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Some pharmacological and microbiological studies on beta-hydroxy thujaplicin Sanders, Harvey David 1961

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SOME PHARMACOLOGICAL AND MICROBIOLOGICAL STUDIES ON BETA-HYDROXY THUJAPLICIN HARVEY DAVID SANDERS A thesis submitted i n p a r t i a l f u l f i l m e n t of the requirements f o r the degree of MASTER OP SCIENCE IN PHARMACY i n the D i v i s i o n of Pharmacology of the Faculty of Pharmacy We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1961 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference•and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, .Canada. Date i SOME PHARMACOLOGICAL AND MICROBIOLOGICAL STUDIES ON BETA-HYDROXY THUJAPLICIN by HARVEY DAVID SANDERS ABSTRACT Beta-hydroxy t h u j a p l i c i n (BHT) i s a natur a l l y occurring tropolone found i n the heartwood of the Western Red Cedar (Thu ja  p l i c a t a D.Don.). I t has been found to possess both stimulatory and depressant components, depending on the dose, when adminis-tered to mice. I t has also been shown to increase the t o x i c i t y of lead and i n l i m i t e d microbiological studies, i t was found to be both b a c t e r i o s t a t i c and fu n g i s t a t i c i n v i t r o . The stimulant effects of BHT were manifest i n mice by a hy p e r e x c i t a b i l i t y and i n l a r g e r doses by convulsions. In rab-b i t s , the convulsive pattern was obtained i n EEG studies. Mice which exhibited convulsions or an intense degree of hyperexcita-b i l i t y i n the absence of convulsions, invariably died. In this respect BHT d i f f e r s from gamma-thujaplicin, where doses just s u f f i c i e n t to produce convulsions are not l e t h a l . The i n t r a -peritoneal LDr^o a n d G D j ? 0 (convulsive dose) i n mice were found to be 1$$ + mgm/Kg. and I 6 3 + 6 . 2 mgm/Kg. respectively, which indicates that a convulsive dose d i f f e r s l i t t l e from a l e t h a l dose. A period of depression always followed the convulsions and was manifest by ptosis, decreased r e s p i r a t i o n and hypoki-nesia. Death occurred i n this stage. Convulsions were pre-i i vented by thiopental and hexobarbital but barbiturate sleeping time was not decreased - i n f a c t i t was increased. Hexobarbital exerted a protective e f f e c t against l e t h a l doses of BHT whereas thiopental delayed but did not prevent death. The a b i l i t y of BHT to prolong sleeping time was not extended to ethanol. In smaller doses, BHT exhibited depressant effects mani-fested by hypothermia, hypokinesia and ease In handling. The hypothermic action was most pronounced at room temperatures and sedation was found to be proportional to the magnitude of the temperature f a l l . A synergistic action as to hypothermia and hypokinesia was observed with chlorpromazine and to a l e s s e r ex-tent with reserpine. At elevated ambient temperatures, animals treated with BHT exhibited hyperthermia and hyperkinesia. At normal temperatures BHT further increased the hyperthermia pro-duced by amphetamine, but antagonized the hyperkinetic e f f e c t , ultimately, however, the temperature of these animals was de-pressed to a greater degree than was the a c t i v i t y . The point of maximum antagonism to the hyperkinetic e f f e c t of amphetamine occurred approximately one hour following administration of BHT, while the maximum hypothermic e f f e c t of BHT was delayed an addi-t i o n a l two hours. BHT-Na (sodium s a l t of BHT) when administered intravenously or i n t r a - a r t e r i a l l y i n rabbits, caused a precipitous r i s e i n blood pressure. This pressor response appeared to be unrelated to the low voltage-fast a c t i v i t y pattern observed on the ESG. BHT exhibited both b a c t e r i o s t a t i c and f u n g i s t a t i c proper-t i e s when tested i n v i t r o against a wide var i e t y of organisms. i i i However, i t was found to be inactive against D. pneumoniae when tested i n vivo. A p o s s i b i l i t y exists that BHT i s inactivated by whole blood. The t o x i c i t y of lead s a l t s was found to be increased by BHT. This action i s probably due to the formation of an i n s o l u -ble chelate, promoting lead retention i n the tissues and i n -creasing i t s uptake from the body f l u i d s . The t o x i c i t y of lead acetate was increased by two-thirds In the presence of BHT. Pre-formed lead chelate of BHT produced no observable toxic symptoms i n the doses tested. Signatures of Examiners i x ACMOWLEDGMENT The w r i t e r i s indebted to Dr. J . E. H a l l i d a y f o r the guidance he so f r e e l y c o n t r i b u t e d throughout the course of t h i s work. A s p e c i a l note of thanks i s tendered to Dr. G. M. L i n g of the Department o f Pharmacology f o r h i s a i d i n the pr e p a r a -t i o n and i n t e r p r e t a t i o n o f the EEG r e c o r d s used i n t h i s study, and to Dr. J . A. P. Gardner and Mr. G. Barton of the Vancouver F o r e s t Products L a b o r a t o r i e s f o r the generous supply of b e t a -hydroxy t h u j a p l i c i n which was made a v a i l a b l e . The work of t h i s p r o j e c t was supported I n p a r t by a gr a n t o f f e r e d by the Canadian Foundation f o r the Advancement of Pharmacy. i v TABLE OP CONTENTS Page ABSTRACT i LIST OP TABLES v i LIST OP FIGURES v i i LIST OF PLATES v i i i INTRODUCTION 1 HISTORICAL REVIEW 3 PROPERTIES OF BETA-HYDROXY" THUJAPLICIN 12 EXPERIMENTAL PROCEDURES AND RESULTS 17 Action of BHT i n Intact Animals 17 Determination of the Nature of the Stretching of the Hind Limbs Following Administration of of BHT i n Mice 21 Acute Toxi c i t y and Convulsive Dose Studies 22 Dosage Studies - Temperature Depression 23 Hypokinesia 25 Action of BHT on Central Nervous System 30 Interaction with Barbiturates 30 Potentiation of Alcohol Depression 35 Potentiation of the Depressant Effects of Chlorpromazine and Reserpine 36 Interaction with Amphetamine ij.2 E f f e c t of Environmental Temperature on BHT-induced Depression 117 Electroencephalographie Studies 52 Preparation of Sodium Salt of BHT $2 Lead T o x i c i t y Studies 59 Preparation of Lead Chelate of BHT 59 V Page Ba c t e r i o l o g i c a l and Mycological Studies 63 Comparison of BHT and Gamma-Thujaplicin 6f? In Vivo Studies 69 DISCUSSION 70 SUMMARY AND CONCLUSIONS 80 BIBLIOGRAPHY 83 v i LIST OP TABLES TABLE P^ge I Summary of Acute To x i c i t y and Convulsive Dose Data 2 3 I I E f f e c t of BHT on Temperature Depression i n Mice 2$ III E f f e c t of BHT on Duration of Sleeping Times Produced by Barbiturates i n Mice. E f f e c t of Barbiturates on L e t h a l i t y of BHT 3J4. IV E f f e c t of BHT on Ethanol-Induced Depression 3 6 V E f f e c t of Ambient Temperature on BHT and Chiorpromazine-Induced Hypothermia $1 VI E f f e c t of BHT on Lead Toxi c i t y 6 2 VII Fungistatic A c t i v i t y of BHT 6 6 VIII B a c t e r i o s t a t i c A c t i v i t y of BHT 6 7 IX Comparison of the Bacteriostatic A c t i v i t y of BHT and T-Na 6 8 v i i LIST OP FIGURES FIGURE Page 1 Structures of the Thujaplicins l± 2 Resonance Contribution t 6 A c i d i t y of Tropolone XLj. 3 Chelation of Tropolones with Metals l£ k Chelation of BHT with Metals 16 5> Percentage Absolute Decrease i n A c t i v i t y i n Mice Measured at D i f f e r e n t Time Intervals at Various Doses 28 6 Interaction of BHT with Chlorpromazine (Temp era ture) 38 7 Interaction of BHT with Chlorpromazine (A c t i v i t y ) 39 8 Interaction of BHT with Reserpine .('Temperature) . lj.0 9 Interaction of BHT with Reserpine ( A c t i v i t y ) I4I 10 Interaction of BHT with Amphetamine (Ac t i v i t y ) k$ 11 Interaction of BHT with Amphetamine (Temperature) I4.6 v i i i LIST OP PLATES PLATE Page I Resting and Activated EEG Pattern i n Normal Rabbit 55 II EEG Pattern of Rabbit Following Intravenous Administration of I4.O mgm/Kg. of BHT 56 III Whistle Blast Pattern Following Intravenous Administration of J4.O mgm/Kg. of BHT. Convulsant Pattern Following Administration of 20 mgm/Kg. of BHT I n t r a - a r t e r i a l l y 57 17 Convulsant Patterns Following I n t r a - a r t e r i a l Administration of 60 mgm/Kg. of BHT 58 1 1. INTRODUCTION Beta-hydroxy t h u j a p l i c i n (BHT) 2,7-dihydroxy-_|-isopropyl-2,-4-, 6-cycloheptatriene -1 -one Is a na t u r a l l y occurring tropolone found i n the Western Red Cedar (Thuja p l i c a t a D.Don,). The tropolones have been well investigated as to t h e i r chemical and physical properties (1)(2) but r e l a t i v e l y l i t t l e information i s available with regards to t h e i r b i o l o g i c a l a c t i v i t y . Pharmacological studies on other Isomers of t h u j a p l i c i n have been c a r r i e d out by Lee (3) O-l.) (f>) and others (6) (7) on h i n o k i t i o l (beta-thujaplicin) and by Halliday (8) on the gamma isomer. A n t i b a c t e r i a l and antifungal studies have also been performed on both the beta and gamma isomer of t h u j a p l i c i n (9) (10) and to a l i m i t e d extent on BHT (11)• Other tropolones have also been s i m i l a r l y investigated (9)(12). The tropolones have also been shown to be strong chela-tin g agents (13) but no information seems to be available r e l a t -ing t h i s property to any b i o l o g i c a l action that these compounds may have. Since BHT has not been previously Investigated with respect to the aforementioned b i o l o g i c a l phenomena, i t was f e l t that a preliminary i n v e s t i g a t i o n of thi s compound was desirable. The following study was designed to investigate these aspects of the drug» s action. (1) The determination of i t s central nervous system a c t i v i t y . (2) The determination of i t s a n t i b a c t e r i a l and antifungal a c t i v i t y , (3) The determination of i t s e f f e c t on lead t o x i c i t y . 2 The studies under discussion were i n no way set up to be exhaustive and t h e i r prime purpose was to give some i n d i c a t i o n as to the scope of the action of the drug. Thus, i n a l l respects, the experimentation i s of a preliminary nature only. 3. HISTORICAL REVIEW Beta-hydroxy t h u j a p l i c i n (BHT) 2,7-dihydroxy-if-isopropyl-2,ii, 6-cycloheptatriene-l-one i s one of several tropolones found i n the heartwood of the Western Red Cedar (Thuja p l i c a t a D.Don.). The generic name tropolone, o r i g i n a l l y suggested for compounds of t h i s type by Dewar (li|.), has enjoyed common usage f o r the past f i f t e e n years and i s used to designate 2-hydroxy-2,lj-, 6-cycloheptatriene-l-one. In accordance with this usage, BHT i s also c a l l e d 7-hydroxy-lj.-isopropyltropolone. Together with BHT, there occur i n the heartwood of the Western Red Cedar three isomeric tropolones v i z . alpha, beta and gamma t h u j a p l i c i n s . (Figure 1 ) . Although the Western Red Cedar has proven to be a r i c h source of the aforementioned compounds, na t u r a l l y occurring tropolones are r e l a t i v e l y rare. Other t r o -polones found i n nature include s t i p i t a t i c acid from P e n i c i l l i u m  stipitatum (l£), puberulic acid (16) and puberulonic acid (17) from P e n i c i l l i u m puberulum. colch i c i n e (18) from Colchicum  autumnale and purpurogallin (19) from Quercas i n f e c t o r i a . The l i t e r a t u r e on the chemistry of these substances and also that of the synthetic tropolone derivatives has been reviewed by Pauson ( 1 ) . Information concerning the b i o l o g i c a l a c t i v i t y of the tropolones i s meager indeed. Both the gamma and beta isomers of t h u j a p l i c i n (8)(2Q) have undergone l i m i t e d pharmacological study and various tropolones including the th u j a p l i c i n s have been tested by various workers for a n t i b a c t e r i a l and antifungal a c t i -v i t y ( 9 ) ( 1 0 ) ( 1 1 ) . 5. Pharmacological Studies on Beta-Thujaplicin Most of the information concerning the pharmacology of .beta-thujaplicin comes from Japan where the compound i s known as h i n o k i t i o l and i s obtained from the tree known as Pormosan hinoki (Chamaecyparis taiwensis, Masamune et Suzuki- Chaemaecy- paris obtusa. Sieb et Zucco. f . formosana, Hayata), The early work on h i n o k i t i o l was done by Lee ( 3)(ij . ) ( 5 ) and was reported In 1951• In his f i r s t paper, Lee reported on the t o x i c i t y and the l o c a l action of h i n o k i t i o l and i t s sodium s a l t . The r e s u l t s of the t o x i c o l o g i c a l studies indicated that subcutaneous i n -j e c t i o n of the drug i n mice produced either convulsions or t r e -mors. In some cases these manifestations were followed by de-pression, while i n others the depression occurred f i r s t and was then followed by a terminal convulsion. In some animals, a d e f i n i t e degree of ataxia was observed and i n these instances the depression appeared to be les s than i n aforementioned ex-periments. The e f f e c t on r e s p i r a t i o n by the drug was always depressant although i n some few cases there was an i n i t i a l period of stimulation. This period of i n i t i a l stimulation, how-ever, was always b r i e f . In a second report, Lee showed that h i n o k i t i o l caused a f a l l i n blood pressure i n guinea pigs and In rabbits. A de-crease i n pulse rate was also noted i n these animals. Occur-r i n g together with these depressant manifestations was severe resp i r a t o r y depression, Lee noted that neither the blood pres-sure nor the pulse rate were affected i n doses that did not pro-duce resp i r a t o r y depression. These actions were not considered to be due to any vagal mechanism. On the i s o l a t e d rabbit heart, 6. small doses produced cardiac arrest but the l e t h a l time was prolonged by the addition of serum. The drug had a vasocon-s t r i c t o r action on the blood vessels of the i s o l a t e d rabbit ear but the addition of serum to the perfusion f l u i d (Ringer's) abolished t h i s action. Lee's t h i r d report was concerned mainly with the action of b e t a - t h u j a p l i c i n on smooth and s k e l e t a l muscle and on p e r i -pheral nerves. In the i s o l a t e d rabbit uterus, the drug ex-h i b i t e d a stimulatory e f f e c t i n low concentrations but produced a paralyzing e f f e c t i n high concentrations. On the rabbit small i n t e s t i n e , the e f f e c t was Inhibitory and Lee believed this to be due to a musculotropic action. Some of the work with t h i s drug was done on frogs using nerve-muscle prepara-tions to show the e f f e c t of the substance on s k e l e t a l muscle. He could show that h i n o k i t i o l sodium had a paralyzing e f f e c t on both motor and sensory f i b e r s , the l a t t e r being affected f i r s t . On the basis of these r e s u l t s , Lee concluded that hino-k i t i o l i s a nerve-muscle poison having a si m i l a r action i n th i s regard to the terpenes. The fate of h i n o k i t i o l i n the organism was studied by Sakiyama (6). He found that the compound i s excreted i n the free form when given to rabbits, guinea pigs, dogs and goats but i n the human subject, 39-71$ i s excreted as a probable ethereal sulfate conjugate. When O.lj. g. of h i n o k i t i o l i s fed to man, 33-klfo i s excreted i n lj.fJ-187 minutes and 93$ i n 12 hours. Several minutes aft e r administration, the drug i s con-centrated mostly i n the l i v e r , kidney and spleen of animals. 