Use of plitidepsin in the preparation of a medicament for the treatment of osteoporosis due to estrogen deficiency

Abstract

This invention discloses the application of Plitidepsin in the preparation of drugs for treating osteoporosis caused by estrogen deficiency, belonging to the field of biomedicine. This cyclic oligopeptide inhibits osteoclast differentiation, maturation, and bone resorption activity by reducing intracellular reactive oxygen species levels in osteoclasts, activating the Nrf2 / HO-1 antioxidant pathway, and inhibiting the NF-κB and MAPK pathways. Experiments have shown that Plitidepsin at doses of 10–40 μg / kg, administered subcutaneously, can downregulate osteoclast-specific gene expression and reduce bone loss.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Plitidepsin in the preparation of drugs for treating osteoporosis caused by estrogen deficiency. Background Technology

[0002] Osteoporosis is a chronic metabolic skeletal disease characterized by decreased bone mass and destruction of bone microstructure. Postmenopausal osteoporosis is the most common type. Its pathogenesis is due to the decline in estrogen levels, which leads to excessive activation of osteoclasts. The rate of bone resorption far exceeds the rate of bone formation by osteoblasts, which in turn leads to increased bone fragility and an increased risk of fracture.

[0003] Currently, commonly used anti-osteoporosis drugs in clinical practice include bisphosphonates, parathyroid hormone, calcitonin, and RANKL receptor activator ligand (RANKL) inhibitors, but they have significant limitations: bisphosphonates are prone to causing serious side effects such as osteonecrosis of the jaw and atypical fractures; RANKL inhibitors are expensive and have limited applicability; and long-term use of some drugs can lead to decreased patient tolerance. Therefore, developing novel anti-osteoporosis drugs with precise targeting, high safety, and significant efficacy is an urgent clinical challenge.

[0004] Plitidepsin (abbreviated Pli) is a cyclic oligopeptide isolated from tunicates. Existing studies have confirmed its antitumor, antiviral, and antioxidant activities, and it has shown good efficacy in the treatment of diseases such as multiple myeloma and SARS-CoV-2 infection, with good clinical tolerability and no significant hematologic or cardiovascular toxicity. However, to date, no studies have reported the application and mechanism of action of plitidepsin in the treatment of osteoporosis. Summary of the Invention

[0005] The purpose of this invention is to provide new uses for Plitidepsin, clarify its application value in the treatment of osteoporosis caused by estrogen deficiency, and provide applications and pharmaceutical compositions based on Plitidepsin to overcome the shortcomings of existing anti-osteoporosis drugs.

[0006] The technical solution of the present invention is as follows:

[0007] Applications of Plitidepsin:

[0008] The application of Plitidepsin in the preparation of drugs for treating osteoporosis caused by estrogen deficiency: Plitidepsin reduces osteoclast differentiation, maturation and bone resorption activity by lowering the level of reactive oxygen species (ROS) in osteoclasts, activating the Nrf2 / HO-1 antioxidant signaling pathway, and inhibiting the NF-κB (antibody) and MAPK (antibody) signaling pathways, thereby reducing bone loss and improving the integrity of bone microstructure.

[0009] Furthermore, the osteoporosis caused by estrogen deficiency is postmenopausal osteoporosis.

[0010] Includes the following steps:

[0011] Administer an effective dose of Plitidepsin to mammalian subjects.

[0012] (1) The dosage of Plitidepsin is 10~40 μg / kg / time;

[0013] (2) The method of administration of Plitidepsin is subcutaneous injection.

[0014] Plitidepsin inhibits osteoclast-mediated bone resorption by downregulating the transcription and protein expression of osteoclast-specific genes (NFATc1, Ctsk, c-Fos, Atp6v0d2), thereby suppressing osteoclast multinucleation, F-actin ring assembly, and acid secretion.

[0015] Beneficial effects of the present invention

[0016] (1) Significant novelty: The effect of Plitidepsin on osteoporosis caused by estrogen deficiency was discovered for the first time, and its mechanism of action through multi-pathway synergistic regulation of osteoclast function mediated by ROS was clarified, breaking through the limitations of this compound in the fields of anti-tumor and antiviral applications.

[0017] (2) High safety: Plitidepsin is well tolerated in clinical applications, with no significant blood or cardiovascular toxicity. Compared with traditional anti-osteoporosis drugs, it has fewer side effects and is suitable for a wider range of people.

