This continuing education activity is supported through an unrestricted educational grant from Bausch & Lomb Incorporated.

THE ROLE OFNitric Oxide

IN GLAUCOMA

FACULTY

Murray Fingeret, OD (Co-Chair)

Ben Gaddie, OD (Co-Chair)

Louis R. Pasquale, MD, FARVO

W. Daniel Stamer, PhD

Distributed with

Administrator

Sponsored by

CE MONOGRAPH

ORIGINAL RELEASE: OCTOBER 1, 2017

EXPIRATION: OCTOBER 31, 2018

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COPE approved for 2.0 credits for optometristsCOPE Course ID: 55170-GLCOPE Course Category: Glaucoma

2

LEARNING METHOD AND MEDIUMThis educational activity consists of a supplement and twenty (20) study questions. The participant should, in order, read the learning objectives contained at the beginning of this supplement, read the supplement, answer all questions in the post test, and complete the Activity Evaluation/Credit Request form. To receive credit for this activity, please follow the instructions provided on the post test and Activity Evaluation/Credit Request form. This educational activity should take a maximum of 2.0 hours to complete.

CONTENT SOURCEThis continuing education (CE) activity captures content from a regional dinner meeting series.

ACTIVITY DESCRIPTIONDespite the plethora of therapeutic options currently used to lower intraocular pressure (IOP) in patients with glaucoma, disease progression occurs with the increased risk of vision loss. Novel therapies with unique mechanisms of action are being developed, which may serve as alternatives or adjuncts to current therapies. One new therapeutic target for glaucoma is the role of nitric oxide and its metabolic pathway in regulating IOP. The purpose of this activity is to update optometrists on the role of nitric oxide in the management of glaucoma.

TARGET AUDIENCEThis educational activity is intended for optometrists.

LEARNING OBJECTIVESUpon completion of this activity, participants will be better able to:• Discuss the role of nitric oxide in IOP regulation• Describe the mechanism of action of current and emerging topical glaucoma therapies• Evaluatetheclinicalrelevanceofsafetyandefficacydatafor emerging topical therapies for the treatment of glaucoma

ACCREDITATION STATEMENTCOPE approved for 2.0 CE credits for optometrists. COPE Course ID 55170-GLCOPE Course Category: Glaucoma

Administrator:

DISCLOSURESMurray Fingeret, OD, hadafinancialagreementoraffiliationduring the past year with the following commercial interests in the form of Consultant/Advisory Board: Aerie Pharmaceuticals, Inc; Alcon; Allergan; Bausch & Lomb Incorporated; Carl Zeiss Meditec, Inc; Heidelberg Engineering; and Topcon Medical Systems, Inc.

Ben Gaddie, OD, hadafinancialagreementoraffiliationduringthe past year with the following commercial interests in the form of Consultant/Advisory Board: Abbott Medical Optics; Aerie Pharmaceuticals, Inc; Akorn, Inc; Alcon; Allergan; Bausch & Lomb Incorporated; Reichert, Inc; Shire; and TearScience.

Louis R. Pasquale, MD,hadafinancialagreementoraffiliationduring the past year with the following commercial interests in the form of Consultant/Advisory Board: Eyenovia; Honoraria from promotional, advertising or non-CME services received directly from commercial interests or their Agents (eg, Speakers Bureaus): Bausch & Lomb Incorporated.

W. Daniel Stamer, PhD,hadafinancialagreementoraffiliationduring the past year with the following commercial interests in the form of Consultant/Advisory Board: Glauconix, Inc; and Precision Biosciences; Contracted Research: Aerie Pharmaceuticals, Inc; Allergan; Inotek Pharmaceuticals Corporation; Ironwood Pharmaceuticals, Inc; and Precision Biosciences.

EDITORIAL SUPPORT DISCLOSURESTony Realini, MD, MPH, hadafinancialagreementoraffiliationduring the past year with the following commercial interests in the form of Consultant/Advisory Board: Aerie Pharmaceuticals, Inc; Alcon; Bausch & Lomb Incorporated; Inotek Pharmaceuticals Corporation; Intelligent Retinal Imaging Systems; New World Medical, Inc; and Smith & Nephew.

Diane McArdle, PhD; Cynthia Tornallyay, RD, MBA, CHCP; and Michelle Ong have no relevant commercial relationships to disclose.

DISCLOSURE ATTESTATIONThe contributing physicians listed above have attested to the following:1) thattherelationships/affiliationsnotedwillnotbiasor otherwiseinfluencetheirinvolvementinthisactivity;2) that practice recommendations given relevant to the companieswithwhomtheyhaverelationships/affiliations will be supported by the best available evidence or, absent evidence, will be consistent with generally accepted medical practice; and3) that all reasonable clinical alternatives will be discussed when making practice recommendations.

PRODUCT USAGE IN ACCORDANCE WITH LABELINGPleaserefertotheofficialprescribinginformationforeachdrug discussed in this activity for approved indications, contraindications, and warnings.

GRANTOR STATEMENTThis continuing education activity is supported through an unrestricted educational grant from Bausch & Lomb Incorporated.

TO OBTAIN CE CREDITWeofferinstantcertificateprocessingandsupportGreenCE.Please take this post test and evaluation online by going to http://tinyurl.com/NOglaucoma. Upon passing, you will receive yourcertificateimmediately.Youmustanswer14outof20questions correctly in order to pass, and may take the test up to 2 times. Upon registering and successfully completing the post test,yourcertificatewillbemadeavailableonlineandyoucanprintitorfileit.Pleasemakesureyoutaketheonlineposttestand evaluation on a device that has printing capabilities. There are no fees for participating in and receiving CE credit for this activity. DISCLAIMERThe views and opinions expressed in this educational activity are those of the faculty and do not necessarily represent the views of TheStateUniversityofNewYorkCollegeofOptometry,MedEdicusLLC, Bausch & Lomb Incorporated, or Optometry Times.

Cover photograph courtesy of Murray Fingeret, OD.This CE activity is copyrighted to MedEdicus LLC ©2017. All rights reserved.

