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Role of Alpha-2 Adrenergic Receptors in Neuroprotection and Glaucoma
SURVEY OF OPHTHALMOLOGY VOLUME 45 • SUPPLEMENT 3 • MAY 2001
CURRENT RESEARCH
Role of Alpha-2 Adrenergic Receptors in Neuroprotection and Glaucoma Larry A. Wheeler, PhD, Daniel W. Gil, PhD, and Elizabeth WoldeMussie, PhD Department of Biological Sciences, Allergan, Inc., Irvine, California, USA Abstract. The loss of retinal ganglion cells (RGCs) in glaucoma occurs progressively over many years. A neuroprotective drug should enhance survival of RGCs in the presence of chronic stress/injury. Four criteria are proposed for assessing the likely therapeutic utility in human glaucoma of drugs that have demonstrated neuroprotective activity in animal models: 1) A specific receptor target must be in the retina/optic nerve; 2) Activation of the target must trigger pathways that enhance a neuron’s resistance to stress/injury and/or suppresses toxic insults; 3) The drug must reach the retina/vitreous at pharmacologic doses; and 4) The neuroprotective activity should be demonstrated in clinical trials. Data are presented that illustrate how the specific and potent alpha-2 agonist, brimonidine, meets these criteria. The alpha-2A receptor was localized in the inner rat retina by immunohistochemistry. Brimonidine reduced the rate of RGC loss in the calibrated rat optic nerve injury model even when dosed 12 and 24 hours before injury, consistent with a long-term enhancement of RGC resistance to stress. Brimonidine was also neuroprotective in the lasered chronic hypertensive rat model, reducing RGC loss over three weeks from 33% to 15%. A clinical trial has been initiated to determine brimonidine’s neuroprotective activity in patients with non-arteritic ischemic optic neuropathy. (Surv Ophthalmol 45(Suppl 3):S290– S294, 2001. © 2001 by Elsevier Science Inc. All rights reserved.) Key words. alpha-2 adrenergic receptors • alpha-2 agonists • brimonidine • cell death pathways • cell survival pathways • glaucoma • neuroprotection • neuroprotective signaling pathways
Chronic and progressive retinal ganglion cell loss is a central feature of the glaucomas. The loss of RGC axons in the optic nerve occurs over many years. It is postulated that the RGCs and neighboring cells maintain a balance between intrinsic cell survival signals and neurotoxic signals. Over time, because of risks such as ischemia, elevated IOP, genetics, etc., the balance is shifted and cells enter a programmed cell death pathway, called apoptosis. One of the hallmarks of apoptosis has been demonstrated by terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate (UTP)-biotin nick end-labeling (TUNEL)-positive cells in histologic sections from glaucoma patients.12 Thus, a neuroprotective drug in glaucoma prevents the progres-
sive loss of RGCs by maintaining and/or enhancing their ability to resist stress and survive. It may do this by either enhancing cell survival pathways or interfering with neurotoxic signals. The alpha-2 receptor is one of the targets of the natural stress hormone norepinephrine, so it is likely to be involved in modulating cellular resistance and/or adaptation to counter stress/injury. In fact, alpha-2 agonists have been demonstrated to be neuroprotective in rat models of stroke involving cerebral ischemia.9,17 Activation of alpha-2 receptors by agonists such as brimonidine have more recently been shown to enhance survival of retinal neurons after many types of injuries and insults (Table 1). These models include transient ischemia,5,8,13,19 calibrated
S290 © 2001 by Elsevier Science Inc. All rights reserved.
