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The effect of meta-iodo benzyl cinnamate on the course of experimental tuberculosis in the guinea pig Horn, Howard J. 1935

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T i l l mWBGf OF IIETA-iODO BEHZYI ClfflMMAT OH THE COURSE OF EXPERlEEIfAI fUBERCULOSIS IK THE GUI SEA PIG. hj Hofard J. Horn* B'.A. A t h e s i s submitted f o r the "Degree of Master of A r t s i n the ' Department of Ba c t e r i o l o g y September 30., 1955. 1.—Introduction* In a discussion on various attempts at chemotherapy in Tuberculosis Galmette makes the following statements; "It must he recognized, that un to the present, despite the great number of attempts made to discover among chemical agents a substance capable of arresting the developement of experimental tuberculosis in the -guinea pig a»d the rabbit, these efforts have been i n vain, But- this i s not a reason for discouragement. Certain attempts among those which we have cited deserve further attention*-- I t w i l l be desirable, for example, to give further consideration -to those with iodized compounds which, i f they do not appear f u l l of promise, give, nevertheless, some definitely favor-able results. tf-7e must always remember that i t i s useless, perhaps i t may be, dangerous, to ia.|ectat random, as has been too often dose, such and such a.chemical into patients with the vague hope of discovering a specific activity, This is a practise- which should be condemned. Experimentation alone, methodically conducted upon animals sensitive to tuberculosis, w i l l enable us to explore with profit the im-mense perspectives that chemotherapy offers." • The Department of Chemistry at this University has for several years, been building up different combinations of certain of the compounds which seem to have given -prom-ise of cheraotherapeutic value i n tuberculosis. These compounds Include .urea, benzyl a l c o h o l , -oinnamyl.-.-- c h l o r i d e , iodine, and a l l i e d compounds. The papers of the Department of Chemistry discuss i n f u l l the eojapottnds -synthesized -and the methods of synthesis employed. . i'Jae Department of'Bacteriology was then approached with a view to determining the therapeutic value of the drugs synthesized. This paper.being, i t i s hoped, the f i r s t of a series on the behavior in vivo of these compounds, is largely a description of the.technique and methods employed, although a complete description of the behavior of one drug i s included. Bearing in mind the tenets of Calciette i t was deter-mines to cover the ground slowly and thoroughly. It was decided to use large populations of guiaea pigs f o r the ex-periments , rather than inoculating a few at random, and a l s o to use a number of controls equal to that of the test population. There v/as- f i r s t to "be discovered the best method by which to administer the drugs* Method of Administration of Chemicals: The chemicals to he used in a preliminary series were benzyl einnamate, aeta-iodo benzyl cincarnate, and ethyl meta-iodo cinnamate. -These compounds (as can be found by reference to the papers of the Chemistry Department on their preparations) are a l l solids at room temperature (2S6C) and Insoluble in water. Their solubility in sub-stances usually employed as solvents for injection is very low. EmuIs i f i ea t i on without the use of an emulsifying agent vns not possible: thus i t was thought best not to use the drug in emulsified form as the protective colloid used as the emulsifying agent might very easily interfere with the action of the drug i t s e l f * It was found, however., that although the drugs did not melt until considerably above Dlood heat (37°C) they could be cooled while molten to blood heat without solid-ifying. It was therefore decided to inoculate the pure drug in molten state at 38°C. thus making sure that the effects, i f any, would be due to the chemical and to that alone, Intracardiac inoculation was f i r s t tried as being the most direct method. Two d i f f i c u l t i e s were encountered, however which rendered this system impractical. The f i r s t d i f f i c u l t y was that although recovery of uninoculated guinea p i g s from the anaesthetic