Molecular and Cellular Pharmacology
Palonosetron triggers 5-HT3 receptor internalization and causes prolonged inhibition of receptor function

https://doi.org/10.1016/j.ejphar.2009.10.002Get rights and content

Abstract

Palonosetron is a 5-HT3 receptor antagonist that has demonstrated superiority in preventing both acute and delayed emesis when compared to older first generation 5-HT3 receptor antagonists. The objective of this work was to determine if palonosetron exhibits unique molecular interactions with the 5-HT3 receptor that could provide a scientific rationale for observed clinical efficacy differences. Previously, we showed that palonosetron exhibits allosteric binding and positive cooperativity to the 5-HT3 receptor in contrast to ondansetron and granisetron which exhibit simple bimolecular binding. The present work shows, through several independent experiments, that palonosetron uniquely triggers 5-HT3 receptor internalization and induces prolonged inhibition of receptor function. After 24 h incubation followed by dissociation conditions, [3H]palonosetron remained associated with whole cells but not to cell-free membranes (P < 0.001). [3H]Palonosetron's binding to cells was resistant to both protease and acid treatments designed to denature cell surface proteins suggesting that the receptor complex was inside the cells rather than at the surface. Cells pretreated with unlabeled palonosetron subsequently exhibited reduced cell surface 5-HT3 receptor binding. Palonosetron-triggered receptor internalization was visualized by confocal fluorescence microscopy using cells transfected with 5-HT3 receptor fused to enhanced cyan fluorescent protein. In contrast, granisetron and ondansetron showed minimal to no effect on receptor internalization or prolonged inhibition of receptor function. These experiments may provide a pharmacological basis for differences noted in published clinical trials comparing palonosetron to other 5-HT3 receptor antagonists.

Introduction

Serotonin 5-HT3 receptor antagonists are widely used alone or in combination with other agents to prevent or treat chemotherapy-induced nausea and vomiting and postoperative nausea and vomiting. 5-HT3 receptor antagonists are competitive antagonists of serotonin, the naturally occurring ligand for the 5-HT3 receptor; their therapeutic activity is thought to be the result of a similar mechanism of action (Hesketh, 2008). Interestingly, palonosetron, the latest 5-HT3 receptor antagonist, introduced in 2003, has demonstrated superiority for the prevention of emesis in multiple randomized, prospective clinical trials when compared to first generation 5-HT3 receptor antagonists. Palonosetron was superior to ondansetron in preventing acute (0–24 h) and delayed (24–120 h) emesis in one randomized phase III clinical trial, and superior to dolasetron in a second phase III trial of patients receiving moderately emetogenic chemotherapy (Eisenberg et al., 2003, Gralla et al., 2003). Palonosetron was also significantly more effective in preventing emesis vs. ondansetron throughout the delayed post-chemotherapy period in a third phase III randomized trial in patients receiving highly emetogenic agents (Aapro et al., 2006). In a recent fourth phase III trial, palonosetron provided superior delayed emesis prevention vs. granisetron in patients receiving highly emetogenic chemotherapy (Yoshizawa et al., 2008). Additionally, palonosetron has significantly reduced the severity of nausea to a greater extent than other 5-HT3-receptor antagonists (Decker et al., 2006, Eisenberg et al., 2003, Gralla et al., 2003).

Palonosetron's clinical results could be partly due to its potent binding affinity (Wong et al., 1995) and long half-life (Stoltz et al., 2004). However, these characteristics do not entirely explain palonosetron's clinical differentiation throughout the 5 days following emetogenic chemotherapy. Higher doses of a drug with lower binding affinity for a receptor could make-up for increased binding affinity of another drug. However, palonosetron exhibits greater efficacy against acute emesis associated with moderately emetogenic chemotherapy compared to higher doses of dolasetron or ondansetron (Eisenberg et al., 2003, Gralla et al., 2003). Similarly, more frequent administration of a drug with a short half-life could compensate for the longer-half-life of another. Interestingly, when ondansetron is administered beyond 24 h after chemotherapy, it does not provide substantial protective action in delayed emesis (Geling and Eichler, 2005).

Since palonosetron also differs in chemical structure from other 5-HT3-receptor antagonists (Thompson and Lummis, 2007), we wondered whether improved clinical efficacy could be the result of differences at the molecular level. Recent work in our laboratory has shown that palonosetron exhibits allosteric interactions and positive cooperativity with the 5-HT3 receptor, characteristics that are not displayed by ondansetron and granisetron. We chose ondansetron and granisetron for our comparative studies because together with palonosetron they constitute the majority of the 5-HT3 receptor antagonists used in worldwide clinical practice (Rojas et al., 2008). In the present work we show that palonosetron's differential 5-HT3 receptor binding results in 5-HT3 receptor internalization and prolonged inhibition of receptor function. These actions were minimal in the case of granisetron and non-existent in the case of ondansetron. These molecular interactions provide a rationale that may help explain palonosetron's differentiation in the clinic.

Section snippets

Plasmid preparation and cell transfections

Plasmid preparation and cell transfections have been described before (Rojas et al., 2008).

Dissociation of antagonists from cells and from cell-free membranes

5-HT3A-HEK293 cells in 35 mm dishes were incubated with 5-fold Kd concentrations of [3H]ondansetron (30 nM), [3H]granisetron (5 nM) or [3H]palonosetron (1 nM) for 24 h. At the end of this period, antagonist-containing media were replaced with antagonist-free media and dissociation of radiolabeled antagonist at 37 °C was followed at 0, 2, 4, 6, 8, 15, 30, 60 and 120 min. After removing the medium, cells were

[3H]Palonosetron dissociates completely from cell-free membranes whereas dissociation from whole cells is significantly reduced

Earlier work had suggested the possibility that palonosetron triggered receptor internalization into cells (Rojas et al., 2008). If this was the case in binding studies, the [3H]palonosetron that went inside the cells as part of a [3H]palonosetron-5HT3 receptor complex would not be available for dissociation. Accordingly, we studied dissociation of [3H]palonosetron from 5-HT3A-HEK293 cells after allocating time for receptor internalization (Section 2.2). The amount of [3H]palonosetron that

Discussion

Palonosetron is a 5-HT3 receptor antagonist used for the prevention of chemotherapy-induced nausea and vomiting and postoperative nausea and vomiting that has shown an improved clinical profile over older 5-HT3 receptor antagonists (Aapro et al., 2006, Eisenberg et al., 2003, Gralla et al., 2003). The mechanism behind palonosetron's apparent clinical differentiation has been difficult to ascertain given that all these drugs act on the same receptor. Recent work in our laboratory has shown that

Acknowledgement

We thank Dr. Solomon H. Snyder at The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, School of Medicine for helpful advice during the course of the work and during the preparation of the manuscript.

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