0039-6257/01/$–see front matter PII S0039-6257(01)00206-5
S291
ROLE OF ALPHA-2 ADRENERGIC RECEPTORS TABLE 1
Animal Models in Which Brimonidine Showed Neuroprotective Activity Model
Reference
Optic nerve crush Ocular hypertensive rat Pressure induced ischemia Vascular ischemia SOD-1 over expressing mice Light-induced photoreceptor damage Photoreceptor degeneration—frog
Ruiz et al, 200018 Yoles et al, 199924 Ahmed et al, 19991 WoldeMussie et al, 200023 Donello et al, 20015 Hasson et al, 19978 Villegas-Perez et al, 200019 Levkovitch-Verbin et al, 200015 Lai et al, 199814 Wen et al, 199620 Ervin et al, 19996
optic nerve crush,18,24 photoreceptor degeneration,6,14,20 chronic ocular hypertension,1,23 and transgenic mice overexpressing superoxide dismutase (SOD).15 There are many pathways activated by alpha-2 receptors that might be involved in raising a neuron’s resistance to stress/injury. These include the activation of intracellular kinases that enhance cell survival and the inhibition of glutamate release5 and calcium influx into cells.22 The studies mentioned above suggest that the alpha-2 receptor and its signaling pathways are good drug targets for retinal neuroprotection. The studies also raise the question of what criteria can be used to compare or evaluate current and new drugs to determine potential usefulness for testing in human clinical trials (Table 2). For a drug to be neuroprotective in glaucoma, there is a specific target in the retina/ optic nerve; the drug must reach the retina at levels sufficient to activate its target; it must have a mechanism of action that enhances a neuron’s resistance to stress or suppresses toxic insults; and there should also be demonstrated activity in human clinical trials. Presented in this article is an evaluation of how brimonidine meets these four criteria. Also presented are two new findings that help to evaluate the mechanism of action of brimonidine. In particular, TABLE 2
Criteria for Neuroprotective Drugs in the Retina • Has a specific target in the retina/optic nerve • Reaches the retina at pharmacological levels • Has mechanism of action that enhances a neuron’s survival to stress or block toxic insult • Demonstrates activity in human clinical trials
we demonstrate that brimonidine is neuroprotective in the laser-induced chronic ocular hypertensive rat. Secondly, a single dose of brimonidine at 24 and 14 hours before injury is neuroprotective, supporting the working hypothesis that activation of the alpha-2 receptor results in a neuroprotective signaling pathway that enhances neuronal survival even after brimonidine has been cleared from the body.
Methods IMMUNOHISTOCHEMISTRY
Immunohistochemistry for the alpha-2A receptor was done on paraformaldehyde fixed, frozen sections of rat retinas. Tissue sections were incubated overnight at 4C with antibodies against the alpha2A adrenergic receptor subtype (generously supplied by Dr. J. Regan, University of Arizona). Sections were washed several times and incubated with secondary antibody followed by Vectastain® ABC for color development. They were counterstained with methyl green. In control samples, primary antibody was omitted. CALIBRATED OPTIC NERVE INJURY
Sprague Dawley rats were anesthetized with a mixture of ketamine (15mg/kg), acepromazine (1.5 mg/kg) and xylazine (0.3 mg/kg). Calibrated optic nerve injury was applied as described previously.24 Brimonidine at 0.1 mg/kg was given by intraperitoneal injection, at 24 or 14 hours before optic nerve injury or immediately after injury (time 0). Control animals received phosphate buffered saline (PBS) vehicle. Ganglion cell survival was evaluated 12 days later. The control group received the same anesthetic protocol of ketamine and xylazine. Therefore, the neuroprotective effect of brimonidine was the difference between control and treated animals. CHRONIC OCULAR HYPERTENSION
Intraocular pressure (IOP) was raised by laser photocoagulation of episcleral and limbal veins on day 0 and day 8. Drugs were applied by osmotic pump (Alzet) implanted subcutaneously on the animal’s back at the time of first laser treatment. Brimonidine was given at 1mg/kg/day and timolol 2 mg/kg/day. Control animals received PBS vehicle in a pump. Drug treatment was done for the 3-week duration of the experiment. IOP was measured weekly with tonopen. At the end of 3 weeks, ganglion cell loss was evaluated following retrograde transport of rhodamine labeled dextran. RETINAL GANGLION CELL ANALYSIS
Retinal ganglion cell loss in both models was evaluated by retrograde labeling at the optic nerve with
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WHEELER ET AL
Fig. 1. Right: Immunoreactivity of the alpha-2A adrenergic receptors is localized mainly in the somas of the cells in the ganglion cell layer and some cells in the inner nuclear cell layer. Left: Control staining where primary antibody was omitted. RGC ganglion cells; IPL inner plexiform layer; INL inner nuclear layer; PR photoreceptors. Scale bar 50 m.
rhodamine-labeled dextran.7,24 Briefly, rats were deeply anesthetized and the optic nerve exposed. A longitudinal incision was made on the meningis and the optic nerve was completely cross-sectioned at about 2 to 3 mm from the globe. Crystals of tetramethyl rhodamine, 3000 mw (Mol. Probes, OR), were applied to the cut end of the optic nerve. Twentyfour hours later the eyes were enucleated and fixed with 4% paraformaldehyde and the retinas were flatmounted. Cell counts were done in eight regions, two in each quadrant, at 400 magnification in areas starting 0.66 mm from the edge of the optic disk. This was 1.56% of the total ganglion cell count, and it provided representative counts of ganglion cells in relation to total cell number. Counting additional
areas 1–2 mm more peripheral to these regions did not alter the assessment of the extent of injury.
