Two-dimensional electrophoresis protein profiling and identification in rat bronchoalveolar lavage fluid following allergen and endotoxin challenge.

The protein content of bronchoalveolar lavage fluid (BALF) from actively sensitised Brown Norway (BN) rats challenged with allergen (ovalbumin, OA) and from naïve Brown Norway rats challenged with endotoxin (lipopolysaccharide, LPS) was analyzed and compared to healthy controls treated with vehicle only. BALF proteins were analyzed by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis and identified by peptide mass fingerprinting matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or nanoliquid chromatography-tandem MS (nanoLC-MS/MS) after in-gel trypsin digestion of selected 2-D gel spots. Our study shows that the BALF protein profile is significantly different in animals after allergen (OA) or endotoxin (LPS) challenge as compared to controls, concerning the content of proteins derived from plasma or produced locally in the lung.

Coordinated activation of notch, Wnt, and transforming growth factor-beta signaling pathways in bone morphogenic protein 2-induced osteogenesis. Notch target gene Hey1 inhibits mineralization and Runx2 transcriptional activity.

To examine early events in osteoblast differentiation, we analyzed the expression of about 9,400 genes in the murine MC3T3 cell line, whose robust differentiation was documented cytochemically and molecularly. The cells were stimulated for 1 and 3 days with the osteogenic stimulus containing bone morphogenic protein 2. Total RNA was extracted and analyzed by Affymetrix GeneChip oligonucleotide arrays.

SAR of benzoylpyridines and benzophenones as p38alpha MAP kinase inhibitors with oral activity.

Benzoylpyridines and benzophenones were synthesized and evaluated in vitro as p38alpha inhibitors and in vivo in several models of rheumatoid arthritis. Oral activity was found to depend upon substitution: 1,1-dimethylpropynylamine substituted benzophenone 10b (IC50: 14 nM) and pyridinoyl substituted benzimidazole 17b (IC50: 21 nM) showed highest efficacy and selectivity with ED50s of 9.5 and 8.

Novel p38 inhibitors with potent oral efficacy in several models of rheumatoid arthritis.

A library of trisubstituted oxazoles, thiazoles, imidazoles (1,2,4- and 2,4,5-substituted) and imidazo[1,2-b]pyridines was prepared and evaluated in vitro as p38alpha inhibitors and in vivo in several models of rheumatoid arthritis. Four structures--32, 37, 45 and 59--were identified as potent inhibitors of p38alpha with high efficacy in the LPS induced TNFalpha release model in the mouse, the adjuvant induced arthritis and the collagen induced arthritis in the rat with ED50s between 1.0 and 9.

SRA880, in vitro characterization of the first non-peptide somatostatin sst(1) receptor antagonist.

This report describes the in vitro features of the first somatostatin sst(1) receptor selective non-peptide antagonist, SRA880 ([3R,4aR,10aR]-1,2,3,4,4a,5,10,10a-Octahydro-6-methoxy-1-methyl-benz[g] quinoline-3-carboxylic-acid-4-(4-nitro-phenyl)-piperazine-amide, hydrogen malonate). SRA was evaluated in a number of in vitro systems of various species, both at native and recombinant receptors, using radioligand binding and second messenger/transduction studies. SRA880 has high affinity for native rat, mouse, monkey and human cerebral cortex somatostatin sst(1) receptors (pK(d) = 7.

Habenula lesions cause impaired cognitive performance in rats: implications for schizophrenia.

Cognitive impairment is a prominent feature of schizophrenia. Currently there is no well-accepted explanation of the aetiology of this disorder, but recent evidence indicates that dysfunction of the habenula may be involved. We therefore examined whether habenula lesions in Sprague-Dawley rats cause behavioural changes resembling those of schizophrenia.

Behavioral characterization of the novel GABAB receptor-positive modulator GS39783 (N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine): anxiolytic-like activity without side effects associated with baclofen or benzodiazepines.

The role of GABAB receptors in various behavioral processes has been largely defined using the prototypical GABAB receptor agonist baclofen. However, baclofen induces sedation, hypothermia and muscle relaxation, which may interfere with its use in behavioral paradigms. Although there is much evidence for a role of the inhibitory neurotransmitter GABA in the pathophysiology of anxiety, the role of GABAB receptors in these disorders is largely unclear.

Discovery and SAR of potent, orally available and brain-penetrable 5,6-dihydro-4H-3-thia-1-aza-benzo[e]azulen- and 4,5-dihydro-6-oxa-3-thia-1-aza-benzo[e]azulen derivatives as neuropeptide Y Y5 receptor antagonists.

