Gefitinib Metabolites Research Articles

Cellular photo(geno)toxicity of gefitinib after biotransformation.

Gefitinib (GFT) is a selective epidermal growth factor receptor (EGFR) inhibitor clinically used for the treatment of patients with non-small cell lung cancer. Bioactivation by mainly Phase I hepatic metabolism leads to chemically reactive metabolites such as O-Demethyl gefitinib (DMT-GFT), 4-Defluoro-4-hydroxy gefitinib (DF-GFT), and O-Demorpholinopropyl gefitinib (DMOR-GFT), which display an enhanced UV-light absorption. In this context, the aim of the present study is to investigate the capability of gefitinib metabolites to induce photosensitivity disorders and to elucidate the involved mechanisms. According to the neutral red uptake (NRU) phototoxicity test, only DF-GFT metabolite can be considered non-phototoxic to cells with a photoirritation factor (PIF) close to 1. Moreover, DMOR-GFT is markedly more phototoxic than the parent drug (PIF = 48), whereas DMT-GFT is much less phototoxic (PIF = 7). Using the thiobarbituric acid reactive substances (TBARS) method as an indicator of lipid photoperoxidation, only DMOR-GFT has demonstrated the ability to photosensitize this process, resulting in a significant amount of TBARS (similar to ketoprofen, which was used as the positive control). Protein photooxidation monitored by 2,4-dinitrophenylhydrazine (DNPH) derivatization method is mainly mediated by GFT and, to a lesser extent, by DMOR-GFT; in contrast, protein oxidation associated with DMT-GFT is nearly negligible. Interestingly, the damage to cellular DNA as revealed by the comet assay, indicates that DMT-GFT has the highest photogenotoxic potential; moreover, the DNA damage induced by this metabolite is hardly repaired by the cells after a time recovery of 18h. This could ultimately result in mutagenic and carcinogenic effects. These results could aid oncologists when prescribing TKIs to cancer patients and, thus, establish the conditions of use and recommend photoprotection guidelines.

Single-cell mass spectrometry studies of drug metabolism heterogeneity and primary resistance to gefitinib in non-small cell lung cancer cells

Patients with epidermal growth factor receptor (EGFR) wild-type non-small cell lung cancer (NSCLC) often show primary resistance to gefitinib therapy. It is thus necessary to study the metabolism of gefitinib in NSCLC cells to comprehensively reveal the reasons for the primary resistance of tumors. Herein, we develop a platform for studying drug metabolism heterogeneity based on single-cell mass spectrometry (sDMH-scMS) by integrating living-cell electrolaunching ionization MS (ILCEI-MS) and high-performance liquid chromatography-MS (HPLC-MS) analysis, and the primary resistance of NSCLC cells to gefitinib was studied using this platform. The ILCEI-MS analysis showed that approximately 11.9% of NSCLC single cells contained the gefitinib metabolite M11; HPLC-MS detection diluted the intensity of M11 in subpopulations and concealed the heterogeneity of drug metabolism in tumor single cells. The intensity of gefitinib in EGFR wild-type A549 cells was markedly lower than in mutant PC9 cells, and the intensity of gefitinib metabolites was significantly higher than in PC9 cells, suggesting that the primary resistance of NSCLC cells is related to gefitinib metabolism. Moreover, the combination of gefitinib and the drug-metabolizing enzyme inhibitor α-naphthoflavone was shown to overcome the primary resistance of the NSCLC cells. Overall, the results of this study are expected to be applicable for clinical drug resistance diagnosis and treatment at the single-cell level.

Computational prediction for the metabolism of human UDP-glucuronosyltransferase 1A1 substrates

UDP-glucuronosyltransferase (UGT) 1A1, one of the most important isoforms in UGTs superfamily, has attracted increasing concerns for its special role in the clearance and detoxification of endogenous and exogenous substances. To avoid the clinical drug-drug interactions, it is of great importance to have the knowledge of the metabolic profile of UGT1A1 substrates early. Herein, we purposed to establish machine learning models to predict the metabolic propeties of UGT1A1 substrates. On the basis of the literature-derived substrates database of UGT1A1, automatic metabolism prediction models for the aromatic hydroxyl (ArOH) and carboxyl (COOH) groups were developed with eight machine learning methods, among which, three methods, i.e. Random Forest, Random Subspace and J48, illustrated the best performance either for the aromatic hydroxyl and the carboxyl model. The models illustrated good robustness when they were evaluated with functions like “Precision”, “Recall”, “F-Measure”, “AUC”, “MCC”, etc. Nice accuracy was observed for the aromatic hydroxyl and carboxyl model of these methods, whose AUCs ranged from 0.901 to 0.997. Additionally, the ArOH model was applied to predict the UGT1A1-mediated metabolism of an external set. Two new unknown substrates, cytochrome P450 (CYPs)-mediated metabolites of gefitinib, were predicted and identified, which were validated by in vitro assays. In summary, this study provides a reliable and robust strategy to predict UGT1A1 metabolites, which will be helpful either in rational-optimization of drug metabolism or in avoiding drug-drug interactions in clinic.

