SURVEY OF OPHTHALMOLOGY VOLUME 41 ,, SUPPLEMENT 1 ° NOVEMBER 1996 Preclinical Evaluation of Brimonidine JAMES BURKE, BSc, ~ AND MICHAL SCHWARTZ, PHD 2 1Department of Biological Sciences, Allevgan, Inc., Irvine, California, USA, and 2Department of Neurobiology, The Weizman Institute of Science, Rehovot, Israel Abstract. Preclinical studies of brimonidine show that it is a potent c%-adrenoceptor agonist that is 1000- fold more selective for the (~2" vs. the %-adrenoceptor, and is 7-12-fold more c~2-selective than clonidine and 23- to 32-fold more oL2-selective than apraclonidine (p-aminoclonidine). Brimonidine decreased in- traocular pressure (IOP) in various animal models but, unlike apraclonidine, brimonidine was not mydri- atic. The site and pharmacology of the IOP response depends on the animal species. In rabbits, the IOP response to brimonidine is mediated by an ocular c%-adrenoceptor while in monkeys, a central nervous system (CNS) 'imidazoline' receptor appears to be involved. Brimonidine decreased IOP by suppressing the rate of aqueous humor flow and enhancing uveoscleral outflow. Topical brimonidine resulted in poste- rior segment drug levels adequate to activate %-adrenoceptors, but was not vasoconstrictive in a model designed to assess the vasoactivity of the human retinal microvasculature. Brimonidine protected the rat optic nerve from secondary damage following mechanical injury to the optic nerve and was nontoxic in an array of experiments designed to evaluate ocular and organ toxicity. Taken together, the high (x2-adrenoceptor selectivity, ocular hypotensive efficacy, retinal bioavailability and neuroprotective properties make brimonidine an important addition to the field of antiglaucoma agents. (Surv Ophthalmol 41 [Suppl 1]: $9-S 18, 1996) Key words. AGN 190342 • alpha 2 adrenoceptor ° apraclonidine • brimonidine clonidine • glaucoma ° intraocular pressure ° Iopidine • neuroprotection • p-arninoclonidine • UK-14,304 Alpha-adrenoceptor agonists have played a sig- nificant role in the medical management of glau- coma. Epinephrine, a nonselective adrenoceptor agonist, has been the mainstay of glaucoma ther- apy for decades. 5~ Clonidine, the first %-adreno- ceptor agonist to be marketed for the treatment of glaucoma, has been available for over 15 years in some countries. It is a highly effective IOP- lowering agent, but its use has been greatly lim- ited by significant systemic side effects, which include sedation and systemic hypotension. The systemic side effects that are observed with cloni- dine appear to be directly related to its ability to penetrate into the central nervous system. 26-'-'s'34 Apraclonidine, also known as p-aminoclonidine and Iopidine®, is a hydrophilic analog of clonidine. It is the first e%-adrenoceptor agonist approved for the treatment of intraocular pressure (IOP) elevation associated with anterior segment laser surgery, and for short-term therapy to delay sur- gery in patients with uncontrolled IOP receiving maximally tolerated medical therapy? v It is also an effective IOP-lowering agent. Apraclonidine has fewer of the central nervous system effects asso- ciated with clonidine; however, apraclonidine has a high affinity for the oq-adrenoceptor, which results in ocular side effects such as mydriasis, ~s'21 conjunc- tival blanching, 1,2~,~9 ciliary vasoconstriction, 2~,24,5s eyelid retraction 21 and reduction in conjunctival oxy- gen tension. 5° Long-term use of apraclonidine has been associated with a high incidence of ocular al- lergies] 6'4s which may be related to its oxidative la- bility and the reactivity of hapten-forming interme- diates. 43 Brimonidine, also known as UK-14,304 and AGN 190342, is a less lipophilic analog of clonidine, 19 and, like apraclonidine, provides clinically signifi- cant lowering of IOP in humans. Brimonidine's molecular structure, however, differs from that of apraclonidine and this gives brimonidine higher e~ 2- adrenoceptor selectivity and a lower potential for hapten formation via oxidative metabolism3 ~ This review will focus on the receptor and ocular pharmacology of brimonidine, its mode of ocular hypotensive action, and posterior segment effects. Comparisons will be made to clonidine and apra- $9 © 1996. Survey of Ophthahnology. (7 Kent Street, Brookline, MA 02146. Tel: 617-566-2138. Fax: 617-566-4019)
