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A study of the mode of action of isoidide dinitrate. Clark, Stewart Cecil 1963

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A STUDY OF THE MODE OP ACTION OP ISOIDIDE DINITRATE by STEWART CECIL CLARK 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 OP 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 f u r t h e r agree that per-mission for extensive copying of t h i s t h e s i s for 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 for 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. 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. i A STUDY OP THE MODE OF ACTION OP ISOIDIDE DINITRATE fey STEWART CECIL CLARK ABSTRACT Isoidide d i n i t r a t e (IIDN), a new n i t r a t e ester, an isomer of isosorbide d i n i t r a t e (ISDN) and isomannide d i n i t r a t e (IMDN), has been studied on the i s o l a t e d rabbit intestine and on the anesthetized r a t blood pressure. Potency was observed as was tachyphylaxis and the e f f e c t of c e r t a i n blocking agents. On the i s o l a t e d rabbit i n t e s t i n e similar r e s u l t s were obtained when n i t r o g l y c e r i n was substituted f o r IIDN. In both preparations IIDN was more potent than ISDN or IMDN and ISDN more potent than IMDN. The potency dependence on con-f i g u r a t i o n suggests a possible action on s p e c i f i c receptor s i t e s . A type of tachyphylaxis to IIDN which was prominent i n the i s o l a t e d rabbit intestine was almost absent i n the anesthetized ra t blood pressure. This i s probably due to metabolism of the drug i n the intact animal and further suggests an a c t i o n on receptors. In these preparations the n i t r a t e action was not blocked by: the beta adrenergic blocking agents DCI or nethalide, the alpha adrenergic blocking agent dibenzyline, or a combination of an alpha and a beta blocking agent. Drugs that are s e l e c t i v e l y i i blocked by these agents were used as controls to indicate the presence of the desired blocking action. Neither the primary bretylium-like action nor the secondary reserpine-like action of guanethidine blocked the e f f e c t of IIDN on the i s o l a t e d rabbit i n t e s t i n e . Depletion of catechol amines by pretreating the animals with reserpine did not a l t e r the response of e i t h e r preparation to the n i t r a t e esters. The antihistamine diphenhydramine did not block the vasodepressor a c t i o n of IIDN on the anesthetized rat blood pressure. It was concluded that although IIDN probably does not exert i t s e f f e c t through combination with adrenergic or histaminergic receptors, i t possibly acts on receptors which are s p e c i f i c f o r the n i t r i t e or n i t r a t e group. Signatures of Examiners i i i TABLE OF CONTENTS Page ABSTRACT. i LIST OF TABLES v LIST OF FIGURES . v i INTRODUCTION a P h a r m a c o l o g i c a l A c t i o n s . 1 General • 1 Isomannide d i n i t r a t e . . . . . . . . . . . . . . . 1 I s o s o r b i d e d i n i t r a t e . . . . . 3 I s o i d i d e d i n i t r a t e If. S t r u c t u r e A c t i v i t y R e l a t i o n s h i p if. Mechanism of A c t i o n . . . . . . . . . . . 8 METHODS AND MATERIALS I s o l a t e d Rabbit I n t e s t i n e . . . . . . . 10 A n e s t h e t i z e d Rat Blood P r e s s u r e . . . 11 RESULTS AND DISCUSSION Potency llf. T a c h y p h y l a x i s . 17 I n t e r a c t i o n w i t h B l o c k i n g Drugs . . . . . . . . . . . 18 DCI and N e t h a l i d e 20 D i b e n z y l i n e 22 D i b e n z y l i n e and a Beta B l o c k i n g Agent 22. Guanethidine 2% Reserpine 28 Diphenhydramine . . . . . 3 3 SUMMARY AND CONCLUSIONS BIBLIOGRAPHY V LIST OP TABLES TABLE Page I I n t e r a c t i o n of IIDN w i t h B l o c k i n g Drugs. . . . . . 19 A I s o l a t e d Rabbit I n t e s t i n e B A n e s t h e t i z e d Rat Blood P r e s s u r e v i LIST OP FIGURES FIGURE Page 1 S t r u c t u r e o f 2 , 5 - d i n i t r a t e s of 1,it; 3 , 6 -d i a n h y d r o h e x i t o l s 2 2 Potency of Isomers on A n e s t h e t i z e d Rat Blood P r e s s u r e . . l£ 3 Absence of Tachyp h y l a x i s t o IIDN i n A n e s t h e t i z e d Rat Blood P r e s s u r e 15> If. T a c h y p h y l a x i s t o IIDN i n I s o l a t e d Rabbit I n t e s t i n e . . . 16 $ A c t i o n of DCI on. IIDN Response i n Is'olated Rabbit I n t e s t i n e . 21 6 A c t i o n of N e t h a l i d e on IIDN Response i n A n e s t h e t i z e d Rat B l o o d P r e s s u r e . . . . . 23 7 A c t i o n of DCI and D i b e n z y l i n e on IIDN Response i n I s o l a t e d Rabbit I n t e s t i n e 2f? 8 A c t i o n of DCI and D i b e n z y l i n e on IIDN Response i n I s o l a t e d Rabbit I n t e s t i n e (No pr e v i o u s IIDN given) . . . 26 9 A c t i o n of N e t h a l i d e and D i b e n z y l i n e on IIDN Response i n A n e s t h e t i z e d Rat Blo o d P r e s s u r e 27 10 A c t i o n of Guanethidine (100 t%) on IIDN Response i n I s o l a t e d Rabbit I n t e s t i n e . . ... . 29 11 A c t i o n of Guanethidine ( 1 mg$) on IIDN Response i n I s o l a t e d Rabbit I n t e s t i n e . 30 12 A c t i o n of IIDN i n R e s e r p i n i z e d R a b b i t . . . . . . . . . . . 31 13 A c t i o n of IIDN i n R e s e r p i n i z e d Rat 32 llj. A c t i o n of Diphenhydramine on IIDN Response i n A n e s t h e t i z e d Rat Blo o d P r e s s u r e . 