Medicine for promoting fracture healing and application thereof

Abstract

The invention discloses a medicine for promoting fracture healing and application thereof, relates to the field of biology and medicine, and solves the technical problems that in the prior art, a fracture patient has complications, cannot heal normally and causes heavy burden to the patient, and the existing medicine is weak in effect and large in side effect. The effective component of the medicine for promoting fracture healing is a high-selectivity inhibitor TMG with OGA, the CAS number is 1009816-48-1, the molecular formula is C9H16N2O4S, the molecular weight is 248.299, and the medicine has the performance of increasing the O-GlcNAc glycosylation modification level. The TMG is locally injected at the fracture position to promote fracture healing, including increase of callus formation and promotion of mineralization degree, the process is accompanied by increase of the number of osteoclasts at the same time, an experimental mouse does not show abnormal reaction, the medicine is non-toxic to main organs, and it is indicated that the TMG can be applied to promotion of fracture healing and has a good application prospect. And a new treatment target is provided for fracture healing delay.

Description

Technical Field

[0001] The present invention relates to the fields of biology and medicine, and specifically relates to a drug for promoting fracture healing and its application. Background Art

[0002] Fracture is the most common traumatic injury of large organs in humans. Approximately 5%-10% of fracture patients will develop complications and cannot heal normally, imposing a heavy burden on patients. Currently, the most common clinical fracture treatment methods are surgical interventions, including external fixation, internal fixation, bone grafting (autologous bone, allogeneic bone, and bone graft substitutes), etc. There are also non-surgical therapies to accelerate normal physiological fracture healing, such as low-intensity pulsed ultrasound, subcutaneous injection of the parathyroid hormone analogue Teriparatide, local injection of Bone morphogenetic protein (BMP), etc. Among them, the US Food and Drug Administration approved BMP-7 for the treatment of refractory long bone nonunion in 2001 and BMP-2 for the treatment of acute open tibial fractures in 2004. Some studies have confirmed that injecting autologous mesenchymal stem cells (MSCs) at the nonunion site can promote bone healing. Bone marrow is extracted from the patient's iliac crest to obtain MSCs, which are concentrated in vitro and then injected into the same patient to effectively treat nonunion; the combination of plaster fixation or intramedullary nailing with MSCs injection also has the same promoting effect.

[0003] Although the above-mentioned surgical treatments, drug injections and other means can cure fractures to a certain extent, there are still many problems, such as surgical complications and the risk of postoperative infection; BMP is expensive and its efficacy is unstable. Some studies have shown that BMP-2 cannot significantly accelerate the closed or open tibial fractures fixed with intramedullary nails; there are also adverse reactions and potential side effects of drugs on the body. For example, some studies have shown that the cancer risk is slightly higher when using BMP-2. Although vitamin D and calcium supplements can increase bone density and reduce the risk of fractures, they also increase the risk of kidney stones; stem cell therapy is highly technically sensitive and costly. Therefore, seeking more efficient and safe drugs for fracture healing treatment remains the research focus in this field. Summary of the Invention

[0004] In view of the deficiencies and drawbacks existing in the prior art, the present invention provides an efficient and safe drug for promoting fracture healing and its application.

[0005] To achieve the above object, the present invention is realized through the following technical solutions: The drug for promoting fracture healing provided by the present invention has an active ingredient of Thiamet-G (TMG), a highly selective inhibitor of OGA, with a CAS number of 1009816-48-1, molecular formula: C9H16N2O4S, and molecular weight: 248.299.

[0006] Preferably, it has the property of increasing the level of O-GlcNAc glycosylation modification.

[0007] Preferably, the dosage form is an injection.

[0008] For the application of the above-mentioned drug for promoting fracture healing, TMG is intramuscularly injected locally at the fracture site, where the dosage of TMG is 5 mg / kg / day; the specific injection method is: starting from the first day after the fracture, inject continuously for 10 - 14 days.

[0009] The application of the above-mentioned drug for promoting fracture healing is used for treating fractures or for promoting fracture healing.

[0010] The present invention provides a drug for promoting fracture healing, its preparation and application. It has the following beneficial effects:

[0011] (1) The present invention first discovers that using the small molecule inhibitor TMG of OGA to increase the level of O-GlcNAc glycosylation can be used for treating fractures, promoting fracture healing and has good biosafety.

