European Journal o f Pharmacology, 32 (1975) 108--115
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
C O M P A R A T I V E S T U D I E S OF A NEW 5 H T - U P T A K E I N H I B I T O R A N D S O M E TRICYCLIC THYMOLEPTICS JORGEN BUUS LASSEN, ERLING PETERSEN, BENGT KJELLBERG and SVEN O. OLSSON Research Laboratories of Ferrosan, Copenhagen, Denmark and Malta6, Sweden
Received 23 December 1974, revised MS received 6 February 1975, accepted 17 February 1975
J. BUUS LASSEN, E. PETERSEN, B. KJELLBERG and S.O. OLSSON, Comparative studies o f a new 5HTuptake inhibitor and some tricyclic thymoleptics, European J. Pharmacol. 32 (1975) 108--115. The new 5HT-uptake inhibitor, FG 4963, and some tricyclic thymoleptics antagonized p-chloroamphetamine (PCA)-induced hypermotility in rats. FG 4963 was active in about the same s.c. and p.o. doses as chlorimipramine. FG 4963, imipramine and chlorimipramine potentiated hypermotility induced in mice by the 5HT precursor 5HTP, FG 4963 being slightly more active than chlorimipramine. In contrast to the tricyclic thymoleptics FG 4963 did not potentiate the heart rate increasing effect of NA in pithed rats. The peripheral anticholinergic effect of FG 4963 and of desipramine was almost identical while the other imipramine derivatives were more active. All tricyclic thymoleptics were strong peripheral antihistaminics, but FG 4963 was almost devoid of this action. Acute tests for ECG changes in guinea pigs and toxicity in mice and rats showed that FG 4963 and chlorimipramine were less toxic than imipramine and amitriptyline. FG 4963 is presumably a selective 5HT-uptake inhibitor producing much less potentiation of peripheral sympathetic mechanisms than do the tricyclic antidepressants. 5HT-uptake inhibition
Peripheral autonomic effects
1. I n t r o d u c t i o n An i m p a i r e d f u n c t i o n o f t h e n e u r o t r a n s m i t ters n o r a d r e n a l i n e ( N A ) a n d s e r o t o n i n ( 5 H T ) m a y be i n v o l v e d in p a t h o g e n e s i s o f depressive illness ( S c h i l d k r a u t , 1 9 6 5 , 1 9 7 3 ; L a p i n a n d O x e n k r u g , 1969). Depressive states are bioc h e m i c a l l y h e t e r o g e n o u s : s e r o t o n e r g i c funct i o n s e e m s t o b e d i s t u r b e d in s o m e p a t i e n t s , w h e r e a s in o t h e r s , t h e r e m a y b e a d i s o r d e r e d n o r a d r e n e r g i c f u n c t i o n ( V a n Praag a n d Korf, 1 9 7 1 ; ~,sberg et al., 1 9 7 3 ; SjSstrSm, 1 9 7 3 ) . Tricyclic a n t i d e p r e s s a n t s inhibit n e u r o n a l u p t a k e o f t h e biogenic a m i n e s released f r o m p r e s y n a p t i c stores t h u s increasing t h e c o n c e n t r a t i o n o f t h e s e a m i n e s at p o s t s y n a p t i c recept o r s (Carlsson e t al., 1 9 6 9 a , b , c ) . T h e tricyclic t h y m o l e p t i c s inhibit t h e u p t a k e m e c h a n i s m s f o r 5 H T a n d N A to a v a r y i n g e x t e n t a n d in
Toxicity
Antidepressant effect
addition have different peripheral effects ( T h e o b a l d e t al., 1 9 6 4 ; Cairncross, 1 9 6 5 ; Hoffm e i s t e r , 1 9 6 9 ) . S o m e side e f f e c t s m a y result f r o m p e r i p h e r a l a c t i o n s ( K l e r m a n a n d Cole, 1 9 6 5 ; B o s t o n C o l l a b o r a t i v e d r u g surveillance program, 1972). D e v e l o p m e n t o f m o r e selective 5HT- and NA-uptake inhibitors may contribute to our knowledge concerning the mechanisms which m e d i a t e depressive states a n d m a y result in i m p r o v e d t r e a t m e n t . Using v a r i o u s b i o c h e m i cal a n d p h a r m a c o l o g i c a l tests t h e s u b s t a n c e ( + )- trans- 3-( 4 - m e t h o x y p h e n o x y m e t h y l )-1 -methyl- 4- phenyl- piperidine hydrochloride (FG 4 9 6 3 , see fig. 1) has b e e n f o u n d t o be a m o r e selective i n h i b i t o r o f 5 H T u p t a k e t h a n seven of t h e m o s t c o m m o n l y used tricyclic a n t i d e p r e s sants (Buus Lassen et al., 1975). In t h e p r e s e n t investigation the effects of these substances
NEW 5HT-UPTAKE INHIBITOR
~
CI-120.-~OCH
3 , HCI
I CH 3
Fig. 1. Structural formula of F G 4963.
were studied in two behavioral tests for 5HTuptake inhibitors. Furthermore some peripheral, cardiac and acute toxic effects were evaluated to try to predict possible side effects.
2. Materials a n d m e t h o d s
2.1. Animals NMRI mice (18--20 g), Wistar rats {120-300 g), guinea pigs (300--500 g) and Beagle dogs (10--13 kg) were used.
2.2. Substances FG 4963, imipramine, chlorimipramine, desipramine, amitriptyline, nortriptyline, doxepine and protriptyline were administered as hydrochlorides; d,l-p-chloroamphetamine (PCA) was given as sulphate and d , l - 5 - h y d r o x y t r y p t o p h a n (5HTP) as such. The doses refer to the forms mentioned. All substances were administered in physiological saline in volumes of 1--10 ml/kg body weight.
2. 3. Statistics LDs 0 values were determined by the method of Litchfield and Wilcoxon {1949). EDs 0 or ED, 00 values were evaluated by means of the log dose--response curves.
2. 4. Potentiation o f 5HTP-induced hypermotility in mice This potentiation was studied by means of the m e t h o d described previously (Buus Lassen,
109
1972). The controls (30 groups of 2 female mice) received 5HTP 150 mg/kg i.p. Test substances were administered s.c. 30 min after 5HTP in 3--4 doses (6 groups of 2 mice per dose). Motility was measured 45--75 min after 5HTP injection. The results are expressed in terms of the dose doubling the motility of controls.
2. 5. Poten tia tion o f NA-induced tachycardia in pithed rats Male rats were anaesthetized with hexobarbital sodium (120 mg/kg i.p.) and pithed according to the m e t h o d of $hipley and Tilden (1947 ). Immediately afterwards artificial ventilation was carried out through a cannula inserted into the trachea. A. carotis comm. dext. and v. jugularis dext. were catheterized. These vessels were ligated on the left side. N. vagus cerv. and n. sympaticus were cut on both sides. A heart rate meter was triggered by arterial blood pressure via a pressure transducer. NA 1--5 pg/kg was injected i.v. The heart rate (HR) response was integrated using an electronic integrator. In each experiment the NA dose giving a standard response was determined. The test substance was then injected i.v. and 30 min later the same NA dose was given again. 3--4 doses of each test substance were investigated (2--4 rats/dose). The results are expressed as the dose (ED,00), which increased the area under the HR curve by 100%.
