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.

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.

Allosteric potentiators increase the affinity of endogenousagonist, in effect amplifying physiological control circuits. Because a potentiator should depend onendogenous tone, its effects are predicted to be self‐limiting and less proneto rapid tolerance development compared to direct‐acting agonists. Our objective was to test this hypothesis using DETQ, a novel allosteric potentiator of the dopamine D1 receptor.Although DETQ has high affinity for the human D1 receptor, it is 70‐fold less potent at the mouse and rat D1 receptors, limiting its use as a pharmacological tool in rodents. To overcome this limitation, we created a transgenic knock‐in mouse expressing the human D1 receptor (hD1). Homozygotes showed normal behavior and breeding.After oral dosing, DETQ caused a dose‐dependent 10‐fold increase in locomotor activity in habituated hD1 mice but not in wild‐type mice, implying a requirement for the human D1 receptor. The increase in locomotor activity was blocked by the D1 antagonist SCH39166 and by pretreatment with a high dose of reserpine, indicating that the behavioral response is dependent on endogenous dopamine release. At higher doses, the response to DETQ reached a plateau even though brain concentrations of unbound drug continued to rise. In contrast, the D1 agonists SKF82958 and A‐77636 showed bell‐shaped dose‐response curves, with a profound decrease in locomotor activity at the highest doses. The suppression of locomotor activity at high doses was due to engagement of competing stereotyped behaviors such as intense grooming. The stereotyped behaviors were not seen with DETQ, providing evidence that the response to DETQ is less liable to cause over stimulation. In repeated dosing over four days, the locomotor response to DETQ was maintained, whereas the response to A‐77636 showed rapid tolerance.In hD1 mice treated with a low dose of reserpine, DETQ restored locomotor activity to untreated control levels; this model is relevant to stand‐alone therapy in mild‐to‐moderate Parkinson's disease. Versus a higher dose of reserpine, DETQ acted synergistically with L‐DOPA in restoring locomotor activity. DETQ also increased wakefulness and decreased sleep and was found to be effective in a behavioral despair model. We also compared DETQ with SKF82958 for efficacy in the Y‐maze. While both compounds enhanced the number of arm entries in a dose–dependent fashion, mice treated with DETQ maintained spontaneous alternation even at high doses, whereas mice treated with higher doses of SKF82958 showed an increased prevalence to return to the same arm (asign of cognitive dysfunction). Finally, DETQ increased spontaneous eye blink rate in the rhesus monkey, a response related to central D1 activation.These results confirm that D1 potentiators may possess advantages over D1 agonists for the treatment of Parkinson's disease and other CNS disorders.

DETQ is a potent and selective dopamine D1 receptor potentiator that is active in several behavioral models (see abstracts by Heinz et al and Svensson et al at this meeting). We wished to explore the neurochemical basis for the behavioral effects of DETQ. Due to low affinity of DETQ in rodents, the current studies were carried out in transgenic mice in which both copies of the murine D1 receptor were replaced with its human counterpart (hD1 mice) (see Svensson et al). Receptor expression in hD1 mice was characterized using autoradiography of the D1‐selective antagonist 3H‐SCH23390. The pattern of D1 receptor distribution was very similar in wild‐type and hD1 mice, but the absolute level of expression was about 50% lower in hD1 mice. Using microdialysis in freely moving hD1 mice, DETQ and the D1 agonist SKF82958 increased extracellular levels of acetylcholine and histamine in cortical and subcortical areas. At a high dose we also observed enhanced extracellular levels of norepinephrine in the prefrontal cortex. Furthermore, a low dose of DETQ elevated hippocampal acetylcholine levels in an additive fashion with the acetylcholinesterase inhibitor rivastigmine. Brain levels of the histamine metabolites tele‐methylhistamine and tele‐methylimidazole acetic acid were also elevated in both microdialysate and post‐mortem tissue samples. The increases in brain histamine metabolites produced by DETQ were not present in wild‐type mice, indicating that the effects were mediated via the D1 receptor. Overall, these neurochemical changes correlated with brain exposure of DETQ and also with the behavioral effects (see Svensson et al.) Effects on brain cyclic nucleotide levels were studied after microwave fixation of the forebran. Levels of cGMP were elevated in the striatum after both DETQ and SKF82958. Further changes in downstream signaling were explored measuring phosphorylation of the transcription factor CREB and the AMPA receptor GluR1. Both DETQ and SKF82958 increased pCREB and pGluR1 in the brain. Together, these data provide neurochemical evidence for enhanced synaptic plasticity of DETQ and SKF82958. In conclusion, our in vivo neurochemical data support the proposal that the D1 potentiator DETQ selectively enhances D1 receptor function in the brain. Studies of neurotransmitter release, second messengers, and downstream signaling support a potential utility for D1 potentiators to enhance cognitive function in CNS disorders. Support or Funding Information All authors are current or past employees of Eli Lilly & Co