3lj. v i i ACKNOWLEDGEMENTS The writer expresses his sincere appreciation to Dr. J.E. Halliday for the guidance and encouragement given during the course of this work. Thanks i s also extended to the following companies for the drugs supplied: Parke Davis & Co. Ltd. f o r N i t r o g l y c e r i n Imperial Chemical Industries Ltd. f o r Nethalide (Alderlin) Ciba Co. fo r Guanethidine (Israelin) Reserpine (Serpasil) INTRODUCTION Pharmacological Actions General The basic pharmacological a c t i o n of the n i t r i t e - n i t r a t e group of drugs i s to relax smooth muscle, e s p e c i a l l y those of the finer blood vessels. The most c h a r a c t e r i s t i c pharmacodynamic ef f e c t , therefore, i s a f a l l i n blood pressure. Goodman and Gilman 1 suggest the action of the n i t r i t e s and n i t r a t e s i s non-s p e c i f i c as the spasmolytic action i s independent of innervation, the vasodilator action i s not blocked by atropine and the muscles are not paralyzed as muscular stimulants and motor nerve impulses can s t i l l exert t y p i c a l constrictor e f f e c t s . A new n i t r a t e ester, i s o i d i d e d i n i t r a t e , has been synthesized. A description of t h i s drug, and i t s isomers isosorbide d i n i t r a t e and isomannide d i n i t r a t e , w i l l follow. Isommanide Di n i t r a t e Isomannide d i n i t r a t e (IMDN), l,lj . j3 ,6-dianhydro-D-mannitol-2 , 5 - d i n i t r a t e , molecular weight 2 3 6 . 1 , i s a white c r y s t a l l i n e compound, stable i n a i r , melting point 6jp.5°C, soluble i n water 1 . 7 : 1 * 0 0 0 , f r e e l y soluble i n alcohol, o i l s and e t h e r l a l solvents. In a study of a new series of organic n i t r a t e s Krantz and associates synthesized IMDN and selected i t f o r study as a 2 p o t e n t i a l therapeutic agent. They found from a more det a i l e d pharmacological study that t h i s compound was a potent vasodepressor i n the anesthetized dog, relaxed i s o l a t e d coronary Figure 1. Structure of 2,5-dinitrates of l,4}3,6-dianhydrohexitols. I Ri = 0N02, R2 = H , R3 = ONO2. R4 = H : isomannide dinitrate I I Ri = 0N02, R2 = H , R3 = H , R4 = 0N02 : . isosorbide dinitrate I I I Ri = H , R2 = 0N02, R3 = H , R4 = 0N02 : isoidide dinitrate ( After : Jackson and Hayward, Can. J . Chem., ^ 8:497, I960 ) 3 artery rings, increased the rate of outflow an average of 21% i n perfused i s o l a t e d rabbit hearts and caused an increase of i n s i t u coronary flow i n dogs. They also found, when taken o r a l l y by normal men, i t caused a f a l l i n blood pressure within ten minutes which lasted f o r f i v e to s i x hours. Although they suggested IMDN lends i t s e l f to medication i n tablet form i n the treatment of hypertension and angina pectoris, no c l i n i c a l studies 3 have been reported. Isosorbide D i n i t r a t e Isosorbide d i n i t r a t e (ISDN), 1,1}.;3,6-dianhydro-D-glucitol-2 , 5 - d i n i t r a t e , molecular weight 236.1, i s a white c r y s t a l l i n e compound, stable i n a i r , melting point 10°Ct soluble i n water 1.125:1,000, f r e e l y soluble i n alcohol and ether and i n arachis o i l to about 50-100:1,000 at 50°C. ISDN i s another member of the 2 series synthesized by Krantz et. a l . Some years l a t e r Goldberg reported that ISDN was a potent vasodepressor and coronary vaso-d i l a t o r i n laboratory animals and e f f e c t i v e l y lowered the blood pressure i n hypertensive patients. He found i t invar i a b l y had a k somewhat stronger action than isomannide d i n i t r a t e . Studies i n t h i s laboratory have generally shown similar r e s u l t s i n 5 laboratory animals. The numerous c l i n i c a l reports on ISDN although mostly subjective i n nature, indicate an excellent 6,7,8 response by angina pectoris patients. Some workers have already placed i t among the most active of the long-acting n i t r i t e s and nit r a t e s f o r the treatment o f angina pectoris i n terras of dose, time of onset of ac t i o n and duration of action. Buckley and associates have shown ISDN Is an e f f e c t i v e vasodilator and causes a greater reduction i n coronary vascular resistance 9 than does pentaerythritol t e t r a n i t r a t e . Isoidide D i n i t r a t e Isoidide d i n i t r a t e (IIDN), 3 , 6 - d i a n h y d r o - L - i d i t o l - 2 , £ -d i n i t r a t e , molecular weight 2 3 6 . 1 , i s a white c r y s t a l l i n e compound, stable i n a i r , melting point 6 7 - 6 9 ° G , s o l u b i l i t y i n water 0 . 8 7 f ? : 1 , 0 0 0 , f r e e l y soluble i n alcohol, o i l s and e t h e r i a l solvents. IIDN was f i r s t synthesized by Jackson and Hayward i n the Depart-1 0 ment of Chemistry, University of B r i t i s h Columbia. I n i t i a l pharmacological studies indicate t h i s drug i s considerably more potent than either of i t s isomers ISDN or IMDN. It has a greater vasodepressor action when administered intravenously i n the cat, rabbit, and dog, and intraduodenally i n the dog. It also produces a much greater increase i n the blood flow of the dog hindleg and i n the outflow from the perfused i s o l a t e d heart.^ Structure A c t i v i t y Relationship L i t t l e work has been done to determine the structure a c t i v i t y r e l a t i o n s h i p of the n i t r a t e drugs. Marshall feels that the vasodilating a c t i o n of the n i t r i c esters of polyhydric alcohols i s connected s o l e l y with the n i t r a t e group. He points out that mannitol hexanitrate, d u l c i t o l hexanitrate and s o r b i t a l hexanitrate a l l have the same action thus i t appears the arrange-ment of the n i t r a t e groups i n the n i t r i c esters of hexahydric alcohols or sugars appears to be without influence. Further, arabinose t e t r a n i t r a t e , rhamnose t e t r a n i t r a t e , glucose pentanitrate, fructose pentanitrate, quercite pentanitrate and 5 saccharose octonitrate show no difference i n a c t i v i t y which can-11 not be attributed to differences i n s t a b i l i t y and s o l u b i l i t y . Prom his study of a series of glucose n i t r a t e s O'Meara agrees that as f a r as he can see the p o s i t i o n of the n i t r a t e group has l i t t l e or no influence on a c t i v i t y . However a l l active glucosides had at l e a s t one pair of n i t r a t e groups attached to adjacent carbon atoms and carbon three and/or four n i t r a t e d . The work of Pranz and associates on a series of f i v e pentanediol d i n i t r a t e s indicates the s p a t i a l configuration of the n i t r a t e groups i s an important factor influencing physiological a c t i v i t y . In terms of smooth muscle a c t i v i t y , 1 ,5-pentanediol d i n i t r a t e and trans-l,2-cyclopentanediol d i n i t r a t e were generally most potent whereas 2,3- and 2,ij.