JPET most recent issues

[HIGHLIGHTED PAPERS] A Dual Endothelin Receptor Blocker for Treating Cardiovascular Disease

2008-11-12

[HIGHLIGHTED PAPERS] A New Approach to Increasing HDL Levels and Cholesterol Excretion

2008-11-12

[HIGHLIGHTED PAPERS] A Combination Therapy to Improve Pancreatic Islet Function

2008-11-12

[PERSPECTIVES IN PHARMACOLOGY] New Insights into Metabolic Signaling and Cell Survival: The Role of {beta}-O-Linkage of N-Acetylglucosamine

Ngoh, G. A., Jones, S. P. — 2008-11-12

The involvement of glucose in fundamental metabolic pathways represents a core element of biology. Late in the 20th century, a unique glucose-derived signal was discovered, which appeared to be involved in a variety of cellular processes, including mitosis, transcription, insulin signaling, stress responses, and potentially, Alzheimer's disease, and diabetes. By definition, this glucose-fed signaling system was a post-translational modification to proteins. However, unlike classical cotranslational N-glycosylation occurring in the endoplasmic reticulum and Golgi apparatus, this process occurs elsewhere throughout the cell in a highly dynamic fashion, similar to the quintessential post-translational modification, phosphorylation. This more recently described post-translational modification, the β-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to nucleocytoplasmic proteins, represents an under-investigated area of biology. This signaling system operates in all of the tissues examined and seems to have persisted throughout all multicellular eukaryotes. Thus, it comes with little surprise that O-GlcNAc signaling is an integral system and viable target for biomedical investigation. This system may be a boundless source for insight into a variety of diseases and yield numerous opportunities for drug design. This Perspective will address recent insights into O-GlcNAc signaling in the cardiovascular system as a paradigm for its involvement in other biological systems.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Pamapimod, a Novel p38 Mitogen-Activated Protein Kinase Inhibitor: Preclinical Analysis of Efficacy and Selectivity

2008-11-12

P38 is a protein kinase that regulates the expression of inflammatory cytokines, suggesting a role in the pathogenesis of diseases such as rheumatoid arthritis (RA) or systemic lupus erythematosus. Here, we describe the preclinical pharmacology of pamapimod, a novel p38 mitogen-activated protein kinase inhibitor. Pamapimod inhibited p38 and p38β enzymatic activity, with IC₅₀ values of 0.014 ± 0.002 and 0.48 ± 0.04 µM, respectively. There was no activity against p38 or p38 isoforms. When profiled across 350 kinases, pamapimod bound only to four kinases in addition to p38. Cellular potency was assessed using phosphorylation of heat shock protein-27 and c-Jun as selective readouts for p38 and c-Jun NH₂-terminal kinase (JNK), respectively. Pamapimod inhibited p38 (IC₅₀, 0.06 µM), but inhibition of JNK was not detected. Pamapimod also inhibited lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) production by monocytes, interleukin (IL)-1β production in human whole blood, and spontaneous TNF production by synovial explants from RA patients. LPS- and TNF-stimulated production of TNF and IL-6 in rodents also was inhibited by pamapimod. In murine collagen-induced arthritis, pamapimod reduced clinical signs of inflammation and bone loss at 50 mg/kg or greater. In a rat model of hyperalgesia, pamapimod increased tolerance to pressure in a dose-dependent manner, suggesting an important role of p38 in pain associated with inflammation. Finally, an analog of pamapimod that has equivalent potency and selectivity inhibited renal disease in lupus-prone MRL/lpr mice. Our study demonstrates that pamapimod is a potent, selective inhibitor of p38 with the ability to inhibit the signs and symptoms of RA and other autoimmune diseases.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Characterization of N-(Adamantan-1-ylmethyl)-5-[(3R-aminopyrrolidin-1-yl)methyl]-2-chloro-benzamide, a P2X7 Antagonist in Animal Models of Pain and Inflammation

2008-11-12

Recent evidence suggests that the P2X₇ receptor may play a role in the pathophysiology of preclinical models of pain and inflammation. Therefore, pharmacological agents that target this receptor may potentially have clinical utility as anti-inflammatory and analgesic therapy. We investigated and characterized the previously reported P2X₇ antagonist N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, hydrochloride salt (AACBA; GSK314181A). In vitro, AACBA was a relatively potent inhibitor of both human P2X₇-mediated calcium flux and quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triemethylammonio)propyl]-diiodide (YO-PRO-1) uptake assays, with IC₅₀ values of approximately 18 and 85 nM, respectively. Compared with the human receptor, AACBA was less potent at the rat P2X₇ receptor, with IC₅₀ values of 29 and 980 nM in the calcium flux and YO-PRO-1 assays, respectively. In acute in vivo models of pain and inflammation, AACBA dose-dependently reduced lipopolysaccharide-induced plasma interleukin-6 release and prevented or reversed carrageenan-induced paw edema and mechanical hypersensitivity. In chronic in vivo models of pain and inflammation, AACBA produced a prophylactic, but not therapeutic-like, prevention of the clinical signs and histopathological damage of collagen-induced arthritis. Finally, AACBA could not reverse L₅ spinal nerve ligation-induced tactile allodynia when given therapeutically. Consistent with previous literature, these results suggest that P2X₇ receptors do play a role in animal models of pain and inflammation. Further study of P2X₇ antagonists both in preclinical and clinical studies will help elucidate the role of the P2X₇ receptor in pain and inflammatory mechanisms and may help identify potential clinical benefits of such molecules.

[TOXICOLOGY] Gene Expression Profiles in Livers from Diclofenac-Treated Rats Reveal Intestinal Bacteria-Dependent and -Independent Pathways Associated with Liver Injury

2008-11-12

Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is associated with idiosyncratic adverse drug reactions in humans. Previous studies revealed a crucial role for intestine-derived bacteria and/or lipopolysaccharide (LPS) in DCLF-induced hepatotoxicity. We further explored this mechanism by conducting gene expression analysis of livers from rats treated with a hepatotoxic dose of DCLF (100 mg/kg) with or without oral antibiotic pretreatment. Genes for which expression was altered by DCLF were divided into two groups: genes with expression altered by antibiotic treatment and those unaffected by antibiotics. The former group of genes represented the ones for which DCLF-induced alterations in expression depended on intestinal bacteria. The expression of the latter group of genes was probably changed by direct effect of DCLF rather than by intestinal bacteria. Functional analysis of genes in the former group revealed LPS-related signaling, further suggesting a role for bacterial endotoxin in the liver injury. Functional analysis of genes in the latter group revealed changes in signaling pathways related to inflammation, hypoxia, oxidative stress, the aryl hydrocarbon receptor, and peroxisome proliferator-activated receptor . Neutrophil depletion failed to protect from DCLF-induced hepatotoxicity, suggesting that intestinal bacteria contribute to liver injury in a neutrophil-independent manner. Hypoxia occurred in the livers of rats treated with DCLF, and hypoxia in vitro rendered hepatocytes sensitive to DCLF-induced cytotoxicity. These results support the hypothesis that intestinal bacteria are required for DCLF-induced hepatotoxicity and suggest that hypoxia plays an important role in the pathogenesis.

[CELLULAR AND MOLECULAR] Bortezomib-Induced Survival Signals and Genes in Human Proximal Tubular Cells

2008-11-12

Bortezomib has been introduced recently in the therapy of multiple myeloma (MM), a disease that is frequently associated with progressive renal failure. Because bortezomib-based therapy has been reported to lead to a rapid recovery of kidney function in patients with MM, we decided to study its direct effects in proximal tubular epithelial cells (PTCs) compared with glomerular mesangial cells (GMCs). After 24 h of stimulation, 50 nM bortezomib led to a 6.37-fold induction of apoptosis and markedly activated caspase-9 and -3 in GMCs but not in PTCs. In PTCs but not in GMCs, bortezomib led to a strong time-dependent degradation of IB- and to a long-lasting phosphorylation of both NF-Bp65 and extracellular signal-regulated kinase 1/2. Microarray analysis in bortezomib-treated PTCs revealed a time-dependent predominance of antiapoptotic genes compared with proapoptotic genes. Bortezomib (50 nM) induced heat shock protein (Hsp) 70 mRNA and protein levels in PTCs, whereas basal and bortezomib-stimulated Hsp70 protein expression was much weaker in GMCs. Moreover, bortezomib induced Bcl-2-associated athanogene (BAG) 3 mRNA and protein expression but inhibited BAG5 mRNA levels in PTCs. These data suggest that the reduced susceptibility of PTCs to bortezomib-induced cell apoptosis is because of cell type-specific effects of this compound on apoptosis/survival genes and pathways. The concept of bortezomib representing a blocker of both NF-B activation and cell survival should be carefully examined in particular renal cell types.

