UBC Theses and Dissertations

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UBC Theses and Dissertations

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 t h e s i s submitted i n p a r t i a l f u l f i l m e n t o f t h e 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 F a c u l t y  o f Pharmacy  We accept t h i s t h e s i s as conforming t o t h e r e q u i r e d  THE UNIVERSITY OP BRITISH COLUMBIA October, 1963  standard  In presenting  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and m i s s i o n f o r extensive purposes may  study.  I f u r t h e r agree that per-  copying of t h i s t h e s i s f o r s c h o l a r l y  be granted by the Head of my Department or  his representatives.  I t i s understood that copying, or  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 g a i n s h a l l not be without my w r i t t e n p e r m i s s i o n .  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.  by publi-  allowed  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 isosorbide dinitrate  (IIDN), a new  n i t r a t e e s t e r , an isomer of  (ISDN) and isomannide  dinitrate  (IMDN), has  been 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 anesthetized r a t blood pressure.  Potency was  observed as  t a c h y p h y l a x i s and the e f f e c t o f c e r t a i n b l o c k i n g agents.  was 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 s i m i l a r r e s u l t s were o b t a i n e d when n i t r o g l y c e r i n was  s u b s t i t u t e d f o r IIDN.  In both p r e p a r a t i o n s 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 p o s s i b l e a c t i o n on s p e c i f i c r e c e p t o r s i t e s . A type o f t a c h y p h y l a x i s t o IIDN which was prominent i s o l a t e d r a b b i t i n t e s t i n e was r a t blood pressure.  i n the  almost absent i n the a n e s t h e t i z e d  T h i s i s probably due t o metabolism of the  drug i n the i n t a c t animal and f u r t h e r suggests an a c t i o n on receptors. In these p r e p a r a t i o n s the n i t r a t e a c t i o n was  not b l o c k e d by:  the b e t a a d r e n e r g i c b l o c k i n g agents DCI or n e t h a l i d e , the a l p h a a d r e n e r g i c b l o c k i n g agent d i b e n z y l i n e , or a combination of an alpha and a beta b l o c k i n g agent.  Drugs that are s e l e c t i v e l y  ii b l o c k e d by these agents were used as c o n t r o l s to i n d i c a t e the presence of the d e s i r e d b l o c k i n g a c t i o n .  N e i t h e r the primary  b r e t y l i u m - l i k e a c t i o n nor the secondary r e s e r p i n e - l i k e a c t i o n of guanethidine intestine.  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 D e p l e t i o n of c a t e c h o l amines by p r e t r e a t i n g the  animals w i t h r e s e r p i n e 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 the n i t r a t e e s t e r s .  The  antihistamine  diphenhydramine d i d not b l o c k the vasodepressor a c t i o n of IIDN on the a n e s t h e t i z e d r a t b l o o d It was  concluded  pressure.  that although  IIDN probably  i t s e f f e c t through combination w i t h a d r e n e r g i c  Signatures  o f Examiners  exert  or h i s t a m i n e r g i c  r e c e p t o r s , i t p o s s i b l y a c t s on r e c e p t o r s which are the n i t r i t e or n i t r a t e group.  does not  specific for  iii  TABLE OF CONTENTS  Page  ABSTRACT.  i  L I S T OF TABLES  v  L I S T OF FIGURES  .  vi  INTRODUCTION a  Pharmacological  Actions  .  1  General  •  1  Isomannide d i n i t r a t e .  . . . . . . . . . . . . . .  1  Isosorbide  . . . .  3  dinitrate.  If.  Isoidide dinitrate  if.  Structure A c t i v i t y Relationship Mechanism o f A c t i o n METHODS AND  . . . .  . . . . . . .  8  . . . . . . .  10  MATERIALS  I s o l a t e d Rabbit Anesthetized  Intestine  Rat B l o o d  Pressure  . . .  11  RESULTS AND DISCUSSION llf.  Potency Tachyphylaxis Interaction with  . B l o c k i n g Drugs  . . . . . . . . . . .  17 18  DCI a n d N e t h a l i d e  20  Dibenzyline  22  Dibenzyline  and a B e t a B l o c k i n g Agent  22.  Guanethidine  2%  Reserpine  28  Diphenhydramine  . . . . .  33  SUMMARY AND CONCLUSIONS BIBLIOGRAPHY  V  L I S T OP  TABLES  TABLE I  Page 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 A  I s o l a t e d Rabbit  Intestine  B  A n e s t h e t i z e d Rat B l o o d  Pressure  Drugs.  . . . . .  19  vi  L I S T OP  FIGURES  FIGURE 1  Page Structure of 2,5-dinitrates dianhydrohexitols  o f 1,it; 3 , 6 -  2  2  P o t e n c y o f Isomers on A n e s t h e t i z e d R a t B l o o d  3  Absence of T a c h y p h y l a x i s Blood  Pressure.  t o IIDN i n A n e s t h e t i z e d  .  l£  Rat 15>  Pressure  If.  Tachyphylaxis  $  A c t i o n o f DCI on. IIDN R e s p o n s e i n I s ' o l a t e d R a b b i t Intestine. A c t i o n o f N e t h a l i d e o n IIDN R e s p o n s e 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  A c t i o n o f DCI and D i b e n z y l i n e I s o l a t e d Rabbit I n t e s t i n e  2f?  6 7  8 9 10 11  t o IIDN i n I s o l a t e d R a b b i t  Intestine . . .  16  21  o n IIDN R e s p o n s e i n  A c t i o n o f DCI a n d D i b e n z y l i n e o n IIDN R e s p o n s e i n I s o l a t e d R a b b i t I n t e s t i n e (No p r e v i o u s IIDN g i v e n ) A c t i o n o f N e t h a l i d e and D i b e n z y l i n e 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  . . .  on IIDN R e s p o n s e i n  26 27  A c t i o n of Guanethidine (100 t%) o n IIDN R e s p o n s e i n I s o l a t e d R a b b i t I n t e s t i n e . . ... .  29  A c t i o n of Guanethidine ( 1 mg$) I s o l a t e d Rabbit I n t e s t i n e .  30  o n IIDN R e s p o n s e i n  12  A c t i o n o f IIDN i n R e s e r p i n i z e d R a b b i t .  13  A c t i o n o f IIDN i n R e s e r p i n i z e d R a t  32  llj.  A c t i o n o f D i p h e n h y d r a m i n 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 .  3lj.  . . . . . . . . . .  