Marsha A. McLaughlin

Identification and characterization of the ocular hypotensive efficacy of travoprost, a potent and selective FP prostaglandin receptor agonist, and AL-6598, a DP prostaglandin receptor agonist

The structure-activity studies that led to the identification of travoprost, a highly selective and potent FP prostaglandin analog, and AL-6598, a DP prostaglandin analog, are detailed. In both series, the 1-alcohol analogs are very effective and are thought to be acting as prodrugs for the biologically active carboxylic acids. The efficacy of amide prodrugs depends on the degree of substitution and the size of the substituents. Selected compounds are profiled in vitro and in vivo preclinically. Clinical studies show that travoprost 0.004% (isopropyl ester) provided intraocular pressure control superior to timolol 0.5% when used as monotherapy in patients with open-angle glaucoma or ocular hypertension. In clinical studies, AL-6598 0.01% provided a sustained intraocular pressure reduction with q.d. application; b.i.d. provided greater intraocular pressure control. The acute and, apparently, conjunctival hyperemia associated with topical ocular AL-6598 can be attenuated while maintaining intraocular pressure-lowering efficacy by formulating with brimonidine.

Levobetaxolol (Betaxon™) and Other β-Adrenergic Antagonists: Preclinical Pharmacology, IOP-Lowering Activity and Sites of Action in Human Eyes

The pharmacological characteristics of levobetaxolol, a single active isomer of betaxolol, were determined and compared with activities of other beta-adrenoceptor antagonists. Levobetaxolol (43-fold beta1-selective) exhibited a higher affinity at cloned human beta1 (Ki = 0.76 nM) than at beta2 (Ki = 32.6 nM) receptors, while dextrobetaxolol was much weaker at both receptors. Levobetaxolol potently antagonized functional activities at cloned human beta1 and beta2 receptors, and also at guinea pig atrial beta1, tracheal beta2 and rat colonic beta3 receptors (IC50s = 33.2 nM, 2970 nM and 709 nM, respectively). Thus, levobetaxolol was 89-times beta1-selective (vs beta2). Levobetaxolol (Ki = 16.4 nM) was more potent than dextrobetaxolol (Ki = 2.97 microM) at inhibiting isoproterenol-induced cAMP production in human non-pigmented ciliary epithelial cells. Levobunolol and (l)-timolol had high affinities at beta1 and beta2 receptors but were considerably less beta1-selective than levobetaxolol. Levo-, dextro- and racemic-betaxolol exhibited little or no affinity, except at sigma sites and Ca2+-channels (IC50s > 1 microM), at 89 other receptor/ligand binding sites. Levobetaxolol exhibited a micromolar affinity for L-type Ca2+-channels. In conscious ocular hypertensive cynomolgus monkeys, levobetaxolol was more potent than dextrobetaxolol, reducing intraocular pressure by 25.9+/-3.2% at a dose of 150 microg/eye (n = 15-30). Quantitative [3H]-levobetaxolol autoradiography revealed high levels of binding to human ciliary processes, iris, choroid/retina, and ciliary muscles. In conclusion, levobetaxolol is a potent, high affinity and beta1-selective IOP-lowering beta-adrenoceptor antagonist.

Effects of travoprost on aqueous humor dynamics in monkeys.

