Metabolism Of Gefitinib Research Articles

Gefitinib metabolism-related lncRNAs for the prediction of prognosis, tumor microenvironment and drug sensitivity in lung adenocarcinoma

The complete compound of gefitinib is effective in the treatment of lung adenocarcinoma. However, the effect on lung adenocarcinoma (LUAD) during its catabolism has not yet been elucidated. We carried out this study to examine the predictive value of gefitinib metabolism-related long noncoding RNAs (GMLncs) in LUAD patients. To filter GMLncs and create a prognostic model, we employed Pearson correlation, Lasso, univariate Cox, and multivariate Cox analysis. We combined risk scores and clinical features to create nomograms for better application in clinical settings. According to the constructed prognostic model, we performed GO/KEGG and GSEA enrichment analysis, tumor immune microenvironment analysis, immune evasion and immunotherapy analysis, somatic cell mutation analysis, drug sensitivity analysis, IMvigor210 immunotherapy validation, stem cell index analysis and real-time quantitative PCR (RT-qPCR) analysis. We built a predictive model with 9 GMLncs, which showed good predictive performance in validation and training sets. The calibration curve demonstrated excellent agreement between the expected and observed survival rates, for which the predictive performance was better than that of the nomogram without a risk score. The metabolism of gefitinib is related to the cytochrome P450 pathway and lipid metabolism pathway, and may be one of the causes of gefitinib resistance, according to analyses from the Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Immunological evasion and immunotherapy analysis revealed that the likelihood of immune evasion increased with risk score. Tumor microenvironment analysis found most immune cells at higher concentrations in the low-risk group. Drug sensitivity analysis found 23 sensitive drugs. Twenty-one of these drugs exhibited heightened sensitivity in the high-risk group. RT-qPCR analysis validated the characteristics of 9 GMlncs. The predictive model and nomogram that we constructed have good application value in evaluating the prognosis of patients and guiding clinical treatment.

Tetracyclic triterpenoids as inhibitors of cytochrome P450 3A4 and their quantitative structure activity relationship analysis

The interaction between herbs and clinical drugs is one of the risk factors for adverse drug reactions. In the present study, the inhibitory effects of 47 tetracyclic triterpenoids toward the main drug-metabolizing CYPs in humans were investigated. Most of the evaluated tetracyclic triterpenoids (TT) exhibited strong inhibitory effects toward CYP3A4 compared with other CYP subtypes. The inhibition kinetics of compounds 9 (23-acetyl alisol C), 44 (hemslecin A), and 47 (cucurbitacin E) against CYP3A4 were studied, and inhibition constant (Ki) was determined as 2.12, 0.196, and 0.162 µM, respectively. Some representative TT exhibited significant inhibitory effects toward the metabolism of gefitinib, atorvastatin, and quetiapine, indicating a potential interaction between TT derivatives and clinical drugs. Additionally, a quantitative structure–activity relationship (QSAR) study of a series of TT as inhibitors of CYP3A4 was performed. The present study provided key information to guide the rational use of herbs rich in TT.

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.

Pharmacokinetics of gefitinib in elderly patients with EGFR-mutated advanced non-small cell lung cancer: a prospective study

BackgroundGefitinib is recommended as a first-line treatment option for elderly patients with non-small cell lung cancer (NSCLC). Because no pharmacokinetics of gefitinib have been examined, we prospectively assessed the pharmacokinetics of gefitinib in patients with epidermal growth factor receptor gene-mutated advanced NSCLC who were 75 years or older.MethodsGefitinib was orally administered once daily at a dose of 250 mg. The concentrations of gefitinib and its major metabolite O-desmethyl gefitinib in plasma were measured by high-performance liquid chromatography. The area under the plasma concentration–time curve from time 0 to 48 h (AUC0–48) was calculated. Polymorphisms in CYP3A5, CYP2D6, ABCG2, ABCB1, and OATP1B1 were analyzed by direct sequencing.ResultsEighteen patients with a median age of 80.5 years (range, 75–89) with adequate liver and kidney functions were examined. AUC0–48 values of gefitinib and O-desmethyl gefitinib in this population were 9.49 ± 3.5 and 10.6 ± 14 µM h, respectively. Compared to the gefitinib pharmacokinetics observed in a previous phase I study in Japan, systemic exposure to gefitinib in elderly patients was slightly higher than that in younger patients. Three patients experienced grade 3 diarrhea, increases in alanine aminotransferase, and aspartate aminotransferase levels 30 days after starting gefitinib treatment. The CYP2D6 genotype was associated with CYP2D6-mediated metabolism of gefitinib to O-desmethyl gefitinib.ConclusionsWe demonstrated for the first time the systemic exposure to gefitinib in elderly patients with NSCLC.Trial registration: The study was registered with the University Hospital Medical Information Network-Clinical Trials Registry Japan (UMIN000026409) on November 8, 2013.

