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ACS Chem Neurosci. 2017 Jul 19;8(7):1570-1576. doi: 10.1021/acschemneuro.7b00079. Epub 2017 Mar 30.

Metabolism and Distribution of Clozapine-N-oxide: Implications for Nonhuman Primate Chemogenetics.

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Yerkes National Primate Research Center , Atlanta, Georgia 30329, United States.
Sano Informed Prescribing, Inc. Franklin, Tennessee 37067, United States.
Department of Psychiatry and Behavioral Sciences, School of Medicine, Emory University , Atlanta, Georgia 30322, United States.
Department of Psychology, Emory University , Atlanta, Georgia 30322, United States.
Department of Neurology, School of Medicine, Emory University , Atlanta, Georgia 30322, United States.
Udall Center of Excellence for Parkinson's Disease Research, Emory University , Atlanta, Georgia 30329, United States.


The use of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in neuroscience has rapidly expanded in rodent studies but has lagged behind in nonhuman primate (NHP) experiments, slowing the development of this method for therapeutic use in humans. One reason for the slow adoption of DREADD technology in primates is that the pharmacokinetic properties and bioavailability of clozapine-n-oxide (CNO), the most commonly used ligand for human muscarinic (hM) DREADDs, are not fully described in primates. We report an extensive pharmacokinetic study using subcutaneous (SC) administration of CNO in five adult rhesus monkeys. CNO reached maximal plasma and cerebrospinal fluid (CSF) concentrations within 2 h after injection, with an observed dose-dependent increase in levels following a 3 and 10 mg/kg SC dose. Since CSF concentrations were below values predicted from unbound plasma concentrations, we investigated whether CNO was restricted from the CNS through active transport at the blood-brain barrier. In vitro assessment demonstrated that CNO is a substrate for P-glycoprotein (Pgp; efflux ratio, 20), thus providing a likely mechanism limiting CNO levels in the CNS. Furthermore, CNO is metabolized to the psychoactive compounds clozapine and n-desmethylclozapine in monkeys. The concentrations of clozapine detected in the CSF are sufficient to activate several types of receptor (including the hM-DREADDs). Our results suggest that CNO metabolism and distribution may interfere with reproducibility and interpretation of DREADD-related experiments in NHPs and calls for a re-evaluation of the use of CNO in DREADD-related experiments in NHPs along with the need to test alternative compounds.


DREADDs; Rhesus macaque; blood−brain barrier; clozapine; pharmacokinetics

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