In vivo optimization of 2,3-diaminopyrazine Rho Kinase inhibitors for the treatment of glaucoma.

A series of 2,3,6-pyrazine Rho Kinase inhibitors were optimized for in vivo activity for topical ocular dosing. Modifications of the 2-(piperazin-1-yl)pyrazine derivatives produced compounds with improved solubility and physicochemical properties. Modifications of the 6-pyrazine substituent led to improvements in in vitro potency. Compound 9 had the best in vitro and in vivo potency of EC50=260 nM with a 30% reduction of IOP in a non-human primate model at a dose of 0.33%.

Dose response for travoprost® in the glaucomatous beagle.

OBJECTIVE: To evaluate the changes in intraocular pressure (IOP) and pupil size in 12 Beagles with inherited glaucoma after instillations of 0.033, 0.0033, 0.001, 0.00033, and 0.0001% travoprost (Travatan®-Alcon Laboratories, Inc., Ft Worth, TX, USA) in multiple single-dose studies. PROCEDURES: Intraocular pressure and pupil diameter (PD) measurements were obtained at 9 am, 12 pm, 3 pm, and 9 am the following day (24 h) in two groups of six glaucoma dogs. After 7 days, the vehicle or concentration was repeated in the contralateral eye of the same animals. RESULTS: Concentrations of 0.00033, 0.001, and 0.0033% travoprost significantly lowered IOP and PD, but the 0.0001% concentration provided limited IOP changes, although PD changes were still significant. This suggests travoprost is effective in the dog to lower IOP and reduce pupil size at concentrations starting between 0.0001 and 0.00033%. CONCLUSIONS: The dose response for travoprost in the glaucomatous Beagle indicates this model is highly sensitive to this group of drugs, even at concentrations as low as 0.00033% (1/12 the commercially available concentration).

Aqueous humor dynamics in inbred rhesus monkeys with naturally occurring ocular hypertension.

This study evaluates aqueous humor dynamics in rhesus monkeys from the University of Florida inbred colony with ocular normotension and naturally occurring ocular hypertension. Eight monkeys with untreated intraocular pressures (IOPs) of less than 18 mmHg in one eye (ONT group) and seven with untreated IOPs of greater than or equal to 18 mmHg in one eye (OHT group) were included in the study. Assessments included central cornea thickness by ultrasound pachymetry, IOP by tonometry, aqueous flow and outflow facility by fluorophotometry, and uveoscleral outflow by mathematical calculation. Animals were sedated with ketamine for all measurements. Values from the two eyes of each animal were averaged, with the exception of one animal that had only one good eye. Comparisons between groups were made by Student's two-tailed unpaired t-tests. Compared to the ONT group, the OHT group had higher IOPs at all times measured (4:00 PM the day before the study, 21.2 ± 6.5 versus 14.4 ± 1.5 mmHg, p = 0.01; 9:00 AM the day of the study, 20.7 ± 6.6 versus 14.8 ± 1.2 mmHg, p = 0.03; 11:00 AM the day of the study, 16.0 ± 1.6 versus 13.3 ± 2.9 mmHg, p = 0.05) and lower aqueous flow (2.12 ± 0.40 versus 4.54 ± 1.11 µl/min, p = 0.0001), outflow facility (0.17 ± 0.10 versus 0.33 ± 0.07 µl/min/mmHg, p = 0.01) and uveoscleral outflow (p < 0.05). The elevated IOP in inbred Florida rhesus monkeys is a result of significantly reduced outflow facility and uveoscleral outflow. These animals also have slower aqueous flow than the ONT animals which does not contribute to the higher IOP.

2,3-Diaminopyrazines as Rho kinase inhibitors.

Inhibition of rho kinase (ROCK) has been recognized as an important target for a number of diseases, including glaucoma. Herein we report SAR development around two hits from a kinase library that led to the discovery of the ROCK inhibitor compound 38. In vitro and in vivo analysis of this compound, including its effects in a monkey model of glaucoma will be discussed.

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.

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 cabergoline's 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.

Discovery of 13-oxa prostaglandin analogs as antiglaucoma agents: synthesis and biological activity.

