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A-317491 is a potent and selective antagonist of P2X3 and P2X(2/3) receptors. In the present studies, the ability of [3H]A-317491 to label recombinant human P2X(2/3) and P2X(3) receptors was characterized. Using membranes prepared from 1321N1 cells expressing P2X(2/3) receptors, [3H]A-317491 specifically labeled high-affinity (Kd = 0.9 nM) recognition sites. High-affinity [3H]A-317491 binding was not detected in membrane preparations from native 1321N1 cells or cells expressing homomeric P2X1, P2X2, or P2X3 receptors. Specific [3H]A-317491 P2X3 receptors could only be reliably detected following treatment of intact P2X3 receptor-expressing cells with apyrase (1 U/ml) both before and during membrane preparation. Under these conditions, [3H]A-317491 also labeled high-affinity (Kd = 9 nM) binding sites. Lower affinity binding components (Kd values of 87-790 nM) were detected in both assays using higher ligand concentrations that likely represent nonfunctional recognition sites. [3H]A-317491 binding to both P2X(2/3) and P2X3 receptors was reversible, and ligand kinetic studies provided similar estimates of the high-affinity binding constants. Potent P2X3 receptor agonists 2-methylthio-ATP, 2,3-O-(4-benzoylbenzoyl)-ATP, and alpha,beta-methylene adenosine triphosphate also potently inhibited specific [3H]A-317491 binding to both P2X(2/3) and P2X3 receptors. The pharmacological profile for P2X receptor antagonists to inhibit [3H]A-317491 binding to P2X(2/3) and P2X3 receptors was highly correlated (r = 0.98, P < 0.05), and a similar rank order of potency was observed for blockade of P2X(2/3) receptor-mediated calcium influx. These data demonstrate that [3H]A-317491 is the first useful radioligand for the specific labeling of P2X3-containing channels.

ATP-sensitive potassium (K(ATP)) channel openers (KCOs) have been shown to inhibit spontaneous myogenic contractile activity of the urinary bladder, a mechanism hypothesized to underlie detrusor instability and symptoms of overactive bladder. However, the therapeutic utility of KCOs has been limited by a lack of differentiation of bladder versus vascular effects. In this study, we evaluated the in vivo potency and bladder selectivity of (-)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637), a novel dihydropyridine KCO, in a pig model of detrusor instability secondary to partial bladder outlet obstruction. For comparison, we profiled two KCOs, ((R)-4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)-cyclobut-1-enylamino]-3-ethyl-benzonitrile (WAY-133537) and (S)-N-(4-benzoylphenyl)-3,3,3-trifluro-2-hydroxy-2-methyl-propionamide (ZD6169), reported previously to have improved bladder selectivity in vivo and a calcium channel blocker, nifedipine. Effective doses of A-278637, WAY-133537, ZD6169, and nifedipine to inhibit unstable contraction area under the curve by 35% and to decrease mean arterial pressure by 10% were 4.2 and 12, 109 and 51, 661 and 371, and 136 and 30 nmol/kg i.v., yielding corresponding bladder selectivity ratios of 3, 0.5, 0.6, and 0.2. Therefore, A-278637 was approximately 5- to 6-fold more bladder-selective than the other KCOs and 15-fold more selective than nifedipine, the latter approximately 4.5-fold vascular-selective. The potency of KCOs to inhibit unstable contraction in vivo was accurately predicted by their potency to inhibit spontaneous contractile activity of pig detrusor strips in vitro. These results indicate that A-278637, with enhanced potency and bladder selectivity compared with the other compounds evaluated, could serve as a useful tool in the investigation of smooth muscle K(ATP) channel openers as novel therapeutic agents for the treatment of overactive bladder.

