REVIEW

Targeting Schlemm’s Canal in the Medical Therapyof Glaucoma: Current and Future Considerations

Vanessa Andres-Guerrero . Julian Garcıa-Feijoo . Anastasios Georgios Konstas

Received: February 2, 2017� The Author(s) 2017. This article is an open access publication

ABSTRACT

Schlemm’s canal (SC) is a unique, complexvascular structure responsible for maintainingfluid homeostasis within the anterior segmentof the eye by draining the excess of aqueoushumour. In glaucoma, a heterogeneous groupof eye disorders afflicting approximately 60million individuals worldwide, the normaloutflow of aqueous humour into SC is progres-sively hindered, leading to a gradual increase inoutflow resistance, which gradually results in

elevated intraocular pressure (IOP). By and largeavailable antiglaucoma therapies do not targetthe site of the pathology (SC), but rather aim todecrease IOP by other mechanisms, eitherreducing aqueous production or by divertingaqueous flow through the unconventional out-flow system. The present review first outlinesour current understanding on the functionalanatomy of SC. It then summarizes existingresearch on SC cell properties; first in the con-text of their role in glaucoma development/progression and then as a target of novel andemerging antiglaucoma therapies. Evidencefrom ongoing research efforts to develop effec-tive antiglaucoma therapies targeting SC sug-gests that this could become a promising site offuture therapeutic interventions.

Keywords: Actin polymerization inhibitors;Adenosine receptor agonists; Antiglaucomadrug development; Aqueous humour;Glaucoma; Intraocular pressure; Nitric oxidedonors; Ophthalmology; Rho Kinaseinhibitors; Schlemm’s canal

INTRODUCTION

Glaucoma is a group of chronic, multifactorial,and progressive eye disorders that eventuallylead to damage of the optic nerve and irre-versible visual loss. Given the progressive ageing

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V. Andres-Guerrero � J. Garcıa-FeijooDepartment of Ophthalmology, Sanitary ResearchInstitute of the San Carlos Clinical Hospital, Madrid,Spain

V. Andres-Guerrero � J. Garcıa-FeijooOcular Pathology National Net OftaRed of theInstitute of Health Carlos III, Madrid, Spain

J. Garcıa-FeijooDepartment of Ophthalmology, Faculty ofMedicine, Complutense University of Madrid,Madrid, Spain

A. G. Konstas (&)1st and 3rd University Departments ofOphthalmology, AHEPA Hospital, AristotleUniversity of Thessaloniki, 1 Kyriakidi Street, 546 36Thessaloniki, Greecee-mail: konstas@med.auth.gr

Adv Ther

DOI 10.1007/s12325-017-0513-z

of the world’s population, the number of indi-viduals affected by glaucoma is projected toincrease to almost 80 million by 2020 [1].Consequently, there is a pressing need for thedevelopment of novel therapies, which will notonly reduce intraocular pressure (IOP) as cur-rent therapies generally do but which will alsoprevent glaucoma development and slow downits progression. To succeed this requires inno-vative biological interventions that will posi-tively modify the various pathophysiologicalprocesses involved in glaucoma pathogenesis.

In glaucoma, it is established that there is animpaired balance between deposition anddegradation of extracellular matrix material(ECM) within the trabecular meshwork (TM).Gradual accumulation of ECM within the out-flow system is thought to result in a functionalobstruction leading to a gradual, asymptomaticelevation of intraocular pressure (IOP) [2]. Ele-vated IOP is at present the most importantmodifiable risk factor for the onset and pro-gression of glaucoma. The elevation of IOP maycompress TM and result in a collapse of Sch-lemm’s canal (SC), pathological steps whichalso increase outflow resistance and contributeto the pathobiology of glaucoma development[3].

At the present time, available glaucomatherapies aim to obtain a meaningful IOPreduction to a predetermined target IOP level.This is achieved by the selection of one or moreclasses of topical medications, which areadministered every day in order to meaning-fully reduce 24-h IOP. Existing antiglaucomatherapies either reduce IOP by decreasingaqueous synthesis (b-blockers, carbonic anhy-drase inhibitors, alpha agonists) or target andenhance the unconventional uveoscleral out-flow pathway (prostaglandins). Sadly, contem-porary glaucoma medications do not target theconventional outflow system (TM and SC),which is the site of glaucomatous pathobiologyand the site of anatomical and functional out-flow obstruction. Drugs that have been shownto improve conventional outflow have eitherbeen abandoned due to unacceptable sideeffects (cholinergics, e.g. pilocarpine), or have aminimal impact on this outflow site (e.g. bri-monidine) [4]. It would therefore appear logical

to develop in the future therapies that target theconventional outflow system. Moreover, it isnot uncommon for available medical therapiesto not sufficiently control IOP. This is due totheir inability to reach an optimal balancebetween long-term efficacy, tolerability andadherence. New medications targeting theconventional outflow system may provideadjunctive IOP reduction with superior tolera-bility allowing a more successful medicalantiglaucoma therapy. This review first sum-marizes recent advancements in our unde-standing on SC cell properties and theircontributing role in glaucoma development andprogression. Then, it discusses and explores theclinical value of targeting SC to better manageglaucoma in the future.

This article is based on previously conductedstudies, and does not involve any new studies ofhuman or animal subjects performed by any ofthe authors.

REGULATION OF AQUEOUSHUMOUR DYNAMICS

Aqueous humour fills the space between thecornea and the lens, providing nutrients tothese avascular structures that must remainclear to allow light transmission. It stabilizesocular structures, transports neurotransmitters,provides nutrition, and removes metabolicby-products, significantly contributing to theregulation of ocular tissue homeostasis. Theaverage rate of production of AH is 2.0–2.5lL min-1, and the turnover rate for aqueousvolume is approximately 1% per minute [5].Fluctuations in either the synthesis, or morecommonly, the drainage of aqueous can beresponsible for an elevation in IOP. The aque-ous is constantly produced by three mecha-nisms: active secretion, diffusion andultrafiltration [6]. The most important mecha-nism is active secretion, mediated by proteintransporters and enzymes, such as Na?–K?-AT-Pase and carbonic anhydrase, both located inthe non-pigmented and pigmented ciliaryepithelia [7]. This process is responsible forapproximately 80–90% of the total aqueousformation, and occurs through selective

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trans-cellular movement of anions, cations, andother molecules across a concentration gradientin the blood–aqueous barrier. The synthesis bydiffusion comprises the pass of solutes—mainlyhydrophobic—through the lipid portions of themembrane of the tissues, between the capillar-ies and the posterior chamber, this processbeing proportional to a concentration gradientacross the membrane [8]. The ultrafiltrationmechanism refers to the flow of water andhydrophilic substances across fenestrated ciliarycapillary endothelia into the ciliary stroma,depending on an osmotic gradient or hydro-static pressure [8].

The continuous synthesis of aqueoushumour must be balanced by sufficient aque-ous drainage through the TM into SC and thenback to the systemic circulation. This system-atic continuous drainage is named the con-ventional outflow pathway and comprisesbeyond the TM, the juxtacanalicular connec-tive tissue, the endothelial lining of SC, thecollecting channels, and the aqueous veins.This pathway provides resistance to aqueousoutflow; in response to this resistance the IOPgradually rises to a level sufficient to allow theflow of aqueous across the TM into SC [9].Thus, aqueous passes through the TM as a bulkflow driven by the pressure gradient. At stea-dy-state IOP, aqueous flow rate across the TMresistance equals the rate of aqueous synthesisby the ciliary body. Alternative outflow isprovided by the unconventional uveoscleralpathway where the flow is pressure-indepen-dent and a proportion of aqueous humourleaves the eye through the interstitial spaces ofthe ciliary body.