[TOXICOLOGY] The Molecular Mechanism of "Ryegrass Staggers," a Neurological Disorder of K+ Channels

2008-11-12

"Ryegrass staggers" is a neurological condition of unknown mechanism that impairs motor function in livestock. It is caused by infection of perennial ryegrass pastures by an endophytic fungus that produces neurotoxins, predominantly the indole-diterpenoid compound lolitrem B. Animals grazing on such pastures develop uncontrollable tremors and become uncoordinated in their movement. Lolitrem B and the structurally related tremor inducer paxilline both act as potent large conductance calcium-activated potassium (BK) channel inhibitors. Using patch clamping, we show that their different apparent affinities correlate with their toxicity in vivo. To investigate whether the motor function deficits produced by lolitrem B and paxilline are due to inhibition of BK ion channels, their ability to induce tremor and ataxia in mice deficient in this ion channel (Kcnma1^-/-) was examined. Our results show that mice lacking Kcnma1 are unaffected by these neurotoxins. Furthermore, doses of these substances known to be lethal to wild-type mice had no effect on Kcnma1^-/- mice. These studies reveal the BK channel as the molecular target for the major components of the motor impairments induced by ryegrass neurotoxins. Unexpectedly, when the response to lolitrem B was examined in mice lacking the β4 BK channel accessory subunit (Kcnmb4^-/-), only low-level ataxia was observed. Our study therefore reveals a new role for the accessory BK β4 subunit in motor control. The β4 subunit could be considered as a potential target for treatment of ataxic conditions in animals and in humans.

[CARDIOVASCULAR] Niacin-induced "Flush" Involves Release of Prostaglandin D2 from Mast Cells and Serotonin from Platelets: Evidence from Human Cells in Vitro and an Animal Model

2008-11-12

Niacin lowers serum cholesterol, low-density lipoprotein, and triglycerides, and it raises high-density lipoprotein. However, most patients experience cutaneous warmth and vasodilation (flush). Acetylsalicylic acid (ASA) can reduce this flush, presumably by decreasing prostaglandin D₂ (PGD₂) release from macrophages. Here, we show that methylnicotinate induces significant PGD₂ release from human mast cells and serotonin from human platelets. Intradermal injection of methylnicotinate induces rat skin vasodilation and vascular permeability. Niacin increases plasma PGD₂ and serotonin in a rat model of flush. The phenothiazine prochlorperazine, the H₁, serotonin receptor antagonist cyproheptadine, and the specific serotonin receptor-2A antagonist ketanserin inhibit niacin-induced temperature increase by 90% (n = 5, p < 0.05), 90 and 50% (n = 3, p < 0.05), and 85% (n = 6, p = 0.0008), respectively, in this animal model. These results indicate that niacin-induced flush involves both PGD₂ and serotonin, suggesting that drugs other than ASA are required to effectively inhibit niacin-induced flush.

[CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY] Tumor-Penetrating Microparticles for Intraperitoneal Therapy of Ovarian Cancer

Lu, Z., Tsai, M., Lu, D., Wang, J., Wientjes, M. G., Au, J. L.-S. — 2008-11-12

Intraperitoneal chemotherapy prolongs survival of ovarian cancer patients, but its utility is limited by treatment-related complications and inadequate drug penetration in larger tumors. Previous intraperitoneal therapy used the paclitaxel/Cremophor EL (polyethoxylated castor oil) formulation designed for intravenous use. The present report describes the development of paclitaxel-loaded microparticles designed for intraperitoneal treatment (referred to as tumor-penetrating microparticles or TPM). Evaluation of TPM was performed using intraperitoneal metastatic, human ovarian SKOV3 xenograft tumor models in mice. TPM were retained in the peritoneal cavity and adhered to tumor surface. TPM consisted of two biocompatible and biodegradable polymeric components with different drug release rates; one component released the drug load rapidly to induce tumor priming, whereas the second component provided sustained drug release. Tumor priming, by expanding interstitial space, promoted transport and penetration of particulates in tumors. These combined features resulted in the following advantages over paclitaxel/Cremophor EL: greater tumor targeting (16-times higher and more sustained concentration in omental tumors), lower toxicity to intestinal crypts and less body weight loss, greater therapeutic efficacy (longer survival and higher cure rate), and greater convenience (less frequent dosing). TPM may overcome the toxicities and compliance-related problems that have limited the utility of intraperitoneal therapy.

[ENDOCRINE AND DIABETES] Combination of the Dipeptidyl Peptidase IV Inhibitor LAF237 [(S)-1-[(3-Hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] with the Angiotensin II Type 1 Receptor Antagonist Valsartan [N-(1-Oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl]methyl]-L-valine] Enhances Pancreatic Islet Morphology and Function in a Mouse Model of Type 2 Diabetes

Cheng, Q., Law, P. K., de Gasparo, M., Leung, P. S. — 2008-11-12

LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] is an inhibitor of dipeptidyl peptidase IV that delays the degradation of glucagon-like peptide-1 (GLP-1). Valsartan [N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-l-valine] is an antagonist of the angiotensin II type 1 receptor (AT1R) that reduces the incidence of type 2 diabetes mellitus. LAF237 and valsartan act on a common target through separate pathways to improve pancreatic islet cell function. We hypothesize that the combination of these two drugs acts in a synergistic or additive manner on islet function and structure. To test this hypothesis, we performed in vitro and in vivo studies. To measure the acute effect of the treatment, pancreatic islets of db/db mice were isolated and stimulated in vitro with glucose in the presence of valsartan (1 µM) and exendin-4 (100 nM), a GLP-1 receptor agonist. Combination treatment with valsartan and exendin-4 significantly enhanced glucose-stimulated insulin secretion from isolated islets. For studies of chronic effect, db/db mice received LAF237 (1 mg/kg/day) and/or valsartan (10 mg/kg/day). Islet cell reactive oxygen species (ROS), proliferation, apoptosis, fibrosis, β-cell area, and glucose homeostasis were evaluated after 8 weeks of treatment, which showed that combination treatment resulted in a significant increase in pancreatic islet β-cell area compared with monotherapy. This beneficial effect correlated with an increase in β-cell proliferation and a decrease in ROS-induced islet apoptosis and fibrosis. These in vitro and in vivo data indicate that combination treatment with LAF237 and valsartan has significant beneficial additive effects on pancreatic β-cell structure and function compared with their respective monotherapeutic effects.

