The antiinflammatory agent SC-41930 inhibits granulocyte infiltration of the rodent dermis induced by 6-trans-leukotriene B4.

Granulocyte diapedesis in response to the generation of defined chemotaxins such as leukotriene B4 (LTB4), 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE], C5a, platelet activating factor and others is a hallmark of the inflammatory process that is thought to contribute to the tissue pathology seen in a number of diseases. 6-trans-LTB4 arises through the myeloperoxidase (MPO)-dependent metabolism of sulfidopeptide leukotrienes and through the action of 5-lipoxygenase on 12(R)-HETE. The intradermal (i.d.) injection of 6-trans-LTB4 induces a dose and time dependent influx of granulocytes into the guinea-pig (Hartley) dermis. When various doses of the LTB4 receptor antagonist and antiinflammatory agent, SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro- 8-propyl-2H-1-benzopyran-2-carboxylic acid) given 30 min ahead of i.d. injection of 6-trans-LTB4 (10 micrograms/i.d. site), granulocyte infiltration, as assessed by dermal levels of the neutrophil marker enzyme MPO was inhibited with an ED50 value of 9.8 mg/kg in the guinea-pig. When various doses (10-25 micrograms) 6-trans-LTB4 were injected in the mouse (CD-1) dermis, there was a dose-related increase in granulocyte accumulation at 4 h. Furthermore when mice were pretreated (-30 min) with SC-41930 (1 mg/kg) orally, the trafficking of granulocytes was inhibited (p less than .01) as assessed by dermal MPO levels. SC-41930 orally inhibits 6-trans-LTB4-induced granulocyte accumulation in the guinea-pig more potently than against the response to 12(R)-HETE(ED50:13.4 mg/kg) but less potently than against LTB4 (ED50:0.6 mg/kg). These multiple activities may contribute to this compound's potential as an inflammatory agent.     

SC-41930: an inhibitor of leukotriene B4-stimulated human neutrophil functions

SC-41930 was evaluated for effects on human neutrophil chemotaxis and degranulation. At concentrations up to 100 microM, SC-41930 alone exhibited no effect on neutrophil migration, but dose-dependently inhibited neutrophil chemotaxis induced by leukotriene B4 (LTB4) in a modified Boyden chamber. Concentrations of SC-41930 from 0.3 microM to 3 microM competitively inhibited LTB4-induced chemotaxis with a pA2 value of 6.35. While inactive at 10 microM against C5a-induced chemotaxis, SC-41930 inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, with 10 times less potency than against LTB4-induced chemotaxis. SC-41930 inhibited [3H]LTB4 and [3H]fMLP binding to their receptor sites on human neutrophils with KD values of 0.2 microM and 2 microM, respectively. SC-41930 also inhibited neutrophil chemotaxis induced by 20-OH LTB or 12(R)-HETE. At concentrations up to 10 microM, SC-41930 alone did not cause neutrophil degranulation, but inhibited LTB4-induced degranulation in a noncompetitive manner. SC-41930 also inhibited fMLP- or C5a-induced degranulation, but was about 8 and 10 times less effective for fMLP and C5a, respectively. The results indicate that SC-41930 is a human neutrophil LTB4 receptor antagonist with greater specificity for LTB4 than for fMLP or C5a receptors.     

SC-41930 inhibits neutrophil infiltration of the cavine dermis induced by 12(R)-hydroxyeicosatetraenoic acid

Psoriasis is a disease state characterized by epidermal proliferation, neutrophil infiltration, along with release of the proinflammatory mediators leukotriene-B4 (LTB4) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE]. LTB4 and 12(R)-HETE are chemoattractant to the neutrophil, the latter approximately 1000X less potent. LTB4 and 12(R)-HETE are present in psoriatic scale, the latter in quantities so much greater than LTB4 that it is proposed as a primary mediator of neutrophil infiltration in psoriasis. 12(R)-HETE, synthesized in optically pure form by a new, shorter route, was injected into the cavine dermis. At a dose of 25 micrograms m per intradermal site, 12(R)-HETE was a significant chemoattractant to the neutrophil (as assessed by dermal myeloperoxidase levels). SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]- 3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, given intragastrically inhibited 12(R)-HETE-induced neutrophil infiltration of the cavine dermis with an ED50 value of 13.5 mg/kg. Compounds such as SC-41930 may well have utility for treating human psoriasis.     

