Histamines and Cortisone in Experimental Anaphylaxis

i__ Med.Pharmacol.exp. (Basel) 3.3,189-198 (1965) LSD _ i

From the Laboratori Ricerche Farmacelogiche, Farmitalia S.p.A., Milano, Italy _

Synergistic Action of an Anti-5-Hydroxytryptamine, Anti- Histamines and Cortisone in Experimental Anaphylaxis

By G. B. FREGNAN and A. H. GLXSSER I

The mechanism of anaphylaxis is not yet clearly understood. Symptoms and lesions have been found to be constant for a given animal species but to i differ from one species to another irrespective of the nature of the sensitizing il antigen. The anaphylactic shock can he considered as the result of reactions initiated by the union of antigen and cellular antibody with release of sub- :' stances (histamine, 5-hydroxytryptamine, acetyleholine, bradykinin, slow- reacting substances, heparin, anaphylatoxin, trypsin, etc.), elevation of some electrolyte values (Na, K, CI) in muscle, viscera and bone, and changes of the lipid metabolism of lung tissue. The reactions may vary qualitatively or quanti- i: tatively with the animal species. Among the substances released during anaphylactic shock, 5-hydroxytryptamine (5-HT) and histamine have been supposed to play an important role together in several animals species (such _: as rabbit, rat, and mice) and some attempts to prevent the anaphylactic shock !i with anti-5-HT or anti-histamine drugs have been, at least partially, successful f_ (1, 8, I0, 11, 13, 17, 19, 21, 22, 23). Histamine and slow-reacting substances (SRS-A) are quite important in guinea.pig anaphylaxig, so that anti-histamine and anti-SRS.A drugs had some protective effects (12, 6). However, a stronger protection has been observed when animals have been pretrealed with a drug E or a mixture of drugs having manifold properties so to counteract all the _

cutaneousalterations anaphylaxisseen duringinanamice,phylwhicaxis.h seemsHalpernto beet theal. result(13) shofowedthe simultaneothat passiveus I release of 5-HT and histamine, is almost completely abolished by the simul- _ taneous injection of lysergic acid diethylamide (LSD 25) and mepyramine with a considerable mutual reinforcement of theix: anti-anaphylactic properties. Other evidences of synergism in protecting guinea-pigs from anaphylaxis have been given by Goadb 9 ond SmitA (12) with cortisone and mepyramine and by Collier et al. (6) with acetylsalicylic acid and mepyramine. Owing to. these results, we have studied the anti-anaphylactic effects of associations of drugs, each one counteracting, as specifically as possible, a particular aspect of the anaphylaxis in rats and mice. In consideration of the fact that the anaphylactic shock in rats and in mice is possibly due to 5-HT, histamine, electrolytes and lipids changes we pretreated the animals with anti- 5-HT, anti-histamines and cortisone given either alone or in combination.

Received: February 18, 1965.

.... _'"..... r'_ r ...... 7 i 190 Ftegnan and GlOsser, Synergistic Action of an

Antl-5-HT and anti-histamines block the effect of 5-HT and histamine on some • receptors, while cortisone besides lowering the tissue reserves of 5-HT and histamine (18) would exert an action on the electrolytes and on the lipid meta- ! bolism (0). Among the anti-5.HT, the new drug 1-methyl-8_-carbobenzyloxy. aminomethyl-10a-ergoline maleate (MCE), synthetized in our laboratories (3), has been used because it has a very specific and long-lasting action (2).

Materials and Methods

Female white mice (15-18 g in weight) and rats (80-100 g in weight) were : used. The mice were sensitized by two i.p. injections (2 ml/mouse) of a mixture ! containing 1 ml of undiluted horse serum and 1 ml of a saline suspension of tIemophilus pertussis vaccine (1,000 millions HP organisms) at two days ! i: interval. The rats received only one i.p. injection (2 ml/rat) of the same _ mixture. The animals were challenged intravenously: mice with 1 ml of un- i: diluted horse serum 26 days after the last sensitizing injection and rats with 0.5 mi of antigen 14-16 days after the sensitizing antigen. These challenging doses consistently produced fatal anaphylactic shock in 84-88Jg of sensitized mice within 10-40 minutes and hypotcnsion in 75.8_g of sensitized rats within 2-5 minutes. The same amounts caused no toxic manifestations in normal unsensitized mice and rats. MCE, given subcutaneously, and cortisone acetate, intramuscularly,were injected 24 hours prior to the challengingdose of antigen. i LSD 25 maleate, mepyramine hydrochloride, chlor-pheniramine maleate, and promethazine hydrochloride were injected subcutaneously 2-3 hours prior to challenge. Acetylsalicylic acid was given intraperitoneally 30-45 minutes prior to challenge. Rats, before the intravenous administration of the antigen, were anesthetized with sodium amobarbital (100 g/kg h.w. intraperitoneally) i and their carotid pressure was recorded on a smoked drum by a mercury manometer. The rectal temperature was measured by a thermocouplc. The probe was inserted into the rectum to a constant depth of 2.5 cm and was . removed after each reading.

