Pharmaceutical composition for treating or preventing lower gastrointestinal diseases such as inflammatory bowel disease, comprising specific self-assembling peptide RADA16

Abstract

An object of the present invention is to provide a pharmaceutical composition for treating or preventing a lower gastrointestinal disease such as inflammatory bowel disease. The present invention provides a pharmaceutical composition for treating or preventing a lower gastrointestinal disease such as inflammatory bowel disease, which comprises the specific self-assembling peptide RADA16.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for treating or preventing lower gastrointestinal diseases such as inflammatory bowel disease, which comprises the specific self-assembling peptide RADA16. [Background technology]

[0002] Inflammatory bowel disease includes ulcerative colitis and Crohn's disease. Both are diseases of unknown cause that repeatedly worsen and remit chronic inflammation in the intestines, and are designated as intractable diseases by the Ministry of Health, Labor and Welfare with no definitive treatment. Treatments include corticosteroids, anti-inflammatory drugs such as salazosulfapyridine and mesalazine, immunosuppressants such as azathioprine, and antibody preparations such as anti-TNF-α antibodies, administered alone or in combination depending on the condition, but none of these are specific treatments for inflammatory bowel disease. In addition, corticosteroids have many side effects, so long-term administration is not recommended, and anti-TNF-α antibodies carry the risk of serious side effects such as opportunistic infections.

[0003] The peptide having the amino acid sequence shown in SEQ ID NO: 1 (RADARADARADARADA, hereafter referred to as RADA16) is known to have specific self-organizing properties. In recent years, it has attracted attention as a material that can be used for medical purposes due to its physical, chemical, and biological properties. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2016-515113 [Patent Document 2] International Publication No. 2020 / 071527 Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to provide a pharmaceutical composition for treating or preventing inflammatory bowel disease in the lower gastrointestinal tract. [Means for solving the problem]

[0006] The present inventors conducted an intensive search for an effective method for treating inflammatory bowel disease and surprisingly found that a composition containing a specific peptide (RADA16) was effective, thereby completing the present invention.

[0007] That is, in one embodiment, the present invention relates to a pharmaceutical composition for treating or preventing a lower gastrointestinal disease such as inflammatory bowel disease, characterized in that the pharmaceutical composition contains a therapeutically effective amount of a specific peptide (RADA16).

[0008] In one embodiment of the present invention, the lower gastrointestinal disease is characterized in that it is inflammatory bowel disease.

[0009] In one embodiment of the present invention, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0010] In one embodiment of the invention, the pharmaceutical composition is topically applied to the affected area of ​​the subject using an endoscope or an anoscope.

[0011] In one embodiment of the invention, the pharmaceutical composition is administered in the form of a suppository.

[0012] In one embodiment of the present invention, the pharmaceutical composition is characterized in that it contains a specific peptide having an amino acid sequence shown in SEQ ID NO: 1: RADARADARADARADA at a concentration of 0.1% by weight to 10.0% by weight.

[0013] In one embodiment of the present invention, the pharmaceutical composition further comprises another drug effective for the treatment or prevention of a lower gastrointestinal disease, such as inflammatory bowel disease, or is used in combination with another drug effective for the treatment or prevention of a lower gastrointestinal disease, such as inflammatory bowel disease.

[0014] In one embodiment of the present invention, the pharmaceutical composition is used in combination with one or more drugs selected from the group consisting of a duodenal ulcer therapeutic drug, an antidiarrheal drug, an anti-inflammatory drug, a corticosteroid, an immunomodulator or immunosuppressant, an intestinal mucosa protective agent, and a blood flow promoter. Preferably, the duodenal ulcer therapeutic drug is rebamipide or teprenone, which are commercially available as Mucosta (registered trademark) or Selbex (registered trademark).

[0015] In one embodiment of the present invention, the pharmaceutical composition is used in combination with a cell sheet, intestinal stem cells, hematopoietic stem cells, adipose stem cells, or mesenchymal stem cells.

[0016] In one embodiment of the present invention, the pharmaceutical composition is characterized in that it gels by self-assembly when applied to a site of lower gastrointestinal disease, such as inflammatory bowel disease, in a subject.

[0017] In one embodiment of the present invention, the specific peptide is a peptide comprising SEQ ID NO: 1: RADARADARADARADA.

