Journal of Pharmacology and Experimental Therapeutics

Published by American Society for Pharmacology and Experimental Therapeutics
Online ISSN: 1521-0103
Print ISSN: 0022-3565
Publications
The effects of the cardioprotective, vasorelaxant agent NS-004 [1-(2'-hydroxy-5'-chlorophenyl)-5-trifluoromethyl-2(3H) benzimidazolone] on K+ currents of freshly isolated canine coronary artery smooth muscle cells were examined using the suction pipette method. The Ca(++)-sensitive K+ current (IK, Ca) was isolated by using a holding potential of 0 mV and 5 mM ATP in the internal perfusate. The voltage-dependent delayed rectifier K+ current (IK, DR) was recorded at potentials below 20 mV in internal solution containing 5 mM ATP. The ATP-sensitive K+ current (IK, ATP) was induced by removing ATP in the pipette solution and was measured at potentials negative to -30 mV where IK, Ca and IK, DR were absent. External application of NS-004 activated IK, Ca, which, at 80 mV, was increased from 0.28 +/- 0.03 to 1.1 +/- 0.2, to 3.2 +/- 1.0, to 5.6 +/- 1.6 and to 8.5 +/- 1.6 nA, respectively, by 12.5, 25, 50 and 100 microM NS-004. Structurally related flufenamic, niflumic and mefenamic acids, at 100 microM, also enhanced IK, Ca. The order of potency for IK, Ca activation was NS-004 > flufenamic = niflumic > mefenamic. In contrast, NS-004 blocked IK, DR, beginning at 2 microM with 50% inhibition at 22 microM. NS-004 also inhibited IK, ATP with a Ki value of 2.4 microM and a Hill coefficient of 1.3 in 0 mM intracellular ATP. These results demonstrate that NS-004 is a more potent activator of IK, Ca than the fenamates and that it is also a relatively potent blocker of IK, DR and IK, ATP in canine coronary artery smooth muscle cells.
 
A series of compounds have been reported which open the large conductance calcium activated potassium channel (maxi-K). By utilizing the most potent compound, NS-004 [1-(5-chloro-2-hydroxyphenyl)-5-trifluromethyl-1,3-dihydro-2-be nzimidazol-2- one], we studied the role of maxi-K channels in ischemic myocardium. Isolated rat hearts were pretreated with vehicle or NS-004 (6-36 microM). NS-004 caused a concentration-dependent reduction in left ventricular developed pressure and an increase in coronary flow. In global ischemia (25 min), a concentration-dependent increase in time to contracture was found in NS-004 (6-20 microM)-treated hearts (EC25 = 8.6 microM). Neither iberiotoxin (50 nM), a maxi-K blocker, nor glyburide (1 microM), an adenosine triphosphate-sensitive potassium channel blocker, reversed the preischemic or ischemic effects of 20 microM NS-004. NS-004 relaxed phenylephrine- and KCl- contracted rat aortic smooth muscle (IC50 = 9.2 microM). This relaxation was unaffected by 50 and 200 nM iberiotoxin. Whole cell potassium currents in ventricular myocytes demonstrated no significant increases in outward potassium current after treatment with NS-004 (1-20 microM). A small, but significant, increase in outward potassium current was observed with 50 microM NS-004. When peak inward L-type calcium currents were measured in ventricular myocytes, a concentration-dependent inhibition was observed in the presence of NS-004 (1-50 microM). Iberiotoxin (50 nM) did not alter the inhibition of inward calcium current observed in the presence of NS-004.(ABSTRACT TRUNCATED AT 250 WORDS)
 
A new inhibitor of acyl CoA:cholesterol acyltransferase (ACAT), HL-004 [N-(2, 6-diisopropylphenyl)tetradecylthioacetamide], suppressed the synthesis of cholesterol [14C]oleate at 10(-9) approximately 10(-7) M in a concentration-dependent manner in both THP-1 cell-derived macrophages and foam cells prepared from aortic intima of rabbits fed a high cholesterol diet. Incorporation of [3H]cholesterol oleate-beta very low density lipoproteins was not inhibited by HL-004 at 10(-9) approximately 10(-7) M. Release of radioactivity from the cells loaded with [3H]cholesterol oleate-beta very low density lipoproteins was increased by the inhibition of ACAT activity with HL-004. HL-004 did not affect on acid and neutral cholesterol esterases. As a result, cholesterol ester content in foam cells decreased. These data suggested that HL-004 decreases cholesterol ester in foam cells by increasing the release of cholesterol and therefore might suppress atherosclerotic lesions.
 
Structures of marizomib and its analogs and mechanism of proteasome inhibition . A, marizomib and analogs with structural modifications at the C-2 substituent (R) are shown. LG compounds include marizomib (NPI-0052), NPI-2151, and NPI-2150 (LG Cl, OTosyl, and OMesyl, respectively). NLG analogs (NPI- 2063, NPI-0047, and NPI-2080) are substituted with unbranched aliphatic side chains. Structures of [ 3 H]marizomib (NPI- 0052) and [ 3 H]NPI-0047 show the position of the tritium label at C-5. B, mechanism for reversible and irreversible inhibition of the proteasome by NLG and LG analogs, respectively. The -lactone of the ligand (I) acylates the proteasome Thr1O (pathway A), giving rise to adduct of general structure II. In the case of NLG analogs, the ligand can be removed from the active site by Thr1NH 2 -catalyzed reformation of the -lactone ring (pathway B) or hydrolysis of the ester (pathway C); the latter pathway gives rise to deactivated product III. Cumulatively , proteasome activity is restored, i.e., inhibition is slowly reversible. In the case of LG analogs (adduct IIa, r CH 2 CH 2 LG), Thr1NH 2 -catalyzed nucleophilic displacement of the LG by C-3O gives stable adduct IIIa (pathway D), which is irreversibly bound.  
Cytotoxicity of LG and NLG analogs against RPMI 8226 and PC-3 cells Compound R IC 50 Value S.D.
Uptake characteristics of marizomib and NPI-0047 in RPMI 8226 and PC-3 cells
The present study was undertaken to compare the cellular transport characteristics of [(3)H]NPI-0052 (1R,4R,5S)-4-(2-chloroethyl)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione (marizomib; salinosporamide A) and [(3)H]NPI-0047 (1R,4R, 5S)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-4-ethyl-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione in RPMI 8226 multiple myeloma and PC-3 prostate adenocarcinoma cells to determine whether these properties explain differences in the cytotoxic potencies of these chemical analogs. The results indicate that marizomib, which possesses a chemical-leaving group, is more cytotoxic to both cell lines and inhibits proteasome activity more completely at lower concentrations than NPI-0047, a nonleaving-group analog. Moreover, it was found that both compounds accumulate in these cells by simple diffusion and the same carrier-mediated transport system. Although the rate of uptake is similar, the cellular efflux, which does not seem to be mediated by a major ATP-binding cassette (ABC)-efflux transporter, is more rapid for NPI-0047 than for marizomib. Experiments revealed that the irreversible binding of marizomib to the proteasome is responsible for its slower efflux, longer duration of action, and greater cytotoxicity compared with NPI-0047. The discovery that major ABC transporters of the multidrug resistance-associated protein family do not seem to be involved in the accumulation or removal of these agents suggests they may not be affected by multidrug resistance mechanisms during prolonged administration.
 
The benztropine analog N-(n-butyl)-3α-[bis(4'-fluorophenyl)methoxy]-tropane (JHW 007) displays high affinity for the dopamine transporter (DAT), but unlike typical DAT ligands, has relatively low abuse liability and blocks the effects of cocaine, including its self-administration. To determine sites responsible for the cocaine antagonist effects of JHW 007, its in vitro binding was compared with that of methyl (1R,2S,3S,5S)-3-(4-fluorophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (WIN 35428) in rats, mice, and human DAT (hDAT)-transfected cells. A one-site model, with K(d) values of 4.21 (rat) and 8.99 nM (mouse) best fit the [(3)H]WIN 35428 data. [(3)H]JHW 007 binding best fit a two-site model (rat, 7.40/4400 nM; mouse, 8.18/2750 nM), although a one-site fit was observed with hDAT membranes (43.7 nM). Drugs selective for the norepinephrine and serotonin transporters had relatively low affinity in competition with [(3)H]JHW 007 binding, as did drugs selective for other sites identified previously as potential JHW 007 binding sites. The association of [(3)H]WIN 35428 best fit a one-phase model, whereas the association of [(3)H]JHW 007 best fit a two-phase model in all tissues. Because cocaine antagonist effects of JHW 007 have been observed previously soon after injection, its rapid association observed here may contribute to those effects. Multiple [(3)H]JHW 007 binding sites were obtained in tissue from mice lacking the DAT, suggesting these as yet unidentified sites as potential contributors to the cocaine antagonist effects of JHW 007. Unlike WIN 35428, the binding of JHW 007 was Na(+)-independent. This feature of JHW 007 has been linked to the conformational status of the DAT, which in turn may contribute to the antagonism of cocaine.
 
The pharmacological properties of cyclohexanecarboxylic acid, {2-[4-(2-bromo-5-methoxybenzyl)piperazin-1-yl]ethyl}-(2-trifluoromethoxyphenyl)amide (Rec 27/0224), and cyclohexanecarboxylic acid, (2-methoxy-phenyl)-{2-[4-(2-methoxyphenyl)-piperazin-1-yl]ethyl}amide (Rec 27/0074), were characterized using radioligand displacement and guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding assays, as well as electrophysiological experiments, in rat hippocampal and dorsal raphe nucleus (DRN) slices. Both compounds showed a high affinity (Ki, approximately 1 nM) and selectivity (>70-fold) at human 5-hydroxytryptamine (5-HT)1A receptors versus other 5-HT receptors. In [35S]GTPgammaS binding assays on HeLa cells stably expressing human 5-HT1A receptors, Rec 27/0224 and Rec 27/0074 inhibited basal [35S]GTPgammaS binding by 44.8 +/- 1.7% (pEC50 = 8.58) and 25 +/- 2.5% (pEC50 = 8.86), respectively. In intracellularly recorded CA1 pyramidal cells, 5-HT1A (hetero)receptor-mediated hyperpolarization, elicited by 100 nM 5-carboxamidoytryptamine (5-CT), was partially antagonized by Rec 27/0224 (approximately 50%; IC50 = 18.0 nM) and Rec 27/0074 (74%; IC50 = 0.8 nM). In extracellularly recorded DRN serotonergic neurons, Rec 27/0224 and Rec 27/0074 fully antagonized the inhibition of firing caused by the activation of 5-HT1A (auto)receptors by 30 nM 5-CT with an IC50 of 34.9 nM and 16.5 nM, respectively. The antagonism had a slow time course, reaching a steady state within 60 min. Both compounds also antagonized the citalopram-elicited, endogenous 5-HT-mediated inhibition of cell firing. In conclusion, Rec 27/0224 and Rec 27/0074 exhibited inverse agonism in [35S]GTPgammaS binding assays and differential antagonistic properties on 5-HT1A receptor-mediated responses in the hippocampus but not in the DRN. Whether this differential effect is causally related to inverse agonist activity is unclear. The qualitatively different nature of the antagonism in the hippocampus versus the DRN clearly distinguishes the compounds from neutral antagonists, such as N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylcyclo-hexanecarboxamide (WAY-100635).
 
