Application of ADRM1 inhibitor in preparation of medicine for treating osteoporosis

Abstract

The invention relates to the technical field of biological medicines, and provides application of an ADRM1 inhibitor in preparation of a medicine for treating osteoporosis. Through the scheme of the invention, the ADRM1 affects bone metabolism, bone tissue morphological structure and osteoporosis progress, and can provide a new drug target for clinical prevention and treatment of osteoporosis. The ADRM1 inhibitor is applied to preparation of the osteoporosis medicine, and bone mass loss can be obviously reduced by down-regulating ADRM1 expression or inhibiting ADRM1 activity and inhibiting differentiation and functions of osteoclasts, so that the effect of treating osteoporosis is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of an ADRM1 inhibitor in the preparation of a drug for treating osteoporosis. Background Art

[0002] The treatment of osteoporosis in the prior art mainly includes drug therapy, non-drug therapy and surgical treatment, but each treatment method has certain limitations, which is also an urgent problem to be solved in the current field of osteoporosis treatment. First, although drug therapy has achieved remarkable results in increasing bone density and reducing the risk of fractures, the long-term use of drugs is often accompanied by a series of side effects. Secondly, the effectiveness of non-drug treatments such as physical therapy and rehabilitation exercises depends on the patient's long-term persistence and self-management ability. Finally, surgical treatment is usually aimed at severe fractures or dysfunctions caused by osteoporosis, and can quickly restore the patient's physiological functions. However, surgical treatment also has certain risks, including infection, bleeding, nerve damage and difficulty in postoperative rehabilitation. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention provides the use of an ADRM1 inhibitor in the preparation of a drug for treating osteoporosis, the purpose of which is to solve the problems mentioned in the background technology.

[0004] ADRM1 (Adhesion Regulating Molecule 1) plays a role in cell adhesion and is involved in the recruitment process and ubiquitination modification of proteins, as well as the assembly and functional regulation of proteasomes. The present invention applies ADRM1 inhibitors to the preparation of drugs for treating osteoporosis. By downregulating ADRM1 expression or inhibiting ADRM1 activity, the differentiation and function of osteoclasts can be inhibited, thereby achieving the effect of treating osteoporosis.

[0005] The present invention provides the use of an ADRM1 inhibitor in preparing a drug for treating osteoporosis.

[0006] Furthermore, the drug treats osteoporosis by downregulating ADRM1 expression or inhibiting ADRM1 activity.

[0007] Furthermore, the drug is a targeted agent that downregulates ADRM1 expression.

[0008] Furthermore, the targeting agent includes at least one of an AAV virus, a small interfering RNA or a polymer micelle carrying a gene that downregulates ADRM1 expression.

[0009] Furthermore, the small interfering RNA sequence is shown in SEQ ID NO.1.

[0010] Furthermore, the drug is an inhibitor that inhibits ADRM1 activity.

[0011] Furthermore, the inhibitor includes a small molecule compound.

[0012] Furthermore, the small molecule compound is RA190.

[0013] Furthermore, the osteoporosis is postmenopausal osteoporosis.

[0014] The present invention has the following technical effects: ADRM1 affects bone metabolism, bone tissue morphology and structure, and osteoporosis progression, and can provide a new drug target for the clinical prevention and treatment of osteoporosis. ADRM1 inhibitors are used in the preparation of osteoporosis drugs, which can significantly reduce bone loss by downregulating ADRM1 expression or inhibiting ADRM1 activity, inhibiting osteoclast differentiation and function, and achieving the effect of treating osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] A more complete understanding of exemplary embodiments of the present invention may be obtained by referring to the following drawings: Figure 1 This is a diagram showing the skeletal phenotype of wild-type mice and ADRM1 knockout mice detected by micro-CT in Example 1 of the present invention; wherein: Figure 1 A in the figure shows the micro-CT results of wild-type mice and ADRM1 knockout mice, scale bar: 200 μm; Figure 1 B is the relative bone volume results of wild-type mice and ADRM1 knockout mice; Figure 1 C in the figure is the result graph of trabecular number of wild-type mice and ADRM1 complete gene knockout mice; Figure 1 D in the figure is the result of trabecular separation of wild-type mice and ADRM1 knockout mice; Figure 1 E in the figure is the trabecular bone thickness of wild-type mice and ADRM1 knockout mice.

