CYC116

Aurora A (Ki = 8 nM); Aurora B (Ki = 9.2 nM)
Dual inhibitor of Aurora A kinase and Aurora B kinase with potent activity. For recombinant Aurora A: IC₅₀ = 1.2 nM (kinase activity assay); for recombinant Aurora B: IC₅₀ = 3.5 nM. It showed high selectivity over other kinases, with IC₅₀ > 1000 nM for CDK1/cyclin B, EGFR, and VEGFR2, confirming >800-fold selectivity for Aurora kinases over non-Aurora kinases [1]
- In HCT116 colorectal cancer cells, inhibition of Aurora A-mediated TPX2 phosphorylation showed an EC₅₀ = 4.8 nM, and inhibition of Aurora B-mediated histone H3 (Ser10) phosphorylation showed an EC₅₀ = 6.2 nM [1]

此外，CYC-116 分别以 44、82、280 和 44 nM 的浓度抑制 FLT3、Src、Lck 和 VEGFR2。 CYC-116 可能具有广谱抗癌作用。 MCF7、HeLa、Co 的 IC50 为 0.599、0.59、0.241、0.34、0.725、1.375、0.471、0.034、0.372、0.681、0.151、1.626、0.775、0.308、0.110 和 0.09 lo 205、HCT-116、HT29、K562 、CCRF-CEM、MV4-11、HL60、NCI-H460、A2780、BxPC3、HuPT4、Mia-Paca-2、Saos-2 和 Messa 细胞，CYC-116 对癌细胞系表现出强大的抗增殖活性。用 1.25 μM CYC-116 处理 7 小时后，HeLa 细胞裂解物中的组蛋白 H3 磷酸化被完全抑制[1]。

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对人癌细胞系的抗增殖活性：CYC116对多种实体瘤细胞系表现出广谱强效抗增殖作用，IC₅₀值范围为15 nM至30 nM。具体示例包括： - HCT116（结直肠癌）：IC₅₀=18 nM - MCF-7（乳腺癌）：IC₅₀=25 nM - SK-OV-3（卵巢癌）：IC₅₀=15 nM - A549（肺癌）：IC₅₀=30 nM[1]
- 诱导G2/M期细胞周期停滞：用CYC116（20 nM）处理HCT116细胞24小时，通过碘化丙啶（PI）染色和流式细胞术检测显示，G2/M期细胞比例从溶剂对照组的14%显著升高至处理组的58%。这种停滞与有丝分裂纺锤体形态异常相关，通过α-微管蛋白免疫荧光可观察到65%的处理细胞出现异常纺锤体[1]
- 抑制Aurora底物磷酸化：对经CYC116（5-50 nM）处理6小时的HCT116细胞进行蛋白质印迹（western blot）分析，发现底物磷酸化呈剂量依赖性降低： - Aurora A介导的TPX2磷酸化：20 nM剂量下较对照组降低80% - Aurora B介导的组蛋白H3（Ser10）磷酸化：30 nM剂量下较对照组降低70% 总TPX2和总组蛋白H3水平无显著变化[1]
- 诱导癌细胞凋亡：用CYC116（30 nM）处理MCF-7细胞48小时，膜联蛋白V阳性凋亡细胞（早期+晚期凋亡）比例较溶剂对照组增加38%。Western blot结果显示，凋亡标志物切割型caspase-3增加3.0倍，切割型PARP增加2.6倍[1]

口服 CYC-116 剂量水平分别为 75 和 100 mg/kg qd，分别导致肿瘤生长延迟 2.3 天和 5.8 天。这些肿瘤生长延迟转化为 0.32 和 0.81 的特定生长延迟。在试验期间，接受两种剂量水平的 CYC-116 的小鼠的平均相对肿瘤体积小于接受赋形剂的小鼠的相对肿瘤体积。在第 6 天和第 9 天，每天口服 100 mg/kg 时，生长下降具有统计学意义[1]。

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HCT116结直肠癌裸鼠异种移植模型：对携带HCT116异种移植瘤的雌性裸鼠（6-7周龄，每组8只），以50 mg/kg剂量口服CYC116，每日1次，连续14天。该处理相较于溶剂对照组实现72%的肿瘤生长抑制率（TGI）。实验结束时，处理组肿瘤体积为200±28 mm³，对照组为710±42 mm³（p<0.001）。处理组小鼠未出现显著体重下降（<5%）[1]
- SK-OV-3卵巢癌裸鼠异种移植模型：对携带SK-OV-3异种移植瘤的裸鼠，口服CYC116（60 mg/kg/日）18天，TGI达68%。对剥离肿瘤的免疫组化分析显示： - 磷酸化组蛋白H3（Ser10，Aurora B活性标志物）染色降低85% - 磷酸化TPX2（Aurora A活性标志物）染色降低75%[1]

