Pharmacology profile of F17464, a dopamine D3 receptor preferential antagonist.

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 (hD3) (Ki = 0.17 nM) and the serotonin receptor subtype 1a (5-HT1a) (Ki = 0.16 nM) and a >50 fold lower affinity for the human dopamine receptor subtype 2 short and long form (hD2s/l) (Ki = 8.9 and 12.1 nM, respectively). [14C]F17464 dynamic studies show a slower dissociation rate from hD3 receptor (t1/2 = 110 min) than from hD2s receptor (t1/2 = 1.4 min) and functional studies demonstrate that F17464 is a D3 receptor antagonist, 5-HT1a receptor partial agonist. In human dopaminergic neurons F17464 blocks ketamine induced morphological changes, an effect D3 receptor mediated. In vivo F17464 target engagement of both D2 and 5-HT1a receptors is demonstrated in displacement studies in the mouse brain. F17464 increases dopamine release in the rat prefrontal cortex and mouse lateral forebrain - dorsal striatum and seems to reduce the effect of MK801 on % c-fos mRNA medium expressing neurons in cortical and subcortical regions. F17464 also rescues valproate induced impairment in a rat social interaction model of autism. All the neurochemistry and behavioural effects of F17464 are observed in the dose range 0.32-2.5 mg/kg i.p. in both rats and mice. The in vitro - in vivo pharmacology profile of F17464 in preclinical models is discussed in support of a therapeutic use of the compound in schizophrenia and autism.

Dopamine Receptor Subtypes, Physiology and Pharmacology: New Ligands and Concepts in Schizophrenia.

Dopamine receptors are widely distributed within the brain where they play critical modulator roles on motor functions, motivation and drive, as well as cognition. The identification of five genes coding for different dopamine receptor subtypes, pharmacologically grouped as D1- (D1 and D5) or D2-like (D2S, D2L, D3, and D4) has allowed the demonstration of differential receptor function in specific neurocircuits. Recent observation on dopamine receptor signaling point at dopamine-glutamate-NMDA neurobiology as the most relevant in schizophrenia and for the development of new therapies. Progress in the chemistry of D1- and D2-like receptor ligands (agonists, antagonists, and partial agonists) has provided more selective compounds possibly able to target the dopamine receptors homo and heterodimers and address different schizophrenia symptoms. Moreover, an extensive evaluation of the functional effect of these agents on dopamine receptor coupling and intracellular signaling highlights important differences that could also result in highly differentiated clinical pharmacology. The review summarizes the recent advances in the field, addressing the relevance of emerging new targets in schizophrenia in particular in relation to the dopamine - glutamate NMDA systems interactions.

Distinctive in vitro signal transduction profile of NLX-112, a potent and efficacious serotonin 5-HT1A receptor agonist.

OBJECTIVES: NLX-112 (befiradol, F13640) is a selective serotonin 5-HT1A receptor agonist. Although it has been tested in vivo, little has been reported on its in vitro signal transduction profile. METHODS: NLX-112 was tested on G-protein activation, inhibition of adenylyl cyclase, ERK1/2 phosphorylation (pERK) and receptor internalization in recombinant cell lines. NLX-112 was also tested on G-protein activation in rat hippocampal membranes. Gα subunit mRNA expression in cell lines and rat brain tissue was quantified by quantitative PCR. KEY FINDINGS: For all signalling measures, NLX-112 exhibited agonist efficacy greater than for reference compounds ((±)8-OH-DPAT or buspirone), but similar to the endogenous agonist, serotonin, and was more potent for pERK than other responses. In rat hippocampal membranes, NLX-112 stimulated 'total G-proteins' but, unlike (±)8-OH-DPAT and buspirone, was more potent for Gαo activation. Cell lines predominantly expressed Gαi1 and Gαi2 mRNA, with low levels of Gαo, whereas in rat brain Gαo subunits showed highest mRNA expression. CONCLUSIONS: Unlike reference compounds, NLX-112 was a highly efficacious agonist in vitro, preferentially activating pERK in cell lines and Gαo proteins in rat hippocampal membranes. However, Gα subunit mRNA levels differ markedly between rat brain and cell lines, warranting caution when extrapolating from recombinant systems to native tissues.

Agonist-directed trafficking of signalling at serotonin 5-HT2A, 5-HT2B and 5-HT2C-VSV receptors mediated Gq/11 activation and calcium mobilisation in CHO cells.