ONO-4057, a novel, orally active leukotriene B4 antagonist: effects on LTB4-induced neutrophil functions.

ONO-4057(5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E- hexenyl]oxyphenoxy]valeric acid), an orally active leukotriene B4(LTB4) antagonist, displaced the binding of [3H] LTB4 to the LTB4 receptor in human neutrophil (Ki = 3.7 +/- 0.9 nM). ONO-4057 inhibited the LTB4-induced rise in cytosolic free calcium (the concentration causing 50% inhibition (IC50) = 0.7 +/- 0.3 microM) and inhibited human neutrophil aggregation, chemotaxis or degranulation induced by LTB4 (IC50 = 3.0 +/- 0.1, 0.9 +/- 0.1 and 1.6 +/- 0.1 microM) without showing any agonist activity at concentration up to 30 microM. ONO-4057 did not inhibit fMLP or C5a-induced neutrophil activation at concentrations up to 30 microM. In the in vivo study, ONO-4057 given orally, prevented LTB4-induced transient neutropenia or intradermal neutrophil migration in guinea pig (the dose causing 50% efficacy (ED50) = 25.6mg/kg or 5.3mg/kg). Furthermore, ONO-4057 given topically, suppressed phorbol-12-myristate-13-acetate (PMA)-induced neutrophil infiltration in guinea pig ear (the effective dose = 1 mg/ear). These results indicate that ONO-4057 is a selective and orally active LTB4 antagonist and may be a potential candidate for the treatment of various inflammatory diseases.     

Effects of two leukotriene B4 (LTB4) receptor antagonists (LY255283 and SC-41930) on LTB4-induced human neutrophil adhesion and superoxide production

Leukotriene B4 (LTB4) induces a number of functional changes in human neutrophils, including both superoxide release and CD11b/CD18 (Mo1)-mediated adherence to various substrates, such as keyhole limpet hemocyanin (KLH). These effects are both time- and concentration-dependent. Neutrophil adhesion was at least 10-fold more sensitive to the stimulatory action of LTB4 than superoxide production. Two LTB4 receptor antagonists, LY255283 (1-(5-ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)heptyloxy )- phenyl)ethanone) and the sodium salt of SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H- 1-benzopyran-2-carboxylic acid) were evaluated for effects on human neutrophil superoxide production and adhesion. Despite being more sensitive to LTB4-induced stimulation, neutrophil adhesion was at least 100-fold less sensitive to inhibition by LY255283 and SC-41930 than superoxide production. Both LTB4 receptor antagonists behaved similarly in these models. These compounds did not inhibit neutrophil responses induced by granulocyte/macrophage colony-stimulating factor (GM-CSF).     

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.     

In vitro and in vivo pharmacological characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with anti-inflammatory activity.

Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.6 nM) and to membranes of RBL 2H3 cells expressing the LTB4 receptor (RBL 2H3-LTB4R; IC50 = 154 nM). This compound demonstrated competitive antagonism of LTB4- and 12-(R)-HETE-induced Ca2+ mobilization responses in human neutrophils (IC50s of 131 nM and 105 nM, respectively) and inhibited LTB4-induced Ca2+ mobilization in human cultured keratinocytes (IC50 = 61 nM), RBL 2H3-LTB4R cells (IC50 = 255 nM) and mouse neutrophils (IC50 = 410 nM). SB 201993 showed weak LTD4-receptor binding affinity (Ki = 1.9 microM) and inhibited 5-lipoxygenase (IC50 of 3.6 microM), both in vitro and ex vivo. In vivo, SB 201993 inhibited LTB4-induced neutrophil infiltration in mouse skin and produced dose-related, long lasting topical anti-inflammatory activity against the fluid and cellular phases of arachidonic acid-induced mouse ear inflammation (ED50 of 580 microg/ear and 390 microg/ear, respectively). Similarly, anti-inflammatory activity was also observed in the murine phorbol ester-induced cutaneous inflammation model (ED50 of 770 and 730 microg/ear, respectively, against the fluid and cellular phases). These results indicate that SB 201993 blocks the actions of LTB4 and 12-(R)-HETE and inhibits a variety of inflammatory responses; and thus may be a useful compound to evaluate the role of these mediators in disease models.     

