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DRUG DISCOVERY

TODAY

DISEASEMODELS

Drug discovery in glaucoma and therole of animal modelsSara McNally*, Colm J. O’BrienCatherine McAuley Clinical Research Centre, Institute of Ophthalmology, Mater Misericordiae Hospital, 21 Nelson Street, Dublin, Ireland

Drug Discovery Today: Disease Models Vol. xxx, No. xx 2014

Editors-in-Chief

Jan Tornell – AstraZeneca, Sweden

Andrew McCulloch – University of California, SanDiego, USA

Models for eye disorders

Glaucoma is a neurodegenerative disorder charac-

terised by damage to inner layers of the retina and

the optic nerve (ON). The slow degeneration of retinal

ganglion cells (RGCs) and their axons results in a

progressive loss of vision. To date, a wide variety of

animal models have been used to study glaucoma dis-

ease mechanisms and these include monkey, dog, and

rodent models. However, there remains no ideal

model for studying glaucoma disease and this is largely

due to its complexity. Here, we review common animal

models in use for glaucoma research and highlight

discoveries which relate to potential new therapeutic

avenues.

Introduction

The discovery of therapeutic drug targets relies heavily on

animal models which represent a valuable tool for under-

standing both the progression and cause of human disease.

However, glaucoma is not one single disease entity and

rather, comprises a heterogeneous group of disorders; this

makes drug discovery difficult. The brainstem, the visual

cortex and the visual pathway are impacted in the disease

and this results in progressive blindness due to chronic optic

nerve damage and a loss of RGCs [1]. Worldwide, glaucoma is

a leading cause of irreversible blindness and visual field

defects and it is estimated that the disease will affect over

80 million people by 2020 [2]. Similar to other neurodegen-

erative disorders, glaucoma correlates with age and as

improvements in healthcare denote an increased lifespan

Please cite this article in press as: McNally S, O’Brien CJ. Drug discovery in glaucoma and

*Corresponding author.: S. McNally ([email protected]), ([email protected])

1740-6757/$ � 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ddmod.201

Section editors:Ian Jackson – Medical and Developmental Genetics,University of Edinburgh Western General Hospital,Edinburgh, UK.Marcela Votruba – School of Optometry & Vision Sciences,Cardiff University, Cardiff, UK.

for our population, a larger economic burden will result from

an increasing number of people at risk of developing visual

impairment.

Glaucoma – a complex disorder

Human glaucoma is generally classified into three major sub-

groups: Primary Open Angle Glaucoma (POAG), Primary

Angle Closure Glaucoma (PACG), and Primary Congenital

Glaucoma (PCG) and in most populations, POAG is the most

common form of the disease [3]. Currently, three causative

genes (Myocilin, Optineurin and WDR36) have been identi-

fied for POAG although more than 20 genetic loci have been

reported [4]. Despite there being different forms of the dis-

ease, progressive visual impairment (and eventual blindness)

caused by atrophy of the optic nerve and axonal damage

represents a ‘final common pathway’ of tissue damage in all

types of glaucoma.

As mentioned, the most common form of glaucoma is

open-angle which is a multi-factorial optic neuropathy

defined by open anterior chamber angles, elevated intra-

ocular pressure (IOP), progressive optic nerve fiber loss and

visual field defects [5]. It is the chronic elevation of IOP in

glaucoma which leads to the death of RGCs and IOP is, in fact,

a major risk factor for the disease [6]. As such, a primary focus

for intervention is lowering IOP. So what causes IOP elevation

the role of animal models, Drug Discov Today: Dis Model (2014), http://dx.doi.org/

3.12.002 e1

http://dx.doi.org/10.1016/j.ddmod.2013.12.002



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Drug Discovery Today: Disease Models | Models for eye disorders Vol. xxx, No. xx 2014


in glaucoma disease? A reduction in the outflow of aqueous

humor (AH) (which is produced by the cilliary body and

moves into the anterior chamber before leaving the eye)

generally gives rise to an IOP increase. Exit of AH can occur

either via the conventional pathway (trabecular meshwork

(TM)/Schlemm’s canal) or via the non-conventional (uveoscl-

eral) pathway [7]. As such IOP homeostasis depends on the

balance between AH production in the ciliary body and its

drainage [8,9].

