The Role of Rho Kinase Inhibitors in Corneal Diseases

Introduction

The cornea, the transparent outermost layer of the eye, plays a crucial role in refracting and focusing light onto the retina, making clear vision possible. Various diseases, injuries, and surgical procedures can compromise the integrity and function of the cornea, leading to vision impairment and blindness.^1,2 The cornea is composed of five layers, including the epithelium, Bowman’s layer, stroma, Descemet’s membrane, and endothelium.

The corneal endothelium is a single layer of cells that lines the posterior surface of the cornea and is essential for maintaining the corneal transparency by regulating the flow of fluids into and out of the cornea. Dysfunction of the corneal endothelium, as seen in Fuchs’ endothelial corneal dystrophy (FECD), can lead to corneal edema, opacification, and ultimately, vision loss.^3,4 As corneal endothelial cells have very limited capacity to regenerate, current therapies for corneal endothelial diseases focus on managing the symptoms rather than restoring the function of the damaged endothelium. The mainstay of treatment is corneal transplantation. However, these procedures are associated with several limitations, such as the shortage of donor corneas and the risk of graft rejection.

Rho-associated protein kinase (ROCK) is a serine/threonine kinase that regulates various cellular processes.^5–7 Dysregulation of ROCK signaling has been implicated in various pathological conditions, such as cancer, cardiovascular diseases, and neurological disorders.^8,9

ROCK inhibitors refer to a class of pharmacological agents that target and inhibit the activity of ROCK and in the realm of ophthalmology, the application of ROCK inhibitors is well-established in glaucoma.^10–14 Recent research has shown that ROCK inhibitors may have therapeutic potential in managing corneal diseases by increasing cell proliferation and adhesion and by reducing apoptosis.^15–20

This review paper aims to provide an overview of the current state of knowledge regarding the application of ROCK inhibitors in corneal diseases. We will discuss the mechanism of action of ROCK inhibitors, summarize the preclinical and clinical studies that have evaluated their efficacy and safety, and highlight potential future directions for research in this field.

The ROCK Pathway in Corneal Endothelial Cells

ROCK is a serine/threonine kinase that is modulated by the activity of RhoA, an intracellular GTPase.⁵ It regulates several cellular processes, including actin cytoskeleton dynamics, cell adhesion and proliferation.^21–28 Dysregulation of ROCK signaling has been implicated in various pathological conditions, such as cancer, cardiovascular diseases, diabetes, neurological disorders and glaucoma.^8,29–34

Several downstream signaling pathways have been identified to mediate the effects of ROCK activation. One of the key pathways is the myosin light chain (MLC) phosphorylation pathway, which results in the activation of myosin II and subsequent cytoskeletal rearrangement, contractility and extracellular matrix synthesis.^5,35 This leads to changes in cell shape, adhesion, and migration, all of which are important for corneal endothelial cell function. Another important pathway downstream of ROCK is the LIMK/cofilin pathway leading to Rho-induced actin polymerization^5,22,36 (see Figure 1).

Figure 1 The ROCK signaling pathway simplified from references. Abbreviations: ECM, extracellular matrix; LIMK, Lin-11, Islet-1, Mec-3 kinase; MLC, myosin light chain; RhoA, Ras homolog family member A; ROCK, Rho-associated coiled-coil containing kinase. Notes: Data from these studies.5,26,30,36

The activation of the RhoA/ROCK pathway in corneal endothelial cells has been found to play a crucial role in the pathogenesis of corneal endothelial dysfunction. Several mechanisms have been proposed for the activation of the ROCK pathway in corneal endothelial cells which cause increased cellular contractility, decreased cell proliferation, and reduced cell migration, ultimately leading to endothelial cell dysfunction.^7,37–39

ROCK inhibitors are a class of drugs that target the ROCK pathway by inhibiting the enzymatic activity of both ROCK isoforms ROCK1 and ROCK2.⁴⁰ By doing so, these drugs can modulate various cellular processes, leading to beneficial effects in several disease models.³⁰ Preclinical and clinical studies have shown that ROCK inhibitors can improve endothelial function by improving cell adhesion, proliferation and inhibiting apoptosis.^6,37,41–43

In recent years, several studies have investigated the application of ROCK inhibitors in corneal diseases, including corneal endothelial dystrophies, corneal injury and post-operative regeneration. These studies have reported promising results, with ROCK inhibitors showing efficacy in improving corneal transparency.^{17,18,44–46}

ROCK Inhibitors as Potential Therapeutics for Corneal Diseases

Comparison of Different ROCK Inhibitors and Their Pharmacological Properties

Several ROCK inhibitors have been studied in preclinical and clinical settings. Here, we provide a comparative analysis of the pharmacological properties of four prominent ROCK inhibitors: Y-27632, fasudil, ripasudil, and netarsudil (for an overview see Table 1 and Figure 2).

