962
Specialia
a n a l o g o u s m a n n e r to t h a t d e s c r i b e d for ( I I I a ) t h e n f u r n i s h e d (IVb) ( D M P D ) a s a c r y s t a l l i n e m a l e i c a c i d s a l t [ m p 131-132] 14.
la We wish to thank Abbott Labs. for sample of pargyline; Geigy Pharmaceutical Co. for imipramine; Hoffmann-La Roche, Inc. for ehlordiazepoxide and diazepam; Merck Sharp and Dohme for amitriptyline; Pfizer Labs. for doxepin; and Smith Kline and French Labs. for chlorpromazine. le The authors wish to express their gratitude to DAGMARJ. KuNc for her expert technical assistance. 17 This paper represents contribution No. 436 from the Syntex Institute of Organic Chemistry.
EXPERIENTIA 31/8
Summary. A n e w t r i c y c l i c a g e n t w i t h a n a l l e n y l s i d e chain experimentally shows antidepressant activity s i m i l a r to a m i t r i p t y l i n e a n d i m i p r a m i n e b u t a l s 0 ~ e x e r t s m a r k e d C N S d e p r e s s i o n . S u c h d u a l a c t i v i t y s h o u f d be of clinical i n t e r e s t for t r e a t m e n t of m i x e d a n x i e t y a n d depression. A. P. ROSZKOWSKI, M. E. SCHULER, M. MARX a n d J. A. EDWARDS 15-17
Institutes o/ Clinical Medicine and Organic Chemistry, Syntex Research, Stan/ord Industrial Park, Palo Alto (California 94304, USA), 26 March 1974.
I n h i b i t i o n of D r u g M e t a b o l i z i n g E n z y m e s by D i a z e p a m in Rat Liver H y p n o t i c s a n d s e d a t i v e s a r e k n o w n to s t i m u l a t e or i n h i b i t t h e d r u g - m e t a b o l i z i n g e n z y m e s in liver I, 2. B e n z o d i a z e p i n e s are w i d e l y u s e d a n d are f r e q u e n t l y p r e s c r i b e d in c o m b i n a t i o n w i t h o t h e r d r u g s . C h l o r d i a z e p o x i d e h a s b e e n s u g g e s t e d t o be a n e n z y m e i n d u c e r 3-e. i n one s t u d y 7 it w a s s h o w n t h a t d i a z e p a m d e c r e a s e d t h e pentobarbital sleeping time and s e r u m concentration, b u t t h e i n d u c i n g e f f e c t could n o t be c o n f i r m e d in a n o t h e r s t u d y 6. C h l o r d i a z e p o x i d e h a s b e e n s h o w n t o s t i m u l a t e its o w n m e t a b o l i s m in r a t 3, a n d a t o l e r a n c e to d i a z e p a m h a s b e e n d e s c r i b e d in c a t s . E t h a n o l s e e m s to e n h a n c e t h e a c t i o n of d i a z e p a m ~ b u t t h e m e c h a n i s m s of t h e s e effects of d i a z e p a m are n o t k n o w n . W e i n v e s t i g a t e d m i c r o s o m a l m e t a b o l i s m of h e x o b a r b i t a l , N - m e t h y l a n i l i n e a n d pn i t r o b e n z o i c a c i d as well as t h e c o n t e n t of c y t o c h r o m e P - 4 5 0 in r a t liver a f t e r d i a z e p a m t r e a t m e n t . F u r t h e r we s t u d i e d w h e t h e r d i a z e p a m c a n s t i m u l a t e its o w n m e t a b olism and t h u s explain the developing tolerance. We also c o m p a r e d d i a z e p a m t o t w o w e l l - k n o w n s t i m u l a t o r s of m i c r o s o m a l m e t a b o l i s m , p h e n o b a r b i t a l a n d 3,4benzpyrene. Materials and methods. 