Schizophrenia is a complex, progressive, and debilitating disorder of early onset that afflicts approximately 1% of the population. It is characterized by disorganized thought and a constellation of symptoms generally classified as positive (delusions and hallucinations), negative (social withdrawal, blunted affect, and mutism), and cognitive (deficits in attention, working and verbal memory, social cognition, and executive function) (Kapur and Mamo, 2003; Meltzer, 2004; Lieberman et al., 2005).

Conventional neuroleptics like haloperidol, which behaves principally as a dopaminergic antagonist, moderate positive symptoms. However, some 20 to 30% of patients are resistant, and efficacy against negative and cognitive symptoms is limited. Furthermore, there is only a modest therapeutic window to doses provoking extrapyramidal motor symptoms (EPSs), and long-term utilization may be associated with the development of irreversible tardive dyskinesia (Dean and Scarr, 2004; Lieberman et al., 2005; Margolese et al., 2005). The “atypical” antipsychotic, clozapine, which potently interacts with several classes of monoaminergic receptor, is active in certain neuroleptic-refractory patients, does not elicit EPS, and is more effective than haloperidol against negative symptoms (Dean and Scarr, 2004; Meltzer, 2004; Lieberman et al., 2005). However, its advantages are offset by the risk of agranulocytosis. Furthermore, clozapine elicits sedation, obesity, cardiovascular-autonomic, and metabolic side effects via actions at α₁-adrenoceptors (ARs), histamine (H₁) receptors, and muscarinic receptors (Cunningham-Owens, 1996; Millan et al., 2000a; Pacher and Kecskemeti, 2004). A number of other multireceptorial antipsychotics have been introduced with similar, although not identical, broad-based receptor-binding profiles: notably, olanzapine and risperidone (Millan et al., 2000a; Dean and Scarr, 2004; Lieberman et al., 2005; McCue et al., 2006). However, despite their undeniable utility, they do not fully reproduce the clinical benefits of clozapine, and therapeutic indexes to doses eliciting undesirable actions are limited; in particular, EPS for risperidone and obesity for olanzapine (Dean and Scarr, 2004; Lieberman et al., 2005; Margolese et al., 2005). Moreover, although the more recently launched partial dopaminergic agonist, aripiprazole, is well tolerated, its efficacy is not superior to that of other agents (Abi-Dargham and Laruelle, 2005; Burstein et al., 2005; McCue et al., 2006; Urban et al., 2007b). Clearly, there remains a need for innovative drugs possessing improved efficacy and favorable side-effect profiles.

It remains to be clinically demonstrated that antipsychotics that act independently of dopaminergic mechanisms are robustly and consistently effective, and several authorities have recently reasserted the importance of dopamine D₂ receptors in the management of schizophrenia (Kapur and Mamo, 2003; McGowan et al., 2004; Abi-Dargham and Laruelle, 2005). Nonetheless, no clinically employed antipsychotic differentiates D₂ from D₃ receptors (Vanhauwe et al., 2000; Dean and Scarr, 2004; Burstein et al., 2005; Joyce and Millan, 2005), so their respective importance remains uncertain. Interestingly, several arguments support current interest in D₃ receptors as a target for the potentially improved treatment of schizophrenia (Joyce and Millan, 2005; Boeckler and Gmeiner, 2006; Sokoloff et al., 2006).

First, although the density of D₃ versus D₂ receptors in rodent central nervous system is modest, they are better represented in primates and in man (Joyce, 2001; Sokoloff et al., 2006). In all species, in contrast to the enrichment of D₂ sites in striatal regions, D₃ receptors are concentrated in the nucleus accumbens and corticolimbic structures implicated in the control of mood, cognition, and the pathogenesis of schizophrenia (Joyce, 2001; Joyce and Millan, 2005; Sokoloff et al., 2006). Second, the density of D₃, but not D₂, receptors was elevated in (off-treatment) psychotic patients (Gurevich et al., 1997). Similar increases were seen in individuals chronically exposed to cocaine, which is known to aggravate and precipitate psychotic states (Richtand et al., 2001; Heidbreder et al., 2005). Third, the S9G polymorphism of D₃ receptors [which more potently binds dopamine (DA)] has been correlated to a higher risk for schizophrenia. Although this association was not seen in all patient populations, studies of the interaction between D₃ receptors and other genes support a relationship between this D₃ receptor polymorphism and vulnerability to schizophrenia (Dubertret et al., 1998; Jonsson et al., 2003; Joyce and Millan, 2005). Fourth, long-term administration of D₃ receptor antagonists reduces the spontaneous activity of mesolimbic but not nigrostriatal dopaminergic pathways (Ashby et al., 2000). Fifth, selective blockade of D₃ versus D₂ receptors improves cognitive performance and enhances frontocortical cholinergic transmission (Laszy et al., 2005; Millan et al., 2007a). Furthermore, consistent with an improvement in negative symptoms, pharmacological or genetic deletion of D₃ receptors enhances social interaction in rodents (Millan, 2003). Finally, gene knockout and pharmacological studies in rodents and primates have shown that antagonism of D₃ receptors favors motor function and counters its disruption by D₂ receptor blockade (Millan et al., 2004b; Joyce and Millan, 2005; Sokoloff et al., 2006).

Clinical evidence that selective blockade of D₃ receptors moderates the core symptoms of schizophrenia is awaited. Nonetheless, the above observations suggest that antipsychotics displaying a relative preference for D₃ over D₂ sites may conserve activity against positive symptoms, display strengthened efficacy against cognitive (and negative) symptoms and have a low EPS potential (Joyce and Millan, 2005). On this basis, we selected the benzopyranopyrrolidine derivative, S33138 (Fig. 1) (Dubuffet et al., 1999), for clinical evaluation. In the accompanying article (Millan et al., 2007), we characterize the actions of S33138 in a broad range of experimental models in vivo. The present article comprises a foundation for these studies in evaluating the interactions of S33138 with cloned, dopamine hD₃, hD₂, and hD_2S receptors and with several other classes of G-protein-coupled receptor.