2.6. In vitro inhibition o f guinea pig ileum contraction produced by acetylcholine, serotonin and histamine Isotonic contractions were registered with a strain gauge transducer connected to a recorder. Spasmogens were applied several times at intervals of 3 min. At least 3 isotonic contractions were obtained before the test substance was added. Acetylcholine 20 ng/ml, serotonin 220 ng/ml and histamine 200 ng/ml were used as agonists. Atropine, promethazine and diphenhydramine were used as standard antagonists. The results are expressed as the
110 c o n c e n t r a t i o n ( I C s 0 ) r e d u c i n g the i n d u c e d c o n t r a c t i o n s b y 50%.
2. 7. Cardiac toxicity in guinea pigs and dogs Male guinea pigs were a n a e s t h e t i z e d with p e n t o b a r b i t a l s o d i u m (40 mg/kg i.p.) and artificially v e n t i l a t e d t h r o u g h a tracheal tube. A c a t h e t e r was inserted into the v. jugularis. The ECG was r e c o r d e d f r o m s t a n d a r d leads and 3 p r e c o r d i a l leads. T h e test s u b s t a n c e was cont i n u o u s l y infused at a rate o f 3 m g / k g / m i n . The dose giving right b u n d l e b r a n c h b l o c k or left b u n d l e b r a n c h b l o c k was used as p a r a m e t e r for t o x i c i t y . Beagle dogs o f b o t h sexes were anaest h e t i z e d with h e x o b a r b i t a l s o d i u m (30 mg/kg i.v.). Artificial v e n t i l a t i o n was i n s t i t u t e d t h r o u g h a tracheal tube. V. jugularis was catheterized and a Walton strain gauge t r a n s d u c e r was fixed o n the right ventricle with 50% s t r e t c h o f t h e fibers to measure t h e isometric force of contraction (dp/dtmax). The heart rate was r e c o r d e d f r o m t h e m y o c a r d i a l contractions b y a t a c h o g r a p h . Test substances were infused i.v. at a rate o f 1 m g / k g / m i n . T h e results were r e c o r d e d as p e r c e n t o f initial values.
J. BUUS LASSEN ET AL. TABLE 1 Doses (EDs0) giving 50% inhibition of PCA-induced hypermotility in rats. Test drugs were administered s.c. simultaneously with PCA. Substance FG4963 Imipramine Chlorimipramine Desipramine Amitriptyline Nortriptyline Doxepine Protriptyline
EDs0 (95% confidence limits) (mg/kg) 2.0( 1.0-- 3.9) 3.] ( 2.4-- 4.2) 0.9 ( 0.5-- 1.6) 26 (19 --35 ) 15 ( 8 --26 ) :>50 1.8 ( 1.2-- 2.6) :>50
High doses o f t h e 5 H T p r e c u r s o r 5 H T P elicit a characteristic j e r k y h y p e r m o t i l i t y in mice. Thymoleptics blocking the neuronal re-uptake o f 5 H T p r o d u c e the same behavioural synd r o m e in mice t r e a t e d with a small, inactive dose o f 5 H T P (Carlsson et al., 1 9 6 9 c ; Buus Lassen, 1972). FG 4 9 6 3 , i m i p r a m i n e and chlorimipramine potentiated 5HTP-induced hyperm o t i l i t y (table 3). F G 4 9 6 3 was s o m e w h a t m o r e active t h a n c h l o r i m i p r a m i n e in this test.
2.8. Acute toxicity in mice and rats 3.2. Peripheral autonomic effects Test substances ( 4 - 7 doses) were administ e r e d via d i f f e r e n t r o u t e s to mice and rats. Each dose o f F G 4 9 6 3 was given t o 10 animals o f e i t h e r sex and t h e tricyclic t h y m o l e p t i c s t o half t h e n u m b e r . Deaths which o c c u r r e d within 48 hr o f drug a d m i n i s t r a t i o n were r e c o r d e d .
3. Results
Peripheral NA p o t e n t i a t i o n was evaluated in p i t h e d rats. Five tricyclic t h y m o l e p t i c s p o t e n t i a t e d N A - i n d u c e d t a c h y c a r d i a in the dose
TABLE 2 Doses (EDs0) giving 50% inhibition of PCA-induced hypermotility in rats. Test drugs were administered p.o. 1 and 2 hr before PCA.
3.1. Behavioral tests for 5HT-uptake inhibition Substance In rats PCA p r o d u c e s h y p e r m o t i l i t y , which p r e s u m a b l y is d e p e n d e n t on P C A - u p t a k e into 5 H T n e u r o n s f o l l o w e d b y 5HT-release (Buus Lassen, 1 9 7 4 ) . F G 4 9 6 3 , i m i p r a m i n e and chlori m i p r a m i n e were inhibitors o f PCA-induced h y p e r m o t i l i t y at a l m o s t t h e same s.c. (table 1) and p.o. (table 2) doses.
FG 4963 Imipramine Chlorimipramine
EDso (95% confidence limits) (mg/kg p.o.) 1 hr
2 hr
22 (18--28) 29 (23--38) 28 (24--32)
22 (13--34) 28 (22--36) 26 (21--31)
Iii
NEW 5HT-UPTAKE INHIBITOR TABLE 3 Doses (EDloo) doubling the motility of 5HTP-treated mice. Test drugs were administered 30 rain after 5HTP. Substance FG 4963 Imipramine Chlorimipramine Desipramine Amitriptyline Nortriptyline Doxepine Protriptyline
EDI00 (95% confidence limits) (mg/kg s.c.) 2.7 ( 1.5-- 5.7) 31 (15 --67 ) 7.2 ( 5.4-- 9.7) ~50 ~20 ~40" ~50 ~50
* About 50% potentiation at 40 mg/kg.
range 0 . 0 1 - - 0 . 3 mg/kg. FG 4 9 6 3 was inactive in doses u p t o 5 m g / k g (table 4). C o n t r a c t i o n o f guinea pig ileum i n d u c e d b y a c e t y l c h o l i n e , s e r o t o n i n and h i s t a m i n e was investigated in vitro. The i n h i b i t o r y activity o f F G 4 9 6 3 was c o m p a r e d with t h a t o f tricyclic t h y m o l e p t i c s and o f k n o w n antagonists (table 5). F G 4 9 6 3 , i m i p r a m i n e and desipramine s h o w e d a b o u t t h e same anticholinergic e f f e c t (ICs 0 1--3 pg/ml), while a m i t r i p t y l i n e , nort r i p t y l i n e , d o x e p i n e and p r o t r i p t y l i n e were m o r e active (ICs 0 0 . 1 - - 0 . 6 #g/ml). F G 4963, six t h y m o l e p t i c s and p r o m e t h a z i n e e x h i b i t e d s e r o t o n i n a n t a g o n i s m at 0.2--1 pg/ml, desipra-
TABLE 4 Potentiation of NA-induced heart rate increase in pithed rats. NA was injected i.v. before and 30 min after i.v. administration of test drugs. The results are presented as doses (EDi00) producing 100% potentiation of the NA response. Substance FG 4963 Imipramine Chlorimipramine Desipramine Amitriptyline Protriptyline
EDloo (mg/kg i.v.) ~5 0.1 0.3 0.01 0.1 0.01
TABLE 5 In vitro concentrations (ICs0) giving 50% inhibition of guinea pig ileum contractions produced by acetylcholine, serotonin or histamine. Antagonist
FG 4963 Imipramine Chlorimipramine Desipramine Amitriptyline Nortriptyline Protriptyline Doxepine Atropine Promethazine Diphenhydramine
ICs0 (#g/ml) Acetylcholine
Serotonin
Histamine
2 1 0.35 2.5 0.16 0.41 0.35 0.6 0.005
O.6 1 0.35 3.5 0.35 0.35 1 0.2
6 0.035 0.035 0.39 0.029 0.1 0.35 0.006
0.27 0.05
mine was weaker. D i p h e n h y d r a m i n e and the tricyclic antidepressants were strong antihistaminics (ICs 0 0 . 0 0 6 - - 0 . 4 pg/ml), whereas F G 4 9 6 3 was m u c h w e a k e r (ICs 0 6 pg/ml).
