Application of LC-NMR for Characterization of Rat Urinary Metabolites of Zonampanel Monohydrate (YM872)

Abstract

Zonampanel monohydrate (YM872, Fig. 1) was discovered to be a novel competitive a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, which is a highly water-soluble agent with the selectivity and potency for AMPA receptors. 1) YM872 has been shown to provide neuroprotection in a variety of animal models of ischemia. [2][3][4] In this study, 14 C-labeled YM872 ( 14 C-YM872) was intravenously administered to rats in order to measure urinary excretion of radioactivity and metabolic fingerprinting in urine. Furthermore, two metabolites were semi-purified by preparative HPLC from rat urine after a single intravenous administration of non-labeled YM872, and their structures were elucidated by various instrumental analyses using high-resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (HR-MALDI-TOFMS), LC-MS, LC-MS/MS, and LC-NMR.LC-NMR has been recently used for metabolite structure determination.5-7) LC-NMR elucidates the structure of trace components in mixtures and unstable compounds which are easily decomposed during purification and evaporation to dryness. LC-NMR is more suitable for the two YM872 metabolites than conventional NMR because they are trace components in urine and one of them is likely to be unstable at room temperature. For these reasons, LC-NMR was selected as the method of choice. Results and DiscussionUrinary excretion of total radioactivity was 75.6Ϯ7.4% of dose [meanϮstandard deviation (SD) of 4 rats] from 0 to 8 h after a single intravenous administration of 14 C-YM872. On the HPLC radiochromatogram of the rat urine collected from 0 to 8 h after administration, a major peak corresponding to the parent compound was observed at about 26 min and small metabolite peaks were observed at about 17 and 19.5 min (Fig. 2). Urinary excretion of radioactivity for each peak at about 17 (R1), 19.5 (R2), and 26 min (the parent compound) were 0.7Ϯ0.2%, 0.8Ϯ0.5%, and 71.5Ϯ6.3% of dose (meanϮSD of 4 rats), respectively. In the fingerprinting study, storage temperature was shown to influence urinary metabolites (data not shown). HPLC radiochromatogram of the rat urine after storage for about 24 h at room temperature showed that the peak of R1 decreased, whereas the peak of R2 increased. In contrast, there was no change after storage for about 24 h at 4°C. The observation suggests that R1 is likely to be unstable at room temperature.The HPLC-UV chromatogram of the rat urine collected from 0 to 6 h after administration of non-labeled YM872 also showed a major peak corresponding to the parent compound and small peaks corresponding to the metabolites, R1 and R2. These metabolites were semi-purified by repeated preparative HPLC as described in the experimental section, followed by the elucidation of their structures.Based on LC-MS analysis, the parent compound had a protonated molecule at m/z 332. MS/MS analysis of the protonated molecule revealed fragment ions at m/z 286 and 242, which were derived from the loss of NO 2 and the loss of NO 2 and CO 2 , r...

Role of Organic Anion Transporters in the Pharmacokinetics of Zonampanel, an α-Amino-3-hydroxy-5-methylisoxazole-4-propionate Receptor Antagonist, in Rats

“…Although zonampanel is metabolized only slightly in both rats and humans (Sohda et al, 2004;Minematsu et al, 2005), fecal excretion of […”

“…Nonrenal clearance represents biliary clearance (CL bile, b ), because zonampanel is hardly metabolized (Sohda et al, 2004). 2.…”

“…In addition, urinary excretions of radioactivity and unchanged zonampanel were 75.6 and 71.5% of the dose, respectively (Sohda et al, 2004). The molecular mechanisms of the excretion of zonampanel in rats as well as the reasons for the interspecies differences remain unknown.…”

Ibuprofen metabolite profiling using a combination of SPE/column-trapping and HPLC–micro-coil NMR

“…However, because of the poor sensitivity of NMR at that time, the method was somewhat impractical for broader analytical applications. Later improvements in LC-NMR technology expanded its use to a variety of analytical applications such as stereo-chemical studies [3], combinatorial [4] and environmental [5] chemistry, analyses of natural products [6] and phytochemistry [7], drug discovery [8], proteomics [9] and metabolite studies [10][11][12][13]. The inherently low sensitivity associated with NMR measurements is a major challenge for the development of LC-NMR methods suitable for isolating and detecting low concentration analytes such as those observed in metabolite profiling.…”

Application of liquid chromatography–nuclear magnetic resonance spectroscopy for the identification of ethyldimethylpyrazine, a food flavouring agent

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