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`S10 Surv Ophthalmol 41 (Suppl 1) November 1996 BURKE, SCHWARTZ clonidine where data are available. Comparisons of clonidine and brimonidine or clonidine and apra- clonidine are available in the literature, but not among all three agents. In addition, binding con- stants can be altered by a number of variables. The following studies compared all three agents under identical conditions to better evaluate similarities and differences. Brimonidine RECEPTOR PHARMACOLOGY Brimonidine's ot2-adrenoceptor selectivity is well known. Hundreds of scientific articles have been published in which brimonidine was used as a refer- ence ~2-adrenoceptor agonist. It is considered a stan- dard reference compound because of its high o~ 2- adrenoceptor selectivity. 17 Brimonidine's receptor selectivity and affinity were compared with apraclonidine and clonidine in radioligand bind- ing and tissue bath bioassays. ~°,11,~5,6J Radioligand binding assays were conducted with [3H]prazosin and [SH]rauwolscine to label %- and %-adreno- ceptors in the human cerebral cortex and the hu- man colonic cell line (HT-29), respectively. Tissue bath bioassays were used to measure %-adrenoceptor activation in the isolated rabbit iris dilator muscle and ot2-adrenoceptor stimulation in the isolated rab- bit vas deferens. Stimulation of the %-receptors in the rabbit iris dilator muscle causes mydriasis? 3 Stimulation of the prejunctional c~2-receptors in the rabbit vas deferens inhibits the electrically-induced contractile response? 5 The combined data from these assays show that brimonidine is 23-32-fold more %-adrenoceptor selective than apracionidine and is 7-12-fold more selective than clonidine (means are depicted in Fig. 1). The affinity ofbrimonidine was highest (1-2 nM) for the ~2 receptor and lowest (1850-2650 nM) for the % receptor. These results suggest that com- pared to clonidine and apraclonidine, brimonidine would be the least likely to produce oq-adrenoceptor- mediated side effects, such as mydriasis and ocular vasoconstriction. OCULAR PHARMACOLOGY The ocular hypotensive effect ofbrimonidine has been demonstrated in animal models including nor- motensive and ocular hypertensive rabbits and mon- keys and in cats. In some of the following studies, brimonidine concentrations are expressed as the tar- trate salt, as in the clinical studies. In others, the concentration of brimonidine is expressed as the base, which is 66% of the salt. Normotensive Rabbits: Single-Drop Studies In normotensive rabbits, brimonidine concentra- 0 200 400 600 800 1000 1200 1400 1600 1800 2000 ~l/e.2-Adrenoceptor Selectivity Fig. 1. Mean az- vs %-adrenoceptor selectivity or brimoni- dine, clonidine and apraclonidine in radioligand binding and tissue bath functional assays. 1°,11,14,~° Data are derived from ratios of % and a2 affinity constraints (Ki) in binding assays and EC50's in functional assays. In binding assays, %-adreuoceptors in the human cerebral cortex and %-adrenoceptors in the human colonic (HT-29) cell line were labeled with [3H]prazosin and [3H]rauwolscine, respectively. In functional assays, %-adrenoceptor activity was determined by contraction of the rabbit iris dilator muscle; % activity was measured by inhibition of electrically-induced contractions in the rabbit vas deferens. Brimonidine was 28 times more %-selective than apraclonidine and 10 times more selective than clonidine. tions ranging from 0.0001% to 0.5% applied in single drop studies, resulted in concentration- related IOP lowering. 9,~4 Brimonidine 0.1% was at the top of the concentration response curve, and produced a peak ocular hypotensive response of 5.1 _+ 0.6 mm Hg (mean -+ sem). A contralateral IOP-lowering response occurred at concentrations of 0.1% or greater. The concentration response curves for brimonidine, clonidine and apracloni- dine show that brimonidine is at least 100-fold more potent in decreasing IOP than apraclonidine. 15 At equivalent concentrations (0.1%), brimonidine had a longer duration of ocular hypotensive activity than apraclonidine (Fig. 2A). The marginal ocular hypotensive response to apraclonidine in the rabbit is due, in part, to the unique sensitivity of this spe- cies to OLz-adrenoceptor stimulation. In rabbits, oq- adrenoceptor stimulation increases IOP while OLz-adrenoceptor stimulation decreases IOP. 12,29,44 Apraclonidine has high affinity for %-adrenoceptors and consequently has a lesser ocular hypotensive re- sponse. Normotensive Rabbits: Chronic Administration The chronic administration of brimonidine in
