Use of rasagiline mesylate for the preparation of a medicament for the treatment of diabetic ulcers

Abstract

The present application relates to the technical field of medicine, in particular to application of rasagiline mesylate in preparation of medicine for treating diabetic ulcer, the present application first proposes that rasagiline mesylate has the effect of treating diabetic ulcer, adopts mouse diabetic ulcer model, studies the effect of rasagiline mesylate on the healing and ROS expression of diabetic ulcer model mice.The results show that rasagiline mesylate has safe and significant therapeutic effect on diabetic ulcer, which provides important basis for future clinical development of efficient strategy for treating diabetic ulcer and preparation of medicine for treating diabetic ulcer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of rasagiline mesylate in the preparation of drugs for treating diabetic ulcers. Background Technology

[0002] Diabetic foot ulcers (DFU) are a common complication of poorly managed diabetes. Because they frequently occur in the feet, they are also known as diabetic foot ulcers. It is estimated that there are approximately 537 million people with diabetes worldwide, of whom 19% to 34% will develop DFU, resulting in a 20% lifetime lower limb amputation rate. The 1-year, 5-year, and 10-year mortality rates are 13.1%, 49.1%, and 76.9%, respectively. Furthermore, the recurrence rate of DFU within 3-5 years is as high as 65%. In recent years, a series of innovative therapies have emerged, including traction-activated payloads, local delivery of short interfering RNA, and hydrogels incorporating bioactive agents or cells. These methods have improved the treatment of DFU to some extent. However, after 3 months of drug treatment, only about 30%-40% of diabetic foot ulcers healed, and about 25% of diabetic foot ulcers remained after 1 year of treatment. (ARMSTRONG DG, BOULTON AJM, BUS S A. Diabetic Foot Ulcers and Their Recurrence[J]. The New England journal of medicine, 2017, 376(24): 2367-75. CHEN L, SUN S, GAO Y, et al. Global mortality of diabetic foot ulcer: A systematic review and meta-analysis of observational studies[J]. Diabetes, obesity & metabolism, 2023, 25(1): 36-45. PETERSEN BJ, ROTHENBERG GM, LAKHANIP J, et al. Ulcer metastasis? Anatomical locations of recurrence for patients in diabetic foot remission[J]. Journal of foot and ankle research, 2020, 13: 1. DIXON) D,EDMONDS M.Managing Diabetic Foot Ulcers:Pharmacotherapy for Wound Healing[J].Drugs,2021,81(1):29-56.VOELKER R.WhatAre Diabetic Foot Ulcers? [J].Jama,2023,330(23):2314.). Therefore, there is an urgent clinical need to develop highly effective and convenient drugs for the prevention and treatment of diabetic foot ulcers to address the limitations of current treatments.

[0003] Rasagiline is an irreversible monoamine oxidase-B (MAO-B) inhibitor primarily used to treat Parkinson's disease. It works by inhibiting MAO-B, reducing dopamine breakdown, thereby increasing dopamine levels in the brain and improving motor symptoms in Parkinson's patients. Rasagiline mesylate is a salt form of rasagiline; this conversion helps improve the drug's stability and solubility, making it more suitable for pharmaceutical manufacturing and human absorption. Recent studies have revealed that rasagiline exerts neuroprotective effects through multiple mechanisms, making it potentially valuable in the treatment of Parkinson's disease and other neurodegenerative diseases. Rasagiline promotes neuronal survival by regulating mitochondrial function and cell signaling pathways, activating the PI3K-Akt survival pathway. Furthermore, it can upregulate the gene expression of neurotrophic factors (such as BDNF and GDNF), regulate the non-amyloid formation pathway of amyloid precursor protein, and reduce the formation of β-amyloid protein, thus preventing the occurrence of neurodegenerative diseases. Recent studies have shown that rasagiline can also provide neuroprotection for ischemic neuronal cultures by inhibiting α-synuclein and glyceraldehyde-3-phosphate dehydrogenase-mediated cell necrosis, and by increasing mitochondrial-specific antioxidant enzymes through mitochondrial protection, involving the Akt / Nrf2 redox signaling pathway. (SCHAPIRA A, BATE G, KIRKPATRICK P. Rasagiline[J]. Nature reviews Drug discovery, 2005, 4(8): 625-6. WEINREB O, AMIT T, BAR-AM O, et al. Rasagiline: a novel anti-Parkinsonian monoamine oxidase-Binhibitor with neuroprotective activity[J]. Progress in neurobiology, 2010, 92(3): 330-44. LECHTS, LAHIANI A, KLAZAS M, et al. Rasagiline Exerts Neuroprotection towards Oxygen-Glucose-Deprivation / Reoxygenation-Induced GAPDH-Mediated Cell Death byActivating Akt / Nrf2 Signaling[J]. Biomedicines, 2024, 12(7).).

