ANTI-ANAPHYLACTIC ACTION OF CHLORPROMAZINE
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1 Keio Journal of Medicine Vol. 6. No. 1. March ANTI-ANAPHYLACTIC ACTION OF CHLORPROMAZINE KYUHEI NAKADATE*, KIYOSHI NAKAYAMA and SHIGEAKI KAMIKATAHIRA Department of Legal Medicine, School of Medicine, Keio University (Received on February 9, 1957) Chlorpromazine, that is chemically 3-chlor-10-(3-dimethylaminopropyl)-phenothiazine hydrochloride, first synthesized by Rhone-Poulenc, Specia has been very recently employed for artificial hibernation therapy, making this drug important in the field of psychiatry. Ever since, this drug has been used in clinical fields with successful therapeutic effects all over the world as well as in France on account of its various effects such as anticonvulsive, analgetic, sedative and anti-hypertention effects which are ascribed to its blocking ability of autonomous nerve system(1,2). The pharmacologic mode of action of Chlorpromazine, however, has not yet been fully elucidated partly due to its complicated and multi-directional effects, although it has been studied very extensively. It was believed that this drug would offer a powerful clue to the understanding of the importance of autonomous nerve system which still has many unsolved problems and also it Was felt of interest to do some experimental studies with Chlorpromazine on the vital defense reactions among which special emphasis has been given on secondary shock by the author's laboratory in the past years(3). The present studies were undertaken to investigate the effects of this drug on the so-called anaphylaxis (both systemic and local). As a result, anti-anaphylactic action of Chlorpromazine was found and the present paper will discuss on its mode of action and the genesis of anaphylaxis. MATERIALS AND METHODS As the experimental animals, white rabbits weighing about 2.5kg and guinea pigs of around 350g were used. Rabbits were immunized 6 times by intravenous injection of 2ml of horse serum each time and the precipitin titers above 1:10,000 were obtained. Guinea pigs were passively immunized with anti- * Professor of Leagl Medicine.
2 2K. NAKADATE, K. NAKAYAMA and S. KAMIKATAHIRA horse-serum rabbit serum by intraperitoneal inoculation on a previous day of the following experiments. As for Chlorpromazine, the 0.5% solution of Yoshitomi Seiyaku's product was used. EXPERIMENTAL RESULTS I. Inhibitory effect of Chlorpromazine against the lethal, A. Preliminary Experiments. passive anaphylactic shock in guinea pigs In the first phase, the minimal dose of horse serum and anti-horse-serum rabbit serum required to kill guinea pigs by causing complete passive anaphylactic shock was determined (Table 1). When 0.2ml of antigen was intravenously administered to the animal to which 0.3ml of anti-serum per 100g of body weight had been given intraperitoneally, only two out of 5 animals died of typical anaphylactic shock (lethality 40%). When 0.3ml of anti-serum and 0.3ml of antigen per 100g of body weight were injected to guinea pigs, three out of 5 succumed to typical anaphylactic shock (lethality 60%). In all of these experiments, anti-serum with precipition titer 1:10,000 was used. When 0.5ml of anti-serum and 0.3ml of antigen per 100g of body weight were administered, Table 1 Inhibitory effect of Chlorpromazine against the death of guinea pigs caused by anaphylactic shock
3 ANTI-ANAPHYLACTIC ACTION OF CHLORPROMAZINE3 typical anaphylactic shock killed three out of 5 (lethality 60%). When 0.5ml of anti-serum and the same amount of antigen were given, all of the 5 animals tested died, causing typical anaphylactic shock. By these preliminary experiments, it was found that when 0.5ml of antihorse-serum rabbit immune serum per 100g of body weight was injected intraperitoneally to guinea pigs weighing about 350g and 0.5ml of horse serum was intravenously injected after 24 hours, all of the animals died, developing complete passive anaphylactic shock. Accordingly, in the following experiments, both anti-serum and antigen were given in 0.5ml respectively per 100g of body weight in guinea pigs. B. Test Experiments. In order to study the effect of Chlorpromazine on the passive anaphylactic shock death, 0.5ml of horse serum was intravenously injected 24 hours after the sensitizing treatment of guinea pigs by intraperitoneal injection of antihorse-serum rabbit immune serum in the dose of 0.5ml per 100g body weight. Survival rate of animals