-pentanediol d i n i t r a t e were least a c t i v e . While the r e l a t i v e potency of cis - 1 ; 2-cyclopentanediol d i n i t r a t e varied between' these extremes, both c i s - and t r a n s - l , 2 -13 cyclopentanediol d i n i t r a t e proved to be potent convulsants.• The studies of ISDN and IMDN, i n which Goldberg found ISDN considerably more active, and more recent studies i n t h i s laboratory of IIDN also indicate that i n t h i s series the p o s i t i o n of the n i t r a t e group i s a v i t a l consideration when studying potency. Thus in v e s t i g a t i o n of the potency of the series of n i t r a t e esters, IMDN, ISDN and IIDN, seemed s i g n i f i c a n t apart from the c l i n i c a l value i n obtaining a more potent drug. In considering the structure of these isomers (Figure 1) i t i s observed that the increased potency i n the order IMDN ISDN IIDN i s associated with greater freedom of r o t a t i o n of the n i t r a t e groups. That 6 i s , i n IMDN b o t h n i t r a t e g r o u p s a r e i n t h e e n d o p o s i t i o n t h u s a r e h e l d somewhat r i g i d l y . W h e r e a s o n l y o n e i s i n t h i s p o s i t i o n i n I S D N a n d b o t h a r e i n t h e e x o p o s i t i o n i n I I D N a n d h a v e m o r e 10 f r e e d o m o f r o t a t i o n . T h e s e f a c t o r s c o u l d i n d i c a t e i n t e r a c t i o n w i t h a s p e c i f i c r e c e p t o r s i t e i n t h e m u s c l e , w i t h w h i c h I I D N c a n m o r e r e a d i l y c o n f o r m d u e t o g r e a t e r f r e e d o m o f r o t a t i o n o f t h e n i t r a t e g r o u p s . I t i s h o w e v e r g e n e r a l l y a c c e p t e d t h a t t h e n i t r a t e d r u g s , w h i c h h a v e v e r y s i m i l a r a c t i o n b u t a r e o f d i v e r s e s t r u c t u r e , d e p e n d o n n o n - s p e c i f i c r e c e p t o r s f o r t h e i r a c t i o n . T h e d i f f e r e n c e i n p o t e n c y may a l s o b e a c c o u n t e d f o r e x c l u s i v e l y b y d i f f e r e n c e s i n s o l u b i l i t y . K r a n t z , G a r r a n d a s s o c i a t e s p l a c e c o n s i d e r a b l e s t r e s s o n t h e i m p o r t a n c e o f t h e o i l / w a t e r c o e f f i c i e n t i n d e t e r m i n i n g t h e r e l a t i v e p o t e n c y o f members o f t h e v a s o d e p r e s s o r n i t r a t e d r u g s . T h e y h a v e f o u n d i n t h i s s e r i e s o f c o m p o u n d s w a t e r s o l u b i l i t y a n d o i l s o l u b i l i t y s e e m t o b e i n i n v e r s e r a t i o t o e a c h o t h e r . S t u d i e s o f v a r i o u s n i t r a t e s i n d i c a t e d e p r e s s o r p o t e n c y i s d i r e c t l y r e l a t e d t o o i l s o l u b i l i t y , 2 h o w e v e r d e c r e a s e d w a t e r s o l u b i l i t y may b e a l i m i t i n g f a c t o r . I n some o f t h e i r s t u d i e s h o w e v e r t h e s e w o r k e r s f o u n d a s t r u c t u r a l 15 f a c t o r w h i c h c o u l d n o t b e e x p l a i n e d b y s o l u b i l i t y c o n s i d e r a t i o n s . I n t h e s e r i e s o f n i t r a t e s b e i n g s t u d i e d , t h e o r d e r o f w a t e r s o l u b i l i t y i s I I D N I S D N IMDN t h e r e f o r e a c c o r d i n g t o K r a n t z a n d C a r r t h e o r d e r o f o i l s o l u b i l i t y w o u l d b e IMDN ISDN I I D N . T h u s m e r e l y o n t h e b a s i s o f r e l a t i v e o i l s o l u b i l i t y , d e p e n d i n g o n s u f f i c i e n t w a t e r s o l u b i l i t y , o n e c a n p r e d i c t t h e o b s e r v e d o r d e r o f p o t e n c y : IMDN ISDN I I D N . 7 Marshall c r i t i c i z e s Krantz, and Garr's emphasis on oil/water d i s t r i b u t i o n on the grounds that i n pharmacology the p r i n c i p l e of p a r t i t i o n c o e f f i c i e n t s i s concerned mainly with absorption and not with a s s i m i l a t i o n and as he i s ce r t a i n that i n producing t h e i r predominant action n i t r i c esters undergo changes within the c e l l s , p a r t i t i o n c o e f f i c i e n t s cannot be so important. Further the l i p o i d content of smooth muscle i s not very d i f f e r e n t from that of blood plasma, therefore why would l i p o i d s o l u b i l i t y 11 play so important a role? He therefore places greater emphasis on water s o l u b i l i t y , which i n the series IIDN, ISDN, and IMDN would not explain the differences i n potency as IIDN which i s most potent i s least water soluble. Generally throughout the n i t r a t e group of drugs the number of n i t r a t e groups cannot be correlated with a c t i v i t y , as compounds with a varied number of n i t r a t e groups are active. Recent evidence supporting t h i s concept l i e s i n the demonstration that replacing one of the n i t r a t e groups of n i t r o g l y c e r i n with 16 chlorine does not a l t e r the a c t i v i t y . However, O'Meara found the degree of n i t r a t i o n to be a c r i t i c a l factor i n determining the vasodilator a c t i v i t y of glucose derivatives. Whereas none of the mono or d i n i t r a t e s were active, methyl beta-D-glucoside t e t r a n i t r a t e was the most active compound studied. These r e s u l t s would suggest that at l e a s t f o r some carbon skeletons a minimum number of n i t r a t e groups i s necessary to endow the compound with 12 vasodilator a c t i v i t y . This information further indicates the possible reaction of at least some of the nitrates with s p e c i f i c receptor s i t e s . 