Use of small molecule compounds containing disulfide bonds for the preparation of a medicament for the treatment of AD-HIES-mediated bone development disorders

Abstract

This invention relates to the application of a disulfide-containing small molecule compound in the preparation of a drug for treating AD-HIES-mediated osteodevelopmental diseases. The structural formula of the small molecule compound is shown in Formulas I to III. Targeting key cell types in the bone development process, this invention discovers a new STAT3 agonist that is more effective than the existing agonist, and its synthetic route is easier than that of colivelin, providing a new perspective for targeted treatment of osteoporosis in AD-HIES patients.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of a small molecule compound containing disulfide bonds in the preparation of a medicament for treating AD-HIES-mediated bone developmental disorders. Background Technology

[0002] AD-HIES (autosomal dominant HIES) is an autosomal dominant genetic disorder primarily caused by mutations in the signal transduction and transcription activator 3 (STAT3) gene. Typical AD-HIES patients present with recurrent staphylococcal abscesses of the skin, lungs, joints, and internal organs. Sinusoidal lung infections, pneumopulmonary edema, and severe pruritic eosinophilic dermatitis may also be present. Patients exhibit coarse facial features, delayed loss of primary teeth, osteopenia, and recurrent fractures.

[0003] Treatment for AD-HIES includes continuous prophylactic anti-staphylococcal antibiotic therapy (usually trimethoprim / sulfamethoxazole), with dosage adjustments made according to the patient's condition. Skin moisturizing, emollient creams, and antihistamines are used to treat dermatitis. Early treatment of lung infections with aggressive antibiotic therapy is also crucial. According to current guidelines, patients without primary immunodeficiency should be screened for and treated for significant osteopenia; patients with skeletal abnormalities such as scoliosis or fractures require orthopedic surgery. The current treatment methods have the following drawbacks: antibiotics against staphylococci are prone to developing resistance; treatment of skeletal abnormalities is only symptomatic, with generally limited efficacy and low patient acceptance due to the use of conventional drugs and surgical procedures. Therefore, there is an urgent need to develop a radical cure strategy targeting the abnormal genes.

[0004] Osteopenia is one of the most significant manifestations of skeletal abnormalities in patients with AD-HIES. Currently, there are no medication guidelines specifically for osteoporosis in AD-HIES; treatment is limited to treating osteoporosis. Previous studies have shown that the STAT3 agonist colivelin can effectively treat osteoporosis caused by STAT3 heterozygosity. Colivelin, the most widely used STAT3 agonist, is a neuroprotective peptide with brain permeability that inhibits neuronal death in vitro by activating STAT3. However, commercially available colivelin is expensive and difficult to synthesize.

[0005] Bone development can be traced back to three sources from an embryological perspective: cranial neural crest cells, paraaxial mesoderm, and somatic mesoderm. Cranial neural crest cells form some of the flat bones of the skull and clavicle; cells from the paraaxial mesoderm develop into the axial skeleton; and cells from the somatic mesoderm primarily form long bones. Bone development is a continuous process of osteogenesis and ossification. This process begins in the sixth to seventh week of embryonic development and continues throughout the entire growth and development process, ultimately forming a mature skeleton through a series of complex processes.

[0006] Osteoblastic lineage cells include osteoblasts, osteocytes, and chondrocytes. These cells secrete extracellular matrix proteins such as type I collagen (COL1), osteopontin, osteocalcin (OCN), and alkaline phosphatase (ALP). Calcium and COL1 deposited in the form of hydroxyapatite promote the continuous mineralization of osteoid and the formation of osteoblasts. Osteoblast differentiation involves multiple steps from stem cells to mature osteoblasts. Current research suggests that mesenchymal stem cells (MSCs) and skeletal stem cells (SCCs) are two sources, but the relationship between these two cell populations remains to be clarified.

[0007] MSCs are a class of self-renewing and multi-lineage cells capable of differentiating into osteoblasts, adipocytes, and chondrocytes. Currently, MSCs are identified based on their surface markers and their ability to form colonies. Surface markers of human MSCs include positive markers such as CD105, CD73, and CD90, and negative markers of hematopoietic and endothelial cell lineages such as CD45, CD34, CD14 / CD11b, CD79α / CD19, and HLA-DR. [1] Osteogenesis and adipogenesis of MSCs are regulated by key signaling pathways and transcription factors. The microenvironment surrounding MSCs, composed of specific extracellular matrix components, growth factors, cytokines, and chemokines, activates a sophisticated regulatory network. During osteoogenesis, signaling pathways primarily utilize SRY-box containing gene 9 (Sox9), runt-related transcription factor 2 (Runx2), and zinc finger protein 7 (osterix / SP7, Osx) as major transcription factors. Sox9 is the earliest expressed transcription factor in the anterior mesenchyme and regulates MSC differentiation into chondrocytes. Runx2 is an early determinant of MSC differentiation into immature osteoblasts while inhibiting MSC differentiation into adipocyte lineages. Osx can induce MSC differentiation into mature and functional osteoblasts, and further differentiate from osteoblasts into osteocytes during bone formation.

