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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 th 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 of 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 Faculty of Pharmaceutical Sciences We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1972 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . 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 e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date KINETICS AND CELLULAR CONTROL MECHANISMS FOR IMIPRAMINE METABOLISM IN THE ISOLATED PERFUSED RAT LIVER b y M. J . MOLDOWAN ABSTRACT An i n v e s t i g a t i o n was undertaken to study the k i n e t i c s and possible 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 perfused r a t l i v e r . The isotope C-imipramme 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 s c i n t i l l a t i o n counting. Analysis f o r imipramine (IMI), desmethy1imipramine (DMI), f r e e hydroxy (OH), glucuronide (G) and N-oxide (N-0) metabolites was done on the perfusate, b i l e and l i v e r . The rate of IMI metabolism was found to be dependent on two major enzymatic routes, N-demethylation (formation o£ DMI) and aromatic hydroxylation (formation of G, OH) of imipramine and one minor enzymatic route, N-oxidation (N-0) . The rate of aromatic hydroxylation of IMI was found to be i n h i b i t e d a f t e r t h i r t y minutes, with IMI concentration 2 X 10 "*M. This i n h i b i t i o n of aromatic hydroxylation could not be detected i f the perfusate h a l f - l i f e f o r IMI (t%=18.5 minutes) was the only parameter monitored. A f t e r incubation periods of f i f t e e n , t h i r t y and s i x t y minutes the per cent of imipramine i n the l i v e r was 70, 75 and X X 80 per cent and the remainder of IMI was i n the perfusate. The -5 -5 -5 dose of IMI was varied (0.5 X 10 M, 1 X 10 M and 2 X 10 M) f o r metabolism by the perfused r a t l i v e r . The incubation time was kept constant at f i f t e e n minutes. The rate of imipramine metabolism (formation of 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. Increasing -5 the dose of IMI to 2 X 10 M s l i g h t l y suppressed the formation of DMI and the formation of GOH followed zero order k i n e t i c s . I t was found that the endogenous DMI formed from IMI metabo-l i s m i n h i b i t e d the formation 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 . - 6 DMI (1.65, 3.33, 6.66 or 13.32 X 10 M) was preincubated p r i o r -6 to a d d i t i o n of IMI. DMI (1.65 or 3.33 X 10 M) was found to s p e c i f i c a l l y i n h i b i t aromatic hydroxylation of IMI. Higher con-- 6 centration of DMI (6.66 or 13.32 X 10 M) i n h i b i t e d the formation of GOH and DMI. E t h y l alcohol (1 mM) preincubated p r i o r to 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 formation. No i n h i b i t i o n of GOH occurred. E t h y l alcohol (1 mM) caused i n h i b i t i o n of formation of DMI from IMI metabolism when the dose of IMI was -5 2 X 10 M. The incubation time for IMI metabolism was f i f t e e n and s i x t y minutes. With t h i s decrease of DMI formation, the formation of GOH increased a f t e r f i f t e e n * or s i x t y minutes of incubation time. From these experiments i t was concluded that suppression of aromatic hydroxylation of imipramine was due to the formation of endogenous DMI formed from IMI metabolism. Optimal conditions were found to study possible c e l l u l a r I l l c o n t r o l mechanisms f o r IMI metabolism i n the i s o l a t e d perfused -5 r a t l i v e r . The dose of IMI was 1 X 10 M and the incubation -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 of IMI metabolism. DMI formation was i n h i b i t e d 28 per cent while GOH formation was i n h i b i t e d 29 per cent. NADPH (1.1 X -6 -6 10 M) or NADH (1.3 X 10 M) was found to i n h i b i t imipramine metabolism. GOH and DMI were both i n h i b i t e d . Succinic a c i d -3 (1.6 X 10 M) was found to i n h i b i t DMI formation but not GOH. Signatures of Examiners i v TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES v i i LIST OF FIGURES x INTRODUCTION 1 Iso 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 I Q Magnesium I Q Dihydronicotinamide j.1 Hormones and Drug Metabolism 1 3 MATERIALS AND METHODS 15 L i v e r Perfusion Techniqe and Apparatus . 15 Su 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 Analysis 2 0 E x t r a c t i o n of Imipramine and Metabolites 20 Reagents 2 1 E x t r a c t i o n Procedure 2 2 Thin Layer Chromatography 25 Qu a n t i f i c a t i o n of Extracted Imipramine and Metabolites 26 L i q u i d S c i n t i l l a t i o n .Counter, Settings and Quench 27 Corrections Procedures f o r Counting and C a l c u l a t i o n of Unknown Samples Comparison Between Ex t e r n a l and I n t e r n a l Quench Corrections 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 Metabolites Imipramine De smethy1imip ramine Chromatography of 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 of Imipramine Metabolism i n the I s o l a t e d Perfused Rat L i v e r Metabolism of Imipramine i n the Is o l a t e d Perfused L i v e r A f t e r Various Incubation Times Perfusate Concentration Rate of Metabolism and D i s t r i b u t i o n Metabolism of Imipramine HCl at Various Concentra-tions of Imipramine Rate of Metabolism and D i s t r i b u t i o n E f f e c t of Desmethylimipramine on Aromatic Hydroxy-l a t i o n and N-demethylation of Imipramine D i s t r i b u t i o n E f f e c t of E t h y l Alcohol on Aromatic Hydroxylation and N-demethylation D i s t r i b u t i o n Discussion of Imipramine Metabolism C e l l u l a r Control Mechanisms f o r Imipramine Metaboli E f f e c t of Magnesium on Imipramine Metabolism E f f e c t of Di 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 of NADH, NADPH or Succinic Acid on Imipramine Metabolism Discussion of C e l l u l a r Control Mechanisms f o r Imipramine Metabolism SUMMARY AND CONCLUSIONS BIBLIOGRAPHY LIST OF TABLES V l l Page Table I Rf Values f o r Imipramine and I t s Known Metabolites. 26 I I Comparison of 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. 30 I I I Q u a n t i f i c a t i o n of Desmethylimipramine and Imipramine by Solvent E x t r a c t i o n and Thin Layer Chromatography. 34 IV Solvent E x t r a c t i o n of Imipramine and I t s Major Metabolites from Aqueous Sol u t i o n s . 39 V S t a b i l i t y of Imipramine 41 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 So l u t i o n 42 VII Calculated and Experimental 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 of Imipramine (2 X 10 M) Metabolism. 50 VI I I The Average Per Cent Metabolism and D i s t r i b u t i o n of Imipramine and Metabolites. The Incubation Time was F i f t e e n , T h i r t y and Si x t y Minutes, With Imipramine HC1 2 X 10 _ 5M. 52 IX The Total Quantity of Imipramine Remaining and Metabolites Formed. The Incubation 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. * 53 X E f f e c t s of Imipramine Concentration on Formation of Metabolites. The Incubation Time was F i f t e e n Minutes. 57 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 Metabolites. -5 Imipramine HC1, 0.5 X 10 M; Incubation Time F i f t e e n Minutes. 68 V l l l Table Page XII 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 Metabolites. -5 Imipramine HC1 1 X 10 M; Incubation Time F i f t e e n Minutes. 69 XI 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 Metabolites. -5 Imipramine HC1, 2 X 10 M; Incubation Time fo r F i f t e e n Minutes. 70 XIV E f f e c t of E t h y l Alcohol on Imipramine Metabolism With the I s o l a t e d Perfused Rat L i v e r ; F i f t e e n Minutes Incubation, 1 X 10 was the Substrate Concentration. 74 XV E f f e c t of E t h y l Alcohol on Imipramine Metabolism With the Is o l a t e d Perfused Rat L i v e r ; F i f t e e n Minutes Incubation With 0.5 X 10~5M Imipramine. 77 XVI E f f e c t of E t h y l Alcohol on Imipramine Metabolism With the Is 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 Concentration was 2 X 10~5M. 79 XVII E f f e c t of E t h y l Alcohol on the D i s t r i b u t i o n of Imipramine and Metabolites. Imipramine HC1 -5 (1 X 10 M); Incubation Time F i f t e e n Minutes. 80 XVIII E f f e c t of E t h y l Alcohol on the D i s t r i b u t i o n of Imipramine and Metabolites. Imipramine HCl (0.5 X 10 _ 5M), Incubation Time F i f t e e n Minutes. 81 XIX E f f e c t of Magnesium on Imipramine Metabolism i n the Iso l a t e d Perfused Rat L i v e r . The Incubation Time was F i f t e e n Minutes and the Substrate Concentration was 1 X 10 ~*M. 90 Table xx Page XX E f f e c t of D i b u t y r y l C y c l i c AMP or Glucagon on Imipramine Metabolism. Incubation Time was f o r F i f t e e n Minutes With the Is o l a t e d Perfused Rat L i v e r . The Dose of Imipramine was 0.5 X 10 ^ M or 1 X 10~5M. 92 XXI E f f e c t of Glucagon or 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 of Imipramine and Metabolites. Imipramine was Incubated f o r F i f t e e n Minutes With the Is o l a t e d Perfused Rat L i v e r . 94 XXII E f f e c t of NADH, NADPH or Succinic A c i d on -5 Imipramine Metabolism (1 X 10 M). The Incubation Time was f o r F i f t e e n Minutes. 96 XXIII The E f f e c t of NADH, NADPH or Suc c i n i c A c i d on the D i s t r i b u t i o n of Imipramine and Metabolites. Imipramine (1 X 10 M) was Incubated f o r F i f t e e n Minutes. 97 X LIST OF FIGURES Page 60 Figure 1. Mechanism of cytochrome P-450 enzyme systems. 4 2. Microsomal metabolism of imipramine. 6 3. Perfusate concentration of imipramine and metabolites. The dose of imipramine was 2 X 10~5M. 46 4. Semi log r i t h m i c p l o t of perfusate imipramine concentration versus time. Imipramine metabolism (2 X 10~ 5M). 48 5. The t o t a l formation of metabolites a f t e r imipramine metabolism f o r f i f t e e n minutes with the perfused r a t l i v e r . 6. E f f e c t of desmethylimipramine on the t o t a l formation of glucuromide, fr e e hydroxy and N-oxide metabolites of imipramine. Desmethyl-imipramine was preincubated f i v e minutes p r i o r -5 -5 to a d d i t i o n of imipramine 0 . 5 X10 M, 1 X 10 M and 2 X 10 M. Incubation f o r imipramine metabolism was f i f t e e n minutes. 7. E f f e c t of desmethylimipramine on the t o t a l formation of glucuronide, free hydroxy or N-oxide metabolites of imipramine. The incubation time was f i f t e e n minutes f o r -5 imipramine metabolism (1 X 10 M). Desmethyl-imipramine was preincubated f i v e minutes p r i o r to imipramine metabolism. 65 8. E f f e c t of desmethylimipramine on endogenous formation of 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 of- imipramine 0.5 X 10~5M, 1 X 10~5M and 2 X 10~5M. 64 Incubation time was f i f t e e n minutes 66 XI ACKNOWLEDGEMENT Dr. G. D. Bellward's guidance and personal encouragement was of great assistance to the author. The author wishes to express h i s appreciation to the fo l l o w i n g people: Mr. B. Virgo and Mrs. C. Moldowan who so generously donated t h e i r time f o r assistance i n t h i s work. Miss Joan Beedle and Mr. K. Grunenberg who chose to a s s i s t i n t h i s p r o j e c t . Canadian Red Cross f o r p r o v i s i o n of supplies and Ciba-Geigy for supplies of chemicals. DEDICATED to C a r o l , Sharleen and Brent who made i t worthwhile. 1 INTRODUCTION The purpose of t h i s research was to study the k i n e t i c s of imipramine metabolism and pos s i b l e c e l l u l a r c o n t r o l mechanisms involved i n the metabolism of drugs by the i s o l a t e d perfused rat l i v e r . I s o lated Perfused Rat L i v e r Technique The use of microsomal f r a c t i o n s i s o l a t e d from the l i v e r has demonstrated at l e a s t four v a r i a b l e s which can change the rate of drug metabolism. These were magnesium, NADPH (abbrevia-t i o n f o r dihydronicotinamide adenine dinucleotide phosphate) and NADH (abbreviation f o r dihydronicotinamide adenine dinucleotide) concentration as w e l l as microsomal drug induction. The l i m i t a -t i o n to these studies has been the inherent a r t i f i c i a l i t y of i n v i t r o techniques. The second widely used technique i n the study of drug me-tabolism was the use of the whole animal. The l i m i t a t i o n to t h i s approach was that due to mu l t i p l e v a r i a b l e s , the c e l l u l a r factors involved i n regulation of drug metabolism could not be completely evaluated. I t was d i f f i c u l t to estimate the dose of each drug to which the l i v e r was exposed because such v a r i a b l e s as pre-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 other organs or l i v e r blood flow can not be c o n t r o l l e d . These 2 disadvantages are eliminated when the i s o l a t e d perfused r a t l i v e r i s used as the experimental model. The perfused l i v e r appeared to be a s a t i s f a c t o r y physio-l o g i c a l preparation 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 rate of b i l e production. Moreover, the contents of metabolic i n t e r -mediates i n freeze-clamped perfused r a t l i v e r s were very s i m i l a r to those obtained i n l i v e r s i n s i t u . In summary, the i n v i t r o studies consisted of using i s o l a t -ed l i v e r microsomal f r a c t i o n s with various co-factors added; these co-factors were required f o r optimal drug metabolism. I t i s not known to what extent these co-factors c o n t r o l the rate 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 perfused r a t l i v e r . The various f a c t o r s which influence i n vivo drug metabolism are t i s s u e binding, 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 induction. Since the l i v e r i s the prime organ which transforms drugs i n t o metabolites, i t i s important to f i n d out what factors a l t e r hepatic 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 oxidation catalyzed by the microsomal enzyme system includes a wide range of r e a c t i o n s , most of which may be ascribed to one common mechanism namely hydroxylation ( 2 ) . The 3 components present i n the microsomes which are necessary f o r drug metabolism have been determined by spectrophotometric techniques which were s i m i l a r to the technique used to study the components of the mitochondrial e l e c t r o n transport chain. I t was found that l i v e r cytochrome P-450 redox system i n the microsomes was responsible f o r most of the drug metabolism (3,4). The pigment cytochrome P-450 i n i t s reduced form r e a d i l y combines with carbon monoxide to form a complex, having a maximum absorp-t i o n at 450 nm and a minimum at 405 nm. For t h i s reason i t has been named P-450 (5). In the absence of carbon monoxide, but i n the presence of a i r , cytochrome P-450 must e x i s t mainly i n the oxidized form or as a complex with oxygen. This pigment i s also present i n adrenal microsomes and mitochondria and i n mi-crosomes of the kidney, lung and i n t e s t i n a l mucosa where i t may play a r o l e i n metabolism of s p e c i f i c agents. The proposed electron flow pathway, cytochrome P-450 redox system, for most hydroxylation i n l i v e r microsomes i s shown i n Figure 1 (6,7). In the metabolism of drugs NADPH serves as an electron donor functioning v i a a r e s p i r a t o r y chain d i r e c t to cytochrome P-450. At one time i t was proposed that the reduced cytochrome P-450 reacted with molecular oxygen to form an " a c t i v e oxygen" complex. This ac t i v e oxygen was^ then t r a n s f e r r e d to the drug substrate which r e s u l t e d i n hydroxylation of a drug (8). However, i t has been shown that the drug substrate f i r s t formed a complex with the oxidized form of cytochrome P-450 (8,9). This complex was then reduced to form reduced cytochrome P-450 sub-4 s t r a t e complex which r a p i d l y combined with molecular oxygen to form an C^-cytochrome P-450 substrate complex. This q u i c k l y decomposed to form the hydroxylated substrate and the ox i d i z e d form of the cytochrome P-450. According to t h i s view, the rate l i m i t i n g step was the reduction of the complex of the substrate and the oxi d i z e d cytochrome. I t was found that NADH could stimu-l a t e drug metabolism further even when NADPH was present i n sat u r a t i n g conditions (10). To explain t h i s e f f e c t NADH must act through a NADH-flavoprotein or cytochrome b5 to donate the + 2 second e l e c t r o n i n the reduction of cytochrome P-450 "^2 su^~ s t r a t e complex. I t seems that NADPH donates the f i r s t e l e c t r o n +3 to cytochrome P-450 substrate complex through NADPH cytochrome c reductase. Then cytochrome b5 or NADH-flavoprotein (11) or NADPH cytochrome c reductase can donate the second e l e c t r o n (9,10). Cytochrome P-450 j Substrate +3 Cytochrome P-450+ 3-O Substrate Cytochrome P-450 -O Cytochrome b5? NADPH Cyt c Reductase? NADH-flavoprotein? e- H„0 Figure 1 Mechanism of cytochrome P-450 enzyme systems. 