[CARDIOVASCULAR] Improvement of Endothelial Function of the Corpus Cavernosum in Apolipoprotein E Knockout Mice Treated with Irbesartan

Baumhakel, M., Custodis, F., Schlimmer, N., Laufs, U., Bohm, M. — 2008-11-12

Angiotensin receptor blockers enhance endothelial function and are suggested to improve erectile function. The effects and underlying mechanisms of treatment with the angiotensin receptor blocker irbesartan on penile endothelial function in apolipoprotein E (ApoE)^-/- mice were determined. Wild-type (C57/B6) and ApoE^-/- mice were fed with a high-fat, cholesterol-rich diet for 7 weeks and treated with irbesartan (50 mg/kg · day) or hydralazine (250 mg/l). Vital parameters were measured with the tail-cuff method. Endothelial (aortic rings) and erectile function (corpora cavernosa) were assessed by pharmacological stimulation in an organ bath chamber. Oxidative stress and angiotensin receptor expression were determined. Blood pressure was significantly decreased in irbesartan- and hydralazine-treated ApoE^-/- mice (p < 0.05) compared with controls and wild-type mice. Endothelial function of the aorta and corpus cavernosum was significantly impaired in ApoE^-/- mice (p < 0.05) and could be restored by treatment with irbesartan (p < 0.05). Consistently, nitric oxide production of corpora cavernosa was impaired in ApoE^-/- mice (p < 0.01), with a restoration in irbesartan- but not hydralazine-treated mice. Dihydroethidium-stained sections and lipid peroxidase assay revealed a reduction of superoxide production in irbesartan (p < 0.05) compared with hydralazine-treated and control ApoE^-/- mice. In summary, irbesartan improves penile endothelial function in ApoE^-/- mice by reduction of vascular and cavernosal oxidative stress. This result emphasizes the beneficial effect of inhibition of the renin-angiotensin system even in terms of erectile function.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] NIM811 (N-Methyl-4-isoleucine Cyclosporine), a Mitochondrial Permeability Transition Inhibitor, Attenuates Cholestatic Liver Injury but Not Fibrosis in Mice

2008-11-12

Cholestasis causes hepatocyte death, possibly because of mitochondrial injury. This study investigated whether NIM811 (N-methyl-4-isoleucine cyclosporine), an inhibitor of the mitochondrial permeability transition (MPT), attenuates cholestatic liver injury in vivo. Cholestasis was induced in mice by bile duct ligation (BDL). NIM811 was gavaged (20 mg/kg) before BDL and daily (10 mg/kg) afterward. Mitochondrial depolarization, cell death, and MPT onset were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. After BDL, serum alanine aminotransferase (ALT), hepatic necrosis, and apoptosis all increased. NIM811 decreased ALT, necrosis, and apoptosis by 60 to 86%. In vehicle-treated mice at 6 h after BDL, viable hepatocytes with depolarized mitochondria were 18/high-power field (hpf), and nonviable cells were ~1/hpf, showing that depolarization preceded necrosis. Calcein entered mitochondria after BDL, indicating MPT onset in vivo. NIM811 decreased depolarization by 72%, prevented calcein entry into mitochondria, and blocked release of cytochrome c. Hepatic tumor necrosis factor , transforming growth factor-β1, procollagen 1(I) mRNA, -smooth muscle actin, and Sirius red staining for collagen increased after BDL but were not different in vehicle- and NIM811-treated mice. Taken together, NIM811 decreased cholestatic necrosis and apoptosis but did not block fibrosis, indicating that the MPT plays an important role in cholestatic cell death in vivo.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Inhibition of Soluble Epoxide Hydrolase Does Not Protect against Endotoxin-Mediated Hepatic Inflammation

2008-11-12

Epoxyeicosatrienoic acids (EETs) are derived from cytochrome P450-catalyzed epoxygenation of arachidonic acid and have emerged as important mediators of numerous biological effects. The major elimination pathway for EETs is through soluble epoxide hydrolase (sEH)-catalyzed metabolism to dihydroxyeicosatrienoic acids (DHETs). Based on previous studies showing that EETs have anti-inflammatory effects, we hypothesized that chronic inhibition of sEH would attenuate a lipopolysaccharide (LPS)-induced inflammatory response in vivo. Continuous dosing of the sEH inhibitors 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA), a polyethylene glycol ester of AUDA, and 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy)ethoxy)-pentyl)urea resulted in robust exposure to the inhibitor and target engagement, as evidenced by significant increases in plasma EET/DHET ratios following 6 days of inhibitor treatment. However, sEH inhibitor treatment was not associated with an attenuation of LPS-induced inflammatory gene expression in the liver, and AUDA did not protect from LPS-induced neutrophil infiltration. Furthermore, Ephx2-/-mice that lack sEH expression and have significantly increased plasma EET/DHET ratios were not protected from LPS-induced inflammatory gene expression or neutrophil accumulation in the liver. LPS did have an effect on sEH expression and function, as evident from a significant down-regulation of Ephx2 mRNA and a significant shift in plasma EET/DHET ratios 4 h after LPS treatment. In conclusion, there was no evidence that increasing EET levels in vivo could modulate an LPS-induced inflammatory response in the liver. However, LPS did have significant effects on plasma eicosanoid levels and hepatic Ephx2 expression, suggesting that in vivo EET levels are modulated in response to an inflammatory signal.

[CARDIOVASCULAR] Novel Peroxisome Proliferator-Activated Receptor {alpha} Agonists Lower Low-Density Lipoprotein and Triglycerides, Raise High-Density Lipoprotein, and Synergistically Increase Cholesterol Excretion with a Liver X Receptor Agonist

2008-11-12

The first generation peroxisome proliferator-activated receptor (PPAR) agonist gemfibrozil reduces the risk of major cardiovascular events; therefore, more potent PPAR agonists for the treatment of cardiovascular diseases have been actively sought. We describe two novel, potent oxybenzylglycine PPAR-selective agonists, BMS-687453 [N-[[3-[[2-(4-chlorophenyl)-5-methyl-4-oxazolyl]methoxy]phenyl]methyl]-N-(methoxycarbonyl)-glycine] and BMS-711939 N-[[5-[[2-(4-chlorophenyl)-5-methyl-4-oxazolyl]methoxy]-2-fluorophenyl]methyl]-N-(methoxycarbonyl)-glycine], that robustly increase apolipoprotein (Apo) A1 and high-density lipoprotein cholesterol in human ApoA1 transgenic mice and lower low-density lipoprotein-cholesterol and triglycerides in fat-fed hamsters. These compounds have much lower potency against mouse PPAR than human PPAR; therefore, they were tested in PPAR-humanized mice that do not express murine PPAR but express human PPAR selectively in the liver. We developed hepatic gene induction as a novel biomarker for efficacy and demonstrate hepatic gene induction at very low doses of these compounds. BMS-711939 induces fecal cholesterol excretion, which is further increased upon cotreatment with a liver X receptor (LXR) agonist. It is surprising that this synergistic increase upon coadministration is also observed in mice that express PPAR in the liver only. BMS-711939 also prevented the LXR agonist-induced elevation of serum triglycerides. Such PPAR agonists could be attractive candidates to explore for the treatment of cardiovascular diseases, especially in combination with a suitable LXR agonist.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Radiodefluorination of 3-Fluoro-5-(2-(2-[18F](fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), a Radioligand for Imaging Brain Metabotropic Glutamate Subtype-5 Receptors with Positron Emission Tomography, Occurs by Glutathionylation in Rat Brain

2008-11-12

Metabotropic glutamate subtype-5 receptors (mGluR5) are implicated in several neuropsychiatric disorders. Positron emission tomography (PET) with a suitable radioligand may enable monitoring of regional brain mGluR5 density before and during treatments. We have developed a new radioligand, 3-fluoro-5-(2-(2-[¹⁸F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ([¹⁸F]SP203), for imaging brain mGluR5 in monkey and human. In monkey, radioactivity was observed in bone, showing release of [¹⁸F]-fluoride ion from [¹⁸F]SP203. This defluorination was not inhibited by disulfiram, a potent inhibitor of CYP2E1. PET confirmed bone uptake of radioactivity and therefore defluorination of [¹⁸F]SP203 in rats. To understand the biochemical basis for defluorination, we administered [¹⁸F]SP203 plus SP203 in rats for ex vivo analysis of metabolites. Radio-high-performance liquid chromatography detected [¹⁸F]fluoride ion as a major radiometabolite in both brain extract and urine. Incubation of [¹⁸F]SP203 with brain homogenate also generated this radiometabolite, whereas no metabolism was detected in whole blood in vitro. Liquid chromatography-mass spectrometry analysis of the brain extract detected m/z 548 and 404 ions, assignable to the [M + H]⁺ of S-glutathione (SP203Glu) and N-acetyl-S-l-cysteine (SP203Nac) conjugates of SP203, respectively. In urine, only the [M + H]⁺ of SP203Nac was detected. Mass spectrometry/mass spectrometry and multi-stage mass spectrometry analyses of each metabolite yielded product ions consistent with its proposed structure, including the former fluoromethyl group as the site of conjugation. Metabolite structures were confirmed by similar analyses of SP203Glu and SP203Nac, prepared by glutathione S-transferase reaction and chemical synthesis, respectively. Thus, glutathionylation at the 2-fluoromethyl group is responsible for the radiodefluorination of [¹⁸F]SP203 in rat. This study provides the first demonstration of glutathione-promoted radiodefluorination of a PET radioligand.

