APR-246 overcomes resistance to cisplatin and doxorubicin in ovarian cancer cells.

Two main causes of platinum resistance are mutation in the tumor suppressor gene TP53 and drug-induced increase in intracellular glutathione concentration. Mutations in TP53 occur in about 50% of human tumors. APR-246 (PRIMA-1(MET)) is the first clinical-stage compound that reactivates mutant p53 and induces apoptosis. APR-246 is a prodrug that is converted to the active compound methylene quinuclidinone (MQ), a Michael acceptor that binds to cysteine residues in mutant p53 and restores its wild-type conformation. Here, we show that MQ also binds to cysteine in glutathione, thus decreasing intracellular free glutathione concentration. We also show that treatment with APR-246 completely restores the cisplatin and doxorubicin sensitivity to p53-mutant drug-resistant ovarian cancer cells. We propose that this unique ability of APR-246/MQ to bind to cysteines in both mutant p53 and glutathione has a key role in the resensitization as well as in the outstanding synergistic effects observed with APR-246 in combination with platinum compounds in ovarian cancer cell lines and primary cancer cells. However, MQ binding to cysteines in other targets, for example, thioredoxin reductase, may contribute as well. Strong synergy was also observed with the DNA-damaging drugs doxorubicin and gemcitabine, while additive effects were found with the taxane docetaxel. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with platinum-based therapy in patients with p53-mutant recurrent high-grade serous (HGS) ovarian cancer. More than 96% of these patients carry TP53 mutations. Combined treatment with APR-246 and platinum or other DNA-damaging drugs could allow dramatically improved therapy of a wide range of therapy refractory p53 mutant tumors.

The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine.

RATIONALE: Clozapine is a unique antipsychotic, with efficacy against positive symptoms in treatment-resistant schizophrenic patients, and the ability to improve cognition and treat the negative symptoms characteristic of this disease. Despite its unique clinical actions, no specific molecular mechanism responsible for these actions has yet been described. OBJECTIVES AND METHODS: To comprehensively profile a large library of neuropsychiatric drugs, including most antipsychotics, at human monoamine receptors using R-SAT, an in vitro functional assay. RESULTS: Profiling revealed that N-desmethylclozapine (NDMC), the principal metabolite of clozapine, but not clozapine itself, is a potent and efficacious muscarinic receptor agonist, a molecular property not shared by any other antipsychotic. To further explore the role of NDMC muscarinic receptor agonist properties in mediating the physiological actions of clozapine, systemically administered NDMC was found to stimulate the phosphorylation of mitogen-activated protein kinase (MAP kinase) in mouse CA1 hippocampal neurons, an effect that was blocked by scopolamine, confirming central M1 muscarinic receptor agonist activity in vivo. Lastly, an analysis of clozapine and NDMC serum levels in schizophrenic patients indicated that high NDMC/clozapine ratios better predicted improvement in cognitive functioning and quality of life than the levels of either compound alone. CONCLUSIONS: The muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy.

The pharmacological profile of (R)-3,4-dihydro-N-isopropyl-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide, a selective 5-hydroxytryptamine(1A) receptor agonist.

The pharmacological properties of the 5-hydroxytryptamine (HT)(1A) receptor agonist (R)-3,4-dihydro-N-isopropyl-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide (NAE-086) were examined with in vitro and in vivo techniques. Receptor binding studies demonstrated that NAE-086 was a high-affinity and selective 5-HT(1A) receptor ligand with a K(i) value of 4.5 nM in membranes from rat hippocampus. Of 32 other receptors examined NAE-086 had a modest affinity only for the 5-HT(7) receptor (K(i) = 240 nM). NAE-086 inhibited VIP-stimulated adenylyl cyclase activity in GH(4)ZD10 cells with 79% of the efficacy of 5-HT. This inhibition was blocked by the 5-HT(1A) receptor (and beta-adrenoceptor) antagonist (-)alprenolol. A minor metabolite of NAE-086 in rats, (R)-3,4-dihydro-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide had a similar receptor profile but had 17 times higher affinity for the 5-HT(1A) receptor (K(i) = 0.26 nM). In vivo, NAE-086 induced all the typical effects of a 5-HT(1A) receptor agonist in rats: it decreased 5-HT synthesis (5-HTP accumulation) and 5-HT turnover (measured as the ratio of 5-hydroxyindoleacetic acid/5-HT), increased corticosterone secretion, induced the 5-HT(1A) syndrome (flat body posture and forepaw treading), inhibited the cage-leaving response, and caused hypothermia. All the responses mediated by postsynaptic 5-HT(1A) receptors were attenuated after single or repeated treatment of the rats with NAE-086. Simultaneously with the development of the tolerance to 5-HT(1A) receptor-mediated responses, 5-HT(2A) receptor-mediated responses were enhanced, as judged from the increased number of spontaneous and/or agonist [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]-induced wet-dog shake responses. The significance of this behavioral effect in relation to clinical observations is discussed.

