41

INTRODUCTION

Endothelin (ET) is an endogenous neuropeptide and one of the most potent vasoconstrictors known. Endothelins (ET-1 and ET-3) and their receptors (ETA

and ETB) are present in most of the ocular tissues, including iris, choroid, retina, optic nerve head, cili-ary body, lens, and corneal endothelium.1 These sub-stances have numerous ocular actions that include the regulation of intraocular pressure (IOP); the regulation of choroidal, retinal and optic nerve blood circulation; and the tone of iris smooth muscle and ciliary muscle.1,2 When present in excess, these substances may contrib-ute to the development of the glaucomatous optic neu-ropathy. This is supported by numerous pre-clinical and clinical studies.3,4 A significant correlation between

Current Eye Research, 36(1), 41–46, 2011Copyright © 2011 Informa Healthcare USA, Inc.ISSN: 0271-3683 print/ 1460-2202 onlineDOI: 10.3109/02713683.2010.512695

ORIGINAL ARTICLE

Effect of SPP 301, an Endothelin Antagonist, on Intraocular Pressure in Glaucomatous

Monkey Eyes

Rong-Fang Wang1, Steven M. Podos1,2, Janet B. Serle1, and Ovidiu C. Baltatu3

1Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York, USA2Department of Neuroscience, Mount Sinai School of Medicine, New York, New York, USA

3Gr. T. Popa University of Medicine and Pharmacy, Iasi, Romania

ABSTRACT

Purpose: To evaluate the effect of topical application of avosentan (SPP 301), endothelin receptor type A antagonist, on intraocular pressure (IOP) in monkey eyes with laser-induced unilateral glaucoma.Materials and Methods: A multiple-dose study was performed in eight glaucomatous monkey eyes that were topically treated with SPP 301 by applying a 50 µl drop (25 µl × 2) at 9:30 a.m. and 3:30 p.m. for 5 consecutive days at three concentrations (0.003%, 0.03%, or 0.3%). IOP was measured hourly for 6 hrs on each day of the study beginning at 9:30 a.m. for one baseline day, one vehicle-treated day, and treatment days 1, 3, and 5.Results: Twice daily administration of each of the three concentrations of SPP 301 for 5 days sig-nificantly (p < 0.05) reduced IOP. The maximum reduction in IOP occurred 2 or 3 hrs after morning dosing and was 1.8 ± 0.8 (mean ± SEM) mmHg (6%) for 0.003% SPP 301, 4.1 ± 0.7 mmHg (13%) for 0.03% SPP 301, and 7.1 ± 1.3 mmHg (21%) for 0.3% SPP 301. The longest duration of IOP reduction was for 2 hrs with 0.003% SPP 301, and was for at least 6 hrs with 0.03% and 0.3% concentrations. Compared to 0.03% or 0.003% concentrations, 0.3% SPP 301 produced a greater (p < 0.05) IOP reduc-tion. IOP was reduced in fellow untreated normal eyes 2 hr after morning dosing with 0.3% SPP 301, maximum reduction in IOP (11%) occurred on day 1. Of the eyes treated with 0.3% SPP 301, one eye demonstrated mild conjunctival discharge and one eye was closed for 5 min after dosing.Conclusion: Topically applied SPP 301, an endothelin antagonist, reduced IOP in glaucomatous monkey eyes in a dose-dependent manner. Endothelin antagonists, a novel class of compound, may have potential for the treatment of glaucoma.

KEYWORDS: Avosentan (SPP 301); Endothelin Antagonist; Glaucoma monkey; Intraocular pressure

Received 20 April 2010; accepted 28 July 2010

Correspondence: Rong Fang Wang, M.D., Department of Ophthalmology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1183, New York, NY 10029. E-mail: rong-fang.wang@mssm.edu

20 April 2010

28 July 2010

© 2011 Informa Healthcare USA, Inc.

2011

Current Eye Research

0271-36831460-2202

10.3109/02713683.2010.512695

36

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512695

NCER

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42 R. F. Wang et al.

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aqueous/vitreous ET-1 levels and retinal damage was seen in dogs with spontaneous hypertensive glaucoma.5 ET-1 caused contraction of human trabecular meshwork cells and a proposed mechanism of IOP elevation may be through decreased trabecular outflow facility.6 In patients with normal tension glaucoma (NTG), primary open angle glaucoma (POAG) and exfoliation syndrome, elevated levels of ET-1 in the systemic circulation and in the aqueous humor have been reported.7–10 A correla-tion between IOP level and the concentration of ET-1 in aqueous humor was observed in patients with POAG and pseudo-exfoliation glaucoma.11 Ocular blood flow in the choroid, retina, and optic nerve head decreased in healthy human volunteers following intravenous administration of ET-1.12,13

The effect of topical administration of ET antagonist on IOP has not yet been reported. This study evaluated the effect of SPP 301, an ET receptor type A antagonist, on IOP at three concentrations in laser-induced glau-comatous monkey eyes.

