Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

I. GLAUCOMA: IOP AS

A RISK FACTOR

Glaucoma is a heterogeneous group of optic neuropathies that share characteristic pathognomonic changes to the optic disc and visual field. Glaucoma is a leading cause of irreversible visual impairment and blindness, affecting approximately 70 million individuals worldwide (Quigley, 1996; Weinreb and Khaw, 2004). There are a number of risk factors associated with glaucoma, including age, ethnicity, and family history. However, the most important causative risk factor for the development and progression of glaucoma is elevated intraocular pressure (IOP). Although not all patients with elevated IOP ( 21 mmHg) develop glaucoma, the prevalence of glaucoma increases significantly with increased IOP. The elevated IOP associated with glaucoma is due to increased aqueous humor outflow resistance and is associated with biochemical (Babizhayev and Brodskaya, 1989; Knepper et al., 1996) and morphological (Lütjen-Drecoll et al., 1986; Rohen et al., 1993) changes in the trabecular meshwork (TM).

The current standard of care for treating glaucoma patients is therapeutic IOP lowering by topical ocular medicines, laser trabeculoplasty, and glaucoma filtration surgery. Several well-controlled clinical trials have clearly demonstrated the importance of IOP-lowering therapy in all phases of the disease. (1) In the Ocular Hypertension Treatment Study (OHTS) (Kass et al., 2002), approximately one half of the enrolled ocular hypertensive patients received IOP lowering therapy (with very modest goals of a 20% IOP decrease or IOP 24mmHg), while the other half were untreated. The patients were followed for 5 years and examined for the development of glaucoma. Those individuals receiving IOP lowering therapy were two-fold less likely to develop glaucoma, therefore, IOP lowering prevented or delayed the onset of glaucoma. (2) The Early Manifest Glaucoma Trial (EMGT) evaluated the effect

of IOP lowering in patients with early disease (Leske et al., 2003). The treated patients had half the risk for glaucoma progression compared to the untreated group. (3) The Collaborative Initial Glaucoma Treatment Study (CIGTS) randomized newly diagnosed glaucoma patients to initial treatment with topical ocular medicines or to glaucoma filtration surgery (Lichter et al., 2001). There was little disease progression over the course of 5 years in those patients with the greatest degree of IOP lowering. (4) The Advanced Glaucoma Intervention Study (AGIS) showed that patients with higher average IOPs progressed more than patients with lower IOPs. In fact, the subgroup of patients with IOPs below 18 at all study visits did not progress over the course of 6 year follow-ups (AGIS-Investigators, 2000). (5) A significant fraction of glaucoma patients have IOPs in what is considered the normal range (IOP 21 mmHg), and therefore are classified as having normal tension glaucoma (NTG). The Collaborative Normal Tension Glaucoma Study (CNTGS) determined that IOP lowering was also beneficial in this patient population (CNTGS-Group, 1998). The conclusion from all of these studies is that lowering IOP is associated with reduced risk of glaucomatous damage.

II. BASIC MECHANISMS OF AQUEOUS HYDRODYNAMICS

Intraocular pressure is delicately maintained by the production and outflow rates of aqueous humor. Under normal conditions, they are at equilibrium. Departure from this equilibrium changes IOP.

A. Aqueous Production

Aqueous humor is generated in the posterior chamber of the eye by the ciliary processes. The capillaries in the stroma of the ciliary processes are highly permeable. Hence, the stromal fluid is chemically

Schlemm’s canal

Cornea

Trabecular Meshwork

Trabecular Outflow

FIGURE 3.1 Aqueous humor outflow pathways

very similar to the plasma of the blood. The stroma and the aqueous humor are separated by a tight boundary consisting of a bilayer of two types of ciliary epithelial cells: the pigmented epithelial cells, adjacent to the stroma, and the non-pigmented epithelial cells, adjacent to the aqueous humor. These cells are functionally coupled through intercellular gap junctions and effectively form the blood–aqueous barrier. Aqueous humor is produced from the stromal fluid, mainly by energy-dependent active ion transport across the ciliary epithelium, followed by osmotic water movement, ultrafiltration, and diffusion. Aqueous production rate varies significantly between the waking and sleeping hours. In a healthy awake person, the production rate is approximately 3 μL/min, which is twice that of a sleeping subject (1.5 μL/min).