31  vii  ACKNOWLEDGEMENTS  The w r i t e r expresses h i s s i n c e r e a p p r e c i a t i o n t o Dr. J.E. H a l l i d a y f o r the guidance and encouragement g i v e n during the course o f t h i s work. Thanks i s a l s o extended t o the f o l l o w i n g companies f o r t h e drugs s u p p l i e d : Parke Davis & Co. L t d . f o r N i t r o g l y c e r i n Imperial Chemical I n d u s t r i e s L t d . f o r Nethalide ( A l d e r l i n ) Ciba Co. f o r Guanethidine (Israelin) Reserpine ( S e r p a s i l )  INTRODUCTION  Pharmacological  Actions  General The  b a s i c p h a r m a c o l o g i c a l a c t i o n of the  nitrite-nitrate  group of drugs i s to r e l a x smooth muscle, e s p e c i a l l y those o f the f i n e r b l o o d v e s s e l s .  The  most c h a r a c t e r i s t i c pharmacodynamic  e f f e c t , therefore, i s a f a l l i n blood pressure. Gilman  1  suggest the a c t i o n of the n i t r i t e s  s p e c i f i c as the spasmolytic  by a t r o p i n e  are not p a r a l y z e d as muscular s t i m u l a n t s  and the muscles  and motor nerve impulses  s t i l l exert t y p i c a l c o n s t r i c t o r e f f e c t s . 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 , has  A d e s c r i p t i o n of t h i s drug, and and  and n i t r a t e s i s non-  a c t i o n i s independent of i n n e r v a t i o n ,  the v a s o d i l a t o r a c t i o n i s not b l o c k e d  can  Goodman and  isomannide d i n i t r a t e , w i l l  been  synthesized.  i t s isomers i s o s o r b i d e  dinitrate  follow.  Isommanide D i 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 ,  p o i n t 6jp.5°C, s o l u b l e i n water  compound, s t a b l e i n a i r , m e l t i n g 1.7:1*000,  f r e e l y s o l u b l e i n a l c o h o l , o i l s and  In a study of a new associates  i s a white c r y s t a l l i n e  s e r i e s of organic  synthesized  IMDN and 2  p o t e n t i a l t h e r a p e u t i c agent.  etherlal  n i t r a t e s Krantz  solvents.  and  s e l e c t e d i t f o r study as a They found from a more d e t a i l e d  p h a r m a c o l o g i c a l study t h a t t h i s compound was vasodepressor i n the a n e s t h e t i z e d  dog,  a potent  r e l a x e d i s o l a t e d coronary  Figure 1. Structure of 2,5-dinitrates of l,4}3,6-dianhydrohexitols.  I  R i = 0N02,  R2 = H  ,  R3 = ONO2.  R4 = H  :  II  R i = 0N02,  R2 = H  ,  R3 = H  ,  R4 = 0N02  : . isosorbide d i n i t r a t e  III  Ri = H  R2 = 0N02,  R3 = H  ,  R4 = 0N02  :  ,  isomannide d i n i t r a t e  isoidide d i n i t r a t e  ( A f t e r : Jackson and Hayward, Can. J . Chem., ^8:497, I960 )  3 a r t e r y r i n g s , i n c r e a s e d t h e r a t e o f outflow an average o f 21% i n p e r f u s e d i s o l a t e d r a b b i t h e a r t s and caused an i n c r e a s e o f i n s i t u coronary flow i n dogs.  They a l s o found, when taken  o r a l l y by normal men, i t caused a f a l l i n b l o o d pressure w i t h i n ten  minutes which l a s t e d f o r f i v e t o s i x hours.  Although they  suggested IMDN l e n d s i t s e l f t o m e d i c a t i o n i n t a b l e t form i n the treatment o f h y p e r t e n s i o n and angina p e c t o r i s , no c l i n i c a l s t u d i e s have been r e p o r t e d .  3  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 compound, s t a b l e i n a i r , m e l t i n g p o i n t 10°C  t  crystalline  s o l u b l e i n water  1.125:1,000, f r e e l y s o l u b l e i n a l c o h o l and ether and i n a r a c h i s o i l t o about 50-100:1,000 a t 50°C.  ISDN i s another member o f the  2 s e r i e s s y n t h e s i z e d by Krantz e t . a l .  Some years l a t e r  Goldberg  r e p o r t e d t h a t ISDN was a potent vasodepressor and coronary vasod i l a t o r i n l a b o r a t o r y animals and e f f e c t i v e l y lowered t h e b l o o d pressure i n h y p e r t e n s i v e p a t i e n t s .  He found i t i n v a r i a b l y had a k  somewhat s t r o n g e r a c t i o n than isomannide d i n i t r a t e . Studies i n t h i s l a b o r a t o r y have g e n e r a l l y shown s i m i l a r r e s u l t s i n l a b o r a t o r y animals.  5  The numerous c l i n i c a l r e p o r t s on ISDN  although mostly s u b j e c t i v e i n nature, i n d i c a t e an e x c e l l e n t  6,7,8 response by angina p e c t o r i s p a t i e n t s .  Some workers have  a l r e a d y p l a c e d i t among t h e most a c t i v e o f the l o n g - a c t i n g n i t r i t e s and n i t r a t e s f o r the treatment o f angina p e c t o r i s i n terras o f dose, time o f onset o f a c t i o n and d u r a t i o n of a c t i o n . Buckley and a s s o c i a t e s have shown ISDN I s an e f f e c t i v e v a s o d i l a t o r  and  causes a g r e a t e r r e d u c t i o n i n coronary v a s c u l a r r e s i s t a n c e  than does p e n t a e r y t h r i t o l t e t r a n i t r a t e .  9  Isoidide Dinitrate Isoidide dinitrate  3,6-dianhydro-L-iditol-2,£-  (IIDN),  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 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 s o l u b l e i n a l c o h o l , o i l s and e t h e r i a l IIDN was f i r s t  compound,  solvents.  s y n t h e s i z e d by Jackson and Hayward i n the Depart-  1 0 ment o f Chemistry, U n i v e r s i t y o f B r i t i s h Columbia. pharmacological potent  Initial  s t u d i e s i n d i c a t e t h i s drug i s c o n s i d e r a b l y more  t h a n e i t h e r o f i t s isomers ISDN or IMDN.  vasodepressor a c t i o n when a d m i n i s t e r e d  I t has a g r e a t e r  i n t r a v e n o u s l y i n the c a t ,  r a b b i t , and dog, and i n t r a d u o d e n a l l y i n t h e dog.  It also  produces a much g r e a t e r i n c r e a s e i n the b l o o d flow o f t h e dog h i n d l e g and i n the outflow  from the p e r f u s e d  Structure A c t i v i t y  isolated  heart.^  Relationship  L i t t l e work has been done to determine the 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 o f the n i t r a t e drugs. the v a s o d i l a t i n g a c t i o n o f the n i t r i c  Marshall  feels  esters of polyhydric  a l c o h o l s i s connected s o l e l y with the n i t r a t e group. out t h a t m a n n i t o l h e x a n i t r a t e ,  that  He p o i n t s  d u l c i t o l h e x a n i t r a t e and s o r b i t a l  h e x a n i t r a t e a l l have the same a c t i o n thus i t appears the arrangement o f t h e n i t r a t e groups i n the n i t r i c  e s t e r s o f hexahydric  a l c o h o l s o r sugars appears t o be without i n f l u e n c e . 