Use of exemestane for the preparation of a medicament for the protection against radiation-induced intestinal damage

Abstract

The application provides an application of exemestane or a derivative thereof in preparation of a medicine for protecting against radiation-induced intestinal injury, and further provides a medicine composition for protecting against radiation-induced intestinal injury, which is composed of exemestane or a derivative thereof and pharmaceutically acceptable adjuvants. The application first screens and locks the exemestane from a high-throughput screening of 3067 compounds approved by FDA. Through a mouse model of radiation-induced intestinal injury, it is verified that the survival rate of mice can be significantly improved by intragastrically treating the mice with 6.25 mg / kg of exemestane 2 hours before whole body 8 Gy irradiation. HE histology shows that the intestinal crypt-villus structure integrity of the mice in the exemestane administration group is recovered, the bone marrow cavity emptiness degree is reduced, and the spleen injury is reduced. The above results prove that the exemestane can protect the intestine, hematopoietic and immune organs at the whole animal level, and is a rare small molecule compound for protecting multiple organs. The application provides a new basis for exemestane or a derivative thereof for protecting against radiation-induced intestinal injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of ethoxzolamide or its derivatives in the preparation of drugs to protect against radiation-induced intestinal injury (RIII). Background Technology

[0002] With the continuous development of nuclear energy in the energy field, the widespread application of radiotherapy in clinical oncology, and the increasing demand for emergency medical rescue in nuclear emergencies, the threat of ionizing radiation to human health has attracted widespread global attention. Radiation-induced intestinal injury (RIII) is one of the most common and serious complications following radiotherapy for abdominal or pelvic malignant tumors, or accidental exposure to high doses of ionizing radiation throughout the body. Its pathological features mainly include massive apoptosis of intestinal crypt stem cells, severe damage to the intestinal epithelial barrier structure, translocation of intestinal flora and their toxins into the circulatory system, and further triggering systemic inflammatory response syndrome and even multiple organ failure. Currently, there are no specific and highly effective drugs for the prevention and treatment of RIII in clinical practice, existing supportive care methods have limited effectiveness, and patients have a poor prognosis.

[0003] In clinical oncology radiotherapy, despite continuous advancements in precision radiotherapy techniques, the small intestine, as a dose-limiting organ in abdominal and pelvic radiotherapy, remains highly susceptible to radiation damage. Studies have shown that in patients receiving conventional fractionated irradiation with a total dose of 45-50 Gy, the incidence of acute radiation enteritis is as high as 60%, with approximately 20% of these patients further developing chronic intestinal injury, manifesting as intestinal wall fibrosis, intestinal stenosis, intractable diarrhea, or intestinal obstruction, severely impacting quality of life and the effectiveness of cancer treatment (Radiother Oncol, 2022). On the other hand, in nuclear accident scenarios, such as long-term follow-up data from the Chernobyl and Fukushima nuclear accidents, over 70% of deaths among patients receiving whole-body irradiation of 8-12 Gy are attributed to intestinal syndrome characterized by severe diarrhea, dehydration, and infection, with most deaths occurring within two weeks of irradiation (Int J Radiat Biol, 2020). Furthermore, in the field of deep space exploration, astronauts will be exposed to galactic cosmic rays (GCR) for extended periods. High-LET carbon and iron ions can cause significant cumulative damage to intestinal tissues. Models predict that during a multi-year Mars mission, the equivalent cumulative dose to the intestines could exceed 0.6 Gy, leading to chronic functional impairment and posing a significant threat to the health and mission safety of astronauts on long-duration missions (NPJ Microgravity, 2021). Therefore, developing a safe, effective, and applicable drug for preventing and treating RIII radiation exposure has become an urgent need for military medicine, radiation oncology, and nuclear emergency medicine.

