Pharmaceutical composition, preparation method and application thereof and medicine for promoting wound healing

Abstract

The invention relates to the technical field of biological medicines, and discloses a pharmaceutical composition, a preparation method and application thereof, and a medicine for promoting wound healing. The pharmaceutical composition comprises a cathepsin K inhibitor and an exosome. Through combined use of the cathepsin K inhibitor and the exosome, especially the exosome derived from mesenchymal stem cells, the defects of an existing diabetes wound treatment method are overcome, collagen degradation is reduced, and collagen deposition is promoted; angiogenesis is enhanced, and wound blood supply is improved; the cell survival rate is improved; energy metabolism is recovered, and cell functions are enhanced. Moreover, the purposes of repairing and healing wounds more efficiently can be achieved on the premise that the dosage is smaller, the production cost is reduced, and the treatment feasibility and clinical popularization value are improved.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a pharmaceutical composition, its preparation method and application, and a medicine for promoting wound healing. Background Technology

[0002] The skin is the largest organ in the human body. Once its integrity is damaged, the normal repair process is a complex and orderly biological process, typically including the inflammatory phase, the proliferative phase (granulation tissue formation phase), and the remodeling phase (matrix formation and scarring phase). The successful completion of this process depends on the precise regulation of various cells (such as immune cells, fibroblasts, vascular endothelial cells, and keratinocytes) and bioactive factors (such as growth factors and cytokines). However, various factors can lead to imbalances in this process, resulting in healing disorders.

[0003] Clinically, wounds are mainly classified into acute wounds and chronic wounds. Acute wounds (such as surgical incisions, trauma, and burns) usually follow a normal healing sequence, but their healing quality is crucial, with the goal of rapid closure, pain reduction, and minimal scar formation. Chronic wounds (such as diabetic foot ulcers, venous leg ulcers, pressure sores, and difficult-to-heal wounds caused by severe infection or ischemia) are stalled in the inflammatory or proliferative phase and cannot enter the normal healing process. The microenvironment of chronic wounds is typically characterized by a persistent inflammatory state, high levels of protease activity, recurrent or persistent bacterial biofilm infection, tissue hypoxia, and cellular dysfunction.

[0004] Taking diabetic wounds as an example, diabetes is a global chronic metabolic disease characterized by persistently elevated blood glucose levels, leading to microvascular complications and decreased tissue repair capacity. Diabetic patients often experience difficulty healing skin wounds, particularly diabetic foot ulcers (DFU), one of the most serious complications of diabetes. Due to its chronic and slow-healing nature, DFU severely impacts patients' quality of life and can even lead to amputation and death. Current treatments for diabetic wounds mainly include: blood glucose control (such as insulin therapy); infection prevention and debridement (such as antibiotics and surgery); and novel biomaterials (such as hydrogel dressings and tissue engineering techniques). However, these methods cannot effectively improve the healing speed and quality of diabetic wounds, and patients still face a high risk of amputation. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a pharmaceutical composition and a method for preparing the same, such that the pharmaceutical composition can significantly reduce collagen degradation, promote angiogenesis, and restore cellular energy metabolism, thereby accelerating wound healing; Another objective of this application is to provide the application of the above-mentioned pharmaceutical composition in the preparation of wound repair and healing products, and to provide a medicine for promoting wound healing based on the above-mentioned pharmaceutical composition.

[0006] In order to solve the above-mentioned technical problems / achieve the above-mentioned objectives, or at least partially solve the above-mentioned technical problems / achieve the above-mentioned objectives, as a first aspect of this application, a pharmaceutical composition is provided, comprising a cathepsin K inhibitor and exosomes.

[0007] Optionally, the mass ratio of the cathepsin K inhibitor to exosomes is 1:3500 to 1:5.

[0008] Further optionally, the cathepsin K inhibitor includes one or more of RNA inhibitors, natural product inhibitors, and small molecule compound inhibitors. The small molecule compound inhibitor includes cathepsin K inhibitor 2.

[0009] Optionally, the exosomes include mesenchymal stem cell exosomes, fibroblast exosomes, and one or more engineered exosomes thereof; the engineered modification includes loading mesenchymal stem cell exosomes and fibroblast exosomes with a small nucleic acid molecule inhibitor of cathepsin K, such as miRNA, siRNA, shRNA, antisense oligonucleotide, or any combination thereof. The mesenchymal stem cell exosomes include, but are not limited to, one or more of umbilical cord mesenchymal stem cell exosomes, bone marrow mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes.

[0010] As a second aspect of this application, a method for preparing the pharmaceutical composition as described in this application is provided, comprising: Exosomes were isolated from cells using differential ultracentrifugation or density gradient centrifugation. The exosomes and cathepsin K inhibitor are combined to form the pharmaceutical composition.

[0011] As a third aspect of this application, the use of the pharmaceutical composition described herein in the preparation of products for wound repair and / or prevention of wound deterioration is provided.

