Chromatographic Characterization of Fezolinetant Pharmacokinetics and Metabolic Pathways Using HPLC–Radioactivity Detection and LC–MS/MS

Researchers set out to characterize the pharmacokinetics, mass balance, and metabolism of fezolinetant, a non-hormonal selective neurokinin-3 receptor antagonist for the treatment of moderate to severe vasomotor symptoms (VMS) associated with menopause, in postmenopausal women. Quantitative metabolite profiling and metabolite structure elucidation were performed on samples collected from the mass balance study by high performance liquid chromatography (HPLC) with radioactivity detector or liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. A paper based on this work was published in the European Journal of Drug Metabolism and Pharmacokinetics.¹

VMS associated with menopause are primarily characterized by hot flashes and night sweats and arise from the activity of kisspeptin, neurokinin B (NKB) and dynorphin (KNDy) neurons in the hypothalamus.²Normally, KNDy neurons are kept in balance by signals that activate them and estrogen, which calms them down. However, during menopause when estrogen levels drop, this balance is lost. Without enough estrogen to keep them in check, these cells become overactive. These overactive cells then send signals to the part of the brain that controls body temperature. This changes how the body cools itself down, causing it to overreact to signals from temperature-sensing nerves.^2-5 Fezolinetant is a hormone-free pill that works by blocking the specific signals that overstimulate those KNDy neuron cells. By calming these cells down, the medication helps reset the brain's temperature control center and keeps it from overreacting.⁶

A single dose of 180 mg ¹⁴C-fezolinetant solution was administered to healthy postmenopausal women (n = 5) for this study to evaluate mass balance and pharmacokinetics. Following a single administration of ¹⁴C-fezolinetant, the average recovery of radioactivity was 90.9%, where most of the radioactivity recovered in urine (mean: 76.9%) and to a lesser extent in feces (mean: 14.0%). Fezolinetant was well absorbed and primarily metabolized to the hydroxylated metabolite, ES259564, which was eliminated mainly in urine. Fezolinetant accounted for nearly 29% of exposure for total radioactivity in plasma. In addition to the parent drug, only ES259564 was detected as a circulating metabolite and accounted for approximately 52% of total drug-related exposure. Additional minor metabolites (< 3.5% of dose for each metabolite) were only detected in urine or feces. Fezolinetant metabolic pathways included hydroxylation of the methyl group of the 3-methyl-1,2,4-thiadiazole moiety (M9, ES259564), further oxidation of M9 to the carboxylic acid metabolite M4, further glucuronidation of the hydroxyl group of M9 to the glucuronide metabolite M5, direct glucuronidation of fezolinetant to the glucuronide M6, and cleavage of the 1,2,4-thiadiazole moiety to the ring-opened metabolite M1.¹

“This study,” wrote the authors of the paper,¹ “successfully characterized the overall pathways of metabolism and excretion of fezolinetant, identified the circulating metabolites, and provided key data to support the development of fezolinetant. Fezolinetant is mainly metabolized to yield ES259564, and primarily excreted into urine as ES259564. In plasma, only fezolinetant and ES259564 were detected, accounting for approximately 81% of total radioactivity, indicating that most circulating drug-related material was quantitatively characterized, with the remaining radioactivity attributable to multiple low-abundance components below the threshold for a major circulating metabolite.”

References

1. Iwai, M.: Souda, K.; Miyagawa, M. et al. Clinical Pharmacokinetics, Mass Balance and Metabolism of Fezolinetant in Postmenopausal Women. Eur J Drug Metab Pharmacokinet. 2026. DOI: 10.1007/s13318-026-00995-2
2. Lehman, M. N.; Coolen, L. M.; Goodman, R. L. Minireview: Kisspeptin/Neurokinin B/Dynorphin (KNDy) Cells of the Arcuate Nucleus: A Central Node in the Control of Gonadotropin-Releasing Hormone Secretion. Endocrinology 2010, 151 (8), 3479-3489. DOI: 10.1210/en.2010-0022
3. Ruka, K. A.; Burger, L. L.; Moenter, S. M. Both Estrogen and Androgen Modify the Response to Activation of Neurokinin-3 and κ-Opioid Receptors in Arcuate Kisspeptin Neurons From Male Mice. Endocrinology 2016, 157 (2), 752-763. DOI: 10.1210/en.2015-1688
4. Padilla, S. L.; Johnson, C. W.; Barker, F. D. et al. A Neural Circuit Underlying the Generation of Hot Flushes. Cell Rep. 2018, 24 (2), 271-277. DOI: 10.1016/j.celrep.2018.06.037
5. Rance, N. E.; Dacks, P. A.; Mittelman-Smith, M. A. et al. Modulation of Body Temperature and LH Secretion by Hypothalamic KNDy (Kisspeptin, Neurokinin B and Dynorphin) Neurons: A Novel Hypothesis on the Mechanism of Hot Flushes. Front Neuroendocrinol. 2013, 34 (3), 211-227. DOI: 10.1016/j.yfrne.2013.07.003
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