7. Yaraaki (7) has studied the effects of h i n o k i t i o l on renal function. The drug was compared with theophylline and a mer-c u r i a l d i u r e t i c (Igrosin) as to i t s e f f e c t on ur i n a t i o n and renal blood flow. H i n o k i t i o l sodium d i s t i n c t l y increased both these e f f e c t s . Theophylline acted i n a s i m i l a r manner while Igrosin increased the renal blood flow only s l i g h t l y . The increase of the renal blood flow by h i n o k i t i o l sodium was d i s t i n c t l y aug-mented by combination with theophylline and, to a l e s s e r degree, by Igrosin. The combined doses were smaller than those s u f f i -c i e n t l y e f f e c t i v e alone. Pharmacological Studies on Gamma Thu.japlicin The f i r s t pharmacological study of gamma t h u j a p l i c i n was ca r r i e d out by Halliday (8) i n Canada. Using the sodium s a l t of this compound (T-Na), Investigations were c a r r i e d out both i n i n t a c t animals and i n i s o l a t e d preparations. In in t a c t , conscious mice, r a t s , rabbits, dogs and cats, T-Na was found to produce convulsions of a mixed c l o n i c and tonic type i n doses ranging from 75-100 mgm/Kg. of body weight i n mice and rats injected i n t r a p e r i t o n e a l l y , to 30 and lj.0 mgm/Kg. i n dogs i n -jected intravenously, with doses intermediate between these two extremes f o r the other animals. The convulsions were preceded by a b r i e f period of ataxia and followed by symptoms of depre-ssion which included slowing of reflexes, decrease i n muscle tone and voluntary movement, and absence of c e r t a i n placing re-actions. During this period the animals would l i e either prone or on one side, with no attempt to move or regain a normal posi-t i o n . The duration of the postconvulsive depression varied with 8 . the dose, most signs of depression disappearing within twenty-minutes following the l a s t seizure. In rodents, r e s p i r a t i o n was the l a s t function to return to normal, often taking one to three hours. When death 'occurred from the lar g e r doses, i t was always during the postconvulsive depressive stage and never during the seizure proper as i s the case with metrazol. The acute t o x i c i t y (LD^ 0) and the convulsive dose (CD^Q) were determined i n mice. A l l deaths were recorded which occur-red during a period of four days following administration of ".'T^'Na injected i n t r a p e r i t o n e a l l y . The mice i n which death was delayed were i n a state of depression following convulsions. The intraperitoneal LD^Q was found to be 162- -+ 3 . 2 mgm/Kg. and the intraperitoneal CD^Q 73*5 + 1«8 mgm/Kg. The e f f e c t of T-Na on the sleeping times Induced by i n -j e c t i o n of two barbiturates and c h l o r a l hydrate was Inves t i -gated f o r possible analeptic properties which were found lacking. In some cases there even appeared to be a prolongation of the sleeping time. The s i t e of the convulsive action was investigated i n rats following decerebration. Massive doses of T-Na f a i l e d to e l i c i t any convulsive a c t i v i t y . Decerebrate r i g i d i t y was not altered s i g n i f i c a n t l y by T-Na. Similar r e s u l t s were also obtained In decerebrate cats, and on the basis of these r e s u l t s , Halliday has postulated a cerebral s i t e f o r the convulsant a c t i v i t y of T-Na. Intravenous i n j e c t i o n of T-Na i n doses of 2$-$0 mgm/Kg. produced a temporary f a l l i n blood pressure accompanied by a 9. decrease i n heart rate i n cats and dogs. Large doses caused complete pa r a l y s i s of r e s p i r a t i o n but respiratory function returned i n l£ - 2 0 minutes. The hypotensive e f f e c t remained unchanged following a t r o p i n i z a t i o n or b i l a t e r a l section of the vagus. Responses to c a r o t i d occlusion and to injected epine-phrine were not altered by an immediately previous i n j e c t i o n of T-Na. Halliday has suggested that these effects are due to a d i r e c t action on the myocardium. An t i b a c t e r i a l and Antifungal Studies of Thujaplicins The f i r s t i n d i c a t i o n that the heartwood of the Western Red Cedar contained p r i n c i p l e s toxic to fungi was proposed by Sowden (21) who treated f i n e l y ground samples of various woods with hot and cold water. He then mixed the extractives with malt agar i n various concentrations i n p e t r i dishes and then inoculated each p e t r i d i s h with a vigorous growing culture of a wood-destroying fungus. The heartwood and hot water extracts were found to be more toxic than the sapwood and the cold water extracts of the f l o u r of the Western Red Cedar. He also showed that k i l n drying temperatures do not destroy the toxic proper-t i e s of the heartwood. Farther testing against bacteria was c a r r i e d out by Southam (22) who found that the hot water extract of the heartwood of the Western Red Cedar was toxic to many bac-t e r i a and molds i n v i t r o but had no e f f e c t against the same organisms i n vivo, using mice as the test animal. The urine of the t e s t animals, however, could be shown to exhibit antibac-t e r i a l action i n v i t r o tests, following administration of the hot water extract. 1 0 . In Sweden, Rennerfelt ( 1 0 ) tested the thujapl i c i n s against a v a r i e t y of decay producing fungi. The most toxic of a l l the thuj a p l i c i n s was the beta isomer but even gamma- t h u j a p l i c i n was found to be 1 0 0 times as fu n g i c i d a l as phenol f o r these organ-isms. He also showed that the thu j a p l i c i n s tend to be more fun-g i c i d a l than f u n g i s t a t i c , B a i l l i e et a l . ( 1 2 ) found that i n concentrations of $ 0 mgm/litre, gamma-thujaplicin had no a n t i b a c t e r i a l e f f e c t against S. aureus. E. c o l i or A. aerogenes t although i t was ef f e c t i v e against a v a r i e t y of fungi, Erdtman and Gripenberg • ( 2 3 ) also showed gamma-thujaplicin to be active against P u l l u - l a r i a pullulans. Katsura and h i s group ( 9 ) showed the e f f e c t of many tropo-lones and the th u j a p l i c i n s on a large v a r i e t y of organisms. Of the t h u j a p l i c i n s , the beta isomer received the most attention, although i t was not the most e f f e c t i v e of the tropolones tested i n i t s a n t i b a c t e r i a l action. He found that development of bac-t e r i a l resistance did not occur, and that according to the meth^ . ods used i n the testing, very low concentrations of h i n o k i t i o l were required f o r the majority of the organisms to produce i n -h i b i t i o n . The degree of i n h i b i t i o n was independent of gram-staining c h a r a c t e r i s t i c s of the organisms. In a l a t e r paper, Katsura used h i n o k i t i o l c l i n i c a l l y i n two cases of lung gang-rene (21}.). In an independent investigation, he had found that although h i n o k i t i o l was e f f e c t i v e i n vivo, i t proved to be In-e f f e c t i v e i n the presence of blood. Thus i n the therapeutic use of the drug f o r lung gangrene, he applied i t t o p i c a l l y with good r e s u l t s . The h i n o k i t i o l was used i n both cases because therapy 11. with p e n i c i l l i n and aureomycin was i n e f f e c t i v e i n a l l e v i a t i n g the symptoms of the condition. Recently, Roff and Whittaker (11) investigated the t o x i -c i t y of BHT on several rot-producing fungi. Their r e s u l t s suggested that BHT plays only a minor role i n the protective mechanisms of the cedar against r o t . 12. PROPERTIES OF BETA-HYDROXY THUJAPLICIN Structure Following I s o l a t i o n of BHT i n the c r y s t a l l i n e form by Gardner et a l . (25), the compound was shown to be one of the t h u j a p l i c i n s with an additional hydroxyl group. Infra red spectroscopy Indicated that the two hydroxyl groups of the cam-pound were symmetrically placed about the carbonyl function and thus the name suggested f o r this new product was 2,7-dihydroxy-l|-isopropyl-2,I|,6-cycloheptatriene-l-one. The preparation of suitable derivatives of BHT plus extensive oxidative degrada-tions have been submitted as further evidence f o r the v a l i d i t y of t h i s structure. Persulphate oxidation of gamma-thujaplicin to 2 ,7-dihydroxy- lj.-isopropyl-2 , l^,6-cycloheptatriene-l-one has established the correctness of this structure. The syntheses of alpha, beta and gamma t h u j a p l i c i n have a l l been accomplished by several routes (1)(26)(27). Source Beta-hydroxy t h u j a p l i c i n i s found i n the heartwood of the Western Red Cedar, Thuja p l i c a t a D.Don (25). The genus Thuja i s one of the important members of the family Cupressaceae. The Western Red Cedar abounds i n the P a c i f i c Northwest of the United States and i n the province of B r i t i s h Columbia i n Canada. A l -though also found i n Europe, i t i s not native to that part of the world and i s known only i n Sweden. The Swedish and North American v a r i e t i e s d i f f e r somewhat i n the compounds extracted from the heartwood. The North American v a r i e t y y i e l d s mostly gamma- and beta-thujaplicins although some alpha has been re-13. ported ( 2 8 ) , while the Swedish v a r i e t y contains the alpha- and gamma- isomers but no beta ( 2 3 ) . To the best of the author's knowledge, no beta-hydroxy t h u j a p l i c i n has been obtained from the Swedish cedar. However, the Pormosan hinoki (Chamaecyparis  Taiwanensis Masamune et Suzuki) has been reported to y i e l d an ether of beta-hydroxy t h u j a p l i c i n (7-hydroxy h i n o k i t i o l ) (29). I s o l a t i o n Although BHT may be obtained by d i r e c t steam d i s t i l l a t i o n of the heartwood of the cedar as are the other t h u j a p l i c i n s (28) , a method has been found which gives a higher y i e l d of BHT with respect to the other t h u j a p l i c i n s (25) . Copper-bronze screen was stretched over wooden frames i n the free space of a k i l n used f o r drying cedar. After f i v e weeks, the screens had be-come l i g h t green In colour. They were removed from the k i l n and washed with chloroform. The resultant chloroform sol u t i o n was f i l t e r e d , concentrated, saturated with hydrogen sulphide and f i n a l l y the o i l was d i s t i l l e d under reduced pressure which crys-t a l l i z e d spontaneously to l i g h t , yellow c r y s t a l s . The y i e l d of BHT obtained by this method was b%%. Physical Properties Beta-hydroxy t h u j a p l i c i n i s a yellow c r y s t a l l i n e material forming long, f l a t , l i g h t yellow c r y s t a l s when r e c r y s t a l l i z e d from cold hexane. The melting point has been reported as 57 .5 -58° C , and the r e f r a c t i v e index n 7 D° 1 .6170. I t i s o p t i c a l l y i n a c t i v e , steam d i s t i l l a b l e , very soluble i n organic solvents and only s l i g h t l y soluble i n water. I t i s r e a d i l y soluble i n 11+. arachis o i l i n which form i t may be prepared f o r i n j e c t i o n . Unlike the other t h u j a p l i c i n s , BHT can be r e c r y s t a l l i z e d from ethanol. Alpha-, beta-, and gamma- thuj a p l i c i n s are a l l very soluble i n ethanol making r e c r y s t a l l i z a t i o n of these compounds d i f f i c u l t . Chemical Properties Tropolone derivatives in v a r i a b l y possess the properties c h a r a c t e r i s t i c of enols and phenols since the hydroxyl group i s an enolic hydroxyl. In p a r t i c u l a r they are ac i d i c and the ef f e c t of the carbonyl i s to enhance the a c i d i t y as i t does i n the hydroxyquinones and beta-diketones. In common with these, tropolones may be regarded as vinylogs of carboxylic acids ( 1 ) . Par each of the tropolones, removal of a p a i r of electrons from a r i n g carbon permits resonance of the type present i n benzene. Hence tropolone i s more stable as the dipolar ion ( 3 0 ) • o O H o o + F i g . 2 Resonance Contribution to A c i d i t y of Tropolone The l o c a t i o n of the p o s i t i v e charge on a l l seven atoms of the r i n g can be represented by the general symbol of a seven-membered r i n g with the po s i t i v e charge i n the center. 'In common with the other tropolones, the thu j a p l i c i n s are. 15. also a c i d i c . Since BHT i s the only one of the t h u j a p l i c i n s with an additional hydroxy! group, i t would be expected that i t would have a greater a c i d i t y than the other t h u j a p l i c i n s . Such i s indeed the case: the pK of h i n o k i t i o l Is 7«2, of alpha-t h u j a p l i c i n i s 7 .0 (1) while that of BHT i s 6.111*. BHT forms s a l t s r e a d i l y with the a l k a l i metals. The t h u j a p l i c i n s , i n common with other tropolones, form chelates with various metals. Formation constants f o r eight of these metals with various tropolones including alpha- and beta-t h u j a p l i c i n have been established by Bryant and Fernelius (13) . BHT also forms chelates with metals (25) , but the complex i s of a d i f f e r e n t type. The t h u j a p l i c i n s , and indeed a l l the tro-polones save BHT, form chelates of the type shown i n Figure 3 , while BHT forms Its chelate as shown by the structure i n Figure I}., which has been confirmed by various methods ( 2 5 ) . F i g . 