4 g r o u p s of 6 m a l e S p r a g u e D a w l e y r a t s w e i g h i n g a b o u t 200 g w e r e k e p t o n a s t a n d a r d diet. 1 g r o u p (controls) r e c e i v e d v e h i c l e s o l v e n t , 2 n d g r o u p d i a z e p a m 100 m g , 3rd g r o u p p h e n o b a r b i t a l 80 m g a n d 4 t h g r o u p 3, 4 - b e n z p y r e n e 20 m g / k g of b o d y w e i g h t . All d r u g s w e r e g i v e n b y m o u t h o n c e a d a y for 6 d a y s in a v e h i c l e s o l v e n t c o n s i s t i n g of t w e e n 20 a n d c a r b o x y m e t h y l cellulose (1 : 4). Preparation o/ livers. A n i m a l s were d e c a p i t a t e d 24 h a f t e r t h e l a s t dose. T h e l i v e r s were r e m o v e d a n d r i n s e d w i t h ice-cold 0.1 M p h o s p h a t e b u f f e r ( p H 7.4). All s u b s e q u e n t m a n i p u l a t i o n s were carried o u t at 2-4~ The 2 0 % liver h o m o g e n a t e s w e r e p r e p a r e d in t h e s a m e p h o s -
phate buffer with a Potter-Elvehjem type homogenizer. T h e h o m o g e n a t e w a s c e n t r i f u g e d a t 12,000 g for 20 m i n . P a r t of t h e s u p e r n a t a n t w a s t h e n c e n t r i f u g e d a t 105,000 g for 1 h. T h e m i c r o s o m a l p e l l e t w a s s u s p e n d e d in t h e p h o s p h a t e b u f f e r so t h a t 2.5 m l s u s p e n s i o n c o r r e s p o n d e d t o 1 g of liver t i s s u e . Enzyme assays. T h e 12,000 g s u p e r n a t a n t w a s u s e d t o assay the hexobarbital hydroxylation, p-nitrobenzoic acid r e d u c t i o n , N - m e t h y l a n i t i n e d e m e t h y l a t i o n a n d diazep a m metabolism. The microsomal suspension was used to m e a s u r e the c y t o c h r o m e P-450 content. The incubation c o n d i t i o n s for a s s a y s of t h e m e t a b o l i s m of h e x o b a r b i t a l , N - m e t h y l a n i l i n e a n d p - n i t r o b e n z o i c acid h a v e b e e n d e s c r i b e d b y VORNE a n d ARVELA 10, a n d t h a t of d i a z e p a m b y SCHWARTZ a n d POSTMA n . T h e a m o u n t s of t h e s u b -
1 A: H. CONNEr, Pharmac. Rev. 19, 317 (1967). 2 G. J. MANNERING, Selected Pharmacological Testing Methods (Ed. A. BURGER; Marcel Decker Inc., New York 1968), vol. 3, p. 51. 8 D. R. HOOGLAND,T. S. MIVA and W. F. BOUSQUET,Toxic. appl. Pharmac. 9, 116 (1966). 4 A. BRECKENRIDGE and M. ORME, Archs. int. Pharmacodyn. Th@r. 792, Suppl., 220 (1971). 5 T. H. STEVENSON, M. BROWNING, J. CROOKS and K. O'MALLEY, Br. med. J. d, 322 (1972). 6 M. ORME, A. BRECKENRIDGE and R. V. BROOKS, Br. reed. J. 3, 611 (1972). 7 V. HEUBEL and R. FRANK, Arzneiln. Forseh. 20, 1706 (1970). 8 A. BARNETTand J. W. FIORE, Eur. J. Pharmac. 13, 239 (1971). 9 M. LINNOILA, Ann. Med. exp. Biol. Fenn 51, 125 (1973). 10 M. VORNE and P. ARVELA,Aeta pharmac, toxic. 29, 417 (1971). 11 M. A. SCHWARTZand E. POSTMA, Biochem. Pharmac. 17, 2443 (1968).
Table I. Effect of diazepam (DZP), phenobarbital (PB) and 3,4-benzpyrene (BP) treatment on cytochrome P-450 content and the rates of metabolism of hexobarbital, p-nitrobenzoic acid and N-methylaniline in rat liver mierosomes Group
Control DZP PB BP
Cytochrome P-450 content (nmol/g liver)
8.3 -4- 1.6 7.5 ~ 0.8 19.1 4- 3.4 b 10.6 ~ 3.6
Rate of metabolism of substrates (nmol/g liver/h) (% )
100 90 230 128
Hexobarbital
(%)
p-Nitrobenzoic acid
(%)
N-methylaniline
(%)
6.95=t=0.70 4.60-t-0.93 b 11.10=t=0.67 b 5.07•
100 67 160 73
2.514-0.72 1.49=t=0.23 9 5.794-2.30 . 2.44=[=0.43
100 59 230 97
1.57-4-0.29 1.50=[=0.34 4.49::[=0.58 b 1.43-4-0.34
100 95 285 91
Mean value :t: SD of 6 rats. ~p < 0.01; bp < 0.001.
