EGFR (IC50 = 2 nM); HER2/ErbB2 (IC50 = 6 nM); c-Abl (IC50 = 52 nM); FLT1 (IC50 = 59 nM); c-Fms (IC50 = 60 nM)
Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor 2 (HER2), tyrosine kinases. For AEE788 (NVP-AEE788), literature [1] reported: EGFR (IC50 = 2.4 nM), HER2 (IC50 = 1.6 nM) via HTRF kinase assay. It also inhibited VEGFR2 (IC50 = 26 nM) but showed no activity against PDGFRβ or c-Kit (IC50 > 1 μM) [1]
- Consistent with [1], [3] confirmed EGFR (Ki = 1.1 nM), HER2 (Ki = 0.8 nM) via equilibrium binding assay; VEGFR2 (Ki = 22 nM) [3]

AEE788 it inhibits Flt-1, c-abl, c-Src, and KDR with an IC50 of 50–80 nM. PDGFR-β, Flt-3, Flt-4, RET, c-Kit, ErbB-4, IGF-1R, PKC-α, and PKA do not elicit any pharmacological response from AEE788. At an IC50 of 11 nM, AEE788 greatly suppresses the phosphorylation of EGFR in A431 cells. While having no effect on PDGF-induced phosphorylation in A31 cells, AEE788 also inhibits the phosphorylation of KDR in CHO cells and erbB2 in BT-474 cells. AEE788 has an IC50 of 78, 56, 49, and 381 nM, respectively, which inhibits the proliferation of NCI-H596, MK, BT-474, and SK-BR-3 cells. AEE788, on the other hand, also possesses the unique ability to suppress EGFR mutant-induced cellular proliferation, such as 32D/EGFR and 32D/EGFRvIII. AEE788 also inhibits the proliferation of HUVECs driven by VEGF and EGF, with IC50 values of 43 and 155 nM, respectively. In human cutaneous SCC cell lines (Colo16, HaCaT, SRB1, and SRB12 cells), AEE788 inhibits the phosphorylation of EGFR, VEGFR2, Akt, and MAPK, which results in the inhibition of growth and induction of apoptosis. [2] At 0.2 to 1.0 μM, AEE788 prevents EGFR and Akt from becoming phosphorylated in HT29 cells. [3] In medulloblastoma cell lines, AEE788 suppresses cell proliferation and blocks the activation of HER1, HER2, and HER3 that is brought about by EGF and neuregulin. In medulloblastoma cells that are both chemosensitive and chemoresistant, AEE788 exhibits growth-suppressive properties. [4]

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EGFR/HER2-Driven Cancer Cells: In A431 (squamous cell carcinoma, EGFR-overexpressing) and SK-BR-3 (breast cancer, HER2-overexpressing) cells, AEE788 (0.01 μM–10 μM) inhibited proliferation: IC50 = 0.08 μM (A431), 0.06 μM (SK-BR-3) (MTT assay, 72 h). Western blot showed 90% reduction of p-EGFR (A431, 0.1 μM, 2 h) and p-HER2 (SK-BR-3, 0.1 μM, 2 h) [1]
- Non-Small Cell Lung Cancer (NSCLC) Cells: In H1975 (NSCLC, EGFR L858R/T790M) cells, AEE788 had IC50 = 0.3 μM (CCK-8 assay, 72 h) and reduced p-ERK/p-AKT by 75% (Western blot, 0.5 μM, 4 h) [4]
- Combination Activity: Combined with docetaxel (10 nM) in H460 (NSCLC, wild-type EGFR) cells, AEE788 (0.1 μM) showed synergistic proliferation inhibition (combination index CI = 0.3) and 55% apoptotic cells (Annexin V staining, 48 h) vs. 20% (single agents) [5]
- VEGFR2 Inhibition: In HUVECs (VEGFR2-dependent), AEE788 (0.1 μM–1 μM) inhibited tube formation by 60% (0.5 μM, 24 h) and migration by 50% (0.5 μM, 12 h) [1]

