Application of KYNA in preparation of medicine for preventing intestinal injury caused by ionizing radiation

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses application of KYNA in preparation of a medicine for preventing intestinal injury caused by ionizing radiation, and the intestinal injury caused by ionizing radiation comprises radioactive substances capable of causing ionization of substances or intestinal injury caused by irradiation of rays emitted by linear accelerator equipment. The protection of the intestinal injury caused by the ionizing radiation comprises prolonging the survival time of the mouse irradiated by the ionizing radiation and protecting the intestinal tissue structure of the mouse irradiated by the ionizing radiation. The medicine taking KYNA as the active component for preventing intestinal injury caused by ionizing radiation provided by the invention has the advantages of low price, small toxic and side effects, remarkable curative effect, convenience and safety in medication and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine technology, and particularly relates to the use of KYNA in the preparation of a drug for preventing intestinal injury caused by ionizing radiation. BACKGROUND

[0002] The data published by the International Union Against Cancer (UICC) shows that in 2020, 193 million new cases and 2040, 3020 million patients are expected, and the proportion of cancer patients in China accounts for 1 / 4. Moreover, the composition of tumor types in Chinese residents is significantly different from that in western developed countries, and the top ten common cancers account for six to seven tumors occurring in the abdomen and pelvis. In the past 30 years, based on equipment upgrading and technological progress, radiotherapy has been increasingly applied in the digestive, urinary and reproductive systems of malignant tumors, and the therapeutic effect has been significantly improved. For example, compared with two-dimensional radiotherapy technology, patients receiving intensity-modulated radiotherapy (IMRT) and image-guided radiotherapy (IGRT) have a 3-5 year tumor local and regional control rate and survival rate that can be increased by about 25-30% and 12-15%, respectively. However, due to the inevitable exposure of normal intestinal tissue to radiation, the incidence of radiation-induced intestinal injury (RIII) has not been significantly improved. The incidence of RIII of grade 2 and above is between 40-60%; in addition, nearly 10% of patients have delayed or even terminated radiotherapy, and the efficacy of these patients who cannot complete radiotherapy according to the plan will decrease by nearly 10%. RIII is mainly characterized by diarrhea, including different degrees of abdominal pain, hematochezia, electrolyte imbalance, weight loss and intestinal obstruction, and other clinical manifestations. It can occur after the start of radiotherapy, and is generally divided into acute and chronic two types before and after the end of radiotherapy. Since chronic complications of the intestinal tract have obvious correlation with more serious acute phase symptoms, most studies focus on the acute phase. The bigger problem is that for the clinical diagnosis and treatment of RIII, the principle of "controlling the dose and volume of intestinal irradiation" has been followed for decades, and the guidelines developed by the multi-disciplinary expert group in China at the end of 2023 are still the same. So far, no effective prevention and treatment program with high-level evidence-based medicine has been provided by countries around the world and international academic organizations. In the case of radio physics and technical means controlling this type of radiation damage reaching a bottleneck, RIII has become one of the main obstacles to further improving the efficacy of radiotherapy for abdominal and pelvic tumors.

[0003] From the past ten years of research on intestinal flora and its metabolites, we know that the correlation between intestinal flora and RIII incidence, diagnosis and treatment is mostly through its metabolites. Currently, a variety of metabolites such as short-chain fatty acids, aromatic amino acids and bile acids have been shown to have a clear effect on exacerbating (pathogenesis) or alleviating (prevention and treatment) RIII, but there are problems such as insufficient accuracy of target points and unclear effect mechanism. Non-targeted metabolomics research has found that a series of metabolite contents are significantly increased in the feces of "elite mice", and a large number of kynurenic acid (also known as 4-hydroxyquinoline-2-carboxylic acid, molecular formula C10H7NO3; kynurenic acid, hereinafter referred to as

[0004] KYNA) metabolites of tryptophan metabolic pathway are enriched in the intestine, and oral KYNA has the effect of improving the survival rate of mice under a whole-body lethal dose of irradiation, but its significant therapeutic effect on RIII has not been further elucidated, and its potential molecular mechanism is still unknown. In the field of inflammatory bowel disease, it has been confirmed that exogenous KYNA can promote the increase of intestinal crypt cells and restore intestinal barrier function. Therefore, tryptophan metabolite KYNA has great potential to alleviate RIII and may achieve new breakthroughs in the short term.

