The genetics of a pharma merger.

The 1990s and early years of this century have seen a series of large-scale mergers and acquisitions in the Pharmaceutical and Biotech arena. These activities each required integration at multiple levels. One of the most important activities is the integration of the R&D pipelines of the participants.

Decreased nuclear factor-kappaB DNA binding activity following permanent focal cerebral ischaemia in the rat.

Many factors implicated in the pathogenesis of cerebral ischaemia such as glutamate, tumour necrosis factor and interleukin-1 have also been shown to activate nuclear factor-kappaB (NF-kappaB). In the present study we have investigated NF-kappaB activity at various times following permanent focal cerebral ischaemia in rats using immunohistochemistry, western blotting and electrophoretic mobility shift assay (EMSA). Three hours following middle cerebral artery occlusion nuclear translocation of NF-kappaB was detected using immunohistochemical and western blotting techniques.

Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells.

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible.

In vitro and in vivo pharmacological characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with anti-inflammatory activity.

Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.

cDNA sequence and characterization of the gene that encodes human myotrophin/V-1 protein, a mediator of cardiac hypertrophy.

The predominant response of the heart to sustained increased work load is development of ventricular hypertrophy, principally as a result of hypertrophy of cardiomyocytes. The molecular mechanisms and factors involved in cardiomyocyte hypertrophy are poorly understood. Myotrophin is a novel 12-kilodalton protein recently implicated as a factor associated with and able to induce cardiac hypertrophy.

Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T cells and inhibits HT29 cell growth.

Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-kappaB and AP-1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF-related proteins derived from an expressed sequence tag data base.

Effect of staurosporine on transcription factor NF-kappaB in human keratinocytes.

Activation of the transcription factor NF-kappaB is known to be important in the regulated expression of a large number of pro-inflammatory genes including interleukin-8 (IL-8). Previously, we showed that the protein kinase inhibitor staurosporine potentiates IL-1-stimulated IL-8 production in human keratinocytes. Moreover, recent studies by other investigators demonstrated that staurosporine treatment alone results in a concentration-dependent increase in IL-8 mRNA and protein production.

Evaluation of the cutaneous anti-inflammatory activity of azaspiranes.

Objective And Design: The ability of azaspiranes to modulate the acute inflammatory response in models of skin inflammation was examined.

Material: The in vivo experiments involved the use of 5-6 age-matched male Balb/c inbred mice (22-25 g) per treatment group and a control group of 8-10 animals. In vitro mechanistic studies used RBL-1 and U937 cells lines and freshly isolated human monocytes.

Inhibition of NFkappaB-mediated interleukin-1beta-stimulated prostaglandin E2 formation by the marine natural product hymenialdisine.

Exposure of human rheumatoid synovial fibroblasts (RSF) to interleukin 1beta (IL-1beta) results in the coordinate up-regulation of 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX II) and subsequent biosynthesis of prostaglandin E2 (PGE2). We have recently demonstrated, through the use of oligonucleotide decoys and antisense, the participation of the proinflammatory transcription factor, nuclear factor kappaB (NFkappaB), in the regulation of the prostanoid-metabolizing enzymes. Hymenialdisine, a marine natural product has recently been characterized as an inhibitor of NFkappaB activation and exposure of IL-1-stimulated RSF-inhibited PGE2 production in a concentration-dependent manner (IC50 approximately 1 microM).

The natural product hymenialdisine inhibits interleukin-8 production in U937 cells by inhibition of nuclear factor-kappaB.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors have been implicated in the inducible expression of genes involved in inflammatory and immune responses. As such, a specific inhibitor of NF-kappaB would be a useful therapeutic agent in a variety of inflammatory disorders. The marine natural product hymenialdisine was evaluated as an inhibitor of NF-kappaB in U937 cells.

Bicyclic N-hydroxyurea inhibitors of 5-lipoxygenase: pharmacodynamic, pharmacokinetic, and in vitro metabolic studies characterizing N-hydroxy-N-(2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl)urea.

A series of N-hydroxyurea derivatives have been prepared and examined as inhibitors of 5-lipoxygenase. Oral activity was established by examining the inhibition of LTB4 biosynthesis in an ex vivo assay in the mouse. The pharmacodynamic performance in the mouse of selected compounds was assessed using an ex vivo LTB4 assay and an adoptive peritoneal anaphylaxis assay at extended pretreat times.

Manipulation of distinct NFkappaB proteins alters interleukin-1beta-induced human rheumatoid synovial fibroblast prostaglandin E2 formation.

Interleukin 1beta (IL-1beta) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFkappaB consensus site. Immunoblot analysis identified NFkappaB1 (p50), RelA (p65), and c-Rel in RSF.

Production of biologically active human RelA (p65) in baculovirus-infected insect cells.

A recombinant baculovirus was constructed to express a cDNA encoding RelA (p65), a member of the NF-kappa B/Rel family of proteins. Infection of Spodoptera frugiderda insect cells with the recombinant baculovirus resulted in the production of the biologically active protein as measured by immunoblotting using RelA-specific antisera and by electrophoretic mobility shift assays. The recombinant protein bound specifically to an oligonucleotide containing the NF-kappa B consensus motif but not to that containing the unrelated Oct-1 consensus motif.

