Interference of PCA 4248, a novel PAF receptor antagonist, with antigen-induced paw edema in mice

European Journal of Pharmacology, 236 (1993) 301-304

301

© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00

EJP 53083

Interference of PCA 4248, a novel PAF receptor antagonist, with antigen-induced paw edema in mice C l a u d i a Z u a n y A m o r i m , R e n a t o S6rgio B. C o r d e i r o a n d B. B o r i s V a r g a f t i g

a

Funda~o Oswaldo Cruz, IOC, Departamento de Fisiologia e Farmacodindmica, Avenida Brasil 4365, Caixa Postal 926, CEP 21040, Rio de Janeiro, Brazil and a Unit~de Pharmacologie Cellulaire, Unit~Associ~e Institut Pasteur, INSERM No. 285, 25 Rue du Dr. Roux, 75015Paris, France

Received 30 July 1992, revised MS received 23 February 1993, accepted 2 March 1993

The interference of the novel platelet-activating factor (PAF) receptor antagonist compound PCA 4248 with paw edema induced by antigen in sensitized boosted or unboosted mice was studied. Although PAF-induced edema was of similar intensity in non-sensitized and in both groups of sensitized mice, PCA 4248 was less effective in inhibiting paw edema induced by PAF in boosted mice. Paw edema induced by antigen in unboosted mice was refractory to PCA 4248 under conditions where edema in boosted mice was inhibited. Our results demonstrate that the PCA 4248 displays an anti-PAF activity in non-sensitized mice that is reduced by the booster injection of antigen. PAF plays an important role in the anaphylactic edema in boosted but not in unboosted mice. PAF (platelet-activating factor, PAF-acether); PCA 4248; Paw edema (mouse); Anaphylaxis

1. Introduction

2. Materials and methods

The involvement of platelet-activating factor (PAF) in allergic reactions is supported by physiopathological and pharmacological evidence (Pinckard et al., 1979 and reviewed by Vargaftig and Braquet, 1987). Our group recently demonstrated that the PAF-unrelated hetrazepinic antagonist W E B 2170 (Heuer et al., 1990) fails to suppress the effects of P A F in sensitized and antigen-boosted mice (Amorim et al., 1992a,b), as was also reported by Pretolani et al. (1989) for guinea pigs, and suppresses anaphylactic paw e d e m a in boosted but not in unboosted mice (Amorim et al., 1991, 1992a,b). The mechanism by which the booster injection of antigen suppresses P A F antagonism by W E B 2170 may have clinical relevance, particularly for patients in frequent contact with allergens. This led us to study whether the booster injection of antigen might also interfere with the ability of another P A F antagonist to antagonize the action of P A F itself. To do so, we evaluated the effect of the novel P A F antagonist compound P C A 4248, a 1,4-dihydropyridine derivative, on mouse paw e d e m a induced by P A F or antigen in sensitized boosted and unboosted animals,

2.1. A n i m a l s and sensitization procedure

Correspondence to: R.S.B. Cordeiro, Fundaq~o Oswaldo Cruz, IOC, Departamento de Fisiologia e Farmacodin~mica, Avenida Brasil 4365, Caixa Postal 926, CEP 20040, Rio de Janeiro, Brazil.

Male Swiss-Webster mice raised at the Oswaldo Cruz Foundation and weighing 18-20 g were actively sensitized by s.c. injection of 0.2 ml 0.9% NaC1 (saline) containing 10 /xg ovalbumin dispersed in 1 mg aluminium hydroxide. Fourteen days later, the animals were boosted with the same dose of antigen but without aluminium hydroxide (Andersson and Brattsand, 1982). This group of animals was used seven days after the booster injection and was n a m e d 14 + 7. In another set of experiments, the booster injection was omitted and the animals were used on the 14th or on the 21st day of sensitization, these groups being called 0 - 1 4 and 0-21, respectively. 2.2. Mice p a w edema

Mice paw edema was induced by injecting 50 tzl of saline containing 1 t~g of P A F or 0.01-20 txg of ovalbumin into one of the hind paws. As a control, 50 tzl of saline was injected into the contralateral paw. E d e m a was measured plethysmographically (Ferreira, 1979) after 30 min, and the results are expressed as the increase in paw volume (~zl) of the challenged paws minus the volume of the saline-injected paws.

302

2.3. Drug administration The animals were treated with the P A F antagonist compound P C A 4248 (25-100 m g / k g ) , given i.p. or orally 1 h before the challenge. The drug was solubilized with a mixture of saline and Tween-80 (2%, v / v ) . The antagonist was replaced by its vehicle in control groups.

2.4. Materials P A F (1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphoryl-choline) was from Bachem (Switzerland). Ovalbumin (5 × crystallized) was from Miles Scientific, U S A . P C A 4248 (2-(phenylthio)ethyl-5-methoxycarbonyl-2,4,6-trimethyl-l,4-dihydropyridine-3-carboxylate) was synthesized by Alter S.A., Spain (Sunkel et al., 1988).

