Synthesis and Pharmacological Characterization of 2-(2,6-Dichlorophenyl)-1-((1S,3R)-5-(3-hydroxy-3-methylbutyl)-3-(hydroxymethyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one (LY3154207), a Potent, Subtype Selective, and Orally Available Positive Allosteric Modulator of the Human Dopamine D1 Receptor.

Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (3) as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.

Preclinical profile of a dopamine D1 potentiator suggests therapeutic utility in neurological and psychiatric disorders.

DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L-DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinson's disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y-maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced-swim test, a model for antidepressant-like activity. In rhesus monkeys, DETQ increased spontaneous eye-blink rate, a measure that is known to be depressed in Parkinson's disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinson's disease, Alzheimer's disease, cognitive impairment in schizophrenia, and major depressive disorder.

Electroencephalographic, cognitive, and neurochemical effects of LY3130481 (CERC-611), a selective antagonist of TARP-γ8-associated AMPA receptors.

6-[(1S)-1-[1-[5-(2-hydroxyethoxy)-2-pyridyl]pyrazol-3-yl]ethyl]-3H-1,3-benzothiazol-2-one (LY3130481 or CERC-611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) γ-8. This molecule has been characterized as a potent and efficacious anticonvulsant in an array of acute and chronic epilepsy models in rodents. The present set of experiments was designed to assess the effects of LY3130481 on the electroencephelogram (EEG), cognitive function, and neurochemical outflow. LY3130481 disrupted food-maintained responding in rats and spontaneous alternation in a Y-maze in mice. In rat fear conditioning, LY3130481 caused a deficit in trace (hippocampal-dependent), but not in delay fear conditioning. Although these effects on cognitive performances were observed, the known cognitive-impairing anticonvulsant, topiramate, did not always produce deficits under these assay conditions. LY3130481 produced modest increases in wake times in rats. In addition, LY3130481 was able to attenuate some impairing effects of standard antiepileptic drugs. The motor-impairing effects of the lacosamide were attenuated by LY3130481 as was the decrease in non-rapid-eye movement sleep induced by carbamazepine. Evaluation of the effect of LY3130481 on neurotransmitter and metabolite efflux in the rat medial prefrontal cortex, using in vivo microdialysis, revealed significant increases in the pro-cognitive and wake-promoting neurotransmitters, histamine and acetylcholine, as well as in serotonin, telemethylhistamine, 5-HIAA, HVA and MHPG. LY3130481 thus presents a novel behavioral profile that will have to be evaluated in patients to fully appreciate its implications for therapeutics. LY3130481 is currently under clinical development as CERC-611 as an antiepileptic.

Fornix deep brain stimulation enhances acetylcholine levels in the hippocampus.

Deep brain stimulation (DBS) of the fornix has gained interest as a potential therapy for advanced treatment-resistant dementia, yet the mechanism of action remains widely unknown. Previously, we have reported beneficial memory effects of fornix DBS in a scopolamine-induced rat model of dementia, which is dependent on various brain structures including hippocampus. To elucidate mechanisms of action of fornix DBS with regard to memory restoration, we performed c-Fos immunohistochemistry in the hippocampus. We found that fornix DBS induced a selective activation of cells in the CA1 and CA3 subfields of the dorsal hippocampus. In addition, hippocampal neurotransmitter levels were measured using microdialysis before, during and after 60 min of fornix DBS in a next experiment. We observed a substantial increase in the levels of extracellular hippocampal acetylcholine, which peaked 20 min after stimulus onset. Interestingly, hippocampal glutamate levels did not change compared to baseline. Therefore, our findings provide first experimental evidence that fornix DBS activates the hippocampus and induces the release of acetylcholine in this region.

Temporally distinct cognitive effects following acute administration of ketamine and phencyclidine in the rat.

