The role of 5-HT receptor subtypes in the anxiolytic effects of selective serotonin reuptake inhibitors in the rat ultrasonic vocalization test

 We evaluated whether the anxiolytic effects of selective serotonin reuptake inhibitors (SSRIs) in the rat ultrasonic vocalization (USV) test are preferentially mediated by (indirect) activation of 5-HT_1A, 5-HT_1B/1D, 5-HT_2A, 5-HT₃ or 5-HT₄ receptors. The SSRIs, paroxetine (ED₅₀ in mg/kg, IP: 6.9), citalopram (6.5), fluvoxamine (11.7) and fluoxetine (>30), dose dependently reduced shock-induced USV. The effects of paroxetine (3.0 mg/kg, IP) were not blocked by the selective 5-HT_1A receptor antagonist, WAY-100635 (3.0 mg/kg, IP), the 5-HT_1B/1D receptor antagonist, GR 127935 (30 mg/kg, IP), the nonselective 5-HT_2A receptor antagonists, ritanserin (3.0 mg/kg, IP) and ketanserin (1.0 mg/kg, IP), the 5-HT₃ receptor antagonist, ondansetron (0.1 mg/kg, IP), or the 5-HT₄ receptor antagonist, GR 125487D (3.0 mg/kg, SC). In contrast, the selective 5-HT_2A receptor antagonist, MDL 100,907 (0.1 mg/kg, IP), completely prevented the paroxetine-induced reduction of USV. Under similar conditions, WAY-100635 blocked the anxiolytic-like effects of the selective 5-HT_1A receptor agonist, 8-OH-DPAT [(±)-8-hydroxy-2-(di-n-propylamino)tetralin, 1.0 mg/kg, IP], and ritanserin, ketanserin, and MDL 100,907 blocked the anxiolytic-like effects of the mixed 5-HT_2A/2C receptor agonist, DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, 3.0 mg/kg, IP]. WAY-100635 (1.0 mg/kg, IP) in combination with ritanserin (3.0 mg/kg, IP), but not ondansetron (0.1 mg/kg, IP), GR 125487D (3.0 mg/ kg, SC), or GR 127935 (30 mg/kg, IP), attenuated the USV reducing effects of paroxetine. Although the results suggest that selective stimulation of 5-HT_1A and 5-HT_2A receptors produces a decrease of USV, we postulate that only 5-HT_2A receptors play a pivotal role in the effects of SSRIs in this model of anxiety. Received: 19 May 1997 / Final version: 21 July 1997     

RU24969-induced behavioural syndrome requires activation of both 5HT1A and 5HT1B receptors

The behavioural profiles of the mixed 5HT_1A/B agonist RU24969 and the more selective 5HT_1B agonist anpirtoline were compared. Both compounds induce an increase in activity as measured in photocell activity cages. The behaviours displayed by the rats receiving each treatment differed markedly, with RU24969 inducing flat body posture and circling of the cage perimeter (1.25−10 mg/kg SC), whereas anpirtoline increased ambulation characterised by a hopping motion (1.25−5.0 mg/kg SC). The effects of RU24969 were attenuated by both the 5HT_1A antagonist WAY100635 (0.03−1.25 mg/kg SC) and the 5HT_1B/D antagonist GR127935 (1.0−5.0 mg/kg SC). Anpirtoline-induced behaviour was attenuated by GR127935 across the same dose range but was largely unaffected by WAY100635 even at doses above those which had blocked the effects of RU24969. Coadministration of the selective 5HT_1A agonist 8-OH-DPAT (0.03−1.25  mg/kg SC) with anpirtoline (2.5 mg/kg) induced a dramatic increase in locomotor activity and a behavioural syndrome identical to that produced by RU24969. Thus it would appear that a synergistic effect of stimulation of both 5HT_1A and 5HT_1B receptors underlies the behavioural effects of RU24969, while anpirtoline acts mainly via stimulation of 5HT_1B receptors only. Received: 31 October 1996/Final version: 24 February 1997     

Model structure–activity relationship studies of potential tropane 5HT1A, 5HT2A, and D2 receptor ligands

The two-stages studies of structure–activity relationship for model ligands of 5HT_1A, 5HT_2A, and D₂ receptors were performed. On the first stage, the pharmacophores of two potential ligands of known in vitro binding to 5HT_1A, 5HT_2A, D₂ receptors and model pharmacophore of strongly interacting D₂ receptor ligands were found and their parameters were related to affinity data. The analyzed parameters were hydrophobic, hydrophilic, aromatic, donor and acceptor of proton centers. The geometry of spatial distribution of these properties was also investigated in comparative analysis. The studied, model compounds were two 3β-acylamine derivatives of tropane. The second stage includes docking of studied compounds to D₂ receptor model and the comparison of its quality with in vivo binding data. The obtained results are consistent with in vitro binding data and applied procedure accurate estimates the affinity of potential ligands to D₂ receptors.     