15.8. 1975
963
Specialia
Table II. Effect of diazepam (DZP), phenobarbital (PB) and 3,4-benzpyrene (BP) treatment on the metabolism of diazepam in rat liver microsomes Group
Control DZP PB BP
Substrate and metabolites recovered after incubation (nmol) Diazepam
N-methyloxazepanl
N-demethyldiazepam
28.10 28.31 14.92 29.62
2.89 2.92 10.35 2.84
2.83 i 0.62 2.46 =1=0.28 8.25 ~ i.89 ~ 2.30 ~- 0.34
~- 0.62 J_ 0.73 + 1.53 a ~ 0.72
• 0.62 • 0.53 J_ 2.19~ ~- 0.39
Mean value :L SD of 6 rats. ~p < 0.001.
s t r a t e s , t h e a m o u n t s of liver t i s s u e s a n d t h e i n c u b a t i o n t i m e s w e r e as follows: H e x o b a r t i t a l 2 ~xmol, 400 rag, 15 m i n , N - m e t h y l a n i l i n e 5 ~zmol, 400 m g , 30 m i n , d i a z e p a m C 14 35 n m o l , 60 m g , 60 rain, a n d p - n i t r o b e n z o i c acid 3 ~mol, 400 rag, 60 m i n . T h e r a t e of m e t a b o l i s m of s u b s t r a t e s a n d t h e c o n t e n t of c y t o c h r o m e P-450 w e r e m e a s u r e d as d e s c r i b e d p r e v i o u s ly 13-1s. S t u d e n t ' s t - t e s t w a s u s e d in s t a t i s t i c a l a n a l y s i s .
Results. Effect o/ diazepam on some drug-metabolizing microsomal enzymes. T h e c o n t e n t of c y t o c h r o m e P - 4 5 0 a n d t h e a c t i v i t i e s of h e x o b a r b i t a l , p - n i t r o b e n z o i c acid a n d N - m e t h y l a n i l i n e - m e t a b o l i z i n g e n z y m e s in r a t liver are s h o w n in T a b l e I. D i a z e p a m h a d no s i g n i f i c a n t effect o n t h e c o n t e n t of c y t o c h r o m e P-450 or N - d e m e t h y l a t i o n o n m e t h y l a n i l i n e . O n t h e o t h e r h a n d , t h e o x i d a t i o n of h e x o b a r b i t a l a n d r e d u c t i o n of p - n i t r o b e n z o i c a c i d were significantly inhibited by diazepam. Lack o/ diazepam to change its own metabolism. T h e a m o u n t s of d i a z e p a m a n d its m e t a b o l i t e s a f t e r i n c u b a t i o n c a n be s e e n in T a b l e II. D i a z e p a m h a d n o effect on its o w n m e t a b o l i s m as c o m p a r e d w i t h c o n t r o l s . I t is c o n s p i c u o u s t h a t n e i t h e r 3 , 4 - b e n z p y r e n e h a d a n y effect on t h e d i a z e p a m m e t a b o l i s m . O x a z e p a m , one of t h e m e t a b o l i t e s of d i a z e p a m , w a s n o t f o u n d a l t h o u g h p h e n o b a r b i t a l i n c r e a s e d m a r k e d l y t h e r a t e of m e t a b o l i s m of d i a z e p a m . Discussion. I n t h e p r e s e n t s t u d y , d i a z e p a m d e l a y e d t h e r a t e s of m e t a b o l i s m of h e x o b a r b i t a l a n d p - n i t r o b e n z o i c acid, w h e r e a s it h a d n e i t h e r i n h i b i t i n g n o r s t i m u l a t i n g effect o n its o w n m e t a b o l i s m . T h e c a u s e of t o l e r a n c e t o d i a z e p a m r e m a i n e d u n s o l v e d in o u r s t u d y . H o w e v e r , o n e p o s s i b i l i t y is t h a t s o m e m e t a b o l i t e of d i a z e p a m a c t s as a c o m p e t i t i v e i n h i b i t o r of d i a z e p a m , p o s s i b l y N - d e s m e t h y l d i a z e p a m as s u g g e s t e d earlier s. A n o t h e r p o s s i b i l i t y , n o t e x c l u d e d in o u r s t u d y , is t h a t t h e r e m a y be c h a n g e s in t h e m i n o r p a t h w a y s of d i a z e p a m m e t a b o l