European Journal of Pharmacology, 213 (1992) 183-188 © 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
183
EJP 52368
Interference of azelastine with anaphylaxis induced by ovalbumin challenge in actively sensitized rats H u g o C. C a s t r o - F a r i a - N e t o , Patrlcia T. B o z z a , A d r i a n a R. Silva, P a t r l c i a M . R . Silva, Mfircia C.R. Lima, M a r c o A. Martins, R e n a t o S.B. C o r d e i r o and B. Boris V a r g a f t i g a Departamento de Fisiologia e Farmacodindmica, Fundaqdo Oswaldo Cruz, Avenida Brasil 4365, Caixa Postal 926, Rio de Janeiro, Brazil and ~ Unit~ de Pharmacologie Cellulaire, Unitd Associde lnstitut Pasteur INSERM 285, 25 Rue Dr. Roux, Paris 75015, France Received 5 September 1991, revised MS received 18 December 1991, accepted 7 January 1992
The inhibition of the haematological alterations and prevention of death due to systemic anaphylaxis after antigen challenge were investigated in rats after various drug treatments. The i.v. injection of ovalbumin (250/zg/kg) into actively sensitized rats induced marked thrombocytopenia and haemoconcentration within 5 min and significant leukocytosis within 30 min, lasting for 2 h after the challenge. Pretreatment with meclizine or terfenadine (15-30 mg/kg i.p.) inhibited antigen-induced haemoconcentration, whereas WEB 2086 (2-10 mg/kg i.p.) and PCA 4248 (5-10 mg/kg p.o.), two platelet-activating factor (PAF) antagonists, interfered with thrombocytopenia only. Azelastine (1-20 mg/kg p.o.) dose dependently inhibited antigen-induced haemoconcentration and thrombocytopenia but failed to block leukocytosis. Azelastine also inhibited the thrombocytopenia observed after the i.v. administration of PAF (4 /zg/kg). Administration of ovalbumin at a dose of 1.5 mg/kg resulted in a lethal anaphylactic reaction in about 85% of the rats. Pretreatment with WEB 2086 (10 mg/kg i.p.), meclizine (30 mg/kg i.p.) or both increased the survival rate from 15 to 57, 68 and 87%, respectively. Azelastine alone (20 mg/kg p.o.) completely blocked the lethal reaction. It was concluded that the ability of azelastine to antagonize histamine and PAF is important for its effectiveness against anaphylactic shock. Anaphylaxis; Haematological alterations; Death; Azelastine
1. Introduction
The phthalazinone derivative, azelastine, is a newly developed anti-allergic drug which potently inhibits the allergic and non-allergic release of histamine and leukotrienes from mast cells and leukocytes (Katayama et al., 1987; Kurosawa, 1989). In addition, azelastine blocks the effects of histamine and leukotrienes in vitro and in vivo (Chand et al., 1986a, b) and also the effects of platelet-activating factor (PAF) (Achterrath-Tuckermann et al., 1988). We recently reported that azelastine inhibits ovalbumin-induced plasma leakage and mast cell degranulation in actively sensitized rats (Lima et al., 1991). Furthermore, this drug also inhibits exercise-induced bronchoconstriction (Motojima et al., 1989) and in vitro acute lung anaphylaxis (Chand et al., 1987). One recent clinical assay with azelastine has
Correspondence to: M.A. Martins, Departamento de Fisiologia e Farmacodin~mica, F u n d a ~ o Oswaldo Cruz, Avenida Brasil 4365, Manguinhos CEP 20010, Caixa Postal 926, Rio de Janeiro, RJ, Brazil.
shown its efficacy and safety in the treatment of asthmatic subjects (Mue et al., 1989). On the basis of these broad pharmacological activities, the question arose as to whether azelastine interferes with systemic anaphylaxis. In the present study we compared its effects with those of anti-histamines and PAF antagonists on the haematological alterations and death following systemic anaphylaxis in actively sensitized rats.
2. Materials and methods
2.1. Sensitization procedure Male Wistar rats weighing 150-180 g were sensitized by a s.c. injection (0.2 ml) of a mixture containing 5 0 / z g of ovalbumin and 5 mg of aluminium hydroxide. Fourteen days later the animals, under light ether anaesthesia, were challenged with an i.v. injection of the antigen (ovalbumin at 250 /zg/kg) through the penial vein. At that time, immunoglobulin (Ig) E titres were 1 : 20 and no IgG levels could be detected using
184
the passive cutaneous anaphylaxis technique (Brocklehurst, 1967).
2.2. Haematological analysis Blood obtained from the tail vein several times after the i.v. injection of PAF (4 /xg/kg) or antigen was diluted with buffered EDTA/formalin solution (1 200 x ) or with 2% acetic acid solution (40 × ) for platelet and leukocyte counts, respectively. The counts were performed in a Neubauer chamber under light microscopy. A buffered EDTA/formalin solution was prepared by adding 3 ml of EDTA 0.077 M, 2 ml of 10 x concentrated phosphate-buffered saline solution (PBS), 150/zl of 40% formalin and distilled water to a final volume of 20 ml. The haematocrit was determined in heparinized microtubes (Inlab) centrifuged for 8 min in a microcentrifuge (Fanem, model 211).
2.3. Treatments WEB 2086 (2-10 mg/kg) (Casal-Stenzel et al., 1986), meclizine (15-30 mg/kg) and terfenadine (15-30 mg/kg) were given by the i.p. route, 1 h before the challenge. The PAF antagonist, PCA 4248 (5-10 mg/kg) (Fernandez-Gallardo et al., 1990), and azelastine (1, 10 and 20 mg/kg) were administered p.o. 2 h before the challenge to animals fasted for 12 h.
3. Results
3.1. Evaluation of antigen-induced acute haematological alterations As shown in fig. 1A, the i.v. injection of ovalbumin (250/xg/kg) into actively sensitized rats induced leukocytosis from 30 min up to 2 h, returning to basal levels after 24 h. This phenomenon was mainly due to an increase in blood neutrophil counts, while monocyte, lymphocyte and eosinophil numbers were not significantly altered (table 1). Figure 1 also shows that 5 min after the antigen, platelet counts decreased by 40% (fig. 1C), while the haematocrit values were signifi-
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2.4. Antigen-induced death
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Ovalbumin, 1.5 mg/kg, was administered i.v. as a single bolus injection. Meclizine (30 mg/kg) or WEB 2086 (20 mg/kg) were given i.p. and azelastine (20 mg/kg) was given p.o. 1 h and 2 h before the challenge, respectively. Survival rate was recorded 24 h after the administration of ovalbumin.