3 Chelation of Tropolones with Metals. tf- Determined from pH measurements. 16 w o F i g , h Chelation of BHT with Metals. Barton (31) has stated that the structure In Figure I4. i s probably an o v e r s i m p l i f i c a t i o n of the true structure of the chelate and that polymerization i s probably involved. The 1:1 r a t i o of divalent metal ion to BHT, however, has been e s t a b l i s h -ed (25). 17. EXPERIMENTAL PROCEDURES AND RESULTS  Action of BHT i n Intact Animals  Methods and Materials Swiss albino mice of weight 20 - 30 gms. were used throughout the course of these experiments. The BHT was pre-pared i n peanut o i l i n a concentration of 10 mgm/ml. and i n a l l cases injected i n t r a p e r i t o n e a l l y through a 23 gauge needle. In a l l cases v a r i a t i o n i n the dosage was accomplished by an increase or decrease i n the volume of i n j e c t i o n , control animals receiv-ing equivalent volumes of the o i l alone. Observations were f o r the most part v i s u a l and subjective, and two pieces of appara-tus were set up, i n order that some objective r e s u l t s could be obtained. The f i r s t of these was the "rotarod" which i s an apparatus o r i g i n a l l y designed to compare sk e l e t a l muscle relax-ants, convulsants and central nervous system (CNS) depressants ( 3 2 ) . The rotarod consists of a round rod 1 inch i n diameter and 36 inches long divided by cardboard p a r t i t i o n s every 9 inches, wherein i s placed the animal to be tested. The rod i s rotated by a simple kymograph motor and turns at the rate of l£ revolu-tions per minute. The rod i s situated at a height of 20. inches from the surface of the table and a sawdust bed i s placed im-mediately below the animals. The test i s considered p o s i t i v e when the animal f a l l s from the rod within 60 seconds. The second piece of apparatus was the sloping screen of Stephenson ( 3 3 ) . An ordinary window screen i s tacked to a simple wooden frame and i n c l i n e d at an angle of 60° from the h o r i z o n t a l . Following i n j e c t i o n , the animals are observed f o r 18. a period of two minutes, and the test i s considered p o s i t i v e when an animal f a l l s from the screen during this period. Results In the i n i t i a l experiments, four dose ranges were chosen, of 5 0 , 100, 1^0 and 200 mgm/Kg. BHT injected i n t r a p e r i t o n e a l l y , the animals being i n t a c t and conscious. Each dose range was administered to 10 mice, 50 mgm/Kg, The f i r s t sign following i n j e c t i o n was the stretching of the hind limbs and the dragging of the underside of the abdomen along the f l o o r of the cage. This manifestation was present i n a l l of the animals receiving BHT regardless of dose and always appeared within two minutes following i n j e c t i o n . The control animals, receiving peanut o i l alone did not exhibit t h i s sign. After $ minutes, 9 of 10 animals were quiescent and would move only when prodded. When placed on the rotarod, none of the animals f e l l , i n d i c a t i n g that muscle coordination and tone were i n t a c t . I f any of the mice l o s t i t s footing on the rotarod, recovery was rapid and i t was able to maintain i t s balance, when placed on the sloping screen, they showed no i n -c l i n a t i o n to explore, but did not f a l l from the steep slope. The period of hypokinesia remained f o r some 3 - k hours f o r most of the animals and two of the animals remained i n a state of lethargy f o r 7 hours. During the period of depression, food was refused although they had been fasted f o r 12 hours p r i o r to the tes t . Respiration seemed to be unaffected. No deaths occurred following t h i s test. 19. 100 mgm/Kg. The same dragging of the hind limbs and the rubbing of the abdomen on the f l o o r of the cage occurred as with the smaller dose. Depression occurred within about 5 minutes and included a l l of the animals. Hypokinesia was pronounced and the mice f e l t cold to the touch. Respiration was slowed but not to a marked extent. The animals did not f a l l from either the rotarod or the sloping screen either l£ minutes following i n j e c -t i o n or 60 minutes following i n j e c t i o n . Recovery from the l e -thargic state took an average of 7 hours although one of the animals took 12 hours. lfpO mgm/Kg. Again the dragging of the limbs i n the im-mediate period following Injection was observed. However, no depression was observed following t h i s i n i t i a l period. A l l the mice i n thi s group showed increased a c t i v i t y f o r about 10 min-utes following the i n j e c t i o n . This was manifested i n an attempt to stand on the hind legs with l i t t l e success as they toppled rather r a p i d l y . S> of 10 animals f e l l from rotarod when tested 5 minutes following i n j e c t i o n of BHT. After 30 minutes 3 of 10 f e l l from the rod. After 8 minutes, 3 of 10 animals developed convulsions (these were among the f i v e that f e l l from the rod) which was preceded by a period where the body of the mouse s t i f f e n s , the t a i l becomes erect, and the animal assumes a straddling p o s i t i o n . After some 30 seconds i n this p o s i t i o n , the head becomes sharply retracted, the back arches, and the limbs either convulse c l o n i c a l l y or are r i g i d l y extended. A l l animals that exhibited convulsions showed both the clonic and tonic phases of the convulsion. A seizure would l a s t f o r periods of 1^-30 seconds and would recur at l e a s t once within 2 minutes. 20. A l l animals that convulsed, died within 20 hours hut did not die i n spasm. Two of the other animals died also and both of these were ones that had f a l l e n from the rotarod. One of these animals died within 20 hours along with those that had con-vulsed and the other died within I4.8 hours. With a l l of the mice i n t h i s dosage range, r e s p i r a t i o n was increased i n the f i r s t 20 minutes and then became depressed. Dyspnea was evident i n the f i n a l post-seizure period u n t i l death. These animals n e i -ther ate nor drank although food and water were ava i l a b l e . 200 mgm/Kg. In th i s dosage range a l l of the animals except one developed convulsions of the type previously describ-ed. The remaining mouse showed an I n i t i a l stimulatory period but did not develop a seizure. A l l of the animals f e l l from the rotarod and a l l died within I4.8 hours. The symptoms manifest i n the 1^0 mgm/Kg. range were evident i n thi s range also, but some were aggravated. The most severe aggravation was that death i n $ of 1 0 mice occurred within 5 hours from the time of the i n j e c -t i o n . Summary BHT when injected into mice i n large doses, produced a combination of stimulant and depressant e f f e c t s . In a l l cases where excessive stimulation occurred, the ultimate r e s u l t was death within I4.8 hours regardless of whether the animal had con-vulsed or not. In the depressive state there seemed to be no lo s s of muscle control while this was very evident i n those a n i -mals that exhibited the stimulatory effects of the drug. Re-spiratory depression was found only with the higher doses a l -21. though this appeared to be the p r i n c i p a l cause of death. Whether the convulsions are of a central or peripheral o r i g i n or both, remains to be determined. Determination of the Nature of the Stretching of the Hind Limbs  Following Administration of BHT i n Mice When BHT Is administered i n t r a p e r i t o n e a l l y i n mice, the f i r s t abnormal sign to appear i s the stretching of the hind limbs and the rubbing of abdomen on the bottom of the cage. In order to determine whether this was due to some systemic e f f e c t of the drug acting on the spinal cord or to some l o c a l i r r i t a -t i o n of the drug i n the peritoneal cavity, the following experi-ment was performed. Four mice were injected with 0.1 cc. of a 10 mgm/cc. solu-t i o n of BHT i n peanut o i l i n t r a p e r i t o n e a l l y , while four other mice were injected at the same time with 10 mgm/Kg. of morphine sulphate. Following the f i r s t f l a g t a i l response from the action of the morphine, the same dose of BHT was then adminis-tered to these animals. While i n the animals that received no morphine the stretching of the hind limbs was manifest within two minutes; In the animals receiving the morphine the response was absent. In order to confirm these r e s u l t s , four more mice were injected with 80 mgm/Kg. i n the scapular region and these animals exhibited none of the aforementioned symptoms but attempted to b i t e the skin at the point of i n j e c t i o n and rub i t o f f on the side of the cage. On the basis of these r e s u l t s , i t i s concluded that the stretching of the hind legs and the drag,-22. ging of the abdomen on the bottom of the cage following i n t r a -peritoneal i n j e c t i o n of BHT i n o i l , i s due to l o c a l i r r i t a t i o n and i s not concerned with any systemic e f f e c t . Acute Toxi c i t y and Convulsive Dose Studies The LD^Q f o r Intraperitoneal i n j e c t i o n i n mice was de-termined. A l l mice used weighed from 2I4.-26 gms. and were of both sexes. A l l of the animals were fasted f o r 12 hours p r i o r to the test and weighed immediately before i n j e c t i o n . Doses of BHT ranged from 120-220 mgm/Kg.. Each group of 10 mice was sepa-rated from the next by an increment of 20 mgm/Kg.. A l l deaths occurring within 7 2 hours were considered to be due to the toxic e f f e c t s of the drug although a f t e r this period no deaths were observed. At the same time that the LD^Q was being recorded, a l l convulsions that occurred following the various doses ad-ministered were also observed and tabulated. In this way both the LD^Q and the CD^Q could be reported simultaneously. This procedure was made possible by the f a c t that the preliminary experimentation indicated that the DD^Q and the CD^Q were very nearly the same, hence the dosage range used provided data f o r both. After about 2l+ hours I t became very obvious which ani-mals would survive and which would not. The majority of the animals died within I4.8 hours and the LD^Q f o r 1+8 hours was 175 + 1+.19 mgm/Kg. while the LD^Q f o r 72 hours was l£ 5 + 1+.5 mgm/Kg.. The animals i n which death was imminent, remained prone and exhibited no i n c l i n a t i o n to move throughout the en-t i r e period preceding death, while the others showed normal 23. a c t i v i t y within 4-12 hours following the administration of the drug. The C D ^ Q was found to be 163 + 6.2 mgm/Kg.. A l l calcu-l a t i o n s f o r the L D ^ Q and CDCJQ and t h e i r respective standard errors were calculated according to the method of M i l l e r and Tainter (34). TABLE I Summary of Acute T o x i c i t y and Convulsive Dose Data Intraperitoneal LD^ Q i n miee— Lj.8 hours: 175 +4*2 mgm/Kg. Intraperitoneal L D ^ Q i n m i c e — 7 2 hours: 155 + 4»5 mgm/Kg. Intraperitoneal C D ^ Q i n mice: 163 +^  6.2 mgm/Kg. Dosage Studies In order to study the depressant e f f e c t s of the drug noted e a r l i e r i n the preliminary experiments, an e f f e c t i v e dos-age range was required i n which depressant effects could be noted without the stimulant and l e t h a l effects exhibited by BHT i n high doses. Preliminary experimentation indicated that temperature depression and hypokinesia were depressant effects due to the action of the drug i n low doses and these two signs were further investigated. Temperature Depression Five groups of mice, weighing 22-21+ gms. and consisting of 10 mice per group, were injected i n t r a p e r i t o n e a l l y with 20, 30, 4°» 50 and 6 0 mgra/Kg. of BHT. Immediately before i n j e c t i o n , the temperature of each animal was recorded and again l£ minutes 2l| . following i n j e c t i o n . A 2°C. f a l l i n r e c t a l temperature with-i n the 1$ minute period as measured with a YSI Model I4.3 Tele-Thermometer (Yellow Springs Instrument Co.) was considered a p o s i t i v e e f f e c t . The re s u l t s were plotted on logarithmic-probit paper and the SD^Q (effective dose) and the standard error were determined according to the method of M i l l e r and Tainter (3I4.). Results The intraperitoneal ED^Q i n mice calculated on the basis of the temperature depression a f t e r 1$ minutes as outlined i n the procedure was 35> + 3«2 mgm/Kg.. Doses of JO and 80 mgm/Kg. of BHT were administered to two other groups of mice and a l -though the expected temperature depression was observed, i t was considerably greater than i n the lower doses and persisted f o r a longer period of time. Preliminary testing indicated that the greatest decrease i n temperature i n a l l dose ranges occurred within 15 minutes following administration of the drug by the intraperitoneal route and an increase i n the temperature to-ward normal values was observed a f t e r 60 minutes i n a l l dose ranges tested. (See Table II.) 2£ TABLE I I The E f f e c t of BHT on Temperature Depression i n Mice Temperature Depression and Standard Error (°C.) Dose i n mam/Kg. 15 mins. 60 mins. 120 mins. 2k0 mins. 10 0.38 + .18 0 0 -20 1.02 + . 3 5 O.kO + .18 0 0 30 1 .00 + .30 0.51 + .18 0.33 + .07 0 ko 2.01 + .30 1 .82 + .10 1.02 + ,k3 0.62 + .51 50 2.k6 + .26 1.1k + .35 - 0.36 + .20 60 3.62 + .k8 1 .50 + .35 - 1.50 + .55 70 3.91 + .35 2.08 + .k0 - 1.32 + .k8 80 k . 3 0 + .23 2.56 + .67 2.0k + .87 1.60 + . k 5 A l l times are i n minutes following i n j e c t i o n of the drug. - indicates no test. Hypokinetic Studies Eight groups of mice, weighing 22-2k gms. and consisting of 10 mice per group, were injected i n t r a p e r i t o n e a l l y with BHT i n doses ranging from 10-80 mgm/Kg. i n increments of 10 mgm/Kg.. The mobility of each group of animals was determined immediately p r i o r to i n j e c t i o n and 15 minutes following i n j e c t i o n i n an Actophotometer (Metro Industries). The instrument employs 6 photoelectric c e l l s situated i n the perimeter of a cage which .