JPET current issue

[HIGHLIGHTED PAPERS] Polyphenols and Cannabinoid Receptor Functional Agonism

2009-06-22

[HIGHLIGHTED PAPERS] Thiazolidinedione Modulation of Farnesoid X Receptor Signaling

2009-06-22

[NEUROPHARMACOLOGY] Donepezil Improves Cognitive Function in Mice by Increasing the Production of Insulin-Like Growth Factor-I in the Hippocampus

Narimatsu, N., Harada, N., Kurihara, H., Nakagata, N., Sobue, K., Okajima, K. — 2009-06-22

Insulin-like growth factor-I (IGF-I) exerts beneficial effects on cognitive function. The selective acetylcholinesterase inhibitor donepezil increases serum IGF-I levels in elderly subjects. Because stimulation of sensory neurons induces IGF-I production by releasing calcitonin gene-related peptide (CGRP) in the mouse brain, we hypothesized that donepezil increases IGF-I production by sensory neuron stimulation to improve the cognitive function in mice. Donepezil, but not tacrine, increased the CGRP release from dorsal root ganglion neurons isolated from wild-type (WT) mice. Pretreatment with the protein kinase A inhibitor KT5720 [(9S,10S,12R)-2,3,9,10,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg: 3',2',1'-kl]pyrrolo[3,4-i][1,6]-benzo-diazocine-10-carboxylic acid hexyl ester] reversed the effects induced by donepezil. Increase in tissue levels of CGRP, IGF-I, and IGF-I mRNA in the hippocampus was observed at 4 weeks after oral administration of donepezil in WT mice. In these animals, c-fos expression in spinal dorsal horns, parabrachial nuclei, the solitary tract nucleus, and the hippocampus was increased. Enhancement in angiogenesis and neurogenesis was observed in the dentate gyrus of the hippocampus of WT mice after donepezil administration. Improvement of spatial learning was observed in WT mice after donepezil administration. Oral administration of tacrine for 4 weeks produced none of the aforementioned effects induced by donepezil in WT mice. However, none of the effects observed in WT mice was seen after donepezil administration in CGRP-knockout mice and WT mice subjected to functional denervation. These observations suggest that donepezil may improve cognitive function in mice by increasing the hippocampal production of IGF-I through sensory neuron stimulation. These effects of donepezil may not be dependent on its acetylcholinesterase inhibitory activity.

[NEUROPHARMACOLOGY] Combination Treatment with Normobaric Hyperoxia and Cilostazol Protects Mice against Focal Cerebral Ischemia-Induced Neuronal Damage Better Than Each Treatment Alone

2009-06-22

Normobaric hyperoxia (NBO) and cilostazol (6-[4-(1-cyclohexy-1H-tetrazol-5-yl)butoxyl]-3,4-dihydro-2-(1H)-quinolinone) (a selective inhibitor of phosphodiesterase 3) have each been reported to exert neuroprotective effects against acute brain injury after cerebral ischemia in rodents. Here, we evaluated the potential neuroprotective effects of combination treatment with NBO and cilostazol against acute and subacute brain injuries after simulated stroke. Mice subjected to 2-h filamental middle cerebral artery (MCA) occlusion were treated with NBO (95% O₂, during the ischemia) alone, with cilostazol (3 mg/kg i.p. after the ischemia) alone, with both of these treatments (combination), or with vehicle. The histological and neurobehavioral outcomes were assessed at acute (1 day) or subacute (7 days) stages after reperfusion. We measured regional cerebral blood flow (rCBF) during and after ischemia by laser-Doppler flowmetry and recovery (versus vehicle) in the combination therapy group just after reperfusion. Mean acute and subacute lesion volumes were significantly reduced in the combination group but not in the two monotherapy groups. The combination therapy increased endothelial nitric-oxide synthase (eNOS) activity in the lesion area after ischemia versus vehicle. Combination therapy with NBO plus cilostazol protected mice subjected to focal cerebral ischemia by improvement of rCBF after reperfusion, in part in association with eNOS activity.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] The Expression of Human Microsomal Epoxide Hydrolase Is Predominantly Driven by a Genetically Polymorphic Far Upstream Promoter

2009-06-22

Microsomal epoxide hydrolase (EPHX1) biotransforms epoxide derivatives of pharmaceuticals, including metabolites of certain antiepileptic medications, such as phenytoin and carbamazepine, and many environmental epoxides, such as those derived from butadiene, benzene, and carcinogenic polyaromatic hydrocarbons. We previously identified a far upstream promoter region, designated E1-b, in the EPHX1 gene that directs expression of an alternatively spliced EPHX1 mRNA transcript in human tissues. In this investigation, we characterized the structural features and expression character of the E1-b promoter region. Results of quantitative real-time polymerase chain reaction analyses demonstrated that the E1-b variant transcript is preferentially and broadly expressed in most tissues, such that it accounts for the majority of total EPHX1 transcript in vivo. Comparative genomic sequence comparisons indicated that the human EPHX1 E1-b gene regulatory region is primate-specific. Direct sequencing and genotyping approaches in 450 individuals demonstrated that the E1-b promoter region harbors a series of transposable element cassettes, including a polymorphic double Alu insertion. Results of reporter assays conducted in several human cell lines demonstrated that the inclusion of the Alu(+/+) insertion significantly decreases basal transcriptional activities. Furthermore, using haplotype block analyses, we determined that the E1-b polymorphic promoter region was not in linkage disequilibrium with two previously identified nonsynonomous single nucleotide polymorphisms (SNPs) in the coding region or with functional SNPs previously identified in the proximal promoter region of the gene. These results demonstrate that the upstream E1-b promoter is the major regulator of EPHX1 expression in human tissues and that polymorphism in this region may contribute an interindividual risk determinant to xenobiotic-induced toxicities.

[CELLULAR AND MOLECULAR] The Dietary Polyphenols trans-Resveratrol and Curcumin Selectively Bind Human CB1 Cannabinoid Receptors with Nanomolar Affinities and Function as Antagonists/Inverse Agonists

Seely, K. A., Levi, M. S., Prather, P. L. — 2009-06-22

The dietary polyphenols trans-resveratrol [5-[(1E)-2-(4-hydroxyphenyl)ethenyl]-1,3-benzenediol; found in red wine] and curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1E,6E-heptadiene-3,5-dione] (found in curry powders) exert anti-inflammatory and antioxidant effects via poorly defined mechanisms. It is interesting that cannabinoids, derived from the marijuana plant (Cannabis sativa), produce similar protective effects via CB1 and CB2 receptors. We examined whether trans-resveratrol, curcumin, and ASC-J9 [1,7-bis(3,4-dimethoxyphenyl)-5-hydroxy-1E,4E,6E-heptatriene-3-one] (a curcumin analog) act as ligands at cannabinoid receptors. All three bind to human (h) CB1 and mouse CB1 receptors with nanomolar affinities, displaying only micromolar affinities for hCB2 receptors. Characteristic of inverse agonists, the polyphenols inhibit basal G-protein activity in membranes prepared from Chinese hamster ovary (CHO)-hCB1 cells or mouse brain that is reversed by a neutral CB1 antagonist. Furthermore, they competitively antagonize G-protein activation produced by a CB1 agonist. In intact CHO-hCB1 cells, the polyphenols act as neutral antagonists, producing no effect when tested alone, whereas competitively antagonizing CB1 agonist mediated inhibition of adenylyl cyclase activity. Confirming their neutral antagonist profile in cells, the polyphenols similarly attenuate stimulation of adenylyl cyclase activity produced by a CB1 inverse agonist. In mice, the polyphenols dose-dependently reverse acute hypothermia produced by a CB1 agonist. Upon repeated administration, the polyphenols also reduce body weight in mice similar to that produced by a CB1 antagonist/inverse agonist. Finally, trans-resveratrol and curcumin share common structural motifs with other known cannabinoid receptor ligands. Collectively, we suggest that trans-resveratrol and curcumin act as antagonists/inverse agonists at CB1 receptors at dietary relevant concentrations. Therefore, these polyphenols and their derivatives might be developed as novel, nontoxic CB1 therapeutics for obesity and/or drug dependence.

[NEUROPHARMACOLOGY] Differential Regulation of Receptor Activation and Agonist Selectivity by Highly Conserved Tryptophans in the Nicotinic Acetylcholine Receptor Binding Site

Williams, D. K., Stokes, C., Horenstein, N. A., Papke, R. L. — 2009-06-22

We have shown previously that a highly conserved Tyr in the nicotinic acetylcholine receptor (nAChR) ligand-binding domain (LBD) (7 Tyr188 or 4 Tyr195) differentially regulates the activity of acetylcholine (ACh) and the 7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) in 4β2 and 7 nAChR. In this study, we mutated two highly conserved LBD Trp residues in human 7 and 4β2 and expressed the receptors in Xenopus laevis oocytes. 7 Receptors with Trp55 mutated to Gly or Tyr became less responsive to 4OH-GTS-21, whereas mutation of the homologous Trp57 in β2 to Gly, Tyr, Phe, or Ala resulted in 4β2 receptors that showed increased responses to 4OH-GTS-21. Mutation of 7 Trp55 to Val resulted in receptors for which the partial agonist 4OH-GTS-21 became equally efficacious as ACh, whereas 4β2 receptors with the homologous mutation remained nonresponsive to 4OH-GTS-21. In contrast to the striking alterations in agonist activity profiles that were observed with mutations of 7 Trp55 and β2 Trp57, mutations of 7 Trp149 or 4 Trp154 universally resulted in receptors with reduced function. Our data support the hypothesis that some conserved residues in the nAChR LBD differentially regulate receptor activation by subtype-selective agonists, whereas other equally well conserved residues play fundamental roles in receptor activation by any agonist. Residues like 7 Trp149 (4 Trp154) may be considered pillars upon which basic receptor function depends, whereas 7 Trp55 (β2 Trp57) and 7 Tyr188 (4 Tyr195) may be fulcra upon which agonists may operate differentially in specific receptor subtypes, consistent with the hypothesis that ACh and 4OH-GTS-21 are able to activate nAChR in distinct ways.