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 ,  Further,  glucose  p e n t a n i t r a t e , f r u c t o s e p e n t a n i t r a t e , q u e r c i t e p e n t a n i t r a t e and  5  saccharose o c t o n i t r a t e  show no  difference  i n a c t i v i t y which  can-  11 not be  a t t r i b u t e d to d i f f e r e n c e s  i n s t a b i l i t y and  solubility.  Prom h i s study of a s e r i e s of glucose n i t r a t e s O'Meara agrees t h a t as f a r as he l i t t l e or no had  see  influence  at l e a s t one  carbon atoms and The  can  the p o s i t i o n of the n i t r a t e group  on a c t i v i t y .  However a l l a c t i v e  glucosides  p a i r of n i t r a t e groups a t t a c h e d to adjacent carbon three and/or four  work of Pranz and  associates  p e n t a n e d i o l d i n i t r a t e s i n d i c a t e s the  nitrated.  on a s e r i e s o f f i v e spatial configuration  n i t r a t e groups i s an important f a c t o r i n f l u e n c i n g  of  the  physiological  In terms o f smooth muscle a c t i v i t y , 1 , 5 - p e n t a n e d i o l  activity.  d i n i t r a t e and  t r a n s - l , 2 - c y c l o p e n t a n e d i o l d i n i t r a t e were  most potent whereas 2,3active.  has  and  generally  2,ij.-pentanediol d i n i t r a t e were l e a s t  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 v a r i e d between' these extremes, b o t h c i s - and  trans-l,2-  13 c y c l o p e n t a n e d i o l d i n i t r a t e proved t o be potent convulsants.• The  studies  o f ISDN and  IMDN, i n which Goldberg found ISDN  c o n s i d e r a b l y more a c t i v e , and more recent s t u d i e s laboratory  in this  of IIDN a l s o i n d i c a t e t h a t i n t h i s s e r i e s the  of the n i t r a t e group i s a v i t a l c o n s i d e r a t i o n  position  when s t u d y i n g  potency. Thus i n v e s t i g a t i o n of the potency of the esters,  IMDN, ISDN and  clinical the  increased  IIDN, seemed s i g n i f i c a n t apart from  value i n obtaining  structure  s e r i e s of n i t r a t e  a more potent drug.  of these isomers (Figure  potency i n the  order IMDN  1)  ISDN  In  the  considering  i t i s observed that IIDN i s  associated  w i t h g r e a t e r freedom of r o t a t i o n of the n i t r a t e groups.  That  the  6  is,  i n  held  IMDN b o t h  somewhat  nitrate  rigidly.  ISDN a n d b o t h  are  in  groups  are  Whereas  the  exo  i n the  only  one  position  endo  is  in  in  position this  thus  position  IIDN and have  are in  more  10 freedom  of  rotation.  with  a  specific  more  readily  receptor  c o n f o r m due  nitrate  groups.  nitrate  drugs,  structure, The  oil/water  this to  in  however I n some  factor  of  to  greater  in  ratio  depressor  stress  the  nitrate  solubility Carr  the  Thus  merely  series is  order  IIDN of  oil  water  potency:  that are  of  their  They  and o i l  diverse  for  Garr  and  importance  relative  of  potency have  directly  related  to  oil  being  ISDN  one IIDN.  seem  nitrates  solubility,  2  15  solubility  studied,  would be  relative  in  be a l i m i t i n g factor. workers found a s t r u c t u r a l  IMDN t h e r e f o r e  of  of  solubility  is  by  the  found  various  explained  the  action.  accounted Krantz,  can  the  of  solubility, IMDN  but  of  Studies  solubility  basis  IIDN  other.  nitrates  ISDN  on the  sufficient of  of  be  which  rotation  for  drugs.  solubility  interaction  each  potency  c o u l d not  be  on the  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 o f t h e i r s t u d i e s however t h e s e  which  of  accepted  receptors  may a l s o  water  to  with  similar action  determining  vasodepressor compounds  freedom  solubility.  considerable  indicate  muscle,  generally  i n potency  in  could  i n the  however  differences  inverse  In the  order  site  on n o n - s p e c i f i c  coefficient the  factors  which have v e r y  place  of  indicate  on  by  series  be  is  difference  associates  members  It  depend  exclusively  These  oil  the  considerations. order  according IMDN  to  ISDN  solubility,  can predict  the  of  water  Krantz  and  IIDN. depending observed  7 Marshall  c r i t i c i z e s Krantz, and Garr's emphasis on o i l / w a t e r  d i s t r i b u t i o n on the grounds t h a t i n pharmacology t h e p r i n c i p l e o f 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 w i t h a b s o r p t i o n and not w i t h a s s i m i l a t i o n and as he i s c e r t a i n t h a t t h e i r predominant a c t i o n n i t r i c the  i n producing  e s t e r s undergo changes  within  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.  F u r t h e r the l i p o i d content of smooth muscle i s not v e r y d i f f e r e n t from that  o f b l o o d plasma, t h e r e f o r e  why would l i p o i d  solubility  11 play  so important a r o l e ?  He t h e r e f o r e  places  greater  emphasis  on water s o l u b i l i t y , which i n the s e r i e s IIDN, ISDN, and IMDN would not e x p l a i n t h e d i f f e r e n c e s i n potency as IIDN which i s most potent i s l e a s t water Generally  soluble.  throughout the n i t r a t e group o f drugs the number  o f n i t r a t e groups cannot be c o r r e l a t e d w i t h a c t i v i t y , as compounds w i t h a v a r i e d number o f n i t r a t e groups a r e a c t i v e .  Recent  evidence s u p p o r t i n g t h i s concept l i e s i n t h e demonstration that r e p l a c i n g one o f the n i t r a t e groups o f n i t r o g l y c e r i n w i t h  16 c h l o r i n e does not a l t e r the a c t i v i t y . the  However, O'Meara found  degree of n i t r a t i o n t o be a c r i t i c a l f a c t o r i n determining  the v a s o d i l a t o r a c t i v i t y o f g l u c o s e d e r i v a t i v e s .  Whereas none o f  the mono or d i n i t r a t e s were a c t i v e , methyl b e t a - D - g l u c o s i d e t e t r a n i t r a t e was the most a c t i v e compound s t u d i e d .  These r e s u l t s  would suggest that a t l e a s t f o r some carbon s k e l e t o n s a minimum number o f n i t r a t e groups i s necessary t o endow the compound w i t h  12 vasodilator a c t i v i t y .  This information  further indicates the  p o s s i b l e r e a c t i o n o f a t l e a s t some o f the n i t r a t e s w i t h s p e c i f i c receptor  sites.  