Small molecule compound L28H15 for preventing and treating posterior cataract and application of small molecule compound L28H15

Abstract

The invention relates to a small molecule compound L28H15 for preventing and treating posterior cataract, which is used as a multi-target inhibitor, is mainly applied to prevention of posterior cataract caused by crystal epithelial intercellular interstitial transformation, provides a new target for clinical prevention and treatment of posterior cataract, and researches the action mechanism of the small molecule compound L28H15 for preventing and treating PCO. The action effects of different drug concentrations on the lens epithelial cells are explored; in animal experiments, in order to overcome the bottleneck of eye administration, a biodegradable PLGA material is adopted to construct a drug-loaded implant, and the purpose of long-acting and local slow release of L28H15 in eyes is achieved. In the later stage, the effectiveness and safety of the L28H15-PLGA implant in preventing PCO are comprehensively evaluated by constructing a rabbit eye model, so that a brand new candidate drug is provided for overcoming the clinical problem of PCO, and an administration approach with a great clinical transformation prospect is also researched.

Description

Technical Field

[0001] This invention relates to the field of prevention and treatment technology for posterior cataracts, specifically to a small molecule compound L28H15 for the prevention and treatment of posterior cataracts and its applications. Background Technology

[0002] Posterior capsular opacification (PCO) is the most common complication after cataract surgery. Studies show that 20-40% of patients will experience vision loss due to PCO 2-5 years later. [1] Despite continuous advancements in surgical techniques and intraocular lens (IOL) design, the incidence of petechial occlusion (PCO) remains high. Its pathological mechanism involves a complex network of multiple processes, including abnormal proliferation and migration of residual lens epithelial cells (LECs), epithelial-mesenchymal transition (EMT), and fibrosis.

[0003] Currently, Nd:YAG laser posterior capsulotomy is the only effective treatment for PCO (peripheral occlusion). However, this invasive procedure not only increases the financial burden on patients but also carries risks such as increased intraocular pressure, increased macular thickness, retinal detachment, IOL damage, and dislocation. Therefore, finding a safe and effective intraoperative or postoperative preventative approach to inhibit the abnormal behavior of LECs (Leg-Oxygen Flow Markers) at its source has become an important and unmet clinical need in ophthalmology. According to research, current research focuses primarily on drugs and delivery systems. Drug research includes antitumor and antimetabolite drugs such as fluorouracil (5-FU), methotrexate (MTX), gefitinib, erlotinib, rapamycin, and mitomycin C; anti-inflammatory drugs such as dexamethasone, succinic acid, cyclosporine A, aspirin, and indomethacin; and natural drugs such as chlorpheniramine, resveratrol, curcumin, and capsaicin. Drugs prevent posterior capsule opacification by inhibiting key pathways of LEC proliferation or directly interfering with the cell cycle, reducing LEC migration to the posterior capsule, inhibiting LEC epithelial-mesenchymal transition (EMT) and related signaling pathways (Smad, PI3K / AKT, MAPK, etc.), and regulating autophagy to induce apoptosis. While drug research has made some progress, it remains largely confined to in vitro or animal experiments, and any drug with potential ocular toxicity is difficult to apply clinically. Many candidate drugs have failed due to serious complications such as corneal endothelial decompensation and retinal toxicity, and drug concentrations are difficult to determine in clinical trials. This necessitates drugs with extremely high safety windows and excellent tissue selectivity. Summary of the Invention

[0004] To address the technical deficiencies of existing technologies, this invention provides a small molecule compound, L28H15, for the prevention and treatment of secondary cataracts and its applications. It is used to prevent secondary cataracts caused by mesenchymal cataracts of lens epithelial cells and to provide a new target for the clinical prevention and treatment of secondary cataracts.

[0005] The technical solution adopted in this invention is: a small molecule compound L28H15 for the prevention and treatment of secondary cataracts, wherein the small molecule compound L28H15 is (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one, and its structural formula is: This molecule possesses a suitable rigid framework and potential amphiphilicity, and its structural features enable it to effectively enter cells and exert its effects.

