Rapid detection and characterization of reactive drug metabolites <i>in vitro</i> using several isotope‐labeled trapping agents and ultra‐performance liquid chromatography/time‐of‐flight mass spectrometry

Rousu, Timo; Pelkonen, Olavi; Tolonen, Ari

doi:10.1002/rcm.3953

Cited by 83 publications

(98 citation statements)

References 33 publications

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“…Occasional cases of liver injury in patients treated with CLZ might be the result of local bioactivation to a reactive nitrenium ion by hepatic P450s (Maggs et al, 1995). In addition, bioactivation of CLZ to a reactive iminium ion, which can be trapped by cyanide, has been demonstrated in microsomal incubations (Argoti et al, 2005;Rousu et al, 2009;Li et al, 2011;Barbara and Castro-Perez JM, 2011) and might contribute to hepatotoxicity. Although these reactive CLZ metabolites have been demonstrated in many in vitro studies using HLM, only very limited information is available on the role of individual P450s in bioactivation of CLZ .…”

Section: Discussionmentioning

confidence: 99%

“…In addition, formation of iminium ions resulting from dehydrogenation of the piperazine-ring of CLZ, DMCLZ, and CLZ-NO were demonstrated using cyanide, which is able to trap hard electrophiles (Rousu et al, 2009;Argoti et al, 2005;Li et al, 2011, Barbara andCastro-Perez, 2011). Theoretically, two different iminium ions can be formed by dehydrogenation of the two different pairs of nitrogen-methylene bonds of the piperazine-ring (Fig.…”

Section: Introductionmentioning

confidence: 98%

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Characterization of Human Cytochrome P450s Involved in the Bioactivation of Clozapine

et al. 2013

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Clozapine is known to cause hepatotoxicity in a small percentage of patients. Oxidative bioactivation to reactive intermediates by hepatic cytochrome P450s (P450s) has be proposed as a possible mechanism. However, in contrast to their role in formation of N-desmethylclozapine and clozapine N-oxide, the involvement of individual P450s in the bioactivation to reactive intermediates is much less well characterized. The results of the present study show that 7 of 14 recombinant human P450s were able to bioactivate clozapine to a glutathione-reactive nitrenium ion. CYP3A4 and CYP2D6 showed the highest specific activity. Enzyme kinetical characterization of these P450s showed comparable intrinsic clearance of bioactivation, implicating that CYP3A4 would be more important because of its higher hepatic expression, compared with CYP2D6. Inhibition experiments using pooled human liver microsomes confirmed the major role of CYP3A4 in hepatic bioactivation of clozapine. By studying bioactivation of clozapine in human liver microsomes from 100 different individuals, an 8-fold variability in bioactivation activity was observed. In two individuals bioactivation activity exceeded N-demethylation and Noxidation activity. Quinidine did not show significant inhibition of bioactivation in any of these liver fractions, suggesting that CYP2D6 polymorphism is not an important factor in determining susceptibility to hepatotoxicity of clozapine. Therefore, interindividual differences and drug-drug interactions at the level of CYP3A4 might be factors determining exposure of hepatic tissue to reactive clozapine metabolites.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Characterization of Human Cytochrome P450s Involved in the Bioactivation of Clozapine

et al. 2013

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“…However, as only one mercapturate and GSH adduct [NVP-3-mercapturate (NVP-M2) and NVP-3-GSH (NVP-G2), respectively] was formed by HLM (Table 1), either the purely oxidative reactions of unsupplemented HLM fail to reproduce metabolic activation of NVP in vivo or HLM do not trap the reactive metabolite(s) with GSH or NAC via both the addition reactions that operate in vivo. A coincubation of HLM and GSH or a GSH derivative is now a standard preparation used in screens for reactive metabolites that are soft electrophiles (Gan et al, 2009), but it frequently yields more conjugates than can be accounted for in vivo (Bu et al, 2007;Rousu et al, 2009). NVP is an exception and emphasizes the need for whole animal studies.…”

