Pharmacological profile of 48740 R.P., a PAF-acether antagonist

European Journal of Pharmacology, 150 (1988) 257-268 257 Elsevier

EJP 50273

Pharmacological profile of 48740 R.P., a PAF-acether antagonist

Jean Lefort 1, Pierre Sedivy 2,., Strphanie Desquand 1, Jacques Randon 1, Eliane Co~ffier 3,4, Isabelle Maridonneau-Parini 1, Anne Floch 2, Jacques Benveniste 3 and B. Boris Vargaftig a

I Unit~ des Venins, Unit~ associ~e lnstitut Pasteur/INSERM U 285, D~partement de Physiopathologie exp~rimentale, Institut Pasteur, 28, Rue du Dr Roux, 75015 Paris, 2 RhOne-Poulenc Sant~, Centre de Recherches de Vitry, 13, Quai Jules Guesde,

94403 Vitry-sur-Seine C~dex, and 3 U 200 INSERM, 32, Rue des Carnets, 92140 Clamart, France

Received 15 December 1987, accepted 1 March 1988

The pyrrolo-thiazole derivative 48740 R.P. inhibited the platelet-activating factor (PAF-acether)-induced aggregation of human and rabbit platelets and was poorly effective against ADP- and arachidonic acid-induced platelet aggregation. 48740 R.P. prevented the activation of guinea-pig alveolar macrophages by PAF-acether, and the PAF-acether-induced thromboxane B 2 production from guinea-pig lungs. 48740 R.P. (3 mg/kg i.v.) antagonized selectively in anaesthetized guinea-pigs the bronchoconstriction due to PAF-acether without affecting that due to acetylcholine, histamine, serotonin, thromboxane A 2 analogue U-46,619 and arachidonic acid. A higher dose of 48740 R.P. (10 mg/kg i.v.) was required to block the thrombocytopenia and the leucopenia induced by PAF-acether in the propranolol-treated guinea-pigs. 48740 R.P. (30 mg/kg i.v.) antagonized the PAF-acether effects when bronchoconstriction was induced by aerosolized PAF-acether. 48740 R.P. is a selective antagonist of PAF-acether under in vitro and in vivo conditions.

48740 R.P.; Platelet-activating factor (PAF-acether); Platelets; Bronchoconstriction; Macrophages (alveolar); Thromboxane; (Guinea-pig)

1. Introduction

PAF-acether (platelet-activating factor, 1-O-al- ky l -2-O-ace ty l - sn-g lycero-3-phosphory lchol ine) (Benveuiste et al., 1972) is a potential mediator of acute inflammation and hypersensitivity. Forma- tion of PAF-acether accompanies the stimulation of various cell types, such as neutrophils (Camussi et al., 1981; Lotner et al., 1980; Jouvin-Marche et al., 1984), vascular endothelial cells (Camussi et al., 1983), platelets (Chignard et al., 1979) and

* To whom all correspondence should be addressed. 4 Present address: Unit6 des Venins, Unit6 associre Institut

Pasteur/INSERM U 285, Drpartement de Physiopathologie exprrimentale, Institut Pasteur, 28, Rue du Dr Roux, 75015 Paris, France.

macrophages (Mencia-Huerta et al., 1978). Fur- thermore, PAF-acether is released into the rabbit circulation during anaphylactic shock (Pinckard et al., 1979) and sensitized rabbit (Kravis and Hen- son, 1975) and guinea-pig lungs (Rotilio et al., 1984; Parente et al., 1985; Fitzgerald et al., 1986) challenged with antigen release PAF-acether or lyso-PAF, one of its metabolites.

A role for PAF-acether in the immediate hypersensitivity reaction (Basran et al., 1984), the late- response to allergen (Morley et al., 1985) and shock (Pinckard et al., 1977; Bessin et al., 1983; Page et al., 1983) was suggested on the basis that this substance induced platelet migration to the proximity of respiratory smooth muscle (Lellouch- Tubiana et al., 1985), bronchoconstriction, thrombopenia (Vargaftig et al., 1980) and leukopenia in the guinea-pig. On the other hand, platelet thrombi

0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

258

C O N H z

© Fig. 1. Structure of 48740 R.P.

and migration of leucocytes were obtained in the same species by PAF-acether superfusion on arteries (Bourgain et al., 1985).

The role of PAF-acether in diseases is not yet known due to the lack of a reliable chemical assay. The first antagonists described were CV-3988 (Terashita et al., 1983), BN 52021 (Braquet et al., 1985; Nunez et al., 1986), kadsurenone (Shen et al., 1985) and 48740 R.P. (Sedivy et al., 1985a,b). PAF-acether antagonists belonging to other chemical families and which have appeared since 1983 are the triazolobenzodiazepines (Kornecki et al., 1984), thienotriazolodiazepine (Casals-Stenzel, 1987), tetrahydrofuranyl derivatives (Wu et al., 1986; Handley et al., 1986; 1987) and glyceryl derivatives (Miyamoto et al., 1985; Burri et al., 1985). PAF-acether antagonists protect rats against endotoxin shock (Terashita et al., 1985; Inarrea et al., 1985; Adnot et al., 1986) and endotoxin-induced gastric ulcers (Wallace and Whittle, 1986). Thus this phospholipid may intervene in a variety of pathophysiological conditions. We now present the pharmacological properties of 48740 R.P. (fig. 1) which belongs to the family of pyrrolo [1,2-c] thiazole derivatives. This compound antagonizes the effects of PAF-acether on platelets and behaves as a selective inhibitor of the bronchopulmonary effects of PAF-acether in the guinea-pig.

