IGF-IR (IC50 = 0.15 ±0.036 μM); InsR (IC50 = 0.14±0.039 μM); Flt-3 (IC50 = 0.42±0.11 μM); PDGFR (IC50 = 2±0.61 μM); c-Src (IC50 = 2.4±0.38 μM); c-Kit (IC50 = 3.3±1.4 μM)
Insulin-like growth factor I receptor (IGF-IR) [2]

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

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In a panel of 10 neuroblastoma cell lines positive for IGF-IR expression, NVP-AEW541 inhibited in vitro proliferation in a submicromolar/micromolar range of concentrations (IC50 values: GI-CA-N 6.80 μM, SH-EP 3.00 μM, HTLA-230 0.50 μM, SK-N-BE2c 1.10 μM, SK-N-BE 2 3.00 μM, SY-SY (N) 2.40 μM, LAN-5 0.40 μM, KCNR 0.40 μM, RN-GA 1.30 μM, SK-N-AS 2.80 μM). [2]
NVP-AEW541 inhibited IGF-II-mediated stimulation of IGF-IR and Akt. Cells were serum-starved for 18 hours, then treated for 10 minutes with recombinant IGF-II (100 ng/mL) with or without a 2-hour pretreatment with NVP-AEW541 at concentrations 20% greater than the IC50 of each cell line. Pretreatment dramatically reduced phosphorylation of IGF-IR and caused a reduction of phosphorylation of Akt in all cell lines tested, but not of Erk1 and Erk2. [2]
NVP-AEW541 induced apoptosis in vitro. Neuroblastoma cells treated with NVP-AEW541 were pyknotic and showed membrane blebbing. Flow cytometry showed an increase in the hypodiploid fraction in all cell lines tested (e.g., KCNR: untreated 1.14%, 72h treatment 3.18%; GI-CA-N: untreated 0.71%, 72h treatment 4.92%; SK-N-BE2c: untreated 2.62%, 72h treatment 26.73%; SY-SY(N): untreated 4.85%, 72h treatment 46.17%) that proceeded in parallel with the depletion of S and G2-M compartments. In some cases (GI-CA-N, HTLA-230, and KCNR), a block in G0-G1 was also detected. [2]
NVP-AEW541 treatment induced activation of caspase-3/7 (relative luminometric units, RLU: KCNR untreated 563 ± 23, treated 1,347 ± 31; GI-CA-N untreated 264 ± 13, treated 53,266 ± 1,173; HTLA-230 untreated 363 ± 24, treated 545 ± 16; SK-N-BE2c untreated 698 ± 3, treated 1,007 ± 29; SY-SY (N) untreated 504 ± 30, treated 19,152 ± 1,273). [2]
NVP-AEW541 inhibited the ability of neuroblastoma cells to form clones in semisolid agar (e.g., KCNR: 0.5 μM 42.6% inhibition, p NS; GI-CA-N: 0.5 μM 58.8% inhibition, p<0.01; HTLA-230: 0.5 μM 96.2% inhibition, p<0.001; SK-N-BE2c: 0.5 μM 38.8% inhibition, p<0.001; SY-5Y(N): 0.5 μM 27.7% inhibition, p<0.05). [2]
Quantitative real-time PCR detected a significant down-regulation of mRNA for vascular endothelial growth factor (VEGF) caused by NVP-AEW541 in vitro. In HTLA-230 cells, relative VEGF mRNA expression was reduced to approximately 25% of untreated control (p<0.001). In SK-N-BE2c cells, it was reduced to approximately 40-45% of untreated control (p<0.01 and p<0.001). [2]
NVP-AEW541 significantly decreased invasion in Matrigel-coated chambers in all cell lines tested (e.g., KCNR: untreated ~100% invasion, 0.5 μM ~70%, 2.0 μM ~40%, 8.0 μM ~10%; GI-CA-N: untreated ~100% invasion, 0.5 μM ~60%, 2.0 μM ~20%, 8.0 μM ~10%; HTLA-230: untreated ~100% invasion, 0.5 μM ~90%, 2.0 μM ~60%, 8.0 μM ~20%). [2]

