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

Abstract

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.

The use of chemogenetics in behavioural neuroscience: receptor variants, targeting approaches and caveats

“…A recent study by Raper et al . () examined the pharmacokinetics of s.c. CNO administration in rhesus monkeys. They found that CNO readily metabolizes to clozapine in monkeys, which may interfere with the behavioural interpretation of DREADD‐based experiments in both humans and non‐human primates.…”

“…The solubility of CNO varies depending on concentration and source. For example, CNO obtained from the National Institutes of Health (NIH) appears to be less soluble, requiring DMSO concentrations of up to 15% in a 10 mg·mL À1 solution (Raper et al, 2017). However, 2 mg·mL À1 CNO obtained from NIH has been dissolved in sterile PBS (Carvalho Poyraz et al, 2016).…”

Functional perturbation of forebrain principal neurons reveals differential effects in novel and well-learned tasks

“…Similarly, it was recently shown that CNO is rapidly metabolized into, clozapine and N-desmethylclozapine (NDMC), atypical antipsychotics that act on numerous endogenous neurotransmitter receptors. Finally, when delivered systemically to Rhesus monkeys, CNO was largely restricted from CNS entry at the blood-brain barrier, drawing into question prior reports of DREADD effects on cognitive abilities after systemic CNO delivery (Raper et al, 2017). Moreover, the AlstR system may provide better temporal control over neural activity than DREADDs, in both latency to onset and return to baseline, as explained Alexander and colleagues in their study of DREADD effects on principle neuron physiology in transgenic mice (2009).…”

Combining designer receptors exclusively activated by designer drugs and neuroimaging in experimental models: A powerful approach towards neurotheranostic applications

“…While CNO has been repeatedly used in numerous studies, recent evidence demonstrated that CNO suffers from its limited ability to cross the blood-brain barrier (BBB) and exerts its effects by conversion to clozapine (Figure 2), an atypical antipsychotic drug with numerous endogenous receptors (Chang et al, 1998;Gomez et al, 2017;Jann, Lam, & Chang, 1994;Raper et al, 2017). that could replace CNO (Chen et al, 2015).…”

“…The DREADDs technology as well as the PSAM 4 technology seem to be good candidates for clinical applications. The translational potential of DREADDs has already been suggested by its use in several nonhuman primate studies Grayson et al, 2016;Nagai et al, 2016;Raper et al, 2017; see Section 2 for a detailed discussion). Moreover, AAV technology has proven safe and promising in several clinical studies such as those targeting coagulation disorders, inherited blindness, and Parkinson's disease (Christine et al, 2009;Colella, Ronzitti, & Mingozzi, 2018;Nathwani et al, 2011;Pierce & Bennett, 2015).…”