2. Materials and methods

2.1. Drugs and chemicals

48740 R.P., 3-(3-pyridyl)-lH,3H-pyrrolo [1,2-c] thiazole-7-carboxamide, hydrochloride was dissolved in 0.9% NaC1 (saline) at a final concentration of 10 mg/ml. PAF-acether (the octadecyl derivative purchased from Bachem, Switzerland, or provided by Prof. J.J. Godfroid, Universit6 de

Paris VII) was kept at - 6 0 °C in a stock solution of saline containing 0.25% bovine serum albumin (BSA) (Sigma). Daily dilutions were made with 0.25% BSA-saline. Other drugs were as follows, the solvents being indicated except when saline was used: sodium pentobarbital (Nembutal, Lathrvet, France); superoxide dismutase (SOD, from bovine erythrocytes, 3500 UI/mg, Serva, Germany); 5-hydroxytryptamine (serotonin, 5- HT), acetylcholine, histamine, lidocaine, N-for- myl-methionyl-leucyl-phenylalanine (FMLP, dissolved first in DMSO at 50 mg/ml, then diluted in saline), ferricytochrome c, arachidonic acid (dissolved in absolute ethanol at 100 mg/ml, then diluted with a Na2CO 3 solution of 20 mg/ml), creatine phosphate (CP), creatine phosphokinase (CPK) (Sigma); leukotriene D 4 (LTD4) in its methylester form (Paesel, Frankfurt, F.R.G.) was hydrolyzed with K2CO 3 solution and diluted in H20; U-46,619 (15(S)-hydroxy-ll,9 (epoxy- methano) prosta-5Z, 13E-dienoic acid) (Upjohn, Kalamazoo, U.S.A.); ADP (Stago, France); mepyramine maleate, gallamine triethiodide (Rhrne-Poulenc); methysergide hydrogen maleate (Sandoz); propranolol (ICI); aspirin as lysine acetylsalicylate (Asprgic, Labs. Egic, France); urethane (Prolabo, France); pancuronium (courte- sy of Dr. D. Savage, Organon, Motherwell, Scot- land); Eagle's Minimal Essential Medium (MEM) (Eurobio, France).

2.2. Platelet aggregation

Male New Zealand rabbits (hybrid Hy 2000, Achard de la Vente, France) weighing 2.5-3 kg had their marginal ear artery punctured and blood was collected in sodium citrate 3.1% (1 ml for 9 ml blood). Citrated blood was centrifuged at 70 x g (15°C, 20 min) to prepare platelet-rich plasma (PRP). The platelet number was adjusted to 4 × 105//~1 with platelet-poor plasma prepared after centrifugation at 2 250 x g (15 o C, 10 min) of the blood pellet.

A volume of 200/tl of PRP was mixed with 25 /tl of 0.1 mM aspirin and 25 #1 of ADP scavenging system (116/~M creatine phosphate and 1.8 U / m l creatine phosphokinase) and 25 /~1 of 48740 R.P. solution or saline and wanned for 2 min at 37 o C.

The mixture was preincubated for 1-3 and 10 min at 37 °C in an Icare aggregometer without stirring to examine the influence of time on platelet cyclo-oxygenase inhibition evoked by aspirin as described by Rosenberg et al. (1971). PAF-acether solution (25/~1, submaximal concentrations of 5 or 10 nM) were added and stirring was started in the aggregometer (1000 r.p.m.). We performed control experiments to detect any partial agonist activity of 48740 R.P. Mixtures of 200/xl of PRP, 25/~1 of aspirin, 25/xl of C P / C P K and 25/~1 of saline were preincubated at 37°C without stirring. We then added 25 /~1 of 48740 R.P. solutions or saline instead of the aggregating agent in the aggregometer. We also tested ADP- and arachidonic acid-induced platelet aggregation with 200 /~1 of PRP mixed with 50/~1 of saline and 25 btl of 48740 R.P. solution or saline and warmed for 2 min at 37 o C. The mixture was then preincubated without stirring 1-3 and 10 min at 37 °C to obtain conditions under which any aspirin-like activity of 48740 R.P. could be detected (Rosenberg et al., 1971). ADP solution (25 /tl, submaximal concentrations of 1.25 or 2.5 /~M) or 25 /~1 of arachidonic acid solution (submaximal concentrations of 0.1 or 0.2 mM) were then added in the aggregometer. The ICs0 ___ S.E.M. were the concentrations of 48740 R.P. providing 50 % of the variation of transmission of the controls.