[0012] Optionally, the wound repair and healing includes acute wound repair and healing and chronic wound repair and healing; the chronic wound repair and healing includes diabetic wound repair and healing, pressure ulcer wound repair and healing, and ulcerative wound repair and healing.

[0013] As a fourth aspect of this application, a medicament is provided that promotes wound healing and / or prevents wound deterioration, comprising the pharmaceutical composition described in this application, and pharmaceutically acceptable excipients.

[0014] As a fifth aspect of this application, a wound healing and / or wound deterioration prevention kit is provided, comprising: (a) A container containing the pharmaceutical composition described in this application; and (b) A device for administering the pharmaceutical composition described in this application; and / or (c) A description of the method of using the kit to repair wounds and / or prevent wound deterioration.

[0015] This application overcomes the shortcomings of existing diabetic wound treatments by combining cathepsin K inhibitors and exosomes, particularly mesenchymal stem cell-derived exosomes, to reduce collagen degradation and promote collagen deposition; enhance angiogenesis and improve wound blood supply; increase cell survival rate; restore energy metabolism and enhance cell function. Furthermore, it achieves more efficient wound repair and healing with lower dosage, reducing production costs and improving the feasibility and clinical application value of the treatment. Attached Figure Description

[0016] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments of this application and their descriptions are used to explain this application and do not constitute an undue limitation of this application. Figure 1 The following are the evidentiary results of hUMSC-EVs in this application: (A) Schematic diagram of the process of extracting hUMSC-EVs by ultracentrifugation; (B) Microscopic morphology image of hUMSC-EVs captured by transmission electron microscopy (TEM), scale bar: 200 nm; (C) Three positive markers and one negative marker of hUMSC-EVs detected by Western blot (WB); (D) Particle size distribution of hUMSC-EVs detected by nanoparticle tracking analysis (NTA). Figure 2 The images show the effects of CTSKI and hUMSC-EVs alone or in combination on wound healing in normal WT mice (C57BL / 6J control group) and db / db mice (diabetic group); (A) Images of wound healing changes over time in normal WT mice and db / db mice (arrows indicate wound area); (B) Curves showing the change in wound area over time in mice. N=6 mice; Statistical significance was assessed using one-way ANOVA followed by Tukey post-hoc test; Data are expressed as mean ± standard error, and significance was marked as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Among them, WT represents the wild-type C57BL / 6J control group, db / db represents the diabetes group, db / db+CTSKI (50pmol, dose 0.0294μg) represents the diabetes + CTSKI group, db / db+hUMSC-EVs (100μg) represents the diabetes + hUMSC-EVs group, and db / db+CTSKI (25pmol, dose 0.0147μg)+hUMSC-EVs (50μg) represents the diabetes + CTSKI + hUMSC-EVs group. Figure 3 The effects of CTSKI and hUMSC-EVs alone or in combination on staining of skin tissue sections are shown; (A) H&E-stained sections of mouse wound skin tissue, scale bar: 500 μm, 50 μm; (B) Immunohistochemistry and Masson staining to label CD31, SMA and collagen in mouse wound skin tissue, scale bar: 100 μm, 50 μm; (CE) The expression of CD31, SMA and collagen was quantified by calculating the area ratio of positive regions, N=3 mice; Statistical significance was assessed by one-way ANOVA followed by Tukey post-hoc test. Data are expressed as mean ± standard error. Significance was marked as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Among them, WT represents the wild-type C57BL / 6J control group, db / db represents the diabetes group, db / db+CTSKI (50pmol, dose 0.0294μg) represents the diabetes + CTSKI group, db / db+hUMSC-EVs (100μg) represents the diabetes + hUMSC-EVs group, and db / db+CTSKI (25pmol, dose 0.0147μg)+hUMSC-EVs (50μg) represents the diabetes + CTSKI + hUMSC-EVs group. Figure 4The images show the effects of CTSKI and hUMSC-EVs alone or in combination on CTSK activity and collagen deposition in HDF and HUVECs damaged by high glucose; (A) Images of the effects of CTSKI and hUMSC-EVs on CTSK activity in HDF and HUVECs observed under a fluorescence microscope, where red represents CTSK activity and blue represents DAPI-labeled cell nuclei, scale bar: 50 μm; (BC) Quantitative analysis of CTSK activity in HDF and HUVECs by calculating the average fluorescence intensity of red fluorescence in the images, N=9 images; Statistical significance was assessed by one-way ANOVA followed by Tukey post-hoc test, data are expressed as mean ± standard error, significance markers are: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; (DE) Detection of extracellular collagen I and III secreted by HDFs using an ELISA kit, N=6 independent replicates; Statistical significance was assessed by one-way ANOVA followed by Tukey post-hoc test. Data are expressed as mean ± standard error. Significance was defined as: *p<0.05, **p<0.01, ***p<0.001. Among them, Control represents the control group, HG represents the high glucose group, HG+CTSKI (0.294μg) represents the high glucose + CTSKI group, HG+hUMSC-Evs (50μg) represents the high glucose + hUMSC-Evs group, and HG+CTSKI (0.147μg)+hUMSC-Evs (25μg) represents the high glucose + CTSKI + hUMSC-Evs group. Figure 5 The images show the effects of CTSKI and hUMSC-EVs