Nucleoside-derived antagonists to A₃ adenosine receptors lower mouse intraocular pressure and act across species

Abstract

The purpose of the study was to determine whether novel, selective antagonists of human A₃ adenosine receptors (ARs) derived from the A₃-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A₃AR antagonists lowered mouse IOP measured with SNMS tonometry by 3–5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A₃AR agonists and the dihydropyridine A₃AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A₃AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A₃ARs derived from the potent, selective A₃ agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A₃AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly-selective A₃AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A₃ antagonists for study in multiple animal models.

Introduction

Intraocular pressure (IOP) is commonly elevated in glaucoma, leading to death of retinal ganglion cells and optic nerve atrophy. Reducing IOP is the only intervention known to delay the onset and slow progression of blindness, even in patients with normotensive disease (Collaborative Normal-Tension Glaucoma Study Group, 1998a, Collaborative Normal-Tension Glaucoma Study Group, 1998b, Kass et al., 2002, The AGIS investigators, 2000). IOP can be reduced by lowering either the rate of inflow or the resistance to outflow of aqueous humor.

Among novel strategies for lowering IOP, focus on adenosine receptors (ARs) has seemed promising because knockout of A₃-subtype ARs reduces IOP in the living mouse (Avila et al., 2002a), likely through a reduction in inflow. Several observations obtained with isolated cells have suggested that A₃ARs physiologically increase inflow of aqueous humor by activating Cl⁻ channels of the nonpigmented ciliary epithelial (NPE) cells at the aqueous surface of the ciliary epithelium (Carré et al., 1997, Carré et al., 2000, Mitchell et al., 1999). In contrast to the robust effects of A₃AR agonists on isolated cells from the inflow pathway, A₃AR agonists have been found to exert relatively modest actions on whole-cell currents of cells cultured from the trabecular meshwork (Fleischhauer et al., 2003) and from Schlemm's canal inner wall (Karl et al., 2005).

Based on the results obtained with isolated NPE cells, adenosine and selective A₃AR agonists would be expected to increase inflow and IOP, and A₃AR antagonists would be expected to exert opposite effects. The predicted changes in IOP triggered by A₃AR agonists and antagonists have been confirmed in the living mouse (Avila et al., 2001b, Avila et al., 2002b, Yang et al., 2005). While these responses in mice suggest a potential relevance of A₃AR-selective antagonists to humans, the binding affinities of these antagonists display substantial species variation (Jacobson et al., 1997, Linden, 2001). For instance, the binding inhibition constants (Ki) of some antagonists can vary by more than 30,000-times between rat and human A₃ARs (Yang et al., 2005). Interestingly, the responses of A₃ARs to selective agonists are much more highly conserved across species (Yang et al., 2005). We previously tested one A₃AR-selective antagonist, MRS 1292 (Yang et al., 2005), generated by modifying the A₃AR agonist IB-MECA (Gao et al., 2002), and observed antagonist activity in both the living mouse and immortalized human NPE cells (Yang et al., 2005).

Compared to the parent agonist previously modified (IB-MECA), the A₃AR agonist Cl-IB-MECA displays 3–4–fold greater potency and a 50-fold greater selectivity for A₃ receptors than for A₁ and A_2A receptors in rat brain (Kim et al., 1994). In the present work, we have examined the effects of five new A₃AR-selective antagonists constructed (Gao et al., 2006, Jeong et al., 2007) by modifying the far more selective agonist Cl-IB-MECA (Fig. 1), with the hope that this strategy might lead to the generation of even more selective A₃AR antagonists that are active across species. Measurements of mouse IOP have been conducted with both invasive SNMS (Avila et al., 2001a) and non-invasive pneumotonometric techniques (Avila et al., 2005). Similar baseline IOP values are obtained by applying the two techniques to the same eyes (Avila et al., 2005). However, under experimental conditions, the measurements can be complementary, rather than identical, because the intact mouse eye can present a substantial barrier to drug penetration (Wang et al., 2007). The fine-tipped micropipettes used for SNMS tonometry enhance entry of topically-applied drugs into the mouse eye (Wang et al., 2007), so that drug efficacy can be detected even if topical drug permeation is too slow to alter IOP measured non-invasively over similar periods.