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Research Area
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Description
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Cancer |
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Biological Activity
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Description
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Pelitinib (EKB-569) is a potent EGFR inhibitor with IC50 of 38.5 nM. |
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Targets
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EGFR |
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IC50 |
38.5 nM [1] |
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In Vitro
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Pelitinib displays much higher inhibitory activity against EGFR, compared with the closely related c-erbB-2, as well as other kinases such as Src, Cdk4, c-Met, Raf, and MEK/ERK, with IC50 ranging from 282 nM for Src to >20 μM for Cdk4. Consistently, Pelitinib treatment significantly inhibits the autophosphorylation of EGFR but not c-Met in A431 cells. [1] Pelitinib potently inhibits the proliferation of normal human keratinocytes (NHEK), as well as A431 and MDA-468 tumor cells with IC50 of 61 nM, 125 nM, and 260 nM, respectively, while displaying little activity against MCF-7 cells with IC50 of 3.6 μM. Pelitinib inhibits EGF-induced phosphorylation of EGFR in A431 and NHEK cells with IC50 of 20-80 nM, as well as the phosphorylation of STAT3 with IC50 of 30-70 nM. Pelitinib at 75-500 nM also specifically inhibits the activation of AKT and ERK1/2, without affecting NF-κB pathway. In NHEK cells, Pelitinib also potently inhibits TGF-α mediated EGFR activation with IC50 of 56 nM, as well as activation of STAT3 and ERK1/2 with IC50 of 60 nM and 62 nM, respectively. [2] |
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In Vivo
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A single oral dose of 10 mg/kg Pelitinib potently inhibits the EGFR phosphorylation in A431 xenografts with over-expressed EGFR, by 90% within 1 hour, and by >50% after 24 hours. Administration of Pelitinib at 20 mg/kg/day inhibits tumorigenesis in APCMin/+ mice by 87%, equivalent to the effect of used with 2 times doses of EKI-785 (40 mg/kg/day), consistent with greater in vivo potency. [1] Pelitinib selectively inhibits EGFR signaling in airway epithelial cells in vivo. In the mouse model of airway epithelial remodeling that is inducible by viral infection and features a delayed but permanent switch to goblet cell metaplasia, Pelitinib treatment at 20 mg/kg/day corrects all 3 aspects of epithelial remodeling, by completely blocking the increase of ciliated cells and decrease of Clara cells, and significantly inhibiting the metaplasia of goblet cells. [3] |
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Clinical Trials
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A Phase II study of Pelitinib in subjects with advanced non-small cell lung cancer has been completed. |
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Features
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More improved than EKI-785. |
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Combination Therapy
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Description
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Although, sulindac treatment alone at 5 mg/kg/day provides little effect on the protection from polyp formation in APCMin/+ mice, Pelitinib (20 mg/kg) in combination of sulindac (5 mg/kg) remarkably prevents the development of intestinal neoplasia by >95%, and 47% of APCMin/+ mice treated with the combination therapy have no tumors at all. [1] A Phase I study of Pelitinib in combination with CCI-779 in patients with advanced solid tumors has been completed. |
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Protocol
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Kinase Assay
[1]
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| Autophosphorylation of EGFR in cells |
For experiments using cells in culture, A431 cells are treated with various concentrations of Pelitinib for 2.75 hours before co-incubation with 100 ng/mL EGF for 0.25 hour. Cells are washed twice with cold phosphate-buffered saline (PBS) before adding to lysis buffer (10 mM Tris, pH 7.5, 5 mM ethylenediamine tetra-acetic acid (EDTA), 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycolate, 0.1 % SDS, 1 mM PMSF, 10 mg/mL pepstatin A, 10 mg/mL leupeptin, 20 KIU/mL aprotinin, 2 mM sodium orthovanadate, and 100 mM sodium fluoride) for 20 minutes on ice, before immunoprecipitation and SDS-PAGE-immunoblotting. For immunoprecipitation, cultured cells are placed in cold lysis buffer and immediately homogenized on ice with a polytron with several pulses. The homogenate is first centrifuged at 2500 rpm (20 minutes, 4 °C) and then again at 14,000 rpm in a microcentrifuge (10 minutes, 4 °C). Supernatants (1000 μg protein) are incubated for 2 hours at 4 °C with 15 mL of EGFR polyclonal antibody. After 2 hours, 50 μL of protein G plus/protein A agarose beads is added and incubated with constant rotation for 2 hours at 4 °C. After washing with lysis buffer, beads are boiled for 2 minutes in Laemmli sample buffer. Proteins are then resolved by SDS-PAGE, transferred to immobilon membrane and probed overnight with an anti-phosphotyrosine antibody conjugated with horseradish peroxidase (HRP). Membranes are developed using the ECL reagent. Total EGFR protein is determined by stripping membranes and re-probing with receptor-specific antibodies. Quantitation of bands is done by densitometry, using ImageQuant software with a Molecular Dynamics laser transmittance scanner. |
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Cell Assay
[2]
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| Cell Lines |
NHEK, A431, MCF-7, and MDA-468 |
| Concentrations |
Dissolved in DMSO, final concentrations ~10 μM |
| Incubation Time |
5 days |
| Methods |
Cells are seeded in 96-well dishes, and after 2 hours, Pelitinib is added and incubated for 5 days. After incubation, the medium is removed from each well and fresh medium (150 μL) + 1 mg/mL MTT solution (50 μL) is added. After incubation for 2 hours at 37 °C, the medium is replaced with 150 μL DMSO, and absorbance at 540 nm in each well is determined. The IC50 is calculated by linear regression of the data. |
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Animal Study
[1]
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| Animal Models |
Athymic nu/nu female mice bearing subcutaneous A431 tumors, or APCMin/+ male mice, a murine model of human familial adenomatous polyposis (FAP) |
| Formulation |
Dissolved in pH 2.0 water |
| Doses |
10, or 20 mg/kg/day |
| Administration |
Oral gavage |
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References |
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[1] Torrance CJ, et al. Nat Med, 2000, 6(9), 1024-1028.
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[2] Nunes M, et al. Mol Cancer Ther, 2004, 3(1), 21-27.
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[3] Tyner JW, et al. J Clin Invest, 2006, 116(2), 309-321.
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