Materials and Methods

Drug Evaluation, Salts, and Sources. S33138 was fully characterized in all experimental procedures. In addition, its binding profile at hD₃, hD_2L, and hD_2S receptors and at sites underlying metabolic, cardiovascular, and autonomic side effects was compared with those of the clinically established antipsychotics, haloperidol, clozapine, olanzapine, and risperidone. The majority of protocols employed have been extensively characterized in previous publications from this laboratory (see citations below). However, for those protocols that we have not, as yet, documented in detail, EC₅₀s for endogenous agonists are given together with pK_B values for prototypical antagonists evaluated in parallel as “internal” validators. In this regard, haloperidol was used as an internal reference agent for hD₃, hD_2L, and hD_2S receptors. Efficacies of S33138 were determined at all subtypes of dopaminergic receptor, with the exception of D₄ receptors, where its affinity (<5.0) was too low for study, and at all sites for which its affinities were within 2 “logs” (100-fold) of its affinity at hD₃ receptors. In all studies, results are based on at least three independent determinations performed in triplicate. S33138 and MDL100,907 were synthesized by G. Lavielle (Servier); SB269,970, olanzapine base, and risperidone base were synthesized by J.-L. Péglion (Servier); clozapine base, RX821,002, dopamine HCl, SCH23390, (–)quinpirole diHCl, forskolin base, noradrenaline ditartrate, serotonin creatinine sulfate, and haloperidol base were purchased from Sigma-Aldrich (St. Quentin-Fallavier, France); [³H]spiperone and [¹²⁵I]iodosulpride were purchased from GE Healthcare Europe (Orsay, France); and methiothepin maleate was purchased from Hoffman-LaRoche (Basel, Switzerland).

Evaluation of the Affinities of S33138 at Dopamine hD₃,hD_2L, and D_2S Receptors and at Other Classes of Binding Site. All protocols used for determination of the affinities of S33138 at multiple classes of native and cloned receptor have been documented previously (Millan et al., 2000a,b, 2004a; Newman-Tancredi et al., 2002; for summaries, see tables). Isotherms were analyzed by non-linear regression using PRISM (GraphPad Software, San Diego, CA) to generate IC values. They were transformed into K_i values according to Cheng-Prussof: K_i = IC₅₀/(1 + L/K_d), where L is the radioligand concentration, and K_d is the dissociation constant.

Antagonist Properties of S33138 at hD₃, hD_2L, and hD_2S Receptors: Guanosine-5′-O-(3-[³⁵S]thio)-Triphosphate Binding. The procedures used for determination of the functional actions of S33138 at Chinese hamster ovary (CHO) cell line-expressed hD₃, hD_2L, and hD_2S receptors (15, 2.2, and 1.6 pmol/mg protein, respectively) by guanosine-5′-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPγS) (PerkinElmer Life and Analytical Sciences, Boston, MA) binding have been described in detail elsewhere (Newman-Tancredi et al., 1999; Millan et al., 2000b, 2004a). In brief, [³⁵S]GTPγS was employed at a concentration of 1.0 nM for hD₃ receptors and at a concentration of 0.1 nM for hD_2L and hD_2S receptors. In each case, the pH was 7.4, the temperature was 22°C, and the incubation period was 40 min. The buffer comprised HEPES (20 mM), NaCl (150 mM for hD₃ receptors and l00 mM for hD_2L and hD_2S receptors), GDP (3 μM), and MgCl₂ (3 mM for D₃ receptors and 10 mM for hD_2L and hD_2S receptors). For evaluation of the actions of S33138 alone, membranes were incubated with incremental concentrations for 15 min before the addition of [³⁵S]GTPγS. For evaluation of the antagonist properties of S33138, interaction studies were undertaken with a fixed concentration of DA (1 μM for hD₃ sites, 10 μM for hD_2L sites, and 3 μM for hD_2S sites). In addition, the concentration-response curve for DA-induced [³⁵S]GTPγS binding at hD₃ receptors was examined in the presence of incremental, fixed concentrations of S33138 and the pA₂ value derived by Schild analysis. Experiments were terminated by rapid filtration through Unifilter-96 GF/B filters using a filter mate harvester (PerkinElmer Life and Analytical Sciences). The radioactivity retained on the filters was quantified using a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences). K_B values for inhibition of DA-stimulated [³⁵S]GTPγS binding by S33138 were calculated according to Cheng-Prusoff: K_B = IC₅₀/(1 + (agonist/EC₅₀)), where IC₅₀ is the IC₅₀ of S3318, agonist is the concentration of DA, and EC₅₀ is the EC₅₀ of DA alone.

Antagonist Properties of S33138 at hD₃ and hD_2L Receptors: Antibody-Capture/Scintillation Proximity Assay Studies of Coupling to Gα_i3. The influence of S33138 upon DA-induced activation of Gα_i3 subunits coupled to CHO-transfected hD₃ and hD_2L receptors was determined essentially as described previously (Millan et al., 2004a). In brief, [³⁵S]GTPγS binding was performed, as outlined above for filtration protocols, in 96-well optiplates (PerkinElmer Life and Analytical Sciences). After incubation, 20 μl of Nonidet P-40 (Roche Diagnostics, Mannheim, Germany, 0.27% final concentration) was added to each well, and plates were incubated for 30 min. Then, 10 μl of mouse anti-Gα_i3 monoclonal antibody (SA-281; Biomol, San Diego, CA) was added, and incubation was continued for 30 min. Scintillation proximity assay (SPA) beads coated with secondary, anti-mouse antibodies (GE Healthcare, Les Ulis, France) were added in a volume of 50 μl, and plates were incubated for 3 h with gentle agitation. They were then centrifuged (10 min, 1300g), and radioactivity was determined on a Top Count microplate scintillation counter (PerkinElmer Life and Analytical Sciences). Membranes were incubated with S33138 alone or with S33138 plus DA (1 μM for hD₃ and 10 μM for hD_2L receptors) for 15 min before addition of [³⁵S]GTPγS. K_B values were calculated according to the Cheng-Prussoff equation (see above).

