Visualization of extrastriatal dopamine D2 receptors in the human brain.

[123I]Epidepride, a potent and selective dopamine D2 radioligand, was administered to a 27 year old normal male volunteer. Single photon tomography revealed that peak striatal uptake occurred at 4 h after injection with a striatal:cerebellar ratio of 7.8 rising to over 100 at 18 h post injection. Uptake above the levels seen in cerebellum was also noted in the thalamus, pituitary, hypothalamus and temporal lobe, particularly medially. Single photon tomography with [123I]epidepride allows visualization of extrastriatal dopamine D2 receptors in man.     

Visualization of dopamine D3-like receptors in human brain with [125I]epidepride.

In sections of human brain containing the striatum (caudate, nucleus, putamen, nucleus accumbens) the competition for binding of [125I]epidepride by compounds with differing selectivity for dopamine D2 and D3 receptors was examined. Domperidone showed higher affinity for D2-like than D3-like sites whereas 7-OH-DPAT (7-hydroxy-2-(N,N-di-n-propylamino)tetralin) and quinpirole demonstrated the reverse selectivity. The pattern of [125I]epidepride binding in the presence of a high concentration of domperidone was negligible in the dorsal striatum but indicated islands of dense binding to D3-like receptors in the nucleus accumbens and ventral putamen.     

Occupancy of striatal and extrastriatal dopamine D2/D3 receptors by olanzapine and haloperidol.

There have been conflicting reports as to whether olanzapine produces lower occupancy of striatal dopamine D(2)/D(3) receptor than typical antipsychotic drugs and preferential occupancy of extrastriatal dopamine D(2)/D(3) receptors. We performed [(18)F] fallypride PET studies in six schizophrenic subjects treated with olanzapine and six schizophrenic subjects treated with haloperidol to examine the occupancy of striatal and extrastriatal dopamine receptors by these antipsychotic drugs. [(18)F] setoperone PET studies were performed in seven olanzapine-treated subjects to determine 5-HT(2A) receptor occupancy. Occupancy of dopamine D(2)/D(3) receptors by olanzapine was not significantly different from that seen with haloperidol in the putamen, ventral striatum, medial thalamus, amygdala, or temporal cortex, that is, 67.5-78.2% occupancy; olanzapine produced no preferential occupancy of dopamine D(2)/D(3) receptors in the ventral striatum, medial thalamus, amygdala, or temporal cortex. There was, however, significantly lower occupancy of substantia nigra/VTA dopamine D(2)/D(3) receptors in olanzapine-treated compared to haloperidol-treated subjects, that is, 40.2 vs 59.3% (p=0.0014, corrected for multiple comparisons); in olanzapine-treated subjects, the substantia nigra/VTA was the only region with significantly lower dopamine D(2)/D(3) receptor occupancy than the putamen, that is, 40.2 vs 69.2% (p<0.001, corrected for multiple comparison). Occupancy of 5-HT(2A) receptors was 85-93% in the olanzapine- treated subjects. The results of this study demonstrated that olanzapine does not produce preferential occupancy of extrastriatal dopamine D(2)/D(3) receptors but does spare substantia nigra/VTA receptors. Sparing of substantia nigra/VTA dopamine D(2)/D(3) receptor occupancy may contribute to the low incidence of extrapyramidal side effects in olanzapine-treated patients.     