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FACULTY

Murray Fingeret, OD (Co-Chair)Clinical Professor

State University of New York College of OptometryNew York, New York

Ben Gaddie, OD (Co-Chair)Owner and Director

Gaddie Eye CentersLouisville, Kentucky

Louis R. Pasquale, MD, FARVOProfessor of Ophthalmology

Harvard Medical SchoolDirector, Glaucoma Service

Massachusetts Eye and EarBoston, Massachusetts

W. Daniel Stamer, PhDJoseph A. C. Wadsworth

Professor of OphthalmologyProfessor of Biomedical Engineering

Duke UniversityDurham, North Carolina

INTRODUCTION

Currently, many therapies lower intraocular pressure (IOP) and prevent progressive vision loss from glaucoma. The availability of highly effective medications with excellentsafetyprofilesandconvenientdosingpermitsthe development of treatment regimens that are personalized to the needs and desires of each individual patient with this disease. Despite this plethora of therapeutic options, some people with glaucoma continue to develop visual loss and dysfunction attributable to the disease. An unmet need remains for novel therapies with unique mechanisms of action for use as alternatives and adjuncts to current therapies in achieving IOP reduction and progression prevention. The role of nitric oxide (NO) and its metabolic pathway as a potential new therapeutic target for glaucoma are emerging as hot topics in glaucoma. Nitric oxide plays a key role in the regulation ofconventional(alsocalledtrabecular)outflow,1siteofglaucomatousoutflowimpairment.Thus,targetingtheNOpathway provides an opportunity to treat glaucoma at its site of pathology. Herein, the NO pathway, its role in IOP regulation, and early clinical data on a new NO-based therapy for glaucoma, latanoprostene bunod (LBN), are discussed.

OCULAR HYPERTENSION IN GLAUCOMA: A TRABECULAR OUTFLOW PROBLEM

Primary open-angle glaucoma (POAG) is a chronic, progressive optic neuropathy, in which IOP is often elevated.1 Along with age, elevated IOP is a primary risk factor for POAG. Elevated IOP is also a causal factor in the pathophysiology of glaucoma. Many studies have demonstrated that reduction of IOP reduces the risk of disease progression.2-4

Intraocular pressure is primarily regulated through the circulation of aqueous humor in the eye. Aqueous humor production and drainage is a dynamic process. Aqueous

THE ROLE OFNitric Oxide

IN GLAUCOMA

4

humor is produced by the epithelium of the ciliary processesoftheciliarybody,flowsfromtheposteriorchamber through the pupil to the anterior chamber, and then exits the globe primarily through the trabecular meshwork(TM)oftheconventionaloutflowpathway,with a small component exiting through the uveoscleral pathway.5,6Trabecularoutflowisalsoopposedby episcleral venous pressure, a source of distal resistancetooutflow.Theuveoscleraloutflowpathwayis incompletely understood and involves passage of aqueous humor through the face of the ciliary body via theextracellularmatrix–filledspacebetweentheciliarymuscle bundles into the suprachoroidal space, which then exits the eye via several routes, including choroidal veins and ocular lymphatics. The balance of aqueous humor production and egress by both the trabecular and uveoscleral routes determines the level of IOP. Intraocular pressure can be altered by changing either the rate of aqueous production or its drainage.

In POAG, IOP is elevated because of increased resistancetoflowthroughtheTM.TheTMconsistsof3 distinct layers. Innermost is the uveal layer, then the corneosclerallayer,andfinallythejuxtacanalicularlayerlocated adjacent to Schlemm canal. It is this outermost layer, the juxtacanalicular layer, that is the site of aqueous humoroutflowresistance,bothinnormaleyesand,toagreater extent, in glaucomatous eyes.7 The reasons for increasedTMoutflowresistanceineyeswithglaucomaare as yet poorly understood.

CURRENT GLAUCOMA MEDICATIONS MISS THE MESHWORK

There are currently 5 main classes of IOP-lowering medications. Each works by altering 1 or more aspects ofaqueoushumorflowthroughtheeye.

The beta blockers and carbonic anhydrase inhibitors reduce the rate of aqueous production. Prostaglandin analogues(PGAs)increaseoutflowprimarilythroughthe uveoscleral pathway. The alpha-adrenergic agonists lower IOP by a dual mechanism: primarily by reducing aqueous humor production and secondarily by increasing uveoscleraloutflow.Noneofthesedrugs,however,worksprimarilyatthesiteofoutflowimpairment—theTM.Themioticclassofdrugsdoesincreasetrabecularoutflow,but only indirectly through actions on the ciliary muscle

(and not through any direct effects on the TM itself); this class of drugs is generally poorly tolerated and has only limited use in modern practice.

There remains an unmet need for an IOP-lowering medicationthatworksattheTM—themainsiteofoutflowobstructioninglaucomatouseyes.LBNisanIOP-lowering drug in late-stage clinical development thatmaybethefirsttotargettheTMdirectly.LBNisaprodrug that is metabolized to latanoprost and an NO molecule by ocular esterase enzymes.8 Latanoprost is a PGAandlowersIOPbyenhancinguveoscleraloutflow.Nitric oxide, a novel therapy for glaucoma, lowers IOP by directlyimprovingtrabecularoutflowviadirecteffectsonTM cells.