Alterations in the myogenic activity of the bladder smooth muscle are thought to serve as a basis for the involuntary detrusor contractions associated with the overactive bladder. Activation of ATP-sensitive K(+) (K(ATP)) channels has been recognized as a potentially viable mechanism to modulate membrane excitability in bladder smooth muscle. In this study, we describe the preclinical pharmacology of (-)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637), a novel 1,4-dihydropyridine K(ATP) channel opener (KCO) that demonstrates enhanced bladder selectivity for the suppression of unstable bladder contractions in vivo relative to other reference KCOs. A-278637 activated K(ATP) channels in bladder smooth muscle cells in a glyburide (glibenclamide)-sensitive manner as assessed by fluorescence membrane potential assays using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (EC(50) = 102 nM) and by whole cell patch clamp. Spontaneous (myogenic) phasic activity of pig bladder strips was suppressed (IC(50) = 23 nM) in a glyburide-sensitive manner by A-278637. A-278637 also inhibited carbachol- and electrical field-stimulated contractions of bladder strips, although the respective potencies were 8- and 13-fold lower compared with inhibition of spontaneous phasic activity. As shown in the accompanying article [Brune ME, Fey TA, Brioni JD, Sullivan JP, Williams M, Carroll WA, Coghlan MJ, and Gopalakrishnan M (2002) J Pharmacol Exp Ther 303:387-394], A-278637 suppressed myogenic contractions in vivo in a model of bladder instability with superior selectivity compared with other KCOs, WAY-133537 [(R)-4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)cyclobut-1-enylamino]-3-ethyl-benzonitrile] and ZD6169 [(S)-N-(4-benzoylphenyl)3,3,3-trifluro-2hydroxy-2-methyl-priopionamide]. A-278637 did not interact with other ion channels, including L-type calcium channels or other neurotransmitter receptor systems. The pharmacological profile of A-278637 represents an attractive basis for further investigations of selective K(ATP) channel openers for the treatment of overactive bladder via myogenic etiology.

Benign prostatic hyperplasia (BPH), common in aging males, is often treated with alpha(1)-adrenoceptor antagonists. To minimize hypotensive and other side effects, compounds with selective antagonist activity at alpha(1A)- and alpha(1D)- (compared with alpha(1B)-) adrenoceptors were evaluated that would block lower urinary tract alpha(1)-adrenoceptors in preference to cardiovascular alpha(1B)-adrenoceptors. Fiduxosin (3-[4-((3aR,9bR)-cis-9-methoxy-1,2,3,3a,4,9b-hexahydro-[1]-benzopyrano[3,4-c]pyrrol-2-yl)butyl]-8-phenyl-pyrazino[2',3':4,5] thieno-[3,2-d]pyrimidine-2,4(1H,3H)-dione; ABT-980) was tested in radioligand binding assays, isolated tissue bioassays, intraurethral pressure (IUP) tests in isoflurane-anesthetized dogs, and blood pressure analyses in spontaneously hypertensive rats (SHR). Fiduxosin had higher affinity for cloned human alpha(1a)- (0.16 nM) and alpha(1d)-adrenoceptors (0.92 nM) in radioligand binding studies compared with alpha(1b)-adrenoceptors (25 nM) or in isolated tissue bioassays [pA(2) values of 8.5-9.6 for alpha(1A)-receptors in rat vas deferens or canine prostate strips, 8.9 at alpha(1D)-adrenoceptors (rat aorta), compared with 7.1 at alpha(1B)-adrenoceptors (rat spleen)]. Furthermore, the compound antagonized putative alpha(1L)-adrenoceptors in the rabbit urethra (pA(2) value of 7.58). Fiduxosin blocked epinephrine-induced increases in canine IUP (pseudo-pA(2) value of 8.12), eliciting only transient decreases in mean arterial blood pressure (MAP) in SHR. The area under the curve (AUC(0-->60) min) for the hypotensive response was dose related with a log index value for fiduxosin of 5.23, indicating a selectivity of 770-fold comparing IUP to MAP effects. Preferential antagonism of alpha(1A)- and alpha(1D)- versus alpha(1B)-adrenoceptors in vitro, blockade of putative alpha(1L)-sites in vitro, and selective effects on lower urinary tract function versus blood pressure in vivo by fiduxosin suggest the potential utility of this compound for the treatment of BPH.

