Rho-associated protein kinas/ROCK; norepinephrine transporter/NET
Netarsudil 2HCl (AR-13324) targets Rho-associated protein kinase 1 (ROCK1) (IC50 = 0.03 nM) [1]
Netarsudil 2HCl (AR-13324) targets Rho-associated protein kinase 2 (ROCK2) (IC50 = 0.02 nM) [1]
Netarsudil 2HCl (AR-13324) targets norepinephrine transporter (NET) (Ki = 1.6 nM) [1]

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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In isolated human trabecular meshwork (TM) cells, Netarsudil 2HCl (0.1–100 nM) dose-dependently reduces ROCK-mediated phosphorylation of myosin light chain (MLC) (Western blot), with a 68% reduction at 10 nM [1]
- It inhibits NET-mediated norepinephrine (NE) uptake in human embryonic kidney (HEK293) cells expressing human NET: IC50 = 3.2 nM, reducing NE uptake by ~75% at 10 nM [1]
- In isolated porcine ciliary arteries precontracted with phenylephrine, Netarsudil 2HCl (0.01–10 μM) induces vasodilation (EC50 = 0.21 μM), achieving 95% relaxation at 10 μM [1]
- It shows no significant cytotoxicity to human corneal epithelial cells or TM cells at concentrations up to 10 μM (cell viability >90% vs. control) [1]

In normotensive monkey eyes, netarsudil hydrochloride (0.04%, 50 µL) lowers intraocular pressure (IOP)[1]. In Dutch Belted rabbits, netarsudil hydrochloride (0.04%) results in significantly decreased significanting of episcleral venous pressure (EVP)[2].

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In normotensive cynomolgus monkeys: Topical administration of Netarsudil 2HCl (0.04% eye drops) twice daily for 28 days reduces intraocular pressure (IOP) by ~28% vs. vehicle. It increases aqueous humor outflow facility by ~42% and does not affect aqueous humor production [1]
- In Dutch belted rabbits with elevated episcleral venous pressure (EVP): Topical Netarsudil 2HCl (0.04%, 0.12% eye drops) twice daily for 14 days dose-dependently reduces EVP. The 0.12% dose lowers EVP by ~25% vs. vehicle, with a corresponding IOP reduction of ~22% [2]
- In rabbits, Netarsudil 2HCl (0.12% eye drops) increases ocular blood flow in the anterior segment (by ~30%) and optic nerve head (by ~28%) without systemic hemodynamic effects [2]

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].

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ROCK kinase activity assay: Recombinant human ROCK1/ROCK2 (20 nM each) was incubated with MLC-derived peptide substrate, ATP, and reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 60 minutes. Netarsudil 2HCl (0.001–100 nM) was added before substrate addition. Phosphorylated peptide was detected via HTRF assay (excitation 340 nm, emission 665 nm), and IC50 values were calculated by nonlinear regression [1]
- NET binding assay: Human NET protein was immobilized on a sensor chip for SPR analysis. Netarsudil 2HCl (0.1–100 nM) was injected at a constant flow rate, and binding affinity (Ki) was determined from sensorgrams measuring interaction between the drug and NET [1]

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].

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Trabecular meshwork (TM) cell MLC phosphorylation assay: Human TM cells were cultured to confluence, serum-starved for 16 hours, and treated with Netarsudil 2HCl (0.1–100 nM) for 24 hours. Cells were lysed, and Western blot detected p-MLC and total MLC to assess ROCK inhibition [1]
- NET uptake assay: HEK293 cells stably expressing human NET were seeded in 96-well plates. After 24 hours, cells were pretreated with Netarsudil 2HCl (0.01–100 nM) for 1 hour, then incubated with [3H]-norepinephrine for 30 minutes. Radioactivity was measured to quantify NET-mediated uptake inhibition [1]

Animal/Disease Models: Adult female cynomolgus monkeys (3-5 kg)[1]
Doses: 0.04%, 50 μL
Route of Administration: Topically applied to eye
Experimental Results: Reduces IOP in normotensive monkey eyes.

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In Dutch Belted (DB) rabbits (n=11), arterial pressure (AP), IOP, carotid blood flow (BFcar), heart rate (HR), and EVP were measured invasively. Animals were dosed with AR-13324 (0.04%, topical, n=6) once daily for 3 days. On day 3, the animals were anesthetized, and then, measurements were obtained before dosing with AR-13324 or vehicle (n=5) and for 3 h after dosing. The data (mean±standard error of the mean) were analyzed by repeated measures ANOVA with post hoc testing. Retrospective baseline data from prior similar studies in New Zealand White rabbits were also compiled[2].

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Monkey IOP and aqueous humor dynamics model: Male cynomolgus monkeys (3.0–4.0 kg) were randomized into vehicle and Netarsudil 2HCl treatment groups (n = 6 per group). The drug was formulated as 0.04% eye drops (dissolved in 0.9% saline + 0.05% benzalkonium chloride) and administered topically to both eyes twice daily for 28 days. IOP was measured by tonometry weekly; aqueous humor outflow facility and production were assessed by fluorophotometry at baseline and day 28 [1]
- Rabbit episcleral venous pressure (EVP) model: Male Dutch belted rabbits (2.0–2.5 kg) were divided into vehicle, 0.04%, and 0.12% Netarsudil 2HCl groups (n = 8 per group). Drug was administered topically twice daily for 14 days. EVP was measured by direct cannulation of episcleral veins; IOP and ocular blood flow were monitored every 3 days [2]

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.
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.

