The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice

Daniel F Manvich,James C Ritchie,Kevin A Webster,David Weinshenker,Stephanie L Foster,Joseph H Porter,Martilias S Farrell

doi:10.1038/s41598-018-22116-z

Abstract

Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). However, recent studies have challenged the long-held assertion that CNO is otherwise pharmacologically inert. The present study aimed to 1) determine whether CNO is reverse-metabolized to its parent compound clozapine in mice (as has recently been reported in rats), and 2) determine whether CNO exerts clozapine-like interoceptive stimulus effects in rats and/or mice. Following administration of 10.0 mg/kg CNO, pharmacokinetic analyses replicated recent reports of back-conversion to clozapine in rats and revealed that this phenomenon also occurs in mice. In rats and mice trained to discriminate 1.25 mg/kg clozapine from vehicle, CNO (1.0–20.0 mg/kg) produced partial substitution for the clozapine stimulus on average, with full substitution being detected in some individual animals of both species at doses frequently used to activate DREADDs. The present demonstration that CNO is converted to clozapine and exerts clozapine-like behavioral effects in both mice and rats further emphasizes the need for appropriate control groups in studies employing DREADDs, and highlights the utility of the drug discrimination procedure as a tool with which to screen the off-target effects of novel DREADD agonists.

Highlights

Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), a series of engineered human muscarinic receptors that respond exclusively to the synthetic ligand clozapine N-oxide (CNO)[1], have emerged as a popular tool among neuroscience researchers
It has been reported that CNO can bind to non-DREADD receptors at concentrations required for DREADD activation[3], and undergoes reverse-metabolism to its parent compound clozapine, an atypical antipsychotic that acts at a variety of pharmacological targets and produces numerous physiological and behavioral effects

Summary

Introduction

Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Back-conversion of CNO to clozapine has been detected in blood in rats[8], and a new study found that clozapine, but not CNO, can cross the blood-brain barrier and activate DREADDs following CNO administration in rats and mice[3] These recent findings would suggest that the reverse-metabolism of CNO to clozapine may be an important determinant of CNO-induced activation of DREADDs in the mouse, but pharmacokinetic data in support of this conclusion are lacking. In light of these collective findings, we sought to rigorously assess whether CNO is pharmacokinetically converted to clozapine in mice, and secondarily, to determine whether CNO administration produces clozapine-like physiological or behavioral effects in rats and/or mice that lack DREADD expression To the latter aim, rather than test the effects of CNO across a battery of behavioral and physiological assessments previously reported to be sensitive to clozapine, we instead chose to employ the drug discrimination procedure, a singular operant-behavioral assay in which animals are trained to use the interoceptive Using the drug discrimination procedure, we sought to examine whether CNO would produce clozapine-like interoceptive stimulus effects in mice and rats trained to discriminate a low dose of clozapine

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Conclusion