Use of alpha-ketoglutarate in improving corneal endothelial damage and dysfunction

Abstract

The application provides an application of alpha-ketoglutaric acid in improving corneal endothelial injury and dysfunction, and belongs to the technical field of biological medicine. The application finds that, as a key metabolic intermediate in the tricarboxylic acid cycle, alpha-ketoglutaric acid can significantly reduce corneal endothelial cell injury and dysfunction induced by various causes such as high glucose, genetic defects, high intraocular pressure and surgical trauma by improving mitochondrial energy metabolism, reducing oxidative stress, stabilizing mitochondrial structure and membrane potential. Experiments prove that alpha-ketoglutaric acid can be used in the form of local administration such as eye drops, anterior chamber perfusion, systemic oral administration or combined administration, effectively reduces corneal edema, reduces central corneal thickness, shortens the recovery time of corneal transparency, and has a clear protection and repair effect on diabetic-related corneal endothelial lesions, Fuchs corneal endothelial dystrophy, glaucoma high intraocular pressure injury and intraocular surgery-related endothelial injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of α-ketoglutarate in improving corneal endothelial damage and dysfunction. Background Technology

[0002] The cornea is an important transparent refractive tissue in the anterior part of the eyeball, and its normal transparency is fundamental to maintaining visual function. Corneal endothelial cells (CECs) are located in the innermost layer of the cornea, arranged as a single layer of squamous epithelium. Through ion pump and barrier functions, they actively remove excess water from the stroma, maintaining a relatively dehydrated state and normal thickness of the corneal stroma, and are the core structure ensuring corneal transparency. Human corneal endothelial cells have almost no proliferative capacity in the body. After damage, they cannot effectively regenerate through cell division, and can only rely on the increase in volume and migration of surrounding healthy cells for limited compensation. When the damage exceeds the compensatory range, it will directly cause corneal edema, opacity, bullous keratopathy, and in severe cases, blindness.

[0003] The causes of corneal endothelial damage and dysfunction in clinical practice are complex and diverse, mainly including the following types:

[0004] Metabolic and hereditary corneal endothelial diseases are represented by diabetic keratopathy and Fuchs' endothelial dystrophy (FECD). A high-glucose environment can cause abnormal mitochondrial function, energy metabolism disorders, and oxidative stress accumulation in corneal endothelial cells, forming "metabolic memory" that leads to decreased cell pump function and weakened damage repair capacity. As a common hereditary corneal endothelial disease, the pathological mechanism of FECD is highly correlated with mitochondrial defects, oxidative stress damage, and metabolic disorders of the tricarboxylic acid cycle (TCA cycle), eventually progressing to endothelial decompensation.

[0005] Physical and mechanical injuries, acute high intraocular pressure in glaucoma, and the impact of irrigation fluid, ultrasound energy, and instrument contact during intraocular surgeries (such as phacoemulsification of cataracts, vitrectomy, and corneal transplantation) can all directly cause acute damage and loss of corneal endothelial cells, which are the main causes of postoperative corneal edema and dysfunction in clinical practice.

[0006] Other pathogenic factors, such as age-related decrease in the number and function of corneal endothelial cells, chemical burns, viral infections, and intraocular inflammation, can also continuously damage the structure and function of the endothelium, inducing irreversible damage.

[0007] Current clinical treatments for corneal endothelial damage and dysfunction have significant limitations:

[0008] 1. Corneal transplantation: It is the only way to cure endothelial decompensation, but it faces problems such as a severe shortage of donor materials, large surgical trauma, high risk of postoperative immune rejection, and complex long-term follow-up and anti-rejection treatment, making it difficult to widely popularize and intervene in the early stage.

[0009] 2. Current symptomatic supportive treatment: mainly using hypertonic dehydration agents and anti-inflammatory drugs to control complications, which can only relieve corneal edema and inflammation in the short term, but cannot repair cell metabolic damage from the root cause, nor can it stop the progressive loss and functional deterioration of endothelial cells.

[0010] 3. Drugs under development: Most focus on single targets such as promoting cell proliferation, anti-inflammation, and anti-oxidation, lacking systematic intervention programs for core metabolic defects in endothelial cells, and lacking universal protective effects against endothelial damage caused by various etiologies such as diabetes, genetic defects, and surgical trauma.

