Application of SRGN in the treatment and diagnosis of osteoporosis

Abstract

This invention relates to the application of SRGN in the treatment and diagnosis of osteoporosis, belonging to the field of biomedical technology. This invention reveals for the first time the key role of SRGN in the pathogenesis of osteoporosis, suggesting its potential as a novel therapeutic or diagnostic target for osteoporosis. Furthermore, this invention designs a novel neutralizing antibody against SRGN, 12E2, which effectively blocks the interaction between SRGN and SIGLEC15, showing significant inhibitory effects on osteoclast formation in both in vivo and in vitro models. In animal experiments, SRGN neutralizing antibody 12E2 or... Srgn Knockout significantly improved bone mineral density in mice. This invention provides novel molecular targets and strategies for the treatment or diagnosis of osteoporosis, and has promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of SRGN in the treatment and diagnosis of osteoporosis. Background Technology

[0002] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass, destruction of bone microstructure, and increased bone fragility, making patients prone to fractures. Its pathogenesis involves multiple factors, including decreased estrogen levels, calcium and phosphorus metabolism disorders, and an imbalance between osteoblast and osteoclast activity. In the early stages, there are often no obvious symptoms. As the disease progresses, back pain, decreased height, and kyphosis may occur. In severe cases, even minor external forces can cause fragility fractures of the hip, vertebrae, or other parts of the body, significantly reducing the patient's quality of life and increasing the risk of disability and death.

[0003] Glucocorticoids (GCs) are widely used clinically due to their potent immunosuppressive effects to treat various autoimmune and highly inflammatory diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. However, long-term glucocorticoid therapy is often accompanied by various adverse reactions, among which glucocorticoid-induced osteoporosis (GIOP) is one of the most common complications of GC treatment. GIOP is characterized by significantly suppressed bone formation, with a transient increase in bone resorption in the early stages, primarily affecting cancellous bone (such as the vertebral body). Bone loss is most rapid in the initial stages of treatment (the first 3-6 months). Currently, the coverage of preventive treatment for GIOP in clinical practice remains low; therefore, bone health assessment and early intervention should be emphasized when initiating GC treatment.

[0004] It is widely believed in the academic community that glucocorticoids (GCs) directly disrupt bone homeostasis by enhancing osteoclastogenesis while simultaneously inhibiting osteoblastogenesis. Based on this, a series of anti-osteoclastogenic drugs (such as denosumab and bisphosphonates) and osteoproliferative drugs (such as vitamin D and calcium supplements) have been used to treat glucocorticoid-induced osteoporosis (GIOP). However, due to various limitations, such as drug side effects, limited efficacy, and poor patient compliance, these drugs cannot fully meet current clinical needs. Therefore, there is an urgent need to develop new therapeutic targets for osteoporosis, especially glucocorticoid-induced osteoporosis.

[0005] Serglycin (SRGN) is composed of a 17.6 kDa core protein and glycosaminoglycan chains, and is typically expressed in immune cells, endothelial cells, and tumor cells. Recent studies have revealed that SRGN plays an important regulatory role in various diseases, but its mechanism of action in osteoporosis, particularly GIOP, remains unclear. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the lack of an effective product for treating osteoporosis in the prior art.

[0007] To address the aforementioned technical problems, this invention provides an application of SRGN in the treatment and diagnosis of osteoporosis. This invention is the first to discover that SRGN promotes osteoclast formation by binding to sialic acid-binding immunoglobulin-like lectin 15 (SIGLEC15) on the surface of osteoclast precursor cells. Therefore, SRGN can serve as a novel therapeutic target for osteoporosis, and blocking the interaction between SRGN and SIGLEC15 can represent a new direction for osteoporosis treatment. Simultaneously, SRGN is highly expressed in glucocorticoid-induced osteoporosis. Applying SRGN to the diagnosis of GIOP yields an AUC of 0.796, demonstrating good diagnostic value. Furthermore, this invention designs a novel SRGN neutralizing antibody, 12E2, which effectively blocks the interaction between SRGN and SIGLEC15, showing significant inhibitory effects on osteoclast formation in both in vivo and in vitro models. In a GIOP mouse model, SRGN neutralizing antibody 12E2 significantly improves bone mineral density (BMD). This invention provides new molecular targets and strategies for the diagnosis and treatment of osteoporosis, and has promising clinical application prospects.