i s m , n o t d e t e c t e d in our methods. P h e n o b a r b i t a l s t r o n g l y i n d u c e d t h e r a t e of m e t a b o l i s m of d i a z e p a m , w h i c h is in g o o d a g r e e m e n t w i t h o t h e r s t u d i e s n , lL No o x a z e p a m w a s n o t e d e v e n a f t e r p h e n o b a r b i t a l t r e a t m e n t . T h i s a g r e e s well w i t h t h e s t u d y of MARCUCCI et al. ~7 m a d e b y t h e s a m e s t r a i n of r a t s as ours, w h e r e a s it differs f r o m t h e s t u d y of SCHWARTZ a n d POSTMAn w h o u s e d a n o t h e r s t r a i n of r a t s in t h e i r s t u d y . T h i s s u g g e s t s t h a t t h e m e t a b o l i c p a t h w a y s of d i a z e p a m d i f f e r in d i f f e r e n t s t r a i n s of r a t s . T h e effects of d i a z e p a m o n d r u g m e t a b o l i s m differed e n t i r e l y f r o m t h o s e of p h e n o b a r b i t a l , b u t t h e y w e r e to s o m e e x t e n t s i m i l a r t o t h e p o l y c y c l i c h y d r o c a r b o n 3, 4benzpyrene. D i a z e p a m inhibited some enzyme activities b u t had no effect o n s o m e o t h e r e n z y m e s . T h i s f i n d i n g a g r e e s well w i t h s o m e earlier r e s u l t s 6. C h l o r d i a z e p o x i d e h a s b e e n
s u g g e s t e d to be a n e n z y m e - i n d u c e r in r a t 3, 6, is a n d m a n 4, w h e r e a s n i t r a z e p a m in r a t a a n d t h e e f f e c t of p r a z e p a m is s i m i l a r to d i a z e p a n 19. I n t h e p r e s e n t s t u d y , d i a z e p a m h a d no effect o n its o w n m e t a b o l i s m , w h i c h is s i m i l a r t o t h e f i n d i n g w i t h p r a z e p a m 2~ b u t d i f f e r s t r o m t h a t of chlord i a z e p o x i d e w h i c h h a s b e e n s u g g e s t e d t o s t i m u l a t e its o w n m e t a b o l i s m 3. T h e r e f o r e it is l i k e l y t h a t t h e b e n z o d i a z e p i n e s differ f r o m e a c h o t h e r in r e g a r d to t h e i r effect on drug metabolizing enzymes.
Zusammen/assung. N a c h w e i s , d a s s V o r b e h a n d l u n g v o n R a t t e n mit Diazepam den Stoffwechsel yon Hexobarbital u n d p - N i t r o b e n z o e s / ~ u r e in d e r R a t t e n l e b e r in v i t r o h e m m t , o h n e d e n G e h a l t d e s C y t o c h r o m s P-450 o d e r d e n e i g e n e n M e t a b o l i s m u s zu v e r X n d e r n . MARTTI VORNE a n d JUHANA IDANPAXN-HEIKKILA 21
Department o/ Pharmacology, University of Oulu, Oulu (Finland), 20 February 7975.
12 j. R. COOPERand B. B. BRODIE, J. Pharmac. exp. Ther. 114, 409 (1955). lz B. B. BRODIE and J. AXELROO, J. Pharmac. exp. Ther. 94, 22 (1948). 14 j. R. FonTs and B. B. BRODIE, J. Pharmac. exp. Ther. llg, 197 (1957). is j . E. ID~,NP~N-HEIKKtL~, P. I. JOUPPILA, J. O. PUOLAKKAand M. S. VORNE, Am. J. Obstet, Gynee. 109, 1011 (1971). is T. OMURAand R. SATO, J. biol. Chem. 239, 2370 (1964). 17 V. MAR~UCCI, R. FANELLI, n. MUSSINI and E. S. GARATTINI, Biochem. Pharmae. 79, 1771 (1970).. 18 B. BALLINGER, K. O'MALLEY, I. H. STEVENSON and M. J. TURNBULL, Br. J. Pharmac. 41,383 P (1971). 19 E. S. VESELL, G. T. PASSANANTI, J. P. VIAU, J. E. EPPS and F. J. DI CARLO, Pharmacology 7, 197 (1972). 2o j. p. VIAU, J. E. EPPs and F. J. oI CARLO, Xenobiotica 3, 581 (1973). ~1 Present address: National Board of Health, Siltasaarenkatu 18 A, SF-00530 Helsinki 53, Finland.