i Results

io Anti-5-HT, anti-histamines, cortisone, and other drugs were studied for their property to protect sensitized rats and mice from anaphylactic shock. For this purpose the hypothermia and the hypotension have been controlled in sensitized rats and the mortality in sensitized mice following the intravenous administration of shockdosesof antigen.

E[lect of chronic treatment tuith MCE on the sensitization o[ -.:. - ! mice and rats ..... : The animals were treated subcutaneously with 5 mg/kg/day of MCE for 12 days beginning 5 days before the first sensitizing dose :;_ I and ending 5 days after the last sensitizing dose. This treatment did 'C i

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• t t i Anti-5-hydroxytryptamlne, Anti-histamines, and Cortisone... 191

not protect the animals exposed to the challenge demonstrating that MCE did not interfere with the production of antibody by the organism. _,

Effect of acute treatment with MCE on the rectal temperature ! o] sensitized rat beIore or alter the shock dose 5-10 mg/kg of MCE given subcutaneously to rats lowered their rectal temperature for 0.8°-1.5°C within the first two hours. The temperature gradually returned to the normal level in about 5-6 _ hours. At the twenty-fourth hour, when the rat temperature was !: quite normal, the ehaUange was given. 90-100_ of the sensitized control animals showed a decrease of their rectal temperature (2°-5°C below the normal temperature within the first two hours} while only 20-30 % of the MCI 0:pretreated animals showed a small decrease (1.5°-2°C}.

Effect of acute treatment with MCE, mepyramine and cortisone on anaphylaxis in rats (blood pressure changes) Table I shows that the MCE (1-10 mg/kg) and mepyramine (25 mg/kg) were scarcely active when given alone, but strongly protected the rats when administered simultaneously: in fact only 28% of the rats gave hypotension. Cortisone (t00 mg/kg) also afforded a protection of the same degree as that given by MCE associated to mepyramine.

TABLE I Reduction of the hypotension following the anaphylaetic shock in sensitized rats by pretreotment with MCE, Mepltramine, and Cortisone

% of rats giving • Interval between hypotension

N* of Antagonists Dose Route the administration within 2-5 mln ruts mg/kg of antagonists and after i.v. ehall- the challenge enge with 0.5 ml of horse serum* 57 Control - - - 75.8 r 30 MCE 1.0 s.c. 24 h 64.0 _r 28 MCE 10.0 s.c. 24 h 56.0 14 Mepyramine 25.0 s.c. 2-3 h 66.6 12 MCE 1.0 s.c. 24 h 28.0 Mepyramine 25.0 s.c. 2--3 h 11 Cortisone 100.0 i.m. 24 h 36".2

* Rats giving a hypotension ranging from 1 to 10 mm Hg were considered protected. ,_

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192 Fregnan and GlOsser, Synergistic Action of an : TABLE II i_ Anti-anaphglactic eHects o/parious drugs in sensitized mice .4

_. % mortality " Interval between within

, N ° of Antagonists Dose Route the administration a0-60 rain after mice mg/kg of antagonists and challenge with the challenge I ml of horse serum

