Application of Gemmadone in the Preparation of Drugs for Treating Diabetic Ulcers

Abstract

This invention relates to the field of pharmaceutical technology, specifically the application of gemmaconazole in the preparation of drugs for treating diabetic ulcers. This invention is the first to propose that gemmaconazole has a therapeutic effect on diabetic ulcers. Using a mouse model of diabetic ulcers and the human vascular endothelial cell line HUVECs, the effects of gemmaconazole on wound healing in diabetic ulcer model mice, as well as its effects on in vitro cell-mediated diabetic ulcer healing indices, were studied. The results show that gemmaconazole has a significant therapeutic effect on diabetic ulcers, providing important evidence for future clinical development of efficient strategies for treating diabetic ulcers and the preparation of drugs for treating diabetic ulcers.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of gemmaconazole in the preparation of drugs for treating diabetic ulcers. Background Technology

[0002] The global burden of diabetes is rising rapidly, with the number of people living with diabetes now exceeding 800 million, more than four times the number in 1990. This expanding epidemic has led to a significant increase in diabetes-related complications, particularly diabetic ulcers. Diabetic ulcers are a major precursor to lower limb amputation, with an extremely poor prognosis. The mortality rate within five years of developing a diabetic ulcer is approximately 30%, and can exceed 50% if accompanied by a major amputation, a mortality rate comparable to many common malignant tumors. Diabetic ulcers impose a heavy economic burden; it is estimated that in the United States alone, the direct medical costs for treating diabetic ulcers amount to between $9 billion and $13 billion annually. Although multidisciplinary intervention strategies have made some progress in recent years (including surgical debridement, systemic anti-infective therapy, and decompression therapy), their efficacy in treating diabetic ulcers remains limited. Therefore, there is an urgent need to develop new, low-cost, safe, and effective treatment strategies to address this increasingly serious global health challenge.

[0003] In the normal skin wound repair process, vigorous angiogenesis is a hallmark event of the proliferative phase, and the proliferation, migration, and luminal remodeling of vascular endothelial cells are crucial for wound repair. However, multiple inflammatory signals are abnormally activated in diabetic ulcers, leading to a significant increase in the expression of pro-inflammatory cytokines such as IL-6 and IL-1β. This amplifies and maintains the local inflammatory response, causing the wound to remain in the inflammatory phase for a prolonged period. This results in endothelial cell dysfunction, significantly inhibits angiogenesis, and further delays the wound healing process.

[0004] Natural products possess unique therapeutic advantages and relatively mild adverse reactions, demonstrating significant therapeutic potential in various disease models. Myrrh, a classic resin-based medicine in traditional Chinese medicine, is rich in various bioactive components in its resin-gel mixture, such as furanones-1,3-diene, turmericene, β-elemene, and gemmaconone. Among these components, gemmaconone has attracted widespread attention due to its outstanding anti-inflammatory and antioxidant activities. Previous studies have shown that germacrone exhibits significant anti-inflammatory effects in various disease models, including acute lung injury, rheumatoid arthritis, and cardiac remodeling. It can improve disease progression by regulating inflammatory signaling pathways and reducing tissue damage. (Wang Y, He X, Zhang H, Hu W. Germacrone ameliorates acute lung injury induced by intestinal ischemia-reperfusion by regulating macrophage M1 polarization and mitochondrial defects. Actabiochimica et biophysica Sinica 2024;57(2):261-73. Tan T, Huang Q, Chu W, LiB, Wu J, Xia Q, Cao X. Delivery of germacrone (GER) using macrophages-targeted polymeric nanoparticles and its application in rheumatoid arthritis. Drug delivery 2022;29(1):692-701. Fang Z, Yushanjiang F, Wang G, Zheng X, Jiang X. Germacrone mitigates cardiac remodeling by regulating PI3K / AKT-mediated oxidative stress, inflammation, and apoptosis. Internationalimmunopharmacology 2023;124(Pt A):110876.).

[0005] However, there are no reports in this field regarding the correlation between gemmadone and the treatment of diabetic ulcers, and the development of new drugs for the treatment of diabetic ulcers is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide a novel use of gemmaconazole in the preparation of medicaments for treating diabetic ulcers.