[0011] In summary, there is an urgent clinical need for a novel metabolic intervention drug and treatment plan that can directly regulate the energy metabolism of corneal endothelial cells, fundamentally improve mitochondrial function, enhance the cells' resistance to damage and repair capabilities, and is applicable to endothelial injuries of multiple causes. Summary of the Invention

[0012] In view of this, the present invention provides the application of α-ketoglutaric acid in improving corneal endothelial damage and dysfunction. Through metabolomics studies, the inventors have confirmed that the tricarboxylic acid cycle (TCA cycle) is significantly inhibited in various corneal endothelial damage models induced by different etiologies, including diabetes, FECD, intraocular hypertension, and surgical trauma. Specifically, the level of the key metabolic intermediate α-ketoglutaric acid (AKG) shows a consistent and significant decrease. Based on this key finding, the inventors propose that exogenous supplementation of AKG can restore TCA cycle metabolic flux, improve mitochondrial energy production, and reduce oxidative stress damage, thereby achieving broad-spectrum protection and functional repair of corneal endothelial cells, providing a novel metabolic therapeutic pathway for corneal endothelial damage and dysfunction.

[0013] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0014] This invention provides the use of α-ketoglutarate or a pharmaceutically acceptable salt thereof in the preparation of medicaments for improving, treating or preventing corneal endothelial damage and dysfunction.

[0015] Preferably, the α-ketoglutaric acid includes one or more of the following forms: free acid, sodium salt, potassium salt, calcium salt, and magnesium salt.

[0016] Preferably, the corneal endothelial injury and dysfunction include at least one of the following:

[0017] (1) Metabolic or hereditary corneal endothelial diseases, including diabetic endothelial disease and Fuchs endothelial dystrophy;

[0018] (2) Physical or mechanical injury, including corneal endothelial injury caused by high intraocular pressure and corneal endothelial injury caused by intraocular surgical trauma;

[0019] (3) Age-related or corneal endothelial dysfunction after corneal transplantation.

[0020] Preferably, the drug is administered via local administration, systemic administration, or a combination of local and systemic administration.

[0021] Preferably, the topical medication is an eye drop, ophthalmic gel, ophthalmic ointment, or anterior chamber infusion solution, wherein the concentration of α-ketoglutarate is 0.1~5 mM.

[0022] Preferably, the systemic administration is oral administration, and the formulation is tablets, capsules, oral liquid, or added to drinking water. The preferred concentration of α-ketoglutarate is 1~3 w / v.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects: The research of the present invention has found that α-ketoglutarate, as a key metabolic intermediate in the tricarboxylic acid cycle, can significantly alleviate corneal endothelial cell damage and dysfunction induced by various etiologies such as high glucose, genetic defects, high intraocular pressure, and surgical trauma by improving mitochondrial energy metabolism, reducing oxidative stress, and stabilizing mitochondrial structure and membrane potential. Experiments have confirmed that α-ketoglutarate can be administered topically (e.g., eye drops, anterior chamber infusion solution), or orally, or in combination, effectively reducing corneal edema, decreasing central corneal thickness, and shortening corneal transparency recovery time. It also has a clear protective and repairing effect on diabetic endothelial lesions, Fuchs' corneal endothelial dystrophy, glaucoma with high intraocular pressure, and intraocular surgery-related endothelial damage. Attached Figure Description

[0024] Figure 1 The study aimed to demonstrate the protective effect of α-KG against high glucose-induced corneal endothelial cell damage. A represents cell viability assay results, B represents mitochondrial membrane potential (TMRE) staining, C represents TMRE quantitative fluorescence analysis results, D represents mitochondrial ROS (MitoSox) staining results, E represents MitoSox quantitative fluorescence analysis, F represents TOMM20 immunofluorescence staining, and H represents dynamic transendothelial resistance (TEER) assay.

[0025] Figure 2 The protective effect of α-KG on corneal endothelial damage in db / db diabetic mice is shown in Figure A, which is the result of dynamic observation of corneal morphology and OCT; Figure B is the result of quantitative analysis of central corneal thickness (CCT); and Figure C is the ultrastructure of corneal endothelial cells mitochondria under transmission electron microscopy.

