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.

Profiling and Identification of Omeprazole Metabolites in Mouse Brain and Plasma by Isotope Ratio-Monitoring Liquid Chromatography-Mass Spectrometric Method

“…Accurate mass measurement is an essential process to elucidate the elemental composition and structural information in the identification of metabolites. Especially, the usage of either radio-labeled or stable-isotope labeled compound would be helpful for the identification of novel metabolites [17][18][19]. In this study, we administered omeprazole and its stable isotope D3-omeprazole concomitantly in a dose level of 50 mg/kg (1:1 ratio of omeprazole and D3-omeprazole mixture) to mice and compared the metabolite production according to the route of administration in the mouse plasma and brain.…”

“…In this study, an approach using concomitant administration of omeprazole with a stable-isotope labeled omeprazole (D3–omeprazole) was used. The concomitant administration of stable isotope is a well-known method to identify characteristic metabolites of a certain compound [ 17 , 18 , 19 ]. Especially, the brain matrix contains various proteins, lipids and other endogenous materials, which can interfere the accurate detection of drug–related metabolites [ 20 ].…”

The Development Process for Discovery and Clinical Advancement of Modern Antimalarials

“…Proposed mechanism for the formation of metabolite M40 and the metabolic pathway of major metabolites detected in rats after oral and intravenous dosing of 14 C-labeled KAF156. 92 a micronucleus assay KAF156 was negative, suggesting a low risk of genotoxicity in humans. ADME profiling of 14 C-and 13 C-labeled KAF156 in rats was undertaken as part of the preclinical assessment studies.…”

“…ADME profiling of 14 C-and 13 C-labeled KAF156 in rats was undertaken as part of the preclinical assessment studies. 92 The primary goal was to pharmacokinetically characterize KAF156 and identify metabolites that may display toxicity. In this study, KAF156 was dosed orally and intravenously to rats at 10 and 3 mg/kg, respectively.…”

“…Figure 12. Proposed mechanism for the formation of metabolite M40 and the metabolic pathway of major metabolites detected in rats after oral and intravenous dosing of 14 C-labeled KAF156 92. …”