KR20230080578A - Compositions for preventing or treating radiation-induced intestinal injury - Google Patents

Abstract

The present invention relates to a composition for preventing or treating intestinal damage caused by radiation irradiation containing stemregenin1 as an active ingredient. More specifically, in intestinal organoids in which damage was induced by radiation irradiation, the regrowth of intestinal organoids and the enhancement of intestinal epithelial stem cells were confirmed by treatment with stemregenin1. In a mouse gastrointestinal tract injury model following high-dose radiation irradiation, an increase in the number and length of crypt-villi, an intestinal-specific histological structure, was confirmed by treatment with stemregenin1. Therefore, the composition containing stemregenin1 as an active ingredient is intended to be provided as a pharmaceutical composition and health food for preventing or treating intestinal damage caused by radiation irradiation.

Description

Compositions for preventing or treating intestinal damage caused by radiation {Compositions for preventing or treating radiation-induced intestinal injury}

The present invention relates to a composition for preventing or treating intestinal damage caused by irradiation containing StemRegenin1 as an active ingredient.

Radiation is one of the widely used treatment methods for cancer treatment of various tissues and for the purpose of extending the life of cancer patients by removing cancer cells or inhibiting their growth. Reactive oxygen species (ROS) generated by radiation It indirectly induces cell damage and cell death, such as DNA, protein and cell membrane.

However, as a complication of radiation treatment, an inflammatory reaction occurs in the mucous membranes of the stomach, small intestine, and large intestine, resulting in clinical symptoms such as nausea, vomiting, abdominal pain or diarrhea, and in severe cases, ulcers, intestinal bleeding, perforation, etc. However, studies on radiation side effects, including radiation-induced intestinal damages, have been very limited, and acute gastrointestinal syndrome (GIS) can be induced. After exposure to this high dose of radiation, there is currently no effective treatment, resulting in 100% death within about two weeks.

Therefore, research is being conducted to develop a new efficient medical technology for measuring, diagnosing, or treating damage during radiation exposure, and to use it for treatment efficiency enhancement technology through protection of normal tissue damage during radiation therapy. are radioprotectants applied before radiation exposure; radiomitigators applied prior to the onset of obvious signs during radiation exposure or within a short period after exposure; And research on a therapeutic agent that is applied after a clear sign appears due to radiation exposure is being conducted.

As a representative drug for such radiation exposure, there is amifostine, an aminothiols derivative. Aminothiol is a radioprotective agent that acts as a free radical scavenger as a chemical analogue of cysteine. It was developed by the Research program, and more than 4,000 aminothiol derivative compounds have been developed and tested so far. These aminothiols are currently used in many patients after radiation chemotherapy for tumors, but they are used in a limited way because of their weak antioxidant effect and side effects such as nausea, vomiting, and hypotension. Studies and reports on drugs exhibiting enhancing efficacy are absent.

Korean Patent Publication No. 10-2011-0016722 (published on February 18, 2011)

As described above, although high-dose radiation gastrointestinal syndrome is a major cause of death when exposed to radiation, there is no clinically applicable treatment to date. Therefore, the present invention provides a composition containing stemreginin 1 as an active ingredient for high-dose radiation It is intended to be provided as a pharmaceutical composition for preventing or treating intestinal damage and a health food for improvement.

The present invention provides a pharmaceutical composition for preventing or treating intestinal damage caused by irradiation containing stemreginin 1 as an active ingredient.

In addition, the present invention provides a health food for preventing or improving intestinal damage caused by radiation containing stemreginin 1 as an active ingredient.

According to the present invention, it was confirmed that intestinal organoid regrowth and intestinal epithelial stem cell enhancement were confirmed by stemreginin 1 treatment in intestinal organoids damaged by irradiation, and in a mouse gastrointestinal injury model caused by high-dose irradiation, stem As the increase in the number and length of crypt-villi, which are intestinal-specific histological structures, was confirmed by treatment with Reginin 1, the composition containing Stem Reginin 1 as an active ingredient is suitable for preventing or treating intestinal damage caused by radiation. It can be provided as a pharmaceutical composition and health food.