HISTORY OF NITRIC OXIDE AND ITS ROLES IN HUMAN PHYSIOLOGY

JosephPriestleyfirstdiscoveredNOinthe1770s.9 The molecule was not immediately recognized as important to human health and was generally considered a toxic gasandairpollutant.Nitroglycerin—akeycomponentofdynamite—wasfoundtorelieveanginapectoris,akeysymptomofcardiacischemia.Thisbeneficialresponse is mediated by nitroglycerin’s well-known vasodilatory effects, which occur through relaxation of smooth muscle in the walls of both arteries and veins. The mechanism by which nitroglycerin and other nitrates produce this vasodilation was not fully appreciated until 1977, when it was discovered that nitrates liberate NO, which in turn produces the vasodilation. A decade later, in 1987, endogenous NO synthesis was demonstrated in endothelial cells.10 Soon thereafter, NO was rapidly recognized to have important regulatory roles in many biologic systems, including the cardiovascular and neurologic systems. Just 5 years later, the American Association for the Advancement of Science (publisher of Science)namedNOthe1992“MoleculeoftheYear.”11 The research that promoted this molecule from pollutant to“MoleculeoftheYear”in15shortyearsearned3 scientists the 1998 Nobel Prize in Medicine to honor their work in demonstrating NO’s role as a signaling molecule in the cardiovascular system. (As an interesting historical footnote, it was Alfred Nobel who invented dynamite, among other explosives.12 Upon his brother Ludvig’s death, a newspaper erroneously published [Alfred] Nobel’s obituary, proclaiming him the “merchant

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ofdeath.”Distraught,heestablishedtheNobelPrizesto leave a more positive legacy to the world. There is an unmistakable irony that 103 years after Nobel’s death, nitrate researchers would receive the Prize that was endowed as an apology for Nobel’s work with nitrates.)

Nitric oxide is synthesized by an enzyme called, appropriately enough, nitric oxide synthase (NOS). Nitric oxide synthase exists in 3 distinct isoforms, each encoded by a different gene, of which NOS3, or endothelial NOS (eNOS),13 is the most relevant to glaucoma. Nitric oxide synthase produces NO in 2 steps by oxidizing the amino acid L-arginine to L-hydroxyarginine and then to NO.

Once synthesized, NO binds soluble guanylate cyclase (sGC), which then converts guanosine triphosphate to cyclic guanosine monophosphate (cGMP).13 cGMP is a second messenger that modulates smooth muscle relaxation and vasodilation and many other important biologic processes, such as platelet inhibition and cell growth and differentiation (Figure 1).

NITRIC OXIDE IN NONOCULAR PATHOPHYSIOLOGY

As shown in Figure 1, inhibition of eNOS will reduce the production of NO, which in turn will reduce the production of cGMP. Reduction of cGMP by dysregulation of eNOS plays a causal role in many human diseases and disorders related to vasoconstriction and/or vasospasm.

These include angina pectoris, pulmonary hypertension, erectile dysfunction, thrombosis, and atherosclerosis.13 Therefore, the NOS-NO-cGMP pathway is an important therapeutic target for human physiology and pharmacology.

Strategies to increase cGMP levels have yielded numerous important treatments for diseases and disorders involving the NO pathway. Among these are the phosphodiesterase (PDE) inhibitors, which increase cGMP levels in cells by blocking its degradation. The resulting pharmacodynamic effect is smooth muscle relaxation (Figure 2). Phosphodiesterase inhibitors are commonly used to treat erectile dysfunction, in which they induce local vasodilation that in turn increases the blood supply to the penis.14 Other PDE inhibitors, such as roflumilastandcilomilast,andnonspecificPDEinhibitors,such as theophylline and theobromine, produce bronchodilation in the treatment of asthma.15 Also, the sGC activator riociguat is approved for use in the management of pulmonary arterial hypertension.16 Other potential applications may include myocardial failure and endotoxic shock.17

Figure 1. Nitric oxide metabolic pathway and its physiologic effectsAbbreviations: cGMP, cyclic guanosine monophosphate; eNOS, endothelial nitric oxide synthase; GTP, guanosine triphosphate.

Figure 2. Nitric oxide–cGMP pathway for relaxation of smooth muscle

Abbreviations: ADP, adenosine diphosphate; ARG, arginine; ATP, adenosine triphosphate; cGMP, cyclic guanosine monophosphate; eNOS, endothelial nitric oxide synthase; GC, guanylate cyclase; GMP, guanosine monophosphate; GTP, guanosine triphosphate; LBN, latanoprostene bunod; NO, nitric oxide; P, phosphate; PDE5, phosphodiesterase type 5; PKG, protein kinase G.

eNOS

Guanylate Cyclase

NitricOxide

L-arginine

GTP

cGMP

Smooth MuscleRelaxation

Platelet Inhibition

Cell Growthand

Differentiation

• Angina Pectoris• Pulmonary Hypertension• Erectile Dysfunction

• Thrombosis• Atherosclerosis

• Atherosclerosis• Angiogenesis

eNOS

SildenafilLBN

Riociguat

ARG

GTP cGMP

PKG

5’-GMP

Protein

Protein

Ca2+

Relaxation of Vascular Smooth Muscle and

Trabecular Meshwork

ATP

ADP

NO

PDE5

GC-1 or GC-2

P

6

NITRIC OXIDE IN THE HEALTHY EYE

For more than 150 years, the likely roles for both IOP and ocularbloodflowhavebeenrecognizedasimportantin the pathophysiology of glaucoma. Nitric oxide has an important physiologic role in the regulation of both optic nerveheadbloodflowandIOP.

The optic nerve has a complex blood supply fed by 4distinctcirculatorybeds,thecentralretinalartery(a branch of the ophthalmic artery) and posterior ciliary arteries as well as both the choroidal and retinal vascular beds (Figure 3). In the optic nerve head, NO donors decrease vascular resistance by relaxing smooth muscle, resulting in local vasodilation and increased optic nerve headbloodflow.17,18 Impairment of the NO pathway converselyreducesopticnerveheadbloodflow,resulting in ischemia.19,20

In the healthy eye, as described above, IOP is determined by the balance between aqueous humor productionandoutflow.ThemajorityofaqueoushumoroutflowisthroughtheTMandintoSchlemmcanal,where it then courses through collector channels and scleral vessels to enter the episcleral circulation. The TM can be thought of as a network of beams composed of a core of extracellular matrix lined with TM cells. As are vascular smooth muscle cells, trabecular cells are contractile in nature.21 Trabecular cell contraction involves the interaction of actin and myosin and results in dense

packing oftheTMtissue,whichconstrictsflowpathwaysandreducesaqueousoutflow.