115 Control - - - 84.0 27 MCE 1.0 s.c. 24 h 88.8 38 MCE 1.0 s.c. 24 h 73.6 MCE 1.0 s.c. 2-3 h 70 MCE 10.0 s.c. 24 h 71.4 20 LSD 10.0 s.c. 2--3h 70.0 48 Mepyramine 25.0 s.c. 2-3 h 70.8 21 Promethazine 1.0 s.c. 2-3 h 71.4 20 Promethazine 10.0 s.c. 2-3 h 45.0 22 Promethazine 25.0 s.c. 2-3 h 22.7 41 Cortisone 10.0 i.m. 24 h 82.2 20 Cortisone 100.0 i.m. 24 h 0 20 Acetylsalicylic acid 2.5 i.p. 30-45 rain 85.0 22 Acetylsalicylic acid 10.0 i.p. 30--45 rain 82.0 34 Acetylsalicylic acid 100.0 i.p. 30-45 rain 82.3 20 MCE 1.0 s.c. 24 h 75.0

Mepyramine 25.0 s.c. 2-3 h 56 MCE 10.0 s.c. 24 h 50.0 ,, Mepyramine 25.0 s.c. 2-3 h 20 MCE 1.0 s.c. 24 h 55.5 MCE 1.0 s.c. 2--,3 h i Promethazine 1.0 s.c. 2-3 h 23 MCE 10.0 s.c. 24 b 39.2 Prometh_zine 10.0 s.c. 2-3 h 22 MCE 10.0 s.c. 24 h 13.6 - Promethazine 25.0 s.c. 2-3 h . 25 MCE 10.0 s.c. 24 h 0 :: Promethazine 10.0 s.c. 2-3 h ( Cortisone 10.0 i.m. 24 h t 20 MCE 1.0 s.c. 24 h 15.0 ( MCE 1.0 s.c. 2-3 h Promethazine 1.O s.c. 2-3 h -' ": Cortisone 10.0 i.m. 24 h _'_-:" .- - _' .- _ 7"," ._7.- ..... _ .C.,"£.'.,_",.-. .,W"."..5 _'2. '_.'_ ::'-;G,;_i:_'&_-_-'J_:k_; " _o _';:A_ _- :? _ "_,..'

Anti-5-hydroxytryptamine, Anti-histamines, and Cortisone... 193

TABLE III

Anti-anaphylactic activity o[ MCE, Chlor-phenlramlne, and Cortisone in mice

% mortality Interval l)etween ,atithin

N ° of Antagonists Dose Route the administration 30-60 rain alter mice mg/kg of antagonists and challenge with the challenge 1 ml of horse Berunl

66 Control - - - 88 40 MCE 1.0 s.c. 24 h 88 MCE 1.0 s.c. 2-3 h

27 Chlor-pheniramine 1.0 s.c. 2-3 h 92

45 Chlor-pheniramine 10.0 s.c. 2-3 h 67 t 63 Chlor-pheniramine 25.0 s.c. 2-3 h 63 36 Cortisone 10.0 i.m. 24 h 78 35 MCE 1.0 s.c. 24 h 51 MCE 1.0 s.c. 2-3 h

Chlor-pheniramine 25.0 s.c. 2--3 h 36 MCE 1.0 s.c. 24 h 33 MCE 1.0 s.c. 2---3 h

Chlor-pheniraraine 25.0 s.c, 2--3 h Cortisone 10.0 i.m, 24 h 35 MCE 1.0 s,c, 24 h 71 MCE 1.0 s.c. 2--3 h _ Cortisone 10.0 i.m. 24 h

35 Chlor-pheniramine 25.0 s.c. 2--3 h 58 Cortisone 10.0 i.m. 24 h

E][ect of acute treatment with MCE and other drugs on fatal anaphylaxis in mice Table II and III summarize the results of these experiments. The two anti-5-HT, MCE and LSD 25 (10 mg/kg) and the two anti-histamines mepyramine (25 mg/kg} and chlor-phenir._mine (1-25 mg/kg), each injected alone, afforded negligible protection from fatal anaphylatic shock and the incidence of mortality was only slightly less than in untreated sensitized mice. On the other hand promethazine, which is a strong anti-histamine with. some anti-5-HT properties, protected the animals almost completely but at doses giving sedation (25 mg/kg). At smaller doses (1 mg/kg)