-. are continuously excited by 6 individual l i g h t sources. Inter-ruption of any of the beams of l i g h t that c r i s s - c r o s s the f l o o r of the cage r e s u l t s i n the recording of the interru p t i o n by a mechanical counter. Thus, the greater the a c t i v i t y of the ani-mals i n the cage, the higher w i l l be the recorded count. A l l 26. determinations were made f o r f i v e minute periods following a 30 second or i e n t a t i o n period. The e f f e c t i v e dose i s reported as that dose of BHT that w i l l cause an absolute decrease i n mobility of $0% as measured by the Actophotometer 1^ minutes f o l l o T t f i n g the Injection of the drug. The re s u l t s are shown i n Figure Calculation of Absolute Decreased A c t i v i t y One of the problems encountered i n the determination of the degree of decrease i n a c t i v i t y following administration of BHT was that of the decrease i n mobility of the control animals on repeated exposure to the Actophotometer. Calculation of the absolute decreased a c t i v i t y (the decrease i n a c t i v i t y due to the action of the drug) then required the subtracting of the per-centage decrease i n mobility of the controls for any one period of time from the percentage decrease i n the test animals at that time. Therefore, i f C° = t o t a l count of co ntrols at time 0 C = t o t a l count of controls at time t C° = t o t a l count of test animals at time 0 C-j = t o t a l count of test animals at time t then absolute decrease i n a c t i v i t y expressed as percent equals 0° - C 1 - 0° - 0 100 C° C° = 100 C?0 - 0,0° = R . P l o t t i n g R against dose i n mgm/Kg. allows f o r the d i r e c t deter-mination of the dose required to produce a %Q% decrease i n 27. a c t i v i t y . Results The r e s u l t s are shown graphically i n Figure $. The effec-t i v e dose (the dose required to produce a $0% decrease i n a c t i -v i t y ) i s k£ mgm/Kg. calculated from the 1$ minute time curve. The sharp decrease recorded between kO and $0 mgm/Kg. i s re-producible within an error of + 10%. At 60 minutes, the maxi-mum e f f e c t of the. BHT has passed and the curve f l a t t e n s out es p e c i a l l y i n the range of 30-70 ragm/Kg.. There i s a sharp r i s e i n the curve at 70 mgm/Kg. at th i s time, i n d i c a t i n g a greater decrease i n a c t i v i t y at this dose but the decrease i s f a r l e s s than the same dose at 1$ minutes. The curves for 120 and k80 minutes show a correspondingly l e s s e r decrease i n a c t i v i t y which i s proportional to the time. Discussion The r e s u l t s of the hypothermic and hypokinetic studies indicate that there exists some c o r r e l a t i o n between the two ef f e c t s . The largest decrease In temperature occurring between two successive dose ranges f a l l s between 30 and kO mgm/Kg. and between $0 and 60 mgm/Kg. and the greatest decrease i n a c t i v i t y between two doses l i e s between kO and $0 mgm/Kg.. Since re-producible e f f e c t s could be produced at a dose of kO mgm/Kg. both of temperature depression and hypokinesia, this dosage of kO mgm/Kg., which l i e s midway between the ED^Q calculated on the basis of temperature depression and that of a $0% decrease 28. Dose in mgm/Kg. Figure 5. Absolute percentage decrease in activity in mice measured at different time intervals at various doses. 29. i n a c t i v i t y , was chosen f o r the majority of the experiments that follow. Moreover, i t was desirable to f i n d a dose of BHT considerably smaller than the L D ^ Q * Continued administra-t i o n of BHT of t h i s dosage f o r a period of nine days resulted i n no observable toxic e f f e c t s (see Table V I ) . 30. The Action of Beta-Hydroxy Thujaplicin  on the Central Nervous System The hypokinetic and hypothermic ef f e c t s of BHT suggest that the drug exerts a central action i n the i n t a c t animal. Moreover, Halliday (8) has shown that the convulsive effects of gamma-thujaplicin sodium are central i n o r i g i n since decerebrate animals could not be stimulated even with large doses of gamma-th u j a p l i c i n . Although the CD^Q of gamma-thujaplicin and that of BHT vary widely, the type of convulsion i s much the same and thus i t would he expected that the convulsive action of BHT i s also of central o r i g i n . Therefore, experiments were performed i n order to elucidate further the extent of involvement of the central nervous system i n the production of the aforementioned e f f e c t s . Interaction with Central Depressants  Interaction with Barbiturates The convulsions produced i n in t a c t mice by high doses of BHT suggested the p o s s i b i l i t y that the drug would exhibit ana-l e p t i c properties when tested against the barbiturates. A known analeptic, metrazol was used f o r comparison. Method BHT i n doses of I4.0, 8 0 , 1^0 and 200 mgm/Kg. was tested f o r i t s e f f e c t on the duration of the sleeping time i n female mice 22-26 gms. produced by thiopental (Pentothal Sodium -Abbott) $0 mgm/Kg. and hexobarbital sodium (Evipal - Winthrop) 100 mgm/Kg.. Although both drugs are c l a s s i f i e d as ult r a - s h o r t 31. acting barbiturates, preliminary investigations indicated that the interaction, of BHT with each produced d i f f e r e n t r e s u l t s . The dose of thiopental was chosen as the minimum dose that would prevent convulsions due to BHT i n a l l animals tested, and that of hexobarbital, as that dose that would produce the same dura-t i o n of sleeping time as that of thiopental. A l l drugs were given i n t r a p e r i t o n e a l l y . The barbiturates were administered f i r s t followed by BHT as soon as the ri g h t i n g r e f l e x had been l o s t . In subsequent experiments, BHT was administered f i r s t , followed i n 15> minutes time by the barbiturate. Sleeping time was measured from the time of the loss of the r i g h t i n g r e f l e x u n t i l this r e f l e x was regained permanently and was performed spontaneously from the supine to the prone po s i t i o n . The number of f a t a l i t i e s and the time of death were recorded f o r each group. Metrazol was injected i n t r a p e r i t o n e a l l y i n a dose of 100 mgm/Kg. when used f o r comparison. Results The expected analeptic action of BHT was not observed. When the barbiturate and metrazol were injected together, the sleeping time was markedly reduced, but when the barbiturate and BHT were administered together, the sleeping times were pro-longed depending on the dosage of BHT used. Moreover, the i n -crease i n sleeping time was much more marked with the thiopental administration than for the hexobarbital when administered with BHT. Both barbiturates prevented convulsions i n the test animals but the e f f e c t on l e t h a l i t y produced by the tropolone i n high 32. doses was found to be quite different. Although thiopental was found to delay the death time by as much as 30 hours, the animals nevertheless died. The mice receiving hexobarbital, however, showed a rapid recovery from the effects of the BHT. There were no f a t a l i t i e s in this group after 30 minutes, and only 1 of 8 animals died within the 30 minute period. A simi-la r percentage of animals died within the f i r s t 30 minute period with thiopental also. When the dose of thiopental was increased to 100 mgm/Kg., the same degree of delay i n the lethal time was observed as for the smaller dose of the barbiturate and there was no increase in the number of survivors. The results of these experiments are tabulated i n Table III. In a l l of the preceding cases, the BHT was administered as soon as the righting reflex was lost. In another experiment the BHT was administered f i r s t , followed in l£ minutes by the barbi-turate. This sequence was possible only with the lower doses of BHT i.e. kO and 80 mgm/Kg., since higher doses produced convul-sions. No significant alteration i n the sleeping time, when compared to the administration of BHT and the barbiturate to-gether, was observed. The excitatory period, however, that usu-al l y precedes the loss of the righting reflex following admini-stration of barbiturates, was found to be absent i f the BHT was given f i r s t . The muscle weakness In those animals receiving a combina-tion of BHT in doses above 80 mgm/Kg. and thiopental was pro-nounced. Although many of the animals appeared to regain the righting reflex after various periods of time, when returned to their backs 30-60 minutes often elapsed before the animals 33. righted themselves again. This manifestation was only present i n high doses of BHT and was not evident i n the same degree when hexobarbital was used as the hypnotic. When the animals began to regain the r i g h t i n g r e f l e x following the combination of hexobarbital and BHT i n high doses, they were ataxic but were f a r more a l e r t than with the thiopental. These actions resembled much more the effects produced by the barbiturates alone, while those animals receiving thiopental and BHT resemble much more those receiving BHT alone but accentuated. Regardless of the barbiturate used, r e s p i r a t i o n was mark-edly depressed with doses of BHT above 80 mgm/Kg. during the sleeping state. Improvement i n r e s p i r a t i o n occurred on regain-ing the ri g h t i n g r e f l e x but the degree of improvement was much greater i n those animals receiving hexobarbital as the anes-t h e t i c . In those animals that died following thiopentone-BHT administration, r e s p i r a t i o n was markedly depressed. In some animals the respiratory rate decreased to 12 per minute as opposed to a normal value of 130-160. Some of the animals receiving thiopental did not lose the ri g h t i n g r e f l e x . These animals were nevertheless injected with BHT within 3 minutes (average time f o r loss of ri g h t i n g reflex) following administration of the thiopental. Some of these a n i -mals subsequently l o s t the ri g h t i n g r e f l e x and some did not hut, regardless of this l a t e manifestation, these animals a l l died within a shorter period of time than those that had o r i g i n a l l y l o s t the ri g h t i n g r e f l e x and had received BHT i n doses of IjpO mgm/Kg. or greater. Although Table I I I shows that generally there i s an 3k. increase i n the sleeping time with an increase i n the dose of BHT, nevertheless with thiopental the sleeping time with 200 mgm/Kg. of BHT i s l e s s than that at lj?0 mgm/Kg.. A possible explanation of this decrease i n sleeping time, i s that at these high doses of BHT, the stimulatory component increases more rap i d l y than the depressant component. TABLE III E f f e c t of BHT on Duration of Sleeping Times Produced by Hypnotic and Dose BHT mgm/Kg. Metrazol mgm/Kg. Deaths i n 72 hours Sleeping Time (min. + S.D.) Thiopental Na 50 mgm/Kg. ft tt t! tl 100 ii tt ti Hexobarbital Na 100 mgm/Kg. it ti it it tt fo 1^ 0 200 150 200 fo l£0 200 100 100 100 0/10 0/10 0/10 6/10 9/10 0/10 6/7 0/8 1/5 0/10 0/10 0/10 0/10 1/10 0/8 l5.k + 5.2 1 8 . 9 " k . 8 50.8 + 20.1 163.1 + 52.5 116.2 + 5k.8 2.3 + 2.9 10.7 + 6 .6 39.1 + 16.1 I8.k + 5.9 2 8 . k £ 7.7 29. k + k«2 39.3 + 16 .6 k 7 . 8 + Ik.9 k.5 + 3.9 sleeping time undetermined 35. Potentiation of Alcohol Depression The depressant e f f e c t s of barbiturate and ethanol admini-stered concomitantly are additive i n nature (35)• Since the eff e c t of BHT appears to be that of potentiating the e f f e c t of the barbiturates, i t was thought that an experiment to determine whether or not BHT would also potentiate the e f f e c t of ethanol depression should be attempted. Method Ten mice of both sexes and weighing 25 gms. each were injected with 1.0 cc. of a 10$ aqueous ethanol solution. This dose was found to be s u f f i c i e n t to produce a loss of a b i l i t y to stay on the rotarod but not s u f f i c i e n t to produce a loss of the ri g h t i n g r e f l e x . Ten more mice were injected with ethanol and with l+O mgm/Kg. of BHT and another 10 with 80 mgm/Kg.. In another experiment, three groups each made up of 5 mice and weighing 2l|-25 gms. were injected with 1 .5 cc. of a 10% aqueous, ethanol solution. This dose was found to be s u f f i c i e n t to produce a loss of the r i g h t i n g r e f l e x i n a l l of the animals. Into two of the groups, doses of 1+0 and 80 mgm/Kg. of BHT were injected 5 minutes following the administration of ethanol. A l l injecti o n s were int r a p e r i t o n e a l . The time of the return of the ri g h t i n g r e f l e x was recorded. Results The r e s u l t s are summarized i n Table IV. Although 1+ of 10 animals l o s t the rig h t i n g r e f l e x with the lar g e r dose of BHT, this e f f e c t was of short duration. Prom the re s u l t s , i t would 36. appear that BHT i n the doses administered, does not potentiate the depressant e f f e c t of ethanol. TABLE IV E f f e c t of BHT on Ethanol-Induced Depression Ethanol BHT Dose Loss of Sleeping Time Dose mgm/Kg. Righting Reflex (min. + S.D.) 1.0 cc. - 0/10 1.0 cc. kO 0/10 1.0 cc. 80 k/lO 1 - 2 mins, .1.5 cc. - 10/10 2k.0 + 11 .3 1 . 5 cc. kO 10/10 23.2 + 8.2 1 . 