Use of prmt6 inhibitors in the manufacture of a medicament for treating a retinal vascular damaging disease

Abstract

The application discloses application of a PRMT6 inhibitor in preparation of a drug for treating retinal vascular diseases. The application takes PRMT6 as a treatment target, and studies a treatment effect of the PRMT6 inhibitor on retinal vascular injury. The research result shows that the PRMT6 inhibitor can significantly reduce retinal vascular leakage caused by the retinal vascular diseases, maintain retinal structure integrity and protect visual function, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of PRMT6 inhibitors in the preparation of drugs for treating retinal vascular injury diseases. Background Technology

[0002] Retinal vascular diseases are a very important category of ophthalmic diseases in clinical practice. They are characterized by damage and dysfunction of retinal blood vessels. The core of these diseases is the disruption of the blood-retinal barrier, which manifests as (1) vascular leakage, causing retinal edema (especially macular edema that threatens central vision) and hard exudates; (2) vascular occlusion, leading to retinal ischemia and hypoxia; and (3) compensatory neovascularization, which is prone to rupture, causing vitreous hemorrhage and potentially tractional retinal detachment, ultimately leading to blindness. Retinal vascular diseases include diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, hypertensive retinopathy, retinopathy of prematurity, and retinal vasculitis. Diabetic retinopathy (DR) is one of the most common and serious ocular complications of diabetes and an important cause of visual impairment in adults. Its pathogenesis is accompanied by persistent retinal vascular dysfunction, decreased barrier stability, and abnormal retinal structure and function. Clinically, fundus photography and fluorescein angiography are typically used to assess vascular leakage and perfusion disorders, optical coherence tomography (OCT) is used to monitor macular edema and changes in the thickness of different retinal layers, and electrophysiological testing is combined to assess visual function status, in order to comprehensively judge disease progression and treatment effectiveness. Currently, clinical treatment mainly relies on laser photocoagulation and anti-angiogenic factor drugs, but these methods suffer from unstable efficacy, high recurrence rates, and difficulty in halting the ongoing neurodegenerative process, potentially leading to further visual decline. Furthermore, there is a lack of precise regulatory targets that effectively address the pathogenesis of the disease.

[0003] Currently, the main clinical treatments include anti-vascular endothelial growth factor (VEGF) drugs and glucocorticoids. Representative anti-VEGF drugs include ranibizumab, aflibercept, conbercept, buxizumab, and farexizumab. These drugs directly reduce vascular leakage and macular edema by antagonizing VEGF. Representative glucocorticoids include triamcinolone acetonide and dexamethasone. These drugs have strong anti-inflammatory and anti-vascular leakage effects and can inhibit VEGF expression.

[0004] Although these drugs are currently first-line treatments used clinically, they have significant drawbacks. Anti-VEGF drugs are antibody drugs, which are expensive. Approximately 10-20% of patients do not respond well to initial anti-VEGF treatment, with little reduction in edema or no improvement in vision. Some patients respond initially, but later experience a decline in efficacy, requiring increased dosage or switching to other drugs. Furthermore, there are potential systemic and ocular risks: VEGF plays a role in maintaining normal vascular function throughout the body (such as wound healing and vascular endothelial integrity). Although intravitreal injections have minimal systemic absorption, there is theoretically a risk for patients with severe atherosclerosis, a recent history of stroke, or myocardial infarction, requiring careful evaluation. The use of glucocorticoids inevitably leads to hormonal side effects, such as steroid-induced glaucoma and steroid-induced cataracts. Approximately 20-60% of patients experience a significant increase in intraocular pressure after use, and some may develop drug-resistant glaucoma, leading to irreversible optic nerve damage. Long-term or repeated use almost 100% accelerates the formation and progression of cataracts, ultimately requiring cataract surgery. In addition, hormones have a wide range of targets and non-specifically inhibit various inflammatory factors, which may interfere with the normal physiological environment inside the eye.

[0005] Therefore, researching new therapeutic targets and discovering novel therapeutic mechanisms is a pressing issue for the clinical drug treatment of retinal vascular injury diseases. Protein arginine methyltransferases (PRMTs) are a class of enzymes widely distributed within cells that regulate protein methylation. They participate in physiological activities by transferring a methyl group from the S-adenosylmethionine group to the guanidino nitrogen atom on the arginine residue of the target protein, thereby regulating the biological activity of the target protein. The arginine N-methyltransferase 6 (PRMT6) gene is located on chromosome 1p13.3. EPZ020411 is a small molecule inhibitor of PRMT6 that selectively inhibits the protein arginine methylation function of PRMT6 and has good bioavailability in vivo. As an important protein arginine methyltransferase, PRMT6 participates in the regulation of various cellular functions. The role of the PRMT6 inhibitor EPZ020411 in the treatment of glioblastoma, neuropathic pain, and hearing loss has been reported in the literature.

