Dose-response titration of clozapine as an alternative ligand for Designer Receptors Exclusively activated by Designer Drugs(DREADD) technology

Abstract

Clozapine N-oxide (CNO) is an inactive form of clozapine (CLZ) which activates G-protein-coupled receptors (GPCRs). CNO has been widely used in chemogenetics as a synthetic ligand for human muscarinic designer receptors (DREADDs). However, recent researches raised concerns regarding effectiveness of CNO as it exhibit limited ability to penetrate the Blood Brain Barrier(BBB) and back-converted to CLZ which show higher affinity to DREADDs than CNO.

The aim of the study was to validate CLZ as alternative DREADD agonist and determine safe margin of dose with minimal side effects. Titration was performed on eights naive rats which were given varying dose of CLZ and saline to detect the functional and behavioral changes due to pharmacological effects of CLZ depending on the dosage. Electrophysiology and immunohistology were performed on DREADD expressed rats to detect neuronal activity changes due to varying dosage of CLZ. Extracellular spikes were recorded from the DREADD expressed rats.

In wild type animals, CLZ 1mg/kg injection promoted brain activity pattern with significantly higher (P <0.001) off target effects compared to CLZ 0.01 and CLZ 0.1 mg/kg in T scans. Openfield test results show that injection of CLZ above 0.1 mg/kg results in significant (P <0.001) decrease in travel distance and increase in immobility. In DREADD expressed animals, electrophysiology and immunohistology results of CLZ 0.01 and CLZ 0.1 mg/kg show recruitment of both excitatory and inhibitory neurons. In conclusion, CLZ does of 0.01 and 0.1 mg/kg are valid parameters for chemogenetic stimulation and requires further investigation for tradeoff between DREADD action and off-target effects.