[CARDIOVASCULAR] Effects of Roflumilast, a Phosphodiesterase-4 Inhibitor, on Hypoxia- and Monocrotaline-Induced Pulmonary Hypertension in Rats

2009-06-22

Phosphodiesterase type 4 (PDE4) is involved in the hydrolysis of cAMP in pulmonary vascular smooth muscle (PA-SMC) and immune inflammatory cells. Given that intracellular cAMP accumulation inhibits contraction and growth of PA-SMCs as well as inflammatory cell functions, we investigated the effects of the PDE4 inhibitor 3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide (roflumilast), on pulmonary hypertension (PH) in rats. Treatment with roflumilast (0.5 or 1.5 mg · kg^-1 day^-1) from day 1 to day 21 after monocrotaline (MCT) injection (60 mg · kg^-1 s.c.) attenuated PH development: pulmonary artery pressure, right ventricular hypertrophy, and muscularization of distal vessels on day 21 were decreased compared to control MCT-treated rats. Roflumilast (1.5 mg · kg^-1 day^-1) also reduced the increases in interleukin-6 and monocyte chemotactic protein-1 mRNAs observed in lung tissue on day 21 without affecting the rise in interleukin-1β mRNA on days 1 and 21. Roflumilast (1.5 mg · kg^-1 day^-1) from day 21 to day 42 after MCT reversed established PH, almost normalizing pulmonary artery pressure and structure, and suppressing proliferating cell nuclear antigen-positive cells in pulmonary vascular walls. Treatment with roflumilast similarly attenuated PH development due to chronic hypoxia. Treatment of human PA-SMCs with roflumilast N-oxide, the active metabolite of roflumilast, at concentrations up to 10^-6 M, potentiated PA-SMC growth inhibition induced by prostacyclin (10^-6 M) or interleukin-1β (10 ng · ml^-1) but was inactive on its own. In conclusion, the PDE4 inhibitor roflumilast significantly attenuates pulmonary vascular remodeling and hypertension induced by chronic hypoxia or MCT and reverses established PH after MCT administration.

[CARDIOVASCULAR] Monitoring White Blood Cell Mitochondrial Aldehyde Dehydrogenase Activity: Implications for Nitrate Therapy in Humans

2009-06-22

Recent animal data suggest that reduced lipoic acid (LA) prevents oxidative inhibition of the nitrate bioactivating enzyme, the mitochondrial aldehyde dehydrogenase (ALDH-2), and that pentaerythritol tetranitrate (PETN) does not induce nitrate tolerance because of its intrinsic antioxidative properties, thereby preserving ALDH-2 activity. We sought to determine whether ALDH-2 activity in circulating white blood cells (WBCs) can be used to monitor nitrate tolerance and whether LA can prevent nitroglycerin tachyphylaxis in humans. Eight healthy male volunteers received, in randomized order, a single dose of glyceryl trinitrate (GTN; 0.8 mg), PETN (80 mg), or GTN plus LA (600 mg) orally. GTN (30 min) and PETN (120 min) administration lead to a comparable dilation of the brachial artery (15 ± 1%). In contrast to PETN, acute GTN treatment resulted in a 60% decrease in WBC ALDH-2 activity (high-performance liquid chromatography), 30% reduction of nitrate bioactivation, and 25% decrease in serum antioxidant capacity (fluorescence assay), which all were prevented by pretreatment with LA. Mechanistic studies in rats identified oxidative stress, ALDH-2 inactivation, and vascular dysfunction as common features in acute and chronic nitrate tolerance. Treatment with GTN, but not PETN, acutely inhibits ALDH-2 activity and nitrate bioactivation in healthy volunteers. These effects were prevented by LA pretreatment, emphasizing the role of oxidative stress-triggered ALDH-2 dysfunction. Assessment of WBC ALDH-2 activity could be used as an easily accessible marker for the detection of nitroglycerin-induced tachyphylaxis in humans and may be of high clinical interest because recent data suggest that ALDH-2 activity correlates with protection from ischemic heart damage in infarct models.

[TOXICOLOGY] Trovafloxacin Enhances the Inflammatory Response to a Gram-Negative or a Gram-Positive Bacterial Stimulus, Resulting in Neutrophil-Dependent Liver Injury in Mice

Shaw, P. J., Ganey, P. E., Roth, R. A. — 2009-06-22

Trovafloxacin (TVX), a fluoroquinolone antibiotic, has been strongly linked with several cases of idiosyncratic hepatotoxicity in humans. Previous studies showed that a modest inflammatory stress induced by a Gram-negative bacterial stimulus [i.e., lipopolysaccharide (LPS)] rendered nontoxic doses of TVX hepatotoxic in mice. This study compared the interaction of TVX with Gram-negative and Gram-positive stimuli. Mice were given TVX 3 h before LPS (Gram-negative stimulus) or a peptidoglycan-lipoteichoic acid (PGN-LTA) mixture isolated from Staphylococcus aureus (Gram-positive stimulus). Administration of TVX, LPS, or PGN-LTA alone was nonhepatotoxic. However, TVX administration before PGN-LTA or LPS resulted in significant liver injury that occurred with similar time courses. TVX/PGN-LTA-induced hepatocellular necrosis was primarily localized to centrilobular regions, whereas that caused by TVX/LPS was predominantly midzonal. Administration of either LPS or PGN-LTA alone led to increased plasma concentrations of several cytokines and chemokines at a time near the onset of liver injury. TVX administration before LPS enhanced the concentrations of all of these cytokines, whereas TVX treatment before PGN-LTA increased all of the cytokines except tumor necrosis factor (TNF)- and interferon-. Liver injury was reduced in TVX/LPS- and TVX/PGN-LTA-treated mice given an antibody to CD18 and also in mice deficient in neutrophil [polymorphonuclear neutrophil (PMN)] elastase. Hepatic PMN accumulation and TNF- production after TVX/PGN-LTA-, but not after TVX/LPS-coexposure, was CD18-dependent. In summary, TVX significantly enhanced the murine inflammatory response to either a Gram-negative or a Gram-positive stimulus and caused hepatotoxicity that developed similarly and was dependent on PMN activation in mice but that differed in lesion location and cytokine profile.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Overexpression of Peroxiredoxin 6 Does Not Prevent Ethanol-Mediated Oxidative Stress and May Play a Role in Hepatic Lipid Accumulation

Roede, J. R., Orlicky, D. J., Fisher, A. B., Petersen, D. R. — 2009-06-22

Oxidative stress is implicated in the etiology of many diseases, including alcoholic liver disease (ALD). Peroxiredoxin 6 is a cytosolic peroxidase that has been demonstrated to protect various tissues, such as skin, lung, and cardiac muscle, against acute oxidative insults. Consequently, peroxiredoxin 6 was hypothesized to also protect the liver from oxidative stress generated during the process of chronic ethanol ingestion. To test this, wild-type peroxiredoxin 6 knockout mice (KO), and transgenic peroxiredoxin 6 overexpressing mice (TG) were fed an ethanol-containing diet. Various biomarkers of ALD were assessed, along with the effects of chronic ethanol consumption on the antioxidant defenses. After 9 weeks of ethanol consumption, all backgrounds exhibited elevations of plasma alanine aminotransferase activity, hepatosteatosis, CYP2E1 induction, and lipid peroxidation; however, hepatic triglyceride accumulation seemed to be exacerbated in ethanol-fed TG mice. Differences in antioxidant protein expression and activity in response to chronic ethanol consumption were also observed. Examples include significant inductions of catalase and glutathione transferase activity in ethanol-fed KO and TG mice, along with elevated levels of glutathione peroxidase activity. These alterations in antioxidant defenses could be attributed to either compensatory responses due to the genetic manipulations or ethanol-mediated responses. In conclusion, both ethanol-fed KO and ethanol-fed TG mice developed early stage ALD and peroxiredoxin 6 may play a role in ethanol-mediated hepatic lipid accumulation.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Advanced Glycation End Products Subspecies-Selectively Induce Adhesion Molecule Expression and Cytokine Production in Human Peripheral Blood Mononuclear Cells

2009-06-22

Advanced glycation end products (AGEs) are proteins or lipids that become glycated after exposure to diverse reducing sugars. Accumulation of AGEs induces diabetes complications. Microinflammation is a common major mechanism in the pathogenesis of diabetic vascular complications. Activation of monocytes/macrophages and T cells plays roles in the pathogenesis of atherosclerosis. The activation of T cells requires the enhanced expression of adhesion molecules on monocytes. AGEs activate monocytes by engaging the receptor for AGE (RAGE); however, little is known about the profile of agonist activity of diverse AGE moieties on monocytes. We investigated the effect of four distinct AGE subtypes (AGE-modified bovine serum albumin; AGE-2, AGE-3, AGE-4, and AGE-5) at concentrations ranging from 0.1 to 100 µg/ml on the expression of intercellular adhesion molecule-1, B7.1, B7.2, and CD40 on monocytes and its impact on the production of interferon- and tumor necrosis factor- in human peripheral blood mononuclear cells. Among the AGEs examined, AGE-2 and AGE-3 selectively induced adhesion molecule expression and cytokine production. Antagonism experiments using antibodies against adhesion molecules demonstrated that cell-to-cell interaction between monocytes and T/natural killer cells was involved in AGE-2- and AGE-3-induced cytokine production. AGE-2 and AGE-3 up-regulated the expression of RAGE on monocytes. The effects of AGE-2 and AGE-3 were inhibited by nuclear factor-B and p38 mitogen-activated protein kinase inhibitors. These results indicated that AGE-2 and AGE-3 activated monocytes via RAGE, leading to the up-regulation of adhesion molecule expression and cytokine production.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] A2A Adenosine Receptor Deficiency Leads to Impaired Tracheal Relaxation via NADPH Oxidase Pathway in Allergic Mice

