Application of nano-selenium in treatment of drug-related osteonecrosis of the jaw

Abstract

The application provides application of nano selenium in treatment of drug-related jaw bone necrosis. Through a large amount of research, it is found that nano selenium with a particle size of 90 nm-110 nm can significantly promote osteoclast differentiation in an in-vitro induction system and a drug-related jaw bone necrosis model animal in vivo, effectively promote bone regeneration and wound healing of alveolar bone after tooth extraction of the drug-related jaw bone necrosis model animal, significantly improve the jaw bone healing state, effectively treat the drug-related jaw bone necrosis, and has important clinical transformation value and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, especially bone tissue repair, and specifically relates to the application of nano-selenium in the treatment of drug-related osteonecrosis of the jaw. Background Technology

[0002] Medication-related osteonecrosis of the jaw (MRONJ) is a serious oral complication caused by long-term use of anti-bone resorption drugs. It mainly manifests as jawbone exposure, pain, infection, non-healing wounds, and even pathological fractures, severely impacting patients' quality of life. Bisphosphonates (BPs), such as zoledronic acid (ZA), are widely used to treat osteoporosis, multiple myeloma, and bone metastases from malignant tumors, and are the most common cause of MRONJ. Clinical data show that the incidence of MRONJ in patients using intravenous BPs long-term can reach 1.6%-20%, and it often occurs after alveolar surgery such as tooth extraction or implantation. Currently, there is no specific treatment.

[0003] One of the core pathological mechanisms of MRONJ is the excessive suppression of osteoclast function, leading to an imbalance in bone remodeling and impaired clearance of necrotic tissue. Studies have shown that bisphosphonates can inhibit the differentiation of osteoclast precursor cells into mature osteoclasts by blocking the RANKL / RANK / NFATc1 signaling pathway, while simultaneously inducing local oxidative stress and chronic inflammatory responses, further exacerbating bone healing disorders. Therefore, restoring the normal differentiation function of osteoclasts is one of the key strategies for intervening in MRONJ.

[0004] Currently, there is a need in this field to develop more drugs and technological solutions that can effectively treat drug-related osteonecrosis of the jaw. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide the application of nano-selenium in the treatment of drug-induced osteonecrosis of the jaw. Nano-selenium can significantly promote osteoclast differentiation in vitro and in vivo, and promote bone regeneration and wound healing of alveolar bone.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] In a first aspect, the invention provides the use of nano-selenium in the preparation of medicaments for the prevention and treatment of drug-associated osteonecrosis of the jaw.

[0008] In some embodiments, the nano-selenium has a particle size of 90 nm-110 nm.

[0009] In some embodiments, the drug-related osteonecrosis of the jaw is bisphosphonate-related osteonecrosis of the jaw.

[0010] In some embodiments, the drug-related osteonecrosis of the jaw is zoledronic acid-related osteonecrosis of the jaw.

[0011] In some implementations, the application includes upregulating the expression of selenoprotein R.

[0012] In some implementations, the application includes reducing the methionine sulfoxide / methionine ratio.

[0013] In some implementations, the application includes promoting alveolar bone regeneration and wound healing.

[0014] In some embodiments, the dosage form of the drug is an oral topical preparation or an oral preparation.

[0015] In some embodiments, the oral topical drug delivery formulation includes injections, gels, absorbable oral dressings, oral mucosal patches, bone repair filling materials, periodontal dressings, mouthwashes, and oral ointments.

[0016] In some embodiments, the oral dosage form includes granules, tablets, pills, capsules, and solutions.

[0017] Compared with the prior art, the present invention has the following beneficial effects.

[0018] This invention, based on extensive research, has discovered that selenium nanoparticles with a particle size of 90 nm-110 nm can significantly promote osteoclast differentiation in in vitro induction systems and in vivo in drug-induced osteonecrosis of the jaw models. It effectively promotes bone regeneration and wound healing of alveolar bone after tooth extraction in drug-induced osteonecrosis of the jaw models, significantly improves the healing status of the jaw, and effectively treats drug-induced osteonecrosis of the jaw. It has important clinical translational value and broad application prospects. Attached Figure Description

[0019] Figure 1 Characterization of nano-selenium; where A: particle size distribution and appearance; B: PDI index and Zeta potential; C: EDS energy spectrum.

[0020] Figure 2 To enhance the osteoclast differentiation-inducing activity of RANKL by nano-selenium; where A: TRAP staining image; B: bar chart of TRAP-positive cell quantitative analysis; C: TRAP staining image; D: multinucleated osteoclast count.

[0021] Figure 3To illustrate the mechanism by which nano-selenium synergistically regulates osteoclast differentiation with RANKL; where A: TRAP staining image; B: Western blot detection of SelR protein expression level during RAW264.7 differentiation into osteoclasts; CD: Retention time and response intensity curves of MetSO and Met; EG: Concentrations of MetSO and Met and the MetSO / Met ratio.

