Antagonism of PAF-induced death in mice

PROSTAGLANDINS

ANTAGONISM

OF PAF-INDUCED

Adam K. Myers, Taichi Nakanishi

DEATH IN MICE and Peter Ramwell

Department of Physiology and Biophysics Georgetown University Medical Center Washington, D.C. 20007 ABSTRACT The ability of three platelet activating factor (PAF) antagonists, BN52021, L652,731 and 48740RP, and the leukotriene antagonist FPL55712 to block iv PAF-induced PAF-induced sudden death has death was tested in mice. been previously characterized as a model of systemic anaphylaxis and circulatory shock related its hypotensive actions. Of the drugs, BN52021 and L652,731 provided dose-dependent protection against PAF toxicity, whereas the others had no effect. 48740RP was, however active against PAF-induced rabbit platelet aggregation. BN52021 was inactive in three other mouse sudden death models in which arachidonic acid, U46619 or collagen combined with epinephrine is injected iv to provoke a In contrast, the TXA2 thrombotic/ischemic sudden death. antagonist SQ29548 inhibited the acute toxicity of two of these latter challenges (arachidonic acid and thromboxane agonist U46619), but was inactive against PAF lethality. These results suggest that PAF toxicity in mice is a specific model for PAF agonism, and is not mediated by TXA or peptido-leukotrienes. Further, PAF-induced mor z ality should be a simple and useful technique for testing potential PAF antagonists for in vivo activity by various routes of administration. INTRODUCTION Platelet activating factor (PAF; 1-0-alkyl 2-acetyl sn glyceryl phosphoryl choline) is currently the focus of intensive research due to its potential importance as an endogenous mediator of inflammatory, allergic and shock states (1). Much of this research effort involves the development and testing of PAF antagonists, which might be clinically useful in pathological states involving PAF. Thus, there is a need for a rapid method for in vivo screening of such compounds. Desirable characteristics of a model for testing for PAF antagonistic activity and in vivo efficacy of compounds include economy, rapidity and specificity. Furthermore, it is important that the usually small

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quantities of experimental drugs available are used One model which might meet these conservatively. characteristics is PAF-induced mortality in mice, first reported by Lanara et al. (2) and described by this laboratory (3,4). The acute toxicity of PAF in mice, as opposed to rabbits (5), is not thrombotic in nature but is apparently related to the potent hypotensive effects of PAF, which produces a state resembling systemic anaphylaxis or circulatory shock (6). Unlike other mammalian species, mouse and rat platelets are not sensitive to PAF (2,7), and thus other cardiovascular actions predominate. Intravenous PAF in mice produces dose-dependent mortality, presenting an opportunity in a small species to test in w activity of anti-PAF compounds. The dose-mortality relationship has been previously published (3). Based on a number of studies performed in our laboratory (3,4,6) the doses of iv PAF producing 20, 50 and 80% mortality (LD20, LD50 and LD80) are approximately 10, 15 and 40 ug/kg, respectively. In the studies below, this model was characterized by examination of the effects of three PAF antagonists on the acute toxicity of PAF. Further, one of the PAF antagonists was tested in a series of related acute toxicity tests in mice, and its effects were compared to a TXA2 antagonist, as well as a leukotriene antagonist. This work was presented in preliminary form at the Sixth International Prostaglandin Conference (4). METHODS Animal Studies Male CD-1 mice, 42 days of age, were purchased from Charles River Breeding Laboratories and maintained on standard rodent diet and tap water. Animals were assigned randomly to experimental and control groups, below (numbers per group were 15 or greater, except where indicated in the results). For each drug treatment group, a separate control group was included. Groups of mice were treated ip with various doses of one of three PAF antagonists or a leukotriene antagonist 45 min before challenge with iv PAF. The PAF antagonists tested were BN52021 (8), L652,731 (9) and 48740RP (10). BN52021 (Institut Henri Beaufour) and L652,731 (Merck, Sharp and Dohme) were administered at doses of 2, IO and 20 mg/kg; 48740RP (Rhone-Polenc) was injected at doses of The doses of FPL55712 (Fisons 2, 20 and 40 mg/kg. Pharmaceuticals), the SRS-A (LTC4/LTD4) antagonist (ll), given 45 min before PAF were 40 and 80 mg/kg. An additional group received 10 mg/kg ip 5 min before PAF, as in a previous report (12). Each of the four drugs was