UCN-01 is a staurosporine-related compound that was isolated from the culture broth of Streptomyces sp. and shows potent and selective inhibitory activity against protein kinase C. Cellular inhibitory activity of UCN-01 against protein kinase C and cytotoxicity of UCN-01 were compared with those of staurosporine. When the mechanism of inhibitory activity was investigated in vitro, UCN-01 as well as staurosporine inhibited the activity of the catalytic domain of protein kinase C. In spite of direct inhibition against the catalytic domain of protein kinase C, cytotoxicity of UCN-01 was much lower than that of staurosporine. In addition, UCN-01 showed more selective inhibitory activity against protein kinase C than did staurosporine because of the sole structural difference at C-7. Therefore, a series of 7-O-alkyl derivatives of UCN-01 was synthesized and investigated. Interestingly, one of the compounds, the beta-methoxy derivative, showed 3-fold greater potency and 17-fold more selective inhibitory activity against protein kinase C than did UCN-01.
 
Tolamolol (UK-6558-01) is a relatively cardioselective beta receptor antagonist which is effective clinically in the treatment of supraventricular and ventricular arrhythmias. Animal studies suggest tolamolol may have significant antifibrillatory properties. The effect of tolamolol on the passive membrane properties of sheep cardiac Purkinje fibers was studied using multiple microelectrode techniques. In sodium-deficient Tyrode's solution, we measured the resting transmembrane voltage (Vr) at differing external potassium concentrations (K+]0, investigated current-voltage relationships and per formed cable analysis. Tolamolol was found to hyperpolarize the membrane in the presence of a large driving force [i.e., when (Vr - VK) was significantly greater than zero, where VK is the potassium equilibrium voltage ]; to hyperpolarize fibers with a Vr decreased due to stretch at [K+]0 = 4.0 mM; to increase membrane chord and slope potassium conductance; to decrease the membrane length constant; and to decrease the membrane time constant. In normal sodium Tyrode's solution, tolamolol could suppress automaticity. These results suggest that tolamolol increases membrane potassium conductance within the range of the pacemaker potential. This action can account, in part, for its ability to suppress automaticity and for its ability to prevent reentrant arrhythmias. In normal sodium Tyrode's solution, tolamolol suppressed oscillatory and sustained rhythmic activity occurring at a low transmembrane voltage which may be related to its alleged antifibrillatory effects.
 
Maximal responses of CDD-0102A at muscarinic receptor subtypes. The data are expressed as a percentage of the maximal response produced by the full agonist carbachol for each receptor subtype. Stimulation of phosphoinositide metabolism provided a measure of agonist activity at M 1 , M 3 , and M 5 receptors expressed in A9 L cells, whereas inhibition of forskolin-stimulated cAMP formation assessed agonist activity at M 2 and M 4 receptors expressed in A9 L and Chinese hamster ovary cells, respectively. Data represent the mean S.E.M. from at least three experiments, each performed in triplicate. Data for M 1 and M 3 receptors are summarized from previous publications (Messer et al., 1997b).
The effect of CDD-0102A on place acquisition and switch to a visual cue discrimination. Each rat received an intraperitoneal injection of saline (SAL) or one of three doses of CDD-0102A (CD) 30 min before acquisition and switch phases. The treatments shown represent the treatment received before acquisition (top rows) followed by the treatment received before the switch phase (bottom rows). A, mean Ϯ S.E.M. trials to criterion on acquisition of a place discrimination. Injection of CDD-0102A had no effect on acquisition. B, mean Ϯ S.E.M. trials to criterion on switch to a visual cue strategy. CDD-0102A at 0.03 and 0.1 mg/kg facilitated a shift to a visual cue strategy. ءء , P Ͻ 0.01 versus SAL-SAL. C, mean Ϯ S.E.M. perseverative errors committed in the switch to visual cue discrimination. CDD-0102A at 0.03 and 0.1 mg/kg significantly decreased perseverative errors. ء , P Ͻ 0.05 versus SAL-SAL; ءء , P Ͻ 0.01 versus SAL-SAL. D, mean Ϯ S.E.M. regressive errors committed in the switch to visual cue discrimination. CDD-0102A at 0.03 and 0.1 mg/kg significantly decreased regressive errors. ءء , P Ͻ 0.01 versus SAL-SAL. E, mean Ϯ S.E.M. never-reinforced errors committed in the switch to visual cue discrimination. CDD-0102A at 0.03 and 0.1 mg/kg significantly decreased never- reinforced errors. ء , P Ͻ 0.05 versus SAL-SAL; ءء , P Ͻ 0.01 versus SAL-SAL. 
The effect of CDD-0102A on visual cue acquisition and switch to place discrimination. Each rat received an intraperitoneal injection of saline (SAL) or one of three doses of CDD-0102A (CD) 30 min before acquisition and switch phases. The treatments shown represent the treatment received before acquisition (top rows) followed by the treatment received before the switch phase (bottom rows). A, mean Ϯ S.E.M. trials to criterion on acquisition of a visual cue discrimination. Injection of CDD-0102A had no effect on acquisition. B, mean Ϯ S.E.M. trials to criterion on switch to a place strategy. CDD-0102A at 0.03 and 0.1 mg/kg facilitated a shift to a place strategy. ءء , P Ͻ 0.01 versus SAL-SAL. C, mean Ϯ S.E.M. perseverative errors committed in the switch to visual cue discrimination. CDD-0102A at 0.03 and 0.1 mg/kg significantly decreased perseverative errors. ءء , P Ͻ 0.01 versus SAL-SAL. D, mean Ϯ S.E.M. regressive errors committed in the switch to visual cue discrimination. CDD-0102A at 0.03 and 0.1 mg/kg significantly decreased regressive errors. ءء , P Ͻ 0.01 versus SAL-SAL. E, mean ( Ϯ S.E.M.) never-reinforced errors committed in the switch to visual cue discrimination. CDD-0102A treatment did not affect never-reinforced errors. 
Various neurodegenerative diseases and psychiatric disorders are marked by alterations in brain cholinergic function and cognitive deficits. Efforts to alleviate such deficits have been limited by a lack of selective M(1) muscarinic agonists. 5-(3-Ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine hydrochloride (CDD-0102A) is a partial agonist at M(1) muscarinic receptors with limited activity at other muscarinic receptor subtypes. The present studies investigated the effects of CDD-0102A on working memory and strategy shifting in rats. CDD-0102A administered intraperitoneally 30 min before testing at 0.1, 0.3, and 1 mg/kg significantly enhanced delayed spontaneous alternation performance in a four-arm cross maze, suggesting improvement in working memory. In separate experiments, CDD-0102A had potent enhancing effects on learning and switching between a place and visual cue discrimination. Treatment with CDD-0102A did not affect acquisition of either a place or visual cue discrimination. In contrast, CDD-0102A at 0.03 and 0.1 mg/kg significantly enhanced a shift between a place and visual cue discrimination. Analysis of the errors in the shift to the place or shift to the visual cue strategy revealed that in both cases CDD-0102A significantly increased the ability to initially inhibit a previously relevant strategy and maintain a new, relevant strategy once selected. In anesthetized rats, the minimum dose required to induce salivation was approximately 0.3 mg/kg i.p. Salivation increased with dose, and the estimated ED(50) was 2.0 mg/kg. The data suggest that CDD-0102A has unique memory and cognitive enhancing properties that might be useful in the treatment of neurological disorders at doses that do not produce adverse effects such as salivation.
 
The present study characterized the effects of TS-011 [N-(3-chloro-4-morpholin-4-yl) phenyl-N'-hydroxyimido formamide], a new selective inhibitor of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), on the metabolism of arachidonic acid by human and rat renal microsomes and the inhibitory effects of this compound on hepatic cytochrome P450 enzymes involved in drug metabolism. The effects of TS-011 on the fall in cerebral blood flow following subarachnoid hemorrhage (SAH) and in reducing infarct size in ischemic stroke models were also examined since 20-HETE may contribute to the development of cerebral vasospasm. TS-011 inhibited the synthesis of 20-HETE by human renal microsomes and recombinant CYP4A11 and 4F2, 4F3A, and 4F3B enzymes with IC50 values around 10 to 50 nM. It had no effect on the activities of CYP1A, 2C9, 2C19, 2D6, or 3A4 enzymes. TS-011 inhibited the synthesis of 20-HETE by rat renal microsomes with an IC50 of 9.19 nM, and it had no effect on epoxygenase activity at a concentration of 100 microM. TS-011 (0.01-1 mg/kg i.v.) reversed the fall in cerebral blood flow and the increase in 20-HETE levels in the cerebrospinal fluid of rats after SAH. TS-011 also reduced the infarct volume by 35% following transient ischemic stroke and in intracerebral hemorrhage in rats. Injection of 20-HETE (8 or 12 mg/kg) into the carotid artery produced an infarct similar to that seen in the ischemic stroke model. These studies indicate that blockade of the synthesis of 20-HETE with TS-011 opposes cerebral vasospasm following SAH and reduces infarct size in ischemic models of stroke.
 
IC 50 values of LCB 03-0110 for the inhibition of protein tyrosine kinases.
Wound healing generally induces an inflammatory response associated with tissue fibrosis in which activated macrophage and myofibroblast cells are primarily involved. Although this is known to be the underlying mechanism for scarring and various fibrotic pathologies, no effective intervention is currently available. We identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol (LCB 03-0110), a thienopyridine derivative, as a potent inhibitor of discoidin domain receptor family tyrosine kinases and discovered that this compound strongly inhibits several tyrosine kinases, including the c-Src family, spleen tyrosine kinase, Bruton's tyrosine kinase, and vascular endothelial growth factor receptor 2, which are important for immune cell signaling and inflammatory reactions. LCB 03-0110 suppressed the proliferation and migration of primary dermal fibroblasts induced by transforming growth factor β1 and type I collagen, and this result correlated with the inhibition ability of the compound against enhanced expression of α-smooth muscle actin and activation of Akt1 and focal adhesion kinase. In J774A.1 macrophage cells activated by lipopolysaccharide LCB 03-0110 inhibited cell migration and nitric oxide, inducible nitric-oxide synthase, cyclooxygenase 2, and tumor necrosis factor-α synthesis. LCB 03-0110 applied topically to full excisional wounds on rabbit ears suppressed the accumulation of myofibroblast and macrophage cells in the healing wound and reduced hypertrophic scar formation after wound closing, without delaying the wound closing process. Taken together, the pharmacological activities of LCB 03-0110 suggest that it could be an effective agent for suppressing fibroinflammation by simultaneously targeting activated fibroblasts and macrophages.
 
Affinity of MRK-016 for the benzodiazepine site of recombinant human and native rat brain GABA A receptors Data shown are the mean S.E. (n 3 separate determinations). K i for Human Recombinant GABA A Receptors Containing 3, 2 Plus K i for Native Rat Brain Receptors 
Predicted hepatic clearance values compared with those measured in vivo 
Comparison of the structures of MRK-016 (compound 13, Chambers et al., 2004), and 5IA (Dawson et al., 2006). Note that the 5-methyl- 3-isoxazolyl and triazolylmethoxy groups are found in both compounds, albeit that the triazole group is in a 1,2,4 configuration in MRK-016 and a 1,2,3 arrangement in 5IA.  
3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. It has inverse agonist efficacy selective for the alpha5 subtype, and this alpha5 inverse agonism is greater than that of the prototypic alpha5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (alpha5IA). Consistent with its greater alpha5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than alpha5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC(50) value of 15 ng/ml that was similar to the rhesus monkey plasma EC(50) value of 21 ng/ml obtained using [(11)C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3-0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species ( approximately 3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.
 