[0018] Another embodiment of the present invention relates to a method for the treatment of lower gastrointestinal tract diseases, such as inflammatory bowel disease, comprising the step of applying to the site of the disease in a patient a pharmaceutical composition comprising a therapeutically effective amount of a specific peptide. Preferably, said method comprises the simultaneous or sequential application of another drug effective in the treatment or prevention of lower gastrointestinal tract diseases, such as inflammatory bowel disease.

[0019] Another embodiment of the present invention relates to the use of specific peptides for the manufacture of a medicament for the treatment or prevention of lower gastrointestinal tract diseases, such as inflammatory bowel disease.

[0020] Yet another embodiment of the present invention relates to a pharmaceutical composition for the treatment or prevention of lower gastrointestinal tract diseases, such as inflammatory bowel disease, comprising a duodenal ulcer therapeutic agent in combination with a peptide comprising the amino acid sequence set forth in SEQ ID NO:1.

[0021] Any combination of one or more of the above features is also included within the scope of the present invention. Effect of the Invention

[0022] According to the present invention, a safe therapeutic drug and treatment method are provided which induces remission of lower gastrointestinal diseases such as inflammatory bowel disease, maintains remission for a long period of time, prevents recurrence, and has extremely few side effects. Furthermore, according to the present invention, even in pathological conditions involving lower gastrointestinal diseases such as inflammatory bowel disease, there is a tendency to suppress weight loss, reduce increases in DAI score, and suppress shortening of the length of the large intestine, and thus has the effect of reducing the progression of the pathological condition. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows the results of the histological scores in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention relates to a pharmaceutical composition for treating or preventing lower gastrointestinal diseases. The present invention has been developed for the treatment of inflammatory bowel diseases, particularly inflammatory bowel diseases caused by ulcerative colitis and Crohn's disease.

[0025] The pharmaceutical composition of the present invention can be used for the treatment or prevention of various pathological conditions of inflammatory bowel disease, and can also be suitably used for the treatment or prevention of refractory inflammatory bowel disease. In this specification, "refractory" inflammatory bowel disease may mean, for example, a disease that shows resistance to general conservative treatments for inflammatory bowel disease (e.g., nutritional therapy, drug therapy, etc.).

[0026] The subject of the present invention may be a human or a non-human. The non-human subject may be, for example, a non-human animal, such as a non-human mammal, a bird, a reptile, an amphibian, or a fish. Examples of the non-human mammal include rodents (e.g., mice, rats), dogs, cats, horses, pigs, cows, sheep, goats, primates, etc.

[0027] The lower digestive tract in which inflammatory bowel disease occurs as referred to in the present invention includes the small intestine and large intestine, and also includes the colon, rectum and anus.

[0028] Although the method and route of administration of the pharmaceutical composition of the present invention are not limited, it is particularly preferable that the method and route of administration are capable of local administration to the affected area, for example, local administration using an endoscope or an anoscope, administration using a nasoenteric tube or an enterostomy tube, administration from a surgical incision site in the intestinal tract, etc. can be used.

[0029] In one embodiment of the invention, the pharmaceutical composition is administered in the form of a suppository.

[0030] The dosage and frequency of administration of the pharmaceutical composition of the present invention can be appropriately determined by those skilled in the art (for example, physicians) depending on the pathology of the subject.

[0031] The concentration of the specific peptide in the pharmaceutical composition of the present invention may be 0.1% by weight to 10.0% by weight, preferably 0.3% by weight to 8.0% by weight, more preferably 0.5% by weight to 5.0% by weight, and most preferably 1.0% by weight to 3.0% by weight.

[0032] The pharmaceutical composition of the present invention may further contain other drugs effective for treating or preventing lower gastrointestinal diseases such as inflammatory bowel disease, or may be used in combination with other drugs effective for treating or preventing inflammatory bowel disease and other lower gastrointestinal diseases. The mode of combination of the pharmaceutical composition of the present invention with other drugs is not limited, and may be, for example, a mode in which the drugs prepared separately are administered to a subject at the same time, or a mode in which the drugs are administered to a subject at different times.

[0033] Other drugs that may be used together with the pharmaceutical composition of the present invention may surprisingly be duodenal ulcer treatments, such as, but not limited to, rebamipide or teprenone, which are commercially available as Mucosta® or Selbex®.

