KR20240055254A - Composition for treating radiation induced enteritis comprising of dorsmorphin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or food composition for preventing, improving or treating radiation-induced enteritis containing dorsomorphin.

Description

Composition for treating radiation induced enteritis comprising of dorsmorphin}

The present invention relates to a composition for preventing or treating radiation-induced enteritis containing dorsomorphin.

Recently, as the number of elderly people increases due to aging, the number of cancer patients increases domestically and internationally, and cancer treatment using radiation is also on the rise. Additionally, as the survival rate of cancer patients doubled compared to the 1970s, interest in treatment side effects, which determine the quality of life after cure, is increasing.

Gastrointestinal syndrome, which occurs during abdominal exposure during radiation therapy, presents various symptoms such as proctitis, rectal bleeding, intestinal obstruction, vomiting, dehydration, and abdominal distension, and the increased survival rate of cancer patients is an important cause of the decline in quality of life in the current situation. It is known that the mechanism of radiation-induced abdominal side effects is that intestinal epithelial cell necrosis occurs during radiation exposure, and permeability increases accordingly, causing intestinal bacteria to cause inflammation.

Korean Patent No. 1676946 relates to an anti-inflammatory implant surface coating composition and an implant manufactured using the same with an anti-inflammatory effect. It discloses dorsomorphin as a type of autophagy enhancing substance with an anti-inflammatory effect, and is registered in Korea. Registration Patent No. 1386601 relates to a method for preventing, treating, and improving radiation-induced enteritis, and discloses a composition for treating radiation-induced enteritis containing a therapeutically effective amount of balsalazid as a pharmaceutical active ingredient.

However, the composition for preventing or treating radiation-induced enteritis containing dorsomorphin of the present invention has not yet been disclosed.

Korean Patent No. 1676946 Korean Patent No. 1386601

The purpose of the present invention is to solve the above problems by confirming the preventive or therapeutic effect of dorsomorphin on radiation-induced enteritis, and to prevent or treat radiation-induced enteritis containing dorsomorphin. The object is to provide a therapeutic composition.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating radiation-induced enteritis, comprising dorsomorphin or a pharmaceutically acceptable salt thereof represented by the following formula (1) as an active ingredient.

[Formula 1]

The radiation-induced enteritis includes digestive system cancer, including colorectal cancer, appendix cancer, anal cancer, or small intestine cancer; Genitourinary cancer, including prostate, bladder, testicular or penile cancer; Gynecological cancer, including cervical, endometrial, ovarian, vaginal, or vulvar cancer; It may be caused by one or more radiation treatments selected from the group consisting of treatment of osteogenic cancer or sarcoma cancer associated with the pelvic structures.

The radiation-induced enteritis may be acute radiation enteritis.

The dorsomorphine is administered in an amount of 0.001 to 10 g per day, and the composition may additionally contain ingredients effective for radiation-induced enteritis.

As another example, the present invention provides a food composition for preventing or improving radiation-induced enteritis, comprising dorsomorphin represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

As another example, the present invention provides a method for preventing or treating radiation-induced enteritis in non-human animals, comprising administering dorsomorphin or a pharmaceutically acceptable salt thereof represented by the following formula (1): .

[Formula 1]

The present invention relates to a composition for preventing or treating radiation-induced enteritis containing dorsomorphin, and it is expected that the drug can be reinvented as a treatment for radiation-induced enteritis.

Figure 1 shows a graph showing the anti-inflammatory effect of dorsomorphine at the gene level in a cell line experiment according to the present invention.
Figure 2 shows a graph showing the anti-inflammatory effect of dorsomorphin at the protein level in a cell line experiment according to the present invention.
Figure 3 shows a graph showing the effect of dorsomorphine on body weight in the radiation enteritis model according to the present invention.
Figure 4 shows images and graphs showing the effect of dorsomorphine on the pathology of intestinal tissue in the radiation enteritis model according to the present invention (magnification x200).
Figure 5 shows images and graphs showing the effect of dorsomorphine on the inflammatory response of intestinal tissue in the radiation enteritis model according to the present invention. Each value is expressed as mean ± SEM. n = 5-8 in each group. *p <0.05, **p <0.01, **p <0.001.
Figure 6 shows images and graphs showing the effect of dorsomorphine on the inflammatory response of intestinal tissue in the radiation enteritis model according to the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details, such as specific components, are shown, but this is provided to facilitate a more general understanding of the present invention, and it is known in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge. Additionally, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.