Identified Gefitinib Metabolism-Related lncRNAs can be Applied to Predict Prognosis, Tumor Microenvironment, and Drug Sensitivity in Non-Small Cell Lung Cancer.

Gefitinib has shown promising efficacy in the treatment of patients with locally advanced or metastatic EGFR-mutated non-small cell lung cancer (NSCLC). Molecular biomarkers for gefitinib metabolism-related lncRNAs have not yet been elucidated. Here, we downloaded relevant genes and matched them to relevant lncRNAs. We then used univariate, LASSO, and multivariate regression to screen for significant genes to construct prognostic models. We investigated TME and drug sensitivity by risk score data. All lncRNAs with differential expression were selected for GO/KEGG analysis. Imvigor210 cohort was used to validate the value of the prognostic model. Finally, we performed a stemness indices difference analysis. lncRNA-constructed prognostic models were significant in the high-risk and low-risk subgroups. Immune pathways were identified in both groups at low risk. The higher the risk score the greater the value of exclusion, MDSC, and CAF. PRRophetic algorithm screened a total of 58 compounds. In conclusion, the prognostic model we constructed can accurately predict OS in NSCLC patients. Two groups of low-risk immune pathways are beneficial to patients. Gefitinib metabolism was again validated to be related to cytochrome P450 and lipid metabolism. Finally, drugs that might be used to treat NSCLC patients were screened.

Inhibitory effects of Thai herbal extracts on the cytochrome P450 3A-mediated the metabolism of gefitinib, lapatinib and sorafenib

Herbal products are widely used in cancer patients via co−administration with chemotherapy. Previous studies have demonstrated that pharmacokinetic interactions between herbs and anticancer drugs exist due to inhibition of drug−metabolizing enzymes, particularly cytochrome P450s (CYPs). The aim of this study was to determine the inhibitory effects of Andrographis paniculata, Curcuma zedoaria, Ganoderma lucidum, Murdannia loriformis and Ventilago denticulata extracts on the metabolism of gefitinib, lapatinib and sorafenib. The activities of CYP3A in human liver microsome on the metabolism of gefitinib, lapatinib and sorafenib in the absence and presence of Thai herbal extracts were assayed using high-performance liquid chromatography analysis. Curcuma zedoaria extract potently inhibited CYP3A−mediated lapatinib and sorafenib metabolism with IC50 values of 4.18 ± 3.20 and 7.59 ± 1.23 μg/mL, respectively, while the metabolism of gefitinib was strongly inhibited by Murdannia loriformis and Ventilago denticulata extracts with IC50 values of 7.53 ± 2.87 and 7.06 ± 1.23 μg/mL, respectively. Andrographis paniculata and Ganoderma lucidum extracts had less effect on the metabolism of the tested anticancers (IC50 values >10 μg/mL). In addition, kinetic analysis of the ability of Curcuma zedoaria extract to inhibit CYP3A-mediated metabolism of anticancer drugs was best described by the noncompetitive and competitive inhibition models with Ki values of 20.08 and 11.55 μg/mL for the metabolism of gefitinib and sorafenib, respectively. The present study demonstrated that there were potential pharmacokinetic interactions between tyrosine kinase inhibitors and herbal extracts.

