1 A hallmark for unstable bladder contractions is hyperexcitability and changes in the nature of spontaneous phasic activity of the bladder smooth muscle. In this study, we have characterized the spontaneous activity of the urinary bladder smooth muscle from the pig, a widely used model for studying human bladder function. 2 Our studies demonstrate that phasic activity of the pig detrusor is myogenic and is in¯uenced by the presence of urothelium. Denuded strips exhibit robust spontaneous activity measured as mean area under the contraction curve (AUC=188.9+15.63 mNs) compared to intact strips (AUC=7.3+1.94 mNs). 3 Spontaneous phasic activity, particularly the amplitude, is dependent on both calcium entry through voltage-dependent calcium channels and release from ryanodine receptors as shown by inhibition of spontaneous activity by nifedipine and ryanodine respectively. 4 Inhibition of BK Ca channels by iberiotoxin (100 nM) resulted in an increase in contraction amplitude (89.1+20.4%) and frequency (92.5+31.0%). The SK Ca channel blocker apamin (100 nM) also increased contraction amplitude (69.1+24.3%) and frequency (53.5+13.6%) demonstrating that these mechanisms are critical to the regulation of phasic spontaneous activity. 5 Inhibition of K ATP channels by glyburide (10 mM) did not signi®cantly alter myogenic contractions (AUC=18.5+12.3%). However, K ATP channel openers (KCOs) showed an exquisite sensitivity for suppression of spontaneous myogenic activity. KCOs were generally 15 fold more potent in suppressing spontaneous activity compared to contractions evoked by electrical ®eld-stimulation. These studies suggest that potassium channel modulation, particularly K ATP channels, may o er a unique mechanism for controlling spontaneous myogenic activity especially those associated with the hyperexcitability occurring in unstable bladders.

Several 3-acylindoles with high affinity for the CB(2) cannabinoid receptor and selectivity over the CB(1) receptor have been prepared. A variety of 3-acyl substituents were investigated, and the tetramethylcyclopropyl group was found to lead to high affinity CB(2) agonists (5, 16). Substitution at the N1-indole position was then examined. A series of aminoalkylindoles was prepared and several substituted aminoethyl derivatives were active (23-27, 5) at the CB(2) receptor. A study of N1 nonaromatic side chain variants provided potent agonists at the CB(2) receptor (16, 35-41, 44-47, 49-54, and 57-58). Several polar side chains (alcohols, oxazolidinone) were well-tolerated for CB(2) receptor activity (41, 50), while others (amide, acid) led to weaker or inactive compounds (55 and 56). N1 aromatic side chains also afforded several high affinity CB(2) receptor agonists (61, 63, 65, and 69) but were generally less potent in an in vitro CB(2) functional assay than were nonaromatic side chain analogues.

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A series of potent indol-3-yl-tetramethylcyclopropyl ketones have been prepared as CB 2 cannabinoid receptor ligands. Two unsubstituted indoles (5, 32) were the starting points for an investigation of the effect of indole ring substitutions on CB 2 and CB 1 binding affinities and activity in a CB 2 in vitro functional assay. Indole ring substitutions had varying effects on CB 2 and CB 1 binding, but were generally detrimental to agonist activity. Substitution on the indole ring did lead to improved CB 2 /CB 1 binding selectivity in some cases (i.e., 7-9, 15-20). All indoles with the morpholino-ethyl side chain (32-43) exhibited weaker binding affinity and less agonist activity relative to that of their tetrahydropyranyl-methyl analogs (5-31). Several agonists were active in the complete Freund's adjuvant model of chronic inflammatory thermal hyperalgesia (32, 15).

Studies demonstrating the antihyperalgesic and antiallodynic effects of cannabinoid CB 2 receptor activation have been largely derived from the use of receptor-selective ligands. Here, we report the identification of A-836339 [2,2,3,3-tetramethyl-cyclopropanecarboxylic acid [3-(2-methoxy-ethyl)-4,5-dimethyl-3H-thiazol-(2Z)-ylidene]-amide], a potent and selective CB 2 agonist as characterized in in vitro pharmacological assays and in in vivo models of pain and central nervous system (CNS) behavior models. In radioligand binding assays, A-836339 displays high affinities at CB 2 receptors and selectivity over CB 1 receptors in both human and rat. Likewise, A-836339 exhibits high potencies at CB 2 and selectivity over CB 1 receptors in recombinant fluorescence imaging plate reader and cyclase functional assays. In addition A-836339 exhibits a profile devoid of significant affinity at other G-protein-coupled receptors and ion channels. A-836339 was characterized extensively in various animal pain models. In the complete Freund's adjuvant model of inflammatory pain, A-836339 exhibits a potent CB 2 receptor-mediated antihyperalgesic effect that is independent of CB 1 or -opioid receptors.A-836339 has also demonstrated efficacies in the chronic constrain injury (CCI) model of neuropathic pain, skin incision, and capsaicin-induced secondary mechanical hyperalgesia models. Furthermore, no tolerance was developed in the CCI model after subchronic treatment with A-836339 for 5 days. In assessing CNS effects, A-836339 exhibited a CB 1 receptor-mediated decrease of spontaneous locomotor activities at a higher dose, a finding consistent with the CNS activation pattern observed by pharmacological magnetic resonance imaging. These data demonstrate that A-836339 is a useful tool for use of studying CB 2 receptor pharmacology and for investigation of the role of CB 2 receptor modulation for treatment of pain in preclinical animal models.It is estimated that as high as 50% of the population will experience chronic pain during their lifetime, and the prevalence is likely to rise with the continued aging of the population (Markman and Philip, 2007). As a consequence, there exists an ever-growing demand for new therapies to provide safe and effective pain management. Despite intensive research to identify novel therapeutic approaches, there have been few major advances in pain therapy over the past sev-