[0012] Fracture healing is a complex and continuous process that requires the regulation of multiple signaling molecules and cell - cell communication. Post - translational modification (PTM) of proteins plays an important role. Glycosylation is a complex and diverse PTM that involves complex metabolic networks and different types of glycosylation pathways. Among them, most nuclear and cytoplasmic proteins undergo dynamic O - linked N - acetylglucosamine (O - GlcNAc) modification. This modification adds or removes O - GlcNAc modification on serine or threonine residues of target proteins through a pair of specific enzymes, O - GlcNAc transferase (OGT) and O - GlcNAcase (OGA). OGT and OGA maintain the dynamic balance of intracellular O - GlcNAc modification levels, which is crucial for cells to respond to changes in the external environment and maintain normal physiological functions. The research of this invention shows that O - GlcNAc glycosylation affects bone homeostasis. In human knee osteoarthritis, the O - GlcNAc glycosylation modification of cartilage proteins is dysregulated. The pro - inflammatory environment changes the expression of OGT and OGA subtypes, and the proteins with O - GlcNAc glycosylation accumulate abnormally. Using the OGA inhibitor TMG to increase the level of O - GlcNAc in MC3T3 - E1 cells increases the expression of osteocalcin and promotes osteogenic differentiation.

[0013] (2) TMG is a highly selective inhibitor of OGA. By inhibiting OGA, the O - GlcNAc level of intracellular proteins can be increased, and the O - GlcNAc glycosylation level in vertebrates can be improved.

[0014] This invention first discovers the existence of O - GlcNAc glycosylation modification during the fracture healing process. Injecting TMG to increase the overall O - GlcNAc glycosylation modification level at the fracture site in the early stage can promote fracture healing by increasing callus formation, promoting bone regeneration, and increasing the degree of callus mineralization. This process is accompanied by an increase in the number of osteoclasts, so it has good application prospects in the treatment of bone repair - related diseases such as delayed fracture healing.

[0015] This invention proves the new use of the TMG drug and provides a drug for promoting fracture healing. The TMG analogues obtained through structural modification have great potential clinical application and therapeutic value for the treatment of fractures.

[0016] (3) In addition, prior art has proved that the application of TMG in vivo has no obvious organ toxicity, so it has high biological safety when used for the treatment of fractures. Description of the Drawings

[0017] Figure 1 It is the immunohistochemical staining result diagram of RL2 after femoral fracture in mice. In the diagram, area 1 is marked as the hard callus area; area 2 is marked as the soft callus area; area bs is marked as bone surface; area bm is marked as bone marrow cavity; area c is marked as chondrocyte; area hc is marked as hypertrophic chondrocyte. The scales are 100μm and 20μm respectively;

[0018] Figure 2 It is the chemical structural formula of Thiamet G (TMG);

[0019] Figure 3 It is the schematic diagram of continuous injection of TMG in mice;

[0020] Figure 4 It is the data diagram of Micro-CT reconstruction and analysis of fracture healing after continuous injection of TMG. Among them, (A)-(D): respectively represent the three-dimensional reconstruction diagrams of the callus 10 days and 14 days after drug injection after fracture; (E)-(H): respectively represent the BMD and BV / TV analysis results of the callus 10 days and 14 days after drug injection after fracture, and the threshold is 200; t-test is used for statistical analysis, n = 3, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001. The scale is 1mm.

[0021] Figure 5 It is the Masson staining diagram of the callus after continuous injection of TMG. Among them, it is the Masson staining result of the callus 10 days after injection of TMG; the dotted area indicates the callus formation area, and the scales are 500μm respectively.

[0022] Figure 6 It is the TRAP staining diagram of the callus after continuous injection of TMG. Among them, it is the TRAP staining result of the callus 10 days after injection of TMG; the dotted area indicates the callus formation area; the scales are 500μm and 20μm respectively, and the number of TRAP cells per unit area + The number of cells is statistically analyzed by t-test, n = 3.

[0023] Figure 7 It is the HE staining result diagram of the main organs (heart, liver, spleen, lung, kidney) after injection of TMG (the scale is 50μm) 。 Detailed implementation manners

[0024] The present invention will be elaborated in detail below in combination with the accompanying drawings and embodiments. Operations not described in the embodiments are all carried out according to the conventional operations in the art, and reagents not described are all conventional commercially available reagents.

[0025] Example 1. Experimental proof of the existence of O-GlcNAc glycosylation modification during fracture healing

[0026] To investigate the expression of O-GlcNAc glycosylation modification during fracture healing, a mouse fracture model was constructed. Femoral samples of the fractured bones were collected on the 7th, 10th, and 14th days after surgery, and the overall O-GlcNAc glycosylation modification level in the callus area was detected using the anti-O-GlcNAc antibody RL2.