Abstract 419: Presence of pulmonary tuberculosis impair survival in advanced EGFR mutation lung adenocarcinoma patients receiving epidermal growth factor receptor-tyrosine kinase inhibitor

Abstract Background Although studies have identified several clinicopathological factors that can predict the response to and survival on epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), few studies have clearly demonstrated the effect of EGFR-TKI in the treatment of patients with underlying pulmonary disease, such as pulmonary tuberculosis (PTB). In addition, cytochrome P450 (CYP) enzyme, CYP3A4, is involved in the metabolism of gefitinib. Previous study reported that in the presence of the anti-tuberculosis drug rifampicin, a potent CYP3A4 inducer, the geometric mean maximum concentration and area under the plasma concentration-time curve of gefitinib were significantly reduced. Thus, we conducted the study to evaluate the impact of PTB on survival for pulmonary adenocarcinoma (PAD) patients that underwent EGFR-TKI treatment. Methods Clinicopathologic data of PAD patients harboring EGFR mutations from the Guangzhou Chest hospital in China between 2017 and 2019 were reviewed retrospectively. Patients receiving EGFR-TKI treatment were divided into PTB and non-PTB groups. The differences in response to EGFR-TKIs and survival between the two groups were assessed. Formalin-fixed paraffin-embedded (FFPE) tumor tissues were used for the detection of alterations in the EGFR, ALK, ROS1, KRAS, BRAF, RET, MET, HER2, NRAS, and PIK3CA genes. Results A total of 1448 patients with PAD were reviewed. 223 patients (15.40%) had co-existing PTB, and 317 (21.89%) patients had EGFR-mutated tumors. Among the 223 patients with PTB, 21 (9.42%) harboring EGFR mutations (PTB group). From 296 non-PTB patients with EGFR mutations, 43 patients (non-PTB group) were randomly selected for subsequent analysis. The frequency of former smoker (57.14% vs. 13.95%, p=0.001) and poor performance status (23.81% vs. 6.98%, p=0.007) were significantly higher in the PTB group compared with the non-PTB group. After EGFR-TKIs treatment, the objective response rate (ORR, 58.14% vs.47.62%) as well as disease control rate (DCR, 97.67% vs.85.71) were higher in the non-PTB group than in the PTB group, but there was no statistical difference. In the survival analysis, both the median progression-free survival (PFS, 7.47 months vs. 11.77 months, p=0.038) and the overall survival (OS, 13.00 months vs. 20.00 months, p=0.0017) after EGFR-TKIs were significantly shorter in the PTB group than in the non-PTB group. Secondary data analysis showed that, for patients with 19Del mutation, or metastases sites less than 3, or using first-line EGFR-TKI, EGFR-TKIs treatment significantly prolonged the median PFS and OS in patients without PTB. Conclusion Presence of PTB may be associated with more frequent former smoker and poor performance status. PAD patients on EGFR-TKI treatment had a worse survival outcome if patients had co-existing PTB. Citation Format: Yalin Xie, Wei Zhou, An Lei, Zhan Huang, Li Weiwei, Changbin Zhu, Guanshan Zhu, Wenchang Cen. Presence of pulmonary tuberculosis impair survival in advanced EGFR mutation lung adenocarcinoma patients receiving epidermal growth factor receptor-tyrosine kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 419.

Rapid determination of the pharmacokinetics and metabolic fate of gefitinib in the mouse using a combination of UPLC/MS/MS, UPLC/QToF/MS, and ion mobility (IM)-enabled UPLC/QToF/MS

The metabolism and pharmacokinetics of gefitinib (Iressa®, N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholino-propoxy)quinazolin-4-amine), a selective thymidylate kinase inhibitor for the epidermal growth factor receptor (EGFR), was studied after IV and PO administration to male C57BL6 mice at 10 and 50 mg/kg respectively. The pharmacokinetics and metabolism of gefitinib were investigated using a range of rapid UHPLC-MS and UHPLC-IM-HRMS methods, using both reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC), to rapidly determine the drugs pharmacokinetics and metabolic fate. Rapid oral absorption resulted in peak plasma concentrations at 1 h of ca. 7 µg/mL, that declined with a half-life of 3.8 h (2.6 h for the IV route), and providing an estimated oral bioavailability of 53%. Gefitinib itself was the major circulating drug-related compound in plasma extracts, with a total of 11 metabolites identified. The urinary profiles determined using both HILIC and RP-UPLC-IM-MS detected gefitinib and 10 metabolites or 15 metabolites respectively including the detection of a number of novel glucuronide conjugates. Despite rapid, sub 5 min, LC profiling methods being employed metabolite coverage was shown to be high and compared well with that of previous studies.