Inhibitors of coenzyme A-independent transacylase induce apoptosis in human HL-60 cells.

ET-18-O-CH3 (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine) is an antiproliferative agent, blocking the growth of cancer cells both in vitro and in vivo. However, there is controversy regarding the mechanism leading to its antiproliferative effects. CoA-independent transacylase (CoA-IT) is an enzyme that remodels arachidonate between specific phospholipid donor and acceptor molecules in a variety of mammalian cells.

1-substituted 4-aryl-5-pyridinylimidazoles: a new class of cytokine suppressive drugs with low 5-lipoxygenase and cyclooxygenase inhibitory potency.

A series of 1-alkyl- or -aryl-4-aryl-5-pyridinylimidazoles (A) were prepared and tested for their ability to bind to a recently discovered protein kinase termed CSBP and to inhibit lipopolysaccharide (LPS)-stimulated TNF production in mice. The kinase, CSBP, appears to be involved in a signaling cascade initiated by a number of inflammatory stimuli and leading to the biosynthesis of the inflammatory cytokines IL-1 and TNF. Two related imidazole classes (B and C) had previously been reported to bind to CSBP and to inhibit LPS-stimulated human monocyte IL-1 and TNF production.

Human keratinocytes lack the components to produce leukotriene B4.

The cellular origin of leukotriene B4 (LTB4), a potent pro-inflammatory molecule present in psoriatic lesions, has yet to be determined. In the present study, cultured human keratinocytes were evaluated for their ability to produce LTB4. Keratinocytes stimulated under a variety of conditions did not produce detectable amounts of LTB4, as measured by enzyme immunoassay and liquid chromatographic techniques.

Effects of CoA-independent transacylase inhibitors on the production of lipid inflammatory mediators.

The enzyme CoA-independent transacylase (CoA-IT) has been proposed to mediate the movement of arachidonate between specific phospholipid subclasses, and we have shown that two inhibitors of CoA-IT (SK&F 98625 and SK&F 45905) block this movement. In this report, we use these inhibitors to further characterize the role of CoA-IT in the production of lipid mediators. SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydro-imidazol-1-yl)heptane- phosphonate) and SK&F 45905 [2(-)[3-(4-chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethyl phenoxy]-4,5-dichlorobenzenesulfonic acid) inhibited CoA-IT activity (IC50 values of 9 microM and 6 microM, respectively).

SB 203347, an inhibitor of 14 kDa phospholipase A2, alters human neutrophil arachidonic acid release and metabolism and prolongs survival in murine endotoxin shock.

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 fatty acyl group [predominantly arachidonic acid (AA)] of membrane phospholipids, the products of which are further metabolized, forming a variety of eicosanoids and/or platelet-activating factor. PLA2 activity is significantly enhanced during inflammation and therefore offers an intriguing target in designing anti-inflammatory drugs. SB 203347 (2-[2-[3,5-bis (trifluoromethyl) sulfonamido]-4- trifluoromethylphenoxy] benzoic acid) potently inhibits rh type II 14 kDa PLA2 (IC50 = 0.

Pharmacological characterization of SB 202235, a potent and selective 5-lipoxygenase inhibitor: effects in models of allergic asthma.

The peptidoleukotrienes and leukotriene B4, formed from arachidonic acid through the action of 5-lipoxygenase (5-LO), exert a spectrum of biological effects. It has been proposed that potent and selective 5-LO inhibitors will be effective therapy in diseases in which the peptidoleukotrienes and leukotriene B4 have been implicated, such as asthma and arthritis. The novel compound (S)-N-hydroxy-N-(2,3-dihydro-6-phenylmethoxy-3-benzyofuranyl )urea (SB 202235) was evaluated as a selective inhibitor of 5-LO in a cell-free system as well as in various cellular assays.

Interleukin-8 production is regulated by protein kinase C in human keratinocytes.

Interleukin-8 (IL-8) is a potent pro-inflammatory molecule present in high amounts in psoriatic skin. Here it may play an important role in the keratinocyte hyperproliferation and the neutrophil and T-lymphocyte infiltration associated with the disease. In this study the effect of protein kinase C inhibitors on IL-8 production by human keratinocytes in vitro was investigated.

Human keratinocytes possess an sn-2 acylhydrolase that is biochemically similar to the U937-derived 85-kDa phospholipase A2.

The phospholipase A2 (PLA2) activities that are localized in the keratinocyte cytosolic and microsomal fractions were biochemically and pharmacologically characterized. The cytosol and to a lesser extent the microsome were sensitive to heat treatment and stable in the presence of sulfhydryl reducing agents. Both fractions were almost totally inactivated by reduction of pH to 2.

Use of a continuous assay of oxygen consumption to evaluate the pharmacology of 5-lipoxygenase inhibitors.

A variety of assay systems have been utilized to evaluate the inhibition of the key enzyme in leukotriene (LT) biosynthesis, 5-lipoxygenase (5-LO). We have developed an assay utilizing a cytosolic preparation of 5-LO from rat basophilic leukemia (RBL-1) cells. Enzyme activity was monitored by continuous measurement of oxygen consumption.