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F17464 (N-(3-{4-[4-(8-Oxo-8H-[1,3]-dioxolo-[4,5-g]-chromen-7-yl)-butyl]-piperazin-1-yl}-phenyl)-methanesulfonamide, hydrochloride) is a new potential antipsychotic with a unique profile. The compound exhibits high affinity for the human dopamine receptor subtype 3 (hD

Agonist and antagonist properties of antipsychotics at human (h) recombinant 5-hydroxytryptamine (h5-HT(1A)) receptor have been examined previously in transfected Chinese hamster ovary (CHO) cells using 5'-O-(3-[(35)S]thio)-triphosphate ([(35)S] GTP gamma S) binding. Na(+)-dependent [35S] GTP gamma S binding to membranes from human epithelioid carcinoma (HeLa) cells, expressing 500 fmol/mg protein of h5-HT(1A) receptor (HA7 cells), appears suitable to characterize not only agonist and antagonist properties of 5-HT(1A) receptor ligands, but also inverse agonist properties. We therefore examined agonist, antagonist, and inverse agonist activity of antipsychotics at h5-HT(1A) receptor in HA7 cells. Some antipsychotics had agonist activity and stimulated [(35)S] GTP gamma S binding with the following order of efficacy: nemonaprideziprasidoneclozapineocaperidone. Tiospirone and trans-5-chloro-2-methyl-2,3,3a,12b-tetrahydro-1H-dibenz[2,3:6,7,5]-oxepino-[4,5c]pyrrole (ORG 5222), were more potent h5-HT(1A) receptor antagonists than raclopride, olanzapine, and risperidone. Haloperidol, chlorpromazine, thioridazine, pimozide, and sertindole showed Na(+)-dependent inverse agonist activity at h5-HT(1A) receptor that could be antagonized by (s)-N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide [(s)-WAY 100135]. These results are further evidence that interactions with h5-HT(1A) receptors could play a role in the pharmacological profile of certain antipsychotics, and that Na(+) affects the ability to detect inverse agonist activity at h5-HT(1A) receptors, likely by influencing receptor precoupling. Also, the manner in which compounds interact with 5-HT(1A) receptors appears to be related to their K(b)/K(i) ratio.

G protein-coupled receptors exist in G protein-coupled and -uncoupled forms that exhibit high and low affinity for agonists, respectively. Consequently, affinity differences of a compound for the high vs. the low affinity state of a receptor have been used to estimate its intrinsic activity at that receptor. We examined the affinity of a series of compounds for 5-hydroxytryptamine 1A (5-HT 1A ) receptor sites labeled with 0.2 nM [ 3 H](±)-8-hydroxy-2-(di- n -propylamino)tetralin ([ 3 H]8-OH-DPAT) (high affinity), or with 0.25 nM [ 3 H]4-(2′-methoxy-)-phenyl-1-[2′-( N -2″-pyridyl)- p -fluorobenzamido]ethyl-piperazine ([ 3 H] p -MPPF) in the presence of 100 μM guanylylimidodiphosphate (Gpp(NH)p) (low affinity) in rat hippocampal membranes. For a variety of 5-HT 1A receptor ligands, the low/high affinity ratio (ranging from 110 for 5-HT to 0.12 for spiperone) was in good agreement with their reported intrinsic activity. Positive rank correlations were found between low/high affinity ratios and intrinsic activities ( E max values) reported in the literature. The high efficacy 5-HT 1A receptor agonists, 1[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl)piperazine (S-14506) and dihydroergotamine, however, had similar, high affinity for both G protein-coupled and -uncoupled forms of the receptor. The Hill coefficients for both compounds were markedly higher than 1.0, suggesting that positive cooperativity could be responsible for the unexpected results. The 5-HT 1A receptor agonist activity of dihydroergotamine and S-14506, assessed by measuring the inhibition of forskolin-stimulated cAMP accumulation, was blocked completely by pertussis toxin, reinforcing the suggested involvement of an inhibitory G protein in their effects. Taken together, the results suggest that, although the low/high affinity ratio of a ligand for 5-HT 1A receptors generally covaries with its intrinsic activity, dihydroergotamine and S-14506 may interact with 5-HT 1A receptors in a manner different from that of other 5-HT 1A receptor agonists. Their effects, however, appear to be G i protein-dependent.

5-HT1A receptor agonists are thought to enhance the antipsychotic-like effects of dopamine D2 receptor antagonists while reducing their potential to produce extrapyramidal side effects. Thus, 5-HT1A receptor agonist properties of mixed 5-HT1A receptor agonists/D2 receptor antagonists might be of clinical importance. The antipsychotics, clozapine and nemonapride, and the putative antipsychotic, bromerguride, have intermediate to high affinity for 5-HT1A receptors. The present study examined the 5-HT1A receptor agonist activity of nemonapride and bromerguride, in comparison with clozapine, which has partial 5-HT1A receptor agonist properties in vitro. Here, 5-HT1A receptor activation was examined in vitro, by measuring forskolin-stimulated cAMP accumulation in HeLa cells expressing human 5-HT1A receptors, and in vivo, by using microdialysis to measure the extracellular concentration of hippocampal 5-hydroxytryptamine (5-HT) in rats. Nemonapride markedly decreased both forskolin-stimulated cAMP accumulation and the extracellular concentration of 5-HT; both effects were antagonized by the 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide (WAY100635). In contrast, clozapine only partially decreased forskolin-stimulated cAMP accumulation and extracellular 5-HT, and only its effects on cAMP accumulation were attenuated by WAY100635. Bromerguride decreased neither forskolin-stimulated cAMP accumulation nor extracellular 5-HT; instead, it antagonized the decrease of cAMP accumulation produced by 5-HT and the decrease of extracellular 5-HT produced by the 5-HT1A agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The selective D2 receptor antagonist, raclopride, affected neither forskolin-stimulated cAMP in vitro nor extracellular 5-HT in vivo. Thus, in contrast with clozapine and bromerguride, only the novel antipsychotic, nemonapride, exhibited marked 5-HT1A receptor agonist properties both in vitro and in vivo; conceivably, these properties may play a role in its preclinical and clinical effects.