Pre-clinical drug-drug interaction (DDI) of gefitinib or erlotinib with Cytochrome P450 (CYP) inhibiting drugs, fluoxetine and/or losartan

ObjectiveTo evaluate drug-drug interactions (DDIs) between gefitinib or erlotinib with fluoxetine, and/or losartan. MethodsHuman pooled microsomes, supersomes, and cryopreserved human hepatocytes were used to monitor DDIs in vitro. RED (Rapid Equilibrium Dialysis) protein binding was employed to investigate other pharmacokinetics. ResultsGefitinib is significantly metabolized by Cytochrome P450 (CYP) 2D6 and CYP3A4, with less than 80% of the drug remaining. Erlotinib is significantly metabolized by CYP3A4, CYP2D6, and CYP1A2. Although gefitinib and erlotinib were metabolized by the same CYP isoenzymes, the metabolites formed from degradation of the two drugs were different.Fluoxetine inhibited CYP2D6 and CYP3A4 metabolism of gefitinib with an IC50 of 65.12 ± 1.88 µM and 4.11 ± 2.26 µM, respectively. Fluoxetine also inhibited CYP2D6 and CYP3A4 metabolism of erlotinib with an IC50 of 7.06 ± 1.54 µM and 4.57 ± 1.22 µM, respectively.For hepatocytes, fluoxetine affected the metabolism of gefitinib or erlotinib, while losartan had no effect. Gefitinib and erlotinib inhibited the metabolism of fluoxetine and losartan. Two-drug combinations involving gefitinib or erlotinib with fluoxetine or losartan yielded insignificant (p-value ≥ 0.05) differences in metabolism. However, combinations involving three drugs yielded significant degrees of inhibition (p-value ≤ 0.05). Three drug combinations involving fluoxetine and losartan with gefitinib or erlotinib yielded significant degrees of inhibition of the metabolism of gefitinib, but not for that of erlotinib. ConclusionAs could be predicted by previous studies involving the inhibitory effect of fluoxetine on CYP3A4 and CYP2D6, and studies involving CYP metabolism of gefitinib and erlotinib, the tests performed here confirmed that fluoxetine has an inhibitory effect on metabolism of gefitinib or erlotinib by the main CYP isoenzymes involved. This study suggests a variable inhibitory effect of fluoxetine particularly on CYP2D6 activity towards gefitinib or erlotinib; erlotinib metabolism is less affected. Likewise, the combination of fluoxetine and losartan does not significantly affect hepatocyte metabolism of erlotinib, but does for that of gefitinib. The results presented in this study thus indicate a need for DDI assays to involve multiple drugs to properly study multidrug regimens.

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.

Functional characterization of wild-type and 24 CYP2D6 allelic variants on gefitinib metabolism in vitro

BackgroundCytochrome P450 2D6 (CYP2D6), a member of the CYP450 enzyme super family, is a polymorphic enzyme that metabolizes ~25% of therapeutic drugs. CYP2D6 exhibits significant genetic polymorphisms which might cause adverse effects and therapeutic failures of some drugs.ObjectiveThe purpose of this study was to evaluate the catalytic activities of 22 novel CYP2D6 alleles (CYP2D6*87, *88, *89, *90, *91, *92, *93, *94, *95, *96, *97, *98, R25Q, F164L, E215K, F219S, V327M, D336N, V342M, R344Q, R440C, R497C) on the metabolism of gefitinib in vitro.Methods and resultsCYP2D6 variants were incubated with 1–100 μM gefitinib for 60 min at 37°C and the reaction was terminated by cooling to −80°C immediately. Gefitinib and its metabolite O-desmethyl gefitinib were analyzed by an ultra-performance liquid chromatography-tandem mass spectrometry system. Compared to CYP2D6.1, most CYP2D6 variants exhibited significantly decreased relative clearance values (from 3.11% to 79.35%), whereas CYP2D6.92 and CYP2D6.96 displayed no detectable enzyme activity. Only CYP2D6.94 exhibited a markedly increased intrinsic clearance value, and eight variants (CYP2D6.88, CYP2D6.89, CYP2D6.91, CYP2D6.97, V342M, R344Q, F219S, and F164L) showed no significant difference. In addition, 23 CYP2D6 allelic isoforms exhibited substrate inhibition trend toward gefitinib.ConclusionAs the first study of all the aforementioned alleles for gefitinib metabolism, these comprehensive data may help in the clinical assessment of the metabolism of gefitinib, and may also offer a reference for personalized treatment with gefitinib in clinical settings.