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`PRECLINICAL EVALUATION OF BRIMONIDINE S 11 0 Saline • Brimonidine O. 1% V Clonidine O. 1% • Apraclonidine O. 1% =i= 24- A m 22- -1- E E 20- EL 0 18- 16- 26- =!= 1 4 [ I I I I I I -1 0 1 2 3 4 5 6 TIME (Hrs.) Fig. 2A. The IOP responses to saline (~; n = 16), and 0.1% concentrations ofbrimonidine (Q; n = 28), clonidine (V; n = 21) and apraclonidine (A; n = 10) in conscious normotensive New Zealand white (albino) rabbits. Data are expressed as mean --- sem. Drugs were applied unilaterally as a single 50 Ixl eyedrop. Intraocular pressure was measured noninvasively with a 30R model Digilab pneumatonometer. Twenty-five microliters of an anesthetic (proparacaine) was topically applied before IOP measure- ments to minimize ocular discomfort due to tonometry. Two baseline measurements were made prior to instillation of the drugs, followed by periodic measurements up to 6 hours post-instillation. Asterisks indicate a significant difference from saline control; p < 0.05, unpaired Student's t test. Brimonidine was more effective at lowering IOP than clonidine or apraclonidine. normotensive rabbits does not result in a loss of the ocular hypotensive response over time. The ocular hypotensive response to concentrations ranging from 0.08-0.8% at the end of 6 months of twice-daily dos- ing was equivalent to or greater than the initial re- sponse. ~2 The maximum IOP decreases for the 0.08%, 0.2%, 0.5% and 0.8% concentrations were 3.9 + 0.4, 5.1 +_ 0.4, 6.9 _ 0.3, and 7.1 _+ 0.4 mm Hg, respectively. Maintenance of the ocular hypoten- sive response is supported by biochemical evidence, which shows that ~x2-receptor-linked inhibition of cyclic adenosine monophosphate (cAMP) in rabbit iris/ciliary body does not undergo receptor desensi- tization with the chronic administration of brimoni- dine. Taken together, these results show that toler- ance does not develop to the chronic administration ofbrimonidine in rabbits. 12 Ocular Hypertensive Rabbits In addition to its ocular hypotensive effect in nor-
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`S12 Surv Ophthalmol 41 (Suppl 1) November 1996 BURKE, SCHWARTZ 0 Saline • Brimonidine O. 1% V Clonidine O. 1% • Apmclonidine O. 1% _ _ i, 6 , I I I I I I I 0 1 2 3 4 5 6 TIME (Hrs.) Fig. 2B. The pupil diameter responses to saline (,2~; n = 16), and 0.1% concentrations ofbrimonidine (0; n = 28), clonidine (V; n = 21) and apraclonidine (A; n = 10) in conscious normotensive New Zealand White (albino) rabbits. Data are expressed as mean + sere. Drugs were applied unilaterally as a single 50 Ixl eyedrop. Pupil diameter was measured noninvasively with a millimeter ruler. Two baseline measurements were made prior to instillation of the drugs, followed by periodic measurements up to 6 hours post-instillation. Asterisks indicate a significant difference from saline control; p < 0,05, unpaired Student's t test. Apraclonidine produced a profound and long-lasting mydriatic effect. Clonidine increased pupil size transiently and brimonidine was without effect. motensive rabbits, brimonidine was effective in a rab- bit model of ocular hypertension. ~ Brimonidine 0.1% suppressed the rise in IOP induced by 60 mL/kg of water loading administered orally for more than 5 hours. Normotensive Monkeys The IOP response to unilaterally-applied brirnoni- dine was investigated in normotensive capuchin and cynomolgus monkeys. In capuchin monkeys,14 a single application of 0.1% and 1.0% brimonidine decreased IOP bilaterally. The maximum IOP drop with the 0.1% concentration was 2.5 -+ 0.6 mm Hg and with the 1.0% concentration, it was 4.1 _+ 0.8 mm Hg. In ketarnine-sedated cyno- molgus monkeys, brimonidine 0.3% decreased IOP by a maximum of 4.9 + 2 mm Hg, brimonidine 0.5% decreased IOP by 9.9 + 1.2 mm Hg, and brimonidine 1.0% decreased it by 7.3 + 1.5 mm Hg. 52 In ketamine-sedated cynomolgus monkeys, 2~ brimonidine 0.1%, 1%, and 2% suppressed IOP by a maximum of 12 mm Hg. In conscious cynomolgus monkeys, brimonidine 0.03%, 0.1% and 0.3% de-