Section: Discussionmentioning

confidence: 99%

Quantifying the Metabolic Activation of Nevirapine in Patients by Integrated Applications of NMR and Mass Spectrometries

Srivastava¹,

Lian²,

Maggs³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Nevirapine (NVP), an antiretroviral drug, is associated with idiosyncratic hepatotoxicity and skin reactions. Metabolic pathways of haptenation and immunotoxicity mechanisms have been proposed. NVP is metabolized by liver microsomes to a reactive intermediate that binds irreversibly to protein and forms a GSH adduct. However, no reactive metabolite of NVP, trapped as stable thioether conjugates, has hitherto been identified in vivo. This study has defined the metabolism of NVP with respect to reactive intermediate formation in patients and a rat model of NVP-induced skin reactions. An integrated NMR and mass spectrometry approach has been developed to discover and quantify stable urinary metabolite biomarkers indicative of NVP bioactivation in patients. Two isomeric NVP mercapturates were identified in the urine of HIV-positive patients undergoing standard antiretroviral chemotherapy. The same conjugates were found in rat bile and urine. The mercapturates were isolated from rat bile and characterized definitively by NMR as thioethers substituted at the C-3 and exocyclic C-12 positions of the methylpyrido ring of NVP. It is proposed that NVP undergoes bioactivation to arene oxide and quinone methide intermediates. The purified major mercapturate was quantified by NMR and used to calibrate a mass spectrometric assay of the corresponding metabolite in patient urine. This is the first evidence for metabolic activation of NVP in humans, and only the second minimum estimate in patients of bioactivation of a widely prescribed drug associated with idiosyncratic toxicities. The method can be used as a template for comparative estimations of bioactivation of any drug in patients.

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“…The task can be readily accomplished with HR-MS-based approached. Rousu et al reported a screening method utilizing stable isotope labeled GSH, KCN, and semicarbazide as trapping agents and TOF MS as analytical tool [30]. MDF processing of high resolution full MS data set provided unambiguous identification of both hard and soft reactive metabolites.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

Jian

Liu²,

Zhao

et al. 2012

J. Am. Soc. Mass Spectrom.

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Drugs can be metabolically activated to soft and hard electrophiles, which are readily trapped by glutathione (GSH) and cyanide (CN), respectively. These adducts are often detected and structurally characterized using separate tandem mass spectrometry methods. We describe a new method for simultaneous screening of GSH and CN adducts using precursor ion (PI) and neutral loss (NL) scansdependent product ion spectral acquisition and data mining tools on an triple quadrupole linear ion trap mass spectrometry. GSH, potassium cyanide, and their stable isotope labeled analogues were incubated with liver microsomes and a test compound. Negative PI scan of m/z 272 for detection of GSH adducts and positive NL scans of 27 and 29 Da for detection of CN adducts were conducted as survey scans to trigger acquisition of enhanced resolution (ER) spectrum and subsequent enhanced product ion (EPI) spectrum. Post-acquisition data mining of EPI data set using NL filters of 129 and 27 Da was then performed to reveal the GSH adducts and CN adducts, respectively. Isotope patterns and EPI spectra of the detected adducts were utilized for identification of their molecular weights and structures. The effectiveness of this method was evaluated by analyzing reactive metabolites of nefazodone formed from rat liver microsomes. In addition to known GSH-and CN-trapped reactive metabolites, several new CN adducts of nefazodone were identified. The results suggested that current approach is highly effective in the analysis of both soft and hard reactive metabolites and can be used as a high-throughput method in drug discovery.

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Rapid detection and characterization of reactive drug metabolites in vitro using several isotope‐labeled trapping agents and ultra‐performance liquid chromatography/time‐of‐flight mass spectrometry

Cited by 83 publications

References 33 publications

Characterization of Human Cytochrome P450s Involved in the Bioactivation of Clozapine

Characterization of Human Cytochrome P450s Involved in the Bioactivation of Clozapine

Quantifying the Metabolic Activation of Nevirapine in Patients by Integrated Applications of NMR and Mass Spectrometries

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

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