[0027] Intramedullary pin fixation model for open transverse femoral fracture in mice:

[0028] Mice were anesthetized by intraperitoneal injection (0.2 - 0.3 mL / 10 g body weight) of 12 mg / mL avertin solution (preparation of 10 mL avertin solution: dissolve 0.12 g tribromoethane in 9.88 mL deionized water containing 0.12 mL tert-amyl alcohol) to provide approximately 60 min of deep anesthesia. The right hindlimb was depilated and prepared for surgery, and scrubbed with 5% povidone iodine solution. The knee joint position was determined in the flexed state of the right limb. A 5-mm longitudinal skin and muscle incision was made on the right hindlimb of the mouse, and the muscle was directly dissected to fully expose the lateral side of the femur. A transverse fracture was made at the midpoint of the femoral shaft using an anatomical scissors, and a sterile needle with a diameter of 0.6 mm was inserted from the intercondylar fossa to connect the fracture ends. After the needle reached the femoral head, the needle was clamped off at the plane of the intercondylar fossa. The muscle and skin were sutured in layers with 5-0 absorbable sutures. At the experimental design time, the mice were sacrificed by cervical dislocation, and the femoral samples were collected and fixed in 4% paraformaldehyde at 4 °C for pathological specimen preparation.

[0029] Experimental methods, results, and analysis of callus histology:

[0030] Sectioning and baking: The paraffin-embedded tissue was sectioned continuously at 5 μm, and baked at 65 °C (in an electrothermal constant temperature drying oven, the same below) for 2 h.

[0031] Immunohistochemical staining:

[0032] 1) Dewaxing to water: Bake the slices for 1 h, xylene Ⅰ for 10 min → xylene Ⅱ for 10 min → absolute ethanol Ⅰ for 5 min → absolute ethanol Ⅱ for 5 min → 95% ethanol for 5 min → 85% ethanol for 5 min → wash repeatedly with tap water.

[0033] 2) Antigen retrieval: Immerse the slices in the antigen retrieval solution, place them in a 95 °C water bath for 10 min, then let them cool naturally to room temperature, and wash 3 times with PBS, 5 min each time;

[0034] 3) Permeabilization: Immerse the slices in PBST containing 0.5% Triton-100 for 10 min, and wash 3 times with PBS, 5 min each time;

[0035] 4) Use a histochemical pen to circle the tissue area. After blotting dry the moisture, block endogenous peroxidase and incubate at room temperature for 10 min, wash 3 times with PBS, 3 min each time;

[0036] 5) Dropwise add 5% BSA and block at room temperature for 30 min;

[0037] 6) Primary antibody incubation: Place the sections in a wet box and incubate with the primary antibody (RL2, diluted 1:200) at 4°C overnight;

[0038] 7) Remove the primary antibody, wash 3 times with PBS, 5 min each time;

[0039] 8) Dropwise add the reaction enhancer solution and incubate at 37°C for 20 min, wash 3 times with PBS, 3 min each time;

[0040] 9) Secondary antibody incubation: Incubate at 37°C for 30 min, wash 3 times with PBS, 3 min each time;

[0041] 10) Develop color with DAB working solution and observe under a microscope;

[0042] 11) Terminate color development: Wash 3 times with PBS, 5 min each time;

[0043] 12) Counterstain with hematoxylin for 1 min, rinse with running water, blue with ammonia water for 10 s, and rinse with tap water;

[0044] 13) Dehydrate with gradient alcohol, clear in xylene I and II for 2 min each, mount with neutral gum, cover the slide and examine under a microscope.

[0045] As Figure 1 shown, the immunohistochemical staining results of RL2 after mouse femoral fracture surgery. The immunohistochemical staining results show that during the normal healing process, callus has O-GlcNAc glycosylation modification. In the hard callus area, there is weak positive expression on the surface of the newly formed bone; while in the newly formed bone marrow cavity, there is strong positive expression; in the cartilage callus area, the O-GlcNAc glycosylation modification level in chondrocytes and hypertrophic chondrocytes is relatively low and can hardly be labeled by RL2.

[0046] Example 2. Experimental proof that local injection of TMG can promote fracture healing

[0047] In this example, the effect of TMG, with the structural formula as Figure 2 shown, in the treatment of fracture healing was studied. The used TMG has a CAS number of 1009816-48-1, was purchased from APE×BIO Company, stored at a concentration of 20 mg / mL in DMSO (purchased from Sigma), and diluted to a concentration of 5 mg / mL when used.

