240 mg daily resulted in steady-state neratinib concentrations that may have been insufficient to inhibit exon 19 deletions or T790M mutations based on the concentrations required for inhibition in preclinical models (60 nmol/L for exon 19 deletion and 90–800 nmol/L for T790M mutation). In contrast, the much lower dose of neratinib required to inhibit the G719S mutation (3 nmol/L) may have been achievable, leading to the PRs Recentin observed in that small subgroup of patients refractory to reversible TKIs [61]. Similar to neratinib, the half-maximal inhibitory concentration of PF00299804 required for growth inhibition in NSCLC cell lines with the T790M resistance mutation is 100– 900 nM. The inability to achieve these concentrations with doses administered clinically may explain the lack of efficacy in tumors with a T790M mutation [83]. Because T790M-mutant EGFR has an affinity for ATP that is similar to the affinity of wild-type EGFR for ATP, concentrations of irreversible inhibitors that overcome the resistance mutation in vitro are not clinically achievable because of toxicities related to systemic wild-type EGFR inhibition,

such as diarrhea and rash. EGFR T790M mutations notwithstanding, there are glimpses into the potential for irreversible inhibitors in gefitinib- or erlotinib-refractory disease. The PRs and SD seen in PF00299804-treated NSCLC patients with exon 20 insertions (typically resistant to reversible EGFR TKIs) and the PRs seen in neratinib-treated NSCLC patients with exon 18 G719X-mutant tumors previously treated with a Recentin VEGFR inhibitor reversible EGFR TKI suggest that specific EGFR mutations have differential sensitivities to TKI inhibition and that, similar to the situation noted for exon 19 deletions and L858R mutations, irreversible inhibitors are better able to address those relative sensitivities [61, 63]. One approach to expand upon the utility of clinically available 4-anilinoquinazoline irreversible EGFR inhibitors is to pair them with downstream pathway inhibitors or other types of EGFR inhibitors.

For instance, afatinib has been combined in vitro with a PI3K/mammalian target of rapamycin (mTOR) inhibitor, a mitogen-activated protein kinase/extracellular signal– related kinase kinase (MEK) inhibitor, and a v-src sarcoma viral oncogene homolog (Src) inhibitor, yielding greater apoptosis in T790M cell lines than with afatinib alone [84]. In another experiment, the combination of afatinib plus the mTOR inhibitor rapamycin was studied in a mouse model of de novo EGFR L858R/T790M-driven lung cancer. Although single-agent afatinib produced a 50% reduction in tumor volume, the addition of Recentin 288383-20-0 rapamycin to afatinib led to nearly complete tumor regression [71]. In the clinic, the combination of an irreversible inhibitor and mTOR inhibitor is being explored in a phase I study of neratinib plus temsirolimus [85].

Results from a phase Ib/II trial of afatinib in combination with the anti-EGFR antibody cetuximab (Erbitux
; Bristol-Myers Squibb, New York) [86] were recently reported. In that trial, patients with mutant EGFR NSCLC and clinically defined acquired resistance to reversible EGFR TKIs were treated with daily afatinib (40 mg) plus biweekly cetuximab (500 mg/m2). Confirmed PRs were observed in 36% of evaluable patients (eight of 22; 95% CI, 0.17– 0.59) including in 29% of patients with T790M-mutant tumors (four of 13). These promising results will be further explored in a larger study. Reported AEs included rash (grade 1, 35%; grade 2, 46%; grade 3, 11.5%) and diarrhea (grade 1, 50%; grade 2, 19%). Despite the potential of drug combinations, the 4-anilinoquinazoline core structure that is common to the clinically available irreversible inhibitors may not provide optimal molecular interactions or binding kinetics in the setting of T790M mutation. Nonetheless, new structurally distinct irreversible HER family inhibitors, such as the pyrimidine-based inhibitors described by Zhou et al. [87], indicate that the concept of irreversibl