SCHLEMM’S CANAL ANATOMY

SC is located at the inner space of the cor-neoscleral junction, encircling the cornea, andis responsible for controlling the balancebetween secretion and drainage of the aqueousafter it crosses the TM. Filled with blood,deriving from the ipsilateral episcleral or jugularvein, SC appears as an irregular red line with a

varying diameter, divided by partitions intosmall tributaries, which join together in aplexiform appearance. In a cross-section, SCappears as a flattened oval structure between thesclera spur and Descemet’s membrane, boundedanterolaterally by the sclera and posteriorly bythe corneoscleral trabeculae. The canal can bedivided into three zones: the endothelial lining,the basal lamina, and the peri- orjuxta-canalicular connective tissue [10]. Theendothelial lining is formed by a single layer ofdelicate spindle-shaped endothelial cells withwedge-shaped, rounded ends, which are locatedparallel to the circumference of the canal. Theendothelial cells show small microvilli on theirapical area, a few projections extended into thebasement membrane in the basal area, and gapjunctions and complex interdigitations betweenadjacent and lateral cells [11]. Endothelial cellscontain an elongated nucleus, mitochondria,rough and smooth endoplasmic reticulum,golgi apparatus, centrosomes, and pinocyticvesicles, as well as fine cytoplasmic filamentsand giant vacuolar configurations, which arebelieved to play a major role in the bulk outflowof aqueous across the endothelial barrier [12].

The basement membrane is composed of anamorphous matrix of an irregular network ofthin microfibril loose sheets, provides anchor-age to SC endothelial lining. The basementmembrane is separated from the outermost partof the TM by the pericanalicular connectivetissue [13]. The extracellular spaces of the con-nective tissue are narrower than those of the TMand contain hydrophilic glycosaminoglycansand collagenous material, causing aqueousoutflow resistance and obstructing particulatematerial [9]. The basement membrane consistsof loosely arranged mesothelial cells and fixedendothelial cells, whose ultrastructure appearssimilar to that of the endothelial lining andfibrous tissues, such as collagen fibrils, elasticfibers, basement membrane material, microfib-rils, and curly collagen. Fixed endothelial cellshave active phagocytic function and showshrinking and swelling properties, being able toalter the porosity of this region and thus par-ticipating in IOP modulation [12].

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SCHLEMM’S CANALBIOMECHANICS IN GLAUCOMA

As discussed above, the conventional aqueouspathway consists of the TM, the juxtacanalicu-lar connective tissue, the endothelial lining ofSC, the collecting channels, and the aqueousveins, in this order. Among these structures, it isestablished that SC offers the greatest resistanceto aqueous outflow, providing a promisingoption for the development of IOP-loweringtherapies. The resistance induced at the SC levelis predominantly due to the compact endothe-lial cell lining, composed by a single layer ofspindle-shaped cells joined with numerous tightjunctions and complex interdigitations betweenadjacent and lateral cells [11]. Although eventoday the precise mechanism delineatingaqueous drainage is not completely understood,a key process involved is cytoplasmic vacuoleformation and migration, which drains aqueousfrom the TM through the juxtacanalicular tissueand the endothelial monolayer, into the lumenof SC [14]. Giant vacuoles are formed inresponse to the pressure gradient created byaqueous flow. Most vacuoles demonstrate alarge opening on the meshwork side of thevacuole and around one-third also have a distalopening, forming transcellular and paracellularpores [15]. Transcellular pores pass from thebasal side of the cell to the apical side at alocation on the cell surface where the inner andouter surfaces of the cell are joined together andare fused. The high hydraulic conductivity of SCappears to be related to the presence of a largenumber of these pores, whose diameter rangesbetween 0.1 and 2 lm [14]. With regard to theparacellular pores, they appear to correlate wellwith the paracellular flow through SCendothelium [9]. Small minipores, covered witha thin diaphragm and with a diameter rangingfrom 60 to 65 nm, have also been identified inthe SC inner wall endothelium. Minipores havesimilar ultrastructural characteristics to thoseforming the fenestrated capillaries in the ciliarybody, or choroid, and thus it has been hypoth-esized that they are the origin of large tran-scellular pores [16].

A number of researchers have investigatedthe relationship between pore density in SCendothelium and aqueous humour facility, inorder to determine a possible relationshipbetween pore number in glaucomatous andnormal eyes [17–19]. However, due to method-ological differences arising from the fixationprocedure employed, and the fact that poredensity increases with the volume of fixativeperfused through the outflow pathway [20],results in glaucomatous eyes have been incon-sistent. This has generated controversy as to theprecise role of inner wall pores in outflowfacility. Despite this, recent studies haveattempted to compare pore density in normaland glaucomatous eyes. Johnson et al. [19]established a correlation in the density ofintracellular and border pores between normaland glaucomatous eyes after correction for theeffects of fixation conditions. They reportedthat, even after accounting for the volume offixative perfused, glaucomatous eyes exhibitonly 20% of the pore density seen in normaleyes (160 vs. 835 pores/mm2, respectively).Consequently, these authors suggested that thisphenomenon may explain the IOP elevationseen in glaucoma [19]. In 2014, Braakman et al.[21] perfused fluorescent tracer nanospheres innon-glaucomatous human donor eyes toexperimentally describe local patterns of out-flow segmentation through the conventionalroute. By confocal and scanning electronmicroscopy, they examined the spatial rela-tionship between inner wall pore density andtracer intensity. Their results suggested that theaqueous passed through micron-sized pores inthe inner wall endothelium of SC, and thatparacellular pores played a major role intransendothelial filtration across the inner wallin comparison to transcellular pores. The spatialcorrelation between pores and tracer suggestedthat pores influenced outflow segmentation,but no insight was offered into the precise nat-ure of this relationship. These investigators [21]speculated that the physiological situation rep-resented a coupled interaction between localfiltration demands and the cellular biome-chanics involved in giant vacuole and poreformation.

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In SC cells, vacuole and pore formation isdependent on pressure and is most likely relatedto tissue stiffness. Tissue and cell stiffness arefactors that may significantly modify theresponsiveness of the TM and SC to normalfluid flow, pressure, and shear stress. Overbyet al. [22] reported that pore formation corre-lated with the stiffness of the subcorticalcytoskeleton in SC cells as well as that glauco-matous SC cells exhibited both a stiffer subcor-tical cytoskeleton and a reduced ability to formpores. It has been recognized that SC stiffness isinfluenced by drug therapies affecting outflow.Specifically, when a drug stiffened SC cells,resistance increased, and when a drug therapyrelaxed these cells, outflow resistance decreased.These observations suggest that those drugs thatwill directly or indirectly modify the cytoskele-ton will decrease cell stiffness and result inreduced outflow resistance [23–27].

The endothelial lining of SC demonstratesboth blood and lymphatic vascular characteris-tics, calling into question the longstandingdogma that the eye lacks lymphatic vessels.Several years ago, lymphatic markers wereidentified in the anterior segment and ciliarybody of the human eye. Nevertheless, it was notclear whether they were actively involved in themaintenance of ocular fluid homeostasis[28, 29]. In a recent study, Hoopes et al. [30]found that temporal delection of Calcrl, ahighly expressed receptor in lymphaticendothelial cells, resulted in dilated cor-neoscleral lymphatic vessels that preceded andwere associated with the formation of cornealedema and inflammation. Hence, these resultssuggested that lymphatic vessels may con-tribute to the maintenance of fluid homeostasisand corneal hydration [30]. More recently,Truong et al. [31] provided the first evidenceshowing the expression of the lymphatic mar-ker Prox-1 on SC endothelium, indicating itscloser similarities with lymphatic endotheliumthan with blood endothelium. Likewise,authors employed a panel of protein markersthat allowed a better identification, isolation,and characterization of SC endothelial cells,resulting in improved endothelial cell harvest-ing efficiency. Therefore, stimulation of SC withlymphatic-specific therapies may result in a

significant lowering of IOP and could constitutea promising approach in future glaucomatreatment.

