The Soluble Guanylate Cyclase Stimulator IWP-953 Increases Conventional Outflow Facility in Mouse Eyes

Abstract

Purpose: The nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway regulates aqueous humor outflow and therefore, intraocular pressure. We investigated the pharmacologic effects of the soluble guanylate cyclase (sGC) stimulator IWP-953 on primary human trabecular meshwork (HTM) cells and conventional outflow facility in mouse eyes.

Methods: Cyclic GMP levels were determined in vitro in HEK-293 cells and four HTM cell strains (HTM120/HTM123: predominantly myofibroblast-like phenotype, HTM130/HTM141: predominantly endothelial-like phenotype), and in HTM cell culture supernatants. Conventional outflow facility was measured following intracameral injection of IWP-953 or DETA-NO using a computerized pressure-controlled perfusion system in enucleated mouse eyes ex vivo.

Results: IWP-953 markedly stimulated cGMP production in HEK-293 cells in the presence and absence of DETA-NO (half maximal effective concentrations: 17 nM, 9.5 μM). Similarly, IWP-953 stimulated cGMP production in myofibroblast-like HTM120 and HTM123 cells, an effect that was greatly amplified by the presence of DETA-NO. In contrast, IWP-953 stimulation of cGMP production in endothelial-like HTM130 and HTM141 cells was observed, but was markedly less prominent than in HTM120 and HTM123 cells. Notably, cGMP was found in all HTM culture supernatants, following IWP-953/DETA-NO stimulation. In paired enucleated mouse eyes, IWP-953 at 10, 30, 60, and 100 μM concentration-dependently increased outflow facility. This effect (89.5%) was maximal at 100 μM (P = 0.002) and in magnitude comparable to DETA-NO at 100 μM (97.5% increase, P = 0.030).

Conclusions: These data indicate that IWP-953, via modulation of the sGC-cGMP pathway, increases aqueous outflow facility in mouse eyes, suggesting therapeutic potential for sGC stimulators as novel ocular hypotensive drugs.

Figure 1

IWP-953 stimulates cGMP production in HEK-293 cells. Human embryonic kidney-293 cells were seeded at a density of 6 × 10⁴ cells/well in 96-well plates and incubated with a concentration–response curve of IWP-953 ranging from 10⁻¹¹ M to 3 × 10⁻⁵ M in the presence or absence of fixed concentration of DETA-NO (10 μM), and in the presence or absence of a fixed concentration of ODQ (10 μM), and IBMX (0.5 mM) for 20 minutes at 37°C. Following cell lysis (50 μL ice cold 10% acetic acid), 150 ng/mL internal cGMP standard was added to each well, and samples were further incubated on ice for 30 minutes. Subsequent to centrifugation, cell supernatants were analyzed for cGMP content by LC/MS/MS. Data are expressed as the mean ± SD (N = 2) and are representative of two independent assys.

Figure 2

Cyclic GMP production in primary cultures of HTM cells stimulated by IWP-953 in the presence of DETA-NO and by DETA-NO alone. Myofibroblast-like HTM120 (A) and HTM123 (B) cultures and endothelial-like HTM130 (C) and HTM141 (D) cell cultures were seeded at a density of 1 × 10⁵ cells/well in 24-well plates and incubated with DMEM containing IBMX (0.5 mM) for 15 minutes at 37°C. Human trabecular meshwork cell cultures were then incubated with increasing concentrations of IWP-953 ranging from 0.1 μM to 10 μM in the presence of a fixed concentration of DETA-NO (10 μM) or vehicle (0.5% DMSO), or DETA-NO (10 μM, 100 μM) for 15 minutes at 37°C. Following cell lysis, cleared HTM cell supernatants were acetylated and the amount of cGMP was measured using a cGMP enzyme immunoassay. Data are expressed as the mean ± SD (N = 3; except for HTM120, HTM130 cultures at IWP-953 [0.1 μM] with DETA-NO [10 μM]: N = 1). *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 3

Cyclic GMP secretion by primary cultures of HTM cells, following treatment with IWP-953 in the presence of DETA-NO and DETA-NO alone. Myofibroblast-like HTM120 (A) and HTM123 (B) cell cultures and endothelial-like HTM130 (C) and HTM141 (D) cell cultures were treated with increasing concentrations of IWP-953 (0.1–10 μM) in the presence of a fixed concentration of DETA-NO (10 μM) or vehicle (0.5% DMSO), or DETA-NO (10 μM, 100 μM). HTM cell culture supernatants were acetylated, and cGMP content was measured using a cGMP enzyme immunoassay. Data are expressed as the mean ± SD (N = 3; except for supernatants from HTM120, HTM130 cell cultures at IWP-953 [0.1 μM] with DETA-NO [10 μM]: N = 1). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Representative perfusion traces showing flow data over time from paired eyes at sequential pressure steps. The gray trace shows the raw flow data obtained from the vehicle treated eye and the black trace shows flow data from the contralateral eye perfused with 100 μM IWP-953. Red and blue highlighted regions of traces represent data used to calculate average flow rate at each corresponding pressure step for control and IWP-953-treated eyes, respectively. Calculated outflow facilities for this pair are 0.027 and 0.041 μL/min/mm Hg for the control and IWP-953-treated eyes, respectively. Trace spikes indicate rapid increases in flow rate (that subside over time) from syringe pump that is attempting to maintain user-defined perfusion pressure.

Figure 5

Pressure versus flow plots showing impact of escalating concentrations of IWP-953 on conventional outflow function of enucleated mouse eyes. Ex vivo perfusions were conducted using (A) 10 μM, (B) 30 μM, (C) 60 μM, and (D) 100 μM IWP-953, or vehicle as control for comparisons. Eyes were subjected to four pressure steps (4, 8, 12, and 16 mm Hg), and flow was continuously monitored. Linear regression was performed on mean flow data at each pressure step (±SD) to estimate outflow facility. As a positive control (E), 100 μM DETA-NO was perfused into a separate cohort of eyes, with contralateral eyes perfused with vehicle. A summary of outflow facility calculations at each concentration is shown in panel F as a box-and-whisker plot (*P < 0.05).