used, whether ether of.chloro-form , was normal, the recovery of guinea pigs inoculated, w i t h the drug while under anaesthetic was very slow, and o f t e n unsuccessful. Since these drugs are highly soluble i n both ether and chloroform this phenomenon may have been due to some sort of loose .chemical., or even physical, combination between the drug and the anaesthetic. Certain individual guinea, pigs, however, did recover from inoc-ulations of as much as 0,» 75c. c, benzyl cinnamate or ineta-iodo benzyl cinnamate or 0.5 e.c. ethyl raeta-iodo• cinnamate-* The other d i f f i c u l t y encountered., however, was such as to majce this system of inoculation unsatisfactory. The needle of gauge large enough to pass the solten chemicals was not sssall enough i n outside diameter to ensure that the heart muscle wou!.d close the puncture, and the deaths by hem-orrhage were thus far too frequent to laajfce practicable inoculation by this method. The necessity for a needle of large, gauge also made intravenous inoculation, impossible. Intraperitoneal inoculation of the various compounds was then atteiapted and found to be highly satisfactory.. The guinea pigs stood inoculations of up to Locc* each of the chemicals without any loss of weight or other.:symptoms of distress., The technique was as follows." the chemical to he inoculated was p.laced in a sterile container and heated at 60°C» until liquid* The molten drug was then drawn up into a sterile syringe with a needle of 22 gauge and inoculated intraperitoneally into the guinea pig. The guinea pig had previously been prepared by clipping a space about one inch square on i t s abdomen and wiping with both alcohol and ..iodine solution* In f i n a l l y determining the route by which the drug was to be administered, consideration was also given to the fact that the drugs are highly insoluble. If the intra-cardiac or intravenous route were chosen, the total amount of comparatively insoluble material which could safely be given was obviously very small and the resulting assimilation of the soluble portion, i f any, into the system would be negligible.', On the ether hand, i f injections were given iatraperitonealiy large quantities of the drug could he administered, with the result that a greater total amount might possibly be; assimilated into the system. Care of Experimental Guinea Pigs. The experimental guinea pigs were jcept In individual cages and .weighed,, fed, and cleaned at regular intervals. In this group of experiments i t was decided for convenience' salce to perform these operations on Hon days, 9A.M. Vfed-needays 1 :%r> i>.M,, and Fridays 5:30 P.M. .The ration, given a f t e r weighing consisted of SQOgms. green feed (moist)-~lettuce, cabbage, and cauliflower leaves-. an'd':85 gms. crushed oats. Bo water was given as the fresh"*"*'"" greens were found to be sufficient as a source of moisture. The cages in every case ivere cleaned and washed thoroughly after each.weighing but before feeding. It was found that by feeding at longer intervals than one day, the weights did not show as great a day-to-day variation, since there was time for evacuation of the intest-ines after feeding. Preparation of Myc. tuberculosis for Inoculation. The t u b e r c l e b a c i l l i used were from a virulent cult-ure , freshly isolated from sputum at the Vancouver General Hospital through the kindness of Dr. H . K . 7- i t t s , Director of the laboratories. The cultures were a l l grown on Petra-gnani's medium at 37 C . A 15--day culture was washed off the medium•with sterile saline <0>87?f-) » using, a sterile Pasteur pipette.. The suspension wss well mixed by continued drawing up and ex-pelling with the pipette, and then allowing the large lumps to settle out before removal from the tube. The resulting suspension was then transferred to another sterile tube and diluted with sterile saline to a turbidity equivalent to Mac Par land Standard #4- After much experimentation i t was found that 0.25 e.c, of a 1/20 dilution of t h i s t u r b i d i t y i n o c u l a t e d i n t r a o e r i t oneally would cause the death of a guinea p i g a f t e r ap-proxilately 6 weeks. This, then, was the i n f e c t i n g dose used f o r the p r e l i m i n a r y t