were examined, when 1.0, 2.0 or 4.0mg/kg of Chlorpromazine was injected into femoral muscles 10 or 30 minutes prior to the antigen injection and also when the mixture of this drug of these doses with the antigen was injected (Table 1). antigen a) When Chlorpromazine was intramuscularly injected 30 minutes before injection. As mentioned above, 1.0, 2.0 or 4.0mg/kg of Chlorpromazine was injected into femoral muscles of guinea pigs 30 minutes before the antigen was intravenously injected to the animals which had been previously sensitized with antiserum. When 1.0mg/kg was administered, 2 cases out of 5 died of typical anaphylactic shock. In other words, when 1.0mg/kg of Chlorpromazine was given 30 minutes before the antigen injection, the survival rate was as low as 60%. When the animals were pretreated with 2.0mg/kg, 4 cases out of 5 showed slight shock symptoms such as erection of hails and scrubbing the nose but none of them succmbed and they became quiet after a short time and recovered to normal after about 5 hours' stuporous state. In other words, intramuscular injection of 2.0mg/kg of Chlorpromazine 30 minutes before antigen injection was 100% of survival rate and is able to protect the animal from death due to passive anaphylactic shock. When as much as 4.0mg/kg was administered, 2 cases out of 4 showed slight shock symptoms and all of them recovered to normal from stuporous state for 24 hours, showing again 100% of survival rate. It is concluded, therefore, that the intramusclar injection of more than 2.0mg/kg
4 4K. NAKADATE, K. NAKAYAMA and S. KAMIKATAHIRA of Chlorpromazine is able to completely prevent the animals from the death due to passive anaphylactic shock. antigen b) When Chlorpromazine was intramuscularly injected 10 minutes before injection. Chlorpromazine in the dose of 1.0, 2.0 or 4.0mg/kg was injected into femoral muscles of guinea pigs 10 minutes before the antigen was intravenously injected to the animals which had been previously sensitized with anti-serum. When 1.0mg/kg was administered, all of 5 cases showed anaphylactic shock symptoms within 5 minutes after antigen injection. When I out of them died of typical anaphylactic shock and the others became after a short time stoporous state and recovered to normal after about 3 hours. In other words, intramuscular injection of 1.0mg/kg of Chlorpromazine 10 minutes before antigen injection was 80% of survival rate. When the animals were pretreated with 2.0mg/kg, all of 5 cases showed shock symptoms within 5 minutes after antigen injection but only I out of them died. The others recovered to normal after 5 hour's stoporous state. In other words, the survival rate of 2.0mg/kg intramuscular injection of Chlorpromazine 10 minutes before antigen injection was 80%. When as much as 4.0mg/kg was administered, 2 cases out of 4 showed slight symptoms within 5 minutes after antigen injection and all of them recovered to normal after 24 hour's stoporous state. In other words, intramuscular injection of 4.0mg/kg of Chlorpromazine 30 minutes before antigen injection is able to completely protect the animals from death due to passive anaphylactic shock. In the present experiments, however, the administration of the same dose of Chlorpromazine 30 minutes before antigen injection was affarently more effective. antigen c) When Chlorpromazine was intravenously injected at the same time as injection. Chlorpromazine in the dose of 1.0, 2.0 and 4.0mg/kg respectively was mixed with antigen and intravenously injected to guinea pigs previously sensitized with anti-serum and the influence of this drug on passive anaphylactic shock was observed. When 1.0mg/kg was given, 4 cases out of 5 developed slight anaphylactic symptoms within 5 minutes after antigen injection but all of them fell into stuporous state and recovered to normal after 3 hours. It is seen that in the injection of 1.0mg/kg of Chlorpromazine at the same time as antigen the survival rate was 100%. In the group of which 2.0mg/kg of the drug was given, 3 out of 4 showed slight anaphylactic symptoms but all of them recovered within 5 hours after stuporous state, showing again 100% of survival rate. When as much as 4.0mg/kg was administered, 4 out of 5 showed only slight