[0008] Research found [2]Heterozygia and loss of function of STAT3 can lead to hyper-IgE syndrome (HIES), also known as Jobs syndrome. Its clinical symptoms are mainly manifested in the skeletal system, including osteoporosis, recurrent fractures, scoliosis, and premature closure of cranial sutures.

[0009] Numerous studies have already explored the key regulatory role of Stat3 in bone development using animal models. [3-5] Zebrafish are a well-established animal model for studying musculoskeletal system diseases because their osteoblasts and osteoclasts, as well as their precursor cell lineages and cellular markers, are highly conserved compared to humans. Furthermore, all functional domains of the zebrafish Stat3 protein are preserved, and its sequence and upstream / downstream signaling pathways are well conserved. Sobah et al. [3] By constructing and observing zebrafish mutants with complete Stat3 knockout, it was found that the growth rate of the knockout mutants was significantly restricted, and spinal deformities were observed. Specifically, the clavicle, operculum, cercarial bone, and cercarial bone were all significantly smaller, and the expression of key osteogenic markers Runx2b, Col10a, and Spp1 was reduced. Stat3-mediated Runx2 expression is crucial for osteoblast differentiation; therefore, Stat3 is essential for osteoblast maturation and function during bone development. Complete Stat3 knockout in mice leads to embryonic lethality; therefore, osteoblast / osteoclast / chondrogenic lineage-specific knockout is often used in mouse models to study the function of Stat3 in bone development and bone-related diseases. Yadav et al. [4] Studies have found that Stat3 inactivation in mice leads to severely impaired osteoblast differentiation. Whole-genome analysis, transcriptome analysis, and histomorphological results indicate that Stat3 inactivation in mice results in downregulation of the osteogenic-related signaling pathway Wnt / β-catenin. Activation of the Wnt / β-catenin signaling pathway can largely rescue the osteoporotic phenotype induced by Stat3 inactivation. This reveals that Stat3 plays an important role in early-stage mesenchymal cells or osteoprogenitor cells. In addition, Yadav et al. [4] Studies have shown that bone defects in Jobs syndrome are caused by reduced Stat3 activity during bone development, leading to impaired Wnt / β-catenin signaling. Activating Wnt / β-catenin signaling may be a treatment approach to alleviate skeletal symptoms in patients with Jobs syndrome. Zhou et al. [5] Further research has validated the crucial role of Stat3 in bone development and osteogenic processes. Stat3 deficiency impairs osteogenic differentiation of mesenchymal progenitor cells both in vivo and in vitro. These studies demonstrate that Stat3 regulates bone development by promoting osteogenic processes. Therefore, drugs targeting the STAT3 pathway hold promise for treating osteoporosis associated with high IgE syndrome.

[0010] The references are as follows:

[0011] [1] Dominici, M. et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8, 315–317 (2006).

[0012] [2] Minegishi, Y. et al. Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome. Nature 448, 1058–1062 (2007).

[0013] [3] Sobah, M. L., Liongue, C. & Ward, A. C. Contribution of Signal Transducer and Activator of Transcription 3 (STAT3) to Bone Development and Repair. Int. J. Mol. Sci. 25, 389 (2023).

[0014] [4] Yadav, P. S. et al. Stat3 loss in mesenchymal progenitors causes Job syndrome-like skeletal defects by reducing Wnt / β-catenin signaling. Proc. Natl. Acad. Sci. U.S.A. 118, e2020100118 (2021).

[0015] [5] Zhou, S. et al. STAT3 is critical for skeletal development and bone homeostasis by regulating osteogenesis. Nat. Commun. 12, 6891 (2021). Summary of the Invention

[0016] The purpose of this invention is to provide the application of a disulfide-containing small molecule compound in the preparation of a drug for treating AD-HIES-mediated bone developmental disorders. Targeting key cell types in bone development, this invention discovers a novel STAT3 agonist that is more effective than existing agonists, and its synthetic route is easier than that of colivelin, providing a completely new perspective for targeted treatment of osteoporosis in AD-HIES patients.

[0017] The objective of this invention can be achieved through the following technical solutions:

[0018] The first objective of this invention is to provide the use of a disulfide-containing small molecule compound in the preparation of a medicament for treating AD-HIES-mediated bone developmental disorders, wherein the structural formula of the small molecule compound is shown in Formulas I to III.

[0019]

[0020] Furthermore, the bone development disorders include osteoporosis, recurrent fractures, scoliosis, and craniosynostosis.

[0021] Furthermore, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

[0022] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0023] Furthermore, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0024] Furthermore, the drug can be administered via one or more of the following methods: intravenous injection, subcutaneous injection, intramuscular injection, oral administration, topical application, and transdermal absorption.

[0025] A second objective of this invention is to provide the use of a disulfide-bonded small molecule compound in the preparation of a medicament for treating bone development disorders by activating the STAT3 signaling pathway, wherein the structural formula of the small molecule compound is shown in Formulas I to III.

[0026]

[0027] Furthermore, the bone development disorders include osteoporosis, recurrent fractures, scoliosis, and craniosynostosis.

[0028] Furthermore, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

[0029] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0030] Furthermore, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0031] Furthermore, the drug can be administered via one or more of the following methods: intravenous injection, subcutaneous injection, intramuscular injection, oral administration, topical application, and transdermal absorption.

