Application of dasatinib and quercetin in prevention and treatment of osteomyelitis

Abstract

The invention discloses application of dasatinib and quercetin in prevention and treatment of osteomyelitis. The dasatinib and the quercetin can effectively eliminate cell senescence of Adipoq + myofibroblasts around medullary cavity abscess. Therefore, blood perfusion and antibiotic permeation around the abscess are increased, local blood perfusion around the abscess is improved, the bacterial load is reduced, the bone mass and bone microstructure of the staphylococcus aureus osteomyelitis are improved, and the method is a potential effective treatment strategy for preventing and treating the pathological progress of the staphylococcus aureus osteomyelitis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of dasatinib and quercetin in preventing and treating osteomyelitis. Background Art

[0002] Infectious osteomyelitis is an inflammation of bone and soft tissue caused by pathogenic bacteria, usually manifested as local bacterial invasion and progressive bone destruction of bone, bone marrow and adjacent tissues. The early and acute states of osteomyelitis are characterized by strong inflammatory immune responses associated with bacterial colonization in the bone, abscess formation, insufficient blood supply to the infected area, fever, pain and swelling. However, if not effectively controlled in time, the infection will gradually enter the chronic stage, manifested by the persistence of bacteria, the production of dead bone and bone shells, and the formation of fistulas and sinus tracts. Staphylococcus aureus (abbreviated as "S. aureus") is the main pathogen of infectious osteomyelitis. Implant-related osteomyelitis caused by S. aureus is a serious complication after orthopedic surgery. The treatment cycle is long, the infection is often prolonged, and the recurrence rate is high. It is often complicated by the risk of bone nonunion and pathological fractures, which brings great psychological pressure and heavy economic burden to patients and their families. At present, the treatment of infectious osteomyelitis mainly relies on the early use of sensitive antibiotics, thorough surgical debridement and local defect reconstruction, but the efficacy is limited. The treatment of Staphylococcus aureus-induced osteomyelitis is particularly difficult because during the long process of host-pathogen interaction, Staphylococcus aureus has evolved a variety of mechanisms to evade immune system monitoring and elimination, including forming biofilms on the surface of implant materials, secreting abundant virulence factors, modifying cell wall components to enhance antibiotic resistance, and internalizing into host cells or osteocytic canalicular network (OLCN) in bone tissue.

[0003] Before the advent of antibiotics, the treatment of osteomyelitis was based on the debridement and drainage principles of Dr. Hiram Winnett Orr. The invention of antibiotics has significantly changed the treatment of osteomyelitis. With the advancement of medical technology and the understanding of the disease, the mainstream treatment of infectious osteomyelitis has developed into targeted antimicrobial therapy, source control, and correction of medical complications. Targeted antimicrobial therapy requires the specific use of sensitive antibiotics based on the site of infection or the microorganisms identified by culture; source control requires the removal of necrotic bone and tissue, and drainage and flushing of the infected site. At the same time, correcting the patient's hyperglycemia and improving medical complications such as peripheral vascular disease are the key to treating chronic infectious osteomyelitis. However, pharmacological studies in healthy tissues cannot simulate factors such as biofilm, abscess, and necrotic bone formation in chronic osteomyelitis, and debridement cannot completely and thoroughly eliminate latent pathogens in the tissue. For these reasons, infectious osteomyelitis still has a high recurrence rate and is still a major problem in orthopedic clinical treatment.

[0004] Studies have shown that factors such as trauma and infection can initiate local inflammatory responses in tissues, leading to the continuous activation of myofibroblasts around abscess lesions and promoting local tissue fibrosis. Abnormally increased fibrotic tissue can lead to vascular occlusion around abscess lesions, reduced blood flow, and thus insufficient local antibiotic concentration and the formation of persistent bacteria. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide the use of dasatinib and quercetin in preventing and treating osteomyelitis.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] One aspect of the present invention provides the use of dasatinib and quercetin in the preparation of a drug for preventing and treating osteomyelitis.

