Several human malignancies, including non-small-cell lung cancer, are associated with dysregulation of the epidermal growth factor receptor (HER1/EGFR). This dysregulation appears to play a pivotal role in tumor growth and progression. Thus, HER1/EGFR and its associated downstream signaling pathways are attractive targets for development of novel anticancer therapies. Erlotinib (Tarceva) is an orally available, potent HER1/EGFR tyrosine kinase inhibitor that shows activity against a range of solid tumors, including non-smallcell lung cancer. This article reviews preclinical data providing the rationale for the clinical development of erlotinib in non-small-cell lung cancer, as well as ongoing preclinical investigations and the additional insights they provide into the mechanisms underlying antitumor effects of HER1/ EGFR tyrosine kinase inhibition. Erlotinib Characteristics Erlotinib is an orally available quinazoline small-molecule inhibitor of HER1/EGFR tyrosine kinase. The molecule is highly selective for HER/ EGFR tyrosine kinase; studies with purified kinase show that the erlotinib 50% inhibitory concentration (IC-50) for HER1/EGFR TK is 2 nM, compared with IC-50 values of 350 to > 10,000 nM for other receptorassociated or cytoplasmic tyrosine kinases. The erlotinib IC-50 in cell-based assays is 20 nM. The HER1/EGFR is associated with two important downstream signaling pathways, the MAPK pathway and the Akt pathway. Both of these pathways directly induce various cellular functions; in addition, the Akt pathway activates the PI3K pathway, which also induces cellular functions. For inhibition of HER1/EGFR tyrosine kinase to produce an antitumor effect, it appears that inhibition of one or both of these downstream pathways is necessary. Recent studies in vitro show that erlotinib inhibits MAPK phosphorylation, but does so at concentrations higher than those required for inhibition of HER1/EGFR TK phosphorylation (Figure 1); further, the concentration required for inhibition is related to cytoplasmic concentrations of HER1/EGFR found in different tumor cell lines. Similar findings were made for inhibition of Akt phosphorylation (Figure 1). These data suggest that inhibition of downstream signaling pathways is likely to require inhibition of the majority or nearly all of the HER1/EGFR receptors in the cell membrane and that this effect is likely to require exposure to high concentrations of erlotinib. Erlotinib Effects in Tumor Models The relationship between higher erlotinib concentrations and antitumor effect is more readily apparent in some human tumor models than in others. For example, assessment of the effect of various oral doses of erlotinib on tumor growth in the HN5 head and neck tumor xenograft model indicated a marked improvement in antitumor effect between doses of 1.6 and 12.5 mg/kg; since the 12.5 mg/kg dose resulted in no substantive tumor growth, relative improvements in antitumor effect at higher doses are not readily apparent (Figure 2). However, evaluation of oral erlotinib in the A549 non-small-cell lung cancer xenograft model shows clear improvement of antitumor effect with an increase in dose from 25 to 100 mg/kg (Figure 2). Experience with other tumor models indicates that optimal antitumor effect can be achieved only at such dose levels in many. Additional studies in tumor models have confirmed that the degree of antitumor effect of erlotinib is related to the degree of inhibition of the receptor target; for example, measurements in vivo in HN5 head and neck cancer xenografts showed that percentage tumor growth inhibition during erlotinib multiple-dose treatment was correlated with percentage HER1/EGFR phosphotyrosine inhibition measured at 1 hour after individual doses. Further confirmation that the antitumor effect is associated with degree of inhibition comes from studies in HN5 xenografts showing a dose-related increase in induced tumor cell apoptosis (Figure 3); this effect is abolished with exposure to insulin- like growth factor-1, indicating that the apoptotic effect is indeed dependent on growth factor inhibition. These findings again point out that more complete receptor inhibition is necessary to optimal antitumor effect and indicate the need for high erlotinib doses to achieve maximal inhibition. Effects of Erlotinib in Combination The rationale for using erlotinib or other HER1/EGFR inhibitors in combination with chemotherapeutic agents, radiation therapy, and/or other targeted agents is that combining mo-dalities that have different mechanisms of action or that affect different pathways may augment antitumor efficacy, as well as prevent development of tumor resistance. Preclinical studies of such combinations with erlotinib are ongoing. Initial findings with combinations of erlotinib with chemotherapeutic agents in xenograft models showed significant tumor growth inhibition and no increase in toxicity when erlotinib was combined with cisplatin(Drug information on cisplatin), doxorubicin(Drug information on doxorubicin), paclitaxel, gemcitabine (Gemzar), and capecitabine(Drug information on capecitabine) (Xeloda)[3,5,6]; no interaction was observed when erlotinib was combined with fluorouracil(Drug information on fluorouracil) and vinorelbine (Navelbine) tartrate in the head and neck cancer model. Figure 4 shows the additive effect on tumor growth with the combination of erlotinib and cisplatin in the HN5 head and neck cancer xenograft model. Recent evaluation of the combination of erlotinib and radiation therapy shows that erlotinib increases the radiosensitivity of non-small-cell lung cancer cells and produces a significant decrease in proportion of surviving cells per dose of radiation (Figure 5). Investigation of the combination of erlotinib with other novel targeted agents has included studies with the angiogenesis inhibitor bevacizumab(Drug information on bevacizumab) (Avastin), an agent that has demonstrated clinical activity in human colon cancer. Augmented activity of the erlotinib/bevacizumab combination was found in human colon cancer xenografts (Figure 6), and has been confirmed in the clinical setting. Augmented activity of the combination is also observed in other tumor models. Erlotinib Activity Against HER1/EGFRvIII Mutant The EGFRvIII mutant is a truncated HER1/EGFR variant (in-frame deletions of exons 2-7) with constitutively active tyrosine kinase that is present in a high percentage of primary glial tumors and also is found in non- small-cell lung cancer, breast, and ovarian tumors. Studies in vitro with erlotinib indicate that EGFRvIII levels were minimally affected by 2-hour drug exposure but markedly reduced over 24-hour exposure, with phosphorylation of the receptor being reduced at both 2 and 24 hours. Such findings suggest that for tumors with the EGFRvIII variant, and possibly other tumor types, prolonged drug exposure as well as high drug concentrations may be necessary for optimal antitumor effect. Studies in tumor cell lines that overexpress the wild-type HER1/EGFR or the EGFRvIII variant show that effects of erlotinib in inhibiting EGFRvIII receptor phosphorylation and cell proliferation are dose dependent, but with inhibition requiring higher erlotinib concentrations compared with the wild-type receptor (Figure 7). Clonogenic assays with EGFRvIII-overexpressing cell lines also showed a dose-dependent effect of erlotinib inhibition, with marked inhibition of colony formation occurring at concentrations above 1 μM. Conclusion Erlotinib is an orally available, ATP-competitive, selective and reversible inhibitor of HER1/EGFR TK. In human cancer xenografts, it produces stasis or regression of tumor growth. Although the 50% effective dose is in the 10-mg/kg range for some of these models, accumulating data indicate that higher drug concentrations are necessary in others to achieve optimum receptor inhibition. This finding is in accordance with data indicating that higher doses and consequent maximal receptor inhibition are necessary for adequate inhibition of downstream signaling pathways. Erlotinib exhibits additive antitumor effects in combination with chemotherapeutic agents, radiation therapy, and other targeted agents. Erlotinib is also active against EGFRvIII-transformed tumor cells, with inhibition requiring higher levels of drug exposure compared with cells expressing the wild-type receptor.