Application of ferristatin-1 in preparation of medicine for treating radiation osteoporosis

Abstract

The invention relates to the technical field of medicine, in particular to application of sidelonin-1 in preparation of a medicine for treating radiation osteoporosis. In animal experiments, it is found that a radiation osteoporosis mouse model is constructed through X-ray irradiation on the right posterior limb of a mouse, and it is detected that the radiation mouse is serious in bone mass loss and reduced in bone mineral density; the bone volume fraction and the number of bone trabecula are obviously reduced, and after intraperitoneal injection of the ferristatin 1, the bone volume fraction and the number of the bone trabecula of the irradiated mouse are partially recovered, and the bone mass is increased. The result shows that the ferristatin 1 can significantly relieve bone mass loss caused by ionizing radiation. The preparation prepared from the siderostatin-1 or the pharmaceutically acceptable carrier of the siderostatin-1 has the application prospect of treating the radiation osteoporosis.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine, and in particular to the application of ferrostatin-1 in the preparation of a drug for treating radioactive osteoporosis. Background Art

[0002] Radiation-induced osteoporosis is a pathological syndrome characterized by decreased bone density and microarchitectural disruption following ionizing radiation damage to bone tissue, resulting in increased bone fragility and fracture risk. It is common in cancer patients undergoing radiotherapy (e.g., for pelvic or spinal tumors) or those with long-term exposure to radioactive substances. Ionizing radiation inhibits osteoblast activity, reducing new bone formation; it also activates osteoclasts, accelerating bone resorption and leading to an imbalance in bone metabolism. Radiation damages mesenchymal stem cells in the bone marrow, impairing their ability to differentiate into osteoblasts and reducing angiogenesis, leading to decreased bone repair capacity. Radiation triggers the release of inflammatory cytokines (e.g., TNF-α, IL-6), further exacerbating bone loss.

[0003] Current mainstays of treatment include: 1. Bisphosphonates (such as alendronate and zoledronic acid), which inhibit osteoclast activity, reduce bone resorption, and increase bone density. However, these drugs are prone to gastrointestinal side effects, musculoskeletal pain, and joint pain. 2. Calcitonin, which slows the progression of osteoporosis by regulating blood calcium levels, may cause hypocalcemia, necessitating regular monitoring of blood calcium, phosphorus, and electrolyte levels. 3. Estrogen / androgen replacement therapy, which regulates bone metabolism and prevents bone loss. However, long-term, high-dose use may induce malignancies such as endometrial and breast cancer. 4. RANKL inhibitors (such as denosumab), which inhibit the binding of RANKL to its receptor, RANK, reduce osteoclast formation and function. However, long-term use may overinhibit bone resorption, leading to mandibular osteonecrosis or atypical femoral fractures. While bisphosphonates / denosumab inhibit bone loss, they also inhibit bone turnover, further weakening already impaired bone regeneration. Bone marrow adiposity and a decrease in stem cells in the irradiated area lead to reduced bone formation. Bone-forming agents (such as teriparatide) may be less effective in areas of severe radiation damage. Regarding the current treatment of ionizing radiation-induced osteoporosis, conventional anti-osteoporosis drugs offer basic protection but lack the ability to target radiation-specific pathological mechanisms. Therefore, there is an urgent need for a specific treatment for radiation-induced osteoporosis with improved efficacy and minimal side effects.

[0004] Ferrostatin-1 (Fer-1 for short), its chemical name is 3-amino-4-cyclohexylaminobenzoic acid ethyl ester, and its molecular formula is C 15 H 22 N2O2, molecular weight 262.35, its chemical structure is as follows:

[0005] .

[0006] Ferrostatin-1 is a lipid peroxide scavenger with an N-cyclohexyl moiety that acts as a lipophilic anchor within biological membranes. While it does not affect physiological functions such as cell proliferation and differentiation, it can inhibit the accumulation of lipid peroxides in the cytoplasm and cells induced by other drugs. Ferrostatin-1 is a white powder that is insoluble in water but readily soluble in alcohol and dimethyl sulfoxide (DMSO). However, there are currently no published studies on the use of ferrostatin-1 in the treatment of radiation-induced osteoporosis.