Results and Discussion LOCALIZATION OF ALPHA-2A RECEPTORS IN THE RETINA
A specific receptor target in the retina offers the best chance for a drug to be neuroprotective and minimize the potential for side effects. The alpha-2A receptor subtype is the most abundant of the three alpha-2 receptors in the nervous system.16 Antibodies generated against the third cytoplasmic loop of the alpha-2A receptor10 were used to localize the presence of the subtype in the retina. As shown in Fig. 1,
Fig. 2. Fluorescent micrograph of labeled retinal ganglion cells of vehicle-treated (A) or brimonidine-treated (B) retinas 12 days after injury. Brimonidine increased cell survival when injected at the time of optic nerve injury. Scale bar 50 m.
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ROLE OF ALPHA-2 ADRENERGIC RECEPTORS
there was immunoreactivity in the ganglion cell layer, which has both ganglion and amacrine cells. Some cells in the inner nuclear layer, possibly amacrine cells, were also immunopositive. The presence of alpha-2 receptors has been shown in human,4 bovine,2 and porcine21 retinas by radioligand binding studies and/or functionally by the use of alpha-2 antagonists to block the retinal neuroprotection induced by agonists such as brimonidine.24 OPTIC NERVE INJURY
With optic nerve crush, 80–85% of the ganglion cells degenerated by day 12 after injury. If brimonidine upregulates pathways that enhance survival, then determining the dosing window that is neuroprotective would help clarify this mechanism of action. Rapid mechanisms would have a short or narrow window of protection before injury whereas gene expression regulation would have a longer or larger window of protection. Brimonidine at 0.1 mg/kg when injected intraperitoneally at time 0 resulted in ganglion cell survival of about two-fold (Figs. 2 and 3). Administration of brimonidine 14 hours before injury also resulted in RGC survival that was similar to that when it was administered at time 0. Significant cell survival was also obtained when brimonidine was administered 24 hours earlier (data not shown). Brimonidine levels in the retina after 0.1 mg/kg peaked at 30 minutes at 37 3 nM and stayed above 2 nM for 6 hours (authors’ unpublished data). Analysis was done using gas chromatograph/mass spectrometry (GC-MS/ MS). Alpha-2 receptors are activated by brimonidine at a concentration of 2 nM.3
Fig. 3. Intraperitoneal administration of brimonidine at the time of optic nerve injury resulted in a two-fold survival of RGC (p 0.01). Pretreatment with brimonidine 14 hours before injury resulted in RGC survival that was equivalent to that administered at time 0. Values are represented as mean SEM from 5 to 10 animals.
CHRONIC OCULAR HYPERTENSION
The chronic ocular hypertensive model represents a model that mimics gradual death of the RGCs seen in the glaucomas. Laser treatment of the episcleral and limbal veins, as described in the methods section, resulted in a two-fold elevation of IOP. This resulted in progressive loss of RGCs. At the end of 3 weeks of elevated IOP, there was a 34 7% decrease in ganglion cells in the vehicle-treated rats. Decrease in IOP by either brimonidine or timolol was less than 10%. In rats treated with 1 mg/kg/day brimonidine ganglion cell loss was reduced by more than half, to 16 3% (Fig. 4). Ganglion cell loss in rats treated with 2 mg/kg/day timolol was similar to those treated with vehicle (Fig. 4). Thus, the protection of RGCs by brimonidine in this model was not due to the lowering of IOP. Two of the criteria for evaluating brimonidine’s potential as a therapeutic neuroprotective agent have been met. First, the specific target—the alpha-2 receptor—is present in the retina, and, second, the activation of the receptor results in ganglion cell protection under conditions of acute and chronic stress and/or injury (Table 1). The third criterion is that the drug reaches the target in the retina at pharmacological doses. Brimonidine is a highly potent and selective alpha-2 adrenergic agonist with the ability to activate its targets at concentrations above 2 nM. It does not significantly activate alpha-1 adrenergic receptors at concentrations below 2000 nM.3 Thus, the drug has the potential to be neuroprotective without causing side
Fig. 4. Ganglion cell loss was evaluated in the different treated groups after IOP was elevated for three weeks. There was a 33 7% decrease in RGC number in vehicletreated group. This was reduced to 16 3% in the presence of brimonidine, which is a 50% protection (p 0.05). With systemic timolol cell loss was 32 3%. Values are represented as mean SEM from 10 animals.
