Discovery of TAK-041: a Potent and Selective GPR139 Agonist Explored for the Treatment of Negative Symptoms Associated with Schizophrenia.

The orphan G-protein-coupled receptor GPR139 is highly expressed in the habenula, a small brain nucleus that has been linked to depression, schizophrenia (SCZ), and substance-use disorder. High-throughput screening and a medicinal chemistry structure-activity relationship strategy identified a novel series of potent and selective benzotriazinone-based GPR139 agonists. Herein, we describe the chemistry optimization that led to the discovery and validation of multiple potent and selective in vivo GPR139 agonist tool compounds, including our clinical candidate TAK-041, also known as NBI-1065846 (compound 56). The pharmacological characterization of these GPR139 agonists in vivo demonstrated GPR139-agonist-dependent modulation of habenula cell activity and revealed consistent in vivo efficacy to rescue social interaction deficits in the BALB/c mouse strain. The clinical GPR139 agonist TAK-041 is being explored as a novel drug to treat negative symptoms in SCZ.

Analyzing the effects of psychotropic drugs on metabolite profiles in rat brain using 1H NMR spectroscopy.

The mechanism of action of standard drug treatments for psychiatric disorders remains fundamentally unknown, despite intensive investigation in academia and the pharmaceutical industry. So far, little is known about the effects of psychotropic medications on brain metabolism in either humans or animals. In this study, we investigated the effects of a range of psychotropic drugs on rat brain metabolites. The drugs investigated were haloperidol, clozapine, olanzapine, risperidone, aripiprazole (antipsychotics); valproate, carbamazapine (mood stabilizers) and phenytoin (antiepileptic drug). The relative concentrations of endogenous metabolites were determined using high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy. The results revealed that different classes of psychotropic drugs modulated a range of metabolites, where each drug induced a distinct neurometabolic profile. Some common responses across several drugs or within a class of drug were also observed. Antipsychotic drugs and mood stabilizers, with the exception of olanzapine, consistently increased N-acetylaspartate (NAA) levels in at least one brain area, suggesting a common therapeutic response on increased neuronal viability. Most drugs also altered the levels of several metabolites associated with glucose metabolism, neurotransmission (including glutamate and aspartate) and inositols. The heterogenic pharmacological response reflects the functional and physiological diversity of the therapeutic interventions, including side effects. Further study of these metabolites in preclinical models should facilitate the development of novel drug treatments for psychiatric disorders with improved efficacy and side effect profiles.

Increased expression of the NR2A NMDA receptor subunit in the prefrontal cortex of rats reared in isolation.

A hypofunction of the N-methyl-D-aspartate (NMDA) receptor has been implicated in the pathophysiology of schizophrenia. Compelling evidence of altered NMDA receptor subunit expression in the schizophrenic brain has not, however, so far emerged. Rats reared in isolation exhibit several characteristics, including disturbed sensory gating, which resemble those seen in schizophrenia. To explore the possibility that NMDA receptor dysfunction may contribute to the behavioral and neurochemical consequences of rearing rats in isolation, we compared NMDA receptor subunit expression in brains of rats which were housed in isolation and which displayed a deficit in prepulse inhibition of the acoustic startle response with that of socially housed controls. An initial microarray analysis revealed a 1.26-fold increase in NR2A transcript in the prefrontal cortex, but not in the nucleus accumbens, of rats reared in isolation compared with those housed socially. In contrast, NR1, NR2B, NR2C, NR2D, NR3A, and NR3B subunit expression was unchanged in either brain area. In a second cohort of animals, in situ hybridization revealed increased NR2A mRNA expression in the medial prefrontal cortex, an observation that was substantiated by increased [(3)H]CGP39653 binding suggesting that NR2A receptor subunit protein expression was also elevated in the medial prefrontal cortex of the same animals. No changes in expression of NR1 or NR2B subunits were observed at both mRNA and protein level. Altered NR2A subunit expression in the medial prefrontal cortex of rats reared in isolation suggests that NMDA receptor dysfunction may contribute to the underlying pathophysiology of this preclinical model of aspects of schizophrenia.

Antipsychotic treatment alters protein expression associated with presynaptic function and nervous system development in rat frontal cortex.

Haloperidol and olanzapine are widely used antipsychotic drugs in the treatment of schizophrenia and other psychotic disorders. Despite extensive research efforts within the biopharmaceutical industry and academia, the exact molecular mechanisms of their action remain largely unknown. Since the response of patients to existing medications can be variable and often includes severe side effects, it is critical to increase our knowledge on their mechanism of action to guide clinical usage and new drug development. In this study, we have employed the label-free liquid chromatography tandem mass spectrometry (LC-MSE) to identify differentially expressed proteins in rat frontal cortex following subchronic treatment with haloperidol or olanzapine. Subcellular fractionation was performed to increased proteomic coverage and provided insight into the subcellular location involved in the mechanism of drug action. LC-MSE profiling identified 531 and 741 annotated proteins in fractions I (cytoplasmic-) and II (membrane enriched-) in two drug treatments. Fifty-nine of these proteins were altered significantly by haloperidol treatment, 74 by olanzapine and 21 were common to both treatments. Pathway analysis revealed that both drugs altered similar classes of proteins associated with cellular assembly/organization, nervous system development/function (particularly presynaptic function) and neurological disorders, which indicate a common mechanism of action. The top affected canonical signaling pathways differed between the two treatments. The haloperidol data set showed a stronger association with Huntington's disease signaling, while olanzapine treatment showed stronger effects on glycolysis/gluconeogenesis. This could either relate to a difference in clinical efficacy or side effect profile of the two compounds. The results were consistent with the findings reported previously by targeted studies, demonstrating the validity of this approach. However, we have also identified many novel proteins which have not been found previously to be associated with these drugs. Further study of these proteins could provide new insights into the etiology of the disease or the mechanism of antipsychotic medications.

