Toni D. Wolinsky

The Novel Neuropeptide Y Y5 Receptor Antagonist Lu AA33810 [N-[[trans-4-[(4,5-Dihydro[1]benzothiepino[5,4-d]thiazol-2-yl)amino]cyclohexyl]methyl]-methanesulfonamide] Exerts Anxiolytic- and Antidepressant-Like Effects in Rat Models of Stress Sensitivity

Neuropeptide Y (NPY) regulates physiological processes via receptor subtypes (Y1, Y2, Y4, Y5, and y6). The Y5 receptor is well known for its role in appetite. Based on expression in the limbic system, we hypothesized that the Y5 receptor might also modulate stress sensitivity. We identified a novel Y5 receptor-selective antagonist, Lu AA33810 [N-[[trans-4-[(4,5-dihydro[1]-benzothiepino[5,4-d]thiazol-2-yl)amino]cyclohexyl]methyl]-methanesulfonamide], that bound to cloned rat Y5 receptors (Ki = 1.5 nM) and antagonized NPY-evoked cAMP and calcium mobilization in vitro. Lu AA33810 (3-30 mg/kg p.o.) blocked feeding elicited by intracerebroventricular injection of the Y5 receptor-selective agonist [cPP1-7,NPY19-23,Ala31,Aib32,Gln34]-hPancreatic Polypeptide in Sprague-Dawley rats. In vivo effects of Lu AA33810 were correlated with brain exposure ≥ 50 ng/g and ex vivo Y5 receptor occupancy of 22 to 95%. Lu AA33810 was subsequently evaluated in models of stress sensitivity. In Fischer 344 rats, Lu AA33810 (30 mg/kg p.o.) attenuated increases in plasma ACTH and corticosterone elicited by intracerebroventricular injection of [cPP1-7,NPY19-23,Ala31,Aib32,Gln34]-hPancreatic Polypeptide. In Sprague-Dawley rats subjected to the social interaction test, Lu AA33810 (3-30 mg/kg p.o.) produced anxiolytic-like effects after acute or chronic treatment. In Flinders sensitive line rats, chronic dosing of Lu AA33810 (10 mg/kg/day i.p.) produced anxiolytic-like effects in the social interaction test, plus antidepressant-like effects in the forced swim test. In Wistar rats exposed to chronic mild stress, chronic dosing of Lu AA33810 (3 and 10 mg/kg/day i.p.) produced antidepressant-like activity, i.e., normalization of stress-induced decrease in sucrose consumption. We propose that Y5 receptors may function as part of an endogenous stress-sensing system to mediate social anxiety and reward or motivational deficits in selected rodent models.

Pharmacological characterization of a novel positive modulator at α4β3δ-containing extrasynaptic GABAA receptors

The in vitro and in vivo pharmacological effects of [2-amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504), which is a close analogue of retigabine, have been investigated. AA29504 induced a rightward shift of the activation threshold at cloned KCNQ2, 2/3 and 4 channels expressed in Xenopus oocytes, with a potency 3-4fold lower than retigabine. AA29504 (1 muM) had no agonist activity when tested at alpha(1)beta(3)gamma(2s) or alpha(4)beta(3)delta GABA(A) receptors expressed in Xenopus oocytes, but left-shifted the EC(50) for GABA and gaboxadol (THIP) at both receptors. The maximum GABA response at alpha(1)beta(3)gamma(2s) receptors was unchanged by AA29504 (1 muM), but increased 3-fold at alpha(4)beta(3)delta receptors. In slices prepared from the prefrontal cortex of adult rats AA29504 had no effect alone on the average IPSC or the tonic current in layer II/III pyramidal neurons, but potentiated the effect of gaboxadol on both phasic and tonic currents. Thus, the effects of gaboxadol could be positively modulated by AA29504. Systemic administration of AA29504 at doses relevant for modulating GABA transmission produced anxiolytic effects and reduced motor coordination consistent with activity at GABA(A) receptors. We conclude that AA29504 exerts a major action via alpha(4)beta(3)delta-containing GABA(A) receptors, which will be important for interpreting its effect in vivo.

