Molecule

Description

Cancer

Description

Erlotinib HCl(CP-358774, OSI-774, NSC 718781, Tarceva) is a HER1/EGFR inhibitor with IC50 of 2 nM.

Targets

IC₅₀

In Vitro

Erlotinib HCl potently inhibits EGFR autophosphorylation in HNS human head and neck tumor cells, a cell line that expresses high levels of EGFR. At the higher concentrations of Erlotinib HCl, the extent of autophosphorylation after EGFR stimulation is lower than that in the controls without EGFR. ^[1] Erlotinib HCl has no effect on the proliferation of SBC-5 cells. On the contrary, Erlotinib HCl significantly inhibits the proliferation of PC-9 cells, which harbor a deletion mutation on exon 19 of the EGFR gene, in a dose-dependent fashion. Cell migration of SBC-5 cells is also not affected by Erlotinib HCl treatment. ^[2] Erlotinib HCl inhibits the growth of HPAC cells and Capan-1 cells with IC50 of 1.1 μM and 3 μM, respectively. The effects of Erlotinib HCl in combination with gemcitabine are considered additive in KRAS-mutated pancreatic cancer cells. Ten micromolar of Erlotinib HCl inhibits EGFR phospho-rylation at the Y845 (Src-dependent phosphorylation) and Y1068 (auto-phosphorylation) sites. ^[3] Erlotinib HCl can inhibit extracellular signal-regulated kinase, Akt, and S6 only in cell lines that are the most sensitive. However, combination with Erlotinib HCl could down-modulate rapamycin-stimulated Akt activity. Combination of Erlotinib HCl and rapamycin produced a synergistic effect on cell growth inhibition. ^[4]

In Vivo

Erlotinib HCl monotherapy dose-dependently inhibits tumor growth in the H460a tumor model, correlating with circulating levels of drug. ^[5]

Clinical Trials

Erlotinib HCl plus BKM120 has entered in a phase II clinical trial in the treatment of non small cell lung cancer.

Features

Description

In cell lines derived from a variety of tissue types (non–small-cell lung, pancreatic, colon, and breast), combination of Erlotinib HCl and Rapamycin results in a synergistic inhibition effect on cell growth by inhibition of both S6 and Akt. In a Calu6 xenograft model, Rapamycin and Erlotinib HCl combination shows synergistic effects in inhibiting tumor growth. ^[6] In NCI-H720 AC and SHP-77 LCNEC cells, Erlotinib HCl combined with Everolimus produces synergistic effects on induction of caspase-3-dependent apoptosis and leads to synergistic inhibition of EGFR/AKT/mTOR pathway. ^[7]

Kinase assays

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 washing 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 μM ATP, 1.6 μg/mL EGF, and 15 ng of EGFR, affinity purified from A431 cell membranes. Erlotinib HCl 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 washing 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 washing buffer. The colonmetric signal is developed by addition of TMB Microwell Peroxidase Substrate, 50μL 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 back ground in wells without AlP, EGFR, or PGT and is proportional to the time of incubation for 10 minutes.

Cell Lines

HPAC cell lines and Capan-1 cells

Concentrations

0.01-10 μM

Incubation Time

4 days or 6 days

Methods

To evaluate cell proliferation inhibition, the MTT assay is performed. Cells are precultured overnight at 37 oC in 96-well clear plates, and Erlotinib HCl and gemcitabine are added alone or in combination. After treatment for 4 (HPAC cells) or 6 days (Capan-1 cells) at 37 oC, 10 mL of MTT is added to each well and incubated for 2-5 hours at 37 oC. The optical density of each well is measured at 450 and 600 nm with a Benchmark Plus microplate reader. Each experiment is performed in duplicate or triplicate for each drug concentration and is independently performed two or three times. The percentage of cell proliferation is calculated as: [(mean absorbance of drug-treated wells mean absorbance of cell-free wells)/(mean absorbance of vehicle wells - mean absorbance of cell-free wells)] × 100. The effects of the Erlotinib HCl and gemcitabine combination are evaluated using a combination index (CI) interpreted as: <1.0, synergistic;="" 1.0,="" additive="" and="">1.0, antagonistic. The CI for each fraction-affected value representing the percentage of proliferation inhibited by a drug is calculated using the Chou-Talalay method [the isobologram equation is used mutually non-exclusive (α =1)].

Animal Models

Male 5-week-old BALB-nu/nu with HPAC cells

Formulation

6% Captisol

Doses

50 mg/kg

Administration

Oral administration