Effect of azelastine on platelet-activating factor and antigen-induced pleurisy in rats

Lkrupeutt Journul o/ Phurnracolo~~. 197 (1991) 201-207 s 1991 Elsevier Science Publishers B.V. 0014-2999/91/!f.O3.50 ADONIS ~14299~~~2917

FJl’ 51822

Effect of azelastine on platelet-activating factor and antige~oinduc~ Marcia CR.

Lima, Marco A. Martins,

li~stif~~oOsu,aIdo

Cm:,

De~urIu~lc~to

and ’ Unir& de Phurmorologie

Sandra A.C. Perez, Patricia and B. Boris Vargaftig 1

de Fisiolog~ff e Fur~ac~in~m~c~

Cellulairc,

Unir& Asso&e

Insrim

in rats

M.R. Silva. Renato S.B. Cordeiro

Acenidu Brush 4365

Pasteur. iNSERM

~~~~~sy

Cab

Posrul9,76.

Rio de Janeiro, Brad

285, 25 Rue Dr. Roux. Paris 75015. France

Received 8 October 1990, revised MS received 3 December 1990. accepted 29 January I991

The interference of azelastine with pleurisy induced by antigen was investigated in actively sensitized rats. The antigenic challenge (ovalbumin, 12 irg,Icavity) caused early plasma leakage, which peaked within 4 h. accompanied by intense neutrophil infiltration. Pleural exudate decayed 24 h after antigen prov~ation, when a Iong-Iasting increase in the number of resident eosinophiis was observed. Oral pretreatment with azelastine (l-10 mg/kg) dose dependently inhibited the vasopermeation (ED,, = 4.2 mg/kg) and reduced the pleural exudate (ED5, = 5.8 mg/kg) induced by the antigen. In contrast, azelastine (10 mg/kg) failed to modify the neutrophil influx observed at 4 h and the eosinophil accumulation detected at 24 h. Azelastine was aiso effective against rat pleurisy induced by either platelet-activating factor (PAF-acether& histamine or serotonin. It reduced exudation and the increase in the number of mononuclear cells, neutrophils and eosinophils observed 6 h after PAF-acether. Nevertheless, antagonism of PAF-acether may not be relevant to the inhibition observed in the present model of allsrgic pleurisy, as the inhibition was refractory to three distinct PAF-acether receptor antagonists. In contrast. like azelastine. the histamine ii, receptor antagonist meclizine and the dual histamine and serotonin receptor antagonist cyproheptadine blocked antigen-induced exudation and failed to interfere rt’ithcell influx. We conclude that the anti-exudatory activity of oral azelastine on antigen-induced pleurisy is consistent with it exerting direct effects against vasoactive amines, but is not related to an effect against leucocyte infiltration nor to its ability to inhibit PAF-acether. Azelasline; Allergic pleurisy: Vasopermeation: Pleural oedema: Cell recruitment

Anaphylatis in passively (Smith et al., 1983) and actively (Sharp and Smith, 1979; Spicer et al., t985) sensitized rats, and on isolated rat mast cells (Kusner et al., 1972; Fields et al., 1984) is largely used to study the pharmacological properties and the potential mechanism of action of anti-allergic drugs. As in humans, h~ersensitivity type-l reactions in rats seem to be predominantly mediated by IgE antibodies, whereas IgG antibodies mainly mediate these actions in guinea pigs and rabbits (Drews, 1990). Studies Ey Sharp et al. (1979) have demonstrated that the acute inflammation following ..:ntigen challenge in the peritoneal cavity of actively sensitized rats is characterized by early vasopermeation associated with the release of histamine and sulphydopeptide leukotrienes. In the same study, an intense neutrophil infiltration was noted at times rang-

Correspondence co: M.A. Martins, lnstituto Oswald0 Crux. Depnrtamento de Fisiologia e FarmacoditGtmica. Avenida Brasil 4365. Caixa Postal 926. Rio de Janeiro, Brazil.

