Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist

L-660,711 (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid is a potent and selective competitive inhibitor of [3H]leukotriene D4 binding in guinea pig (Ki value, 0.22 nM) and human (Ki value, 2.1 nM) lung membranes but is essentially inactive versus [3H]leukotriene C4 binding (IC50 value in guinea pig lung, 23 microM). Functionally it competitively antagonized contractions of guinea pig trachea and ileum induced by leukotriene (LT) D4 (respective pA2 values, 9.4 and 10.5) and LTE4 (respective pA2 values, 9.1 and 10.4) and contractions of human trachea induced by LTD4 (pA2 value, 8.5). L-660,711 (5.8 x 10(-8)M) antagonized contractions of guinea pig trachea induced by LTC4 in the absence (dose ratio = 28) but not in the presence of 45 mM L-serine borate (dose ratio less than 2). L-660,711 (1.9 x 10(-5)M) did not block contractions of guinea pig trachea induced by histamine, acetylcholine, 5-hydroxytryptamine, PGF2 alpha, U-44069, or PGD2. In the presence of atropine, mepyramine, and indomethacin, L-660,711 (1.9 x 10(-5)M) inhibited a small component of the response to antigen on guinea pig trachea but completely blocked anti-IgE-induced contractions of human trachea. L-660,711 (i.v.) antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. LTC4, LTD4, and LTE4 but did not block bronchoconstriction to arachidonic acid, U-44069, 5-hydroxytryptamine, histamine, or acetylcholine. Intraduodenal L-660,711 antagonized LTD4 (0.2-12.8 micrograms/kg)-induced bronchoconstriction in guinea pigs, and p.o. L-660,711 blocked LTD4- and Ascaris-induced bronchoconstriction in conscious squirrel monkeys and ovalbumin-induced bronchoconstriction in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile of L-660,711 indicates that it is a potent, selective, orally active leukotriene receptor antagonist which is well suited to determine the role played by LTD4 and LTE4 in asthma and other pathophysiologic conditions.     

Effects of a newly synthesized leukotriene antagonist, NZ-107, on immediate-type hypersensitivity reaction in rats and guinea-pigs.

The effects of 4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone (NZ-107) on immediate type hypersensitivity reactions in rats and guinea-pigs were studied. 1. When NZ-107, at a dose of 50 mg/kg (i.p.) or 100 mg/kg (orally), was administered to rats, 48-h homologous passive cutaneous anaphylaxis (PCA) reaction and histamine-, leukotriene C4 (LTC4)- and leukotriene D4 (LTD4)-induced skin reactions were suppressed by the agent. 2. NZ-107 (10(-6) g/ml) inhibited both LTC4- and LTD4-induced contractions of isolated rat stomach smooth muscle. 3. NZ-107 inhibited antigen-induced histamine release from rat peritoneal mast cells by 26% at a concentration of 10(-4) g/ml. 4. NZ-107, at doses of 25 and 50 mg/kg (orally), significantly inhibited guinea-pig 3-h heterologous PCA reaction. 5. NZ-107 inhibited antigen-induced histamine release from guinea-pig lung tissue by 17% and 48% at concentrations of 5 x 10(-5) and 10(-4) g/ml, respectively. 6. NZ-107, at doses of 25 and 50 mg/kg (i.p.), inhibited antigen-induced bronchoconstriction and eosinophil accumulation in the bronchoalveolar lavage fluid (BALF) of guinea-pigs. These results suggest that NZ-107 has anti-allergic action including inhibition of eosinophil accumulation in an antigen-challenged airway lesion in rats and guinea-pigs. The anti-allergic action of this agent is thought to be due to its action as a histamine and LT antagonist and its consequent inhibition of antigen-induced histamine release.     