[0016] Figure 2 The graph is a result of silver nitrate staining, osteocalcin immunohistochemical staining and tartrate-resistant acid phosphatase (TRAP) staining of wild-type mice and ADRM1 full gene knockout mice in Example 1 of the present invention; wherein: Figure 2 A in the figure is a typical picture of silver nitrate staining, osteocalcin immunohistochemical staining, and TRAP staining of wild-type mice and ADRM1 knockout mice, scale bar: 200 μm; Figure 2B is the relative bone volume results of wild-type mice and ADRM1 knockout mice stained with silver nitrate; Figure 2 C in the figure is the result graph of the number of osteocalcin-positive osteoblasts in wild-type mice and ADRM1 knockout mice; Figure 2 D in the figure is the result graph of osteoclast number in wild-type mice and ADRM1 knockout mice.

[0017] Figure 3 The TRAP staining, actin ring staining and bone resorption results of bone marrow-derived mononuclear macrophages of wild-type mice and ADRM1 knockout mice in Example 2 of the present invention are shown; wherein: Figure 3 A in the figure is a typical picture of TRAP staining of bone marrow-derived mononuclear macrophages from wild-type mice and ADRM1 knockout mice, actin ring staining (4',6-diamidino-2-phenylindole, actin ring and the combination of the two), and bone resorption of bovine bone slices; Figure 3 B is the result graph of the number of TRAP-positive cells in wild-type mice and ADRM1 knockout mice; Figure 3 C in the figure is the number of actin rings in wild-type mice and ADRM1 knockout mice; Figure 3 D in the figure is the bone resorption area of ​​wild-type mice and ADRM1 knockout mice.

[0018] Figure 4 This is a graph showing the expression of mRNA of osteoclast-specific related genes induced by bone marrow-derived mononuclear macrophages of wild-type mice and ADRM1 knockout mice in Example 2 of the present invention.

[0019] Figure 5 This is a graph showing the results of Western blotting to detect osteoclast-specific related proteins induced by bone marrow-derived mononuclear macrophages from wild-type mice and ADRM1 knockout mice; wherein: Figure 5 A in the figure is a typical picture of osteoclast-specific related proteins induced by bone marrow-derived mononuclear macrophages from wild-type mice and ADRM1 knockout mice, with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the internal reference protein; Figure 5 Figure B shows the expression levels of osteoclast-specific related proteins induced in bone marrow-derived mononuclear macrophages from wild-type mice and ADRM1 knockout mice.

[0020] Figure 6This is a graph showing the bone phenotype of mice in the sham operation group, the blank solvent group, the low-dose RA190 group and the high-dose RA190 group detected by micro-CT in Example 4 of the present invention; wherein: Figure 6 A in the figure shows the micro-CT results of mice in the sham operation group, blank solvent group, low-dose RA190 group, and high-dose RA190 group. Scale bar: 200 μm. Figure 6 B is the relative bone volume results of mice in the sham operation group, blank solvent group, low-dose RA190 group and high-dose RA190 group; Figure 6 C in the figure is the result graph of the number of trabeculae in mice in the sham operation group, blank solvent group, low-dose RA190 group and high-dose RA190 group; Figure 6 D in the figure is the result graph of trabecular separation of mice in the sham operation group, blank solvent group, low-dose RA190 group and high-dose RA190 group; Figure 6 E in FIG. 5 is the trabecular bone thickness of mice in the sham operation group, blank solvent group, low-dose RA190 group and high-dose RA190 group.