[0026] Figure 3 The study aimed to improve corneal endothelial damage in db / db diabetic mice by oral administration of α-KG in combination. In the figure, A is the blood glucose change curve, B is the corneal morphology and OCT dynamic observation results, and C is the quantitative analysis results of central corneal thickness (CCT).

[0027] Figure 4 The protective effect of α-KG on corneal damage in Fuchs corneal endothelial dystrophy model mice is shown in Figure A, which is the result of dynamic observation of corneal morphology and OCT, and Figure B is the quantitative analysis of central corneal thickness (CCT). Detailed Implementation

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] The db / db diabetic mice used in this embodiment of the invention were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and the Col8a2 gene knockout mutant mice were purchased from The Jackson Laboratory in the United States.

[0030] Example 1. Protective effect of α-ketoglutarate against high glucose-induced in vitro corneal endothelial cell damage.

[0031] (1) Human corneal endothelial cells (B4G12 human corneal endothelial cell line) in the logarithmic growth phase after passage were seeded into 24-well culture plates at an appropriate density and cultured in a 37℃, 5% CO2 incubator. After 24 h of culture, when the cells were fully attached and their growth was stable, the medium was replaced with serum-free medium and starved for 12 h to synchronize the cell cycle and ensure uniform physiological state in each group, thereby reducing experimental errors.

[0032] (2) Grouping and Model Building:

[0033] After synchronization is complete, process the data in the following groups:

[0034] Blank control group: cultured in standard complete culture medium without any additional treatment;

[0035] High glucose model group: Sterile glucose solution was added to the culture medium to a final concentration of 30 mM;

[0036] Hypertonic control group: Isotonic mannitol was added to the high glucose model to eliminate osmotic pressure interference;

[0037] AKG administration group: Based on the high glucose model, α-ketoglutarate (AKG) was added at concentrations of 0.01, 0.05, 0.1, 0.5, 1, and 2 mM, respectively.

[0038] (3) Cell culture and indicator detection

[0039] Cells from each group were cultured continuously at 37℃ and 5% CO2 for 72 h, with the corresponding culture medium replaced every 24 h. After culture, cells were collected and subjected to Seahorse cell metabolic analysis, transendothelial electrical resistance (TEER) detection, and Na+ analysis. + / K + Functional assays such as ATPase activity detection, as well as molecular biological assays such as metabolomics and transcriptomics, yielded results as follows: Figure 1 As shown.

[0040] Cell viability assay results showed that, under normal conditions, low to moderate concentrations of α-KG (≤ 0.5 mM) could partially enhance cell viability, while high concentrations (≥1 mM) showed a certain inhibitory trend. Figure 1 (A). Mitochondrial membrane potential (TMRE) staining results showed that high glucose treatment significantly reduced mitochondrial membrane potential, while α-KG intervention significantly increased membrane potential levels, showing a concentration-dependent improvement trend. Figure 1 (B, C). Meanwhile, MitoSOX assay results showed that high glucose stimulation significantly increased mitochondrial reactive oxygen species (ROS) levels, while α-KG supplementation significantly decreased ROS levels. Figure 1 (D, E). Further evaluation of corneal endothelial cell function revealed that α-KG intervention could, to some extent, delay the decline in TEER caused by a high-glucose environment. Figure 1 (F). Furthermore, Na⁺ / K⁺-ATPase activity assays showed that, compared to the high-glucose group, the α-KG treatment group exhibited significantly increased enzyme activity (F). Figure 1 (G). In summary, α-KG supplementation can improve high glucose-induced mitochondrial dysfunction, reduce oxidative stress levels, and to some extent restore barrier and pump function-related indicators of corneal endothelial cells.

[0041] Example 2. Protective effect of topical administration of α-ketoglutarate on corneal endothelial damage in db / db diabetic mice.