[0004] The main pathological manifestations of diabetic ulcers are: impaired reepithelialization; chronic inflammation and impaired angiogenesis; and neuropathy. Whether rasagiline can promote the healing of diabetic ulcers warrants further investigation.

[0005] However, there are currently no studies exploring the potential use of rasagiline mesylate in the treatment of diabetic ulcers, and the development of new drugs for the treatment of diabetic ulcers is essential. Summary of the Invention

[0006] The purpose of this invention is to provide a novel use of rasagiline mesylate in the treatment of diabetic ulcers, which has significant therapeutic effects.

[0007] A first aspect of the present invention provides the use of rasagiline mesylate in the preparation of a medicament for treating diabetic ulcers.

[0008] Furthermore, the molecular formula of the rasagiline mesylate is C2. 13 H 17 NO3S has the chemical structure shown in Formula I below:

[0009]

[0010] The source of rasagiline mesylate in this invention is not particularly limited. It can be prepared by chemical synthesis or purchased through commercial channels.

[0011] Furthermore, in the aforementioned drug, rasagiline mesylate exerts its effects by promoting the healing and reepithelialization of diabetic ulcer wounds and reducing ROS expression in diabetic ulcer wounds.

[0012] Furthermore, the application of rasagiline mesylate in the preparation of drugs that promote healing and reepithelialization of diabetic ulcers is mentioned.

[0013] Furthermore, in the aforementioned applications, rasagiline mesylate is used in the preparation of drugs that reduce ROS expression in diabetic ulcer wounds.

[0014] In a second aspect, the present invention provides a medicament for treating diabetic ulcers, said medicament having rasagiline mesylate as the sole active ingredient.

[0015] Furthermore, the drug is a topical preparation, and even further, the drug also includes excipients commonly used in topical preparations.

[0016] 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.

[0017] Furthermore, the effective concentration of rasagiline mesylate in vivo is 5 μM.

[0018] The advantages of this invention are:

[0019] 1. This invention is the first to discover that rasagiline mesylate has significant efficacy in treating diabetic ulcers and has no toxic side effects on the liver, spleen and kidneys, providing a solid basis for future clinical development of strategies for the effective treatment of diabetic ulcers.

[0020] 2. This invention uses a mouse diabetic ulcer model to study the effects of rasagiline mesylate on the healing and ROS expression of diabetic ulcers in mice. The results show that rasagiline mesylate has a safe and significant therapeutic effect on diabetic ulcers, providing important experimental evidence for the future clinical development of efficient strategies for treating diabetic ulcers and the preparation of drugs for treating diabetic ulcers. Attached Figure Description

[0021] Figure 1 The effect of rasagiline mesylate on wound healing in diabetic ulcer mice is shown in Figure A, which shows the photographic results of wound healing and the calculated wound healing rate; Figure B shows the HE staining results and the HE quantitative calculation results. Scale bar = 1 mm.

[0022] Figure 2 The effect of rasagiline mesylate on ROS expression in the wounds of diabetic ulcer mice is shown in the left figure, which is the result of ROS detection in the wound under fluorescence microscopy, and the right figure is the result of quantitative calculation of ROS detected in the wound under fluorescence microscopy.