Site-directed mutagenesis of the 5-HT1B receptor increases the affinity of 5-HT for the agonist low-affinity conformation and reduces the intrinsic activity of 5-HT.

The antagonist radioligand [3H]GR125743 and the agonist radioligand [3H]5-HT were used to investigate the pharmacological characteristics of the G protein uncoupled agonist low-affinity and G protein coupled agonist high-affinity conformations of the wild-type and mutant human 5-hydroxytryptamine 1B (5-HT1B) receptors. We found that substitution of phenylalanine 185 in transmembrane region IV by alanine or methionine resulted in a reduced number of receptors in the coupled conformation, as well as a reduced affinity of 5-HT for the uncoupled conformation. In contrast, substitution of phenylalanine 331 in transmembrane region VI by alanine increased the affinity of 5-HT for the uncoupled conformation 11-fold thus reducing the agonist low-affinity to agonist high-affinity (K(il)/K(ih)) ratio 5-fold. This reduced ratio was correlated with a significantly reduced intrinsic activity of 5-HT previously determined by its ability to inhibit forskolin-stimulated cAMP production. In conclusion, these results show that single amino acid substitutions can selectively change the affinity of 5-HT for the G protein uncoupled conformation of the 5-HT1B receptor and alter the intrinsic activity of the ligand.

Serotonergic and dopaminergic activities of rigidified (R)-aporphine derivatives.

Novel rigidified (R)-aporphine derivatives were synthesized from (R)-1,11-carbonylaporphine by ring expansion reactions. The structures of the novel analogues were assigned by NMR spectroscopy and X-ray crystallography. The compounds showed moderate affinities and selectivities at serotonin S-HT1A and 5-HT7 and dopamine D2A receptors.

Synthesis of novel 5-substituted 3-amino-3,4-dihydro-2H-1-benzopyran derivatives and their interactions with the 5-HT1A receptor.

A series of new enantiomerically pure 3-amino-3,4-dihydro-2H-1-benzopyrans (3-aminochromans) has been synthesized from (R)- and (S)-5-methoxy-3-amino-3,4-dihydro-2H-1-benzopyran. The absolute configuration of the respective (R)- and (S)-enantiomers was deduced from X-ray crystallography of (R)-3-(N-isopropylamino)-5-methoxy-3,4-dihydro-2H-1-benzopyran, (R)-9a. Various 5-substituents were introduced via palladium-catalyzed carbonylation of N-substituted 3-amino-5-trifluoromethanesulfonyloxy-3,4-dihydro-2H-1-benzopyran. The effect of N- and 5-substitution on affinity for the 5-HT1A receptor was evaluated in competition experiments using rat hippocampal membranes and [3H]8-OH-DPAT as radioligand. Selected compounds were also tested for their affinity to the D1 (rat striatum), D2 (rat striatum), D2A (human cloned), and 5-HT2A (rat cortex) receptors. The intrinsic activity of the compounds was evaluated by measuring their effect on VIP-stimulated cAMP production in GH4ZD10 cells stably transfected with the 5-HT1A receptor. High-affinity compounds with high selectivity for the 5-HT1A receptor were found among structures substituted with carboxylate esters, amides, and ketones in the 5-position. Primary and secondary amines bound with lower affinity than tertiary amines. Larger substituents were well-tolerated by the receptor, but the smaller N-ethyl-N-isopropyl bound with lower affinity. Generally, the (R)-enantiomers displayed higher affinity for the 5-HT1A receptor than the corresponding (S)-enantiomers. In the present series of compounds, both full and partial agonists were found.

