Differential effects of the PAF receptor antagonist UK-74,505 on neutrophil and eosinophil accumulation in guinea-pig skin.

1. The effect of the dihydropyridine, platelet activating factor (PAF) receptor antagonist, UK-74,505, on leucocyte accumulation and oedema formation in guinea-pig skin was investigated. The inflammatory reactions studied were elicited by exogenous mediators, a passive cutaneous anaphylactic (PCA) reaction and zymosan particles. 2. Leucocyte accumulation and oedema formation were measured as the local accumulation of i.v. administered 111In-labelled neutrophils or eosinophils together with 125I-labelled albumin. UK-74,505 was either administered i.v. or used to pretreat the radiolabelled leucocytes in vitro prior to their last wash and injection into recipient animals. 3. In vitro, UK-74,505 inhibited PAF-induced elevations in cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2-loaded guinea-pig neutrophils and eosinophils with IC50 values of 10(-9) M and 7 x 10(-9) M respectively. Neutrophils and eosinophils pretreated with 10(-7) M and 10(-6) M UK-74,505 respectively, and maintained at 37 degrees C, were unresponsive to PAF for the 4 h period investigated. 4. In vivo, using 2 h test periods, i.v. UK-74,505 (0.5 and 2.5 mg kg-1) inhibited the accumulation of 111In-neutrophils, 111In-eosinophils and oedema formation induced by intradermal PAF, but had no effect on responses elicited by leukotriene B4 (LTB4) and zymosan-activated plasma (ZAP, used as a source of C5a des Arg). UK-74,505 (2.5 mg kg-1) was also without an effect on response induced by a PCA reaction but significantly suppressed the 111In-eosinophil accumulation following the intradermal administration of zymosan particles. The 111In-neutrophil accumulation induced by zymosan particles was not, however, affected by UK-74,505.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of platelet-activating factor (PAF) in platelet accumulation in rabbit skin: effect of the novel long-acting PAF antagonist, UK-74,505.

1. The contribution of platelet-activating factor (PAF) to platelet deposition and oedema formation induced by exogenous soluble mediators, zymosan particles and associated with a reversed passive Arthus (RPA) reaction in rabbit skin was investigated by use of a novel long-acting PAF receptor antagonist, UK-74,505. 2. Oedema formation and platelet accumulation were simultaneously measured by i.v. injection of [125I]-albumin and 111In-labelled rabbit platelets. UK-74,505 was either administered i.v. or used to pretreat radiolabelled platelets in vitro before their injection into recipient animals. Platelets pretreated with UK-74,505 were also labelled with the fluorescent calcium indicator, Fura-2, to assess their ex vivo reactivity to PAF at the end of the in vivo experiment. 3. UK-74,505 (0.5 mg kg-1), administered i.v., inhibited PAF-induced oedema formation, but did not affect oedema induced by zymosan particles, bradykinin (BK), histamine, formyl-methionyl-leucylphenylalanine (FMLP), zymosan-activated plasma (ZAP, as a source of C5a des Arg), leukotriene B4 (LTB4) or interleukin-8 (IL-8). 4. UK-74,505, administered i.v. also suppressed the small platelet accumulation induced by exogenous PAF, but had no effect on accumulation induced by IL-8 or ZAP. Although oedema induced by zymosan was not affected by i.v. UK-74,505, zymosan-induced platelet accumulation was significantly attenuated by the antagonist. 5. The RPA reaction in rabbit skin was associated with marked oedema formation and platelet accumulation which were both inhibited by i.v. UK-74,505.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological activities of a novel thienodiazepine derivative as a platelet-activating factor antagonist