[0006] Chinese patent document CN120771279A discloses the use of PRMT6 inhibitors in the preparation of anti-bone resorption products. PRMT6, as a novel metabolic checkpoint, mediates the metabolic switch from fatty acid oxidation to glycolysis, translating RANK signaling into metabolic switching during osteoclast differentiation.

[0007] Chinese patent document CN118903115A discloses the application of PRMT6 inhibitors in the preparation of drugs for treating thyroid-associated ophthalmopathy (TAO). PRMT6 inhibitors can significantly inhibit the proliferation of orbital adipose tissue in TAO mice and significantly improve ocular symptoms of TAO by inhibiting adipogenic differentiation of preadipocytes through macrophages.

[0008] There are currently no reports of a correlation between PRMT6 inhibitors and retinal vascular injury. Summary of the Invention

[0009] This invention is the first to discover that PRMT6 inhibitors can be used to treat retinal vascular injury diseases, especially diabetic retinopathy. Specifically, it achieves comprehensive intervention for retinal vascular injury diseases by improving abnormal retinal vascular permeability, abnormal angiogenesis, and damage to visual function-related structures.

[0010] The specific technical solution of this invention is as follows:

[0011] Application of PRMT6 inhibitors in the preparation of drugs for treating retinal vascular injury.

[0012] The retinal vascular injury diseases described in this invention include diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, hypertensive retinopathy, retinopathy of prematurity, and retinal vasculitis.

[0013] It can be used to regulate retinal pathological conditions, improve abnormal retinal vascular permeability, and maintain the physiological stability of retinal structure and visual function.

[0014] In the application described in this invention, the PRMT6 inhibitor is one or more of the following: protein inhibitors of the PRMT6 gene, nucleic acid aptamers, and / or interfering RNA, gRNA, microRNA, and small molecule compound inhibitors of the PRMT6 encoding gene.

[0015] The small molecule compound inhibitor described in this invention can be a compound that has an inhibitory effect on PRMT6 disclosed in the prior art, such as PRMT6 inhibitors disclosed in EPZ020411, Chinese patent documents CN119080685A, CN113582864A, CN105339351A, CN112469441A, etc.

[0016] In a specific example of the present invention, the PRMT6 inhibitor is EPZ020411, a compound with the following structure or a pharmaceutically acceptable salt thereof: .

[0017] The drug of this invention uses a PRMT6 inhibitor as its active ingredient. It can be an oral or injectable formulation. Preferably, the drug is an intravenous injection. The drug also includes one or more of a buffer, an isotonic adjuster, a surfactant, a stabilizer, and a preservative.

[0018] Buffers can be phosphate buffers (such as sodium dihydrogen phosphate, disodium hydrogen phosphate), acetate buffers, histidine buffers, and citrate buffers. Isotonic adjusters can be sodium chloride, mannitol, glycerol, etc. Surfactants can be polysorbate 20, polysorbate 80, poloxamer, etc. Stabilizers can be sugars: sucrose, trehalose; amino acids: arginine, histidine, glycine, methionine; antioxidants: disodium edetate, etc.

[0019] The dosage of PRMT6 inhibitors can be adjusted according to factors such as patient weight and disease severity. In a specific example of this invention, when the PRMT6 inhibitor is EPZ020411, the recommended dose is 20 mg / kg.

[0020] Beneficial effects

[0021] This invention discovers a novel therapeutic mechanism for retinal vascular injury. The results of this study indicate that the PRMT6 inhibitor EPZ020411 can maintain retinal structural integrity to a certain extent, particularly the stability of the inner layers related to visual function, and improve impaired retinal functional indicators in diabetic patients, thereby protecting against retinal pathological changes. Simultaneously, the PRMT6 inhibitor can improve abnormal retinal vascular conditions, reduce abnormal vascular permeability, and significantly decrease retinal vascular leakage. The PRMT6 inhibitor demonstrates therapeutic efficacy in maintaining retinal structural integrity and protecting visual function, showing promising clinical application prospects in the treatment of retinal vascular injury. Attached Figure Description

[0022] Figure 1 To establish and administer drugs to mice with diabetic retinopathy (DR). A is a schematic diagram of mouse experimental grouping; B is a schematic diagram of intraperitoneal injection of PRMT6 inhibitor into DR mice; C is the blood glucose level of mice in each group.