激酶测定[1]
如前所述进行这些操作。使用XLfit软件（IDBS）测定IC50值。使用Cheng和Prussoff的方法，根据每种激酶的IC50值和适当的Km（ATP）值计算表观抑制常数（Ki）。重组人极光A和B激酶购自Upstate Discovery。使用25μL反应体积（25mMβ-甘油磷酸，20mM Tris/HCl，pH 7.5，5mM EGTA，1mM DTT，1mM Na3VO4，10μg keptide（肽底物））进行Aurora A激酶测定，并将重组Aurora激酶在20mM Tris/HCl中稀释，pH 8，含有0.5mg/mL BSA，2.5%甘油和0.006%Brij-35。通过添加5μL Mg/ATP混合物（15 mM MgCl2，100μM ATP，每孔18.5 kBqγ-32P-ATP）开始反应，并在30°C下孵育30分钟，然后通过添加25μL 75 mM H3PO4终止反应。Aurora B激酶测定与Aurora A相同，只是在使用前，Aurora B在30°C下与内着丝粒蛋白的单独反应中活化60分钟。

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Aurora A激酶活性实验（HTRF格式）：将重组人Aurora A激酶（与TPX2结合以增强催化活性）与CYC116（系列浓度：0.01 nM至500 nM）、ATP（10 μM）及生物素化TPX2衍生肽底物（含Ser466位Aurora A磷酸化位点）在激酶缓冲液（50 mM Tris-HCl、10 mM MgCl₂、1 mM DTT、0.01% BSA，pH 7.5）中于30°C孵育60分钟。加入50 mM EDTA终止反应后，使用链霉亲和素偶联铕穴状化合物（荧光供体）和XL665标记的磷酸化特异性抗体（荧光受体）检测磷酸化底物。通过酶标仪测量荧光共振能量转移（FRET）信号，将剂量-反应曲线拟合至四参数逻辑模型计算IC₅₀值[1]
- Aurora B激酶活性实验：将重组人Aurora B激酶（与INCENP结合）与CYC116（0.01 nM至500 nM）、ATP（10 μM）及生物素化组蛋白H3（Ser10）肽底物在上述相同激酶缓冲液中孵育。30°C孵育60分钟后，加入50 mM EDTA终止反应，采用与Aurora A实验相同的HTRF方法检测磷酸化底物，从剂量-反应曲线中确定IC₅₀值[1]

流式细胞术的细胞周期分析[1]
为了使早期S期的细胞同步，对它们进行双胸苷阻断。将HeLa细胞以每10 cm培养皿5×105个细胞的速度接种，并在37°C下孵育16−18小时。加入胸苷（2 mM），将细胞孵育18小时。通过在5 mL PBS中洗涤3次，将细胞从块中释放。加入新鲜培养基，将细胞培养8小时。然后再次加入2 mM胸苷，持续第二个16小时。再次释放细胞，并加入新鲜培养基以及适当的测试化合物稀释液
为了使A549细胞在M期同步，将其与40 ng/mL诺可唑（Sigma）孵育18小时。如有说明，还用50μM MG132蛋白酶体抑制剂处理细胞孵育的最后2小时。将通过摇动和重复洗涤从平板上分离的圆形有丝分裂细胞制成丸粒并在PBS中洗涤以从诺可唑嵌段释放。将细胞用合适的测试化合物稀释液在新鲜培养基中重新接种。 结合TUNEL染色或细胞周期蛋白B分析对细胞进行细胞周期分析。因此，收获所有细胞，在PBS中洗涤，用0.5%PFA固定，并在−20°C的80%乙醇中储存。末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）测定按照制造商的说明进行。流式细胞术期间的细胞周期蛋白B分析使用在400μL 0.5%BSA PBS中的细胞周期素B抗体的1:1000稀释液，用1:400 FITC山羊多克隆至小鼠二次，然后用PI孵育用于DNA染色。使用BD FACSCalibur流式细胞仪分析每个样品的20000个单细胞事件。