Non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonists such as phencyclidine (PCP) and ketamine are commonly and interchangeably used to model aspects of schizophrenia in animals. We compared here the effects of acute administration of these compounds over a range of pre-treatment times in tests of instrumental responding (VI 30s response schedule), simple reaction time (SRT) and cognitive flexibility (reversal learning and attentional set shifting digging task) in rats. At standard pre-treatment times (15-30 min), both ketamine and PCP produced overall response suppression in VI 30 and increased reaction times in SRT suggesting that any concomitant cognitive performance deficits are likely to be confounded by motor and/or motivational changes. However, the use of extended pre-treatment times produced deficits in cognitive flexibility measured up to 4h after drug administration in the absence of motor/motivational impairment. Generally, PCP increased impulsive responding in the SRT indicating a possible loss of inhibitory response control that may have contributed to deficits observed in reversal learning and attentional set-shifting. In contrast to PCP, ketamine did not have the same effect on impulsive responding, and possibly as a consequence produced more subtle cognitive deficits in attentional set-shifting. In summary, acute treatment with NMDAR antagonists can produce cognitive deficits in rodents that are relevant to schizophrenia, provided that motor and/or motivational effects are allowed to dissipate. The use of longer pre-treatment times than commonly employed might be advantageous. Also, ketamine, which is more frequently used in clinical settings, did not produce as extensive cognitive deficits as PCP.

Biochemical and morphological consequences of human α-synuclein expression in a mouse α-synuclein null background.

A consensus about the functions of human wild-type or mutated α-synuclein (αSYN) is lacking. Both forms of αSYN are implicated in Parkinson's disease, whereas the wild-type form is implicated in substance abuse. Interactions with other cellular proteins and organelles may meditate its functions. We developed a series of congenic mouse lines containing various allele doses or combinations of the human wild-type αSYN (hwαSYN) or a doubly mutated (A30P*A53T) αSYN (hm(2) αSYN) in a C57Bl/6J line spontaneously deleted in mouse αSYN (C57BL/6JOla). Both transgenes had a functional role in the nigrostriatal system, demonstrated by significant elevations in striatal catecholamines, metabolites and the enzyme tyrosine hydroxylase compared with null-mice without a transgene. Consequences occurred when the transgenes were expressed at a fraction of the endogenous level. Hemizygous congenic mice did not exhibit any change in the number or size of dopaminergic neurons in the ventral midbrain at 9 months of age. Human αSYN was predominantly located in neuronal cell bodies, neurites, synapses, and in intraneuronal/intraneuritic aggregates. The hm(2) αSYN transgene resulted in more aggregates and dystrophic neurites than did the hw5 transgene. The hwαSYN transgene resulted in higher expression of two striatal proteins, synaptogamin 7 and UCHL1, compared with the levels of the hm(2) αSYN transgene. These observations suggest that mutations in αSYN may impair specific functional domains, leaving others intact. These lines may be useful for exploring interactions between hαSYN and environmental or genetic risk factors in dopamine-related disorders using a mouse model.

Mapping the central effects of ketamine in the rat using pharmacological MRI.

RATIONALE: Ketamine induces, in both humans and rodents, behaviours analogous to some of the symptoms of schizophrenia. OBJECTIVES: To utilise pharmacological magnetic resonance imaging (phMRI) techniques that identify changes in blood-oxygenation-level-dependent (BOLD) contrast to determine the temporal and spatial neuronal activation profile of ketamine in the rat brain. METHOD: To obtain a pharmacodynamic profile of the drug, we assessed changes in locomotor activity after vehicle and 10 and 25 mg/kg ketamine. Separate animals were then anaesthetised and placed in a 4.7-T magnetic resonance (MR) system before receiving the same doses of ketamine during serial MR image acquisition. Subsequent statistical parametric mapping of the main effect of the drug was then undertaken to identify changes in BOLD contrast. Levels of gamma-aminobutyric acid (GABA) and dopamine (DA) in brain areas showing localised changes in BOLD contrast were then assessed via microdialysis. RESULTS: Both doses of ketamine produced increases in BOLD image contrast in frontal, hippocampal, cortical and limbic areas. A further investigation of the release of DA and its metabolites in the nucleus accumbens, both in anaesthesised and freely moving rats, corroborated these findings. However, an investigation of GABA and DA levels in the ventral pallidum gave no indication of changes in activity. CONCLUSIONS: Ketamine produced localised dose-dependent alterations in BOLD MR signal, which correlate with the pharmacodynamic profile of the drug. These results can be, at least, partially substantiated with complementary techniques but consideration must be given to the input function applied to the MR signal and the use of anaesthesia during phMRI experimentation.

Investigation of the SSRI augmentation properties of 5-HT(2) receptor antagonists using in vivo microdialysis.

