Rho-associated protein kinas/ROCK; norepinephrine transporter/NET
Rho kinase (Ki = 0.2 nM); norepinephrine transporter [1]
Rho kinase; norepinephrine transporter [2]
Rho kinase; norepinephrine transporter [3]

Netarsudil has been demonstrated in prior research to be able to cause TM cells' extracellular matrix composition to alter, focal adhesions to disappear, actin stress fiber loss, and cell shape alterations.

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Ex vivo perfusion of enucleated mouse eyes with netarsudil mesylate (100 nM) increased outflow facility compared to vehicle (0.001% DMSO) treatment. For C57BL/6 mice (n=8), the treatment led to a significant average increase in outflow facility (P=0.006), and for CD1 mice (n=6), a significant increase was also observed (P=0.025). The flow-pressure relationship was analyzed through 9 sequential pressure steps after 45-60 min of perfusion with the drug or vehicle [2]
Perfusion of enucleated human eyes with 0.3 μM netarsudil-M1 (active metabolite) at constant pressure (15 mmHg) for 3 hours significantly increased outflow facility (C) by 51% compared to baseline (P<0.01) and by 102% compared to paired vehicle controls (P<0.01). It also significantly increased the percentage effective filtration length (PEFL) in the inner wall (IW) of Schlemm's canal (SC) (P<0.05) and episcleral veins (ESVs) (P<0.01). In treated eyes, PEFL in ESVs was significantly higher than in IW (P<0.01) and positively correlated with the percentage change in C (R²=0.58, P=0.01). Additionally, the cross-sectional area of ESVs (P<0.01) and juxtacanalicular connective tissue (JCT) thickness (P<0.05) were significantly increased compared to controls [3]

Netarsudil effectively lowers intraocular pressure (IOP) in the eyes of both humans and non-human primates by primarily targeting cells in the conventional outflow tract. Furthermore, it has been demonstrated that netarsudil lowers episcleral venous pressure in rabbit eyes and increases outflow facility in non-human primate eyes[2].

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In Dutch Belted rabbits, netarsudil mesylate (ester 60, prodrug of parent ROCK inhibitor 29) demonstrated effective and sustained intraocular pressure (IOP) reduction for 24 hours after dosing [1]
Topical administration of 10 μl of 0.04% netarsudil mesylate to right eyes of 10-week-old C57 mice and 6-14 week-old CD1 mice (5 mice/group) significantly lowered IOP compared to placebo (CF324-01) treatment (P<0.05 or P<0.01 for different strains) [2]
Intracameral preloading of 100 nM netarsudil mesylate into contralateral eyes of living mice (n=8) enhanced IOP recovery after artificial elevation to 40 mmHg. The rate constant α (characterizing pressure decay) was significantly increased compared to vehicle (0.001% DMSO) treatment (P<0.01) [2]
Topical netarsudil mesylate treatment in living C57 mice led to widening of the trabecular meshwork (TM) and a significant increase in the cross-sectional area of SC, as visualized by optical coherence tomography (OCT) imaging 45 min post-treatment. It also increased speckle variance intensity of outflow vessels, enhanced tracer deposition in conventional outflow tissues, and decreased IOP [2]
In living mice with elevated IOP, topical netarsudil mesylate treatment (10 μl of 0.04%) increased the cross-sectional area of SC lumen when IOP was controlled at 10, 15, and 30 mmHg (P<0.05 or P<0.01). OCT imaging showed significant changes in SC area relative to baseline (10 mmHg pre-treatment) in both C57 and CD1 mice (n=11) [2]
Topical netarsudil mesylate treatment in C57 and CD1 mice increased the cross-sectional area and speckle variance intensity of scleral vessels involved in aqueous humor outflow, as analyzed by OCT speckle variance images 30-60 min post-treatment (P<0.05) [2]

Netarsudil (formerly known as AR-13324) is an inhibitor of ROCK having a Ki of 0.2-10.3 nM. Moreover, it suppresses norepinephrine transport activity, which may lessen aqueous humor production.

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A total of 23 ROCK structures were found in the PDB. The maximum and minimum resolutions were 3.4 Å and 2.93 Å, respectively. Seven ROCK-I and two ROCK-II non-redundant structures were selected for the binding assay. Out of 46 compounds tested (20 isoquinolines, 15 aminofurazan, 6 benzodiazepine, 4 indazoles, and 1 amide), 34 presented a significantly higher docking score for ROCK-1, when compared to Y-27632 (p < 0.0001). All ROCKi classes presented a stronger mean docking score than Y-27632 (p < 0.0001). The frequency of compounds presenting highest docking score was higher in the isoquinoline, aminofurazan, and benzodiazepine classes for ROCK-I; and in isoquinolines and amides for ROCK-II (Supplementary Figure S2A). The top ten compounds that presented the highest mean docking scores for ROCK-I and II are shown in Supplementary Figure S2B. The isoquinoline class represented 70% of the drugs within the top ten highest docking scores, with three compounds presenting a docking score stronger than 􀀀12. There were no significant differences among ROCK inhibitors other than Y-27632. Interestingly, in silico molecular docking simulation showed that the majority of the molecules evaluated, specifically fromthe isoquinoline, benzodiazepine, and amide classes, had higher binding strength for ROCK-1 and ROCK-2 than Y-27632 (Supplementary Figure S2B). In silico molecular docking simulation was performed, coupling isoforms found for AR-13324 and Y-27632 inhibitors in the PDB to high-resolution ROCK proteins. All of the AR-13324 molecules tested had a higher docking score for ROCK-1 and -2 than Y-27632. In addition, PDB molecules from the isoquinoline, benzodiazepine, and amide classes also showed superior mean docking scores than Y-27632 isoforms (Supplementary Figure S2B)[4].

