Wnt-C59

PORCN (IC50 = 74 pM)[1]

通过阻断 Wnt 棕榈酰化、Wnt 与载体蛋白 Wntless/WLS 的相互作用、Wnt 分泌以及 Wnt β-连环蛋白报告活性的激活，确定 Wnt-C59 (C59) 在纳摩尔浓度下可在体外降低 PORCN 功能。 Wnt-C59 的 IC50 为 74 pM，可阻断 WNT3A 介导的驱动荧光素酶的多聚化 TCF 结合位点的激活[1]。

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Wnt-C59（C59）是PORCN酶活性的强效抑制剂[1]
诺华公司最近开发并获得了小分子2-（4-（2-甲基吡啶-4-基）苯基）-N-（4-（吡啶-3-基）苯）乙酰胺作为Wnt信号调节剂的专利。它以C59的名称从至少2个来源商购，并声称在纳摩尔浓度下抑制PORCN酶活性。然而，目前还没有关于其疗效和分子靶点的同行评审的公开信息。由于目前还没有一种有效、生物可利用和稳定的PORCN抑制剂，我们评估了C59。我们发现，使用许多基于细胞的检测，C59确实可以作为真正的PORCN抑制剂发挥作用。C59抑制WNT3A介导的驱动萤光素酶（Super8xTopFlash；STF）的多聚TCF结合位点的激活，IC50为74 pmol/L（图1A）。正如对PORCN抑制剂的预期，C59处理完全消除了Wnt分泌到培养基中的现象（图1A，插图）。与靶向PORCN的C59一致，PORCN过表达可以缓解WNT3A介导的STF活性的抑制，类似于无关的PORCN抑制剂IWP-1（参考文献21、22；图1B）。Wnt酰化是与载体蛋白WLS结合所必需的。WNT3A和WNT8A与WLS共免疫沉淀，但当细胞用C59预处理时，这种相互作用被阻断（图1C）。使用炔棕榈酸和点击化学，我们发现C59可以防止棕榈酸盐掺入WNT3A，这与抑制PORCN活性是一致的（图1D）。C59抑制小鼠PORCN所有剪接变体的活性（图2A）。在初步研究中，我们发现在爪蟾胚胎发生中产生发育表型需要非常高浓度的C59。与此一致，虽然非洲爪蟾PORCN在PORCN缺失的人类细胞中表达时具有活性，但其活性对C59的抑制具有抗性（图2A）。由于爪蟾蛋白与人类PORCN有77%的相同性，这提供了PORCN是C59分子靶标的遗传证据，表明了C59耐药性出现的机制，并表明相关性较低的MBOAT蛋白也不受C59的影响。表明抑制PORCN可能会阻止所有Wnt介导的信号传导，我们发现，当细胞用C59处理时，9个激活β-catenin的Wnts中有9个和4个额外的非正则Wnts中的4个失去了活性（图2B和C）。总之，C59是哺乳动物PORCN酰基转移酶活性的纳摩尔抑制剂，可阻断所有评估的人类Wnts的激活。因此，我们预计C59给药将阻止所有人类和小鼠Wnt依赖性信号传导。

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SUNE1细胞表现出对Wnt-C59（C59）的强耐受性，正如在许多其他人类癌症细胞系中报道的那样。在较高的Wnt-C59浓度（20μM）下，CNE1和HNE1细胞的生长能力都降低了，而HK1细胞对所有浓度（5μM、10μM和20μM）的Wnt-C5 9处理都很敏感。
SUNE1和HNE1细胞都显示出更大的形成球体的能力（图1A和1B）。它们在48小时对Wnt-C59治疗的IC50大于60μM。此外，这两种细胞系具有明显不同的体内细胞生长动力学，为确定Wnt-C59在鼻咽癌细胞中的生物学效应提供了良好的动物模型。
低浓度（1μM）的Wnt-C59（C59）处理抑制了SUNE1细胞，与未处理的对照细胞相比，3D生长明显受到抑制（图3A）。Wnt-C59对鼻咽癌细胞的抑制作用呈剂量依赖性；5μM和20μM Wnt-C59处理都可以阻止HNE1和SUNE1细胞系中球体的形成，但不能阻止CNE1细胞。然而，在第1至3周观察到，用Wnt-C59处理显示单层生长的细胞持续扩增，3D抑制和单层生长都是一致的（图3B）。
经过三周的治疗后，我们撤回了Wnt-C59（C59），并在对照细胞培养条件下继续培养细胞10至20天。HNE1细胞中没有明显的3D生长恢复或检测到，尽管细胞密度和孵育期足以使未处理的细胞产生3D生长。然而，在未经处理的培养条件下，单层生长的SUNE1细胞在20天内逐渐形成巨大的球体（图3C）。这些发现表明，Wnt-C59对茎干的抑制作用是不可逆的，如HNE1所示。Wnt抑制剂可以安全地根除整个细胞群中具有干性特性的HNE1细胞。