Differential effects of olanzapine at dopamine D1 and D2 receptors in dopamine depleted animals

We investigated the degree of striatal dopamine-2 (D₂) receptor occupancy in six schizophrenic patients receiving clinically effective antipsychotic treatment with olanzapine 10–25 mg/day in comparison to patients treated with clozapine 300–600 mg/day (n = 6) or haloperidol 5–20 mg/day (n = 10). ¹²³I Iodobenzamide (IBZM) and single photon emission computerized tomography (SPECT) were used for the visualization of striatal D₂ receptors. For the quantification of striatal D₂ receptor occupancy, striatal IBZM binding in patients treated with antipsychotics was compared to that in untreated healthy controls (n = 8) reported earlier. Olanzapine led to a mean striatal D₂ receptor occupancy rate of 75% (range 63–85). Haloperidol-treated patients showed dose-dependently (Pearson r = 0.64; P < 0.05) a significantly higher (P < 0.05) mean occupancy rate of 84% (range 67–94). During clozapine treatment, the mean D₂ receptor occupancy of 33% (range < 20–49) was significantly lower than with olanzapine (P < 0.005). The higher striatal D₂ receptor occupancy of haloperidol was correlated with the incidence and severity of extrapyramidal motor side-effects (EPS). No clinical relevant EPS occurred during treatment with olanzapine or clozapine. There was no correlation between the degree of striatal D₂ receptor occupancy and clinical improvement. Received: 18 March 1998/Final version: 10 June 1998     

Central 5-HT2A and D2 dopamine receptor occupancy after sublingual administration of ORG 5222 in healthy men

It has been suggested that a combined blockade of 5-HT_2A and D₂-dopamine receptors improves efficacy and reduces the risk for extrapyramidal symptoms when treating schizophrenic patients with antipsychotic drugs. ORG 5222 is a new potential anti-psychotic drug which has high affinity for 5-HT_2A, D₂-dopamine and α₁ adrenergic receptors in vitro. The objective of this study was to examine if ORG 5222 occupies 5-HT_2A and D₂-dopamine receptors in human subjects in vivo. Central receptor occupancy was measured by positron emission tomography (PET) in three healthy subjects after sublingual administration of 100 μg ORG 5222. [¹¹C]N-methylspiperone ([¹¹C] NMSP) was the radioligand used to measure 5-HT_2A receptor binding in the neocortex and [¹¹C]raclopride to measure D₂-dopamine receptor binding in the striatum. The 5-HT_2A occupancy was 15–30% and the D₂-dopamine receptor occupancy was 12–23%. The study confirms that ORG 5222 binds to 5-HT_2A and D₂-dopamine receptors in human brain. Since receptor occupancy of ORG 5222 is rather low, doses higher than 100 μg are suggested in future clinical trials to evaluate the antipsychotic drug effect of ORG 5222. Received: 9 September 1996 / Final version: 2 January 1997     

Time course of central nervous dopamine-D2 and 5-HT2 receptor blockade and plasma drug concentrations after discontinuation of quetiapine (Seroquel®) in patients with schizophrenia