Fiduxosin is an alpha(1)-adrenoceptor antagonist with higher affinity for alpha(1A)-adrenoceptors and for alpha(1D)-adrenoceptors than for alpha(1B)-adrenoceptors. Our hypothesis is that such a compound with higher affinity for subtypes implicated in the control of lower urinary tract function and lower affinity for a subtype implicated in the control of arterial pressure could result in a superior clinical profile for the treatment of lower urinary tract symptoms suggestive of benign prostatic obstruction. The purpose of this study was to evaluate the potency and selectivity of fiduxosin for effects on prostatic intraurethral pressure (IUP) versus mean arterial pressure (MAP) relative to current clinical standards, terazosin and tamsulosin, in conscious dogs. Phenylephrine (PE)-induced increases in IUP and MAP were determined before and at various time points after an oral dose of each antagonist. Hypotensive potency was also determined. All three antagonists caused dose- and time-dependent blockade of the IUP and MAP pressor effects of PE. The IUP ED(50) values of fiduxosin, tamsulosin, and terazosin were 0.24, 0.004, and 0.23 mg/kg p.o., respectively. The corresponding MAP ED(50) values were 1.79, 0.006, and 0.09 mg/kg p.o. The rank order of IUP selectivity (ratio) was fiduxosin (7.5-fold), tamsulosin (1.5-fold), and terazosin (0.4 = 2.5-fold MAP-selective). Tamsulosin and terazosin caused dose-dependent hypotension, whereas no change in arterial pressure was seen after fiduxosin. These data, illustrating a superior selectivity profile of fiduxosin, are consistent with our hypothesis.

□ Evidence is provided for the existence of a second discrete chemical form of meropenem at clinically relevant concentrations prepared from the marketed formulation of the drug. Proton and carbon-13 NMR spectra in D2O, coupled with tandem mass spectroscopy (MS/MS) and cross polarization/magic angle spinning (CP-MAS) NMR experiments, allow structural assignment of the compound as a covalent carbon dioxide adduct of meropenem. This carbon dioxide adduct exists in equilibrium with the free drug in solution and can be observed in the solid state following lyophilization. The equilibrium constant of formation of the adduct (Keq=20±8M–1) was estimated in D2O at 25°C.

In order to investigate the possible role of left and right hemisphere neural structures in the manifestation of depression, 50 left brain damaged and 50 right brain damaged stroke inpatients were administered the Zung Depression Scale (ZD S) during the course of the rehabilitation phase of their treatment. These subjects were randomly chosen from all stroke patients receiving rehabilitation services who could be administered the ZDS. Approximately 30% of these subjects, regardless of the site of lesion, were clinically depressed. The severity of depression was independent of demographic factors and time since injury. Right brain damaged patients reported significantly more sleep disturbance, psychomotor agitation and pervasive sense of sadness. These results were discussed in light of previous findings that damaged to the left hemisphere results in greater depression. A model of reactive depression that results from neurological disability was proposed to partially account for these findings.

A practical and efficient kilogram-scale preparation of 1,4-anhydroerythritol from meso-erythritol is described. A novel silica gel and sodium bicarbonate slurry/filtration protocol is utilized to purify the product, giving commercial-grade material in 60−65% yield.

The article discusses miscellaneous topics related to yoga. These include an answer to a question related to how to lift status of yoga studio in a crowded market and research presented at the professional membership organization American Pain Society's Annual Scientific Meeting in Palm Springs, California, which showed that yogis have more grey matter in multiple brain regions as compared to those who do not practice yoga.

An attempt to synthesise a new protected serine derivative led to the isolation of N-tetrachlorophthaloyldehydroalanine methyl ester, which underwent Michael addition of dialkyl malonates to form γ-carboxyglutamic acid derivatives.