and molecules. As discussed earlier in this chapter, TM cells and Schlemm’s canal endothelial cells in glaucoma eyes were shown to have abnormal cytoskeletal structures, which may be responsible, at least partially, for the reduction in aqueous outflow facility seen in POAG patients. Therefore, compounds that disrupt the cytoskeleton may modify these cell functions and local topography of the outflow pathway and consequently aVect aqueous outflow (Tian et al., 2000b). Indeed, drugs with this pharmacological action were shown eVectively lower IOP in animal studies (Table II).

1. Cytochalasins

Cytochalasins block the polymerization and elongation of actin microfilaments by capping the barbed ends of the filaments. In perfused human eyes, cytochalasin D increased outflow facility with a duration of action of at least 14 h (Johnson, 1997). Perfusion of the anterior chamber of anesthetized monkeys with cytochalasin B doubled the aqueous outflow (Kaufman and Ba´ra´ny, 1977; Robinson and Kaufman, 1991). Morphological evaluation of the treated eyes showed that these compounds caused TM distension and ruptures in the inner wall of Schlemm’s canal, thereby enhancing outflow and washout of ECM (Svedbergh et al., 1978).

2. Latrunculins

The latrunculins bind to monomeric G actin and cause the disorganization of actin filaments. In human ocular tissues and cells, these compounds induced many cytoskeletal changes, such as reorganization of intermediate filaments in Schlemm’s canal inner wall cells, disruption of actin microfilament integrity in TM cells, and substantial expansion of the space between the of Schlemm’s canal inner wall and the trabecular collagen beams (Cai et al., 1999; Cai et al., 2000; Sabanay et al., 2006). In addition, latrunculin B dose dependently relaxed the ciliary muscle (Okka et al., 2004a). All these actions can contribute to the enhanced outflow eVect of latrunculins. In anesthetized monkeys and cultured porcine and human eyes, latrunculin A and/or B significantly improved aqueous humor outflow and decreased IOP for up to 24 h (Okka et al., 2004b; Fan et al., 2005; Ethier et al., 2006).

3. Swinholide A

Swinholide A is a marine macrolide that severs actin filaments and sequesters actin dimers. Perfusion of this compound in the anterior chamber increased aqueous outflow facility in anesthetized monkeys (Tian et al., 2001).

4. Ethacrynic Acid

Ethacrynic acid inhibits microtubule assembly and reduces phosphorylation of focal adhesion kinase and paxillin, both focal adhesion proteins. Focal adhesion kinase and paxillin are important components in the integrin mediated cell adhesion signaling pathways. In cultured TM cells, ethacrynic acid and analogs disrupted cytoskeleton, altered cell shape irreversibly, and decreased of focal adhesion (Shimazaki et al., 2004b; Rao et al., 2005c). Furthermore, ethacrynic acid inhibited the Na–K–Cl cotransport mechanism on TM cell membrane, which aVected cell volume and

permeability of the TM (O’Donnell et al., 1995). Intracameral administration of ethacrynic acid lowered IOP in rabbits, monkeys, and advanced glaucoma patients (Melamed et al., 1992). It increased outflow facility in ex vivo bovine and human eyes. Unfortunately, this compound does not penetrate the cornea well; its eYcacy after topical ocular administration was minimal. And most importantly, its long term use caused significant local untoward eVects in animals. Recently, new and more eYcacious derivatives of ethacrynic acid were synthesized and shown to lower IOP in cats and monkeys after intracameral injection (Shimazaki et al., 2004a).

5. Protein Kinase Inhibitors

Even though many protein kinase inhibitors lower IOP in animal studies, their mechanism of action has not been conclusively demonstrated. Histological assessment suggests that they aVect cytoskeleton of the TM or Schlemm’s canal endothelial cells and increase outflow rate of aqueous humor. Initial work employed kinase inhibitors that have broad spectrum activity, inhibiting many kinases. Recently, a family of rho associated coiled coil forming kinase (ROCK) inhibitors was found to eVectively lower IOP (Honjo et al., 2001b).

