Agonist activity of N-desmethylclozapine at δ-opioid receptors of human frontal cortex

The clozapine metabolite N-desmethylclozapine (NDMC) has been recently shown to act at different neurotransmitter receptors and to display both antagonist and agonist activities. We have previously reported that in cells over-expressing the recombinant δ-opioid receptor NDMC behaved as partial agonist with high intrinsic activity, but its action at the receptors naturally expressed in human brain remained to be investigated. In the present study, we examined whether NDMC was able to bind to and activate δ-opioid receptors in membranes of post-mortem human frontal cortex. In radioligand binding assays, NDMC competition curves displayed high- (Ki = 26 nM) and low-affinity (Ki = 3 µM) components, whose proportion was regulated by guanine nucleotides in an agonist-like fashion. In functional assays, NDMC stimulated [35S]GTPγS binding (EC50 = 905 nM) and inhibited cyclic AMP formation (EC50 = 590 nM) as effectively as δ-opioid agonists, whereas clozapine was much less potent and efficacious and clozapine N-oxide was completely inactive. The NDMC agonist activity was potently antagonized by the δ-opioid antagonist naltrindole, but not by the µ-opioid receptor antagonist CTAP (D-phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) or the κ-opioid antagonist nor-binaltorphimine. Moreover, blockade of either acetylcholine muscarinic, dopamine D2 or serotonin 5HT1A receptors failed to affect NDMC agonist activity. These data demonstrate that at clinically relevant concentrations NDMC behaves as an efficacious agonist at δ-opioid receptors of human frontal cortex.