Comprehensive identification, fragmentation pattern, and metabolic pathways of gefitinib metabolites via UHPLC-Q-TOF-MS/MS: in vivo study of rat plasma, urine, bile, and faeces

Gefitinib, the first approved inhibitor for oral epidermal growth factor receptor (EGFR), has been proved to be effective in non-small cell lung cancer with EGFR mutation. However, there are many metabolites of gefitinib that have not been identified in vivo. This study aims to identify the metabolites of gefitinib and its metabolic pathways in rats using ultra-high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) detector. Protein precipitation, solid-phase and ultrasonic extraction were used for the pre-treatment of plasma, urine, bile and faeces samples. In this study, a total of 28 compounds were identified in rat plasma, 29 in bile, 20 in urine and 16 in faeces. 20 new compounds were firstly reported as metabolites of gefitinib. Reduction, hydroxylation, dealkylation and dehalogenation were the major metabolic pathways in phase I. For phase II, the main pathways were sulphate and glucuronide conjugation. The fragment ions of gefitinib and its metabolites were usually generated via the fracture of C1–O bond of propoxy on the C6 position of aniline quinazoline ring. The results may be valuable and important for understanding the metabolic process of gefitinib in clinical application and drug safety.

Tentative identification of gefitinib metabolites in non-small-cell lung cancer patient plasma using ultra-performance liquid chromatography coupled with triple quadrupole time-of-flight mass spectrometry.

BackgroundGefitinib is an orally potent and selective ATP-competitive inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase and is commonly used to treat locally advanced or metastatic non-small-cell lung cancer (NSCLC) with sensitive EGFR mutations. Multiple adverse effects associated with gefitinib, including liver and lung injuries, severe nausea, and diarrhea, have limited its clinical application. Xenobiotic-induced bioactivation is thought to be an important reason for gefitinib toxicity, which encouraged us to clarify the metabolism of gefitinib in NSCLC patients.Materials and methodsAn ultra-performance liquid chromatography coupled with triple quadrupole time-of-flight mass spectrometry (UPLCQ-TOF-MS) method was established to tentatively identify the metabolites of gefitinib in human plasma. The extracted ion chromatogram peak intensity threshold was set at 1500 cps with minimum MS and MS/MS peak intensities of 400 and 100 cps, respectively.ResultsA total of 18 tentative metabolites were identified. Eight novel tentative metabolites with metabolic changes in dechlorination, defluorination, and hydrogenation on the quinazoline skeleton; removal of a partial or complete 3-chloro-4-fluoroaniline-substituted group; and sulfate conjugation and taurine conjugation were newly discovered in human plasma. Based on structural analysis of the tentative metabolites, the metabolic pathways were proposed. In addition, the pathways of dechlorination, defluorination, and hydrogenation on the quinazoline skeleton; removal of partial or complete 3-chloro-4-fluoroaniline-substituted groups; and sulfate conjugation and taurine conjugation in humans in vivo indicate that novel metabolic pathways exist in humans.ConclusionsIn summary, the metabolism of gefitinib in humans in vivo is extensive and complex. Based on in vivo evidence, the propoxy-morpholine ring side chain and O-methyl group are the critical metabolic regions of gefitinib in humans. The novel metabolic pathways differ from those of in vitro studies, suggesting that intestinal floral metabolism might be involved.

