Agonist and antagonist properties of antipsychotics at human dopamine D4.4 receptors: G-protein activation and K+ channel modulation in transfected cells.

Interaction at dopamine D4 receptors may improve cognitive function, which is highly impaired in individuals with schizophrenia, but comparative studies of recent antipsychotics in cellular models of D4 receptor activation are lacking. Here, we report the in-vitro profile of over 30 ligands at recombinant hD4.4 receptors. In [35S]GTPgammaS binding experiments using membranes of CHO-hD4.4 cells, apomorphine, preclamol and the selective D4 agonists, ABT724, CP226269, Ro-10-5824 and PD168077, behaved as partial agonists (Emax 20-60% vs. dopamine), whereas L745870 and RBI257, displayed antagonist properties. The 'conventional' antipsychotic, haloperidol and the 'atypicals', clozapine and risperidone, exhibited antagonist properties, while 'third generation' compounds bifeprunox, SLV313 and F15063, acted as partial agonists (10-30%). Aripiprazole and SSR181507 slightly stimulated [35S]GTPgammaS binding at micromolar concentrations. In Xenopus laevis oocytes co-expressing hD4.4 receptors with G-protein-coupled inwardly rectifying potassium (GIRK) channels, apomorphine, preclamol, ABT724, CP226269, and PD168077 stimulated GIRK currents (Emax 70-80%). The 5-HT1A receptor ligands, WAY100635 and flibanserin, also exhibited partial agonist activity (30% and 15%, respectively). Haloperidol, clozapine, olanzapine and nemonapride did not stimulate GIRK currents, whereas aripiprazole, bifeprunox, SLV313 and F15063, but not SSR181507, exhibited partial agonism (Emax 20-35%). In-vitro responses depended on experimental conditions: increasing NaCl concentration (30 mm to 100 mm) reduced agonist efficacy in [35S]GTPgammaS binding, whereas decreasing the amount of hD4.4 cRNA injected into oocytes (from 2.0 to 0.5 ng/oocyte) reduced agonist efficacy of several compounds. These data indicate that, unlike conventional or 'atypical' antipsychotics, several 'third generation' agents display D4 receptor partial agonism that may be sufficient to influence physiological D4 receptor activity in vivo.

WAY-100635 has high selectivity for serotonin 5-HT(1A) versus dopamine D(4) receptors.

The serotonin 5-HT(1A) receptor antagonist WAY-100635 was recently reported to have potent agonist properties at dopamine D(4) receptors (Chemel et al., 2006, Psychopharmacology 188, 244-251.). Herein WAY-100635 (pK(i) at human (h) serotonin 5-HT(1A) receptors=9.51; pK(i) at dopamine hD(4.4) receptors=7.42) stimulated [(35)S]GTPgammaS incorporation in membranes of Chinese Hamster Ovary cells expressing dopamine hD(4.4) receptors with only moderate potency and modest efficacy (pEC(50)=6.63; E(max)=19% of dopamine). Moreover, in antagonism experiments, WAY-100635 had a much lower potency at dopamine hD(4.4) receptors (pK(B)=7.09), than at serotonin h5-HT(1A) receptors (pK(B)=9.47). These data demonstrate that WAY-100635 has high selectivity for serotonin h5-HT(1A)versus dopamine hD(4.4) receptors.

Native rat hippocampal 5-HT1A receptors show constitutive activity.

Previous studies have shown that human 5-hydroxytryptamine (5-HT)1A receptors stably expressed in transfected cell lines show constitutive G-protein activity, as revealed by the inhibitory effect of inverse agonists, such as spiperone, on basal guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding. In the present study, we evaluated the constitutive activity of native rat 5-HT1A receptors in hippocampal membranes. Using anti-Galphao-antibody capture coupled to scintillation proximity assay under low sodium (30 mM) conditions, we observed high basal [35S]GTPgammaS binding to Galphao subunits (defined as 100%). Under these conditions, 5-HT and the prototypic selective 5-HT1A agonist (+)8-hydroxy-2-(di-n-propylamino)tetralin [(+)-8-OH-DPAT] both stimulated [35S]GTPgammaS binding to Galphao to a similar extent, raising binding to approximately 130% of basal with pEC50 values of 7.91 and 7.87, respectively. The 5-HT1A-selective neutral antagonist [O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) could block these effects in a competitive manner with pKb values (5-HT, 9.57; (+)-8-OH-DPAT, 9.52) that are consistent with its pKi value at r5-HT1A receptors (9.33). In this native receptor system, spiperone and methiothepin reduced basal [35S]GTPgammaS binding to Galphao in a concentration-dependent manner to 90% of basal with pIC50 values of 7.37 and 7.98, respectively. The inhibition of basal [35S]GTPgammaS binding induced by maximally effective concentrations of spiperone (10 microM) or methiothepin (1 microM) was antagonized by WAY100,635 in a concentration-dependent manner (pKb, 9.52 and 8.87, respectively), thus indicating that this inverse agonism was mediated by 5-HT1A receptors. These data provide the first demonstration that native rat serotonin 5-HT1A receptors can exhibit constitutive activity in vitro.

Partial agonist properties of the antipsychotics SSR181507, aripiprazole and bifeprunox at dopamine D2 receptors: G protein activation and prolactin release.