Flow cytometric evaluation of the effects of leukotriene B4 receptor antagonists (LY255283 and SC-41930) on calcium mobilization and integrin expression of activated human neutrophils

Leukotriene B4 (LTB4) is a naturally occurring eicosanoid mediator which chemoattracts and stimulates human neutrophils to an activated state. In an attempt to identify novel antiinflammatory drugs, synthetic LTB4 receptor antagonists have been developed in several laboratories. In this study, the effects of two such LTB4 receptor antagonists were examined for their influences on two elements of human neutrophil activation using flow cytometric techniques. Quantitative flow cytometric assays of human neutrophil intracellular calcium mobilization and up-regulation of integrin (CD11b/CD18) cell surface expression were developed and used to determine the potency and selectivity of compounds LY255283 and SC-41930 on these activities. Our results indicate that both compounds preferentially block these functions of LTB4-induced human neutrophil activation in a concentration dependent manner and fall in the 1-2 microM range of antagonist activity. Compound SC-41930 was approximately twice as potent as LY255283 in blocking the targeted agonist effects. Both compounds were approximately 100-fold less potent in blocking the same functions of interleukin-8-induced human neutrophil activation.     

Bioisosteres of 9-carboxymethyl-4-oxo-imidazo[1,2-a]indeno-[1,2-e]pyrazin-2-carboxylic acid derivatives. Progress towards selective, potent in vivo AMPA antagonists with longer durations of action

A novel series of 2- and 9-disubstituted heterocyclic-fused 4-oxo-indeno[1,2-e]pyrazin derivatives was synthesized. One of them, the 9-(1H-tetrazol-5-ylmethyl)-4-oxo-5,10-dihydroimidazo[1,2-a]indeno[1,2-e]pyrazin-2-yl phosphonic acid 4i exhibited a strong and a selective binding affinity for the AMPA receptor (IC50 = 13 nM) and demonstrated potent antagonist activity (IC50 = 6nM) at the ionotropic AMPA receptor. This compound also displayed good anticonvulsant properties against electrically-induced convulsions after ip and iv administration with ED50 values between 0.8 and 1 mg/kg. Furthermore, a strong increase in potency was observed when given iv 3 h before test (ED50 = 3.5 instead of 25.6 mg/kg for the corresponding 9-carboxymethyl-2-carboxylic acid analogue). These data confirmed that there is an advantage in replacing the classical carboxy substituents by their bioisosteres such as tetrazole or phosphonic acid groups.     

Mediation by platelet-activating factor of 12-hydroxyeicosatetraenoic acid-induced cytosolic free calcium concentration elevation in neutrophils

12(R)-hydroxyeicosatetraenoic acid (HETE) shows biphasic increase in cytosolic free calcium concentration ([Ca2+]i) in rabbit and human neutrophils; the initial transient phase and the continuous falling phase. 12(S)-HETE was less potent in both species. BN50739, a platelet-activating factor (PAF) receptor antagonist, inhibited both phases of 12(R)-HETE-induced [Ca2+]i rise but did not affect leukotriene B4 (LTB4)-induced [Ca2+]i rise. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a PAF synthesis inhibitor, and manoalide, a phospholipase A2 inhibitor, reduced 12(R)-HETE-induced [Ca2+]i rise. These blockers inhibited the continuous phase of [Ca2+]i rise induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) with little effect on the initial phase. It had no significant effect on LTB4-induced [Ca2+]i rise. SC-41930, a LTB4-receptor antagonist, did not block 12-HETE-induced [Ca2+]i rise. In 12(R)-HETE-, FMLP- and LTB4-stimulated cells, accumulations of cell-associated PAF and released PAF were detected but not in unstimulated cells. BN50739 did not affect the accumulation of cell-associated PAF and release of PAF in 12(R)-HETE-stimulated cells. These results suggest that 12(R)-HETE-induced and partially, FMLP-induced, but not LTB4-induced [Ca2+]i rise are mediated by PAF, which is produced and released by stimulation of the cells by 12(R)-HETE and FMLP, respectively.     

