Use of nitrate in preparation of medicament for treating diabetic ulcers

Abstract

A use of nitrate in the preparation of a medicament for treating diabetic ulcers. The nitrate includes, but is not limited to, sodium nitrate, potassium nitrate, lithium nitrate, zinc nitrate, magnesium nitrate, iron nitrate, calcium nitrate or copper nitrate.

Description

Uses of nitrates in the preparation of drugs for treating diabetic ulcers

[0001] This application claims priority to Chinese Patent Application No. 202510365649.1, filed on March 26, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application provides the use of nitrate compounds in the preparation of drugs for treating diabetic ulcers. Background Technology

[0003] Diabetic ulcers are a common and highly disabling complication of diabetes, and have always been a challenge in clinical treatment. The mortality rate after amputation in patients with diabetic ulcers increases significantly with the progression of the disease (1. Armstrong DG, Tan TW, Boulton AJM, Bus SA. Diabetic Foot Ulcers: A Review. Jama. 2023; 330(1):62-75.). The persistently high rates of disability and death from diabetic ulcers have a serious negative impact on patients and society, placing a heavy burden on patients and global public health. Therefore, therapies that minimize amputation and promote wound healing are of great significance.

[0004] Peripheral nerve and lower extremity vascular lesions caused by long-term hyperglycemia, as well as delayed wound healing caused by a combination of factors such as chronic inflammation, ischemia-hypoxia, oxidative stress and repeated infection, are typical characteristics of tissue damage in diabetic ulcers. The wound healing process, such as inflammation reduction and tissue regeneration, is disordered or stalled in diabetic ulcers, ultimately manifesting as long-term local chronic inflammation and persistent wound healing (2. Jeffcoate W, Boyko EJ, Game F, Cowled P, Senneville E, Fitridge R. Causes, prevention, and management of diabetes-related foot ulcers. Lancet Diabetes Endocrinol. 2024; 12(7):472-82.).

[0005] Traditional treatments for diabetic foot ulcers mainly focus on wound management, infection control, and blood glucose management. New drugs and dressings, such as Xianglei Tang Foot Ointment and hydrocolloids, have shown certain effects in the treatment of diabetic foot ulcers (3. Chinese Society of Endocrinology, Chinese Alliance of Endocrinology and Metabolic Diseases. Expert consensus on the treatment of diabetic foot ulcers (2024). Chinese Journal of Endocrinology and Metabolism, 2024; 40(7):565-569.). Literature reports that silver ion salts (including silver nitrate) can also treat diabetic ulcers (although meta-analysis studies show that the clinical evidence for the treatment of diabetic ulcers with silver nanoparticles is not conclusive (4. Hosseini RS, Hasanpour K, Khoshnevis M, Fakhr MS, Derin E, Ghaffarian A, Kement C. Therapeutic Effect of Silver Nanoparticles in the Management of Diabetic Ulcers: A Systematic Review and Meta-Analysis on RCTs. Int J Low Extrem Wounds. 2024:15347346241241836.), but current evidence suggests that the mechanism of silver ion salts in treating ulcer-like diseases is mainly based on the antibacterial and proliferative effects of silver ions (5. Lansdown AB. Silver in health care: antimicrobial effects and safety in use. Curr Probl Dermatol. 2006; 33:17-34; 9. Tian J, Wong KK, Ho CM, Lok CN, Yu WY, Che CM, Chiu JF, Tam PK. Topical delivery of silver nanoparticles promotes wound healing. Chem Med Chem. 2007; 2(1):129-36.). Summary of the Invention

[0006] The inventors of this application have surprisingly discovered that nitrates such as potassium nitrate and zinc nitrate, despite not containing silver ions, can still exert a therapeutic effect on diabetic ulcers based on the activity of nitrate ions. Therefore, this invention proposes that nitrates, as a single active ingredient, can effectively treat diabetic ulcers. The nitrates of this invention can also be used in combination with other therapeutic agents.

[0007] According to one aspect of this application, the use of nitrates in the preparation of medicaments for treating diabetic ulcers is provided.

[0008] According to another aspect of this application, this application provides nitrates for the treatment of diabetic ulcers.