1 is a result of searching for drugs for treating radiation damage using intestinal organoids, FIG. 1A is the result of confirming the effect of reproducing the radiation response of the intestine in vivo, and FIG. (1, 10 and 20uM) These are the experimental procedures and MTT analysis results confirming the regeneration enhancing effect.
Figure 2 is a result of confirming the SR1 regeneration efficacy and intestinal epithelial stem cell expression in irradiated intestinal organoids, Figure 2A is a schematic diagram showing the treatment process of SR1 (10uM) for irradiated intestinal organoids, 2C is the result of confirming the regeneration enhancing effect by SR1 by morphology and MTT-stained colony, and FIGS. 2D and 2E are the results of E-dU staining and Lgr5 gene expression analysis confirming the number and expression of intestinal epithelial stem cells.
Figure 3 is a result of confirming the expression of signaling factors (WNT and NOTCH) mediators related to intestinal stem cell regeneration by SR1 (10uM) treatment in irradiated intestinal organoids. Figure 3A shows Ascl2 (WNT mediator) and Hes1 (NOTCH Mediator) is the result of real-time PCR analysis confirming the expression, Figure 3B is the result of confirming the level of intestinal stem cell regeneration according to ABC99 (notum inhibitor, 0, 50 and 500 nM) treatment, Figure 3C is Ascl2 (ABC99 (50 nM) treatment by treatment WNT mediator) is the result of confirming the expression level, Figure 3D is the result of confirming the change in the notum expression level by SR1 (10uM), and Figure 3E is the result of confirming the regeneration level by SR1 when the recombinant notum protein is treated.
Figure 4 is a result of confirming the effect of SR1 and ABC99 (notum inhibitor) on regeneration of intestinal epithelial tissue in the irradiated mouse intestine, Figure 4A is the result of hematoxylin and eosin (H & E) staining in the small intestine tissue, Figure 4B is the result of confirming the effect of enhancing intestinal tissue regeneration by quantifying the number of crypts and villi length in the H&E staining results, and Figures 4C and 4D are quantification of changes in intestinal epithelial stem cells by SR1 and ABC99 after ki-67 staining 4E is a result confirming the increase in intestinal epithelial stem cells through Lgr5 gene expression analysis.

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

The inventors of the present invention confirmed that re-proliferation of intestinal organoids was increased in a radiation damage model of intestinal organoids treated with stemreginin 1 (SR1), and in a mouse animal injury experiment to confirm the in vivo therapeutic effect of SR1 As it was confirmed that the intestinal tissue regeneration effect of the SR1 treatment group was promoted, the present invention was completed to provide a composition containing SR1 as an active ingredient as a new treatment for radiation exposure to gastrointestinal syndrome.

The present invention can provide a pharmaceutical composition for preventing or treating intestinal damage caused by radiation containing StemRegenin1 as an active ingredient.

The intestinal damage may be gastrointestinal syndrome (GIS), but is not limited thereto.

The irradiation may be high-dose irradiation, and more specifically, the high-dose radiation may be 5 to 15 Gy, but is not limited thereto.

The stemreginin 1 may increase regeneration of intestinal tissue damaged by irradiation.

According to an embodiment of the present invention, after dispensing into 4 well plates for irradiation of intestinal organoids, they were cultured for 24 hours. Immediately before irradiation, it was replaced with a new culture medium and irradiated with a single dose of 5 Gy (3.25 Gy/min) using a Gamma-Cell 3000 (Nordion, Cadana) using 137 Cs as a source. In addition, after anesthetizing mice using Alfaxalone (Alfaxan ^® , Careside, Korea) and Xylazine (Rompun ^® , Bayer Korea, Korea) for mouse abdominal irradiation, 7-8 mice were irradiated at a time in the irradiation correction frame. corrected to be able to. Using an X-ray irradiator (X-RAD 320; Softex Korea, Korea), 13.5 Gy (2 Gy/min) was irradiated locally to the abdomen. From the day of investigation, StemRegenin1 (SR1; 100 μg/kg/day) drug was administered by intraperitoneal injection once a day along with fluid administration, and autopsy was performed on the 6th day.

As a result, as shown in FIG. 1A, it was confirmed that intestinal organoids showed similar results to radiation-induced cell damage and regeneration responses in vivo, and as shown in FIG. It was confirmed that regeneration on day 1 was enhanced by SR1 concentration.