5 Therefore NADPH plays an important r o l e i n microsomal drug metabolism. I t i s not known whether the formation of NADPH i s the rate l i m i t i n g step i n the hepatic c e l l s . I t was found that NADH could play a minor r o l e i n microsomal drug metabolism, however i t i s not known to what extent t h i s co-factor can i n f l u -ence drug metabolism i n the i n t a c t c e l l . Imipramine Metabolism The antidepressant drug, imipramine, i s one of a few drugs whose metabolism i s comprehensively known i n the r a t (12,13,14) and i n man (15, 16). The majority of imipramine metabolism occurred i n the l i v e r due to the microsomal enzyme system r e q u i r i n g NADPH and molecular oxygen. The metabolites formed from 2 umolar imip-ramine incubated with r a t microsomal enzymes were (14)t amount Q. ~6 (substrate 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 reaction f o r the degradation of imipramine was N-demethylation while aromatic hydroxylase and 6 N-oxidase were minor enzymatic r e a c t i o n s . The primary metabolites of imipramine were 2-hydroxyimipramine, desmethylimipramine and imipramine N-oxide (Figure 2 ) . I t therefore appeared that imip-ramine metabolism would be very s u i t a b l e to study f o r the f o l l o w -ing reasons: three enzymatic r e a c t i o n s , aromatic hydroxylation, N-oxidation and N-demethylation, can be studied from the same substrate; and the metabolic products are s i m i l a r i n r a t and man. Experiments were designed to study the factors which a f f e c t the three enzymatic reactions and f i n d out i f more than one microsomal enzyme or rate l i m i t i n g step would be involved i n the metabolism of imipramine. C H 3 C H 3 H C H 3 C H 3 2-hydroxyimipramine Desmethylimipramine Imipramine N-oxide Figure 2 Microsomal metabolism of imipramine-7 The microsomal enzymatic metabolism of imipramine can be influenced by many f a c t o r s . I t was found that magnesium concen-t r a t i o n , imipramine concentration and SKF 525A (abbreviation f o r 2-diethylaminoethyl-2, 2-diphenylvalerate HC1) had d i f f e r e n t e f f e c t s on the three enzymatic reactions involved i n imipramine metabolism (14,17,18,19). Increasing the imipramine concentra-t i o n to 1 mM increased the v e l o c i t y of N-demethylation to a maximum; however, a fur t h e r increase i n imipramine concentration to 2 mM decreased the v e l o c i t y of N-demethylation. Increasing the imipramine concentration to 2 mM d i d not i n h i b i t N-oxidation; however, aromatic hydroxylation was i n h i b i t e d to a small extent. The a d d i t i o n of magnesium to the microsomal enzyme f r a c t i o n (100,000 g and 9,000 g) increased the rate of N-demethylation of imipramine and i n h i b i t e d N-oxidation of imipramine (18). The rate of aromatic hydroxylation was unaffected (16). SKF 525A was found to i n h i b i t N-demethylation and aromatic hydroxylation but not N-oxidation. Phenobarbital pretreatment was found to induce microsomal N-demethylation and aromatic hydroxylation of imipramine but not N-oxidation (16,18). I t therefore seems that N-oxidation was c o n t r o l l e d by a d i f f e r e n t rate l i m i t i n g step or metabolized by a d i f f e r e n t enzyme. I t also seems that aromatic hydroxylase and N-demethylation were also c o n t r o l l e d by d i f f e r e n t enzymes or rate l i m i t i n g steps since the rate of formation of desmethylimipramine and 2-hydroxyimipramine was d i f f e r e n t , the degree of 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 reactions. The enzymatic aromatic hydroxylation of imipramine i n 8 microsomal preparations from r a t l i v e r was very low compared to i t s a c t i v i t y i n vivo i n the r a t (13) and humans (16). The major excretory products of imipramine were hydroxylated pro-ducts of the drug. Imipramine and desmethylimipramine were not excreted to a s i g n i f i c a n t degree. From these experiments i t can be seen that microsomal preparations had lower enzymatic a c t i v i t y f o r aromatic hydroxylation of imipramine. The i s o l a t e d perfused r a t l i v e r may be more representative of aromatic hydroxylation i n vivo f o r the following reasons. The i s o l a t e d r a t l i v e r per-fusion technique was used to study imipramine metabolism and i t was found that 47 per cent of the dose of imipramine i n three hours was metabolized to hydroxylated products (20). In the same study 3 8 per cent of the dose of desmethylimipramine i n three hours was metabolized to hydroxylated metabolites. However, only 2.5 per cent of the dose of desmethylimipramine was metab-o l i z e d i n one hour with the microsomal f r a c t i o n from the r a t l i v e r (12). I t was also found that microsomal enzyme preparations could not hydroxylate d-amphetamine, however, the preparation could N-demethylate aminopyrine and hydroxylate hexobarbital (21). I t was found that the i s o l a t e d perfused l i v e r d i d hydroxylate d-amphetamine. There are very few published reports i n the l i t e r a t u r e which study the e f f e c t of the substrate concentration on the rate of drug metabolism. This 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 reports which suggest that plasma h a l f - l i f e for drugs i n vivo was dose dependent. Some of these drugs were strepto-9 mycin (22) , dicumarol (23) , diphenylhydantoin (24) and probeni-c i d (25) . Dicumarol (26) and diphenylhydantoin (27) perfusion h a l f - l i f e was also found to be dose dependent with the i s o l a t e d perfused r a t l i v e r technique. Dose dependent k i n e t i c s may mean that the rate of formation of some metabolites i s changing. Reasons f o r the rate of change i n drug metabolism could be that the enzyme responsible f o r metabolism of a drug approaches sat-u r a t i o n or as the dose increases the f r a c t i o n of the drug going to the l i v e r could be l e s s , as demonstrated with dicumarol (26). Before any experiments are designed to study various e f f e c t s on drug metabolism the k i n e t i c s of drug metabolism should be f u l l y understood i n order to choose the proper substrate concen-t r a t i o n . In most studies of drug metabolism i n vivo or i n the i s o l a t e d perfused r a t l i v e r the dose of the drug was us u a l l y decided upon a r b i t r a r i l y and not va r i e d . The p r i n c i p a l metabolite formed from imipramine was desmethylimipramine i n the r a t i n vivo (13) and i n i s o l a t e d r a t l i v e r perfusion studies (20). In these studies only one dose of imipramine was used. U t i l i z a t i o n of r a t l i v e r microsomes and varying dosages of imipramine (12, 19) showed that N-demethylation was the predominant enzymatic reac-t i o n with imipramine. Experiments were done to determine whether various substrate concentrations of imipramine would a l t e r i t s metabolic pattern 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 concentration, above a concentration of 2 mM, which contributed to maximum N-demethylation a c t i v i t y , r e s u l t e d i n a decrease of imipramine metabolism (12). When mag-nesium concentration was reduced below 2 mM there was a decrease i n N-demethylation enzymatic a c t i v i t y and therefore a decrease i n drug metabolism. In a recent p u b l i c a t i o n (28) i t was demon-s t r a t e d , that at magnesium concentration up to a maximal enzy-matic a c t i v i t y , cytochrome P-450 reductase was rate l i m i t i n g f o r benzphetamine and a n i l i n e metabolism. Since imipramine me-tabolism was dependent on magnesium i t could be that magnesium caused an increase i n cytochrome P-450 reductase and therefore an increase i n metabolism. Magnesium concentration up to 5 mM caused an increase i n cytochrome P-450 reductase a c t i v i t y along with an increase i n NADPH oxidase and NADPH-cytochrome c reduct-ase a c t i v i t y . The enzymatic a c t i v i t y of NADPH oxidase was deter-mined by the t o t a l decrease i n NADPH concentration i n the micro-somal incubation mix. This increase i n enzymatic a c t i v i t y r e s u l t -ed i n an increase i n drug metabolism since there was an increase i n enzymatic a c t i v i t y which was rate l i m i t i n g . Higher concentra-tions of magnesium (5 mM) further increased microsomal NADPH-cytochrome c and cytochrome P-450 reductase a c t i v i t y but decreased 11 NADPH oxidase 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 hexobarbital and apparently NADPH oxidase became rate l i m i t i n g f o r the el e c t r o n transport-ing system. Higher magnesium concentrations r e s u l t e d i n a fu r t h e r decrease i n NADPH oxidase a c t i v i t y which i n turn r e s u l t e d i n a decrease of drug metabolism. Since imipramine metabolism (12) was i n h i b i t e d by higher magnesium concentration i t may w e l l be that 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 of a l i v e r one should.be able to de-monstrate the above hypothesis. I f magnesium concentration i n the l i v e r of an animal was lower than required for maximal cyto-chrome P-450 reductase a c t i v i t y , then the rate l i m i t i n g r e a c t i o n should be cytochrome P-450 reductase. Increases i n l i v e r mag-nesium concentrations should increase drug metabolism to maximum i f cytochrome P-450 reductase was rate l i m i t i n g , f u r t h e r increases i n l i v e r magnesium should decrease drug metabolism. This decrease i n drug metabolism would occur i f NADPH oxidase 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 derive some information on the d i r e c t e f f e c t of magnesium on the drug metabolizing enzymes, i n the research 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 subjected to an excess or a deficiency of magnesium i n the perfusion medium. Dihydronicotinamide Adenine Dinucleotide Phosphate In ad d i t i o n to s u i t a b l e magnesium concentration, excess 12 NADPH was required f o r maximum i n v i t r o microsomal enzymatic a c t i v i t y . Microsomal demethylation of imipramine was g r e a t l y enhanced by increasing the concentration of glucose-6-phosphate dehydrogenase i n the presence of NADP (abbreviation f o r n i c o t i n a -mide adenine dinucleotide phosphate). This suggests that the amount of NADPH formed was l i m i t i n g the reac t i o n rate (12) . The p r i n c i p a l e l e c t r o n donor i s NADPH fo r the cytochrome P-450 redox chain. The main source of NADPH i n the l i v e r i s the pentose phosphate shunt (29) . This cycle provides NADPH for b i o s y n t h e t i c processes such as f a t t y a c i d synthesis and pentose f o r nucleotides 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 of the reduced nucleotide to the i n vivo metabolism of drugs i n the smooth endoplasmic reticulum. I t i s possible to f i n d to what degree NADPH functions i n drug metabolism by varying i t s con-centration i n the c e l l . I f one of the enzymes i n the cytochrome P-450 redox system i s rate l i m i t i n g , f o r example cytochrome P-450 reductase, an increase i n c e l l content of NADPH should not a f f e c t the rate of metabolism of a drug. Some studies have been done on the e f f e c t of NADPH a v a i l -a b i l i t y on the rate of s t e r o i d metabolism (30). These studies may apply to drug metabolism since s t e r o i d microsomal metabolism was i n h i b i t e d or induced by the same chemicals that influence microsomal drug metabolism (12,13,14) . The A4 -double bond of steroids such as cortisone and C o r t i s o l was hydrogenated by microsomal -<i-hydrogenase. In c e r t a i n circumstances NADPH a v a i l a b i l i t y might be rate l i m i t i n g i n vivo and not enzymes of 13 the cytochrome P-450 redox system. Two methods used i n these studies consisted of administration of t r i - i o d o t h y r o n i n e , 50 ug/ gm. of body weight, f o r two days to r a t s . This l e d to an increase i n production of NADPH which i n turn r e s u l t e d i n an increase i n theZ^4-5-*-hydrogenase a c t i v i t y (30) . Further evidence to suggest that 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 that fasted animals show a decrease inZ^-5-Ot-hydrogenase a c t i v i t y along with a decrease i n NADPH and glucose-6-phosphate i n r a t l i v e r (30). I f these fasted r a t s were administered glucose the hydrogenation of these st e r o i d s returned to c o n t r o l values. From these studies i n v i t r o and i n v i v o , i t seems that an i n -crease i n NADPH i n the l i v e r c e l l s may increase the rate of some microsomal drug metabolism re a c t i o n s . Therefore, i n c e r t a i n circumstances, the enzymes of cytochrome P-450 redox system may not be the rate l i m i t i n g step f o r the i n vivo metabolism of drugs. Various experiments were c a r r i e d out to determine the e f f e c t of changes i n NADPH concentration on the metabolism of imipramine i n the perfused l i v e r as reported i n t h i s t h e s i s . Hormones and Drug Metabolism There i s very l i t t l e information 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 concern-ed with gluconeogenesis (30) and qu 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 the e f f e c t of c y c l i c AMP (abbreviation i t for adenosine 3 , 5 monophosphate) and glucagon on gluconeo-14 genesis i n the r a t l i v e r . I t has been found that glucagon caused an increase i n c y c l i c AMP l e v e l s i n the l i v e r and stimulated gluconeogenesis from l a c t a t e i n the perfused r a t l i v e r (31). Direct a d d i t i o n of c y c l i c AMP to the perfusion f l u i d caused gluconeogenesis i n the perfused r a t l i v e r . I t i s not known what e f f e c t these hormones or gluconeogenesis has on drug metabolism, although these processes are c o n t i n u a l l y occurring i n the l i v e r . I t i s known that s t a r v a t i o n and diabetes increase the l e v e l of 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 stimulated or depressed. I t also seems that microsomal drug metabolism due to s t a r v a t i o n may not represent a true account of i n vivo metabolism since hexobarbital microsomal metabolism increased due to s t a r v a t i o n ; however the hexobarbital hypnotic time was increased (33). The fa c t o r which contributed to the prolonged sleeping time i n starved r a t s may be reduction of NADPH generation. To summarize, the primary objects of the present research thesi s were: a) to study the r e l a t i o n s h i p of substrate concentration to aromatic hydroxylation and N-demethylation of imipramine b) to determine i f the two enzymatic reactions involved i n the metabolism of imipramine, aromatic hydroxylation and N-demethylation, are c o n t r o l l e d to the same extent by c e l l u l a r factors 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 Perfusion 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 perfusion studies were s i m i l a r to those described by M i l l e r (35) The apparatus was made e n t i r e l y of glass . The temperature of the medium and the l i v e r was maintained at 37 degrees centigrade 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 walled, multi-bulb tube t r a n s f e r r i n g the perfusate from the c o l l e c t i n g vessel i n the oxygenator. To oxygenate the red blood c e l l s a mixture of 95 per cent oxygen and 5 per cent carbon dioxide gas was f i r s t humidified at 37 degrees centigrade, then passed i n t o the multi-bulb-oxygen ator. The perfusion medium was pumped from the c o l l e c t i n g vessel v i a a s e r i e s of one-way valves, by a Rodent Respirator Model 680, Harvard Apparatus Co. L t d . , to the oxygenator. Once the perfusate was oxygenated, the f l u i d entered the l i v e r by free flow through a cannula i n the p o r t a l vein of the l i v e r . The hydrostatic pressure to maintain flow through the l i v e r was constant at 14 to 15 cm. water. The f l u i d returned to the reser-v o i r from the hepatic vein and from an overflow o u t l e t i n the multi-bulb oxygenator. The perfusate then was r e c i r c u l a t e d to 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 ether anes-t h e s i a . The abdomen was opened. The gastrohepatic and gastro-duodenal ligaments were divided and the b i l e duct was cannulated, d i s t a l to the branch entering the l i v e r , with a PE-50 polyethy-lene tube (Clay Adams Inc., New York, N.Y.). The p o r t a l vein was then i s o l a t e d and t i e s were placed l o o s e l y around i t . The hepatic 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 placed around t h i s a r tery p r i o r to cannulation of the p o r t a l vein. The i n f e r i o r vena cava was l i g a t e d and divided between the l i v e r and the r i g h t kidney j u s t p r i o r to removal of the l i v e r . The p o r t a l vein was then r a p i d l y cannulated with a 3 mm. glass cannula. P r i o r to i n s e r t i o n , the glass cannula was f i l l e d with Krebs Hensleit s o l u t i o n and a f t e r cannulation 0.5 Ttil. sodium heparin 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 chest was then opened and the hepatic vein clamped and cut between the clamp and the heart. The l i v e r , with diaphragm, was then c a r e f u l l y dissected out and removed, together with 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 perfusion was approximately ten minutes and the time elapsed from cannula-t i o n of the l i v e r to l i v e r perfusion was between two and three 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 perfused f o r one hour for e q u i l i b r a t i o n ; then imipramine was 17 added to the r e c i r c u l a t i n g perfusate. Perfusion F l u i d Red blood c e l l s were i s o l a t e d from human blood. Blood stored four to f i v e weeks at 4 degrees centigrade and no longer s u i t a b l e for blood transfusion was obtained from the Canadian Red Cross. Whole blood (50 ml.) was centrifuged at 2,000 g f o r ten minutes. The plasma and buffy layer were removed by suction. The red blood c e l l s were washed twice with 20 ml. Krebs Hen 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 red 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 at 2,000 g and removed by suction. The washed c e l l s were then made up to 30 ml. with Krebs Hensleit s o l u t i o n , and hemoglobin concentration was determined. This stock s o l u t i o n was used on the day of prepara-t i o n and stored at 4 degrees centigrade. For each perfusion, 100 ml. of medium was required and was prepared on the day of l i v e r perfusion. This perfusion medium was very s i m i l a r to that described by Krebs (37). To the con-centrated Krebs Hensleit 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., St. Louis, Missouri) and 200 mg. glucose. Since the albumin was a c i d , the pH was adjusted to 7.4 by the ad d i t i o n of not more than 0.5 ml. of IN NaOH. A s u f f i c i e n t volume of stock red blood c e l l suspen-sion was then added to give a hemoglobin concentration of 2.5 gm. 100 ml. I f required, the pH was again adjusted to 7.4 with 18 sodium bicarbonate s o l u t i o n . Five ml. of phosphate b u f f e r pH 7.4 (Na 2HP0 4 0.103 gm. , and KH2PC>4 0.024 gm. i n 10 ml. H20) was added. Approximately 2 ml. of glass d i s t i l l e d water was used to make up to 100 ml. Ninety nine ml. of t h i s s o l u t i o n was tr a n s f e r r e d to the c o l l e c t i n g vessels of the l i v e r perfusion apparatus then a f t e r one hour of perfusion of the l i v e r 1 ml. of imipramine s o l u t i o n was added. Perfusion Medium gm./ 100 ml. NaCL 0.687 KC1 0.04 MgS04.7H20 0.014 Ca C l 2 0.028 Na 2HP0 4 0.103 KH 2P0 4 0.0246 NaHC03 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 of cyanmet-hemoglobin 80 mg./ 100 ml. (Hycel Inc., Houston, Texas). Drabkins s o l u t i o n was prepared i n t h i s laboratory (38). 