(S)-(+)-6-(2-Chlorophenyl)-3-cyclopropanecarbonyl-8,11- dimethyl - 2,3,4,5 - tetrahydro - 8H - pyrido[4',3':4,5] thieno[3,2-fl-[1,2,4]triazolo]4,3-a][1,4]diazepine (E-6123) is a newly synthesized platelet-activating factor (PAF) antagonist. The effects of E-6123 on in vitro and in vivo PAF-induced responses were investigated. The IC50 values of E-6123 on 3H-PAF binding to human and guinea pig platelets were 2.7 and 3.0 nmol/l, respectively, and those on PAF-induced platelet aggregation in platelet-rich plasma of human, guinea pig and beagle dog were 10.1, 14.7 and 16 nmol/l, respectively. Oral administration of E-6123 at 3 and 10 micrograms/kg to dogs inhibited ex vivo PAF-induced platelet aggregation in a dose-dependent manner. In guinea pigs, E-6123 at 3 micrograms/kg completely inhibited ex vivo PAF-induced platelet aggregation up to 8 h and the inhibition was still significant at 24 h after administration. Occupancy of the platelet PAF receptor by E-6123 at 3 h and 24 h after administration amounted to 80% and 56%, respectively. Bronchoconstriction induced by PAF injection in guinea pigs was inhibited dose-dependently by oral or intravenous administration of E-6123 at similar doses. The IC50 value of E-6123 at 3 h after oral administration was 1 microgram/kg. Oral administration of E-6123 at 3 micrograms/kg inhibited the bronchoconstriction by more than 90% up to 8 h. Hemato-concentration induced by PAF injection in guinea pigs was inhibited by oral administration of E-6123 at 10 micrograms/kg. E-6123 also protected mice from PAF injection-induced death in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet activating factor antagonists: synthesis and structure-activity studies of novel PAF analogues modified in the phosphorylcholine moiety

New analogues of platelet activating factor (PAF), in which the phosphate and trimethylammonium moieties were replaced with an acylcarbamoyl moiety and a quaternary cyclic ammonium group, were synthesized. Their biological activities as PAF antagonists were evaluated by the inhibition of PAF-induced rabbit platelet aggregation in vitro and protective effects on PAF-induced hypotension in rats and PAF-induced death in mice. Investigation of structure-activity relationships revealed that PAF antagonist activity is strongly influenced by the acyl substituent of the nitrogen atom on the carbamoyl group and the nature of the polar head group at the 3-position of the glycerin backbone. Among the compounds tested, 2-[[N-acetyl-N-[[2-methoxy-3-[ (octadecylcarbamoyl)oxy]propoxy]-carbonyl]amino] methyl]-1-ethylpyridinium chloride (21, CV-6209) was one of the most potent compounds in the in vitro assay (IC50 = 7.5 X 10(-8) M) and the most potent and long-lasting in the in vivo assays. (R)-(-)-21 and (S)-(+)-21 were also synthesized, and no significant differences were observed in PAF antagonist activity in vitro and an inhibitory effect on PAF induced hypotension in vivo between (RS)-21 and its enantiomers.     

Development, synthesis, and biological evaluation of (-)-trans-(2S,5S)-2-[3-[(2-oxopropyl)sulfonyl]-4-n-propoxy-5-(3- hydroxypropoxy)-phenyl]-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran, a potent orally active platelet-activating factor (PAF) antagonist and its water-soluble prodrug phosphate ester.

(-)-trans-(2S,5S)-2-[3-[(2-Oxopropyl)sulfonyl]-4-n-propoxy-5-(3- hydroxypropoxy)phenyl]-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran (10) is one of the most potent platelet-activating factor (PAF) antagonists in vitro and in vivo developed to date. This diaryltetrahydrofuran derivative evolved from modifications of MK 0287 which has been evaluated in clinical studies for asthma. Two structural modifications of MK 0287 were made: (1) elaboration of the 3'-[(hydroxyethyl)sulfonyl] group to a beta-keto propylsulfonyl, and (2) replacement of the 5'-methyl ether by a 3-hydroxypropyl ether. Compound 10 potently and specifically inhibits the binding of [3H]-C18-PAF to human platelet membranes (Ki 1.85 nM) and PMN membranes (Ki 2.89 nM). In vivo, 10 inhibits PAF-induced plasma extravasation and elevated N-acetyl-beta-D-glucosaminidase (NAGA) levels in male rats with ED50 values of 60 micrograms/kg, po and 4 micrograms/kg, iv respectively, and inhibits PAF-induced bronchoconstriction in guinea pigs with an ED50 value of 15 micrograms/kg after intraduodenal administration. Compound 15, a water-soluble phosphate ester prodrug derivative of 10 is at least equipotent to 10 in the in vivo models. Compound 19S, the primary and major metabolite of 10 and 15, is equipotent in in vitro and in vivo models.     