[0007] This invention utilizes network pharmacology analysis to discover potential targets for the treatment of diabetic ulcers with gemmadone. Through KEGG enrichment analysis, two classic angiogenesis-related pathways (VEGF signaling pathway and Hedgehog signaling pathway) were significantly enriched. Studies have confirmed that impaired VEGF signaling is a significant cause of impaired angiogenesis and poor healing in diabetic ulcers (Huang K, Mi B, Xiong Y, Fu Z, Zhou W, Liu W, Liu G, Dai G. Angiogenesis during diabetic wound repair: from mechanism to therapy opportunity. Burns & Trauma 2025;13:tkae052.). Meanwhile, the Hedgehog signaling pathway plays a crucial regulatory role in angiogenesis and wound repair, and its activation helps promote endothelial cell proliferation, migration, and angiogenesis (Wang J, Zhan H, Wang M, Song H, Sun J, Zhao G. Sonic hedgehog signaling promotes sangiogenesis of endothelial progenitor cells to improve pressure ulcershealing by PI3K / AKT / eNOS signaling. Aging 2023;15(19):10540-8. Luo JD, Hu TP, Wang L, Chen MS, Liu SM, Chen AF. Sonic hedgehog improves delayed woundhealing via enhancing Cutaneous nitric oxide function in diabetes. American Journal of Physiology Endocrinology and Metabolism 2009;297(2):E525-31.). This suggests that gemmaconazole may exert its therapeutic effect on diabetic ulcers by promoting angiogenesis and inhibiting local inflammatory responses.

[0008] A first aspect of the present invention provides the use of gemmaconazole in the preparation of a medicament for treating diabetic ulcers.

[0009] Furthermore, the molecular formula of the said gemmaconone is C2 15 H 22 O, its chemical structural formula is shown in Formula I below:

[0010]

[0011] Formula I.

[0012] This invention does not specifically limit the source of gemmaconazole; it can be obtained through extraction and preparation or purchased commercially.

[0013] Furthermore, gemmaconazole works by promoting the healing and reepithelialization of diabetic ulcer wounds, promoting the proliferation of human vascular endothelial cells, inhibiting the expression of inflammatory factors in human vascular endothelial cells, and promoting angiogenesis in diabetic ulcer wounds.

[0014] Furthermore, in the aforementioned applications, gemmaconazole is used in the preparation of drugs that promote the healing and reepithelialization of diabetic ulcer wounds.

[0015] Furthermore, in the aforementioned applications, gemmaconazole is used in the preparation of drugs that promote the proliferation of human vascular endothelial cells.

[0016] Furthermore, in the aforementioned applications, gemmaconazole is used in the preparation of drugs that inhibit the expression of inflammatory factors in human vascular endothelial cells.

[0017] Furthermore, in the aforementioned applications, gemmaconazole is used in the preparation of drugs that promote angiogenesis in diabetic ulcer wounds.

[0018] In a second aspect, the present invention provides a medicament for treating diabetic ulcers, wherein the medicament uses gemmaconazole as the sole active ingredient.

[0019] Furthermore, the drug is a topical preparation, and even further, the drug also includes excipients commonly used in topical preparations.

[0020] This invention does not impose any special limitations on the types and specific sources of excipients commonly used in topical preparations; any excipients commonly used in topical preparations in this field may be used.

[0021] Furthermore, the effective concentration of gemcitabine in the drug is 2.5 mM in vivo and 5 μM in vitro.

[0022] The advantages of this invention are:

[0023] 1. This invention is the first to propose that the traditional Chinese medicine monomer gemcitabine has the effect of treating diabetic ulcers and has achieved significant therapeutic effects, providing a basis for the future clinical development of efficient strategies for treating diabetic ulcers.

[0024] 2. This invention uses a mouse diabetic ulcer model and human vascular endothelial cell line HUVECs to study the effect of gemmaconazole on wound healing in diabetic ulcer model mice, as well as its effect on diabetic ulcer healing indicators in vitro, providing important experimental evidence for the preparation of drugs to treat diabetic ulcers. Attached Figure Description

[0025] Figure 1 The effect of different concentrations of gemmadone on wound healing in diabetic ulcer mice. p <0.001, p <0.01, p <0.05;

[0026] Figure 2 The effect of gemmaconazole on wound healing in diabetic ulcer mice is shown in Figure a, where Figure a shows the results of wound healing photographs and the calculated wound healing rate, and Figure b shows the results of HE staining and the quantitative calculation of HE (scale bar = 250 μm). p <0.001, p <0.01, p <0.05;

[0027] Figure 3 To predict potential targets of gemmaconazole using network pharmacology, Figure a shows the intersection of gemmaconazole targets and diabetic ulcer-related targets, Figure b shows the "compound-target-disease" interaction network constructed from the intersection targets, and Figure c shows the KEGG pathway enrichment analysis results of the intersection targets.

