Netupitant and palonosetron trigger NK1 receptor internalization in NG108-15 cells

Abstract

Current therapy for chemotherapy-induced nausea and vomiting includes the use of both 5-HT3 and NK1 receptor antagonists. Acute emesis has largely been alleviated with the use of 5-HT3 receptor antagonists, while an improvement in preventing delayed emesis has been achieved with NK1 receptor antagonists. Delayed emesis, however, remains a problem with a significant portion of cancer patients receiving highly emetogenic chemotherapy. Like other drugs in its class, palonosetron, a 5-HT3 receptor antagonist, has shown efficacy against acute emesis. However, palonosetron has also shown consistent improvement in the suppression of delayed emesis. Since both 5-HT3 and NK1 receptor antagonists are often simultaneously administered to patients, the question remains if palonosetron's effect on delayed emesis would remain distinct when co-administered with an NK1 receptor antagonist. Recent mechanistic studies using NG108-15 cells have shown that palonosetron and netupitant, an NK1 receptor antagonist currently in phase 3 clinical trials, exhibited synergistic effects when inhibiting the substance P response. The present studies showed that both netupitant and palonosetron-induced NK1 receptor internalization in NG108-15 cells and that when used together receptor internalization was additive. Palonosetron-induced NK1 receptor internalization was dependent on the presence of the 5-HT3 receptor. Results provide a possible explanation for palonosetron's enhancement of the inhibition of the SP response and suggest that the effect of palonosetron and NK1 receptor antagonists on prevention of delayed emesis could be additive.

Fig. 1

Reduction in binding of [³H] netupitant after incubation of cells with NK₁ and 5-HT₃ receptor antagonists. Reduction in binding in a NG108-15 cells expressing both NK₁ and 5-HT₃ receptors and b NK₁-HEK-293 cells expressing NK₁ receptors only—cells were preincubated ± antagonist (s) for 24 h as indicated. Netu: netupitant (5 nM); Palo: palonosetron (1 nM); Netu + Palo: netupitant (5 nM) plus palonosetron (1 nM); Ond: ondansetron (30 nM); Netu + Ond: netupitant (5 nM) plus ondansetron (30 nM). Cells were washed to remove the antagonists and subsequently incubated with [³H] netupitant for 40 min at room temperature. At the end of the incubation period, unbound [³H] netupitant was removed, and the radioactivity associated with the cells was measured (methods). Radioactivity associated with control cells was normalized to 100 %. Percent binding with respect to control cells for each treatment was subtracted from 100 to obtain the reduction in binding shown on the y-axis. Error bars correspond to standard deviation of three independent experiments run in triplicate. ***p < 0.001 when compared to netupitant; student’s t test was used for statistical analysis

Fig. 2

Dissociation of [³H]-netupitant ± palonosetron or ondansetron from NG108-15 cells (a) and cell-free membranes (b). Cells were preincubated for 24 h with [³H]-netupitant (5 nM), [³H]-netupitant (5 nM) plus palonosetron (1 nM) or [³H]-netupitant (5 nM) plus ondansetron (30 nM). At the end of this incubation, antagonist-containing media were replaced with prewarmed HEPES-buffered saline containing excess unlabeled netupitant (5 µM) and dissociation of [³H]-netupitant at 37 °C was followed at different times as shown. After removing medium, cells were scraped into 200 µl of fresh ice-cold buffer, and the radioactivity present in the scraped material at each time point was measured using a scintillation counter. When using cell-free membranes the association phase was carried out for 90 min (24 h gave same results) at 37 °C. The dissociation phase was then initiated by addition of excess unlabeled netupitant (1 μM). The amount of [³H]-netupitant bound to the receptor was measured at various times during the first hour after addition of displacer. Prism (GraphPad Software Inc, San Diego, CA) was used to obtain half-life values

Fig. 3

Effects of netupitant and palonosetron in NG108-15 cells—Receptor internalization can be induced through direct binding of palonosetron to the 5-HT₃ receptor (1) and of netupitant to the NK₁ receptor (2). Internalization of either receptor could lead to alterations in receptor signaling cross talk (3). NK₁ receptor internalization would result in lower NK₁ receptor density at the cell surface that in turn desensitizes NK₁ signaling (4). NK₁ receptor internalization can be induced by netupitant (directly and possibly indirectly) or by palonosetron (indirectly)