6. Broad Spectrum Kinase Inhibitors

H 7, a broad spectrum protein kinase inhibitor eVective in inhibiting the activities of many kinases, including protein kinase A, protein kinase C, protein kinase G, and ROCK, was shown to increase aqueous outflow in perfused human anterior segments (Bahler et al., 2004) and in anesthetized monkey eyes (Tian et al., 2004). In the TM of perfused eyes, cytoskeleton reorganization and cell relaxation were observed. The Schlemm’s canal inner wall also exhibited protrusion and partial loss of endothelial cells (Bahler et al., 2004; Sabanay et al., 2004; Hu et al., 2006). Other broad spectrum kinase inhibitors, HA1077, ML 7, ML 9, and chelerythrine, stimulated outflow facility in various animal models (Tian et al., 2000a; Honjo et al., 2001a, 2002).

7. ROCK Inhibitors

Recently, ROCK inhibitors, such as Y 27632, Y 39983, and H 1152, have been found to lower IOP eYcaciously. Y 27632 and H 1152 increased outflow facility of aqueous humor in enucleated porcine eyes (Rao et al., 2001; Rao et al., 2005a). Y 27632 and Y 39983 also lowered IOP in the rabbit and monkey (Honjo et al., 2001b; Waki et al., 2001; Tian and Kaufman, 2005; Tokushige et al., 2007).

ROCKs are kinases that can be activated by a cell signaling molecule Rho. Once activated, ROCKs modify functions of various proteins by phosphorylation. These target proteins, such as adducin, ezrin–radixin–moesin proteins, intermediate filament proteins, LIM kinases, myosin light chain phosphatase, sodium hydrogen exchanger NHE1, etc, play significant roles in cell shape, contractility, and focal adhesion. Consequently, compounds that inhibit ROCK activity are expected to aVect these cell functions. In cultured human TM and Schlemm’s canal cells, ROCK inhibitors were reported to decrease actin stress fibers, reduce myosin light chain phosphorylation, and change cell shape, leading to a widening of the extracellular spaces in the TM, especially of the JCT (Rao et al., 2005b; Rosenthal et al., 2005; Koga et al., 2006). These cellular changes likely contribute to the ocular hypotensive eVect of ROCK inhibitors. It is important to note that ROCKs are present in many other tissues, notably vascular cells, and ROCK inhibitors may aVect these other tissues which may produce side eVects. In fact, conjunctival hyperemia and sporadic punctate subconjunctival hemorrhage have been observed in animals receiving topical administration of ROCK inhibitors (Tokushige et al., 2007).

B. Activators of ECM Hydrolysis

As described earlier in this chapter, an excessive accumulation of ECM in the TM may be responsible for ocular hypertension seen in glaucoma patients. Reduction of the excessive ECM by stimulating its degradation should improve aqueous outflow and consequently lower IOP. ECM turnover in the TM is regulated by a family of zinc containing extracellular neutral proteinases, called matrix metalloproteinases (MMPs) (Alexander et al., 1991; Acott, 1992; Samples et al., 1993). These enzymes are involved in normal development, reproduction, wound healing, and tissue remodeling, as well as in disease conditions, such as angiogenesis, tumor metastasis, arthritis, Sorsby’s fundus dystrophy, and age related macular degeneration (Clark, 1998). MMPs, synthesized as proenzymes, require proteolytic cleavage for activation. Their enzymatic activities are inhibited by endogeneous peptides known as tissue inhibitors of metalloproteinases (TIMPs). In the TM, several MMPs, e.g., MMP 1, MMP 2, MMP 3, and MMP 9, as well as TIMPs, such as TIMP 1 and TIMP 2, were detected (Alexander et al., 1991; Samples et al., 1993; Parshley et al., 1996; Alexander et al., 1998; Pang et al., 2003b). MMPs are also present in human aqueous humor (Ando et al., 1993).

The involvement of MMPs in the regulation of aqueous outflow has been demonstrated in numerous studies. Ex vivo perfusion of the human eye with purified MMP 3 alone or together with MMP 2 and MMP 9 increased

444 Pang and Clark

outflow facility considerably (Bradley et al., 1998). Correspondingly, inhibitors of MMPs, such as the TIMPs, minocylcine, or L tryptophan hydroxa-

and PGAs (Parshley et al., 1995, 1996; Lindsey et al., 1996, 1997).