In the present invention, the term “prevention” or “prevention” means suppressing or delaying the occurrence of a disease from its cause.

In this specification, “treatment” means stopping the progression of damage by suppressing the progression and/or worsening of symptoms even without complete cure, or improving some or all of the symptoms and leading toward healing.

In the present invention, the term “improvement” refers to any action that improves or beneficially changes symptoms.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating radiation-induced enteritis containing Dorsomorphin or a pharmaceutically acceptable salt thereof represented by the following formula (1) as an active ingredient: do.

[Formula 1]

Dorsomorphin is a compound having the structure of Formula 1, Cas No. 866405-64-3 is a compound. Dorsomorphine is a pyrazolo[1,5-a]pyrimidine substituted with pyridin-4-yl and p-[2-(piperidin-1-yl)ethoxy]phenyl groups at positions 3 and 6, respectively. It is zolopyrimidine. It is a potent, selective, reversible, ATP-competitive inhibitor of AMP activated protein kinase (AMPK) and a selective inhibitor of bone morphogenetic protein (BMP) signaling.

The compounds of the present invention may include pharmaceutically acceptable salt forms, which can be prepared by methods conventional in the art, for example, hydrochloric acid, hydrogen bromide, sulfuric acid, sodium bisulfate, phosphoric acid. , salts with inorganic acids such as carbonic acid, or with organic acids such as formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gestisic acid, fumaric acid, lactobionic acid, salicylic acid, or acetylsalicylic acid (aspirin). It means forming a salt of an acid together, or reacting with alkali metal ions such as sodium and potassium to form metal salts thereof, or reacting with ammonium ions to form another type of pharmaceutically acceptable salt.

The radiation-induced enteritis includes digestive system cancer, including colorectal cancer, appendix cancer, anal cancer, or small intestine cancer; Genitourinary cancer, including prostate, bladder, testicular or penile cancer; Gynecological cancer, including cervical, endometrial, ovarian, vaginal, or vulvar cancer; It may be caused by one or more radiation treatments selected from the group consisting of treatment of osteogenic cancer or sarcoma cancer associated with the pelvic structures.

The radiation-induced enteritis may be acute radiation enteritis.

The composition of the present invention can be manufactured in any form by further including appropriate carriers, excipients and diluents commonly used in the preparation of the composition, and is preferably manufactured in the form of a pharmaceutical composition or (health function) food composition. However, it is not limited to this.

The pharmaceutical composition of the present invention can be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods. , tablets, capsules, injections and liquid formulations are more preferable. This formulation can be performed by a method commonly performed in the pharmaceutical field, and can be preferably formulated according to each disease or ingredient using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, it can be prepared by additionally using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous agents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween, cacao, laurin, glycerogeratin, etc. can be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method, and the preferred dosage is determined by the patient's condition and weight, and the severity of the disease. It varies depending on the degree, drug type, administration route and period, but can be appropriately selected by a person skilled in the art. However, for desirable effects, the pharmaceutical composition of the present invention may be included in an amount of 0.001 to 99.9% by weight, preferably 0.01 to 99% by weight, per day. The daily dosage may be about 0.01 to 1,000 mg/kg, preferably 100 to 300 mg/kg. If necessary, the active ingredient may be administered in an amount of 0.001 to 10 g per day, but is not limited thereto.

Additionally, in the composition according to one embodiment of the present invention, the composition may contain additional ingredients useful for preventing, treating, or improving radiation-induced enteritis, and the additional ingredients include all ingredients known to have effects, including compounds and natural products. may include.

As another example, the present invention provides a food composition for preventing or improving radiation-induced enteritis, comprising dorsomorphin represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Food compositions to which the above composition can be added (health function) can be manufactured in the form of powders, granules, tablets, capsules or beverages. For example, there are various general foods, beverages, gum, tea, vitamin complexes, or health supplements.