[0022] Figure 4 To illustrate the antagonistic effect of nano-selenium on drug-related osteonecrosis of the jaw; A: Body weight changes in SD rats from 0 to 10 weeks; B: Wound healing status; C: Hematoxylin-eosin staining of jawbone wound; D: MicroCT cross-sectional scan of the jawbone; E: MicroCT detection indicators of the jawbone. Detailed Implementation

[0023] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0024] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0025] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0026] The following description is based on specific implementation methods.

[0027] Example 1: Preparation of Nano Selenium This embodiment uses the vitamin C reduction method to prepare nano-selenium. The specific method is as follows: 4.75 mL of poloxamer (0.01%) solution was added to a beaker, followed by 50 μL of Na₂SeO₃ (100 mM) solution. The mixture was stirred thoroughly on a magnetic stirrer. 200 μL of vitamin C solution (100 mM) was slowly added dropwise to the beaker, and the mixture was reacted at 4°C for 12 h. After the reaction was complete, the reaction solution was dialyzed against deionized water for 24 h using a 22 mm dialysis bag. The dialysis process was carried out on a magnetic stirrer, and the deionized water was changed three times during the dialysis process to ensure the removal of unreacted Na₂SeO₃ and vitamin C. After dialysis, the product obtained in the dialysis bag was nano-selenium (SeNPs), which was collected and used for subsequent experiments.

[0028] The prepared SeNPs solution has the following appearance: Figure 1 As shown in Figure A, the liquid is a clear orange-red color. The particle size distribution of SeNPs was detected using a particle size scattering analyzer, and the results are as follows. Figure 1 A shows that SeNPs are mainly distributed in the 90 nm-110 nm range. Further analysis of the dispersion index (PDI) and Zeta sites of the SeNPs yielded the following results: Figure 1 As shown in Figure B, the PDI of the nano-selenium is 0.1, and the Zeta site is -20 mV, demonstrating that the prepared SeNPs have a uniform distribution, good stability, and are not prone to aggregation. Results obtained using field emission transmission electron microscopy coupled with EDS spectroscopy are shown below. Figure 1 As shown in C, SeNPs are spheres with a dense distribution of selenium.

[0029] Example 2: Nano-selenium synergistically enhances the osteoclast differentiation induction activity of RANKL. RANKL-induced differentiation of RAW264.7 cells is one of the most classic in vitro cell models for studying osteoclast formation and bone resorption mechanisms. This example studies the effect of SeNPs obtained in Example 1 on RANKL-induced osteoclast differentiation of RAW264.7 cells.

[0030] RAW264.7 cells were divided into 5x10 groups. 3 Cells / wells were seeded at a density of 24-well plates and cultured adherently in 10% fetal bovine serum medium for 24 h. The medium was then changed, and different concentrations of RANKL (purchased from R&D Company, USA, catalog number 462-TR-010) were added: 0 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, and 50 ng / mL. Simultaneously, different concentrations of SeNPs were added: 0 μM, 0.5 μM, 1 μM, and 2 μM. Cells were cultured for 5–7 days, with the medium and treatment reagents changed every 2 days. The medium was discarded, and the cells were washed three times with PBS. They were then fixed and permeabilized sequentially with 4% paraformaldehyde and PBS buffer containing 0.2% (v / v) Triton X-100. TRAP staining solution was then added, and the cells were incubated at 37°C in the dark for 30 min. After washing with PBS, the cells were photographed and images acquired using a cell imaging system.

[0031] The results are as follows Figure 2 A- Figure 2 As shown in Figure B, when the RANKL concentration gradually increased from 10 ng / mL to 50 ng / mL, the number of TRAP-positive cells in all SeNPs-treated groups was significantly increased compared to the RANKL-treated group alone, indicating that SeNPs can significantly promote osteoclast differentiation in the in vitro induction system. Example 3

[0032] Effects of selenium compounds of different valence states on osteoclast differentiation in an in vitro induced system.

[0033] RAW264.7 cells were treated with four different valence states of selenium in combination with RANKL in a medium containing 50 ng / mL: The selenium nanoparticles (SeNPs; Se) prepared in Example 1 were obtained. 0 Sodium selenite (Na2SeO3; Se) 4+ ), methyl selenoic acid (MSA; Se) 6+ ) and Ebselen; Se 2+ The concentration of each reagent was 1 μM, and the other treatment conditions were the same as in Example 2. The culture was continued for 5-7 days, with the culture medium and treatment reagents changed every 2 days, followed by TRAP staining.

