Application of ferroptosis inhibitor in preparation of medicine for treating diabetes dry eye

Abstract

The invention provides application of a ferroptosis inhibitor in preparation of a medicine for treating diabetic dry eye, the ferroptosis inhibitor is ethyl ester formed by formal condensation of carboxyl of 3-amino-4-(cyclohexyl amino) benzoic acid and ethanol, the molecular formula is C15H22N2O2, the molecular weight is 262.35, the purity is 99.98%, and the cas number is 347174-05-4.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of a ferroptosis inhibitor in the preparation of a drug for treating diabetic dry eye. Background Technology

[0002] Diabetic dry eye can cause symptoms such as dry eyes, pain, and blurred vision, severely impacting patients' quality of life and potentially leading to corneal ulcers and vision impairment. Currently, treatments for diabetic dry eye are limited, mostly symptomatic rather than specific, and their effectiveness is not ideal. Therefore, developing novel drugs for the treatment of diabetic dry eye is of great significance.

[0003] The pathogenesis of diabetic dry eye is complex, involving multiple aspects such as reduced tear secretion and decreased tear film stability. It affects not only the cornea but also tissues crucial for tear secretion and tear film stability, such as the lacrimal glands and meibomian glands. Recent studies have found that ferroptosis, a cell death mechanism closely related to excessive lipid accumulation and lipid peroxidation, plays a key role in the development of other diabetic complications. However, its application in the treatment of diabetic dry eye is currently limited, and whether inhibiting ferroptosis can treat diabetic dry eye remains a highly intriguing question. Summary of the Invention

[0004] The main objective of this invention is to provide an application of a ferroptosis inhibitor in the preparation of a drug for treating diabetic dry eye.

[0005] To achieve the above objectives, the technical solution adopted in this invention is: the application of ferroptosis inhibitors in the preparation of drugs for treating diabetic dry eye, wherein the ferroptosis inhibitor Ferrostatin-1 (Fer-1) is an ethyl ester formed by the formal condensation of the carboxyl group of 3-amino-4-(cyclohexylamino)benzoic acid with ethanol, with the molecular formula: C 15 H 22 N₂O₂, molecular weight: 262.35, purity: 99.98%, CAS number: 347174-05-4, structural formula as follows:

[0006]

[0007] Preferably, the ferroptosis inhibitor injection is prepared by the following method:

[0008] Step 1: Add a certain amount of 5mg / ml clarified DMSO mother liquor to a certain amount of PEG300, mix well and let it become clear;

[0009] Step 2: Add a certain amount of TWEEN80 to the product obtained in Step 1, mix well and let it become clear;

[0010] Step 3: Add a certain amount of ddH2O to the product obtained in Step 2 to make up the volume, and obtain a certain amount of injection solution, wherein DMSO, purity is 99%, CAS number: 67-68-5; TWEEN 80, purity is 99%, CAS number: 9005-65-6; PEG300, purity is 99%, CAS number: 25322-68-3.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] This invention applies the ferroptosis inhibitor (Fer-1) to diabetic dry eye. Diabetes can increase the osmotic pressure of tears, placing many ocular surface tissues (such as the cornea, conjunctiva, lacrimal glands, and meibomian glands) in a high-glycemic environment, which easily induces the generation of reactive oxygen species (ROS), leading to oxidative stress and endoplasmic reticulum stress. Fer-1 can reduce oxidative stress by lowering ROS levels in tissues, effectively improving cell death, and salvaging the structure and function of many ocular surface tissues, thereby alleviating dry eye caused by their damage. Simultaneously, as a biological therapeutic agent, Fer-1 has high biocompatibility, with no significant adverse reactions observed. Once-daily administration is simple and convenient, providing a new strategy for the treatment of diabetic-related dry eye. It allows for individualized treatment plans based on the patient's specific condition, improving treatment efficacy. Attached Figure Description

[0013] Figure 1 This is a comparison chart of the changes in body weight of diabetic mice in the ferroptosis inhibitor group and the control group (n=3).

[0014] Figure 2 This is a comparison chart of the survival rate (n=6) of diabetic mice in the ferroptosis inhibitor group and the control group at the intervention time;

[0015] Figure 3 This is a control group showing the improvement in tear secretion in diabetic mice (n=3) after treatment with ferroptosis inhibitors. ** indicates p<0.01.

[0016] Figure 4 The images show representative fluorescein staining of the cornea in diabetic mice after treatment with ferroptosis inhibitors and the improvement in corneal fluorescein staining scores (n=3), ** indicates p<0.01;

[0017] Figure 5 This is a representative image of HE staining of the cornea of ​​diabetic mice after treatment with ferroptosis inhibitors (scale bar: 50 μm);

[0018] Figure 6 This is a representative image of PAS staining of the conjunctiva in diabetic mice after treatment with ferroptosis inhibitors (scale bar: 50 μm).

