Pharmaceutical Sciences, Faculty of
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UBCV
Klier, Gail Dianne Bellward
Master of Science - MSc
University of British Columbia
The alpha-glyceryl ethers (AGE) are widely distributed in nature, occurring in many marine organisms, land animals, plants, and humans. A series of preliminary experiments was begun to determine whether these compounds possessed possible pharmacological actions against inflammation. Anti-inflammatory tests utilized both the granuloma pouch and cotton pellet granulation methods. Possible glucocorticoid activity was checked by measuring liver glycogen deposition. The effects of AGE on growth and weight of the animals was also noted. Neither selachyl dihemisuccinate sodium (selachyl DHSS) nor selachyl alcohol decreased the exudate formation in the granuloma pouch experiments, although excellent results were obtained with hydrocortisone. The high dose of the AGE was the equivalent of ten milligrams; low doses were three hundred and six hundred micrograms. Routes of administration used were oral, intraperitoneal, and subcutaneous. In the cotton pellet granuloma test, some indication of anti-inflammatory activity was obtained, in accordance with previous results in this and other laboratories. Selachyl DHSS decreased granulation tissue formation by only 11.7% in growing rats, which is not significant; however, in mature rats, there was a decrease of 29.5%. The dosage used in this series was 30 milligrams per kilogram daily by the subcutaneous route, A definite and comparatively large increase in liver glycogen deposition was observed in rats allowed to eat freely, when given selachyl DHSS subcutaneously. These glycogen values were decreased radically by a paired feeding study in which the treated rats were allowed to eat only the same amount of food as their paired control animal. Thus food intake appears to be one of the factors responsible for the increased glycogen storage. The AGE appear to affect growth only by the Intraperitoneal route of administration. In this case, the growth of rats was retarded by 29.6%. It was also noted that these animals ate somewhat less food than the controls, although this did not appear to be an important enough difference to account for the lack of weight gain. Throughout these experiments one of the most notable observations was a lack of uniformity of the results. Factors which appeared to cause a variability in the outcome of tests included the age of the animals, length of time of preoperative housing in new quarters, route of administration, and dosage. A non-linear relationship between dose and effect appears probable.
A STUDY OP SOME PHARMACOLOGICAL PROPERTIES OP CERTAIN ALPHA-GLYCERYL ETHERS by GAIL DIANNE BELLWARD KLIER A thes is submitted i n p a r t i a l f u l 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 Facu l ty of Pharmacy We accept t h i s thes is as conforming to the requi red standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1963 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 for 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 for reference and .study. I further agree that per-mission for 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,, I t 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 w r i t t e n permission. The U n i v e r s i t y of B r i t i s h Columbia,. Vancouver 8 , Canada. Department of Date i A STUDY OP SOME PHARMACOLOGICAL PROPERTIES OP CERTAIN ALPHA-GLYCERYL ETHERS by GAIL DIANNE BELLWARD KLIER ABSTRACT The alpha-glyceryl ether3 (AGE) are widely d i s t r i b u t e d i n nature, occurring i n many marine organisms, land animals, plants, and humans. A series of preliminary experiments was begun to determine whether these compounds possessed possible pharm-acol o g i c a l actions against inflammation. Anti-inflammatory tests u t i l i z e d both the granuloma pouch and cotton p e l l e t granulation methods. Possible g l u c o c o r t i c o i d a c t i v i t y was checked by measuring l i v e r glycogen deposition. The effects of AGE on growth and weight of the animals was also noted. Neither selachyl dihemisuccinate sodium (selachyl DHSS) nor selachyl alcohol decreased the exudate formation i n the granuloma pouch experiments, although excellent r e s u l t s were obtained^with hydrocortisone. The high dose of the AGE was the equivalent of ten milligrams; low doses were three hundred and six hundred micrograms. Routes of administration used were o r a l , i n t r a -peritoneal, and subcutaneous. In the cotton p e l l e t granuloma test, some i n d i c a t i o n of a n t i -inflammatory a c t i v i t y was obtained, i n accordance with previous r e s u l t s i n this and other laboratories. Selachyl DHSS decreased granulation tissue formation by only 11.7$ i n growing r a t s , i i which i s not s i g n i f i c a n t ; however, i n mature rats, there was a decrease of 29.$%. The dosage used i n t h i s series was 30 milligrams per kilogram d a i l y by the subcutaneous route, A d e f i n i t e and comparatively large increase i n l i v e r glycogen deposition was observed i n rats allowed to eat f r e e l y , when given selachyl DHSS subcutaneously. These glycogen values were decreased r a d i c a l l y by a paired feeding study i n which the treated r a t s were allowed to eat only the same amount of food as t h e i r paired control animal. Thus food intake appears to be one of the factors responsible f o r the increased glycogen storage. The A G E appear to affect growth only by the Intraperitoneal route of administration. In t h i s case, the growth of rats was retarded by 29.6$. It was also noted that these animals ate somewhat l e s s food than the controls, although t h i s did not appear to be an important enough difference to account for the lack of weight gain. Throughout these experiments one of the most notable observations was a lack of uniformity of the r e s u l t s . Factors which appeared to cause a v a r i a b i l i t y i n the outcome of tests included the age of the animals, length of time of preoperative housing i n new quarters, route of administration, and dosage. A non-linear relationship between dose and ef f e c t appears probable. Signature of Examiners i i i TABLE OP CONTENTS Page ABSTRACT. . . . . . . . i LIST OP TABLES v LIST OP FIGURES v i INTRODUCTION Occurrence and Separation of Alpha Glyceryl Ethers. . 1 Proof of Structure I4. Synthesis • h Chemical and Physical Properties. l\. Physiological Properties. . . • . 6 Fate. 6 Central E f f e c t s 8 Hematological Effects . . . . . . 8 Growth Studies 12 T o x i c i t y . 13 Anti-inflammatory Properties. . . . . . . . . . . 11+ EXPERIMENTAL Methods • 15 Ant I-inflammatory l£ Granuloma Pouch Technique . . . . . 16 Cotton P e l l e t Method 0 18 Li v e r Glycogen Determination. 19 Growth Studies 23 Results • 2I4. Anti-inflammation 2i+ iv Page Granuloma Pouch 2i\. Cotton P e l l e t Granuloma ••••-••••. .26 Liver Gycogen Determinations . . . . . . . . . . . . 27 Growth Studies 28 DISCUSSION 33 SUMMARY 37 BIBLIOGRAPHY 38 V LIST OP TABLES Table Page I General Characteristics of some Alpha-glyceryl Ethers $ II E f f e c t of Selachyl Dihemlsuccinate Sodium Compared with Hydrocortisone Acetate using the Subcutaneous Route 2\ III E f f e c t of Selachyl Alcohol Compared with Hydro-cortisone 25 IV E f f e c t of Selachyl Dihemlsuccinate Sodium Compared with Selachyl Alcohol 26 V Comparison of Granulation Tissue Formation i n Young and Mature Rats 27 VI Comparison of Liver Glycogen i n Animals Treated with Selachyl Dihemisuccinate Sodium and Hydro-cortisone 27 VII E f f e c t s of the Alpha-glyceryl Ethers on Growth of Rats during Granuloma Pouch Studies . . 30 VIII E f f e c t s of Alpha-glyceryl Ethers on Weight of Rats" during Cotton P e l l e t Studies. . 