B. Aqueous Outflow

Aqueous humor journeys from the posterior chamber between the iris and lens, through the pupil, into the anterior chamber. It exits the anterior chamber via two main outflow pathways (Figure 3.1). A fraction of aqueous humor flows through the

trabecular meshwork (TM) located at the anterior chamber angle. The TM is a mesh formed by strands of collagenous beams and sheets populated with specialized TM cells, with open spaces between the beams. The aqueous humor then enters the juxtacanalicular tissue (JCT) and inner wall endothelium of Schlemm’s canal, and subsequently drains into the episcleral veins. This outflow pathway is called the trabecular or conventional outflow. The principal source of resistance in this pathway is at the JCT and inner wall of Schlemm’s canal.

In addition to the trabecular pathway, the remaining aqueous humor leaves the anterior chamber through the intercellular spaces of the iris root, ciliary muscle, sclera and eventually empties into the episcleral tissues. This outflow pathway is generally known as the uveoscleral or unconventional outflow.

C. Pathological Changes of Aqueous

Hydrodynamics in Glaucoma

In theory, ocular hypertension can be a result of an excessive production of aqueous humor and/or a reduction of its outflow.

However, there are no substantial differences in the rates of aqueous humor production between glaucomatous and nonglaucomatous individuals that would have a clinically meaningful effect on IOP. In glaucoma and ocular hypertensive patients, decreased aqueous outflow is responsible for the elevation in IOP. Quantitative morphological studies showed a significant increase in extracellular matrix (ECM) material in the TM of primary open angle glaucoma eyes. The excessive accumulation of ECM in the TM may be a result of a reduction in the phagocytic function of the TM cells, a decrease in other TM cell functions, and/or a decrease in the number of TM cells in the outflow pathway. In addition to the TM, the ciliary muscle, especially the anterior tip and the surrounding elastic fibers, also appears to have a higher amount of ECM. Furthermore, the Schlemm’s canal of glaucomatous human eye was reported to

have a smaller cross-sectional area, smaller perimeter, and shorter inner wall length compared to normal eyes, which may account for the diminished outflow (Gabelt and Kaufman, 2005). All of these changes can contribute to the reduction in aqueous outflow facility.

III. OVERVIEW OF CURRENTLY AVAILABLE GLAUCOMA MEDICATIONS

For the treatment of glaucoma, IOP can be lowered by three basic mechanisms: suppression of aqueous humor formation, increase of trabecular outflow, and increase of uveoscleral outflow. Currently, five classes of pharmacological compounds employing these mechanisms are being used clinically (Table 3.1) (Clark and Pang, 2002).

IV. PROSTAGLANDIN ANALOGS

(PGAS)

Prostaglandin analogs, such as latanoprost, travoprost, and bimatoprost, are very effective ocular hypotensives, and thus popular compounds useful for the treatment of glaucoma. Topical administration of latanoprost once daily is effective in decreasing IOP in open angle glaucoma, chronic angle closure glaucoma, normal tension glaucoma, and ocular hypertensive patients. It is also useful as post-iridectomy IOP control in acute angle closure glaucoma. Its therapeutic efficacy is independent of the awake– sleep stages of the patient. When used in conjunction with other topical glaucoma medications, such as timolol, dipivefrin, dorzolamide, or pilocarpine, prostanoids produce a significant additional reduction in pressure.

Latanoprost, travoprost, and bimatoprost are prodrugs of potent agonists of the FP prostaglandin receptor, whereas it is controversial whether unoprostone activates the FP receptor (Bhattacherjee et al., 2001; Griffin et al., 1997). Unoprostone is less efficacious than the other prostanoids; its mean IOP reduction was consistently less than that of latanoprost. Latanoprost, travoprost, and unoprostone lower IOP mainly by enhancing the uveoscleral outflow without significantly affecting trabecular outflow or aqueous production. In contrast, bimatoprost was reported to mildly accelerate both the trabecular outflow and aqueous production in addition to enhancing uveoscleral outflow.

FP receptor agonists stimulate the expression of matrix metalloproteinases (MMPs) in human and monkey ciliary muscle cells. Matrix metalloproteinases hydrolyze excessive ECM, which should open up extracellular spaces and decrease fluid resistance flowing through these spaces. In addition, FP agonists have also been shown to induce relaxation of the TM and ciliary muscle (Thieme et al., 2006), which reduces tension and changes topography of the outflow

pathways. The combined actions of MMP activation and tissue relaxation can provide an explanation of the observation that in the ciliary muscle of monkeys treated with prostanoids for a year, there was a significant increase in optically empty spaces between muscle bundles compared with untreated and vehicle-treated control animals (Richter et al., 2003). These morphological changes presumably contribute to the improvement of uveoscleral outflow.