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`PRECLINICAL EVALUATION OF BRIMONIDINE S 13 creased IOP by a maximum of 3.5 _+ 0.8 mm Hg, 6.3 -+ 1.4 mm Hg and 9.3 -+ 1.3 mm Hg, respectively. 9 The IOP response in monkeys was without evidence of the transient initial rise, which contrasts with the rabbit and underlines a species difference to %- adrenoceptor agonists? Ocular Hypertensive Monkeys Brimonidine was evaluated in monkeys made uni- laterally ocular hypertensive by argon laser photoco- agulation of the trabecular meshwork. This experi- mental model is characterized by features commonly seen in human glaucoma. 4~ In one study)'-' one drop of 0.5% brimonidine re- duced IOP in ketamine-sedated monkeys for 24 hours. The maximum IOP decrease was 22.3 + 3.2 mm Hg, which occurred 4 hours after dosing. In the same study, 0.5% brimonidine was administered twice daily for 5 days, with a maximum reduction in IOP of ap- proximately 15 mm Hg. No tolerance of the ocular hypotensive response was noted. In another study comparing the concentration-re- sponse relationship in conscious monkeys, ~5 the ranked order of potency for decreasing IOP in the hypertensive eye was brimonidine = clonidine > apraclonidine. Brimonidine was 10-fold more potent as an ocular hypotensive agent than apra- clonidine in this animal model and produced a maxi- mum IOP reduction of 12.3 _+ 0.8 mm Hg with the 0.3% concentration. Clonidine and apraclonidine lowered IOP by 1 1.8 +_ 2 mm Hg (0.3% concentra- tion) and 8.5 _+ 2 mm Hg (3% concentration), respec- tively. Cats The IOP response to brimonidine was also investi- gated in cats. 14 Unilateral dosing with the 0.1% con- centration produced a delayed ocular hypotensive response starting at 3 hours post-dose. A maximum hypotensive effect of 6.9 +- 1.1 mm Hgwas seen at 5 hours. A contralateral IOP response of 5.2 --- 1.4 mm Hg occurred at 1 hour. This cat IOP profile was simi- lar to that of the rabbit. PUPIL SIZE The effect ofbrimonidine on pupil size was evalu- ated in rabbits, monkeys and cats. Rabbits Brimonidine had very little effect on pupil size in rabbits. Concentration response curves in rabbits show that the ranked order of potency for eliciting a mydriatic effect was apraclonidine (high effect) > > clonidine > brimonidine (no effect). 15 The pupillary responses to brimonidine, clonidine and apraclonidine at the 0.1% concentration in rabbits at 6 hours post-dosing are shown in Fig. 2B. Apraclonidine produced a profound and long-last- ing mydriasis. Mydriasis is related to stimulation of %-adrenoceptors in the iris dilator muscle. Apra- clonidine has high affinity for the c~-receptors in this organ (see Receptor Pharmacology, supra). The low a~-receptor affinity ofbrimonidine results in the ab- sence of a mydriatic response at therapeutic concen- trations. Monkeys and Cats In monkeys 9,25 and cats, 14 ocularly administered brimonidine produced miosis, which is mediated by prejunctional %-adrenoceptors on sympathetic nerves innervating the dilator muscle.~4 Withdrawal of sympathetic tone to the iris dilator muscle allows greater contribution of the iris sphincter to pupil size and, thus, miosis. The iris dilator muscle of higher mammals such as cats and monkeys is less sensitive to ~-receptor stimulation than rabbits, 57 which may explain the relatively smaller mydriatic response to apraclonidine in monkeys ''5 and man ~,49 compared to rabbits. Vasoactivity The vascular response to 0t2-adrenoceptor agonists can vary according to species, tissue and location within a given vascular bed. 45 az-adrenoceptors can mediate vasoconstriction and play a role in the au- toregulation of capillary pressure and tissue oxygen delivery. 22"4° On the other hand, brimonidine can produce vasodilation 5'7 via ~2-adrenoceptors on en- dothelial cells, which release endothelial-derived relaxing factor. To assess the potential for vasoactivity in functional human retinal microvasculature, brimonidine, clonidine and apraclonidine were applied topically to retinal tissue transplanted into the hamster cheek pouch membrane, an immunologically tolerant tis- sue. 54 The hamster cheek pouch model is widely used to study the effects of drugs on human microvascu- lature. 