Use of uch37 inhibitors for the preparation of a medicament for the treatment of osteoporosis

Abstract

The present application relates to the technical field of gene therapy and drug development, and provides application of UCH37 inhibitor in preparation of medicine for treating osteoporosis. The expression level of UCH37 in blood of patients with osteoporosis is significantly higher than that of healthy people, and is positively correlated with markers such as NFATC1 and MMP9 of osteoclast activity. The UCH37 inhibitor is applied to preparation of medicine for treating osteoporosis, directly targets UCH37, and the curative effect is independent of expression change of ADRM1. Whether ADRM1 of patients with osteoporosis is in low or high expression state, the osteoclast activation pathway can be stably blocked, the consistency of treatment effect of different individuals is ensured, the scope of suitable population of the medicine is expanded, the UCH37 inhibitor has the characteristics of clear mechanism of action and wide application range, and provides important theoretical basis and conversion prospect for development of new and efficient medicine for treating osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of gene therapy and drug development technology, and in particular relates to the application of UCH37 inhibitors in the preparation of drugs for treating osteoporosis. Background Technology

[0002] Osteoporosis (OP) is a prevalent chronic skeletal disease worldwide, seriously threatening the health of middle-aged and elderly people. Osteoporosis is often accompanied by serious complications such as nonunion and bone defects, leading to persistent pain and motor dysfunction, significantly reducing patients' quality of life. Currently, the pathogenesis of osteoporosis is not fully understood, and there is a lack of effective prevention and treatment methods in clinical practice. Therefore, in-depth research into its pathogenesis and the development of novel treatment strategies are of great significance. Existing osteoporosis treatments (such as bisphosphonates and calcitonin) mostly focus on broad-spectrum regulation of "inhibiting bone resorption" or "promoting bone formation," lacking precise targeting of core molecular targets that overactivate osteoclasts, resulting in limited efficacy and a high risk of off-target effects. Furthermore, long-term use of commonly used osteoporosis treatments can easily lead to serious adverse reactions (such as osteonecrosis of the jaw and atypical fractures caused by bisphosphonates, and cardiovascular risks caused by hormone replacement therapy), and some patients develop drug resistance, leading to treatment failure.

[0003] In the pathological process of osteoporosis, osteoclasts, as the only multinucleated giant cells with bone resorption function, are the core factor leading to hyperactive bone resorption due to their overactivation. An abnormal increase in the number of osteoclasts or an enhanced differentiation and bone resorption capacity can both cause an imbalance in bone metabolism. Therefore, identifying new therapeutic targets by targeting key regulatory nodes in osteoclast differentiation and bone resorption processes is of great value for the development of drugs for osteoporosis. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides the application of UCH37 inhibitors in the preparation of drugs for treating osteoporosis, with the aim of solving the problems mentioned in the background art.

[0005] UCH37 belongs to the ubiquitin C-terminal hydrolase family. It is a deubiquitinating enzyme containing a conserved C12 domain and regulates cellular protein turnover and proteasome function. Currently, the research and application of UCH37 are mainly focused on the field of oncology. It is highly expressed in various malignant tumor tissues and cancer cell lines, such as liver cancer. Its abnormal expression is closely related to the occurrence, development, metastasis and recurrence of tumors.

[0006] This invention provides the application of UCH37 inhibitors in the preparation of drugs for treating osteoporosis.

[0007] Furthermore, the UCH37 inhibitor is a siRNA that targets UCH37, and the sequence of the siRNA is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] Furthermore, the drug treats osteoporosis by inhibiting the expression of the UCH37 gene and reducing the synthesis of the protein encoded by the UCH37 gene.

[0009] Furthermore, the drug treats osteoporosis by blocking the osteoclast activation pathway and inhibiting osteoclast activity.

[0010] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0011] Furthermore, the carrier comprises liposomes or nanoparticles.

[0012] Furthermore, the dosage form of the drug is any clinically or pharmaceutically acceptable dosage form.

[0013] Furthermore, the dosage form includes tablets, capsules, granules, injections, patches, or gels.

[0014] Furthermore, the osteoporosis mentioned is oophorectomy-induced osteoporosis.