The quest for Eldorado: development of radioligands for in vivo imaging of nicotinic acetylcholine receptors in human brain

Neuronal nicotinic acetylcholine receptors (nAChRs), ubiquitously distributed in the human brain, are implicated in various neurophysiological processes and in the pathophysiology and/or treatment strategies of Alzheimer's and Parkinson's diseases, Tourette's syndrome, epilepsy, schizophrenia, depression, and anxiety, as well as being particularly affected in tobacco dependence/withdrawal. In the past two decades, researchers have developed an extensive series of radioligands for the assessment of nAChRs in vivo through emission tomography, PET and SPECT. Several radioligands, derivatives of A-85380: 2-[(18)F]FA, 6-[(18)F]FA and 5-[(123)I]IA, are now being employed for the evaluation of nAChR in humans with PET and SPECT. Displaying better imaging properties than (11)C-nicotine and a better toxicity profile than epibatidine analogs, they have allowed quantification of thalamic nAChR in the human brain. Nevertheless, A-85380 derivatives still exhibit slow brain kinetics and a moderate signal-to-noise ratio. Current research efforts on the part of PET/SPECT radiochemists, therefore, have focused on development of new, highly specific and highly selective nAChR radioligands with improved brain kinetics that are able to localize high-affinity nAChRs in vivo. Key examples of new PET/SPECT ligands that are derived from several different structural classes are discussed along with a review of their chemical as well as their in vitro and/or in vivo properties. In particular, new PET nAChR radioligands will be examined that either present faster brain kinetics allowing simple and reliable quantification approaches or higher binding potentials suitable for the evaluation of extrathalamic nAChR.     

Striatal vs extrastriatal dopamine D2 receptors in antipsychotic response--a double-blind PET study in schizophrenia.

Blockade of dopamine D2 receptors remains a common feature of all antipsychotics. It has been hypothesized that the extrastriatal (cortical, thalamic) dopamine D2 receptors may be more critical to antipsychotic response than the striatal dopamine D2 receptors. This is the first double-blind controlled study to examine the relationship between striatal and extrastriatal D2 occupancy and clinical effects. Fourteen patients with recent onset psychosis were assigned to low or high doses of risperidone (1 mg vs 4 mg/day) or olanzapine (2.5 mg vs 15 mg/day) in order to achieve a broad range of D2 occupancy levels across subjects. Clinical response, side effects, striatal ([11C]-raclopride-positron emission tomography (PET)), and extrastriatal ([11C]-FLB 457-PET) D2 receptors were evaluated after treatment. The measured D2 occupancies ranged from 50 to 92% in striatal and 4 to 95% in the different extrastriatal (frontal, temporal, thalamic) regions. Striatal and extrastriatal occupancies were correlated with dose, drug plasma levels, and with each other. Striatal D2 occupancy predicted response in positive psychotic symptoms (r=0.62, p=0.01), but not for negative symptoms (r=0.2, p=0.5). Extrastriatal D2 occupancy did not predict response in positive or negative symptoms. The two subjects who experienced motor side effects had the highest striatal occupancies in the cohort. Striatal D2 blockade predicted antipsychotic response better than frontal, temporal, and thalamic occupancy. These results, when combined with the preclinical data implicating the mesolimbic striatum in antipsychotic response, suggest that dopamine D2 blockade within specific regions of the striatum may be most critical for ameliorating psychosis in schizophrenia.     

Development of radioligands for in vivo imaging of GABA(A)-benzodiazepine receptors

Changes in the biochemical integrity and function of the GABA(A)-benzodiazepine receptor (BZR) complex have been implicated in various neurological and psychiatric disorders. The development of specific radioligands for the GABA(A)-BZR have not only contributed to the elucidation of the receptor's biochemical functions, but also provided a means by which these changes are correlated to disease states when studied with the functional imaging modalities of positron emission tomography (PET) and single photon emission computed tomography (SPECT).     

