Application of ethozolamide in preparation of medicine for preventing radioactive intestinal injury

Abstract

The invention provides an application of ixozolamide or a derivative thereof in preparation of a medicine for preventing radioactive intestinal injury, and also provides a pharmaceutical composition for preventing radioactive intestinal injury, which is composed of ixozolamide or a derivative thereof and pharmaceutically acceptable auxiliary materials. According to the invention, high-throughput screening and locking of ethozolamide are carried out from 3067 compound libraries approved by FDA for the first time. A radioactive intestinal injury mouse model verifies that 25 or 50 mg / kg of ethofenzolamide is used for intragastric therapy 2 hours before the whole body is irradiated by 8 Gy, and the survival rate of 30 days can be remarkably increased. HE histology shows that the integrity of intestinal crypt-villus structures of mice in an exozolamide administration group is recovered, the empty degree of marrow cavities is relieved, and spleen injury is relieved. The results prove that the esozolamide can protect intestinal tracts, hematopoietic organs and immune organs at the same time at the whole animal level, and the esozolamide is a rare multi-organ integrated protection small-molecule compound so far. The invention provides a new basis for the protection of radioactive intestinal injury by the ixozolamide or the derivative thereof.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of Ethoxzolamide or a derivative thereof in preparation of a drug for preventing radiation-induced intestinal injury (RIII). BACKGROUND

[0002] Ionizing radiation exposure is a major health threat in nuclear power applications, radiotherapy, nuclear accidents, and space and deep space missions. Radiation-induced intestinal injury (RIII) is one of the earliest and highest mortality complications after abdominal / pelvic tumor radiotherapy, nuclear accidents or wartime nuclear exposure, mainly manifested as intestinal crypt stem cell apoptosis, intestinal epithelial barrier destruction, bacterial translocation and systemic inflammatory response, and there is currently no specific and effective prevention and treatment drug.

[0003] In precise radiotherapy of abdominal or pelvic tumors, small intestine as a dose-limiting organ, about 60% of patients develop acute radiation enteritis after receiving 45-50 Gy irradiation, and some patients can progress to chronic injury (Radiother Oncol, 2022). Nuclear accident data shows that more than 70% of deaths after whole body irradiation of 8-12 Gy are caused by intestinal syndrome (Int J Radiat Biol, 2020). In addition, high-energy heavy ion radiation in space missions can also cause progressive intestinal function damage, threatening the health of astronauts (NPJ Microgravity, 2021). Therefore, it is of great clinical and military value to develop safe and effective, easy-to-use radiation intestinal injury prevention drugs.

[0004] The core pathology of radiation-induced intestinal injury is that intestinal crypt stem cells (Lgr5 + ISCs) are extremely sensitive to ionizing radiation. High-energy rays ionize water molecules, instantaneously generating a large amount of ROS (·OH, O2 - ·, H2O2), which directly attacks ISCs DNA, causes double-strand breaks (DSB), activates the p53 / Bax / caspase-3 pathway, induces stem cell apoptosis, and causes the collapse of villus-crypt structure. In the prior art, some drugs that have been proven to be effective in protection have obvious defects: free radical scavengers such as WR-2721 (amifostine) are limited in clinical use due to their high toxicity and side effects; growth factor drugs are expensive and have short half-lives; anti-inflammatory drugs such as 5-aminosalicylic acid have limited efficacy for acute radiation damage. Therefore, there is an urgent need to discover new radiation protection drugs that are safe, effective, and can be quickly translated into clinical use.

[0005] Ethoxzolamide is a classic carbonic anhydrase inhibitor, which was first synthesized by American Wyeth Company in the 1950s, and is mainly used for the treatment of glaucoma. It reduces aqueous humor production and intraocular pressure by inhibiting carbonic anhydrase isozymes (mainly CA-II, CA-IV, etc.). Subsequent studies have shown that ethoxzolamide also has certain antiepileptic, diuretic and anti-high altitude disease effects. Ethoxzolamide can be absorbed after oral administration, and the bioavailability is about 70%, the plasma half-life is about 5-6 hours, and it is mainly excreted by the kidney. Ethoxzolamide has been used in clinical for decades, and the safety data is relatively complete. The common adverse reactions include paresthesia, metabolic acidosis, gastrointestinal discomfort, etc., and most of the symptoms are mild and controllable. However, as of now, the role of ethoxzolamide in the protection of radiation-induced intestinal injury has not been reported. SUMMARY

[0006] The present application aims to provide a new medical use of ethoxzolamide or its derivative, and a pharmaceutical composition containing ethoxzolamide or its derivative.

