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1. The presence and properties of K+ channels activated by arachidonic acid were studied in neuronal cells cultured from the mesencephalic and hypothalamic areas of rat brain.2. Arachidonic acid produced a concentration-dependent (5-50 #UM) and reversible activation of whole-cell currents. 3. In excised membrane patches, arachidonic acid applied to the cytoplasmic or extracellular side of the membrane caused opening of three types of channels whose current-voltage relationships were slightly outwardly rectifying, inwardly rectifying and linear, and whose single channel slope conductances at +60 mV were 143, 45 and 52 pS, respectively.4. All three currents were K+ selective and blocked by 2 mm Ba2+ but not by other K+ channel blockers such as tetraethylammonium chloride, 4-aminopyridine and quinidine. The outwardly and inwardly rectifying currents were slightly voltage dependent with higher channel activity at more depolarized potentials.5. Arachidonic acid activated the K+ channels in cells treated with cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and nordihydroguaiaretic acid), indicating that arachidonic acid itself can directly activate the channels. Alcohol and methyl ester derivatives of arachidonic acid failed to activate the K+ channels, indicating that the charged carboxyl group is important for activation. 6. Certain unsaturated fatty acids (linoleic, linolenic and docosahexaenoic acids), but not saturated fatty acids (myristic, palmitic, stearic acids), also reversibly activated all three types of K+ channel. 7. All three K+ channels were activated by pressure applied to the membrane (i.e. channels were stretch sensitive) with a half-maximal pressure of -18 mmHg. The K+ channels were not blocked by 100 ,UM GdCl3. 8. A decrease in intracellular pH (over the range 5 6-7 2) caused a reversible, pH-dependent increase in channel activity whether the channel was initially activated by arachidonic acid or stretch.9. Glutamate, a neurotransmitter reported to generate arachidonic acid in striatal neurons, did not cause activation of the K+ channels when applied extracellularly in cell-attached patches. 10. It is suggested that the K+ channels described here belong to a distinct family of ion channels that are activated by either fatty acids or membrane stretch. Although the physiological roles of these K+ channels are not yet known, they may be involved in cellular processes such as cell volume regulation and ischaemia-induced elevation of K+ loss.

Optimization of a novel series of pyridazin-3-one histamine H(3) receptor (H(3)R) antagonists/inverse agonists identified 6-{4-[3-(R)-2-methylpyrrolidin-1-yl)propoxy]phenyl}-2H-pyridazin-3-one (8a, CEP-26401; irdabisant) as a lead candidate for potential use in the treatment of attentional and cognitive disorders. 8a had high affinity for both human (K(i) = 2.0 nM) and rat (K(i) = 7.2 nM) H(3)Rs with greater than 1000-fold selectivity over the hH(1)R, hH(2)R, and hH(4)R histamine receptor subtypes and against an in vitro panel of 418 G-protein-coupled receptors, ion channels, transporters, and enzymes. 8a demonstrated ideal pharmaceutical properties for a CNS drug in regard to water solubility, permeability and lipophilicity and had low binding to human plasma proteins. It weakly inhibited recombinant cytochrome P450 isoforms and human ether-a-go-go-related gene. 8a metabolism was minimal in rat, mouse, dog, and human liver microsomes, and it had good interspecies pharmacokinetic properties. 8a dose-dependently inhibited H(3)R agonist-induced dipsogenia in the rat (ED(50) = 0.06 mg/kg po). On the basis of its pharmacological, pharmaceutical, and safety profiles, 8a was selected for preclinical development. The clinical portions of the single and multiple ascending dose studies assessing safety and pharmacokinetics have been completed allowing for the initiation of a phase IIa for proof of concept.

The rat novel object recognition (NOR) assay is a relatively high-throughput, robust, and sensitive procedure for evaluating compounds for cognition-enhancing activity. For the test, rats are given the opportunity to explore two identical objects for a predetermined period of time. After a delay, the animals are then presented with two objects to explore, one of which is the same as in the first exploration trial, the other a new object. Depending on the length of the delay between the two trials, the rats will either explore the novel object for a greater time period, indicating memory for the familiar object, or will explore the novel and familiar objects for the same amount of time, indicating a lack of recall or loss of memory for the familiar object presented during the initial trial. The protocol described in this unit can be used to evaluate the effects of a compound on the short-term/working memory of adult male rats following a 24-hr inter-trial interval.

The histamine H 3 receptor (H 3 R) modulates the release of neurotransmitters that are involved in vigilance, cognition, and sleepwake regulation. H 3 R antagonism has been proposed as a novel approach to the treatment of cognitive and attention deficit as well as sleep disorders. It is apparent that H 3 R antagonists produce pharmacological effects in preclinical animal models across a wide dose range. Several H 3 R antagonists were reported to be effective at producing cognitive enhancing effects at low doses, while producing robust wake enhancement at higher doses. To better understand the effect of H 3 R antagonists across a broad dose range, an ex vivo receptor binding assay has been used to estimate the degree of H 3 R occupancy in vivo. The H 3 R antagonists ciproxifan, thioperamide, GSK189254 (6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride), and ABT-239produced wake-promoting activity in vivo and a dose-dependent inhibition of H 3 R binding ex vivo. For ciproxifan, thioperamide, and GSK189254, a relatively low level of cumulative wake activity was linearly correlated with up to 80% of the receptor occupancy. In contrast, an abrupt break from linearity and a robust increase of waking activity was observed at doses that produce greater than 80% occupancy. Our results suggest a relatively small increase of waking activity at low levels of receptor occupancy that may be consistent with reported enhancement of attention and cognitive function. Robust waking activity at higher levels of H 3 R occupancy may be mechanistically different from activities at low levels of H 3 R occupancy.The neurotransmitter histamine is synthesized by neurons originating in the tuberomammillary nucleus of the posterior hypothalamus. These neurons project widely throughout the cortex, hippocampus, amygdala, and striatum (Brown et al., 2001). Histamine exerts its action by interacting with a group of G protein-coupled histamine receptors and plays important roles in mediating a variety of functions in the central nervous system (Brown et al., 2001). The H 3 R is presynaptically localized and functions both as an autoreceptor and a heteroreceptor by modulating the release of neurotransmitters, including histamine (Arrang et al., 1985), dopamine, acetylcholine, serotonin, and norepinephrine (Schlicker et al., 1994;Blandina et al., 1996;Brown et al., 2001). Activation of the H 3 R results in the inhibition of neurotransmitter release. In contrast, blockade of the H 3 R by selective antagonists or inverse agonists can reverse the histamine-mediated inhibition of neurotransmitter release. By virtue of the unique central nervous system localization of the H 3 R (Drutel et al., 2001;Leurs et al., 2005) and its ability to regulate a variety of neurotransmitters that are thought to be involved in vigilance, cognition, and wakefulness, H 3 R antagonists and inverse agonists are suggested to hold promise for a number of therapeutic applications (Hancock and Fox, 2004).Selectiv...