Specialia
a n a l o g o u s m a n n e r to t h a t d e s c r i b e d for ( I I I a ) t h e n f u r n i s h e d (IVb) ( D M P D ) a s a c r y s t a l l i n e m a l e i c a c i d s a l t [ m p 131-132] 14.
la We wish to thank Abbott Labs. for sample of pargyline; Geigy Pharmaceutical Co. for imipramine; Hoffmann-La Roche, Inc. for ehlordiazepoxide and diazepam; Merck Sharp and Dohme for amitriptyline; Pfizer Labs. for doxepin; and Smith Kline and French Labs. for chlorpromazine. le The authors wish to express their gratitude to DAGMARJ. KuNc for her expert technical assistance. 17 This paper represents contribution No. 436 from the Syntex Institute of Organic Chemistry.
EXPERIENTIA 31/8
Summary. A n e w t r i c y c l i c a g e n t w i t h a n a l l e n y l s i d e chain experimentally shows antidepressant activity s i m i l a r to a m i t r i p t y l i n e a n d i m i p r a m i n e b u t a l s 0 ~ e x e r t s m a r k e d C N S d e p r e s s i o n . S u c h d u a l a c t i v i t y s h o u f d be of clinical i n t e r e s t for t r e a t m e n t of m i x e d a n x i e t y a n d depression. A. P. ROSZKOWSKI, M. E. SCHULER, M. MARX a n d J. A. EDWARDS 15-17
Institutes o/ Clinical Medicine and Organic Chemistry, Syntex Research, Stan/ord Industrial Park, Palo Alto (California 94304, USA), 26 March 1974.
I n h i b i t i o n of D r u g M e t a b o l i z i n g E n z y m e s by D i a z e p a m in Rat Liver H y p n o t i c s a n d s e d a t i v e s a r e k n o w n to s t i m u l a t e or i n h i b i t t h e d r u g - m e t a b o l i z i n g e n z y m e s in liver I, 2. B e n z o d i a z e p i n e s are w i d e l y u s e d a n d are f r e q u e n t l y p r e s c r i b e d in c o m b i n a t i o n w i t h o t h e r d r u g s . C h l o r d i a z e p o x i d e h a s b e e n s u g g e s t e d t o be a n e n z y m e i n d u c e r 3-e. i n one s t u d y 7 it w a s s h o w n t h a t d i a z e p a m d e c r e a s e d t h e pentobarbital sleeping time and s e r u m concentration, b u t t h e i n d u c i n g e f f e c t could n o t be c o n f i r m e d in a n o t h e r s t u d y 6. C h l o r d i a z e p o x i d e h a s b e e n s h o w n t o s t i m u l a t e its o w n m e t a b o l i s m in r a t 3, a n d a t o l e r a n c e to d i a z e p a m h a s b e e n d e s c r i b e d in c a t s . E t h a n o l s e e m s to e n h a n c e t h e a c t i o n of d i a z e p a m ~ b u t t h e m e c h a n i s m s of t h e s e effects of d i a z e p a m are n o t k n o w n . W e i n v e s t i g a t e d m i c r o s o m a l m e t a b o l i s m of h e x o b a r b i t a l , N - m e t h y l a n i l i n e a n d pn i t r o b e n z o i c a c i d as well as t h e c o n t e n t of c y t o c h r o m e P - 4 5 0 in r a t liver a f t e r d i a z e p a m t r e a t m e n t . F u r t h e r we s t u d i e d w h e t h e r d i a z e p a m c a n s t i m u l a t e its o w n m e t a b olism and t h u s explain the developing tolerance. We also c o m p a r e d d i a z e p a m t o t w o w e l l - k n o w n s t i m u l a t o r s of m i c r o s o m a l m e t a b o l i s m , p h e n o b a r b i t a l a n d 3,4benzpyrene. Materials and methods. 