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Kinetics and cellular control mechanisms for imipramine metabolism in the isolated perfused rat liver Moldowan, Mervin John 1973

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KINETICS AND CELLULAR CONTROL MECHANISMS FOR IMIPRAMINE METABOLISM IN THE ISOLATED PERFUSED RAT LIVER by M. J . MOLDOWAN A t h e s i s submitted i n p a r t i a l f u l f i l l m e n t o f the requirements f o r the degree of DOCTOR OF PHILOSOPHY i n the D i v i s i o n of Pharmacology of the F a c u l t y of P h a r m a c e u t i c a l Sciences  We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA November 1972  In p r e s e n t i n g t h i s an  thesis  in p a r t i a l f u l f i l m e n t of  a d v a n c e d d e g r e e at t h e U n i v e r s i t y  the  Library  of  British  s h a l l make i t f r e e l y a v a i l a b l e  I f u r t h e r agree t h a t p e r m i s s i o n f o r s c h o l a r l y p u r p o s e s may  be g r a n t e d by  the  his  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  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  Date  Columbia, I agree r e f e r e n c e and  Head o f my  It i s understood that  permission.  Department  requirements  for extensive copying of  by  written  representatives.  for  the  Columbia  be  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  KINETICS AND CELLULAR CONTROL MECHANISMS FOR IMIPRAMINE METABOLISM IN THE ISOLATED PERFUSED RAT LIVER  by  M. J . MOLDOWAN  ABSTRACT An i n v e s t i g a t i o n was undertaken t o study t h e k i n e t i c s and p o s s i b l e c e l l u l a r c o n t r o l mechanisms f o r imipramine HCl metabolism 14 i n the i s o l a t e d p e r f u s e d r a t l i v e r . The i s o t o p e was used and q u a n t i f i c a t i o n was done by l i q u i d c o u n t i n g . A n a l y s i s f o r imipramine  C-imipramme scintillation  (IMI), desmethy1imipramine  (DMI), f r e e hydroxy (OH), g l u c u r o n i d e  (G) and N-oxide (N-0)  m e t a b o l i t e s was done on the p e r f u s a t e , b i l e and l i v e r . The r a t e o f IMI metabolism was found t o be dependent on two major enzymatic  r o u t e s , N-demethylation  (formation o£ DMI)  and aromatic h y d r o x y l a t i o n (formation of G, OH) o f imipramine and one minor enzymatic  route, N-oxidation  (N-0) . The r a t e o f  aromatic h y d r o x y l a t i o n of IMI was found t o be i n h i b i t e d a f t e r t h i r t y minutes, w i t h IMI c o n c e n t r a t i o n 2 X 10 "*M. This i n h i b i t i o n of aromatic h y d r o x y l a t i o n could n o t be detected i f the p e r f u s a t e h a l f - l i f e f o r IMI (t%=18.5 minutes) was the o n l y parameter monitored.  A f t e r i n c u b a t i o n p e r i o d s of f i f t e e n , t h i r t y and s i x t y  minutes t h e per cent o f imipramine  i n t h e l i v e r was 70, 75 and  XX  80 per cent and the remainder of IMI was i n the p e r f u s a t e . The -5 -5 -5 dose of IMI was v a r i e d (0.5 X 10 M, 1 X 10 M and 2 X 10 M) f o r metabolism by the p e r f u s e d r a t l i v e r . The i n c u b a t i o n time was kept constant at f i f t e e n minutes. The r a t e of imipramine metabolism (formation o f DMI and GOH)  followed f i r s t order k i n e t -5 -5 i c s when the dose of IMI was 0.5 X 10 M or 1 X 10 M. I n c r e a s i n g -5  the dose of IMI t o 2 X 10  M s l i g h t l y suppressed the f o r m a t i o n  o f DMI and the f o r m a t i o n of GOH  f o l l o w e d zero o r d e r k i n e t i c s .  I t was found t h a t the endogenous DMI formed from IMI metabol i s m i n h i b i t e d the f o r m a t i o n of GOH  a f t e r f i f t e e n minutes and  t h i r t y minutes of IMI metabolism as shown by the f o l l o w i n g r e s u l t s . DMI  (1.65, 3.33, 6.66 o r 13.32 X 10  -6  M) was p r e i n c u b a t e d p r i o r -6 to a d d i t i o n of IMI. DMI (1.65 o r 3.33 X 10 M) was found t o s p e c i f i c a l l y i n h i b i t aromatic h y d r o x y l a t i o n of IMI. Higher conc e n t r a t i o n of DMI  (6.66 or 13.32 X 10  -6  M) i n h i b i t e d the f o r m a t i o n  of GOH and DMI. E t h y l a l c o h o l (1 mM) p r e i n c u b a t e d p r i o r t o a d d i t i o n -5 of 1 X 10 M of IMI s p e c i f i c a l l y i n h i b i t e d DMI f o r m a t i o n . No i n h i b i t i o n of GOH o c c u r r e d . E t h y l a l c o h o l (1 mM)  caused i n h i b i t i o n  of f o r m a t i o n of DMI from IMI metabolism when the dose of IMI was -5 2 X 10  M. The i n c u b a t i o n time f o r IMI metabolism was  fifteen  and s i x t y minutes. With t h i s decrease o f DMI f o r m a t i o n , the formation of GOH  i n c r e a s e d a f t e r f i f t e e n * or s i x t y minutes  of i n c u b a t i o n time. From these experiments i t was concluded t h a t suppression of aromatic h y d r o x y l a t i o n of imipramine was due t o the formation of endogenous DMI formed from IMI metabolism. Optimal c o n d i t i o n s were found t o study p o s s i b l e c e l l u l a r  Ill  c o n t r o l mechanisms f o r IMI metabolism i n the i s o l a t e d p e r f u s e d -5  r a t l i v e r . The dose o f IMI was 1 X 10  M and the i n c u b a t i o n -6  time was f i f t e e n minutes. D i b u t y r y l c y c l i c AMP (2 X 10  M) caused  i n h i b i t i o n o f IMI metabolism. DMI f o r m a t i o n was i n h i b i t e d 28 p e r cent w h i l e GOH f o r m a t i o n was i n h i b i t e d 29 p e r cent. NADPH (1.1 X -6 -6 10 M) o r NADH (1.3 X 10 M) was found t o i n h i b i t imipramine metabolism. GOH and DMI were both i n h i b i t e d . S u c c i n i c a c i d -3 (1.6 X 10 M) was found t o i n h i b i t DMI f o r m a t i o n b u t n o t GOH.  S i g n a t u r e s o f Examiners  iv  TABLE OF CONTENTS Page ABSTRACT  i  LIST OF TABLES  v i i  LIST OF FIGURES  x  INTRODUCTION  1  I s o l a t e d Perfused Rat L i v e r Technique  i  Microsomal Drug Metabolism  2  Imipramine Metabolism  5  Co-Factors Necessary f o r Drug Metabolism  IQ  Magnesium  IQ  Dihydronicotinamide  j.1  Hormones and Drug Metabolism MATERIALS AND METHODS L i v e r P e r f u s i o n Techniqe and Apparatus  13 15 . 15  S u r g i c a l Procedure  15  Perfusion F l u i d  17  V i a b i l i t y of the L i v e r  19  Lactate-Pyruvate  19  Magnesium A n a l y s i s  20  E x t r a c t i o n of Imipramine and M e t a b o l i t e s  20  Reagents  21  E x t r a c t i o n Procedure  22  Thin Layer Chromatography  25  Q u a n t i f i c a t i o n of E x t r a c t e d Imipramine and M e t a b o l i t e s L i q u i d S c i n t i l l a t i o n .Counter, S e t t i n g s and Quench Corrections  26 27  Procedures f o r Counting and C a l c u l a t i o n o f Unknown Samples Comparison Between E x t e r n a l and I n t e r n a l Quench C o r r e c t i o n s E x t r a c t i o n S p e c i f i c i t y and E f f i c i e n c y f o r Imipramine and M e t a b o l i t e s Imipramine De smethy1imip ramine Chromatography o f Imipramine and Desmethyl imipramine 2-hydroxydesmethylimipramine and Imipramine N-oxide 2-hydroxydesmethylimipramine Imipramine N-oxide S t a b i l i t y of Imipramine RESULTS AND DISCUSSION K i n e t i c s o f Imipramine Metabolism i n the I s o l a t e d P e r f u s e d Rat L i v e r Metabolism o f Imipramine i n t h e I s o l a t e d P e r f u s e d L i v e r A f t e r V a r i o u s I n c u b a t i o n Times Perfusate Concentration Rate o f Metabolism and D i s t r i b u t i o n Metabolism of Imipramine HCl a t V a r i o u s Concentrat i o n s o f Imipramine Rate o f Metabolism and D i s t r i b u t i o n E f f e c t o f Desmethylimipramine on Aromatic Hydroxyl a t i o n and N-demethylation o f Imipramine Distribution  E f f e c t of E t h y l A l c o h o l on Aromatic H y d r o x y l a t i o n and N-demethylation Distribution D i s c u s s i o n o f Imipramine Metabolism C e l l u l a r C o n t r o l Mechanisms f o r Imipramine M e t a b o l i E f f e c t o f Magnesium on Imipramine Metabolism E f f e c t o f D i b u t y r y l C y c l i c AMP and Glucagon on Imipramine Metabolism E f f e c t o f NADH, NADPH o r S u c c i n i c A c i d on Imipramine Metabolism D i s c u s s i o n o f C e l l u l a r C o n t r o l Mechanisms f o r Imipramine Metabolism SUMMARY AND CONCLUSIONS BIBLIOGRAPHY  V l l  LIST OF TABLES Page  Table I  Rf Values f o r Imipramine and I t s Known Metabolites.  II  26  Comparison o f Per Cent Counting E f f i c i e n c y  III  IV V VI VII  VIII  Between I n t e r n a l Standard and E x t e r n a l Standard Methods.  30  Q u a n t i f i c a t i o n o f Desmethylimipramine and Imipramine by S o l v e n t E x t r a c t i o n and T h i n Layer Chromatography.  34  S o l v e n t E x t r a c t i o n o f Imipramine and I t s Major M e t a b o l i t e s from Aqueous S o l u t i o n s .  39  S t a b i l i t y o f Imipramine  41  S t a b i l i t y and P e r Cent Recovery of Imipramine With and Without Red Blood C e l l s i n S o l u t i o n  42  C a l c u l a t e d and E x p e r i m e n t a l Imipramine Remaining A f t e r F i f t e e n , T h i r t y and S i x t y -5 Minutes o f Imipramine (2 X 10 M) Metabolism. The Average P e r Cent Metabolism and D i s t r i b u t i o n o f Imipramine and M e t a b o l i t e s . The I n c u b a t i o n Time was F i f t e e n , T h i r t y and S i x t y M i n u t e s , With Imipramine HC1 2 X 1 0 M . _5  IX  X  XI  The T o t a l Q u a n t i t y o f Imipramine Remaining and M e t a b o l i t e s Formed. The I n c u b a t i o n Time was F i f t e e n , T h i r t y and S i x t y Minutes f o r Imipramine 2 X 10 M. * E f f e c t s o f Imipramine C o n c e n t r a t i o n on Formation o f M e t a b o l i t e s . The I n c u b a t i o n Time was F i f t e e n Minutes.  50  52  53  57  E f f e c t of Desmethylimipramine on t h e D i s t r i b u t i o n of Imipramine and M e t a b o l i t e s . Imipramine HC1, 0.5 X 10-5M; Incubation Time F i f t e e n Minutes.  68  V l l l  Table XII  XIII  XIV  XV  Page E f f e c t of Desmethylimipramine on t h e D i s t r i b u t i o n o f Imipramine and M e t a b o l i t e s . -5 Imipramine HC1 1 X 10 M; I n c u b a t i o n Time F i f t e e n Minutes. E f f e c t of Desmethylimipramine on t h e D i s t r i b u t i o n of Imipramine and M e t a b o l i t e s . -5 Imipramine HC1, 2 X 10 M; I n c u b a t i o n Time f o r F i f t e e n Minutes. E f f e c t o f E t h y l A l c o h o l on Imipramine Metabolism With the I s o l a t e d P e r f u s e d Rat L i v e r ; F i f t e e n Minutes I n c u b a t i o n , 1 X 10 was t h e S u b s t r a t e C o n c e n t r a t i o n .  XVIII  E f f e c t o f E t h y l A l c o h o l on Imipramine Metabolism With the I s o l a t e d P e r f u s e d Rat L i v e r . The I n c u b a t i o n Time was F i f t e e n and S i x t y M i n u t e s , S u b s t r a t e C o n c e n t r a t i o n was 2 X 10~ M.  79  E f f e c t o f E t h y l A l c o h o l on the D i s t r i b u t i o n of Imipramine and M e t a b o l i t e s . Imipramine HC1 -5 (1 X 10 M); I n c u b a t i o n Time F i f t e e n Minutes. E f f e c t of E t h y l A l c o h o l on the D i s t r i b u t i o n of Imipramine and M e t a b o l i t e s . Imipramine HCl (0.5 X 1 0 M ) , I n c u b a t i o n Time F i f t e e n Minutes.  81  E f f e c t o f Magnesium on Imipramine Metabolism i n the I s o l a t e d P e r f u s e d Rat L i v e r . The I n c u b a t i o n Time was F i f t e e n Minutes and the S u b s t r a t e C o n c e n t r a t i o n was 1 X 10 ~*M.  90  _5  XIX  74  77  5  XVII  70  E f f e c t of E t h y l A l c o h o l on Imipramine Metabolism With the I s o l a t e d P e r f u s e d Rat L i v e r ; F i f t e e n Minutes I n c u b a t i o n With 0.5 X 10~ M Imipramine. 5  XVI  69  80  xx  Page  Table XX  E f f e c t of D i b u t y r y l C y c l i c AMP o r Glucagon on Imipramine Metabolism. I n c u b a t i o n Time was f o r F i f t e e n Minutes With t h e I s o l a t e d P e r f u s e d Rat L i v e r . The Dose o f Imipramine was 0.5 X 10 ^M o r 1 X 10~ M.  92  5  XXI  E f f e c t o f Glucagon o r D i b u t y r y l  Cyclic  AMP on the D i s t r i b u t i o n o f Imipramine and M e t a b o l i t e s . Imipramine was Incubated f o r F i f t e e n Minutes With the I s o l a t e d P e r f u s e d Rat L i v e r . XXII  94  E f f e c t of NADH, NADPH o r S u c c i n i c A c i d on -5  XXIII  Imipramine Metabolism (1 X 10 M). The I n c u b a t i o n Time was f o r F i f t e e n Minutes.  96  The E f f e c t of NADH, NADPH o r S u c c i n i c A c i d on t h e D i s t r i b u t i o n o f Imipramine and M e t a b o l i t e s . Imipramine (1 X 10 M) was Incubated f o r F i f t e e n Minutes.  97  X  LIST OF FIGURES Page  Figure 1.  Mechanism o f cytochrome P-450 enzyme systems.  4  2.  Microsomal metabolism o f imipramine.  6  3.  P e r f u s a t e c o n c e n t r a t i o n o f imipramine and m e t a b o l i t e s . The dose o f imipramine was 2 X 10~ M.  46  Semi l o g r i t h m i c p l o t of p e r f u s a t e imipramine c o n c e n t r a t i o n versus time. Imipramine metabolism (2 X 10~ M).  48  The t o t a l f o r m a t i o n of m e t a b o l i t e s a f t e r imipramine metabolism f o r f i f t e e n minutes w i t h the p e r f u s e d r a t l i v e r .  60  5  4.  5  5.  6.  E f f e c t o f desmethylimipramine on the t o t a l f o r m a t i o n o f glucuromide, f r e e hydroxy and N-oxide m e t a b o l i t e s o f imipramine. Desmethylimipramine was p r e i n c u b a t e d f i v e minutes p r i o r -5 -5 to a d d i t i o n o f imipramine 0 . 5 X 1 0 M, 1 X 10 M and 2 X 10 M. I n c u b a t i o n f o r imipramine metabolism 64  was f i f t e e n minutes. 7.  8.  E f f e c t of desmethylimipramine on t h e t o t a l f o r m a t i o n o f g l u c u r o n i d e , f r e e hydroxy o r N-oxide m e t a b o l i t e s o f imipramine. The i n c u b a t i o n time was f i f t e e n minutes f o r -5 imipramine metabolism (1 X 10 M). Desmethylimipramine was p r e i n c u b a t e d f i v e minutes p r i o r t o imipramine metabolism. E f f e c t of desmethylimipramine on endogenous f o r m a t i o n o f desmethylimipramine from imipramine metabolism. Desmethylimipramine was p r e i n c u b a t e d f i v e minutes p r i o r t o a d d i t i o n of- imipramine 0.5 X 10~ M, 1 X 10~ M and 2 X 10~ M. I n c u b a t i o n time was f i f t e e n minutes 5  5  65  5  66  XI  ACKNOWLEDGEMENT  Dr. G. D. B e l l w a r d ' s guidance and p e r s o n a l encouragement was o f g r e a t a s s i s t a n c e t o t h e author.  The author wishes t o express h i s a p p r e c i a t i o n t o t h e f o l l o w i n g people:  Mr. B. V i r g o and Mrs. C. Moldowan who so generously  donated  t h e i r time f o r a s s i s t a n c e i n t h i s work.  Miss Joan Beedle and Mr. K. Grunenberg who chose t o a s s i s t in this project.  Canadian Red Cross f o r p r o v i s i o n of s u p p l i e s and Ciba-Geigy f o r s u p p l i e s o f chemicals.  DEDICATED to  C a r o l , Sharleen and Brent who made i t worthwhile.  1  INTRODUCTION  The purpose o f t h i s r e s e a r c h was t o study t h e k i n e t i c s o f imipramine metabolism and p o s s i b l e c e l l u l a r c o n t r o l mechanisms i n v o l v e d i n the metabolism o f drugs by the i s o l a t e d p e r f u s e d rat  liver.  I s o l a t e d P e r f u s e d Rat L i v e r Technique  The use o f microsomal f r a c t i o n s i s o l a t e d from t h e l i v e r has demonstrated a t l e a s t f o u r v a r i a b l e s which can change the r a t e of drug metabolism. These were magnesium, NADPH ( a b b r e v i a t i o n f o r d i h y d r o n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate) and NADH ( a b b r e v i a t i o n f o r d i h y d r o n i c o t i n a m i d e adenine d i n u c l e o t i d e ) c o n c e n t r a t i o n as w e l l as microsomal drug i n d u c t i o n . The l i m i t a t i o n t o these s t u d i e s has been t h e i n h e r e n t a r t i f i c i a l i t y o f i n v i t r o techniques. The second w i d e l y used technique i n the study o f drug met a b o l i s m was the use o f the whole a n i m a l . The l i m i t a t i o n t o t h i s approach was t h a t due t o m u l t i p l e v a r i a b l e s , t h e c e l l u l a r  factors  i n v o l v e d i n r e g u l a t i o n o f drug metabolism c o u l d n o t be c o m p l e t e l y e v a l u a t e d . I t was d i f f i c u l t t o e s t i m a t e the dose of each drug to which t h e l i v e r was exposed because such v a r i a b l e s as p r e f e r e n t i a l t i s s u e accumulation, metabolic a l t e r a t i o n , excretion by o t h e r organs o r l i v e r b l o o d f l o w can n o t be c o n t r o l l e d . These  2  disadvantages are e l i m i n a t e d when the i s o l a t e d p e r f u s e d l i v e r i s used as the experimental The perfused  rat  model.  l i v e r appeared t o be a s a t i s f a c t o r y p h y s i o -  l o g i c a l p r e p a r a t i o n by a number of c r i t e r i a  ( 1 ) , such as,  the  h i s t o l o g i c a l appearance, the oxygen consumption and the r a t e of b i l e p r o d u c t i o n . Moreover, the contents of m e t a b o l i c mediates i n freeze-clamped perfused t o those obtained  inter-  r a t l i v e r s were very s i m i l a r  in livers in situ.  In summary, the i n v i t r o s t u d i e s c o n s i s t e d of u s i n g  isolat-  ed l i v e r microsomal f r a c t i o n s w i t h v a r i o u s c o - f a c t o r s added; these c o - f a c t o r s were r e q u i r e d f o r o p t i m a l drug metabolism. I t i s not known to what e x t e n t these c o - f a c t o r s c o n t r o l the r a t e of r e a c t i o n i n the i n t a c t l i v e r c e l l or the i s o l a t e d p e r f u s e d rat l i v e r . The v a r i o u s f a c t o r s which i n f l u e n c e i n v i v o drug metabolism are  t i s s u e b i n d i n g , plasma d i s t r i b u t i o n of drugs w i t h i n the  organism and drug i n d u c t i o n . Since the l i v e r i s the prime organ which transforms drugs i n t o m e t a b o l i t e s , i t i s important t o f i n d out what f a c t o r s a l t e r h e p a t i c c e l l drug d i s t r i b u t i o n and  me-  tabolism. Microsomal Drug Metabolism  B i o l o g i c a l o x i d a t i o n c a t a l y z e d by the microsomal enzyme system i n c l u d e s a wide range of r e a c t i o n s , most o f which may a s c r i b e d to one common mechanism namely h y d r o x y l a t i o n  be  ( 2 ) . The  3 components p r e s e n t i n the microsomes which are  necessary f o r  drug metabolism have been determined by s p e c t r o p h o t o m e t r i c techniques which were s i m i l a r t o the technique used t o study the components o f the m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t c h a i n . I t was found t h a t l i v e r cytochrome P-450 redox system i n the microsomes was r e s p o n s i b l e f o r most of the drug metabolism The pigment cytochrome P-450 i n i t s reduced form r e a d i l y  (3,4).  combines  w i t h carbon monoxide t o form a complex, h a v i n g a maximum absorpt i o n at 450 nm and a minimum a t 405 nm. For t h i s reason i t has been named P-450 ( 5 ) . I n the absence of carbon monoxide, but i n the  presence of a i r , cytochrome P-450 must e x i s t m a i n l y i n the  o x i d i z e d form or as a complex w i t h oxygen. T h i s pigment i s a l s o p r e s e n t i n a d r e n a l microsomes  and m i t o c h o n d r i a and i n mi-  crosomes of the k i d n e y , lung and i n t e s t i n a l mucosa where i t may p l a y a r o l e i n metabolism of s p e c i f i c agents. The proposed e l e c t r o n f l o w pathway, cytochrome P-450 redox system, f o r most h y d r o x y l a t i o n i n l i v e r microsomes  i s shown i n  F i g u r e 1 (6,7). In the metabolism o f drugs NADPH serves as an e l e c t r o n donor f u n c t i o n i n g v i a a r e s p i r a t o r y chain d i r e c t t o cytochrome P-450. A t one time i t was proposed t h a t the reduced cytochrome P-450 r e a c t e d w i t h m o l e c u l a r oxygen t o form an " a c t i v e oxygen" complex. T h i s a c t i v e oxygen was^ then t r a n s f e r r e d t o the drug s u b s t r a t e which r e s u l t e d i n h y d r o x y l a t i o n of a drug ( 8 ) . However, i t has been shown t h a t the drug s u b s t r a t e f i r s t formed a complex w i t h the o x i d i z e d form of cytochrome P-450 (8,9). T h i s complex was then reduced t o form reduced cytochrome P-450 sub-  4  s t r a t e complex which r a p i d l y combined w i t h m o l e c u l a r oxygen t o form an C^-cytochrome P-450 s u b s t r a t e complex. T h i s q u i c k l y decomposed t o form t h e h y d r o x y l a t e d s u b s t r a t e and t h e o x i d i z e d form of t h e cytochrome P-450. A c c o r d i n g t o t h i s view, t h e r a t e l i m i t i n g step was t h e r e d u c t i o n o f the complex o f the s u b s t r a t e and t h e o x i d i z e d cytochrome. I t was found t h a t NADH c o u l d s t i m u l a t e drug metabolism f u r t h e r even when NADPH was p r e s e n t i n s a t u r a t i n g c o n d i t i o n s (10). To e x p l a i n t h i s e f f e c t NADH must a c t through a NADH-flavoprotein o r cytochrome b5 t o donate t h e +2  second e l e c t r o n i n t h e r e d u c t i o n of cytochrome P-450 "^2 ^~ s t r a t e complex. I t seems t h a t NADPH donates the f i r s t e l e c t r o n su  +3  t o cytochrome P-450  s u b s t r a t e complex through NADPH cytochrome  c r e d u c t a s e . Then cytochrome b5 o r NADH-flavoprotein (11) o r NADPH cytochrome c reductase can donate t h e second e l e c t r o n +3  Cytochrome P-450 j Substrate  Cytochrome P-450  -O  Cytochrome P-450 -O Substrate +3  H„0  Cytochrome b5? eNADPH Cyt c Reductase? NADH-flavoprotein? Figure 1  Mechanism o f cytochrome P-450 enzyme systems.  (9,10).  5 Therefore NADPH p l a y s an important r o l e i n microsomal drug metabolism. I t i s n o t known whether t h e f o r m a t i o n o f NADPH i s the  r a t e l i m i t i n g step i n t h e h e p a t i c c e l l s . I t was found t h a t  NADH c o u l d p l a y a minor r o l e i n microsomal drug metabolism, however i t i s n o t known t o what e x t e n t t h i s c o - f a c t o r can i n f l u ence drug metabolism i n t h e i n t a c t c e l l .  