Ocular Hypotensive Action of Topical Flunarizine in the Rabbit: Role of ς₁Recognition Sites

Abstract

In a previous study we ascertained the presence of 1 and 2 recognition sites in the rabbit iris-ciliary body, an ocular structure involved in aqueous humor production and drainage. We characterized the 1 sites using the preferential ligand (ϩ)-pentazocine, which caused a significant reduction of intraocular pressure (IOP). In the present study, flunarizine, a calcium channel blocker with a complex pharmacological profile, bound to 1 sites expressed in the iris-ciliary body with moderate affinity (K i ϭ 68 nM). Unilateral topical flunarizine (0.01-0.1%) caused a dose-related reduction of IOP in ocular normotensive rabbits and in the ␣-chymotrypsin model of ocular hypertension, without altering the IOP of the contralateral eye. This activity was blocked by the 1 site antagonist NE-100 [N,Ndipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine HCl] which, by itself, had no effect on IOP. Detection of flunarizine in rabbit iris-ciliary body homogenates, after topical instillation, showed that it adequately penetrates the rabbit eye. To investigate mechanisms that may contribute to ocular hypotension induced by 1 agonists, we carried out in vitro studies on the isolated rabbit iris-ciliary body. Sigma () recognition sites are a unique class of binding sites, heterogeneously distributed in the nervous system and in peripheral organs, that presumably serve as receptors for some unidentified endogenous ligand (Walker et al., 1990;Quiron et al., 1992). The recognition sites bind an array of structural classes of compounds including haloperidol, 1,3-di-O-tolylguanidine (DTG), and (ϩ)-benzomorphans, such as (ϩ)-pentazocine and (ϩ)-N-allylnormetazocine (Su and Junien, 1994). On the basis of biochemical and radioligand binding data, recognition sites have been classified into at least two types, 1 and 2 (Quiron et al., 1992). The 1 recognition sites display preferential affinity and stereoselectivity for (ϩ)-benzomorphans . A 1 binding protein has been cloned (Hanner et al., 1996), and its sequence shows significant similarities with sterol C 8 -C 7 isomerases from fungi.The functional role of recognition sites and the cellular mechanisms responsible for the effects produced by -site ligands have not been clearly determined, although these compounds may act as neuromodulators. Previous reports have associated -site ligands with calcium homeostasis (Brent et al., 1996;Hayashi et al., 2000;Hayashi and Su, 2001). -site ligands can influence [ 3 H]dopamine and [ 3 H]norepinephrine (NE) release from rat brain slices, acting, at least partially, presynaptically (Gonzalez-Alvear and Werling, 1995;Gonzalez and Werling, 1997).Several -site ligands influence electrically evoked contractions in the guinea pig longitudinal muscle/myenteric plexus preparation (Campbell et al., 1989). These latter findings add evidence to the theory that recognition sites participate in the regulation of autonomic functions and that -site ligands may interfere with neurotransmitter release, modulating their action on innervated tissue (Su and J...

Study Design and Methodologies for Evaluation of Anti-glaucoma Drugs

“…Commonly reported doses of α-chymotrypsin include: 45 UAE in 100 μl [62], 50 UAE in 100 μl of saline [131], 50 UAE in 200 μl of saline [142], and 150 units in 0.5 ml of sterile saline [126]. In the latter study stable increased IOP was achieved at 15 days (instead of at 1 month in the other studies).…”

“…the model is generally not used to assess drug tolerability, the effect of the test article on intraocular structures or to investigate the pathophysiology of glaucoma). Common complications that may render the animal not useful for the study include lens luxation, severe intraocular inflammation (5-10 % of animals, [62,142]), severe corneal inflammation (17 % in one study, [126]), and failure to develop a sustained increase in IOP (up to 50 % of animals). Most studies wait 4 weeks after administration of α-chymotrypsin to evaluate the utility of a given animal, and only use rabbits with an IOP which is at least 15 mmHg higher than the fellow control eye or has an IOP of 25 mmHg or more.…”

“…A single injection of α-chymotrypsin, typically into the posterior chamber, also results in a chronic elevation in IOP which may last a year or more [62,126,131,[140][141][142]. IOP is usually increased into the 30's, and is seldom greater than 50 mmHg [139].…”

“…One approach is to constrict the pupil with one to two drops of pilocarpine 15 min prior to the injection [62], induce anesthesia with intramuscular ketamine HCL (35 mg/kg) and xylazine (5 mg/kg), enter the anterior chamber with a 25-30 g needle and inject α-chymotrypsin into the posterior chamber through the pupil with a 27-30 g needle or a comparably sized blunt cannula. The tip of the needle/cannula should be swept so as to distribute the enzyme evenly throughout the posterior chamber and left in the posterior chamber for at least 1 min before being carefully withdrawn to avoid the enzyme coming into contact with the cornea (especially the corneal stroma which may melt if the enzyme contacts it, [142]). The external surface of the eye should then be rinsed with 10 ml of sterile saline to remove any residual enzyme [142].…”

“…The tip of the needle/cannula should be swept so as to distribute the enzyme evenly throughout the posterior chamber and left in the posterior chamber for at least 1 min before being carefully withdrawn to avoid the enzyme coming into contact with the cornea (especially the corneal stroma which may melt if the enzyme contacts it, [142]). The external surface of the eye should then be rinsed with 10 ml of sterile saline to remove any residual enzyme [142]. Some authors inject 60 units in 0.2 ml of saline into the anterior chamber and leave the needle in the anterior chamber for 2 min to minimize leak back [141], but corneal injury may be increased with this technically simpler approach.…”