[0023] Figure 2To investigate the protective effect of PRMT6 inhibitors on retinal structure and improvement of visual function in DR mice. A: Optical coherence tomography (OCT) assessment of overall retinal thickness; B: HE staining assessment of changes in the structure and thickness of each retinal layer; C: Quantitative statistical analysis of inner retinal thickness; D: Electroretinography (ERG) detection of changes in the electrophysiological function of retinal cells in each group of mice. Data are expressed as mean ± SEM (N = 10). Significance level: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns indicates no statistical difference. Scale bar = 50 μm.

[0024] Figure 3 To investigate the effect of PRMT6 inhibitors on the abnormal vascular phenotype in DR mice. A shows color fundus photography of each group (N=3, scale bar 200 μm, white arrows indicate abnormal vascular hemorrhage); B shows fundus fluorescein angiography of each group (N=3, scale bar 200 μm; inset below is magnified 20 times; white arrows indicate vascular perfusion disorders, white boxes indicate high fluorescence bright spots indicating capillary leakage); C shows the effect of EPZ020411 on the expression of retinal vascular damage markers detected by qPCR. Data are expressed as mean ± SEM (N = 5). Significance level: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Detailed Implementation

[0025] The specific steps of the present invention are illustrated below through examples, but are not limited to these examples.

[0026] Unless otherwise stated, the terms used in this invention generally have the meanings commonly understood by those skilled in the art.

[0027] The present invention will now be described in further detail with reference to specific examples and data. It should be understood that these embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention in any way.

[0028] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art.

[0029] Example 1: Protective effect of PRMT6 inhibitors on retinal structure and visual function in a mouse model of diabetic retinopathy.

[0030] 1. Experimental materials and animals

[0031] Experimental animals: C57BL / 6J male mice, 8-10 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (China).

[0032] Experimental Materials: A diabetic retinopathy model was established by intraperitoneal injection of streptozotocin (STZ, S0130, powder, Sigma-Aldrich, USA). A blood glucose meter (C7-0A2) and blood glucose test strips (KF-A01-C type) were purchased from Kefu Medical Technology Co., Ltd. (China). Anesthesia medications Sutamethasone (teletamine hydrochloride and zolazepam hydrochloride for injection) and Sumatrazepine were purchased from Virbac (France) and Saint-Gobain Animal Pharmaceutical Co., Ltd. (China), respectively. Mydriatic medications Metoprolol (compound tropicamide eye drops) and levofloxacin (Clonax) were purchased from Santen Pharmaceutical Co., Ltd. (China), and topical anesthetic promecaine hydrochloride eye drops (Alcarein) were purchased from Alcon Eye Products Co., Ltd. (China). Ultrasound coupling gel was purchased from Haisheng Health Technology Co., Ltd. (China). HE staining solution (G1005) for section staining was purchased from Wuhan Sewell Biotechnology Co., Ltd. (China).

[0033] Experimental equipment: The visual electrophysiology instrument (OPTO-ERG) and ophthalmic ultramicroscopic imaging system (ISOCT) were purchased from Optoprobe Science LTD (UK). The visual electrophysiology instrument was a Diagnosys D315, and the OCT image analysis software was OCT Image Analysis (Version 2.0).

[0034] 2. Experimental Grouping and Procedure

[0035] Male C57BL / 6J mice aged 8-10 weeks were randomly divided into 3 groups (N=10 / group): normal control group (NC), DR model group (STZ + PBS), and DR + PRMT6 inhibitor treatment group (STZ + EPZ020411). Figure 1 A). DR model mice were injected with STZ at a dose of 50 mg / kg for 5 consecutive days. After one week, blood glucose and body weight were measured. A blood glucose level >16.7 mmol / L on two consecutive measurements was considered a successful model. After successful modeling, blood glucose and body weight were measured weekly to confirm that the blood glucose level in the model group was within the hyperglycemic standard. Mice below the hyperglycemic standard were discarded. Figure 1 C). Four weeks after model establishment, 5 mg of PRMT6 inhibitor (EPZ020411, S7842, Selleck, China) was dissolved in 5 ml of pure water to a concentration of 1 mg / ml. Mice in the DR + EPZ group were intraperitoneally injected with PRMT6 inhibitor 20 mg / kg once a week for 12 weeks; mice in the DR + PBS group were injected with an equal volume of PBS ( Figure 1 B).