e s t s . For the aere conclusive experiment the culture used was f r o a the Tranquilie Sanatorium, isolated from pleural f l u i d , third subeultui-e. This was a more slowly growing organism and necessitated the use of an 86 day culture for maximum growth. A f t e r further txperimentatior i t was found best to al l o w the suspension of t u r b i d i t y #4 to settle for exactly ]0 minutes and then make the 1/20 d i l u t i o n from the super-natant suspension. This removed the majority of clumps of b a c i l l i , and gave a much finer suspension, thus i n c r e a s i n g the p e r i o d before death to approximately-12 weeks-, This therefore gave any possible therapeutic powers of the drug a much b e t t e r opportunity. Inoculation, whether of drug or tubercle b a c i l l i , was for convenience' sake always given before feeding, a period at which the intestines would be comparatively empty. Preliminary Experiments. To determine i f the drugs had any p r o p h y l a c t i c Prop-e r t i e s i t was decided to inoculate small numbers of guinea p i g s with the drug and tubercle b a c i l l i siauItanenasiy. I t 8. -was thought that t h i s perhaps would give some lead as to •' the r e l a t i v e value of the drugs and help to decide which -:f s e v e r a l should he used i n the snore extensive • experiment • .. 4$h& f o l l o w i n g table shows the detail® of the p r e l i m i n a r y experiments- . • ' jaurobcr of guinea (jigs Co impound i n o c u l a t e d Quantity of compound quantity of suspension of tubercle b a c i l l i i n o c u l a t e d Benzyl einnamate 1o Oc.c . 0,<> S5 c. e. 4 " Meta-iodo benzyl einnamate 1. 0 6*0* ' 0 •». &j «5 c * c » 4. E t h y l meta-iodo einn-aaiate Hone—C on t r o 1 s Of the controls, three averaged 47. days before death; the remaining control, however, remained alive for 77 days. 'Theguinea' pigs inoculated with ethyl meta-iodo oincarnate also lived for an average of 47 days after inoculation. The animals inoculated with benzyl einnamate averaged 62 days, one remaining alive for 73 days. The guinea pigs inoculated with metafiodo benzyl eiimamate gave by f a r the most promising results, averaging 62 days, one however, re-maining alive for 93 days. Due 'co the one control which was living at 77. days after inoculation these results did not seem to be even in-dicative of any conclusions. This guinea pig, however, on autopsy, ear-puncture, proving that at some time this animal had been ear-tagged. After a check-up of animals i t was found that this was a guinea pig which had been tagg-ed when inoculated intracardialiy 71 clays previous to the inoculation with tubercle b a c i l l i with 0.7 c.c. benzyl einn-amate for a toxicity test. This, then, caused the eliminat-ion of this animal: thus the experiments gave some Indications of slightly better prophylaxis from meta-Iodo benzyl cirumin-ate than from benzyl airnamate. Such inconclusive results definitely upheld, the necess-ity of employing much larger populations for the next ex-periment For the large scale experiment i t was decided to try aeta-iodo benzyl eizmamate as i t had given some indication of slightly better prophylaxis then benzyl cinnaraate. It was also thought that i t would perhaps combine the supposed thera-peutic properties of both benzyl ciunamate and iodine. The f i r s t method used by the Chemistry Department for the synthesis of t h i s drag was successful f o r small quantities o n l y . As i t was c a l c u l a t e d that at l e a s t 100 gn;s. of the compound would be r e q u i r e d , the w r i t e r a s s i s t e d the i n -v e s t i g a t o r i n the Department of Chemistry f o r s e v e r a l months i n d e v i s i n g a new method f o r quantity production. P i n a l Experiment with Meta-iodo Benzyl Gianaifiaie. Mgthpu of .Becording uinea I;ig Weights^ lue to the h i g h l y i n c o n c l u s i v e r e s u l t s with s i a a l l e r populations i n the p r e l i m i n a r y worfe i t was decided t o use ^population of 20 guinea pigs f o r the f i n a l experiment, with an a d d i t i o n a l £0 guinea pigs f o r the c o n t r o l . Each pop-u l a t i o n c o n s i s t e d of S males and 12 females, the control animals averaging 424 gms. and the t e s t