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Purity: ≥98%
Netarsudil (formerly known as AR-13324; AR13324; trade name Rhopressa) is ROCK inhibitor approved in 2017 for the treatment of glaucoma and ocular hypertension. It inhibits ROCK with Ki of 0.2-10.3 nM. Netarsudil also inhibits norepinephrine transport activity which can reduce the production of aqueous humor. As of 2018, Netarsudil has been approved by FDA for the treatment of glaucoma and ocular hypertension. Previous study showed that at the cellular level, netarsudil was 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. Netarsudil primarily targets cells in the conventional outflow tract, efficiently decreasing IOP in both human and non-human primate eyes. In addition, netarsudil has been shown to increase outflow facility in non-human primate eyes and to decrease episcleral venous pressure in rabbit eyes.
| Targets |
Rho-associated protein kinas/ROCK; norepinephrine transporter/NET
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| Enzyme Assay |
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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| Cell Assay |
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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| References |
[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. |
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| Additional Infomation |
A Rho kinase inhibitor with norepinephrine transport inhibitory activity that reduces production of aqueous As of December 18, 2017 the FDA approved Aerie Pharmaceutical's Rhopressa (netarsudil ophthalmic solution) 0.02% for the indication of reducing elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Acting as both a rho kinase inhibitor and a norepinephrine transport inhibitor, Netarsudil is a novel glaucoma medication in that it specifically targets the conventional trabecular pathway of aqueous humour outflow to act as an inhibitor to the rho kinase and norepinephrine transporters found there as opposed to affecting protaglandin F2-alpha analog like mechanisms in the unconventional uveoscleral pathway that many other glaucoma medications demonstrate.
Netarsudil is a Rho Kinase Inhibitor. The mechanism of action of netarsudil is as a Rho Kinase Inhibitor. Netarsudil is an amino-isoquinoline amide and inhibitor of Rho kinase (ROCK) and norepinephrine transporter (NET), with potential intraocular pressure (IOP)-lowering activity. Upon ocular administration, netarsudil inhibits ROCK and the Rho pathway, increases aqueous humor (AH) outflow via the trabecular pathway, and lowers IOP. In addition, netarsudil may lower IOP by decreasing episcleral venous pressure and decreasing the production of aqueous humor through inhibition of NET. |
| Molecular Formula |
C28H27N3O3
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| Molecular Weight |
453.54
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| Exact Mass |
453.205241
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| Elemental Analysis |
C, 74.15; H, 6.00; N, 9.27; O, 10.58
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| CAS # |
1254032-66-0
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| Related CAS # |
1422144-42-0 (mesylate);1254032-66-0;1253952-02-1 (HCl);
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| PubChem CID |
66599893
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| Appearance |
Typically exists as solids (or liquids in special cases) at room temperature
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
711.9±60.0 °C at 760 mmHg
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| Flash Point |
384.3±32.9 °C
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| Vapour Pressure |
0.0±2.3 mmHg at 25°C
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| Index of Refraction |
1.667
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| LogP |
4.6
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| tPSA |
94.3Ų
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| SMILES |
O=C(OCC1=CC=C([C@@H](CN)C(NC2=CC3=C(C=NC=C3)C=C2)=O)C=C1)C4=CC=C(C)C=C4C
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| InChi Key |
OURRXQUGYQRVML-AREMUKBSSA-N
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| InChi Code |
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
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| Chemical Name |
Benzoic acid, 2,4-dimethyl-, (4-((1S)-1-(aminomethyl)-2-(6-isoquinolinylamino)-2-oxoethyl)phenyl)methyl ester
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2049 mL | 11.0244 mL | 22.0488 mL | |
| 5 mM | 0.4410 mL | 2.2049 mL | 4.4098 mL | |
| 10 mM | 0.2205 mL | 1.1024 mL | 2.2049 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Netarsudil lowered intraocular pressure (IOP) in both pigmented and nonpigmented mice.
Netarsudil mesylate enhanced IOP recovery in living mouse eyes.Eur J Pharmacol.2016 Sep 15;787:20-31. |
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Netarsudil mesylate increased outflow facility in perfused mouse eyes ex vivo.Eur J Pharmacol.2016 Sep 15;787:20-31. |
 Enhanced tracer deposition in outflow tissues of living mice subjected to netarsudil mesylate treatment.Eur J Pharmacol.2016 Sep 15;787:20-31. |
 Netarsudil-induced changes in conventional outflow tissue morphology of living mice visualized by optical coherence tomography (OCT).Eur J Pharmacol.2016 Sep 15;787:20-31. |
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Netarsudil increased cross-sectional area of Schlemms canal (SC) lumen in living mice with elevated intraocular pressure (IOP) visualized by optical coherence tomography (OCT).Eur J Pharmacol.2016 Sep 15;787:20-31. |
 Netarsudil-induced changes in flow area and intensity in scleral vessels visualized on OCT speckle variance images.Eur J Pharmacol.2016 Sep 15;787:20-31. |
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