Quetiapine (Seroquel) is a novel antipsychotic with an atypical profile in animal models and a relatively short plasma half-life of 2.5–5 h. In the present study, we used PET to compare the time course of blockade of dopamine D₂ and serotonin 5HT₂ receptors of quetiapine using C¹¹-raclopride and C¹¹-N-methyl-spiperone as ligands, parallel to monitoring plasma drug concentrations. It was an open study in 11 schizophrenic men using a fixed dose of 450 mg quetiapine. Eight men completed the 29 days treatment, followed by four PET scans performed over a 26-h period after withdrawal of the compound. Quetiapine was shown to bind to dopamine D₂ receptors in striatum and 2 h (t_max) after the last dose, 44% receptor occupancy was calculated. After 26 h it had dropped to the same level as was found in untreated healthy volunteers. Serotonin 5HT₂ receptor blockade in the frontal cortex was 72% after 2 h, which declined to 50% after 26 h. The terminal plasma half-life of quetiapine was 5.3 h. Clinically, our eight patients had good antipsychotic effect without any extrapyramidal side-effects. Our data shows that quetiapine has a relatively low affinity for dopamine D₂ receptors, with an occupancy half-life (10 h), which was about twice as long as that for plasma. A more prolonged blockade of the serotonin 5HT₂ receptors was found in the frontal cortex, with receptor occupancy half-life of 27 h. Compared to clozapine, as demonstrated in other studies, quetiapine has much the same ratio of D₂/5HT₂ occupancy. This could suggest that the combination of D₂/5HT₂ receptor blockade contributes to the antipsychotic effect and a low incidence of EPS seen with quetiapine in comparative phase three trials. Our results also confirm the clinical data that quetiapine can be administered twice daily. Received: 13 December 1996/Final version: 13 June 1997     

In vivo occupancy of dopamine D2 receptors by antipsychotic drugs and novel compounds in the mouse striatum and olfactory tubercles

Interaction with dopamine D₂-like receptors plays a major role in the therapeutic effects of antipsychotic drugs. We examined in vivo dopamine D₂ receptor occupancy of various established and potential antipsychotics in mouse striatum and olfactory tubercles 1 h after administration of the compound, using [³H]nemonapride as a ligand. All the compounds reduced in vivo binding of [³H]nemonapride in the striatum. When administered systemically, conventional antipsychotics, D₂ antagonists, nemonapride (ID₅₀: 0.034 mg/kg), eticlopride (0.047), haloperidol (0.11) and raclopride (0.11) potently inhibited [³H]nemonapride binding. The ‘atypical’ antipsychotics, risperidone (0.18), ziprasidone (0.38), aripiprazole (1.6), olanzapine (0.99), and clozapine (11.1) were less potent for occupying D₂-like receptors. New compounds, displaying marked agonism at 5-HT_1A receptors in addition to D₂ receptor affinity, exhibited varying D₂ receptor occupancy: bifeprunox (0.25), SLV313 (0.78), SSR181507 (1.6) and sarizotan (6.7). ID₅₀ values for inhibition of [³H]nemonapride binding in the striatum correlated with those in the olfactory tubercles (r=0.95, P<0.0001). These values also correlated with previously-reported in vitro affinity of the compounds at rat D₂ receptors (r=0.85, P=0.0001) and with inhibition of apomorphine-induced climbing in mice (r=0.79 P=0.0005). In contrast, there was no significant correlation between ID₅₀ values herein and previously-reported ED₅₀ values for catalepsy in mice. These data indicate that: (1) there is no difference in D₂ receptor occupancy in limbic versus striatal regions between most classical and atypical or potential antipsychotics; and (2) high occupancy of D₂ receptors can be dissociated from catalepsy, if the drugs also activate 5-HT_1A receptors. Taken together, these data support the strategy of simultaneously targeting D₂ receptor blockade and 5-HT_1A receptor activation for new antipsychotics.     

Evaluation of dopamine D2/D3 and serotonin 5-HT2A receptor occupancy for a novel antipsychotic, lurasidone, in conscious common marmosets using small-animal positron emission tomography

Rationale

Lurasidone is a novel antipsychotic drug with potent binding affinity for dopamine D₂ and serotonin (5-hydroxytryptamine, 5-HT)_2A, 5-HT₇, and 5-HT_1A receptors. Previous pharmacological studies have revealed that lurasidone exhibits a preferable profile (potent antipsychotic activity and lower incidence of catalepsy) to other antipsychotic drugs, although the contribution of receptor subtypes to this profile remains unclear.

Objectives

To compare target engagements of lurasidone with those of an atypical antipsychotic, olanzapine, we performed evaluation of dopamine D₂/D₃ and serotonin 5-HT_2A receptor occupancy in vivo by positron emission tomography (PET) with conscious common marmosets.

Methods

We measured brain receptor occupancies in conscious common marmosets after oral administrations of lurasidone or olanzapine by PET with [¹¹C]raclopride and [¹¹C]R-(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine methanol (MDL 100907) for D₂/D₃ and 5-HT_2A receptors, respectively.

Results

Increases in brain D₂/D₃ receptor occupancies of both lurasidone and olanzapine, which reached >80 % at maximum, were observed in the striatum with significant correlations to plasma drug levels. However, lurasidone showed lower 5-HT_2A receptor occupancy in the frontal cortex within the same dose range, while olanzapine showed broadly comparable 5-HT_2A and D₂/D₃ receptor occupancies.

