Effects of COX-2 inhibitors on aortic prostacyclin production in cholesterol-fed rabbits.

Prostacyclin (PGI(2)) is a potent vasodilator and inhibitor of platelet aggregation that is produced by prostacyclin synthase via the cyclooxygenase (COX) pathway of arachidonic acid metabolism. We investigated the potential role of COX-2 in the production of vasoactive prostanoids by aortic tissue in a rabbit model of dietary cholesterol-induced atherosclerosis. COX-1 was detected as the major isoform by immunoblot analysis in extracts from aortas of normal and 8 week cholesterol-fed animals with COX-2 being induced in atherosclerotic plaques from cholesterol-fed animals. Aortic tissue from cholesterol-fed animals showed decreased levels of basal 6-keto-PGF(1 alpha) and PGE(2) production as compared to the normal controls but showed no difference with respect to their ability to synthesize these prostanoids in response to exogenous arachidonic acid. The highly selective COX-2 inhibitors rofecoxib and the furanone DFP at concentrations of up to 10 micromol/l had no effect on the arachidonic acid-dependent production of 6-keto-PGF(1 alpha), in contrast to indomethacin, which caused a complete inhibition at 0.5 micromol/l. Celecoxib caused a significant inhibition of 6-keto-PGF(1 alpha) at 10 micromol/l but had little effect when the dose was lowered to 1 micromol/l. Similar effects of these inhibitors were observed with respect to the production of PGE(2) and no major difference was observed between aortic tissues from normal and cholesterol-fed animals with regard to inhibitor sensitivity. These results indicate that in a rabbit model of early stage cardiovascular disease, the basal production of 6-keto-PGF(1 alpha) and PGE(2) by aortic tissue is decreased. Furthermore, COX-2 expression is induced in atherosclerotic plaques and may play a role in altering localized synthesis of prostanoids in these lesions but does not appear to significantly impact the arachidonic acid-dependent prostacylin production of aortic tissues, which is largely mediated by COX-1.

Substituted indoles as potent and orally active 5-lipoxygenase activating protein (FLAP) inhibitors.

This paper reports on the SAR investigation of inhibitors of 5-lipoxygenase activating protein (FLAP) based on MK-0591. Emphasis was made on modifications to the nature of the link between the indole and the quinoline moieties, to the substitution pattern around the two heterocycles and to possible replacements of the quinoline moiety. Lead optimization culminated in (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(pyridin-2-ylmethoxy)-ind ol-2-yl]-2,2-dimethylpropanoic acid (18k), as a potent inhibitor of leukotriene biosynthesis that is well absorbed and active in functional models.

Analysis of prostaglandin G/H synthase-2 inhibition using peroxidase-induced luminol luminescence.

The inducible form of the heme-protein prostaglandin G/H synthase (PGHS-2 or COX-2) has been established as a pivotal enzyme in the cascade of events leading to inflammation, hyperalgesia, and pyresis and represents a major therapeutic target in inflammatory disease. Accordingly, we have exploited the heme-catalyzed hydroperoxidase activity of recombinant hCOX-2 to generate luminescence in the presence of luminol, or a cyclic naphthalene hydrazide, and the substrate arachidonic acid. Arachidonate-induced luminescence was shown to be an index of real-time catalytic activity and demonstrated the turnover inactivation of the enzyme. Luminol luminescence was proportional to hCOX-2 concentration and gave accurate Km determinations for arachidonate. Inhibition of hCOX-2 activity, measured by luminescence, by a variety of selective (for COX-2) and nonselective inhibitors showed rank orders of potency similar to those observed with other in vitro and whole cell methods using the recombinant protein. The sensitivity of the luminescence assay also allowed determination of inhibitor potency at substrate concentrations below Km, distinguishing competitive inhibitors such as ibuprofen from time-dependent inhibitors such as DuP-697. Finally the use of higher quantum-yielding luminol analogues allowed measurement of cyclooxygenase activity at extremely low substrate and protein concentrations, enabling a variety of novel assay formats.

Quinolines as potent 5-lipoxygenase inhibitors: synthesis and biological profile of L-746,530.

