European Journal o f Pharmacology, 35 (1976) 35--43 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
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COMPARATIVE ACTION OF FENFLURAMINE ON THE UPTAKE AND RELEASE OF SEROTONIN AND DOPAMINE MARIE-HI~LI~NE KANNENGIESSER, PETER F. HUNT and JEAN-PIERRE RA Y N A U D
Centre de Recherches Roussel-Uclaf, 93230 Romainville, France Received 24 September 1974, revised MS received 14 March 1975, accepted 12 August 1975 6
M.-H. KANNENGIESSER, P.F. HUNT and J.-P. RAYNAUD, Comparative action o f fenfluramine on the uptake and release ofserotonin and dopamine, European J. Pharmacol. 35 (1976) 35--43. The anorectic agent, fenfluramine, proves to be a good inhibitor of serotonin uptake in vitro, in synaptosomes from rat whole brain (ICs0 = 8.5 ± 0.6 x 10 -7 M). After administration in vivo, its inhibitory activity in vitro equals that of chlorimipramine and in contrast to the latter, its effect is of long duration. Fenfluramine is also effective in promoting the release of serotonin from pre-loaded synaptosomes. In comparison, the structurally related compound, amphetamine, has little activity with respect to these serotonin mechanisms. It is, however, active both in inhibiting the uptake of dopamine and in promoting its release, whereas fenfluramine is inactive. The implication of these mechanisms in the serotonin-depleting capacity as well as in the anorectic activity of fenfluramine is discussed. Fenfluramine
Synaptosomes
Amphetamine
1. Introduction Compared with amphetamine, the structurally related anorectic agent, fenfluramine, has a number of distinct pharmacological (Boissier et al., 1965; Le Douarec and Neveu, 1970) and clinical features (Woodward, 1970). Thus it is sedative and exhibits neither the marked locomotor stimulating properties nor the sideeffects characteristic of the former. While noradrenaline (NA) (Weissman et al., 1966; Booth, 1968) and more recently dopamine (DA) (Fibiger et al., 1973; Groppetti, 1973; Kruk, 1973; Baez, 1974) have been implicated as mediators of the anorectic effect of amphetamine, most pharmacological evidence suggests that serotonin (hilT) (Jespersen and Scheel-Kr/iger, 1970 and 1973; Funderburk et al., 1971; Samanin et al., 1972; Clineschmidt, 1973; Ghezzi et al., 1973) may play a
Serotonin
Anorectic
Dopamine
more important role in the action of fenfiuramine. Biochemically, fenfluramine causes a significant and long-lasting depletion of brain 5HT and 5-hydroxy-indole acetic acid (hHIAA) (Duhault and Verdavainne, 1967; Opitz, 1967), which has led to the suggestion that the compound may release 5HT from nerve-endings. More recent studies in vitro and in vivo would seem to support this idea in that fenfluramine has been reported to inhibit neither 5HT synthesis (Le Douarec and Neveu, 1970} nor its re-uptake (Morgan et al., 1972), but to cause an increase in 5HT turnover (Costa et al., 1971). We have further investigated the biochemical mechanism of action of fenfluramine by comparing its effects with those of amphetamine, on the uptake as well as the release of 5HT and DA, in synaptosome preparations.
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2. Materials and methods
2.1. General All reagents used were of analytical grade. DL-fenfluramine was kindly supplied by Laboratoires Servier. Amphetamine refers to DLamphetamine base. ~ 4 C-serotonin (5-hydroxy-3-indolyl (ethyl-2-amino-114 C) creatinine sulphate) was prepared at Roussel--Uclaf. Its melting point and UV-spectrum were identical to those of the authentic c o m p o u n d and on thin-layer chromatography both c o m p o u n d s migrated together as a single peak in two different solvent systems (n-butanol--acetic acid--water, 65 : 13 : 22; methylene chloride-acetone--acetic acid--water, 40 : 40 : 20 : 5). Specific activity was 53.3 mCi/mmole calculated by weight or by UV absorption, thus confirming purity. [ Ring (G)]- [ 3H] -dopamine (500 mCi/mmole) was purchased from the Radiochemical Centre (Amersham, Bucks, England) and was used without further purification. Radioactivity measurements were made by liquid scintillation. Immature, female rats {19--21 days), Sprague--Dawley strain from Charles River, were used for all experiments.
2. 2. Up take experiments 2.2.1. Uptake o f 14C-5HTand 3H-DA into non-purified synaptosomes from whole brain or corpus striatum Non-purified synaptosomes were prepared according to the method of Whittaker (1969) and the preparation was checked by electron microscopy. As previously described for 5HT uptake (Kannengiesser et al., 1973), one whole rat brain was homogenised in 20 ml 0.32 M sucrose. For DA uptake, striata from two rats were homogenised in 6 ml 0.32 M sucrose. Supernatants from a 900 g debris centrifugation were diluted 5 times with 40 mM Na phosphate buffer pH 7.0 containing (mM) KC1 3, NaC1 100, D-glucose 11 and in the case of DA, 0.2 mg/ml Na ascorbate. Aliquots (3 ml for whole brain, 1.5 ml for striata) were incubated at 37°C for 5 min with 14C.5H T (10-7 M) or
M.-H. K A N N E N G I E S S E R ET AL.
3 H-DA (5 • 10-8 M) in the presence or absence of inhibitors. The incubation was terminated by cooling in ice. Aliquots (2 × 100 pl) were removed for radioactivity measurement and the remainder was centrifuged (Ecco, type E2/12 centrifuge) at 7000 g for 20 min at 4°C (Hunt et al., 1974). The pellets (P~) were rinsed twice with incubation buffer and extracted with 0.4 N HClO4. After removal of precipitated proteins by centrifugation, the radioactivity in the extract was measured. In the case of iproniazid-treated rats, the c o m p o u n d (200 mg/kg) was administered i.p., as an aqueous solution of the phosphate, 18 hr beforehand. ICs 0 values were determined from logarithmic probability plots of inhibitor concentration against percentage inhibition of 14C5HT or 3H-DA active uptake. In all experiments non-specific binding of ~4C-5HT or 3 H-DA was estimated by incubation with the synaptosome suspension at 0 ° C which prevents active uptake. Non-specific binding amounts to approximately 35% of the total in the case of 5HT and 20% in the case of DA.