[0032] A third objective of this invention is to provide the use of a disulfide-bonded small molecule compound in the preparation of a medicament for treating bone development disorders by promoting the proliferation and differentiation of osteoblasts, wherein the structural formula of the small molecule compound is shown in Formulas I to III.

[0033]

[0034] Furthermore, the bone cells include osteoblasts and bone resorbing cells.

[0035] Furthermore, the bone development disorders include osteoporosis, recurrent fractures, scoliosis, and craniosynostosis.

[0036] Furthermore, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

[0037] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0038] Furthermore, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0039] Furthermore, the drug can be administered via one or more of the following methods: intravenous injection, subcutaneous injection, intramuscular injection, oral administration, topical application, and transdermal absorption.

[0040] The fourth objective of this invention is to provide the application of a disulfide-bonded small molecule compound in the preparation of a medicament for treating bone development disorders by promoting osteogenic differentiation of osteocytes and enhancing osteogenic activity, wherein the structural formula of the small molecule compound is shown in Formulas I to III.

[0041]

[0042] Furthermore, the bone development disorders include osteoporosis, recurrent fractures, scoliosis, and craniosynostosis.

[0043] Furthermore, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

[0044] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0045] Furthermore, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0046] Furthermore, the drug can be administered via one or more of the following methods: intravenous injection, subcutaneous injection, intramuscular injection, oral administration, topical application, and transdermal absorption.

[0047] The fifth objective of this invention is to provide the use of a disulfide-bonded small molecule compound in the preparation of a medicament for treating bone development disorders by increasing the expression levels of osteogenic genes Alp, Col1a1, Ocn, Sp7, and Runx2 mRNA, wherein the structural formula of the small molecule compound is shown in Formulas I to III.

[0048]

[0049] Furthermore, the bone development disorders include osteoporosis, recurrent fractures, scoliosis, and craniosynostosis.

[0050] Furthermore, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

[0051] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0052] Furthermore, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0053] Furthermore, the drug can be administered via one or more of the following methods: intravenous injection, subcutaneous injection, intramuscular injection, oral administration, topical application, and transdermal absorption.

[0054] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0055] 1. This invention utilizes high-throughput screening technology to rapidly evaluate the impact of a large number of compounds on STAT3 activity within a small molecule drug library containing disulfide bonds, thereby identifying potential STAT3 agonists. High-throughput screening technologies include cell fluorescence detection, enzyme activity assays, and biosensors.

[0056] 2. This invention uses structural biology techniques to resolve the structure of the STAT3 protein, particularly the binding sites of STAT3 with its ligands or regulatory factors. This facilitates the design of agonists with higher selectivity and affinity.

[0057] 3. This invention uses molecular dynamics simulations to simulate the interaction between STAT3 protein and agonists, thereby gaining a better understanding of their binding patterns and mechanisms of action.

[0058] 4. After drug synthesis, the present invention completes the initial screening through in vitro bone biology experiments, selects three highly effective drugs to carry out in vivo bone biology experiments, disease model treatment, epichromatomics research, etc., and finds that the drugs can promote osteogenic differentiation of cells and enhance osteogenic activity.

[0059] 5. This study investigates STAT3 agonists targeting key cell types in bone development, such as osteoblasts and bone resorbent cells. By modulating the STAT3 signaling pathway, this invention influences bone cell proliferation and differentiation, thereby affecting bone mineral density and bone strength, and holds promise for treating osteoporosis and other bone development disorders. Attached Figure Description

[0060] Figure 1 This is a schematic diagram of the chemical structure of Colivelin;

[0061] Figure 2 To detect the cytotoxicity of different drugs on bone marrow mesenchymal stem cells using CCK8 assay;

[0062] Figure 3 To detect the effects of different drug concentrations on alkaline phosphatase activity in bone marrow mesenchymal stem cells using a semi-quantitative alkaline phosphatase assay.

[0063] Figure 4 To detect the effects of different drugs on osteogenic activity of bone marrow mesenchymal stem cells using alkaline phosphatase staining;

[0064] Figure 5 To detect changes in p-STAT3 expression in bone marrow mesenchymal stem cells caused by different drugs using protein imprinting;

[0065] Figure 6 To detect the expression changes of osteogenic-related genes Alp, Col1, Ocn, Sp7 and Runx2 in bone marrow mesenchymal stem cells under the action of the drug GKB6-P4 by qPCR;

[0066] Figure 7 To detect the expression changes of osteogenic-related genes Alp, Col1, Ocn, Sp7 and Runx2 in bone marrow mesenchymal stem cells under the action of the drug GKB6-P13 by qPCR;

[0067] Figure 8 To detect the expression changes of osteogenic-related genes Alp, Col1, Ocn, Sp7 and Runx2 in bone marrow mesenchymal stem cells under the action of the drug GKB6-P23 by qPCR. Detailed Implementation

[0068] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0069] In the following embodiments, the sources of the experimental instruments and reagents involved are as follows:

[0070] Scissors (Guozhong, China); 1.5mL centrifuge tubes (Axygen, USA); 0.2mL qPCR eight-tube bundle (Beyotime, China); isopropanol (Sinopharm, China); 75% ethanol (Sinopharm, China); PCR premix (TIANDZ, China); PCR primers (Sangon Biotech, China); electronic balance (Mettler Toledo, Switzerland); dimethyl sulfoxide (DMSO) (Beyotime, China); 4% paraformaldehyde (Saiwell, China); biosafety cabinet (Thermo Fisher, USA); cell culture incubator (Thermo Fisher, USA); phosphate buffer (Gibco, USA); fetal bovine serum (FBS) (Gibco, USA); α-MEM medium (Gibco, USA); penicillin-streptomycin antibiotics (Gibco, USA); 0.25% EDTA trypsin (Gibco, USA); Osteogenic induction medium (Cyagen Biosciences, China); Alkaline phosphatase staining kit (Beyotime, China); Alizarin red staining solution (Beyotime, China); Inverted optical microscope (Olympus, Germany); Polymerase chain reaction (PCR) instrument (Thermo Fisher, USA); Tabletop centrifuge (Thermo Fisher, USA); SDS-PAGE electrophoresis system (Bio-Rad, USA); Protein lysate (Beyotime, China); Protease inhibitor (Beyotime, China); BCA protein quantification kit (Beyotime, China); 5× Loading buffer (Beyotime, China); 5× SDS electrophoresis buffer (Beyotime, China); Nitrocellulose membrane (Sigma-Aldrich, USA); 10× Transfer buffer (Beyotime, China); 10× TBST washing solution (Beyotime, China); Blotting apparatus (Bio-Rad, USA); Trizol (Invitrogen, USA); Chloroform (Shanghai National Pharmaceutical, China); Isopropanol (Shanghai National Pharmaceutical, China); DEPC water (Sangon Biotech, China); Prime Script TM RT Master Mix reverse transcription reagent (TAKARA, Japan); 2× Universal SYBR Green Fast qPCR Mix (ABclonal, USA); p-Stat3 antibody (CST9138) (Cell Signaling Technology, USA); GAPDH antibody (CST2118) (Cell Signaling Technology, USA); Horseradish peroxidase-labeled goat anti-mouse IgG (Beyotime, China); Horseradish peroxidase-labeled goat anti-rabbit IgG (Beyotime, China).

[0071] In the following examples, the sources of the experimental animals involved are as follows:

[0072] 4-week-old male C57BL6 mice (SPF grade), purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., Experimental Animal Production License: SCXK (Shanghai) 2018-0004; housed in the Experimental Animal Center of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (SPF grade).

[0073] The above mice were housed in an environment with a temperature of (24±2)°C and a humidity of (60±5)%, with a 12-hour day-night cycle, and could drink water and eat freely. The treatment methods for animals in this study strictly complied with the "Guiding Opinions on the Humane Treatment of Laboratory Animals" issued by the Ministry of Science and Technology of the People's Republic of China.

[0074] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0075] Example 1

[0076] This embodiment provides a GKB6-P4, specifically CAS number 30912-76-6.

[0077] Example 2

[0078] This embodiment provides a method for preparing GKB6-P13, the specific steps of which are as follows:

[0079] S1. Dissolve sodium hydroxide (2.40 g, 60 mmol) in water (10 mL), then add thiomorpholine (3.09 g, 30 mmol) dissolved in DCM (20 mL) to obtain a mixed solution.

[0080] S2. Dissolve dichlorodisulfide (2.02 g, 15 mmol) in DCM (5 mL) and slowly add it dropwise to the mixed solution obtained in step S1. After the addition is complete, stir in an ice-water bath for half an hour.

[0081] S3. The reaction process was monitored by TLC (petroleum ether / ethyl acetate). After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product.

[0082] S4. A yellow oily liquid (2-(dodecyl dithio)-1H-isoindole-1,3(2H)-dione, 3.22 g, 80.0%) was obtained by silica gel column chromatography and named GKB6-P13.

[0083] The reaction process of GKB6-P13 is shown below:

[0084]

[0085] The NMR data of GKB6-P13 are consistent with the literature reports (Bioorganic & Medicinal Chemistry, 2000, 8(11), 2549-2560.).

[0086] Example 3

[0087] This embodiment provides a method for preparing GKB6-P23, the specific steps of which are as follows:

[0088] S1. Dissolve sodium hydroxide (240 mg, 6 mmol) in water (1 mL), and add dibenzylamine (591 mg, 3.0 mmol) dissolved in DCM (2 mL) to obtain a mixed solution.

[0089] S2. Dissolve dichlorodisulfide (202 mg, 1.5 mmol) in DCM (0.5 mL) and slowly add it dropwise to the mixed solution obtained in step S1. After the addition is complete, stir in an ice-water bath for half an hour.

[0090] S3. The reaction process was monitored by TLC (petroleum ether / ethyl acetate). After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product.

[0091] S4. A white solid (2-(o-chlorophenyl dithio)-1H-isoindole-1,3(2H)-dione, 0.31 g, 47.4%) was obtained by silica gel column chromatography and named GKB6-P23.

[0092] The reaction process of GKB6-P23 is shown below:

[0093]

[0094] The NMR data of GKB6-P23 are consistent with the literature reports (ACS Catal. 2019, 9(12), 11426-11430.).