XANTHINES INHIBIT HUMAN PLATELET AGGREGATION INDUCED BY PLATELET-ACTIVATING FACTOR

1. Platelet-activating factor (PAF) may be involved in the pathogenesis of asthma, and therefore the effects of the anti-asthma drugs theophylline and enprofylline on human platelet aggregation and adenosine triphosphate (ATP) release induced by PAF and adenosine diphosphate (ADP) were studied. 2. Enprofylline (50% inhibitory concentration [IC50] = 94.8 +/- 13.2 mumol/L) was more potent than theophylline (IC50 = 934.1 +/- 40.1 mumol/L) as an inhibitor of PAF-induced aggregation, and the xanthines were twice as potent as inhibitors of PAF-induced aggregation when compared with ADP-induced aggregation. ATP release was 1.4 times more sensitive to inhibition by the xanthines than aggregation. 3. Although high concentrations of xanthines inhibited platelet aggregation and ATP release induced by PAF, therapeutic concentrations are unlikely to inhibit PAF-induced effects.     

PAF antagonism in vitro and in vivo by aglafoline from Aglaia elliptifolia Merr.

Aglafoline, isolated from Aglaia elliptifolia Merr, inhibited in a selective and concentration-dependent manner the aggregation and ATP release reaction induced in washed rabbit platelets by PAF (platelet-activating factor). The IC50 values of aglafoline, BN52021 and kadsurenone on PAF (3.6 nM)-induced platelet aggregation were about 50, 12 and 18 microM, respectively. Aglafoline also inhibited [3H]PAF (3.6 nM) binding to washed rabbit platelets with an IC50 value of 17.8 +/- 2.6 microM. The concentration-response curve of PAF-induced platelet aggregation was shifted to the right by aglafoline with pA2 and pA10 values of 5.97 and 5.04, respectively. Although thromboxane B2 formation caused by collagen and thrombin was partially suppressed by aglafoline, thromboxane B2 formation caused by ionophore A23187 and arachidonic acid was not affected. Aglafoline inhibited the [3H]inositol monophosphate formation caused by PAF but not that caused by collagen or thrombin in the presence of indomethacin (20 microM). The cAMP content of washed rabbit platelets was not affected by aglafoline. Rat femoral intravenous administration of aglafoline (10 mg/kg) did not affect blood pressure. However, aglafoline (10 mg/kg) both prophylactically and therapeutically antagonized PAF (2.5 micrograms/kg)-induced hypotensive shock in rats. Intravenous PAF (30 ng/kg) caused severe bronchoconstriction in guinea pigs. This effect was completely blocked by aglafoline. This implies aglafoline is an effective PAF antagonist not only in vitro, but also in vivo.     

Inhibitory effects of the novel platelet activating factor receptor antagonist, 1-ethyl-2-[N-(2-methoxy)benzoyl-N-[(2R)-2-methoxy-3-(4- octadecylcarbamoyloxy) piperidinocarbonyloxypropyloxy]carbonyl] aminomethyl-pyridinium chloride, in several experimentally induced shock models.

E5880 (1-ethyl-2-[N-(2-methoxy)benzoyl-N-[(2R)-2-methoxy-3-(4- octadecylcarbamoyloxy) piperidinocarbonyloxypropyloxy] carbonyl]aminomethylpyridinium chloride, CAS 128420-61-1) is a novel analog-type antagonist of platelet activating factor (PAF). This paper describes the in vitro PAF antagonistic activity of E5880 and its in vivo effect in various experimentally induced shock models. Inhibition by E5880 of [3H]platelet activating factor (PAF) binding to human platelet PAF receptor was extremely potent; its IC50 value was 0.27 nmol/l, so that it was about 5 times more potent than PAF itself. Its IC50 value in inhibition of washed human platelet aggregation induced by PAF was 0.66 nmol/l. Intravenous treatment with E5880 dose-dependently reversed PAF-induced hypotension in rats and protected mice from lethality caused by PAF. Lipopolysaccharide (LPS)-induced hypotension in rats was inhibited by both pre- and post-treatment with E5880. It was also confirmed that blood PAF level, measured by the GC-NICI-MS method, was increased after LPS challenge in this model. Furthermore, E5880 was extremely effective in preventing passive anaphylactic lethality in mice. Blood PAF level in this model was also increased immediately after antigen challenge, and this was coincident with the time at which signs of shock became apparent. These findings support the concept that PAF is an important mediator in the development of LPS-induced shock and anaphylactic shock, and suggest that E5880, a novel and potent PAF antagonist, may be effective in clinical treatment for shock states.     