[0006] Application of a small molecule compound L28H15 in the preparation of drugs for the prevention and treatment of secondary cataracts.

[0007] The aforementioned secondary cataracts include secondary cataracts caused by mesenchymal cataracts of the lens epithelial cells.

[0008] The concentrations of the small molecule compound L28H15 in the drug are 0.1 μM and 0.2 μM.

[0009] Application of a small molecule compound L28H15 in the preparation of materials for preventing secondary cataracts.

[0010] The material for preventing secondary cataracts is an artificial lens modified with a coating of the small molecule compound L28H15.

[0011] The aforementioned material for preventing secondary cataracts is an intraocular lens modified with a coating of polylactic acid-glycolic acid copolymer (PLGA) and the small molecule compound L28H15.

[0012] The artificial lens is prepared by the following steps: first, the artificial lens is irradiated in ultraviolet light for 30 minutes, then PLGA and L28H15 are mixed and dropped onto the edge of the lens loop to obtain an artificial lens modified with PLGA and the small molecule compound L28H15 coating.

[0013] The beneficial effects of this invention are as follows: This invention provides a small molecule compound, L28H15, for the prevention and treatment of secondary cataracts and its applications. It develops a novel small molecule compound, L28H15, as a multi-target inhibitor and studies its mechanism of action in preventing PCO. Firstly, at the in vitro cellular level, this invention explores the effects of different drug concentrations on lens epithelial cells. In animal experiments, to overcome the bottleneck of ocular drug delivery, a drug-loaded implant is constructed using biodegradable PLGA material, aiming to achieve long-term, locally sustained release of L28H15 within the eye. Later, by constructing a rabbit eye model, the efficacy and safety of the L28H15-PLGA implant in preventing PCO are comprehensively evaluated. This not only provides a novel candidate drug for overcoming the clinical challenges of PCO but also explores a drug delivery route with great potential for clinical translation. Attached Figure Description

[0014] Figure 1 The chemical structural formula is L28H15.

[0015] Figure 2 The effect of L28H15 on cell viability; (A): CCK-8 assay results analysis; (B): Cell viability and death detection, results of the effect of different concentrations of L28H15 on lens epithelial cell viability.

[0016] Figure 3 The results are RNA sequencing results of LECs after L28H15 treatment; (A): Correlation plot of gene expression profiles among samples (correlation coefficient R²); (B, C): Volcano plot of gene expression changes (screening criteria: pvalue < 0.05 and |FoldChange| > 2); (D): GSEA-GO enrichment analysis (TOP5); (E): KEGG pathway enrichment analysis.

[0017] Figure 4 The results show the effects of L28H15-induced apoptosis in lens epithelial cells. (A): The effects of 0.1 and 0.2 μM L28H15 on the morphology of lens epithelial cells were observed by transmission electron microscopy at different magnifications. Microvilli (Mv), nucleus (N), nucleolus (Nu), mitochondria (M), Golgi apparatus (Go), rough endoplasmic reticulum (RER), primary lysosome (Ly), autophagosome (AP), autophagolysosome (ASS), cytoplasmic lysis forming vacuoles (red arrow), nuclear fragmentation forming more apoptotic bodies (black arrow). ×2.0k=2000 times, ×8.0k=8000 times, ×20.0k=20000 times. (B): Quantitative analysis of early and late apoptosis of lens epithelial cells by flow cytometry after treating cells with different concentrations of L28H15 for 24 h.

