Rho-associated protein kinas/ROCK; norepinephrine transporter/NET
Rho kinase; norepinephrine transporter [1]
Rho kinase; norepinephrine transporter [2]

In vitro activity: Previous study showed that at the cellular level, netarsudil had been shown to be able to induce loss of actin stress fibers, cell shape changes, loss of focal adhesions, as well as changes in extracellular matrix composition of TM cells

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Kinase Assay: Netarsudil (formerly known as AR-13324) is ROCK inhibitor with Ki of 0.2-10.3 nM. It also inhibits norepinephrine transport activity which can reduce the production of aqueous humor.

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Cell Assay: Previous study showed that at the cellular level, netarsudil had been shown to be able to induce loss of actin stress fibers, cell shape changes, loss of focal adhesions, as well as changes in extracellular matrix composition of TM cells.

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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 [1]
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 [2]

Animal efficacy study found that the topical treatment of netarsudil was able to affect both proximal (trabecular meshwork and Schlemms Canal) and distal portions (intrascleral vessels) of the mouse conventional outflow tract.

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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 intraocular pressure (IOP) compared to placebo (CF324-01) treatment (P<0.05 or P<0.01 for different strains) [1]
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) [1]
Topical netarsudil 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 [1]
In living mice with elevated IOP, topical netarsudil 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) [1]
Topical netarsudil 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) [1]

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)[3].

The proliferation rates of primary CECs were assessed using the EdU incorporation Click-iT cell proliferation assay as per the manufacturer’s instructions. Two ROCK inhibitors, AR-13324 and AR-13503, were assessed for their capacity to enhance proliferation of CECs, with two concentrations (100 nM or 1 M for AR-13324 and 1 M or 10 M for AR-13503). Donor-matched CECs with no ROCKi added served as negative control, whereas CECs with Y-27632 added served as positive control. Briefly, cultured CECs, passaged using TS, were seeded onto FNC-coated glass slides at a density of 5  103 cells per cm2 and maintained in M5-Endo for 24 h (Day 1). On the second day (Day 2), the medium was switched to each respective condition, and cells were cultured for another 24 h. On the third day, cells were incubated in M4-F99 containing 10mMof EdU for 24 h. Subsequently, samples were rinsed once with PBS before they were fixed in freshly prepared 4% PFA for 15 min at room temperature. Next, Samples were rinsed twice with 3% BSA in PBS and were incubated in 0.5% Triton X-100 in PBS for 20 min at room temperature for blocking and permeabilization. Incorporated EdU was detected by fluorescent-azide-coupling Click-iT reaction where samples were incubated for 30 min in the dark with a reaction mixture containing Click-iT EdU reaction buffer, CuSO4, azide-conjugated Alexa Fluor 488 dye, and reaction buffer additive. Following that, samples were rinsed with 3% BSA before incubating in 5 g/mL Hoechst 33,342 for 10 min at room temperature in the dark. Finally, samples were washed twice in PBS and mounted in Vectashield containing 4,6-diamidino-2-phenylindole (DAPI). Labelled proliferative cells were examined under a Zeiss Axioplan 2 fluorescence microscope. At least 250 nuclei were analyzed for each experimental condition[3].

Topical application
\nMice with elevated intraocular pressure (IOP) \nVisual impairment due to glaucoma currently impacts 70 million people worldwide. While disease progression can be slowed or stopped with effective lowering of intraocular pressure, current medical treatments are often inadequate. Fortunately, three new classes of therapeutics that target the diseased conventional outflow tissue responsible for ocular hypertension are in the final stages of human testing. The rho kinase inhibitors have proven particularly efficacious and additive to current therapies. Unfortunately, non-contact technology that monitors the health of outflow tissue and its response to conventional outflow therapy is not available clinically. Using optical coherence tomographic (OCT) imaging and novel segmentation software, we present the first demonstration of drug effects on conventional outflow tissues in living eyes. Topical netarsudil (formerly AR-13324), a rho kinase/ norepinephrine transporter inhibitor, affected both proximal (trabecular meshwork and Schlemm's Canal) and distal portions (intrascleral vessels) of the mouse conventional outflow tract. Hence, increased perfusion of outflow tissues was reliably resolved by OCT as widening of the trabecular meshwork and significant increases in cross-sectional area of Schlemm's canal following netarsudil treatment. These changes occurred in conjunction with increased outflow facility, increased speckle variance intensity of outflow vessels, increased tracer deposition in conventional outflow tissues and decreased intraocular pressure. This is the first report using live imaging to show real-time drug effects on conventional outflow tissues and specifically the mechanism of action of netarsudil in mouse eyes. Advancements here pave the way for development of a clinic-friendly OCT platform for monitoring glaucoma therapy.[1]

