The Effect of Brimonidine on Intraocular Pressure Via the Central Nervous System in the Conscious Pigmented Rabbit

Showa Univ J Med Sci 15(4), 313•`322, December 2003

Original The Effect of Brimonidine on Intraocular Pressure via the Central Nervous System in the Conscious Pigmented Rabbit

Mitsutaka SODA1), Masato YOSHIDA1), Hldetoshl ONDA1), Guo SHI-YU2), Takako NAKANISHI-UEDA3), Toshihiko UEDA1), Noriko FUJISHIRO3), Tadashi HISAMITSU2), Makoto INATOMI1), Hajime YASUHARA3), Katsuji OGUCHI3) and Ryohei KOIDE1)

Abstract : The central effects of brimonidine on intraocular pressure (IOP) in

male pigmented rabbits were investigated. Twenty ƒÊL brimonidine was administered to the lateral eye, or intraorally. For intracerebroventricular

(icy) treatment, the guide cannula was inserted two weeks before injection into the lateral ventriculus following Fifkova's brain map. Ten ƒÊL brimonidine

was injected (5ƒÊL/ min, icy). After the treatments, IOP was measured by

pneumatonometer and pupil diameter (PD) was measured using a ruler. Heart rate (HR) and mean arterial blood pressure (MABP) at metatarsus

were also monitored after topical administration. IOPs were significantly

decreased bilaterally by 0.05% brimonidine administered topically to the lateral

eye (p< 0.001, n=6), or 0.1% administered orally (p•ƒ0.001, n=11)

compared to a saline treatment control group. After 0.0001 % brimonidine icy treatment, IOP was significantly decreased bilaterally (p•ƒ0.05, n=4). PD, HR and MABP were not changed by 0.01% topical administration of

brimonidine. The decrease in IOP which was induced by 0.01 % brimonidine

icy treatment was inhibited by pretreatment with 0.1% yohimbin icy (p•ƒ0.05,

n=4). These results suggest that topical use of brimonidine might decrease

IOP in the pigmented rabbit by a central ƒ¿2-receptor effect in addition to its local effect.

Key words : ƒ¿2-adrenergic agonist, brimonidine, ocular hypertension, central

nervous system, intracerebroventricular injection

Introduction

Sympathomimetic and sympatholytic agents have been used for reducing intraocular pressure (lOP) in the treatment of glaucoma. Some of these agents lower IOP bilaterally even when administered unilaterally. By measuring IOP of pigmented rabbits following the administration of ƒ¿ 2-agonists (clonidine and apraclonidine) and ƒÀ-blockers (timolol, befunolol and cartelol ), we previously demonstrated that central a2-receptors are involved in the regulation of IOP and that the sympathetic nerves are involved in the centrifugal 1) Department of Ophthalmology, Showa University, School of Medicine. 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142- 8666, Japan. 2 ) Department of Physiology, Showa University, School of Medicine. 3)Department of Pharmacology, Showa University, School of Medicine. 314 Mitsutaka SODA, et al pathway of this action1-4) Brimonidine is a more potent and highly selective ƒ¿2-adrenergic agonist than clonidine or apraclonidine. In the United States, these agents have already been introduced clinically as a drug for reducing IOP5-7). The mechanism of action is said to be suppression of aqueous humor production and increase in uveoscleral outflow8,9). The drug, when administered by topical instillation, may be distributed via conjunctival, scleral and nasal mucosal absorption and manifest its effect on a central site. The pharmacology of action of brimonidine has been studied by several investigators9-12), but still remains under debate. In this study, we administered brimonidine by topical, oral and intracerebroventricular (icy) injection and measured the change in IOP, pupil diameter (PD), mean arterial blood pressure (MABP ) and heart rate (HR) to investigate the effect of brimonidine on lowering IOP via the central nervous system. In addition, we evaluated the effect of pretreatment with yohimbine, an ƒ¿2-adrenoceptor antagonist, on altering ocular hypotension induced by brimonidine.