[0048] Observation of healing in a fracture model by local injection of TMG: 8-week-old male C57BL / 6J mice were subjected to fracture modeling and then divided into 2 groups, namely the experimental group (TMG) and the control group (DMSO solvent). In the experimental group, 5 mg / kg TMG was locally injected into the muscle at the fracture site, and in the control group (DMSO), the same volume of DMSO solvent was injected once a day for 10 or 14 consecutive days. Then the mice were sacrificed by cervical dislocation, and the fracture segments were taken for Micro-CT scanning and reconstruction to analyze callus; Masson staining and tartrate-resistant acid phosphatase (TRAP) staining were used to explore the therapeutic effect of O-GlcNAc glycosylation modification on the healing of femoral fractures in mice. The specific operation steps are as follows:

[0049] First, 8-week-old male C57BL / 6J mice were established with a fracture model according to Example 1 and divided into two major groups according to the injection duration, namely continuous injection for 10 days and 14 days. In each group, equal volumes of DMSO and TMG (5 mg / kg / day) were intramuscularly injected at the fracture site for the control group and the experimental group respectively starting from the first day after surgery. Samples were collected on the 10th and 14th days after fracture, as Figure 3 shown.

[0050] I. Experimental methods, results and analysis of bone histomorphometry of callus:

[0051] Micro-CT scanning and reconstruction analysis:

[0052] 1) Micro-CT scanning: The key parameters for femoral scanning using a μCT 45 desktop Micro-CT scanner (Scanco Medical AG, USA) were as follows: voxel size (13.1 μm 3 ), X-ray tube potential (55 kVp), X-ray intensity (145 μA), integration time (250 ms).

[0053] 2) Micro-CT reconstruction: The most swollen part of the callus was selected as the fracture center according to the cross-section. A circle was drawn counterclockwise along the outer side of the callus, and a circle was drawn clockwise along the outer side of the cortical bone. The volume within the two circles was the callus volume. Cross-sections were selected on both sides until the outer callus image disappeared, and three-dimensional reconstruction of the callus was performed with a threshold of 200; a circle was drawn counterclockwise along the outermost side of the callus, and a circle was drawn counterclockwise along the outermost side of the cortical bone where there was no callus, and three-dimensional reconstruction of the entire femur was performed with a threshold of 200.

[0054] 3) Micro-CT analysis: Select the thickest part of the callus as the fracture center according to the cross-section. Draw a counterclockwise circle along the outside of the callus and a clockwise circle along the outside of the cortical bone. The volume within the two circles is the callus volume. Select cross-sections on both sides until the image of the outer callus disappears, and analyze the bone mineral density (BMD) and bone volume fraction (BV / TV) of the callus, with a threshold of 200.

[0055] Perform Micro-CT reconstruction analysis on the callus samples of mice continuously injected with the small molecule inhibitor TMG of OGA for 10 days and 14 days after fracture. The three-dimensional Micro-CT reconstruction images show that 10 days after fracture, the control group is still in the soft callus stage, while increasing O-GlcNAc glycosylation modification, the callus at the distal end of the fracture in the TMG group has begun to mineralize, as Figure 4 A, Figure 4 B; 14 days after fracture, the control group also shows that the soft callus begins to mineralize and the fracture ends are still obvious. While increasing O-GlcNAc glycosylation modification, the degree of callus mineralization in the TMG group is increased. The reconstruction images show that the fracture ends are covered by mineralized hard callus, as Figure 4 C, Figure 4 D. The Micro-CT bone tissue parameter analysis is shown in the figure. 10 days after fracture, increasing O-GlcNAc glycosylation modification, both BMD and BV / TV of the callus increase, as Figure 4 E, Figure 4 F, with statistical significance (p < 0.05); 14 days after fracture, increasing O-GlcNAc glycosylation modification, BMD increases but without statistical significance, BV / TV increases, with statistical significance (p < 0.05), as Figure 4 G, Figure 4 H. The above data results indicate that increasing O-GlcNAc glycosylation modification in the early stage of fracture healing promotes the mineralization of soft callus.

[0056] II. Experimental methods, results and analysis of callus morphology research:

[0057] Masson staining:

[0058] The steps of baking the slides and dewaxing and rehydrating are the same as in Example 1. The remaining steps are strictly operated according to the instructions of the Masson staining kit. The specific steps are as follows:

[0059]

[0060] Dehydration, clearing, and mounting: Perform in the following order successively, 95% ethanol (3 s) → 100% ethanol I and II (each 5 s) → xylene I and II (each 1 min), seal with neutral gum, and take pictures.