NVP-AEW541 (20, 30, or 50 mg/kg) administered orally causes the NWT-21 tumor xenograft to lose its basal and IGF-I-induced receptors, as well as its phosphorylation of PKB and MAPK[1]. Using oral gavage, NVP-AEW541 is given twice a day for 14 days in a row at a dose of 50 mg/kg in 0.2 mL of 25 mM L-(+)-tartaric acid. A 0.2 mL carrier [25 mM L-(+)-tartaric acid] administered twice a day is the same treatment given to the control group. Measurements of tumor volume and animal weight are made three times a week until the course of treatment is completed. Tumors are gathered, formalin-fixed, and analyzed histologically and immunohistochemically at that point after the animals have been sacrificed. Tissue shrinkage resulting from NVP-AEW541 treatment reaches statistical significance in both cases (P=0.0156 and P=0.0111 for HTLA-230 and SK-N-BE2c, respectively)[2].

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Oral administration of NVP-AEW541 (50 mg/kg twice daily) inhibited tumor growth of neuroblastoma xenografts (HTLA-230 and SK-N-BE2c) in nude mice. Tumor volumes in treated animals were significantly smaller than controls (HTLA-230: P = 0.0156; SK-N-BE2c: P = 0.0111). [2]
Analysis of tumors from NVP-AEW541-treated animals revealed a marked apoptotic pattern (many pyknotic cells with frequent presence of micronuclei) and a decrease in microvascularization (scant or no microvascularization) compared with controls (highly cellular and with a rich network of blood microvessels). [2]
Immunohistochemical analysis using a phospho-IGF-IR-specific antibody showed strong membrane-associated staining in tumors from controls, whereas tumors from NVP-AEW541-treated animals were mostly negative (showing strong inhibition of IGF-IR phosphorylation). [2]
Quantitative real-time PCR from RNA of NVP-AEW541-treated tumors (SK-N-BE2c xenografts) showed significantly lower amounts of VEGF mRNA compared to controls (relative VEGF mRNA expression reduced to approximately 15% of untreated control, p<0.001). [2]
In a pseudometastatic model (tail vein injection of HTLA-230 cells in SCID mice), NVP-AEW541 (50 mg/kg twice daily for 14 days) substantially reduced tumor cell burden in target organs (adrenals, kidneys, liver, and lungs) compared with controls. A significant reduction of the involved areas in adrenals and kidneys was clearly detectable in treated animals. The number and size of the metastatic foci were strongly diminished in treated animals. [2]

The incorporation of ³³P from [γ³³P]ATP (1000 Ci/mmol) into suitable substrates is used to measure the activities of protein kinases in the presence or absence of inhibitors. The protein kinase assays are conducted in 96-well plates at room temperature (RT) using the parameters outlined below. The assays are concluded with the addition of 20 μL of 125 mM EDTA. The reaction mixture is then transferred onto Immobilon-PVDF, which has been pre-soaked for five minutes with methanol, rinsed with water, and soaked for five minutes with 0.5% H₃PO₄ before being mounted on a vacuum manifold in 30 μL (c-Abl, c-Src, and IGF-1R) or 40 μL (all other kinases). Once every sample has been spotted, the vacuum is connected, and 200 μL of 0.5% H₃PO₄ is rinsed through each well. Membranes are taken out and given a 4× shake on 1% H₃PO₄ after being once again cleaned with ethanol. Membranes are counted after drying, mounting in a Packard TopCount 96-well frame, and adding 10 μL of Microscint per well. By performing a linear regression analysis on the percentage inhibition of each compound in duplicate at four different concentrations (typically 0.01, 0.1, 1, and 10 μM), IC50 values are determined. A single unit of protein kinase activity is measured as one nanomole of ³³P transferred per minute per milligram of protein at 37C from [γ³³P]ATP to the substrate protein[1].