5 cc. 80 10/10 21.k + lk.2 Potentiation of the Depressant Ef f e c t s of Chlorpromazine and  Reserplne Both reserpine and chlorpromazine have the a b i l i t y to pro-duce a state of hypothermia and hypokinesia i n mammals (36) . In this they resemble BHT which also has this a b i l i t y although i n much higher doses. Thus i t was f e l t that some i n t e r a c t i o n between BHT and these t r a n q u i l l i z e r s was l i k e l y and the following experiments were designed to determine the extent of the i n t e r -action. Method The test dose of chlorpromazine was determined on the basis of the dose required to produce a loss of mobility of between 30$ and 50$ and a f a l l i n temperature (absolute) of more than 1°G. but not more than 3 ° C . This was found to be 2 mgm/Kg.. 37. The dose of reserpine was determined i n the same manner and found to be 0.75 mgm/Kg.. The test dose of BHT was kO mgm/Kg.. A l l i njections were i n t r a p e r i t o n e a l . Six groups of 10 mice each and weighing 2k-26 gms. of both sexes were used i n the deter-mination. Pour of the s i x groups served as controls v i z . abso-l u t e , chlorpromazine, reserpine and BHT. The other two groups received BHT and one or the other a t a r a c t i c . Injections of BHT were made k5 minutes following the administration of chlorpro-mazine, since the maximum e f f e c t of chlorpromazine i s manifest approximately 60 minutes following i t s administration by this route, and the maximum e f f e c t of BHT occurs 15 minutes following i t s i n j e c t i o n i n t r a p e r i t o n e a l l y . BHT was injected 225 minutes following administration of reserpine f o r the above stated rea-sons. Rectal temperatures were recorded with the Tele-Thermo-meter and a c t i v i t y measured with the Actophotometer. The re s u l t s are shown i n Figures 6 - 9 . Results The f a l l i n temperature produced by the combination of chlorpromazine and BHT was very marked and greater than the ef f e c t produced by either agent alone. Although the absolute f a l l i n temperature with reserpine was greater than that of the chlorpromazine - BHT combination, the control values f o r reser-pine alone were also lower. The degree of difference between the chlorpromazine control and i t s combination with BHT was much lar g e r than that between the reserpine control and i t s BHT combination. (See Figures 6 and 8 .) The same observation obtains f o r the decrease i n a c t i v i t y shown i n Figures 7 and 9 . 38. O O Chlorpromazine O O Chlorpromazine + BHT • • BHT Figure 6. Interaction of BHT with chlorpromazine (Temperature) 39 Figure 7. Interaction of BHT with chlorpromazine (Activity) O Reserpine 0 Reserpine + BHT % BHT Figure 8. Interaction of BHT with reserpine (Temperature) Time in hours Figure 9. Interaction of BHT with reserpine (Activity) 42. An i n t e r e s t i n g observation was the prolongation of the depressant action of chlorpromazine. This was true for both the decrease i n temperature and the hypokinetic e f f e c t . A l -though the experiment was a r b i t r a r i l y stopped aft e r 8 hours, i t i s evident from the graphs that the animals receiving the chlor-promazine-BHT combination had not recovered. Those animals re-ceiving BHT alone had recovered at the end of f i v e hours, and at the end of s i x hours f o r the animals receiving chlorpromazine alone. The experiment was not continued f o r a long enough p e r i -od to make any such statement about the reserpine-BHT combina-t i o n . A further observation was the e f f e c t of BHT on i n d i v i d u a l animals receiving chlorpromazine or reserpine. Following ad-m i n i s t r a t i o n of BHT to animals receiving chlorpromazine, a l l of the animals so treated appeared to be affected equally regard-l e s s of the extent of the temperature depression p r i o r to In-j e c t i o n of BHT. In opposition to this observation, animals re-ceiving reserpine followed by BHT, did not show t h i s generali-zed . f a l l i n temperature. Those animals who had the l e a s t amount of temperature depression p r i o r to i n j e c t i o n of BHT showed the greatest f a l l following administration of the tropolone. Interaction with Amphetamine Animals receiving s u f f i c i e n t l y large amounts of amphetamine exhibit tremors, restlessness, increased motor a c t i v i t y , agi-t a t i o n , and hyperthermia. These effects are central i n o r i g i n and thus i t was f e l t that a p o s s i b i l i t y existed that the central 1*3. depressant action of BHT could antagonize these e f f e c t s . Two reproducible parameters were chosen f o r the determination of the extent of the antagonism, i f any. These were (1) Hyperthermic action; (2) Hyperkinetic action. Method Preliminary t r i a l s with amphetamine alone showed that a dose of 8 mgm/Kg. would produce i n mice a marked increase i n a c t i v i t y and a s l i g h t r i s e i n r e c t a l temperature. The peak f o r these actions occurred between 1+5 and 90 minutes. In none of the animals tested (20) d i d this dosage produce convulsions and there were no f a t a l i t i e s . Three groups of female mice, consisting of 10 mice per group, and weighing 21+-26 gms. each, were injected i n t r a p e r i -toneally with 8 mgm/Kg. of amphetamine sulphate i n aqueous solu-t i o n . 1+5 minutes following the administration of the amphet-amine, BHT i n a dose of 1+0 mgm/Kg. was administered to one of the groups and 80 mgm/Kg. to the other. Core temperatures and a c t i v i t i e s were recorded as previously described at the times Indicated i n Figures 10 and 11. Recordings of BHT controls were also made as indicated on the same graphs. Results Although antagonism to the hyperkinetic e f f e c t of amphet-amine was exhibited by BHT, potentiation of the i n i t i a l hyper-thermic e f f e c t was observed. The antagonism to the hyperkinetic e f f e c t of amphetamine was observed 15 minutes following adminis-t r a t i o n of BHT and reached a maximum 60 minutes l a t e r . The kk. potentiation of the amphetamine-induced hyperthermia occurred 15> minutes following administration of BHT and the maximum temperature depression produced by BHT alone was delayed by three hours when administered In combination with amphetamine* Furthermore, the groups that had received amphetamine plus BHT eventually attained the same degree of hypothermia as was exhibi ted by the BHT controls but d i d not a t t a i n the same degree of hypokinesia. Figure 10. Interaction of BHT with amphetamine (Activity) 1 © g -i. _ — ! ! ! | | 1 r ^ 1 1 2 3 4 5 6 7 Time i n hours Figure 11. Interaction of BHT with amphetamine (Temperature) 4 7 . E f f e c t of Environmental Temperature on BHT-induced Depression It has been shown that chlorpromazine and reserpine both lower body temperature i n mice ( 3 & H 3 7 ) . Moreover, i t has also been shown that the hypothermia i s accompanied by sedation at ambient temperatures below 30°C. ( 3 8 ) . At temperatures of 30°C. and above, hypothermia i s not produced with either drug and the sedative e f f e c t i s absent i n the reserpine treated animals. Since BHT also produces hypothermia and sedation at room temper-atures (21-23°C) i t was f e l t that information concerning the eff e c t of the environmental temperature on i t s hypothermic and sedative action should be obtained. Method Two groups of male mice weighing 28-32 gms. and consist-ing of 5 mice per group were injected with 2 and k mgm/Kg. of chlorpromazine and another two groups of the same weight and size were injected with kO and 80 mgm/Kg. of BHT. A l l i n -jections were i n t r a p e r i t o n e a l . Rectal temperatures were re-corded immediately preceding i n j e c t i o n and the animals were placed i n cages p r e c h i l l e d at 5>°C. and maintained at this temper-ature. Temperatures were recorded at l£, 60 and 120 minutes and the actions of the animals noted subjectively, since the Actophotometer could not be brought to the above stated temper-ature. Uninjected animals were also placed i n the same environ-ment as controls. In four d i f f e r e n t groups of mice, the above procedure was repeated at 3k°C, and another four groups at 2 2 ° C k8. R e s u l t s The r e s u l t s o f the experiment are t a b u l a t e d i n Table V. Chlorpromazine, i n a dose o f 2 mgm/Kg., d i d not produce a marked f a l l i n temperature i n animals a t room temperature (22°C.) showed even a g r e a t e r f a l l a t 60 minutes. The l a t t e r , however, e x h i b i t e d a r e c o v e r y t r e n d a t the end of 120 minutes, w h i l e those animals exposed to the c o l d showed l i t t l e change from the 60-minute r e a d i n g . At t h i s dosage the degree of seda-t i o n appeared to be g r e a t e r a t room temperature than i n the c o l d environment, e x p e c i a l l y a f t e r one hour. On r e t u r n i n g the animals from the c o l d to room temperatures, there was a s h o r t p e r i o d o f s e d a t i o n of about l£ minutes d u r a t i o n . At k mgm/Kg. of chlorpromazine, however, the hypothermic a c t i o n , i n the c o l d was pronounced and was three times g r e a t e r than those animals r e c e i v i n g the same dose a t room temperature. Moreover, s e d a t i o n was observed i n b o t h groups but those animals i n the c o l d en-vironment e x h i b i t e d a c a t a l e p t i c c o n d i t i o n a t the end of two hours t h a t was absent i n the animals maintained a t room temper-a t u r e . The animals which had r e c e i v e d BHT r e a c t e d somewhat d i f f e r e n t l y . Temperature d e p r e s s i o n i n the animals maintained at room temperature was g r e a t e r than i n those h e l d at f>°C. a f t e r 15> minutes i r r e s p e c t i v e o f the dosage used. In the c o l d en-vironment the animals t h a t had r e c e i v e d the l a r g e r dose of BHT showed no g r e a t e r temperature d e p r e s s i o n and no i n c r e a s e d degree of s e d a t i o n over those t h a t h ad r e c e i v e d kO mgm/Kg.. However, as the time i n the c o l d i n c r e a s e d , the temperature i n b o t h groups c o n t i n u e d to f a l l , and although the exte n t o f temperature 49. f a l l was s t i l l approximately the same for both groups, at the end of 120 minutes exposure to the cold the temperature depres-sion of these animals was greater than those maintained at room temperature that had received equivalent doses of BHT. Even though the animals i n the cold exhibited this greater f a l l In body temperature, they nevertheless appeared to be less se-dated than those at 22°C.. P i l o e r e c t i o n and ptosis s t i l l were observed e s p e c i a l l y i n those mice that had received the 80 mgm/Kg. dose, but t h e i r mobility was f a r greater than those at room temperature that had received this dose. When the animals were removed from the cold and held at room temperature, seda-t i o n became very marked i n both groups but those that had re-ceived the la r g e r dose of BHT were affected to a greater degree. No deaths were observed i n either group a f t e r k8 hours, although depression and loss of appetite were observed i n the group that had received 80 mgm/Kg. BHT and that had been held i n the cold f o r two hours. These depressant e f f e c t s were observed f o r ap-proximately 2k hours. In a preliminary experiment with 80 mgm/ Kgi of BHT and exposure to cold, 2 of 10 animals exhibited body temperatures of 20 and 21°G. aft e r 120 minutes. These two ani-mals subsequently died. At ambient temperatures of 3k°C, the f a l l i n temperature following chlorpromazine administration d i d not occur. More-over, i n the animals that received 2 mgm/Kg. there was no ob-served sedative e f f e c t , while i n those that had received k mgm/ Kg., there was a short period of exc i t a t i o n (3-5 minutes) f o l -lowed by a mild sedation. At this temperature, animals that had received BHT i n a dose of kO mgm/Kg. showed a s l i g h t r i s e i n 5 0 . temperature at a l l times tested and no sedation. The depres-sant action of the drug appeared to be t o t a l l y l a c k i n g . At 80 mgm/Kg. there was a sharp r i s e In temperature accompanied by a marked h y p e r e x c i t a b i l i t y that appeared to be preconvulsive i n nature. These animals were d i f f i c u l t to handle and i n the f i r s t 60 minutes following administration of the drug, never stopped moving. Respiration was increased p r e c i p i t o u s l y . During the next 60-minute period, a stage of depression was observed that resembled more one of fatigue than one of depression due to the drug. Respiration was s t i l l rapid and s a l i v a t i o n profuse a l -though the animals refused water. During this period one of the animals died and four hours following the termination of the ex-periment another died. On removal of the animals to room temper-ature, depression was very pronounced and r e s p i r a t i o n remained elevated t i l l the body temperature f e l l . In the k8-hour period following the termination of the experiment, the animals refused to eat and drink. 1 Only on the t h i r d day did some semblance of normal a c t i v i t y return. The animals that had received kO mgm/Kg. returned to normal almost immediately following a short period of depression (30 minutes) when they were returned to room temperature. 51 TABLE V Ef f e c t of Ambient Temperature on BHT and  Chlorpromazine-Induced Hypothermia  Ambient Temperature 5°C. Agent and Dosage Chlorpromazine 2 mgm/Kg. k mgm/Kfe. BHT kO mgm/Kg. 80 mgm/Kg. Temperature Depression i n 0°C. + S.D. 15 mlns. 60 mins. 120 mins. 0.18 + 0.30 2.60 + 1.63 1.57 + 0.50 1.80 + 0.92 l.l±6 + O.33 1.3k. + 1.11 5.60 + 2.< ' ,0k 2.12 + 0.16 2.30 + 0.82 13.15 + 5.33 3.35 + O.ko 3.kl + 1.70 Chiorproma z ine 2 mgm/Kg. k mgm/Kg. BHT kO mgm/Kg. 80 mgm/Kg. Ambient Temperature 22°C. Temperature Depression i n 0°C. + S.D. 15 mins. 60 mins. 120 mins. 0.70 + 0.19 2.2k + 1.01 2.01 + 0.30 k.30 + 0.23 .kk + 0.73 ..li.6 + 1.35 1.82 + 0.10 2.56 + 0.67 1.56 + 0.k3 3.86 + 1.32 1.02 + 0.k3 2.0k + 0.87 Chlorpromazine 2 mgm/Kg. k mgm/Kg. BHT kO mgm/Kg. 80 Ambient Temperature 3k°C. Temperature Elevation i n 0°C. + S.D. 15 mlns. 0.12 + 0.10 0.12 + 0.28 0.58 + 0.56 2.05 + 0.69 60 mins. 0.21 + o.ko 0.0k + 0.23 0.1k + o«6o 3.72 + 0.30 120 mins. 0.11 + 0.11 0.30 + 0.05 0.59 + 1.0k 3.68 + 1.35* mgm/Kg. •3:- Temperature elevation of survivors - k /5 A l l values l i s t e d are absolute, calculated against untreated controls at the temperature stated. 52. ELeotroencephalographic Studies The preceding r e s u l t s strongly suggest the involvement of the central nervous system i n the sedative and convulsive actions produced by BHT. Thus i t was f e l t that the recording of some EEG patterns might prove h e l p f u l i n the further e l u c i d a t i o n of these c e n t r a l actions. Since the following series of experi-ments could be more e f f e c t i v e l y performed with intravenous and i n t r a - a r t e r i a l administration, a more soluble form of the drug was required. Preparation of the Sodium S a l t of BHT - (BHT-Na) The sodium s a l t of BHT was prepared according to the method of Halliday ( 8 ) ( 3 9 ) . The sodium s a l t was formed by add-ing an a l c o h o l i c solution of sodium ethylate, f r e s h l y prepared by reacting m e t a l l i c sodium with an excess of absolute alcohol, to a s o l u t i o n of BHT also In absolute alcohol. The quantity of sodium used was just enough to combine with the amount of BHT with which i t was to be mixed. Mixing of the two solutions pro-duced a b r i g h t yellow, alcohol insoluble p r e c i p i t a t e of BHT-Na. The p r e c i p i t a t e was c o l l e c t e d by suction f i l t r a t i o n and washed with several 30 cc. portions of anhydrous ether. The excess ether was removed under reduced pressure i n a dessicator over anhydrous calcium chloride. Y i e l d by this procedure - 89$ by weight of the o r i g i n a l quantity of BHT. pH of a \\.