NVP-AEW541 (AEW541) is a potent inhibitor of IGF-1R with IC₅₀ of 0.15 μM, also inhibits InsR, with IC₅₀ of 0.14 μM^[1].

IC50: 0.15 ±0.036 μM (IGF-IR), 0.14±0.039 μM (InsR), 0.42±0.11 μM (Flt-3), 2±0.61 μM (PDGFR), 2.4±0.38 μM (c-Src), 3.3±1.4 μM (c-Kit)^[1]

NVP-AEW541 inhibits the in vitro kinase activity of the recombinant IGF-IR kinase domain with an IC50 value of 0.15 μM and to be equipotent against the recombinant InsR kinase domain. NVP-AEW541 is confirmed active toward the IGF-IR kinase (IC₅₀=86 nM) and shown to be selective at the cellular level. Indeed, NVP-AEW541 is found to be 27-fold more potent toward the native IGF-IR, as compared to the structurally related native InsR (IC₅₀=2.3 μM). NVP-AEW541 suppresses the IGF-I-mediated survival, soft agar and proliferation of MCF-7 cells with IC₅₀ of 0.162 μM, 0.105 μM and 1.64 μM, respectively^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Oral administration of NVP-AEW541 (20, 30, or 50 mg/kg) results in abrogation of basal and IGF-I-induced receptor, and PKB and MAPK phosphorylation in the NWT-21 tumor xenograft^[1]. NVP-AEW541 is administered by oral gavage [50 mg/kg in 0.2 mL of 25 mM L-(+)-tartaric acid] twice a day for 14 consecutive days. The control group is similarly treated with 0.2 mL carrier [25 mM L-(+)-tartaric acid] twice a day. Tumor volume and animal weight are measured thrice a week till the end of the treatment. At that time, animals are sacrificed and tumors are collected and formalin fixed for histologic and immunohistochemical analyses. In both cases, NVP-AEW541 treatment causes tumor shrinkage that reached the statistical significance (P=0.0156 and P=0.0111 for HTLA-230 and SK-N-BE2c, respectively)^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

439.55

C₂₇H₂₉N₅O

475489-16-8

Room temperature in continental US; may vary elsewhere.

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

In Vitro: 

DMSO : 50 mg/mL (113.75 mM; Need ultrasonic)

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (5.69 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (5.69 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: 2.5 mg/mL (5.69 mM); Suspended solution; Need ultrasonic

  此方案可获得 2.5 mg/mL (5.69 mM) 的均匀悬浊液，悬浊液可用于口服和腹腔注射。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (5.69 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (5.69 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. García-Echeverría C, et al. In vivo antitumor activity of NVP-AEW541-A novel, potent, and selective inhibitor of the IGF-IR kinase. Cancer Cell. 2004 Mar;5(3):231-9.

  [2]. Tanno B, et al. Down-regulation of IGF-1 receptor activity by NVP-AEW541 has an antitumor effect on neuroblastoma cells in vitro and in vivo. Clin Cancer Res. 2006, 12(22), 6772-6780.

The activities of protein kinases are assayed in the presence or absence of inhibitors by measuring the incorporation of ³³P from [γ³³P]ATP (1000 Ci/mmol) into appropriate substrates. The protein kinase assays are carried out in 96-well plates at RT under conditions described in details below andterminated by the addition of 20 μL of 125 mM EDTA. Subsequently, 30 μL (c-Abl, c-Src, IGF-1R) or 40 μL (all other kinases) of the reaction mixture are transferred onto Immobilon-PVDF pre-soaked for 5 min with methanol, rinsed with water, then soaked for 5 min with 0.5 % H₃PO₄ and mounted on vacuum manifold. After spotting all samples, vacuum is connected and each well rinsed with 200 μL 0.5 % H₃PO₄. Membranes are removed and washed 4× on a shaker with 1% H₃PO₄, once with ethanol. After drying, mounting in Packard TopCount 96- well frame, and adding of 10 μL/well of Microscint, membranes are counted. IC₅₀ values are calculated by linear regression analysis of the percentage inhibition of each compound in duplicate, at four concentrations (usually 0.01, 0.1, 1, and 10 μM).One unit of protein kinase activity is defined as 1 nmole of ³³P transferred from [γ³³P]ATP to the substrate protein per minute per mg of protein at 37C^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Between 3000 and 6000 cells/well are seeded in 96-well plates with a total media volume of 100 μL/well. Increasing concentrations of the compound are added 24 hr thereafter in quadruplicate. 72 hr later, cells are fixed by addition of 25 μL/well Glutaraldehyde (20%) and incubation for 10 min at RT. Cells are then washed 2× with 200μL/well H2O and 100μL Methylene Blue (0.05%) is added. After incubation for 10 min at RT, cells are washed 3× with 200 μL/well H₂O. 200 μL/well HCl (3%) is added, and following incubation for 30 min at RT on a plate shaker, absorbance is measured at 650 nm^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Female Harlan athymic nude mice are used NWT-21 cells are grown in DMEM (high glucose, 4.5 g/L), 10% FCS, 1% L-glutamine, and 1% Na-pyruvate. 5×10⁶ cells/animal are initially injected s.c. into the right flank of five mice. For the in vivo efficacy experiment, tumors of 500 to 800 mm³ are excised and nonnecrotic areas are cut to fragments of 3×3×3 mm. Tumor fragments are washed in sterile PBS and one tumor fragment per animal is trans- planted s.c. into the right flank. Tumor volumes (length×width ×height×π/6) and body weights are determined three times weekly. At the first day of treatment (day 0), the therapy group (NVP-AEW541) and the control group (vehicle only) are selected by stratification (8 animals per group, average tumor volume of about 95 mm³ per group). Animals are treated p.o. twice daily, 7 days/week either with NVP-AEW541 (20, 30, or 50 mg/kg; 10 mL/kg dissolved in 25 mM L(+)-tartaric acid, therapy group) or with 25 mM L(+)-tartaric acid (control group). Antitumor activity is expressed as T/C%(mean increase of tumor volumes of treated animals divided by the mean increase of tumor volumes of control animals multiplied by 100). The experiment is terminated when the mean tumor volume is about 1500 mm³.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. García-Echeverría C, et al. In vivo antitumor activity of NVP-AEW541-A novel, potent, and selective inhibitor of the IGF-IR kinase. Cancer Cell. 2004 Mar;5(3):231-9.

  [2]. Tanno B, et al. Down-regulation of IGF-1 receptor activity by NVP-AEW541 has an antitumor effect on neuroblastoma cells in vitro and in vivo. Clin Cancer Res. 2006, 12(22), 6772-6780.