[0009] According to another aspect of this application, this application provides a method for treating diabetic ulcers with nitrates, comprising the step of administering an effective dose of nitrates to a patient requiring treatment.

[0010] According to some embodiments of this application, the nitrates in this application include, but are not limited to, sodium nitrate, potassium nitrate, lithium nitrate, zinc nitrate, magnesium nitrate, ferric nitrate, calcium nitrate, or copper nitrate.

[0011] According to some embodiments of this application, the nitrate in this application is preferably potassium nitrate or zinc nitrate, and more preferably zinc nitrate.

[0012] According to some embodiments of this application, the nitrates of this application are used to treat diabetic ulcers.

[0013] Nitrates can be administered topically or systemically, with an effective dose range of 0.1 mg / kg body weight to 50 mg / kg body weight.

[0014] The diabetic ulcer in this application refers to one or more of the following symptoms on the skin, feet, and other parts of the body caused by distal nerve abnormalities and varying degrees of vascular lesions in diabetic patients: infection, ulceration, and deep tissue destruction.

[0015] The nitrates in this application refer to salts formed by the reaction of nitric acid (HNO3) with metals, which are composed of metal ions and nitrate ions. They include sodium nitrate, potassium nitrate, lithium nitrate, zinc nitrate, magnesium nitrate, iron nitrate, calcium nitrate, or copper nitrate, with zinc nitrate being preferred.

[0016] The beneficial effects of this invention are: nitrates have a therapeutic effect on diabetic ulcers, can improve the wound healing rate of diabetic ulcers, reduce the size of the wound, increase the thickness of the newly formed tissue in the wound, and can promote the expression of CD31 in the wound tissue and promote angiogenesis; nitrates have a protective effect on microvascular endothelial cells damaged by high glucose, can improve cell vitality, and promote cell migration and tube formation, thereby playing a beneficial role in cells. Attached Figure Description

[0017] Figure 1 includes Figure 1A and Figure 1B, which show representative images and statistical results of the wound healing rate in diabetic ulcer mice, respectively, illustrating the effects of potassium nitrate and zinc nitrate on wound healing. Mean ± standard deviation was used; one-way ANOVA was employed, and Dunnett's t-test was used for comparisons between groups. Figure 1A shows photographs of the wound; Figure 1B shows a statistical graph of the wound area. * p<0.05, **P<0.01, compared with the STZ group. D0 is day 1, D6 is day 6, D12 is day 12; Control is normal + surgery group, STZ is diabetes + surgery group, STZ + PN is diabetes + potassium nitrate group, STZ + ZN is diabetes + zinc nitrate group.

[0018] Figure 2, comprising Figures 2A, 2B, and 2C, shows the effects of potassium nitrate and zinc nitrate on the histopathological effects of wounds in diabetic ulcer mice. The method involved local tissue samples undergoing HE staining, followed by microscopic imaging of the HE-stained areas. The length of unhealed wounds and the thickness of newly formed tissue were statistically analyzed. Figure 2A shows representative images from each group; Figure 2B shows the quantitative analysis of the length of the unhealed portion of the wound; and Figure 2C shows the quantitative analysis of the thickest newly formed tissue at the wound site. Mean ± standard deviation were used, and one-way ANOVA was employed. Dunnett's t-test was used for inter-group comparisons. * p<0.05, ** P<0.01, *** P<0.001, compared with the STZ group. The English abbreviations of the group names are the same as in Figure 1. Gap is the length of the unhealed part of the wound, and Thickness is the thickness of the newly formed tissue in the wound.

[0019] Figure 3, including Figures 3A and 3B, shows the effects of potassium nitrate and zinc nitrate on CD31 (also known as platelet endothelial cell adhesion molecule-1, PECAM-1) protein expression in wound tissue of diabetic ulcer mice. The method involved immunohistochemical staining of local tissue blocks, microscopic imaging, and statistical analysis of the percentage of CD31-positive expression area. Figure 3A shows representative images of each group of immunohistochemically stained wound tissue; Figure 3B is a statistical graph showing the percentage of CD31-positive (brown) expression area. Mean ± standard deviation were used, and one-way ANOVA was performed. Dunnett's t-test was used for intergroup comparisons. * p<0.05, ** P<0.01, *** P<0.001, compared with the STZ group. The English abbreviations of the group names are the same as in Figure 1.