[CARDIOVASCULAR] Pharmacology of Macitentan, an Orally Active Tissue-Targeting Dual Endothelin Receptor Antagonist

2008-11-12

Macitentan, also called Actelion-1 or ACT-064992 [N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4-yl]-N'-propylaminosulfonamide], is a new dual ET_A/ET_B endothelin (ET) receptor antagonist designed for tissue targeting. Selection of macitentan was based on inhibitory potency on both ET receptors and optimization of physicochemical properties to achieve high affinity for lipophilic milieu. In vivo, macitentan is metabolized into a major and pharmacologically active metabolite, ACT-132577. Macitentan and its metabolite antagonized the specific binding of ET-1 on membranes of cells overexpressing ET_A and ET_B receptors and blunted ET-1-induced calcium mobilization in various natural cell lines, with inhibitory constants within the nanomolar range. In functional assays, macitentan and ACT-132577 inhibited ET-1-induced contractions in isolated endothelium-denuded rat aorta (ET_A receptors) and sarafotoxin S6c-induced contractions in isolated rat trachea (ET_B receptors). In rats with pulmonary hypertension, macitentan prevented both the increase of pulmonary pressure and the right ventricle hypertrophy, and it markedly improved survival. In diabetic rats, chronic administration of macitentan decreased blood pressure and proteinuria and prevented end-organ damage (renal vascular hypertrophy and structural injury). In conclusion, macitentan, by its tissue-targeting properties and dual antagonism of ET receptors, protects against end-organ damage in diabetes and improves survival in pulmonary hypertensive rats. This profile makes macitentan a new agent to treat cardiovascular disorders associated with chronic tissue ET system activation.

[CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY] Chemotherapy-Induced CXC-Chemokine/CXC-Chemokine Receptor Signaling in Metastatic Prostate Cancer Cells Confers Resistance to Oxaliplatin through Potentiation of Nuclear Factor-{kappa}B Transcription and Evasion of Apoptosis

2008-11-12

Constitutive activation of nuclear factor (NF)-B is linked with the intrinsic resistance of androgen-independent prostate cancer (AIPC) to cytotoxic chemotherapy. Interleukin-8 (CXCL8) is a transcriptional target of NF-B whose expression is elevated in AIPC. This study sought to determine the significance of CXCL8 signaling in regulating the response of AIPC cells to oxaliplatin, a drug whose activity is reportedly sensitive to NF-B activity. Administration of oxaliplatin to PC3 and DU145 cells increased NF-B activity, promoting antiapoptotic gene transcription. In addition, oxaliplatin increased the transcription and secretion of CXCL8 and the related CXC-chemokine CXCL1 and increased the transcription and expression of CXC-chemokine receptors, especially CXC-chemokine receptor (CXCR) 2, which transduces the biological effects of CXCL8 and CXCL1. Stimulation of AIPC cells with CXCL8 potentiated NF-B activation in AIPC cells, increasing the transcription and expression of NF-B-regulated antiapoptotic genes of the Bcl-2 and IAP families. Coadministration of a CXCR2-selective antagonist, AZ10397767 (Bioorg Med Chem Lett 18:798–803, 2008), attenuated oxaliplatin-induced NF-B activation, increased oxaliplatin cytotoxicity, and potentiated oxaliplatin-induced apoptosis in AIPC cells. Pharmacological inhibition of NF-BorRNA interference-mediated suppression of Bcl-2 and survivin was also shown to sensitize AIPC cells to oxaliplatin. Our results further support NF-B activity as an important determinant of cancer cell sensitivity to oxaliplatin and identify the induction of autocrine CXCR2 signaling as a novel mode of resistance to this drug.

[CELLULAR AND MOLECULAR] Distinct Interactions of Human {beta}1- and {beta}2-Adrenoceptors with Isoproterenol, Epinephrine, Norepinephrine, and Dopamine

Weitl, N., Seifert, R. — 2008-11-12

Fluorescence studies with purified human β₂-adrenoceptor (β₂AR) revealed that the endogenous catecholamines, (-)-epinephrine (EPI), (-)-norepinephrine (NE), and dopamine (DOP), stabilize distinct active receptor conformations. However, the functional relevance of these ligand-specific conformations is as yet poorly understood. We addressed this question by studying fusion proteins of the β₁-adrenoceptor (β₁AR) and β₂AR with the short and long splice variants of G_s (G_s_S and G_s_L), respectively. Fusion proteins ensure efficient receptor/G-protein coupling and defined stoichiometry of the coupling partners. EPI, NE, DOP, and the prototypical synthetic βAR agonist, (-)-isoproterenol (ISO), showed marked differences in their efficacies at stabilizing the high-affinity ternary complex at β₁AR-G_s and β₂AR-G_s fusion proteins. Ternary complex formation was more sensitive to disruption by GTP with the β₂AR than with the β₁AR. Generally, in steady-state GTPase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. Exceptionally, at β₁AR-G_s_L, NE was only a partial agonist. Generally, in adenylyl cyclase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. At β₂AR-G_s_L, NE was only a partial agonist. There was no correlation between efficacy at stabilizing the ternary complex and activating GTPase, and there were also dissociations between K_i values for high-affinity agonist binding and EC₅₀ values for GTPase activation. In contrast to synthetic partial agonists, DOP did not exhibit increased efficacy at βAR-G_s_L versus βAR-G_s_S fusion proteins. In conclusion, our data with βAR-G_s fusion proteins show that endogenous catecholamines and ISO stabilize distinct conformations in the β₁AR and β₂AR.

[TOXICOLOGY] Dehydroalanine Analog of Glutathione: An Electrophilic Busulfan Metabolite That Binds to Human Glutathione S-Transferase A1-1

2008-11-12

Elimination of hydrogen sulfide from glutathione (GSH) converts a well known cellular nucleophile to an electrophilic species, -glutamyldehydroalanylglycine (EdAG). We have found that a sulfonium metabolite formed from GSH and busulfan undergoes a facile β-elimination reaction to give EdAG, which is an ,β-unsaturated dehydroalanyl analog of GSH. EdAG was identified as a metabolite of busulfan in a human liver cytosol fraction. EdAG condenses with GSH in a Michael addition reaction to produce a lanthionine thioether [(2-amino-5-[[3-[2-[[4-amino-5-hydroxy-5-oxopentanoyl]amino]-3-(carboxymethylamino)-3-oxopropyl]sulfanyl-1-(carboxymethylamino)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid); GSG], which is a nonreducible analog of glutathione disulfide. EdAG was less cytotoxic than busulfan to C6 rat glioma cells. GSH and EdAG were equally effective in displacing a glutathione S-transferase (GST) isozyme (human GSTA1-1) from a GSH-agarose column. The finding of an electrophilic metabolite of GSH suggests that alteration of cellular GSH concentrations, irreversible nonreducible glutathionylation of proteins, and interference with GST function may contribute to the toxicity of busulfan.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] A Cross-Link between Protein Kinase A and Rho-Family GTPases Signaling Mediates Cell-Cell Adhesion and Actin Cytoskeleton Organization in Epithelial Cancer Cells

Leve, F., de Souza, W., Morgado-Diaz, J. A. — 2008-11-12

Disassembly of the apical junctional complex (AJC) together with actin cytoskeleton alterations are among the initial events for the development of epithelial cancer. The cell signaling pathways for these processes have been analyzed separately. However, the existence of a link between these two events has not been defined. In this study, using the extracellular calcium depletion model, we analyzed the signaling pathways regulating AJC disassembly together with actin cytoskeleton organization in colon adenocarcinoma cells (Caco-2). Changes in the location of AJC proteins were examined by immunofluorescence and immunoblotting, and tight junction (TJ) functionality was observed by measuring the transepithelial electrical resistance and permeation to ruthenium red. The actin cytoskeleton was stained with rhodamine-phalloidin and analyzed by confocal microscopy. Rho-GTPase activation was assessed by its translocation to the membrane (a hallmark of RhoA activation) and immunoblotting. Pharmacological inhibition of protein kinase A (PKA) with H-89 [N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)] prevented AJC disassembly and actin disorganization at the apical and medial regions caused by calcium depletion. Rho inhibition using toxin A induced AJC disassembly and actin cytoskeleton reorganization. Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-ciclohexanecarboxamide], a Rho-associated kinase inhibitor, reversed redistribution of E-cadherin, but not of TJ proteins and actin disorganization caused by calcium depletion. Calcium depletion and forskolin treatment caused activation of Rho, as evidenced by their translocation to the membrane, an event concurrent to Rac and RhoGDI translocation, and this effect was also reverted by H-89. Thus, our findings demonstrate a central role of a regulatory cascade that integrates PKA and Rho-family GTPases in the AJC disassembly and actin organization in tumor epithelial cells.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Involvement of ST1B Subfamily of Cytosolic Sulfotransferase in Kynurenine Metabolism to Form Natriuretic Xanthurenic Acid Sulfate

