CY 09

NLRP3

CY-09 在浓度为 1 至 10 μM 的 LPS 攻击的骨髓源性巨噬细胞 (BMDM) 中表现出对尿酸钠 (MSU)、MSU、ATP 诱导的 caspase-1 激活和 IL-1β 行走的反应性。死亡依赖性抑制。 BMDM 中细胞质 LPS 诱导的非典型 NLRP3 激活也可以通过 CY-09 给药而肿胀。 CY-09 缓冲液激活 NLRP3 炎症小体，对 LPS 诱导的启动效应没有影响。 CY-09处理主要抑制因子，发现CY-09处理HEK-293T细胞中Flag-NLRP3和mCherry-NLRP3的相互作用，表明CY-09阻止NLRP3寡聚化[1]。

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CY-09特异性阻断巨噬细胞中NLRP3的激活。CY-09阻断NLRP3炎性小体激活。 CY-09抑制NLRP3寡聚和炎性小体组装。CY-09直接与NLRP3结合并抑制其ATP酶活性。 CY-09与NLRP3-NACHT结构域的ATP结合位点结合。CY-09抑制NLRP3-ATP酶活性[1]。

在用 CY-09 处理的腔室中，注射尿酸钠 (MSU) 诱导的 IL-1β 生成和中性粒细胞流入显着受到抑制，表明 CY-09 可以阻止腔室内 MSU 诱导的 NLRP3 炎性体激活。即使治疗在第 25 天终止，CY-09 治疗也将 NLRP3 突变的均匀率提高到 30 至 48 天。 CY-09 还抑制 caspase-1 (caspase-1) 通道的均匀加工，这在高脂饮食 (HFD) 的脂肪组织中有所体现 [1]。

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CY-09在体内抑制NLRP3激活，并在CAPS小鼠模型中防止新生儿死亡。 CY-09抑制MSU诱导的腹膜炎和MWS小鼠模型中NLRP3的激活。 CY-09通过抑制NLRP3依赖性炎症来逆转糖尿病小鼠的代谢紊乱。 CY-09治疗HFD诱导的糖尿病小鼠代谢紊乱。CY-09抑制糖尿病小鼠NLRP3依赖性代谢炎症。 CY-09对健康人或痛风患者的细胞具有体外活性。CY-09对健康人或痛风患者的细胞具有活性[1]。

MST测定[1]
KD值使用Monolith NT.115仪器测量。在测定缓冲液（50 mM Hepes、10 mM MgCl2、100 mM NaCl、pH 7.5和0.05%吐温20）中，将不同浓度的CY-09（0.025 mM至1.2 nM）与200 nM纯化的His-GFP-NLRP3蛋白一起孵育40分钟。将样品装入NanoTemper玻璃毛细管中，使用100%LED功率和80%MST功率进行MST。KD值是通过NanoTemper软件从实验的重复读取中使用质量作用方程计算的。

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微粒体稳定性[1]
在一家CRO公司中确定了微软的稳定性CY-09或用50%甲醇稀释至100µM浓度的对照化合物DMSO储备溶液（10 mM）。将6µl 100µM化合物溶液与534µl肝微粒体溶液（0.7mg蛋白质/ml磷酸钾缓冲液）混合，制备化合物工作溶液。在加入NADPH再生系统（10µl）开始反应之前，将90µl等分的该工作溶液在37°C下孵育10分钟。通过加入300µl冷乙腈（含有500 nM的甲磺丁脲作为内标）在0、10、30和60分钟停止反应，并在4000 rpm下离心20分钟。将上清液（100µl）加入水（300µl）中，通过液相色谱/串联质谱法进行分析。使用试验化合物剩余峰面积/内标的比率来确定试验化合物浓度随时间的降低。然后计算t1/2和肝清除率（CLhep）。