Antagonist Properties of S33138 at hD₃ and hD_2L Receptors: Immunoblot Studies of Coupling to Extracellular-Regulated Kinase. Extracellular-regulated kinase [ERK; also known as mitogen-activated protein (MAP) kinase] phosphorylation was quantified as previously (Cussac et al., 1999; Millan et al., 2000b, 2004a). In brief, CHO cells expressing hD₃ or hD_2L receptors were grown in 24-well plates until 90% confluent and then starved overnight in serum-free medium. S33138 was diluted in the medium and added to each well at the desired concentration. For antagonist studies, cells were preincubated with S33138 for 20 min, then exposed to DA (0.1 μM for hD₃ and hD_2L receptors in each case) for 5 min. After incubation, 0.25 ml of Laemmli sample buffer containing 200 mM dithiothreitol was added. Whole-cell lysates were boiled for 3 min at 95°C, and 14 μl of cell extracts was loaded onto 15-well, 10% polyacrylamide gels. Phosphorylated forms of pp42^MAP-KINASE (ERK 2) and pp44^MAP-KINASE (ERK 1) were revealed using a monoclonal antibody (NanoTools, Denzlingen, Germany) followed by chemiluminescence detection with horseradish peroxidase coupled to the secondary antibody (GE Healthcare). Immunoblots shown are from representative experiments. Autoradiograms were analyzed, and phosphorylated ERK1/2 was quantified by computerized densitometry using AIS software (Imaging Research Inc., ON, Canada). Isotherms were analyzed by nonlinear regression with PRISM. K_B values were calculated according to the Cheng-Prusoff equation (see above).

Antagonist Actions of S33138 at hD₃, hD_2L, hD₃/hD_2L, and hD_3i3(D2) Receptors: Adenylyl Cyclase Assays. The methods used have been detailed previously (Maggio et al., 2003; Novi et al., 2007). In brief, COS-7 cells, grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin, were seeded at a density of 5 × 10⁵ per 100-mm dish. Twenty-four hours later, they were transiently cotransfected by the DEAE-dextran chloroquine method with plasmid DNA encoding adenylyl cyclase (AC)-V and hD₃ receptors or with chimeric AC-V/VI plus hD₃ receptors, hD_2L receptors, hD₃ and hD_2L receptors, or chimeric hD_3i3(D2) receptors. In this chimeric construct, kindly provided by Dr. Daniel Lévesque, the hD₂ receptor sequence, corresponding to amino acids 327 to 338 (KTMSR-RKLSQQK) of the C-terminal portion of the “i3” loop, was introduced into the hD₃ receptor, replacing amino acids 312 to 323. This substitution changes neither ligand recognition profile nor cell surface expression compared with wild-type hD₃ receptors (Novi et al., 2007). Twenty-four hours after transfection, cells were trypsinized, recultured in 24-well plates, and, after an additional 24 h, the cells were assayed for AC activity. In brief, the cells were incubated for 2 h with 0.25 ml/well fresh growth medium containing 5 μCi/ml [³H]adenine. This medium was replaced with 0.5 ml/well Dulbecco's modified Eagle's medium containing 20 mM HEPES, pH 7.4, 0.1 mg of bovine serum albumin, and the phosphodiesterase inhibitors, 1-methyl-3-isobutylxanthine (0.5 mM) and RO 20-1724 (0.5 mM). AC activity was stimulated by addition of 1 μM forskolin in the presence or absence of S33138 and the D₂/D₃ receptor agonist, quinpirole, at concentrations indicated in the legend to Fig. 4. After 10 min of incubation at 30°C, the medium was removed, and the reaction was terminated by the addition of perchloric acid containing 0.1 mM unlabeled cAMP, followed by neutralization with KOH. The amount of [³H]cAMP formed was determined by a two-step column separation procedure, and disintegrations per minute values were expressed with respect to the protein content of each sample; the average value (mean ± S.E.M.) was 56.7 ± 4.2 μg. K_B values were calculated according to the Cheng-Prussoff equation (see above).

Antagonist Properties of S33138 at hD₁ Receptors: SPA Studies of Coupling to Gα_s. Activation of hD₁ receptor-coupled Gα_s-protein was determined essentially as previously (Cussac et al., 2004). In brief, membranes of L cells expressing hD₁ receptors (11 pmol/mg protein) were preincubated with S33138 or assay buffer containing 20 mM HEPES (pH 7.4, 1 μM GDP, 50 mM MgCl₂, and 100 mM NaCl). The reaction was started by adding DA (1.0 μM) and [³⁵S]GTPγS(∼0.3 nM) to 96-well optiplates (PerkinElmer Life and Analytical Sciences) for 60 min at room temperature. At the end of incubation, 20 μl of Nonidet P-40 (0.27% final concentration) was added to each well, and plates were incubated with gentle agitation for 30 min. Polyclonal antibodies against Gα_s/olf (C18; Santa Cruz Biotechnology Inc., Santa Cruz, CA) were added to each well in a volume of 10 μl before 30 min of additional incubation. After addition of SPA beads coated with secondary antibodies (50 μl; GE Healthcare) and incubation for 3 h, plates were centrifuged (10 min, 1300g), and radioactivity was detected on a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences). K_B values were calculated according to the Cheng-Prussoff equation (see above).

Antagonist Properties of S33138 at hD₅ Receptors: Fluorescence Quantification of cAMP Production. GH4 cells stably expressing human D₅ receptors (0.34 pmol/mg protein) were resuspended in Hanks' balanced salt solution buffer containing 20 mM HEPES/NaOH, pH 7.4, 138 mM NaCl, 5.3 mM KCl, 0.4 mM KH₂PO₄, 0.3 mM Na₂HPO₄, 1.25 mM CaCl₂, 0.5 mM MgCl₂, 0.4 mM MgSO₄, and 0.1% glucose and preincubated for 30 min at 37°C with 0.5 mM 3-isobutyl-1-methylxanthine. Cells were distributed at a density of 1.10⁴ cells/well in microplates and preincubated for 10 min at room temperature with S33138, the D₅ receptor antagonist, SCH23390, or buffer (control). In antagonist studies, DA (30 nM) was added, and the mixture was incubated for 20 min at room temperature. The fluorescence acceptor (XL665-labeled cAMP) and the fluorescence donor (anti-cAMP antibody labeled with europium cryptate) were then added. After 60 min at room temperature, fluorescence transfer was measured at λ_ex = 337 nm, λ_em = 620 nm, and λ_em = 665 nm using a “high time-resolved fluorescence” microplate reader (RUBY-star; BMG, Offenburg, Germany). The production of cAMP was determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results were expressed as percentage inhibition of the level of cAMP induced by 30 nM DA. K_B values were calculated according to the Cheng-Prussoff equation (see above).