[0018] (3) Definite therapeutic effect: In vitro and in vivo experiments have confirmed that Plitidepsin can effectively inhibit osteoclast differentiation and bone resorption activity, significantly improve bone trabecular density and bone structure integrity in ovariectomized mice, and reduce bone loss.

[0019] (4) Great industrialization potential: The drug composition has a variety of dosage forms, which can meet different clinical drug administration needs and provide solid technical support for subsequent new drug research and development and market transformation. Attached Figure Description

[0020] Figure 1 A schematic diagram illustrating the mechanism by which Plitidepsin improves osteoporosis caused by estrogen deficiency;

[0021] Figure 2 Analysis of the effect of Plitidepsin on osteoclast differentiation; where A is the molecular structure of Plitidepsin, B and C are the cell proliferation after 48 h and 96 h of Plitidepsin treatment by CCK-8 analysis, respectively, D is the TRAP staining of osteoclasts after exposure to different doses of Plitidepsin, E is the statistical analysis of osteoclasts, F is the TRAP staining image of osteoclasts after Plitidepsin treatment at different time intervals, and G is the quantitative result of multinucleated osteoclast count. *p < 0.05, **p < 0.01, ***p < 0.001;

[0022] Figure 3 Analysis of the inhibitory activity of Plitidepsin on osteoclast-mediated bone degradation; A shows representative TRAP-stained micrographs of osteoclasts and resorption pits on bone slices after treatment with different concentrations of Plitidepsin; B shows the measurement of bone resorption degree; C shows the statistical comparison of osteoclast counts per well; D shows the fluorescence visualization of the F-actin ring structure after Plitidepsin treatment; E shows the fluorescence image after acid staining; F shows the bar chart of the average osteoclast area of ​​total F-actin; G shows the analysis of the ratio of red to green fluorescence intensity after acid staining. *p < 0.05, **p < 0.01, ***p < 0.001;

[0023] Figure 4 This study analyzed how Plitidepsin blocks osteoclast differentiation and function by inhibiting NFATC1 nuclear translocation and downstream gene expression. A represents representative immunofluorescence images; B represents statistical evaluation of NFATC1-positive cell nuclei; CF represents the relative mRNA abundance of osteoclast-related markers (NFATc1, Ctsk, c-Fos, and Atp6v0d2) normalized to β-actin; G represents Western blot analysis showing NFATC1, Ctsk, and c-Fos protein levels; HJ represents density-based measurements of NFATC1, Ctsk, and c-Fos protein levels with β-actin as the loading control. *p < 0.05, **p < 0.01, ***p < 0.001;

[0024] Figure 5This study analyzed how Plitidepsin inhibits ROS production in osteoclasts by activating the Nrf2 / HO-1 / Cat antioxidant pathway. A shows fluorescence visualization of ROS levels in RANKL-stimulated osteoclasts treated with different concentrations of Plitidepsin (0, 50, and 200 pM) for 48 hours, as detected by DCFH-DA. B shows the quantitative assessment of DCF (strong green fluorescence)-derived fluorescence intensity. C shows the immunoblotting analysis of Keap1, Nrf2, HO-1, and Cat expression after Plitidepsin exposure. DG shows the density quantification of Cat, Keap1, HO-1, and Nrf2 relative to β-actin. *p < 0.05, **p < 0.01, ***p < 0.001.

[0025] Figure 6 Analysis of the effect of plitidepsin on the inhibition of NF-κB and MAPK signaling activation; A shows the effect of plitidepsin on p65 phosphorylation and iκB-α degradation as shown by Western blot analysis; B shows the quantification of the p-P65 / total p65 ratio; C shows the relative expression of iκB-α degradation to β-actin; D shows the changes in p38, JNK and ERK phosphorylation in the MAPK pathway induced by Western blot analysis. Figures E and G show the quantification of the density of phosphorylated p38, JNK and ERK proteins relative to total protein, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001;

[0026] Figure 7 Molecular docking of Plitidepsin with osteoclast-associated target proteins showed the first four binding affinities;

[0027] Figure 8 Analysis of the effect of Plitidepsin treatment on reducing bone loss in OVX mice; where A is a representative micro-CT image of the proximal tibia, BE is the quantitative analysis of trabecular microstructure, B is the bone volume fraction (BV / TV), C is the trabecular thickness (Tb.Th), D is the number of trabeculae (Tb.N) and E is the trabecular separation (Tb.Sp), * p < 0.05, ** p < 0.01, *** p < 0.001;

[0028] Figure 9Analysis of the effects of Plitidepsin on trabecular microstructure and osteoclast activity in OVX mice; A shows histological and immunohistochemical visualization of H&E, TRAP, and HO-1 stained tibial sections between experimental groups; B shows quantitative analysis of trabecular bone area by H&E staining; C shows quantitative analysis of TRAP-reactive osteoclast surface area; D shows quantitative analysis of HO-1 positive areas. *p < 0.05, **p < 0.01, ***p < 0.001. Detailed Implementation

[0029] To make the above-mentioned objectives, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not deviate from the concept of the invention, they should all fall within the protection scope of the present invention.

