Application of CXCL12 / CXCR4 signaling pathway as a target in the preparation of drugs for the treatment or prevention of heterotopic ossification

Abstract

The present invention belongs to the field of biomedicine and relates to the application of CXCL12 / CXCR4 signaling pathway in the preparation of drugs for the treatment or prevention of heterotopic ossification. The present invention first discovered that CXCL12 is highly expressed in the site of ectopic new bone formation in ankylosing spondylitis (AS) patients and AS-related animal models, and after plerixafor was used to inhibit CXCR4, the ectopic osteogenesis of mice was significantly reduced. The present invention also proposes the application of CXCL12 detection reagents in the preparation of heterotopic ossification detection reagents or kits, as well as the application of CXCL12 / CXCR4 signaling pathway inhibitors in the preparation of drugs for the treatment or prevention of heterotopic ossification. Taking heterotopic ossification of ankylosing spondylitis as an example, plerixafor has a good inhibitory effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of CXCL12 / CXCR4 signaling pathway as a target in the preparation of a drug for treating or preventing heterotopic ossification. Background Art

[0002] Heterotopic ossification (HO) is characterized by pathological ectopic bone formed in soft tissues such as muscles, tendons, ligaments, etc. adjacent to bone tissue and joints. Heterotopic ossification is most commonly associated with trauma such as spinal cord injury, brain injury, head injury, burns, fractures, muscle contusions, combat-related trauma, and surgery (particularly arthroplasty). In addition, HO may occur in patients receiving neuromuscular blockade to control adult respiratory distress syndrome and in patients with non-traumatic myelopathy. In rare cases, HO may appear as part of a genetic disease and is sometimes associated with lower motor neuron disorders. The consequences of HO can include, among others, joint contractures and ankylosis, pain, spasms, swelling, fever, neurovascular compression, pressure sores, and severe disability. The range of damage ranges from small lesions that are not clinically apparent to large deposits throughout the body.

[0003] Heterotopic ossification is generally divided into two types: the first is traumatic heterotopic ossification, which is also a common ossification phenomenon, often after severe burns, injuries, and operations such as hip replacement. The second type of hereditary heterotopic ossification includes progressive muscular ossification and progressive bone dysplasia. Hereditary diseases are good experimental models for studying the formation mechanism of heterotopic ossification. Heterotopic ossification is an extremely common and serious health problem. However, there is still a lack of effective treatment for heterotopic calcification in clinical practice.

[0004] Ankylosing spondylitis (AS) is a chronic inflammatory connective tissue disease that primarily affects the axial skeleton, including the sacroiliac joints, and can also affect peripheral joints and extra-articular organs. Its characteristic pathological changes are inflammation of the tendon and ligament attachment points accompanied by chronic inflammatory changes [1]. Chronic inflammatory pain and spinal ankylosis are the two key factors causing disability in this disease [2]. Previous research and treatment have focused on controlling inflammatory activity and relieving inflammatory pain. Due to the lack of sufficient understanding of the etiology and treatment of spinal ankylosis, there is currently no recognized effective method to inhibit the progression of spinal ankylosis [3]. The progression of spinal ankylosis ultimately leads to spinal stiffness and kyphosis, and a significant decrease in the range of motion, which seriously affects the patient's ability to work and quality of life, and imposes a heavy burden on patients, their families, and society [1,3]. The main cause of spinal ankylosis is the formation of ectopic new bone at the attachment point, which gradually spreads to the ligaments and joint capsules, resulting in bony connections between active parts, and a serious decrease or even disappearance of mobility [2]. The mechanism leading to ectopic new bone formation at the enthesis remains unclear. Summary of the invention

[0005] In one aspect, the present invention provides use of a CXCL12 detection reagent in the preparation of a heterotopic ossification detection reagent or kit.

[0006] In one aspect, the present invention provides a heterotopic ossification detection kit, wherein the reagent contains a CXCL12 detection reagent.

[0007] In some embodiments, the CXCL12 detection reagent is selected from one or more of Western blot reagents and / or kits, immunohistochemistry detection reagents and / or kits, immunofluorescence detection reagents and / or kits, ELISA detection reagents and / or kits, RNA probe reagents and / or kits, or nucleic acid reagents and / or kits.

[0008] In one aspect, the present invention provides a system for detecting heterotopic ossification, the system comprising:

[0009] a. a detection component for CXCL12, and

[0010] b. Result judgment system.