In addition, as a result of analyzing hematoxylin and eosin (H&E) staining and crypt number and villi length in the small intestine tissue on the 6th day after SR1 administration, as shown in FIGS. 4A and 4B in the intestinal injury mouse model irradiated with high-dose radiation, Enhancement of intestinal tissue regeneration by In addition, ki-67 staining analysis and Lgr5 gene expression analysis were performed as shown in FIGS. 4C to 4E to confirm an increase in intestinal epithelial stem cells.

From the above results, the effect of enhancing intestinal tissue regeneration by SR1 in vivo was confirmed.

The stemreginin 1 may activate WNT signaling by suppressing notum expression induced by irradiation.

According to another embodiment of the present invention, in order to analyze the regenerative efficacy of SR1 in irradiated intestinal organoids, the expression of signaling factors (WNT and NOTCH) mediators related to intestinal stem cell regeneration was analyzed.

As a result, as shown in FIG. 3A, it was confirmed that Ascl2 (WNT mediator) expression was increased by SR1, whereas Hes1 (NOTCH mediator) expression was not affected. In addition, as a result of confirming the efficacy of notum, a WNT ligand-independent WNT activator, it was confirmed that regeneration was promoted and WNT signal activity was induced when ABC99 (notum inhibitor) was treated, as shown in FIGS. 3B and 3C, and SR1 and Notum As a result of correlation analysis, as shown in FIGS. 3D and 3E , it was confirmed that notum expression was reduced by SR1, and in particular, regeneration by SR1 was reduced when the recombinant notum protein was treated.

From the above results, it was confirmed that SR1 enhances intestinal regeneration by re-activating WNT signals through suppression of notum (WNT activity inhibitor) expression increased by radiation.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating intestinal damage caused by irradiation containing stemreginin 1 as an active ingredient is an injection, granule, powder, tablet, pill, capsule, Any one formulation selected from the group consisting of suppositories, gels, suspensions, emulsions, drops, and solutions may be used.

In another embodiment of the present invention, a pharmaceutical composition for preventing or treating intestinal damage caused by irradiation containing stemreginin 1 as an active ingredient includes suitable carriers, excipients, disintegrants, sweeteners, One or more additives selected from the group consisting of a coating agent, an expanding agent, a lubricant, a flavoring agent, an antioxidant, a buffer, a bacteriostatic agent, a diluent, a dispersing agent, a surfactant, a binder, and a lubricant may be further included.

Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for internal use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As the base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

According to one embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject as

The preferred dosage of stemreginin 1 may vary depending on the condition and body weight of the subject, the type and severity of the disease, the type of drug, the route and duration of administration, and may be appropriately selected by a person skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200mg/kg, specifically 0.1 to 200mg/kg, and more specifically 0.1 to 100mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

In addition, the present invention can provide a health food for preventing or improving intestinal damage caused by radiation containing stemreginin 1 as an active ingredient.

The health food is used together with other foods or food additives in addition to the stemreginin 1, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used according to the effective dose of the therapeutic agent, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control. Since there is no problem in terms of safety, it is certain that the components can be used in an amount greater than the above range.

There is no particular limitation on the type of health food, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes; and the like.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Mouse Preparation

The mice used in the experiment were 7-week-old male C57BL/6 mice weighing around 20 ± 2 g. After checking the object monitoring data and appearance at the supplier, after acclimatization in the quarantine room for about a week, general symptoms were observed and only healthy objects were provided for the test. Animal specifications are maintained at 22±1℃ temperature, 50±10% humidity, lighting time 12 Light: 12 Day cycle by an auto controller in the SPF room of the Institute of Nuclear Medicine, and sterilized feed is sterilized in water fed with Animal experiments were approved by The Animal Investigation Committee of the Korea Institute of Radiological and Medical Sciences (KIRAMS), and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (1996, USA).

2. Isolation of small intestinal crypts from mice and culture of intestinal organoids

In order to isolate crypts from the small intestine of C57/B6 mice (9 weeks old), an area of about 10 cm was cut except for 2 cm below the duodenum. The cylindrical intestinal region was split and spread widely, and each region was cut into 20 mm pieces. After putting the slices in 10ml of 2mM EDTA and maintaining them in a 37° C. water bath for 5 minutes, the slices were put in 10ml of PBS, shaken for 1 minute, and the supernatant was used. The separated sections were centrifuged at 100 g for 3 minutes, the supernatant was discarded, and 10 ml of 2% D-sorbitol was added, and the sections were passed through a 70 μm cell strainer. The pellet was centrifuged again, washed 4 to 5 times, and the separated crypts were counted under an optical microscope.