3.3. Cardiac effects and toxicity Several cardiac side effects have b e e n rep o r t e d d u r i n g t r e a t m e n t with tricyclic t h y m o leptics, particularly various t y p e s o f c o n d u c t i o n defects. The acute c a r d i o t o x i c e f f e c t o f F G 4 9 6 3 and 7 tricyclic t h y m o l e p t i c s was studied b y i.v. infusion t o guinea pigs (table 6). F G 4 9 6 3 p r o d u c e d b u n d l e b r a n c h b l o c k at m u c h higher d o s e levels t h a n the six t h y m o l e p tics, o n l y c h l o r i m i p r a m i n e s h o w e d a b o u t t h e same c a r d i o t o x i c i t y as FG 4963. 2 o u t o f 8 guinea pigs receiving F G 4 9 6 3 did n o t develop b r a n c h b l o c k even at a dose o f 180 mg/kg. T h e e f f e c t o f F G 4963, i m i p r a m i n e and a m i t r i p t y l i n e o n m y o c a r d i a l c o n t r a c t i l i t y and h e a r t rate was investigated b y c o n t i n u o u s i.v. infusion to dogs. The results are s h o w n in fig. 2. Initially, all t h r e e substances increased contractility. A f t e r i m i p r a m i n e and amitriptyline, b o t h 6 mg/kg, c o n t r a c t i l i t y r e t u r n e d t o c o n t r o l values; higher doses strongly r e d u c e d contrac-
112
J. B U U S L A S S E N E T AL.
TABLE 6
I O FG 4 9 6 3 Z~ IMIPRAMINE t , AMITRIPTYLINE
C a r d i o t o x i c i t y in a n a e s t h e t i z e d g u i n e a pigs. T e s t drugs were infused c o n t i n u o u s l y (3 m g / k g / m i n ) . T h e results are p r e s e n t e d as t h e dose giving right or left b u n d l e b r a n c h block.
2°° I
t
|
Number of animals
Block- p r o d u cing dose ( m g / k g i.v. ) ( m e a n ± S.D.)
FG 4963
6 2 3 6 2 3 2 3 3
57 + 18 180" 20 i 13 57 -+ 43 18 + 3 13 ± 7 11 +- 2 7 ± 2 18 ± 5
Imipramine Chlorimipramine Desipramine Amitriptyline Nortriptyline Doxepine Protriptyline
HEART RATE
'
150"
Substance
:
N ¢
"- 200 150
100
* T h e i n f u s i o n was s t o p p e d a f t e r a d m i n i s t r a t i o n o f 180 m g / k g a n d n o b l o c k s were o b t a i n e d at this dose.
tility. FG 4963 induced a prolonged increase in contractility. At 25 mg/kg contractility returned to the original value and higher doses elicited a slowly developing reduction. The three test substances initially produced some tachycardia and, at higher dose levels, varying degrees of
5O
20
40
A c u t e t o x i c i t y in mice. Route
rng/!
Fig. 2. T h e e f f e c t o f c o n t i n u o u s i.v. i n f u s i o n (1 m g / k g / m i n ) o n h e a r t rate a n d m y o c a r d i a l c o n t r a c t i l i t y in a n a e s t h e t i z e d dogs. F G 4 9 6 3 was given to t w o dogs, i m i p r a m i n e a n d a m i t r i p t y l i n e to o n e dog each. T h e results are s h o w n as p e r c e n t o f values b e f o r e i n f u s i o n o f t h e test drug.
TABLE 7
Substance
60
LDs0 (95% c o n f i d e n c e limits) ( m g / k g ) Females
Males
FG 4963
i.v. s.c. p.o.
48 941 1408
41-- 56 731--1210 1184--1674
45 723 1687
38-53) 563-- 929) 1404--2026)
Imipramine
i.v. s.c. p.o.
26 385 412
21-- 33 307-- 485 328-- 519
28 356 413
22-35) 283-- 447) 328-- 519)
Chlorimipramine
i.v. s.c. p.o.
29 484 1172
25-- 32 233--1005 613--2243
22 663 1316
17-28) 414--1062) 883--1963)
Amitriptyline
i.v. s.c. p.o.
19 147 280
21-- 24 104-- 209 224-- 351
20 112 223
18-22) 8 9 - - 141) 1 7 8 - - 281)
113
NEW 5HT-UPTAKE INHIBITOR TABLE 8 Acute toxicity in rats. Substance
Route
LDs0 (95% confidence limits) Females
Males
FG 4963
i.v. s.c. p.o.
48 ( 41-- 56) 1600 (1357--1886) 1600 (1357--1886)
48( 41-- 53) 1120(904--1398) 1473(1286--1658)
Imipramine
i.v. s.c. p.o.
28 ( 24-- 34) 292 ( 222-- 384) 562 ( 448-- 706)
25( 21-- 31) 4 9 2 ( 440-- 551) 5 2 9 ( 473-- 592)
Chlorimipramine
i.v. s.c. p.o.
29 ( 25-- 32) 2031 (1005--4102) 914 (608--1375)
26( 21-- 33) 2052 (1457--2932) 1068(770--1482)
Amitriptyline
i.v. s.c. p.o.
18 ( 14-- 22) 385 ( 307-- 485) 455 ( 311-- 666)
bradycardia. Imipramine and amitriptyline produced bradycardia at the same doses which reduced myocardial contractility. The decrease in heart rate after FG 4963 was much smaller and a positive inotropic effect was observed even at doses which reduced the heart rate. This suggests that FG 4963 may exert a direct positive inotropic effect. The acute toxicity of FG 4963, imipramine, amitriptyline and chlorimipramine was studied by i.v., s.c., and p.o. administration to mice and rats (tables 7, 8). FG 4963 was less toxic than imipramine and amitriptyline, whereas FG 4963 and chlorimipramine showed about equal toxicity. Toxic doses of all four substances produced clonic convulsions and cyanosis lasting 2--20 min before death occurred.