[0020] Figure 4 shows the effects of potassium nitrate and zinc nitrate on the viability of high glucose-induced human microvascular endothelial cells (HMEC-1). Cell viability was detected using the CCK-8 (Cell Counting Kit-8) assay, with OD as the metric. 450 As an indicator. Mean ± standard deviation, one-way ANOVA, and Dunnett's t-test were used for comparisons between groups. * p<0.05, ** P<0.01, ***P<0.001, compared with the HG group. OD 450 The absorbance is measured at 450 nm. Control represents the normal control group, HG represents the high glucose model group, Mannitol represents the mannitol control group, HG+PN 50 represents the high glucose + potassium nitrate 50 μM group, HG+PN100 represents the high glucose + potassium nitrate 100 μM group, HG+PN200 represents the high glucose + potassium nitrate 200 μM group, HG+ZN 4 represents the high glucose + zinc nitrate 4 group, HG+ZN 8 represents the high glucose + zinc nitrate 8 μM group, and HG+ZN 16 represents the high glucose + zinc nitrate 16 μM group.

[0021] Figure 5, including Figures 5A and 5B, shows the effects of potassium nitrate and zinc nitrate on the migration ability of high glucose-induced HMEC-1 cells. The method used was a scratch healing assay, with the scratch healing rate as the indicator. Mean ± standard deviation was analyzed using one-way ANOVA, and Dunnett's t-test was used for intergroup comparisons. * p<0.05, ** P<0.01, *** P<0.001, compared with the HG group. Wound healing rate is the rate of wound healing; the English abbreviations of the group names are the same as in Figure 4. Figure 5A shows representative images of each group, and Figure 5B shows the statistical results.

[0022] Figure 6, comprising Figures 6A and 6B, shows the effects of potassium nitrate and zinc nitrate on the tube-forming ability of high glucose-induced HMEC-1 cells. The method used was the matrix gel method, with total tube length as the indicator. Mean ± standard deviation were analyzed using one-way ANOVA, and Dunnett's t-test was used for intergroup comparisons. * p<0.05, ** P<0.01, *** P<0.001, compared with the HG group. Total length refers to the total length of cell tube formation, and the English abbreviations of the group names are the same as in Figure 4. Figure 6A shows representative images of each group, and Figure 6B shows the statistical results. Detailed Implementation

[0023] To make the objectives, technical solutions, and features of this invention clearer, the technical solutions of this application will be described by way of example through embodiments below, but this application is not limited to the following embodiments.

[0024] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0025] Example 1: The therapeutic effect of nitrates on diabetic ulcers

[0026] (1) Animal preparation. Six male C57BL / 6J mice, aged 6-8 weeks, were acclimatized for one week and then fasted for at least 12 hours to establish a diabetic model. Streptozocin (STZ), the modeling drug, was dissolved in citrate-sodium citrate buffer (pH 4.2) and injected intraperitoneally at a dose of 150 mg / kg body weight. The streptozocin solution was kept at low temperature and protected from light; the solution was prepared fresh and injected within 30 minutes. Mice were then allowed free access to food. Blood glucose levels were measured via tail vein sampling 72 hours later. A blood glucose level of 16.67 mmol / L was considered a successful establishment of the diabetic model.

[0027] (2) Wound preparation. Prepare the skin on the mouse's back (by shaving the back hair). Using an 8mm diameter punch, prepare two full-thickness skin wounds on both sides of the mouse's back, with a distance of more than 1cm between the wounds. If no bleeding is observed and the wounds are of roughly the same size after preparation, the wound preparation is considered successful.

[0028] (3) The therapeutic effect of nitrates on diabetic ulcers. Potassium nitrate solution (40 mM) and zinc nitrate solution (8 mM) were prepared using deionized water. Mice were divided into a blank control group, a diabetic ulcer group, a diabetic ulcer + potassium nitrate group, and a diabetic ulcer + zinc nitrate group. The drugs were applied to the ulcer sites once daily, and the wounds were bandaged after application. The experimental period was 12 days. After full-thickness skin removal, the drugs were applied to the wounds once daily, and photographs were taken on days 0, 6, and 12.