The cardiac actions of a number of prostaglandins and their modification by the prostaglandin antagonist sodium p-benzyl-4-[1-oxo-2-(4-chlorobenzyl)-3-phenyl propyl]phenyl phosphonate (N-0164) was studied in the isolated guinea-pig heart. Arachidonic acid, prostaglandin (PG)E2 (0.01--1 micrograms) and prostacyclin (0.01--10 micrograms), administered by bolus injection, caused dose-dependent increases in coronary flow rate, whereas PGD2, PGF2 alpha and the stable PG enderoperoxide analog U46619 (0.01--100 micrograms) caused dose-dependent decreases in coronary flow rate. Over the dose range studied, PGE2, arachidonic acid, prostacyclin and PGF2 alpha increased the sinus rate and PGD2 decreased the sinus rate, whereas U46619 had no consistent effect. Arachidonic acid, PGF2 alpha, PGD2 and U46619 produced a decrease in ventricular contractile force, whereas PGE2 had no effect and prostacyclin produced a modest increase in ventricular contractile force. N-0164 (10 ng and 100 ng/ml) selectively antagonized the coronary vasoconstrictor effects of PGD2 and PGF2 alpha. N-0164, at higher concentrations (1 micrograms/ml), antagonized the coronary vasodilator actions of PGE2; however, it did not modify the coronary vasodilator action of prostacyclin. N-0164 (10--100 ng/ml) also antagonized as a function of its concentration sinus rate changes produced by either PGD2 or PGF2 alpha, whereas at higher concentrations (1 microgram/ml), rather than antagonizing it potentiated sinus rate increases produced by either PGE2 or prostacyclin. These results suggest that the cardiac actions of prostaglandins are complex and are not readily elucidated by the use of the prostaglandin antagonist, N-0164.
 
Time course (0-120 min) of the Ro 60-0175-induced increase in plasma levels of ACTH (A), corticosterone (B), oxytocin (C), and prolactin (D). , significant effect comparing the Ro 60-0175-injected group (filled circles) with the uninjected control (filled triangle), p 0.01 (one-way ANOVA, Newman-Keuls multiple range test). F, significant effect when comparing the saline-injected group (open circle) with uninjected group, p 0.01 (one-way ANOVA, Newman-Keuls multiple range test). 
Effect of various doses of Ro 60-0175 on plasma levels of ACTH (A), corticosterone (B), oxytocin (C), and prolactin (D). , significant effect of Ro 60-0175-injected group (filled circles) compared with saline-injected group (open circle at 0 mg/kg), p 0.01 (one-way ANOVA, Newman-Keuls multiple range test). 
Effect of SB 242084 pretreatment (0.1, 0.5, 1.0, and 5.0 mg/kg i.p.) on Ro 60-0175 (5.0 mg/kg s.c.)-induced increases in plasma levels of ACTH (A), corticosterone (B), oxytocin (C), and prolactin (D). , significant effect comparing the Ro 60-0175-injected group (filled circles) with saline-injected group (open circles), p 0.01 (two-way ANOVA, Newman-Keuls multiple range test). 
Effect of MDL 100,907 pretreatment (1, 5, and 10 g/kg s.c.) on Ro 60-0175 (5.0 mg/kg s.c.)-induced increases in plasma levels of ACTH (A), corticosterone (B), oxytocin (C), and prolactin (D). , significant effect comparing the Ro 60-0175-injected group (filled circles) with their respective saline-injected group (open circles), p 0.01 (two-way ANOVA, Newman-Keuls multiple range test). 
The 5-hydroxytryptamine(2A) and (2C) (5-HT(2A) and 5-HT(2C)) receptors are so closely related that selective agonists have not been developed until recently with the advent of (S)-2-(chloro-5-fluoro-indol-l-yl)-1-methylethylamine fumarate (Ro 60-0175), a putatively selective 5-HT(2C) receptor agonist. In the present study, Ro 60-0175 was used to analyze the importance of 5-HT(2C) receptors in hormone secretion. Injection of Ro 60-0175 (5 mg/kg s.c.) produced a maximum increase in plasma levels of adrenocorticotrophic hormone, oxytocin, and prolactin at 15 min postinjection and a maximum increase in plasma corticosterone levels at 60 min postinjection. Ro 60-0175-mediated increases in plasma hormone levels were dose-dependent (corticosterone ED(50) = 2.43 mg/kg; oxytocin ED(50) = 4.19 mg/kg; and prolactin ED(50) = 4.03 mg/kg). To assess the role of 5-HT(2C) and 5-HT(2A) receptors in mediating the hormone responses to Ro 60-0175, rats were pretreated with the 5-HT(2C) antagonist 6-chloro-5-methyl-1-[2-(2-methylpyridyl-3-oxy)-pyrid-5-yl carbonyl] indoline (SB 242084) or 5-HT(2A) antagonists (+/-)-2,3-dimethoxyphenyl-1-[2-4-(piperidine)-methanol] (MDL 100,907) before injection of Ro 60-0175 (5 mg/kg s.c.). Neither SB 242084 (0.1, 0.5, 1, and 5 mg/kg i.p.) nor MDL 100,907 (1, 5, and 10 microg/kg s.c.) significantly inhibited the Ro 60-0175-induced increases in plasma hormone levels. The data suggest that Ro 60-0175 increases hormone secretion by mechanisms independent of the activation of 5-HT(2C) and/or 5-HT(2A) receptors and suggest that Ro 60-0175 is not a highly selective 5-HT(2C) receptor agonist.
 
Marijuana substitutes often contain blends of multiple psychoactive synthetic cannabinoids (SCBs), including the prevalent SCBs JWH-018 and JWH-073. Because SCBs are frequently used in combinations, we hypothesized that co-administering multiple SCBs induces synergistic drug-drug interactions. Drug-drug interactions between JWH-018 and JWH-073 were investigated in vivo for Δ(9)-tetrahydrocannabinol (Δ(9)-THC)-like discriminative stimulus effects, analgesia, task disruption, and hypothermia by administering combinations (JWH-018: JWH-073) of these drugs to mice in assays of Δ(9)-THC discrimination, tail-immersion, and food-maintained responding, and measuring rectal temperature, respectively. Synergism occurred in the Δ(9)-THC discrimination assay for two constant dose ratio combinations (1:3 and 1:1). A 1:1 and 2:3 dose ratio induced additivity and synergy, respectively, in the tail-immersion assay. Both 1:1 and 2:3 dose ratios were additive for hypothermia, while a 1:3 dose ratio induced sub-additive suppression of food-maintained responding. In vitro drug-drug interactions were assessed using competition receptor binding assays employing mouse brain homogenates and CB1R-mediated inhibition of adenylyl cyclase activity in Neuro2A wild-type (Neuro2AWT) cells. Interestingly, synergy occurred in the competition receptor binding assay for two dose ratios (1:5 and 1:10), but not in the adenylyl cyclase activity assay (1:5). Altogether, these data indicate that drug-drug interactions between JWH-018 and JWH-073 are effect- and ratio-dependent and may increase the relative potency of marijuana substitutes for subjective Δ(9)-THC-like effects. Combinations may improve the therapeutic profile of cannabinoids, considering that analgesia, but not hypothermia or task disruption, was potentiated. Importantly, synergy in the competition receptor binding assay suggests multiple CB1R-SCB binding sites.
 
Discriminative stimulus effects of 9 -THC (left), JWH-018 (center), and JWH-073 (right): antagonism by rimonabant. Abscissae: dose in milligram per kilogram of body weight or vehicle (VEH). Ordinates: mean ( S.E.M.) percentage of responses on the 9 -THC lever (top) and mean ( S.E.M.) response rate expressed as a percentage of control (VEH training days) rate [rate (% control)]. The control dose-response curves for 9 -THC, JWH-018, and JWH-073 are replotted from Fig. 1.  
Schild plots for 9 -THC (top) and JWH-018 (middle) and a log (DR-1) value for JWH-073 (bottom) constructed from the mean data shown in Fig. 2. Abscissae: negative logarithm of the dose in moles per kilogram. Ordinates: mean ( S.E.M.) logarithm of the dose ratio 1. Schild plots were constructed from the unconstrained slopes (dashed lines) and by constraining the slopes to 1 (solid lines).  
Products containing naphthalen-1-yl-(1-pentylindol-3-yl) methanone (JWH-018) and naphthalen-1-yl-(1-butylindol-3-yl) methanone (JWH-073) are emerging drugs of abuse. Here, the behavioral effects of JWH-018 and JWH-073 were examined in one behavioral assay selective for cannabinoid agonism, rhesus monkeys (n = 4) discriminating Δ⁹-tetrahydrocannabinol (Δ⁹-THC; 0.1 mg/kg i.v.), and another assay sensitive to cannabinoid withdrawal, i.e., monkeys (n = 3) discriminating the cannabinoid antagonist rimonabant (1 mg/kg i.v.) during chronic Δ⁹-THC (1 mg/kg s.c. 12 h) treatment. Δ⁹-THC, JWH-018, and JWH-073 increased drug-lever responding in monkeys discriminating Δ⁹-THC; the ED₅₀ values were 0.044, 0.013, and 0.058 mg/kg, respectively and the duration of action was 4, 2, and 1 h, respectively. Rimonabant (0.32-3.2 mg/kg) produced surmountable antagonism of Δ⁹-THC, JWH-018, and JWH-073. Schild analyses and single-dose apparent affinity estimates yielded apparent pA₂/pK(B) values of 6.65, 6.68, and 6.79 in the presence of Δ⁹-THC, JWH-018, and JWH-073, respectively. In Δ⁹-THC-treated monkeys discriminating rimonabant, the training drug increased responding on the rimonabant lever; the ED₅₀ value of rimonabant was 0.20 mg/kg. Δ⁹-THC (1-10 mg/kg), JWH-018 (0.32-3.2 mg/kg), and JWH-073 (3.2-32 mg/kg) dose-dependently attenuated the rimonabant-discriminative stimulus (i.e., withdrawal). These results suggest that Δ⁹-THC, JWH-018, and JWH-073 act through the same receptors to produce Δ⁹-THC-like subjective effects and attenuate Δ⁹-THC withdrawal. The relatively short duration of action of JWH-018 and JWH-073 might lead to more frequent use, which could strengthen habitual use by increasing the frequency of stimulus-outcome pairings. This coupled with the possible greater efficacy of JWH-018 at cannabinoid 1 receptors could be associated with greater dependence liability than Δ⁹-THC.
 