2.5. Statistical analysis The data were analyzed statistically by a microcomputer program using as analysis of variance (ANOVA), followed by Newman-Keuls Student's test. P values of 0.05 or less were considered significant. Results are presented as means + S.E.M.

3. Results

3.1. Ovalbumin-induced mice paw edema

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26.4 + 6 13.9+8 20.5+_8

54.4 + 3 55.9_+9 60.6+7

18.6 + 6 31.9+3 16.7+7

29.8 _ 7 31.1+4 33.6+_6

As seen in table 1, P A F (0.1-2 txg/paw) induced e d e m a in all groups of control or sensitized mice. The dose of 1 t z g / p a w induced an e d e m a of 54.4 + 3/xl in non-sensitized mice, of 56 + 9 /xl in sensitized unboosted and of 61 + 7/xl in boosted mice. This dose was thus selected for further studies.

E d e m a induced by 1 /xg of P A F injected to nonsensitized mice was reduced by 42 and 75% following the oral administration of P C A 4248 at 50 and 100 m g / k g , respectively, and by 44 and 65% in sensitized and unboosted mice, respectively (fig. 2). PCA 4248 was less effective in inhibiting PAF-induced e d e m a in

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E d e m a was of similar intensity in unboosted (0-14) and in boosted mice (14 + 7), in doses up to 1 ~ g of ovalbumin, whereas 10 /zg induced e d e m a only in boosted mice, and 20 ~ g was inactive in both groups of mice. Non-sensitized animals did not respond to ovalbumin in doses up to 20/~g. Based on these results, 1 and 10 /xg of ovalbumin were selected for further experiments on unboosted and boosted mice, respectively.

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3.3. Interference of compound PCA 4248 with paw edema induced by PAF

Figure 1 shows that, within 30 min, the intraplantar injection of ovalbumin (0.01-20 p,g) was followed by paw e d e m a in both groups of immunized animals,

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TABLE 1 Increase in paw volume (~1) measured 30 min after the intraplantar injectionof PAF (0.1-2.0 ~g). Each point represents the mean +-S.E.M. from at least six animals.

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DOSE (/~g/paw) Fig. 1. Increase in mice paw volume (#.1) measured 30 rain after the intraplantar injection of ovalbumin (0.01-20 /~g/paw). Open bars refer to unboosted animals on the 14th day of immunization and hatched bars to boosted mice. Each point represents the mean+_ S.E.M. f r o ~ at least six animals. Closed circle refers to 20 # g ovalbumin injected to non-sensitized mice.

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DOSE (mg/kg) Fig. 2. Inhibition by PCA 4248 (25-100 mg/ kg p.o.) of PAF-induced edema in mice. (~) Non-sensitized; ( ~ ) 0-21; and (©) 14+ 7. Each point represents the mean +- S.E.M. from at least six animals. SignifJcance is indicated as * P < 0.01 and * * P < 0.05.

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T h e hetrazepinic P A F antagonist W E B 2170 (Heuer et al., 1991) fails to suppress the effects of P A F in

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~os~(m~/~ Fig. 3. Inhibition by P C A 4248 (25-100 m g / k g i.p.) of PAF-induced e d e m a in mice. (~,) Non-sensitized; and (e) 1 4 + 7 mice. Each point represents the mean_+ S.E.M. from at least six animals. Significance is indicated as * P < 0.01 and * * P < 0.05).

sensitized and boosted animals, with 32% inhibition being observed at 100 m g / k g . PCA 4248 was equally effective when given by i.p. and oral routes (fig. 3).

3.4. Interference of PCA 4248 with allergic paw edema PCA 4248 administered orally (fig. 4) or i.p. (table 2) failed to interfere with the inflammatory response to 50. z

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OOSE(~n~/~) Fig. 4. Inhibition by P C A 4248 (25-100 m g / k g p.o.) of mice paw e d e m a induced by the injection of ovalbumin. (e) 0 - 1 4 (1 ~ g / p a w ) ; (,x) 0-21 (1 /~g/paw) and (©) 1 4 + 7 ( 1 0 / x g / p a w ) ovalbumin. Each point indicates the m e a n +_S.E.M. from at least six animals. Significance is indicated as * P < 0.01.

TABLE 2 Effect of the i.p. administration of P C A 4248 on the paw e d e m a induced b y o v a l b u m i n in the 1 4 + 7 g r o u p . Results are expressed as m e a n s + S.E.M. from at least six animals, Treatment

Dose

(mg/kg)

Vehicle PCA4248 25 PCA4248 50 PCA 4248 100 a P<0.01 and b P<0.05.

Discussion

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ovalbumin in unboosted mice (0-14 and 0-21 groups), but was effective in boosted mice.