[0034] Other drugs that may be used together with the pharmaceutical composition of the present invention may be drugs commonly used to treat lower gastrointestinal diseases such as inflammatory bowel disease (ulcerative colitis, Crohn's disease), etc. Drugs commonly used to treat inflammatory bowel disease (ulcerative colitis, Crohn's disease) include, for example, antidiarrheals, anti-inflammatory agents, corticosteroids, immunomodulators or immunosuppressants, intestinal mucosa protective agents, and blood flow promoters.

[0035] Antidiarrheal drugs that may be used with the pharmaceutical composition of the present invention include, for example, diphenoxylate, loperamide, deodorized opium tincture, and codeine. Anti-inflammatory drugs that may be used with the pharmaceutical composition of the present invention include, for example, salazosulfapyridine and related drugs (mesalazine, olsalazine, balsalazide, etc.). Corticosteroids that may be used with the pharmaceutical composition of the present invention include, for example, prednisolone, budesonide, and hydrocortisone. Immunomodulators or immunosuppressants that may be used with the pharmaceutical composition of the present invention include, for example, tacrolimus, azathioprine, mercaptopurine, cyclosporine, infliximab, adalimumab, and golimumab.

[0036] When the pharmaceutical composition of the present invention is used together with other drugs, the ratio of the pharmaceutical composition of the present invention to other drugs may be any ratio.

[0037] Other drugs that may be used together with the pharmaceutical composition of the present invention may be, for example, compositions containing cell sheets, intestinal stem cells, hematopoietic stem cells, adipose stem cells, or mesenchymal stem cells. Mucosal regeneration therapy for patients with inflammatory bowel disease using cell sheets, stem cells, etc. is being developed, and those skilled in the art can combine treatment with the pharmaceutical composition of the present invention and mucosal regeneration therapy using cell sheets, stem cells, etc. In addition, a synergistic effect can be expected by mixing the pharmaceutical composition of the present invention with cell sheets, stem cells, etc. and applying the mixture to a subject.

[0038] The specific peptide RADA16 used in the present invention may be modified (or labeled) as long as the main properties of the peptide intended by the present invention are not lost, and such modified (or labeled) peptides are also included in the "specific peptide" of the present invention. The method of modifying (or labeling) the specific peptide used in the present invention can be selected arbitrarily by those skilled in the art, and may be, for example, the addition of a functional group or the like, the addition of a chemical substance, or the addition of an additional protein or peptide. Examples of the addition of the functional group or the like include acylation, acetylation, alkylation, amidation, biotinylation, formylation, carboxylation, glutamylation, glycosylation (addition of a sugar chain), glycylation, hydroxylation, isoprenylation, lipoylation, the addition of a nucleotide or a derivative thereof, polyethylene glycol (PEG)ization, and the addition of a lipid chain. Examples of the addition of a chemical substance include the addition of a suitable labeling agent, for example, a radioisotope (e.g., 125 1. 131 I, 3 H, 14 Examples of the addition of further proteins or peptides include addition of ISG conjugates, SUMO conjugates, and ubiquitin conjugates. Examples of the addition of further proteins or peptides include addition of ISG conjugates, SUMO conjugates, and ubiquitin conjugates. Examples of the addition of further proteins or peptides include addition of ISG conjugates, SUMO conjugates, and ubiquitin conjugates. Examples of the addition of further proteins or peptides include addition of ISG conjugates, SUMO conjugates, and ubiquitin conjugates. Examples of the addition of further proteins or peptides include addition of ISG conjugates, SUMO conjugates, and ubiquitin conjugates.

[0039] It should be noted that the terms used in this specification are used to describe particular embodiments and are not intended to limit the invention.

[0040] Furthermore, the term "comprise" as used in this specification intends that the described items (components, steps, elements, numbers, etc.) are present, unless the context clearly indicates otherwise, and does not exclude the presence of other items (components, steps, elements, numbers, etc.).

[0041] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in the present specification and related technical fields, and should not be interpreted in an idealized or overly formal sense, unless otherwise defined.

[0042] Although terms such as first, second, etc. may be used to describe various elements, it is understood that these elements should not be limited by these terms, and these terms are used only to distinguish one element from another element, and for example, a first element can be described as a second element, and similarly, a second element can be described as a first element, without departing from the scope of the present invention.