[0004] A deeper exploration at the molecular level reveals that the core pathological link in radiation-induced intestinal injury lies in the explosive release of massive reactive oxygen species (ROS) induced by ionizing radiation. High-energy rays act on intracellular water molecules, instantly generating a large number of hydroxyl radicals (·OH) and superoxide anions (O2). - Radiation can also emit reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). These highly reactive molecules directly attack the DNA of intestinal crypt stem cells, causing severe double-strand breaks (DSBs), which in turn continuously activate the p53 / Bax / caspase-3-mediated endogenous apoptosis pathway, ultimately leading to stem cell population depletion, crypt structure destruction, and impaired intestinal epithelial regeneration. Simultaneously, radiation can also activate inflammatory signaling pathways such as NF-κB, promoting the excessive release of various pro-inflammatory factors such as TNF-α, IL-1β, and IL-6, further exacerbating local tissue damage and systemic inflammatory responses.

[0005] Faced with this severe challenge, several strategies have been attempted for radiation protection using existing technologies. Amifostine (WR-2721), as a broad-spectrum free radical scavenger, has some protective effect, but its severe toxic side effects such as nausea, vomiting, and hypotension greatly limit its clinical application. Various growth factor drugs, such as keratinocyte growth factor (KGF) or fibroblast growth factor (FGF), can promote epithelial repair, but they suffer from problems such as formulation instability, short in vivo half-life, and high treatment costs, making widespread clinical application difficult. Traditional anti-inflammatory drugs, such as 5-aminosalicylic acid, have very limited protective effects against acute radiation-induced damage. Therefore, there is an urgent clinical need for a novel radiation protection drug that combines high protective activity, good safety, convenient administration, and controllable cost. Its core focus should be on regulating ROS levels, protecting stem cells from apoptosis, and promoting DNA damage repair.

[0006] Exemestane is an irreversible steroidal aromatase inhibitor that significantly inhibits peripheral estrogen synthesis by irreversibly binding to the active site of aromatase. Since its market launch, it has been widely used as adjuvant endocrine therapy for hormone receptor-positive breast cancer, and its clinical safety, pharmacokinetic characteristics, and long-term use data are well-established. It is known to have good oral bioavailability, is primarily metabolized by the hepatic CYP3A4 enzyme, and common adverse reactions include hot flashes and arthralgia, which are usually mild and tolerable. Notably, some recent studies suggest that aromatase inhibitors, in addition to their hormonal regulatory effects, may also have potential pleiotropic effects, such as regulating oxidative stress, influencing cell cycle progression, and even participating in DNA damage responses. However, to date, the potential value of exemestane, or other aromatase inhibitors, in the field of radiation damage protection remains unexplored, and their specific protective effects and mechanisms against radiation-induced intestinal injury are unknown.

[0007] In summary, given the significant unmet clinical need for radiation-induced intestinal injury, the limitations of existing protective strategies, and the potential pleiotropic pharmacological properties of exemestane, this invention proposes for the first time a novel use for protecting against radiation-induced intestinal injury, aiming to provide a new drug option with rapid translational potential for clinical radiation protection. Summary of the Invention

[0008] The present invention aims to provide a new pharmaceutical use of exemestane or its derivatives, and a pharmaceutical composition containing exemestane or its derivatives.

[0009] The research approach of this invention is as follows: Through in vitro cell experiments, compounds from a library of 3067 FDA-approved compounds were selected as screening targets. The screening criteria included a ≥1.5-fold HIEC survival rate in intestinal epithelial cells (P<0.01). High-throughput screening of the top 10% of compounds revealed several compounds, including exemestane, which demonstrated excellent performance and significantly improved cell proliferation; these were selected as candidate compounds. Further experiments using irradiation hemorrhage and animal models confirmed that exemestane showed superior efficacy.

[0010] Based on the above research, the specific technical solution of the present invention is as follows:

[0011] A first aspect of the present invention provides the use of exemestane or a derivative thereof in the preparation of a medicament for protection against radiation-induced intestinal injury.

[0012] According to the experimental results of this invention, exemestane has the following technical effects:

[0013] (1) Improve the survival rate of irradiated animals ( Figure 2 );

[0014] (2) Reduce pathological damage to bone marrow, spleen, and intestinal tissues ( Figure 3-4 );

[0015] (3) Reduces apoptosis of small intestinal crypt stem cells after irradiation and promotes intestinal epithelial regeneration. Figure 5 );

[0016] (4) Reduces DNA double-strand breaks (γ-H2AX level) in irradiated intestinal epithelial cells and restores cell proliferation capacity. Figure 6-7 ).