[0028] Figure 4 Figure a shows the effect of gemmaconazole on the proliferation of HUVECs cells; Figure b shows the effect of gemmaconazole on the expression of inflammatory factors in HUVECs cells. p <0.001, p <0.01, p <0.05;

[0029] Figure 5 Effects of gemmaconazole on the expression of F4 / 80, NF-κB p50, PCNA, PTCH1, SHH, GSK3B, CD31 and VEGF-A in the wound tissue of diabetic mice with ulcers (scale bar = 50 μm). p <0.001, p<0.01, p <0.05. Detailed Implementation

[0030] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0031] Unless otherwise specified, the following embodiments are all conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] Example 1: Effect of Gemmadone on Wound Healing in Diabetic Ulcers in Mice

[0034] 1. Animals: Forty male C57BL / 6J mice, aged 6-8 weeks, were used in the animal experiments. Twenty mice were used for the preliminary experiments, and twenty for the formal experiments. The experimental animals were housed at the Experimental Animal Center of Shanghai Skin Disease Hospital and purchased from GemPharmatech Co., Ltd. All animal experiments were conducted according to the protocol approved by the Animal Ethics Committee of Shanghai Skin Disease Hospital (Approval No.: 2024-106(Animal)Preliminary). All animals were housed in a light-dark cycle environment, with the temperature maintained at 23±2℃, and had free access to water and food.

[0035] Animal Model: Thirty-five C57BL / 6J mice used for establishing a diabetes model were first fed a high-fat diet for four weeks, followed by intraperitoneal injections of streptozotocin (STZ) every other day. Four hours after injection, they were given free access to 5% glucose solution. Seven days after injection, blood glucose levels were measured and typical symptoms of diabetes (polyuria, polydipsia, and hyperglycemia) were observed. Mice with blood glucose concentrations higher than 16.7 mmol / L were considered to have successfully established a diabetes model. Hair was shaved from the backs of normal controls and STZ-induced diabetic mice, with a shaved area of ​​approximately 4 × 4 cm. Before ulcer modeling, mice were anesthetized with isoflurane, and then the skin on the back was pinched, and 6 × 6 mm ulcer models were created using a punch, with four ulcers on the back of each mouse. The experiment was conducted under sterile conditions.

[0036] 2. Grouping and Treatment:

[0037] (1) Control group (n=5 animals / group): Ulcer modeling was performed, and no drug treatment was used; (2) Diabetic group (STZ, n=5 animals / group): Ulcer modeling was performed, and no drug treatment was used; (3) Diabetic gimazone treatment group (STZ+GM, n=5 animals / group): Different concentrations (1.5 mM, 2.5 mM, 3.5 mM) of gimazone monomer were dissolved in a solvent system (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline). Based on the healing rate analysis of the wound healing pre-experiment, it was confirmed that the 2.5 mM concentration did not have a therapeutic disadvantage compared to the 3.5 mM concentration (e.g., Figure 1 ), and in subsequent experiments, 2.5 mM was used as the drug concentration. The mice were wet-applied with 80 μl / mouse / day for 9 consecutive days; (4) Diabetes positive treatment group (STZ+rb-bFGF, n=5 mice / group): Bovine basic fibroblast growth factor external solution, specification 63000 IU / bottle, was applied locally to the mice ulcers with 80 μl / mouse / day for 9 consecutive days.

[0038] 3. Experimental methods:

[0039] (1) Wound healing analysis

[0040] Macroscopic observation: Photos were taken on days 1, 3, 5, 7, and 9 after drug treatment. Image-J software was used to process the photos to calculate the ulcer area, and the wound closure status was calculated using the following formula:

[0041] Wound healing rate (%) = 100 - (observed area / initial area) × 100

[0042] (2) Histological hematoxylin-eosin staining (HE staining)

[0043] Mice were sacrificed on day 9 after induction, and skin specimens were fixed in 4% neutral formaldehyde buffer for 24 hours. Routine histological examination was performed, including paraffin embedding, sectioning, and HE staining. The sections were observed using a digital slide scanner.

[0044] (3) Network pharmacology analysis

[0045] Potential targets for gimazone were predicted using the TCMSP database, while targets related to diabetic ulcers were retrieved from the OMIM and GeneCards databases, respectively. The intersection of these potential gimazone targets with those related to diabetic ulcers was used to obtain a set of potential gimazone targets for treating diabetic ulcers. Subsequently, KEGG signaling pathway enrichment analysis was performed on the intersection targets of gimazone and diabetic ulcers using the DAVID database platform, with p < 0.05 as a statistically significant screening criterion.