在小鼠中，wnt-C59 表现出良好的生物利用度。 Wnt-C59 下调 Wnt/β-catenin 靶基因，同时预防 MMTV-WNT1 转基因小鼠中乳腺癌的生长[1]。在人类癌症中，Wnt-C59 具有消除癌症干细胞的能力。 Wnt-C59 阻止 HNE1 和 SUNE1 细胞中球体的形成，从而以剂量依赖性方式抑制 NPC 细胞的干性特征 [2]。

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Wnt-C59（C59）可以给小鼠服用并防止肿瘤生长[1]
为了测试Wnt信号在体内的作用，我们评估了C59在小鼠体内的生物利用度和体内半衰期。静脉注射（2.5mg/kg）或口服（5mg/kg）后，化合物在血液中的半衰期约为1.94小时。值得注意的是，单次口服给药后至少16小时，C59浓度仍高于体外IC50 10倍以上（图3A）。根据药代动力学分析，每天给药一次C59，以测试其治疗既定Wnt驱动肿瘤的疗效。在携带小鼠乳腺肿瘤病毒（MMTV）-WNT1转基因的小鼠中，小鼠WNT1的过表达导致从10周龄开始的乳腺腺癌发病率很高。值得注意的是，这些小鼠中出现的肿瘤仍然依赖于Wnt，但具有不同的分子表型和生长速率，这与WNT1扩大弱势群体然后经历二次打击的假设相一致
为了测试Wnt-C59（C59）的体内疗效，我们将2个独立的原发性MMTV-WNT1肿瘤的片段原位移植到裸鼠体内。在出现可触及的肿瘤后，用赋形剂或C59，10mg/kg/d治疗小鼠17天。C59给药可阻止或逆转所有接受治疗的小鼠的肿瘤生长（n=22；图3B）。经过17天的治疗，肿瘤被切除并进一步分析。最终肿瘤重量存在显著差异（图3C）。为了证实C59在免疫功能正常的小鼠中具有活性，我们监测了雌性未产妇Bl6-MMTV-WNT1小鼠的肿瘤发展情况。当肿瘤变得可触及时，用赋形剂或C59（5mg/kg/d）治疗小鼠。虽然参与这项研究的小鼠数量较少，但即使是较低剂量的C59也能显著抑制肿瘤生长（图3D）。最终肿瘤重量如补充图S2A所示。

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肿瘤生长抑制与肿瘤中Wnt/β-catenin信号传导减少有关[1]
为了确定肿瘤生长的抑制是否伴随着Wnt/β-catenin信号传导的抑制，我们通过定量逆转录聚合酶链式反应（qRT-PCR）检测了同种异体移植物和原发性肿瘤中选定靶基因的表达。C59治疗的小鼠肿瘤中Axin2、Ccnd1、c-Myc和Tcf7转录物显著减少（图4A和补充图S2B）。Ki67染色显示，与c-Myc和CyclinD的减少相一致，经治疗的肿瘤增殖也显著降低（图4B）。

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Wnt-C59（C59）在体内抑制Wnt通路[2]
正如最近报道的那样，基质中的Wnt信号传导在干细胞生态位中起着至关重要的作用。因此，我们检测了肿瘤组织中活性β-catenin和Axin2的蛋白表达。这两种蛋白质在癌症巢周围的基质细胞中明显积累，尤其是在肿瘤组织的快速生长区域。对照组和注射SUNE1细胞的Wnt-C59处理的小鼠之间没有检测到这两种蛋白质的表达有明显差异，表明SUNE1（图4A）和HNE1细胞（图4B）中的肿瘤微环境具有良好的上调Wnt通路活性。Wnt-C59对注射SUNE1细胞的小鼠体内这些生长中的肿瘤的影响有限。 重要的是，与对照组小鼠相比，注射Wnt-C59细胞的HNE1小鼠肝脏（图4C）和肾脏（图4D）中活性β-catenin和Axin2的表达明显下调。这些发现证实，在41天的测定期内，Wnt-C59的给药对受试动物的Wnt通路产生了系统性影响，从而诱导了受试小鼠的肿瘤抑制。