Comparative study of the pulmonary effects of intravenous leukotrienes and other bronchoconstrictors in anaesthetized guinea pigs

Pulmonary responses to intravenous leukotrienes C4, D4 and E4 administered as a bolus injection and by continuous infusion were studied in anesthetized guinea pigs. LTD4, LTC4 and LTE4 (respective ED50 of 0.21 +/- .1, 0.64 +/- .2 and 2.0 +/- .1 microgram kg-1) produced dose-dependent increases in insufflation pressure when given as a bolus injection to anesthetized guinea pigs (Konzett-R?ssler). Bronchoconstriction was antagonized by FPL-55712 (50-200 micrograms kg-1), and indomethacin (50-200 micrograms kg-1) but was not significantly altered by mepyramine (1.0 mg kg-1), methysergide (0.1 mg kg-1), intal (10 mg kg-1) mepacrine (5 mg kg-1) or dexamethasone (10 mg kg-1). The beta adrenoceptor blocker, timolol (5 micrograms kg-1) produced a significantly greater potentiation of the responses to the leukotrienes than to arachidonic acid, histamine and acetylcholine. Responses to bolus injection of LTE4 but not LTD4 or LTC4 were partially antagonized by atropine (100 micrograms kg-1) and bilateral vagotomy. In experiments of a different design, continuous infusion of LTD4 and LTE4 (2.8-3.2 micrograms kg-1 min-1) into indomethacin-treated animals produced slowly developing increases in pulmonary resistance and decreases in compliance. The increase in resistance produced by LTE4 and LTD4 was partly reversed by intravenous FPL-55712 (1.0 mg kg-1) and atropine (100 micrograms kg-1) but was almost completely reversed by FPL-55712 (3 - 10 mg kg-1). These findings indicate that leukotrienes can produce bronchoconstriction in guinea pigs through cyclooxygenase-dependent and cyclooxygenase independent mechanisms both of which are blocked by FPL-55712. Cholinergic mechanisms are involved in the mediation of part of the response to bolus injection of LTE4 as well as a small part of the initial response to continuous infusion of LTD4 and LTE4. Intrinsic beta adrenoceptor activation serves to down modulate responses to the leukotrienes to a greater extent than responses to arachidonic acid, histamine and acetylcholine.     

Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methane sulfonamide) an orally active leukotriene D4 antagonist: pharmacological characterization in vitro and in vivo in the guinea pig

The following communicates the pharmacology of Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methanesulfonamide) a chemically novel and orally potent leukotriene (LT) D4 receptor antagonist. In the isolated guinea-pig trachea pretreated with indomethacin (5 microM) and L-cysteine (10 mM), Wy-48,252 antagonized TD4-induced contraction with a pKB = 7.6. Against LTC4 on tissues pretreated with IND and glutathione (10 mM), Wy-48,252 had a pKB greater than 5. Wy-48,252 (10 microM) did not antagonize pilocarpine-, histamine- or PGF2 alpha-induced tracheal contraction. Further, in the presence of indomethacin and chlorpheniramine (1 microM), Wy-48,252 dose-dependently inhibited the antigen-induced contraction of guinea-pig trachea in a manner consistent with antagonism at the LTD4 receptor and inhibition of LT synthesis. In the Konzett-Rossler model of i.v. LTD4-induced bronchoconstriction in indomethacin treated guinea pigs, intragastric Wy-48,252 (2 hr) had an ID50 of 100 micrograms/kg and a functional half-life of 5 hr. Against i.v. antigen-induced bronchoconstriction in guinea pigs treated with indomethacin and chlorpheniramine, intragastric Wy-48,252 (2 hr) had an ID50 of 0.6 mg/kg and a 5 hr half life. Intragastric Wy-48,252 also selectively blocked the cutaneous wheal reaction to intradermal LTD4 but not histamine. We conclude that Wy-48,252 is distinguished from other selective LTD4 receptor antagonists by its oral potency and should be useful in ascertaining the role of LTD4 mediated processes in asthma, allergy and animal models.     

Identification and characterization of leukotriene C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1

We studied the characteristics of the leukotriene (LT) C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1. Specific [3H]LTC4 binding to the cell membrane was greater than 80% of total binding and saturable at a density of 3.96 +/- 0.39 pmol/mg protein, with an apparent dissociation constant (Kd) of 14.3 +/- 2.0 nM (n = 9). The association and dissociation of [3H]LTC4 binding were rapid and apparent equilibrium conditions were established within 5 min. Calculated Kd value of [3H]LTC4 binding from the kinetic analysis was 9.9 nM. From the competition analysis, calculated Ki value of unlabeled LTC4 to compete for the specific binding of [3H]LTC4 was 9.2 nM and was in good agreement with the Kd value obtained from the Scatchard plots or kinetic analysis. The rank order of potency of the unlabeled competitors for competing specific [3H]LTC4 binding was LTC4 much greater than LTD4 greater than LTE4 greater than FPL-55712. The maximum number of binding sites (Bmax) of [3H]LTD4 in the membrane of BC3H-1 cell line was about 11 times lower than that of the [3H]LTC4. The calculated values of Kd and Bmax of [3H]LTD4 binding were 9.3 +/- 0.8 nM and 0.37 +/- 0.04 pmol/mg protein, respectively (n = 3). The rank order of potency or the unlabeled competitors for competing specific [3H]LTD4 binding was LTD4 = LTE4 greater than FPL-55712 much greater than LTC4. These findings demonstrate that BC3H-1 cell line possess both LTC4 and LTD4 receptors with a predominance of LTC4 receptors. Thus BC3H-1 cell line is a good model to study the regulation of LTC4 and LTD4 receptors.     