Senggunprai, L., Yoshinari, K., Shimada, M., Yamazoe, Y. — 2008-11-12

Natriuretic substances are a group of molecules affecting sodium homeostasis in the body. Recently, two new molecules having natriuresis effects, xanthurenic acid 8-O-β-d-glucoside and xanthurenic acid 8-O-sulfate (XA sulfate), have been isolated from human urine. In the present study, we have investigated the sulfation of xanthurenic acid (XA) in mouse tissues to assess the contribution of specific sulfotransferases (STs) to the reaction. Cytosols from tissues of both sexes of C57BL/6N mice (liver, stomach, jejunum, colon, and kidney) were capable of forming XA sulfate, with various K_m values. Jejunum cytosol showed the lowest K_m value, and its V_max/K_m value was much greater than those of other tissues. The kinetic analyses with recombinant mouse (m) STs (Sult1a1, Sult1b1, Sult1c2, and Sult1d1) showed the lowest K_m value for mSult1b1, and the value was comparable with that for jejunum cytosol. The highest expression of mSult1b1 in small intestine was confirmed at the mRNA and protein levels. mSult1b1 is thus suggested as a major enzyme responsible for XA sulfation in jejunum. Similar to mSult1b1, human SULT1B1 and rat Sult1b1 mediated XA sulfation efficiently. Thus, XA is likely to be an endogenous substrate for ST1B members. In contrast to XA, an XA-related compound, kynurenic acid strongly inhibited mSult1b1-mediated sulfations, with IC₅₀ values at a micromolar range. These results indicate the functional role of ST1B subfamily of ST in XA sulfate formation in the body.

[CELLULAR AND MOLECULAR] Identification and Characterization of Novel Small-Molecule Protease-Activated Receptor 2 Agonists

2008-11-12

We report the first small-molecule protease-activated receptor (PAR) 2 agonists, AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide], each representing a distinct chemical series. AC-55541 and AC-264613 each activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca²⁺ mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 and 30 to 100 nM for AC-264613. In comparison, the PAR2-activating peptide 2-furoyl-LIGRLO-NH₂ had similar potency, whereas SLIGRL-NH₂ was 30 to 300 times less potent. Neither AC-55541 nor AC-264613 had activity at any of the other PAR receptor subtypes, nor did they have any significant affinity for over 30 other molecular targets involved in nociception. Visualization of EYFP-tagged PAR2 receptors showed that each compound stimulated internalization of PAR2 receptors. AC-55541 and AC-264613 were well absorbed when administered intraperitoneally to rats, each reaching micromolar peak plasma concentrations. AC-55541 and AC-264613 were each stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 and 2.5 h, respectively. Intrapaw administration of AC-55541 or AC-264613 elicited robust and persistent thermal hyperalgesia and edema. Coadministration of either a tachykinin 1 (neurokinin 1) receptor antagonist or a transient receptor potential vanilloid (TRPV) 1 antagonist completely blocked these effects. Systemic administration of either AC-55541 or AC-264613 produced a similar degree of hyperalgesia as was observed when the compounds were administered locally. These compounds represent novel small-molecule PAR2 agonists that will be useful in probing the physiological functions of PAR2 receptors.

[CELLULAR AND MOLECULAR] S32826, A Nanomolar Inhibitor of Autotaxin: Discovery, Synthesis and Applications as a Pharmacological Tool

2008-11-12

Autotaxin catalyzes the transformation of lyso-phosphatidylcholine in lyso-phosphatidic acid (LPA). LPA is a phospholipid possessing a large panel of activity, in particular as a motility factor or as a growth signal, through its G-protein coupled seven transmembrane receptors. Indirect evidence strongly suggests that autotaxin is the main, if not the only source of circulating LPA. Because of its central role in pathologic conditions, such as oncology and diabetes/obesity, the biochemical properties of autotaxin has attracted a lot of attention, but confirmation of its role in pathology remains elusive. One way to validate and/or confirm its central role, is to find potent and selective inhibitors. A systematic screening of several thousand compounds using a colorimetric assay and taking advantage of the phosphodiesterase activity of autotaxin that requires the enzymatic site than for LPA generation, led to the discovery of a potent nanomolar inhibitor, [4-(tetradecanoylamino)benzyl]phosphonic acid (S32826). This compound was inhibitory toward the various autotaxin isoforms, using an assay measuring the [¹⁴C]lyso-phosphatidylcholine conversion into [¹⁴C]LPA. We also evaluated the activity of S32826 in cellular models of diabesity and oncology. Nevertheless, the poor in vivo stability and/or bioavailability of the compound did not permit to use it in animals. S32826 is the first reported inhibitor of autotaxin with an IC₅₀ in the nanomolar range that can be used to validate the role of autotaxin in various pathologies in cellular models.

[BEHAVIORAL PHARMACOLOGY] Inactivation of the Maternal Fragile X Gene Results in Sensitization of GABAB Receptor Function in the Offspring

Zupan, B., Toth, M. — 2008-11-12

Fragile X syndrome is an X-linked disorder caused by the inactivation of the FMR1 gene, with symptoms ranging from impaired cognitive functions to seizures, anxiety, sensory abnormalities, and hyperactivity. Although fragile X syndrome is considered a typical Mendelian disorder, we have recently reported that the environment, specifically the fmr1^+/- or fmr1^-/- [H or knockout (KO)] maternal environment, elicits on its own a partial fragile X-like phenotype and can contribute to the overall phenotype of fmr1^-/0 (KO) male offspring. Genetically fmr1^+/0 (WT) males born to H females (H_maternal > WT_offspring), similar to KO male offspring born to H and KO mothers (H > KO and KO > KO), exhibit locomotor hyperactivity. These mice also showed reduced D₂ autoreceptor function, indicating a possible diminished feedback inhibition of dopamine (DA) release in the nigrostriatal and mesolimbic systems. The GABAergic system also regulates DA release, in part via presynaptic GABA_B receptors (Rs) located on midbrain dopaminergic neurons. Here, we show that the locomotor inhibitory effect of the GABA_BR agonist baclofen [4-amino-3-(4-chlorophenyl)-butanoic acid] is enhanced in all progeny of mutant mothers (H > WT, H > KO, and KO > KO) compared with WT > WT mice, irrespective of their own genotype. However, increased sensitivity to baclofen was selective and limited to the locomotor response because the muscle-relaxant and sedative effects of the drug were not altered by the maternal environment. These data show that GABA_BR sensitization, traditionally induced pharmacologically, can also be elicited by the fmr1-deficient maternal environment.

[NEUROPHARMACOLOGY] ADX47273 [S-(4-Fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]-oxadiazol-5-yl]-piperidin-1-yl}-methanone]: A Novel Metabotropic Glutamate Receptor 5-Selective Positive Allosteric Modulator with Preclinical Antipsychotic-Like and Procognitive Activities

2008-11-12

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca²⁺ assays (EC₅₀ = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 µM) and competed for binding of [³H]2-methyl-6-(phenylethynyl)pyridine (K_i = 4.3 µM), but not [³H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.