alone or in combination on the proliferation of HDF and HUVECs damaged by high glucose; (AB) CCK8 kit was used to detect the cell viability of HDF and HUVECs, with N=6 independent replicates; statistical significance was assessed by one-way ANOVA followed by Tukey post-hoc test, and data are expressed as mean ± standard error. Significance was marked as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; (C) Images of HDF and HUVEC proliferation detected by EdU acquired by fluorescence microscopy, where red indicates the nuclei of newly proliferating cells and blue indicates the nuclei of all cells, scale bar: 250 μm; In this context, Control represents the control group, HG represents the high-sugar group, HG+CTSKI (0.294μg) represents the high-sugar + CTSKI group, HG+hUMSC-Evs (50μg) represents the high-sugar + hUMSC-Evs group, HG+CTSKI (0.147μg) + hUMSC-Evs (25μg) and HG+CTSKI (0.294μg) + hUMSC-Evs (50μg) both represent the high-sugar + CTSKI + hUMSC-Evs group; Figure 6 The figure shows the effects of CTSKI and hUMSC-EVs alone or in combination on the proliferation of HDF and HUVECs under high glucose damage; (AB) by calculation Figure 5 The ratio of red to blue cell nuclei in image C was used to quantify the proliferation of HDF and HUVECs. N=9 images were used. Statistical significance was assessed using one-way ANOVA followed by Tukey's post-hoc test. Data are expressed as mean ± standard error. Significance was defined as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. In this context, Control represents the control group, HG represents the high-sugar group, HG+CTSKI (0.294μg) represents the high-sugar + CTSKI group, HG+hUMSC-Evs (50μg) represents the high-sugar + hUMSC-Evs group, HG+CTSKI (0.147μg) + hUMSC-Evs (25μg) and HG+CTSKI (0.294μg) + hUMSC-Evs (50μg) both represent the high-sugar + CTSKI + hUMSC-Evs group; Figure 7 The image shows the migration of HDF and HUVEC detected by scratch assay using CTSKI and hUMSC-Evs alone or in combination. Scale bar: 500 μm. Among them, Control represents the control group, HG represents the high glucose group, HG+CTSKI (0.294μg) represents the high glucose + CTSKI group, HG+hUMSC-Evs (50μg) represents the high glucose + hUMSC-Evs group, and HG+CTSKI (0.147μg)+hUMSC-Evs (25μg) represents the high glucose + CTSKI + hUMSC-Evs group. Figure 8 The figures show the effects of CTSKI and hUMSC-Evs alone or in combination on the migration of HDF and HUVECs in scratch experiments; (A and B) are calculated. Figure 7The rate of reduction in scratch area quantified the migration of HDF and HUVEC, N=18 images; (C and D) Area under the curve in figures A and B, N=18 images; Statistical significance was assessed using one-way ANOVA followed by Tukey's post-hoc test. Data are expressed as mean ± standard error, and significance was marked as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Among them, Control represents the control group, HG represents the high glucose group, HG+CTSKI (0.294μg) represents the high glucose + CTSKI group, HG+UC-EVs (50μg) represents the high glucose + UC-EVs group, and HG+CTSKI (0.147μg)+UC-EVs (25μg) represents the high glucose + CTSKI + hUMSC-EVs group. Figure 9 The effects of CTSKI and hUMSC-EVs alone or in combination on angiogenesis in HUVECs after high glucose treatment are shown. (A) Images from the angiogenesis experiment show the angiogenesis potential of HUVECs, scale bar: 500 μm; (BD) Quantification of angiogenesis parameters: number of newly formed junctions, number of newly formed nodes, and total branch length in the angiogenesis experiment (N=6 independent experiments). Statistical significance was assessed using one-way ANOVA followed by Tukey post-hoc test. Data are expressed as mean ± standard error, and significance was marked as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. In this context, Control represents the control group, HG represents the high glucose group, HG+CTSKI (0.294μg) represents the high glucose + CTSKI group, HG+hUMSC-Evs (50μg) represents the high glucose + hUMSC-Evs group, and HG+CTSKI (0.147μg)+hUMSC-Evs (25μg) represents the high glucose + CTSKI + hUMSC-Evs group. Detailed Implementation

[0017] This application discloses a pharmaceutical composition, its preparation method, its application, and a medicament for promoting wound healing. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this application. The products, processes, and applications described in this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit, and scope of this application to realize and apply the technology of this application. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.

[0018] It should be noted that, in this document, relational terms such as "first" and "second," "step 1" and "step 2," and "(1)" and "(2)" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. Moreover, the embodiments and features described in this application can be combined with each other without conflict.

[0019] Compared to other types of wound repair, diabetic wounds face more challenges in repair due to their complex pathological environment, such as high glucose-induced oxidative stress, persistent inflammation, impaired angiogenesis, and accelerated collagen degradation. Current treatments for diabetic wounds mainly include: drug therapy (such as antibiotics, anti-inflammatory drugs, and growth factors); physical therapy (such as negative pressure wound therapy and hyperbaric oxygen therapy); and biomaterials (such as hydrogels and dressings). However, these methods cannot effectively improve the healing speed and quality of diabetic wounds, and patients still face a high risk of amputation.