[0019] Figure 7This is a representative HE staining image of the lacrimal glands of diabetic mice after treatment with ferroptosis inhibitors (red arrows indicate damage to acinar vesicles in the control group, scale bar: 50 μm);

[0020] Figure 8 This is a representative image of TUNEL staining of the lacrimal glands of diabetic mice treated with ferroptosis inhibitors (scale bar: 50 μm).

[0021] Figure 9 This is a representative image of mitochondrial damage in the lacrimal glands of diabetic mice after treatment with ferroptosis inhibitors (scale bar: 200nm).

[0022] Figure 10 This is a representative immunofluorescence image of AQP5 in the lacrimal glands of diabetic mice treated with ferroptosis inhibitors (scale bar: 20 μm).

[0023] Figure 11 The image shows the representative Western blot (WB) of the lacrimal glands in diabetic mice treated with ferroptosis inhibitors and its quantification (n=3). * indicates p<0.05.

[0024] Figure 12 This is a representative image of meibomian gland opening obstruction in diabetic mice after treatment with ferroptosis inhibitors.

[0025] Figure 13 This is a representative image of the meibomian glands in diabetic mice treated with ferroptosis inhibitors.

[0026] Figure 14 This is a representative image of HE staining of the meibomian gland ducts in diabetic mice treated with ferroptosis inhibitors.

[0027] Figure 15 Yes. Representative image of meibomian gland TUNEL staining in diabetic mice treated with ferroptosis inhibitors (scale bar: 20 μm);

[0028] Figure 16 This is a representative image of mitochondrial changes in the meibomian glands of diabetic mice after treatment with ferroptosis inhibitors (scale bar: 200nm). Detailed Implementation

[0029] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0030] Example 1

[0031] The application of ferroptosis inhibitors in the preparation of drugs for treating diabetic dry eye, wherein the ferroptosis inhibitor Ferrostatin-1 (Fer-1) is an ethyl ester formed by the formal condensation of the carboxyl group of 3-amino-4-(cyclohexylamino)benzoic acid with ethanol, with the molecular formula: C 15 H22 N₂O₂, molecular weight: 262.35, purity: 99.98%, CAS number: 347174-05-4, is a synthetic antioxidant and a selective ferroptosis inhibitor. The structural formula of the ferroptosis inhibitor is as follows:

[0032]

[0033] Fer-1 mainly works through the following mechanisms: 1. Inhibiting lipid peroxidation: Fer-1 protects cells from damage caused by lipid peroxidation by scavenging free radicals and stabilizing lipid peroxides, thus preventing further oxidation reactions; 2. Regulating iron metabolism: Fer-1 can also regulate intracellular iron metabolism, inhibiting the accumulation and release of iron ions, preventing excessive free iron ions from participating in oxidation reactions, thereby reducing oxidative stress.

[0034] Example 2

[0035] This example describes the preparation of Fer-1 injection solution, as follows:

[0036] Taking a 1.5ml working solution as an example, add 60ul of 5mg / ml clarified DMSO stock solution to 500ul of PEG300 and mix thoroughly until clarified; add 50ul of TWEEN80 to the above system and mix thoroughly until clarified; then add 930ul of ddH2O to bring the volume to 1.5ml, resulting in an injection concentration of 0.2mg / ml and a DMSO content of 4%. The DMSO (Dimethyl Sulfoxide, Cell Culture Regent) has a purity of 99% and CAS number: 67-68-5; TWEEN 80 has a purity of 99% and CAS number: 9005-65-6; PEG300 has a purity of 99% and CAS number: 25322-68-3.

[0037] Experimental Example 1

[0038] This experiment used db / db mice as a diabetic mouse model to observe the therapeutic effect of the injection in Example 2 on dry eye in diabetic mice.

[0039] 1. Laboratory animals

[0040] Animal quality: 8w db / db mice, male, SPF grade, 12 mice, weighing approximately 40-50g, random blood glucose concentration >16.7mmol / L, purchased from Jicui Pharmaceutical Biotechnology Co., Ltd., certificate number: B202406020076.

[0041] Animal housing environment: air-conditioned, constant temperature and humidity, ventilation 10 times / hour with fresh air, artificial lighting 12 hours a day, free access to food and water.

[0042] Pre-experimental adaptation period: After the animals are introduced and tested as required, they are housed in separate cages of 3 animals each. They are observed for 7 days to assess their general condition. At the end of the adaptation period, their weight is measured.

[0043] 2. Animal model establishment and drug administration

[0044] (1) Twelve db / db mice were randomly divided into two groups of six mice each, based on their weight, fasting blood glucose, and ocular surface condition.

[0045] (2) Each group of animals was intraperitoneally injected with 1 mg / kg of Fer-1 and an equal dose of solvent according to their grouping.

[0046] Blank control group: equal dose of solvent;

[0047] Fer-1 group: 1 mg / kg, the dosage is estimated based on the mouse's body weight, for example: 250 μL for a 50 g mouse;

[0048] Starting on day 8, administer the medication intraperitoneally according to the above grouping criteria, once daily at the same time, for 21 consecutive days;

[0049] (3) The mice were sacrificed 24 hours after the last administration, and samples were collected for index detection.

