Etoricoxib (MK-0663): preclinical profile and comparison with other agents that selectively inhibit cyclooxygenase-2.

We report here the preclinical profile of etoricoxib (MK-0663) [5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonylphenyl) pyridine], a novel orally active agent that selectively inhibits cyclooxygenase-2 (COX-2), that has been developed for high selectivity in vitro using whole blood assays and sensitive COX-1 enzyme assays at low substrate concentration. Etoricoxib selectively inhibited COX-2 in human whole blood assays in vitro, with an IC(50) value of 1.1 +/- 0.1 microM for COX-2 (LPS-induced prostaglandin E2 synthesis), compared with an IC(50) value of 116 +/- 8 microM for COX-1 (serum thromboxane B2 generation after clotting of the blood). Using the ratio of IC(50) values (COX-1/COX-2), the selectivity ratio for the inhibition of COX-2 by etoricoxib in the human whole blood assay was 106, compared with values of 35, 30, 7.6, 7.3, 2.4, and 2.0 for rofecoxib, valdecoxib, celecoxib, nimesulide, etodolac, and meloxicam, respectively. Etoricoxib did not inhibit platelet or human recombinant COX-1 under most assay conditions (IC(50) > 100 microM). In a highly sensitive assay for COX-1 with U937 microsomes where the arachidonic acid concentration was lowered to 0.1 microM, IC(50) values of 12, 2, 0.25, and 0.05 microM were obtained for etoricoxib, rofecoxib, valdecoxib, and celecoxib, respectively. These differences in potency were in agreement with the dissociation constants (K(i)) for binding to COX-1 as estimated from an assay based on the ability of the compounds to delay the time-dependent inhibition by indomethacin. Etoricoxib was a potent inhibitor in models of carrageenan-induced paw edema (ID(50) = 0.64 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 0.34 mg/kg), LPS-induced pyresis (ID(50) = 0.88 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.6 mg/kg/day) in rats, without effects on gastrointestinal permeability up to a dose of 200 mg/kg/day for 10 days. In squirrel monkeys, etoricoxib reversed LPS-induced pyresis by 81% within 2 h of administration at a dose of 3 mg/kg and showed no effect in a fecal 51Cr excretion model of gastropathy at 100 mg/kg/day for 5 days, in contrast to lower doses of diclofenac or naproxen. In summary, etoricoxib represents a novel agent that selectively inhibits COX-2 with 106-fold selectivity in human whole blood assays in vitro and with the lowest potency of inhibition of COX-1 compared with other reported selective agents.

The human prostanoid DP receptor stimulates mucin secretion in LS174T cells.

This study demonstrates the localization of the prostaglandin (PG)D(2) receptor (DP) within the mucous-secreting globlet cells of the human colon by in situ hybridization, which suggests a role for DP in mucous secretion. Selective high affinity ligands were used, therefore, to evaluate DP regulation of mucous secretion in LS174T human colonic adenocarcinoma cells. The expression of hDP in LS174T cells was confirmed at the mRNA level by reverse transcriptase-polymerase chain reaction, and at the protein level by radioligand binding assays and signal transduction (cyclic AMP accumulation) assays. PGD(2) and the highly selective DP-specific agonist L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)), but not PGE(2) competed for [(3)H]-PGD(2)-specific binding to LS174T cell membranes (K:(i) values of 0.4 nM and 7 nM, respectively). The DP-specific agonists PGD(2), PGJ(2), BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropylhydantoin)), and L-644,698 showed similar potencies in stimulating cyclic AMP accumulation (EC(50) values: 45 - 90 nM) and demonstrated the expected rank order of potency. PGE(2) also elicited cyclic AMP production in this cell line (EC(50) value: 162 nM). The activation of cyclic AMP production by PGD(2) and L-644,698, but not PGE(2), was inhibited by the selective DP antagonist BW A868C. Thus, PGD(2) and L-644,698 act through hDP in LS174T cells. PGD(2), L-644,698 and PGE(2) (an established mucin secretagogue) potently stimulated mucin secretion in LS174T cells in a concentration-dependent manner (EC(50)<50 nM). However, BW A868C effectively antagonized only the mucin secretion mediated by PGD(2) and L-644,698 and not PGE(2). These data support a role for the DP receptor in the regulation of mucous secretion.

