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Pseudo-irreversible antagonist profile of osanetant at human NK3 receptors in an IP scintillation proximity assay
Osanetant (SR 142801) is a tachykinin NK3 receptor antagonist that is in clinical development for the treatment of schizophrenia. Previous studies have reported that osanetant displays competitive antagonism at the human cloned NK3 (hNK3) receptor (Oury-Donat et al., 1995; Thomas, et al., 2004) and is non-competitive at NK3 receptors in the guinea pig ileum (Beaujouan et al., 1997). Given this apparent discrepancy in pharmacological profile the present study was undertaken to evaluate the activity of osanetant at the hNK3 receptor using a scintillation proximity assay that measures inositol phosphates. Inositol phosphate assays were performed as described by Brandish et al, (2003). Briefly, HEK293 cells stably expressing hNK3 receptors were incubated with 1 m Ci 3H inositol for 16 h. Cells were then washed with inositol free (IF)-DMEM and pre-incubated with or without osanetant (30 min, 37 oC). For washout experiments cells were then exposed to another wash with IF-DMEM. The NK3 receptor-selective agonist, neurokinin B (NKB), was added and cells incubated for 30 min at 37 oC in the presence of 5 mM LiCl. Assay buffer was removed and cells lysed with 0.1 M formic acid. Aliquots of the lysate were incubated with yttrium silicate SPA beads and levels of radioactivity determined using a Packard TopcountTM. NKB stimulated IP accumulation in a concentration dependent manner with a pEC50 (mean ± standard error mean (n=5) of 8.81 ± 0.08. Osanetant induced a concentration-dependent rightward-shift of the NKB concentration response curve shifting the pEC50 to 8.13 ± 0.14 ,7.13 ± 0.06 and 5.9 ± 0.13, at 0.1 µM, 1 µM and 10 µM, respectively, (n=5). At all concentrations osanetant produced a 50% reduction of the maximal NKB response. Repeated cell washing (up to 4 washes) following pre-incubation with osanetant did not prevent the antagonism afforded by osanetant. These data suggest that the non-competitive profile of osanetant may be due to pseudo-irreversible antagonism of hNK3 receptors. The reason for the difference in profile of antagonism of hNK3 receptors observed here compared to previous studies is unclear but may be explained by an allosteric interaction at the NK3 receptor which is probe and assay dependent (Christopoulos & Kenakin 2002). Beaujouan, J. et al., 1997. Eur. J. Pharmacol., 319, 307-316.
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