Figure 2 Chemical structures for Y-27632,49 fasudil,50 ripasudil51 and netarsudil.52 Note the similarities between fasudil and ripasudil.

Table 1 Comparison of ROCK Inhibitors for Corneal Diseases

Y-27632 was one of the first ROCK inhibitors identified and has been extensively studied in vitro and in vivo. Y-27632 is a dual ROCK-1/2 inhibitor with a K_i (inhibition constant) value of 140–220 nM for ROCK-1 and 300 nM for ROCK-2.^31,53 However, its clinical use has been limited since in vitro studies have revealed that Y-27632 exhibits comparatively low potency when compared to other ROCK inhibitors.^29,54,55 Moreover, Li et al reported a serum half-life ranging from 1 to 1.5 hours,⁵⁶ while data on the ophthalmic application of this ROCK inhibitor is currently unavailable.

Fasudil, another dual ROCK-1/2 inhibitor and formerly known as HA-1077, is a clinically approved ROCK inhibitor for the treatment of cerebral vasospasm in Japan. It has also been studied for its potential use in diabetic macular edema⁵⁷ and glaucoma.⁵⁸ Fasudil has demonstrated promising pharmacological properties with a K_i value of 76 nM for ROCK-1 and 47 nM for ROCK-2.³¹ Fasudil has a plasma half-life of 0.3 h and its active metabolite hydroxyfasudil has a half-life of 2.9 h.^9,59 Pharmacokinetic studies regarding its application in ophthalmology are yet to be done. However, similar to Y-27632, fasudil exhibits relatively low in vitro potency³¹ and therefore its clinical use in ophthalmology has been limited so far.

Ripasudil, a derivative of fasudil (Figure 2), and netarsudil are two newer ROCK inhibitors that have been approved for the treatment of glaucoma. Ripasudil, also known as K-115 from various clinical trials, has since 2014 been available in Japan for glaucoma treatment as a 0.4% formulation twice daily under the brand name Glanatec.⁶⁰ It acts as a dual ROCK-1/2 inhibitor, with an IC50 (half maximal inhibitory concentration) of 19 nM for ROCK-1 and 51 nM for ROCK-2.^12,61–65 Comparative K_i values with the other ROCK inhibitors presented in this review are unfortunately not available. The plasma half-life of ripasudil is 0.6–0.7 h and rabbit studies showed good corneal penetration when applied as eye drops.^60,66

Netarsudil, in clinical studies known as AR-13324 and marketed as Rhopressa, is a dual ROCK-1/2 and norepinephrine transport inhibitor. It has a K_i value of 1 nM for both ROCK-1 and ROCK-2, indicating high affinity for ROCK inhibition.³¹ Its active metabolite netarsudil-M1 (AR-13503) has a five times higher potency against both ROCK-1 and ROCK-2. Furthermore, the inhibition of norepinephrine transport by netarsudil contributes to the reduction of aqueous humor production.⁶⁷ For netarsudil maximum concentration in the cornea after topical administration has been found to be after 0.25–0.5 h with a half-life of 13–14 h.³¹ Netarsudil is available as a 0.02% formulation and administered once daily for the treatment of glaucoma. It has been approved in the United States since 2017, with subsequent authorization in the European Union in 2021.^7,39

In conclusion, the pharmacological properties of Y-27632, fasudil, ripasudil, and netarsudil provide valuable insights into their potential for treating corneal endothelial disease. While Y-27632 and fasudil exhibit lower in vitro potency, both netarsudil and ripasudil’s clinical availability as an ophthalmic solution offers promising prospects for research on its application in corneal disease. Further research and clinical investigations are warranted to fully explore the therapeutic potential of these ROCK inhibitors in the management of corneal endothelial disease.