We have reported recently that seizure model and experimental protocol may influence the anticonvulsant tolerance and the withdrawal characteristics of benzodiazepine (BDZ) receptor ligands so that predictions on tolerance and dependence liability of novel drugs should be based on a battery of chronic experiments. In the present study, we evaluated two novel BDZ receptor ligands, i.e., NNC 14-0185 ¿3-(3-cyclopropyl-5-isoxazolyl)-6-fluoro-5-morpholino-imidazo [1,5-a]quinazoline¿ and NNC 14-0189 ¿3-(5-cyclopropyl-1, 2,4-oxadiazol-3-yl)-7-fluoro-5-(4-methyl-1-piperazinyl)-imidazol[1 , 5-a]quinazoline¿, which seem to act as partial agonists at BDZ receptors, in two seizure models by using different experimental approaches to assess the tolerance and dependence liability. In one approach, mice were chronically treated with either NNC 14-0185 or NNC 14-0189 for 4 weeks at doses which were about equipotent to increase the threshold for myoclonic seizures induced by pentylenetetrazole. Anticonvulsant activity was determined several times during the period of chronic treatment as well as up to 2 weeks after termination of treatment in the same group of animals per drug. The threshold for electroshock-induced tonic seizures was used as a second seizure model in separate groups of mice. In another approach, the drug treatment protocols were the same, but the seizures were induced only twice during the 4-week period of treatment to reduce the number of trials which could lead to "learned" tolerance. In additional groups of mice, the seizure thresholds were only determined before and after the period of treatment to assess whether repeated seizure induction during chronic treatment affect the development of dependence. Only moderate tolerance was seen with the two drugs in the pentylenetetrazole seizure threshold experiments and with NNC 14-0185 in the electroshock-induced tonic seizure threshold experiments, whereas NNC 14-0189 did not lose any activity in the latter model during chronic treatment. There was no indication for a significant involvement of learned tolerance during repeated drug testing. With respect to the withdrawal symptoms, i.e., measures of physical dependence-inducing properties of the two drugs, moderate but significant decreases in the seizure threshold were seen in the withdrawal period. Both in terms of tolerance- and dependence-inducing properties and adverse effects seen during chronic treatment in mice, NNC 14-0185 and NNC 14-0189 seem to offer clear advantages compared to the more traditional BDZ receptor ligands.
 
Amiloride improves mucociliary clearance (MC) by blocking airway epithelial sodium channels (ENaC) and expanding airway surface liquid (ASL). However, the low potency and rapid absorption of amiloride by airway epithelia translated into a short duration of efficacy as an aerosolized therapy for cystic fibrosis (CF) patients. To improve ENaC blocker CF pharmacotherapy, a more potent and durable ENaC blocker tailored for aerosol delivery was synthesized. Parion compound N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02) was tested for potency and reversibility of ENaC block, epithelial absorption and biotransformation, selectivity, durability of ASL expansion under isotonic and hypertonic conditions in canine and human CF bronchial epithelial cells, and drug dissociation on ENaC in Xenopus oocytes. Short-circuit current assessed compound potency and reversibility, patch-clamp recordings of ENaC current assessed drug off-rate (k(off)), a gravimetric method and confocal microscopy measured mucosal water retention and ASL height, and drug absorption and biotransformation were assessed using liquid chromatography-mass spectrometry. Amiloride and 552-02 were tested in vivo for MC activity in sheep immediately and 4 to 6 h after aerosol dosing. Compared with amiloride, compound 552-02 was 60 to 100-fold more potent, it was 2 to 5-fold less reversible, it was slower at crossing the epithelium, and it exhibited a 170-fold slower k(off) value. 552-02 exhibited greater ASL expansion over 8 h in vitro, and it was more effective than amiloride at increasing MC immediately and 4 to 6 h after dosing. When combining hypertonic saline and 552-02, a synergistic effect on ASL expansion was measured in canine or CF bronchial epithelia. In summary, the preclinical data support the clinical use of 552-02 +/- hypertonic saline for CF lung disease.
 
The authors studied the pharmacological properties of N-(6-(2-(5-bromopyrimidin-4-yl)-4-(2-hydroxy-1, 1-dimethylethyl)benzensulfonamide sodium salt sesquihydrate (T-0201), a new nonpeptide endothelin (ET) receptor antagonist, in vitro and in vivo. In binding studies, T-0201 competitively antagonized the specific binding of [125I]-ET-1 to human cloned ETA receptors (the Ki value was 0.015 +/- 0.004 nM). T-0201 weakly inhibited [125I]-ET-1-binding to human cloned ETB receptors; the Ki value was 41 +/- 21 nM. T-0201 shifted the concentration-response curve of ET-1-induced contraction of the isolated rat aorta (ETA receptors) to the right (pA2 = 9.0 +/- 0.2). In the isolated rat trachea, a selective ETB agonist sarafotoxin S6c-induced contraction was inhibited by T-0201 (pA2 = 6.8 +/- 0.3). T-0201 also caused the inhibition of ET-1-induced contraction of the isolated rabbit pulmonary artery (pA2 = 5.7 +/- 0.3). In anesthetized rats, T-0201 (0.01-1 mg/kg) inhibited the pressor response to exogenous big ET-1 (1 nmol/kg i.v.), after both i.v. and p.o. administration, in a dose-dependent manner. The significant inhibitory effect of orally administered T-0201 on big ET-1-induced pressor response lasted for 4 hr at 0.1 mg/kg and for 8 hr at 1 mg/kg. Thus the present study demonstrates that T-0201 is a highly potent, long-lasting, orally active and selective ETA receptor antagonist.
 
The prostaglandin (PG) EP(4) receptor subtype is expressed by peripheral sensory neurons. Although a potential role of EP(4) receptor in pain has been suggested, a limited number of selective ligands have made it difficult to explore the physiological functions of EP(4) or its potential as a new analgesic target. Here, we describe the in vitro and in vivo pharmacology of a novel EP(4) receptor antagonist, N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo [4,5-c] pyridin-1-yl) phenyl]ethyl}amino) carbonyl]-4-methylbenzenesulfonamide (CJ-023,423). In vitro, CJ-023,423 inhibits [(3)H]PGE(2) binding to both human and rat EP(4) receptors with K(i) of 13 +/- 4 and 20 +/- 1 nM, respectively. CJ-023,423 is highly selective for the human EP(4) receptor over other human prostanoid receptor subtypes. It also inhibits PGE(2)-evoked elevation in intracellular cAMP at the human and rat EP(4) receptors with pA(2) of 8.3 +/- 0.03 and 8.2 +/- 0.2 nM, respectively. In vivo, oral administration of CJ-023,423 significantly reduces thermal hyperalgesia induced by intraplantar injection of PGE(2) (ED(50) = 12.8 mg/kg). CJ-023,423 is also effective in models of acute and chronic inflammatory pain. CJ-023,423 significantly reduces mechanical hyperalgesia in the carrageenan model. Furthermore, CJ-023,423 significantly reverses complete Freund's adjuvant-induced chronic inflammatory pain response. Taken together, the present data indicate that CJ-023,423, a highly potent and selective antagonist of both human and rat EP(4) receptors, produces antihyperalgesic effects in animal models of inflammatory pain. Thus, specific blockade of the EP(4) receptor signaling may represent a novel therapeutic approach for the treatment of inflammatory pain.
 
Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-me thylpyridine)] was evaluated in vivo in rat brain for effects on neurotransmitter turnover and inhibition of ex vivo binding of muscarinic radioligands. Xanomeline produced dose-related increases in the metabolite of dopamine, dihydroxyphenylacetic acid (DOPAC), in striatum. The increases in striatal DOPAC levels produced by xanomeline were antagonized by the relatively selective M1 antagonist trihexyphenidyl, suggesting that xanomeline interacts with M1 heteroreceptors on dopamine nerve terminals. Xanomeline produced small increases in striatal acetylcholine levels and did not antagonize the large increases in acetylcholine produced by the nonselective muscarinic agonist oxotremorine, indicating that xanomeline did not block M2 autoreceptors. Xanomeline inhibited ex vivo binding of muscarinic radioligands to homogenates of brain and the inhibition of ex vivo binding occurred in the same dose range as increases in DOPAC levels. Xanomeline did not appreciably induce salivation or antagonize oxotremorine-induced salivation indicating that xanomeline does not interact with M3 receptors. The effects of xanomeline on ex vivo binding and DOPAC levels lasted for about 3 hr and were evident after oral administration. An analog of xanomeline with similar in vivo effects did not inhibit acetylcholinesterase or choline acetyltransferase and inhibited choline uptake only at concentrations much higher than those required to inhibit binding. These data indicate xanomeline is selective agonist for M1 over M2 and M3 receptors in vivo in rat. It is not known whether xanomeline interacts with m4 or m5 receptors in vivo.
 
The effects of PF-02367982 on progesterone-induced endometrial differentiation. A and B, chronic treatment with progesterone alone resulted in differentiation and thickening of the endometrial epithelial layer (Epi), resulting in tortuous arborization and thickening of the endometrium, compaction of the lamina propria layer (LP), and a dense arteriole plexus within the extracellular matrix (arrows). C and D, inhibition of progesterone-induced endometrial arborization by the nonsteroidal PR antagonist PF-02367982 (3 mg/kg q.d.), resulting in a distinct endometrial lumen and undifferentiated epithelium.
PR antagonists PF-02367982 and RU-486 in the rabbit McPhail test. Chronic dosing of either PF-02367982 [3, 1, 0.1 mg/kg q.d., or 2 mg/kg/day (1 mg/kg b.i.d.)] or RU-486 (3, 1, 0.3 mg/kg q.d.) with progesterone treatment induced a significant reduction in endometrial differentiation compared with progesterone (P 4 ) treatment alone. The assessment of inhibitory effects was performed according to the McPhail classification system (0 no progestagenic activity to 4 maximal progestagenic activity) (n 4 animals per group, mean S.E.M., representative data of n 2 experiments; , p 0.01 compared with P 4 alone group by ANOVA).
Results of PK/PD analysis of McPhail test data using a categorical logistic regression method. McPhail test scores were converted to categorical binary scores of 1 or 0 according to antiprogestagenic efficacy (McPhail score 2 or 2, respectively), regardless of dose, and plotted with respect to unbound drug treatment exposure for the 96 h of drug dosing (AUC 0 –96 h ). Units on x-axis are in nmol h/l. The values of fractional free drug used to calculate daily unbound AUC 0 –24 for PF-02367982 and RU-486 were 0.254 and 0.03, respectively. The PK/PD model was used to calculate the required exposure to elicit such antiprogestagenic activity in the endometrium in the presence of mid-luteal phase levels of progesterone.  
The effects of PF-02367982 on endometrial differentiation in the cynomolgus macaque. Histological staining of endometrium from untreated (A), PF-02367982-treated (B), or RU-486-treated (C) cynomolgus monkeys at 22 days after first dose (hematoxylin and eosin stain). D, change in histological progestagenic score (McPhail score) of endometrial biopsy samples taken from cynomolgus macaques at pretreatment and day 22 after treatment (4 progestagenic, 0 estrogenic). Data represent the reduction in progestagenic score in response to treatment between days 22 and 0 (n 5 animals per treatment group for vehicle and PF-02367982; n 7 for RU-486 treatment; scale bar represents 100 m; , p 0.05 compared with vehicle control by ANOVA).  
Pharmacokinetic analysis of PF-02367982 in the female cynomolgus monkey. Doses were selected on the basis of the rabbit PK/PD model output on required daily exposure needed to elicit an antiprogestagenic response on the endometrium.  
The progesterone receptor (PR) is an important regulator of endometrial function. Blockade of PR function has been recognized as the potential basis for preventing gynecological conditions such as endometriosis and uterine fibroids. In this study, we examine the in vitro and in vivo properties of a nonsteroidal PR antagonist, 2-[4-(4-cyano-phenoxy)-3,5-dicyclopropyl-1H-pyrazol-1-yl]-N-methylacetamide (PF-02367982) in comparison with the nonselective steroidal antagonist RU-486 (mifepristone). PF-02367982 was found to be a potent PR antagonist with far greater selectivity over the glucocorticoid receptor than RU-486. Both PF-02367982 and RU-486 blocked progesterone-induced arborization of the rabbit endometrium in a dose-dependent manner at unbound drug exposures that were commensurate with their potencies as PR antagonists in vitro. Translation of this pharmacology to a clinically relevant system was required to bridge the pharmacological gap between nonmenstruating rabbits and humans. Thus, the pharmacokinetic (PK) and pharmacodynamic (PD) data from the rabbit were combined to predict pharmacological effects on the naturally cycling cynomolgus macaque endometrium. PF-02367982 blocked the effect of progesterone on the cynomolgus macaque endometrium to the same degree as RU-486 and at exposures predicted by the rabbit PK-PD model. With such an efficacious and superior selectivity profile to the nonselective RU-486, PF-02367982 may have significant therapeutic value in the treatment of gynecological conditions such as endometriosis.
 
