A medicine for treating glucocorticoid-induced glaucoma
By using the Rho kinase inhibitor RKI1447 to regulate the trabecular meshwork cytoskeleton, the limitations of existing drugs in reducing the efficacy of glucocorticoid-induced glaucoma were overcome, resulting in a significant reduction in intraocular pressure and improved safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 王腾文
- Filing Date
- 2026-04-27
- Publication Date
- 2026-05-29
AI Technical Summary
Existing medications are ineffective in lowering intraocular pressure when treating glucocorticoid-induced glaucoma, and they also have limitations in efficacy and tolerability issues, affecting long-term treatment adherence.
By using the Rho kinase inhibitor RKI1447, the intraocular pressure is reduced by regulating the trabecular meshwork cytoskeleton, altering cell morphology, inhibiting cell migration and pathological extracellular matrix deposition, and reducing aqueous humor outflow resistance.
RKI1447 significantly reduces intraocular pressure, reverses pathological cytoskeleton remodeling, improves therapeutic efficacy and safety, and provides a good balance between efficacy and safety, making it suitable for use alone or in combination with other drugs.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of medicine, specifically relating to a drug for treating glucocorticoid-induced glaucoma. Background Technology
[0002] Glaucoma is a leading cause of irreversible blindness worldwide. Clinically, it is characterized by localized loss of optic disc rim tissue, progressive enlargement and deepening of the optic cup, and is accompanied by typical glaucomatous visual field defects. Glucocorticoid-induced intraocular pressure is a common and serious complication of glucocorticoid (GC) use. Given that intraocular pressure remains the only modifiable risk factor in glaucoma treatment, developing new drugs that target the aqueous humor outflow pathway to lower intraocular pressure is of significant clinical importance.
[0003] Under physiological conditions, aqueous humor drains through the trabecular meshwork, juxtatubular tissue, and Schlemm's canals. Obstruction or increased resistance to aqueous humor outflow through this pathway is a major cause of elevated intraocular pressure (IOP). Studies have shown that specific small-molecule compounds can disrupt the trabecular meshwork cytoskeleton and reduce the contractility of trabecular meshwork cells, effectively reducing aqueous humor outflow resistance and thus lowering IOP. As the primary pathway for aqueous humor drainage, the trabecular meshwork has become an important target for glaucoma treatment. Therefore, it is essential to seek novel IOP-lowering drugs that act on the trabecular meshwork pathway to reduce IOP.
[0004] The Rho family is an important component of the Ras superfamily of small guanosine triphosphate (GTPases), which mainly consists of three isoforms: RhoA, RhoB, and RhoC. As a crucial downstream signal transduction node for Rho GTPases, ROCK belongs to the serine / threonine protein kinase family. ROCK has two isoforms: ROCK1, containing a 1354-amino acid sequence, and ROCK2, containing a 1388-amino acid sequence. Once activated, ROCK catalyzes phosphorylation modifications of various substrate proteins, such as myosin light chain hosphatase (MLCP), LIM kinase, and myosin phosphatase-targeting subunit-1 (MYPT-1), thereby mediating biological processes related to actin cytoskeleton dynamics, smooth muscle contraction, and ECM remodeling.
[0005] Traditional intraocular pressure-lowering drugs often fail to exert a direct effect on trabecular meshwork lesions. Rho-associated kinase (ROCK) inhibitors, as a new class of intraocular pressure-lowering drugs, have shown unique advantages by regulating cell contractility and biomechanical properties, but there is still a need to explore new molecules with greater efficacy and fewer side effects.
[0006] RKI1447, whose structural formula is shown in Formula 1, is a highly effective small molecule inhibitor of ROCK1 and ROCK2 in the RhoA / ROCK family.
[0007] Summary of the Invention
[0008] The purpose of this invention is to provide the use of Rho kinase inhibitor RKI1447 in the preparation of drugs for treating glucocorticoid-induced glaucoma.
[0009] This invention demonstrates through experiments that RKI-1447 reversibly alters the morphology of trabecular meshwork cells, inhibits cell migration, disrupts glucocorticoid-induced cytoskeleton remodeling, and reduces extracellular matrix deposition. These results suggest that RKI1447 may regulate aqueous humor drainage and reduce intraocular pressure by modulating the trabecular meshwork cytoskeleton, thereby potentially treating glucocorticoid-induced glaucoma.