A novel biological role for prostaglandin D2 is suggested by distribution studies of the rat DP prostanoid receptor.

We report the cloning, functional expression and cell-specific localization of the rat homologue of the prostaglandin D2 receptor (DP). In situ hybridization, utilizing multiple digoxigenin-labelled riboprobes and their complementary sense controls, was performed to determine the detailed distribution of DP receptor mRNA in the central nervous system and the gastrointestinal tract. Within the brain, the leptomeninges and choroid plexus expressed DP receptor mRNA. Transcripts detected in the spinal cord were localized to the sensory and motor neurons of the dorsal and ventral horns, respectively, suggesting a role for the DP receptor in the modulation of central nervous system processes, including pain transmission. Within the gastrointestinal tract (stomach, duodenum, ileum and colon) signals were highly localized to the mucous-secreting goblet cells and the columnar epithelium. These findings suggest a novel biological role for prostaglandin D2-mediated activity at the DP receptor, namely mucous secretion. In addition, radioligand binding assays (saturation analyses and equilibrium competition assays) and functional assays (measuring cAMP accumulation) were performed to characterize the recombinant rat DP receptor expressed in human embryonic kidney (HEK) 293(EBNA) cells. A single site of binding (K(D) = 14 nM, Bmax = 115 fmol/mg protein) was measured for prostaglandin D2-specific binding to the rat DP receptor. Prostaglandin D2 proved to be a potent agonist at the rat DP receptor (EC50 = 5 nM). The rank order of efficacy for DP receptor specific agonists [prostaglandin D2 = prostaglandin J2 = BW 245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropylhydantoin)) > L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)] reflected the affinity with which the ligands bound to the receptor.

Rofecoxib [Vioxx, MK-0966; 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone]: a potent and orally active cyclooxygenase-2 inhibitor. Pharmacological and biochemical profiles.

The discoveries that cyclooxygenase (COX)-2 is an inducible form of COX involved in inflammation and that COX-1 is the major isoform responsible for the production of prostaglandins (PGs) in the gastrointestinal tract have provided a rationale for the development of specific COX-2 inhibitors as a new class of anti-inflammatory agents with improved gastrointestinal tolerability. In the present study, the preclinical pharmacological and biochemical profiles of rofecoxib [Vioxx, also known as MK-0966, 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone], an orally active COX-2 inhibitor, are described. Rofecoxib is a potent inhibitor of the COX-2-dependent production of PGE(2) in human osteosarcoma cells (IC(50) = 26 +/- 10 nM) and Chinese hamster ovary cells expressing human COX-2 (IC(50) = 18 +/- 7 nM) with a 1000-fold selectivity for the inhibition of COX-2 compared with the inhibition of COX-1 activity (IC(50) > 50 microM in U937 cells and IC(50) > 15 microM in Chinese hamster ovary cells expressing human COX-1). Rofecoxib is a time-dependent inhibitor of purified human recombinant COX-2 (IC(50) = 0.34 microM) but caused inhibition of purified human COX-1 in a non-time-dependent manner that could only be observed at a very low substrate concentration (IC(50) = 26 microM at 0.1 microM arachidonic acid concentration). In an in vitro human whole blood assay, rofecoxib selectively inhibited lipopolysaccharide-induced, COX-2-derived PGE(2) synthesis with an IC(50) value of 0.53 +/- 0.02 microM compared with an IC(50) value of 18.8 +/- 0.9 microM for the inhibition of COX-1-derived thromboxane B(2) synthesis after blood coagulation. Using the ratio of the COX-1 IC(50) values over the COX-2 IC(50) values in the human whole blood assay, selectivity ratios for the inhibition of COX-2 of 36, 6.6, 2, 3, and 0.4 were obtained for rofecoxib, celecoxib, meloxicam, diclofenac, and indomethacin, respectively. In several in vivo rodent models, rofecoxib is a potent inhibitor of carrageenan-induced paw edema (ID(50) = 1.5 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 1.0 mg/kg), lipopolysaccharide-induced pyresis (ID(50) = 0.24 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.74 mg/kg/day). Rofecoxib also has a protective effect on adjuvant-induced destruction of cartilage and bone structures in rats. In a (51)Cr excretion assay for detection of gastrointestinal integrity in either rats or squirrel monkeys, rofecoxib has no effect at doses up to 200 mg/kg/day for 5 days. Rofecoxib is a novel COX-2 inhibitor with a biochemical and pharmacological profile clearly distinct from that of current nonsteroidal anti-inflammatory drugs and represents a new therapeutic class of anti-inflammatory agents for the treatment of the symptoms of osteoarthritis and rheumatoid arthritis with improved gastrointestinal tolerability.