Evidence Supporting the Use of ROCK Inhibitors in Corneal Diseases

In this section, we present a comprehensive summary of relevant studies that demonstrate the efficacy of ROCK inhibitors in treating corneal diseases, with a specific focus on four main conditions: FECD, pseudophakic bullous keratopathy (PBK), corneal neovascularization (CNV), and iridocorneal endothelial (ICE) syndrome. Table 2 provides a condensed overview of the key findings from these studies, shedding light on the therapeutic potential of ROCK inhibitors in addressing corneal disease.

Table 2 Evidence Supporting the Use of ROCK Inhibitors in Corneal Diseases

FECD is a progressive and bilateral corneal disease characterized by the loss of endothelial cells and the presence of guttae, which are excrescences of Descemet’s membrane.⁷⁴ In Western countries, FECD is the predominant cause of corneal endothelial dysfunction and represents the most prevalent indication for corneal transplantation.⁷⁵ The pathophysiological mechanisms of FECD involve channelopathies, oxidative stress, apoptosis, and abnormal cellular-matrix interactions.^74,76 In less severe cases of Fuchs’ corneal edema, conservative treatment involves the use of hyperosmotic saline drops.^6,77 Surgical management options vary depending on the stage of the disease and may include penetrating keratoplasty (PK), Descemet membrane endothelial keratoplasty (DMEK), Descemet stripping automated endothelial keratoplasty (DSAEK), or Descemet stripping only (DSO).⁷⁸

Numerous preclinical and clinical studies have employed ROCK inhibitors in the context of corneal endothelial diseases. Okumura et al conducted a study using the ROCK inhibitor Y-27632 in a rabbit corneal endothelial dysfunction model. The injection of cultivated corneal endothelial cells (CECs) supplemented with Y-27632 resulted in enhanced adhesion properties of the CECs, as evidenced by the expression of ZO-1 and Na/K-ATPase.⁶⁸

The first clinical study involving ROCK inhibitors for endothelial dysfunction was conducted by Okumura et al in 2013 and included a mixed sample of seven cynomolgus monkeys and eight patients with corneal endothelial dysfunction, specifically with diffuse or central corneal edema as in advanced stages of FECD.⁴⁷ The study revealed the promotion of corneal endothelial wound healing with the use of 10 mM Y-27632 eye drops 6 times daily for 7 days. Notably, the treated group exhibited significantly higher corneal endothelial cell density, along with improved corneal clarity and reduced corneal thickness. Morphologically, the regenerated corneal endothelium displayed a restoration of normal hexagonal cell morphology and increased cell density.⁴⁷

In a trial involving 18 patients diagnosed with FECD, the utilization of the ROCK inhibitor ripasudil in combination with DSO resulted in enhanced recovery of endothelial cells.⁷⁰ Specifically, the administration of ripasudil 0.4% eye drops four times daily for two months following DSO resulted in superior outcomes. These included enhanced visual recovery, and elevated corneal endothelial cell density after one year.⁷⁰ Another clinical study by Moloney et al reported corneal clearance and improvement in best corrected visual acuity in 22 out of 23 eyes with FECD following DSO with the use of ripasudil 0.4% eye drops 6 times daily after 4 weeks.⁶⁹

Netarsudil’s effectiveness was assessed by Price and Price in a double-masked, randomized study involving 29 eyes with symptomatic FECD. The study involved randomized administration of placebo or netarsudil 0.02% eye drops once daily for three months.⁷¹ Subsequent examinations demonstrated a significant decrease in central corneal thickness at both 1 and 3 months, along with significant improvement in scotopic corrected distance visual acuity (CDVA) after 3 months.⁷¹

PBK, a complication after cataract surgery, is the second most prevalent cause of corneal endothelial dysfunction and stands out as the most frequent secondary cause of corneal edema.⁷⁹ In the course of cataract surgery, the rise in local temperature generated by the phacoemulsification probe can result in thermal damage to the adjacent corneal tissue⁸⁰ and it is estimated that PBK may occur in approximately 1–2% of cataract surgeries.⁸¹ Furthermore, damage to endothelial tissue during cataract surgery can be inflicted by a combination of factors including elevated irrigation or aspiration rates, and the presence of air bubbles or lens particles making contact with the endothelium.⁸⁰