[0020] To address the shortcomings of existing treatments for diabetic wounds, this application provides a pharmaceutical composition comprising a cathepsin K inhibitor (CTSKI) and exosomes (EVs) in its first aspect. During the research process of this application, it was found that CTSKI can only partially improve wound repair but cannot address the problem of insufficient vascular supply, resulting in slow wound healing. Exosomes, due to their heterogeneity and limitations in isolation and purification technology leading to poor batch-to-batch consistency and high costs for large-scale production, are also prone to degradation during storage, have short in vivo half-lives, and insufficient targeting, affecting the stability of therapeutic efficacy and requiring frequent or high-dose use. For example, in the diabetic wound repair experiments using mesenchymal stem cell exosomes (MSC-EVs) in this application, it was found that although they can promote angiogenesis, they are costly, have poor stability, and cannot address the problem of collagen degradation when used alone, nor can they effectively improve the cell repair issues caused by abnormal energy metabolism in diabetic patients.

[0021] Surprisingly, this application showed that when CTSKI and EVs were used in combination, with reduced dosages, they produced higher CTSK inhibition, collagen deposition, and enhanced activity, proliferation, and migration of human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs). This resulted in more efficient promotion of angiogenesis and tissue regeneration at the wound site, accelerating wound healing. A series of experimental results fully demonstrate that the combined use of CTSKI and EVs has a synergistic effect, enhancing each other's capabilities and exhibiting superior repair effects compared to single-component applications.

[0022] In some embodiments of this application, the mass ratio of the cathepsin K inhibitor to exosomes is 1:3500 to 1:5, for example, 1:3500, 1:3400, 1:3300, 1:3200, 1:3100, 1:3000, 1:2900, 1:2800, 1:2700, 1:2600, 1:2500, 1:2400, 1:2300, 1:2200, 1:2100, 1:2000, 1:1900, 1:1800, 1:1700, etc. 1:1600, 1:1500, 1:1400, 1:1300, 1:1200, 1:1100, 1:1000, 1:900, 1:800, 1:700, 1:600, 1:500, 1:400, 1:300, 1:200, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, or any value between any two.

[0023] In some embodiments of this application, the cathepsin K inhibitor includes one or more of RNA inhibitors, natural product inhibitors, and small molecule compound inhibitors. The RNA inhibitors include, but are not limited to, microRNA, siRNA, shRNA, and other RNA molecules and antisense oligonucleotides that can effectively inhibit CTSK generation; the natural product inhibitors include, but are not limited to, plant extracts, mainly including flavonoids such as quercetin and rutin; the small molecule compound inhibitors are chemically synthesized CTSK inhibitors, whose structures typically contain small molecule structures capable of binding to the active site of CTSK, including nitrile groups, ketone groups, and heterocyclic structures (such as pyridine and pyrimidine) such as Odanacatib, Relacatib, and ASP-1706. In other embodiments of this application, the small molecule compound inhibitor includes cathepsin K inhibitor 2, whose chemical structure is as follows:

[0024] In some embodiments of this application, the concentration of the CTSKI solution ranges from 0.0588 mg / L to 1.176 mg / L. In some experiments of this application, suitable concentrations, such as 0.294 mg / L and 0.147 mg / L, were selected for a series of experimental verifications. The solution is typically dissolved in an organic solvent such as DMSO and diluted with PBS to the required working concentration. The concentration of the exosomes ranges from 0.01 g / L to 0.2 g / L.

[0025] In some embodiments of this application, the dose of CTSKI is not higher than 0.5 μg, for example 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.06 μg, 0.07 μg, 0.08 μg, 0.09 μg, 0.10 μg, 0.11 μg, 0.12 μg, 0.13 μg, 0.14 μg, 0.15 μg, 0.16 μg, 0.17 μg, 0.18 μg, 0.19 μg, 0.20 μg, 0.21 μg. The exosome dosage is 0.22 μg, 0.23 μg, 0.24 μg, 0.25 μg, 0.26 μg, 0.27 μg, 0.28 μg, 0.29 μg, 0.30 μg, 0.31 μg, 0.32 μg, 0.33 μg, 0.34 μg, 0.35 μg, 0.36 μg, 0.37 μg, 0.38 μg, 0.39 μg, 0.40 μg, 0.50 μg, or any value between two of these values, wherein the dose of the exosome is not higher than 0.5 mg, for example, 0.01 mg. g, 0.02mg, 0.03mg, 0.04mg, 0.05mg, 0.06mg, 0.07mg, 0.08mg, 0.09mg, 0.10mg, 0.11mg, 0.12mg, 0.13mg, 0.1 4mg, 0.15mg, 0.16mg, 0.17mg, 0.18mg, 0.19mg, 0.20mg, 0.21mg, 0.22mg, 0.23mg, 0.24mg, 0.25mg, 0.26mg, 0 0.27mg, 0.28mg, 0.29mg, 0.30mg, 0.31mg, 0.32mg, 0.33mg, 0.34mg, 0.35mg, 0.36mg, 0.37mg, 0.38mg, 0.39mg, 0.40mg, 0.41mg, 0.42mg, 0.43mg, 0.44mg, 0.45mg, 0.46mg, 0.47mg, 0.48mg, 0.49mg, 0.50mg, or any value between any two.