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NLRP3-ATP酶活性和ATP结合测定[1]
对于ATP酶活性测定，纯化的重组人蛋白（1.4 ng/µl）在37°C下与指示浓度的CY-09在反应缓冲液中孵育15分钟。然后加入ATP（25µm，超纯ATP），将混合物在37°C下进一步孵育40分钟。根据制造商的方案，使用ADP-Glo激酶检测试剂盒通过发光ADP检测来测定转化为二磷酸腺苷（ADP）的ATP量。结果以残留酶活性与载体处理酶的百分比表示。 对于ATP结合测定，纯化的NLRP3蛋白（0.1 ng/µl）与ATP结合琼脂糖一起孵育1小时，然后加入不同浓度的CY-09，在4°C下运动孵育2小时。将珠子在装载缓冲液中洗涤并煮沸。对样本进行免疫印迹分析。

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ASC低聚分析[1]
BMDM以1×106/ml的浓度接种在6孔板中。第二天，更换培养基，用50 ng/ml LPS预处理细胞3小时。用CY-09处理细胞30分钟，然后用黑曲霉素刺激30分钟。去除上清液，用冰冷的PBS冲洗细胞，然后用NP-40裂解细胞30分钟。裂解物在4°C下以330 g离心10分钟。将颗粒在1ml冰冷的PBS中洗涤两次，然后重新悬浮在500µl PBS中。将2mM二琥珀酰亚胺酯加入重新悬浮的颗粒中，在室温下旋转培养30分钟。然后将样品在4°C下以330 g离心10分钟。将交联颗粒重新悬浮在30µl样品缓冲液中，然后煮沸并通过免疫印迹进行分析。

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细胞色素P450抑制[1]
在无锡应用科技有限公司测定了CY-09对五种主要CYP同工酶（1A2、2C9、2C19、2D6和3A4）的细胞色素P450抑制作用。使用磷酸盐缓冲液将CY-09的工作储备溶液（DMSO中的10 mM）稀释至100µM的浓度。在DMSO中制备了五种浓度为3 mM的抑制剂储备溶液：α-萘黄酮、磺胺苯唑、N-3-苄基尼凡诺、奎尼丁和酮康唑。使用磷酸盐缓冲液将抑制剂储备稀释至30µM的浓度。通过使用磷酸盐缓冲液稀释甲醇储备溶液，制备了五种主要CYP同工酶（1A2、2C9、2C19、2D6和3A4）的底物混合物（非那西丁、双氯芬酸、S-美芬妥英、右美沙芬和咪达唑仑）CY-09、已知抑制剂或空白溶液（20µl工作储备溶液）和底物鸡尾酒溶液（20μl）在37°C下与人肝微粒体（158µl 0.253mg/ml磷酸盐缓冲液）一起孵育10分钟，然后加入辅因子NADPH（20µl10mM溶液，溶于33mM MgCl2中）。继续孵育，在0、5、10、20、30和60分钟时取出等分试样，然后与冷乙腈（含有甲苯磺丁脲作为内标，浓度为200 ng/ml）混合，以4000 rpm离心20分钟。通过液相色谱/串联质谱分析上清液，以确定代谢物和内标的峰面积。在存在和不存在试验化合物的情况下测定的峰面积用于测定抑制百分比。SigmaPlot v.11用于绘制对照活性百分比与试验化合物浓度的关系图，并对数据进行非线性回归分析。使用三参数逻辑斯谛方程确定IC50值。当最高浓度（50µM）下的抑制百分比<50%时，IC50值报告为“>50µM”。

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hERG通道测定[1]
一家CRO公司使用手动膜片钳法（QPatchHTX）来评估CY-09对hERG钾通道的影响。使用来自Aviva Biosciences的稳定表达hERG钾通道的CHO细胞。测定了CY-09和阿米替林作为阳性对照对全细胞hERG电流的抑制作用。