Combination of structural elements from a potent Y5 antagonist (2) with thiazole fragments that exhibit weak Y5 affinities followed by lead optimisation led to the discovery of (5,6-dihydro-4H-3-thia-1-aza-benzo[e]azulen-2-yl)-piperidin-4-ylmethyl-amino and (4,5-dihydro-6-oxa-3-thia-1-aza-benzo[e]azulen-2-yl)-piperidin-4-ylmethyl-amino derivatives. Both classes of compounds are capable of delivering potent and selective orally and centrally bioavailable NPY Y5 receptor antagonists.

A simple, rapid and sensitive method for simultaneous determination of rivastigmine and its major metabolite NAP 226-90 in rat brain and plasma by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry.

A simple and sensitive reversed-phase liquid chromatography coupled with electrospray-mass spectrometry was developed and validated for the simultaneous determination of rivastigmine, a cholinesterase inhibitor, and its major metabolite NAP 226-90 in rat plasma and brain homogenates. Rivastigmine and NAP 226-90 were extracted from plasma and brain by ethyl acetate and, after drying under nitrogen, re-dissolved in acetonitrile and separated isocratic by HPLC on a C(18) column and quantified by single ion monitoring mass spectrometer. The mean (+/-SD) extraction efficiency for rivastigmine in plasma and brain was 93 +/- 2 and 95 +/- 2% (n = 5) of NAP 226-90 in a drug range of 10-100 pmol/mL or pmol/g.

In vivo antitumor activity of NVP-AEW541-A novel, potent, and selective inhibitor of the IGF-IR kinase.

IGF-IR-mediated signaling promotes survival, anchorage-independent growth, and oncogenic transformation, as well as tumor growth and metastasis formation in vivo. NVP-AEW541 is a pyrrolo[2,3-d]pyrimidine derivative small molecular weight kinase inhibitor of the IGF-IR, capable of distinguishing between the IGF-IR (IC50 = 0.086 microM) and the closely related InsR (IC50 = 2.

Comparison of functional profiles at human recombinant somatostatin sst2 receptor: simultaneous determination of intracellular Ca2+ and luciferase expression in CHO-K1 cells.

1. Somatostatin (somatotropin release inhibiting factor; SRIF) acts via five G protein-coupled receptors (sst(1)-sst(5)) that modulate multiple cellular effectors. The aim of this study was to compare two functional effects of the human sst(2) receptor stably expressed in CHO-K1 cells in a single experiment using a duplex assay for intracellular calcium and serum response element (SRE)-driven luciferase expression.

Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay.

Osteoclasts are cells of hematopoietic origin with a unique property of dissolving bone; their inhibition is a principle for treatment of diseases of bone loss. Protocols for generation of human osteoclasts in vitro have been described, but they often result in cells of low activity, raising questions on cell phenotype and suitability of such assays for screening of bone resorption inhibitors. Here we describe an optimized protocol for the production of stable amounts of highly active human osteoclasts.

Advances in the structural biology, design and clinical development of VEGF-R kinase inhibitors for the treatment of angiogenesis.

Initial studies with angiogenesis inhibitors showed little clinical benefit. However, recently reported clinical studies in colorectal cancer have shown that bevacizumab, a vascular endothelial growth factor (VEGF) monoclonal antibody, in combination with cytotoxic therapy has positive effects on patient survival. Furthermore, the VEGF receptor kinase (VEGF-R) tyrosine kinase inhibitor, vatalanib, has also shown encouraging results in colorectal cancer, with molecular resonance imaging providing evidence that the anti-tumor efficacy was indeed the result of anti-angiogenic activity.

Interrupting apoptosis in neurodegenerative disease: potential for effective therapy?

Current treatment options for neurodegenerative diseases are limited and mainly affect only the symptoms of disease. Because of the unknown and probably multiple causes of these diseases, they cannot be readily targeted. However, it has been established that apoptosis contributes to neuronal loss in most neurodegenerative diseases.

Magnetic resonance imaging in drug discovery: lessons from disease areas.

Imaging technologies are presently receiving considerable attention in the pharmaceutical area owing to their potential to accelerate the drug discovery and development process. One of the principal imaging modalities is magnetic resonance imaging (MRI). The multiparametric nature of MRI enables anatomical, functional and even molecular information to be obtained non-invasively from intact organisms at high spatial resolution, thereby enabling a comprehensive characterization of a disease state and the corresponding drug intervention.