Synthesis and biological activities of leukotriene F4 and leukotriene F4 sulfone

Leukotriene F4 (LTF4) and LTF4 sulfone have been synthesized and their biological activities determined in the guinea pig. In vitro LTF4 displayed comparable activity to LTD4 on guinea pig trachea and parenchyma but was less active on the ileum. When injected intravenously into the guinea pig, LTF4 induced a bronchoconstriction (ED50 16 micrograms Kg-1) which was blocked by indomethacin and FPL-55712 and was 50-100 X less potent than LTD4 in this assay. LTF4 sulfone was approximately 2-5 times less active than LTF4 in vitro and in vivo. When injected into guinea pig skin with PGE2 (100 ng); LTF4 and LTF4 sulfone (10-1000 ng) induced changes in vascular permeability. The order of potency in this assay was LTE4 sulfone = LTD4 = LTD4 sulfone greater than LTE4 greater than LTF4 = LTF4 sulfone.     

ATP-dependent leukotriene export from mastocytoma cells

The biosynthesis of leukotrienes (LT) C4 and B4 is followed by an export of these mediators into the extracellular space. This transport was characterized using plasma membrane vesicles prepared from mastocytoma cells and identified as an ATP-dependent primary active process. The apparent Km-values were 110 nM for LTC4 and 48 microM for ATP. The transport rate was highest for LTC4, whereas LTD4, LTE4, and N-acetyl-LTE4 were transported with relative rates of 31, 12 and 8%, respectively, at a concentration of 10 nM. LTB4 transport was also dependent on ATP. LTC4 transport was inhibited by LTD4 receptor antagonists (IC50 = 1.0 microM for MK-571 and 1.3 microM for LY245769) and by the inhibitor of leukotriene biosynthesis MK-886 (IC50 = 1.8 microM). The ATP-dependent export carrier for leukotrienes in leukotriene-synthesizing cells represents a novel member of the family of ATP-dependent exit pumps.     

Prolonged pulmonary hypertension caused by platelet-activating factor and leukotriene C4 in the rat lung.

Platelet-activating factor (PAF) and leukotrienes (LTs) are potent pulmonary hypertensive and inflammatory mediators produced by the lung. Previously we showed that a rapid injection of PAF into the pulmonary artery of an isolated rat lung produced an extended elevation in mean pulmonary arterial pressure (PAP). The objective of the present study was to determine whether the extended pressor response induced by PAF was caused by prolonged activation of the 5-lipoxygenase pathway or slow clearance of LTs from the lung parenchyma. Rat lungs were perfused with a nonrecirculating physiological salt solution that contained indomethacin and albumin. Five minutes after a rapid injection of PAF into the pulmonary artery catheter, the following elevations (mean % above baseline) were observed: PAP (83%), LTB4 (3,260%), LTC4 (1,490%), LTD4 (970%), and LTE4 (1,500%). At 20 min these levels declined but were still significantly elevated above baseline. The 5-lipoxygenase inhibitor diethylcarbamazine (DEC), administered before the PAF injection, inhibited the elevations of PAP and all LTs. DEC administration that began 5 min after PAF reduced PAP and only LTC4 levels at 20 min in comparison to lungs with no DEC. The 5-lipoxygenase-activating protein inhibitor MK886, administered orally 2-6 h before perfusion, also inhibited the pressor response to PAF as well as LT production, as did DEC. We conclude that 1) the extended pulmonary hypertension induced by PAF was caused mainly by prolonged activation of 5-lipoxygenase with LTC4 production, 2) the relative overall lung clearance of LTB4, LTD4, and LTE4 was slower than that of LTC4, and 3) LTB4, LTD4, and LTE4 had no appreciable pressor effect.     