[0018] Figure 5Results of L28H15-induced cell proliferation via the mitochondrial pathway; (A) Transmission electron microscopy observation of mitochondrial morphological changes in lens epithelial cells, magnified 20×=20000 times; (B) L28H15-induced mitochondrial dysfunction: after 24 h of treatment with L28H15 (control, 0.1, 0.2 μM), mitochondrial membrane potential was measured using JC-1 mitochondrial membrane potential assay; green fluorescence showed monomers, indicating the presence of depolarized mitochondria. Red fluorescence showed J-aggregates, indicating mitochondrial functional polarization; (C) Results of Western blotting analysis of apoptosis-related protein expression levels; (D) Detection of intracellular ROS levels after 24 h of L28H15 (control, 0.1, 0.2 μM) treatment with the fluorescent probe DHE; (E) Quantitative analysis of ROS levels in cells by flow cytometry.

[0019] Figure 6 The effect of L28H15 on the proliferation of lens epithelial cells was investigated. (A): Cell cycle distribution was detected by flow cytometry; (B): Cell count (%) at different cell cycle phases; (C): Cell proliferation was determined by EDU colorimetric assay, with EDU staining showing green fluorescence and Hoechst 33342 staining showing blue fluorescence.

[0020] Figure 7 The effect of L28H15 on the migration ability of lens epithelial cells; where (A): the effect of different concentrations of L28H15 on cell migration; (B): wound healing rate quantified by migration area.

[0021] Figure 8 Results of the effect of L28H15 on TGFβ-2-induced EMT transformation of human lens epithelial cells in vitro; results of gene expression levels of VIM, MMP9, MMP2, FN1, COL1A2, and CDH2 in different treatment groups.

[0022] Figure 9 To observe the artificial lens under a slit lamp, fibrotic folds and numerous scattered cells were observed in the posterior capsule (black arrow).

[0023] Figure 10 Images of frozen sections of tissue; (A): control group with implanted intraocular lenses; (B): L28H15+PLGA coated intraocular lenses; anterior capsule (AC), posterior capsule (PC), Soemmerring ring (SR). Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Effects of L28H15 on cell viability In cultured lens epithelial cells, L28H15 drug at concentrations of 0.05, 0.1, 0.2, 0.4, 0.5, 1.0, 2.0, and 4.0 μM were added, respectively. After 12 h of treatment, cell viability was quantitatively and qualitatively assessed using the CCK-8 assay and cell viability / death staining, respectively. The results showed that, compared with the control group, the effect of L28H15 drug on cell viability decreased in a concentration-dependent manner. Figure 2 A). Observation of cell number using a fluorescence microscope showed that as the drug concentration increased, the number of live cells decreased, the number of dead cells increased, the number of cells floating in the culture medium increased, and the cell adhesion ability decreased. Figure 2 B). Combining CCK-8 and cell viability assays, L28H15 was found to have an inhibitory effect on LECs. The results also showed that 0.1 μM L28H15 had an inhibitory effect on lens epithelial cells, with a cell survival rate of 80% compared to the control group; the inhibitory effect was more pronounced at 0.2 μM, with a cell survival rate of 71%. Considering the efficacy and cytotoxicity, subsequent experiments selected 0.1 μM and 0.2 μM concentrations for treatment.

