Use of small molecule inhibitor BAY-593 for the preparation of a medicament for the treatment and / or prevention of keloids

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to application of small molecule inhibitor BAY-593 in preparation of a drug for treating and / or preventing keloid. The BAY-593 can significantly promote the apoptosis of fibroblasts in keloid tissue and inhibit cell proliferation, and the volume of the keloid tissue can be significantly reduced by local injection of the BAY-593 in animals, so that the BAY-593 is expected to become an effective drug selection for treating keloid.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of the small molecule inhibitor BAY-593 in the preparation of drugs for the treatment and / or prevention of keloids. Background Technology

[0002] Keloids are pathological scars caused by abnormal healing after skin injury. Their core pathological features include excessive fibroblast proliferation and massive extracellular matrix deposition. Unlike ordinary scars, keloids often extend beyond the original wound boundaries, exhibiting a continuous progression. Clinically, they present as nodular, cord-like, or sheet-like masses raised above the skin surface, with a firm texture, significant congestion, and often accompanied by marked pain and itching. Besides affecting appearance, keloids can also have a significant negative impact on a patient's psychological state and quality of life; in severe cases, local contracture can lead to functional impairment or deformity. Due to poor tissue elasticity and fragile texture, the scar surface is prone to cracking, increasing the risk of local infection, such as folliculitis or boils. These infections are not only difficult to control but may also further stimulate scar hyperplasia, forming a vicious cycle of "infection-hyperplasia."

[0003] Currently, commonly used clinical treatments include surgical resection, local injection of glucocorticoids, anti-proliferative drug therapy, and physical therapy. However, these methods generally have certain limitations: simple surgical resection has a high recurrence rate, usually requiring postoperative radiotherapy; while drug therapy faces problems such as large individual differences in efficacy and a limited range of available drugs. Therefore, developing more effective and controllable local treatment strategies has become an urgent clinical need. Summary of the Invention

[0004] To develop a treatment strategy for keloids, this invention provides the application of the small molecule inhibitor BAY-593 in the preparation of drugs for the treatment and / or prevention of keloids. In this invention, BAY-593 can significantly promote apoptosis of fibroblasts in keloid tissue and inhibit cell proliferation. Local injection of BAY-593 into animals can significantly reduce the volume of keloid tissue, and it shows promise as an effective drug option for the treatment of keloids.

[0005] This invention provides the use of the small molecule inhibitor BAY-593 in the preparation of drugs for treating and / or preventing keloids, wherein the structural formula of the small molecule inhibitor BAY-593 is as follows: .

[0006] In this invention, BAY-593 can significantly promote apoptosis of fibroblasts in keloid tissue and inhibit cell proliferation. Local injection of BAY-593 in animals can significantly reduce the volume of keloid tissue, and it is expected to become an effective drug option for the treatment of keloids.

[0007] Furthermore, the drug is used to inhibit the proliferation of keloid fibroblasts, induce cell cycle arrest in keloid fibroblasts, and / or promote apoptosis in keloid fibroblasts.

[0008] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0009] Furthermore, the drug dosage form is a buffered salt solution, gel, ointment, cream, or patch.

[0010] Furthermore, the active ingredient of the solution-type drug is the small molecule inhibitor BAY-593, and the concentration of the small molecule inhibitor BAY-593 in the solution-type drug is 0.1 μM to 50 μM.

[0011] Furthermore, the concentration of the small molecule inhibitor BAY-593 in the solution-type drug is 1 μM to 20 μM.

[0012] Furthermore, the concentration of the small molecule inhibitor BAY-593 in the solution-type drug is 10 μM.

[0013] Furthermore, the solvent for the solution-type drug is 1×PBS.

[0014] Furthermore, the drug is administered via local injection or topical application to the skin.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention investigates the therapeutic effect of BAY-593 on keloid fibroblasts. The results show that BAY-593 significantly promotes apoptosis and inhibits cell proliferation in keloid fibroblasts. Local injection of BAY-593 into animals significantly reduces the volume of keloid tissue, suggesting it may be an effective drug option for keloid treatment. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The effect of different concentrations of the small molecule inhibitor BAY-593 on cell viability.

[0018] Figure 2 The effects of the small molecule inhibitor BAY-593 on fibroblast cell morphology and density.

[0019] Figure 3 The effect of the small molecule inhibitor BAY-593 on the cell cycle of fibroblasts; In the figure, A is a flow cytometry scatter plot of the cell cycle of fibroblasts treated with the small molecule inhibitor BAY-593; B represents the effect of treatment with the small molecule inhibitor BAY-593 on the proportion of fibroblasts at different cell cycles.

[0020] Figure 4 The effect of the small molecule inhibitor BAY-593 on apoptosis in fibroblasts.

[0021] In the figure, A is a flow cytometry scatter plot of apoptosis in fibroblasts treated with the small molecule inhibitor BAY-593; B is a statistical graph showing the apoptosis rate of fibroblasts treated with the small molecule inhibitor BAY-593.