Materials and Methods

Experiment Animals Male Dutch pigmented rabbits (2.0-3.0 kg) were maintained on a 12-hour light! dark cycle during these experiments. For icy treatment, rabbits were anesthetized with a single intravenous injection of pentobarbiturate at 30 mg/kg. The guide cannula (0.5 mm diameter) was inserted into a lateral ventricle following the brain map of Fifkova, and fixed in place with dental cement. To reduce surgery stress, drug administrations were performed after two weeks of stabilization. Rabbits were conscious during all measurement-recording series. The study protocol was approved by the Showa University Animal Care Ethics Committee.

Drug preparations and administration Brimonidine (UK 14, 304, 5-bromo-N- [2-imidazoline-2-yl] -6-quinoxalinamine, Sigma Chemi- cal Co, St. Louis, MO. USA) and yohimbine hydrochloride (Wako Pure Chemical Industries Ltd, Osaka, Japan) were used in these experiments.

Brimonidine was suspended in absolute saline with 0.1N HCl at pH 6.5. Twenty pL of brimonidine solution (0.01%, 0.05%, and 0.1%) or of saline control was applied to the right eye or given orally. For icy injection, 10ƒÊL of brimonidine (0.0001%, 0.001% and

0.01%) or saline control was injected from the guide cannula using a microsyringe at a rate of 5ƒÊL/min. For pretreatment with yohimbine hydrochloride (10 ƒÊL of a 0.1% solution), icy injection was performed 30 min prior to brimonidine treatment.

Intraocular pressure IOP was measured by a pneumatonometer (RE Medical Co, Osaka, Japan) at 0, 15, 30, 60, 90, 120, 180, 240 and 300 minutes after unilateral topical brimonidine treatment. Rabbits were conscious and gently restrained during the course of measurements. Before tonometry, of 0.4% oxybuprocaine (Santen Chemical Co, Osaka, Japan) was applied to the cornea as a local anesthetic to minimize discomfort to the animal. The Central Effect of Brimonidine on Intraocular Pressure 315

Pupil Diameter PD was determined with a ruler at 0, 30, 60, 90 and 120 minutes after unilateral topical brimonidine treatment under conscious conditions.

Heart rate and mean arterial blood pressure Metatarsal HR and MABP were measured with a sphygmomanometer (Softron BP-98E, Softron, Tokyo, Japan) at 0, 30, 60, 90 and 120 minutes as above.

Statistics

Statistical analysis was performed using the Student's t-test. All data are expressed as a mean•}standard error (S.E.) and a probability level of 0.05 was selected as the minimum level of significance.

Results The effect of topical administration of brimonidine on IOP

The maximum decrease in IOP was recorded in the treated eye as 8.5•}0.7 mmHg

(p•ƒ0.001) 120 min after treatment with 0.1% brimonidine. At 90 min after 0.05% brimonidine treatment, IOP was decreased by 8.3•}0.9 mmHg (p•ƒ0.001) and at 60 min after treatment with 0.01% brimonidine, it was decreased by 5.8•}0.7 mmHg (p•ƒ0.001)

(Fig. 1A). In the left (non-treated) eye, the maximum decrease in IOP was recorded at 7.3•}0.4 mmHg (p•ƒ0.00l) after 60 min of 0.1% brimonidine treatment and 7.2•}0.9 mmHg

(p•ƒ0.001) after 60 min of treatment with 0.05% brimonidine (Fig. 1B). The average IOP in saline groups in right and left eyes was 18.8•}0.3 mmHg and 19.0•}0.2 mmHg, respectively.

The effect of topical administration of brimonidine on PD, HR and MABP

PD decreased by 0.15•}0.04 mm (p•ƒ0.05) in the treated eye (right eye) at 90 min after

0.1% brimonidine treatment, as compared to saline control (Fig. 2A). In the left eye (not

treated), the influence of the drug was not observed by unilateral topical administration of brimonidine (Fig. 2B). HR (Fig. 3A) was not changed by topical administration of brimonidine (0.01%, 0.05% and 0.1%). MABP (Fig. 3B) was decreased by 27.9 mmHg

(p•ƒ0.05) after 60 min of 0.1% brimonidine topical administration, as compared to saline control.

