David Shaw

LY2456302 is a novel, potent, orally-bioavailable small molecule kappa-selective antagonist with activity in animal models predictive of efficacy in mood and addictive disorders

Kappa opioid receptors and their endogenous neuropeptide ligand, dynorphin A, are densely localized in limbic and cortical areas comprising the brain reward system, and appear to play a key role in modulating stress and mood. Growing literature indicates that kappa receptor antagonists may be beneficial in the treatment of mood and addictive disorders. However, existing literature on kappa receptor antagonists has used extensively JDTic and nor-BNI which exhibit long-lasting pharmacokinetic properties that complicate experimental design and interpretation of results. Herein, we report for the first time the in vitro and in vivo pharmacological profile of a novel, potent kappa opioid receptor antagonist with excellent selectivity over other receptors and markedly improved drug-like properties over existing research tools. LY2456302 exhibits canonical pharmacokinetic properties that are favorable for clinical development, with rapid absorption (t(max): 1-2 h) and good oral bioavailability (F = 25%). Oral LY2456302 administration selectively and potently occupied central kappa opioid receptors in vivo (ED₅₀ = 0.33 mg/kg), without evidence of mu or delta receptor occupancy at doses up to 30 mg/kg. LY2456302 potently blocked kappa-agonist-mediated analgesia and disruption of prepulse inhibition, without affecting mu-agonist-mediated effects at doses >30-fold higher. Importantly, LY2456302 did not block kappa-agonist-induced analgesia one week after administration, indicating lack of long-lasting pharmacodynamic effects. In contrast to the nonselective opioid antagonist naltrexone, LY2456302 produced antidepressant-like effects in the mouse forced swim test and enhanced the effects of imipramine and citalopram. LY2456302 reduced ethanol self-administration in alcohol-preferring (P) rats and, unlike naltrexone, did not exhibit significant tolerance upon 4 days of repeated dosing. LY2456302 is a centrally-penetrant, potent, kappa-selective antagonist with pharmacokinetic properties favorable for clinical development and activity in animal models predictive of efficacy in mood and addictive disorders.

Effects of drugs that potentiate GABA on extinction of positively-reinforced operant behaviour.

Extinction following positively reinforced operant conditioning reduces response frequency, at least in part through the aversive or frustrative effects of non-reinforcement. According to J.A. Grays theory, non-reinforcement activates the behavioural inhibition system which in turn causes anxiety. As predicted, anxiolytic drugs including benzodiazepines affect the operant extinction process. Recent studies have shown that reducing GABA-mediated neurotransmission retards extinction of aversive conditioning. We have shown in a series of studies that anxiolytic compounds that potentiate GABA facilitate extinction of positively reinforced fixed-ratio operant behaviour in C57B1/6 male mice. This effect does not occur in the early stages of extinction, nor is it dependent on cumulative effects of the compound administered. Potentiation of GABA at later stages has the effect of increasing sensitivity to the extinction contingency and facilitates the inhibition of the behaviour that is no longer required. The GABAergic hypnotic, zolpidem, has the same selective effects on operant extinction in this procedure. The effects of zolpidem are not due to sedative action. There is evidence across our series of experiments that different GABA-A subtype receptors are involved in extinction facilitation and anxiolysis. Consequently, this procedure may not be an appropriate model for anxiolytic drug action, but it may be a useful technique for analysing the neural bases of extinction and designing therapeutic interventions in humans where failure to extinguish inappropriate behaviours can lead to pathological conditions such as post-traumatic stress disorder.

