Molecular and cellular pharmacology
Inhibition of substance P-mediated responses in NG108-15 cells by netupitant and palonosetron exhibit synergistic effects

https://doi.org/10.1016/j.ejphar.2012.05.037Get rights and content

Abstract

Netupitant is a potent and selective NK1 receptor antagonist under development in combination with a fixed dose of palonosetron for the prevention of chemotherapy induced nausea and vomiting. Palonosetron is a 5-HT3 receptor antagonist approved for both the prevention of acute and delayed chemotherapy induced nausea and vomiting after moderately emetogenic chemotherapy. Accumulating evidence suggests that substance P (SP), a ligand acting largely on tachykinin (NK1) receptors, is the dominant mediator of delayed emesis. Interestingly, palonosetron does not bind to the NK1 receptor so that the mechanism behind palonosetron's unique efficacy against delayed emesis is not clear. Palonosetron exhibits a distinct ability among 5-HT3 receptor antagonists to inhibit crosstalk between NK1 and 5-HT3 receptor signaling pathways. The objective of the current work was to determine if palonosetron's ability to inhibit receptor signaling crosstalk would influence netupitant's inhibition of the SP-mediated response when the two drugs are dosed together. We first studied the inhibition of SP-induced Ca2+ mobilization in NG108-15 cells by palonosetron, ondansetron and granisetron. Unexpectedly, in the absence of serotonin, palonosetron inhibited the SP-mediated dose response 15-fold; ondansetron and granisetron had no effect. Netupitant also dose-dependently inhibited the SP response as expected from an NK1 receptor antagonist. Importantly, when both palonosetron and netupitant were present, they exhibited an enhanced inhibition of the SP response compared to either of the two antagonists alone. The results further confirm palonosetron's unique pharmacology among 5-HT3 receptor antagonists and suggest that it can enhance the prevention of delayed emesis provided by NK1 receptor antagonists.

Introduction

Current therapy for the treatment of chemotherapy induced nausea and vomiting includes the use of both 5-HT3 and NK1 receptor antagonists along with dexamethasone (Basch et al., 2011, Ettinger et al., 2011, Roila et al., 2010). Acute emesis has been largely associated with activation of 5-HT3 receptors by serotonin while delayed emesis is thought to occur mainly through the activation of NK1 receptors by substance P (SP) (Hesketh et al., 2003). While treatment of acute emesis was largely resolved with the introduction of 5-HT3 receptor antagonists in the 1990s, nausea and delayed emesis are lingering problems (Feyer and Jordan, 2011, Oo and Hesketh, 2005). The introduction of NK1 receptor antagonists has been shown to improve overall antiemetic efficacy including delayed emesis when used along with 5-HT3 receptor antagonists and dexamethasone (Darmani and Ray, 2009, Feyer and Jordan, 2011, Hesketh et al., 2003). Even though a majority of patients are fully protected against chemotherapy induced nausea and vomiting by the use of these therapies, there are still a significant number of patients that experience nausea and delayed emesis, especially following highly or moderately emetogenic chemotherapies (Feyer and Jordan, 2011). Netupitant is a potent and selective NK1 receptor antagonist currently under development in combination with a fixed dose of palonosetron for the prevention of chemotherapy induced nausea and vomiting. Palonosetron is the only 5-HT3 receptor antagonist that has been found to be effective in both acute and delayed chemotherapy induced nausea and vomiting after moderate emetogenic chemotherapy (Aapro et al., 2006, Eisenberg et al., 2003, Gralla et al., 2003, Saito et al., 2009). Since palonosetron does not bind to the NK1 receptor, the mechanism behind palonosetron's unique efficacy among 5-HT3 receptor antagonists against nausea and delayed emesis is obscure. Recent mechanism of action studies showed that palonosetron could inhibit SP mediated responses in vitro and in vivo possibly as a result of inhibition of 5-HT3/NK1 receptor crosstalk (Rojas et al., 2010b). It is not clear however, if palonosetron's effect on 5-HT3/NK1 receptor crosstalk would enhance or negate the inhibition of the SP response when used in combination with NK1 antagonists. In the present work, we used NG108-15 cells known to express both the 5-HT3 and NK1 receptors (Emerit et al., 1993, Reiser and Hamprecht, 1989), to study the inhibition of the SP response by netupitant and palonosetron. We report that netupitant and palonosetron exhibit a synergistic effect in the prevention of the SP-mediated response in these cells.

Section snippets

Calcium-ion release measurements in NG108-15 cells

NG108-15 cells were grown to 95% confluence in high-glucose Dulbecco's modified Eagle's medium. Medium was supplemented with a mixture of sodium hypoxanthine, aminopterin, and thymidine, 10% heat-inactivated fetal bovine serum, glutamine (2 mM), penicillin (100 units), streptomycin (100 μg), and amphotericin B (0.25 μg). All media exchanges were preceded by a 2–5 min plate spin at 168×g, to prevent loosely adherent cells from coming off the plate surface. When determining the effect of 5-HT3

Palonosetron inhibited the SP response in the absence of serotonin

When cells were preincubated with a saturating concentration of palonosetron (6 nM) in the absence of serotonin followed by media changes to remove antagonist remaining on the cell surface, the SP-induced response was significantly inhibited. The SP response after preincubation with palonosetron exhibited an EC50 of 30±6 μM which corresponded to a 15-fold shift to the right compared to the SP response in control cells i.e., when there was no preincubation with palonosetron (EC50=2±0.2 μM) (Fig. 1

Discussion

Recent studies showed that palonosetron inhibits SP mediated responses in vitro and in vivo possibly as a result of inhibition of 5-HT3/NK1 receptor crosstalk. Specifically, palonosetron was found to inhibit the serotonin enhancement of the SP response in NG108-15 cells and the cisplatin enhancement of the neuronal response to SP in rat nodose ganglia. This inhibition was not observed when using ondansetron or granisetron, two other commonly used 5-HT3 receptor antagonists (Rojas et al., 2010b

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