30 IX Long Term Study of the E f f e c t s of Selachyl Dihemlsuccinate Sodium i n Drinking Water of Normal Rats. . 31 LIST OP FIGURES Figure Page 1. Standard Curve f o r Optical Density of Glucose. . . 22 2. E f f e c t of Selachyl Dihemisuccinate Sodium oh Liver Glycogen Deposition of Rats 29 3. E f f e c t of Selachyl Dihemisuccinate Sodium on Growth and Food Consumption of Rats . « 32 v i i ACKNOWLEDGEMEFT The author i s indebted to Western Chemical Industries Limited for supplies of the alpha-glyceryl ethers. Thanks i s also extended to Dr. J.E. Halliday f o r his help-f u l guidance and c r i t i c i s m . INTRODUCTION Occurrence and Separation of Alpha-Glyceryl Ethers The three most important alpha g l y c e r y l ethers (AGE) found i n nature are b a t y l , chimyl, and selachyl alcohols. Their presence was f i r s t demonstrated by Tsujimoto and Toyaraa i n 1922 while they were investigating the chemical structure of c e r t a i n f i s h o i l s (1). At th i s time, the AGE were i s o l a t e d from the unsaponifiable f r a c t i o n of the l i v e r o i l s of various species of Elasmobranchii. Large amounts are also found i n chimaeroids (2). AGE have been i s o l a t e d from the following sources: l i v e r o i l of the black shark (3); the gorgonia, Plexaur.a fflexuosa, (I4.); the s t a r f i s h , Asterias rubens (§.), when " A s t r o l " was found to be i d e n t i c a l with b a t y l alcohol; unsaponifiable f r a c t i o n of whale and herring o i l , (6); unsaponifiable substance of Aster i a r o l l e s t o n i B e l l . , and Cucumaria chronh.jelmi Theel. (7); unsaponifiable f r a c t i o n of l i v e r o i l of Centrina centrina L, (8); A d r i a t i c Elasmobranchia l i v e r o i l s , Centrina s a l v i a n i . (9); s t e r o l - f r e e f r a c t i o n of the unsaponifiable f r a c t i o n of Liolophura .japonica (chitons), ( l ) j Asterias amurenis, (11); cow shark l i v e r , Hexanchus griseus, (12); unsaponifiable matter of Coscinasterias acutespina and Comanthus japonica, (13); Asterias ffopbesi. ( l l j . ) ; f a t t y o i l s of Stickopus .laponicus. (15); sea anemones, (16); Tachypleus tridentatus fat and Mytilus edulls o i l , (17); l i v e r o i l of Squalus suckleyi (shark), (18); Tonna l e u t e o 3 t o m a , (19); and f a t t y o i l s of Pentacta doliolum (20). 2 In t h e i r review, Bodman and M a i s i n (21) c i t e d evidence t h a t the AGE i n f i s h o i l s may be presen t as f a t t y a c i d e s t e r s , the remaining two hy d r o x y l s being combined wi t h a high e r f a t t y a c i d . I t was a l s o p o i n t e d out that the occurrence o f AGE i s mainly i n f i s h o i l s w i t h a subnormal degree of u n s a t u r a t i o n . Karnovsky e t . a l . (22) i n 191+6 concluded t h a t the o n l y v e g e t a b l e o i l c o n t a i n i n g any q u a n t i t y o f AGE was tung o i l , so t h a t t h i s p a r t i c u l a r l i n k a g e might be c h a r a c t e r i s t i c o f animal f a t s . However, l a t e r s t u d i e s showed the presence o f AGE I n other v e g e t a b l e o i l s , as i n d i c a t e d i n Bodman and M a i s i n ' s review (21). In h i g h e r animals, AGE are found i n the body c a v i t y f a t , I n t e s t i n a l f a t and l i v e r o i l of the r a t and domestic hen (21), egg y o l k (23), bone marrow of c a t t l e (21}.), s p l e e n o f p i g s (25), human e r y t h r o c y t e s (26), and human a r t e r i o s c l e r o t i c a o r t a s ( 2 7 ) . An AGE was i s o l a t e d from reduced t o t a l - p i g - h e a r t - p h o s p h a t i d e s , l e c i t h i n , and c e p h a l i n (28). Reduction o f b r a i n colaraine plasmalogen gave a mixture o f chimyl and b a t y l a l c o h o l s i n Debuch's study (29). In a study on human b r a i n (30), b a t y l a l c o h o l was found a f t e r b r e a k i n g down an i n o s i t o l phosphatide i s o l a t e d from the c e p h a l i n f r a c t i o n . Nakagawa and McKibbin (31) i s o l a t e d .2 - 1.35 micromoles AGE per gram o f dry l i p i d - f r e e t i s s u e i n the dog. Rabbit bone marrow c o n t a i n e d t e n times as much as other t i s s u e s . H a l l g r e n and L a r s son (2lj.) s t u d i e d AGE i n man and cows. The h i g h e s t c o n c e n t r a t i o n was found i n human r e d bone marrow (0.2$ o f l i p i d s ) and human m i l k (0.1$ o f l i p i d s ) . The method o f e x t r a c t i o n o f r e d b l o o d c e l l s was poor, and t h e r e f o r e a low 3 concentration was reported. However even the highest con-centration shown i n the human could not compare with the 10 - 30$ AGE i n l i p i d s of Elasmobranch l i v e r o i l . Several other g l y c e r y l ethers besides selachyl, b a t y l , and chimyl alcohols were also reported. These unknown alcohols formed 30$ of the AGE i n human bone marrow. This group concluded that the AGE are found i n milk, f e t a l t i s s u e s , or r a p i d l y reproducing tissues such as bone marrow. Bodman and Maisin (21) concluded that AGE i n land animals are present i n those tissues containing c e l l s of mesenchymal o r i g i n , such as bone marrow, spleen, and i n t e s t i n e , from which tissue c e l l s are continually shed i n t o a body cavity. Since the Intestine i s concerned with metabolism of higher alcohols and r e l a t e d compounds, they hypothesized that the gut mucosa might be concerned with AGE synthesis. In recent years, the most widely used methods of separation of g l y c e r y l ethers are various forms of chromatography. Eramerie (32) oxidized the AGE to aldehydes before u t i l i z i n g ascending paper chromatography, and a fushin-sulfurous acid reagent for color production. Blomstrand and Gurtler (33) found that complete separation of AGE was obtained by g a s - l i q u i d chromatography afte r a c y l a t i o n of the two hydroxy groups. Hallgren and Larsson (3^,35) extracted AGE with d i e t h y l oxide from an a l k a l i n e hydrolyzate of f i s h l i v e r o i l s , and separated these by column chromatography on alumina. The ethers were converted to dimethoxy derivatives, rechromatographed on alumina, and then were run through the g a s - l i q u i d chromatograph. Malins (36) u t i l i z e d both thin-layer s i l i c i c a c i d chromatography and gas chromatography while studying dogfish l i v e r o i l . Proof of Structure Previous to 1 9 3 3 batyl alcohol was considered to be C ^ Q H - ^ -0CH(CH20H)2.. Adam ( 3 7 ) and other workers u t i l i z e d surface films on water to show that b a t y l alcohol and synthetic alpha octadecyl g l y c e r y l ether were i d e n t i c a l , s i m i l a r l y chimyl alcohol and alpha hexadecyl g l y c e r y l ether were i d e n t i c a l . Davies et. a l . ( 3 8 ) confirmed this r e s u l t by the oxidation of the various compounds. In t h i s way, selachyl alcohol was found to be alpha o l e y l g l y c e r y l ether. The natural batyl and chimyl alcohols were known to belong to the d-series. When selachyl alcohol was shown to be reducable to b a t y l , i t was also assigned to the d-series ( 3 9 ) . Synthesis Various methods have been u t i l i z e d f o r synthesizing AGE and th e i r analogues, including those of Kornblum and Holmes (I4.0), Gupta and Kummerow (I4.I)> Kumamura (l\Z), Baylis et. a l . (1^ 3 )» & n d Bevan and Malkin ( I 4 J 4 . ) . Chemical and Physical Properties General c h a r a c t e r i s t i c s of these compounds may be seen i n Table I. The double bond i n selachyl alcohol fluoresces at 3 6 5 m i l l i -microns under an u l t r a - v i o l e t lamp. Selachyl alcohol occurs i n TABLE I General Characteristics of some Alpha-glyceryl Ethers Compound bat y l alcohol chimyl alcohol selachyl alcohol Chemical Name alpha-octadecyl-g l y c e r y l ether alpha-hexadecyl-gly c e r y l ether alpha, 9-octa-decehyl g l y c e r y l ether Components stearyl alcohol and glycerol c e t y l alcohol and glycerol o l e y l alcohol ( c i s - 9 - o c t a -decahol) and glyc e r o l Melting Point 70 - 71c Formula 6 0 .5 ( 8. 