[0011] In some embodiments, the CXCL12 detection component comprises one or more of: Western blot reagents and / or kits, immunohistochemistry detection reagents and / or kits, immunofluorescence detection reagents and / or kits, ELISA detection reagents and / or kits, RNA probe reagents and / or kits, or nucleic acid reagents and / or kits.

[0012] In some embodiments, the result judgment component is used to output the risk of heterotopic ossification and / or the type of heterotopic ossification according to the CXCL12 expression result detected by the detection system.

[0013] In some embodiments, the disease risk is based on comparing the CXCL12 expression levels of the test sample with those of the normal sample. When the CXCL12 nucleic acid level and / or protein expression level of the test sample and the normal sample are significantly different or extremely significantly different, the result determines that the disease risk of the test sample is high.

[0014] In some embodiments, when the CXCL12 nucleic acid level and / or protein expression level of the test sample is greater than 2.5 times that of the normal sample, the result indicates that the test sample has a high risk of disease.

[0015] The "to be significantly different or extremely significantly different" mentioned in the present invention means that the corresponding value of the sample to be tested is greater than 2.5 times that of the normal sample.

[0016] In one aspect, the present invention provides the use of the CXCL12 / CXCR4 signaling pathway as a target in the preparation of a drug for treating or preventing heterotopic ossification.

[0017] Chemokine receptors are a superfamily of G protein-coupled receptors (GPCRs) with seven transmembrane domains, usually expressed on the membranes of various types of cells, such as immune cells, epithelial cells, vascular endothelial cells, and neural cells. One type of CXC chemokine receptor, CXCR4, and its natural ligand chemokine CXCL12 (stromal cell-derived factor-1, SDF-1), are widely expressed in a variety of cells and tissues.

[0018] In one aspect, the present invention provides use of a CXCL12 / CXCR4 signaling pathway inhibitor in the preparation of a medicament for treating or preventing heterotopic ossification.

[0019] In some embodiments, the CXCL12 / CXCR4 signaling pathway inhibitor is selected from a CXCL12 inhibitor or a CXCR4 inhibitor.

[0020] In some embodiments, the CXCL12 inhibitor is selected from a substance that inhibits CXCL12 activity, a substance that degrades CXCL12, or a substance that reduces CXCL12 levels.

[0021] In some embodiments, the substance that inhibits the activity of CXCL12 is selected from one or more of a CXCL12 neutralizing antibody, a neutralizing protein, and a chemical antagonist.

[0022] In some embodiments, the CXCR4 inhibitor is selected from a substance that inhibits CXCR4 activity, a substance that degrades CXCR4, or a substance that reduces CXCR4 levels.

[0023] In some embodiments, the substance that inhibits CXCR4 activity is selected from compounds.

[0024] In some embodiments, the compound is selected from one or more of plerixafor, AMD3329, or bis-tetraazamacrocycles and their derivatives, optical isomers, chelated forms, hydrated forms, or pharmaceutically acceptable salts or esters.

[0025] Plerixafor (AMD3100) acts on chemotaxis regulated by CXCR4 and CXCL12, with IC50 of 44nM and 5.7nM respectively. Its main function is to inhibit the migration of CXCR4+ cells to areas with high CXCL12 concentrations. In many early studies, it has been shown to quickly and effectively increase the number of stem cells in the blood circulation of leukemia patients.

[0026] In some embodiments, the plerixafor has the structural formula shown in Formula I:

[0027]

[0028] In some embodiments, the pharmaceutically acceptable salt of plerixafor is selected from plerixafor octahydrochloride.

[0029] In some embodiments, the plerixafor octahydrochloride has the structural formula shown in Formula II:

[0030]

[0031] In some embodiments, the substance that reduces the level of plerixafor is selected from RNA interference, microRNA, gene editing or gene knockout materials.

[0032] In some embodiments, the dosage form of the drug is selected from any pharmaceutically allowed dosage form.

[0033] In some embodiments, the dosage form is selected from one or more of tablets, granules, capsules, pills, oral solutions, injection solutions, and liposomes.

[0034] In some embodiments, the administration of the drug or pharmaceutical composition is selected from one or more of oral administration, topical administration, intranasal administration, systemic administration, intravenous administration, subcutaneous administration, intramuscular administration, intracerebroventricular administration, intrathecal administration or transdermal administration.