[0007] The research idea of the present application is as follows: through in vitro cell experiments, the compounds in the FDA-approved 3067 compound library are taken as screening objects, the survival rate of intestinal epithelial cells HIEC ≥ 1.5 times, P < 0.01 are taken as screening conditions, and the top 10% of compounds are screened through high-throughput screening. A few compounds including ethoxzolamide perform excellently and can significantly improve the cell proliferation ability, and are selected as candidate compounds. Further through irradiation bleeding and animal model experiments, it is determined that ethoxzolamide has better effect.

[0008] According to the above research, the specific technical scheme of the present application is as follows:

[0009] In a first aspect of the present application, the use of ethoxzolamide or its derivative in the preparation of a drug for preventing radiation-induced intestinal injury is provided.

[0010] According to the experimental results of the present application, ethoxzolamide has the following technical effects:

[0011] (1) Improving the survival rate of irradiated animals ( Figure 2 );

[0012] (2) Reducing the pathological damage of bone marrow, spleen and intestinal tissue ( Figures 3-4 );

[0013] (3) Reducing the apoptosis of crypt stem cells in the small intestine after irradiation and promoting the regeneration of intestinal epithelium ( Figure 5 );

[0014] (4) Reducing the DNA double-strand break (γ-H2AX level) of intestinal epithelial cells after irradiation and restoring the cell proliferation ability ( Figures 6-7 ).

[0015] Preferably, the drug is a drug for reducing bone marrow, spleen and intestinal tissue pathological damage, reducing small intestinal crypt stem cell apoptosis or promoting intestinal epithelial cell regeneration.

[0016] Further, the drug for promoting intestinal epithelial cell regeneration is a drug for promoting intestinal epithelial cell proliferation and promoting DNA repair.

[0017] The chemical structure core of Ethoxzolamide is a benzothiophene and thiopyran ring system containing a sulfonamide group. Ethoxzolamide derivatives refer to Ethoxzolamide compounds that can help Ethoxzolamide effectively exert pharmacological effects or improve drug acceptance, such as pharmaceutically acceptable salts (Ethoxzolamide sodium salt, Ethoxzolamide lysine salt, Ethoxzolamide magnesium salt, etc.), hydrates, metabolites, prodrugs, etc. Related derivatives are within the protection scope of the present application without reducing the efficacy.

[0018] Preferably, the drug for protecting against radiation-induced intestinal injury according to the present application is Ethoxzolamide or its derivatives as the only active ingredient or as part of the efficacy of other components.

[0019] In the second aspect of the present application, a drug composition for protecting against radiation-induced intestinal injury is provided, which comprises a therapeutically effective amount of Ethoxzolamide or its pharmaceutically acceptable salt, hydrate, prodrug or active metabolite, and a pharmaceutically acceptable excipient.

[0020] The composition can be prepared into tablets, capsules, powders, granules, oral solutions, enteric-coated pellets, suppositories, enemas, injections, liposomes or nanocrystal preparations to adapt to oral, rectal or intravenous administration routes.

[0021] Ethoxzolamide or its derivatives according to the present application are used for protecting against radiation-induced intestinal injury, and are particularly suitable for protecting against radiation-induced injury in nuclear war, nuclear accidents, radiation workers, patients undergoing abdominal tumor radiotherapy treatment, etc. They are administered to people who may suffer from radiation-induced intestinal injury in advance or during treatment.

[0022] Effects and advantages of the present application

[0023] The present application first screens and locks Ethoxzolamide from the FDA-approved 3067 compound library at high throughput. Administration of 50 or 100 mg / kg Ethoxzolamide by gavage 2h before whole body 8 Gy irradiation can significantly improve the survival rate of mice.

[0024] Histological analysis showed that the intestinal crypt-villi structure of animals treated with exozide was more intact, bone marrow hematopoietic tissue damage was reduced, and splenic white pulp atrophy was improved, indicating that the drug has multi-organ radiation protection effects. At the cellular level, exozide pretreatment enhanced the proliferative activity of intestinal epithelial cells (HIEC) after irradiation and accelerated DNA damage repair, as evidenced by a significant decrease in the number of γ-H2AX focal points.