Human blood (90 ml) was collected from the forearm vein of informed healthy donors in 10 ml of ACD solution (citric acid, 38 mM, trisodium citrate, 75 mM and dextrose, 136 mM). PRP was obtained by centrifugation (100 × g , 15 min, 20 ° C) and was used to prepare washed platelets according to the method of Lagarde et al. (1980) modified by Lalau-Keraly et al. (1984) in which PRP is acidified to pH 6.4 before centrifugation. The platelet pellet was resuspended into Tyrode- HEPES-ACD buffer containing, in mM: 2.6 KC1, 137 NaC1, 0.56 dextrose, 5 HEPES, 12 NaHCO3, 0.4 NaH2PO 4. H20, 1 /9 (v/v) of ACD at pH 6.4. The pellet was resuspended in Tyrode-HEPES- ACD buffer after two washes and centrifugations (900 × g, 10 rain, 20 o C). The suspension was then divided into three aliquots. The first was incubated with 0.1 mM aspirin (to block cyclo-oxygenase) and was used for ADP-induced aggregation. The

259

second was incubated with 0.1 mM ADP (to desensitize to ADP effects) before using it for arachidonic acid-induced aggregation. The third aliquot was incubated with aspirin and ADP before PAF-acether- induced aggregation. The aliquots were centrifuged after 1 h at room temperature they diluted in Tyrode-HEPES-ACD buffer to a final concentration of 21 × 105 cells//~l. Aggregation was tested with 50 /~1 of the platelet suspension mixed with 300 /~1 ACD-free Tyrode buffer pH 7.4, containing 1.3 mM CaC12, 0.16 m g / m l fibrinogen and 48740 R.P. or the solvent. The mixture containing 3 × 105 platelets//~l was then incubated at 37°C for 10 min without stir bars. Stirring was then started (1 100 r.p.m.) in an Icare aggregometer and 1-10 /~1 of solution of aggregating agent were added. A dose-response curve was drawn for each aggregating agent in the presence of the solvent to determine the concentration inducing submaximal aggregation (ap- proximately 80% of variation of transmission). The inhibition experiments were performed with the following concentrations + S.D. of aggregating agents: 16.8 + 7/~M ADP, 1.6 + 1/xM arachidonic acid and 3.3 + 4.5 nM PAF-acether. The IC50 + S.D. were the concentrations of 48740 R.P. for which the variation in transmission was 50% of the control value.

2.3. Bronchoconstriction by i.v. and aerosolized PAF-acether

Two different protocols were used, in order to study the selectivity of 48740 R.P. and its depen- dence on fl-adrenergic inhibition. In the first protocol in which 48740 R.P. was tested against PAF-acether and unrelated bronchoconstrictor agents, male Hartley guinea-pigs (400-500 g) were anaesthetized with urethane (1.25 g /kg i.p.) and injected with gallamine triethiodide (3 mg total dose/animal). In the second protocol the guinea- pigs were anaesthetized with pentobarbitone (30 m g / k g i.p.), paralyzed with pancuronium and injected with propranolol (1 m g / k g i.v. and 3 mg /kg i.p.). In all instances, the animals were prepared for the recording of bronchial resistance to inflation (in cm H20 ) as described by Lefort and Vargaftig (1978). The right jugular vein was

260

catheterized for drug injections. Both carotid arteries were cannulated, one for blood sampling and the other to record arterial blood pressure (in cm Hg) with a Statham-Gould P23Db transducer in the second protocol. Bronchial reactivity was tested 1 h after surgery, with i.v. serotonin (1-3 gg /kg) or acetylcholine (10-40 t tg/kg) until similar bronchial responses were obtained. The propranolol-treated animals were given a first injection of PAF-acether (30-60 ng/kg) to obtain a control response. We initially verified in a separate group of 12 animals that the bronchial resistance to inflation was practically the same following a first and a second injection of PAF-acether at 1 h interval i.e. 10.8 _+ 1.1 and 9.8 _+ 1.0 cm H 2 0 respectively. The solvent of 48740 R.P. was given 2 min before the second injection of PAF- acether. The thrombocytopenia and leukopenia were the same for the two injections of PAF- acether. This allowed us to test 48740 R.P. using each animal as its own control. 48740 R.P. (1, 3 or 10 mg/kg) was therefore given 2 min before the same dose of PAF-acether as in the control. The effects of 48740 R.P. against the bronchoconstriction induced by i.v. PAF-acether, acetylcholine (30 gg/kg) , histamine (10 #g/kg) , serotonin (3 gg/kg) , compound U-46,619 (2 #g /kg) and arachidonic acid (750 t tg/kg) were investigated in animals prepared according to the first protocol.

For the aerosol experiments, a solution of PAF-acether (300 t tg/ml) was placed in the re- servoir of an aerosolator connected to an air com- pressor and the flow was directed for two min into the inlet tube of the respiratory pump (Lefort et al., 1984). Bronchoconstriction evaluated according to the pulmonary resistance to inflation and arterial blood pressure were recorded on a Beck- man R 511 or on a Gemini recorder. The intensity of bronchoconstriction was always measured at its height, i.e. 2-3 rain after the injections of PAF- acether.