Novel azo derivatives as prodrugs of 5-aminosalicylic acid and amino derivatives with potent platelet activating factor antagonist activity

This paper describes the synthesis of a series of azo compounds able to deliver 5-aminosalicylic acid (5-ASA) and a potent platelet activating factor (PAF) antagonist in a colon-specific manner for the purpose of treating ulcerative colitis. We found it possible to add an amino group on the aromatic moiety of our reported 1-[(1-acyl-4-piperidyl)methyl]-1H-2-methylimidazo[4,5-c]pyridine derivatives or on British Biotech compounds BB-882 and BB-823 maintaining a high level of activity as PAF antagonist. A selected compound UR-12715 (49c) showed an IC(50) of 8 nM in the in vitro PAF-induced aggregation assay, and an ID(50) of 29 microg/kg in the in vivo PAF-induced hypotension test in normotensive rats. Through attachment of 49c to the 5-ASA via azo functionality we obtained UR-12746 (70). Pharmacokinetics experiments with [14C]-70 allow us to reach the following conclusions, critical in the design of these new prodrugs of 5-ASA. Neither the whole molecule 70 nor the carrier 49c were absorbed after oral administration of [14C]-70 in rat as was demonstrated by the absence of plasma levels of radioactivity and the high recovery of it in feces. Effective cleavage of azo bond (84%) by microflora in the colon is achieved. These facts ensure high topical concentrations of 5-ASA and 49c in the colon. Additionally, 70 exhibited a potent anticolitic effect in the trinitrobenzenesulfonic acid-induced colitis model in the rat. This profile suggests that UR-12746 (70) provides an attractive new approach to the treatment of ulcerative colitis.     

An antagonistic activity of etizolam on platelet-activating factor (PAF). In vitro effects on platelet aggregation and PAF receptor binding

The antagonistic effect of etizolam, an anti-anxiety drug, on platelet-activating factor (PAF) was investigated in rabbit platelets in vitro. Etizolam inhibited PAF-induced aggregation in a dose-dependent manner, with an IC50 of 3.8 microM, about one tenth that of triazolam (IC50 = 30 microM). At 300 microM, it inhibited both ADP and arachidonic acid-induced aggregation only slightly, while the other anti-anxiety drugs tested had no effect on PAF-induced aggregation even at this concentration. Etizolam and triazolam inhibited the specific binding of 3H-PAF to PAF receptor sites on washed rabbit platelets with IC50 values of 22 nM and 320 nM, respectively. Diazepam and estazolam were inactive even at 1 microM. These results indicate that etizolam is a specific antagonist of PAF.     

Platelet-activating factor stimulates phosphoinositide turnover in neurohybrid NCB-20 cells: involvement of pertussis toxin-sensitive guanine nucleotide-binding proteins and inhibition by protein kinase C.