Subtype-selective GABAergic drugs facilitate extinction of mouse operant behaviour

Several recent studies have shown that reducing gamma-aminobutyric acid (GABA)-mediated neurotransmission retards extinction of aversive conditioning. However, relatively little is known about the effect of GABA on extinction of appetitively motivated tasks. We examined the effect of chlordiazepoxide (CDP), a classical benzodiazepine (BZ) and two novel subtype-selective BZs when administered to male C57Bl/6 mice during extinction following training on a discrete-trial fixed-ratio 5 (FR5) food reinforced lever-press procedure. Initially CDP had no effect, but after several extinction sessions CDP significantly facilitated extinction, i.e. slowed responding, compared with vehicle-treated mice. This effect was not due to drug accumulation because mice switched from vehicle treatment to CDP late in extinction showed facilitation immediately. Likewise, this effect could not be attributed to sedation because the dose of CDP used (15 mg/kg i.p.) did not suppress locomotor activity. The two novel subtype-selective BZ partial agonists, L-838417 and TP13, selectively facilitated extinction in similar fashion to CDP. The non-GABAergic anxiolytic buspirone was also tested and found to have similar effects when administered at a non-sedating dose. These studies demonstrate that GABA-mediated processes are important during extinction of an appetitively motivated task, but only after the animals have experienced several extinction sessions.

Synthesis and Pharmacological Characterization of C4-Disubstituted Analogs of 1S,2S,5R,6S-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylate: Identification of a Potent, Selective Metabotropic Glutamate Receptor Agonist and Determination of Agonist-Bound Human mGlu2 and mGlu3 Amino Terminal Domain Structures.

As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4β-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure-activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein-ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.

The effect of mGlu8 deficiency in animal models of psychiatric diseases

The metabotropic glutamate receptor subtype 8 (mGlu8) is presynaptically located and regulates the release of the transmitter. Dysfunctions of this mechanism are involved in the pathophysiology of different psychiatric disorders. mGlu8 deficient mice have been previously investigated in a range of studies, but the results are contradictory and there are still many open questions. Therefore, we tested mGlu8‐deficient animals in different behavioral tasks that are commonly used in neuropsychiatric research. Our results show a robust contextual fear deficit in mGlu8‐deficient mice. Furthermore, novel object recognition, chlordiazepoxide‐facilitated extinction of operant conditioning and the acoustic startle response were attenuated by mGlu8 deficiency. We found no changes in sensory processing, locomotor activity, prepulse inhibition, phencyclidine‐induced changes in locomotion or prepulse inhibition, operant conditioning, conditioned fear to a discrete cue or in animal models of innate fear and post‐traumatic stress disorder. We conclude that mGlu8 might be a potential target for disorders with pathophysiological changes in brain areas where mGlu8 modulates glutamate and gamma‐amino butyric acid (GABA) transmission. Our data especially point to anxiety disorders involving exaggerated contextual fear, such as generalized anxiety disorders, and to conditions with disturbed declarative memory.

Chlordiazepoxide and lavender oil alter unconditioned anxiety-induced c-fos expression in the rat brain

Lavender oil has a long history of use for treating anxiety, but only recent research has examined its effects using standard behavioural methods used to test novel drugs. The aim of this study was to investigate the effects of inhaled lavender oil on anxiety related behaviour of rats in the open field and to compare them with the effects of chlordiazepoxide (CDP), a typical anxiolytic drug. Additionally c-fos immunochemistry was used to investigate whether lavender oil produced the same pattern of c-fos expression as CDP in eight different brain areas associated with anxiety. As previously found, lavender oil showed anxiolytic properties in the open field similar to but not as extensive as those of CDP. Immunochemistry results indicated that exposure to the open field increased c-fos expression, while CDP reversed the effects of this behavioural stressor on c-fos expression in all brain regions examined except the central nucleus of the amygdala, where c-fos expression increased. Lavender oil had similar effects to CDP on the paraventricular nucleus of the hypothalamus, the dorsomedial hypothalamic nucleus and the central nucleus of the amygdala. These results strengthen the suggestion that inhaling lavender oil has anxiolytic behavioural effects, but they are weaker than the effects of benzodiazepines, and there is limited evidence that they are mediated by the same neural processes.

Synthesis and Pharmacological Characterization of C4-(Thiotriazolyl)-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1R,2S,4R,5R,6R)-2-Amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2812223), a Highly Potent, Functionally Selective mGlu2 Receptor Agonist.

Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.

Behavioral and neurochemical mechanisms of the action of mild stress in the enhancement of feeding

Rats were trained to respond under a cyclic-ratio schedule of reinforcement composed of an ascending, followed by a descending, sequence of ratio values. Subjects were trained while exposed to 70 dB white noise, then tested while exposed to 70 or 90 dB white noise. Exposure to 90 dB white noise elevated the response function (p<.02). Naloxone was then administered intraperitoneally at 0.3. 1.0. and 3.0 mg/kg under 70 dB and 90 dB white noise. Naloxone administration (1.0 and 3.0 mg/kg) significantly depressed the response function obtained under 90 dB white noise (ps<.01) but did not affect the function obtained under 70 dB white noise. These findings suggest that mild stress increases food intake through a mechanism affecting palatability enhanced by the release of endogenous opioids.

Facilitation of extinction of operant behaviour in C57Bl/6 mice by chlordiazepoxide and D-cycloserine.

Rationale and objectiveEffects on the extinction of GABAergic drug, chlordiazepoxide (CDP), and glutamatergic drug, d-cycloserine (DCS), in C57BL/6 mice were compared.Materials and methodsFollowing a palatability test (Experiment 1), Experiments 2–6 involved food-reinforced lever press training followed by extinction sessions at 1- or 4-day intervals. The effects of drugs were examined. Experiment 7 involved a two-lever task.ResultsCDP did not affect food palatability (Experiment 1), but facilitated extinction when administered prior to extinction sessions via intracerebral (Experiment 2) or peripheral administration at 1-day (Experiments 3–7) or 4-day intervals (Experiment 6). Reducing the amount of training prior to extinction reduced the delay in the effect of CDP typically seen, and CDP had a larger effect in early sessions on mice that had received less training (Experiment 3). There was some evidence that CDP could be blocked by flumazenil (Experiment 4), and CDP withdrawal reversed extinction facilitation (Experiments 5 and 7). With 4-day intervals, DCS administered immediately following extinction sessions, or pre-session CDP, facilitated extinction with 48-trial sessions (experiment 6B). With six-trial sessions, the co-administration of post-session DCS enhanced facilitation produced by pre-session CDP (experiment 6A). Finally, CDP facilitated extinction in a dose-related fashion following training on a two-lever food-reinforced task (Experiment 7).ConclusionsThe findings are consistent with the hypotheses that two neurotransmitter systems have different roles in operant extinction and that glutamatergic systems are involved in extinction learning and GABAergic systems involved in the expression of that learning. This parallels findings with extinction following Pavlovian conditioning, which has been more extensively investigated.

Effects of reinforcement magnitude and ratio values on behaviour maintained by a cyclic ratio schedule of reinforcement

In Experiments 1 and 2, lever pressing by rats was reinforced on a cyclic ratio schedule of food reinforcement, comprising a repeated sequence of fixed-ratio component schedules. Reinforcement magnitude was varied, on occasional sessions in Experiment 1 and across blocks of sessions in Experiment 2, from one to two or three 45-mg food pellets. In the one-pellet condition, post-reinforcement pauses increased with component schedule value. At higher magnitudes, post-reinforcement pauses increased, and overall response rates declined. Response rate on component schedules was a decreasing linear function of the obtained rate of reinforcement in all conditions. Plotted against component schedule value, response rate increased exponentially to an asymptote that decreased when reinforcement magnitude increased. These findings are consistent with regulatory accounts of food reinforced behaviour. In Experiment 3, rats were trained under a cyclic ratio schedule comprising fixed-ratio components including higher values, and some inverted U-shaped response functions were obtained. Those rats that did not showthis relationship were trained on cyclic ratios with even higher values, and all showed inverted U-shaped response functions. This suggests that behaviour on cyclic ratio schedules can reflect activating of reinforcement as well as the satiating effects seen in Experiments 1 and 2.