Conclusions

Compared with olanzapine, lurasidone preferentially binds to D₂/D₃ receptors rather than 5-HT_2A receptors in common marmosets. These results suggest that the contribution of in vivo 5-HT_2A receptor blocking activity to the pharmacological profile of lurasidone might differ from olanzapine in terms of the low risk of extrapyramidal syndrome and efficacy against negative symptoms.     

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 5HT_2A receptors and D₂-type receptors. Predominant 5HT_2A 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 5HT_2A than for D₂ receptors. Subnanomolar affinity for human 5HT_2A 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 5HT_2A than D₂ receptors. Risperidone and ORG-5222 had 5HT_2A versus D₂ potency ratio of about 20. Highest potency for 5HT_2A receptor occupancy was observed for ORG-5222 followed by risperidone and olanzapine. Ziprasidone exclusively occupied 5HT_2A 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 ₁ receptors, except fluspirilene and ziprasidone. Clozapine, zotepine, ORG-5222 and sertindole occupied even more ₁ than D₂ receptors. Clozapine showed predominant occupancy of H₁ receptors and occupied cholinergic receptors with equivalent potency to D₂ receptors. A stronger predominance of 5HT_2A 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 5HT_2A 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 5HT_2A and D₂ occupancy and the avoidance of D₂ receptor overblockade are believed to reduce the risk for extrapyramidal symptoms.     

Neurochemical evidence for antagonism by olanzapine of dopamine,serotonin, α1-adrenergic and muscarinic receptors in vivo in rats

The ability of the atypical antipsychotic drug candidate olanzapine to antagonize dopamine, serotonin, -adrenergic and muscarinic receptors in vivo was assessed by various neurochemical measurements in rat brain. Olanzapine increased the concentrations of the dopamine metabolites DOPAC and HVA in striatum and nucleus accumbens. Olanzapine antagonized the pergolide-induced decrease of striatal DOPA concentrations in rats treated with gammabutyrolactone and NSD1015 and increased striatal 3-methoxytyramine concentrations in nomifensine-treated rats (but not after gammabutyrolactone administration), suggesting that olanzapine blocked terminal and somatodendritic autoreceptors on dopamine neurons. Inactivation of dopamine D₁ and D₂ receptors by EEDQ was antagonized by olanzapine. The ex vivo binding of the 5HT₂ radioligand [³H]-ketanserin was inhibited by olanzapine treatment, as was quipazine-induced increases in MHPG-SO₄, evidence suggesting that olanzapine antagonized 5HT₂ receptors. At higher doses, olanzapine increased the concentration of the norepinephrine metabolite, MHPG-SO₄, probably by blocking ₁-adrenergic receptors. Olanzapine inhibited ex vivo binding of the muscarinic antagonist radioligand [³H]-pirenzepine and lowered concentrations of striatal, but not hippocampal, acetylcholine levels. The findings provide evidence that olanzapine antagonized dopamine, serotonin, -adrenergic and muscarinic receptors in vivo, consistent with its high affinity for these receptor sites in vitro.     

7-OH-DPAT,a dopamine D3-selective receptor agonist,produces contralateral rotation in 6-hydroxydopamine-lesioned rats

The purpose of the present study was to characterize the rotational behavior in unilateral 6-OHDA-lesioned rats produced by the high affinity and selective dopamine D₃ receptor ligand 7-OH-DPAT. Qualitatively similar to the direct-acting DA agonist apomorphine, 7-OH-DPAT causes rats to rotate in a direction contralateral to the side of the nigrostriatal DA pathway lesion. This effect is dose-dependent and the minimum effective dose is 0.03 mg (0.12 m?mol)/kg. 7-OH-DPAT-induced rotation is blocked in a dose-dependent manner by oral pretreatment with the “D₂-like” receptor antagonists haloperidol, eticlopride, or clozapine, but not by the “D₁-like” antagonist SCH 23390. The rank order potency for inhibition of 7-OH-DPAT rotation for haloperidol [ID₅₀ = 0.067 mg (0.18 m?mol)/kg], eticlopride [ID₅₀ = 0.41 mg (1.2 m?mol)/kg], clozapine [ID₅₀ = 13 mg (40 m?mol)/kg], and SCH 23390 [ID₅₀ > 90 mg (313 m?mol)/kg] closely parallels their rank order affinity for binding to either the D₂ or the D₃ receptor. Pretreatment with the non-DA receptor antagonists ritanserin (serotonin 5HT₂), scopolamine (muscarinic cholinergic), propranolol (betaadrenergic), or naltrexone (opiate), each at relevant pharmacological doses, failed to reduce 7-OH-DPAT rotation. Taken together, these results are consistent with mediation of 7-OH-DPAT-induced rotational behavior via an agonist interaction with one or more DA receptors. ©1995 Wiley-Liss, Inc.     