8 Mechanism of Action Although considerable work has been done i n an attempt to elucidate the mechanism of action of the n i t r i t e / n i t r a t e group of drugs, a d e f i n i t e mechanism has not been established. For many years i t was assumed that the organic n i t r a t e s exerted t h e i r eardio-vascular effects following hydrolysis and subsequent reduction to n i t r i t e . However the work of Krantz and associates suggests that the vasodepressor e f f e c t i s produced by the i n t a c t X*7 18 19 molecule. ' ' It i s possible however that the organic n i t r a t e molecule r a p i d l y enters the c e l l s and releases pharmacologically e f f e c t i v e concentrations of n i t r i t e at an 1 1 , 2 0 , 2 1 i n t r a c e l l u l a r locus. More experimental data i s required before a conclusion can be reached concerning the molecular e n t i t y responsible for the cardiovascular effects of the organic n i t r a t e s . In concluding a review of h a l f a century of his own work on the pharmacology of the n i t r a t e esters, Marshall suggests that the i n i t i a l pharmacological action, at l e a s t on blood, i s associated with a very mobile oxygen. He states however that he 11 was unable to obtain supporting evidence for this concept. Recent work i n Hayward's laboratory on the hemolytic cleavage of 22 oxygen from n i t r a t e i n photolysis supports Marshall's suggestion. Raab and Humphreys have proposed a biochemical mechanism to explain the c l i n i c a l a c t i o n of n i t r o g l y c e r i n . They found that the cardiac acceleration and ECG T-wave depression produced i n atropinized cats by i n j e c t i o n s of adrenaline, faradic stimulation 9 of the s t e l l a t e ganglia i n cats with the adrenals t i e d o f f , and faradic stimulation of the splanchnic nerves were abolished by simultaneous injections of n i t r o g l y c e r i n . However the drug i n t e r f e r e d only to a moderate extent with the accumulation of adrenaline-like material i n the myocardium. These facts suggest a s p e c i f i c d e s e n s i t i z a t i o n of the heart muscle to the sympathomimetic amines and support the concept that the anginal syndrome i s due to excessive influxes of adrenaline and nor-adrenaline into the heart muscle during exercise, emotions, etc. Thus Raab and Humphreys suggest the therapeutic a c t i o n of n i t r o -g l y c e r i n i n angina pectoris i s not due to coronary d i l a t i o n alone but also to a s p e c i f i c counteraction against the myocardial metabolic effects of an excess accumulation of catechol amines 23,2 i j . ,25 xn the heart. From the previous consideration of the structure a c t i v i t y r e l a t i o n s h i p of the n i t r i t e / n i t r a t e group of drugs and because of the implication of the adrenergic mechanism i n t h e i r action, the p o s s i b i l i t y arises that the n i t r i t e s and n i t r a t e s act s p e c i f i c a l l y on the adrenergic receptors. Since there are now available various types of adrenergic blocking agents i t was f e l t t h i s possible action would be investigated p a r t i c u l a r l y i n terms of the beta adrenergic receptors, as, l i k e drugs which activate the beta receptors, the n i t r a t e s ^ i n v a r i a b l y relax smooth muscle. 10 METHODS AND MATERIALS Isolated Rabbit Intestine Albino rabbits of either sex were used throughout these studies. Animals weighing 1-3 kg. were employed however best res u l t s were obtained with young rabbits weighing around 1 kg. The animals, previously fasted f o r at l e a s t 12 hours, were k i l l e d by a blow on the back of the neck. The abdomen was opened and a portion of duodenum and ileum was removed, placed i n Tyrode's solut i o n and the lumen washed out using a syringe. Duodenum, taken from the p y l o r i c sphincter area, was employed i n most experiments as i t gave a more stable, longer l a s t i n g preparation than ileum. Four 3-lf cm. s t r i p s were cut from the p y l o r i c sphincter end of the duodenal section, or from the ileum, t i e d to a holder and placed i n an i s o l a t e d tissue bath (Metro Industries). They were l e f t 1$ minutes to equilibrate i n Tyrode's solution maintained at 38°C and oxygenated with 9$% oxygen and carbon dioxide. The s t r i p s were then attached to writing levers with a minimum counterbalance and l e f t a further 15 minutes. The writing points were arranged to record contractions on smoked kymograph drums. Af t e r discovery of the development of a type of tachyphylaxis to repeated doses of the n i t r a t e s , each test s t r i p was compared with a control s t r i p which had received no blocking drug. The washing procedure which followed the use of most drugs consisted of complete replacement of the bathing f l u i d at l e a s t four times. A useful feature of the smooth muscle baths employed i s that the 11 bathing solution can be replaced while the tissue remains f u l l y immersed. In some of the e a r l i e r experiments the nit r a t e s were administered by replacing the perfusion f l u i d with fresh Tyrode's containing the desired concentration of drug. This procedure was abandoned as i n the absence of drug the state of contraction and the tone of the s t r i p were s t i l l a l t e r e d considerably. In some of the experiments the ni t r a t e s were added i n