In the context of PBK, Kinoshita et al conducted a single-group study involving 11 eyes treated with cultured human corneal endothelial cells supplemented with the ROCK inhibitor Y-27632, which were injected into the anterior chamber. The study reported improvements in important parameters such as corneal transparency, reduction of corneal thickness to normal values, and increased visual acuity in all eyes of the sample.¹⁶ Follow-up controls conducted two years after the injection revealed maintenance of corneal transparency in all eyes and corneal thickness values below 630 μm in 10 out of 11 eyes.¹⁶

Ripasudil has been tested as additional medical therapy in challenging cataract surgeries for patients with FECD in a clinical series involving four patients.⁸² The use of ripasudil prophylactically or postoperatively resulted in improvements in corneal edema and symptoms, demonstrating its possible role for the reduction of PBK after cataract surgery.⁸²

CNV is a serious threat to vision, often associated with inflammatory, infectious, and traumatic ocular conditions. This condition occurs when pro-angiogenic factors, such as inflammatory cytokines and reactive oxygen species, outweigh anti-angiogenic factors, particularly in corneal burns. The imbalance leads to the formation of new blood vessels in the cornea, posing a significant risk to eyesight.⁸³

The application of ROCK inhibitors for CNV has been explored through several studies, providing valuable insights into the therapeutic potential.^72,84 In the study conducted by Zeng et al, the ROCK inhibitor fasudil exhibited significant efficacy in inhibiting CNV in mice following alkali burns.⁷² Administration of 100 μM fasudil eye drops four times daily led to a marked reduction in CNV incidence, correlating with decreased inflammatory cell infiltration, lowered production of reactive oxygen species, and an upregulation of heme oxygenase-1 protein. Moreover, the study highlighted the healing promotion of corneal epithelial defects. These findings collectively underscore the promise of ROCK inhibitors in mitigating pathological wound healing and neovascularization after corneal trauma.

The ICE syndrome encompasses a distinctive cluster of ocular pathologies, including Chandler syndrome, progressive iris atrophy, and Cogan-Reese syndrome. Characterized by the abnormal proliferation of corneal endothelial cells migrating towards the iridocorneal angle and iris surface, it leads to varying degrees of corneal edema, decompensation, and secondary glaucoma. Primarily affecting young women unilaterally, diagnosis relies on ocular findings, aided by in vivo confocal microscopy. Management focuses on addressing corneal edema and decompensation through endothelial keratoplasty and tackling secondary glaucoma with surgical interventions.⁸⁵

Rho-kinase inhibitors have shown positive effects on patients with ICE syndrome. Davies reported a case of an 80-year-old woman suffering from corneal edema due to ICE syndrome, in which a 4-weeks therapy with netarsudil eye drops led to significant regression of the corneal edema and improved visual acuity.⁷³

Safety Considerations and Potential Side Effects of ROCK Inhibitors in the Cornea

The safety of ROCK inhibitors in the treatment of corneal diseases is of paramount importance. Investigating the safety profiles of ophthalmic ROCK inhibitors, both netarsudil and ripasudil have been approved for clinical use, supported by numerous studies. However, limited data of small case studies exists for the safety profile of fasudil,⁴⁸ which has gained approval solely for cerebral vasospasm treatment. Similarly, due to its lack of approval, Y-27632 has only garnered limited attention in terms of clinical safety studies. Therefore, we will here focus on the two drugs netarsudil and ripasudil, where data from larger clinical trials is available.

A 2016 Phase III trial conducted by Tanihara et al with 388 patients examined the safety of ripasudil 0.4% when applied twice daily over a 52-week period.¹² Overall, the study found that 94.1% of patients experienced adverse events undergoing treatment with ripasudil. The most frequent adverse events included conjunctival hyperemia (74.6%), blepharitis (20.6%), allergic conjunctivitis (17.2%), eye irritation (10.2%), and conjunctivitis (7.3%). A total of 14.4% of patients withdrew from the study as a result of adverse events and long-term studies showed that blepharitis and conjunctival hyperemia were the most common reasons for discontinuation.^86,87