2009-06-22

A_2A adenosine receptor (A_2AAR) has been shown to suppress superoxide generation in leukocytes via the cAMP-protein kinase A (PKA) pathway. However, no study has yet explored the role of A_2AAR in relation to NADPH oxidase in murine tracheas in vitro, which may lead to altered smooth muscle relaxation in asthma. Therefore, the present study evaluated the effects of A_2AAR deficiency on the NADPH oxidase pathway in tracheas of A_2A wild-type (WT) and A_2A knockout (KO) mice. A_2AWT mice were sensitized with ovalbumin (30 µg i.p.) on days 1 and 6, followed by 5% ovalbumin aerosol challenge on days 11, 12, and 13. A_2AAR (gene and protein expression), cAMP, and phosphorylated PKA (p-PKA) levels were decreased in A_2AWT sensitized mice compared with controls. A_2AKO mice also showed decreased cAMP and p-PKA levels. A_2AWT sensitized and A_2AKO control mice had increased gene and protein expression of NADPH oxidase subunits (p47phox and gp91phox) compared with the controls. Tracheal relaxation to specific A_2AAR agonist, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS 21680), decreased in A_2AWT sensitized mice compared with the controls, although it was absent in A_2AKO mice. Pretreatment with NADPH oxidase inhibitors apocyanin/diphenyliodonium reversed the attenuated relaxation to CGS 21680 in A_2AWT sensitized tracheas, whereas specific PKA inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i] [1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) blocked CGS 21680-induced relaxation. Tracheal reactive oxygen species (ROS) generation was also increased in A_2AWT sensitized and A_2AKO control mice compared with the controls. In conclusion, this study shows that A_2AAR deficiency causes increased NADPH oxidase activation leading to decreased tracheal relaxation via altered cAMP-PKA signaling and ROS generation.

[CELLULAR AND MOLECULAR] Rab1 GTPase and Dimerization in the Cell Surface Expression of Angiotensin II Type 2 Receptor

2009-06-22

The physiological function of angiotensin II (Ang II) is mediated through the Ang II type 1 (AT1R) and type 2 (AT2R) receptors. Our previous studies have demonstrated that cell surface targeting of AT1R is regulated by Rab and Sar1 GTPases and the F(x)₆LL motif in the membrane-proximal C terminus. However, the molecular mechanisms underlying the export of nascent AT2R remain poorly defined. In this report, we determined the role of Rab1 GTPase, which specifically controls protein transport from the endoplasmic reticulum (ER) to the Golgi, and receptor dimerization in the biosynthesis of AT2R. Cell surface expression of AT2R was augmented by transient expression of Rab1 and attenuated by dominant-negative Rab1 mutants and small interfering RNA-mediated knockdown of Rab1. Consistently, AT2R inhibition of epidermal growth factor-activated extracellular signal-regulated kinase 1/2 was significantly reduced by the Rab1 mutants, indicating that endogenous Rab1 modulates the cell surface targeting and signaling of AT2R. It is of interest to note that Rab1 augmented the overall expression of AT2R and its mRNA, whereas the Rab1 mutants attenuated the total AT2R expression and enhanced ubiquitin-dependent AT2R degradation. Furthermore, our previously characterized ER export-deficient AT1R mutant in which the F(x)₆LL motif was mutated formed both homodimers and heterodimers with AT2R. Dimerization of the AT1R mutant with AT2R blocked AT2R trafficking to the cell surface, suggesting constitutive dimerization of both receptors in the ER and an important role of dimerization in ER export of the receptors. These data demonstrate for the first time that Rab1 GTPase and dimerization modulate export traffic from the ER to the cell surface of newly synthesized AT2R.

[CELLULAR AND MOLECULAR] Angiotensin II-Induced Cyclooxygenase 2 Expression in Rat Aorta Vascular Smooth Muscle Cells Does Not Require Heterotrimeric G Protein Activation

Morinelli, T. A., Kendall, R. T., Luttrell, L. M., Walker, L. P., Ullian, M. E. — 2009-06-22

Angiotensin II (AngII) initiates cellular effects via its G protein-coupled angiotensin 1 (AT₁) receptor (AT₁R). Previously, we showed that AngII-induced expression of the prostanoid-producing enzyme cyclooxygenase 2 (COX-2) was dependent upon nuclear trafficking of activated AT₁R. In the present study, mastoparan (an activator of G proteins), suramin (an inhibitor of G proteins), 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122; a specific inhibitor of phospholipase C), and sarcosine¹-Ile⁴-Ile⁸-AngII (SII-AngII; a G protein-independent AT₁R agonist) were used to determine the involvement of G proteins and AT_1AR trafficking in AngII-stimulated COX-2 protein expression in human embryonic kidney-293 cells stably expressing AT_1A/green fluorescent protein receptors and cultured vascular smooth muscle cells, respectively. Mastoparan alone stimulated release of intracellular calcium and increased COX-2 expression. Preincubation with mastoparan inhibited AngII-induced calcium signaling without altering AngII-induced AT_1AR trafficking, p42/44 extracellular signal-regulated kinase (ERK) activation, or COX-2 expression. Suramin or U73122 had no significant effect on their own; they did not inhibit AngII-induced AT_1AR trafficking, p42/44 ERK activation, or COX-2 expression; but they did inhibit AngII-induced calcium responses. SII-AngII stimulated AT_1AR trafficking and increased COX-2 protein expression without activating intracellular calcium release. These data suggest that G protein activation results in increased COX-2 protein expression, but AngII-induced COX-2 expression seems to occur independently of G protein activation.

[CELLULAR AND MOLECULAR] Differential Modulation of Farnesoid X Receptor Signaling Pathway by the Thiazolidinediones

Kaimal, R., Song, X., Yan, B., King, R., Deng, R. — 2009-06-22

Thiazolidinediones (TZD), including troglitazone, rosiglitazone, and pioglitazone, are agonists of peroxisome proliferator-activated receptor (PPAR)- and belong to a class of insulin-sensitizing drugs for type 2 diabetes mellitus. However, member-specific, PPAR-independent activities and toxicity have been reported, especially for troglitazone. Currently, the underlying mechanisms are not fully understood. In this study, we demonstrated that troglitazone but not rosiglitazone or pioglitazone modulated expression of farnesoid X receptor (FXR) target genes bile salt export pump (BSEP) and small heterodimer partner (SHP) in Huh-7 cells. More specifically, troglitazone acted as a partial agonist of FXR to weakly increase BSEP and SHP expression but functioned as a potent antagonist to significantly suppress bile acid-induced expression. Consistent with the finding, troglitazone partially induced but markedly antagonized bile acid-mediated BSEP promoter transactivation. However, such modulating effects were not detected with rosiglitazone or pioglitazone. Using the crystal structure of ligand-bound FXR ligand binding domain (LBD), molecular docking predicted that troglitazone, but not rosiglitazone or pioglitazone, could form a stable complex with FXR LBD. The specific -tocopherol side chain of troglitazone significantly contributed to the formation of such a stable complex through extensive interactions with FXR LBD. The docking model was further validated by functional analyses of a series of docking-guided FXR mutants. In summary, the data demonstrated that troglitazone, but not rosiglitazone or pioglitazone, was an FXR modulator and potently antagonized bile acid-induced expression of FXR target genes. Such differential modulation of FXR signaling pathway by TZDs may represent one of the mechanisms for member-specific, PPAR-independent activities and toxicity.

[BEHAVIORAL PHARMACOLOGY] Pharmacokinetics and Behavioral Effects of an Extended-Release, Liposome-Encapsulated Preparation of Oxymorphone in Rhesus Macaques

2009-06-22

The objectives of the study were to determine the pharmacokinetics of oxymorphone (oxy) and of ammonium sulfate-loaded, liposome-encapsulated oxymorphone (LE-ASG oxy) and to evaluate the behavioral effects of both opioid preparations by using ethographic evaluation specific to rhesus monkeys. Rhesus monkeys (n = 8) were injected with 2.0 mg/kg LE-ASG oxy s.c.. Blood samples were collected at serial time points up to 144 h in six monkeys and up to 456 h in two monkeys. Separate groups of monkeys were injected with 0.1 mg/kg oxy s.c. (n = 4) or i.v. (n = 5). Blood samples were collected at serial time points up to 24 h after injection. Pharmacokinetic parameters were calculated by using commercially available software. Behavior was recorded in a different group of 10 monkeys administered LE-ASG oxy (2.0 mg/kg s.c.) or oxy (0.1 mg/kg s.c.) on separate occasions. Behavioral evaluations were made at serial time points while monkeys were in an extended cage with a compatible stimulus animal. Oxymorphone was rapidly eliminated from the serum in the oxy group. Measurable drug was present in serum for up to 4 h after oxy was administered subcutaneously or intravenously. LE-ASG oxy was present in serum in measurable concentrations for more than 2 weeks. Neither oxy nor LE-ASG oxy produced observable sedation. LE-ASG oxy decreased some environmentally directed behaviors, but this drug formulation increased watchfulness, decreased self-directed and elimination behaviors, increased nonspecific social contact, and decreased threat behaviors. LE-ASG oxy persisted for an extended period in rhesus monkey serum and produced behavioral changes consistent with this opioid.