Platelet-activating factor (PAF) is an unusually potent phospholipid known to be produced by neuronal cells and to modulate cerebral blood flow and metabolism. In previous studies with NCB-20 cells, we reported that PAF induced a significant mobilization of intracellular free Ca2+ ([Ca2+]i), which was inhibited by PAF antagonists. The increase was the result of release from intracellular stores and influx from extracellular sources. The present study was designed to characterize further PAF receptor-mediated cellular signal-transduction mechanisms in myo-[3H]inositol-labeled cells. PAF induced a concentration-dependent increase in phosphatidylinositol (Pl) metabolism, with EC50 values of 1.96 +/- 0.62 nM and 1.12 +/- 0.50 nM for inositol trisphosphate (IP3) and inositol monophosphate (IP1) formation, respectively (four experiments). The maximal production of IP3 and IP1 induced by 50 nM PAF was 254 +/- 34% and 178 +/- 25% over the basal, respectively (four experiments). PAF-induced Pl metabolism was concentration-dependently inhibited by the PAF antagonist BN50739, with an IC50 value of 6.48 +/- 0.52 nM (four experiments). The protein kinase C (PKC) activator phorbol 12,13-dibutyrate concentration-dependently inhibited PAF-induced Pl metabolism and [Ca2+]i mobilization in NCB-20 cells, of NCB-20 cells with pertussis toxin (PTX) resulted in a concentration-dependent inhibition of PAF-induced IP3 production and intracellular Ca2+ release, with a maximal reduction of 66.9 +/- 3.5% and 63 +/- 6.1%, respectively, at 300 ng/ml PTX. PTX in the presence of [32P]NAD specifically [32P]ADP-ribosylated a 38-kDa protein in membranes prepared from NCB-20 cells. Pretreatment of the cells with PTX resulted in a concentration-dependent inhibition of subsequent 32P-labeling of the toxin substrate in the membranes and correlated with the uncoupling of PAF-induced IP3 formation. PAF (0.01-10 nM) elicited a concentration-related stimulation in guanosine 5'-O-(3-[35S]) triphosphate ([35S]GTP gamma S) binding to G alpha i(1,2) proteins, which was inhibited by the PAF antagonist BN50739. PAF at 10 nM also increased [35S]GTP gamma S binding to G alpha s and G alpha o. PAF-evoked activation of G alpha i(1,2) and G alpha o was reduced by preincubation with PTX. Our results reveal that neuronal cells possess PAF receptors linked through guanine nucleotide-binding proteins to phospholipase C and receptor-operated Ca2+ channels that are regulated by PKC. Both PTX-sensitive and -insensitive guanine nucleotide-binding proteins appear to couple the PAF receptor to activation of phospholipase C and the increase in [Ca2+]i. These results contribute to the further understanding of the mechanisms behind PAF actions on neuronal cells.     

Platelet-activating factor drives eotaxin production in an allergic pleurisy in mice

1. The activation of eosinophils via G-protein-coupled seven transmembrane receptors play a necessary role in the recruitment of these cells into tissue. The present study investigates a role for PAF in driving eotaxin production and eosinophil recruitment in an allergic pleurisy model in mice. 2. The intrapleural injection of increasing doses of PAF (10(-11) to 10(-9) moles per cavity) induced a dose- and PAF receptor-dependent recruitment of eosinophils 48 h after stimulation. 3. Intrapleural injection of PAF induced the rapid (within 1 h) release of eotaxin into the pleural cavity of mice and an anti-eotaxin antibody effectively inhibited PAF-induced recruitment of eosinophils. 4. Eosinophil recruitment in the allergic pleurisy was markedly inhibited by the PAF receptor antagonist UK-74,505 (modipafant, 1 mg kg(-1)). Moreover, recruitment of eosinophils in sensitized and challenged PAF receptor-deficient animals was lower than that observed in wild-type animals. 5. Blockade of PAF receptors with UK-74,505 suppressed by 85% the release of eotaxin in the allergic pleurisy. 6. Finally, the injection of a sub-threshold dose of PAF and eotaxin cooperated to induce eosinophil recruitment in vivo. 7. In conclusion, the production of PAF in an allergic reaction could function in multiple ways to facilitate the recruitment of eosinophils -- by facilitating eotaxin release and by cooperating with eotaxin to induce greater recruitment of eosinophils.     