In addition to laser treatment and prostaglandins, there are other means to increase MMP activity in ocular tissues (Table III). Recently, it was discovered that stimulation of the activator protein 1 (AP 1) pathway in cultured human TM cell upregulated MMP 3 expression (Fleenor et al., 2003). Subsequently, tert butylhydroquinone, a small molecule AP 1 stimulator, was found to improve aqueous outflow in glaucoma and non glaucoma donor eyes (Pang et al., 2003a). This outflow eVect correlated with an increase in MMP 3 levels in the TM cells. These data suggest that small molecules that can increase MMP expression are potentially valuable approaches to IOP regulation. It is interesting to note that the AP 1 pathway is by no means the only cell signaling pathway involved in MMP production. JNK and p38 MAP kinases were also shown to play important roles in the modulation of MMP expression (Kelley et al., 2007b). Compounds that stimulate these molecular mechanisms may also prove useful.

A subset of ECM molecules, the glycosaminoglycans (GAGs), can be hydrolyzed by GAG degrading enzymes. GAGs comprise hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and others. The GAGs likely contribute to IOP elevation in glaucoma. For example, the GAG profile in human glaucoma TM is significantly diVerent

TABLE III

Activators of Extracellular Matrix Hydrolysis

from that of normal donors (Knepper et al., 1996a,b). Similarly, experimental ocular hypertension caused changes in GAG profiles in the TM of several animal species (Knepper et al., 1978, 1985a). Hence, Francois hypothesized that an increase in GAG in the TM is a causative factor of IOP elevation in POAG patients (Francois, 1975). This hypothesis is further supported by GAG degrading enzymes, such as hyaluronidases and chondroitinases, that produced a decrease in IOP when perfused into bovine (Ba´ra´ny and Scotchbrook, 1954; Pedler, 1956; Sawaguchi et al., 1993), rabbit (Knepper, et al.,1985b), guinea pig (Melton and DeVille, 1960), dog (Van Buskirk and Brett, 1978), or monkey (Peterson and Jocson, 1974; Sawaguchi et al., 1992) eyes. However, Hubbard and colleagues were unable to detect any outflow eVect of intracameral injection of chrondroitinase or hyaluronidase in the monkey (Hubbard et al., 1997).

Degradation of GAGs can also be catalyzed by metal ions in the presence of ascorbate. For example, a small molecule with a chelated ferric ion, sodium ferri ethylenediaminetetraacetate (AL 3037A), together with ascorbate, accelerated GAG depolymerization (Pang et al., 2001). In perfused bovine, normal human, or glaucomatous human eyes, AL 3037A plus ascorbate produced marked increases in aqueous outflow (Pang et al., 2001). In normal or dexamethasone induced hypertensive rabbits, topical ocular administration of AL 3037A was eVective in lowering IOP (Pang et al., 2001). Addition of ascorbate is not necessary in rabbit studies, because the aqueous humor already contains approximately 1.1 mM of ascorbate. These results suggest that stimulation of GAG degradation may represent another new and practical method to treat glaucoma.

C. Adenosine Receptor Agonists and Antagonists

Three adenosine receptor subtypes, the A1, A2A, and A3 receptors, have been shown to participate in the regulation of aqueous production and outflow. Compounds that activate or antagonize the activation of these receptors aVect IOP in various species (Table IV). For example, A1 receptor agonists, such as N 6 cyclohexyladenosine (CHA) and (R) phenylisopropyladenosine (R PIA), lower IOP in the mouse (Avila et al., 2001), rabbit (Crosson, 1992, 1995, 2001; Crosson and Gray, 1994), and monkey (Tian et al., 1997). In some studies, the ocular hypotensive eVects of these compounds were preceded by a transient increase in IOP (Crosson and Gray, 1994; Tian et al., 1997; Crosson, 2001). This was likely a result of non-specific activation of the A2A receptor, because A2A receptor antagonists abolished the initial ocular hypertensive eVect without aVecting the longer lasting hypotensive eVect of CHA and R PIA (Tian et al., 1997; Crosson, 2001).

Agonists of A1 receptor lower IOP by increasing aqueous outflow, as demonstrated in perfused bovine (Crosson et al., 2005) and monkey eyes (Tian et al., 1997). The outflow eVect in bovine eyes was reduced by a non-selective MMP inhibitor GM 6001, suggesting that MMP may play a role in the outflow eVect of A1 receptor agonists (Crosson et al., 2005). In addition, A1 receptor agonists also increase whole cell currents in cultured human Schlemm’s canal inner wall cells (Karl et al., 2005) and increase calcium influx and decrease cell volume in cultured human TM cells (Fleischhauer et al., 2003). Changes in functions of these cells may also contribute to the decrease in outflow resistance induced by the compounds.