8  Mechanism o f A c t i o n Although c o n s i d e r a b l e  work has  been done i n an attempt t o  e l u c i d a t e the mechanism of a c t i o n o f the n i t r i t e / n i t r a t e group o f drugs, a d e f i n i t e mechanism has many years i t was eardio-vascular  not been e s t a b l i s h e d .  assumed that the organic  n i t r a t e s exerted  e f f e c t s f o l l o w i n g h y d r o l y s i s and  r e d u c t i o n to n i t r i t e .  For their  subsequent  However the work o f Krantz and  associates  suggests t h a t the vasodepressor e f f e c t i s produced by the i n t a c t 18  X*7  molecule.  '  19  '  I t i s p o s s i b l e however t h a t the  n i t r a t e molecule r a p i d l y enters the c e l l s and pharmacologically  e f f e c t i v e concentrations  organic  releases  of n i t r i t e a t  an  11,20,21  i n t r a c e l l u l a r locus.  More experimental data i s r e q u i r e d  b e f o r e a c o n c l u s i o n can be reached concerning entity responsible  f o r the c a r d i o v a s c u l a r  the m o l e c u l a r  e f f e c t s o f the  organic  nitrates. In c o n c l u d i n g  a r e v i e w of h a l f a century  o f h i s own  the pharmacology of the n i t r a t e e s t e r s , M a r s h a l l  suggests that  the i n i t i a l p h a r m a c o l o g i c a l a c t i o n , at l e a s t on b l o o d , a s s o c i a t e d w i t h a very mobile oxygen.  He  work on  is  s t a t e s however that  he  11  was unable t o o b t a i n supporting evidence f o r t h i s concept. Recent work i n Hayward's l a b o r a t o r y on the hemolytic cleavage o f 22  oxygen from n i t r a t e i n p h o t o l y s i s supports M a r s h a l l ' s Raab and Humphreys have proposed a b i o c h e m i c a l  suggestion.  mechanism t o  e x p l a i n 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 t h a t the c a r d i a c a c c e l e r a t i o n and ECG  T-wave d e p r e s s i o n  a t r o p i n i z e d c a t s by i n j e c t i o n s o f a d r e n a l i n e ,  produced i n  faradic stimulation  9  o f the s t e l l a t e g a n g l i a i n c a t s w i t h the adrenals  t i e d o f f , and  f a r a d i c s t i m u l a t i o n of the s p l a n c h n i c nerves were a b o l i s h e d by simultaneous i n j e c t i o n s o f 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 o n l y to a moderate extent with the accumulation a d r e n a l i n e - l i k e m a t e r i a l i n the myocardium.  of  These f a c t s 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 h e a r t muscle to the sympathomimetic amines and syndrome i s due  support  the concept that the a n g i n a l  to e x c e s s i v e i n f l u x e s o f a d r e n a l i n e and  nor-  a d r e n a l i n e i n t o the h e a r t muscle during e x e r c i s e , emotions, e t c . Thus Raab and Humphreys suggest the t h e r a p e u t i c a c t i o n of n i t r o g l y c e r i n i n angina p e c t o r i s i s not due  to coronary  dilation  alone but a l s o to a s p e c i f i c c o u n t e r a c t i o n a g a i n s t the metabolic xn the  e f f e c t s o f an excess accumulation 23,2ij.,25  myocardial  of c a t e c h o l amines  heart.  From the p r e v i o u s  c o n s i d e r a t i o n of the 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 of the n i t r i t e / n i t r a t e group o f drugs and because of the i m p l i c a t i o n of the a d r e n e r g i c mechanism i n t h e i r a c t i o n , the p o s s i b i l i t y a r i s e s t h a t the n i t r i t e s and n i t r a t e s a c t s p e c i f i c a l l y on the a d r e n e r g i c r e c e p t o r s . a v a i l a b l e v a r i o u s types  S i n c e there are  of a d r e n e r g i c b l o c k i n g agents i t was  now felt  t h i s p o s s i b l e a c t i o n would be i n v e s t i g a t e d p a r t i c u l a r l y i n terms of the b e t a a d r e n e r g i c r e c e p t o r s , as, l i k e drugs which a c t i v a t e the beta r e c e p t o r s , the n i t r a t e s ^ i n v a r i a b l y r e l a x smooth muscle.  10 METHODS AND  MATERIALS  I s o l a t e d Rabbit I n t e s t i n e A l b i n o r a b b i t s o f e i t h e r sex were used throughout these studies.  Animals weighing 1-3  kg. were employed  however b e s t  r e s u l t s were o b t a i n e d w i t h young r a b b i t s weighing around 1  kg.  The animals, p r e v i o u s l y f a s t e d f o r a t 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  p o r t i o n o f duodenum and ileum was removed, p l a c e d i n Tyrode's s o l u t i o n and the lumen washed out u s i n g a s y r i n g e .  Duodenum,  taken from the p y l o r i c s p h i n c t e r a r e a , was  i n most  employed  experiments as i t gave a more s t a b l e , l o n g e r l a s t i n g p r e p a r a t i o n than i l e u m . Four 3-lf cm. of  s t r i p s were cut from the p y l o r i c s p h i n c t e r  the duodenal s e c t i o n , or from the ileum, t i e d t o a h o l d e r and  p l a c e d i n an i s o l a t e d t i s s u e b a t h (Metro I n d u s t r i e s ) . left at  end  They were  1$ minutes to e q u i l i b r a t e i n Tyrode's s o l u t i o n maintained  38°C and oxygenated w i t h 9$% oxygen and  carbon d i o x i d e .  The s t r i p s were t h e n a t t a c h e d t o w r i t i n g l e v e r s w i t h a minimum counterbalance and l e f t  a f u r t h e r 15 minutes.  The w r i t i n g p o i n t s  were arranged t o r e c o r d c o n t r a c t i o n s on smoked kymograph drums. A f t e r d i s c o v e r y o f the development to  o f a type o f t a c h y p h y l a x i s  r e p e a t e d doses o f the n i t r a t e s , each t e s t s t r i p was  compared  w i t h a c o n t r o l s t r i p which had r e c e i v e d no b l o c k i n g drug.  The  washing procedure which f o l l o w e d the use of most drugs c o n s i s t e d of  complete replacement o f the b a t h i n g f l u i d a t l e a s t four times.  A u s e f u l f e a t u r e o f the smooth muscle baths employed  i s that the  11 b a t h i n g s o l u t i o n can be r e p l a c e d while t h e t i s s u e remains  fully  immersed. In some of the e a r l i e r experiments t h e n i t r a t e s were a d m i n i s t e r e d by r e p l a c i n g t h e p e r f u s i o n f l u i d w i t h f r e s h Tyrode's c o n t a i n i n g the d e s i r e d c o n c e n t r a t i o n o f drug.  