[NEUROPHARMACOLOGY] Blockade of Orexin-1 Receptors Attenuates Orexin-2 Receptor Antagonism-Induced Sleep Promotion in the Rat

2009-06-22

Orexins are peptides produced by lateral hypothalamic neurons that exert a prominent role in the maintenance of wakefulness by activating orexin-1 (OX1R) and orexin-2 (OX2R) receptor located in wake-active structures. Pharmacological blockade of both receptors by the dual OX1/2R antagonist (2R)-2-[(1S)-6,7-dimethoxy-1-{2-[4-(trifluoromethyl)phenyl]ethyl}-3,4-dihydroisoquinolin-2(1H)-yl]-N-methyl-2-phenylethanamide (almorexant) has been shown to promote sleep in animals and humans during their active period. However, the selective distribution of OX1R and OX2R in distinct neuronal circuits may result in a differential impact of these receptors in sleep-wake modulation. The respective role of OX1R and OX2R on sleep in correlation with monoamine release was evaluated in rats treated with selective antagonists alone or in combination. When administered in either phase of the light/dark cycle, the OX2R antagonist 1-(2,4-dibromophenyl)-3-[(4S,5S)-2,2-dimethyl-4-phenyl-1,3-dioxan-5-yl]urea (JNJ-10397049) decreased the latency for persistent sleep and increased nonrapid eye movement and rapid eye movement sleep time. Almorexant produced less hypnotic activity, whereas the OX1R antagonist 1-(6,8-difluoro-2-methylquinolin-4-yl)-3-[4-(dimethylamino)phenyl]urea (SB-408124) had no effect. Microdialysis studies showed that either OX2R or OX1/2R antagonism decreased extracellular histamine concentration in the lateral hypothalamus, whereas both OX1R and OX1/2R antagonists increased dopamine release in the prefrontal cortex. Finally, coadministration of the OX1R with the OX2R antagonist greatly attenuated the sleep-promoting effects of the OX2R antagonist. These results indicate that blockade of OX2R is sufficient to initiate and prolong sleep, consistent with the hypothesis of a deactivation of the histaminergic system. In addition, it is suggested that simultaneous inhibition of OX1R attenuates the sleep-promoting effects mediated by selective OX2R blockade, possibly correlated with dopaminergic neurotransmission.

[NEUROPHARMACOLOGY] L-Type Calcium Channels and Calcium/Calmodulin-Dependent Kinase II Differentially Mediate Behaviors Associated with Nicotine Withdrawal in Mice

Jackson, K. J., Damaj, M. I. — 2009-06-22

Smoking is a widespread health problem. Because the nicotine withdrawal syndrome is a major contributor to continued smoking and relapse, it is important to understand the molecular and behavioral mechanisms of nicotine withdrawal to generate more effective smoking cessation therapies. Studies suggest a role for calcium-dependent mechanisms, such as L-type calcium channels and calcium/calmodulin-dependent protein kinase II (CaMKII), in the effects of nicotine dependence; however, the role of these mechanisms in nicotine-mediated behaviors is unclear. Thus, the goal of this study was to elucidate the role of L-type calcium channels and CaMKII in nicotine withdrawal behaviors. Using both pharmacological and genetic methods, our results show that L-type calcium channels are involved in physical, but not affective, nicotine withdrawal behaviors. Although our data do provide evidence of a role for CaMKII in nicotine withdrawal behaviors, our pharmacological and genetic assessments yielded different results concerning the specific role of the kinase. Pharmacological data suggest that CaMKII is involved in somatic signs and affective nicotine withdrawal, and activity level is decreased after nicotine withdrawal, whereas the genetic assessments yielded results suggesting that CaMKII is involved only in the anxiety-related response, yet the kinase activity may be increased after nicotine withdrawal; thus, future studies are necessary to clarify the precise behavioral specifics of the relevance of CaMKII in nicotine withdrawal behaviors. Overall, our data show that L-type calcium channels and CaMKII are relevant in nicotine withdrawal and differentially mediate nicotine withdrawal behaviors.

[CELLULAR AND MOLECULAR] Gi-Dependent Cell Signaling Responses of the Human P2Y14 Receptor in Model Cell Systems

Fricks, I. P., Carter, R. L., Lazarowski, E. R., Harden, T. K. — 2009-06-22

Eight G protein-coupled receptors comprise the P2Y receptor family of cell signaling proteins. The goal of the current study was to define native cell signaling pathways regulated by the uridine nucleotide sugar-activated P2Y₁₄ receptor (P2Y₁₄-R). The P2Y₁₄-R was stably expressed in human embryonic kidney (HEK) 293 and C6 rat glioma cells by retroviral infection. Nucleotide sugar-dependent P2Y₁₄-R activation was examined by measuring inhibition of forskolin-stimulated cAMP accumulation. The effect of P2Y₁₄-R activation on mitogen-activated protein kinase signaling also was studied in P2Y₁₄-HEK293 cells and in differentiated HL-60 human myeloid leukemia cells. UDP-Glc, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine promoted inhibition of forskolin-stimulated cAMP accumulation in P2Y₁₄-HEK293 and P2Y₁₄-C6 cells, and this signaling effect was abolished by pretreatment of cells with pertussis toxin. Inhibition of cAMP formation by nucleotide sugars also was observed in direct assays of adenylyl cyclase activity in membranes prepared from P2Y₁₄-C6 cells. UDP-Glc promoted concentration-dependent and pertussis toxin-sensitive extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y₁₄-HEK293 cells. P2Y₁₄-R mRNA was not observed in wild-type HL-60 cells but was readily detected in dimethyl sulfoxide-differentiated cells. Consistent with this observation, no effect of UDP-Glc was observed in wild-type HL-60 cells, but UDP-Glc-promoted pertussis toxin-sensitive activation of ERK1/2 occurred after differentiation. These results illustrate that the human P2Y₁₄-R signals through G_i to inhibit adenylyl cyclase, and P2Y₁₄-R activation also leads to ERK1/2 activation. This work also identifies two stable P2Y₁₄-R-expressing cell lines and differentiated HL-60 cells as model systems for the study of P2Y₁₄-R-dependent signal transduction.

[ENDOCRINE AND DIABETES] Mechanism-Based Pharmacokinetic/Pharmacodynamic Model of Parathyroid Hormone-Calcium Homeostasis in Rats and Humans

Abraham, A. K., Mager, D. E., Gao, X., Li, M., Healy, D. R., Maurer, T. S. — 2009-06-22

The purpose of this study was to develop a mechanism-based pharmacokinetic/pharmacodynamic model that describes the regulation of the parathyroid hormone (PTH)-Ca²⁺ system in rats and humans. Temporal concentration data for endogenous PTH and Ca²⁺ were extracted from literature for rats (normal adult males) and humans. In addition, exogenous PTH was administered subcutaneously to male Sprague-Dawley rats with jugular vein catheters, and plasma concentrations were measured over time. A mathematical model was developed and fitted simultaneously to endogenous PTH, Ca²⁺, and exogenous PTH concentrations in rats. Ca²⁺ concentrations were described using a turnover model, with its depletion being induced by a chelating agent, and PTH concentrations were characterized using a precursor-dependent indirect response model. The same structural model was used for fitting data obtained in humans. PTH stimulation was driven by occupancy of the Ca²⁺ sensing receptor, and lowering of physiological Ca²⁺ concentrations increased PTH secretion, with PTH profiles being adequately described by the model. PTH stimulatory capacity was baseline-dependent in rats [S_{max_rats} = 34.8 x PTH₀] and humans [S_{max_humans} = 392/PTH₀]. Modeling results suggest that normal rats are twice as sensitive to Ca²⁺-induced PTH stimulation compared with humans. In conclusion, the developed model adequately characterizes the PTH-Ca²⁺ regulation across species and may be useful in the development of therapeutic drugs targeting this system.

[NEUROPHARMACOLOGY] Unique Antipsychotic Activities of the Selective Metabotropic Glutamate Receptor 1 Allosteric Antagonist 2-Cyclopropyl-5-[1-(2-fluoro-3-pyridinyl)-5-methyl-1H-1,2,3-triazol-4-yl]-2,3-dihydro-1H-isoindol-1-one

2009-06-22

A newly discovered metabotropic glutamate receptor (mGluR) 1 allosteric antagonist, 2-cyclopropyl-5-[1-(2-fluoro-3-pyridinyl)-5-methyl-1H-1,2,3-triazol-4-yl]-2,3-dihydro-1H-isoindol-1-one (CFMTI), was tested both in vitro and in vivo for its pharmacological effects. CFMTI demonstrated potent and selective antagonistic activity on mGluR1 in vitro and in vivo after oral administration. CFMTI inhibited l-glutamate-induced intracellular Ca²⁺ mobilization in Chinese hamster ovary cells expressing human and rat mGluR1a, with IC₅₀ values of 2.6 and 2.3 nM, respectively. The selectivity of CFMTI to mGluR1 over mGluR5 was >2000-fold, and CFMTI at 10 µM showed no agonistic or antagonistic activities toward other mGluR subtypes and other receptors. It antagonized face-washing behavior in mice induced by (S)-3,5-dihidroxyphenylglycine at a dose range of 3 to 30 mg/kg, for which receptor occupancy was 73 to 94%. As with the classical neuroleptic haloperidol and an atypical antipsychotic, clozapine, orally administered CFMTI reduced methamphetamine-induced hyperlocomotion and disruption of prepulse inhibition (PPI) at the same dose range as required to antagonize the face-washing behavior. CFMTI and clozapine improved ketamine-induced hyperlocomotion, PPI disruption and (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801)-induced social withdrawal without any cataleptogenic activities, whereas haloperidol only improved ketamine-induced hyperlocomotion. CFMTI, unlike clozapine, caused neither hypolocomotion nor motor incoordination at therapeutic doses. In c-fos expression studies, CFMTI and clozapine increased the number of fos-positive neurons in the nucleus accumbens and medial prefrontal cortex but not in the dorsolateral striatum. These results suggest that the antipsychotic activities of mGluR1 antagonists are more similar to those of atypical antipsychotics than those of typical antipsychotics.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Renal Organic Anion Transporter-Mediated Drug-Drug Interaction between Gemcabene and Quinapril