Wnt分泌、TOPFlash分析、Dvl2移动转移和PORCN救援[1]
为了测定C59 IC50，用稀释到培养基中1/1000的化合物处理STF3a细胞，48小时后测量荧光素酶活性。为了监测Wnt分泌到培养基中，用C59处理STF3a细胞，C59稀释在以一半体积添加的含有1%FBS的新鲜培养基中。24小时后在4%SDS中收集裂解物。对于HT1080细胞中的SuperTOPflash测定，用50 ng Wnt、100 ng mCherry和650 ng SuperTOPflash在24孔板中转染实验。对于PORCN拯救实验，添加了100 ng 3xHA-mPORCN-D。为了测量Dvl2迁移率偏移，用200ng的每个Wnt质粒和600ng的mCherry转染。在100mM磷酸钠、pH 7.5、150mM NaCl、1%IGEPAL-CA630、完全蛋白酶抑制剂混合物中制备裂解物，并进行蛋白质印迹以检测Dvl2

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Wnt WLS交互[1]
用C末端V5标记的WNT3A或WNT8A转染6孔培养皿中的HeLa细胞。转染后6小时，用0.1%DMSO作为对照或加入终浓度为10nM的C59代替培养基。孵育过夜后，用缓冲液裂解细胞：50mM HEPES、150mM NaCl、1mM EDTA、0.5%NP-40和完全蛋白酶抑制剂）。用兔抗WLS C端多克隆抗体免疫沉淀共500μg裂解物。对V5和WLS进行蛋白质印迹（使用针对WLS的小鼠抗体）

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Alk-C16代谢标记和点击化学[1]
用C-末端V5标记的WNT3A转染在10cm板中以3×106细胞密度铺板的HT1080细胞。转染6小时后，用DMEM代替培养基，DMEM含有5%不含脂肪酸的BSA（Sigma），含有或不含有100μMω-炔基棕榈酸（Alk-C16），如先前报道所合成（3，4）。同时用0.1%DMSO作为对照或100nM C59处理细胞。过夜孵育后，用冷PBS洗涤细胞两次，并在缓冲液中制备细胞裂解物：50mM HEPES、150nM NaCl、1mM EDTA、1%NP-40和蛋白酶抑制剂（Roche）。将蛋白裂解物与抗V5抗体孵育过夜，然后加入30μL蛋白A/G加琼脂糖（50%浆液）2小时。将颗粒用裂解缓冲液洗涤四次，并重悬于磷酸钠缓冲液（100mM磷酸钠，pH 7.5，1%Nonidet P-40，150mM NaCl）中。然后将免疫沉淀的蛋白质在50μl体积中进行点击标记反应1小时RT，最终浓度依次加入以下试剂：0.1 mM生物素叠氮化物（Life Technologies）、1 mM Tris（2-羧乙基）膦盐酸盐（TCEP）、0.2 mM Tris[（1-苄基-1H-1,2,3-三唑-4-基）甲基]胺（TBTA）溶于二甲基亚砜/叔丁醇（20%/80%）和1 mM CuSO4（在水中新制备）。标记的免疫沉淀蛋白通过SDS–PAGE解析，转移到PVDF膜上，用抗V5一级抗体印迹，然后用Dylight 680缀合的抗小鼠抗体和Dylight 800缀合的链亲和素印迹，然后在各自的通道中显现。

体外C59毒性研究[1]
HCT116、K562、HL60、A549、U-2-OS、U266、HCC1937、HCC38、MCF7、BT-20、MB157、MDA-MB-435S、MDA-MB-231、MDA-MBA-468、DLD1、SW480、T98G、A172、U-87MG、KATO III、SNU1、SNU5、SNU16、SK-MEL-5、SK-MEL-28、HS294T、A375、A2058、MEWO、FU97、IM95、IM95M、NUGC-3、NUGC-4、OCUM-1、SCH、MKN1、MKN7、MKN45 MKN74、AZ521、JHH2、SNU216、YCC-3（韩国细胞系库，韩国）、EOL-1在供应商规定的培养条件下生长。对于测定，将75μl培养基中的5000个细胞接种在黑色96孔板的每个孔中，并在37°C下孵育过夜。C59在培养基中以4倍原液的形式连续稀释，然后将25μl的每种稀释液加入细胞中，得到50μM至1.5 nM的最终浓度。处理2天后，向每个孔中加入100μl CellTiter-Glo®发光细胞活力测定试剂，并在室温下孵育10分钟。使用Tecan Safire仪器测量发光。

