N-{4-Chloro-2-[(1-oxidopyridin-4-yl)carbonyl]phenyl}-4-(propan-2-yloxy)benzenesulfonamide (MLN3126) is an orally available chemokine C-C motif receptor 9 selective antagonist. In nonclinical pharmacokinetic studies of MLN3126, nonextractable radioactivity was observed in plasma after oral administration of C-labeled MLN3126 ([C]MLN3126) to Sprague-Dawley (SD) rats. In this study, the nonextractable radioactive component was digested with trypsin or a nonspecific protease, pronase, after chemical reduction to obtain drug-peptide adducts or drug-amino acid adducts. The chemical structure of these adducts was characterized by liquid chromatography/mass spectrometry. The results demonstrated that the major part of the nonextractable radioactivity was accounted for by covalent binding via the Schiff base formed specifically between the -amino group of lysine residue 199 in rat serum albumin and the carbonyl group of MLN3126. The half-life () of the total radioactivity in plasma during and after 21 daily multiple oral administrations of [C]MLN3126 to SD rats was approximately 5-fold shorter than the reported of albumin in rats. The data indicated that the covalent binding was reversible under physiologic conditions. The formation of the covalent binding was also confirmed in in vitro incubations with serum albumins from rats, humans, and dogs in the same manner, indicating that there are no qualitative interspecies differences in the formation of the Schiff base.
Orexin-A (OXA) and -B (OXB) are involved in the regulation
of multiple
physiological functions including the sleep–wake states; therefore,
it is critical to monitor their levels under various conditions. Unfortunately,
the widely used radioimmunoassay has insufficient specificity for
OXA. Although liquid chromatography–tandem mass spectrometry
(LC–MS/MS) has higher specificity for OXA, previously reported
OXA levels in human cerebrospinal fluid (CSF) measured using this
technique are still inconsistent. Moreover, to the best of our knowledge,
OXB has not been detected in the CSF. In this study, we established
a novel method for OXA and OXB measurement. We noticed that OXA and
OXB in the CSF was sticky; thus, citric acid and Tween 80 were used
to prevent their nonspecific binding. Then, highly specific and sensitive
nanoflow liquid chromatography–high-resolution mass spectrometry
(nanoLC-HRMS) was used to measure OXA and OXB levels. Evaluation of
the diurnal fluctuations of OXA and OXB in cisternal and lumbar CSF
samples from cynomolgus monkeys revealed a sharp increase in the early
light period, followed by a gradual increase to the maximum levels
at the end of the light period, and then a sharp drop to the minimum
levels during the early dark period. OXB levels were lower than OXA
levels in cisternal CSF. Although basal OXA levels in individual monkeys
showed substantial variations, the ratios between the maximum and
minimum OXA levels of each monkey were similar. Our method for accurate
OXA and OXB measurement should help improve our knowledge of orexin
biology.
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