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4.5.
40 10.
2.5. Drugs 0
The drugs were obtained from the following sources: meclizine (Pfizer); terfenadine (Teldane-Merrel Lepetit); PCA 4248 ((2-phenylthioethyl)-5-methoxycarbonyl-2,4,6-trimethyl-1,4-dihydropyridine-3-carboxylate) was from Alter S.A.; WEB 2086 (3-4-(2-chlorophenyl-9-methyl-6H-tieno-3,2-f-l,2,4-triazolo-4,3-1,4-diazepin-2yl-l-(4-morpholinyl)-l-propanone) was from Boehringer Ingelheim; azelastine was kindly donated by Dr. J. Roux (Laboratories Sarget, France).
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2.6. Statistical analysis The data were analysed statistically by means of Student's t-test for unpaired samples; P values of 0.05 or less were considered significant.
Fig. 1. Kinetics of changes in blood leukocyte numbers (A); haematocrit (B); and platelet numbers (C) induced by the i.v. administration of ovalbumin (250 /~g/kg) to actively sensitized rats. Closed circles represent the control values for each haematological parameter. The results are the m e a n s + S.E.M. from six to eight animals. Statistically significant differences are indicated by an asterisk.
185 30.
TABLE 1 Differential leukocyte count alterations induced by the i.v. injection of ovalbumin (Ova 250 /xg/kg) into actively sensitized rats. Blood samples were obtained 1 h after the challenge. Saline (Sal)-injected animals were used as controls. Each value represents the mean_+ S.E.M. from at least six animals
~e,,.
5.6_+0.6 5.9±0.8
4.1 ±2.0 8.3-+2.1
1.2-+0.3 11.2+1.2 a
20.
X I
II
15. 10.
v
Lymphocytes Monocytes Neutrophils Eosinophils ( × 1 0 3 / m m 3) ( × 1 0 2 / m m 3) ( × 1 0 3 / m m 3) ( × 1 0 ] / m m 3) Sal Ova
A
m'E E 25.
3.0±2.0 1.0± 1.0
" Statistics significant difference (P < 0.01).
B
41
++
cantly increased (fig. 1B). Both values returned to their basal levels after 2 h.
3.2. Inhibition by azelastine of antigen- and PAF-induced thrombocytopenia and haemoconcentration
4O
Azelastine (1-20 mg/kg) dose dependently inhibited antigen-induced thrombocytopenia and haemoconcentration, under conditions in which leukocytosis was not significantly affected (fig. 2). Azelastine also blocked the thrombocytopenia observed 1 h after the i.v. administration of PAF (4/zg/kg) (fig. 3).
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3.3. Interference of histamine and PAF antagonists with haematological alterations induced by the antigen
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Table 2 shows that the pretreatment with two different H 1 antagonists, meclizine and terfenadine, at doses of 15 and 30 mg/kg, inhibited only the antigen-induced haemoconcentration without interfering with thrombocytopenia and leukocytosis. In contrast, the PAF antagonists, WEB 2086 (2-10 mg/kg) and PCA 4248 (5-10 mg/kg), inhibited antigen-induced thrombocytopenia but failed to modify leukocytosis and haemoconcentration (table 3).
10
20
DOSE (mglkg)
Fig. 2. Interference of azelastine with ovalbumin (250/xg/kg i.v.)-induced changes in blood leukocyte counts (A); haematocrit (B); and platelet numbers (C). Saline-injected animals were used as controls (closed circle). The results are the means±S.E.M, from six to eight animals and statistically significant differences from the untreated group are indicated by an asterisk.
10
~E E
TABLE 2 Interference of meclizine (Mec) and terfenadine (Ter) with the haematological alterations induced by ovalbumin (Ova 250 tzg/kg) in actively sensitized rats. Saline (SaD-stimulated rats were used as controls. Each value represents the mean + S.E.M. from six to eight animals Treatment Stimulus Leukocytes Platelets Haematocrit (mg/kg) (X 103/mm 3) ( × 105/mm 3) (%) None None Mec 15 Mec 30 Ter 15 Ter30 a
Sal Ova Ova Ova Ova Ova
9.5 ± 0.6 18.3 ± 0.9 21.7±1.0 17.2 ± 1.2 18.8±0.6 19.0_+2.6
8.0 + 0.03 4.5 ± 0.03 4.1±0.03 4.8 ± 0.03 5.9_+0.07 5.5_+0.04
46.8 ± 1.2 60.8 ± 1.4 56.0±1.2 54.1 -+ 1.5 a 52.8_+0.8a 51.8_+1.1a
Statistically significant difference (P < 0.05) compared to untreated group stimulated with Ova.
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CONT
PAF
PAF +
AZE Fig. 3. Interference of azelastine (20 mg/kg, hatched column) with PAF (4/~g/kg i.v., black column)-induced thrombocytopenia. Platelet counts were performed 1 h after the i.v. injection of PAF. Saline-injected animals were used as controls (white column). Each bar represents the mean+ S.E.M. from six animals and statistically significant differences are indicated by an asterisk.
186 TABLE 3
TABLE 4
Interference of PAF antagonists with the haematological alterations induced by ovalbumin (Ova 250/~g/kg). Saline (Sal)-stimulated rats were used as controls. Each value represents the mean _+S.E.M. from at least six animals
Analysis of the haematological parameters 24 h after challenge with a lethal dose of ovalbumin (Ova 1.5 mg/kg), in azelastine (20 mg/kg)-treated rats. A saline (SaD-stimulated group was taken as control. Each value is the mean-+ S.E.M. from at least six animals
Treatment Stimulus Leukocytes Platelets Haematocrit (mg/kg) ( × 1 0 3 / m m 3) ( × 1 0 5 / m m 3) (%)
Treatment Stimulus Leukocytes Platelets Haematocrit ( x l 0 3 / m m 3) ( x l 0 5 / m m 3) (%)
None None WEB 2086 2 10 PCA4248 5 10 20
None Sal Azelastine Ova
Sal Ova
9.4_+0.5 21.0_+1.1
7.9_+0.02 4.5_+0.02
46.2_+0.7 57.6_+1.0
Ova Ova
20.5_+2.8 19.6±2.0
8.0±0.13 a 8.3_+0.02a
54.7_+2.7
Ova Ova Ova
22.2_+1.9 21.1±1.9 19.9±1.5
6.8_+0.04a 7.0_+0.04~ 7.5_+0.06a
60.6_+0.7 55.0±1.1 62.6_+1.4
9.8+0.7 10.7_+0.5
7.7_+0.02 7.6-+0.03
50.2+1.9 49.3+0.8
ters including platelet and total leukocyte counts, as well as the haematocrit levels, from the azelastinetreated group were not different from those of unchallenged animals (table 4).
a Statistically significant difference (P < 0.01) compared to untreated group stimulated with Ova.