Evidence that the enhancement of dopamine function by repeated electroconvulsive shock requires concomitant activation of D1-like and D2-like dopamine receptors

In this study, the behavioural response to dopamine D₁-like receptor agonists (SKF 38393, SKF 81297 and SKF 77434) and D₂-like receptor agonists (quinpirole and RU 24213), administered alone and in combination to rats treated repeatedly with electroconvulsive shock (five ECS over 10 days) or sham, was tested. Agonist-induced behaviour was monitored by automated activity meters and direct observation using a checklist scoring method. Repeated ECS (compared to sham controls) had no significant effect on the behavioural response to SKF 38393 (7.5 mg/kg SC), SKF 81297 (0.2 mg/kg SC), SKF 77434 (0.1 mg/kg SC), quinpirole (0.1 and 0.25 mg/kg SC) or RU 24213 (0.3 mg/kg SC), when administered alone. In contrast, repeated ECS markedly increased locomotion (activity counts and scores) induced by the non-selective dopamine agonist apomorphine (0.5 mg/kg SC) and by co-administration of a D₁-like agonist plus a D₂-like agonist [SKF 38393 (7.5 mg/kg SC) plus quinpirole (0.25 mg/kg SC), SKF 81297 (0.2 mg/kg SC) plus quinpirole (0.1 mg/kg SC), and SKF 77434 (0.1 mg/ kg SC) plus RU 24213 (0.3 mg/kg SC)]. This ECS-induced enhancement of dopamine-mediated behaviour was observed for up to 3 weeks after cessation of ECS treatment. In addition, ECS also enhanced the locomotor response to intra-accumbens SKF 38393 plus quinpirole (0.4 and 1.0 μg/side, respectively). These results provide evidence that the enhancement of dopamine function by repeated ECS requires concomitant stimulation of both D₁-like and D₂-like receptors, and that this effect is long-lasting. Received: 24 January 1997 /Final version: 5 March 1997     

The effects of dopamine D2 and D3 antagonists on spontaneous motor activity and morphine-induced hyperactivity in male mice

Rationale: Dopaminergic neurotransmission, in particular the mesolimbic pathway, is involved in spontaneous locomotor activity and in morphine-induced hyperactivity, since the drugs acting on DA receptors can modify the action of morphine and this effect could be dependent on the type of DA receptor affected. Objective: In this study, the action of U-99194A maleate, haloperidol, sulpiride and morphine (5, 10, 20, 40 mg/kg) on locomotor activity in male mice was evaluated. Likewise, the effects of these dopaminergic antagonists on morphine-induced hyperactivity were studied. Methods: Animals treated with U-99194A maleate (2.5, 5, 10, 20 mg/kg), haloperidol (0.075, 0.1 mg/kg), sulpiride (20, 40 mg/kg), or morphine (5, 10, 20, 40 mg/kg), and animals treated with these neuroleptics plus morphine were tested in an actimetre at different time points. Results: It was found that an increase in locomotor activity was produced between 0 and 30 min after the administration of 20 mg/kg U-99194A maleate and between 30 and 60 min after the administration of 20 and 40 mg/kg morphine. This dose of U-99194A maleate and the high dose of sulpiride reverts the hyperactivity induced by 20 mg/kg morphine. Haloperidol reversed the hyperactivity induced by all doses of morphine. Conclusions: Our results confirm that the action of DA D₂ and D₃ receptors could be dependent on the dopaminergic state, in this case modified by the action of morphine. Received: 19 June 1998/Final version: 17 October 1998     

The synthetic cathinone psychostimulant α‐PPP antagonizes serotonin 5‐HT2A receptors: In vitro and in vivo evidence