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In vitro kinase assays were performed with purified recombinant kinase domains or purified kinases and synthetic peptide substrates. The compound was dissolved in DMSO (10 mM) and dilutions were made fresh in DMSO/water 1:1. The final DMSO concentration in enzyme assays was <0.5%. Kinase activity was assayed by measuring the incorporation of 33P from [γ-33P] ATP into appropriate substrates in 96-well plates at room temperature. The reaction was terminated by adding EDTA. An aliquot of the reaction mixture was transferred onto PVDF membrane pre-soaked with methanol, rinsed with water, then soaked with 0.5% H3PO4. After washing, the membranes were dried and counted. IC50 values were calculated by linear regression analysis of percentage inhibition at four concentrations (usually 0.01, 0.1, 1, and 10 μM). [1]
For specific kinases: PKC-α was assayed in a buffer containing Tris-HCl, Mg(NO3)2, protamine sulfate, ATP, and [γ-33P]ATP. Cdk1/cycB was assayed with histone H1 as substrate in a buffer containing β-glycerophosphate, nitrophenylphosphate, MOPS, EGTA, MgCl2, DTT, Na3VO4, ATP, and [γ-33P]ATP. PKA was assayed using Kemptide as peptide substrate in a buffer containing MES-NaOH, Mg(OAc)2, NaCl, ATP, BSA, and [γ-33P]ATP. c-Raf-1 was assayed with His-tagged IκB as substrate. c-Abl and c-Src were assayed using poly-AEKY or poly(Glu,Tyr) as substrate. IGF-IR and Ins-R kinase assays used the cytoplasmic domains expressed as GST fusions, with poly(Glu,Tyr) as substrate. KDR, Fit-1, Tek, PDGFR-β, FGFR-1, c-Kit, c-Met, Flt-3, and Flt-4 kinase assays were performed using GST-fusion proteins of their cytoplasmic domains, with poly(Glu,Tyr) as substrate in buffers containing Tris-HCl, MnCl2, MgCl2, DTT, Na3VO4, and ATP. [1]

In 96-well plates, 3000–6000 cells are seeded per well, with a 100 μL media volume per well overall. Twenty-four hours later, the compound is added in quadruplicate at increasing concentrations. After 72 hours, cells are fixed by adding 25 μL/well of 20% glutaraldehyde and letting them sit at room temperature for 10 minutes. After two rounds of washing with 200μL/well H2O, 100μL of Methylene Blue (0.05%) is added to the cells. Following a 10-minute RT incubation period, cells are triple-washed using 200 μL/well H₂O. After adding 200 μL/well of HCl (3%) and incubating on a plate shaker for 30 minutes at room temperature, absorbance at 650 nm is measured[1].