为了诱导NLRP3炎性体激活，将5×105/ml BMDM和6×106/ml PBMCs接种在12孔板上。第二天早上，更换培养基，用50 ng/ml LPS或400 ng/ml Pam3CSK4（用于非正常炎性体激活）刺激细胞3小时。之后，将CY-09或其他抑制剂加入培养物中30分钟，然后用MSU（150µg/ml）、鼠伤寒沙门氏菌（多重感染）刺激细胞4小时，或用ATP（2.5 mM）或黑曲霉素（10µM）刺激细胞30分钟。使用Lipofectamine 2000用聚（dA:dT）（0.5µg/ml）转染细胞4小时或用LPS（500 ng/ml）转染细胞过夜。通过免疫印迹分析细胞提取物和沉淀的上清液。

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共聚焦显微镜[1]
如前所述进行共聚焦分析（Yan等人，2015）。简而言之，2×105/ml BMDM被镀在盖玻片上。第二天，将培养基替换为含有LPS（50 ng/ml）的Opti-MEM（1%FBS）3小时，然后再加入指定剂量的CY-0930分钟。然后使用BMDM进行刺激，并用MitoTracker Red（50 nM）或MitoSOX（5µM）染色。用冰冷的PBS洗涤三次后，在室温下用4%PFA的PBS溶液固定细胞15分钟，然后用含吐温20的PBS洗涤3次。使用蔡司LSM700进行共聚焦显微镜分析。

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细胞内钾或氯化物检测[1]
为了准确测量细胞内钾，将BMDM在6孔板上放置过夜，然后用50 ng/ml LPS预处理3小时。之后，用CY-09处理细胞30分钟，然后用黑曲霉素刺激30分钟。彻底吸出培养基，用65%超纯HNO3裂解。使用PerkinElmer Optima 2000 DV光谱仪，以钇为内标，通过电感耦合等离子体发射光谱法进行细胞内K+测量。
为了准确测量细胞内氯化物，将BMDM在12孔板上放置过夜，然后用50 ng/ml LPS预处理3小时。之后，用CY-09或MCC950处理细胞30分钟，然后用黑曲霉素刺激15分钟。去除12孔板的上清液，加入ddH2O（200µl/孔），将上清液在37°C下保持15分钟。将裂解物转移到1.5ml EP管中，在10000g下离心5分钟。然后将160-µl上清液转移到新的1.5ml EP试管中，并与40µl MQAE（10µM）混合。使用BioTek多模微孔板读数器（Synergy2）测试吸光度。在每个实验中都设置了一个对照，以确定抽吸后剩余的细胞外氯化物量，并减去该值。

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免疫沉淀（IP）和下拉试验[1]
对于内源性IP测定，用含有完全蛋白酶抑制剂的NP-40裂解缓冲液刺激和裂解BMDM。将细胞裂解物与一抗和Protein G Mag-Sepharose（GE Healthcare）在4°C下孵育过夜。用G蛋白珠沉淀抗体结合的蛋白质，并进行免疫印迹分析。对于外源性IP测定，HEK-293T细胞（3×105/ml）通过聚乙烯亚胺在6孔板中用质粒转染。24小时后，收集细胞并用NP-40裂解缓冲液裂解。用抗Flag抗体包被的珠子对蛋白质提取物进行免疫沉淀，然后通过免疫印迹分析进行评估。
对于下拉分析，收集BMDM或293T裂解物，并在8000 rpm下离心。将上清液转移到另一个试管中，彻底丢弃细胞碎片。将预洗的链霉抗生物素蛋白珠加入上清液中，在4°C下运动预孵育2小时，以8000 rpm离心。将上清液转移到另一个试管中，丢弃链霉抗生物素蛋白珠以去除非特异性结合蛋白。将预处理的上清液和纯化的人重组NLRP3蛋白（溶解在裂解缓冲液中）与指定剂量的游离CY-09一起孵育，然后与指定浓度的生物素-CY-09一起孵育1小时。之后，将样品与预洗的链霉抗生物素蛋白珠一起孵育过夜。分别用0.1%吐温20的PBS溶液和1%NP-40的PBS溶液洗涤珠子两次，以去除非特异性结合蛋白，并在SDS缓冲液中煮沸。

