Receptor distribution studies.

Receptor distribution studies have played a key role in the characterization of receptor systems (e.g. GABAB, NMDA (GluNRs), and Neurokinin 1) and in generating hypotheses to exploit these systems as potential therapeutic targets. Distribution studies can provide important information on the potential role of candidate receptors in normal physiology/disease and alert for possible adverse effects of targeting the receptors. Moreover, they can provide valuable information relating to quantitative target engagement (e.g. % receptor occupancy) to drive mechanistic pharmacokinetic/pharmacodynamic (PK/PD) hypotheses for compounds in the Drug Discovery process. Finally, receptor distribution and quantitative target engagement studies can be used to validate truly translational technologies such as PET ligands and pharmacoEEG paradigms to facilitate bridging of the preclinical/clinical interface and thus increase probability of success.

The novel mu-opioid antagonist, GSK1521498, reduces ethanol consumption in C57BL/6J mice.

RATIONALE: Using the drinking-in-the-dark (DID) model, we compared the effects of a novel mu-opioid receptor antagonist, GSK1521498, with naltrexone, a licensed treatment of alcohol dependence, on ethanol consumption in mice. OBJECTIVE: We test the ability of GSK1521498 to reduce alcohol consumption and compare its intrinsic efficacy to that of naltrexone by comparing the two drugs at doses matched for equivalent receptor occupancy. METHODS: Thirty-six C57BL/6J mice were tested in a DID procedure. In 2-day cycles, animals experienced one baseline, injection-free session, and one test session when they received two injections, one of test drug and one placebo. All animals received GSK1521498 (0, 0.1, 1 and 3 mg/kg, i.p., 30 min pre-treatment) and naltrexone (0, 0.1, 1 and 3 mg/kg, s.c. 10 min pre-treatment) in a cross-over design. Receptor occupancies following the same doses were determined ex vivo in separate groups by autoradiography, using [3H]DAMGO. Binding in the region of interest was measured integrally by computer-assisted microdensitometry and corrected for non-specific binding. RESULTS: Both GSK1521498 and naltrexone dose-dependently decreased ethanol consumption. When drug doses were matched for 70-75% receptor occupancy, GSK1521498 3 mg/kg, i.p., caused a 2.5-fold greater reduction in alcohol consumption than naltrexone 0.1 mg/kg, s.c. Both GSK1521498 and naltrexone significantly reduced sucrose consumption at a dose of 1 mg/kg but not 0.1 mg/kg. In a test of conditioned taste aversion, GSK1521498 (3 mg/kg) reduced sucrose consumption 24 h following exposure to a conditioning injection. CONCLUSIONS: Both opioid receptor antagonists reduced alcohol consumption but GK1521498 has higher intrinsic efficacy than naltrexone.

Occupancy of brain dopamine D3 receptors and drug craving: a translational approach.

Selective dopamine D(3) receptor (D(3)R) antagonists prevent reinstatement of drug-seeking behavior and decrease the rewarding effects of contextual cues associated with drug intake preclinically, suggesting that they may reduce drug craving in humans. GSK598809 is a selective D(3)R antagonist recently progressed in Phase I trials. The aim of this study was to establish a model, based on the determination of the occupancy of brain D(3)Rs (O(D(3))(R)) across species, to predict the ability of GSK598809 to reduce nicotine-seeking behavior in humans, here assessed as cigarette craving in smokers. Using ex vivo [(125)I](R)-trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino] tetralin ([(125)I]7OH-PIPAT) autoradiography and [(11)C]PHNO positron emission tomography, we demonstrated a dose-dependent occupancy of the D(3)Rs by GSK598809 in rat, baboon, and human brains. We also showed a direct relationship between O(D(3))(R) and pharmacokinetic exposure, and potencies in line with the in vitro binding affinity. Likewise, GSK598809 dose dependently reduced the expression of nicotine-induced conditioned place preference (CPP) in rats, with an effect proportional to the exposure and O(D(3))(R) at every time point, and 100% effect at O(D(3))(R) values 72%. In humans, a single dose of GSK598809, giving submaximal levels (72-89%) of O(D(3))(R), transiently alleviated craving in smokers after overnight abstinence. These data suggest that either higher O(D(3))(R) is required for a full effect in humans or that nicotine-seeking behavior in CPP rats only partially translates into craving for cigarettes in short-term abstinent smokers. In addition, they provide the first clinical evidence of potential efficacy of a selective D(3)R antagonist for the treatment of substance-use disorders.