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.     

The human renin infused rat: use as an in vivo model for the biological evaluation of human renin inhibitors

The human renin infused rat model (HRIRM) was used as an in vivo small-animal model for evaluating the efficacy of a collection of inhibitors of human renin. The intravenous infusion of recombinant human renin (2.4 microg x kg(-1) x min(-1)) in the ganglion-blocked, nephrectomized rat produced a mean blood pressor response of 47+/-3 mm Hg (1 mm Hg = 133.3 Pa), which was reduced by captopril, enalkiren, and losartan in a dose-dependent manner following oral administration, with ED50 values of 0.3+/-0.1, 2.5+/-0.9, and 5.2+/-1.6 mg/kg, respectively. A series of peptidomimetic P2-P3 butanediamide renin inhibitors inhibited purified recombinant human renin in vitro in a concentration-dependent manner, with IC50 values ranging from 0.4 to 20 nM at pH 6.0, with a higher range of IC50 values (0.8-80 nM) observed at pH 7.4. Following i.v. administration of renin inhibitors, the pressor response to infused human renin in the HRIRM was inhibited in a dose-dependent manner, with ED50 values ranging from 4 to 600 microg/kg. The in vivo inhibition of human renin following i.v. administration in the rat correlated significantly better with the in vitro inhibition of human renin at pH 7.4 (r = 0.8) compared with pH 6.0 (r = 0.5). Oral administration of renin inhibitors also resulted in a dose-dependent inhibition of the pressor response to infused human renin, with ED50 values ranging from 0.4 to 6.0 mg/kg and the identification of six renin inhibitors with an oral potency of <1 mg/kg. The ED50 of renin inhibitors for inhibition of angiotensin I formation in vivo was highly correlated (r = 0.9) with the ED50 for inhibition of the pressor response. These results demonstrate the high potency, dose dependence, and availability following oral administration of the butanediamide series of renin inhibitors.     

Antagonism of peptidoleukotrienes and inhibition of systemic anaphylaxis by RG 12525 in guinea pigs

RG 12525 was determined to be a specific, competitive and orally effective antagonist of the peptidoleukotrienes, LTC4, LTD4 and LTE4, in several assays utilizing guinea pigs. In vitro, RG 12525 competitively inhibited 3H-LTD4 binding to lung membranes (Ki = 3.0 +/- 0.3 nM) and competitively antagonized the spasmogenic activity of LTC4, LTD4 and LTE4 on lung strips (KB values = 3 nM) with greater than 8000 fold selectivity. In vivo, RG 12525 orally inhibited LTD4 induced wheal formation (ED50 = 5 mg/kg with a t1/2 = 10 hrs at 9 mg/kg), LTD4 induced bronchoconstriction (ED50 = 0.6 mg/kg), and anaphylactic death (ED50 = 2.2 mg/kg with a t1/2 = 7 hrs at 10 mg/kg) and antigen induced bronchoconstriction (ED50 = 0.6 mg/kg). RG 12525 represents a significant improvement in receptor affinity and oral efficacy and thus, is a valuable pharmacological tool to evaluate peptidoleukotrienes in allergic diseases.     

Single-step organic extraction of leukotrienes and related compounds and their simultaneous analysis by high-performance liquid chromatography