ing from 2 to 8 h; infiltration was not accompa~~d by significant alterations in the peritoneal fluid content of fi-glucuronidase and lacticodehydrogenase. We reported recently that rats actively sensitized with ova~bu~n in alu~nium hydroxide show a drastic pleural inflammatory response when challenged with 12 pg of antigen (Lima et al., 1990). This model has now been used to study the potential interference of the anti-allergic compound azelastine on allergen-induced inflammation. Azelastine displays a large spectrum of pharmacological properties, including the ability to inhibit the biosynthesis andl’or release of histamine (Katayirma et al., 1981; Chand et al. 1985a.b) and leukotdenes C, and I& (Katayama et al., 1987). as well as the direct antagonism of histamine (Katayama et al., 1981: Zechel et al., 1981). serotonin (Zechel et al., 1981), leukotrienes C4 and D4 (Diamantis et al., 1982; Katayama et al., 1987) and platelet-activating factor (PAF-acether) (Achterrath-Tuckermann. 1988). When administered ora%y, azelastine suppresses passive cutaneous anaphylaxis in rats and guinea pigs (Katayama et al., 1981) and has bronchodilator properties in humans (Storm et al., 1985; Kemp et al.. 1986).

t-e&e I~~~~;~~~ist~~ts~ for the anti-allergic activity of ~~~e~a~t~~~~ is not fully elucidated. but the calcium chanockrng activity of arelastine (Chand et al., 1983: ama et al.. 1987) and its ability to increase the ~~~~~~~~~~~~~a~ cyclic AMP levels (Kstagama et al.. 1987) $3 a~ a critical role in its complex mode of action. present study. oral treatment with azelastine t suppressed pleurisy induced in rats by intrathoracical (it.) injection of allergen. The effect was unrelated to a~tag~~~isnl of PAF-acether and is consistent with direct actions agamst biogenic amines.

W&tar rats of both sexes. weighin 150-200 g. were sensitized with a dorsal subcntnneous injection (0.2 ml) of a mixture containing 50 $g of ovalbumin and 5 mg of alumin~um hydroxide. Ovalbumin dissolved in 0.9% NaCl solution (saline) was administered i.t. (12 pg/cavity) 14 days after sensitization. The antigen was introduced into the pleural cavity of concious animals. in a final volume of 0.1 ml, using a 27.S-gauge needle about 3 mm in length. The animals were killed at different times, ranging from 5 min to 5 days. with a terminal ether anaesthesia. The thoracic cavity was opened, via diaphragm, and rinsed with 3 ml of saline containing heparin (20 U/ml). The pleural wash was collected and the exudate co!ume measured with a plastic graduated syringe. The exudate volume (~1) was shown as the difference between the recovered volume from control znd antigen-injected animals. Sham-challenged animals (antigen was replaced by saline as challenge) and sham-sensitized animals (sensitization in which ovalbumin was omitted) were used as negative control groups. 2.2. Rate o/plasma protein leakage The kinetics of exudation were evaluated by measuring the amount of dye-labelled plasma proteins leaked, at distinct periods of time, after antigen stimulation. Four periods were analysed: the first from zero to 10 mm: the second from 10 to 30 mm; the third from 30 to 60 min and the fourth and last from 60 to 240 mm. The animals were injected with an i.v. injection of 1% Evans b1ue (20 mg/kg) 1 min before the beginning of each period and then killed, as described above, at the end of each period. The pleural cavity was washed out with 3 ml of heparinized saline; the concentration of Evans blue in the cell-free pleural wash was evaluated spectrophotometrically at 650 nm. Exudation rate was expressed as the difference between the amount of Evans

blue dye (pg/cavity) recovered from antigen-injected sensitized and antigen-injected sham-sensitized rats. 2.3. Pleurisy triggered ly* hiogertic awhes acether

atld

PA F-