[0023] Figure 3 Representative HE images of the liver, spleen, and kidneys of mice in each group. Scale bar = 100 μm. Detailed Implementation

[0024] The specific embodiments provided by the present invention will be described in detail below with reference to the examples and accompanying drawings. Unless otherwise specified, the methods described in the following embodiments are conventional. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0025] Example 1: Effect of rasagiline mesylate on wound healing in diabetic ulcer mice

[0026] 1. Animals: Male, 8 weeks old, m / m control mice and db / db diabetic mice were used in animal experiments. They were provided by the Experimental Animal Center of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, affiliated to Shanghai University of Traditional Chinese Medicine, and purchased from Jiangsu GemPharmatech Co., Ltd. All animals were kept at a standard temperature of 23±2℃ and subjected to SPF-grade 12-hour light / 12-hour dark treatment. The animals had free access to high-fat or standard diets and water.

[0027] Animal model: Twelve db / db diabetic mice were randomly divided into a diabetic group, a diabetic rasagiline mesylate treatment group, and a diabetic positive treatment group. Hair was shaved from the backs of m / m normal controls and db / db diabetic mice, with a shaved area of ​​approximately 4×4 cm. Before ulcer model creation, mice were anesthetized with isoflurane, and then 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.

[0028] 2. Grouping and Treatment:

[0029] (1) Normal group (m / m): Ulcer modeling was performed, and no drug treatment was used; (2) Diabetes group (db / db): Ulcer modeling was performed, and no drug treatment was used; (3) Diabetes rasagiline mesylate treatment group (db / db+RM): Rasagiline mesylate was dissolved in solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline) at a concentration of 5μM, and applied to the ulcers of mice at a dosage of 80ul / mouse / day for 9 consecutive days; (4) Diabetes positive treatment group (db / db+rb-bFGF): Bovine basic fibroblast growth factor topical solution, specification 63000IU / bottle, was applied topically to the ulcers of mice at a dosage of 80μl / mouse / day for 9 consecutive days.

[0030] 3. Experimental methods:

[0031] (1) Wound healing analysis

[0032] Macroscopic observation: Photos were taken on days 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:

[0033] Wound healing rate (%) = [(Initial wound area - Remaining wound area) / Initial wound area] × 100%

[0034] (2) Histological hematoxylin-eosin staining (HE staining)

[0035] 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.

[0036] 4. Experimental Results

[0037] Rasagiline mesylate can improve wound healing in diabetic ulcer mice.

[0038] The effects of rasagiline mesylate on the healing of diabetic ulcers in vivo were observed using wound healing analysis and HE staining. The results are as follows: Figure 1 As shown in Figure A, compared with the diabetic ulcer group, the diabetic rasagiline mesylate treatment group had a more significant wound healing effect on day 9 post-injury, and the effect was better than the positive treatment group. HE staining results ( Figure 1 B) shows that on day 9, the rasagiline mesylate treatment group for diabetes showed significantly smaller wounds compared to the diabetic ulcer group, and the effect was superior to the positive treatment group for diabetes. This result indicates that wound healing is slowed in db / db diabetic mice, and rasagiline mesylate intervention can effectively promote wound healing and re-epithelialization in diabetic mice, with a more significant effect than the positive treatment group.

[0039] Example 2: Effect of rasagiline mesylate on ROS expression in diabetic ulcer mouse wounds

[0040] 1. Animal models: m / m control mice and db / db diabetic mice.

[0041] 2. Grouping and Treatment:

[0042] (1) Normal group (m / m): Ulcer modeling was performed, and no drug treatment was used; (2) Diabetes group (db / db): Ulcer modeling was performed, and no drug treatment was used; (3) Diabetes rasagiline mesylate treatment group (db / db+RM): Rasagiline mesylate was dissolved in solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline) at a concentration of 5μM, and applied to the ulcers of mice at a dosage of 80ul / mouse / day for 9 consecutive days; (4) Diabetes positive treatment group (db / db+rb-bFGF): Bovine basic fibroblast growth factor topical solution, specification 63000IU / bottle, was applied topically to the ulcers of mice at a dosage of 80μl / mouse / day for 9 consecutive days.