i k 194 Fregnan and Ghisser, Synergistic Action of an promethazine had little or no effect in preventing anaphylactic sal shock. 100 mg/kg of cortisone also afforded complete protection, So while 10 mg/kg were not effective. No protective activity was ob- th_ served with acetylsalicylic acid from 2.5 to 100 m-/kg_ . gi_ re_ Sinergism of cu_li-cmaph!llactic e/[ecls o/ 3ICE, mepgramine," is chlor-pheniramine, promelhc_zine, and cortisone 5-] MCE, mepyramine, clHor-pheniramine, prometazine, cortisone, ev, given in doses scarcely or not active when injected alone, showed Co a considerable mutual reinforcement of their ability to inhibit tic fatal anaphylaxis in mice when 'given together. Table II shows that Scso: mice pretreated with MCE (1-10 m,,'/kg) were highly protected ad when also injected with either mepyramine (25 mg/kg) or pro- of methazine (1 mg/kg). However by increasing the dose of prome- TI thazine (10-25 mg/kg) in the associations only a slight improve- w:, ment has been observed. The best protection was achieved when cortisone (10m-/ke/_,_, was sin-mttaneously administered with MCE ar (1-10 mg/kg) and promethazine (1-10 mg/kg), tocr A further demonstration of the synergistic action of anti-5-IiT, anti-histamine, and corticosteroid is given in Table III. In this w case chlor-pheniramine is the anti-histaminic drug used. it can be m seen again that the best protection against fatal anaphylaxis in ti' mice is afforded by the triple association of 3ICE, chlor-phenira- w mine, and cortisone (always administered at doses which by tbem- d_ seh, es are almost inactive). The association of two drugs either s( MCE mad chlor-pheniramine or chlor-pheniramine and cortisone d, also affords some protection which is however inferior to that p givenby the tripleassociation, sl P a Di.}cussion ti

It is well known that anaphylaxis is the result of a complex x_ h mechanism involving numerous factors; and for this reason a mixture of drugs or a single drug antagonizing these changes can afford the best protection. Good evidence of this point of view is given by Halrmrn et al. (13) in mice, Goadbtj and Smith (12), and Collier ct al. (6) in guinea-pigs, who observed that a mixture (: of two specific antagonists with a different action (mepyramine and LSD 25, mepyramine and cortisone, mepyramine and acetyl- t, Anti-5-hydroxytryptamiAnne,tl-histamineasnd, Cortisone... I{}5 I salicylic acid) is much more active than the single antagonists. Some drugs with less specific mechanism of action (i.e.; prome- is thazine and cortisone) are found to be quite active, even when [t given alone, but at high doses which usually have harmful side reactions. In fact the protective effect of promethazine given alone i is probably due to its manifold properties: anti-histaminic, anti- i 5-HT, and to other non specific effects on capillaries (13). How- ! ever promethazine is active with doses which give sedation (7). Cortisone seems to afford a very good anti-anaphylactic protection either by decreasing the electrolyte values of Na, K, C1 in i some tissues (7) or by influencing the metabolism of the lipids (9). !, Schayer eL al. (18) and Hicks and West (14) also suggested that _ adrenal cortical hormones and cortisone lower the tissue reserves of histamine and 5-HT by decreasing the rate of their formation, i:r. The anti-anaphylactic effect of cortisone given alone is present wRh doses which may give serious untoward reactions in animals and in human beings, specially those associated with edema, increased blood pressure, increased glycosuria, lowered resistance to certain infectiousdiseases,peptic ulcers (5,15, 16, 20). _" Our data strenghten these facts and show that single drugs with a specific mechanism of action (such as MCE, LSD 25, :: mepyramine, and chlor-pheniramine) are inactive or scarcely active when given alone but that the same drugs become quite active when administered simultaneously. We also confirmed that other drugs with numerous actions (such as promethazine and cortisone) afford good protection, even when given alone, but at high doses. On the other hand wheia MCE, promethazine (or chlor- pheniramine), and cortisone are administered together, even in such doses which by themselves affect the development of anaphylaxis only moderately or not at all, they become greatly active and show a strong mutual reinforcement of their anti-anaphylactic properties. This allowed us to use low doses of promethazine which did not give sedation and low doses of cortisone which may

havefewerharmfulside-effects. zF i: Summary _ .