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dissolved in DMSO at a concentration such that the appropriate dose was delivered in an injection volume of Groups of control mice received the 2.5 ul/g body weight. DMSO vehicle. Subsequent to the PAF or leukotriene antagonist treatment, mice were anesthetized with sodium amytal (100 mg/kg ip), the jugular vein was exposed, and an iv injection of 40 ug/kg PAF, the approximate LD80 was This standard dose was used in all in vivo administered. experiments involving PAF. PAF (Behring Diagnostics) was dissolved in 0.9% NaCl solution at a concentration such that the injection volume was 5 ul/g body weight. Mice were observed until death or full recovery of the righting reflex, indicating recovery from PAF-induced shock as well as anesthesia. At the dose of PAF used, mortality usually occurs within 30 min, whereas survivors are fully recovered within 4 hr. Mortality was compared between experimental and control groups by the Chi-square test, with statistical significance assumed at pcO.05. Additional groups of mice were treated with 20 mg/kg BN52021, or with the TXA receptor antagonist, SQ29548 (13) before challenge wi+ h various agents. BN52021 was administered as above by ip injection 45 min before challenge, whereas 5429548 was injected iv 2 min before SQ29548 (E.R. Squibb and Sons) was dissolved challenge. in 100 mM Na2C03 and injected in a volume of 5 ul/g body weight, and BN52021 was prepared and administered as Control groups received vehicle injections. above. The challenges consisted of the approximate LD80 of arachidonic acid (75 mg/kg), the thromboxane agonist U46619 (0.8 mg/kg) or a collagen/epinephrine (1.0 and 0.12 The use of these mg/kg, respectively) combination. challenges has been more thoroughly described elsewhere (14,15,16). Arachidonic acid (Nu-Chek-Prep) and U46619 (Upjohn) were prepared in 1OOmM Na2C03 solution: the collagen (Hormon Chemie)/epinephrine (Parke-Davis) combination and PAF were dissolved in 0.9% NaCl. All challenges were prepared at concentrations such that the injection volume was 5 ul/g body weight. Mice were observed and mortality was recorded, as above, and compared between drug treatment groups and the individual vehicle control groups. Platelet aqqreqation studies. These were performed by an adaption of the method described by Terashita et al. (17). Using a two channel aggregometer (Payton Associates). The purpose of these studies was to confirm activity of PAF antagonists. Blood was collected in 3.8% sodium citrate (1 ml for 9 ml of blood) by ear artery puncture of male New Zealand white rabbits (Buckshire Corporation) anesthetized with Inovar-vet. Platelet rich plasma (PRP) was obtained by

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centrifuging the blood at 150 X g for 15 min at room temperature. Platelet poor plasma (PPP) was obtained after PRP was decanted by centrifugation of the pellet at 1500 X g for 15 min. The platelet concentration in PRP was adjusted to 450,000 ~1-1 with PPP. Two concentrations of the PAF antagonist 48740RP were tested (10 ug/ml and 100 ug/ml). 48740RP was dissolved in DMSO and this PAF antagonist or the solvent (DMSO) was added two minutes before aggregation. Platelet aggregation was initiated by adding one of three differ:qt doses and 4_5 of x PAF ,,_Q;fnal concentration 4:5 X 10°8M, 4.5 X 10 M These concentrations of PAF Induced approximately 40, 90 and 100% of maximal aggregation in control experiments. The maximum amplitude was measured and the inhibition percentage was calculated by comparison with the maximum amplitude obtained in the control aggregation performed in the presence of DMSO. PAF (Behring Diagnostics) was dissolved daily in 0.9% NaCl and placed on ice until used. Albumin was not used for preparing the PAF solution.

100

Mortality (XI

501 BN5202

1

1652.731 .

r

10

2

.

20

.

