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 mor- tality 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 , > , ii • 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 corti- sone 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 af- forded 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. ,_ $_" I :-_',:_z_ o,= _, _z____,_<_:_'_._£'._): ' _=_:,_L_¢_.:,X,.-:_'_,._,-_,j,_ ,, :,,,_:__ i 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.