[0035] In some embodiments, the heterotopic ossification is selected from one or more of inflammatory heterotopic ossification and traumatic heterotopic ossification.

[0036] In some embodiments, the heterotopic ossification is selected from inflammatory heterotopic ossification.

[0037] In some embodiments, the inflammatory heterotopic ossification is selected from one or more of ankylosing spondylitis heterotopic ossification, osteoarthritis heterotopic ossification, rheumatic disease heterotopic ossification, and gout heterotopic ossification.

[0038] In some embodiments, the inflammatory heterotopic ossification is selected from ankylosing spondylitis heterotopic ossification.

[0039] As used herein, "prevention" means that when used for a disease or condition, the drug can reduce the frequency of symptoms of a medical condition or delay the onset of the condition in a subject compared to a subject not administered the drug.

[0040] In the present application, the term "treat" refers to alleviating, relieving or improving the symptoms of a disease or condition, improving symptoms caused by potential metabolism, inhibiting a disease or symptom, such as preventing the development of a disease or condition, alleviating a disease or condition, causing regression of a disease or condition, alleviating the condition caused by a disease or condition, or preventing the symptoms of a disease or condition.

[0041] As used in this application, the term "and / or" refers to and encompasses any and all possible combinations of one or more associated listed items. When used in a list of two or more items, the term "and / or" means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a composition, combination, construction, etc. is described as including (or comprising) components A, B, C, and / or D, the composition can include A alone; B alone; C alone; D alone; A and B in combination; A and C in combination; A and D in combination; B and C in combination; B and D in combination; C and D in combination; or A, B, C, and D in combination.

[0042] In the present application, the "one or more" means at least one; and "multiple" means a combination of any number of 2 or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the structural formula of plerixafor;

[0044] Figure 2AS patients and non-AS patients with matching age, gender, and general condition were selected from surgical patients. During the operation, bone tissues were collected from the side of the spine, followed by tissue RNA extraction and RNA high-throughput chip sequencing. (a) Schematic diagram of the sampling site; (b) Sequencing results showed differences in the nucleic acid levels of various cells and inflammatory factors, among which CXCL12 was significantly overexpressed in the ligament tissues of AS patients; (c) Volcano plot showing the differences in CXCL12; (d) Immunohistochemistry staining (IHC) showed that CXCL12 was highly expressed in the ligament specimens of AS patients.

[0045] Figure 3a In the DBA1 spontaneous inflammation ectopic osteogenesis model, HE and SOFG staining showed that obvious ectopic bone blocks appeared in the soles of mice after 20 weeks of modeling, and immunohistochemical staining showed high expression of CXCL12 in a large number of bone blocks. (CB: cortical bone, NB: new bone).

[0046] Figure 3b In the PGIS spinal fusion model, HE and SOFG staining showed that obvious ectopic bone blocks appeared in the spine of mice after 20 weeks of modeling, and immunohistochemical staining showed high expression of CXCL12 in a large number of bone blocks. (CB: cortical bone, AF: intervertebral disc fiber ring).

[0047] Figure 3c In the SMTS Achilles tendon hemisection model, HE and SOFG staining showed obvious ectopic calcification at the insertion of the Achilles tendon in mice after 12 weeks of modeling, and immunohistochemical staining showed high expression of CXCL12 in the calcified area. (SMTS: Achilles tendon hemisection; AT: Achilles tendon; UF: uncalcified fibrocartilage; CF: calcified fibrocartilage; CB: cortical bone).

[0048] Figure 4Micro-CT showed that in the DBA / 1 mouse inflammatory osteogenesis model (modeling week 32), plerixafor significantly reduced ectopic bone formation in the sole of the DBA1 mouse, and in the ectopic bone tissue, the number of Prx1+ osteoblast precursor cells in the plerixafor group was significantly reduced. (a) Micro-CT showed that in the mouse PGIS model (modeling week 32), plerixafor significantly reduced ectopic bone formation in the paradiscal ligament of the spine; (b) HE staining showed that in the mouse spinal paradiscal ligament osteogenesis model (modeling week 32), plerixafor significantly reduced ectopic bone formation in the paradiscal ligament of the spine. In the ectopic osteoblastic tissue, the number of Sca1+ osteoblast precursor cells in the plerixafor group was significantly reduced; (c) Micro-CT showed in the mouse SMTS model (12th week of modeling); (d) Plerixafor significantly reduced ectopic bone formation in the Achilles tendon of mice with hemisection of the Achilles tendon (SMTS: hemisection of the Achilles tendon); (e) HE and toluidine blue staining showed that in the mouse Achilles tendon hemisection inflammatory osteogenesis model (12th week of modeling), plerixafor significantly reduced ectopic bone formation in the Achilles tendon of mice with hemisection of the Achilles tendon (SMTS: hemisection of the Achilles tendon; AT: Achilles tendon; UF: uncalcified fibrocartilage; CF: calcified fibrocartilage; CB: cortical bone). In the ectopic osteoblastic tissue, the number of Prx1+ osteoblast precursor cells in the plerixafor group was significantly reduced.