Leukotriene biosynthesis inhibitors have potential as new therapeutic agents for asthma and inflammatory diseases. A series of novel substituted 2-cyanoquinolines have been synthesized and the structure activity relationships were evaluated with respect to their ability to inhibit the formation of leukotrienes via the 5-lipoxygenase enzyme. [1S,5R]-2-Cyano-4-(3-furyl)-7-¿3-fluoro-5-[3-(3 alpha-hydroxy-6,8-dioxabicyclo[3.2.1]-octanyl)]phenoxymethyl ¿quinoline (L-746,530) 3 represents a distinct class of inhibitors and possesses in vitro and in vivo potency comparable or superior to naphthalenic analog (L-739,010) 2.

Substituted (pyridylmethoxy)naphthalenes as potent and orally active 5-lipoxygenase inhibitors; synthesis, biological profile, and pharmacokinetics of L-739,010.

Dioxabicyclooctanyl naphthalenenitriles have been reported as a class of potent and nonredox 5-lipoxygenase (5-LO) inhibitors. These bicyclo derivatives were shown to be metabolically more stable than their tetrahydropyranyl counterparts but were not well orally absorbed. Replacement of the phenyl ring in the naphthalenenitrile 1 by a pyridine ring leads to the potent and orally absorbed inhibitor 3g (L-739,010, 2-cyano-4-(3-furyl)-7-[[6-[3-(3-hydroxy-6,8-dioxabicyclo[3.2.1] octanyl)]-2-pyridyl]methoxy]naphthalene). Compound 3g inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50S of 20, 1.6, and 42 nM, respectively). Derivative 3g is orally active in the rat pleurisy model (inhibition of LTB4, ED50 = 0.3 mg/kg) and in the anesthetized dog model (inhibition of ex vivo whole blood LTB4 and urinary LTE4, ED50 = 0.45 and 0.23 microgram/kg/min, respectively, i.v. infusion). In addition, 3g shows excellent functional activity against ovalbumin-induced dyspnea in rats (60% inhibition at 0.5 mg/kg, 4 h pretreatment) and Ascaris-induced bronchoconstriction in conscious sheep (50% and > 85% inhibition in early and late phases, respectively at 2.5 micrograms/kg/min, i.v. infusion) and, more particularly in the conscious antigen sensitive squirrel monkey model (53% inhibition of the increase in RL and 76% in the decrease of Cdyn, at 0.1 mg/kg, po). In rats and dogs, 3g presents excellent pharmacokinetics (estimated half-lives of 5 and 16 h, respectively) and bioavailabilities (26% and 73% when dosed as its hydrochloride salt at doses of 20 and 10 mg/kg, respectively, in methocel suspension). Based on its overall biological profile, compound 3g has been selected for preclinical animal toxicity studies.

Mechanism of selective inhibition of human prostaglandin G/H synthase-1 and -2 in intact cells.

Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess potent anti-inflammatory, antipyretic, and analgesic properties but demonstrate reduced side-effects (e.g. gastrotoxicity) when compared with nonselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS-2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) and examined factors that contribute to the increased potency of PGHS inhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS isozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrates by both CHO [hPGHS-1] and CHO [hPGHS-2] cell lines. However, differences were observed in the ability of the hPGHS isoforms to utilize endogenous arachidonic acid released intracellularly following calcium ionophore stimulation or released by human cytosolic phospholipase A2 transiently expressed in the cells. Cell-based screening of PGHS inhibitors demonstrated that the selectivities and potencies of PGHS inhibitors determined using intact cells are affected by substrate concentration and differ from that determined in cell-free microsomal or purified enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved difference in their time-dependent inhibition. Indomethacin displayed time-dependent inhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS-2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 but time-dependent inhibition of hPGHS-2 in intact cells. Reversible inhibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS-2 inhibitor DuP-697 was reversible against hPGHS-1 but irreversible against hPGHS-2.

Eicosanoids modulate hyperpnea-induced bronchoconstriction in canine peripheral airways.