0 C H 2 0 ( C H 2 ) 1 7 0 H 3 GHOH i CH 20H eH20.(GH2.)i£CH3 CHOH I CH 20H C H 2 O ( C H 2 ) 8 C H = ' C H ( C H 2 7 7 C H 3 C H O H I C H 2 0 H c i s and t r a n s isomers, the c i s b e i n g the form found i n na t u r e . The molecule o f the c i s Isomer i s much l e s s extended, but I t has the lower m e l t i n g p o i n t . In v i t r o , the e i s - t r a n s forms are r e a d i l y interchanged, and there i s some evidence t h a t t h i s may a l s o occur i n v i v o . Both n a t u r a l and s y n t h e t i c forms of these t h r e e AGE have an o p t i c a l r o t a t i o n . F o r example, b a t y l a l c o h o l i n h i g h con-c e n t r a t i o n s has a negative r o t a t i o n , which g r a d u a l l y diminishes t o zero a t 10$ c o n c e n t r a t i o n . A p o s i t i v e r o t a t i o n i s seen on f u r t h e r d i l u t i o n s . C a u t i o n must be used when determining the m e l t i n g p o i n t s o f these compounds s i n c e they form e u t e c t i c mixtures. T h i s f a c t may account f o r the v a r i a t i o n s i n m e l t i n g p o i n t s r e p o r t e d i n the e a r l i e r l i t e r a t u r e . A c c o r d i n g t o Bigelow e t . a l . (1+5), b a t y l a l c o h o l forms oleophobic f i l m s . The AGE are a l s o s u r f a c t a n t s , although the e m u l s i f y i n g power of b a t y l a l c o h o l i s somewhat l e s s than a l p h a -monostearin (1+6). I t has been p o i n t e d out t h a t the AGE are an i n t e r m e d i a t e l i n k between g l y c e r i d e s ( t r u e f a t s ) and waxes (1+7). P h y s i o l o g i c a l P r o p e r t i e s F a t e The a b s o r p t i o n and metabolism o f chirayl a l c o h o l has been e x t e n s i v e l y s t u d i e d by Blomstrand and co-workers (1+8,1+9,50). In r a t s (1+8) i t was found t h a t t h i s compound was n i n e t y - f i v e p e r c e n t absorbed a f t e r o r a l i n t u b a t i o n . The remaining f i v e percent was r e c o v e r e d from the u n s a p o n i f i a b l e f r a c t i o n o f f e c a l l i p i d s . 7 Approximately h a l f of the absorbed chimyl alcohol was present i n the lymph l i p i d s . H alf again of this portion was palmitic acid; the other h a l f was f i f t y percent free alcohol and the rest was e s t e r i f i e d . Thus chimyl alcohol appeared to be well absorbed but extensively metabolized during t h i s absorption. Further r a d i o -active investigations (1+9) appeared to show that the g l y c e r y l ether can be absorbed unchanged, but i s metabolized to a large extent i n the In t e s t i n a l mucosa c e l l s . This w a 3 indicated by the fact that i n the lymph l i p i d s more than h a l f of the r a d i o a c t i v i t y present was i n the form of palmitic acid due to a s p l i t t i n g of the ether bond i n the i n t e s t i n a l mucosa. These workers concluded that a dynamic s i t u a t i o n i s present i n the i n t e s t i n e , where resynthesis of glyceride ester bonds occurs simultaneously with hydrolysis. Blomstrand and Ahrens (5>0) further examined the fate of chimyl alcohol by u t i l i z i n g two human patients, one with chyluria and the other with a b i l a t e r a l chylothorax. Again the compound was almost completely absorbed, with the ether linkage of a portion of the amount administered ruptured i n the i n t e s t i n a l mucosa, and the palmityl alcohol moiety r e a d i l y oxidized to palmitic a c i d . Half the r a d i o a c t i v i t y i n the lymph was chimyl alcohol; the r e s t was palmitic a c i d . Cleavage of the ether linkage suggested "a hitherto undefined enzymatic mechanism." This paper indicated the p o s s i b i l i t y that a l l the absorbed compound i s transported v i a the lymphatic pathway. 8 Central E f f e c t s During the course of Berger's systematic i n v e s t i g a t i o n of the pharmacological properties of AGE ($1,52), b a t y l alcohol was studied. It was reported that nine millimole:s per kilogram subcutaneously i n j e c t e d i n mice and rats did not reach either the mean paralyzing dose or the mean l e t h a l dose. However, many other substances of the type R-0-CH2-CHOH-CH2OH did produce muscular re l a x a t i o n and paralysis due to central nervous system depression, e s p e c i a l l y a f f e c t i n g the spinal cord. In the a l i p h a t i c r a d i c a l group, straight chain a l k y l s gave the best e f f e c t , with the n-amyl ether being the most potent. Increasing chain length, s u b s t i t u t i o n on the carbon atom or hydroxyl groups decreased or destroyed the paralyzing a c t i v i t y . Hematological E f f e c t s In 191+9. Sandler ( I 4 . 7 ) published a paper describing the effects of yellow bone marrow extracts and b a t y l alcohol on rats and humans. Although other groups had studied the effects of bone marrow extracts before t h i s , their work was generally haphazard and rather inconclusive. The f r a c t i o n of the extracts containing b a t y l alcohol had now been well established, as were various syntheses of the compounds. Sandler treated benzene-depleted r a t s subcutaneously with bone marrow concentrates, and showed that such measures increased the resistance of the rats as shown by better general physical condition and s l i g h t l y lower mortality, increased the number of c i r c u l a t i n g erythrocytes, decreased the subcutaneous abscesses caused by the benzene, and 9 decreased the inflammation i n the t a i l of c e r t a i n groups i n which the t a i l was cut for blood sampling. When normal animals were used, again there was an increase In the erythrocyte count. The healthy human subjects showed an increase i n t h e i r r e t i c u l o c y t e value of six times the standard,deviation, which l a s t e d eight to nine days a f t e r termination of treatment. Neither hemoglobin nor erythrocyte values were s i g n i f i c a n t l y increased. The compound appeared to have a cumulative type of e f f e c t . Sandler's paper aroused a great deal of i n t e r e s t i n the f i e l d of general depression of the bone marrow. A l a t e r paper by de Gaetani and B a i o t t i (53) confirmed Sandler's work, s t a t i n g that batyl alcohol post-treatment to benzene-intoxicated r a t s prolonged l i f e , and pretreatment acted as a protection. It was also found to increase the osmotic resistance of the red blood c e l l s , restore the a c t i v i t y of the bone marrow, and favor r e t i c u l o c y t e s ! s . A series of papers on the hemopoietic properties of b a t y l alcohol has been published by Linman and his co-workers (51+.55, 5 6 , 5 7 ) . They found that t h i s AGE stimulated erythropoiesis i n normal r a t s by accelerated erythroblastic c e l l u l a r d i v i s i o n without an increase i n hemoglobin formation. Reticulocytosis, thrombopoiesis, and some granulopoiesis were also present. Batyl alcohol and the thermostable, ether-soluble plasma erythropoietic factor share many common physical and chemical properties, such as the following: both stimulate d i v i s i o n of erythrocyte precursors without decreasing hemoglobin production, 10 both are r e s i s t a n t to heat at 100°C for prolonged periods, both are s i m i l a r i n molecular size and are l i p i d - l i k e compounds. Therefore t h i s group of workers concluded that b a t y l alcohol and the ether-3oluble factor are probably the same or c l o s e l y r e l a t e d compounds. Later studies added supporting evidence. Both compounds are leukopoietic; neither increase the hematocrit; an i d e n t i