This  procedure  was abandoned as i n the absence o f drug t h e s t a t e o f c o n t r a c t i o n and t h e tone o f the s t r i p were s t i l l a l t e r e d c o n s i d e r a b l y .  In  some o f the experiments the n i t r a t e s were added i n c o n c e n t r a t e d a l c o h o l i c s o l u t i o n , however best r e s u l t s were o b t a i n e d with a d d i t i o n s o f c o n c e n t r a t e d aqueous s o l u t i o n .  A l l other drugs were  added to the b a t h i n g f l u i d as c o n c e n t r a t e d aqueous s o l u t i o n s . A n e s t h e t i z e d Rat Blood Male Wistar r a t s weighing experiments.  Pressure  3 0 0 - 5 0 0 gm. were used f o r these  The technique  employed was s i m i l a r t o t h a t 26 27 o r i g i n a l l y d e s c r i b e d by Landgrebe and m o d i f i e d by Dekanski. I n i t i a l l y urethane,  ( 1 7 5 mg./lOO gm. body weight) g i v e n ' i n t r a -  p e r i t o n e a l l y was employed as an a n e s t h e t i c however h i g h e r b l o o d p r e s s u r e s were o b t a i n e d i n l a t e r experiments u s i n g 5 0 mg./kg. p e n t o b a r b i t a l sodium, a d m i n i s t e r e d  intraperitoneally.  The h i n d l e g s o f t h e animal were secured w i t h s c o t c h tape to a heated  t a b l e and the r e c t a l temperature was maintained a t  33 0. a  Operative  Procedures:  a) One c a r o t i d a r t e r y was d i s s e c t e d ready f o r c a n n u l a t i o n . b) The t r a c h e a was c a n n u l a t e d w i t h a s h o r t p o l y e t h y l e n e tube w i t h o u t s i d e 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 c l o s e to the i n g u i n a l ligament.  I f care was taken i t was not necessary  o f f the branches, and c a n n u l a t i o n was e a s i e r .  to t i e  A  p o l y e t h y l e n e cannula o f o u t s i d e diameter about 1 (PE 50) was employed, and the adapter  m.m.  was connected t o a  three-way stop cock t o which a s y r i n g e c o u l d be connected f o r intravenous d) H e p a r i n  (1.5  the venous  injections.  gm./lOO gm. body weight) was i n j e c t e d through cannula.  e) The c a r o t i d cannula,  a l s o 1 m.m.  o u t s i d e diameter,  was  t i e d i n and connected by a column o f normal s a l i n e t o a Condon manometer. To reduce f l u c t u a t i o n s o f the blood p r e s s u r e an intravenous dose o f 0.125 mg. p e n t o l i n i u m t a r t r a t e per r a t was i n j e c t e d 28 immediately  a f t e r cannulation.  f o r about one hour to permit  The p r e p a r a t i o n was then  the b l o o d pressure  left  to s t a b i l i z e .  Drugs were g e n e r a l l y i n j e c t e d i n 0.05 or 0.10 ml. o f s a l i n e and washed i n with s u f f i c i e n t warm s a l i n e such that the t o t a l volume o f f l u i d g i v e n a t any one time was 0.5 m l .  Great  taken not t o i n j e c t a i r I n the venous cannula bubble can be f a t a l . saline containing The  c a r e must be  as t h e s m a l l e s t a i r  The n i t r a t e s were i n j e c t e d i n 0.1 ml. o f alcohol.  f o l l o w i n g drugs were employed i n t h i s study:  dinitrate, isosorbide dinitrate,  isomannide d i n i t r a t e ,  isoidide nitro-  g l y c e r i n , p e n t o b a r b i t a l sodium, e t h y l carbamate, h e p a r i n sodium, pentolinium t a r t r a t e , dichloroisoproterenol hydrochloride, nethalide hydrochloride, dibenzyline hydrochloride,  guanethidine,  13  r e s e r p i n e , i s o p r o t e r e n o l h y d r o c h l o r i d e , phenylephrine hydroc h l o r i d e , a d r e n a l i n e h y d r o c h l o r i d e , tyramine monohydrochloride, n o r - a d r e n a l i n e b i t a r t r a t e and papaverine. are expressed i n terras o f the s a l t  The doses of drugs  employed.  lit.  RESULTS AND DISCUSSION  Potency In a c o n c e n t r a t i o n o f 1-2 mg°/o IIDN was i n v a r i a b l y a more potent r e l a x a n t o f the i s o l a t e d r a b b i t i n t e s t i n e than t h e same dose o f ISDN or HON and was g e n e r a l l y equipotent w i t h 0.5 mg$ nitroglycerin. IMDN.  ISDN was l i k e w i s e a more potent r e l a x a n t than  I n a d d i t i o n t o t h e r e l a x a n t e f f e c t o f the n i t r a t e drugs  on t h e i s o l a t e d r a b b i t  i n t e s t i n e , they a l s o produced  a marked  decrease or t o t a l i n h i b i t i o n o f spontaneous c o n t r a c t i o n s . On t h e r a t b l o o d p r e s s u r e a dose o f 0.05 mg IIDN per r a t was a more potent vasodepressor potent than IMDN.  than ISDN or IMDN and ISDN was more  ( F i g u r e 2)  I t has been shown that ISDN has a somewhat g r e a t e r r e l a x a n t e f f e c t on the smooth muscle of the i n t e s t i n e and v a s c u l a r system than has IMDN.  T h i s i s i n agreement w i t h r e s u l t s o b t a i n e d by  Goldberg on t h e r a b b i t b l o o d p r e s s u r e , p e r f u s e d i s o l a t e d r a b b i t 'line a r t and i s o l a t e d r a b b i t ileum.  The isomer  IIDN has been shown  to possess even g r e a t e r potency than ISDN on both p r e p a r a t i o n s used.  T h i s agrees w i t h p r e v i o u s o b s e r v a t i o n s i n t h i s l a b o r a t o r y  on the b l o o d p r e s s u r e o f the c a t , dog and r a b b i t , on the b l o o d f l o w o f the dog h i n d l e g and on t h e outflow from the p e r f u s e d  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 c o n f i g u r a t i o n o f the n i t r a t e groups c o u l d be s i g n i f i c a n t i n the d e t e r m i n a t i o n of potency.  A l s o , they  suggest  that IIDN which i s the most potent of the s e r i e s , can c o n c e i v a b l y  s 0.05  Figure 2 .  I 0.05  mg  I  mg  0.05  Anesthetized rat blood pressure. potency of 0 . 0 5 mg ISDN (s),  mg  Comparison of  IMDN (M), and IIDN ( i ) .  Figure 11. Anesthetized rat blood pressure.  Absence of  tachyphylaxis development to repeated doses of 0 . 0 5 mg IIDN  (I).  Ad 5Vt  w  Figure 4.  W 2 mg#  2 mg#  Isolated rabbit duodenum.  Development of tachyphylaxis to 2 mg# IIDN ( i ) .  before and after tachyphylaxis.  W = wash.  Response to 5  Ad 5**  adrenaline(Ad)  17 conform moat r e a d i l y w i t h r e c e p t o r s due t o g r e a t e r freedom o f r o t a t i o n o f the n i t r a t e groups, b o t h of which are i n t h e exo position. Tachyphylaxis 4 type o f t a c h y p h y l a x i s was  to IIDN, ISDN, IMDN and n i t r o g l y c e r i n  demonstrated 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 .  (Figure  Once the t a c h y p h y l a x i s developed the i n t e s t i n e no longer responded to  any of t h e n i t r a t e drugs employed however i t was s t i l l  by i s o p r o t e r e n o l , a d r e n a l i n e and papaverine.  relaxed  I f the second  a d d i t i o n of t h e drug was made w i t h i n a few minutes o f the f i r s t , the decrease i n response was v e r y marked, however doses g i v e n every t h i r t y minutes a l s o produced t a c h y p h y l a x i s .  Repeated  washing seemed t o decrease the degree o f t a c h y p h y l a x i s .  Pre-  treatment o f the animal with r e s e r p i n e d i d not a l t e r the development o f t a c h y p h y l a x i s . Tachyphylaxis  d i d not occur 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 p r e p a r a t i o n even when IIDN was a d m i n i s t e r e d to  t e n minutes o f a p r e v i o u s The  dose.  within five  ( F i g u r e 3)  t a c h y p h y l a x i s which develops so r e a d i l y 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 i s l i k e l y caused e i t h e r by o c c u p a t i o n o f r e c e p t o r s i t e s by the drug o r exhaustion which the a c t i o n of the drug i s mediated.  o f t h e mechanism by I t has been suggested  that t h e n i t r i t e s and n i t r a t e s , l i k e papaverine,  a c t d i r e c t l y on  the muscle, that i s , depend on " 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 2  action.  S i n c e t h e i n t e s t i n e s t i l l responds t o papaverine during  nitrate' tachyphylaxis  i t would seem t h a t d i f f e r e n t mechanisms a r e  18  i n v o l v e d i n the a c t i o n s of the two  drugs.  Normal responses to  a d r e n a l i n e and i s o p r o t e r e n o l while the i n t e s t i n e i s  unresponsive  to the n i t r a t e s , i n d i c a t e s t h a t the n i t r a t e s do not a c t on adrenergic  receptors.  The  absence o f t a c h y p h y l a x i s  development  i n the a n e s t h e t i z e d r a t b l o o d pressure p r e p a r a t i o n c o u l d be to metabolism of the drug i n the i n t a c t animal. would f r e e the r e c e p t o r s o f drug thus supports  the  This  due  process  the concept t h a t  the n i t r a t e s a c t on r e c e p t o r s r a t h e r than through a d e p l e t a b l e intermediate. I n t e r a c t i o n w i t h B l o c k i n g Drugs Considerable  experimentation  was  c a r r i e d out t o o b t a i n t h e  r e q u i r e d i n f o r m a t i o n , i n a d d i t i o n t o t h a t a v a i l a b l e i n the l i t e r a t u r e , concerning experimental  the use  of the b l o c k i n g agents.  The  c o n d i t i o n s , such as c o n c e n t r a t i o n of and time o f  exposure t o the drugs, determined to be most s u i t a b l e f o r these s t u d i e s are l i s t e d i n Table 1-A and Table  I-B  f o 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 o r 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  "Time" column l i s t s the l e n g t h of time, f o l l o w i n g a d m i n i s t r a t i o n o f the b l o c k i n g drug, f o r the b l o c k i n g a c t i o n t o develop. blocking action  1 3  The  demonstrated by the use o f a c o n t r o l drug the  a c t i o n of which i s b l o c k e d by the b l o c k i n g agent. Throughout the experiments 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 the e f f e c t of the b l o c k i n g 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 w i t h IIDN, none of the drugs employed b l o c k e d  the n i t r a t e response.  TABLE I I n t e r a c t i o n o f IIDN with B l o c k i n g Drugs  BLOCKING DRUG Concentration A.  CONTROL DRUG Concentration  Time (min.)  EFFECT on 2 mg$ IIDN RESPONSE  I s o l a t e d Rabbit Intestine  DCI Nethalide Dibenzyline D i b e n z y l i n e and DCI or n e t h a l i d e Guanethidine Reserpine  50 1% 200 %% 100 \%  20 - 30 10 - 20 lj.0 - 60  100 1 W>%. 0.5  15 - 30 90  as above  as above  isoproterenol isoproterenol phenylephrine  5  tt  2 mg$ it  adrenaline tyramine tyramine  nil  2 2  ti ii  £- G i v e n subcutaneously on the day previous to the experiment or f o r two days previous, B.  A n e s t h e t i z e d Rat Blood Pressure  Nathalide Dibenzyline D i b e n z y l i n e and nethalide Reserpine Diphenhydramine  5-10 mg/kg 5 mg/kg as above 2 mg/kg* 2 mg/kg  10-15 20 - 30  isoproterenol adrenaline  10 - 15  adrenaline tyramine histamine  as above  0.25 ^ 0.5-1.0 * o.5  5.0 * 2.0 t  * Given i n t r a p e r i t o n e a l l y f o r two days previous t o the experiment.  nil ti n  ti  tt  20 Due  to the marked t a c h y p h y l a x i s development i n the r a b b i t  i n t e s t i n e i t was  necessary  to compare the r e s u l t s o b t a i n e d  the b l o c k i n g drugs w i t h responses i n a c o n t r o l , u n t r e a t e d I t was  also d i f f i c u l t  i n both the r a b b i t i n t e s t i n e and  after strip.  the  a n e s t h e t i z e d r a t b l o o d pressure p r e p a r a t i o n s to o b t a i n r e g u l a r responses to the n i t r a t e s over a p e r i o d of time.  The  del-  e t e r i o u s e f f e c t s o f the b l o c k i n g agents on the normal s t a t e o f the p r e p a r a t i o n s f u r t h e r complicated I t i s now there are two  g e n e r a l l y accepted  i n t e r p r e t a t i o n of the r e s u l t s .  t h a t i n smooth muscle f i b r e s  types o f a d r e n e r g i c r e c e p t o r s - - a l p h a  and  beta.  S t i m u l a t i o n of the alpha r e c e p t o r s causes c o n t r a c t i o n of smooth muscle and  s t i m u l a t i o n o f the beta r e c e p t o r s r e s u l t s i n r e l a x a t i o n  o f smooth muscle and an i n c r e a s e i n the r a t e and cardiac contractions.  30  strength of  However s t i m u l a t i o n of both the  alpha  31 and b e t a r e c e p t o r s i n i s o l a t e d r a b b i t duodenum  and I n t a c t  32 canine  ileum  results i n relaxation.  