[0043] The present invention will be described in more detail below with reference to examples. However, the present invention can be embodied in various forms and should not be construed as being limited to the examples set forth herein. EXAMPLES

[0044] Example 1 Anal administration experiment on mice with inflammatory bowel disease Mice were given a 2.5 w / v% aqueous solution of dextran sulfate sodium, and the efficacy of a specific peptide having the amino acid sequence shown in SEQ ID NO: 1 against an induced ulcerative colitis model was examined using the Disease Activity Index. Similarly, duodenal ulcer treatment agents Mucosta (registered trademark) and Selbex (registered trademark) were administered alone or in combination with a peptide having the amino acid sequence shown in SEQ ID NO: 1, and ulcerative colitis treatment agents Pentasa (registered trademark) and Rectaburu (registered trademark) were administered alone to compare their efficacy. Furthermore, the plasma and excised colon collected on the final day of the experiment were sent to the test contractor. Dextran sulfate sodium aqueous solution drinking period (preparation of dextran sulfate sodium induced ulcerative colitis model): November 16th ~ November 25th, 2019 Treatment period: November 22, 2019 to November 24, 2019 Disease Activity Index (DAI: evaluation of body weight, stool condition and fecal occult blood): November 16, 22 and 25, 2019 Blood collection: November 25, 2019 Colon removal and length measurement: November 25, 2019 Test End Date: November 25th, 2019

[0045] 1. Materials and Methods 1.1 Test Substance Name: A specific peptide having the amino acid sequence shown in SEQ ID NO:1 (hereafter abbreviated as RADA) Peptide + Mucosta (hereinafter referred to as RADA / Mu) Peptide + Selvex (hereinafter referred to as RADA / Se) Provided by: Test sponsor Pre-mixed liquid was obtained. Properties: Liquid Storage conditions: Protect from light, airtight, refrigerated (tolerance range: 1.0 to 8.0°C) Storage location: Cooled storage room for test substances Disposition of remainder: Discarded.

[0046] 1.2. Control substances 1.2.1 Negative control substances Name: Japanese Pharmacopoeia Saline Solution (hereafter abbreviated as Saline Solution) Manufacturer: Otsuka Pharmaceutical Factory, Inc. Lot number: K9D85 Storage conditions: Room temperature (tolerance range: 1.0 to 30.0°C) Storage location: Test substance storage room

[0047] 1.2.2 Control substances Name: 1) Mucosta (registered trademark) Ophthalmic Solution UD 2% (hereinafter abbreviated as Mucosta or Mu) 2) Selbex (registered trademark) Granules 10% (hereinafter abbreviated as Selbex or Se) 3) Pentasa® Enema 1g (hereinafter abbreviated as Pentasa or Pen) 4) Rectabular (registered trademark) 2 g enema foam, 14 times (hereinafter abbreviated as Rectabular or Rec) Manufacturer: 1) Otsuka Pharmaceutical Co., Ltd. 2) Eisai Co., Ltd. 3) Kyorin Pharmaceutical Co., Ltd. 4) Kissei Pharmaceutical Co., Ltd. Lot number: 1), 2) None 3) R11655A 4) K037A Properties: Liquid Storage conditions: 1), 2) Light-proof, airtight, refrigerated (allowable range: 1.0 to 8.0°C) 3), 4) Light-shielded, airtight, room temperature (tolerance range: 1.0 to 30.0°C) Storage location: 1) 2) Refrigerated storage room for test substances 3) and 4) Test substance storage room Disposition of remainder: Discarded.

[0048] 1.3 Ulcerative colitis inducers Name: Dextran Sodium Sulfate 5000 (hereinafter abbreviated as DSS) Manufacturer: Fujifilm Wako Pure Chemical Corporation Lot number: PTF0313 Storage conditions: Room temperature (tolerance range: 1.0 to 30.0°C) Storage location: Test substance storage room Treatment of the remainder: Excluded from the study.

[0049] 1.4 Dosing solution 1.4.1 Test substance, control substance and negative control substance administration solutions Preparation method: No preparation was performed as the preparation was used as is. Disposal of residual liquid after administration: Disposed of in an industrial waste container for incineration.