[0025] Exozopramide, a carbonic anhydrase inhibitor with a long history of clinical use, boasts well-established pharmacokinetic and safety data, convenient oral administration, and suitability for rapid application in various scenarios such as nuclear emergency response and tumor radiotherapy. Therefore, this invention not only expands the medical applications of exozopramide but also provides a candidate drug with high translational potential for the prevention and treatment of radiation-induced intestinal injury. Attached Figure Description

[0026] Figure 1 The FDA has approved the results of high-throughput screening of the compound library.

[0027] Figure 2 Survival rate of animals after treatment with exozide.

[0028] Figure 3 Figure showing the effects of estozonide on the femoral head and medullary cavity of mice after irradiation.

[0029] Figure 4 The effect of exozolamide on the spleen of mice after irradiation.

[0030] Figure 5 The results of the effects of estrazoride on the intestines of mice after irradiation.

[0031] Figure 6 The effect of erythrazole on the proliferation of intestinal epithelial cells after irradiation is shown in the figure.

[0032] Figure 7 The effect of exozolamide on the γ-H2AX index of DNA damage repair in intestinal epithelial cells after irradiation. Detailed Implementation

[0033] The following embodiments further illustrate the present invention and should not be construed as limiting the invention. The embodiments do not include a detailed description of conventional methods, which are well known to those skilled in the art and described in numerous publications.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention, and the preferred embodiments and materials described in the specific implementation are for illustrative purposes only.

[0035] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.

[0036] Example 1: Screening for exolotl

[0037] Intestinal epithelial cells (HIECs) were used as experimental cells, and HIECs were seeded in 96-well plates (2 × 10³ / well). The following day, the cells were pretreated with either a candidate compound from the compound library (10 μM) or 0.1% DMSO (control group) for 2 h, followed by 8 Gy¹³ 7 Cs were irradiated with gamma rays (1 Gy / min) and then cultured for another 48 h. The survival rate was then determined by CellTiter-Glo.

[0038] Screening criteria: survival rate increase ≥1.5-fold, P<0.01. See results below. Figure 1 Exozide was selected as a candidate compound because it significantly enhances cell proliferation. Further experiments will be conducted to further elucidate its protective effect against intestinal injury.

[0039] Example 2: Exozopramide can improve the survival rate of mice irradiated with whole body.

[0040] Eight-week-old male C57BL / 6J mice were randomly divided into three groups (n=5): ① irradiation alone; ② irradiation plus exozolamide 50 mg / kg; ③ irradiation plus exozolamide 100 mg / kg. In groups ② and ③, exozolamide was dissolved in 0.5% CMC-Na and administered by gavage 2 hours before irradiation; group ① received an equal volume of CMC-Na. Subsequently, groups ①–③ underwent systemic TBI at a dose rate of 1 Gy / min.

[0041] The survival rate of mice after irradiation was observed, and the results are shown in [the table below]. Figure 2 The survival rate of the exozolamide-treated group was significantly higher than that of the irradiation-only group, but this was not drug concentration-dependent. The mice in the 500 mg / kg exozolamide-treated group had the highest survival rate (40% survival rate until day 15), while all mice in the 100 mg / kg exozolamide-treated group died by day 11.

[0042] Example 3: Exozopramide can reduce bone marrow, spleen, and intestinal damage in mice after irradiation.

[0043] (1) Staining of femoral and medullary cavity pathological sections

[0044] The femur was harvested on day 3.5 post-irradiation, fixed in 10% neutral formalin, decalcified with EDTA, embedded in paraffin, and stained with H&E. Results are shown below. Figure 3 Irradiation resulted in a reduction in the bone marrow cavity and a decrease in fat vacuoles; the degree of bone marrow cavity damage in the exozolamide-treated group was less than that in the irradiation-only group.

[0045] (2) Staining of spleen pathological sections

[0046] Spleen weight was measured and spleen index was calculated on day 3.5 post-irradiation; spleen hematologic and epithelial eosinophil (H&E) was used to observe white pulp lymphocyte apoptosis. Results are shown in […]. Figure 4 Irradiation caused atrophy of the white pulp of the spleen; this phenomenon was improved in the exozopramide administration group.