3~ The arteriolar caliber in the retinal xe- nografts was measured by intravital microscopy (mi- croscopy in living systems). Vessel caliber was unchanged by brimonidine at concentrations up to 10 -5 M. A high concentration (10 .4 M) reduced vessel caliber by 11.1 + 4.2% at 1 minute, 1.4 _+ 3.0% at 5 nfinutes, and 2.3 -+ 2.6% at 10 minutes (n = 5). Clonidine evoked a marked con- centration-dependent decrease in arteriolar caliber of up to 34.8 + 6.3% with 10 -4 M concentration (n = 6) at 1 minute. At the 10 -7 M concentration, caliber size was decreased by 13.5 _+ 3.7%. The ef- fects for clonidine were similar at 5 minutes, and less at 10 minutes. Apraclonidine was the most po- tent compound in this model, producing a response
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`S14 Surv Ophthalmol 41 (Suppl 1) November 1996 BURKE, SCHWARTZ A 70 B Fig. 3. Low-power photomicrographs from whole-mounted retinas showing the distribution of surviving retinal ganglion cells 2 weeks after a calibrated partial crush to the optic nerve of an adult rat, taken from control (Panel A, upper montage) and 100 p,g/kg brimonidine, administered intraperitoneally (ip; Panel B, lower montage). Brimonidine was administered at the time of injury. The retinal ganglion cells were retrogradely labelled with the dye 4 Di-10 ASP applied at the site of injury. Brimonidine enhanced the survival of retinal ganglion cells as depicted by a 4-fold increase in labelling compared to vehicle control. of 15.9 _+ 4.5% at the 10 ~ M concentration at 5 min- utes (n = 6) up to a maximum of 28.4 _+ 6.2% with the 10 .5 M concentration. Pharmacokinetic studies in rabbits and monkeys show that the vitreous hu- mor concentration following 0.2% brimonidine BID administration for 2 weeks was N 10-7 M.2,20 These data suggest that brimonidine ocular therapy in hu- mans is not likely to be associated with vasoconstric- tion in the posterior pole. NEUROPROTECTION o~2-Adrenoceptor agonists are neuroprotective in a variety of animal models, including focal models of cerebral ischemia? °,47 A recent report
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`PRECLINICAL EVALUATION OF BRIMONIDINE S 15 showed that brimonidine protected the optic nerve and retinal ganglion cells from secondary degenera- tion produced by a calibrated partial crush lesion to the adult rat optic nerve. 62 Secondary de- generation is the consequence of autodestructive events (membrane damage, energy depletion, acti- vation ofproteases, lipases and nucleases), which lead to the death of neurons that escaped the primary injury as well as death of retinal ganglion cells of directly injured optic nerve. The model, although not mimicking the human glaucomatous neuropathy itself, sustains damage resembling that seen in the disease. Moreover, the progression of the damage seen in glaucoma, re- gardless of the primary trigger, is similar to the pro- gression of the damage secondary to any neuronal trauma caused by pressure and/or ischemic/hypoxic injuries? 8 Brimonidine 100 Ixg/kg, given intraperi- toneally immediately after infliction of the crush le- sion, increased optic nerve axonal excitability 2.5- fold (measured by the compound action potential: 364 + 51 to 857 + 194 IxV) and survivability of reti- nal ganglion cells 4-fold (assessed morphometrically by retrograde transport [Fig. 3]) compared to vehicle- treated injured animals. The findings show that brimonidine is neuroprotective in an ocular model of neurodegeneration. The mechanism of action for the neuroprotective effect may be up-regulation of neuronal survival factors, such as basic fibroblast growth factor. %- Adrenoceptor agonists increased by 3-5-fold the mRNA for retinal basic fibroblast growth factor and prevented photoreceptor and ganglion cell degen- eration in rats exposed to excessive light. 59'6° Ba- sic fibroblast growth factor protects photorecep- tors from light-induced damage. A neuroprotective effect of brimonidine for glau- coma treatment is possible only with adequate retinal concentration of drug following ocular appli- cation. Pharmacokinetic studies show that brimoni- dine distributes not only to the anterior segment but also to the posterior segment. 2,2° Acute or chronic (twice daily for 2 weeks) ocular instillation of brimonidine 0.2% in rabbits and cynomolgus mon- keys produced maximal concentrations of approxi- mately 450-1300 nM in the aqueous humor and 100- 170 nM in the vitreous humor. 