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effects if levels greater than 2 nM and less than 2000 nM can be achieved in the human retina following topical application. Initial results from a study by Kent et al11 in patients who received 0.2% Alphagan® BID for 4–14 days prior to undergoing elective pars plana vitrectomy demonstrated that vitreous levels of brimonidine were in the pharmacological range. Because brimonidine has demonstrated a broad neuroprotective profile in a variety of different kinds of injury and meets the first three criteria for a retinal/ optic nerve neuroprotective agent, Allergan has initiated clinical trials in nonarteritic ischemic optic neuropathy (NAION) to determine whether the drug’s neuroprotective effect can be measured in patients. In NAION, there is injury to the axons of the retinal ganglion cells, and this trial will provide a proof of principle for neuroprotective therapy in humans.
WHEELER ET AL
11. 12. 13.
14. 15.
16. 17.
References 1. Ahmed F, Hegazy K, Chaudhray P, Sharma S: Neuroprotective effect of brimonidine on adult rat retinal ganglion cells after increased intraocular pressure [abstract]. Invest Ophthalmol Vis Sci 40(Suppl):S265, 1999 2. Berlie JR, Iversen LJ, Blaxall HS, et al: Alpha-2 adrenergic receptors in the bovine retina. Presence of only the alpha-2D subtype. Invest Ophthalmol Vis Sci 36:1885–92, 1995 3. Burke J, Manlapaz C, Kharlamb A, et al: Therapeutic use of 2-adrenceptor agonists in glaucoma, in Lanier SM, Limbird LE (eds): Alpha-2-Adrenergic Receptors Structure, Function and Therapeutic Implications. Reading, UK, Harwood Academic Publishers, 1996, pp 179–187 4. Bylund DB, Chacko DM: Characterization of alpha2 adrenergic receptor subtypes in human ocular tissue homogenates. Invest Ophthalmol Vis Sci 40:2299–306, 1999 5. Donello JE, Padillo EU, Webster ML, et al: 2-Adrenoceptor agonists inhibit vitreal glutamate and aspartate accumulation and preserve retinal function after transient ischemia. J Pharmacol Exp Ther 296:216–23, 2001 6. Ervin C, Wohabrebbi A, Iannaccone A, Jablonski M: Brimonidine rescues photoreceptors from degeneration induced by retinal pigment epithelium removal in vitro. [abstract]. Invest Ophthalmol Vis Sci 40(Suppl):S160, 1999 7. Fritzsch B: Fast axonal diffusion of 3000 molecular weight dextran amines. J Neurosci Methods 50:95–103, 1993 8. Hasson D, Lai R, Chun T, Wheeler L: Functional protection of rat retina from ischemic injury by brimonidine [abstract]. Soc Neurosci Abstracts 23:168, 1997 9. Hoffman WE, Kochs E, Werner C, et al: Dexmedetomidine improves neurologic outcome from incomplete ischemia in the rat. Reversal by the alpha-2-adrenergic antagonist atipamezole. Anesthesiology 75:328–32, 1991 10. Huang Y, Gil DW, Vanscheeuwijck P, et al: Localization of al-
18.
19.
20.
21.
22. 23. 24.