Yuan, H., Feng, B., Yu, Y., Chupka, J., Zheng, J. Y., Heath, T. G., Bond, B. R. — 2009-06-22

In humans and rats, a synergistic blood pressure reduction was observed when the fibrate gemcabene (CI-1027) was coadministered with the angiotensin-converting enzyme inhibitor quinapril. In a quinapril (3 mg/kg) pharmacokinetic rat study, there was a 40% decrease in urinary excretion and a 53% increase in plasma area under the curve from 0 to 24 h of the active metabolite quinaprilat when coadministered with gemcabene (30 mg/kg). This observation revealed a possible transporter-mediated drug-drug interaction (DDI) between gemcabene and quinapril. This led to a series of studies investigating the underlying clearance mechanisms associated with these compounds intended to elucidate renal transporter interactions between quinapril and gemcabene. In vitro transporter studies using human embryonic kidney 293 cells transfected with human or rat organic anion transporter 3 (hOAT3, rOat3) revealed that quinaprilat is a substrate in both species, with a K_m value of 13.4 µM for hOAT3. Subsequent studies discovered that gemcabene inhibited quinaprilat uptake by hOAT3 and rOat3 at IC₅₀ values of 35 and 48 µM, respectively. Moreover, gemcabene acylglucuronide, the major metabolite of gemcabene glucuronidation, also inhibited hOAT3- and rOat3-mediated uptake of quinaprilat at IC₅₀ values of 197 and 133 µM, respectively. High plasma concentrations of gemcabene (>100 µM) achieved in humans and rats upon oral dosing corroborate with gemcabene inhibition of renal OAT3-mediated secretion of quinaprilat in vitro. This investigation established that a DDI between gemcabene and quinapril involving inhibition of renal transporters and subsequent elevation in plasma concentrations of quinaprilat is responsible for the apparent synergistic blood pressure reduction observed with these compounds.

[NEUROPHARMACOLOGY] Single-Channel Analysis of Ethanol Enhancement of Glycine Receptor Function

Welsh, B. T., Goldstein, B. E., Mihic, S. J. — 2009-06-22

The glycine receptor (GlyR) is a ligand-gated ion channel and member of the nicotinic acetylcholine receptor superfamily. Acting as allosteric modulators of receptor function, drugs such as alcohol and volatile anesthetics enhance the function of GlyRs. The actions of these drugs at inhibitory receptors in the brain and spinal cord are thought to produce many of the physiological effects associated with their use. The actions of ethanol on the GlyR have been well studied on the macroscopic, whole cell level. We examined the effects of 3 µM glycine ± 50 or 200 mM ethanol on outside-out patches pulled from Xenopus laevis oocytes expressing wild-type 1 GlyR, to determine the effects of alcohol at the single-channel level. Alcohol enhanced GlyR function in a very specific manner. It had minimal effects on open and closed dwell times and likelihood. Instead, ethanol potentiated GlyR function almost exclusively by increasing burst durations and increasing the number of channel openings per burst, without affecting the percentage of open time within bursts. Kinetic modeling suggests that ethanol increases burst durations by decreasing the rate of glycine unbinding.

[CARDIOVASCULAR] Mechanical Strain Induces Expression of C-Reactive Protein in Human Blood Vessels

Huang, G., Luo, C., Gu, X., Wu, Z., Wang, Z., Du, Z., Hu, C., Tang, L. — 2009-06-22

C-reactive protein (CRP) is a powerful independent risk factor for cardiovascular diseases. Elevated mechanical strain on vessels induces the local expression of proinflammatory cytokines. We hypothesized that mechanical strain on vessels may induce local CRP expression. Human saphenous vein and internal mammary artery (IMA) rings were stretched in vitro with a mechanical strength of 1, 3, or 5 g. Reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay results showed that mechanical stretching significantly induced CRP mRNA and protein expression in the saphenous vein and IMA rings in a strength-dependent manner reaching a maximum at a mechanical strength of 3 g, but CRP expression returned at strengths of >5 g. In vessels, mechanical strain-induced CRP expression was blocked by two stretch-activated ion channel (SAC) blockers: GdCl₃ and streptomycin. Mechanical strain also increased activation of nuclear factor B (NF-B), which was detected with a nonradioactive NF-B p50/p65 EZ-TFA transcription factor assay. Mechanical strain-induced NF-B activation was blocked by SAC blockers and the NF-B inhibitor (SN50, H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH). SN50 also blocked mechanical strain-induced CRP expression in vessels. In conclusion, mechanical strain induces CRP expression in IMAs and saphenous veins by activating the SAC-induced NF-B pathway.

[CELLULAR AND MOLECULAR] The Neuroactive Peptide N-Acetylaspartylglutamate Is Not an Agonist at the Metabotropic Glutamate Receptor Subtype 3 of Metabotropic Glutamate Receptor

Chopra, M., Yao, Y., Blake, T. J., Hampson, David. R., Johnson, E. C. — 2009-06-22

The peptide N-acetylaspartylglutamate (NAAG) is present in high concentrations in the mammalian central nervous system. Various mechanisms have been proposed for its action, including selective activation of the metabotropic glutamate receptor (mGluR) subtype 3, its action at the N-methyl-d-aspartate receptor, or the production of glutamate by its hydrolysis catalyzed by an extracellular protease. To re-examine its agonist activity at mGluR3, we coexpressed human or rat mGluR3 with G protein inward rectifying channels in Xenopus laevis oocytes. High-performance liquid chromatography analysis of commercial sources of NAAG showed 0.38 to 0.48% glutamate contamination. Although both human and rat mGluR3 were highly sensitive to glutamate, with EC₅₀ values of 58 and 28 nM, respectively, purified NAAG (100 µM) had little activity (7.7% of full activation by glutamate). Only in the millimolar range did it show significant activity, possibly due to residual traces of glutamate remaining in the purified NAAG preparations. In contrast, the unpurified NAAG sample did produce a full agonist response with mGluR3 coexpressed with G₁₅, with an EC₅₀ of 120 µM, as measured by a calcium release assay. This response can be explained by the 0.38 to 0.48% glutamate contamination. Our results suggest that NAAG may not have a direct agonist activity at the mGluR3 receptor. Thus, several in vivo and in vitro published results that did not address the issue of glutamate contamination of NAAG preparations may need to be re-evaluated.

[NEUROPHARMACOLOGY] Use-Dependent Block of Voltage-Gated Cav2.1 Ca2+ Channels by Petasins and Eudesmol Isomers

Horak, S., Koschak, A., Stuppner, H., Striessnig, J. — 2009-06-22

Migraine is a frequent and often disabling disease. Treatment is unsatisfactory in many patients. A disturbed dynamic balance between excitatory and inhibitory signal processing with enhanced cortical activity probably underlies common forms of migraine. Presynaptic voltage-gated Ca²⁺ channels are critical determinants of neurotransmitter release and also contribute to trigeminovascular signal transduction. Because clinical evidence exists for migraine-prophylactic actions of Petasites hybridus extracts, we investigated whether petasins comprising the main constituents of the extract inhibit currents through presynaptic Ca_v2.1 channels expressed in Xenopus laevis oocytes. P. hybridus extract (0.02 mg/ml), petasin, neopetasin, isopetasin, S-petasin, and iso-S-petasin (50 µM) were weak tonic blockers of Ca_v2.1-mediated barium currents (I_Ba) during infrequent depolarizations (0.1 Hz), but their inhibitory potency increased at higher stimulation rates (1 Hz), indicating preferential block of open and/or inactivated channels. Sulfur-containing compounds (S-petasin, Iso-S-petasin) were the most potent significantly promoting the accumulation of Ca_v2.1 channel in inactivated states during pulse trains (I_Ba decrease during 1-Hz pulse trains: control, 45%, S-petasin, 79%; iso-S-petasin, 80%). For the Eucalyptus williamsiania sesquiterpenes - and -eudesmol, a comparable use-dependent inhibition was found in addition to a tonic block component. -Eudesmol and petasins accelerated the voltage-dependent inactivation of Ca_v2.1 channels during depolarizations. We demonstrate that S-petasin, iso-S-petasin, and eudesmol are Ca_v2.1 channel inhibitors preferentially acting as use-dependent channel blockers and with the sulfur-containing substituent in position 3 of the petasins serving as important functional feature. The Ca_v2.1-inhibitory properties of these petasins may contribute to migraine-prophylactic properties described for P. hybridus extracts.