[0008] The first objective of this invention is to provide an application of SRGN and its combination with SIGLEC15 in the development and utilization of osteoporosis-related products.

[0009] A second objective of this invention is to provide a neutralizing antibody against SRGN, wherein the heavy chain variable region of the neutralizing antibody includes heavy chain complementarity-determining regions VH-CDR1, VH-CDR2 and VH-CDR3, the amino acid sequences of which are shown in SEQ ID NO.1-3, respectively.

[0010] The light chain variable region of the neutralizing antibody includes light chain complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3, with amino acid sequences as shown in SEQ ID NO.4-6, respectively.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of the neutralizing antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region of the neutralizing antibody is shown in SEQ ID NO.8.

[0012] Furthermore, the amino acid sequence of the heavy chain constant region of the neutralizing antibody is shown in SEQ ID NO.9, and the amino acid sequence of the light chain constant region of the neutralizing antibody is shown in SEQ ID NO.10.

[0013] A third objective of this invention is to provide the use of a substance that blocks the binding of SRGN to SIGLEC15 in the preparation of osteoporosis treatment products.

[0014] Furthermore, the substance includes an antibody / ligand capable of blocking the binding of SRGN to SIGLEC15, or a polypeptide capable of blocking the binding of SRGN to SIGLEC15, or one or more of small interfering RNA (siRNA), miRNA, or shRNA capable of blocking the binding of SRGN to SIGLEC15.

[0015] Furthermore, the substance includes the aforementioned neutralizing antibody.

[0016] Furthermore, the osteoporosis mentioned includes glucocorticoid-induced osteoporosis.

[0017] A fourth object of the present invention is to provide a pharmaceutical composition for treating osteoporosis, the pharmaceutical composition comprising a substance that blocks the binding of SRGN to SIGLEC15.

[0018] Furthermore, the substance includes the aforementioned neutralizing antibody.

[0019] Furthermore, the pharmaceutical composition also includes a neutralizing antibody against receptor activator of nuclear factor κB (RANKL).

[0020] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0021] Furthermore, the excipients include pharmaceutically acceptable salts, excipients, or carriers.

[0022] Furthermore, the osteoporosis mentioned includes glucocorticoid-induced osteoporosis.

[0023] A fifth objective of this invention is to provide an application of an SRGN inhibitor in the preparation of osteoporosis treatment products, wherein the SRGN inhibitor targets and reduces the expression or secretion of SRGN.

[0024] A sixth objective of this invention is to provide the use of the above-mentioned neutralizing antibody in the preparation of products that block the binding of SRGN to SIGLEC15.

[0025] The seventh objective of this invention is to provide a reagent for detecting SRGN levels and its application in the preparation of a diagnostic kit for glucocorticoid-induced osteoporosis.

[0026] Furthermore, in the glucocorticoid-induced osteoporosis diagnostic kit, the sample is derived from serum.

[0027] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0028] This invention reveals for the first time the crucial role of SRGN in the pathogenesis of osteoporosis, discovering that SRGN promotes osteoclast formation by binding to SIGLEC15 on the surface of osteoclast precursor cells. Therefore, SRGN can serve as a novel therapeutic and diagnostic target for osteoporosis. Furthermore, this invention designs a novel SRGN neutralizing antibody, 12E2, which effectively blocks the interaction between SRGN and SIGLEC15, demonstrating significant inhibitory effects on osteoclast formation in both in vivo and in vitro models. This invention provides new molecular targets and strategies for the treatment and diagnosis of osteoporosis, showing promising clinical application prospects. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0030] Figure 1 yes Srgn The experimental results of knockout mice are shown in the figure, where ns indicates no significant difference;

[0031] Figure 2 This is a graph validating the efficacy of the SRGN neutralizing antibody 12E2.