V. β-BLOCKERS

The β-adrenergic antagonists are some of the most commonly used therapeutic agents in the treatment of glaucoma. Examples of this class include betaxolol, carteolol, levobunolol, metipranolol, and timolol. After topical ocular administration, they are effective in lowering IOP, but usually less efficacious than prostanoids. They are used for both primary open angle glaucoma and angle closure glaucoma.

β-Blockers are competitive antagonists of the β-adrenergic receptors. They inhibit the activation of these receptors in the ciliary processes by blocking the binding of endogenous adrenergic neurotransmitters, i.e. norepinephrine and epinephrine. It is interesting to note that because the endogenous adrenergic activity is minimal during sleep, the IOP-lowering effect of β-blockers is prominent only during waking hours. The β-adrenergic receptors are coupled to adenylyl cyclase via a stimulatory G-protein. Hence, blockade of the receptor activation leads to a decrease in cyclic AMP levels in the ciliary epithelial cells and consequently suppression of aqueous humor production. The precise cellular mechanism(s) involved in the regulation of aqueous production by cyclic AMP is still elusive. Nonetheless, β-blockers were shown to inhibit the Na-K- ATPase and Na-K-Cl cotransport in the ciliary epithelium, reduce the blood–aqueous flux of ascorbate, as well as inhibit plasma flow to the ciliary processes.

VI. α2-AGONISTS

α2-Adrenergic agonists, such as apraclonidine and brimonidine, are another important class of compounds for glaucoma therapy. These compounds are effective IOPlowering agents for both open and closed angle glaucomas. They have rapid onset and generally reach their maximal ocular hypotensive effect in 2 to 3 hours after topical ocular administration.

They selectively activate the α2-adrener- gic receptor of the ciliary epithelium, which leads to a reduction in intracellular cyclic AMP levels and, eventually, suppressed aqueous humor production. Interestingly, apraclonidine was also reported to increase trabecular outflow and brimonidine was shown to increase uveoscleral outflow. The molecular mechanisms of their outflow effects are unclear, but are speculated to involve changes in contractility of the TM and ciliary muscle.

VII. TOPICAL CARBONIC ANHYDRASE INHIBITORS

Oral administration of carbonic anhydrase inhibitors (CAIs), such as acetazolamide, lowers IOP effectively and was used to treat glaucoma for many years. CAIs inhibit the carbonic anhydrase in the ciliary epithelium and reduce the production of bicarbonate ion, which is a critical component for active ion transport in aqueous formation. A reduction in bicarbonate by CAIs limits sodium and fluid transport across the ciliary epithelium, and decreases aqueous humor production.

Unfortunately, oral CAIs could not be tolerated by many patients because of their various systemic adverse effects. The discovery of compounds such as brinzolamide and dorzolamide, which successfully inhibit carbonic anhydrase in the ciliary epithelium after topical ocular application, provides an important improvement in this drug class.

Topical CAIs have minimal systemic side effects. Topical CAIs are useful IOP-lower- ing compounds and their effect is not influenced by the circadian rhythm. However, they are generally less efficacious compared to other glaucoma therapies. Topical CAIs are not usually used as a first-line medication. They are typically indicated as an adjunctive remedy when the primary treatment, such as a β-blocker or PGA, does not control IOP adequately.

VIII. CHOLINERGICS

Cholinergic compounds including muscarinic cholinergic agonists, such as pilocarpine and carbachol, as well as cholinesterase inhibitors, such as physostigmine and echothiophate iodide, are effective IOP-lowering agents. They are mainly employed as supplementary treatment for primary open angle glaucoma and are also useful to treat glaucoma attack in primary angle closure glaucoma. Cholinergics cause contraction of the ciliary muscle and iris sphincter via activation of the muscarinic cholinergicreceptorinthesetissues.Contraction of the ciliary muscle pulls the TM posteriorly, opens up the extracellular spaces in the TM, and results in an increase in trabecular aqueous outflow. Contraction of the iris sphincter induces miosis, which breaks the iris–lens contact during glaucoma attack of primary angle closure glaucoma.

IX. EPINEPHRINE AND

ANALOGS

Epinephrine and its prodrug dipivefrin bind to and activate various adrenergic receptor subtypes. They lower IOP by suppressing aqueous production and increasing its outflow. The multiple cellular mechanisms involved in these actions are yet to be precisely elucidated.