Nitric oxide has important physiologic effects in several of the tissues relevant to the maintenance of IOP. Evidence for the role of NO in IOP regulation comes from several lines of research. Importantly, eNOS is present in the endothelium of uveal vasculature, Schlemm canal, and ciliary body.22,23 Nitric oxide is known to increasetrabecularoutflowfacilityinthehumananteriorsegment,24 and NO donors lower IOP in animal models.8

cGMPsupplementationalsoincreasesoutflowfacilityinlive monkey eyes.25 Furthermore, mice overexpressing eNOS have lower IOP.26 In contrast, eNOS knockout mice (animals with no functional eNOS gene and thus no endogenous eNOS) have elevated IOP,27 and sGC knockout mice have both elevated IOP and optic nerve degeneration.28 The mechanism by which NO lowers IOP appears to be via relaxation of cells in the TM and Schlemm canal via inhibition of actin-myosin interactions, whichleadstoincreasedaqueousoutflowandIOPreduction.21,22

NITRIC OXIDE IN THE GLAUCOMATOUS EYE

Significantevidenceexistsshowingthatdysfunctionwithin the NO pathway plays a causal role in the pathophysiology of POAG. In the optic nerve head, the NO system promotes vasodilation and increased blood flowtothisimportanttissuebed.Conversely,inhibitionof the NO pathway would be expected to reduce optic nerveheadbloodflow.

Evidence for these hypotheses in humans has been reported. In a small clinical trial involving 12 patients with glaucoma and 12 healthy subjects, NOS inhibition accomplished by intravenous administration of a known inhibitorofNOSreducedopticnerveheadbloodflowinhealthy eyes more than in glaucomatous eyes (P = .03),29 suggesting that the NO pathway is already impaired in glaucomatous eyes.

Support for the role of NO pathway impairment in humans with ocular hypertension comes from several distinct lines of research. The substrate for NOS, L-arginine levels are high in the aqueous humor of patients with glaucoma30 and in the vitreous humor of monkeys with experimental glaucoma.31 This suggests that NOS impairment has reduced the conversion of L-arginine to NO, resulting

Figure 3. Blood supply to the optic nerve headAbbreviations: A, arachnoid; C, choroid; Col. Br., collateral branches supplying the optic nerve pial plexus; CRA, central nerve artery; CRV, central retinal vein; D, dura; LC, lamina cribrosa; ON, optic nerve; PCA, posterior ciliary artery; PR, prelaminar region; R, retina; S, sclera; SAS, subarachnoid space.Ischemic Optic Neuropathies, Pathogenesis of posterior ischemic opticneuropathy,2011,427-436,HayrehSS,©Springer-VerlagBerlinHeidelberg 2011 with permission of Springer.

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in a surplus of L-arginine (see Figure 1). Furthermore, NO levels are low in the aqueous humor of patients with glaucoma.32,33 Additionally, eNOS gene variants have been described in patients with POAG,34,35 further suggesting a role for NOS impairment in glaucoma.

Other data support a role for impaired NO signaling in the pathophysiology of glaucoma. Acetylcholine (ACh) is known to mediate vasodilation via the generation of NO. Therefore, ACh delivered to healthy subjects would beexpectedtoincreasebloodflowviaNOsignaling,whereasareducedorabsentincreaseinbloodflowwould be expected in subjects with impairment of the NO signaling pathway. In a clinical trial, intravenous administration of ACh in subjects with untreated normal-tensionglaucomaproducedasignificantlysmallerincreaseinforearmbloodflowthanthatinhealthy controls, as measured by venous occlusion plethysmography.36 Likewise, peripheral limb ischemia should induce brachial artery vasodilation, but this response is impaired in patients with POAG across the spectrum of IOP.37 These studies suggest an underlying impairment of the NO pathway in these patients.

NITRIC OXIDE PATHWAY AS A THERAPEUTIC TARGET IN GLAUCOMA

In light of the data supporting the role of an impaired NO signaling pathway in open-angle glaucoma, it is reasonable to investigate potential therapeutic targets within the NO signaling pathway that might prove effective in the treatment of glaucoma. The NO pathway represents a novel therapeutic target for glaucoma, and an NO-based therapy would offer a unique mechanism of action among existing treatment options.

Multiple potential approaches are currently being investigated. Among these, agonists of sGC are being evaluated to increase production of cGMP and result invasodilationandincreasedoutflowfacility.38 Dietary supplementation of the cGMP precursor L-arginine represents a potential upstream intervention for conditions associated with NO pathway impairment. Such an approach may have value in mediating endothelial dysfunction associated with diabetes, smoking, and obesity, although evidence to date is limited to animal and early clinical studies.13 An analysis of the Nurses’ Health Study and Health Professionals Follow-up Study suggests that higher dietary nitrate intake may be related to a lower incidence of POAG.39 This study

evaluated the relationship between consumption of leafy greens—animportantdietarysourceofnitrates—andthedevelopmentofPOAG,findingthathealthcareprofessionals who consumed the highest levels of dietary nitrates(1.45servings/d)wereapproximately18%lesslikely to develop POAG than those consuming the lowest daily levels (0.3 servings/d).

An alternate approach is to increase intraocular NO directly, eg, via NO donor compounds. An NO-donating formulation of bimatoprost is in preclinical development and may be entering clinical development in 2018. In various animal models of glaucoma, this formulation produced mean IOP reductions on the order of 5 to 8 mm Hg in rabbits, dogs, and monkeys, in each case in excess of bimatoprost alone.40 Similarly, NO-donating formulations of the carbonic anhydrase inhibitors dorzolamide and brinzolamide lower IOP in animal models.41

LBN, another NO-donating compound, is in late-stage development, and a new drug approval application is currently undergoing a review by the US Food and Drug Administration. Following topical ocular administration, LBN undergoes carboxyl ester hydrolysis to latanoprost acid and butanediol mononitrate, which is subsequently reducedto1,4-butanediolandNO.42Preclinical studies evaluated the effects of LBN vs latanoprost alone on human TM cell contractility and the NO-cGMP signaling pathway.43Inthesestudies,LBNsignificantlyincreasedcGMP levels and reduced TM cell contractility compared with latanoprost. In animal models of glaucoma involving beaglesandmonkeys,LBNloweredIOPby35%to44%,comparedwithonly26%to27%withlatanoprostalone.44Taken together, these results suggested that LBN led to greatertrabecularoutflowandthusgreaterIOPreductionthan did latanoprost.