4. Discussion
Tricyclic thymoleptics have been shown to inhibit amine uptake at the neuronal cell membrane. Tertiary amines, such as imipramine and chlorimipramine, act preferentially on 5HT neurons, whereas secondary amines, such as desipramine and protriptyline, preferentially inhibit the membrane pump of NA-neurons
16( 455( 453(
13-- 21) 312-- 667) 295-- 633)
(Carlsson et al., 1969a,b,c; Carlsson, 1970; Fuxe and Ungerstedt, 1968; Lidbrink et al., 1971). The imipramine derivatives antagonize PCA-induced 5HT depletion from rat brain, and the strongest 5HT-uptake inhibitors are the most potent PCA antagonists (Squires, 1972). Presumably PCA is taken up by the neuronal membrane group for 5HT. Inhibition of this pump prevents PCA entry into 5HT neurons and thus protects against 5HT depletion. In rats, PCA produces hypermotility, which is antagonized by the 5HT-synthesis inhibitor p-chlorophenylalanine and 5HT-uptake inhibitors (Buus Lassen, 1974). This suggests that 5HT-release is involved in the PCA-induced hypermotility. The new 5HT-uptake inhibitor, FG 4963, was also found to inhibit PCA hypermotility. Previously the substance has been shown to antagonize PCA-induced 5HT depletion from rat brain (Buus Lassen et al., 1975). In both PCA tests, FG 4963 was active in about the same doses as chlorimipramine. The 5HT precursor 5HTP elicits jerky hypermotility in mice. Imipramine and chlorimipramine potentiate this effect of 5HTP (Carlsson et al., 1969c). The behavioral effect of 5HTP + chlorimipramine is antagonized by the central decarboxylase inhibitor 3-hydroxybenzylhy-
114 d r a z i n e (NSD 1 0 1 5 ) b u t n o t b y the catechola m i n e synthesis i n h i b i t o r a - m e t h y l - p - t y r o s i n e (H 4 4 / 6 8 ) (Buus Lassen, 1 9 7 2 ) . Thus, t h e e f f e c t o f a 5 H T - - u p t a k e i n h i b i t o r in 5HTPt r e a t e d mice is n o t d e p e n d e n t o n catechola m i n e release, b u t requires d e c a r b o x y l a t i o n o f 5 H T P t o 5HT. T h e r e f o r e , the behavioral synd r o m e p r o b a b l y is d u e to highly facilitated s e r o t o n e r g i c n e u r o t r a n s m i s s i o n . As a 5 H T P p o t e n t i a t o r F G 4 9 6 3 was slightly m o r e active than chlorimipramine. T h e i m i p r a m i n e derivatives p r o d u c e a strong p e r i p h e r a l N A p o t e n t i a t i o n (Sigg et al., 1963; H a e f e l y et al., 1 9 6 4 ; T h e o b a l d et al., 1964; Westfall, 1 9 7 3 ) , which p r o b a b l y is caused b y i n h i b i t i o n o f N A u p t a k e into peripheral symp a t h e t i c n e r v e endings ( A x e l r o d et al., 1 9 6 1 ; Iversen, 1 9 6 5 ; H a m b e r g e r , 1 9 6 7 ; Sachs and J o n s s o n , 1 9 7 2 ) . In t h e p r e s e n t investigation several tricyclic t h y m o l e p t i c s , b u t n o t F G 4 9 6 3 , were f o u n d to p o t e n t i a t e strongly the N A - i n d u c e d h e a r t rate increase in p i t h e d rats. This is in g o o d a g r e e m e n t with t h e very weak i n h i b i t i o n o f NA u p t a k e into isolated rat iris p r o d u c e d b y F G 4 9 6 3 in previous e x p e r i m e n t s (Buus Lassen e t al., 1 9 7 5 ) . T h e tricyclic a n t i d e p r e s s a n t s have peripheral anticholinergic e f f e c t ( D o m e n j o z and Theobald, 1 9 5 9 ; T h e o b a l d et al., 1 9 6 4 ; Cairncross, 1 9 6 5 ; V o n R i b b e n t r o p and S c h a u m a n n , 1965; V o n H o f f m e i s t e r , 1969). Desipramine was f o u n d t o be t h e w e a k e s t anticholinergic a m o n g t h e i m i p r a m i n e derivatives and F G 4 9 6 3 had a b o u t t h e same activity. B o t h N A p o t e n t i a t i o n and anticholinergic e f f e c t result in an increased s y m p a t h e t i c activity. F G 4 9 6 3 has n o p e r i p h e r a l N A - p o t e n t i a t i n g e f f e c t and a r a t h e r w e a k p e r i p h e r a l anticholinergic effect. T h e r e f o r e , F G 4 9 6 3 p r e s u m a b l y p r o d u c e s m u c h less s y m p a t h e t i c d o m i n a n c e t h a n d o t h e tricyclic antidepressants. This m a y o f f e r advantages in cardiac diseases with symp a t h e t i c h y p e r f u n c t i o n or increased b l o o d conc e n t r a t i o n o f c a t e c h o l a m i n e s ( M c D o n a l d et al., 1 9 6 9 ; J e w i t t et al., 1 9 6 9 ; Siggers et al., 1 9 7 1 ) , which m a y be aggravated b y tricyclic antidepressants {Leon and Abrams, 1 9 7 1 ) . A c u t e i n t o x i c a t i o n s with tricyclic t h y m o leptics in p a t i e n t s ( F r e e m a n et al., 1969; Fournier, 1 9 7 3 ) and in e x p e r i m e n t a l animals (Bois-
J. BUUS LASSEN ET AL. sier et al., 1965; Marno, 1 9 7 0 ) have been r e p o r t e d to i n d u c e several t y p e s o f h e a r t blocks. The a c u t e c a r d i o t o x i c i t y o f FG 4 9 6 3 was f o u n d t o be less t h a n t h a t o f i m i p r a m i n e and a m i t r i p t y l i n e . In dogs, FG 4 9 6 3 even s h o w e d a p r o l o n g e d positive i n o t r o p i c effect, which m a y l o w e r the risk for negative i n o t r o p y .
Acknowledgement The authors wish to thank Dr. Richard F. Squires for helpful suggestions. References .~sberg, M., L. Bertilsson, D. Tuck, B. Cronholm and F. Sjhqvist, 1973, Indoleamine metabolites in the cerebrospinal fluid of depressed patients before and during treatment with nortriptyline, Clin. Pharmacol. Therap. 14, 277. Axelrod, J., L.G. Whitby and G. Hefting, 1961, Effect of psychotropic drugs on the uptake of 3H. norepinephrine by tissues, Science 133, 383. Boissier, J.-R., P. Simon and S. Witchitz, 1965, Etude chez le cobaye de la toxicit~ cardiaque de l'imipramine, de l'amitriptyline et de leurs d~riv6s monodesm6thyl~s, Th~rapie 20, 67. Boston Collaborative drug surveillance program, 1972. Adverse reactions to the tricyclic-antidepressant drug. A report, Lancet I, 529. Buus Lassen, J., 1972, Behavioural effect of tricyclic thymoleptics and chlorpheniramine in mice after pretreatment with 5 hydroxytryptophan (5HTP), Acta Pharmacol. (Kbh.) 31, Suppl. I, 11. Buus Lassen, J., 1974, The effect of p-chloroamphetamine on motility in rats after inhibition of monoamine synthesis, storage, uptake and receptor interaction, Psychopharmacologia (Berlin) 34, 243. Buus Lassen, J., R.F. Squires, J.A. Christensen and L. Molander, 1975, Neurochemical and pharmacological studies on a new 5HT-uptake inhibitor, FG 4963, with potential antidepressant properties, Psychopharmacologia (in press). Cairncross, K.D., 1965, On the peripheral pharmacology of amitriptyline, Arch. Intern. Pharmacodyn. 154, 438. Carlsson, A., H. Corrodi, K. Fuxe and T. Hhkfelt, 1969a, Effect of antidepressant drugs on the depletion of intraneuronal brain 5-hydroxytryptamine stores caused by 4-methyl-c~-ethyl-metatyramine, European J. Pharmacol. 5, 357. Carlsson, A., H. Corrodi, K. Fuxe and T. H6kfelt, 1969b, Effect of some antidepressant drugs on the depletion of intraneuronal brain catecholamine stores caused by 4,(x-dimethyl-meta-tyramine, European J. Pharmacol. 5, 367.