Stable expression and functional characterization of a human nicotinic acetylcholine receptor with α6ß2 properties: discovery of selective antagonists.

BACKGROUND AND PURPOSE: Despite growing evidence that inhibition of α6ß2-containing (α6ß2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of α6ß2*-nAChRs has delayed the discovery of α6ß2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with α6ß2 properties, to enable pharmacological characterization and the identification of novel α6ß2-selective antagonists. EXPERIMENTAL APPROACH: Different combinations of the α6, ß2, ß3, chimeric α6/3 and mutant ß3(V273S) subunits were transfected in human embryonic kidney cells and tested for activity in a fluorescent imaging plate reader assay. The pharmacology of rat immune-immobilized α6ß2*-nAChRs was determined with ¹²5I-epibatidine binding. KEY RESULTS: Functional channels were detected after co-transfection of α6/3, ß2 and ß3(V273S) subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed α6/3ß2ß3(V273S)-nAChR pharmacology was unique, and clearly distinct from α4ß2-, α3ß4-, α7- and α1ß1δε-nAChRs. Antagonist potencies in inhibiting α6/3ß2ß3(V273S) -nAChRs was similar to their binding affinity for rat native α6ß2*-nAChRs. Agonist affinities for α6ß2*-nAChRs was higher than their potency in activating α6/3ß2ß3(V273S)-nAChRs, but their relative activities were equivalent. Focussed set screening at α6/3ß2ß3(V273S)-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel α6ß2-selective antagonists. CONCLUSIONS AND IMPLICATIONS: We generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native α6ß2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, α6ß2-selective antagonists. We also propose a pharmacophore model of α6ß2 antagonists, which offers a starting point for the development of new smoking cessation agents.

Imaging dopamine D3 receptors in the human brain with positron emission tomography, [11C]PHNO, and a selective D3 receptor antagonist.

BACKGROUND: Dopamine D(3) receptors are involved in the pathophysiology of several neuropsychiatric conditions. [(11)C]-(+)-PHNO is a radiolabeled D(2) and D(3) agonist, suitable for imaging the agonist binding sites (denoted D(2HIGH) and D(3)) of these receptors with positron emission tomography (PET). PET studies in nonhuman primates documented that, in vivo, [(11)C]-(+)-PHNO displays a relative selectivity for D(3) compared with D(2HIGH) receptor sites and that the [(11)C]-(+)-PHNO signal is enriched in D(3) contribution compared with conventional ligands such as [(11)C] raclopride. METHODS: To define the D(3) contribution (f(PHNO)(D3)) to [(11)C]-(+)-PHNO binding potential (BP(ND)) in healthy humans, 52 PET scans were obtained in 19 healthy volunteers at baseline and following oral administration of various doses of the selective D(3) receptor antagonist, GSK598809. RESULTS: The impact of GSK598809 on [(11)C]-(+)-PHNO was regionally selective. In dorsal regions of the striatum, GSK598809 did not significantly affect [(11)C]-(+)-PHNO BP(ND) (f(PHNO)(D3) approximately 0%). Conversely, in the substantia nigra, GSK598809 dose-dependently reduced [(11)C]-(+)-PHNO binding to nonspecific level (f(PHNO)(D3) approximately 100%). In ventral striatum (VST), globus pallidus and thalamus (THA), [(11)C]-(+)-PHNO BP(ND) was attributable to a combination of D(2HIGH) and D(3) receptor sites, with f(PHNO)(D3) of 26%, 67% and 46%, respectively. D(3) receptor binding potential (BP(ND)(D3)) was highest in globus pallidus (1.90) and substantial nigra (1.39), with lower levels in VST (.77) and THA (.18) and negligible levels in dorsal striatum. CONCLUSIONS: This study elucidated the pharmacologic nature of the [(11)C]-(+)-PHNO signal in healthy subjects and provided the first quantification of D(3) receptor availability with PET in the living human brain.

A comparative study of the effects of the intravenous self-administration or subcutaneous minipump infusion of nicotine on the expression of brain neuronal nicotinic receptor subtypes.

