J. E. Leysen

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.

Evidence for functional 5-HT2 receptor sites on human blood platelets.

The aggregation of normal human platelets by 5-hydroxytryptamine (5-HT) is the result of a specific interaction of the monoamine with a platelet receptor since it is not influenced by adrenergic receptor blockade, inhibition of fatty acid cyclo-oxygenase or ADP-scavenging. The 5-HT induced platelet reaction is inhibited in a concentration-dependent way by various serotonergic antagonists; the potency of these compounds in this respect correlates strongly with their potential to inhibit the specific binding of [3H] ketanserin, a selective label for 5-HT2 binding sites, to rat prefrontal cortex and striatum and to cat platelet membranes. This study thus provides evidence for a functional role as true receptor initiating a physiological response of the 5-HT2 receptor on human platelets.

Serotonergic receptor subtypes and vascular reactivity

Two distinct binding sites for serotonin have been identified in brain tissue and labeled 5-HT1 and 5-HT2 binding sites. The receptor mediating contractions of several types of vascular smooth muscle (including the canine basilar artery), aggregation of blood platelets, and contractions of tracheal muscle can be defined as a 5-HT2 serotonergic receptor. The prejunctional inhibitory effect of serotonin on adrenergic neurotransmission is mediated by receptors which have the same affinity for agonists as the 5-HT1 binding sites. The other peripheral actions of serotonin, in particular its vasodilator effects and its excitatory effects on gastrointestinal smooth muscle, do not involve 5-HT2 serotonergic receptors.

OPPOSITE REGULATION OF SEROTONIN-S2 AND DOPAMINE-D2 RECEPTORS IN RAT BRAIN FOLLOWING CHRONIC RECEPTOR BLOCKADE

Rats were chronically treated with setoperone, a mixed serotonin and dopamine antagonist. Alterations in serotonin-S2 and dopamine-D2 receptors in the brain and changes in behavioural responses to tryptamine and apomorphine were studied along with duration of treatment and drug withdrawal. As with neuroleptics, behavioural supersensitivity to apomorphine and increase in the number of striatal dopamine-D2 receptor sites were apparent after 2 days setoperone treatment, both effects were maximal with 14 days treatment and were maintained over more than 20 days drug withdrawal. In contrast to the changes in the dopaminergic system, the rats showed a decreased response to tryptamine and serotonin-S2 receptor sites in the frontal cortex were significantly reduced in numbers. Both effects developed in parallel over 14 days treatment and extinguished over 10 days drug withdrawal. KD-values of radioligand binding to dopamine-D2 and serotonin-S2 receptor sites were unchanged by the setoperone treatment. The concomitant development and extinction of the in vivo and in vitro effects suggests a causal relationship between them. Chronic treatment with a selective histamine-H1 antagonist (levocabastine) or the tranquilizer diazepam did not affect dopamine-D2 or serotonin-S2 receptor sites. These observations demonstrate that in contrast to the receptor regulation theory, serotonin-S2 receptors are down regulated following persistent receptor blockade. Implications for the clinical use of serotonin antagonists and possible molecular mechanisms involved in the receptor regulation have been discussed.

Receptor Interactions of Dopamine and Serotonin Antagonists: Binding In Vitro and In Vivo and Receptor Regulation

The advent of receptor binding techniques has provided new ways of studying the mechanism of action of drugs. In vitro radioligand binding is now currently applied to investigate the specificity or multiple action of compounds. By using the same technique, the binding affinity of a drug can be measured for a variety of neurotransmitter, drug, peptide and ion channel receptor binding sites, providing the drugs receptor binding profile (LEYSEN et al. 1981; LEYSEN 1984). However, in vitro receptor binding is only the initial step in the investigation of drug-receptor interactions. Investigations in vivo are required to allow evaluation of how and where a drug acts. In fact, the study of drug-receptor interactions comprises three main stages: (a) in vitro radioligand receptor binding; (b) in vivo receptor binding, providing information on the accessibility of the drugs to the receptors localized in various central and peripheral tissues, on the drug potency for occupying various receptors, on the duration of receptor occupation and on the relationship between the degree of receptor occupation and pharmacological effects; and (c) the study of receptor regulation: the effect of chronic drug treatment on receptor alterations compared with alterations in functional responses in vivo. In this article, we will illustrate the three stages of investigation of receptor interactions and discuss the relevance and importance of the findings, using as examples three drugs known in psychopharmacological research: (a) the neuroleptic haloperidol, a prototype of a dopamine D2 antagonist: (b) Setoperone, a potential antipsychotic agent with very potent serotonin S2 and moderate D2 antagonistic activity (CEULEMANS et al. 1985; LEYSEN et al. 1986); and (c) ritanserin, a potent and long-acting S2 antagonist (LEYSEN et al. 1985), which has revealed therapeutic activity in dysthymia and negative symptoms of schizophrenia (REYNTJENS et al. 1986; GELDERS et al. 1986). Particular attention will be paid to the problem of receptor regulation. We challenge the general applicability of the receptor regulation theory, which states that persistent receptor stimulation causes desensitisation and receptor downregulation, whereas chronic deprivation of receptor stimulation leads to supersensitivity and receptor upregulation. Recent research has revealed that the theory does not hold for S2 receptor alterations, which were found to downregulate following chronic receptor blockade.

The signal transducing system coupled to serotonin-S2 receptors

The signal transducing system coupled to the serotonin-S2 receptor on platelets involves metabolism of inositolcontaining phospholipid, elevation of intracellular free Ca2+ and activation of protein kinase C. Evidence for coupling of the serotonin-S2 receptor to the same signal transducing system in brain and smooth muscle tissue is reviewed.

Thromboxane A2/prostaglandin endoperoxide (TXA2/PG-END) receptor binding properties in human platelets of ridogrel, a combined TXA2 synthase inhibitor--TXA2/PG-END receptor antagonist.

Ridogrel, a potent thromboxane A2 (TXA2) synthase inhibitor, also has thromboxane A2 prostaglandin endoperoxide (TXA2/PG-END) receptor antagonistic properties as documented in functional studies of human platelets. In the present study, the binding affinities of the TXA2 synthase inhibitors, ridogrel, dazoxiben, dazmegrel and pirmagrel, and the TXA2/PG-END receptor antagonists, GR32191, L670596, SQ29548, ICI159995, AH69212 and sulotroban, for the TXA2/PG-END receptor labelled with [3H]SQ29548 on intact human platelets were assessed. The potencies of the TXA2/PG-END receptor antagonists to inhibit specific [3H]SQ29548 binding to intact human platelets ranged between 1.2 nM and 6 200 nM and corresponded to the ability of the drugs to suppress human platelet aggregation induced by TXA2/PG-END receptor stimulation with U46619 and collagen. The TXA2 synthase inhibitors dazoxiben, dazmegrel and pirmagrel could not inhibit specific [3H]SQ29548 binding to intact human platelets, tested up to 10-5 M, nor suppress human platelet aggregation, indicating lack of any receptor antagonistic properties. Ridogrel, however, directly bound to the TXA2/PG-END receptor with micromolar affinity (IC50 = 5.2 μM) and inhibited U46619–27, or collagen-induced platelet aggregation, with ED50-values of 27 μM and 4.7 μM respectively. The present study thus demonstrates that antagonism by ridogrel of TXA2/PG-END receptor activation on platelets as denned in functional tests, coincides with inhibition of specific ligand binding to the receptors.