Synthetic cathinones (SCs) are β‐keto analogs of amphetamines. Like amphetamines, SCs target monoamine transporters; however, unusual neuropsychiatric symptoms have been associated with abuse of some SCs, suggesting SCs might possess additional pharmacological properties. We performed radioligand competition binding assays to assess the affinities of nine SCs at human 5‐HT_2A receptors (5‐HT_2AR) and muscarinic M₁ receptors (M₁R) transiently expressed in HEK293 cells. None of the SCs exhibited affinity at M₁R (minimal displacement of [~K_d] [³H]scopolamine up to 10 μM). However, two SCs, α‐pyrrolidinopropiophenone (α‐PPP) and 4‐methyl‐α‐PPP, had low μM K_i values at 5‐HT_2AR. In 5‐HT_2AR–phosphoinositide hydrolysis assays, α‐PPP and 4‐methyl‐α‐PPP displayed inverse agonist activity. We further assessed the 5‐HT_2AR functional activity of α‐PPP, and observed it competitively antagonized 5‐HT_2AR signaling stimulated by the 5‐HT₂R agonist (±)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI; K_b = 851 nM). To assess in vivo 5‐HT_2AR activity, we examined the effects of α‐PPP on the DOI‐elicited head‐twitch response (HTR) in mice. α‐PPP dose‐dependently blocked the HTR with maximal suppression at 10 mg/kg (P < 0.0001), which is a moderate dose used in studies investigating psychostimulant properties of α‐PPP. To corroborate a 5‐HT_2AR mechanism, we also tested 3,4‐methylenedioxy‐α‐PPP (MDPPP) and 3‐bromomethcathinone (3‐BMC), SCs that we observed had 5‐HT_2AR K_is > 10 μM. Neither MDPPP nor 3‐BMC, at 10 mg/kg doses, attenuated the DOI HTR. Our results suggest α‐PPP has antagonist interactions at 5‐HT_2AR in vitro that may translate at physiologically‐relevant doses in vivo. Considering 5‐HT_2AR antagonism has been shown to mitigate effects of psychostimulants, this property may contribute to α‐PPPs unpopularity compared to other monoamine transporter inhibitors.     

Blockade of phencyclidine-induced hyperlocomotion by olanzapine, clozapine and serotonin receptor subtype selective antagonists in mice

In humans, phencyclidine (PCP) is known to produce a syndrome of behavioral effects which have many characteristics in common with schizophrenia. Therefore, antagonism of PCP effects might be evidence for antipsychotic efficacy of a compound. In the present studies, the effects of the D₂-like antagonist haloperidol, the mixed D₂-like/5-HT₂ antagonists olanzapine and clozapine, and a series of 5-HT receptor subtype selective antagonists on the hyperlocomotion produced by PCP were evaluated in mice. PCP (0.3–10 mg/kg) produced a dose-related increase in locomotor activity, with a peak effect at 3.0 mg/kg. The D₂-like antagonist haloperidol produced a dose-related decrease in locomotor activity when administered alone, and blocked the hyperactivity effects of PCP over the same dose-range (minimal effective dose, MED = 0.3 mg/kg for both effects). In contrast, olanzapine and clozapine reversed the hyperlocomotion effects of PCP at doses (MED = 0.03 and 0.3 mg/kg, respectively) approximately 30-and 10-fold, respectively, below those that decreased activity when administered alone (MED = 1.0 and 3.0 mg/kg, respectively). The selective 5-HT₂ antagonist LY53857 (0.3–3.0 mg/kg) administered alone had no effect on locomotor activity but reversed (MED = 0.1 mg/kg) the effects of PCP. Similarly, the selective 5-HT_2A/2C antagonist ritanserin (0.001–1.0 mg/kg) alone had no effect on locomotor activity, but reversed (MED = 0.01 mg/kg) the effects of PCP. The selective 5-HT_2A antagonists ketanserin (MED = 3.0 mg/kg) and MDL 100,907 (MED = 0.3 mg/kg) produced dose-related decreases in locomotor activity and ketanserin (MED = 0.1 mg/kg) and MDL 100,907 (MED = 0.003 mg/kg) reversed the effects of PCP. The selective 5-HT₃ antagonist zatosetron (0.01–10 mg/kg) and the selective 5-HT_1A antagonist WAY 100,635 (0.001–3 mg/kg) were without effects on spontaneous locomotor activity. Zatosetron reversed the effects of 3.0 mg/kg PCP at the nonselective dose of 10 mg/kg whereas WAY 100,635 (0.001–1 mg/kg) did not affect PCP-induced hyperlocomotion. The present results indicate that PCP increases locomotor activity, at least in part, due to actions at 5-HT_2A, but not 5-HT₃ or 5-HT_1A, receptors. Further, the present findings support the hypothesis that antagonism at 5-HT_2A receptors contributes to the in vivo actions of atypical antipsychotics such as olanzapine and clozapine. Received: 27 June 1996/Final version: 20 August 1996     