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.

Characterization of the human cysteinyl leukotriene CysLT1 receptor.

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.

Demonstration of cell-specific phosphorylation of LTC4 synthase.

PMA-induced leukotriene C4 synthase (LTC4S) phosphorylation was investigated over a period of 8 h in a monocytic cell line (THP-1). The level of LTC4S phosphorylation was increased 3-5 fold over a 4 h period decreasing to basal levels after 8 h. This phosphorylation event was found to be specific to THP-1 cells as there was a lack of LTC4S phosphorylation in both COS-7 and K-562 cells, and was also found to be dependent on the cellular confluency. In the presence of specific protein kinase C (PKC) inhibitors, a dose-dependent inhibition of the phosphorylation of LTC4S became evident, an effect not seen with PKA and tyrosine kinase inhibitors. This represents the first direct demonstration of LTC4S phosphorylation in whole cells.

A random rapid equilibrium mechanism for leukotriene C4 synthase.

Kinetic studies performed on the conjugation reaction catalyzed by LTC4 synthase proved to conform to a random rapid equilibrium mechanism which was further substantiated by competition patterns ruling out other possible mechanisms. Most cytosolic Gst's investigated to date appear to follow a random kinetic mechanism although are mainly responsible for detoxification purposes. Conversely, LTC4 synthase possesses a very different biological role yet still follows a similar mechanism. Therefore, it can be concluded that most GSTs function in a consistent manner regardless of their biological function. Of interest are the mechanisms of the other members of the MAPEG family which in some respects are closer to conventional GSTs than to LTC4 synthase, yet they remain to be deciphered.

Molecular cloning and functional expression of a Caenorhabditis elegans aminopeptidase structurally related to mammalian leukotriene A4 hydrolases.

In a search of the Caenorhabditis elegans DNA data base, an expressed sequence tag of 327 base pairs (termed cm01c7) with strong homology to the human leukotriene A4 (LTA4) hydrolase was found. The use of cm01c7 as a probe, together with conventional hybridization screening and anchored polymerase chain reaction techniques resulted in the cloning of the full-length 2.1 kilobase pair C. elegans LTA4 hydrolase-like homologue, termed aminopeptidase-1 (AP-1). The AP-1 cDNA was expressed transiently as an epitope-tagged recombinant protein in COS-7 mammalian cells, purified using an anti-epitope antibody affinity resin, and tested for LTA4 hydrolase and aminopeptidase activities. Despite the strong homology between the human LTA4 hydrolase and C. elegans AP-1(63% similarity and 45% identity at the amino acid level), reverse-phase high pressure liquid chromatography and radioimmunoassay for LTB4 production revealed the inability of the C. elegans AP-1 to use LTA4 as a substrate. In contrast, the C. elegans AP-1 was an efficient aminopeptidase, as demonstrated by its ability to hydrolyze a variety of amino acid p-nitroanilide derivatives. The aminopeptidase activity of C. elegans AP-1 resembled that of the human LTA4 hydrolase/aminopeptidase enzyme with a preference for arginyl-p-nitroanilide as a substrate. Hydrolysis of the amide bond of arginyl-p-nitroanilide was inhibited by bestatin with an IC50 of 2.6 +/- 1.2 microM. The bifunctionality of the mammalian LTA4 hydrolase is still poorly understood, as the physiological substrate for its aminopeptidase activity is yet to be discovered. Our results support the idea that the enzyme originally functioned as an aminopeptidase in lower metazoa and then developed LTA4 hydrolase activity in more evolved organisms.