X. RECENT DEVELOPMENT IN FUTURE OCULAR HYPOTENSIVE MEDICATIONS

The availability of the above effective medications has contributed greatly to the treatment of glaucoma. Unfortunately, there still exist patients whose IOP cannot be satisfactorily controlled by these agents, either alone or in combination. Furthermore, many of these drugs have untoward effects that limit their universal acceptance. Therefore, research and development of novel and improved remedies are still a continuous and considerable need. In the past decades, many exciting discoveries of new IOPlowering compounds and mechanisms have been described. Regrettably, due to the constraint in space, this chapter will only focus on new pharmacological agents that have made important recent progress (Table 3.1). Other agents, such as forskolin, compounds

involved in the renin–angiotensin hormone system, and endothelin-related compounds, will not be discussed, even though they were once very important topics in glaucoma drug discovery.

XI. CYTOSKELETON ACTING

AGENTS

The cytoskeleton is a complex system of cytoplasmic fibers responsible for many vital cellular functions, such as the maintenance of cell shape, cell adhesion, contractility, motility, and intracellular transport. Dependent on their size, molecular components, and structure, cytoskeleton are classified as microfilaments, microtubules, and intermediate filaments. Cells within the aqueous outflow pathway, such as the TM cells and the endothelial cells lining the Schlemm’s canal, have an extensive

Identification of Possible Targets

Confirmation of Target

Validation of Target

Drug Discovery Target

Proteomics, genomics, molecular genetics, and basic disease research are being used to discover potential new therapeutic targets (see “XVIII. Identification of New Therapeutic Targets Based on Understanding Disease Pathogenesis”). The next step is to

confirm these new targets. For example, if gene chips were used to find altered expression of a specific gene in a diseased tissue compared to normal tissue, then many more samples need to be evaluated using an independent assay (such as RT-QPCR). The potential therapeutic target must then be validated. Does altered expression of that specific gene or protein (or in the case of molecular genetics – a specific mutation in the target gene) lead to a glaucoma phenotype? Once the new target is validated, an in vitro drug screening assay relevant for that target or target pathway is developed to look for agents that have the desired activity. In addition, a relevant in vivo model needs to be generated as a secondary drug screening model.

cytoskeleton. Compounds that disrupt the cytoskeleton can affect the cell shape, contractility and motility, and these changes may be sufficient to alter the local geometry of the outflow pathway and consequently aqueous outflow (Tian et al., 2000b). Several compounds of this pharmacological class were shown to be effective in lowering IOP in animal studies.

Cytochalasins prevent the elongation of normally dynamic actin filaments, causing distension of the TM and ruptures in the inner wall of Schlemm’s canal, which can lead to the enhancement of outflow facility. In anesthetized monkey, perfusion of the anterior chamber with cytochalasin B produces more than a 100% increase in aqueous outflow (Kaufman and Bárány, 1977; Robinson and Kaufman, 1991). Similarly, cytochalasin D increases outflow facility by approximately 40% in human eye perfused organ culture, with a peak effect 2 to 6 hours after infusion and a duration of action of 14 hours (Johnson, 1997).

Ethacrynic acid inhibits microtubule assembly and affects phosphorylation of certain other cytoskeletal molecules, which triggers cell contraction, irreversible alteration of cell shape, and reduction of focal adhesion (Rao et al., 2005a; Shimazaki et al., 2004b). In addition, ethacrynic acid also inhibits the Na-K-Cl cotransport mechanism in the cell membrane, which affects intracellular volume and, consequently, permeability of the TM (O’Donnell et al., 1995). These cellular events explain its outflow-enhancing effect in perfused human and calf eyes, and in eyes of anesthetized monkeys. Correspondingly, intracameral administration of this compound lowers IOP in rabbits and monkeys. In advanced glaucoma patients, intracameral injection of ethacrynic acid produces a dose-dependent reduction in IOP of 9 to 31mmHg, and the effect lasts for 3 days (Melamed et al., 1992). Unfortunately, this compound does not penetrate the cornea very well; its topical efficacy is limited. And most importantly, long-term use of ethacrynic acid in animals causes

significant local untoward effects, such as edema of the eye lid, conjunctival hyperemia, and corneal erosion. These side effects have limited its clinical utility as a glaucoma therapeutic agent.

Recently, new derivatives of ethacrynic acid were synthesized and evaluated. One of them, SA9000, was found to lower IOP in cats and monkeys after injection into the anterior chamber. Its efficacy is reported to be better than ethacrynic acid (Shimazaki et al., 2004a). At the present time, it is not clear if these new analogs of ethacrynic acid have an improved side effect profile and/or better corneal penetrability.