[0030] Example

[0031] S1. Experimental Materials:

[0032] S11. Reagents: Plitidepsin (purity ≥98%, MedChemExpress), α-MEM medium, RANKL, M-CSF, CCK-8 kit, TRAP staining kit, ROS detection kit, NF-κB, MAPK, Nrf2 / HO-1 pathway-related antibodies.

[0033] S12. Experimental animals: 6-8 week old female C57BL / 6J mice;

[0034] S13. Instruments: Microplate reader, fluorescence microscope, confocal microscope, micro-CT (μCT), Western blot imaging system, real-time quantitative PCR instrument.

[0035] S2. Experimental Methods:

[0036] Extraction and culture of bone marrow-derived macrophages (BMMs): Female C57BL / 6J mice aged 6-8 weeks were euthanized with cervical dislocation. The femur and tibia were separated, and the bone marrow cavity was rinsed with sterile PBS. The cell suspension was collected and filtered through a 100 μm cell filter. The filtered cell suspension was centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in α-MEM medium containing 30 ng / mL M-CSF. The pellet was seeded in culture dishes and cultured at 37℃ in a 5% CO2 incubator. After 48 h, the medium was changed, and non-adherent cells were removed. Culture was continued until cell confluence reached 80%. Osteoclast differentiation was induced by adding 50 ng / mL RANKL + 30 ng / mL M-CSF. Simultaneously, 0, 50, 100, and 200 pM Plitidepsin treatment groups were established. The medium was changed every 48 h, and the cells were cultured continuously for 7 days.

[0037] S3. Cell viability assay:

[0038] BMMs are divided into 7×10 3 Cells were seeded at a density of 1 cell per well in 96-well plates. When the cell confluence reached 80%, 0–200 pm of Plitidepsin was added for treatment, with DMSO as the solvent control. After 48 h and 96 h of treatment, 10 μL of CCK-8 reagent was added to each well. After incubation for 2 h, the absorbance at 450 nm was measured using a microplate reader to assess cell viability.

[0039] S4. Osteoclast differentiation detection:

[0040] Cells induced and cultured for 7 days were fixed with 4% paraformaldehyde for 30 min and washed 3 times with PBS. They were stained according to the TRAP staining kit instructions. TRAP-positive multinucleated cells with ≥3 nuclei were observed and counted under a microscope. The inhibition rate of osteoclast differentiation by different concentrations of Plitidepsin was calculated.

[0041] Simultaneously, a time gradient experiment was set up: 200 pm of Plitidepsin was added on days 1-3, 3-5, 5-7, and 1-7 of osteoclast differentiation to clarify the key periods during which Plitidepsin exerts its effect.

[0042] S5. Bone resorption activity test:

[0043] Bovine bone slices were placed at the bottom of a 24-well plate, seeded with BMMs, and 50 ng / mL RANKL + 30 ng / mL M-CSF were added to induce differentiation. Simultaneously, 0, 50, and 200 pM Plitidepsin treatment groups were set up. After 7 days of culture, the bone slices were removed, washed with PBS, and the cells on the surface of the bone slices were removed by sonication. After staining, the morphology of bone resorption pits was observed under a microscope, and the area and number of bone resorption pits were analyzed using ImageJ software.

[0044] S6. Cytoskeleton analysis:

[0045] BMMs were seeded in 6-well plates, induced to differentiate, and treated with Plitidepsin. Cells were then fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with 3% BSA. Cells were incubated with the phalloidin fluorescent probe, stained with DAPI, and the morphology and integrity of the F-actin ring were observed under a confocal microscope. The diameter and fluorescence intensity of the ring were measured using ImageJ software.

[0046] S7. Intracellular ROS level detection:

[0047] BMMs were seeded in confocal culture dishes, induced to differentiate, and treated with Plitidepsin for 48 h. Then, DCFH-DA probe (final concentration 10 μM) was added and incubated at 37°C in the dark for 30 min. After washing with PBS, the intracellular fluorescence intensity was observed using a fluorescence microscope, and the ROS level was quantitatively analyzed using ImageJ software.