[0046] 4. Experimental Results:

[0047] (1) Gemmadone can improve wound healing in diabetic ulcer mice.

[0048] The effects of gemcitabine on the healing of diabetic ulcers in vivo were observed using wound healing analysis and HE staining. The results are as follows: Figure 2 As shown, by Figure 2 As can be seen, wound healing was significantly impaired in the diabetic group mice, while the gimazone treatment group showed significantly improved wound healing, with a faster healing rate compared to the positive control group. HE staining ( Figure 2 (b) The results showed that on day 9, the wound width in STZ-induced diabetic mice was significantly increased compared to that in normal mice. After treatment with gemmadone, the wound width was significantly reduced, and the effect was superior to that in the positive treatment group. These results indicate that STZ-induced wound healing in diabetic mice is slowed down, and gemmadone intervention can effectively promote wound healing and re-epithelialization in diabetic mice, with a more significant effect than that in the positive treatment group.

[0049] (2) Gemmazone may exert its therapeutic effect on diabetic ulcers by promoting angiogenesis.

[0050] A total of 1859 genes related to diabetic ulcers were retrieved from the GeneCards and OMIM databases, and 120 potential targets of gemmaconazole were obtained from the TCMSP database. Intersection analysis of the two databases revealed 43 targets that are commonly associated with both gemmaconazole and diabetic ulcers. Figure 3 (a and 3b). KEGG enrichment analysis of these 43 intersecting targets showed that two classic angiogenesis-related pathways (VEGF signaling pathway and Hedgehog signaling pathway) were significantly enriched. Figure 3 c). Based on this, it is speculated that gemmaconazole may exert its therapeutic effect on diabetic ulcers by promoting angiogenesis.

[0051] Example 2: Effect of Gemmadone on HUVECs Cell Proliferation in Vitro

[0052] 1. Cell model:

[0053] Human vascular endothelial cell line HUVECs. Cells were cultured in immortalized human umbilical vein endothelial cell culture medium at a constant temperature of 37°C.

[0054] 2. Grouping:

[0055] (1) Control group; (2) Gemarone treatment group (GM): HUVECs cells were treated with different concentrations of gemmarone.

[0056] 3. Experimental methods:

[0057] Gemmazone stock solution was diluted in human umbilical vein endothelial cell immortalization medium at final concentration gradients of 0, 2, 5, 10, 20, and 50 μM. After 48 hours, HUVEC cell proliferation was detected using the Cell Counting Kit-8 (CCK-8). In short, cells were seeded at a density of 2000 cells / 100 μL in 96-well plates in three separate seedings. CCK-8 solution was added at specified time points, and absorbance at 450 nm was measured. Data from four independent experiments were used as the mean standard deviation.

[0058] 4. Experimental Results:

[0059] Gemmadone can promote the proliferation of HUVECs cells.

[0060] The effect of different concentrations of gemcitabine on the proliferation of HUVECs was detected using the CCK-8 assay. Figure 4 a). We found that gimazone at in vitro concentrations of 2, 5, and 10 μM significantly promoted the proliferation of HUVECs cells, with 5 μM being the most significant.

[0061] Example 3: Effects of Gemmadone on the Expression of Inflammatory Factors in HUVECs Cells in Vitro

[0062] 1. Cell model:

[0063] Human vascular endothelial cell line HUVECs. Cells were cultured in immortalized human umbilical vein endothelial cell culture medium at a constant temperature of 37°C. To simulate the characteristic inflammatory microenvironment of diabetic ulcers, HUVECs were stimulated with 10 μg / mL lipopolysaccharide (LPS) for 6 hours to construct an in vitro inflammation model.

[0064] 2. Grouping:

[0065] (1) Normal group (NC); (2) Inflammation model group (LPS): HUVECs were stimulated with LPS for 6 hours; (3) Gemmadone intervention group (LPS+GM): LPS and 5 μM gemmadone (GM) were used simultaneously for 6 hours.

[0066] 3. Experimental methods:

[0067] After HUVEC cell intervention, total RNA was extracted using Trizol reagent, and then reverse transcribed into complementary DNA (cDNA) using the PrimeScript RTMaster Mix kit. Subsequently, SYBR Premix Ex Taq was used. TM Real-time quantitative PCR amplification was performed using reagent II. Inflammatory factors. IL -6 and IL-1β The relative expression level was calculated using the ∆∆Ct method.