[0026] Cell RNA sequence analysis results Lens epithelial cells were treated with different concentrations (control, 0.1 μM, 0.2 μM) of L28H15 for 24 h, and then the cells were collected for sequencing analysis. The correlation coefficient (R0.05) of gene expression profiles among all samples was used as the basis for analysis. 2 This was used to evaluate experimental repeatability and grouping effectiveness, and to plot correlation diagrams. Sequencing results are as follows: Figure 3 As shown, all biological replicates exhibited high intragroup correlation (R0). 2 >0.99), forming a clear block distribution. Simultaneously, there were significant distinctions between different treatment groups (Group A: control; Group B: 0.1 μM; Group C: 0.2 μM), indicating that drug treatment caused global changes in gene expression. (See volcano plot). Figure 3As shown in B and C), compared with group A, group B revealed a large number of significantly differentially expressed genes, with 97 upregulated and 198 downregulated (screening criteria: pvalue < 0.05 and |FoldChange| > 2); group C revealed a large number of significantly differentially expressed genes, with 240 upregulated and 208 downregulated. These results indicate that changes in the concentration of the drug L28H15 alter gene expression in the cellular transcriptome, providing a reliable basis for subsequent differential analysis. GSEA enrichment results analysis ( Figure 3 D) showed that group A genes were significantly enriched in pathways related to cell survival, proliferation, and signal transduction, including DNA replication, inflammatory response, receptor ligand activity, and signal receptor activator activity. This phenomenon indirectly confirms that after cells are treated with the drug L28H15, the core processes maintaining normal cellular life activities are significantly inhibited, which are closely related to the inhibition of DNA replication and cell proliferation; while the drug's interference with receptor ligand signaling pathways may block external signals essential for cell survival. KEGG pathway enrichment analysis ( Figure 3 E) The final results that validated the drug's effects showed that drug treatment significantly enriched the apoptosis pathway. Multi-level bioinformatics analysis in this study consistently demonstrated that the drug L28H15 can induce apoptosis in lens epithelial cells by massively reshaping gene expression profiles, inhibiting cell proliferation and survival signals (such as DNA replication and receptor signaling), and simultaneously activating the classical apoptosis pathway and its upstream inflammation-related signaling networks (such as TNF / NF-κB).

[0027] Effects of L28H15 on cell structure and apoptosis Lens epithelial cells were treated with different concentrations (control, 0.1 μM, 0.2 μM) of L28H15 for 24 h, and the results were observed using transmission electron microscopy as follows: Figure 4A): Compared with the control group, apoptosis was observed in both the 0.1 μM and 0.2 μM groups. The cell membrane was blurred and discontinuous, and the cytoplasm dissolved unevenly; in the 0.2 μM group, cytoplasmic dissolution and vacuolation were visible (red arrow). The nuclear membrane of the cell nucleus (N) was damaged and disappeared, heterochromatin condensed in large quantities, and the nucleus fragmented to form apoptotic bodies (black arrow); the 0.2 μM group had a greater number of apoptotic bodies; the number of mitochondria (M) was reduced, and the damage was obvious, with blurred and damaged membranes, diluted matrix dissolution, and broken and disappeared cristae; the rough endoplasmic reticulum (RER) was obviously expanded, and ribosome degranulation was visible on the surface; a large number of primary lysosomes (Ly) were visible in the field of view; a few autophagosomes (AP) and a large number of autolysosomes (ASS) were present. Flow cytometry was used to observe L28H15-induced apoptosis in LECs. Compared with the control group, the early apoptosis rate (Annexin V-iFluor 488) (4.367±0.4309%, 9.233±0.8650%) and late apoptosis rate (PI) (7.823±0.8593%, 12.27±0.1528%) of LECs treated with L28H15 (0.1μM, 0.2μM) both increased in a concentration-dependent manner. Figure 4 B) L28H15 induces apoptosis via the mitochondrial pathway. Lens epithelial cells were treated with different concentrations (control, 0.1 μM, 0.2 μM) of L28H15 for 24 h, and mitochondrial morphology was observed under a 20,000x transmission electron microscope. Figure 5 A) It was found that the mitochondria in the control group were mostly oval or rod-shaped, with intact membranes and normal structure. Mitophagy was observed in both the 0.1 μM and 0.2 μM groups, with the disappearance of damaged mitochondrial cristae and matrix swelling, encapsulated by autophagosomes formed by a double-membrane structure (black arrows). The JC-1 experiment showed that ( Figure 5 B), LECs treated with L28H15 (0.1 μM, 0.2 μM) showed significantly reduced red fluorescence intensity and significantly enhanced green fluorescence, indicating that L28H15 significantly reduced mitochondrial transmembrane potential (Δψm). Western blot results showed ( Figure 5 C): Compared with the control group, after treatment with L28H15, the ratio of the anti-apoptotic protein Bcl-2 to the pro-apoptotic protein Bax was significantly reduced, while the expression level of the apoptosis-executing protein Cleaved Caspase-3 was significantly increased. This indicates that the drug L28H15 promotes apoptosis signaling by regulating the key mitochondrial apoptosis "switch" of Bcl-2 / Bax, ultimately activating the Caspase-3-dependent apoptosis program. Oxidative stress is another typical feature of mitochondrial dysfunction. The changes in intracellular ROS after treatment with L28H15 (control, 0.1 μM, 0.2 μM) were detected using the fluorescent probe DHE, such as ( Figure 5As shown in E), the intracellular ROS fluorescence intensity increased in a concentration-dependent manner. To further investigate the effect of icariin on the intracellular oxidative stress response of LECs, flow cytometry was used to detect the intracellular oxidative stress intensity. The results showed that L28H15 significantly increased the intracellular ROS level (E). Figure 5 (D and E). In summary, L28H15 can promote apoptosis through the mitochondrial pathway.