2.4. Blood cell counts

Human and guinea-pig platelets were counted with a Coulter Counter ZBI (France) and rabbit platelets with a Linson 431 A Counter (Sweden). The in vivo assays in guinea-pig were done with

blood (200 ttl) collected from the carotid artery for counting leukocytes or platelets with the Coulter Counter before and 1 min after the administration of PAF-acether. Repeated measurements were performed 1 h later.

2.5. Activation of alveolar macrophages

Alveolar macrophages were prepared as described by Maridonneau-Parini et al. (1985). Briefly, they were obtained by 7-10 repeated lavages of lungs of anaesthetized guinea-pigs with 5 ml of sterile phosphate buffered saline, pH 7.4 at 37 ° C, containing 10 mM lidocaine. The cells were then washed and resuspended in MEM sup- plemented with 20 mM HEPES at a final concentration of 3 × 106/ml cells and were left to rest at 37 °C for 2 h in a Coming tube. Aliquots were then transferred into the 1 ml cuvette of a spectrophotometer at 37 °C and incubated either with the drug solvent or with 48740 R.P. (0.3 mM) for 5 min. Cytochrome c (100 /xM) was added and followed by either PAF-acether or FMLP (0.01 and 1 /~M). Production of superoxide anion was measured by the superoxide dismutase-inhibitable reduction of cytochrome c, the absorbance changes being recorded at 550 nm at one min intervals for 4 min in a Kontron spectrophotometer type Uvikon 810. The amounts of reduced cytochrome c were calculated with an extinction coefficient of 21.1 mM -1 (Massey, 1959). No reduction of cytochrome c was observed when superoxide dismutase was at 50/~g/ml.

2.6. Lung perfusion experiments

The lungs were prepared as described by Lefort et al. (1984). Briefly, guinea-pigs were anaesthetized with sodium pentobarbitone, a midthoraco- tomy was performed and the pulmonary artery was exposed, cannulated and perfused for 10 min with 50 ml of Krebs solution containing 2.5 g/1 of BSA (Krebs-BSA). The lungs were removed and suspended in a plastic chamber and the trachea was connected to a respiratory pump. Perfusion with Krebs-BSA was started immediately at 8-10 m l /m in (37°C) and the lung effluent was collected for the measurement of thromboxane B 2

(TXB2) 10 min after the beginning of the perfusion. PAF-acether or LTD 4 (10, 30 and 100 ng) was administered sequentially as bolus through the pulmonary artery at 10 min intervals. 48740 R.P. was added to the Krebs-BSA solution 10 rain before the first injection of PAF-acether.

2. 7. Radioimmunoassay of thromboxane B 2 (TXB2)

TXB 2 was assayed according to the method of Sors et al. (1978). Aliquots (100 /~1) of the lung perfusate or of Krebs solution from the organ bath were collected before and at various times after each addition of PAF-acether. Aliquots were left at room temperature for 10 min (to transform TXA 2 to TXB2) in tubes containing 0.1 mM of indomethacin and were then stored at - 2 0 ° C . The samples were thawed for the radioimmunoassay (RIA) then incubated overnight at 4 ° C with 125I-labeled TXB 2 and anti-TXB 2 serum in a 3'- globulin buffer. The next day, bound and free fractions were separated by polyethyleneglycol 6000 precipitation followed by centrifugation at 2000 × g and 4 ° C for 10 rain. The radioactivity of the pellet corresponding to the bound fraction was counted for 1 min with an Intertechnique gamma counter connected directly to a Hewlett- Packard calculator.

2.8. Data analysis

The data were expressed as means + S.E.M. or S.D. Statistical differences between two means for data obtained in the same animal were evaluated by the paired Student's t-test, whereas the un- paired test was used to compare parallel values from different animals.

3. Results

3.1. Antagonism of platelet activation

3.1.1. Rabbit platelets Submaximal concentrations, 5 and 10 nM

(mean: 8.0 + !.2 nM), of PAF-acether evoked a less than 50% increase in transmission in the aggregometer in the presence of aspirin and

261

C P / C P K . Under these conditions, 48740 R.P. inhibited platelet aggregation between 10 /~M and 5 0 / tM with an ICs0 of 24.9 __ 3.9/xM after 1 min preincubation with PRP. When 48740 R.P. was preincubated with PRP for 3 min there was no significant increase of activity (ICso = 21.1 _+ 1.8 #M). After 10 min preincubation of 48740 R.P. with PRP there was a slight increase of activity (ICs0 = 16.6 _ 2.7/~M) with respect to 1 rain preincubation. The mean value for the anti-aggregating activity of 48740 R.P. after 10 min preincubation was statistically different from the mean value obtained after 1 min (P < 0.05) but not from the value obtained after 3 min preincubation (table 1). Submaximal concentrations of ADP of 1.25 and 2.5/xM (mean: 2.25 _+ 0.25 #M) evoked a less than 50% increase in transmission. 48740 R.P. only produced approximatively 7% inhibition at 100 /xM after 10 min preincubation and showed little activity at higher concentrations at all preincubation times studied (table 1). The activity of 48740 R.P. was evaluated in the presence of submaximal concentrations, 0.1 and 0.2 mM (mean: 0.16 -1- 0.02 mM), of arachidonic acid which induced a less than 70% increase in transmission. 48740 R.P., again demonstrated little activity at the high concentration of 400 #M at all preincubation times studied (table 1).