[0032] Figure 3 This is the AUC curve of SRGN in the diagnosis of GIOP. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0034] Example 1: Srgn Effects of knockout on bone

[0035] 1. Srgn Construction of knockout mice

[0036] Srgn ( Serglycin The construction of knockout mice (encoding serine proteoglycan) was achieved using CRISPR-Cas9 gene editing technology, specifically clustered regularly spaced short palindromic repeats-Cas9 protein. Firstly, the mice were targeted with... SerglycinA specific sgRNA target was designed for the gene (located on chromosome 10), selecting exon regions or key functional domain sequences. The Cas9 protein-coding gene and sgRNA were co-microinjected into the pronucleus of fertilized eggs from C57BL / 6 mice. The injected fertilized eggs were then transplanted into the oviducts of pseudopregnant mice. The genotypes of the newborn mice were identified by polymerase chain reaction (PCR) amplification of the target sequence and dideoxy chain termination (Sanger) sequencing. F0 generation chimeric mice carrying biallelic knockout were selected. These chimeric mice were backcrossed with wild-type mice to obtain heterozygous F1 generation mice. Homozygous knockout mice were then obtained through heterozygous crosses. Srgn - / - Mice.

[0037] Table 1 sgRNA

[0038]

[0039] Wild-type C57BL / 6 mice were divided into two groups and treated as follows, while... Srgn - / - The mice were divided into two groups and treated as follows:

[0040] Wild-type + dimethyl sulfoxide group: Wild-type C57BL / 6 mice were injected with dimethyl sulfoxide for 28 consecutive days, with each mouse receiving 200 µg.

[0041] Wild-type + dexamethasone group: Wild-type C57BL / 6 mice were injected with dimethyl sulfoxide for 28 consecutive days, with each mouse receiving 200 µg.

[0042] Srgn Knockout mice + dimethyl sulfoxide group: to Srgn Knockout mice were injected with dimethyl sulfoxide for 28 consecutive days, with each mouse receiving 200 µg.

[0043] Srgn Knockout mice + dexamethasone group: to Srgn Knockout mice were injected with dimethyl sulfoxide for 28 consecutive days, with each mouse receiving a dose of 200 µg.

[0044] Treatment with dexamethasone can simulate the pathogenesis environment of GIOP and is a commonly used treatment method in the construction of GIOP mouse models. On day 28, mice in each group were euthanized and their femurs and tibias were removed. Micro-computed tomography (Micro-CT, SkyScan) was used to analyze the femurs and tibias. The bones were fixed with 4% paraformaldehyde and stained with a tartrate-resistant acid phosphatase (TRAP) staining kit (Catalog No. G1050, Servicebio). Results are as follows: Figure 1 As shown, SrgnKnockout significantly increases bone density and reduces the number of osteoclasts, therefore Srgn Knockout can alleviate GIOP.

[0045] Example 2: Preparation of SRGN neutralizing antibody 12E2

[0046] Rabbits were immunized with two different polypeptides from SRGN (amino acid sequences DRILTEQNQDQPEDD and LGDMEWEYQPTDESN, respectively), and then B cells isolated from their peripheral blood were cultured as single cells to isolate and identify the monoclonal antibody 12E2 with the highest affinity for SRGN. The amino acid sequence of the SRGN neutralizing antibody 12E2 is shown in Table 2.

[0047] Table 2. Amino acid sequence information of SRGN neutralizing antibody 12E2

[0048]

[0049] Example 3: SRGN neutralizing antibody 12E2 blocks the binding of SRGN to SIGLEC15

[0050] SIGLEC15 (sialic acid-binding immunoglobulin-like lectin 15) is involved in the regulation of actin cytoskeleton organization, modulating bone resorption and osteoclast development. Therefore, recombinant mouse-derived SIGLEC15 protein was used to verify the efficacy of the SRGN neutralizing antibody 12E2.

[0051] Recombinant mouse-derived SIGLEC15 protein (catalog number HY-P70744, brand name MedChemExpress) was coated into 96-well plates at a concentration of 0.5 µg / mL (100 µL / well), followed by blocking with 3% skim milk (w / v). Recombinant mouse-derived SRGN protein (catalog number 10190-SN, brand name R&D, concentration range 10) was also coated into 96-well plates. -2 ng / mL to 10 4Biotin-labeled SRGN (5 µg / mL) was prepared using kit ab201795 (Abcam) and premixed with 5 µg / mL SRGN neutralizing antibody 12E2. This mixture was then added to the pretreated plate and incubated at 37 °C for 1 hour. After washing three times with TBST buffer containing 0.05% Tween 20, 100 µL / pore of horseradish peroxidase (HRP)-labeled streptavidin (Catalog No. G3431, Servicebio) was added and incubated at 37 °C for 1 hour. Finally, after washing with TBST buffer, the binding of SRGN to SIGLEC15 was quantitatively detected using a universal enzyme-linked immunosorbent assay (ELISA) kit (Catalog No. D601059, Sangon), and the absorbance was read at 450 nm on a Cytation™ 5 microplate reader (BioTek). Rabbit IgG antibody was used as a control antibody and the same procedure was performed.