The  d i f f e r e n t i a t i o n of  these r e c e p t o r s has been made p o s s i b l e by the use  of drugs t h a t  s e l e c t i v e l y b l o c k or s t i m u l a t e e i t h e r type o f r e c e p t o r . DCI and The  nethalide d i c h l o r o analog  of i s o p r o t e r e n o l (DCI)  originally  33 d e s c r i b e d by Powell and S l a t e r  has been c h a r a c t e r i z e d by Morah  3k and P e r k i n s as an a d r e n e r g i c b l o c k i n g agent that s e l e c t i v e l y b l o c k s the beta adrenergic r e c e p t o r s . Isoproterenol s e l e c t i v e l y  32 a c t i v a t e s b e t a r e c e p t o r s thus i t s a c t i o n i s b l o c k e d by A c o n c e n t r a t i o n of DCI  DCI.  s u f f i c i e n t to b l o c k the a c t i o n of i s o -  p r o t e r e n o l on the r a b b i t i n t e s t i n e d i d not b l o c k the a c t i o n of IIDN or n i t r o g l y c e r i n .  (Figure 5)  N e t h a l i d e , a new  adrenergic  Figure 5.  Isolated rabbit duodenum.  Blocking action by 50 Tf $ D C I of 5  but not of 2 mg# IIDN ( i ) response.  W = wash.  isoproterenol (IP) response  22  b e t a r e c e p t o r b l o c k i n g compound, d i d not a l t e r the response to IIDN o f the i s o l a t e d r a b b i t i n t e s t i n e or the a n e s t h e t i z e d r a t blood pressure.  (Figure 6)  T h i s compound e x h i b i t s l e s s  intrinsic  sympathomimetic a c t i v i t y t h e r e f o r e f a c i l i t a t e s the demonstration 35 of the presence or absence of b l o c k i n g a c t i o n . The r e s u l t s o b t a i n e d w i t h DCI agree w i t h those r e c e n t l y r e p o r t e d by Abboud and E c k s t e i n . dog,  7 mg/kg o f DCI b l o c k e d  proterenol but  They found i n the  anesthetized  the vasodepressor ..action of i s o 36  d i d not e f f e c t the response to n i t r o g l y c e r i n .  Dibenzyline D i b e n z y l i n e , a cogener o f the o r i g i n a l a d r e n e r g i c haloalkylamine 3*7  b l o c k i n g agent, was  beta-  used as an a l p h a b l o c k i n g  38  agent. ' Phenylephrine was employed as the c o n t r o l drug 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 experiments as i t s e l e c t i v e l y 32 a c t i v a t e s alpha r e c e p t o r s .  The r e v e r s a l of the  p r e s s o r e f f e c t to a depressor  e f f e c t was  blood pressure  experiments.  adrenaline  used as a c o n t r o l i n the  In both cases b l o c k i n g doses o f  d i b e n z y l i n e had no e f f e c t on the n i t r a t e a c t i o n . D i b e n z y l i n e and a Beta B l o c k i n g Agent Since the i n h i b i t o r y e f f e c t o f a d r e n a l i n e on r a b b i t i n t e s t i n e could not be b l o c k e d Furchgott  isolated  e i t h e r by DCI  or dibenamine,  p o s t u l a t e d the presence of d e l t a r e c e p t o r s .  Activation  of these r e c e p t o r s 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 i n t a c t canine ileum A h l q u i s t and Levy antagonized  the e f f e c t s of a d r e n a l i n e w i t h a combination of 32 an alpha and a beta b l o c k i n g agent. F u r c h g o t t confirmed these  0.05 mg  Figure 6.  IP  N  0.25 *  7.5 mg/kg  Anesthetized rat blood pressure.  IP  0.25 *  0.05 mg  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  results  i n i s o l a t e d segments o f r a b b i t  investigate a l p h a and  of b l o c k i n g drugs.  b l o c k e d by a c o m b i n a t i o n  o f IIDN was (Figures (Figure  of the n i t r a t e  beta r e c e p t o r s experiments  both types was  the p o s s i b i l i t y  8)  31  To  esters acting  were p e r f o r m e d  Although  on  both  utilizing  the a c t i o n of a d r e n a l i n e  o f the b l o c k i n g agents,  not b l o c k e d i n e i t h e r  7 and  intestine.  the  the i s o l a t e d r a b b i t  or the a n e s t h e t i z e d r a t b l o o d  action  intestine  pressure.  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 o f He  suggested  like  t h a t the  a c t i o n , may  guanethidine.  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 -  account  f o r at l e a s t  p a r t of the a c t i o n  of  i|.0 guanethidine. agree and  Cass and  that guanethidine  a secondary  S p r i g g s and  possesses  reserpine-like  and Green b r e t y l i u m p r o b a b l y impulses  more r e c e n t w o r k e r s g e n e r a l l y  a primary b r e t y l i u m - l i k e a c t i o n  action.  a c t s by  According  impairing conduction  i n a d r e n e r g i c neurones w i t h consequent k3  a d r e n a l i n e and a c t i o n was  adrenaline release.  produced  by  exposing  The  nerve  has  the rabbit  mg% g u a n e t h i d i n e  its  intestine.  a c t i o n was  exposing  f o r n i n e t y minutes.  r e p o r t e d by K a d z i e l a w a rabbit  by  to a  T h i s dose  of  block  the  nor-  guanethidine con-  sympathetic kl,k-2  d e p l e t i o n of nor-adrenaline  S e c o n d a r y e f f e c t s were p r o d u c e d  of  of  intestine  been r e p o r t e d to completely  s t i m u l a t i o n w i t h no  failure  primary  c e n t r a t i o n o f 100*6$ f o r f i f t e e n m i n u t e s . guanethidine  to Boura  content.  intestine  T h i s treatment  has  to 1 been  t o d e p l e t e t h e c a t e c h o l amine c o n t e n t  T y r a m i n e was  b l o c k e d by  this  used  as t h e c o n t r o l  treatment.  As  drug  originally  of  and  Figure 7.  Isolated rabbit duodenum.  Blocking action by 50  response but not of 2 mg$ IIDN ( i ) response.  DCI and 100 t% dibenzyline (D) of 5 o# adrenaline(Ad)  W = wash.  w Ad 5  Figure 8.  D 100  x$  h - 20 min.  x2  ¥  DCI  x2  50 20 min.  Isolated rabbit duodenum.  10 min.  2 mg# 20 min.  Blocking action by 50 %$> DCI and 100  5 o*$ adrenaline (Ad) response but not of 2 mg# IIDN ( i ) response. W = wash.  Ad 5X#  dibenzyline ( D ) of No previous I given. §>  0.05 mg  Figure 9  0.5 "K  Anesthetized rat blood pressure.  5 mg/kg  10 mg/kg  0.05 mg  0.5 "*  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  s u g g e s t e d by  Burn and  Rand, and  now  generally  accepted,  tyramine  1{5,1}.6,1}.7 acts  i n d i r e c t l y by  releasing  catechol  amines.  Therefore  absence o f a tyramine response i n d i c a t e s c a t e c h o l However n e i t h e r  the  i d i n e b l o c k e d the intestine.  p r i m a r y nor  e f f e c t of  (Figures  10  and  the  the  amine  depletion.  secondary a c t i o n of  n i t r a t e s on  the  guaneth-  isolated rabbit  11)  Reserpine Burn and reserpine  Rand p r e s e n t e d t h e  acts  by  depleting  the  first  evidence i n d i c a t i n g  nor-adrenaline  content  of  tissues.  