Philip J. Vickers

The discovery of rofecoxib, [MK 966, VIOXX®, 4-(4′-methylsulfonylphenyl)-3-phenyl-2(5H)-furanone], an orally active cyclooxygenase-2 inhibitor

The development of a COX-2 inhibitor rofecoxib (MK 966, Vioxx) is described. It is essentially equipotent to indomethacin both in vitro and in vivo but without the ulcerogenic side effect due to COX-1 inhibition.

5-lipoxygenase-activating protein is an arachidonate binding protein.

5‐Lipoxygenase‐activating protein (FLAP) is an 18‐kDa integral membrane protein which is essential for cellular leukotriene (LT) synthesis, and is the target of LT biosynthesis inhibitors. However, the mechanism by which FLAP activates 5‐LO has not been determined. We have expressed high levels of human FLAP in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus, and used this system to demonstrate that FLAP specifically binds [125I]L‐739,059, a novel photoaffinity analog of arachidonic acid. This binding is inhibited by both arachidonic acid and MK‐886, an LT biosynthesis inhibitor which specifically interacts with FLAP. These studies suggest that FLAP may activate 5‐LO by specifically binding arachidonic acid and transferring this substrate to the enzyme.

FROM INDOMETHACIN TO A SELECTIVE COX-2 INHIBITOR Development of Indolalkanoic Acids as Potent and Selective Cyclooxygenase-2 Inhibitors

Abstract A series of potent and highly selective cyclooxygenase-2 inhibitors have been prepared by replacing the benzoyl group of indomethacin with a 4-bromobenzyl group, and by extending the acetic acid side chain. These compounds show anti-inflammatory activity in rats with no evidence of GI toxicity, even at high doses.

Eicosanoids and their role in immune modulation in fish—a brief overview

Abstract Eicosanoids have been demonstrated to play a central role in immune regulation in mammals brought about by their direct effects on cells such as macrophages and lymphocytes or by their indirect effects via cytokines. Studies have shown that fish mononuclear phagocytes, granulocytes and thrombocytes synthesize and release both cyclooxygenase- and lipoxygenase-derived products such as prostaglandin E2, leukotriene B4 and lipoxin A4. Whether lymphocytes have the ability to generate leukotrienes and lipoxins is still unclear but they do appear to have 12-lipoxygenase activity that leads to the generation of 12-hydroxy fatty acid derivatives. As in mammals, leukotriene and lipoxin biosynthesis requires the presence of a 5-lipoxygenase activating protein-like molecule that is sensitive to the action of the specific inhibitor, MK-886. The prostaglandin-generating ability of trout macrophages can be altered by incubation with lipopolysaccharide suggesting the possible presence of an inducible cyclooxygenase activity. Prostaglandins have been found to suppress the mitogen-induced proliferation of trout leucocytes and the generation of humoral antibody and plasma cells both in vivo and in vitro. The lipoxygenase products, leukotriene B4 and lipoxin A4 have more variable effects ranging from inhibition to stimulation depending on the assay system employed. Overall, there is clear evidence that eicosanoids play a role in immune regulation in fish in a similar way to that reported in mammals.

Mutation of serine‐516 in human prostaglandin G/H synthase‐2 to methionine or aspirin acetylation of this residue stimulates 15‐R‐HETE synthesis

Prostaglandin G/H synthase (PGHS) is a key enzyme in cellular prostaglandin (PG) synthesis and is the target of non‐steroidal anti‐inflammatory agents. PGHS occurs in two isoforms, termed PGHS‐1 and PGHS‐2. These isoforms differ in several respects, including their enzymatic activity following acetylation by aspirin. While PG synthesis by both isoforms is inhibited by aspirin, 15‐R‐hydroxyeicosatetraenoic acid (15‐R‐HETE) synthesis by PGHS‐2, but not PGHS‐1, is stimulated by preincubation with aspirin. We have mutated the putative aspirin acetylation site of hPGHS‐2, and expressed the mutants in COS‐7 cells using recombinant vaccinia virus. Enzyme activity and inhibitor sensitivity studies provide evidence that Ser516 is the aspirin acetylation site of human PGHS‐2 and that substitution of a methionine residue at this position can mimic the effects of aspirin acetylation on enzyme activity.