% aqueous so l u t i o n was found to be 1 0 . 0 . Method Two unanesthetized rabbits curarized with 10 mgm/Kg. of 53. gallamine t r i e t h i o d i d e (Flaxedil - Poulenc) and maintained by a r t i f i c i a l r e s p i r a t i o n were used throughout the course of these experiments. One of the animals had been prepared f o r i n t r a -venous i n j e c t i o n by cannulation of the femoral artery, while the other, i n an attempt to observe d i r e c t c e n tral e f f e c t s , was prepared with a cannula implanted into the innominate artery so that i t s t i p l a y at the b i f u r c a t i o n of the r i g h t and l e f t common carotids. I n i t i a l doses of BHT-Na, whether intravenous or i n t r a -a r t e r i a l were 20 mgm/Kg. and i n cases where additional doses were administered, these were also 20 mgm/Kg.. An Offner Electroencephalograph, Type lkO-A and c a l i b r a -ted at 100 microvolts per 1.5 cm. was used throughout. The EEG-records were recorded on a dynograph inkwriter at a track speed of 3 cm./sec. Bipolar recordings were c a r r i e d out between "ri g h t f r o n t a l - r i g h t p a r i e t a l " , " r i g h t p a r i e t a l - r i g h t o c c i p i t a l " and between corresponding positions on the co n t r a l a t e r a l side. Audiogenic s t i m u l i were produced by means of a whistle b l a s t . Simultaneous blood pressure recordings were also made from the femoral artery with a mercury manometer. Results Following intravenous administration of 20 mgm/Kg. of BHT-Na, no discernible changes were observed i n the EEG even after a la t e n t period of 35 minutes. The subsequent adminis-t r a t i o n of a si m i l a r dose of BHT-Na produced Intense desynchro-n i z a t i o n of the EEG af t e r a l a t e n t period of approximately 15 minutes. This activated pattern was accompanied by a p r e c i p i -tous pressor response of 65-125 mm. Hg which persisted f o r 5k. s e v e r a l minutes. (See P l a t e I I . ) In the same p r e p a r a t i o n , EEG- a c t i v a t i o n and s u b s t a n t i a l i n c r e a s e s i n b l o o d p r e s s u r e were e l i c i t e d immediately f o l l o w i n g an audiogenic stimulus ( w h i s t l e b l a s t ) . (See P l a t e I I I . ) Once ag a i n the r i s e i n b l o o d p r e s s u r e was p r e c i p i t o u s . An i n t e r e s t -i n g o b s e r v a t i o n was t h a t the d u r a t i o n of the e f f e c t as e x h i b i t -ed by the a c t i v a t e d EEG p a t t e r n and the i n c r e a s e d p r e s s o r r e -sponse, exceeded the d u r a t i o n o f the a c t i v a t i n g stimulus by a c o n s i d e r a b l e p e r i o d of time. Another o b s e r v a t i o n was that the a c t i v a t e d p a t t e r n f o l l o w -i n g an audiogenic s t i m u l u s appeared to be u n r e l a t e d to the p r e s s o r response s i n c e a marked and p r e c i p i t o u s r i s e i n b l o o d p r e s s u r e , 7I4—120 mm. Hg, s t i l l o ccurs i n the presence o f an i n t e n s e l y desynchronized EEG f o l l o w i n g a s i m i l a r s t i m u l u s . I n t r a - a r t e r i a l a d m i n i s t r a t i o n o f the same dose o f BHT-Na produced h i g h v o l t a g e - s l o w wave a c t i v i t y i n t e r r u p t e d a t i n t e r -v a l s o f 5-7 c y c l e s per second which i s ma n i f e s t e d i n a l l f o u r c o r t i c a l l e a d s . (See P l a t e I I I . ) Again t h i s i s accompanied hy a p r e s s o r response. F o l l o w i n g ah a d d i t i o n a l dose of BHT-Na admi n i s t e r e d by the same r o u t e , the s p i n d l e a c t i v i t y i n c r e a s e d but the r i s e i n b l o o d p r e s s u r e was minimal. A t h i r d dose of BHT-Na produced more i n t e n s e s p i n d l i n g and c o n v u l s i v e a c t i v i t y w i t h a l a t e n c y o f 20 seconds and a d u r a t i o n o f more than 70 seconds b e f o r e r e t u r n i n g to i t s p r e - i n j e c t i o n c o n t r o l p a t t e r n . (See P l a t e IV.) The r i s e i n b l o o d p r e s s u r e was 7 5 - 1 5 ° mm. Hg. The p r e - i n j e c t i o n c o n t r o l p a t t e r n i s seen i n P l a t e I. 55. PLATE I RP-0 LP-0 S a W 1 B P 10 s 1 Whitfl. R e s t i n g and A c t i v a t e d EEG P a t t e r n i n Normal Rabbit 56. PLATE II 1 M U ' p g 1 [ *"/*v m 5 •WW ] EEG- Pattern of Rabbit Following Intravenous Administration of kO mgm/Kg. of BHT 57. PLATE III IPO . L F - P t Vlluttl. Blast [ AW SO Kin aR.r l.v. 40mom/kjBHT B P no KP-o LF-P tU. JDwgm/fo) BHT « B P . H 100 *.s MC. I U iio iuno Whistle Blast Pattern Following Intravenous Administration of kO mgm/Kg. of BHT. Convulsant Pattern Following Administration of 20 mgm/Kg. of BHT Intra-arterially. 5 8 . PLATE IV Convulsant Patterns Following Intra-arterial Administration of 60 mgm/Kg. of BHT 5 9 . Lead T o x i c i t y Studies The a b i l i t y of the t h u j a p l i c i n s to chelate with heavy metals (13) i s shared by BHT (25) . Lead sal t s administered to mice i n s u f f i c i e n t l y large doses r e s u l t i n the death of these animals i n a r e l a t i v e l y short period of time. Thus i t was f e l t that BHT would possibly be capable of preventing or delaying the l e t h a l e f f e cts of lead poisoning through the mechanism of chelation. To this end the following experiments were directed. Method Preparation of the Lead Chelate of BHT This compound was prepared according to the method of •Gardner et a l (25) f o r the copper chelate of BHT. Chloroform solutions of BHT were shaken with 5$ aqueous lead acetate solu-tions, washing with bicarbonate solu t i o n and water and removing the solvent. The lead-BHT chelate (BHT-Pb) r e c r y s t a l l i z e d from chloroform appeared as bright yellow, short needles. The melt-ing point was Indeterminate - decomposition occurred before the melting point was reached, even when the .compound was placed i n a melting point block preheated to k O O ° C Y i e l d f o r this meth-od calculated on the basis of C 1 0 H 1 0 ° 3 P D W a s 98$. (See Pig. k.) The compound was found to be insoluble i n water, ether, ethyl alcohol, propylene g l y c o l , cottonseed o i l and peanut o i l . Thus, i n order that i t could be put i n a suitable form f o r i n -j e c t i o n into mice, i t was t r i t u r a t e d to a f i n e powder i n a mor-tar and suspended i n cold .peanut o i l where the v i s c o s i t y of the o i l was s u f f i c i e n t to maintain the BHT-Pb i n suspension f o r a 60. long enough period of time so that intraperitoneal i n j e c t i o n could be accomplished. Toxic i t y Testing Four groups of female mice, 2k-26 gms. i n weight and con-s i s t i n g of 10 mice per group were used i n this series of experi-ments. The f i r s t group received kO mgm/Kg. of BHT d a i l y . The second group received 200 mgm/Kg. of lead acetate s o l u t i o n i n d i s t i l l e d water; the t h i r d was injected with kO mgm/Kg. of BHT followed i n 30 minutes by an i n j e c t i o n of 200 mgm/Kg. of lead acetate. • Injections i n groups two and three continued d a i l y t i l l death ensued. The fourth group received 86 mgm/Kg. of BHT-Pb (equivalent to kO mgm/Kg. of BHT) f o r eight days. A l l injections were intraperitoneal and were administered at the same hour every day t i l l the experiment was terminated. Results The r e s u l t s are tabulated i n Table VI. The animals that had received BHT alone, showed no f a t a l i t i e s following 9 days of d a i l y administration of the drug. Moreover, there seemed to be no development of tolerance on the part of the mice to the ef-~ fects of the drug, since they exhibited the same depressant signs every time the BHT was administered. Recovery was always rapid (k-6 hours) and the general condition of the animals seem-ed good. The animals that received the lead acetate injections, ex-hi b i t e d signs of the expected lead poisoning 2k hours following 61 the f i r s t administration of the lead s a l t . This was manifested i n l o s s of appetite, black, hard stools and hypokinesia. Fur-ther administration of lead acetate, however, seemed to produce l i t t l e worsening of the condition u n t i l about the seventh day, when ptosis was marked and movements were minimal. A l l the mice receiving this course of injections were dead on the ninth day and only 1 of 10 died before the ninth i n j e c t i o n was given. The expected protective e f f e c t of BHT on lead t o x i c i t y was not observed - i n f a c t the t o x i c i t y of the combination of BHT and lead acetate seemed to be greater than that of the lead acetate alone. A l l of the mice receiving both BHT and lead acetate i n the aforementioned doses were dead by the t h i r d day with deaths occurring as early as 30 hours following adminis-t r a t i o n of the f i r s t p a i r of i n j e c t i o n s . The animals exhibited the t y p i c a l depression pattern following the BHT i n j e c t i o n , but never recovered from th i s state at any time p r i o r to death. As i n the case of the animals that had received the lead s a l t alone, appetite was lacking although food was available and the stools were black and hard. Administration of the BHT-Pb i n the above stated dose, produced l i t t l e depression, no loss of appetite, and l i t t l e loss of a c t i v i t y . The stools of these animals appeared normal at the end of the f i r s t three days of injections and became s l i g h t l y darker i n the l a s t f i v e days of i n j e c t i o n . No f a t a l i t i e s occur-red during the eight day test period. During the l a t t e r part of the i n j e c t i o n course, the animals exhibited a s l i g h t degree of depression but no toxic symptoms were manifested. 62 TABLE VI The E f f e c t of BHT on Lead Toxicity Agent and Daily Number of Survivors aft e r Dose per Mouse 1 Day 2 -Days 3 Days 8 Days 9 Days BHT kO mgm/Kg. 10/10 10/10 10/10 10/10 10/10 Lead Acetate 200 mgm/Kg. 10/10 10/10 10/10 9/10 0/10 BHT kO mgm/Kg. + Lead Acetate 10/10 7/10 0/10 200 mgm/Kg. BHT-Pb 86 mgm/Kg. 10/10 10/10 10/10 10/10 6 3 . B a c t e r i o l o g i c a l and Mycological Studies A l l of the t h u j a p l i c i n s and some of the tropolones have been shown to possess either b a c t e r i o s t a t i c or f u n g i s t a t i c a c t i v i t y or both (9) (10)(11) (12)(23)• I t has also been shown that the a n t i b i o t i c a c t i v i t y of the hot water extract of the heart wood of the Western Red Cedar i s absent under i n vivo conditions ( 2 2 ) . H i n o k i t i o l , however, has been used success-f u l l y i n the treatment of lung gangrene ( 2 k ) . BHT i t s e l f has been shown to contribute l i t t l e to the decay r e s i s t a n t proper-t i e s of Thuja p l i c a t a compared to other heart wood constituents when tested against rot producing fungi (11). Thus i t was f e l t that BHT should be tested against a variety of bacteria and fungi i n an attempt to determine the range of the antimicrobial a c t i v i t y of the drug. Method 100 mgm. of BHT were dissolved i n 1000 cc. of nutrient broth (Difco) and buffered to pH 7.k with sodium hydroxide. S e r i a l d i l u t i o n s were made with the highest d i l u t i o n being 6.3 mgm/litre. A l l d i l u t i o n s were made with nutrient broth buffered at pH 7.k and a l l tubes contained a t o t a l of k.O ml.. B a c t e r i a l suspensions were prepared In this l i q u i d medium from an 18 hour-old broth culture. For b a c t e r i a that required an enriched medium, brain-heart infusion broth (Difco) plus human serum was used f o r growth, d i l u t i o n s and solution of BHT and was also buffered at pH 7.k. Incubation was c a r r i e d out at 3 7°C. The introduction of bacteria into the test media was 6k. accomplished by means of Pasteur pipettes cal i b r a t e d to d e l i v e r exactly 0.02 ml. of suspension per drop at room temperature. The concentration of the b a c t e r i a l suspensions was determined according to the method of Brown and Kirwan (kO) using a #2 opacity tube. Although by use of this method the numbers of each organism Innoculated vary widely ( k l ) , the t o t a l proto-plasm content i s approximately the same f o r a l l species (k2). Thus, 0.02 ml. of b a c t e r i a l suspension was used throughout the b a c t e r i o l o g i c a l studies. A l l tubes were incubated at 37°C. and read at the end of 2k and k8 hours. I f no growth was observed i n any tube at the end of k8 hours, 0.1 ml. of the mixture was removed and placed i n 10 ml. of the appropriate broth to deter-mine whether BHT was b a c t e r i o s t a t i c or b a c t e r i c i d a l . P a r a l l e l determinations were also made on s o l i d media. Homogeneous suspensions of the fungi were accomplished by reducing the mycelia to powder by t r i t u r a t i o n and suspending the p a r t i c l e s i n Sabouraud's cerelose broth. A l l of the moulds used i n these tests were grown, d i l u t e d and tested i n thi s broth while the yeasts were treated s i m i l a r l y i n Sabouraud's maltose broth. BHT i n a concentration of 100 mgm/litre was also pre-pared i n these media which were buffered at pH 6.0. 