115 Carlsson, A., J.J. Jonason, M. Lindqvist and K. Fuxe, 1969c, Demonstration of extraneuronal 5-hydroxytryptamine accumulation in brain following membrane-pump blockade by chlorimipramine, Brain Res. 12,456. Carlsson, A., 1970, Structural specificity for inhibition and ( 14 C)-5-hydroxytryptamine uptake by cerebral slices, J. Pharm. Pharmacot. 22, 729. Domenjoz, R. and W. Theobald, 1959, Zur Pharmakologie des Trofranil (R) (N.(3
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
C O M P A R A T I V E S T U D I E S OF A NEW 5 H T - U P T A K E I N H I B I T O R A N D S O M E TRICYCLIC THYMOLEPTICS JORGEN BUUS LASSEN, ERLING PETERSEN, BENGT KJELLBERG and SVEN O. OLSSON Research Laboratories of Ferrosan, Copenhagen, Denmark and Malta6, Sweden
Received 23 December 1974, revised MS received 6 February 1975, accepted 17 February 1975
J. BUUS LASSEN, E. PETERSEN, B. KJELLBERG and S.O. OLSSON, Comparative studies o f a new 5HTuptake inhibitor and some tricyclic thymoleptics, European J. Pharmacol. 32 (1975) 108--115. The new 5HT-uptake inhibitor, FG 4963, and some tricyclic thymoleptics antagonized p-chloroamphetamine (PCA)-induced hypermotility in rats. FG 4963 was active in about the same s.c. and p.o. doses as chlorimipramine. FG 4963, imipramine and chlorimipramine potentiated hypermotility induced in mice by the 5HT precursor 5HTP, FG 4963 being slightly more active than chlorimipramine. In contrast to the tricyclic thymoleptics FG 4963 did not potentiate the heart rate increasing effect of NA in pithed rats. The peripheral anticholinergic effect of FG 4963 and of desipramine was almost identical while the other imipramine derivatives were more active. All tricyclic thymoleptics were strong peripheral antihistaminics, but FG 4963 was almost devoid of this action. Acute tests for ECG changes in guinea pigs and toxicity in mice and rats showed that FG 4963 and chlorimipramine were less toxic than imipramine and amitriptyline. FG 4963 is presumably a selective 5HT-uptake inhibitor producing much less potentiation of peripheral sympathetic mechanisms than do the tricyclic antidepressants. 5HT-uptake inhibition
Peripheral autonomic effects
1. I n t r o d u c t i o n An i m p a i r e d f u n c t i o n o f t h e n e u r o t r a n s m i t ters n o r a d r e n a l i n e ( N A ) a n d s e r o t o n i n ( 5 H T ) m a y be i n v o l v e d in p a t h o g e n e s i s o f depressive illness ( S c h i l d k r a u t , 1 9 6 5 , 1 9 7 3 ; L a p i n a n d O x e n k r u g , 1969). Depressive states are bioc h e m i c a l l y h e t e r o g e n o u s : s e r o t o n e r g i c funct i o n s e e m s t o b e d i s t u r b e d in s o m e p a t i e n t s , w h e r e a s in o t h e r s , t h e r e m a y b e a d i s o r d e r e d n o r a d r e n e r g i c f u n c t i o n ( V a n Praag a n d Korf, 1 9 7 1 ; ~,sberg et al., 1 9 7 3 ; SjSstrSm, 1 9 7 3 ) . Tricyclic a n t i d e p r e s s a n t s inhibit n e u r o n a l u p t a k e o f t h e biogenic a m i n e s released f r o m p r e s y n a p t i c stores t h u s increasing t h e c o n c e n t r a t i o n o f t h e s e a m i n e s at p o s t s y n a p t i c recept o r s (Carlsson e t al., 1 9 6 9 a , b , c ) . T h e tricyclic t h y m o l e p t i c s inhibit t h e u p t a k e m e c h a n i s m s f o r 5 H T a n d N A to a v a r y i n g e x t e n t a n d in
Toxicity
Antidepressant effect
addition have different peripheral effects ( T h e o b a l d e t al., 1 9 6 4 ; Cairncross, 1 9 6 5 ; Hoffm e i s t e r , 1 9 6 9 ) . S o m e side e f f e c t s m a y result f r o m p e r i p h e r a l a c t i o n s ( K l e r m a n a n d Cole, 1 9 6 5 ; B o s t o n C o l l a b o r a t i v e d r u g surveillance program, 1972). D e v e l o p m e n t o f m o r e selective 5HT- and NA-uptake inhibitors may contribute to our knowledge concerning the mechanisms which m e d i a t e depressive states a n d m a y result in i m p r o v e d t r e a t m e n t . Using v a r i o u s b i o c h e m i cal a n d p h a r m a c o l o g i c a l tests t h e s u b s t a n c e ( + )- trans- 3-( 4 - m e t h o x y p h e n o x y m e t h y l )-1 -methyl- 4- phenyl- piperidine hydrochloride (FG 4 9 6 3 , see fig. 1) has b e e n f o u n d t o be a m o r e selective i n h i b i t o r o f 5 H T u p t a k e t h a n seven of t h e m o s t c o m m o n l y used tricyclic a n t i d e p r e s sants (Buus Lassen et al., 1975). In t h e p r e s e n t investigation the effects of these substances
NEW 5HT-UPTAKE INHIBITOR
~
CI-120.-~OCH
3 , HCI
I CH 3
Fig. 1. Structural formula of F G 4963.
were studied in two behavioral tests for 5HTuptake inhibitors. Furthermore some peripheral, cardiac and acute toxic effects were evaluated to try to predict possible side effects.
2. Materials a n d m e t h o d s
2.1. Animals NMRI mice (18--20 g), Wistar rats {120-300 g), guinea pigs (300--500 g) and Beagle dogs (10--13 kg) were used.
2.2. Substances FG 4963, imipramine, chlorimipramine, desipramine, amitriptyline, nortriptyline, doxepine and protriptyline were administered as hydrochlorides; d,l-p-chloroamphetamine (PCA) was given as sulphate and d , l - 5 - h y d r o x y t r y p t o p h a n (5HTP) as such. The doses refer to the forms mentioned. All substances were administered in physiological saline in volumes of 1--10 ml/kg body weight.
2. 3. Statistics LDs 0 values were determined by the method of Litchfield and Wilcoxon {1949). EDs 0 or ED, 00 values were evaluated by means of the log dose--response curves.
2. 4. Potentiation o f 5HTP-induced hypermotility in mice This potentiation was studied by means of the m e t h o d described previously (Buus Lassen,
109
1972). The controls (30 groups of 2 female mice) received 5HTP 150 mg/kg i.p. Test substances were administered s.c. 30 min after 5HTP in 3--4 doses (6 groups of 2 mice per dose). Motility was measured 45--75 min after 5HTP injection. The results are expressed in terms of the dose doubling the motility of controls.
2. 5. Poten tia tion o f NA-induced tachycardia in pithed rats Male rats were anaesthetized with hexobarbital sodium (120 mg/kg i.p.) and pithed according to the m e t h o d of $hipley and Tilden (1947 ). Immediately afterwards artificial ventilation was carried out through a cannula inserted into the trachea. A. carotis comm. dext. and v. jugularis dext. were catheterized. These vessels were ligated on the left side. N. vagus cerv. and n. sympaticus were cut on both sides. A heart rate meter was triggered by arterial blood pressure via a pressure transducer. NA 1--5 pg/kg was injected i.v. The heart rate (HR) response was integrated using an electronic integrator. In each experiment the NA dose giving a standard response was determined. The test substance was then injected i.v. and 30 min later the same NA dose was given again. 3--4 doses of each test substance were investigated (2--4 rats/dose). The results are expressed as the dose (ED,00), which increased the area under the HR curve by 100%.