19 V i a b i l i t y of the L i v e r The metabolic status of the i s o l a t e d perfused r a t l i v e r has been examined i n several studies by measurement of perfu-sate and b i l e flow rates (39), and by study of biochemical func-t i o n (40) . The perfusate flow rates 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 incubation period. I f the rate f e l l below 2 ml./ 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 rate was 0.3 to 0.55 ml. per hour f o r the f i r s t hour of perfusion which was also maintained during the second hour when drug metabolism studies were done. These flow rates were s i m i l a r to b i l e flow rates i n v i v o . Lactate-Pyruvate The lactate-pyruvate r a t i o was monitored i n a l l perfusion experiments to 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 state then the lactate-pyruvate r a t i o increased to 25 from the normal r a t i o of 8 to 12 (41). In a l l experiments the lactate-pyruvate r a t i o was w i t h i n these normal l i m i t s . The method f o r l a c t a t e and pyruvate analysis was described i n the Sigma Technical B u l l e t i n no. 826-UV, Sigma Chemical Co. The enzyme, l a c t i c dehydrogenase, catalyzes the f o l l o w i n g revers-20 i b l e r e a c t i o n . Pyruvic Acid + NADH *" j . L a c t i c Acid + NAD In the presence of an excess of NADH a l l of the pyruvic aci d was converted to l a c t i c a c i d . The amount of NADH which was converted to NAD (abbreviation f o r nicotinamide adenine dinucleo-tide) was measured spectrophotometrically at 340 nm and became a measure of the amount of pyruvic 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 to l e f t with an excess of NAD and hydrazine. The hydrazine complex-ed with the pyruvic 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 to near completion. The amount of NADH formed was measured spectrophotometrically at 340 nm and became a measure of the amount of L (+) l a c t i c a c i d . Magnesium Analysis Magnesium was determined i n the perfusate of each experi-ment ( 4 2 ) . This method i s based on the formation of a red color produced by a t h i a z o l e yellow- Mg. (OH)^ complex i n a l k a l i n e s o l u t i o n . E x t r a c t i o n of Imipramine and Metabolites The separation of imipramine and i t s metabolites was done according to a modified method developed by Moody, T a i t 21 and Todrick (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 of 1 4C-imipramine and the metabolites was by l i q u i d s c i n t i l l a t i o n using a Picker Liquimat. Reagents a) heptane-3 per cent (v/v) isoamyl alcohol b) 1,2-dichloroethane Heptane or 1,2-dichloroethane was shaken twice with equal volumes of 0. 5N NaOH, then with equal volumes of 0.5N H2SO^ and f i n a l l y , three times with equal volumes of d i s t i l l e d water c) a c e t i c anhydride d) 0.1N H„SO. 2 4 e) IN NaOH f) Chloroform, n-propanol, saturated ammonia, 100:100:2 This was the solvent system used f o r t h i n l a y e r chroma-tography. 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. NaN02 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 solutions were mixed 1:1:1 p r i o r to spraying of t h i n layer chromatography p l a t e . h) concentrated HC1 Concentrated HCl was used to spray the t h i n layer chroma-tography plates a f t e r g) . 22 i ) triethanolamine HC1 buf f e r pH 7.4 triethanolamine 13.7 gm. 2N HC1, s u f f i c i e n t volume to adjust to pH 7.4 d i s t i l l e d water to 1,000 ml. j) c i t r a t e b u f f e r c i t r i c acid 21.01 gm. i n 1,000 ml. sodium c i t r a t e 29.91 gm. i n 1,000 ml. c i t r i c acid 16 ml. t i t r a t e d with sodium c i t r a t e to pH 5.4 and d i l u t e d to 100 ml. d i s t i l l e d water E x t r a c t i o n Procedure Five ml. of perfusate was withdrawn from the l i v e r perfusion apparatus and centrifuged f o r f i v e minutes at 2,000 g to remove the red blood c e l l s . Three ml. of the supernatant was transferred to centrifuge t e s t tubes to which 2 ml. of triethanolamine HC1 buffer 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. syringe. The b i l e was then removed from the syringe to a t e s t tube. The syringe was washed with 3 ml. of Krebs Hensleit s o l u t i o n pH 7.4. The contents were then made up to 5 ml. with triethanolamine buffer. One ml. was removed f o r counting, then 1 ml. of NaOH was added. The l i v e r was b l o t t e d dry with paper, weighed and placed i n a glass container to which three parts of cold triethanolamine HCl buffer pH 7.4 was added to one part l i v e r . This was immediately homogenized with a Potter Elvehjem homogenizer with a t e f l o n p e s t l e . 23 Four ml. of t h i s homogenate, which represents 1 gm. of t i s s u e , was t r a n s f e r r e d to a t e s t tube to which 1 ml. triethanolamine and 1 ml. IN NaOH was added. The remainder of the e x t r a c t i o n was the same f o r b i l e , plasma and l i v e r . To each sample, 6 ml. heptane-3 per cent isoamyl alcohol was used to ex t r a c t imipramine and desmethylimipramine. This mixture was shaken by hand for f i f t e e n minutes to twenty minutes and l e f t f o r phase separation or centrifuged t i l l complete phase separation occurred. Four ml. of the organic layer was tra n s f e r r e d to another t e s t tube which contained two or three drops of a c e t i c anhydride. Five ml. 0.IN ^SO^ was added, and the tubes shaken again. The acetylated desmethylimipramine remained i n the organic phase while the unacetylated imipramine was tr a n s f e r r e d to the H2SO4 phase. One ml. was removed from each phase f o r counting of imipramine i n IN ^SO^ and desmethylimipramine i n heptane-3 per cent isoamyl alcohol. One ml. of the basic aqueous phase remaining a f t e r e x t r a c t i o n with heptane-3 per cent isoamyl a l c o h o l was also removed f o r l i q u i d s c i n t i l l a t i o n counting. This phase contained imipramine N-oxide, free hydroxylated and glucuronide metabolites of imipramine. Imipramine N-oxide was separated from the basic aqueous phase by e x t r a c t i n g with 10 ml. of 1,2-dichloroethane. A f t e r t h i s e x t r a c t i o n 1 ml. of the basic aqueous phase was used f o r q u a n t i f i c a t i o n of the free hydroxylated and glucuronide metabo-l i t e s of imipramine. This phase represented the t o t a l enzymatic hydroxylation of imipramine since the hydroxylated metabolites were present i n the glucuronide form. The quantity of imipramine 24 N-oxide which was extracted i n t o 1,2-dichloroethane could be 14 found since the amount of C present before and a f t e r extrac-t i o n of imipramine N-oxide was known. The free hydroxylated metabolites were separated from the glucuronide metabolites by removing 4 ml. of the o r i g i n a l NaOH aqueous phase to another tube and t i t r a t i n g to pH 10 with IN HC1 The free hydroxylated metabolites were then extracted with 10 ml 1,2-dichloroethane. This mixture was shaken c a r e f u l l y to prevent emulsion formation and centrifuged at 2,000 g f o r approximately one h a l f to one hour. The organic layer contained the free hydroxylated metabolites of imipramine while the basic aqueous phase contained the glucuronide metabolites of imipramine. One ml. sample of the basic aqueous phase was removed to determine the quantity of glucuronide formation. The quantity of free hydroxylated metabolites could be found since the amount of 14 C present before and a f t e r e x t r a c t i o n of free hydroxylated me-t a b o l i t e s was known. The organic phase which contained the free hydroxy metabolites was evaporated to dryness under reduced pressure and the residue dissolved i n 500 u l methanol, 50 u l of which was used f o r t h i n layer chromatography f o r i d e n t i f i c a -t i o n of free hydroxy metabolites. Two ml. of the basic aqueous phase which was extracted with 1,2-dichloroethane, was removed and a c i d i f i e d to pH 5.5 with IN HCl. C i t r a t e b u f f e r , pH 5.5 was then added to make the t o t a l volume to 5 ml. Glusulase (Boeh-ringer) was then added, 0.15 ml., and incubated for twenty four hours at 37 degrees centigrade. The aqueous phase was then t i t r a t e d with IN NaOH to pH 10. The free hydroxy-25 l a t e d metabolites were extracted with 1,2-dichloroethane and prepared as described above to 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 hydroxylated and glucuronide metabolite t h i n l a y e r chromatography was used (13). Methanol was used to redissolve the metabolites obtained from the ex-t r a c t i o n procedure. The methanol s o l u t i o n containing the un-known metabolites were placed on t h i n layer plates and developed with chloroform/ n-propanol/ saturated ammonia, 100:100:2. Known reference metabolites, imipramine, desmethylimipramine, imipra-mine N-oxide, iminodibenzyl, 2-hydroxydesmethy1imipramine and 2-hydroxyimipramine were dissolved i n methanol. This s o l u t i o n was always run on t h i n layer plates along with unknown metabo-l i t e s . The Rf values were compared to Rf values of known r e f e r -ence substances. The reference metabolite 2-hydroxyimipramine was synthesized i n t h i s laboratory (4 4) and other metabolites were obtained from Ciba-Geigy Pharmaceuticals. The t h i n l a y e r plates used were Baker-Flex S i l i c a - G e l 1 BF obtained from Baker Chemical Co. Ltd. , P h i l l i p s b u r g h , Penn.. The Rf value and c o l o r reference of known metabolites separated on s i l i c a gel t h i n l a y e r plates are given below. Table I Rf Values f o r Imipramine and I t s Known Metabolites Metabolite Rf and Color L i t e r a t u r e Experimentally Report(13) 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 that the Rf values changed s i g n i f i c a n t l y between determinations, so known reference metabolites were done along with unknown metabolites on each t h i n l a y e r chroma-tography p l a t e . Q u a n t i f i c a t i o n of Extracted Imipramine and Metabolites 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 fo l l o w i n g Toluene 3 1 PPO (2,5-diphenyloxazole) 21 gm. POPOP (1,4-Bis-(2-(5-phenyloxazolyl))-benzene)1.08 gm. BBS-3 ( S o l u b i l i z e r ) 600 ml. 27 14 Imipramine- C HC1 (Amersham-Searle 8.05 m C/ mM) used i n a l l experiments was d i l u t e d with imipramine HC1 (Ciba-Geigy) to a s p e c i f i c a c t i v i t y of 5 0 DPM/ ug to 50 0 DPM/ ug. The com-pound was then r e c r y s t a l l i z e d , using the solvent 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 obtained. A s o l u t i o n was then prepared with d i s t i l l e d water and stored at 0 to 4 degrees centigrade. L i q u i d S c i n t i l l a t i o n Counter, Settings and Quench Corrections The maximum counting e f f i c i e n c y was determined on a Picker Liquimat which was the instrument used f o r counting samples. A standard toluene-"*"4C, (99,000 DPM) sample was used to determine the s e t t i n g s . The counting time was one minute for the standard sample and twenty minutes to determine background counts. I t was found that the highest 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 set at 700 and the lower l e v e l d i s c r i m i n a t o r was set at 150 f o r channel A and B. The counting e f f i c i e n c y of the Picker standard toluene-14 C was 94.2 per cent with a background count of 34. An external standard method was developed f o r quench cor r e c t i o n s . Channel A and B discriminators were set at 150 to 700. Picker standard quench series samples were used. These samples contained toluene-14 C with an a c t i v i t y of 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 of the quenched 137 series of samples to the counting rate of Cs as the external standard source (with, the d i s c r i m i n a t o r s set at 700 and 900 for channel C) was found. The quench c o r r e c t i o n curve was over a narrow range with counting e f f i c i e n c y between 84 and 95 per cent. I f an unknown sample had counting 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 toluene- C was added to each sample. Each sample i n the cycle was counted f o r ten minutes f o r three cycles and the average count, standard deviation and per cent error were determined. The per cent counting e f f i c i e n c y was then determin-ed f o r each unknown sample i n the usual 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 set 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 cycles. Each cycle of unknown samples was preceded by a standard quench s e r i e s to 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 external standard. The standards were counted e i t h e r f o r ten minutes or the time nec-essary for the counts to reach one m i l l i o n counts i n channel A, 137 whichever occurred f i r s t . The e x t e r n a l Cs counting time was fo r one minute. A best f i t polynomial equation was used to correlate the per cent counting e f f i c i e n c y from channel A and the external standard count f o r each cycle. 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 sol v i n g 29 f o r the external standard count of an unknown sample i n the corresponding cycle. To f i n d the per cent counting 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 variance, standard de 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 conversion of CPM to DPM. A l l the information required was programmed f o r and computed by an IBM 360 computer. The program was i n Fortran 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 l i q u i d s c i n t i l l a t i o n counter automatically controls punching of a l l data other than experiment, date, e t c . The information on the punched tape was then transferred to 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. This information was stored 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 External and I n t e r n a l Quench Corrections A comparison was made between the external and i n t e r n a l method to determine the per cent counting 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 metabolites were done from the perfusion medium. The external 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 analysis as l i s t e d i n Table 11.The r e s u l t s of four such experiments are l i s t e d i n Table 1 1 . 30 Table 11 Comparison of Per Cent Counting E f f i c i e n c y Between Int e r n a l Standard and External Standard Methods Per Cent Counting E f f i c i e n c y Phase Experiment I n t e r n a l External Number Standard Standard 0.1N H 2S0 4 1 86.6 87 2 86 87 3 85 87 4 88 88 Heptane-3% 1 88 90 Isoamyl-alcohol 2 89.6 88 3 90 90 4 89 .90 0 . IN NaOH 1 83. 8 88 2 86 87 3 85.6 87 4 86 85 Aqueous Phase 1 82 83 pH 7.4 2 88 85.5 3 86.5 87 4 85 85 The per cent counting e f f i c i e n c y determined by the exter-nal or i n t e r n a l stadard method was v i r t u a l l y the same. In a l l experiments the external standard method was used i f the per cent counting e f f i c i e n c y was between 84 and 95 per cent. I n t e r -n a l standards were used for e f f i c i e n c i e s below 84 per cent. 3 1 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 Metabolites Imipramine Imipramine was added to Krebs Hen s l e i t s o l u t i o n containing 2.5 per cent albumin i n order to determine the per cent recovery 14 of C-imipramine from the basic aqueous phase i n t o 0.1N H 2S0 4 by solvent e x t r a c t i o n . The concentration of imipramine was 14 ug/ ml. Imipramine was then extracted i n t o heptane-3 per cent isoamyl alcohol and then i n t o 0.1N H2SO^. One ml. of 0.1N H2SO^ was analyzed by l i q u i d s c i n t i l l a t i o n counting. The per cent of dosage recovered i n the 0.1N H 2S0 4 phase was 96.6 per cent i n an average of four experiments. The range was 92.5 to 99 per cent. The ex t r a c t i o n procedure used f o r the analysis of imipramine was adequate f o r our purposes. Desmethylimipramine A spectrophotometry analysis was developed to quantitate desmethylimipramine since t h i s r a d i o a c t i v e drug was not 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 so l u t i o n s of desmethylimipramine were made with 0.1N H 2S0 4 ranging i n concen-t r a t i o n from 2.04 ug/ ml. to 20.4 ug/ ml. The o p t i c a l density of these solutions was determined at 250 nm, the maximum absorbance peak. The blank used was 0.1N H 2S0 4. The o p t i c a l density was then p l o t t e d against known concentrations of desmethylimipramine. From t h i s graph the concentration of unknown samples of des-methylimipramine could be determined. The per cent recovery of desmethylimipramine by solvent e x t r a c t i o n was then determined i n the fo l l o w i n g manner. Solutions were made, ranging from 8.52 ug to 6 8.13 ug/ ml. i n phosphate buffer (as i n metabolic experiments) pH 7.4, to a t o t a l volume of 5 ml. One ml. IN NaOH and 6 ml. heptane-3 per cent isoamyl-alcohol were added. The t e s t tubes were shaken f o r ten to f i f t e e n minutes then 5 ml. of the organic layer was tr a n s f e r r e d to another t e s t tube containing three drops of a c e t i c anhydride. Five ml. 0.1N E2^Q^ w a s added a f t e r two minutes and the tubes shaken. Four ml. of the organic layer and 4 ml. of 0.1N H2S0^ were removed to separate t e s t tubes and evaporated to dryness. The contents of the organic phase was redissol v e d i n 10 ml. 0.1N E2^0^ a n <^ t n e contents of the 0.1N H2S0 4 phase was r e d i s s o l v -ed i n 0.1N H2S0 4. Five ml. of the basic aqueous phase (phosphate buffer + IN NaOH) was tran s f e r r e d to another container, evapo-rated and redissolved i n 5 ml. 0.1N ^SO^. The absorbance from each phase, heptane-3 per cent isoamyl a l c o h o l , 0.1N ^SO^, and basic aqueous phase was found and thus the concentration of desmethylimipramine determined. The per cent recovery of desmethylimipramine from s i x experiments was: heptane-3 per cent isoamyl alcohol 76 per cent, 0.IN H2SO^ 0.2 per cent and the basic aqueous phase 2.2 per cent (see Table V). The t o t a l recovery was 7 8.4 per cent and almost a l l the desmethylimipramine that could be accounted for was i n the heptane-3 per cent isoamyl alcohol phase while 2.2 per cent was i n the basic aqueous phase. 