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]

体外活性：先前的研究表明，在细胞水平上，netarsudil 已被证明能够诱导肌动蛋白应力纤维的损失、细胞形状的改变、粘着斑的损失以及 TM 细胞的细胞外基质组成的变化。 Netarsudil（以前称为 AR-13324）是 ROCK 抑制剂，Ki 为 0.2-10.3 nM。它还抑制去甲肾上腺素转运活性，从而减少房水的产生。细胞测定：先前的研究表明，在细胞水平上，netarsudil 已被证明能够诱导肌动蛋白应力纤维的损失、细胞形状的改变、粘着斑的损失以及 TM 细胞的细胞外基质组成的变化。

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对去核小鼠眼球进行体外灌注实验，使用甲磺酸奈他舒地尔（100 nM）处理后，与溶媒（0.001% DMSO）组相比，房水流出系数显著增加。C57BL/6小鼠（n=8）中，药物处理组的流出系数平均增幅具有统计学意义（P=0.006）；CD1小鼠（n=6）中，同样观察到显著的流出系数增加（P=0.025）。在药物或溶媒灌注45-60分钟后，通过9个连续的压力梯度测定流速（Q）与压力（P）的关系，进而计算流出系数 [2]
以恒定压力（15 mmHg）对去核人眼球进行体外灌注，使用0.3 μM 奈他舒地尔-M1（活性代谢产物）处理3小时后，与基线相比，房水流出系数（C）显著增加51%（P<0.01），与配对溶媒对照组相比显著增加102%（P<0.01）。同时，施莱姆管（SC）内壁（IW）和巩膜外静脉（ESVs）的有效滤过长度百分比（PEFL）显著增加（分别为P<0.05和P<0.01）。在药物处理组眼中，巩膜外静脉的PEFL显著高于内壁（P<0.01），且与流出系数的百分比变化呈正相关（R²=0.58，P=0.01）。此外，与对照组相比，巩膜外静脉的横截面积（P<0.01）和邻管结缔组织（JCT）厚度（P<0.05）均显著增加 [3]

动物功效研究发现，奈塔舒地尔的局部治疗能够影响小鼠常规流出道的近端部分（小梁网和施累姆斯管）和远端部分（巩膜内血管）。

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在荷兰带兔中，甲磺酸奈他舒地尔（酯类化合物60，母体Rho激酶抑制剂29的前药）给药后，实现了有效且持续24小时的眼内压（IOP）降低 [1]
向10周龄C57小鼠和6-14周龄CD1小鼠（每组5只）的右眼局部给予10 μl 0.04%甲磺酸奈他舒地尔，与安慰剂（CF324-01）处理组相比，显著降低了眼内压（IOP）（不同品系的P值分别为P<0.05或P<0.01） [2]
向活体小鼠（n=8）的对侧眼玻璃体内预加载100 nM甲磺酸奈他舒地尔，在人工将眼内压升高至40 mmHg后，药物处理组的眼内压恢复能力增强。表征压力衰减速率的常数α与溶媒（0.001% DMSO）处理组相比显著增加（P<0.01） [2]
对活体C57小鼠进行局部甲磺酸奈他舒地尔处理后，通过光学相干断层扫描（OCT）成像观察到，处理后45分钟小梁网（TM）增宽，施莱姆管（SC）横截面积显著增加。同时，流出血管的散斑方差强度增加，传统流出组织中的示踪剂沉积增强，眼内压降低 [2]
在眼内压升高的活体小鼠中，局部给予甲磺酸奈他舒地尔（10 μl 0.04%）后，当眼内压被控制在10、15和30 mmHg时，施莱姆管腔的横截面积增加（P<0.05或P<0.01）。OCT成像显示，C57和CD1小鼠（n=11）的施莱姆管面积相对于基线（处理前10 mmHg）发生显著变化 [2]
对C57和CD1小鼠进行局部甲磺酸奈他舒地尔处理后，通过OCT散斑方差图像分析发现，参与房水流出的巩膜血管的横截面积和散斑方差强度在处理后30-60分钟增加（P<0.05） [2]

Netarsudil（以前称为 AR-13324）是一种 ROCK 抑制剂，Ki 为 0.2-10.3 nM。此外，它还能抑制去甲肾上腺素转运活性，从而减少房水的产生。