There is considerable ongoing investment in the research and development of selective progesterone receptor (PR) modulators for the treatment of gynecological conditions such as endometriosis. Here, we provide the first report on the clinical evaluation of a nonsteroidal progesterone receptor antagonist 4-[3-cyclopropyl-1-(mesylmethyl)-5-methyl-1H-pyrazol-4-yl]oxy,-2,6-dimethylbenzonitrile (PF-02413873) in healthy female subjects. In in vitro assays, PF-02413873 behaved as a selective and fully competitive PR antagonist, blocking progesterone binding and PR nuclear translocation. The pharmacological mode of action of PF-02413873 seems to differ from the founding member of the class of steroidal PR antagonists, 11β-4-dimethylaminophenyl-17β-hydroxy-17α-propinyl-4,9-estradiene-3-one (RU-486; mifepristone). Exposure-effect data from studies in the cynomolgus macaque, however, demonstrated that PF-02413873 reduced endometrial functionalis thickness to a comparable degree to RU-486 and this effect was accompanied by a decrease in proliferation rate (as measured by bromodeoxyuridine incorporation) for both RU-486 and high-dose PF-02413873. These data were used to underwrite a clinical assessment of PF-02413873 in a randomized, double-blinded, third-party open, placebo-controlled, dose-escalation study in healthy female volunteers with dosing for 14 days. PF-02413873 blocked the follicular phase increase in endometrial thickness, the midcycle lutenizing hormone surge, and elevation in estradiol in a dose-dependent fashion compared with placebo. This is the first report of translational efficacy data with a nonsteroidal PR antagonist in cynomolgus macaque and human subjects.
 
The antithrombotic and bleeding time (BT)-prolonging effects of TAK-029, a novel glycoprotein IIb/IIIa antagonist, were characterized and compared with those of conventional antithrombotic agents in guinea pigs. TAK-029 potently inhibited the binding of fibrinogen and von Willebrand factor to purified human GPIIb/IIIa with IC50 values of 0.67 +/- 0.03 and 0.33 +/- 0.04 nM; it also inhibited human platelet aggregation induced by various aggregating agents with IC50 values of 29 to 38 nM. The in vitro antiplatelet effect of TAK-029 was potent in humans, guinea pigs and monkeys. When TAK-029 was given p.o. to guinea pigs, severe prolonging of BT (>1800 sec) was not observed with plasma concentrations of TAK-029 that inhibited ex vivo platelet aggregation by < 100%. The p.o. administration of TAK-029, ticlopidine and clopidogrel prolonged BT to the same extent, in parallel with their inhibition of ex vivo platelet aggregation. TAK-029 inhibited ex vivo platelet adhesion and thrombus formation in an arteriovenous shunt model more strongly than ticlopidine, clopidogrel and aspirin at doses causing similar prolongations of BT. In a balloon catheter-induced carotid thrombosis model, i.v. administration of TAK-029 significantly inhibited thrombus formation without prolonging BT. At doses that caused an incomplete antithrombotic effect, PGE1-alpha-cyclodextrin and argatroban produced hypotension and prolongation of BT, respectively. TAK-029 may be effective in patients suffering from arterial thrombotic diseases, which are refractory to these conventional antithrombotic agents.
 
5-Hydroxytryptamine (5-HT)(4) receptor agonists reportedly stimulate brain acetylcholine (ACh) release, a property that might provide a new pharmacological approach for treating cognitive deficits associated with Alzheimer's disease. The purpose of this study was to compare the binding affinities, functional activities, and effects on neuropharmacological responses associated with cognition of two highly selective 5-HT(4) receptor agonists, prucalopride and 6,7-dihydro-4-hydroxy-7-isopropyl-6-oxo-N-[3-(piperidin-1-yl)propyl]thieno[2,3-b]pyridine-5-carboxamide (PRX-03140). In vitro, prucalopride and PRX-03140 bound to native rat brain 5-HT(4) receptors with K(i) values of 30 nM and 110 nM, respectively, and increased cAMP production in human embryonic kidney-293 cells expressing recombinant rat 5-HT(4) receptors. In vivo receptor occupancy studies established that prucalopride and PRX-03140 were able to penetrate the brain and bound to 5-HT(4) receptors in rat brain, achieving 50% receptor occupancy at free brain exposures of 330 nM and 130 nM, respectively. Rat microdialysis studies revealed that prucalopride maximally increased ACh and histamine levels in the prefrontal cortex at 5 and 10 mg/kg, whereas PRX-03140 significantly increased cortical histamine levels at 50 mg/kg, failing to affect ACh release at doses lower than 150 mg/kg. In combination studies, donepezil-induced increases in cortical ACh levels were potentiated by prucalopride and PRX-03140. Electrophysiological studies in rats demonstrated that both compounds increased the power of brainstem-stimulated hippocampal θ oscillations at 5.6 mg/kg. These findings show for the first time that the 5-HT(4) receptor agonists prucalopride and PRX-03140 can increase cortical ACh and histamine levels, augment donepezil-induced ACh increases, and increase stimulated-hippocampal θ power, all neuropharmacological parameters consistent with potential positive effects on cognitive processes.
 
ED 50 and E max values of WIN 55,212-2 in stimulating [ 35 S]GTPγS binding in the presence or 
The compound N-piperidinyl-[8-chloro-1-(2,4-dichlorophenyl)-1,4,5,6-tetrahydrobenzo [6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide] (NESS 0327) was synthesized and evaluated for binding affinity toward cannabinoid CB1 and CB2 receptor. NESS 0327 exhibited a stronger selectivity for CB1 receptor compared with N-piperidinyl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A), showing a much higher affinity for CB1 receptor (Ki = 350 +/- 5 fM and 1.8 +/- 0.075 nM, respectively) and a higher affinity for the CB2 receptor (Ki = 21 +/- 0.5 nM and 514 +/- 30 nM, respectively). Affinity ratios demonstrated that NESS 0327 was more than 60,000-fold selective for the CB1 receptor, whereas SR 141716A only 285-fold. NESS 0327 alone did not produce concentration-dependent stimulation of guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding in rat cerebella membranes. Conversely, NESS 0327 antagonized [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (WIN 55,212-2)-stimulated [35S]GTPgammaS binding. In functional assay, NESS 0327 antagonized the inhibitory effects of WIN 55,212-2 on electrically evoked contractions in mouse isolated vas deferens preparations with pA2 value of 12.46 +/- 0.23. In vivo studies indicated that NESS 0327 antagonized the antinociceptive effect produced by WIN 55,212-2 (2 mg/kg s.c.) in both tail-flick (ID50 = 0.042 +/- 0.01 mg/kg i.p.) and hot-plate test (ID50 = 0.018 +/- 0.006 mg/kg i.p.). These results indicated that NESS 0327 is a novel cannabinoid antagonist with high selectivity for the cannabinoid CB1 receptor.
 
5-Hydroxytryptamine (5-HT) receptors and dopamine(2) (D(2)) receptor modulate gastrointestinal motility. Gastroprokinetic agents that act on several 5-HT receptor subtypes and/or D(2) receptors are used clinically. Although the 5-HT(4) receptor is known to mediate the gastroprokinetic effects of these agents, the absence of highly selective 5-HT(4) receptor agonists has made it difficult to confirm the physiological consequences of selective 5-HT(4) receptor stimulation. In this study, we report the in vitro pharmacological profiles and the in vivo gastroprokinetic effects of 5-amino-6-chloro-N-[(1-isobutylpiperidin-4-yl)methyl]-2-methylimidazo[1,2-alpha]pyridine-8-carboxamide (CJ-033,466), a novel, potent, and selective 5-HT(4) partial agonist. Compared with preceding 5-HT(4) agonists such as cisapride, mosapride, and tegaserod, CJ-033,466 had a superior in vitro profile, with nanomolar agonistic activities for the 5-HT(4) receptor and 1000-fold greater selectivity for the 5-HT(4) receptor over other 5-HT and D(2) receptors. In vivo studies in conscious dogs showed that CJ-033,466 dose-dependently stimulated gastric antral motility in both the fasted and postprandial states at the same dose range and that it was 30 times more potent than cisapride. Furthermore, CJ-033,466 accelerated the gastric emptying rate in a gastroparesis dog model at the minimally effective dose established in the gastric motility study. In conclusion, CJ-033,466 is a potent and highly selective 5-HT(4) agonist that stimulates physiologically coordinated gastric motility, and it has no activity on other 5-HT receptor subtypes and D(2) receptors. Therefore, CJ-033,466 could be used to treat gastroparesis, providing better gastroprokinetics and reduced side effects mediated by the other receptors.
 
Chemical structures of ACAT inhibitors, Sah 58-035, 447C88, and TMP-153; estrogen receptor ligands, ICI 164,384, E 2 , and tamoxifen; and a non-antiestrogen tamoxifen derivative that binds selectively to the microsomal AEBS, PBPE. In these molecules, the chemical groups that may be superimposable with the diphenyl ethane part of Sah 58-035 are in boldface. A, Sah 58-035 is very similar to the pure estrogen antagonist ICI 164,384. Three-dimensional structures of the calculated minimal energy conformation of Sah 58-035 and the conformation of ICI 164,384 taken in the crystallographic structure of rat ER-ICI 164,384 are shown. Calculated minimal energy was carried out using the Discover module of Insight II (version 2000) as described under Materials and Methods. Overlay of Sah 58-035 and ICI 164,384 as well as van der Waals volume calculations and intersection measurements were done using the Search-Compare module. The van der Waals volume intersection is depicted as the gray grid and illustrates the structural similarities between Sah 58-035 and ICI 164,384. The van der Waals volume of Sah 58-035 and of ICI 164,384 are 425.03 and 468.77 Å 3 , respectively. Seventy percent of the van der Waals volume of Sah 58-035 is in common with that of ICI 164,384.
Effect of the ACAT inhibitor Sah 58-035 on PR expression in MCF-7 cells. Cells were treated for 48 h with solvent vehicle, 10 nM E 2 , or 1 and 10 ␮ M Sah 58-035. Analyses were performed as described under 
Effect of Sah 58-035 on estrogen-regulated growth. A, MCF-7 cells were treated with 10 nM E 2 and 5 ␮ M Sah 58-035, with or without 100 nM ICI 164,384 (ICI) or with the solvent vehicle. Cell density was deter- 
A, chemical structures of pure antiestrogens, ICI 164,384, ICI 182,780, EM-319 (Jordan, 2003b), and ZK-164,015 (Biberger and von Angerer, 1996). The chemical groups involved in pure antagonist activity are highlighted in gray. B, agonists and partial agonists 2,3-bis (4-hydroxyphe- nyl)pentyl 3-( N , N -dimethylaminopropylsulfide) (Zablocki et al., 1987), Sah 58-035, 11-(3,17 ␤ -hydroxy-estra-1,3,5(10)-triene-7-yl)-undecanoic acid (Wakeling and Bowler, 1988), and ICI 160,325 (Wakeling and Bowler, 1988). 
of Sah 58-035, compound 447C88, and TMP-153 on ER, ER, and AEBS Binding of radiolabeled E 2 on the ER and ER, or tritiated tamoxifen on the AEBS were measured at different concentrations of E 2 , tamoxifen, PBPE, Sah 58-035, compound 447C88, and TMP-153 as described under Materials and Methods. Values are the mean S.E.M. from three independent experiments.
We have shown recently that estrogen receptor (ER) ligands share a diphenyl ethane pharmacophore with Sah 58-035 [3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]-propanamide], a prototypical inhibitor of the acyl-cholesterolacyl-transferase (ACAT), which enabled us to establish that ER ligands were potent inhibitors of ACAT and blocked the formation of foam cells. In the present study, we have tested whether this structural similarity means that Sah 58-035 is an ER modulator. We report that Sah 58-035 bound to ERalpha and ERbeta with an IC(50) of 2.9 and 3.1 microM, respectively. Docking studies using molecular modeling of Sah 58-035 with the X-ray structure of the ER showed that Sah 58-035 fits well into the ligand binding site known for 4-hydroxy-tamoxifen. Despite having high three-dimensional structural similarities with the pure antiestrogen ICI 164,384 [(N-n-butyl-N-methyl-11-[3,17beta-di-hydroxyestra-1,3, 5(10)-trien-7alpha-yl]-undecanamide], we showed that Sah 58-035 is an agonist of ER for transcription and cellular proliferation. These data showed that Sah 58-035 was an estrogen receptor agonist and that the size and the chemical nature of the side chain were critical for agonist versus antagonist activity on ER. This new molecular mechanism of action for Sah 58-035 has to be taken into account in understanding better its pharmacological activities. Moreover, these data give new structural insights into the understanding of agonist versus antagonist activities of ER ligands and also for the conception of new drugs with a dual ACAT inhibition and ER modulation potential and their evaluation in different pathologies where both targets are involved, such as atherosclerosis, Alzheimer's disease, and cancer.
 