Purpose:To determine the mechanism by which travoprost, a prodrug of a prostaglandin F2α analog, reduces intraocular pressure (IOP) in cynomolgus monkey eyes. Methods:One eye each of 12 monkeys was treated with laser burns to the trabecular meshwork to elevate IOP. At least 4 months later (Baseline Day), IOP was measured by pneumatonometry (9:00 AM and 11:45 AM), and aqueous flow and outflow facility were determined by a fluorophotometric method. Uveoscleral outflow was calculated. Both eyes were treated with travoprost 0.004% at 9:00 AM and 5:00 PM for two days and at 9:30 AM on the third day (Treatment Day), when measurements were repeated as on Baseline Day. Statistical analyses were performed using two-tailed, paired t tests. Results:On Treatment Day compared with Baseline Day, IOP in hypertensive eyes was reduced at 2.25 hours (25.8 ± 11.2 vs 33.7 ± 13.2 mm Hg; mean ± standard error of the mean [SEM]; P = 0.02) and 16 hours (26.3 ± 10.2 vs 35.1 ± 13.6 mm Hg; P = 0.02) after treatment. The increase in uveoscleral outflow was not significant. In normotensive eyes, IOP was reduced at 2.25 hours (19.0 ± 3.7 vs 23.0 ± 4.0 mm Hg; P = 0.03) and 16 hours (20.7 ± 5.4 vs 23.4 ± 5.3 mm Hg; P = 0.01) after treatment, and uveoscleral outflow was significantly (P = 0.02) increased (1.02 ± 0.43 vs 0.35 ± 0.72 μL/min). Conclusion:Travoprost reduces IOP in normotensive monkey eyes by increasing uveoscleral outflow. The IOP reduction in hypertensive eyes is probably via the same mechanism, although the increased uveoscleral drainage did not reach statistical significance. Travoprost had no effect on aqueous flow or outflow facility.

Studies on the Involvement of Melatonergic Mechanism in Intraocular Pressure Regulation

It has been reported that diurnal changes in intraocular pressure (IOP) may be related to the fluctuation of melatonin levels in the eye. In order to test this hypothesis, effects of methysergide, tranylcypromine, and p-chlorophenylalanine (PCPA) on IOP recovery were studied in rabbits infused with hypertonic saline. Methysergide, a serotonin blocker at receptor sites, did not affect IOP recovery, presumably because it did not affect melatonin synthesis. Tranylcypromine, a monoamine oxidase inhibitor which preserves serotonin and eventually increases the level of melatonin in tissues, facilitated IOP recovery. PCPA, known to inhibit serotonin synthesis and to lower the melatonin level in tissues, markedly delayed IOP recovery. These results indicate that a chemical which decreases melatonin content in the eye also lowers the IOP and vice versa. It is suggested that some melatonergic mechanism is involved in the circadian rhythm of IOP.

3-Oxa-15-cyclohexyl Prostaglandin DP Receptor Agonists as Topical Antiglaucoma Agents

A series of prostaglandin DP agonists containing a 3-oxa-15-cyclohexyl motif was synthesized and evaluated in several in vitro and in vivo biological assays. The reference compound ZK 118.182 (9beta-chloro-15-cyclohexyl-3-oxa-omega-pentanor PGF(2alpha)) is a potent full agonist at the prostaglandin DP receptor. Saturation of the 13,14 olefin affords AL-6556, which is less potent but is still a full agonist. Replacement of the 9-chlorine with a hydrogen atom or inversion of the carbon 15 stereochemistry also reduces affinity. In in vivo studies ZK 118.182 lowers intraocular pressure (IOP) upon topical application in the ocular hypertensive monkey. Ester, 1-alcohol, and selected amide prodrugs of the carboxylic acid enhance in vivo potency, presumably by increasing bioavailability. The clinical candidate AL-6598, the isopropyl ester prodrug of AL-6556, produces a maximum 53% drop in monkey IOP with a 1 microg dose (0.003% w/w) using a twice-daily dosing regime. Synthetically, AL-6598 was accessed from known intermediate 1 using a novel key sequence to install the cis allyl ether in the alpha chain, involving a selective Swern oxidative desilylation of a primary silyl ether in the presence of a secondary silyl ether. In this manner, 136 g of AL-6598 was synthesized under GMP conditions for evaluation in phase I clinical trials.

Cabergoline: Pharmacology, ocular hypotensive studies in multiple species, and aqueous humor dynamic modulation in the Cynomolgus monkey eyes.