33 Chromatography of Imipramine and Desmethylimipramine Plasma, b i l e and l i v e r samples from metabolic experiments where ra d i o a c t i v e imipramine HC1 was metabolized f o r one h a l f hour were used to determine whether imipramine and desmethyl-imipramine were s p e c i f i c a l l y extracted i n t o heptane-3 per cent isoamyl alcohol. The e x t r a c t i o n procedure and t h i n layer chroma-tography techniques f o r t h i s experiment were reported prev i o u s l y . The sample prepared f o r t h i n layer chromatography was from 1 to 2 ml. of heptane-3 per cent isoamyl alcohol a f t e r i t was shaken with the basic aqueous phase. This phase should only contain imipramine and desmethylimipramine. Five ml. 0.1N H^SO^ was added to the remaining 4 ml. of heptane-3 per cent isoamyl alcohol 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 counting. The organic 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 active count i n the methanol, 0.25 ml. was used for 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 layer chromatography. The spots that appeared on the t h i n layer chromatography p l a t e were removed and placed i n a v i a l . One h a l f ml. methanol was added to the v i a l , twenty four 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 count-ed. In summary, imipramine and desmethylimipramine have been quantitated by two methods (a) by t h i n layer chromatography (b) by solvent 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 of Desmethylimipramine and Imipramine by Solvent E x t r a c t i o n and Thin Layer Chromatography Experiment Number Drug % by TLC2 % by solvent ext. 3 % by TLC 2 % by solvent ext. % by TLC 3 % by solvent ext. t % by TLC t % by solvent ext. Imipramine 27.2 19 .4 35.6 24 32.7 37.4 27.6 17.1 Desmethyl- 83 imipramine 73.2 82.6 52.8 76 67.4 62.5 73 U n i d e n t i f i e d *4 11.6 * * % Recovered"*" 68 70 73.8 73. 8 i 1 Per cent recovered= IMI(PPM) + DMI(PPM) + unidentified(PPM) X 100 DPM i n 0.5 ml. methanol 2 Per cent imipramine or desmethylimipramine by t h i n l ayer chroma tography= DPM X 100 t o t a l DPM recovered 3 Per cent imipramine or desmethylimipramine= IMI (DPM) extracted i n H 2S0 4 or DMI (DPM) inorganic phase X 100 1 4C t o t a l DPM 4 * below background (34 CPM) 35 The counting 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 per cent. The per cent of imipramine found by t h i n layer chromatography was always 6 to 11 per cent higher than the content of imipramine found by solvent e x t r a c t i o n f o r the same sample. This could mean that t h i s amount of imipramine was not extracted i n t o 0.IN P^SO^ but remained i n the organic phase. However, t h i s was u n l i k e l y since i t has been shown that 95 to 100 per cent imipramine present i n the organic phase was extracted i n t o the H2SC>4 phase. The per cent of desmethylimipramine recovered by t h i n layer chromatography was 9 and 11 per cent lower than the amount found by solvent e x t r a c t i o n i n two experiments, the same i n one and 24 per cent lower i n another experiment. This could mean that some desmethylimipramine (at l e a s t 9 and 10 per cent) was e x t r a c t -ed out of the organic phase by 0.1N E2SO^. This again seems u n l i k e l y since i t was found by spectrophotometric analysis that 0.2 per cent desmethylimipramine could be extracted from the organic phase i n t o 0.IN H^SO^. In a l l four experiments an u n i d e n t i -f i e d red spot appeared with an Rf value of 0.920. This compound may be the u n i d e n t i f i e d metabolite which B i c k e l (13) has mentioned i n h i s studies. 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 chromato-graphy. In three experiments the compound with an Rf value of 0.920 was w e l l below background count, while i n the other 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 alcohol phase. This compound with an Rf value 36 of <0.920 appeared i n l i v e r samples but not i n plasma or b i l e sam-ples. The per cent recovery of r a d i o a c t i v i t y from t h i n layer chromatography was low, 68 to 73.8 per cent. Using known qu a n t i t i e s of radioactive imipramine twice r e c r y s t a l l i z e d , i t was found that only 60 per cent could be accounted f o r when extracted o f f the t h i n layer chromatography p l a t e . Therefore, the q u a n t i t a t i o n of metabolites using t h i n layer chromatography i s not a very s u i t a b l e method since the per cent recovery of the metabolites from the pl a t e was low. The heptane-3 per cent isoamyl alcohol e x t r a c t from nine plasma samples, four b i l e samples and four l i v e r samples, taken from three experiments were subjected to t h i n layer chromato-graphy. Most of these samples contained only imipramine and desmethylimipramine, except one sample contained the above drugs plus iminodibenzyl which appeared as a very f a i n t blue spot. These samples d i d not contain any hydroxylated metabolites or imipramine N-oxide. In conclusion i t has been shown that e x t r a c t i o n of imipra-mine and desmethylimipramine from b i o l o g i c a l samples with hep-tane-3 per cent isoamyl alcohol was adequate and that the ex-t r a c t i o n of imipramine from the organic layer using 0.1N I^SO^ was also s u i t a b l e . 2-hydroxydesmethylimipramine and Imipramine N-oxide The purpose of these experiments was (a) to f i n d whether 2-hydroxydesmethylimipramine or imipramine N-oxide was ext r a c t -37 ed i n t o heptane-3 per cent isoamyl a l c o h o l when the basic aqueous phase i s greater than pH 12, (b) to determine the per cent recovery of imipramine N-oxide by e x t r a c t i o n of the basic aqueous phase pH 12 with 10 ml. 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 the spectrophotometric analysis (see recovery of DMI) have been noted. Standard solutions of 2-hydroxydesmethylimipramine were made i n IN NaOH ranging i n concentration from 6.8 8 ug to 6 8.7 ug/ ml. and t h e i r absorbance p l o t t e d . The aqueous phase con-t a i n i n g the metabolite was made basic (pH 12.5 to 13) before heptane-3 per cent isoamyl alcohol was added. A f t e r e x t r a c t i o n with heptane-3 per cent isoamyl a l c o h o l , the aqueous phase was t i t r a t e d to pH 10 with IN HC1, then 10 ml. of 1,2-dichloroethane was added. For spectrophotometric analysis a l l solutions were evaporated to dryness under reduced pressure and the contents dissolved with appropriate volumes of IN NaOH. The r e s u l t s r e -ported are from eleven observations from each phase. The per cent, 2-hydroxydesmethylimipramine, found i n the heptane-3 per 1 cent isoamyl alcohol phase was 1.2 per cent, the 1,2-dichloro-ethane phase 78.3 per cent and the basic aqueous phase 7.6 per cent. The t o t a l per cent recovery was 87.1 per cent. Samples were removed from a l l phases and placed on t h i n i 38 layer chromatography plates (see experimental method). 2-hy-droxydesmethylimipramine could not be detected i n heptane-3 per cent isoamyl alcohol phase or the aqueous phase, but spots appeared with corresponding Rf values to 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 also extracted i n t o the 1,2-dichloroethane phase with 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 analysis f o r imipramine N-oxide was s i m i l a r to that f o r 2-hydroxydesmethylimipramine The pH of the aqueous phase was greater 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 alcohol or 1,2-dichloroethane. The concentration of the aqueous phase varied from 4.3 to 25.8 ug/ ml. The concentration i n the standard s o l u t i o n was 1.72 to 21.5 ug/ ml. imipramine N-oxide prepared i n phosphate buffer pH 7.4. The per cent of imipramine N-oxide recovered i n the heptane-3 per cent isoamyl alcohol 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 102.5. per cent. The r e s u l t s are an average from s i x observations i n each phase. I t therefore seems that most of the imipramine N-oxide can be extracted from the aqueous phase at pH greater than 12 by 10 ml. of 1,2-dichloroethane and very l i t t l e was Table IV Solvent Ex t r a c t i o n of Imipramine and I t s Major Metabolites from Aqueous Solutions Heptane-3% Isoamyl A l c o h o l 1 1,2-dichloro-ethane*^ 0.1N H 2S0 4 3 Remaining Aqueous 4 Phase 5 % Recovery pH of per pH of per per Metabolite n s o l u t i o n cent s o l u t i o n cent cent Imipramine 4 12.5 96.6 12.5 96.6 96.6 Desmethylimipramine 6 12.5 76 0.2 2.2 78.4 2-hyd roxy de sme thy1-imipramine 11 12.5 1.2 10 78.3 7.6 87.1 Imipramine N-oxide 6 12.5 8.6 12.5 92.0 1.9 102.5 1 per cent extracted i n t o heptane-3% isoamyl alcohol from aqueous phase 2 per cent extracted 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 with heptane-3% isoamyl alcohol 3 per cent extracted i n t o 0.1N H 2S0 4 from heptane-3% isoamyl al c o h o l 4 per cent remaining i n aqueous sample a f t e r e x t r a c t i o n with heptane-3% isoamyl a l c o h o l 5 % recovery= t o t a l ug i n -each phase x ug added to aqueous phase 40 extracted by the heptane-3 per cent isoamyl a l c o h o l . Since only 8.6 per cent of the imipramine N-oxide was extracted by heptane-3 per cent isoamyl alcohol and i t appears as a very minor metabolite, 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 of Imipramine To each of three t e s t tubes, 24 ml. of perfusion f l u i d and 1 ml. of imipramine s o l u t i o n was added. Imipramine HC1 concen-t r a t i o n i n each tube was 28 ug/ ml. which was equivalent to 14 1421 DPM of imipramine- C HC1. These solutions were then i n -cubated at 37 degrees centigrade f o r f i f t e e n minutes. Five ml. was withdrawn from each sample, centrifuged and analyzed. 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 at 2,000 g f o r ten minutes was 80 per cent of the t o t a l quantity of imipramine HC1. Twenty per cent of the dose of imipramine HC1 could not be accounted for and appears to be absorbed i n t o the red 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 could be extracted i n t o the organic layer then into 0.1N ^SO^. There was no s i g n i f i c a n t amount of a c t i v i t y i n the other phases . 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) E x t r a c t i n g Phase 0.1N H-SO.1 2 4 Heptane-3% Isoamyl Alcohol 0.1N Na OH2 Total A c t i v i t y i n Aqueous Solution Before E x t r a c t i o n 1 the per cent r a d i o a c t i v i t y was the per cent of radio-a c t i v i t y i n the aqueous phase 2 0 means counts which appeared were e i t h e r h or less than h of background count The r e s u l t s are s i m i l a r to those found by B i c k e l and Weder (7). They found that when the sample was made basic (pH 8 to 12.4) a l l imipramine could be extracted with heptane-3 per cent isoamyl a l c o h o l . This e x t r a c t i o n was also very s i m i l a r to that done by Todrich (43). To f i n d out whether imipramine was absorbed onto the red 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 Hensleit s o l u t i o n with albumin, the other group contained Krebs Hensleit s o l u t i o n , albumin and red blood 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 Per Cent R a d i o a c t i v i t y Experiments 1 2 3 100 98 94.5 0 0 0 0 0 0 80.5 80 82.5 42 of 1 4C-imipramine, 1 ml. was removed immediately a f t e r mixing 14 the contents of each tube with C-imipramine and 1 ml. was removed a f t e r three hours. The r e s u l t s of these experiments appear i n Table VI. 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 Solution . 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 1 per cent recovered from aqueous phase 2 per cent of dose 3 Krebs Hensleit s o l u t i o n In both s o l u t i o n s , with and without red blood c e l l s , c o n t r o l and a f t e r three hours incubation, the amount recovered i n the 0.IN H2S04 phase was s i m i l a r which indi c a t e s that imipramine HC1 was stable under these conditions. Analysis 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 Hensleit s o l u t i o n with albumin accounted f o r 100 per cent of the dose administered while only 80 to 85 per cent of the dose was i n the supernatant when red Phase Per Ce 3 KH s o l u t i o n and albumin Time 0 hours 3 hours 0.1N H^C^ 1 97.5,97.5 92.5,99 2 supernatant 99,100 100,100 n= 2 43 blood c e l l s were present i n the s o l u t i o n . I t therefore was apparent that the 15 to 18 per cent l o s s of imipramine HC1 was due to the red blood c e l l s , i n an a l y s i s of perfusate imipramine an e r r o r of 15 to 18 per cent could r e s u l t . However, i n metabolic experiments l a t e r conducted, there was 90 to 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 that t h i s p r e d icted e r r o r d i d not occur. Two experiments were designed to determine whether imipra-mine HC1 was stable when c i r c u l a t e d through the l i v e r perfusion apparatus. Krebs Hensleit s o l u t i o n was prepared with 2.5 gm. of albumin. 99 ml. of t h i s was added to the l i v e r perfusion appara-tus along with 1 ml. imipramine HC1 s o l u t i o n (84,000 DPM/ ml.) equivalent to 1.665 mg. imipramine HC1. This s o l u t i o n was c i r c u -l a t e d through the l i v e r perfusion apparatus without the r a t l i v e r . A c o n t r o l sample of 5 ml. was removed p r i o r to c i r c u l a t i o n , then 5 ml. every hour f o r four hours. The c i r c u l a t i n g s o l u t i o n was maintained at 37 degrees centigrade. A f t e r each hour up to four hours 87 and 90 per cent of the imipramine HC1 was recovered. From these r e s u l t s i t seems that imipramine was stable under the experimental conditions planned to study imipramine metabolism. 44 RESULTS AND DISCUSSION K i n e t i c s of Imipramine Metabolism i n the Isolated Perfused Rat L i v e r In the presentation of these r e s u l t s , reference i s made to N-demethylation, aromatic hydroxylation or N-oxidation. The meaning of these terms are as f o l l o w s . The t o t a l quantity of desmethylimipramine (abbreviated form DMI) formed from imipra-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 free hydroxylated metab-o l i t e s (abbreviated form OH.) plus the t o t a l quantity of glucuron-ide (abbreviated form G) metabolites 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 hydroxylation. 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-oxidation. Metabolism of Imipramine i n the Isolated Perfused L i v e r A f t e r Various Incubation Times To determine whether the rate of N-demethylation, aromatic 1 hydroxylation or N-oxidation varies with incubation time, imipra-mine HC1 2 X 10 M^ (6.336 mg.) was incubated f o r various time periods with 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 perfusing f l u i d was 110 ml. Two 5 ml. samples were removed p r i o r to addition of imipramine, to determine l a c t a t e and pyruvate 1 the time during which the drug was present i n the perfusion f l u i d and i n contact with the i s o l a t e d r a t l i v e r . 45 perfusate concentrations. A f t e r the l i v e r was perfused f o r one hour, 1 ml. of imipramine (.6.336 mg./ ml.) was added to 100 ml. of perfusing f l u i d . The perfusate concentration of imipramine and the metabolites were determined by withdrawing 5 ml. from the perfusion apparatus at the appropriate 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 of incubation with imipramine. The l i v e r , perfusate and b i l e were analyzed f o r imipramine, desmethylimipramine and the more polar metabolites such as glucuronide, free hydroxy and N-oxide metabolites of imipramine. From these analyses, the t o t a l metabolism of imipramine as w e l l as the formation of desmethylimipramine and the aromatic hydroxylated metabolites could be determined. This information could then be r e l a t e d to the perfusate h a l f - l i f e of imipramine. The d i s t r i b u t i o n of imipramine and i t s metabolites i n the b i l e , l i v e r and perfusate could also be determined. In a l l experiments the lactate/pyruvate r a t i o was not great-er than twelve and the blood flow through the l i v e r was not l e s s than 2 ml./ gm. l i v e r per minute and not greater than 3 ml./ 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 perfusate concentration of imipramine and the metabolic products of imipramine HC1 (2 X 10 M^) metabolism are presented 5 10 15 30 60 Time (minutes) Figure 3 . Perfusate concentration of imipramine and metabolites. The dose of imipramine was 2 X 10 M^. * ug equivalent to ug imipramine 47 i n Figure 3. The polar metabolites of imipramine, glucuronide, free hydroxy and N-oxide appeared r a p i d l y i n the perfusate with a s i m i l a r combined concentration to desmethylimipramine up to f i f -teen minutes of incubation. These polar metabolites (G, OH, N-0) were the composite of aromatic hydroxylation of the iminodi-benzyl r i n g , i t s glucuronide products and imipramine N-oxide. The concentration of these polar metabolites was greater than the concentration of desmethylimipramine a f t e r f i f t e e n minutes of incubation. Very l i t t l e free hydroxylated metabolite appear-ed i n the perfusate. For example, a f t e r f i f t e e n minutes of imip-ramine metabolism the concentration of glucuronide metabolites was 3.2 ug/ ml. whereas the concentration of glucuronide and free hydroxy metabolites was 3.3 ug/ ml. I t appeared that the hydroxylated metabolites formed from imipramine were q u i c k l y conjugated with glucuronide to form the glucuronide metabolite. The perfusate concentration of imipramine N-oxide remained very low and e s s e n t i a l l y the same during the e n t i r e incubation period. The perfusate concentration of desmethylimipramine increas-ed l i n e a r l y to f i f t e e n minutes. The concentration of desmethyl-imipramine a f t e r t h i r t y and s i x t y minutes of imipramine metabo-li s m d i d not increase f u r t h e r . I t was evident upon examining Figure 4 that the disappear-ance of imipramine from the perfusing f l u i d into the l i v e r was 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 while the second phase was between f i f t e e n and t h i r t y minutes. The perfusate decline of imipramine between zero and f i f t e e n minutes 40 48 30 (10) 20 (10) Cn 3 H 10 (4) o •H •P rd M -p a CD o cs o o <D -P rd cn 4-1 u cu A 4 :4) 1.5 60 ( ) 5 1 0 15 30 Time(minutes) number of observations per point Figure 4 Semi logarithmic p l o t of perfusate imipramine concentration (ug/ ml.) versus time. Imipramine metabolism (2 X 10~5M). Each point represents the average imipramine concentration. 