Imipramine Metabolism  The a n t i d e p r e s s a n t drug, imipramine, i s one o f a few drugs whose metabolism i s comprehensively known i n t h e r a t (12,13,14) and i n man (15, 16). The m a j o r i t y o f imipramine metabolism o c c u r r e d i n t h e l i v e r due t o t h e microsomal enzyme system r e q u i r i n g NADPH and m o l e c u l a r oxygen. The m e t a b o l i t e s formed from 2 umolar i m i p ramine i n c u b a t e d w i t h r a t microsomal enzymes were (14)t  amount  Q. ~6  ( s u b s t r a t e 100%)  Imipramine  35  Desmethylimipramine  45  2-hydroxyimipramine  10  Imipramine N-oxide  5  2-hydroxydesmethylimipramine  3  Iminodibenzyl  2  The p r i n c i p a l enzymatic r e a c t i o n f o r the d e g r a d a t i o n o f imipramine was N-demethylation w h i l e aromatic hydroxylase and  6  N-oxidase were minor enzymatic r e a c t i o n s . The primary m e t a b o l i t e s of imipramine were 2-hydroxyimipramine, desmethylimipramine and imipramine N-oxide  (Figure 2 ) . I t t h e r e f o r e appeared t h a t i m i p -  ramine metabolism would be v e r y s u i t a b l e t o study f o r the f o l l o w i n g reasons: t h r e e enzymatic r e a c t i o n s , aromatic h y d r o x y l a t i o n , N - o x i d a t i o n and N-demethylation, can be s t u d i e d from t h e same s u b s t r a t e ; and the m e t a b o l i c products a r e s i m i l a r i n r a t and man. Experiments were designed t o study the f a c t o r s which a f f e c t the t h r e e enzymatic r e a c t i o n s and f i n d out i f more than one microsomal enzyme o r r a t e l i m i t i n g s t e p would be i n v o l v e d i n t h e metabolism of imipramine.  CH  3  CH  H  3  2-hydroxyimipramine  CH  CH  3  Desmethylimipramine  3  Imipramine  Figure 2 Microsomal metabolism o f imipramine-  N-oxide  7  The microsomal enzymatic metabolism o f imipramine can be i n f l u e n c e d by many f a c t o r s . I t was found t h a t magnesium concent r a t i o n , imipramine c o n c e n t r a t i o n and SKF 525A ( a b b r e v i a t i o n f o r 2 - d i e t h y l a m i n o e t h y l - 2 , 2 - d i p h e n y l v a l e r a t e HC1) had d i f f e r e n t e f f e c t s on t h e t h r e e enzymatic r e a c t i o n s i n v o l v e d i n imipramine metabolism (14,17,18,19). I n c r e a s i n g the imipramine c o n c e n t r a t i o n t o 1 mM i n c r e a s e d the v e l o c i t y o f N-demethylation t o a maximum; however, a f u r t h e r i n c r e a s e i n imipramine c o n c e n t r a t i o n to 2 mM decreased t h e v e l o c i t y o f N-demethylation. I n c r e a s i n g the imipramine c o n c e n t r a t i o n t o 2 mM d i d n o t i n h i b i t N - o x i d a t i o n ; however, aromatic h y d r o x y l a t i o n was i n h i b i t e d t o a s m a l l e x t e n t . The a d d i t i o n o f magnesium t o the microsomal enzyme f r a c t i o n (100,000 g and 9,000 g) i n c r e a s e d the r a t e o f N-demethylation of imipramine and i n h i b i t e d N - o x i d a t i o n o f imipramine (18). The r a t e o f aromatic h y d r o x y l a t i o n was u n a f f e c t e d (16). SKF 525A was found t o i n h i b i t N-demethylation and aromatic h y d r o x y l a t i o n but not N - o x i d a t i o n . P h e n o b a r b i t a l pretreatment was found t o induce microsomal N-demethylation and aromatic h y d r o x y l a t i o n o f imipramine b u t not N - o x i d a t i o n (16,18). I t t h e r e f o r e seems t h a t N - o x i d a t i o n was c o n t r o l l e d by a d i f f e r e n t r a t e l i m i t i n g step o r metabolized by a d i f f e r e n t enzyme. I t a l s o seems t h a t aromatic hydroxylase and N-demethylation were a l s o c o n t r o l l e d by d i f f e r e n t enzymes o r r a t e l i m i t i n g steps s i n c e the r a t e o f f o r m a t i o n o f desmethylimipramine and 2-hydroxyimipramine was d i f f e r e n t , the degree o f i n h i b i t i o n by SKF 525A was d i f f e r e n t and magnesium had a d i f f e r e n t e f f e c t on the two r e a c t i o n s . The enzymatic aromatic h y d r o x y l a t i o n o f imipramine i n  8 microsomal p r e p a r a t i o n s from r a t l i v e r was v e r y low compared to i t s a c t i v i t y i n v i v o  i n the r a t (13) and humans (16). The  major e x c r e t o r y products of imipramine were h y d r o x y l a t e d p r o ducts of the drug. Imipramine and desmethylimipramine were not e x c r e t e d to a s i g n i f i c a n t degree. From these experiments i t can be seen t h a t microsomal p r e p a r a t i o n s had lower enzymatic a c t i v i t y f o r aromatic h y d r o x y l a t i o n o f imipramine. The i s o l a t e d p e r f u s e d r a t l i v e r may be more r e p r e s e n t a t i v e of aromatic h y d r o x y l a t i o n i n v i v o f o r the f o l l o w i n g reasons. The i s o l a t e d r a t l i v e r p e r f u s i o n technique was used t o study imipramine metabolism and i t was found t h a t 47 per cent of the dose o f imipramine i n t h r e e hours was m e t a b o l i z e d t o h y d r o x y l a t e d products (20). In the same study 3 8 per cent o f the dose of desmethylimipramine i n t h r e e hours was m e t a b o l i z e d t o h y d r o x y l a t e d m e t a b o l i t e s . However, only 2.5 per cent o f the dose of desmethylimipramine was metabo l i z e d i n one hour w i t h the microsomal f r a c t i o n from the r a t liver  (12). I t was a l s o found t h a t microsomal enzyme p r e p a r a t i o n s  c o u l d not h y d r o x y l a t e d-amphetamine, however, the p r e p a r a t i o n c o u l d N-demethylate aminopyrine and h y d r o x y l a t e h e x o b a r b i t a l (21). I t was found t h a t the i s o l a t e d p e r f u s e d l i v e r d i d h y d r o x y l a t e d-amphetamine. There are very few p u b l i s h e d r e p o r t s i n the l i t e r a t u r e which study the e f f e c t of the s u b s t r a t e c o n c e n t r a t i o n on the r a t e of drug metabolism. T h i s i s p a r t i c u l a r l y of i n t e r e s t  be-  cause of some r e p o r t s which suggest t h a t plasma h a l f - l i f e f o r drugs i n v i v o was dose dependent. Some of these drugs were s t r e p t o -  9 mycin  (22) , dicumarol (23) , d i p h e n y l h y d a n t o i n (24) and p r o b e n i -  c i d (25) . Dicumarol (26) and d i p h e n y l h y d a n t o i n (27) p e r f u s i o n h a l f - l i f e was a l s o found t o be dose dependent w i t h t h e i s o l a t e d p e r f u s e d r a t l i v e r technique. Dose dependent k i n e t i c s may mean t h a t the r a t e o f f o r m a t i o n o f some m e t a b o l i t e s i s changing. Reasons f o r t h e r a t e o f change i n drug metabolism c o u l d be t h a t the enzyme r e s p o n s i b l e f o r metabolism o f a drug approaches  sat-  u r a t i o n o r as the dose i n c r e a s e s the f r a c t i o n o f the drug g o i n g t o the l i v e r c o u l d be l e s s , as demonstrated w i t h d i c u m a r o l (26). Before any experiments are designed t o study v a r i o u s e f f e c t s on drug metabolism the k i n e t i c s o f drug metabolism should be f u l l y understood i n order t o choose t h e proper s u b s t r a t e concent r a t i o n . I n most s t u d i e s o f drug metabolism i n v i v o o r i n t h e i s o l a t e d p e r f u s e d r a t l i v e r t h e dose o f t h e drug was u s u a l l y decided upon a r b i t r a r i l y and not v a r i e d . The p r i n c i p a l m e t a b o l i t e formed from imipramine was desmethylimipramine i n t h e r a t i n v i v o (13) and i n i s o l a t e d r a t l i v e r p e r f u s i o n s t u d i e s (20). I n these s t u d i e s o n l y one dose o f imipramine was used. U t i l i z a t i o n o f r a t l i v e r microsomes and v a r y i n g dosages o f imipramine (12, 19) showed t h a t N-demethylation was the predominant enzymatic r e a c t i o n w i t h imipramine. Experiments were done t o determine whether v a r i o u s s u b s t r a t e c o n c e n t r a t i o n s o f imipramine would a l t e r i t s metabolic p a t t e r n i n the i s o l a t e d perfused r a t l i v e r .  10  Co-Factors Necessary f o r Drug Metabolism  Magnesium  This i o n i s  necessary f o r maximum i n v i t r o  microsomal  drug metabolism. Magnesium c o n c e n t r a t i o n , above a c o n c e n t r a t i o n of 2 mM, which c o n t r i b u t e d t o maximum N-demethylation a c t i v i t y , r e s u l t e d i n a decrease o f imipramine metabolism (12). When magnesium c o n c e n t r a t i o n was reduced below 2 mM t h e r e was a decrease i n N-demethylation enzymatic a c t i v i t y and t h e r e f o r e a decrease i n drug metabolism. I n a r e c e n t p u b l i c a t i o n (28) i t was demons t r a t e d , t h a t a t magnesium c o n c e n t r a t i o n up t o a maximal enzymatic a c t i v i t y , cytochrome P-450 reductase was r a t e f o r benzphetamine  limiting  and a n i l i n e metabolism. Since imipramine me-  t a b o l i s m was dependent on magnesium i t c o u l d be t h a t magnesium caused an i n c r e a s e i n cytochrome P-450 reductase and t h e r e f o r e an i n c r e a s e i n metabolism. Magnesium c o n c e n t r a t i o n up t o 5 mM caused an i n c r e a s e i n cytochrome P-450 reductase a c t i v i t y along w i t h an i n c r e a s e i n NADPH oxidase and NADPH-cytochrome c r e d u c t ase a c t i v i t y . The enzymatic a c t i v i t y o f NADPH o x i d a s e was d e t e r mined by the t o t a l decrease i n NADPH c o n c e n t r a t i o n i n the m i c r o somal i n c u b a t i o n mix. This i n c r e a s e i n enzymatic a c t i v i t y  result-  ed i n an i n c r e a s e i n drug metabolism s i n c e there was an i n c r e a s e i n enzymatic a c t i v i t y which was r a t e l i m i t i n g . Higher concentrat i o n s o f magnesium (5 mM) f u r t h e r i n c r e a s e d microsomal NADPHcytochrome c and cytochrome P-450 reductase a c t i v i t y but decreased  11  NADPH o x i d a s e a c t i v i t y . Under t h i s c o n d i t i o n drug metabolism decreased f o r c e r t a i n drugs such as h e x o b a r b i t a l and a p p a r e n t l y NADPH o x i d a s e became r a t e l i m i t i n g f o r the e l e c t r o n t r a n s p o r t i n g system. Higher magnesium c o n c e n t r a t i o n s r e s u l t e d i n a f u r t h e r decrease i n NADPH o x i d a s e a c t i v i t y which i n t u r n r e s u l t e d i n a decrease o f drug metabolism. S i n c e imipramine metabolism (12) was i n h i b i t e d by h i g h e r magnesium c o n c e n t r a t i o n i t may w e l l be t h a t NADPH oxidase was l i m i t i n g imipramine metabolism. In the i n t a c t c e l l s o f a l i v e r one should.be a b l e t o demonstrate the above h y p o t h e s i s . I f magnesium c o n c e n t r a t i o n i n the l i v e r o f an animal was lower than r e q u i r e d f o r maximal c y t o chrome P-450 reductase a c t i v i t y , then the r a t e l i m i t i n g r e a c t i o n should be cytochrome P-450 r e d u c t a s e . Increases i n l i v e r magnesium c o n c e n t r a t i o n s should i n c r e a s e drug metabolism t o maximum i f cytochrome P-450 reductase was r a t e l i m i t i n g , f u r t h e r i n c r e a s e s i n l i v e r magnesium s h o u l d decrease drug metabolism. T h i s decrease i n drug metabolism would occur i f NADPH o x i d a s e a c t i v i t y was i n h i b i t e d and imipramine metabolism was dependent on i t s a c t i v i t y . To d e r i v e some i n f o r m a t i o n on the d i r e c t e f f e c t o f magnesium on t h e drug m e t a b o l i z i n g enzymes, i n the r e s e a r c h f o r t h i s t h e s i s the i s o l a t e d perfused r a t l i v e r was s u b j e c t e d t o an excess o r a d e f i c i e n c y of magnesium i n the p e r f u s i o n medium.  Dihydronicotinamide Adenine D i n u c l e o t i d e Phosphate  In a d d i t i o n t o s u i t a b l e magnesium c o n c e n t r a t i o n , excess  12 NADPH was r e q u i r e d f o r maximum i n v i t r o microsomal enzymatic a c t i v i t y . Microsomal d e m e t h y l a t i o n o f imipramine was g r e a t l y enhanced by i n c r e a s i n g t h e c o n c e n t r a t i o n o f glucose-6-phosphate dehydrogenase i n t h e presence o f NADP ( a b b r e v i a t i o n f o r n i c o t i n a mide adenine d i n u c l e o t i d e phosphate). T h i s suggests t h a t the amount o f NADPH formed was l i m i t i n g t h e r e a c t i o n r a t e (12) . The p r i n c i p a l e l e c t r o n donor i s NADPH f o r t h e cytochrome P-450 redox c h a i n . The main source o f NADPH i n the l i v e r i s the pentose phosphate shunt (29) . T h i s c y c l e p r o v i d e s NADPH f o r b i o s y n t h e t i c processes such as f a t t y a c i d s y n t h e s i s and pentose f o r n u c l e o t i d e s  and n u c l e i c a c i d b i o s y n t h e s i s . I t i s  not known what c o n t r i b u t i o n t h i s shunt has on the supply o f the reduced n u c l e o t i d e t o t h e i n v i v o metabolism o f drugs i n the smooth endoplasmic r e t i c u l u m . I t i s p o s s i b l e t o f i n d t o what degree NADPH f u n c t i o n s i n drug metabolism by v a r y i n g i t s conc e n t r a t i o n i n the c e l l . I f one o f the enzymes i n t h e cytochrome P-450 redox system i s r a t e l i m i t i n g , f o r example cytochrome P-450 r e d u c t a s e , an i n c r e a s e i n c e l l content o f NADPH should not a f f e c t t h e r a t e o f metabolism o f a drug. Some s t u d i e s have been done on the e f f e c t o f NADPH a v a i l ability  on t h e r a t e o f s t e r o i d metabolism (30). These s t u d i e s  may apply t o drug metabolism s i n c e s t e r o i d microsomal metabolism was i n h i b i t e d o r induced by the same chemicals t h a t i n f l u e n c e microsomal drug metabolism (12,13,14) . The A  4  s t e r o i d s such as c o r t i s o n e and microsomal  C o r t i s o l  -double bond o f  was hydrogenated by  -<i-hydrogenase. I n c e r t a i n circumstances NADPH  a v a i l a b i l i t y might be r a t e l i m i t i n g i n v i v o and n o t enzymes o f  13  the  cytochrome P-450 redox system. Two methods used i n these  studies consisted of administration of tri-iodothyronine, 50 ug/ gm. of body w e i g h t , f o r two days t o r a t s . T h i s l e d t o an i n c r e a s e i n p r o d u c t i o n o f NADPH which i n t u r n r e s u l t e d i n an i n c r e a s e i n theZ^ -5-*-hydrogenase a c t i v i t y 4  (30) . F u r t h e r evidence  to suggest t h a t NADPH a v a i l a b i l i t y may a f f e c t s t e r o i d metabolism was t h a t f a s t e d animals show a decrease inZ^-5-Ot-hydrogenase a c t i v i t y along w i t h a decrease i n NADPH and glucose-6-phosphate i n r a t l i v e r (30). I f these f a s t e d r a t s were a d m i n i s t e r e d glucose the hydrogenation o f these s t e r o i d s r e t u r n e d t o c o n t r o l v a l u e s . From these s t u d i e s i n v i t r o and i n v i v o , i t seems t h a t an i n crease i n NADPH i n t h e l i v e r c e l l s may i n c r e a s e the r a t e o f some microsomal drug metabolism r e a c t i o n s . T h e r e f o r e , i n c e r t a i n c i r c u m s t a n c e s , the enzymes o f cytochrome P-450 redox system may not be the r a t e l i m i t i n g step f o r t h e i n v i v o metabolism o f drugs. V a r i o u s experiments  were  c a r r i e d out t o determine the  e f f e c t o f changes i n NADPH c o n c e n t r a t i o n on the metabolism o f imipramine i n the p e r f u s e d l i v e r as r e p o r t e d i n t h i s  thesis.  Hormones and Drug Metabolism  There i s very l i t t l e i n f o r m a t i o n on the e f f e c t of hormones on g l y c o l y s i s i n the l i v e r c e l l . The l i v e r i s p r i m a r i l y concerned w i t h gluconeogenesis (30) and q u i t e a few i n v e s t i g a t i o n s have been c a r r i e d out on t h e e f f e c t o f c y c l i c AMP ( a b b r e v i a t i o n i  t  f o r adenosine 3 , 5 monophosphate) and glucagon on gluconeo-  14 genesis i n t h e r a t l i v e r . I t has been found t h a t glucagon caused an i n c r e a s e i n c y c l i c AMP l e v e l s i n t h e l i v e r and s t i m u l a t e d gluconeogenesis from l a c t a t e i n the p e r f u s e d r a t l i v e r (31). D i r e c t a d d i t i o n of c y c l i c AMP t o the p e r f u s i o n f l u i d  caused  gluconeogenesis i n t h e p e r f u s e d r a t l i v e r . I t i s n o t known what e f f e c t these hormones o r gluconeogenesis has on drug metabolism, although these processes a r e c o n t i n u a l l y o c c u r r i n g i n t h e l i v e r . I t i s known t h a t s t a r v a t i o n and d i a b e t e s i n c r e a s e t h e l e v e l o f c y c l i c AMP i n the l i v e r c e l l (32) and as a r e s u l t microsomal drug metabolism can e i t h e r be s t i m u l a t e d o r depressed. I t a l s o seems t h a t microsomal drug metabolism due t o s t a r v a t i o n may n o t r e p r e s e n t a t r u e account o f i n v i v o metabolism s i n c e h e x o b a r b i t a l microsomal metabolism i n c r e a s e d due t o s t a r v a t i o n ; however the h e x o b a r b i t a l h y p n o t i c time was i n c r e a s e d (33). The f a c t o r which c o n t r i b u t e d t o the prolonged s l e e p i n g time i n s t a r v e d r a t s may be r e d u c t i o n o f NADPH g e n e r a t i o n . To summarize, the primary o b j e c t s o f the p r e s e n t r e s e a r c h t h e s i s were: a)  t o study the r e l a t i o n s h i p o f s u b s t r a t e c o n c e n t r a t i o n  to aromatic h y d r o x y l a t i o n and N-demethylation o f imipramine b)  t o determine i f the two enzymatic r e a c t i o n s i n v o l v e d  i n the metabolism of imipramine, aromatic h y d r o x y l a t i o n and N-demethylation, a r e c o n t r o l l e d t o the same e x t e n t by c e l l u l a r f a c t o r s such as magnesium, NADPH, NADH, d i b u t y r y l c y c l i c AMP and glucagon.  15 MATERIALS AND METHODS  L i v e r P e r f u s i o n Technique and Apparatus  The techniques and apparatus f o r the i s o l a t e d r a t l i v e r p e r f u s i o n s t u d i e s were s i m i l a r t o those d e s c r i b e d by M i l l e r (35) The apparatus was made e n t i r e l y o f g l a s s . The temperature o f the medium and the l i v e r was m a i n t a i n e d a t 37 degrees c e n t i g r a d e by c i r c u l a t i n g t h e r m o s t a t i c a l l y c o n t r o l l e d water through the double w a l l e d , m u l t i - b u l b tube t r a n s f e r r i n g the p e r f u s a t e from the  c o l l e c t i n g v e s s e l i n the oxygenator. To oxygenate the r e d b l o o d c e l l s a mixture o f 95 p e r cent  oxygen and 5 per cent carbon d i o x i d e  gas was f i r s t h u m i d i f i e d  at 37 degrees c e n t i g r a d e , then passed i n t o the multi-bulb-oxygen a t o r . The p e r f u s i o n medium was pumped from the c o l l e c t i n g v e s s e l v i a a s e r i e s o f one-way v a l v e s , by a Rodent R e s p i r a t o r Model 680, Harvard Apparatus Co. L t d . , t o the oxygenator. Once the p e r f u s a t e was oxygenated, the f l u i d entered the l i v e r by f r e e flow through a cannula i n the p o r t a l v e i n o f the l i v e r . The h y d r o s t a t i c pressure t o m a i n t a i n f l o w through the l i v e r was constant a t 14 t o 15 cm. water. The f l u i d r e t u r n e d t o the r e s e r v o i r from the h e p a t i c v e i n and from an o v e r f l o w o u t l e t i n the m u l t i - b u l b oxygenator. The p e r f u s a t e then was r e c i r c u l a t e d t o the  l i v e r v i a the oxygenator.  16  S u r g i c a l Procedure  S u r g i c a l procedures were performed under l i g h t e t h e r anest h e s i a . The abdomen was opened. The g a s t r o h e p a t i c and g a s t r o duodenal ligaments were d i v i d e d and the b i l e duct was c a n n u l a t e d , d i s t a l t o the branch e n t e r i n g the l i v e r , w i t h a PE-50 p o l y e t h y lene tube (Clay Adams I n c . , New York, N.Y.). The p o r t a l v e i n was then i s o l a t e d and t i e s were p l a c e d l o o s e l y around i t . The h e p a t i c a r t e r y was i s o l a t e d and a l i g a t u r e p l a c e d around a r t e r y p r i o r t o c a n n u l a t i o n of the p o r t a l v e i n . The  this  inferior  vena cava was l i g a t e d and d i v i d e d between the l i v e r and the r i g h t k i d n e y j u s t p r i o r t o removal of the l i v e r . The p o r t a l v e i n was then r a p i d l y cannulated w i t h a 3 mm.  g l a s s cannula.  P r i o r t o i n s e r t i o n , the g l a s s cannula was f i l l e d w i t h Krebs H e n s l e i t s o l u t i o n and a f t e r c a n n u l a t i o n 0.5 Ttil. sodium h e p a r i n s o l u t i o n (500 U.S.P. u n i t s / ml.) was i n j e c t e d through the cannula. The c h e s t was then opened and the h e p a t i c v e i n clamped and cut between the clamp and the h e a r t . The l i v e r , w i t h diaphragm, was then c a r e f u l l y d i s s e c t e d out and removed, t o g e t h e r w i t h the two cannulas, to the l i v e r chamber. The t o t a l time f o r the procedure, from opening the abdomen to s t a r t i n g the p e r f u s i o n was approximately t e n minutes and the time elapsed from cannulat i o n of the l i v e r t o l i v e r p e r f u s i o n was between two and t h r e e minutes. Extended i n t e r r u p t i o n of the l i v e r c i r c u l a t i o n beyond three minutes may cause i r r e v e r s i b l e d e t e r i o r a t i o n of the t i s s u e as evidenced by uneven c o l o r i n g of the l i v e r . The l i v e r  was  p e r f u s e d f o r one hour f o r e q u i l i b r a t i o n ; then imipramine was  17 added t o t h e r e c i r c u l a t i n g p e r f u s a t e .  Perfusion Fluid  Red blood c e l l s were i s o l a t e d from human b l o o d . Blood s t o r e d four t o f i v e weeks a t 4 degrees c e n t i g r a d e and no l o n g e r s u i t a b l e f o r blood t r a n s f u s i o n was obtained from the Canadian Red Cross. Whole b l o o d (50 ml.) was c e n t r i f u g e d a t 2,000 g f o r t e n minutes. The plasma and b u f f y l a y e r were removed by s u c t i o n . The r e d blood c e l l s were washed t w i c e w i t h 20 m l . Krebs H e n s l e i t s o l u t i o n pH 7.4 (36). The washing f l u i d was separated from the r e d blood c e l l s by c e n t r i f u g a t i o n f o r f i v e minutes a t 2,000 g and removed by s u c t i o n . The washed c e l l s were then made up t o 30 m l . w i t h Krebs H e n s l e i t s o l u t i o n , and hemoglobin c o n c e n t r a t i o n was determined.  