title: Utilization of Stable Isotope Labeling to Facilitate the Identification of Polar Metabolites of KAF156, an Antimalarial Agent.
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 ([(14)C]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., [(12)C]/[(14)C]) and mass difference that serve as the basis for a mass filter using accurate mass spectrometry. Unfortunately, [(14)C]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 [(13)C6]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 [(14)C]KAF156-1 (specific activity 3.65 μCi/mg) and [(13)C6]KAF156-1 with a mass ratio of [(12)C]/[(13)C6] 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, [(14)C]KAF156-2 and [(13)C2]KAF156-2 were synthesized to allow the detection of any putative polar metabolites that may have lost labeling during biotransformations using the previous [(14)C]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.
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey Su.huskey@novartis.com.
authors.forename: Su-Er W
authors.initials: SE
authors.lastname: Huskey
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey.
authors.forename: Ry R
authors.initials: RR
authors.lastname: Forseth
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Li
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey.
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Jian
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Catoire
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey.
authors.forename: Tapan
authors.initials: T
authors.lastname: Ray
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey.
authors.forename: Handan
authors.initials: H
authors.lastname: He
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey.
authors.forename: Jimmy
authors.initials: J
authors.lastname: Flarakos
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey.
authors.forename: James B
authors.initials: JB
authors.lastname: Mangold
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology* / Q000652:urine; D002853:Chromatography, Liquid; D007093:Imidazoles / Q000494:pharmacology* / Q000652:urine; D007553:Isotope Labeling; D008297:Male; D010879:Piperazines / Q000494:pharmacology* / Q000652:urine; D051381:Rats; D017208:Rats, Wistar; D053719:Tandem Mass Spectrometry
chemical_list: D000962:Antimalarials; D007093:Imidazoles; D010879:Piperazines; C000599914:ganaplacide