TARGETING SCHLEMM’S CANALTO REDUCE IOP

The precise biomechanics of the resistance toaqueous humour flow through the conven-tional outflow pathway is a subject still underinvestigation. However, the most popularhypothesis is that the contractile status of cellsin the aqueous pathway regulate outflow resis-tance with a key role played by cell stiffness[32–37]. It is established that drugs such asbradykinin, dexamethasone, or triamcinoloneare capable of increasing cell stiffness and bydoing so augment outflow resistance, whileother drugs decrease outflow resistance byrelaxing SC cells [12]. In glaucomatous eyes, SCcells are more sensitive to substrate stiffnessthan normal, age-matched SC cells [38], modi-fying the expression of genes implicated inoutflow obstruction and glaucoma in responseto changes in substrate stiffness, such as theconnective tissue growth factor CTGF [33].Consequently, targeting the SC level of stiffnessand enhancing outflow facility would appear tobe a promising anti-glaucoma strategy. Further,this would appear a logical step: decreasingoutflow resistance at the conventional outflowpathway which is known to be responsible forregulating the majority of aqueous humouroutflow. The effectiveness of several IOP-low-ering therapies that focus on the mechanismsunderlying ECM turnover and cell stiffness hasalready been successfully demonstrated. Theseinclude adenosine A1 receptor agonists [39–45],nitric oxide donors [12, 46–48], Rho Kinaseinhibitors [49] and actin depolymerizers(Tables 1, 2) [23, 25, 26, 50–53].

Adenosine Receptor Agonists

Adenosine is a nucleoside modulator and acommon signaling molecule associated withcellular responses to stressful situations, such aseye ischemia that can lead to a rapid elevation

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of adenosine concentration [54]. In the cell,adenosine is detected in the cytosol; in theextracellular space adenosine is produced fromATP, which gets dephosphorylated to AMP andsubsequently to adenosine [55]. The enzymesinvolved in the production of adenosine areecto-nucleotidases, which are expressed in thechoroid, ciliary body processes and TM [56–58].Elevated ATP levels in the aqueous humourhave been associated with primary angle-clo-sure glaucoma and chronic angle-closure glau-coma [59, 60]. While the mean aqueousadenosine concentration in healthy eyes isaround 200 nM, in ocular hypertension itsconcentration rises to 500 nM, showing a sig-nificant correlation with elevated IOP [59, 61].Studies have shown that the effect of adenosinereceptor stimulation on IOP is species-specific.However, in general, the stimulation of

adenosine A1 receptors lowers IOP in all species[62]. In rabbits, mice, and monkeys, the use ofadenosine A1 agonists has effectively reducedIOP [39–43], generating both a decrease inaqueous flow and an increase in outflow facility[40, 41, 43]. Further, Shearer and Crosson [44]provided convincing evidence for the presenceof adenosine A1 receptors on trabecular cells.These authors concluded that the activation ofthese receptors leads to a rapid secretion ofMMP-2, and supported the notion that theincrease in outflow facility involves the activa-tion of adenosine A1 receptors on TM cells.

In vivo studies in rabbits have shown thatthe topical administration of the relativelyselective A1 adenosine receptor agonistR-phenylsopropyladenosine (R-PIA) produced abiphasic effect, with a 8.4-mm Hg rise at30 min and a maximum IOP reduction of

Table 1 Summary of new drugs included in the review that have been employed in preclinical studies to reduce intraocularpressure in glaucoma by targeting the conventional outflow system

Type Proposed mechanism ofaction

Effect Experimentaldrug

References

Adenosine

receptor

agonists

Stimulation of adenosine

receptors on trabecular cells.

The activation of these

receptors leads to secretion

of matrix-degrading

enzymes.

R-PIA [40, 41]

Trabodenoson [92, 93]

Nitric oxide

donors

Homeostatic signaling

function on trabecular and

Schlemm’s canal cells.

Modulation of cell response

to shear stress during times

of elevated IOP.

NCX 125 [46]

BOL-303259-X [47]

NCX 470 [48]

Rho Kinase

inhibitors

Inhibition of myosin light

chain phosphatase on

trabecular and Schlemm’s

canal cells.

Actin cytoskeletal changes in

cells that influence the

contractile properties of

tissues.

Y-27632 [79, 80]

HA-1077 [81]

H-1152P [82]

Compound 38 [83]

Compound 1y [84]

AMA0076 [85]

K-115 [101]

Actin

polymerization

inhibitors

Actin depolymerizers on

trabecular and Schlemm’s

canal cells.

Cell morphology alterations

that enlarge openings

between cells.

Cytochalasin D [89]

Latrunculin [25, 26, 50, 52, 89]

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5.8 mm Hg 5 h after administration [40]. Thesame was observed in normotensive cynomol-gus monkeys, where a single dose of the ade-nosine receptor agonists R-PIA (100 lg) andCHA (N6-cyclohexyladenosine, 500 lg) resultedin an IOP rise of 4 mm Hg within 30 min,followed by an IOP reduction of 2–3.5 mm Hg2–6 h after administration [41]. Studies in rab-bits and monkeys have explained the biphasiceffect observed in IOP, by a mechanism thatinvolves the activation of both adenosine A1and A2 receptors, the latter being responsiblefor the initial IOP rise. With regard to adverseevents, the use of adenosine receptor agonistscan produce a mild transient anterior chamberflare, this effect being observed in monkeystreated with 250 lg of R–PIA, and indicating apossible breakdown of the blood-aqueous bar-rier [41].

Nitric Oxide Donors

Nitric oxide (NO) is a mediator generatedendogenously from L-arginine by a family ofenzymes named NO synthases (NOS), and sig-naled via the second messenger cGMP [47]. Inhealthy eyes, the aqueous humour outflowpathway and ciliary muscle are sites of NOsynthesis from endothelial NOS. This mecha-nism, however, is implicated in the abnormalIOP homeostasis in primary open-angle glau-coma (POAG) patients as well as in animalglaucoma models [63]. In POAG eyes, NO syn-thase activity is decreased in the TM, SC andciliary muscle tissues suggesting a relationshipwith IOP elevation [64]. In contrast to normaleyes, lower levels of both NO end-products andcGMP are detected in the aqueous humour ofPOAG patients [65]. Although the site of NO

Table 2 Summary of clinical trials included in the review to evaluate new medications targeting the conventional outflowsystem, for the treatment of ocular hypertension in glaucoma

Type Drug Clinical trial identifier Number of subjects References

Adenosine receptor agonists Trabodenoson NCT01123772 70 [94]

NCT01123785 144 [95]

NCT01917383 101 –

NCT02565173 303 –

NCT02829996 165 –

OPA-6566 NCT01410188 160

Nitric oxide donors BOL-303259-X NCT01223378 396 [96]

NCT01749904 421 –

Rho Kinase inhibitors AR-13324 NCT01528787 85 [98]

NCT01731002 213 [99]

PG324 NCT02207491 292 [100]

NCT02674854 Recruiting –

K-115 JAPIC090708 28 [103]

JAPIC111700 205 [104]

– 92 [105]

Actin polymerization inhibitors INS115644 NCT00443924 14 [106]

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activity responsible for increasing conventionaloutflow facility is currently unknown, a numberof studies have proposed that TM cells and SCcells might be involved in this process [66–68].