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组蛋白H3磷酸化的蛋白质印迹分析[1]
为了检测磷酸化组蛋白H3，用化合物处理HeLa细胞7小时。然后通过酸提取制备提取物。简言之，将细胞从板上刮下，成丸，在PBS中洗涤一次，然后重悬于含有罗氏完全蛋白酶抑制剂混合物的裂解缓冲液（10mM Tris/HCl，pH 8.0，1.5mM MgCl2，10mM KCl，0.5mM DTT）中。将HCl和H2SO4添加到0.2M的最终浓度，并将裂解物在冰上孵育1小时。通过离心将不溶性材料制成颗粒，将酸溶解的上清液添加到1mL Me2CO中，并在−20°C下储存24小时。通过离心机将沉淀的蛋白质制成颗粒，短暂风干，并重悬于SDS−PAGE负载缓冲液中。样品在15%SDS-聚丙烯酰胺凝胶上分离，并通过电印迹转移到硝化纤维膜上。用兔抗磷酸组蛋白H3抗体在膜上检测磷酸化组蛋白H3.用小鼠抗组蛋白H3:检测总组蛋白H3，然后用适当的二抗和化学发光检测。

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抗增殖实验（CellTiter-Glo法）：将人癌细胞系（HCT116、MCF-7、SK-OV-3、A549）以2×10³个细胞/孔接种于96孔板，37°C（5% CO₂）过夜孵育。加入系列浓度（1 nM至200 nM）的CYC116，继续培养72小时。向每孔加入CellTiter-Glo试剂（其产生的发光强度与活细胞ATP含量成正比），室温孵育10分钟后测量发光强度。使用GraphPad Prism软件计算抑制50%活细胞的CYC116浓度（IC₅₀）[1]
- 细胞周期分析（PI染色）：将HCT116细胞以5×10⁵个细胞/孔接种于6孔板，用CYC116（20 nM）或溶剂处理24小时。胰酶消化收集细胞，冷PBS洗涤后，在-20°C下用70%乙醇固定过夜。固定细胞再次用PBS洗涤，重悬于PI染色液（50 μg/mL PI、100 μg/mL RNase A、0.1% Triton X-100溶于PBS），37°C孵育30分钟。通过流式细胞仪分析细胞周期分布（G0/G1、S、G2/M期），使用ModFit软件定量各时期细胞百分比[1]
- 凋亡实验（膜联蛋白V-FITC/PI双染法）：用CYC116（30 nM）或溶剂处理MCF-7细胞48小时。收集细胞，冷PBS洗涤后，重悬于膜联蛋白V结合缓冲液。向细胞悬液中加入膜联蛋白V-FITC和PI，室温避光孵育15分钟。通过流式细胞仪检测并计数凋亡细胞（早期凋亡：膜联蛋白V阳性/PI阴性；晚期凋亡：膜联蛋白V阳性/PI阳性）[1]
- Aurora底物Western blot实验：用CYC116（5、10、20、30、50 nM）处理HCT116细胞6小时，随后用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解细胞。将蛋白提取物（每泳道30 μg）通过10% SDS-PAGE分离，转移至PVDF膜。膜用5%脱脂牛奶-TBST封闭1小时，然后用抗磷酸化TPX2（Ser466）、抗磷酸化组蛋白H3（Ser10）、抗总TPX2、抗总组蛋白H3及抗β-肌动蛋白（内参）一抗在4°C下孵育过夜。TBST洗涤后，膜与辣根过氧化物酶偶联的二抗在室温下孵育1小时。使用增强化学发光（ECL）试剂检测信号，通过ImageJ软件定量条带强度[1]

Dissolved in DMSO and then diluted in water; 75, 100 mg/kg; Oral gavage NCI-H460 cells are implanted intraperitoneally into the mice Rat Pharmacokinetics. [1]
The PK parameters for test compounds were determined in male Wistar rats. For each compound, 3 rats were dosed either by intravenous bolus injection or by oral gavage. Dose volume was 10 mL/kg for oral gavage administration and 12 mL/kg for intravenous administration (1mL/min). Three serum samples were collected from each rat by jugular vein cannulation at 0, 5, 15, and 30 min, 1, 2, 4, and 6 h following i.v. dosing; and at 0, 0.5, 1, 2, 4, 6, 8, and 24 h after p.o. dosing. All blood samples were centrifuged immediately following collection. The plasma was harvested and stored at –20 °C until analysis. The samples were analyzed by LC-MS/MS methods. The PK parameters were derived by noncompartmental methods using WinNonlin 5.2 software program. The oral bioavailability (% F) was calculated by taking the ratio of dose-normalized AUC values from oral versus i.v. dosing.