Iron deficiency decreases dopamine D1 and D2 receptors in rat brain

Iron deficiency (ID) in early life is known to alter neurological development and functioning, but data regarding specific effects on dopamine biology are lacking. The objective of this study was to determine the extent of functional alterations in dopamine receptors in two dopaminergic tracts in young, growing, iron-deficient rats. Forty male and 40 female weanling Sprague-Dawley rats were fed either an iron-deficient (ID) diet or control (CN) diet for 6 weeks. ID decreased densities of D(1) and D(2) receptors in the caudate-putamen and decreased D(2) receptor densities in the nucleus accumbens. There were no apparent effects of ID on the affinities for the ligands in either receptor in several brain regions. In situ hybridization studies for both dopamine receptors revealed no significant effect of ID on mRNA expression for either receptor. Iron-deficient rats had a significantly higher ED(50) for raclopride-induced hypolocomotion in male and female rats compared to control rats of each sex. The loss of iron in the striatum due to dietary ID was significantly correlated with the decrease in D(2) receptor density; however, this relationship was not apparent in other brain regions. These experiments thus demonstrate abnormal dopamine receptor density and functioning in several brain regions that are related to brain regional iron loss. Importantly, the impact of ID on dopamine was more pronounced in males than females, demonstrating sex-related different sensitivities to nutrient deprivation.     

Rotigotine is a potent agonist at dopamine D1 receptors as well as at dopamine D2 and D3 receptors

Background and Purpose

Rotigotine acts as a dopamine receptor agonist with high affinity for the dopamine D₂, D₃, D₄ and D₅ receptors but with a low affinity for the dopamine D₁ receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson''s disease (PD).

Experimental Approach

The binding of rotigotine to human dopamine D₁, D₂, D₃, D₄ and D₅ receptors was determined in radioligand binding studies using [³H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined.

Key Results

[³H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D₁, D₂ and D₃ receptors and, to a lesser extent, D₄ and D₅ receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors.

Conclusions and Implications

Rotigotine is a high-potency agonist at human dopamine D₁, D₂ and D₃ receptors with a lower potency at D₄ and D₅ receptors. These studies differentiate rotigotine from conventional dopamine D₂ agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D₁ and D₅ receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties.     

Syntheses of 2-amino and 2-halothiazole derivatives as high-affinity metabotropic glutamate receptor subtype 5 ligands and potential radioligands for in vivo imaging

The structure of the potent selective mGlu(5) ligand, SP203 (1, 3-fluoro-5-[[2-(fluoromethyl)thiazol-4-yl]ethynyl]benzonitrile), was modified by replacing the 2-fluoromethyl substituent with an amino or halo substituent and by variation of substituents in the distal aromatic ring to provide a series of new high-affinity mGlu(5) ligands. In this series, among the most potent ligands obtained, the 2-chloro-thiazoles 7a and 7b and the 2-fluorothiazole 10b showed subnanomolar mGlu(5) affinity. 10b also displayed >10000-fold selectivity over all other metabotropic receptor subtypes plus a wide range of other receptors and binding sites. The 2-fluorothiazoles 10a and 10b were labeled using [(18)F]fluoride ion (t(1/2) = 109.7 min) in moderately high radiochemical yield to provide potential radioligands that may resist troublesome radiodefluorination during the imaging of brain mGlu(5) with position emission tomography. The iodo compound 9b has nanomolar affinity for mGlu(5) and may also serve as a lead to a potential (123)I-labeled ligand for imaging brain mGlu(5) with single photon emission computed tomography.     

Interaction of simple tetrahydroisoquinolines with opiate and high-affinity dopamine (D3) receptors in the rat corpus striatum

The pharmacological activities of salsolinol (Sal) and salsoline (San) were compared by their abilities to displace 3H-dopamine and 3H-D-ala2-met-enkephalinamide from specific binding sites of rat striatal membrane preparations. Sal was more potent in receptor tests than San. Consequently, methylation by using catechol-O-methyltransferase is the process of its partial inactivation. Sal affinity for D3-receptors was higher than for opiate receptors (IC50 was 1.1 mumol/l for the former and 88 mumol/l for the latter). It was concluded that dopamine-like mechanisms of action of simple tetrahydroisoquinolines are possible to exist.     