2.6. In vitro inhibition o f guinea pig ileum contraction produced by acetylcholine, serotonin and histamine Isotonic contractions were registered with a strain gauge transducer connected to a recorder. Spasmogens were applied several times at intervals of 3 min. At least 3 isotonic contractions were obtained before the test substance was added. Acetylcholine 20 ng/ml, serotonin 220 ng/ml and histamine 200 ng/ml were used as agonists. Atropine, promethazine and diphenhydramine were used as standard antagonists. The results are expressed as the
110 c o n c e n t r a t i o n ( I C s 0 ) r e d u c i n g the i n d u c e d c o n t r a c t i o n s b y 50%.
2. 7. Cardiac toxicity in guinea pigs and dogs Male guinea pigs were a n a e s t h e t i z e d with p e n t o b a r b i t a l s o d i u m (40 mg/kg i.p.) and artificially v e n t i l a t e d t h r o u g h a tracheal tube. A c a t h e t e r was inserted into the v. jugularis. The ECG was r e c o r d e d f r o m s t a n d a r d leads and 3 p r e c o r d i a l leads. T h e test s u b s t a n c e was cont i n u o u s l y infused at a rate o f 3 m g / k g / m i n . The dose giving right b u n d l e b r a n c h b l o c k or left b u n d l e b r a n c h b l o c k was used as p a r a m e t e r for t o x i c i t y . Beagle dogs o f b o t h sexes were anaest h e t i z e d with h e x o b a r b i t a l s o d i u m (30 mg/kg i.v.). Artificial v e n t i l a t i o n was i n s t i t u t e d t h r o u g h a tracheal tube. V. jugularis was catheterized and a Walton strain gauge t r a n s d u c e r was fixed o n the right ventricle with 50% s t r e t c h o f t h e fibers to measure t h e isometric force of contraction (dp/dtmax). The heart rate was r e c o r d e d f r o m t h e m y o c a r d i a l contractions b y a t a c h o g r a p h . Test substances were infused i.v. at a rate o f 1 m g / k g / m i n . T h e results were r e c o r d e d as p e r c e n t o f initial values.
J. BUUS LASSEN ET AL. TABLE 1 Doses (EDs0) giving 50% inhibition of PCA-induced hypermotility in rats. Test drugs were administered s.c. simultaneously with PCA. Substance FG4963 Imipramine Chlorimipramine Desipramine Amitriptyline Nortriptyline Doxepine Protriptyline
EDs0 (95% confidence limits) (mg/kg) 2.0( 1.0-- 3.9) 3.] ( 2.4-- 4.2) 0.9 ( 0.5-- 1.6) 26 (19 --35 ) 15 ( 8 --26 ) :>50 1.8 ( 1.2-- 2.6) :>50
High doses o f t h e 5 H T p r e c u r s o r 5 H T P elicit a characteristic j e r k y h y p e r m o t i l i t y in mice. Thymoleptics blocking the neuronal re-uptake o f 5 H T p r o d u c e the same behavioural synd r o m e in mice t r e a t e d with a small, inactive dose o f 5 H T P (Carlsson et al., 1 9 6 9 c ; Buus Lassen, 1972). FG 4 9 6 3 , i m i p r a m i n e and chlorimipramine potentiated 5HTP-induced hyperm o t i l i t y (table 3). F G 4 9 6 3 was s o m e w h a t m o r e active t h a n c h l o r i m i p r a m i n e in this test.
2.8. Acute toxicity in mice and rats 3.2. Peripheral autonomic effects Test substances ( 4 - 7 doses) were administ e r e d via d i f f e r e n t r o u t e s to mice and rats. Each dose o f F G 4 9 6 3 was given t o 10 animals o f e i t h e r sex and t h e tricyclic t h y m o l e p t i c s t o half t h e n u m b e r . Deaths which o c c u r r e d within 48 hr o f drug a d m i n i s t r a t i o n were r e c o r d e d .
3. Results
Peripheral NA p o t e n t i a t i o n was evaluated in p i t h e d rats. Five tricyclic t h y m o l e p t i c s p o t e n t i a t e d N A - i n d u c e d t a c h y c a r d i a in the dose
TABLE 2 Doses (EDs0) giving 50% inhibition of PCA-induced hypermotility in rats. Test drugs were administered p.o. 1 and 2 hr before PCA.
3.1. Behavioral tests for 5HT-uptake inhibition Substance In rats PCA p r o d u c e s h y p e r m o t i l i t y , which p r e s u m a b l y is d e p e n d e n t on P C A - u p t a k e into 5 H T n e u r o n s f o l l o w e d b y 5HT-release (Buus Lassen, 1 9 7 4 ) . F G 4 9 6 3 , i m i p r a m i n e and chlori m i p r a m i n e were inhibitors o f PCA-induced h y p e r m o t i l i t y at a l m o s t t h e same s.c. (table 1) and p.o. (table 2) doses.
FG 4963 Imipramine Chlorimipramine
EDso (95% confidence limits) (mg/kg p.o.) 1 hr
2 hr
22 (18--28) 29 (23--38) 28 (24--32)
22 (13--34) 28 (22--36) 26 (21--31)
Iii
NEW 5HT-UPTAKE INHIBITOR TABLE 3 Doses (EDloo) doubling the motility of 5HTP-treated mice. Test drugs were administered 30 rain after 5HTP. Substance FG 4963 Imipramine Chlorimipramine Desipramine Amitriptyline Nortriptyline Doxepine Protriptyline
EDI00 (95% confidence limits) (mg/kg s.c.) 2.7 ( 1.5-- 5.7) 31 (15 --67 ) 7.2 ( 5.4-- 9.7) ~50 ~20 ~40" ~50 ~50
* About 50% potentiation at 40 mg/kg.
range 0 . 0 1 - - 0 . 3 mg/kg. FG 4 9 6 3 was inactive in doses u p t o 5 m g / k g (table 4). C o n t r a c t i o n o f guinea pig ileum i n d u c e d b y a c e t y l c h o l i n e , s e r o t o n i n and h i s t a m i n e was investigated in vitro. The i n h i b i t o r y activity o f F G 4 9 6 3 was c o m p a r e d with t h a t o f tricyclic t h y m o l e p t i c s and o f k n o w n antagonists (table 5). F G 4 9 6 3 , i m i p r a m i n e and desipramine s h o w e d a b o u t t h e same anticholinergic e f f e c t (ICs 0 1--3 pg/ml), while a m i t r i p t y l i n e , nort r i p t y l i n e , d o x e p i n e and p r o t r i p t y l i n e were m o r e active (ICs 0 0 . 1 - - 0 . 6 #g/ml). F G 4963, six t h y m o l e p t i c s and p r o m e t h a z i n e e x h i b i t e d s e r o t o n i n a n t a g o n i s m at 0.2--1 pg/ml, desipra-
TABLE 4 Potentiation of NA-induced heart rate increase in pithed rats. NA was injected i.v. before and 30 min after i.v. administration of test drugs. The results are presented as doses (EDi00) producing 100% potentiation of the NA response. Substance FG 4963 Imipramine Chlorimipramine Desipramine Amitriptyline Protriptyline
EDloo (mg/kg i.v.) ~5 0.1 0.3 0.01 0.1 0.01
TABLE 5 In vitro concentrations (ICs0) giving 50% inhibition of guinea pig ileum contractions produced by acetylcholine, serotonin or histamine. Antagonist
FG 4963 Imipramine Chlorimipramine Desipramine Amitriptyline Nortriptyline Protriptyline Doxepine Atropine Promethazine Diphenhydramine
ICs0 (#g/ml) Acetylcholine
Serotonin
Histamine
2 1 0.35 2.5 0.16 0.41 0.35 0.6 0.005
O.6 1 0.35 3.5 0.35 0.35 1 0.2
6 0.035 0.035 0.39 0.029 0.1 0.35 0.006
0.27 0.05
mine was weaker. D i p h e n h y d r a m i n e and the tricyclic antidepressants were strong antihistaminics (ICs 0 0 . 0 0 6 - - 0 . 4 pg/ml), whereas F G 4 9 6 3 was m u c h w e a k e r (ICs 0 6 pg/ml).