This stock s o l u t i o n was used on the day o f prepara-  t i o n and s t o r e d a t 4 degrees c e n t i g r a d e . For each p e r f u s i o n , 100 ml. o f medium was r e q u i r e d and was prepared on t h e day of l i v e r p e r f u s i o n . T h i s p e r f u s i o n medium was very s i m i l a r t o t h a t d e s c r i b e d by Krebs (37). To the conc e n t r a t e d Krebs H e n s l e i t s o l u t i o n was added 2.5 gm. of bovine serum albumin powder f r a c t i o n V (Sigma Chemical Co., S t . L o u i s , M i s s o u r i ) and 200 mg. glucose. Since the albumin was a c i d , the pH was a d j u s t e d t o 7.4 by t h e a d d i t i o n o f n o t more than 0.5 m l . of IN NaOH. A s u f f i c i e n t volume of stock r e d blood c e l l suspens i o n was then added t o g i v e a hemoglobin c o n c e n t r a t i o n o f 2.5 gm. 100 m l . I f r e q u i r e d , t h e pH was again a d j u s t e d t o 7.4 w i t h  18 sodium b i c a r b o n a t e s o l u t i o n . F i v e m l . o f phosphate b u f f e r pH 7.4 (Na HP0 2  4  0.103 gm. , and KH PC> 2  4  0.024 gm. i n 10 m l . H 0) was 2  added. Approximately 2 m l . o f g l a s s d i s t i l l e d water was used to make up t o 100 m l . N i n e t y n i n e ml. o f t h i s s o l u t i o n was t r a n s f e r r e d t o the c o l l e c t i n g v e s s e l s o f the l i v e r p e r f u s i o n apparatus then a f t e r one hour of p e r f u s i o n o f the l i v e r 1 ml. of imipramine s o l u t i o n was added.  P e r f u s i o n Medium  gm./ 100 ml.  NaCL  0.687  KC1  0.04  MgS0 .7H 0  0.014  CaCl  0.028  4  2  2  Na HP0 2  4  0.103  KH P0 2  4  0.0246  NaHC0  3  0.21  Glucose  0.20  Albumin  2.5  Hemoglobin  2.5  Hemoglobin was determined as cyanmethemoglobin (38). The standard curve was prepared from a standard s o l u t i o n o f cyanmethemoglobin 80 mg./ 100 ml. (Hycel I n c . , Houston, Texas). Drabkins s o l u t i o n was prepared i n t h i s l a b o r a t o r y (38).  19 V i a b i l i t y of t h e L i v e r  The metabolic  s t a t u s of t h e i s o l a t e d perfused r a t l i v e r  has been examined i n s e v e r a l s t u d i e s by measurement o f p e r f u sate and b i l e flow r a t e s ( 3 9 ) , and by study o f b i o c h e m i c a l  func-  t i o n (40) . The p e r f u s a t e f l o w r a t e s were between 2 and 3 ml./ minute per gm. wet weight l i v e r . This r a t e was maintained  throughout  the e n t i r e i n c u b a t i o n p e r i o d . I f the r a t e f e l l below 2 m l . / minute per gm. wet weight l i v e r the metabolism of imipramine was d r a s t i c a l l y i n h i b i t e d . The b i l e flow r a t e was 0.3 t o 0.55 ml. per hour f o r the f i r s t hour of p e r f u s i o n which was a l s o maintained  d u r i n g the  second hour when drug metabolism s t u d i e s were done. These f l o w r a t e s were s i m i l a r t o b i l e flow r a t e s i n v i v o .  Lactate-Pyruvate  The l a c t a t e - p y r u v a t e r a t i o was monitored i n a l l p e r f u s i o n experiments t o e s t a b l i s h the v i a b i l i t y of the l i v e r . I f the l i v e r was i n an anaerobic  s t a t e then the l a c t a t e - p y r u v a t e  ratio  i n c r e a s e d t o 25 from the normal r a t i o o f 8 to 12 (41). I n a l l experiments the l a c t a t e - p y r u v a t e r a t i o was w i t h i n these normal limits. The method f o r l a c t a t e and pyruvate a n a l y s i s was  described  i n the Sigma T e c h n i c a l B u l l e t i n no. 826-UV, Sigma Chemical Co. The enzyme, l a c t i c dehydrogenase, c a t a l y z e s the f o l l o w i n g r e v e r s -  20  ible reaction.  P y r u v i c A c i d + NADH *" j . L a c t i c A c i d + NAD  In the presence o f an excess o f NADH a l l o f the p y r u v i c a c i d was converted t o l a c t i c a c i d . The amount o f NADH which was converted t o NAD ( a b b r e v i a t i o n f o r n i c o t i n a m i d e adenine d i n u c l e o t i d e ) was measured s p e c t r o p h o t o m e t r i c a l l y a t 340 nm and became a measure o f the amount o f p y r u v i c a c i d o r i g i n a l l y  present. To  measure L (+) l a c t i c a c i d the r e a c t i o n was run from r i g h t t o l e f t w i t h an excess o f NAD and h y d r a z i n e . The hydrazine  complex-  ed w i t h the p y r u v i c a c i d formed. This r e s u l t e d i n the r e a c t i o n going t o near completion. The amount o f NADH formed was measured s p e c t r o p h o t o m e t r i c a l l y a t 340 nm and became a measure o f the amount o f L (+) l a c t i c a c i d .  Magnesium A n a l y s i s  Magnesium was determined i n the p e r f u s a t e o f each e x p e r i ment ( 4 2 ) . This method i s based on the formation o f a red c o l o r produced by a t h i a z o l e y e l l o w - Mg. (OH)^ complex i n a l k a l i n e solution.  E x t r a c t i o n o f Imipramine and M e t a b o l i t e s  The s e p a r a t i o n of imipramine and i t s m e t a b o l i t e s was  done according t o a m o d i f i e d method developed by Moody, T a i t  21 and T o d r i c k (43) and B i c k e l and Weder (13). Q u a n t i f i c a t i o n o f 14  C-imipramine  and the m e t a b o l i t e s was by l i q u i d  scintillation  using a Picker Liquimat.  Reagents  a)  heptane-3 p e r cent (v/v) i s o a m y l a l c o h o l  b)  1,2-dichloroethane Heptane o r 1,2-dichloroethane was shaken t w i c e w i t h equal volumes o f 0. 5N NaOH, then w i t h equal volumes of 0.5N H SO^ and f i n a l l y , t h r e e times w i t h equal 2  volumes o f d i s t i l l e d water c)  a c e t i c anhydride  d) e)  0.1N H„SO. 2 4 IN NaOH  f)  Chloroform, n-propanol, s a t u r a t e d ammonia, 100:100:2 This was t h e s o l v e n t system used f o r t h i n l a y e r chromatography.  g)  0.5 gm. p - n i t r o a n i l i n e i n 50 ml. IN HC1 0.5 gm. NaN0  2  i n 50 ml. water  0.5 gm. s u l f a n i l i c a c i d i n 50 ml. water The three s o l u t i o n s were mixed 1:1:1 p r i o r t o s p r a y i n g of t h i n l a y e r chromatography p l a t e . h)  concentrated HC1 Concentrated HCl was used t o spray the t h i n l a y e r chromatography p l a t e s a f t e r g) .  22  i)  t r i e t h a n o l a m i n e HC1 b u f f e r pH 7.4 t r i e t h a n o l a m i n e 13.7 gm. 2N HC1, s u f f i c i e n t volume t o a d j u s t t o pH 7.4 d i s t i l l e d water t o 1,000 m l .  j)  citrate buffer c i t r i c a c i d 21.01 gm. i n 1,000 m l . sodium c i t r a t e 29.91 gm. i n 1,000 ml. c i t r i c a c i d 16 ml. t i t r a t e d w i t h sodium c i t r a t e t o pH 5.4 and d i l u t e d t o 100 ml. d i s t i l l e d water  E x t r a c t i o n Procedure  F i v e ml. o f p e r f u s a t e was withdrawn from the l i v e r p e r f u s i o n apparatus and c e n t r i f u g e d f o r f i v e minutes a t 2,000 g t o remove the red blood c e l l s . Three ml. o f the supernatant was t r a n s f e r r e d to c e n t r i f u g e t e s t tubes t o which 2 ml. o f t r i e t h a n o l a m i n e HC1 b u f f e r pH 7.4 and 1 ml. I N NaOH had been added. At each time i n t e r v a l b i l e was c o l l e c t e d i n a 1 ml. s y r i n g e . The b i l e was then removed from the s y r i n g e t o a t e s t tube. The s y r i n g e was washed w i t h 3 ml. o f Krebs H e n s l e i t s o l u t i o n pH 7.4. The contents were then made up t o 5 ml. w i t h t r i e t h a n o l a m i n e b u f f e r . One ml. was  removed f o r c o u n t i n g , then 1 ml. o f NaOH was added. The  l i v e r was b l o t t e d d r y w i t h paper, weighed and placed i n a g l a s s c o n t a i n e r t o which three p a r t s of c o l d t r i e t h a n o l a m i n e HCl b u f f e r pH 7.4 was added t o one p a r t l i v e r . This was immediately homogenized w i t h a P o t t e r Elvehjem homogenizer w i t h a t e f l o n p e s t l e .  23 Four ml. of t h i s homogenate, which r e p r e s e n t s 1 gm. o f t i s s u e , was  t r a n s f e r r e d t o a t e s t tube t o which 1 ml. t r i e t h a n o l a m i n e  and 1 ml. IN NaOH was added. The remainder o f t h e e x t r a c t i o n was t h e same f o r b i l e , plasma and l i v e r . To each sample, 6 m l . heptane-3 p e r cent isoamyl a l c o h o l was used to e x t r a c t imipramine and desmethylimipramine.  T h i s mixture was shaken by hand f o r  f i f t e e n minutes t o twenty minutes and l e f t f o r phase s e p a r a t i o n or c e n t r i f u g e d t i l l complete phase s e p a r a t i o n o c c u r r e d . Four ml. of t h e o r g a n i c l a y e r was t r a n s f e r r e d t o another t e s t tube which contained two o r t h r e e drops o f a c e t i c anhydride. F i v e ml. 0.IN ^SO^  was added, and the tubes shaken a g a i n . The a c e t y l a t e d  desmethylimipramine  remained i n the o r g a n i c phase w h i l e the  u n a c e t y l a t e d imipramine was t r a n s f e r r e d t o t h e H 2 S O 4 phase. One ml. was removed from each phase f o r counting o f imipramine i n IN ^ S O ^ and desmethylimipramine  i n heptane-3 p e r cent i s o a m y l  a l c o h o l . One ml. of the b a s i c aqueous phase remaining  after  e x t r a c t i o n w i t h heptane-3 p e r cent isoamyl a l c o h o l was a l s o removed f o r l i q u i d s c i n t i l l a t i o n c o u n t i n g . T h i s phase contained imipramine N-oxide, f r e e h y d r o x y l a t e d and g l u c u r o n i d e m e t a b o l i t e s of  imipramine. Imipramine N-oxide was separated from the b a s i c aqueous  phase by e x t r a c t i n g w i t h 10 ml. o f 1,2-dichloroethane.  After  t h i s e x t r a c t i o n 1 ml. o f the b a s i c aqueous phase was used f o r q u a n t i f i c a t i o n o f the f r e e h y d r o x y l a t e d and glucuronide metabol i t e s of imipramine. This phase represented the t o t a l  enzymatic  h y d r o x y l a t i o n o f imipramine s i n c e the h y d r o x y l a t e d m e t a b o l i t e s were present i n the g l u c u r o n i d e form. The q u a n t i t y o f imipramine  24 N-oxide which was e x t r a c t e d i n t o 1,2-dichloroethane 14 found s i n c e the amount o f t i o n o f imipramine  c o u l d be  C present before and a f t e r e x t r a c -  N-oxide was known.  The f r e e h y d r o x y l a t e d m e t a b o l i t e s were separated from the g l u c u r o n i d e m e t a b o l i t e s by removing 4 ml. o f the o r i g i n a l NaOH aqueous phase t o another tube and t i t r a t i n g t o pH 10 w i t h IN HC1 The f r e e h y d r o x y l a t e d m e t a b o l i t e s were then e x t r a c t e d w i t h 10 ml 1,2-dichloroethane.  This mixture was shaken c a r e f u l l y t o prevent  emulsion formation and c e n t r i f u g e d a t 2,000 g f o r approximately one h a l f t o one hour. The o r g a n i c l a y e r c o n t a i n e d t h e f r e e h y d r o x y l a t e d m e t a b o l i t e s o f imipramine w h i l e t h e b a s i c aqueous phase contained the g l u c u r o n i d e m e t a b o l i t e s o f imipramine. One ml. sample o f the b a s i c aqueous phase was removed t o determine the q u a n t i t y o f g l u c u r o n i d e f o r m a t i o n . The q u a n t i t y of f r e e h y d r o x y l a t e d m e t a b o l i t e s c o u l d be found s i n c e the amount o f 14 C present before and a f t e r e x t r a c t i o n o f f r e e h y d r o x y l a t e d met a b o l i t e s was known. The o r g a n i c phase which contained t h e f r e e hydroxy m e t a b o l i t e s was evaporated  t o dryness under reduced  pressure and the r e s i d u e d i s s o l v e d i n 500 u l methanol, 50 u l of which was used f o r t h i n l a y e r chromatography f o r i d e n t i f i c a t i o n of f r e e hydroxy m e t a b o l i t e s . Two ml. of the b a s i c aqueous phase which was e x t r a c t e d w i t h 1,2-dichloroethane, was removed and a c i d i f i e d t o pH 5.5 w i t h IN HCl. C i t r a t e b u f f e r , pH 5.5 was then added t o make the t o t a l volume t o 5 ml. Glusulase  (Boeh-  r i n g e r ) was then added, 0.15 ml., and incubated f o r twenty f o u r hours a t 37 degrees c e n t i g r a d e . The aqueous phase was then t i t r a t e d w i t h IN NaOH t o pH 10. The f r e e hydroxy-  25 l a t e d m e t a b o l i t e s were e x t r a c t e d w i t h 1,2-dichloroethane  and  prepared as d e s c r i b e d above t o be i d e n t i f i e d by t h i n l a y e r chromatography.  Thin Layer Chromatography  For i d e n t i f i c a t i o n of each h y d r o x y l a t e d and g l u c u r o n i d e m e t a b o l i t e t h i n l a y e r chromatography was used (13). Methanol was used to r e d i s s o l v e the m e t a b o l i t e s o b t a i n e d from the ext r a c t i o n procedure. The methanol s o l u t i o n c o n t a i n i n g the unknown m e t a b o l i t e s were p l a c e d on t h i n l a y e r p l a t e s and  developed  w i t h c h l o r o f o r m / n-propanol/ s a t u r a t e d ammonia, 100:100:2. Known r e f e r e n c e m e t a b o l i t e s , imipramine, desmethylimipramine,  imipra-  mine N-oxide, i m i n o d i b e n z y l , 2-hydroxydesmethy1imipramine and 2-hydroxyimipramine were d i s s o l v e d i n methanol. T h i s s o l u t i o n was  always run on t h i n l a y e r p l a t e s along w i t h unknown metabo-  l i t e s . The Rf values were compared to Rf values o f known r e f e r ence substances. The r e f e r e n c e m e t a b o l i t e 2-hydroxyimipramine was s y n t h e s i z e d i n t h i s l a b o r a t o r y (4 4) and o t h e r m e t a b o l i t e s were o b t a i n e d from Ciba-Geigy P h a r m a c e u t i c a l s . The t h i n l a y e r p l a t e s used were Baker-Flex S i l i c a - G e l 1 BF o b t a i n e d from Baker Chemical Co. L t d . , P h i l l i p s b u r g h , Penn.. The Rf v a l u e and c o l o r r e f e r e n c e of known m e t a b o l i t e s separated on s i l i c a g e l t h i n l a y e r p l a t e s are given below.  Table I Rf Values f o r Imipramine and I t s Known M e t a b o l i t e s Metabolite  Rf and C o l o r Literature Report(13)  Experimentally Found  Imipramine  0.71 b  0.54 b  De smethy1imipramine  0.31 b  0.30 b  Imipramine N-oxide  0.13 b  0.10 b  2-hydroxyimipramine  0.53 r  0.40 r  2-hydroxydesmethylimipramine  0.17 r  0.14 r  Iminodibenzy1  1.00 b  0.80 b  b=blue  r=red  I t was found t h a t the Rf values changed s i g n i f i c a n t l y between d e t e r m i n a t i o n s , so known r e f e r e n c e m e t a b o l i t e s were done along w i t h unknown m e t a b o l i t e s on each t h i n l a y e r chromatography  plate.  Quantification  o f E x t r a c t e d Imipramine and M e t a b o l i t e s  The l i q u i d s c i n t i l l a t i o n s o l u t i o n contained the f o l l o w i n g Toluene PPO (2,5-diphenyloxazole)  3 1 21 gm.  POPOP (1,4-Bis-(2-(5-phenyloxazolyl))-benzene)1.08 BBS-3 ( S o l u b i l i z e r )  gm.  600 ml.  27 Imipramine-  14 C HC1 (Amersham-Searle  8.05 m C/ mM) used i n  a l l experiments was d i l u t e d w i t h imipramine HC1 (Ciba-Geigy) to a s p e c i f i c a c t i v i t y o f 5 0 DPM/ ug t o 50 0 DPM/ ug. The compound was then r e c r y s t a l l i z e d , u s i n g the s o l v e n t system e t h y l acetate-acetone u n t i l constant s p e c i f i c a c t i v i t y was o b t a i n e d . A s o l u t i o n was then prepared w i t h d i s t i l l e d water and s t o r e d a t 0 t o 4 degrees c e n t i g r a d e .  L i q u i d S c i n t i l l a t i o n Counter, S e t t i n g s and Quench C o r r e c t i o n s  The maximum counting e f f i c i e n c y was determined on a P i c k e r Liquimat which was the instrument used f o r counting samples. A standard toluene-"*" C, (99,000 DPM) sample was used t o determine 4  the s e t t i n g s . The counting time was one minute f o r the standard sample and twenty minutes to determine background counts. I t was found t h a t the h i g h e s t e f f i c i e n c y and lowest background count was obtained when the upper l e v e l d i s c r i m i n a t o r was s e t a t 700 and the lower l e v e l d i s c r i m i n a t o r was s e t a t 150 f o r channel A and B. The counting e f f i c i e n c y of the P i c k e r standard t o l u e n e 14 C was 94.2 per cent w i t h a background count o f 34. An e x t e r n a l standard method was developed f o r quench c o r r e c t i o n s . Channel A and B d i s c r i m i n a t o r s were s e t a t 150 t o 700. P i c k e r standard quench s e r i e s samples were used. These samples contained t o l u e n e 14 C w i t h an a c t i v i t y o f 197,000 DPM per v i a l . The quenching agent used was chloroform. The c o r r e l a t i o n curve o f the quenched s e r i e s o f samples to the counting r a t e of 137 Cs as the e x t e r n a l  standard source (with, the d i s c r i m i n a t o r s s e t a t 700 and  900  f o r channel C) was found. The quench c o r r e c t i o n curve was  over  a narrow range w i t h c o u n t i n g e f f i c i e n c y between 84 and 95 per cent. I f an unknown sample had c o u n t i n g e f f i c i e n c y below 84 per cent then the i n t e r n a l standard method was used and 10 u l 14 or 4260 DPM of t o l u e n e -  C was added t o each sample. Each sample  i n the c y c l e was counted f o r t e n minutes f o r t h r e e c y c l e s and the average count, standard d e v i a t i o n and per cent e r r o r were determined. The per cent counting e f f i c i e n c y was then determined f o r each unknown sample i n the u s u a l manner. Procedure f o r Counting and C a l c u l a t i o n of Unknown Samples  The l i q u i d s c i n t i l l a t i o n counter d i s c r i m i n a t o r s were s e t at 150 and 700 f o r channel A and B and 700 and 900 f o r channel C. A l l unknown samples were counted f o r ten minutes  through  three c y c l e s . Each c y c l e of unknown samples was preceded by a standard quench s e r i e s t o determine the c o r r e l a t i o n between per 137 cent e f f i c i e n c y versus CPM of  Cs e x t e r n a l standard. The  standards were counted e i t h e r f o r ten minutes o r the time necessary f o r the counts to reach one m i l l i o n counts i n channel A, 137 whichever o c c u r r e d f i r s t . The e x t e r n a l  Cs c o u n t i n g time was  f o r one minute. A best f i t p o l y n o m i a l equation was used to c o r r e l a t e the per cent counting e f f i c i e n c y from channel A and the e x t e r n a l standard count f o r each c y c l e . This equation was then used to f i n d the per cent counting e f f i c i e n c y by s o l v i n g  29  f o r the e x t e r n a l standard count of an unknown sample i n the corresponding c y c l e . To f i n d the per cent c o u n t i n g e f f i c i e n c y of an unknown sample which was below 84 per cent the i n t e r n a l standard method was  used.  The v a r i a n c e , standard d e v i a t i o n and per cent e r r o r i n counting of each sample was c a l c u l a t e d , as w e l l as c o n v e r s i o n of CPM to DPM.  A l l the i n f o r m a t i o n r e q u i r e d was programmed f o r  and computed by an IBM 360 computer. The program was i n F o r t r a n IV.  The data p r i n t o u t from each experiment was prepared on the  l i q u i d s c i n t i l l a t i o n system's paper tape punch. The s c i n t i l l a t i o n counter a u t o m a t i c a l l y c o n t r o l s  liquid  punching of a l l  data o t h e r than experiment, d a t e , e t c . The i n f o r m a t i o n on the punched tape was then t r a n s f e r r e d t o magnetic tape and read by the computer. Conversion of CPM to DPM/  t o t a l b i l e and  DPM/  gm. l i v e r was c a l c u l a t e d by the computer. T h i s i n f o r m a t i o n was s t o r e d on a d i s c f o r f u r t h e r c a l c u l a t i o n .  Comparison Between E x t e r n a l and I n t e r n a l Quench C o r r e c t i o n s  A comparison was made between the e x t e r n a l and i n t e r n a l method t o determine the per cent c o u n t i n g e f f i c i e n c y f o r unknown samples. E x t r a c t i o n of imipramine and m e t a b o l i t e s were done from the p e r f u s i o n medium. The e x t e r n a l standard method and i n t e r n a l standard method was done on each sample. One ml. of each phase was used f o r a n a l y s i s as l i s t e d i n Table 11.The r e s u l t s of f o u r such experiments are l i s t e d i n Table 1 1 .  30 Table 11 Comparison o f Per Cent Counting E f f i c i e n c y Between I n t e r n a l Standard and E x t e r n a l Standard Methods Per Cent Counting E f f i c i e n c y Phase 0.1N H S 0 2  4  Heptane-3% Isoamyl-alcohol  0 . IN NaOH  Aqueous Phase pH 7.4  Experiment Number  Internal Standard  External Standard  1 2 3 4  86.6 86 85 88  87 87 87 88  1 2 3 4  88 89.6 90 89  90  1 2 3 4  83. 8 86 85.6 86  88 87 87 85  1 2 3 4  82 88 86.5 85  83 85.5 87 85  88 90 .90  The per cent c o u n t i n g e f f i c i e n c y determined by t h e e x t e r n a l o r i n t e r n a l s t a d a r d method was v i r t u a l l y the same. In a l l experiments the e x t e r n a l standard method was used i f the p e r cent counting e f f i c i e n c y was between 84 and 95 p e r cent. I n t e r n a l standards were used f o r e f f i c i e n c i e s below 84 per cent.  31  E x t r a c t i o n S p e c i f i c i t y and E f f i c i e n c y f o r Imipramine and M e t a b o l i t e s  Imipramine  Imipramine was added t o Krebs H e n s l e i t s o l u t i o n c o n t a i n i n g 2.5 p e r cent albumin i n order t o determine the p e r cent r e c o v e r y 14 of  C-imipramine from the b a s i c aqueous phase i n t o 0.1N H S 0 2  4  by s o l v e n t e x t r a c t i o n . The c o n c e n t r a t i o n o f imipramine was 14 ug/ ml. Imipramine was then e x t r a c t e d i n t o heptane-3 p e r cent isoamyl a l c o h o l and then i n t o 0.1N H SO^. One ml. of 0.1N 2  H SO^ was analyzed by l i q u i d s c i n t i l l a t i o n c o u n t i n g . The p e r 2  cent of dosage recovered i n the 0.1N H S 0 2  4  phase was 96.6 p e r  cent i n an average o f f o u r experiments. The range was 92.5 t o 99 p e r cent. The e x t r a c t i o n procedure used f o r t h e a n a l y s i s of imipramine was adequate f o r our purposes. Desmethylimipramine  A s p e c t r o p h o t o m e t r y a n a l y s i s was developed t o q u a n t i t a t e desmethylimipramine s i n c e t h i s r a d i o a c t i v e drug was n o t a v a i l able f o r l i q u i d s c i n t i l l a t i o n a n a l y s i s . Standard s o l u t i o n s o f desmethylimipramine were made w i t h 0.1N H S 0 2  4  r a n g i n g i n concen-  t r a t i o n from 2.04 ug/ ml. t o 20.4 ug/ ml. The o p t i c a l d e n s i t y o f these s o l u t i o n s was determined a t 250 nm, the maximum absorbance peak. The blank used was 0.1N H S 0 . The o p t i c a l d e n s i t y was 2  4  then p l o t t e d a g a i n s t known c o n c e n t r a t i o n s o f desmethylimipramine.  From t h i s graph the c o n c e n t r a t i o n of unknown samples o f desmethylimipramine c o u l d be determined. The p e r cent r e c o v e r y of desmethylimipramine by s o l v e n t e x t r a c t i o n was then determined i n t h e f o l l o w i n g manner. S o l u t i o n s were made, r a n g i n g from 8.52 ug t o 6 8.13 ug/ ml. i n phosphate b u f f e r (as i n m e t a b o l i c experiments) pH 7.4, t o a t o t a l volume o f 5 m l . One ml. IN NaOH and 6 m l . heptane-3 p e r cent i s o a m y l a l c o h o l were added. The t e s t tubes were shaken f o r t e n t o f i f t e e n minutes then 5 ml. o f the o r g a n i c l a y e r was t r a n s f e r r e d t o another t e s t tube c o n t a i n i n g t h r e e drops o f a c e t i c anhydride. F i v e ml. 0.1N E2^ ^ Q  w  a  s  added a f t e r two minutes and t h e tubes  shaken. Four ml. o f the o r g a n i c l a y e r and 4 ml. o f 0.1N H2S0^ were removed t o separate t e s t tubes and evaporated t o dryness. The contents o f t h e o r g a n i c phase was r e d i s s o l v e d i n 10 m l . 