Effects of Angelica gigas extract on the oral pharmacokinetics of gefitinib in rats

This study was performed to evaluate the potential drug–herb interaction between the conventional anti-cancer drug, gefitinib, and the herbal medication, Angelica gigas (AG) extract. One group of rats received a single intravenous (IV) dose of gefitinib (10 mg/kg) to determine its basic pharmacokinetics. Another group received a single oral dose of gefitinib (10 mg/kg) after pretreatment with saline or AG extract (500 mg/kg). The third group received an oral dose of gefitinib after pretreatment with saline or AG extract daily for 7 days. In addition, fecal and urinary recovery of gefitinib after IV and oral administration was evaluated. Gefitinib concentrations were determined using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and the pharmacokinetic parameters were estimated by non-compartmental analysis. In addition, inhibition by AG of CYP3A4, the primary enzyme responsible for the metabolism of gefitinib, was evaluated. The results indicated that the pharmacokinetic parameters of gefitinib following single and multiple doses were not significantly affected by pretreatment with AG extract. Fecal and urinary recovery of gefitinib was not significantly different between groups, and AG extract did not inhibit CYP3A4 activity until the concentration reached 600 μg/mL. Therefore, the data indicated that gefitinib was not significantly affected by co-treatment with AG extract.

Pharmacokinetics of Gefitinib: Roles of Drug Metabolizing Enzymes and Transporters.

Gefitinib (Iressa, AstraZeneca) has been widely used for the treatment of locally advanced or metastatic non-small cell lung cancer. A number of studies have been reported on its pharmacokinetics profiles, especially on the metabolism. In this review, we have comprehensively summarized the pharmacokinetic characteristics of gefitinib: absorption, distribution, metabolism and excretion (ADME). Overall, gefitinib reached the maximum plasma level relatively fast and distributed extensively. It underwent extensive biotransformation and predominantly excreted in feces, with less than 7% in the urine. CYP450 enzymes played critical roles in the process of gefitinib metabolism. The major enzyme involved in the metabolism was CYP3A4, with other CYP450 enzymes playing a secondary role. A high clearance of gefitinib might result in drug resistance by lowering drug concentration. The enhanced efflux and decreased uptake by transporters were important resistance mechanisms. The transporters involved in pharmacokinetics of gefitinib consist of the ATP-binding cassette and the solute carrier superfamily. Understanding the pharmacokinetics property of gefitinib may provide valuable and new information for dealing with drug resistance and making personalized therapy regarding their interindividual variability.

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.

Marsdenia tenacissima extract inhibits gefitinib metabolism in vitro by interfering with human hepatic CYP3A4 and CYP2D6 enzymes

Ethnopharmacological relevanceThe stem of Marsdenia tenacissima (Roxb.) Wight et Arn. is mainly produced in Yunnan China and has long been used as a medicine to treat cancer in China. Xiao-Ai-Ping injection, the water-soluble part of the stem of Marsdenia tenacissima, is administrated as an anti-cancer agent in clinics for decades. Our previous study showed that Marsdenia tenacissima extract (MTE) restored gefitinib sensitivity in gefitinib-resistant non-small cell lung cancer (NSCLC) cells, but the mechanism involved is unknown. Gefitinib undergoes hepatic metabolism predominantly through human cytochrome P450 (CYP) 3A4 and CYP2D6 enzymes. This study aims to evaluate whether MTE interferes with gefitinib metabolism via human hepatic P450 enzymes. Material and methodsA cocktail-substrate assay was used to test the effect of MTE on major CYP enzyme activities by incubation of pooled human liver microsomes with specific substrate probes of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in the absence and presence of MTE. Recombinant human CYP450 enzymes were used to predict in vitro gefitinib metabolic clearance in the absence and presence of MTE. The metabolites of the substrate probes and gefitinib were detected by high-performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS). Human hepatoma HepG2 cells were used to investigate the effect of gefitinib alone or in combination with MTE on CYP3A4 and CYP2D6 mRNA and protein expression. ResultsThe cocktail-substrate assay showed that MTE inhibited CYP450 activities in human liver microsomes with the inhibition rate of 3A4>2C9>2C19>1A2>2D6. The co-administration of MTE with gefitinib significantly decreased the in vitro intrinsic clearance (Clint) of gefitinib by 2.6 and 4.0-fold for CYP2D6 and CYP3A4, respectively, but did not affect other CYP450s. CYP2D6 and CYP3A4 mRNA and protein expression in human hepatoma HepG2 cells were greatly reduced in the combined gefitinib and MTE treatment. ConclusionWe demonstrate that MTE inhibits gefitinib metabolism by interfering with CYP3A4 and CYP2D6. Meanwhile, MTE combined with gefitinib down-regulates the mRNA and protein expression of CYP3A4 and CYP2D6 in the HepG2 cells. Thus, these data suggest that MTE is a promising herbal medicine to enhance gefitinib efficacy through improving its metabolic stability.