0.1 ml. of innoculum, prepared according to a #2 Brown's tube, was i n t r o -duced into each tube i n the series which were of the same d i l u -tions as stated above f o r the ba c t e r i a . Incubation was ca r r i e d out at 25°C f o r four weeks, a period necessary f o r some of the moulds. Tubes were read every day f o r the f i r s t week and then once weekly t i l l the termination of the experiment. At the end of the four week period, 0.5 ml. was removed from a l l tubes 65 showing no growth and innoculated into 10 ml. of suitable broth to determine f u n g i s t a t i c or f u n g i c i d a l e f f e c t . P a r a l l e l deter-minations were also done on s o l i d media. Results The re s u l t s of this series of experiments are reported i n Tables VII and VIII. For most organisms, f"0 mgm/litre of BHT i n h i b i t e d t h e i r growth but i n no case was the tropolone shown to be b a c t e r i c i d a l or f u n g i c i d a l , even i n the highest concen-trations used. The Pseudomonas genus, which showed such a mark-ed resistance to the drug, was also tested against concentrations of 100 mgm/litre of BHT.and produced good growth. As a general statement, BHT i n the concentrations used appeared to be a better f u n g i s t a t i c agent than a b a c t e r i o s t a t i c agent. Comparison of BHT and Gamma-Thujaplicin Since gamma-thujaplicin 'sodium (T-Na) was available f o r use, a l i m i t e d comparison of t h e i r respective a n t i b a c t e r i a l a c t i v i t y was c a r r i e d out. Six organisms were chosen at random and the complete procedure c a r r i e d out as i n the preceding ex-periment. The re s u l t s are reported i n Table IX. Results The r e s u l t s Indicate that T-Na i n h i b i t s growth at a higher d i l u t i o n than does BHT. However, i t too was found to be only b a c t e r i o s t a t i c at the concentrations tested. 66. TABLE VII  Fungistatic A c t i v i t y of BHT Organism Concentration of BHT i n mgm/litre. 96 hours ft weeks 50 25 1 2 . 5 6.3 0 50 25 12 .5 6.3 0 Aspergillus c h e v a l i e r i 0 0 0 k ft 0 0 ft k ft Aspergillus nidulans 0 3 k ft k 1 k k k ft Aspergillus niger 0 0 ft ft li- 0 0 k k ft Aspergillus fumigatus 0 0 0 0 ft 0 0 1 k ft Aspergillus giganteus 0 0 ft ft ft 0 2 k k ft Trichophyton asteriodes 0 0 0 0 ft 0 0 0 k ft Trichophyton mentagrophytes 299.2 0 0 ft li- ft 0 0 k- k ft 299.k 0 0 2 ft ft 0 0 k ft Trichophyton l a c t i c o l o r 0 0 2 ft ft 0 0 k k ft Trichophyton epilans 0 0 0 0 0 0 0 0 k ft Microsporum gypseum 0 0 1 3 ft 0 0 k h ft Microsporum fulvum 0 0 0 ft ft 0 0 k k ft P e n i c i l l i u m notaturn 0 0 2 ft k 0 0 k k ft P e n i c i l l i u m lilacenium 0 1 2 k k 0 k k k ft Candida albicans 0 0 0 0 k 0 1 k k ft Candida p a r a p s i l l o s i s rO 0 0 1 0 0 2 k ft Candida guilliermondi 0 0 0 0 k 0 0 0 k ft Candida intermedia 0 0 0 0 k 0 0 k k ft Candida s t e l l a r o i d e a 0 0 0 1 k 0 1 3 k ft Saccharorayces cerivisae baker 0 0 0 k k 0 k k k ft capilano 0 0 0 k k 0 1 k k ft Y-25 v.O 0 3 ft k 0 k k k k Y-27 0 0 2 ft k 0 k k k ft Y-3k 0 0 0 k k 0 1 k k ft Y-75 0 0 0 k k 0 k k ft ft Y-91 0 0 0 ft k 0 k k ft ft Key: 0 = no growth 1 = trace of growth 2 = s l i g h t growth 3 = good growth 4 = abundant growth as control 67 TABLE VIII  Bacte r i o s t a t i c A c t i v i t y of BHT Organism Concentration of BHT i n mgm/litre. 2k hours k8 hours 50 25 12 .5 6 .3 0 50 25 12 .5 6 .3 0 Alcaligenes f e c a l i s 0 0 2 k k 0 0 2 k k Aerobacter aerogenes #1 0 1 2 k k k k k k k Aerobacter aerogenes #5 0 1 2 3 k 1 k k k k B a c i l l u s anthracis 0 0 1 2 k 0 0 1 3 k Ba c i l l u s cereus 0 1 2 3 k 0 1 2 3 k B a c i l l u s brevis 0 0 2 k k 0 0 2 k k B a c i l l u s f i r m i s 0 0 2 k k 0 0 3 k k B a c i l l u s megaterium 0 0 0 1 k 0 0 0 3 k B a c i l l u s s u b t i l i s 0 1 2 3 k 0 1 2 3 k Brucella suis 0 1 2 3 k 0 2 3 k k Diplococcus pneumoniae-» 0 1 k k k 0 k k k k Escherichia c o l i VG-H 0 2 3 k k 1 2 3 k k K l e b s i e l l a pneumoniae 1 2 3 k k 1 2 3 k k Paracolon coliforme 0 0 1 3 k 0 0 1 3 k Paracolon aerogenoid.es 0 2 3 3 k 0 2 3 3 k Proteus morganii 0 0 1 3 k 0 1 k k k Proteus vulgaris 0 0 1 2 k 0 1 k k k Pseudomonas aeruginosa B9k 2 3 k k k 3 k k k k Pseudomonas aeruginosa B97 0 3 k k k 1 k k k k Pseudomonas aeruginosa B101 k k k k k k k k ii k Pseudomonas aeruginosa B106 3 k k k k k k k k k Pseudomonas ambigua 1 2 3 k k 1 2 3 k k Pseudomonas fluorescens k k k k k k k k k k Pseudomonas milden B95 1 2 3 k k 1 2 3 k k Pseudomonas putrefasciens 0 0 2 3 k 0 1 2 k k Pseudomonas pyocyaneus k k k k k k k k k k Salmonella s a l t 1 2 3 k k 1 2 3 h k Salmonella paratyphi B 0 1 2 k k 0 1 2 k k Salmonella typhi 0 1 2 k k 0 1 2 k k Sarcina lutea 0 0 0 0 k 0 0 0 l k Serr a t i a indica 0 1 2 k k 1 3 k k Shigella sonnei 0 0 1 3 k 0 0 2 k k Staphylococcus albus 0 0 1 2 k 0 1 2 3 k Staphylococcus aureus E 0 0 1 3 k 0 1 2 3 k Staphylococcus aureus #6 0 0 1 2 k 0 0 1 k k Staphylococcus citreus 0 0 1 3 k 0 0 2 3 k Streptococcus f e c a l i s 0 1 1 2 k 0 2 2 k Streptococcus hemolyticus*- 0 1 k k 0 1 k h Streptococcus l a c t i s 0 1 2 3 k 1 1 2 2 k Streptococcus pyogenes*- 3 k k k k 3 k k k Streptococcus viridanstf- 0 1 2 2 k 1 1 2 2 k Streptococcus zymogenes 0 1 1 2 k. 1 1 1 2 k •Jf- indicates brain-heart infusion broth and serum. Key: same as Table VII. 68. TABLE IX Comparison of the Ba c t e r i o s t a t i c A c t i v i t y of BHT and T-Na Organism Lowest concentration of agent showing absence of growth i n mgm/litre. 2k hours S. aureus E 25 S. albus 25 P. vulgaris 25 A. aerogenes #L 50 E. c o l i 50 Strep, l a c t i s 50 ! T-Na k8 hours 2k hours k8 hours 50 12.5 25 50 25 25 50 12.5 25 ioo 25 5o ioo 50 5o 50 50 69. In Vivo Studies Since i n v i t r o studies have shown that BHT at a concen-t r a t i o n of $0 mgm/litre prevented the growth of Diplococcus pneumoniae Type I, i t was f e l t that some preliminary work should be done i n an attempt to determine whether i t would delay or prevent the death of animals challenged with this organism. Method A suspension of Diplococcus pneumoniae Type I was pre-pared according to a #2 Brown's opacity tube. 0.02 ml. of thi s suspension, administered i n brain-heart infusion broth, was found to be l e t h a l f o r 25 gm. male mice when injected i n t r a p e r i -toneally within 2k hours. Ten mice were injected with this suspension 30 minutes following i n j e c t i o n i n t r a p e r i t o n e a l l y of 80 mgm/Kg. of BHT i n o i l . Assuming about 80% of the body weight of the mouse to be water, this dose corresponds to a concen-t r a t i o n of about 100 mgm/litre of BHT. In another group of 10 mice, the same procedure was carri e d out but 80 mgm/Kg. of BHT was administered every s i x hours f o r 2k hours. Results BHT exhibited no protective e f f e c t on any of the animals treated. Control and test animals were a l l dead within 2k + 6 hours. 70. DISCUSSION The preceding experiments have indicated that beta-hydroxy t h u j a p l i c i n , both as the acid and as the sodium s a l t , has cen-t r a l nervous system effects i n mice and rabbits. Moreover, i t exhibits a b a c t e r i o s t a t i c and fu n g i s t a t i c action on various organisms and potentiates lead t o x i c i t y . The e f f e c t of BHT on the central nervous system i s both stimulatory and depressant i n nature depending on the dosage administered. At normal room temperatures (22-2i|.0C.), doses of 100 mgm/Kg. of BHT or l e s s , produced only depression when ad-ministered i n t r a p e r i t o n e a l l y i n mice. At doses of lfjO mgm/Kg. and above, the stimulant effects were manifest f i r s t and i n some cases convulsions were exhibited as the component of the stimulant phase. As the dosage of BHT increased, the convulsant stage included more and more of the animals. A l l the animals that convulsed, eventually died as did many of those that did not convulse but showed signs of preconvulsant behaviour. In this the e f f e c t of BHT d i f f e r s from that of gamma-thujaplicin. The l a t t e r i n the form of the sodium s a l t (T-Na), always pro-duced convulsions at doses of 100 mgm/Kg. i n mice and the ani-mals always recovered ( 8 ) . There i s a wide spread existing between the C D ^ Q and LDcjo of T-Na, while those of BHT are almost the same. As i n the case of T-Na, however, a depressant stage followed the stimulant phase following large doses of BHT, but i n the l a t t e r case death was always the r e s u l t . That the e f f e c t of BHT i s central i n o r i g i n as f a r as the convulsive a c t i v i t y i s concerned, i s supported by the EEG records obtained following intravenous and i n t r a - a r t e r i a l 71. administration of the sodium s a l t of BHT. However, on the basis of only two animals and the f a c t that no further experi-mentation was done i n th i s f i e l d , the exact locus of the con-vulsive a c t i v i t y can not be stated d e f i n i t e l y . I f , at some future date, lesions are made i n the mid-brain and si m i l a r r e s u l t s are obtained, then i t would be assumed that the s i t e of action i s r o s t r a l . That the drug af f e c t s the e l e c t r i c a l a c t i v i t y of the b r a i n cannot be denied. In low doses a change toward low voltage-fast a c t i v i t y was produced while i n high doses, spike a c t i v i t y was produced which preliminary r e s u l t s suggest a s i m i l a r i t y to metrazol spiking. From the res u l t s of BHT-barbiturate experiments, i t would appear that this s i m i l a r i t y to metrazol i s s u p e r f i c i a l . Whereas metrazol produced a sharp decrease i n the sleeping time of the barbiturates tested, BHT prolonged the sleeping time despite the f a c t that i n the doses used, both metrazol and BHT produced convulsions when administered alone. In the presence of an adequate dose of barbiturate, BHT and metrazol-induced convul-sions are prevented. I t would appear from these re s u l t s that BHT i n high doses produces a mixture of depressant and stimulant actions, one superimposed upon the other. When the stimulant action i s prevented by the barbiturate, the depressant action i s uppermost and thus adds to the depressant action of the barbi-turate. This seemed to be the case e s p e c i a l l y i n the i n t e r -action with thiopentone, where the sleeping time was increased more at l£0 mgm/Kg. than at 200 mgm/Kg. of BHT, but at l £ 0 mgm/ Kg. i t was nevertheless greater than at any other lower dose 72. tested. Although thiopentone i n the dose used afforded a cer-t a i n amoxint of protection to the animal from BHT t o x i c i t y , i t only delayed the onset of death but did not prevent i t . The in t e r a c t i o n with hexobarbitone was somewhat d i f f e r e n t . The longest sleeping time recorded was at 200 mgm/Kg. of BHT but this was only an increase of 2l§- times that recorded for hexo-barbitone alone, whereas f o r thiopentone, the increase at the maximum was approximately 11 times. Despite this difference, hexobarbitone afforded almost complete protection of the ani-mals that had received doses of 200 mgm/Kg. of BHT. BHT i t -s e l f produced no lo s s of r i g h t i n g r e f l e x as did the barbiturates tested and thus the prolongation of sleeping time i s one of potentiation of the barbiturate action rather than one of syn-ergism. At present the reason f o r the protective action of hexo-barbitone and the r e l a t i v e l y short Increase i n sleeping time compared with the large increase i n sleeping time and the lack of protection with thiopentone i s unknown. Some postulates, however, may be offered i n an attempt to elucidate this action although the experimental proof f o r this theory i s lacking. I t has been shown that the u l t r a short action of hexo-barbitone and thiopentone i s not due to thei r rapid metabolism but to the i r rapid uptake by l i p o i d a l tissue (k3)(kk)(k5). Hermann and Wood (k6) have also shown that the duration of thio-pental anesthesia i s prolonged when body f a t i s decreased. Thus i t would be expected that any substance that would prevent or slow the uptake of the barbiturate into f a t would necessarily prolong the sleeping time. Similar results to those observed 73. with BHT have been reported by Giarman et a l (I4.7) f o r the i n t e r -action of various barbiturates and alpha-tocopherol. These workers have shown that while alpha-tocopherol prolonged thi o -pental sleeping time i t had only a l i m i t e d e f f e c t on hexobarbi-t a l sleeping time. Preliminary r e s u l t s indicated that alpha-tocopheral tended to impede the transfer of thiopental into and out of the b r a i n c e l l s , the net r e s u l t being a prolongation of the thiobarbiturate action. The p o s s i b i l i t y that BHT a f f e c t s the d i s t r i b u t i o n of thiopental i n the same manner as does alpha-tocopherol cannot be Ignored. A t t r a c t i v e as this evidence may be, however, i n the explanation of the prolongation of the sleeping times due to BHT and barbiturate in t e r a c t i o n , i t does l i t t l e to c l a r i f y the s i t u a t i o n with respect to the protective action of hexobarbital. Since both thiopental and hexobarbital prevented convulsions i n mice administered convulsive doses of BHT, the delay i n the onset i n death i s r e a d i l y appreciated, as convulsions have been shown to contribute to the l e t h a l e f f e cts of the tropolone. In the l i g h t of the information available at present, however, the reason f o r the protection from the l e t h a l effects of BHT afforded by hexobarbital and lacking i n the case of thiopental must remain obscure. Although the depressant e f f e c t s of ethanol and barbiturate administered concomitantly are additive i n nature, the depressant effects of BHT and ethanol are not, despite the f a c t that BHT potentiates barbiturate depression. Although no reason f o r this phenomenon i s offered here, i t appears that a d i f f e r e n t mecha-nism i s involved i n ethanol-barbiturate i n t e r a c t i o n than i n BHT-ethanol i n t e r a c t i o n . 