[0007] Research results from the inventors' team show that in animal experiments (mice), a model of radiation-induced osteoporosis was established by X-raying the right hind limb of mice. Testing revealed that the irradiated mice experienced severe bone loss, decreased bone density, and significantly reduced bone volume fraction and trabecular number. However, intraperitoneal injection of ferrostatin-1 partially restored the bone volume fraction and trabecular number in the irradiated mice, and bone mass increased. Consequently, the inventors have proposed a new method for treating radiation-induced osteoporosis. Summary of the Invention

[0008] The present invention provides the use of ferrostatin-1 in the preparation of a drug for treating radioactive osteoporosis. The drug has a low dosage and small side effects. Ferrostatin-1 can significantly alleviate bone loss caused by ionizing radiation.

[0009] The technical solutions provided by the present invention are as follows:

[0010] The use of ferrostatin-1 in the preparation of a drug for treating radiation-induced osteoporosis, wherein the chemical structural formula of ferrostatin-1 is as follows:

[0011] .

[0012] Furthermore, the dosage of ferrostatin-1 in the drug is 1-5 μmol / kg / day.

[0013] Furthermore, the medicine is an injection.

[0014] Furthermore, the drug comprises ferrostatin-1 with a purity of more than 95%.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the pharmaceutically acceptable excipients include one or more of a buffer, a surfactant, an antioxidant, a preservative, a filler and a stabilizer.

[0017] Furthermore, the drug is used to alleviate bone loss caused by ionizing radiation.

[0018] Furthermore, the drug is used to increase the levels of osteocalcin and alkaline phosphatase after ionizing radiation, thereby improving bone formation function.

[0019] Furthermore, the drug is used to increase bone density, bone volume fraction and trabecular number after ionizing radiation.

[0020] Furthermore, the drug is used to improve the quality of cancellous bone after ionizing radiation.

[0021] Furthermore, ferrostatin-1 is administered by subcutaneous injection.

[0022] According to the results of animal experiments on ferrostatin-1 for the treatment of radiation-induced osteoporosis, the injection dose of ferrostatin-1 in the present invention for the treatment of radiation-induced osteoporosis can be varied according to other relevant factors such as the administration method, the patient's age, weight, and severity of the disease. Our recommended dose is 1-5 (μmol / kg) / day.

[0023] The research results showed that in animal experiments, the radiation-induced osteoporosis mouse model suffered severe bone loss. By injecting the irradiated mice with an appropriate amount of ferrostatin-1, the mice's bone mass was restored.

[0024] Ferrostatin-1 has been shown to have a modest anti-osteoporotic effect in an animal model of radiation-induced osteoporosis. This discovery has significant implications for the research and treatment of radiation-induced osteoporosis and has potential clinical utility, opening up new clinical applications for ferrostatin-1. However, to circumvent the potential for ferrostatin-1 to denature in air, we chose to administer it via intraperitoneal (subcutaneous) injection.

[0025] In the above technical solution, the ferrostatin-1 pharmaceutical composition can be prepared by adding common pharmaceutical auxiliary ingredients to ferrostatin-1 with a purity of more than 95% (mass percentage) to prepare a preparation in the form of a solution, suspension, emulsion, lyophilized product or sterile powder.

[0026] Beneficial effects

[0027] In animal experiments, a radioactive osteoporosis mouse model was established by X-raying the right hind limbs of mice. Testing revealed severe bone loss, decreased bone density, and a significant decrease in bone volume fraction and trabecular number in the irradiated mice. However, intraperitoneal injection of ferrostatin-1 partially restored the bone volume fraction and trabecular number in the irradiated mice, and bone mass increased. Thus, the present invention provides the use of ferrostatin-1 in the preparation of a drug for treating radioactive osteoporosis, expanding the application of ferrostatin-1 in various diseases. This drug has a low dosage and minimal side effects, and ferrostatin-1 can significantly alleviate ionizing radiation-induced bone loss. As a specific inhibitor of ferroptosis, ferrostatin-1 exhibits unique advantages in treating diseases induced by ionizing radiation, particularly bone damage and hematopoietic system damage. Its core value lies in precisely targeting the radiation-triggered ferroptosis pathway, overcoming the limitations of traditional drugs. Preparations prepared with ferrostatin-1 or a pharmaceutically acceptable carrier thereof have promising applications in treating radioactive osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Immunofluorescence staining was used to detect the levels of osteocalcin (OCN) and alkaline phosphatase (ALP) in the femur of the mice in each group of experiments. Toluidine blue staining was used to detect the osteogenic function of the femur tissue of the mice in each group of experiments. Figure 1 A in the middle is the stained image; Figure 1 Middle B shows the osteocalcin (OCN) level in the femur of mice in each group; Figure 1 Middle C shows the alkaline phosphatase (ALP) levels of mice in each group; Figure 1 Middle D is the toluidine blue staining method to detect the osteogenic function of femoral tissue of mice in each experimental group;