The biotransformation in vitro of cysteinyl leukotrienes in blood of normal and asthmatic subjects

The metabolism of exogenous leukotriene C4 (LTC4), LTD4 and LTE4 (10(-8) M) was studied in vitro in blood of normal and asthmatic subjects for up to 2 hr by reverse-phase high performance liquid chromatography. In whole blood, incubation of LTC4 (T1/2 = 11.5 min) resulted in the formation of LTD4 and LTE4 whose biosynthesis was inhibited by serine borate (30 mM). Similar experiments performed with LTD4 (T1/2 = 5 min) produced a single metabolite (LTE4) which was inhibited by L-cysteine (10 mM). On the other hand, LTE4 represented a highly stable product in our in vitro system. The bioconversion of LTC4 or LTD4 was slower in plasma but this effect appeared more pronounced for the cysteinylglycinyl derivative. The bioconversion of LTD4 in whole blood or plasma was almost twice as rapid as LTC4. Experiments performed with asthmatic blood showed no significant difference in the survival of LTC4. These results suggest that blood may play a role in regulating the bioavailability of cysteinyl-containing LTs which could be of relevance to their excretion in man.     

Role of leukotrienes during oleic acid-induced lung injury in pigs

We hypothesized that leukotrienes might contribute to the pathophysiology of acute lung injury induced by oleic acid. Oleic acid (2-20 mg.kg-1.h-1), LY171883 [leukotriene (LT) D4/LTE4 receptor antagonist, 10 mg/kg + 1 mg.kg-1.h-1] + oleic acid (10 mg.kg-1. h-1), or triolein (20 mg.kg-1.h-1) were infused intravenously into anesthetized pigs. Treatment with the cyclooxygenase inhibitor was designed to possibly enhance LT release. Bronchoalveolar lavage fluid concentrations of LTB4, LTC4, LTD4, and LTE4 were measured by reverse-phase high-performance liquid chromatography and radioimmunoassay. Oleic acid caused dose-related hypoxemia and pulmonary hypertension and increased pulmonary vascular resistance, lung water, and alveolar-capillary membrane permeability. Bronchoalveolar lavage fluid levels of LTB4, LTC4, LTD4, and LTE4 showed no significant changes in oleic acid- or indomethacin + oleic acid-treated pigs, compared with triolein-treated controls. Indomethacin modestly attenuated the oleic acid-induced hypoxemia and the early increases (i.e., 0-0.5 h) in mean pulmonary arterial pressure and pulmonary vascular resistance. In contrast, LY171883 provided no protection against any oleic acid-induced cardiopulmonary effect (measured or calculated). We conclude that LTs are not likely to be important mediators of oleic acid-induced lung injury in the pig.     

Sulfidopeptide leukotrienes mediate acrolein-induced bronchial hyperresponsiveness

The sulfidopeptide leukotrienes are bronchoconstrictive lipid mediators thought to have an important role in the pathophysiology of asthma. The objective of this study was to determine if treatment with a leukotriene receptor antagonist and 5-lipoxygenase inhibitors could diminish acrolein-induced bronchial hyperresponsiveness and to determine whether leukotriene (LT) C4 generation is augmented by acrolein exposure. Guinea pigs (groups of 6-7) were exposed to 1.3 ppm acrolein for 2 h and bronchial responsiveness to intravenous acetylcholine determined twice before, and once 1, 2, 6, and 24 h after exposure. Immediately after acrolein exposure (5 min) specific total airway resistance (sRt) increased from 0.86 +/- 0.01 to 1.29 +/- 0.07 ml.cmH2O.ml-1.s. Within 1 h after exposure, the effective dose of acetylcholine sufficient to double sRt (ED200) decreased from 114.0 +/- 6.6 to 58.5 +/- 6.5 micrograms.kg-1.min-1. Bronchial hyperresponsiveness became maximal at 2 h with ED200 = 44.7 +/- 4.2 and persisted for up to 24 h after exposure (24 h ED200 = 60.2 +/- 11.6 micrograms.kg-1.min). A LTC4/LTD4 receptor antagonist, L-649,923 (10 mg/kg iv), and two putative inhibitors of 5-lipoxygenase, L-651,392 (10 mg/kg po) and U-60,257 (5 mg/kg i.v.), diminished the immediate bronchoconstriction and markedly inhibited bronchial hyperresponsiveness. Analysis of bronchoalveolar lavage fluid obtained from guinea pigs after acrolein exposure revealed a significant increase in immunoreactive LTC4 concentrations (control LTC4 = 8.8 +/- 0.3, n = 7; exposed LTC4 = 15.9 +/- 2.4 pg/ml, n = 6). Treatment with L-651,392 inhibited this response (acrolein exposed = 9.4 +/- 2.4 pg/ml, n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of LTC4 to LTD4 and LTE4 in isolated guinea pig lung lobes