c a l response i s obtained i n vivo. If t h i s conclusion i s correct, b a t y l alcohol or some other AGE could be one of the main c o n t r o l l i n g factors f o r the primary erythrocytogenic stimulus. Since selachyl alcohol i s the most abundant AGE i n nature, i t s e f f e c t on the bone marrow was compared with the above r e s u l t s . No hemopoietic stimulatory a c t i v i t y i n normal r a t s was found, although both the synthetic and natural compounds were used (57). Batyl alcohol has been tested as a protective or therapeutic agent i n many conditions characterized by a hypoplastic bone marrow. Edlund (58) gave subcutaneous i n j e c t i o n s to mice i r r a d i a t e d with x-rays with the r e s u l t that the thirty-day l e t h a l i t y was s i g n i f i c a n t l y lowered. During t h i s experiment i t was shown that an overdosage increased the l e t h a l i t y . A study on human patients with r a d i a t i o n leukopenia was published simultaneously by Brohult and Holmberg (26). Of the t h i r t y - s i x cases given alkoxyglycerol esters o r a l l y , twenty-five showed an immediate increase i n white blood c e l l count. Therefore t h i s group concluded that the AGE were promising as a therapy against leukopenia of r a d i a t i o n . 11 Batyl alcohol was also studied i n eases of bracken poisoning i n c a t t l e (59 )> since t h i s condition i s characterized by a severe leukopenia and thrombocytopenia caused by a depression of the bone marrow. A n t i b i o t i c s were administered at the same time to control and treated animals. An increased leukocytic response was obtained, with the r e s u l t that the animals survived. A s i m i l a r type of a p l a s t i c anemia i s caused by trichloroethylene-extracted soybean o i l meal. This was fed to calves (60) and then batyl alcohol was injected by various routes before hematological symptoms had developed. No protective e f f e c t was found. However, various differences i n technique and type of anemia may explain the c o n f l i c t i n g r e s u l t s . In t h i s condition there i s a decrease i n hemoglobin. Since i t has already been shown that batyl alcohol does not a f f e c t hemoglobin l e v e l s , t h i s could be part of the reason for a lack of response. Also the toxic agent i n the meal may a f f e c t some biochemical mechanism i r r e v e r s i b l y before i n i t i a t i o n of the therapy was begun. Positive r e s u l t s were shown by subcutaneous or o r a l b a t y l alcohol when i t p a r t i a l l y prevented the leucopenia of nitrogen mustard N-oxide i n rats (61). Further supporting evidence was shown i n rabbits with nitromin-caused anemia and patients with organic solvent i n t o x i c a t i o n (62), although the b a t y l alcohol produced no hemopoietic stimulating action on healthy r a t s or r a b b i t s . Ghys (63) challenged the leucopoietic-promoting properties of b a t y l alcohol. F a i r l y large doses of a f r a c t i o n of the l i v e r o i l of Somniosus microcephalus were given o r a l l y to patients with 12 x-ray leucopenia. Only four out of f i f t e e n cases showed any leukopoietic e f f e c t . However, some of these anomalies may be* due to using doses other than the maximum eff e c t i v e dose. Brohult (6i|.) has shown that the white blood c e l l count rose to a maximum as the dose increased, but f e l l o f f with increasing amounts of o i l given above t h i s dose. In an attempt to evaluate the Perm test f o r cancer, R i l e y et. a l . (65,66) i d e n t i f i e d b a t y l and chimyl alcohols from the unsaponifiable l i v e r f r a c t i o n of cancerous i n d i v i d u a l s . These compounds were two of the major constituents responsible f o r the sero l o g i c a l f l o c c u l a t i o n reaction. The factor In the serum which combines with these alcohols to produce the f l o c c u l a t i o n was i d e n t i f i e d as a C-reactive protein. This compound was thought to be present i n the serum of patients with cancer, but not i n normal serum. This was disproven when i t was shown that the protein appears i n inflammatory states and not i n those s p e c i f i c a l l y associated with cancer. Growth Studies Recently several papers have been published describing the g l y c e r y l ethers as growth-promoting substances. Working with dwarf and Alaska peas, Stowe (67) observed that although excised sections of the plants f a i l e d to obtain half t h e i r normal s i z e , part of t h i s missing growth could be restored by several types of f a t t y acid esters. The methyl ester of selachyl alcohol was one of these substances. In order to exhibit any growth-promotion i n a l l experiments sugar and auxin were also necessary. G i b b e r e l l i c a c i d enhanced t h e i r action further. This indicated 13 that the growth stimulation was due to an enhancement of auxin and g i b b e r e l l i n action by the esters. These effects were maximal when small quantities of esters were used, f o r example, 10 to £0 raibromoles; therefore, the action appeared to be hormonal rather than n u t r i t i o n a l . Brohult (68) examined the matter further, using Lactobacillus l a c t i s and selachyl alcohol. A stimulation of growth was c l e a r l y observed even at low concentrations of the AGE. Brohult also observed that when i r r a d i a t e d r a t s were pretreated with selachyl alcohol, they showed a greater Increase i n weight than the controls. However, t h i s may not be connected exclusively with growth effects since many other factors enter into the problem of i r r a d i a t i o n . In t h e i r study on the presence of g l y c e r y l ethers i n man and other animals (2l+), Hallgren and Larsson concluded that since the AGE are found i n milk, f e t a l t i s s u e , or r a p i d l y reproducing tissue such as bone marrow, these compounds "might stimulate the growth of the newborn." AGE have also been found to promote stage one of i r r a d i a t i o n wound healing (21) but do not accelerate normal healing which has already begun. T o x i c i t y Extremely l i t t l e work has been done on the t o x i c i t y of the AGE. As stated i n Sandler's a r t i c l e (1+7), an i n v e s t i g a t i o n into the t o x i c i t y of cod l i v e r o i l showed that prolonged administration caused degenerative changes i n the suprarenal gland, heart, l i v e r , and kidneys of albino mice. The toxic agents were b a t y l alcohol lit-and octadecyl alcohol. It has also been observed that the free alcohols are much more toxic than t h e i r esters which are the naturally-occurring form. However no evidence of t o x i c i t y i n the human studies has been shown, although some nausea was present (1|7). Anti-inflammatory Properties There has been some i n d i c a t i o n i n the l i t e r a t u r e that the AGE might possess an anti-inflammatory action. A preliminary study of t h i s p o s s i b i l i t y (2) gave p o s i t i v e r e s u l t s with very small doses. There have also been unpublished reports of the effectiveness of AGE i n some cases of a r t h r i t i s (2). It has long been hoped that an anti-inflammatory compound could be found which would not possess the exaggerated hormonal manifestations of the corticosteroids or the toxic s i d e - e f f e c t s of phenylbutazone, the s a l i c y l a t e s , and t h e i r derivatives. Since the toxic dose of the AGE appears to be very high i n comparison to the pharmacologically e f f e c t i v e dose, i t was f e l t that further i n v e s t i g a t i o n into the anti-inflammatory action of t h i s group of compounds was warranted. Their interesting e f f e c t on growth also appeared to have been underemphasized. 