animals 419 gms. It say be w e l l to g&y here that the p r o p o r t i o n of males t© f e -males was not due to any s t a t i s t i c a l requirement, but to the f a c t -that the ; young animals on hand happened to be so d i v i d e d . The animals were to be fed and weighed., f o l l o w i n g the normal r o u t i n e f o r a considerable p e r i o d without any i n o c u l a t i o n s . The average. weight of each p o p u l a t i o n at each weighing was t o be c a r r i e d i n t o a moving average. This was done i n the f o l l o w i n g manner—for each population the t o t a l weight I s d i v i d e d by the number of guinea p i g s , g i v i n g the weight of the average guinea pig f o r that day. These weights wore then .1.1.. added i n over lap M ng groups rf three and the mean taren, thus g i v i n g a moving average. This method tends to smooth out the curve, since I t e l i m i n a t e s to a large extent the d a i l y f l u c t u a t i ons,, The moving averages of the two pop-u l a t i o n s were p l o t t e d against the time on semilogarithmic paper. The complete experiment l a s t e d from Oct. 22, 19S4 Ino c u l a t i ons. With Meta-iodo Benzyl Cinnamate, In order to give the drug every p o s s i b l e chance i t was decided to ino c u l a t e the drug i n 0.5 c c . q u a n t i t i e s at several d i f f e r e n t p eriods. The guinea pigs were tons i n o c u l a t e d with the drug 14 days, 7 days, and 3 days before r e c e i v i n g the i n o c u l a t e n of tne tubercle baci H i and 2, 9. and 16 days a f t e r . I t i s I n t e r e s t i n g to note on trie curve that the drug aaa seemingly no effect, up to the time of the i n o c u l a t i o n w i t h My c. t u b e r c u l o s i s * .On r e c e i v i n g the tubercle b a c i l l i . , the animals immediately,began to drop i n weight, recovering s l i g h t l y and then dropping again at each f u r t h e r i n o c u l a t i o n w i t h the drug*. I t was f o r t h i s reason that the drug was a i s c o n t i n u e a 16 days a f t e r the tubercle b a c i l l i i n o c u l a t i o n , as the guinea pigs were becoming rough-haired and emaciated and shewing f u r t h e r signs of d i s t r e s s . WItb "tPuberele B a c i l l i -As described before the i n o c u l a t i o n used f o r both 12. • populations was 0.25 c„c. of a 1/20 d i l u t i o n of a t u r b i d i t y! to Mac Parland #4, u s i n g tiie supernatant l i q u i d f o r d i l u t i o n a f t e r 10 minutes s e t t l i n g . R e s ults S t a t i s t i c a l l y the r e s u l t s were e n t i r e l y negative. Eleven of the t r e a t e d animals died of t u b e r c u l o s i s before m days, whereas only e i g h t of the c o n t r o l s d i e d over the same per i o d . The remaining guinea pigs were then i c i l l e d and aut op-s l e d , a l l showing d e f i n i t e symptoms of t u b e r c u l o s i s . The .knowledge that these deaths were s p e c i f i c a l l y due to t u b e r c u l o s i s i n f e c t i o n , i s owing to the kind co-operation of Dr. JI . i l . P i t t s , P a t h o l o g i s t and D i r e c t o r of l a b o r a t o r i e s at the Vancouver General H o s p i t a l . Dr. i - i t t s performed autopsy and h i s t o l o g i c a l examination on a l l guinea pigs used In the experiments« I t would seem therefore,, from t h i s , that the drug Is of no value i n t u b e r c u l o s i s p r o p h y l a x i s . In f a c t , from a study of the weight curves i t would seem th a t the drug has a d e t r i -mental e f f e c t i n the presence of t u b e r c u l o s i s , as explained i n the previous s e c t i o n . In every case, i t vias found that the treated guinea pigs on autopsy showed extensive i n t r a p e r i t o n e a l adhesions., whereas the uhdrugged guinea pigs d i d not. The adhesions therefore were doubtless due to the i r r i t a t i n g a c t i o n of the drug. Discussion. The one v a l i d conclusion which may be d r a m from t h i s -experiment i s that aeta-iodo benzyl cinnamate i s of no thera-ypeutie value i n experimental t u b e r c u l o s i s , l a r g e populations were used; a minimal inoculum of tubercle b a c i l l i was given; drug inoculations were so spaced as to give ample opport-u n i t y f o r both