Several laboratories demonstrated that A2A receptor agonists, such as 2 p (2 carboxyethyl) phenethylamino 50 benzyl) adenosine (CGS 21680) and 2 (1 hexyn 1 yl) adenosine (2 H Ado), increase IOP in the mouse (Avila et al., 2001), rabbit (Crosson, 1995; Crosson and Gray, 1996; Konno et al., 2005b), and cat (Crosson and Gray, 1996), accompanied by an increase in aqueous production (Crosson and Gray, 1996; Konno et al., 2005b). However, Konno and colleagues showed that CGS 21680 and other A2A receptor agonists enhanced aqueous outflow and consequently lowered IOP in rabbits (Konno et al., 2004, 2005a). The reason for this discrepancy in results is currently not understood. A2A agonists have opposite eVect compared to the

A1 agonists in whole cell currents in Schlemm’s canal inner wall cells (Karl et al., 2005), but increase calcium influx and decrease cell volume, similar to A1 agonists, in cultured human TM cells (Fleischhauer et al., 2003).

Agonists of the A3 receptor, such as N6 (3 iodobenzyl) adenosine 50 N methyl uronamide (IB MECA), have been shown to increase IOP in the mouse (Avila et al., 2001, 2002; Civan, 2003; Civan and Macknight, 2004; Yang et al., 2005; Do and Civan, 2006). The cellular mechanism of action involves activation of chloride channels on the plasma membrane of non pigmented ciliary epithelial cells, which leads to stimulation of aqueous

humo r formati on (Mitetchella,. 1999;Ci van, 2003; Civan and Macknight , 2004; Do and Civan 2004). Apparently, the A3 receptor mediated aqueous humor formation is active in normal eyes, because antagonists of the A3 receptor lower IOP in the mouse (Avila et al., 2002; Do and Civan, 2006;

Yang et al., 2005) and monkey (Okamura et al., 2004). Similarly, A3 receptor knockout mice had a significantly lower IOP than their wild type controls (Avila et al., 2002).

D. Serotonergic Agonists

Serotonergic receptor agonists and antagonists have long been studied for their IOP eVects. However, because of the multiple serotonergic receptor subtypes and the lack of specificity of many of the agents tested, pharmacological actions of many of these compounds on IOP and aqueous hydrodynamics are unclear and controversial. Recently, a 5 HT2 agonist, R ( ) 1 (4 iodo 2,5 dimethoxyphenyl) 2 aminopropane (R DOI), was shown to lower IOP in laser induced ocular hypertensive and normotensive monkeys (May et al., 2003a; Gabelt et al., 2005). The eVect of this compound was largely mediated by an increase in uveoscleral outflow (Gabelt et al., 2005). These findings, together with the discoveries of 5 HT2 receptors in the human ciliary body (Chidlow et al., 2004) and cultured human TM cells (Sharif et al., 2006), sparked a renewed interest in this pharmacological class of compounds. A series of selective 5 HT2 agonists, such as S (þ) 1 (2 aminopropyl) 8,9 dihydropyrano[3,2 e]indole, the 1R,2R isomer of 1 (4 bromo 2, 5 dimethoxyphenyl) 2 aminopropan 1 ol, 1 ((S) 2 aminopropyl) 1H indazol 6 ol, and tetrahydrobenzodifuran analogs, were synthesized and demonstrated to have high binding aYnities for the 5 HT2A, 5 HT2B, and 5 HT2C receptor subtypes, but not other receptors (May et al., 2003a, 2006; Glennon et al., 2004; Feng et al., 2007). They were highly eYcacious in lowering IOP in lasered monkey eyes (May et al., 2003b, 2006; Glennon et al., 2004; Feng et al., 2007). Although their mechanism of action is still unknown, they are speculated to improve uveoscleral outflow analogous to R DOI.