[0043] 3. Experimental methods:

[0044] (1) ROS fluorescence staining and semi-quantitative analysis in wound

[0045] Mice were sacrificed on day 9 after induction, and skin specimens from the wounds were collected. ROS expression in the wounds was detected using a dihydroethidium ROS detection kit. 200 μl of washing buffer was added to a 10 μm frozen section for 5 min, and the washing buffer was removed. A 50 μM dihydroethidium fluorescent probe was diluted 1000-fold with PBS and then added to the frozen section. The section was incubated in a humidified chamber at 37°C for 30 min, protected from light. The dihydroethidium fluorescent probe was removed, washed three times with PBS, counterstained with DAPI, and washed three times with PBS. The section was mounted with an anti-fluorescence quenching mounting medium. ROS was detected under a fluorescence microscope. The average fluorescence intensity was semi-quantitatively analyzed using Image-J software, and the average fluorescence intensity was calculated using the following formula:

[0046] Average fluorescence intensity = Sum of fluorescence intensities in the region / Area of ​​the region

[0047] 4. Experimental Results:

[0048] Rasagiline mesylate can reduce ROS expression in the wounds of diabetic mice with ulcers.

[0049] ROS content was detected using a dihydroethidium ROS detection kit. The effect of rasagiline mesylate on ROS expression in diabetic ulcer mouse wounds was observed. The results are as follows: Figure 2 As shown, compared with the diabetic ulcer group, the ROS content in the wound of the diabetic rasagiline mesylate treatment group was lower, and also lower than that of the diabetic positive control group. This result indicates that ROS expression is abnormally increased in the wounds of db / db diabetic mice, and that rasagiline mesylate intervention can effectively reduce ROS expression in diabetic wounds, with a more significant therapeutic effect than the positive control betamethasone.

[0050] Example 3: HE staining to determine pathological changes in the liver, kidneys, and spleen after treatment with rasagiline mesylate.

[0051] 1. Animal models: m / m control mice and db / db diabetic mice.

[0052] 2. Grouping and Treatment:

[0053] (1) Normal group (m / m): Ulcer modeling was performed, and no drug treatment was used; (2) Diabetes group (db / db): Ulcer modeling was performed, and no drug treatment was used; (3) Diabetes rasagiline mesylate treatment group (db / db+RM): Rasagiline mesylate was dissolved in solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline) at a concentration of 5μM, and applied to the ulcers of mice at a dosage of 80ul / mouse / day for 9 consecutive days; (4) Diabetes positive treatment group (db / db+rb-bFGF): Bovine basic fibroblast growth factor topical solution, specification 63000IU / bottle, was applied topically to the ulcers of mice at a dosage of 80μl / mouse / day for 9 consecutive days.

[0054] 3. Experimental methods:

[0055] (1) HE staining of liver, spleen and kidney of mice in each group

[0056] Mice were sacrificed on day 9 after induction, and liver, spleen, and kidney tissues were harvested. The tissue samples were fixed in 4% neutral formaldehyde buffer for 24 hours. Routine histological examination was performed, including paraffin embedding, sectioning, and hematoxylin and eosin (HE) staining. The sections were then observed using a digital slide scanner.

[0057] 4. Experimental Results:

[0058] Rasagiline mesylate did not cause liver, spleen, or kidney damage in diabetic ulcer mice.

[0059] The effects of rasagiline mesylate on the liver, spleen, and kidneys of diabetic ulcer mice were observed using HE staining. The results are as follows: Figure 3 As shown in the figure, after treatment with rasagiline mesylate, diabetic ulcer mice showed no significant differences in the gross appearance of the liver, spleen, and kidneys compared to the normal group in the diabetic group and the diabetic rasagiline mesylate treatment group, with no significant differences in histopathology. This suggests that rasagiline mesylate did not produce any toxic side effects on the liver, spleen, and kidneys during the treatment period.

[0060] 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 rasagiline mesylate in the preparation of drugs for treating diabetic ulcers.

2. The application according to claim 1, characterized in that, Application of rasagiline mesylate in the preparation of drugs that promote healing and reepithelialization of diabetic ulcers.

3. The application according to claim 1, characterized in that, Application of rasagiline mesylate in the preparation of drugs that reduce ROS expression in diabetic ulcer wounds.

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