[0049] Figure 5 Micro-CT and safranin fast green staining showed that in the Achilles tendon hemisection inflammatory osteogenesis model established in conditional CXCR4 knockout mice (modeling week 12), ectopic bone formation in the Achilles tendon of CXCR4 knockout mice was significantly reduced, and its effect was consistent with that of plerixafor. (SMTS: hemisection of the calcaneus; AT: Achilles tendon; UF: uncalcified fibrocartilage; CF: calcified fibrocartilage; CB: cortical bone). In the ectopic bone tissue, the Prx1+ osteoblast precursor cells of CXCR4 knockout mice were significantly reduced. DETAILED DESCRIPTION

[0050] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0051] Example 1 Model establishment and general experimental methods

[0052] All animal experiments were approved by the ethics committee of the applicant's institution and conducted in strict accordance with regulatory requirements.

[0053] 1. Establishment of Spontaneous Arthritis DBA / 1 Mouse Model

[0054] Male 8-week-old DBA / 1 mice (Beijing Weitonglihua Experimental Animal Company) were obtained and bred in cages of 9 mice per cage. They were divided into experimental and control groups, with 3 cages in each group.

[0055] The inflammation score was recorded and the clinical signs of arthritis were observed. The mice were scored twice a week, 0: no symptoms; 1: redness and swelling of one toe; 2: redness and swelling of more than one toe; 3: toe deformity; 4: whole foot deformity or ankle involvement. Both hind paws were evaluated, and the maximum score was 8, divided by the number of feet 2, and the average was recorded.

[0056] μCT scans were performed on the bone formation site at 8, 20, and 32 weeks, and the values ​​were recorded.

[0057] Mice were sacrificed at 8, 20, and 32 weeks, and samples were collected for sectioning and staining. For histological examination, the hind feet were fixed with formalin, decalcified using Decal (Serva), embedded in paraffin, and sectioned in the sagittal plane. Histological scores were used, 1: inflammatory cell infiltration; 2: induction of fibroblast-like cell proliferation; 3: cartilage formation; 4: bone formation; and 5: occurrence of ankylosis. In each mouse, the total score was calculated by each joint score.

[0058] 2. Establishment of the PGIS (proteoglycan-induced spondylitis, PGIS) mouse model

[0059] (1) Inducing collagen PG to prepare proteoglycan (PG) is the immune inducer of this model. PG is a protein component in human articular cartilage, which is composed of keratan sulfate end (KS), core protein (Core protein) and chondroitin sulfate end (CS). The presence of the KS end will cover some T cell markers, so it is necessary to extract the core protein part during the preparation process. The cartilage used to make PG in this experiment was derived from adult knee replacement cartilage, removing the knee joint with rheumatic disease (due to the extensive degeneration of cartilage in patients with rheumatoid arthritis). When taking the material, the subchondral bone was removed, and the cartilage was cut into thin slices after being obtained. It was frozen and ground into powder by liquid nitrogen, and PG was extracted in guanidine hydrochloride and a standard protease inhibitor formula. After centrifugation to obtain the PG stock solution, it was layered by cesium chloride ultracentrifugation to obtain six layers. The proteins of the bottom two layers were dialyzed and then freeze-dried to obtain purified PG powder. The CS end of the purified PG was removed by chondroitinase, and then the KS end was removed by β-lactose endonuclease to obtain the core protein, which was then freeze-dried for storage. (2) PG injection to induce ankylosing spondylitis The prepared PG core protein was dissolved in PBS solution and injected intraperitoneally into mice with an equal amount of Freund's adjuvant. In the first week (W0), 100ul PG and 100ul FCA (Freund's complete adjuvant, FCA) were used for intraperitoneal injection, and 100ul PG and 100ul FIA (Freund'simcomplete adjuvant, FIA) were used for intraperitoneal injection at W3 and W6. The mice were killed during the time period of spondylitis (8-16W), and the samples were taken for section staining.