Pharmacokinetic Analysis[1]
C57/BL6 mice were used. C59 was dissolved in 30% propylene glycol for intravenous tail-vein administration or a mixture of 0.5% methylcellulose and 0.1% tween-80 for oral administration. After single doses (volume 5 ml/kg IV, 10 ml/kg po), mice were sacrificed at indicated times. Blood samples were collected tubes. Plasma was obtained and analyzed using validated bioanalytical LC/MS/MS method. To determine the concentration of C59 in samples, the standard curve of C59 was established in the linear-quadratic manner (r2 = 0.99) over the range of 1 – 1000 ng/ml in mouse plasma. The accuracy and precision of the triplicate quality control samples were within 15% of deviation and 25% of relative standard deviation, respectively. The lower limit of quantification of C59 in mouse plasma was 1 ng/ml. Pharmacokinetic parameters were calculated by the non-compartmental method (Gibaldi and Peter, 1982) using Phoenix WinNonlin 6.0 software.

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Animal studies [1]
MMTV-WNT1 mice were obtained from Jackson Laboratories and backcrossed at least six generations to C57/Bl6 mice. Female virgin mice were placed into control or treatment groups at random as tumors appeared. C59 treatment or vehicle administration was daily by oral gavage, with alternate day caliper-based tumor measurement. The study was conducted for 21 days or till the tumors reached preset size limits. At sacrifice, tumors were resected and weighed, then separated for RNA isolation and histology.

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Dissolved in 0.5% methylcellulose and 0.1% Tween-80; 10 mg/kg; Oral gavage

Female nude mice orthotopically transplanted with independent MMTV-WNT1 tumors

[1]. Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer. Cancer Res. 2013 Jan 15;73(2):502-7.

[2]. Wnt-C59 arrests stemness and suppresses growth of nasopharyngeal carcinoma in mice by inhibiting the Wnt pathway in the tumor microenvironment. Oncotarget. 2015 Jun 10;6(16):14428-39.

Porcupine (PORCN) is a membrane bound O-acyltransferase that is required for Wnt palmitoylation, secretion, and biologic activity. All evaluable human Wnts require PORCN for their activity, suggesting that inhibition of PORCN could be an effective treatment for cancers dependent on excess Wnt activity. In this study, we evaluated the PORCN inhibitor Wnt-C59 (C59), to determine its activity and toxicity in cultured cells and mice. C59 inhibits PORCN activity in vitro at nanomolar concentrations, as assessed by inhibition of Wnt palmitoylation, Wnt interaction with the carrier protein Wntless/WLS, Wnt secretion, and Wnt activation of β-catenin reporter activity. In mice, C59 displayed good bioavailability, as once daily oral administration was sufficient to maintain blood concentrations well above the IC(50). C59 blocked progression of mammary tumors in MMTV-WNT1 transgenic mice while downregulating Wnt/β-catenin target genes. Surprisingly, mice exhibit no apparent toxicity, such that at a therapeutically effective dose there were no pathologic changes in the gut or other tissues. These results offer preclinical proof-of-concept that inhibiting mammalian Wnts can be achieved by targeting PORCN with small-molecule inhibitors such as C59, and that this is a safe and feasible strategy in vivo. [1]
In this study, we confirm that the small-molecule C59 is a nanomolar inhibitor of the acyltransferase activity of PORCN, and show that small-molecule–mediated inhibition of PORCN is an effective means for preventing WNT1-driven tumor growth in mice. C59 inhibits palmitoylation of Wnts and is not active against Xenopus PORCN. Thus, changes in the primary sequence of PORCN confer resistance to C59, confirming genetically that PORCN is the target of C59. C59 is more than100-fold more potent than the previously reported PORCN inhibitor IWP1. We find no apparent toxicity to cells or mice at a drug concentration that effectively inhibits MMTV-WNT1–driven tumor growth. Intestinal architecture of treated mice appears normal. A similar lack of intestinal toxicity was seen when Wnt signaling was inhibited with Fzd8CRD-Fc. We speculate that Wnt-addicted tumors are hypersensitive to small reductions in Wnt activity, whereas normal tissues such as intestine are more tolerant of decreases in Wnt signals and/or have alternative pathways for self-renewal.
The Wnt pathways contribute to the progression of various cancers, via both β-catenin–activating mutations and by paracrine and autocrine Wnt signaling. Increased Wnt production has also been identified in diverse nonmalignant diseases. In many cases, the implicated Wnts may be working via non-β-catenin pathways. PORCN inhibitors may therefore have efficacy even in diseases without activated β-catenin. Thus, it is a longstanding goal to identify therapeutics that can effectively target this pathway. Our recent work has confirmed that PORCN is a key node for fine control of total Wnt-dependent cell signaling, further supporting its use as a target. As such, specific and bioavailable inhibitors of PORCN represent attractive new molecules that may be of value in the treatment of various cancers, in addition to other Wnt-stimulated diseases.[1]