Reduced CYP2D6 function is associated with gefitinib-induced rash in patients with non-small cell lung cancer

BackgroundRash, liver dysfunction, and diarrhea are known major adverse events associated with erlotinib and gefitinib. However, clinical trials with gefitinib have reported different proportions of adverse events compared to trials with erlotinib. In an in vitro study, cytochrome P450 (CYP) 2D6 was shown to be involved in the metabolism of gefitinib but not erlotinib. It has been hypothesized that CYP2D6 phenotypes may be implicated in different adverse events associated with gefitinib and erlotinib therapies.MethodsThe frequency of each adverse event was evaluated during the period in which the patients received gefitinib or erlotinib therapy. CYP2D6 phenotypes were determined by analysis of CYP2D6 genotypes using real-time polymerase chain reaction techniques, which can detect single-nucleotide polymorphisms. The CYP2D6 phenotypes were categorized into 2 groups according to functional or reduced metabolic levels. In addition, we evaluated the odds ratio (OR) of the adverse events associated with each factor, including CYP2D6 activities and treatment types.ResultsA total of 232 patients received gefitinib therapy, and 86 received erlotinib therapy. Reduced function of CYP2D6 was associated with an increased risk of rash of grade 2 or more (OR, 0.44; 95% confidence interval [CI], 0.21–0.94; *p = 0.03), but not diarrhea ≥ grade 2 (OR, 0.49; 95% CI, 0.17–1.51; *p = 0.20) or liver dysfunction ≥ grade 2 (OR, 1.08; 95% CI, 0.52–2.34; *p = 0.84) in the gefitinib cohort. No associations were observed between any adverse events in the erlotinib cohort and CYP2D6 phenotypes (rash: OR, 1.77; 95% CI, 0.54–6.41; *p = 0.35/diarrhea: OR, 1.08; 95% CI, 0.21–7.43; *p = 0.93/liver dysfunction: OR, 0.93; 95% CI, 0.20–5.07; *p = 0.93).ConclusionsThe frequency of rash was significantly higher in patients with reduced CYP2D6 activity who treated with gefitinib compared to patients with functional CYP2D6. CYP2D6 phenotypes are a risk factor for the development of rash in response to gefitinib therapy.

189P - Reduced CYP2D6 Function Potentiates the Gefitinib-Induced Rash in Patients with Non-Small Cell Lung Cancer

ABSTRACT Background Rash, liver dysfunction, and diarrhea are known as adverse events of erlotinib and gefitinib. However, clinical trials with gefitinib have reported different proportion of adverse events compared to those with erlotinib. In an in vitro study, cytochrome P450 (CYP) 2D6 was shown to be involved in the metabolism of gefitinib and not of erlotinib. It has been hypothesized that CYP2D6 phenotypes may be implicated in different adverse events between in the gefitinib and erlotinib therapies. Methods The frequency of each adverse event was evaluated during the period that the patients received EGFR-TKI therapy. CYP2D6 phenotypes were determined from the CYP2D6 genotype using real-time polymerase chain reaction methods, which are able to determine single nucleotide polymorphisms. The CYP2D6 phenotypes were categorized into 2 groups according to functional or reduced metabolic levels. In addition, we evaluated the odds ratio (OR) of adverse events with each factor, including CYP2D6 activities as well as treatment types. Results Patients were identified through a query of patient information for subjects prospectively enrolled in the Medical Information System within Osaka City University Hospital between January 1999 and February 2012 that tracks all of the patients referred for CYP2D6 sequencing from our hospital. A total of 232 patients received gefitinib therapy, and 86 patients received erlotinib therapy. Reduced function of CYP2D6 was associated with an increased risk of rash of grade 2 or more (OR 0.44, 95% confidence interval [CI] 0.21-0.94, p = 0.03), and not of diarrhea ≥ grade 2 (OR 0.49, 95%CI 0.17-1.51, p = 0.20) and liver dysfunction ≥ grade 2 (OR 1.08, 95%CI 0.52-2.34, p = 0.84) in gefitinib cohort. No associations were observed between any adverse events in erlotinib cohorts and CYP2D6 phenotypes (rash: OR 1.77, 95%CI 0.54-6.41, p = 0.35; diarrhea: OR 1.08, 95%CI 0.21-7.43, p = 0.93; and liver dysfunction: OR 0.93, 95%CI 0.20-5.07, p = 0.93). Conclusions CYP2D6 may relate to the metabolism of gefitinib and not of erlotinib. CYP2D6 phenotypes are one of promising factors for the development of rash in gefitinib therapy. Disclosure All authors have declared no conflicts of interest.