4 g r o u p s of 6 m a l e S p r a g u e D a w l e y r a t s w e i g h i n g a b o u t 200 g w e r e k e p t o n a s t a n d a r d diet. 1 g r o u p (controls) r e c e i v e d v e h i c l e s o l v e n t , 2 n d g r o u p d i a z e p a m 100 m g , 3rd g r o u p p h e n o b a r b i t a l 80 m g a n d 4 t h g r o u p 3, 4 - b e n z p y r e n e 20 m g / k g of b o d y w e i g h t . All d r u g s w e r e g i v e n b y m o u t h o n c e a d a y for 6 d a y s in a v e h i c l e s o l v e n t c o n s i s t i n g of t w e e n 20 a n d c a r b o x y m e t h y l cellulose (1 : 4). Preparation o/ livers. A n i m a l s were d e c a p i t a t e d 24 h a f t e r t h e l a s t dose. T h e l i v e r s were r e m o v e d a n d r i n s e d w i t h ice-cold 0.1 M p h o s p h a t e b u f f e r ( p H 7.4). All s u b s e q u e n t m a n i p u l a t i o n s were carried o u t at 2-4~ The 2 0 % liver h o m o g e n a t e s w e r e p r e p a r e d in t h e s a m e p h o s -
phate buffer with a Potter-Elvehjem type homogenizer. T h e h o m o g e n a t e w a s c e n t r i f u g e d a t 12,000 g for 20 m i n . P a r t of t h e s u p e r n a t a n t w a s t h e n c e n t r i f u g e d a t 105,000 g for 1 h. T h e m i c r o s o m a l p e l l e t w a s s u s p e n d e d in t h e p h o s p h a t e b u f f e r so t h a t 2.5 m l s u s p e n s i o n c o r r e s p o n d e d t o 1 g of liver t i s s u e . Enzyme assays. T h e 12,000 g s u p e r n a t a n t w a s u s e d t o assay the hexobarbital hydroxylation, p-nitrobenzoic acid r e d u c t i o n , N - m e t h y l a n i t i n e d e m e t h y l a t i o n a n d diazep a m metabolism. The microsomal suspension was used to m e a s u r e the c y t o c h r o m e P-450 content. The incubation c o n d i t i o n s for a s s a y s of t h e m e t a b o l i s m of h e x o b a r b i t a l , N - m e t h y l a n i l i n e a n d p - n i t r o b e n z o i c acid h a v e b e e n d e s c r i b e d b y VORNE a n d ARVELA 10, a n d t h a t of d i a z e p a m b y SCHWARTZ a n d POSTMA n . T h e a m o u n t s of t h e s u b -
1 A: H. CONNEr, Pharmac. Rev. 19, 317 (1967). 2 G. J. MANNERING, Selected Pharmacological Testing Methods (Ed. A. BURGER; Marcel Decker Inc., New York 1968), vol. 3, p. 51. 8 D. R. HOOGLAND,T. S. MIVA and W. F. BOUSQUET,Toxic. appl. Pharmac. 9, 116 (1966). 4 A. BRECKENRIDGE and M. ORME, Archs. int. Pharmacodyn. Th@r. 792, Suppl., 220 (1971). 5 T. H. STEVENSON, M. BROWNING, J. CROOKS and K. O'MALLEY, Br. med. J. d, 322 (1972). 6 M. ORME, A. BRECKENRIDGE and R. V. BROOKS, Br. reed. J. 3, 611 (1972). 7 V. HEUBEL and R. FRANK, Arzneiln. Forseh. 20, 1706 (1970). 8 A. BARNETTand J. W. FIORE, Eur. J. Pharmac. 13, 239 (1971). 9 M. LINNOILA, Ann. Med. exp. Biol. Fenn 51, 125 (1973). 10 M. VORNE and P. ARVELA,Aeta pharmac, toxic. 29, 417 (1971). 11 M. A. SCHWARTZand E. POSTMA, Biochem. Pharmac. 17, 2443 (1968).
Table I. Effect of diazepam (DZP), phenobarbital (PB) and 3,4-benzpyrene (BP) treatment on cytochrome P-450 content and the rates of metabolism of hexobarbital, p-nitrobenzoic acid and N-methylaniline in rat liver mierosomes Group
Control DZP PB BP
Cytochrome P-450 content (nmol/g liver)
8.3 -4- 1.6 7.5 ~ 0.8 19.1 4- 3.4 b 10.6 ~ 3.6
Rate of metabolism of substrates (nmol/g liver/h) (% )
100 90 230 128
Hexobarbital
(%)
p-Nitrobenzoic acid
(%)
N-methylaniline
(%)
6.95=t=0.70 4.60-t-0.93 b 11.10=t=0.67 b 5.07•
100 67 160 73
2.514-0.72 1.49=t=0.23 9 5.794-2.30 . 2.44=[=0.43
100 59 230 97
1.57-4-0.29 1.50=[=0.34 4.49::[=0.58 b 1.43-4-0.34
100 95 285 91
Mean value :t: SD of 6 rats. ~p < 0.01; bp < 0.001.