Kinetic mechanism of glutathione conjugation to leukotriene A4 by leukotriene C4 synthase.

The kinetic mechanism for human leukotriene (LT) C4 synthase, a membrane-bound glutathione S-transferase, which catalyzes the conjugation of glutathione (GSH) to 5,6-oxido-7,9,11, 14-eicosatetraenoic acid (LTA4), to form 5(S)-hydroxy-6(R)-S-glutathionyl-7,9,trans-11, 14-cis-eicosatetraenoic acid (LTC4) was investigated by initial rate kinetic studies in which concentrations of both substrates and the reversible dead-end inhibitor, 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)- methoxy]- 4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]ethoxy]butanoic acid (L-699,333) were varied. Analysis of the initial velocities of LTC4 formation in the absence of the inhibitor using non-linear regression fits of various models to the data favoured a random, rapid equilibrium mechanism, with strong substrate inhibition by LTA4, over both a compulsory ordered mechanism and a ping-pong mechanism. The estimated parameters were calculated to be Vmax = 14 +/- 4 microM/min, KLTA4 = 40 +/- 18 microM, KGSH = 0.4 +/- 0.2 mM, and a KiLTA4 = 2.3 +/- 1.7 microM for the rapid equilibrium random model. Inhibition of enzymatic activity by L-699,333 was found to be reversible as assessed by the ability of the enzyme to restore its activity by 95% upon dilution. L-699,333 was found to be a competitive inhibitor against GSH and non-competitive against LTA4. Non-linear least squares regression analysis yielded estimated parameters of Km = 0.7 +/- 0.1 mM, Vmax = 2.5 +/- 0.1 microM/min, and Ki = 0.7 +/- 0.1 microM for GSH at a fixed LTA4 concentration of 20 microM, and Km = 45 +/- 3 microM, Vmax = 4.9 +/- 0.2 microM/min, and a Ki = 5.8+/-0.4 microM for LTA4 at a fixed GSH concentration of 2 mM. The rate equation for the random equilibrium mechanism accommodates the inhibition patterns observed for L-699,333 against both substrates as revealed by kinetic fits of the inhibition data to the overall rate equation.

Characterization of the recombinant human prostanoid DP receptor and identification of L-644,698, a novel selective DP agonist.

1. A human embryonic kidney cell line [HEK 293(EBNA)] stably expressing the human recombinant prostaglandin D2 (PGD2) receptor (hDP) has been characterized with respect to radioligand binding and signal transduction properties by use of prostanoids and prostanoid analogues. Radioligand binding studies included saturation analyses, the effects of nucleotide analogues, the initial rate of ligand-receptor association and equilibrium competition assays. In addition, adenosine 3':5'-cyclic monophosphate (cyclic AMP) generation in response to ligand challenge was also measured, as this is the predominant hDP signalling pathway. 2. L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)) was identified as a novel ligand having high affinity for hDP with an inhibitor constant (Ki) of 0.9 nM. This Ki value was comparable to the Ki values obtained in this study for ligands that have previously shown high affinity for DP: PGD2 (0.6 nM), ZK 110841 (0.3 nM), BW245C (0.4 nM), and BW A868C (2.3 nM). 3. L-644,698 was found to be a full agonist with an EC50 value of 0.5 nM in generating cyclic AMP following activation of hDP. L-644,698 is, therefore, comparable to those agonists with known efficacy at the DP receptor (EC50): PGD2 (0.5 nM), ZK 110841 (0.2 nM) and BW245C (0.3 nM). 4. L-644,698 displayed a high degree of selectivity for hDP when compared to the family of cloned human prostanoid receptors: EP1 (> 25,400 fold), EP2 (approximately 300 fold), EP3-III (approximately 4100 fold), EP4 (approximately 10000 fold), FP (> 25,400 fold), IP (> 25,400 fold) and TP (> 25,400 fold). L-644,698 is, therefore, one of the most selective DP agonists as yet described. 5. PGJ2 and delta12-PGJ2, two endogenous metabolites of PGD2, were also tested in this system and shown to be effective agonists with Ki and EC50 values in the nanomolar range for both compounds. In particular, PGJ2 was equipotent to known DP specific agonists with a Ki value of 0.9 nM and an EC50 value of 1.2 nM.