[0008] In some embodiments of the present invention, the osteomyelitis includes osteomyelitis caused by bacterial infection.

[0009] In some embodiments of the present invention, the osteomyelitis includes osteomyelitis caused by Staphylococcus aureus infection.

[0010] In the present invention, the abnormally increased fibrotic tissue around the abscess lesion of Staphylococcus aureus osteomyelitis can lead to vascular occlusion around it, reduce blood flow, and then cause insufficient local antibiotic concentration and the formation of retained bacteria, which ultimately aggravates the pathology of osteomyelitis. Dasatinib + quercetin can effectively remove the cell senescence of Adipoq + myofibroblasts around the medullary abscess, thereby increasing blood perfusion and antibiotic penetration around the abscess, improving local blood perfusion around the abscess, reducing bacterial load and improving bone mass and bone microstructure of Staphylococcus aureus osteomyelitis, which is a potential effective treatment strategy for preventing and treating the pathological progression of Staphylococcus aureus osteomyelitis.

[0011] Another aspect of the present invention provides the use of dasatinib and quercetin in the preparation of a drug for preventing and treating infectious diseases caused by Staphylococcus aureus.

[0012] In some embodiments of the present invention, the infectious disease caused by Staphylococcus aureus is specifically an infectious disease of bone and surrounding soft tissue caused by Staphylococcus aureus.

[0013] In some embodiments of the present invention, the diseases associated with Staphylococcus aureus infection include osteomyelitis, necrotizing fasciitis, endocarditis, peritonitis, septic arthritis, sepsis, bacteremia, sepsis, pneumonia, abscess and toxic shock syndrome.

[0014] In some embodiments of the present invention, the molar ratio of dasatinib to quercetin in the drug is 1 to 8:100, such as 5 to 6:100.

[0015] In some embodiments of the present invention, the therapeutically effective amount of dasatinib and quercetin in the drug is 0.5-2 mg / kg / d and 5-25 mg / kg / d, respectively. The therapeutically effective amount is for humans. When animals such as mice are used as treatment objects, the therapeutically effective amount is 5-20 mg / kg / d and 50-250 mg / kg / d.

[0016] In some embodiments of the present invention, the drug contains dasatinib and quercetin as the only active ingredients.

[0017] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient or carrier.

[0018] In some embodiments of the present invention, pharmaceutically acceptable excipients or carriers may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0019] In some embodiments of the present invention, the dosage form of the drug is: injection, powder, capsule, tablet, ointment, suppository, aerosol, oral agent, pill, drop, sustained-release tablet, suspension, granule, lozenge, granule, drop, pill, powder, solution, cream, patch, lozenge or any combination thereof.

[0020] In some embodiments of the invention, oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0021] The beneficial effects of the present invention are:

[0022] In the present invention, dasatinib and quercetin can effectively remove senescent Adipoq+ myofibroblasts around medullary abscesses, thereby increasing blood perfusion and antibiotic penetration around abscesses, reducing bacterial load, and improving bone mass and bone microstructure of Staphylococcus aureus osteomyelitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The treatment time and method of dasatinib and quercetin in Example 1.

[0024] Figure 2 This is a diagram of the senescent phenotype of Adipoq+ myofibroblasts around the bone marrow abscess foci of mice after treatment with dasatinib and quercetin in Example 1.

[0025] Figure 3 for Figure 2 Quantification result diagram of .

[0026] Figure 4 The results of the number and inner diameter of blood vessels around the bone marrow abscess in mice after treatment with dasatinib and quercetin in Example 1.

[0027] Figure 5 for Figure 4 Quantification result diagram of .

[0028] Figure 6 This is a graph showing the Staphylococcus aureus load in the medullary cavity after treatment with dasatinib and quercetin in Example 1.

[0029] Figure 7 for Figure 6 Quantification result diagram of .

[0030] Figure 8 This is a diagram showing the changes in bone structure of mice after treatment with dasatinib and quercetin in Example 1.