[0010] Therefore, this invention provides the use of Rho kinase inhibitors in the preparation of drugs for treating glucocorticoid-induced glaucoma.
[0011] Preferably, the inhibitor of Rho kinase is RKI1447.
[0012] A second objective of this invention is to provide a medicament for treating glucocorticoid-induced glaucoma, characterized in that it contains an inhibitor of Rho kinase as an active ingredient.
[0013] Preferably, the inhibitor of Rho kinase is RKI1447.
[0014] The third objective of this invention is to provide the application of RKI1447 in the preparation of drugs that alter the morphology of trabecular meshwork cells and inhibit cell migration, thereby regulating aqueous humor drainage and reducing intraocular pressure.
[0015] In current glaucoma treatment, lowering intraocular pressure remains the core strategy; however, existing drugs still have limitations in efficacy, tolerability, or dosing frequency. Some patients face problems such as insufficient response, eye irritation, or the need for frequent medication, affecting long-term treatment adherence and efficacy. This invention has found that the ROCK inhibitor RKI-1447 induces reversible morphological changes in HTM cells and destroys pathological stress fibers at the cellular level, directly reducing aqueous humor outflow resistance by enhancing tissue compliance. At the molecular and phenotypic levels, RKI-1447, by blocking the MYPT-1 / MLCP / MLC signaling cascade, not only reverses the DEX-induced myofibroblast phenotypic transformation (marked by α-SMA downregulation) but also significantly inhibits the pathological accumulation of ECM components (such as COL IV, FN, and LN). The results of this experiment suggest that RKI-1447 exerts significant intraocular pressure-lowering and TM-protective effects mainly by antagonizing the abnormal cytoskeleton remodeling mediated by the RhoA / ROCK signaling axis.
[0016] The RKI-1447 of this invention targets the RhoA / ROCK signaling cascade, altering the molecular processes of actin system relaxation and cytoskeleton depolymerization, loosening TM cells, reversing hormone-induced pathological cytoskeleton remodeling and ECM pathological deposition, thereby reducing aqueous humor outflow resistance. This drug has been shown to exhibit a good balance between efficacy and safety. Unlike currently used antiglaucoma drugs, it can be used alone to lower intraocular pressure or in combination with other drugs to achieve better efficacy. Attached Figure Description
[0017] Figure 1 The phosphorylation levels of MLC and MYPT-1 in human trabecular meshwork cells and HTM cells treated with DEX, DEX and RKI1447, and RKI1447, respectively.
[0018] Figure 2 These are the results of cell viability measurements using CCK-8. 50 μM RKI-1447 significantly inhibited cell viability, while concentrations ≤ 10 μM showed no significant toxicity.
[0019] Figure 3 (A) Representative images of COL IV, FN, LN, and α-SMA expressed by immunofluorescence. (B) Quantitative analysis of COL IV immunofluorescence intensity. (C) Quantitative analysis of FN immunofluorescence intensity. (D) Quantitative analysis of LN immunofluorescence intensity. (E) Quantitative analysis of α-SMA immunofluorescence intensity.
[0020] Figure 4The changes in intraocular pressure in SD rats after treatment with DEX and DEX+2μM, DXX+10μM, and DEX+50μM RKI1447 are shown. Detailed Implementation
[0021] The following embodiments are further illustrations of the present invention, but not limitations thereof.
[0022] Example 1
[0023] 1. Main reagents
[0024] RKI1447 (Beyotime Biotechnology Co., Ltd.); Dexamethasone Sodium Phosphate Injection (Chenxin Pharmaceutical Co., Ltd.); Phosphate Buffered Salt (Thermo Fisher Scientific); Trypsin (Gibco); MLC-2 (CST); MYPT-1 (CST); PBS Buffer (phosphate-buffered saline); CCK-8 Kit (APE×BIO); Reverse Transcription Kit (Takara).
[0025] 2. Experimental Procedure
[0026] 2.1 Human ocular trabecular meshwork cell culture
[0027] Human trabecular meshwork cells (CyberKang (Shanghai) Biotechnology Co., Ltd.) were cultured in vitro in primary human trabecular meshwork cell-specific complete culture medium (primary trabecular meshwork cell basal medium + primary trabecular meshwork cell culture additive + 10% fetal bovine serum + antibiotics) at 37℃ in a CO2 incubator with a 5% CO2 concentration. Cells cultured to passages 3-6 were used in the experiments.