Antagonist Properties of S33138 at hα_2C-ARs: [³⁵S]GTPγS Binding. [³⁵S]GTPγS binding was determined as documented in detail elsewhere (Audinot et al., 2002; Newman-Tancredi et al., 2002). In brief, membranes of CHO cells expressing hα_2C-AR (1.9 pmol/mg protein) were incubated for 60 min at 22°C with noradrenaline (NA) and [³⁵S]GTPγS at a concentration of 0.1 nM in a buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 3 μM GDP, and 3 mM MgSO₄. Antagonist properties of S33138 and the selective α₂-AR antagonist, RX821,002, were evaluated against a fixed concentration of NA (10 μM). Incubations were terminated by rapid filtration through Whatman GF/B filters using a Filter Harvester (Perkin-Elmer Life and Analytical Sciences). Radioactivity retained on the filters was quantified by liquid scintillation counting. K_B values were calculated according to the Cheng-Prussoff equation (see above).

Antagonist Properties of S33138 at hα_2C-ARs: SPA Studies of Coupling to Gα_i3 and Gα_o. The coupling of hα_2C-ARs to Gα_i3 was evaluated using an SPA procedure essentially as described outlined above for hD₃ receptors. The protocol used for Gα_o was the same as for Gα_i3. In brief, membranes were incubated on 96-well plates with NA (10 μM) and [³⁵S]GTPγS (0.2 nM) for 1 h at 22°C following preincubation with S33138, RX821,002, or assay buffer for 30 min. The buffer contained 20 mM HEPES, pH 7.4, 0.3 μMGDP, 3 mM MgCl₂, and 150 mM NaCl. The reaction was stopped by solubilizing membranes with detergent, Nonidet P-40 (0.27% final concentration). After gentle agitation for 30 min, 10 μl of mouse monoclonal anti-Gα_i3 antibodies (see above) or anti-Gα_o antibodies (SA-280; Biomol) were then added, and plates were incubated for a further 1 h. At the end of the incubation period, SPA beads coated with anti-mouse secondary antibodies (GE Healthcare) were added. They were incubated with gentle agitation overnight before counting radioactivity on a TopCount microplate scintillation counter. Nonspecific binding was defined with 10 μM GTPγS. K_B values were calculated according to the Cheng-Prussoff equation (see above).

Antagonist Properties of S33138 at hα_2C-ARs: ERK Phosphorylation. The procedure used was essentially that outlined above for hD₃ receptors. CHO cells expressing hα_2C-ARs were grown in six-well plates until confluent. Cells were then washed twice with serum-free medium and incubated overnight in this medium. Cells were preincubated for 20 min with S33138, RX821,002, or buffer and then stimulated with NA (1 μM) for 5 min. Phosphorylated ERK 1/2 was measured in cell extracts using a monoclonal antibody against phosphorylated pp42^MAP-Kinase (ERK 2) and pp44^MAP-Kinase (ERK 1) (NanoTools). Autoradiograms were analyzed, and K_B values were calculated according to the Cheng-Prusoff equation (see above).

Antagonist Properties of S33138 at h5-HT_1D Receptors: [³⁵S]GTPγS Binding. Membranes of CHO cells expressing h5HT_1D receptors (1.6 pmol/mg protein) were incubated for 30 min at 22°C with 5-HT and [³⁵S]GTPγS at a concentration of 0.1 nM in a buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 3 μM GDP, and 3 mM MgCl₂. Incubations were terminated by rapid filtration through Whatman GF/B filters using a Filter Harvester (PerkinElmer Life and Analytical Sciences). Radioactivity retained on the filters was quantified by liquid scintillation counting. Antagonist properties of S33138 and the 5-HT_1D receptor antagonist, methiothepin, were evaluated against a fixed concentration of 5-HT (10 nM). K_B values were calculated according to the Cheng-Prussoff equation (see above).

Antagonist Properties at h5-HT_2A Receptors: SPA Studies of Coupling to Gα_q. Efficacy at h5-HT_2A receptors was determined by Gα_q activation in CHO cells expressing h5-HT_2A receptors (4.4 pmol/mg protein) employing an SPA protocol previously used for study of Gα_q-coupled h5-HT_2C receptors (Cussac et al., 2002). Cells were preincubated with S33138, the selective 5-HT_2A receptor antagonist, MDL100,987, or buffer for 30 min at 22°C. They were then incubated for 1 h with 5-HT (at a concentration of 100 nM in antagonist studies) and [³⁵S]GTPγS (0.3 μM). K_B values were calculated according to the Cheng-Prussoff equation (see above).

Antagonist Properties at h5-HT_2A Receptors: Studies of Inositol Phosphate Production. Determination of the accumulation of inositol phosphate (IP) in CHO cells expressing h5-HT_2A receptors was performed in 96-well plates (0.25 × 10⁶ cells/well) after overnight labeling with [³H]myoinositol (0.5 μCi/well). Stimulation by 5-HT (0.3 μM) was conducted for 30 min in a medium containing 10 mM LiCl and S33138, MDL100,907, or buffer. The reaction was stopped by adding formic acid (0.1 M). Supernatants were recovered, and IPs were purified by ion-exchange chromatography using DOWEX AG1-X8 resin (Bio-Rad, Hercules, CA) in 96-well filter plates (Millipore, Bedford, MA). The total radioactivity remaining in the membrane fraction was counted after treatment of the cells by a mixture of 10% Triton X-100 and 0.1 N NaOH. Radioactivity was quantified using a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences). Data are expressed (percentage) as (amount of total IP produced/amount of radioactivity remaining in membranes) × 100. K_B values were calculated according to the Cheng-Prussoff equation (see above).

Antagonist Properties of S33138 at h5-HT₇ Receptors: Fluorescence Quantification of cAMP Production. Actions of S33138 were determined in CHO cells stably expressing human 5-HT₇ receptors (0.25 pmol/mg protein) (1.10⁴ cells/well in microplates) as described above for hD₅ receptors. Cells were preincubated for 10 min at room temperature with S33138, the selective 5-HT₇ receptor antagonist, SB269,970, or buffer. In antagonist studies, 5-HT (100 nM) was added, and incubations were performed for 20 min at room temperature. Fluorescence was quantified as described above for hD₅ receptors. The results are expressed as percent inhibition of the level of cAMP induced by 5-HT. K_B values were calculated according to the Cheng-Prussoff equation (see above).

