Prolonged inhibition of 5‐HT3 receptors by palonosetron results from surface receptor inhibition rather than inducing receptor internalization

J Daniel Hothersall,Christopher N Connolly,Christopher Moffat

doi:10.1111/bph.12204

J Daniel Hothersall, Christopher N Connolly + Show 1 more

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https://doi.org/10.1111/bph.12204

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Abstract

Background and PurposeThe 5-HT3 receptor antagonist palonosetron is an important treatment for emesis and nausea during cancer therapy. Its clinical efficacy may result from its unique binding and clearance characteristics and receptor down-regulation mechanisms. We investigated the mechanisms by which palonosetron exerts its long-term inhibition of 5-HT3 receptors for a better understanding of its clinical efficacy.Experimental ApproachCell surface receptors (recombinantly expressed 5HT3A or 5HT3AB in COS-7 cells) were monitored using [3H]granisetron binding and ELISA after exposure to palonosetron. Receptor endocytosis was investigated using immunofluorescence microscopy.Key ResultsChronic exposure to palonosetron reduced the number of available cell surface [3H]granisetron binding sites. This down-regulation was not sensitive to either low temperature or pharmacological inhibitors of endocytosis (dynasore or nystatin) suggesting that internalization did not play a role. This was corroborated by our observation that there was no change in cell surface 5-HT3 receptor levels or increase in endocytic rate. Palonosetron exhibited slow dissociation from the receptor over many hours, with a significant proportion of binding sites being occupied for at least 4 days. Furthermore, our observations suggest that chronic receptor down-regulation involved interactions with an allosteric binding site.Conclusions and ImplicationsPalonosetron acts as a pseudo-irreversible antagonist causing prolonged inhibition of 5-HT3 receptors due to its very slow dissociation. In addition, an irreversible binding mode persists for at least 4 days. Allosteric receptor interactions appear to play a role in this phenomenon.

Highlights

The 5-HT3 receptor is a cationic ligand-gated ion channel that mediates neuronal depolarization in both the central and1252 British Journal of Pharmacology (2013) 169 1252–1262 peripheral nervous system in response to 5-HT
We found that palonosetron induced a long-term down-regulation in the number of available 5-HT3 receptor binding sites that persisted for greater than 24 h, and that recovery from this inhibition did not require the delivery of new surface receptors, suggesting that there was instead slow dissociation from the receptor
To determine whether prior exposure to palonosetron causeds a long-term loss of 5-HT3 receptor binding sites, as reported previously (Rojas et al, 2008; 2010b) we incubated 5-HT3A receptor expressing cells with palonosetron (1 nM) for 150 min at 37°C followed by removal of unbound ligand and incubation in drug-free media for 150 min to permit palonosetron unbinding

Summary

Introduction

The 5-HT3 receptor is a cationic ligand-gated ion channel that mediates neuronal depolarization in both the central and1252 British Journal of Pharmacology (2013) 169 1252–1262 peripheral nervous system in response to 5-HT (receptor nomenclature follows Alexander et al, 2011). The 5-HT3 receptor antagonists are important tools in cancer therapy to control emesis and nausea during chemotherapy, radiotherapy, surgery or anaesthesia (Aapro, 2007; Muchatuta and Paech, 2009). The antiemetic activity of 5-HT3 receptor antagonists is believed to involve inhibition of these peripheral receptors, a role of 5-HT3 receptor antagonism in the CNS has been proposed (Costall and Naylor, 1992; Hornby, 2001; Minami et al, 2003). AND PURPOSE The 5-HT3 receptor antagonist palonosetron is an important treatment for emesis and nausea during cancer therapy. We investigated the mechanisms by which palonosetron exerts its long-term inhibition of 5-HT3 receptors for a better understanding of its clinical efficacy.

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