[0050] 1.4.2 DSS aqueous solution (for drinking water) Preparation method: 1) 125.00 g of DSS was weighed out and transferred to a glass beaker. 2) 4000 mL (80% of the final volume of the solution) of drinking water used for animals in the breeding room was added, and the solution was stirred with a magnetic stirrer to dissolve. 3) After confirming that the solution had dissolved, the solution was transferred to a measuring cylinder and water was added to make up to 5000 mL (DSS 2.5 w / v% aqueous solution). 4) Stir with a magnetic stirrer. Storage conditions: Room temperature (tolerance range: 1.0 to 30.0°C) Although the expiration date was 7 days after preparation, the product was used within 4 days after preparation. Storage location: Test substance preparation room and breeding room Disposal of remaining liquid after administration: Disposed of in an industrial waste container dedicated to incineration.

[0051] 1.5 Test System Species: Mouse (SPF) Strain: BALB / cAnNCrlCrlj Source: Charles River Japan Co., Ltd. (Atsugi Breeding Center) Reason for selecting this test system: This mouse strain is widely used for toxicity and drug efficacy tests. Gender and number ordered (obtained): Male: 60 Age when obtained: 9 weeks

[0052] Quarantine and Acclimatization: The animals were quarantined for 6 days from the day of acquisition. During the quarantine period, their general condition was observed every day, and their body weight was measured on the first day (the day of acquisition), the third day, and the seventh day (the day of end of quarantine). In addition, the period from the day of acquisition to the day before the animals started drinking the DSS aqueous solution was set as the acclimatization period, and even after the quarantine period, their general condition was observed once a day, and their body weight was measured on the day of grouping. Age at start of test substance administration: 11 weeks

[0053] 1.6 Rearing conditions Temperature: Allowable range: 21.0~25.0℃ Humidity: Tolerance range: 40.0~70.0% Ventilation rate: 15-17 times / hour Lighting hours: 12 hours / day (artificial lighting from 7am to 7pm) Feeding method: Feed was provided in stainless steel feeders and was available ad libitum. Water supply method: Before starting to drink the DSS aqueous solution: Water was freely available from a glass water dispenser equipped with a touch-drink tip. After starting drinking DSS solution: Untreated group: Water was freely available from a glass or polycarbonate water dispenser equipped with a touch-drinking tip. Groups other than the untreated group: Mice were allowed to freely consume 2.5 w / v% DSS aqueous solution through a glass or polycarbonate water dispenser equipped with a touch-drinking tip tube. Housing in cages: Mice were individually housed in polycarbonate cages (17.5W x 24.5D x 12.5H cm, with bedding). Cage replacement frequency: At least once every four days (bedding and water dispensers were also replaced) Environmental enrichment: During the rearing period, chew sticks (wood chips) were provided in the cages.

[0054] 1.7 Breeding materials and analysis 1.7.1 Feed Name: CRF-1 (solid) Supplier: Oriental Yeast Co., Ltd. (Chiba)

[0055] 1.7.2 Drinking water Type: Municipal water supply

[0056] 1.7.3 Wood chips for environmental enrichment Name: Wood chip (approx. 5 x 2 x 2 cm, high pressure steam sterilization: 121°C, 20 minutes) Source: Chiba Animal Materials Co., Ltd. Material: Fir Replacement frequency: At least once a week

[0057] 1.8 Individual Identification Animals: On the day of acquisition, animals were assigned identification numbers (1 to 60) by ear punching. Cage: Using cage cards, indicate the test number, lineage, sex, and identification number on the day of animal acquisition, and indicate the group name, animal number, and group color when dividing the animals into groups. a) was added. a) Untreated group: white RADA / Se group: purple Control group: Black Mu group: Pink RADA group: blue Se group: green Pen group: Orange RADA / Mu group: Red Rec group: Light blue

[0058] 1.9 Grouping [Table 2] Handling of non-study animals: The remaining animals after group division: 10 animals were used for the study in Section 1.11.3 "Study of administration volume."