4. Discussion
3.4. Effect of drugs on antigen-induced death The i.v. injection of ovalbumin at a dose of 1.5 mg/kg induced lethal shock in about 85% of the untreated rats. It is noteworthy that 61% of the deaths occurred within 1 h, 32% between 1 and 4 h and 7% between 4 and 24 h. Meclizine (30 mg/kg), WEB 2086 (20 mg/kg) and azelastine (20 mg/kg) were tested against this lethal reaction. As shown in fig. 4, the survival rate analysed 24 h after the antigenic challenge was shown to be 58% in rats receiving WEB 2086 and 64% in meclizine-treated animals, under conditions where the WEB 2086/meclizine combination increased the survival rate to 87%. The same figure shows that azelastine completely impaired the lethal reaction in this period, a protective effect that persisted for at least 7 days (data not shown). It was noted that 24 h after challenge, the haematological parame-
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(9) (9)
~ 60_J ~ 40~
20-
(23)
[--1 CONT
WEB
MEC
MEC +
AZE
WEB
Fig. 4. Interference of WEB 2086 20 mg/kg (WEB); meclizine 30 mg/kg (MEC); the combination of WEB 2086 20 mg/kg with meclizine 30 mg/kg (MEC+WEB); and of azelastine 20 mg/kg (AZE) with ovalbumin (1.5 mg/kg)-induced lethality. Control group (CONT), unprotected animals injected with ovalbumin. Survival rate was recorded 24 h after the challenge. The number of animals used is indicated in parentheses.
It has been previously reported that rats actively sensitized with a mixture of aluminium hydroxide and ovalbumin develop a local immediate hypersensibility response following the intrathoracic injection of ovalbumin 14 days after the initial sensitization (Lima et al., 1990). This reaction was characterized by an intense increase in vascular permeability and pleural oedema which was blocked by histamine antagonists or by azelastine (Lima et al., 1991). In the present study we demonstrated that the i.v. injection of a sub-lethal dose of ovalbumin (250 ~g/kg) into actively sensitized rats induces a systemic anaphylactic reaction leading to neutrophilia, haemoconcentration and thrombocytopenia. Furthermore, lethal anaphylaxis was observed after challenge with high doses of the antigen. This model was used to investigate the interference of the anti-allergic drug, azelastine, with the haematological changes and death observed during systemic anaphylaxis. The oral administration of azelastine dose dependently inhibited both the antigen-induced haemoconcentration and thrombocytopenia without affecting the rise in blood leukocyte counts. It has been reported that azelastine inhibits IgE-mediated histamine release from rat mast ceils (Chand et al., 1985). In addition, azelastine also directly antagonizes the effects of this amine in a number of models in vitro and in vivo (Katayama et al., 1981; Lima et al., 1991). Histamine was long been recognized as an important mediator of anaphylaxis (Dale, 1929), and the effect of azelastine in our model might be attributed to its anti-histamine property. Nevertheless, while two H~ antagonists, meclizine and terfenadine, were effective against antigen-induced haemoconcentration, they failed to inhibit thrombocytopenia, contrary to azelastine. Histamine is thus involved only in haemoconcentration and, accordingly, azelastine operates via histamine-independent
187
mechanisms when inhibiting antigen-induced thrombocytopenia. Although rat platelets are refractory to in vitro stimulation with PAF (Ifiarrea et al., 1984), we reported that the i.v. injection of this lipid into rats induces a marked thrombocytopenia, which is blocked by the PAF antagonists, WEB 2086 and BN 52021 (Martins et al., 1988). In the experiments described here, the oral administration of azelastine also completely inhibited PAF-induced thrombocytopenia in rats. This finding is in agreement with those of Achterrath-Tuckermann et al. (1988) and Lima et al. (1991) who demonstrated that azelastine potently inhibits PAF-induced platelet aggregation, bronchoconstriction and pleurisy, indicating that it interferes with the effects of PAF. In the present study we also showed that the structurally unrelated PAF antagonists, PCA 4248 and WEB 2086, blocked antigen-induced thrombocytopenia, suggesting that PAF may play a role in this phenomenon. Together our results indicate that the inhibition of antigen-induced thrombocytopenia by azelastine may involve its ability to interfere with PAF. Nevertheless, since azelastine also induces calcium channel block (Chand et al., 1983) and raises intracellular cyclic AMP levels (Katayama et al., 1987), an unspecific inhibitory effect on platelet aggregation in general is also possible. It is of interest to note that antigen-induced leukocytosis was accounted for mainly by an increase in blood neutrophil counts. This alteration was accompanied by a concomitant decrease in the number of bone marrow nucleated cells (not shown), suggesting that the pool of marrow neutrophils was mobilized to enter the circulation after the antigenic challenge. An identical pattern of response was observed by Bozza et al. (in press) after the i.v. administration of PAF to rats. In this case, both blood neutrophilia and the decrease in bone marrow cell counts were blocked by the pretreatment with PAF antagonists as well as by general anaesthesia (Bozza et al., in press). The similarity between the leukocyte count alterations induced by PAF and those induced by antigen suggests that this lipid may mediate the leukocytosis elicited by the antigen. Yet, as PAF antagonists and anesthetics (not shown) failed to inhibit antigen-induced leukocytosis, these mechanisms are indeed distinct. Interleukin-1 and tumor necrosis factor a were also shown to induce acute blood neutrophilia concomitant with a reduction of bone marrow cell counts in rats (Ulich et al., 1987). The potential involvement of these cytokines in antigen-induced leukocytosis is now under investigation. The administration of ovalbumin at a dose of 1.5 m g / k g into actively sensitized rats induced death in approximately 85% of the animals, which was prevented by azelastine and by the combination of WEB 2086 and meclizine. In contrast, lethality was only
partially reduced by either WEB 2086 or meclizine alone. These results agree with those obtained using passively sensitized guinea pigs, in which the association of an anti-histamine and WEB 2086 inhibited the lethal anaphylactic reaction (Casals-Stenzel, 1987). We do not yet know the major cause of death from systemic anaphylaxis in the IgE rat model. Whatever the cause, our results indicate that synergism between PAF and histamine should be involved. From this point of view, the combined anti-histamine and anti-PAF properties of azelastine might explain its remarkable effect against anaphylactic mortality. Nevertheless, other pharmacological properties of azelastine, such as the inhibition of leukotriene synthesis and effects (Katay~ma et al., 1987), cannot be excluded and deserve further study. To summarize and conclude, our findings indicate that azelastine blocks antigen-induced haemoconcentration, thrombocytopenia and death, probably due to its combined effects against histamine and PAF. These properties may implicate azelastine as a potential candidate for the therapy of systemic anaphylaxis. New assays are now underway in order to investigate this possibility.