[0048] S8. Gene expression detection (qRT-PCR):

[0049] Cells from different treatment groups were collected, total RNA was extracted, and RNA purity (A260 / A280 = 1.8~2.0) was detected. RNA was reverse transcribed into cDNA, and the mRNA expression levels of osteoclast-specific genes (NFATc1, Ctsk, c-Fos, Atp6v0d2) were detected using real-time quantitative PCR with β-actin as an internal reference gene.

[0050] S9. Protein expression detection:

[0051] Cells from different treatment groups were collected, and cells were lysed with RIPA buffer containing pMSF and protease / phosphatase inhibitors. Total protein was extracted and quantified. SDS-PAGE electrophoresis was performed, and proteins were transferred to PVDF membranes and blocked with 5% skim milk powder for 1 h. Primary antibodies (Nrf2, HO-1, Keap1, p-P65, P65, p-JNK, JNK, p-ERK, ERK, p-P38, P38, NFATc1, Ctsk, c-Fos, β-actin) were added and incubated overnight at 4°C. After washing with TBST, fluorescently labeled secondary antibodies were added and incubated for 1 h. Protein bands were detected using an imaging system, and the gray values ​​of the bands were analyzed using ImageJ software. The relative expression level of protein was calculated with β-actin as an internal reference.

[0052] S10 and NFATC1 core localization detection:

[0053] BMMs were seeded in confocal culture dishes, induced to differentiate, treated with Plitidepsin, fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with 3% BSA. The cells were then incubated overnight at 4°C with NFATC1 primary antibody, followed by incubation with Alexa Fluor 488-labeled secondary antibody for 1 h, and stained with DAPI. The localization of NFATC1 in the cells was observed under a confocal microscope, and the proportion of NFATC1-positive cells in the nucleus was counted.

[0054] S11, Molecular docking experiment:

[0055] The three-dimensional structure of Plitidepsin was obtained from the PubChem database, and the three-dimensional structures of osteoclast-related target proteins (Nrf2, Keap1, NFATc1, Ctsk) were obtained from the Protein Data Bank (PDB) database. Molecular docking simulations were performed using AutoDockTools-1.5.7 software, docking parameters were set, and the binding energies of Plitidepsin with each target protein were calculated. The binding modes of Plitidepsin with target proteins were visualized using PyMOL software, and key intermolecular interactions were analyzed.

[0056] S12. Construction of an ovariectomized (OVX) mouse osteoporosis model and evaluation of drug efficacy:

[0057] Ten-week-old female C57BL / 6J mice were acclimatized for one week and then randomly divided into five groups (n=6): sham operation group (Sham), model group (OVX+PBS), positive control group (OVX+estrogen), low-dose Plitidepsin group (OVX+10 μg / kg), and high-dose Plitidepsin group (OVX+40 μg / kg).

[0058] Except for the sham surgery group, mice in the other groups underwent bilateral ovariectomy to establish an osteoporosis model; the corresponding drugs were injected subcutaneously every other day after the operation and continued to be administered for 8 weeks; after the last administration, the mice were weighed, sacrificed and the tibia was separated.

[0059] (1) Micro-CT (μCT) detection: Scan the proximal tibia to reconstruct the three-dimensional structure of bone tissue and analyze bone microstructure parameters: trabecular volume fraction (BV / TV), number of trabecular bones (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp);

[0060] (2) Histological staining: The tibia was decalcified, embedded in paraffin, and sectioned. H&E staining was performed to observe the bone tissue morphology, TRAP staining was used to count the number of osteoclasts, and HO-1 immunohistochemical staining was used to assess the activation of antioxidant pathways. Image J software was used to perform quantitative analysis of the staining results.

[0061] Experimental results

[0062] Figure 1 A schematic diagram illustrating the mechanism by which Plitidepsin improves osteoporosis caused by estrogen deficiency.

[0063] Figure 2 A represents the molecular structure of Plitidepsin. Assessment of BMM activity using a CCK-8 assay showed that Plitidepsin concentrations up to 200 pMol / L did not induce significant cytotoxicity. Figure 2 B, 2C). In a concentration gradient experimental design, TRAP staining and quantification of multinucleated cells showed that the inhibitory effect of plitidepsin on OC differentiation was dose-dependent in the range of 0-200 pMol / L (p < 0.05), with the maximum inhibitory effect at 200 pMol / L. Figure 2 D, 2E). Furthermore, time analysis further indicated that plitidepsin exerted the most effective anti-osteoclast effect during the intermediate stage of differentiation (3-5 days). Figure 2 F, 2G).