Synthesis and evaluation of radioligands for imaging brain nociceptin/orphanin FQ peptide (NOP) receptors with positron emission tomography

Positron emission tomography (PET) coupled to an effective radioligand could provide an important tool for understanding possible links between neuropsychiatric disorders and brain NOP (nociceptin/orphanin FQ peptide) receptors. We sought to develop such a PET radioligand. High-affinity NOP ligands were synthesized based on a 3-(2'-fluoro-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)-2(2-halobenzyl)-N-alkylpropanamide scaffold and from experimental screens in rats, with ex vivo LC-MS/MS measures, three ligands were identified for labeling with carbon-11 and evaluation with PET in monkey. Each ligand was labeled by (11)C-methylation of an N-desmethyl precursor and studied in monkey under baseline and NOP receptor-preblock conditions. The three radioligands, [(11)C](S)-10a-c, gave similar results. Baseline scans showed high entry of radioactivity into the brain to give a distribution reflecting that expected for NOP receptors. Preblock experiments showed high early peak levels of brain radioactivity, which rapidly declined to a much lower level than seen in baseline scans, thereby indicating a high level of receptor-specific binding in baseline experiments. Overall, [(11)C](S)-10c showed the most favorable receptor-specific signal and kinetics and is now selected for evaluation in human subjects.     

Saturation of striatal D(2) dopamine receptors by clozapine

Positron emission tomography (PET) studies have demonstrated low striatal D2 dopamine receptor occupancy in clozapine-treated schizophrenic patients. The aim of this pilot study was to explore if this low receptor occupancy indeed represents partial saturability of striatal D2 dopamine receptors by clozapine. Three anaesthetized Cynomolgus monkeys were examined during one day with PET and [11C]raclopride at baseline and after intravenous injections of clozapine 1.5 mg/kg followed by 18.5 mg/kg. The estimated corresponding human oral doses were approx. 210 mg/d and 2800 mg/d. D2 dopamine receptor occupancy was calculated using an equilibrium-ratio analysis and ranged from 54 to 58% after 1.5 mg/kg and 87 to 89% after the total dose 20 mg/kg. The calculated maximal occupancy was 93%. We conclude that PET-measured D2 dopamine receptor occupancy by clozapine can be described using a model based on the law of mass action, previously validated for conventional antipsychotics. Therefore, sufficiently high doses of clozapine are expected to produce complete striatal D2 dopamine receptor occupancy. The findings further support our previous findings of low D2 dopamine receptor occupancy in patients treated with standard doses of clozapine.     

Structural determinants of pharmacological specificity between D(1) and D(2) dopamine receptors

To test the hypothesis that pharmacological differentiation between D(1) and D(2) dopamine receptors results from interactions of selective ligands with nonconserved residues lining the binding pocket, we mutated amino acid residues in the D(2) receptor to the corresponding aligned residues in the D(1) receptor and vice versa and expressed the receptors in human embryonic kidney 293 cells. Determinations of the affinity of the 14 mutant D(2) receptors and 11 mutant D(1) receptors for D(1)- and D(2)-selective antagonists, and rhodopsin-based homology models of the two receptors, identified two residues whose direct interactions with certain ligands probably contribute to ligand selectivity. The D(1) receptor mutant W99(3.28)F showed dramatically increased affinity for several D(2)-selective antagonists, particularly spiperone (225-fold), whereas the D(2) receptor mutant Y417(7.43)W had greatly decreased affinity for benzamide ligands such as raclopride (200-fold) and sulpiride (125-fold). The binding of the D(1)-selective ligand R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) was unaffected, indicating that SCH23390 makes little contact with these ancillary pocket residues. Mutation of A/V(5.39) caused modest but consistent and reciprocal changes in affinity of the receptors for D(1) and D(2)-selective ligands, perhaps reflecting altered packing of the interface of helices 5 and 6. We also obtained some evidence that residues in the second extracellular loop contribute to ligand binding. We conclude that additional determinants of D(1)/D(2) receptor-selective binding are located either in that loop or in the transmembrane helices but, like residue 5.39, indirectly influence the interactions of selective ligands with conserved residues by altering the shape of the primary and ancillary binding pockets.     