15.8. 1975
963
Specialia
Table II. Effect of diazepam (DZP), phenobarbital (PB) and 3,4-benzpyrene (BP) treatment on the metabolism of diazepam in rat liver microsomes Group
Control DZP PB BP
Substrate and metabolites recovered after incubation (nmol) Diazepam
N-methyloxazepanl
N-demethyldiazepam
28.10 28.31 14.92 29.62
2.89 2.92 10.35 2.84
2.83 i 0.62 2.46 =1=0.28 8.25 ~ i.89 ~ 2.30 ~- 0.34
~- 0.62 J_ 0.73 + 1.53 a ~ 0.72
• 0.62 • 0.53 J_ 2.19~ ~- 0.39
Mean value :L SD of 6 rats. ~p < 0.001.
s t r a t e s , t h e a m o u n t s of liver t i s s u e s a n d t h e i n c u b a t i o n t i m e s w e r e as follows: H e x o b a r t i t a l 2 ~xmol, 400 rag, 15 m i n , N - m e t h y l a n i l i n e 5 ~zmol, 400 m g , 30 m i n , d i a z e p a m C 14 35 n m o l , 60 m g , 60 rain, a n d p - n i t r o b e n z o i c acid 3 ~mol, 400 rag, 60 m i n . T h e r a t e of m e t a b o l i s m of s u b s t r a t e s a n d t h e c o n t e n t of c y t o c h r o m e P-450 w e r e m e a s u r e d as d e s c r i b e d p r e v i o u s ly 13-1s. S t u d e n t ' s t - t e s t w a s u s e d in s t a t i s t i c a l a n a l y s i s .
Results. Effect o/ diazepam on some drug-metabolizing microsomal enzymes. T h e c o n t e n t of c y t o c h r o m e P - 4 5 0 a n d t h e a c t i v i t i e s of h e x o b a r b i t a l , p - n i t r o b e n z o i c acid a n d N - m e t h y l a n i l i n e - m e t a b o l i z i n g e n z y m e s in r a t liver are s h o w n in T a b l e I. D i a z e p a m h a d no s i g n i f i c a n t effect o n t h e c o n t e n t of c y t o c h r o m e P-450 or N - d e m e t h y l a t i o n o n m e t h y l a n i l i n e . O n t h e o t h e r h a n d , t h e o x i d a t i o n of h e x o b a r b i t a l a n d r e d u c t i o n of p - n i t r o b e n z o i c a c i d were significantly inhibited by diazepam. Lack o/ diazepam to change its own metabolism. T h e a m o u n t s of d i a z e p a m a n d its m e t a b o l i t e s a f t e r i n c u b a t i o n c a n be s e e n in T a b l e II. D i a z e p a m h a d n o effect on its o w n m e t a b o l i s m as c o m p a r e d w i t h c o n t r o l s . I t is c o n s p i c u o u s t h a t n e i t h e r 3 , 4 - b e n z p y r e n e h a d a n y effect on t h e d i a z e p a m m e t a b o l i s m . O x a z e p a m , one of t h e m e t a b o l i t e s of d i a z e p a m , w a s n o t f o u n d a l t h o u g h p h e n o b a r b i t a l i n c r e a s e d m a r k e d l y t h e r a t e of m e t a b o l i s m of d i a z e p a m . Discussion. I n t h e p r e s e n t s t u d y , d i a z e p a m d e l a y e d t h e r a t e s of m e t a b o l i s m of h e x o b a r b i t a l a n d p - n i t r o b e n z o i c acid, w h e r e a s it h a d n e i t h e r i n h i b i t i n g n o r s t i m u l a t i n g effect o n its o w n m e t a b o l i s m . T h e c a u s e of t o l e r a n c e t o d i a z e p a m r e m a i n e d u n s o l v e d in o u r s t u d y . H o w e v e r , o n e p o s s i b i l i t y is t h a t s o m e m e t a b o l i t e of d i a z e p a m a c t s as a c o m p e t i t i v e i n h i b i t o r of d i a z e p a m , p o s s i b l y N - d e s m e t h y l d i a z e p a m as s u g g e s t e d earlier s. A n o t h e r p o s s i b i l i t y , n o t e x c l u d e d in o u r s t u d y , is t h a t t h e r e m a y be c h a n g e s in t h e m i n o r p a t h w a y s of d i a z e p a m m e t a b o l