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Cell growth was evaluated by seeding cells in triplicate in complete medium. After 18 hours, fresh medium or medium containing NVP-AEW541 (0.5, 2.0, and 8.0 μmol/L) was added. Cell counts were carried out after 72 hours. IC50 values were calculated by regression analysis. [2]
Anchorage-independent growth was assessed in semisolid agar. Base agar (0.5% agar, 1× RPMI 1640, and 10% FCS) was added and let solidify. An equal volume of top agar (0.35% agar, 1× RPMI 1640, and 10% FCS) containing cells at 10^3 cells/cm^2 was added. NVP-AEW541 (0.5, 2.0, and 8.0 μmol/L) was included where needed. Plates were incubated for 11 days and stained with crystal violet. Single cells (≤20 cells) and clones (>20 cells) were scored. [2]
Cell cycle analysis and evaluation of the hypodiploid peak were carried out using propidium iodide staining (50 μg/mL) followed by flow cytometric analysis. Cells were treated with NVP-AEW541 at a concentration 20% greater than the calculated IC50 of each cell line for 24, 48, and 72 hours. [2]
Caspase-3 and caspase-7 activities were measured using a luminescent assay based on detection of a luminescent caspase-3/7 substrate following caspase cleavage. 1.5 × 10^4 cells per well were seeded in RPMI 1640 with 10% FCS. NVP-AEW541 at concentrations 20% above the calculated IC50 of each cell line was added where needed. [2]
For protein analysis, cellular proteins were extracted, separated on SDS-polyacrylamide gels, and Western blot analyses were carried out. Antibodies used included anti-IGFIRβ, anti-phospho-IGF-IR (Tyr1131)/insulin receptor (Tyr1146), anti-Akt, anti-phospho-Akt (Ser473), anti-p44/42 MAPK, anti-phospho-p44/42 MAPK (Thr202/Tyr204), anti-cleaved-caspase-9 (Asp315; human specific), anti-Bim, anti-Bax, and anti-β-actin. For testing IGF-II-mediated activation, cells were serum-starved for 18 hours, then treated for 10 minutes with recombinant IGF-II (100 ng/mL) with or without a 2-hour pretreatment with NVP-AEW541 at concentrations 20% greater than the IC50. [2]
For real-time PCR, total RNA was prepared and cleaned from DNA contaminants. 500 ng was reverse transcribed. Real-time PCR was carried out using TAQMAN technology and Assays-On-Demand kit for human VEGF. TAQMAN predeveloped kit for human β-actin was used to normalize. Reactions were run in triplicate in two independent experiments. [2]
For invasion assay, cells were pretreated with scalar concentrations (0.5, 2.0, and 8.0 μmol/L) of NVP-AEW541 for 18 hours and replated in Matrigel-coated invasion chambers. After 24 hours, the number of cells that migrated into the lower part of the chamber was counted. [2]

Mice: The Harlan athymic female nude mice are utilized. DMEM (high glucose, 4.5 g/L), 10% FCS, 1% L-glutamine, and 1% Na-pyruvate are used to cultivate NWT-21 cells. Five mice are first given a subcutaneous injection of 5×10⁶ cells/animal into their right flank. Tumors ranging in size from 500 to 800 mm³ are removed, and non-crotic regions are divided into 3 x 3 x 3 mm pieces for the in vivo effectiveness trial. One tumor fragment per animal is transplanted s.c. into the right flank after being cleaned in sterile PBS. Body weights and tumor volumes (length × width × height ×π/6) are calculated three times a week. The therapy group (NVP-AEW541) and the control group (vehicle only) are chosen by stratification on the first day of treatment (day 0) (8 animals per group, average tumor volume of about 95 mm³ per group). The animals receive oral treatment twice a day, seven days a week, using either 25 mM L(+)-tartaric acid (control group) or NVP-AEW541 (20, 30, or 50 mg/kg; 10 mL/kg dissolved in 25 mM L(+)-tartaric acid, therapy group). T/C%, or the mean increase in tumor volumes of treated animals divided by the mean increase in tumor volumes of control animals multiplied by 100, is the expression used to express antitumor activity. When the mean tumor volume approaches 1500 mm3, the experiment comes to an end.

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For xenograft studies, 4-week-old male athymic nude mice were injected subcutaneously with 20 million cells (HTLA-230 or SK-N-BE2c) per animal into the right flank. When tumor volume reached ~100 mm^3 (7-10 days), animals were treated by oral gavage twice daily, 7 days/week either with NVP-AEW541 (50 mg/kg) dissolved in 0.2 mL of 25 mmol/L L-(+)-tartaric acid (treated group) or with 0.2 mL of 25 mmol/L L-(+)-tartaric acid (control group). The experiments concluded 14 days after start of treatment. Tumor volumes (length × width × height × π/6) and body weights were determined three times weekly. [2]
For pseudometastatic studies, 4-week-old CB-17/IcrHsd severe combined immunodeficiency (SCID) mice were injected in the tail vein with 3 × 10^6 HTLA-230 cells. Oral NVP-AEW541 treatment was started the same day (50 mg/kg dissolved in 0.2 mL of 25 mmol/L L-(+)-tartaric acid for treated group or with 0.2 mL of 25 mmol/L L-(+)-tartaric acid for control group twice a day). Treatment was given for 14 days. All animals were sacrificed when the first death occurred (35th day). Autopsy was done for macroscopic assessment and organs were collected for histologic analysis. [2]