[NEUROPHARMACOLOGY] Pharmacological Modulation of Glutamate Transmission in a Rat Model of L-DOPA-Induced Dyskinesia: Effects on Motor Behavior and Striatal Nuclear Signaling

Rylander, D., Recchia, A., Mela, F., Dekundy, A., Danysz, W., Cenci, M. A. — 2009-06-22

l-DOPA-induced dyskinesia (LID) in Parkinson's disease has been linked to altered dopamine and glutamate transmission within the basal ganglia. In the present study, we compared compounds targeting specific subtypes of glutamate receptors or calcium channels for their ability to attenuate LID and the associated activation of striatal nuclear signaling and gene expression in the rat. Rats with 6-hydroxydopamine lesions were treated acutely or chronically with l-DOPA in combination with the following selective compounds: antagonists of group I metabotropic glutamate receptors (mGluR), (2-methyl-1,3-thiazol-4-yl) ethynylpyridine (MTEP) for mGluR5 and (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methane sulfonate (EMQMCM) for mGluR1; an agonist of group II mGluR, 1R,4R,5S,6R-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268); N-methyl-d-aspartate (NMDA)-R2B subunit (NR2B)-selective NMDA receptor antagonists 1-[2-(4-hydroxyphenoxy)ethyl]-4-[(4-methylphenyl)methyl]-4-piperidinol hydrochloride (Ro631908) and (±)-(R^*,S^*)--(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)1-piperidine propanol (Ro256981); and an L-type calcium channel antagonist, 4-(4-benzofurazanyl)-1,-4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid methyl 1-methylethyl ester (isradipine). Dyskinesia and rotarod performance were monitored during chronic drug treatment. The striatal expression of phospho-extracellular signal-regulated kinase (ERK) 1/2 and mitogen- and stress-activated kinase (MSK)-1, or prodynorphin mRNA was examined after acute or chronic treatment, respectively. In the acute treatment studies, only MTEP and EMQMCM significantly attenuated l-DOPA-induced phospho-ERK1/2 and/or phospho-MSK-1 expression, with MTEP being the most effective (70–80% reduction). In the chronic experiment, only MTEP significantly attenuated dyskinesia without adverse motor effects, whereas EMQMCM and LY379268 inhibited the l-DOPA-induced improvement in rotarod performance. The NR2B antagonist had positive antiakinetic effects but did not reduce dyskinesia. Only MTEP blocked the up-regulation of prodynorphin mRNA induced by l-DOPA. Among the pharmacological treatments examined, MTEP was most effective in inhibiting LID and the associated molecular alterations. Antagonism of mGluR5 seems to be a promising strategy to reduce dyskinesia in Parkinson's disease.

[NEUROPHARMACOLOGY] Preclinical Evaluation of the Abuse Potential of the Analgesic Bicifadine

2009-06-22

The abuse liability of the analgesic bicifadine was investigated in animal models used to predict the abuse potential of psychostimulants in humans. Bicifadine, cocaine, d-amphetamine, bupropion, and desipramine were evaluated for the production of cocaine-like discriminative stimulus effects in rats. Cocaine, d-amphetamine, and bupropion dose-dependently and fully substituted for cocaine. Bicifadine and desipramine produced a maximum mean cocaine-lever selection of 80 and 69%, respectively, but doses yielding peak substitution strongly suppressed response rates. Microdialysis studies in normal waking rats indicated that d-amphetamine increased dopamine levels in the nucleus accumbens and striatum to a much greater degree than bicifadine, but bicifadine increased 5-hydroxytryptamine levels in the nucleus accumbens and striatum more than d-amphetamine. Bicifadine was also tested for intravenous self-administration in rhesus monkeys experienced with cocaine administration. Reinforcing effects of bicifadine were observed in only two of four subjects, whereas cocaine, d-amphetamine, and bupropion served as reinforcers in all four monkeys. When evaluated under a progressive ratio procedure, no dose of bicifadine maintained responding to the extent of cocaine, d-amphetamine, or bupropion. The discriminative stimulus effects associated with bicifadine were similar, but not identical, to those of psychostimulants. Although bicifadine maintained self-administration behavior in some subjects, its reinforcing efficacy was very low relative to cocaine, d-amphetamine, and bupropion. These results are consistent with the microdialysis findings of lower dopamine levels and higher 5-hydroxytryptamine levels after administration of bicifadine relative to d-amphetamine. Overall, the current findings support a low abuse potential of bicifadine, more resembling that of antidepressants than psychostimulants.

[CELLULAR AND MOLECULAR] 5,5-Dimethyl-3-(5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1-phenyl-3-(trifluoromethyl)-3,5,6,7-tetrahydro-1H-indole-2,4-dione, a Potent Inhibitor for Mammalian Elongase of Long-Chain Fatty Acids Family 6: Examination of Its Potential Utility as a Pharmacological Tool

2009-06-22

Long-chain fatty acid elongases reside in the endoplasmic reticulum and are responsible for the rate-limiting step of the elongation of long-chain fatty acids. The elongase of long-chain fatty acids (ELOVL) family 6 (ELOVL6) is involved in the elongation of saturated and monosaturated fatty acids. Increased expression of ELOVL6 in ob/ob mice suggests a role for ELOVL6 in metabolic disorders. Furthermore, ELOVL6-deficient mice are protected from high-fat diet-induced insulin resistance, which suggests that ELOVL6 might be a new therapeutic target for diabetes. As reported previously, we developed a high-throughput screening system for fatty acid elongases and discovered lead chemicals that possess inhibitory activities against ELOVL6. In the present study, we examined in detail the biochemical and pharmacological properties of 5,5-dimethyl-3-(5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1-phenyl-3-(trifluoromethyl)-3,5,6,7-tetrahydro-1H-indole-2,4-dione (Compound-A), a potent inhibitor of ELOVL6. In in vitro assays, Compound-A dose-dependently inhibited mouse and human ELOVL6 and displayed more than 30-fold greater selectivity for ELOVL6 over the other ELOVL family members. In addition, Compound-A effectively reduced the elongation index of fatty acids of hepatocytes, suggesting that Compound-A penetrates the cell wall and inhibits ELOVL6. More importantly, upon oral administration to mice, Compound-A showed high plasma and liver exposure and potently reduced the elongation index of the fatty acids of the liver. This is the first study to report a potent and selective inhibitor of mammalian elongases. Furthermore, Compound-A seems to be a useful tool to further understand the physiological roles of ELOVL6 and to evaluate the therapeutic potential of an ELOVL6 inhibitor.

[NEUROPHARMACOLOGY] In Vitro Pharmacological Characterization of a Novel Allosteric Modulator of {alpha}7 Neuronal Acetylcholine Receptor, 4-(5-(4-Chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), Exhibiting Unique Pharmacological Profile

2009-06-22

Targeting 7 neuronal acetylcholine receptors (nAChRs) with selective agonists and positive allosteric modulators (PAMs) is considered a therapeutic approach for managing cognitive deficits in schizophrenia and Alzheimer's disease. In this study, we describe a novel type II 7 PAM, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), that exhibits a unique pharmacological profile. In oocytes expressing 7 nAChRs, A-867744 potentiated acetylcholine (ACh)-evoked currents, with an EC₅₀ value of ~1 µM. At highest concentrations of A-867744 tested, ACh-evoked currents were essentially nondecaying. At lower concentrations, no evidence of a distinct secondary component was evident in contrast to 4-naphthalen-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), another type II 7 PAM. In the presence of A-867744, ACh concentration responses were potentiated by increases in potency, Hill slope, and maximal efficacy. When examined in rat hippocampus CA1 stratum radiatum interneurons or dentate gyrus granule cells, A-867744 (10 µM) increased choline-evoked 7 currents and recovery from inhibition/desensitization, and enhanced spontaneous inhibitory postsynaptic current activity. A-867744, like other 7 PAMs tested [1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)urea (NS1738), TQS, and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596)], did not displace the binding of [³H]methyllycaconitine to rat cortex 7^* nAChRs. However, unlike these PAMs, A-867744 displaced the binding of the agonist [³H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539) in rat cortex, with a K_i value of 23 nM. A-867744 neither increased agonist-evoked responses nor displaced the binding of [³H]A-585539 in an 7/5-hydroxytryptamine₃ (7/5-HT₃) chimera, suggesting an interaction distinct from the 7 N terminus or M2-3 loop. In addition, A-867744 failed to potentiate responses mediated by 5-HT_3A or 3β4 and 4β2 nAChRs. In summary, this study identifies a novel and selective 7 PAM showing activity at recombinant and native 7 nAChRs exhibiting a unique pharmacological interaction with the receptor.

[NEUROPHARMACOLOGY] Discovery and Pharmacological Characterization of a Small-Molecule Antagonist at Neuromedin U Receptor NMUR2

2009-06-22

Neuromedin U (NMU), through its cognate receptor NMUR2 in the central nervous system, regulates several important physiological functions, including energy balance, stress response, and nociception. By random screening of our corporate compound collection with a ligand binding assay, we discovered (R)-5'-(phenylaminocarbonylamino)spiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine] (R-PSOP), a highly potent and selective NMUR2 antagonist. R-PSOP is a nonpeptidic small-molecule with the chemical composition C₂₀N₄O₂H₂₂. In competition binding experiments, this compound was found to bind to NMUR2 with high affinity; the K_i values were determined to be 52 and 32 nM for the human and rat NMUR2, respectively. Moreover, in functional assays measuring phosphoinositide turnover or intracellular calcium mobilization, R-PSOP strongly inhibited the responses stimulated by peptide agonists NMU-25, NMU-23, and NMU-8 in human embryonic kidney 293 cells expressing NMUR2. From Schild analyses, the functional K_b values for R-PSOP were determined to be 92 and 155 nM at human and rat NMUR2, respectively. Highly selective for NMUR2, R-PSOP exhibited low affinity to the other subtype of NMU receptor, NMUR1, with a K_i value >10 µM. R-PSOP in vivo attenuated NMU-23-evoked nociceptive responses in a rat spinal reflex preparation. To our knowledge, this is the first antagonist ever reported for NMU receptors. This compound could serve as a valuable tool for further understanding the physiological and pathophysiological roles of NMU system, while providing a chemical starting point that may lead to development of new therapeutics for treatment of eating disorders, obesity, pain, and stress-related disorders.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Heat Shock Protein 90 Inhibitor Induces Apoptosis and Attenuates Activation of Hepatic Stellate Cells

Myung, S. J., Yoon, J.-H., Kim, B. H., Lee, J.-H., Jung, E. U., Lee, H.-S. — 2009-06-22