40

Dose (mg/kg)

Figure 1. Effects of three PAF antagonists or vehicle on mortality following LD80 challenge by PAF (40 ug/kg, iv). The antagonists were administered ip 45 min before PAF injection. **p
450

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RESULTS Animal Studies Both BN52021 and L652,731 provided dose-dependent protection against the acute toxicity of iv PAF (fig. 1). In both cases, doses of 10 and 20 mg/kg of the PAF antagonists were significantly protective against the PAF LD80 (~~0.01). Mortality in vehicle treated control groups ranged from 67 to 100% (not illustrated). The extrapolated ED50 doses of the antagonists (doses reducing mortality to 50% of the level observed in controls) were 13 mg/kg and 8 mg/kg for BN52021 and 48740RP had no effect on PAF L652,731, respectively. toxicity at the doses tested (fig. 1). Similarly, FPL55712 was not protective against PAF at the doses or pretreatment intervals used (table 1).

Table 1.

FPL55712 (mg/kg,ip)

10 40 80

FPL55712

and PAF-induced

Pretreatment Time (min)

5 45 45

Death in Micea

No. Dead/ No. Tested, Pretreated

g/g 8/10 lO/ll

No. Dead/ No. Tested, Control

8/8 lO/lO lO/ll

aGroups of mice were pretreated with indicated FPL55712 doses or the DMSO vehicle (controls) prior to challenge with the approximate LD80 of PAF.

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PROSTAGLANDINS

O-

5-

o-

5-

AA

U46619

C/E

PAF

Challenge

Figure 2. Effect of BN52021, a PAF antagonist, on mortality following LD80 challenges in mouse acute toxicity tests. BN52021 (hatched bars) or vehicle (open bars) was administered ip, 45 min prior to iv arachidonic acid (75 mg/kg), a TX agonist (U46619, 0.8 mg/kg), collagen/epinephrine combination (C/E, 1 mg/kg and 0.12 mg/kg, respectively) or PAF (40 ug/kg). **p
BN52021 (20 mg/kg ip) had no protective actions against the other challenges tested (fig. 2). The acute toxicity of arachidonic acid, the thromboxane agonist U46619 and collagen/epinephrine combination were unaffected. In contrast, pretreatment with the TXA2 antagonist 5429,548 (2 mg/kg iv) reduced mortality following arachidonic acid (~~0.05) or U46619 (p
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PROSTAGLANDINS

o-

5-

Q-

5-

l.

AA

U46619

C/E

PAF

Challenge

Figure 3. Effect of SQ 29,548, a thromboxane antagonist, on mortality following LD80 challenges in mouse acute toxicity tests. SQ 29,548 (hatched bars) or vehicle (open bars) was administered iv, 2 min prior to iv arachidonic acid (75 mg/kg), a thromboxane agonist (U46619, 0.8 mg/kg) , collagen/epinephrine combination (C/E, 1 mg/kg and 0.12 mg/kg, respectively) or PAF (40 ug/kg). *p
Platelet aqqreaation studies 48740RP effectively inhibited PAF-induced platelet aggregation in rabbit PRP (table 2). At a concentration of 10 ug/ml, aggregation induced by 4.5 X 10 -8M PAF was nearly abolished, whereas 100 ug/ml 48740RP coxnfjletelyblocked7the aggregation produced by 4.5 X 10 and 4.5 X 10 M PAF. Higher PAF concentrations, however, overcame the inhibition.

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Inhibition of PAF-induced Aggregation by 48740 RPa

Table 2.

4.5 4.5 4.5 4.5 4.5 4.5

Rabbit Platelet

PAF (M)

48740 RP (ug/ml)

n

Inhibition (%+SE)