[0095] Example 4

[0096] This embodiment provides a method for extracting bone marrow mesenchymal stem cells, the specific steps of which are as follows:

[0097] (1) Mice were euthanized by cervical dislocation and then immersed in ethanol for 10 minutes.

[0098] (2) Using ophthalmic scissors, separate the skin and muscles of the mouse leg under sterile operating table, expose and separate the femur and tibia of the mouse, and place them in a 6cm dish containing sterile PBS. Hold the femur or tibia of the mouse with forceps and cut off both ends of the femur and tibia. Rinse the bone marrow cavity repeatedly with 5mL of injection solution containing α-MEM medium (containing 2% penicillin-streptomycin) until the bone turns white.

[0099] (3) Collect the culture medium containing mouse bone marrow into a 15 mL centrifuge tube, centrifuge at 300 g for 3 min, and discard the supernatant. Resuspend the cells in 1 mL of α-MEM complete medium (containing 10% FBS and 2% penicillin-streptomycin antibiotics), place them in a 10 cm dish, and add complete medium to 10 mL. Gently mix the cells in a cross-shaped manner and incubate in a cell culture incubator at 37°C and 5% CO2. Replace half of the complete medium after 3 days, and replace all of the complete medium after 6 days.

[0100] (4) After 7 days of culture, when the cells are 75%-80% confluent, remove the original culture medium, dilute the remaining FBS in the dish with 2 mL PBS, add 1 mL 0.25% trypsin to digest the cells, then add 2 mL of complete culture medium to stop the digestion, collect the cells, centrifuge, resuspend, and passage at a ratio of 1:3 for cell experiments.

[0101] Example 5

[0102] This embodiment provides a method for inducing osteogenic formation of bone marrow mesenchymal stem cells, the specific steps of which are as follows:

[0103] The bone marrow mesenchymal stem cells obtained in Example 4 were used at a rate of 2.5 × 10⁻⁶. 5 The seeds were seeded at a density of [missing value] mL in well plates and cultured for 1 day. The culture medium was then replaced with osteogenic induction medium (containing 1% glutamine, 0.20% ascorbic acid, 1% β-glycerophosphate, and 0.01% dexamethasone) and cultured for another day, changing the osteogenic induction medium every 1 day. ALP staining was performed after 7 days of culture. Alizarin red staining was performed after 14 days of culture. The samples were observed and photographed under a stereomicroscope.

[0104] Example 6

[0105] This embodiment provides a method for preparing a drug, the specific steps of which are as follows:

[0106] Dissolve the drug in DMSO to prepare concentrations of 0.01 mM, 0.1 mM, and 1 mM. Dilute 1:1000 in αMEM medium before use.

[0107] Example 7

[0108] This embodiment provides a CCK-8 experimental method, the specific steps of which are as follows:

[0109] (1) Typically, 2000 cells are added to each well for cell proliferation experiments and 5000 cells are added to each well for cytotoxicity experiments (the specific number of cells used per well depends on factors such as cell size and cell proliferation rate). Culture the cells according to experimental requirements and administer 0-10 μL of a specific drug for stimulation.

[0110] (2) Add 10 μL of CCK-8 solution to each well. If the initial culture volume is 200 μL, then add 20 μL of CCK-8 solution, and so on. Wells with the corresponding amount of cell culture medium and CCK-8 solution but without cells can be used as blank controls. If you are concerned that the drug used may interfere with the detection, you should set up wells with the corresponding amount of cell culture medium, drug, and CCK-8 solution but without cells as blank controls.

[0111] (3) Continue incubation in the cell culture incubator for 0.5-4 hours. In most cases, 1 hour is sufficient. The duration depends on the cell type and cell density, etc. For the first experiment, you can use an ELISA reader to detect the absorbance after 0.5, 1, 2 and 4 hours, and then select a time point with a suitable absorbance range for subsequent experiments.

[0112] (4) Measure absorbance at 450 nm. If a 450 nm filter is unavailable, a 420-480 nm filter can be used. A wavelength greater than 600 nm, such as 650 nm, can be used as a reference wavelength for dual-wavelength measurement.

[0113] (5) Different numbers of HeLa cells were seeded into 96-well plates at 100 μL of culture medium per well. After the cells were fully adhered, 10 μL of CCK-8 solution was added to each well and incubated for 2 hours before measuring A450.

[0114] Example 8

[0115] This embodiment provides a method for detecting alkaline phosphatase, the specific steps of which are as follows:

[0116] (1) Reagent preparation: Remove all reagents and bring them to room temperature before use.

[0117] a. Chromogenic substrate solution: For P0321S packaging, take one tube of chromogenic substrate and dissolve it in 2.5 ml of detection buffer (you can first dissolve it in 1 ml of detection buffer, then transfer it to a 15 ml centrifuge tube and add 1.5 ml of detection buffer). Dissolve and mix thoroughly, and place on ice. For P0321M packaging, take one tube of chromogenic substrate and dissolve it in 5 ml of detection buffer (you can first dissolve it in 2 ml of detection buffer, then transfer it to a 15 ml centrifuge tube and add 3 ml of detection buffer). Dissolve and mix thoroughly, and place on ice. Freshly prepared chromogenic substrate solutions should be used within 6 hours.