[0031] Fig. 9 It is a graph showing the effect of osteomyelitis in mice after treatment with dasatinib and quercetin in Example 1; wherein A is a graph showing the amount of cortical bone loss, B is a graph showing reactive new bone formation, C is a graph showing bone density (BMD), D is a graph showing the ratio of bone volume to total volume (BV / TV), E is a graph showing changes in the number of trabeculae (Tb.N), F is a graph showing changes in trabecular thickness (Tb.Th), and G is a graph showing changes in trabecular pattern factor (Tb.Pf). DETAILED DESCRIPTION

[0032] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0033] Example 1

[0034] This example studies the effect of dasatinib and quercetin in preventing and treating the course of infectious osteomyelitis caused by Staphylococcus aureus infection. The specific process is as follows:

[0035] 1. Animal farming

[0036] All the experimental personnel in this study have been trained at the Animal Center of Southern Medical University and obtained the "Animal Experiment Operation Qualification Certificate". The animal experiment protocol was approved by the Experimental Animal Ethics Committee of Zhujiang Hospital. The C57BL / 6 mice used in this study were all from units with experimental animal production licenses and issued the "Experimental Animal Quality Certificate". Mouse breeding was carried out in accordance with the "Regulations on the Management of Experimental Animals in Zhujiang Hospital of Southern Medical University". Specific Pathogen Free (SPF) mice were raised at 25±3℃ and relative humidity of 40-60%, with a 12-hour light and 12-hour dark cycle, and provided with sufficient food and pure water. The number of animals was controlled at an appropriate density, and new bedding was replaced every 3 days to maintain a clean and dry environment. The 3R principle was followed when selecting animals: reduce the use of animals, or use the same animals to obtain more data; optimize all experimental steps to reduce the pain of experimental animals; try to replace animal experiments with cell experiments or other methods. Animals were collected after being anesthetized and cervical dislocation, in accordance with international practice or with reference to the latest guidelines for euthanasia formulated by the American Veterinary Medical Association.

[0037] 2. Isolation, preservation, recovery and quantification of Staphylococcus aureus

[0038] The Staphylococcus aureus strain used in this study was isolated from a patient diagnosed with methicillin-sensitive Staphylococcus aureus osteomyelitis in the Department of Traumatology and Orthopedics of Nanfang Hospital. Pick a Staphylococcus aureus colony from the identified culture medium, add it to a shaking glass tube containing 5 mL of TSB culture medium, place it in a constant temperature oscillator at an angle of 45°, and culture it aerobically at 37°C and 200 rpm for 16 to 18 hours. Take out the glass tube, transfer the bacterial solution to a 50 mL centrifuge tube, add 45 mL of DMSO to mix, and then freeze it at -80°C for later use. When used, take out the frozen bacterial solution, pipette 10 μL of the bacterial solution into a glass tube containing 5 mL of TSB culture medium, and culture it aerobically at 37°C and 200 rpm in a constant temperature oscillator for 16 to 18 hours until the bacteria reach the logarithmic growth phase. Centrifuge it at 2500g for 5 minutes, remove the supernatant, and resuspend it with 1 mL of PBS. Repeat 3 times. Place the bacterial inoculation loop in the outer flame of an alcohol lamp and burn it. After cooling, dip the resuspended bacterial solution and inoculate the bacteria on the TSA agar plate using the plate streak method. Place it in a 37°C bacterial incubator. After the colony grows to an appropriate size, seal it with a sealing film and store it at 4°C. Use it within 2 weeks. Before modeling, pick a colony on the agar plate with a bacterial inoculation loop and add it to a glass tube containing 5mL TSB. Perform bacterial amplification as described above to ensure that the amplified bacteria come from a single colony. After amplification, wash it three times with PBS and adjust the concentration of the bacterial solution with PBS so that its OD at 600nm is 0.5. The results of the previous experiments in this study showed that the bacterial content in the bacterial solution was about 1×10 8CFU / mL. Then the bacterial solution was diluted 100 times to make the bacterial content about 1×10 6 CFU / mL.