X x X X x x

10 10 10 100 100 100

4 5 5 5 5 5

96.5k2.2 15.lt1.2 7.022.8 100 99.3k1.3 20.9L3.0

10-g 10-7 10-6 10-a 10-7 10-6

aRabbit PRP was incubated 2 min with 48740 RP prior to addition of PAF. Inhibition is relative to control aggregations performed after incubation with the vehicle for 48740 RP (DMSO). DISCUSSION These studies suggest that acute PAF toxicity in mice is a useful model for in vivo evaluation of PAF antagonists. The use of this small species allows testing in relatively large numbers of animals while conserving experimental drugs. Furthermore, both the toxicity of PAF and the actions of PAF antagonists displayed a high degree of specificity in the model. Of the three PAF antagonists tested, only BN52021 and L652,731 had beneficial effects against PAF lethality. In both cases, dose-dependency was observed. The failure of 48740RP, a known PAF antagonist (lo), to protect against iv PAF in mice might reflect lack of drug bioavailability, metabolism of the drug, drug toxicity, Interestingly, this compound dosage or other factors. reportedly does not block PAF-induced rat paw edema (18), which is blocked by other antagonists (18,19). In order to test for PAF antagonistic activity of our 48740RP sample, the rabbit platelet aggregation Under these circumstances the experiments were performed. compound was clearly active against PAF. The antagonism is apparently competitive, since it was overcome by high Obviously, the use of concentrations of the agonist. multiple methods for testing the activity of experimental drugs is desirable. The specificity of PAF agonism and antagonism in mice is demonstrated by the failure of the leukotriene

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antagonist FPL55712 or the thromboxane antagonist SQ29548 In previous studies in to protect against PAF toxicity. this laboratory, the lack of efficacy of the cyclooxygenase inhibitor indomethacin, a thromboxane synthetase inhibitor and calcium channel blockers was demonstrated (3). Although one previous report suggested that FPL55712 was beneficial (12), this was not confirmed in the present experiment or in another study (20). Thus, the systemic actions of PAF in this mouse model are apparently quite specific, not involving secondary effects It should be noted, of TXA2 or peptido- leukotrienes. (3,12) and high doses of however, that glucocorticoids some lipoxygenase inhibitors (12,ZO) are protective, indicating that lipoxygenase products other than LTC4 or LTD4, which are antagonized by FPL55712, might participate in PAF toxicity. Further evidence for specificity in this in vivo paradigm of PAF agonism and antagonism is the contrasting spectrum of activity of BN52021 and the TXA2 antagonist SQ29548 in the four challenge models. The protective efficacy of 5429548 apparently corresponds directly to the degree of involvement of TXA in the challenge models; it was most protective against z he thromboxane agonist U46619, moderately beneficial against arachidonate, and insignificantly protective against collagen combined with epinephrine. No effect of SQ29548 was seen against PAF. The former three challenge models have been previously associated with platelet and TXA2-dependent phenomena (14,15,21). In the same four acute toxicity tests, the PAF antagonist BN52021 had a completely different spectrum of action, with no protective efficacy except against PAF itself. It should be noted that the PAF solutions were not prepared with 0.25% albumin in these studies, unlike most experiments involving PAF injections. PAF solutions were prepared instead in 0.9% NaCl without albumin, in accord with previous studies in the toxicity model performed in this and other laboratories (3,4,6,12,20). However, PAF was prepared and used within 2 hr. We have obtained consistently reproducible results with this method, and chose it for the present study for comparability to previous reports. Because PAF solutions prepared with albumin might be more stable, it is possible that PAF toxicity is actually somewhat greater than reported in this model. This point should be addressed in subsequent work in the mouse toxicity test. Thus, in summary, PAF toxicity in mice appears to be a simple, economical and specific test in which in vivo activity of PAF antagonists can be tested. The technique should be easily adapted for testing such drugs by various routes of administration and treatment regimens. Of the

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three PAF antagonists evaluated in the present study, the Ginkolide BN52021 and the lignan L652,731 were effective in blocking PAF lethality in the mouse. ACKNOWLEDGMENTS This work was supported in part by a grant from the BN52021, L652,731 and 5429548 were gifts NIH (HL31498). of Institut Henri Beaufour, Merck Sharp and Dohme, and E.R. Squibb and Sons, respectively. FPL55712 was a gift of Fisons. REFERENCES 1.

2.

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8.