[0042] (1) Laboratory animals and grouping

[0043] Eight-week-old male db / db diabetic mice and normal wild-type mice were randomly divided into three groups: normal mouse ocular hypertension injury group (wild-type mice + ocular hypertension model), diabetic mouse ocular hypertension injury group (db / db mice + ocular hypertension model), and diabetic mouse ocular hypertension injury combined with local drug administration group (db / db mice + ocular hypertension model + AKG local intervention (the local drug administration group used physiological saline perfusion solution containing 0.5 mM α-KG for anterior chamber perfusion).

[0044] (2) Animal anesthesia and preoperative preparation

[0045] Mice were anesthetized by intraperitoneal injection of sodium pentobarbital; tropicamide eye drops were instilled on the ocular surface to fully dilate the pupils; after pupil dilation, 0.5% promecaine hydrochloride eye drops were instilled for corneal surface anesthesia.

[0046] (3) Construction of corneal endothelial injury model

[0047] Under a surgical microscope, a smooth tunnel was created from the limbus into the corneal stroma using a 34G microneedle, and then the needle was slowly inserted into the anterior chamber with the tip pointing downwards. The needle tip was connected to an infusion set into a 500 mL saline bottle, and the anterior chamber was continuously perfused for 30 minutes to establish a corneal endothelial injury model. During the perfusion, intraocular pressure changes were monitored in real time using a tonometer, and the perfusion height was kept consistent with each injection to maintain the stability and consistency of the model. Postoperatively, antibiotic eye drops were routinely administered to prevent infection.

[0048] On postoperative days 2, 4, and 7, slit-lamp anterior segment photography was used to record corneal morphological changes, and optical coherence tomography (OCT) was used to measure central corneal thickness to objectively assess the degree of corneal endothelial damage.

[0049] Slit-lamp and anterior segment OCT dynamic observations showed that diabetic mice rapidly developed significant corneal edema and opacity after surgery, with a significant increase in central corneal thickness, indicating damage to the corneal endothelial barrier and pump function. Compared with the control group, the α-KG intervention group showed significantly improved corneal transparency and reduced edema. Figure 2 (A) Quantitative analysis further confirmed that α-KG local intervention could significantly reduce the increase in central corneal thickness after high-pressure stimulation and shorten the time required for edema recovery (A). Figure 2 (B in the text). Transmission electron microscopy further confirmed that high intraocular pressure injury can lead to ultrastructural changes such as mitochondrial swelling and cristae destruction in corneal endothelial cells, while mitochondrial morphology remained relatively intact and cristae structure was well preserved after α-KG intervention. Figure 2 (C in the middle).

[0050] In summary, topical administration of α-ketoglutarate can significantly reduce corneal edema and endothelial damage caused by acute intraocular pressure in diabetic mice, effectively protect the structure and function of corneal endothelial cells and accelerate corneal repair after injury.

[0051] Example 3. Systemic administration of α-ketoglutarate on corneal endothelial damage in db / db diabetic mice

[0052] Eight-week-old db / db diabetic mice were selected as the starting group and randomly divided into the following two groups:

[0053] In the AKG oral administration group: Drinking water containing 2% α-KG was administered before modeling and continued for 8 weeks, with blood glucose monitored weekly. When blood glucose levels returned to near normal, the anterior chamber was perfused with physiological saline to establish a corneal endothelial injury model.

[0054] Diabetic model group: Anterior chamber perfusion was performed using physiological saline solution, and the same amount of physiological saline was given to the drinking water.

[0055] On postoperative days 2, 4, and 7, the degree of corneal opacity was assessed using a slit lamp, and central corneal thickness (CCT) was measured using OCT to assess the recovery of corneal endothelial edema. Results are as follows: Figure 3 As shown.

[0056] Continuous monitoring results showed that blood glucose levels in the α-KG oral group gradually decreased over time, and statistical differences appeared after week 4. Figure 3 (A). After 6 weeks of oral intervention, an acute corneal endothelial injury model was established in mice by anterior chamber hyperbaric perfusion. Slit-lamp and anterior segment OCT observations showed that the db / db group exhibited significant corneal edema and opacity after perfusion, with a marked decrease in corneal transparency; while the α-KG treatment group showed milder corneal opacity and a relatively faster recovery of transparency. Figure 3 (B) Quantitative analysis of central corneal thickness showed that the cornea in the db / db group was significantly thickened on days 2 and 4 after perfusion, while the increase in thickness in the α-KG oral group was significantly reduced, suggesting that it has a certain alleviating effect on post-injury corneal edema; by day 7, the difference in corneal thickness between the two groups gradually narrowed (B). Figure 3 (C)

[0057] In summary, α-ketoglutarate (α-KG) can reduce blood glucose in db / db diabetic mice, alleviate corneal opacity and edema after intraocular pressure perfusion, and promote the recovery of corneal endothelial damage. It has a significant effect on improving and protecting corneal endothelial damage in diabetic mice, with the intervention method of systemic combined with local administration showing the best effect.