In a phase 2 study, LBN provided an approximately 1- to 1.5-mm Hg greater IOP reduction than did latanoprost (P≤.009).45 Results from 2 phase 3 clinical trials of LBN (APOLLO and LUNAR) have recently been reported. The APOLLO study was a randomized, multicenter, double-maskedstudy,inwhich420patientswitheitherocularhypertension or POAG were randomized to receive either LBN,0.024%,everyeveningortimolol,0.5%,twicedaily for 3 months.46 The primary outcome measure was postbaseline IOP measured at 8 am, 12 pm,and4pm at weeks 2 and 6 and month 3. The mean IOP at all

8

9timepointswassignificantlylower with LBN than with timolol (P≤.002).SignificantlymorepatientstreatedwithLBNachievedanIOPof≤18mmHg(22.9%vs11.3%,respectively; P=.005)anda≥25%reductioninIOP(34.9%vs19.5%;P = .001) than did those treated with timolol. The most common treatment-emergent adverse events were mild to moderate and included eye pain upon instillation in both treatment groups, with 1 case of severe eye pain in the timolol group.

The LUNAR study was identical in design to APOLLO and included 387 subjects.47 In this trial, the mean IOP at 8ofthe9timepoints(allbutthefirsttimepoint,week2at 8 am)wassignificantlylowerwithLBNthanwithtimolol(P≤.025).SignificantlymorepatientstreatedwithLBNachieveda≥25%reductioninIOPthanthosetreatedwith timolol (P = .007), whereas the proportion achieving anIOP≤18mmHgwasinsignificantlydifferentbetweenthe2groups(17.7%vs11.1%,favoringLBN;P=.084).The most common treatment-emergent adverse events wereconjunctivalhyperemia(9%)andeyeirritation(7.2%)intheLBNgroupandeyeirritation(4.4%)andeyepain(3.7%)inthetimololgroup.

Following the initial 3-month study periods, subjects in APOLLO and LUNAR entered an open-label extension study for an additional 3 months (LUNAR) or 9 months (APOLLO), in which they all received LBN regardless of initial randomization.48 Subjects who switched from timolol toLBNgainedanadditional6%to8%IOPreduction,with overall reductions in mean diurnal IOP from baseline intherangeof32%to34%(P < .001 vs baseline). The rate and nature of adverse events in the extension did not differ from those reported in the initial studies.

Two studies of LBN in Japanese patients have also been reported.49,50ThefirstwasanuncontrolledtrialconductedinhealthyJapanesesubjectswhoreceivedLBN,0.024%, oncedailyfor14days.49 Intraocular pressure was measuredat9timepointsspanningthefull24-hourcircadian period both before and after the 2-week course oftreatment.Inthiscohortof24youngsubjects(meanage,27years),mean24-hourIOPwasreducedfrom13.6 ± 1.3 mm Hg before treatment to 10.0 ± 1.0 mm Hg aftertreatment.ThisIOPreduction—3.6mmHgor27%—in eyes with low baseline IOP may suggest a role for LBN in the management of normal-tension glaucoma.

The second Japanese study of LBN was the JUPITER study, which was conducted in 130 subjects with POAG or ocular hypertension.50 In this uncontrolled open-label

study, subjects (mean age, 62.5 years) received LBN, 0.024%,oncedailyfor12months,andtheprimaryendpoint was long-term safety. Mean IOP at baseline was 19.6±2.9mmHg,andbyweek4,wasreducedto15.3±3.0mmHg,a22%reductionthatwasexceededat every subsequent monthly visit through 12 months of therapy,withafinalIOPreductionof26.3%seenatmonth 12. The most common treatment-emergent adverse eventswereconjunctivalhyperemia(17.7%),eyelashgrowth (16.2%),eyeirritation(11.5%),andeyepain(10.0%).Noneof the hyperemia cases was graded as severe, and at any given monthly visit, no more than 2 hyperemia cases were graded as moderate, clearly indicating that hyperemia is mild in the vast majority of eyes treated with LBN.

The results of these clinical studies support the investigators’ conclusionsthatLBN,0.024%,oncedailywassafeandeffective,withsignificantlygreaterIOP-loweringeffectsthantimolol.49,50 If LBN should garner approval by the US Food andDrugAdministration,itwouldrepresentthefirstnewdrug with a novel mechanism of action since the debut of latanoprost in the mid-1990s.

CLINICAL RELEVANCE

Expert Commentary

Q: What is your current preferred first-line therapy for glaucoma?

Dr Fingeret: IwillusePGAsasmyfirstlineoftherapybecause of their ability to reduce IOP with few side effects. Generic latanoprost is often the medication I use because this is the medication found on the formulary for the facility I am associated with.

Dr Gaddie: Myfirst-linetherapytodayforapatientwithmild-to-moderate glaucoma is a PGA. PGAs offer once- daily treatment, with little to no systemic side effects and only mild cosmetic side effects. They are a solid primary therapywithfewdownsides.However,>50%ofpatientswith glaucoma require more than primary therapy with a PGA. Clearly, there is a gap for better primary therapy.

Dr Pasquale: It depends on what type of glaucoma the patient has. Currently for patients with paracentral open-angle glaucoma with frequent disc hemorrhages, I start with timolol and switch to brimonidine/timolol if the patient’s insurance allows for such use. I do this because I feel such a patient has an inherent problem with NO signaling and typically presents with an IOP that is in the

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upper teens and low 20s. I think that there is considerable evidencethatthebrimonidineinthisfixed-combinationagent enhances NO signaling, whereas the combined effect of timolol and brimonidine provides reasonable IOP lowering. I am excited about the possibility LBN holds for this type of patient because NO donation and the combined IOP lowering of latanoprost and NO might be tailored to the patient’s needs.

Q: What are the biggest clinical challenges for glaucoma therapy today?

Dr Fingeret: One challenge is with the few individuals being managed for open-angle glaucoma who are getting worse despite their IOP being at an acceptable range of pressure. We have discussed IOP-independent causes of glaucoma, but to date, we have not had the ability to manage this particular mechanism. A medication that mayimprovebloodflowindependentofloweringIOPwould be valuable.