[0118] b. Standard working solution: Take 10 μl of p-nitrophenol solution (10 mM) and dilute it to 0.2 ml with detection buffer to obtain a final concentration of 0.5 mM.

[0119] (2) Sample preparation:

[0120] a. Preparation of cell or tissue lysis buffer: Lyse cells or tissues using appropriate cell or tissue lysis buffer. Use Beyotime's P0013J Western and IP cell lysis buffer (inhibitor-free) to lyse the relevant samples. If necessary, homogenize appropriately, then centrifuge and collect the supernatant for alkaline phosphatase detection.

[0121] Note: The lysis buffer must not contain phosphatase inhibitors. Samples can be stored at -80°C, but repeated freeze-thaw cycles should be avoided.

[0122] b. Preparation of Plasma, Serum, and Urine: Plasma and serum can be directly used for this experiment after preparation using standard methods. However, to eliminate interference from the sample's own color, a control containing plasma or serum but without substrate should be included. Anticoagulant tubes containing EDTA and citrate should not be used when preparing plasma. Urine can usually be used directly for the assay. The above samples can be stored at -80°C, but repeated freeze-thaw cycles should be avoided.

[0123] c. Sample dilution: If the sample contains highly active alkaline phosphatase, it can be diluted using the original lysis buffer or PBS, or the detection buffer provided in the kit. If using the detection buffer provided in the kit, ensure that sufficient detection buffer is reserved for the kit's detection process.

[0124] (3) Refer to Table 1 to set up blank control wells, standard wells, and sample wells using a 96-well plate. The amounts of standards used are 4, 8, 16, 24, 32, and 40 μL, respectively. Samples can usually be added directly at 50 μL. If the alkaline phosphatase activity in the sample is too high, the sample amount can be reduced or the sample can be diluted appropriately before measurement.

[0125] Table 1 Additives for blank control wells, standard wells, and sample wells in 96-well plates

[0126]

[0127] (4) Gently blow and mix with the nozzle, or use a shaker to mix.

[0128] (5) Incubate at 37℃ for 5-10 minutes (Note: If the alkaline phosphatase activity in the sample to be tested is low, the incubation time can be extended to 30 minutes).

[0129] (6) Add 100 μl of reaction stop solution to each well to terminate the reaction. At this time, the wells containing the standard or alkaline phosphatase will show different shades of yellow.

[0130] (7) Measure absorbance at 405 nm. If 405 nm cannot be measured, absorbance can be measured in the range of 400-415 nm. If it cannot be measured immediately, it can be completed within several hours, and the yellow color that appears will be stable within several hours.

[0131] (8) Definition of alkaline phosphatase activity unit: In diethanolamine (DEA) buffer at pH 9.8 and 37°C, the amount of alkaline phosphatase required per minute to hydrolyze the para-nitrophenyl phosphate chromogenic substrate to produce 1 micromolar p-nitrophenol is defined as one unit of enzyme activity, also known as one DEA enzyme activity unit. In glycine buffer at pH 9.6 and 25°C, the amount of alkaline phosphatase required per minute to hydrolyze the para-nitrophenyl phosphate chromogenic substrate to produce 1 micromolar p-nitrophenol is defined as one unit of enzyme activity, also known as one Glycine enzyme activity unit. One Glycine enzyme activity unit is approximately equivalent to 3 DEA enzyme activity units.

[0132] (9) Calculate the alkaline phosphatase activity in the sample according to the definition of enzyme activity.

[0133] Example 9

[0134] This embodiment provides a protein blotting (WB) method, the specific steps of which are as follows:

[0135] (1) Protein sample preparation: Aspirate the cell culture medium, wash the cells once with PBS, add 150 μL of SDS lysis buffer (containing 2% protease phosphatase inhibitor mixture) to each well of a 6-well plate, scrape the cells off the culture plate with a cell scraper, transfer them to centrifuge tubes using a pipette, add SDS-PAGE protein loading buffer, centrifuge, and boil at 100℃ for 10 min. The obtained protein samples can be stored at -80℃.

[0136] (2) Electrophoresis: Fix the SDS-PAGE precast gel in the electrophoresis tank, add the prepared electrophoresis buffer, and load the sample. Turn on the power and run at a constant voltage of 80V for 30 minutes, then switch to 100V and run for 90 minutes.

[0137] (3) Transfer: Place the sponge, transfer filter paper, SDS-PAGE gel, nitrocellulose (NC) membrane, transfer membrane, filter paper and sponge in sequence on the transfer clamp. After ensuring that the air bubbles are removed, close the transfer clamp and transfer it into the electroporation apparatus. Pour in the transfer solution, place the ice box in the electroporation apparatus and turn on the power. Set the constant current to 0.3A and transfer for 75 minutes.

[0138] (4) Sealing: Immerse the NC membrane in 5% skim milk and incubate in a shaker at room temperature for 45 minutes.

[0139] (5) Primary antibody incubation: Wash the NC membrane with TBST, shake for 5 min, and wash 3 times. Cut the NC membrane according to the molecular weight of the target protein, and immerse it in the primary antibody diluted in the specified proportion. Incubate overnight at 4°C with shaking.