TABLE 1

Effects of 48740 R.P. on aggregation of rabbit platelet-rich plasma induced by several agonists: IC50 calculated from dose-response curves 5: S.E.M. (n = 5). Rabbit PRP were preincubated for 1-3 and l0 min at 3 7 ° C without stirring, with various concentrations of 48740 R.P. Submaximal concentrations of agonists as indicated in parentheses 5: S.E.M. were then added.

Preincu- 48740 R.P. IC50 5: S.E.M. (/~M) bation time of PRP at 3 7 ° C without stirring

PAF-acether A D P Arachidonic acid (8.05:1.2 nM) (2.25+0.25 #M) (0.16+0.02 mM)

1 min 3 min

10 min

24.9 -I- 3.9 > 400 > 400 21.1 + 1.8 > 400 > 400 16.6+2.7 >/399+0.09 > 400

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TABLE 2

Effects of 48740 R.P. on platelets in rabbit PRP in comparison with effects of PAF-acether, ADP and arachidonic acid under aggregation conditions. Saline and 48740 R.P. were studied in the presence of aspirin and CP/CPK with the PRP of five rabbits. These PRP were also used with PAF-acether (in the presence of aspirin and CP/CPK) and with ADP and arachidonic acid to test their aggregability. The results are means+ S.E.M. of the maximal decrease in light transmission (n = 5) (submaximal concentrations of agonists as indicated in parentheses 5: S.E.M.).

Preincubation time Saline 48740 R.P. (/~M) PAF-acether ADP Arachidonic of PRP at 37 o C 50 100 200 400 (8.0 5:1.2 nM) (2.25 + 0.25 # M) acid without stirring (0.16 5:0.02 mM)

I min 0.90 0.85 0.85 1.35 0.83 40.66 37.26 67.22 +0.41 -t-0.38 5 :0 .55 5:0.94 5:0.48 5:0.62 +2.95 5:3.20

3 min 1.41 1.94 1.39 1.73 1.75 41.26 36.64 68.04 5:0.27 5 :0 .33 +0.23 +0.46 +0.50 +1.54 5:4.14 5:2.45

10 rain 1.57 1.50 1.20 1.15 1.29 42.40 37.58 63.34 5:0.29 5:0 .42 5:0.20 + 0.55 + 0.44 + 0.17 5:5.43 5:3.07

Even at a high concen t ra t ion (400 /~M), 48740 R.P. fai led to evoke p la te le t aggrega t ion in the presence of asp i r in and C P / C P K , af ter 1-3 and 10 min p re incuba t ion ( tab le 2). These var ious pre- i ncuba t ion t imes had no effect on PAF-ace the r - , A D P - and a rach idon ic ac id - induced aggrega t ion ( tab le 2).

3.1.2. Human platelets A t the high concen t ra t ion of 500 /~M, 48740

R.P. inh ib i ted A D P - i n d u c e d p la te le t -aggrega t ion b y 46.3-t-24.7% ( + S.D.) bu t the inh ib i t ion was on ly 15.0 + 10.3% at 100 / tM. A r a c h i d o n i c ac id - induced p la te le t aggregat ion was inh ib i ted by 57.8 + 32.8% at 500 /~M and by 18.0% at 100 /~M (IC50 = 390 + 180 /~M). 48740 R.P. inh ib i ted P A F - a c e t h e r - i n d u c e d p la te le t aggrega t ion by 97.0 + 6.0% at 500 / zM and b y 68.5 + 27.3% at 100/LM wi th an IC50 of 38.0 + 22 .0 /~M (n = 4).

3.2. Antagonism of bronchoconstriction, thrombocytopenia and leukopenia induced by PAF-acether

In the first p ro toco l wi th u re thane -anaes the - t ized guinea-pigs , 48740 R.P. an tagon ized the b ronchocons t r i c t i on caused by P A F - a c e t h e r (fig. 2). This an t agon i sm was selective since the b ronchocons t r i c t i on caused by h i s tamine (Hist) , ace ty lchol ine (ACh) , se ro tonin (5-HT), a rach idon ic ac id (AA) or c o m p o u n d U-46,619 was unaf fec ted by up to 10 m g / k g of 48740 R.P., a dose which ent i re ly suppressed the effects of P A F - a c e t h e r (fig. 2).

Examples (14 cases) f rom the second p ro toco l are p resen ted (fig. 3) tha t inc lude controls f rom o ther exper iments . Signif icance was es t imated by means of the unpa i r ed t- test wi th the four an imals t reated. Signif icance was also es t imated with a pa i r ed t- test app l i ed to values f rom only the four t rea ted animals . In bo th cases the differences were ident ical .

