br Materials and methods br Results br
Materials and methods
Discussion In this study, we employed a liquid chromatography mass spectrometry (LC–MS)-based metabolomic approach to profile GEF metabolism and bioactivation in human and mouse liver microsomes. Metabolomic approaches rendered us readily screen out the GEF-related metabolites and reactive intermediates from the biomatrix. We identified 34 GEF metabolites and adducts, including 16 previously reported (M1–M4, M7, M12, M16, M19, M26–M32, and M34) and 18 newly identified ones using this strategy , , . Most of the metabolites were presented in both HLM and MLM (Table 1). GEF was mainly metabolized to M1-M6 in HLM, while in MLM M1–M3, M10, M13 and M16 were major metabolites (Fig. 2). Among these metabolites, three potential reactive metabolites, two aldehydes (M18 and M20), one iminium (M33), were detected and characterized for the first time. The known GSH–GEF adduct (M34) and two primary amines (M29 and M30) were also detected in our study. M1, the major metabolite, was present in both HLM (28%) and MLM (25% of whole metabolites). The observation of alcohols (M17 and M21) and am 630 (M19) in HLM indicated the generation of aldehydes (M18 and M20) in the metabolism of GEF. The existence of aldehydes (M18 and M20) was confirmed by the formation of oximes (M18_oxime and M20_oxime) with methoxylamine (Fig. 6) in HLM. In addition, the formation of 4-morpholinobutanal is proportional to the major metabolite M1 (Fig. 2A), so the level of 4-morpholinobutanal is high . The aldehydes (M18 and M20) and 4-morpholinobutanal belong to the alkanals (hard electrophiles). Previous studies indicated that alkanals could induce the formation of deoxynucleoside-protein amino acid cross-links by interacting with exocyclic amino groups of DNA and ϵ-amino groups of lysine residues , . These interactions may lead to toxicity by impairing the function of macromolecules . The aldehydes produced from the GEF metabolism may play a role in GEF-induced toxicity. Notably, the alcohols M17 and M21 in HLM were not observed in MLM, which suggested species difference existed. In general, the formation of acid may contribute to the detoxification of aldehyde, because the resulting acid may conjugate with glycine in vivo and excrete from the body . However, the alcohol can be further sulfated to generate a leaving group in vivo, which reacts with nucleophiles (e.g., GSH) and then may result in toxicity . The metabolic fate of alcohols (M17 and M21), may require metabolism in vivo which will be pursued in future studies. The formation of cyanide–GEF adduct (M33) suggested that a reactive iminium intermediate ion has been generated in the metabolism of GEF. Iminium intermediates have been proposed to play crucial, adverse roles in several drugs bearing tertiary amine structures. For instance, iminium may contribute to nicotine toxicity and addiction . Extensive studies have shown that iminium ion intermediate from nicotine covalently binds to cellular macromolecules . In addition, iminium has been trapped in the metabolism of nefazodone  and clozapine . Liver toxicities caused by both drugs have been reported in clinical practice and iminium intermediates participated in these adverse effects . The iminium from GEF possibly contributes to the GEF-induced toxicity. The primary amines, M29 and M30, from dealkyaltion may also be related to GEF toxicity. Previous studies suggest that primary amines can be further oxidized to nitroso compounds, which can irreversibly inhibit enzymes leading to drug–drug interaction or toxicity . We have demonstrated that CYP3A4 was the primary enzyme contributing to aldehyde intermediates (M18 and M20), pathway of M1 (indicator of the formation of 4-morpholinobutanal), and primary amines (M29 and M30) (Table 2 and Fig. 9). The formation of iminium from GEF was mediated by multiple enzymes including CYP1A2, CYP2C, CYP2D6 and CYP3A4 (Table 2). Inhibition experiment in HLM indicated that CYP3A plays a role in the iminium formation (Fig. 9A), although not exclusively. Previous studies suggested that the formation of GSH–GEF adducts is mediated by CYP1A1 in lung and CYP3A4 in liver . Generally, CYP2D6 is not inducible. To this end, CYP3A4, CYP1A1, CYP2C inducers will increase the formation of these reactive metabolites, which may augment GEF-related toxicity. Clinically, an increase of adverse pulmonary toxicity has been noted in patients who continued smoking . Cigarette smoke can significantly induce CYP1A1 in lung . In line with the clinical data, a 12-fold increase in GSH adduct formation was observed in human pulmonary microsomes from smokers over nonsmokers . Recent studies suggested that GEF frequently induced liver damage in advanced adenocarcinoma patients who had previously undergone other chemotherapies . The significant liver injuries induced by GEF were observed in patients pretreated with cisplatin (CPT)+docetaxel and CPT+pemetrexed sodium. CPT is a strong pregnane X receptor (PXR) activator , , which transcriptionally regulates CYP3A4 and CYP2C , . It is possible that CPT induces CYP3A4 and CYP2C expression via PXR activation, which accelerates GEF metabolism to reactive metabolites, such as aldehyde, primary amines, and iminium, leading to liver injury. Taken together, our data and clinical findings suggest that GEF should be used with caution for patients receiving CYP3A4, CYP1A1, and CYP2C inducers.