SYNTHESIS AND BIOLOGICAL EVALUATION OF 2,3-DIARYLTHIOPHENES AS SELECTIVE COX-2 AND COX-1 INHIBITORS

Abstract A series of 2,3-diarylthiophene compounds was prepared and their biological activities were evaluated against human Cox-1 and Cox-2 enzymes. It appears that the methylsulfone group is essential for both the activity and selectivity for the Cox-2 enzyme. Removal of the methylsulfone group gave relatively selective Cox-1 inhibitors.

Synthesis and biological evaluation of 2,3-diarylthiophenes as selective cox-2 inhibitors. part II: Replacing the heterocycle

Abstract The thiophene ring of DuP 697 was replaced by a variety of heterocycles and the products were tested for their ability to inhibit human Cox-2 and Cox-1, the isozymes of cyclooxygenase.

CHARACTERIZATION OF AUTOCRINE INDUCIBLE PROSTAGLANDIN H SYNTHASE-2 (PGHS-2) IN HUMAN OSTEOSARCOMA CELLS

Abstract. The human osteosarcoma 143.98.2 cell line was found to express high levels of prostaglandin synthase-2 (PGHS-2) without detectable levels of prostaglandin synthase-1 (PGHS-1) as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Maximal levels of PGHS-2 induction were attained when the cells were grown beyond confluence. The osteosarcoma cells also secrete IL-1, IL-1 and TNF in the culture medium. PGHS-2 expression was inducible by the exogenous addition of these cytokines as well as conditioned media from auto-induced cultures and inhibitable by treatment with dexamethasone. In contrast, undifferentiated U937 cells selectively express PGHS-1 as analyzed by RT-PCR and Western blotting. The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the cellular PGE2 production mediated by each isoform of human PGHS were determined using osteosarcoma and undifferentiated U937 cells. When cells were p tors to allow time-dependent inhibition prior to arachidonic acid stimulation, NS-398, CGP 28238, L-745,337, SC-58125 all behaved as potent (IC50 = 1 - 30 nM) and selective inhibitors of PGHS-2, in contrast to indomethacin, flurbiprofen or diclofenac which are potent inhibitors of both enzymes. DuP-697 and sulindac sulfide were also potent inhibitors of PGHS-2 but both compounds inhibited cellular PGHS-1 activity at higher doses (IC50 = 0.2 = 0.4 M). Time-dependent inhibition of PGE2 production in osteosarcoma cells was observed for indomethacin, diclofenac and etodolac. The synthesis of PGE2 by U937 cells was strongly dependent on exogenous arachidonic acid (100-fold stimulation) whereas confluent osteosarcoma cells also produced PGE2 without exogenous stimulus (7-fold stimulation by arachidonic acid). Osteosarcoma cells grown beyond confluence released m ore PGE2 from endogenous substrate than arachidonic acid stimulated undifferentiated U937 cells. These results indicate that osteosarcoma cells selectively express PGHS-2 with an autocrine regulation and effective utilization of endogenous arachidonic acid for PGE2 synthesis.

SYNTHESIS AND BIOLOGICAL EVALUATION OF BOTH ENANTIOMERS OF L-761,000 AS INHIBITORS OF CYCLOOXYGENASE 1 AND 2

Abstract Both enantiomers of L-761,000 were prepared and evaluated for their cyclooxygenase activities.

Structural requirements for the binding of fatty acids to 5-lipoxygenase-activating protein

5-Lipoxygenase-activating protein is required for cellular leukotriene synthesis and is the target of the leukotriene biosynthesis inhibitors MK-886 (3-[1-(p-chlorophenyl)-5-isopropyl-3-tert-butylthio-1H- indol-2-yl]-2,2-dimethylpropanoic acid) and MK-591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)-indol-2-yl] - 2,2-dimethylpropanoic acid). Recent studies demonstrate that 5-lipoxygenase-activating protein binds arachidonic acid and stimulates the utilization of this substrate by 5-lipoxygenase. The present study utilizes a radioligand binding assay to assess the affinity of 5-lipoxygenase-activating protein for arachidonic acid and the specificity of the fatty acid binding site on 5-lipoxygenase-activating protein. Our findings demonstrate that the presence of a free carboxyl group on fatty acids or leukotriene biosynthesis inhibitors which interact with 5-lipoxygenase-activating protein is not required for specific binding to the protein. However, the degree of saturation significantly affects the affinity of fatty acids for 5-lipoxygenase-activating protein.