Web-based cheminformatics and molecular property prediction tools supporting drug design and development at Novartis.

Web-based tools offer many advantages for processing chemical information, most notably ease of use and high interactivity. Therefore more and more pharmaceutical companies are using web technology to deliver sophisticated molecular processing tools directly to the desks of their chemists, to assist them in the process of designing and developing new drugs. In this paper, the web-based cheminformatics system developed at Novartis and currently used by more than thousand users is described.

Chronic antidepressants reverse cerebrocortical allopregnanolone decline in the olfactory-bulbectomized rat.

Olfactory bulbectomy is one of the most validated models of depression. We demonstrate that bilateral removal of the olfactory bulbs in rats produced a significant decline of allopregnanolone content in a select cerebrocortical area which was reversed by chronic (3-week) treatment with three different classes of antidepressant (desipramine, fluoxetine, and sertraline, and venlafaxine). The effects of the chronic antidepressant treatments on allopregnanolone cortical content are observed at a time which typically coincides with the drug's abilities to reverse the behavioral deficits of the bulbectomy syndrome.

Selected amino acids, dipeptides and arylalkylamine derivatives do not act as allosteric modulators at GABAB receptors.

Based on recent reports describing enhancing actions of arylalkylamines (fendiline [N-(3,3-diphenylpropyl)-alpha-methylbenzylamine] and prenylamine [N-(3,3-diphenylpropyl)-alpha-methylphenethylamine]), amino acids (L-phenylalanine, L-leucine and L-isoleucine), and dipeptides (L-Phe-Phe and L-Phe-Leu) on baclofen-induced responses in cortical slices, we have examined whether these compounds might act as positive allosteric modulators at GABA(B) receptors. Unlike the previously described allosteric GABA(B) receptor modulator CGP7930 (2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol), these compounds did not enhance GABA(B) receptor-mediated guanosine 5'-O-(3-thiotriphosphate) [GTP(gamma)35S] binding in native or recombinant cell membrane preparations. Similarly, in a competition binding assay using the antagonist radioligand [3H]CGP62349, CGP7930, but not the other compounds, enhanced the affinities of gamma-aminobutyric acid (GABA) for native GABA(B) receptors from rat brain cortex.

A novel Pd-catalyzed cyclization reaction of ureas for the synthesis of dihydroquinazolinone p38 kinase inhibitors.

A series of potent p38 inhibitors based on the dihydroquinazoline scaffold was synthesized using a novel Pd-catalyzed cyclization reaction of aryl-benzyl ureas. Optimization of this compound class led to compound 20, which inhibits p38alpha in vitro with IC(50)=14 nM and is active in the mouse TNFalpha-release model.

SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands.

A high-throughput screening methodology tailored to the discovery of ligands for known and orphan proteins is presented. With this method, labeling of neither target protein nor screened compounds is required, as the ligands are affinity selected by incubation of the protein with mixtures of compounds in aqueous binding buffer. Unbound small-molecular-weight compounds are removed from the target protein:ligand complex by rapid size-exclusion chromatography in the 96-well format.

Non-nicotinic neuropharmacological strategies for nicotine dependence: beyond bupropion.

Smoking is a major health problem and is propelled, at least in part, by the addictive properties of nicotine. Two types of pharmacological therapies have been approved for smoking cessation by the US Food and Drug Administration. The first therapy consists of nicotine replacement, substituting the nicotine from cigarettes with safer nicotine formulations.

Effects of a mitogen-activated protein kinase inhibitor on allergic airways inflammation in the rat studied by magnetic resonance imaging.

We recently described a new model to study non-invasively with magnetic resonance imaging (MRI) the effects of compounds to prevent and/or resolve airway inflammation induced by ovalbumin in the lungs of actively sensitised rats. We report here the effects of 4-(4-fluorophenyl)-2-(1-methylpiperidin-4-yl)-5-(2-(1-(S)-phenylethyl)amino-4-pyridinyl)thiazole fumarate (Compound 1), which exhibits inhibitory activity against p38alpha and p38beta2 and residual activity on c-Jun amino-terminal kinase (JNK)2 mitogen-activated protein (MAP) kinases, on the oedematous signals detected by MRI and generated by antigen challenge in the lungs of sensitized rats. Compound 1 (10 mg kg(-1)) given orally 1 h prior to allergen challenge significantly reduced the oedematous signal measured at 24 h.