0.1N E2^ ^ 0  an<  ^  t n e  contents o f the 0.1N H2S0 phase was r e d i s s o l v 4  ed i n 0.1N H2S0 . F i v e ml. o f the b a s i c aqueous phase 4  (phosphate  b u f f e r + IN NaOH) was t r a n s f e r r e d t o another c o n t a i n e r , evapor a t e d and r e d i s s o l v e d i n 5 ml. 0.1N ^SO^. The absorbance  from  each phase, heptane-3 p e r cent isoamyl a l c o h o l , 0.1N ^SO^, and b a s i c aqueous phase was found and thus the c o n c e n t r a t i o n of desmethylimipramine determined. The p e r cent recovery of desmethylimipramine from s i x experiments was: heptane-3 per cent isoamyl a l c o h o l 76 per cent, 0.IN H2SO^ 0.2 per cent and the b a s i c aqueous phase 2.2 per cent (see Table V ) . The t o t a l recovery was 7 8.4 p e r cent and almost a l l the desmethylimipramine t h a t could be accounted f o r was i n the heptane-3 per cent isoamyl a l c o h o l phase w h i l e 2.2 p e r cent was i n the b a s i c aqueous phase.  33 Chromatography  of Imipramine and Desmethylimipramine  Plasma, b i l e and l i v e r samples from m e t a b o l i c experiments where r a d i o a c t i v e imipramine HC1 was m e t a b o l i z e d f o r one h a l f hour were used to determine whether imipramine and desmethylimipramine were s p e c i f i c a l l y e x t r a c t e d i n t o heptane-3 per cent isoamyl a l c o h o l . The e x t r a c t i o n procedure and t h i n l a y e r chromatography techniques f o r t h i s experiment were r e p o r t e d p r e v i o u s l y . The sample prepared f o r t h i n l a y e r chromatography was  from  1 t o 2 ml. of heptane-3 per cent i s o a m y l a l c o h o l a f t e r i t was shaken w i t h the b a s i c aqueous phase. T h i s phase should only c o n t a i n imipramine and desmethylimipramine. F i v e ml. 0.1N  H^SO^  was added to the remaining 4 ml. of heptane-3 per cent isoamyl a l c o h o l and shaken. One ml. from each phase was used f o r l i q u i d s c i n t i l l a t i o n c o u n t i n g . The o r g a n i c phase  (1 or 2 ml.)  was  evaporated to dryness, then 0.5 ml. methanol added. To f i n d the t o t a l r a d i o a c t i v e count i n the methanol, 0.25 ml. was used f o r l i q u i d s c i n t i l l a t i o n counting and 50 u l was used f o r t h i n l a y e r chromatography. The spots t h a t appeared on the t h i n l a y e r chromatography p l a t e were removed and p l a c e d i n a v i a l . One  half  ml. methanol was added to the v i a l , twenty f o u r hours l a t e r the c o c k t a i l was added, then the contents of the v i a l was counted. In summary, imipramine and desmethylimipramine have been q u a n t i t a t e d by two methods (a) by t h i n l a y e r  chromatography  (b) by s o l v e n t e x t r a c t i o n . The r e s u l t s appear i n Table 111.  34 Table I I I Q u a n t i f i c a t i o n o f Desmethylimipramine and Imipramine by S o l v e n t E x t r a c t i o n and T h i n Layer Chromatography Experiment Number  Drug  tt  3  2  % by % by % by % by TLC s o l v e n t TLC s o l v e n t ext. ext.  % by % by TLC2 s o l v e n t ext. 3  % by % by TLC s o l v e n t ext.  Imipramine  27.2  19 .4  35.6  24  32.7  37.4  27.6  17.1  Desmethylimipramine  73.2  82.6  52.8  76  67.4  62.5  73  83  Unidentified  *4  11.6  *  *  % Recovered"*"  68  70  73.8  73. 8 i  1 2  3  Per cent recovered= IMI(PPM) + DMI(PPM) + u n i d e n t i f i e d ( P P M ) X 100 DPM i n 0.5 ml. methanol Per cent imipramine o r desmethylimipramine by t h i n l a y e r chroma tography= DPM X 100 t o t a l DPM r e c o v e r e d Per cent imipramine o r desmethylimipramine= IMI (DPM) e x t r a c t e d i n H S 0 o r DMI (DPM) i n o r g a n i c phase X 100 2  C t o t a l DPM * below background (34 CPM) 1 4  4  4  35 The c o u n t i n g e f f i c i e n c y of a l l samples analyzed by l i q u i d s c i n t i l l a t i o n was between 85 and 88 p e r cent. The p e r cent o f imipramine found by t h i n l a y e r chromatography  was always 6 t o  11 p e r cent h i g h e r than the content of imipramine found by s o l v e n t e x t r a c t i o n f o r the same sample. T h i s c o u l d mean t h a t t h i s amount o f imipramine was n o t e x t r a c t e d i n t o 0.IN P^SO^ but remained i n the o r g a n i c phase. However, t h i s was u n l i k e l y s i n c e i t has been shown t h a t 95 t o 100 p e r cent imipramine present i n the o r g a n i c phase was e x t r a c t e d i n t o the H2SC> phase. 4  The per cent of desmethylimipramine recovered by t h i n l a y e r chromatography  was 9 and 11 p e r cent lower than the amount found  by s o l v e n t e x t r a c t i o n i n two experiments, t h e same i n one and 24 per cent lower i n another experiment. T h i s c o u l d mean t h a t some desmethylimipramine (at l e a s t 9 and 10 p e r cent) was e x t r a c t ed out o f the o r g a n i c phase by 0.1N E2SO^. T h i s a g a i n seems u n l i k e l y s i n c e i t was found by s p e c t r o p h o t o m e t r i c a n a l y s i s t h a t 0.2 per cent desmethylimipramine c o u l d be e x t r a c t e d from the o r g a n i c phase i n t o 0.IN H^SO^. I n a l l f o u r experiments an u n i d e n t i f i e d r e d spot appeared w i t h an Rf v a l u e of 0.920. This compound may be the u n i d e n t i f i e d m e t a b o l i t e which B i c k e l (13) has mentioned i n h i s s t u d i e s . A l t e r n a t i v e l y , i t may be a decomposed product from desmethylimipramine or imipramine caused by the chromatography. In t h r e e experiments the compound w i t h an Rf v a l u e o f 0.920 was w e l l below background count, w h i l e i n the o t h e r sample i t accounted f o r 11 per cent of the a c t i v i t y i n the heptane-3 per cent isoamyl a l c o h o l phase. This compound w i t h an Rf v a l u e  36 o f <0.920 appeared i n l i v e r samples but not i n plasma or b i l e samp l e s . The per cent r e c o v e r y o f r a d i o a c t i v i t y from t h i n l a y e r chromatography was low, 68 t o 73.8 per cent. U s i n g known q u a n t i t i e s o f r a d i o a c t i v e imipramine t w i c e r e c r y s t a l l i z e d , i t was found t h a t only 60 p e r cent c o u l d be accounted f o r when e x t r a c t e d o f f the t h i n l a y e r chromatography p l a t e . T h e r e f o r e , the q u a n t i t a t i o n o f m e t a b o l i t e s u s i n g t h i n l a y e r  chromatography  i s n o t a very s u i t a b l e method s i n c e the p e r cent r e c o v e r y of the m e t a b o l i t e s from t h e p l a t e was low. The heptane-3 per cent isoamyl a l c o h o l e x t r a c t from n i n e plasma samples, f o u r b i l e samples and f o u r l i v e r samples, taken from t h r e e experiments were s u b j e c t e d t o t h i n l a y e r  chromato-  graphy. Most o f these samples c o n t a i n e d only imipramine and desmethylimipramine, except one sample c o n t a i n e d the above drugs p l u s i m i n o d i b e n z y l which appeared as a very f a i n t b l u e spot. These samples d i d not c o n t a i n any h y d r o x y l a t e d m e t a b o l i t e s or imipramine N-oxide. In c o n c l u s i o n i t has been shown t h a t e x t r a c t i o n o f i m i p r a mine and desmethylimipramine from b i o l o g i c a l samples w i t h heptane-3 p e r cent isoamyl a l c o h o l was adequate and t h a t the ext r a c t i o n o f imipramine from the o r g a n i c l a y e r u s i n g 0.1N I^SO^ was a l s o s u i t a b l e .  2-hydroxydesmethylimipramine and Imipramine N-oxide  The purpose o f these experiments was (a) t o f i n d whether 2-hydroxydesmethylimipramine o r imipramine N-oxide was e x t r a c t -  37 ed i n t o heptane-3 per cent isoamyl a l c o h o l when the b a s i c aqueous phase i s g r e a t e r than pH 12, (b) t o determine  t h e p e r cent  recovery o f imipramine N-oxide by e x t r a c t i o n o f t h e b a s i c aqueous phase pH 12 w i t h 10 m l . 1,2-dichloroethane. The r e s u l t s appear i n Table IV.  2-hydroxydesmethy1imipramine  The e x t r a c t i o n method (see experimental methods) and t h e spectrophotometric a n a l y s i s (see recovery o f DMI) have been noted. Standard s o l u t i o n s o f 2-hydroxydesmethylimipramine were made i n IN NaOH ranging i n c o n c e n t r a t i o n from 6.8 8 ug t o 6 8.7 ug/ ml. and t h e i r absorbance p l o t t e d . The aqueous phase cont a i n i n g the m e t a b o l i t e was made b a s i c (pH 12.5 t o 13) before heptane-3 per cent isoamyl a l c o h o l was added. A f t e r e x t r a c t i o n w i t h heptane-3 p e r cent isoamyl a l c o h o l , the aqueous phase was t i t r a t e d t o pH 10 w i t h IN HC1, then 10 ml. o f 1,2-dichloroethane was added. F o r spectrophotometric a n a l y s i s a l l s o l u t i o n s were evaporated t o dryness under reduced pressure and the contents d i s s o l v e d w i t h a p p r o p r i a t e volumes o f IN NaOH. The r e s u l t s r e p o r t e d a r e from e l e v e n o b s e r v a t i o n s from each phase. The p e r cent, 2-hydroxydesmethylimipramine, 1  found i n the heptane-3 p e r  cent isoamyl a l c o h o l phase was 1.2 per c e n t , t h e 1 , 2 - d i c h l o r o ethane phase 78.3 per cent and the b a s i c aqueous phase 7.6 p e r cent. The t o t a l per cent recovery was 87.1 per cent. Samples were removed from a l l phases and p l a c e d on t h i n  i  38 l a y e r chromatography p l a t e s (see e x p e r i m e n t a l method). 2-hydroxydesmethylimipramine c o u l d not be d e t e c t e d i n heptane-3 p e r cent i s o a m y l a l c o h o l phase or the aqueous phase, but spots appeared w i t h corresponding Rf v a l u e s t o a standard s o l u t i o n of 2-hydroxydesmethylimipramine i n the 1,2-dichloroethane phase. An u n i d e n t i f i e d compound appeared which was a l s o e x t r a c t e d i n t o the 1,2-dichloroethane phase w i t h Rf valueO.930, which probably was a breakdown product of 2-hydroxydesmethylimipramine  Imipramine N-oxide  The method of e x t r a c t i o n and a n a l y s i s f o r imipramine N-oxide was s i m i l a r t o t h a t f o r  2-hydroxydesmethylimipramine  The pH of the aqueous phase was g r e a t e r than twelve f o r the e x t r a c t i o n of imipramine N-oxide i n t o heptane-3 per cent i s o amyl a l c o h o l or 1,2-dichloroethane. The c o n c e n t r a t i o n of the aqueous phase v a r i e d from 4.3 t o 25.8 ug/ ml. The c o n c e n t r a t i o n i n the standard s o l u t i o n was 1.72 t o 21.5 ug/ ml. imipramine N-oxide prepared i n phosphate b u f f e r pH 7.4. The per cent o f imipramine N-oxide recovered i n the heptane-3 per cent i s o a m y l a l c o h o l phase was 8.6, the 1,2-dichloroethane phase 92.0 and the aqueous phase 1.9 per cent. The t o t a l recovery was per  102.5.  cent. The r e s u l t s are an average from s i x o b s e r v a t i o n s i n  each phase. I t t h e r e f o r e seems t h a t most o f the imipramine N-oxide can be e x t r a c t e d from the aqueous phase a t pH g r e a t e r than 12 by 10 ml. of 1,2-dichloroethane and very l i t t l e  was  Table IV Solvent E x t r a c t i o n of Imipramine and I t s Major M e t a b o l i t e s from Aqueous S o l u t i o n s Heptane-3% Isoamyl A l c o h o l  Metabolite  n  1,2-dichloroethane*^  1  pH o f solution  pH o f per s o l u t i o n cent 12.5  96.6  Desmethylimipramine 6  12.5  76  2-hyd roxy de sme thy111 imipramine  12.5  1.2  10  Imipramine N-oxide  12.5  8.6  12.5  6  2  3 4  Remaining % Recovery Aqueous 4 Phase  5  per cent 96.6  96.6  12.5  4  Imipramine  per cent  0.1N H S 0  2.2  78.4  78.3  7.6  87.1  92.0  1.9  102.5  0.2  1  per cent e x t r a c t e d i n t o heptane-3% i s o a m y l a l c o h o l from aqueous phase  2  per cent e x t r a c t e d i n t o 1,2-dichloroethane from aqueous sample a f t e r e x t r a c t i o n  3 4 5  w i t h heptane-3% isoamyl a l c o h o l per cent e x t r a c t e d i n t o 0.1N H S 0 from heptane-3% i s o a m y l a l c o h o l per cent remaining i n aqueous sample a f t e r e x t r a c t i o n w i t h heptane-3% i s o a m y l a l c o h o l % recovery= t o t a l ug i n -each phase ug added t o aqueous phase 2  4  x  40 e x t r a c t e d by the heptane-3 per cent i s o a m y l a l c o h o l . Since o n l y 8.6 per cent of the imipramine N-oxide was e x t r a c t e d by heptane-3 per cent i s o a m y l a l c o h o l and i t appears as a very minor m e t a b o l i t e , t h i s procedure i s s u i t a b l e f o r e x t r a c t i o n of imipramine N-oxide.  S t a b i l i t y o f Imipramine  To each of t h r e e t e s t t u b e s , 24 ml. of p e r f u s i o n f l u i d and 1 ml. of imipramine s o l u t i o n was added. Imipramine HC1 concent r a t i o n i n each tube was 28 ug/ ml. which was e q u i v a l e n t t o 14 1421 DPM o f imipramine-  C HC1. These s o l u t i o n s were then i n -  cubated a t 37 degrees c e n t i g r a d e f o r f i f t e e n minutes. F i v e ml. was withdrawn from each sample, c e n t r i f u g e d and a n a l y z e d . The r e s u l t s appear i n Table V. The t o t a l r a d i o a c t i v i t y which appeared i n the supernatant a f t e r c e n t r i f u g a t i o n a t 2,000 g f o r t e n minutes was 80 per cent o f the t o t a l q u a n t i t y of imipramine HC1. Twenty per cent of the dose of imipramine HC1 could not be accounted f o r and appears t o be absorbed i n t o the r e d blood c e l l s . However, 94.5 to 100 per cent of the t o t a l r a d i o a c t i v i t y which appeared i n the aqueous phase c o u l d be e x t r a c t e d i n t o the o r g a n i c l a y e r then i n t o 0.1N ^SO^. i n the o t h e r phases .  There was no s i g n i f i c a n t amount o f a c t i v i t y  41 Table V S t a b i l i t y of Imipramine (Per Cent R a d i o a c t i v i t y Recovered From Each Phase) Per E x t r a c t i n g Phase 1  Cent R a d i o a c t i v i t y Experiments 2 3  0.1N H-SO. 2 4  100  98  94.5  Heptane-3% Isoamyl A l c o h o l  0  0  0  0  0  0  80.5  80  82.5  1  0.1N Na OH  2  T o t a l A c t i v i t y i n Aqueous S o l u t i o n Before E x t r a c t i o n 1 2  t h e per cent r a d i o a c t i v i t y was t h e per cent o f r a d i o a c t i v i t y i n the aqueous phase 0 means counts which appeared were e i t h e r h o r l e s s than h of background count  The r e s u l t s are s i m i l a r t o those found by B i c k e l and Weder (7). They found t h a t when the sample was made b a s i c (pH 8 t o 12.4) a l l imipramine c o u l d be e x t r a c t e d w i t h heptane-3 per cent isoamyl a l c o h o l . T h i s e x t r a c t i o n was a l s o very s i m i l a r t o t h a t done by T o d r i c h (43). To f i n d out whether imipramine was absorbed onto t h e r e d 14 blood c e l l s  C-imipramine was added to.two groups of t e s t tubes.  One group of t e s t tubes contained Krebs H e n s l e i t s o l u t i o n w i t h albumin, the o t h e r group contained Krebs H e n s l e i t s o l u t i o n , albumin and r e d b l o o d c e l l s  (2.5 per cent hemoglobin). For q u a n t i f i c a t i o n  42 of the  14  C - i m i p r a m i n e , 1 ml. was removed immediately a f t e r m i x i n g 14 contents of each tube w i t h  C-imipramine and 1 ml. was  removed a f t e r t h r e e hours. The r e s u l t s o f these experiments appear i n Table V I . Table VI S t a b i l i t y and Per Cent Recovery of Imipramine With and Without Red Blood C e l l s i n S o l u t i o n Phase  3  Per Ce  KH  0.1N  H^C^  s o l u t i o n and albumin Time 0 hours 3 hours 97.5,97.5 92.5,99  1  2 supernatant  99,100 n= 2  1 2 3  100,100  . Recovery 3 KH s o l u t i o n , albumin and red blood c e l l s Time 0 hours  3 hours  95.5  103  81.7  85  n= 1  per cent recovered from aqueous phase per cent of dose Krebs H e n s l e i t s o l u t i o n  In both s o l u t i o n s , w i t h and w i t h o u t red b l o o d c e l l s , c o n t r o l and a f t e r three hours i n c u b a t i o n , the amount recovered i n the 0.IN H2S0 phase was s i m i l a r which i n d i c a t e s t h a t imipramine HC1 4  was s t a b l e under these c o n d i t i o n s . A n a l y s i s of the aqueous phase a f t e r c e n t r i f u g a t i o n of Krebs H e n s l e i t s o l u t i o n w i t h albumin accounted f o r 100 per cent of the dose a d m i n i s t e r e d w h i l e o n l y 80 to 85 per cent of the dose was i n the supernatant when red  43 b l o o d c e l l s were p r e s e n t i n the s o l u t i o n . I t t h e r e f o r e was apparent t h a t the 15 to 18 per cent l o s s of imipramine HC1  was  due t o the red blood c e l l s , i n a n a l y s i s o f p e r f u s a t e imipramine an e r r o r of 15 to 18 per cent c o u l d r e s u l t . However, i n m e t a b o l i c experiments l a t e r conducted, t h e r e was 90 t o 100 per cent recovery of r a d i o a c t i v i t y which i n d i c a t e s t h a t t h i s p r e d i c t e d e r r o r d i d not occur. Two experiments were designed to determine whether i m i p r a mine HC1 was s t a b l e when c i r c u l a t e d through the l i v e r p e r f u s i o n apparatus. Krebs H e n s l e i t s o l u t i o n was prepared w i t h 2.5 gm.  of  albumin. 99 ml. of t h i s was added to the l i v e r p e r f u s i o n apparatus  along w i t h 1 ml. imipramine HC1 s o l u t i o n (84,000 DPM/  e q u i v a l e n t to 1.665  mg.  imipramine HC1. This s o l u t i o n was  ml.) circu-  l a t e d through the l i v e r p e r f u s i o n apparatus w i t h o u t the r a t l i v e r . A c o n t r o l sample o f 5 ml. was removed p r i o r t o c i r c u l a t i o n , then 5 ml. every hour f o r f o u r hours. The c i r c u l a t i n g s o l u t i o n was maintained a t 37 degrees c e n t i g r a d e . A f t e r each hour up to f o u r hours 87 and 90 per cent of the imipramine HC1 was recovered. From these r e s u l t s i t seems t h a t imipramine was s t a b l e under the experimental c o n d i t i o n s planned to study imipramine metabolism.  44  RESULTS AND  DISCUSSION  K i n e t i c s of Imipramine Metabolism i n the I s o l a t e d Perfused  Rat  Liver  In the p r e s e n t a t i o n of these r e s u l t s , r e f e r e n c e i s made t o N-demethylation, aromatic h y d r o x y l a t i o n or N - o x i d a t i o n .  The  meaning of these terms are as f o l l o w s . The t o t a l q u a n t i t y of desmethylimipramine (abbreviated form DMI)  formed from i m i p r a -  mine (abbreviated form IMI) i n a s p e c i f i c time i s r e f e r r e d to as N-demethylation. The t o t a l amount of f r e e h y d r o x y l a t e d metabolites ide  (abbreviated form OH.) p l u s the t o t a l q u a n t i t y of  glucuron-  (abbreviated form G) m e t a b o l i t e s formed i n a s p e c i f i c time  i s r e f e r r e d to as aromatic h y d r o x y l a t i o n . The t o t a l amount of imipramine N-oxide (abbreviated form N-0)  formed i n a s p e c i f i c  time i s r e f e r r e d to as N - o x i d a t i o n .  Metabolism of Imipramine i n the I s o l a t e d Perfused L i v e r A f t e r Various Incubation Times  To determine whether the r a t e of N-demethylation, aromatic 1 h y d r o x y l a t i o n or N - o x i d a t i o n v a r i e s w i t h i n c u b a t i o n time, i m i p r a mine HC1  2 X 10 ^M  (6.336 mg.)  was  incubated f o r v a r i o u s time  periods w i t h the i s o l a t e d perfused l i v e r . The i n i t i a l volume of p e r f u s i n g f l u i d was  110 ml. Two  5 ml. samples were removed  p r i o r to a d d i t i o n of imipramine, to determine l a c t a t e and pyruvate 1 the time d u r i n g which the drug was present i n the p e r f u s i o n f l u i d and i n contact w i t h the i s o l a t e d r a t l i v e r .  45 p e r f u s a t e c o n c e n t r a t i o n s . A f t e r the l i v e r was p e r f u s e d f o r one hour, 1 ml. of imipramine (.6.336 mg./ ml.) was added t o 100 ml. of p e r f u s i n g f l u i d . The p e r f u s a t e c o n c e n t r a t i o n o f imipramine and the m e t a b o l i t e s were determined by withdrawing 5 m l . from the p e r f u s i o n apparatus a t the a p p r o p r i a t e time. These e x p e r i ments were terminated e i t h e r a f t e r f i f t e e n ,  t h i r t y or s i x t y  minutes o f i n c u b a t i o n w i t h imipramine. The l i v e r , p e r f u s a t e and b i l e were analyzed f o r imipramine, desmethylimipramine and the more p o l a r m e t a b o l i t e s such as g l u c u r o n i d e , f r e e hydroxy and N-oxide m e t a b o l i t e s o f imipramine. From these a n a l y s e s , the t o t a l metabolism of imipramine as w e l l as the f o r m a t i o n o f desmethylimipramine and the aromatic h y d r o x y l a t e d m e t a b o l i t e s c o u l d be determined. This i n f o r m a t i o n c o u l d then be r e l a t e d to the p e r f u s a t e h a l f - l i f e of imipramine. The d i s t r i b u t i o n o f imipramine and i t s m e t a b o l i t e s i n the b i l e , l i v e r and p e r f u s a t e c o u l d a l s o be determined. In a l l experiments the l a c t a t e / p y r u v a t e r a t i o was n o t greater than twelve and the blood f l o w through the l i v e r was not l e s s than 2 m l . / gm. l i v e r per minute and not g r e a t e r than 3 m l . / gm. 14 l i v e r per minute. The per cent recovery of  C was between 90  and 100 per cent i n a l l experiments. Perfusate Concentration  The p e r f u s a t e c o n c e n t r a t i o n of imipramine and the m e t a b o l i c products o f imipramine HC1 (2 X 10 ^M) metabolism are presented  5  10  15  30 Time (minutes)  60  Figure 3 . P e r f u s a t e c o n c e n t r a t i o n of imipramine and m e t a b o l i t e s . The dose of imipramine was 2 X 10 ^M. * ug e q u i v a l e n t t o ug imipramine  47  i n F i g u r e 3. The p o l a r m e t a b o l i t e s of imipramine, f r e e hydroxy and N-oxide appeared r a p i d l y  glucuronide,  i n the p e r f u s a t e w i t h  a s i m i l a r combined c o n c e n t r a t i o n to desmethylimipramine up t o f i f teen minutes of i n c u b a t i o n . These p o l a r m e t a b o l i t e s  (G, OH,  N-0)  were the composite o f aromatic h y d r o x y l a t i o n of the i m i n o d i b e n z y l r i n g , i t s g l u c u r o n i d e products and imipramine The c o n c e n t r a t i o n of these p o l a r m e t a b o l i t e s was  N-oxide.  