Identification and characterization of a novel microsomal enzyme with glutathione-dependent transferase and peroxidase activities.

5-Lipoxygenase activating protein (FLAP), leukotriene-C4 (LTC4) synthase, and microsomal glutathione S-transferase II (microsomal GST-II) are all members of a common gene family that may also include microsomal GST-I. The present work describes the identification and characterization of a novel member of this family termed microsomal glutathione S-transferase III (microsomal GST-III). The open reading frame encodes a 16.5-kDa protein with a calculated pI of 10.2. Microsomal GST-III has 36, 27, 22, and 20% amino acid identity to microsomal GST-II, LTC4 synthase, microsomal GST-I, and FLAP, respectively. Microsomal GST-III also has a similar hydrophobicity pattern to FLAP, LTC4 synthase, and microsomal GST-I. Fluorescent in situ hybridization mapped microsomal GST-III to chromosomal localization 1q23. Like microsomal GST-II, microsomal GST-III has a wide tissue distribution (at the mRNA level) and is predominantly expressed in human heart, skeletal muscle, and adrenal cortex, and it is also found in brain, placenta, liver, and kidney tissues. Expression of microsomal GST-III mRNA was also detected in several glandular tissues such as pancreas, thyroid, testis, and ovary. In contrast, microsomal GST-III mRNA expression was very low (if any) in lung, thymus, and peripheral blood leukocytes. Microsomal GST-III protein was expressed in a baculovirus insect cell system, and microsomes from Sf9 cells containing either microsomal GST-II or microsomal GST-III were both found to possess glutathione-dependent peroxidase activity as shown by their ability to reduce 5-HPETE to 5-HETE in the presence of reduced glutathione. The apparent Km of 5-HPETE was determined to be approximately 7 microM for microsomal GST-II and 21 microM for microsomal GST-III. Microsomal GST-III was also found to catalyze the production of LTC4 from LTA4 and reduced glutathione. Based on these catalytic activities it is proposed that this novel membrane protein is a member of the microsomal glutathione S-transferase super family, which also includes microsomal GST-I, LTC4 synthase, FLAP, and microsomal GST-II.

Characterization of microsomal GST-II by western blot and identification of a novel LTC4 isomer.

Protein expression of microsomal GST-II and LTC4 synthase was analyzed by Western blot. Correlation between a 17 kDa band and LTC4 formation was observed for both enzymes. The expression of microsomal GST-II was several fold more efficient than the expression of LTC4 synthase. In addition to catalyzing the biosynthesis of LTC4, microsomal GST-II also produces another product, which has been subjected to mass spectrometric analysis. This analysis demonstrates that the novel product is an isomer of LTC4.

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor.