As shown in fig. 3, 48740 R.P. also b locked P A F - a c e t h e r - i n d u c e d b ronchocons t r i c t i on in pro- p r a n o l o l - p r e t r e a t e d animals . Thus, 48740 R.P. at 3

i i

i

* 5 0

10

o

- 5 0

- 1 ~

4 8 7 4 0 R.R ( m g / k g i.v. )

10 10 1 3 10 10 10

Agonist : Ach Hist 5-HT P A F - a c e t h e r U-46,619 A A

Fig. 2. Failure of i.v. 48740 R.P. (dotted columns, 1 mg/kg; hatched columns, 3 mg/kg and open columns, 10 mg/kg) to interfere with bronchoconstriction caused by i.v. acetylcholine (30 /xg/kg), histamine (10 /~g/kg), serotonin (3 /zg/kg), arachidonic acid (750 /~g/kg) or compound U-46,619 (2 /xg/kg), as compared to its effectiveness against PAF-acether (50 ng/kg). Bronchoconstrictor responses are expressed as % of control responses (n=4-5 animals/group). ** P <0.01;

* * * P < 0.001.

263

2 0 _

I

+ -II + • ~ <

0 _ 0 O-

- 6 0 2 60 min - 6 0 2 60 min - 6 0 2 60 min.

Fig. 3. Effects of 48740 R.P. (n = 4) 2-3 rain after the injection of PAF-acether (33 ng/kg i.v.) on bronchoconstriction (BC: cm H20 + S.E.M.) (left), and 1 rain after PAF-acether injection on thrombopenia (middle) and leukopenia (right), (percent of cell count decrease+S.E.M.) in propranolol-treated animals. * P<0.05; ** P<0.01 (14 control animals and 4 animals treated with

PAF-acether 60 rain before and 2 and 60 min after the administration of 48740 R.P.). • Control; [] 3 mg/kg; • 10 mg/kg.

m g / k g inhibited by 66 + 11% (n = 4) the bronchoconstriction induced by PAF-acether and this effect was no longer present 60 rain later. However, the reduction in the number of circulating platelets and leukocytes induced by PAF- acether was not prevented by this dose of 48740 R.P. Both bronchoconstriction and the decrease in the number of circulating platelets and leukocytes due to PAF-acether were suppressed by 10 m g / k g of 48740 R.P. 48740 R.P. had no effect on the peripheral blood cell count at 10 m g / k g i.v. The numbers of peripheral blood cells counted 1 rain before and 1 min after the administration of 48740 R.P. were 473 000 + 60 000 and 464 000 + 56 000 for the platelets and 8900 + 1 700 and 7300-F 1 000 for the leukocytes. These differences are not statistically significant.

PAF-acether-induced hemoconcentration did not change the absolute cell count and the extent of thrombocytopenia and leukopenia. The number of platelets before the first injection of PAF- acether was 498 000 + 53 000 and 473 000 + 60 000 1 h after this injection. The number of leukocytes was 4800 ± 1100 before the first injection of PAF-acether and 8 900 ± 1700 1 h later. The increase in the number of leukocytes could be explained in part by the injection of PAF-acether,

but we noted in other experiments that there was a spontaneous increase of the number of leukocytes 2 h after the injection of anaesthetic. Furthermore, in another group of experiments with seven guinea-pigs, three repeated injections of PAF- acether at 1 h intervals induced reproducible leukopenia and thrombocytopenia which allowed us to test 48740 R.P, using each animal as its own control.

The inhibition of bronchoconstriction and of leukopenia had ceased 1 h after the administration

15

10 S

o 5 I i | !

0 10 30 (mg/kg)

Fig. 4. Effects of 48740 R.P. on bronchoconstriction induced by aerosolized PAF-acether. The doses of i.v. 48740 R.P. are indicated under each column. * P < 0.05 (n = 4-5 animals/

group).

264

of 48740 R.P. whereas the inhibition of PAF- acether-induced thrombocytopenia persisted in part (fig. 3). This dose of 48740 R.P. reduced the PAF-acether-induced thrombocytopenia by 64% (fig. 3).

Aerosolized PAF-acether induces bronchoconstriction however the animals rapidly develop tachyphylaxis to this effect (Lefort et al., 1984). Separate groups of animals were thus used, in which PAF-acether was aerosolized for 2 min after pretreatment with an i.v. injection of 48740 R.P. or its solvent. As seen in fig. 4, only 30 mg /kg of 48740 R.P. suppressed the bronchoconstrictor activity of aerosolized PAF-acether.

3.3. Interference of 48740 R.P. with the superoxide production induced by PAF-acether and FMLP in macrophages

Cell viability was monitored in presence of 0.3 mM of 48740 R.P. by Trypan blue exclusion. Macrophages of four guinea-pigs were incubated for 5 min in MEM and the percent of dead macrophages was 13.5 -t- 1.9%. Other macrophages were incubated for 5 rain with 0.3 mM of 48740 R.P. and the percent of dead macrophages was 14.0 + 5.3% which is not statistically different from that in control experiments.

As seen in fig. 5, the stimulating effect of PAF-acether on the formation of superoxide ions was suppressed by 48740 R.P. whereas the effect of FMLP was only marginally reduced. The reduction of ability of the treated macrophages to generate superoxide anion observed with FMLP seems to have been due to a non-specific effect of 48740 R.P. rather than a toxic effect of 48740 R.P.