[0026] In some embodiments of this application, the exosomes include mesenchymal stem cell exosomes, fibroblast exosomes, and one or more of the aforementioned engineered cell exosomes; the engineered modification includes loading the mesenchymal stem cell exosomes and fibroblast exosomes with a small nucleic acid molecule inhibitor of cathepsin K, such as miRNA, shRNA, antisense oligonucleotide, or any combination thereof, for example, any combination of two, three, or four.

[0027] In other embodiments of this application, the mesenchymal stem cell exosomes include, but are not limited to, one or more of umbilical cord mesenchymal stem cell exosomes, bone marrow mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes.

[0028] In some embodiments of this application, the CTSKI and exosomes in the pharmaceutical composition can be stored separately, or they can be directly mixed and stored using a medium that does not impair the activity of CTSKI and exosomes. Stabilizers and other excipients can be added according to the requirements for maintaining the activity of CTSKI and exosomes.

[0029] In a second aspect of this application, a method for preparing a pharmaceutical composition as described in this application is provided, comprising: Exosomes were isolated from cells using differential ultracentrifugation or density gradient centrifugation. The exosomes and cathepsin K inhibitor are combined to form the pharmaceutical composition.

[0030] In some embodiments of this application, human umbilical cord mesenchymal stem cells are used as an example, and exosomes are isolated and extracted from them by differential ultracentrifugation. The human umbilical cord mesenchymal stem cells are cultured at 37°C and 5% CO2 without exotropic fetal bovine serum until 80% confluence, and then centrifuged sequentially at 300g, 2000g, and 10000g, collecting the supernatant at each step. Finally, the cells are centrifuged at 100000g, and the precipitate is collected as human umbilical cord mesenchymal stem cell exosomes (hUMSC-EVs).

[0031] In the third aspect of this application, through in vivo animal experiments (mouse wound model) and in vitro cell experiments, it has been demonstrated that the drug composition method provided in this application can reduce collagen degradation, promote angiogenesis, and restore cellular energy metabolism. It can regulate the wound microenvironment in multiple dimensions and improve the quality of repair. Its wound repair and healing effect is significantly better than that of the single-component experimental group even with reduced dosage, and it greatly saves drug costs.

[0032] In certain embodiments of this application, in a mouse diabetic wound model (db / db mice), the wound healing rate after 10 days of combined treatment with CTSKI and hUMSC-EVs was significantly higher than that of the groups treated with CTSKI or hUMSC-EVs alone (p<0.01). The wound healing rate in the control group (diabetic + PBS) was only 40%–50%, while the healing rate in the combined treatment group reached 85%–90%.

[0033] In other embodiments of this application, immunohistochemical detection of CD31 (a vascular endothelial marker) revealed that the number of CD31-positive vessels in the db / db group (PBS) was lower. The microvessel density in the CTSKI+hUMSC-EVs combined treatment group increased by 35% compared to the hUMSC-EVs-only group and by 2.5 times compared to the control group (p<0.001). This indicates that although CTSKI has no angiogenic capacity, its combination with hUMSC-EVs helps enhance hUMSC-EVs' ability to promote angiogenesis, improve tissue blood supply, further optimize the tissue microenvironment, and make angiogenesis more stable and persistent.

[0034] Based on the aforementioned superior technical effects, the application of the pharmaceutical composition described in this application is provided in the preparation of products for wound repair and / or prevention of wound deterioration.

[0035] In some embodiments of this application, the wound repair and healing includes acute wound repair and healing and chronic wound repair and healing. The wound type has wide applicability and is particularly suitable for various types of chronic, acute and difficult-to-heal wounds in diabetic and non-diabetic individuals. The chronic wound repair and healing includes diabetic wound repair and healing, pressure ulcer wound repair and healing, and ulcerative wound repair and healing.

[0036] In a fourth aspect of this application, a medicament is provided that promotes wound healing and / or prevents wound deterioration, comprising the pharmaceutical composition described in this application, and pharmaceutically acceptable excipients.

[0037] In some embodiments of this application, the drug may be an injectable drug (e.g., lyophilized powder, injection solution), a topical drug (e.g., gel), or a microneedle patch for releasing the pharmaceutical composition of the present invention, etc.; the pharmaceutically acceptable excipients are selected according to the dosage form prepared, such as thickeners, crosslinking agents, stabilizers, pH adjusters, preservatives, etc. The thickeners include, but are not limited to, hydrogels, carbomer, etc.; the crosslinking agents include, but are not limited to, sodium alginate, gelatin, etc.; the stabilizers include, but are not limited to, glycerin, hyaluronic acid, human serum albumin, etc.; the pH adjusters include, but are not limited to, sodium hydroxide, citric acid, etc.; and the preservatives include, sodium benzoate, propylene glycol, etc. By selecting suitable excipients to prepare the drug, the stability, sustained release, and targeting of the pharmaceutical composition of this application can be improved, the administration method optimized, the sustained release of the drug ensured, and the therapeutic effect enhanced.