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在PDB中总共发现了23个ROCK结构。最大和最小分辨率分别为3.4Å和2.93Å。选择7个ROCK-I和2个ROCK-II非冗余结构用于结合测定。在测试的46种化合物（20种异喹啉、15种氨基呋咱、6种苯二氮卓、4种吲唑和1种酰胺）中，与Y-27632相比，34种化合物的ROCK-1对接得分显著更高（p<0.0001）。所有ROCKi类的平均对接得分均高于Y-27632（p＜0.0001）。ROCK-I的异喹啉、氨基呋咱和苯二氮卓类化合物呈现最高对接得分的频率更高；以及ROCK-II的异喹啉和酰胺（补充图S2A）。ROCK-I和II平均对接得分最高的前十种化合物如补充图S2B所示。异喹啉类药物占前十个最高对接得分内药物的70%，其中三种化合物的对接得分强于Ş12。除Y-27632外，ROCK抑制剂之间没有显著差异。有趣的是，计算机分子对接模拟显示，大多数评估的分子，特别是异喹啉、苯二氮卓和酰胺类分子，对ROCK-1和ROCK-2的结合强度高于Y-27632（补充图S2B）。进行了计算机分子对接模拟，将PDB中发现的AR-13324和Y-27632抑制剂的异构体与高分辨率ROCK蛋白偶联。所有测试的AR-13324分子对ROCK-1和-2的对接得分都高于Y-27632。此外，异喹啉、苯二氮卓和酰胺类的PDB分子也显示出比Y-27632异构体更高的平均对接得分（补充图S2B）[3]。

先前的研究表明，netarsudil 可能会导致 TM 细胞的细胞外基质组成发生改变，以及粘着斑、肌动蛋白应力纤维和细胞形状的丧失。

Topical application
Mice with elevated intraocular pressure (IOP)

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Rabbit 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]
Mouse 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]
Mouse 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]
Mouse 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]
Mouse 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]
Mouse 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]
Mouse 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]
Human 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
The systemic exposure of netarsudil and its active metabolite, AR-13503, after topical ocular administration of netarsudil opthalmic solution 0.02% once daily (one drop bilaterally in the morning) for eight days in 18 healthy subjects demonstrated no quantifiable plasma concentrations of netarsudil (lower limit of quantitation [LLOQ] 0.100 ng/mL) post dose on Day 1 and Day 8. Only one plasma concentration at 0.11 ng/mL for the active metabolite was observed for one subject on Day 8 at 8 hours post dose.
Route of Elimination
Clinical studies assessing the *in vitro* metabolism of netarsudil using corneal tissue from humans, human plasma, and human liver microsomes and microsomal S9 fractions demonstrated that netarsudil metabolism occurs through esterase activity. Subsequent metabolism of netarsudil's esterase metabolite, AR-13503, was not detectable. In fact, esterase metabolism in human plasma was not detected during a 3 hour incubation.
Volume of Distribution
As netarsudil and its active metabolite demonstrate a high degree of protein binding, it is expected to exhibit a low volume of distribution.
Clearance
The clearance of netarsudil is strongly influenced by its low plasma concetrations following topical administration and absorption and high protein binding in human plasma inn.

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

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

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

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of netarsudil during breastfeeding. Because netarsudil poorly absorbed by the mother after administration to the eye, it is unlikely to adversely affect the breastfed infant. Until more data become available, netarsudil should be used with caution during breastfeeding, especially while nursing a newborn or preterm infant. To decrease the amount of drug that reaches the breastmilk after using eye drops, place pressure over the tear duct by the corner of the eye for 1 minute or more, then remove the excess solution with an absorbent tissue.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[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: Netarsudil (has active moiety); Latanoprost; netarsudil mesylate (component of).

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Netarsudil mesylate (formerly AR-13324) is a dual inhibitor of Rho kinase and norepinephrine transporter, developed for the treatment of open-angle glaucoma and ocular hypertension [1][2][3]
Inhibition of Rho kinase by netarsudil mesylate improves fluid outflow through the trabecular meshwork, thereby lowering intraocular pressure, which is a key strategy for anti-glaucoma therapy [1]
In living mouse eyes, netarsudil mesylate affects both proximal (trabecular meshwork and Schlemm's Canal) and distal portions (intrascleral vessels) of the conventional outflow tract, increasing perfusion of outflow tissues through widening of TM and increasing SC cross-sectional area, which is associated with increased outflow facility, enhanced speckle variance intensity of outflow vessels, and decreased IOP [2]
The mechanism of netarsudil mesylate in human eyes involves acute expansion of JCT and dilation of ESVs, leading to redistribution of aqueous outflow through a larger area of IW and ESVs, thereby increasing outflow facility [3]
This is the first report using live imaging (OCT) to show real-time drug effects of netarsudil mesylate on conventional outflow tissues in living eyes, paving the way for the development of a clinic-friendly OCT platform for monitoring glaucoma therapy [2]
Netarsudil mesylate was identified from alpha-aryl-beta-amino isoquinoline analogs, which were found to be potent ROCK inhibitors with the ability to inhibit norepinephrine transporter and provide a longer duration of IOP reduction [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

220Ų

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (3.87 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (3.87 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (3.87 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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