Synthesis,docking studies,and pharmacological evaluation of 5HT2C ligands containing the N′‐cyanoisonicotinamidine or N′‐cyanopicolinamidine nucleus

N′‐Cyanoisonicotinamidine and N′‐cyanopicolinamidine derivatives, linked to an arylpiperazine moiety, were prepared and their affinities to the 5‐HT_1A, 5‐HT_2A, and 5‐HT_2C receptors were evaluated. Several of the newly synthesized compounds, tested by binding studies, showed nanomolar affinity at the 5‐HT_1A and 5‐HT_2C receptors and moderate or no affinity for other relevant receptors (D₁, D₂, α₁, and α₂). Compound 8e (K_i = 21.4 nM) was the most affine for the 5‐HT_2C receptor, showing, at the same time, a high selectivity with respect to the other receptors analyzed. Compounds 4a and 4c , instead, showed an interesting mixed 5‐HT_1A/5‐HT_2C activity with K_i values of 21.3/11.5 and 23.2/6.48 nM, respectively. The compounds with better affinity and selectivity binding profiles toward 5‐HT_2C ( 4a , 4c , 8b , and 8e ) were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that compounds 4a , 8b , and 8e exerted antidepressant‐like effects and 4a and 8e revealed also significant anxiolytic properties. Among the developed derivatives, the most promising compound seems to be 4a , which displayed antipsychotic‐, antidepressant‐ and anxiolytic‐like properties. No side effects, like catalepsy, motor‐impairment or ethanol‐potentiating effects, were observed after the injection of the tested compounds.     

Oxotremorine suppresses thalamocortical oscillations via thalamic muscarinic acetylcholine receptors

We investigated whether the local intrathalamic infusion of a muscarinic acetylcholine receptor agonist (oxotremorine) at either the reticular nucleus of thalamus (NRT) or the ventroposteromedial nucleus of thalamus (VPM) suppresses thalamocortically generated neocortical high-voltage spindles (HVSs). In addition, we studied whether the intracerebroventricular (ICV) infusion of a selective muscarinic M₂ acetylcholine receptor antagonist (methoctramine) could block the suppression of HVSs induced by either systemic (IP) administration of an anticholinesterase drug [tetrahydroaminoacridine (THA)] or ICV infusion of oxotremorine in rats. Intrathalamic administration of oxotremorine at 3 and 15 μg in the NRT, and at 15 μg in the VPM suppressed HVSs. ICV oxotremorine at 30 and 100 μg and IP THA at 3 mg/kg decreased HVSs. ICV methoctramine at 100 μg increased HVSs and completely blocked the decrease in HVSs produced by oxotremorine 100 μg and THA 3 mg/kg. The results suggest that activation of muscarinic M₂ acetylcholine receptors in thalamic nuclei (NRT and VPM) can suppress thalamocortical oscillations and that ICV or systemically administered drugs that activate either directly (oxotremorine and methoctramine) or indirectly (THA) the muscarinic M₂ acetylcholine receptors may modulate neocortical HVSs via the thalamus. Received: 13 November 1997/Final version: 18 April 1998     

Activation of acetylcholine receptors and 5-HT2 receptors have additive effects in the suppression of neocortical high-voltage spindles in aged rats

We investigated if activation of the muscarinic or nicotinic acetylcholine receptors and serotonin (5-hydroxytryptamine; 5-HT) subtype 2 receptors would have additive or synergistic effects on the suppression of thalamocortically generated rhythmic neocortical high-voltage spindles (HVSs) in aged rats. The 5-HT₂ receptor antagonist, ketanserin, at a moderate dose (5 mg/kg) prevented the ability of a muscarinic acetylcholine receptor agonist, (oxotremorine 0.1 mg/kg), and a nicotinic acetylcholine receptor agonist (nicotine 0.1 mg/kg), to decrease HVSs. At a higher dose (20 mg/kg), ketanserin completely blocked the decrease in HVSs produced by moderate doses of muscarinic acetylcholine receptor agonists (pilocarpine 1 mg/kg and oxotremorine 0.1 mg/kg), and by a high dose of nicotine (0.3 mg/kg), though not that produced by high doses of pilocarpine (3 mg/kg) and oxotremorine (0.9 mg/kg). The ability of a 5-HT₂ receptor agonist, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.1–1.0 mg/kg), to suppress HVSs was non-significantly modulated by the nicotinic acetylcholine receptor antagonist, mecamylamine (1–15 mg/kg), and the muscarinic acetylcholine receptor antagonist, scopolamine (0.03–0.3 mg/kg). The effects of the drugs on behavioral activity could be separated from their effects on HVSs. The results suggest that activation of the muscarinic or nicotinic acetylcholine receptors plus 5-HT₂ receptors has additive effects in the suppression of thalamocortical oscillations in aged rats. Received: 2 November 1996 /Final version: 7 February 1997     