The food composition of the present invention may include allitiamine, fursultiamine, benfotiamine, or salts thereof, as well as ingredients commonly added during food production, such as proteins, carbohydrates, fats, nutrients, and seasonings. Includes agents and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink, in addition to allitiamine, fursultiamine, benfotiamine or salts thereof, citric acid, high-fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, cephalic acid extract, jujube extract, and licorice. Extracts, etc. may additionally be included.

Additionally, the composition can be added to food or beverages for the purpose of preventing diseases. At this time, the amount of the active ingredient in the food or beverage can be added at 0.001 to 15% by weight of the total food weight, and the food composition can be added at a rate of 0.002 to 5 g, preferably 0.03 to 1 g, based on 100 g. However, it is not limited to this.

As another example, the present invention provides a method for preventing or treating radiation-induced enteritis in non-human animals, comprising administering dorsomorphin or a pharmaceutically acceptable salt thereof represented by the following formula (1): .

[Formula 1]

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms, and the present embodiments are merely intended to ensure that the disclosure of the present invention is complete and to include common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the invention is only defined by the scope of the claims.

<Example 1> Anti-inflammatory effect of dorsomorphine in vitro (mRNA)

Dorsomorphine was purchased from Selleck (https://www.selleckchem.com).

To culture mouse-derived macrophages, bone marrow cells were collected from the femur of mice. To differentiate into macrophages, cells were cultured in 10% FBS DMEM + mCSF at 37°C and 5% CO2 for 5 days after collection. The cultured cells were cultured at 37°C and 5% CO2 for an additional day after replacing the medium with 5% FBS DMEM. Dorsomprhin or DMSO pretreatment was administered 1 hour before radiation exposure. 1 hour after irradiation, RNA was isolated from macrophages using Trizol. Changes in inflammatory cytokines (IL-6 and IL-1β) were confirmed using the extracted RNA. In the dorsomphin pretreatment group, the effect of reducing inflammatory cytokines during radiation treatment was confirmed (Figure 1).

RNA extraction and qPCR were performed as described below. Total RNA from macrophages or intestinal tissue was isolated using TRIzol reagent (Thermo Fisher Scientific). cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen, 18064). qPCR was performed using cDNA, primers, and SYBR Green PCR kits (Qiagen, 204074) using the Real-time PCR Cycler RotorGene Q 2plex system (Qiagen GmbH, 9001620, Hilden, Germany). Samples were amplified for 50 cycles as follows: 95°C for 5 seconds and 60°C for 10 seconds. To analyze qPCR data, relative quantification was performed using the 2ΔΔCt method using Gapdh as an internal control gene. Data were expressed as relative fold change.

The primer sequences used in the experiment are shown in Table 1 below.

Gene name Primer Sequences IL6 Forward
Reverse 5'-ACAAAGCCAGAGTCCTTCAGA-3'
5'-TGGTCCTTAGCCACTCCTTC-3' Il1b Forward
Reverse 5'-TGACGGACCCCCAAAAGATGA-3'
5'-AAAGACACAGGTAGCTGCCA-3' actb Forward
Reverse 5'-CCACCATGTACCCAGGCATT-3'
5'-AGGGTGTAAAACGCAGCTCA-3'

<Example 2> Anti-inflammatory effect of dorsomorphin in vitro (protein)

1 hour after irradiation, proteins were separated from macrophages using Ripa Buffer. Using the extracted proteins, changes in MAPK signaling, a sub-mechanism of the inflammatory response, were confirmed. In the dorsomphin pretreatment group, the effect of reducing phosphorylation of MAPK signaling during radiation treatment was confirmed (Figure 2).