Non-sensitized rats were stimulated i.t. with histamine (200 pg/cavity). serotonin (100 pg/cavity) or PAF-acether (1 pg/cavity) in a final volume of 0.1 ml. The animals were ki!led 1 h after administration of the biogenic amines and 6 h after PAF-acether administration. The reasor for the difference in time is because, contrasted wi!h the former agonists, PAF-acether induces an early exudation followed by a marked cellular influx, which is only apparent 6 h after stimulation (Tarayre et al.. 1986; Martins et al.. 1989a). The pleural fluid was collected and its volume measured as described above. 2.4. Cell coli~lts Total leucocytes were counted in Neubauer chambers by means of an optical microscope, after diluting the pleural fluid in Turk solution. Differential analysis was performed under an oil immersion objective, in cytocentrifuged smears stained with May-Grunwald-Giemsa dye. 2.5. Treatments Meclizine (30 mg/kg), cyproheptadine (2 mg/kg), BW755C (50 mg/kg) and the PAF-acether antagonists BN 52021. WEB 2170 and WEB 2086 (20 mg/kg) were given i-p. 1 h before the antigen. LY 171883 (10 pg/ cavity) was administered i.t. 5 mm before pleural stimulation. Azelastine (l-10 mg/kg) was given orally (p.0.) 2 h before the agonists or antigen. In another set of experiments, either azelastine or cyproheptadine (10 to 50 pg/cavity) were injected i.t. 5 min before the agonist or antigen stimulation. In this case, the intereference with the exudate volume elicited by either histamine (200 pg/cavity), serotonin (100 pg/cavity) or ovalbumin (12 pg/cavity) was expressed as percentage of inhibition, which was calculated based on the following formula: [lo0 - (exudate volume of antagonist-treated rats X lOO/mean of exudate volume from vehicle-treated rats)]. Drugs were diluted in sterile saline, except meclizinc which was dissolved in Tween 80 and further diluted with saline. In control groups, the antagonists were replaced by their vehicles. 2.6. Drugs Ovalbumin was provide:? by Biochemika Fluka (Switzerland); PAF-acether (l-0-hexadecyl-2-acetylsn-glyceryl-3-phosphorylcholine) was purchased from Bachem (Switzerland); histamine and serotonin were

203

purchased from Sigma (USA); meclizine was from Pfizer (Brazil); cyproheptadine was from Merck, Sharp % Dohme (USA); azelastine (4-(p-chlorobenzyl)-2-(hexahydro-l-methyl-l H-azepine-4-yl)-1-(2H)-phthalasinone hydrochloride was from Asta-Werke AG, Chemical Manufacture (FRG); BW 755C (3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride) was kindly provided by Dr. S. Moncada, Wellcome Research Laboratories (Beckenham. UK); LY 171883 (I-[2-hydroxy3-propy1-4-[4-(1H-tetrazo1-5-yl)butoxy]phenyl]ethanone) was from Eli Lilly and Company (Indianapolis, U.S.A.); BN 52021 (3-(l,l-dimethylethyl)hexahydro-1.4.7b-trihydroxy-8-methyl-9H-l,7a-(epoxy-methanol)-lH,6aH-cyclopenta(c)furo-(2,3-b)furo(3’,2’ : 3,4)cyclopenta(l,2-d)furan-5,9,12(4H)-trione) was from Dr. P. Braquet, Institute Henri Beaufour (France); WEB 2170 (6-(2-chlorophenyl)-8,9-dihydro-l-methy1-8-(4-morpholinyl-carbony1)-4H,7H-cyclopenta1(4,5)t~en~3,2-~(1, 2,4)-triazolo-(4,3-a)( 1,4)-diazepine and WEB 2086 (3-(4-(2-chlo:opheny1)-9-methy1-6H-thieno2,2-f-(1,2,4)triazolo(4,3-aK1,4)-diazepin-2-y1-1-(4-morpholinyl)-lpropanone) were provided by Dr. H. Heuer. Boehringer-Ingrlhcim (F.R.G.). 2.7. Statistical analysis The data were analysed statistically by means of the Newman-Keuls-Student test. P values of 0.05 or less were considerd signifii,dr;t.

3. Results 3.1. Exudation and cellular alterations induced bJ*antigen As illustrated in fig. 1, the i.t. injection of ovalbumin (12 pg/cavity) into sensitized rats induced an intense

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1200

200

t .900

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3 -600 -500

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Fig. 1. Kinetics of the leakage of Evans blue dye and exudate volume ah#er antigen stimulation (ovalbumin, 12 /.rg/cavity) in sensitized animals. Four distinct time periods, including the first 10 min. the interval ranging from 10 to 30 min. from 30 to 60 mitt and from 60 to 240 min. were analysed. Each point represents the mean f S.E.M. from at least six animals; * statistically significant as compared to the respective sham-sensitized group.

