Walter Gommeren

Risperidone compared with new and reference antipsychotic drugs: in vitro and in vivo receptor binding

Risperidone and its active metabolite 9-OH-risperidone were compared to reference antipsychotic drugs (haloperidol, pipamperone, fluspirilene, clozapine, zotepine) and compounds under development (olanzapine, seroquel, sertindole, ORG-5222, ziprasidone) for in vitro binding to neurotransmitter receptors in brain tissue and on membranes of recombinant cells expressing cloned human receptors and for in vivo occupancy of neurotransmitter receptors in rat and guinea-pig brain following acute treatment (2 h., s.c.). An ex vivo autoradiography technique was applied to determine the receptor occupancy by the drugs administered in vivo. Of particular interest are the central 5HT2A receptors and D2-type receptors. Predominant 5HT2A receptor antagonism is supposed to add to an atypical profile of the antipsychotics (treatment of the negative symptoms, low incidence of extrapyramidal side effects). D2 antagonism is required for the treatment of positive symptoms. A contribution of the new dopamine receptor subtypes D3 and in particular D4 receptors has been proposed.In vitro, all compounds, except the ‘typical’ antipsychotics haloperidol and fluspirilene, showed higher affinity for 5HT2A than for D2 receptors. Subnanomolar affinity for human 5HT2A receptors was observed for ORG-5222, sertindole, resperidone, 9-OH-risperidone and ziprasidone. Fluspirilene, ORG-5222, haloperidol, ziprasidone, risperidone, 9-OH-risperidone and zotepine displayed nanomolar affinity for human D2 receptors. Sertindole and olanzapine were slightly less potent. Pipamperone, clozapine and seroquel showed 2 orders of magnitude lower D2 affinity in vitro. Clozapine, but even more so pipamperone, displayed higher affinity for D4 than for D2 receptors. For most other compounds, D4 affinity was only slightly lower than their D2 affinity. Seroquel was totally devoid of D4 affinity. None of the compounds had nanomolar affinity for D1 receptors; their affinity for D3 receptors was usually slightly lower than for D2 receptors.In vivo, ORG-5222, risperidone, pipamperone, 9-OH-risperidone, sertindole, olanzapine, zotepine and clozapine maintained a higher potency for occupying 5HT2A than D2 receptors. Risperidone and ORG-5222 had 5HT2A versus D2 potency ratio of about 20. Highest potency for 5HT2A receptor occupancy was observed for ORG-5222 followed by risperidone and olanzapine. Ziprasidone exclusively occupied 5HT2A receptors. ORG-5222, haloperidol, fluspirilene and olanzapine showed the highest potency for occupying D2 receptors. No regional selectivity for D2 receptor occupancy in mesolimbic versus nigrostriatal areas was detected for any of the test compounds. Risperidone was conspicuous because of its more gradual occupancy of D2 receptors; none of the other compounds showed this property. The various compounds also displayed high to moderate occupancy of adrenergic α1 receptors, except fluspirilene and ziprasidone. Clozapine, zotepine, ORG-5222 and sertindole occupied even more α1 than D2 receptors. Clozapine showed predominant occupancy of H1 receptors and occupied cholinergic receptors with equivalent potency to D2 receptors. A stronger predominance of 5HT2A versus D2 receptor occupancy combined with a more gradual occupancy of D2 receptors differentiates risperidone and its 9-OH-metabolite from the other antipsychotic compounds in this study. The predominant 5HT2A receptor occupancy probably plays a role in the beneficial action of risperidone on the negative symptoms of schizophrenia, whereas maintenance of a moderate occupancy of D2 receptors seems adequate for treating the positive symptoms of schizophrenia. A combined 5HT2A and D2 occupancy and the avoidance of D2 receptor overblockade are believed to reduce the risk for extrapyramidal symptoms.