Synthesis and in vitro evaluation of 2,3-dimethoxy-5-(fluoroalkyl)-substituted benzamides: high-affinity ligands for CNS dopamine D2 receptors.

A number of 2,3-dimethoxy-5-(fluoroalkyl)-N-[(1-ethyl-2- pyrrolidinyl)methyl]benzamides (with or without a 6-hydroxy group) were synthesized and evaluated as dopamine D2 receptor ligands. The parent acids were synthesized via the Claisen rearrangement of the appropriate O-allyl ethers, which were derived from o-vanillic acid or 2,3-dimethoxysalicylic acid. A decrease in reactivity was found to be characteristic of pentasubstituted benzoates, and difficulties were encountered with the introduction of fluorine onto the ethyl side chains. The (fluoroethyl)- and (fluoropropyl)salicylamides were 5 times more potent than the corresponding benzamides in inhibiting [3H]spiperone binding to the D2 receptor. These (fluoroalkyl)salicylamides are of potential value for in vivo positron emission tomography (PET) studies upon the basis of their relatively selective, high potency binding affinity for the D2 receptor.     

In vivo extrastriatal and striatal D2 dopamine receptor blockade by amisulpride in schizophrenia

Amisulpride, a substituted benzamide with high affinity for dopamine D2 and D3 receptors only, has been reported to have therapeutic effects on both negative and positive schizophrenic symptoms, although at distinct dose ranges (50-300 mg/day vs. 400-1,200 mg/day). The purpose of this study was to investigate the binding of amisulpride to extrastriatal (i.e., thalamus and temporal cortex) and striatal D2 dopamine receptors with respect to plasma amisulpride determinations. Ten patients with schizophrenia treated with amisulpride over a wide range of doses (25-1,200 mg/day) were studied. Positron emission tomography images were acquired by using 76Br-FLB-457, a highly specific antagonist of the D2 and D3 dopamine receptors. Binding indexes (BI) in the regions studied were estimated with reference to values from six healthy subjects. A curvilinear relationship was demonstrated between plasma concentration of amisulpride and the BI in extrastriatal regions. The BI also varied as a function of plasma concentration in striatum. Furthermore, the data provide evidence for different binding profiles: low plasma concentrations (28-92 ng/mL) induced marked extrastriatal binding and low striatal binding, whereas higher plasma concentrations (>153 ng/mL) induced marked binding both in extrastriatal and striatal regions. Dose-dependent differential binding profiles of amisulpride to D2 receptors in extrastriatal and striatal regions were demonstrated, and two therapeutic ranges of plasma concentrations for negative and positive schizophrenic symptoms, respectively, are suggested.     

In vitro pharmacology of aripiprazole, its metabolite and experimental dopamine partial agonists at human dopamine D2 and D3 receptors