Ashpole et al. [69] established that humanSC cells respond to shear stress in a similarfashion to that of other vascular endothelia. Intheir study, SC cells reacted to physiologicallevels of shear stress by aligning with thedirection of flow and by increasing productionof NO. Interestingly, these investigators alsofound that SC cells isolated from glaucomatouseyes were either shear-unresponsive, or liftedfrom their substrate in the presence of shearstress, suggesting that NO production by SCcells exhibited a homeostatic signaling functionduring times of elevated IOP, when SC narrowsand shear stress on SC cells increases [69].Accordingly, NO-donating drugs may be ofinterest as they could provide greater and moreefficient IOP control in glaucoma. To date, ithas been demonstrated that NO-donatingcompounds effectively increase conventionaloutflow facility and lower IOP in mice, rabbits,pigs, dogs, monkeys, and humans [46, 67, 70].

Borghi t al. [46] evaluated the use of NCX125, a NO-donating latanoprost acid synthe-sized compound, in the rabbit, dog, and pri-mate experimental glaucoma models. Thehypotensive efficacy obtained with NCX 125was compared with a commercial formulationof latanoprost [46]. Notably, NCX 125 exhibitedgreater IOP-lowering efficacy in all cases. Inrabbits, the effects were maximal between 30and 90 min with an IOP difference versuscommercial latanoprost of 10.1 ± 2.3 mm Hg.The pharmacokinetics of latanoprost-free acidin the aqueous and iris ciliary body after theinstillation of NCX 125 compared with latano-prost did not differ significantly between studygroups. In dogs, the maximum effect occurred4 h after administration of NCX 125, which wassignificantly greater in comparison to thatobserved after latanoprost treatment(-6.7 ± 1.2 vs. -10.6 ± 2.3 mm Hg, respec-tively). In non-human primates, NCX 125therapy reduced IOP by -16.7 ± 2.2 mm Hg(Tmax 240 min), while the equivalent dose oflatanoprost resulted in a smaller decline in IOP(-11.9 ± 3.7 mm Hg, Tmax 360 min).

In a different study with NO-donating lata-noprost, Krauss et al. [47] investigated the effi-cacy of BOL-303259-X in preclinical models ofhypertensive glaucoma. The levels of latano-prost acid were measured in rabbit and primateaqueous humour, corneas, and iris/ciliary bodyfollowing the topical administration ofBOL-303259-X and latanoprost. Levels of lata-noprost acid in all ocular compartments wereelevated to a similar extent regardless of thetreatment received in both rabbits and mon-keys, although the limited sample size in mon-keys (n = 2) did not allow a proper statisticalevaluation of differences in this species. In thesame study, IOP-lowering activity was alsoevaluated in an experimental canine glaucomamodel, in which the peak IOP reduction was-13.2 ± 1.5 mmHg and the effect was evidentfor at least 6 h post-instillation. A similar doseof latanoprost whilst significantly reducing IOPwas not as effective as the NO-donating com-pound (-7.1 ± 1.8 mmHg), and demonstrated aslower onset of action. In laser-induced ocularhypertensive primates, BOL-303259-X was tes-ted with 3.6-lg (0.012%), 9-lg (0.030%) and36-lg (0.12%) dosing. While the lowest dosewas inactive when compared to the respectivevehicle, doses of 9 and 36 lg significantlydecreased IOP with a peak reduction relative tobaseline of -15.2 ± 4.9 and -13.5 ± 3.0 mmHg, respectively. In contrast, the highest IOPreduction obtained with latanoprost, with a30-lg (0.10%) dosing regimen, was still inferiorto that of the novel compound(-11.9 ± 3.8 mm Hg) [47].

In a more recent study, Impagnatiello et al.[48] compared the IOP-lowering activity of NCX470, a novel NO-donating bimatoprost, withequivalent dosage of bimatoprost in ocularhypertensive rabbits, ocular normotensive dogs,and hypertensive non-human primates. Thisnovel compound was effective in lowering IOPin all 3 animal models, and a repeated dailydosing of NCX 470 in dogs resulted in sustainedIOP-lowering activity over time. In general,NCX 470 was around 3 times more potent thanbimatoprost, as the 0.014% dose of NCX 470was as effective as the 0.03% dose of bimato-prost (-4.8 ± 0.5 vs. -3.2 ± 0.9 mm Hg,respectively). The tested NO-donating

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bimatoprost compound lowered IOP progres-sively and reached maximum efficacy between6 and 24 h after administration. In hypertensivenon-human primates, the maximum IOP-low-ering activity was obtained 24 h post-dosing(-6.3 ± 1.7 mm Hg) and was superior to that ofbimatoprost (-2.1 ± 1.6 mm Hg).

To date, cumulative evidence with availableexperimental glaucoma models supports thenotion that the IOP-lowering activity ofNO-donating prostaglandin analogs exceedsthat of commercially available prostaglandinanalogs. This is thought to be owed to theconcomitant action of the NO and pros-taglandin moieties. However, although thetherapeutic efficacy of these compounds seemsbeyond doubt, further research is needed toaddress their potential ocular toxicity and theirclinical efficacy and safety in controlled studiesin humans.

Rho Kinase Inhibitors

Rho Kinases (ROCKs) are serine/theroninekinases that exist as two isoforms, ROCK1 andROCK2, and appear to have several actincytoskeletal-related targets, which directlyinfluence the contractile properties of TM andSC tissues. ROCKs inhibit myosin light chainphosphatase, mediating actin cytoskeletalchanges and induce vasoconstriction [71].Moreover, they stabilize filamentous actin andreduce cell migration by activating LIM kinasesvia phosphorylation [72]. Other targets ofROCKs comprise the phosphoprotein phos-phatase inhibitor CPI-17 and the ezrin/radix-in/moesin family of actin-binding proteins [27].

In humans, ROCK1 and ROCK2 tend to beexpressed in the majority of tissues, includingTM and ciliary muscle cells [73]. The catalyticactivity of ROCKs is activated in response toRho binding as well as binding of certain lipids,such as arachidonic acid, which is able toinduce a five- to sixfold activation of theenzyme [73, 74]. RhoA is a small guanosinetriphosphate GTP-binding protein that belongsto the Rho family and regulates aspects of cellshape, motility, proliferation, and apoptosisthroughout the body [72]. By binding to GTP,

RhoA activates ROCK1 and ROCK2, which sig-nal downstream molecules to polymerize actinfibers in the cardiovascular, pulmonary, andrenal systems [75, 76]. In the eye, RhoA hasbeen detected in the optic nerve of glaucoma-tous eyes, supporting the relationship betweenRho proteins and glaucoma pathophysiology[77]. Therefore, ROCKs may play a role inglaucoma pathogenesis, and Rho Kinase inhi-bitors may become a new therapeutic tool inour efforts to reduce IOP by modifying acto-myosin activity [78].

The effectiveness of RhoA kinase inhibitorY-27632 in increasing aqueous humour outflowfacility was evaluated by Honjo et al. [79]. Incultured human TM cells, Y-27632 successfullyinduced retraction and thinning of cells after30–60 min, as well as a high alteration of F-actindistribution. Outflow facility, IOP anduveoscleral outflow were evaluated after topical,intracameral, and intravitreal administration inJapanese rabbits. By topical administration,authors reported a significant IOP decrease after30 min. Intracameral and intravitreal injectionsprovided significant IOP lowering between 30min and 24 h. Eyes treated with Y-27632 expe-rienced a twofold increase in outflow facility incomparison with control (0.24 ± 0.02 vs.0.12 ± 0.01 lL min-1, respectively). With regardto safety these authors did not observe anteriorchamber, lens, or fundus abnormalities by slitlamp examination for any of the three admin-istration routes employed. In another experi-mental study Rosenthal et al. [80] incubatedsmall TM strips, dissected from freshly enucle-ated bovine eyes, with Y-27632 to evaluate itseffect on the contractility of the tissue. Theinitial contraction produced by endothelin-1and carbachol was significantly reduced with a20-min pre-incubation of the tissue with theROCK inhibitor.