[0017] Preferably, the drug is a drug that reduces pathological damage to bone marrow, spleen and intestinal tissues, reduces apoptosis of small intestinal crypt stem cells or promotes regeneration of intestinal epithelial cells.

[0018] Furthermore, the drug that promotes intestinal epithelial cell regeneration is a drug that promotes intestinal epithelial cell proliferation and DNA repair.

[0019] The exemestane involved in this invention, chemically named 6-methylandrost-1,4-diene-3,17-dione, has a core structure of a modified sterane skeleton. Exemestane derivatives refer to exemestane compounds that can contribute to the effective pharmacological action of exemestane or improve its acceptability, such as its hydrates, metabolites, and prodrugs. Related derivatives are all within the scope of protection of this invention as long as they do not reduce the efficacy of the drug.

[0020] Preferably, the protective drug against radiation-induced intestinal injury of the present invention uses exemestane or its derivatives as the sole active ingredient or as part of its active ingredient, in combination with other components.

[0021] In a second aspect, the present invention provides a pharmaceutical composition for protecting against radiation-induced intestinal injury, comprising a therapeutically effective amount of exemestane or its hydrate, a prodrug or an active metabolite, and pharmaceutically acceptable excipients.

[0022] The composition can be formulated into tablets, capsules, powders, granules, oral liquids, enteric-coated microcapsules, suppositories, enemas, injections, liposomes, or nanocrystal formulations to suit oral, rectal, or intravenous administration routes.

[0023] The present invention relates to exemestane or its derivatives for protection against radiation-induced intestinal injury, particularly suitable for radiation protection in cases of nuclear war, nuclear accidents, radiation workers, and patients undergoing radiotherapy for abdominal tumors. It is administered beforehand or during treatment to individuals at risk of radiation-induced intestinal injury.

[0024] The role and effect of invention

[0025] This invention is the first to use high-throughput screening from a library of 3067 FDA-approved compounds to identify exemestane. Validation using a mouse model of radiation-induced intestinal injury (RIII) showed that administration of 6.25 mg / kg exemestane by gavage 2 hours before whole-body 8 Gy irradiation improved survival rates in mice to some extent. HE histology revealed that, compared to the control group, mice in the exemestane-treated group showed restored intestinal crypt-villus structure integrity, reduced bone marrow cavity emptiness, and reduced spleen damage. These results demonstrate that exemestane can simultaneously protect the intestine, hematopoietic, and immune organs at the whole-animal level, representing a rare example of a small molecule compound providing "multi-organ integrated protection." In vitro cellular mechanism validation showed that exemestane significantly enhanced the proliferation capacity of normal intestinal epithelial cells (HIEC) and reduced DNA damage after irradiation. Therefore, this invention provides new evidence for the protection of exemestane or its derivatives against radiation-induced intestinal injury.

[0026] Furthermore, exemestane has been safely used in patients with gastric ulcers for over 30 years, with an oral bioavailability of 20%. It is primarily metabolized by hepatic CYP3A4 and has mild toxic side effects. Oral administration can meet the diverse needs of nuclear emergency response and tumor radiotherapy. In summary, exemestane represents a promising new indication for an FDA-approved drug, supported by robust experimental evidence. Its target closely matches the core pathological processes of radiation-induced intestinal injury, demonstrating significant clinical potential for "discovery-to-translation." Attached Figure Description

[0027] Figure 1 The FDA has approved the results of high-throughput screening of the compound library.

[0028] Figure 2 Animal survival rate after exemestane treatment.

[0029] Figure 3 Figure showing the effects of exemestane on the femoral head and medullary cavity of mice after irradiation.

[0030] Figure 4 Figure showing the effect of exemestane on the spleen of mice after irradiation.

[0031] Figure 5 The results of the effects of exemestane on the intestines of mice after irradiation.

[0032] Figure 6 Figure showing the effect of exemestane on the proliferation of intestinal epithelial cells after irradiation.