[0029] Figure 2 Micro-CT scans were performed on the lower femurs of mice in each experimental group and three-dimensional images were reconstructed; Figure 2 Middle A is the control group (Sham); Figure 2 Middle B is the right hind limb irradiated group (Irradiated); Figure 2 Middle C is the treatment group (Irradiated + Fer-1);

[0030] Figure 3 To quantitatively analyze the bone density, bone volume fraction, trabecular number, trabecular thickness and trabecular space of each experimental mouse using three-dimensional images reconstructed from micro-CT scans; Figure 3 A in the middle is the bone density of mice in each experimental group; Figure 3 Middle B is the bone volume fraction of mice in each experimental group; Figure 3 Middle C is the number of trabeculae in each experimental group of mice; Figure 3 Middle D is the trabecular space of mice in each experimental group; Figure 3 Middle E is the trabecular bone thickness of mice in each experimental group;

[0031] Figure 4 Hematoxylin-eosin (H&E) staining was used to detect femoral trabeculae in each group of mice; Figure 4 Middle A is the control group (Sham); Figure 4 Middle B is the right hind limb irradiated group (Irradiated), Figure 4 Center C is the treatment group (Irradiated+Fer-1). DETAILED DESCRIPTION

[0032] The above scheme is further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. The implementation conditions adopted in the examples can be further adjusted according to the conditions of the specific manufacturer's laboratory. The implementation conditions not specified are generally those in routine experiments.

[0033] Example 1

[0034] 1. Materials

[0035] 1. Experimental Animals

[0036] Fifteen male C57 / B6J mice, 6–8 weeks old, weighing 21–37 g, SPF grade, were provided by Shanghai Slake Laboratory Animal Co., Ltd. (Laboratory Animal Production License No. SCXK (Shanghai) 2012-0002).

[0037] During the experiment, the temperature in the feeding room was 20±3.2℃ and the relative humidity was 65-75%. The animals were allowed to eat and drink freely.

[0038] 2. Reagents and instruments:

[0039] 2.1 Reagents

[0040] Ferrostatin-1 (Fer-1), white powder, purity ≥95%, 1 mg / bottle, provided by Sigma-Aldrich, USA, product number SML0583, stored in a refrigerator at 4°C away from light.

[0041] Osteocalcin (OCN) antibody was provided by Affinity Biotechnology (USA), product number DF12303.

[0042] Alkaline phosphatase (ALP) antibody was provided by Affinity Biotechnology (USA), product number DF6225.

[0043] Toluidine blue dye was provided by Wuhan Sevier Biotechnology Co., Ltd., China, with product number G1032.

[0044] Hematoxylin-eosin stain was provided by Wuhan Sevier Biotechnology Co., Ltd., China, with product number G1076.

[0045] 2.2 Instruments

[0046] Micro-CT scanner (model: GE explore Locus SP), instrument number: PT004938, was provided by the Institute of Bone Research, Soochow University.

[0047] Microtome, model: RM-2016, instrument number: 14045741506, manufactured in Germany, provided by Tang Zhongying Medical Research Institute of Soochow University.

[0048] An upright fluorescence microscope (Nikon Eclipse C1, instrument number 2501758) was manufactured in Japan and provided by the Tang Zhongying Medical Research Institute of Soochow University.

[0049] 2. Experimental Methods

[0050] 1. Modeling and drug administration

[0051] The experimental groups were as follows: control group (Sham), right hind limb irradiation group (Irradiated) and treatment group (Irradiated + Fer-1), with 3 mice in each group.

[0052] Control group: 6-8 week old normal C57 mice were used as controls.

[0053] Right hind limb irradiation group: mice aged 6-8 weeks were irradiated on their right hind limbs and used for the radiation osteoporosis model. The irradiation dose was 24 Gy, the dose rate was 2 Gy / min, and the number of irradiations was 1.

[0054] Treatment group: 6-8 week-old mice were irradiated on the right hind limb and given intraperitoneal injection of ferrostatin-1 at a dose of 2.5 (μmol / kg) three times a week for a total of 4 weeks.

[0055] 2. The mouse samples of each group were collected

[0056] Preparation of mouse femoral specimens: Mice were sacrificed by cervical dislocation, and the skin and muscles of the proximal hind limbs of the mice were cut open to fully expose the femur and hip joint. The femur was carefully separated and removed intact, and the muscles and other soft tissues were removed. The right femur was fixed with 4% PFA and preserved for micro-CT, immunofluorescence staining, toluidine blue staining, and hematoxylin-eosin (H&E) staining.