Spiperone: A ligand of choice for neuroleptic receptors: 1. Kinetics and characteristics of in vitro binding

Abstract A binding assay for neuroleptic receptors has been developed with spiperone as the labelled ligand. As compared to haloperidol, spiperone showed a 2-times higher ratio of specific versus aspecific binding, a 10-fold greater association constant and a slower dissociation of the receptor ligand complex. The receptor sites labelled by spiperone appeared to be for a great deal similar to those of haloperidol, however certain differences were apparent; the number of receptor sites per gram of tissue was found to be higher for the former; spiperone showed a biphasic receptor ligand dissociation curve which was not observed for haloperidol; also a slight difference in physical stability between spiperone and haloperidol binding sites was noted. Inhibition studies using antagonists and agonists in comparison with the pharmacological profile of the compounds showed that the receptor sites labelled by both ligands are mainly of dopaminergic nature, but also a serotonergic and to a minor extent a noradrenergic component should be involved. Within the striatum haloperidol binding sites seemed to be relatively more related to dopaminergic sites whilst the spiperone binding sites appeared to comprise a higher serotonergic component. It is concluded that spiperone is a more suitable ligand than haloperidol for studying the neuroleptic receptors. The use of different labelled ligands provided evidence for the heterogeneity of the neuroleptic binding sites in the striatum.

[3H]sufentanil, a superior ligand for μ-opiate receptors: Binding properties and regional distribution in rat brain and spinal cord

Stereospecific [3H]sufentanil binding, inhibited by dextromoramide, represents 90% of the total binding in membrane preparations of rat brain and spinal cord. Scatchard plots of the binding in the forebrain, at 37 degrees C in Tris-HCl buffer without and with 120 mM NaCl, were rectilinear; KD = 0.13 nM and 0.31 nM, Bmax = 13 fmol/mg tissue and 9.9 fmol/mg tissue in the absence and the presence of sodium ions respectively. The reduction in binding affinity in the presence of sodium ions was found to be due to a 9.7 fold enhancement of the initial dissociation rate from t1/2 = 2.1 min in the absence to 13 s in the presence of sodium ions. The [3H]sufentanil binding properties were superior to those of [3H]fentanyl, [3H]dihydromorphine and [3H]naloxone; [3H]sufentanil showed an unmatched favourable ratio of stereospecific versus non-specific binding; it had a 7.7, 20 and 40 fold binding affinity than the above ligands respectively. Due to its relatively slow dissociation rate, a more accurate estimation of the Bmax value was obtained with [3H]sufentanil than with the other, fast dissociating 3H-ligands (t1/2 less than 10 s). A total of 37 narcotic analgesic agonists and antagonists belonging to 5 different major structural classes all inhibited stereospecific [3H]sufentanil binding in a competitive way. There was no relationship between binding affinities and lipophilicity and degree of ionization of the compounds. Binding affinities correlated highly significantly with the analgesic potency measured in vivo, demonstrating that [3H]sufentanil labels mu-opiate receptor sites which mediate narcotic analgesia. Moreover, the binding affinity of sufentanil for delta-type binding sites labelled by [3H] [D-Ala2,D-Leu5]enkephalin was found to be 100 times lower than its binding affinity for the mu-receptor sites. [3H]Sufentanil was used for a detailed investigation of the regional distribution of mu-opiate receptor sites in the brain; Bmax and KD values were measured in the dorsal and ventral spinal cord.

Optimal Conditions for [3H]Apomorphine Binding and Anomalous Equilibrium Binding of [3H]Apomorphine and [3H]Spiperone to Rat Striatal Membranes: Involvement of Surface Phenomena Versus Multiple Binding Sites