MATERIALS AND METHODS

Animals

Ten adult female cynomolgus monkeys each weighing 3–5 kg, in which glaucoma had been induced unilater-ally by repeated diode laser photocoagulation of the mid-trabecular meshwork, were used in this study.14 All experimental studies complied with the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research and were approved by the Institutional Animal Care and Utilization Committee of Mount Sinai School of Medicine (New York, NY).

IOP Measurement and Slit-Lamp Examination

IOP was measured hourly for 6 hrs on each day of the study beginning at 9:30 a.m. for one baseline day, one vehicle-treated day, and treatment days 1, 3, and 5. All IOP measurements were obtained with a calibrated pneumatonometer (Model 30 Classic; Mentor Inc., Norwell, Massachusetts, USA). The monkeys were sedated with intramuscular ketamine hydrochloride (2–5 mg/kg), and one drop of 0.5% proparacaine hydrochloride was administered topically 5 min before all measurements of IOP.

Slit-lamp examination for the detection of aqueous humor flare and cells was performed in a dark room before SPP 301 administration and at 1, 3, and 5 hr after treatment on days 1, 3, and 5.

Preparation and Instillation of Testing Compounds

SPP 301 and vehicle solution was prepared and provided by Speedel Experimenta Ltd. (Basel, Switzerland). A multiple-dose study was performed in eight unilaterally glaucomatous monkeys. The ani-mals were randomly divided into two groups. Each concentration was initially tested in groups of four glaucomatous monkey eyes, subsequently crossed over and then each concentration was repeated in another four glaucomatous monkeys. After one baseline day (untreated) and one vehicle-treated day, two 25 µl drops of SPP 301 0.003%, 0.03%, or 0.3% were topically applied to the glaucomatous eye at 9:30 a.m. and 3:30 p.m. The washout period between each concentration of SPP 301 tested was 2 to 4 weeks.

Statistics

Repeated measures ANOVA test was used for analysis of the multiple-dose study, and one-way ANOVA was used for analysis of the differences between groups. P < 0.05 was considered statistically significant. Data were calculated as the mean ± SEM.

RESULTS

Twice daily administration of 0.003% SPP 301 to eight glaucomatous monkey eyes for 5 days significantly (p < 0.05) reduced IOP only at 2 hrs after the first dose, and at 2 and 3 hrs after the 9th dose. The maximum reduction in IOP was 1.8 ± 0.8 (mean ± SEM) mmHg (6%), after the morning dosing on treatment day 5. No enhancement of IOP reduction was observed after repeated dosing (Figure 1A).

Twice daily administration of 0.03% SPP 301 to eight glaucomatous monkeys reduced (p < 0.05) IOP from 2–5 hrs after the first dose on day 1. The maxi-mum reduction in IOP occurred 2 or 3 hrs after each morning dosing and was 2.5 ± 1.0 mmHg (8%) on day 1, 4.0 ± 0.5 mmHg (13%) on day 3, and 4.1 ± 0.7mmHg (13%) on day 5. The longest duration of IOP reduction was for at least 6 hrs after the 5th dose (Figure 1B). IOP reductions were greater (p < 0.05) on day 3 than on day 1 at 1 hr and at 4–6 hrs after dosing (Figure 1B). IOP reductions were similar (p > 0.05) on days 3 and 5.

Twice daily administration of 0.3% SPP 301 to eight glaucomatous monkeys significantly (p < 0.05) reduced IOP from 1–5 hrs after the first dose. The maximum reductions in IOP occurred 3 hrs after each morn-ing dosing and were 4.4 ± 0.8 mmHg (13%) on day 1, 5.3 ± 0.7 mmHg (16%) on day 3, and 7.1 ± 1.3 mmHg

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New Class of Compound for Treating Glaucoma 43

© 2011 Informa Healthcare USA, Inc.

(21%) on day 5. The longest duration of IOP reduction was at least for 6 hrs after the 5th dose (Figure 1C). Enhancement of IOP reduction was observed from day 1 to day 5 with repeated dosing. IOP reduction was greater (p < 0.05) on day 3 than on day 1 at 1, 3, and 5 hrs, and was greater on day 5 than on day 3 at 1 and 3 hrs.

A significant (p < 0.05) reduction in IOP was observed only with the highest concentration of SPP 301, 0.3% in fellow untreated normal eyes at 2 hr after each morning dosing, with a reduction in IOP of 2.0 ± 0.5 mmHg (11%) on day 1, 1.3 ± 0.5 mmHg (7%) on day 3, and 1.1 ± 0.4 mmHg (6%) on day 5.