The anti-anaphylactic effects of a new highly specific anti-5-tIT drug __I' (MCE) has been investigated in actively sensitized rats and mice. t MCE, LSD 25, mepyramine, and chlor-pheniramine have negligible pro- { tective activity when administered alone; while, when an anti-5-HT and an _: i'

h_ J 196 Fregnan and Gldsser, Synergistic Action of an

anti-histamine are given together, they afford a good protection by a mechanism _' of synergism•

l Promethazine and cortisone (which have many properties and are less specific), protect rats and mice even when given alone, but at high dosages which may have harmful side reactions. MCE, promeihazine (or chlor-pheniramine), and cortisone given simul- ! taneously in doses, which by themselves are inactive or scarcely active, afford an excellent protection against fatal anaphylaxis in mice. , Acetylsalicylic acid is without any protective effect in mice.

Z usamme nf assunff

Die anti-anaphylakfische Wirkung eines neuen hoch spezifiseh wirksamen Serotoninantagonisten (MCE) wurde an sensibilisierten Ratten und Miiusen ' untersucht. i: ! MCE, LSD 25, Mepyramin und Chlorpheniramin allein verabreicht, be- j sitzen keine Schutzwirkung. Werden jedoch ein Serotoninantagonist und ein _,_ Antihistamin gleichzeitig verabreicht, so zeigt sich, wohl auf Grund einer • synergistisehen Wirkung, ein guter protektiver Effekt. Promethazin und Cortison, die mehrere Wirkungsqualifiiten besitzen und weniger spezifisch sind, allein verabreicht, wirken anti-anaphylaktisch; die hierzu ben6tigten Dosen rufen aber schon nachteilige Nebenwirkungen hervor. ! MCE, Promethazin (oder Chlorpheniramin) und Cortison in Dosen, die ! sclbstunwirksam oder nut schwach wirksam sind, zusammen appliziert,be. _. wirken bei Miiusen einen ausgezeichneten Schutz gegen die Anaphylaxie. _ Acetylsalicylsiiure war bei M[iusen wirkungslos.

'!," t' ' Rdsumd , ' , Nous avons dtudid Faction anti-anaphylactique d'un nouveau mddieament iJ anti-5 HT (MCE) hautement spdcifique sur les rats et les souris activement ! sensibilis6s. . MCE, LSD 25, mdpyramine et chlor-pheniramine administrds seuls donnent _: une protection n_.gligeable ; alors que si on administre anti-5-HT et anti-hista- ! mine ensemble, on obtient une bonne protection par un mdcanisme synergique. i Promethazine et cortisone (qui ont beaucoup de propridt_s et sont moins I, spdcifiques) prot_gent les rats et les souris m6me s'ils sont administrds seuls

_- _ mais 'h doses 61evdes ce qui peut entrainer des r6actions secondaires dange- : reuses. MCE, promdthazine (on chlor-ph_niramine), et cortisone administrds simultandment ._ des doses qui par elles-m_mes sont inactives ou h peine actives, donnent une protection excellente contre ranaphylaxie mortelle des souris.

L'acide acdtylsalicylique ne protege pas les souris.

i Anti-5-bydroxytryptamine, Anti-histan_ines, and Cortisone... 197

]-_CfcFcnces

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i 198 Fregnan and Gldsser

19. Tokuda, S. and Weiser, R. S.: Studies on the role of serotonin and mast _ cells in anaphylaxis of the mouse produced with soluble antigen-antibody complexes. J. Immunol. 86:292-301 (1961). 20. Treadwell, B. L. J.; Sever, E. D.; Savage, O. and Copeman, W. S. C.: Side effects of long-term treatment with corticosteroids and corticotrophin. I; Lancet 1:1121-1123 (1964). I ! 21. Udenlriend, S. and WaaUces, T. P.: On the role of serotonin in anaphylaxis, i Mechanism of hypersensitivity, p. 219. Henry Ford Hospital, Intern. Sym- i_ posium (Little Brown. Boston/Toronto 1959). i 22. Waalkes, T. P. and Coburn, H.: The role of platelets and the release of serotonin and histamine during anaphylaxis in the rabbit. 3. Allergy 30: 394-407 (1959). _ 23. West, G. B.: A pharmacological approach to allergy. Progress in drug ] research, Vol. 3, p. 409-449 (Birkh_user, Basel/Stuttgart 1961). ,.! Authors' address: Dr. B. G. Fregnan and Prof. A. H. GlOsser, Farmitalia, Laboratori I:ticerche, i via dei Gracchi 35 Mitano (Italy).

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