[0029] (4) Results presentation. In Figures 1 to 3, Control is the normal control group, STZ is the diabetic ulcer group, STZ+PN is the potassium nitrate treatment group for diabetic ulcers, and STZ+ZN is the zinc nitrate treatment group for diabetic ulcers.

[0030] Figure 1A shows representative images of the wound at 0, 6, and 12 days. Figure 1B shows the statistical results of the wound healing rate at 12 days. The method involved analyzing and measuring the wound area using the Image Pro Plus 6.0 image analysis system. The calculation formula is: Wound healing rate = (Wound area at 0 days - Wound area at 12 days) / Wound area at 0 days × 100%.

[0031] Figure 2 shows representative HE-stained images of the wound site (semi-circular section) and statistical results of wound size and new tissue thickness under a microscope. Figure 2A shows representative images of each group obtained under a microscope at 10× after fixation, dehydration, embedding, sectioning, and HE staining; Figure 2B shows the quantitative analysis of the unhealed portion of the wound, representing the size of the gap; Figure 2C shows the quantitative analysis of the thickest part of the newly formed epidermal tissue at the wound site, representing the thickness of the new tissue; the units in Figures 2B and 2C are millimeters (mm).

[0032] Figures 1 and 2 show the efficacy results of nitrate treatment for diabetic ulcers. Figure 1 shows that both nitrates exhibited higher wound healing rates at 12 days compared to the diabetic ulcer group, suggesting a therapeutic effect of nitrates on diabetic ulcers. Figure 2 shows that both nitrates reduced wound size and increased the thickness of newly formed tissue compared to the diabetic ulcer group, suggesting that nitrates can improve the severity of wounds in diabetic ulcer mice.

[0033] Figure 3 shows the statistical results of immunohistochemical detection of CD31 protein expression, a marker of angiogenesis, in wound tissue sections. CD31 (also known as platelet endothelial cell adhesion molecule-1, PECAM-1) is mainly expressed on the surface of vascular endothelial cells, and its expression reflects the status of angiogenesis. Immunohistochemical staining was used to detect the percentage of CD31-positive expression in the total tissue area; a high percentage indicates a higher degree of ulcer tissue repair. Figure 3A shows representative images of each group of wound tissue blocks after dewaxing, repair, sealing, primary antibody incubation, secondary antibody incubation, and staining, taken under a microscope at 20×. Figure 3B shows the percentage of CD31-positive (brown in Figure 3A) expression area in the total area, representing the status of angiogenesis.

[0034] The results in Figure 3 show that both nitrates promoted CD31 expression in the model mice, suggesting that both nitrates can promote angiogenesis in diabetic ulcer tissue and thus promote tissue repair.

[0035] Example 2: Effects of nitrate on a high glucose-induced human microvascular endothelial cell line (HMEC-1) cell damage model

[0036] Endothelial cell damage caused by diabetes is one of the main pathological damages of diabetes and a key factor in the development of diabetic ulcers. HMEC-1 is one of the commonly used cell lines for studying endothelial cell damage caused by hyperglycemia. This example observed the effects of nitrate on the viability, migration, and tube formation of a hyperglycemia-induced HMEC-1 cell damage model. Each experiment was divided into 9 groups: normal group (Control), hyperglycemia model group (HG), mannitol control group (Mannitol), hyperglycemia + potassium nitrate 50 μM group (HG+PN 50), hyperglycemia + potassium nitrate 100 μM group (HG+PN 100), hyperglycemia + potassium nitrate 200 μM group (HG+PN 200), hyperglycemia + zinc nitrate 4 μM group (HG+ZN 4), hyperglycemia + zinc nitrate 8 μM group (HG+ZN 8), and hyperglycemia + zinc nitrate 16 μM group (HG+ZN 16), with 3 replicates per group. The results were statistically analyzed using GraphPad 8.0.2 software.

[0037] (1) Effects of nitrate on HMEC-1 cell viability

[0038] HMEC-1 cells in logarithmic growth phase were digested and seeded at 10,000 cells per well in 96-well plates and cultured in a 5% CO2 incubator. After 24 hours, glucose solution (50 mM) was added to the model group and each drug-treated group to induce cell damage. Mannitol was added to the Mannitol group at a concentration of 50 mM, serving as a high-glucose control. After another 24 hours of culture, the corresponding drug concentration was added to each drug-treated group. After another 48 hours of culture, cell viability was assessed using the CCK-8 assay.