2.2° This means that drug concentrations available to the retina from the vitreous humor was 50 to 80 times higher than what is required to bind and activate the a~-adreno- ceptor. 4k42 Interestingly, a comparison of tissue concentra- tions in pigmented and albino rabbits shows that, except for aqueous humor where turnover plays a large role in drug elimination, the concentrations of brimonidine were markedly higher in pigment-con- taining tissues. In the choroid/retina it was 22-fold, in the iris 43-fold and in ciliary body 25-fold higher in pigmented than in albino animals. This held true, also, for the optic nerve and vitreous humor. 2,2° Like timolol, 6 brimonidine binds reversibly to ocular mela- nin 55 and may be acting as a depot for sustained re- lease into the vitreous and neural retina. Taken together, these data indicate that ocular dosing ofbrimonidine results in pharmacologically relevant concentrations in the posterior pole that may contribute to a neuroprotective effect. These findings, along with the findings from the nerve crush studies, suggest a potential beneficial eft~ct in the management of glaucoma beyond the lower- ing of IOP. MODE OF ACTION Site of Action The pharmacology and site of action of the lOP response to brimonidine in animals is species- dependent. In rabbits, pretreatment with e~2-selec- tive antagonists inhibited the IOP response to brimonidine, indicating %-adrenoceptor agonism.14 The site of action is peripheral, independent of cen- tral nervous system mediation, given that ocular hypotension could be achieved with topicaP ~ or intracameral 8 dosing without a concomitant de- crease in blood pressure or a contralateral re- sponse. 9 The contralateral decrease in IOP in rab- bits that is produced by higher concentrations is due to systemic absorption and is reflected by plasma concentrations high enough to activate central ~2-adrenoceptors. 4 In monkeys, the IOP response to brimonidine may be mediated, in part, by the imidazoline recep- tor because of preferential inhibition by idazoxan.13.3~.32 The data also suggest that the IOP response to brimonidine in normotensive monkeys may have a central origin because of a contralateral response at low concentrations and concomitant cardiovas- cular depression. 9,2~ In man, unilateral application ofbrimonidine pro- duced unilateral IOP decreases without concomitant cardiovascular changes, indicating a peripheral site of action? 5 Mechanism of Action Brimonidine decreases IOP by suppressing aque- ous humor flow and increasing uveoscleral outflow. In rabbits, 0.1% brimonidine reduced flow rate by N 70% using a recovery rate method.14 Tracer experi- ments in rabbits show an enhancement ofuveoscleral outflow up to 57% (treated 0.33 _+ 0.05 ixl/min, con- trol 0.21 + 0.04 tzl/min) following dosing with 0.1% brimonidine? ~,5~ The same investigators demon- strated that 0.5% brimonidine reduced flow by 67%
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`S16 Surv Ophthalmol 41 (Suppl 1) November 1996 BURKE, SCHWARTZ (treated 0.5 + 0.2 ~l/min, baseline 1.4 -+ 0.1 txl/min) in monkeys without affecting outflow facilityJ 2 In another study in monkeys, 2:5 a dose-related reduc- tion in flow of up to 45% was produced by small vol- umes (10 tzl) ofbrimonidine 0.5% and 2.5%. These animal data correlate very well with a mechanism of action study in humans, showing an effect on both aqueous humor and uveoscleral outflow. 56 An additional mechanism of action is implied from a comparison of flow and IOP data for brimonidine and beta-blockers in monkeys. ~5 The extent of aque- ous flow suppression in normotensive monkeys and IOP reduction in ocular hypertensive monkeys by brimonidine, timolol, and betaxolol was comparable (32-44%). However, unlike the beta-blockers, brimonidine also lowered IOP in normotensive eyes, suggesting that it may have mechanisms of action other than aqueous humor suppression. TOXICOLOGY Brimonidine has been evaluated extensively for ocular and systemic toxicity in a number of ani- mal models, as required to support an NDA. 3,4 Ocular (0.08-0.8%, twice daily) and systemic (0.1- 2.5 mg/kg/day by oral/diet) administration for up to one year produced no organ toxicity, despite plasma levels that were 4 to 