References Achterrath-Tuckermann, U., C.H. Weisher and I. Szelenyi, 1988, Azelastine, a new antiallergic/antiasthmatic agent, inhibits PAF-acether induced platelet aggregation, paw oedema and bronchoconstriction, Pharmacology 36, 265. Bozza, P.T., P.M.R. Silva, H.C. Castro-Faria-Neto, M.A. Martins and R.S.B. Cordeiro, Increase in the rat blood leukocyte counts induced by PAF-acether is suppressed by general anaesthesia, J. Leukocyte Biol. (in press). Brocklehurst, W.E., 1967, Passive cutaneous anaphylaxis, in: Handbook of Experimental Immunology, ed. D.M. Weir (Science Publications, Oxford) p. 745. Casals-Stenzel, J., 1987, Effects of WEB 2086, a novel antagonist of platelet activating factor, in active and passive anaphylaxis, Immunopharmacology 13, 117. Casals-Stenzel, J., G. Muacevic and K.H. Weber, 1986, WEB 2086 a new and specific antagonist of platelet activating factor (PAF), Arch. Pharmacol. 334 (Suppl.), R44. Chand, N., J. Pillar, W. Diamantis, J.L. Perhach and R.D. Sofia, 1983, Inhibition of calcium ionophore (A23187)-stimulated histamine release from rat peritoneal mast cell by azelastine: lmplications for its mode of action, Eur. J. Pharmacol. 96, 227. Chand, N., J. Pillar, W. Diamantis and R.D. Sofia, 1985, Inhibition of IgE-mediated allergic histamine release from rat peritoneal mast cell by azelastine and selected anti-allergic drugs, Agents Actions 16, 318. Chand, N., W. Diamantis and R.D. Sofia, 1986a, Modulation of in vitro anaphylaxis of guinea-pig isolated tracheal segments by azelastine, inhibitors of arachidonic acid metabolism and selected antiallergic drugs, Br. J. Pharmacol. 87, 443. Chand, N., K. Nolan, W. Diamantis, J.L. Perhach, Jr. and R.D. Sofia, 1986b, Inhibition of leukotriene (SRS-A)-mediated acute lung anaphylaxis by azelastine in guinea-pigs, Allergy 41,473.
188 Chand, N., K. Nolan, W. Diamantis and R.D. Sofia, 1987, Inhibition of acute lung anaphylaxis by aerosolized azelastine in guinea-pigs sensitized by three different procedures, Ann. Allergy 58, 344. Dale, H.H., 1929, Croonian lectures on some chemical factors in the control of the circulation, Lancet 1, 1285. Fernandez-Gallardo, S., M.P. Oretega, J.G. Priego, M.F. Casa-Juana, C. Sunkel and M. Sanchez Crespo, 1990, Pharmacological actions of PCA 4248, a new platelet-activating factor receptor antagonist: in vivo studies, J. Pharmacol. Exp. Ther. 255, 34. Inarrea, P., J. Gomez-Cambronero, M. Nieto and M. Sanchez-Crespo, 1984, Characteristics of the binding of platelet-activating factor to platelets of different animal species, Eur. J. Pharmacol. 105, 309. Katayama, S., N. Akimoto, H. Shionoya, T. Morimoto and Y. Katoh, 1981, Anti-allergic effects of azelastine hydrochloride on immediate type hypersensitivity reactions in vivo and in vitro, Arzneim. Forsch. 31, 1196. Katayama, S., H. Tsunoda, Y. Sakuma, H. Kai, I. Tanaka and K. Katayama, 1987, Effect of azelastine on the release and action of leukotriene C4 and D4, Int. Arch. Allergy Appl. Immunol. 83, 284. Kurosawa, M., 1989, Inhibitory mechanism of chemical mediator release by azelastine, Drugs Today 25 (Suppl. 6), 9. Lima, M.C.R., M.A. Martins, S.A.C. Perez, J.M. Peixoto, P.M.R.
Silva and R.S.B. Cordeiro, 1990, Kinetics of pleural exudation and cellular alterations induced by antigen in actively sensitized rats, Braz. J. Med. Biol. Res. 23, 857. Lima, M.C.R., M.A. Martins, S.A.C. Perez, P.M.R. Silva, R.S.B. Cordeiro a n d B.B. Vargafitig, 1991, Effect of azelastine on platelet-activating factor and antigen-induced pleurisy in rats, Eur. J. Pharmacol. 197, 201. Martins, M.A., P.M.R. Silva, H.C. Castro-Faria-Neto, P.T. Bozza, P.M.F.L. Dias, R.S.B. Cordeiro and B.B. Vargaftig, 1988, Intravenous injections of PAF-acether induce platelet aggregation in rats, Eur. J. Pharmacol. 149, 89. Motojima, S., N. Ando, Y. Ohashi, T. Otsuka, T. Yamai, T. Fukuda and S. Makino, 1989, The effects of azelastine on allergen-, exercise- and LTD4-induced bronchoconstriction, Drugs Today 25 (Suppl. 6), 15. Mue, S., G. Tamura, K. Yamauchi, H. Ohtsu and T. Takishima, 1989, Efficacy and safety of azelastine in the treatment of asthmatics: Japanese experience, Drugs Today 25 (Suppl. 6), 29. Ulich, T.R., J. Castillo, M. Keys, G.A. Granger and R.-X. Ni, 1987, Kinetics and mechanisms of recombinant human interleukin 1 and tumor necrosis factor alpha-induced changes in circulating numbers of neutrophils and lymphocytes, J. Immunol. 139, 3406.