[0059] 1.10 Dosage [Table 3]

[0060] 1.11 Administration 1.11.1 Route of Administration Route of administration: Anal 1.11.2 Reason for selection: The same route as the clinical application route was selected. Method of administration: Administration method: The subjects were anesthetized by inhalation of Japanese Pharmacopoeia isoflurane (Mylan Pharmaceuticals Co., Ltd.; hereafter abbreviated as isoflurane). If there was any retained fecal mass in the rectum, it was massaged to remove as much feces as possible. The administration liquid was drawn into a 1 mL polypropylene syringe (sterilized disposable product), inserted into the anus using a mouse probe (approximately 1 cm from the tip), and slowly administered via the anus. The mouse probe and the anus were held down with the hand during administration and for approximately 10 seconds after administration to prevent the administration liquid from leaking out of the anus. The probe was then slowly removed, and any liquid that had leaked out of the anus was wiped away. The test substance administration liquid and the rectable administration liquid were mixed gently by inversion with the hand to avoid the inclusion of large air bubbles, and the other control substance administration liquids were mixed to avoid foaming, before being drawn into the syringe. Volume administered: 0.7mL Reason for selection: This is a common and appropriate administration method for mice.

[0061] 1.11.3 Consideration of volume of administered fluid Target animals: Of the 10 animals excluded from group allocation, the 7 animals with the lowest identification numbers were used for the study. Study method: Under isoflurane inhalation anesthesia, 0.7mL of saline solution colored with India ink (calculated based on colon diameter: approx. 3mm, length from terminal ileum to administration site (rectum): approx. 10cm) was administered into the rectum and held for approx. 10 seconds to prevent leakage from the anus. After that, the abdomen was opened to confirm whether saline solution filled the colon. 0.5mL was also examined in the same way. Since it was confirmed that physiological saline adequately filled the colon when 0.7 mL was administered, 0.7 mL was used as the administration volume for the experiment. Animal treatment: Three surviving animals that were excluded from grouping and were not used in the study were euthanized by carbon dioxide inhalation on the day of starting administration of the test substance. After the end of the study, the seven animals used in the study were killed by exsanguination by cutting the posterior vena cava and abdominal aorta under isoflurane inhalation anesthesia.

[0062] 1.12 Experiments The day when the subjects started drinking the DSS aqueous solution was designated as Day 1, the following day as Day 2, and the subsequent days were designated as Day 2.

[0063] 1.12.1 Preparation of DSS-induced ulcerative colitis model Target animals: All test animals after group division (excluding the untreated group) Model creation period: Day 1~10 Model creation method: On the day when the mice started drinking the DSS aqueous solution (between 13:00 and 14:00), the water in the glass or polycarbonate water dispenser placed in the cage was replaced with a 2.5 w / v% DSS aqueous solution, and the mice were allowed to drink the water ad libitum.

[0064] 1.12.2 Treatment of Test Substances Target animals: All test animals after group division (excluding the untreated group) Treatment days: Day 7, 8 and 9 Treatment frequency: Once a day Treatment time: Between 1pm and 4pm Special note: When the volume of fluid administered was as specified in Section 1.11.3 “Consideration of the volume of fluid administered”, it was determined that the entire volume of fluid was administered even if some fluid leaked from the anal area.

[0065] 1.12.3 General Condition [Table 4]

[0066] 1.12.4 Weight Measurement target: All cases Measurement time and frequency: Day 1, 4, 5, 7 and 10 (before administration) Equipment used: Electronic balance UX4200H (Shimadzu Corporation)

[0067] 1.12.5 Collection of Plasma Samples Blood collection subjects: All cases except for deceased cases Blood collection time: Day 10 Blood sampling site: Posterior vena cava Amount of blood collected: 0.4mL Plasma volume: 100μL or more (50μL or more x 2 bottles) Blood collection method: Animals were anesthetized with isoflurane inhalation, and blood was collected using a 1 μL polypropylene syringe containing approximately 2.5″L of heparin sodium (10,000 units / 10 mL) and a 25-gauge needle (both sterilized disposable products). Blood processing: Collected blood was placed in a polypropylene tube, immediately cooled on ice, and centrifuged (1600×g, 10 min, 4℃) within 30 min after collection to obtain plasma. Plasma was dispensed into 2 polypropylene tubes (50 μL or more) and frozen until shipment. Plasma storage conditions: Frozen (allowable range: -95.0℃~-65.0℃) Plasma storage location: Ultra-low temperature freezer in the freezer room

[0068] 1.12.6 Removal of the large intestine and measurement of its length Target of removal: All cases except for deceased cases Extraction time: Day 10 Extraction method: Under isoflurane inhalation anesthesia, after blood collection, the posterior vena cava and abdominal aorta were cut to exsanguinate the animals, and the large intestine was extracted (from the terminal ileum to the anus). Measurement of colon length: The length (mm) of the excised colon was measured using a caliper and the colon was photographed. The excised colon was then fixed in 10 vol% neutral buffered formalin. Length of large intestine: Terminal ileum to just before rectum, terminal ileum to anus

[0069] 1.12.7 Sample Delivery Date sent: December 17, 2019 Shipping method: Plasma samples were labeled with the test number, animal number, and blood collection date, and sent frozen (packed on dry ice) along with a detailed sample shipping slip. The large intestines, fixed in 10 vol% neutral buffered formalin, were sent at room temperature to the test client.