[0050] Key data analysis:

[0051] 1) Mouse growth status:

[0052] Animals were weighed every 7 days to observe the effect of the drug on their body weight. The results are shown in [the table below]. Figure 1 . Figure 1 The results showed no significant difference in body weight between the Fer-1 group and the control group. At the end of the experiment, mouse survival rate and any adverse events were recorded. Figure 2 The results showed no significant difference in body weight between the Fer-1 group and the control group, all diabetic mice survived normally, and no significant adverse events occurred during the experiment.

[0053] 2) Ophthalmic examination of mice:

[0054] Tear secretion tests and corneal fluorescein staining scores were performed. Figure 3 As can be seen, tear secretion in the Fer-1 group mice was significantly improved. In contrast, tear secretion in the control group did not change significantly over time and even slightly worsened. Figure 4The results showed that at the end of the experiment, corneal fluorescein staining in the Fer-1 group was significantly improved compared with that in the control group. The quantitative score of corneal fluorescein staining also indicated this result, and there was a statistically significant difference between the two groups (score criteria: the score used a 12-point method: the cornea was divided into 4 quadrants, each quadrant was scored from 0 to 3 points, 0 points: no staining; 1 point: 1-30 punctate staining; 2 points: >30 punctate staining, staining not fused; 3 points: corneal punctate staining appeared and fused).

[0055] 3) Damage to the cornea and conjunctiva of mice:

[0056] Figure 5 The results showed that, compared with the blank control group, the number and morphology of corneal epithelial cells in diabetic mice in the Fer-1 group were improved. Figure 6 The results showed that, compared with the blank control group, the Fer-1 group of diabetic mice had an increased number of conjunctival goblet cells (PAS staining) and improved conjunctival function.

[0057] 4) Improvement in lacrimal gland function:

[0058] Figure 7 The results showed that, compared with the blank control group, the morphology of lacrimal gland secretory vesicles was improved in the Fer-1 group mice. Figure 8 TUNEL staining results showed that, compared with the blank control group, the apoptosis level of lacrimal gland cells in the Fer-1 group mice was significantly improved. Figure 9 Transmission electron microscopy results showed that, compared with the blank control group, the mitochondrial damage and ferroptosis-induced mitochondrial-specific damage (such as mitochondrial cristae damage) in the lacrimal gland cells of mice in the Fer-1 group were significantly improved. Figure 10 Immunofluorescence results of AQP5, a specific protein for lacrimal gland function, showed that the expression level of AQP5 in lacrimal gland cells of Fer-1 group mice was significantly increased compared with the blank control group. Figure 11 Immunofluorescence results of AQP5 showed that, compared with the blank control group, Fer-1 improved the expression of AQP5, a functional protein of the lacrimal gland in diabetic mice.

[0059] 5) Improvement in meibomian gland function

[0060] Figure 12 and Figure 13 Results from meibomian gland orifices and gross photographs of the meibomian glands showed that, compared with the blank control group, the ferroptosis inhibitor improved meibomian gland orifice obstruction and acinar atrophy in diabetic mice. Figure 14 HE staining results of the meibomian gland ducts showed that ferroptosis inhibitors, used to prepare related drugs, improved the abnormal dilation of the meibomian gland ducts in diabetic mice. Figure 15 and Figure 16TUNEL staining and transmission electron microscopy results suggest that ferroptosis inhibitors can improve meibomian gland cell apoptosis and mitochondrial damage in diabetic mice.

[0061] The above results indicate that intraperitoneal administration of 1 mg / kg Fer-1 injection significantly improved ocular surface damage in diabetic mice, reducing damage to the cornea, conjunctiva, lacrimal gland, and meibomian gland tissues, without causing additional damage or affecting the survival rate of diabetic mice. Therefore, Fer-1 injection has a good therapeutic effect on dry eye in diabetic mice and can be considered as a novel treatment method.

[0062] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. The application of ferroptosis inhibitors in the preparation of drugs for treating diabetic dry eye, characterized in that, The ferroptosis inhibitor is an ethyl ester formed by the formal condensation of the carboxyl group of 3-amino-4-(cyclohexylamino)benzoic acid and ethanol, with the molecular formula: C 15 H 22 N₂O₂, molecular weight: 262.35, purity: 99.98%, CAS number: 347174-05-4, structural formula as follows:

2. The application of the ferroptosis inhibitor according to claim 1 in the preparation of a drug for treating diabetic dry eye, wherein the ferroptosis inhibitor injection solution is prepared by the following method: Step 1: Add a certain amount of 5mg / ml clarified DMSO mother liquor to a certain amount of PEG300, mix well and let it become clear; Step 2: Add a certain amount of TWEEN80 to the product obtained in Step 1, mix well and let it become clear; Step 3: Add a certain amount of ddH2O to the product obtained in Step 2 and bring the volume to a certain amount to obtain an injection solution. DMSO, purity 99%, CAS No.: 67-68-5; TWEEN 80, purity 99%, CAS No.: 9005-65-6; PEG300, purity 99%, CAS No.: 25322-68-3.

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