Glaucoma patients with chronic high IOP who are left

untreated develop aberrations in the retinal inner layers

and the optic nerve head (ONH) which clinically manifests

as visual field loss [5]. Given that IOP is the only proven

treatable risk factor for glaucoma disease, IOP-reduction stra-

tegies remain the major approach for patient management.

However, owing to the complexity of glaucoma disease (and

adding to the difficulty of treatment) many patients continue

to suffer visual field defects despite IOP management. Addi-

tionally, some patients develop normal tension glaucoma

(NTG); highlighting that IOP-independent mechanisms for

the development and progression of optic neuropathy exist

[10].

What drugs are currently in use for treatment of

disease?

The current mode of treating glaucoma comprises an IOP

reduction strategy involving laser therapy, surgical operation

or pharmacology (typically treatment with eye drops). Even

for glaucoma patients suffering from impaired vision and

having low IOP (normal tension or low-tension glaucoma),

therapies which regulate IOP are advised [11–14]. Both open-

angle and closed angle patients are initially managed with

IOP-lowering therapies which act by decreasing the rate of AH

inflow and/or increasing outflow. There are five major classes

of drugs administered as eye-drops that are approved for

lowering IOP in glaucoma patients (these are reviewed in

[15]). b-Adrenergic receptor blockers (timolol, betaxol, car-

teolol, levobunolol) decrease inflow by regulating AH forma-

tion [16]. Cholinergic drugs (pilocarpine, carbachol) increase

TM outflow through ciliary muscle contraction [17]. a-Adre-

nergic receptor agonists (apraclonidine, brimonidine)

decrease inflow by inactivating adenylel cyclase [18]. Prosta-

glandins (PGF2a analogues: latanaprost, travoprost, bima-

trost, tafluprost) increase outflow by increasing matrix

metalloproteinase expression [19] and carbonic anyhydrase

inhibitors (dorzolamide, brinzolamide, acetazolamide,

methazolamide) decrease AH formation [20].

Animal models for glaucoma disease

As previously mentioned, increased IOP is a major risk factor

for glaucoma onset and progression. It is evident that the

relevant animal models for glaucoma would comprise RGC

and ON damage brought about by chronic or transient ocular


e2 www.drugdiscoverytoday.com

hypertension. The best glaucoma animal models require that

frequent IOP measurements be done and easy assessment of

retinal neuronal damage should be possible. However, the

field describes a lack of validated animal models for glaucoma

neuroprotection and this negatively impacts drug develop-

ment. Glaucoma rodent models used to investigate pressure-

independent factors are reviewed in [21].

The relevance, usefulness and validity of any animal model

must, in part, be based on its similarity to the human disease

in question but there is large variation in glaucoma research

in terms of reliance on different model species. For example,

rodent models possess similarity to human ocular anatomy

and other factors such as affordability, genetic manipulation,

availability and short life span all form the basis of decision

making in terms of experimental design [22].

And yet, currently incurable diseases are plighted, in part,

by the use of research models which fail to mimic the human

disorder completely. Separately, glaucoma damage at time of

clinical diagnosis precludes the study of disease onset and this

poses a problem. However, the development of animal mod-

els has been necessary for the study of the pathophysiology of

human glaucoma. Good animal models are also essential for

pharmacological studies and should be characterised by low

cost, reproducibility, easy disease induction and limited side

effects to neighbouring tissue. For an up-to date review of

current animal models for PACG, PCG and other forms of

glaucoma, see [23].