[0038] In a fifth aspect of this application, a kit for wound healing and / or prevention of wound deterioration is also provided, comprising: (a) A container containing the pharmaceutical composition described in this application; and (b) A device for administering the pharmaceutical composition described in this application; and / or (c) A description of the method of using the kit to repair wounds and / or prevent wound deterioration.

[0039] In a sixth aspect of this application, a method is also provided for repairing and healing a wound or preventing wound deterioration using a preventively effective or therapeutically effective amount of the pharmaceutical composition described in this application, wherein the effective amount refers to any amount that improves the treatment, healing, prevention, or reduction of the disease, condition, or side effects, or slows the rate of disease or condition progression, compared to a corresponding subject who did not receive such a dose. The term also includes, within its scope, amounts that effectively enhance normal physiological function.

[0040] The prevention described in this application includes prevention, and refers to measures or procedures designed to prevent rather than cure or treat a disease. Prevention means reducing the risk that a subject who may be exposed to a pathogenic factor or is susceptible to the disease will acquire or develop at least one clinical symptom of the disease before the onset of the disease.

[0041] The pharmaceutical compositions described in this application are intended for use in mammals, including but not limited to humans, mice, and pet cats.

[0042] In the comparative experiments provided in this application, unless otherwise specified, all experimental conditions and materials remain consistent to ensure comparability. Furthermore, all materials used in this application are commercially available.

[0043] The following provides further details regarding a pharmaceutical composition, its preparation method, its application, and a medicine for promoting wound healing provided in this application.

[0044] Example 1: Pharmaceutical composition of this application 1. Cathepsin K inhibitors The structure of cathepsin K inhibitor 2 (CTSKI2) is as follows:

[0045] The CTSKI2 solution concentration range is 0.0588 mg / L to 1.176 mg / L. Appropriate concentrations such as 0.294 mg / L and 0.147 mg / L are used for validation. CTSKI2 is dissolved in DMSO (final concentration ≤ 0.1%), and then diluted with PBS to the working dose.

[0046] 2. Preparation of human umbilical cord mesenchymal stem cell exosomes (1) Cell culture Cell source: human umbilical cord mesenchymal stem cells (hUC-MSCs); culture medium: exosome-free fetal bovine serum (FBS); culture conditions: 37°C, 5% CO2, cultured until 80% confluence.

[0047] (2) Exosome isolation 300g, 10min: Remove residual cells and collect supernatant; 2000g, 30min: Remove cell debris and collect supernatant; 10000g, 1h: Remove large vesicles and collect supernatant; 100000g, 2h: Ultracentrifuge to collect precipitate and exosomes. Protein concentration was determined using a BCA kit to ensure hUMSC-EVs purity. Concentration was diluted to 0.01g / L to 0.2g / L and stored at -80℃. The preparation flowchart is shown below. Figure 1 A.

[0048] (3) Exosome identification Morphology of UC-EVs (diameter 30 ~ 150 nm) observed by transmission electron microscopy (TEM) is shown in [see image]. Figure 1 B. Western blot detection of exosome markers (CD63, Alix, TSG101, negative control GAPDH), see [link to Western blot analysis]. Figure 1 C. Nanoparticle tracking analysis (NTA) measures particle size distribution, see... Figure 1 D.

[0049] according to Figure 1 The results show that this application successfully isolated and extracted exosomes from human umbilical cord mesenchymal stem cells.

[0050] Example 2: In vivo animal experiments (1) Laboratory animals C57BL / 6J wild-type mice (control group); db / db diabetic mice (experimental group). Mice age: 7 weeks, weight 20-25g. Husbandry conditions: 25°C, 12h light / dark cycle, free access to food.

[0051] (2) Wound model After anesthesia, a 0.8 cm diameter full-thickness skin wound was created on the back of the mouse by using a 6 mm skin biopsy device.

[0052] (3) Processing grouping Table 1

[0053] In vivo experiments used wild-type C57BL / 6J mice as the control group, and db / db mice as the other four groups. The wounds of the control group and one group of db / db mice were treated with 50 μL PBS at the same time daily. The other three groups of db / db mice were administered CTSKI, hUMSC-EVs, and a mixture of CTSKI and hUMSC-EVs at half the dose (total injection volume 50 μL for each group) daily using the same method. Male C57BL / 6J and db / db mice were anesthetized, and a circular wound with a diameter of 0.8 cm was created on the back of the mice. Administered medication intradermally around the wound daily using a microsyringe. Mouse weight was measured every two days, and the wound was photographed. On day 10 of modeling, three mice from each group were randomly selected and euthanized. Skin wounds and surrounding skin tissue were collected for paraffin sectioning, immunohistochemistry, and HE staining.