[0034] The results are as follows Figure 2 C- Figure 2 As shown in Figure D, the number of TRAP-positive cells in the MSA and Ebselen treatment groups was not significantly different from that in the control group (PBS), indicating that neither group promoted osteoclast differentiation. The number of osteoclasts in the Na2SeO3 group was reduced and the cell density was significantly decreased, suggesting that it may have an inhibitory effect. Only SeNPs at a concentration of 1 μM significantly enhanced RANKL-induced osteoclast formation and significantly improved differentiation efficiency.

[0035] The above results indicate that SeNPs specifically promote the differentiation of RANKL-induced osteoclast precursor cells into mature osteoclasts, and the effect depends on the chemical form of selenium, suggesting that SeNPs may play a unique role in osteoclastogenesis as an important regulatory molecule of the RANKL signaling pathway.

[0036] Example 4: Mechanism of Nano-Selenium Regulating Osteoclast Differentiation Construction of an osteoclast differentiation cell model.

[0037] RAW264.7 cells in logarithmic growth phase were cultured adherently in 10% fetal bovine serum medium for 24 h and then treated as follows: Control group: 50 ng / mL RANKL only; Zolpidem group (ZA group): 50 ng / mL RANKL + 5 μM ZA; ZA + SeNPs group: 50 ng / mL RANKL + 5 μM ZA + 1 μM SeNPs. The medium and treatment reagents were changed every 2 days, and the cells were cultured for 5-7 days.

[0038] TRAP staining was used to assess the regulatory effect of RANKL combined with SeNPs on osteoclast differentiation.

[0039] The results are as follows Figure 3 As shown in Figure A, the Control group developed a large number of TRAP-positive multinucleated osteoclasts after RANKL stimulation, indicating that the differentiation process proceeded normally. The ZA group showed a significant decrease in the number of TRAP-positive cells, suggesting that ZA strongly inhibited the differentiation potential of osteoclast precursor cells. However, in the ZA+SeNPs group, the number of TRAP-positive cells significantly recovered, with no statistically significant difference compared to the Control group. These results indicate that SeNPs can effectively reverse ZA-mediated osteoclast differentiation inhibition.

[0040] SeNPs enhance the inhibitory effect on ZA by upregulating selenoprotein R (SelR).

[0041] To further investigate the mechanism by which SeNPs promote osteoclast differentiation, the expression of SelR in cells was first detected by Western blot. The results are as follows: Figure 3 As shown in Figure B, SelR protein expression was significantly downregulated in the ZA group compared to the Control group; however, after combined intervention with SeNPs, SelR expression levels significantly rebounded. This suggests that SeNPs may alleviate ZA-induced osteoclastogenesis disorders by specifically upregulating SelR expression.

[0042] SeNPs regulate methionine redox homeostasis to mitigate oxidative stress damage.

[0043] SelR is a selenocysteine-dependent methionine sulfoxide reductase that catalyzes the reduction of oxidatively damaged methionine residues (MetO) to functional methionine (Met), thereby repairing the structure and function of key signaling proteins and maintaining cellular redox balance.

[0044] To further verify whether SeNPs regulate oxidative stress through the SelR-MetO / Met axis, total protein was extracted from cells in each group, and free methionine (Met) and methionine sulfoxide (MetSO) were separated and quantified. High-performance liquid chromatography (HPLC) was used for detection. The characteristic elution peaks of MetSO and Met appeared at 8.748 min (…). Figure 3 C) and 35.962 min ( Figure 3 D). Based on the peak area and normalized to the total protein concentration, the relative contents of MetSO and Met in each group were calculated, and the results are as follows. Figure 3 E- Figure 3 As shown in Figure F, the intracellular MetSO level in the ZA group was significantly increased, while the Met content decreased, indicating accumulated oxidative damage. However, after SeNPs intervention, the MetSO content decreased significantly, and the Met level rebounded. Further calculation of the MetSO / Met ratio was performed based on the results. Figure 3G found that ZA treatment significantly increased this ratio, reflecting enhanced intracellular oxidative stress; while SeNPs treatment significantly reduced this ratio, bringing it closer to normal physiological levels.

[0045] Based on the results of this embodiment, SeNPs upregulate SelR expression, promote the reduction of MetSO to Met, restore protein function, and regulate redox homeostasis, thereby antagonizing ZA-induced inhibition of osteoclast differentiation.

[0046] Example 5: Antagonistic effect of nano-selenium on drug-induced osteonecrosis of the jaw. A drug-related osteonecrosis of the jaw (MRONJ) animal model was established using 7-8 week old male Sprague Dawley (SD) rats weighing 180-200 g. To simulate osteoporosis and facilitate the establishment of the MRONJ model, all rats were intraperitoneally injected with dexamethasone (5 mg / kg) once a week for 10 weeks, starting from week 1 of the experiment. Simultaneously, rats in the MRONJ and SeNPs groups received tail vein injections of Zazone (0.125 μg / g) twice a week for 10 weeks; the SeNPs group received additional tail vein injections of SeNPs (0.4 μg / g) twice a week for 10 weeks; the Control group received neither Zazone nor SeNPs.