FP-Class prostaglandin analogs have demonstrated utility for the treatment of glaucoma and ocular hypertension. A series of novel FP prostaglandin analogs was designed to optimize topical ocular activity and reduce ocular side-effects by replacing 13-carbon with oxygen. A facile synthesis was successfully developed for synthesis of the 13-oxa prostaglandins from the commercially available Corey aldehyde benzoate. Among the compounds synthesized, AL-16082 was the most potent prostaglandin FP agonist in vitro. In a prostaglandin FP receptor-linked second-messenger assay, phosphoinositide (PI) turnover, it exhibited a potency value (EC(50)) of 1.9 nM (78% max. response relative to fluprostenol). The isopropyl ester of AL-16082, compound AL-16049, significantly lowered intraocular pressure (IOP) in the ocular hypertensive monkey eyes by 30%. In the study of acute ocular irritation response in New Zealand albino rabbits, AL-16049 produced lower incidence of hyperemia, swelling, and discharge than PGF(2alpha) (1 microg), and a similar incidence of hyperemia, swelling, and discharge to latanoprost (1.8 microg). AL-16049 also produced no signs of ocular irritation or discomfort in the cat at the doses evaluated.

AL-34662: a potent, selective, and efficacious ocular hypotensive serotonin-2 receptor agonist.

PURPOSE AND METHODS: The aim of this study was to determine the ocular pharmacological characteristics of AL-34662 (1-((S)-2-aminopropyl)-1H-indazole-6-ol), a new synthetic serotonin-2 (5-HT2) receptor-agonist ocular hypotensive agent. A variety of well-documented in vitro and in vivo procedures were utilized to study the pharmacological attributes of AL-34662. RESULTS: AL-34662 exhibited a high affinity for the rat and human 5-HT2 receptor (IC50=0.8-1.5 nM) and for cloned human 5-HT2A-C receptors (IC50=3-14.5 nM). AL-34662 stimulated phosphoinositide turnover in human ciliary muscle (h-CM; EC50=289+/-80 nM) and in human trabecular meshwork (h-TM; EC50=254+/-50 nM) cells. AL-34662 also mobilized intracellular Ca2+ ([Ca2+]i) in h-CM (EC50=140+/-23 nM) and h-TM (EC50=38+/-8 nM) cells, being a full agonist like 5-HT itself. AL-34662's effects in the h-CM (and h-TM) cells were potently antagonized by 5-HT2A-antagonist M-100907 (IC50=1.8+/-0.7 nM), but weakly by 5-HT2B-antagonist (RS-127445 IC50>10 microM), 5-HT2B/C- antagonist (SB-242084 IC50=2.08 microM) and 5-HT2C antagonist (RS-102221 IC50>1 microM). AL-34662 caused relatively minimal ocular discomfort and hyperemia in rabbit and guinea pig eyes. It efficaciously lowered intraocular pressure (IOP) in the conscious ocular hypertensive monkey eyes (33% at 300 microg). The (R)-enantiomer (AL-34707) and the racemate (AL-34497) were less potent and/or efficacious than AL-34662 in all of these assays. CONCLUSIONS: AL-34662 is a high-affinity 5-HT2 receptor agonist that potently mobilizes [Ca2+]i in h-CM and h-TM cells, and which efficaciously lowers IOP in conscious ocular hypertensive cynomolgus monkey eyes through a local effect with minimal side-effects.

1-((S)-2-aminopropyl)-1H-indazol-6-ol: a potent peripherally acting 5-HT2 receptor agonist with ocular hypotensive activity.

Serotonin 5-HT(2) receptor agonists have been identified as a potential new class of agents for the treatment of ocular hypertension and glaucoma. The initially reported tryptamine analogues displayed either poor solution stability, potent central nervous system activity, or both of these undesirable characteristics and were unacceptable for clinical evaluation. A series of 1-(2-aminopropyl)-1H-indazole analogues was synthesized and evaluated for their suitability for consideration as clinical candidates. 1-((S)-2-Aminopropyl)-1H-indazol-6-ol (9) was identified as a peripherally acting potent 5-HT(2) receptor agonist (EC(50) = 42.7 nM, E(max) = 89%) with high selectivity for the 5-HT(2) receptors relative to other serotonergic receptor subtypes and other families of receptors and has significantly greater solution stability than alpha-methyl-5-hydroxytryptamine. Additionally, 9 potently lowers intraocular pressure in conscious ocular hypertensive monkeys (-13 mmHg, 33%); this reduction appears to be through a local rather than a centrally mediated effect. Compound 9 appears to be an excellent 5-HT(2) receptor agonist for conducting further studies directed toward a clinical proof-of-concept study for this class of ocular hypotensive agents.