I. Binding of [3H]apomorphine to dopaminergic receptors in rat striatum was most reproducible and clearly detectable when incubations were run at 25°C in Tris‐HCl buffer, pH 7.5, containing 1 mM‐EDTA and 0.01% ascorbic acid, using a washed total‐membrane fraction. The receptor binding was stereospecifically inhibited by (+)‐butaclamol, and dopamine agonists and antagonists showed high binding affinity for these sites. Unlabelled apomorphine inhibited an additional nonstereospecific binding site, which was unrelated to dopamine receptors. EDTA in the incubation mixture considerably lowered nonstereospecific [3H]apomorphine binding, apparently by preventing the complexation of the catechol moiety with metal ions which were demonstrated in membrane preparations. Stereospecific [3H]apomorphine binding was not detectable in the frontal cortex, whereas in the absence of EDTA much saturable nonstereospecific binding occurred. II. Kinetic patterns of stereospecific [3H]spiperone and [3H] apomorphine binding to rat striatal membranes and the inhibition patterns of a dopamine antagonist and an agonist were evaluated at different temperatures in high‐ionic‐strength Tris buffer with salts added and low‐ionic‐strength Tris buffer with EDTA. Apparent KD, values of spiperone decreased with decreasing tissue concentrations. KD, values of both spiperone and apomorphine were little influenced by temperature changes. Scatchard plots of the stereospecific binding changed from linear to curved; the amount of nonstereospecific binding of the 3H ligands varied considerably, but in opposite directions for spiperone and apomorphine in the different buffers. In various assay conditions, interactions between agonists, and between antagonists, appeared fully competitive, but agonist‐antagonist interactions were of mixed type. The anomalous binding patterns are interpreted in terms of surface phenomena occurring upon reactions of a ligand with complex physicochemical properties and nonsolubilized sites on membranes suspended in a buffered aqueous solution. It is concluded that anomalous binding patterns are not necessarily an indication of binding to multiple sites or involvement of distinct receptors for high‐affinity agonist and antagonist binding.

Structure of the Human Serotonin 5‐HT4 Receptor Gene and Cloning of a Novel 5‐HT4 Splice Variant

Abstract: Several variants of the serotonin 5‐HT4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5‐HT4 gene. Based on sequence analysis seven C‐terminal variants (a‐g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C‐terminal b variant; therefore, we call this receptor 5‐HT4(hb). This novel receptor variant was expressed transiently in COS‐7 cells, and its pharmacological profile was compared with those of the previously cloned 5‐HT4(a) and 5‐HT4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5‐HT4 ligands, no significant differences were detected. However, the broadly used 5‐HT4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5‐HT4(a) and 5‐HT4(b) variant but showed partial agonistic activity on the 5‐HT4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5‐HT4 ligands in different model systems.

Demonstration of S2-receptor binding sites on cat blood platelets using [3H]ketanserin

[3H]Ketanserin showed substantial binding to cat platelet membranes; 40% of this was specific binding, characterized by a plateau in serotonin antagonist inhibition curves, and another 40% was non-specific binding to structural recognition sites displaceable by unlabelled ketanserin. Specific [3H]ketanserin binding to platelets shows the features of serotonin S2-receptor binding sites previously identified in rat pre-frontal cortex and in striatum. In platelets, KD = 1.02 nM and Bmax = 86 fmol/10(9) platelets; the Ki values of 21 compounds were similar in the three different tissues.

Mapping of serotonin 5‐HT4 receptor mRNA and ligand binding sites in the post‐mortem human brain

The anatomical localization of 5‐HT4 receptor mRNA and 5‐HT4 receptor protein was examined in sections of post‐mortem human brain by in situ hybridization histochemistry and radioligand receptor autoradiography. In the in situ hybridization study, the highest levels of 5‐HT4 receptor mRNA were found in caudate nucleus, putamen, nucleus accumbens, and in the hippocampal formation. No 5‐HT4 receptor mRNA was detected in globus pallidus and substantia nigra. For receptor autoradiography, two new and highly selective radioligands were compared: [3H]prucalopride, which preferentially labels the G‐protein coupled fraction of receptors, and [3H]R116712, which labels the entire receptor population at subnanomolar concentrations. [3H]Prucalopride and [3H]R116712 binding was performed on human brain hemisphere sections. The highest densities for both radioligands were found in the basal ganglia (caudate nucleus, putamen, nucleus accumbens, globus pallidus, substantia nigra). Moderate to low densities were detected in the hippocampal formation and in the cortical mantle. Mismatches between 5‐HT4 receptor mRNA and binding sites in the globus pallidus and the substantia nigra suggested that the binding sites may be localized on axonal projections originating from the striatum. To compare densities of binding sites, concentration binding curves with [3H]prucalopride, [3H]R116712 and [3H]GR113808 were performed on membranes from homogenates of several human brain regions. Comparison of Bmax‐values obtained with [3H]prucalopride and [3H]R116712 indicated that the G‐protein coupled fraction of 5‐HT4 receptors in the substantia nigra was exceptionally high (54%) in comparison with percentages (16–27%) found in the frontal cortex, the striatum and the hippocampus. Such a high percentage (40%) of [3H]prucalopride vs. [3H]R116712 binding was also observed in the substantia nigra in the receptor autoradiography experiments. The [3H]prucalopride binding was GppNHp‐sensitive, whereas [3H]R116712 and [3H]GR113808 was not. These data indicate that in the substantia nigra 5‐HT4 receptors are more strongly coupled to their signal transduction pathway than in other brain regions. Synapse 36:35–46, 2000.