In 2009, Fukunaga et al. [81] evaluated theeffect of the ROCK inhibitor HA-1077 in ananimal model of ocular hypertension. For theevaluation, HA-1077 was dissolved in phos-phate-buffered saline to be instilled on rabbitsat 1, 2 or 3 mM. These authors reported amaximal concentration-dependent IOP reduc-tion that occurred 120 min after the instillation(-17.4%, -27.3% and -46.4%, for 1, 2, and

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3 mM, respectively). Although no abnormalitiesof the anterior chamber or fundus wereobserved, minor conjunctival hyperaemia wasnoticed in a few cases of eyes treated with 3 mMHA-1077.

In a similar study, the hypotensive effect of adifferent ROCK inhibitor, named H-1152P, wasevaluated in normal and hypertensive rabbit eyes[82]. H-1152P significantly reduced both normaland elevated IOP in these animals in a time- anddose-dependent manner. The maximumIOP-lowering effect was observed within60–90 min after the instillation of this compoundand was -3.6 ± 0.9, -5.4 ± 0.7, -6.8 ± 0.7, and-7.2 ± 1.9 mm Hg, respectively, for the 0.1, 1.0,10, and 28 mM dosing of H-1152P. As regards theduration on IOP-lowering action this variedbetween 90 min and 7 h depending on the con-centration employed and was longer with higherconcentrations. No serious side effects wereobserved in ocular tissues, except conjunctivalhyperemia observed in some cases, which disap-peared within 3 h of application.

Following the evidence of IOP-lowering effi-cacy with the so-far tested ROCK inhibitors, thesynthesis of new molecules is under investiga-tion. In that respect, Henderson et al. [83] haverecently published a SAR development aroundtwo hits from a kinase library that led to anumber of ROCK inhibitors with sub-500 nMactivity against ROCK 1 and sub-100 nM activ-ity against ROCK2. One of these compounds,named 38, had a suitable profile for in vivodeterminations similar to the one found forH1152. Compound 38 has demonstrated anaverage IOP reduction of 33% at the 300-lg doseand 37% at the 600-lg dose in a hypertensivemodel of cymologous monkey. The topicaladministration of compound 38 was also asso-ciated with mild hyperemia, but no discharge orswelling were evident with this compound.

In order to improve the pharmaceuticalproperties of ROCK inhibitors, Yin et al. [84]have recently designed a series of urea-basedROCK2 inhibitors with SAR studies performedon the central phenyl ring and on the urea bondof the molecules. Importantly, the introductionof both electron-donating groups and elec-tron-withdrawing groups to the central phenylring, at the position ortho to the urea group,

generated high ROCK2 potency (1 mM orbelow) and high cellular activity. The instilla-tion of one of these compounds, named 1y, onBrown Norway rats, reduced IOP around 7 mmHg compared with that of the vehicle. The IOPreduction was observed from 1 to 4 h andreturned to baseline levels approximately 8 hlater. According to this study, the use of urea-based ROCK inhibitors may offer a promisingoption for the future treatment of glaucoma.However, there is still a need to develop ocularappropriate toxicology studies to evaluate,among others, conjunctival hyperemia, a com-mon adverse event described in long-termtherapies with other ROCK inhibitors. Thishyperemia is believed to derive from a smoothmuscle cell relaxation of conjunctival bloodvessels. It is worth noting that research effortsunder way on the design and synthesis of ROCKinhibitors aim to convert them into a pre-dictable, nontoxic metabolite by controlledmetabolic inactivation. In this sense, Van deVelde and et al. [85] have evaluated the in vivosafety profile and IOP-lowering potency ofAMA0076, a ROCK1 and ROCK2 inhibitor thatcan be inactivated by esterases, thus minimizingits effect on the ocular surface. Studies todetermine the rate of conversion of AMA0076into its functionally inactive metabolite wereperformed in cornea, conjunctiva, sclera, andaqueous humour, confirming that the highestspecific activity was found in cornea, followedby the conjunctiva, sclera, and aqueous humour(4.81, 1.93, 1.25, 0.14 pmol min-1 mg-1,respectively). Maximum IOP reduction com-pared with baseline values after a single dose ofAMA0076 at a concentration of 0.5%, 0.3%, and0.1% was 48 ± 0.32% (p = 0.03), 39 ± 0.41%(p = 0.003), and 23 ± 0.17% (p = 0.0006),respectively. IOP-lowering efficacy lasted 2 hpost-dosing for the 0.5% and 0.3% concentra-tions, and 4 h post-dosing for the 0.1% con-centration. In a hypertensive rabbit model,induced by the injection of a viscous agent inthe anterior chamber of the eye, the topicalapplication of AMA0076 0.3% and 0.5%, com-pletely inhibited IOP elevation (p\0.0001)with minimal conjunctival hyperaemia.

Following the well-documented IOP-lower-ing efficacy of ROCK inhibitors in multiple

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animal models, a number of clinical trials havebeen developed. Further information is pro-vided in the clinical trials section.

Actin Polymerization Inhibitors

Actin comprises a monomeric unit of microfil-aments able to spontaneously polymerize intohelical filaments. It is an integral part of thecytoskeleton and provides mechanical struc-ture, motility, and contributes to a series ofcomplex cellular activities in amoeboid andanimal cells [86]. The use of molecules whichcan inhibit actin polymerization in conven-tional pathway cells is currently of greatresearch interest. Research efforts have concen-trated on exploring novel mechanisms bywhich either the spaces between the inner wallof SC and the trabecular collagen beams can beexpanded, and/or openings between inner wallcells can be enlarged. In this sense, studiesemploying actin-disrupting agents, such ascytochalasans and latrunculin, have shownpromising results [26, 50–52, 87].

Cytochalasans are a diverse group of fungalpolyketide-amino acid hybrid metabolites withdiversified distinctive biological functions. Thebest known property of cytochalasans is thecapping of actin filaments, preventing theirelongation and affecting several actin-depen-dent cellular events, such as cell morphogene-sis, motility, and endocytosis [88]. Latrunculinsare macrolides isolated from the marine spongeLatrunculia magnifica that sequester monomericG-actin, leading to the disassembly of actin fil-aments and morphological changes in cells [50].Sanka et al. [89] investigated the activity ofcytochalasin D and latrunculin A on MMP-2activation in cultured primary human conflu-ent TM cells. Cells treated with both agentsdemonstrated the greatest degree of cell mor-phology alterations, with rounding-up of cells,cell–cell separation, and cell detachment, aswell as amendments in staining patterns forF-actin. A dramatic effect on MMP-2 activationwas also observed by these authors and wasattributed to a reduction of the MMP-2 proform.It has also been suggested that there is a rela-tionship between substrate compliance and cell

actin polymerization that might influence theresponse of human TM to latrinculin-B [90]. Innon-human primates, the topical administra-tion of latrinculin-B considerably increasedoutflow facility by 123 ± 67% and 272 ± 45%,respectively with the 0.8- and 4.0-lg doses overa 90-min period [91]. Further studies incynomolgus monkeys and human enucleatedpostmortem eyes demonstrated latrunculin B tosignificantly enhance the conventional outflow[25, 26, 50, 52].

As demonstrated by a number of investiga-tors, the use of actin depolymerizers results in ameaningful enhancement in outflow activityand significant improvement in effective filtra-tion area compared with the controls. Thesefindings have been corroborated in vivo byobserving a meaningful IOP reduction after thetopical administration of latrinculin-B innon-human primates. Subsequent to theseexperimental results, clinical trials have beeninitiated evaluating the safety, tolerability, andIOP-lowering efficacy of these actin depoly-merizers in patients with ocular hypertension,or early POAG. For further information, see thenext section.

