norclozapine and Neuroblastoma

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 delta-opioid receptor (CHO/DOR), NDMC behaved as a full agonist both in stimulating [(35)S]GTPgammaS binding (pEC(50)=7.24) and in inhibiting cyclic AMP formation (pEC(50)=6.40). NDMC inhibited [(3)H]naltrindole binding to CHO/DOR membranes with competition curves that were modulated by guanine nucleotides in an agonist-like manner. Determination of intrinsic efficacies by taking into consideration both the maximal [(35)S]GTPgammaS 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 delta-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 kappa-opioid receptor and was inactive at mu-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 delta-opioid receptor. Moreover, in membranes of different brain regions, NDMC stimulated [(35)S]GTPgammaS 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 delta-opioid receptor agonist and suggest that this unique property may contribute, at least in part, to the clinical actions of the atypical antipsychotic clozapine.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Antipsychotic Agents; CHO Cells; Clozapine; Cricetinae; Cricetulus; Cyclic AMP; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Mice; Neuroblastoma; Protein Binding; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Recombinant Proteins

The goal of the present study was to determine the effects of clozapine (Cloz) and its metabolites norclozapine (Norcloz) and clozapine-N-oxide (Cloz-N-oxide) on the 5-HT(2) receptor system on the levels of protein and gene expression in in vitro systems of primary cortical cells of the rat and human hippocampal SHS5Y5 neuroblastoma cells.. Clinically relevant concentrations of Cloz (200/400 ng/ml) and its metabolites (200 ng/ml) were used for the examination of the effects of Cloz and its metabolites on serotoninergic 5-HT(2) receptor parameters (density, affinity and mRNA levels) as well as on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels in primary cortical cells of the rat after treatment for 24 h under in vitro conditions. To compare the results to human cells, we also measured treatment-induced changes in 5-HT(2) and GAPDH mRNA levels in human hippocampal SHS5Y5 cells.. A significant decrease was found in primary cortical cells for 5-HT(2) receptor density (Cloz 200/Cloz 400/Norcloz 200 and Cloz-N-oxide 200 vs. control) and 5-HT(2A) receptor mRNA levels (Cloz 200 vs. control). 5-HT(2A) receptor mRNA levels were also significantly reduced (Norcloz 200 vs. control) in SHS5Y5 cells. GAPDH mRNA levels were not affected.. The results of the present study show that Cloz and Norcloz induce significant alterations on the 5-HT(2) receptor system in primary cortical cells of the rat and in human hippocampal cells.

Topics: Analysis of Variance; Animals; Cells, Cultured; Cerebral Cortex; Clozapine; Dose-Response Relationship, Drug; Gene Expression Regulation; Hippocampus; Humans; In Vitro Techniques; Neuroblastoma; Neurons; Protein Binding; Radioligand Assay; Rats; Receptors, Serotonin, 5-HT2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serotonin Antagonists