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

BACKGROUND AND PURPOSE: The 5-HT₃ receptor antagonist palonosetron is an important pharmacotherapy for the treatment of emesis and nausea during cancer therapy. Its clinical efficacy is thought to 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 in order to better understand its clinical efficacy. EXPERIMENTAL APPROACH: Cell 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 RESULTS: Chronic exposure to palonosetron reduces the number of available cell surface [³H]granisetron binding sites. This downregulation is not sensitive to either low temperature or pharmacological inhibitors of endocytosis (dynasore or nystatin) suggesting that internalisation does not play a role. This was corroborated by our observation that there is no change in cell surface 5-HT₃ receptor levels or increase in endocytic rate. We find that palonosetron exhibits 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 downregulation involves interactions with an allosteric binding site. CONCLUSIONS AND IMPLICATIONS: Our results indicate that palonosetron 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 at least four days. Allosteric receptor interactions appear to play a role in this phenomenon.