Human trabecular meshwork cells express functional serotonin-2A (5HT2A) receptors: role in IOP reduction.

PURPOSE: To apply a multidisciplinary approach to the identification and pharmacological characterization of the serotonin (5HT) receptors that mediate functional responses in human trabecular meshwork (h-TM) cells. To correlate in vitro findings with intraocular pressure (IOP) changes in conscious ocular hypertensive cynomolgus monkeys. METHODS: Documented methods were used, including reverse transcription-polymerase chain reaction (RT-PCR), phosphoinositide (PI) turnover, and intracellular Ca2+ ([Ca2+]i) mobilization. IOP was measured using standard applanation pneumatonometry. RESULTS: h-TM cells expressed robust mRNA signals for 5HT2A and 5HT2B receptors. 5HT and its analogues stimulated PI turnover and [Ca2+]i mobilization in h-TM cells from multiple donors (20/24 donors' TM cells responded). The agonist potencies (EC50) of compounds in mobilizing [Ca2+]i were (nM): 5-methoxy tryptamine, 8 +/- 4; (R)-DOI, 18 +/- 6; alpha-methyl-5HT, 22 +/- 3; 5HT, 40 +/- 7; 5-methoxy-dimethyl tryptamine, 64 +/- 27; and BW-723C86, 1213 +/- 210. These effects were potently blocked by the 5HT2A-receptor-selective antagonist, M-100907 (Ki = 1 +/- 0.3 nM), but weakly by antagonists of 5HT2B and 5HT2C receptors. Only 5HT2 receptor agonists such as (R)-DOI (300 microg lowered IOP 34.4% from baseline of 38.2 mm Hg; P < 0.001) and alpha-methyl-5HT (250 microg lowered IOP 30.8% from baseline of 41.8 mm Hg; P < 0.001) lowered IOP in ocular hypertensive cynomolgus monkeys. CONCLUSIONS: Strong signals for 5HT2A and 5HT2B receptor mRNAs were detected in h-TM cells. The receptors that coupled to PI hydrolysis and [Ca2+]i mobilization in h-TM cells were the 5HT2A receptor subtype, which also significantly lowered IOP in a primate model. These receptors may mediate the ocular hypotensive actions of 5HT2A agonists.

Effects of a prostaglandin DP receptor agonist, AL-6598, on aqueous humor dynamics in a nonhuman primate model of glaucoma.

This study examines, in 11 cynomolgus monkeys with unilateral laser-induced glaucoma, the ocular hypotensive mechanism of action of AL-6598, partial agonist at the DP and EP prostanoid receptors. In a crossover fashion, both eyes of each monkey were dosed twice daily with 25 microL of either AL-6598 0.01% or vehicle for 2 days and on the morning of the 3rd day. Measurements were made on day 3 of each treatment. Alternative treatments were separated by at least 2 weeks. Intraocular pressures (IOPs) were measured by pneumatonometry and aqueous flow and outflow facility by fluorophotometry. Uveoscleral outflow was calculated mathematically. In the normotensive eyes, compared to vehicle treatment, AL-6598 decreased IOP from 22.5 +/- 0.7 to 18.7 +/- 0.9 mmHg (P = 0.006), increased uveoscleral outflow from 0.47 +/- 0.17 to 1.22 +/- 0.17 microL/min (P = 0.03), and increased aqueous flow from 1.49 +/- 0.10 to 1.93 +/- 0.13 microL/min (P = 0.01). No measurement in AL-6598-treated hypertensive eyes was significantly different from vehicle treatment. It is concluded that AL-6598 reduces IOP by increasing uveoscleral outflow in normotensive eyes of ketamine-sedated monkeys, despite an increase in aqueous flow. This effect is different from that of PGD(2), which decreases aqueous flow, and of the selective DP receptor agonist, BW245C, which increases both outflow facility and uveoscleral outflow in addition to decreasing aqueous flow.