2.2.2. Uptake after in vivo administration of compounds Drugs were administered by i.p. injection of a 0.02 N HC1 solution (0.5 ml/100 g) at various periods of time before sacrifice. Controls received carrier only. Uptake experiments were carried o u t in vitro as above using pooled whole brains for 5HT and striata for DA, from 2 rats. 2.2. 3. Assay of labelled amines Following incubation with fenfluramine or amphetamine at 10 -s M, 14C.5H T and 3H-DA were assayed in the rinsed P2 pellets, after extraction with ethanol (2 ml) containing carrier 5HT or DA (40 pg/ml) and subsequent thin-layer chromatography on silica gel (Merck F 240) in an acetic acid : n-butanol : water (3 : 12 : 5) solvent system. Plates were scraped and silica gel fractions were counted directly. Labelled amine in the corresponding supernatants ($2) was separated from acid metabolites by the method of Colburn and Kopin
ACTION OF F E N F L U R A M I N E ON MONOAMINES
37
(1972). Dowex resin (50 W × 8; 200--400 mesh, Na ÷ form) (50 mg dry weight) was added to 0.5 ml of supernatant. After brief mixing, the resin was removed by centrifugation and the radioactivity left in solution, which corresponds to acid metabolites, was measured.
lease were, similar to those described for uptake, that is, 5 min at 37°C.
2.3. Release experiments 2.3.1. Loading of synaptosomes with labelled amine The striata ( ~ 5 0 0 mg) from 5 rats were homogenised in 6 ml 0.32 M sucrose. The supernatants from the 900 g debris centrifugation were diluted 5 times with the same buffer as above (2.2.1). The suspension was incubated with 14C_5H T (10-7 M) or 3H-DA ( 5 . 1 0 -8 M) for 10 min at 37°C. After centrifugation as before, the pellet (P2) loaded with labelled amine was washed by resuspension in the same cold phosphate buffer containing unlabelled 5HT (2 × 10 -8 M) or DA (10 -8 M) and re-centrifuged. 2.3.2. Release of la belled am ine To measure release of amine from the pellet, the latter was resuspended, alone or with various concentrations of drug, in the same phosphate buffer (1.5 ml/50 mg wet striatal tissue) containing 0.75 mM CaC12. Aliquots (2 × 100 pl) were removed for radioactivity measurement. The remainder of the suspension in aliquots of 1.5 ml was incubated at 37°C for 5 min (DA) or for 20 min (5HT) and centrifuged at 7000 g at 4°C for 20 min. Total radioactivity was measured in the pellet by extraction with 0.4 N HClO4 (0.3 ml) and in the supernatant. The striatum is used for measuring 5HT release, as well as for DA release, since it is a region relatively rich in serotoninergic nerve endings, where the releasing effect is more clearly seen than with whole brain. In the case of 5HT only, a release experiment was carried o u t exactly as above, in the presence of fenfluramine (10 -s M), b u t the P2 pellets used were prepared as described for 5HT uptake and the incubation conditions for re-
3. Results 3.1. Inhibition of 14C.5HTuptak e 3.1.1. In vitro Fenfluramine was tested in vitro as an inhibitor of ' 4C-5HT uptake in a non-purified synaptosome suspension from whole brain. The IC50, the concentration required for 50% inhibition, is compared to that of amphetamine and of the tricyclic antidepressant, chlorimipramine which is a strong inhibitor of 5HT uptake. As shown in table 1, fenfluramine proved to be less active than chlorimipramine b u t is more active than amphetamine by at least an order of magnitude. Pretreatment of the rats with a monoamine oxidase inhibitor (MAOI), iproniazid, did not appreciably modify the ICs 0 values. 3.1.2. In vivo--in vitro At 1 hr after in vivo administration of the drugs (15 mg/kg i.p.), fenfluramine was as effective as chlorimipramine in inhibiting the in vitro uptake of 14C.5H T (table 1). Under the same conditions, amphetamine at 10 mg/kg was significantly less active than fenfluramine at the same dose. At 4 hr, whereas the action of chlorimipramine was considerably reduced (Ross et al., 1972), that of fenfluramine was still high and remained high even at 18 hr. 3.2. Inhibition o f 3H-DA uptake 3.2.1. In vitro As shown in table 2 fenfluramine, at concentrations up to 10 -s M, had no inhibitory effect on the uptake of 3H-DA in a non-purified striatal synaptosome preparation. Amphetamine and benztropine are both strong inhibitots o f this process, the latter being 3--4 times more active than the former (Hunt et al., 1974).
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M.-H. K A N N E N G I E S S E R ET AL.