Long-term nicotine exposure changes neuronal acetylcholine nicotinic receptor (nAChR) subtype expression in the brains of smokers and experimental animals. The aim of this study was to investigate nicotine-induced changes in nAChR expression in two models commonly used to describe the effects of nicotine in animals: operant (two-lever presses) intravenous self-administration (SA) and passive subcutaneous nicotine administration via an osmotic minipump (MP). In the MP group, alpha4beta2 nAChRs were up-regulated in all brain regions, alpha6beta2* nAChRs were down-regulated in the nucleus accumbens (NAc) and caudate-putamen, and alpha7 nAChRs were up-regulated in the caudal cerebral cortex (CCx); the up-regulation of alpha4beta2alpha5 nAChRs in the CCx was also suggested. In the SA group, alpha4beta2 up-regulation was lower and limited to the CCx and NAc; there were no detectable changes in alpha6beta2* or alpha7 nACRs. In the CCx of the MP rats, there was a close correlation between the increase in alpha4beta2 binding and alpha4 and beta2 subunit levels measured by means of Western blotting, demonstrating that the up-regulation was due to an increase in alpha4beta2 proteins. Western blotting also showed that the increase in the beta2 subunit exceeded that of the alpha4 subunit, suggesting that a change in alpha4beta2 stoichiometry may occur in vivo as has been shown in vitro. These results show that nicotine has an area-specific effect on receptor subtypes, regardless of its administration route, but the effect is quantitatively greater in the case of MP administration.

Nicotinic acetylcholine receptors in the mesolimbic pathway: primary role of ventral tegmental area alpha6beta2* receptors in mediating systemic nicotine effects on dopamine release, locomotion, and reinforcement.

alpha6* nicotinic acetylcholine receptors (nAChRs) are highly and selectively expressed by mesostriatal dopamine (DA) neurons. These neurons are thought to mediate several behavioral effects of nicotine, including locomotion, habit learning, and reinforcement. Yet the functional role of alpha6* nAChRs in midbrain DA neurons is mostly unknown. The aim of this study was to determine the composition and in vivo functional role of alpha6* nAChR in mesolimbic DA neurons of male rats. Immunoprecipitation and immunopurification techniques coupled with cell-specific lesions showed that the composition of alpha6* nAChR in the mesostriatal system is heterogeneous, with (non-alpha4)alpha6beta2* being predominant in the mesolimbic pathway and alpha4alpha6beta2* in the nigrostriatal pathway. We verified whether alpha6* receptors mediate the systemic effects of nicotine on the mesolimbic DA pathway by perfusing the selective antagonists alpha-conotoxin MII (CntxMII) (alpha3/alpha6beta2* selective) or alpha-conotoxin PIA (CntxPIA) (alpha6beta2* selective) into ventral tegmental area (VTA). The intra-VTA perfusion of CntxMII or CntxPIA markedly decreased systemic nicotine-elicited DA release in the nucleus accumbens and habituated locomotion; the intra-VTA perfusion of CntxMII also decreased the rate of nicotine infusion in the maintenance phase of nicotine, but not of food, self-administration. Overall, the results of these experiments show that the alpha6beta2* nAChRs expressed in the VTA are necessary for the effects of systemic nicotine on DA neuron activity and DA-dependent behaviors such as locomotion and reinforcement, and suggest that alpha6beta2*-selective compounds capable of crossing the blood-brain barrier may affect the addictive properties of nicotine and therefore be useful in the treatment of tobacco dependence.

1,2,4-Triazolyl azabicyclo[3.1.0]hexanes: a new series of potent and selective dopamine D(3) receptor antagonists.

The discovery of new highly potent and selective dopamine (DA) D(3) receptor antagonists has recently allowed the characterization of the DA D(3) receptor in a range of preclinical animal models of drug addiction. A novel series of 1,2,4-triazol-3-yl-azabicyclo[3.1.0]hexanes, members of which showed a high affinity and selectivity for the DA D(3) receptor and excellent pharmacokinetic profiles, is reported here. Members of a group of derivatives from this series showed good oral bioavailability and brain penetration and very high in vitro affinity and selectivity for the DA D(3) receptor, as well as high in vitro potency for antagonism at this receptor. Several members of this series also significantly attenuate the expression of conditioned place preference (CPP) to nicotine and cocaine.

Transient forebrain over-expression of CRF induces plasma corticosterone and mild behavioural changes in adult conditional CRF transgenic mice.