Cloning and expression of a human serotonin 5-HT4 receptor cDNA

Abstract: Using a combination of library screening and nested PCR based on a partial human serotonin 5‐HT4 receptor sequence, we have cloned the complete coding region for a human 5‐HT4 receptor. The sequence shows extensive similarity to the published porcine 5‐HT4A and rat 5‐HT4L receptor cDNA; however, in comparison with the latter, we find an open reading frame corresponding to only 388 amino acids instead of 406 amino acids. This difference is due to a frame shift caused by an additional cytosine found in the human sequence after position 1,154. Moreover, we also found the same additional cytosine in the rat 5‐HT4 sequence. We confirmed the occurrence of the sequence by examining this part of the sequence in genomic DNA of 10 human volunteers and in rat genomic DNA. Based on a part of the genomic 5‐HT4 receptor sequence that was identified in the cloning process, there seem to be at least two possible splice sites in the coding region of the gene. The human 5‐HT4 receptor, transiently expressed in COS‐7 cells, showed radioligand binding properties similar to 5‐HT4 receptors in guinea pig striatal tissue. [3H]GR 113808 revealed KD values of 0.15 ± 0.01 nM for the human receptor and 0.3 ± 0.1 nM in the guinea pig tissue. Binding constants were determined for four investigated 5‐HT4 antagonists and three agonists, and appropriate binding inhibition constants were found in each case. Stimulation of transfected COS‐7 cells with 5‐HT4‐specific agonists caused an increase in cyclic AMP levels.

Quantitative autoradiographic distribution of gamma-hydroxybutyric acid binding sites in human and monkey brain

gamma-Hydroxybutyric acid (GHB), a naturally occurring metabolite of GABA, is present in micromolar concentrations in various areas of the mammalian brain. Specific GHB binding sites, uptake system, synthetic and metabolizing enzymes have been identified in CNS. The present study shows the anatomical distribution of GHB binding sites in sections of primate (squirrel monkey) and human brain by radioligand quantitative autoradiography. In both species the highest densities of binding sites were found in the hippocampus, high to moderate densities in cortical areas (frontal, temporal, insular, cingulate and entorhinal) and low densities in the striatum; no binding sites were detected in the cerebellum. High density of GHB binding was found in the monkey amygdala. In addition the binding characteristics of [(3)H]GHB to membrane preparations of human brain cortex were examined. Scatchard analysis and saturation curves revealed both a high (K(d1) 92+/-4.4 nM; B(max1) 1027+/-110 fmol/mg protein) and a low-affinity binding site (K(d2) 916+/-42 nM; B(max2) 8770+/-159 fmol/mg protein). The present study is the first report on the autoradiographic distribution of specific GHB binding sites in the primate and human brain: such distribution is in both species in good agreement with the distribution found in the rat brain.

Autoradiographic evidence for the occlusion of rat brain dopamine D3 receptors in vivo.

[125I]Iodosulpride binding was studied in frontal rat brain sections by quantitative autoradiography. Using preincubated (= washed) sections, selective labelling and identification of dopamine D3 receptors was obtained using 0.2 nM [125I]iodosulpride in the presence of 100 nM domperidone for the occlusion of the D2 receptors. A high density of D3 receptors was noticed in the islands of Calleja. When preincubation of the sections was omitted, no D3 receptor labelling could be achieved, indicating tight binding to the receptor of an endogenous inhibitor. Such a tight receptor occupancy was not observed for the D2 receptor and various other neurotransmitter receptors. The occlusion of the D3 receptor could be prevented by tetrabenazine-induced monoamine depletion of the rats. It can be concluded, therefore, that D3 receptors are massively occupied by a monoamine, likely to be dopamine. This observation prompts the question to what extent dopamine D3 receptors can become occupied in vivo by systematically applied exogenous compounds.