concentrated alcoholic solution, however best r e s u l t s were obtained with additions of concentrated aqueous solution. A l l other drugs were added to the bathing f l u i d as concentrated aqueous solutions. Anesthetized Rat Blood Pressure Male Wistar r a t s weighing 3 0 0 - 5 0 0 gm. were used f o r these experiments. The technique employed was similar to that 26 27 o r i g i n a l l y described by Landgrebe and modified by Dekanski. I n i t i a l l y urethane, (175 mg./lOO gm. body weight) given'intra-pe r i t o n e a l l y was employed as an anesthetic however higher blood pressures were obtained i n l a t e r experiments using 50 mg./kg. pentobarbital sodium, administered i n t r a p e r i t o n e a l l y . The hind legs of the animal were secured with scotch tape to a heated table and the r e c t a l temperature was maintained at 3 3 a 0 . Operative Procedures: a) One ca r o t i d artery was dissected ready for cannulation. b) The trachea was cannulated with a short polyethylene tube with outside diameter between 2 and 2 . 5 m.m. (PE 2lj.O or 205). 12 c) The femoral v e i n was i s o l a t e d close to the inguinal ligament. I f care was taken i t was not necessary to t i e of f the branches, and cannulation was easier. A polyethylene cannula of outside diameter about 1 m.m. (PE 50) was employed, and the adapter was connected to a three-way stop cock to which a syringe could be connected for intravenous i n j e c t i o n s . d) Heparin (1.5 gm./lOO gm. body weight) was in j e c t e d through the venous cannula. e) The c a r o t i d cannula, also 1 m.m. outside diameter, was t i e d i n and connected by a column of normal saline to a Condon manometer. To reduce fluctuations of the blood pressure an intravenous dose of 0.125 mg. pentolinium tartrate per rat was inj e c t e d 28 immediately a f t e r cannulation. The preparation was then l e f t f o r about one hour to permit the blood pressure to s t a b i l i z e . Drugs were generally i n j e c t e d i n 0.05 or 0.10 ml. of saline and washed i n with s u f f i c i e n t warm saline such that the t o t a l volume of f l u i d given at any one time was 0.5 ml. Great care must be taken not to i n j e c t a i r In the venous cannula as the smallest a i r bubble can be f a t a l . The nit r a t e s were inj e c t e d i n 0.1 ml. of saline containing alcohol. The following drugs were employed i n t h i s study: i s o i d i d e d i n i t r a t e , isosorbide d i n i t r a t e , isomannide d i n i t r a t e , n i t r o -g l y c e r i n , pentobarbital sodium, ethyl carbamate, heparin sodium, pentolinium t a r t r a t e , dichloroisoproterenol hydrochloride, nethalide hydrochloride, dibenzyline hydrochloride, guanethidine, 13 reserpine, isoproterenol hydrochloride, phenylephrine hydro-chloride, adrenaline hydrochloride, tyramine monohydrochloride, nor-adrenaline b i t a r t r a t e and papaverine. The doses of drugs are expressed i n terras of the salt employed. lit. RESULTS AND DISCUSSION Potency In a concentration of 1-2 mg°/o IIDN was inv a r i a b l y a more potent relaxant of the i s o l a t e d rabbit intestine than the same dose of ISDN or HON and was generally equipotent with 0.5 mg$ n i t r o g l y c e r i n . ISDN was likewise a more potent relaxant than IMDN. In addition to the relaxant effect of the n i t r a t e drugs on the i s o l a t e d rabbit i n t e s t i n e , they also produced a marked decrease or t o t a l i n h i b i t i o n of spontaneous contractions. On the rat blood pressure a dose of 0.05 mg IIDN per rat was a more potent vasodepressor than ISDN or IMDN and ISDN was more potent than IMDN. (Figure 2) It has been shown that ISDN has a somewhat greater relaxant e f f e c t on the smooth muscle of the intestine and vascular system than has IMDN. This i s i n agreement with r e s u l t s obtained by Goldberg on the rabbit blood pressure, perfused i s o l a t e d rabbit 'li-ne art and i s o l a t e d rabbit ileum. The isomer IIDN has been shown to possess even greater potency than ISDN on both preparations used. This agrees with previous observations i n t h i s laboratory on the blood pressure of the cat, dog and rabbit, on the blood flow of the dog hindleg and on the outflow from the perfused 5 i s o l a t e d rabbit heart. These r e s u l t s support the concept that the s p a t i a l configuration of the nit r a t e groups could be s i g n i f i c a n t i n the determination of potency. Also, they suggest that IIDN which i s the most potent of the series, can conceivably s 0.05 mg I 0.05 mg I 0.05 mg Figure 2 . Anesthetized rat blood pressure. Comparison of potency of 0.05 mg ISDN (s), IMDN (M), and IIDN ( i ) . Figure 11. Anesthetized rat blood pressure. Absence of tachyphylaxis development to repeated doses of 0.05 mg IIDN ( I ) . w Ad 5Vt 2 mg# W 2 mg# Ad 5** Figure 4. Isolated rabbit duodenum. Development of tachyphylaxis to 2 mg# IIDN ( i ) . Response to 5 adrenaline(Ad) before and after tachyphylaxis. W = wash. 17 conform moat r e a d i l y with receptors due to greater freedom of ro t a t i o n of the n i t r a t e groups, both of which are i n the exo po s i t i o n . Tachyphylaxis 4 type of tachyphylaxis to IIDN, ISDN, IMDN and n i t r o g l y c e r i n was demonstrated i n the i s o l a t e d rabbit i n t e s t i n e . (Figure Once the tachyphylaxis developed the inte s t i n e no longer responded to any of the n i t r a t e drugs employed however i t was s t i l l relaxed by isoproterenol, adrenaline and papaverine. If the second addition of the drug was made within a few minutes of the f i r s t , the decrease i n response was very marked, however doses given every t h i r t y minutes also produced tachyphylaxis. Repeated washing seemed to decrease the degree of tachyphylaxis. Pre-treatment of