Leukotrienes are known to be easily metabolized to other substances. But the metabolic fates of LTC4 and LTD4 have not been established in the intact lung. In this investigation we perfused isolated guinea pig lung lobes and injected synthesized LTC4 and LTD4. The effluent was assayed by HPLC. LTD4 and LTE4 were detected following perfusion of LTC4, and LTE4 was detected following perfusion of LTD4. These results suggest that perfused guinea pig lung lobes may metabolize LTC4 to LTD4 and LTE4, and LTD4 to LTE4.     

The in vivo production of peptide leukotrienes after pulmonary anaphylaxis in the rat

Inbred hyper-reactive rats, actively sensitized to OVA, were anesthetized, cannulated, and ventilated with room air. Tracheal instillation of Ag (OVA) resulted in an elevation of airways pressure (14.4 +/- 0.6 cm H2O). Measurement of biliary peptide leukotriene levels before and after Ag challenge using reverse phase HPLC and RIA techniques showed significant elevations in leukotriene (LT) levels, the amounts released being LTC4 (3.65 +/- 0.78), LTD4 (2.8 +/- 1.11), and N-Ac LTE4 (3.87 +/- 1.15) expressed as ng/100 g of body weight, n = 13. Identification of these metabolites were confirmed by HPLC/RIA techniques and LTC4 was further characterized by UV spectroscopy and its enzymatic conversion by gamma-glutamyl transpeptidase to LTD4. [3H]LTC4 (16 ng) administration by tracheal instillation resulted in a 31.4 +/- 4.3% recovery of radioactivity through the bile over 4 h (n = 3) with the major identified metabolite being N-Ac LTE4. [3H]LTC4 (16 ng) plus synthetic LTC4 (5 micrograms) showed a 30.8 +/- 3.1% recovery through the bile after tracheal instillation (3-h collection, n = 4) with significant amounts of LTC4 as well as N-Ac LTE4 present. [3H]LTC4 administration by the portal vein resulted in a 37.4 +/- 8.8% biliary recovery over 60 min (n = 6), the metabolites present in the bile being LTC4, LTD4, LTE4, and N-Ac LTE4. Pretreatment with the 5-lipoxygenase inhibitor L-656,224 (15 mg/kg, 3.5 h pre-p.o.) before Ag challenge resulted in a significant inhibition (greater than 90%, p less than 0.05) of biliary leukotriene levels in this model. Our study demonstrates that peptide leukotrienes are produced in the anesthetized rat after pulmonary anaphylaxis and that biliary leukotriene measurement is suitable for showing the biochemical efficacy of leukotriene inhibitors in vivo. In vivo tracer experiments suggest that the biliary metabolic profile of the peptide leukotrienes is dependent on the site and levels of release as well as the efficiency of the vascular clearance of the various metabolites.     

Biological activity of leukotriene sulfones on respiratory tissues

The biological activity of synthetic leukotriene C4, D4 and E4 sulfone has been determined in respiratory smooth muscle in vitro and in vivo. The sulfones of LTC4, LTD4 and LTE4 were potent contractile agonists on indomethacin-treated guinea pig tracheal chains with respective pD2-values of 8.2, 8.0 and 7.9. Contractions were submaximal (75-85% of the cholinergic maximum), slow in onset, prolonged in duration, slowly reversed by washing (compared to acetylcholine or histamine) and were partially reversed by 2 muM FPL-55712. The sulfones of LTC4, LTD4 and LTE4 also contracted indomethacin-treated guinea pig parenchyma (respective pD2's of 7.9 8.2 and 7.8) and rat parenchyma (respective pD2's of 7.1, 7.2 and 7.2) but were inactive on rat trachea (0.01-2.0 muM). When administered intravenously to anaesthetized guinea pigs, the sulfones of LTD4, LTE4 and to a lesser degree LTC4 (respective ED50's - 0.5; 2.0 and 4.6 microgram/kg) elicited dose-dependent increases in inflation pressure which were antagonized by FPL-55712 and indomethacin. Leukotriene C4, D4 and E4 sulfones display a qualitatively similar profile of biological activity to that of their corresponding sulfides.     