With the formulation of DMA:EL:HP-β-CD (10%, wt/vol) = 5:5:90 (vol/vol/v), CY-09 achieved a concentration of 1 mg/ml at pH 7.4. Using the formulation of DMSO:Solutol HS 15:saline = 10%:10%:80% (vol/vol/v), CY-09 reached a concentration of 5 mg/ml at pH 9.0. For the in vivo experiments, CY-09 was formulated in a vehicle containing 10% DMSO, 10% Solutol HS 15, and 80% saline.[1]

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MSU-induced peritonitis [1]
C57BL/6J mice were injected i.p. with 40 mg/kg CY-09 or vehicle 30 min before i.p. injection of MSU (1 mg MSU crystals dissolved in 0.5 ml sterile PBS). After 6 h, mice were killed, and peritoneal cavities underwent lavage with 10 ml ice-cold PBS. Peritoneal lavage fluid was assessed by flow cytometry with the neutrophil markers Ly6G and CD11b for analysis of the recruitment of polymorph nuclear neutrophils. IL-1β production in serum or peritoneal lavage fluid was determined using ELISA.

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Determination of the pharmacokinetic properties of CY-09 in mice [1]
The pharmacokinetics of CY-09 were determined after single i.v. and oral administration in C57BL/6J mice (n = 3 at each time point) at doses of 5 and 10 mg/kg, respectively. Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, 10, and 24 h (i.v.) and 0.25, 0.5, 1, 2, 4, 8, 10, and 24 h (oral) after administration. Then the samples were quantified by liquid chromatography/tandem mass spectrometry, and data analysis was conducted using WinNonlin v.6.3.

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MWS mouse model [1]
Nlrp3A350VneoR mice were crossed with LysM-Cre mice (B6.129P2-Lyz2tm1(cre)Ifo/J). CY-09 (20 mg/kg) or MCC950 (20 mg/kg) were administered orally every day starting at day 4 after birth. The weight and survival of mice were monitored every day.

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HFD and CY-09 treatment [1]
WT or Nlrp3−/− mice at the age of 6 wk, with similar plasma glucose levels and body weights were randomized into different groups. For generation of HFD-induced diabetic mice, mice were fed with HFD for 14 wk. The diabetic mice were treated with CY-09 (i.p.) at a dose of 2.5 mg/kg once a day for 6 wk. The mice were maintained with HFD when used for CY-09 treatment and the subsequent experiments.

We next examined the pharmacokinetic profile of this compound before assessing the therapeutic potential of CY-09 in vivo. The metabolic stability of CY-09 was first evaluated using human and mouse liver microsomes, exhibiting favorable stability with the half-life >145 min for both human and mouse microsomes (Table S1). CY-09 was tested against the five major cytochrome P450 enzymes 1A2, 2C9, 2C19, 2D6, and 3A4 with half maximal inhibitory concentration (IC50) values of 18.9, 8.18, >50, >50, and 26.0 µM, respectively (Table S2), which exhibited low risk of drug–drug interactions. To evaluate the potential for cardiotoxicity, we examined the effect of CY-09 on the human ether-a-go-go (hERG) potassium channel using the automated patch clamp method (QPatchHTX), and CY-09 showed no activity for hERG at 10 µM (Table S3). Then, the pharmacokinetic properties of CY-09 were further evaluated in C57BL/6J mice administered a single i.v. or oral dose. CY-09 exhibited favorable pharmacokinetics, with a half-life of 2.4 h, an area under the curve of 8,232 (h·ng)/ml, and bioavailability of 72% (Table S4). With these data in hand, the in vivo efficacy of CY-09 was then evaluated. [1]

[1]. Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders. J Exp Med. 2017 Nov 6;214(11):3219-3238.

