Two cyclooxygenase isozymes catalyze conversion of arachidonic acid to prostaglandin H2: constitutive COX-1 and inducible COX-2. To assess the role of COX-2 in colorectal tumorigenisis, we determined the effects of COX-2 gene (Ptgs2) knockouts and a novel COX-2 inhibitor on Apc delta716 knockout mice, a model of human familial adenomatous polyposis. A Ptgs2 null mutation reduced the number and size of the intestinal polyps dramatically. Furthermore, treating Apc delta716 mice with a novel COX-2 inhibitor reduced the polyp number more significantly than with sulindac, which inhibits both isoenzymes. These results provide direct genetic evidence that COX-2 plays a key role in tumorigenesis and indicate that COX-2-selective inhibitors can be a novel class of therapeutic agents for colorectal polyposis and cancer.

The cysteinyl leukotrienes-leukotriene C4(LTC4), leukotriene D4(LTD4) and leukotriene E4(LTE4)-are important mediators of human bronchial asthma. Pharmacological studies have determined that cysteinyl leukotrienes activate at least two receptors, designated CysLT1 and CysLT2. The CysLT1-selective antagonists, such as montelukast (Singulair), zafirlukast (Accolate) and pranlukast (Onon), are important in the treatment of asthma. Previous biochemical characterization of CysLT1 antagonists and the CysLT1 receptor has been in membrane preparations from tissues enriched for this receptor. Here we report the molecular and pharmacological characterization of the cloned human CysLT1 receptor. We describe the functional activation (calcium mobilization) of this receptor by LTD4 and LTC4, and competition for radiolabelled LTD4 binding to this receptor by the cysteinyl leukotrienes and three structurally distinct classes of CysLT1-receptor antagonists. We detected CysLT1-receptor messenger RNA in spleen, peripheral blood leukocytes and lung. In normal human lung, expression of the CysLT1-receptor mRNA was confined to smooth muscle cells and tissue macrophages. Finally, we mapped the human CysLT1-receptor gene to the X chromosome.

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In this study, PGE2 levels in lipopolysaccharide (LPS)-challenged human whole blood and TxB2 levels following blood coagulation were measured as biochemical index for cyclooxygenase (Cox)-2 and Cox-1 activity respectively. Incubation of human mononuclear cells isolated from whole blood with LPS (100 mu g/mL) induced a time-dependent increase in the expression of Cox-2 protein (>100 fold at 24 hr). This is associated with increases in PGE2 production and free arachidonate release in the plasma. Cox-1 protein was detected in the human mononuclear cells at time zero but was not induced by either LPS or PBS. Most non-steroidal anti-inflammatory drugs (NSAIDs) are more potent at inhibiting Cox-1 than Cox-2. Five experimental compounds CGP-28238, Dup-697, NS-398, SC-58125 and L-745,337, have a greater selectivity for Cox-2. Indomethacin at a single oral dose (25 mg) inhibited approximately 90% the whole blood Cox-2 and Cox-1 activities ex vivo in healthy subjects. These results support the use of this assay to assess the biochemical efficacy of selective Cox-2 inhibitors in clinical trials.

identi®ed as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2 In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic aciddependent production of prostaglandin E 2 (PGE 2 ) with at least a 1,000 fold selectivity for COX-2 (IC 50 =41+14 nM) over COX-1 (IC 50 450 mM). Indomethacin was a potent inhibitor of both COX-1 (IC 50 =18+3 nM) and COX-2 (IC 50 =26+6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B 2 (TXB 2 ) by Ca 2+ ionophore-challenged human platelets (IC 50 450 mM and 4.1+1.7 nM, respectively). 3 DFU caused a time-dependent inhibition of puri®ed recombinant human COX-2 with a K i value of 140+68 mM for the initial reversible binding to enzyme and a k 2 value of 0.11+0.06 s 71 for the ®rst order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62+26 mM and 0.06+0.01 s 71 , respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t 1/2 =1 ± 3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4 Inhibition of puri®ed recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC 50 =63+5 mM at 0.1 mM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5 DFU inhibited lipopolysaccharide (LPS)-induced PGE 2 production (COX-2) in a human whole blood assay with a potency (IC 50 =0.28+0.04 mM) similar to indomethacin (IC 50 =0.68+0.17 mM). In contrast, DFU was at least 500 times less potent (IC 50 497 mM) than indomethacin at inhibiting coagulationinduced TXB 2 production (COX-1) (IC 50 =0.19+0.02 mM). 6 In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 mM), DFU inhibited COX-1 with an IC 50 value of 13+2 mM as compared to 20+1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac4indomethacin*naproxen4nimesulide* meloxicam*piroxicam4NS-398*SC-576664SC-581254CGP 28238*etodolac4L-745,3374DFU. 7 DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED 50 of 1.1 mg kg 71 vs 2.0 mg kg 71 for indomethacin) and hyperalgesia (ED 50 of 0.95 mg kg 71 vs 1.5 mg kg 71 for indomethacin). The compound was also e ective at reversing LPS-induced pyrexia in rats (ED 50 =0.76 mg kg 71 vs 1.1 mg kg 71 for indomethacin). 8 In a sensitive model in which 51 Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no signi®cant e ect was detected after oral...

Motilin is a 22-amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract of humans and other species. It affects gastric motility by stimulating interdigestive antrum and duodenal contractions. A heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptor for motilin was isolated from human stomach, and its amino acid sequence was found to be 52 percent identical to the human receptor for growth hormone secretagogues. The macrolide antibiotic erythromycin also interacted with the cloned motilin receptor, providing a molecular basis for its effects on the human GI tract. The motilin receptor is expressed in enteric neurons of the human duodenum and colon. Development of motilin receptor agonists and antagonists may be useful in the treatment of multiple disorders of GI motility.

The cysteinyl leukotrienes (CysLTs) are important mediators of human asthma. Pharmacologic and clinical studies show that the CysLTs exert most of their bronchoconstrictive and proinflammatory effects through activation of a putative, 7-transmembrane domain, G-protein-coupled receptor, the CysLT1 receptor. The initial molecular characterization of the CysLT1 receptor showed by in situ hybridization, the presence of CysLT1 receptor messenger RNA (mRNA) in human lung smooth-muscle cells and lung macrophages. We confirmed the results of these in situ hybridization analyses for the CysLT1 receptor, and produced the first immunohistochemical characterization of the CysLT1 receptor protein in human lung. The identification of the CysLT1 receptor in the lung is consistent with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists. We also report the expression of CysLT1 receptor mRNA and protein in most peripheral blood eosinophils and pregranulocytic CD34+ cells, and in subsets of monocytes and B lymphocytes.

Caspase-3 is synthesized as a dormant proenzyme and is maintained in an inactive conformation by an Asp-Asp-Asp ''safetycatch'' regulatory tripeptide contained within a flexible loop near the large-subunit͞small-subunit junction. Removal of this ''safety catch'' results in substantially enhanced autocatalytic maturation as well as increased vulnerability to proteolytic activation by upstream proteases in the apoptotic pathway such as caspase-9 and granzyme B. The safety catch functions through multiple ionic interactions that are disrupted by acidification, which occurs in the cytosol of cells during the early stages of apoptosis. We propose that the caspase-3 safety catch is a key regulatory checkpoint in the apoptotic cascade that regulates terminal events in the caspase cascade by modulating the triggering of caspase-3 activation.