Results

Binding Profile of S33138 at Multiple Subtypes of Dopamine Receptor (Table 1;Fig. 2). S33138 concentration-dependently and potently displaced the binding of [³H]spiperone to hD₃ receptors (Fig. 2A). S33138 similarly competed with [³H]spiperone at hD_2L receptors (long isoform) and hD_2S receptors (short isoform), but its affinity was markedly (approximately 25-fold) lower compared with hD₃ receptors (Fig. 2A). A similar pattern of data was acquired with [¹²⁵I]iodosulpride (used at 0.2 nM for hD₃ receptors and 0.1 nM for hD_2L receptors); this radioligand yielded affinities (pK_i values) of S33138 of 8.66 ± 0.09 and 7.43 ± 0.02 at hD₃ and hD_2L receptors, respectively. The affinity of S33138 for native, rat striatal D₂ receptors labeled by [³H]spiperone was close to that seen at recombinant hD₂ receptors (Table 1). In contrast to hD₃ sites, the affinity of S33138 for cloned hD₄ receptors was low, although it displayed modest affinity for hD₁ and hD₅ receptors. S33138 displayed low affinity for native, rat, and cloned, human DA reuptake sites (pK_i values, <5.0 and 4.91 ± 0.03, respectively).

Binding Profile of S33138 at Nondopaminergic Receptors (Table 1). S33138 displayed significant affinity for h5-HT₇ receptors as well as for h5-HT_2A receptors, although its affinity for native, rat 5-HT_2A receptors was slightly lower than for h5-HT_2A sites. Modest affinity of S33138 also was seen at h5-HT_1D and, less markedly, at h5-HT_1A, h5-HT_1B, h5-HT_2B, and h5-HT_2C receptors. The affinity of S33138 was, however, negligible at h5-HT₃, h5-HT₄, h5-HT_5A, and h5-HT₆ receptors. S33138 also displayed low affinity for cloned, human 5-HT reuptake sites ([lt]5.0). The affinity of S33138 for multiple subtypes of hα₁-ARs was modest, and it manifested negligible affinity for hα_2A- and hα_2B-ARs. Conversely, S33138 showed greater affinity for hα_2C-ARs. The affinity of S33138 for hβ₁ and hβ₂-ARs was negligible, and it also showed low affinity for cloned, human NA reuptake sites (pK_i, 5.34 ± 0.14). S33138 showed negligible (<5.0) affinity for numerous other sites listed in the legend to Table 1.

Low Affinity of S33138 as Compared with Prototypical Antipsychotics for Receptors Underlying Metabolic, Cardiovascular, and Autonomic Side Effects (Table 2). Compared with its high affinity for hD₃ receptors, S33138 displayed only low affinities for h5-HT_2C receptors, hα_1A-ARs, hα_1B-ARs, hα_1C-ARs, and hH₁ receptors. It displayed negligible affinity for hM₁, hM₂, hM₃, and hM₄ receptors and weak affinity (K_i, 525 ± 11, n = 3) for hM₅ receptors. The affinity of haloperidol for h5-HT_2C compared with hD₃ receptors was low, although it showed marked affinities at α_1A-ARs, hα_1B-ARs, and hα_1C-ARs. It only weakly recognized hH₁ sites and multiple subtypes of muscarinic receptor. Compared with hD₃ receptors, clozapine manifested high affinity for h5-HT_2C receptors, for each subtype of hα₁-AR, and for hH₁ receptors. Its affinities at hM₁, hM₃, and hM₄ receptors were also high, and it displayed modest affinity for hM₂ receptors. Olanzapine revealed high affinities for h5-HT_2C receptors, for each subtype of hα₁-AR, for hH₁ receptors, and for all subtypes of muscarinic receptor, except hM₃, for which its affinity was modest. The affinities of risperidone for h5-HT_2C receptors, hα₁-AR subtypes, and hH₁ receptors were high, but, in contrast to clozapine and olanzapine, it only weakly interacted with muscarinic receptors.

Antagonist Properties of S33138 at hD₃ versus hD_2L and hD_2S Receptors: Filtration Assays of [³⁵S]GTPγS Binding (Fig. 2;Table 3). In line with previous studies (Newman-Tancredi et al., 1999; Millan et al., 2000b, 2004a), DA concentration-dependently (approximately 2-fold) stimulated [³⁵S]GTPγS binding at hD₃, hD_2L, and hD_2S receptors, actions blocked by haloperidol with pK_B values of 8.51 ± 0.06, 9.49 ± 0.16, and 8.90 ± 0.16, respectively. S33138 did not modify [³⁵S]GTPγS binding when applied alone. Indeed, it potently suppressed the stimulation of [³⁵S]GTPγS binding at hD₃ receptors by DA with a pK_B of 8.85 ± 0.10; this value corresponds well to its affinity (8.68) at these sites. Furthermore, in the presence of incremental concentrations of S33138 (3–100 nM), the concentration-response curve for DA-induced [³⁵S]GTPγS binding at hD₃ receptors was shifted in parallel to the right with no loss of maximal effect (Fig. 2E). Schild analysis (Fig. 2F) yielded a slope not significantly different to unity (1.18, r = 0.88) and a pA₂ (8.69) very close to its pK_B and pK_i values at hD₃ sites. These observations indicate that S33138 behaves as a competitive and reversible antagonist at hD₃ receptors. S33138 likewise suppressed DA-induced [³⁵S]GTPγS binding at hD_2L and hD_2S receptors, indicating that it is also an antagonist at these sites (Fig. 2, C and D). However, pK_B values of 7.82 ± 0.03 and 7.79 ± 0.06 at hD_2L and hD_2S receptors, respectively, were lower than for hD₃ sites.

Antagonist Properties of S33138 at hD₃ versus hD_2L Receptors: SPA Studies of Coupling to Gα_i3 (Table 3). At hD₃ and hD_2L receptors, in line with previous work (Millan et al., 2004a), DA enhanced [³⁵S]GTPγS binding to Gα_i3 by approximately 2-fold; its actions were blocked by haloperidol, with pK_B values of 8.25 ± 0.01 and 9.18 ± 0.26, respectively. In contrast, S33138 alone was without activity. At hD₃ receptors, S33138 concentration-dependently suppressed DA-induced stimulation of Gα_i3 with a pK_B of 8.24 ± 0.07. S33138 also behaved as an antagonist at hD_2L receptor-coupled Gα_i3 in blocking its activation by DA with a pK_B of 7.76 ± 0.14.