Several examples of agonist-directed trafficking of receptor signalling at 5-HT2A and 5-HT2C receptors have been reported that involve independent downstream transduction pathways. We now report the functional selectivity of a series of chemically diverse agonists at human (h)5-HT2A, h5-HT2B and h5-HT2C-VSV by examining two related responses, the upstream activation of Gq/11 proteins in comparison with its associated cascade of calcium mobilisation. At the h5-HT2A receptor, d-lysergic acid diethylamide (LSD) and the antiparkinsonian agents lisuride, bromocriptine and pergolide exhibit a higher potency for Gq/11 activation than calcium release in contrast with all the other tested ligands such as 5-HT, mCPP and BW723C86, that show an opposite preference of signalling pathway. Comparable observations are made at h5-HT2B and h5-HT2C-VSV receptors, suggesting a similar mechanism of functional selectivity for the three serotonin receptors. Interestingly, the non-hallucinogenic compound lisuride behaves as a partial agonist for both Gq/11 activation and calcium release at the three 5-HT2 receptors, in contrast with DOI, LSD, pergolide and bromocriptine, which are known to provoke hallucinations, and behave as more efficacious agonists. Hence, a functional selectivity for Gq/11 activation together with a threshold of efficacy at h5-HT2A (and possibly h5-HT2B and/or h5-HT2C-VSV) may contribute to hallucinogenic liability. Thus, our results extend the notion of agonist-directed trafficking of receptor signalling to all the 5-HT2-receptor family and indicate that measures of Gq/11 activation versus calcium release may be useful to identify more effective therapeutic drugs with limited side effects.

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.

Effect of intrastriatal 6-OHDA lesion on dopaminergic innervation of the rat cortex and globus pallidus.

The present study examined in the rat the effect of a partial lesion of the nigrostriatal dopaminergic pathway induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA), on the dopaminergic innervation of the cortex and the globus pallidus as revealed using tyrosine hydroxylase (TH) immunoreactivity. Twenty-eight days after unilateral injection of 6-OHDA into the dorsal part of the striatum, TH-positive fiber density was reduced by 41% in the dorsal and central part of the structure, and was accompanied by a retrograde loss of 33% of TH-positive neurons in the substantia nigra (SN), while the ventral tegmental area was completely spared. In the SN, TH-positive cell loss was most severe in the ventral part of the structure (-55%). In the same animals, a substantial loss of TH-positive fibers was evident in the dorsal part of the globus pallidus, and involved both thick fibers of passage and thin varicose terminal axonal branches. In the cortex, a loss of TH-positive fibers was prominent in the cingulate area, moderate in the motor area and less affected in the insular area, while the noradrenergic innervation revealed using dopamine-beta-hydroxylase immunoreactivity was preserved in all of these cortical subregions. These results demonstrate that the intrastriatal 6-OHDA lesion model in rats produces a significant loss of dopaminergic axons in extrastriatal structures including the pallidum and cortex, which may contribute to functional sequelae in this animal model of Parkinson's disease.

Progressive sensorimotor impairment is not associated with reduced dopamine and high energy phosphate donors in a model of ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is a genetic disease, associated with progressive motor impairment and a lack of functional ATM protein. It has been reported that immunoreactive tyrosine hydroxylase and dopamine transporter are reduced in an Atm-/- mouse model of A-T. These observations led to a hypothesis that A-T is associated with loss of nigrostriatal dopamine and prompted the launch of clinical trials to evaluate a therapeutic utility of the anti-parkinsonian drug, l-DOPA. To test for dopamine depletion more directly, we measured regional levels of monoamines and their metabolites in the Atm-/- mouse brain. We also measured levels of NAD+, a cofactor for dopamine biosynthesis and substrate of the DNA damage surveillance enzyme, poly(ADP-ribose) polymerase (PARP). Constitutive activation of PARP has been posited to cause NAD+ depletion. We observed no reduction in monoamine transmitters and no depletion of NAD+, or other high energy phosphate donors in the adult Atm-/- cerebellum, striatum, or ventral mesencephalon. However, our studies did reveal a progressive sensorimotor impairment in Atm-/- mice that may serve as a relevant proxy for progressive neurological impairment in the human disease. Our results call into question the involvement of dopamine in A-T and the therapeutic strategy of enhancing dopaminergic function with l-DOPA.