4

-lo 24

72 TIME

4

120 (h)

Fig. 2. Kinetics of the pleural infiltration of total leucocytes and mononuclear cells (A) and of neutrophils and eosinophils (B) after intrathoracic injection of ovalbumin (12 rg/cavity) into sensrtized (closed symbols) and sham-sensitized (open symbols) animals. Each point represents the mean + S.E.M. from at least six animals; l statistically significant as compared to the respective sham-sensitized group.

and early leakage of plasma proteins, as evaluated by the Jye content in the pleura1 fluid, which peaked within 10 min and decayed drastically thereafter. This rapid increase in vasopermeation was followed by a marked accumulation of liquid in the pleural cavity. Vasopermeation was apparent within 10 min. peaked from 1 to 4 h and declined to undetectable levels within 48 h. Figure 2 shows the kinetics of the change in leucocyte count after injection of antigen. There was a marked increase in the total leucocyte count 4 h after ovalbumin (fig. 2A). when a predominant influx of neutrophils (fig. 2B) and a smaller but significant augmentation in the mononnr!ear ce!l count (fig. 2A) were noted. The pleural leucocyte accumulation peaked within 24 h, but the neutrophil population was smaller than before and disappeared 48 h later. As indicated in fig. 2B, the reduction in the neutrophil count occurred in parallel to a long-lasting accumulation of eosinophils. which was apparent at 24 h and remained elevated until 96 h post challenge. In both fig. 2A and B, the control values, indicated as open symbols, were obtained from non-sensitized ovalbumin-injected animals in each analysed period (see legend fig. 2). These values were not different from the basal values (normal resident population) nor from those of sensitized saline-injected animals.

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Fts. .3 it;terfs~en~~ af <~r.tlaJmimatr.tti0n of azclastitte t I-10 mg/kg) with Fvans blue :e&rge (A) and oedema t B) evaluated 10 mitt af:er ,~vatbumm 412 pp c.w~r>)challenge. Columns reprrscnt t’he mean ? S.E.XI. fn~m at least ai\ animals; * ztatistically significant as compurredt,> the msptitivr

sham-sensitized group.

Oral treatment with azelastine. 2 h before the challenge. dose dependently inhibited both the plasma leakage (fig. 3A) and the pleural oedema (fig. 3B) observed within 10 min. The anti-exudatory effect of azelastine was apparent in doses ranging from 1 to 10 mg/kg. with ED,, values of 4.2 mg/kg for vasopermeation and of 6.8 mg/kg for oedema. As indicated in fig. 4 , azelastine (10 mg/kg) also reduced the accumulation of pleural liquid obsened 1 and 4 h after antigen. In contrast. the increase in the total leucocyte count noted at 4 h (fig. 5A) and the eosinophil accumulation observed 24 h after the challenge (fig. 5B) were refractory to azelastine. The interference of azelastine with the pleurisy induced by PAF-acether was analysed 6 h after injection of the iipid. when. as reported (Martins et al., 1989a). exudation and leucoeyte infiltration are observed. As illustrated in fig. 6A. azelastine inhibited exudation and

w J---M+0

1

4

TIME (h) Fig. 4. Interference of oral administration of azelastine (10 mg/kg) (closed symbols) or its vehicle (open symbols) with exudation induced by ova!bumin (12 pg/cavtty) at 1 and 4 h after its iqection. Each point represents the mean * S.E.‘M. from at least six animals: l strtis tically significant as compared to the respective sham-sensitized group,