Novel derivatives of 3-(dipropylamino)chroman. Interactions with 5-HT1A and D2A receptors.

Novel 8-aryl and 8-aroyl substituted derivatives of 3-(dipropylamino)chroman are described. The compounds have been prepared by a palladium catalyzed reaction of iodoarenes and a stannylated derivative of [eta6-3-(dipropylamino)chroman]Cr(CO)3. Several of the compounds have high affinity for 5-HT1A receptors whereas the affinity for D2A receptors is lower, the 8-arylated derivatives being slightly more potent than the 8-aroylated analogues.

Structural analogues of 5-OMe-BPAT: synthesis and interactions with dopamine D2, D3, and serotonin 5-HT1A receptors.

Several structural analogues of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1), a representative of a series of 2-aminotetralin-derived benzamides with potential atypical antipsychotic properties, were synthesized and evaluated for their ability to bind to dopamine D2A, D3, and serotonin 5-HT1A receptors in vitro. The structure affinity relationships revealed that the aromatic ring of the benzamide moiety of 1 contributes to the high affinities for all three receptor subtypes. Furthermore, 1 may interact with the dopamine D2 and D3 receptors through hydrogen bond formation with its carbonyl group. Investigation of the role of the amide hydrogen atom by amide N-alkylation was not conclusive, since conformational aspects may be responsible for the decreased dopaminergic affinities of the N'-alkylated analogues of 1. The effects of the amide modifications on the serotonin 5-HT1A receptor affinity were less pronounced, suggesting that the benzamidoethyl side-chain of 1 as a whole enhances the affinity for this receptor subtype probably through hydrophobic interactions with an accessory binding site. The structural requirements for the substituents at the basic nitrogen atom supported the hypothesis that the 2-aminotetralin moieties of the 2-aminotetralin-derived substituted benzamides may share the same binding sites as the 2-(N,N-di-n-propylamino)tetralins.

Receptor binding characteristics of [3H]NAD-299, a new selective 5-HT1A receptor antagonist.

In vitro receptor binding properties of the novel tritiated 5-hydroxytryptamine1A (5-HT1A) receptor antagonist (R)-3-N,N-dicyclobutylamino-8-fluoro-[6-3H]-3,4-dihydro-2H-1-benzo pyran-5-carboxamide ([3H]NAD-299, generic name robalzotan) were evaluated and compared with those of the agonist 8-hydroxy-2-[2,3-3H]di-n-(propylamino)tetralin ([3H]8-OH-DPAT). [3H]NAD-299 binding displayed a Kd value of 0.17 nM and a Bmax value of 26.7 pmol/g wet weight of rat hippocampus. Same binding affinity (Kd = 0.16 nM) was found to cloned human 5-HT1A receptors. Addition of the nonhydrolyzable GTP analog guanylylimidodiphosphate had no effect on the binding characteristics of [3H]NAD-299, while it significantly decreased both the affinity and density of receptors labeled with [3H]8-OH-DPAT. The rank order of potency of various compounds to inhibit [3H]NAD-299 binding is consistent with the labeling of 5-HT1A receptors. This newly developed high-affinity and selective antagonist radioligand provides a valuable tool for studies of 5-HT1A receptors both in vitro and in vivo.

Interactions of ligands with active and inactive conformations of the dopamine D2 receptor.

The affinities of 19 pharmacologically diverse dopamine D2 receptor ligands were determined for the active and inactive conformations of cloned human dopamine D2 receptors expressed in Ltk cells. The agonist [3H]quinpirole was used to selectively label the guanine nucleotide-binding protein-coupled, active receptor conformation. The antagonist [3H]raclopride, in the presence of the non-hydrolysable GTP-analogue Gpp(NH)p and sodium ions and in the absence of magnesium ions, was used to label the free inactive receptor conformation. The intrinsic activities of the ligands were determined in a forskolin-stimulated cyclic AMP assay using the same cells. An excellent correlation was shown between the affinity ratios (KR/KRG) of the ligands for the two receptor conformations and their intrinsic activity (r=0.96). The ligands included eight structurally related and enantiopure 2-aminotetralin derivatives; the enantiomers of 5-hydroxy-2-(dipropylamino)tetralin, 5-methoxy-2-(dipropylamino)tetralin, 5-fluoro-2-(dipropylamino)tetralin and 2-(dipropylamino)tetralin. The (S)-enantiomers behaved as full agonists in the cyclic AMP assay and displayed a large KR/KRG ratio. The (R)-enantiomers were classified as partial agonists and had lower ratios. The structure-affinity relationships of these compounds at the active and the inactive receptor conformations were analysed separately, and used in conjunction with a homology based receptor model of the dopamine D2 receptor. This led to proposed binding modes for agonists, antagonists and partial agonists in the 2-aminotetralin series. The concepts used in this study should be of value in the design of ligands with predetermined affinity and intrinsic activity.