The generation of superoxide anion by macrophages obtained from three guinea-pigs and incubated with 0.3 mM of 48740 R.P. and cytochrome c in the absence of stimuli were measured. A slight reduction of cytochrome c could be observed in presence of 48740 R.P. but this reduction was about 10 times less than the reduction induced by agonists like PAF-acether or FMLP. Therefore 48740 R.P. has no agonist effect on superoxide anion production (1.8 +_ 0.3-1.6 + 0.4 and 1.4 + 0.6/~mol of reduced cytochrome C / 1 0 6

cells were detected in the presence of 48740 R.P.

. . J W 0

o 0

i "l

! :0 d

W

I .

P A F - A C E T H E R

16

I0

|

F - r n e t - l e u - 1 1 -

1 1 -

I S -

5 -

• , 1 l Z O

I t ~1 . T i l e l i l l l

~he

i i t i T I l t I n l | l

Fig. 5. Upper panel: inhibition by 48740 R.P. (0.3 mM: n = 3) of the in vitro product ion of superoxide anion by PAF-acether-stimulated macrophages (1 /~M: left panel and 0.01 /~M: right panel). The results are expressed as nmol of reduced cytochrome c/106 cells-t-S.E.M. The spontaneous reduction of cytochrome c in the absence of the stimulating agent was below the limits of measurements (n = 4). * P < 0.05; • * P < 0.01. Lower panel: failure of 48740 R.P. (0.3 mM: n = 3) and its solvent (n = 4) to block the in vitro production of superoxide anion by FMLP-stimulated macrophages (1/~M: left panel and 0.01 ~tM: right panel). Black dots: control; black squares, in presence of 48740 R.P.; black triangles, basal levels.

after 1-2 and 3 min incubation respectively).

3.4. Interference of 48740 R.P. with PAF-acether- induced thromboxane A 2 formation by perfused lungs

T X A 2 is formed when PAF-acether, leukotriene D 4 or arachidonic acid are injected intra-arterially into isolated guinea-pig lungs (Lefort et al., 1984). As seen in table 3, 48740 R.P. (100/xM) added to the solution used to perfuse the lung suppressed

265

TABLE 3

Effects of 48740 R.P. 100 btM on thromboxane B 2 released from isolated guinea-pig lungs. The results are m e a n s + S.E.M. of the amounts of thromboxane B 2 (ng /ml ) formed during the first 3 n'fin after the intra-arterial injections of PAF-acether or LTD 4 (10, 30 and 100 ng) or 1 min after the injection of 100 /tg of arachidonic acid to perfused guinea-pig lungs. Number of experiments in parentheses.

PAF-acether (ng) LTD 4 (ng) Arachidonic

10 30 100 10 30 100 acid 100/~g

Control

4 .9+1 10 + 2 2 5 + 4 1 0 + 4 70+18 2 5 0 + 4 4 95+ 8 (6) (6) (6) (5) (6) (6) (3)

48740 R.P.

0.7+0.2 a 1 .2+0.2 b 4 + 1 b 16-/-6 101+25 240 83+13 (3) (3) (5) (5) (5) (2) (3)

a P < 0.05, b P < 0.01.

the effects of PAF-acether whereas the TXA2-re- leasing effects of leukotriene D 4 and of arachidonic acid were unaffected.

4. Discussion

The pharmacological profile of 48740 R.P., an antagonist of PAF-acether-binding on rabbit platelets and PAF-acether-induced platelet aggregation in animals and humans (Sedivy et al., 1985a, b) was further extended in the present study.

Terashita et al. (1983) described the first PAF- antagonist, CV-3988, and reported that this compound inhibited aggregation of rabbit platelets in PRP stimulated with 30 nM PAF-acether (IC50 = 7.8 #M) without aspirin and ADP scavenger. Shen et al. (1985) described the inhibition by 2.4 /~M kadsurenone of PAF-acether-induced platelet aggregation with 2.2 nM PAF-acether under similar conditions. Braquet et al. (1986) obtained an IC50 of 0.188 #M for BN 52021 (Ginkgolide B) using 2.5 nM of PAF-acether without aspirin and ADP scavenger. We used a PAF-acether concentration of 8.0 + 1.2 nM in the presence of aspirin and ADP scavenger in order to be sure to study exclu- sively PAF-acether-induced platelet activation. Furthermore, in the study of the influence of preincubation time of PRP with 48740 R.P. in PAF-acether-, ADP- and arachidonic acid-induced platelet aggregation we did not detect any anti-cyclo-oxygenase property of 48740 R.P. but