[0052] The results are as follows Figure 2 As shown in A, the SRGN neutralizing antibody 12E2 can effectively inhibit the interaction between SRGN and SIGLEC15.

[0053] Example 4: In vitro osteoclast formation experiment

[0054] Mouse mononuclear macrophage leukemia cells (RAW264.7 cells) were pretreated in DuPont modified Eagle medium (DMEM) containing 10% fetal bovine serum (F0193, Sigma-Aldrich), 100 U / mL penicillin, and 100 mg / mL streptomycin (C100C5, New Cell and Molecular Biotechnology Co., Ltd.).

[0055] For osteoclast acquisition, α-modified Eagle Minimum Essential Medium (α-MEM) containing 20% ​​fetal bovine serum (product number JYK-FBS-301, Jinyuankang Biotechnology) was used as the basal medium. Pretreated mouse mononuclear macrophage leukemia cells were seeded into the basal medium. 12.5 ng / mL SRGN neutralizing antibody 12E2 (with rabbit IgG antibody added as a control antibody) was added to the basal medium. After 5 to 7 days of differentiation, mature osteoclasts were stained using an anti-tartrate acid phosphatase (TRAP) staining kit (G1050, Servicebio). The number of osteoclasts was assessed by counting the number of multinucleated TRAP-positive cells in each field of view.

[0056] Mouse mononuclear macrophage leukemia cells were directly seeded into basal culture medium without pretreatment, and 12.5 ng / mL SRGN neutralizing antibody 12E2 was added to the basal culture medium as a control (rabbit IgG antibody was added as a control antibody). After 5 to 7 days of differentiation, mature osteoclasts were stained using an anti-tartrate acid phosphatase (TRAP) staining kit (Catalog No. G1050, Servicebio). The number of osteoclasts was assessed by counting the number of multinucleated TRAP-positive cells in each field of view.

[0057] The results are as follows Figure 2 As shown in B and C, the addition of SRGN neutralizing antibody 12E2 to the in vitro osteoclast formation induction system significantly inhibited osteoclast formation.

[0058] Example 5: Evaluation of the therapeutic effect in a GIOP mouse model

[0059] Establishment of the GIOP mouse model: C57BL / 6 mice were divided into four groups, and each group received subcutaneous injections of dexamethasone (product number D8040, brand name Solarbio) at a dose of 25 mg / kg body weight daily for 28 consecutive days, and were treated as follows:

[0060] Dexamethasone + control antibody group: Starting from day 7 of dexamethasone injection, mice were injected intraperitoneally with an additional 200 µg of control antibody (rabbit IgG antibody) every other day.

[0061] Dexamethasone + 12E2 group: Starting from day 7 of dexamethasone injection, mice were given an additional intraperitoneal injection of 200 µg of SRGN neutralizing antibody 12E2 every other day;

[0062] Dexamethasone + RANKL neutralizing antibody group: 100 µg mouse RANKL neutralizing antibody (product number IK22 / 5, brand name BioXCell) was injected subcutaneously once a week;

[0063] Dexamethasone + combination therapy group: Starting from day 7 of dexamethasone injection, mice were given an additional intraperitoneal injection of 200 µg of SRGN neutralizing antibody 12E2 every other day, and a subcutaneous injection of 100 µg of mouse RANKL neutralizing antibody once a week.

[0064] In addition, C57BL / 6 mice were subcutaneously injected with dimethyl sulfoxide for 28 consecutive days as a control group.

[0065] On the 28th day, each group of mice was euthanized and their femurs and tibias were removed. The femurs and tibias were analyzed using Micro-CT (brand: SkyScan). The bones were fixed with 4% paraformaldehyde and stained with a TRAP staining kit (product number: G1050, brand: Servicebio).