Propenyl carboxamide derivatives as antagonists of platelet activating factor

A series of N-[4-(3-pyridinyl)butyl] 3-substituted propenyl carboxamide derivatives bearing an unsaturated bicyclic moiety in the 3-position was prepared and evaluated for PAF (platelet activating factor) antagonist activity. These compounds represent conformationally constrained direct analogues of the corresponding potent 5-aryl-pentadienecarboxamides (5). Most of the new compounds were active in a PAF-binding assay employing whole, washed dog platelets as the receptor source and inhibited PAF-induced bronchoconstriction in guinea pigs after intravenous administration. However, oral activity in the PAF-induced bronchoconstriction model was highly sensitive to the nature and substitution of the bicyclic ring system. The most interesting compounds included [R-(E)]-(1-butyl-6-methoxy-2-naphthyl)-N-[1-methyl-4-(3- pyridinyl)butyl]-2-propenamide (4b), [R-(E)]-(3-butyl-6-methoxy-2- benzo[b]thiophene-yl)-N-[1-methyl-4-(3-pyridinyl)butyl]-2-propenamide (4k), and [R-(E)]-(3-butyl-6-methoxy-1-methyl-2-indoly)-N-[1-ethyl-4- (3-pyridinyl)butyl]-2-propenamide (4l) which inhibited PAF-induced broncho-constriction in guinea pigs with IC50s of 3.0-5.4 mg/kg, when the animals were challenged 2 h after drug treatment. They were also highly effective 6 h after a 50 mg/kg oral dose. This study supports the notion that the key remote aromatic ring present in the 5-arylpentadienecarboxamides (5) is preferentially coplanar with the diene system for good PAF antagonist activity.     

Methoxytetrahydropyrans. A new series of selective and orally potent 5-lipoxygenase inhibitors.

Investigation of the SAR of the lead (methoxyalkyl)thiazole 1-[3-(naphth-2-ylmethoxy)phenyl]-1-thiazol-2-ylprop yl methyl ether (1, ICI 211965) led to the methoxytetrahydropyrans, a new series of 5-lipoxygenase (5-LPO) inhibitors exemplified by the parent compound 4-[3-(naphth-2-ylmethoxy)phenyl]-4- methoxy-3,4,5,6-tetrahydro-2H-pyran (4f). In vitro 4f inhibited leukotriene C4 (LTC4) synthesis in zymosan-stimulated plasma-free mouse macrophages and LTB4 synthesis in A-23187-stimulated human whole blood (IC50s 0.5 nM and 0.07 microM, respectively). In the rat 4f inhibited LTB4 synthesis in blood ex vivo and in zymosan-inflamed air pouch exudate with an ED50 3 h after oral dosing of 10 mg/kg in each system. In seeking more potent orally active compounds, strategies were explored in congeners of 4f for reducing lipophilicity without sacrificing potency. For example, replacement of 2-naphthyl of 4f by various aza- and oxoheterocycles afforded compounds in which log P is reduced by 1.7-2.3 units while potency in human whole blood in vitro was maintained or enhanced relative to 4f. In addition, the oxoheterocyclic replacements provided compounds with improved oral potency and the preferred compound from this group is 6-[[3-fluoro-5-(4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4- yl)phenoxy]methyl]-1-methylquinol-2-one (4y). In the in vitro systems, 4y inhibited LT formation with IC50s in mouse macrophages and human whole blood of 3 nM and 0.02 microM, respectively. 4y did not inhibit the synthesis of cyclooxygenase (CO) products at concentrations up to 500 microM in human blood, a selectivity for 5-LPO over CO of greater than 20,000-fold. In the rat 4y inhibited the formation of LTB4 in blood ex vivo and in inflammatory exudate with ED50s 3 h after oral dosing of 0.9 and 0.3 mg/kg, respectively. 4y was more potent in vitro in human whole blood and in rat blood ex vivo at 3 h than either the 5-LPO inhibitor A-64077 or the FLAP antagonist MK-886. Based on these data 4y (ICI D2138) has been entered into development as an orally active, selective 5-LPO inhibitor for clinical evaluation in inflammatory conditions in which LTs are believed to play a role.     