Preclinical pharmacology of AL-12182, a new ocular hypotensive 11-oxa prostaglandin analog.

PURPOSE: The aim of this study was to determine selected in vivo ocular properties of AL-12182 (5,6-dihydro-4,5-didehydro-11-deoxy-11-oxa-16-(3-chlorophenoxy)-omega-tetranor-PGF(2alpha) isopropyl ester) and the in vitro profile of its free acid, AL-12180. METHODS: Previously documented radioligand binding and functional assays involving human ciliary muscle cells (h-CM), human trabecular meshwork (h-TM) and other cells, and porcine ocular arteries were utilized. For in vivo procedures, we utilized rabbits, cats, and nonhuman primates to measure hyperemia, pupil diameter, and intraocular pressure (IOP), respectively. RESULTS: AL-12180 exhibited the highest affinity for the FP-receptor (K(i) = 143 +/- 36 nM) and much lower affinity for DP-, EP(3)-, IP-, and TP-receptors, and for several nonprostanoid receptors, enzymes, neurotransmitter uptake sites, ion channels, and other regulatory sites. AL-12180 activated phospholipase C-mediated phosphoinositide hydrolysis (potency, EC(50) = 13.7-42.7 nM) through the FP-receptor in a variety of cells, such as h-CM, h-TM cells, human embryonic kidney cells expressing the cloned human ciliary body FP-receptor (HEK-FP), mouse 3T3 cells, and rat vascular smooth muscle cells. AL-8810, an FP-antagonist, blocked the effects of AL-12180 in h-CM cells (IC(50) = 8.7 microM). AL-12180 also stimulated the mobilization of intracellular Ca(2+) ([Ca(2+)](i)) in h-TM cells (EC(50) = 111 +/- 36 nM), h-CM cells (EC(50) = 11 nM), and in host cells expressing the cloned human ciliary body FP-receptor (EC(50) = 5.9 +/- 3.1 nM). AL-12180 lacked significant agonist activity at DP-, EP(2)-, EP(4)-, IP-, and TP-receptors in cell-based assays. However, AL-12180 contracted porcine central retinal and short posterior ciliary arteries in vitro with micromolar potencies that appeared to involve TP-receptor activation. in vivo, AL-12182 elicited dose-related hyperemia in the rabbit eye, miosis in the cat eye, and ocular hypotension in the nonhuman primate eye. CONCLUSIONS: AL-12180 is a relatively potent and selective FP-receptor agonist whose isopropyl ester prodrug (AL-12182) lowers IOP by as much as 40% following topical ocular dosing in a laser-induced nonhuman primate model of ocular hypertension.

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.

Effects of travoprost on aqueous humor dynamics in monkeys.

PURPOSE: To determine the mechanism by which travoprost, a prodrug of a prostaglandin F2alpha 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 microL/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.

Ocular Hypotensive Response in Nonhuman Primates of (8R)-1-[(2S)-2-Aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-ol a Selective 5-HT2 Receptor Agonist.

Recently, it has been reported that 5-HT2 receptor agonists effectively reduce intraocular pressure (IOP) in a nonhuman primate model of glaucoma. Although 1-[(2S)-2-aminopropyl]indazol-6-ol (AL-34662) was shown to have good efficacy in this nonhuman primate model of ocular hypertension as well as a desirable physicochemical and permeability profile, subsequently identified cardiovascular side effects in multiple species precluded further clinical evaluation of this compound. Herein, we report selected structural modifications that resulted in the identification of (8R)-1-[(2S)-2-aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-ol (13), which displayed an acceptable profile to support advancement for further preclinical evaluation as a candidate for proof-of-concept studies in humans.

Beta-oxygenated analogues of the 5-HT2A serotonin receptor agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane.