[0058] Example 4. Therapeutic effect of α-ketoglutarate on Col8a2 mutant Fuchs corneal endothelial dystrophy mouse model

[0059] To evaluate the protective effect of α-KG against corneal endothelial damage in Col8a2 mutant mice, Col8a2 gene knockout mutant mice (Fuchs model mice) were randomly divided into 3 groups:

[0060] Mut-Ctrl model control group: No α-KG intervention was given;

[0061] Mut-Perf local intervention group: 0.5 mM α-KG added to the perfusion fluid;

[0062] Mut-Oral+Perf combined intervention group: oral administration of 2% α-KG in drinking water for 1 month + α-KG added to perfusion fluid.

[0063] All mutant mice underwent a systematic evaluation of corneal morphology and endothelial function after intraocular pressure infusion. (Specific methods are the same as in Example 2).

[0064] Central corneal thickness (CCT) was measured on postoperative days 2, 4, and 7. Corneal tissue was harvested on postoperative day 7 for immunofluorescence staining to detect the expression and distribution of tight junction protein (ZO-1), Na⁺ / K⁺-ATPase, and endoplasmic reticulum stress marker protein (GRP78). Results are as follows: Figure 4 As shown.

[0065] Central corneal thickness measurements showed that corneal edema was most severe in the Mut-Ctrl group on postoperative day 2, with a significantly increased central corneal thickness. In contrast, corneal edema was significantly reduced in both the Mut-Perf and Mut-Oral+Perf groups, and central corneal thickness was significantly lower in the latter group. With prolonged recovery time, corneal thickness recovery was fastest in the Mut-Oral+Perf group on postoperative days 4 and 7, and significantly lower than in the Mut-Ctrl group. Figure 4 ).

[0066] In summary, α-ketoglutarate (α-KG) has significant protective and therapeutic effects on corneal endothelial damage in Col8a2 mutant Fuchs corneal endothelial dystrophy mouse model; among them, the combined intervention of oral administration of 2% α-KG in drinking water and perfusion solution with added α-KG has the best effect.

[0067] As can be seen from the above embodiments, the present invention provides the application of α-ketoglutarate in improving corneal endothelial damage and dysfunction. Topical administration of α-ketoglutarate can significantly improve corneal edema and endothelial damage.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of α-ketoglutarate or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the improvement, treatment or prevention of corneal endothelial damage and dysfunction.

2. The application according to claim 1, characterized in that, The α-ketoglutaric acid includes one or more of the following forms: free acid, sodium salt, potassium salt, calcium salt, and magnesium salt.

3. The application according to claim 1, characterized in that, The corneal endothelial damage and dysfunction include at least one of the following: (1) Metabolic or hereditary corneal endothelial diseases, including diabetic endothelial disease and Fuchs endothelial dystrophy; (2) Physical or mechanical injury, including corneal endothelial injury caused by high intraocular pressure and corneal endothelial injury caused by intraocular surgical trauma; (3) Age-related or corneal endothelial dysfunction after corneal transplantation.

4. The application according to claim 1, characterized in that, The drug can be administered locally, systemically, or in combination.

5. The application according to claim 4, characterized in that, The topical medication is an eye drop, ophthalmic gel, ophthalmic ointment, or anterior chamber infusion solution, wherein the concentration of α-ketoglutarate is 0.01~2 mM.

6. The application according to claim 4, characterized in that, The systemic administration is oral administration, and the formulation is tablets, capsules, oral liquid, or added to drinking water. The preferred concentration of α-ketoglutarate is 1~3 w / v.

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