These new potential therapeutic compounds, cytoskeleton disrupting agents, kinase inhibitors, ECM hydrolysis stimulators, adenosine analogs, and serotonin agonists, are exciting innovations in the arsenal for glaucoma treatment. Their pharmacological actions diVer from the existing therapies, hence may provide additional or complementary advantages to the existing therapies. For example, these new drug classes may lower IOP in glaucoma patients who are refractory to the currently available medications. Alternatively, they may induce further reduction in IOP when used as an adjunctive agent. Furthermore, some of these compounds, such as the ECM hydrolysis stimulators, are designed to correct pathological changes (e.g., excessive accumulation of ECM) in the TM. They should be able to modify the underlying disease process instead of just managing the symptoms. Nevertheless, these new compounds may have their limitations as well. Since most of them have not been tested in humans, it is uncertain what untoward eVects they may produce. Their pharmacological actions are unlikely specific to only the tissues involved in IOP regulation. This is especially true for the cytoskeleton disrupting agents, kinase inhibitors, and ECM hydrolysis stimulators. These cellular targets are ubiquitous in most tissues. Chronic exposure to these compounds may generate unacceptable ocular or systemic toxicity. The adenosine analogs and serotonergic agonists, depending on the distribution of their respective receptors, may be more specific in their eVects. However, they are also known to aVect cardiovascular and neurological functions. Their future clinical value will be determined by the balance between beneficial and possible side eVects.

It is important to note that, in addition to the pharmacological agents described here, others, such as compounds related to the angiotensin renin system, cyclic AMP and cyclic GMP stimulating agents, have also been explored extensively and shown to regulate IOP. Nonetheless, because there is no or only minimal new development reported in recent years, they are not discussed in this chapter. Interested readers are encouraged to consult earlier publications.

The above mentioned new therapeutic approaches have demonstrated their proof of concept and usefulness with specific compounds in animal models. Most recently, other novel, potential approaches for glaucoma treatment have been unveiled. They are still at the ‘‘therapeutic target’’ developmental stage. In other words, at the present time, no or only limited pharmacological agents are recognized to selectively modify these targets and proven to aVect IOP in animals. These targets are identified as probably involved in the pathogenesis of glaucoma. Functional modification of them may correct the underlying disease etiology and/or pathology. Future studies

on these new targets (described below), including growth factors, cytokines, and other cell signaling molecules, are expected to lead to revolutionary, innovative treatment principles.

E. Growth Factors

The TM makes and secretes a wide variety of growth factors and cytokines (Wordinger et al., 1998; Wordinger and Clark, 2008). The TM is often a target of these growth factors and cytokines, which regulate a number of cellular functions and activities. Many growth factors and cytokines are involved in controlling ECM metabolism (both synthesis and degradation) as well as regulating TM cell proliferation and phagocytosis. Altered growth factor signaling has important implications in the pathogenesis of glaucomatous damage to the aqueous outflow system (Table V).

1. TGFb

A key central mediator that regulates TM cell function is transforming growth factor beta (TGFb). TM cells make and secrete both TGFb1 and TGFb2 (Tripathi et al., 1993a,b), as well as express functional TGFb receptors (Wordinger et al., 1998). TGFb also appears to play important roles in glaucoma pathogenesis. A number of studies have shown that aqueous humor levels of TGFb2 are elevated in POAG patients (Lu¨tjen Drecoll, 2005), while aqueous humor levels of TGFb1 are elevated in patients with pseudoexfoliation syndrome (Schlotzer Schrehardt et al., 2001). Mechanical stress (e.g., cellular stretch or elevated IOP) can induce TGFb expression in the TM (Liton et al., 2005). Overall, TGFb modifies TM cell ECM metabolism promoting ECM deposition, making it an interesting candidate for mediating glaucomatous damage to the TM.

The eVects of TGFb on the TM are manifold. Treating TM cells with TGFb1 or TGFb2 alters the expression of hundreds of genes (Zhao et al., 2004) (Shepard AR et al. personal communication). TGFb2 increases the expression of TM cell fibronectin, PAI 1, collagen types I, III, IV, thrombospondin 1 (TSP 1) (Fuchshofer et al., 2007; Wordinger et al., 2007), tissue transglutaminase (Welge Lussen et al., 2000; Tovar Vidales et al., 2007), hyaluronan synthase (Usui et al., 2003), and proteoglycans such as versican (Zhao and Russell, 2005). TSP 1 activates latent TGFb in vivo, and its induction by TGFb further amplifies TGFb eVects. The increased synthesis of ECM components, their cross linking by transglutaminase, and decreased degradation via elevated PAI 1 would lead to increased ECM deposition, which is a key feature in glaucomatous TM tissues (Rohen, 1983). In addition, TGFb can change the composition of the TM ECM by diVerentially