y-39983 and Glaucoma

To discover novel therapies that lower IOP by increasing aqueous humor outflow facility, ex vivo ocular perfusion systems provide a valuable tool. However, currently available designs are limited by their throughput. Here we report the development of a compact, scalable perfusion system with improved throughput and its validation using bovine and porcine eyes.. At a fixed IOP of 6 mm Hg, flow rate was measured by flow sensors. We validated the system by measuring the outflow responses to Y-39983 (a Rho kinase inhibitor), endothelin-1 (ET-1), ambrisentan (an antagonist for endothelin receptor A [ETA]), sphigosine-1-phosphate (S1P), JTE-013 (antagonist for S1P receptor 2 [S1P2]), S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide [NO] donor), and 3-Morpholino-sydnonimine (SIN-1, another NO donor).. The instrument design enabled simultaneous measurements of 20 eyes with a footprint of 1 m2. Relative to vehicle control, Y-39983 increased outflow by up to 31% in calf eyes. On the contrary, ET-1 decreased outflow by up to 79%, a response that could be blocked by pretreatment with ambrisentan, indicating a role for ETA receptors. Interestingly, the effect of ET-1 was also inhibited by up to 70% to 80% by pretreatment with NO donors, SNAP and SIN-1. In addition to testing in calf eyes, similar effects of ET-1 and ambrisentan were observed in adult bovine and porcine eyes.. The compact eye perfusion platform provides an opportunity to efficiently identify compounds that influence outflow facility and may lead to the discovery of new glaucoma therapies.

Topics: Animals; Aqueous Humor; Cattle; Computer-Aided Design; Disease Models, Animal; Endothelin-1; Equipment Design; Glaucoma; Intraocular Pressure; Perfusion; Pyrazoles; Pyridines; Swine; Trabecular Meshwork

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%.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Glaucoma; Guinea Pigs; Humans; Inhibitory Concentration 50; Models, Molecular; Molecular Structure; Protein Kinase Inhibitors; Pyrazines; Pyridines; rho-Associated Kinases

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.

Topics: Animals; Disease Models, Animal; Glaucoma; Haplorhini; HeLa Cells; Humans; Intraocular Pressure; Ocular Hypertension; Protein Kinase Inhibitors; rho-Associated Kinases; Thiophenes

The discovery of a pyrrolopyrimidine class of LIM-kinase 2 (LIMK2) inhibitors is reported. These LIMK2 inhibitors show good potency in enzymatic and cellular assays and good selectivity against ROCK. After topical dosing to the eye in a steroid induced mouse model of ocular hypertension, the compounds reduce intraocular pressure to baseline levels. The compounds also increase outflow facility in a pig eye perfusion assay. These results suggest LIMK2 may be an effective target for treating ocular hypertension and associated glaucoma.

Topics: Administration, Topical; Animals; Antihypertensive Agents; Glaucoma; Guanidines; In Vitro Techniques; Intraocular Pressure; Lim Kinases; Mice; Nitriles; Ocular Hypertension; Piperazines; Pyrimidines; Pyrroles; Structure-Activity Relationship; Swine; Urea