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Rho-Associated Protein Kinase Inhibitors and Primary Open-Angle Glaucoma

Pratap Challa, MD, MS and John J. Arnold, PhD

Rho-associated protein kinase (ROCK) inhibitors have been broadly investigated over the past 15 years as potential agents for the treatment of elevated intraocular pressure (IOP) secondary to primary open-angle glaucoma (POAG).^1,2 Preclinical and clinical evidence has suggested that ROCK inhibitors can lower IOP through a unique mechanism of action heretofore untargeted by current ocular hypotensive drugs.^3–9 Mechanistically, ROCK inhibitors can lower IOP through their ability to induce changes in the tissue of the conventional outflow pathway of the anterior chamber, which includes: (1) disassembly of cell-cell junctions and subsequent loosening of the paracellular spaces between cells and (2) general relaxation of contractile tone. Both physiologic actions can improve outflow facility of aqueous humor from the anterior chamber.^3,4,10 Furthermore, ROCK inhibitors have been postulated to enhance blood flow to the optic nerve, which serves to ameliorate nerve destruction and potentially preserve sight.¹¹ The purpose of this chapter is to analyze the evolving role of ROCK inhibitors in the treatment of POAG. While the authors recognize that ROCK inhibitors have been studied for the treatment of other forms of glaucoma, the primary focus of this chapter will be their use in POAG.

When considering the utility of ROCK inhibitors in the treatment of POAG, one must recall the physiology of aqueous humor outflow from the anterior chamber. Aqueous humor primarily exits the anterior chamber through the conventional outflow pathway, which is composed of the trabecular meshwork (TM) and Schlemm’s canal.^12,13 Anatomically, the TM is composed of the uveal meshwork, the corneoscleral meshwork, and the juxtacanalicular connective tissue (JCT), with the JCT being immediately adjacent to Schlemm’s canal.^14,15 Morphologically, the regions of the conventional outflow pathway are distinct. Generally, the TM is composed of collagen beams arranged in a regular, lattice-like structure with extracellular matrix located in paracellular spaces between cells of the tissue. The JCT is less regular in structure with cells loosely embedded with extracellular matrix and a ground substance consisting of proteoglycans and hyaluron filling in the spaces of the tissue. For aqueous humor to exit the anterior chamber, it must traverse the TM and JCT and enter Schlemm’s canal through either endothelial cells or vacuoles present in the inner wall of Schlemm’s canal. From there, aqueous humor is collected and funneled through the tube-like structure of Schlemm’s canal into the episcleral vein and back into general blood circulation.^12–16

Studies have demonstrated that the tissue of the conventional outflow pathway is extremely dynamic with an architecture that functionally can adapt and conform to maintain homeostatic conditions in aqueous humor outflow.^17–20 Additionally, the functional properties of the outflow pathway can be affected more temporally by factors such as age and in patients with ocular diseases such as POAG.²¹ Changes in cellular morphology of the TM can occur transiently in response to physiological conditions or pharmacological intervention, which can enhance or diminish outflow facility. For instance, muscarinic activation induced by pilocarpine can result in a contraction of the ciliary muscle, which causes a posterior pull on the scleral spur and a widening of the paracellular spaces in the TM.¹⁹ Consequently, outflow resistance is reduced in the TM and aqueous humor flow is increased, resulting in reduced IOP. The intrinsic contractility of the outflow pathway can also influence outflow facility. Cells of contractile tissue such as muscle have highly evolved actomyosin systems composed of actin and myosin, which are instrumental in contraction and relaxation. In nonmuscle cells, the cellular actomyosin system is responsible for altering cellular shape and morphology. In the conventional outflow pathway, the cellular actomyosin system is highly structured and possesses a profound influence on contractile tone of the tissue, which would also affect aqueous humor flow. Nonetheless, the region of the conventional outflow pathway, which provides the most resistance to aqueous humor outflow, has not been completely illuminated. Studies in various ex vivo models have suggested that resistance to aqueous humor flow is greatest in the inner wall of Schlemm and JCT.12–15,22

Whatever the source of resistance to aqueous humor flow in nondiseased or diseased conventional outflow pathway tissue, pharmacologic disruption of actin microfilaments resulting in actomyosin system down regulation should result in cellular relaxation and shape alterations. Such changes could result in widening of paracellular spaces and an overall decrease in cellular contractile tone, which could increase overall outflow facility of aqueous humor. It is possible that a lack of appropriate contractile tone in the outflow pathway could play a major role in the etiology of POAG. Indeed, studies have demonstrated fewer functioning TM cells, fibrosis, and increased extracellular matrix deposition in the JCT region of glaucomatous cells. Patients with glaucoma have also been shown to possess increased levels of endogenous contractile mediators such as endothelin-1 and transforming growth factor β2.^23–26