183
EJP 52368
Interference of azelastine with anaphylaxis induced by ovalbumin challenge in actively sensitized rats H u g o C. C a s t r o - F a r i a - N e t o , Patrlcia T. B o z z a , A d r i a n a R. Silva, P a t r l c i a M . R . Silva, Mfircia C.R. Lima, M a r c o A. Martins, R e n a t o S.B. C o r d e i r o and B. Boris V a r g a f t i g a Departamento de Fisiologia e Farmacodindmica, Fundaqdo Oswaldo Cruz, Avenida Brasil 4365, Caixa Postal 926, Rio de Janeiro, Brazil and ~ Unit~ de Pharmacologie Cellulaire, Unitd Associde lnstitut Pasteur INSERM 285, 25 Rue Dr. Roux, Paris 75015, France Received 5 September 1991, revised MS received 18 December 1991, accepted 7 January 1992
The inhibition of the haematological alterations and prevention of death due to systemic anaphylaxis after antigen challenge were investigated in rats after various drug treatments. The i.v. injection of ovalbumin (250/zg/kg) into actively sensitized rats induced marked thrombocytopenia and haemoconcentration within 5 min and significant leukocytosis within 30 min, lasting for 2 h after the challenge. Pretreatment with meclizine or terfenadine (15-30 mg/kg i.p.) inhibited antigen-induced haemoconcentration, whereas WEB 2086 (2-10 mg/kg i.p.) and PCA 4248 (5-10 mg/kg p.o.), two platelet-activating factor (PAF) antagonists, interfered with thrombocytopenia only. Azelastine (1-20 mg/kg p.o.) dose dependently inhibited antigen-induced haemoconcentration and thrombocytopenia but failed to block leukocytosis. Azelastine also inhibited the thrombocytopenia observed after the i.v. administration of PAF (4 /zg/kg). Administration of ovalbumin at a dose of 1.5 mg/kg resulted in a lethal anaphylactic reaction in about 85% of the rats. Pretreatment with WEB 2086 (10 mg/kg i.p.), meclizine (30 mg/kg i.p.) or both increased the survival rate from 15 to 57, 68 and 87%, respectively. Azelastine alone (20 mg/kg p.o.) completely blocked the lethal reaction. It was concluded that the ability of azelastine to antagonize histamine and PAF is important for its effectiveness against anaphylactic shock. Anaphylaxis; Haematological alterations; Death; Azelastine
1. Introduction
The phthalazinone derivative, azelastine, is a newly developed anti-allergic drug which potently inhibits the allergic and non-allergic release of histamine and leukotrienes from mast cells and leukocytes (Katayama et al., 1987; Kurosawa, 1989). In addition, azelastine blocks the effects of histamine and leukotrienes in vitro and in vivo (Chand et al., 1986a, b) and also the effects of platelet-activating factor (PAF) (Achterrath-Tuckermann et al., 1988). We recently reported that azelastine inhibits ovalbumin-induced plasma leakage and mast cell degranulation in actively sensitized rats (Lima et al., 1991). Furthermore, this drug also inhibits exercise-induced bronchoconstriction (Motojima et al., 1989) and in vitro acute lung anaphylaxis (Chand et al., 1987). One recent clinical assay with azelastine has
Correspondence to: M.A. Martins, Departamento de Fisiologia e Farmacodin~mica, F u n d a ~ o Oswaldo Cruz, Avenida Brasil 4365, Manguinhos CEP 20010, Caixa Postal 926, Rio de Janeiro, RJ, Brazil.
shown its efficacy and safety in the treatment of asthmatic subjects (Mue et al., 1989). On the basis of these broad pharmacological activities, the question arose as to whether azelastine interferes with systemic anaphylaxis. In the present study we compared its effects with those of anti-histamines and PAF antagonists on the haematological alterations and death following systemic anaphylaxis in actively sensitized rats.
2. Materials and methods
2.1. Sensitization procedure Male Wistar rats weighing 150-180 g were sensitized by a s.c. injection (0.2 ml) of a mixture containing 5 0 / z g of ovalbumin and 5 mg of aluminium hydroxide. Fourteen days later the animals, under light ether anaesthesia, were challenged with an i.v. injection of the antigen (ovalbumin at 250 /zg/kg) through the penial vein. At that time, immunoglobulin (Ig) E titres were 1 : 20 and no IgG levels could be detected using
184
the passive cutaneous anaphylaxis technique (Brocklehurst, 1967).
2.2. Haematological analysis Blood obtained from the tail vein several times after the i.v. injection of PAF (4 /xg/kg) or antigen was diluted with buffered EDTA/formalin solution (1 200 x ) or with 2% acetic acid solution (40 × ) for platelet and leukocyte counts, respectively. The counts were performed in a Neubauer chamber under light microscopy. A buffered EDTA/formalin solution was prepared by adding 3 ml of EDTA 0.077 M, 2 ml of 10 x concentrated phosphate-buffered saline solution (PBS), 150/zl of 40% formalin and distilled water to a final volume of 20 ml. The haematocrit was determined in heparinized microtubes (Inlab) centrifuged for 8 min in a microcentrifuge (Fanem, model 211).
2.3. Treatments WEB 2086 (2-10 mg/kg) (Casal-Stenzel et al., 1986), meclizine (15-30 mg/kg) and terfenadine (15-30 mg/kg) were given by the i.p. route, 1 h before the challenge. The PAF antagonist, PCA 4248 (5-10 mg/kg) (Fernandez-Gallardo et al., 1990), and azelastine (1, 10 and 20 mg/kg) were administered p.o. 2 h before the challenge to animals fasted for 12 h.
3. Results
3.1. Evaluation of antigen-induced acute haematological alterations As shown in fig. 1A, the i.v. injection of ovalbumin (250/xg/kg) into actively sensitized rats induced leukocytosis from 30 min up to 2 h, returning to basal levels after 24 h. This phenomenon was mainly due to an increase in blood neutrophil counts, while monocyte, lymphocyte and eosinophil numbers were not significantly altered (table 1). Figure 1 also shows that 5 min after the antigen, platelet counts decreased by 40% (fig. 1C), while the haematocrit values were signifi-
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Ovalbumin, 1.5 mg/kg, was administered i.v. as a single bolus injection. Meclizine (30 mg/kg) or WEB 2086 (20 mg/kg) were given i.p. and azelastine (20 mg/kg) was given p.o. 1 h and 2 h before the challenge, respectively. Survival rate was recorded 24 h after the administration of ovalbumin.