We examined the role of leukotrienes (LTs) in the development of dry air-induced bronchoconstriction (AIB) in canine peripheral airways. Airway reactivity to exogenous LTs was first tested by using an LTD4 aerosol challenge: peripheral airway resistance increased approximately 130 +/- 51% (n = 4) above baseline when compared with its vehicle control. AIB was then assessed by measuring peripheral airway resistance after, and airway wall temperature during, a dry air challenge (DAC). Treatment with a peptidoleukotriene biosynthesis inhibitor (MK-0591) attenuated AIB by approximately 65% without altering airway wall temperature. The fact that MK-0591 did not alter airway reactivity to aerosolized acetylcholine and completely inhibited Ca2+ ionophore-induced LTB4 generation in canine whole blood attests to the specificity of the drug. Treatment with MK-0591 did not affect the increased number of epithelial cells recovered in bronchoalveolar lavage fluid 5 min after DAC. Concentrations of LTs and other eicosanoids in bronchoalveolar lavage fluid from vehicle-treated DAC airways were increased above baseline values; only LTs were reduced by MK-0591. Before MK-0591, AIB was significantly correlated with the dry air-induced generation of LTC4, LTD4, and LTE4. After treatment with MK-0591, AIB was correlated with thromboxane B2, prostaglandin (PG) F2 alpha, and PGE2. We conclude that hyperpnea with dry air stimulates local production and release of LTs in canine bronchi and, alone with the generation of bronchoconstricting and bronchodilating PGs, plays a central role in the modulation of AIB.

Epitope-labeled soluble human interleukin-5 (IL-5) receptors. Affinity cross-link labeling, IL-5 binding, and biological activity.

The human receptor for the potent eosinophilopoietic cytokine interleukin-5 (IL-5) consists of two components: a 60-kDa ligand-binding alpha chain (IL-5 alpha R) and a 130-kDa beta chain (IL-5 beta R). Three ectodomain constructs of the alpha chain (alpha RED) bearing C-terminal epitope tags were engineered and expressed in baculovirus-infected Sf9 cells. Each recombinant alpha chain was secreted into the medium, maximum expression occurring 72 h post-infection. The various soluble alpha chains were shown by affinity cross-link labeling and competition with unlabeled IL-5 to bind recombinant human (rh) 125I-IL-5 specifically with an ED50 of 2-5 nM. The epitope tag provided a simple purification of the receptor from conditioned medium using immunoaffinity chromatography. The purified material had an apparent molecular mass of 43 kDa and was heterogeneously glycosylated. Sedimentation analysis revealed a 1:1 association of the purified epitope-tagged soluble receptor with its ligand, resulting in the formation of a 70-74-kDa complex. Circular dichroism analysis revealed that the soluble alpha chain existed with a significantly ordered structure consisting of 42% beta-sheet and 6% alpha-helix. Such analyses combined with fluorescence spectrometry suggested that ligand-receptor complex formation in solution resulted in minimal conformational changes, consistent with the suggestion that the membrane-associated form of the alpha chain itself has minimal signal transduction capability. Surface plasmon resonance studies of the interaction of the purified alpha RED with immobilized rhIL-5 revealed a specific, competable interaction with a dissociation constant of 9 nM. Preincubation of an IL-5-dependent cell line with the epitope-tagged alpha RED also dose-dependently neutralized rhIL-5-induced proliferation. These data demonstrate that biologically active epitope-tagged recombinant soluble IL-5 receptors are facile to produce in large quantities and may have therapeutic utility in the modulation of IL-5-dependent eosinophilia in man.

Levels of peptidoleukotriene E4 are elevated in active Crohn's disease.

Proinflammatory mediators, including leukotriene (LT) B4, are elevated in the intestinal mucosa in active chronic inflammatory bowel diseases (IBD). LTE4 is the major peptidoleukotriene metabolite and it is stable in urine. The aim of this study was to measure LTE4 levels in the urine of 27 children with Crohn's disease and 27 control subjects including 12 children with functional recurrent abdominal pain and 15 unaffected siblings of IBD patients. LTE4 levels were measured in urine using high-performance liquid chromatography separation and radioimmunoassay with specific antibody. The Pediatric Crohn's Disease Activity Index and physician global assessment were used to categorize patient groups. C-reactive protein, orosomucoid, and erythrocyte sedimentation rate were employed as laboratory markers of mucosal inflammation. Urinary LTE4 levels were elevated in the 13 children with active Crohn's disease (160.5 +/- 59.4 pg/ml; mean +/- SEM) compared with both levels in the 14 patients with inactive disease (67.1 +/- 18.1 pg/ml; p < 0.05) and controls (45.0 +/- 10.9 pg/ml; p < 0.05). We conclude that measurement of urinary LTE4 is a useful test for monitoring the activation of peptidoleukotrienes in patients with Crohn's disease. It provides a noninvasive, objective adjunct for assessment of disease activity and could be employed in future trials examining the role of the leukotriene inhibitors in the medical therapy of IBD.

Effect of altitude on urinary leukotriene (LT) E4 excretion and airway responsiveness to histamine in children with atopic asthma.