Dopamine D2 receptor antagonists induce hyperprolactinemia depending on the extent of D2 receptor blockade. We compared the effects of the new antipsychotic agents SSR181507 ((3-exo)-8-benzoyl-N-[[(2 s)7-chloro-2,3-dihydro-1,4-benzodioxin-1-yl]methyl]-8-azabicyclo[3.2.1]octane-3-methanamine monohydrochloride), bifeprunox (DU127090: 1-(2-Oxo-benzoxazolin-7-yl)-4-(3-biphenyl)methylpiperazinemesylate) and SLV313 (1-(2,3-dihydro-benzo[1,4]dioxin-5-yl)-4-[5-(4-fluorophenyl)-pyridin-3-ylmethyl]-piperazine) with those of aripiprazole (7-{4-[4-(2,3-dichlorophenyl)-1-piperazinyl]-butyloxy)-3,4-dihydro-2(1 H)-quinolinone), clozapine and haloperidol, on functional measures of dopamine D2 receptor activity in vitro and in vivo: [35S]-GTPgammaS binding to membranes from Sf9 insect cells expressing human dopamine D2 Long (hD2 L) receptors, and serum prolactin levels in the rat. All compounds antagonized apomorphine-induced G protein activation at dopamine hD2 L receptors. Antagonist potencies of aripiprazole, bifeprunox and SLV313 were similar to haloperidol (pK(b) = 9.12), whereas SSR181507 (8.16) and clozapine (7.35) were less potent. Haloperidol, SLV313 and clozapine were silent antagonists but SSR181507, bifeprunox and aripiprazole stimulated [35S]-GTPgammaS binding by 17.5%, 26.3% and 25.6%, respectively, relative to 100 microM apomorphine (Emax = 100%). pEC50s were: SSR181507, 8.08; bifeprunox, 8.97; aripiprazole, 8.56. These effects were antagonized by raclopride. Following oral administration in vivo, the drugs increased prolactin release to different extents. SLV313 and haloperidol potently (ED50 0.12 and 0.22 mg/kg p.o., respectively) stimulated prolactin release up to 86 and 83 ng/ml. Aripiprazole potently (ED50 0.66 mg/kg p.o.) but partially (32 ng/ml) induced prolactin release. SSR181507 (ED50 4.9 mg/kg p.o.) also partially (23 ng/ml) enhanced prolactin release. Bifeprunox only weakly increased prolactin at high doses (13 ng/ml at 40 mg/kg) and clozapine only affected prolactin at the highest dose tested (41 ng/ml at 40 mg/kg). Prolactin levels of the corresponding vehicle-treated animals were <4.3 ng/ml. These data show that (1) SSR181507, aripiprazole and bifeprunox, but not SLV313, are partial agonists at dopamine hD2 L receptors in vitro; (2) SSR181507, bifeprunox and aripiprazole exhibit reduced prolactin release in vivo compared with drugs that are neutral antagonists at dopamine D2 receptors.

Novel antipsychotics activate recombinant human and native rat serotonin 5-HT1A receptors: affinity, efficacy and potential implications for treatment of schizophrenia.

Serotonin 5-HT1A receptors are promising targets in the management of schizophrenia but little information exists about affinity and efficacy of novel antipsychotics at these sites. We addressed this issue by comparing binding affinity at 5-HT1A receptors with dopamine rD2 receptors, which are important targets for antipsychotic drug action. Agonist efficacy at 5-HT1A receptors was determined for G-protein activation and adenylyl cyclase activity. Whereas haloperidol, thioridazine, risperidone and olanzapine did not interact with 5-HT1A receptors, other antipsychotic agents exhibited agonist properties at these sites. E(max) values (% effect induced by 10 microM of 5-HT) for G-protein activation at rat brain 5-HT1A receptors: sarizotan (66.5), bifeprunox (35.9), SSR181507 (25.8), nemonapride (25.7), ziprasidone (20.6), SLV313 (19), aripiprazole (15), tiospirone (8.9). These data were highly correlated with results obtained at recombinant human 5-HT1A receptors in determinations of G-protein activation and inhibition of forskolin-stimulated adenylyl cyclase. In binding-affinity determinations, the antipsychotics exhibited diverse properties at r5-HT1A receptors: sarizotan (pK(i)=8.65), SLV313 (8.64), SSR181507 (8.53), nemonapride (8.35), ziprasidone (8.30), tiospirone (8.22), aripiprazole (7.42), bifeprunox (7.19) and clozapine (6.31). The affinity ratios of the ligands at 5-HT1A vs. D2 receptors also varied widely: ziprasidone, SSR181507 and SLV313 had similar affinities whereas aripiprazole, nemonapride and bifeprunox were more potent at D2 than 5-HT1A receptors. Taken together, these data indicate that aripiprazole has low efficacy and modest affinity at 5-HT1A receptors, whereas bifeprunox has low affinity but high efficacy. In contrast, SSR181507 has intermediate efficacy but high affinity, and is likely to have more prominent 5-HT1A receptor agonist properties. Thus, the contribution of 5-HT1A receptor activation to the pharmacological profile of action of the antipsychotics will depend on the relative 5-HT1A/D2 affinities and on 5-HT1A agonist efficacy of the drugs.