For organoid culture, about 100 to 200 crypts are mixed with Matrigel, and 50 μl per well is dispensed into a 24 well plate, and then ENR medium (10 mM Hepes, 0.5 Unit Penicilin/Streptomycin, N ₂ , B ₂₇ , 0.5 M NAC, 50 ng /ml EGF, 100ng/ml Noggin, 500ng/ml R-spondin 1 supplemented with DMEM/F12 GlutaMAX-1media) and cultured in a 37°C incubator. The culture medium was exchanged every 3-4 days. On day 7, the cultured mini-organs were subcultured using enzyme-free cell dissociation buffer (STEMCELL Technologies Inc, Vancouver, BC, Canada).

3. Irradiation of intestinal organoids and mice

For irradiation of intestinal organoids, the cells were divided into 4 well plates and cultured for 24 hours. Immediately before irradiation, a new culture medium was exchanged, and a single irradiation was performed at 5 Gy (3.25 Gy/min) using a Gamma-Cell 3000 (Nordion, Cadana) using 137Cs as a source. The culture medium (ENR) was treated at a final concentration of 10 uM, and the medium was exchanged every 3 days. SR1 was purchased from ApexBio (cat. no. A8224).

For mouse abdominal irradiation, mice are anesthetized using Alfaxalone (Alfaxan ^® , Careside, Korea) and Xylazine (Rompun ^® , Bayer Korea, Korea), and then 7-8 mice are irradiated at a time in the irradiation correction frame. Corrected. Using an X-ray irradiator (X-RAD 320; Softex Korea, Korea), 13.5 Gy (2 Gy/min) was irradiated locally to the abdomen. From the day of investigation, StemRegenin1 (SR1; 100 μg/kg/day) drug was administered by intraperitoneal injection once a day along with fluid administration, and autopsy was performed on the 6th day.

4. Analysis of intestinal organoid viability and intestinal epithelial stem cell proliferation (MTT and E-dU staining)

To analyze the viability of intestinal organoids, 10% MTT solution was added and cultured in an incubator at 37 ° C for about 3 hours. After adding 2% SDS and DMSO, respectively, except for the medium, the OD value was measured using a multi-channel optical analyzer. .

To analyze the proliferation pattern of intestinal epithelial stem cells, 5-ethynyl-2′-deoxyuridine (EdU) staining was used. After adding 10 μM EdU solution (Molecular probes) to intestinal organoids, they were cultured in an incubator at 37°C for 30 minutes, the solution was removed, and 4% paraformaldehyde solution was added and fixed at 4°C. Thereafter, nuclear staining was performed at 1:2000 Hoechst (Molecular probes) and analyzed with an immuno-fluorescence microscope (Olympus).

5. Tissue staining

Intestinal tissues fixed in 10% neutral formalin were paraffin-embedded and sectioned at 4 μm thickness, and tissue changes were confirmed by hematoxylin and eosin (H&E) staining and Ki-67 immunostaining.

6. Real-time polymerase chain reaction (qPCR)

Total RNA was extracted from intestinal organoids and small intestine tissues using RNA minikit (Qiagen, Inc.), and cDNA was synthesized using Accupower RT mix reagent (Bioneer Corp., Seoul, Korea). Real-time PCR was performed using the FastStart Essential DNA Green Master (Roche, Indianapolis, IN, USA).

< Example 1> irradiated in intestinal organoids of StemRegenin1 Check the effect

The effect of each concentration of spamreginin 1 (SR1) on the damage and proliferation of irradiated intestinal organoids was confirmed.

As a result, as shown in FIG. 1A, it was confirmed that intestinal organoids showed results similar to those of radiation-induced cell damage and regeneration in vivo. More specifically, as shown in FIG. 1B, it was confirmed that regeneration was enhanced by SR1 concentration on the 9th day after irradiation in the search for radiation damage treatment drugs using intestinal organoids.