g r e a t e r than  the c o n c e n t r a t i o n of desmethylimipramine a f t e r f i f t e e n minutes of i n c u b a t i o n . Very l i t t l e f r e e h y d r o x y l a t e d m e t a b o l i t e appeared i n the p e r f u s a t e . For example, a f t e r f i f t e e n minutes of i m i p ramine metabolism the c o n c e n t r a t i o n of glucuronide m e t a b o l i t e s was  3.2 ug/ ml. whereas the c o n c e n t r a t i o n of g l u c u r o n i d e  f r e e hydroxy m e t a b o l i t e s was  and  3.3 ug/ ml. I t appeared t h a t the  h y d r o x y l a t e d m e t a b o l i t e s formed from imipramine were q u i c k l y conjugated w i t h g l u c u r o n i d e to form the g l u c u r o n i d e m e t a b o l i t e . The p e r f u s a t e c o n c e n t r a t i o n of imipramine N-oxide remained very low and e s s e n t i a l l y  the same d u r i n g the e n t i r e  incubation  period. The p e r f u s a t e c o n c e n t r a t i o n of desmethylimipramine i n c r e a s ed l i n e a r l y to f i f t e e n minutes. The c o n c e n t r a t i o n of imipramine  desmethyl-  a f t e r t h i r t y and s i x t y minutes of imipramine  metabo-  l i s m d i d not i n c r e a s e f u r t h e r . I t was  e v i d e n t upon examining F i g u r e 4 t h a t the disappear-  ance of imipramine was  from the p e r f u s i n g f l u i d i n t o the l i v e r  composed of two d i s t i n c t phases. The f i r s t phase occurred  between zero and f i f t e e n minutes of imipramine metabolism w h i l e the second phase was between f i f t e e n and t h i r t y minutes.  The  p e r f u s a t e d e c l i n e of imipramine between zero and f i f t e e n minutes  48  40  30  (10) 20  (10)  Cn  3  10  (4)  H  o  •H  •P rd M  -p a  :4)  CD  o cs o o <D  -P rd  cn 4-1 u cu A4  1.5 5  10  15  30  60  Time(minutes) ( ) number o f o b s e r v a t i o n s per p o i n t Figure 4 Semi l o g a r i t h m i c p l o t of p e r f u s a t e imipramine c o n c e n t r a t i o n (ug/ ml.) versus time. Imipramine metabolism (2 X 1 0 ~ 5 M ) . Each p o i n t represents the average imipramine c o n c e n t r a t i o n .  49 was extremely r a p i d . The r a t e of d e c l i n e of imipramine i n the f i r s t phase was a composite of a r a p i d i n i t i a l d i s t r i b u t i o n between the p e r f u s a t e and the l i v e r and metabolism o f imipramine, s i n c e the per cent o f imipramine m e t a b o l i z e d i n the f i r s t  fif-  teen minutes was 43 per cent (Table V I I I ) . The second phase o f imipramine d e c l i n e from the p e r f u s a t e between f i f t e e n and s i x t y minutes was slower. This second phase was probably a composite of an e q u i l i b r i u m r a t e between the l i v e r and p e r f u s a t e f o r imipramine and the r a t e o f imipramine metabolism. The r a t e of imipramine p e r f u s a t e c o n c e n t r a t i o n d e c l i n e seemed t o f o l l o w f i r s t o r d e r k i n e t i c s w i t h time, s i n c e a s e m i - l o g r i t h m i c p l o t of p e r f u s a t e imipramine c o n c e n t r a t i o n versus time produced a s t r a i g h t l i n e between f i f t e e n and s i x t y minutes phase of imipramine d e c l i n e h a l f - l i f e  (Figure 4 ) . The (t%) was 18.5  second  minutes  which was c a l c u l a t e d by the d i r e c t g r a p h i c a l method (45).  t  t  2  3  = s  2  t  l  t  i s the time when the c o n c e n t r a t i o n was twice t h a t a t time t ^  Rate o f Metabolism and D i s t r i b u t i o n To determine whether the p e r f u s a t e h a l f - l i f e  (t%) f o r i m i p -  ramine was an accurate e s t i m a t i o n of the r a t e of imipramine metabolism the l i v e r , b i l e and p e r f u s a t e were analyzed f o r imipramine a f t e r f i f t e e n , t h i r t y and s i x t y minutes of i m i p r a -5 mine metabolism. The dose of imipramine was 2 X 10 adequately study the p r o p o r t i o n of N-demethylation  M. To  50 to aromatic h y d r o x y l a t i o n and N - o x i d a t i o n the m e t a b o l i c products of imipramine were analyzed i n the b i l e , l i v e r and p e r f u s a t e . The r e s u l t s from the t o t a l a n a l y s i s o f imipramine and i t s m e t a b o l i t e s i n the b i l e , l i v e r and p e r f u s a t e a f t e r f i f t e e n , t h i r t y and -5 s i x t y minutes o f imipramine metabolism  (2 X 10  M) w i t h the  i s o l a t e d p e r f u s e d r a t l i v e r are presented i n Table V I I I . Table V I I C a l c u l a t e d and E x p e r i m e n t a l Imipramine Remaining  After -5  F i f t e e n , T h i r t y and S i x t y Minutes o f Imipramine Metabolism.  Incubation Time  Calculated % IMI Remaining  Found % IMI Remaining  (2 X 10  Deviation From C a l c u l a t e d IMI  15  59.4  56.911.5  2.5  30  34.4  32.1+5.7  2.3  60  11.0  19.7+2.1  8.7  1 2  c a l c u l a t e d u s i n g th = 18.5 f o r imipramine metabolism the amount o f imipramine found a f t e r a n a l y s i s o f l i v e r , b i l e and p e r f u s a t e d i v i d e d by t o t a l C recovered . The dose o f IMI was 2 X 10~ M. value o f 2 - value of 1 = d e v i a t i o n 1 4  5  3  M)  Assuming f i r s t order k i n e t i c s f o r imipramine metabolism and determining the th from the p e r f u s a t e imipramine concentrt i o n change from F i g u r e 4 the per cent o f imipramine remaining can be c a l c u l a t e d a f t e r f i f t e e n , t h i r t y and s i x t y minutes o f  51 metabolism. A comparison was made between the c a l c u l a t e d (Table VII)  p e r cent imipramine remaining from p e r f u s a t e th and t h e  amount o f imipramine found from a n a l y s i s o f p e r f u s a t e , b i l e and l i v e r (Table V I I I ) . The per cent imipramine remaining a f t e r f i f t e e n and t h i r t y minutes o f metabolism o f imipramine are s i m i l a r , however, t h e d e v i a t i o n between t h e c a l c u l a t e d and e x p e r i mental value f o r s i x t y minutes of metabolism was 8.7 p e r c e n t . I t t h e r e f o r e seems t h a t f i r s t order r a t e occurs f o r imipramine metabolism f o r the f i r s t t h i r t y minutes o f i n c u b a t i o n w i t h the i s o l a t e d p e r f u s e d r a t l i v e r because t h e th f o r t h e change i n t h e p e r f u s a t e imipramine c o n c e n t r a t i o n was an accurate e s t i m a t i o n of the r a t e o f imipramine metabolism. The c a l c u l a t e d per cent metabolism between t h i r t y and s i x t y minutes o f imipramine metab o l i s m was much h i g h e r than the per cent metabolism which was e x p e r i m e n t a l l y found. I t thus appears t h a t the r a t e o f imipramine metabolism changed and t h i s c o u l d not be d e t e c t e d when c o n s i d e r ing the p e r f u s a t e c o n c e n t r a t i o n o f imipramine. I t was found t h a t there was s l i g h t i n h i b i t i o n o f imipramine metabolism which was not r e f l e c t e d i n the p e r f u s a t e imipramine c o n c e n t r a t i o n (Table V I I I ) . Consequently experiments which o n l y monitor p e r f u s a t e imipramine c o n c e n t r a t i o n d e c l i n e would not be an accurate e s t i m a t i o n o f imipramine metabolism. The f a c t o r which c o n t r i b u t e d t o the i n h i b i t i o n o f imipramine -5 (2 X 10  M) metabolism a f t e r t h i r t y minutes was the i n h i b i t i o n o f  formation o f g l u c u r o n i d e , f r e e h y d r o x y l a t e d m e t a b o l i t e s as w e l l as imipramine N-oxide  (Table V I I I ) . The per cent o f the dose o f  Table V I I I The Average P e r Cent Metabolism and D i s t r i b u t i o n o f Imipramine and M e t a b o l i t e s . The Incubation Time was F i f t e e n , T h i r t y and S i x t y M i n u t e s , With Imipramine HC1 2 X 10 M. _5  Per Cent Metabolized Incubation Time(minutes)  IMI  3  DMI  ±S.E  G,0H,N-0  4  Per Cent D i s t r i b u t i o n Perfusate Bile IMI DMI G,OH,N-0 G,OH,N-0 IMI 4  15 n=4  56.9 ±1.5  25.4 ±1.5  19.1 ±1.41  30.1 ±2.0  30 n=3  32.1 ±5.7  41.0 ±2.7  27 ±0.15  60 n=4  19.7 ±2.1  50.4 ±5.5  29.8 ±4.5  20.9 ±0.6  Liver DMI G ,OH,N-0  4  37.1 ±4.2  11.7 ±4.03  70.0 ±2.3  24.7 20.7 ±4.08 ±2.4  64.5 ±4.90  7.4 ±1.4  74.7 78.6 ±4.08 ±2.5  26.7 ±5.7  15.8 ±3.6  46.4 ±2.44  28.5 ±1.7  80.2 ±5.2  21.3 ±2.2  n= number o f o b s e r v a t i o n s  18.3 ±6.2  79.0 ±2.17  80. 8 ±6.4  50.6 ±2.5  £>.E.= standard e r r o r  per cent= t o t a l ug IMI, DMI o r G, OH, N-0 formed ug o f C recovered 1 4  3 4  ^  per cent= ug o f IMI, DMI o r G, OH, N-0 i n l i v e r , p e r f u s a t e o r b i l e X 100 t o t a l ug o f IMI, DMI o r G, OH, N-0 formed per cent o f t h e dose o f IMI remaining Ul G, OH, N-0 (glucuronide, f r e e hydroxy and N-oxide m e t a b o l i t e s o f imipramine)= t t o t a l t o • C remaining a f t e r e x t r a c t i o n o f aqueous phase pH 12.5 w i t h heptane-3 p e r cent isoamyl a l c o h o l . L4  53 imipramine which was m e t a b o l i z e d t o g l u c u r o n i d e , f r e e hydroxy and N-oxide m e t a b o l i t e s was 27 p e r cent a f t e r t h i r t y minutes and 29.8 p e r cent a f t e r s i x t y minutes of imipramine -5 The dose o f imipramine was 2 X 10  metabolism.  M. Although i n h i b i t i o n o f  aromatic h y d r o x y l a t i o n and N - o x i d a t i o n o c c u r r e d ,  N-demethylation  continued over the e n t i r e i n c u b a t i o n p e r i o d . Table IX The T o t a l Quantity o f Imipramine Remaining and M e t a b o l i t e s Formed. The I n c u b a t i o n Time was F i f t e e n , T h i r t y and S i x t y -5 Minutes f o r Imipramine 2 X 10 M. M e t a b o l i t e s Formed I n c u b a t i o n Time(minutes) Metabolites  15 mg.iS.E.  30 mg.iS.E.  60 mg.iS.E.  IMI remaining  3.5610.11  2.0310.32  1.23±0.11  DMI  1.59±0.05  2.5810.25  3.13+0.37  1.1910.06  1.7310.08  1.84±0.26  1  G^R^N-O ' 1  1 2  2  mg. formed e q u i v a l e n t t o mg. IMI 14 G, OH, N-0 t o t a l C remaining a f t e r e x t r a c t i o n o f aqueous phase pH 12.5 w i t h heptane-3 p e r cent isoamyl alcohol.  The k i n e t i c s o f imipramine metabolism was complicated by the f a c t t h a t i n h i b i t i o n of aromatic h y d r o x y l a t i o n and N-oxidat i o n had occurred. The formation o f desmethylimipramine  continu-  ed t o i n c r e a s e over the e n t i r e i n c u b a t i o n p e r i o d . The t o t a l  54  q u a n t i t y of desmethylimipramine formed a f t e r imipramine metabol i s m f o r t h i r t y minutes was 2.5 mg.  (Table IX) and  after  s i x t y minutes was 3.1 mg. The amount o f imipramine decreased i n the same p e r i o d from 2 mg. t o 1.2 mg. There was more desmethylimipramine than imipramine i n the i s o l a t e d p e r f u s e d r a t l i v e r apparatus. Reports i n the l i t e r a t u r e suggested t h a t desmethylimipramine could i n h i b i t the metabolism of o t h e r drugs (23,24,25,26,27), t h e r e f o r e i t was thought t h a t the i n h i b i t i o n of aromatic h y d r o x y l a t i o n and N - o x i d a t i o n was  due  to desmethylimipramine formed from imipramine metabolism. The drug imipramine was very l i p o p h i l i c s i n c e 70 per cent had accumulated i n the l i v e r a f t e r f i f t e e n minutes of i n c u b a t i o n i n the i s o l a t i o n p e r f u s i o n apparatus. The amount o f i m i p r a mine i n c r e a s e d w i t h time t o 80 per cent (Table V I I I ) . T h i s g r a d u a l i n c r e a s e i n imipramine c o n c e n t r a t i o n i n the l i v e r may e x p l a i n the g r a d u a l d e c l i n e of imipramine p e r f u s a t e c o n c e n t r a t i o n , which f o l l o w s f i r s t order k i n e t i c s , between t h i r t y and s i x t y minutes of metabolism d e s p i t e the i n h i b i t i o n of aromatic h y d r o x y l a t i o n and N - o x i d a t i o n . The per cent of imipramine remaining i n the p e r f u s a t e a f t e r f i f t e e n minutes was 30 per cent which d e c l i n e d to 16 per cent a f t e r s i x t y minutes of imipramine metabolism. The p e r f u s a t e d e c l i n e of imipramine between t h i r t y and s i x t y minutes was probably a r e s u l t of imipramine metabolism and a new e q u i l i b r i u m e s t a b l i s h e d w i t h the l i v e r . Less than 1 per cent of imipramine was found i n the b i l e . The m e t a b o l i t e of i m i p r a mine, desmethylimipramine, was a l s o very l i p o p h i l i c s i n c e 80 per  55 cent of the desmethylimipramine remained i n the l i v e r , the remaini n g 20 per cent was i n the p e r f u s a t e w i t h no e x c r e t i o n of t h i s drug i n the b i l e . The d i s t r i b u t i o n of desmethylimipramine between the l i v e r and p e r f u s a t e was very s i m i l a r to t h a t found by Von Bohr (46). The d i s t r i b u t i o n of more p o l a r m e t a b o l i t e s o f imipramine, g l u c u r o n i d e , f r e e hydroxy and N-oxide d e r i v a t i v e s s t e a d i l y  de-  creased i n the l i v e r w i t h time from 50 per cent to 21 per cent. The per cent of these p o l a r m e t a b o l i t e s i n c r e a s e d i n the p e r fusate between f i f t e e n and t h i r t y minutes, f o l l o w e d by a decrease a f t e r t h i r t y minutes of imipramine metabolism. T h i s decrease of p e r f u s a t e c o n c e n t r a t i o n ( g l u c u r o n i d e , f r e e hydroxy and N-oxide) was r e f l e c t e d  i n an i n c r e a s e of b i l i a r y e x c r e t i o n .  Metabolism of Imipramine HC1 at V a r i o u s C o n c e n t r a t i o n s o f Imipramine  The metabolism of imipramine i n the i s o l a t e d p e r f u s e d r a t l i v e r was dependent on t h r e e enzymatic r e a c t i o n s , namely aromat i c h y d r o x y l a t i o n , N-demethylation and N - o x i d a t i o n . To p r o v i d e some i n f o r m a t i o n on whether imipramine metabolism, f o l l o w e d f i r s t order k i n e t i c s or was dose dependent, imipramine concent r a t i o n was v a r i e d . Since the metabolism of imipramine was  de-  pendent on three enzymatic r e a c t i o n s , changing the s u b s t r a t e c o n c e n t r a t i o n could p r o v i d e i n f o r m a t i o n on the r e l a t i v e  acti-  v i t i e s of these enzymes i n r e l a t i o n t o imipramine c o n c e n t r a t i o n . This type of i n f o r m a t i o n was found necessary i n order t o choose the best dose of imipramine f o r experiments designed t o f i n d  56 p o s s i b l e c e l l u l a r c o n t r o l mechanisms f o r imipramine metabolism. The dose o f imipramine HC1 chosen was 0.5 X 10  (1.5 mg.)  and 1 X 10~ M (3.17 mg.) s i n c e these were the approximate amounts 5  of imipramine remaining a f t e r f i f t e e n and t h i r t y minutes of -5 i n c u b a t i o n w i t h 2 X 10  M (Table I X ) . The i n c u b a t i o n times f o r  these experiments were f i f t e e n minutes s i n c e a l o n g e r i n c u b a t i o n time c o u l d c o m p l i c a t e aromatic h y d r o x y l a t i o n o f imipramine as was demonstrated i n the l a s t s e r i e s o f experiments. Imipramine -5 0.5 X 10  -5 M o r 1 X 10  M was added t o the p e r f u s i o n apparatus  a f t e r the l i v e r was p e r f u s e d f o r one hour. F i v e ml. of t h e p e r f u s a t e was withdrawn f i v e , t e n and f i f t e e n minutes a f t e r a d d i t i o n of imipramine, f o r a n a l y s i s of imipramine and m e t a b o l i t e s . The experiments were terminated a f t e r f i f t e e n minutes, the l i v e r and b i l e were a n a l y z e d f o r imipramine and i t s m e t a b o l i t e s . The r e s u l t s appear i n Table X. Rate o f Metabolism and D i s t r i b u t i o n I f a drug i s m e t a b o l i z e d by f i r s t order k i n e t i c s and the substrate concentration i s  changed, the per cent o f a drug  metabolized i n a s p e c i f i c time should be the same because the h a l f - l i f e o f a drug i s independent of the drug c o n c e n t r a t i o n (46)  th  = 0.693 k  k= constant f o r f i r s t order k i n e t i c s  Table X E f f e c t s of Imipramine Concentration on Formation o f M e t a b o l i t e s . The I n c u b a t i o n Time was F i f t e e n Minutes Dose o f IMI Incubated f o r F i f t e e n Minutes 1  0.5 X 10~ M  76.7 ±3.3  29.9 ±1.8  41.9 ±2.6  5.6 ±0.6  4. 85 ±0.8  1 X 10" M  67.6 ±3.3  34.3 ±4.0  25.5 ±2.6  3.8 ±1.2  5.6 ±1.2  2 X 10" M  43.1 ±1.5  20.9 ±0.6  13. 8 ±2.1  1.3 ±0.2  7.0 ±0.1  5  5  5  1 2  2 ± S.E. Per Cent Metabolized N-0 OH GOH DMI IMI  f o u r experiments per dose of imipramine per cent. IMI metabolized or m e t a b o l i t e formed= t o t a l IMI, DMI, __GOH, OH or N-0 formed X 100 • — total C recovered  58 In these experiments the dose o r s u b s t r a t e c o n c e n t r a t i o n of  imipramine was changed, however, t h e i n c u b a t i o n time was  kept constant a t f i f t e e n minutes. The p e r cent o f imipramine m e t a b o l i z e d and the per cent o f the m e t a b o l i c products formed was c a l c u l a t e d as a per cent o f the o r i g i n a l c o n c e n t r a t i o n o f imipramine.The  r e s u l t s o f these experiments are expressed i n  Table X. The per cent o f imipramine m e t a b o l i z e d had decreased as the dose o f imipramine was i n c r e a s e d . F o r example, when i m i p r a -5 -5 -5 mine HC1 0.5 X 10 M, 1 X 10 M o r 2 X 10 M was incubated w i t h the i s o l a t e d p e r f u s e d r a t l i v e r 76, 67 and 43 per cent o f the dose of  imipramine was m e t a b o l i z e d . The metabolism o f imipramine d i d  not f o l l o w f i r s t order k i n e t i c s but was dose dependent because the per cent o f imipramine HC1 m e t a b o l i z e d t o g l u c u r o n i d e and f r e e hydroxy m e t a b o l i t e s a t the d i f f e r e n t c o n c e n t r a t i o n o f imipramine was dose dependent. The p e r cent of g l u c u r o n i d e and f r e e hydroxy m e t a b o l i t e s o f imipramine formed was 42, 25 and -5 -5 14 per cent f o r imipramine c o n c e n t r a t i o n o f 0.5 X 10 M, 1 X 10 M -5 and 2 X 10  M r e s p e c t i v e l y . The r a t e o f f o r m a t i o n o f desmethyl-  imipramine f o l l o w e d f i r s t order k i n e t i c s s i n c e the per cent o f desmethylimipramine formed was s i m i l a r f o r imipramine concentr-5 -5 t i o n o f 0.5 X 10 M and 1 X 10 M. However, t h e r e was a decrease i n the r a t e o f desmethylimipramine metabolism when the c o n c e n t r -5 t i o n o f imipramine was 2 X 10  M. The r a t e o f formation o f  imipramine N-oxide f o l l o w e d f i r s t order k i n e t i c s throughout the e n t i r e range of imipramine c o n c e n t r a t i o n s used; t h a t i s , the per cent of imipramine N-oxide formed was s i m i l a r .  The reason drug plasma d e c l i n e was dose dependent c o u l d be t h a t the enzymatic r e a c t i o n s i n v o l v e d i n the d e g r a d a t i o n o f the drug were s a t u r a t e d a t h i g h e r doses. At s a t u r a t i o n of the enzyme, zero order k i n e t i c s o c c u r r e d . The r e s u l t s of the dose dependent metabolism of imipramine were expressed more c l e a r l y when the t o t a l micrograms  formed per gram l i v e r of the m e t a b o l i t e  was p l o t t e d versus the s u b s t r a t e c o n c e n t r a t i o n (Figure 5 ) . The V max or zero order k i n e t i c s was a t t a i n e d f o r aromatic hydroxyl a t i o n of imipramine s i n c e i n c r e a s i n g the dose of imipramine -5 t o 2 X 10  M d i d not l i n e a r l y i n c r e a s e the amount of g l u c u r o n -  ide and h y d r o x y l a t e d m e t a b o l i t e s of imipramine formed  (Figure 5)„  There can be two reasons f o r the zero o r d e r k i n e t i c s of the aromatic hydroxylase enzymatic r e a c t i o n . In terms of M i c h a e l i s Menten k i n e t i c s the enzyme might have reached the t r u e V max and the s u b s t r a t e c o n c e n t r a t i o n exceeded the c o n c e n t r a t i o n of aromatic h y d r o x y l a s e . In t h i s case the amount of imipramine would have exceeded the amount of aromatic hydroxylase enzyme necessary t o form 2-hydroxyimipramine. T h i s was the p r i n c i p a l enzymatic r e a c t i o n s i n c e 2-hydroxyimipramine was the o n l y h y d r o x y l a t e d m e t a b o l i t e i d e n t i f i e d by t h i n l a y e r  chromatography.  The second reason f o r zero order k i n e t i c s might have been t h a t i n h i b i t i o n of the aromatic h y d r o x y l a t i o n of Imipramine occurred due t o the desmethylimipramine  formed.  -5 When the dose of imipramine was decreased from 2 X 10 M -5 -5 to 1 X 10 M or 0.5 X 10 M the amount of desmethylimipramine formed became equal to or l e s s than the amount of g l u c u r o n i d e  60  300 n= 4 r a t s per p o i n t standard e r r o r  200 to  U  <u  CD  -M •H  > -H  rH  rH  o  iH  *tnL00 3  0 0.5 Dose  1 ( X 10~ M)  2  5  F i g u r e 5. The t o t a l formation of m e t a b o l i t e s a f t e r imipramine metabolism f o r f i f t e e n minutes w i t h the perfused r a t liver.  61  and h y d r o x y l a t e d m e t a b o l i t e s of imipramine. When 0.5 X 10  -5  M  of imipramine was m e t a b o l i z e d by the i s o l a t e d p e r f u s e d r a t l i v e r the amount of desmethylimipramine was l e s s than the q u a n t i t y of g l u c u r o n i d e and h y d r o x y l a t e d m e t a b o l i t e s of i m i p r a mine, 51 ± 4.06 to 70 ± 5.4 ug/ gm. l i v e r . At lower doses o f imipramine, N-demethylation was no l o n g e r the predominant enzymatic pathway f o r d e g r a d a t i o n of imipramine.  E f f e c t o f Desmethylimipramine on Aromatic H y d r o x y l a t i o n and N-demethylation o f Imipramine  I t was found t h a t the formation of g l u c u r o n i d e and f r e e h y d r o x y l a t e d m e t a b o l i t e s of imipramine was  inhibited after -5  t h i r t y minutes of imipramine metabolism (2 X 10  M) w i t h the  i s o l a t e d p e r f u s e d r a t l i v e r . I t was a l s o found t h a t aromatic h y d r o x y l a t i o n of imipramine f o l l o w e d zero o r d e r k i n e t i c s a f t e r -5 imipramine metabolism a t s u b s t r a t e c o n c e n t r a t i o n o f 1 X 10  M.  