BACKGROUND: Converging findings support a role for extra-hypothalamic CRF in the mediation of the stress response. The influence of CRF in the amygdala is well established, while less is known of its role in other areas of the forebrain where CRF and CRF(1) receptors are also expressed. In the present study CRF was genetically induced to allow forebrain-restricted expression in a temporally-defined manner at any time during the mouse lifespan. This mouse model may offer the possibility to establish a model of the pathogenesis of recurrent episodes of depression. METHODS: Mice were engineered to carry both the rtTA transcription factor driven by the CamKII alpha promoter and the doxycycline-regulated operator (tetO) upstream of the CRF coding sequence. Molecular, biochemical and behavioural characterisation of this mouse is described. RESULTS: Following a three-week period of transcriptional induction, double transgenic mice showed approximately 2-fold increased expression of CRF mRNA in the hippocampus and cortex, but not hypothalamus. These changes were associated with 2-fold increase in morning corticosterone levels, although responses to the dexamethasone suppression test or acute stress were unaffected. In contrast, induced mice displayed modestly altered behaviour in the Light and Dark test and Forced Swim test. CONCLUSIONS: Transient induction of CRF expression in mouse forebrain was associated with endocrine and mild anxiety-like behavioural changes consistent with enhanced central CRF neurotransmission. This mouse allows the implementation of regimens with longer or repeated periods of induction which may model the initial stages of the pathology underlying recurrent depressive disorders.

1,2,4-triazol-3-yl-thiopropyl-tetrahydrobenzazepines: a series of potent and selective dopamine D(3) receptor antagonists.

The discovery of new highly potent and selective dopamine D3 receptor antagonists has recently permitted characterization of the role of the dopamine D3 receptor in a wide range of preclinical animal models. A novel series of 1,2,4-triazol-3-yl-thiopropyl-tetrahydrobenzazepines demonstrating a high level of D3 affinity and selectivity with an excellent pharmacokinetic profile is reported here. In particular, the pyrazolyl derivative 35 showed good oral bioavailability and brain penetration associated with high potency and selectivity in vitro. In vivo characterization of 35 confirmed that this compound blocks the expression of nicotine- and cocaine-conditioned place preference in the rat, prevents nicotine-triggered reinstatement of nicotine-seeking behavior in the rat, reduces oral operant alcohol self-administration in the mouse, increases extracellular levels of acetylcholine in the rat medial prefrontal cortex, and potentiates the amplitude of the relative cerebral blood volume response to d-amphetamine in a regionally specific manner in the rat brain.

The use of knock-out mice unravels distinct roles for mGlu2 and mGlu3 metabotropic glutamate receptors in mechanisms of neurodegeneration/neuroprotection.

Dual metabotropic glutamate 2/3 (mGlu2/3) receptor agonists have been examined with success in the clinic with positive proof of efficacy in several tests of anxiety and schizophrenia. Moreover, a large body of evidence has accumulated that these drugs have significant neuroprotective potential. An important discussion in the field deals with dissecting effects on mGlu2 versus effects on mGlu3 receptors, which is relevant for the potential use of subtype-selective agonists or allosteric activators. We addressed this issue using mGlu2 and mGlu3 receptor knock-out mice. We used mixed cultures of cortical cells in which astrocytes and neurons were plated at different times and could therefore originate from different mice. Cultures were challenged with NMDA for the induction of excitotoxic neuronal death. The mGlu2/3 receptor agonist, (-)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), was equally neuroprotective in cultures containing neurons from wild-type, mGlu2-/-, or mGlu3-/- mice. Neuroprotection was instead abolished when astrocytes lacked mGlu3 receptors, unless neuronal mGlu2 receptors were also absent. The latter condition partially restored the protective activity of LY379268. Cultures in which neurons originated from mGlu2-/- mice were also intrinsically resistant to NMDA toxicity. In in vivo experiments, systemic administration of LY379268 protected striatal neurons against NMDA toxicity in wild-type and mGlu2-/- mice but not in mGlu3-/- mice. In addition, LY379268 was protective against nigrostriatal degeneration induced by low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine only in mice lacking mGlu2 receptors. We conclude that neuroprotection by mGlu2/3 receptor agonists requires the activation of astrocytic mGlu3 receptors, whereas, unexpectedly, activation of mGlu2 receptors might be harmful to neurons exposed to toxic insults.

The role of central dopamine D3 receptors in drug addiction: a review of pharmacological evidence.

