PURPOSE: Rho-associated protein kinase (ROCK) inhibitors lower intraocular 
pressure (IOP) by increasing aqueous outflow through the trabecular meshwork 
(TM). The preclinical characterization of netarsudil, a new ROCK/norepinephrine 
transporter (NET) inhibitor currently in clinical development, is presented 
herein.
METHODS: The kinase inhibitory activity of netarsudil was compared to its 
esterase metabolite, netarsudil-M1, and 3 other ROCK inhibitors using a 
commercially available kinase assay kit. Disruption of actin stress fibers was 
measured in primary porcine TM cells and disruption of focal adhesions in 
transformed human TM (HTM) cells. Induction of fibrosis markers after exposure 
to transforming growth factor-β2 (TGF-β2) was conducted in primary HTM cells. 
Ocular hypotensive activity and tolerability of topical formulations were 
evaluated in normotensive Dutch Belted rabbits and Formosan Rock monkeys. In 
vitro corneal metabolism assays were conducted using dog, pig, rabbit, monkey, 
and human corneas. In vivo ocular pharmacokinetics was studied in Dutch Belted 
rabbits.
RESULTS: Netarsudil inhibited kinases ROCK1 and ROCK2 with a Ki of 1 nM each, 
disrupted actin stress fibers and focal adhesions in TM cells with IC50s of 79 
and 16 nM, respectively, and blocked the profibrotic effects of TGF-β2 in HTM 
cells. Netarsudil produced large reductions in IOP in rabbits and monkeys that 
were sustained for at least 24 h after once daily dosing, with transient, mild 
hyperemia observed as the only adverse effect.
CONCLUSION: Netarsudil is a novel ROCK/NET inhibitor with high potency in 
biochemical and cell-based assays, an ability to produce large and durable IOP 
reductions in animal models, and favorable pharmacokinetic and ocular 
tolerability profiles.