5 ANTI-ANAPHYLACTIC ACTION OF CHLORPROMAZINE5 symptoms and recovered to normal from stuporous state for 24 hours. It is concluded, therfore, that the intravenous injection of the mixture of antigen and more than 1.0mg/kg of Chlorpromazine is able to prevent the death of guinea pigs due to passive anaphylactic shock. As mentioned above, the protective effect of Chlorpromazine against the death of guinea pigs caused by passive anaphylactic shock. The results obtained are summarized as follows. When this drug is given 30 minutes before antigen injection, more than 2.0mg/kg will protect the animals and when 10 minutes before, 4.0mg/kg will also do. When this drug is mixed with antigen, 1.0mg/kg will be sufficient. 2. Inhibitory effect of Chlorpromazine on local anaphylaxis visualized by Magnus test with small intestine About 5cm long piece of ileocaecal portion was cut out from the rabbits guinea pigs which had been either actively or passively sensitized by the above mentioned procedures and the movement of the intestinal piece that was kept in 100ml Tyrode solution in Magnus apparatus with proper temperature and oxygen supply was recorded with kymographion. When 0.5ml of antigen horse serum was added to the medium, marked activation of intestinal movement and increased tonus are caused by local anaphylaxis, which fact is already well known. In the presnet studies, Chlorpromazine was added before, after or with antigen addition and the inhibitory effect of this drug on local anaphylaxis was examined. In all of the experiments reported below, 1.0mg of Chlorpromazine was added to Tyrode solution. and A. Influence of Chlorpromazine on passive local anaphylaxis in guinea pigs. When 0.5ml of horse serum was added to Tyrode solution in which intestinal segment was kept, the segment which was removed from a guinea pig that had been sensitized before 24 hours by intraperitoneal injection of antihorse-serum rabbit serum showed remarkable increased intestinal tonus and peristalsis (Figure 1). This results shows the passive anaphylaxis in removed Fig. 1 Anaphylaxis in guinea pig intestine.
6 6K. NAKADATE, K. NAKAYAMA and S. KAMIKATAHIRA Fig. 2 Influence of Chlorpromazine on the anaphylaxis in guinea pig intestine. Fig. 3 Influence of Chlorpromazine on the anaphylaxis in guinea pig intestine. intestine. In the next place, Chlorpromazine was added before or after such local anaphylaxis developed. a) When Chlorpromazine was added after antigen. When Chlorpromazine was added to Tyrode solution in 0.1mg/ml where intestinal piece under passive anaphylaxis caused by the addition of antigen horse serum was dipped, intestinal movement and tonus suddenly disappeared (Figure 2). This effect was probably brought about by the suppression of essential intestinal movement and tonus by a large dose of the drug and it is considered that Chlorpromazine has symptomatic inhibitory effect on local anaphylaxis. b) When Chlorpromazine was added before antigen. When removed intestine was showing normal movement, 0.5mg of Chlorpromazine was added. In this case, normal movement was not affected almost at all or contractility apparently showed slight increase (Figure 3). When
7 ANTI-ANAPHYLACTIC ACTION OF CHLORPROMAZINE7 another 0.5mg of the drug was added to the intestine in such a state, both tonus and contractility disappeared almost completely. The following addition of 0.5mg of antigen horse serum did not cause any expected anaphylactic signs. In this fashion, Chlorpromazine showed a prophylactic effect against local anaphylaxis. Furthermore, when 0.05mg of Pilocarpin which stimulates parasympathic nerves was administered to this intestine, intestinal tonus and contraction increased, showing that its excitability was not yet affected. From what has been mentioned above, Chlorpromazine is considered to have an inhibitory effect against passive local anaphylaxis in guinea pigs both symptomatically and prophylactically. B. Effect of Chlorpromazine on active local anaphylaxis in rabbits. When 0.5ml of antigen horse serum was added to the Tyrode solution where intestinal segment was dipped that had been cut out from a rabbit with precipitin titer higher than 1:10,000 immunized as usual with horse serum, its tonus and peristalsis were clearly increased as shown in Figure 4. This fact proves the development of active local anaphylaxis in isolated intestinal segment. In the next step, Chlorpromazine was added to the solution before or after such local anaphylaxis took place in order to observed the effect of the drug. Fig. 4 Influence of Chlorpromazine on the anaphylaxis in rabbit intestine. Fig. 5 Influence of Chlorpromazine on the anaphylaxis in rabbit intestine.