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\nPaired human eyes (n = 5) were perfused with either 0.3 μM netarsudil-M1 or vehicle solution at constant pressure (15 mm Hg). After 3 hours, fluorescent microspheres were added to perfusion media to trace the outflow patterns before perfusion-fixation. The percentage effective filtration length (PEFL) was calculated from the measured lengths of tracer distribution in the trabecular meshwork (TM), episcleral veins (ESVs), and along the inner wall (IW) of Schlemm's canal after global and confocal imaging. Morphologic changes along the trabecular outflow pathway were investigated by confocal, light, and electron microscopy.\nResults: Perfusion with netarsudil-M1 significantly increased C when compared to baseline (51%, P < 0.01) and to paired controls (102%, P < 0.01), as well as significantly increased PEFL in both IW (P < 0.05) and ESVs (P < 0.01). In treated eyes, PEFL was significantly higher in ESVs than in the IW (P < 0.01) and was associated with increased cross-sectional area of ESVs (P < 0.01). Percentage effective filtration length in ESVs positively correlated with the percentage change in C (R2 = 0.58, P = 0.01). A significant increase in juxtacanalicular connective tissue (JCT) thickness (P < 0.05) was found in treated eyes compared to controls.[2]

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\nFor IOP lowering assessment in mice: 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 of placebo (CF324-01) eye drops. IOP was measured in both eyes before administration, and ΔIOP was compared between groups using Mann Whitney U-test [1]
\nFor IOP recovery assessment in living mice: 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, and IOP was monitored over time using pressure transducers. The rate constant α was calculated and compared between groups using student t-test (n=8) [1]
\nFor ex vivo outflow facility measurement in mice: 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.随后, 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 [1]
\nFor tracer deposition assessment in mice: 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 [1]
\nFor OCT imaging of conventional outflow tissues in mice: Living C57 mice were treated with topical netarsudil 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 [1]
\nFor OCT imaging of SC in mice with elevated IOP: C57 and CD1 mice (n=11) were treated with topical netarsudil 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 [1]
\nFor ex vivo perfusion of human eyes: 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 [2]

Absorption, Distribution and Excretion
In 18 healthy subjects, systemic exposure to nertasudil and its active metabolite AR-13503 was assessed after 8 consecutive days of topical administration of 0.02% nertasudil eye drops (one drop in each eye in the morning). 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 was the active metabolite detected in the plasma of one subject at a concentration of 0.11 ng/mL. Clinical studies using human corneal tissue, human plasma, human liver microsomes, and their S9 fraction demonstrated that nertasudil is metabolized via esterase activity. No further metabolism of the nertasudil esterase metabolite AR-13503 was detected. In fact, no esterase metabolism was detected in human plasma during a 3-hour incubation period.
Due to the high protein binding rate of neltasudil and its active metabolites, its volume of distribution is expected to be small.
The clearance rate of neltasudil is affected by its low plasma concentration after topical administration and absorption, as well as its high protein binding rate in human plasma.
Metabolism/Metabolites

Following topical ocular administration, neltasudil is metabolized in the eye by esterases to the active metabolite neltasudil-M1 (or AR-13503).
Biological Half-Life