Dr Gaddie: We lack contemporary agents that work on thetrabecularoutflowpathwaythataresystemicallysafeand easy to dose. In addition, getting patients access to the medicines we prescribe is becoming more time consuming and frustrating.

Dr Pasquale: The patient with combined high myopia, dysmorphic optic nerves, and TM dysfunction is a real challenge. Such a patient may have IOP-independent functional visual loss due to severe optic nerve tilt or torsion that is compounded by mildly elevated IOP. Even if one is successful in reducing IOP in this case, progression can continue, which is frustrating for the patient and physician.

Q: Where might NO-donating IOP-lowering drugs fit into the current treatment regimen? Is there a particular patient subgroup in whom this approach may be most beneficial?

Dr Fingeret: If effective, I can see NO-donating drugs evolvingintofirst-linetherapy.Oneparticularsubgroupwould be patients with low-tension glaucoma because IOP may not be the primary mechanism of damage. Thus, amedicationthatimprovesbloodflowmaybevaluableinthis group.

Dr Gaddie: I would envision NO-donating agents to be appropriateinvariouspatientgroups,withfirst-line/newtreatment.LBNhasthebenefitofboththePGAandtheNO-donating effects. If you can get even slightly better

IOP reduction than with a PGA, why would you not want to start all patients on this class of medication? I could also see it being appropriate for established patients on a PGA who need some additional IOP lowering but do not want to add a second agent or be exposed to the additional side effects of a second agent.

Dr Stamer: Because NO dysregulation underlies vascular disease and likely some forms of glaucoma, NOsupplementationholdspromisetoprovidebenefitforpatients with glaucoma both at the optic nerve head and the TM. The challenge will be effective delivery over time to these 2 affected tissues.

Q: Should we be encouraging our patients with glaucoma to consume more nitrate-rich diets?

Dr Fingeret: This is an interesting question, and the obvious answer is, of course we should stress anything that may improve our patients’ health. Saying that, I am notconvincedthatapeptalkintheofficewillchangeapatient’s behavior, such as modifying his/her diet. This is acomplexissuethatrequiressignificanteffortonthepartof the doctor and patient.

Dr Gaddie: I think any organic consumption of nitrate-rich foodcouldbebeneficial.

Dr Pasquale: Yes,thisisahealthydietsolongasthenitrates are sourced from root vegetables.

CONCLUSION

Glaucoma is a multifactorial disease with a complex pathophysiology and many known risk factors. Reduction of IOP remains the only established therapy. Intraocular pressure regulation is incompletely understood and involves many diverse physiologic pathways. A better understanding of the NO-cGMP pathway in human health and disease has resulted in novel therapeutic targets for many disease states, including glaucoma. The NO-cGMP pathway plays a role in the regulation of both opticnerveheadbloodflowandIOP,bothofwhicharecritically important in the glaucomatous disease state. TheNOdonorLBNshowssignificantpromiseasanoveltherapeutic agent for glaucoma. LBN and other emerging drugs that directly target the diseased tissues have the potential to alter clinical glaucoma management in the near future, providing ever more tools to enhance the individualization of glaucoma therapy for our patients.

10

REFERENCES1. American Academy of Ophthalmology. Preferred Practice Pattern®. Primary Open-Angle Glaucoma. San Francisco, CA: American Academy of Ophthalmology; 2015.2. Heijl A, Leske MC, Bengtsson B, Hyman L, Bengtsson B, Hussein M; Early Manifest Glaucoma Trial Group. Reduction of intraocular pressure and glaucoma progression: results from the Early Manifest Glaucoma Trial. Arch Ophthalmol. 2002;120(10):1268-1279.3. Garway-Heath DF, Crabb DP, Bunce C, et al. Latanoprost for open-angle glaucoma (UKGTS): a randomised, multicentre, placebo-controlled trial. Lancet.2015;385(9975):1295-1304.4. CollaborativeNormal-TensionGlaucomaStudyGroup.Comparisonof glaucomatous progression between untreated patients with normal-tension glaucoma and patients with therapeutically reduced intraocular pressures. Am J Ophthalmol.1998;126(4):487-497.5. Goel M, Picciani RG, Lee RK, Bhattacharya SK. Aqueous humor dynamics: a review. Open Ophthalmol J. 2010;4:52-59.6. NilssonSF.Theuveoscleraloutflowroutes. Eye (Lond). 1997;11(Pt 2): 149-154.7. JohnsonM.‘Whatcontrolsaqueoushumouroutflowresistance?’ Exp Eye Res.2006;82(4):545-557.8. Cavet ME, Vittitow JL, Impagnatiello F, Ongini E, Bastia E. Nitric oxide (NO): an emerging target for the treatment of glaucoma. Invest Ophthalmol Vis Sci. 2014;55(8):5005-5015.9. SteinhornBS,LoscalzoJ,MichelT.Nitroglycerinandnitricoxide—arondo of themes in cardiovascular therapeutics. N Engl J Med. 2015;373(3): 277-280.10. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987; 327(6122):524-526.11. Koshland DE Jr. The molecule of the year. Science. 1992;258(5090):1861.12. Encyclopaedia Britannica. Alfred Nobel. https://www.britannica.com/ biography/Alfred-Nobel. Accessed July 6, 2017.13. Murad F. Shattuck Lecture. Nitric oxide and cyclic GMP in cell signaling and drug development. N Engl J Med. 2006;355(19):2003-2011.14.HuangSA,LieJD.Phosphodiesterase-5(PDE5)inhibitorsinthemanagement of erectile dysfunction. P T.2013;38(7):407-419.15.SpinaD.PDE4inhibitors:currentstatus.Br J Pharmacol. 2008;155(3): 308-315.16. Adempas [package insert]. Whippany, NJ: Bayer HealthCare Pharmaceuticals, Inc; 2013.17. Papapetropoulos A, Hobbs AJ, Topouzis S. Extending the translational potential of targeting NO/cGMP-regulated pathways in the CVS. Br J Pharmacol. 2015;172(6):1397-1414.18.HaefligerIO,FlammerJ,BényJL,LüscherTF.Endothelium-dependent vasoactive modulation in the ophthalmic circulation. Prog Retin Eye Res. 2001;20(2):209-225.19.AdachiK,FujitaY,MorizaneC,etal.InhibitionofNMDAreceptorsand nitric oxide synthase reduces ischemic injury of the retina. Eur J Pharmacol. 1998;350(1):53-57.20.GeyerO,AlmogJ,Lupu-MeiriM,LazarM,OronY.Nitricoxidesynthase inhibitors protect rat retina against ischemic injury. FEBS Lett. 1995; 374(3):399-402.21. Wiederholt M, Thieme H, Stumpff F. The regulation of trabecular meshwork and ciliary muscle contractility. Prog Retin Eye Res. 2000;19(3):271-295.22.BecquetF,CourtoisY,GoureauO.Nitricoxideintheeye:multifacetedroles and diverse outcomes. Surv Ophthalmol. 1997;42(1):71-82.23.NathansonJA,McKeeM.Identificationofanextensivesystemofnitric oxide-producingcellsintheciliarymuscleandoutflowpathwayofthe human eye. Invest Ophthalmol Vis Sci. 1995;36(9):1765-1773.24.DismukeWM,MbadughaCC,EllisDZ.NO-inducedregulationoftrabecular meshworkcellvolumeandaqueoushumoroutflowfacilityinvolvetheBKCa ion channel. Am J Physiol Cell Physiol. 2008;294(6):C1378-C1386.25. Kee C, Kaufman PL, Gabelt BT. Effect of 8-Br cGMP on aqueous humor dynamics in monkeys. Invest Ophthalmol Vis Sci. 1994;35(6):2769-2773.26.StamerWD,LeiY,Boussommier-CallejaA,OverbyDR,EthierCR.eNOS, a pressure-dependent regulator of intraocular pressure. Invest Ophthalmol Vis Sci.2011;52(13):9438-9444.27. LeiY,ZhangX,SongM,WuJ,SunX.Aqueoushumoroutflowphysiologyin NOS3 knockout mice. Invest Ophthalmol Vis Sci.2015;56(8):4891-4898.28.BuysES,KoYC,AltC,etal.Solubleguanylatecyclasea1-deficient mice: a novel murine model for primary open angle glaucoma. Ann Neurosci. 2013;20(2):65-66.29. Polak K, Luksch A, Berisha F, Fuchsjaeger-Mayrl G, Dallinger S, Schmetterer L. Altered nitric oxide system in patients with open-angle glaucoma. Arch Ophthalmol. 2007;125(4):494-498.

