Use of roflumilast in the treatment of bone defect repair

Abstract

The application belongs to the technical field of medicine, and discloses application of roflumilast in bone defect repair treatment. The application firstly finds that roflumilast can significantly promote cell osteogenic differentiation, and up-regulate the expression of osteogenic differentiation genes and proteins of in-vitro cultured mouse embryonic mesenchymal stem cells, human umbilical cord mesenchymal stem cells and rat bone marrow mesenchymal stem cells, improve cell alkaline phosphatase activity and promote cell mineralization nodule formation; finds that roflumilast can significantly promote bone repair at a rat skull defect, restore bone structure and increase bone mass, and can effectively treat bone defects; and provides experimental data support for further clinical use and popularization.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to the application of roflumilast in the treatment of bone defect repair. Background Technology

[0002] Roflumilast is a selective and potent phosphodiesterase 4 (PDE4) inhibitor approved by the FDA for the treatment of severe chronic obstructive pulmonary disease (COPD) with chronic bronchitis and frequent exacerbations. The most common adverse drug reactions to roflumilast in clinical use include diarrhea, nausea, vomiting, indigestion, and dizziness, primarily affecting the gastrointestinal and nervous systems. Most adverse reactions are mild to moderate, usually transient, and resolve within the first few weeks of treatment. They are dose-related; gradually increasing the dose over time from a low level can help avoid more adverse reactions. Topical application of roflumilast, such as skin application, has a lower incidence and milder adverse reactions than oral administration. In addition to COPD, roflumilast currently shows some therapeutic effects in other diseases primarily characterized by inflammation, such as ulcerative colitis, psoriasis, and periodontitis. Recent studies have also reported therapeutic effects in diabetes, neurological disorders, and cardiovascular diseases, but whether roflumilast has bone-promoting properties remains unresolved.

[0003] The skeleton is a dynamic organ, maintaining bone mass and homeostasis through two distinct processes: bone building and remodeling. It can also repair minor injuries through bone regeneration. However, bone regeneration capacity is limited. Defects exceeding the critical range, caused by severe trauma, infection, tumors, or congenital malformations, cannot heal spontaneously, placing a significant burden on patients. These defects often manifest as limb dysfunction and pain at the defect site, severely impacting their physical and mental health and subsequent quality of life. External intervention is necessary to accelerate healing through repair and reconstruction. While many drugs are currently used clinically, they all have certain side effects and adverse reactions. Therefore, finding drugs with fewer adverse effects and higher efficacy to treat bone defects holds great promise, offering a new approach to bone defect repair. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention provides the application of roflumilast in the treatment of bone defect repair, wherein roflumilast can effectively promote osteogenic differentiation of mesenchymal stem cells and effectively promote the repair of bone defects.

[0005] The first aspect of this invention provides the use of roflumilast in the preparation of a drug that promotes the repair of bone defects.

[0006] A second aspect of this invention provides the use of roflumilast in the preparation of a drug that promotes osteogenic differentiation of mesenchymal stem cells.

[0007] According to some embodiments of the present invention, the mesenchymal stem cells include at least one of embryonic mesenchymal stem cells, umbilical cord mesenchymal stem cells, and bone marrow mesenchymal stem cells.

[0008] According to some embodiments of the present invention, the osteogenic differentiation includes at least one of the following a)-c):

[0009] a) Promotes the expression of osteogenic-related genes and proteins;

[0010] b) Increase the activity of alkaline phosphatase in the early stages of osteogenic differentiation;

[0011] c) Increase the area of ​​mineralized nodules that appear in the later stages of osteogenic differentiation.

[0012] According to some embodiments of the present invention, the osteogenic-related genes and proteins include at least one of ALP, RUNX2, OCN, and COL1.

[0013] A third aspect of the present invention provides a drug for promoting the repair of bone defects, the drug comprising the ingredient: roflumilast.

[0014] According to some embodiments of the present invention, roflumilast is the sole active ingredient in the drug that promotes bone defect repair.

[0015] According to some embodiments of the present invention, the dosage concentration of roflumilast is 500 μM.

[0016] According to some embodiments of the present invention, the drug for promoting bone defect repair is administered locally.

[0017] A fourth aspect of the present invention provides a drug for promoting osteogenic differentiation of mesenchymal stem cells, the drug comprising the ingredient: roflumilast.