Aripiprazole is the first dopamine D₂/D₃ receptor partial agonist successfully developed and ultimately approved for treatment of a broad spectrum of psychiatric and neurological disorders. Aripiprazole's dopamine D₂ and serotonin 5-HT_1A receptor partial agonist activities have been postulated to confer clinical efficacy without marked sedation, and a relatively favorable overall side-effect profile. Using aripiprazole's unique profile as a benchmark for new dopamine partial agonist development may facilitate discovery of new antipsychotics. We conducted an in vitro comparative analysis between aripiprazole, and its human metabolite OPC-14857 (7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl)butoxy)-2(1H)-quinolinone)); RGH-188 (trans-1-[4-[2-[4-(2,3-dichlorophenyl)piperazine-1-yl]ethyl]cyclohexyl]-3,3-dimethylurea), and its metabolite didesmethyl-RGH-188 (DDM-RGH-188); as well as bifeprunox, sarizotan, N-desmethylclozapine (NDMC; clozapine metabolite), and SDZ 208-912 (N-[(8α)-2-chloro-6-methylergolin-8-yl]-2,2-dimethylpropanamide). In vitro pharmacological assessment included inhibition of forskolin-stimulated cAMP accumulation and the reversal of dopamine-induced inhibition in clonal Chinese hamster ovary cell lines expressing D_2S, D_2L, D₃ Ser-9 and D₃ Gly-9 for human dopamine receptors. All test compounds behaved as dopamine D₂/D₃ receptor partial agonists. Aripiprazole's intrinsic activity at dopamine D_2S and D_2L receptors was similar to that of OPC-14857 and RGH-188; lower than that of dopamine and bifeprunox; and higher than that of DDM-RGH-188, SDZ 208-912, sarizotan, and NDMC. Aripiprazole's intrinsic activity at dopamine D₃ Ser-9 and D₃ Gly-9 receptors was similar to that of OPC-14857 and sarizotan; lower than that of dopamine, bifeprunox, RGH-188 and DDM-RGH-188; and higher than that of SDZ 208-912 and NDMC. A consolidated assessment of these findings may help defining the most appropriate magnitude of intrinsic activity at dopamine D₂/D₃ receptors for clinical efficacy and safety.     

PET and SPECT imaging of the opioid system: receptors, radioligands and avenues for drug discovery and development

As we celebrate the bicentennial of the isolation of morphine by Sertürner, opioids continue to dominate major sectors of the analgesic market worldwide. The pharmaceutical industry stands to benefit greatly from molecular imaging in preclinical and early clinical trials of new or improved opioid drugs. At this juncture, it seems fitting to summarize the past twenty or so years of research on molecular imaging of the opioid system from the viewpoint of drug discovery and development. Opioid receptors were first imaged in human volunteers by positron emission tomography (PET) in 1984. Now, quantitative PET imaging of the major opioid receptor types (micro, delta , kappa) is possible in the brain and peripheral organs of healthy persons and patient populations. Radioligands are under development for single photon emission computed tomography (SPECT) of opioid receptors as well. These functional, nuclear imaging techniques can trace the fate of radiolabeled molecules directly, but non-invasively, and allow precise pharmacokinetic and pharmacodynamic measurements. Molecular imaging provides unique data that can aid in selecting the best drug candidates, determining optimal dosing regimens, clearing regulatory hurdles and lowering risks of failure. Using a historical perspective, this review touches on opioid receptors as drug targets, and focuses on the status and use of radiotracers for opioid receptor PET and SPECT. Selected studies are discussed to illustrate the power of molecular imaging for facilitating opioid drug discovery and development.     

Psychomotor-activating effects mediated by dopamine D(2) and D(3) receptors in the nucleus accumbens

The contribution made by specific dopamine receptor subtypes to the induction of motor behaviors has not been firmly established. Here, we first characterized the behavioral effects induced by a D(2)-class receptor agonist, bromocriptine, following injections into the nucleus accumbens (Acb). Bromocriptine showed an atypical D(2)-class receptor agonist profile, having no observable effect on a range of motor behaviors. However, when coadministered with the D(1)-class receptor agonist SKF 38393, bromocriptine showed a typical D(2)-class receptor agonist profile, enhancing locomotor activity and suppressing spontaneous yawning. We then administered the dopamine receptor antagonists L-741626 and nafadotride, which possess relative selectivity for D(2) and D(3) receptors, respectively, prior to injections of dopamine agonists into the Acb. Nafadotride significantly reduced the locomotor-enhancing effects elicited by the coadministration of SKF 38393 and the D(2)-class receptor agonist (+)-PD 128907 into the Acb, and also attenuated the effects induced by the combination of SKF 38393 and bromocriptine, although not significantly so. L-741626 mildly attenuated the locomotor effects elicited by both drug combinations. Taken together, these results suggest that both D(2) and D(3) receptors in the Acb contribute to the expression of heightened psychomotor activation.