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furan one) was identified 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 acid-dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX-2 (IC50 = 41 +/- 14 nM) over COX-1 (IC50 > 50 microM). Indomethacin was a potent inhibitor of both COX-1 (IC50 = 18 +/- 3 nM) and COX-2 (IC50 = 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 B2 (TXB2) by Ca2+ ionophore-challenged human platelets (IC50 > 50 microM and 4.1 +/- 1.7 nM, respectively). 3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a Ki, value of 140 +/- 68 microM for the initial reversible binding to enzyme and a kappa 2 value of 0.11 +/- 0.06 s-1 for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62 +/- 26 microM and 0.06 +/- 0.01 s-1, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1:1 stoichiometry and to dissociate only very slowly (t1/2 = 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 purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC50 = 63 +/- 5 microM at 0.1 microM 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 PGE2 production (COX-2) in a human whole blood assay with a potency (IC50 = 0.28 +/- 0.04 microM) similar to indomethacin (IC50 = 0.68 +/- 0.17 microM). In contrast, DFU was at least 500 times less potent (IC50 > 97 microM) than indomethacin at inhibiting coagulation-induced TXB2 production (COX-1) (IC50 = 0.19 +/- 0.02 microM). 6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 microM), DFU inhibited COX-1 with an IC50 value of 13 +/- 2 microM 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 diclofenac > indomethacin approximately naproxen > nimesulide approximately meloxicam approximately piroxicam > NS-398 approximately SC-57666 > SC-58125 > CGP 28238 approximately etodolac > L-745,337 > DFU. 7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED50 of 1.1 mg kg-1 vs 2.0 mg kg-1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg-1 vs 1.5 mg kg-1 for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED50 = 0.76 mg kg-1 vs 1.1 mg kg-1 for indomethacin). 8. In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg-1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg-1), meloxicam (3 mg kg-1) or etodolac (10-30 mg kg-1). A 5 day administration of DFU in squirrel monkeys (100 mg kg-1) did not affect chromium leakage in contrast to diclofenac (1 mg kg-1) or naproxen (5 mg kg-1). 9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti-inflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor.

Production of leukotriene C4 in different human tissues is attributable to distinct membrane bound biosynthetic enzymes.

Microsomal glutathione S-transferase-II (GST-II) has recently been discovered and characterized as a member of the 5-lipoxygenase-activating protein (FLAP)/5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11, 14-cis-eicosatetraenoic acid (LTC4) synthase gene family, which also includes microsomal glutathione S-transferase-I (GST-I) as a distant member of this gene family. This new enzyme is unique as it is the only member of this family capable of efficiently conjugating reduced glutathione to both 5,6-oxido-7,9,11,14-eicosatetraenoic acid (LTA4) and 1-chloro-2,4-dinitrobenzene. Although microsomal GST-II has been demonstrated to display both general glutathione S-transferase (GST) and specific LTC4 synthase activities, its biological function remains unknown. In this study, we investigated the physiological location of microsomal GST-II as well as the relative importance of this enzyme versus LTC4 synthase for the production of LTC4 in various human tissues and cells that have been previously demonstrated to possess LTC4 synthase activity. As determined by Western blot, microsomal GST-II was predominantly expressed in human liver microsomes, human endothelial cell membranes, and sparsely detected in human lung membranes. In contrast, LTC4 synthase was prevalent in human lung membranes, human platelet homogenates, and human kidney tissue. Concomitant to the formation of LTC4, microsomal GST-II also produces a new metabolite of LTA4, a postulated LTC4 isomer. This isomer was used to distinguish between microsomal GST-II and LTC4 synthase activities involved in the biosynthesis of LTC4. Based on the relative production of LTC4 to the LTC4 isomer, microsomal GST-II was demonstrated to be the principal enzyme responsible for LTC4 production in human liver microsomes and human endothelial cells and played a minor role in the formation of LTC4 in human lung membranes. In comparison, LTC4 synthase was the main enzyme capable of catalyzing the conjugation of reduced glutathione to LTA4 in human lung membranes and human platelet homogenates. Therefore, microsomal GST-II appears to be an integral component in the detoxification of biological systems due to its marked presence in human liver, in accordance with its known GST activity. Microsomal GST-II, however, may also be pivotal for cysteinyl leukotriene formation in endothelial cells, and this could change our current understanding of the regulation of leukotriene biosynthesis in inflammatory disorders such as asthma.