Agonist and inverse agonist activity at the dopamine D3 receptor measured by guanosine 5'--gamma-thio-triphosphate--35S- binding.

In this study, the ligand-receptor-G protein interactions of the dopamine D3 receptor expressed in Chinese hamster ovary cells were investigated using guanosine 5'-[gamma-thio]triphosphate-[35S] ([35S]GTPgammaS) and receptor binding experiments. Dopamine stimulated the [35S]GTPgammaS binding in a guanine nucleotide, magnesium and sodium-dependent manner. Dopamine and quinpirole produced maximal stimulation of the [35S]GTPgammaS binding whereas (+)-7-OH-DPAT and (-)-3-PPP were partial agonists. Interestingly, several compounds previously classified as D2 receptor antagonists behaved as inverse agonists at the D3 receptor, i.e., they inhibited the basal [35S]GTPgammaS binding in a dose dependent fashion. Haloperidol, (+)-UH-232, (+)-AJ-76 and raclopride were full inverse agonists but clozapine was a partial inverse agonist. Pertussis toxin treatment abolished the D3 receptor-mediated agonist as well as inverse agonist responses, indicating the involvement of Gi/Go proteins in both processes. According to the ternary complex model, agonists should bind with higher affinity to the G protein coupled receptor (RG) and thereby shift the equilibrium from free receptor (R) toward RG, which produces a biological response. However, an inverse agonist should bind with higher affinity to R than to RG and thereby inhibit the basal activity of the cell. We found that the high affinity agonist binding site (RG) was abolished by pertussis toxin treatment of the cells. However, the inverse agonists bound with the same affinity to untreated and pertussis toxin treated D3 receptor membranes. Thus, we found no evidence for the hypothesis that inverse agonists would shift the equilibrium from RG toward R by binding with higher affinity to R than to RG.

2-aminotetralin-derived substituted benzamides with mixed dopamine D2, D3, and serotonin 5-HT1A receptor binding properties: a novel class of potential atypical antipsychotic agents.

A new chemical class of potential atypical antipsychotic agents, based on the pharmacological concept of mixed dopamine D2 receptor antagonism and serotonin 5-HT1A receptor agonism, was designed by combining the structural features of the 2-(N,N-di-n-propylamino)tetralins (DPATs) and the 2-pyrrolidinylmethyl-derived substituted benzamides in a structural hybrid. Thus, a series of 35 differently substituted 2-aminotetralin-derived substituted benzamides was synthesized and the compounds were evaluated for their ability to compete for [3H]-raclopride binding to cloned human dopamine D2A and D3 receptors, and for [3H]-8-OH-DPAT binding to rat serotonin 5-HT1A receptors in vitro. The lead compound of the series, 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (12a), displayed high affinities for the dopamine D2A receptor (Ki = 3.2 nM), the dopamine D3 receptor (Ki = 0.58 nM) as well as the serotonin 5-HT1A receptor (Ki = 0.82 nM). The structure-affinity relationships of the series suggest that the 2-aminotetralin moieties of the compounds occupy the same binding sites as the DPATs in all three receptor subtypes. The benzamidoethyl side chain enhances the affinities of the compounds for all three receptor subtypes, presumably by occupying an accessory binding site. For the dopamine D2 and D3 receptors, this accessory binding site may be identical to the binding site of the 2-pyrrolidinylmethyl-derived substituted benzamides.

The pharmacological characterization of a novel selective 5-hydroxytryptamine1A receptor antagonist, NAD-299.