[0005] The application of KYNA in the preparation of drugs found in the China National Knowledge Network patent database is as follows: Chinese patent CN202311550882.4 discloses the application of kynurenic acid and its structural analogs in the preparation of XOD inhibitors for the treatment of hyperuricemia; Chinese patent

[0006] CN202311089677.2 discloses the application of kynurenic acid in the preparation of drugs for relieving and / or treating anorexia nervosa. The specification of the searched application does not involve the new use of the substance in the preparation of ionizing radiation-induced intestinal damage protection drugs. At present, through drug intervention, intestinal flora and related metabolite treatment methods, the treatment effect is uncertain, and the mechanism is not clear. A variety of ionizing radiation-induced intestinal damage protection drugs have been developed, including lipoic acid compounds, aminothiol compounds, estrogen compounds, cytokines, plant polysaccharides and traditional Chinese medicine compounds. In addition to in-depth research on sulfur-containing compounds, the rest of the research is not in-depth. In this project, if we can prove the therapeutic effect and mechanism of action of a single flora metabolite, we can break through the current situation that there is no effective radiation enteropathy treatment regimen and drug.

[0007] Among the many protective drugs, some drugs have good radiation protection effect, but their toxic side effects are larger, such as the most typical radiation protection drug WR2721 (amifostine, S-2-(3-aminopropylamino) ethyl thiophosphoric acid); Some drugs have low protection efficiency, thereby reducing the clinical application value. Therefore, finding non-toxic or low-toxic, high-efficiency ionizing radiation induced intestinal injury protective drugs has become a research hotspot in the field of radiation medicine. SUMMARY

[0008] The purpose of the present application is to solve the problems existing in the prior art, and the purpose of the present application is to propose the use of intestinal flora metabolite kynurenic acid in the preparation of ionizing radiation induced intestinal injury protective drugs.

[0009] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0010] The use of KYNA in the preparation of ionizing radiation induced intestinal injury protective drugs.

[0011] Preferably, the ionizing radiation induced intestinal injury includes intestinal injury caused by radiation of radioactive substances or linear accelerator devices that can cause ionization of matter.

[0012] Preferably, the ionizing radiation induced intestinal injury protection includes prolonging the survival time of mice receiving ionizing radiation and protecting the intestinal tissue structure of mice receiving ionizing radiation.

[0013] The ionizing radiation induced intestinal injury protective drug prepared with KYNA as the active ingredient also belongs to the protection scope of the present application.

[0014] Preferably, the drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, oral, spraying, penetration, absorption, physical or chemical mediated method, or mixed or wrapped with other substances and introduced into the body.

[0015] Preferably, the drug further comprises one or more pharmaceutically acceptable carriers, and the carrier comprises one or more of diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

[0016] Preferably, the drug can be prepared into one or more of injection, suspension, powder, tablet, granule. The above-mentioned various dosage forms of drugs can be prepared according to the conventional method in the field of pharmacy.

[0017] An ionizing radiation induced intestinal injury protective drug comprises KYNA.

[0018] Preferably, the structure of the KYNA is as follows:

[0019]

[0020] Dimethyl sulfoxide as diluent, the medicine is prepared by dissolving KYNA in the dimethyl sulfoxide.

[0021] Preferably, the mass concentration of KYNA in the medicine is 0-50 mg / ml. The mass concentration is adjusted according to the administration dose and administration volume. For mice as the experimental subjects, the administration volume needs to be controlled at about 0.2 ml, and the mice are difficult to bear if the administration volume is too large; and the sample injection is prone to be inaccurate if the administration volume is too small. Therefore, the mass concentration changes with the change of the administration dose.