Currently, commercially available ocular hypotensive drugs target IOP reductions by reducing aqueous humor production by the ciliary process (ie, beta-adrenergic antagonists) or increasing aqueous humor outflow through the unconventional (or uveoscleral) pathway (ie, prostaglandin analogs).^27–29 As mentioned, pilocarpine can increase aqueous humor drainage through the conventional outflow pathway by contracting the ciliary muscle and pulling on the scleral spur to increase the amount of paracellular space in the tissues of the conventional outflow pathway. However, the pharmacological effects of pilocarpine are transient and require frequent dosing to maintain reductions in IOP.³⁰ Additionally, epinephrine analogs target the trabecular (conventional) outflow, but they are used infrequently in clinical practice due to an undesirable side effect profile and relatively weaker potency for IOP lowering compared to other ocular hypotensive agents. Currently, ROCK inhibitors represent the only available ocular hypotensive agents that specifically target the conventional outflow pathway with sufficient potency, duration of action, and minimal side effects. To appreciate how ROCK inhibition results in clinically relevant effects in the eye, a review of the molecular actions of ROCK activation and the potential role of antagonism in a broad array of diseases is warranted.

PHYSIOLOGICAL EFFECTS OF THE ROCK PATHWAY

The Rho signaling pathway plays an important role in the contraction of smooth muscle.^31–33 The contractility of smooth muscle is generated by the cellular influx of extracellular calcium (Ca++) into receptor-gated and voltage-gated Ca++ channels. Concurrent G-coupled protein receptor activation of protein kinase C and the phosphatidylinositol cascade promotes release of intracellular Ca++ from the sarcoplasmic reticulum (Figure 17-1A). Rising intracellular concentrations of Ca++ activate myosin light chain kinase (MLCK). The myosin light chain (MLC) of myosin II is phosphorylated by MLCK with results in muscle contraction. Later, as intracellular concentrations of Ca++ decrease, MLC is dephosphorylated by myosin phosphatase to its inactive form.^34–37

Historically, evidence has suggested that increasing intracellular Ca++ is not the only physiological mechanism responsible for eliciting smooth muscle contraction. Indeed, evidence suggested that force of smooth muscle contraction induced by G-protein agonist activation is greater than would be predicted from increasing intracellular Ca++ concentrations alone.^32,33 Consequently, the search for a Ca++-independent pathway for smooth muscle contractility led to the discovery of the G protein Rho, which is a member of the Ras family of G proteins (Figure 17-1B). After activation, Rho guanine nucleotide exchange factor (RhoGEF) mediates the cycling of Rho from its inactive state (Rho-GDP) to its active state (Rho-GTP). Rho-GTP activates serine-threonine kinases known as Rho-associated kinases (ROCKs). ROCK isoforms found in mammalian tissues include ROCK1 (1354 amino acids) and ROCK2 (1388 amino acids). ROCK1 and ROCK2 share an overall homology in amino sequence of 65% and within their kinase domain a homology in amino acid sequence of 92%.³⁸ Although the differential downstream targets and functions of ROCK1/ROCK2 is an area of continued research, for the purposes of the chapter they will primarily be referred to collectively as ROCK. Thus, MLC can be activated independently of increasing intracellular Ca++ concentrations as ROCK can phosphorylate the same serine residue phosphorylated by MLCK, which leads to smooth muscle contraction. In addition, to induce smooth muscle contraction, ROCK can promote other cellular activities that increase cytoskeletal stability. ROCK has been shown to activate LIM kinase-1 and LIM kinase-2, which function to phosphorylate and inactivate cofilin, which functions intracellularly by binding to actin and stimulating its turnover.^39,40 As a result, ROCK activation can indirectly decrease actin breakdown and promote a more stable cytoskeleton. Accordingly, ROCK, through control of cellular contractility, can regulate many cellular processes including growth, migration, and life cycle.

Genetic experimentation has been conducted to determine the physiological implications of ROCK inhibition. Genetic deletion (ie, knock-out experiments of or either ROCK1 or ROCK2) in mice has provided evidence of phenotypic changes consistent with defects in epithelial cell motility.41–46 Mice deficient in ROCK have demonstrated developmental defects, including lack of eyelid development and protrusion of internal organs due to lack of ventral body wall closure.⁴¹ An absence of epithelial cell migration has been proposed as a possible explanation for these findings. ROCK inhibition presents a tantalizing potential treatment modality for several diseases. It has been proposed that ROCK inhibition could be clinically useful in the treatment of diseases as varied as asthma, cancer, cardiovascular hypertrophy, diabetes mellitus, erectile dysfunction, hyperproliferative diseases, hypertension, inflammatory diseases, pulmonary hypertension, renal disease, and vasospasm.^47–76 ROCK-deficient mice have been shown to have reduced cardiac fibrosis and reductions of cadiomyocyte apoptosis secondary to cardiac pressure overload.^44,45 These findings coupled with the vasodilatory actions of ROCK inhibition suggest a potentially substantial role in the treatment of cardiovascular diseases. Indeed, the ROCK inhibitor, fasudil (originally known as HA-1077), has been used clinically in Japan in the prevention of cerebral vasospasms.⁷⁷