Recent evidence that 5-HT(2) receptors exert a negative influence on central 5-hydroxytryptamine (5-HT) neurones suggests that 5-HT(2) receptor antagonists may augment the effects of serotonin selective reuptake inhibitors (SSRIs). The present study investigated whether pre-treatment with 5-HT(2) receptor antagonists enhances the effect of SSRI administration on hippocampal extracellular 5-HT of freely moving rats. Administration of the SSRI citalopram at a low (2mg kg(-1)) and higher (4 mg kg(-1)) dose, increased dialysate 5-HT by 5- and 8-fold, respectively. Pre-treatment with the 5-HT(2) receptor antagonist ketanserin (4 mg kg(-1)) augmented the effect of 4 mg kg(-1) but not 2mg kg(-1) citalopram. The effect of 4 mg kg(-1) citalopram was also augmented by pre-treatment with either the 5-HT(2C) receptor antagonist SB 242084 (0.5mg kg(-1)) or the 5-HT(2A) receptor antagonist MDL 100907 (0.5mg kg(-1)). As with citalopram, fluoxetine elevated dialysate 5-HT at both a low (5mg kg(-1)) and higher (20mg kg(-1)) dose. However, neither dose of fluoxetine was augmented by ketanserin (4 mg kg(-1)). These results confirm recent findings that 5-HT(2) receptor antagonists augment the effect of citalopram on extracellular 5-HT, and indicate the involvement of 5-HT(2C) and possibly 5-HT(2A) receptors. The lack of augmentation of fluoxetine might reflect the intrinsic 5-HT(2) receptor antagonist properties of this drug.

The role of the 5-HT1D receptor as a presynaptic autoreceptor in the guinea pig.

The present study investigated the role of the 5-hydroxytryptamine (5-HT, serotonin)1D receptor as a presynaptic autoreceptor in the guinea pig. In keeping with the literature, the 5-HT1B selective antagonist, 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydrospiro [furo[2,3-f]indole-3,4'-piperidine]oxalate (SB224289) potentiated [3H]5-HT outflow from pre-labelled slices of guinea pig cerebral cortex confirming its role as a presynaptic autoreceptor in this species. In addition, the 5-HT1D receptor-preferring antagonists, 1-[2-[4-(6-fluoro-1H-indol-3-yl)-3,6-dihydro-2H-pyridin-1-yl]-ethyl]-3-pyridin-4-yl-methyl-tetrahydro-pyrimidin-2-one (LY367642), (R)-1-[2-(4-(6-fluoro-1H-indol-3-yl-)-3,6-dihydro-1(2H)-pyridinyl)ethyl]-3,4-dihydro-1H-2-benzopyran-6-carboxamide (LY456219), (S)-1-[2-(4-(6-fluoro-1H-indol-3-yl-)-3,6-dihydro-1(2H)-pyridinyl)ethyl]-3,4-dihydro-1H-2-benzopyran-6-carboxamide (LY456220) and 1-[2-[4-(4-fluoro-benzoyl)-piperidin-1-yl]-ethyl]-3,3-dimethyl-1,2-dihydro-indol-2-one (LY310762), potentiated [3H]5-HT outflow from this preparation with potencies (EC50 values=31-140 nM) in the same range as their affinities for the guinea pig 5-HT1D receptor (Ki values=100-333 nM). The selective 5-HT1D receptor agonist, R-2-(4-fluoro-phenyl)-2-[1-[3-(5-[1,2,4]triazol-4-yl-1H-indol-3-yl)-propyl]-piperidin-4-ylamino]-ethanol dioxylate (L-772,405), inhibited [3H]5-HT outflow. In microdialysis studies, administration of either SB224289 or LY310762 at 10 mg/kg by the intraperitoneal (i.p.) route, potentiated the increase in extracellular 5-HT concentration produced by a maximally effective dose of the selective serotonin re-uptake inhibitor, fluoxetine (at 20 mg/kg i.p.). In addition, the 5-HT1D receptor-preferring antagonist and 5-HT transporter inhibitor, LY367642 (at 10 mg/kg i.p.), elevated extracellular 5-HT concentrations to a greater extent than a maximally effective dose of fluoxetine. It is concluded that the 5-HT1D receptor, like the 5-HT1B receptor, may be a presynaptic autoreceptor in the guinea pig.