only a slight increase of activity with PAF- acether-induced aggregation which could not be explained. As 48740 R.P. has no agonist properties, we can conclude that it is a specific PAF- acether antagonist which is less active in vitro than CV-3988, kadsurenone and BN 52021 on rabbit platelets. On the other hand, Hwang et al. (1986) have recently graded the activity of these compounds based on their order of potency in an in vitro binding assay with [3H]PAF-acether and rabbit platelet membranes as follows: kadsurenone > CV-3988 > BN 52021 > 48740 R.P. Again 48740 R.P. was less active than the three other compounds. Nunez et al. (1986) described the inhibition of PAF-acether-induced platelet aggregation by CV-3988, kadsurenone and BN 52021 with washed human platelets insensitive to ADP and arachidonic acid (IC50 respectively 1-0.8 and 0.86 /~M) after 1 min preincubation of the compounds at 37°C with platelets. A PAF-acether concentration of 2.5 nM was used in these experiments. 48740 R.P. was studied after 10 min preincubation with PAF-acether concentrations of 3.3 _ 4.5 nM (IC50 = 38 /~M). 48740 R.P. was less active than the three other compounds and inhibited at high concentrations (500 #M) the ADP- and arachidonic acid-induced aggregation after 10 min preincubation at 37°C. After 1 min preincubation, BN 52021 (220 /~M) inhibited slightly the arachidonic acid-induced aggregation but CV- 3988 had an IC50 of 22 /~M (Nunez et al., 1986). We tested the activity of 48740 R.P. on PAF-

266

acether-stimulated guinea-pig alveolar macrophages and found that the production of superoxide anion was inhibited by 48740 R.P. 300/~M. This activity has been described for BN 52021 at the same concentration (Desquand et al., 1986). As BN 52021, a specific PAF-acether antagonist and 48740 R.P. are both inactive against FMLP- induced activation of the same alveolar macrophages, we can conclude that 48740 R.P. is capable of blocking the PAF-acether effects selectively in platelet-independent systems. Previous experiments (Sedivy et al., 1985a) showed that 48740 R.P., 20 mg /kg p.o., was capable of inhibiting PAF-acether-induced hypotension in conscious spontaneously hypertensive rats 2 h after its administration. Platelets are one of the targets of PAF-acether in the bronchoconstriction induced by i.v. administration of PAF-acether to guinea- pigs. In this technique, BN 52021 (3 mg /kg i.v.) has been described as an inhibitor of bronchoconstriction, in which fl-adrenergic blockade was obtained by propranolol treatment, 2 and 60 min after administration (Desquand et al., 1986). The activity of 48740 R.P. at the same dose was not maintained 60 min after i.v. administration, sug- gesting rapid elimination of the compound when given i.v. in this species.

PAF-acether-induced thrombopenia was inhibited 60 min after the administration of 48740 R.P. to the same level as with BN 52021 (Des- quand et al., 1986). The leukopenia due to injection of PAF-acether was inhibited 2 min after administration of 48740 R.P. (3 mg /kg i.v.) as described for BN 52021 (Desquand et al., 1986). BN 52021 (3 mg /kg i.v.) given to pentobarbital- anaesthetized guinea-pigs pretreated with propranolol did not block the bronchoconstriction evoked by serotonin, arachidonic acid, collagen or FMLP. The same dose of BN 52021 given to urethane-anaesthetized guinea-pigs not pretreated with propranolol blocked the PAF-acether-induced bronchoconstriction but had no effect against the action of acetylcholine (Desquand et al., 1986). This specific PAF-acether antagonistic property of BN 52021 was also observed with 48740 R.P. at the same dose. 48740 R.P. was capable of inhibit ing PAF-ace ther - induced bronchoconstriction but was inactive against the

bronchoconstrict ion evoked by acetylcholine, serotonin, histamine, arachidonic acid or the en- doperoxide analogue U-46,619 in urethane- anaesthetized guinea-pigs not pretreated with propranolol. Like BN 52021, 48740 R.P. acts on the bronchoconstriction induced by PAF-acether in the presence and in the absence of propranolol and its action is therefore not due to stimulation of bronchodilator fl2-adrenoceptors. Platelets are not the main target in the bronchoconstriction induced by PAF-acether administered by aerosol and alveolar macrophages may be involved in the process (Maridonneau-Parini et al., 1985). BN 52021, 3 m g / k g i.v., was capable of blocking the action of aerosolized PAF-acether (Desquand et al., 1986) and 30 m g / k g i.v. of 48740 R.P. were needed to obtain an inhibition of the bronchoconstriction.

Intra-arterial injections of PAF-acether to the perfused guinea-pig lung induce a dose-related release of thromboxane A 2 (Lefort et al., 1984). In in vitro experiments with guinea-pig lungs infused intra-arterially with 48740 R.P. and then with PAF-acether, 48740 R.P. (100/xM) inhibited selectively the PAF-acether-induced TXB 2 release and was ineffective against arachidonic acid and LTD 4. BN 52021 was effective at 30 /~M and was as specific as 48740 R.P. against PAF-acether effects (Desquand et al., 1986).

Recent experiments have shown that 48740 R.P. was capable of inhibiting the hemoconcentration induced by endotoxin in the rat. Preliminary re- suits have evidenced a protective effect of 48740 R.P. in anaphylactic shock studied in mice according to the technique used by Casals-Stenzel (1987) in the study of another new PAF-acether antagonist, WEB 2086. Further work with 48740 R.P. is in progress to help to explain the physio- logical role of PAF-acether in inflammation and shock models.

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Pharmacological profile of 48740 R.P., a PAF-acether antagonist

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