Abstract
Metabolism catalyzed by the cytochrome P450 enzymes (CYPs) represents the most important clearance pathways for most drugs in humans. However, CYP-mediated metabolism can also lead to drug bioactivation resulting to formation of reactive metabolites that can potentially induce idiosyncratic toxicity by covalently binding to endogenous proteins and nucleic acids. Therefore, it has become imperative to implement strategies for screening and identifying bioactivation liability of drug candidates as an integrated approach to reduce the attrition rate in drug discovery and development. This chapter describes a detailed protocol for the in-vitro stable isotopic trapping and screening for reactive metabolites using common LC-MS/MS methodologies such as neutral loss scan and precursor ion scan.
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References
Ulrich RG (2007) Idiosyncratic toxicity: a convergence of risk factors. Annu Rev Med 58:17–34
Boelsterli UA (2003) Idiosyncratic drug hepatotoxicity revisited: new insights from mechanistic toxicology. Toxicol Mech Methods 13:3–20
Caldwell GW, Yan Z (2006) Screening for reactive intermediates and toxicity assessment in drug discovery. Curr Opin Drug Discov Devel 9:47–60
Prakash C, Sharma R, Gleave M, Nedderman A (2008) In vitro screening techniques for reactive metabolites for minimizing bioactivation potential in drug discovery. Curr Drug Metab 9:952–964
Kalgutkar AS, Gardner I, Obach RS, Shaffer CL, Callegari E, Henne KR, Mutlib AE, Dalvie DK, Lee JS, Nakai Y et al (2005) A comprehensive listing of bioactivation pathways of organic functional groups. Curr Drug Metab 6:161–225
Yan Z, Maher N, Torres R, Huebert N (2007) Use of a trapping agent for simultaneous capturing and high-throughput screening of both “soft” and “hard” reactive metabolites. Anal Chem 79:4206–4214
Yan Z, Caldwell GW (2004) Stable-isotope trapping and high-throughput screenings of reactive metabolites using the isotope MS signature. Anal Chem 76:6835–6847
Yan Z, Maher N, Torres R, Caldwell GW, Huebert N (2005) Rapid detection and characterization of minor reactive metabolites using stable-isotope trapping in combination with tandem mass spectrometry. Rapid Commun Mass Spectrom 19:3322–3330
Wen B, Ma L, Nelson SD, Zhu M (2008) High throughput screening and characterization of reactive metabolites using polarity switching of hybrid triple quadrupole linear ion trap mass spectrometry. Anal Chem 80:1788–1799
Yan Z, Caldwell GW, Maher N (2008) Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition. Anal Chem 80:6410–6422
Ma L, Wen B, Ruan Q, Zhu M (2008) Rapid screening of glutathione-trapped reactive metabolites by linear ion trap mass spectrometry with isotope pattern-dependent scanning and postacquisition data mining. Chem Res Toxicol 21:1477–1483
Liao S, Ewing NP, Boucher B, Materne O, Brummel CL (2012) High-throughput screening for glutathione conjugates using stable-isotope labeling and negative electrospray ionization precursor-ion mass spectrometry. Rapid Commun Mass Spectrom 26:659–669
Rousu T, Pelkonen O, Tolonen A (2009) 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. Rapid Commun Mass Spectrom 23:843–855
Jian W, Liu H-F, Zhao W, Jones E, Zhu M (2012) Simultaneous screening of glutathione and cyanide adducts using precursor ion and neutral loss scans-dependent product ion spectral acquisition and data mining tools. J Am Soc Mass Spectrom 23:964–976
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Yan, Z., Caldwell, G.W. (2014). In Vitro Trapping and Screening of Reactive Metabolites Using Liquid Chromatography-Mass Spectrometry. In: Caldwell, G., Yan, Z. (eds) Optimization in Drug Discovery. Methods in Pharmacology and Toxicology. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-742-6_28
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DOI: https://doi.org/10.1007/978-1-62703-742-6_28
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-741-9
Online ISBN: 978-1-62703-742-6
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