[0047] (3) Staining of small intestinal pathological sections

[0048] Methods: Jejunum was harvested on day 3.5 post-irradiation and stained with H&E; results are shown in […]. Figure 5 The degree of intestinal damage in the exozide-treated group was less than that in the irradiation-only group.

[0049] Example 4: Exozide promotes intestinal cell proliferation

[0050] Intestinal epithelial cells (HIEC) were seeded in 96-well plates (2 × 10³ / well). The following day, the cells were pretreated with exozide (10 μM) or 0.1% DMSO for 2 h, followed by 8 Gy¹³. 7 Cs γ-ray irradiation (1 Gy / min). CCK8 reagent was added at 24 h, 48 h, and 72 h post-irradiation, and the results were detected by a microplate reader after 2 h. Results are shown below. Figure 6 The cell proliferation capacity of the exozide-treated group was significantly higher than that of the irradiation-only group, and the difference between the group and the control group (DMSO) increased with the duration of treatment.

[0051] Example 5: Exorazolamide promotes DNA damage repair (γ-H2AX)

[0052] Intestinal epithelial cells (HIEC) were seeded in glass dishes (5 × 10⁵ / well). The following day, the cells were pretreated with exolotlamide (10 μM) or 0.1% DMSO for 2 h, followed by 8 Gy¹³. 7 Cs gamma-ray irradiation (1 Gy / min). Fixation was performed at 2 h, 8 h, and 24 h post-irradiation, followed by γ-H2AX immunofluorescence staining and focal point counting using confocal microscopy. Results are shown below. Figure 7 At 0.5 h after irradiation, the number of focal points in the two groups was similar; as time progressed, compared with the control group, the number of γ-H2AX foci decreased in the itazolidinium group at 8 h and 24 h, suggesting accelerated DNA repair.

[0053] in conclusion:

[0054] Exozide, screened by the FDA database and validated in vitro and in vivo, significantly improves the survival rate of irradiated animals, protects bone marrow, spleen, and intestinal stem cells, and promotes DNA damage repair. Given that it is an already marketed drug with known and minimal side effects, it possesses the potential for rapid clinical translation.

[0055] The above embodiments used exozolamide as an example to explore the effects of exozolamide on intestinal injury and bleeding after irradiation in mice, inhibition of proliferation of intestinal epithelial cells after HIEC irradiation, and mitigation of DNA damage. However, the present invention is not limited to exozolamide, and exozolamide derivatives that can achieve the same pharmacological effects are also within the scope of protection of the present invention.

[0056] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. Application of exozolamide or its derivatives in the preparation of drugs for protection against radiation-induced intestinal injury.

2. The use of exozolamide or its derivatives according to claim 1 in the preparation of drugs for protecting against radiation-induced intestinal injury, characterized in that: in, The aforementioned drug for protecting against radiation-induced intestinal injury is a drug that reduces pathological damage to bone marrow, spleen, and intestinal tissues, decreases apoptosis of small intestinal crypt stem cells, or promotes the regeneration of intestinal epithelial cells.

3. The use of exozolamide or its derivatives according to claim 2 in the preparation of drugs for protecting against radiation-induced intestinal injury, characterized in that: in, The drug that promotes the regeneration of intestinal epithelial cells is a drug that promotes the proliferation of intestinal epithelial cells and promotes DNA repair.

4. The use of exozolamide or its derivatives according to any one of claims 1 to 3 in the preparation of drugs for protecting against radiation-induced intestinal injury, characterized in that: in, The drug for protecting against radiation-induced intestinal injury is either exozolamide or a derivative thereof as the sole active ingredient, or a pharmaceutical composition containing exozolamide or a derivative thereof.

5. The use of exozolamide or its derivatives according to any one of claims 1 to 3 in the preparation of drugs for protecting against radiation-induced intestinal injury, characterized in that: in, Exozolamide derivatives include pharmaceutically acceptable salts, hydrates, prodrugs, or active metabolites of exozolamide.

6. The use of exozolamide or its derivatives according to claim 1 in the preparation of drugs for protecting against radiation-induced intestinal injury, characterized in that: in, The protective drugs against radiation-induced intestinal injury are tablets, capsules, powders, granules, oral liquids, enteric-coated microcapsules, suppositories, enemas, injections, liposomes, or nanocrystal preparations.

7. A pharmaceutical composition for protecting against radiation-induced intestinal injury, characterized in that: It consists of exozide or its derivatives and pharmaceutically acceptable excipients.

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