pha-2-adrenergic receptor subtypes in the anterior segment of the human eye with selective antibodies. Invest Ophthalmol Vis Sci 36:2729–39, 1995 Kent AR, Nussdorf JD, David R, et al: Vitreous concentration of topically applied 0.2% brimonidine tartrate. Ophthalmology, 108:784–7, 2001 Kerrigan LA, Zack DJ, Quigley HA, et al: TUNEL-positive ganglion cells in human primary open-angle glaucoma. Arch Ophthalmol 115:1031–5, 1997 Lai R, Chun T, Hasson D, Wheeler L: Activation of cell survival signaling pathway in the retina by selective apha-2 adrenoceptor agonist brimonidine [abstract]. Invest Ophthalmol Vis Sci 40:S763, 1999 Lai R, Chun T, Hasson D, et al: Brimonidine protects photoreceptors and visual function after light damage [abstract]. Invest Ophthalmol Vis Sci 39(Suppl): S876, 1998 Levkovitch-Verbin H, Harris-Cerruti C, Groner Y, et al: RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci 41:4169–74, 2000 MacDonald E, Kobilka BK, Scheinin M: Gene targeting— homing in on alpha-2-adrenoceptor-subtype function. Trends Pharmacol Sci 18:211–9, 1997 Maier C, Steinberg GK, Sun GH, et al: Neuroprotection by the alpha-2-adrenoreceptor agonist dexmedetomidine in a focal model of cerebral ischemia. Anesthesiology 79:306–12, 1993 Ruiz G, Wheeler L, WoldeMussie E, Schwartz M: Time course of pre or post treatment by brimonidine on neuroprotection in rat optic nerve injury model [abstract]. Invest Ophthalmol Vis Sci 41(Suppl):S830, 2000 Villegas-Perez M, Lafuente M, Mayor-Torroglosa S, et al: Short and long term neuroprotection of a selective alpha-2 agonist (AGN 191103) on retinal ganglion cell survival after ischemia induced by selective ligature of the ophthalmic vessels [abstract]. Invest Ophthalmol Vis Sci 41(Suppl):S14, 2000 Wen R, Cheng T, Li Y, et al: Alpha-2-adrenergic agonists induce basic fibroblast growth factor expression in photoreceptors in vivo and ameliorate light damage. J Neurosci 16: 5986–92, 1996 Wikberg-Matsson A, Wikberg JE, Uhlen S: Characterization of alpha-2-adrenoceptor subtypes in the porcine eye: identification of alpha-2A-adrenoceptors in the choroid, ciliary body and iris, and alpha-2A- and alpha-2C-adrenoceptors in the retina. Exp Eye Res 63:57–66, 1996 Williams JT, North RA: Catecholamine inhibition of calcium action potentials in rat locus coeruleus neurones. Neuroscience 14:103–9, 1985 WoldeMussie E, Ruiz G, Wijono M, Wheeler L: Neuroprotective effect of brimonidine in chronic ocular hypertensive rats [abstract]. Invest Ophthalmol Vis Sci 41(Suppl):S830, 2000 Yoles E, Wheeler L, Schwartz M: Alpha2-adrenoreceptor agonists are neuroprotective in a rat model of optic nerve degeneration. Invest Ophthalmol Vis Sci 40:65–73, 1999
The authors are employed by Allergan, Inc. Reprint address: Larry A. Wheeler, PhD, Department of Biological Sciences, Allergan, Inc., 2525 Dupont Dr., Irvine, CA 92612.
CURRENT RESEARCH
Role of Alpha-2 Adrenergic Receptors in Neuroprotection and Glaucoma Larry A. Wheeler, PhD, Daniel W. Gil, PhD, and Elizabeth WoldeMussie, PhD Department of Biological Sciences, Allergan, Inc., Irvine, California, USA Abstract. The loss of retinal ganglion cells (RGCs) in glaucoma occurs progressively over many years. A neuroprotective drug should enhance survival of RGCs in the presence of chronic stress/injury. Four criteria are proposed for assessing the likely therapeutic utility in human glaucoma of drugs that have demonstrated neuroprotective activity in animal models: 1) A specific receptor target must be in the retina/optic nerve; 2) Activation of the target must trigger pathways that enhance a neuron’s resistance to stress/injury and/or suppresses toxic insults; 3) The drug must reach the retina/vitreous at pharmacologic doses; and 4) The neuroprotective activity should be demonstrated in clinical trials. Data are presented that illustrate how the specific and potent alpha-2 agonist, brimonidine, meets these criteria. The alpha-2A receptor was localized in the inner rat retina by immunohistochemistry. Brimonidine reduced the rate of RGC loss in the calibrated rat optic nerve injury model even when dosed 12 and 24 hours before injury, consistent with a long-term enhancement of RGC resistance to stress. Brimonidine was also neuroprotective in the lasered chronic hypertensive rat model, reducing RGC loss over three weeks from 33% to 15%. A clinical trial has been initiated to determine brimonidine’s neuroprotective activity in patients with non-arteritic ischemic optic neuropathy. (Surv Ophthalmol 45(Suppl 3):S290– S294, 2001. © 2001 by Elsevier Science Inc. All rights reserved.) Key words. alpha-2 adrenergic receptors • alpha-2 agonists • brimonidine • cell death pathways • cell survival pathways • glaucoma • neuroprotection • neuroprotective signaling pathways