[0070] 1.12.8 Handling of dead animals Deceased animals: Animal numbers KX03M1 and KX04M5 Immediately after discovery, the animal was weighed and autopsied. The large intestine was removed and fixed in 10 vol% neutral buffered formalin together with the ear (for individual identification), and sent to the test contractor.

[0071] 1.13 Statistical analysis The average values ​​and standard deviations for each group were calculated for the following items. Weight (actual value and rate of change) Change rate (%, reported to one decimal place) = (value at each time point - value on administration day) / value on administration day x 100 Disease Activity Index (DAI) The DAI score was calculated by adding up the scores for body weight, stool condition, and occult blood in stool on Days 1, 7, and 10 according to the score table below. [Table 5] -Length of excised colon For the above three items, a test for equality of variance was performed between the control group and other groups using the F test. If the variances were equal, a Student's t-test was performed, and if the variances were not equal, an Aspin-Welch t-test was performed. All tests were two-sided, and differences observed at a significance level of 5% were considered significant; these are shown in the table separately for 5% and 1%.

[0072] 2. Results and Discussion 2.1 Death (Table 1, Appendices 1, 2 and 3) One death was noted in the RADA group (animal number KX03M1) as follows: <Animal number KX03M1> Observation of the animal's general condition while alive revealed irregular breathing from Day 6, small amounts of feces and occasional piloerection from Day 8, and death was discovered on Day 10. When death was discovered, the animal was in a prone position and had black stains around the anus, but no abnormalities were observed in the excreted feces. Body weight was measured and showed a 21.6% decrease on Day 7 compared to Day 1. Autopsy revealed black stains on the hair around the anus. Based on these findings and the results of the surviving cases described below, the cause of death in the above case was thought to be the progression of DSS-induced ulcerative colitis. The results below are for surviving cases.

[0073] 2.2 General condition In the untreated group, no abnormalities were observed during the observation period. The following changes were observed in the animals with the DSS-induced ulcerative colitis model. Small amounts of feces were observed in 1 to 3 cases in the control group, RADA group, RADA / Se group, Se group, Pen group, and Rec group; irregular breathing was observed in 1 to 3 cases in the RADA group, RADA / Mu group, RADA / Se group, Mu group, Se group, Pen group, and Rec group; piloerection was observed in 1 case each in the RADA group and Rec group; black stool was observed in 1 case each in the RADA group and Se group; and stained perianal hair (pale red) and bleeding from the anus were observed in 1 case each in the RADA group. In addition, in terms of stool characteristics as determined by the DAI score, one patient in the control group was scored 1 (loose stool (mild)) on Day 10, and in terms of stool occult blood (visual inspection), one patient in the RADA group was scored 1 (+) on Day 10. These changes were thought to be due to the progression of DSS-induced ulcerative colitis.

[0074] 2.3 Weight In the untreated group, no notable changes were observed during the observation period. In the control group, weight loss was observed from Day 7 onwards, and compared to the untreated group, both the actual body weight measurements and the rate of weight change were significantly lower on Days 7 and 10. Furthermore, the values ​​on Day 10 were lower than those on Day 7, confirming that DSS-induced ulcerative colitis was progressing during this period as well. In the test substance administration group and the control substance administration group, weight loss was observed from Day 5 onwards, and no significant difference was observed in either the actual weight measurement or the weight change rate compared to the control group, except for the actual weight measurement on Day 5 in the Mu group. However, unlike the control group, no further weight loss was observed on Day 10 in the RADA group and Mu group compared to Day 7, which may have suppressed the progression of DSS-induced ulcerative colitis. The significant low value in the actual weight measurement on Day 5 in the Mu group was considered to be due to the earlier progression of DSS-induced ulcerative colitis, since it was before the administration of Mu.