[0068] 4. Experimental Results:

[0069] Gemmadone can inhibit the expression of inflammatory factors in HUVECs cells.

[0070] The effect of gemcitabine on the expression of inflammatory factors in HUVECs cells was detected by RT-qPCR. Figure 4 b). The results showed that LPS stimulation significantly increased inflammatory factors in HUVEC cells. IL-6 and IL-1β The expression of [something] was significantly alleviated after treatment with gemmadone. IL-6 and IL-1β The expression of was significantly downregulated.

[0071] Example 4: Effects of Gemmaconazole on the expression of F4 / 80, NF-κB p50, PCNA, PTCH1, SHH, GSK3B, CD31 and VEGF-A in diabetic ulcer mouse wound tissue

[0072] 1. Animals: Same as in Example 1

[0073] 2. Grouping and treatment: Same as in Example 1

[0074] 3. Experimental methods:

[0075] Immunohistochemical staining (IHC) was performed on serial paraffin sections. After dewaxing with xylene and rehydration to water with graded ethanol, the sections underwent antigen retrieval and blocking according to standard procedures. Subsequently, primary antibodies against PTCH1 (1:2400; Abcam, Ab53715), SHH (1:1600; Proteintech, 20697-1-ap), GSK3B (1:1200; Proteintech, 51065-1-ap), CD31 (1:600; Cell Signalling Technology, 77699), VEGF-A (1:50; Abcam, Ab52917), F4 / 80 (1:1200; Cell Signalling Technology, 70076), NF-κB p50 (1:600; Abcam, ab32360), and PCNA (1:6400; Abcam, ab29) were added and incubated overnight at 4°C. The following day, the appropriate secondary antibodies were incubated and developed according to the kit instructions, followed by hematoxylin counterstaining, dehydration, clearing, and mounting. Finally, the slides were observed and images were acquired under an optical microscope, and quantitative analysis was performed using ImageJ software.

[0076] 4. Experimental Results:

[0077] Gemmadone promotes angiogenesis and accelerates the healing of diabetic ulcers by activating the Hedgehog signaling pathway.

[0078] IHC staining was used to assess the effects of gemcitabine on the Hedgehog and VEGF signaling pathways in the wound tissue of diabetic mice with ulcers. Figure 5 As shown, the Hedgehog signaling pathway was significantly suppressed and angiogenesis was impaired in STZ-induced diabetic mouse wounds. However, after gemmadone treatment, the expression of key Hedgehog signaling pathway molecules PTCH1 and SHH was significantly upregulated, while the expression of the negative regulator of this pathway, GSK3B, was significantly downregulated. Simultaneously, angiogenesis-related markers CD31 and VEGF-A were also significantly enhanced. In addition to its pro-angiogenic effects, gemmadone also significantly reduced wound inflammatory infiltration (F4 / 80, NF-κB p50) and promoted re-epithelialization (PCNA). In conclusion, gemmadone can accelerate the healing of diabetic ulcers by activating the Hedgehog signaling pathway, promoting angiogenesis, inhibiting inflammation, and promoting cell proliferation.

[0079] 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. Application of gemmaconazole in the preparation of drugs for treating diabetic ulcers.

2. The use of gemmaconazole according to claim 1 in the preparation of a drug for treating diabetic ulcers, characterized in that, Application of gemmaconazole in the preparation of drugs that promote healing and reepithelialization of diabetic ulcers.

3. The use of gemmaconazole according to claim 1 in the preparation of a drug for treating diabetic ulcers, characterized in that, Application of gemmaconazole in the preparation of drugs that promote the proliferation of human vascular endothelial cells.

4. The use of gemmaconazole according to claim 1 in the preparation of a drug for treating diabetic ulcers, characterized in that, Application of gemmaconazole in the preparation of drugs that inhibit the expression of inflammatory factors in human vascular endothelial cells.

5. The use of gemmaconazole according to claim 1 in the preparation of a drug for treating diabetic ulcers, characterized in that, Application of gemmaconazole in the preparation of drugs that promote angiogenesis in diabetic ulcer wounds.

6. A drug for treating diabetic ulcers, characterized in that, The drug uses gemcitabine as its sole active ingredient.

7. The drug according to claim 6, characterized in that, The drug is a topical preparation.

8. The medicament according to claim 7, characterized in that, The drugs mentioned also include excipients commonly used in topical preparations.

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