The effects of stressful stimuli and hypothalamic–pituitary–adrenal axis activation are reversed by the melanin-concentrating hormone 1 receptor antagonist SNAP 94847 in rodents

Melanin-concentrating hormone (MCH) is an orexigenic and dipsogenic neuropeptide that has been reported to mediate acute behavioral and neuroendocrine stress-related responses via MCH(1) receptor activation in rodents. The purpose of the present investigation was to use the MCH(1) receptor antagonist SNAP 94847 (N-(3-{1-[4-(3,4-difluoro-phenoxy)-benzyl]-piperidin-4-yl}-4-methyl-phenyl)-isobutyramide) to determine the effects of MCH(1) receptor blockade on MCH-evoked adrenocorticotropic hormone (ACTH) release, chronic mild stress-induced anhedonia, stress-induced hyperthermia and forced swim stress-induced immobility. The appropriate dose range for testing SNAP 94847 was determined by measuring MCH-evoked water drinking. The corresponding occupancy of MCH(1) receptors in rat striatum was also measured across a broad dose range. Orally administered (p.o.) SNAP 94847 (1-10 mg/kg) corresponds to 30-60% occupancy at MCH(1) receptors and significantly blocks water drinking induced by the intracerebroventricular (i.c.v.) injection of MCH. MCH (i.c.v.) significantly elevates plasma levels of ACTH in rats, and SNAP 94847 (2.5 mg/kg, p.o.) blocks MCH-evoked ACTH release. Using the chronic mild stress paradigm, we show that repeated daily exposure to environmental stressors for 5 weeks significantly suppresses sucrose intake in rats, and that SNAP 94847 (1 mg/kg, BID) for 1-5 weeks restores baseline sucrose intake. Moreover, a single administration of SNAP 94847 attenuates stress-induced hyperthermia and the behavioral effects of forced swim stress with minimal effective doses of 2.5 and 30 mg/kg (p.o.), respectively. The regulation of ACTH release and reversal of the effects of chronic and acute stress by SNAP 94847 are suggestive of a role for MCH(1) receptor blockade in the treatment of disorders characterized by high allostatic load.

How Good Are Current Approaches to Nonclinical Evaluation of Abuse and Dependence

Nonclinical assessment of drug abuse and dependence is the subject of several recent regulatory guidelines, which are generally aligned on the methods to be employed. The most direct approach to assessing reinforcing properties of a drug is the self-administration procedure whereby animals can initiate intravenous injections of the test substance, something they readily do with prototypic drugs of abuse. Complications arise because there is no standardized procedure for evaluating substances with differing potencies, reinforcement properties, or pharmacokinetics. Moreover, the choice of training substance, species, and procedural parameters can radically affect the outcome. Apart from the lower cost of rats, primates present several advantages for self-administration studies with similarity to human pharmacokinetics in particular. The most powerful method for assessing similarities between a test substance and a prototypic drug of abuse is the drug discrimination procedure. In contrast to self-administration, drug discrimination is pharmacologically very specific, often reflecting functional activity at receptor level. Dependence is assessed by the occurrence of withdrawal effects on drug discontinuation. Although conceptually simple, interpretation can be complicated by factors such as duration and frequency of administration and observations as well as the choice of end points. Telemetry allows continuous observation of multiple parameters during withdrawal, thereby increasing sensitivity. Presently available tools identify all substances known to cause abuse or dependence, with little risk of false-positives. It remains unclear, however, how predictive these models are with entirely novel substances. Nonetheless, drug abuse/dependence is an area of safety pharmacology where the predictive value of animal models is remarkably high.

Positive modulation of δ-subunit containing GABAA receptors in mouse neurons

δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) receptors in mouse neurons in vitro and in vivo. Whole-cell patch-clamp recordings were carried out in the dentate gyrus in mouse brain slices. In granule cells, AA29504 (1 μM) caused a 4.2-fold potentiation of a tonic current induced by THIP (1 μM), while interneurons showed a potentiation of 2.6-fold. Moreover, AA29504 (1 μM) increased the amplitude and prolonged the decay of miniature inhibitory postsynaptic currents (mIPSCs) in granule cells, and this effect was abolished by Zn²⁺ (15 μM). AA29504 (1 μM) also induced a small tonic current (12.7 ± 3.2 pA) per se, and when evaluated in a nominally GABA-free environment using Ca²⁺ imaging in cultured neurons, AA29504 showed GABA(A) receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo. We propose that AA29504 potentiates δ-containing GABA(A) receptors to enhance tonic inhibition, and possibly recruits perisynaptic δ-containing receptors to participate in synaptic phasic inhibition in dentate gyrus.

Discovery of Lu AA33810: a highly selective and potent NPY5 antagonist with in vivo efficacy in a model of mood disorder.

The structure-activity relationship of a series of tricyclic-sulfonamide compounds 11-32 culminating in the discovery of N-[trans-4-(4,5-dihydro-3,6-dithia-1-aza-benzo[e]azulen-2-ylamino)-cyclohexylmethyl]-methanesulfonamide (15, Lu AA33810) is reported. Compound 15 was identified as a selective and high affinity NPY5 antagonist with good oral bioavailability in mice (42%) and rats (92%). Dose dependent inhibition of feeding was observed after i.c.v. injection of the selective NPY5 agonist ([cPP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]-hPP). In addition, ip administration of Lu AA33810 (10 mg/kg) produced antidepressant-like effects in a rat model of chronic mild stress.