It has been reported that both gefitinib and its des methyl metabolite formed through CYP2D6, inhib this website ited with a similar potency and selectivity subcellular EGFR tyrosine kinase activity. However, M3 was 15 times less active in a cell based assay and consequently it was assumed that this metabolite was unlikely to con tribute to the activity of gefitinib in vivo due to poor cell penetration. On the contrary, when metabolites M1, M2 and M3 were tested in our responsive cell models at concentra tions equivalent to that of gefitinib, they exhibited a signif icant inhibition of EGFR autophosphorylation and proliferation in intact cells, indicating their ability to enter cells and to interact with the catalytic domain of EGFR.

Finally, in gefitinib resistant cell lines M1, M2 and M3 metabolites were poorly effective indicating that at least these metabolites did not produce additive toxic effects in NSCLC cell lines. In contrast to its abundant hepatic expression, CYP3A4 seems to play a minor role in lung metabolism, being expressed in only about 20% Inhibitors,Modulators,Libraries of cases. Real time PCR analysis confirmed the lack of expression of this isoform in our NSCLC cell models, as reported for A549 cells. CYP2D6 was detected in all cell lines, whereas both CYP1A1 and CYP1A2 were expressed at significant levels in sensitive cells. Inducibility of CYP1A1 and CYP1A2 transcripts by gefitinib was clearly demonstrated in sensitive cell lines, while induction of CYP1A1 mRNA was not detected in resistant cell lines. EROD activity demonstrated a 3 6 fold induction of CYP1A1 elicited by gefitinib in sensitive cells.

To the best of our knowledge, this is the first time that the Inhibitors,Modulators,Libraries induction by gefitinib Inhibitors,Modulators,Libraries of relevant metabolic enzyme has been demonstrated. Inhibitors,Modulators,Libraries The reason why gefitinib induces CYP expression Inhibitors,Modulators,Libraries and activity only in sensitive cells could be ascribed to the ability of gefitinib to inhibit signal transduction pathway downstream EGFR. It has been recently demonstrated that EGF represses the dioxin mediated induction of CYP1A1 in normal human keratinocytes preventing recruitment of the p300 coactivator. Therefore, EGFR signalling is a repressor of the aryl hydrocarbon receptor and regulates the transcription of numerous genes including CYP1A1. In this context, EGFR inhibi tors such as gefitinib, erlotinib, lapatinib or cetuximab might affect the induction of CYP1A1 in those cell types in which the drug effectively inhibits signalling controlled by EGFR.

The inhibition of MAPK pathway might repre sent a link between EGFR inhibition and CYP1A1 induc tion since PD98059 and U0126, well known MEK1/2 inhibitors, induced CYP1A1 activity as did gefitinib in H322 cells, while none of PI3K/AKT/mTOR selleck chem inhibitor inhibitors tested was effective. It is noteworthy that constitutive activation of signaling pathways downstream of EGFR is a recognized mechanism or resistance against reversible EGFR tyrosine kinase inhibitors.