[0022] Figure 5 The effect of the small molecule inhibitor BAY-593 on the volume of keloid-like tissue; In the figure, A represents the status of keloid xenografts after treatment with the small molecule inhibitor BAY-593; B is a statistical graph showing the effect of the small molecule inhibitor BAY-593 on the volume of keloid xenografts. Detailed Implementation

[0023] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0024] Example 1: Application of the small molecule inhibitor BAY-593 in the preparation of drugs for treating keloids.

[0025] I. Experimental Methods 1. Primary cell culture and cell passage This invention uses fibroblasts derived from keloid scars to obtain primary cells. The specific steps are as follows: Surgically excised keloid tissue was aseptically preserved, transferred to an ultra-clean workbench, and rinsed three times with 1× phosphate-buffered saline (1×PBS). After removing the epidermis and subcutaneous tissue with a sterile scalpel, the tissue was rinsed again. Ophthalmic scissors were used to cut the tissue into small pieces approximately 1mm × 1mm × 1mm in size. These small tissue pieces were evenly placed in 100mm culture dishes pre-coated with fetal bovine serum (F102-01, Nanjing Novizan Biotechnology Co., Ltd.), with approximately 5mm spacing between the pieces. Subsequently, DMEM high-glucose complete medium (SH30022.01, Hyclone) containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to the culture dishes, and the dishes were incubated at 37°C in a 5% CO2 saturated humidity incubator. Regularly observe the cell migration from the tissue block and the cell state. When the migrated cells reach 60% confluence, digest them with 0.25% trypsin, centrifuge at 1000 rpm for 5 min, collect the cell pellet, resuspend the cells in DMEM high-glucose complete medium, and passage the cells at a 1:3 ratio. Use the 6th generation cells in the logarithmic growth phase for subsequent experiments.

[0026] 2. Effects of different concentrations of BAY-593 on fibroblast proliferation In a preliminary experiment comparing the effects of different concentrations of BAY-593 on fibroblast proliferation, cells were seeded in 96-well plates. After cell attachment, they were divided into a control group and a BAY-593 treatment group. The BAY-593 treatment group had different concentration gradients, with the culture medium replaced with DMEM complete medium containing different working concentrations (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, and 20 μM) of BAY-593. The control group was supplemented with DMEM complete medium. Both groups were cultured for 48 hours. After 48 hours, the medium was discarded, and the medium was replaced entirely with DMEM complete medium containing 10% CCK8. The cells were incubated at 37°C for 1 hour, and the absorbance at 450 nm was measured using a microplate reader for subsequent analysis.

[0027] 3. CCK-8 detection With approximately 1×10 per hole 4 Cells were seeded into 96-well plates. Two experimental groups were set up: a control group and a BAY-593 experimental group. The control group received 100 μL of DMEM complete medium per well, while the experimental group received medium containing 10 μM BAY-593 (molecular formula: C). 26 H 31F3N2O3 (molecular weight: 476.53, CAS number: 2413020-56-9, purity: 99.94%) was cultured in DMEM complete medium, with 6 replicates per group, and incubated at 37°C in a 5% CO2 saturated humidity incubator for 48 hours. The medium was then discarded, and 100 μL of DMEM complete medium containing 10% CCK-8 reagent (C000, TargetMol Chemicals) was added to each well. The cells were incubated at 37°C in the dark for 2 hours. The absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0028] 4. Cell cycle assay Cells from the control and experimental groups (treated with 10 μM BAY-593 for 48 hours) were collected, centrifuged at 1000 rpm for 5 min, resuspended in pre-chilled 75% ethanol, and stored overnight at 4°C. After centrifugation at 1000 rpm for 5 min, the liquid was discarded, and cell cycle analysis was performed using a cell cycle assay kit (KGA512, KGI Biotechnology). 500 μL of pre-prepared PI:RNase = 9:1 solution was added, and after thorough mixing, cell cycle analysis was performed using a NovoCyte flow cytometer.

[0029] 5. Apoptosis assay Cells from the control and experimental groups (treated with 10 μM BAY-593 for 48 hours) were collected and centrifuged at 1000 rpm for 5 min. Apoptosis was detected using a cell apoptosis assay kit (559763, BD). Cells were resuspended in 100 μL of 1× Binding Buffer, and the cell suspension was transferred to flow cytometry tubes. 5 μL of Annexin V-PE and 5 μL of AAD were added, and the cells were incubated at room temperature in the dark. Then, 400 μL of 1× Binding Buffer was added and mixed thoroughly. Apoptosis was detected using a NovoCyte flow cytometer (NovoCyte, Agilent Technologies).