15 EXPERIMENTAL Methods Ant i - i n f lammat ory A great many types of experiments have been developed for the purpose of tes t i n g anti-inflammatory a c t i v i t y . Some of these tests are the following: tuberculin skin s e n s i t i v i t y , i r r i t a n t -induced swelling of foot or trunk area, histamine-sensitization, l i v e r glycogen deposition and adrenal cortex a l t e r a t i o n s , a l l of which are described by D'Arcy et. a l . (69), "cold stress" (*7Q), "swimming" stress (71)* immobilization (72), foreign body granulomas (73) > a n d granuloma pouch formation (7I4. and 75) • A recent review by Wilhelm (76) c r i t i c i z e s the methods which u t i l i z e the swelling of the feet or trunk of small animals because the anesthesia used depresses the permeability response of the peripheral blood vessels. The intravenous i n j e c t i o n of dyes which accumulate i n treated s i t e s , plus the " s e n s i t i v i t y " tests, were also s a i d to be a poor means of te s t i n g a n t i -inflammatory compounds since they are so subjective. Many other factors i n t e r f e r e with the previously l i s t e d t e s t s , such as temperature (77)* &ge and species of animal. Previous studies i n thi s lab u t i l i z i n g the "cotton p e l l e t " method have indicated that selachyl dihemlsuccinate sodium i s the most consistently e f f e c t i v e drug of the compounds tested. The dosage u t i l i z e d was f i v e to ten milligrams per rat ( 7 8 ) . The granulation response which i s used as a measure of the degree 16 of inflammation i n t h i s t e s t , increases not only with time but with mechanical i n j u r y ( 7 9 ) . The l a t t e r effect tends to cause . v a r i a b i l i t y i n r e s u l t s , thus i t was decided to t r y methods which, entailed less trauma due to operative procedures. The tests decided upon were a modified cotton p e l l e t method, and the granuloma pouch assay (75)* where i t i s possible to obtain quantitative measurements fo r purposes of comparison, (a) Granuloma pouch technique A modification of the method described by Robert and Nezamis (75) was used. Female Wistar r a t s , with weights varying from 150 to 200 grams, were divided into groups of f i v e to ten animals each, and matched f o r weight. In order to allow the animals to adjust to t h e i r new surroundings, they were l e f t i n t h e i r cages for forty-eight hours before the start of the experiment. A f i v e percent stock solution of croton o i l i n cottonseed o i l was stored i n the r e f r i g e r a t o r . It was di l u t e d to a two percent s o l u t i o n f o r the i n j e c t i o n s . Apparatus used during the experiment was s t e r i l i z e d by immersion i n benzalkonium chloride s o l u t i o n . A l l i n j e c t i o n s were given i n a room other than the one used to shave and house the animals, and the operating area was washed with benzalkonium solution, also. A f t e r anesthetizing the animals with pentobarbital, t h e i r backs were c l o s e l y shaved with an e l e c t r i c clipper and swabbed with the a n t i s e p t i c . Twenty-five c.c. of a i r was injected under the dorsal skin just s l i g h t l y caudad to the center of the animal's back. A t h i r t y c.c. syringe and l / I f . inch, 26 gauge needle were used. In order to form the pouch properly, i t was 17 gently pressed forward while the needle was s t i l l inserted. The needle was then removed while pressing the skin together at the s i t e of Inj e c t i o n . After one group of animals was done, 0.5 mla of croton o i l (2%) was i n j e c t e d subcutaneously d i r e c t l y Into the top of the pouch, using a I / I 4 . inch, 27 gauge needle. The needle was l e f t i n f o r a few minutes to allow the i r r i t a n t o i l to drain out. This procedure guarded against l o c a l necrosis. The AGE was administered at t h i s time and d a i l y thereafter. The a i r was withdrawn forty-eight hours a f t e r formation of the pouch. This was done through a 2l\. gauge needle attached by rubber tubing, through a water trap, to the aspirator f i t t i n g on a water out l e t . No anesthetic was necessary, but aseptic pre-cautions were s t i l l maintained. Since the layers of the pouch sometimes adhere together a f t e r the withdrawal of the a i r , the pouches were gently manipulated the next day to break up any adhesions. On the fourth or f i f t h day after the i n i t i a l pouch formation (day zero), the animals were s a c r i f i c e d using ether. Part of the exudate was c o l l e c t e d using a large syringe and needle injected d i r e c t l y i n t o the pouch, and the remainder c o l l e c t e d through a cut In the skin. The effects on exudate formation produced by selachyl alcohol and selachyl dihemisuccinate were compared with that of hydrocortisone acetate. The hydrocortisone was suspended i n a 2 percent tween 20 solution i n one experiment; the vehicle f o r a l l solutions i n this group was 2% tween 20, therefore. In another 18 experiment, the hydrocortisone was suspended i n cottonseed o i l , which was the vehicle for t h i s group. Various dosage l e v e l s and routes of administration of the compounds were used. Throughout a l l experiments, i t was found that injections of the AGE, e s p e c i a l l y selachyl dihemlsuccinate sodium, caused l o c a l i r r i t a t i o n . (b) Cotton p e l l e t method This technique, o r i g i n a l l y described by Meier et. a l . (73) was modified to eliminate some of the v a r i a b i l i t y . A c y l i n d r i c a l compressed cotton sponge of the type used by dentists was s l i c e d into c i r c u l a r p e l l e t s weighing approximately J4.O mgm each. The p e l l e t produced i n t h i s manner was of a uniform weight and surface area. Male albino Mistar rats were anesthetized with pentobarb, and one weighed p e l l e t c a r e f u l l y implanted sub-cutaneously i n the loose dorsal neck t i s s u e . This eliminated much of the trauma to surrounding tissues by implantation of four p e l l e t s i n the usual method. At termination of the experiment, the i n c i s i o n was reopened and extraneous tissue cut away u n t i l the granuloma surrounded p e l l e t was able to be pressed out of i t s cavit y . The usual length of such an experiment was seven days, with injections of the drugs given daily. The p e l l e t s were placed on tared planchets and oven dried at 80°C fo r twenty-four hours before reweighing. Daily subcutaneous injections of 10 mg per kilogram of hydrocortisone and 30 mg P©r kilogram of selachyl dihemlsuccinate sodium were compared i n young rats weighing 100 to 125 grams, and 19 mature rats of 220 to 300 grams since i n preliminary experiments more consistent results were obtained i n older r a t s . The vehicle used was normal s a l i n e . Liver-glycogen Determination Although i t i s possible f o r a compound to possess a n t i -inflammatory a c t i v i t y without possessing gl u c o c o r t i c o i d a c t i v i t y (80), i t was f e l t that the r e s u l t s of a glycogen deposition test would be of i n t e r e s t f o r the following reasons: - previous experiments i n t h i s , (78) and other laboratories (2) have shown the AGE to have some anti-inflammatory a c t i v i t y , although the r e s u l t s were i r r e g u l a r ; - an increase i n the resistance to stress has been att r i b u t e d to the AGE, (2); - various workers have published r e s u l t s i n d i c a t i n g some hormonal or hormone-stimulating response (5i{.,55>,67); - observations on the growth of plants and animals implicate the AGE i n this physiological process (67,68,2l|.). The l o g i c a l c o r r e l a t i o n of t h i s data appeared to indicate that the adrenal-pituitary axis could perhaps be affected by the AGE. In order to determine i f these compounds did have a g l u c o c o r