[NEUROPHARMACOLOGY] Nanomolar Concentrations of Pregnenolone Sulfate Enhance Striatal Dopamine Overflow in Vivo

Sadri-Vakili, G., Janis, G. C., Pierce, R. C., Gibbs, T. T., Farb, D. H. — 2008-11-12

The balance between GABA-mediated inhibitory and glutamate-mediated excitatory synaptic transmission represents a fundamental mechanism for controlling nervous system function, and modulators that can alter this balance may participate in the pathophysiology of neuropsychiatric disorders. Pregnenolone sulfate (PS) is a neuroactive steroid that can modulate the activity of ionotropic glutamate and GABA_A receptors either positively or negatively, depending upon the particular receptor subtype, and modulates synaptic transmission in a variety of experimental systems. To evaluate the modulatory effect of PS in vivo, we infused PS into rat striatum for 20 min via a microdialysis probe while monitoring local extracellular dopamine (DA) levels. The results demonstrate that PS at low nanomolar concentrations significantly increases extracellular DA levels. The PS-induced increase in extracellular DA is antagonized by the N-methyl-d-aspartate (NMDA) receptor antagonist, d-AP5 [d-(-)-2-amino-5-phosphonopentanoic acid], but not by the receptor antagonist, BD 1063 [1(-)[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine]. The results demonstrate that exogenous PS, at nanomolar concentrations, is able to increase DA overflow in the striatum through an NMDA receptor-mediated pathway.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Do Desipramine [10,11-Dihydro-5-[3-(methylamino) propyl]-5H-dibenz[b,f]azepine monohydrochloride] and Fluoxetine [N-Methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]-propan-1-amine] Ameliorate the Extent of Colonic Damage Induced by Acetic Acid in Rats?

Guemei, A. A., El Din, N. M. N., Baraka, A. M., El Said Darwish, I. — 2008-11-12

The present study was designed to compare the anti-inflammatory and antioxidant effects of two antidepressant drugs, desipramine [10,11-dihydro-5-[3-(methylamino) propyl]-5H-dibenz-[b,f]azepine monohydrochloride] and fluoxetine [N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]-propan-1-amine], administered with variable doses, on experimentally induced colitis in rats. Two doses for each drug (10 and 20 mg/kg/day i.p.) were injected in 48 adult male albino rats for 2 weeks after induction of colitis by intracolonic administration of 2 ml of 3% acetic acid. Several parameters, including macroscopic (ulcer score index) and biochemical such as myeloperoxidase (MPO), reduced glutathione (GSH), tumor necrosis factor (TNF)-, and interleukin (IL)-1β, were measured using standard assay procedures. The study demonstrates that both desipramine and fluoxetine significantly attenuated the extent and the severity of the macroscopic signs of cell damage. Both drugs significantly reduced tissue MPO activity in a dose-dependent manner. Both desipramine and fluoxetine, at either dose, significantly increased GSH in colonic tissue. Desipramine and fluoxetine, at either dose, significantly reduced TNF- and IL-β. Desipramine at the dose of 20 mg/kg produced more decrease in the level of TNF- compared with the effect of the smaller dose, but fluoxetine at 10 mg/kg diminished more in the level of IL-1β compared with the effect of the larger dose. The present data indicate that both desipramine and fluoxetine have anti-inflammatory and antioxidants effects in experimentally induced colitis in rats, opening the avenue to their possible protective role in patients with inflammatory bowel disease.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Comparison of Cigarette Smoke-Induced Acute Inflammation in Multiple Strains of Mice and the Effect of a Matrix Metalloproteinase Inhibitor on These Responses

Morris, A., Kinnear, G., Wan, W.-Y. H., Wyss, D., Bahra, P., Stevenson, C. S. — 2008-11-12

The activities of proteases in the lung, specifically matrix metalloproteinases (MMPs), have been implicated in driving the inflammation and lung destruction observed in smokers with chronic obstructive pulmonary disease. Here, our aims were to compare the acute response with cigarette smoke exposure (CSE) in four mouse strains to identify common and distinguishing features and to assess the effect of an MMP inhibitor on this response. To do this, we exposed mice (BALB/C, C57BL/6, A/J, or 129/Sv) to whole-body CSE (1 h/day) for 3 days. CSE induced dose- and time-dependent increases in neutrophils and keratinocyte chemoattractant levels in the airways of all strains; however, the proportion of the neutrophilia differed among strains. In the two most contrasting strains, BALB/C and C57BL/6, we examined MMP gene expression and found only small changes apart from MMP-12, which was highly expressed in both strains. Both strains were then treated with a broad-spectrum MMP inhibitor, PKF242-484 [(2S,3R)-N⁴-((S)-2,2-dimethyl-1-methylcarbamoyl-propyl)-N¹-hydroxy-2-hydroxymethyl-3-(4-methoxy-phenyl)-succinimide] (0.5–10 mg/kg) either orally or intranasally 1 h before and 5 h after CSE for 3 days. PKF242-484 dose-dependently reduced neutrophilia in BALB/C mice when dosed orally (p < 0.01) or intranasally (p < 0.01) but had no clear effect in C57BL/6 by either route. PKF242-484 reduced BAL macrophages when dosed intranasally (p < 0.05) but had no dose-dependent effect when dosed orally in both strains. These data suggest the inflammation induced by CSE is similar, but not identical, in different mouse strains. In addition, the ability of broad-spectrum MMP inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, whereas its ability to limit macrophage infiltration may be route dependent.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Heme Oxygenase-1 Inhibits the Proliferation of Pancreatic Stellate Cells by Repression of the Extracellular Signal-Regulated Kinase1/2 Pathway

2008-11-12

Activation of pancreatic stellate cells (PSCs) is the key process in the development of pancreatic fibrosis, a common feature of chronic pancreatitis and pancreatic cancer. In recent studies, curcumin has been shown to inhibit PSC proliferation via an extracellular signal-regulated kinase (ERK)1/2-dependent mechanism. In addition, curcumin is a potent inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1) in other cell types. Therefore, the aims of this study were to 1) characterize the effect of curcumin on HO-1 gene expression in PSCs, 2) explore whether HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation, and 3) clarify the involvement of the mitogen-activated protein kinase (MAPK) family in this context. Cultured rat PSCs were incubated with curcumin and assessed for HO-1 up-regulation by Northern blot analysis, immunoblotting, and activity assays. The effect of HO-1 on platelet-derived growth factor (PDGF)-induced PSC proliferation and MAPK activation was determined by immunoblotting, cell proliferation assays, and cell count analyses. Curcumin induced HO-1 gene expression in PSCs in a time- and dose-dependent manner and inhibited PDGF-mediated ERK1/2 phosphorylation and PSC proliferation. These effects were blocked by treatment of PSCs with tin protoporphyrin IX, an HO inhibitor, or transfection of HO-1 small interfering RNA. Our data provide evidence that HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation. Therefore, therapeutic up-regulation of HO-1 could represent a mode for inhibition of PSC proliferation and thus may provide a novel strategy in the prevention of pancreatic fibrosis.

[NEUROPHARMACOLOGY] Chronic Benzodiazepine Administration Potentiates High Voltage-Activated Calcium Currents in Hippocampal CA1 Neurons

2008-11-12

Signs of physical dependence as a consequence of long-term drug use and a moderate abuse liability limit benzodiazepine clinical usefulness. Growing evidence suggests a role for voltage-gated calcium channel (VGCC) regulation in mediating a range of chronic drug effects from drug withdrawal phenomena to dependence on a variety of drugs of abuse. High voltage-activated (HVA) calcium currents were measured in whole-cell recordings from acutely isolated hippocampal CA1 neurons after a 1-week flurazepam (FZP) treatment that results in withdrawal-anxiety. An ~1.8-fold increase in Ca²⁺ current density was detected immediately after and up to 2 days but not 3 or 4 days after drug withdrawal. Current density was unchanged after acute desalkyl-FZP treatment. A significant negative shift of the half-maximal potential of activation of HVA currents was also observed but steady-state inactivation remained unchanged. FZP and diazepam showed use- and concentration-dependent inhibition of Ca²⁺ currents in hippocampal cultured cells following depolarizing trains (FZP, IC₅₀ = 1.8 µM; diazepam, IC₅₀ = 36 µM), pointing to an additional mechanism by which benzodiazepines modulate HVA Ca²⁺ channels. Systemic preinjection of nimodipine (10 mg/kg), an L-type (L)-VGCC antagonist, prevented the benzodiazepine-induced increase in -amino-3-hydroxy-5-methylisoxasole-4-propionic acid receptor (AMPAR)-mediated miniature excitatory postsynaptic current in CA1 neurons 2 days after FZP withdrawal, suggesting that AMPAR potentiation, previously linked to withdrawal-anxiety may require enhanced L-VGCC-mediated Ca²⁺ influx. Taken together with prior work, these findings suggest that enhanced Ca²⁺ entry through HVA Ca²⁺ channels may contribute to hippocampal AMPAR plasticity and serve as a potential mechanism underlying benzodiazepine physical dependence.

