Application of rasagiline mesylate in preparation of medicine for treating diabetic ulcer

Abstract

The invention relates to the technical field of medicines, in particular to application of rasagiline mesylate in preparation of a medicine for treating diabetic ulcer. The invention proposes that rasagiline mesylate has the effect of treating diabetic ulcer for the first time, and by adopting a mouse diabetic ulcer model, the influence of rasagiline mesylate on the healing and ROS expression of a diabetic ulcer model mouse is researched. The result shows that the rasagiline mesylate has a safe and remarkable treatment effect on the diabetic ulcer, and an important basis is provided for clinically developing an efficient strategy for treating the diabetic ulcer and preparing the medicine for treating the diabetic ulcer in the future.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, in particular to application of rasagiline mesylate in preparing medicines for treating diabetic ulcers. Background Art

[0002] Diabetic ulcers are common complications of long-term improper management of diabetes. Because they often occur in the feet, they are also called diabetic foot (DFU). It is estimated that there are about 537 million diabetic patients worldwide, of which 19% to 34% will suffer from DFU, resulting in a lifetime lower limb amputation incidence of 20%, and 1-year, 5-year and 10-year mortality rates of 13.1%, 49.1% and 76.9%, respectively. In addition, the 3-5 year recurrence rate of DFU 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 with added bioactive agents or cells, which have improved the treatment of DFU to some extent. However, after 3 months of drug treatment, only about 30%-40% of diabetic foot ulcers heal, and about 25% of diabetic foot ulcers still exist 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 need to develop efficient and simple drugs for the prevention and treatment of diabetic ulcers in clinical practice to address the limitations of current treatments.

[0003] Rasagiline is an irreversible monoamine oxidase-B (MAO-B) inhibitor, mainly used to treat Parkinson's disease. It inhibits MAO-B and reduces the decomposition of dopamine, thereby increasing the level of dopamine in the brain and improving the motor symptoms of Parkinson's patients. Rasagiline mesylate is the salt form of rasagiline. This conversion helps to improve the stability and solubility of the drug, making it more suitable for pharmaceutical preparation and human absorption. Recent studies have revealed that rasagiline can exert neuroprotective effects through multiple mechanisms, making it potentially useful in the treatment of Parkinson's disease and other neurodegenerative diseases. Rasagiline promotes neuronal survival by regulating mitochondrial function and cell signaling pathways and activating the PI3K-Akt survival pathway. In addition, it can also upregulate the gene expression of neurotrophic factors (such as BDNF and GDNF), regulate the non-amyloidogenic pathway of amyloid precursor protein, and reduce the formation of β-amyloid protein, thereby 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 and the mechanism 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 re-epithelialization, chronic inflammation and impaired angiogenesis, and neuropathy. Whether rasagiline can promote the healing of diabetic ulcers deserves further study.

[0005] However, no studies have investigated the potential application of rasagiline mesylate in the treatment of diabetic ulcers, and the development of new drugs for the treatment of diabetic ulcers is highly necessary. Summary of the invention

[0006] The purpose of the present invention is to provide a new use of rasagiline mesylate in treating diabetic ulcers. Rasagiline mesylate has a significant therapeutic effect in treating diabetic ulcers.

[0007] The 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 rasagiline mesylate is C 13 H 17 NO3S, the chemical structure is shown in the following formula I:

[0009]

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

[0011] Furthermore, in the drug, the rasagiline mesylate plays a role by promoting the healing and re-epithelialization of diabetic ulcer wounds and reducing the expression of ROS in diabetic ulcer wounds.

[0012] Furthermore, in the application, rasagiline mesylate is used in the preparation of a drug for promoting wound healing and re-epithelialization of diabetic ulcers.

[0013] Furthermore, in the application, rasagiline mesylate is used in the preparation of a drug for reducing the expression of ROS in diabetic ulcer wounds.

[0014] The second aspect of the present invention provides a drug for treating diabetic ulcers, wherein the drug contains rasagiline mesylate as the sole active ingredient.

[0015] Furthermore, the medicine is an external preparation, and further, the medicine also includes excipients commonly used in external preparations.

[0016] The present invention has no particular limitation on the types and specific sources of the commonly used auxiliary materials for external preparations, and the common auxiliary materials for external preparations in the art can be used.

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

[0018] The advantages of the present invention are:

[0019] 1. The present invention discovers for the first time that rasagiline mesylate has significant therapeutic effects in treating diabetic ulcers and has no toxic side effects on the liver, spleen and kidneys, providing a solid basis for the future clinical development of efficient strategies for treating diabetic ulcers.