8 8K. NAKADATE, K. NAKAYAMA and S. KAMIKATAHIRA Fig. 6 Influence of Chlorpromazine on the anaphylaxis in rabbit intestine. a) When Chlorpromazine was added after antigen. When 1.0mg of Chlorpromazine was added to the Tyrode solution where intestinal segment had been undergoing active anaphylaxis caused by the addition of antigen horse serum, increased tonus and peristalsis were suppressed to nearly normal state as shown in Figure 4 or 6. From this results, it is considered that Chlorpromazine in dose larger than this is symptomatically effective in suppressing the already caused local anaphylaxis. b) When Chlorpromazine was added at the same time as antigen. When 0.5ml of antigen and 1.0mg of Chlorpromazine were added at the same time to the intestine showing normal movement, the normal movement became slightly irregular as shown in Figure 5, the expected anaphylactic signs were not developed but the intestinal movement rather decreased. Chlorpromazine is considered to have a prophylactic effect against local anaphylaxis of intestine, if it is administered in the dose larger than used in this experiment. c) When Chlorpromazine was added before antigen. When 1.0mg of Chlorpromazine was added to intestinal segment in normal movement, the movement is strongly suppressed as shown in Figure 6. The intestine thus treated did not show any expected anaphylactic signs, when 0.5ml of antigen was added. Chlorpromazine is considered to have a prophylactic effect against active anaphylaxis of intestine, when it is used in the dose larger than used in this experiment. As mentioned above the inhibitory effect of Chlorpromazine against passive local anaphylaxis in guinea pigs and active one in rabbits was studied using Magnus technique. In all of the cases tested, the addition of more than 1.0mg
9 of Chlorpromazine ANTI-ANAPHYLACTIC ACTION OF CHLORPROMAZINE9 to the Tyrode solution is very effective against local anaphylaxis of intestine both prophylactically and symptomatically. 3. Inhibitory effect of Chlorpromazine on Arthus reaction in rabbits Abdominal skin of rabbits immunized as usual with horse serum and with precipitin titers higher than 1:10,000 and 0.1, 0.2 and 0.3ml respectively of antigen horse serum was intradermally injected and 0.5mg of Chlorpromazine or 0.25mg of the drug solved in physiologic saline was mixed with antigen and injected on the opposite side. The inhibitory effect of Chlorpromazine on Arthus reaction such as local reddening, induration, edema, necrosis and so on which develop after 24 hours was studied. A. Preliminary experiment. Rabbit immunized with horse serum and with precipitin titer of 1:16,000 was used. In order to determine the minimal antigen dose of intradermal injection required for causing Arthus reaction in 100%, antigen horse serum was injected in 0.3, 0.2 and 0.1ml respectively as shown in Figure 7. When the animal was examined after 24 hours, fairly intensive edema was generally found (a) (b) Fig. 7 Arthus reaction in rabbit. (a) Fig. 8 Influence of Chlorpromazine on the Arthus reaction in rabbit. (b)
10 10K. NAKADATE, K. NAKAYAMA and S. KAMIKATAHIRA in the abdominal subcutaneous tissue. At the sites of injection of 0.3, 0.2 and respectively on the left side. From this preliminary experiment, the intradermal injection dose of antigen necessary for causing Arthus reaction is considered to be around 0.2ml in rabbits with about 1:10,000 precipitin titers. B. Test experiments. a) Into the abdominal skin of a rabbit with the precipitin titer of 1:10,000, 0.2ml of antigen was injected on the left side and the mixture of 0.2ml of the drug and 0.5mg of Chlorpromazine on the right. As shown in Figure 8, redden- whereas no abnormal finding was obstained on the right. In this fashion, local injection of 0.5mg of Chlorpromazine is considered to inhibit Arthus reaction. b) Into the abdominal skin of a rabbit with the precipitin titer of 1:10,000, 0.3, 0.2 and 0.1ml of antigen only were injected on the left side as shown in Figure 9, and the mixture of 0.5mg of Chlorpromazine with 0.3, 0.2 and 0.1ml Fig. 9 Influence of Chlorpromazine on the Arthus reaction in rabbit. Fig. 10 Influence of Chlorpromazine on the Arthus reaction in rabbit.