Selective action of (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), a group II metabotropic glutamate receptor agonist, on basal and phencyclidine-induced dopamine release in the nucleus accumbens shell.

The effect of the group II metabotropic receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), on basal and phencyclidine-induced dopamine efflux were measured in the shell and core subdivisions of the nucleus accumbens--regions which are associated with limbic and motor functions, respectively. Extracellular levels of dopamine were measured using microdialysis in conscious animals, and LY379268 was delivered locally by inclusion in the artificial cerebrospinal fluid (aCSF) flowing through the microdialysis probe. Local administration of LY379268 in the concentration range 10 nM-10 microM reduced basal levels of dopamine in the nucleus accumbens shell, whilst having no effect in the nucleus accumbens core. In the nucleus accumbens shell, basal levels were reduced to approximately 60% compared to the pre-injection control, with a maximal reduction occurring at concentrations of LY379268 > or =100 nM. The response to LY379268 (100 nM) was reversible, with levels returning to baseline following its removal from the aCSF. In a separate experiment, local perfusion of the nucleus accumbens shell with LY379268 (at both 1 and 10 microM) reduced the magnitude of the response to a subsequent systemic administration of phencyclidine (5 mg/kg i.p.). The reduction in the peak dopamine response was only evident with doses of LY379268 that also reduced basal dopamine efflux--LY379268 being ineffective against PCP at 10 nM. However, in animals pre-treated with LY379268 at 1 or 10 microM, PCP still evoked a dopamine response, and in these animals the relative extent of the response was not significantly different between the respective treatment groups. In contrast, in the nucleus accumbens core the magnitude of the dopamine response to PCP was unaffected by local application of LY379268 (at 1 or 10 microM). Our data suggest that within the nucleus accumbens, there exists a distinct regional difference in the control of dopamine release by group II mGluRs, with the nucleus accumbens shell being preferentially affected. Moreover, the selective action of LY379268 on dopamine levels in the nucleus accumbens shell may have implications for the potential antipsychotic activity of group II mGluR agonists.

Role of muscarinic receptors in the activation of the ventral subiculum and the consequences for dopamine release in the nucleus accumbens.

The nucleus accumbens receives limbic inputs from a number of brain regions, including the ventral subiculum. In rats, activation of the ventral subiculum following microinjection of N-methyl-D-aspartate (NMDA) or carbachol increases locomotor activity, whilst ventral subiculum application of NMDA also increases dopamine efflux in the ipsilateral nucleus accumbens. Microdialysis experiments were therefore conducted to ascertain the consequences for dopamine release in the nucleus accumbens following ventral subiculum administration of carbachol, and to explore the acetylcholine receptor subtype(s) that might be involved. We report that, in anaesthetised rats, ventral subiculum administration of carbachol increased dopamine levels in the nucleus accumbens. The response was attenuated by co-administration with atropine, whilst administration of nicotine and the alpha-7 nicotinic acetylcholine receptor agonist AR-R17779 (spiro[1-azabicyclo[2,2,2]octane-3,5'-oxazolidine]-2'-one monohydrochloride) failed to evoke a response. Oxotremorine-M produced a dose-dependent increase in dopamine efflux confirming sensitivity to muscarinic receptor stimulation. However, the ventral subiculum was insensitive to xanomeline and pilocarpine, muscarinic M(1) receptor-preferring agonists, but sensitive to BuTAC ([5R-[exo]-6-[butylthio]-1,2,5-thiadiazol-3-yl]-1-azabicyclo[3.2.1])octane), a muscarinic M(2)/M(4) receptor agonist. The dopamine response to oxotremorine-M was significantly attenuated, although not abolished by co-administration with the M(2)/M(4) receptor antagonist methoctramine, and studies combining oxotremorine-M with (-)-bicuculline, indicated a dual action in the ventral subiculum that was dependent and independent of reduced GABA neurotransmission. The data presented indicates that activation of the ventral subiculum by carbachol increases dopamine efflux in the nucleus accumbens by stimulation of muscarinic receptors, and that the ventral subiculum-nucleus accumbens projection system is sensitive to muscarinic M(2)/M(4) receptor stimulation.