IOP

( m

mH

g ±

SE

M)

0 1 2 3 4 5 624

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**

0.003% SPP 301 VehicleDay 1Day 3Day 5

0 1 2 3 4 5 6

0 1 2 3 4 5 6

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*

* ****** ** **

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0.03% SPP 301

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38C

B

***

******

****

****** **

****

**

0.3% SPP 301

Hours

VehicleDay 1Day 3 Day 5

VehicleDay 1Day 3Day 5

FIGURE 1 Effects of twice-daily administration of SPP 301, 0.003%, 0.03%, and 0.3%, for 5 days on intraocular pressure (IOP) in eight glaucomatous monkey eyes. Points represent mean ± SEM mmHg of IOP on the vehicle-treated day and days 1, 3, and 5 of treatment. Asterisks indicate a significant reduction in IOP (*p < 0.05, **p < 0.005) of treated eyes with glaucoma compared with values in the same eyes treated with vehicle (repeated- measures ANOVA).

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44 R. F. Wang et al.

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The duration and magnitude of IOP reductions were different (p < 0.05) when comparing three concentra-tions of SPP 301, 0.3%, 0.03%, and 0.003%. The 0.03% and 0.3% concentrations produced a longer duration of action (6 hrs vs. 2 hrs) than the 0.003% concentration. SPP 301 0.3% produced a greater (p < 0.05) IOP reduc-tion than 0.03% or 0.003% SPP 301 (Figure 2).

IOP on baseline and vehicle treatment days was similar (p > 0.80) in all three treatment groups. The

mean baseline and vehicle-only treated IOPs of the three treatment groups were similar (p > 0.90).

Ocular side effects were observed in two of the eyes treated with the 0.3% concentration. One eye exhibited mild conjunctival discharge on treatment days 3 and 5, and another eye was closed after dosing for about 5 min on days 1 and 3. No anterior chamber cellular response was observed in any eye during the 5 days of treatment by slit-lamp examination.

Cha

nge

In IO

P (M

ean

mm

Hg

± S

EM

)

Hours0 1 2 3 4 5 6

**

****

*

Day 5

SPP 301 0.003%SPP 301 0.03%SPP 301 0.3%

SPP 301 0.003%SPP 301 0.03%SPP 301 0.3%

SPP 301 0.003%SPP 301 0.03%SPP 301 0.3%

0 1 2 3 4 5 6

−8

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−6

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2

*****

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*

Day 3

FIGURE 2 Comparison of reduction in intraocular pressure (IOP) of three concentrations of SPP301 0.003%, 0.03%, and 0.3% on days 1, 3, and 5 of dosing, in eight glaucomatous monkey eyes. Points represent mean change ± SEM mmHg of IOP from vehicle-treated values. Asterisks indicate significant difference of IOP reduction (*p < 0.05, **p < 0.005) comparing SPP301 0.003%, 0.03%, and 0.3% (one-way ANOVA).

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New Class of Compound for Treating Glaucoma 45

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DISCUSSION

Recent accumulated data indicate that the endothelin system is involved in the pathogenesis of glaucoma.1–8 ET-1, a potent vasoconstrictor peptide produced by vascular endothelin cells, has an important role in vascular homeostasis and in numerous vasospastic diseases, including diabetic retinopathy, retinal arte-riovenous occlusions, choroidal ischemia, amaurosis fugax, and glaucoma.2,15 Several physiological or pathophysiological stimuli cause the release of ET, including possibly elevated IOP and reduced ocular blood flow. Elevated levels of ET-1 in the eye have been shown to decrease trabecular outflow facility, to elevate IOP, to reduce ocular blood flow, and to dam-age the optic nerve head.3–12 Thus, application of an endothelin antagonist would be expected to reduce IOP and improve ocular blood flow by inhibiting ET activity. Pretreatment with an ETA receptor antago-nist has been shown to protect against the acute IOP elevation induced by argon laser trabeculoplasty in rabbits.16 An orally administered dual ETA and ETB receptor antagonist has been shown to increase retinal, choroidal, and optic nerve head blood flow in patients with glaucoma and in healthy subjects.17 Intravenous administration of a selective ETA receptor antagonist blunted a decrease in retinal blood flow in patients with NTG.18 These observations suggest that ET-1 may be a contributing factor to increased IOP and optic nerve damage in glaucoma.

This study is the first to demonstrate that the topi-cal application of an ET antagonist reduces IOP. In this study, 5 days of twice daily dosing with SPP 301, an ETA receptor antagonist, significantly reduced IOP in laser-induced glaucomatous monkey eyes. Enhancement of the ocular hypotensive effect was observed with repeated dosing. The reduction in IOP appears to be dose-dependent; IOP reductions were greater with 0.3% SPP 301 than with the 0.03% and 0.003% concen-trations (21% vs. 13% vs. 6%). Longer durations of IOP reduction were recorded with 0.3% and 0.03% concen-trations than with the 0.003% concentration (6 hrs vs. 2 hrs, respectively). A reduction in IOP in the fellow untreated control eyes, which was of lesser magnitude than in the treated eyes, and which was only observed at the time of peak IOP reduction with 0.3% SPP 301 suggests an effect due to systemic absorption follow-ing topical application in these small animals. SPP 301 was well tolerated, causing minimal, transient exter-nal ocular side effects in only two of the animals at the highest concentration. Intraocular inflammation was not observed.