TABLE 1 I n h i b i t i o n o f 14 C-5HT uptake. In vitro. 14C-5HT (10 -7 M) w i t h or w i t h o u t i n h i b i t o r was i n c u b a t e d at 37°C for 5 min with t h e s y n a p t o s o m e p r e p a r a t i o n f r o m n o n - t r e a t e d rats or rats p r e t r e a t e d w i t h Iproniazid (200 m g / k g i.p.) 18 hr b e f o r e h a n d . I n h i b i t o r s were used at 3 c o n c e n t r a t i o n s b e t w e e n 10 -s and 10 -4 M. ICs 0 values (+S.E.M.) were d e t e r m i n e d f r o m logarithmic p r o b a b i l i t y plots o f i n h i b i t o r c o n c e n t r a t i o n against p e r c e n t a g e i n h i b i t i o n o f 14 C-5HT u p t a k e after s u b t r a c t i o n of ' n o n - s p e c i f i c ' i n c o r p o r a t i o n , e s t i m a t e d by an identical i n c u b a t i o n o f 14C_5H T at 0°C. F o r t h e controls, total uptake o f 14C.5H T at 37°C was 0.130 + 0.025 p m o l e / m g t i s s u e / m i n and at 0°C was 0.046 -+ 0.004 p m o l e / m g tissue/rain. In vivo - - in vitro. Drugs were a d m i n i s t e r e d i.p. in 0.02 N HCl. I n c u b a t i o n o f 14C.5H T w i t h s y n a p t o s o m e s f r o m d r u g - t r e a t e d or carrier-treated rats were carried o u t as above. The values ( - + S . E . M . ) r e p r e s e n t t h e average o f at least 3 s e p a r a t e d e t e r m i n a t i o n s . In vivo - - in vitro
In vitro IC50 (M) Non t r e a t e d
Pretreated with MAOI
Dose sacrifice
% Inhibition
(mg/kg)
(hr)
Fenfluramine
8.5 ± 0.6 x 10 -7
5.5 ± 0.6 x 10 -7
5 10 15 15 15
1 1 1 4 18
35.5 50.8 59.1 53.5 49.0
± ± ± ± ±
1.2 3.6** 1.9 1.4 1.7
Amphetamine
2.3 ± 0.3 x 10 -s
3.0 ± 0.8 X 10 -s
10
1
29.9 ± 1.7
Chlorimipramine
4.1 + 0.4 X 10 -s
4.2 ± 0.6 X 10 -s
15 15
1 4
64.9 ± 1.9 34.1 ± 4.5
** Significantly d i f f e r e n t (at least p < 0.01) f r o m c o r r e s p o n d i n g value for a m p h e t a m i n e . S t u d e n t ' s t-test.
TABLE 2 I n h i b i t i o n o f a H-DA uptake. In vitro. 3H-DA (5 x 10 -8 M) w i t h or w i t h o u t i n h i b i t o r was i n c u b a t e d at 37°C for 5 m i n w i t h t h e striatal syna p t o s o m e p r e p a r a t i o n . I n h i b i t o r s were at 10 -7, 10 -6 or 10 -s M. ICs 0 values were d e t e r m i n e d as for 54 C-5HT. F o r the c o n t r o l s , u p t a k e at 37°C was 0.360 + 0.09 p m o l e / m g t i s s u e / m i n and at 4°C was 0.068 + 0.008 p m o l e / m g t i s s u e / m i n . In vivo - - in vitro. Drugs dissolved in 0.02 N HCl were a d m i n i s t e r e d i.p. and sacrifice was at 30 min. I n c u b a t i o n o f 3 H-DA w i t h striatal s y n a p t o s o m e s f r o m drug-treated or carrier-treated rats was as above. In vitro ICs 0 (M)
In vivo - - in vitro Dose (mg/kg)
% Inhibition
Fenfluramine
Inactive at 10 -s M
15
0
Amphetamine
8.8 ± 1.5 x 10 -7
2 5 10 20
52.8 63.5 72.0 77.0
Benztropine
2.6 ± 0.2 X 10 -7
5 20 50
0 45.3 ± 7.1 83.0 ± 11.4
± ± ± ±
5.5 5.5 4.7 5.7
A C T I O N O F F E N F L U R A M I N E ON M O N O A M I N E S
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TABLE 3 M e t a b o l i s m o f labelled amines. Labelled 5HT and DA were e x t r a c t e d f r o m P2 pellets and f r o m the c o r r e s p o n d i n g s u p e r n a t a n t s ($2) following inc u b a t i o n w i t h o u t drug or with f e n f l u r a m i n e or a m p h e t a m i n e at 10 -s M. Values are e x p r e s s e d as the p e r c e n t a g e o f total radioactivity in each fraction. In brackets: values o b t a i n e d after p r e t r e a t m e n t o f rats with iproniazid (200 mg/kg, i.p.) 18 hr b e f o r e h a n d . % A m i n e remaining
14 C-5HT
3 H-DA
P2
$2
P2
$2
Control
53.4 + 1.7 (86.3 ± 1.7)
89.8 ± 0.9 (97.5 -+ 0.2)
55.8 ÷ 2.8
64.4 + 1.5
Fenfluramine 10 -s M
49.4 ± 5.1 (91.5 ± 1.3)
91.7 ± 0.5 (97.2 ± 0.1)
58.2 ± 5.3
64.5 ± 1.0
Amphetamine 10 -s M
67.4 ± 2.2* (87.8 ± 0.5)
92.8 ± 0.2* (97.6 ± 0.3)
44.4 ± 4.1
69.1 ± 1.4
* Significantly d i f f e r e n t (at least p < 0.05) f r o m c o r r e s p o n d i n g c o n t r o l value. S t u d e n t ' s t-test.
3.2.2. In vivo - - in vitro
Administered in vivo at a dose of 15 mg/kg (i.p.), fenfluramine was inactive in inhibiting the in vitro uptake of 3H-DA measured 30 min after the injection. Amphetamine, on the other hand, even at the lowest dose of 2 mg/kg, already showed appreciable inhibition (table 2). Benztropine was markedly less active, and a dose of more than 20 mg/kg was required to approach the activity of amphetamine at 2 mg/kg. 3.3. M e t a b o l i s m o f labelled a m i n e s
To determine the extent of metabolism of 4 C-5HT and 3H-DA following incubation with a high concentration of fenfluramine or amphetamine, the radioactive amines were extracted from the P2 pellets and the corresponding supernatants ($2). The a m o u n t of unchanged amine remaining after incubation with drug is compared to that in an identical incubation w i t h o u t drug (table 3). Fenfluramine modified neither the a m o u n t of ~4C.5HT recovered nor the a m o u n t of 3H-DA, with respect to control values. In the case of amphetamine, a significant difference from control values was seen only for ~4 C-5HT.