Prior research revealed that netarsudil could cause TM cells' extracellular matrix composition to alter, as well as the loss of focal adhesions, actin stress fibers, and cell shape.

Topical application
\nMice with elevated intraocular pressure (IOP)

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\nRabbit IOP reduction duration assessment: Dutch Belted rabbits were administered netarsudil mesylate (ester 60) via appropriate topical dosing. IOP was measured at various time points up to 24 hours post-dosing to evaluate the duration of IOP-lowering effect [1]
\nMouse IOP lowering assessment: 10-week-old C57 mice and 6-14 week-old CD1 mice were divided into age and gender-matched groups (5 mice/group). Each strain had two groups: one group received 10 μl of 0.04% netarsudil mesylate topically to the right eye, and the other received 10 μl placebo (CF324-01) eye drops. IOP was measured in both eyes before administration, and ΔIOP was compared between groups using Mann Whitney U-test [2]
\nMouse IOP recovery assessment: Vehicle (0.001% DMSO) or 100 nM netarsudil mesylate was preloaded into perfusion needles and inserted intracamerally into contralateral eyes of living mice. Both eyes were exposed to 15 mmHg IOP for 30 min to allow drug/vehicle entry, then IOP was artificially raised to 40 mmHg for 5 min. The fluid reservoir was closed, but remained open to pressure transducers, which monitored IOP in both eyes over time. The rate constant α was calculated and compared between groups using student t-test (n=8) [2]
\nMouse ex vivo outflow facility measurement: Paired enucleated eyes of C57BL/6 (n=8) and CD1 (n=6) mice were perfused with netarsudil mesylate or vehicle (0.001% DMSO) via microneedles for 45-60 min. Subsequently, eyes were exposed to 9 sequential pressure steps, and flow rate (Q) vs pressure (P) was measured using an iPerfusion system to calculate outflow facility. Percentage change in facility was analyzed using paired weighted t-test [2]
\nMouse tracer deposition assessment: Fluorescent microbeads were loaded into microneedles with or without netarsudil mesylate. Anterior chambers of paired eyes from C57 and CD1 mice (n=5/group) were cannulated and perfused at a constant flow rate of 0.167 μl/min for 1 hour. Mice were maintained for another hour before euthanasia, and anterior segments were flat-mounted and visualized by epifluorescence microscopy. Fluorescence intensity, width, and area in conventional outflow regions were quantified and compared using student t-test [2]
\nMouse conventional outflow tissue OCT imaging: Living C57 mice were treated with topical netarsudil mesylate or placebo. Averaged OCT images from 200 B-scans of iridocorneal angles were acquired before and 45 min post-treatment. SC was segmented using Schlemm II software, and speckle variance images were analyzed with Schlemm III software to quantify SC area, speckle variance intensity of scleral vessels, and TM width (n=5/group). Student t-test was used for statistical analysis [2]
\nMouse elevated IOP SC OCT imaging: C57 and CD1 mice (n=11) were treated with topical netarsudil mesylate or placebo. A glass needle was inserted into the anterior chamber to control IOP at 10, 15, and 30 mmHg sequentially before and 30-60 min after treatment. OCT images of iridocorneal angles were acquired at the same location, and SC cross-sectional area was quantified using Schlemm II software, expressed relative to baseline (10 mmHg pre-treatment). Mann Whitney U-test was used for statistical comparison [2]
\nHuman eye ex vivo perfusion: Paired human eyes (n=5) were perfused with 0.3 μM netarsudil-M1 or vehicle solution at constant pressure (15 mmHg) for 3 hours. Fluorescent microspheres were added to the perfusion media to trace outflow patterns before perfusion-fixation. Global and confocal imaging were performed to calculate PEFL in TM, ESVs, and IW of SC. Morphologic changes were investigated by confocal, light, and electron microscopy. Outflow facility was measured over time, and parameters including ESV cross-sectional area, JCT thickness were quantified and compared between groups [3]