Platelet-activating factor induces the production of leukotrienes by human monocytes

The aim of this study was to evaluate the role of platelet-activating factor (PAF) as a stimulator of leukotriene production by human monocytes. The production of leukotrienes was time- and concentration-dependent. Release of leukotrienes was half-maximal after 2 min and reached a maximum after 10 min. At a concentration of 10(-8) M, PAF induced the production of 0.14 +/- 0.01 ng LTB4/10(6) cells (mean +/- S.E., n = 8). At concentrations of 10(-6) M, PAF induced the production of 1.0 +/- 0.04 ng LTB4 and 0.22 +/- 0.03 ng peptidoleukotrienes (mean +/- S.E., n = 16). There was no metabolism of LTB4 as judged from stability of [3H]LTB4 added to the incubations. LTC4 was slowly metabolized by human monocytes to LTD4 and LTE4. The two specific PAF-receptor antagonists BN 52021 and WEB 2086 in concentrations of 10(-4) and 10(-6) M, respectively, inhibited the PAF (10(-6) M) stimulated LTB4 production completely. In this study, we demonstrate that nanomolar concentrations of PAF can stimulate the production of LTB4 and peptidoleukotrienes in human monocytes by a receptor-mediated mechanism.     

Adenosine prevents phorbol ester injury in rabbit lungs: role of leukotrienes and TNF.

The objective of this study was to determine whether adenosine (ADO) prevents phorbol myristate acetate- (PMA) induced lung injury by modulating peptidoleukotrienes (LT) and/or tumor necrosis factor (TNF) production. PMA significantly increased pulmonary vascular resistance (PVR, 275 +/- 4 to 447 +/- 30 cmH2O.1-1.min) and microvascular filtration coefficient.(Kf, 0.024 +/- 0.002 to 0.040 +/- 0.006 g.min-1.cmH2O-1) in isolated blood-perfused rabbit lungs. ADO (5 mumol/min) blocked the increases in PVR (257 +/- 9 to 283 +/- 26) and Kf (0.028 +/- 0.005 to 0.018 +/- 0.002). After PMA (30 min), perfusate levels of LTC4 + LTD4 increased by 15.3 +/- 2.1 pg/ml; LTE4 increased by 15.1 +/- 4.1 pg/ml. ADO reduced the increase in LTC4 + LTD4 to 2.7 +/- 6.1 pg/ml, but total LT increased by 31.9 +/- 16.6 pg/ml, implying that ADO enhanced the conversion of LTC4 and LTD4 to LTE4. MK-886 (L663,536), an LT synthesis inhibitor, blocked the increase in total LT (6.1 +/- 13.9 pg/ml) but did not reduce the PMA-induced increase in Kf (0.022 +/- 0.003 to 0.035 +/- 0.005) or PVR (238 +/- 11 to 495 +/- 21). After PMA administration, perfusate TNF levels were not different from the 10-fold increase observed in control experiments and were not reduced by ADO or MK-886. TNF production was independent of perfusate blood components and presumably due to low levels of endotoxin in the perfusate (70-90 ng/ml). These results indicate that ADO does not protect against PMA-induced acute lung injury by altering circulating levels of LT or TNF.     

L-663,536 (MK-886) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2 - dimethylpropanoic acid), a novel, orally active leukotriene biosynthesis inhibitor

L-663,536 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2, 2-dimethylpropanoic acid) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human polymorphonuclear leukocytes (PMN) (IC50, 2.5 nM). Similarly, L-663,536 inhibited A23187-induced LTB4 formation by rat peripheral blood and elicited PMN. At concentrations where inhibition of leukotriene biosynthesis occurred in human whole blood (1.1 microM), no effect was seen on cyclooxygenase or 12-lipoxygenase, an effect also observed in washed human platelets. The compound had no effect on rat or porcine 5-lipoxygenase indicating that L-663,536 is not a direct 5-lipoxygenase inhibitor. When administered in vivo L-663,536 was a potent inhibitor of antigen-induced dyspnea in inbred rats pretreated with methysergide (ED50, 0.036 mg/kg p.o.) and of Ascaris-induced bronchoconstriction in squirrel monkeys (1 mg/kg p.o.). The compound inhibited leukotriene biosynthesis in vivo in a rat pleurisy model (ED50, 0.2 mg/kg p.o.), an inflamed rat paw model (ED50, 0.8 mg/kg), a model of leukotriene excretion in rat bile following antigen provocation, and a model in the guinea-pig ear where leukotriene synthesis was induced by topical challenge with ionophore A23187 (ED50, 2.5 mg/kg p.o. and 0.6 micrograms topically). The results indicate that L-663,536 is a potent inhibitor of leukotriene biosynthesis both in vitro and in vivo indicating that the compound is suitable for studying the role of leukotrienes in a variety of pathological situations.     