k9 The To  mechanism o f a c t i o n has further  releasing  i n v e s t i g a t e the catechol  intestinal pressure  strips  of  rats  13)  (Figure  50  response to  was  IIDN was  (Figures  c o n f i r m s the  nor-adrenaline  depletion  They f o u n d t h a t  M e l v i l l e and  type  the  i f anything, reserpine  blood  depressor 12  and the  of  action  13) reserpinized  catechol increase  sypmathomimetic consistent  pretreatment  responses to n i t r o g l y c e r i n .  on  tyramine  Varma i n i s o l a t e d r a b b i t  51 the  observed  b l o c k a d e produce an  T h e s e r e s u l t s o b t a i n e d w i t h IIDN a r e by  on  r e s p o n s e shown I n  means o f a d r e n e r g i c the  nitrates i n  In these animals the  unchanged.  adrenaline  further  various  those r e p o r t e d  increased  the  o r much r e d u c e d however t h e  the  the r e s p o n s e t o  amines.  action of  numerous w o r k e r s .  from r e s e r p i n i z e d r a b b i t s and  absent  Potentiation  in  amines, the  n i t r a t e esters  amines as  possible  of r e s e r p i n i z e d r a t s .  r e p o n s e was of the  b e e n c o n f i r m e d by  with  hearts.  somewhat  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.  2 mg#  Figure 8 .  T  90 min.  Isolated rabbit duodenum.  V  W  ¥  2 m  g  $  2 m  g  $  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  31  I 2 mg#  Figure 9.  W T  W  w I  mg# NORMAL RABBIT 2  2  Isolated rabbit intestine.  mg$  T mg# RESERPINIZED RABBIT 2  Comparison of 2 mg$ tyramine (T)  response and 2 mg$ IIDN ( i ) response i n normal and reserpinized ( 0 . 5 mg/kg) animals.  W = wash.  mm Hg  NORMAL RAT  Figure 13.  Anesthetized rat blood pressure.  RESERPINIZED RAT  Comparison of 5 J tyramine ( T ) , 0 . 0 5 * adrenaline (Ad) and 0 . 0 5 mg  IIDN ( i ) responses i n normal and reserpinized (2 mg/kg) animals.  ro  33  Diphenhydramine Although'it activate  the  r a t where i t was prevent  depressor  As  p r e v i o u s l y b e e n shown t h a t  histaminergic  confirmed with  not  has  the  receptors,  new  isomer.  i t was  T h i s was  vasodepressor response to  response  to histamine  n o t e d p r e v i o u s l y the  was  fact  i n t e s t i n e f u r t h e r suggests  adrenergic indicate and  the  support  receptors.  any  In fact  connection  adrenergic  as p r o p o s e d by  In t h e i r  n i t r o g l y c e r i n on even l a r g e  consumption,  the  (Figure  IIDN has  the  1I4.)  exerts i t s  developed i n  IIDN does n o t results  a c t i o n of the  did  act  on  the the  obtained nitrate  esters not  mechanism o f a c t i o n f o r n i t r o g l y c e r i n  Humphreys as  studies dog  d o s e s do  i n systolic  cardiogram which are  the  r e a s o n f o r the  effect  However t h e r e s u l t s u p o n w h i c h  of the  antiadrenergic  h e a r t , E c k s t e i n and not modify vigor,  induced  R e p e a t i n g Raab's work on t h e  by  i n heart  of adrenaline  and  co-workers  r a t e o r i n the  adrenergic  cat heart  f o u n d t h a t n i t r o g l y c e r i n does n o t  e f f e c t s of found  changes i n m y o c a r d i a l  o x y g e n c o n s u m p t i o n w h i c h Raab d i d n o t ,  effects  anesthetized  Humphreys b a s e t h i s mechanism have p r e v i o u s l y b e e n  questioned.  that  be  These r e s u l t s t h e r e f o r e would  of t h i s drug i n angina p e c t o r i s . Raab a n d  not  diphenhydramine  adrenaline  none o f t h e  receptors.  Raab a n d  that  between the  an a n t i a d r e n e r g i c  to  should  IIDN a l t h o u g h  blocked.  that  this  done i n t h e  shown t h a t p r e t r e a t m e n t w i t h  t y p i c a l r e s p o n s e when t a c h y p h y l a x i s rabbit  felt  n i t r a t e s do  and  a c t u a l l y measuring  P o p o v i c h and  53  electro-  stimulation.  significantly  1-arterenol.  oxygen  associates  neutralize  the  Figure 14.  Anesthetized 2  mg/kg  but  not  rat  blood  pressure.  Blocking action  diphenhydramine  (d)  of  of  (i)  response.  0.05  mg I I D N  histamine  (H)  by  response  35  SUMMARY AND  The nitrate rabbit The  p h a r m a c o l o g y and ester, isoidide  intestine  and  CONCLUSIONS  a p o s s i b l e mechanism o f a c t i o n o f a  dinitrate,  on t h e  anesthetized rat blood  f o l l o w i n g c o n c l u s i o n s were 1.  In both its  2.  A type the  preparations  isomers  ISDN o r  more p o t e n t  IIDN w h i c h was  isolated rabbit intestine  blocked  pressure.  than e i t h e r  of  IMDN.  of tachyphylaxis to  In the p r e p a r a t i o n s  isolated  reached:  IIDN was  anesthetized rat blood 3.  were s t u d i e d on t h e  new  was  not  prominent  observed  in  i n the  pressure.  employed, t h e a c t i o n o f IIDN was  not  by:  i ) Beta  adrenergic  b l o c k i n g a g e n t s s u c h as DCI  and  nethalide. 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  dibenzyline. iii)  A  combination  blocking [j..  Neither the guanethidine rabbit  5.  o f an a l p h a  and  a beta  adrenergic  agent.  primary blocked  nor the  the  secondary b l o c k i n g a c t i o n of  effect  o f IIDN on t h e  isolated  intestine.  Pretreatment catechol  o f the  animals  amines, d i d not  p r e p a r a t i o n to  IIDN.  with reserpine, to  alter  the r e s p o n s e o f  deplete either  36  6.  The a n t i h i s t a m i n e diphenhydramine d i d not b l o c k vasodepressor blood  7.  the  a c t i o n of IIDN on the a n e s t h e t i z e d r a t  pressure,  IIDN probably does not a c t on the a d r e n e r g i c  or  h i s t a r a i n e r g i c r e c e p t o r s o r the r e c e p t o r s upon which papaverine  acts.  However a c o n s i d e r a t i o n of f a c t o r s  . such as potency i n terms of s t r u c t u r e , and the  develop-  ment of t a c h y p h y l a x i s p o i n t s to a p o s s i b l e a c t i o n on some type of r e c e p t o r s u r f a c e . demonstration  A more p o s i t i v e  of an a c t i o n w i t h r e c e p t o r s as  definitive  as the a d r e n e r g i c or a n a l g e s i c r e c e p t o r s must i n v o l v e b l o c k i n g the a c t i o n of IIDN w i t h an a n t a g o n i s t , a c t i o n of which i s s p e c i f i c to these  drugs.  the  37 BIBLIOGRAPHY  1.  Goodman, L.S., and G i l m a n , A., of Therapeutics. 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