Antagonist Properties of S33138 at hD₃ versus hD_2LReceptors: Immunoblot Studies of ERK Phosphorylation (Fig. 3;Table 3). By analogy to previous studies (Cussac et al., 1999; Millan et al., 2004a), in CHO cells transfected with hD₃ or hD_2L receptors, DA triggered a transient yet marked activation (phosphorylation) of ERK 1/2 (MAP kinase). In distinction, S33138 did not increase levels of the phosphorylated forms of ERK. Furthermore, S33138 concentration-dependently abolished induction of ERK 1/2 at hD₃ receptors with a pK_B of 9.08 ± 0.02 (Fig. 3A). S33138 similarly behaved as an antagonist of DA-stimulated ERK 1/2 phosphorylation at hD_2L receptors with a pK_B of 8.03 ± 0.02 (Fig. 3B).

Antagonist Properties of S33138 at hD₃ Receptors, hD_2L Receptors, Cotransfected hD₃ and hD_2L Receptors, and hD_3i3(D2) Receptors: Enzymatic Studies of AC Activity (Fig. 4). In COS-7 cells transfected with a chimeric AC-V/VI, forskolin elicited a marked (approximately 6-fold) increase in cAMP accumulation. This AC isoform is insensitive to D₃ receptors, and, confirming previous work (Maggio et al., 2003; Novi et al., 2007), quinpirole did not modify the influence of forskolin in cells cotransfected with hD₃ receptors. On the other hand, quinpirole (EC₅₀, 1.2 nM) robustly and potently suppressed forskolin-induced cAMP accumulation in cells cotransfected with AC-V/VI and hD_2L receptors. With similar potency, it reduced the activity of AC-V/VI in cells cotransfected with both hD_2L and hD₃ receptors (EC₅₀, 0.97 nM). S33138 alone did not modify the effects of forskolin in hD₃, hD_2L, or hD₃/hD_2L cotransfected cells. However, it concentration-dependently abolished the inhibitory influence of quinpirole both in cells expressing hD_2L receptors (pK_B, 7.57 ± 0.12) and, more potently, in cells cotransfected with hD₃ and hD_2L receptors (pK_B, 8.69 ± 0.14) (Fig. 4A). In COS-7 cells transfected with AC-V/VI and chimeric hD_3i3(D2) receptors, where a 12-amino acid sequence of the third intracellular loop was substituted by the corresponding sequence of hD_2L receptors to enhance coupling efficacy to AC-V/VI, quinpirole (EC₅₀, 1.01 nM) markedly suppressed cAMP formation. Its action was blocked by S33138 with pK_B of 8.71 ± 0.16 (Fig. 4B). In contrast to AC-V/VI, the AC-V isoform is responsive to D₃ receptors, although effects of agonists are less robust than at hD_2L receptors (Robinson and Caron, 1997; Maggio et al., 2003). Quinpirole significantly reduced the influence of forskolin upon cAMP accumulation in cells transfected with hD₃ receptors and AC-V; this effect was reversed by S33138, which was inactive alone (Fig. 4C).

Antagonist Properties of S33138 at hD₁ and hD₅ Receptors. In L cells transfected with hD₁ receptors, DA (EC₅₀, 11.5 nM) elicited an approximately 2-fold increase in [³⁵S]GTPγS binding to Gα_s as quantified using an SPA protocol. In contrast, S33138 was ineffective and concentration-dependently eliminated the action of DA with a pK_B of 6.21 ± 0.11. In CHO cells transfected with hD₅ receptors, DA elevated cAMP accumulation with an EC₅₀ of 41.7 nM. This action of DA was blocked by the D₅ receptor antagonist, SCH23390, with a pK_B of 8.82 ± 0.08. It was also concentration-dependently abrogated by S33138 (pK_B, 6.46 ± 0.16), which was inactive alone. S33138 then behaves as an antagonist of hD₁ and hD₅ receptors with modest potency.

Antagonist Properties of S33138 at hα_2C-ARs (Fig. 5). Corroborating previous studies at CHO-transfected hα_2C-ARs (Audinot et al., 2002; Newman-Tancredi et al., 2002), NA elicited an approximately 2-fold enhancement in [³⁵S]GTPγS binding, whereas S33138 was without effect. The action of NA (10 μM) was abolished by S33138 with a pK_B of 7.23 ± 0.15. In SPA assays, NA robustly (approximately 1.5-fold in each case) enhanced the binding of [³⁵S]-GTPγStoGα_i3 (EC₅₀, 513 nM) and Gα_o (EC₅₀, 145 nM), actions blocked by the prototypical α₂-AR antagonist, RX821,002, with pK_B values of 9.04 ± 0.09 and 8.57 ± 0.05, respectively. S33138, which was inactive alone, also abolished the effects of NA with pK_B values of 6.85 ± 0.07 and 7.29 ± 0.14 (Fig. 5, A and B), respectively. Likewise, in CHO cells transfected with hα_2C-ARs, NA (EC₅₀, 27.7 nM) elicited a robust increase in ERK 1/2 phosphorylation. This effect of NA (1 μM) was blocked by RX821,002 (pK_B, 8.32 ± 0.10) and dose-dependently abolished by S33138 (pK_B, 7.11 ± 0.15) (Fig. 5C). In line with its weak affinity for hα_2A- and hα_2B-ARs, S33138 showed no antagonist properties (up to 10 μM) in [³⁵S]GTPγS binding studies of these sites (data not shown).

Antagonist Properties of S33138 at 5-HT Receptor Subtypes (Fig. 5). Serotonin (EC₅₀, 1.3 nm) evoked an approximately 1.3-fold increase in [³⁵S]GTPγS binding at h5-HT_1D receptors. The antagonist, methiothepin, antagonized the action of 5-HT with a pK_B of 7.55 ± 0.07. S33138 also blocked the influence of 5-HT upon [³⁵S]GTPγS binding at h5-HT_1D receptors with a pK_B of 6.79 ± 0.18. It was inactive alone at these sites. At h5-HT_2A receptors, an SPA assay revealed activation by 5-HT (EC₅₀, 27.8 nM) of [³⁵S]GTPγS binding to Gα_q with a maximal 2-fold increase versus basal values. The effect of 5-HT (0.3 μM) was abolished by the prototypical 5-HT_2A antagonist, MDL100,907, with a pK_B of 9.26 ± 0.20. Activation of Gα_q coupled to h5-HT_2A receptors was also abolished by S33138 with a pK_B of 6.86 ± 0.02 (Fig. 5D). S33138 did not influence Gα_q alone. Serotonin (EC₅₀, 31.9 nM) also activated phospholipase C coupled to h5-HT_2A receptors as reflected in a marked increase in IP formation. This action of 5-HT (0.3 μM) was abolished by MDL100,907 with a pK_B of 8.9 ± 0.1. It was also blocked by S33138 with a pK_B of 6.79 ± 0.07 (Fig. 5E). At h5-HT₇ receptors, 5-HT enhanced the activity of AC with an EC₅₀ of 30.0 nM. The 5-HT₇ antagonist, SB269,970, blocked the action of 5-HT with a pK_B of 8.85 ± 0.12. S33138 was inactive alone and blocked this action of 5-HT with a pK_B of 7.06 ± 0.25 (Fig. 5F).

