The A431 epidermoid carcinoma and the H125 NSCLC tumor lines were maintained by serial passage in nude mice (NCr nu/nu). Nude mice were also used as tumor hosts for anticancer agent evaluations against these tumor models. In each experiment for anticancer activity evaluation, test mice weighing 18-22 g were randomized and implanted with tumor fragments in the region of the right axilla on day 0. Treatment with PD168393 was initiated when tumors reached approximately 100-150 mg in mass. PD168393 was administered itraperitoneally twice a day at dose 47 mg/kg, or orally at 150 mg/kg twice a day.

Enzyme assays for IC50 determinations were performed in 96-well filter plates. The total volume was 0.1 mL containing 20 mM Hepes, pH 7.4, 50 mM sodium vanadate, 40 mM magnesium chloride, 10 mM adenosine triphosphate (ATP) containing 0.5 mCi of [32P]ATP, 20 mg of poly(glutamic acid)/tyrosine, 20 ng of EGFR tyrosine kinase (for a calculated final concentration of 1.18 nM), and appropriate dilutions of inhibitor. All components except the ATP were added to the well, and the plate was incubated with shaking for 10 min at 25 °C. The reaction was started by adding [32P]ATP, and the plate was incubated at 25 °C for 10 min. The reaction was terminated by addition of 0.1 mL of 20% trichloroacetic acid (TCA). The plate was kept at 4 °C for at least 15 min to allow the substrate to precipitate. The wells were then washed five times with 0.2 mL of 10% TCA, and 32P incorporation was determined with a Wallac beta plate counter. PD-168393 had IC50 of 0.7 nM against EGFR kinase.