Activated hepatic stellate cells (HSCs) are major participants in hepatic fibrosis; thus, the induction of HSC apoptosis has been proposed as an antifibrotic treatment strategy. Heat shock protein (Hsp) 90 is a molecular chaperone that stabilizes major signal transduction proteins, and its inhibitors have antitumor activity. In this study, the susceptibility of HSCs to an Hsp90 inhibitor was evaluated. LX-2 cells, an immortalized human HSC line, 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an Hsp90 inhibitor, and monensin, an acidic sphingomyelinase inhibitor, were used in this study. Cellular apoptosis was quantified by 4',6-diamidino-2-phenylindole dihydrochloride staining, and signaling cascades were explored using immunoblotting and immunoprecipitation techniques. Nuclear factor (NF) B activities were evaluated by immunofluorescent microscopy and enzyme-linked immunosorbent assay. Collagen 1 and -smooth muscle actin expressions were determined by real-time reverse transcription-polymerase chain reaction and immunoblotting, respectively. It was found that 17AAG induced HSC apoptosis and that caspase 8 cleavage preceded the downstream activation of apoptotic signaling cascades. Furthermore, this caspase 8 activation was dependent on ceramide generation by acidic sphingomyelinase. In addition, 17AAG prevented NFB nuclear translocation and activation, specifically by inducing complex formation between NFB and the glucocorticoid receptor. In accordance, NFB-dependent cellular FLICE-like inhibitory protein expression level was found to be reduced by 17AAG. Finally, 17AAG down-regulated collagen 1 and -smooth muscle actin expression levels in HSCs before inducing apoptosis. These results demonstrate that the Hsp90 inhibitor induces HSC apoptosis via a sphingomyelinase- and NFB-dependent mechanism. Because this inhibitor also reduces HSC activation before apoptosis, Hsp90 inhibitor treatment might be therapeutically useful as an antifibrotic strategy in a variety of liver diseases.

[ENDOCRINE AND DIABETES] The Intracellular II-III Loops of Cav1.2 and Cav1.3 Uncouple L-Type Voltage-Gated Ca2+ Channels from Glucagon-Like Peptide-1 Potentiation of Insulin Secretion in INS-1 Cells via Displacement from Lipid Rafts

2009-06-22

L-type Ca²⁺ channels play a key role in the integration of physiological signals regulating insulin secretion that probably requires their localization to specific subdomains of the plasma membrane. We investigated the role of the intracellular II-III loop domains of the L-type channels Ca_v1.2 and 1.3 in coupling of Ca²⁺ influx with glucose-stimulated insulin secretion (GSIS) potentiated by the incretin hormone glucagon-like peptide (GLP)-1. In INS-1 cell lines expressing the Ca_v1.2/II-III or Ca_v1.3/II-III peptides, GLP-1 potentiation of GSIS was inhibited markedly, coincident with a decrease in GLP-1-stimulated cAMP accumulation and the redistribution of Ca_v1.2 and Ca_v1.3 out of lipid rafts. Neither the Ca_v1.2/II-III nor the Ca_v1.3/II-III peptide decreased L-type current density compared with untransfected INS-1 cells. GLP-1 potentiation of GSIS was restored by the L-type channel agonist 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL-64176). In contrast, potentiation of GSIS by 8-bromo-cAMP (8-Br-cAMP) was inhibited in Ca_v1.2/II-III but not Ca_v1.3/II-III cells. These differences may involve unique protein-protein interactions because the Ca_v1.2/II-III peptide, but not the Ca_v1.3/II-III peptide, immunoprecipitates Rab3-interacting molecule (RIM) 2 from INS-1 cell lysates. RIM2, and its binding partner Piccolo, localize to lipid rafts, and they may serve as anchors for Ca_v1.2 localization to lipid rafts in INS-1 cells. These findings suggest that the II-III interdomain loops of Ca_v1.2, and possibly Ca_v1.3, direct these channels to membrane microdomains in which the proteins that mediate potentiation of GSIS by GLP-1 and 8-Br-cAMP assemble.

[NEUROPHARMACOLOGY] Characterization of the Potent and Highly Selective A2A Receptor Antagonists Preladenant and SCH 412348 [7-[2-[4-2,4-Difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] in Rodent Models of Movement Disorders and Depression

2009-06-22

The adenosine A_2A receptor has been implicated in the underlying biology of various neurological and psychiatric disorders, including Parkinson's disease (PD) and depression. Preladenant and SCH 412348 [7-[2-[4-2,4-difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] are potent competitive antagonists of the human A_2A receptor (K_i = 1.1 and 0.6 nM, respectively) and have >1000-fold selectivity over all other adenosine receptors, making these compounds the most selective A_2A receptor antagonists reported to date. Both compounds attenuate hypolocomotion induced by the A_2A receptor agonist CGS-21680 [2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine], suggesting that they inhibit A_2A receptor activity in vivo. Their high degree of selectivity and robust in vivo activity make preladenant and SCH 412348 useful tools to investigate the role of the A_2A receptor system in animal models of PD and depression. Oral administration of preladenant and SCH 412348 (0.1–1 mg/kg) to rats potentiated 3,4-dihydroxy-l-phenylalanine (l-Dopa)-induced contralateral rotations after 6-hydroxydopamine lesions in the medial forebrain bundle and potently attenuated the cataleptic effects of haloperidol. Preladenant (1 mg/kg) inhibited l-Dopa-induced behavioral sensitization after repeated daily administration, which suggests a reduced risk of the development of dyskinesias. Finally, preladenant and SCH 412348 exhibited antidepressant-like profiles in models of behavioral despair, namely the mouse tail suspension test and the mouse and rat forced swim test. These studies demonstrate that preladenant and SCH 412348 are potent and selective A_2A receptor antagonists and provide further evidence of the potential therapeutic benefits of A_2A receptor inhibition in PD (with reduced risk of dyskinesias) and depression (one of the primary nonmotor symptoms of PD).

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] What Are Next Generation Innovative Therapeutic Targets? Clues from Genetic, Structural, Physicochemical, and Systems Profiles of Successful Targets

Zhu, F., Han, L., Zheng, C., Xie, B., Tammi, M. T., Yang, S., Wei, Y., Chen, Y. — 2009-06-22

Low target discovery rate has been linked to inadequate consideration of multiple factors that collectively contribute to druggability. These factors include sequence, structural, physicochemical, and systems profiles. Methods individually exploring each of these profiles for target identification have been developed, but they have not been collectively used. We evaluated the collective capability of these methods in identifying promising targets from 1019 research targets based on the multiple profiles of up to 348 successful targets. The collective method combining at least three profiles identified 50, 25, 10, and 4% of the 30, 84, 41, and 864 phase III, II, I, and nonclinical trial targets as promising, including eight to nine targets of positive phase III results. This method dropped 89% of the 19 discontinued clinical trial targets and 97% of the 65 targets failed in high-throughput screening or knockout studies. Collective consideration of multiple profiles demonstrated promising potential in identifying innovative targets.

[BEHAVIORAL PHARMACOLOGY] A Receptor Mechanism for Methamphetamine Action in Dopamine Transporter Regulation in Brain

Xie, Z., Miller, G. M. — 2009-06-22

This study reveals a novel receptor mechanism for methamphetamine action in dopamine transporter (DAT) regulation. Trace amine-associated receptor 1 (TAAR1) is expressed in brain dopaminergic nuclei and is activated by methamphetamine in vitro. Here, we show that methamphetamine interaction with TAAR1 inhibits [³H]dopamine uptake, enhances or induces [³H]dopamine efflux, and triggers DAT internalization. In time course assays in which methamphetamine and [³H]dopamine were concurrently loaded into cells or synaptosomes or in pretreatment assays in which methamphetamine was washed away before [³H]dopamine loading, methamphetamine caused a distinct inhibition in [³H]dopamine uptake in TAAR1 + DAT-cotransfected cells and in wild-type mouse and rhesus monkey striatal synaptosomes. This distinct uptake inhibition was not observed in DAT-only transfected cells or in TAAR1 knockout mouse striatal synaptosomes. In [³H]dopamine efflux assays using the same cell and synaptosome preparations, methamphetamine enhanced [³H]dopamine efflux at a high loading concentration of [³H]dopamine (1 µM) or induced [³H]dopamine efflux at a low loading concentration of [³H]dopamine (10 nM) in a TAAR1-dependent manner. In DAT biotinylation assays using the same cell and synaptosome preparations, we observed that 1 µM methamphetamine induced DAT internalization in a TAAR1-dependent manner. All these TAAR1-mediated effects of methamphetamine were blocked by the protein kinase inhibitors H89 [N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline] and/or 2-{8-[(dimethylamino) methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methylindol-3-yl)maleimide (Ro32-0432), suggesting that methamphetamine interaction with TAAR1 triggers cellular phosphorylation cascades and leads to the observed effects of methamphetamine on DAT. These findings demonstrate a mediatory role of TAAR1 in methamphetamine action in DAT regulation and implicate this receptor as a potential target of therapeutics drugs for methamphetamine addiction.