Agonist activity of LSD and lisuride at cloned 5HT2A and 5HT2C receptors

Evidence from studies with phenylisopropylamine hallucinogens indicates that the 5HT_2A receptor is the likely target for the initiation of events leading to hallucinogenic activity associated with LSD and related drugs. Recently, lisuride (a purported non-hallucinogenic congener of LSD) was reported to be a potent antagonist at the 5HT_2C receptor and an agonist at the 5HT_2A receptor. LSD exhibited agonist activity at both receptors. These data were interpreted as indicating that the 5HT_2C receptor might be the initiating site of action for hallucinogens. To test this hypothesis, recombinant cells expressing 5HT_2A and 5HT_2C receptors were used to determine the actions of LSD and lisuride. LSD and lisuride were potent partial agonists at 5HT_2A receptors with EC₅₀ values of 7.2 nM and 17 nM, respectively. Also, LSD and lisuride were partial agonists at 5HT_2C receptors with EC₅₀ values of 27 nM and 94 nM, respectively. We conclude that lisuride and LSD have similar actions at 5HT_2A and 5HT_2C receptors in recombinant cells. As agonist activity at brain 5HT_2A receptors has been associated with hallucinogenic acitivity, these results indicate that lisuride may possess hallucinogenic activity, although the psychopharmacological effects of lisuride appear to be different from the hallucinogenic effects of LSD. Received: 19 September 1997/Final version: 31 October 1997     

5‐HT modulates the rat mesenteric vasopressor outflow by 5‐HT1D sympatholytic receptors

5‐hydroxytryptamine (5‐HT) modulates noradrenergic activity in different cardiovascular territories, but its effect on the mesenteric vasopressor outflow has not yet been clarified. This study investigated the in vivo serotonergic influence, characterizing 5‐HT receptors implicated, in sympathetic innervation of mesenteric vasculature. Wistar rats were anaesthetised and prepared for the in situ autoperfused rat mesentery, monitoring systemic blood pressure (SBP), heart rate (HR) and mesenteric perfusion pressure (MPP). Electrical stimulation of mesenteric sympathetic nerves resulted in frequency‐dependent increases in MPP (9 ± 1.6, 25.7 ± 3.9 and 60.2 ± 5 mmHg for 2, 4 and 8 Hz, respectively), without altering SBP or HR. 5‐HT (1‐25 μg/kg), 5‐carboxamidotryptamine (5‐HT_1/7 agonist; 25 μg/kg) or L‐694,247 (5‐HT_1D agonist; 1‐25 μg/kg) i.a. bolus inhibited vasopressor responses by mesenteric nerves electrical stimulation, unlike i.a. bolus of agonists 8‐OH‐DPAT (5‐HT_1A), CGS‐12066B (5‐HT_1B), BRL 54443 (5‐HT_1e/1F), α‐methyl‐5‐HT (5‐HT₂), 1‐PBG (5‐HT₃), cisapride (5‐HT₄) or AS‐19 (5‐HT₇) (25 μg/kg each). Interestingly, i.a. L‐694,247 (25 μg/kg) also reduced the exogenous norepinephrine‐induced vasoconstrictions. Pretreatment with selective 5‐HT_1D receptor antagonist, LY310762 (1 mg/kg, i.v.), completely abolished L‐694,247‐ and 5‐HT‐induced mesenteric sympathoinhibition. Furthermore, ELISA analysis confirmed 5‐HT_1D receptors expression in mesenteric artery. These findings suggest that serotonergic mechanisms‐induced sympathoinhibition of mesenteric noradrenergic outflow is mediated by pre and/or postjunctional 5‐HT_1D receptors.