We describe a novel, potent and selective orexin-2 (OX2)/hypocretin-2 receptor antagonist with in vivo activity in an animal model predictive of antidepressant-like efficacy. N-biphenyl-2-yl-4-fluoro-N-(1H-imidazol-2-ylmethyl) benzenesulfonamide HCl (LSN2424100) binds with high affinity to recombinant human OX2 receptors (Ki = 4.5 nM), and selectivity over OX1 receptors (Ki = 393 nM). LSN2424100 inhibited OXA-stimulated intracellular calcium release in HEK293 cells expressing human and rat OX2 receptors (Kb = 0.44 and 0.83 nM, respectively) preferentially over cells expressing human and rat OX1 (Kb = 90 and 175 nM, respectively). LSN2424100 exhibits good exposure in Sprague-Dawley rats after IP, but not PO, administration of a 30 mg/kg dose (AUC0-6 h = 1300 and 269 ng(*)h/mL, respectively). After IP administration in rats and mice, LSN2424100 produces dose-dependent antidepressant-like activity in the delayed-reinforcement of low-rate (DRL) assay, a model predictive of antidepressant-like efficacy. Efficacy in the DRL model was lost in mice lacking OX2, but not OX1 receptors, confirming OX2-specific activity. Importantly, antidepressant-like efficacy of the tricyclic antidepressant, imipramine, was maintained in both OX1 and OX2 receptor knock-out mice. In conclusion, the novel OX2 receptor antagonist, LSN2424100, is a valuable tool compound that can be used to explore the role of OX2 receptor-mediated signaling in mood disorders.

Acute administration of atypical antipsychotic drugs, such as clozapine, produces an increase in the efflux of catecholamines preferentially in the prefrontal cortex (PFC) and a characteristic expression of Fos‐like immunoreactivity (Fos‐li) in the forebrain of experimental animals. We examined the effects of the CB‐1 receptor antagonist rimonabant on clozapine‐induced increases in the efflux of dopamine (DA) and norepinephrine (NE) in the PFC, and Fos‐li in the PFC, nucleus accumbens shell (NAc‐shell) and core (NAc‐core) and dorsolateral striatum (DL‐Str) in the rat. Rimonabant and clozapine dose‐dependently increased cortical efflux of DA and NE. Pretreatment (30min) with rimonabant (3mg/kg, IP) significantly enhanced the clozapine (10mg/kg, SC)‐induced increase in catecholamine efflux in the PFC. Clozapine (8mg/kg, SC) significantly increased the Fos‐li in the PFC and NAc‐shell. Rimonabant (10mg/kg, IP) significantly increased the Fos‐li in the PFC. Rimonabant pretreatment (30min) significantly enhanced the clozapine‐induced Fos‐li in the PFC and NAc‐shell. Clozapine alone and the combination of rimonabant and clozapine demonstrated a positive atypical index (based on the difference in the Fos‐li in the NAc‐shell versus the DL‐Str) indicating atypicality of antipsychotic‐like activity. The present results indicate that rimonabant enhances the neurochemical and histochemical effects of clozapine and that this combined administration reveals an atypical antipsychotic‐like profile of drug action.