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2.5. Drugs 0
The drugs were obtained from the following sources: meclizine (Pfizer); terfenadine (Teldane-Merrel Lepetit); PCA 4248 ((2-phenylthioethyl)-5-methoxycarbonyl-2,4,6-trimethyl-1,4-dihydropyridine-3-carboxylate) was from Alter S.A.; WEB 2086 (3-4-(2-chlorophenyl-9-methyl-6H-tieno-3,2-f-l,2,4-triazolo-4,3-1,4-diazepin-2yl-l-(4-morpholinyl)-l-propanone) was from Boehringer Ingelheim; azelastine was kindly donated by Dr. J. Roux (Laboratories Sarget, France).
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2.6. Statistical analysis The data were analysed statistically by means of Student's t-test for unpaired samples; P values of 0.05 or less were considered significant.
Fig. 1. Kinetics of changes in blood leukocyte numbers (A); haematocrit (B); and platelet numbers (C) induced by the i.v. administration of ovalbumin (250 /~g/kg) to actively sensitized rats. Closed circles represent the control values for each haematological parameter. The results are the m e a n s + S.E.M. from six to eight animals. Statistically significant differences are indicated by an asterisk.
185 30.
TABLE 1 Differential leukocyte count alterations induced by the i.v. injection of ovalbumin (Ova 250 /xg/kg) into actively sensitized rats. Blood samples were obtained 1 h after the challenge. Saline (Sal)-injected animals were used as controls. Each value represents the mean_+ S.E.M. from at least six animals
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Lymphocytes Monocytes Neutrophils Eosinophils ( × 1 0 3 / m m 3) ( × 1 0 2 / m m 3) ( × 1 0 3 / m m 3) ( × 1 0 ] / m m 3) Sal Ova
A
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3.0±2.0 1.0± 1.0
" Statistics significant difference (P < 0.01).
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41
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cantly increased (fig. 1B). Both values returned to their basal levels after 2 h.
3.2. Inhibition by azelastine of antigen- and PAF-induced thrombocytopenia and haemoconcentration
4O
Azelastine (1-20 mg/kg) dose dependently inhibited antigen-induced thrombocytopenia and haemoconcentration, under conditions in which leukocytosis was not significantly affected (fig. 2). Azelastine also blocked the thrombocytopenia observed 1 h after the i.v. administration of PAF (4/zg/kg) (fig. 3).
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3.3. Interference of histamine and PAF antagonists with haematological alterations induced by the antigen
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Table 2 shows that the pretreatment with two different H 1 antagonists, meclizine and terfenadine, at doses of 15 and 30 mg/kg, inhibited only the antigen-induced haemoconcentration without interfering with thrombocytopenia and leukocytosis. In contrast, the PAF antagonists, WEB 2086 (2-10 mg/kg) and PCA 4248 (5-10 mg/kg), inhibited antigen-induced thrombocytopenia but failed to modify leukocytosis and haemoconcentration (table 3).
10
20
DOSE (mglkg)
Fig. 2. Interference of azelastine with ovalbumin (250/xg/kg i.v.)-induced changes in blood leukocyte counts (A); haematocrit (B); and platelet numbers (C). Saline-injected animals were used as controls (closed circle). The results are the means±S.E.M, from six to eight animals and statistically significant differences from the untreated group are indicated by an asterisk.
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TABLE 2 Interference of meclizine (Mec) and terfenadine (Ter) with the haematological alterations induced by ovalbumin (Ova 250 tzg/kg) in actively sensitized rats. Saline (SaD-stimulated rats were used as controls. Each value represents the mean + S.E.M. from six to eight animals Treatment Stimulus Leukocytes Platelets Haematocrit (mg/kg) (X 103/mm 3) ( × 105/mm 3) (%) None None Mec 15 Mec 30 Ter 15 Ter30 a
Sal Ova Ova Ova Ova Ova
9.5 ± 0.6 18.3 ± 0.9 21.7±1.0 17.2 ± 1.2 18.8±0.6 19.0_+2.6
8.0 + 0.03 4.5 ± 0.03 4.1±0.03 4.8 ± 0.03 5.9_+0.07 5.5_+0.04
46.8 ± 1.2 60.8 ± 1.4 56.0±1.2 54.1 -+ 1.5 a 52.8_+0.8a 51.8_+1.1a
Statistically significant difference (P < 0.05) compared to untreated group stimulated with Ova.
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AZE Fig. 3. Interference of azelastine (20 mg/kg, hatched column) with PAF (4/~g/kg i.v., black column)-induced thrombocytopenia. Platelet counts were performed 1 h after the i.v. injection of PAF. Saline-injected animals were used as controls (white column). Each bar represents the mean+ S.E.M. from six animals and statistically significant differences are indicated by an asterisk.
186 TABLE 3
TABLE 4
Interference of PAF antagonists with the haematological alterations induced by ovalbumin (Ova 250/~g/kg). Saline (Sal)-stimulated rats were used as controls. Each value represents the mean _+S.E.M. from at least six animals
Analysis of the haematological parameters 24 h after challenge with a lethal dose of ovalbumin (Ova 1.5 mg/kg), in azelastine (20 mg/kg)-treated rats. A saline (SaD-stimulated group was taken as control. Each value is the mean-+ S.E.M. from at least six animals
Treatment Stimulus Leukocytes Platelets Haematocrit (mg/kg) ( × 1 0 3 / m m 3) ( × 1 0 5 / m m 3) (%)
Treatment Stimulus Leukocytes Platelets Haematocrit ( x l 0 3 / m m 3) ( x l 0 5 / m m 3) (%)
None None WEB 2086 2 10 PCA4248 5 10 20
None Sal Azelastine Ova
Sal Ova
9.4_+0.5 21.0_+1.1
7.9_+0.02 4.5_+0.02
46.2_+0.7 57.6_+1.0
Ova Ova
20.5_+2.8 19.6±2.0
8.0±0.13 a 8.3_+0.02a
54.7_+2.7
Ova Ova Ova
22.2_+1.9 21.1±1.9 19.9±1.5
6.8_+0.04a 7.0_+0.04~ 7.5_+0.06a
60.6_+0.7 55.0±1.1 62.6_+1.4
9.8+0.7 10.7_+0.5
7.7_+0.02 7.6-+0.03
50.2+1.9 49.3+0.8
ters including platelet and total leukocyte counts, as well as the haematocrit levels, from the azelastinetreated group were not different from those of unchallenged animals (table 4).
a Statistically significant difference (P < 0.01) compared to untreated group stimulated with Ova.