p r o p h y l a c t i c and therapeutic a c t i o n . I t i s I n t e r e s t i n g , however, to speculate upon the mechanism of the r e a c t i o n of the drugged pigs f o l l o w i n g i n -o c u l a t i o n of the tubercle b a c i l l i . It- i s obvious that t h i s cannot be the r e a c t i o n of the drug on tuberculous l e s i o n s as such. It might be explained by accumulative traumatic effect of continued i n o c u l a t i o n s upon the peritoneum of the guinea p i g . On the other hand i t i s p o s s i b l e that the drug had s e n s i t i z e d the animals to the tuberculous toxins or- even to the a c t u a l p r o t e i n of the organism. " S e n s i t i z e d " i s used here i n the sense of 'a chemical sensitization rather than w i t h any reference to a l l e r g y . Beferenees. Since t h i s paper, i n the main, concerns the a c t i o n of a chemical compound Which has not been h i t h e r t o prepared, r e f e r e n c e s - a l l u d i n g d i r e c t l y to the subject matter are n a t u r a l l y unobtainable, For references to work of a s i m i l a r nature, the reader i s r e f e r r e d to the f o l l o w i n g s o u r c e s ; — 14. Index-Catalogue of the L i b r a r y of the Surgeon General's O f f i c e , United States Array: T h i r d S e r i e s , ¥.pl.Z5 1932 See Tuberculosis (Experimental, Treatment of.) rs.527. Tuberculosis {Pulmonary., Treatment of )p.5Sl—583. Tuberculosis (Buinonary., Treatment o f ) w i t h calcium u. 592. Tuberculosis (Pulmonary, Treatment of) by ohemotherapy p. 592—593. Tuberculosas (Pulmonary, Treatment of) by gold s o l u t i o n s p. 593 Tuberculosis -(Pulmonary, Treatment of) by i n h a l a t i o n therapy, p.596 .Tuberculosis (Pulmonary, Treatment of) with ie&ine.-p. 59 6 Tuberculosis (Pulmonary, Treatment of) with sanocrysin. p.. 599. Tuberculosis (Pulmonary, Treatment of) w i t h s i l i c i c acid. ;p.60Q» Tuberculosis (Pulmonary, Treatment of) with sugar p.. 600* Authorised E n g l i s h l a i t i o n - - f f T u b e r e l e B a c i l l u s -Infection and Tuberculosis i n Man and Animals 1 1 by A l b e r t c a l r o e t t e — Williams and Wll k l n s Go., Baltimore, 1923.—with s p e c i a l reference to - Chapter XKEII- "Attempts at chemotherapy In Tuberculosis" Zur Jodbehandlung der lungentubericulose ; A* S y l l a ; B e i t r . a. K l i n . d» Tuberlc. May, 1932, lxxx,51.. The use of Iodine and c e r t a i n Iodine compounds i n exper-XI3 • imental t u b e r c u l o s i s . G. Kaiaysi, J . I n f . M s . March, 1932 r l s 261. Treatment of pulmonary t u b e r c u l o s i s on l i n e s of mineral' d e f i c i e n c y . IC. Fraser, Brit. M. J. May 24, 1930, #3620, 946. Affpgndix^ Since there appear to be several modifications of P e t r a g n a n l ! s medium f o r the c u l t i v a t i o n of Hye. tubereulosi i t was &®emea advisable to give here the formula followed i n t h i s particular experiment, i'he s t r a i n s of Iiy_c. tuberc-ulosis used would give satisfactory growth on t h i s medium 1 l e s s than one-half the time required on any of the more common media for tubercle bacillus cultivation. JrVL3- JLIC* . t? e • P & » » 9 c « & « » » * * » n ,« * « #- 9 0 0 0 < G fl Potato ' f l o u r . . .. 5 6 grass Peptone ......... 6 grams 1 otato — e g g - s i z e d pieces...,-. 6 Eggs--who.] e ......... . 24 Egg y o l i c s . . . . . . . . . . . . . . . . . . . . . 6 0 o © x i'i© • * « DO* o * * J. . £1***1 i' 70 o «o * Malachite green-—aqueous s oXufcJ. 0 2 1««»w *»«*•«•-*» »•«0- * » « * > « « 6 0 c«c p Grind potatoes with fine meat g r i n d e r . Mix a l l in-gredients. This mixture i s fcept i n a b o i l i n g water bath : 16. with, frequent s t i r r i n g u n t i l i t "becomes sticky. After thi i t is l e f t In the water hath for 1 to 2 hours. A f t e r , c o o l i n g to*.50 O.add 24 whole eggs and 6 egg yolks and 70 c glycerine-- Then add 60 c c , 2<P aqueous s o l u t i o n malachite green. The whole mixture Is f i l t e r e d through s t e r i l e gauge, .tubed f o r slants and placed i n an Arnold in a s l a n t -ing position. I n e u b a t e — f i r s t day 1/2 hour at ?Q to 75 i„ -next day 1/2 hour a t 80 Q* l a s t day 1/2 hour a t 80 0-


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