3.3. Cardiac effects and toxicity Several cardiac side effects have b e e n rep o r t e d d u r i n g t r e a t m e n t with tricyclic t h y m o leptics, particularly various t y p e s o f c o n d u c t i o n defects. The acute c a r d i o t o x i c e f f e c t o f F G 4 9 6 3 and 7 tricyclic t h y m o l e p t i c s was studied b y i.v. infusion t o guinea pigs (table 6). F G 4 9 6 3 p r o d u c e d b u n d l e b r a n c h b l o c k at m u c h higher d o s e levels t h a n the six t h y m o l e p tics, o n l y c h l o r i m i p r a m i n e s h o w e d a b o u t t h e same c a r d i o t o x i c i t y as FG 4963. 2 o u t o f 8 guinea pigs receiving F G 4 9 6 3 did n o t develop b r a n c h b l o c k even at a dose o f 180 mg/kg. T h e e f f e c t o f F G 4963, i m i p r a m i n e and a m i t r i p t y l i n e o n m y o c a r d i a l c o n t r a c t i l i t y and h e a r t rate was investigated b y c o n t i n u o u s i.v. infusion to dogs. The results are s h o w n in fig. 2. Initially, all t h r e e substances increased contractility. A f t e r i m i p r a m i n e and amitriptyline, b o t h 6 mg/kg, c o n t r a c t i l i t y r e t u r n e d t o c o n t r o l values; higher doses strongly r e d u c e d contrac-
112
J. B U U S L A S S E N E T AL.
TABLE 6
I O FG 4 9 6 3 Z~ IMIPRAMINE t , AMITRIPTYLINE
C a r d i o t o x i c i t y in a n a e s t h e t i z e d g u i n e a pigs. T e s t drugs were infused c o n t i n u o u s l y (3 m g / k g / m i n ) . T h e results are p r e s e n t e d as t h e dose giving right or left b u n d l e b r a n c h block.
2°° I
t
|
Number of animals
Block- p r o d u cing dose ( m g / k g i.v. ) ( m e a n ± S.D.)
FG 4963
6 2 3 6 2 3 2 3 3
57 + 18 180" 20 i 13 57 -+ 43 18 + 3 13 ± 7 11 +- 2 7 ± 2 18 ± 5
Imipramine Chlorimipramine Desipramine Amitriptyline Nortriptyline Doxepine Protriptyline
HEART RATE
'
150"
Substance
:
N ¢
"- 200 150
100
* T h e i n f u s i o n was s t o p p e d a f t e r a d m i n i s t r a t i o n o f 180 m g / k g a n d n o b l o c k s were o b t a i n e d at this dose.
tility. FG 4963 induced a prolonged increase in contractility. At 25 mg/kg contractility returned to the original value and higher doses elicited a slowly developing reduction. The three test substances initially produced some tachycardia and, at higher dose levels, varying degrees of
5O
20
40
A c u t e t o x i c i t y in mice. Route
rng/!
Fig. 2. T h e e f f e c t o f c o n t i n u o u s i.v. i n f u s i o n (1 m g / k g / m i n ) o n h e a r t rate a n d m y o c a r d i a l c o n t r a c t i l i t y in a n a e s t h e t i z e d dogs. F G 4 9 6 3 was given to t w o dogs, i m i p r a m i n e a n d a m i t r i p t y l i n e to o n e dog each. T h e results are s h o w n as p e r c e n t o f values b e f o r e i n f u s i o n o f t h e test drug.
TABLE 7
Substance
60
LDs0 (95% c o n f i d e n c e limits) ( m g / k g ) Females
Males
FG 4963
i.v. s.c. p.o.
48 941 1408
41-- 56 731--1210 1184--1674
45 723 1687
38-53) 563-- 929) 1404--2026)
Imipramine
i.v. s.c. p.o.
26 385 412
21-- 33 307-- 485 328-- 519
28 356 413
22-35) 283-- 447) 328-- 519)
Chlorimipramine
i.v. s.c. p.o.
29 484 1172
25-- 32 233--1005 613--2243
22 663 1316
17-28) 414--1062) 883--1963)
Amitriptyline
i.v. s.c. p.o.
19 147 280
21-- 24 104-- 209 224-- 351
20 112 223
18-22) 8 9 - - 141) 1 7 8 - - 281)
113
NEW 5HT-UPTAKE INHIBITOR TABLE 8 Acute toxicity in rats. Substance
Route
LDs0 (95% confidence limits) Females
Males
FG 4963
i.v. s.c. p.o.
48 ( 41-- 56) 1600 (1357--1886) 1600 (1357--1886)
48( 41-- 53) 1120(904--1398) 1473(1286--1658)
Imipramine
i.v. s.c. p.o.
28 ( 24-- 34) 292 ( 222-- 384) 562 ( 448-- 706)
25( 21-- 31) 4 9 2 ( 440-- 551) 5 2 9 ( 473-- 592)
Chlorimipramine
i.v. s.c. p.o.
29 ( 25-- 32) 2031 (1005--4102) 914 (608--1375)
26( 21-- 33) 2052 (1457--2932) 1068(770--1482)
Amitriptyline
i.v. s.c. p.o.
18 ( 14-- 22) 385 ( 307-- 485) 455 ( 311-- 666)
bradycardia. Imipramine and amitriptyline produced bradycardia at the same doses which reduced myocardial contractility. The decrease in heart rate after FG 4963 was much smaller and a positive inotropic effect was observed even at doses which reduced the heart rate. This suggests that FG 4963 may exert a direct positive inotropic effect. The acute toxicity of FG 4963, imipramine, amitriptyline and chlorimipramine was studied by i.v., s.c., and p.o. administration to mice and rats (tables 7, 8). FG 4963 was less toxic than imipramine and amitriptyline, whereas FG 4963 and chlorimipramine showed about equal toxicity. Toxic doses of all four substances produced clonic convulsions and cyanosis lasting 2--20 min before death occurred.