Chronic and progressive retinal ganglion cell loss is a central feature of the glaucomas. The loss of RGC axons in the optic nerve occurs over many years. It is postulated that the RGCs and neighboring cells maintain a balance between intrinsic cell survival signals and neurotoxic signals. Over time, because of risks such as ischemia, elevated IOP, genetics, etc., the balance is shifted and cells enter a programmed cell death pathway, called apoptosis. One of the hallmarks of apoptosis has been demonstrated by terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate (UTP)-biotin nick end-labeling (TUNEL)-positive cells in histologic sections from glaucoma patients.12 Thus, a neuroprotective drug in glaucoma prevents the progres-
sive loss of RGCs by maintaining and/or enhancing their ability to resist stress and survive. It may do this by either enhancing cell survival pathways or interfering with neurotoxic signals. The alpha-2 receptor is one of the targets of the natural stress hormone norepinephrine, so it is likely to be involved in modulating cellular resistance and/or adaptation to counter stress/injury. In fact, alpha-2 agonists have been demonstrated to be neuroprotective in rat models of stroke involving cerebral ischemia.9,17 Activation of alpha-2 receptors by agonists such as brimonidine have more recently been shown to enhance survival of retinal neurons after many types of injuries and insults (Table 1). These models include transient ischemia,5,8,13,19 calibrated
S290 © 2001 by Elsevier Science Inc. All rights reserved.
0039-6257/01/$–see front matter PII S0039-6257(01)00206-5
S291
ROLE OF ALPHA-2 ADRENERGIC RECEPTORS TABLE 1
Animal Models in Which Brimonidine Showed Neuroprotective Activity Model
Reference
Optic nerve crush Ocular hypertensive rat Pressure induced ischemia Vascular ischemia SOD-1 over expressing mice Light-induced photoreceptor damage Photoreceptor degeneration—frog
Ruiz et al, 200018 Yoles et al, 199924 Ahmed et al, 19991 WoldeMussie et al, 200023 Donello et al, 20015 Hasson et al, 19978 Villegas-Perez et al, 200019 Levkovitch-Verbin et al, 200015 Lai et al, 199814 Wen et al, 199620 Ervin et al, 19996
optic nerve crush,18,24 photoreceptor degeneration,6,14,20 chronic ocular hypertension,1,23 and transgenic mice overexpressing superoxide dismutase (SOD).15 There are many pathways activated by alpha-2 receptors that might be involved in raising a neuron’s resistance to stress/injury. These include the activation of intracellular kinases that enhance cell survival and the inhibition of glutamate release5 and calcium influx into cells.22 The studies mentioned above suggest that the alpha-2 receptor and its signaling pathways are good drug targets for retinal neuroprotection. The studies also raise the question of what criteria can be used to compare or evaluate current and new drugs to determine potential usefulness for testing in human clinical trials (Table 2). For a drug to be neuroprotective in glaucoma, there is a specific target in the retina/ optic nerve; the drug must reach the retina at levels sufficient to activate its target; it must have a mechanism of action that enhances a neuron’s resistance to stress or suppresses toxic insults; and there should also be demonstrated activity in human clinical trials. Presented in this article is an evaluation of how brimonidine meets these four criteria. Also presented are two new findings that help to evaluate the mechanism of action of brimonidine. In particular, TABLE 2
Criteria for Neuroprotective Drugs in the Retina • Has a specific target in the retina/optic nerve • Reaches the retina at pharmacological levels • Has mechanism of action that enhances a neuron’s survival to stress or block toxic insult • Demonstrates activity in human clinical trials
we demonstrate that brimonidine is neuroprotective in the laser-induced chronic ocular hypertensive rat. Secondly, a single dose of brimonidine at 24 and 14 hours before injury is neuroprotective, supporting the working hypothesis that activation of the alpha-2 receptor results in a neuroprotective signaling pathway that enhances neuronal survival even after brimonidine has been cleared from the body.