[0075] 2.4 Disease Activity Index (DAI) The mean DAI scores for each group on days 7 and 10 were as follows: [Table 6] In the untreated group, the scores on both Day 7 and Day 10 were 0.0. The DAI score on Day 10 in the control group was elevated compared to Day 7, which was considered to be due to further progression of ulcerative colitis caused by drinking the DSS aqueous solution. On Day 10, there was no increase in the DAI score in the RADA, RADA / Se, and Mu groups compared to Day 7. This suggests that, unlike the control group, the progression of ulcerative colitis caused by drinking DSS aqueous solution may have been suppressed. On the other hand, on Day 10, the RADA / Mu, Se, Pen and Rec groups showed an increase in DAI scores compared to Day 7, similar to the control group.

[0076] 2.5 Length of the large intestine The average length (mm) of the large intestine in each group from the "terminal ileum to just before the rectum" and "terminal ileum to anus" was as follows. [Table 7] In the control group, the length of the large intestine from the "terminal ileum to just before the rectum" and from the "terminal ileum to the anus" both showed statistically significant decreases compared to the untreated group. In the test substance and control substance groups, the lengths of the large intestine from the "terminal ileum to just before the rectum" and "terminal ileum to anus" in the Mu and Pen groups, and from the "terminal ileum to anus" in the Rec group, were significantly higher than in the negative control group. In the other groups, the length of the large intestine showed a tendency to suppress shortening. The comparative control substances Mucosta, Selvex and Rectable were found to have a clear inhibition of shortening of the colon length, and therefore demonstrated the effect of reducing the progression of DSS-induced ulcerative colitis.

[0077] 3. Conclusion Under these experimental conditions, the negative control group showed a decrease in body weight, an increase in DAI score, and a decrease in colon length as a result of drinking DSS aqueous solution, compared with untreated animals. This indicates that the DSS-induced ulcerative colitis model was appropriately created. In this model animal, the self-assembling peptide (RADA) was administered anally for 3 days, and showed a tendency to suppress weight loss, reduce the increase in DAI score, and suppress the shortening of the colon length, suggesting that it may have an effect of reducing the progression of DSS-induced ulcerative colitis. In addition, based on a comparison of the colon length, it was thought that this reducing effect may be more effective when used in combination with other drugs (Mucosta (registered trademark), Selbex (registered trademark)), but the effect was higher when used alone with Mucosta (registered trademark), Selbex (registered trademark), and Rectab (registered trademark). In addition, when other peptides known to have self-organizing properties were tested in parallel, their effectiveness could not be confirmed.

[0078] Example 2 Histopathological evaluation of HE-stained colonic specimens in mice with ulcerative colitis The HE-stained colon specimen prepared in Example 1 was subjected to histopathological evaluation. 1. Sample (slide specimen) Sample number: 1-18 Assign the above numbers to each slide and create a separate table of correspondence with the numbers displayed on the slides. 2. Histopathological Evaluation For two sections from the upper and lower parts of the colon of each specimen, a pathological score was evaluated in a blinded manner according to the scores below (36 sections in total). Each score of (1), (2), and (3) was multiplied by (4), and the sum of these was determined as the Histological Colitis Score. 1) The lower part of the large intestine is on the frosted side, and the upper part of the large intestine is on the right side. [Table 8] The results of the histological scores are shown in Figure 1. The results showed that administration of RADA alone had an inhibitory effect on intestinal tissue inflammation compared to the negative control, but that combined administration of Mucosta and Selvex produced a greater effect than administration of either drug alone.

Claims

1. A pharmaceutical composition for treating or preventing lower gastrointestinal diseases, which comprises a peptide having the amino acid sequence shown in SEQ ID NO: 1 and is administered by enema.

2. 2. The pharmaceutical composition according to claim 1, wherein the lower gastrointestinal disease is inflammatory bowel disease.

3. 3. The pharmaceutical composition according to claim 2, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

4. The pharmaceutical composition according to any one of claims 1 to 3, for rectal or endoscopic administration in the form of a suppository.

5. 5. The pharmaceutical composition according to any one of claims 1 to 4, which is used in combination with another drug effective in treating or preventing lower gastrointestinal disorders.

6. 6. The pharmaceutical composition according to claim 5, wherein the other drug effective in treating or preventing a lower gastrointestinal disease is rebamipide or teprenone.

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