The literature classifies glaucoma models as ‘naturally

occurring’ or ‘induced/experimental’ models. Naturally

occurring models of glaucoma arise spontaneously and the

caveat with these is the difficulty in regulating disease onset

and subsequently, obtaining a homogenous experimental

group. In contrast, induced glaucoma models provide the

correct conditions for controlled experiments and therefore

allow the examination of disease onset and pathological

progression. However, induction of glaucoma disease can

be somewhat unpredictable and whilst experimental models

are important for testing responses to pharmacologic agents,

genetic models developed to address specific hypotheses

generate further insight into pathophysiology of glaucoma

and potentially lead to the discovery of new drug targets.

Table 1 highlights the major (and historic) animal models

that exist in both the natural and spontaneous classifications,

and segregates models according to species. Because of their

contribution to knowledge about hypertension and sponta-

neous or induced glaucoma, animal models have facilitated

the development of therapeutic strategies [24]. As Table 1

shows, in glaucoma, a wide variety of animal models of

different species have been used to study the disease

[24,25]. These include large animals such as monkeys [26],

dogs [27,28], pigs [29], and small animals such as rodents [21].

However, no ideal animal model for glaucoma exists and

Table 2 lists the advantages and disadvantages associated



Vol. xxx, No. xx 2014 Drug Discovery Today: Disease Models | Models for eye disorders


Please cite this article in press as: McNally S, O’Brien CJ. Drug discovery in glaucoma and the role of animal models, Drug Discov Today: Dis Model (2014), http://dx.doi.org/

Table 1. Natural and induced models of glaucoma

Species Naturally occurring glaucoma models Induced glaucoma models

Mouse - DBA/2J mouse; develops progressive increase of IOP; death of

ganglion cells [62]

- Increase in IOP appears at 8 months, pressure remains chronically

high until death; model of spontaneous, chronic, high IOP; suitable

for studying cause of pathology

- Transgenic mouse strain expressing the Tyr423His myocilin point

mutation corresponding to the human MYOC Tyr427His mutation

developed to study POAG [63,64]; loss of RGCs in peripheral retina,

axonal degeneration on ON, moderate and persistent elevation of

IOP

- Transgenic mouse; a1 collagen type 1 mutation. POAG model. open

angles, progressive ON axonal loss, gradual elevation of IOP [65,66]

Rabbit - 1960s; albino New Zealand rabbits, open-angle glaucoma, alteration

in trabecular meshwork development [67]

- Reduction in structural support of the trabeculae could be the cause

of elevated IOP

- Corticosteroid model; topical steroid application, increase in IOP in

albino rabbits [68]; mimics human chronic open angle glaucoma

Dog - Inherited POAG in lab beagles [69]; autosomal recessive, bilateral

elevation of IOP, reduction in AH outflow

- Closed angle glaucoma in Beagles, Cockers, and Basset hounds

- Cocker race develops glaucoma from an early age; Beagles and

Bassets the process is progressive and expressed between 6-12

months of age [70]

- Autosomal recessive phenotype in Beagles, present a pre-glaucoma

stage

- Glaucoma treated pharmacologically (pilocarpine, epinephrine,

acetozolamide, dichlorphenamide)

Non-human

primates

- First described in quay/Cayo Santiago Macque/Macaca monkeys in

Puerto Rico; maternal inheritance, 40% prevalence of elevated IOP;

loss of retina ganglion cells, excavation of the optic nerve and

electrophysiological evidence of damages in the retina peripheral

field [71]

- Earliest models of induced glaucoma

- Alpha chymotrypsin model (Lessell and Kuwabara, 1969); atrophy in

ciliary body. Blockage of anterior chamber by drug-induced lysates.