Absorption
In 18 healthy subjects, following 8 consecutive days of topical administration of 0.02% nertasudil eye drops (one drop in each eye in the morning), systemic exposure to nertasudil and its active metabolite AR-13503 was assessed. On days 1 and 8, nertasudil was undetectable in plasma (lower limit of quantification [LLOQ] 0.100 ng/mL). Only 8 hours after administration on day 8, the active metabolite was detected in the plasma of one subject at a concentration of 0.11 ng/mL.
Elimination Pathway
Clinical studies using human corneal tissue, human plasma, human liver microsomes, and their S9 fraction demonstrated that nertasudil is metabolized via esterase activity. Subsequent metabolism of the nertasudil esterase metabolite AR-13503 was not detected. In fact, esterase metabolism was not detected in human plasma during a 3-hour incubation period.
Volume of Distribution
Due to the high protein binding of neltasudil and its active metabolite, its volume of distribution is expected to be small.
Clearance
The clearance of neltasudil is affected by its low plasma concentration after topical administration and absorption, as well as its high protein binding in human plasma.

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Metabolites/Metabolites
After topical ocular administration, neltasudil is metabolized in the eye by esterases to the active metabolite neltasudil-M1 (or AR-13503).

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Biological Half-Life
The half-life of neltasudil in vitro with human corneal tissue is 175 minutes.

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Nettasudil mesylate (ester 60) is a prodrug that enhances the bioavailability of its parent Rho kinase inhibitor (compound 29)[1].

Use of Netasudil During Pregnancy and Lactation ◉ Overview of Use During Lactation
There is currently no information regarding the use of netasudil during lactation. Because netasudil is poorly absorbed in the mother after instillation, it is unlikely to have adverse effects on breastfed infants. Until more data are available, netasudil should be used with caution during lactation, especially when breastfeeding newborns or premature infants. To reduce the amount of medication that enters breast milk after instillation, press your finger against the tear duct near the corner of your eye for at least 1 minute, then blot away any excess medication with absorbent paper.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Breastfeeding and Breast Milk
As of the revision date, no relevant published information was found.

[1]. Bioorg Med Chem Lett. 2016 May 15;26(10):2475-2480.

[2]. Eur J Pharmacol. 2016 Sep 15:787:20-31.

[3]. Invest Ophthalmol Vis Sci. 2016 Nov 1;57(14):6197-6209.

[4]. Cells. 2023 May 3;12(9):1307.

See also: Netasudil (with active ingredient); Latanoprost; Netasudil mesylate (ingredient).

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Netasudil mesylate (formerly known as AR-13324) is a dual inhibitor of Rho kinase and norepinephrine transporter for the treatment of open-angle glaucoma and ocular hypertension[1][2][3]
The inhibitory effect of netasudil mesylate on Rho kinase can improve the outflow of aqueous humor through the trabecular meshwork, thereby reducing intraocular pressure, which is a key strategy for anti-glaucoma treatment[1]
In live mouse eyes, netasudil mesylate can affect the proximal (trabecular meshwork and Schlem's canals) and distal (intrascleral vessels) of the conventional aqueous humor outflow tracts, increasing perfusion of outflowing tissues by dilating the trabecular meshwork and increasing the cross-sectional area of Schlem's canals, which is associated with increased aqueous humor outflow rate and enhanced speckle variance intensity. Aqueous outflow vessels dilate, intraocular pressure decreases [2]
In the human eye, the mechanism of action of netarsudil mesylate involves acute dilation of the JCT and ESV, leading to redistribution of aqueous outflow through larger IW and ESV areas, thereby increasing the outflow rate of aqueous fluid [3]
This is the first report demonstrating the real-time pharmacological effect of netarsudil mesylate on routine aqueous outflow tissue in a living eye using real-time imaging (OCT) technology, paving the way for the development of a clinically friendly OCT platform for monitoring glaucoma treatment [2]
Netarsudil mesylate was discovered from α-aryl-β-aminoisoquinoline analogs, which have been found to be potent ROCK inhibitors that inhibit norepinephrine transporters and provide a more sustained intraocular pressure-lowering effect [1]

C30H35N3O9S2

645.74

645.181

C, 55.80; H, 5.46; N, 6.51; O, 22.30; S, 9.93

1422144-42-0

Netarsudil hydrochloride;1253952-02-1;AR-13324 analog mesylate; 1422144-42-0; 1254032-66-0

90410375

White to yellow solid powder

4

11

8

44

770

1

S(C)(=O)(=O)O.S(C)(=O)(=O)O.O=C([C@@H](CN)C1C=CC(COC(C2C=CC(C)=CC=2C)=O)=CC=1)NC1C=CC2C=NC=CC=2C=1

QQDRLKRHJOAQDC-FBHGDYMESA-N

InChI=1S/C28H27N3O3.2CH4O3S/c1-18-3-10-25(19(2)13-18)28(33)34-17-20-4-6-21(7-5-20)26(15-29)27(32)31-24-9-8-23-16-30-12-11-22(23)14-24;2*1-5(2,3)4/h3-14,16,26H,15,17,29H2,1-2H3,(H,31,32);2*1H3,(H,2,3,4)/t26-;;/m1../s1

[4-[(2S)-3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl]phenyl]methyl 2,4-dimethylbenzoate;methanesulfonic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)