The pharmacological properties of a novel selective 5-hydroxytryptamine1A (5-HT1A) receptor antagonist, NAD-299 [(R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate] were examined in vitro and in vivo and compared with the reference 5-HT1A receptor antagonist, WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazin-yl))ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride]. The new compound had high affinity for 5-HT1A receptors in vitro with a Ki value of 0.6 nM. The only other receptors for which NAD-299 had affinity less than 1 microM were alpha-1 and beta adrenoceptors with Ki values of 260 and 340 nM, respectively. Thus, the selectivity of NAD-299 for 5-HT1A receptors was more than 400 times. WAY-100635 had considerably higher affinity than NAD-299 for alpha-1 adrenoceptors (Ki = 45 nM) and dopamine D2 and D3 receptors (Ki = 79 and 67 nM, respectively). Like WAY-100635, NAD-299 competitively blocked 5-HT-induced inhibition of vasoactive intestinal peptide-stimulated cAMP production in GH4ZD10 cells and had no intrinsic activity. Both compounds were therefore 5-HT1A receptor antagonists in vitro and also behaved as such in in vivo experiments. Thus, they competitively antagonized the 8-hydroxy-2-(di-n-propylamino)tetralin-induced 5-HT behavioral effects, hypothermia, corticosterone secretion and inhibition of passive avoidance behavior without causing any actions of their own. The effective dose of NAD-299 varied between 0.03 and 0.35 micromol/kg s.c., depending on the test and the dose of 8-hydroxy-2-(di-n-propylamino)tetralin.

Derivatives of (R)- and (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin: synthesis and interactions with 5-HT1A receptors.

Analogs of the 5-HT1A receptor antagonist (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin [(S)-1,(S)-UH301] have been prepared. The C8-substituent has been varied, and in some derivatives one of the N-propyl groups has been exchanged for a 4-(8-aza-7,9-dioxospiro[4.5]decan-8-yl)-butyl group. The novel compounds have been evaluated for affinity to rat brain 5-HT1A receptors in competition experiments with [3H]-8-OH-DPAT. In addition, the efficacy of the compounds was assessed by their ability to inhibit the VIP-stimulated cAMP formation in GH4ZD10 cells expressing rat 5-HT1A receptors. Varying degrees of intrinsic activity was revealed among the compounds tested, i.e., the profiles ranged from full agonists to antagonists. All R-enantiomers are characterized as full agonists at 5-HT1A receptors, whereas partial agonists or antagonists were found among the corresponding S-enantiomers. Substitution of one of the N-propyl groups for a 4-(8-aza-7,9-dioxospiro[4.5]decan-8-yl)butyl group seems to increase efficacy as well as affinity for 5-HT1A receptors. A favorable interaction with an accessory binding site by the N-4-(8-aza-7,9-dioxospiro[4.5]decan-8-yl)butyl group may contribute to the increased affinity.

Critical reevaluation of spiperone and benzamide binding to dopamine D2 receptors: evidence for identical binding sites.

There are several inconsistencies in the literature as regards the characteristics of benzamide and butyrophenone binding to dopamine D2-like receptors. The variations observed in Bmax, Kd and Ki values have led to hypotheses, such as the existence of a specific "benzamide binding site' and that dopamine D2 receptors exist in a monomer-dimer equilibrium, where benzamides are supposed to bind receptor monomers and butyrophenones receptor dimers. We have previously suggested that the discrepant results may instead be related to methodological difficulties associated with the use of very high-affinity radioligands (e.g. ligand depletion and failure to achieve equilibrium). The present study was designed to reinvestigate and critically reevaluate the binding characteristics of [3H]spiperone, [3H]nemonapride, [125I](S)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6- dimethylsalicylamide ([125I]NCQ-298) and [3H]raclopride to cloned human dopamine D2A and rat striatal dopamine D2 receptors in order to establish whether they label the same receptor population. We found that the Kd values of [3H]spiperone, [125I]NCQ-298 and [3H]nemonapride were about 20 pM and that of [3H]raclopride about 1 nM. We did not find any significant differences between the Bmax values determined with the various radioligands. Furthermore, the Ki values of spiperone and NCQ-298 (derived from cross-competition studies) for dopamine D2 receptors labelled with either [3H]spiperone or [125I]NCQ-298 were in good agreement with the corresponding Kd values. In conclusion, our results clearly demonstrate that when studied under correct experimental conditions, all four radioligands label an identical receptor population.

Differential serotoninergic and dopaminergic activities of the (R)- and the (S)-enantiomers of 2-(di-n-propylamino)tetralin.