[0039] 3. Establishment of the Staphylococcus aureus Osteomyelitis Mouse Model

[0040] Each mouse was intraperitoneally injected with tribromoethanol at a dose of 250 mg / kg. After successful anesthesia, the skin was disinfected with iodine. The hair on the right lower limb and back of the mouse was plucked, the skin was lifted in the middle of the femur, and an incision of about 0.5 cm long was made. After separating the muscles, the femoral bone surface was exposed. A 1 mL syringe needle was used to drill a hole, and then a stainless steel screw (diameter 0.3 mm) about 2 mm long and sterilized at high temperature and high pressure was placed into the bone marrow cavity through the drilled cortical bone hole. A microsyringe was used to inject 3 μL of 1×10 6 The control group was injected with 3 μL of sterile PBS. The cortical bone holes were then sealed with bone wax to prevent the bacterial solution from spilling out, and the muscles and skin were sutured layer by layer.

[0041] 4. SA-β-gal staining

[0042] On the 14th day after surgery, the tissues were fixed with 4% paraformaldehyde for 1 day after routine cardiac perfusion, decalcified with EDTA solution for 7 days, and then precipitated with 20% + 30% sucrose for 1 day, then embedded in OCT and frozen sections were made. On the day of staining, the tissues were gently washed three times with PBS, 5 minutes each time; PBS was aspirated, and an appropriate amount of SA-β-gal staining working solution was added to fully immerse the tissue sections, and incubated in a 37°C carbon dioxide-free incubator in the dark overnight; the staining working solution was removed, PBS was rinsed twice, and the sections were sealed with DAPI anti-fluorescence quencher; LSM980 Zeiss confocal microscope was used for observation and photography.

[0043] 5. Immunofluorescence Staining

[0044] On the 14th day after surgery, the cells were fixed with 4% paraformaldehyde for 1 day after routine cardiac perfusion, decalcified with EDTA solution for 7 days, and then precipitated with 20% + 30% sucrose for 1 day, then embedded in OCT and frozen sections were made. On the day of staining, the cells were first aspirated twice with 0.1% PBST and PBS, each for 5 minutes each time; PBS was aspirated, and a blocking solution containing 10% goat serum was added, and the cells were blocked at room temperature for 1 hour; the blocking solution was aspirated, and the primary antibody staining solution was added, and the cells were incubated at 4°C overnight; the primary antibody staining solution was aspirated, and 0.1% PBST and PBS were aspirated twice, each for 5 minutes each time; the secondary antibody staining solution was added, and the cells were incubated at room temperature in the dark for 1 hour; the secondary antibody staining solution was aspirated, and 0.1% PBST and PBS were aspirated twice, each for 5 minutes each time; the cells were sealed with DAPI anti-fluorescence quencher, and the cells were observed and photographed with LSM980 Zeiss confocal microscope.

[0045] 6. Micro CT analysis

[0046] Fourteen days after infection, the right femurs of mice were scanned using high-resolution micro-CT (Scanco Medical, Switzerland). The voxel size of all images was 6 μm, the current was 145 mA, the voltage was 55 kV, and the integration time was 400 ms. The three-dimensional structural parameters of cortical bone and trabecular microstructure parameters were analyzed using standard methods. Bone density (BMD) was calibrated using ceramic standard samples. Briefly, the three-dimensional structural parameters of cortical bone, including cortical bone loss and reactive bone formation, were calculated within 1200 layers (7.2 mm) centered on a single cortical hole for self-tapping screws. In addition, the evaluation range of trabecular microstructure was defined as a longitudinal distance extending from 0.444 mm from the growth plate to 1.2 mm proximal to the end, and these parameters included bone density (BMD), bone volume to total volume ratio (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular pattern factor (Tb.Pf).