[0140] The primary antibody information is as follows: p-Stat3 antibody (CST9138) (Cell Signaling Technology, USA), GAPDH antibody (CST2118) (Cell Signaling Technology, USA).

[0141] (6) Secondary antibody incubation: Wash the NC membrane with TBST, shake for 5 min, and wash 3 times. Immerse the NC membrane in the secondary antibody diluted in proportion and incubate at room temperature on a shaker for 45 min.

[0142] The secondary antibody information is as follows: horseradish peroxidase-labeled goat anti-mouse IgG (Beyotime, China), horseradish peroxidase-labeled goat anti-rabbit IgG (Beyotime, China).

[0143] (7) Development: Clean the NC membrane with TBST, shake for 5 minutes, and wash 3 times. Prepare the developer solution at a 1:1 ratio, place the NC membrane covered with the developer solution in the instrument for development, and save the image.

[0144] Example 10

[0145] This embodiment provides a method for alkaline phosphatase staining, the specific steps of which are as follows:

[0146] Aspirate the cell culture medium, fix with 4% PFA for 15 min, and gently wash three times with PBS. Prepare the BCIP / NBT staining working solution in the following proportions in the dark: 3 mL alkaline phosphatase chromogenic buffer, 10 μL BCIP solution (300×), and 20 μL NBT solution (150×). Add the working solution to the wells of the plate and incubate at 37°C in the dark for 20-30 min until staining is complete. Discard the working solution, wash three times with PBS, air dry, and observe and photograph.

[0147] Example 11

[0148] This embodiment provides a real-time quantitative polymerase chain reaction method, the specific steps of which are as follows:

[0149] (1) Aspirate the culture medium from the well plate, rinse the cells twice with PBS, then add 1 mL of Trizol and transfer to a centrifuge tube without RNase.

[0150] (2) Pre-cool the centrifuge to 4°C, add chloroform at a ratio of 5:1, shake vigorously, let stand for 5 minutes, and centrifuge at 12000 rpm for 15 minutes.

[0151] (3) Take the colorless, clear supernatant and place it in a new centrifuge tube. Add an equal amount of isopropanol, shake vigorously, and let stand for 10 minutes. Centrifuge at 12,000 rpm for 15 minutes.

[0152] (4) Discard the supernatant, add 1 mL of DEPC water to dilute to 75% ethanol, shake vigorously, and centrifuge at 12000 rpm for 15 min. Repeat this step once.

[0153] (5) Carefully aspirate the supernatant, invert the centrifuge tube onto a clean paper towel, and let it air dry at room temperature.

[0154] (6) Add 20 μL of DEPC water to dissolve the RNA and use an ultra-micro UV spectrophotometer to determine the RNA concentration.

[0155] (7) Reverse transcribe RNA into cDNA: 500 ng RNA, 2 μL 5×ABScriptⅡRT Mix, and DEPC water to make up to 10 μL.

[0156] The reverse transcription reaction conditions were as follows: 25℃ for 5 min, 42℃ for 15 min, 85℃ for 5 s, and 4℃ to finish.

[0157] (8) Quantitative real-time polymerase chain reaction (qPCR): Each well contains 0.5 μL cDNA, 3.8 μL double-distilled water, 5 μL 2×Universal SYBR Green Fast qPCR Mix, and a 10 μL system. Three auxiliary wells are set for each sample. After signing the 96 qPCR plate, centrifuge at 1000 rpm for 1 min, place it in the PCR instrument, and start the program. The program settings are as follows: 95℃ pre-denaturation for 1 min, 95℃ for 5 s, and 60℃ for 30 s to collect fluorescence signals (a total of 50 cycles).

[0158] Primer information is shown in Table 2:

[0159] Table 2 Primer sequences

[0160]

[0161] Example 12

[0162] This embodiment provides a statistical analysis method, the specific steps of which are as follows:

[0163] Quantitative data are expressed as mean ± standard deviation. Independent samples t-tests were performed using SPSS 24.0 software to analyze differences between groups. One-way ANOVA was used to compare data from multiple groups. Pairwise comparisons were performed between groups with significant differences. P < 0.05 was considered statistically significant.

[0164] Example 13

[0165] This embodiment uses the CCK8 assay to detect the effect of the drug on cell proliferation and differentiation. The specific steps are as follows:

[0166] The GKB6-P4, GKB6-P13, and GKB6-P23 prepared in Examples 1-3, and the existing drug colivelin were tested using the CCK-8 assay method described in Example 7. The cytotoxicity results of different drugs on bone marrow mesenchymal stem cells are as follows: Figure 2 As shown, GKB6-P4 had the highest cell concentrations at 0.1 μM and 1 μM. Subsequent experiments were conducted at the following concentrations: 1 μM for GKB6-P4; 0.1 μM for GKB6-P13; 1 μM for GKB6-P23; and 1 nM for colivelin.