[0060] 3. Establishment of the Achilles Tendon Hemisection (SMTS) Mouse Model

[0061] Male 8-week-old B6J mice were obtained (Beijing Weitonglihua Experimental Animal Company) and housed 5 mice / cage.

[0062] After anesthesia, a small incision is made above the calcaneus, the Achilles tendon is separated, 1 / 2 of the Achilles tendon is cut off, and then the incision is sutured.

[0063] 4. Cxcr4 conditional knockout mouse model strategy

[0064] This model uses CRISPR / Cas9 technology to edit the Cxcr4 gene. The Cxcr4 gene has two transcripts. According to the Cxcr4 gene structure, the Cxcr4-201 (ENSMUST00000052172.6) transcript exon2 is selected as the knockout region. The exon2 coding region is 1056bp, and the flox region is about 1.7kb. Knocking out this region is predicted to cause the Cxcr4 gene to lose its function. The present invention uses CRISPR / Cas9 technology to modify the Cxcr4 gene. The brief process is as follows: in vitro transcription of sgRNA, construction of Donor vector, microinjection of Cas9, sgRNA and Donor into the fertilized eggs of C57BL / 6J mice for homologous recombination, and obtain F0 generation mice. The positive F0 generation mice verified by PCR & sequencing, the positive F0 mice are mated with C57BL / 6J mice to obtain a stable and heritable positive F1 generation mouse model. When the flox mouse is mated with mice that tissue-specifically express Cre recombinase, its flox region is knocked out, resulting in the loss of function of the target gene in specific tissues and cell types.

[0065] 5. Plerixafor intervention

[0066] Starting from the second week after cage integration, a plerixafor physiological saline solution was prepared and injected intraperitoneally into the model mice at a dose of 1 mg / kg, 3 times a week for 4 weeks.

[0067] 6. μCT Scanning

[0068] The sample was fixed on a bracket and scanned by μCT using Scanco μCT40 scanner, and three-dimensional reconstruction and analysis were performed using MicroCT Ray V3.0 software.

[0069] 7. HE staining

[0070] Place the slices in a 37°C oven overnight, take them out and place them in a 60°C oven for 30 minutes, soak them in two portions of xylene for 10 minutes each; then soak them in 100% ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 5 minutes each, and rinse them with distilled water for 5 minutes to complete dewaxing. Place the dewaxed slices in hematoxylin for 10 minutes, rinse them with running water 3 times, and rinse them with distilled water for 2 minutes. Place them in 1% hydrochloric acid ethanol differentiation solution for 10 seconds, and rinse them with distilled water for 2 minutes. Place them in the blue staining solution for 2 minutes, and rinse them with distilled water for 2 minutes. Soak them in 95% ethanol for 1 minute and then place them in eosin staining solution for 15 seconds to complete the staining. Then place the slices in 80% ethanol, 95% ethanol, and 100% ethanol for 2 minutes each to complete the dehydration, and soak them in xylene for 5 minutes to make them transparent. Finally, seal the slices with neutral resin.

[0071] 8. SOFG (Safranin O-FastGreen) staining

[0072] Place the slices in a 37°C oven overnight, take them out and place them in a 60°C oven for 30 minutes, soak them in two portions of xylene for 10 minutes each; then soak them in 100% ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 5 minutes each, and rinse them with distilled water for 5 minutes to complete dewaxing. Place the dewaxed slices in hematoxylin for 10 minutes, rinse them with running water 3 times, and rinse them with distilled water for 2 minutes. Place them in 1% hydrochloric acid ethanol differentiation solution for 10 seconds and rinse them with distilled water for 2 minutes. Soak the slices in 0.05% fast green staining solution for 5 minutes, then soak them in 1% acetic acid for 15 seconds, and finally soak them in 0.1% safranin O staining solution for 5 minutes to complete the staining. Then place the slices in 80% ethanol, 95% ethanol, and 100% ethanol for 2 minutes each to complete the dehydration, and soak them in xylene for 5 minutes to make them transparent. Finally, seal the slices with neutral resin.