the animal with reserpine did not a l t e r the develop-ment of tachyphylaxis. Tachyphylaxis did not occur i n the anesthetized r a t blood pressure preparation even when IIDN was administered within f i v e to ten minutes of a previous dose. (Figure 3) The tachyphylaxis which develops so r e a d i l y i n the iso l a t e d rabbit intestine i s l i k e l y caused either by occupation of receptor sites by the drug or exhaustion of the mechanism by which the action of the drug i s mediated. It has been suggested that the n i t r i t e s and n i t r a t e s , l i k e papaverine, act d i r e c t l y on the muscle, that i s , depend on "non-specific" receptors for t h e i r 2 action. Since the inte s t i n e s t i l l responds to papaverine during nitrate' tachyphylaxis i t would seem that d i f f e r e n t mechanisms are 18 involved i n the actions of the two drugs. Normal responses to adrenaline and isoproterenol while the intestine i s unresponsive to the n i t r a t e s , indicates that the n i t r a t e s do not act on the adrenergic receptors. The absence of tachyphylaxis development i n the anesthetized rat blood pressure preparation could be due to metabolism of the drug i n the i n t a c t animal. This process would free the receptors of drug thus supports the concept that the n i t r a t e s act on receptors rather than through a depletable intermediate. Interaction with Blocking Drugs Considerable experimentation was c a r r i e d out to obtain the required information, i n addition to that available i n the l i t e r a t u r e , concerning the use of the blocking agents. The experimental conditions, such as concentration of and time of exposure to the drugs, determined to be most suitable f o r these studies are l i s t e d i n Table 1-A f o r the i s o l a t e d rabbit i n t e s t i n e and Table I-B for the anesthetized r a t blood pressure. The "Time" column l i s t s the length of time, following administration of the blocking drug, f o r the blocking a c t i o n to develop. The blocking action 1 3 demonstrated by the use of a control drug the action of which i s blocked by the blocking agent. Throughout the experiments on the i s o l a t e d rabbit intestine the e f f e c t of the blocking drugs on the a c t i o n of n i t r o g l y c e r i n was observed. As with IIDN, none of the drugs employed blocked the n i t r a t e response. TABLE I Interaction of IIDN with Blocking Drugs BLOCKING DRUG Concentration A. Isolated Rabbit Intestine DCI Nethalide Dibenzyline Dibenzyline and DCI or nethalide Guanethidine 50 1% 200 %% 100 \% as above 100 1 W>%. 0.5 Time (min.) 20 - 30 10 - 20 lj.0 - 60 as above 15 - 30 90 CONTROL DRUG Concentration EFFECT on 2 mg$ IIDN RESPONSE Reserpine £- Given subcutaneously on the day previous B. Anesthetized Rat Blood Pressure Nathalide Dibenzyline Dibenzyline and nethalide Reserpine Diphenhydramine 5-10 mg/kg 5 mg/kg as above 2 mg/kg* 2 mg/kg 1 0 - 1 5 20 - 30 as above 10 - 15 isoproterenol isoproterenol phenylephrine adrenaline tyramine tyramine 5 2 mg$ 2 2 n i l tt it ti ii to the experiment or for two days previous, isoproterenol adrenaline adrenaline tyramine histamine 0.25 ^ n i l 0.5-1.0 * ti o . 5 n 5.0 * ti 2.0 t tt * Given in t r a p e r i t o n e a l l y for two days previous to the experiment. 20 Due to the marked tachyphylaxis development i n the rabbit intestine i t was necessary to compare the r e s u l t s obtained a f t e r the blocking drugs with responses i n a control, untreated s t r i p . It was also d i f f i c u l t i n both the rabbit intestine and the anesthetized rat blood pressure preparations to obtain regular responses to the n i t r a t e s over a period of time. The d e l -eterious effects of the blocking agents on the normal state of the preparations further complicated i n t e r p r e t a t i o n of the r e s u l t s . It i s now generally accepted that i n smooth muscle f i b r e s there are two types of adrenergic receptors--alpha and beta. Stimulation of the alpha receptors causes contraction of smooth muscle and stimulation of the beta receptors r e s u l t s i n r e l a x a t i o n of smooth muscle and an increase i n the rate and strength of 30 cardiac contractions. However stimulation of both the alpha 31 and beta receptors i n i s o l a t e d rabbit duodenum and Intact 32 canine ileum r e s u l t s i n relaxation. The d i f f e r e n t i a t i o n of these receptors has been made possible by the use of drugs that s e l e c t i v e l y block or stimulate either type of receptor. DCI and nethalide The dichloro analog of isoproterenol (DCI) o r i g i n a l l y 33 described by Powell and Slater has been characterized by Morah 3k and Perkins as an adrenergic blocking agent that s e l e c t i v e l y blocks the beta adrenergic receptors. Isoproterenol s e l e c t i v e l y 32 activates beta receptors thus i t s action i s blocked by DCI. A concentration of DCI s u f f i c i e n t to block the action of i s o -proterenol on the rabbit intestine did not block the action of IIDN or n i t r o g l y c e r i n . (Figure 5) Nethalide, a new adrenergic Figure 5. Isolated rabbit duodenum. Blocking action by 50 Tf $ D C I of 5 isoproterenol (IP) response but not of 2 mg# IIDN (i) response. W = wash. 22 beta receptor blocking compound, did not a l t e r the response to IIDN of the i s o l a t e d rabbit intestine or the anesthetized r a t blood pressure. (Figure 6) This compound exhibits l e s s i n t r i n s i c sympathomimetic a c t i v i t y therefore f a c i l i t a t e s the demonstration 35 of the presence or absence of blocking action. The r e s u l t s obtained with DCI agree with those recently reported by Abboud and Eckstein. They found i n the anesthetized dog, 7 mg/kg of DCI blocked the vasodepressor ..action of