49 was extremely rapid. The rate of decline 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 perfusate and the l i v e r and metabolism of imipramine, since the per cent of imipramine metabolized i n the f i r s t f i f -teen minutes was 43 per cent (Table V I I I ) . The second phase of imipramine decline from the perfusate 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 rate between the l i v e r and perfusate f o r imipramine and the rate of imipramine metabolism. The rate of imipramine perfusate concentration decline seemed to f o l l o w f i r s t order k i n e t i c s with time, since a semi-logrithmic p l o t of perfusate imipramine concentration 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 (Figure 4). The second phase of imipramine decline h a l f - l i f e (t%) was 18.5 minutes which was calcu l a t e d by the d i r e c t graphical method (45). t3 s = t2 tl t 2 i s the time when the concentration was twice that at time t ^ Rate of Metabolism and D i s t r i b u t i o n To determine whether the perfusate h a l f - l i f e (t%) f o r imip-ramine was an accurate estimation of the rate of imipramine metabolism the l i v e r , b i l e and perfusate were analyzed for 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 imipra--5 mine metabolism. The dose of imipramine was 2 X 10 M. To adequately study the proportion of N-demethylation 50 to aromatic hydroxylation and N-oxidation the metabolic products of imipramine were analyzed i n the b i l e , l i v e r and perfusate. The r e s u l t s from the t o t a l analysis of imipramine and i t s metabolites i n the b i l e , l i v e r and perfusate 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 of imipramine metabolism (2 X 10 M) with the i s o l a t e d perfused r a t l i v e r are presented i n Table V I I I . Table VII Calculated and Experimental Imipramine Remaining A f t e r -5 F i f t e e n , T h i r t y and S i x t y Minutes of Imipramine (2 X 10 M) Metabolism. Incubation Time 15 30 60 Calculated % IMI Remaining 59.4 34.4 11.0 Found % IMI Remaining 56.911.5 32.1+5.7 19.7+2.1 Deviation From Calculated IMI 2.5 2.3 8.7 1 calcul a t e d using th = 18.5 f o r imipramine metabolism 2 the amount of imipramine found a f t e r a n a l y s i s of l i v e r , b i l e and perfusate divided by t o t a l 1 4C recovered . The dose of IMI was 2 X 10~5M. 3 value of 2 - value of 1 = d e v i a t i o n 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 perfusate imipramine concentr-t i o n change from Figure 4 the per cent of imipramine remaining can be calculated 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 51 metabolism. A comparison was made between the c a l c u l a t e d (Table VII) per cent imipramine remaining from perfusate th and the amount of imipramine found from analysis of perfusate, 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 of metabolism of imipramine are s i m i l a r , however, the deviation between the cal c u l a t e d and ex p e r i -mental value for s i x t y minutes of metabolism was 8.7 per cent. I t therefore seems that f i r s t order rate occurs f o r imipramine metabolism f o r the f i r s t t h i r t y minutes of incubation with the i s o l a t e d perfused r a t l i v e r because the th f o r the change i n the perfusate imipramine concentration was an accurate estimation of the rate of imipramine metabolism. The ca 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 of imipramine meta-bolism was much higher than the per cent metabolism which was experimentally found. I t thus appears that the rate of imipramine metabolism changed and t h i s could not be detected when consider-ing the perfusate concentration of imipramine. I t was found that there was s l i g h t i n h i b i t i o n of imipramine metabolism which was not r e f l e c t e d i n the perfusate imipramine concentration (Table V I I I ) . Consequently experiments which only monitor perfusate imipramine concentration decline would not be an accurate es-timation of imipramine metabolism. The f a c t o r which contributed to the i n h i b i t i o n of 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 of formation of glucuronide, free hydroxylated metabolites as w e l l as imipramine N-oxide (Table V I I I ) . The per cent of the dose of Table V I I I The Average Per Cent Metabolism and D i s t r i b u t i o n of Imipramine and Metabolites. The Incubation Time was F i f t e e n , T h i r t y and Six t y Minutes, With Imipramine HC1 2 X 10 _ 5M. Per Cent Metabolized ±S.E Per Cent D i s t r i b u t i o n Incubation Perfusate B i l e L i v e r Time(minutes) IMI 3 DMI G,0H,N-04 IMI DMI G,OH,N-0 G,OH,N-04 IMI DMI G ,OH,N-04 15 56.9 25.4 19.1 30.1 20.9 37.1 11.7 70.0 79.0 50.6 n=4 ±1.5 ±1.5 ±1.41 ±2.0 ±0.6 ±4.2 ±4.03 ±2.3 ±2.17 ±2.5 30 32.1 41.0 27 24.7 20.7 64.5 7.4 74.7 78.6 26.7 n=3 ±5.7 ±2.7 ±0.15 ±4.08 ±2.4 ±4.90 ±1.4 ±4.08 ±2.5 ±5.7 60 19.7 50.4 29.8 15.8 18.3 46.4 28.5 80.2 80. 8 21.3 n=4 ±2.1 ±5.5 ±4.5 ±3.6 ±6.2 ±2.44 ±1.7 ±5.2 ±6.4 ±2.2 3 4 n= number of observations £>.E.= standard e r r o r per cent= t o t a l ug IMI, DMI or G, OH, N-0 formed ug of 1 4C recovered ^ per cent= ug of IMI, DMI or G, OH, N-0 i n l i v e r , perfusate or b i l e t o t a l ug of IMI, DMI or G, OH, N-0 formed per cent of the dose of IMI remaining G, OH, N-0 (glucuronide, free hydroxy and N-oxide metabolites of imipramine)= t •L4C remaining a f t e r e x t r a c t i o n of aqueous phase pH 12.5 with heptane-3 per cent isoamyl alcohol. X 100 Ul t o t a l to 53 imipramine which was metabolized to glucuronide, f r e e hydroxy and N-oxide metabolites was 27 per cent a f t e r t h i r t y minutes and 29.8 per cent a f t e r s i x t y minutes of imipramine metabolism. -5 The dose of imipramine was 2 X 10 M. Although i n h i b i t i o n of aromatic hydroxylation and N-oxidation occurred, N-demethylation continued over the e n t i r e incubation period. Table IX The Total Quantity of Imipramine Remaining and Metabolites Formed. The Incubation 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. Metabolites Formed Incubation Time(minutes) Metabolites 15 30 60 mg.iS.E. mg.iS.E. mg.iS.E. IMI remaining 3.5610.11 2.0310.32 1.23±0.11 DMI 1 1.59±0.05 2.5810.25 3.13+0.37 G^R^N-O1'2 1.1910.06 1.7310.08 1.84±0.26 1 mg. formed equivalent to mg. IMI 14 2 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 of aqueous phase pH 12.5 with heptane-3 per cent isoamyl alcohol. The k i n e t i c s of imipramine metabolism was complicated by the f a c t that i n h i b i t i o n of aromatic hydroxylation and N-oxida-t i o n had occurred. The formation of desmethylimipramine continu-ed to increase over the e n t i r e incubation period. The t o t a l 54 quantity of desmethylimipramine formed a f t e r imipramine metabo-l i s m f o r t h i r t y minutes was 2.5 mg. (Table IX) and a f t e r s i x t y minutes was 3.1 mg. The amount of imipramine decreased i n the same period from 2 mg. to 1.2 mg. There was more des-methylimipramine than imipramine i n the i s o l a t e d perfused r a t l i v e r apparatus. Reports i n the l i t e r a t u r e suggested that desmethylimipramine could i n h i b i t the metabolism of other drugs (23,24,25,26,27), therefore i t was thought that the i n h i b i t i o n of aromatic hydroxylation and N-oxidation was due to desmethylimipramine formed from imipramine metabolism. The drug imipramine was very l i p o p h i l i c since 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 incuba-t i o n i n the i s o l a t i o n perfusion apparatus. The amount of imipra-mine increased with time to 80 per cent (Table V I I I ) . This gradual increase i n imipramine concentration i n the l i v e r may explain the gradual decline of imipramine perfusate concentration, which follows 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 despite the i n h i b i t i o n of aromatic hydroxylation and N-oxidation. The per cent of imipramine remaining i n the perfusate a f t e r f i f t e e n minutes was 30 per cent which declined to 16 per cent a f t e r s i x t y minutes of imipramine metabolism. The perfusate decline 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 established with the l i v e r . Less than 1 per cent of imipramine was found i n the b i l e . The metabolite of imipra-mine, desmethylimipramine, was also very l i p o p h i l i c since 80 per 55 cent of the desmethylimipramine remained i n the l i v e r , the remain-ing 20 per cent was i n the perfusate with no excretion 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 perfusate was very s i m i l a r to that found by Von Bohr (46). The d i s t r i b u t i o n of more polar metabolites of imipramine, glucuronide, free 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 with time from 50 per cent to 21 per cent. The per cent of these polar metabolites increased i n the per-fusate between f i f t e e n and t h i r t y minutes, followed by a decrease a f t e r t h i r t y minutes of imipramine metabolism. This decrease of perfusate concentration (glucuronide, free hydroxy and N-oxide) was r e f l e c t e d i n an increase of b i l i a r y e x cretion. Metabolism of Imipramine HC1 at Various Concentrations of Imipramine The metabolism of imipramine i n the i s o l a t e d perfused r a t l i v e r was dependent on three enzymatic reactions, namely aroma-t i c hydroxylation, N-demethylation and N-oxidation. To provide some information on whether imipramine metabolism, followed f i r s t order k i n e t i c s or was dose dependent, imipramine concen-t r a t i o n was varied. Since the metabolism of imipramine was de-pendent on three enzymatic reactions, changing the substrate concentration could provide information on the r e l a t i v e a c t i -v i t i e s of these enzymes i n r e l a t i o n to imipramine concentration. This type of information was found necessary i n order to choose the best dose of imipramine for experiments designed to f i n d 56 possible c e l l u l a r c o n t r o l mechanisms f o r imipramine metabolism. The dose of imipramine HC1 chosen was 0.5 X 10 (1.5 mg.) and 1 X 10~5M (3.17 mg.) since these were the approximate amounts 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 incubation with 2 X 10 M (Table IX). The incubation times f o r these experiments were f i f t e e n minutes since a longer incubation time could complicate aromatic hydroxylation of imipramine as was demonstrated i n the l a s t s e r i e s of experiments. Imipramine -5 -5 0.5 X 10 M or 1 X 10 M was added to the perfusion apparatus a f t e r the l i v e r was perfused f o r one hour. Five ml. of the per-fusate was withdrawn f i v e , ten and f i f t e e n minutes a f t e r addi-t i o n of imipramine, f o r analysis of imipramine and metabolites. 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 analyzed f o r imipramine and i t s metabolites. The r e s u l t s appear i n Table X. Rate of Metabolism and D i s t r i b u t i o n I f a drug i s metabolized 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 of 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 of a drug i s independent of the drug concentration (46) th = 0.693 k k= constant for f i r s t order k i n e t i c s Table X Effe c t s of Imipramine Concentration on Formation of Metabolites. The Incubation Time was F i f t e e n Minutes Dose of IMI 1 Incubated Per Cent 2 Metabolized ± S.E. for F i f t e e n Minutes IMI DMI GOH OH N-0 0.5 X 10~5M 76.7 29.9 41.9 5.6 4. 85 ±3.3 ±1.8 ±2.6 ±0.6 ±0.8 1 X 10"5M 67.6 34.3 25.5 3.8 5.6 ±3.3 ±4.0 ±2.6 ±1.2 ±1.2 2 X 10"5M 43.1 20.9 13. 8 1.3 7.0 ±1.5 ±0.6 ±2.1 ±0.2 ±0.1 1 four experiments per dose of imipramine 2 per cent. IMI metabolized or metabolite formed= t o t a l IMI, DMI, GOH, OH or N-0 formed X 100 __ • — t o t a l C recovered 58 In these experiments the dose or substrate concentration of imipramine was changed, however, the incubation time was kept constant at f i f t e e n minutes. The per cent of imipramine metabolized and the per cent of the metabolic products formed was ca l c u l a t e d as a per cent of the o r i g i n a l concentration of imipramine.The r e s u l t s of these experiments are expressed i n Table X. The per cent of imipramine metabolized had decreased as the dose of imipramine was increased. For example, when imipra--5 -5 -5 mine HC1 0.5 X 10 M, 1 X 10 M or 2 X 10 M was incubated with the i s o l a t e d perfused r a t l i v e r 76, 67 and 43 per cent of the dose of imipramine was metabolized. The metabolism of imipramine d i d not fol 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 of imipramine HC1 metabolized to glucuronide and free hydroxy metabolites at the d i f f e r e n t concentration of imipramine was dose dependent. The per cent of glucuronide and free hydroxy metabolites of imipramine formed was 42, 25 and -5 -5 14 per cent f o r imipramine concentration of 0.5 X 10 M, 1 X 10 M -5 and 2 X 10 M re s p e c t i v e l y . The rate of formation of desmethyl-imipramine followed f i r s t order k i n e t i c s since the per cent of desmethylimipramine formed was s i m i l a r f o r imipramine concentr--5 -5 t i o n of 0.5 X 10 M and 1 X 10 M. However, there was a decrease i n the rate of desmethylimipramine metabolism when the concentr--5 t i o n of imipramine was 2 X 10 M. The rate of formation of imipramine N-oxide followed f i r s t order k i n e t i c s throughout the en t i r e range of imipramine concentrations used; that i s , the per cent of imipramine N-oxide formed was s i m i l a r . The reason drug plasma decline was dose dependent could be that the enzymatic reactions involved i n the degradation of the drug were saturated at higher doses. At sa t u r a t i o n of the enzyme, zero order k i n e t i c s occurred. 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 metabolite was p l o t t e d versus the substrate concentration (Figure 5). The V max or zero order k i n e t i c s was attained for aromatic hydroxy-l a t i o n of imipramine since increasing the dose of imipramine -5 to 2 X 10 M did not l i n e a r l y increase the amount of glucuron-ide and hydroxylated metabolites of imipramine formed (Figure 5)„ There can be two reasons f o r the zero order k i n e t i c s of the aromatic hydroxylase enzymatic r e a c t i o n . In terms of Michaelis Menten k i n e t i c s the enzyme might have reached the true V max and the substrate concentration exceeded the concentration of aromatic hydroxylase. In t h i s case the amount of imipramine would have exceeded the amount of aromatic hydroxylase enzyme necessary to form 2-hydroxyimipramine. This was the p r i n c i p a l enzymatic reaction since 2-hydroxyimipramine was the only hydroxylated metabolite i d e n t i f i e d by t h i n layer chromatography. The second reason for zero order k i n e t i c s might have been that i n h i b i t i o n of the aromatic hydroxylation of Imipramine occurred due to 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 less than the amount of glucuronide 60 300 200 to U <u CD -M > •H -H rH rH o i H *tnL00 3 n= 4 rats per point standard e r r o r 0 0.5 1 2 Dose ( X 10~5M) Figure 5. The t o t a l formation of metabolites a f t e r imipramine metabolism f o r f i f t e e n minutes with the perfused r a t l i v e r . 