Following pretreatment of rats with iproniazid, more than 85% of unchanged 14C.5H T was found in the P2 fraction and more than 97% in the $2 fraction. 3.4. R e l e a s e o f ~ 4 C - 5 H T
At concentrations of 10 -s M both fenfluramine and amphetamine released radioactivity* from striatal synaptosome preparations preloaded with 14C.5H T (table 4). At 10 -6 M only fenfluramine had an effect. To show that the processes of uptake inhibition and release can be distinguished, a release experiment using 14C-5HT was carried out under 'uptake conditions', in the presence of 10 -s M fenfluramine and using P2 pellets pre-
* Total released radioactivity is taken as the m e a s u r e of amine release since the same relative o r d e r o f activity was o b t a i n e d w h e n released a m i n e was sep a r a t e d f r o m its acid m e t a b o l i t e s o n D o w e x resin as described in M e t h o d s ( S e c t i o n 2.2.3.). F o r m a t i o n o f acid m e t a b o l i t e s in the p r e s e n c e o f f e n f l u r a m i n e or a m p h e t a m i n e at 10 -s M was n o t significantly d i f f e r e n t f r o m the c o n t r o l w i t h o u t drug and repres e n t e d , for 5 HT, 30--40% o f the total radioactivity in the s u p e r n a t a n t and for DA, 10--20%.
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M.-H. KANNENGIESSER ET AL.
TABLE 4 Release of 14 C-5HT and 3 H-DA from synaptosomes. Synaptosomes from corpus striatum, pre-loaded with 14C.5HT or 3 H-DA were resuspended and incubated at 37°C for 20 min or 5 min respectively with drugs as shown. Release is expressed as the percentage of radioactivity lost with respect to an identical incubation without drug and is measured both in the pellet and in the supernatant. In controls, the radioactivity in washed P2 pellets was 22% of the total incubated for 5HT and 28% for DA. Of this activity, spontaneous release amounted to approximately 66% and 40% respectively. Results represent the average (-+ S.E.M.) of pellet and supernatant values from at least 2 experiments. Concentration (M) Fenfluramine
10-4 10-s 10 -6
Amphetamine
10-7 10-4 10-s 1 0 -6
10-7
% Release 5HT 40.6 ± 3.6 31.8 ± 2.6
32.2 ± i.I 6.2 ± 2.6
DA 22.0 5.0 2.0 0 42.8 38.1 21.8 2.7
± 2.4 ± 1.8 ± 1.3 ± 3.5 +-2.2 t 1.2 +- 1.5
p a r e d as d e s c r i b e d f o r 5 H T u p t a k e . N o differe n c e in r a d i o a c t i v i t y release was d e t e c t e d , c o m p a r e d w i t h an identical c o n t r o l w i t h o u t drug. 3.5. Release o f 3H-DA T h e o r d e r o f activity was reversed w h e n striatal s y n a p t o s o m e s p r e - l o a d e d w i t h 3H-DA were used. F e n f l u r a m i n e o n l y s h o w e d a releasing e f f e c t at a v e r y high c o n c e n t r a t i o n ( 1 0 -4 M), w h e r e a s a m p h e t a m i n e was active d o w n t o 10 -6 M (table 4). 4. Discussion I t has b e e n s h o w n t h a t f e n f l u r a m i n e , unlike a m p h e t a m i n e , inhibits 5 H T u p t a k e in a syna p t o s o m a l p r e p a r a t i o n f r o m w h o l e brain. A d m i n i s t e r e d in vivo, f e n f l u r a m i n e is as e f f e c t i v e in inhibiting in v i t r o 5 H T u p t a k e as c h l o r i m i p r a m i n e at the s a m e dose, b u t unlike
c h l o r i m i p r a m i n e , the e f f e c t o f f e n f l u r a m i n e is long-lasting and a p p r e c i a b l e i n h i b i t i o n is still o b s e r v e d a f t e r 18 hr. This result is in c o n t r a s t t o t h a t o f M o r g a n et al. ( 1 9 7 2 ) w h o f o u n d fenf l u r a m i n e inactive in r a t brain slices f r o m h y p o t h a l a m u s , brain s t e m or s t r i a t u m 4 hr a f t e r a d m i n i s t r a t i o n o f a c o m p a r a b l e dose. T h e diff e r e n c e is p r o b a b l y d u e t o the higher c o n c e n t r a t i o n o f 5 H T (10 -6 M) used b y these a u t h o r s , which w o u l d t e n d to m a s k a specific i n h i b i t o r y effect. With r e s p e c t to D A nerve-endings fenfiuram i n e is t o t a l l y inactive, while on the o t h e r h a n d a m p h e t a m i n e is a g o o d i n h i b i t o r o f D A u p t a k e b o t h in v i t r o and in vivo - - in vitro. B o t h f e n f l u r a m i n e and a m p h e t a m i n e a p p e a r to be m o r e e f f e c t i v e u p t a k e i n h i b i t o r s a f t e r in vivo a d m i n i s t r a t i o n , p r o b a b l y o w i n g to their e f f i c i e n t passage and r e t e n t i o n in the brain. Similar o b s e r v a t i o n s have b e e n m a d e for chlorp h e n t e r m i n e ( R o s s et al., 1 9 7 2 ) and 4-chloroa m p h e t a m i n e (Wong et al., 1973) a n d like f e n f l u r a m i n e , these t w o drugs also h a v e a relatively long-lasting effect. T h a t the o b s e r v e d d i f f e r e n c e s in in v i t r o inhibition of 5HT and DA uptake caused by f e n f l u r a m i n e and a m p h e t a m i n e are n o t d u e t o d i f f e r e n c e s in t r a n s m i t t e r m e t a b o l i s m i n d u c e d b y the drugs themselves, is d e m o n s t r a t e d b y e x t r a c t i o n o f the r a d i o a c t i v e a m i n e s f r o m b o t h the P2 and $2 f r a c t i o n s a f t e r i n c u b a t i o n w i t h high c o n c e n t r a t i o n s o f the drugs. A l t h o u g h m e t a b o l i s m was q u i t e extensive, p a r t i c u l a r l y in the P2 f r a c t i o n , a significant d i f f e r e n c e f r o m c o n t r o l values was seen o n l y for 5 H T a f t e r i n c u b a t i o n with a m p h e t a m i n e . In this case, p r e t r e a t m e n t with iproniazid, which r e d u c e s m e t a b o l i s m to a u n i f o r m l y l o w level, did n o t m o d i f y the ICs 0 values, e x c e p t for fenfluram i n e , the ICs 0 o f w h i c h was slightly lower. As p r e v i o u s l y discussed b y K a n n e n g i e s s e r et al. ( 1 9 7 3 ) and b y C o s t a {1973), this result m a y indicate m o r e o v e r t h a t f e n f l u r a m i n e , unlike reserpine, d o e s n o t inhibit u p t a k e at the vesicle level, since, in the case of reserpine, i m p a i r e d u p t a k e is ' n o r m a l i s e d ' b y M A O I p r e t r e a t m e n t , w h i c h w o u l d t h e r e f o r e lead to a higher ICs 0 value.