30. Hannappel E, Pankow G, Grassl F, Brand K, Naumann GO. Amino acid pattern in human aqueous humor of patients with senile cataract and primary open-angle glaucoma. Ophthalmic Res.1985;17(6):341-343.31. Dreyer EB, Zurakowski D, Schumer RA, Podos SM, Lipton SA. Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma. Arch Ophthalmol.1996;114(3):299-305.32.DoganayS,EverekliogluC,TurkozY,ErH.Decreasednitricoxide production in primary open-angle glaucoma. Eur J Ophthalmol. 2002; 12(1):44-48.33. Galassi F, Renieri G, Sodi A, Ucci F, Vannozzi L, Masini E. Nitric oxide proxies and ocular perfusion pressure in primary open angle glaucoma. Br J Ophthalmol.2004;88(6):757-760.34.KangJH,WiggsJL,RosnerBA,etal.Endothelialnitricoxidesynthasegene variants and primary open-angle glaucoma: interactions with sex and postmenopausal hormone use. Invest Ophthalmol Vis Sci. 2010;51(2):971-979.35. Magalhães da Silva T, Rocha AV, Lacchini R, et al. Association of polymorphisms of endothelial nitric oxide synthase (eNOS) gene with the risk of primary open angle glaucoma in a Brazilian population. Gene.2012;502(2):142-146.36. Henry E, Newby DE, Webb DJ, O’Brien C. Peripheral endothelial dysfunction in normal pressure glaucoma. Invest Ophthalmol Vis Sci. 1999;40(8): 1710-1714.37. Su WW, Cheng ST, Ho WJ, Tsay PK, Wu SC, Chang SH. Glaucoma is associated with peripheral vascular endothelial dysfunction. Ophthalmology. 2008;115(7):1173-1178.e1.38. Buys ES, Potter LR, Pasquale LR, Ksander BR. Regulation of intraocular pressure by soluble and membrane guanylate cyclases and their role in glaucoma. Front Mol Neurosci. 2014;7:38.39. Kang JH, Willett WC, Rosner BA, Buys ES, Wiggs JL, Pasquale LR. Association of dietary nitrate intake with primary open-angle glaucoma: a prospective analysis from the Nurses’ Health Study and Health Professionals Follow-up Study. JAMA Ophthalmol. 2016;134(3):294-303.40. ImpagnatielloF,TorisCB,BatugoM,etal.Intraocularpressure-lowering activityofNCX470,anovelnitricoxide-donatingbimatoprostinpreclinical models. Invest Ophthalmol Vis Sci.2015;56(11):6558-6564.41.SteeleRM,BenediniF,BiondiS,etal.Nitricoxide-donatingcarbonic anhydrase inhibitors for the treatment of open-angle glaucoma. Bioorg Med Chem Lett. 2009;19(23):6565-6570.42.CavetME,DeCoryHH.Theroleofnitricoxideintheintraocularpressure loweringefficacyoflatanoprostenebunod:reviewofnonclinicalstudies [published online ahead of print August 7, 2017]. J Ocul Pharmacol Ther. doi:10.1089/jop.2016.0188.43.CavetME,VollmerTR,HarringtonKL,VanDerMeidK,RichardsonME. Regulation of endothelin-1-induced trabecular meshwork cell contractility by latanoprostene bunod. Invest Ophthalmol Vis Sci. 2015;56(6):4108-4116.44.KraussAH,ImpagnatielloF,TorisCB,etal.Ocularhypotensiveactivityof BOL-303259-X,anitricoxidedonatingprostaglandinF2αagonist,in preclinical models. Exp Eye Res. 2011;93(3):250-255.45.WeinrebRN,OngT,ScassellatiSforzoliniB,VittitowJL,SinghK,KaufmanPL; VOYAGERStudyGroup.Arandomised,controlledcomparisonof latanoprostenebunodandlatanoprost0.005%inthetreatmentofocular hypertensionandopenangleglaucoma:theVOYAGERstudy.Br J Ophthalmol. 2015;99(6):738-745.46.WeinrebRN,ScassellatiSforzoliniB,VittitowJ,LiebmannJ.Latanoprostene bunod0.024%versustimololmaleate0.5%insubjectswithopen-angle glaucoma or ocular hypertension: the APOLLO study. Ophthalmology. 2016;123(5):965-973.47.MedeirosFA,MartinKR,PeaceJ,ScassellatiSforzoliniB,VittitowJL, WeinrebRN.Comparisonoflatanoprostenebunod0.024%andtimolol maleate0.5%inopen-angleglaucomaorocularhypertension:theLUNAR study. Am J Ophthalmol. 2016;168:250-259.48.VittitowJ,LiebmannJM,KaufmanPL,MedeirosF,MartinK,WeinrebRN. Long-termefficacyandsafetyoflatanoprostenebunod0.024%forintraocular pressure lowering in patients with open-angle glaucoma or ocular hypertension: APOLLO and LUNAR studies. Paper presented at: The Association for Research in Vision and Ophthalmology 2016 Annual Meeting; May 1-5, 2016; Seattle, WA.49.AraieM,SforzoliniBS,VittitowJ,WeinrebRN.Evaluationoftheeffectof latanoprostenebunodophthalmicsolution,0.024%inloweringintraocular pressureover24hinhealthyJapanesesubjects.Adv Ther. 2015;32(11): 1128-1139.50. Kawase K, Vittitow JL, Weinreb RN, Araie M; JUPITER Study Group. Long-termsafetyandefficacyoflatanoprostenebunod0.024%inJapanese subjects with open-angle glaucoma or ocular hypertension: the JUPITER study. Adv Ther. 2016;33(9):1612-1627.