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sensitized and boosted mice (Amorim et al., 1992a), as reported by Pretolani et al. (1989) for guinea pigs, but suppresses anaphylactic paw edema in boosted and not in unboosted mice (Amorim, et al., 1991, 1992a). PCA 4248, a 1,4-dihydropyridine belonging to a new class of PAF receptor antagonists (Sunkel et al., 1988), is highly effective in inhibiting PAF-induced systemic hypotension and plasma extravasation in r a t s a s well as PAFinduced death in mice (Ortega et al., 1990; Fernandez-Gallardo et al., 1990). We now demonstrate that PCA 4248 inhibits edema caused by PAF in non-immunized and in immunized non-boosted mice, but is less effective in boosted mice. Since it has been demonstrated that the booster injection of antigen enhances the subsequent release of inflammatory mediators from guinea pig lungs, augments the lung sensitivity to PAF and to leukotriene D 4 (Pretolani et al., 1988), and reduces the interference of PAF antagonists with the effects of PAF itself (Pretolani et al., 1989), the question is raised whether the booster injection modifies the paw sensitivity to PAF. In fact, a large range of doses of PAF induced edema of similar intensity in non-sensitized and in sensitized mice, suggesting that the limited interference of PCA 4248 with PAF-induced paw edema is not due to different paw sensitivities to PAF. Although 1/xg ovalbumin induced edema of similar intensity, in both immunized groups, 10/zg of antigen failed to trigger edema in non-boosted mice. This could result from antigen leakage into the circulation triggering systemic hypotension, thus reducing the leakage. This lack of effect of antigen was evident when unboosted mice were challenged with 1 / ~ g (right paw) and 10/~g ovalbumin (left paw) and failed to develop edema in either paws (data not shown). The fact that systemic hypotension and a reduced leakage did not occur in the boosted group, may result from the higher levels of circulating immunoglobulin (Ig) G in these mice, which might prevent antigen leakage into the systemic circulation (Amorim et al., 1992a). Even though PCA 4248 was less effective against paw edema induced by PAF in boosted mice than in unboosted mice, it inhibited edema induced by antigen in boosted mice~ Paradoxically, in the unboosted group (0--14 and 0-21), PCA 4248 maintained its ability to antagonize PAF, but failed to inhibit anaphylactic edema. Furthermore, the observation that PCA 4248 prevented this edema in boosted animals, but was

304

clearly less effective against PAF itself, is consistent with the interpretation that the amount of endogenous PAF generated during the anaphylactic reaction in the paw might not be very high. We demonstrated that different mediators, including histamine, serotonin and leukotrienes, are involved in anaphylactic edema in boosted mice (Amorim et al., 1992b), whereas paw edema in non-boosted mice involves histamine and serotonin (Amorim et al., 1992a), and that PCA 4248 was inactive in inhibiting the edema, Thus PCA 4248 does not have anti-amine properties, The most likely mechanism of action of PCA 4248 seems to be receptor antagonism, since it blocks PAF binding to platelets and polymorphonuclear leukocytes, and does not cause non-selective inhibition of platelet activation (Fernandez-Gallardo et al., 1990); however, we cannot rule out other pharmacologic properties of PCA 4248 that could interfere with inflammation~ Consistent with this, no single antagonist of known mediat o r s supressed edema, and thus it is n o t surprising that PCA 4248 exerted only a moderate protective effect against ovalbumin-induced paw edema, Taken together, our results show that PCA 4248 inhibits PAF-induced edema in non-immunized mice and that, like the chemically unrelated PAF antagonist compound WEB 2170 (Amorim et al., 1992a), it has a diminished inhibitory effect on PAF-induced p a w edema in sensitized and boosted mice. In contrast, both PCA 4248 and WEB 2170 are effective against anaphylactic edema in boosted animals, where PAF may play a n important role. Acknowledgements The authors thank Dr. C. Sunkel (ALTER S.A., Spain) for providing PCA 4248 and CNPq for the fellowship of C.Z. Amorim.

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255, 962. M. Sanchez-Crespoand C. Sunkel, 1990, Pharmacologicalactions of PCA 4248, a new Platelet-Activating factor receptor antagonist: in vivo studies, J. Pharmacol. Exp. Ther. 255, 34.

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Pinckard, N.R., R.S. Farr and D.J. Hanahan, 1979, Physiochemical and functional identity of rabbit platelet-activating factor (PAF) release in vivo during IgE-anaphylaxis with PAF released in vitro from IgE-sensitized basophils, J. Immunol. 123, 1847. Pretolani, M., J. Lefort and B.B. Vargaftig, 1988, Active immunization induces lung hyperresponsiveness in the guinea-pig: Pharmacological modulation and triggering role of the booster injection, Am. Rev. Respir. Dis. 138, 1572. Pretolani, M., J. Lefort and B.B. Vargaftig, 1989, Limited interference of specific PAF antagonist with hyperresponsiveness to PAF itself of lungs from actively sensitized guinea-pigs, Br. J. Pharmacol. 97, 433. Sunkel, C.E., C.J.M. Eau, F.J. Cillero, J.G. Priego and M.P. Ortega, 1988, Synthesis platelet-aggregation inhibitory activity and in vivo anti-thrombotic activity of new 1,4-dihydropyridines, J. Med. Chem. 33, 3205. Vargaflig, B.B. and P.G. Braquet, 1987, PAF-acether today - Relevance for acute experimental anaphylaxis, Br. Med. Bull. 43, 312.