[0073] Example 2 High expression of CXCL12 at the site of ectopic new bone formation in the DBA / 1 mouse model

[0074] like Figure 3a-3c As shown, CXCL12 is highly expressed in ectopic bone formation sites in AS-related animal models (including the DBA1 spontaneous inflammatory osteogenesis model, the (proteoglycan-induced spondylitis) PGIS model, and the (semi-Achilles tendon transection) SMTS mouse model).

[0075] In the AS-related animal model DBA1 spontaneous inflammatory osteogenesis model, the expression of CXCL12 in the ectopic osteogenesis site in the plantar foot was significantly increased at 20 weeks of model construction; in the PGIS model, the expression of CXCL12 in the ectopic osteogenesis site in the paradisk ligament of the spine was significantly increased at 20 weeks of model construction; in SMTS mice, CXCL12 was highly expressed in the ectopic osteogenesis site at the origin and insertion of the Achilles tendon at 12 weeks of model construction.

[0076] Example 3

[0077] like Figure 4As shown, Micro-CT showed that in the DBA / 1 mouse inflammatory osteogenesis model (20th week of modeling), plerixafor significantly reduced ectopic bone formation in the sole of the DBA1 mouse, and in the ectopic osteoblastic tissue, the number of Prx1+ osteoblast precursor cells in the plerixafor group was significantly reduced. Micro-CT showed that in the mouse PGIS model, plerixafor significantly reduced ectopic bone formation in the paradiscal ligament of the spine. SOFG staining showed that in the mouse paradiscal ligament osteogenesis model, plerixafor significantly reduced ectopic bone formation in the paradiscal ligament of the spine. And in the ectopic osteoblastic tissue, the number of Sca1+ osteoblast precursor cells in the plerixafor group was significantly reduced. Micro-CT showed that in the mouse SMTS model, plerixafor significantly reduced ectopic bone formation in the Achilles tendon of mice with hemisection of the Achilles tendon. SOFG staining showed that in the mouse hemisection inflammatory osteogenesis model, plerixafor significantly reduced ectopic bone formation in the Achilles tendon of mice with hemisection of the Achilles tendon. In the ectopic osteoblastic tissue, the number of Prx1+ osteoblast precursor cells in the plerixafor group was significantly reduced.

[0078] Sca1+ and Prx1+ cells are both important bone marrow osteoblast precursor stem cells. Plerixafor significantly inhibited the migration and adhesion of Prx1+ osteoblast precursor cells in ectopic osteoblastic tissue, suggesting that the possible mechanism by which inhibition of CXCL12 / CXCR4 reduces ectopic osteogenesis is that it inhibits the migration and differentiation of osteoblast precursor cells to the osteogenic site.

[0079] Example 4

[0080] like Figure 5 As shown in the figure, Micro-CT and safranin fast green staining showed that in the Achilles tendon hemisection inflammatory osteogenesis model constructed in conditional CXCR4 knockout mice (modeling week 12), the ectopic bone formation in the Achilles tendon of CXCR4 knockout mice induced by Tamoxifen was significantly reduced compared with the control group mice that were not induced by Tamoxifen, and its effect was consistent with that of plerixafor. In the ectopic bone tissue, the Prx1+ osteoblast precursor cells of CXCR4 knockout mice were significantly reduced.

[0081] This indicates that the CXCL12 / CXCR4 signaling pathway can be used as a target for preventing or treating ectopic osteogenesis, and that inhibiting the receptors of this pathway can effectively inhibit heterotopic ossification.

[0082] References:

[0083] 1. Taurog JD, Chhabra A, Colbert RA. Ankylosing Spondylitis and AxialSpondyloarthritis. NEngl J Med. 2016; 375:1302-03.

[0084] 2.Ranganathan V,Gracey E,Brown M A.Pathogenesis of ankylosingspondylitis-recent advances and future directions.[J].Nat Rev Rheumatol.2017;13(6):359.

[0085] 3.Sieper J,Poddubnyy D.Axial spondyloarthritis.Lancet.2017;Jul 1,390(10089):73-84.

Claims

1. Application of CXCL12 detection reagent in the preparation of ankylosing spondylitis (AS) heterotopic ossification detection reagent.

2. The use according to claim 1, wherein the detection reagent of CXCL12 is also used to prepare a kit comprising the detection reagent for ankylosing spondylitis (AS) heterotopic ossification.