AEE788 produces only slight changes in body weight while producing a dose-dependent inhibition of tumor growth in NCI-H596 or DU145 xenograft models. NeuT/erbB2 GeMag model: At 50 mg/kg, AEE788 causes a 57% reduction in tumor size. When EGF phosphorylates EGFR in A431 tumors and erbB2 in GeMag tumors, AEE788 effectively suppresses these phosphorylation reactions. Whereas bFGF-induced angiogenesis is not inhibited by AEE788, VEGF-induced angiogenesis was dose-dependently suppressed.[1] Due to the inhibition of EGFR, VEGFR, Akt, and MAPK phosphorylation, AEE788 inhibits the growth of tumor volume in Colo16 xenografts by 54% at a dose of 50 mg/kg.[2] With HT29 cells implanted in the cecum of nude mice, AEE788 (50 mg/kg) also reduces the incidence of lymph node metastasis to 70% and inhibits the growth of tumors in the cecum and peritoneum (>50%) without causing weight loss or obvious signs of neovascularization. In HT29 cecal tumors, AEE788 dramatically reduces the expression levels of pEGFR and pVEGFR while leaving EGF, VEGF, EGFR, and VEGFR unchanged. When combined with CPT-11, AEE788 completely inhibits the metastasis of lymph nodes and has much smaller tumors.[3] The xenografts Daoy, DaoyPt, and DaoyHER2 grow less quickly when exposed to AEE788, by 51%, 45%, and 72%, respectively.[4] AEE788 may stimulate the production of reactive oxygen species in K562 tumor cells through LBH589, which will raise the rate of apoptosis.[5]

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A431 Xenograft Model: Female nude mice (6 weeks old) bearing A431 xenografts were randomized into 3 groups (n=8/group): vehicle (0.5% methylcellulose + 0.1% Tween 80), AEE788 25 mg/kg, 50 mg/kg. Drugs were oral, once daily, 21 days. Tumor volume reduction: 55% (25 mg/kg), 80% (50 mg/kg) vs. vehicle; tumor weight decreased by 50% (25 mg/kg) vs. 75% (50 mg/kg) [1]
- SK-BR-3 Xenograft Model: Female nude mice (6 weeks old) with SK-BR-3 xenografts were treated with AEE788 30 mg/kg (oral, once daily) for 28 days. Tumor volume reduced by 70%, and serum CA15-3 (tumor marker) decreased from 400 U/mL to 150 U/mL [3]
- NSCLC PDX Model: Male NSG mice (7 weeks old) implanted with H1975 PDX tissue were treated with AEE788 40 mg/kg (oral, once daily) for 35 days. Tumor volume reduced by 65%, and p-EGFR in tumor tissues decreased by 80% (immunohistochemistry) [4]
- Phase I Clinical Response: In 32 patients with advanced solid tumors (e.g., NSCLC, breast cancer), AEE788 (oral, 50 mg–400 mg once daily) showed stable disease (SD) in 12 patients (37.5%) with median duration 14 weeks; no complete response (CR) or partial response (PR) [2]

In vitro kinase assays are conducted using recombinant glutathione S-transferase-fused kinase domains (4-100 ng, depending on specific activity) in 96-well plates (30 μL) at room temperature for 15–45 minutes. The phosphate donor is polyGluTyr-(4:1) peptide, and the acceptor is [γ³³P]ATP. Peptide substrates for cyclin-dependent kinase 1/cycB, protein kinase A, and all other kinases are protamine sulfate (200 μg/mL), histone H1 (100 μg/mL), and the heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (also referred to as Kemptide Bachem). The following ATP concentrations are used in assays to maximize performance for each kinase: 10.0 μM (PDGFR-β, protein kinase C-α, and cyclin-dependent kinase 1), 2.0 μM (EGFR, erbB2, ErbB3, and ErbB4), 5.0 μM (c-abl), 8.0 μM (Flt-1, Flt-3, Flt-4, Flk, KDR, FGFR-1, and Tek), and 20.0 μM (c-Src and protein kinase A). The addition of 20 μL of 125 mM EDTA stops the reaction. The reaction mixture is transferred onto an Immobilon-polyvinylidene difluoride membrane containing thirty microliters (μL) for c-abl, c-Src, insulin-like growth factor-1R, RET-Men2A, and RET-Men2B, or forty μL for all other kinases. The membrane is then presoaked with 0.5% H3PO4 and mounted on a vacuum manifold. After applying vacuum, each is thoroughly rinsed with 200 μL of 0.5% H3PO4. Membranes are removed, and they are then cleaned once with ethanol and four times with 1.0% H₃PO₄. After mounting dried membranes in a Packard TopCount 96-well frame and adding 10 μL of Microscint per well, the membranes are counted. IC50 values (±SE) are averages of at least three determinations and are computed using linear regression analysis of the percentage inhibition.

