Rho-associated protein kinas/ROCK; norepinephrine transporter/NET

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].

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].

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].

[1]. Wang RF, et al. 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]. Kiel JW, et al. Effect of AR-13324 on episcleral venous pressure in Dutch belted rabbits. J Ocul Pharmacol Ther. 2015 Apr;31(3):146-151.
[3]. Effects of Rho-Associated Kinase (Rock) Inhibitors (Alternative to Y-27632) on Primary Human Corneal Endothelial Cells. Cells . 2023 May 3;12(9):1307.

(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds-AR-13324 (Netarsudil) and its active metabolite, AR-13503-and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 μM; * p < 0.05); (ii) untreated vs. AR-13503 (10 μM; *** p < 0.001); (iii) Y-27632 vs. AR-13503 (10 μM; ** p < 0.005); (iv) AR-13324 (1 μM) vs. AR-13503 (10 μM; ** p < 0.005); and (v) AR-13324 (0.1 μM) vs. AR-13503 (10 μM; * p < 0.05). Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans.[3]

C28H27N3O3.2HCL

526.45

525.15856

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

94.3Ų

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.[H]Cl.[H]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.)