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Research ArticleArticle

Utilization of Stable Isotope Labeling to Facilitate the Identification of Polar Metabolites of KAF156, an Antimalarial Agent

Su-Er W. Huskey, Ry R. Forseth, Hongmei Li, Zhigang Jian, Alexandre Catoire, Jin Zhang, Tapan Ray, Handan He, Jimmy Flarakos and James B. Mangold
Drug Metabolism and Disposition October 2016, 44 (10) 1697-1708; DOI: https://doi.org/10.1124/dmd.116.072108
Su-Er W. Huskey
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey
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Ry R. Forseth
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey
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Hongmei Li
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey
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Zhigang Jian
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey
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Alexandre Catoire
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey
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Jin Zhang
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey
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Tapan Ray
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey
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Handan He
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey
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Jimmy Flarakos
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey
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James B. Mangold
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey
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Abstract

Identification of polar metabolites of drug candidates during development is often challenging. Several prominent polar metabolites of 2-amino-1-(2-(4-fluorophenyl)-3-((4-fluorophenyl)amino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)ethanone ([14C]KAF156), an antimalarial agent, were detected in rat urine from an absorption, distribution, metabolism, and excretion study but could not be characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) because of low ionization efficiency. In such instances, a strategy often chosen by investigators is to use a radiolabeled compound with high specific activity, having an isotopic mass ratio (i.e., [12C]/[14C]) and mass difference that serve as the basis for a mass filter using accurate mass spectrometry. Unfortunately, [14C]KAF156-1 was uniformly labeled (n = 1–6) with the mass ratio of ∼0.1. This ratio was insufficient to be useful as a mass filter despite the high specific activity (120 μCi/mg). At this stage in development, stable isotope labeled [13C6]KAF156-1 was available as the internal standard for the quantification of KAF156. We were thus able to design an oral dose as a mixture of [14C]KAF156-1 (specific activity 3.65 μCi/mg) and [13C6]KAF156-1 with a mass ratio of [12C]/[13C6] as 0.9 and the mass difference as 6.0202. By using this mass filter strategy, four polar metabolites were successfully identified in rat urine. Subsequently, using a similar dual labeling approach, [14C]KAF156-2 and [13C2]KAF156-2 were synthesized to allow the detection of any putative polar metabolites that may have lost labeling during biotransformations using the previous [14C]KAF156-1. Three polar metabolites were thereby identified and M43, a less polar metabolite, was proposed as the key intermediate metabolite leading to the formation of a total of seven polar metabolites. Overall this dual labeling approach proved practical and valuable for the identification of polar metabolites by LC-MS/MS.

Footnotes

    • Received June 17, 2016.
    • Accepted August 1, 2016.
  • dx.doi.org/10.1124/dmd.116.072108.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

  • Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 44 (10)
Drug Metabolism and Disposition
Vol. 44, Issue 10
1 Oct 2016
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Research ArticleArticle

Identification of Polar Metabolites of KAF156

Su-Er W. Huskey, Ry R. Forseth, Hongmei Li, Zhigang Jian, Alexandre Catoire, Jin Zhang, Tapan Ray, Handan He, Jimmy Flarakos and James B. Mangold
Drug Metabolism and Disposition October 1, 2016, 44 (10) 1697-1708; DOI: https://doi.org/10.1124/dmd.116.072108

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Research ArticleArticle

Identification of Polar Metabolites of KAF156

Su-Er W. Huskey, Ry R. Forseth, Hongmei Li, Zhigang Jian, Alexandre Catoire, Jin Zhang, Tapan Ray, Handan He, Jimmy Flarakos and James B. Mangold
Drug Metabolism and Disposition October 1, 2016, 44 (10) 1697-1708; DOI: https://doi.org/10.1124/dmd.116.072108
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