Pharmacological cross-reactivity between 5-lipoxygenase-activating protein, 5-lipoxygenase, and leukotriene C4 synthase.

Leukotriene (LT) C4 synthase, an integral membrane protein, catalyzes the first committed step in the biosynthesis of the peptidyl leukotrienes, which have been implicated in various inflammatory disorders, including human bronchial asthma. To identify possible inhibitors of LTC4 synthase, synthetic compounds known to inhibit other proteins in the leukotriene biosynthetic pathway (5-lipoxygenase-activating protein, FLAP, and 5-lipoxygenase, 5-LO) or to antagonize leukotriene receptors (cys LT1) were tested for activity against LTC4 synthase. These assays were performed on enriched fractions of human LTC4 synthase purified from the human monocytic cell line THP-1. LTA4 and glutathione were used as substrates, and LTC4 product formation was monitored by reverse-phase high pressure liquid chromatography. Representative compounds from distinct structural classes were tested over a concentration range of 40 nM to 100 microM. The most potent inhibitor was found to be a previously established nanomolar 5-lipoxygenase inhibitor, 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)- methoxy]-4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]ethoxy]but anoic acid (L-699.333) of the phenylpyridine structural class of compounds. L-699.333 inhibited LTC4 synthase activity in vitro with an IC50 value of 3.9 microM and a Ki value of 0.25 microM, making it the most potent synthetic inhibitor known of this enzyme. Structure-activity analyses of other phenylpyridines indicated that the inhibition imparted by L-699.333 was retained following the replacement of the carboxylic acid group with other equivalents. Structurally diverse FLAP inhibitors tested against LTC4 synthase were all micromolar inhibitors of the enzyme over a 10-fold range, with MK-886 at 11 microM. These results implicate that compounds that bind competitively to arachidonic acid binding sites on FLAP and 5-LO recognize motifs that are also weakly conserved on the binding site of LTC4 synthase.

Identification and characterization of a novel human microsomal glutathione S-transferase with leukotriene C4 synthase activity and significant sequence identity to 5-lipoxygenase-activating protein and leukotriene C4 synthase.

5-Lipoxygenase-activating protein (FLAP) and leukotriene C4 (LTC4) synthase, two proteins involved in leukotriene biosynthesis, have been demonstrated to be 31% identical at the amino acid level. We have recently identified and characterized a novel member of the FLAP/LTC4 synthase gene family termed microsomal glutathione S-transferase II (microsomal GST-II). The open reading frame encodes a 16.6-kDa protein with a calculated pI of 10.4. Microsomal GST-II has 33% amino acid identity to FLAP, 44% amino acid identity to LTC4 synthase, and 11% amino acid identity to the previously characterized human microsomal GST (microsomal GST-I). Microsomal GST-II also has a similar hydrophobicity pattern to FLAP, LTC4 synthase, and microsomal GST-I. Fluorescent in situ hybridization mapped microsomal GST-II to chromosomal localization 4q28-31. Microsomal GST-II has a wide tissue distribution (at the mRNA level) and was specifically expressed in human liver, spleen, skeletal muscle, heart, adrenals, pancreas, prostate, testis, fetal liver, and fetal spleen. In contrast, microsomal GST-II mRNA expression was very low (when present) in lung, brain, placenta, and bone marrow. This differs from FLAP mRNA, which was detected in lung, various organs of the immune system, and peripheral blood leukocytes, and LTC4 synthase mRNA, which could not be detected in any tissues by Northern blot analysis. Microsomal GST-II and LTC4 synthase were expressed in a baculovirus insect cell system, and microsomes from Sf9 cells containing microsomal GST-II or LTC4 synthase were both found to catalyze the production of LTC4 from LTA4 and reduced glutathione. Microsomal GST-II also catalyzed the formation of another product, displaying a conjugated triene UV absorption spectra with a maximum at 283 nm, suggesting less catalytic stereospecificity compared with LTC4 synthase. Also, the apparent Km for LTA4 was higher for microsomal GST-II (41 microM) than LTC4 synthase (7 microM). In addition, unlike LTC4 synthase, microsomal GST-II was able to catalyze the conjugation of 1-chloro-2, 4-dinitrobenzene with reduced glutathione. Therefore, it is proposed that this novel membrane protein is a member of the microsomal glutathione S-transferase family, also including LTC4 synthase, with significant sequence identities to both LTC4 synthase and FLAP.