[0015] The present invention has the following technical effects: (1) The expression level of UCH37 in the blood of patients with osteoporosis is significantly higher than that in healthy individuals, and it is positively correlated with osteoclast activity markers such as NFATC1 and MMP9. Silencing UCH37 with siRNA targeting UCH37 can effectively inhibit osteoclast differentiation and bone resorption, and this inhibitory effect remains consistent at different ADRM1 expression levels. Inhibiting UCH37 activity can significantly improve bone loss in osteoporosis induced by ovariectomy, further verifying the effectiveness and independence of UCH37 as a therapeutic target.

[0016] (2) UCH37 inhibitors are used in the preparation of drugs for treating osteoporosis. They directly target UCH37. The efficacy does not depend on changes in ADRM1 expression. Regardless of whether the ADRM1 expression of osteoporosis patients is low or high, it can stably block the osteoclast activation pathway, ensuring the consistency of treatment effects among different individuals and expanding the range of applicable populations. It has the characteristics of clear mechanism of action and wide applicability, providing important theoretical basis and translational prospects for the development of new and efficient drugs for treating osteoporosis. Attached Figure Description

[0017] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures: Figure 1The results of HE staining of cancellous bone from osteoporosis patients and osteoporosis patients in Example 1 of this invention are shown. Scale bar: 200 μm.

[0018] Figure 2 The results of qPCR analysis and Western blot detection in osteoporosis patients and osteoporosis patients in Example 1 of this invention are as follows: Figure 2 In the table, A represents the statistical results of mRNA expression levels of UCH37, ALK5, NFATC1, and MMP9 genes. ** indicates P < 0.01, and *** indicates P < 0.001. Figure 2 B in the image is a typical image of UCH37, ALK5, NFATC1, and MMP9 proteins, with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal reference protein. Figure 2 In the table, C represents the quantitative analysis results of the expression levels of UCH37, ALK5, NFATC1, and MMP9 proteins. ** indicates P < 0.01, and *** indicates P < 0.001.

[0019] Figure 3 These are the qPCR analysis and Western blot detection results of the UCH37 siRNA1 group and the negative control siRNA group in Example 2 of this invention, wherein: Figure 3 In the table, A represents the statistical results of the mRNA expression levels of CTSK and MMP9 genes under high and low concentrations of ADRM1 stimulation after UCH37 silencing. ** indicates P < 0.01, and *** indicates P < 0.001. Figure 3 Image B in the image shows typical images of CTSK and MMP9 proteins stimulated with high and low concentrations of ADRM1 after silencing UCH37, with GAPDH as an internal control protein. Figure 3 In the figure, C represents the quantitative analysis results of the expression levels of CTSK and MMP9 proteins under high and low concentrations of ADRM1 stimulation after silencing UCH37. ** indicates P < 0.01, and *** indicates P < 0.001.

[0020] Figure 4 The results of qPCR analysis and Western blot detection for different UCH37 siRNA2 concentration groups (0 μM, 0.05 μM, 0.1 μM and 0.5 μM) in Example 3 of this invention are as follows: Figure 4In the table, A represents the statistical results of the mRNA expression levels of CTSK and MMP9 genes under different UCH37 siRNA2 concentrations, with and without ADRM1 overexpression. ns indicates no statistical significance, * indicates P < 0.5, and ** indicates P < 0.01. Figure 4 B in the image shows typical images of CTSK and MMP9 proteins under different UCH37 siRNA2 concentrations of stimulation, with overexpression and no ADRM1 overexpression, using GAPDH as an internal control protein. Figure 4 In the figure, C represents the quantitative analysis results of the expression levels of CTSK and MMP9 proteins under different UCH37 siRNA2 concentrations and under conditions of overexpression and no ADRM1 overexpression. ns indicates no statistical significance, * indicates P < 0.5, ** indicates P < 0.01, and *** indicates P < 0.001.

[0021] Figure 5 This is a microCT scan result and quantitative analysis of mouse femoral tissue from the sham-operated group, blank solvent group, low-dose group, and high-dose group in Example 4 of the present invention, wherein: Figure 5 In the image, A represents the result of a microCT scan. Scale bar: 1 mm. Figure 5 In the table, B represents the results of microCT quantitative analysis. * indicates P < 0.5, ** indicates P < 0.01, and *** indicates P < 0.001. Detailed Implementation

[0022] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0024] This invention provides the application of UCH37 inhibitors in the preparation of drugs for treating osteoporosis.