The safety profile of netarsudil has been comprehensively investigated in the ROCKET^88,89 and MERCURY¹¹ trials, providing consistent findings regarding the incidence of adverse events associated with netarsudil. In a pooled analysis of the ROCKET studies the safety profile of once-daily netarsudil was assessed in comparison to twice-daily timolol involving 839 patients in each group.¹⁰ The study revealed that 83.3% of patients treated with once-daily netarsudil experienced adverse events with the incidence of ocular adverse events being 79.3%. Overall systemic adverse events occurred in 26.3% of netarsudil-treated patients and no single systemic adverse event occurred in more than 2% of patients. Serious adverse events were reported in 3.3% of twice-daily netarsudil-treated patients and no serious ocular adverse event was reported among patients administered netarsudil once daily. The most frequent ocular adverse event in the netarsudil group was conjunctival hyperemia, occurring in 54.4% of patients. The severity of conjunctival hyperemia was mostly graded as mild (77.6%) and led to treatment discontinuation in 6% of netarsudil patients. Other ocular adverse events associated with netarsudil included cornea verticillata, reported in 20.9% of patients, and conjunctival hemorrhage, reported in 17.2% of patients. Cornea verticillata did not significantly affect visual acuity and resulted in treatment discontinuation in 3.7% of patients. Conjunctival hemorrhage was typically mild and self-limiting. So far only in case series reticular bullous epithelial edema has been observed with netarsudil use, which resolved after discontinuation.^90,91

All in all, adverse events associated with ROCK inhibitors include conjunctival hyperemia, conjunctival hemorrhage, cornea verticillata, conjunctivitis, and blepharitis. Comparing the two inhibitors, ripasudil was found to have a higher prevalence of adverse events (94.1%) compared to netarsudil (83.3%) with conjunctival hyperemia being the most frequent in both drugs. Notably, cornea verticillata is specifically observed with netarsudil, while blepharitis is more commonly associated with ripasudil.

Further investigation is needed to comprehensively evaluate the safety profiles of ROCK inhibitors in the treatment of corneal diseases. Although both ripasudil and netarsudil have shown promising therapeutic effects, their associated adverse events should be carefully considered in clinical practice. It is crucial for clinicians to be aware of these potential risks and monitor patients closely during treatment with ROCK inhibitors to ensure optimal therapeutic outcomes and patient safety.

Future Directions and Challenges

Limitations and Challenges in the Clinical Application of ROCK Inhibitors for Corneal Diseases

While the positive outcomes of ROCK inhibitors in addressing corneal diseases are well-documented, it is essential to acknowledge the existing limitations constraining their applications.

There are concerns regarding the use of ROCK inhibitors for patients with FECD associated corneal guttae-induced reduction in CEC density. It has not yet been shown whether ROCK inhibitors are effective in cases where the CEC layer is intact and not disrupted.¹⁷

DSO with the use of ROCK inhibitors is a surgical option that yields comparable visual outcomes to DMEK.⁴⁵ While patients undergoing DSO may experience a longer recovery period than those undergoing DMEK, they benefit from reduced adverse effects and the elimination of long-term immunosuppression or the need for donor corneal tissue.⁴⁵ Nevertheless, in cases of advanced stromal edema or significant peripheral loss of endothelial cells, endothelial keratoplasty remains the established surgical approach. Furthermore, there are reports indicating that the proliferation and migration of remaining corneal endothelial cells occurs at a faster rate over areas with intact Descemet’s membrane compared to areas where the Descemet’s membrane was removed, as in DSO. This suggests that the presence of Descemet’s membrane aids in the proliferation of CECs in cases of corneal endothelial disease.⁹²

In the context of ICE syndrome, only a single case has demonstrated successful corneal edema treatment using a ROCK inhibitor.⁷³ As a result, the effectiveness of this intervention awaits confirmation through larger-scale clinical trials.

There is also an ongoing discussion regarding the optimal frequency of the administration of ROCK inhibitor eye drop therapy. Published data indicate that the application of ripasudil 0.4% ophthalmic solution on the cornea exhibits an action lasting approximately 6 hours,¹⁷ while netarsudil 0.02% has a half-life of 13–14 hours in the cornea.³¹ Yet, further studies are needed to investigate the most effective frequency of administration of ROCK inhibitors eye drops for corneal diseases.