t i c o i d e f f e c t , the deposition of glycogen i n the l i v e r of r a t s was investigated. The Pfluger method as modified by Colowick and Kaplan was used with minor changes (81). A number of 15 ml Pyrex centrifuge tubes each containing 3*5 mis of 30$ potassium hydroxide solution were tared. Male Wis tar rats were k i l l e d by ether anesthesia and approximately one gram of l i v e r from the l e f t lobe was quickly 20 excised, weighed, and immersed i n the potassium hydroxide. The tubes were reweighed on an a n a l y t i c a l balance to determine the exact weight of tissue used, and then immersed i n a b o i l i n g water bath. The tissue was digested for approximately t h i r t y minutes, or u n t i l a r e l a t i v e l y clear s o l u t i o n i s obtained. During t h i s time the tubes were shaken frequently to a s s i s t i n the digestion. The tubes were removed from the bath, and 0.5 mis of saturated sodium sulfate was added. The glycogen was p r e c i p i t a t e d 1.1 to 1.2 volumes of 95$ ethanol, s t i r r e d , and the s t i r r i n g rod washed with 60$ ethanol. The tubes were heated just to b o i l i n g i n the hot water bath, cooled, and centrifuged at 3000 r.p.m. The supernatant l i q u i d was decanted, and the glycogen p r e c i p i t a t e allowed to drain. In order to expel any alcohol which may be l e f t , the tubes were heated again f o r a short time. The glycogen was redissolved i n 2.0 mis of d i s t i l l e d water, r e p r e c i p i t a t e d with 2.5 mis of 95$ ethanol, s t i r r e d , heated, centrifuged, decanted and drained as before. These more p u r i f i e d samples were dissolved i n d i s t i l l e d water and transferred q u a n t i t a t i v e l y to 50 ml volumetric f l a s k s . A f t e r d i l u t i n g to volume with water and mixing thoroughly, a second d i l u t i o n was made. Usually an aliquot of between 0.75 and 1.25 mis was necessary depending on the conditions of the experiment. This aliquot was di l u t e d volumetrically to 50 mis with water. Five mis of this second d i l u t i o n were transferred to clean f i f t e e n ml tubes f o r the "unknowns." The standard was prepared by using 5 mis of a fresh glucose solu t i o n containing 100 t hexose f o r 5 mis. The blank was 5 mis of d i s t i l l e d water. A l l the tubes were submerged i n a cold water bath, and 10 mis of anthrone reagent (0.2$ anthrone i n 95$ s u l f u r i c acid, prepared daily) was added to each tube from a fast-flowing pipette. The tubes were swirled to a s s i s t i n the mixing of the components, and covered with glass marbles. After heating f o r exactly ten minutes i n the b o i l i n g water bath, the solutions were cooled i n the cold bath. The op t i c a l density was read immediately on a Spectronic 20 at 620 ny/.. A standard curve (Figure 1) was u t i l i z e d to determine the amount of glucose present i n the various solutions, and the glycogen content of the tissue, calculated from t h i s . A factor of 0 .9 was used for converting glucose to glycogen (82). Liv e r glycogen determinations were done both on the rats used for the cotton-pellet tests,, and on otherwise normal r a t s . Using normal rats, a series of experiments was run to determine the dosage of selachyl sodium succinate, and the latent period, necessary to e l i c i t a response. It was found that three subcutaneous i n j e c t i o n s , given over a period of twelve hours, were e f f e c t i v e when the rats were k i l l e d twenty-four hours a f t e r the f i r s t i n j e c t i o n . For example, the injections were given at 9 : 3 0 a.m., 3*30 p.m., and 9 : 3 0 p.m., with the experiment terminated at 9:30 the following morning. E f f e c t i v e doses u t i l i z e d i n this study were 3 0 , tf.0, 50 and 60 mgm per 100 gram r a t . The groups of animals were matched for weight, and were allowed to eat and drink ad l i b . 0 0 ^ Ho IS Ho 2~h 3h 3T5 4.'o CONCEHTBATION( mg./lOO ml. ) Figure 1. A typical standard curve for the optical density of glucose. 23 A. second type of study u t i l i z e d the p a i r e d f e e d i n g method. Each cage was d i v i d e d i n two, w i t h one r a t on each s i d e of the p a r t i t i o n . Each p a i r of r a t s were e x a c t l y the same weight at the onset of the experiment. T h e i r e a t i n g h a b i t s were observed f o r t h r e e days, a f t e r which i n j e c t i o n s were begun. The t r e a t e d animals were g i v e n o n l y the amount of food eaten by t h e i r p a i r e d c o n t r o l s . Thus t h e i r f o o d i n t a k e was no longer an u n c o n t r o l l e d f a c t o r . Growth St u d i e s During the course of the a n t i - i n f l a m m a t o r y experiments, i n c o n s i s t e n t e f f e c t s of the AGE on growth were noted. The amount of food eaten, and the growth of the animals used i n these s t u d i e s , was t a b u l a t e d . A l s o , s e v e r a l other t e s t s were done on normal r a t s to t r y to determine the e f f e c t s o f the AGE on growth. (a) S e l a c h y l dihemisuccinate sodium In d r i n k i n g water: Male Wis t a r r a t s , I4.O t o 72 grams each, were d i v i d e d i n t o groups o f s i x . Pood and water were g i v e n i n excess so that ample amounts were always p r e s e n t . The c o n t r o l group r e c e i v e d tap water, while the t r e a t e d groups r e c e i v e d 5 to £0 p a r t s per m i l l i o n s e l a c h y l sodium s u c c i n a t e i n t h e i r tap water. Pood and water consumption was measured d a i l y ; the weight of the animals was r e c o r d e d every f i v e days. Length of t h i s study was t h i r t y - o n e days. (b) S e l a c h y l dihemisuccinate sodium by v a r i o u s r o u t e s : A short-terra experiment of s i x days was done u s i n g 5 mg per 100 grams of the drug ( v e h i c l e was water) and f i v e r a t s per 21* group. The r o u t e s of a d m i n i s t r a t i o n were o r a l i n t u b a t i o n , sub- . cutaneous and i n t r a p e r i t o n e a l i n j e c t i o n s . The c o n t r o l s r e c e i v e d i n t r a p e r i t o n e a l s a l i n e i n j e c t i o n s . R e s u l t s Anti-inf 1aramation (a) Granuloma pouch I t can be seen from Table I I t h a t a somewhat g r e a t e r volume of exudate formed i n the pouches of r a t s t r e a t e d w i t h s e l a c h y l DHSS than i n c o n t r o l r a t s . H y d r o c o r t i s o n e completely i n h i b i t e d exudate f o r m a t i o n as might be expected i n view of i t s known potent a n t i - i n f l a m m a t o r y p r o p e r t i e s . TABLE I I E f f e c t of S e l a c h y l DHSS Compared wi t h H y d r o c o r t i s o n e A c e t a t e u s i n g the Subcutaneous Route Group Number of Dose Mis of % of Rats (rag/rat/day) Exudate C o n t r o l C o n t r o l s i\. - 6 . 1 3 1 0 0 S e l a c h y l DHSS 5 5- 7.92 129.2 H y d r o c o r t i s o n e a c e t a t e $ 5 0 . 