The NLRP3 inflammasome has been implicated in the pathogenesis of a wide variety of human diseases. A few compounds have been developed to inhibit NLRP3 inflammasome activation, but compounds directly and specifically targeting NLRP3 are still not available, so it is unclear whether NLRP3 itself can be targeted to prevent or treat diseases. Here we show that the compound CY-09 specifically blocks NLRP3 inflammasome activation. CY-09 directly binds to the ATP-binding motif of NLRP3 NACHT domain and inhibits NLRP3 ATPase activity, resulting in the suppression of NLRP3 inflammasome assembly and activation. Importantly, treatment with CY-09 shows remarkable therapeutic effects on mouse models of cryopyrin-associated autoinflammatory syndrome (CAPS) and type 2 diabetes. Furthermore, CY-09 is active ex vivo for monocytes from healthy individuals or synovial fluid cells from patients with gout. Thus, our results provide a selective and direct small-molecule inhibitor for NLRP3 and indicate that NLRP3 can be targeted in vivo to combat NLRP3-driven diseases. [1]

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In this study, we describe a potent, selective, and direct inhibitor of NLRP3 with remarkable inhibitory activity for NLRP3 inflammasome in mice in vivo and in human cells ex vivo. CY-09 will serve as a versatile tool to pharmacologically interrogate NLRP3 biology and study its role in inflammatory diseases.
A few compounds have shown potent inhibitory activity for the NLRP3 inflammasome and have been tested in animal models, but the unspecific effects of these compounds have limited their clinical potential. The inhibitory effects of sulforaphane on AIM2 or NLRC4 inflammasome suggest that it might impair the role of these inflammasomes in host defense (Greaney et al., 2016). The broad anti-inflammatory activity of sulforaphane, isoliquiritigenin, BHB, parthenolide, BAY 11-7082, and INF39 (Heiss et al., 2001; Yip et al., 2004; Honda et al., 2012; Strickson et al., 2013; Fu et al., 2015; Cocco et al., 2017) suggests that these compounds might cause immunosuppressive side effects and increase the risk for infection. The effects of flufenamic acid and mefenamic acid on chloride efflux and BHB on potassium efflux indicate that these compounds target the upstream signaling event of NLRP3 and have other unavoidable biological activities (Youm et al., 2015; Daniels et al., 2016). MCC950 has shown strong inhibitory activity and beneficial effects in several mice models of NLRP3-related diseases (Coll et al., 2015; Dempsey et al., 2017), but the mechanism is not understood. Here we showed that MCC950 could block NLRP3 agonist–induced chloride efflux, a proposed upstream signaling event of NLRP3 activation (Daniels et al., 2016), suggesting that it might target the volume-regulated anion channel or other chloride channels to inhibit NLRP3 inflammasome activation and might have unspecific effects. Here we describe CY-09 as a specific NLRP3 inflammasome inhibitor that directly targeted NLRP3 itself and found that CY-09 had remarkable preventive or therapeutic effects on the mice models of CAPS, T2D, and gout. Thus, our study describes a direct and specific NLRP3 inhibitor with the potential to treat NLRP3-driven diseases.
Our results demonstrate that pharmacological inhibition of NLRP3 ATPase activity is efficient to treat NLRP3-driven diseases. Previous studies have reported that MNS, parthenolide, BAY 11-7082, and INF39 can inhibit the ATPase activity of NLRP3 and show inhibitory activity for NLRP3 inflammasome in vitro. However, these compounds are not specific NLRP3 inhibitors and have multiple biological activities, such as the inhibitory activity for tyrosine kinases or NF-κB signaling pathway (Yip et al., 2004; Wang et al., 2006; Strickson et al., 2013; Cocco et al., 2017). In addition, MNS, parthenolide, and BAY 11-7082 have not been tested in vivo in the animal models of NLRP3-driven diseases. CY-09 directly bound to the NACHT domain of NLRP3 and inhibited its ATPase activity, which is essential for NLRP3 oligomerization and inflammasome assembly (Duncan et al., 2007). Furthermore, the mutation of Walker A motif in the NACHT domain, which is required for ATP binding to NLRP3 (MacDonald et al., 2013), impaired the ability of CY-09 binding to NLRP3. In addition, our results clearly demonstrate that CY-09 competes with ATP to bind to NLRP3 and inhibits its ATPase activity and the subsequent NLRP3 oligomerization and inflammasome assembly. Importantly, our results show that suppression of NLRP3 ATPase activity by CY-09 has remarkable effects to reduce NLRP3 inflammasome activation and symptoms in mice models of T2D and CAPS. Thus, our results suggest the ATPase activity could be targeted to screen drug candidates for treatment of NLRP3-drive diseases.
The current available clinical treatment for NLRP3-related diseases is the use of agents that target IL-1β, but targeting NLRP3 itself with small-molecule inhibitors with high specificity, such as CY-09, might have certain advantages. In the CAPS mouse model, CY-09 was effective to prevent lethality, but blocking IL-1β alone could not (Brydges et al., 2009). The possible reason is that the IL-18 production or pyroptosis caused by inflammasome activation might also contribute to the pathology. In addition, IL-1β is also produced by other inflammasomes or in an inflammasome-independent way (Davis et al., 2011; Netea et al., 2015), so inhibition of NLRP3 itself might have less immunosuppressive side effects than blockade of IL-1β. Indeed, our results showed that CY-09 had no effect on AIM2 or NLRC4 inflammasomes, suggesting that CY-09 might not impair the role of these inflammasomes in host defense. Moreover, the small-molecule compounds are in general more cost effective than biological agents (Fautrel, 2012).
T2D is characterized by insulin resistance and hyperglycemia and can cause several complications, including nerve and kidney damage. However, the drugs available currently are not effective in correcting the underlying cause of insulin resistance, and most patients need pharmacotherapy for the rest of their lives (Nathan et al., 2009; Qaseem et al., 2012). Our study demonstrates that inhibition of NLRP3-dependent metainflammation by CY-09 is efficient to reverse the metabolic disorders in diabetic mice. CY-09 treatment had remarkable beneficial effects for metainflammation, hyperglycemia, and insulin resistance in diabetic mice. Thus, this study suggests that correcting NLRP3-dependent metainflammation might be an effective approach to treat T2D. Considering the role of NLRP3-dependent inflammation in the progression of gout, Alzheimer’s disease, and atherosclerosis, CY-09 or its derivatives could be used for the development of new NLRP3-targeted therapeutics for these diseases. [1]