Activation of 5-HT(2A) serotonin receptors represents a novel approach to lowering intraocular pressure. Because 5-HT(2A) serotonin receptor agonists might also produce undesirable central effects should sufficient quantities enter the brain, attempts were made to identify 5-HT(2) serotonin receptor agonists with reduced propensity to penetrate the blood-brain barrier. 1-(4-Bromo-2,5-dimethoxyphenyl)-2-aminopropan-1-ol (6), an analogue of the 5-HT(2) serotonin receptor agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB; 1a) bearing a benzylic hydroxyl group, was identified as a candidate structure. Of the four optical isomers of 6, the 1R,2R-isomer (6d; K(i) = 0.5 nM) was found to bind at 5-HT(2A) receptors with an affinity similar to that of R(-)DOB (K(i) = 0.2 nM). Like R(-)DOB, 6d behaved as a partial agonist (efficacy ca. 50%) in a 5-HT(2)-mediated calcium mobilization assay. However, in an in vivo test of central action (i.e., stimulus generalization with rats as subjects), 6d was >15 times less potent than R(-)DOB. O-Methylation of 6d (i.e., 7d; 5-HT(2A) K(i) = 0.3 nM) resulted in an agent that behaved as a full (93% efficacy) agonist. Intraocular administration of 300 microg of 6d and 7d to ocular hypertensive monkeys was shown to reduce intraocular pressure by 20-27%. Given the route of administration (i.e., topical), and concentrations necessary to reduce intraocular pressure, compounds such as 6d should demonstrate minimal central effects at potentially useful therapeutic doses and offer useful leads for further development.

A novel and selective 5-HT2 receptor agonist with ocular hypotensive activity: (S)-(+)-1-(2-aminopropyl)-8,9-dihydropyrano[3,2-e]indole.

Serotonin 5-HT2 receptor agonists have recently been shown to be effective in lowering intraocular pressure in nonhuman primates and represent a potential new class of antiglaucoma agents. As part of an effort to identify new selective agonists at this receptor, we have found that (S)-(+)-1-(2-aminopropyl)-8,9-dihydropyrano[3,2-e]indole (AL-37350A, 11) has high affinity and selectivity (>1000-fold) for the 5-HT(2) receptor relative to other 5-HT receptors. More specifically, 11 is a potent agonist at the 5-HT2A receptor (EC50 = 28.6 nM, E(max) = 103%) that is comparable to serotonin. Evaluation of 11 in conscious ocular hypertensive cynomolgus monkeys showed this compound to be efficacious in reducing intraocular pressure (13.1 mmHg, -37%). Thus, 11 is a potent full agonist with selectivity for the 5-HT2 receptor and is anticipated to serve as a useful tool in exploring the role of the 5-HT2 receptor and its effector system in controlling intraocular pressure.

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.

A new method and device to induce transient retinal ischemia in the rat.

PURPOSE: To describe and characterize a novel, non-invasive method to induce retinal ischemia in the rat. METHODS: Retinal ischemia was produced by applying a compression force with a suture thread anchored about 2mm behind the limbus of the eye in order to increase intraocular pressure and reduce ocular blood flow. Both ends of the suture thread were connected to weights via a pulley system. The effect of various weights (20, 25, or 35 g) on retinal damage was tested in animals of different body weights (350 and 574 g). Scotopic ERG measurements and histological sections of retinal tissue were used to evaluate possible functional and morphological changes to the retina as a consequence of ischemia followed by reperfusion. RESULTS: Retinal ischemia was achieved by this method with good reproducibility. The degree of retinal damage, as quantified by ERG measurement, correlated with the amount of weight connected to the suture thread. Seven days after 60 minutes of retinal ischemia, the amplitudes of A- and B-waves of eyes compressed with a 20 g weight were 84 +/- 11% and 86 +/- 16% (mean +/- SEM, n = 4, amplitudes before ischemia defined 100%), respectively. The A- and B-wave amplitudes of eyes compressed with 25 g were 69 +/- 7% and 76 +/- 6% (n = 11), respectively. A 35 g treatment produced A- and B-wave amplitudes of 55 +/- 3.9% and 52 +/- 4% (n = 35), respectively. There was no significant difference in the level of ischemic damage in the two groups of animals with different body weights evaluated in this study. At two weeks after 60-minute retinal ischemia, there was a significant decrease (by 31.1%) in the cell density in the retinal ganglion cell layer. CONCLUSIONS: The pulley method is an easy, non-invasive, and highly reproducible technique for inducing retinal ischemia in rats. This procedure may provide a useful animal model of ischemic retinopathy.

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.