3. The use according to any one of claims 1 to 2, characterized in that: The CXCL12 detection reagent is selected from one or more of Western blot reagent, immunohistochemistry detection reagent, immunofluorescence detection reagent, ELISA detection reagent, RNA probe reagent, or nucleic acid reagent.

4. The use according to any one of claims 1 to 2, characterized in that: The CXCL12 detection reagent is selected from one or more of a Western blot kit, an immunohistochemistry detection kit, an immunofluorescence detection kit, an ELISA detection kit, an RNA probe kit, or a nucleic acid kit.

5. A detection system for heterotopic ossification of ankylosing spondylitis (AS), characterized in that: The system comprises: a. a detection component for CXCL12, and b. Result judgment system.

6. The detection system according to claim 5, characterized in that: The CXCL12 detection component contains one or more of: Western blot reagent, immunohistochemistry detection reagent, immunofluorescence detection reagent, ELISA detection reagent, RNA probe reagent, or nucleic acid reagent.

7. The detection system according to claim 5, characterized in that: The CXCL12 detection component comprises: one or more of a Western blot kit, an immunohistochemistry detection kit, an immunofluorescence detection kit, an ELISA detection kit, an RNA probe kit, or a nucleic acid kit.

8. The detection system according to claim 5, characterized in that: The result judgment system is used to output the risk of heterotopic ossification according to the CXCL12 expression results detected by the detection system.

9. The detection system according to claim 8, characterized in that: The disease risk is based on comparing the CXCL12 expression levels of the test sample with those of the normal sample through the results. When the CXCL12 nucleic acid level and / or protein expression of the test sample and the normal sample are significantly different or extremely significantly different, the results judge that the disease risk of the test sample is high.

10. The detection system according to claim 9, characterized in that: When the CXCL12 nucleic acid level and / or protein expression of the test sample is greater than 2.5 times that of the normal sample, the result indicates that the test sample has a high risk of disease.

11. Application of CXCL12 / CXCR4 signaling pathway as a target in the preparation of drugs for the treatment or prevention of heterotopic ossification in ankylosing spondylitis (AS).

12. Use of CXCL12 / CXCR4 signaling pathway inhibitors in the preparation of drugs for treating or preventing heterotopic ossification in ankylosing spondylitis (AS).

13. The use according to claim 12, characterized in that The CXCL12 / CXCR4 signaling pathway inhibitor is selected from a CXCL12 inhibitor or a CXCR4 inhibitor.

14. The use according to claim 13, characterized in that The CXCL12 inhibitor is selected from substances that inhibit CXCL12 activity, substances that degrade CXCL12, or substances that reduce CXCL12 levels.

15. The use according to claim 14, characterized in that The substance that inhibits the activity of CXCL12 is selected from one or more of CXCL12 neutralizing antibodies and chemical antagonists.

16. The use according to claim 13, characterized in that The CXCR4 inhibitor is selected from substances that inhibit CXCR4 activity, substances that degrade CXCR4, or substances that reduce CXCR4 levels.

17. The use according to claim 16, characterized in that The substance that inhibits CXCR4 activity is selected from diazabicyclic compounds, and their derivatives, optical isomers, chelated forms or hydrated forms.

18. The use according to claim 17, characterized in that The diazabicyclic compound is selected from one or more of plerixafor, AMD3329, or pharmaceutically acceptable salts or esters.

19. The use according to claim 18, characterized in that The plerixafor has a structural formula shown in Formula I: 。 20. The use according to claim 18, characterized in that The pharmaceutically acceptable salt of plerixafor is selected from plerixafor octahydrochloride.

21. The use according to claim 20, characterized in that The plerixafor octahydrochloride has the structural formula shown in Formula II: 。 22. The use according to any one of claims 11 to 13, characterized in that: The dosage form of the drug is selected from any pharmaceutically permitted dosage form.

23. The use according to claim 22, characterized in that The dosage form is selected from one or more of tablets, granules, capsules, pills, oral solutions, injections, and liposomes.

24. The use according to claim 23, characterized in that The administration mode of the drug or pharmaceutical composition is selected from one or more of oral administration, intranasal administration, systemic administration, intravenous administration, subcutaneous administration, intramuscular administration, intracerebroventricular administration, intrathecal administration or transdermal administration.

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