The effect of oral administration of brimonidine on IOP

IOP was decreased bilaterally from 15 min until 120 min after 0.1% oral administration of brimonidine. The maximum decrease of IOP was 5.3•}0.5 mmHg (p•ƒ0.001) in the

right eye and 5.6•}0.4 mmHg (p•ƒ0.001) in the left eye after 60 min, as compared to saline

control (Fig. 4). The average IOP in the saline group was 19.7•}0.2 mmHg in the right

eye and 19.8•}0.2 mmHg in the left eye.

The effect of icy treatment with brimonidine and yohimbine on IOP

IOP was decreased bilaterally by icy treatment with brimonidine (Fig. 5). The maximum

decrease in IOP in the right eye was 5.3•}1.3 mmHg (p•ƒ0.05) by 0.01% brimonidine at 30

min, 3.5•}0.8 mmHg (p•ƒ0.05) by 0.001% brimonidine at 60 min and 4.5•}1.2 (p•ƒ0.05) by 316 Mitsutaka SODA, et al

Fig. 1. The effect of unilateral topical administration of brimonidine on IOP Changes in IOP by unilateral topical administration (-•›-: saline, n=7, -•œ-: 0.01%

brimonidine, n=6, -• -: 0.05% brimonidine, n=6, -•£-: 0.1% brimonidine, n=7) are shown in A : treated (right) eye, and B : untreated (left) eye. Data represent mean f S.E.

(mmHg). *p•ƒ005, #p•ƒ0.001 v.s. saline-treated group.

0.0001% brimonidine at 30 min after icy treatment (Fig. 5A). In the left eye , IOP was decreased by 5.5•}0.6 mmHg (p•ƒ 0.001) by 0.01% brimonidine at 15 min , 4.0•}0.3 mmHg (p•ƒ0.01) by 0.001% brimonidine at 60 min and 3.6•}0.9 mmHg (p•ƒ 0.05) by 0.0001% brimonidine at 30 min after icy treatment (Fig. 5B). The average IOP in the saline group was 20.5•}0.4 mmHg in the right eye, and 20.4•}0.4 mmHg in the left eye . Pretreatment with 0.1% yohimbine (icy) bilaterally inhibited ocular hypotension induced by 0 .01% The Central Effect of Brimonidine on Intraocular Pressure 317

Fig. 2. The effect of unilateral topical administration of brimonidine on PD Changes in PD by unilateral topical administration (-•›-: saline, n=4, -•œ-: 0.01% brimonidine, n=4, -• - : 0.05% brimonidine, n=4, -•£-: 0.1% brimonidine, n=4) are shown in A : treated (right) eye, and B : untreated (left) eye. Data represent mean•}S.E.

(mm). * p•ƒ0.05 v.s. saline-treated group. brimonidine icy treatment (Fig. 6), however no significant change in IOP was seen (data not shown).

Discussion

In this study, IOP measurements in control animals (mean =18.8•}0.3 mmHg for the right eye and 19.0•}0.2 mmHg for the left eye) were very reproducible. 318 Mitsutaka SODA, et al

Fig. 3. The effect of unilateral topical administration of brimonidine on HR and MABP Changes in HR and MABP by unilateral topical administration (-•›- : saline, n=4, -•œ-: 0 .01% brimonidine, n=4, -• -: 0.05% brimonidine, n=4, -•£-: 0.1%

brimonidine, n=4) are shown in A and B, respectively. MABP was significantly decreased by topical administration of 0.1% brimonidine. Data represent mean•}S.E. (mm). *p•ƒ005 v.s. saline-treated group.

Dose-response curves for topical ( Fig.1 A/B) or oral administration (Fig. 4) were highly significant. We have also demonstrated that topical use of brimonidine might decrease IOP by a central ƒ¿2-receptor effect in addition to a local effect in the pigmented rabbit. The finding of bilateral reduction in IOP after unilateral topical and oral administration of brimonidine suggests that the local administration of brimonidine exerts actions on a central site. From the literature it would appear that brimonidine administered topically is carried The Central Effect of Brimonidine on Intraocular Pressure 319

Fig. 4. The effect of oral administration of brimonidine on IOP Changes in IOP by oral administration (right eye ; -•›-: saline, n=11, -• - : 0.1% brimonidine, n=11, left eye: -•œ-: saline, n=11, -•¡-: 0.1% brimonidine, n=11). Data represent mean•}S.E. (mmHg). *p•ƒ0.05, **p•ƒ0.001 v.s. saline-treated group (right eye), #p•ƒ005 .##p•ƒ0.001, v.s. saline-treated group (left eye).

to a central site via the blood brain barrier (BBB). Therefore, we administered brimonidine directly into the lateral ventricle to demonstrate the central effect of brimonidine.