Elevated IOP

- Experimental monkey model of POAG. Argon laser photocoagulation

[72]. First laser model of glaucoma. Argon laser-induced scar forma-

tion of trabecular meshwork (Gaasterland and Kupfer, 1974); tem-

porary increase in IOP caused by fibrin mesh obstructing the

trabecular meshwork. IOP elevation in 70% of animals. ON cupping,

loss of RGCs, thinning of nerve fiber layer

- Model of chronic IOP elevation developed using latex microspheres

into anterior chamber, inexpensive [40]

- Model of chronic IOP elevation developed using autologous fixed red

blood cells/ghost blood cells injected into anterior chamber; cant

visualise fundus [38,39]

- Model of acute elevation of IOP [41]

Rats - 1995; cauterisation of epi-scleral veins, induce chronically high IOP

[up to 6 months, 25% rat life]. Trabecula protected [31]; pressure-

reducing and neuron-protecting drugs tested in this model. Less

invasive than laser photo-coagulation

- Long-lived glaucomatous rats present optic disc changes similar to

those in late stage human glaucoma [73]

- Rat glaucoma model, induced by topical application of dexametha-

sone; to study myocilin expression. Shows elevated IOP [74]

- Hypertonic saline solution injection to episcleral vein [32]; increase

IOP by reducing AH drainage

- Injection of magnetic microspheres; directed by handheld magnet [75]

Zebra-fish - wdr36 mutant; used to characterise wdr36 function but does not

show typical glaucoma phenotype [76]

- bug eye mutant; RGC death and high IOP [77,78]; model used to

identify mutation in low-density lipoprotein receptor-related protein

2 (Lrp2) which is important for myopia and other glaucoma risk

factors [79]

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Table 2. Comparison of animal species used in glaucoma models

Model species Pros Cons

Mouse - Few ethical restrictions

- Sample numbers for study can be large

- Eyes are easy to obtain

- Easy to house and handle

- Genetic manipulation

- High degree of conservation between mice and human

genomes

- Inexpensive

- Availability of specific models may be limited

- Very small size of ocular globe; hard to access clinically

- Absence of lamina cribrosa in the ON

Rat - Easy to maintain in the lab

- Enable genetic manipulation

- Can be used in large numbers

- Main progress in the study of glaucoma was driven by

development of rat models

- Economic

- Small ethical issues

- increased IOP easily induced

- Chronic high IOP progressively damages RGCs (as in humans)

[80]

- The rat shares similar anatomical and developmental char-

acteristics of the anterior chamber, especially in the aqueous

outflow pathway, with the human [81–84]

- Anatomical similarities with primates regarding anterior

segment blood supply and aqueous humor drainage [85]

- Size of its eye limits its use

Non-human primates - Close phylogeny and high homology of the monkey with

humans; retinal and ON anatomy almost identical

- Broadly used for improving clinical indicators of initial optic

nerve damages in glaucoma

- Resemblance between human and primate glaucoma [36]

- Very expensive

- Limited availability

- Difficult to handle, special housing facilities

- Ethical and economical factors

- Fixed midriasis occurs; ciliar nerve damage

- Large fluctuations in IOP

- Several laser sessions needed to obtain continuous high IOP

- Severe inflammation in ocular globe; trabecular alterations

Pigs and mini-pigs - Epi-scleral cauterisation system

- Vascular and retinal studies possible as the eyes are larger

than rodent models [29].

- Pig eye/retina shares many similarities with the human [29]

- Diagnostic tools can be applied

- Mini pigs are easy to handle and grow slowly

- Visualisation of lamina cribrosa in mini pig is more difficult

than in humans 4 to central venous ring [86]

Dogs - Spontaneous inheritance of the disease without congenital

abnormalities

- Availability of genome sequence

- Relatively large eyes

- Can be aggressive and difficult to handle

- Availability may be limited

- Intrascleral plexas rather than a Schlemm’s canal

Rabbits - Inadequate model for studying alterations in the retina or its

vascularisation in glaucoma

- Absence of lamina cribrosa

- Partial myelinisation (by oligodendrocytes) of optic axons

within the retina

- Prominent vasculous sac

- Steroid model; IOP measurements are difficult to standardise

as rabbit eye dries variably depending on stress

Zebra-fish - Short generation time

- Well supported genomic infrastructure

- Can be maintained in a small space

- Attractive model for genetic manipulation





with various animal species for their inclusion as an experi-

mental model.