[0066] The results were as Figure 2 shown in D, E, and F of

[0067] Example 6: Preparation of a pharmaceutical composition

[0068] The SRGN neutralizing antibody 12E2 was mixed with a pharmaceutically acceptable carrier to prepare a pharmaceutical composition suitable for injection. The pharmaceutically acceptable carrier includes buffers (such as PBS), stabilizers (such as human serum albumin), preservatives, etc. The pharmaceutical composition can be administered by intravenous injection, subcutaneous injection, or intraperitoneal injection.

[0069] Example 7: Application of SRGN in the diagnosis of glucocorticoid-induced osteoporosis

[0070] This example included two groups of subjects:

[0071] (1) The GIOP patient group: It included 16 GIOP patients (recruited from the Third Affiliated Hospital of Soochow University). The diagnostic criteria were as follows: They had a previous long-term history of glucocorticoid use (glucocorticoid treatment ≥ 2.5 mg / d for more than 3 months) and met one of the following three criteria:

[0072] (a) Hip or vertebral fragility fracture;

[0073] (b) The T value of axial bone mineral density or distal 1 / 3 radial bone density measured by dual-energy X-ray absorptiometry (DXA) ≤ -2.5;

[0074] (c) Bone mineral density measurement met low bone mass (-2.5 < T value < -1.0) + proximal humerus, pelvis, or distal forearm fragility fracture.

[0075] (2) The healthy control group: It included 15 healthy control individuals from the Third Affiliated Hospital of Soochow University.

[0076] The subjects in this example were excluded for having a history of cancer, severe infection, recent use of antibiotics, or use of probiotics.

[0077] All participants signed informed consent forms before participating in the study. Venous blood was collected from participants in the morning on an empty stomach, serum was separated, and stored at -80°C for later use.

[0078] 2. The expression level of SRGN in serum was quantitatively detected by enzyme-linked immunosorbent assay (ELISA).

[0079] The expression level of SRGN in serum was detected using a commercially available ELISA kit and a microplate reader. The results showed that the SRGN levels in the GIOP patient group were significantly higher than those in the healthy control group (see...). Figure 3 (A) A model was constructed using logistic regression, the receiver operating characteristic (ROC) curve was plotted, and the area under the curve (AUC) was calculated. The results are as follows: Figure 3 As shown in B, its AUC value is 0.796, and the 95% confidence interval (CI) is 0.6357-0.9559.

[0080] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Application of serine proteoglycan and its binding with sialic acid to immunoglobulin-like lectin 15 in the development and utilization of osteoporosis-related products.

2. A neutralizing antibody against serine proteoglycan, characterized in that, The heavy chain variable region of the neutralizing antibody includes heavy chain complementarity-determining regions VH-CDR1, VH-CDR2 and VH-CDR3, with amino acid sequences as shown in SEQ ID NO.1-3, respectively. The light chain variable region of the neutralizing antibody includes light chain complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3, with amino acid sequences as shown in SEQ ID NO.4-6, respectively.

3. The neutralizing antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the neutralizing antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region of the neutralizing antibody is shown in SEQ ID NO.

8.

4. Application of substances that block the binding of serine proteoglycans to sialic acid and immunoglobulin-like lectin 15 in the preparation of osteoporosis treatment products.

5. The application according to claim 4, characterized in that, The substance includes the neutralizing antibody as described in claim 2 or 3.

6. A pharmaceutical composition for treating osteoporosis, characterized in that, The pharmaceutical composition includes a substance that blocks the binding of serine proteoglycan to sialic acid-binding immunoglobulin-like lectin 15.

7. The pharmaceutical composition according to claim 6, characterized in that, The substance includes the neutralizing antibody as described in claim 2 or 3.

8. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also includes an antibody against nuclear factor κB receptor activator ligand.

9. The application of serine proteoglycan inhibitors in the preparation of osteoporosis treatment products, characterized in that, The serine inhibitor targets and reduces the expression or secretion of serine.

10. The use of the neutralizing antibody of claim 2 or 3 in the preparation of a product that blocks the binding of serine proteoglycan to sialic acid-binding immunoglobulin-like lectin 15.

11. Application of reagents for detecting serine proteoglycan content in the preparation of diagnostic kits for glucocorticoid-induced osteoporosis.

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