[0054] (4) Evaluation indicators Wound healing rate (photographs taken, wound area calculated using ImageJ).

[0055] Histological analysis: HE staining (to observe tissue morphology); Masson staining (to assess collagen deposition); immunohistochemistry (CD31) to assess angiogenesis.

[0056] (5) Results and Analysis Wound healing rate: such as Figure 2 A and Figure 2 As shown in Figure B, regular monitoring of the wound area on the back of mice indicated that, without intervention, the wound healing process in db / db mice (diabetic group) was significantly delayed compared to normal WT mice (C57BL / 6J control group). The back wounds in db / db mice healed completely by day 16, while the wounds in normal WT mice healed completely by day 12. Administration of CTSKI or hUMSC-EVs alone accelerated wound healing in db / db mice. Back wounds in db / db mice treated with either CTSKI or hUMSC-EVs alone healed completely by day 14. Combined administration of CTSKI and hUMSC-EVs further accelerated wound healing in db / db mice, with a stronger promoting effect than either treatment alone. Wounds in db / db mice treated with a combined half-dose of CTSKI and UC-EVs reached complete closure by day 12.

[0057] Wound area was calculated using ImageJ. In a mouse diabetic wound model, the wound healing rate was significantly higher after 10 days of combined treatment with half-dose CTSKI and hUMSC-EVs than that of CTSKI or hUMSC-EVs alone (p<0.01). The wound healing rate in the diabetic group was only 40%–50%, while the healing rate in the half-dose combined treatment group reached 85%–90%, comparable to the repair and healing level in normal WT mice.

[0058] Histological analysis: Figure 3 As shown, in addition to observing changes in wound area visually, this application also collected wound skin tissue for histological section staining and observed the changes in skin caused by different treatments at the tissue level. HE staining of mouse wound skin tissue revealed that, compared to normal C57BL / 6J mice, db / db mice had thinner granulation tissue with numerous dense vacuoles; treatment with CTSKI or hUMSC-EVs alone increased granulation tissue thickness and reduced vacuoles. When CTSKI and hUMSC-EVs were used in combination, the thickness of the granulation tissue increased compared to treatment with either drug alone, and the vacuoles became even more sparse (E). CD31 immunohistochemical staining revealed reduced angiogenesis in db / db mice compared to normal C57BL / 6J mice, while angiogenesis increased after intervention with CTSKI or hUMSC-EVs alone. Combined use of half-dose CTSKI and hUMSC-EVs resulted in slightly more angiogenesis in the granulation tissue than treatment with either CTSKI or hUMSC-EVs alone. Compared with normal C57BL / 6J mice, administration of CTSKI and hUMSC-EVs alone or in combination with half the dose of CTSKI and hUMSC-EVs resulted in decreased and increased α-SMA expression in granulation tissue of db / db mice, with no statistically significant differences among the three groups. Masson staining showed that collagen deposition in db / db mice was significantly lower than in normal C57BL / 6J mice, and collagen deposition was significantly improved after administration of CTSKI and hUMSC-EVs alone. The collagen deposition effect was even more pronounced when half the dose of CTSKI and hUMSC-EVs was used in combination. These findings indicate that the combined use of CTSKI and hUMSC-EVs also has a more significant promoting effect on granulation tissue, CD31, α-SMA, and collagen deposition in the skin tissue of diabetic mice than its use alone. (Reduced CD31 in diabetic wounds leads to insufficient angiogenesis; while abnormal expression of SMA may affect wound contraction.) Example 3: In vitro cell experiments (1) Cell culture Human dermal fibroblasts (HDF): To observe the skin matrix repair and extracellular matrix (ECM) remodeling process and to verify the effect of treatment on fibrosis regulation and tissue regeneration in diabetic wounds; Human umbilical vein endothelial cells (HUVECs): To assess angiogenesis and endothelial barrier function, and to verify whether treatment improves blood supply and oxygen / nutrient delivery in diabetic wounds by promoting vascularization. Culture media: HDF: RPMI-1640 + 10% FBS; HUVEC: ECM-specific medium; High glucose treatment: 50mM glucose incubation for 24h to simulate a diabetic environment. For a 24-well plate, each well contains 0.5 ml of culture medium.

[0059] (2) Group processing Table 2

[0060] In in vitro experiments, HDFS and HUVECS were divided into 5 groups. The control group did not receive high glucose treatment, while the other 4 groups used a culture medium containing 50 mM glucose to simulate the hyperglycemic environment of diabetes. When drug intervention was performed, the cell culture medium of the control group and one high glucose treatment group was supplemented only with PBS, while the other 3 groups were supplemented with CTSKI, hUMSC-EVs, and a mixture of CTSKI and hUMSC-EVs at half the dose, respectively. The total injection volume was the same for all groups.

[0061] (3) Evaluation indicators: cell proliferation capacity (CCK-8, EdU test); cell migration capacity (scratch test); angiogenesis capacity (angiogenesis test); CTSK activity detection.