The physiological implications of ROCK inhibition on contractile tone are intriguing for a disease state such as POAG. Research has demonstrated the pivotal role of the cellular actomyosin system in altering TM morphology and influencing aqueous humor facility in response to pressure changes.^3–6 In vitro and ex vivo evidence has suggested that the Rho signaling pathway plays an important role in controlling the cellular morphology and the contractility of the conventional outflow pathway. Elements of the Rho signaling pathway (RhoA, ROCK1, ROCK2, MLC, MLCK, and MLCP) have been shown to be present in the TM and ciliary muscle tissue of animals and humans. Indeed, experimental evidence suggests that the contractile tone of the conventional outflow can be diminished through ROCK inhibition.^78–80 The ROCK inhibitor, Y-27632, has been shown to prevent carbachol and endothelin-1–mediated calcium-independent contraction in excised bovine TM tissue.⁸¹ Further, the ROCK inhibitor H-1152 has been shown to reduce the phosphorylation MLC in TM tissue.⁶ These experimental findings highlight the potential importance that the Rho signaling system plays in reducing the contractile tone of the conventional outflow pathway, thus increasing outflow facility.

Molecularly, the effects of ROCK inhibition on the cellular cytoskeleton have been well characterized (Table 17-1). In vitro studies in TM and Schlemm’s canal cells of animals and humans have demonstrated the ability of Y-27632 to affect cytoskeletal integrity through various mechanisms. In cultured monkey Schlemm’s canal cells, Y-27632 treatment decreased formation of giant vacuoles intended for aqueous humor transport and expression of the cytoskeletal proteins including ZO-1 and claudin-5.84 Morphologically, these ROCK-treated Schlemm’s canal cells displayed rounding and detachment resulting in paracellular widening and increased permeation of fluorescein and horseradish peroxidase.

To correlate these cellular effects to changes in the outflow pathway, ex vivo perfusion experiments have been conducted in excised anterior segments or enucleated eyes.3,7,82–87 These experiments have demonstrated a significant increase in outflow facility in perfused eyes of various species (porcine, bovine, monkey), which can be demonstrably tied to changes in the morphology of the outflow pathway. Microscopic analysis in perfused bovine eyes have shown the space between the JCT and inner wall of Schlemm to be distended in the presence of ROCK inhibition, which could contribute to increases in outflow facility. One particularly novel perfusion experiment in human eyes was conducted to ascertain the effect of genetically disrupting the Rho signaling pathway of outflow facility. In cadaver eyes, an adenovirus vector equipped with a gene expressing a dominant negative Rho binding domain was transfected into TM cells.⁸³ Perfusion studies in these genetically modified eyes demonstrated a significant increase in outflow facility vs those eyes not modified. These results unequivocally demonstrate the importance of the ROCK pathway in modulating conventional outflow facility.

PRECLINICAL EVIDENCE OF ROCK INHIBITION’S ROLE IN TREATMENT

It is clear that in vitro and ex vivo findings have explicitly demonstrated the importance of the ROCK pathway in modulating the physiology of the conventional outflow facility at the molecular level (see Table 17-1). Whether sufficient concentrations of Rho kinase inhibitors can be achieved at the site of action to elicit changes in aqueous humor outflow has been tested in many animal models (Table 17-2). Obviously, achieving therapeutic concentrations in the eye is not guaranteed. Administered drug has a tortuous pathway to navigate to achieve clinically relevant concentrations in the eye. Nevertheless, many animal studies in various models have demonstrated that the principle of ROCK inhibition leading to increased aqueous humor outflow is sound. Data have indicated that various ROCK inhibitors can induce rapid (within 30 to 60 minutes) and relatively sustained reductions in IOP following administration in different animal models by different means of administration.^{4,6,85,88–91} Previous studies with the ROCK inhibitor Y-39983 demonstrated a dramatic reduction in IOP (maximal reduction = -13.2 ± 0.6 mm Hg) after administration to normotensive rabbits.⁸⁸ However, when Y-39983 was administered to normotensive monkeys, the effect on IOP reduction was not as dramatic (maximal reduction = -2.5 ± 0.8 mm Hg).

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