Preclinical investigations into the antipsychotic potential of the novel histamine H3 receptor antagonist GSK207040.

OBJECTIVES: To test the novel nonimidazole histamine H3 receptor antagonist 5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazapin-7-yl)oxy]-N-methyl-2-pyrazinecarboxamide (GSK207040) in a series of behavioral and neurochemical paradigms designed to evaluate its antipsychotic potential. MATERIALS AND METHODS: Acute orally administered GSK207040 was investigated for its capacity to reverse a 24-h-induced deficit in novel object recognition memory, deficits in prepulse inhibition (PPI) induced by isolation rearing, and hyperlocomotor activity induced by amphetamine. The acute neurochemical effects of GSK207040 were explored by analyzing rat anterior cingulate cortex microdialysates for levels of dopamine, noradrenaline, and acetylcholine and by c-fos immunohistochemistry. The potential for interaction with the antipsychotic dopamine D2 receptor antagonist haloperidol was explored behaviorally (spontaneous locomotor activity and catalepsy), biochemically (plasma prolactin), and via ex vivo receptor occupancy determinations. RESULTS: GSK207040 significantly enhanced object recognition memory (3 mg/kg) and attenuated isolation rearing-induced deficits in PPI (1.0 and 3.2 mg/kg) but did not reverse amphetamine-induced increases in locomotor activity. There was no evidence of an interaction of GSK207040 with haloperidol. GSK207040 (3.2 mg/kg) raised extracellular concentrations of dopamine, noradrenaline, and acetylcholine in the anterior cingulate cortex and c-fos expression in the core of the nucleus accumbens was increased at doses of 3.2 and 10.0 mg/kg. CONCLUSIONS: The behavioral and neurochemical profile of GSK207040 supports the potential of histamine H3 receptor antagonism to treat the cognitive and sensory gating deficits of schizophrenia. However, the failure of GSK207040 to reverse amphetamine-induced locomotor hyperactivity suggests that the therapeutic utility of histamine H(3) receptor antagonism versus positive symptoms is less likely, at least following acute administration.

Chandelier cartridges in the prefrontal cortex are reduced in isolation reared rats.

Chandelier neurons are a subset of parvalbumin containing cortical interneurons characterised by their preferential targeting of the axon initial segments of pyramidal neurons. They have been the focus of recent interest after evidence that the arrays of boutons are reduced in the prefrontal cortex of schizophrenic patients, post mortem. Since one chandelier neuron may innervate the axon initial segments of several hundred pyramidal neurons, it is hypothesized that their special connectivity might facilitate synchronisation of cortical outputs and play a key role in working memory. Disruption in their function is therefore thought to play a potentially important role in cortically associated symptoms of schizophrenia. Using the isolation rearing animal model of schizophrenia, we examined immunolabelling for GABA-transporter 1, a marker of chandelier cartridges. We show that the numbers of arrays of chandelier axons are reduced by 36% in the ventral prelimbic cortex of isolation-reared rats, compared with their socially-housed litter mates. This mimics findings in the PFC of schizophrenic patients where GAT-1-positive cartridges are reduced by 40% and is the first study to demonstrate changes in chandelier cartridges in an animal model of schizophrenia.

(+/-) Ketamine-induced prepulse inhibition deficits of an acoustic startle response in rats are not reversed by antipsychotics.

Prepulse inhibition (PPI) is the reduction in the startle response caused by a low intensity non-startling stimulus (the prepulse) which is presented shortly before the startle stimulus and is an operational measure of sensorimotor gating. PPI is impaired in psychiatric disorders such as schizophrenia. Ketamine, a non-competitive N-methyl-D-aspartate antagonist has been shown to induce schizophrenia-like behavioural changes in humans and PPI deficits in rats, which can be reversed by antipsychotics. Thus, ketamine-induced PPI deficits in rats may provide a translational model of schizophrenia. The aim of this study was to investigate the effects of antipsychotic drugs and drugs known to alter the glutamate system upon ketamine-induced PPI deficits in rats. Rats were habituated to the PPI procedure [randomized trials of either pulse alone (110 dB/50 ms) or prepulse + pulse (80 dB/10 ms)]. Animals were assigned to pre-treatments based on the level of PPI on the last habituation test and balanced across startle chambers. Ketamine (1-10 mg/kg s.c; 15 min ptt) increased startle amplitude and induced PPI deficits at 6 and 10 mg/kg. PPI deficits induced by ketamine at 6 mg/kg were not attenuated by clozapine (2.5-10 mg/kg s.c.; 60 min ptt), risperidone (0.1-1 mg/kg i.p.; 60 min ptt), haloperidol (0.1-1 mg/kg i.p.; 60 min ptt), lamotrigine (3-30 mg/kg p.o.; 60 min ptt), or SB-271046-A (5-20 mg/kg p.o.; 2 hour ptt) nor potentiated by 2-methyl-6-(phenylethynyl)-pyridine (3-10 mg/kg i.p.; 30 min ptt). These results suggest that under these test conditions ketamine-induced PPI deficits in rats is relatively insensitive and does not represent a translational model for drug discovery in schizophrenia.