[0047] 7. Statistical methods

[0048] All quantitative data are expressed as mean ± standard deviation, and independent t-test was used for comparison between two groups of data. For multiple comparisons, one-way analysis of variance (ANOVA) and Bonferroni test were used. Statistical analysis was performed using SPSS version 20 software (International Business Machines Corporation, IBM Corp.), and the significance level was P < 0.05.

[0049] like Figure 1 As shown, the implant-related Staphylococcus aureus osteomyelitis mouse model established above (Yihuang Lin, et al, Infect Immun, 2021, 89 (5): e00814-20) was used. Dasatinib (Dasatinib, S1021, Selleck Chemicals, 5 mg / kg / d) and quercetin (Quercetin, S2391, Selleck Chemicals, 50 mg / kg / d) were given by oral gavage for 2 consecutive days (Day-2, -1) before surgery. The two drugs were suspended in 100 μL of thickener (60% Phosal50PG + 10% Ethanol + 30% PEG-400), and the same drugs and doses were given for 14 consecutive days (Day 1-14) after surgery. The results are shown in the figure. Figures 2 to 9 shown.

[0050] The results of SA-β-gal staining showed that the senescent phenotype of Adipoq+ myofibroblasts around the bone marrow abscess foci in mice was significantly reduced after treatment with dasatinib and quercetin (D+Q). Figure 2 , Figure 3 Immunofluorescence staining results showed that the number and inner diameter of blood vessels around the bone marrow abscess foci in mice were significantly increased after treatment with dasatinib and quercetin ( Figure 4 , Figure 5 ); at the same time, the Staphylococcus aureus load in the medullary cavity was significantly reduced ( Figure 6 , Figure 7 Micro-CT scan results showed that the bone structure of mice treated with dasatinib and quercetin was significantly improved ( Figure 8 ), expressed as the amount of cortical bone loss ( Fig. 9 A) and reactive new bone ( Fig. 9 B) decreased, while bone mineral density (BMD), the ratio of bone volume to total volume (BV / TV), the number of trabeculae (Tb.N), and the thickness of trabeculae (Tb.Th) increased significantly ( Fig. 9 CF), trabecular bone pattern factor (Tb.Pf) decreased ( Fig. 9 G). The combination of dasatinib and quercetin can effectively remove senescent myofibroblasts around abscess lesions, thereby improving local fibrosis, enhancing local antibiotic penetration in abscesses, and ultimately delaying the pathological progression of osteomyelitis.

[0051] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of dasatinib and quercetin in the preparation of drugs for preventing and treating osteomyelitis.

2. The use according to claim 1, characterized in that: The osteomyelitis includes osteomyelitis caused by bacterial infection.

3. The use according to claim 1, characterized in that: The osteomyelitis includes osteomyelitis caused by Staphylococcus aureus infection.

4. Application of dasatinib and quercetin in the preparation of drugs for preventing and treating infectious diseases caused by Staphylococcus aureus.

5. The use according to claim 4, characterized in that: The infectious disease caused by Staphylococcus aureus is specifically an infectious disease of bone and surrounding soft tissue caused by Staphylococcus aureus.

6. The use according to claim 5, characterized in that: The diseases associated with Staphylococcus aureus infection include osteomyelitis, necrotizing fasciitis, endocarditis, peritonitis, septic arthritis, sepsis, bacteremia, sepsis, pneumonia, abscess and toxic shock syndrome.

7. The use according to any one of claims 1 to 6, characterized in that: The molar ratio of dasatinib to quercetin in the drug is 1 to 8:

100.

8. The use according to any one of claims 1 to 6, characterized in that: The therapeutically effective amounts of dasatinib and quercetin in the drug are 0.5-2 mg / kg / d and 5-25 mg / kg / d, respectively.

9. The use according to any one of claims 1 to 6, characterized in that: The drug contains dasatinib and quercetin as the only active ingredients.

10. The use according to any one of claims 1 to 6, characterized in that: The drug further comprises a pharmaceutically acceptable excipient or carrier.

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