11 ANTI-ANAPHYLACTIC ACTION OF CHLORPROMAZINE11 respectively were injected on the right. When the animal was examined after 24 hours, almost no abnormalities were found in the skin on the right side, respectively were observed. But at the site of injection of 0.1ml antigen, there was no visible reaction either on the left side (Figure 9). Local injection of 0.5mg of Chlorpromazine is considered to be able to inhibit Arthus reaction completely. c) Into the abdominal skin of a rabbit with the precipitin titer of 1:10,000, 0.3, 0.2 and 0.1ml respectively of antigen only were injected on the left side and the mixtures of 0.3, 0.2 and 0.1ml respectively of antigen with 0.25mg of Chlorpromazine two times diluted with physiologic saline were injected on the right side in the craniocaudal order. After 24 hours, reddening and induration and induration with obscure surrounding at the contral site. While no abnormal change was observed at the caudal site. On the right side, reddening and indura- found at the other two sites on this side (Figure 10). When Chlorpromazine was used in this type of experiment, it seemed to be capable of preventing Arthus raction in the dose of 0.25mg but the inhibitory effect was weaker than when 0.5mg was given. The inhibitory effect of Chlorpromazine against the Arthus reaction in rabbit skin was studied as mentioned above. From the results obtained, the injection of more than 0.5mg of Chlorpromazine mixed with antigen into local skin is considered to show complete and consistant inhibitory effect against Arthus reaction. DISCUSSION In the present experimental studies on the influence of Chlorpromazine on anaphylactic shock, it was elucidated that this drug protects the animals from death due to anaphylactic shock, suppresses local anaphylaxis in intestine both prophylactically and symptomatically and also inhibits the Arthus reaction in skin. In other words, Chlorpromazine was found to have a remarkable antianaphylactic effect. It has been disclosed that Chlorpromazine is effective to the other secondary shocks in various aspects of defence mechanism. It leaves no doubt that the participation of the so-called hypophysis-adrenocortical system as well as of the autonomous nerve system is very important in the mechanism of the development of shock and, assuming also from the complexity of the
12 12K. NAKADATE, K. NAKAYAMA and S. KAMIKATAHIRA cause of shock, it is very difficult to discuss on shock and its inhibition. problems reported in the present paper are merely concerned with the systemic and local effects of Chlorpromazine on the anaphylactic shock caused by the antigen-antibody reaction with horse serum antigen and so it would not be reasonable to extend the discussion to the other shocks. Concerning the mechanism of the development of anaphylactic shock, histamine (Schachter(4)), peptone (Mita(5)), acethylcholine (Nakamura(6)) and myoglobin (Ueno(7)) have been ascribed to the causative poisons and especially the histamine theory accumulated much evidence in these years but still none of these substances is sufficient to disclose the mechanism of anaphylactic shock. Also from the present experimental results, it is impossible to discuss on the mechanism of anaphylactic shock but when we compare them with a series of symptoms shown by the hyperfunction of the