Tissue homogenates were incubated in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-HCl) supplemented with protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). 40, 0.5% DOC (deoxycholic acid), 0.1% SDS, 1 mM PMSF) (ELPIS Bio, Lexington, MA, USA). Equal amounts of protein were mixed with 5X SDS sample buffer (ELPIS) and heated for 10 minutes. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Membranes were blocked in 5% skim milk in TBS-T (Trisbuffered saline-Tween 20) for 1 hour at room temperature and incubated overnight at 4°C with the following specific primary antibodies: p-anti-pJNK, anti-p38. , anti-pERK 1/2, anti-pAMPK (1:2,000, 4668S, 4511, 4370, 2535; Cell Signaling Technology), anti-IKB-α (1:4,000,sc-371; Santa Cruz Biotechnology, anti-ACTB) (1: 2000,sc-47778, Santa Cruz Biotechnology. After washing with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technologies) for 1 hour at room temperature. Blots were imaged using a chemiluminescence assay kit (Millipore) on a UVitec Alliance mini chemiluminescence device (BioSPX, Abcoude, Netherlands).

<Example 3> Effect of dorsomorphine (body weight) in radiation enteritis model

C57BL/6 wild-type mice were used to evaluate the effect of Dorsomrphin on body weight changes in mice induced with acute radiation gastrointestinal syndrome. A group irradiated to 6-week-old C57BL/6 wild-type mice (normal control group, normal) with 16.5 gray (Gy) of radiation (test control group, IR) and a group irradiated with 16.5 gray (Gy) of radiation after Dorsomrphin pretreatment (test group). The results of comparing body weight changes are shown in Figure 3. As can be seen in Figure 3, mice in the test control group treated only with radiation and those treated with 16.5 gray (Gy) of radiation after Dorsomrphin pretreatment showed a statistically significantly smaller change in body weight on day 7 (Figure 3). .

<Example 4> Effect of dorsomorphine in radiation enteritis model (tissue staining)

Small intestine hematoxylin of 6-week-old C57BL/6 wild-type mice (normal control group, normal) irradiated with 16.5 Gray (Gy) (test control group, IR) and the group irradiated with 16.5 Gray (Gy) after Dorsomrphin pretreatment. and eosin staining results. The experimental group showed a greater number of crypts and higher villi height compared to the control group (Figure 4).

Changes in the crypts and crypts of the small intestine were examined 5 days after irradiation. Intestinal tissue was dissected, fixed in 10% formalin, embedded in paraffin, and then subjected to normal processing for histological examination. 4 μm thick tissue sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin.

<Example 5> Effect of dorsomorphine in radiation enteritis model (mRNA)

The levels of IL-1β, IL-6, and TNFα were measured in small intestine extracted RNA collected from C57BL/6J mice 5 days after irradiation. The level of inflammation-related gene expression in the group irradiated with 16.5 gray (Gy) of radiation after dorsomrphin pretreatment decreased compared to the control group (Figure 5).

Intestinal tissues were individually transferred to Eppendorf tubes containing RNAlater (Thermo Fisher Scientific, Wilmington, DE, USA) and stored at -80°C until processing. RNA extraction and qPCR were performed as described below. Total RNA from macrophages or intestinal tissue was isolated using TRIzol reagent (Thermo Fisher Scientific). cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen, 18064). qPCR was performed using cDNA, primers, and SYBR Green PCR kits (Qiagen, 204074) using the Real-time PCR Cycler RotorGene Q 2plex system (Qiagen GmbH, 9001620, Hilden, Germany). Samples were amplified for 50 cycles as follows: 95°C for 5 seconds and 60°C for 10 seconds. To analyze qPCR data, relative quantification was performed using the 2ΔΔCt method using Gapdh as an internal control gene. Data were expressed as relative fold change.

The primer sequences used in the experiment are shown in Table 2 below.