[0061] TRAP staining:

[0062] The steps of baking the slices, dewaxing and rehydrating are the same as those in Example 1. The remaining steps are operated according to the instructions of the TRAP staining kit. The specific steps are as follows:

[0063] 1) Fix with the pre-cooled TRAP fixative at 2°C - 8°C for 1 min;

[0064] 2) Wash with ddH2O, drain off the excess water, and pay attention not to dry it too much;

[0065] 3) Prepare the TRAP incubation solution and use it immediately after preparation;

[0066] 4) Drop the TRAP incubation solution to cover the slice samples, place them in a wet box, incubate at 37°C for 1 h, and wash with distilled water;

[0067] 5) Counterstain with methyl green staining solution for 1 min and wash slightly with ddH2O;

[0068] After slightly air-drying, seal the slices with an aqueous mounting medium, observe and take pictures.

[0069] TMG was continuously injected for 10 days after fracture. The results of Masson staining showed that increasing O-GlcNAc glycosylation modification would increase the cartilage components in the callus, as Figure 5 shown. The results of TRAP staining showed that increasing O-GlcNAc glycosylation modification would increase the TRAP-positive cells in the callus, as Figure 6 shown. Increasing O-GlcNAc glycosylation modification would promote bone formation, increase the mineralization degree of the callus, and promote fracture healing.

[0070] III. In vivo biosafety evaluation of TMG

[0071] After the local injection treatment of TMG was completed, the main organs of the mice (heart, liver, spleen, lung and kidney) were taken, fixed with 4% paraformaldehyde by volume for 24 h, dehydrated, embedded, made into tissue wax blocks, and the cell morphology of each group was observed by HE staining method to analyze its pathological changes.

[0072] HE staining:

[0073] The steps of baking the slices, dewaxing and rehydrating are the same as those in Example 1 → stain with hematoxylin solution for 5 min → wash off the floating color with ddH2O → differentiate with the differentiating solution for 30 s, immerse in tap water 2 times, 3 min / time → eosin for 30 s, pour off the excess staining solution, wash off the floating color → 95% alcohol Ι for 1 min → 95% alcohol ΙΙ for 1 min → absolute alcohol Ι for 1 min → absolute alcohol ΙΙ for 1 min → seal with neutral gum and examine under the microscope.

[0074] HE staining showed that, as Figure 7In each treatment group, no obvious pathological changes were observed in the main organs (heart, liver, spleen, lung, and kidney) of the mice. After the treatment, the mice were observed for any abnormalities, indicating that the drug has good in vivo safety when used for fracture treatment.

[0075] In summary, the present invention proves that O-GlcNAc glycosylation modification exists during the fracture healing process. The drug provided for promoting fracture healing, the reason for the significant application effect of TMG in treating fractures and promoting fracture repair is that TMG can increase O-GlcNAc glycosylation modification. Local injection of TMG at the fracture site has a promoting effect on fracture healing, including increasing callus formation and promoting its mineralization degree. This process is accompanied by an increase in the number of osteoclasts, and no abnormal reactions were observed in the experimental mice. This drug is non-toxic to the main organs, indicating that TMG can be applied to promote fracture healing and provide a new treatment target for delayed fracture healing.

[0076] The above are only examples of the present invention. For example, the application of a highly selective inhibitor TMG of OGA as the active ingredient in treating bone repair-related diseases such as delayed fracture healing belongs to the protection scope of the present invention.

[0077] The above is only the preferred specific implementation manner of the present invention. However, the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered by the protection scope of the present invention.

Claims

1. A drug for promoting fracture healing, characterized in that: The active ingredient is Thiamet-G (TMG), a highly selective inhibitor of OGA, with CAS No. 1009816-48-1, molecular formula: C9H16N2O4S, and molecular weight: 248.

299.

2. A drug for promoting fracture healing according to claim 1, characterized in that: It has the performance of increasing the level of O-GlcNAc glycosylation modification.

3. A drug for promoting fracture healing according to claim 1, characterized in that: The dosage form is injection.

4. The use of a drug for promoting fracture healing according to claim 3, characterized in that: TMG was injected locally into the muscle at the fracture site, with a TMG dosage of 5 mg / kg / day. The specific injection method was: starting from the first day after the fracture and continuing for 10-14 days.

5. The use of a drug for promoting fracture healing according to claim 1, characterized in that: Used to treat fractures or to promote fracture healing.

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