Abstract
Although ketoconazole (KCZ) has been used worldwide for 30 years, its metabolic characteristics are poorly described. Moreover, the hepatotoxicity of KCZ limits its therapeutic use. In this study, we used liquid chromatography–mass spectrometry-based metabolomics to evaluate the metabolic profile of KCZ in mouse and human and identify the mechanisms underlying its hepatotoxicity. A total of 28 metabolites of KCZ, 11 of which were novel, were identified in this study. Newly identified metabolites were classified into three categories according to the metabolic positions of a piperazine ring, imidazole ring, and N-acetyl moiety. The metabolic characteristics of KCZ in human were comparable to those in mouse. Moreover, three cyanide adducts of KCZ were identified in mouse and human liver microsomal incubates as “flags” to trigger additional toxicity study. The oxidation of piperazine into iminium ion is suggested as a biotransformation responsible for bioactivation. In summary, the metabolic characteristics of KCZ, including reactive metabolites, were comprehensively understood using a metabolomics approach.
Highlights
Ketoconazole (KCZ; Figure 1A), an imidazole-containing antifungal drug, was approved by the US Food and Drug Administration (FDA) in 1981 as the first orally available azole antifungal agent [1]
KCZ (≥98%), Krebs–Henseleit buffer, β-nicotinamide adenine dinucleotide phosphate (NADP), the reduced form of NADP (NADPH), glucose-6-phosphate, glucose-6-phophate dehydrogenase, magnesium chloride, potassium phosphate, GSH, potassium cyanide (KCN), semicarbazide, dimethyl sulfoxide (DMSO), and methyl cellulose were purchased from Sigma-Aldrich
The residue was reconstituted in 100 μL of 10% methanol; 5 μL aliquots were injected into the liquid chromatography-high resolution mass spectrometry (LC-high resolution mass spectrometry (HRMS)) system to perform metabolite profiling
Summary
Ketoconazole (KCZ; Figure 1A), an imidazole-containing antifungal drug, was approved by the US Food and Drug Administration (FDA) in 1981 as the first orally available azole antifungal agent [1]. Identified nine metabolites of KCZ in mouse liver and reported that N-deacetylation was the primary metabolic pathway. TThhee aaccqquuiirreedd MMSS ssppeeccttrraa wweerree pprroocceesssseedd ffoorr cchheemmoommeettrriicc aannaallyyssiiss aass ddeessccrriibbeedd iinn SSeeccttiioonn 44..77,, aanndd mmeeaanniinnggffuull vvaarriiaabblleesswwereereexetxratrcatectdedbybpyarptiaarltilaeal sltesaqsut asrqeusadriesscrdimisicnraimntinaannatlyasinsa(lPyLsiSs-D(PAL)So-Dr oAr)thoorgoonrathl pogarotniaall lpeaarsttiasql uleaarsets sdqiuscarriems indaisnctriamnianlyansits a(nOaPlyLsSi-sD(AO)P. SSchchememataitcic wwoorkrkflflooww ffoorr tthhee mmeettaabboolliittee pprrooffiilliinngg ooff kkeettooccoonnaazzoollee uussiinngg lliiqquuiidd cchhrroommaattooggrraapphhyy––hhiigghh rerseosolulutitoionn mamssassspecstproemctreotrmyetr(LyC-H(LRCM-HS)R-bMasSe)d-bamseedtabomloemtaibcso.lomKCicZs.: KkCetZoc: okneatzoocloen, aNzoAleD,PNHA: DrePdHuc: erdedfuocremd ofof rmnicootfinnaimcoidtienaamdiedneinaedednininuecledointiudcelepohtiodsephpahtoe,spKhCatNe,: KpCotNas:spiuomtascsyiaunmidcey, aMnSid/Me,SM: tSa/nMdeSm: tmanadssemspemctarsosmseptercyt,roMmDeAtr:ym, MulDtivAa:rmiatueltdivaatariaantealdyastisa, aPnLaSl-yDsiAs,: PpLaSrt-iDalAl:eapsatrstqiaulalreeassdt issqcuriamreinsadnitsacrniamlyinsiasn, tOaPnLaSl-yDsiAs,: oOrPthLoSg-DonAa:l opratrhtioaglolenaaslt psqarutaiarel sledaisstcrsiqmuianraenst dainsacrlyimsiisn. KCZ produced several characteristic fragment ions at m/z 489.1448, m/z 446.1028, m/z 267.0082, m/z 255.0082, m/z 219.1125, m/z 177.1021, and m/z 112.0757 (Figure 3A). MLM: mouse liver microsomes; HLM: human liver microsomes; MHP: mouse hepatocytes; HHP: human hepatocytes; MF: mouse feces
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