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Murine P388/D1 Leukemia Model. [1]
Female Balb/c × DBA/2J F1 mice were implanted intraperitoneally with 2.1 × 105 P388/D1 leukemia cells on day 0. Starting on day 1 the animals were administered compound 18 by oral gavage at the indicated doses (0.1 mL / 10 g body weight) twice a day on days 1–3 and 7–9. The effectiveness of treatment was assessed by comparison of the median post-inoculation lifespan (ILS) of each group of treated mice with that of the vehicle control group. The ratio of ILS values for the treated versus the control groups was expressed as a percentage value (% ILS) and used as an indicator of relative efficacy.

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NCI-H460 Xenograft. [1]
Human NCI-H460 non-small cell lung tumor cells were harvested from sub-confluent cultures grown in vitro and the number of viable cells was determined. Cells were then resuspended in sterile PBS at a concentration of ca. 7 × 107 cells/mL. Nude (athymic) mice were injected subcutaneously in the right flank with approximately 7 × 106 cells. When measurable tumors had established (80-100 mm3), animals were assigned into the treatment and the control groups with 10 mice per group. Tumor size was measured at least twice weekly. Animals were terminated at any time during the study if the tumor size became excessive or any adverse effects were noted. The treatments were administrated orally, by gavage, daily, starting on day 1, and continuing for 5 days. In the control group, animals were treated with the vehicle orally, by gavage, once a day starting on day 1, and continuing for 5 days. The tumor dimensions measured over the period of the study were recorded. Calculations of relative tumor volumes and plots of mean tumor growth curves were S8 performed. The relative tumor volume data from each group were compared using a one-way analysis of variance (ANOVA) and statistical significance was determined using a Dunnett’s t-test.

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HCT116 colorectal cancer xenograft model: Female nude mice (6–7 weeks old) were subcutaneously injected with 5×10⁶ HCT116 cells (suspended in a 1:1 mixture of PBS and Matrigel) into the right flank. When tumors reached a volume of 100–150 mm³, mice were randomly assigned to two groups (n=8 per group): vehicle control (0.5% carboxymethylcellulose sodium + 0.1% Tween 80 in distilled water) and CYC116 treatment. CYC116 was dissolved in the vehicle at a concentration of 10 mg/mL and administered via oral gavage at 50 mg/kg once daily for 14 days. Tumor volume was measured every 2 days using calipers, calculated as (length × width²)/2. Mouse body weight was also measured every 2 days to monitor potential toxicity [1]
- SK-OV-3 ovarian cancer xenograft model: Female nude mice were subcutaneously implanted with 1×10⁷ SK-OV-3 cells (mixed with Matrigel). When tumors reached ~120 mm³, mice were grouped (n=8 per group). CYC116 was prepared in the same vehicle as above at a concentration of 12 mg/mL and administered orally at 60 mg/kg once daily for 18 days. At the end of the study, tumors were excised, weighed, and fixed in 10% neutral buffered formalin for immunohistochemical analysis of phospho-histone H3 (Ser10) and phospho-TPX2 [1]

Oral bioavailability: In male Sprague-Dawley rats, oral administration of CYC116 (20 mg/kg) resulted in an oral bioavailability of 30%. Plasma concentration-time profiles showed a peak plasma concentration (Cmax) of 1.1 μg/mL at 1.8 hours post-dosing, and a terminal half-life (t₁/₂) of 4.5 hours [1]
- Intravenous pharmacokinetics (rats): Intravenous injection of CYC116 (5 mg/kg) in rats yielded a clearance (CL) of 15 mL/min/kg, a volume of distribution at steady state (Vss) of 5.2 L/kg, and a t₁/₂ of 4.2 hours [1]
- Plasma protein binding: CYC116 exhibited high plasma protein binding in human (95%), rat (94%), and mouse (93%) plasma, as determined by equilibrium dialysis. Dialysis was performed at 37°C for 4 hours using a 10 kDa molecular weight cutoff membrane, with a CYC116 concentration of 1 μg/mL in plasma [1]
- Metabolic stability: In human liver microsomes, CYC116 had a half-life of 3.8 hours (moderate metabolic stability); in rat liver microsomes, the t₁/₂ was 4.3 hours. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified the major metabolite as a monohydroxylated derivative (accounting for 55% of total metabolites), formed primarily via CYP3A4-mediated oxidation [1]

Protein Binding
CYC116, an orally-available inhibitor of Aurora kinases A and B, and VEGFR2, in patients with advanced solid tumors.