[0018] According to some embodiments of the present invention, roflumilast is the sole active ingredient in the drug that promotes osteogenic differentiation of mesenchymal stem cells.

[0019] According to some embodiments of the present invention, the dosage of roflumilast is 0.5 μM.

[0020] According to some embodiments of the present invention, the drug for promoting bone defect repair, or the drug for promoting osteogenic differentiation of mesenchymal stem cells, further includes pharmaceutically acceptable excipients.

[0021] According to some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, antibacterial agents, or buffers.

[0022] According to some embodiments of the present invention, the drug for promoting bone defect repair, or the drug for promoting osteogenic differentiation of mesenchymal stem cells, is in the form of any one of tablets, capsules, granules, injections, powder for injection, eye drops, liniments, suppositories, ointments, aerosols, powders, pellets, emulsions, gels, films, transdermal patches, controlled-release formulations, or nano-formulations.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention is the first to discover that roflumilast can significantly promote osteogenic differentiation of cells and upregulate the expression of osteogenic differentiation genes and proteins in in vitro cultured mouse embryonic mesenchymal stem cells, human umbilical cord mesenchymal stem cells, and rat bone marrow mesenchymal stem cells, increase cellular alkaline phosphatase activity, and promote the formation of cellular mineralization nodules. It also discovers that roflumilast can significantly promote bone repair at the site of skull defects in rats, restore bone structure, increase bone mass, and can effectively treat bone defects.

[0025] The findings of this invention provide a theoretical basis for the treatment of bone defects with roflumilast. In particular, the discovery that roflumilast, a drug that has been used clinically but is limited to chronic obstructive pulmonary disease, can be used to treat bone defects provides experimental data support for its further clinical use and promotion. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Figure 1 This is a qRT-PCR detection image of ALP, RUNX2, OCN, and COL1 gene expression in different cells of each group in Example 1.

[0028] Figure 2 This is a Western blot image showing the expression of ALP, RUNX2, OCN, and COL1 proteins in different cells of each group in Example 1.

[0029] Figure 3 These are alkaline phosphatase staining images of different cells in each group of Example 1.

[0030] Figure 4 These are alizarin red staining images of different cells in each group of Example 1.

[0031] Figure 5These are miniature CT analysis images of skull defects in SD rats from each group in Example 2.

[0032] Figure 6 This is a graph showing the quantitative analysis results of the new bone volume fraction at the skull defect site in each group of SD rats in Example 2. *p<0.05, **p<0.01, ***p<0.001.

[0033] Figure 7 These are HE staining images of skull defects in SD rats from each group in Example 2.

[0034] Figure 8 Masson staining images of skull defects in SD rats from each group in Example 2. Detailed Implementation

[0035] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0037] The main materials and reagents involved in the following examples are as follows:

[0038] Roflumilast was purchased from MedChemExpress, USA.

[0039] SD rats were purchased from the Guangdong Provincial Animal Experiment Center.

[0040] Human umbilical cord mesenchymal stem cells and mouse embryonic mesenchymal stem cells were purchased from the American Collection of Cell Cultures (ATCC); rat bone marrow mesenchymal stem cells were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0041] Example 1: Study on the effect of roflumilast on mesenchymal stem cells

[0042] 1.1 Experimental Methods:

[0043] Human umbilical cord mesenchymal stem cells, mouse embryonic mesenchymal stem cells, and rat bone marrow mesenchymal stem cells were cultured in vitro. Once the cell fusion rate reached 70%-80%, intervention treatment was initiated.

[0044] After the cell confluence reached 70%-80%, the culture medium was replaced with the culture induction solution for each group to induce osteogenic differentiation of the three types of mesenchymal stem cells. The experiment was divided into three groups: CTRL group: ordinary culture medium group; OS group: osteogenic induction solution group; RO group: 0.5μM roflumilast + ordinary culture medium group.

[0045] 1.2 Osteogenesis Indicators Detection Methods:

[0046] ① Cells were collected 7 days after osteogenic induction of three types of mesenchymal stem cells and detected by real-time quantitative polymerase chain reaction (qRT-PCR). Total RNA was extracted from the cells using the Trizol method and reversed to cDNA before being quantified by real-time quantitative PCR to observe changes in early osteogenic genes (ALP, RUNX2, OCN, COL1) after drug administration.

[0047] ② After osteogenic induction of three types of mesenchymal stem cells for 7 days, cells were collected, cell proteins were extracted using cell lysis buffer, and Western blot was used to detect changes in osteogenic proteins (ALP, RUNX2, OCN, COL1) in the early stage after drug administration.