Marsdenia tenacissima extract promotes gefitinib accumulation in tumor tissues of lung cancer xenograft mice via inhibiting ABCG2 activity

Ethnopharmacological relevanceMarsdenia tenacissima extract (MTE) is the water-soluble part of a traditional Chinese medicine Marsdenia tenacissima (Roxb.) Wight & Arn, and is commercially available in China for treating cancers. MTE has been revealed to be effective in improving gefitinib efficacy in treating non-small cell lung cancer (NSCLC). However, the mechanisms remain to be defined. Aim of the studyTo determine the effects of MTE on gefitinib metabolism and accumulation in vivo, and to explore the underlying mechanisms. Materials and methodsMTE or vehicle were intraperitoneally administrated to the H1975 xenograft model, followed by intragastric administration of gefitinib 12 h later. Mice plasma, tumors and liver tissues were harvested for further analysis. Hoechst 33342, a specific substrate of ATP Binding Cassette Subfamily G Member 2 (ABCG2), was used to determine the effects of MTE on activities of ABCG2 in tumor cells. ResultsA higher concentration of plasma gefitinib was detected in MTE-treated mice at 24 h after delivery of gefitinib, however, it became insignificant in another 24 h. By contrast, gefitinib levels were continuously higher in MTE-pretreated mice tumor tissues at 12–48 h post gefitinib administration. MTE suppressed plasma levels of gefitinib metabolites (M523595, M608236 and M537194) in the first 24 h after gefitinib delivery, and inhibited activities of liver CYP2D6 and CYP3A4 at early stage (within 6 h) after gefitinib treatment. Strikingly, the activities of ABCG2, the primary drug transporter for gefitinib, were significantly inhibited by MTE in H1975 lung cancer cells. Further, it was identified that tenacissoside H, but not tenacissoside I, may contribute to the ABCG2-suppressive effects of MTE. ConclusionsMTE pretreatment temporarily elevated plasma concentrations of gefitinib via inhibiting CYP450 enzymes. Most importantly, MTE promoted gefitinib accumulation in tumor tissues in a long-lasting manner via decreasing activities of ABCG2, a drug transporter responsible for gefitinib efflux.

Metabolomics reveals the formation of aldehydes and iminium in gefitinib metabolism

Gefitinib (GEF), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is widely used for the treatment of cancers, particularly non-small cell lung cancer. However, its clinical use is limited by multiple adverse effects associated with GEF, such as liver and lung injuries, severe nausea, and diarrhea. Although, the exact mechanism of GEF adverse effects are still unknown, xenobiotic-induced bioactivation is thought to play a significant role in GEF induced toxicity. Using a metabolomic approach, we investigated the metabolic pathways of GEF in human and mouse liver microsomes. Thirty four GEF metabolites and adducts were identified and half of them are novel. The potential reactive metabolites, two aldehydes and one iminium, were identified for the first time. The previously reported GSH adducts and primary amines were observed as well. The aldehyde and iminium pathways were further confirmed by using methoxylamine and potassium cyanide as trapping reagents. Using recombinant CYP450 isoforms, CYP3A4 inhibitor, and S9 from Cyp3a-null mice, we confirmed CYP3A is the major enzyme contributing to the formation of aldehydes, GSH adducts, and primary amines in liver. Multiple enzymes contribute to the formation of iminium. This study provided us more knowledge of GEF bioactivation and enzymes involved in metabolic pathways, which can be utilized for understanding the mechanism of adverse effects associated with GEF and predicting possible drug-drug interactions. Further studies are suggested to determine the roles of these bioactivation pathways in GEF toxicity.

In vitro metabolism of gefitinib in human liver microsomes

1. The in vitro metabolism of gefitinib was investigated by incubating [14C]-gefitinib, as well as M537194, M387783 and M523595 (the main metabolites of gefitinib observed in man), at a concentration of 100 μM with human liver microsomes (4 mg ml−1) for 120 min. These relatively high substrate and microsomal protein concentrations were used in an effort to generate sufficient quantities of metabolites for identification.2. HPLC with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of a large number of metabolites. Although 16 metabolites were identified, metabolism was restricted to three regions of the molecule.3. The major pathway involved morpholine ring-opening and step-wise removal of the morpholine ring and propoxy side chain. O-demethylation of the quinazoline methoxy group was a quantitatively less important pathway, in contrast to the clinical situation, where O-desmethyl gefitinib (M523595) is the predominant plasma metabolite. The third metabolic route, oxidative defluorination, was only a minor route of metabolism. Some metabolites were formed by a combination of these processes, but no metabolism was observed in other parts of the molecule.4. Incubation of gefitinib produced ten identified metabolites, but the use of the three main in vivo metabolites as additional substrates enabled a more comprehensive metabolic pathway to be constructed and this has been valuable in supporting the more limited data available from the human in vivo study.