Inhibition by CV-3988 of the binding of [3H]-platelet activating factor (PAF) to the platelet

The inhibitory effects of CV-3988, a specific antagonist of PAF, on the binding of [3H]-PAF to washed platelets of various species including human were examined. The dissociation constant (Kd), binding capacity (Bmax), and the number of receptor/platelet for the specific binding site of rabbit platelets were 2.2 +/- 0.2 nM, 93.7 +/- 8.3 fmoles/10(8) platelets, and 568 +/- 50, respectively. CV-3988 selectively inhibited the specific binding of [3H]-PAF to rabbit platelets with an IC50 of 7.9 X 10(-8) M, and it slightly increased the Kd value (2.5 +/- 0.8 nM) and decreased the binding capacity for PAF (Bmax: 54.3 +/- 16.3 fmoles/10(8) platelets). The Ki value of CV-3988 for the specific binding of [3H]-PAF to rabbit platelets was 1.2 X 10(-7) M. CV-3988 had no effects on the binding of [3H]-5-hydroxytryptamine (5-HT) to rabbit platelets and on the shape change of the platelet induced by 5-HT. CV-3988 also inhibited the specific binding of [3H]-PAF to human and guinea-pig platelets with IC50 values of 1.6 X 10(-7) and 1.8 X 10(-7) M, respectively. CV-3988 inhibited the PAF-induced aggregation in rabbit, guinea-pig, and human platelets. These findings show that CV-3988 is a specific antagonist of PAF at the receptor site(s) of platelets and, in these species, inhibits PAF-induced platelet aggregation by inhibiting the binding of PAF to the "PAF receptor". No specific binding of [3H]-PAF to the platelet of rats and mice was observed, indicating that these species lack a PAF receptor.     

Comparison of the reversed passive Arthus and local Shwartzman reactions of rabbit skin: effects of the long-acting PAF antagonist UK-74,505

1. By using the selective, potent and long acting platelet-activating factor (PAF) antagonist, UK-74,505, we investigated the role of PAF in a local Shwartzman reaction (LSR) and a reversed passive Arthus (RPA) reaction in rabbit skin. For comparison, we also studied the effect of the PAF antagonist on neutrophil aggregation in vitro and on acute inflammatory responses induced by intradermally (i.d.) injected lipopolysaccharide (LPS), PAF, bradykinin and zymosan-activated plasma.
2. Neutrophil aggregation was assessed photometrically. Haemorrhage, oedema formation, platelet deposition and neutrophil accumulation were quantified in rabbit skin by measuring the accumulation of i.v. injected ⁵¹Cr-labelled red blood cells (RBC), ¹²⁵I-labelled human serum albumin, ¹¹¹In-labelled platelets and ¹¹¹In-labelled neutrophils respectively.
3. UK-74,505 inhibited in vitro neutrophil aggregation induced by PAF but not by leukotriene B₄. When injected i.v. into rabbits UK-74,505 suppressed oedema formation in response to i.d. PAF for up to 4 h but had no effect on oedema induced by bradykinin or zymosan-activated plasma.
4. Oedema formation, but not neutrophil accumulation, produced during the RPA reaction was significantly inhibited by i.v. UK-74,505. The PAF antagonist also suppressed ¹¹¹In-platelet but not ¹¹¹In-neutrophil accumulation in response to i.d. LPS. UK-74,505 did not affect haemorrhage or oedema formation produced during the LPS-mediated LSR.
5. The results demonstrate that PAF is an important mediator of oedema formation, but not neutrophil accumulation, in the immune-complex mediated RPA reaction in rabbit skin. PAF also appears to be required for platelet, but not neutrophil, accumulation in response to locally injected LPS. Our studies do not suggest a role for PAF in the LPS-mediated LSR.

     

Platelet-activating factor (PAF) inhibitory profile of KO-286011 on blood platelets in vitro and in vivo

Summary A newly synthesised structural analogue of PAF, coded KO-286011 (1-O-hexadecyl-2-O-ethyl-racglycero-3-phosphoric acid 4-(N,N-dimethylamino)pyridinium butylester), was proved for its ability to inhibit PAF-mediated platelet responses in vitro and in vivo. The compound inhibited effectively the PAF-induced aggregation and secretion of human and rabbit platelets. In contrast, there was little influence on ADP-, collagen-, and arachidonic acid-triggered platelet responses. Schild-analysis of aggregation data ascertained in human platelet-rich plasma was consistent with a simple competitive antagonism and yielded a pA₂, of 6.44. Pro-aggregatory activity of KO-286011 was excluded turbidimetrically as well as by means of a single cell counting technique. [³H]PAF binding studies provided evidence that KO-286011 exerts its inhibitory action at the PAF-receptor level. A significant inhibition of the ex vivo PAF-induced platelet aggregation was found after i.v. administration of 0.5 mg/kg KO-286011 to rabbits. The effect was most pronounced 5 min after dosing the inhibitor and detectable over a period of 30 min. Intravenous administration of 10 and 25 μg/kg KO-286011 to guinea pigs prevented dose-dependently the PAF-induced formation of thromboxane A₂. The PAF-inhibitory action of KO-286011 was more potent than that of the ginkgolide BN 52021. Send offprint requests to G. Ostermann at the above address     