In o r d e r to determine i f desmethylimipramine was i n h i b i t i n g the h y d r o x y l a t i o n r e a c t i o n , the f o l l o w i n g experiments were c a r r i e d out. A f t e r t h i r t y minutes of imipramine metabolism (see Table I X ) , 2.5 mg. of desmethylimipramine had accumulated i n the p e r f u s i o n apparatus and the amount of imipramine which remained was 2 mg. Under these c o n d i t i o n s f u r t h e r m e t a b o l i c formation of g l u c u r o n i d e , f r e e hydroxy and N-oxide m e t a b o l i t e s of imipramine was  i n h i b i t e d although N-demethylation continued. The amount of  desmetb.yl imipramine accumulated to. 3.2 mg. w h i l e imipramine d i m i n i s h e d t o 1.2 mg. These changes o c c u r r e d a f t e r an a d d i t i o n a l t h i r t y minutes o f metabolism. Aromatic h y d r o x y l a s e a c t i v i t y was as a c t i v e as N-demethylase a c t i v i t y when the dose o f i m i p r a mine was 0.5 X 10  (Figure 5 ) . One may c o n s i d e r the d i f f e r e n c e  between the two experiments i n the f o l l o w i n g way. I n the former experiment 2.5 mg. o f desmethylimipramine was p r e s e n t i n the i s o l a t e d p e r f u s e d l i v e r apparatus, b e f o r e the metabolism of 2 mg. of imipramine had begun. I f the 2.5 mg. o f desmethylimipramine formed d i d i n h i b i t aromatic h y d r o x y l a t i o n then p r e i n c u b a t i o n of approximately 2.5 mg. o r l e s s of desmethylimipramine t r r i o r t o -5 -5 0.5 X 10  M o r 1 X 10  M of imipramine metabolism should i n h i b i t  aromatic h y d r o x y l a t i o n . I t was a l s o found t h a t the f o r m a t i o n o f g l u c u r o n i d e and -5 f r e e hydroxy m e t a b o l i t e s o f imipramine (between 1 X 10 2 X 10  M and  o f i m i p r a m i n e ) , f o r f i f t e e n minutes, f o l l o w e d zero  order k i n e t i c s . The amount o f desmethylimipramine which accumulated a f t e r f i f t e e n minutes o f i n c u b a t i o n w i t h i m i p r a -5 mine 2 X 10  M was 1.5 mg. and the t o t a l imipramine remaining  was 3.5 mg. The amount of desmethylimipramine which accumulated a f t e r f i f t e e n minutes of metabolism d f imipramine (1 X 10 "*M) was 1 mg. To show t h a t the desmethylimipramine formed c o u l d suppress aromatic h y d r o x y l a t i o n and produce zero o r d e r k i n e t i c s , p r e i n c u b a t i o n of 2 mg. o r l e s s of desmethylimipramine should i n h i b i t aromatic h y d r o x y l a t i o n when the dose o f imipramine was between 1 X 1 0 M o r 2 X 1 0 ~ M . _5  5  63 V a r y i n g amounts o f desmethylimipramine were p r e i n c u b a t e d -5 -5 f i v e minutes p r i o r to the a d d i t i o n o f 0.5 X 10  M, 1 X 10  M  -5 o r 2 X 10  M o f imipramine i n t o the p e r f u s i o n apparatus. The  i n c u b a t i o n time f o r the metabolism of imipramine was f i f t e e n minutes. The r e s u l t s of these experiments are g i v e n i n F i g u r e 6. They are compared t o c o n t r o l experiments w i t h o u t p r e t r e a t ment of desmethylimipramine. P r e i n c u b a t i o n o f 6.6 X 10  (2 mg.) o r l e s s o f desmethy-  imipramine w i t h the p e r f u s e d l i v e r p r i o r to a d d i t i o n of 0.5 X -5 10  -5 M o r 1 X 10  J5f o f imipramine i n h i b i t e d aromatic h y d r o x y l a -  t i o n . The i n c u b a t i o n time was f i f t e e n minutes. The i n h i b i t i o n -5 of aromatic h y d r o x y l a t i o n f o r 1 X 10 M o f imipramine was 45 -5 per cent and f o r 0.5 X 10  M imipramine tne i n h i b i t i o n was 58  per cent. Therefore the 2.5 mg. desmethylimipramine formed a f t e r t h i r t y minutes of imipramine metabolism c o u l d i n h i b i t the format i o n of aromatic h y d r o x y l a t i o n . The t r u e per cent i n h i b i t i o n of imipramine metabolism should be between 45 and 58 per cent s i n c e the imipramine remaining a f t e r t h i r t y minutes of i m i p r a mine metabolism was 2.032 ± 0.3 3 mg. and the e x p e r i m e n t a l i n h i b i t i o n s t u d i e s were done f o r 1.5 and 3 mg. o f imipramine metabolism. To f u r t h e r study the e f f e c t o f desmethylimipramine on aromatic h y d r o x y l a t i o n , desmethylimipramine was p r e i n c u b a t e d -5 -5 p1r iand o r t2o mg. a d d istiinocne otfh i 1s Xwas 10 the M oamount r 2 X 10 M imipramine i n t o the of desmethylimipramine -5 pformed e r f u s i ofrom n apparatus. The1 qXu a10 n t-5 i tMy or of desmethylimipramine imipramine 2 X 10 M incubated fwhich or was l a t e d t o i n h i b i t aromatic h y d r o x y l a t i o n was between f i f t ce ae ln c uminutes.  !  64 c o -H  1.65 Dose  6.6  3.3  (X 10  13.2  M desmethylimipramine)  Figure 6 E f f e c t of desmethylimipramine on the t o t a l formation of g l u c u r o n i d e , f r e e hydroxy and N-oxide m e t a b o l i t e s of i m i p ramine. Desmethylimipramine was preincubated f i v e minutes p r i o r to a d d i t i o n of imipramine 0.5 X 10 MA, 1 X 10 ~*M0, — 5 /~\ and 2 X 10 M O . Incubation f o r imipramine metabolism was f i f t e e n minutes.  1  65 -70  -60  -40 O  u  •P c o  o  -20  +5  I  1.65 Dose  ,  6.6  3.3  —6 (X 10  L_  13 .2  M Desmethylimipramine )  F i g u r e 7on the t o t a l f o r m a t i o n o f E f f e c t of desmethylimipramine g l u c u r o n i d e O , f r e e h y d r o x y ^ o r N-oxide A m e t a b o l i t e s of imipramine. The i n c u b a t i o n time was f i f t e e n minutes -5 f o r imipramine metabolism (1 X 10 M).Desmethylimipramine was p r e i n c u b a t e d f i v e minutes p r i o r to imipramine metabolism.  66  _L  1.65 Dose  3.3  .-6,  (X 10 M U  6.6  13.2  Desmethylimipramine)  Figure 8 E f f e c t of desmethylimipramine on endogenous formation o f desmethylimipramine from imipramine metabolism. Desmethylimipramine was preincubated f i v e minutes p r i o r to a d d i t i o n 5 .-5.1 >, and 2X10 5M O . of imipramine 0.5 X 10 MA, 1 X lO'^M Incubation time was f i f t e e n minutes.  67 Desmethylimipramine  was p r e i n c u b a t e d f i v e m i n u t e s  prior to  a d d i t i o n o f imipramine f o r m e t a b o l i s m by t h e p e r f u s e d r a t l i v e r . The e x p e r i m e n t s w e r e t e r m i n a t e d a f t e r perfusate lites.  bile,  and l i v e r were a n a l y z e d f o r i m i p r a m i n e and i t s metabo-  The r e s u l t s  glucuronide, These r e s u l t s  are presented i n Figure  free hydroxy  glucuronide,  7 f o rinhibition of  o r N-oxide m e t a b o l i t e s o f imipramine.  show t h a t g l u c u r o n i d e ,  m e t a b o l i t e s were i n h i b i t e d of  f i f t e e n m i n u t e s . The  free hydroxy o r N-oxide  t o t h e same e x t e n t .  The f o r m a t i o n  f r e e hydroxy o r N-oxide m e t a b o l i t e s o f i m i p r a -  m i n e was i n h i b i t e d  50 p e r c e n t  (Figure  7) w h e n p r e t r e a t e d  with  -6 d e s m e t h y l i m i p r a m i n e 6.6 of  the effect  tion  X 10  M. I n F i g u r e  8 are the results  o f d e s m e t h y l i m i p r a m i n e on t h e endogenous  forma-  o f desmethylimipramine from imipramine metabolism.  inhibition  of metabolism occurred only  An  at higher substrate  concentrations. Distribution  Alteration  of the concentration of imipramine a t the s i t e  w h e r e i t was m e t a b o l i z e d , i n t h i s its  metabolism.  case t h e l i v e r ,  I t w o u l d be p o s s i b l e  could  that a preincubation  a n o t h e r compound b e f o r e a d d i t i o n o f i m i p r a m i n e c o u l d d i s t r i b u t i o n between t h e l i v e r its  metabolism.  of  alter  In a l lexperiments the per cent d i s t r i b u t i o n  d e t e r m i n e d . The e f f e c t distribution  with  alter i t s  and p e r f u s a t e and i n t u r n  metabolite o r imipramine i n the b i l e ,  the  affect  p e r f u s a t e and l i v e r  of a  was  o f p r e i n c u b a t i o n o f d e s m e t h y l i m i p r a m i n e on  o f i m i p r a m i n e a n d i t s m e t a b o l i t e s when t h e d o s e  i m i p r a m i n e was 0.5  X 10  -5  M , l X 10  -5  M o r 2 X 10  -5  M, a p p e a r s i n  Table XI E f f e c t of Desmethylimipramine on the D i s t r i b u t i o n of Imipramine and M e t a b o l i Imipramine HC1, 0.5 X 10~ M; I n c u b a t i o n Time F i f t e e n Minutes. 5  2 Perfusate GOH DMI  % Distribution Bile IMI GOH N-0  ± S.E. Liver GOH DMI  N-0  87.6 ±1.5  36.1 ±3.1  88.7 ±4.3  65.5  81.9  36.2  12.7  26.0  74.1  86.3  39.4  74.4  —  21.0  65.6  82.0  36.2  —  25.1  28.6  67.9  79.6  --  66.2  Dose of Desmethylimipramine  n  IMI  0  4  19.0 ±1.6  11.6 ±0.5  37.1 ±4.4  15.7 ±1.7  26.2 ±3.1  80.3 ±1.4  1.65 X 10~ M(0.5 mg.)  1  34 .3  17.4  41.4  81.2  20.8  3.3 X 10~ M(1 mg.)  1  25.7  13.2  32.3  23.1  6.6 X 10 M(2 mg.)  1  34 .1  13.2  28.0  13.2 X 10 M(4 mg.)  1  32.0  19.6  43.9  6  6  _6  _6  1  amount of desmethylimipramine added f i v e minutes p r i o r t o imipramine m e t a b o l i  2  %= ug of IMI, DMI, GOH o r N-0 i n l i v e r , p e r f u s a t e o r b i l e t o t a l ug of IMI, DMI, GOH or N-0 n= number of experiments  X 100  Table X I I E f f e c t o f Desmethylimipramine on the D i s t r i b u t i o n o f Imipramine and M e t a b o l i t e s . -5 Imipramine HC1 1 X 10 M; Incubation Time F i f t e e n Minutes. 2 ± S.E. Liver DMI GOH  n  IMI  0  4  20.2 ±2.4  14.3 ±1.2  39 .6 ±7.2  38.8 ±2.6  20.6 ±5.3  79.2 ±2.5  85.1 ±2.0  38.1 ±2.5  5 8.7 ±4.1  1.65 X 10 M(0.5 mg.)  1  29.0  18.8  30.6  17.8  22.1  70. 6  80. 0  47.2  83. 0  3.3 X 10~ M(1 mg.)  3  24.3 ±2.2  15.0 ±1. 4  39.3 ±5.0  34.2 ±2.1  21.3 ±1.0  75.5 + 2.0  84.4 ±1.3  38.8 ±4.3  62.7 ±1.1  6.6 X 10 M(2 mg.)  2  21.2  13. 8  36.6  22.2  22.9  78.4  85.6  39.5  76 .4  13.2 X 10 M(4 mg.)  1  23.1  17.2  41.2  29.3  0.0  76.5  82.3  58.7  70.2  _6  6  _6  _6  1 2  Perfusate DMI GOH  % Distribution Bile GOH N-0 IMI  Dose o f Desmethylimipramine  N-0  amount of desmethylimipramine added f i v e minutes p r i o r t o imipramine metabolism %= ug of IMI, DMI, GOH or N-0 i n l i v e r , p e r f u s a t e o r b i l e X 100 t o t a l ug of IMI, DMI, GOH or N-0 formed n= number of experiments  Table X I I I E f f e c t of Desmethylimipramine on the D i s t r i b u t i o n of Imipramine and M e t a b o l i t e s . Imipramine HC1 ,2 X 10~ M; Incubation Time f o r F i f t e e n Minutes. 5  2 Perfusate GOH DMI  % Distribution Bile IMI GOH  ± S.E. Liver GOH DMI  N-0  Dose of Desmethylimipramine  n  IMI  0  4  30.1 ±2.0  20.9 ±0.6  37.0 ±7.6  21.6 ±0.01  21.9 + 4.0  70.0 ±2.3  79.0 ±2.17  41.1 ±3.6  78.5 ±0.5  3.3 X 10~ M(1 mg.)  2  26.0  20.5  45.4  48.8  20.1  67.2  78.8  31.0  51.2  6.6 X 10~ M(2 mg.)  2  24.5  18.1  25.9  37.5  5.0  70.7  81.2  67.53  62.5  13.2 X 10~ M(4 mg.)  1  28.0  18.4  35.2  4.0  67.0  81.0  61.8  1  6  6  6  1 2  N-0  amount o f desmethylimipramine added f i v e minutes p r i o r t o imipramine metabolism %= ug of IMI, DMI, GOH or N-0 i n l i v e r , p e r f u s a t e o r b i l e X 100 t o t a l ug of IMI, DMI, GOH or N-0 formed n= number of experiments  71 Table X I , X I I and X I I I . Pretreatment o f desmethylimipramine f o r experiments -5 -5 where t h e dose o f imipramine was 1 X 10  M o r 2 X 10  M metabo-  l i z e d f o r f i f t e e n minutes had no e f f e c t on t h e d i s t r i b u t i o n o f imipramine and desmethylimipramine. The per cent d i s t r i b u t i o n of imipramine and desmethylimipramine was t h e same as the c o n t r o l experiments. The i n h i b i t i o n o f imipramine metabolism -5 -5 (1 X 10 M and 2 X 10 M) by d e c r e a s i n g the amount o f imipramine available  t o the l i v e r t o be metabolized was r u l e d out i n  these experiments. Some a l t e r a t i o n i n d i s t r i b u t i o n c o u l d be -5 seen a t t h e s u b s t r a t e c o n c e n t r a t i o n o f 0.5 X 10 M. Imipramine i n c r e a s e d 10 per cent i n the p e r f u s a t e and decreased 10 t o 15 per cent i n the l i v e r when desmethylimipramine was p r e s e n t .  E f f e c t o f E t h y l A l c o h o l on Aromatic H y d r o x y l a t i o n and N-demethylation  I t was found t h a t exogenous desmethylimipramine d i d i n h i b i t aromatic h y d r o x y l a t i o n of imipramine. T h e r e f o r e , i t was thought t h a t the desmethylimipramine formed from imipramine metabolism c o u l d suppress aromatic h y d r o x y l a t i o n of imipramine. To f u r t h e r demonstrate t h a t desmethylimipramine had t h i s e f f e c t ,  inhibi-  t i o n of desmethylimipramine formation from imipramine metabolism at h i g h s u b s t r a t e c o n c e n t r a t i o n should r e s u l t i n an i n c r e a s e i n aromatic h y d r o x y l a t i o n o f imipramine. Therefore, experiments were c a r r i e d out t o demonstrate t h i s e f f e c t .  72 Two  i n t e r e s t i n g p r o p e r t i e s had been reported i n t h e l i t e r a -  t u r e f o r e t h y l a l c o h o l . F i r s t l y e t h y l a l c o h o l was repo-rted t o i n h i b i t aromatic h y d r o x y l a t i o n or N-demethylation (47, 48, 49, 50) of drugs metabolized by microsomes. Therefore i t would be of i n t e r e s t t o f i n d out i f t h i s compound would i n h i b i t N-demethyl a t i o n and aromatic h y d r o x y l a t i o n of imipramine.  I f N-demethyla-  t i o n o f imipramine metabolism was s e l e c t i v e l y suppressed by e t h y l a l c o h o l , then t h i s compound would be used t o f i n d o u t whether i n h i b i t i o n o f desmethylimipramine a t h i g h s u b s t r a t e c o n c e n t r a t i o n o f imipramine metabolism would r e s u l t i n an i n c r e a s e i n aromatic h y d r o x y l a t i o n . Secondly, i n other experiments r e ported i n the l i t e r a t u r e e t h y l a l c o h o l caused an i n c r e a s e i n NADH content i n the l i v e r  (51, 52, 53). This i n c r e a s e i n l i v e r  NADH content c o i n c i d e s w i t h an i n c r e a s e i n the r a t i o o f l a c t i c a c i d t o p y r u v i c a c i d . The normal r a t i o o f l a c t i c a c i d t o p y r u v i c a c i d was 10, a f t e r e t h y l a l c o h o l pretreatment  the p e r f u s a t e  r a t i o i n c r e a s e d t o 125 o r h i g h e r . This i n c r e a s e i n r a t i o was due to a decrease i n p y r u v i c a c i d when the i s o l a t e d perfused r a t l i v e r technique was used (54, 5 5 ) . The f o l l o w i n g experiments were c a r r i e d out t o i n v e s t i g a t e the e f f e c t of e t h y l a l c o h o l on N-demethylation and aromatic h y d r o x y l a t i o n o f imipramine.  The same procedure and dose o f  e t h y l a l c o h o l was used as was r e p o r t e d by Vendsborg and Schanbye (54) t o i n c r e a s e the l a c t a t e / p y r u v a t e r a t i o i n the p e r f u s a t e of the i s o l a t e d r a t l i v e r p r e p a r a t i o n . In one experiment, e t h y l a l c o h o l was added t w i c e , p r i o r t o imipramine metabolism. The dose o f e t h y l a l c o h o l was 3 mM and was added f o r t y f i v e minutes  73 p r i o r t o adding 1 X 10  -5  M imipramine. The second dose of e t h y l  a l c o h o l was 1.5 mM and was added f i f t e e n minutes p r i o r t o i m i p r a mine metabolism. I t was found t h a t the amount of d e s m e t h y l i m i p r a mine formed decreased from 35 per cent t o 10 per cent of the dose of imipramine (Table X I V ) , however t h e r e was l i t t l e change i n aromatic hydroxylase a c t i v i t y . The amount of g l u c u r o n i d e and f r e e h y d r o x y l a t e d m e t a b o l i t e formed were s i m i l a r t o the c o n t r o l v a l u e of 2 8 per cent. The l a c t a t e t o pyruvate r a t i o i n c r e a s e d f o u r f o l d from the c o n t r o l v a l u e of 10. I n another experiment the same e x p e r i m e n t a l procedure was f o l l o w e d except the dose of e t h y l a l c o h o l was reduced t o 1.5 mM and 0.75 mM p r i o r t o a d d i t i o n of imipramine. The reason the dose of e t h y l a l c o h o l was decreased was t o decrease the l a c t a t e - p y r u v a t e r a t i o t o normal l e v e l s of 10 and f i n d out i f i n h i b i t i o n of N-demethylation would occur. Although the dose of e t h y l a l c o h o l was decreased t o one h a l f the dose p r e v i o u s l y used the l a c t a t e t o pyruvate r a t i o was g r e a t e r than the c o n t r o l v a l u e of 10; i n f a c t , p y r u v i c a c i d c o u l d not be d e t e c t e d . In a d d i t i o n t o an i n c r e a s e i n l a c t a t e t o pyruvate r a t i o , s p e c i f i c i n h i b i t i o n of N-demethylation was observed. In both experiments e t h y l a l c o h o l t o t a l l y suppressed b i l e s e c r e t i o n . However, the q u a n t i t y of g l u c u r o n i d e m e t a b o l i t e s of imipramine formed was u n a f f e c t e d . Although i t was found t h a t e t h y l a l c o h o l s e l e c t i v e l y suppress-5 ed N-demethylation of imipramine (1 X 10  M), more experiments  were done t o determine i f the i n c r e a s e i n r a t i o of l a c t i c a c i d or i n c r e a s e i n NADH was r e s p o n s i b l e f o r the i n h i b i t i o n of N-dem e t h y l a t i o n of imipramine. These experiments were designed t o f i n d the maximum q u a n t i t y of e t h y l a l c o h o l .  74  Table  XIV  E f f e c t o f E t h y l A l c o h o l on Iraipramine Me-tabalisa Y the I s o l a t e d Perfused Rat L i v e r ; F i f t e e n Minutes IncubaB  -5  t x o n , 1 X 10  M .was the S u b s t r a t e  Concentration.  Per Cent of Dose M e t a b o l i z e d 1 Dose o f Ethyl Alcohol  n  0  1  0.25 BM  2  0.5 aM  1  1 mM  1  3 am  1  1.5 mM+ 0.75 IBM 3 mM+ 1.5 mM  2  2  Lactate/ Pyruvate mg./lOOal. 55.5 =10 , 5.3  GOH  34.8  27.9  24.2  3.7  6.3  26_ 0  26.2  25.5  22.0  3.5  3.9  26 0  28.9  27.6  24.0  3.4  4.3  22  20.6  26.3  21.2  5.1  3.9  28  15.7  23.2  19.1  4.1  4.3  rt  0  0  1  59 , 1-4  1  41.5 0  Q  G  OH  N-0  DMI  15.5  25.0  21.0  3.92  2.7  10.3  26.8  22.3  4.5  2.9  e t h y l a l c o h o l added t o p e r f u s a t e f o r t y f i v e minutes p r i o r t o imipramine m e t a b o l i s a f i r s t dose o f e t h y l a l c o h o l added to p e r f u s a t e f o r t y f i v e minutes p r i o r to imipramine metabolism then h a l f the f i r s t dose added f i f t e e n iuinutes p r i o r to i m i p r a mine metabolism per cent IMI metabolized or m e t a b o l i t e formed= t o t a l IMI, DMI, GOH, OH or N-0 formed X 100 14 total C recovered  75 which would b r i n g about s i g n i f i c a n t i n h i b i t i o n of desmethylimipramine. I n these experiments the dose o f e t h y l a l c o h o l a d m i n i s t e r ed was decreased t i l l i n h i b i t i o n of N-demethylation was no l o n g er observed. The experiments were done i n the f o l l o w i n g  way.  E t h y l a l c o h o l was added f o r t y f i v e minutes p r i o r t o a d d i t i o n of imipramine 1 X 10 —5 M o r 0.5 X 10 -5M. The i n c u b a t i o n time was f i f t e e n minutes. The doses of e t h y l a l c o h o l used were 3 mM,  1 mM and 0.5 mM.  The r e s u l t s appear i n Table XIV f o r the  e f f e c t of e t h y l a l c o h o l on imipramine metabolism (1 X 10~^M) and Table XV f o r the e f f e c t of e t h y l a l c o h o l on imipramine metabolism (0.5 X 10~ M). 5  E t h y l a l c o h o l i n h i b i t e d N-demethylation of imipramine, when -5 1 X 10  M o f imipramine was incubated f o r f i f t e e n minutes. The  maximum i n h i b i t i o n of N-demethylation was a t t a i n e d w i t h 3 mM of e t h y l a l c o h o l ; however, t h e r e was some decrease i n aromatic h y d r o x y l a t i o n as w e l l . When the dose of e t h y l a l c o h o l was 1 mM „ N-demethylation was i n h i b i t e d 41 per cent. The amount of g l u c u ronide and f r e e hydroxy m e t a b o l i t e s of imipramine were the same as the c o n t r o l v a l u e . Although the amount o f desmethylimipramine decreased from 1 mg. t o 0.6 mg. w i t h 1 mM of e t h y l a l c o h o l a f t e r f i f t e e n minutes o f imipramine metabolism, t h e r e was no e f f e c t on the aromatic h y d r o x y l a t i o n of imipramine. T h e r e f o r e , aromatic h y d r o x y l a t i o n had not been suppressed by the formation of desmethylimipramine from imipramine at t h i s dosage l e v e l . I t was found t h a t the i n c r e a s e i n l a c t a t e - p y r u v a t e r a t i o or an i n c r e a s e i n NADH caused by e t h y l a l c o h o l had no r e l a t i o n s h i p t o the i n h i b i t i o n of N-demethylation s i n c e the formation of desmethylimipramine was not i n h i b i t e d by 0.5 mM of e t h y l  76 alcohol, although the r a t i o of l a c t i c acid t o pyruvic  acid  i n c r e a s e d s i g n i f i c a n t l y . I n f a c t , p y r u v i c a c i d c o u l d n o t be detected i n the perfusate. The r e s u l t s f o r t h e e f f e c t o f e t h y l a l c o h o l on i m i p r a m i n e -5 m e t a b o l i s m when t h e dose o f i m i p r a m i n e was 0.5 X 10  M appear  i n T a b l e XV. I t was f o u n d t h a t when t h e d o s e o f e t h y l  alcohol  was 0.5 mM o r 1 mM t h e r e was no e f f e c t on N - d e m e t h y l a t i o n o f i m i p r a m i n e . A h i g h e r dose o f e t h y l a l c o h o l 3 mM, c a u s e d  inhi-  b i t i o n o f N - d e m e t h y l a t i o n b u t h a d v e r y l i t t l e e f f e c t on a r o m a t i c hydroxylation. -5 When t h e dose o f i m i p r a m i n e was 2 X 10  M i t was t h o u g h t  t h a t t h e d e s m e t h y l i m i p r a m i n e formed f r o m i m i p r a m i n e m e t a b o l i s m a f t e r f i f t e e n m i n u t e s and s i x t y m i n u t e s c a u s e d s u p p r e s s i o n o f a r o m a t i c h y d r o x y l a t i o n o f i m i p r a m i n e . To show t h i s e f f e c t , a d o s e o f e t h y l a l c o h o l was chosen w h i c h w o u l d i n h i b i t t h e f o r m a t i o n o f d e s m e t h y l i m i p r a m i n e . E t h y l a l c o h o l (1 mM) i n h i b i t s N-de-5 m e t h y l a t i o n ( i m i p r a m i n e 1 X 10 M) b y 40 p e r c e n t and had no e f f e c t on a r o m a t i c h y d r o x y l a t i o n . When e t h y l a l c o h o l 1 mM was -5 a d m i n i s t e r e d f o r t y f i v e minutes p r i o r t o imipramine  (2 X 10  and m e t a b o l i s m f o l l o w e d f o r s i x t y m i n u t e s i t was found  M)  (Table  XVI) t h a t t h e amount o f d e s m e t h y l i m i p r a m i n e formed d e c r e a s e d f r o m 421 u g / gm. l i v e r t o 308 u g . / gm. l i v e r . The amount o f i m i p r a m i n e w h i c h was m e t a b o l i z e d t o g l u c u r o n i d e , f r e e h y d r o x y and N - o x i d e i n c r e a s e d f r o m 325 ug t o 366 u g / gm. l i v e r . When 1 mM e t h y l was p r e i n c u b a t e d f o r t y f i v e m i n u t e s p r i o r t o i m i p r a m i n e f o r f i f t e e n minutes  alcohol -5  (2 X 10  ( T a b l e XVI) t h e amount o f d e s m e t h y l i m i p r a -  mine d e c r e a s e d f r o m 211 u g / gm. l i v e r t o 132 u g / gm. l i v e r and  M)  77 Table XV E f f e c t o f E t h y l A l c o h o l on Imipramine Metabolism By the I s o l a t e d Perfused Rat L i v e r ; F i f t e e n Minutes I n c u b a t i o n With -5 0.5 X 10 M Imipramine. 