Gene name Primer Sequences IL6 Forward
Reverse 5'-ACAAAGCCAGAGTCCTTCAGA-3'
5'-TGGTCCTTAGCCACTCCTTC-3' Il1b Forward
Reverse 5'-TGACGGACCCCCAAAAGATGA-3'
5'-AAAGACACAGGTAGCTGCCA-3' TNFa Forward
Reverse 5'-CCCACGTCGTAGCAAACCAC-3'
5'-GCAGCCTTGTCCCTTGAAGA-3' actb Forward
Reverse 5'-CCACCATGTACCCAGGCATT-3'
5'-AGGGTGTAAAACGCAGCTCA-3'

<Example 6> Effect of dorsomorphine in radiation enteritis model (protein)

IL-6 in the small intestine of 6-week-old C57BL/6 wild-type mice (normal control group) irradiated with 16.5 Gray (Gy) (test control group, IR) and the group irradiated with 16.5 Gray (Gy) after Dorsomrphin pretreatment. Immunohistochemical tissue sections. The level of IL-6 expression decreased in the group irradiated with 16.5 gray (Gy) of radiation after dorsomrphin pretreatment compared to the control group (Figure 6).

After embedding in paraffin, the tissue was deparaffinized into 4 μm thick tissue sections. The slides were immersed in REALTarget Retrieval Solution (S2031, Dako, CA, USA) and heated at 95°C for 20-40 minutes for antigen retrieval. Next, the slide was reacted with Peroxidase-Blocking Solution (S2023, Dako) and then incubated with 1% normal goat serum at room temperature for 30 minutes to block non-specific binding. Sections were washed three times with PBS and reacted with primary antibody against IL-6 (1:2000, sc-28343, Santa Cruz) for 2 hours at room temperature, followed by secondary antibody (1:1,000; A28175, Life Technologies). For visualization, DAB + ((D9542, Sigma)) was used to quantify IL-6 immune intensity using the color deconvolution plugin.

Claims (11)

A pharmaceutical composition for preventing or treating radiation-induced enteritis containing dorsomorphin or a pharmaceutically acceptable salt thereof represented by the following formula (1) as an active ingredient:
[Formula 1]
The method of claim 1, wherein the radiation-induced enteritis is cancer of the digestive system, including colorectal cancer, appendix cancer, anal cancer, or small intestine cancer; Genitourinary cancer, including prostate, bladder, testicular or penile cancer; Gynecological cancer, including cervical, endometrial, ovarian, vaginal, or vulvar cancer; Pharmaceutical composition for preventing or treating radiation-induced enteritis, characterized in that it is caused by one or more radiation treatments selected from the group consisting of treatment of osteogenic cancer or sarcoma cancer associated with the pelvic structure The pharmaceutical composition for preventing or treating radiation-induced enteritis according to claim 1, wherein the radiation-induced enteritis is acute radiation enteritis. The pharmaceutical composition for preventing or treating radiation-induced enteritis according to claim 1, wherein the dorsomorphin or a pharmaceutically acceptable salt thereof is administered in an amount of 0.001 to 10 g per day. The pharmaceutical composition for preventing or treating radiation-induced enteritis according to claim 1, wherein the composition further contains an ingredient effective against radiation-induced enteritis. A food composition for preventing or improving radiation-induced enteritis containing dorsomorphin or a pharmaceutically acceptable salt thereof represented by the following formula (1) as an active ingredient:
[Formula 1]
The method of claim 6, wherein the radiation-induced enteritis is cancer of the digestive system, including colorectal cancer, appendix cancer, anal cancer, or small intestine cancer; Genitourinary cancer, including prostate, bladder, testicular, or penile cancer; Gynecological cancer, including cervical, endometrial, ovarian, vaginal, or vulvar cancer; A food composition for preventing or ameliorating radiation-induced enteritis, characterized in that it is caused by one or more radiation treatments selected from the group consisting of treatment of osteogenic cancer or sarcoma cancer associated with the pelvic structure The food composition for preventing or improving radiation-induced enteritis according to claim 6, wherein the radiation-induced enteritis is acute radiation enteritis. The food composition for preventing or improving radiation-induced enteritis according to claim 6, wherein the dorsomorphin or a pharmaceutically acceptable salt thereof is administered in an amount of 0.001 to 10 g per day. The food composition for preventing or improving radiation-induced enteritis according to claim 6, wherein the composition further contains an ingredient effective against radiation-induced enteritis. A method for preventing or treating radiation-induced enteritis in non-human animals comprising administering dorsomorphin or a pharmaceutically acceptable salt thereof represented by the following formula (1):
[Formula 1]