[0025] In some embodiments, the UCH37 inhibitor is a siRNA that targets UCH37, and the sequence of the siRNA is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0026] In some embodiments, the drug treats osteoporosis by inhibiting the expression of the UCH37 gene and reducing the synthesis of the protein encoded by the UCH37 gene.

[0027] In some embodiments, the drug treats osteoporosis by blocking osteoclast activation pathways and inhibiting osteoclast activity.

[0028] In some embodiments, the medicament may also include a pharmaceutically acceptable carrier.

[0029] In some embodiments, the carrier includes liposomes or nanoparticles.

[0030] In some embodiments, the dosage form of the drug is any clinically or pharmaceutically acceptable dosage form.

[0031] In some embodiments, the dosage form includes tablets, capsules, granules, injections, patches, or gels.

[0032] In some embodiments, the osteoporosis is oophorectomy-induced osteoporosis.

[0033] Example 1: 1. Patients who underwent amputation or hip replacement surgery due to trauma or deformity at the First Affiliated Hospital of Nanchang University between September 2022 and December 2024 were included in this study. Bone mineral density values ​​were systematically measured in all enrolled patients using dual-energy X-ray absorptiometry (DXA), and the pathological degree of osteoporosis was simultaneously assessed. Based on the "Chinese Guidelines for the Diagnosis of Osteoporosis by Quantitative CT (QCT) (2018)," 6 non-osteoporosis patients and 6 osteoporosis patients were ultimately included, and cancellous bone specimens (from the same location) and blood specimens were collected from them.

[0034] (1) Histological evaluation of cancellous bone samples from both groups of patients was performed using HE staining (hematoxylin-eosin staining). HE staining results are as follows: Figure 1 As shown, the results revealed that compared with non-osteoporosis patients, osteoporosis patients had a significantly reduced number of trabeculae in their cancellous bone, with disordered arrangement and widened gaps; the trabeculae were incomplete in shape, with some showing fractures, defects, or bifurcation, and the edges exhibiting serrated or even fragmented changes; the medullary cavity was abnormally dilated, with a large proliferation of adipocytes and a significantly reduced tissue density. These characteristic changes suggest that osteoporosis patients have a significant bone metabolic imbalance, and the abnormal activation of osteoclasts may play an important role in disease progression.

[0035] (2) Peripheral blood mononuclear cells (PBMCs) were collected from blood samples of both groups of patients, and RNA and proteins were extracted for detection and analysis. The results of qPCR (real-time quantitative polymerase chain reaction) analysis and Western blot detection are as follows: Figure 2As shown, the results indicated that the mRNA expression level of the UCH37 gene in osteoporosis patients was significantly upregulated compared to that in non-osteoporosis patients; simultaneously, the mRNA expression levels of osteoclast-specific related genes (NFATC1, MMP9) were also significantly increased. Figure 2 (A) Compared to non-osteoporosis patients, the protein expression level of UCH37 was significantly increased in osteoporosis patients, while the protein levels of NFATC1 and MMP9 also showed a synergistic increase. Figure 2 (B and C). Furthermore, the mRNA and protein expression levels of ALK5 showed a high degree of consistency with UCH37, suggesting a possible synergistic regulatory mechanism between them. Figure 2 (A, B, and C in the original text).

[0036] Example 2: A UCH37-specific siRNA silencing system was constructed for RAW264.7 cells. The siRNAs targeting UCH37 included UCH37 siRNA1 and UCH37 siRNA2, with the following nucleotide sequences: UCH37 siRNA1:GGAUUCAAAAGUAUAGUGA (SEQ ID NO.1); UCH37 siRNA2: GCAGGUAAUUAAUAAUGCU (SEQ ID NO. 2).

[0037] UCH37 siRNA1 group and negative control siRNA group were set up; an in vitro model for inducing RAW264.7 cells to differentiate into osteoclasts was constructed, that is, osteoclast differentiation was induced by 50 ng / ml RANKL, and stimulated with high concentration (0.5 μM) and low concentration (0 μM) ADRM1, respectively; the expression levels of osteoclast-specific related proteins and mRNAs (CTSK and MMP9) in each group under high and low concentration ADRM1 stimulation were detected by qPCR and Western blotting.