PHASE I–III CLINICAL TRIALSON NOVEL IOP-LOWERINGCOMPOUNDS

This section is focused on novel, ocularIOP-lowering compounds that are active inPhases I to III of glaucoma clinical trials withinthe past 2 years (until July 2016). More specifi-cally, we briefly examine IOP-lowering thera-pies that focus on the mechanism of theconventional aqueous outflow in relation toECM turnover and cell stiffness in the TM andSC (Table 2). Databases used in these searchesinclude published articles, meeting abstractsand Clinical Trial Registry (http://www.clinicaltrials.gov).

Adenosine Receptor Agonists

Trabodenoson is a highly selective adenosinemimetic targeting the A1 receptor with the

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potential to lower IOP by increasing conven-tional outflow facility directly at the TM level.The administration of this novel molecule hasbeen shown to significantly increase aqueoushumour outflow facility in Dutch-Belted rab-bits, ocular normotensive and hypertensiveNew Zealand white rabbits, and in isolatedperfused porcine anterior segments [92, 93].Subsequently, Laties et al. [94] published thetolerability and safety profile of different dosesof trabodenoson in a randomized, dou-ble-masked, placebo-controlled dose escalationstudy, in which the drug was topically appliedin healthy volunteers (aged 35–76 years) indoses varying from 200 to 3200 lg [94]. Overallresults were very encouraging with minimalocular adverse events encountered that lastedless than 24 h and were generally mild inintensity and self-limiting. Although no ante-rior chamber inflammation or changes in visualacuity were noted in any of the normal subjects,the bilateral topical application of trabo-denoson once daily beginning at 200 lg in 1 eyeand 1600 lg in the fellow eye—with dose esca-lation every other day thereafter—was associ-ated with a greater incidence of adverse eventscompared with placebo (50 vs. 25%, respec-tively). Further analysis indicated that there wasno tissue accumulation with repeated dosing,supporting continued clinical development oftrabodenoson to confirm and better character-ize its safety and efficacy profile in a sufficientsample of patients with POAG and ocularhypertension. Of particular interest in thefuture will be the effectiveness of this novelIOP-lowering compound in secondary glauco-mas with worse 24-h IOP characteristics thanPOAG caused by a functional blockade of theconventional outflow system (e.g. exfoliative orpigmentary glaucoma). The direct effect of tra-bodenoson upon the conventional outflowfacility implies that the efficacy potential of thisadenosine receptor agonist may be greater inthese glaucomas compared with POAG.

In 2016, Myers et al. [95] in a randomized,double-marked, placebo-controlled, dose esca-lation study reported the efficacy and safety of 4trabodenoson doses (50, 100, 200, and 500 lg)administered twice daily over 14 or 28 days insubjects with ocular hypertension, or POAG.

There were few treatment-related adverseevents, mainly ocular and conjunctival hyper-emia detected in 16 subjects treated with tra-bodenoson (18.8%), of whom 10 subjectsexhibited mild and 6 moderate hyperemia. Thehighest dose (500 lg) was associated with thehighest incidence of hyperemia, both beforeand after randomization. Consequently, it wassuggested that the hyperemia may have been apre-existing pathology rather than an effect oftrabodenoson therapy. With regard to efficacy,a dose-dependent reduction in IOP was identi-fied with trabodenoson. For the 500 lg dosinggroup at day 28, the IOP reduction rangedbetween -3.5 and -5.0 mm Hg (mean reduc-tion of -4.1 mm Hg), compared to a reductionbetween -1.0 and -2.5 mm Hg (mean reduc-tion of -1.6 mm Hg) for the correspondingplacebo-treated group. Moreover, IOP loweringseemed to improve with longer exposure totherapy; IOP reduction at day 29 was signifi-cantly greater than that seen at day 14(p = 0.0163). Importantly, in the 500 lg dosinggroup, trabodenoson’s IOP-lowering efficacylasted at least 24 h after the last dose. Never-theless, the 24-h efficacy of trabodenosonremains to be elucidated.

In 2015, Inotek Pharmaceuticals completed aPhase II study evaluating the adjunctive efficacyof trabodenoson to latanoprost in subjects witheither ocular hypertension, or POAG (clinicaltrial identifier NCT01917383). To date, theseresults have not being published. In 2016, thesame company started recruiting participantsfor a Phase III study (clinical trial identifierNCT02565173) that involved the topical appli-cation, in both eyes, of trabodenoson oph-thalmic formulation 3.0% or 6.0% once per day,or trabodenoson 4.5% administered twice dailyin adults with ocular hypertension, or POAG,with the purpose of assessing the efficacy, tol-erability, and safety of trabodenoson treatmentfor 12 weeks, in comparison with timolol, usedas active control in the study. At the same time,they have also started recruiting participants fora Phase II study to evaluate the overall benefit/risk profile of bilateral, once-daily topicalapplication of fixed-dose combinations of tra-bodenoson (3.0% and 6.0%) and latanoprost(0.05% or 0.0025%) in adults with ocular

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hypertension or POAG (clinical trial identifierNCT02829996).

In a separate research approach, Acucela Inc.and Otsuka Pharmaceuticals Co. have per-formed a Phase I–II study to evaluate the safety,tolerability, pharmacokinetics, and IOP-lower-ing efficacy of the adenosine A2 receptor ago-nist OPA-6566, in a dose-escalation study insubjects with ocular hypertension, or POAG(clinical trial identifier NCT01410188). Nostudy results are currently available.

Nitric Oxide Donors

A phase II study to assess the efficacy and safetyof latanoprost bunod, a NO-donating pros-taglandin F2a receptor agonist, has recentlybeen published by Weinreb et al. [96]. Theclinical trial, named the VOYAGER study (clin-ical trial identifier NCT01223378), was a ran-domized, investigator-masked, parallel-group,dose-ranged study designed to compare theefficacy and safety of 4 different concentrationsof latanoprost bunod ophthalmic solutionagainst a commercially available formulation oflatanoprost 0.005% ophthalmic solution, inpatients diagnosed with open-angle glaucoma(including pigmentary or exfoliative glaucoma),or ocular hypertension. A total of 396 subjectswere randomized and completed this study (76on latanoprost bunod 0.006%; 81 on latano-prost bunod 0.012%; 80 on latanoprost bunod0.024%; 80 on latanoprost bunod 0.040%; and79 on latanoprost 0.005%). No difference intreatment effect between equivalent molarconcentrations of either drug was detected. Adose-dependent increase in efficacy wasobserved with latanoprost bunod beyond the0.006% dose and up to the 0.024% dose,revealing the dose–response curve for theNO-donating moiety of latanoprost bunod. Ingeneral, IOP reduction was significantly greaterwith latanoprost bunod compared with com-mercially available latanoprost. The safetyassessment indicated that latanoprost bunod atconcentrations between 0.006% and 0.040%,dosed once daily for a period of 28 days, waswell tolerated but was associated with a higherincidence of ocular adverse events. The most

common adverse events encountered wereocular pain upon instillation and hyperemia,which were both mild to moderate in severity.

Latanoprost bunod entered a Phase 3 clinicaltrial, sponsored by Baush and Lomb that fin-ished in 2015 (clinical trial identifierNCT01749904). In July 2016, the FDA did notidentify any efficacy or safety concerns withrespect to the New Drug Application for lata-noprost bunod 0.024% ophthalmic solution. Ifapproved, latanoprost bunod 0.024%, formula-tion licensed by Nicox to Bauch and Lomb,would be the first once-daily nitric oxide-do-nating prostaglandin analog in the marketwhich will lower IOP by increasing aqueoushumour outflow through both the conven-tional (TM and SC) and the unconventional(uveoscleral pathway) outflow systems.