Asthmatic subjects who are resident at altitude may experience a deterioration in lung function following a stay at sea level. To determine whether measurement of urinary leukotriene E4 (LTE4) reflects changes in asthma severity and airway responsiveness, 14 allergic asthmatic subjects resident at altitude (1560 m, Davos, Switzerland) were studied. Subjects were randomly divided into two groups. Measurements of baseline forced expiratory volume in one second (FEV1), the concentration of histamine producing a 20% decrease in FEV1, (PC20 FEV1), serum total immunoglobulin E (IgE), eosinophil count, and urinary LTE4 concentration were determined prior to and following a 2 week stay in The Netherlands (sea level) in eight subjects (4 males and 4 females, aged 14 +/- 0.5 yrs) (mean +/- SEM) and over a similar time period in six subjects (4 males and 2 females, aged 15 +/- 0.3 yrs) resident in Davos, Switzerland. There was no significant difference in total IgE and eosinophil count, and no significant correlation between urinary LTE4 and PC20FEV1 histamine, FEV1, total IgE, and eosinophil count. In subjects returning to Davos from The Netherlands there was a significant increase in urinary LTE4 from a baseline value of 16.9 pg.mg-1 creatinine (GM, range 0.3-101.7 pg.mg-1 creatinine) to 52.3 pg.mg-1 creatinine (GM, range 8.8-301.6 pg.mg-1 creatinine), a significant decrease in PC20FEV1 from 1.7 mg.ml-1 (GM, range 0.3-16.4 mg.ml-1) to 0.9 mg.ml-1 (GM, range 0.1-->32 mg.ml-1), and a significant fall in FEV1 from 3.0 +/- 0.3 to 2.8 +/- 0.3 l (mean +/- SEM).(ABSTRACT TRUNCATED AT 250 WORDS)

Antiasthmatic effects of a leukotriene biosynthesis inhibitor (MK-0591) in allergic dogs.

Peptidoleukotrienes may be important mediators of human bronchial asthma. Accordingly, the effects of a selective leukotriene (LT) biosynthesis inhibitor (MK-0591) were assessed in allergic dogs characterized by acute bronchoconstriction and subsequent airway hyperresponsiveness induced by inhaled ragweed allergen. Peak acute increases in airway resistance (Rrs) induced by ragweed were associated with increased bronchoalveolar lavage histamine concentration, and neither parameter was inhibited by MK-0591 (8 micrograms.kg-1.min-1 i.v.). However, the duration of the bronchoconstriction was significantly decreased by MK-0591, with a reduction in the area under the curve of 40% (P < 0.05). Associated with the acute bronchoconstriction in placebo-treated animals was a fivefold increase in urinary LTE4 excretion (as seen with allergic asthmatic patients), which was reduced to < 10% of basal values by MK-0591. Similarly, whole blood LTB4 biosynthesis was abolished in the MK-0591-treated animals. Bronchial hyperresponsiveness preallergen (measured as the percent concentration of acetylecholine required to increase Rrs by 5 cmH2O.l-1.s) tended to improve with MK-0591 (0.41 +/- 0.15 vs. 0.23 +/- 0.05%). Five hours after allergen inhalation, the percent concentration declined substantially in the placebo group (0.07 +/- 0.02%; P < 0.01), revealing an increased airway responsiveness that was significantly blunted by MK-0591 (0.26 +/- 0.07%; P < 0.001). These data suggest that selective inhibition of LT biosynthesis by novel compounds such as MK-0591 may modify the airway changes associated with bronchial hyperresponsiveness, as well as offer symptomatic relief in asthma.

A single-step purification of biologically active recombinant human interleukin-5 from a baculovirus expression system.

Recombinant human interleukin-5 (rhIL-5) was expressed in baculovirus-infected insect cells and purified to homogeneity from the culture medium in a single chromatographic step. Beginning with a cDNA encoding the full-length precursor form of human IL-5, including the authentic secretory leader sequence, recombinant baculovirus-infected insect cells expressed high levels of rhIL-5 (5-15 mg/liter culture) of which > 90% was processed to the mature form and secreted into the culture medium. After removing cells by centrifugation, rhIL-5 was purified by first adjusting the culture medium to the calculated pI value of mature IL-5 (pI 7.44) and then passing the conditioned medium through tandem linked anion- and cation-exchange columns. The resulting pass-through fraction contained the rhIL-5 and was devoid of contaminating proteins. An optional hydrophobic-interaction chromatography step effectively concentrated the pure homodimeric N-glycosylated rhIL-5 with a high overall yield (> 90%). N-terminal amino acid sequence determination indicated that cleavage of the human IL-5 leader sequence in insect cells occurred between Ala19 and Ile20. Recombinant human IL-5 prepared by this procedure bound to the high-affinity IL-5 receptor present on an eosinophilic leukemia cell line and elicited a proliferative response in the IL-5-dependent murine B-cell line BCL1. This rapid and simple procedure for the expression and purification of mature rhIL-5 should therefore enable studies requiring large amounts of this cytokine.