A method for the simultaneous single-step organic extraction from biological matrices of peptido- and dihydroxyleukotrienes as well as 5-hydroperoxy- and 5-hydroxyeicosatetraenoic acid followed by separation and quantitation in a single run on reversed-phase high-performance liquid chromatography was evaluated. Using an extraction system comprising 400/1200/4800 (v/v/v) aqueous phase/isopropanol/dichloromethane, pH 3.0, absolute recoveries of 82.3 +/- 2.0, 89.7 +/- 1.0, 93.7 +/- 1.4, 92.8 +/- 1.4, 90 +/- 4, and 90 +/- 4% for prostaglandin B1 (PGB1), leukotriene C4 (LTC4), leukotriene B4 (LTB4), leukotriene D4 (LTD4), 5-hydroperoxyeicosatetraenoic acid (5-HETE), respectively, were achieved. Separation and quantitation of products were performed on a Nucleosil 100 C18 column (5 microns, 4.6 X 250 mm) using, at pH 6.0, a gradient system comprising 72/28/0.02 (v/v/v) methanol/water/glacial acetic acid from 0 to 15 min, followed by a convex gradient to 76/24/0.02 (v/v/v) methanol/water/glacial acetic acid, followed by a 10-min hold at this methanol concentration. The method was used to investigate the profile of leukotrienes synthesized by rat hepatocyte homogenates from 5-HPETE or leukotriene A4 in absence or presence of glutathione (GSH). During a 5-min incubation with 100 microM 5-HPETE, 9.6 ng LTB4/mg protein and 2.2 micrograms 5-HETE/mg protein were formed in the absence of GSH. In the presence of 0.4 mM GSH, 3.7 ng LTB4/mg protein and 11.0 micrograms 5-HETE/mg protein were formed. Using 20 microM LTA4 as a substrate, 17.3 and 324.0 ng LTC4/mg protein X min and 14.3 and 19.3 ng LTB4/mg protein X min were formed in the presence of 0.4 and 10 mM GSH, respectively.     

M&B 28,767: a potent anti-secretory and anti-ulcer PG analogue. A comparative study with 16, 16' dimethyl PGE2 methylester

M&B 28,767 [(+/-)11-deoxy-16-phenoxy-omega-tetranor PGE1] and 16, 16'-dimethyl PGE2 methylester (DMPG) were compared for their effects on gastric acid secretion (GAS) and gastric ulceration (GU), employing various laboratory models. In anaesthetised rats, GAS was stimulated by a continuous i.v. infusion of pentagastrin (30 micrograms/kg/h), and PG analogues were perfused through the stomach for 1 h. M&B 28,767 (3-15 micrograms/kg/h) and DMPG (3-60 micrograms/kg/h) reduced GAS in a dose-related manner, the ED50 values being 4 and 15 micrograms/kg/h respectively. In conscious rats possessing indwelling gastric cannulae, oral doses of M&B 28,767 (0.025-0.1 microgram/kg) and DMPG (0.50-1.0 microgram/kg) caused a prolonged inhibition of pentagastrin-stimulated GAS. M&B 28,767 was 17 times more potent than DMPG; the respective ED50 values were 0.036 and 0.6 microgram/kg. Indomethacin-induced ulceration in rats, was reduced by both M&B 28,767 and DMPG; the respective ED50 values being 3.0 and 0.8 micrograms/kg. Both compounds given orally increased gastrointestinal motility in mice; M&B 28,767 (1-3 mg/kg) and DMPG (0.1-0.3 mg/kg) caused diarrhoea, the former being about 0.1 times as potent as the latter. In another test, M&B 28,767 (0.5-5.0 mg/kg) and DMPG (10-40 micrograms/kg) overcame morphine-induced constipation in a dose-related manner, the respective ED50s being 0.9-1.4 mg/kg and 20-40 micrograms/kg. Thus, M&B 28,767 had a better profile of activity than DMPG as an antisecretory and antiulcer agent.     

Development of receptors for leukotriene B4 on HL-60 cells induced to differentiate by 1 alpha,25-dihydroxyvitamin D3