[0030] 6. Construction of human tissue xenotransplantation model Count the fibroblasts originating from the keloids, approximately 1 × 10⁻⁶ per dish. 6Cells were seeded in 100 mm culture dishes, with 10 mL of DMEM complete medium added to each dish. The dishes were incubated at 37°C with 5% CO2 saturated humidity for 24 hours. 1 mL of 0.25% (w / v) trypsin was added to each dish for digestion. Once cells began to detach from the culture dish, 1 mL of DMEM complete medium was added to each dish to stop digestion. Cells were collected by pipetting into sterile centrifuge tubes and centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in DMEM complete medium to prepare a cell suspension. The cell suspension was then mixed with Mogengel Matrix (ABW, catalog number 082724) at a 1:1 volume ratio to prepare an injection suspension. Each 100 μL of injection suspension contained 2 × 10⁶ cells. 6 Using a 1mL syringe, 100μL of the above-mentioned injection suspension was injected subcutaneously into the dorsal side of the left and right upper limbs of nude mice, respectively, to construct keloid xenografts (hereinafter referred to as grafts). The day of injection was recorded as day 0. After injection, the nude mice were fed normally, and the graft volume was observed and measured every 2 days. When the graft volume reached 100mm², the graft was considered complete. 3 At approximately 26 days, subsequent medication was administered. The transplant volume was approximately 100 mm². 3 The transplanted mice were randomly divided into two groups. The experimental group received a local injection of insulin in a 10 μM buffered saline solution of BAY-593 prepared with 1×PBS, with a total injection volume of 100 μL. The control group received a single local injection of insulin in 100 μL of 1×PBS. Nude mice were kept in the environment, and the volume changes of the transplanted mice were observed regularly. On day 5 post-injection, the mice were sacrificed, the transplanted mice were removed, photographed, and the transplanted volume was calculated.

[0031] II. Experimental Results: 1. Effects of different concentrations of BAY-593 on cell proliferation Experimental results are as follows Figure 1 As shown, different concentrations of BAY-593 (0.1μM, 0.5μM, 1μM, 5μM, 10μM, 15μM, 20μM) all inhibited cell proliferation and showed statistical differences compared with the control group. Considering the further impact on cell apoptosis and the duration of action in in vivo experiments, a concentration of 10μM was selected for subsequent experiments.

[0032] 2. BAY-593 inhibits cell proliferation. like Figure 2As shown, fibroblasts derived from keloids exhibit a typical elongated spindle-shaped morphology under in vitro culture conditions. Sixth-generation logarithmic growth phase fibroblasts were seeded in 6-well plates. After cell attachment, they were randomly assigned to a control group and an experimental group. The experimental group was cultured in DMEM complete medium containing 10 μM BAY-593 for 48 hours, while the control group was cultured in normal DMEM complete medium for 48 hours. Both groups initially showed good cell condition and similar cell density. After treatment with 10 μM BAY-593 for 48 hours, the experimental group showed a significant decrease in cell number, with some cells exhibiting marked morphological changes, becoming rod-shaped, accompanied by increased cell debris. The control group showed a significant increase in cell number after proliferation, suggesting that BAY-593 inhibits fibroblast proliferation and induces apoptosis.

[0033] 3. BAY-593 induces cell cycle arrest like Figure 3 As shown, after treatment with 10 μM BAY-593 for 48 hours, compared with the control group, the proportion of fibroblasts in both the S and G2 phases decreased, indicating that the cells did not undergo sufficient DNA replication, resulting in cell proliferation disorder, cell cycle arrest, and inhibited proliferation capacity.

[0034] 4. BAY-593 promotes apoptosis. like Figure 4 As shown, compared with the control group, the proportions of early apoptotic cells, late apoptotic cells, and early apoptotic + late apoptotic cells were significantly increased after 48 hours of treatment with BAY-593 (P<0.05), indicating that BAY-593 significantly promotes cell apoptosis.

[0035] 5. Local injection of BAY-593 into keloid tissue can significantly reduce its volume. like Figure 5 As shown, a keloid xenograft model was constructed using Balb / c-Nude nude mice. When the graft volume was approximately 100 mm², 3 At the time of treatment, BAY-593 was injected locally into the grafts. Five days after injection, nude mice were sacrificed, the grafts were removed, and photographs were taken. Observation showed that solid keloid-like tissue formed in both groups, with intact capsules and mostly irregular round shapes. The graft volume measurement results showed that the graft volume after BAY-593 treatment was significantly smaller than that in the control group (P<0.05).

[0036] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0037] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The use of the small molecule inhibitor BAY-593 in the preparation of drugs for the treatment and / or prevention of keloids, characterized in that, The drug is used to inhibit the proliferation of keloid fibroblasts, induce cell cycle arrest in keloid fibroblasts, and / or promote apoptosis of keloid fibroblasts. The structural formula of the small molecule inhibitor BAY-593 is as follows: 。 2. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

3. The application according to claim 2, characterized in that, The drug dosage form is a solution, gel, ointment, cream, or patch.

4. The application according to claim 3, characterized in that, The active ingredient in the solution-type drug is the small molecule inhibitor BAY-593, and the concentration of the small molecule inhibitor BAY-593 in the drug is 0.1 μM to 50 μM.

5. The application according to claim 4, characterized in that, The concentration of the small molecule inhibitor BAY-593 in the solution-type drug is 1 μM to 20 μM.

6. The application according to claim 5, characterized in that, The concentration of the small molecule inhibitor BAY-593 in the solution-type drug is 10 μM.

7. The application according to claim 6, characterized in that, The solvent for the solution-type drug is 1×PBS.

8. The application according to claim 1, characterized in that, The medication is administered via local injection or topical application to the skin.

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