[CELLULAR AND MOLECULAR] 1,4-Diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) Enhances the Cytotoxicity of Combretastatin A4 Independently of Mitogen-Activated Protein Kinase Kinase

Quan, H., Liu, H., Li, C., Lou, L. — 2009-06-22

Combretastatin A4 (CA4) is a novel vascular-disrupting agent that has shown promising anticancer effects through its inhibition of microtubule assembly and subsequent disruption of tumor blood flow. In this report, we demonstrate that 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126), a selective inhibitor of mitogen-activated protein kinase kinase (MEK), significantly enhances the cytotoxicity of CA4 in BEL-7402 cells, independently of MEK inhibition. This independence is evidenced by the fact that another, more specific MEK inhibitor, PD0325901 [N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-[2-fluoro-4-iodo-phenylamino]-benzamide], does not have the same effect as U0126. The disassembled microtubules are able to reassemble in the later stages of CA4 treatment, because of the inactivating glucuronidation of CA4. U0126, but not PD0325901, inhibits CA4 glucuronidation, thereby blocking microtubule reassembly and enhancing CA4-induced G₂/M cell-cycle arrest. Consistent with this, U0126 significantly enhances CA4-induced cytotoxicity for cells in which CA4 glucuronidation occurs, but not for cells in which such glucuronidation does not occur. These results suggest that great caution should be exercised when interpreting data obtained using U0126 or when CA4 is combined with inhibitors of glucuronidation in clinical practice. It is most important to note that these findings indicate that the combination of CA4 with inhibitors of glucuronidation may be a novel and rational strategy for cancer therapy.

[CARDIOVASCULAR] Analysis of Pulmonary Vasodilator Responses to SB-772077-B [4-(7-((3-Amino-1-pyrrolidinyl)carbonyl)-1-ethyl-1H-imidazo(4,5-c)pyridin-2-yl)-1,2,5-oxadiazol-3-amine], a Novel Aminofurazan-Based Rho Kinase Inhibitor

Dhaliwal, J. S., Badejo, A. M., Casey, D. B., Murthy, S. N., Kadowitz, P. J. — 2009-06-22

The effects of SB-772077-B [4-(7-((3-amino-1-pyrrolidinyl)carbonyl)-1-ethyl-1H-imidazo(4,5-c)pyridin-2-yl)-1,2,5-oxadiazol-3-amine], an aminofurazan-based Rho kinase inhibitor, on the pulmonary vascular bed and on monocrotaline-induced pulmonary hypertension were investigated in the rat. The intravenous injections of SB-772077-B decreased pulmonary and systemic arterial pressures and increased cardiac output. The decreases in pulmonary arterial pressure were enhanced when pulmonary vascular resistance was increased by U46619 [9,11-dideoxy-11,9-epoxymethanoprostaglandin F₂], hypoxia, or N-nitro-l-arginine methyl ester. SB-772077-B was more potent than Y-27632 [trans-4-[(1R)-1-aminoethyl]-N-4-pyridinyl-cyclohexanecarboxamide dihydrochloride] or fasudil [5-(1,4-diazepane-1-sulfonyl)isoquinoline] in decreasing pulmonary and systemic arterial pressures. The results with SB-772077-B, fasudil, and Y-27632 suggest that Rho kinase is constitutively active and is involved in the regulation of baseline tone and vasoconstrictor responses. Chronic treatment with SB-772077-B attenuated the increase in pulmonary arterial pressure induced by monocrotaline. The intravenous injection of SB-772077-B decreased pulmonary and systemic arterial pressures in rats with monocrotaline-induced pulmonary hypertension. The decreases in pulmonary arterial pressure in response to SB-772077-B in monocrotaline-treated rats were smaller than responses in U46619-infused animals, and the analysis of responses suggests that approximately 60% of the pulmonary hypertensive response is mediated by a Rho kinase-sensitive mechanism. The observation that Rho kinase inhibitors decrease pulmonary arterial pressure when pulmonary vascular resistance is increased by interventions such as hypoxia, U46619, angiotensin II, nitric-oxide synthase inhibition, and Bay K 8644 [S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester] suggest that the vasodilatation is independent of the mechanisms used to increase intracellular calcium and promote vasoconstriction. The present results suggest that SB-772077-B would be beneficial in the treatment of pulmonary hypertensive disorders.

[NEUROPHARMACOLOGY] [N-methyl-3H3]AZ10419369 Binding to the 5-HT1B Receptor: In Vitro Characterization and in Vivo Receptor Occupancy

2009-06-22

Radiotracers suitable for positron emission tomography studies often serve as preclinical tools for in vivo receptor occupancy. The serotonin 1B receptor (5-HT_1B) subtype is a pharmacological target used to discover treatments for various psychiatric and neurological disorders. In psychiatry, 5-HT_1B antagonists may provide novel therapeutics for depression and anxiety. We report on the in vitro and in vivo evaluation of tritiated 5-methyl-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylicacid (4-morpholin-4-yl-phenyl)-amide ([N-methyl-³H₃]AZ10419369), a potent 5-HT_1B radiotracer. [N-methyl-³H₃]-AZ10419369 showed saturable single-site high-affinity in vitro binding (guinea pig, K_d = 0.38 and human, K_d = 0.37) to guinea pig or human 5-HT_1B receptors in recombinant membranes and high-affinity (K_d = 1.9 nM) saturable (B_max = 0.099 pmol/mg protein) binding in membranes from guinea pig striatum. When [N-methyl-³H₃]AZ10419369 was administered to guinea pigs by intravenous bolus, the measured radioactivity was up to 5-fold higher in brain areas containing the 5-HT_1B receptor (striatum/globus pallidus, midbrain, hypothalamus, and frontal cortex) compared with the cerebellum, the nonspecific binding region. Specific uptake peaked 30 min after injection with slow dissociation from target regions, as suggested by the in vitro binding kinetic profile. Pretreatment with 6-fluoro-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylic acid [4-(4-propionyl-piperazin-1-yl)-phenyl]-amide (AZD1134) and 2-aminotetralin (AR-A000002), 5-HT_1B-selective ligands, inhibited [N-methyl-³H₃]AZ10419369-specific binding in a dose-dependent manner. In the guinea pig striatum, AZD1134 (ED₅₀ = 0.017 mg/kg) occupies a greater percentage of the 5-HT_1B receptors at a lower administered dose than AR-A000002 (ED₅₀ = 2.5 mg/kg). In vivo receptor occupancy is an essential component to build binding-efficacy-exposure relationships and compare novel compound pharmacology. [N-methyl-³H₃]AZ10419369 is a useful preclinical tool for investigating 5-HT_1B receptor occupancy for novel compounds targeting this receptor.

[TOXICOLOGY] Neuritogenic Actions of Botulinum Neurotoxin A on Cultured Motor Neurons

Coffield, J. A., Yan, X. — 2009-06-22

Botulinum neurotoxins (BoNTs) are extremely potent neuromuscular poisons that act through soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein cleavage to inhibit neurotransmitter release. The ability of BoNT serotype A (BoNT/A) to eliminate localized transmitter release at extremely low doses is well characterized. In the current study, we investigated the less understood characteristic of BoNT/A to induce nerve outgrowth, sometimes referred to as sprouting. This phenomenon is generally considered a secondary response to the paralytic actions of BoNT/A, and other potential factors that may initiate this sprouting have not been investigated. Alternatively, we hypothesized that BoNT/A induces sprouting through presynaptic receptor activation that is independent of its known intracellular actions on the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) synaptosomal associated protein of 25 kDa (SNAP-25). To test this, the effects of BoNT/A application on neurite outgrowth were examined using primary cultures enriched with motor neurons isolated from embryonic mouse spinal cord. In this system, BoNT/A potently stimulated neuritogenesis at concentrations as low as 0.01 nM. The neuritogenic effects of BoNT/A exposure were concentration dependent and antagonized by Triticum vulgaris lectin, a known competitive antagonist of BoNT. Similar results were observed with the isolated BoNT/A binding domain, revealing that neuritogenesis could be initiated solely by the binding actions of BoNT/A. In addition, the presence or absence of SNAP-25 cleavage by BoNT/A was not a determinant factor in BoNT/A-induced neuritogenesis. Collectively, these results suggest that binding of BoNT/A to the motor neuronal membrane activates neuritogenesis through as yet undetermined intracellular pathway(s), independent of its known action on vesicular release.

[CELLULAR AND MOLECULAR] Mechanisms of Growth Inhibition in Human Papillomavirus Positive and Negative Cervical Cancer Cells by the Chloromethyl Ketone Protease Inhibitor, Succinyl-Alanine-Alanine-Proline-Phenylalanine Chloromethyl Ketone

Duncan, K. J., Eckert, K. A., Clawson, G. A. — 2009-06-22

The chymotrypsin-like serine protease inhibitor, succinyl-alanine-alanine-proline-phenylalanine chloromethyl ketone (AAPF_CMK), has been shown to have anticarcinogenic activity in a number of model systems and to be relatively selective for a nuclear protease. This inhibitor also has substantial effects on growth of tumorigenic human papillomavirus (HPV)-infected keratinocytes in organotypic raft cultures. Here, we examined the effects of AAPF_CMK on cell growth, cell-cycle kinetics, apoptosis induction, and DNA synthesis in two human cervical carcinoma cell lines: SiHa cells, which have integrated high-risk HPV-16; and C33a cells, which do not contain HPV DNA. AAPF_CMK inhibited growth of both cell lines in a time- and dose-dependent manner. Apoptosis studies showed no significant difference in drug-treated versus vehicle-treated cells in the C33a cell line. However, a significant dose-dependent increase in apoptosis occurred at a late time point in SiHa cells. Cell-cycle progression and DNA synthesis assays showed that the cellular mechanisms of growth inhibition by AAPF_CMK differ between the HPV16-positive and HPV-negative tumorigenic cell lines. Drug-treated C33a cells showed a significant accumulation of cells in the G₂ phase of the cell cycle. In SiHa cells, growth inhibition produced by AAPF_CMK seemed to result from a global arrest of the cell cycle. Although the molecular mechanisms involved in AAPF_CMK-induced growth inhibition are distinct between the two tumorigenic cell lines, such differences may ultimately prove to have therapeutic utility. Novel therapies for treating established HPV infections are needed, because HPV is a causative agent in the development of multiple types of cancer.

[ERRATUM] Correction to "Nonpeptidic Allosteric Antagonists Differentially Bind to the CXCR2 Chemokine Receptor"

2009-06-22