The aims of the current studies were to determine the in vitro and in vivo ocular and non-ocular pharmacological properties of cabergoline using well documented receptor binding, cell-based functional assays, and in vivo models. Cabergoline bound to native and/or human cloned serotonin-2A/B/C (5HT(2A/B/C)), 5HT(1A), 5HT(7), alpha(2B), and dopamine-2/3 (D(2/3)) receptor subtypes with nanomolar affinity. Cabergoline was an agonist at human recombinant 5HT(2), 5HT(1A) and D(2/3) receptors but an antagonist at 5HT(7) and alpha(2) receptors. In primary human ciliary muscle (h-CM) and trabecular meshwork (h-TM) cells, cabergoline stimulated phosphoinositide (PI) hydrolysis (EC(50)=19+/-7 nM in TM; 76 nM in h-CM) and intracellular Ca(2+) ([Ca(2+)](i)) mobilization (EC(50)=570+/-83 nM in h-TM; EC(50)=900+/-320 nM in h-CM). Cabergoline-induced [Ca(2+)](i) mobilization in h-TM and h-CM cells was potently antagonized by a 5HT(2A)-selective antagonist (M-100907, K(i)=0.29-0.53 nM). Cabergoline also stimulated [Ca(2+)](i) mobilization more potently via human cloned 5HT(2A) (EC(50)=63.4+/-10.3 nM) than via 5HT(2B) and 5HT(2C) receptors. In h-CM cells, cabergoline (1 microM) stimulated production of pro-matrix metalloproteinases-1 and -3 and synergized with forskolin to enhance cAMP production. Cabergoline (1 microM) perfused through anterior segments of porcine eyes caused a significant (27%) increase in outflow facility. Topically administered cabergoline (300-500 microg) in Dutch-belted rabbit eyes yielded 4.5 microMM and 1.97 microM levels in the aqueous humor 30 min and 90 min post-dose but failed to modulate intraocular pressure (IOP). However, cabergoline was an efficacious IOP-lowering agent in normotensive Brown Norway rats (25% IOP decrease with 6 microg at 4h post-dose) and in conscious ocular hypertensive cynomolgus monkeys (peak reduction of 30.6+/-3.6% with 50 microg at 3h post-dose; 30.4+/-4.5% with 500 microg at 7h post-dose). In ketamine-sedated monkeys, IOP was significantly lowered at 2.5h after the second topical ocular dose (300 microg) of cabergoline by 23% (p<0.02) and 35% (p<0.004) in normotensive and ocular hypertensive eyes, respectively. In normotensive eyes, cabergoline increased uveoscleral outflow (0.69+/-0.7 microL/min-1.61+/-0.97 microL/min, n=13; p<0.01). However, only seven of the eleven ocular hypertensive monkeys showed significantly increased uveoscleral outflow. These data indicate that cabergolines most prominent agonist activity involves activation of 5HT(2), 5HT(1A), and D(2/3) receptors. Since 5HT(1A) agonists, 5HT(7) antagonists, and alpha(2) antagonists do not lower IOP in conscious ocular hypertensive monkeys, the 5HT(2) and dopaminergic agonist activities of cabergoline probably mediated the IOP reduction observed with this compound in this species.

Are β-Adrenergic Mechanisms Involved in Ocular Hypotensive Actions of Adrenergic Drugs?

Specific beta 1- and beta 2-adrenergic agonists and antagonists were studied for their effects on aqueous humor dynamics in a cat model. Tazolol (beta 1-agonist) reduced formation of aqueous humor more than outflow, while metoprolol (beta 1-antagonist) reduced outflow more than formation. Both salbutamol (beta 2-agonist) and butoxamine (beta 2-antagonist) inhibited aqueous humor formation and aqueous humor outflow to an equal extent. It is concluded that the adrenergic mechanism is functioning only slightly, if at all, in affecting the intraocular pressure. On the other hand, receptor binding experiments on iris-ciliary body showed clearly that there are 40-fold and 281-fold differences in binding affinity of D- and L-isomers of timolol and propranolol, respectively. These results indicate that the adrenergic receptor mechanism does exist in the eye tissues but that it does not control intraocular pressure significantly.

