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

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 [1]
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 [2]

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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青光眼造成的视力损害目前影响着全球7000万人。虽然可以通过有效降低眼压来减缓或停止疾病的进展，但目前的医学治疗往往是不够的。幸运的是，针对导致高眼压的病变常规流出组织的三种新疗法已进入人体试验的最后阶段。rho激酶抑制剂已被证明特别有效，并可添加到当前的治疗中。不幸的是，监测流出液组织健康状况及其对常规流出液治疗反应的非接触式技术在临床上尚不可用。利用光学相干层析成像（OCT）和新型分割软件，我们首次展示了药物对活体眼睛常规流出组织的影响。外用奈沙地尔（原AR-13324）是一种rho激酶/去甲肾上腺素转运蛋白抑制剂，影响小鼠常规流出道的近端（小梁网和施莱姆管）和远端（束内血管）。因此，流出组织灌注增加可以通过OCT可靠地分辨为netarsudil治疗后小梁网变宽和Schlemm管横截面积显著增加。这些变化与流出设施增加、流出血管斑点变异强度增加、常规流出组织中示踪剂沉积增加和眼压降低同时发生。这是第一次使用实时成像技术来显示药物对常规流出组织的实时作用，特别是奈沙地尔在小鼠眼睛中的作用机制。这里的进步为开发用于监测青光眼治疗的临床友好型OCT平台铺平了道路。[1]

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配对人眼（n = 5）分别灌注0.3 μM netarsuil - m1或恒压（15 mm Hg）载药溶液。3小时后，在灌注介质中加入荧光微球，在灌注固定前追踪流出模式。通过测量示踪剂在小梁网（TM）、锁膜外静脉（ESVs）和施莱姆管内壁（IW）的分布长度，计算有效滤过长度百分比（PEFL）。通过共聚焦、光学和电子显微镜观察小梁流出通道的形态学变化。结果：与基线（51%,P < 0.01）和配对对照（102%,P < 0.01）相比，灌注netarsudil-M1显著增加了C， IW （P < 0.05）和esv （P < 0.01）的PEFL均显著增加。在治疗组中，esv组PEFL明显高于IW组（P < 0.01），且与esv横截面积增加相关（P < 0.01）。esv有效滤过长度百分比与C变化百分比呈正相关（R2 = 0.58, P = 0.01）。与对照组相比，治疗组的眼关节旁结缔组织（JCT）厚度显著增加（P < 0.05）。[2]

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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） [1]
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） [1]
对活体C57小鼠进行局部奈他舒地尔处理后，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 [1]
在眼内压升高的活体小鼠中，局部给予奈他舒地尔（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 [1]
对C57和CD1小鼠进行局部奈他舒地尔处理后，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） [1]

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]。

根据制造商的说明，使用EdU掺入Click-iT细胞增殖测定法评估原代CEC的增殖率。评估了两种ROCK抑制剂AR-13324和AR-13503在两种浓度（AR-13324为100 nM或1 M，AR-13503为1 M或10 M）下增强CECs增殖的能力。不添加ROCKi的供体匹配CECs作为阴性对照，而添加Y-27632的CECs作为阳性对照。简言之，使用TS传代的培养的CEC以5 103个细胞/cm2的密度接种到FNC涂布的载玻片上，并在M5 Endo中维持24小时（第1天）。第二天（第2天），将培养基切换到各自的条件，并将细胞再培养24小时。第三天，将细胞在含有10mMof-EdU的M4-F99中孵育24小时。随后，用PBS冲洗样品一次，然后将样品在室温下固定在新制备的4%PFA中15分钟。接下来，用PBS中的3%BSA冲洗样品两次，并在室温下在PBS中0.5%Triton X-100中孵育20分钟以进行封闭和透化。通过荧光叠氮化物偶联Click-iT反应检测掺入的EdU，其中将样品与含有Click-iT-EdU反应缓冲液、CuSO4、叠氮化物缀合的Alexa Fluor 488染料和反应缓冲液添加剂的反应混合物在黑暗中孵育30分钟。之后，用3%BSA冲洗样品，然后在室温下黑暗中在5g/mL Hoechst 33342中孵育10分钟。最后，在PBS中洗涤样品两次，并将样品安装在含有4,6-二脒基-2-苯基吲哚（DAPI）的Vectashield中。在Zeiss Axioplan 2荧光显微镜下检查标记的增殖细胞。对于每种实验条件，至少分析了250个细胞核[3]。

Topical application
Mice with elevated intraocular pressure (IOP) Visual 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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Paired 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. Results: 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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For 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]
For 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]
For 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]
For 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]
For 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]
For 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]
For 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
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.
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.
As netarsudil and its active metabolite demonstrate a high degree of protein binding, it is expected to exhibit a low volume of distribution.
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.

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.

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Protein Binding
The active metabolite of netarsudil, AR-13503 is highly protein bound in plasma, at approximately 60% bound. As AR-13503 is considered to bind less extensively to plasma proteins as its parent netarsudil, the % protein binding of netarsudil 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 humour flows out of the eye via two pathways: 1) the conventional trabecular pathway and 2) the unconventional uveoscleral pathway. And, although it has been shown that the conventional trabecular pathway accounts for most aqueous outflow due to various pathologies, most medications available for treating glaucoma target the uveoscleral pathway for treatment and leave the diseased trabecular pathway untreated and unhindered in its progressive deterioration and dysfunction. Netarsudil is subsequently a novel glaucoma medication that is both a rho kinase and norepinephrine transport (NATs)s inhibitor that specifically targets and inhibits rho kinase and NATS found in the conventional trabecular pathway while many of its contemporaries offer therapy that focuses on cell and muscle tissue remodelling

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Netarsudil (formerly AR-13324) is a dual inhibitor of Rho kinase and norepinephrine transporter, being developed for the treatment of glaucoma and ocular hypertension [1][2]
In living mouse eyes, netarsudil 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 [1]
The mechanism of netarsudil 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 [2]
This is the first report using live imaging (OCT) to show real-time drug effects of netarsudil on conventional outflow tissues in living eyes, paving the way for the development of a clinic-friendly OCT platform for monitoring glaucoma therapy [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

94.3Ų

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。