[0167] Example 14

[0168] This embodiment uses a semi-quantitative alkaline phosphatase experiment to observe the effects of different drug concentrations on alkaline phosphatase activity and osteogenic activity. The specific steps are as follows:

[0169] The GKB6-P4, GKB6-P13, and GKB6-P23 prepared in Examples 1-3 and the existing drug colivelin were used to prepare three drug concentration gradients according to molecular weight using DMSO, namely 0.01 / 0.1 / 1 mM. The liquid was changed every two days, and the samples were collected after seven days (the specific experimental methods are as described in Examples 8 and 9). The alkaline phosphatase activity was calculated, and the osteogenic differentiation ability of different drugs was detected.

[0170] The effects of different drug concentrations on alkaline phosphatase activity in bone marrow mesenchymal stem cells, such as Figure 3 As shown, when the concentration of GKB6-P4 was 1 μm, the GKB6-P4, GKB6-P13, and GKB6-P23 prepared in Examples 1-3 showed significantly enhanced alkaline phosphatase activity in bone marrow mesenchymal stem cells compared with the control group without added drugs, demonstrating that the GKB6-P4, GKB6-P13, and GKB6-P23 prepared in Examples 1-3 can effectively promote osteogenic differentiation.

[0171] The effects of different drugs on the osteogenic activity of bone marrow mesenchymal stem cells, such as Figure 4 As shown, when the concentration of GKB6-P4 was 1 μm, the GKB6-P4, GKB6-P13, and GKB6-P23 prepared in Examples 1-3 showed significantly deeper ALP staining compared to the control group, and even deeper staining compared to the colivelin group. This indicates that the GKB6-P4, GKB6-P13, and GKB6-P23 prepared in Examples 1-3 promoted osteogenic differentiation of MSCs, and their effects were superior to those of colivelin.

[0172] Figure 3 and Figure 4This also demonstrates that the concentration of CCK8 selected in Example 13 effectively promotes increased osteogenic activity.

[0173] Example 15

[0174] This embodiment uses a protein imprinting experiment to observe whether the drug can effectively activate STAT3. The specific steps are as follows:

[0175] The GKB6-P4, GKB6-P13, and GKB6-P23 prepared in Examples 1-3, and the existing drug colivelin were detected using the protein imprinting method described in Example 9. The effects of different drugs on p-STAT3 expression in bone marrow mesenchymal stem cells were as follows: Figure 5 As shown, compared with the control group, the p-STAT3 bands of GKB6-P4, GKB6-P13, GKB6-P23 and the existing drug colivelin were all deepened, that is, the p-STAT3 content was increased, indicating that GKB6-P4, GKB6-P13, GKB6-P23 and the existing drug colivelin can effectively activate STAT3.

[0176] Example 16

[0177] This embodiment observes the osteogenic activity of the drug using real-time quantitative polymerase chain reaction. The specific steps are as follows:

[0178] The GKB6-P4, GKB6-P13, and GKB6-P23 prepared in Examples 1-3 and the existing drug colivelin were tested using the real-time fluorescence quantitative polymerase chain reaction method described in Example 11 to observe whether the osteogenic activity of the newly generated drugs was superior to that of the original drug colivelin.

[0179] Experimental results are as follows Figure 6 As shown, compared with the control group, the expression levels of osteogenic genes Alp, Col1a1, Ocn, Sp7 and Runx2 mRNA were significantly increased in the GKB6-P4 group and the existing drug colivelin. Moreover, the expression levels of the above osteogenic gene mRNA were higher in the GKB6-P4 group than in the existing drug colivelin group, indicating that the newly generated drug GKB6-P4 is significantly better than the original drug colivelin in promoting osteogenic activity.

[0180] Experimental results are as follows Figure 7As shown, compared with the control group, the expression levels of osteogenic genes Alp, Col1a1, Ocn, Sp7 and Runx2 mRNA were significantly increased in the GKB6-P13 group and the existing drug colivelin. Moreover, the expression levels of the above osteogenic genes mRNA were higher in the GKB6-P13 group than in the existing drug colivelin group. The newly generated drug GKB6-P13 is significantly better than the original drug colivelin in promoting osteogenic activity.

[0181] Experimental results are as follows Figure 8 As shown, compared with the control group, the expression levels of osteogenic genes Alp, Col1a1, Ocn, Sp7 and Runx2 mRNA were significantly increased in the GKB6-P23 group and the existing drug colivelin. Moreover, the expression levels of the above osteogenic genes mRNA were higher in the GKB6-P23 group than in the existing drug colivelin group. The newly generated drug GKB6-P23 is significantly better than the original drug colivelin in promoting osteogenic activity.

[0182] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of a small molecule compound containing disulfide bonds in the preparation of a drug that promotes osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that, The structural formulas of the small molecule compounds are shown in Formulas I to III. Formula I, Formula II, Formula III.

2. The application according to claim 1, characterized in that, The drug mentioned is one that enhances osteogenic activity.

3. The application according to claim 1 or claim 2, characterized in that, The dosage form of the drug includes any one of the following: suspension, granules, capsules, powders, tablets, emulsions, injections, pills, suppositories, enemas, aerosols, patches, or drops.

4. The application according to claim 1 or claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The excipients include any one or a combination of at least two of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

6. The application according to claim 1 or claim 2, characterized in that, The drug can be administered via one or more of the following methods: intravenous injection, subcutaneous injection, intramuscular injection, oral administration, and transdermal absorption.

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