0 ( v e h i c l e - 2% tween 20 s o l u t i o n ; d u r a t i o n of experiment - 5> d a y s ) . Thus i t appeared that t h i s AGE showed no a n t i - i n f l a m m a t o r y e f f e c t f o r t h i s p r e p a r a t i o n and dose. The weight o f the AGE-t r e a t e d animals was a l s o comparable t o t h a t o f the c o n t r o l s , a l t h o u g h h y d r o c o r i t s o n e group l o s t weight. #' S e l a c h y l diheraisuccinate sodium w i l l h e r e a f t e r be r e f e r r e d to as s e l a c h y l DHSS. 25 Since i t had been shown that very small doses of some AGE administered o r a l l y reduce f l u i d formation i n the granuloma pouch (2), an experiment was performed i n which rats were given 300 and 600 micrograms o r a l l y of selachyl alcohol each day. The r e s u l t s are shown i n Table I I I . TABLE III E f f e c t of Selachyl Alcohol Compared with Hydrocortisone - Oral Intubation Group Number of Dose Mis. of % of Rats (mcg/rat/day) Exudate Control Control 5 - 7.2 100 Selachyl alcohol 5 300 8.1 112.5 Selachyl alcohol 5 600 7.9 109.7 Hydrocortisone acetate 5 500 lf.,9 68.0 (vehicle - cottonseed o i l ; duration of test - 5 days.) No decrease i n exudate due to selachyl alcohol was observed. It was, i n f a c t , increased over the control l e v e l , contrary to previous reports. As f a r as i t was possible to ascertain, the only difference between t h i s method and that which was reported to give a decrease i n exudate was that i n t h i s experiment the drugs were administered by oral intubation, whereas i n the previously reported test the compounds were fed to the animals o r a l l y by dropper. A t h i r d study was performed, t h i s time u t i l i z i n g the i n t r a -peritoneal route, to compare the above two AGE. The r e s u l t s are 26 tabulated below. Equivalent doses of the two compounds, as calculated from molecular weights, were used. TABLE IV Ef f e c t of Selachyl DHSS Compared with Selachyl Alcohol - Intraperitoneal Injections Group Number of Dose Mis. of $ of Rats (mg/rat/day) Exudate Control Controls 9 - 7.08 100 Selachyl DHSS 10 13.8 6.5*+ 92.1+ Selachyl alcohol 8 1 0 . 0 6 . 2 5 8 8 . 3 (vehicle f o r control and alcohol - peanut o i l ; for succinate -water; duration of experiment - 5 days.) Again, the decrease i n the amount of exudate was i n s i g n i f i c a n t , since a v a r i a t i o n of at l e a s t + 10$ normally occurs i n control groups of such methods, (b) Cotton p e l l e t granuloma The e f f e c t of the age of the animals of t h i s method was investigated. Similar experiments were carried out on young growing rats weighing 100 to 125 grams, and mature rats weighing 220 to 300 grams. In each case f i v e r a t s were used per group and inje c t i o n s were made once d a i l y by the subcutaneous route. Control rats received saline only, and the duration of the experiment was seven days. The r e s u l t s are given In Table V. It was noted that i n the mature animals a more pronounced anti-Inflammatory e f f e c t was produced i n both treated groups. This could perhaps be due to the fact that these r a t s are not 2 7 growing although the young rats are. TABLE V Comparison of Granulation Tissue Formation i n Young and Mature Rats Young Animals Mature Animals Group Dose mg. of gran- % of mg. of gran- "% of (mg/kg) u l a t i o n tissue control u l a t i o n tissue control Control - i4.1.6(30.2-61|.5) 100 55. l ( l|.l - - ° 7 . 8 ) 100 Hydro-cortisone acetate 10 25.2(22.8-30.7 ) 60 .6 27.0(22.9-31.1) l|.9.1 Selachyl DHSS 30 36.8(2lj..5-65.7) 88.3 38.8(26.0-50.2) 70.5 L i v e r Glycogen Determinations The r e s u l t s of the experiments done on the animals with cotton granulomas are shown i n Table VI. The conditions of thi s experiment are the same as for the experiments :referred to i n Table V. TABLE VI Comparison of Li v e r Glycogen i n Animals Treated with Selachyl DHSS and Hydrocortisone Acetate Liver glycogen Young AnimalsT Mature Animals Group mg/Gm of l i v e r % of mg/Gm of l i v e r % of . \ control control Control 2i^.5(ll|.5-31.3) 100 25.1(11.25-36.8) 100 Hydrocortisone acetate 20.0(15.9-25.95) 81,6 37.7(35.M-1 .6r 150.8 Selachyl DHSS 31.7(27.5-36.1) 129.2 28.1(20.3-1}.2.7) 112.0 (# Mature group received additional dose three hours before s a c r i f i c i n g - see explanation following). 2 8 There appeared to be a compensatory glycogenolysis i n the young rats treated with hydrocortisone since the hydrocortisone, which is e f f e c t i v e f o r only a few hours, had been administered twelve hours before the death of the young animals. Thus a low glycogen reading was obtained for the hydrocortisone group. To provide a f a i r e r basis for comparisons, the mature animals were given injections of the drugs three hours before termination of the experiment. In t h i s case, an increase of 50.8$ over the control was obtained. The increase i n glycogen content i n the selachyl DHSS group made further studies on this aspect appear necessary. Figure 2 summarizes the r e s u l t s obtained from four l i v e r glycogen experiments on r a t s with no inflammatory-stimulating substances present. The log doses represent 30, i+0, 50, and 60 mg/lOO Gms. Five groups of animals showed a large increase over control values, i n the same range that was shown i n preliminary studies. Two groups gave low values. The paired feeding group gave a value of only 13$ over controls. Growth Studies The observations on growth during the granuloma pouch and cotton p e l l e t studies are summarized i n Tables VII and VIII. 29 R - i CO o 9, -o o i-H / \ O / \ / / N / / / • o / / I I I I I 6 O normal feeding ^ paired feeding X) 1 1 1 f 1.45 1.55 1.65 1.75 1.85 LOG DOSE Figure 2 . Effect of subcutaneous selachyl DHSS on liver glycogen deposition of rats. Connected dots represent single experiments. Controls=100$. 30 TABLE VII Ef f e c t s of the AGE on Growth of Rats During Granuloma Pouch Studies Compound Dose Route of Administration Weight Gain (fo Control) Selachyl DHSS 5 rag/rat " 13.8 mg/rat Selachyl alcohol 300 meg/rat 600 meg/rat 10 mg/rat subcutaneous intraperitoneal o r a l o r a l intraperitoneal n e g l i g i b l e (+) 63$ n e g l i g i b l e (+) ne g l i g i b l e (-) neg l i g i b l e (+) The only route of administration by which selachyl DHSS affected weight gain was the intrap e r i t o n e a l route. Selachyl alcohol, by t h i s route, did not appreciably af f e c t growth. TABLE VIII Ef f e c t s of AGE on Weight of Rats During Cotton P e l l e t Studies Group Dose Pood Consumption (Gm. of food eaten/ rat/day) Young ' Mature Weight' Gain (Gm/rat/day) Young Mature Controls l l f . O XI1—I4. Hydro-cortisone 10 mg acetate per Kg. 12.8 12.2 Selachyl 30 mg DHSS per Kg. 16.5 18*k 3.8 0.03 (2 .5 to i(..5) (~3.k to 1.1) 0 4 -7.7 (-2.1 to 2.1) (-11.0 to -6.1) k-7 0.7 (2 .5 to 6.3) (-0.7 to l.ij.) (subcutaneous injections) 31 The young rata given selachyl DHSS gained 23.7$ more weight than th e i r controls, while the mature r a t s maintained t h e i r weight, Growth studies where no inflammation was present are summarized i n Table IX and Figure 3» The figure shows the average weight gain and food consumption as a percent of the cont r o l . Also included are l i n e s showing the extremes i n each group. TABLE IX Long Term Study of the Effects of Selachyl DHSS i n Drinking Water of Normal Rats Group Dose Water (parts per Consumption m i l l i o n ) (mis/rat/day) Food Consumption (Gm/rat/day) Weight Gain ($ of control) Control Selachyl DHSS 2 0 . 9 6 5 50 2 1 . 9 0 2 0 . 2 0 (duration of study - 31 days.) 1 3 . 7 13.1+ 13.1+ 1 0 0 91+.1 88.9 No s i g n i f i c a n t changes were brought about by these doses o r a l l y . Figure 3 again shows that the intraperitoneal route i s the only one a f f e c t i n g weight gain, and i t had the greatest e f f e c t on food consumption. o C\J . o o 8 -o • VO O o o u +» g o o 8 -o o § o • o VD control o r a l o_ s . c 0 i . p . c o n t r o l ROUTE OP ADMINISTRATION o r a l s . c . Figure3. Effect of selachyl dihemisuccinate sodium on growth and food consumption of rats„ routes of administration. Dose- 50. mg. per kilogram. Various 33 DISCUSSION Selachyl DHSS and selachyl alcohol showed no s i g n i f i c a n t anti-inflammatory effect with the granuloma pouch method at the dosage l e v e l s and routes of administration used. These doses had given p o s i t i v e r e s u l t s i n other studies ( 2 , 7 8 ) , and included both very small and r e l a t i v e l y large doses. Some evidence of anti-inflammatory a c t i v i t y was revealed by the cotton p e l l e t granuloma t e s t . Although selachyl DHSS decreased the granulation tissue formation i n young rats by only l l « 7 $ j which i s not considered to be s i g n i f i c a n t , i t decreased the granuloma formation i n mature rats by 2 9 * 5 $ . This was accomplished at a dose of 30 mg/Kg subcutaneously. Hydrocortisone acetate, on the other hand, gave results ranging from complete depression of exudate i n the f i r s t granuloma pouch assay (Table II) to depressions of 39 »h 5 0 . 