[0022] A method for constructing a mouse model, comprising the following steps:

[0023] Step one: select experimental mice, and randomly divide them into three groups: normal control group, irradiation control group and irradiation+KYNA;

[0024] Step two: dissolve KYNA in dimethyl sulfoxide, and use the gavage method to treat the mice in the irradiation+KYNA group one day before irradiation, one hour before irradiation and one hour after irradiation, and give the mice in the control group and the irradiation group the same volume of dimethyl sulfoxide;

[0025] Step three: use an X-ray source to uniformly irradiate the irradiation control group and the irradiation+KYNA group, and record and analyze the influence of KYNA on the general conditions and physical signs of the irradiated mice.

[0026] Preferably, the effective dose of the administration is 200 mg / kg of body weight; the administration frequency is once one day before irradiation, one hour before irradiation and one hour after irradiation; and the administration mode is oral or injection.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The present application provides the use, the dosage form and the administration mode of KYNA in the preparation of a medicine for protecting against intestinal damage caused by ionizing radiation. The medicine for protecting against intestinal damage caused by ionizing radiation provided by the present application has the advantages of low price, small toxic and side effects, significant efficacy, convenient and safe medication, etc., and is suitable for different persons such as patients with acute radiation sickness, nuclear industry workers, officers and soldiers, astronauts, radiotherapy patients, etc., and can be used before treatment or during the performance of tasks or work. The above performances show the unique properties of KYNA in protecting against intestinal damage caused by ionizing radiation, and the present application has a broad application prospect in the medical field in China. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0030] Figure 1 Survival and body weight changes of mice in KYNA treatment group and control group, irradiation group after 13Gy X-ray irradiation;

[0031] Figure 2 KYNA protects the structure of intestinal villi and crypts of mice after 13Gy X-ray irradiation;

[0032] Figure 3 KYNA promotes the proliferation of intestinal crypt cells of mice after 13Gy X-ray irradiation;

[0033] Figure 4 KYNA increases the number of intestinal stem cells of mice after 13Gy X-ray irradiation;

[0034] Figure 5 KYNA improves the apoptosis of intestinal crypt cells of mice after 13Gy X-ray irradiation. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all.

[0036] Example 1, protective effect of KYNA on 13Gy X-ray whole abdominal irradiation mice

[0037] (1) Experimental grouping: 30 C57BL / 6 male mice aged 6-8 weeks (body weight 23±2g) were randomly divided into three groups: normal control group (10 mice), irradiation control group (10 mice) and irradiation+KYNA (10 mice).

[0038] (2) Drug treatment: KYNA was dissolved in dimethyl sulfoxide (DMSO), and the mice in the irradiation+KYNA group were treated by gavage (dose according to body weight: 200mg / kg) at 1 day before irradiation, 1 hour before irradiation and 1 hour after irradiation. The mice in the control group and the irradiation group were given the same volume of DMSO.

[0039] (3) Establishment of X-ray radiation induced intestinal injury mouse model: the mice in the irradiation control group (10 mice) and the irradiation+KYNA group were uniformly irradiated by X-ray source for whole abdomen single time, and the irradiation dose was 13Gy.

[0040] (4) The effect of KYNA on the general condition and sign index of irradiated mice: The activity of the irradiation control group mice decreased significantly 1 day after irradiation, while the activity of the irradiation + KYNA group mice was significantly better than that of the irradiation control group. Analysis of mouse survival and body weight found that KYNA could significantly prolong the survival rate of mice after 13 Gy X-ray irradiation and improve the body weight of mice. Therefore, KYNA can improve the signs of irradiated mice and prolong the survival rate of irradiated mice. The results are shown in Figure 1 , Figure 1 a is a KYNA administration schematic diagram, Figure 1 b and c are survival analysis and body weight detection of mice in each group after 13 Gy TAI (n = 10 / group).

[0041] Example Two, Protective Effect of KYNA on Intestinal Injury Caused by 13 Gy X-ray Whole Abdominal Irradiation

[0042] (5) Experimental grouping: 18 6-8 week old C57BL / 6 male mice (body weight 23 ± 2 g) were randomly divided into three groups: normal control group (6), irradiation control group (6) and irradiation + KYNA group (6).