[0048] ③ Seven days after osteogenic induction, alkaline phosphatase staining was performed on three types of mesenchymal stem cells. The staining was observed under a microscope and a scanner to detect the level of alkaline phosphatase, an early biomarker of osteogenic differentiation.

[0049] ④ After osteogenic induction of the three types of mesenchymal stem cells for 14-21 days, alizarin red staining was performed, and the formation of mineralized nodules in the cells was observed under a microscope and a scanner to detect the level of osteogenic mineralization.

[0050] 1.3 Experimental Results:

[0051] qRT-PCR results are as follows Figure 1 As shown, Figure 1 In the graph, "BMSC" represents bone marrow mesenchymal stem cells, "UCMSC" represents umbilical cord mesenchymal stem cells, and "C3H10T1 / 2" represents mouse embryonic mesenchymal stem cells. The vertical axis "Relative mRNA expression" represents relative mRNA expression. Figure 1 It can be seen that after inducing the three types of mesenchymal stem cells with roflumilast for 7 days, the expression levels of osteogenic-related genes (ALP, RUNX2, OCN, COL1) in the roflumilast-added group (RO group) were significantly increased compared with the CTRL group, and even more significantly upregulated than in the osteogenic induction solution group (OS group).

[0052] Western blot results are as follows Figure 2As shown, Figure 2 In the graph, "GAPDH" represents glyceraldehyde-3-phosphate dehydrogenase, and the vertical axis "Relative protein expression" represents relative protein expression. From... Figure 2 It can be seen that after inducing the three types of mesenchymal stem cells with roflumilast for 7 days, the expression levels of osteogenic-related proteins (ALP, RUNX2, OCN, COL1) in the roflumilast-added group (RO group) were significantly increased compared with the CTRL group, and even more significantly upregulated than the osteogenic induction solution group (OS group), confirming that roflumilast can promote osteogenic differentiation of cells.

[0053] The results of alkaline phosphatase staining are as follows: Figure 3 As shown. From Figure 3 It can be seen that after inducing the three types of mesenchymal stem cells with roflumilast for 7 days, the alkaline phosphatase staining color of the roflumilast group (RO group) was significantly darker than that of the CTRL group, and even darker than that of the osteogenic induction solution group (OS group), confirming that roflumilast can promote early osteogenic differentiation.

[0054] Alizarin Red staining results as follows Figure 4 As shown. From Figure 4 It can be seen that after inducing the three types of mesenchymal stem cells with roflumilast for 7 days, the roflumilast-added group (RO group) formed more mineralized nodules compared with the CTRL group, and even more than the osteogenic induction fluid group (OS group), confirming that roflumilast can promote late-stage mineralization and osteogenic formation of cells.

[0055] Example 2: Study on the effect of roflumilast on bone defects

[0056] 2.1 Experimental Methods:

[0057] Eight-week-old SD rats were randomly divided into three groups: a blank control group (CTRL group), a gel group (Gel group), and a gel + roflumilast group (Gel + RO group), with three rats in each group. Each rat had two skull defects, for a total of six skull defects. The SD rats were weighed and their weight recorded. The injection volume of 4% chloral hydrate solution (1 mL / 100g) was then calculated based on the rats' weight. The SD rats were fixed with their bellies facing upwards and heads downwards, concentrating the organs in the upper abdomen to avoid damage during injection. The needle was inserted into the abdominal cavity at a 45° angle to the left of the midline in the lower third of the abdomen. Insertion was stopped upon feeling a loss of sensation, and no blood was aspirated. Chloral hydrate solution was then slowly injected. The SD rats were examined for symptoms such as regular and stable breathing and loss of reflexes to determine if they were anesthetized. A 1-1.5 cm longitudinal incision was made along the forehead above the orbit of the SD rat. After skin preparation, the skin and subcutaneous tissue were incised sequentially down to the periosteum. A blunt instrument was then used to dissect and fully expose the frontal and parietal bones, taking care to protect the dura mater. A circular defect area approximately 5 mm in diameter was carefully cut on both sides of the suture in the SD rat skull using a trephine, avoiding damage to the midline suture and dura mater. The surgical area was continuously irrigated and cooled with 0.9% saline to create a skull defect model. The surgical area was then irrigated with saline to remove bone fragments, and gauze was used for hemostasis. The prepared gels were implanted, and the periosteum was sutured first with absorbable sutures, followed by suturing the surface skin tissue with non-absorbable sutures. Postoperatively, the wound area was disinfected with povidone-iodine to keep it clean, and the rat was fed routinely. Tissue samples were harvested at 1 month and 3 months for micro-CT, HE staining, and Masson staining experiments to detect osteogenic parameters.