Racemic 2-(di-n-propylamino)tetralin ((R,S)-DPAT), which lacks phenolic or other aromatic substituents, induces both dopaminergic (sniffing, licking and gnawing) and serotoninergic (forepaw treading and flat body posture) behavioural responses. The present study shows that s.c. administration of (R)-DPAT induces typical 5-HT1A receptor agonist behaviours. These effects are blocked by the 5-HT1A receptor antagonist (S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((S)-UH-301). Administration of (S)-DPAT induces dopaminergic behaviours, which are fully antagonised by raclopride, a dopamine D2 receptor antagonist. Both enantiomers induce hypothermia, (R)-DPAT being antagonised by (S)-UH-301, whereas (S)-DPAT is antagonised by raclopride. The accumulation of 5-hydroxytryptophan and DOPA (3,4-dihydroxyphenylalanine) after decarboxylase inhibition that reflects presynaptic actions on 5-HT (5-hydroxytryptamine, serotonin) and dopamine neurons, respectively, are inhibited by both enantiomers of DPAT. (R)-DPAT is more potent than (S)-DPAT as an inhibitor of 5-hydroxytryptophan accumulation whereas (S)-DPAT is more potent than (R)-DPAT as an inhibitor of DOPA accumulation. Thus, in functional tests of postsynaptic actions (R)-DPAT behaves as a 5-HT1A receptor agonist and (S)-DPAT as a dopamine D2 receptor agonist. Presynaptically, (R)-DPAT shows selectivity for 5-HT1A receptors and (S)-DPAT for dopamine D2 receptors. Receptor binding studies, utilizing [3H]8-hydroxy-2-(di-n-propylamino)tetralin and [3H]quinpirole as radioligands for 5-HT1A and dopamine D2 receptors, respectively, showed (R)-DPAT to have a 3-fold higher affinity than (S)-DPAT for 5-HT1A receptors, whereas (S)-DPAT had a 6-fold higher affinity than (R)-DPAT for dopamine D2 receptors. Thus, the results from receptor binding studies support the conclusion that (R)- and (S)-DPAT are agonists showing selectivity for 5-HT1A and dopamine D2 receptors, respectively. Taken together, these findings may explain previous controversies with regard to the pharmacology of racemic DPAT and re-emphasise the necessity to study pure enantiomers of chiral compounds.

Why does clozapine stimulate the motor activity of reserpine-pretreated rats when combined with a dopamine D1 receptor agonist?

The aim of the present experiments was to investigate the locomotor stimulant effects of the atypical antipsychotic agent, clozapine, in rats depleted of their dopamine by reserpine and alpha-methyl-p-tyrosine pretreatment. Clozapine itself induced a slight but never significant stimulation of locomotor activity which was enhanced by the addition of the selective dopamine D1 receptor agonist, SKF38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3- benzazepine), but not by the selective dopamine D2 receptor agonist, quinpirole. The stimulation produced by clozapine plus SKF38393 was blocked by the selective dopamine D1 receptor antagonist, SCH23390 (7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5- tetrahydro-1H-3-benzapine hydrochloride), while the selective dopamine D2 receptor antagonist, haloperidol, was ineffective. A combination of SCH23390 and haloperidol blocked the clozapine plus SKF38393-induced locomotion. Unlike clozapine, neither the selective 5-HT2 receptor antagonist, ritanserin, nor the dopamine D2 receptor antagonists, haloperidol and remoxipride, caused locomotor activation when given alone or in combination with SKF38393. The indirectly acting sympathomimetic amine, d-amphetamine, was inactive in the monoamine-depleted rats, indicating that no dopamine was available for release by d-amphetamine. The muscarinic receptor antagonist, scopolamine, alone did not alter locomotion, but produced marked stimulation when combined with SKF38393 but not with quinpirole. This stimulation was not affected by haloperidol. However, the scopolamine plus SKF38393-induced stimulation was partially blocked by SCH23390 or by a combination of haloperidol and SCH23390. The data indicate that clozapine, in rats depleted of their dopamine stores, exhibits properties consistent with those of a dopamine receptor agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of [3H]quinpirole binding to human dopamine D2A and D3 receptors: effects of ions and guanine nucleotides.

