Molecule

Description

Cancer

Description

OSI-420 (Desmethyl Erlotinib, CP-473420) is an active metabolite of Erlotinib which is an orally active EGFR tyrosin kinase inhibitor for inhibition of human EGFR and EGFR autophosphorylation with IC50 of 2 nM and 20 nM, respectively.

Targets

IC₅₀

In Vitro

Erlotinib inhibits the proliferation of DiFi human colon tumor cells at submicromolar concentrations in cell culture and blocks cell cycle progression at the G1 phase. Erlotinib produces a marked accumulation of retinoblastoma protein in its underphosphorylated form and accumulation of p27KIP1 in DiFi cells, which may contribute to the cell cycle block. Inhibition of the EGFR also triggers apoptosis in these cells as determined by formation of DNA fragments and other criteria. ^[1] Erlotinib and OSI-420 are equipotent, and the combined concentrations of Erlotinib plus OSI-420 achieved in the CSF exceeded the IC50 (20 nM) for the EGFR tyrosine kinase inhibition in intact tumor cells. ^[2] Formation of O-desmethyl-Erlotinib (OSI-420), the major metabolite of Erlotinib in human plasma, is mediated primarily by CYP3A4, CYP3A5, and CYP1A1, to a lesser extent by CYP1A2 and CYP2D6, and to a negligible extent by CYP1B1. The estimated intrinsic clearance for OSI-420 formation is 0.09, 0.05, 0.04, 0.02, and 0.03 mL/min/nmol CYP by CYP3A4, CYP3A5, CYP2D6, CYP1A1, and CYP1A2, respectively. ^[3] In apoptosis-sensitive AML cells, Erlotinib causes a rapid (within less than 1 hour) nucleocytoplasmic translocation of nucleophosmin-1 (NPM-1) and p14^ARF. ^[4]

In Vivo

At doses of 100 mg/kg, Erlotinib completely prevents EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice and of the hepatic EGFR of the treated mice. ^[1] Erlotinib reduces the growth of xenografted human AML cells. ^[4]

Clinical Trials

Erlotinib has entered in a phase II clinical trial in the treatment of primary sclerosing cholangitis, cholangiocarcinoma and chemoprevention.

Features

OSI 420 is a major active metabolite of Erlotinib.

Kinase Assays

Nunc MaxiSorp 96-well plates are coated by incubation overnight at 37 °C with 100 μL per well of 0.25 mg/mL PGT in PBS. Excess PGT is removed by aspiration, and the plate is washed 3 times with wash buffer (0.1% Tween 20 in PBS). The kinase reaction is performed in 50 μL of 50 mM HEPES (pH 7.3), containing 125 mM sodium chloride, 24 mM magnesium chloride, 0.1 mM sodium orthovanadate, 20 mM AlP, I .6 μg/mL EGF, and 15 ng of EGFR, affinity purified from A431 cell membranes. Erlotinib in DMSO is added to give a final DMSO concentration of 2.5%. Phosphorylation is initiated by addition of ATP and proceeded for 8 minutes at room temperature, with constant shaking. The kinase reaction is terminated by aspiration of the reaction mixture and is washed 4 times with wash buffer. Phosphorylated PGT is measured by 25 minutes of incubation with 50 μL per well HRP-conjugated PY54 antiphosphotyrosine antibody, diluted to 0.2 μg/mL in blocking buffer (3% BSA and 0.05% Tween 20 in PBS). Antibody is removed by aspiration, and the plate is washed 4 times with wash buffer. The colonmetric signal is developed by addition of TMB Microwell Peroxidase Substrate, 50 μL per well, and stopped by the addition of 0.09 M sulfuric acid, 50 μL per well. Phosphotyrosine is estimated by measurement of absorbance at 450 nm. The signal for controls is typically 0.6-1.2 absorbance units, with essentially no background in wells without AlP, EGFR, or PGT and is proportional to the time of incubation for 10 minutes.