[0028] 2.2 Observation of trabecular meshwork cell morphology and cytoskeleton interstitial spaces
[0029] HTM cells in the logarithmic growth phase after resuscitation were seeded at an appropriate density in 6-well plates, with 2 ml per well. RKI-1447 was diluted in complete culture medium at different final concentrations (0.4 μM, 2 μM, 10 μM) and cells were induced for 0 and 2 h consecutively. At each time point before and during drug intervention, the adherent cells were monitored in real time using an inverted phase-contrast microscope, and images were acquired and recorded. After drug intervention, the cells were morphologically evaluated and photographed again 15 h after drug withdrawal.
[0030] 2.3 Effects of RKI1447 on the growth of human trabecular meshwork cells
[0031] HTM cells in the logarithmic growth phase were seeded into 96-well plates at a concentration of approximately 5 × 10³ cells per well, with 100 μl of cell suspension added to the outermost layer of each well. After seeding, the 96-well plates were placed in an incubator. After 24 hours, the original culture medium was discarded and replaced with culture medium containing different concentration gradients of drugs. A control group was set up: the same volume of the corresponding solvent (anhydrous ethanol / DMSO) as the drug treatment group was added. A blank group was set up: no cells were seeded, only an equal volume of complete culture medium was added.
[0032] The cells were placed in a CO2 incubator and exposed to the drug according to a preset time gradient. 110 μl of a pre-mixed reaction mixture consisting of 100 μl of complete culture medium and 10 μl of CCK-8 reagent was added to each well. The cells were incubated in the CO2 incubator in the dark for 4 h. An automated microplate reader was then used to measure the OD value at a wavelength of 450 nm, and cell viability was quantitatively assessed based on the acquired optical density data.
[0033] 2.4 RKI1447 inhibits dexamethasone-induced upregulation of extracellular matrix and α-SMA.
[0034] Primary HTM cells, confluent at a fusion density of 60%-70%, were washed onto coverslips with PBS, fixed with 4% paraformaldehyde at room temperature for 30 min, washed again with PBS, permeabilized with 0.1% Triton X-100 for 20 min, and washed again with PBS. Blocking with 5% BSA for 1 h was performed to inhibit non-specific binding. After discarding the blocking solution, primary antibodies diluted appropriately (COLⅣ 1:200, FN 1:200, α-SMA 1:100, LN 1:50) were added and incubated overnight at 4°C. The next day, the primary antibodies were recovered, washed thoroughly with PBS, and then diluted secondary antibodies (Goat Anti-Mouse IgG H&L, diluted 1:200 or Goat Anti-Rabbit IgG H&L, diluted 1:200) were added and incubated at room temperature in the dark for 1 h.
[0035] Wash again in the dark, and finally mount with a DAPI-containing anti-fluorescence quenching mounting medium. Observe the results using a fluorescence microscope or confocal microscope. Throughout the process, avoid light and keep the sample moist.
[0036] 2.5RKI1447 reduces dexamethasone-induced high intraocular pressure
[0037] Forty SD rats (20 males and 20 females) were randomly divided into two groups based on age and sex: a treatment group (n=20) and a control group (n=20). Baseline intraocular pressure (IOP) was measured at 0, 2, 4, 6, and 8 hours prior to drug administration. A single application of 2 μM RKI-1447 eye drops to the right eye was administered daily at 9:00 AM. An equal volume of the solution was administered to the right eye of one of the remaining 20 rats as a control, for 7 consecutive days. IOP was measured bilaterally using a tonometer at 2 hours (11:00), 4 hours (13:00), 6 hours (15:00), and 8 hours (17:00) after daily drug administration. Changes in IOP over time were observed and recorded, along with changes in conjunctival congestion. A one-week washout period was established between experiments with different concentrations of RKI-1447, following the same procedures.
[0038] 2.6 Statistical Methods
[0039] All data are expressed as mean ± standard deviation or mean ± standard error. Statistical analysis was performed using paired t-tests (comparison with baseline), repeated measures ANOVA (comparison at different time points), and one-way ANOVA (comparison among multiple groups), combined with appropriate post-hoc tests (Bonferroni or Tukey methods). A p-value < 0.05 was considered statistically significant.