[0058] 2.2 Detection Method:

[0059] Micro CT: One and three months after modeling, SD rats were euthanized by overdose anesthesia with chloral hydrate, and their skulls were collected. The skulls were then fixed in 4% paraformaldehyde at 4°C for 24 hours, followed by preservation with 70% alcohol. Micro CT was then used for analysis, employing 70 kV and 110 μA energy at a resolution of 5 μm. The defect area was identified by cylindrical contours, and the bone regeneration volume fraction (BV / TV) within this fixed target volume was calculated, recorded, and statistically analyzed using analysis software.

[0060] Tissue embedding and sectioning: The fixed skull tissue was sequentially placed in 70% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol I → 100% ethanol II → xylene I → xylene II → paraffin I → paraffin II for dehydration and clearing paraffin impregnation. The tissue was then embedded and sectioned to a thickness of 5 μm.

[0061] HE staining: After baking the cut sections in a 60℃ oven for 3-4 hours, remove them and soak them in xylene I, xylene II, 100% ethanol I, 100% ethanol II, 90% ethanol, 80% ethanol, 70% ethanol, and deionized water in sequence. Then, perform hematoxylin staining by soaking the sections in hematoxylin solution and rinsing them with running water until colorless. After that, transfer them to 1% hydrochloric acid ethanol separation solution and soak them for 8-9 seconds. Immediately after that, soak them in deionized water to regain their blue color. Then, transfer them to eosin solution for staining and rinse them with running water until colorless. Dehydrate and clear the sections again, mount them with neutral resin, and then place them in a bellows to air dry naturally. Finally, observe and photograph them under an upright microscope at different magnifications.

[0062] Masson staining: After dewaxing and hydration, sections were stained with hematoxylin and eosin (HE), then stained in hematoxylin solution for 5-10 minutes, rinsed with running water, differentiated in 1% hydrochloric acid for a few seconds, rinsed with running water for 5 minutes, then immersed in Masson's compound staining solution (Orange G, Ponceau S, Acid Fuchsin) for 5-10 minutes, rinsed with running water, treated with 1% phosphotungstic acid solution for about 5 minutes, stained with brilliant green solution for 5 minutes, treated with 1% glacial acetic acid solution for 1 minute, dehydrated repeatedly with 95% ethanol, then dehydrated with anhydrous ethanol, cleared with xylene, mounted with neutral resin, and observed and photographed under a microscope.

[0063] 2.3 Experimental Results:

[0064] MicroCT results of skull defects in SD rats are as follows: Figure 5 As shown. From Figure 5 It can be seen that in the first and third months after treatment, the different groups showed varying degrees of bone repair effects, with the Gel+RO group exhibiting a significantly better effect in promoting new bone formation. Further analysis using the bone volume to tissue volume ratio (BV / TV) reflected the amount of bone, such as... Figure 6 As shown, the results indicate that in the first and third months after treatment, the bone mass increase was more significant in the Gel+RO group, and the BV / TV was significantly increased.

[0065] HE staining of the cranial tissue after sectioning is as follows Figure 7 As shown. From Figure 7 It can be seen from the HE staining results that light pink collagen fibers and muscle fibers were visible in the CTRL group after 1 and 3 months, but there was no obvious new bone formation. In the Gel group and Gel+RO group, new bone formation was visible in addition to fibrous tissue, and the area of ​​new bone formation was the largest in the Gel+RO group.

[0066] Masson staining of cranial tissue after sectioning is as follows Figure 8 As shown. From Figure 8It can be seen that the color development of Masson's three colors is related to the maturity of collagen in bone tissue. Blue-green represents collagen fibers and newly formed bone tissue, while red represents muscle and mature bone tissue. The Masson staining results of this experiment show that after 1 and 3 months, the newly formed bone tissue in the Gel+RO group was significantly more than that in the CTRL group and the Gel group.

[0067] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. The application of roflumilast in the preparation of drugs that promote bone defect repair, characterized in that, Roflumilast is the sole active ingredient in the drug that promotes bone defect repair.

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