Increased urinary LTE4 excretion following inhalation of LTC4 and LTE4 in asthmatic subjects.

Urinary leukotriene E4 (LTE4) increases during exacerbations of asthma and following antigen challenge. We determined whether urinary LTE4 excretion reflects sulphidopeptide leukotrienes in the airways of asthmatic patients. Urinary LTE4 concentration was measured prior to and 1.5 and 3.5 h following inhalation of bronchoconstrictive doses of leukotriene C4 (LTC4) or LTE4 in eight asthmatic subjects. Increasing doses of agonist were inhaled until a 35% fall in specific airways conductance (sGaw) was achieved. There was no significant difference between the 53 +/- 3% (mean +/- SEM) fall in sGaw following inhalation of LTC4 (63.1 ng geometric mean, GM, range 5.8-527.5 ng) and the 43 +/- 4% fall in sGaw following inhalation of LTE4 7.94 ng/GM (range 132-3701 ng). The LTE4 excretion rate increased significantly from 2.95 (range 0.6-17.5) ng.h-1 to 4.67 (range 0.8-20) ng.h-1 at 1.5 h following LTC4 inhalation; and from 1.8 (range 0.07-6.7) ng.h-1 to 6.9 (range 2.9-27.3) ng.h-1 at 1.5 h following LTE4 inhalation; and had returned from baseline by 3.5 h. There was a correlation between the dose of LTC4 inhaled and LTE4 excreted in the urine (r = 0.82 and r = 0.72, respectively). The % recovery of LTE4 in the urine, of the total dose of inhaled LTC4 or LTE4 administered, was 6.9 +/- 4.1% and 0.8 +/- 0.3%, respectively. Thus, inhalation of bronchoconstricting doses of LTC4 or LTE4 alter urinary LTE4 excretion in a dose-dependent fashion. This indicates that urinary LTE4 can be used as a marker of sulphidopeptide leukotriene synthesis in the lungs of patients with asthma.

Measurement of canine urinary thromboxanes by GC-MS and HPLC-RIA.

Immunoaffinity extraction/gas chromatography-mass spectrometry (IA/GC-MS) and high-performance liquid chromatography-radioimmunoassay (HPLC-RIA) methods were developed to analyse a major human urinary thromboxane metabolite, 2,3-dinor thromboxane B2, in the urine of dogs, a species commonly used for functional studies of thromboxane pharmacology. The beta-metabolite 2,3-dinor TXB2 was unequivocally identified in pooled normal canine urine by IA/GC-MS, and its excretion measured in 6 anesthetized dogs over a 7h period as 2001 +/- 132 pg 2,3-dinor TXB2/mg creatinine (range 624-4493 pg/mg). Thromboxane immunoreactivity co-eluting with synthetic 2,3-dinor TXB2 was also identified by HPLC-RIA and similarly determined (2585 +/- 276 pg/mg creatinine). Exogenous 2,3-dinor TXB2 could be quantitatively recovered by both methodologies over a wide range of concentrations (50-5000 pg/mL), although with better precision by IA/GC-MS (added vs recovered; m = 1.05, r = 0.99) compared with HPLC-RIA (added vs recovered; m = 0.89, r = 0.89). The cyclooxygenase inhibitor indomethacin given by infusion in anaesthetized dogs (2.5, 8 and 25 micrograms/kg/min) dose-dependently inhibited 2,3-dinor TXB2 excretion measured by IA/GC-MS, with maximal inhibition (83.0 +/- 4.2%) being achieved after 6h (25 micrograms/kg/min). Similar results were obtained by HPLC-RIA, with a correlation of 0.88 (slope = 0.9) between the methodologies in samples after drug treatment. These data suggest that the profile of metabolism and excretion of thromboxanes in dogs resembles that of man, and provide a useful animal model for the non-invasive in vivo assessment of inhibitors of thromboxane biosynthesis.