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Wnt/β-catenin signaling is responsible for the generation of cancer stem cells (CSCs) in many human tumors, including nasopharyngeal carcinoma (NPC). Recent studies demonstrate that Wnt or PORCN inhibitor, Wnt-C59 (C59), inhibits tumor growth in MMTV-WNT1 transgenic mice. The effect of Wnt-C59 (C59) in human tumors is not clear. In this study, the NPC cell lines investigated manifest heterogeneous responses to Wnt-C59 treatment. Wnt-C59 decreased tumor growth of SUNE1 cells in mice immediately following the administration of Wnt-C59. Mice injected with HNE1 cells did not develop visible tumors after the treatment of Wnt-C59, while control mice developed 100% tumors. Wnt-C59 inhibited stemness properties of NPC cells in a dosage-dependent manner by arresting sphere formation in both HNE1 and SUNE1 cells. Thus, Wnt-C59 has the potential to eradicate CSCs in human tumors. Active β-catenin and Axin2 proteins were strongly expressed in stromal cells surrounding growing tumors, confirming the importance of Wnt signaling activities in the microenvironment being driving forces for cell growth. These novel findings confirm the ability of Wnt-C59 to suppress Wnt-driven undifferentiated cell growth in NPC. Both anti-Wnt signaling and anti-CSC approaches are feasible strategies in cancer therapy. [2]
To detect long-term effects of Wnt-C59 in animals, which have not been reported yet, we investigated tumor growth of HNE1 cells. These cells require a longer latency period to form growing tumors in mice, reflecting greater intra-tumoral heterogeneity in this cell line. In the control group, surviving tumor cells, presumably CSCs, expanded in the injection sites and formed progressively growing tumors very quickly after the latency period. In contrast, injected tumor cells died and did not form visible and progressively growing tumors after the treatment of Wnt-C59 in animals. It was not possible to directly show the presence of CSCs in injected HNE1 cells, but the sphere inhibition assay demonstrates that Wnt-C59 could safely eliminate cells with stemness properties in an irreversible manner. Both in vitro and in vivo assays further confirm these findings and support the notion for the presence of CSCs in this cell line.
Targeting CSCs to suppress tumor growth is a major focus in current cancer research. The current findings demonstrate that Wnt-C59 can effectively suppress Wnt signaling and stemness properties of certain tumor cells. We provide functional evidence demonstrating the use of an anti-CSC agent for tumor growth control is a feasible approach in cancer therapy and may be broadly exploited in human tumors.[2]

C25H21N3O

379.45

379.168

C, 79.13; H, 5.58; N, 11.07; O, 4.22

1243243-89-1

Wnt-C59;1243243-89-1

57519544

White to yellow solid

1.2±0.1 g/cm3

628.3±55.0 °C at 760 mmHg

333.8±31.5 °C

0.0±1.8 mmHg at 25°C

1.648

4.19

54.88

1

3

5

29

508

0

O=C(NC1=CC=C(C2=CC=CN=C2)C=C1)CC3=CC=C(C4=CC(C)=NC=C4)C=C3

KHZOJCQBHJUJFY-UHFFFAOYSA-N

InChI=1S/C25H21N3O/c1-18-15-22(12-14-27-18)20-6-4-19(5-7-20)16-25(29)28-24-10-8-21(9-11-24)23-3-2-13-26-17-23/h2-15,17H,16H2,1H3,(H,28,29)

2-(4-(2-methylpyridin-4-yl)phenyl)-N-(4-(pyridin-3-yl)phenyl)acetamide

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 (6.59 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 (6.59 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 (6.59 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O:5 mg/mL

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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。