[0062] (4) Results and Analysis Figure 4 As shown in the AC experiment, CTSK activity was significantly increased in high-glucose-treated HDF and HUVECs. Intervention with either CTSKI or hUMSC-EVs alone reduced CTSK activity. The combined use of half-dose CTSKI and hUMSC-EVs showed a stronger inhibitory effect on CTSK activity compared to using either drug alone.

[0063] DE used an ELISA kit to detect Col I and Col III collagen secreted by cultured HDF cells. The results showed that high glucose treatment significantly reduced Col I and Col III secretion. Treatment with CTSKI or hUMSC-EVs alone significantly increased Col I and Col III secretion, but failed to return to normal levels (p<0.05). When CTSKI and hUMSC-EVs were used in combination at half the dose, Col I and Col III increased even more than when used alone, essentially returning to normal levels. These results indicate that the combined use of CTSKI and hUMSC-EVs can more effectively and synergistically inhibit CTSK activity, thus promoting collagen deposition.

[0064] Figure 5 and Figure 6The study demonstrated the effects of CTSKI and hUMSC-EVs alone or in combination on the proliferation of HDF and HUVECs damaged by high glucose. CCK8 assays of HDF and HUVEC viability revealed a significant decrease in cell viability after high glucose treatment. However, the combined use of CTSKI and hUMSC-EVs at half the dose significantly enhanced cell viability compared to using either alone, indicating a synergistic effect.

[0065] Red fluorescent labeling of newly proliferating HDF and HUVECs with EdU revealed that high glucose treatment led to a decrease in proliferating cells. The combined use of CTSKI and hUMSC-EVs significantly increased the number of newly proliferating cells compared to either treatment alone. These results indicate that the combined use of CTSKI and hUMSC-EVs, at half the dose, promotes cell proliferation more effectively than either treatment alone, demonstrating a synergistic effect.

[0066] Figure 7 and Figure 8 As shown, the migration ability of cells under different treatments was evaluated using a scratch-and-migration assay. The results of the scratch assay indicated that high glucose treatment significantly reduced cell migration ability. However, the combined use of CTSKI and hUMSC-EVs, at half the dose, significantly promoted cell migration compared to using either treatment alone, demonstrating a synergistic effect.

[0067] Figure 9 As shown, angiogenesis assays were used to detect changes in the tube-forming ability of HUVECs after different treatments. High glucose treatment significantly reduced the number of newly formed junctions, the number of new nodes, and the total branch length. The combined use of CTSKI and hUMSC-EVs also showed a stronger enhancing effect on these three indicators than either treatment alone. These results indicate that the combined use of CTSKI and hUMSC-EVs, even at half dose, has a superior promoting effect on angiogenesis compared to either treatment alone, demonstrating a synergistic effect.

[0068] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A pharmaceutical composition, characterized by, The composition comprises a Cathepsin K inhibitor and an exosome.

2. The pharmaceutical composition of claim 1, wherein, The mass ratio of the Cathepsin K inhibitor and the exosome is 1:3500~1:

5.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The Cathepsin K inhibitor comprises one or more of an RNA inhibitor, a natural product inhibitor, and a small molecule compound inhibitor.

4. The pharmaceutical composition of claim 3, wherein, The small molecule compound inhibitor comprises Cathepsin K inhibitor 2.

5. The pharmaceutical composition according to claim 1 or 2, characterized by, The exosome comprises one or more of a mesenchymal stem cell exosome, a fibroblast cell exosome, and an engineered exosome of the foregoing cells; the engineering comprises loading the mesenchymal stem cell exosome or the fibroblast cell exosome with a nucleic acid small molecule inhibitor of Cathepsin K, including miRNA, siRNA, shRNA, antisense oligonucleotide, or any combination thereof.

6. The pharmaceutical composition of claim 5, wherein, The mesenchymal stem cell exosome comprises one or more of an umbilical cord mesenchymal stem cell exosome, a bone marrow mesenchymal stem cell exosome, and an adipose tissue mesenchymal stem cell exosome.

7. A process for the preparation of a pharmaceutical composition as claimed in claim 1, characterized in that, The composition comprises: The exosome is isolated from the cell by differential ultracentrifugation or density gradient centrifugation; The combination of the exosome and the Cathepsin K inhibitor forms the pharmaceutical composition.

8. Use of the pharmaceutical composition of any one of claims 1-6 in the manufacture of a product for wound repair and / or prevention of wound deterioration.

9. A medicament for promoting wound healing and / or preventing wound deterioration, characterized in that, The pharmaceutical composition of any one of claims 1-6, and a pharmaceutically acceptable excipient.

10. A kit for wound healing and / or prevention of wound deterioration, characterized in that, The composition comprises: (a) a container containing the pharmaceutical composition of any one of claims 1-6; and (b) a device for administering the pharmaceutical composition of any one of claims 1-6; and / or (c) instructions for using the kit for repairing a wound and / or preventing wound deterioration. The composition comprises:

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