Effect of FLAP antagonist MK-0591 on leukotriene production and ozone-induced airway responses in dogs.

We used the 5-lipoxygenase-activating protein (FLAP) antagonist MK-0591 to investigate the importance of leukotrienes (LT) in causing ozone-induced bronchoconstriction, airway inflammation, and airway hyperresponsiveness in dogs. Six random source dogs were studied. On one day, dogs were treated with MK-0591 (2 mg/kg iv) followed by a continuous intravenous infusion of 8 micrograms.kg-1.min-1. On the other day, the diluent was infused. Acetylcholine airway responsiveness was measured before and 1 h after ozone inhalation (3 ppm for 30 min). On each day, whole blood and bronchoalveolar lavage (BAL) cells were challenged with calcium ionophore to stimulate LTB4 production. Urinary LTE4 levels were measured before and after ozone. MK-0591 inhibited LTB4 production in whole blood by 96% (P = 0.001) and that from BAL cells by 91% (P = 0.001). By contrast, MK-0591 had no effect on ozone-induced bronchoconstriction, airway hyperresponsiveness, or influx of neutrophils into BAL. The mean log difference of the pre- to post-acetylcholine provocative concentration was 0.64 +/- 0.40 during MK-0591 treatment and 0.68 +/- 0.40 during diluent treatment (P = 0.71). These results indicate that peptidoleukotrienes are produced during ozone inhalation and that MK-0591 inhibits LT production in dogs. However, LTs do not play a role in ozone-induced bronchoconstriction, airway inflammation, or airway hyperresponsiveness in dogs.

Chronic leukotriene inhibition in the rat fails to modify the toxicological effects of a cyclooxygenase inhibitor.

A 5-week study was carried out in rats using a leukotriene biosynthesis inhibitor (MK-886; 3-[1-(4-chlorobenzyl)-3-t-butylthio-5-isopropylindol-2-yl]-2,2- dimethylpropanoic acid) at a dose of 300 mg.kg-1 x day-1, this being sufficient to produce > 90% inhibition of ex vivo leukotriene B4 synthesis in rat blood, and a cyclooxygenase inhibitor (indomethacin, 4 and 6 mg.kg-1 x day-1) to ascertain whether inhibition of leukotriene biosynthesis would potentiate or inhibit the toxicity associated with the administration of nonsteroidal anti-inflammatory drugs (NSAIDs), in particular the gastrointestinal damage. Treatment with indomethacin alone or in combination with MK-886 resulted in the toxicity normally associated with NSAIDs, including gastrointestinal lesions. No toxicity was associated with the administration of MK-886 alone, and MK-886 had no significant effect on the incidence of gastrointestinal lesions produced by indomethacin. These results indicate that leukotrienes are not significant mediators of NSAID-induced gastroenteropathy in the rat.

Assessment of the in vivo biochemical efficacy of orally active leukotriene biosynthesis inhibitors.

In man, the therapeutic effectiveness of specific inhibitors of leukotriene (LT) biosynthesis against allergen-induced bronchoconstriction appears to be related to the in vivo biochemical efficacy of these compounds, as measured by inhibition of whole blood LTB4 generation (upon A23187 stimulus) and, particularly, urinary LTE4 excretion. Accordingly, we have assessed the ability of two clinically documented LT biosynthesis inhibitors, zileuton and MK-886, and the structurally novel 5-lipoxygenase activating protein antagonist, MK-0591, to inhibit the production of these inflammatory arachidonic acid metabolites in laboratory dogs. Zileuton (2 mg/kg) was extremely bioavailable in dogs (> 10 microM plasma concentrations), and inhibited the A23187-induced ex vivo production of LTB4 by venous blood by > 90%, in concordance with its potency in canine blood in vitro (IC50 = 1.1 microM). Despite this degree of inhibition in whole blood, urinary LTE4 excretion was reduced by only 52%, a profile of activity similar to that seen in clinical studies. MK-886 was less well absorbed, with plasma concentrations of 3 microM being achieved only at 25 mg/kg. These levels resulted in < 45% inhibition of LTB4 production, but a significant (p < 0.05) 47% inhibition of urinary LTE4 excretion. MK-0591 was similarly bioavailable (compared with MK-886), but 10-fold more active in vivo as a 2 mg/kg dose resulted in 41-62% inhibition of urinary LTE4 excretion (p < 0.05 vs controls; n = 4, 28). Significant inhibition of ex vivo LTB4 synthesis was also observed at this dose (49%), in accord with peak plasma concentrations of 0.5 microM and an in vitro potency of 0.2-0.4 microM (IC50) in whole blood from these animals. At higher dose (10 mg/kg), MK-0591 inhibited LTE4 excretion by 69%, with 88% inhibition of the LT biosynthetic capacity of whole blood. These data demonstrate that the biochemical efficacy of structurally diverse leukotriene biosynthesis inhibitors can be assessed in vivo in normal laboratory dogs. Such measurements, combined with bioavailability data from other species, may be useful for predicting biochemical activity in man.