[0039] Figure 4 shows the effect of nitrate on cell viability in a high glucose-induced endothelial cell injury model. As shown in the figure, high glucose inhibited cell viability, while potassium nitrate (50, 100, and 200 μM) and zinc nitrate (8 and 16 μM) all increased cell viability, suggesting that both potassium nitrate and zinc nitrate have a protective effect against high glucose-induced endothelial cell injury.

[0040] (2) Effects of nitrate on HMEC-1 cell migration (scratch healing assay)

[0041] HMEC-1 cells were passaged twice normally before the experiment. First, horizontal lines were drawn on the bottom of a 6-well plate with a marker, approximately 1 cm apart, with at least three lines per well. Cells in logarithmic growth phase were seeded at 300,000 cells per well in the 6-well plate. After 24 hours, glucose solution was added to the model group and each drug-treated group to induce cell mutation at a concentration of 30 mM. After another 24 hours of culture, the corresponding drugs were added to each drug-treated group. After another 24 hours of culture, a 200 μl pipette tip was used, perpendicular to the horizontal lines on the back of the plate, aligned with a ruler. Cells were washed three times with PBS, and the marker lines on the back of the 6-well plate were wiped off. Photos were taken at 0 hours and 24 hours, and the images were processed using ImageJ software to calculate cell migration rate. Cell migration rate = (0-hour scratch area - 24-hour scratch area) / 0-hour scratch area.

[0042] Figure 5 shows the effect of nitrate on the migration ability of a high glucose-induced endothelial cell injury model. Figure 5A shows representative images of each group, and Figure 5B shows the statistical results. As can be seen from the figure, compared with the normal group, high glucose reduced the scratch healing rate and inhibited cell migration, while potassium nitrate 200 μM and zinc nitrate 4, 8, and 16 μM all increased the scratch healing rate and promoted cell migration, suggesting that both drugs promote the migration of high glucose-induced damaged cells, thus producing a beneficial effect on cells.

[0043] (3) Effect of nitrate on tube formation of HMEC-1 cells (Matrix gel method)

[0044] HMEC-1 cells were cultured for 24 hours after high-glucose incubation, followed by 24 hours of additional treatment with the respective drugs. Cells were then digested, resuspended in conditioned medium, and seeded at 100 μl per well in 96-well plates coated with matrix gel. After 4 hours of incubation, images were acquired under a microscope to observe cell tube formation. ImageJ software was used to process and analyze the images, and the total length of junctions was used as an indicator to quantitatively analyze cell tube formation.

[0045] Figure 6 shows the effect of nitrate on tubular formation in a high glucose-induced endothelial cell injury model. Figure 6A shows representative images from each group, and Figure 6B shows the statistical results. As can be seen from the figure, high glucose inhibited cell tubular formation, significantly reducing the total tubular length. In contrast, potassium nitrate (200 μM) and zinc nitrate (16 μM) both increased the total tubular length, suggesting that both drugs can promote tubular formation in the endothelial cell injury model, thus producing a beneficial effect on the cells.

Claims

1. The use of nitrates in the preparation of drugs for treating diabetic ulcers.

2. The use according to claim 1, wherein the nitrate is selected from one or more of sodium nitrate, potassium nitrate, lithium nitrate, zinc nitrate, magnesium nitrate, ferric nitrate, calcium nitrate, and copper nitrate.

3. The use according to claim 2, wherein the nitrate is selected from potassium nitrate or zinc nitrate.

4. The use according to any one of claims 1-3, wherein the diabetic ulcer refers to the skin or feet of a diabetic patient with one or more of the following symptoms: infection, ulceration, and deep tissue destruction.

5. The use according to any one of claims 1-3, wherein the nitrate has one or more of the following effects: improving the wound healing rate of diabetic ulcers, reducing the size of the wound, increasing the thickness of newly formed tissue in the wound, promoting the expression of CD31 in the wound tissue, promoting angiogenesis, protecting microvascular endothelial cells damaged by high glucose, improving cell viability, and promoting cell migration and tube formation.

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