Discussion

Preferential Antagonism by S33138 of hD₃ versus hD_2L and hD_2S Receptors. The higher affinity of S33138 for hD₃ versus hD_2L and hD_2S receptors distinguishes it from haloperidol, clozapine, olanzapine, risperidone, and other clinically available antipsychotics displaying similar affinities for hD₃, hD_2L, and hD_2S sites (Table 2) (Millan et al., 2000a; Vanhauwe et al., 2000; Burstein et al., 2005). Furthermore, the marked D₃ versus D₂ receptor preference of S33138 likewise differentiates it from aripiprazole and bifeprunox, which behave as partial agonists at hD₃ and hD₂ receptors (Shapiro et al., 2003; Burstein et al., 2005; Novi et al., 2007; Urban et al., 2007b). Although the preference of S33138 for hD₃ versus hD₂ sites is less pronounced than the 100-fold selectivity of antagonists like S33084, SB414,796, and A437,203 (Millan et al., 2000b; Reavill et al., 2000; Joyce and Millan, 2005; Boeckler and Gmeiner, 2006), it was selected for clinical development precisely on this basis. That is, rather than “absolute” selectivity, preferential D₃ versus D₂ receptor blockade should allow for antipsychotic activity against positive and cognitive-negative symptoms in the relative absence of extrapyramidal dysfunction (Joyce and Millan, 2005). This preferential interaction of S33138 with D₃ versus D₂ receptors is supported by in vivo findings reported in the accompanying article (Millan et al., 2007b).

hD₃ receptors couple via pertussis-sensitive G-proteins to AC and ERK 1/2 (MAP kinases) (Ahlgren-Beckendorf and Levant, 2004; Beom et al., 2004; Neve et al., 2004; Sokoloff et al., 2006). Activation of hD₃ receptors by DA led to enhanced [³⁵S]GTPγS binding (Newman-Tancredi et al., 1999, 2002; Vanhauwe et al., 2000), an effect potently and reversibly blocked by S33138, indicating competitive antagonist properties. Moreover, using an SPA procedure coupled to a highly selective antibody (Millan et al., 2004a; Neve et al., 2004), the activation of Gα_i3 by DA was also shown to be antagonized by S33138. Coupling of hD₃ receptors to AC is variable and isoform-dependent (Griffon et al., 1997; Robinson and Caron, 1997; Hall and Strange, 1999; Neve et al., 2004). Nonetheless, in COS-7 cells transfected with hD₃ receptors, quinpirole inhibited forskolin-stimulated AC-V activity, and this action was abolished by S33138. Furthermore, S33138 blocked the inhibition of cAMP formation by quinpirole in cells transfected with a chimeric hD_3i3(D2) receptor displaying enhanced coupling efficacy to AC-V/VI (Novi et al., 2007), yet identical ligand recognition profiles and cell surface expression levels versus wild-type hD₃ receptors (Novi et al., 2007). D₃ receptors recruit the ERK pathway by pertussis toxin-sensitive Gα_i (including Gα_i3) proteins and an array of intracellular cascades involving both α and β subunits, a “phosphoinoside-3 kinase,” and an atypical protein kinase C (Cussac et al., 1999; Newman-Tancredi et al., 1999; Beom et al., 2004; Neve et al., 2004). Correspondingly, in CHO cells bearing hD₃ receptors, DA stimulated ERK 1/2 phosphorylation, an effect abolished by S33138. The lack of effect of S33138 alone is important because ERK 1/2 activation is highly sensitive even to weak partial agonist actions; for example, aripiprazole and bifeprunox (M. J. Millan and C. Mannoury la Cour, unpublished observation; Cussac et al., 1999; Shapiro et al., 2003; Burstein et al., 2005; Urban et al., 2007b). In certain cell lines, D₃ receptors couple to G_o, G_q/11, G_z, and (via pertussis-insensitive pathways) phospholipase D (Everett and Senogles, 2004; Neve et al., 2004). Thus, it would be of interest to extend the present work to other intracellular cascades.

D₂ receptors show subtle differences to hD₃ sites as concerns their interactions with signaling pathways (Hall and Strange, 1999; Vanhauwe et al., 1999'02; Ahlgren-Beckendorf and Levant, 2004; Beom et al., 2004; Neve et al., 2004). Nonetheless, like their D₃ counterparts, both hD_2S and hD_2L receptors (which are principally localized presynaptically and postsynaptically to dopaminergic pathways, respectively) couple via G_o/i to AC and ERK 1/2. Across a broad range of measures, [³⁵S]GTPγS binding to hD_2L and hD_2S receptors (Vanhauwe et al., 2000; Newman-Tancredi et al., 2002), as well as hD_2L receptor coupling to Gα_i3 (Millan et al., 2004a; Lane et al., 2007), AC (O'Hara et al., 1996; Hall and Strange, 1999), and ERK 1/2 (Beom et al., 2004; Neve et al., 2004), S33138 behaved as a pure antagonist at both hD_2L and hD_2S receptors with lower potency than at hD₃ receptors. Recent work on “agonist-directed” trafficking and “protean” agonism at hD_2L sites emphasizes that drug efficacy can vary as a function of the intracellular pathway (Neve et al., 2004; Lane et al., 2007; Urban et al., 2007a). Thus, it would be interesting to examine the influence of S33138 upon other D₂ receptor-coupled transduction cascades.