61 -5 and hydroxylated metabolites of imipramine. When 0.5 X 10 M of imipramine was metabolized by the i s o l a t e d perfused r a t l i v e r the amount of desmethylimipramine was l e s s than the quantity of glucuronide and hydroxylated metabolites of imipra-mine, 51 ± 4.06 to 70 ± 5.4 ug/ gm. l i v e r . At lower doses of imipramine, N-demethylation was no longer the predominant enzymatic pathway f o r degradation of imipramine. E f f e c t of Desmethylimipramine on Aromatic Hydroxylation and N-demethylation of Imipramine I t was found that the formation of glucuronide and free hydroxylated metabolites of imipramine was i n h i b i t e d a f t e r -5 t h i r t y minutes of imipramine metabolism (2 X 10 M) with the i s o l a t e d perfused r a t l i v e r . I t was also found that aromatic hydroxylation of imipramine followed zero order k i n e t i c s a f t e r -5 imipramine metabolism at substrate concentration of 1 X 10 M. In order to determine i f desmethylimipramine was i n h i b i t i n g the hydroxylation 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 IX) , 2.5 mg. of desmethylimipramine had accumulated i n the perfusion apparatus and the amount of imipramine which remained was 2 mg. Under these conditions further metabolic formation of glucuronide, free hydroxy and N-oxide metabolites 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. while imipramine diminished to 1.2 mg. These changes occurred a f t e r an a d d i t i o n -a l t h i r t y minutes of metabolism. Aromatic hydroxylase 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 of i m i p r a -mine was 0.5 X 10 (Figure 5). One may consider the di f f e r e n c e between the two experiments i n the fo l l o w i n g way. In the former experiment 2.5 mg. of desmethylimipramine was present i n the i s o l a t e d perfused l i v e r apparatus, before the metabolism of 2 mg. of imipramine had begun. I f the 2.5 mg. of desmethylimipramine formed d i d i n h i b i t aromatic hydroxylation then preincubation of approximately 2.5 mg. or less of desmethylimipramine tr r i o r to -5 -5 0.5 X 10 M or 1 X 10 M of imipramine metabolism should i n h i b i t aromatic hydroxylation. I t was also found that the formation of glucuronide and -5 free hydroxy metabolites of imipramine (between 1 X 10 M and 2 X 10 of imipramine), f o r f i f t e e n minutes, followed zero order k i n e t i c s . The amount of desmethylimipramine which accumulated a f t e r f i f t e e n minutes of incubation with imipra--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 df imipramine (1 X 10 "*M) was 1 mg. To show that the desmethylimipramine formed could suppress aromatic hydroxylation and produce zero order k i n e t i c s , preincubation of 2 mg. or less of desmethylimipramine should i n h i b i t aromatic hydroxylation when the dose of imipramine was between 1 X 10 _ 5M or 2 X 10~ 5 M. 63 Varying amounts of desmethylimipramine were preincubated -5 -5 f i v e minutes p r i o r to the a d d i t i o n of 0.5 X 10 M, 1 X 10 M -5 or 2 X 10 M of imipramine i n t o the perfusion apparatus. The incubation 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 given i n Figure 6. They are compared to c o n t r o l experiments without p r e t r e a t -ment of desmethylimipramine. Preincubation of 6.6 X 10 (2 mg.) or l e s s of desmethy-imipramine with the perfused l i v e r p r i o r to addition of 0.5 X -5 -5 10 M or 1 X 10 J5f of imipramine i n h i b i t e d aromatic hydroxyla-t i o n . The incubation time was f i f t e e n minutes. The i n h i b i t i o n -5 of aromatic hydroxylation f o r 1 X 10 M of 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 could i n h i b i t the forma-t i o n of aromatic hydroxylation. The true per cent i n h i b i t i o n of imipramine metabolism should be between 45 and 58 per cent since the imipramine remaining a f t e r t h i r t y minutes of imipra-mine metabolism was 2.032 ± 0.3 3 mg. and the experimental i n h i b i -t i o n studies were done f o r 1.5 and 3 mg. of imipramine metabolism. To further study the e f f e c t of desmethylimipramine on aromatic hydroxylation, desmethylimipramine was preincubated -5 -5 p r i o r to addition of 1 X 10 M or 2 X 10 M imipramine i n t o the perfusion apparatus. The quantity of desmethylimipramine which was ca l c u l a t e d to i n h i b i t aromatic hydroxylation was between 1 and 2 mg. since t h i s was the amount of desmethylimipramine -5 -5 formed from imipramine 1 X 10 M or 2 X 10 M incubated for f i f t e e n minutes. ! 64 c o -H 1.65 3.3 6.6 1 3 . 2 Dose (X 10 M desmethylimipramine) Figure 6 E f f e c t of desmethylimipramine on the t o t a l formation of glucuronide, free hydroxy and N-oxide metabolites of imip-ramine. Desmethylimipramine was preincubated f i v e minutes p r i o r to addition of imipramine 0.5 X 10 MA, 1 X 10 ~*M0, — 5 /~\ and 2 X 10 M O . Incubation for imipramine metabolism was f i f t e e n minutes. 1 65 -70 -60 -40 O u •P c o o -20 +5 1.65 Dose 3.3 I , 6.6 —6 (X 10 M Desmethylimipramine ) Figure 7 L _ 13 .2 E f f e c t of desmethylimipramine on the t o t a l formation of glu c u r o n i d e O , free hydroxy^or N-oxide A metabolites of imipramine. The incubation time was f i f t e e n minutes -5 for imipramine metabolism (1 X 10 M).Desmethylimipramine was preincubated f i v e minutes p r i o r to imipramine metab-olism. 66 _L 1.65 Dose 3.3 6.6 13.2 . - 6 , (X 10 UM Desmethylimipramine) Figure 8 E f f e c t of desmethylimipramine on endogenous formation of desmethylimipramine from imipramine metabolism. Desmethyl-imipramine was preincubated f i v e minutes p r i o r to addition .-5. of imipramine 0.5 X 10 5MA, 1 X lO'^M1 Incubation time was f i f t e e n minutes. >, and 2X10 5M O . 67 D e s m e t h y l i m i p r a m i n e was p r e i n c u b a t e d f i v e m i n u t e s p r i o r t o a d d i t i o n o f i m i p r a m i n e 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 were t e r m i n a t e d a f t e r f i f t e e n m i n u t e s . The b i l e , p e r f u s a t e 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-l i t e s . The r e s u l t s a r e p r e s e n t e d i n F i g u r e 7 f o r i n h i b i t i o n o f g l u c u r o n i d e , f r e e h y d r o x y o r N - o x i d e m e t a b o l i t e s o f i m i p r a m i n e . These r e s u l t s show t h a t g l u c u r o n i d e , f r e e h y d r o x y o r N - o x i d e m e t a b o l i t e s were i n h i b i t e d t o t h e same e x t e n t . The 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 h y d r o x y o r N - o x i d e m e t a b o l i t e s o f i m i p r a -mine was i n h i b i t e d 50 p e r c e n t ( F i g u r e 7) when p r e t r e a t e d w i t h -6 d e s m e t h y l i m i p r a m i n e 6.6 X 10 M. I n F i g u r e 8 a r e t h e r e s u l t s o f t h e e f f e c t 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 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 f r o m i m i p r a m i n e m e t a b o l i s m . An i n h i b i t i o n o f m e t a b o l i s m o c c u r r e d o n l y a t h i g h e r s u b s t r a t e c o n c e n t r a t i o n s . D i s t r i b u t i o n A l t e r a t i o n o f t h e c o n c e n t r a t i o n o f i m i p r a m i n e a t t h e s i t e where i t was m e t a b o l i z e d , i n t h i s c a s e t h e l i v e r , c o u l d a f f e c t i t s m e t a b o l i s m . I t w o u l d be p o s s i b l e t h a t a p r e i n c u b a t i o n w i t h 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 a l t e r i t s d i s t r i b u t i o n between t h e l i v e r and p e r f u s a t e and i n t u r n a l t e r i t s m e t a b o l i s m . I n a l l e x p e r i m e n t s t h e p e r c e n t d i s t r i b u t i o n o f a m e t a b o l i t e o r i m i p r a m i n e i n t h e b i l e , p e r f u s a t e and l i v e r was d e t e r m i n e d . The e f f e c t 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 t h e d i s t r i b u t i o n o f i m i p r a m i n e and i t s m e t a b o l i t e s when t h e dose -5 -5 -5 o f i m i p r a m i n e was 0.5 X 10 M , l X 10 M o r 2 X 10 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 Metaboli Imipramine HC1, 0.5 X 10~5M; Incubation Time F i f t e e n Minutes. 2 % D i s t r i b u t i o n ± S.E. Dose of Perfusate B i l e L i v e r Desmethylimipramine n IMI DMI GOH N-0 GOH IMI DMI GOH N-0 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 87.6 ±1.5 36.1 ±3.1 88.7 ±4.3 1.65 X 10~6M(0.5 mg.) 1 34 .3 17.4 41.4 81.2 20.8 65.5 81.9 36.2 12.7 3.3 X 10~6M(1 mg.) 1 25.7 13.2 32.3 23.1 26.0 74.1 86.3 39.4 74.4 6.6 X 10 _ 6M(2 mg.) 1 34 .1 13.2 28.0 — 21.0 65.6 82.0 36.2 — 13.2 X 10 _ 6M(4 mg.) 1 32.0 19.6 43.9 25.1 28.6 67.9 79.6 -- 66.2 1 amount of desmethylimipramine added f i v e minutes p r i o r to imipramine metaboli 2 %= ug of IMI, DMI, GOH or N-0 i n l i v e r , perfusate or b i l e X 100 t o t a l ug of IMI, DMI, GOH or N-0 n= number of experiments Table XII 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 Metabolites. -5 Imipramine HC1 1 X 10 M; Incubation Time F i f t e e n Minutes. 2 % D i s t r i b u t i o n ± S.E. Dose of Perfusate B i l e L i v e r Desmethylimipramine n IMI DMI GOH N-0 GOH IMI DMI GOH N-0 0 4 20.2 14.3 39 .6 38.8 20.6 79.2 85.1 38.1 5 8.7 ±2.4 ±1.2 ±7.2 ±2.6 ±5.3 ±2.5 ±2.0 ±2.5 ±4.1 1.65 X 10 _ 6M(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 10~6M(1 mg.) 3 24.3 15.0 39.3 34.2 21.3 75.5 84.4 38.8 62.7 ±2.2 ±1. 4 ±5.0 ±2.1 ±1.0 + 2.0 ±1.3 ±4.3 ±1.1 6.6 X 10 _ 6M(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 _ 6M(4 mg.) 1 23.1 17.2 41.2 29.3 0.0 76.5 82.3 58.7 70.2 1 amount of desmethylimipramine added f i v e minutes p r i o r to imipramine metabolism 2 %= ug of IMI, DMI, GOH or N-0 i n l i v e r , perfusate or 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 Metabolites. Imipramine HC1 ,2 X 10~5M; Incubation Time f o r F i f t e e n Minutes. 2 % D i s t r i b u t i o n ± S.E. Dose of 1 Perfusate B i l e L i v e r Desmethylimipramine n IMI DMI GOH N-0 GOH IMI DMI GOH N-0 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 10~6M(1 mg.) 2 26.0 20.5 45.4 48.8 20.1 67.2 78.8 31.0 51.2 6.6 X 10~6M(2 mg.) 2 24.5 18.1 25.9 37.5 5.0 70.7 81.2 67.53 62.5 13.2 X 10~6M(4 mg.) 1 28.0 18.4 35.2 4.0 67.0 81.0 61.8 1 amount of desmethylimipramine added f i v e minutes p r i o r to imipramine metabolism 2 %= ug of IMI, DMI, GOH or N-0 i n l i v e r , perfusate or 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 XI, XII and X I I I . Pretreatment of desmethylimipramine f o r experiments -5 -5 where the dose of imipramine was 1 X 10 M or 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 the d i s t r i b u t i o n of imipramine and desmethylimipramine. The per cent d i s t r i b u t i o n of imipramine and desmethylimipramine was the same as the con t r o l experiments. The i n h i b i t i o n of imipramine metabolism -5 -5 (1 X 10 M and 2 X 10 M) by decreasing the amount of imipramine a v a i l a b l e to the l i v e r to be metabolized was ruled 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 could be -5 seen at the substrate concentration of 0.5 X 10 M. Imipramine increased 10 per cent i n the perfusate and decreased 10 to 15 per cent i n the l i v e r when desmethylimipramine was present. E f f e c t of E t h y l Alcohol on Aromatic Hydroxylation and N-demethylation I t was found that exogenous desmethylimipramine did i n h i b i t aromatic hydroxylation of imipramine. Therefore, i t was thought that the desmethylimipramine formed from imipramine metabolism could suppress aromatic hydroxylation of imipramine. To further demonstrate that desmethylimipramine had t h i s e f f e c t , i n h i b i -t i o n of desmethylimipramine formation from imipramine metabolism at high substrate concentration should r e s u l t i n an increase i n aromatic hydroxylation of imipramine. Therefore, experiments were c a r r i e d out to demonstrate t h i s e f f e c t . 72 Two i n t e r e s t i n g properties had been reported i n the l i t e r a -ture 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 alcohol was repo-rted to i n h i b i t aromatic hydroxylation 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 to f i n d out i f t h i s compound would i n h i b i t N-demethy-l a t i o n and aromatic hydroxylation of imipramine. I f N-demethyla-t i o n of imipramine metabolism was s e l e c t i v e l y suppressed by et h y l a l c o h o l , then t h i s compound would be used to f i n d out whether i n h i b i t i o n of desmethylimipramine at high substrate concentration of imipramine metabolism would r e s u l t i n an increase i n aromatic hydroxylation. 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 alcohol caused an increase i n NADH content i n the l i v e r (51, 52, 53). This increase i n l i v e r NADH content coincides with an increase i n the r a t i o of l a c t i c a c i d to pyruvic a c i d . The normal r a t i o of l a c t i c a c i d to pyruvic acid was 10, a f t e r e t h y l alcohol pretreatment the perfusate r a t i o increased to 125 or higher. This increase i n r a t i o was due to a decrease i n pyruvic 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, 55). The following experiments were c a r r i e d out to inv e s t i g a t e the e f f e c t of ethyl alcohol on N-demethylation and aromatic hydroxylation of imipramine. The same procedure and dose of eth y l alcohol was used as was reported by Vendsborg and Schanbye (54) to increase the lactate/pyruvate r a t i o i n the perfusate of the i s o l a t e d r a t l i v e r preparation. In one experiment, e t h y l alcohol was added twice, p r i o r to imipramine metabolism. The dose of e t h y l alcohol was 3 mM and was added f o r t y f i v e minutes 73 -5 p r i o r to adding 1 X 10 M imipramine. The second dose of e t h y l alcohol was 1.5 mM and was added f i f t e e n minutes p r i o r to imipra-mine metabolism. I t was found that the amount of desmethylimipra-mine formed decreased from 35 per cent to 10 per cent of the dose of imipramine (Table XIV), however there was l i t t l e change i n aromatic hydroxylase a c t i v i t y . The amount of glucuronide and free hydroxylated metabolite formed were s i m i l a r to the c o n t r o l value of 2 8 per cent. The l a c t a t e to pyruvate r a t i o increased four f o l d from the c o n t r o l value of 10. In another experiment the same experimental procedure was followed except the dose of e t h y l alcohol was reduced to 1.5 mM and 0.75 mM p r i o r to addition of imipramine. The reason the dose of e t h y l alcohol was decreased was to decrease the lactate-pyruvate r a t i o to 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 alcohol was decreased to one h a l f the dose previously used the l a c t a t e to pyruvate r a t i o was greater than the c o n t r o l value of 10; i n f a c t , pyruvic acid could not be detected. In addition to an increase i n l a c t a t e to 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 alcohol t o t a l l y suppressed b i l e secretion. However, the quantity of glucuronide metabolites of imipramine formed was unaffected. Although i t was found that e t h y l alcohol 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 to determine i f the increase i n r a t i o of l a c t i c acid or increase i n NADH was responsible for the i n h i b i t i o n of N-de-methylation of imipramine. These experiments were designed to f i n d the maximum quantity of e t h y l alcohol. 74 Table XIV E f f e c t of E t h y l Alcohol on Iraipramine Me-tabalisa B 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 Incuba--5 txon, 1 X 10 M .was the Substrate Concentration. Per Cent of Dose Metabolized 1 Dose of Lactate/ E t h y l A l c o h o l n Pyruvate DMI GOH G OH N-0 mg./lOOal. 0 1 55.5 ,rt 34.8 27.9 24.2 3.7 6.3 =10 5.3 0.25 BM 2 26_ 26.2 25.5 22.0 3.5 3.9 0 0.5 aM 1 26 28.9 27.6 24.0 3.4 4.3 0 1 mM 1 22 20.6 26.3 21.2 5.1 3.9 0 3 am 1 28 15.7 23.2 19.1 4.1 4.3 0 1.5 mM+ 2 1 59 , Q 15.5 25.0 21.0 3.92 2.7 0.75 IBM 1-4 3 mM+ 2 1 41.5 10.3 26.8 22.3 4.5 2.9 1.5 mM 0 e t h y l a l c o h o l added to perfusate f o r t y f i v e minutes p r i o r to imipramine metabolisa f i r s t dose of e t h y l alcohol added to perfusate 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 imipra-mine metabolism per cent IMI metabolized or metabolite formed= t o t a l IMI, DMI, GOH, OH or N-0 formed X 100 14 t o t a l C recovered 75 which would bring about s i g n i f i c a n t i n h i b i t i o n of desmethylimip-ramine. In these experiments the dose of e t h y l alcohol administer-ed was decreased t i l l i n h i b i t i o n of N-demethylation was no long-er observed. The experiments were done i n the f o l l o w i n g way. E t h y l alcohol was added f o r t y f i v e minutes p r i o r to —5 -5 addition of imipramine 1 X 10 M or 0.5 X 10 M. The incubation 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 alcohol 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 alcohol on imipramine metabolism (0.5 X 10~ 5M). Et h y l alcohol i n h i b i t e d N-demethylation of imipramine, when -5 1 X 10 M of imipramine was incubated for f i f t e e n minutes. The maximum i n h i b i t i o n of N-demethylation was attained with 3 mM of e t h y l a l c o h o l ; however, there was some decrease i n aromatic hydroxylation as w e l l . When the dose of e t h y l alcohol was 1 mM „ N-demethylation was i n h i b i t e d 41 per cent. The amount of glucu-ronide and free hydroxy metabolites of imipramine were the same as the c o n t r o l value. Although the amount of desmethylimipramine decreased from 1 mg. to 0.6 mg. with 1 mM of e t h y l alcohol a f t e r f i f t e e n minutes of imipramine metabolism, there was no e f f e c t on the aromatic hydroxylation of imipramine. Therefore, aromatic hydroxylation had not been suppressed by the formation of des-methylimipramine from imipramine at t h i s dosage l e v e l . I t was found that the increase i n lactate-pyruvate r a t i o or an increase i n NADH caused by e t h y l alcohol had no r e l a t i o n -ship to the i n h i b i t i o n of N-demethylation since 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 a l c o h o l , a lthough the r a t i o of l a c t i c a c i d t o p y r u v i c a c i d 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 not be de t e c t e d i n the p e r f u s a t e . The r e s u l t s 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 -5 metabolism when the dose of imipramine was 0.5 X 10 M appear i n Table XV. I t was found t h a t when the dose of e t h y l a l c o h o l was 0.5 mM or 1 mM t h e r e was no e f f e c t on N-demethylation of imipramine. A h i g h e r dose o f e t h y l a l c o h o l 3 mM, caused i n h i -b i t i o n of N-demethylation but had ve r y l i t t l e e f f e c t on aromatic h y d r o x y l a t i o n . -5 When the dose of imipramine was 2 X 10 M i t was thought t h a t the desmethylimipramine formed from imipramine metabolism a f t e r f i f t e e n minutes and s i x t y minutes caused s u p p r e s s i o n o f aromatic h y d r o x y l a t i o n of imipramine. To show t h i s e f f e c t , a dose of e t h y l a l c o h o l was chosen which would i n h i b i t the forma-t i o n of desmethylimipramine. 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 (imipramine 1 X 10 M) by 40 per cent and had no e f f e c t on aromatic 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 M) and metabolism f o l l o w e d f o r s i x t y minutes i t was found (Table XVI) t h a t the amount of desmethylimipramine formed decreased from 421 ug/ gm. l i v e r t o 308 ug./ gm. l i v e r . The amount of imipramine which was me 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 i n c r e a s e d from 325 ug t o 366 ug/ gm. l i v e r . When 1 mM e t h y l a l c o h o l -5 was p r e i n c u b a t e d f o r t y f i v e minutes p r i o r t o imipramine (2 X 10 M) f o r f i f t e e n minutes (Table XVI) the amount of desmethylimipra-mine decreased from 211 ug/ gm. l i v e r t o 132 ug/ gm. l i v e r and 77 Table XV E f f e c t of E t h y l Alcohol on Imipramine Metabolism By the Isolated Perfused Rat L i v e r ; F i f t e e n Minutes Incubation With -5 0.5 X 10 M Imipramine. 