[0038] Results of qPCR and Western blot assays are as follows Figure 3 As shown, the results indicated that silencing UCH37 significantly reduced the mRNA and protein levels of osteoclast-specific genes CTSK and MMP9, and the decreasing trend was not related to changes in ADRM1 concentration.

[0039] Example 3: The effect of different concentrations of UCH37 siRNA2 on osteoclast formation was observed by inducing RAW264.7 cells to differentiate into osteoclasts in vitro. Four groups were set up according to the concentration of UCH37 siRNA2: 0 μM, 0.05 μM, 0.1 μM and 0.5 μM, and ADRM1 was overexpressed and not overexpressed, respectively. The transcription and translation levels of osteoclast-specific genes CTSK and MMP9 in each group were detected by qPCR and Western blotting under ADRM1 overexpression and ADRM1 non-overexpression conditions.

[0040] Results of qPCR and Western blot assays are as follows Figure 4 As shown, the results indicate that with increasing concentrations of UCH37 inhibitors, the mRNA and protein expression levels of osteoclast-specific genes CTSK and MMP9 showed a dose-dependent downregulation trend, with the 0.5 μM group exhibiting the lowest levels. Furthermore, UCH37 inhibitors effectively inhibited osteoclast activity even under conditions of high ADRM1 expression, and the inhibitory trend remained unchanged. This further demonstrates that UCH37 inhibitors can directly act on the active site of UCH37 itself, and can stably block the osteoclast activation pathway regardless of whether the patient's ADRM1 expression is low or high.

[0041] Example 4: A sham surgery group was set up, and an OVX (oophorectomy)-induced osteoporosis model was constructed in mice. Three days after the osteoporosis surgery, the mice were divided into three groups: blank solvent group: intraperitoneal injection of phosphate buffer solution; low-dose group: intraperitoneal injection of UCH37 siRNA2 (10 mg / kg); high-dose group: intraperitoneal injection of UCH37 siRNA2 (20 mg / kg). The intraperitoneal injection was given three times a week for a total of eight weeks. Femoral tissue specimens were collected after the injection was completed.

[0042] Bone quality in mice of each group was assessed using microCT. The microCT scan results and analysis of femoral tissue in each group are as follows: Figure 5 As shown, there are significant differences between the different treatment groups. MicroCT scan results showed that the blank solvent group had less bone mass than the sham surgery group, while the UCH37 inhibitor intervention group (low / high dose) showed a clear trend of bone mass improvement compared to the blank solvent group. Figure 5(A) MicroCT quantitative analysis results showed that: in the blank solvent group, the trabecular structure model index (SMI), trabecular number (Tb.N), trabecular thickness (Tb.Th), and cortical bone relative volume (BV / TV) were all lower than in the sham surgery group, while trabecular separation (Tb.Sp) was significantly higher; in the UCH37 inhibitor intervention group (low / high dose), the trabecular structure model index, trabecular number, trabecular thickness, and cortical bone relative volume were all significantly higher than in the blank solvent group, while trabecular separation was significantly reduced, and the high-dose group showed more significant results than the low-dose group. Figure 5 (B in the middle).

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of UCH37 inhibitors in the preparation of drugs for treating osteoporosis, characterized in that: The UCH37 inhibitor is a siRNA that targets UCH37, and the sequence of the siRNA is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that: The drug is used to treat osteoporosis induced by oophorectomy.

3. The application as described in claim 2, characterized in that: The drug treats osteoporosis by inhibiting the expression of the UCH37 gene and reducing the synthesis of the protein encoded by the UCH37 gene.

4. The application as described in claim 3, characterized in that: The drug treats osteoporosis by blocking the osteoclast activation pathway and inhibiting osteoclast activity.

5. The application as described in claim 4, characterized in that: The drug also includes a pharmaceutically acceptable carrier.

6. The application as described in claim 5, characterized in that: The carrier includes liposomes or nanoparticles.

7. The application as described in claim 6, characterized in that: The dosage form of the drug is any clinically or pharmaceutically acceptable dosage form.

8. The application as described in claim 7, characterized in that: The dosage forms include tablets, capsules, granules, injections, patches, or gels.

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