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 were4.3 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.

To examine further the hypothesis that the magnitude of the intrinsic activity of agonists at 5-HT1A receptors determines the magnitude of their psychotropic activity, we studied the relationship between the maximal receptor activation produced by various 5-HT1A receptor ligands and their antidepressant-like effects (i.e., decreased immobility in the forced swimming test in rats). Using three different in vitro assays suitable to measure differences among high, intermediate, and low efficacy 5-HT1A receptor agonists, ligands were identified with intrinsic activities ranging from low-negative (i.e., the inverse agonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane-carboxamide (WAY 100635)) to high-positive (i.e., 3-chloro-4-fluorophenyl-(4-fluoro-4-{[(5-methyl-6-methylamino-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone (F 13714)). In addition, novel compounds with intermediate intrinsic activity, like buspirone, but with high selectivity for 5-HT1A receptors, unlike buspirone, were identified. The maximal effects of the 5-HT1A receptor ligands in the forced swimming test correlated positively (rS=0.91, P

Agonist binding to G protein-coupled receptors induces the formation of a receptor-G protein complex and subsequent guanosine 5'-diphosphate/guanosine 5'-triphosphate (GDP/GTP) exchange. Some receptors, however, form receptor-G protein complexes and promote GDP/GTP exchange even when not occupied by agonists. Such receptors preferentially activate pertussis toxin-sensitive G proteins (i.e., G(i)/G(o)), and the interactions of receptors and G proteins are affected by monovalent cations (most notably Na(+)), both in the occupied and unoccupied state. We investigated the effects of Na(+) on the intrinsic activity of 5-hydroxytryptamine(1A) (5-HT(1A)) receptor ligands, measured as maximal effect (E(MAX)), using guanosine 5'-0-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding to membranes prepared from human epithelioid carcinoma (HeLa) cells, expressing 500 fmol/mg protein of cloned human 5-HT(1A) receptor (HA7 cells). A decrease of the NaCl concentration decreased the maximal effect of serotonin, increased basal [35S]GTPgammaS binding, and increased the negative intrinsic activity of spiperone and N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-N-(2-pyridinyl)cyclohexaneca rboxamide (WAY 100635). This ability of WAY 100635 to decrease basal [35S]GTPgammaS binding was antagonized by (s)-N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropa namide ((s)-WAY 100135) (pA(2)=7.77). Further, WAY 100635 was able to antagonize carboxamidotryptamine (5-CT)-stimulated [35S]GTPgammaS binding with a pA(2) of 9.9, in standard NaCl conditions, and of 9.7, in the absence of NaCl. Changes in membrane concentration did not affect the ability of WAY 100635 to decrease [35S]GTPgammaS binding. WAY 100635 did not affect basal [35S]GTPgammaS binding to membranes from untransfected HeLa cells. Pertussis toxin (200 ng/ml) prevented WAY 100635 and spiperone to decrease [35S]GTPgammaS binding, showing that their effects were mediated by G proteins of the G(i)/G(o) family. In conclusion, the constitutive and stimulated activity of human 5-HT(1A) receptors expressed in HA7 cells is sodium-dependent, which allowed to confirm the 5-HT(1A) inverse agonist properties of spiperone, and to show that WAY 100635 is an inverse agonist at 5-HT(1A) receptors that inhibits basal [35S]GTPgammaS binding to a lesser extent than spiperone. [35S]GTPgammaS binding to membranes from HA7 cells under low NaCl conditions appears to be especially suitable to evidence and pharmacologically analyze the inverse agonist properties of 5-HT(1A) receptor ligands.

The effect of N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl) acetamide (BM-5) on acetylcholine release from the cerebral cortex was investigated in unanaesthetized and urethane-anaesthetized rats. BM-5 at doses ranging from 0.3 to 5 mg/kg i.p. enhanced acetylcholine output in both groups of rats. The maximum increase occurred with 0.5 mg/kg in the unanaesthetized and 2 mg/kg in the anaesthetized rats. The effect lasted approximately 60 min. At the largest doses peripheral muscarinic effects including salivation, chromodachryorrhea and rhinorrhea were also seen. These results demonstrate that BM-5 exerts presynaptic antagonistic and postsynaptic agonistic effects on muscarine receptors in vivo also.

The effect of sulfated cholecystokinin (CCK-8S) on acetylcholine turnover (TRACh) and dopamine (DA) release in the rat cerebral cortex and striatum was studied in unanaesthetized animals in vivo. CCK-8S (1 mg/kg s.c.) decreased TRACh in the fronto-parietal cortex but not in the striatum. This effect was prevented by peripheral (10 mg/kg i.p.) but not central (1 microgram i.v.t.) administration of the peripheral CCK receptor antagonist CR 1409. In a separate study, CCK-8S decreased 3-methoxytyramine (3-MT) levels (an index of DA release) in the fronto-parietal cortex and in the striatum. CR 1409 appeared to have a partial agonist action, reducing cortical and striatal 3-MT levels, and only partially reversing the effect of CCK-8S in the striatum. These data indicate that peripheral administration of CCK-8S decrease TRACh in the cortex but not in the striatum and that this action is mediated by peripheral-type CCK receptors possibly located outside the CNS. CCK-8S also reduces DA release in the cortex and in the striatum, and this effect appears to be mediated by a mechanism of action different from that modulating cortical TRACh.