[0036] Twelve weeks after injection of EPZ and control solvents, mice underwent dark adaptation for 12 hours and then underwent visual electrophysiological examination under red light. The procedure was briefly described as follows: Mice were anesthetized by intraperitoneal injection of a mixture of 5 mg / ml cyclophosphamide and 10 mg / ml cyclophosphamide at a 10:1 ratio (5 µl / g). Compound tropicamide eye drops were used for routine mydriasis, followed by topical anesthesia with promecaine hydrochloride eye drops. The mice were then fixed on the operating table, their eyes positioned, and electrodes placed. Electroretinography (ERG) electrode placement: positive electrode to the cornea, negative electrode to the cheek, and ground to the subcutaneous area of ​​the tail. Subsequently, the a-wave, b-wave, and light-adapted negative (PhNR) waves were measured using the OPTOPROBE OPTO-III visual electrophysiology instrument operating system. After the electrophysiological examination, the mice were transferred to an ophthalmic super-microscopic imaging system. The light source was adjusted and focused, and the lens was focused on the retina. Optical coherence tomography (OCT) was then performed on the retina. Both eyes of each experimental animal were photographed. After the experiment, rinse the eyes with saline solution and use levofloxacin eye drops to prevent infection.

[0037] After the above experiments were completed, the mice were euthanized 2-3 days after they recovered from anesthesia, in accordance with ethical guidelines, and eyeball sections were taken for HE staining analysis.

[0038] 3. Experimental Results

[0039] Compared with the STZ + PBS modeling group, the PRMT6 inhibitor treatment group preserved normal retinal thickness. Figure 2 A), HE results showed that the retinal structure of mice in the STZ + EPZ group was well intact ( Figure 2 (B) Further quantitative analysis showed that after EPZ020411 treatment, the thickness of the inner retinal structure related to visual signal transmission was significantly increased compared with the STZ model group, but there was no statistically significant difference compared with the normal control group. Figure 2 C). Functional testing results showed that the EPZ020411 treatment group exhibited varying degrees of improvement in multiple retinal electrophysiological function indicators compared to the model group, suggesting that retinal function had been restored to a certain extent. Figure 2 D).

[0040] Conclusion: The above results indicate that the PRMT6 inhibitor EPZ020411 can maintain the structural integrity of the retina to a certain extent in the STZ-induced diabetic model, especially the stability of the inner layer structure related to visual function, and improve the damaged retinal function indicators in the diabetic state, thereby exerting a protective effect against retinal pathological changes.

[0041] Example 2: PRMT6 inhibitors improve the leakage phenotype in diabetic retinopathy mice

[0042] 1. Laboratory animals and materials

[0043] Experimental animals: C57BL / 6J male mice, 8-10 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (China).

[0044] Experimental Materials: A diabetic retinopathy model was established by intraperitoneal injection of streptozotocin (STZ, S0130, powder, Sigma-Aldrich, USA). Anesthesia: telatamine hydrochloride and zolazepam hydrochloride for injection and sedatives were purchased from Vibac (France) and Saint-Gobain Animal Pharmaceuticals Co., Ltd. (China), respectively. Mydriatic medication: tropicamide compound eye drops and anti-inflammatory medication: levofloxacin (Clonaxetine) were purchased from Santen Pharmaceutical Co., Ltd. (China). Topical anesthetics: promecaine hydrochloride eye drops (Alcarein) and sodium fluorescein injection (107R4, Lissex) were purchased from Alcon Ophthalmic Products Co., Ltd. (China). Ultrasound coupling gel was purchased from Haisheng Health Technology Co., Ltd. (China). Tissue RNA extraction kit (EZB-RN001-plus) was purchased from EZBioscience (China), reverse transcription reagent (R433) and qPCR reagent (R712) were purchased from Novizan Pharmaceutical Co., Ltd. (China).

[0045] Experimental equipment: The retinal imaging system (OPTO-RIS) was purchased from Optoprobe Science LTD (UK).

[0046] 2. Experimental Grouping and Procedure

[0047] Male C57BL / 6J mice aged 8-10 weeks were randomly divided into 3 groups (N=5 / group): normal control group (NC), DR model group (STZ+PBS), and DR+PRMT6 inhibitor treatment group (STZ+EPZ020411). The modeling and drug administration methods were the same as those in the experimental grouping and procedure section of Case 1.