The cDNA for the dopamine D3 receptor was isolated and characterized in 1990. Subsequent studies have indicated that D3 receptors, as well as D3 receptor mRNA, are primarily localized in limbic regions in mammals. This finding led to the postulate that D3 receptors may be involved in drug dependence and addiction. However, this hypothesis has been difficult to test due to the lack of compounds with high selectivity for central D3 receptors. The interpretation of results from studies using mixed D2/D3 agonists and/or antagonists is problematic because these agents have low selectivity for D3 over D2 receptors and it is likely that their actions are primarily related to D2 receptor antagonism and possibly interaction with other neurotransmitter receptors. Currently, with the synthesis and characterization of new highly selective D3 receptor antagonists such as SB-277011-A this difficulty has been surmounted. The purpose of the present article is to review, for the first time, the effects of various putative D3 receptor selective compounds in animal models of drug dependence and addiction. The results obtained with highly selective D3 receptor antagonists such as SB-277011-A, SB-414796, and NGB-2904 indicate that central D3 receptors may play an important role in drug-induced reward, drug-taking, and cue-, drug-, and stress-induced reinstatement of drug-seeking behavior. Provided these results can be extrapolated to human drug addicts, they suggest that selective DA D3 receptor antagonists may prove effective as potential pharmacotherapeutic agents to manage drug dependence and addiction.

Dopamine responsiveness to drugs of abuse: A shell-core investigation in the nucleus accumbens of the mouse.

The existence of subterritories within the nucleus accumbens has now been widely supported by histochemical, neurochemical, electrophysiological, as well as morphological and ultrastructural studies and suggest specific afferent and efferent systems involved in different behavioral aspects. Microdialysis studies in the rat have consistently shown that most drugs of abuse increase extracellular dopamine levels preferentially in the shell subregion of the nucleus accumbens. The study of the relative roles of NAc subregions may considerably help our understanding of the neurobiological basis of drug addiction. Accordingly, the aim of the present work was to extend the outcome of rat studies to the mouse species. Five major drugs of abuse were systemically and acutely administered to mice with a microdialysis probe implanted in either the shell or the core. A statistical comparison was performed on data transformed as percentage values of baseline dopamine vs. logarithmic values with baseline dopamine as a covariate. Results show a significant increase in dopamine levels in both the shell and core subregions following cocaine, amphetamine, nicotine, ethanol, and morphine treatments. A difference between shell and core after cocaine, nicotine, and morphine was evident when data were analyzed as percent values of baseline. However, such a shell-core dichotomy became no longer significant when ANOVA was applied on the statistically more appropriate logarithmic transformation of data with baseline as a covariate. The significant baseline differences among groups of mice (dopamine levels in the shell significantly lower compared with dopamine levels in the core) may have compromised, at least in part, the statistical procedure usually applied in microdialysis studies. These findings suggest that a careful evaluation of the data is required when subtle changes in extracellular levels of DA are measured.

Upregulation of [3H]methyllycaconitine binding sites following continuous infusion of nicotine, without changes of alpha7 or alpha6 subunit mRNA: an autoradiography and in situ hybridization study in rat brain.

It is well established that exposure of experimental animals to nicotine results in upregulation of the alpha4beta2-subtype of neuronal nicotinic acetylcholine receptors (nAChRs). The aim of this study was to determine the effect of nicotine on the levels of alpha7-nAChRs in rat brain, for which only partial information is available. Rats were infused with nicotine (3 mg/kg/day) or saline for 2 weeks and their brains processed for receptor autoradiography with [3H]methyllycaconitine (MLA), a radioligand with nanomolar affinity for alpha7-nAChRs. In control rats binding was high in hippocampus, intermediate in cerebral cortex and hypothalamus, and low in striatum, thalamus and cerebellum. There was high correlation between the distribution of [3H]MLA binding sites and alpha7 subunit mRNA (r = 0.816). With respect to saline-treated controls, nicotine-treated rats presented higher [3H]nicotine binding in 11 out of 15 brain regions analysed (average increase 46 +/- 6%). In contrast, only four regions showed greater [3H]MLA binding, among which the ventral tegmental area (VTA) and cingulate cortex (mean increase 32 +/- 3%). No changes in alpha7 mRNA levels were observed after nicotine treatment. Similarly, there was no variation of alpha6 subunit transcript in the VTA, a region which may contain MLA-sensitive (non-alpha7)-alpha6*-nAChRs (Klink et al., 2001). In conclusion, nicotine increased [3H]MLA binding, although to a smaller extent and in a more restricted regional pattern than [3H]nicotine. The enhancement of binding was not paralleled by a significant change of alpha7 and alpha6 subunit transcription. Finally, the present results provide the first anatomical description of the distribution of [3H]MLA binding sites in rat brain.