[0057] 3. Based on the results of animal experiments on the treatment of radiation-induced osteoporosis with ferrostatin-1 and the clinical use of ferrostatin-1, the present invention can use ferrostatin-1 for subcutaneous injection at a dose of 1-5 μmol / kg / day for the treatment of radiation-induced osteoporosis.

[0058] The levels of osteocalcin and alkaline phosphatase in the femur of mice irradiated on the right hind limb were reduced, and the bone formation function was weakened; while ferrostatin-1 could effectively increase the levels of OCN and ALP in the right hind limb irradiated group (Irradiated) mice and improve the bone formation function.

[0059] We used immunofluorescence staining to detect the levels of osteocalcin and alkaline phosphatase in the femur of mice in each experimental group, and toluidine blue staining to detect the osteogenic function of the femur of mice in each experimental group. Figure 1 As shown in the data, the levels of osteocalcin and alkaline phosphatase in the femur and the bone formation function of the right hind limb irradiated mice (Irradiated) were significantly lower than those of the control group (P<0.05). In addition, the levels of osteocalcin, alkaline phosphatase and bone formation function of the treated mice (Irradiated + Fer-1) were higher than those of the right hind limb irradiated group (Irradiated) (P<0.05). This result indicates that irradiation of the right hind limb of mice can reduce the levels of osteocalcin, alkaline phosphatase and bone formation function, and ferrostatin-1 can effectively increase the low levels of osteocalcin, alkaline phosphatase and bone formation function caused by the right hind limb irradiated group (Irradiated).

[0060] Example 2

[0061] After irradiation of the right hind limb, the bone mass of mice decreased. Ferrostatin-1 can partially restore the bone loss caused by irradiation in mice.

[0062] like Figure 2 、 4 As shown, micro-CT scans of the femurs of the three experimental groups of mice were performed, followed by three-dimensional reconstruction and quantitative analysis. Hematoxylin and eosin staining of the femurs accurately describes bone density and bone microstructure, thereby determining the degree of osteoporosis. Three-dimensional image reconstruction and hematoxylin and eosin staining revealed a significant decrease in the number of trabeculae in the right hind limb of the irradiated group compared to the control group. However, after treatment with ferrostatin-1, the number of trabeculae in the treated group (irradiated + Fer-1) was significantly increased compared to the right hind limb irradiated group.

[0063] Example 3

[0064] To further verify the results of the three-dimensional images, we quantitatively analyzed the bone density, bone volume fraction, trabecular number, trabecular spacing, and trabecular thickness of each experimental mouse. Figure 3 As shown, compared with the control group, the right hindlimb bone density, bone volume fraction, and trabecular number in the irradiated group (Irradiated) were significantly decreased (P < 0.05). Compared with the right hindlimb irradiated group (Irradiated + Fer-1), the femoral bone density, bone volume fraction, and trabecular number in the treated group (Irradiated + Fer-1) were significantly restored (P < 0.05). These results indicate that irradiation can induce osteoporosis in mice, and that ferrostatin-1 treatment significantly increases cancellous bone mass. Therefore, we conclude that ferrostatin-1 is an effective treatment for radiation-induced osteoporosis.

[0065] The specific embodiments described above are only preferred implementations of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements or substitutions can be made without departing from the principles of the present invention. These improvements or substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. Application of ferrostatin-1 in the preparation of a drug for treating radiation-induced osteoporosis, characterized in that: The chemical structural formula of the ferrostatin-1 is as follows: 。 2. The use according to claim 1, characterized in that The dosage of ferrostatin-1 in the medicine is 1-5 μmol / kg / day.

3. The use according to claim 1, characterized in that The medicine is an injection.

4. The use according to claim 1, characterized in that The medicine comprises ferrostatin-1 with a purity of more than 95%.

5. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that The pharmaceutically acceptable excipients include one or more of a buffer, a surfactant, an antioxidant, a preservative, a filler and a stabilizer.

7. The use according to claim 1, characterized in that The drug is used for alleviating bone loss caused by ionizing radiation.

8. The use according to claim 1, characterized in that The medicine is used for increasing the levels of osteocalcin and alkaline phosphatase after ionizing radiation and improving the bone formation function.

9. The use according to claim 1, characterized in that The medicine is used for improving bone density, bone volume fraction and trabecular number after ionizing radiation.

10. The use according to claim 1, characterized in that The medicament is used for improving the quality of cancellous bone after ionizing radiation.

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