[0020] 2. The present invention uses a mouse diabetic ulcer model to study the effects of rasagiline mesylate on the healing and ROS expression of diabetic ulcer model mice. The results show that rasagiline mesylate has a safe and significant therapeutic effect on diabetic ulcers, which provides an important experimental basis for the future clinical development of efficient strategies for the treatment of diabetic ulcers and the preparation of drugs for the treatment of diabetic ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of rasagiline mesylate on wound healing in diabetic ulcer mice, where Figure A shows the photographic results of wound healing and the calculation results of 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 the expression of ROS in the wounds of diabetic ulcer mice, where the left picture shows the results of ROS detection by fluorescence microscopy in the wound surface, and the right picture shows the quantitative calculation results of ROS detection by fluorescence microscopy in the wound surface;

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

[0024] The specific embodiments provided by the present invention are described in detail below in conjunction with the examples and drawings. In the following examples, unless otherwise specified, all are conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0025] Example 1: Effect of Rasagiline Mesylate on Wound Healing in Mice with Diabetic Ulcer

[0026] 1. Animals: Animal experiments used m / m control mice and db / db diabetic mice, male, 8 weeks old, 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℃, with SPF grade 12 hours of light / 12 hours of dark, and the animals had free access to high-fat or standard diets and water.

[0027] Animal model: 12 db / db diabetic mice were randomly divided into a diabetic group, a diabetic rasagiline mesylate treatment group, and a diabetic positive treatment group. The hair on the back of the m / m normal control and db / db diabetic mice was shaved, and the shaved area was about 4×4cm; before the ulcer model was established, the mice were anesthetized with isoflurane, and then a wound model with an area of ​​6×6mm was created using a punch, with 4 wounds on the back of each mouse, and the experiment was carried out under sterile conditions.

[0028] 2. Grouping and treatment:

[0029] (1) Normal group (m / m): ulcer model was established without any drug treatment; (2) Diabetes group (db / db): ulcer model was established without any drug treatment; (3) Diabetes rasagiline mesylate treatment group (db / db+RM): rasagiline mesylate was dissolved in a solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline) at a concentration of 5 μM, and applied to the ulcer of mice at a dosage of 80 ul / mouse / day for 9 consecutive days; (4) Diabetes positive treatment group (db / db+rb-bFGF): bovine basic fibroblast growth factor topical solution with a specification of 63000 IU / bottle was applied topically to the ulcer of mice at a dosage of 80 μl / mouse / day for 9 consecutive days.

[0030] 3. Experimental methods:

[0031] (1) Wound healing analysis

[0032] Macroscopic observation: Pictures were taken on the 3rd, 5th, 7th and 9th days after treatment, and the pictures were processed with Image-J software to calculate the wound ulcer area. The wound closure was calculated using the following formula:

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

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

[0035] The mice were killed on the 9th day after induction, and the skin specimens were fixed in 4% neutral formaldehyde buffer for 24 hours. Routine histological examination, paraffin embedding, sectioning, HE staining were performed, and the sections were observed under a digital section scanner.

[0036] 4. Experimental Results

[0037] Rasagiline mesylate improves wound healing in mice with diabetic ulcers.

[0038] Wound healing analysis and HE staining were used to observe the effect of rasagiline mesylate on diabetic ulcer wound healing in vivo. Figure 1 As shown in A, compared with the diabetic ulcer group, the diabetic rasagiline mesylate treatment group had a more significant wound healing effect on the 9th day after injury, and the effect was better than that of the positive treatment group. HE staining results ( Figure 1 B) shows that on day 9, the wound of the diabetic rasagiline mesylate treatment group was significantly smaller than that of the diabetic ulcer group, and the effect was better than that of the diabetic positive treatment group. This result shows that wound healing is slow in db / db diabetic mice, and rasagiline mesylate intervention can effectively promote diabetic wound healing and re-epithelialization, and the effect is more significant than that of the positive treatment group.

[0039] Example 2: Effect of Rasagiline Mesylate on ROS Expression in Wounds of Mice with Diabetic Ulcer

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

[0041] 2. Grouping and treatment:

[0042] (1) Normal group (m / m): ulcer model was established without any drug treatment; (2) Diabetes group (db / db): ulcer model was established without any drug treatment; (3) Diabetes rasagiline mesylate treatment group (db / db+RM): rasagiline mesylate was dissolved in a solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline) at a concentration of 5 μM, and applied to the ulcer of mice at a dosage of 80 ul / mouse / day for 9 consecutive days; (4) Diabetes positive treatment group (db / db+rb-bFGF): bovine basic fibroblast growth factor topical solution with a specification of 63000 IU / bottle was applied topically to the ulcer of mice at a dosage of 80 μl / mouse / day for 9 consecutive days.