[0047] Tooth extraction was performed in the fifth week of the animal experiment. Patients were fasted and deprived of water for 12 hours prior to surgery, and surgical instruments were autoclaved. The patient was weighed before anesthesia, and fentanyl (250 mg / kg) was administered intraperitoneally. After the anesthesia took effect, the patient was immobilized in a supine position. The perioral area and oral cavity were disinfected with iodine solution. The gingiva of the left maxillary first molar was dissected in a circular motion, and the tooth was removed using miniature extraction forceps. After cleaning the extraction socket, sterile gelatin sponge was applied to control bleeding. Postoperatively, vital signs, food intake, weight, and behavior were closely monitored to ensure animal welfare.

[0048] During the experiment, all rats were housed in an SPF-grade clean-grade animal room with controlled temperature and humidity and a 12-hour light-dark cycle, with free access to feed and sterilized drinking water. The weight of the SD rats was recorded weekly, and the results are as follows: Figure 4 As shown in Figure A, there were significant differences in the weight change trends of the SD rats in each group during the experiment. During the first 5 weeks, the weight of all three groups steadily increased. After tooth extraction (week 5), the weight of all groups experienced a brief decrease, possibly related to postoperative feeding restrictions. From weeks 6 to 10, the weight of the Control and SeNPs groups continued to increase, while the growth of the MRONJ group stagnated, suggesting that wound healing was impaired, affecting feeding function. Overall, the weight of the MRONJ group was significantly lower than that of the Control and SeNPs groups, indicating that normal growth of rats was inhibited during the establishment of this model, and that SeNPs intervention alleviated this effect to some extent.

[0049] The healing process is assessed using photographs of the extraction wound and the affected tooth. Figure 4 As shown in Figure B, the wounds in the Control group healed well with no abnormal exposure; the MRONJ group showed obvious bone exposure and signs of osteonecrosis; although the wounds in the SeNPs intervention group were not completely closed, no bone exposure or necrosis was observed, indicating that the healing status of the jawbone was significantly improved.

[0050] Maxillae were harvested from SD rats after sacrifice. After decalcification, dehydration, and cryo-embedding, sections were prepared using a cryostat to a thickness of 6 μm. The obtained jawbone tissue sections were stained with hematoxylin and eosin. Histological results are as follows: Figure 4 As shown in Figure C, the MRONJ group exhibited bone depression in the jawbone, indicating impaired bone regeneration. The Control and SeNPs groups showed intact trabecular bone structure, adequate bone filling in the wound area, and elevated wound interface, suggesting accelerated bone regeneration. To further quantitatively assess the degree of bone healing, Micro-CT was used for three-dimensional reconstruction analysis of the maxilla. Figure 4 The results showed that the alveolar bone mass in the MRONJ group was significantly lower than that in the Control group, with sparse and disordered trabecular bone structure. The bone mineral density and trabecular bone structure in the SeNPs group were similar to those in the Control group, indicating that its bone regeneration level was significantly better than that in the MRONJ group. These results suggest that SeNPs can effectively promote alveolar bone regeneration and wound healing after tooth extraction, antagonize the pathological process of MRONJ, and can effectively treat MRONJ.

[0051] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0052] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of nano-selenium in the preparation of drugs for the prevention and treatment of drug-related osteonecrosis of the jaw.

2. The application as described in claim 1, characterized in that, The nano-selenium has a particle size of 90 nm-110 nm.

3. The application as described in claim 1, characterized in that, The drug-related osteonecrosis of the jaw is bisphosphonate-related osteonecrosis of the jaw.

4. The application as described in claim 3, characterized in that, The drug-related osteonecrosis of the jaw is zoledronic acid-related osteonecrosis of the jaw.

5. The application as described in claim 1, characterized in that, The application includes upregulating the expression of selenoprotein R.

6. The application as described in claim 1, characterized in that, The application includes reducing the methionine sulfoxide / methionine ratio.

7. The application as described in claim 1, characterized in that, The applications include promoting alveolar bone regeneration and wound healing.

8. The application as described in any one of claims 1 to 7, characterized in that, The drug is available in oral topical formulations or oral formulations.

9. The application as described in claim 8, characterized in that, The oral topical drug delivery preparations include injections, gels, absorbable oral dressings, oral mucosal patches, bone repair filling materials, periodontal dressings, mouthwashes, and oral ointments.

10. The application as described in claim 8, characterized in that, The oral preparations include granules, tablets, pills, capsules, and solutions.

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