The incubation of HL-60 human promyelocytic leukemia cells for 7 days with 100 nM 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced differentiation into monocyte-like cells, as assessed by morphologic and biochemical characteristics. Stereospecific receptors for leukotriene B4 (LTB4) developed on the surface of the HL-60 cell-derived monocytes that had the capacity to transduce LTB4 stimulation of a transient increase in the cytosolic concentration of calcium ([Ca+2]in). HL-60 cell-derived monocytes, but not undifferentiated HL-60 cells, expressed a high affinity subset of 6400 +/- 3700 receptors per cell with a dissociation constant (Kd) of 2.3 +/- 1 nM (mean +/- SD, n = 3) and a low affinity subset of approximately 2.2 X 10(6) receptors per cell with an apparent Kd of 680 +/- 410 nM. Derivatives of LTB4 inhibited the binding of [3H]LTB4 to HL-60 cell-derived monocytes with a rank order of potency of LTB4 greater than 20-OH-LTB4 greater than 3-aminopropyl amide-LTB4, which is similar to the order for LTB4 receptors of human blood PMNL. In contrast, leukotrienes C4 and D4 and formyl-methionyl chemotactic peptides did not inhibit the binding of [3H] LTB4, which demonstrates the specificity of these receptors for isomers of 5,12-dihydroxy-eicosatetraenoic acid. LTB4 stimulated an increase in [Ca+2]in in HL-60 cell-derived monocytes which reached 50% of the maximal level at an LTB4 concentration of 0.5 nM (EC50). Preincubation of HL-60 cell-derived monocytes with 10 nM LTB4 resulted in a selective loss of high affinity receptors, as assessed by binding of [3H]LTB4, and a 200-fold increase in the EC50 for stimulation by LTB4 of increases in [Ca+2]in, without alterations in either the low affinity receptors for LTB4 or the responsiveness of [Ca+2]in to formyl-methionyl chemotactic peptides. HL-60 cells that are induced to differentiate into monocytes thus develop stereospecific receptors for LTB4 with binding and transductional characteristics similar to those of human blood PMNL.     

Pharmacological actions of Y-24180: I. A potent and specific antagonist of platelet-activating factor

The ability of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a] [1,4]diazepine to inhibit platelet-activating factor (PAF)-induced reactions was investigated. Y-24180 (0.0003–0.003 mg/kg, i.v.) dose-dependently inhibited PAF-induced bronchoconstriction in guinea pigs, but even at a high dose of 10 mg/kg, i.v., it was either inactive or weakly active against the bronchoconstriction induced by histamine, serotonin, acetylcholine, arachidonic acid, bradykinin, or leukotriene D₄. Oral doses (0.003–0.1 mg/kg) of Y-24180 also prevented hemoconcentration due to PAF in a dosedependent manner and produced a parallel shift of the PAF dose-response curve. Y-24180 (0.0003–0.1 mg/kg, i.v.) and WEB 2086 (0.03–1 mg/kg, i.v.) dose-dependently reversed PAF-induced hypotension in anesthetized rats. In mice, PAF-induced lethality was inhibited by Y-24180 and WEB 2086 with ED₅₀ values of 0.022 and 1.42 mg/kg, p.o., and 0.023 and 0.12 mg/kg, i.v., respectively. This protective effect of Y-24180 given p.o. persisted for at least 6 hr. In actively sensitized mice lethal anaphylactic shock was prevented by oral doses of Y-24180 and WEB 2086 with ED₅₀ values of 0.095 and 0.69 mg/kg, respectively. These results suggested that Y-24180 is an extremely potent and specific PAF antagonist with a good duration of action.     

Synthesis and 5-hydroxytryptamine (5-HT) activity of 2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5-(6H)ones and 2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoxalines

A series of 2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5-(6H)ones and 2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoxalines was shown to exhibit 5-HT2C agonist binding and functional activity. Compound 21R inhibited food intake over 2 h in fasted, male Sprague Dawley rats with ED50 values of 2 mg/kg (i.p.) and 10 mg/kg (p.o.).     

Specific binding of leukotriene B4 to receptors on human polymorphonuclear leukocytes