There are several general limitations in the reviewed studies that warrant attention. Our bibliographic research did not identify any published studies with long-term outcomes or studies with large sample sizes regarding the application of ROCK inhibitors in corneal diseases. Moreover, there is a need for comparative studies that include subjects receiving conservative therapy with ROCK inhibitors and those undergoing surgical therapy, particularly in patients with FECD. Such studies would provide valuable insights into the effectiveness of ROCK inhibitors. Therefore, we strongly advocate for further investigation of these preliminary results, as they raise important questions about the potential impact of ROCK inhibitors on current standards in the treatment of corneal disease.

Other Potential Therapeutics for Corneal Diseases

Recent advancements in the treatment of corneal diseases have shown promising results with the use of ROCK inhibitors. However, ongoing research is also exploring other potential therapeutics. In particular, gene therapy, bioengineered corneal grafts, and cell therapies have emerged as areas of interest. Numerous studies have focused on therapies for various corneal diseases, including corneal scarring, corneal epithelial wound healing, corneal neovascularization, corneal (endothelial) dystrophies, herpetic keratitis, and dry eye disease.⁹³ Here, we will focus on alternative therapeutic approaches for corneal endothelial diseases.

Gene therapy involves the delivery of genetic material to modify gene expression or correct genetic abnormalities. FECD is a condition commonly characterized by the expansion of CTG trinucleotide repeats within the TCF4 gene, resulting in the formation of toxic RNA foci.⁹⁴ Preclinical studies utilizing antisense oligonucleotide therapy have shown promise in reducing the formation of RNA foci and toxicity markers associated with repeat-expansion-mediated diseases.⁹⁵ A proof-of-concept study demonstrated that antisense oligonucleotides effectively reduced RNA foci formation in patient-derived cells affected by FECD, suggesting their potential for treating corneal tissue in these diseases.⁹⁶ Additionally, targeting the expansion of CTG repeats in FECD using CRISPR/Cas9 has also demonstrated promising results in reducing RNA foci associated with FECD pathophysiology.^97,98

Furthermore, tissue engineering approaches are being explored to develop artificial corneal implants or cell-based therapies that can replace damaged or dysfunctional corneal tissue.^99,100

While the clinical application of ROCK inhibitors for corneal diseases holds promise, these other potential therapeutics also warrant further investigation and development. Future studies will need to compare the efficacy and safety of these different approaches to identify the most effective treatment strategies for corneal diseases.

Conclusion

The role of ROCK inhibitors in corneal diseases, particularly those affecting the corneal endothelium, shows promising potential for therapeutic intervention. The ROCK signaling cascade is involved in various pathological conditions, and recent research has demonstrated that ROCK inhibitors can modulate cellular processes in corneal endothelial cells, leading to improved cell adhesion, proliferation, and reduced apoptosis. These effects make ROCK inhibitors attractive candidates for the management of corneal diseases, such as FECD and PBK.

The comparative analysis of four prominent ROCK inhibitors, namely Y-27632, fasudil, ripasudil, and netarsudil, revealed variations in their pharmacological properties. While Y-27632 and fasudil have exhibited lower potency, netarsudil and ripasudil have shown promising clinical availability as ophthalmic solutions. Netarsudil, in particular, has demonstrated high affinity for ROCK inhibition and long-lasting activity, making it a promising candidate for further investigation in corneal diseases.

Preclinical and clinical studies have provided evidence supporting the use of ROCK inhibitors in corneal diseases. These studies have shown that ROCK inhibitors can improve corneal transparency and enhance endothelial function. However, further research and clinical investigations are needed to evaluate the therapeutic potential and ideal application of ROCK inhibitors in the management of corneal diseases. Additional studies investigating the pharmacokinetics, optimal dosing, and long-term safety of ROCK inhibitors in corneal disease treatment are necessary. Moreover, exploring the combination of ROCK inhibitors with other potential therapeutics, such as bioengineered tissues and gene therapy, may provide synergistic effects and enhance their therapeutic potential.

In conclusion, the application of ROCK inhibitors in corneal diseases represents a promising avenue for future research and clinical translation. By targeting the ROCK pathway, these inhibitors have the potential to address the limitations of current therapies, and offer new strategies for restoring corneal endothelial function. Further studies and clinical trials will provide valuable insights into the optimal use of ROCK inhibitors and their long-term effects in corneal diseases, ultimately leading to improved outcomes for patients with corneal pathologies.