A rapid biochemical method for measuring antigen-induced pulmonary eosinophil margination in allergic guinea pigs.

The ability of purified guinea pig peritoneal eosinophils (EOS) to oxidise 3,3',5,5'-tetramethylbenzidine (TMB) was assessed in the presence/absence of Br- (3 mM), and compared with that of unpurified elicited peritoneal polymorphonuclear leukocytes (PMN). Br- selectively stimulated EOS peroxidase activity in a cell number-dependent manner, which was not significantly affected by the presence of diluted lung homogenate. By comparison with the peroxidase activity of added purified EOS, lung parenchyma homogenate from naive guinea pigs was estimated to contain 1.04 +/- 0.18 x 10(5) cells/mg wet tissue (n = 6), a value comparable to those calculated from published histological analyses. This was not significantly increased by ovalbumin (OA) allergen inhalation in unsensitised guinea pigs (1.4 x 10(5) EOS/mg), but was increased two-fold over the latter control to 3.0 +/- 0.18 x 10(5) cells/mg after 17 h in animals sensitised by a single injection of OA and subsequently exposed to an aerosol of bronchoactive allergen (n = 13, p < 0.05). Similar results were obtained in a parallel study using bronchoalveolar lavage (saline challenge, 20.2 +/- 2.2% EOS in lavage fluid; OA challenge, 47.1 +/- 3.6% EOS; n = 6, p < 0.05). In animals that had been doubly sensitised (two injections) to OA, the pulmonary eosinophilic response measured biochemically was more pronounced (4.9 +/- 0.2 x 10(5) cells/mg) and was significantly greater than both a non-specific protein inhalation in this sensitisation group, and OA inhalation in singly sensitised animals (n = 12, p < 0.05). Sera from the latter group was shown to contain five times less specific anti-OA IgG than the doubly sensitised animals, suggesting that EOS margination in guinea pigs is proportionate to the animals' immune status for a defined immunological challenge. These data demonstrate that in vivo EOS migration into the whole guinea pig lung can be rapidly determined by biochemical methods, and thus facilitate the in vivo assessment of novel therapeutic agents against the eosinophilic inflammation characteristic of human allergic asthma.

Leukotriene generation and metabolism in dogs: inhibition of biosynthesis by MK-0591.

Peptidoleukotriene metabolism in dogs was investigated to determine the suitability of this species for the development of in vivo biochemical models of asthma and inflammation. Circulatory metabolism of [3H]leukotriene (LT)C4 (0.5 microCi/kg, i.v.) to [3H]LTE4 and subsequent clearance was rapid (T1/2 = 100 sec). After 3 h, the major urinary metabolite was [3H]16-carboxydihydrotetranor LTE4 (identified by radiochromatography), with [3H]LTE4 accruing to a significant 1.7 +/- 0.9% (n = 3) of the original [3H]LTC4 dose. Immunoreactive LTE4 was excreted into canine urine at 1.85 +/- 0.35 to 2.35 +/- 0.57 ng/h (n = 4) over a 6-h period, suggesting that this metabolite may be an index of acute in vivo 5-lipoxygenase activity. MK-0591, a high-affinity ligand for the canine homolog of the human 5-lipoxygenase activating protein, dose-dependently inhibited the systemic generation of peptidoleukotrienes as measured by urinary LTE4 excretion (ED50 1 microgram/kg/min), the time course of disappearance of LTE4 from the urine being similar to that of the clearance of [3H]LTE4. Because the therapeutic improvements in human allergic asthmatics treated with LT synthesis inhibitors and challenged with antigen appear to be related to the degree of in vivo inhibition of LT biosynthesis (measured by urinary LTE4), the dog may be an appropriate species for preclinical assessment of LT inhibitors.