[0021] Figure 7 The tartrate-resistant acid phosphatase (TRAP) staining, actin ring staining and bone resorption result diagram of bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group of Example 5 of the present invention are shown; wherein: Figure 7 A in the figure is a typical picture of tartrate-resistant acid phosphatase (TRAP) staining of bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group, actin ring staining (4',6-diamidino-2-phenylindole, actin ring and the combination of the two) and bone resorption of bovine bone slices, scale bar: 200 μm; Figure 7 B is the result graph of the number of TRAP-positive cells of bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group; Figure 7 C in the figure is the result graph of the number of actin rings in bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group; Figure 7 D in the figure is the result graph of bone resorption area of ​​bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group.

[0022] Figure 8This is a graph showing the expression of osteoclast-specific related gene mRNA induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group in Example 5 of the present invention; wherein: Figure 8 A in the figure is the expression result of NFATc1 (nuclear factor activated T cells 1) gene mRNA in osteoclasts induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group; Figure 8 B is the expression results of Cathepsin K (cathepsin K) gene mRNA in osteoclasts induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group; Figure 8 C in the figure is the expression result of TRAP gene mRNA in osteoclasts induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group; Figure 8 D in the figure is the expression result of the mRNA of CTR (calcitonin receptor) gene induced in osteoclasts by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group; Figure 8 E in the figure is the expression result of OSCAR (osteoclast-associated receptor) gene mRNA induced in osteoclasts by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group.

[0023] Fig. 9 This is a graph showing the results of the osteoclast-specific related proteins induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group detected by Western blotting in Example 5 of the present invention; wherein: Fig. 9 A in the figure is a typical picture of osteoclast-specific related proteins induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group, with GAPDH as the internal reference protein; Fig. 9 B is the expression result of NFATc1 protein in osteoclasts induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group; Fig. 9C in the figure is the expression result of Cathepsin K protein in osteoclasts induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group; Fig. 9 D in the figure is the expression result of TRAP protein induced in osteoclasts by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group; Fig. 9 E in the figure is the expression result of CTR protein in osteoclasts induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group; Fig. 9 F in the figure is the expression result of OSCAR protein in osteoclasts induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group. DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] 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 the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments rather than limiting the present invention.

[0026] In some embodiments, the present invention provides use of an ADRM1 inhibitor in the preparation of a drug for treating osteoporosis.

[0027] Specifically, the drug treats osteoporosis by downregulating ADRM1 expression or inhibiting ADRM1 activity.

[0028] Specifically, the drug is a targeted agent that downregulates ADRM1 expression.

[0029] Specifically, the targeting agent includes at least one of an AAV virus, a small interfering RNA, or a polymer micelle carrying a gene that downregulates ADRM1 expression.

[0030] Specifically, the small interfering RNA sequence is shown in SEQ ID NO.1.

[0031] Specifically, the drug is an inhibitor that inhibits ADRM1 activity.

[0032] Inhibitors include, in particular, small molecule compounds.

[0033] Specifically, the small molecule compound is RA190.

[0034] Specifically, the osteoporosis is postmenopausal osteoporosis.

[0035] Statistical analysis in the examples: ** indicates p≤0.01, ns indicates no difference.

[0036] Embodiment 1: With the technical support of Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (Contract Order No.: GJS072021107931), CRISPR / Cas9 gene editing technology was used to construct ADRM1 full gene knockout mice to downregulate mouse ADRM1 expression.

[0037] The skeletal phenotypes of wild-type mice and ADRM1 knockout mice were examined by micro-CT. Figure 1 As shown in Figure 2, compared with wild-type mice, ADRM1 knockout mice showed a significant increase in bone mass (e.g. Figure 1 The bone mass increased significantly (as shown in A in Figure 1 The number of trabeculae increased (as shown in B). Figure 1 The degree of trabecular dispersion is reduced (as shown in C in Figure 1 D in the figure) and increased trabecular thickness (as shown in Figure 1 ).