Brimonidine icy treatment lowered IOP bilaterally in a dose-dependent manner. These results confirm the findings of Campbell and Potter11) who demonstrated that brimonidine

(0.3ƒÊg/20ƒÊL, equal volume is 0.003%/10ƒÊL), injected into the lateral ventricle, lowered IOP bilaterally in conscious pigmented rabbits11). Therefore, since locally administered brimonidine appears to reach the lateral ventricle, the drug probably reduces IOP via the central site. Clonidine also has an ocular hypotensive effect which involves the central nervous system1,2). Innemee et al.13) analyzed ocular autoradiograms in the cat following unilateral topical administration of 14C-clonidine. They found that the concentration of 14C -clonidine detected in the brain was sufficient to explain the bilateral decrease in IOP by a central mechanism. The amount of clonidine detected in the brain was 1 % of the topically administered dose. These previous studies also suggested that brimonidine, administered topically, passes across the BBB, reaches a central site and exerts its effect,

thereby reducing IOP. In the present experiments, the lowest dose of brimonidine of

0.0001% (0.01ƒÊg/ 10ƒÊL) for icy treatment was effective for bilateral IOP reduction. This lowest dose level corresponds to less than 1 % of the amount of brimonidine administered topically (0.1% ; 20ƒÊg/20ƒÊL), and based on the findings of Innemee et al.13), it may be sufficient for the drug to cross the BBB and act via the central nervous system.

0.1% yohimbine, an ƒ¿2-antagonist, given 30 minutes prior to 0.001% brimonidine

inhibited brimonidine-induced ocular hypotension. Campbell and Potter.11) also reported that rauwolsine, another ƒ¿2-antagonist, inhibited ocular hypotension induced by 0.9ƒÊg

brimonidine. These results suggest that the central hypotensive action of brimonidine is due

to a central ƒ¿2-receptor. 320 Mitsutaka SODA, et al

Fig. 5. The effect of icy treatment with brimonidine on IOP Changes in IOP by icy treatment (-•›-: saline, n=4, -•£-: 0.0001% brimonidine, n=4, -•œ-: 0.001% brimonidine, n=4, -• -: 0.01% brimonidine , n=4) are shown in A : right eye, and B : left eye. Data represent mean f S.E. (mmHg). * p•ƒ0.05, #p•ƒ0 .001 vs. saline-treated group.

Topical administration of brimonidine had almost no observable effect on PD . These results were similar to those of other studies9,11). Mydriasis is generally believed to be primarily an ƒ¿1-mediated response in rabbits. It seems unlikely that brimonidine, which is highly ƒ¿2-selective, causes mydriasis, in contrast to clonidine or apraclonidine, which act on

ƒ¿1-receptors. Whether or not systemic blood pressure is correlated with IOP is still controversial14,15). In The Central Effect of Brimonidine on Intraocular Pressure 321

Fig. 6. The effect of yohimbine icy pretreatment on icy brimonidine-induced ocular hypotension Changes in IOP induced by icy treatment (-•›-: 0.01% brimonidine ; n=4, -•œ-: 0.1%

yohimbine+0.01 % brimonidine ; n=4). icy pretreatment with 0.1% yohimbin inhibited ocular hypotension induced by icy treatment with 0.01 % brimonidine. Data represent mean•} S.E. (mmHg). *p•ƒ0.05 v.s. 0.01% brimonidine-treated group.

this experiment we demonstrated changes in MABP and HR when brimonidine was administered topically. Although 0.1% brimonidine reduced MABP, topical administration of 0.05% or 0.01% brimonidine did not have any effect. These results suggest that the bilateral ocular hypotensive effect of brimonidine is not secondary to a decrease of blood pressure. In this study, we have demonstrated the ocular hypotensive mechanism of brimonidine. Topical, oral and icy administration of brimonidine lowered intraocular pressure bilaterally and yohimbine, an a 2-antagonist, inhibited the response. These results suggest that topical administration of brimonidine might lower IOP not only by a local effect, but also by a central a 2-receptor effect in the pigmented rabbit.