[0048] Twelve weeks after injection of EPZ and control solvents, retinal and fundus vascular examinations were performed. The procedure was as follows: Mice were anesthetized with a 5µl / g intraperitoneal injection of a mixture of 5mg / ml of cyclophosphamide and 10mg / ml of cyclophosphamide at a 10:1 ratio. Compound tropicamide eye drops were used for routine mydriasis, followed by topical anesthesia with promecaine hydrochloride eye drops. The mice were then fixed on the operating table, their eyes positioned, and brought into contact with the lens of the small animal fundus imaging system microscope. The stage and lens focus were adjusted, and photographs were taken. Photographs were taken of both eyes of each animal. For FFA angiography: The light source was switched to the Blue channel, the light intensity was adjusted, and the manual filter wheel on the camera was moved. Then, 1.7ml / kg of 2% sodium fluorescein injection was injected intraperitoneally. After 5 minutes, the animal was placed on the adjustable table, its eyes positioned, and after pupil dilation, medical carbomer eye drops gel was applied to the cornea to be tested. The eye was then brought into contact with the lens of the small animal fundus imaging system microscope, and the stage and lens focus were adjusted, and photographs were taken. Photographs were taken of both eyes of each animal. After the experiment, rinse the eyes with saline solution and use levofloxacin eye drops to prevent infection.

[0049] After the above experiments were completed, the mice were euthanized 2-3 days after recovery from anesthesia, in accordance with ethical guidelines. RNA was extracted from the retina according to the reagent instructions, and reverse transcription was performed for qPCR detection of vascular damage-related markers (ZO-1, Occludin). The primer sequences and qPCR program used are as follows:

[0050] ZO-1:

[0051] Forward primer: 5′-ACCTCTGCAGCAATAAAGCAG-3′;

[0052] Reverse primer: 5′-GAAATCGTGCTGATGTGCCA-3′.

[0053] Occludin:

[0054] Forward primer: 5′-TTCAGGTGAATGGGTCACCG-3′;

[0055] Reverse primer: 5′-AGATAAGCGAACCTGCCGAG-3′.

[0056] qPCR amplification conditions: Step 1: 95 ℃ for 30 seconds, 1 cycle; Step 2: 95 ℃ for 10 seconds + 60 ℃ for 30 seconds, 40 cycles; Step 3: Melting curve.

[0057] 3. Experimental Results

[0058] Treatment with the PRMT6 inhibitor EPZ020411 improved STZ-induced retinal vascular pathological changes in diabetic mice to some extent. Color fundus photography showed that, compared with the model group, the EPZ020411 treatment group exhibited reduced retinal vascular abnormalities and hemorrhages. Figure 3 A). Further fluorescence angiography results showed that EPZ020411 treatment reduced retinal vascular perfusion impairment and significantly improved abnormal capillary permeability. Figure 3 B). Molecular-level analysis showed that the expression levels of structural molecules related to blood-retinal barrier stability (including ZO-1 and Occludin) partially recovered compared to the model group. Figure 3 C).

[0059] Conclusion: The above results indicate that the PRMT6 inhibitor EPZ020411 can improve the abnormal state of retinal vessels and reduce abnormal vascular permeability to a certain extent in a diabetic retinal pathology model, thereby helping to maintain the stability of retinal vascular structure and function.

Claims

1. Application of PRMT6 inhibitors in the preparation of drugs for treating retinal vascular injury.

2. The application as described in claim 1, characterized in that... The retinal vascular injury diseases mentioned include diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, hypertensive retinopathy, retinopathy of prematurity, and retinal vasculitis.

3. The application as described in claim 1, characterized in that... The drug reduces vascular leakage and damage, and maintains retinal structure and visual function.

4. The application as described in claim 1, characterized in that... The PRMT6 inhibitor is one or more of the following: protein inhibitors of the PRMT6 gene, nucleic acid aptamers, and / or interfering RNA, gRNA, microRNA, and small molecule compound inhibitors of the PRMT6 encoding gene.

5. The application as described in claim 4, characterized in that... The PRMT6 inhibitor is a compound with the following structure or a pharmaceutically acceptable salt thereof: .

6. The application as described in claim 1, characterized in that... The drug uses a PRMT6 inhibitor as its active ingredient.

7. The application as described in claim 1, characterized in that... The drug is an oral or injectable formulation.

8. The application as described in claim 7, characterized in that... The drug is an intravenous injection.

9. The application as described in claim 8, characterized in that... The drug also includes one or more of the following: buffers, isotonic modifiers, surfactants, stabilizers, and preservatives.

10. The application as described in claim 5, characterized in that... The dosage of the PRMT6 inhibitor is 20 mg / kg.

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