[0033] Figure 7 The effect of exemestane on the γ-H2AX index of DNA damage repair in intestinal epithelial cells after irradiation. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.

[0037] Example 1: Screening for exemestane

[0038] 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.

[0039] Screening criteria: survival rate increase ≥1.5-fold, P<0.01. See results below. Figure 1 Exemestane significantly enhances cell proliferation and was selected as a candidate compound. Further experiments will be conducted to further elucidate its protective effect against intestinal injury.

[0040] Example 2: Exemestane can improve the survival rate of mice irradiated with whole body.

[0041] Eight-week-old male C57BL / 6J mice were randomly divided into three groups (n=10): ① irradiation alone; ② irradiation plus exemestane 25 mg / kg; ③ irradiation plus exemestane 6.25 mg / kg. In groups ② and ③, exemestane was dissolved in physiological saline and administered by gavage 2 hours before irradiation; group ① received an equal volume of physiological saline. Subsequently, groups ①–③ underwent systemic TBI at a dose rate of 1 Gy / min.

[0042] The survival rate of mice after irradiation was observed, and the results are shown in [the table below]. Figure 2 The survival rate of mice in the exemestane-treated group was higher than that in the irradiation-only group, but this was not drug concentration-dependent. The mice in the exemestane 6.25 mg / kg group had the highest survival rate and did not die until the 13th day, while the control mice all died on the 6th day.

[0043] Example 3: Exemestane can reduce bone marrow, spleen, and intestinal damage in mice after irradiation.

[0044] (1) Staining of femoral and medullary cavity pathological sections

[0045] 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 exemestane-treated group was less than that in the irradiation-only group.

[0046] (2) Staining of spleen pathological sections

[0047] 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; exemestane treatment improved this phenomenon.

[0048] (3) Staining of small intestinal pathological sections

[0049] 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 was reduced in the exemestane-treated group compared with the irradiation-only group.

[0050] Example 4: Exemestane promotes intestinal cell proliferation

[0051] Intestinal epithelial cells (HIEC) were seeded in 96-well plates (2 × 10³ / well). The following day, pretreatment with exemestane (10 μM) or 0.1% DMSO for 2 h was performed, followed by 8 Gy¹³. 7Cs 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 exemestane-treated group was significantly higher than that of the irradiation-only group, and the difference between the exemestane-treated group and the control group (DMSO) increased with the duration of treatment.

[0052] Example 5: Exemestane promotes DNA damage repair (γ-H2AX)

[0053] Intestinal epithelial cells (HIEC) were seeded in glass dishes (5 × 10⁵ / well). The following day, pretreatment with exemestane (10 μM) or 0.1% DMSO for 2 h was performed, 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 exemestane group at 8 h and 24 h, suggesting accelerated DNA repair.

[0054] in conclusion:

[0055] Exemestane, 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.

[0056] The above embodiments used exemestane as an example to explore the effects of exemestane on intestinal injury and bleeding after irradiation in mice, inhibition of proliferation of intestinal epithelial cells after HIEC irradiation, and reduction of DNA damage. However, the present invention is not limited to exemestane, and exemestane derivatives that can achieve the same pharmacological effects are also within the scope of protection of the present invention.

[0057] 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 exemestane in the preparation of drugs for protection against radiation-induced intestinal injury.

2. The application of exemestane according to claim 1 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 one that reduces apoptosis of small intestinal crypt stem cells or promotes the regeneration of intestinal epithelial cells.

3. The application of exemestane 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 exemestane according to any one of claims 1 to 3 in the preparation of a drug for protecting against radiation-induced intestinal injury, characterized in that: in, The protective drug against radiation-induced intestinal injury is either exemestane as the sole active ingredient or a pharmaceutical composition containing exemestane.

5. The use of exemestane according to claim 1 in the preparation of a drug for protecting against radiation-induced intestinal injury, characterized in that: in, The drugs for protecting against radiation-induced intestinal injury are tablets, capsules, powders, granules, oral liquids, enteric-coated microcapsules, suppositories, enemas, injections, and liposomes.

Images