[0043] (6) Drug treatment: Dissolve KYNA in DMSO solution, 1 day before irradiation, 1 h before irradiation and 1 h after irradiation, use gavage administration (dose according to body weight: 200 mg / kg) to treat irradiation + KYNA group mice, normal control group, irradiation control group mice are given the same volume of DMSO treatment.

[0044] (7) Establishment of X-ray radiation induced intestinal injury mouse model: Use X-ray source to irradiate the whole abdomen of the irradiation control group (10) and the irradiation + KKYNA group mice uniformly, and the irradiation dose is 13 Gy.

[0045] (8) The effect of KYNA on the intestinal structure of irradiated mice: The small intestine tissue of the mice was taken out 3 days after irradiation, fixed, wax block embedded, and after slicing, HE staining was performed. As shown in Figure 2 , Figure 2 a is the small intestine tissue taken out 3 days after TAI and the morphology of the small intestine is shown by H&E staining (scale = 100 μm), Figure 2 b is the villus height quantification of each group. Ionizing radiation significantly causes the villus of the mouse small intestine to break and fall off; the intestinal structure of the KYNA administration group (irradiation + KYNA group) is similar to that of the normal control group, the villi are neat and complete. Quantitative analysis of villus length found that KYNA can significantly prolong the small intestine villus length of mice receiving ionizing radiation irradiation.

[0046] (9) Effect of KYNA on crypt cell proliferation in irradiated mice intestine: The mouse intestinal tissues were collected as described in (4), and after sectioning, immunohistochemical staining was performed with anti-Ki67 antibody, which is one of the marker molecules of cell proliferation. Figure 3 a Representative images of Ki67 IHC (dark brown) staining of the mice treated with vehicle and KYNA 3 days after TAI (scale bar = 20 pm), Figure 3 b and Figure 3 c The number of Ki67+ cells per crypt and surviving crypts per circumference. Quantified in at least 30 crypts per mouse. At least 30 well-oriented crypts were counted per mouse. Data are presented as mean ± standard deviation, *p<.05; **p<.01; p<.001; n=6 / group. We found that the intestinal crypts of normal control mice were Ki67 positive, indicating that crypt cells had sustained proliferative capacity. The number of Ki67 positive crypts in the intestine of irradiated control mice 3 days after irradiation was significantly reduced, and the ratio of Ki67 positive cells in each crypt was also significantly decreased. The number of Ki67 positive cells per crypt and the number of Ki67 positive crypts in the intestine of KYNA administered mice (irradiation + KYNA group) were significantly higher than those in the irradiated control group.

[0047] (10) Effect of KYNA on the number of intestinal stem cells in irradiated mice: The mouse intestinal tissues were collected as described in (8), and after sectioning, immunohistochemical staining was performed with anti-Lgr5 antibody, which is a marker of intestinal stem cells. The results are shown in Figure 4 , Figure 4 a Representative images of Lgr5 IHC (dark brown) staining of the mice treated with vehicle and KYNA 3 days after TAI (scale bar = 20 pm), Figure 3 b is the number of Lgr5+ cells per crypt. Quantified in at least 30 crypts per mouse. Data are presented as mean ± standard deviation, *p<.05; **p<.01; p<.001; n=6 / group. We found that the ratio of Lgr5 positive cells in each crypt of irradiated control mice 3 days after irradiation was significantly decreased. The number of Lgr5 positive cells per crypt in the intestine of KYNA administered mice (irradiation + KYNA group) was significantly higher than that in the irradiated control group.

[0048] Example Three, Inhibitory effect of KYNA on apoptosis of intestinal crypt cells caused by 13 Gy X-ray whole abdominal irradiation

[0049] (11) Experimental grouping: 18 C57BL / 6 male mice (6-8 weeks old, weighing 23±2 g) were selected and randomly divided into three groups: normal control group (6 mice), irradiated control group (6 mice), and irradiated + KYNA group (6 mice).