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the increase in !he number of mononuclear cells (fig. 6B). neutrophils (fig. 6C) and eosinophils (fig. 6D) induced by PAF-acether (1 pg/cavity). Azelastine (10 mg/kg) was also effective against pleurisy induced by histamine (200 f.tg/cavity) and serotonin (100 $g/cavity) (fig. 7). Exudation was anaiysed 1 h after the it. injection of the agonists. It is noteworthy that, when administered topically, azeiastine (1 to 50 pg/cavity). unlike cyproheptadine. failed to inhibit exudation induced by histamine, serotonin or antigen (fig. 8).

Fig. 6. Interference of azelastine with the pleurisy induced by PAFacether (1 pg/cavity). Exudation (A) and the incredse in the number of mononuclear cells (B). neutrophils (C) and eosinophils (D) were analysed 6 h after administration of the lipid. White and black columns represent vehicle- and PAF-acether-stimulated animals. respectively. Azelastine. 0.5 mg/kg (cross-hatched columns), 1.0 ntg/kg (left diagonal hatched columns) or 5.0 mg/kg (right diagonal columns) was orally administered 2 h before the agonist. Coiumns represent the tneatt * S.E.M. from at least six animals: * statistically significant as compared to the saline-stimulated control group.

205 1200 h -

TABLE 1 I

Interference of PAF-acether antagonists with the rat allergic pIruns? induced by ovalbtrmin (OVA: 12 pg/cavity). Each value represents the mean + S.E.M. from at least six animals.

Fig. 7. Interference of oral administration of azelastine (10 mg/kg) (hatched columns) or its vehicle (white columns) with exudation induced by intrathoracic injection of histamine (200 &cavity) or serotonin (100 &cavity). Columns represent the mean*S.tM. from at least six animals; * statistically significant as compared to the saline-treated control group.

3.3. Interference

of potential

inhibitors

Stimulus

Treatment (20 mg/kg)

Exudation (cl)

Total leucocytes (lO-h/cavity)

OVA = OVA ’ OVA * OVAb OVAh

None None BN 52021 WEB 2086 WEB 2170

o+ 0 890?70’ 827 ?r 41 800* 0 760f80

6.2?0.6 28.2 f 3.2 ’ 33.1 f 2.2 24.4 f 3.9 25.754.5

’ Stimulation performed in sham-sensitized rats. h Stimulation performed in sensitized rats. c‘P -z 0.001 compared to OVAa group.

TABLE 2 Interference of meclizine (MEC) and cyproheptadine (CH) with the allergic pleurisy induced by ovalbumin (OVA; 12 pg/cavity). Fach value represents the mean i S.E.M. from at least six animals.

with antigen-in-

duced rat pleurisy

Three distinct PAF-acether receptor antagonists were clearly effective against pleurisy induced by PAF-acether itself (1 pg/cavity). When administered 1 h before the agonist, BN 52021, WEB 2086 and WEB 2170 (20 mg,/kg) reduced exudation from 840 f 29 p! (mean f S.E.M.) to 273 i 21 pl (P < O.OOl), 306 f 48 ~1 (P < 0.001) and 273 f 31 pl (P < O.OOl), respectively. Nevertheless, the=. drugs failed to modify the exudation and the leuwyte infiltration elicited by antigen (table 1). As indicated in table 2, the histamine H, receptor antagonist meclizine (30 mg/kg) and the dual histamine and serotonin antagonist cyproheptadine (2 mg/kg) inhibited exudation elicited by antigen. As with azelastine, these drugs failed to interfere with the cellular influx triggered by ovalbumin (table 2). Table 3 s1;2r%the effects of both the dual cyclooxygenase and lipoxygenase inhibitor BW 755C (Salmon et al., 1983) (50 mg/kg) and the leukotriene D4 antagonist LY 171883 (Fleisch et al., 1985) (10 pg/cavity) in pleurisy triggered

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Stimulus

Treatment

Exudation (Pi)

Total leucocytes (lo-*/cavity)

OVA = OVA ’ OVA *

None None MEC (30 mg/kg) CH (2 mg/kg)

o+ 0 950;50 c 390+58d 280+40 d

5.3f0.9 26.4 7 2.7 c 29.1 f 3.9 22.7 + 2.5

OVAh

p Stimulation performed in sham-sensitized rats. b Stimulation performed in sensitized rats. ’ P -z i_i.li(ii compared to C&A” group. ’ P c 0.00: compared to OVA h untreated group.

TABLE 3 Interference of BW 755C and LY 171883 with the rat allergic pleurisy induced by ovalbumin (12 fig/cavity). Each value represents the mean rt S.E.M. from at least six animals. The analysis was wrfnrmed 4 h after the antigen challenge. Drug