The cannabinoid-1 receptor (CB1R) has been implicated in the control of energy balance. To explore the pharmacological utility of CB1R inhibition for the treatment of obesity, we evaluated the efficacy of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-[[5-(trifluoromethyl)pyridin-2-yl]oxy]propanamide (MK-0364) and determined the relationship between efficacy and brain CB1R occupancy in rodents. MK-0364 was shown to be a highly potent CB1R inverse agonist that inhibited the binding and functional activity of various agonists with a binding K(i) of 0.13 nM for the human CB1R in vitro. MK-0364 dose-dependently inhibited food intake and weight gain, with an acute minimum effective dose of 1 mg/kg in diet-induced obese (DIO) rats. CB1R mechanism-based effect was demonstrated for MK-0364 by its lack of efficacy in CB1R-deficient mice. Chronic treatment of DIO rats with MK-0364 dose-dependently led to significant weight loss with a minimum effective dose of 0.3 mg/kg (p.o.), or a plasma C(max) of 87 nM. Weight loss was accompanied by the loss of fat mass. Partial occupancy (30-40%) of brain CB1R by MK-0364 was sufficient to reduce body weight. The magnitude of weight loss was correlated with brain CB1R occupancy. The partial receptor occupancy requirement for efficacy was also consistent with the reduced food intake of the heterozygous mice carrying one disrupted allele of CB1R gene compared with the wild-type mice. These studies demonstrated that MK-0364 is a highly potent and selective CB1R inverse agonist and that it is orally active in rodent models of obesity.
 
Inhibition of H(+),K(+)-ATPase is accepted as the most effective way of controlling gastric acid secretion. However, current acid suppressant therapy for gastroesophageal reflux disease, using histamine H(2) receptor antagonists and proton pump inhibitors, does not fully meet the needs of all patients because of their mechanism of action. This study sought to characterize the in vitro and in vivo pharmacology of a novel acid pump antagonist, N-(2-Hydroxyethyl)-N,2-dimethyl-8-{[(4R)-5-methyl-3,4-dihydro-2H-chromen-4-yl]amino}imidazo[1,2-a]pyridine-6-carboxamide (PF-03716556), and to compare it with other acid suppressants. Porcine, canine, and human recombinant gastric H(+),K(+)-ATPase activities were measured by ion-leaky and ion-tight assay. The affinities for a range of receptors, ion channels, and enzymes were determined to analyze selectivity profile. Acid secretion in Ghosh-Schild rats and Heidenhain pouch dogs were measured by titrating perfusate and gastric juice samples. PF-03716556 demonstrated 3-fold greater inhibitory activity than 5,6-dimethyl-2-(4-fluorophenylamino)-4-(1-methyl-1,2,3,4-tetrahydroisoquinoline-2-yl)pyrimidine (revaprazan), the only acid pump antagonist that has been available on the market, in ion-tight assay. The compound did not display any species differences, exhibiting highly selective profile including the canine kidney Na(+),K(+)-ATPase. Kinetics experiments revealed that PF-03716556 has a competitive and reversible mode of action. More rapid onset of action than 5-methoxy-2-{[(4-methoxy-3,5-dimethyl-2-pyridyl)methyl]-sulfinyl}-benzimidazole (omeprazole) and 3-fold greater potency than revaprazan were observed in Ghosh-Schild rats and Heidenhain pouch dogs. PF-03716556, a novel acid pump antagonist, could improve upon or even replace current pharmacological treatment for gastroesophageal reflux disease.
 
Observations were made on respiration, alveolar concentration of CO2, arterial blood pressure and heart rate in barbiturate-anesthetized cats subjected routinely to section of the vagus and carotid sinus nerves and maintained with pure O2 for inhalation. Bolus i.v. injection of angiotensin II (AII), 0.6 to 6.0 micrograms/kg, evoked a prompt shortlasting suppression of breathing, manifested mainly as a reduction in tidal volume susceptible to the development of tachyphylaxis if injections were spaced more closely than 30 min apart. Ablation of the area postrema failed to eliminate the respiratory effect of All. The response also persisted after midbrain transection, spinal cord transection at C8, dorsal rhizotomy of spinal segments C1 to C8 and interruption of cranial nerves V, VII, VIII, IX, X, XI and XII. No influence of All was evident on indirectly evoked tibialis muscle contractions and on pulmonary compliance. A reduction in phrenic nerve impulse traffic coincided with the respiratory response to All. Cranial i.a. injection of All initiated the respiratory effect in its first pass through the brain, before any change occurred in the blood pressure. The degree of All-induced respiratory inhibition was relatively constant at different magnitudes of breathing produced by CO2 inhalation or by electrical stimulation of the respiratory center in the medulla oblongata. It is concluded that All given by bolus intravascular injection causes respiratory inhibition through a direct central action at the final processing step in the CO2-tidal volume controller.
 
Cocaine and heroin often are abused together in a combination known as a "speedball," but relatively little is known about ways in which cocaine and heroin may interact to modify each other's abuse-related effects. The present study evaluated the discriminative stimulus effects of a speedball combination of cocaine and heroin. Three rhesus monkeys were trained to discriminate vehicle from a 10:1 ratio of cocaine (0.4 mg/kg) in combination with heroin (0.04 mg/kg). Both cocaine alone and heroin alone substituted completely for the cocaine/heroin combination, although cocaine and heroin were more potent when administered together than when administered alone. Combined pretreatment with the dopamine antagonist flupenthixol and the opioid antagonist quadazocine dose-dependently antagonized the discriminative stimulus effects of the cocaine/heroin combination, but pretreatment with either antagonist alone was less effective. These findings suggest that either cocaine or heroin alone was sufficient to substitute for the cocaine/heroin training combination. To characterize the discriminative stimulus properties of this speedball more fully, a series of cocaine-like and heroin-like agonists were studied in substitution tests. The indirect dopamine agonists CFT, amphetamine and bupropion and the mu opioid agonists alfentanil, fentanyl and morphine produced high levels of speedball-appropriate responding. However, the indirect dopamine agonist GBR12909, the D1 dopamine agonist SKF82958, the D2 dopamine agonist quinpirole and the partial mu opioid agonist nalbuphine did not substitute for the cocaine/heroin combination. Because these compounds produce discriminative stimulus effects similar to either cocaine or mu opioid agonists alone, these findings suggest that the discriminative stimulus effects of the cocaine/heroin combination do not overlap completely with the effects of cocaine and heroin alone. Finally, a series of compounds that produce partial or no substitution for cocaine or mu agonists alone also did not substitute for the cocaine/heroin combination, which indicates that the discriminative stimulus effects of the combination were pharmacologically selective. Taken together, these findings suggest that a combination of cocaine and heroin produces a pharmacologically selective discriminative stimulus complex that includes aspects of both component drugs.
 
Low, nonreinforcing doses of heroin have been shown to shift the dose- response function of cocaine leftward in rhesus monkeys trained under a progressive-ratio schedule of i.v. drug injection. Our study sought to determine 1) whether a reciprocal enhancement of heroin self-administration would be observed when heroin was combined with low, nonreinforcing doses of cocaine, and 2) whether self-administration of cocaine-heroin combinations could be antagonized by the opioid antagonist naltrexone. Rhesus monkeys (n = 4) were prepared with i.v. catheters and trained to serf-administer cocaine under a progressive-ratio schedule. The initial response requirement of this schedule was fixed-ratio 120, which doubled across the session to a maximum of 1920. Injections were separated by a 30-min time out. Cocaine dose- response functions (6.4-100 μg/kg/injection) for injections/session and breakpoints were monophasic, i.e., increased with dose until responding reached a maximum. Heroin dose-response functions (1.6-25 μg/kg/ injection) either increased to a peak and then decreased or reached an asymptote. When nonreinforcing doses of cocaine (3.2-25 μg/kg/injection) were combined with heroin, the heroin dose-response function was shifted to the left, without change in maximum injections/session. Presession treatments with naltrexone (3.2-1600 μg/kg, i.m., 10-min presession) antagonized self-administration of heroin and heroin + cocaine combinations in a dose-dependent fashion. However, naltrexone treatment had no effect on cocaine self-administration. Antagonism by naltrexone of self-administration of heroin and heroin + cocaine was surmounted by increasing the dose of heroin either alone or in the heroin + cocaine combination. In vivo apparent pA2 and pK(B) analyses of these data revealed values of approximately 8.0, consistent with a role for mu opioid receptors in the self-administration of heroin and cocaine-heroin (i.e., 'speedball') combinations.
 