The in vitro receptor binding characteristics of [3H]quinpirole to cloned human dopamine D2A (long isoform) and D3 receptors were investigated and compared with those of rat striatal dopamine receptors. [3H]Quinpirole selectively labeled the high-affinity state of cloned dopamine D2A and striatal D2 receptors with an affinity of about 4 nM. In the striatum, [3H]quinpirole bound to 70% of the receptors labeled by the antagonist [3H]raclopride, whereas the corresponding value for cloned dopamine D2A receptors was 26%. [3H]Quinpirole labeled both the high- and "low-"affinity states of the dopamine D3 receptor with the affinities of 0.6 nM (36% of the receptors) and 7.3 nM, respectively. At all three receptors, sodium decreased the proportion of receptors labeled by [3H]quinpirole, whereas its affinity for the remaining high-affinity sites was not changed. Further addition of guanine nucleotides completely converted the high-affinity binding into low. Thus, even the dopamine D3 receptor was regulated by sodium and guanine nucleotides. Competition studies for [3H]quinpirole and [3H]raclopride binding revealed that the agonists (+)-(R)-7-hydroxy-2-dipropylaminotetralin and quinpirole, previously claimed to be highly dopamine D3-selective (approximately 100-fold), displayed high affinity for the high-affinity agonist states of both dopamine D2 and D3 receptors. When these values were compared, instead of the apparent affinities from the one-site analysis, the dopamine D3-selectivities were 20-fold for (+)-(R)-7-hydroxy-2-dipropylaminotetralin and 8-fold for quinpirole. Thus, it is of importance to consider both high- and low-affinity agonist states when receptor selectivities are evaluated.

Carbon-11-FLB 457: a radioligand for extrastriatal D2 dopamine receptors.

UNLABELLED: D2 dopamine receptors in extrastriatal brain regions are of central interest for research in schizophrenia and antipsychotic drugs. This article reports the development of [11C]FLB 457 for PET examination of extrastriatal D2 dopamine receptors. METHODS: Carbon-11-FLB 457 was prepared by O-methylation of FLB 604 (2-hydroxy precursor) with [11C]methyl iodide. Total radiochemical yield was 25%-35% within a total synthesis time of 30 min. The specific radioactivity at the end of synthesis was about 1300 Ci/mmole (48 GBq/mumole). RESULTS: FLB 457 bound with high affinity to D2 and D3 dopamine receptors, whereas binding to other putative central receptors was negligible. PET studies in Cynomolgus monkeys demonstrated 15 times higher accumulation of radioactivity in the striatum than in the cerebellum after 60 min. Uptake in the thalamus and neocortex, extrastriatal regions with a low density of D2 dopamine receptors, was, respectively, 4 and 2.5 times higher than in the cerebellum. Radioactivity was displaced by raclopride and haloperidol which confirms the selectivity and reversibility of [11C]FLB 457 binding to D2 dopamine receptors in vivo in the striatum, thalamus and neocortex. CONCLUSION: Carbon-11-FLB 457 should be a useful PET ligand for quantitative examination of D2 dopamine receptors in extrastriatal regions in the human brain.

Does the dopamine receptor subtype selectivity of antipsychotic agents provide useful leads for the development of novel therapeutic agents?

Antipsychotic agents share the ability to antagonize dopamine (DA) receptors, and correlation studies have indicated that the clinical efficacy of neuroleptic agents may be coupled to their affinity for D2 receptors. More recently, a family of DA D2-like receptors has been identified. These receptors include the D2A, D2B, D3 and D4 receptors. On the basis of in vitro receptor-binding studies, it has been suggested that the atypical profile of clozapine might be related to a selective effect on the D4 receptor subtype. We have studied the receptor-binding profiles of a series of antipsychotic agents and evaluated some of the compounds in behavioural assays in the rat. Most of the antipsychotic agents lack selectivity for DA-receptors as well as selectivity for the various DA-receptor subtypes. Because of this lack of selectivity, it is impossible to draw firm conclusions about the role of any particular receptor in the clinical profile of the neuroleptic agents. Furthermore, the pharmacology of potential human metabolites has to be taken into account in a proper analysis of the clinical profile. Consequently, most speculations on the key-target of clinically interesting antipsychotics (including clozapine) may be of little practical value. Clinical studies with receptor (subtype)-selective agents will be more informative.