Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics in mice

Martin Jendryka,Monika Palchaudhuri,Bastiaan Van Der Veen,Wiebke Nissen,Anton Pekcec,Dennis Kätzel,Birgit Liss,Daniel Ursu

doi:10.1038/s41598-019-41088-2

Abstract

Muscarinic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) gated by clozapine-N-oxide (CNO) allow selective G-protein cascade activation in genetically specified cell-types in vivo. Here we compare the pharmacokinetics, off-target effects and efficacy of CNO, clozapine (CLZ) and compound 21 (Cmpd-21) at the inhibitory DREADD human Gi-coupled M4 muscarinic receptor (hM4Di). The half maximal effective concentration (EC50) of CLZ was substantially lower (0.42 nM) than CNO (8.1 nM); Cmpd-21 was intermediate (2.95 nM). CNO was back-converted to CLZ in mice, and CLZ accumulated in brain tissue. However, CNO itself also entered the brain, and free cerebrospinal fluid (CSF) levels were within the range to activate hM4Di directly, while free (CSF) CLZ levels remained below the detection limit. Furthermore, directly injected CLZ was strongly converted to its pharmacologically active metabolite, norclozapine. Cmpd-21 showed a superior brain penetration and long-lasting presence. Although we identified a wide range of CNO and Cmpd-21 off-targets, there was hardly any nonspecific behavioural effects among the parameters assessed by the 5-choice-serial-reaction-time task. Our results suggest that CNO (3–5 mg/kg) and Cmpd-21 (0.4–1 mg/kg) are suitable DREADD agonists, effective at latest 15 min after intraperitoneal application, but both require between-subject controls for unspecific effects.

Highlights

Designer Receptors Exclusively Activated by Designer Drugs (DREADD) enable the direct modulation of cellular activity by activation of the Gi, Gq- or Gs-protein-coupled signalling pathways
It has been suggested that systemically applied CNO is instead converted into CLZ, and it is this CLZ, rather than CNO, that acts as the DREADD activator in brain tissue due to its strong potency at the two DREADDs, human Gq-coupled M3 muscarinic receptor and human Gi-coupled M4 muscarinic receptor, and its substantially higher blood-brain barrier permeability[7]
Our study shows that, in mice, unbound CNO is present in the brain at sufficient levels to activate the DREADDs directly, after injection of a 3.5 mg/kg dose

Summary

Introduction

Designer Receptors Exclusively Activated by Designer Drugs (DREADD) enable the direct modulation of cellular activity by activation of the Gi-, Gq- or Gs-protein-coupled signalling pathways. Www.nature.com/scientificreports base, CLZ8, has been demonstrated in humans[9,10,11], monkeys[12], guinea pigs[10], rats[7,13,14,15] and, recently, in mice[15,16] This back-conversion of CNO to CLZ, an antagonist at a wide range of G-protein coupled receptors (GPCRs), adds another source of possible off-target effects which may confound findings from in vivo studies – a critical issue that has been previously identified[8]. A recent publication demonstrated elegantly that mice trained to report their interoceptive sensation produced by 1.25 mg/kg CLZ report such sensations after being injected with 10 mg/kg (but not 5 mg/kg or lower doses) of CNO15 This back-conversion introduces difficulties in the rational design of in vivo studies, given that drug conversion is a factor that may vary between species, strains and sex, and depends on many additional external and intrinsic factors including metabolic capacity of the liver or health status. It has not been shown that the back-converted CLZ entering the brain is freely available to activate the DREADD (not unspecifically bound), a finding which could be more accurately measured using CNO concentration in cerebral spinal fluid (CSF) rather than in total brain tissue, as done previously[7]

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Results

Conclusion