The mechanism of endothelin-dependent regulatory functions in the eye is complex and not completely understood. The ocular effects may

depend on the amount of ET present, the duration of exposure, the tissues involved, and the ET receptors that are expressed.2 The regulation of the synthesis and release of ET are affected by numerous stimuli including ischemia, hypoxia, and adrenergic- and muscarinic-stimulation, which are often encoun-tered in a number of cardiovascular diseases includ-ing myocardial infarction, hypertension, congestive heart failure, and cerebral vasospasm. These stimuli are also important contributors to the development of glaucoma.1,2 Additional investigations of this class of compounds and their effects in the eye will clarify the role of the endothelin pathways in patients with glaucoma.

In conclusion, topical application of SPP 301, a new ETA receptor antagonist, reduces IOP in glaucomatous monkey eyes in a dose-dependent manner. Endothelin antagonists represent an exciting new potential class of compounds for treating glaucoma possibly by a dual mechanism, reducing IOP and preventing endothelin-mediated optic neuropathy. Further investigations of the ocular effects and mechanism by which this compound and others in this class lower IOP are warranted.

ACKNOWLEDGMENTS

The authors wish to acknowledge support by an unre-stricted grant from Research to Prevent Blindness, Inc., New York, NY and Speedel Experimenta Ltd., Basel, Switzerland.

Declaration of interest: Drs. Wang, Podos, and Serle have no proprietary interest in the drug evaluated in this article. Dr. Baltatu was an employee of Speedel Experimenta, Ltd. The authors report no conflicts of interest. The authors alone are responsible for the con-tent and writing of the paper.

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[2] Yorio T, Krishnamoorthy RR, Prasanna G. Endothelin: Is it a contributor to glaucoma pathophysiology? J Glaucoma. 2002;11:259–270.

[3] Orgul S, Cioffi GA, Bacon DR, et al. An endothelin-1 induced model of chronic optic nerve ischemia in rhesus monkeys. J Glaucoma. 1996;5:135–138.

[4] Balwantray CC. Model of chronic endothelin-induced optic nerve damage in rat. Exp Eye Res. 2000;17:S24.

[5] Kallberg ME, Brooks DE, Allis AE, et al. Correlation of reti-nal damage to endothelin 1 levels in the aqueous humor and vitreous in dogs with spontaneous hypertensive glaucoma. Invest Ophthalmol Vis Sci. 2002;43:E-Abstract 312.

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[7] Sugiyama T, Moriya S, Oku H, et al. Association of endothe-lin-1 with normal tension glaucoma. Surv Ophthalmol. 1995;39:S49–S56.

[8] Kaiser HJ, Flammer J, Wenk M, et al. Endothelin-1 plasma level in normal tension glaucoma: Abnormal response to postural changes. Graefes Arch Clin Exp Ophthalmol. 1995;233:484–488.

[9] Lepple-Wienhues A, Becker M, Stahl F, et al. Endothelin-like immunoreactivity in the aqueous humor and in conditioned medium from cultures ciliary epithelial cells. Curr Eye Res. 1992;11:1041–1046.

[10] Koliakos GG, Konstas AGP, Schlotzer-Schrehardt U, et al. Endothelin-1 concentration in the aqueous humor with exfoliation syndrome. Br J Ophthalmol. 2004;88: 523–527.

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[12] Polak K, Luksch A, Frank B, et al. Regulation of human retinal blood flow by endothelin-1. Exp Eye Res. 2003;76:633–640.

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[14] Wang RF, Schumer RA, Serle SB, et al. A comparison of argon laser and diode laser photocoagulations of the trabe-cular meshwork to produce the glaucoma monkey model. J Glaucoma. 1998;7:45–49.

[15] Flammer J, Pache M, Resink T. Vasospasm: Its role in the pathogenesis of diseases with particular reference to the eyes. Prog Retina Eye Res. 2001;20:319–349.

[16] Hollo G, Kothy P, Lakatos P, et al. Endothelin-A recep-tor antagonist BQ-485 protects against intraocular pres-sure spike induced by laser trabeculoplasty in rabbits. Ophthalmologica. 2002;216:459–462.

[17] Resch H, Karl K, Weigert G, et al. Effect of dual endothe-lin receptor blockade on ocular blood flow in patients with glaucoma and healthy subjects. Invest Ophthalmol Vis Sci. 2009;50:358–363.

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