References

1) Moriwaki Y, Iizuka T, Nakamura A, Nakata K, Masaoka Y, Ueda T, Koide R, Inatomi M, Fukado Y, Uchida E, Oguchi K, Yasuhara H, Hisamitsu T and Takeshige C : Ocular hypotensive effect of ƒ¿-adrenoceptor agonist and antagonist in the conscious pigmented rabbit. Acta Soc Ophthalmol Jpn 96: 152-160 (1992)

(in Japanese) 2) Masaoka Y, Nakata K, Ueda T, Koide R, Inatomi M, Fukado Y, Uchida E, Oguchi K and Yasuhara H : Possible mechanism of the ocular hypotensive effect of clonidine in the pigmented rabbit. Acta Soc Ophthalmol Jpn 92: 1503-1509 (1989) (in Japanese) 3) Nakamura T, Nakata K, Masaoka Y, Ueda T, Koide R, Inatomi M, Fukado Y, Uchida E, Oguchi K, Yasuhara H, Hisamitsu T and Takeshige C : Beta blocker induced hypotension via central nervous system. J Eyes (Atarashii Ganka) 6 : 593-596 (1989) (in Japanese) 4) Fukado S, Ueda T, Koide R, Inatomi M, Fukado Y, Oguchi K, Uchida E, Kobayashi S, Yasuhara H, Hisamitsu T and Takeshige C : Central ocular hypotensive effect of apraclonidine in pigmented eye rabbits. J Eyes (Atarashii Ganka) 9 : 865-868 (1992) (in Japanese) 5) Javitt JC and Schiffman RM : Clinical success and quality of life with brimonidine 0.2% or timolol 0.5% used twice daily in glaucoma or ocular hypertension : A randomized clinical trial. Brimonidine Outcomes Study 322 Mitsutaka SODA, et al

Group 1 J Glaucoma 9 : 224-234 (2000) 6) David R : Brimonidine (ALPHAGAN(R)): A clinical profile four years after launch. Eur J Ophthalmol 11 72-77 (2001) 7) Schuman JS : Clinical experience with brimonidine 0.2% and timolol 0.5% in glaucoma and ocular hypertension. Surv Ophthalmol 41 : 27-37 (1996) 8) Burke J and Schwartz M : Preclinical evaluation of brimonidine. Surv Ophthalmol 41 : 9-18 (1996) 9) Burke JA and Potter DE : Ocular effect of a relatively selective ƒ¿2 agonist (UK-14,304-18) in cats, rabbits and monkeys. Curr Eye Res 5 : 665-675 (1986) 10) Burke J, Crosson C and Potter D : Can UK-14,304-18 lower IOP in rabbits by a peripheral mechanism? Curr Eye Res 8 : 547-552 (1989) 11) Campbell WR and Potter DE : Central mediated ocular hypotension : potential role of imidazoline receptors. Ann NY Acad Sci 763 : 463-485 (1996) 12) Campbell WR, Long S, Strothers R and Potter DE : Centrally-induced ocular hypotension in the rabbit : involvement of alpha-2 and imidazoline receptor subtypes. Abstr Soc Neurosci 19 : 1790 (1993) 13) Innemee HC, Hermans AJ and van Zwisen PA : The influence of clonidine on intraocular pressure after topical application to the eyes of anesthetized cats. Albrecht, Von Graefs Arch Klin Exp Ophthalmol212: 19-27 (1979) 14) Liu JH and Neufeld AH : Study of central Regulation of intraocular pressure using ventriculocisternal perfusion. Invest Ophthalmol Vis Sci 26: 136-143 (1985) 15) Burke J, Kharlamb A, Shan T, Runde E, Padillo E, Manlapaz C and Wheeler L : Adrenergic and imidazoline receptor-mediatedresponses to UK-14,304-18 (brimonidine) in rabbits and monkeys. A special difference. Ann NY Acad Sci 763: 78-95 (1996)

[Received May 27, 2003 : Accepted July 16, 2003]