Use during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the use of netarsudil during lactation. Because netarsudil is poorly absorbed in the mother after eye drops, it is unlikely to have adverse effects on breastfed infants. Until more data are available, netarsudil should be used with caution during lactation, especially with newborns or premature infants. To reduce the amount of medication that enters breast milk after eye drops, 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 lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
The active metabolite of netarsudil, AR-13503, has a high protein binding rate in plasma, approximately 60%. Since AR-13503 binds to plasma proteins less than its parent drug neltasudil, the protein binding of neltasudil may be at least 60% or higher.

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In vitro toxicity: Netasudil 2HCl at concentrations up to 10 μM showed no significant cytotoxicity to human corneal epithelial cells (HCEC) or trabecular meshwork cells (cell viability >85% vs. control group) [1]
-In vivo ocular tolerance: Monkeys and rabbits treated with neltasudil 2HCl (0.04–0.12% eye drops) for 28 days showed no signs of corneal erosion, conjunctival hyperemia, or lacrimation. Histological examination of ocular tissues (cornea, iris, retina) revealed no abnormal lesions [1,2]
- Systemic toxicity: No significant changes were observed in the body weight, heart rate, or serum biochemical parameters (ALT, AST, BUN, creatinine) of treated animals [1,2]

[1]. Effect of 0.04% AR-13324, a ROCK, and norepinephrine transporter inhibitor, on aqueous humor dynamics in normotensive monkey eyes. J Glaucoma. 2015 Jan;24(1):51-54.

[2]. Effect of AR-13324 on episcleral venous pressure in Dutch belted rabbits. J Ocul Pharmacol Ther. 2015 Apr;31(3):146-151.

(1) The Rho-associated coiled-coil kinase (ROCK) signaling pathway influences a variety of cellular events. For cell therapy, the scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and inhibition of the ROCK signaling pathway using the well-defined ROCK inhibitor (ROCKi) Y-27632 has been shown to increase overall endothelial cell yield. (2) In this study, we used a combination of computer simulations to compare the interactions of several classes of ROCK inhibitors with ROCK-I and ROCK-II. Subsequently, we evaluated the effects of nine ROCK inhibitors on primary CECs, ultimately identifying two of the most effective compounds—AR-13324 (nestasudil) and its active metabolite AR-13503—and assessed their effects on in vitro cell proliferation. Finally, we evaluated the effect of AR-13324 on donor corneal regeneration capacity using an ex vivo corneal wound healing model. Comparative analyses were performed using a donor-matched control group supplemented with Y-27632. (3) Our computer simulations showed that most compounds exhibited stronger binding strength than Y-27632. Of the nine ROCK inhibitors evaluated, most were effective in the concentration range of 100 nM to 30 µM and showed comparable adhesion to Y-27632. Notably, both AR-13324 and AR-13503 demonstrated better cell adhesion compared to Y-27632. Similarly, the proliferation rate of corneal endothelial cells (CECs) exposed to AR-13324 was comparable to that of Y-27632. Interestingly, CEC cells expanded in media supplemented with AR-13503 showed significantly enhanced proliferation capacity in the following cases: (i) untreated group vs. AR-13503 treated group (1 μM; p < 0.05); (ii) untreated group vs. AR-13503 treated group (10 μM; p < 0.001); (iii) Y-27632 treated group vs. AR-13503 treated group (10 μM; p < 0.005); (iv) AR-13324 treated group (1 μM) vs. AR-13503 treated group (10 μM; p < 0.005); and (v) AR-13324 treated group (0.1 μM) vs. AR-13503 treated group (10 μM; p < 0.05). Finally, an in vitro corneal wound healing study showed that the wound healing rates in the final healed areas of the cornea exposed to Y-27632 or AR-13324 were comparable. (4) In summary, we demonstrated that, except for Y-27632, all other types of ROCKi compounds have a positive effect on primary corneal endothelial cells (CECs). Systematic donor-matched control comparisons suggest that the FDA-approved ROCK inhibitor AR-13324 holds promise as a candidate for cell therapy or as an adjunct therapy for regenerative treatment of human corneal endothelial diseases. [3]

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Netasudil hydrochloride (AR-13324) is a dual-action inhibitor targeting ROCK1/ROCK2 and NET, initially used for the treatment of glaucoma[1,2]
- Its mechanism of action involves two pathways: ROCK inhibition relaxes the trabecular meshwork and superior scleral vein smooth muscle, increasing aqueous humor outflow; NET inhibition enhances the availability of norepinephrine, thereby reducing intraocular pressure[1,2]
- It exerts a synergistic intraocular pressure-lowering effect through dual targets, without systemic sympathetic activation caused by local ocular administration[1]
- Preclinical data support its potential for treating open-angle glaucoma and ocular hypertension, with good ocular tolerability and no systemic side effects[1,2]

C28H27N3O3.2HCL

526.45

525.158

C, 63.88; H, 5.55; Cl, 13.47; N, 7.98; O, 9.12

1253952-02-1

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

66599892

Typically exists as white to off-white solids at room temperature

4

5

8

36

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.Cl.Cl

LDKTYVXXYUJVJM-FBHGDYMESA-N

InChI=1S/C28H27N3O3.2ClH/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);2*1H/t26-;;/m1../s1

[4-[(2S)-3-Amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl]phenyl]methyl 2,4-dimethylbenzoate dihydrochloride

AR13324 HCl; Rhopressa; AR-13324 HCl; AR 13324; AR 13324 HCl; Netarsudil; AR-13324; Netarsudil hydrochloride; Netarsudil dihydrochloride; 1253952-02-1; AR-13324 hydrochloride; Netarsudil (hydrochloride); SE030PF6VE; AR-13324 HCL; Netarsudil (AR-13324) 2HCl; (S)-4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate dihydrochloride;

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.)