organs which are innervated by parasympathic nerve system as pointed by Wells(8), the following discussion will be allowed. When antigen-antibody reaction arisen in vivo shows a pathologic tendency, the so-called anaphylaxis will be caused, while if it has a protective nature to the body, immunity will result. The cardinal symptoms of smooth muscles (for example, bronchial spasm or abnormal excitation of intestine as shown in the present paper), hyperexsection, vasomotory hyper-function, change of permiability (for example, hypotension, hemoconcentration, decrease of circulating blood volume or Artus reaction in skin as studied above) and so on, showing the process to shock. It is assumed that the anaphylaxis caused brings about dysharmonie of the function of autonomous nerve system (especially parasympathicus) and as a result, if we follow the poison theory, poisonous substances are formed in the body which will develop shock, thus causing a vicious circle. In pharmacology of Chlorpromazine, its central action has been emphasized in blocking the autonomous nerves and in its clinical use it has been mostly used for hibernation therapy, sedative or anti-hypertention and its use as antishock remedy has been very recently started. While its anti-histaminic action has been found fairly strong, its suppressing effect against acethylcholine or parasympathic nerves and especially its peripheral action have not been taken into much consideration. The present studies disclosed that Chlorpromazine has anti-parasympathic effect which is powerful centrally as well as peripherally, because it showed inhibitory effect against the death due to anaphylaxis in intestine and also against Arthus reaction in skin. When Chlorpromazine is administered to intestine which has rather complex innervation partly on account of Auerbach's nerves plexs, this drug stimulates the normal movement of intestine The
13 ANTI-ANAPHYLACTIC ACTION OF CHLORPROMAZINE13 in small doses but suppresses in larger amounts. The blocking of autonomous nerves by Chlorpromazine is accomplished by such a complicated mechanism and the pharmacology of this drug is believed to offer an interesting but complicated problem and it is thought to be a good experimental tool in studying the various aspects of secondary shock, increasing its practical value in the clinical medicine. The effect of Chlorpromazine SUMMARY which is a blocking agent of autonomous nerves upon anaphylactic shock were experimentally studied and the following inhibitory actions of this drug were found. 1. The administration of more than 1.0mg/kg of Chlorpromazine was able to protect guinea pigs from the death caused by passive anaphylactic shock. 2. Chlorpromazine inhibited both prophylactically and symptomatically the passive and active local anaphylaxis of the intestine of guinea pigs and rabbits as observed with Magnus technique. 3. Chlorpromazine prevented the Arthus reaction in rabbit skin. 4. From the above results, the anti-anaphylactic actions of Chlorpromazine are considered to be effected both centrally and locally. A part of the present paper was reported at the 40th General Meeting of the Japanese Society of Legal Medicine in REFERENCES 1. Laborit, H.: Reaction Organique a L'Agression et Choc, Laborit, H. & Huguenard, P.: Pratique de L'Hibernotherapie, Nakadate, K.: Japanese Medical Journal, No. 1672, Schachter, M.: Anaphylaxis and Histamine Release in the Rabbit; Brit. J. Pharmacol., 8: 412, Mita, T.: New Findings in the Field of Serology, 1936 (in Japanese). 6. Nakamura, K.: The Nature of Allergy, 1951 (in Japanese). 7. Ueno, M.: Physiology and Pathology of Myoglobin, 1951 (in Japanese). 8. Wells, H.G.: Chemical Aspects of Immunity, 1929.
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