We tested a novel, orally active inhibitor of plasminogen activator inhibitor-1 (PAI-1) in a canine model of electrolytic injury. Dogs received by oral gavage either vehicle (control) or the PAI-1 inhibitor PAI-039 [{1-benzyl-5-[4-(trifluoromethoxy)phenyl]-1H-indol-3-yl}(oxo)acetic acid] (1, 3, and 10 mg/kg) and were subjected to electrolytic injury of the coronary artery. PAI-039 caused prolongation in time to coronary occlusion (control, 31.7 +/- 6.3 min; 3 mg/kg PAI-039, 66.0 +/- 6.4 min; 10 mg/kg, 56.7 +/- 7.4 min; n = 5-6; p < 0.05) and a reduced thrombus weight (control, 7.6 +/- 1.5 mg; 10 mg/kg PAI-039, 3.6 +/- 1.0 mg; p < 0.05). Although occlusive thrombosis was observed across all groups based upon the absence of measurable blood flow, a high incidence (>60%) of spontaneous reperfusion occurred only in those groups receiving PAI-039. Spontaneous reperfusion in the 10 mg/kg PAI-039 group accounted for total blood flow (area under the curve of coronary blood flow) of 99.6 +/- 11.7 ml after initial thrombotic occlusion (p < 0.05 compared with control). Plasma PAI-1 activity was reduced in all drug-treated groups (percentage of reduction in activity p < 0.05; 10 mg/kg PAI-039), whereas ADP-, 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha) (U46619)-, and collagen-induced platelet aggregation, as well as template bleeding and prothrombin time, remained unaffected by PAI-039. Ex vivo clot lysis analysis revealed normal clot formation but accelerated clot lysis in PAI-039-treated groups. The pharmacokinetic profile of PAI-039 indicated an oral bioavailability of 43 +/- 15.3% and a plasma half-life of 6.2 +/- 1.3 h. In conclusion, PAI-039 is an orally active prothrombolytic drug that inhibits PAI-1 and accelerates fibrinolysis while maintaining normal coagulation in a model of coronary occlusion.
 
Mibefradil is a Ca2+ channel antagonist that inhibits both T-type and high-voltage-activated Ca2+ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (1S, 2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride (NNC 55-0396), that exerts a selective inhibitory effect on T-type channels. The acute IC(50) of NNC 55-0396 to block recombinant alpha(1)G T-type channels in human embryonic kidney 293 cells was approximately 7 microM, whereas 100 microM NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca2+ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca2+ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca2+ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channels, thus rendering it more selective to T-type Ca2+ channels.
 
The ability of a peptidomimetic (SC-67655) to block the peptide binding site of the rheumatoid arthritis-linked human leukocyte antigen encoded by the DRB1*0401 allele was evaluated. The inhibitor bound to purified DRB1*0401 molecules with an affinity similar to that of the well-characterized peptide ligand HA307-319. Cell binding assays demonstrated that, in contrast to the promiscuous HA307-319 peptide, the peptidomimetic was highly specific for DRB1*0401. The inhibitor also blocked functional T cell responses to peptide antigens but did not block T cell proliferation in response to protein antigens. Furthermore, it did not appear to be taken up by cells. An analog of the peptidomimetic that was conjugated to a signal peptide sequence did inhibit a T cell proliferative response to protein antigen. Thus, the peptidomimetic must be taken up by cells to block the presentation of peptides derived from protein antigens. These findings have implications for the rational development of inhibitors that block the class II peptide binding groove for the treatment of autoimmune diseases.
 
Several lines of circumstantial evidence support the assumption that protein kinase C (PKC) activation together with elevated levels of cytosolic Ca++ are necessary for T-cell activation and proliferation in response to a physiological stimulus, i.e., MHC class II restricted antigen presentation. By using a potent, cell-permeable and selective inhibitor of PKC, Ro 32-0432, we have tested this hypothesis. Ro 32-0432 inhibits interleukin-2 (IL-2) secretion, IL-2 receptor expression in, and proliferation of, peripheral human T-cells stimulated with phorbol ester together with phytohemagglutin or anti-CD3, but does not inhibit IL-2 induced proliferation in cells already stimulated to express IL-2 receptors. Proliferation of the influenza peptide antigen HA 307-319-specific human T-cell clone (HA27) after exposure to antigen-pulsed autologous presenting cells was also inhibited by Ro 32-0432. Oral administration of Ro 32-0432 inhibited subsequent phorbol ester-induced edema in rats demonstrating the systemic efficacy of the compound to inhibit PKC-driven responses. Induction of more physiologically T-cell driven responses such as host vs. graft responses and the secondary paw swelling in adjuvant-induced arthritis were also inhibited by Ro 32-0432. These data demonstrate the crucial role for PKC in T-cell activation and that selective p.o. bioavailable PKC inhibitors are efficacious in preventing T-cell driven chronic inflammatory responses in vivo. Inhibition of PKC represents an important mechanistic approach to prevent T-cell activation and compounds of this class may have important therapeutic applicability to chronic inflammatory and autoimmune diseases.
 
N-0437 [2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin], a potent and selective agonist for D-2 dopamine receptors, was used to investigate inhibitory prejunctional dopamine receptors in the rat tail artery and rabbit ear artery. N-0437 inhibited contractile responses to transmural nerve stimulation in a frequency dependent manner. Thus, N-0437 profoundly inhibited responses to nerve stimulation in the rat tail artery at a frequency of 1 Hz (ED50 = 1.6 nM), but had minimal effects when nerves were stimulated at 6 Hz. The D-1/D-2 dopamine agonist, apomorphine, exhibited a similar frequency dependent inhibitory effect but with less potency (ED50 = 30 nM at 1 Hz). In concentrations up to 1 microM, N-0437 had no effect on responses to exogenously applied norepinephrine, but N-0437 inhibited [3H] norepinephrine efflux induced by transmural stimulation. Inhibitory effects of N-0437 were blocked by the D-2 antagonist sulpiride but not by the D-1 antagonist SCH 23390. Furthermore, the selective D-1 agonist SKF 38393 did not inhibit vascular responses to adrenergic nerve stimulation. These data indicate that the inhibitory effects of N-0437 are via activation of D-2 dopamine receptors that inhibit norepinephrine release. Thus, N-0437 shows potency and selectivity of action for prejunctional D-2 dopamine receptors in vascular tissues. The frequency dependence of the actions of N-0437 suggest that the level of sympathetic activity is an important variable in determining effectiveness of prejunctional modulation.
 
Receptor binding and antagonist properties of an endothelin (ET) receptor antagonist, TAK-044 ¿cyclo[D-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl) carbonyl]-L-alanyl-L-alpha-aspartylD-2-(2-thienyl) glycyl-L-leucyl-D-tryptophyl]disodium salt¿, were investigated using recombinant human ETA and ETB receptors expressed in Chinese hamster ovary cells. The membranous ETA receptor was shown to be heterogeneous in ET-3 binding affinity (Hill coefficient = 0.54, Kd1 = 390 pM and Kd2 = 8.1 nM). This heterogeneity disappeared upon the addition of guanosine-5'-O-3-thiotriphosphate (Hill coefficient = 0.95, Kd = 7.8 nM). The Kd (from a computer program LIGAND analysis) and Ki (from Dixon plot analysis) values of TAK-044 were 95 and 120 pM for the membranous ETA receptor and 41 and 60 nM for the ETB receptor, respectively. The Kd values of TAK-044 for the ETA receptor was comparable to that of ET-1. The Ki values of TAK-044 for the cellular ETA and ETB receptors were 130 pM and 130 nM at 5,000 cells/well and 1.3 and 590 nM at 50,000 cells/well, respectively. Dixon plot analysis indicated that TAK-044 is a competitive inhibitor of ET-1 binding. TAK-044 inhibited ET-1-induced phosphatidylinositol hydrolysis and arachidonic acid release at 50,000 cells/well in a competitive manner with respective pA2 values of 8.5 and 8.7 in the ETA-expressing cells and 7.4 and 6.6 in the ETB-expressing cells. TAK-044 suppressed ET-1-induced transient increase in intracellular Ca+2 concentration in the ETA- and ETB-expressing cells with respective IC50 values of 2.8 and 230 nM. TAK-044 is a potent and competitive ETA receptor antagonist which simultaneously exhibits definite antagonist activity at the ETB receptor.
 
The present study describes the pharmacological profile of an endothelin (ET) receptor antagonist, TAK-044, in anesthetized rats. TAK-044 given 10 min before administration of ET-1 (0.3 nmol/kg i.v.) partially inhibited the ET-1-induced pressor response at 0.1 and 1 mg/kg i.v. and almost completely inhibited the response at a dose of 10 mg/kg. The transient depressor response induced by ET-1 was also inhibited by 1 and 10 mg/kg TAK-044. BQ-123 partially inhibited the pressor response at 0.1-10 mg/kg i.v., whereas it did not inhibit the depressor response except at a dose of 10 mg/kg. These inhibitory effects of TAK-044 were longer lasting than those of BQ-123: 3 hr for TAK-044 and 1 hr for BQ-123 at 10 mg/kg. A selective ETB agonist, sarafotoxin S6c (0.3 nmol/kg i.v.), induced both depressor and pressor responses similar to ET-1. The initial depressor response was inhibited by TAK-044 in a dose-dependent manner (0.1-10 mg/kg i.v.) and by BQ-123 at the highest dose, whereas the sustained pressor response was inhibited only by TAK-044 at a dose of 10 mg/kg. Similar differences between TAK-044 and BQ-123 were observed for sarafotoxin S6c-induced renal vasoconstriction: TAK-044 but not BQ-123 inhibited the response at a dose of 3 mg/kg i.v. We conclude that TAK-044 inhibited both ETA- and ETB-mediated blood pressure responses and that these effects were longer lasting than those of BQ-123. In addition, the ETB-mediated vasoconstriction and pressor responses were inhibited by TAK-044 and not by BQ-123.
 
2-Methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242) is a novel κ-opioid receptor (KOR) antagonist with high affinity for human (3 nM), rat (21 nM), and mouse (22 nM) KOR, a ∼ 20-fold reduced affinity for human μ-opioid receptors (MORs; K(i) = 64 nM), and negligible affinity for δ-opioid receptors (K(i) > 4 μM). PF-04455242 also showed selectivity for KORs in vivo. In rats, PF-04455242 blocked KOR and MOR agonist-induced analgesia with ID(50) values of 1.5 and 9.8 mg/kg, respectively, and inhibited ex vivo [(3)H](2-(benzofuran-4-yl)-N-methyl-N-((5S,7R,8R)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]decan-8-yl)acetamide ([(3)H]CI977) and [(3)H](2S)-2-[[2-[[(2R)-2-[[(2S)-2-amino-3-(4-hydroxyphenyl) propanoyl]amino]propanoyl]amino]acetyl]-methylamino]-N-(2-hydroxyethyl)-3-phenylpropanamide ([(3)H]DAMGO) binding to KOR and MOR receptors with ID(50) values of 2.0 and 8.6 mg/kg, respectively. An in vivo binding assay was developed using (-)-4-[(3)H]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(3)H]PF-04767135), a tritiated version of the KOR positron emission tomography ligand (-)-4-[(11)C]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(11)C]GR103545) in which PF-04455242 had an ID(50) of 5.2 mg/kg. PF-04455242 demonstrated antidepressant-like efficacy (mouse forced-swim test), attenuated the behavioral effects of stress (mouse social defeat stress assay), and showed therapeutic potential in treating reinstatement of extinguished cocaine-seeking behavior (mouse conditioned place preference). KOR agonist-induced plasma prolactin was investigated as a translatable mechanism biomarker. Spiradoline (0.32 mg/kg) significantly increased rat plasma prolactin levels from 1.9 ± 0.4 to 41.9 ± 4.9 ng/ml. PF-04455242 dose-dependently reduced the elevation of spiradoline-induced plasma prolactin with an ID(50) of 2.3 ± 0.1 mg/kg, which aligned well with the ED(50) values obtained from the rat in vivo binding and efficacy assays. These data provide further evidence that KOR antagonists have potential for the treatment of depression and addiction disorders.
 
