dialysis.

5. Use of CHP or a pharmaceutically acceptable salt thereof in the preparation of a peritoneal dialysis solution for preventing or treating peritoneal fibrosis.

6. The use according to claim 5, characterized in that CHP or a pharmaceutically acceptable salt thereof shows one or more effects selected from the group consisting of inhibition of epithelial-mesenchymal transition of peritoneum, inhibition of apoptosis of peritoneal cells, and inhibition of infiltration of immune cells into peritoneum.

Images