i s o -36 proterenol but d i d not effect the response to n i t r o g l y c e r i n . Dibenzyline Dibenzyline, a cogener of the o r i g i n a l adrenergic beta-haloalkylamine blocking agent, was used as an alpha blocking 3*7 38 agent. ' Phenylephrine was employed as the control drug i n the i s o l a t e d rabbit intestine experiments as i t s e l e c t i v e l y 32 activates alpha receptors. The reversal of the adrenaline pressor e f f e c t to a depressor effect was used as a control i n the blood pressure experiments. In both cases blocking doses of dibenzyline had no e f f e c t on the n i t r a t e action. Dibenzyline and a Beta Blocking Agent Since the i n h i b i t o r y e f f e c t of adrenaline on i s o l a t e d rabbit i n t e s t i n e could not be blocked either by DCI or dibenamine, Furchgott postulated the presence of delta receptors. A c t i v a t i o n of these receptors supposedly resulte'd i n i n h i b i t i o n of i n t e s t i n a l 30 smooth muscle. Working with intact canine ileum Ahlquist and Levy antagonized the e f f e c t s of adrenaline with a combination of 32 an alpha and a beta blocking agent. Furchgott confirmed these 0.05 mg I P 0.25 * N 7.5 mg/kg I P 0.25 * 0.05 mg Figure 6. Anesthetized rat blood pressure. Blocking action by 7.5 mg/kg nethalide (N) of response of 0.25* isoproterenol ( I P ) but not of 0.05 mg IIDN (i) response. ro 2k 31 r e s u l t s i n i s o l a t e d segments of r a b b i t i n t e s t i n e . To i n v e s t i g a t e the p o s s i b i l i t y of the n i t r a t e e s t e r s a c t i n g on both a l p h a and beta r e c e p t o r s experiments were performed u t i l i z i n g b o t h types o f b l o c k i n g drugs. Although the a c t i o n of a d r e n a l i n e was b l o c k e d by a combination of the b l o c k i n g agents, the a c t i o n of IIDN was not b l o c k e d i n e i t h e r the i s o l a t e d r a b b i t i n t e s t i n e ( F i g u r e s 7 and 8) or the a n e s t h e t i z e d r a t b l o o d p r e s s u r e . ( F i g u r e 9) Guanethidine 39 Maxwell o r i g i n a l l y d e s c r i b e d the pharmacology of guanethidine. He suggested t h a t the d e p l e t i o n of c a t e c h o l amines, a r e s e r p i n e -l i k e a c t i o n , may account f o r at l e a s t p a r t of the a c t i o n o f i|.0 g u a n e t h i d i n e . Cass and Spriggs and more r e c e n t workers generally agree that guanethidine possesses a primary b r e t y l i u m - l i k e a c t i o n and a secondary r e s e r p i n e - l i k e a c t i o n . A c c o r d i n g to Boura and Green b r e t y l i u m probably a c t s by i m p a i r i n g conduction of impulses i n a d r e n e r g i c neurones w i t h consequent f a i l u r e of nor-k3 a d r e n a l i n e and a d r e n a l i n e r e l e a s e . The primary guanethidine a c t i o n was produced by exposing the r a b b i t i n t e s t i n e t o a con-c e n t r a t i o n of 100*6$ f o r f i f t e e n minutes. T h i s dose o f guanethidine has been r e p o r t e d to completely b l o c k sympathetic kl,k-2 nerve s t i m u l a t i o n w i t h no d e p l e t i o n of n o r - a d r e n a l i n e c o n t e n t . Secondary e f f e c t s were produced by exposing the i n t e s t i n e t o 1 mg% guanethidine f o r n i n e t y minutes. T h i s treatment has been r e p o r t e d by Kadzielawa to deplete the c a t e c h o l amine content o f r a b b i t i n t e s t i n e . Tyramine was used as the c o n t r o l drug and i t s a c t i o n was b l o c k e d by t h i s treatment. As o r i g i n a l l y Figure 7. Isolated rabbit duodenum. Blocking action by 50 DCI and 100 t% dibenzyline (D) of 5 o# adrenaline(Ad) response but not of 2 mg$ IIDN (i) response. W = wash. Ad 5 D 100 x$ h - 20 w x2 DCI 50 ¥ x2 min. 20 min. 10 min. 20 min. 2 mg# Ad 5X# Figure 8. Isolated rabbit duodenum. Blocking action by 50 %$> DCI and 100 dibenzyline ( D ) of 5 o*$ adrenaline (Ad) response but not of 2 mg# IIDN (i) response. No previous I given. W = wash. §> 0.05 mg 0.5 "K 5 mg/kg 10 mg/kg 0.05 mg 0.5 "* Figure 9 Anesthetized rat blood pressure. Blocking action by 10 mg/kg nethalide (N) and 5 mg/kg dibenzyline (D) of response to 0.5* adrenaline (Ad) but not of 0.05 mg IIDN (i) response. ro 28 suggested by Burn and Rand, and now g e n e r a l l y accepted, tyramine 1{5,1}.6,1}.7 a c t s i n d i r e c t l y by r e l e a s i n g c a t e c h o l amines. T h e r e f o r e absence o f a tyramine response i n d i c a t e s c a t e c h o l amine d e p l e t i o n . However n e i t h e r the primary nor the secondary a c t i o n of guaneth-i d i n e b l o c k e d the e f f e c t of the n i t r a t e s on the i s o l a t e d r a b b i t i n t e s t i n e . ( F i g u r e s 10 and 11) Reserpine Burn and Rand p r e s e n t e d the f i r s t evidence i n d i c a t i n g r e s e r p i n e a c t s by d e p l e t i n g the n o r - a d r e n a l i n e content of t i s s u e s . k9 The mechanism of a c t i o n has been confirmed by numerous workers. To f u r t h e r i n v e s t i g a t e the p o s s i b l e a c t i o n o f the n i t r a t e s i n r e l e a s i n g c a t e c h o l amines, the response to IIDN was observed on i n t e s t i n a l s t r i p s from r e s e r p i n i z e d r a b b i t s and on the b l o o d p r e s s u r e o f r e s e r p i n i z e d r a t s . In these animals the tyramine reponse was absent or much reduced however the depressor a c t i o n of the n i t r a t e e s t e r s was unchanged. ( F i g u r e s 12 and 13) P o t e n t i a t i o n of the a d r e n a l i n e response shown In the r e s e r p i n i z e d r a t s ( F i g u r e 13) f u r t h e r confirms the d e p l e t i o n o f c a t e c h o l amines as v a r i o u s means of a d r e n e r g i c blockade produce an i n c r e a s e i n the response t o the n o r - a d r e n a l i n e type sypmathomimetic 50 amines. These r e s u l t s o b t a i n e d w i t h IIDN are c o n s i s t e n t w i t h those r e p o r t e d by M e l v i l l e and Varma i n i s o l a t e d r a b b i t h e a r t s . They found t h a t i f anything, r e s e r p i n e pretreatment somewhat 51 i n c r e a s e d the responses to n i t r o g l y c e r i n . 