Rudzinski and Saragovi review rat glaucoma models cur-

rently used in research and categorise each model based on

the (pre-trabecular, trabecular and post-trabecular) mechan-

ism of increased IOP [30]. Episcleral cauterisation models are

used widely in the literature owing to their feasibility and lack

of complications compared to other in vivo models. For the

examination of alterations in IOP, one of the most exten-

sively used animal models is the chronic moderately elevated

IOP rat. This model is based on the obstruction of the veins

responsible for drainage, either by cauterisation of episcleral

veins [31] or by micro-injection of hypertonic solution [32].

Also, pharmacological studies of pressure-lowering and neu-

roprotective agents employ the chronic IOP elevation rat

model based of Sharma’s episcleral vein cauterisation [31],

and this is generally done preceding studies in larger animals

and human clinical trials. Aims to investigate the causes of

increased IOP from anatomic and functional alterations in

the eye and the optic nerve rely heavily on non-human

primate (monkey) models [33–37]. Additionally, models of

chronic IOP elevation have been designed using autologous

fixed red blood cells [38,39] and latex microspheres in experi-

mental monkeys [40]. The mechanism of optic nerve damage

has also been studied in a separate model which develops

acute IOP elevation [41]. Interestingly, the identification of

ADAMTS10 as a candidate gene for POAG arose from a

genome wide SNP array study to map disease genes in a

canine model of POAG (autosomal recessive) [42].

Drug discovery from animal models – areas to watch

The development of new pharmacological interventions

comes from studies of the molecular mechanisms of pathology

and the mechanisms which lead to RGC death, both of which

rely on animal models. Drug discovery for glaucoma is both

enhanced and impeded by unique features of the human eye.

In situ visualisation of the optic nerve and retina is possible

with non-invasive diagnostic techniques (e.g. optical coher-

ence tomography). Because of the clinical accessibility of the

human eye tissues, drug delivery methods such as local injec-

tions or eye drops can be employed. Systemic toxic effects are

minimised in cases of local delivery and exposure to a ther-

apeutic agent and this enhances drug therapeutic index. How-

ever, in spite of the advantage of clinical accessibility, other

challenges exist in relation to ocular barriers of the human eye.

For example, drug efficacy can be reduced and drug transport

impeded by ocular barriers (tear dilution, blood flow, lympha-

tic clearance, blood-ocular barriers) [43]. Because of these

factors and to the complexity of the disease itself, glaucoma

drug discovery is a relatively slow process. In fact, no new

classes of glaucoma drugs have arisen since Latanoprost, a

prostaglandin which was launched a decade ago and is now


considered first-line treatment. We now highlight areas of

interest for potential emerging therapies.

Novel IOP-lowering drugs

Inhibition of actin polymerisation via the action of Latrun-

culins (marine sponge macrolides) is a possible mode of IOP-

reduction which is under current study [44]. A novel mechan-

ism of action of actin cytoskeleton disruption has been

reported for Latrunculins where trabecular meshwork out-

flow is increased in the eyes of male and female adult cyno-

molgus monkeys and post-mortem patient eyes [45].

Unfortunately, clinical trials to date have only yielded mar-

ginal success and this is possibly due, in part, to their poor

solubility. It is hoped that improvements in drug efficacy may

follow changes in the delivery system [46].

Cannabinoid receptor agonists

Intriguingly, historic evidence from observations in the

1970s highlight a transient IOP reduction in response to

smoking marijuana and has prompted interest in the use

of cannabinoid receptor agonists for glaucoma disease [47].

It has subsequently been shown that topical application of a

cannabinoid receptor agonist lowers IOP in a non-human

primate induced model of glaucoma (unilateral induction via

argon or diode laser photocoagulation of the mid-trabecular

meshwork) [48]. This study employed an agonist selective for

cannabinoid receptor 1 using normotensive and glaucoma-

tous adult female Macaca cynamolgus monkeys and IOP was

reduced by a reduction in AH flow.