Leukotriene biosynthesis and metabolism detected by the combined use of HPLC and radioimmunoassay

A radioimmunoassay for leukotriene D4 (LTD4) has been developed which exhibits sufficiently high sensitivity to be useful in conjunction with RP-HPLC in the detection of LTC4, LTD4 and LTE4 in physiological samples. The detection limit of the assay was approximately 240 amoles, using antiserum TG1 at a dilution of 6 X 10(3), with 50% displacement at 70 fmoles. Antiserum NW1, also at a dilution of 6 X 10(3), displayed a detection limit of 9 fmoles with 50% displacement at 100 fmoles. The two antisera have similiar crossreactivities, both manifesting useful affinities for LTE4 and LTC4, and low or negligible affinities for other arachidonic acid metabolites, or their derivatives. The radioimmunoassay was used to detect 1) LTC4, LTD4 and LTE4 released from perfused rat lung in response to platelet-activating factor (PAF) stimulation, 2) conversion of exogenous LTD4 to LTE4 in human blood, and 3) endogenous leukotrienes in human blood samples.     

A novel anti-asthmatic quinone derivative, AA-2414 with a potent antagonistic activity against a variety of spasmogenic prostanoids

The anti-asthmatic activity of AA-2414 [(+/-)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7-phenylheptano ic acid] has been studied in vivo and in vitro. Experimental allergic asthma was inhibited by orally administered AA-2414 in a dose-dependent manner. AA-2414, 0.08-1.25 mg/kg (p.o.), inhibited the bronchconstriction in guinea pigs induced by a prostaglandin endoperoxide analogue (U-46619), leukotriene D4 (LTD4), and platelet activating factor (PAF) with a long duration of action. The compound did not inhibit histamine-induced bronchoconstriction. AA-2414 reduced the induction of pulmonary inflation caused by LTD4 aerosol inhalation. AA-2414 competitively inhibited the contractile response to U-46619 in guinea pig tracheal and parenchymal strips and dog saphenous vein strips with pA2 values of 7.69, 8.29 and 6.79, respectively. Furthermore, the contractile responses of guinea pig tracheal strip to PGD2, 9 alpha, 11 beta-PGF2 and PGF2 alpha were inhibited with pA2 values of 7.20, 7.79 and 5.71, respectively. These results suggest that AA-2414, a quinone derivative, is a novel, potent and orally active antagonist of a variety of spasmogenic prostanoids.     

Specificity of receptors for leukotrienes A4, B4, C4, D4, E4 and histamine on the guinea-pig lung parenchyma. Effect of FPL-55712 and desensitization of the myotropic activity

The novel metabolites of arachidonic acid, leukotriene (LT) A4, B4, C4, D4 and E4 have potent myotropic activity on guinea-pig lung parenchymal strip in vitro. The receptors responsible for their action were characterized using desensitization experiments and the selective SRS-A antagonist, FPL-55712. During the continuous infusion of LTB4, the tissues became desensitized to LTB4 but were still responsive to histamine, LTA4, LTC4, LTD4 and LTE4. When LTD4 was infused continuously, the lung strips contracted to LTB4 and histamine but were no longer responsive to LTA4, LTC4, LTD4 and LTE4. Furthermore, FPL-55712 (10 ng ml-1 - 10 ug ml-1) produced dose-dependent inhibitions of LTA4, LTC4, LTD4 and LTE4 without inhibiting the contraction to LTB4 and histamine. On the basis of these results, it appears that the guinea-pig lung parenchyma may have one type of receptor for LTB4 and another for LTD4; LTA4, LTC4 and LTE4 probably act on the LTD4 receptor.