‘Rinhibition Exudation

Cell influx 30.15 5.6 =

BW 755C

50 mg/k

40.0 f 7.0 p

LY 171883

16 &cavity

36.4 f 8.2 *

5.8k4.0

Ii P-=0.001.

04 : : : : : 010

Dose

so 5a WSE (W/e&)

0010

so 50 ~0% cw/cavity)

Fig. 8. Interference of topical treatment of either azelastine (lo-50 pg/cavity) (closed symbols) or cyproheptadine (5 and 50 Pg/cavitY) (open symbols) with exudation induced by histamine (200 pg/cavity) (A) serotonin (100 pg/cavity) (B) and ovalbumin 112 !%/cavltY) (‘1. Part (A) and (B) sho\r values from non-sensitized animals and part (C) values from sensitized animals. Each point represent the mean f S.E.M. from at least six animals; l statistically significant as compared to the saline-treated control group.

b\ antigen. BW75SC inhibited exudation and the celluu\. whereas the latter was effective only against eriudatinn.

The present study was undertaken to verify the potential interference of the anti-allergic azelastine agdinst pleurisy induced by antigen and also to analyse. in terms of this esperimental model, the relative importance of the already reported activity of azelastine against histamine (Kaiayama et al.. 1981). serotonin (Chand et ai.. 1985b) and PAF-acether (AchterrathTuckermann et al.. 19883. Our findings indicate that intense vasopermeation. attested by the leakage of dye-labelled plasma proteins into the pleural cavity. occurs within 10 min after antigen provocation and decays drastically thereafter. Pleural dcma was already noted at 10 min. but peaked only in the period from 1 to 4 h and faded 48 h later. Antigen also induced a long-lasting increase in the number of Ieucocytes recovered from the pleural fluid. This effect was apparent 4 h after antigen challenge. when a predominant influx of neutrophiis and a smailer but significant influx of mononuclear cells were noted. The number of eosinophils increased within 24 h and remained elevated until 96 h post challenge. Active Skrgk plcilrisyin rats is thus characterized by marked exudation. followed by two consecutive waves of polymorphonuclear hucocyte influx: neutrophils followed by eosinophils. both of which accumulate in the pleural cavity. In addition, a smaller but long-lasting increase in the number of mononuclear cells was noted in the period from 4 to 96 h. This model was used to study the interference of the phthalazinone derivative azelsstine with the allergic reaction. Oral administration of azelastine dose dependently inhibited both the plasma extravasation (ED,, = 4.2 mgikg) and the pleural oedema (ED,, = 6.8 mg/kg) detected 10 min after antigen provocation. Azelastine was also effective against the pleural oedema detected 1 and 4 h later, which shows that azelastine has potent anti-exudatoty activity. In contrast, azelastine failed to interfere with the pleural cellular influx triggered by antigen. The drug was inactive against the early (4 h) neutrophilia and also against the late (24 h) eosinophilia. The results indicated that. at least in this particular model. the anti-exudatory activity of azelastine nqy not be accounted for by interference with leucocyte infiltration. It has been reported previously that i.t. injection of PAF-acether into rats also causes early exudation, followed within 6 h by pleural accumulation of leucocytes (Tarayre et al.. 1986; Martins et at., 1989a); this accumulation is accounted for by a significant increase in