4. Discussion
3.4. Effect of drugs on antigen-induced death The i.v. injection of ovalbumin at a dose of 1.5 mg/kg induced lethal shock in about 85% of the untreated rats. It is noteworthy that 61% of the deaths occurred within 1 h, 32% between 1 and 4 h and 7% between 4 and 24 h. Meclizine (30 mg/kg), WEB 2086 (20 mg/kg) and azelastine (20 mg/kg) were tested against this lethal reaction. As shown in fig. 4, the survival rate analysed 24 h after the antigenic challenge was shown to be 58% in rats receiving WEB 2086 and 64% in meclizine-treated animals, under conditions where the WEB 2086/meclizine combination increased the survival rate to 87%. The same figure shows that azelastine completely impaired the lethal reaction in this period, a protective effect that persisted for at least 7 days (data not shown). It was noted that 24 h after challenge, the haematological parame-
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[--1 CONT
WEB
MEC
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WEB
Fig. 4. Interference of WEB 2086 20 mg/kg (WEB); meclizine 30 mg/kg (MEC); the combination of WEB 2086 20 mg/kg with meclizine 30 mg/kg (MEC+WEB); and of azelastine 20 mg/kg (AZE) with ovalbumin (1.5 mg/kg)-induced lethality. Control group (CONT), unprotected animals injected with ovalbumin. Survival rate was recorded 24 h after the challenge. The number of animals used is indicated in parentheses.
It has been previously reported that rats actively sensitized with a mixture of aluminium hydroxide and ovalbumin develop a local immediate hypersensibility response following the intrathoracic injection of ovalbumin 14 days after the initial sensitization (Lima et al., 1990). This reaction was characterized by an intense increase in vascular permeability and pleural oedema which was blocked by histamine antagonists or by azelastine (Lima et al., 1991). In the present study we demonstrated that the i.v. injection of a sub-lethal dose of ovalbumin (250 ~g/kg) into actively sensitized rats induces a systemic anaphylactic reaction leading to neutrophilia, haemoconcentration and thrombocytopenia. Furthermore, lethal anaphylaxis was observed after challenge with high doses of the antigen. This model was used to investigate the interference of the anti-allergic drug, azelastine, with the haematological changes and death observed during systemic anaphylaxis. The oral administration of azelastine dose dependently inhibited both the antigen-induced haemoconcentration and thrombocytopenia without affecting the rise in blood leukocyte counts. It has been reported that azelastine inhibits IgE-mediated histamine release from rat mast ceils (Chand et al., 1985). In addition, azelastine also directly antagonizes the effects of this amine in a number of models in vitro and in vivo (Katayama et al., 1981; Lima et al., 1991). Histamine was long been recognized as an important mediator of anaphylaxis (Dale, 1929), and the effect of azelastine in our model might be attributed to its anti-histamine property. Nevertheless, while two H~ antagonists, meclizine and terfenadine, were effective against antigen-induced haemoconcentration, they failed to inhibit thrombocytopenia, contrary to azelastine. Histamine is thus involved only in haemoconcentration and, accordingly, azelastine operates via histamine-independent
187
mechanisms when inhibiting antigen-induced thrombocytopenia. Although rat platelets are refractory to in vitro stimulation with PAF (Ifiarrea et al., 1984), we reported that the i.v. injection of this lipid into rats induces a marked thrombocytopenia, which is blocked by the PAF antagonists, WEB 2086 and BN 52021 (Martins et al., 1988). In the experiments described here, the oral administration of azelastine also completely inhibited PAF-induced thrombocytopenia in rats. This finding is in agreement with those of Achterrath-Tuckermann et al. (1988) and Lima et al. (1991) who demonstrated that azelastine potently inhibits PAF-induced platelet aggregation, bronchoconstriction and pleurisy, indicating that it interferes with the effects of PAF. In the present study we also showed that the structurally unrelated PAF antagonists, PCA 4248 and WEB 2086, blocked antigen-induced thrombocytopenia, suggesting that PAF may play a role in this phenomenon. Together our results indicate that the inhibition of antigen-induced thrombocytopenia by azelastine may involve its ability to interfere with PAF. Nevertheless, since azelastine also induces calcium channel block (Chand et al., 1983) and raises intracellular cyclic AMP levels (Katayama et al., 1987), an unspecific inhibitory effect on platelet aggregation in general is also possible. It is of interest to note that antigen-induced leukocytosis was accounted for mainly by an increase in blood neutrophil counts. This alteration was accompanied by a concomitant decrease in the number of bone marrow nucleated cells (not shown), suggesting that the pool of marrow neutrophils was mobilized to enter the circulation after the antigenic challenge. An identical pattern of response was observed by Bozza et al. (in press) after the i.v. administration of PAF to rats. In this case, both blood neutrophilia and the decrease in bone marrow cell counts were blocked by the pretreatment with PAF antagonists as well as by general anaesthesia (Bozza et al., in press). The similarity between the leukocyte count alterations induced by PAF and those induced by antigen suggests that this lipid may mediate the leukocytosis elicited by the antigen. Yet, as PAF antagonists and anesthetics (not shown) failed to inhibit antigen-induced leukocytosis, these mechanisms are indeed distinct. Interleukin-1 and tumor necrosis factor a were also shown to induce acute blood neutrophilia concomitant with a reduction of bone marrow cell counts in rats (Ulich et al., 1987). The potential involvement of these cytokines in antigen-induced leukocytosis is now under investigation. The administration of ovalbumin at a dose of 1.5 m g / k g into actively sensitized rats induced death in approximately 85% of the animals, which was prevented by azelastine and by the combination of WEB 2086 and meclizine. In contrast, lethality was only
partially reduced by either WEB 2086 or meclizine alone. These results agree with those obtained using passively sensitized guinea pigs, in which the association of an anti-histamine and WEB 2086 inhibited the lethal anaphylactic reaction (Casals-Stenzel, 1987). We do not yet know the major cause of death from systemic anaphylaxis in the IgE rat model. Whatever the cause, our results indicate that synergism between PAF and histamine should be involved. From this point of view, the combined anti-histamine and anti-PAF properties of azelastine might explain its remarkable effect against anaphylactic mortality. Nevertheless, other pharmacological properties of azelastine, such as the inhibition of leukotriene synthesis and effects (Katay~ma et al., 1987), cannot be excluded and deserve further study. To summarize and conclude, our findings indicate that azelastine blocks antigen-induced haemoconcentration, thrombocytopenia and death, probably due to its combined effects against histamine and PAF. These properties may implicate azelastine as a potential candidate for the therapy of systemic anaphylaxis. New assays are now underway in order to investigate this possibility.