Neuroprotective agents

As highlighted, current treatment methods for glaucoma

focus on IOP reduction and fail to combat associated retino-

pathy and optic neuropathy. It is clear that there is a require-

ment for neuroprotective treatment of glaucoma. A common

end point in glaucoma and retinal diseases is the death of

retinal neurons. As such, neuroprotective therapies are an

unmet medical need for glaucoma patients but have been a

focus of animal model research for some time. For examples,

agonists of the 5-hydroxy-tryptamine 1A receptor (5-HT1A)

are documented to have neuroprotective qualities. AL-8309B

is a topical selective agonist of 5-HT1A which has been used in

studies using male Wistar and male LongEvans rats as a model

of central nervous system injury [49,50]. AL-8309B represents

a promising next generation therapy as results show neuro-

protective effects against excitotoxic neuronal damage. In

addition, a separate model of excitotoxic neuronal and light

damage using male Sprague-Dawley rats demonstrated a

reduction in neuronal death upon treatment with AL-

8309B [51].

Reduced IOP is reported for agonists of the a-adrenergic

receptor and experimental animal models also reveal RGC

protection but convincing data has yet to emerge from






patient studies [52]. One a2-adrenergic receptor agonist,

brimonidine tartrate, which was originally created for IOP

reduction, has proven promising as a neuroprotective agent

in rodent models. Induction of retinal degeneration by light

damage in male Sprague-Dawley rats reveals that brimoni-

dine is protective for photoreceptors and enhances the pro-

duction of neurotrophic factors [53]. An experimental rodent

model of ocular hypertension (argon laser photocoagulation

used for IOP elevation in male Wistar rats) documents pro-

tection of RGCs in response to systemic administration of

brimonidine [54]. Topical brimonidine is FDA approved for

glaucomatous IOP reduction and its preservation of visual

field loss has been studied.

A role for melatonin in the treatment and management of

glaucoma is slowly emerging. Melatonin in the eye is a

promising agent which has local antioxidant effects due to

the inhibition of nitric oxide synthase and this makes it a

candidate as a neuroprotective factor [55–57]. Animal models

used for the study of melatonin action on IOP are reviewed in

[58]. Studies using a normotensive white rabbit model have

shown that the melatonin derivative, agomelatine, is a hypo-

tensive compound which could also hold promise for glau-

coma treatment [59]. While agomelatine has proven IOP-

lowering ability (equal to that of melatonin), it is also a

neuroprotective agent [60].

Comparisons can be drawn between chronic neurodegen-

erative disorders and glaucoma and this can shed light on

factors responsible for disease progression [61]. For example,

Alzheimer’s disease and glaucoma can both be characterised

by dysregulation of neurotrophic growth factors, caspase

activation and both diseases can be managed via NMDA

(N-methyl-D aspartate) receptor antagonists, neurotrophins

or immune regulators. One NMDA receptor antagonist, mem-

antine, has come to the fore in studies of non-human primate

experimental models. RGCs can become over-loaded with

intracellular calcium if there is sustained activation of the

NMDA signalling pathway, and this results in cell death by

apoptotic means. In an experimental primate model of

induced unilateral glaucoma, RGCs of non-human primates

are conferred protection (as are relay neurons of the lateral

geniculate body) in response to oral administration of mem-

antine [1]. To date, the efficacy of memantine in glaucoma

has failed at Phase III of clinical trials as patients at high risk of

developing glaucoma see no reduction in visual field loss.

Conclusion

Progress in glaucoma treatment has been associated with the

development of animal models and disease prevention and

neurological protection are prime areas of focus [24]. One

major impediment to breakthrough in human studies is that

damage present at diagnosis precludes the study of human

disease development from onset. Comparative animal studies

therefore, are necessary and have broadened understanding



of glaucoma whilst also facilitating development of thera-

peutic strategies which could not have been developed other-

wise. The potential biological divergence between animals

and human is ground for caution in aiming for a direct

translation of preclinical outcomes to patients. Glaucoma

remains incurable.

Conflict of interest

The authors have no conflict of interest to declare.

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