Methods IMMUNOHISTOCHEMISTRY
Immunohistochemistry for the alpha-2A receptor was done on paraformaldehyde fixed, frozen sections of rat retinas. Tissue sections were incubated overnight at 4C with antibodies against the alpha2A adrenergic receptor subtype (generously supplied by Dr. J. Regan, University of Arizona). Sections were washed several times and incubated with secondary antibody followed by Vectastain® ABC for color development. They were counterstained with methyl green. In control samples, primary antibody was omitted. CALIBRATED OPTIC NERVE INJURY
Sprague Dawley rats were anesthetized with a mixture of ketamine (15mg/kg), acepromazine (1.5 mg/kg) and xylazine (0.3 mg/kg). Calibrated optic nerve injury was applied as described previously.24 Brimonidine at 0.1 mg/kg was given by intraperitoneal injection, at 24 or 14 hours before optic nerve injury or immediately after injury (time 0). Control animals received phosphate buffered saline (PBS) vehicle. Ganglion cell survival was evaluated 12 days later. The control group received the same anesthetic protocol of ketamine and xylazine. Therefore, the neuroprotective effect of brimonidine was the difference between control and treated animals. CHRONIC OCULAR HYPERTENSION
Intraocular pressure (IOP) was raised by laser photocoagulation of episcleral and limbal veins on day 0 and day 8. Drugs were applied by osmotic pump (Alzet) implanted subcutaneously on the animal’s back at the time of first laser treatment. Brimonidine was given at 1mg/kg/day and timolol 2 mg/kg/day. Control animals received PBS vehicle in a pump. Drug treatment was done for the 3-week duration of the experiment. IOP was measured weekly with tonopen. At the end of 3 weeks, ganglion cell loss was evaluated following retrograde transport of rhodamine labeled dextran. RETINAL GANGLION CELL ANALYSIS
Retinal ganglion cell loss in both models was evaluated by retrograde labeling at the optic nerve with
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Surv Ophthalmol 45 (Suppl 3) May 2001
WHEELER ET AL
Fig. 1. Right: Immunoreactivity of the alpha-2A adrenergic receptors is localized mainly in the somas of the cells in the ganglion cell layer and some cells in the inner nuclear cell layer. Left: Control staining where primary antibody was omitted. RGC ganglion cells; IPL inner plexiform layer; INL inner nuclear layer; PR photoreceptors. Scale bar 50 m.
rhodamine-labeled dextran.7,24 Briefly, rats were deeply anesthetized and the optic nerve exposed. A longitudinal incision was made on the meningis and the optic nerve was completely cross-sectioned at about 2 to 3 mm from the globe. Crystals of tetramethyl rhodamine, 3000 mw (Mol. Probes, OR), were applied to the cut end of the optic nerve. Twentyfour hours later the eyes were enucleated and fixed with 4% paraformaldehyde and the retinas were flatmounted. Cell counts were done in eight regions, two in each quadrant, at 400 magnification in areas starting 0.66 mm from the edge of the optic disk. This was 1.56% of the total ganglion cell count, and it provided representative counts of ganglion cells in relation to total cell number. Counting additional
areas 1–2 mm more peripheral to these regions did not alter the assessment of the extent of injury.
Results and Discussion LOCALIZATION OF ALPHA-2A RECEPTORS IN THE RETINA
A specific receptor target in the retina offers the best chance for a drug to be neuroprotective and minimize the potential for side effects. The alpha-2A receptor subtype is the most abundant of the three alpha-2 receptors in the nervous system.16 Antibodies generated against the third cytoplasmic loop of the alpha-2A receptor10 were used to localize the presence of the subtype in the retina. As shown in Fig. 1,
Fig. 2. Fluorescent micrograph of labeled retinal ganglion cells of vehicle-treated (A) or brimonidine-treated (B) retinas 12 days after injury. Brimonidine increased cell survival when injected at the time of optic nerve injury. Scale bar 50 m.