Antagonism by S33138 of Cotransfected hD₃ and hD_2L Receptors. D₃ and D₂ receptors are partially colocalized in dopaminergic pathways and postsynaptic neurones (Surmeier et al., 1992; Joyce and Millan, 2005), and they assemble into functional D₃/D_2L heterodimers in vitro (Maggio et al., 2003; Novi et al., 2007). Heterodimers display functional characteristics distinct from monomers, so it is of significance that S33138 abolished the inhibitory influence of quinpirole upon AC in cells expressing both hD₃ and hD_2L receptors. Furthermore, S33138 was more potent in cells expressing hD₃ and hD₂ sites than in cells expressing hD₂ receptors alone. This finding resembles the altered potencies of other drugs at D₃/D₂ heterodimers compared with constituent monomers (Lee et al., 2003; Maggio et al., 2003; Novi et al., 2007). The underlying reasons remain to be defined. Nonetheless, proof that functional heterodimers form in the brain is awaited. Thus, relevance of the actions of S33138 (and other antipsychotics) at D₃/D₂ heterodimers to the control of psychotic states remains to be clarified (Lee et al., 2003; Maggio et al., 2003).

Antagonism by S33138 of hD₁ and hD₅ Receptors. Dopamine D₁ and (closely related) D₅ receptors couple positively to AC via G_s (Cai et al., 1999; Neve et al., 2004). As quantified by SPA coupled to a specific anti-Gα_s antibody (Cussac et al., 2004), recruitment of G_s by hD₁ receptors was blocked by S33138, indicating mild antagonist properties at these sites. Blockade of mesolimbic D₁ receptors may participate in the antipsychotic properties of certain antipsychotics, and equilibrated antagonism of striatal D₁ and D₂ receptors contributes to the low EPS potential of clozapine (Josselyn et al., 1997; Tauscher et al., 2004). However, in vivo studies suggest that blockade of D₁ sites is unlikely to play a major role in the therapeutic actions of S33138 (Millan et al., 2007). Antagonist properties of S33138 at hD₅ receptors were also expressed at concentrations well below those blocking D₃ receptors. Furthermore, although clozapine likewise antagonizes D₅ receptors (Neve et al., 2004), any potential advantage in the management of schizophrenia remains conjectural.

Antagonist Actions of S33138 at hα_2C-ARs. An intriguing finding differentiating S33138 from other antipsychotics was its interaction with hα_2C-ARs despite low affinities for their hα_2A and hα_2B counterparts. All hα₂-AR subtypes couple via G_i/G_o to downstream pathways converging on ERK 1/2 (Hieble et al., 1995), and, by analogy to other α₂-AR antagonists, S33138 abolished activation of hα_2C-ARs in procedures of G-protein activation, recruitment of Gα_i3 and Gα_o, and ERK 1/2 phosphorylation (Alblas et al., 1993; Jasper et al., 1998; Audinot et al., 2002). Few ligands possessing a marked preference for α_2C-versus α_2A/α_2B-ARs have been described previously (Hieble et al., 1995), so S33138 may serve as a useful template for construction of chemically novel selective α_2C-AR antagonists. Exploration of the significance of α_2C-AR blockade to the functional profile of S33138 would be of interest because activation of α_2C-ARs in frontal cortex and hippocampus may compromise cognitive performance (Marcus et al., 2005; Soto-Moyano et al., 2005). Moreover, antagonism of corticolimbic and striatal α_2C-ARs may contribute to the atypical profile of clozapine, including its low EPS potential (Millan et al., 2001'02; Kalkman and Loetscher, 2003; Svensson, 2003).

Antagonist Actions of S33138 at h5-HT_2A and h5-HT₇ Receptors. S33138 behaved as a pure antagonist at h5-HT_2A receptors in protocols quantifying the activation of Gα_q and the turnover of IP (Cussac et al., 2002; Kurrasch-Orbaugh et al., 2003). This observation is of potential importance because the more marked affinity of atypical antipsychotics for 5-HT_2A versus D₂ receptors has been related to improved control of negative symptoms and a low propensity to elicit EPS (Meltzer et al., 2003; Werkman et al., 2006). Activation of 5-HT₇ receptors engages AC via recruitment of G_s (Thomas and Hagan, 2004), and modest antagonist actions of S33138 at h5-HT₇ sites were seen in a protocol of cAMP formation. Blockade of 5-HT₇ receptors by S33138 mimics clozapine and several other antipsychotics (Roth et al., 1994; Thomas and Hagan, 2004). 5-HT₇ receptors are enriched in the cortex, hippocampus, striatum, and suprachiasmatic nucleus, and their blockade may favorably influence mood, sleep, and circadian rhythms, which are perturbed in schizophrenic patients (Thomas and Hagan, 2004; Monti and Monti, 2005).

Low Affinity of S33138 for hH₁, Human Muscarinic, and hα₁-AR Receptors. The weak affinity of S33138 for H₁ receptors suggests a low sedative potential and a low risk of obesity compared with antipsychotics that potently block these sites (Table 2) (Cunningham-Owens, 1996; Kroeze et al., 2003; Kim et al., 2007). A low risk of sedation is consistent with the weak affinity of S33138 for α₁-ARs, which also suggests a low risk of orthostatic hypotension and cardiovascular perturbation (Pacher and Kecskemeti, 2004; Lieberman et al., 2005). Moreover, S33138 did not interact with hM₁-hM₄ muscarinic receptors, suggesting that it should not provoke autonomic side effects such as troubled vision and gastrointestinal discomfort (Cunningham-Owens, 1996). Importantly, moreover, S33138 should not compromise cognitive function by blocking postsynaptic populations of muscarinic and H₁ receptors (Lieberman et al., 2005).

Conclusions

The novel benzopyranopyrrolidine derivative, S33138, behaves as a preferential antagonist of hD₃ versus hD_2L and hD_2S receptors. It also displays modest antagonist properties at hα_2C-ARs, h5-HT_2A receptors, and h5-HT₇ receptors, actions likewise of potential relevance to the treatment of schizophrenia. This distinctive receptor-binding profile of S33138 was corroborated by in vivo studies in rodents and primates (Millan et al., 2007), underscoring its innovative profile for the improved management of psychotic states. Conceivably, low doses may suffice for maintenance therapy, whereas during acute exacerbation of psychosis, doses can be increased to more markedly antagonize D₂ receptors. This remains to be seen. In any case, ongoing (phase II) clinical studies should soon shed light on the long-standing question of how antipsychotics that preferentially block D₃ versus D₂ receptors compare with clinically established agents acting with similar affinity at these sites.