2 Per Cent of Dose Metabolized Dose of 1 Lactate/ E t h y l Alcohol n Pyruvate DMI GOH G OH N-0 mg./100ml. 0 1 21 - i n 2 6 ' 3 4 0 - 2 3 5 , 0 5 ' 4 4 , 8 0.5 mM 1 30 23.9 38.7 33.2 5.6 2.6 0 1 mM 3 25 19.5 23.3 38.7 35.9 2.7 6.5 0 ;"0 ±1.3 ±1.7 ±2.3 ±0.9 ±1.6 3 mM 1 26 12.9 35.8 33.9 1.9 5.3 0 1 e t h y l alcohol added to perfusate f o r t y f i v e minutes p r i o r to imipramine metabolism 2 per cent IMI metabolized or metabolite formed= t o t a l IMI, DMI, GOH, OH or N-0 formed X 100 t o t a l 1 4C recovered 78 the amount of glucurcnide, free hydroxy and N-oxide metabolites of imipramine increased from 158 ug/ gm. l i v e r to 196 ug/ gm. l i v e r . In two of three experiments i n each group of experiments the blood flow through the l i v e r was below 2 ml./ gm. l i v e r per minute and therefore the data was not used. As a r e s u l t only one experiment i n each group was reported. From these r e s u l t s i t can be seen that 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 formation there was an increase i n formation of hydroxylated metabolites. This -5 occurred when 2 X 10 M of imipramine was metabolized 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 hydroxylation. At a l l doses and incubation times t h i n l a y e r chromatography was done to i d e n t i f y the hydroxy metabolites formed from imipra-mine metabolism. The predominant metabolite formed was 2-hydroxy-imipramine. Small amounts of u n i d e n t i f i e d metabolites, as w e l l as 2-hydroxydesmethylimipramine, were formed when the dose of imipramine was 2 X 10 and metabolism was f o r s i x t y minutes. However, the primary hydroxy metabolite formed from imipramine was 2-hydroxyimipramine and very l i t t l e 2-hydroxydesmethylimipra-mine. I f 2-hydroxydesmethylimipramine was a major metabolite, then i n h i b i t i o n of desmethylimipramine formation by e t h y l alcohol would r e s u l t i n a decrease i n the amount of glucuronide and free hydroxy metabolite formed. This did not happen with any experi-ment where desmethylimipramine was i n h i b i t e d . Table XVI Ef f e c t of Ethyl Alcohol on Imipramine Metabolism By the Isolated 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 Dose o f 1 IMI DMI GOH,N-0 Time Ethyl % of dose ug/ % of dose ug/ % of dose ug/ Alcohol Remaining gm. L Metabolized gm. L Metabolized gm. L 60 0.0 19.7 162.1 50.4 421.7 29.8 235.3 n=4 + 2.1 ±21.3 ±5.5 ±80.9 ±4.5 ±23.1 60 n = 1 1 mM 13.2 102 40.1 308.0 46.6 366 15 0.0 56.9 478.9 25.4 211.6 19.1 158.4 n=4 ±1.5 ±43.3 ±1.5 ±14.4 ±1.4 ±8.7 15 1 mM 47.5 365 19.0 132.0 28.48 196 n=l 1 e t h y l alcohol added to perfusate 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 of Ethyl Alcohol on the D i s t r i b u t i o n of Imipramine and Metabolites. Imipramine -5 HC1 (1 X 10 M); Incubation Time F i f t e e n Minutes. % D i s t r i b u t i o n ± S.E. Dose of Perfusate B i l e L i v e r Ethyl Alcohol n IMI DMI GOH N-0 GOH IMI DMI GOH N-0 0 1 22.6 14.1 49.0 35.1 19.4 77.0 85.2 29.5 54.4 0.25 mM 2 28.1 + 4.76 18.93 ±6.52 45.14 ±0.76 41.3: ±3.3 16.04 ±2.24 71.54 ±8.0 80.04 ±6.9 36.61 ±1.65 52.55 ±1.48 0.5 mM 1 23.55 15.84 39.40 15.5] 21.36 76.32 83.25 37.13 84.09 1 mM 1 30.17 15.31 45.89 25.5E 13.18 69.76 83.81 38.9 69.56 3 mM 1 34.79 20.99 65.71 34.0^ 0.81 65.20 77.80 30.54 63. 59 1.5 mM 2 , 0.75 mM 1 24.8 17.2 62.3 55.7 4.8 75.2 81.8 30.1 42.0 3 mM 2 , 1.5 mM 1 25.9 15.4 76.0 63.3 0.0 74.1 83.5 20.7 46.7 1 et h y l alcohol added to perfusate f o r t y f i v e minutes p r i o r to imipramine metabolism. 2 f i r s t dose of et h y l alcohol added to perfusate 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 to imipramine metabolism. 3 %= ug of IMI,DMI, GOH or N-0 i n l i v e r , perfusate or b i l e X 100 t o t a l ug of IMI, DMI, GOH or N-0 formed CO o Table XVIII E f f e c t of E t h y l Alcohol on the D i s t r i b u t i o n of Imipramine and Metabolite. Imipramine HC1 (0.5 X 10~ 5M), Incubation Time F i f t e e n Minutes. % D i s t r i b u t i o n ± S.E. Dose of Perfusate B i l e L i v e r Ethyl Alcohol n IMI DMI GOH N-0 GOH IMI DMI GOH N-0 0 4 19.0 11.6 37.1 15.7 26.2 80.3 87.6 36.1 88.7 + 1.6 ±0.53 ±4.4 + 1.73 ±3.1 +1.4 ±1.5 ±3.1 ±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 14.65 48.4 18.46 19.02 72.57 84.58 27.6 60.12 + 2.1 ±1.21 ±3.7 ±5.7 ±9.8 ±2.9 ±1.25 ±1.5 ±9.52 3 mM 1 32.15 18.63 63.93 44.75 5.68 67.80 80.58 27.92 50. 59 1 et h y l alcohol added to perfusate f o r t y f i v e minutes p r i o r to imipramine metabolism 2 %= ug of IMI, DMI, GOH or N-0 i n l i v e r , perfusate or b i l e X 100 t o t a l ug of IMI, DMI, GOH or N-0 formed 82 D i s t r i b u t i o n I f a l t e r a t i o n of imipramine concentration at the s i t e of metabolism could a f f e c t i t s metabolism then i t would be pos s i b l e that a pretreatment could a l t e r the d i s t r i b u t i o n of imipramine and i n turn a l t e r the metabolism. In a l l experiments the per cent d i s t r i b u t i o n of a metabolite or imipramine i n the b i l e , perfusate and l i v e r was determined. The e f f e c t of e t h y l alcohol on the d i s t r i b u t i o n of imipramine and i t s metabolites (0.5 X -5 -5 -5 10 M, 1 X 10 M and 2 X 10 M imipramine) appears i n Table XVII and XVIII. Compared to controls there was a s l i g h t decrease of imipra-mine and desmethylimipramine i n the l i v e r s which were p r e t r e a t -ed with e t h y l alcohol. The decrease or 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 to a l t e r the metabolism -5 of imipramine since 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 -t i o n pattern. Discussion of Imipramine Metabolism The objective of these experiments was to gain more know-ledge and a better 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 continuing research on possible c e l l u l a r c o n t r o l mechanisms involved i n imipramine metabolism. The decline i n plasma concentration of many drugs i s frequent-l y assumed to follow apparent f i r s t order k i n e t i c s independent 83 of the dose or plasma concentration. Despite reports i n the l i t e r a t u r e that dose dependent k i n e t i c s f o r drug metabolism have been demonstrated i n vivo and i n the i s o l a t e d perfused r a t l i v e r f o r c e r t a i n drugs, research i s done on drug metabolism i n the i s o l a t e d perfused r a t l i v e r without f i r s t acquiring know-ledge about the r e l a t i o n s h i p of substrate concentration to the rate of drug metabolism. Since imipramine at c e r t a i n concentra-tions i n h i b i t s i t s own metabolism i n microsomal studies 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 rate of imipra-mine metabolism was dependent on two p r i n c i p a l enzymatic reac-t i o n s , aromatic hydroxylation and N-demethylation. I t i s possible that at c e r t a i n concentrations of imipramine one enzymatic reac-t i o n could reach saturation k i n e t i c s while the other enzymatic rea c t i o n might not. I t had been demonstrated i n t h i s research that aromatic hydroxylation was i n h i b i t e d at higher concentrations -5 of imipramine (2 X 10 M). I t also had been shown that at longer incubation time (eg. s i x t y minutes) aromatic hydroxylation was suppressed. In most experiments u t i l i z i n g the i s o l a t e d perfused l i v e r technique to study drug metabolism the perfusate h a l f - l i f e (th) of the drug was the only parameter monitored (56,57,58,59,60). This parameter was used to determine the h a l f - l i f e or rate con-stant f o r drug metabolism. This may have led to erroneous r e s u l t s since t h i s parameter (th) may not be a true i n d i c a t i o n of drug metabolism as was indicated i n t h i s t h e s i s . I t was found that over extended incubation time the metabolism of imipramine 84 — 5 (2 X 10 M) 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 incubation time and therefore t h i s i n h i b i t i o n would not be detected. Comparing perfusate drug concentrations to determine i f a d i f f e r e n c e i n drug metabolism had occurred, due to various pretreatments, could lead to f a l s e conclusions. An example of where a change i n perfusate concentra-t i o n had occurred although the rate of 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 alcohol on imipramine metabolism. I t was found that the per cent of imipramine increased i n the perfusate a f t e r treatment with e t h y l alcohol (Table X V I I I ) ; however, the metabolism of imipra-mine was unaltered. Therefore, i t i s necessary to monitor other parameters i n add i t i o n to perfusate concentration, such as the amount of a drug which i s i n the l i v e r . Another reason for analyzing the t o t a l amount of imipramine i n the i s o l a t e d perfused system i s that at higher doses of imipramine the l i v e r may be reaching a saturated condition with respect to imipra-mine and desmethylimipramine binding and therefore the perfu-sate concentration w i l l be higher. The f r a c t i o n of the t o t a l amount of imipramine present i n the l i v e r s t e a d i l y decreased -5 from 80 per cent with a dose of 0.5 X 10 M to 70 per cent . . . -5 when a dose of imipramine 2 X 10 M was used. This means that the per cent i n the perfusate increased from 19 per cent to 30 per cent. The same r e s u l t s were found for desmethylimipramine. From the r e s u l t s of t h i n layer chromatography and i n h i b i -t i o n of desmethylimipramine formation by e t h y l alcohol the 85 primary enzymatic reactions f o r the degradation of imipramine at f i f t e e n minutes was N-demethylation and aromatic hydroxyla-t i o n . Although some 2-hydroxydesmethylimipramine was i s o l a t e d from t h i n layer chromatography studies a f t e r s i x t y minutes, i t does not seem to be a major metabolic route. The other minor metabolic route f o r degradation of imipramine was N-oxidation. Since some of the r e s u l t s f o r the formation of imipramine N-oxide were e r r a t i c and the c o n t r i b u t i o n to imipramine metabolism was minor, very l i t t l e discussion w i l l center around the enzy-matic r e a c t i o n of N-oxidation. The t o t a l amount of free hydroxy metabolites present a f t e r imipramine metabolism was very low. Most of t h i s metabolite was i n the conjugated form as the glucuronide (Table X). The t o t a l amount of free hydroxy metabolite was approximately the same, 0.084, 0.12 and 0.082 mg. while the t o t a l amount of glucuronide metabolite was 0.544, 0.688 and 0.792 mg. which -5 increased as the dose of imipramine increased (0.5 X 10 M, 1 X 10~5M and 2 X 10~ 5M). The incubation time was f i f t e e n minutes for these experiments. This suggests that the conjuga-t i o n reaction was more rapid than the formation of 2-hydroxy-imipramine from imipramine. There appears to be a constant r e s i d u a l f r a c t i o n of free hydroxy metabolites remaining independ-ent of the dose of imipramine. The per cent of the dose of glucuronide and free hydroxy metabolites which was excreted i n the b i l e was the same. This suggests that b i l i a r y excretion of these metabolites was not 86 saturated. When high doses of e t h y l alcohol were used, b i l e flow was i n h i b i t e d . Consequently there was very l i t t l e or no excretion of glucuronide metabolites. The amount of aromatic hydroxy metabolites formed i n these experiments were the same as controls (Table XIV,XVII). Therefore, i n these experiments, hydroxylation of imipramine as w e l l as conjugation of t h i s metabolite i s independent of b i l i a r y excretion. The rate of imipramine metabolism was a l t e r e d with incuba-t i o n time and imipramine concentration. The metabolism of imipra-mine was dose dependent due to decreased aromatic hydroxylase -5 a c t i v i t y . At a higher 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 to f i n d the reason why aromatic hydroxylase was i n h i b i t e d . I t was found that desmethylimipramine i n the same -5 quantity 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 hydroxylation and N-oxida-t i o n . In a d d i t i o n , e t h y l alcohol was found to i n h i b i t the forma-t i o n of desmethylimipramine, and to increase the formation of -5 hydroxylated metabolites (imipramine, 2 X 10 M; incubation time f i f t e e n and s i x t y minutes). I t therefore could be conclud-ed from these r e s u l t s that the desmethylimipramine formed from imipramine metabolism i n h i b i t e d aromatic hydroxylation a f t e r f i f t e e n and s i x t y minutes of metabolism. The rate of aromatic hydroxylation of imipramine was found to be dependent on the concentration of imipramine. A l l of the above experiments l e d to the conclusion that dose dependent k i n e t i c s at higher con-87 centrations of imipramine was due to i n h i b i t i o n of the aromatic hydroxylase caused by the endogenous formation of desmethylimipr mine. There have been reports i n the l i t e r a t u r e that desmethyl-imipramine may i n h i b i t the hydroxylation reaction i n the metabo-l i s m of such drugs as phenobarbital (23), tremorine (24) and amphetamine (25). These studies have been done i n vivo and i n v i t r o on r a t s . Desmethylimipramine i n h i b i t e d amphetamine metabo-li s m i n the i s o l a t e d perfused r a t l i v e r system. In a l l these studies only the disappearance of the drug was measured so i t i s not known i f , f o r example, p-hydroxylation of the aromatic r i n g was i n h i b i t e d i n the metabolism of amphetamines. In our labora-tory i t was found that desmethylimipramine d i d competitively i n h i b i t aromatic hydroxylation of imipramine i n mouse l i v e r homogenate (61). Desmethylimipramine was metabolized mainly by aromatic hydroxylation i n the i s o l a t e d perfused l i v e r (20). I t would then seem that desmethylimipramine could i n h i b i t aromatic hydroxylation i n a competitive manner. More experiments are needed to 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 competitive. E t h y l alcohol has been shown to i n h i b i t the a c t i v i t i e s of a n i l i n e (47) or phenobarbital (48) microsomal hydroxylase and the demethylation of aminopyrine (49) or ethylmorphine (50). However e t h y l alcohol was found to 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 perfused r a t l i v e r system. The highest dose of et h y l alcohol used (Table XIV) reduced the formation of desmethylimipramine from 35 per cent to 10 per cent without s i g n i f i c a n t i n h i b i t i o n of aromatic 88 hydroxylation. This type of i n h i b i t i o n i s very complex and i s not of the competitive type. Evidence f o r t h i s i s that 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 of 1 X 10 - 5M 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 speculation on whether there are one or many d i f f e r e n t drug metabolizing enzymes for d i f f e r e n t substrates. 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 only i n d i r e c t evidence can be supplied f o r one or 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 kinds of observations. There are marked species differences (2), and sex differences (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 kinds of drug metabolism stimulated 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 provided f o r more than one cytochrome P-450 system since e t h y l alcohol was found to i n h i b i t N-demethylation of imipramine without i n h i b i t i n g aromatic hydroxylation. The most s u i t a b l e incubation time f o r imipramine was f i f t e e n minutes since longer incubation times may i n h i b i t aromatic hydroxylation of imipramine. The most s u i t a b l e dose of imipramine used to study the various e f f e c t s on imipramine -5 metabolism was 1 X 10 M. At thxs dose aromatic hydroxylation was not suppressed and adequate amounts of imipramine remained for further metabolism i f a c e r t a i n pretreatment resulted i n stimulation of metabolism. 89 C e l l u l a r C o n t r o l Mechanisms f o r Imipramine Metabolism E f f e c t of Magnesium on Imipramine Metabolism In a l l p r e v i o u s l y reported experiments, the perfusate magne-sium sulphate concentration, f o r imipramine metabolism, i n the i s o l a t e d perfused r a t l i v e r was 0.014 mg./ ml. To determine whether the magnesium concentration i n the l i v e r was a t optimum concentration f o r maximum imipramine metabolism the f o l l o w i n g experiments were done. The c o n t r o l experiments contained the normal perfusate concentration of magnesium (0.014 mg./ ml.) which the l i v e r was perfused with f o r one hour before the a d d i t i o n of imipramine HC1. The dose of imipramine HC1 used i n a l l these -5 experiments was I X 10 M (3.17 mg.) and the incubation time was f i f t e e n minutes.In a l l other experiments magnesium sulphate was e i t h e r omitted from the p e r f u s i o n medium or twice the normal concentration of magnesium sulphate (0.028 mg./ ml.) was used. In these experiments as i n the c o n t r o l experiment the l i v e r was then perfused f o r one hour before imipramine HC1 was added t o the i s o l a t e d perfused r a t l i v e r apparatus. To f i n d out whether omission of magnesium from the p e r f u s i o n medium or twice the normal perfusate magnesium concentration a l t e r e d the magnesium concentration i n the l i v e r , the perfusate magnesium concentra-t i o n was determined. Magnesium was analyzed i n the c i r c u l a t i n g p e r f u s i o n medium before and a f t e r the l i v e r was perfused f o r one hour. The d i f f e r e n c e i n perfusate magnesium concentration before and a f t e r p e r f u s i n g the l i v e r was a t t r i b u t e d to a change i n l i v e r magnesium concentration. Table XIX E f f e c t of Magnesium on Imipramine Metabolism i n the Iso l a t e d Perfused Rat L i v e r . The Incubation Time was F i f t e e n Minutes and the Substrate Concentration was 1 X 10 M. Magnesium Added Magnesium Concentration Per Cent 3 of Dose Metabolized to Perfusion Medium n Before P e r f u s i o n 1 A f t e r P e r f u s i o n 2 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 perfusate magnesium concentration p r i o r to perfusion with l i v e r 2 perfusate magnesium concentration a f t e r perfusion of r a t l i v e r f o r one hour 3 per cent= t o t a l IMI, DMI, GOH, G or N-0 formed x 1 0 Q t o t a l 1 4C recovered VO o 91 I t was found that changing the magnesium perfusate concen-t r a t i o n had no e f f e c t on imipramine metabolism (Table XIX). The reason magnesium d i d not have an e f f e c t on imipramine metabolism was that magnesium i n the l i v e r was not a l t e r e d since the mag-nesium concentration i n the perfusate a f t e r perfusion of the l i v e r f o r one hour was the same as the concentration before per-fusion of the l i v e r . This occurred e i t h e r with an excess or no magnesium i n the perfusate. I t was also found by others (7) that the omission of magnesium from the perfusate d i d not cause mag-nesium to be released by the l i v e r . E f f e c t of Dibu 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 of glucagon HC1 ( E l i L i l l y and Co.) or the monosodium s a l t of 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 perfused 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) or d i b u t y r y l c y c l i c AMP (2 X 10 M^) was added to the perfusion f l u i d ten minutes p r i o r to the addi t i o n of imipramine. Imipra--5 -5 mine (0.5 X 10 M or 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 perfused 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 the foll o w i n g reasons. Desmethylimipramine 0.5 mM has been shown to increase c y c l i c AMP l e v e l s i n brain s l i c e s (68,69) and i n a recent report (60) d i b u t y r y l c y c l i c AMP and glucagon were found to i n h i b i t drug metabolism i n the i s o l a t e d perfused 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 or Glucagon, on Imipramine Metabolism. Incubation Time was f o r F i f t e e n Minutes With the I s o l a t e d Perfused Rat L i v e r . The Dose of Imipramine was 0.5 X 10~5M or 1 X 10~5M. Per Cent of Dose Metabolized 2 Treatment 1 n IMI Remaining DMI GOH G N-0 Control 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 10~5M 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 1 Glucagon (5.7 X 10" 8M), d i b u t y r y l c y c l i c AMP (DBc 2 X 10 M was added ten minutes p r i o r to imipramine metabolism. 