[0028] L28H15 affects cell proliferation by arresting the cell cycle. L28H15 (control, 0.1 μM, 0.2 μM) was used to treat crystalloid epithelial cells for 24 h, followed by staining with propidium iodide (PI) and flow cytometry analysis. The results showed that L28H15 induced G2 / M phase arrest in LECS cells. Figure 6 A, B). G2 / M phase arrest is a prerequisite for cell apoptosis. LECs treated with L28H15 (control, 0.1, 0.2 μM) were labeled with 5-ethyl-2'-deoxyuridine (EDU), and the nuclei were stained with Hoechst 33342. A significant decrease in EDU (red fluorescence) was observed, along with a reduction in the overall number of Hoechst-labeled cells (weakened cell adhesion). Simultaneously, some nuclei exhibited apoptosis-related fragmentation morphology. These results indicate that L28H15 significantly reduces the proportion of EDU+ cells in a concentration-dependent manner, inhibiting cell proliferation. Figure 6 C).

[0029] The impact of L28H15 on LEC migration ability Cells were treated with different concentrations of L28H15 (control, 0.1 μM, 0.2 μM) for 0 h, 12 h, and 24 h, and the wound healing area was observed under a microscope. The results showed that the wound healing rate at 12 h and 24 h was 57.40±5.378% in the control group, 44.48±3.050% in the 0.1 μM group, and 38.57±3.506% in the 0.2 μM group. This indicates that treatment of LECs with L28H15 (control, 0.1, 0.2 μM) significantly slowed the wound healing rate. In conclusion, L28H15 inhibits the migration of LECs in a concentration-dependent manner. Figure 7 A, B).

[0030] Effects of L28H15 on TGFβ2-induced EMT-related gene expression in lens epithelial cells Cells were treated with different concentrations of L28H15 (control, 0.1 μM, 0.2 μM) for 24 h, and cellular mRNA was extracted for quantitative Q-PCR. The results showed that, compared with the control group, the TGFβ2 group significantly increased the mRNA expression of mesenchymal markers (CDH2, VIM, FN1) and extracellular matrix molecules (COL1A2, MMP9, MMP2) (p<0.001). Compared with cells treated with 10 ng / ml TGFβ2 alone, the combined treatment with L28H15 and 10 ng / ml TGFβ2 significantly reduced the mRNA expression levels of VIM, MMP9, and MMP2 (p<0.05), while the reduction in the mRNA expression levels of FN1, COL1A2, and CDH2 was not significant. The effect was more pronounced at 0.2 μM than at 0.1 μM. These results indicate that L28H15 can antagonize the TGFβ2-induced expression of some EMT markers in HLE B-3 cells.