11For instant processing, complete the CE Post Test onlinehttp://tinyurl.com/NOglaucoma

1. Elevated IOP is both a risk factor and a ________ for POAG. a. Protective factor b. Mitigating factor c. Causal factor d. Clotting factor

2. Intraocular pressure is determined by the balance of aqueous humor production and __________.

a. The epithelium of the ciliary body b. The anterior uveal tract c. The suprachoroidal space d. Aqueoushumoroutflow

3. Which of the following is an important structure in the uveoscleral outflowpathway?

a. TM b. Schlemm canal c. Ciliary body face d. Episcleral vessels

4.IntraocularpressureiselevatedinPOAGby: a. Decreasing aqueous production b. Increasinguveoscleraloutflow c. Decreasingtrabecularoutflow d. Increasingtrabecularoutflow

5. Which of the following ocular structures is relevant to the aqueous inflowpathway?

a. Juxtacanalicular TM b. Schlemm canal c. Ciliary body processes d. Episcleral blood vessels

6. PGAs lower IOP primarily by: a. Reducing aqueous production b. Reducingtrabecularoutflow c. Increasinguveoscleraloutflow d. Reducing episcleral venous pressure

7. Beta blockers lower IOP by: a. Reducing aqueous humor production b. Increasingtrabecularoutflow c. Increasinguveoscleraloutflow d. Reducing episcleral venous pressure

8. Which drug class lowers IOP by indirectly increasing trabecular outflow?

a. Beta blocker b. Miotic c. PGA d. Carbonic anhydrase inhibitor

9. The substrate for NOS that generates NO is: a. Nitroglycerin b. sGC c. L-arginine d. Guanosine triphosphate

10. Nitric oxide plays a role in the pathophysiology of many human diseases, including: a. Cancer b. Cerebrovascular accidents c. Multiple sclerosis d. Angina pectoris

11. Nitric oxide’s physiologic effect results in: a. Bradycardia b. Lipid metabolism c. Smooth muscle relaxation d. Norepinephrine reuptake

12. Which of the following is the second messenger that mediates NO’s effects on smooth muscle cells? a. Water b. NOS c. cGMP d. L-arginine

13.TheNOsignalingpathwayregulatesopticnerveheadbloodflow and _______, 2 important functions relevant to glaucoma. a. Reversalofvisualfieldloss b. IOP c. Thepupillarylightreflex d. Corneal biomechanical properties

14.The____________vascularbeddoesNOTcontributetotheblood supply of the optic nerve. a. Central retinal artery b. Posterior ciliary artery c. Anterior ciliary artery d. Choroidal vascular bed

15. Nitric oxide lowers IOP primarily by: a. Decreasing episcleral venous pressure b. Decreasingaqueousfluidproduction c. Increasinguveoscleraloutflow d. Increasingtrabecularoutflow

16. Trabecular cell contraction involves the interaction of: a. Actin and pectin b. Myosin and myoglobulin c. Actin and cGMP d. Actin and myosin

17. In phase 2 and 3 clinical trials, LBN lowered IOP more than: a. Timolol or dorzolamide b. Timolol or latanoprost c. Selective laser trabeculoplasty d. Latanoprost or brimonidine

18. Which of the following is a valid approach to targeting the NO signaling system for glaucoma therapy? a. sGC agonism b. cGMP dietary supplementation c. NOS inhibition d. NO donor compounds

19. Which is the most common adverse event associated with LBN? a. Conjunctival hyperemia b. Shortness of breath c. Dry eye syndrome d. Bradycardia

20. Which of the following is FALSE regarding dietary nitrates? a. They are found in leafy green vegetables b. Theyhavemanysystemichealthbenefits c. Epidemiologic studies suggest that a nitrate-rich diet may be associated with a reduced risk of incident POAG d. One serving of kale or collards per month can lower IOP as much as topical PGAs

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134D

THE ROLE OFNitric Oxide

IN GLAUCOMA