NVP-AEW541 is orally bioavailable. [1]

Signs of systemic toxicity (lethargy, disturbances in feeding behavior) were not observed by daily monitoring during treatment of nude mice with NVP-AEW541 (50 mg/kg twice daily for 14 days). Animal weight was not significantly different in treated and untreated animals. [2]

[1]. 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]. 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.

Neuroblastoma is an IGF-sensitive tumor. Suppression of IGF-II/IGF-IR signaling through the use of antisense expression vectors or blocking antibodies has been shown to be effective in neuroblastoma preclinical models. However, difficulties in inhibiting selectively IGF-IR without simultaneously interfering with the insulin receptor have precluded further exploitation. NVP-AEW541 is capable of discriminating between IGF-IR and insulin receptor. [2]
Analysis of IGF-IR expression in a series of 43 neuroblastoma primary tumors revealed IGF-IR positivity (detectable levels defined as any signal visible by ethidium bromide staining after 30 cycles of PCR amplification) in 86% of cases (37 of 43). [2]
NVP-AEW541 treatment caused inhibition of IGF-IR activation in vivo, as confirmed by immunohistochemistry showing a strong reduction of the active (phosphorylated) form of the receptor in tumors from treated animals. [2]
The drug’s antitumor effect involves not only inhibiting cell growth and stimulating apoptosis, but also interfering with proangiogenic signals (VEGF) released by neuroblastoma cells. [2]
NVP-AEW541 is a pyrrole pyrimidine derivative. For in vitro studies, it was dissolved in DMSO (10 mmol/L). For in vivo applications, it was dissolved in 25 mmol/L L-(+)-tartaric acid (5 mg/mL). [2]

C27H29N5O

439.55

439.237

C, 73.78; H, 6.65; N, 15.93; O, 3.64

475489-16-8

475488-34-7; 475489-16-8 (cis-isomer free base);1618643-96-1 (HCl); 2320261-63-8 (cis-isomer HCl)

11476171

white solid powder

1.3±0.1 g/cm3

669.0±55.0 °C at 760 mmHg

145℃

358.4±31.5 °C

0.0±2.0 mmHg at 25°C

1.710

4.66

1

5

7

33

632

0

NC1=C2C(N([C@@H]3C[C@@H](C3)CN4CCC4)C=C2C5=CC=CC(OCC6=CC=CC=C6)=C5)=NC=N1

AECDBHGVIIRMOI-UHFFFAOYSA-N

InChI=1S/C27H29N5O/c28-26-25-24(21-8-4-9-23(14-21)33-17-19-6-2-1-3-7-19)16-32(27(25)30-18-29-26)22-12-20(13-22)15-31-10-5-11-31/h1-4,6-9,14,16,18,20,22H,5,10-13,15,17H2,(H2,28,29,30)

7-[3-(azetidin-1-ylmethyl)cyclobutyl]-5-(3-phenylmethoxyphenyl)pyrrolo[2,3-d]pyrimidin-4-amine

AEW541 cis-isomer; NVP-AEW541; NVP-AEW 541; NVP-AEW-541; AEW-541; AEW 541; AEW541

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: 50~88 mg/mL(113.8~200.2 mM)
Ethanol: ~24 mg/mL (~54.6 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.69 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.69 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 20 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)