References Achterrath-Tuckermann, U., C.H. Weisher and I. Szelenyi, 1988, Azelastine, a new antiallergic/antiasthmatic agent, inhibits PAF-acether induced platelet aggregation, paw oedema and bronchoconstriction, Pharmacology 36, 265. Bozza, P.T., P.M.R. Silva, H.C. Castro-Faria-Neto, M.A. Martins and R.S.B. Cordeiro, Increase in the rat blood leukocyte counts induced by PAF-acether is suppressed by general anaesthesia, J. Leukocyte Biol. (in press). Brocklehurst, W.E., 1967, Passive cutaneous anaphylaxis, in: Handbook of Experimental Immunology, ed. D.M. Weir (Science Publications, Oxford) p. 745. Casals-Stenzel, J., 1987, Effects of WEB 2086, a novel antagonist of platelet activating factor, in active and passive anaphylaxis, Immunopharmacology 13, 117. Casals-Stenzel, J., G. Muacevic and K.H. Weber, 1986, WEB 2086 a new and specific antagonist of platelet activating factor (PAF), Arch. Pharmacol. 334 (Suppl.), R44. Chand, N., J. Pillar, W. Diamantis, J.L. Perhach and R.D. Sofia, 1983, Inhibition of calcium ionophore (A23187)-stimulated histamine release from rat peritoneal mast cell by azelastine: lmplications for its mode of action, Eur. J. Pharmacol. 96, 227. Chand, N., J. Pillar, W. Diamantis and R.D. Sofia, 1985, Inhibition of IgE-mediated allergic histamine release from rat peritoneal mast cell by azelastine and selected anti-allergic drugs, Agents Actions 16, 318. Chand, N., W. Diamantis and R.D. Sofia, 1986a, Modulation of in vitro anaphylaxis of guinea-pig isolated tracheal segments by azelastine, inhibitors of arachidonic acid metabolism and selected antiallergic drugs, Br. J. Pharmacol. 87, 443. Chand, N., K. Nolan, W. Diamantis, J.L. Perhach, Jr. and R.D. Sofia, 1986b, Inhibition of leukotriene (SRS-A)-mediated acute lung anaphylaxis by azelastine in guinea-pigs, Allergy 41,473.
188 Chand, N., K. Nolan, W. Diamantis and R.D. Sofia, 1987, Inhibition of acute lung anaphylaxis by aerosolized azelastine in guinea-pigs sensitized by three different procedures, Ann. Allergy 58, 344. Dale, H.H., 1929, Croonian lectures on some chemical factors in the control of the circulation, Lancet 1, 1285. Fernandez-Gallardo, S., M.P. Oretega, J.G. Priego, M.F. Casa-Juana, C. Sunkel and M. Sanchez Crespo, 1990, Pharmacological actions of PCA 4248, a new platelet-activating factor receptor antagonist: in vivo studies, J. Pharmacol. Exp. Ther. 255, 34. Inarrea, P., J. Gomez-Cambronero, M. Nieto and M. Sanchez-Crespo, 1984, Characteristics of the binding of platelet-activating factor to platelets of different animal species, Eur. J. Pharmacol. 105, 309. Katayama, S., N. Akimoto, H. Shionoya, T. Morimoto and Y. Katoh, 1981, Anti-allergic effects of azelastine hydrochloride on immediate type hypersensitivity reactions in vivo and in vitro, Arzneim. Forsch. 31, 1196. Katayama, S., H. Tsunoda, Y. Sakuma, H. Kai, I. Tanaka and K. Katayama, 1987, Effect of azelastine on the release and action of leukotriene C4 and D4, Int. Arch. Allergy Appl. Immunol. 83, 284. Kurosawa, M., 1989, Inhibitory mechanism of chemical mediator release by azelastine, Drugs Today 25 (Suppl. 6), 9. Lima, M.C.R., M.A. Martins, S.A.C. Perez, J.M. Peixoto, P.M.R.
Silva and R.S.B. Cordeiro, 1990, Kinetics of pleural exudation and cellular alterations induced by antigen in actively sensitized rats, Braz. J. Med. Biol. Res. 23, 857. Lima, M.C.R., M.A. Martins, S.A.C. Perez, P.M.R. Silva, R.S.B. Cordeiro a n d B.B. Vargafitig, 1991, Effect of azelastine on platelet-activating factor and antigen-induced pleurisy in rats, Eur. J. Pharmacol. 197, 201. Martins, M.A., P.M.R. Silva, H.C. Castro-Faria-Neto, P.T. Bozza, P.M.F.L. Dias, R.S.B. Cordeiro and B.B. Vargaftig, 1988, Intravenous injections of PAF-acether induce platelet aggregation in rats, Eur. J. Pharmacol. 149, 89. Motojima, S., N. Ando, Y. Ohashi, T. Otsuka, T. Yamai, T. Fukuda and S. Makino, 1989, The effects of azelastine on allergen-, exercise- and LTD4-induced bronchoconstriction, Drugs Today 25 (Suppl. 6), 15. Mue, S., G. Tamura, K. Yamauchi, H. Ohtsu and T. Takishima, 1989, Efficacy and safety of azelastine in the treatment of asthmatics: Japanese experience, Drugs Today 25 (Suppl. 6), 29. Ulich, T.R., J. Castillo, M. Keys, G.A. Granger and R.-X. Ni, 1987, Kinetics and mechanisms of recombinant human interleukin 1 and tumor necrosis factor alpha-induced changes in circulating numbers of neutrophils and lymphocytes, J. Immunol. 139, 3406.