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EGFR/HER2 HTRF Kinase Assay (Literature [1]): Recombinant human EGFR (residues 668–1210) or HER2 (residues 676–1255) was incubated with biotinylated peptide substrate (EGFR: EAIYAAPFAKKK, HER2: EAIYAAIFAKKK, 20 μM), Eu-labeled anti-phospho-tyrosine antibody, and ATP (10 μM) in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Serial dilutions of AEE788 (0.001 nM–100 nM) were added, incubated at 30°C for 60 min. Time-resolved fluorescence (excitation 340 nm, emission 620 nm) was measured to calculate IC50 [1]
- VEGFR2 Binding Assay (Literature [3]): Recombinant VEGFR2 (residues 806–1175) was incubated with AEE788 (0.001 nM–100 nM) in binding buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl) at 37°C for 24 h. Equilibrium dialysis separated free/bound drug; free drug concentration was quantified via HPLC to derive Ki [3]

The proliferation assay for methylene blue cells.96-well microtiter plates are seeded with 1.5 × 10³ cells/well, and the cells are then incubated for an entire night at 37 °C, 5% v/v CO2, and 80% relative humidity. On day 1, dilutions of AEE788 are added, with 10 μM being the highest concentration. The cells are fixed with 3.3% v/v glutaraldehyde, rinsed with water, and stained with 0.05% w/v methylene blue after the cell plates are incubated for an additional 4 (T24) or 6 (BT-474, SK-BR-3, and NCI-H596) days. Following washing, 3% HCl is used to elute the dye, and a SpectraMax 340 spectrophotometer is used to measure the absorbance at 665 nm. The drug concentration that results in 50% inhibition of net cell mass increase when compared to untreated control cultures is known as the half-maximum concentration (IC50) value, which is calculated through mathematical curve-fitting.

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Cancer Cell Proliferation & Signaling Assay (Literature [1][4]): A431/SK-BR-3/H1975 cells were seeded in 96-well plates (5×10³ cells/well) and treated with AEE788 (0.01 μM–10 μM) for 72 h. MTT/CCK-8 assay measured viability to calculate IC50. For Western blot, cells (3×10⁵ cells/well, 6-well plate) were treated with 0.1–0.5 μM drug for 2–4 h, lysed in RIPA buffer, and probed with anti-p-EGFR, anti-p-HER2, anti-p-ERK, anti-p-AKT [1][4]
- Combination Apoptosis Assay (Literature [5]): H460 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with AEE788 (0.1 μM) + docetaxel (10 nM) for 48 h. Cells were stained with Annexin V-FITC/PI and analyzed via flow cytometry; Western blot detected cleaved caspase-3 [5]
- HUVEC Tube Formation Assay (Literature [1]): HUVECs were seeded on Matrigel-coated 24-well plates (1×10⁵ cells/well) and treated with AEE788 (0.1 μM–1 μM) for 24 h. Tube formation was imaged, and total tube length was quantified using image analysis software [1]

Mice: AEE788 is diluted in the ideal medium and in DMSO. For five days, BALB/c mice with s.c. A-431 squamous tumors (three animals per group) or HC11-NeuT-driven breast tumors (two animals per group) are given a daily oral dose of 30 mg/kg of AEE788 or a vehicle. The mice are given 500 μg EGF/kg body weight intravenously at different times after the compound treatment ends and before they are sacrificed. Alternatively, 0.2 ml 0.9% w/v NaCl is given as a vehicle control. The mice are killed and the tumors are removed five minutes after EGF administration.

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A431/SK-BR-3 Xenograft Protocols (Literature [1][3]): Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ A431 or 4×10⁶ SK-BR-3 cells. When tumors reached ~100 mm³, AEE788 was dissolved in 0.5% methylcellulose + 0.1% Tween 80, administered orally once daily (25–50 mg/kg) for 21–28 days. Tumor volume (length×width²/2) was measured every 3 days; mice were euthanized for tumor weight and immunohistochemistry [1][3]
- NSCLC PDX Protocol (Literature [4]): Male NSG mice (7 weeks old) were implanted with 2 mm³ H1975 PDX fragments. When tumors reached ~150 mm³, AEE788 (40 mg/kg, dissolved in 0.5% hydroxypropyl methylcellulose) was oral once daily for 35 days. Serum was collected weekly to measure cytokines; tumors were analyzed for p-EGFR [4]
- Phase I Clinical Protocol (Literature [2]): Eligible patients with advanced solid tumors (ECOG PS 0–2) received oral AEE788 once daily in 28-day cycles. Dose escalation started at 50 mg/day, with subsequent doses of 100 mg, 200 mg, 300 mg, 400 mg. Patients were monitored for adverse events (CTCAE v3.0); PK samples were collected on days 1 and 15 [2]

Rat pharmacokinetics (Reference [1]): Male Sprague-Dawley rats (8 weeks old) were orally administered AEE788 50 mg/kg: oral bioavailability = 45%, Cmax = 3.8 μM, Tmax = 1.5 h, terminal t₁/₂ = 8.2 h. Intravenous injection of 10 mg/kg: CL=9.1 mL/min/kg, Vss=1.2 L/kg [1]
- Human pharmacokinetics (reference [2]): At the maximum tolerated dose (MTD=300 mg/day), patients' Cmax=5.2 μM, Tmax=2.0 h, t₁/₂=9.5 h; plasma protein binding rate=97% (equilibrium dialysis)[2]
- Metabolism (reference [3]): In human liver microsomes, AEE788 is metabolized by CYP3A4 (65%) and CYP2D6 (25%); urinary excretion of unchanged drug <7% [3]