2 per cent= ug of IMI, DMI, G or N-0 formed x 1 Q 0 ug of 1 4C recovered 1 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 aro-matic hydroxylation i n the i s o l a t e d r a t perfused l i v e r by des-methylimipramine could be by i n c r e a s i n g c y c l i c AMP l e v e l s i n 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 i n the l i v e r c e l l could then i n h i b i t aromatic hydroxylation of imipramine. I t was shown that 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-chloro-N-methylaniline 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 hydroxylation occur from the same substrate as was found f o r e t h y l alcohol i n h i b i t i o n of aromatic hydroxylation? The d i b u t y r y l analogue of c y c l i c AMP was used because i t has been demonstrated to produce p a r a l l e l e f-fe c t s to those caused by c y c l i c AMP at lower concentration(70)because of increased penetration through c e l l u l a r membranes and decreased rate of destruction by phosphodiesterase. Glucagon was used i n these experiments because i t increased 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 to occur f o r desmethylimipramine. Glucagon or 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 imipra-mine metabolism at 0.5 X 10 5M; however both compounds produced -5 i n h i b i t i o n of imipramine metabolism at 1 X 10 M (Table XX). Imipramine metabolism was i n h i b i t e d 13 per cent by glucagon and 26 per cent by d i b u t y r y l c y c l i c AMP. D i b u t y r y l c y c l i c AMP caused i n h i b i t i o n of aromatic hydroxylation and N-demethylation of imipramine. The decrease, as a per cent of substrate concen-t r a t i o n , for aromatic hydroxylation was from 31 per cent (control) to 22 per cent whereas N-demethylation decreased from 39 per cent (control) to 31 per cent. I t therefore seems that N-de-methylation and aromatic hydroxylation were i n h i b i t e d to the Table XXI Ef f e c t of Glucagon or 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 of Imipramine and Metabolites. Imipramine was Incubated f o r F i f t e e n Minutes With the Isolated Perfused Rat L i v e r . % D i s t r i b u t i o n Dose of 1 Imipramine Treatment 0.5 X 10~5M Control 0.5 X 10~5M Glucagon ft 5.7 X 10 M 0.5 X 10~5M DBc AMPfi 2 X 10 1 X 10"5M Control 1 X 10~5M Glucagon ft 5.7 X 10" a 1 X 10~5M DBc AMPfi 2 X 10 1 Glucagon 5.7 X IMI Perfusate DMI IGOH N-0 B i l e GOH Li v e r IMI DMI GOH N-0 1 1 18.0 19.5 24.2 29.9 27.4 23.3 10.1 7.7 15.3 11.8 16.1 12.6 36.1 44.7 50.7 61.0 43. 3 44.1 45.0 53.6 67.4 49.1 62.5 16.1 15.0 28.5 37.6 34.5 35.2 28.0 31.5 15.6 11.7 16.8 16.0 82.0 80.1 75.6 70.1 72.5 76.7 89.7 88.4 85.4 87.7 83.1 86.9 45.0 23. 4 30.3 24.0 37.5 35. 8 23.5 23.5 26.5 16.5 27.5 19.9 83. 8 84.9 81.1 164.6 61.5 64.4 metabolism. 2 per cent= ug of IMI, DMI, GOH, G or N-0 i n l i v e r , perfusate or b i l e x 1 0 0 t o t a l ug of IMI, DMI, GOH, G or N-0 formed 95 same extent. Glucagon i n h i b i t e d imipramine metabolism but to a le s s e r extent than d i b u t y r y l c y c l i c AMP. E f f e c t of NADH, NADPH or Succinic A c i d on Imipramine Metabolism To increase the l e v e l of NADH or NADPH i n the l i v e r these compounds were added to the c i r c u l a t i n g perfusate i n the i s o l a t -ed r a t l i v e r perfusion apparatus. NADPH or NADH can traverse the membrane of the hepatic c e l l (3,4). The dose of disodium s a l t of NADH (Sigma Chemical Co.) used was 1.33 X 10~6M (10 mg.). The dose of the tetrasodium s a l t of NADPH (Sigma Chemical Co.) —6 used was 1.11 X 10 M (10 mg.). One of the t r i c a r b o x y l i c a c i d -3 cycle intermediates, potassium succinate (1.6 X 10 M), was added to the perfusate. Ten minutes l a t e r imipramine HCl (1 X -5 10 M) was added to the i s o l a t e d perfusion apparatus. The incubation time f o r imipramine metabolism was f i f t e e n minutes. The r e s u l t s of these experiments are expressed i n Table XXII. I t was found that NADH, NADPH and s u c c i n i c a c i d decreased imipramine metabolism to the same extent. The amount of imipra-mine remaining i n the cont r o l experiment a f t e r f i f t e e n minutes metabolism was 23 per cent and a f t e r treatment by a l l three compounds the per cent of the dose of imipramine remaining was 36, 32 and 36 per cent. Aromatic hydroxylation and N-demethyla-t i o n of imipramine were i n h i b i t e d to the same extent by t r e a t -ment with NADH or NADPH. The per cent of the dose of imipramine i n the con t r o l experiment which was metabolized to desmethyl-imipramine was 41 per cent. As a r e s u l t of NADH or NADPH addition Table XXII Effect of NADH, NADPH or Succinic Acid on Imipramine Metabolism (1 X 10 M) . The Incubation Time was Fifteen Minutes. Per Cent of Dose Metabolized ± S.E.2 Treatment n IMI DMI GOH OH N-0 Control 2 23.2 40.8 30.7 4.0 5.2 ±0.7 ±1.6 ±0.1 ±0.2 ±0.8 NADH 2 36.9 33.0 25.5 4.6 5.0 1.3 X 10"6M ±1.3 ±3.0 ±0.7 ±1.2 ±0.4 NADPH 2 32.5 37.2 25.8 5.0 4.2 1.1 X 10 _ 6M ±2.8 ±0.5 ±0.7 ±0.3 ±1.5 Succinic 2 35.9 29.1 29.2 6.5 5.8 Acid ±1.0 ±1.6 ±0.1 ±2.7 ±0.6 1.6 X 10~3M 1 NADH, NADPH or succinic acid was added ten minutes prior to imipramine metabolism 2 per cent= tota l IMI remaining, DMI, GOH, OH, N-0 formed x 1 0 Q 14 i t o t a l C recovered vo Table XXIII The E f f e c t of NADH, NADPH or Succinic Acid on the D i s t r i b u t i o n of Imipramine and Metabolites. Imipramine (1 X 10 - 5M) was Incubated f o r F i f t e e n Minutes 2 Per Cent D i s t r i b u t i o n ±S.E. Treatment 1 n IMI Perfusate DMI GOH OH N-0 B i l e GOH IMI DMI L i v e r GOH OH N-0 Control 2 31.5 15.6 55.3 18.9 39.7 16.9 68.3 83.7 24.6 76.6 61.3 ±1.5 + 3.8 ±5.7 ±1.7 ±2.2 ±5.3 ±1.8 ±4.0 ±0.9 ±6.1 ±4.6 NADH 2 23.3 11.9 40.2 1.7 27.6 17.2 76.5 87.8 42.6 97.8 72.4 1.3 X 10 _ 6M + 2.7 + 0.2 ±11.3 ±1.6 + 6.1 ±0.7 ±2.7 ±0.2 ±10.6 ±1.2 ±6.1 NADPH 2 28.5 15.1 38.3 9.4 39.3 22.4 71.3 84.3 37.6 91.8 60.7 1.1 X 10"6M ±0.2 ±0.1 ±1.4 ±2.5 ±0.8 ±0.1 ±0.1 ±0.1 ±1.6 ±4.3 ±0.8 Succinic 2 21.2 12.6 38.9 11.6 26.4 17.2 78.5 86.9 43.9 88.4 73.5 Acid ±1.4 ±1.3 ±13.7 ±3.2 ±11.4 ±1.6 ±1.6 ±1.5 ±12.2 ±3.2 ±11.9 1.6 X 10~3M 1 NADH, NADPH or su c c i n i c a c i d added ten minutes p r i o r to imipramine metabolism 2 per cent= ug of IMI, DMI, GOH, OH or N-0 i n l i v e r , perfusate or b i l e x 1 Q 0 t o t a l ug of IMI, DMI, GOH, OH or N-0 formed vo 98 to the perfusate the per cent decreased to 3 3 per cent and 37 per cent r e s p e c t i v e l y . The amount of aromatic hydroxylation of imipramine was 31 per cent f o r the c o n t r o l experiment which decreased to 25 and 26 per cent a f t e r NADH or NADPH pretreatment. Although s u c c i n i c a c i d decreased the formation of desmethylimip-ramine from 41 per cent to 29 per cent, aromatic hydroxylation was not changed to a s i g n i f i c a n t extent. The d i s t r i b u t i o n of imipramine (Table XXIII) was changed i n experiments where NADH or s u c c i n i c acid was added p r i o r to imipramine metabolism. The amount of imipramine i n the l i v e r was 6 8 per cent of the imipramine present and due to pretreatment of NADH or s u c c i n i c a c i d the l i v e r content of imipramine increased to 76 and 7 8 per cent. Discussion of C e l l u l a r Control 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 recently published report (60) that glucagon and d i b u t y r y l c y c l i c AMP could i n h i b i t drug metabolism i n the i s o l a t e d perfused r a t l i v e r . Presumably glucagon i n h i b i t s the metabolism of these drugs through elevation of c y c l i c AMP i n the hepatic c e l l . The mechanism of t h i s i n h i b i t i o n was not known, however one of the major actions of d i b u t y r y l c y c l i c AMP and glucagon on the hepatic c e l l i s the m o b i l i z a t i o n of glucose out of the hepatic c e l l and in t o the perfusate (71,75) as w e l l as the breakdown of glycogen. Perhaps the removal of glucose from the 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 decreasing the energy production f o r drug metabolism i n the l i v e r . The glyco-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 or glucagon occurred only i n l i v e r s of fed rats (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 fasted r a t s was gluconeogenesis (77) . I f the i n h i b i t i o n of drug metabolism was due to the glycogenolytic 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 should 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 fasted l i v e r s . I t seems that the mechanism of i n h i b i t i o n of aromatic hydroxylation by desmethylimipramine was not 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 fo 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 of N-demethylation and 29 per cent i n h i b i t i o n of aromatic hydroxylation and there-fore d i d not cause s p e c i f i c i n h i b i t i o n of aromatic hydroxylation. — 6 — 6 Desmethylimipramine at 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 per cent i n h i b i t i o n of aromatic hydroxylation and no i n h i b i t i o n of N-de-methylation. The quantity of d i b u t y r y l c y c l i c AMP used i n these perfusion studies i s known to produce an i n t r a c e l l u l a r concentration of 10 (60,71). Epinephrine i s also known to. produce s i m i l a r hepatic i n t r a c e l l u l a r concentration of c y c l i c AMP (71). I t therefore seems possible that c i r c u l a t i n g epinephrine or sympathetic a c t i v i t y which can cause hyperglycemia could have some i n vivo control on hepatic drug metabolism. To f i n d out whether NADPH content was l i m i t i n g the rate 100 of imipramine metabolism i n the i s o l a t e d perfused r a t l i v e r , NADPH was added to the perfusing medium to increase NADPH l i v e r content. The l i v e r content of NADPH should increase since i t had been reported that absorption occurred a f t e r i n j e c t i o n of t h i s compound to rats (74). In other i n v i t r o preparations such as l i v e r microsomal f r a c t i o n s (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 the reaction of drug metabolism since maximal drug metabolism r e s u l t e d only a f t e r adequate q u a n t i t i e s of t h i s nucleotide was added. Therefore, i f the quantity of NADPH i n the l i v e r was not s u f f i c i e n t to maintain imipramine metabolism an increase i n l i v e r NADPH would r e s u l t i n an increase i n metabolism. In the present 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 of NADPH d i d not increase imipramine metabo-l i s m . A d d i t i o n of NADPH to i n t a c t c e l l s derived from two d i f f e r e n t techniques produced opposite r e s u l t s . Drug metabolism was report-ed by others to be stimulated i n the r a t l i v e r s l i c e s (74) and shown to be i n h i b i t e d i n the i s o l a t e d perfused r a t l i v e r i n t h i s t h e s i s . The reason NADPH increased drug metabolism i n the r a t l i v e r s l i c e s was that the endogenous supply of reducing equivalents 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 rat l i v e r s l i c e s was below the normal content found i n the i n vivo 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 of 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 of the enzymes responsible for drug metabolism since excess NADPH had always been used to study drug metabolism i n l i v e r homogenates (21) or 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 the adrenal cortex have shown that Krebs cycle intermediates (79,80,81) could i n f l u e n c e mitochondrial s t e r o i d metabolism. The metabolism of these s t e r o i d s was by 11^-hydroxylase which required cytochrome P-450. Since cytochrome P-450 was required f o r drug metabolism i n the l i v e r these intermediates may influence drug metabolism. These studies were done on broken c e l l preparations. In a very recent study i t was reported that Krebs cycle intermediates, succinate 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). Addition of succinate to l i v e r homogenate or r a t l i v e r s l i c e s increased the rate of aminopyrine metabolism but an increase i n drug metabolism d i d not occur i n microsomal preparations. I t therefore appeared that succinate did not a f f e c t the drug metabolizing enzymes d i r e c t l y . I t was found that succinate d i d not increase imipramine metabolism i n the i s o l a t e d perfused r a t l i v e r but decreased imipramine metabo-li s m . The type of i n h i b i t i o n of imipramine metabolism seems to be d i f f e r e n t for s u c c i n i c acid than for NADH or NADPH, since 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 addition of NADH to the c i r c u l a t i n g perfusion medium caused a decrease i n imipramine. Addition of t h i s reduced nucleotide to hepatic microsomal f r a c -t i o n (80) and l i v e r homogenates caused an increase i n V max for drug metabolism. This e f f e c t has not been reported i n r a t l i v e r s l i c e s . However, i t has been reported that addition of NADH or NAD caused an increase i n the rate of cytochrome P-450 reduction (78) . I t therefore appears that i f NADH was not i n s u f f i c i e n t 102 quantity i n the i s o l a t e d perfused r a t l i v e r to maintain drug metabolism an increase i n t h i s nucleotide would cause an increase i n imipramine metabolism. This 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 other i n v i t r o preparations. Since an increase i n imipramine metabolism was not observed i n the i s o l a t e d perfused r a t l i v e r , NADH was not l i m i t i n g the rate of drug metabolism or NADH does not contribute an e f f e c t to drug metabolism i n the i s o l a t e d perfused 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 the i s o l a t e d perfused r a t l i v e r i s not known. SUMMARY AND CONCLUSIONS 103 The perfusate h a l f - l i f e of imipramine was not an accurate estimation of imipramine metabolism with long incubation times. The metabolism of imipramine at high substrate concentra--5 t i o n (2 X 10 M) was dose dependent because the endogenous desmethylimipramine formed i n h i b i t e d aromatic hydroxylation. The primary metabolic routes for imipramine metabolism with f i f t e e n minutes incubation was N-demethylation to desmethylimipramine, aromatic hydroxylation to 2-hydroxy-imipramine and N-oxidation of imipramine. The rate of aromatic hydroxylation of imipramine was great-er than the rate of N-demethylation of imipramine a t low -5 doses of imipramine (0.5 X 10 M) . Et 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. Succinic acid i n h i b i t e d N-demethylation of imipramine. Low concentrations of desmethylimipramine (1.65 or 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 hydroxylation of imipramine metabolism when the dose of imipramine was -5 1 or 2 X 10 M. Higher concentration of desmethylimxpramine i n h i b i t e d aromatic hydroxylation and N-demethylation of imipramine. Hydroxylation of imipramine and conjugation of t h i s metabo-l i t e was independent of b i l i a r y excretion. 104 A d d i t i o n a l evidence was provided for more than one cytochrome P-450 system because of s p e c i f i c i n h i b i t i o n of aromatic hydroxylation of imipramine or N-demethylation of imipramine. A l t e r a t i o n of the perfusate magnesium concentration 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 to i n h i b i t aromatic hydroxy-l a t i o n and N-demethylation of imipramine. The nucleotides NADH and NADPH were found to i n h i b i t aro-matic hydroxylation and N-demethylation of imipramine. 105 BIBLIOGRAPHY Neushalme, E.A. and Gevers, W., Control of g l y c o l y s i s and gluconeogenesis i n l i v e r and kidney cortex. Vitamins and Hormones 25-30: 14 1967. Edited by H a r r i s , R., Wool, I. and Loraine, J . , Academic Press. Williams, R.T., Detoxication mechanisms i n man. C l i n Pharmacol. Therap., 4-12: 234, 1963. Conney, A.H., Pharmacological i m p l i c a t i o n of microsomal enzyme induction. Pharmacol. Rev., 19, 317-366, 1967. Kantzman, R., Drugs and enzyme induction. Ann. Rev. Pharmacol., 9: 21-36, 1969. 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