Rho Kinase Inhibitors

AR-13324 (netarsudil) is a new class of ocularhypotensive compounds that inhibits both RhoKinase and the norepinephrine transporter.AR-13324 seems capable of obtaining a signifi-cant IOP reduction in both rabbits and mon-keys, with a longer duration of action thanreported for previously characterized ROCKinhibitors and a mechanism of action thatcombines an increase in outflow facility (byROCK inhibition) and a concomitant decreasein aqueous humour production (by nore-pinephrine transporter inhibition) [97].

In 2012, Aerie Pharmaceuticals sponsoredthe first study in patients with elevated IOP thatreceived ophthalmic solutions of AR-13324(netarsudil), at 3 different concentrations(0.01%, 0.02% and 0.04%), dosed once daily fora period of 7 days (clinical trial identifierNCT01528787). Netarsudil achieved a signifi-cant gradual IOP reduction for a period of 8 hafter administration and its effect lasting forover 24 h. Researchers detected a dose-relatedocular hyperemia that declined in incidenceand severity with repeated dosing [98].

More recently, Bacharach et al. [99] docu-mented the hypotensive efficacy and safety of0.01% and 0.02% netarsudil, compared withlatanoprost ophthalmic solution used as

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positive control, in a 28-day study with PMdosing in subjects with either open-angle glau-coma, or ocular hypertension. On day 14, meandiurnal IOP decreased from 25.8 mm Hg,25.6 mm Hg and 25.5 mm Hg to 19.8 mm Hg,19.5 mm Hg and 18.4 mm Hg in the 0.01%netarsudil, 0.02% netarsudil and 0.005% lata-noprost groups, respectively. On day 28, netar-sudil did not meet the criteria fornon-inferiority compared to commerciallyavailable latanoprost. Latanoprost and 0.02%netarsudil were statistically similarly effective atall timepoints in the subset of patients withbaseline IOPs B26 mm Hg, demonstrating areduction in mean diurnal IOP of 7.7 and5.6 mm Hg, respectively. The most frequentlyreported drug-related event was conjunctival/ocular hyperemia, which was more commonwith netarsudil: its incidence was 52% (39/75),57% (41/72), and 16% (12/77) in the netarsudil0.01%, netarsudil 0.02%, and latanoprostgroups, respectively (p\0.001). Slit-lampexamination conducted during safety visits onday 7 observed mild, or moderate conjunctivalhyperemia in 28% (21/74) and 35% (25/72) ofpatients in the netarsudil 0.01% and 0.02%treatment groups, respectively, in contrast toonly 4% (3/76) of patients in the latanoprost0.005% group. Overall, conjunctival hyperemiawas seen in more than half of the patientstreated with netarsudil, but it was described bythese investigators as transient due to the lowerincidence scored by slit-lamp examination inthe morning compared to the greater incidencereported by patients after dosing in the evening.However, this study did not provide informa-tion on the long-term efficacy of netarsudil, itsefficacy in patients with a corneal thicknessgreater than 600 mm, or in patients with otherforms of glaucoma than open-angle glaucoma.These should be investigated in the future.

Aerie Pharmaceuticals has also evaluated theuse of PG324 ophthalmic solution, a fixedcombination of netarsudil 0.01%, or 0.02% andlatanoprost 0.005%. A total of 292 patients wererandomized to receive: (1) PG324 0.01%, (2)PG324 0.02%, (3) netarsudil 0.02%, or (4)commercially available latanoprost 0.005%[100]. In this study, both PG324 0.02%(p\0.0001) and PG324 0.01% (p = 0.0071 and

0.002) fixed combinations met the criterion forstatistical superiority versus the two monother-apies tested (latanoprost and netarsudil 0.02%).All four treatments provided statistically(p\0.0001) and clinically meaningful IOPdecreases (-6.2 to -9.1 mm Hg) in mean diur-nal IOP from untreated baseline. Moreover,PG324 0.02% fixed combination obtained meanIOP between 15.6 and 17 mm Hg across all tes-ted time points compared to mean IOP between17.7 and 19.6 mm Hg provided by latanoprost.Thus, the novel PG324 0.02% fixed combina-tion was superior to latanoprost monotherapyby 1.6–3.2 mm Hg. The greater IOP-loweringeffect for PG324 persisted 36 h after last dosingwith a difference of 2.2 mm Hg in favor ofPG324 on day 30. The most frequently reportedadverse event was mild and transient conjunc-tival hyperemia with an incidence of 41% and40% for PG324 0.01% and PG324 0.02%,respectively. In 2016, Aerie Pharmaceuticalsinitiated a sponsored phase III study to evaluatethe ocular hypotensive efficacy and safety of thenew fixed combination compared to netarsudilophthalmic solution 0.02% and latanoprostophthalmic solution 0.005% in patients withopen-angle glaucoma or ocular hypertension(clinical trial identifier NCT02674854).

It is noteworthy that, in the last 2 years,another ROCK inhibitor has emerged as a ther-apeutic molecule with potential to become afuture topical antiglaucoma therapy. This K-115compound, named ripasudil, has become thefirst topical ROCK inhibitor developed for thetreatment of glaucoma or ocular hypertensionin Japan. Initially, ripasudil demonstrated ameaningful IOP-lowering effect in rabbits andmonkeys [101]. Subsequently, phase I clinicalstudies showed its efficacy in healthy adultvolunteers [102]. Furthermore, Tanihara et al.[103] conducted a multicenter, prospective,randomized, crossover study for the estimationof its IOP-lowering efficacy over 24 h in patientswith either ocular hypertension or POAG. Theseauthors enrolled 43 patients who receivedripasudil 0.2%, ripasudil 0.4%, or placebo inboth eyes twice a day for 3 periods separated by5–30 days. They established statistically signifi-cant IOP reductions from baseline for bothdosages of ripasudil and for placebo at all

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time-points except at 24 h after the first instil-lation (12 h after the second instillation) for theplacebo group. The IOP lowering obtained var-ied between -2.0 and -4.1 mm Hg in the pla-cebo group, -5.2 and -6.8 mm Hg in the 0.2%ripasudil group, and -6.4 and -7.3 mm Hg inthe 0.4% ripasudil group, respectively, at 2 hafter the first and the second instillation [103].Furthermore, differences in maximum IOPreductions between placebo and ripasudil-ad-ministered patients were -3.4 and -2.9 mm Hgin 0.2% ripasudil group and -4.4 and -3.3 mmHg in the 0.4% ripasudil group, respectively,after the first and the second instillation. Con-junctival hyperemia was detected in 3 (11%), 22(79%) and 27 (96%) of the 28 patients, in pla-cebo, ripasudil 0.2% and ripasudil 0.4%,respectively. In most cases, however, there wasonly mild to moderate conjunctival hyperemiaappearing within 1 h after instillation and onlybeing evident for 1–3 h.

In a phase III study, Tanihara et al. [104]conducted a 56-day, multicenter, randomized,placebo-controlled, double-masked, parallelgroup comparison to evaluate additiveIOP-lowering effects and safety of ripasudil0.4%, in combination with timolol 0.5%, orlatanoprost 0.005%, in patients with ocularhypertension or POAG [104]. Patients wererandomly assigned to two groups and treatedwith ripasudil 0.4% or placebo in addition totimolol 0.5% or latanoprost 0.005%. The meanbaseline IOP at 0900 hours for the populationanalyzed was 19.8 ± 1.8 and 19.8 ± 1.9 mm Hgin the ripasudil–timolol and ripasudil–la-tanoprost studies, respectively.