[0040] 3. Experimental Results
[0041] 3.1 Observation of trabecular meshwork cell morphology
[0042] Under an electron microscope, trabecular meshwork cells cultured after dexamethasone treatment were observed to exhibit irregular, spindle-shaped, pointed, fusiform, and flattened characteristics, with extensive intercellular contact. (e.g.) Figure 1 Treatment with RKI-1447 resulted in cells exhibiting significant loosening, shrinkage, thinning, and increased intercellular spaces. The drug-containing medium was then replaced with complete medium. Restoration of normal cell morphology was observed after 15 hours.
[0043] 3.2 CCK-8 assay for cell viability inhibition
[0044] We used GrafPadPrismer 7 software to process the CCK-8 cell viability data using one-way ANOVA, and the results are as follows: Figure 3As shown in the figure, the growth of trabecular meshwork cells was significantly inhibited in the 10 μM ML141 treatment group compared to the control group (P < 0.001). The growth of trabecular meshwork cells was not inhibited in groups with ML141 concentrations of ≤ 5 μM. These results suggest that concentrations of ML141 below 5 μM do not affect cell growth, have no toxic side effects on trabecular meshwork cells, and can promote aqueous humor drainage by increasing the intercellular spaces of the trabecular meshwork cells, thereby reducing intraocular pressure and treating glaucoma.
[0045] 3.3 RKI-1447 inhibits dexamethasone-induced upregulation of extracellular matrix and α-SMA.
[0046] This experiment aimed to investigate the intervention effect of RKI-1447 on the DEX-induced contractile properties of HTM cells by detecting the expression of the contractile protein α-SMA. Under consistent immunofluorescence staining conditions, antibody concentration, and incubation time, the results showed that DEX treatment significantly increased the expression of COLⅣ, FN, LN, and α-SMA in HTM cells. Conversely, RKI-1447 treatment significantly inhibited the DEX-induced upregulation of these proteins.
[0047] 3.4 RKI-1447 significantly reduced intraocular pressure in normal and glucocorticoid-induced high intraocular pressure rats.
[0048] In normotensive SD rats, a single topical administration of different concentrations of RKI-1447 (2 μM, 10 μM, and 50 μM) showed a significant dose-dependent IOP-lowering effect compared to the solvent control group. After one week of treatment, significant IOP-lowering effects were observed within 2 to 8 hours post-administration, peaking at 2 hours. Compared to the solvent control group, the maximum IOP reductions from 2 μM, 10 μM, and 50 μM RKI-1447 were 1.45 ± 0.32 mmHg, 1.95 ± 0.30 mmHg, and 2.30 ± 0.30 mmHg, respectively. Furthermore, during the one-week treatment period, all concentrations of RKI-1447 (2 μM, 10 μM, and 50 μM) maintained a significant IOP-lowering effect for 2 hours post-administration.
[0049] 4. Conclusion
[0050] RKI1447 inhibits the RhoA-ROCK signaling pathway, disrupts glucocorticoid-induced cytoskeleton remodeling, and reduces extracellular matrix deposition. Simultaneously, at concentrations below 10 μM, it exhibits no toxic side effects on cell growth while significantly inhibiting the RhoA-ROCK signaling pathway. These results suggest that RKI1447 may regulate aqueous humor drainage and reduce intraocular pressure by altering trabecular meshwork cell morphology and inhibiting cell migration, thereby potentially treating glucocorticoid-induced glaucoma.
Claims
1. Application of Rho kinase inhibitor RKI1447 in the preparation of drugs for treating glucocorticoid-induced glaucoma.
2. The application according to claim 1, characterized in that, The inhibitor of Rho kinase is RKI1447.
3. A drug for treating glucocorticoid-induced glaucoma, characterized in that, It contains an inhibitor of Rho kinase as the active ingredient.
4. The medicament for treating glucocorticoid-induced glaucoma according to claim 3, characterized in that, The inhibitor of Rho kinase is RKI1447.
5. The application of Rho kinase inhibitors in the preparation of drugs that alter the morphology of trabecular meshwork cells and inhibit cell migration, thereby regulating aqueous humor drainage and reducing intraocular pressure.
6. The application according to claim 5, characterized in that, The inhibitor of Rho kinase is RKI1447.