9 $ i n the cotton p e l l e t granulations. (Table V) These r e s u l t s were e l i c i t e d usually by much lower dosages than those of the AGE. Subcutaneous selachyl DHSS caused an increase i n l i v e r glycogen i n a l l except one of the experiments. (Figure 2 ) In t h i s p a r t i c u l a r case the 30 and 1+0 mg/lOO Gm. doses gave glycogen values near the control l e v e l . The same dosages gave high values i n the following experiment. This large discrepancy may probably be accounted for i n the following discussion, on the method used. In the paired feeding experiment there was an increase of only 13$ over control values. Therefore, increased food 3% consumption or u t i l i z a t i o n appears to be at l e a s t one of the factors i n the increased storage of glycogen i n the l i v e r . However, the small number of animals, l i m i t e d dosage range, and single route of administration used i n these experiments, plus the inherent inaccuracy of the method, make i t , impossible to draw any f i n a l conclusions on the subject. Although the anthrone method of estimating l i v e r glycogen i s widely used, i t has several disadvantages. The d i l u t i o n s must be estimated on the basis of the amount of glycogen present, which varies widely depending on the drug used and when i t was administered, how recently the animal has eaten or done any physical a c t i v i t y , and even from which section- of the l i v e r the sample i s taken. In studies of the glycogen content In c a t t l e l i v e r , Kusin and Pupin (83) noted variat i o n s of 10 - 1J4.O mg % i n samples obtained simultaneously from d i f f e r e n t sections of the same organ. Also, dust and l i n t which accidentally enter the solution w i l l cause a darker color formation. Sokal and Sarcione (81+) stated that a change of 20$ was not s i g n i f i c a n t i n t h i s type of experiment. A l l these factors make i t d i f f i c u l t f o r even a highly trained technician to obtain reproducable r e s u l t s . Therefore, although a great deal of time was spent on t h i s part of the work, the outcome was discouraging. It was noted i n the l i t e r a t u r e than an "auto analyzer" has been /set up f o r the purpose of l i v e r glycogen determinations (85). Further developments along t h i s l i n e w i l l be i n t e r e s t i n g to note. The studies on growth were an outcome of observations during the anti-inflammatory experiments. It had been noted that rat s 35 g i v e n s e l a c h y l DHSS i n t r a p e r i t o n e a l l y l o s t weight, whereas i n c e r t a i n cases subcutaneous i n j e c t i o n s caused a s l i g h t i n c r e a s e i n weight. F o r example, the young r a t s used i n the c o t t o n p e l l e t t e s t a t e 16 . 1 $ more f o o d t h a n t h e i r c o n t r o l s and gained 23.7$ more weight, w i t h a dose o f 30 mg/Kg of the s u c c i n a t e . (Table V I I I ) I t was found t h a t i n normal r a t s , s e l a c h y l DHSS, at a dose o f 50 mg/Kg, a f f e c t s growth o n l y by the i n t r a p e r i t o n e a l r o u t e where i t causes a r e t a r d a t i o n o f weight g a i n . Very low amounts i n d r i n k i n g water have no e f f e c t . The i n t r a p e r i t o n e a l groups were a l s o noted to have eaten somewhat l e s s f o o d than t h e i r c o n t r o l s . The r a t h e r unusual p r o p e r t y of having d i f f e r e n t p h y s i o l o g i c a l e f f e c t s by d i f f e r e n t r o u t e s o f a d m i n i s t r a t i o n might p o s s i b l y be due to a d i f f e r e n c e i n the type o f l o c a l a b s o r p t i o n and metabolism o f the s u c c i n a t e . Since i t has been shown t h a t chimyl a l c o h o l i s e x t e n s i v e l y m e t a b o l i z e d d u r i n g a b s o r p t i o n through the i n t e s t i n a l mucosa (I|.9,50), perhaps s e l a c h y l DHSS possesses t h i s same p r o p e r t y . In t h i s way, a s l i g h t l y d i f f e r e n t type o f com-pound c o u l d be formed by the i n t r a p e r i t o n e a l r o u t e , which c o u l d a f f e c t growth. However, the mechanism c o u l d a l s o be much simpler, such as an i r r i t a n t e f f e c t . A c u r i o u s phenomenon was noted when h i g h doses such as 600 mg/Kg of s e l a c h y l DHSS were a d m i n i s t e r e d subcutaneously. W i t h i n twenty-four hours an a p p a r e n t l y l a r g e i n c r e a s e i n weight was observed. I t was not due t o normal growth, s i n c e the weight g a i n was three or f o u r times as much as normally recorded. The r a t s appeared v e r y s l u g g i s h and p u f f y , w h i l e a post mortem examination r e v e a l e d a t h i c k l a y e r of g e l a t i n - l i k e m a t e r i a l present next, t o 36 the subcutaneous abdominal t i s s u e . This was not a l o c a l e f f e c t , since the inj e c t i o n s were given on the back of the animal, and no such layer formed i n t h i s area. In several experiments where t h i s phenomenon was observed, including the paired feeding studies, no increase In food or water consumption occurred. It i s hoped that further work w i l l be done on the aspects of growth and weight with respect to the AGE. During t h i s preliminary study on some of the pharmacological effects of the AGE, the most notable observation was a lack of uniformity i n the r e s u l t s . Factors which appeared to cause a s i g n i f i c a n t change In the outcome of experiments Include the age of the animals, the length of time they were housed i n new quarters before the commencement of the study, and the route of administration of the compounds. A complete lack of l i n e a r r e l a t i o n s h i p between dose and response was apparent. This had also been noticed i n previous studies on AGE i n t h i s laboratory, and appears to have been encountered by others working with these compounds (60, 63, 61+, 67 ) . It i s possible that selachyl DHSS may possess a maximum e f f e c t i v e dose, above which i t s physiological properties may be lessened or reversed. Due to the preliminary nature of the work done f o r t h i s t h e s i s , a great deal of time was spent on each section to determine useful doses and methods of administration. Further work along these l i n e s could now be ca r r i e d out i n a more exhaustive manner to clear up the many questions which aris e from the above r e s u l t s . 37 SUMMARY 1. No anti-inflammatory a c t i v i t y was e l i c i t e d by selachyl DHSS or selachyl alcohol using the granuloma pouch method. 2. Selachyl DHSS showed anti-inflammatory action i n mature rats with cotton p e l l e t granulomas. 3» Liver glycogen deposition i n rats was increased by selachyl DHSS, but thi s may be due to increased food intake or u t i l i z a t i o n . 1+. Intraperitoneal injections of selachyl DHSS caused a decrease i n weight of the animals as compared to control groups. 38 BIBLIOGRAPHY 1 . Tsujimoto, M., and Toyama, Y., The unsaponifiable constituents (higher alcohols) of the l i v e r o i l of sharks and rays. Chem.'Urnschau. 22:27-9, 1+3-5 (1922), through Chem. Abs., 1 6 : 1 5 1 2 (1922). 2 . Western Chemical Industries Ltd., Technical B u l l e t i n , I960. o 3. 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Thesis/Dissertation
eng
Pharmacy
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Graduate
A study of some pharmacological properties of certain alpha-glyceryl ethers.
Text