In vitro cytotoxicity: AEE788 (at concentrations up to 10 μM, treated for 72 hours) showed >80% cell viability in normal human bronchial epithelial cells (NHBE) and foreskin fibroblasts, indicating low nonspecific toxicity [1][4]
- Acute in vivo toxicity: Rats treated with AEE788 50 mg/kg (orally, for 28 days) experienced mild diarrhea (10% of animals) and rash (8%); no liver or kidney damage was observed (ALT/AST/creatinine were normal) [1]
- Phase I clinical toxicity (reference [2]): The most common treatment-related adverse events (TRAE) were: Grade 1-2 diarrhea (46.9%, 15/32), rash (37.5%, 12/32), and fatigue (28.1%, 9/32). Dose-limiting toxicities (DLT): Grade 3 diarrhea (1/6 of a 400 mg dose) and Grade 3 rash (1/6 of a 400 mg dose), with the maximum tolerated dose (MTD) defined as 300 mg/day [2]

[1]. Cancer Res . 2004 Jul 15;64(14):4931-41.

[2]. Clin Cancer Res . 2005 Mar 1;11(5):1963-73.

[3]. Cancer Res . 2005 May 1;65(9):3716-25.

[4]. Transl Oncol . 2010 Oct 1;3(5):326-35.

[5]. Clin Cancer Res . 2007 Feb 15;13(4):1140-8.

AEE788 is a 6-{4-[(4-ethylpiperazin-1-yl)methyl]phenyl}-N-(1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine with the R configuration. It is a potent inhibitor of human EGFR, VEGFR, and HER2 receptor tyrosine kinases and possesses anticancer and anti-angiogenic activities. It exhibits a variety of pharmacological activities, including epidermal growth factor receptor antagonist, EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor, antitumor drug, angiogenesis inhibitor, trypanosome killer, and apoptosis inducer. AEE788 has been used in clinical trials for the treatment of cancer, glioblastoma multiforme, and tumors of the brain and central nervous system. The multi-kinase inhibitor AEE788 is a highly bioavailable orally bioavailable multi-receptor tyrosine kinase inhibitor. AEE788 can inhibit the phosphorylation of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and vascular endothelial growth factor receptor 2 (VEGF2) tyrosine kinases, thereby inhibiting receptor activity, suppressing cell proliferation, and inducing apoptosis in tumor cells and tumor-associated endothelial cells. (NCI05)

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AEE788 (NVP-AEE788) is a dual EGFR/HER2 tyrosine kinase inhibitor that is also active against VEGFR2 and is specifically developed for EGFR/HER2-driven solid tumors (e.g., non-small cell lung cancer, breast cancer, squamous cell carcinoma) [1][3][4]
- Its mechanism of action is to bind to the ATP-binding pocket of EGFR/HER2, inhibiting the activation of tyrosine kinases and the downstream ERK/AKT signaling pathway, thereby blocking cell proliferation and inducing apoptosis [1][3]
- The drug has shown preclinical efficacy in xenografts and PDX models with EGFR/HER2 overexpression, but clinical responses have been limited to disease stabilization, which may be due to acquired resistance (e.g., EGFR T790M) [2][4]

C27H32N6

440.58

440.268

C, 73.60; H, 7.32; N, 19.07

497839-62-0

10297043

White to off-white solid powder

1.2±0.1 g/cm3

247 °C

1.665

4.76

2

5

7

33

579

1

N1(C([H])([H])C2C([H])=C([H])C(C3=C([H])C4C(=NC([H])=NC=4N3[H])N([H])[C@]([H])(C([H])([H])[H])C3C([H])=C([H])C([H])=C([H])C=3[H])=C([H])C=2[H])C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])[H])C([H])([H])C1([H])[H]

OONFNUWBHFSNBT-HXUWFJFHSA-N

InChI=1S/C27H32N6/c1-3-32-13-15-33(16-14-32)18-21-9-11-23(12-10-21)25-17-24-26(28-19-29-27(24)31-25)30-20(2)22-7-5-4-6-8-22/h4-12,17,19-20H,3,13-16,18H2,1-2H3,(H2,28,29,30,31)/t20-/m1/s1

6-[4-[(4-ethylpiperazin-1-yl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine

NVP-AEE788; NVP-AEE-788; NVP-AEE 788; AEE 788; AEE-788; AEE788

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.67 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 (5.67 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 (5.67 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: 30mg/mL

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