[0064] Analysis of the formation of absorption pits in bovine bone fragments showed that the absorption area decreased in a concentration-dependent manner after Plitidepsin treatment (p < 0.05), indicating an effective reduction in osteoclast-mediated bone degradation. Figure 3 A-3C). Phallodin-based fluorescence staining showed that Plitidepsin significantly disrupted the assembly of the F-actin loop in mature OCs, indicating that cytoskeleton damage is a mechanism of functional inhibition. Figure 3D, 3E). Simultaneously, extracellular acidification assays showed that Plitidepsin dose-dependently inhibited acid secretion, further confirming its inhibitory effect on bone resorption. Figure 3 F, 3G).

[0065] Immunofluorescence analysis showed that RANKL stimulation promoted NFATC1 nuclear translocation, while Plitidepsin significantly inhibited NFATC1 nuclear translocation. Figure 4 A, 4B). Gene expression profiling by qRT-PCR and Western blot confirmed that Plitidepsin significantly downregulated the expression of osteoclast-specific markers, including NFATc1, Ctsk, c-Fos, and Atp6v0d2. Figure 4 C-4F). In addition... Figure 4 G represents the levels of NFATc1, Ctsk, and c-Fos proteins as shown by Western blot analysis. Figure 4 H-4J shows the density-based measurements of NFATc1, Ctsk, and c-Fos protein levels using β-actin as the load control. Plitidepsin effectively inhibited the protein expression of NFATc1, Ctsk, and c-Fos, with the most significant reduction observed on days 3 and 5 of differentiation.

[0066] Fluorescence assessment using the DCFH-DA probe showed that Plitidepsin treatment significantly reduced intracellular ROS levels in a concentration-dependent manner. Figure 5 A, 5B). Western blot analysis further showed that Plitidepsin downregulated Keap1 expression, promoted Nrf2 accumulation, and enhanced the expression of antioxidant mediators HO-1 and Cat. Figure 5 C-5G).

[0067] RANKL stimulation accelerated iκB-α degradation and enhanced P65 phosphorylation, while Plitidepsin treatment significantly delayed iκB-α degradation and reduced P65 phosphorylation levels, indicating that NF-κB signaling was effectively blocked. Figure 6 A-6C). Furthermore, Plitidepsin significantly inhibited RANKL-induced phosphorylation of JNK, ERK, and p38 (A-6C). Figure 6 D-6G).

[0068] Figure 7 The top four Plitidepsin-protein complexes ranked by affinity are shown, detailing the key intermolecular contacts.

[0069] Bilateral ovariectomy recreated estrogen deficiency-related osteoporotic changes in mice, providing a reliable in vivo system for studying postmenopausal bone degeneration. Microcomputed tomography (MCT) assessment showed that OVX mice exhibited cortical thinning, reduced trabecular number, structural fractures, and overall bone loss. In contrast, the Plitidepsin treatment group showed increased trabecular density, improved structural continuity, and enhanced bone mineral parameters, indicating its potential for treating osteoporosis. Figure 8 A-8E).

[0070] Histological evaluation by H&E staining further indicated that OVX caused trabecular disintegration, while administration of Plitidepsin restored the integrity and alignment of the trabeculae, particularly at higher doses. Figure 9 A, 9B). TRAP staining showed a significant increase in OCs activity in OVX mice, which was significantly reversed by Plitidepsin treatment. Figure 9 Immunohistochemical analysis confirmed that Plitidepsin significantly upregulated HO-1 expression, and extensive immunoreactivity was observed along the trabecular surface and OCs region in the high-dose group. Figure 9 A, 9D).

Claims

1. Use of plitidepsin for the preparation of a medicament for the treatment of osteoporosis due to estrogen deficiency, characterized in that, The osteoporosis caused by estrogen deficiency mentioned above refers to postmenopausal osteoporosis.

2. The application according to claim 1, characterized in that, Plitidepsin inhibits osteoclast differentiation, maturation, and bone resorption activity by reducing intracellular ROS levels, activating the Nrf2 / HO-1 signaling pathway, and inhibiting the NF-κB and MAPK signaling pathways.

3. The application according to claim 1, characterized in that, The method of administration for Plitidepsin is subcutaneous injection.

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