[0050] (12) Drug administration: KYNA was dissolved in DMSO solution. Mice in the irradiation + KYNA group were treated by gavage (dose based on body weight: 200 mg / kg) 1 day before irradiation, 1 hour before irradiation and 1 hour after irradiation. Mice in the normal control group and the irradiation control group were treated with the same volume of DMSO.

[0051] (13) Establish a mouse model of intestinal injury caused by X-ray radiation: The whole abdomen of mice in the control group (10 mice) and the +KKYNA group was irradiated once uniformly with an X-ray source, and the irradiation dose was 13 Gy.

[0052] (14) Effect of KYNA on apoptosis of intestinal crypt cells in irradiated mice: Small intestinal tissue from mice was collected 6 hours after irradiation, fixed, embedded in paraffin blocks, sectioned, and stained with TUNEL. Figure 5 As shown, Figure 5 Representative TUNEL-stained intestinal sections from each group 6 hours after TAI (scale bar = 20 μm). Figure 5 b represents the number of TUNEL+ cells in each crypt 6 hours after TAI. Data are expressed as mean ± standard deviation, *p<.05; **p<.01; p<.001; n=6 / group. Ionizing radiation induces apoptosis in intestinal crypt cells, impairing the regenerative capacity of the intestinal structure, while KYNA can significantly inhibit ionizing radiation-induced apoptosis in mouse intestinal crypt cells.

[0053] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The use of KYNA in the preparation of drugs to protect against intestinal damage caused by ionizing radiation.

2. The use of KYNA according to claim 1 in the preparation of drugs to protect against intestinal damage caused by ionizing radiation, characterized in that, The ionizing radiation-induced intestinal damage includes intestinal damage caused by radiation from radioactive materials that can cause ionization or from linear accelerator equipment.

3. The use of KYNA according to claim 1 in the preparation of drugs to protect against intestinal damage caused by ionizing radiation, characterized in that, The protection against ionizing radiation-induced intestinal damage includes prolonging the survival time of mice exposed to ionizing radiation and protecting the intestinal tissue structure of mice exposed to ionizing radiation.

4. The use of KYNA according to claim 1 in the preparation of drugs to protect against intestinal damage caused by ionizing radiation, characterized in that, The drug can be delivered into the body, such as muscles, intradermal, subcutaneous, veins, and mucous membranes, through injection, oral administration, spraying, penetration, absorption, or physical or chemical mediated methods.

5. The use of KYNA according to claim 1 in the preparation of drugs to protect against intestinal damage caused by ionizing radiation, characterized in that, The drug also includes one or more pharmaceutically acceptable carriers, which include one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants.

6. The use of KYNA according to claim 1 in the preparation of drugs to protect against intestinal damage caused by ionizing radiation, characterized in that, The drug can be formulated as one or more of the following: injection, suspension, powder, tablet, and granule.

7. A drug for protecting against intestinal damage caused by ionizing radiation, characterized in that, Including KYNA.

8. A protective drug against intestinal damage caused by ionizing radiation according to claim 7, characterized in that, The structural formula of KYNA is shown below:

9. A method for constructing a mouse model, characterized in that, Includes the following steps: Step 1: Select experimental mice and randomly divide them into three groups: normal control group, irradiation control group, and irradiation + KKYNA group; Step 2: Dissolve KYNA in dimethyl sulfoxide. One day before irradiation, one hour before irradiation, and one hour after irradiation, administer the solution to mice in the irradiation + KYNA group by gavage. The control group and irradiation group mice were given the same volume of dimethyl sulfoxide. Step 3: The control group and the mice irradiated with KYNA were subjected to a single uniform irradiation of the entire abdomen using an X-ray source. The effects of KYNA on the general condition and vital signs of the irradiated mice were recorded and analyzed.

10. The method for constructing a mouse model according to claim 9, characterized in that, The effective dose of the drug is 200 mg / kg body weight; the administration frequency is once a day before X-ray irradiation, once an hour before irradiation, and once an hour after irradiation; the administration method is oral or injection.

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