Metabolism of the EGFR tyrosin kinase inhibitor gefitinib by cytochrome P450 1A1 enzyme in EGFR-wild type non small cell lung cancer cell lines

BackgroundGefitinib is a tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) especially effective in tumors with activating EGFR gene mutations while EGFR wild-type non small cell lung cancer (NSCLC) patients at present do not benefit from this treatment.The primary site of gefitinib metabolism is the liver, nevertheless tumor cell metabolism can significantly affect treatment effectiveness.ResultsIn this study, we investigated the intracellular metabolism of gefitinib in a panel of EGFR wild-type gefitinib-sensitive and -resistant NSCLC cell lines, assessing the role of cytochrome P450 1A1 (CYP1A1) inhibition on gefitinib efficacy. Our results indicate that there is a significant difference in drug metabolism between gefitinib-sensitive and -resistant cell lines. Unexpectedly, only sensitive cells metabolized gefitinib, producing metabolites which were detected 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 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 the metabolism of gefitinib in the sensitive cells is 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.ConclusionOur findings suggest that gefitinib metabolism in lung cancer cells, elicited by CYP1A1 activity, 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 thus increase gefitinib potency.

Differential Metabolism of Gefitinib and Erlotinib by Human Cytochrome P450 Enzymes

To examine the enzyme kinetics of gefitinib and erlotinib metabolism by individual cytochrome P450 (CYP) enzymes, and to compare their effects on CYP3A activity, with the aim to better understand mechanisms underlying pharmacokinetic variability and clinical effects. Enzyme kinetics were examined by incubating gefitinib or erlotinib (1.5-50 micromol/L) with recombinant human CYP3A4, CYP3A5, CYP2D6, CYP1A1, CYP1A2, and CYP1B1 (10-160 pmol/mL). Their effects on CYP3A activity were examined by comparing midazolam metabolism in the presence and absence of gefitinib or erlotinib in human liver and intestinal microsomes. Parent compounds and metabolites were monitored by high-performance liquid chromatography with a photodiode detector or tandem mass spectrometer. Both drugs were metabolized primarily by CYP3A4, CYP3A5, and CYP1A1, with respective maximum clearance (Cl(max)) values for metabolism of 0.41, 0.39, and 0.57 mL/min/nmol for gefitinib and 0.24, 0.21, 0.31 mL/min/nmol for erlotinib. CYP2D6 was involved in gefitinib metabolism (Cl(max), 0.63 mL/min/nmol) to a large extent, whereas CYP1A2 was considerably involved in erlotinib metabolism (Cl(max), 0.15 mL/min/nmol). Both drugs stimulated CYP3A-mediated midazolam disappearance and 1-hydroxymidazolam formation in liver and intestinal microsomes. Gefitinib is more susceptible to CYP3A-mediated metabolism than erlotinib, which may contribute to the higher apparent oral clearance observed for gefitinib. Metabolism by hepatic and extrahepatic CYP1A may represent a determinant of pharmacokinetic variability and response for both drugs. The differential metabolizing enzyme profiles suggest that there may be differences in drug-drug interaction potential and that stimulation of CYP3A4 may likely play a role in drug interactions for erlotinib and gefitinib.