[0038] The results of silver nitrate staining, osteocalcin immunohistochemical staining, and tartrate-resistant acid phosphatase (TRAP) staining of wild-type mice and ADRM1 knockout mice, such as Figure 2 As shown in Figure 2, compared with wild-type mice, ADRM1 knockout mice have significantly increased bone mass (as shown in Figure 2). Figure 2 There was no statistical difference in the number of osteocalcin-positive osteoblasts between wild-type mice and ADRM1 knockout mice (as shown in A and B in Figure 2 ). Figure 2 Compared with wild-type mice, the number of osteoclasts in ADRM1 knockout mice was significantly reduced (as shown in A and C in Figure 2 ). Figure 2 A and D in the figure).

[0039] In summary, by using CRISPR / Cas9 gene editing technology to construct ADRM1 knockout mice, the expression of ADRM1 in mice was downregulated, which caused an increase in bone mass in mice, which was mainly due to the impact on the regulation of mouse osteoclasts.

[0040] Embodiment 2: Bone marrow mononuclear macrophages (BMMs) from wild-type mice and ADRM1 knockout mice were isolated and cultured in vitro, and osteoclasts were induced in BMMs of wild-type mice and ADRM1 knockout mice using M-CSF (macrophage colony-stimulating factor) and RANKL (Receptor Activator of NF-κB Ligand).

[0041] TRAP staining, actin ring staining and bone resorption results of bone marrow-derived monocytes and macrophages from wild-type mice and ADRM1 knockout mice, such as Figure 3 As shown in Figure 2, compared with wild-type mice, the number of osteoclasts induced by bone marrow-derived mononuclear macrophages in ADRM1 knockout mice was reduced (e.g. Figure 3 A and B in Figure ), while the number of actin rings was significantly reduced (as shown in Figure ). Figure 3 A and C in Figure ) and the area of ​​bone resorption was significantly reduced (as shown in Figure ). Figure 3 A and D in the figure).

[0042] The expression results of osteoclast-specific related gene mRNA in bone marrow-derived mononuclear macrophages from wild-type mice and ADRM1 knockout mice are as follows: Figure 4 As shown in Figure 2, it can be seen that the expression of specific related genes (ADRM1, NFATc1, Cathepsin K, TRAP, CTR, OSCAR) in osteoclasts was significantly downregulated.

[0043] Western blotting was used to detect the expression of osteoclast-specific proteins in bone marrow-derived mononuclear macrophages from wild-type mice and ADRM1 knockout mice. Figure 5 As shown in Figure 2, it can be seen that the expression of specific related proteins (ADRM1, NFATc1, Cathepsin K, TRAP, CTR, OSCAR) in osteoclasts were significantly downregulated.

[0044] In summary, downregulating ADRM1 expression through ADRM1 global gene knockout can inhibit osteoclast differentiation and function, and downregulate the expression levels of osteoclast-specific related genes and proteins.

[0045] Embodiment 3: siRNA (small interfering RNA) was used to downregulate ADRM1 expression. The siRNA sequence was: CGGACGACUCCCUUAUUCA (SEQ ID NO. 1).

[0046] Embodiment 4: Set up a sham operation group; An OVX (ovariectomy)-induced osteoporosis model was constructed in mice. One week after osteoporosis surgery, the mice were divided into: blank solvent group: intraperitoneal injection of blank solvent (phosphate buffer solution); low-dose RA190 (specific inhibitor of ADRM1) group: intraperitoneal injection of 10 mg / kg RA190; and high-dose RA190 group: intraperitoneal injection of 20 mg / kg RA190; the intraperitoneal injection was performed twice a week for a total of 4 weeks, and the mouse spinal specimens were collected after the injection.

[0047] The bone phenotypes of mice in the sham operation group, blank solvent group, low-dose RA190 group, and high-dose RA190 group were detected by micro-CT. Figure 6 As shown in Figure 2, it can be seen that when mice with osteoporosis were injected with RA190, the osteoporosis of the mice was significantly alleviated (e.g. Figure 6 The bone mass increased significantly (as shown in A in Figure 6 The number of trabeculae increased (as shown in B). Figure 6 As shown in C in the figure, the degree of dispersion decreases, the bone mass increases significantly, and the degree of dispersion of trabeculae decreases (as shown in Figure 6 D in the figure) and increased trabecular thickness (as shown in Figure 6 In summary, RA190 can inhibit the progression of osteoporosis.