A C T I O N O F F E N F L U R A M I N E ON M O N O A M I N E S
Using synaptosomes pre-loaded with 14C5HT or 3H-DA in vitro, it is shown that fenfluramine is more effective than amphetamine in releasing 5HT, but unlike amphetamine has no effect on DA release except at very high concentration. In the synaptosome system used, it is possible that such a transmitter-releasing process could be misinterpreted as an apparent inhibition of uptake. As far as amphetamine is concerned, Ferris et al. (1972) have demonstrated that, under the conditions used for measuring inhibition of DA uptake, release of DA is dose-dependent, but weak. For fenfluramine, it has been confirmed that under 'uptake conditions' no detectable release of ' 4C-5HT occurs, thus showing that uptake inhibition and release can be differentiated by the m e t h o d used. These experiments support the original proposition that the in vivo depletion of 5HT by fenfluramine may be caused by 5HT release. However, we now further propose that fenfluramine's effective and long-lasting inhibition of 5HT uptake could play at least an equally important role, whereby 5HT, released from the nerve-ending either physiologically or by fenfluramine itself, is prevented from being re-captured and re-used. Considerable indirect evidence implicates 5HT in the anorectic action of fenfluramine. Thus the activity o f fenfluramine is reduced by lesion of 5HT neurons (Samanin et al., 1972; Clineschmidt, 1973), by blockers of 5HT receptors (Jesperson and Scheel-Krfiger, 1970; Funderburk et al., 1971) and by an inhibitor of 5HT uptake (Ghezzi et al., 1973; Jesperson and Scheel-Krfiger, 1973). The 5HT depletion caused by fenfluramine does not appear to be correlated with its anorectic action. While fenfluramine-induced 5HT depletion only reaches a maximum 4 hr after a single dose and remains low for at least the following 4 hr (Ghezzi et al., 1973), the anorectic effect begins to decline 2--4 hr after administration (Schmitt, 1973). It is possible, however, that stimulatory activity induced only after chronic fenfluramine treatment (Taylor et al., 1973), may be associated
41
with persistent 5HT depletion. The relative importance of 5HT release and uptake inhibition in the anorectic action of fenfluramine remains to be established*. Uptake inhibition is of long-duration compared to the anorectic effect, which would suggest that release is a more likely candidate. However, it is to be noted that a c o m p o u n d such as chlorphentermine which inhibits both 5HT and NA uptake (Ross et al., 1972) but which does not deplete 5HT, suggesting the absence of releasing properties, is nevertheless anorectic (MOller Nielsen and Dubnick, 1970). Ghezzi et al. (1973) have shown that the anorectic effect of fenfluramine can be blocked by chlorimipramine, an inhibitor of 5HT uptake. They suggest, moreover, that fenfluramine may be accumulated within 5HT neurons by means of the amine transport process. Closely analogous results have been reported for the related c o m p o u n d 4-chloroamphetamine, the stimulatory activity of which can be similarly blocked (Lassen, 1974). 4-Chloroamphetamine is actively accumulated by synaptosomes and is a competitive inhibitor of 5HT uptake (Wong et al., 1973). If the same is true for fenfluramine, then a possible role for the uptake process in mediating anorectic activity would be simply to concentrate the c o m p o u n d in 5HT neurons where its releasing effect can come into play. As previously mentioned, 4-chloroamphetamine and fenfluramine show very similar behaviour with respect to 5HT release and uptake inhibition, and in the case of the former this seems to be associated with its stimulatory properties since these can be attenuated by chlorimipramine. Fenfluramine on the other hand is primarily sedative, so this property
* A l t h o u g h an a n o r e c t i c e f f e c t is o n l y a p p a r e n t in a d u l t rats, t h e relative b i o c h e m i c a l effects o f fenf l u r a m i n e a n d a m p h e t a m i n e , d e s c r i b e d here, are identical regardless of w h e t h e r b r a i n s f r o m i m m a t u r e ( 1 9 - - 2 1 d a y s ) or a d u l t rats ( 1 5 0 - - 2 0 0 g) are used as a s o u r c e of s y n a p t o s o m e s ( K a n n e n g i e s s e r , Hunt, unpublished).
42 w o u l d n o t a p p e a r to be related to these 5 H T m e c h a n i s m s . Instead, it was first a t t r i b u t e d to a reserpine-like a c t i o n in N A n e u r o n s (Ziance and Rutledge, 1 9 7 2 ) and indeed at low doses, fenfluramine, like reserpine, increases N A turnover ( G r o p p e t t i et al., 1972). H o w e v e r , D A t u r n o v e r is also increased (Costa et al., 1 9 7 1 ) and r e c e n t l y a neuroleptic-like a c t i o n has been d e m o n s t r a t e d b y Jori et al. ( 1 9 7 4 ) as well as by G r a b o w s k a and Michaluk (1974). Compared to amphetamine, fenfluramine has no e f f e c t o n D A u p t a k e and only a slight e f f e c t on D A release. Considering the implication o f D A in the increased m o t o r activity, the p s y c h o t i c activity and the d e p e n d e n c e i n d u c e d by the f o r m e r , it is possible t h a t the lack o f these effects, to w h i c h f e n f l u r a m i n e owes its low incidence o f serious clinical side-effects, m a y be d u e to its lack o f activity on these D A mechanisms.