20 min. Figure 10. Isolated rabbit duodenum. Action of 2 mg$ IIDN (i) not blocked by 100 t% guanethidine ( G ) . IP = isoproterenol, T = tyramine, W = wash. ¥ W V 2 mg# T 2 m g $ 2 m g $ 90 min. Figure 8 . Isolated rabbit duodenum. Blocking action by 1 mg# guanethidine (G) of response to 2 mg$ tyramine (T) but not of IIDN (i) 2 mg# response. W = wash. o 3 1 W W w I T I T 2 mg# 2 mg# 2 mg$ 2 mg# NORMAL RESERPINIZED RABBIT RABBIT Figure 9. Isolated rabbit intestine. Comparison of 2 mg$ tyramine (T) response and 2 mg$ IIDN (i) response in normal and reserpinized ( 0 . 5 mg/kg) animals. W = wash. mm Hg NORMAL RESERPINIZED RAT RAT Figure 13. Anesthetized rat blood pressure. Comparison of 5 J tyramine (T), 0 . 0 5 * adrenaline (Ad) and 0 . 0 5 mg IIDN (i) responses in normal and reserpinized (2 mg/kg) animals. ro 33 Diphenhydramine A l t h o u g h ' i t has p r e v i o u s l y been shown t h a t n i t r a t e s do not a c t i v a t e h i s t a m i n e r g i c r e c e p t o r s , i t was f e l t t h i s should be confirmed with the new isomer. T h i s was done i n the a n e s t h e t i z e d r a t where i t was shown t h a t pretreatment w i t h diphenhydramine d i d not prevent the vasodepressor response to IIDN although the depressor response to histamine was b l o c k e d . ( F i g u r e 1I4.) As noted p r e v i o u s l y the f a c t t h a t a d r e n a l i n e e x e r t s i t s t y p i c a l response when t a c h y p h y l a x i s to IIDN has developed i n the r a b b i t i n t e s t i n e f u r t h e r suggests that IIDN does not act on the a d r e n e r g i c r e c e p t o r s . In f a c t none of the r e s u l t s o b t a i n e d i n d i c a t e any c o n n e c t i o n between the a c t i o n of the n i t r a t e e s t e r s and the a d r e n e r g i c r e c e p t o r s . These r e s u l t s t h e r e f o r e would not support an a n t i a d r e n e r g i c mechanism of a c t i o n f o r n i t r o g l y c e r i n as proposed by Raab and Humphreys as the r e a s o n f o r the e f f e c t o f t h i s drug i n angina p e c t o r i s . However the r e s u l t s upon which Raab and Humphreys base t h i s mechanism have p r e v i o u s l y been qu e s t i o n e d . In t h e i r s t u d i e s of the a n t i a d r e n e r g i c e f f e c t s o f n i t r o g l y c e r i n on the dog h e a r t , E c k s t e i n and co-workers found that even l a r g e doses do not modify changes i n myocardial oxygen consumption, i n s y s t o l i c v i g o r , i n h e a r t r a t e or i n the e l e c t r o -cardiogram which are induced by a d r e n e r g i c s t i m u l a t i o n . Repeating Raab's work on the cat h e a r t and a c t u a l l y measuring oxygen consumption which Raab d i d not, Popovich and a s s o c i a t e s found t h a t n i t r o g l y c e r i n does not s i g n i f i c a n t l y n e u t r a l i z e the 53 e f f e c t s o f a d r e n a l i n e and 1 - a r t e r e n o l . Figure 14. A n e s t h e t i z e d r a t b l o o d p r e s s u r e . B l o c k i n g a c t i o n b y 2 m g / k g d i p h e n h y d r a m i n e ( d ) o f h i s t a m i n e (H) r e s p o n s e b u t n o t o f 0.05 mg I I D N ( i ) r e s p o n s e . 35 SUMMARY AND CONCLUSIONS The pharmacology and a p o s s i b l e mechanism of a c t i o n of a new n i t r a t e e s t e r , i s o i d i d e d i n i t r a t e , were s t u d i e d on the i s o l a t e d r a b b i t i n t e s t i n e and on the a n e s t h e t i z e d r a t b l o o d p r e s s u r e . The f o l l o w i n g c o n c l u s i o n s were reached: 1. In both p r e p a r a t i o n s IIDN was more potent than e i t h e r o f i t s isomers ISDN or IMDN. 2. A type of t a c h y p h y l a x i s t o IIDN which was prominent i n the i s o l a t e d r a b b i t i n t e s t i n e was not observed i n the a n e s t h e t i z e d r a t b l o o d p r e s s u r e . 3. In the p r e p a r a t i o n s employed, the a c t i o n o f IIDN was not b l o c k e d by: i ) Beta a d r e n e r g i c b l o c k i n g agents such as DCI and n e t h a l i d e . i i ) An alpha a d r e n e r g i c b l o c k i n g agent such as d i b e n z y l i n e . i i i ) A combination of an a l p h a and a b e t a a d r e n e r g i c b l o c k i n g agent. [j.. N e i t h e r the primary nor the secondary b l o c k i n g a c t i o n of guanethidine b l o c k e d the e f f e c t of IIDN on the i s o l a t e d r a b b i t i n t e s t i n e . 5 . Pretreatment o f the animals w i t h r e s e r p i n e , to d e p l e t e c a t e c h o l amines, d i d not a l t e r the response of e i t h e r p r e p a r a t i o n to IIDN. 36 6. The antihistamine diphenhydramine did not block the vasodepressor action of IIDN on the anesthetized r a t blood pressure, 7. IIDN probably does not act on the adrenergic or histarainergic receptors or the receptors upon which papaverine acts. However a consideration of factors . such as potency i n terms of structure, and the develop-ment of tachyphylaxis points to a possible action on some type of receptor surface. A more p o s i t i v e demonstration of an action with receptors as d e f i n i t i v e as the adrenergic or analgesic receptors must involve blocking the action of IIDN with an antagonist, the action of which i s s p e c i f i c to these drugs. 37 BIBLIOGRAPHY 1 . Goodman, L.S., and Gilman, A., The Pharmacological B a s i s of T h e r a p e u t i c s . 2nd. Ed. pp. 730-71+3. 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