The latrunculins are macrolides that sequester monomeric G-actin and cause the disassembly of actin filaments, disorganization and disruption of the actin cytoskeleton in cells, which leads to a change in cell shape,anddecreasescell–cellandcell–matrix adhesion (Cai et al., 2000). Latrunculins produce significant morphological changes in the human TM, such as loss of microfilament integrity especially in TM cells on the collagen beams, formation of cytoplasmic projections of the subcanalicular cells, reorganization of intermediate filaments in Schlemm’s canal inner wall cells, as well as substantial expansion of the space between the Schlemm’s canal inner wall and the trabecular collagen beams (Sabanay et al., 2006). In addition, latrunculin B dosedependently relaxes the ciliary muscle. All these actions are expected to contribute to the enhanced outflow effect of latrunculins.

Latrunculin B increases aqueous outflow rate by up to 72% in perfused porcine eyes and 64% in perfused human eyes (Ethier et al., 2006). Intracameral perfusion or topical administration of latruculin A or B in anesthetized monkeys causes a timeand dose-dependent increase in outflow facility (up to four-fold) (Fan et al., 2005; Okka et al., 2004). This effect is completely reversible at the end of treatment. Multiple doses reduce IOP more than a single dose. The change in outflow facility by latrunculins A and B correlates with their IOP-lowering effect

in the monkey after topical ocular application. It is interesting to note that latrunculin B appears to be 10 times more potent than latrunculin A, and the onset of its IOPlowering effect is also more rapid.

Swinholide A is a marine macrolide that also interferes with normal cytoskeleton function. It severs actin filaments and sequesters actin dimers. In vivo intracameral perfusion of this compound increases aqueous outflow facility in anesthetized monkeys to a similar degree as latrunculin B (Tian et al., 2001).

An important issue of using cytoskeletondisrupting agents as IOP-lowering compounds is that cytoskeleton is present and plays vital roles in essentially all cells. It is not clear what significant untoward effects on other structures in the eye these drugs may have, especially after prolonged use.

XII. PROTEIN KINASE

INHIBITORS

Protein kinase inhibitors lower IOP in various animal studies. Though their exact mechanism of action is not fully understood, they likely increase aqueous outflow by affecting cytoskeleton of the TM or Schlemm’s canal endothelial cells. Early work used kinase inhibitors that were typically non-specific; they inhibited many kinases. Recently, a family of rho-associ- ated coiled coil-forming kinase (ROCK) inhibitors was discovered that are very efficacious in lowering IOP.

A. Broad Spectrum Kinase

Inhibitors

At μM concentrations, H-7 is a broad spectrum protein kinase inhibitor effective in inhibiting the activities of many kinases, including protein kinase A (cyclic AMPdependent protein kinase), protein kinase C, protein kinase G (cyclic GMP-dependent protein kinase), and ROCK. In perfused human anterior segments, H-7 increases

outflow facility and causes a partial loss of endothelial cells of the Schlemm’s canal without disruption of other TM cells (Bahler et al., 2004). However, there is no significant correlation between the amount of endothelial cell loss and outflow facility. In monkey eyes, H-7 perfusion triggers Schlemm’s canal inner wall protrusion, TM cell relaxation and cytoskeleton reorganization, some of which are likely structural bases for H-7-induced increase in outflow facility (Hu et al., 2006; Sabanay et al., 2004). In in vivo studies, topical administration of H-7 increases outflow facility by 135% in normal monkey eyes (Tian et al., 2004). Multiple doses of H-7 produce greater IOP reduction than a single dose.

Similar to H-7, HA1077 also inhibits many kinases, including protein kinase A, MAPK-activated protein kinase 1, mitogen and stress-activated protein kinase 1, p70 ribosomal protein S6 kinase 1, protein kinase C-related kinase 2, and ROCK. HA1077 dose-dependently decreases IOP in the rabbit, accompanied by an increase in outflow facility (Honjo et al., 2001a). Other broad spectrum protein kinase inhibitors, ML-7 and chelerythrine, also increase outflow facility in in vivo perfused monkey eyes (Tian et al., 2000a), while ML-9 lowers rabbit IOP (Honjo et al., 2002).

B. Protein Kinase C Inhibitors

Inhibitors of protein kinase C, such as GF109203X, were found to cause cytoskeletal reorganization and cell shape changes in human TM and Schlemm’s canal cells. GF109203X increases outflow by 46% in perfused porcine eyes (Khurana et al., 2003). In contrast, activators of protein kinase C, phorbol-12-myristate 13-acetate and phor- bol-12,13-dibutyrate (PDBu) increase myosin light chain phosphorylation, formation of actin stress fibers, and focal adhesions of human TM cells. Yet interestingly, PDBu also increases aqueous outflow facility in the pig eye, though only by 27% (Khurana et al., 2003). These results suggest that protein