C19H12F3NO3S2

423.43

423.021

C, 53.90; H, 2.86; F, 13.46; N, 3.31; O, 11.34; S, 15.14

1073612-91-5

44561595

Light yellow to yellow solid powder

4.9

115

1

8

4

28

672

0

S1C(N(C(/C/1=C\C1C=CC(C(=O)O)=CC=1)=O)CC1C=CC=C(C(F)(F)F)C=1)=S

DJTINRHPPGAPLD-DHDCSXOGSA-N

InChI=1S/C19H12F3NO3S2/c20-19(21,22)14-3-1-2-12(8-14)10-23-16(24)15(28-18(23)27)9-11-4-6-13(7-5-11)17(25)26/h1-9H,10H2,(H,25,26)/b15-9-

4-[[4-Oxo-2-thioxo-3-[[3-(trifluoromethyl)phenyl]methyl]-5-thiazolidinylidene]methyl]benzoic acid

CY 09; CY-09; 1073612-91-5; CY 09; 4-[[4-Oxo-2-thioxo-3-[3-(trifluoromethyl)benzyl]thiazolidin-5-ylidene]methyl]benzoic Acid; 4-[[4-oxo-2-sulfanylidene-3-[[3-(trifluoromethyl)phenyl]methyl]-1,3-thiazolidin-5-ylidene]methyl]benzoic acid; MFCD31619349; CY09; DJTINRHPPGAPLD-UHFFFAOYSA-N; (Z)-4-((4-Oxo-2-thioxo-3-(3- (trifluoromethyl)benzyl)thiazolidin-5- ylidene)methyl)benzoic acid; CY09

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)

DMSO : ≥ 150 mg/mL (~354.25 mM)
H2O : ~1.1 mg/mL (~2.60 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.90 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 (5.90 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；
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。