N-Desmethylclozapine, a Major Clozapine Metabolite, Acts as a Selective and Efficacious δ-Opioid Agonist at Recombinant and Native Receptors

Abstract

The present study examined the effects of N-desmethylclozapine (NDMC), a biologically active metabolite of the atypical antipsychotic clozapine, at cloned human opioid receptors stably expressed in Chinese hamster ovary (CHO) cells and at native opioid receptors present in NG108-15 cells and rat brain. In CHO cells expressing the d-opioid receptor (CHO/DOR), NDMC behaved as a full agonist both in stimulating [ 35 S]GTPgS binding (pEC 50 ¼ 7.24) and in inhibiting cyclic AMP formation (pEC 50 ¼ 6.40). NDMC inhibited [ 3 H]naltrindole binding to CHO/DOR membranes with competition curves that were modulated by guanine nucleotides in an agonistlike manner. Determination of intrinsic efficacies by taking into consideration both the maximal [ 35 S]GTPgS binding stimulation and the extent of receptor occupancy at which half-maximal effect occurred indicated that NDMC had an efficacy value equal to 82% of that of the full d-opioid receptor agonist DPDPE, whereas clozapine and the other clozapine metabolite clozapine N-oxide displayed much lower levels of agonist efficacy. NDMC exhibited poor agonist activity and lower affinity at the k-opioid receptor and was inactive at m-opioid and NOP receptors. In NG108-15 cells, NDMC inhibited cyclic AMP formation and stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 by activating the endogenously expressed d-opioid receptor. Moreover, in membranes of different brain regions, NDMC stimulated [ 35 S]GTPgS binding and regulated adenylyl cyclase activity and the effects were potently antagonized by naltrindole. These data demonstrate for the first time that NDMC acts as a selective and efficacious d-opioid receptor agonist and suggest that this unique property may contribute, at least in part, to the clinical actions of the atypical antipsychotic clozapine. Neuropsychopharmacology (2007) 32, 773-785.

Exploring Binding Properties of Agonists Interacting with a δ-Opioid Receptor

“…Within the framework of the message-address model of opioid receptor pharmacology the binding pocket of the receptor can be divided into two regions: the lower, highly conserved, is involved in recognizing the message of the ligand (efficacy), the upper, poorly conserved among opioid subtypes, is responsible of discriminating the address (selectivity). A correspondence between Basin 1 and the upper region of the binding pocket and between Basin 2 and the lower part might account for the enhanced activity of DSM compared to CLZ [37], [38]. Interestingly, although DSM and CLZ sample very close spatial regions associated with Basin 1 (see Figure 4), the list of above-threshold interacting residues extracted from Figure 5 does not overlap, i.e., there are no residues that interact with both compounds in Basin 1.…”

“…Additionally, DSM activates -opioid receptors endogenously expressed in brain while in the brain membrane preparations CLZ displays negligible agonist activity [35], [37]. Determination of intrinsic efficacies by taking into consideration both the maximal [S]GTPS binding stimulation and the extent of receptor occupancy at which half-maximal effect occurred indicated that DSM has an efficacy value equal to 82% of that of the full -opioid-receptor agonist DPDPE, whereas CLZ displays much lower levels of agonist efficacy [37], [38]. Since the two compounds differ in a methyl group, they are particularly well suited for a comparative study, homing in on understanding the microscopic details of their interactions with the target.…”

δ‐Opioid receptors stimulate GLUT1‐mediated glucose uptake through Src‐ and IGF‐1 receptor‐dependent activation of PI3‐kinase signalling in CHO cells

“…CHO/DOR cells were developed by transfecting CHO-K1 cells with pcDNA3.1-Hygro(+)vector encoding the human d-opioid receptor (Onali and Olianas, 2007) using PolyFect (Qiagen GmbH, Hilden, Germany) as transfection reagent following the manufacturer's instructions. Cells were selected by their resistance to 1 mg·mL -1 of hygromycin for 4 weeks and cell clones were isolated by using cloning cylinders.…”

Spatiotemporal Mapping Techniques Show Clozapine Impairs Neurogenic and Myogenic Patterns of Activity in the Colon of the Rabbit in a Dose-Dependent Manner

“…This effect is antagonized by the non-selective opioid antagonist naloxone ( p < 0.05), implying an opioid mechanism of action is involved in clozapine-induced anti-nociception. Norclozapine has also been reported to act as a opioid receptor agonist (Onali and Olianas, 2007; Olianas et al, 2009). Activation of neural mu and kappa subtypes of opioid receptors have been reported to inhibit neurally mediated contraction of both circular and longitudinal muscle in the ileum of the guinea pig (Kosterlitz and Waterfield, 1972) and inhibit ileal peristalsis in vitro (Kromer et al, 1980) and opioids like morphine are potently constipating (Frantzides et al, 1992).…”