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

J. Daniel Hothersall, Christopher Moffat, Christopher N. Connolly (Lead / Corresponding author)

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    14 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)1252-1262
    Number of pages11
    JournalBritish Journal of Pharmacology
    Volume169
    Issue number6
    DOIs
    Publication statusPublished - Jul 2013

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    Receptors, Serotonin, 5-HT3
    Granisetron
    Down-Regulation
    Binding Sites
    Endocytosis
    Allosteric Site
    Serotonin 5-HT3 Receptor Antagonists
    Nystatin
    COS Cells
    Cell Surface Receptors
    Fluorescence Microscopy
    Nausea
    Vomiting
    palonosetron
    Cell Count
    Enzyme-Linked Immunosorbent Assay
    Pharmacology
    Drug Therapy
    Temperature
    Therapeutics

    Cite this

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    title = "Prolonged inhibition of 5-HT3 receptors by palonosetron results from surface receptor inhibition rather than inducing receptor internalization",
    abstract = "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.",
    author = "Hothersall, {J. Daniel} and Christopher Moffat and Connolly, {Christopher N.}",
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    T1 - Prolonged inhibition of 5-HT3 receptors by palonosetron results from surface receptor inhibition rather than inducing receptor internalization

    AU - Hothersall, J. Daniel

    AU - Moffat, Christopher

    AU - Connolly, Christopher N.

    N1 - This article is protected by copyright. All rights reserved.

    PY - 2013/7

    Y1 - 2013/7

    N2 - 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.

    AB - 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.

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