Acknowledgement The technical assistance of Mme D. MassardierCh~ze and of Mr. J.C. Massardier is gratefully acknowledged.
References Baez, L.A., 1974, Role of catecholamines in the anorectic effects of amphetamine in rats, Psychopharmacol. (Berl.) 35, 91. Boissier, J.R., P. Simon, J. Fichelle and F. Hervouet, 1965, Action psychoanaleptique de quelques anorexig~nes d~riv~s de la ph4nyl~thylamine, Th~rapie 20, 297. Booth, D.A., 1968, Amphetamine anorexia by direct action on the adrenergic feeding system of rat hypothalamus, Nature 217,869. Clineschmidt, B.V., 1973, 5,6-dihydroxytryptamine: suppression of the anorexigenic action of fenfluramine, European J. Pharmacol. 24, 405. Colburn, R.W. and I°J. Kopin, 1972, Effects of reserpine and tyramine on release of norepinephrine from synaptosomes, Biochem. Pharmacol. 21,733. Costa, E., 1973, Principles of neurochemistry and drug screening procedures in neuropsychopharmacology, Pure Appl. Chem. 35,439. Costa, E., A. Groppetti and A. Revuelta, 1971, Ac-
M.-H. KANNENGIESSER ET AL. tion of fenfluramine on monoamine stores of rat tissues, Brit. J. Pharmacol. 41, 57. Duhault, J. and C. Verdavainne, 1967, Modification du taux de s~rotonine c~r~brale chez le rat par les trifluorom~thylph~nyl-2-~thyl-aminopropane (fenfluramine 768 S), Arch. Intern. Pharmacodyn. 170, 276. Ferris, R.M., F.L.M. Tang and R.A. Maxwell, 1972, A comparison of the capacities of isomers of amphetamine, deoxypipradol and methylphenidate to inhibit the uptake of tritiated catecholamines into rat cerebral cortex slices, synaptosomal preparations of rat cerebral cortex, hypothalamus and striatum and into adrenergic nerves of rabbit aorta, J. Pharmacol. Exp. Ther. 181,407. Fibiger, H.C., A.P. Zis and E.G. McGeer, 1973, Feeding and drinking deficits after 6-hydroxy-dopamine administration in the rat: similarities to the lateral hypothalamic syndrome, Brain Res. 55, 135. Funderburk, W.H., J.C. Hazelwood, R.T. Ruckart and J.W. Ward, 1971, Is 5-hydroxytryptamine involved in the mechanism of action of fenfluramine? J. Pharm. Pharmacol. 23,468. Ghezzi, D., R. Samanin, S. Bernasconi, G. Tognoni, M. Gerna and S. Garattini, 1973, Effect of thymoleptics on fenfluramine-induced depletion of brain serotonin in rats, European J. Pharmacol. 24, 205. Grabowska, M. and J. Michaluk, 1974, On the antiapomorphine effect of fenfluramine, J. Pharm. Pharmacol. 26,549. Groppetti, A., 1973, Amphetamines and cocaine on amine turnover, Life Sci. 13 lxii. Groppetti, A., A. Misher, M. Naimzada, A. Revuelta and E. Costa, 1972, Evidence than in rats 1-benzyl~-methoxy-3-trifluoromethylphenethylamine (SK & F 1-39728) dissociates anorexia from central stimulation and actions on brain monoamine stores, J. Pharmacol. Exp. Ther. 182, 464. Hunt, P., M.H. Kannengiesser and J.P. Raynaud, 1974, Nomifensine: a new potent inhibitor of dopamine uptake in synaptosomes from rat brain corpus striatum, J. Pharm. Pharmacol. 26,370. Jespersen, S. and J. Scheel-Kr~iger, 1970, Antagonism by methysergide of the 5-hydroxytryptamine-like action of toxic doses of fenfluramine in dogs, J. Pharm. Pharmacol. 22, 637. Jespersen, S. and J. Scheel-Kr/iger, 1973, Evidence for a difference in mechanism of action between fenfluramine- and amphetamine-induced anorexia, J. Pharm. Pharmacol. 25, 49. Jori, A., G. Cecchetti, D. Ghezzi and R. Samanin, 1974, Biochemical and behavioral antagonism between fenfluramine and apomorphine in rats, European J. Pharmacol. 26, 179. Kannengiesser, M.H., P. Hunt and J.P. Raynaud, 1973, An 'in vitro' model for the study of psycho-
ACTION OF FENFLURAMINE ON MONOAMINES tropic drugs and as a criterion of antidepressant activity, Biochem. Pharmacol. 22, 73. Kruk, Z.L., 1973, Dopamine and 5-hydroxy-tryptamine inhibit feeding in rats, Nature New Biol. 246, 52. Lassen, J.B., 1974, The effect of p-chloroamphetamine on motility in rats after inhibition of monoamine synthesis, storage, uptake and receptor interaction, Psychopharmacol. (Berl.) 34, 243. LeDouarec, J.C. and C. Neveu, 1970, Pharmacology and biochemistry of fenfluramine, in: Amphetamines and Related Compounds; Proceedings of the Mario Negri Institute for Pharmacological Research, Milan, Italy, eds. E. Costa and S. Garattini (Raven Press, New York) p. 75. M¢ller Nielsen, I. and B. Dubnick, 1970, Pharmacology of chlorphentermine, in: Amphetamines and Related Compounds, Proceedings of the Mario Negri Institute for Pharmacological Research, Milan, Italy, eds. E. Costa and S. Garattini (Raven Press, New York) p. 63. Morgan, D., S. Lofstrandh and E. Costa, 1972, Amphetamine analogues and brain amines, Life Sci. II, part I, 83. Opitz, K., 1967, Anorexigene Phenylalkylamine und Serotonin-Stoffwechsel, Arch. Pharmakol. Exp. Pathol. 259, 56. Ross, S.B., A.L. Renyi and S.-O. Ogren, 1972, Inhibition of the uptake of noradrenaline and 5-hydroxy-tryptamine by chlorphentermine and chlorimipramine, European J. Pharmacol. 17, 107. Samanin, R., D. Ghezzi, L. Valzelli and S. Garattini, 1972, The effects of selective lesioning of brain
43 serotonin or catecholamine containing neurons on the anorectic activity of fenfluramine and amphetamine, European J. Pharmacol. 19, 318. Schmitt, H., 1973, Influence d'agents interf~rant avec les cat~cholamines et la 5-hydroxytryptamine sur les effets anorexig~nes de l'amph~tamine et de la fenfluramine, J. Pharmacol. (Paris) 4, 17. Taylor, M., A.J. Goudie and A. Williams, 1973, The effects of chronic fenfluramine administration on behaviour and body weight, Psychopharmacol. (Berl.) 31, 63. Weissman, A., B.K. Koe and S.S. Tenen, 1966, Antiamphetamine effects following inhibition of tyrosine hydroxylase, J. Pharmacol. Exp. Ther. 151, 339. Whittaker, V.P., 1969, In: Handbook of Neurochemistry, ed. A. Lajtha (Plenum Press, London) Vot. 2, p. 327. Wong, D.T., J.S. Horng and R.W. Fuller, 1973, Kinetics of serotonin accumulation into synaptosomes of rat brain: effects of amphetamine and chloroamphetamines, Biochem. Pharmacol. 22, 311. Woodward, E., 1970, Clinical experience with fenfluramine in the United States, in: Amphetamines and Related Compounds; Proceedings of the Mario Negri Institute for Pharmacological Research, Milan, Italy, eds. E. Costa and S. Garattini (Raven Press, New York) p. 685. Ziance, R.J. and C.O. Rutledge, 1972, A comparison of the effects of fenfluramine and amphetamine on uptake, release and catabolism of norepinephrine in rat brain, J. Pharmacol. Exp. Ther. 180, 118.