As it was confirmed from the above results that SR1 enhances the regeneration effect of intestinal organoids damaged by irradiation, the SR1 regeneration efficacy and intestinal epithelial stem cell expression were confirmed in various ways in the same process as in FIG. 2A.

As a result, as shown in FIGS. 2B and 2C, the regeneration enhancing effect by SR1 was confirmed by an increase in morphology and MTT-stained colony, and as shown in FIG. 2D, the number and expression of intestinal epithelial stem cells increased by E- It was confirmed by dU staining and Lgr5 gene expression analysis.

< Example 2> irradiated in intestinal organoids of StemRegenin1 Confirmation of regenerative efficacy mechanism

In order to analyze the regenerative efficacy of SR1 in irradiated intestinal organoids, the expression of signaling factors (WNT and NOTCH) mediators related to intestinal stem cell regeneration was analyzed.

As a result, as shown in FIG. 3A, it was confirmed that Ascl2 (WNT mediator) expression was increased by SR1, whereas Hes1 (NOTCH mediator) expression was not affected. In addition, as a result of confirming the efficacy of notum, a WNT ligand-independent WNT activator, it was confirmed that when treated with ABC99 (notum inhibitor), regeneration was promoted and WNT signal activity was induced, as shown in FIGS. 3B and 3C. In addition, as a result of analyzing the correlation between SR1 and Notum, as shown in FIGS. 3D and 3E, notum expression was reduced by SR1, and in particular, when recombinant notum protein was treated, regeneration by SR1 was reduced.

From the above results, it was confirmed that the regeneration enhancement of SR1 is involved in the promotion of intestinal regeneration by re-activating the WNT signal through suppression of the expression of notum (WNT activity inhibitor) increased by radiation.

< Example 3> SR1 in the irradiated mouse intestine and ABC99 (notum inhibitor) Confirmation of intestinal epithelial tissue regeneration enhancement effect

As confirmed in the previous experiment, as SR1 was confirmed to promote intestinal regeneration through suppression of notum (WNT activity inhibitor) expression increased by radiation, in order to confirm whether the same effect appears in vivo, mice were treated with the entire abdomen. A single dose of 13.5 Gy was irradiated, and SR1 (IR+SR1), ABC99 (IR+ABC99), or vehicle (IR) were intraperitoneally injected, and intestinal tissue changes and characteristics were compared and analyzed on day 6.

First, as shown in FIGS. 4A and 4B, hematoxylin and eosin (H&E) staining, crypt number, and villi length were compared and analyzed in the small intestine tissue on day 6 after administration of SR1 or ABC99 to quantify the enhancement of intestinal tissue regeneration by SR1 and ABC99. . In addition, changes in intestinal epithelial stem cells caused by SR1 and ABC99 were quantified by ki-67 staining as shown in FIGS. 4C and 4D, and an increase in intestinal epithelial stem cells was confirmed through Lgr5 gene expression analysis as shown in FIG. 4E.

From the above results, the effect of enhancing intestinal tissue regeneration by SR1 and ABC99 in vivo was confirmed.

From the above results, SR1 improved the regrowth of intestinal organoids, increased the number of intestinal epithelial stem cells, and increased the number and length of crypt-villi, which are intestinal-specific histological structures, in the radiation gastrointestinal injury model. It was confirmed that the intestinal tissue regeneration effect was enhanced by re-activating the WNT signal through suppression of Notum expression, an inhibitory factor.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

A pharmaceutical composition for preventing or treating intestinal damage caused by irradiation containing StemRegenin1 as an active ingredient. The pharmaceutical composition according to claim 1, wherein the intestinal damage is gastrointestinal syndrome (GIS). The pharmaceutical composition according to claim 1, wherein the irradiation is high-dose irradiation. The pharmaceutical composition according to claim 3, wherein the high-dose radiation is 5 to 15 Gy. The pharmaceutical composition according to claim 1, wherein the stemreginin 1 increases regeneration of intestinal tissue damaged by irradiation. The pharmaceutical composition according to claim 1, wherein the stemreginin 1 activates the WNT signal by inhibiting notum expression induced by irradiation. A health functional food composition for preventing or improving intestinal damage caused by irradiation containing StemRegenin1 as an active ingredient.

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