[0031] The role of L28H15 in animal models This experiment used intraocular lenses from Aibonuo Pharmaceuticals. Healthy male New Zealand white rabbits aged 11-14 weeks, weighing 2.5 ± 0.3 kg, were selected. All animals underwent slit-lamp examination before surgery to rule out pre-existing eye diseases and were randomly divided into two groups of five animals each. This animal experimental protocol was approved by the Experimental Animal Ethics Committee of Wenzhou Medical University, and all animal procedures were strictly conducted in accordance with the university's animal experimental guidelines.

[0032] The intraocular lenses (IOLs) were first irradiated with ultraviolet light in a clean bench for 30 minutes. Then, 1% PLGA and 100 μL of L28H15 were mixed, and 1 μL was dropped onto the lens loop edge. The control group received no treatment after ultraviolet irradiation and was stored in a light-proof, sealed container. After phacoemulsification surgery, IOLs modified with the PLGA+L28H15 coating were implanted into the eyes of the experimental animals. No acute inflammation or infection or iris adhesion was observed in any of the operated eyes. Three days postoperatively, both the IOL group and the PLGA+L28H15-coated IOL group showed mild corneal edema and slight inflammation, possibly related to the surgical procedure and residual viscoelastic agent. These symptoms subsided after treatment with anti-inflammatory eye drops. Two weeks postoperatively, pathological proliferation of residual lens endothelial cells was observed, and the differences between the groups gradually became apparent and rapidly developed. In the intraocular lens group, partial fibrosis and wrinkling of the posterior capsule were observed; in contrast, rabbit eyes implanted with PLGA+L28H15-coated modified intraocular lenses at the same time showed no atrophy or fibrosis of the posterior capsule. At 4 weeks post-implantation, significant cell proliferation was observed in the intraocular lens group, with cells migrating towards the central visual area; while the posterior capsule of the PLGA+L28H15-coated modified intraocular lens remained clear and transparent, without proliferation or significant opacity. Figure 9 ).

[0033] The role of L28H15 in animal models After a 4-week observation period, rabbits were anesthetized, and the lens capsule of the rabbit's eye was harvested for frozen section preparation. Results showed that in the intraocular lens group, the Sommering ring, formed by the fusion of the anterior and posterior capsules, surrounded varying numbers of lens cortex and proliferating / degenerated epithelial cells; severe cortical remnants and significant anterior and posterior capsule hyperplasia were observed in the visual area. Figure 10 (As indicated by the arrow at point A). In contrast, the PLGA+L28H15 coated intraocular lens group showed intact capsule structure and minimal cell proliferation and fibrosis. Figure 10 B). Pathological histological analysis confirms that L28H15 inhibits the formation of PCO.

[0034] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of patent protection of this patent.

[0035] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A small molecule compound L28H15 for the prevention and treatment of secondary cataracts, characterized in that, The small molecule compound L28H15 is (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one, and its structural formula is as follows: 。 2. The use of the small molecule compound L28H15 according to claim 1 in the preparation of a drug for preventing and treating secondary cataracts.

3. The application according to claim 2, characterized in that, The aforementioned secondary cataracts include secondary cataracts caused by mesenchymal lesions of the lens epithelial cells.

4. The application according to claim 2, characterized in that, The concentrations of the small molecule compound L28H15 in the drug are 0.1 μM and 0.2 μM.

5. The application of the small molecule compound L28H15 according to claim 1 in the preparation of materials for preventing and treating secondary cataracts.

6. The application according to claim 5, characterized in that, The material for preventing secondary cataracts is an artificial lens modified with a coating of the small molecule compound L28H15.

7. The application according to claim 6, characterized in that, The aforementioned material for preventing secondary cataracts is an intraocular lens modified with a coating of polylactic acid-glycolic acid copolymer (PLGA) and the small molecule compound L28H15.

8. The application according to claim 7, characterized in that, The artificial lens is prepared by the following steps: first, the artificial lens is irradiated in ultraviolet light for 30 minutes, then PLGA and L28H15 are mixed and dropped onto the edge of the lens loop to obtain an artificial lens modified with PLGA and the small molecule compound L28H15 coating.

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