i s m , n o t d e t e c t e d in our methods. P h e n o b a r b i t a l s t r o n g l y i n d u c e d t h e r a t e of m e t a b o l i s m of d i a z e p a m , w h i c h is in g o o d a g r e e m e n t w i t h o t h e r s t u d i e s n , lL No o x a z e p a m w a s n o t e d e v e n a f t e r p h e n o b a r b i t a l t r e a t m e n t . T h i s a g r e e s well w i t h t h e s t u d y of MARCUCCI et al. ~7 m a d e b y t h e s a m e s t r a i n of r a t s as ours, w h e r e a s it differs f r o m t h e s t u d y of SCHWARTZ a n d POSTMAn w h o u s e d a n o t h e r s t r a i n of r a t s in t h e i r s t u d y . T h i s s u g g e s t s t h a t t h e m e t a b o l i c p a t h w a y s of d i a z e p a m d i f f e r in d i f f e r e n t s t r a i n s of r a t s . T h e effects of d i a z e p a m o n d r u g m e t a b o l i s m differed e n t i r e l y f r o m t h o s e of p h e n o b a r b i t a l , b u t t h e y w e r e to s o m e e x t e n t s i m i l a r t o t h e p o l y c y c l i c h y d r o c a r b o n 3, 4benzpyrene. D i a z e p a m inhibited some enzyme activities b u t had no effect o n s o m e o t h e r e n z y m e s . T h i s f i n d i n g a g r e e s well w i t h s o m e earlier r e s u l t s 6. C h l o r d i a z e p o x i d e h a s b e e n
s u g g e s t e d to be a n e n z y m e - i n d u c e r in r a t 3, 6, is a n d m a n 4, w h e r e a s n i t r a z e p a m in r a t a a n d t h e e f f e c t of p r a z e p a m is s i m i l a r to d i a z e p a n 19. I n t h e p r e s e n t s t u d y , d i a z e p a m h a d no effect o n its o w n m e t a b o l i s m , w h i c h is s i m i l a r t o t h e f i n d i n g w i t h p r a z e p a m 2~ b u t d i f f e r s t r o m t h a t of chlord i a z e p o x i d e w h i c h h a s b e e n s u g g e s t e d t o s t i m u l a t e its o w n m e t a b o l i s m 3. T h e r e f o r e it is l i k e l y t h a t t h e b e n z o d i a z e p i n e s differ f r o m e a c h o t h e r in r e g a r d to t h e i r effect on drug metabolizing enzymes.
Zusammen/assung. N a c h w e i s , d a s s V o r b e h a n d l u n g v o n R a t t e n mit Diazepam den Stoffwechsel yon Hexobarbital u n d p - N i t r o b e n z o e s / ~ u r e in d e r R a t t e n l e b e r in v i t r o h e m m t , o h n e d e n G e h a l t d e s C y t o c h r o m s P-450 o d e r d e n e i g e n e n M e t a b o l i s m u s zu v e r X n d e r n . MARTTI VORNE a n d JUHANA IDANPAXN-HEIKKILA 21
Department o/ Pharmacology, University of Oulu, Oulu (Finland), 20 February 7975.
12 j. R. COOPERand B. B. BRODIE, J. Pharmac. exp. Ther. 114, 409 (1955). lz B. B. BRODIE and J. AXELROO, J. Pharmac. exp. Ther. 94, 22 (1948). 14 j. R. FonTs and B. B. BRODIE, J. Pharmac. exp. Ther. llg, 197 (1957). is j . E. ID~,NP~N-HEIKKtL~, P. I. JOUPPILA, J. O. PUOLAKKAand M. S. VORNE, Am. J. Obstet, Gynee. 109, 1011 (1971). is T. OMURAand R. SATO, J. biol. Chem. 239, 2370 (1964). 17 V. MAR~UCCI, R. FANELLI, n. MUSSINI and E. S. GARATTINI, Biochem. Pharmae. 79, 1771 (1970).. 18 B. BALLINGER, K. O'MALLEY, I. H. STEVENSON and M. J. TURNBULL, Br. J. Pharmac. 41,383 P (1971). 19 E. S. VESELL, G. T. PASSANANTI, J. P. VIAU, J. E. EPPS and F. J. DI CARLO, Pharmacology 7, 197 (1972). 2o j. p. VIAU, J. E. EPPs and F. J. oI CARLO, Xenobiotica 3, 581 (1973). ~1 Present address: National Board of Health, Siltasaarenkatu 18 A, SF-00530 Helsinki 53, Finland.