the number of mononuclear cells. neutrophils and eosinophils (Martins et al.. 1989a.b; Silva et al.. 1989). PAF-acether antagonists and auto-desensitization to this lipid inhibit both exudation and pleural leucocyte accumulation induced by zymosan, which suggests that PAF-acether is involved in this inflammatory reaction (Martins et al., 1989a,b). Azelastine was clearly effective against pleurisy induced by exogenous PAF-acether. inhibiting exudation and the increase in the number of mononuclear cellsi neutrophi!s and eosinophils. Xevertheless. the failure of three distinct PAF-acether antagonists to inhibit antigen-induced pleurisy, including the acute exudation and delayed cell migration, shows clearly that the allergic pleurisy in single (nonboosted) actively sensitized rats is PAF-acether-independent. Thus the ability of azelastine to antagonize PAF-acether is not relevant to this particular model of allergic pleurisy. Azelastine is claimed to have a large spectrum of pharmacological activities, including the inhibition of synthesis and/or release of mediators such as leukotrienes C,/D, (Katayama et al., 1987) and histamine (Katayama et al., 1981; Chand et al.. 1985a). In addition, azelastine directly antagonizes different autacoids inciuding histamine, serotonin and leukotrienes C,/D, (Chand et al., 1985b; Katayama et al., 1987). Our study confirms the antagonism against biogenic amines, as oral treatment with azelastine inhibited the rat pleurisy induced by either histamine or serotonin. Since the exudation induced by allergen was inhibited by antihistamine and anti-serotonin drugs, it depends on the local release of these vasoactive amines. Like azelastine, these drugs blocked the antigen-induced exudation without modifying the leucocyte infiltration. This suggests that, in this model, azelastine and biogenic amine receptor antagonists behave similarly. We showed that the exudation evoked by antigen was, although to a lesser extent, also sensitive to both the dual cyclooxygenase and lipoxygenase inhibitor BW 755C and to the leukotriene D4 receptor antagonist LY 171883 under conditions where the leucocyte influx was only weakly inhibited by the former. Therefore, in rat allergic pleurisy, azelastine is probably effective because of its strong anti-histamine and anti-serotonin effects, as confirmed in this study, and also probably because of its ability to inhibit the synthesis/secretion of mediators including vasoactive amines and leukotrienes (Chand et al., 1989). Nevertheless, these effects cannot explain oiher anti-inflammatory properties of azelastine including, for instance, its ability to interfere with allergic bronchial eosinophilia in guinea pigs (Chand et al., 1990) or with PAF-acether-induced rat pleural eosinophilia (Vargaftig, 1990), which is clearly refractory to histamine and serotonin antagonists and also to lipoxygenase inhibitors (data not shown). Thus, we cannot exclude that azelastine interferes with the cellular

207

influx into the inflammatory focus when PAF-acether is implicated in the process. Finally, it is noteworthy that locally administered azelastine failed to interfere with the pleurisy induced by histamine, serotonin or antigen under conditions where cyproheptadine was effective. Topical treatment with azelastine also failed to interfere with the rat pleurisy induced by PAF-acether (data not shown), which raises the possibility that the effects of azelastine may depend on intermediate metabolite(s). Our findings indicate that azelastine interferes with the exudatory and cellular effects of PAF-acether and with the exudation elicited by antigen that is suppressed by anti-histamine and anti-serotonin agents, but not by PAF-acether antagonists. It is thus likely that azelastine inhibits antigen-induced exudation because of its antiamine effects and that its ability to interfere with PAFacether is not relevant to this particular model.

Acknowledgements The authors thank Dr. P. Braquet, I.H.B. Research Labs. (France) for BN 52021. Dr. H. Heuer. Boehringer-Ingelheim (FRG) for WEB 2086 and WEB 2170 and Dr. S. Moncada, Wellcome Research Laboratories (Beckenham. UK) for BW 755C. Tinis study was supported by grants from CNPq, FlNEP and FAPERJ (Brazil).

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