The amount of radioactivity was determined in intracellular (gray), extracellular (white) and intracellular macromolecule-linked (black) compartments. both inside and outside the cells. As a consequence of gefitinib metabolism, the intracellular level of gefitinib was markedly reduced after 12-24 h of treatment. Consistent with this observation, RT-PCR analysis and EROD assay showed that mRNA and activity of CYP1A1 were present at significant levels and were induced by gefitinib only in sensitive Quinine cells. Gefitinib metabolism was elevated in crowded cells, stimulated by exposure to cigarette smoke extract and prevented by hypoxic condition. It is worth noting that this metabolism of gefitinib in the sensitive cells is usually a consequence and not the cause of drug responsiveness, indeed treatment with a CYP1A1 inhibitor increased the efficacy of the drug because it prevented the fall in intracellular gefitinib level and significantly enhanced the inhibition of EGFR autophosphorylation, MAPK and PI3K/AKT/mTOR signalling pathways and cell proliferation. == Conclusion == Our findings suggest that gefitinib metabolism in lung cancer cells, elicited by CYP1A1 activity, Rabbit Polyclonal to OR52D1 might represent an early assessment of gefitinib responsiveness in NSCLC cells lacking activating mutations. On the other hand, in metabolizing cells, the inhibition of CYP1A1 might lead to increased local exposure to the active drug and Quinine thus increase gefitinib potency. Keywords:Lung cancer, EGFR, gefitinib, Quinine metabolism, CYP1A1 == Background == Gefitinib is an orally active, selective EGFR TKI used in the treatment of patients with advanced-NSCLC carrying activating EGFR mutations [1]. In fact, it is well established that gefitinib is usually more active in some patient subgroups, such as Asians, females, never smokers and adenocarcinoma histotypes which have a higher probability of harbouring activating mutations in the tyrosine kinase domain name, the most frequent being L858R in exon 21 and Del (746-750) in exon 19 [1]. As a consequence most of the NSCLCs made up of wild-type EGFR receptor are excluded and hence the role of gefitinib for the treatment of NSCLC is limited. However, some studies have shown that patients without mutations responded to gefitinib with response rates reaching 6.6% [2,3]. In addition to cancer cell genomic determinants of sensitivity, some pharmacokinetic parameters may also play a role in the variable response to gefitinib and other TKIs [4]. When administered at 250 mg/day, gefitinib is usually 60% orally assimilated and 90% plasma protein-bound [5,6]. The very high distribution volume of gefitinib (1400 litres) clearly indicates that this drug is extensively distributed in tissues such as liver, kidney, gastrointestinal tract, lung and in tumors [7]. A tendency to accumulate in the lung was observed with concentrations 10 occasions higher than in plasma [8]. We have recently exhibited in NSCLC cell lines that this uptake of gefitinib is an essentially active process leading to intracellular gefitinib concentrations more than two hundred occasions higher than outside the cells [9]. There are few data on gefitinib intracellular metabolism in tumors, the majority of the available data concerns liver metabolism.In vitroandin vivostudies indicate that in the liver gefitinib is mainly metabolized by cytochrome P450-dependent (CYP) activities, including CYP3A4, CYP3A5 and CYP2D6 Quinine [10-12]. The main metabolic pathway characterized by using human liver microsomes include morpholine ring opening, O-demethylation of the methoxy-substituent around the quinazoline ring structure and oxidative defluorination of the halogenated phenyl group [13,14]. A study investigating the contribution of individual CYPs to gefitinib metabolism exhibited that gefitinib disappeared with comparable clearance when incubated with CYP3A4 or CYP2D6 enzymes, less efficiently with CYP3A5 or CYP1A1, whereas CYP1A2 and CYP1B1 were not involved in the metabolism of the drug [12]. Incubation with CYP3A4 and to a lesser extent CYP3A5, produced a similar range of metabolites as that produced by liver microsomes [11], but the main plasma metabolite, the O-desmethyl derivative present at plasma concentrations similar to gefitinib [10], was formed predominantly through the CYP2D6 enzyme. CYP1A1 is one of the three members of the CYP1 family mainly expressed in extra-hepatic tissue, involved in the metabolism of a large number of xenobiotics as well as a small number of endogenous substrates [15]. Being expressed at a significant level in human lung [16], it might play a role in the metabolism of gefitinib by lung tumor cells and its activity might be involved in the variability of the drug response. In experiments using lung microsomes, CYP1A1 was shown to produce significant amounts of thepara-hydroxyaniline metabolite derived from oxidative defluorination of gefitinib. Hydroxyaniline metabolites produced by CYP1A1 can be oxidized to reactive quinone-imine derivatives that.