[0043] 3. Experimental methods:

[0044] (1) Fluorescence staining and semi-quantitative analysis of ROS in wound surface

[0045] On the 9th day after induction, mice were killed, wound skin specimens were taken, and the expression of ROS in the wound was detected using a dihydroethidium ROS detection kit. 200 μl of cleaning solution was added to 10 μm frozen sections for 5 min, and the cleaning solution was removed; 50 μM dihydroethidium fluorescent probe was diluted 1000 times with PBS, and then added to the frozen sections, protected from light, and the wet box was placed in a 37°C water bath for 30 min; the dihydroethidium fluorescent probe was removed, washed 3 times with PBS, counterstained with DAPI, and washed 3 times with PBS; the sections were sealed with anti-fluorescence quenching sealing agent; ROS was detected under a fluorescence microscope. The mean fluorescence intensity of the fluorescence images was semi-quantitatively analyzed using Image-J software, and the mean fluorescence intensity was calculated using the following formula:

[0046] Average fluorescence intensity = total fluorescence intensity of the region / area of ​​the region

[0047] 4. Experimental results:

[0048] Rasagiline mesylate can reduce the expression of ROS in the wound surface of diabetic ulcer mice.

[0049] The dihydroethidium ROS detection kit was used to detect the ROS content and observe the effect of rasagiline mesylate on the expression of ROS in the wound surface of diabetic ulcer mice. Figure 2 As shown in the figure, compared with the diabetic ulcer group, the ROS content in the wound of the diabetic rasagiline mesylate treatment group was lower and lower than that of the diabetic positive treatment group. This result shows that in the wound of db / db diabetic mice, ROS expression is abnormally increased, and rasagiline mesylate intervention can effectively reduce ROS expression in diabetic wounds, and the therapeutic effect is more significant than the positive control Beifuji.

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

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

[0052] 2. Grouping and treatment:

[0053] (1) Normal group (m / m): ulcer model was established without any drug treatment; (2) Diabetes group (db / db): ulcer model was established without any drug treatment; (3) Diabetes rasagiline mesylate treatment group (db / db+RM): rasagiline mesylate was dissolved in a solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline) at a concentration of 5 μM, and applied to the ulcer of mice at a dosage of 80 ul / mouse / day for 9 consecutive days; (4) Diabetes positive treatment group (db / db+rb-bFGF): bovine basic fibroblast growth factor topical solution with a specification of 63000 IU / bottle was applied topically to the ulcer of mice at a dosage of 80 μl / 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] On the 9th day after induction, the mice were killed, and the liver, spleen and kidney tissues were taken and fixed in 4% neutral formaldehyde buffer for 24 hours. Routine histological examination, paraffin embedding, sectioning, HE staining were performed, and the sections were observed under a digital section scanner.

[0057] 4. Experimental results:

[0058] Rasagiline mesylate has no liver, spleen, and kidney damage in mice with diabetic ulcer.

[0059] HE staining was used to observe the effects of rasagiline mesylate on the liver, spleen and kidney of diabetic ulcer mice. Figure 3 After the diabetic ulcer mice were treated with rasagiline mesylate, the gross appearance of the liver, spleen and kidney of the diabetic group and the diabetic rasagiline mesylate treatment group was normal compared with the normal group, and there was no significant difference in histopathology, indicating that rasagiline mesylate did not produce toxic side effects on the liver, spleen and kidney during the medication period.

[0060] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in 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 use according to claim 1, characterized in that: Application of rasagiline mesylate in the preparation of a drug for promoting wound healing and re-epithelialization of diabetic ulcers.

3. The use according to claim 1, characterized in that: Application of rasagiline mesylate in the preparation of a drug for reducing ROS expression in diabetic ulcer wounds.

4. A drug for treating diabetic ulcer, characterized in that: The drug contains rasagiline mesylate as the only active ingredient.

5. The drug according to claim 4, characterized in that The medicine is an external preparation.

6. The drug according to claim 5, characterized in that The medicine also includes auxiliary materials commonly used in external preparations.

7. The drug according to claim 4, characterized in that The effective concentration of rasagiline mesylate in the drug in vivo is 5 μM.

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