35
COMPARATIVE ACTION OF FENFLURAMINE ON THE UPTAKE AND RELEASE OF SEROTONIN AND DOPAMINE MARIE-HI~LI~NE KANNENGIESSER, PETER F. HUNT and JEAN-PIERRE RA Y N A U D
Centre de Recherches Roussel-Uclaf, 93230 Romainville, France Received 24 September 1974, revised MS received 14 March 1975, accepted 12 August 1975 6
M.-H. KANNENGIESSER, P.F. HUNT and J.-P. RAYNAUD, Comparative action o f fenfluramine on the uptake and release ofserotonin and dopamine, European J. Pharmacol. 35 (1976) 35--43. The anorectic agent, fenfluramine, proves to be a good inhibitor of serotonin uptake in vitro, in synaptosomes from rat whole brain (ICs0 = 8.5 ± 0.6 x 10 -7 M). After administration in vivo, its inhibitory activity in vitro equals that of chlorimipramine and in contrast to the latter, its effect is of long duration. Fenfluramine is also effective in promoting the release of serotonin from pre-loaded synaptosomes. In comparison, the structurally related compound, amphetamine, has little activity with respect to these serotonin mechanisms. It is, however, active both in inhibiting the uptake of dopamine and in promoting its release, whereas fenfluramine is inactive. The implication of these mechanisms in the serotonin-depleting capacity as well as in the anorectic activity of fenfluramine is discussed. Fenfluramine
Synaptosomes
Amphetamine
1. Introduction Compared with amphetamine, the structurally related anorectic agent, fenfluramine, has a number of distinct pharmacological (Boissier et al., 1965; Le Douarec and Neveu, 1970) and clinical features (Woodward, 1970). Thus it is sedative and exhibits neither the marked locomotor stimulating properties nor the sideeffects characteristic of the former. While noradrenaline (NA) (Weissman et al., 1966; Booth, 1968) and more recently dopamine (DA) (Fibiger et al., 1973; Groppetti, 1973; Kruk, 1973; Baez, 1974) have been implicated as mediators of the anorectic effect of amphetamine, most pharmacological evidence suggests that serotonin (hilT) (Jespersen and Scheel-Kr/iger, 1970 and 1973; Funderburk et al., 1971; Samanin et al., 1972; Clineschmidt, 1973; Ghezzi et al., 1973) may play a
Serotonin
Anorectic
Dopamine
more important role in the action of fenfiuramine. Biochemically, fenfluramine causes a significant and long-lasting depletion of brain 5HT and 5-hydroxy-indole acetic acid (hHIAA) (Duhault and Verdavainne, 1967; Opitz, 1967), which has led to the suggestion that the compound may release 5HT from nerve-endings. More recent studies in vitro and in vivo would seem to support this idea in that fenfluramine has been reported to inhibit neither 5HT synthesis (Le Douarec and Neveu, 1970} nor its re-uptake (Morgan et al., 1972), but to cause an increase in 5HT turnover (Costa et al., 1971). We have further investigated the biochemical mechanism of action of fenfluramine by comparing its effects with those of amphetamine, on the uptake as well as the release of 5HT and DA, in synaptosome preparations.
36
2. Materials and methods
2.1. General All reagents used were of analytical grade. DL-fenfluramine was kindly supplied by Laboratoires Servier. Amphetamine refers to DLamphetamine base. ~ 4 C-serotonin (5-hydroxy-3-indolyl (ethyl-2-amino-114 C) creatinine sulphate) was prepared at Roussel--Uclaf. Its melting point and UV-spectrum were identical to those of the authentic c o m p o u n d and on thin-layer chromatography both c o m p o u n d s migrated together as a single peak in two different solvent systems (n-butanol--acetic acid--water, 65 : 13 : 22; methylene chloride-acetone--acetic acid--water, 40 : 40 : 20 : 5). Specific activity was 53.3 mCi/mmole calculated by weight or by UV absorption, thus confirming purity. [ Ring (G)]- [ 3H] -dopamine (500 mCi/mmole) was purchased from the Radiochemical Centre (Amersham, Bucks, England) and was used without further purification. Radioactivity measurements were made by liquid scintillation. Immature, female rats {19--21 days), Sprague--Dawley strain from Charles River, were used for all experiments.