[NEUROPHARMACOLOGY] Inhibition of Native and Recombinant Nicotinic Acetylcholine Receptors by the Myristoylated Alanine-Rich C Kinase Substrate Peptide

Gay, E. A., Klein, R. C., Melton, M. A., Blackshear, P. J., Yakel, J. L. — 2008-11-12

A variety of peptide ligands are known to inhibit the function of neuronal nicotinic acetylcholine receptors (nAChRs), including small toxins and brain-derived peptides such as β-amyloid_1–42 and synthetic apolipoproteinE peptides. The myristoylated alanine-rich C kinase substrate (MARCKS) protein is a major substrate of protein kinase C and is highly expressed in the developing and adult brain. The ability of a 25-amino acid synthetic MARCKS peptide, derived from the effector domain (ED), to modulate nAChR activity was tested. To determine the effects of the MARCKS ED peptide on nAChR function, receptors were expressed in Xenopus laevis oocytes, and two-electrode voltage-clamp experiments were performed. The MARCKS ED peptide completely inhibited acetylcholine (ACh)-evoked responses from 7 nAChRs in a dose-dependent manner, yielding an IC₅₀ value of 16 nM. Inhibition of ACh-induced responses was both activity- and voltage-independent. The MARCKS ED peptide was unable to block -bungarotoxin binding. A MARCKS ED peptide in which four serine residues were replaced with aspartate residues was unable to inhibit 7 nAChR-mediated currents. The MARCKS ED peptide inhibited ACh-induced 4β2 and 2β2 responses, although with decreased potency. The effects of the MARCKS ED peptide on native nAChRs were tested using acutely isolated rat hippocampal slices. In hippocampal interneurons, the MARCKS ED peptide was able to block native 7 nAChRs in a dose-dependent manner. The MARCKS ED peptide represents a novel antagonist of neuronal nAChRs that has considerable utility as a research tool.

[BEHAVIORAL PHARMACOLOGY] The 5-Hydroxytryptamine2A Receptor Antagonist R-(+)-{alpha}-(2,3-Dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl-4-piperidinemethanol (M100907) Attenuates Impulsivity after Both Drug-Induced Disruption (Dizocilpine) and Enhancement (Antidepressant Drugs) of Differential-Reinforcement-of-Low-Rate 72-s Behavior in the Rat

2008-11-12

Previous work has suggested that N-methyl-d-aspartate (NMDA) receptor antagonism and 5-hydroxytryptamine (5-HT)_2A receptor blockade may enhance and attenuate, respectively, certain types of impulsivity mediated by corticothalamostriatal circuits. More specifically, past demonstrations of synergistic "antidepressant-like" effects of a 5-HT_2A receptor antagonist and fluoxetine on differential-reinforcement-of-low-rate (DRL) 72-s schedule of operant reinforcement may speak to the role of 5-HT_2A receptor blockade with respect to response inhibition as an important prefrontal cortical executive function relating to motor impulsivity. To examine the dynamic range over which 5-HT_2A receptor blockade may exert effects on impulsivity, [R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl-4-piperidinemethanol] (M100907) was examined both alone and in combination with the psychotomimetic NMDA receptor antagonist dizocilpine [e.g., (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; MK-801] and two different antidepressants, the tricyclic antidepressant desmethylimipramine (DMI) and the monoamine oxidase inhibitor tranylcypromine in rats performing under a DRL 72-s schedule. MK-801 increased the response rate, decreased the number of reinforcers obtained, and exerted a leftward shift in the inter-response time (IRT) distribution as expected. A dose of M100907 that exerted minimal effect on DRL behavior by itself attenuated the psychotomimetic effects of MK-801. Extending previous M100907-fluoxetine observations, addition of a minimally active dose of M100907 to low doses of DMI and tranylcypromine enhanced the antidepressant-like effect of the antidepressants. Therefore, it may be that a tonic excitation of 5-HT_2A receptors modulates impulsivity and function of corticothalamostriatal circuits over an extensive dynamic range.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Identification and Characterization of NDT 9513727 [N,N-bis(1,3-Benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a Novel, Orally Bioavailable C5a Receptor Inverse Agonist

2008-11-12

The complement system represents an innate immune mechanism of host defense that has three effector arms, the C3a receptor, the C5a receptor (C5aR), and the membrane attack complex. Because of its inflammatory and immune-enhancing properties, the biological activity of C5a and its classical receptor have been widely studied. Because specific antagonism of the C5aR could have therapeutic benefit without affecting the protective immune response, the C5aR continues to be a promising target for pharmaceutical research. The lack of specific, potent and orally bioavailable small-molecule antagonists has limited the clinical investigation of the C5aR. We report the discovery of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a small-molecule, orally bioavailable, selective, and potent inverse agonist of the human C5aR. NDT 9513727 was discovered based on the integrated use of in vitro affinity and functional assays in conjunction with medicinal chemistry. NDT 9513727 inhibited C5a-stimulated responses, including guanosine 5'-3-O-(thio)triphosphate binding, Ca²⁺ mobilization, oxidative burst, degranulation, cell surface CD11b expression and chemotaxis in various cell types with IC₅₀s from 1.1 to 9.2 nM, respectively. In C5a competition radioligand binding experiments, NDT 9513727 exhibited an IC₅₀ of 11.6 nM. NDT 9513727 effectively inhibited C5a-induced neutropenia in gerbil and cynomolgus macaque in vivo. The findings suggest that NDT 9513727 may be a promising new entity for the treatment of human inflammatory diseases.

[NEUROPHARMACOLOGY] Ethanol Decreases Purkinje Neuron Excitability by Increasing GABA Release in Rat Cerebellar Slices

Mameli, M., Botta, P., Zamudio, P. A., Zucca, S., Valenzuela, C. F. — 2008-11-12

Cerebellar Purkinje neurons (PNs) receive inhibitory GABAergic input from stellate and basket cells, which are located in the outer and inner portions of the molecular layer, respectively. Ethanol (EtOH) was recently shown to increase GABAergic transmission at PNs via a mechanism that involves enhanced calcium release from presynaptic internal stores (J Pharmacol Exp Ther 323:356–364, 2007). Here, we further characterized the effect of EtOH on GABA release and assessed its impact on PN excitability. Using whole-cell patch-clamp electrophysiological techniques in cerebellar vermis parasagittal slices, we found that EtOH acutely increases the frequency but not the amplitude or half-width of miniature and spontaneous inhibitory postsynaptic currents (IPSCs). EtOH significantly increased the amplitude and decreased the paired pulse ratio of IPSCs evoked by stimulation in the outer but not inner molecular layer. In current clamp, EtOH decreased both the amplitude of excitatory postsynaptic potentials evoked in PNs by granule cell axon stimulation and the number of action potentials triggered by these events; these effects depended on GABA_A receptor activation because they were not observed in presence of bicuculline. Loose-patch cell-attached PN recordings revealed that neither the spontaneous action potential firing frequency nor the coefficient of variation of the interspike interval was altered by acute EtOH exposure. These findings suggest that EtOH differentially affects GABAergic transmission at stellate cell- and basket cell-to-PN synapses and that it modulates PN firing triggered by granule cell axonal input. These effects could be in part responsible for the cerebellar impairments associated with acute EtOH intoxication.

[CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY] Impact of High and Low Folate Diets on Tissue Folate Receptor Levels and Antitumor Responses Toward Folate-Drug Conjugates

2008-11-12

Herein, we present a detailed analysis on the effects of feeding laboratory mice both high and low folic acid (folate)-containing diets as related to associated changes in serum and red blood cell (RBC) folate levels, tissue-derived folate receptor levels, and the ability of folate-drug conjugates to bind and effectuate activity against folate receptor (FR)-positive tumor xenografts. Our data show that serum and RBC folate concentrations sharply drop immediately after mice are switched to low folate diets; however, both parameters reach steady-state, "human-like" levels after 6 weeks. Interestingly, tissue-related folate binding capacities were also lowered during the dietary modulation period, whereas the net uptake of a radiolabeled folate conjugate was simultaneously increased 2.6- and 5-fold in FR-positive kidney and tumor tissue, respectively. Finally, the performances of several clinically and preclinically relevant folate-drug conjugates were evaluated against tumors in mice that were fed high or low folate diets. Except when administered at a dose level 6-fold less than that required to saturate endogenous FRs, no significant loss of antitumor activity was observed. From these findings, we conclude that lowering the dietary intake of folates in mice has little impact on the biological activity of repetitively dosed folate-targeted agents but that low folate diet regimens will reduce serum and RBC folate levels down to levels that more closely approximate the normal human ranges.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] A Novel CCR5-Specific Pharmacodynamic Assay in Whole Blood Using Phosphoflow Cytometry Highlights Different Ligand-Dependent Responses but Similar Properties of Antagonists in CD8+ and CD4+ T Lymphocytes

Dahl, M. E., Berson, A., Lora, J., Fuentes, M. — 2008-11-12

Chemokine CC motif receptor (CCR) 5 is a major drug target for both inflammation and virology indications. The primary function of CCR5 is to mediate the trafficking of CCR5-expressing lymphocytes to any of the CCR5 ligands, which are often increased during inflammatory responses. In addition, CCR5 is a coreceptor for HIV, mediating R5 tropic HIV infection of CCR5-expressing CD4 T cells. We report the use of a novel method to assay the pharmacodynamic (PD) properties of small-molecule and antibody inhibitors of CCR5 ligand-induced activation by measuring phosphorylation of serine residue 349 in the cytoplasmic tail of human CCR5 using phosphoflow cytometry in whole blood. This assay is highly specific and measures CCR5 phosphorylation in both CD8⁺ and CD4⁺ T cells and allows the calculation of inhibitor IC₅₀ values from both lymphocyte subsets in the presence of CCR5 antagonists. In addition, this assay is cross-reactive to nonhuman primates and allows PD analysis in whole blood from rhesus and cynomolgus macaque. Using this assay, we identified different ligand-dependent response properties between CD8⁺ and CD4⁺ T cells, although CCR5 antagonists behave with similar properties against both cell types. The use of this assay may be of particular benefit to monitor PD effects of CCR5 inhibitors during drug development, preclinical in vivo studies, and in patients currently being treated for HIV or CCR5-mediated inflammatory diseases with CCR5 inhibitors. Similar phosphoflow approaches to other GPCR targets on circulating lymphocytes may prove to be the most reliable PD assay for preclinical and potentially clinical development.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Effect of Erythromycin on Biological Activities Induced by Clostridium perfringens {alpha}-Toxin

2008-11-12

Clostridium perfringens -toxin, an important agent of gas gangrene with inflammatory myopathies, possesses lethal, hemolytic, and necrotic activities. Here, we show that -toxin-induced lethality in mice was inhibited by i.v. preadministration of erythromycin (ERM). Administration of ERM resulted in a drastic reduction in the release of tumor necrosis factor (TNF)-, interleukin (IL)-1β, and IL-6 and systemic hemolysis induced by -toxin, whereas the administration of kitasamycin did not. Furthermore, the lethality and systemic hemolysis caused by -toxin were blocked by the preinjection of anti-TNF-, but not the anti-IL-1β- or anti-IL-6-antibody. In addition, TNF--deficient mice were resistant to -toxin, indicating that TNF- plays an important role in the lethality. ERM inhibited the toxin-induced release of TNF- from neutrophils and phosphorylation of toropomyosin-related kinase receptor A (TrkA) and extracellular-regulated kinase (ERK) 1/2. Furthermore, K252a, a TrkA inhibitor, and PD98059 (2'-amino-3'-methoxyflavone), an ERK1/2 inhibitor, inhibited the toxin-induced release of TNF- from neutrophils. The observation shows that the toxin-induced release of TNF- is dependent on the activation of ERK/mitogen-activated protein kinase signal transduction via TrkA in neutrophils and that ERM specifically blocks the toxin-induced events through the activation of neutrophils.

[NEUROPHARMACOLOGY] Centrally Active Allosteric Potentiators of the M4 Muscarinic Acetylcholine Receptor Reverse Amphetamine-Induced Hyperlocomotor Activity in Rats

2008-11-12

Previous clinical and animal studies suggest that selective activators of M₁ and/or M₄ muscarinic acetylcholine receptors (mAChRs) have potential as novel therapeutic agents for treatment of schizophrenia and Alzheimer's disease. However, highly selective centrally penetrant activators of either M₁ or M₄ have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors. We previously identified VU10010 [3-amino-N-(4-chlorobenzyl)-4, 6-dimethylthieno[2,3-b]pyridine-2-carboxamide] as a potent and selective allosteric potentiator of M₄ mAChRs. However, unfavorable physiochemical properties prevented use of this compound for in vivo studies. We now report that chemical optimization of VU10010 has afforded two centrally penetrant analogs, VU0152099 [3-amino-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide] and VU0152100 [3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide], that are potent and selective positive allosteric modulators of M₄. VU0152099 and VU0152100 had no agonist activity but potentiated responses of M₄ to acetylcholine. Both compounds were devoid of activity at other mAChR subtypes or at a panel of other GPCRs. The improved physiochemical properties of VU0152099 and VU0152100 allowed in vivo dosing and evaluation of behavioral effects in rats. Interestingly, these selective allosteric potentiators of M₄ reverse amphetamine-induced hyperlocomotion in rats, a model that is sensitive to known antipsychotic agents and to nonselective mAChR agonists. This is consistent with the hypothesis that M₄ plays an important role in regulating midbrain dopaminergic activity and raises the possibility that positive allosteric modulation of M₄ may mimic some of the antipsychotic-like effects of less selective mAChR agonists.

[NEUROPHARMACOLOGY] NS11394 [3'-[5-(1-Hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile], a Unique Subtype-Selective GABAA Receptor Positive Allosteric Modulator: In Vitro Actions, Pharmacokinetic Properties and in Vivo Anxiolytic Efficacy

2008-11-12

The novel positive allosteric modulator NS11394 [3'-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile] possesses a functional selectivity profile at GABA_A receptors of ₅ > ₃ > ₂ > ₁ based on oocyte electrophysiology with human GABA_A receptors. Compared with other subtype-selective ligands, NS11394 is unique in having superior efficacy at GABA_A-₃ receptors while maintaining low efficacy at GABA_A-₁ receptors. NS11394 has an excellent pharmacokinetic profile, which correlates with pharmacodynamic endpoints (CNS receptor occupancy), yielding a high level of confidence in deriving in vivo conclusions anchored to an in vitro selectivity profile and allowing for translation to higher species. Specifically, we show that NS11394 is potent and highly effective in rodent anxiety models. The anxiolytic efficacy of NS11394 is most probably mediated through its high efficacy at GABA_A-₃ receptors, although a contributory role of GABA_A-₂ receptors cannot be excluded. Compared with benzodiazepines, NS11394 has a significantly reduced side effect profile in rat (sedation, ataxia, and ethanol interaction) and mouse (sedation), even at full CNS receptor occupancy. We attribute this benign side effect profile to very low efficacy of NS11394 at GABA_A-₁ receptors and an overall partial agonist profile across receptor subtypes. However, NS11394 impairs memory in both rats and mice, which is possibly attributable to its efficacy at GABA_A-₅ receptors, albeit activity at this receptor might be relevant to its antinociceptive effects (J Pharmacol Exp Ther 327:doi;10.1124/jpet.108.144, 2008). In conclusion, NS11394 has a unique subtype-selective GABA_A receptor profile and represents an excellent pharmacological tool to further our understanding on the relative contributions of GABA_A receptor subtypes in various therapeutic areas.