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

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation There is currently no information regarding the use of neltasudil during lactation. Because neltasudil is poorly absorbed in the mother after instillation, it is unlikely to have adverse effects on breastfed infants. Until more data are available, neltasudil should be used with caution during lactation, especially with newborns or premature infants. To reduce the amount of medication that enters breast milk after using eye drops, press the tear duct at the corner of the eye for at least 1 minute, then wipe away any excess medication with absorbent tissue. ◉ Effects on Breastfed Infants As of the revision date, no relevant published information was found. ◉ Effects on Lactation and Breast Milk As of the revision date, no relevant published information was found. Protein Binding The active metabolite of neltasudil, AR-13503, has a high protein binding rate in plasma, approximately 60%. Because AR-13503 binds to plasma proteins less than its parent drug neltasudil, the protein binding rate of neltasudil may be at least 60% or higher.

[1]. Eur J Pharmacol.2016 Sep 15;787:20-31.
[2]. Invest Ophthalmol Vis Sci.2016 Nov1;57(14):6197-6209.
[3]. Cells . 2023 May 3;12(9):1307.

Pharmacodynamics
Aqueous humor flows out of the eye via two pathways: 1) the traditional trabecular meshwork pathway and 2) the non-traditional uveal-scleral pathway. Although studies have shown that the traditional trabecular meshwork pathway is the main pathway for aqueous humor outflow due to various pathological reasons, most drugs currently used to treat glaucoma target the uveal-scleral pathway, while leaving the diseased trabecular meshwork pathway untreated, allowing it to progressively deteriorate and become dysfunctional. Netarsudil is a novel glaucoma drug that is both a Rho kinase inhibitor and a norepinephrine transporter (NAT) inhibitor, specifically targeting and inhibiting Rho kinase and NAT in the traditional trabecular meshwork pathway. Many similar drugs focus on the remodeling of cells and muscle tissue. Netarsudil (formerly AR-13324) is a dual inhibitor of Rho kinase and norepinephrine transporter and is currently being developed for the treatment of glaucoma and ocular hypertension [1][2].
In live mouse eyes, netarsudil affects the proximal (trabecular meshwork and Schlemm's canal) and distal (intrascleral vessels) of conventional aqueous humor outflow tracts, increasing perfusion of aqueous humor outflow tissues by dilating the trabecular meshwork and increasing the cross-sectional area of intrascleral vessels, thereby increasing aqueous humor outflow rate and enhancing speckle variation intensity of aqueous humor outflow vessels. Intraocular pressure reduction [1]
In human eyes, the mechanism of action of netarsudil involves acute dilation of the JCT and dilation of the ESV, resulting in redistribution of aqueous humor outflow through larger IW and ESV regions, thereby increasing aqueous humor outflow rate [2]
This is the first report demonstrating the real-time pharmacological effect of netarsudil on conventional aqueous humor outflow tissues in a live eye using real-time imaging (OCT) technology, paving the way for the development of a clinically friendly OCT platform for monitoring glaucoma treatment [1]

C28H27N3O3

453.54

453.205

C, 74.15; H, 6.00; N, 9.27; O, 10.58

1254032-66-0

1422144-42-0 (mesylate);1254032-66-0;1253952-02-1 (HCl);

66599893

White to off-white solid powder

1.3±0.1 g/cm3

711.9±60.0 °C at 760 mmHg

384.3±32.9 °C

0.0±2.3 mmHg at 25°C

1.667

3.53

2

5

8

34

678

1

O(C(C1C=CC(C)=CC=1C)=O)CC1C=CC(=CC=1)[C@H](C(NC1C=CC2C=NC=CC=2C=1)=O)CN

OURRXQUGYQRVML-AREMUKBSSA-N

InChI=1S/C28H27N3O3/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/h3-14,16,26H,15,17,29H2,1-2H3,(H,31,32)/t26-/m1/s1

Benzoic acid, 2,4-dimethyl-, (4-((1S)-1-(aminomethyl)-2-(6-isoquinolinylamino)-2-oxoethyl)phenyl)methyl ester

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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