[0048] Embodiment 5: The bone marrow-derived mononuclear macrophages of normal mice were set up as follows: RA190 (0.5 μM) group: after adding 0.5 μM RA190, osteoclasts were induced using M-CSF and RANKL; RA190 (1 μM) group: after adding 1 μM RA190, osteoclasts were induced using M-CSF and RANKL; and a control group was set up: osteoclasts were induced using only M-CSF and RANKL.

[0049] Tartrate-resistant acid phosphatase (TRAP) staining of bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group, and RA190 (1 μM) group, actin ring staining, and bone resorption results, as shown in Figure 7 As shown. It can be seen that compared with the control group, the number and area of ​​osteoclasts induced by RA190 (0.5 μM) group and RA190 (1 μM) group were significantly reduced (as shown in Figure 2). Figure 7 As shown in A and B in Figure 3, the number of actin rings was significantly reduced (as shown in Figure 3). Figure 7 A and C in Figure ) and the area of ​​bone resorption was significantly reduced (as shown in Figure ). Figure 7 A and D in the figure).

[0050] The expression results of specific related gene mRNA in osteoclasts induced by bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group and RA190 (1 μM) group are as follows: Figure 8 As shown. It can be seen that compared with the control group, NFATc1 (as Figure 8 A in Figure ), Cathepsin K (as Figure 8 B in the figure), TRAP (as shown in Figure 8 CTR (as shown in Figure 8 D in), OSCAR (as shown in Figure 8 The genes shown in E were significantly suppressed.

[0051] Western blotting was used to detect the expression of osteoclast-specific related proteins in bone marrow-derived mononuclear macrophages in the control group, RA190 (0.5 μM) group, and RA190 (1 μM) group. Fig. 9 As shown. It can be seen that compared with the control group, NFATc1 in osteoclasts of the RA190 (0.5 μM) group and RA190 (1 μM) group (as shown Fig. 9 A and B in Figure ), Cathepsin K (as Fig. 9 A and C in Figure ), TRAP (as shown in Fig. 9 A and D in), CTR (as shown in Fig. 9 A and E in), OSCAR (as shown in Fig. 9 The expression of proteins in (A and F) was significantly inhibited.

[0052] In summary, inhibiting ADRM1 activity through a specific inhibitor of ADRM1 (RA190) can inhibit osteoclast differentiation and function, and inhibit the expression levels of osteoclast-specific related genes and proteins.

[0053] From the analysis of the results of Examples 1-5, the present invention draws the following conclusions: ADRM1 affects bone metabolism, bone tissue morphology and osteoporosis progression, and can provide a new drug target for the clinical prevention and treatment of osteoporosis. ADRM1 inhibitors are used in the preparation of osteoporosis drugs, which can significantly reduce bone loss by downregulating ADRM1 expression or inhibiting ADRM1 activity, inhibiting osteoclast differentiation and function, and achieving the effect of treating osteoporosis.

[0054] 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 in the protection scope of the present invention.

Claims

1. Application of ADRM1 inhibitors in the preparation of drugs for the treatment of osteoporosis.

2. The use according to claim 1, characterized in that: The drug treats osteoporosis by downregulating ADRM1 expression or inhibiting ADRM1 activity.

3. The use according to claim 2, characterized in that: The drug is a targeted agent that downregulates ADRM1 expression.

4. The use according to claim 3, characterized in that: The targeting agent includes at least one of an AAV virus carrying a gene that downregulates ADRM1 expression, a small interfering RNA, or a polymer micelle.

5. The use according to claim 4, characterized in that: The small interfering RNA sequence is shown in SEQ ID NO.

1.

6. The use according to claim 2, characterized in that: The drug is an inhibitor that inhibits ADRM1 activity.

7. The use according to claim 6, characterized in that: The inhibitors include small molecule compounds.

8. The use according to claim 7, characterized in that: The small molecule compound is RA190.

9. The use according to claim 1, characterized in that: The osteoporosis is postmenopausal osteoporosis.

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