The endogenous cannabinoid (endocannabinoid) anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH). Pharmacological blockade of FAAH has emerged as a potentially attractive strategy for augmenting endocannabinoid signaling and retaining the beneficial effects of cannabinoid receptor activation, while avoiding the undesirable side effects, such as weight gain and impairments in cognition and motor control, observed with direct cannabinoid receptor 1 agonists. Here, we report the detailed mechanistic and pharmacological characterization of N-pyridazin-3-yl-4-(3-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzylidene)piperidine-1-carboxamide (PF-04457845), a highly efficacious and selective FAAH inhibitor. Mechanistic studies confirm that PF-04457845 is a time-dependent, covalent FAAH inhibitor that carbamylates FAAH's catalytic serine nucleophile. PF-04457845 inhibits human FAAH with high potency (k(inact)/K(i) = 40,300 M(-1)s(-1); IC(50) = 7.2 nM) and is exquisitely selective in vivo as determined by activity-based protein profiling. Oral administration of PF-04457845 produced potent antinociceptive effects in both inflammatory [complete Freund's adjuvant (CFA)] and noninflammatory (monosodium iodoacetate) pain models in rats, with a minimum effective dose of 0.1 mg/kg (CFA model). PF-04457845 displayed a long duration of action as a single oral administration at 1 mg/kg showed in vivo efficacy for 24 h with a concomitant near-complete inhibition of FAAH activity and maximal sustained elevation of anandamide in brain. Significantly, PF-04457845-treated mice at 10 mg/kg elicited no effect in motility, catalepsy, and body temperature. Based on its exceptional selectivity and in vivo efficacy, combined with long duration of action and optimal pharmacokinetic properties, PF-04457845 is a clinical candidate for the treatment of pain and other nervous system disorders.
 
Developing a population-based pharmacokinetic-pharmacodynamic (PKPD) model is a challenge in ophthalmology due to the difficulty of obtaining adequate pharmacokinetic (PK) samples from ocular tissues to inform the pharmacodynamic (PD) model. Using limited PK data, we developed a preclinical population-based PD model suitable for capturing the time course of dog intraocular pressure (IOP) that exhibited time-dependent sensitization after topical administration of PF-04475270 [5-{3-[(2S)-2-{(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-5-oxopyrrolidin-1-yl]propyl}thiophene-2-carboxylate]. A physiologically relevant PK model was chosen to simultaneously capture the concentration profiles of CP-734432, a potent EP4 agonist and the active metabolite of PF-04475270, sampled from three ocular tissues of the anterior chamber: cornea, aqueous humor, and iris-ciliary body. Two population-based PD models were developed to characterize the IOP lowering profiles: model I, a standard indirect-response model (IRM); and model II, an extension of a standard IRM that empirically incorporated a response-driven positive feedback loop to account for the observed PD sensitization. The PK model reasonably described the PK profiles in all three ocular tissues. As for the PD, model I failed to capture the overall trend in the population IOP data, and model II more adequately characterized the overall data set. This integrated PKPD model may have general utility when PD sensitization is observed and is not a result of time-dependent PK. In addition, the model is applicable in the ophthalmology drug development setting in which PK information is limited but a population-based PD model could reasonably be established.
 
4-[5-(Pyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridine-2-carbonitrile (FYX-051) is a potent inhibitor of bovine milk xanthine oxidoreductase (XOR). Steady-state kinetics study showed that it initially behaved as a competitive-type inhibitor with a K(i) value of 5.7 × 10(-9) M, then after a few minutes it formed a tight complex with XOR via a Mo-oxygen-carbon atom covalent linkage, as reported previously (Proc Natl Acad Sci USA 101:7931-7936, 2004). Thus, FYX-051 is a hybrid-type inhibitor exhibiting both structure- and mechanism-based inhibition. The FYX-051-XOR complex decomposed with a half-life of 20.4 h, but the enzyme activity did not fully recover. This was found to be caused by XOR-mediated conversion of FYX-051 to 4-[5-(2-hydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridine-2-carbonitrile (2-hydroxy-FYX-051), as well as formation of 6-hydroxy-4-[5-(2-hydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridine-2-carbonitrile (dihydroxy-FYX-051) and 4-[5-(2,6-dihydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]-6-hydroxypyridine-2-carbonitrile (trihydroxy-FYX-051) during prolonged incubation for up to 72 h. A distinct charge-transfer band was observed concomitantly with the formation of the trihydroxy-FYX-051-XOR complex. Crystallographic analysis of the charge-transfer complex indicated that a Mo-nitrogen-carbon bond was formed between molybdenum of XOR and the nitrile group of trihydroxy-FYX-051. FYX-051 showed a potent and long-lasting hypouricemic effect in a rat model of potassium oxonate-induced hyperuricemia, and it seems to be a promising candidate for the clinical treatment of hyperuricemia.
 
There is growing evidence that nerve growth factor (NGF) may function as a mediator of persistent pain states. We have identified a novel nonpeptidic molecule, ALE-0540, that inhibits the binding of NGF to tyrosine kinase (Trk) A or both p75 and TrkA (IC50 5.88 +/- 1. 87 microM, 3.72 +/- 1.3 microM, respectively), as well as signal transduction and biological responses mediated by TrkA receptors. ALE-0540 was tested in models of neuropathic pain and thermally-induced inflammatory pain, using two routes of administration, a systemic i.p. and a spinal intrathecal (i.th.) route. Morphine was also tested for comparison in the antiallodynia model using mechanical stimuli. We show that either i.p. or i.th. administration of ALE-0540 in rats produced antiallodynia in the L5/L6 ligation model of neuropathic pain. The calculated A50 values (and 95% confidence intervals) for ALE-0540 administered i.p. and i. th. were 38 (17.5-83) mg/kg and 34.6 (17.3-69.4) microgram, respectively. ALE-0540 given i.th., at doses of 30 and 60 microgram, also blocked tactile allodynia in the thermal sensitization model. Although morphine displayed greater potency [A50 value of 7.1 (5.6-8. 8) mg/kg] than ALE-0540 in anti-allodynic effect when given i.p. to L5/L6-ligated rats, it was not active when administered i.th. These data suggest that a blockade of NGF bioactivity using a NGF receptor antagonist is capable of blocking neuropathic and inflammatory pain and further support the hypothesis that NGF is involved in signaling pathways associated with these pain states. ALE-0540 represents a nonpeptidic small molecule which can be used to examine mechanisms leading to the development of agents for the treatment of pain.
 
The antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide fumarate (LF22-0542), a novel nonpeptidic B1 antagonist, was characterized. LF22-0542 showed high affinity for human and mouse B1 receptors with virtually no affinity for the human B2 receptor; a selectivity index of at least 4000 times was obtained when LF22-0542 was profiled throughout binding or cell biology assays on 64 other G-protein-coupled receptor, 10 ion channels, and seven enzymes. LF22-0542 was a competitive B1 receptor antagonist and elicited significant antinociceptive actions in the mouse acetic acid-induced writhing assay, as well as in the second phases of formalin-induced nociception in mice and in both the first and second phases of the formalin response in rats. LF22-0542 was active after s.c. but not p.o. administration. In B1 receptor knockout (KO) mice, acetic acid and formalin responses were significantly reduced and LF22-0542 had no additional effects in these animals. LF22-0542 alleviated thermal hypersensitivity in both acute (carrageenan) and persistent inflammatory (complete Freund's adjuvant) pain models in rats. LF22-0542 produced a full reversal of experimental neuropathic thermal hypersensitivity but was inactive in reversing nerve injury-induced tactile hypersensitivity in rats. In agreement with this observation, B1 KO mice subjected to peripheral nerve injury did not show thermal hypersensitivity but developed nerve injury-induced tactile hypersensitivity normally. The data demonstrate the antihyperalgesic actions of a selective systemically administered B1 receptor antagonist and suggest the utility of this class of agents for the treatment of inflammatory pain states and for some aspects of neuropathic pain.
 
Flow chart of the studies. PKF275-055 (3 mg/kg) was administered by gavage daily for 1 week to the rats in the prevention group (thick dotted line). Diabetes was induced by injection of 60 mg/kg i.p. STZ, and PKF275-055 (3 mg/ kg) was administered in drinking water for 4 weeks to the rats subjected to the protection protocol (thick solid line). For the therapeutic protocol, PKF275-055 (10 mg/ kg) was administered in drinking water for 5 weeks (thick dotted line). In a small group of animals from the prevention schedule, PKF275-055 (3 mg/kg) was administered in drinking water for an additional 5 weeks (prolonged prevention group; thin dotted line).  
Tail nerve conduction velocity in rats on day 36 (randomization) and day 79 of PKF275-055 treatment under the therapeutic protocol (completed) (see Materials and Methods). Mean core temperatures for the indicated groups are 36.8 0.34°C (CTRL), 36.7 0.10°C (CTRLPKF), 36.6 0.33°C (STZPKF Therapeutic), and 36.9 0.10°C (STZ Untreated). Data are mean S.E.M. ANOVA followed by Turkey-Kramer post-test: , p 0.001 versus CTRL; , p 0.01 versus CTRL; E E E E, p 0.001 versus CTRLPKF; E E, p 0.01 versus CTRLPKF.  
PKF275-055 significantly improved impaired thermal (A) and mechanical (B) sensitivity in diabetic rats. Thermal sensitivity and mechanical allodynia are expressed as withdrawal latency in seconds and grams, respectively . Data are mean S.E.M. ANOVA followed by Turkey-Kramer post-test. , p 0.001 versus CTRL; , p 0.005 versus CTRL; , p 0.01 versus CTRL; , p 0.05 versus CTRL; E E E E, p 0.001 versus CTRLPKF; E E E, p 0.005 versus CTRLPKF; E, p 0,05 versus CTRLPKF; §, p 0.05 versus STZ untreated.  
1-[(2-adamantyl)amino]acetyl-2-cyano-(S)-pyrrolidine, monohydrochloride (PKF275-055), a vildagliptin analog, is a novel, selective, potent, orally bioavailable, and long-acting dipeptidyl peptidase IV inhibitor. We studied the effect of PKF275-055 administration on the prevention, protection, and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. PKF275-055 improved body and muscle weight. Oral glucose tolerance tests showed a marked improvement in glucose metabolism under all treatment schedules. When tested in prevention and protection experiments, PKF275-055 completely averted the decrease of Na⁺/K⁺-ATPase activity and partially counteracted the nerve conduction velocity (NCV) deficit observed in untreated diabetic rats but had no effects on abnormal mechanical and thermal sensitivity. When used in a therapeutic setting, PKF275-055 induced a significant correction in the alteration in Na⁺,K⁺-ATPase activity and NCV present in untreated diabetics. Diabetic rats developed mechanical hyperalgesia within 2 weeks after streptozotocin injection and exhibited significantly longer thermal response latencies. It is noteworthy that PKF275-055 treatment restored mechanical sensitivity thresholds by approximately 50% (p < 0.01) and progressively improved the alteration in thermal responsiveness. In conclusion, PKF275-055 showed an anabolic effect, improved oral glucose tolerance, and counteracted the alterations in Na⁺,K⁺-ATPase activity, NCV, and nociceptive thresholds in diabetic rats. The present data support a potential therapeutic effect of PKF275-055 in the treatment of rodent diabetic neuropathy.
 
Top-cited authors
Aron H Lichtman
  • Virginia Commonwealth University
Yuichi Sugiyama
Hiroyuki Kusuhara
  • The University of Tokyo
William F Elmquist
  • University of Minnesota Twin Cities
David H Rominger