7k. Interaction between chlorpromazine and BHT indicates that synergism occurs both i n the case of the hypothermic action and the hypokinetic action of these agents. Moreover, the point of maximum depression of BHT has been shown to have s h i f t e d from l£ minutes to 3 hours and If? minutes a f t e r a p r i o r i n j e c t i o n of chlorpromazine. Nevertheless, If? minutes following the i n j e c -t i o n of BHT af t e r chlorpromazine, the temperature depression was found to be greater than either drug administered alone at any time. A p o s s i b i l i t y exists that further experimentation may show that rather than a peak a c t i v i t y occurring at any one hour, there may be a plateau continuing from lf> minutes after i n j e c t i o n of BHT to 3 hours. This would mean that instead of the peak of a c t i v i t y being s h i f t e d as has been Indicated i n the preceding experiments, there would be only a prolongation of the maximum e f f e c t . The res u l t s obtained i n these experiments would require l i t t l e change to produce such a condition. The resu l t s of the reserpine-BHT experiments indicate that a si m i l a r condition may also occur In this case although the results are inconclusive. The phenomenon of the s h i f t i n peak a c t i v i t y i s c l e a r l y shown i n the amphetamine-BHT experiments. If? minutes following administration of BHT af t e r a previous i n j e c t i o n of amphetamine, the a c t i v i t y of the animals began to decrease and reached a minimum at k hours (3 hours and l£ minutes aft e r BHT). The BHT minimum occurred If? minutes following i n j e c t i o n both i n the case of temperature depression and decreased a c t i v i t y . The BHT-amphetamine combination showed the maximum f a l l i n temperature also 3 hours and If? minutes following administration of BHT. 75. However, when the temperature of the animals was determined ljp minutes following i n j e c t i o n of BHT, there appeared a further r i s e i n temperature whereas those animals that had received amphetamine alone had begun to return to normal body temperatures and below. This e f f e c t of temperature elevation resembles to a li m i t e d extent that produced by BHT at 3k°C. and the p o s s i b i l i t y that the same mechanism i s involved i n both conditions exists, although the animals i n the Xifarm environment exhibited not only temperature elevation but also increased mobility. In the case of the amphetamine-induced temperature elevation, BHT produced a further elevation i n temperature but this was accompanied by a decrease i n a c t i v i t y . The e f f e c t of BHT on thermoregulation i s marked. At room temperatures (22-2i+°C. ), the maximum temperature depression was reached i n 15 minutes, regardless of dose. In the cold, how-ever, this did not occur. After 1$ minutes, even with large doses of BHT (80 mgm/Kg.), the temperature depression was much le s s than that at room temperature, but i t continued to decrease as the time i n the cold was extended, whereas at room tempera-'. ture, a return toward normal values was seen a f t e r 60 minutes. In this respect i t appeared to act much l i k e chlorpromazine i n a dose that at room temperature produced approximately the same degree of hypothermia (k mgm/Kg. chlorpromazine and 80 mgm/Kg. BHT), although this dose of chlorpromazine i n the cold produced a marked f a l l i n temperature at the end of two hours, while that of BHT was moderate. In the warm environment ( 3 k ° C ) , the animals that had received chlorpromazine were not affected at a l l , i . e . neither l o s t nor gained heat, while those animals that 76. had r e c e i v e d 80 mgm/Kg. of BHT showed a marked temperature i n -cr e a s e . Thus i t appears from these r e s u l t s , t h a t whereas c h l o r -promazine impairs the a b i l i t y o f the mouse to conserve heat i t has no e f f e c t on the mechanisms i n v o l v e d i n the d i s s i p a t i o n o f heat. BHT, on the o t h e r hand, seems to a f f e c t b o t h mechanisms although i t appears t h a t the e f f e c t on h e a t - d i s s i p a t i n g mecha-nism i s more pronounced s i n c e when exposed to the c o l d , there appears to be an i n i t i a l r e s i s t a n c e to the f a l l i n temperature. Since i t has been e s t a b l i s h e d t h a t i n man two separate c e n t r e s e x i s t f o r response to heat and to c o l d , and that these are s i t u -a ted i n the hypothalamus (ft8), the p o s s i b i l i t y t h a t one c e n t r e may be a f f e c t e d by BHT to a g r e a t e r degree than the other e x i s t s . Both the a c t i o n of chlorpromazine and BHT would appear to sup-p o r t t h i s c o n t e n t i o n . A r e l a t i o n between hypothermia and h y p o k i n e s i a i n BHT-t r e a t e d animals a l s o appears to e x i s t . In g e n e r a l i t may be s t a t e d t h a t the lower the body temperature of the animal, the g r e a t e r the s e d a t i o n . T h i s e f f e c t has been shown not o n l y i n a l l dosage ranges a t room temperature but a l s o where the en-vironmental temperature has been a l t e r e d . Those animals i n which the body temperature reached kl°C. showed a marked hyper-e x c i t a b i l i t y and h y p e r k i n e s i a i n the f i r s t hour f o l l o w i n g ad-m i n i s t r a t i o n o f the drug, and a l t h o u g h the body temperature r e -mained e l e v a t e d and a s t a t e of h y p o k i n e s i a was observed a t the end of the second hour, t h i s appeared to be due more to f a t i g u e than the s e d a t i v e a c t i o n of BHT. At e l e v a t e d temperatures the hypothermic a c t i o n of r e s e r p i n e i s a l s o prevented and i s accom-pa n i e d by a concomitant l o s s i n i t s s e d a t i v e p r o p e r t i e s , but no 77. hyperthermia or hyperkinesia i s evident (36)(38). In this re-gard, BHT seems to act l i k e amphetamine where an increase i n body temperature was accompanied by an increased e x c i t a b i l i t y . L i t t l e can be said with respect to the precipitous r i s e i n blood pressure accompanying intravenous and i n t r a - a r t e r i a l administration BHT to rabbits. The pressor response, however, seems to be unrelated to the low voltage-fast a c t i v i t y pattern shown on the EEG since this pressor response can be e l i c i t e d even i n the presence of an already activated pattern. Whether BHT acts peripherally or c e n t r a l l y i n t h i s regard, or whether i t resembles amphetamine, epinephrine or other pressor drugs i s not known. Although i n v i t r o studies with BHT concerning i t s a n t i -b a c t e r i a l and antifungal a c t i v i t y indicate that i n concentra-tions of 50 mgm/litre i t i s b a c t e r i o s t a t i c and f u n g i s t a t i c , t h i s property does not seem to extend to i n vivo conditions. Although testing was ca r r i e d out on one organism only, Katsura (2k) and h i s group have shown that a si m i l a r s i t u a t i o n obtains for h i n o k i t i o l (beta-thujaplicin) unless the drug i s adminis-tered t o p i c a l l y . He found that this was due to the presence of whole blood and concluded that the erythrocytes were responsible f o r the i n a e t i v a t i o n of h i n o k i t i o l . For this reason then, i n hi s treatment of lung gangrene he administered beta-thuj'aplicin by intratracheal i n j e c t i o n and this treatment proved successful. Although extensive studies were not ca r r i e d out on t h i s facet of the work, preliminary experiments with BHT and whole blood (not reported i n the experimental section) indicate that BHT i s inactivated also i n the presence of whole blood but not i n the 78. presence of serum. The p o s s i b i l i t y that the t h u j a p l i c i n chel-ate with i r o n i n the hemoglobin molecule leading to the i n a c t i -vation of these tropolones cannot be ignored. The potentiation of lead t o x i c i t y by BHT lends i t s e l f to a pla u s i b l e explanation although further experimentation i s necessary to v e r i f y the hypothesis outlined below. Urinary ex-cr e t i o n of lead can be augmented by administration of ethylene-diamine t e t r a c e t i c acid, a powerful chelating agent ( I i 9 ) . I t has been shown to be highly e f f e c t i v e i n mobilizing lead from the tissues as well as promoting urinary excretion of the metal. Thus the formation of a soluble complex allows for the removal of the lead from the body. On the other hand, BHT-Pb i s insol u -ble except at high pH values where the complex dissociates. Thus there exists no possible route f o r the excretion of the metal since i n the form of the chelate i t cannot be absorbed. BHT i t s e l f has been shown to be rap i d l y absorbed since i t s maxi-mum effects under normal conditions occurred within 15 minutes following intraperitoneal administration of the durg, and thus, although lead Is normally absorbed very slowly, the presence of a strong chelating agent i n the tissues would be expected to i n -crease the absorption of the lead by the tissues. Preformed BHT-Pb i s i n a l l p r o b a b i l i t i e s not absorbed from the peritoneal cavity because of i t s lack of s o l u b i l i t y and thus no toxic effects were immediately evident. I t must be remembered, how-ever, that the dose of BHT-Pb that was administered, contained only kj? mgm/Kg. of Pb whereas administered as the lead acetate, there was l £ 6 mgm/Kg. of Pb. Thus, the above statement i s made with a good deal of reservation. Urinary studies to determine the magnitude of lead excretion under the above conditions would do much to increase our knowledge of the mechanism of action of this increased toxic e f f e c t . 80. SUMMARY AND CONCLUSIONS A preliminary study has been made of the pharmacological a c t i v i t y of beta-hydroxy t h u j a p l i c i n . In low doses I t has been found to exert a depressant e f f e c t on the central nervous system and In high doses both stimulation and depression. No attempt at c l a s s i f i c a t i o n of th i s compound as a central depressant has been attempted although i n some respects i t s actions resemble those of chlorpromazine and i n other respects those of reserpine. The stimulant action i s also u n c l a s s i f i e d . Some preliminary studies have also been carried out as to the a n t i b a c t e r i a l and antifungal a c t i v i t y of the compound and i t has been found to be b a c t e r i o s t a t i c and f u n g i s t a t i c f o r the ma-j o r i t y of organisms tested. 1. In Intact conscious mice, central stimulation has been manifested by an increased e x c i t a b i l i t y , and, i n s u f f i c i e n -t l y large doses, by convulsions. That these convulsions are of central o r i g i n has been shown by EEG- records. 2. The depression following convulsive doses of BHT was manifested by ptosi s , respiratory depression and hypokinesia and was always followed by death of the animal. 3. BHT lias been found to exert no analeptic action on barbiturate depression - i n f a c t sleeping time was prolonged i n animals receiving a barbiturate and BHT. k. Hexobarbital has been shown to prevent the ef-fects of convulsive doses of BHT as well as preventing the 81. l e t h a l e f f e c t s . Thiopental also prevented the convulsions pro-duced by BHT but only delayed the onset of death. 5>. No prolongation of ethanol sleeping time was ex-h i b i t e d by BHT administered concomitantly. 6. At room temperature, low doses of BHT i n mice produced depressant effects which were manifested by hypothermia and hypokinesia. 7. BHT i n low doses was shown to exhibit a syner-g i s t i c action on chlorpromazine-induced hypothermia and hypo-kine s i a . To a l e s s e r extent t h i s action was observed also with reserpine. With both these drugs, the hypothermic and hypo-k i n e t i c actions are prolonged by BHT. 8. BHT has been shown to a f f e c t the thermoregulatory mechanisms of mice, e s p e c i a l l y that of heat d i s s i p a t i o n . A d i r e c t r e l a t i o n s h i p appeared to e x i s t between hypothermia and sedation. In t h i s respect i t resembled chlorpromazine. 9. In the presence of amphetamine, BHT caused an increase i n body temperature accompanied by a decrease i n mo-b i l i t y . Ultimately, however, the temperature was depressed to a greater degree than the mobility. The point of maximum de-pression was delayed about three hours. 10. The difference i n dosage f o r the L D ^ Q and the G D ^ Q was found to be very small. In this respect BHT d i f f e r s from gamma-thujaplicin i n which there i s a wide spread between 82 the CDcjo a n d L D 5 0 . 11. Convulsive doses of BHT administered i n t r a -venously or i n t r a - a r t e r i a l l y to rabbits caused a precipitous r i s e i n blood pressure which appeared to be unrelated to the low voltage-fast a c t i v i t y pattern which occurred concomitantly on the EEG. 12. In v i t r o tests indicated that BHT i n concen-trations of £0 mgm/litre i s b a c t e r i o s t a t i c and f u n g i s t a t i c f o r the majority of organisms tested. Moreover, BHT appeared to be a more e f f e c t i v e f u n g i s t a t i c agent than a b a c t e r i o s t a t i c one. Preliminary experimentation also indicated that gamma-thuja-p l i c i n sodium i s a more active b a c t e r i o s t a t i c agent than BHT. 13. BHT was found to exert a b a c t e r i o s t a t i c action on D. pneumoniae i n v i t r o but f a i l e d to afford any protection to mice challenged with this organism. This i n vivo phenomenon i s probably due to the i n a e t i v a t i o n of BHT by whole blood. Ik. BHT increased the t o x i c i t y of lead s a l t s proba-b l y through the mechanism of chelation. The survival time of mice receiving BHT and lead acetate was reduced by two-thirds over those receiving lead acetate alone. The preformed chelate of BHT and lead produced no observable toxic symptoms i n the doses tested when administered i n t r a p e r i t o n e a l l y . 83. BIBLIOGRAPHY 1. Pauson, P.L.: Tropones and tropolones. Chem. 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Rubin, M., Gignac, S., Bessman, S.P. and Belknap, E.L.: Enhancement of lead excretion i n humans by d i -sodium calcium ehylenediamine tetracetate. Science, 111, 659(1953). Beta-hydroxy thujaplicin Alpha-thujaplic in 28. Dose in mgm/Kg. Figure 5. Absolute percentage decrease in activity in mice measured at different time intervals at various doses. 1+6. # Amphetamine ® 0 Amphetamine + 40 mgm/Kg BHT © 0 Amphetamine + 80 mgm/Kg BHT O O BHT 40 mgm/Kg O O BHT 80 mgm/Kg T EH W pq i 3 Time in hours Figure 11. Interaction of BHT with amphetamine (Temperature) Figure 10. Interaction of BHT with amphetamine (Activity) 1*1. Figure 9. Interaction of BHT with reserpine (Activity) Ij.0. Figure 8. Interaction of BHT with reserpine (Temperature) 39 Figure 7. Interaction of BHT with chlorpromazine (Activity) 38. Figure 6. Interaction of BHT with chlorpromazine (Temperature) 

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