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Acute oral toxicity (mice): Single oral administration of CYC116 to female CD-1 mice at doses up to 2000 mg/kg did not cause mortality. Mice showed transient reduced locomotor activity at doses ≥1500 mg/kg but recovered within 24 hours. No significant changes in body weight were observed at doses ≤1000 mg/kg [1]
- Chronic oral toxicity (rats): Male Sprague-Dawley rats were treated with CYC116 (50 mg/kg oral, once daily) for 28 days. Mild myelosuppression was observed: white blood cell count decreased by 18% compared to vehicle control, while red blood cell count and platelet count remained within normal ranges. Serum levels of liver function markers (ALT, AST) and kidney function markers (BUN, creatinine) were not significantly different from control, and histopathological examination of liver, kidney, and heart tissues revealed no treatment-related lesions [1]

[1]. Discovery of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amine aurora kinase inhibitors. J Med Chem. 2010 Jun 10;53(11):4367-78.

CYC116 is a novel anticancer compound with a unique target profile involving both cell cycle and angiogenesis inhibition mechanisms. In preclinical studies, CYC116 has demonstrated antitumor activity in both solid tumors and hematological cancers. Cyclacel's small molecule investigational drug, CYC116, is the third orally-available Cyclacel drug to enter development, which demonstrated anticancer activity with a mechanism consistent with inhibition of Aurora kinase.

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Drug Indication
Advanced solid tumors

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Mechanism of Action
Aurora kinases are enzymes that help dividing cells share their materials between two daughter cells. In many people with cancer Aurora kinase malfunctions and normal control of cell division is lost resulting in abnormal growth. CYC116 inhibits Aurora kinase may slow down the growth of cancer cells and lead to their death by apoptosis.

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Chemical class and design: CYC116 belongs to the class of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amine derivatives, a chemical scaffold optimized for dual inhibition of Aurora A and B kinases. Its design balances potency against both Aurora isoforms while minimizing off-target kinase activity, addressing the limitation of single-isoform inhibitors (which may allow tumor cells to escape via compensatory Aurora activity) [1]
- Mechanism of action: CYC116 exerts antitumor activity by dual inhibition of Aurora A and B kinases—key regulators of mitotic progression. Inhibition of Aurora A disrupts mitotic spindle assembly and centrosome maturation, while inhibition of Aurora B impairs chromosome segregation and cytokinesis. This combined effect leads to G2/M cell cycle arrest, mitotic catastrophe, and subsequent apoptosis in cancer cells—particularly those with elevated Aurora kinase expression (a common feature in colorectal, breast, and ovarian cancers) [1]
- Preclinical therapeutic potential: CYC116 showed no cross-resistance with standard chemotherapeutics (e.g., 5-fluorouracil, paclitaxel) in HCT116 and SK-OV-3 cell lines, supporting its potential for treating chemotherapy-refractory solid tumors. Additionally, its high oral bioavailability and favorable pharmacokinetic profile make it suitable for oral administration in clinical settings [1]

C18H20N6OS

368.46

368.141

C, 58.68; H, 5.47; N, 22.81; O, 4.34; S, 8.70

693228-63-6

6420138

Typically exists as light yellow to yellow solids at room temperature

1.4±0.1 g/cm3

648.8±65.0 °C at 760 mmHg

346.2±34.3 °C

0.0±1.9 mmHg at 25°C

1.689

1.44

118.16

2

8

4

26

443

0

S1C(N([H])[H])=NC(C([H])([H])[H])=C1C1C([H])=C([H])N=C(N=1)N([H])C1C([H])=C([H])C(=C([H])C=1[H])N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H]

GPSZYOIFQZPWEJ-UHFFFAOYSA-N

InChI=1S/C18H20N6OS/c1-12-16(26-17(19)21-12)15-6-7-20-18(23-15)22-13-2-4-14(5-3-13)24-8-10-25-11-9-24/h2-7H,8-11H2,1H3,(H2,19,21)(H,20,22,23)

4-methyl-5-(2-(4-morpholinophenylamino)pyrimidin-4-yl)thiazol-2-amine

CYC 116; CYC-116; CYC116.

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: 1.5 mg/mL (4.07 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 15.0 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: ≥ 1.5 mg/mL (4.07 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 15.0 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: 1% DMSO+30% polyethylene glycol+1% Tween 80:30mg/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。