Synthesis and structure-activity relationships of a novel class of 5-lipoxygenase inhibitors. 2-(Phenylmethyl)-4-hydroxy-3,5-dialkylbenzofurans: the development of L-656,224

The synthesis of a series of 2-(phenylmethyl)-4-hydroxy-3,5-dialkylbenzofurans and their inhibitory effects against leukotriene biosynthesis and 5-lipoxygenase activity in vitro are described. Many compounds in this series were found to be potent inhibitors of LTB4 production by human polymorphonuclear leukocytes with IC50 values ranging from 7 to 100 nM. Structure-activity relationships of the series are presented. Within this series, 2-[(4'-methoxyphenyl)methyl]-4-hydroxy-3-methyl-5-propyl-7-chlorobenz ofuran (L-656,224) showed extremely potent activity, inhibiting leukotriene biosynthesis in intact human leukocytes (IC50 = 11 nM), as well as the 5-lipoxygenase reaction catalyzed by cell-free preparations from rat leukocytes (IC50 = 36 nM), human leukocytes (IC50 = 0.4 microM), and the purified enzyme from porcine leukocytes (IC50 = 0.4 microM). The compound also shows oral activity in a number of animal models in vivo.     

4-Alkyl-1,4-dihydropyridines derivatives as specific PAF-acether antagonists

A new series of 4-alkyl-1,4-dihydropyridines (1,4-DHP) were synthesized and evaluated for their ability to inhibit washed rabbit platelet aggregation induced by PAF-acether (1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glycero-3-phosphorylcholine) and to reverse PAF-induced hypotension in anesthetized rats. Additionally, compounds were evaluated for their ability to inhibit the binding of radiolabeled PAF to its receptor on rabbit platelets. Among these compounds, 6I and 6L were the most potent and specific antagonists. At concentrations up to 100 microM, neither compound 6I nor compound 6L caused platelet aggregation nor did they inhibit platelet aggregation induced by collagen or adenosine diphosphate. Compound 6L did not show in vitro calcium channel blocker activity measured on vascular smooth muscle preparations of rabbit aorta and on [3H]nitrendipine binding assays. The compound did not show any cardiovascular effects in anesthetized rat at iv doses up to 1000 micrograms/kg, and the Ki value was 568.62 nmol. These results indicate that compound 6L is a potent and specific PAF antagonist with 1,4-dihydropyridine structure but devoid of a significant cardiovascular activity related to calcium-antagonist properties.     

Prehispanolone, a novel platelet activating factor receptor antagonist from Leonurus heterophyllus.

1. Using an in vitro radioligand binding assay for the platelet activating factor (PAF) receptor, we have identified a novel, specific PAF antagonist, prehispanolone, from a Chinese medicinal herb Leonurus heterophyllus. 2. The presence of sodium ions inhibited specific [3H]-PAF binding to rabbit platelet membrane with an IC50 of 5.2 mM, decreased the inhibitory potency of PAF but increased the inhibitory potency of prehispanolone. 3. Prehispanolone and several of its derivatives inhibited the binding of [3H]-PAF to rabbit platelets with potencies closely resembling that of inhibition of PAF-induced aggregation. 4. The integrity of the tetrahydrofuran ring of prehispanolone is critical for its interaction with the PAF receptor. 5. By hydrogenating the dihydrofuran ring and replacing the keto group of prehispanolone with a hydroxyl group, we obtained a compound, LC5507, that is more stable and more active than prehispanolone as a PAF receptor antagonist.