2 Per Cent of Dose M e t a b o l i z e d Dose o f Ethyl Alcohol 1  0  n  Lactate/ Pyruvate mg./100ml. 21 - i n  1  DMI 2 6  '  3  GOH 4 0  -  2  G 3  5  ,  0  OH 5  '  4  N-0 4 , 8  0.5 mM  1  30 0  23.9  38.7  33.2  5.6  1 mM  3  25 19.5 0 "0  23.3 ±1.3  38.7 ±1.7  35.9 ±2.3  2.7 6.5 ±0.9 ±1.6  26 0  12.9  35.8  33.9  1.9  ;  3 mM  1 2  1  2.6  5.3  e t h y l a l c o h o l added t o p e r f u s a t e f o r t y f i v e minutes p r i o r t o imipramine metabolism per cent IMI metabolized o r m e t a b o l i t e formed= t o t a l IMI, DMI, GOH, OH o r N-0 formed X 100 t o t a l C recovered 1 4  78 the  amount of g l u c u r c n i d e , f r e e hydroxy and N-oxide m e t a b o l i t e s  of imipramine i n c r e a s e d from 158 ug/ gm. l i v e r t o 196 ug/  gm.  l i v e r . In two of t h r e e experiments i n each group o f experiments the  b l o o d flow through the l i v e r was below 2 ml./ gm. l i v e r per  minute and t h e r e f o r e the data was not used. As a r e s u l t o n l y one experiment i n each group was From these r e s u l t s  reported.  i t can be seen t h a t when e t h y l a l c o h o l  caused i n h i b i t i o n of desmethylimipramine f o r m a t i o n t h e r e was an i n c r e a s e i n f o r m a t i o n of h y d r o x y l a t e d m e t a b o l i t e s . T h i s -5 o c c u r r e d when 2 X 10  M o f imipramine was m e t a b o l i z e d f o r f i f -  teen and s i x t y minutes. When the dose of imipramine was  0.5  or 1 X 10 ^M, i n h i b i t i o n of desmethylimipramine by e t h y l a l c o h o l d i d not a f f e c t aromatic h y d r o x y l a t i o n . At a l l doses and i n c u b a t i o n times t h i n l a y e r  chromatography  was done t o i d e n t i f y the hydroxy m e t a b o l i t e s formed from i m i p r a mine metabolism. The predominant m e t a b o l i t e formed was  2-hydroxy-  imipramine. Small amounts of u n i d e n t i f i e d m e t a b o l i t e s , as w e l l as 2-hydroxydesmethylimipramine, were formed when the dose o f imipramine was 2 X 10  and metabolism was f o r s i x t y minutes.  However, the primary hydroxy m e t a b o l i t e formed from imipramine was 2-hydroxyimipramine and very l i t t l e 2-hydroxydesmethylimipramine. I f 2-hydroxydesmethylimipramine was a major m e t a b o l i t e , then i n h i b i t i o n of desmethylimipramine formation by e t h y l a l c o h o l would r e s u l t i n a decrease i n the amount of g l u c u r o n i d e and f r e e hydroxy m e t a b o l i t e formed. T h i s d i d not happen w i t h any e x p e r i ment where desmethylimipramine was  inhibited.  Table XVI E f f e c t of E t h y l A l c o h o l on Imipramine Metabolism By the I s o l a t e d Perfused Rat L i v e r . The Incubation Time was F i f t e e n and S i x t y Minutes, Substrate -5 Concentration was 2 X 10 M. Incubation Time  Dose o f Ethyl Alcohol  1  IMI % o f dose Remaining  ug/ gm. L  DMI % o f dose Metabolized  ug/ gm. L  GOH,N-0 % o f dose Metabolized  ug/ gm. L  60 n=4  0.0  19.7 + 2.1  162.1 ±21.3  50.4 ±5.5  421.7 ±80.9  29.8 ±4.5  235.3 ±23.1  60  1 mM  13.2  102  40.1  308.0  46.6  366  15 n=4  0.0  56.9 ±1.5  478.9 ±43.3  25.4 ±1.5  211.6 ±14.4  19.1 ±1.4  158.4 ±8.7  15 n=l  1 mM  47.5  365  19.0  132.0  28.48  196  n =1  1  e t h y l a l c o h o l added t o p e r f u s a t e f o r t y f i v e minutes p r i o r to imipramine metabolism.  Table XVII E f f e c t o f E t h y l A l c o h o l on the D i s t r i b u t i o n o f Imipramine and M e t a b o l i t e s . -5 HC1 (1 X 10 M); Incubation Time F i f t e e n Minutes. % Distribution Dose of Ethyl Alcohol  Perfusate GOH DMI  n  IMI  0  1  22.6  14.1  49.0  0.25 mM  2  28.1 + 4.76  18.93 ±6.52  0.5 mM  1  23.55  1 mM  1  3 mM  N-0  Bile GOH  N-0  77.0  85.2  29.5  54.4  45.14 41.3: 16.04 ±0.76 ±3.3 ±2.24  71.54 ±8.0  80.04 ±6.9  36.61 ±1.65  52.55 ±1.48  15.84  39.40 15.5] 21.36  76.32  83.25  37.13  84.09  30.17  15.31  45.89 25.5E 13.18  69.76  83.81  38.9  69.56  1  34.79  20.99  65.71 34.0^  0.81  65.20  77.80  30.54  63. 59  1.5 mM , 0.75 mM  1  24.8  17.2  62.3  55.7  4.8  75.2  81.8  30.1  42.0  3 mM , 1.5 mM  1  25.9  15.4  76.0  63.3  0.0  74.1  83.5  20.7  46.7  35.1  19.4  IMI  ± S.E. Liver DMI GOH  Imipramine  2  2  1 2 3  e t h y l a l c o h o l added to p e r f u s a t e f o r t y f i v e minutes p r i o r t o imipramine metabolism. f i r s t dose of e t h y l a l c o h o l added to p e r f u s a t e f o r t y f i v e minutes p r i o r to imipramine metabolism then h a l f f i r s t dose added f i f t e e n minutes p r i o r t o imipramine metabolism. CO %= ug o f IMI,DMI, GOH o r N-0 i n l i v e r , p e r f u s a t e o r b i l e X 100 o t o t a l ug o f IMI, DMI, GOH o r N-0 formed  Table X V I I I E f f e c t o f E t h y l A l c o h o l on the D i s t r i b u t i o n o f Imipramine and M e t a b o l i t e . Imipramine HC1 (0.5 X 10~ M), Incubation Time F i f t e e n Minutes. 5  Dose o f Ethyl Alcohol  n  IMI  Perfusate GOH DMI  N-0  Bile GOH  % Distribution ± S.E. Liver DMI GOH N-0 IMI  0  4 19.0 + 1.6  11.6 ±0.53  37.1 ±4.4  15.7 + 1.73  26.2 ±3.1  80.3 +1.4  87.6 ±1.5  36.1 ±3.1  88.7 ±4.3  0.5 mM  1 27.76  14.85  42.08  87.85  24.18  72.11  84.17  31.44  5.94  1 mM  3 27.5 + 2.1  14.65 ±1.21  48.4 ±3.7  18.46 ±5.7  19.02 ±9.8  72.57 ±2.9  84.58 ±1.25  27.6 ±1.5  60.12 ±9.52  3 mM  1 32.15  18.63  63.93  44.75  5.68  67.80  80.58  27.92  50. 59  1 2  e t h y l a l c o h o l added t o p e r f u s a t e f o r t y f i v e minutes p r i o r t o imipramine metabolism %= ug o f IMI, DMI, GOH o r N-0 i n l i v e r , p e r f u s a t e o r b i l e X 100 t o t a l ug of IMI, DMI, GOH o r N-0 formed  82 Distribution  I f a l t e r a t i o n o f imipramine c o n c e n t r a t i o n a t the s i t e o f metabolism could a f f e c t i t s metabolism then i t would be p o s s i b l e t h a t a pretreatment c o u l d a l t e r the d i s t r i b u t i o n of imipramine and i n t u r n a l t e r the metabolism. In a l l experiments the p e r cent d i s t r i b u t i o n of a m e t a b o l i t e o r imipramine i n the b i l e , p e r f u s a t e and l i v e r was determined. The e f f e c t o f e t h y l a l c o h o l on the d i s t r i b u t i o n o f imipramine and i t s m e t a b o l i t e s (0.5 X -5 -5 -5 10 M, 1 X 10 M and 2 X 10 M imipramine) appears i n Table XVII and X V I I I . Compared t o c o n t r o l s t h e r e was a s l i g h t decrease o f i m i p r a mine and desmethylimipramine i n the l i v e r s which were p r e t r e a t ed w i t h e t h y l a l c o h o l . The decrease o r change i n d i s t r i b u t i o n of imipramine was not s i g n i f i c a n t enough t o a l t e r the metabolism -5 of imipramine s i n c e imipramine metabolism (0.5 X 10  M) was  not a l t e r e d although there was a s l i g h t change i n the d i s t r b u tion pattern. D i s c u s s i o n of Imipramine Metabolism  The o b j e c t i v e of these experiments was t o g a i n more knowledge and a b e t t e r understanding of imipramine metabolism i n the i s o l a t e d perfused r a t l i v e r before c o n t i n u i n g r e s e a r c h on p o s s i b l e c e l l u l a r c o n t r o l mechanisms i n v o l v e d i n imipramine metabolism. The d e c l i n e i n plasma c o n c e n t r a t i o n of many drugs i s f r e q u e n t l y assumed t o f o l l o w apparent f i r s t order k i n e t i c s  independent  83 of the dose o r plasma c o n c e n t r a t i o n . D e s p i t e r e p o r t s i n the l i t e r a t u r e t h a t dose dependent k i n e t i c s f o r drug metabolism have been demonstrated i n v i v o and i n the i s o l a t e d p e r f u s e d r a t l i v e r f o r c e r t a i n drugs, r e s e a r c h i s done on drug metabolism i n the i s o l a t e d p e r f u s e d r a t l i v e r without f i r s t a c q u i r i n g knowledge about the r e l a t i o n s h i p o f s u b s t r a t e c o n c e n t r a t i o n t o the r a t e o f drug metabolism. S i n c e imipramine a t c e r t a i n c o n c e n t r a t i o n s i n h i b i t s i t s own metabolism i n microsomal s t u d i e s i t was necessary to f i n d out i f dose dependent k i n e t i c s would occur i n the  i s o l a t e d perfused r a t l i v e r . .  The r a t e of i m i p r a -  mine metabolism was dependent on two p r i n c i p a l enzymatic r e a c t i o n s , aromatic h y d r o x y l a t i o n and N-demethylation. I t i s p o s s i b l e t h a t a t c e r t a i n c o n c e n t r a t i o n s of imipramine one enzymatic r e a c t i o n c o u l d reach s a t u r a t i o n k i n e t i c s w h i l e the o t h e r enzymatic r e a c t i o n might not. I t had been demonstrated i n t h i s r e s e a r c h t h a t aromatic h y d r o x y l a t i o n was i n h i b i t e d a t h i g h e r c o n c e n t r a t i o n s -5 of imipramine (2 X 10  M). I t a l s o had been shown t h a t a t l o n g e r  i n c u b a t i o n time (eg. s i x t y minutes) aromatic h y d r o x y l a t i o n was suppressed. In most experiments u t i l i z i n g the i s o l a t e d p e r f u s e d l i v e r technique to study drug metabolism the p e r f u s a t e h a l f - l i f e of the drug was the o n l y parameter monitored  (th)  (56,57,58,59,60).  T h i s parameter was used t o determine the h a l f - l i f e o r r a t e cons t a n t f o r drug metabolism. This may have l e d to erroneous r e s u l t s s i n c e t h i s parameter  (th) may not be a t r u e i n d i c a t i o n of drug  metabolism as was i n d i c a t e d i n t h i s t h e s i s . I t was found t h a t over extended i n c u b a t i o n time the metabolism of imipramine  84 (2 X 10 — 5M) was p a r t i a l l y i n h i b i t e d although the h a l f - l i f e  was  the same over the e n t i r e i n c u b a t i o n time and t h e r e f o r e t h i s i n h i b i t i o n would not be d e t e c t e d . Comparing p e r f u s a t e drug c o n c e n t r a t i o n s to determine i f a d i f f e r e n c e i n drug metabolism had o c c u r r e d , due t o v a r i o u s p r e t r e a t m e n t s , c o u l d l e a d to f a l s e c o n c l u s i o n s . An example of where a change i n p e r f u s a t e c o n c e n t r a t i o n had o c c u r r e d although the r a t e o f imipramine metabolism was s i m i l a r was shown i n the study of the e f f e c t of e t h y l a l c o h o l on imipramine metabolism. I t was found t h a t the per cent o f imipramine i n c r e a s e d i n the p e r f u s a t e a f t e r treatment w i t h e t h y l a l c o h o l (Table X V I I I ) ; however, the metabolism of i m i p r a mine was u n a l t e r e d . T h e r e f o r e , i t i s necessary t o monitor o t h e r parameters i n a d d i t i o n t o p e r f u s a t e c o n c e n t r a t i o n , such as the amount o f a drug which i s i n the l i v e r . Another reason f o r a n a l y z i n g the t o t a l amount o f imipramine i n the i s o l a t e d p e r f u s e d system i s t h a t at h i g h e r doses of imipramine the l i v e r may be r e a c h i n g a s a t u r a t e d c o n d i t i o n w i t h r e s p e c t to i m i p r a mine and desmethylimipramine b i n d i n g and t h e r e f o r e the p e r f u sate c o n c e n t r a t i o n w i l l be h i g h e r . The f r a c t i o n of the t o t a l amount of imipramine p r e s e n t i n the l i v e r s t e a d i l y decreased -5 from 80 per cent w i t h a dose of 0.5 X 10 M to 70 per cent . . . -5 when a dose of imipramine 2 X 10  M was used. T h i s means t h a t  the per cent i n the p e r f u s a t e i n c r e a s e d from 19 per cent to 30 perFrom cent. r e s ut lh ti sn were f o r desmethylimipramine. the The r e s usame l t s of l a y e r found chromatography and i n h i b i t i o n of desmethylimipramine formation by e t h y l a l c o h o l the  85 primary enzymatic r e a c t i o n s f o r the degradation of imipramine at f i f t e e n minutes was N-demethylation  and aromatic h y d r o x y l a -  t i o n . Although some 2-hydroxydesmethylimipramine was i s o l a t e d from t h i n l a y e r chromatography s t u d i e s a f t e r s i x t y  minutes,  i t does n o t seem t o be a major m e t a b o l i c r o u t e . The o t h e r minor m e t a b o l i c route f o r degradation of imipramine was N - o x i d a t i o n . Since some of the r e s u l t s f o r the formation o f imipramine were e r r a t i c and the c o n t r i b u t i o n t o imipramine  N-oxide  metabolism  was minor, very l i t t l e d i s c u s s i o n w i l l c e n t e r around t h e enzymatic r e a c t i o n o f N - o x i d a t i o n . The t o t a l amount o f f r e e hydroxy m e t a b o l i t e s p r e s e n t a f t e r imipramine metabolism was very low. Most of t h i s m e t a b o l i t e was i n the conjugated form as the g l u c u r o n i d e (Table X ) . The t o t a l amount o f f r e e hydroxy m e t a b o l i t e was approximately the same, 0.084, 0.12 and 0.082 mg. w h i l e t h e t o t a l amount of g l u c u r o n i d e m e t a b o l i t e was 0.544, 0.688 and 0.792 mg. which -5 i n c r e a s e d as the dose o f imipramine i n c r e a s e d (0.5 X 10  M,  1 X 10~ M and 2 X 10~ M). The i n c u b a t i o n time was f i f t e e n 5  5  minutes f o r these experiments. T h i s suggests t h a t the conjugat i o n r e a c t i o n was more r a p i d than the formation o f 2-hydroxyimipramine from imipramine. There appears to be a constant r e s i d u a l f r a c t i o n o f f r e e hydroxy m e t a b o l i t e s remaining  independ-  ent of the dose o f imipramine. The per cent of the dose of glucuronide and f r e e hydroxy m e t a b o l i t e s which was e x c r e t e d i n the b i l e was the same. This suggests t h a t b i l i a r y e x c r e t i o n o f these m e t a b o l i t e s was not  86 s a t u r a t e d . When h i g h doses o f e t h y l a l c o h o l were used, b i l e flow was i n h i b i t e d . Consequently t h e r e was very l i t t l e o r no e x c r e t i o n o f g l u c u r o n i d e m e t a b o l i t e s . The amount of aromatic hydroxy m e t a b o l i t e s formed i n these experiments were the same as c o n t r o l s (Table X I V , X V I I ) . T h e r e f o r e , i n these experiments, h y d r o x y l a t i o n of imipramine as w e l l as c o n j u g a t i o n of t h i s m e t a b o l i t e i s independent o f b i l i a r y e x c r e t i o n . The r a t e of imipramine metabolism was a l t e r e d w i t h i n c u b a t i o n time and imipramine c o n c e n t r a t i o n . The metabolism of i m i p r a mine was dose dependent due t o decreased aromatic h y d r o x y l a s e -5  a c t i v i t y . At a h i g h e r dose of imipramine (2 X 10  M) aromatic  hydroxylase reached zero order k i n e t i c s . Experiments were then c a r r i e d out t o f i n d the reason why aromatic h y d r o x y l a s e was i n h i b i t e d . I t was found t h a t desmethylimipramine i n the same -5  q u a n t i t y formed from 2 X 10  M imipramine a f t e r f i f t e e n and  s i x t y minutes could i n h i b i t aromatic h y d r o x y l a t i o n and N-oxidat i o n . In a d d i t i o n , e t h y l a l c o h o l was found to i n h i b i t the format i o n of desmethylimipramine, and to i n c r e a s e the f o r m a t i o n of -5  h y d r o x y l a t e d m e t a b o l i t e s (imipramine, 2 X 10  M; i n c u b a t i o n  time f i f t e e n and s i x t y m i n u t e s ) . I t t h e r e f o r e could be c o n c l u d ed from these r e s u l t s t h a t the desmethylimipramine formed from imipramine metabolism i n h i b i t e d  aromatic h y d r o x y l a t i o n a f t e r  f i f t e e n and s i x t y minutes o f metabolism. The r a t e of aromatic h y d r o x y l a t i o n of imipramine was found t o be dependent on the c o n c e n t r a t i o n of imipramine. A l l of the above experiments l e d to the c o n c l u s i o n t h a t dose dependent k i n e t i c s a t h i g h e r con-  87 c e n t r a t i o n s of imipramine was due t o i n h i b i t i o n of the aromatic hydroxylase caused by the endogenous f o r m a t i o n of desmethylimipr mine. There have been r e p o r t s i n the l i t e r a t u r e t h a t desmethylimipramine may i n h i b i t the h y d r o x y l a t i o n r e a c t i o n i n the metabol i s m o f such drugs as p h e n o b a r b i t a l (23), tremorine (24) and amphetamine  (25). These s t u d i e s have been done i n v i v o and i n  v i t r o on r a t s . Desmethylimipramine i n h i b i t e d amphetamine metabol i s m i n the i s o l a t e d p e r f u s e d r a t l i v e r system. I n a l l these s t u d i e s o n l y the disappearance o f the drug was measured so i t i s not known i f , f o r example, p - h y d r o x y l a t i o n o f the aromatic r i n g was i n h i b i t e d i n the metabolism o f amphetamines. I n our l a b o r a t o r y i t was found t h a t desmethylimipramine d i d c o m p e t i t i v e l y i n h i b i t aromatic h y d r o x y l a t i o n o f imipramine i n mouse l i v e r homogenate  (61). Desmethylimipramine was m e t a b o l i z e d mainly  by aromatic h y d r o x y l a t i o n i n the i s o l a t e d p e r f u s e d l i v e r (20). I t would then seem t h a t desmethylimipramine c o u l d i n h i b i t aromatic h y d r o x y l a t i o n i n a c o m p e t i t i v e manner. More experiments are needed t o e s t a b l i s h whether t h i s i n h i b i t i o n was c o m p e t i t i v e . E t h y l a l c o h o l has been shown t o i n h i b i t the a c t i v i t i e s o f aniline  (47) o r p h e n o b a r b i t a l (48) microsomal hydroxylase  and the demethylation of aminopyrine (49) o r ethylmorphine (50). However e t h y l a l c o h o l was found t o be a s p e c i f i c i n h i b i t o r of N-demethylation of imipramine i n the i s o l a t e d p e r f u s e d r a t l i v e r system. The h i g h e s t dose o f e t h y l a l c o h o l used (Table XIV) reduced the formation of desmethylimipramine from 35 per cent to 10 per cent w i t h o u t s i g n i f i c a n t i n h i b i t i o n of aromatic  88 h y d r o x y l a t i o n . T h i s type of i n h i b i t i o n i s very complex and i s not of the c o m p e t i t i v e t y p e . Evidence f o r t h i s i s t h a t a 1 mM dose of e t h y l a l c o h o l i n h i b i t e d N-demethylation when a dose o f 1 X 10 M -5  imipramine was employed; however i n h i b i t i o n d i d not -5  occur when 0.5 X 10  M imipramine was  used.  There has been much s p e c u l a t i o n on whether t h e r e are one or many d i f f e r e n t drug m e t a b o l i z i n g enzymes f o r d i f f e r e n t s u b s t r a t e s . T i l l the cytochrome P-450 redox system i s p u r i f i e d i n a c t i v e enzymatic form o n l y i n d i r e c t evidence can be s u p p l i e d f o r one o r more than one cytochrome P-450 system. Evidence f o r more than one system has been based on the f o l l o w i n g k i n d s of o b s e r v a t i o n s . There are marked s p e c i e s d i f f e r e n c e s ( 2 ) , and sex d i f f e r e n c e s (3). Pretreatment of animals produces r e l a t i v e changes i n the metabolism of d i f f e r e n t drugs  (64,65). S p e c i f i c i t y  i s shown i n the k i n d s o f drug metabolism s t i m u l a t e d or i n h i b i t ed by c e r t a i n agents (66). A d d i t i o n a l evidence i s p r o v i d e d f o r more than one cytochrome P-450 system s i n c e e t h y l a l c o h o l was found t o i n h i b i t N-demethylation of imipramine w i t h o u t i n h i b i t i n g aromatic h y d r o x y l a t i o n . The most s u i t a b l e i n c u b a t i o n time f o r imipramine was f i f t e e n minutes s i n c e longer i n c u b a t i o n times may  inhibit  aromatic h y d r o x y l a t i o n of imipramine. The most s u i t a b l e dose of imipramine used t o study the v a r i o u s e f f e c t s on imipramine -5 metabolism was 1 X 10  M. At thxs dose aromatic h y d r o x y l a t i o n  was not suppressed and adequate amounts of imipramine remained fs ot ri m fu ul ra tt hi eo rn metabolism i f a c e r t a i n pretreatment r e s u l t e d i n of metabolism.  89 C e l l u l a r C o n t r o l Mechanisms f o r Imipramine  E f f e c t o f Magnesium on Imipramine  In  Metabolism  Metabolism  a l l p r e v i o u s l y r e p o r t e d e x p e r i m e n t s , t h e p e r f u s a t e magne-  sium s u l p h a t e c o n c e n t r a t i o n , f o r i m i p r a m i n e m e t a b o l i s m , i s o l a t e d p e r f u s e d r a t l i v e r was 0.014 mg./  i n the  m l . To d e t e r m i n e  whether t h e magnesium c o n c e n t r a t i o n i n t h e l i v e r was a t optimum c o n c e n t r a t i o n f o r maximum i m i p r a m i n e m e t a b o l i s m  the f o l l o w i n g  e x p e r i m e n t s were done. The c o n t r o l e x p e r i m e n t s c o n t a i n e d t h e normal p e r f u s a t e c o n c e n t r a t i o n o f magnesium  (0.014 mg./  ml.)  which t h e l i v e r was p e r f u s e d w i t h f o r one hour b e f o r e t h e a d d i t i o n of  i m i p r a m i n e HC1. The dose o f i m i p r a m i n e HC1 u s e d i n a l l t h e s e -5  e x p e r i m e n t s was I X fifteen  10  M  (3.17 mg.)  and t h e i n c u b a t i o n time was  m i n u t e s . I n a l l o t h e r e x p e r i m e n t s magnesium s u l p h a t e was  e i t h e r o m i t t e d from t h e p e r f u s i o n medium o r t w i c e t h e n o r m a l c o n c e n t r a t i o n o f magnesium s u l p h a t e (0.028 mg./ In  ml.) was  used.  t h e s e e x p e r i m e n t s as i n t h e c o n t r o l e x p e r i m e n t t h e l i v e r  was  then p e r f u s e d f o r one hour b e f o r e i m i p r a m i n e HC1 was added t o the  isolated perfused r a t l i v e r  a p p a r a t u s . To f i n d  o u t whether  o m i s s i o n o f magnesium from t h e p e r f u s i o n medium o r t w i c e t h e normal p e r f u s a t e magnesium c o n c e n t r a t i o n a l t e r e d t h e magnesium c o n c e n t r a t i o n i n the l i v e r ,  t h e p e r f u s a t e magnesium c o n c e n t r a -  t i o n was d e t e r m i n e d . Magnesium was a n a l y z e d i n t h e c i r c u l a t i n g p e r f u s i o n medium b e f o r e and a f t e r t h e l i v e r was p e r f u s e d f o r one hour. The d i f f e r e n c e i n p e r f u s a t e magnesium  concentration  b e f o r e and a f t e r p e r f u s i n g t h e l i v e r was a t t r i b u t e d t o a change i n l i v e r magnesium  concentration.  