1 18 times the sys- temic exposure in humans following ocular ad- ministration with 0.2%. Studies show no oncogenic or teratogenic activity, nor do they show adverse effects on fertility and reproduction. These data indicate that brimonidine is nontoxic and safe. General Discussion The findings of these extensive preclinical stud- ies with brimonidine show that brimonidine pos- sesses high (x2-adrenoceptor selectivity and pro- duces potent ocular hypotensive effects. The high ~2-adrenoceptor selectivity means that patients would benefit by a lowering of IOP at therapeutic doses with fewer ~l-adrenoceptor-mediated side effects, such as mydriasis and ocular vasoconstriction, than apraclonidine or clonidine. The ocular hypotensive response to brimonidine was not associated with tachyphylaxis or ~2- adrenoceptor desensitization. ~2 These observations were borne out in the clinical studies. 49a,5°a,5sa No evidence of tachyphylaxis was seen in long-term studies in humans with glaucoma and ocular hy- pertension. Thus, brimonidine appears to be use- ful for chronic administration. Brimonidine appears to have a dual mechanism of action: suppression of aqueous inflow and increase in uveoscleral outflow. The effect on inflow is expected from this class of compound, but the effect on uveoscleral outflow was unexpected. The mechanism for this effect is unknown at this time. One hypoth- esis tested was a mediation by prostaglandins, but brimonidine's IOP response in animals was not af- fected by pretreatment with cyclo-oxygenase inhibi- tors (J. Burke, unpublished data). Nevertheless, the dual mechanism of action property may prove useful in combination therapy: brimonidine would be expected to be additive to aqueous flow suppressants and/or outflow enhancers. Ocular instillation ofbrimonidine resulted in phar- macologically relevant drug concentrations in the posterior segment of the eye. 2'2° This observation prompted studies to determine the bio- logical relevance of brimonidine in the posterior segment. The first mechanism considered was vasoconstriction, given that oL2-adrenoceptor ago- nists are vasoactive in some vascular beds. 22'4° The results from the human retinal xenograft model ruled out a vasoconstrictive effect for brimonidine in this important vascular bed. 54 The second mechanism considered was a ben- eficial effect, neuroprotection. It is documented in the literature that o~2-adrenoceptor are neuro- protective in models of cerebral ischemia? 9,47 Brim- onidine was studied in an ocular model for pro- tective effects following a pressure-related injury to the optic nerve. 62 The results showed that brimonidine was protective of retinal ganglion cells and optic nerve fibers from the cascade of events that are a consequence of this type of in- jury. Because glaucoma therapy is traditionally directed toward the lowering of IOP, this neuro- protective effect of brimonidine may portend a new approach to the treatment of this disease. In conclusion, brimonidine possesses a mix of interesting properties (high receptor selectivity, chronic ocular hypotension with low side effects, neuroprotection) that should make it a useful addi- tion to the tools available for the chronic treatment of glaucoma. References 1. Abrams DA, Robin AL, Pollack IP: The safety and efficacy of topical 1% ALO 2145 (p-aminoclonidine hydrochloride) in normal volunteers. Arch Ophthalmol 105:1205-1207, 1987 2. Acheampong A, Shackleton M, Tang-Liu D: Comparative ocular pharmacokinetics of brimonidine after a single dose application to the eyes of albino and pigmented rabbits. Drug Metabolism and Disposition 23:708-712, 1995 3. Angelov O, Wiese A, Ismail M, et al: Reproductive and de- velopment safety studies with brimonidine (Alphagan) [ab- stract]. Invest Ophthalmol Vis Sci 37:S1101, 1996 4. Angelov O, Wiese A, Tang-Liu D, et al: Preclinical safety pro- file ofbrimonidine. EurJ Ophthalmol 6:21-25, 1996 5. Angus JA, Cocks TM, Wright CE, et al: Endothelium- dependent responses in large arteries and in the microcircu- lation, in: Vanhoutte PM (ed): Relaxing and Contracting Fac- tors. Clifton, Humana Press, 1988. pp 361-387 6. Araie M, Takase M, Sakai Y, et al: Beta-adrenergic blockers: ocular penetration and binding to the uveal pigment. Jpn J
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