456

Braquet, P., Touquit, L., Shen, T.Y., and B.B. Perspectives in platelet activating Vargaftig. factor research. Pharmacol. Rev. =:97. 1987. Lanara, E., Vakirtzi-Lemonias, C., Kritikou, L., and C.A. Demopoulos. Response of mice and mouse platelets to acetyl glyceryl ether phosphorylcholine. Biochem. Biophys. Res. Comm. 109: 1148. 1981. Myers, A., Ramey, E., and P. Ramwell. Glucocorticoid protection against PAF-acether toxicity in mice. Br. J. Pharmacol. 79:595. 1983. Myers, A., Torres Duarte, A.P., and P. Ramwell. Pharmacological manipulation of platelet-activating factor toxicity in rodents. In: Advances in Prostaglandin, Thromboxane, and Leukotriene Research, vol. 17. (B. Samuelsson, R. Paoletti, and P.W. Ramwell, eds.) Raven Press, New York, 1987, p. 833. Lefer, A.M., Muller, H.F., and J.B. Smith. Pathophysiological mechanisms of sudden death induced by platelet activating factor. Br. J. Pharmacol. =:125. 1984. Role of adrenal steroids Myers, A.K. and T.J. Bader. in the recovery from platelet activating factor challenge. Circ. Shock. 23: 143. 1987. Namm, D.H., Tadepalli, A.S. and J.A. High. Species specificity of the platelet responses to 1-O-alkyl-2Thromb. Res. acetyl-a-glycero-3-phosphocholine. 25:341. 1982. Braquet, P., Etienne, A., Touvay, S., Bourgain, R.H., Lefort, J., and B.B. Vargaftig. Involvement of platelet-activating factor in respiratory anaphylaxis, demonstrated by PAF-acether inhibitor Lancet i: 1501. 1985. BN52021.

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Hwang, S.-B., Lam, M.-H., Biftu, T., Beattie, T.R., and T.-Y. Shen. Trans-2,5-bis (3,4,5An orally active, trimethoxyphenyl) tetrahydrofuran. specific, and competitive receptor antagonist of platelet activating factor. J. Biol. Chem. 260: 15639. 1985. Sedivy, P., Caillard, C.G., Floch, A., Folliard, F., Mondot, S., Robaut, C., and B. Terlain. 48740 R.P.: A specific PAF-acether antagonist. Prostaglandins 30:688. 1985. Augstein, J., Farmer, J.B., Lee, T.B., Sheard, P., Selective inhibitor of slow and M.L. Tattersall. reacting substance of anaphylaxis. Nature (London) New Biol. -:215. 1973. Young, J.M., Maloney, P.J., Jubb, S.N., and J.S. Clark. Pharmacological investigation of the mechanisms of platelet-activating factor induced mortality in the mouse. Prostaglandins 30:545. 1985. Ogletree, M.L., Harris, D.N., Greenberg, R., Pharmacological Haslanger, M.F., and M. Nakane. actions of SQ29,548, a novel selective thromboxane antagonist. J. Pharmacol. Exp. Ther. 234:435. 1985. Myers, A., Penhos, J., Ramey, E. and P. Ramwell. Thromboxane agonism and antagonism in a mouse sudden death model. J. Pharmacol. Exp. Ther. =:369. 1983. Myers, A.K., Forman, G., Torres Duarte, A.P., Penhos, J and P. Ramwell. Comparison of verapamil and nikedipine in thrombosis models. Proc. Sot. Exp. Biol. Med. 183:86. 1986. DiMinno, G., and M.J. Silver. Mouse antithrombotic assay: A simple method for the evaluation of antithrombotic agents in vivo. Potentiation of antithrombotic activity by ethyl alcohol. J. Pharmacol. Exp. Ther. 225:57. 1983. Terashita, Z., Tsushima, S., Yoshioka, Y., Nomura, H ., Inada, Y., and K. Nishikawa. CV-3988 - A specific antagonist of platelet activating factor (PAF). Life Sci. 32 1975. 1983. Martins, M.A., Silva, P.M.R., Neto, H.C.F., Lima, M.C.R., Cordeiro, R.S.B., and B.B. Vargaftig. Interactions between local inflammatory and systemic haematological effects of PAF-acether in the rat. Eur. J. Pharmacol. 36:353. 1987. Hwang, S.-B., Lam, M.-H., Li, C.-L., and T.-Y. Shen. Release of platelet activating factor and its involvement in the first phase of carrageenin-induced rat foot edema. Eur. J. Pharmacol. m:33. 1986.

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Criscuoli, M., and Subissi, A. Paf-acether-induced death in mice: involvement of arachidonate metabolites and B-adrenoceptors. Br. J. Pharmacol. %:203. 1987. Torres Duarte, A.P., Ramwell, P., and A. Myers. Sex differences in mouse platelet aggregation. Thromb. Res. 43:33. 1986. Editor: J.R. Fletcher

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Received:E-29-87

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Accepted: 2-10-88

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