Benzothiophene containing Rho kinase inhibitors: Efficacy in an animal model of glaucoma

We identified a new benzothiophene containing Rho kinase inhibitor scaffold in an ultra high-throughput enzymatic activity screen. SAR studies, driven by a novel label-free cellular impedance assay, were used to derive 39, which substantially reduced intraocular pressure in a monkey model of glaucoma-associated ocular hypertension.

Effects of brinzolamide on aqueous humor dynamics in monkeys and rabbits

This study examines the mechanisms by which brinzolamide reduces intraocular pressure (IOP) in healthy rabbits and in monkeys with unilateral ocular hypertension. Intraocular pressures were measured by pneumatonometry and aqueous flow was determined by fluorophotometry before and after three twice-daily drops of 1% brinzolamide to both eyes per monkey and after similar treatment to one eye per rabbit. In monkeys, outflow facility was determined by fluorophotometry and uveoscleral outflow was calculated. In rabbits, outflow facility was determined by two-level constant pressure infusion and uveoscleral outflow was measured by an intracameral tracer technique. Compared with contralateral vehicle-treated rabbit eyes, IOP was reduced in brinzolamide-treated eyes by 2.5 +/- 1.9 mmHg (mean +/- standard deviation; p =.006) at four hours after the second dose. Aqueous flow was reduced by 0.50 +/- 0.65 microl/min (p =.02). This effect was found in rabbits previously treated with brinzolamide but not in naive rabbits. Treated hypertensive eyes of monkeys had a reduction in IOP of 7.3 +/- 8.8 mmHg (p = 0.01) and aqueous flow of 0.69 +/- 1.10 microL/min (p = 0.05) when compared with baseline. Brinzolamide did not affect outflow facility or uveoscleral outflow in either rabbits or monkeys. It is concluded that, in normotensive eyes of rabbits and hypertensive eyes of monkeys, brinzolamide reduces IOP by reducing aqueous flow and not by affecting aqueous humor drainage.

Effects of Rho Kinase Inhibitors on Intraocular Pressure and Aqueous Humor Dynamics in Nonhuman Primates and Rabbits

PURPOSE This study examines the effects of 2 Rho kinase inhibitors on intraocular pressure (IOP) and aqueous humor dynamics. METHODS IOPs of New Zealand albino rabbits with ocular normotension and cynomolgus macaques (nonhuman primate, NHP) with chronic unilateral laser-induced glaucoma were measured at baseline and periodically after a 9 a.m. dose of H-1152, Y-27632, or vehicle. In a separate group of NHPs, aqueous flow, outflow facility, uveoscleral outflow, and IOP were determined after treatment with Y-27632 or vehicle control. RESULTS Decreases in IOP were found in rabbits (n = 5) at 6 h after one dose of 2% Y-27632 (29%, P = 0.0002) or 1% H-1152 (35%, P = 0.0001), and in hypertensive eyes of NHPs (n = 7-9) at 3 h after one dose of 2% Y-27632 (35%, P = 0.005) or 1% H-1152 (51%, P = 0.0003). With 2 doses of 1% Y-27632 or vehicle in NHP hypertensive eyes (n = 12), significant drug effects were IOP reduction of 28% (P = 0.05) at 2.5 h after the second dose and increases in aqueous flow (36%; P = 0.013), uveoscleral outflow (59%, P = 0.008), and outflow facility (40%; P = 0.01). In normotensive eyes of the same animals, aqueous flow increased by 21% (P = 0.03). No significant change was found in any of the other parameters. CONCLUSIONS Y-27632 and H-1152 lower IOP in rabbits and hypertensive eyes of NHPs for at least 6 h after single doses. The Y-27632 effect on IOP in hypertensive NHP eyes is caused by increases in outflow facility and uveoscleral outflow. An increase in aqueous humor formation attenuates but does not prevent an IOP decrease.