Regulation of leukotriene-biosynthetic enzymes during differentiation of myelocytic HL-60 cells to eosinophilic or neutrophilic cells.

Leukotrienes (LTs) are potent mediators of bronchial inflammation and are predominantly produced by myeloid cells. As myelocytic cells differentiate towards either eosinophils or neutrophils, the profile of leukotrienes they produce upon stimulation diverges. Eosinophils produce mainly cysteinyl leukotrienes whereas neutrophils predominantly synthesize 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (LTB delta). The mechanism by which this change in leukotriene composition occurs is unknown. In this study, we investigated the control of leukotriene biosynthetic enzymes during myeloid cell differentiation. Western-blot analyses of myelocytic leukemia cell lines, HL-60#7 and HL-60, differentiated towards eosinophilic or neutrophilic cell types, respectively, demonstrated that as myelocytic cells differentiate towards eosinophils or neutrophils, the protein levels of cytosolic phospholipase A2 (cPLA2) remain constant, whereas 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) levels are simultaneously elevated. As myelocytic cells become more eosinophil-like, 5(S)-hydroxy- 6(R)-S-glutathionyl-7,9-trans-11, 14-cis-eicosatetraenoic acid (LTC delta) synthase activity and expression of both the protein and messenger RNA in the cells are dramatically increased (approximately 75-fold), while the LTC delta synthase level and activity in neutrophil-like cells remain constant at very low levels. In contrast, in neutrophilic cells, the amount of 5,6-oxido-7,9,11,14-eicosatetraenoic acid (LTA delta) hydrolase was elevated approximately 100-fold greater than the increase in LTA delta hydrolase from eosinophilic cells. These results indicate that as a myeloid cell differentiates towards a granulocyte, similar mechanisms of regulation may be applied to the leukotriene biosynthetic pathway up to the point at which the pathway diverges. At the stage in the leukotriene biosynthetic pathway where LTA delta may be converted to either LTC delta or to LTB delta, specific regulators of transcription may become activated as a myelocytic cell differentiates, thereby causing increased LTA delta hydrolase or LTC delta synthase expression.

Cyclooxygenase-2 inhibitors. Synthesis and pharmacological activities of 5-methanesulfonamido-1-indanone derivatives.

The recent discovery of an alternative form cyclooxygenase (cyclooxygenase-2, COX-2), which has been proposed to play a significant role in inflammatory conditions, may provide an opportunity to develop anti-inflammatory drugs with fewer side effects than existing non-steroidal anti-inflammatory drugs (NSAIDs). We have now identified 6-[(2,4-difluorophenyl)-thio]-5-methanesulfonamido-1-indanone++ + (20) (L-745,337) as a potent, selective, and orally active COX-2 inhibitor. The structure-activity relationships in this series have been extensively studied. Ortho- and para-substituted 6-phenyl substitutents are optimal for in vitro potency. Replacement of this phenyl ring by a variety of heterocycles gave compounds that were less active. The methanesulfonamido group seems to be the optimal group at the 5-position of the indanone system. Compound 20 has an efficacy profile that is superior or comparable to that of the nonselective COX inhibitor indomethacin in animal models of inflammation, pain, and fever and appears to be nonulcerogenic within the dosage ranges required for functional efficacy. Although 20 and its oxygen linkage analog 2 (flosulide) are equipotent in the in vitro assays, compound 20 is more potent in the rat paw edema assay, has a longer t1/2 in squirrel monkeys, and seems less ulcergenic than 2 in rats.