4. Discussion
Tricyclic thymoleptics have been shown to inhibit amine uptake at the neuronal cell membrane. Tertiary amines, such as imipramine and chlorimipramine, act preferentially on 5HT neurons, whereas secondary amines, such as desipramine and protriptyline, preferentially inhibit the membrane pump of NA-neurons
16( 455( 453(
13-- 21) 312-- 667) 295-- 633)
(Carlsson et al., 1969a,b,c; Carlsson, 1970; Fuxe and Ungerstedt, 1968; Lidbrink et al., 1971). The imipramine derivatives antagonize PCA-induced 5HT depletion from rat brain, and the strongest 5HT-uptake inhibitors are the most potent PCA antagonists (Squires, 1972). Presumably PCA is taken up by the neuronal membrane group for 5HT. Inhibition of this pump prevents PCA entry into 5HT neurons and thus protects against 5HT depletion. In rats, PCA produces hypermotility, which is antagonized by the 5HT-synthesis inhibitor p-chlorophenylalanine and 5HT-uptake inhibitors (Buus Lassen, 1974). This suggests that 5HT-release is involved in the PCA-induced hypermotility. The new 5HT-uptake inhibitor, FG 4963, was also found to inhibit PCA hypermotility. Previously the substance has been shown to antagonize PCA-induced 5HT depletion from rat brain (Buus Lassen et al., 1975). In both PCA tests, FG 4963 was active in about the same doses as chlorimipramine. The 5HT precursor 5HTP elicits jerky hypermotility in mice. Imipramine and chlorimipramine potentiate this effect of 5HTP (Carlsson et al., 1969c). The behavioral effect of 5HTP + chlorimipramine is antagonized by the central decarboxylase inhibitor 3-hydroxybenzylhy-
114 d r a z i n e (NSD 1 0 1 5 ) b u t n o t b y the catechola m i n e synthesis i n h i b i t o r a - m e t h y l - p - t y r o s i n e (H 4 4 / 6 8 ) (Buus Lassen, 1 9 7 2 ) . Thus, t h e e f f e c t o f a 5 H T - - u p t a k e i n h i b i t o r in 5HTPt r e a t e d mice is n o t d e p e n d e n t o n catechola m i n e release, b u t requires d e c a r b o x y l a t i o n o f 5 H T P t o 5HT. T h e r e f o r e , the behavioral synd r o m e p r o b a b l y is d u e to highly facilitated s e r o t o n e r g i c n e u r o t r a n s m i s s i o n . As a 5 H T P p o t e n t i a t o r F G 4 9 6 3 was slightly m o r e active than chlorimipramine. T h e i m i p r a m i n e derivatives p r o d u c e a strong p e r i p h e r a l N A p o t e n t i a t i o n (Sigg et al., 1963; H a e f e l y et al., 1 9 6 4 ; T h e o b a l d et al., 1964; Westfall, 1 9 7 3 ) , which p r o b a b l y is caused b y i n h i b i t i o n o f N A u p t a k e into peripheral symp a t h e t i c n e r v e endings ( A x e l r o d et al., 1 9 6 1 ; Iversen, 1 9 6 5 ; H a m b e r g e r , 1 9 6 7 ; Sachs and J o n s s o n , 1 9 7 2 ) . In t h e p r e s e n t investigation several tricyclic t h y m o l e p t i c s , b u t n o t F G 4 9 6 3 , were f o u n d to p o t e n t i a t e strongly the N A - i n d u c e d h e a r t rate increase in p i t h e d rats. This is in g o o d a g r e e m e n t with t h e very weak i n h i b i t i o n o f NA u p t a k e into isolated rat iris p r o d u c e d b y F G 4 9 6 3 in previous e x p e r i m e n t s (Buus Lassen e t al., 1 9 7 5 ) . T h e tricyclic a n t i d e p r e s s a n t s have peripheral anticholinergic e f f e c t ( D o m e n j o z and Theobald, 1 9 5 9 ; T h e o b a l d et al., 1 9 6 4 ; Cairncross, 1 9 6 5 ; V o n R i b b e n t r o p and S c h a u m a n n , 1965; V o n H o f f m e i s t e r , 1969). Desipramine was f o u n d t o be t h e w e a k e s t anticholinergic a m o n g t h e i m i p r a m i n e derivatives and F G 4 9 6 3 had a b o u t t h e same activity. B o t h N A p o t e n t i a t i o n and anticholinergic e f f e c t result in an increased s y m p a t h e t i c activity. F G 4 9 6 3 has n o p e r i p h e r a l N A - p o t e n t i a t i n g e f f e c t and a r a t h e r w e a k p e r i p h e r a l anticholinergic effect. T h e r e f o r e , F G 4 9 6 3 p r e s u m a b l y p r o d u c e s m u c h less s y m p a t h e t i c d o m i n a n c e t h a n d o t h e tricyclic antidepressants. This m a y o f f e r advantages in cardiac diseases with symp a t h e t i c h y p e r f u n c t i o n or increased b l o o d conc e n t r a t i o n o f c a t e c h o l a m i n e s ( M c D o n a l d et al., 1 9 6 9 ; J e w i t t et al., 1 9 6 9 ; Siggers et al., 1 9 7 1 ) , which m a y be aggravated b y tricyclic antidepressants {Leon and Abrams, 1 9 7 1 ) . A c u t e i n t o x i c a t i o n s with tricyclic t h y m o leptics in p a t i e n t s ( F r e e m a n et al., 1969; Fournier, 1 9 7 3 ) and in e x p e r i m e n t a l animals (Bois-
J. BUUS LASSEN ET AL. sier et al., 1965; Marno, 1 9 7 0 ) have been r e p o r t e d to i n d u c e several t y p e s o f h e a r t blocks. The a c u t e c a r d i o t o x i c i t y o f FG 4 9 6 3 was f o u n d t o be less t h a n t h a t o f i m i p r a m i n e and a m i t r i p t y l i n e . In dogs, FG 4 9 6 3 even s h o w e d a p r o l o n g e d positive i n o t r o p i c effect, which m a y l o w e r the risk for negative i n o t r o p y .
Acknowledgement The authors wish to thank Dr. Richard F. Squires for helpful suggestions. References .~sberg, M., L. Bertilsson, D. Tuck, B. Cronholm and F. Sjhqvist, 1973, Indoleamine metabolites in the cerebrospinal fluid of depressed patients before and during treatment with nortriptyline, Clin. Pharmacol. Therap. 14, 277. Axelrod, J., L.G. Whitby and G. Hefting, 1961, Effect of psychotropic drugs on the uptake of 3H. norepinephrine by tissues, Science 133, 383. Boissier, J.-R., P. Simon and S. Witchitz, 1965, Etude chez le cobaye de la toxicit~ cardiaque de l'imipramine, de l'amitriptyline et de leurs d~riv6s monodesm6thyl~s, Th~rapie 20, 67. Boston Collaborative drug surveillance program, 1972. Adverse reactions to the tricyclic-antidepressant drug. A report, Lancet I, 529. Buus Lassen, J., 1972, Behavioural effect of tricyclic thymoleptics and chlorpheniramine in mice after pretreatment with 5 hydroxytryptophan (5HTP), Acta Pharmacol. (Kbh.) 31, Suppl. I, 11. Buus Lassen, J., 1974, The effect of p-chloroamphetamine on motility in rats after inhibition of monoamine synthesis, storage, uptake and receptor interaction, Psychopharmacologia (Berlin) 34, 243. Buus Lassen, J., R.F. Squires, J.A. Christensen and L. Molander, 1975, Neurochemical and pharmacological studies on a new 5HT-uptake inhibitor, FG 4963, with potential antidepressant properties, Psychopharmacologia (in press). Cairncross, K.D., 1965, On the peripheral pharmacology of amitriptyline, Arch. Intern. Pharmacodyn. 154, 438. Carlsson, A., H. Corrodi, K. Fuxe and T. Hhkfelt, 1969a, Effect of antidepressant drugs on the depletion of intraneuronal brain 5-hydroxytryptamine stores caused by 4-methyl-c~-ethyl-metatyramine, European J. Pharmacol. 5, 357. Carlsson, A., H. Corrodi, K. Fuxe and T. H6kfelt, 1969b, Effect of some antidepressant drugs on the depletion of intraneuronal brain catecholamine stores caused by 4,(x-dimethyl-meta-tyramine, European J. Pharmacol. 5, 367.
115 Carlsson, A., J.J. Jonason, M. Lindqvist and K. Fuxe, 1969c, Demonstration of extraneuronal 5-hydroxytryptamine accumulation in brain following membrane-pump blockade by chlorimipramine, Brain Res. 12,456. Carlsson, A., 1970, Structural specificity for inhibition and ( 14 C)-5-hydroxytryptamine uptake by cerebral slices, J. Pharm. Pharmacot. 22, 729. Domenjoz, R. and W. Theobald, 1959, Zur Pharmakologie des Trofranil (R) (N.(3