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ROLE OF ALPHA-2 ADRENERGIC RECEPTORS
there was immunoreactivity in the ganglion cell layer, which has both ganglion and amacrine cells. Some cells in the inner nuclear layer, possibly amacrine cells, were also immunopositive. The presence of alpha-2 receptors has been shown in human,4 bovine,2 and porcine21 retinas by radioligand binding studies and/or functionally by the use of alpha-2 antagonists to block the retinal neuroprotection induced by agonists such as brimonidine.24 OPTIC NERVE INJURY
With optic nerve crush, 80–85% of the ganglion cells degenerated by day 12 after injury. If brimonidine upregulates pathways that enhance survival, then determining the dosing window that is neuroprotective would help clarify this mechanism of action. Rapid mechanisms would have a short or narrow window of protection before injury whereas gene expression regulation would have a longer or larger window of protection. Brimonidine at 0.1 mg/kg when injected intraperitoneally at time 0 resulted in ganglion cell survival of about two-fold (Figs. 2 and 3). Administration of brimonidine 14 hours before injury also resulted in RGC survival that was similar to that when it was administered at time 0. Significant cell survival was also obtained when brimonidine was administered 24 hours earlier (data not shown). Brimonidine levels in the retina after 0.1 mg/kg peaked at 30 minutes at 37 3 nM and stayed above 2 nM for 6 hours (authors’ unpublished data). Analysis was done using gas chromatograph/mass spectrometry (GC-MS/ MS). Alpha-2 receptors are activated by brimonidine at a concentration of 2 nM.3
Fig. 3. Intraperitoneal administration of brimonidine at the time of optic nerve injury resulted in a two-fold survival of RGC (p 0.01). Pretreatment with brimonidine 14 hours before injury resulted in RGC survival that was equivalent to that administered at time 0. Values are represented as mean SEM from 5 to 10 animals.
CHRONIC OCULAR HYPERTENSION
The chronic ocular hypertensive model represents a model that mimics gradual death of the RGCs seen in the glaucomas. Laser treatment of the episcleral and limbal veins, as described in the methods section, resulted in a two-fold elevation of IOP. This resulted in progressive loss of RGCs. At the end of 3 weeks of elevated IOP, there was a 34 7% decrease in ganglion cells in the vehicle-treated rats. Decrease in IOP by either brimonidine or timolol was less than 10%. In rats treated with 1 mg/kg/day brimonidine ganglion cell loss was reduced by more than half, to 16 3% (Fig. 4). Ganglion cell loss in rats treated with 2 mg/kg/day timolol was similar to those treated with vehicle (Fig. 4). Thus, the protection of RGCs by brimonidine in this model was not due to the lowering of IOP. Two of the criteria for evaluating brimonidine’s potential as a therapeutic neuroprotective agent have been met. First, the specific target—the alpha-2 receptor—is present in the retina, and, second, the activation of the receptor results in ganglion cell protection under conditions of acute and chronic stress and/or injury (Table 1). The third criterion is that the drug reaches the target in the retina at pharmacological doses. Brimonidine is a highly potent and selective alpha-2 adrenergic agonist with the ability to activate its targets at concentrations above 2 nM. It does not significantly activate alpha-1 adrenergic receptors at concentrations below 2000 nM.3 Thus, the drug has the potential to be neuroprotective without causing side
Fig. 4. Ganglion cell loss was evaluated in the different treated groups after IOP was elevated for three weeks. There was a 33 7% decrease in RGC number in vehicletreated group. This was reduced to 16 3% in the presence of brimonidine, which is a 50% protection (p 0.05). With systemic timolol cell loss was 32 3%. Values are represented as mean SEM from 10 animals.
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Surv Ophthalmol 45 (Suppl 3) May 2001
effects if levels greater than 2 nM and less than 2000 nM can be achieved in the human retina following topical application. Initial results from a study by Kent et al11 in patients who received 0.2% Alphagan® BID for 4–14 days prior to undergoing elective pars plana vitrectomy demonstrated that vitreous levels of brimonidine were in the pharmacological range. Because brimonidine has demonstrated a broad neuroprotective profile in a variety of different kinds of injury and meets the first three criteria for a retinal/ optic nerve neuroprotective agent, Allergan has initiated clinical trials in nonarteritic ischemic optic neuropathy (NAION) to determine whether the drug’s neuroprotective effect can be measured in patients. In NAION, there is injury to the axons of the retinal ganglion cells, and this trial will provide a proof of principle for neuroprotective therapy in humans.
WHEELER ET AL
11. 12. 13.
14. 15.
16. 17.
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The authors are employed by Allergan, Inc. Reprint address: Larry A. Wheeler, PhD, Department of Biological Sciences, Allergan, Inc., 2525 Dupont Dr., Irvine, CA 92612.