Table XIX E f f e c t o f Magnesium on Imipramine Metabolism i n the I s o l a t e d P e r f u s e d Rat L i v e r . The Incubation Time was F i f t e e n Minutes and the S u b s t r a t e C o n c e n t r a t i o n was 1 X 10 Magnesium Added to P e r f u s i o n Medium  Per Cent  Magnesium C o n c e n t r a t i o n n  Before P e r f u s i o n A f t e r P e r f u s i o n 1  2  M. 3  o f Dose M e t a b o l i z e d  IMI  DMI  GOH  G  N-0  0.014 mg./ ml.  1  0.015 mg./ ml.  0.016 mg./ ml.  37.2  32.0  27.5  22.0  5.2  0.00 mg./ ml.  1  0.001 mg./ ml.  0.002 mg./ ml.  35.0  30.0  30.0  20.0  4.1  0.028 mg./ ml.  1  0.031 mg./ ml.  0.032 mg./ ml.  29.2  35.0  30.7  24.5  2.9  1 2 3  p e r f u s a t e magnesium c o n c e n t r a t i o n p r i o r t o p e r f u s i o n w i t h l i v e r p e r f u s a t e magnesium c o n c e n t r a t i o n a f t e r p e r f u s i o n o f r a t l i v e r f o r one hour per cent= t o t a l IMI, DMI, GOH, G o r N-0 formed t o t a l C recovered x  1 0 Q  1 4  VO  o  91 I t was found t h a t changing t h e magnesium p e r f u s a t e concent r a t i o n had no e f f e c t on imipramine metabolism (Table X I X ) . The reason magnesium d i d n o t have an e f f e c t on imipramine metabolism was t h a t magnesium i n the l i v e r was n o t a l t e r e d s i n c e t h e magnesium c o n c e n t r a t i o n i n the p e r f u s a t e a f t e r p e r f u s i o n o f t h e l i v e r f o r one hour was t h e same as t h e c o n c e n t r a t i o n b e f o r e p e r f u s i o n of the l i v e r . T h i s o c c u r r e d e i t h e r w i t h an excess o r no magnesium i n the p e r f u s a t e . I t was a l s o found by o t h e r s (7) t h a t the  o m i s s i o n o f magnesium from the p e r f u s a t e d i d n o t cause mag-  nesium t o be r e l e a s e d by t h e l i v e r .  E f f e c t of D i b u t y r y l C y c l i c AMP and Glucagon on Imipramine Metabolism  To determine the e f f e c t o f glucagon HC1 ( E l i L i l l y and Co.) or the monosodium s a l t o f d i b u t y r y l c y c l i c AMP (Sigma Chemical Co.) on imipramine metabolism i n the i s o l a t e d p e r f u s e d r a t l i v e r — 8  the f o l l o w i n g experiments were done. Glucagon (5.7 X 10 M) o r d i b u t y r y l c y c l i c AMP (2 X 10 ^M) was added t o the p e r f u s i o n f l u i d t e n minutes p r i o r t o the a d d i t i o n of imipramine. I m i p r a -5 -5 mine (0.5 X 10  M o r 1 X 10  M) was incubated f o r f i f t e e n minutes  i n the i s o l a t e d p e r f u s e d r a t l i v e r apparatus. The r e s u l t s f o r these experiments appear i n Table XX. These experiments were done f o r t h e f o l l o w i n g reasons. Desmethylimipramine 0.5 mM has been shown t o i n c r e a s e c y c l i c AMP l e v e l s i n b r a i n s l i c e s (68,69) and i n a r e c e n t r e p o r t (60) d i b u t y r y l c y c l i c AMP and glucagon were found t o i n h i b i t drug metabolism i n the i s o l a t e d p e r f u s e d  92 Table XX E f f e c t of D i b u t y r y l C y c l i c AMP o r Glucagon, on Imipramine Metabolism. I n c u b a t i o n Time was f o r F i f t e e n Minutes With the I s o l a t e d P e r f u s e d Rat L i v e r . The Dose of Imipramine was 0.5 X 10~ M or 1 X 10~ M. 5  Treatment  1  n  5  Per Cent of Dose M e t a b o l i z e d 2 N-0 G GOH DMI IMI Remaining  C o n t r o l j. 0.5 X 10 M IMI  1  23.8  30.9  39.1  35.3  6.2  Glucagon  1  24.6  32.3  35.3  35.1  6.5  DBc AMP  2  26.3  25.5  42.3  41.6  6.5  Control 1 X 10~ M IMI  1  23.8  39.2  30.8  28.6  6.0  Glucagon  1  33.9  35.6  26.7  21.8  3.9  DBc AMP  1  44.5  30.6  22.5  19.0  3.0  5  1  2  Glucagon (5.7 X 10" M ) , d i b u t y r y l c y c l i c AMP (DBc 2 X 10 M was added t e n minutes p r i o r to imipramine 8  metabolism. per cent= ug of IMI, DMI, G o r N-0 formed ug o f C recovered 1 4  1  x  1 Q 0  93 r a t l i v e r . The mechanism f o r the i n h i b i t i o n of imipramine a r o matic h y d r o x y l a t i o n i n the i s o l a t e d r a t p e r f u s e d l i v e r by desmethylimipramine c o u l d be by i n c r e a s i n g c y c l i c AMP the r a t l i v e r . This e l e v a t i o n of c y c l i c AMP  levels i n  i n the l i v e r  cell  c o u l d then i n h i b i t aromatic h y d r o x y l a t i o n o f imipramine. I t was shown t h a t d i b u t y r y l c y c l i c AMP  could i n h i b i t hexobarbital  and p - c h l o r o - N - m e t h y l a n i l i n e metabolism. However, would s p e c i f i c i n h i b i t i o n of N-demethylation or aromatic h y d r o x y l a t i o n occur from the same s u b s t r a t e as was found f o r e t h y l a l c o h o l  inhibition  of aromatic h y d r o x y l a t i o n ? The d i b u t y r y l analogue of c y c l i c was used because i t has been demonstrated f e c t s t o those caused by c y c l i c AMP  AMP  t o produce p a r a l l e l e f -  a t lower concentration(70)because  of i n c r e a s e d p e n e t r a t i o n through c e l l u l a r membranes and decreased r a t e of d e s t r u c t i o n by phosphodiesterase. Glucagon was used i n these experiments because i t i n c r e a s e d c e l l u l a r l e v e l s of c y c l i c AMP  (71,72) as was presumed t o occur f o r desmethylimipramine. Glucagon o r d i b u t y r y l c y c l i c AMP had no e f f e c t on i m i p r a -  mine metabolism a t 0.5 X 10 M; 5  however both compounds produced -5  i n h i b i t i o n o f imipramine metabolism a t 1 X 10  M (Table XX).  Imipramine metabolism was i n h i b i t e d 13 per cent by and 26 per cent by d i b u t y r y l c y c l i c AMP.  glucagon  Dibutyryl cyclic  caused i n h i b i t i o n of aromatic h y d r o x y l a t i o n and  AMP  N-demethylation  of imipramine. The decrease, as a per cent of s u b s t r a t e concent r a t i o n , f o r aromatic h y d r o x y l a t i o n was from 31 per cent ( c o n t r o l ) to 22 per cent whereas N-demethylation decreased from 39 per cent ( c o n t r o l ) t o 31 per cent. I t t h e r e f o r e seems t h a t N-dem e t h y l a t i o n and aromatic h y d r o x y l a t i o n were i n h i b i t e d to the  Table XXI E f f e c t o f Glucagon o r D i b u t y r y l C y c l i c AMP on the D i s t r i b u t i o n o f Imipramine and M e t a b o l i t e s . Imipramine was Incubated f o r F i f t e e n Minutes With t h e I s o l a t e d Perfused Rat L i v e r .  Dose o f Imipramine  Treatment  1  IMI  0.5 X 10~ M C o n t r o l 5  0.5 X 10~ M Glucagon 5.7 X 10  % Distribution Bile Perfusate GOH N-0 DMI IGOH  IMI  M  N-0  18.0  10.1  36.1  16.1  28.0  82.0  89.7 45.0 23.5 83. 8  19.5  7.7  44.7 45.0 15.0  31.5  80.1  88.4 23. 4 23.5 84.9  5  ft  Liver DMI GOH  0.5 X 10~ M DBc AMP 2 X 10  1  24.2  15.3  50.7 53.6 28.5  15.6  75.6  85.4 30.3 26.5 81.1  1 X 10" M  Control  1  29.9  11.8  61.0 67.4 37.6  11.7  70.1  87.7 24.0 16.5 164.6  1 X 10~ M  Glucagon 5.7 X 1 0 "  27.4  16.1  43. 3 49.1 34.5  16.8  72.5  83.1 37.5 27.5 61.5  1 X 10~ M  DBc AMP 2 X 10  23.3  12.6  44.1 62.5 35.2  16.0  76.7  86.9 35. 8 19.9 64.4  5  fi  5  5  5  1 2  ft  fi  a  Glucagon 5.7 X metabolism. per cent= ug of IMI, DMI, GOH, G o r N-0 i n l i v e r , p e r f u s a t e o r b i l e t o t a l ug o f IMI, DMI, GOH, G or N-0 formed  x  1 0 0  95 same e x t e n t . Glucagon i n h i b i t e d imipramine metabolism b u t t o a l e s s e r e x t e n t than d i b u t y r y l c y c l i c AMP.  E f f e c t o f NADH, NADPH o r S u c c i n i c A c i d on Imipramine  Metabolism  To i n c r e a s e the l e v e l o f NADH o r NADPH i n t h e l i v e r these compounds were added t o t h e c i r c u l a t i n g p e r f u s a t e i n the i s o l a t ed r a t l i v e r p e r f u s i o n apparatus. NADPH o r NADH can t r a v e r s e the membrane o f the h e p a t i c c e l l  (3,4). The dose o f disodium  s a l t of NADH (Sigma Chemical Co.) used was 1.33 X 10~ M (10 mg.). 6  The dose o f the t e t r a s o d i u m s a l t o f NADPH (Sigma Chemical Co.) —6 used was 1.11 X 10 M (10 mg.). One o f the t r i c a r b o x y l i c a c i d -3  c y c l e i n t e r m e d i a t e s , potassium s u c c i n a t e (1.6 X 10 M), was added t o the p e r f u s a t e . Ten minutes l a t e r imipramine H C l (1 X -5 10  M) was added t o the i s o l a t e d p e r f u s i o n apparatus. The  i n c u b a t i o n time f o r imipramine metabolism was f i f t e e n minutes. The r e s u l t s o f these experiments are expressed i n Table X X I I . I t was found t h a t NADH, NADPH and s u c c i n i c a c i d decreased imipramine metabolism t o the same e x t e n t . The amount o f i m i p r a mine remaining i n the c o n t r o l experiment a f t e r f i f t e e n minutes metabolism was 23 p e r cent and a f t e r treatment by a l l t h r e e compounds the p e r cent of the dose o f imipramine remaining was 36, 32 and 36 p e r cent. Aromatic h y d r o x y l a t i o n and N-demethylat i o n of imipramine were i n h i b i t e d t o the same e x t e n t by t r e a t ment w i t h NADH o r NADPH. The p e r cent o f the dose o f imipramine i n the c o n t r o l experiment which was metabolized t o desmethylimipramine was 41 per cent. As a r e s u l t o f NADH o r NADPH a d d i t i o n  Table XXII  E f f e c t o f NADH, NADPH o r S u c c i n i c A c i d on Imipramine Metabolism (1 X 10  M) .  The Incubation Time was F i f t e e n Minutes. Per Cent o f Dose Metabolized ± S.E. OH GOH DMI  2  N-0  Treatment  n  IMI  Control  2  23.2  40.8  30.7  4.0  5.2  ±0.7  ±1.6  ±0.1  ±0.2  ±0.8  36.9  33.0  25.5  4.6  5.0  ±1.3  ±3.0  ±0.7  ±1.2  ±0.4  32.5  37.2  25.8  5.0  4.2  ±2.8  ±0.5  ±0.7  ±0.3  ±1.5  35.9  29.1  29.2  6.5  5.8  ±1.0  ±1.6  ±0.1  ±2.7  ±0.6  2  NADH 1.3 X 10" M 6  2  NADPH 1.1 X 10 M _6  2  Succinic Acid 1.6 X 10~ M 3  1  NADH, NADPH o r s u c c i n i c a c i d was added ten minutes p r i o r to imipramine metabolism  2  per cent= t o t a l IMI remaining, DMI, GOH, OH, N-0 formed 14 i total C recovered  x  1  0  Q  vo  Table X X I I I The E f f e c t of NADH, NADPH o r S u c c i n i c A c i d on t h e D i s t r i b u t i o n o f Imipramine and M e t a b o l i t e s . Imipramine (1 X 10 M) was Incubated f o r F i f t e e n Minutes -5  Perfusate DMI GOH  n  IMI  Control  2  31.5 ±1.5  NADH 1.3 X 1 0 M  2  23.3 11.9 40.2 + 2.7 + 0.2 ±11.3  NADPH 1.1 X 10" M  2  28.5 ±0.2  15.1 ±0.1  Succinic Acid 1.6 X 10~ M  2  21.2 ±1.4  12.6 ±1.3  Treatment  1  _6  6  2 Per Cent D i s t r i b u t i o n ±S.E. Bile IMI DMI GOH OH N-0  Liver GOH  OH  N-0  18.9 ±1.7  39.7 ±2.2  16.9 ±5.3  68.3 83.7 ±1.8 ±4.0  24.6 ±0.9  76.6 ±6.1  61.3 ±4.6  1.7 ±1.6  27.6 17.2 + 6.1 ±0.7  76.5 87.8 ±2.7 ±0.2  42.6 ±10.6  97.8 ±1.2  72.4 ±6.1  38.3 ±1.4  9.4 ±2.5  39.3 ±0.8  22.4 ±0.1  71.3 84.3 ±0.1 ±0.1  37.6 ±1.6  91.8 ±4.3  60.7 ±0.8  38.9 ±13.7  11.6 ±3.2  26.4 ±11.4  17.2 ±1.6  78.5 86.9 ±1.6 ±1.5  43.9 ±12.2  88.4 ±3.2  73.5 ±11.9  15.6 55.3 + 3.8 ±5.7  3  1 2  NADH, NADPH or s u c c i n i c a c i d added ten minutes p r i o r t o imipramine metabolism per cent= ug o f IMI, DMI, GOH, OH o r N-0 i n l i v e r , p e r f u s a t e o r b i l e t o t a l ug of IMI, DMI, GOH, OH o r N-0 formed x  1 Q 0  vo  98 to the p e r f u s a t e the p e r cent decreased t o 3 3 p e r cent and 37 per cent r e s p e c t i v e l y . The amount of aromatic h y d r o x y l a t i o n o f imipramine was 31 p e r cent f o r the c o n t r o l experiment  which  decreased t o 25 and 26 p e r cent a f t e r NADH o r NADPH p r e t r e a t m e n t . Although s u c c i n i c a c i d decreased the f o r m a t i o n o f desmethylimipramine from 41 per cent t o 29 p e r c e n t , aromatic h y d r o x y l a t i o n was n o t changed t o a s i g n i f i c a n t e x t e n t . The d i s t r i b u t i o n o f imipramine  (Table XXIII) was changed  i n experiments where NADH o r s u c c i n i c a c i d was added p r i o r t o imipramine metabolism. The amount of imipramine i n the l i v e r was 6 8 p e r cent o f the imipramine p r e s e n t and due t o pretreatment o f NADH o r s u c c i n i c a c i d the l i v e r content o f imipramine i n c r e a s e d t o 76 and 7 8 per cent.  D i s c u s s i o n o f C e l l u l a r C o n t r o l Mechanisms f o r Imipramine Metabolism  The i n h i b i t i o n of imipramine metabolism by d i b u t y r y l c y c l i c AMP and glucagon support a r e c e n t l y p u b l i s h e d r e p o r t (60) t h a t glucagon and d i b u t y r y l c y c l i c AMP c o u l d i n h i b i t drug  metabolism  i n the i s o l a t e d p e r f u s e d r a t l i v e r . Presumably glucagon  inhibits  the metabolism of these drugs through e l e v a t i o n of c y c l i c AMP i n the h e p a t i c c e l l . The mechanism of t h i s i n h i b i t i o n was n o t known, however one o f the major a c t i o n s o f d i b u t y r y l c y c l i c AMP and glucagon on the h e p a t i c c e l l i s the m o b i l i z a t i o n o f glucose out o f the h e p a t i c c e l l and i n t o the p e r f u s a t e (71,75) as w e l l as the breakdown o f glycogen. Perhaps t h e removal o f glucose  from t h e l i v e r may r e s u l t i n drug i n h i b i t i o n by d e c r e a s i n g the energy p r o d u c t i o n f o r drug metabolism i n the l i v e r . The g l y c o g e n o l y t i c e f f e c t caused by d i b u t y r y l c y c l i c AMP o r glucagon occurred only i n  l i v e r s o f f e d r a t s (71,76). The p r i n c i p a l  a c t i o n of d i b u t y r y l c y c l i c AMP on l i v e r s removed from f a s t e d r a t s was gluconeogenesis (77) . I f the i n h i b i t i o n o f drug metabolism was due t o the g l y c o g e n o l y t i c e f f e c t produced by d i b u t y r y l c y c l i c AMP then drug metabolism s h o u l d be d i f f e r e n t when d i b u t y r y l c y c l i c AMP produces gluconeogenesis i n f a s t e d l i v e r s . I t seems t h a t the mechanism of i n h i b i t i o n o f aromatic h y d r o x y l a t i o n by desmethylimipramine was n o t through s t i m u l a t i o n by d i b u t y r y l c y c l i c AMP f o r the f o l l o w i n g reason. D i b u t y r y l c y c l i c AMP caused 28 per cent i n h i b i t i o n o f N-demethylation and 29 p e r cent i n h i b i t i o n o f aromatic h y d r o x y l a t i o n and t h e r e f o r e d i d n o t cause s p e c i f i c i n h i b i t i o n o f aromatic h y d r o x y l a t i o n . —6 —6 Desmethylimipramine a t a s i m i l a r dose (1.6 X 10  M and 3.3 X 10 ]  to d i b u t y r y l c y c l i c AMP (2 X 10 ^M) caused 30 and 40 p e r cent i n h i b i t i o n o f aromatic h y d r o x y l a t i o n and no i n h i b i t i o n of N-demethylation. The q u a n t i t y o f d i b u t y r y l c y c l i c AMP used i n these p e r f u s i o n s t u d i e s i s known t o produce an i n t r a c e l l u l a r c o n c e n t r a t i o n o f 10 (60,71). E p i n e p h r i n e i s a l s o known to. produce s i m i l a r h e p a t i c i n t r a c e l l u l a r c o n c e n t r a t i o n o f c y c l i c AMP (71). I t t h e r e f o r e seems p o s s i b l e t h a t  c i r c u l a t i n g e p i n e p h r i n e or sympathetic  a c t i v i t y which can cause hyperglycemia c o u l d have some i n v i v o c o n t r o l on h e p a t i c drug metabolism. To f i n d out whether NADPH content was l i m i t i n g the r a t e  100 of imipramine metabolism i n the i s o l a t e d p e r f u s e d r a t l i v e r , NADPH was added t o the p e r f u s i n g medium t o i n c r e a s e NADPH l i v e r content. The l i v e r c o n t e n t o f NADPH should i n c r e a s e s i n c e i t had been r e p o r t e d t h a t a b s o r p t i o n o c c u r r e d a f t e r i n j e c t i o n o f t h i s compound t o r a t s  (74). I n o t h e r i n v i t r o  l i v e r microsomal f r a c t i o n s  p r e p a r a t i o n s such as  (100,000 g) (20), l i v e r homogenate (21)  and r a t l i v e r s l i c e s (74) NADPH l i m i t e d t h e r e a c t i o n o f drug metabolism s i n c e maximal drug metabolism r e s u l t e d o n l y a f t e r adequate q u a n t i t i e s  o f t h i s n u c l e o t i d e was added. T h e r e f o r e , i f  the q u a n t i t y o f NADPH i n the l i v e r was n o t s u f f i c i e n t t o m a i n t a i n imipramine metabolism an i n c r e a s e i n l i v e r NADPH would r e s u l t i n an i n c r e a s e i n metabolism. I n t h e p r e s e n t experiments, NADPH was i n s u f f i c i e n t quantity i n the i s o l a t e d perfused r a t l i v e r since a d d i t i o n a l amounts o f NADPH d i d n o t i n c r e a s e imipramine metabol i s m . A d d i t i o n o f NADPH t o i n t a c t c e l l s d e r i v e d from two d i f f e r e n t techniques produced o p p o s i t e r e s u l t s . Drug metabolism was r e p o r t ed by o t h e r s t o be s t i m u l a t e d i n the r a t l i v e r s l i c e s (74) and shown t o be i n h i b i t e d i n the i s o l a t e d p e r f u s e d r a t l i v e r i n t h i s t h e s i s . The reason NADPH i n c r e a s e d drug metabolism i n the r a t l i v e r s l i c e s was t h a t the endogenous supply o f r e d u c i n g e q u i v a l e n t s was l i m i t i n g drug metabolism (74) because the NADPH content i n l i v e r homogenate and r a t l i v e r s l i c e s was below the normal content found i n the i n v i v o l i v e r  (78) . I t i s not known why NADPH i n h i b i t -  ed imipramine metabolism i n the i s o l a t e d l i v e r but perhaps t h i s i n h i b i t i o n was a r e s u l t o f excess NADPH. This i n h i b i t i o n could not be caused by d i r e c t i n h i b i t i o n o f  the enzymes r e s p o n s i b l e  f o r drug metabolism s i n c e excess NADPH had always been used t o study drug metabolism i n l i v e r homogenates (21) o r l i v e r micro-  101 somal f r a c t i o n s (20). Studies i n s t e r o i d metabolism by t h e adrenal c o r t e x have shown t h a t Krebs c y c l e i n t e r m e d i a t e s  (79,80,81) c o u l d i n f l u e n c e  m i t o c h o n d r i a l s t e r o i d metabolism. The metabolism o f these s t e r o i d s was by 1 1 ^ - h y d r o x y l a s e which r e q u i r e d cytochrome P-450. Since cytochrome P-450 was r e q u i r e d f o r drug metabolism i n t h e l i v e r these i n t e r m e d i a t e s may i n f l u e n c e drug metabolism. These s t u d i e s were done on broken c e l l p r e p a r a t i o n s . I n a very r e c e n t study i t was r e p o r t e d t h a t Krebs c y c l e i n t e r m e d i a t e s , s u c c i n a t e and i s o c i t r a t e , could support drug metabolism i n l i v e r homogenates and i n r a t l i v e r s l i c e s  (74). A d d i t i o n o f s u c c i n a t e t o l i v e r  homogenate o r r a t l i v e r s l i c e s i n c r e a s e d the r a t e o f aminopyrine metabolism but an i n c r e a s e i n drug metabolism d i d n o t occur i n microsomal p r e p a r a t i o n s . I t t h e r e f o r e appeared t h a t s u c c i n a t e d i d n o t a f f e c t the drug m e t a b o l i z i n g enzymes d i r e c t l y . I t was found t h a t s u c c i n a t e d i d not i n c r e a s e imipramine metabolism i n the i s o l a t e d perfused r a t l i v e r b u t decreased imipramine metabol i s m . The type of i n h i b i t i o n o f imipramine metabolism seems t o be d i f f e r e n t f o r s u c c i n i c a c i d than f o r NADH o r NADPH, s i n c e s u c c i n i c a c i d only i n h i b i t e d N-demethylation. In the i s o l a t e d perfused r a t l i v e r a d d i t i o n of NADH t o the c i r c u l a t i n g p e r f u s i o n medium caused a decrease i n imipramine. A d d i t i o n o f t h i s reduced n u c l e o t i d e t o h e p a t i c microsomal f r a c t i o n (80) and l i v e r homogenates caused an i n c r e a s e i n V max f o r drug metabolism. T h i s e f f e c t has not been r e p o r t e d i n r a t l i v e r s l i c e s . However, i t has been reported t h a t a d d i t i o n o f NADH o r NAD caused an i n c r e a s e i n the r a t e of cytochrome P-450 r e d u c t i o n (78) . I t t h e r e f o r e appears t h a t i f NADH was not i n s u f f i c i e n t  102 q u a n t i t y i n the i s o l a t e d p e r f u s e d r a t l i v e r t o m a i n t a i n drug metabolism an i n c r e a s e i n t h i s n u c l e o t i d e would cause an i n c r e a s e i n imipramine metabolism. T h i s would occur i f NADH had a s i m i l a r a c t i o n on drug metabolism as i n o t h e r i n v i t r o p r e p a r a t i o n s . Since an i n c r e a s e i n imipramine metabolism was n o t observed i n the i s o l a t e d p e r f u s e d r a t l i v e r , NADH was not l i m i t i n g t h e r a t e of drug metabolism o r NADH does n o t c o n t r i b u t e an e f f e c t t o drug metabolism i n the i s o l a t e d p e r f u s e d r a t l i v e r . The mechanism whereby NADH caused i n h i b i t i o n of imipramine metabolism i n t h e i s o l a t e d p e r f u s e d r a t l i v e r i s n o t known.  103 SUMMARY AND CONCLUSIONS  The p e r f u s a t e h a l f - l i f e o f imipramine was n o t an a c c u r a t e e s t i m a t i o n o f imipramine metabolism w i t h l o n g i n c u b a t i o n times. The metabolism of imipramine a t h i g h s u b s t r a t e c o n c e n t r a -5 tion  (2 X 10  M) was dose dependent because the endogenous  desmethylimipramine formed i n h i b i t e d aromatic h y d r o x y l a t i o n . The primary m e t a b o l i c routes f o r imipramine metabolism w i t h f i f t e e n minutes i n c u b a t i o n was N-demethylation t o desmethylimipramine, aromatic h y d r o x y l a t i o n t o 2-hydroxyimipramine and N - o x i d a t i o n o f imipramine. The r a t e o f aromatic h y d r o x y l a t i o n o f imipramine was g r e a t er than the r a t e o f N-demethylation o f imipramine a t low -5 doses of imipramine (0.5 X 10 M) . E t h y l a l c o h o l was a s p e c i f i c i n h i b i t o r of N-demethylation of imipramine. S u c c i n i c a c i d i n h i b i t e d N-demethylation o f imipramine. Low c o n c e n t r a t i o n s o f desmethylimipramine (1.65 o r 3.3 —6 X 10  M) s p e c i f i c a l l y i n h i b i t e d aromatic h y d r o x y l a t i o n  of imipramine metabolism when the dose o f imipramine was -5 1 o r 2 X 10  M. Higher c o n c e n t r a t i o n o f desmethylimxpramine  i n h i b i t e d aromatic h y d r o x y l a t i o n and N-demethylation o f imipramine. H y d r o x y l a t i o n o f imipramine and c o n j u g a t i o n o f t h i s metabol i t e was independent of b i l i a r y e x c r e t i o n .  104 A d d i t i o n a l evidence was p r o v i d e d f o r more than one cytochrome P-450 system because of s p e c i f i c  inhibition  of aromatic h y d r o x y l a t i o n of imipramine o r N-demethylation of imipramine. A l t e r a t i o n o f t h e p e r f u s a t e magnesium c o n c e n t r a t i o n had no e f f e c t on imipramine metabolism. D i b u t y r y l c y c l i c AMP was found t o i n h i b i t aromatic hydroxyl a t i o n and N-demethylation o f imipramine. The n u c l e o t i d e s NADH and NADPH were found t o i n h i b i t a r o m a t i c h y d r o x y l a t i o n and N-demethylation o f imipramine.  105  BIBLIOGRAPHY  Neushalme, E.A. and Gevers, W., C o n t r o l of g l y c o l y s i s and gluconeogenesis i n l i v e r and kidney c o r t e x . V i t a m i n s and Hormones 25-30: 14 1967. 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