GSK189254, a novel H3 receptor antagonist that binds to histamine H3 receptors in Alzheimer's disease brain and improves cognitive performance in preclinical models.

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H(3) receptor antagonist with high affinity for human (pK(i) = 9.59 -9.90) and rat (pK(i) = 8.51-9.17) H(3) receptors. GSK189254 is >10,000-fold selective for human H(3) receptors versus other targets tested, and it exhibited potent functional antagonism (pA(2) = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC(50) = 8.20 versus basal guanosine 5'-O-(3-[(35)S]thio)triphosphate binding] at the human recombinant H(3) receptor. In vitro autoradiography demonstrated specific [(3)H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H(3) binding was detected in medial temporal cortex samples from severe cases of Alzheimer's disease, suggesting for the first time that H(3) receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(-)-alpha-methyl[imidazole-2,5(n)-(3)H]histamine dihydrochloride ([(3)H]R-alpha-methylhistamine) binding (ED(50) = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3-3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50) = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimer's disease and other cognitive disorders.

Effect of the selective dopamine D3 receptor antagonist SB-277011-A on regional c-Fos-like expression in rat forebrain.

SB-277011-A is a dopamine D(3) receptor antagonist that exhibits over 100-fold selectivity over dopamine D(2) receptors and a broad spectrum of other receptor, ion channels, and enzymes. We employed c-Fos immunohistochemistry to characterise the functional neuroanatomical effects of acute administration of SB-277011-A and observed a time-dependent increase in the density of c-Fos-like positive nuclei in rat forebrain with maximal effects observed 2 h post-dose. The relative influence of the different brain regions on the overall effect of SB-277011-A was ranked by partial least squares discriminant analysis loadings plot which indicated that sites within the nucleus accumbens exerted the greatest influence on the separation of the vehicle and SB-277011-A treatment groups. At the 2 h time-point, c-Fos-like expression was shown to be significantly elevated (p<0.05) in the core and shell of the nucleus accumbens, at both rostral and caudal levels, and in the lateral septum. No significant changes were detected in the caudate nucleus (lateral or medial) or in the cingulate, infralimbic prefrontal, or somatosensory cortices. The capacity of SB-277011-A to trigger immediate early gene expression in these limbic regions of rat brain adds to a growing consensus of the potential utility of dopamine D(3) receptor antagonism in psychiatric disorders including schizophrenia and drug dependency.

Reversal of isolation-rearing-induced PPI deficits by an alpha7 nicotinic receptor agonist.

RATIONALE: The alpha7 subtype of the nicotinic receptor plays an important role in auditory sensory gating. Schizophrenics show deficient sensory gating and abnormalities in the number and regulation of nicotinic receptors. Prepulse inhibition (PPI) deficits exhibited by isolation-reared rats are thought to model the sensorimotor gating deficits seen in schizophrenia. OBJECTIVE: To examine the role of nicotinic alpha7 receptors in the isolation-rearing rat model, we tested whether the selective alpha7 receptor agonist (R)-N-(1-Azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide) (compound A) could reverse isolation-rearing-induced PPI deficits, and investigated alpha7 receptor RNA expression in the hippocampus, prefrontal cortex, cerebellum, nucleus accumbens and thalamus, and alpha7 receptor protein expression in the hippocampus of isolation- and group-reared rats. METHOD: Rats reared in isolation or groups of five from weaning were tested in the PPI paradigm under conditions of variable inter-stimulus interval (ISI) (pulse = 110 dB/50 ms; prepulse = 75 dB/30 ms; ISI = 30, 100 and 300 ms) 30 min following administration of compound A (3.2-10 mg/kg i.p.). Alpha7 receptor expression was measured by TaqMan RT-PCR (total RNA) and autoradiography (protein). RESULTS: Isolation-rearing-induced PPI deficits were attenuated by both doses of compound A at 100-ms ISI and by 10 mg/kg at 300-ms ISI. Expression of alpha7 receptor RNA and protein was unaltered in isolation-reared rats. CONCLUSION: Although altered alpha7 receptor expression may not underlie the phenotype of isolation-reared rats, the activation of these receptors may be of benefit in re-establishing efficient gating function.