Leukotriene B4 (5(S),12(R)-di-hydroxy-eicosa-6,14-cis-8,10-trans-tetraenoic acid [LTB4]) is a product of the 5-lipoxygenation of arachidonic acid, which elicits human PMN leukocyte chemotactic responses in vitro that are 50% of the maximal level at concentrations of 3 X 10(-9) M to 10(-8) M and are maximal at 2 X 10(-8) M to 10(-7) M. The specific binding of highly purified [3H]LTB4 to human PMN leukocytes was assessed both by extracting the unbound and weakly bound [3H]LTB4 with acetone at -78 degrees C and by centrifuging the PMN leukocytes through cushions of phthalate oil to separate the unbound from bound [3H]LTB4. The levels of total binding of [3H]LTB4 and of nonspecific binding of [3H]LTB4, in the presence of a 1500-fold molar excess of nonradioactive LTB4, were approximately two times higher with the phthalate oil method. Scatchard plots of the concentration dependence of the specific binding (total - nonspecific binding) of [3H]LTB4 to PMN leukocytes were linear for the acetone extraction and phthalate oil methods and revealed dissociation constants of 10.8 X 10(-9) M and 13.9 X 10(-9) M, respectively, and mean of 2.6 X 10(4) and 4.0 X 10(4) receptors per PMN leukocyte. The 5(S),12(S)-all-trans-di-HETE analog of LTB4 and 5-HETE competitively inhibited by 50% the binding of [3H]LTB4 to PMN leukocytes at respective concentrations that evoked half-maximal chemotactic responses, whereas neither N-formyl-methionyl-leucyl-phenylalanine nor chemotactic fragments of C5 inhibited the binding. Human erythrocytes exhibited no specific binding sites for [3H]LTB4. Human PMN leukocytes possess a subset of receptors for LTB4 that are distinct from those specific for peptide chemotactic factors.     

Characterization of the plasma membrane bound GTPase from rabbit neutrophils. I. Evidence for an Ni-like protein coupled to the formyl peptide, C5a, and leukotriene B4 chemotaxis receptors

We have characterized the GTPase activity of the Ni-like guanine-nucleotide-binding regulatory protein in rabbit neutrophil plasma membranes. The low Km (3.64 +/- 0.87 X 10(-7) M) GTPase copurified with the formyl peptide receptor in the plasma membrane fraction obtained by discontinuous sucrose density gradient centrifugation. The Vmax (23.9 +/- 2.91 pmol/mg/min) and Km of the unstimulated enzyme were similar to those reported for Ni in other cell types. The activity of the unstimulated enzyme was both magnesium and sodium dependent and linear over the first 4 min of the assay. The chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and leukotriene B4 (LTB4) stimulated the GTPase in purified neutrophil plasma membrane preparations, whereas other secretagogues, such as A23187 and PMA, were without effect. Lineweaver-Burk analysis showed an fMLP-induced increase in Vmax (31.94 +/- 4.80 pmol/mg/min) (33.1 +/- 9.5%) but not in Km. The dose-response curve for fMLP stimulation showed an ED50 of 4.1 +/- 1.0 X 10(-8) M and an overall 22.2 +/- 3.1% maximal stimulation. C5a (30 micrograms/ml) increased the activity of the GTPase 21.3 +/- 5.7% and 10(-7) M LTB4 produced a 32.2 +/- 5.4% increase. Activated pertussis toxin treatment of neutrophil plasma membranes inhibited by 72.5 +/- 14.3% the stimulation of GTPase activity induced by fMLP; however, activated cholera toxin had no effect on the inhibition of fMLP stimulation, suggesting a direct role for an Ni-like protein in the coupling process. In contrast to the lack of inhibition of fMLP stimulation by activated cholera toxin treatment of plasma membranes, both pertussis toxin and to a lesser extent cholera toxin treatment reduced fMLP, C5a, and LTB4 stimulation of the GTPase in sonicates prepared from pretreated whole cells. Pertussis toxin inhibited fMLP stimulation of the GTPase by 75 +/- 7%, C5a stimulation was inhibited by 83 +/- 13%, and LTB4 stimulation was inhibited completely. Sonicates prepared from neutrophils treated similarly with cholera toxin showed a smaller inhibition of GTPase activity (50 +/- 4% and 14 +/- 9% for fMLP and LTB4, respectively) with the exception of C5a, where CT inhibition (81 +/- 32%) equaled pertussis toxin inhibition. Similarly, pertussis toxin completely inhibited the release of the granule enzyme N-acetyl-glucosaminidase by all three chemoattractants, whereas cholera toxin, except with C5a stimulation, had little or no effect.(ABSTRACT TRUNCATED AT 400 WORDS)