Two hours after instillation (1100 hours)mean IOP reductions for ripasudil and placebowere -2.9 and -1.3 mm Hg and -3.2 and-1.8 mm Hg, respectively, in the ripasudil–ti-molol and the ripasudil–latanoprost studies.Statistically significant IOP differences betweenripasudil and placebo were only detected in theripasudil–timolol study.

The most frequent adverse event describedwith these fixed combinations was conjunctivalhyperemia, with incidence rates of 65.4% and55.9%, respectively, in the ripasudil–timololand ripasudil–latanoprost studies. In almost allcases, the investigators considered hyperemia to

be drug-related. Even though hyperemia wasobserved in most cases after every instillation, itshould be noted that hyperemia was mild in allcases. Further, in most cases, hyperemiaresolved prior to the next instillation (clinicaltrials Japanese identifiers JAPIC111700 andJAPIC111701).

The use of ripasudil was also evaluated in 92glaucoma patients who could not be controlledmedically despite the use of maximal toleratedmedical therapy [105]. A total of 43 subjectswith POAG, 28 with normal-tension glaucoma,10 with secondary glaucoma, 7 with exfoliativeglaucoma, and 4 with developmental glaucomareceived ripasudil as adjunctive therapy. Themean pre-administration IOP and mean per-centage of IOP reduction at the last follow-upwere 19.7 ± 4.9 mm Hg and 6.5 ± 17.0% forPOAG, 15.5 ± 2.0 mm Hg and 2.3 ± 10.4% fornormal-tension glaucoma, 22.8 ± 8.3 mm Hgand 19.1 ± 13.5% for secondary glaucoma,22.5 ± 4.4 mm Hg and 2.1 ± 14.5% for exfolia-tive glaucoma, and 20.2 ± 8.9 mm Hg and11.4 ± 23.1% for developmental glaucoma,respectively.

Actin Polymerization Inhibitors

As previously discussed, the administration oflatrinculin-B alters the structural geometry ofthe TM, by inducing a reduction in cell–cell andcell–matrix adhesion, along with impairment ofcellular contractility. Consequently, outflowfacility is enhanced by a relaxation of the TMand SC, which expands the area available forfluid outflow and reduces resistance to fluidflow across the system [26, 50–52, 91]. Ras-mussen et al. [106] evaluated the administrationof latrinculin-B in 14 subjects diagnosed withocular hypertension or early POAG in a phaseI-II study, sponsored by Merck Sharp & DohmeCorp. and developed by Inspire Pharmaceuticals(clinical trial identifier NCT00443924). Latrin-culin-B ophthalmic solution (designated asINS115644) was evaluated in four concentra-tions: 0.005%, 0.01%, 0.02% and 0.05%. Fivesingle-dose instillations of INS115644 separatedby approximately 12 h were given over a periodof 3 days to evaluate safety, tolerability, and

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efficacy in a multicenter, double-masked, ran-domized, placebo-controlled, ascending dosestudy. The entry requirements for IOP wereuntreated IOP of 22–30 mm Hg at 8 AM (\4 mmHg difference between eyes). Following the firstinstillation, 0.02% INS115644 reduced IOP by3.8 ± 2.25 mm Hg (p = 0.002), and 0.05%INS115644 by 3.9 ± 3.1 mm Hg (p = 0.004)from baseline at 4 h post instillation. After thefifth instillation (day 3), IOP decreased frombaseline by 5.4 ± 2.4 mm Hg (p = 0.004), a 24%reduction in IOP, in the 0.02% cohort and2.8 ± 2.7 mm Hg (p = 0.02), a 12% reduction, inthe 0.05% cohort at 4 h post instillation.INS115644 did not significantly lower IOP inthe treated eyes compared with contralateral,placebo-control eyes at the two lowest dosestested (0.005% and 0.01%).

Regarding safety evaluation, adverse eventsconsisted mainly of mild ocular redness andirritation, although transient changes in centralcorneal thickness were noted 4 h post-instilla-tion. The largest change was an increase ofB2.5% at the 0.05% dose, evaluated by authorsas not statistically significant.

FUTURE DIRECTIONS

As a new strategy for the management of ele-vated IOP adenosine receptor agonists, nitricoxide donors, Rho Kinase inhibitors, and actinpolymerization inhibitors have all been repor-ted to offer a meaningful experimental andclinical IOP reduction, which may facilitateglaucoma management. The main drawback ofcurrent antiglaucoma medications is that theyreduce IOP either by decreasing the synthesis ofaqueous humor or by enhancing unconven-tional outflow facility. In contrast, the agentsdiscussed in the present review act on the con-ventional aqueous humour outflow route. Theyinduce structural or functional changes in theTM, SC, or both these tissues by a cascade ofcomplex biological mechanisms that by andlarge require further elucidation. It is eminentlylogical to target the TM and SC with futuretherapies as these are the sites of functional oranatomical resistance in glaucoma. However, tosuccessfully develop and commercialize agents

that specifically target these tissues, there is aneed first to better understand the molecularchanges they induce in glaucomatous cells andthe responses to substrate stiffness, as well asthe mechanics of the pore formation or con-tractility in SC cells. Progress in these importantareas will facilitate adoption of currently testeddrugs and will facilitate the development of newmolecules.

It should be noted that, over the years, sev-eral candidate drugs targeting SC have failedduring clinical testing, usually due to insuffi-cient efficacy (e.g. candidate drugs Y39983,AR-12286, DE-104, ATS907), or due to safetyand tolerability concerns in human subjects(e.g. candidate drugs INS117548, INS-115644);[107]. Nevertheless, certain molecules describedhere have already provided promising experi-mental data and initial clinical results demon-strating potential as future glaucoma therapies(e.g. trabodenoson, OPA-6566, LTB, netarsudil,ripasudil, latrunculin-B). Nevertheless, there is aneed for more comprehensive efficacy evidence(dosing studies, long-term efficacy, 24-h effi-cacy, efficacy in other glaucomas). Further,more conclusive evidence is needed with largerstudies that should assess long-term safety andtolerability. Initial results to date suggest thatthese agents, although promising, may stillneed improvements in terms of formulation,delivery mechanisms and administration tomeet desirable safety and efficacy requirements.However, the overall positive benefit-to-riskratio and their novel mechanisms of action(targeting conventional outflow and especiallySC) should enable some of them to be launchedcommercially and to become successfuladjunctive therapies in glaucoma management.Additional, well-designed, controlled clinicaltrials are a vital prerequisite for their furtherdevelopment and ultimate clinical success.

ACKNOWLEDGEMENTS

No funding or sponsorship was received for thepublication charges of this article. All namedauthors meet the International Committee ofMedical Journal Editors (ICMJE) criteria for

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authorship for this manuscript, take responsi-bility for the integrity of the work as a whole,and have given final approval for the version tobe published. The authors would like toacknowledge the financial support from theSanitary Research Institute of the San CarlosClinical Hospital, and the Ocular PathologyNational Net OftaRed of the Institute of HealthCarlos III.

Disclosures. Professor A.G. Konstas hasreceived research support from Alcon, Allerganand Santen; honoraria from Alcon, Allergan,Mundipharma, Santen and had congressexpenses covered by Alcon, Allergan, SantenVianex. Authors V. Andres-Guerrero and J.Garcıa-Feijoo have nothing to disclose.

Compliance with Ethics Guidelines. Thisarticle is based on previously conducted studies,and does not involve any new studies of humanor animal subjects performed by any of theauthors.

Data Availability. Data sharing is notapplicable to this article as no datasets weregenerated or analyzed during the current study.

Open Access. This article is distributedunder the terms of the Creative CommonsAttribution-NonCommercial 4.0 InternationalLicense (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommer-cial use, distribution, and reproduction in anymedium, provided you give appropriate creditto the original author(s) and the source, providea link to the Creative Commons license, andindicate if changes were made.

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