2. 2. Up take experiments 2.2.1. Uptake o f 14C-5HTand 3H-DA into non-purified synaptosomes from whole brain or corpus striatum Non-purified synaptosomes were prepared according to the method of Whittaker (1969) and the preparation was checked by electron microscopy. As previously described for 5HT uptake (Kannengiesser et al., 1973), one whole rat brain was homogenised in 20 ml 0.32 M sucrose. For DA uptake, striata from two rats were homogenised in 6 ml 0.32 M sucrose. Supernatants from a 900 g debris centrifugation were diluted 5 times with 40 mM Na phosphate buffer pH 7.0 containing (mM) KC1 3, NaC1 100, D-glucose 11 and in the case of DA, 0.2 mg/ml Na ascorbate. Aliquots (3 ml for whole brain, 1.5 ml for striata) were incubated at 37°C for 5 min with 14C.5H T (10-7 M) or
M.-H. K A N N E N G I E S S E R ET AL.
3 H-DA (5 • 10-8 M) in the presence or absence of inhibitors. The incubation was terminated by cooling in ice. Aliquots (2 × 100 pl) were removed for radioactivity measurement and the remainder was centrifuged (Ecco, type E2/12 centrifuge) at 7000 g for 20 min at 4°C (Hunt et al., 1974). The pellets (P~) were rinsed twice with incubation buffer and extracted with 0.4 N HClO4. After removal of precipitated proteins by centrifugation, the radioactivity in the extract was measured. In the case of iproniazid-treated rats, the c o m p o u n d (200 mg/kg) was administered i.p., as an aqueous solution of the phosphate, 18 hr beforehand. ICs 0 values were determined from logarithmic probability plots of inhibitor concentration against percentage inhibition of 14C5HT or 3H-DA active uptake. In all experiments non-specific binding of ~4C-5HT or 3 H-DA was estimated by incubation with the synaptosome suspension at 0 ° C which prevents active uptake. Non-specific binding amounts to approximately 35% of the total in the case of 5HT and 20% in the case of DA.
2.2.2. Uptake after in vivo administration of compounds Drugs were administered by i.p. injection of a 0.02 N HC1 solution (0.5 ml/100 g) at various periods of time before sacrifice. Controls received carrier only. Uptake experiments were carried o u t in vitro as above using pooled whole brains for 5HT and striata for DA, from 2 rats. 2.2. 3. Assay of labelled amines Following incubation with fenfluramine or amphetamine at 10 -s M, 14C.5H T and 3H-DA were assayed in the rinsed P2 pellets, after extraction with ethanol (2 ml) containing carrier 5HT or DA (40 pg/ml) and subsequent thin-layer chromatography on silica gel (Merck F 240) in an acetic acid : n-butanol : water (3 : 12 : 5) solvent system. Plates were scraped and silica gel fractions were counted directly. Labelled amine in the corresponding supernatants ($2) was separated from acid metabolites by the method of Colburn and Kopin
ACTION OF F E N F L U R A M I N E ON MONOAMINES
37
(1972). Dowex resin (50 W × 8; 200--400 mesh, Na ÷ form) (50 mg dry weight) was added to 0.5 ml of supernatant. After brief mixing, the resin was removed by centrifugation and the radioactivity left in solution, which corresponds to acid metabolites, was measured.
lease were, similar to those described for uptake, that is, 5 min at 37°C.
2.3. Release experiments 2.3.1. Loading of synaptosomes with labelled amine The striata ( ~ 5 0 0 mg) from 5 rats were homogenised in 6 ml 0.32 M sucrose. The supernatants from the 900 g debris centrifugation were diluted 5 times with the same buffer as above (2.2.1). The suspension was incubated with 14C_5H T (10-7 M) or 3H-DA ( 5 . 1 0 -8 M) for 10 min at 37°C. After centrifugation as before, the pellet (P2) loaded with labelled amine was washed by resuspension in the same cold phosphate buffer containing unlabelled 5HT (2 × 10 -8 M) or DA (10 -8 M) and re-centrifuged. 2.3.2. Release of la belled am ine To measure release of amine from the pellet, the latter was resuspended, alone or with various concentrations of drug, in the same phosphate buffer (1.5 ml/50 mg wet striatal tissue) containing 0.75 mM CaC12. Aliquots (2 × 100 pl) were removed for radioactivity measurement. The remainder of the suspension in aliquots of 1.5 ml was incubated at 37°C for 5 min (DA) or for 20 min (5HT) and centrifuged at 7000 g at 4°C for 20 min. Total radioactivity was measured in the pellet by extraction with 0.4 N HClO4 (0.3 ml) and in the supernatant. The striatum is used for measuring 5HT release, as well as for DA release, since it is a region relatively rich in serotoninergic nerve endings, where the releasing effect is more clearly seen than with whole brain. In the case of 5HT only, a release experiment was carried o u t exactly as above, in the presence of fenfluramine (10 -s M), b u t the P2 pellets used were prepared as described for 5HT uptake and the incubation conditions for re-
3. Results 3.1. Inhibition of 14C.5HTuptak e 3.1.1. In vitro Fenfluramine was tested in vitro as an inhibitor of ' 4C-5HT uptake in a non-purified synaptosome suspension from whole brain. The IC50, the concentration required for 50% inhibition, is compared to that of amphetamine and of the tricyclic antidepressant, chlorimipramine which is a strong inhibitor of 5HT uptake. As shown in table 1, fenfluramine proved to be less active than chlorimipramine b u t is more active than amphetamine by at least an order of magnitude. Pretreatment of the rats with a monoamine oxidase inhibitor (MAOI), iproniazid, did not appreciably modify the ICs 0 values. 3.1.2. In vivo--in vitro At 1 hr after in vivo administration of the drugs (15 mg/kg i.p.), fenfluramine was as effective as chlorimipramine in inhibiting the in vitro uptake of 14C.5H T (table 1). Under the same conditions, amphetamine at 10 mg/kg was significantly less active than fenfluramine at the same dose. At 4 hr, whereas the action of chlorimipramine was considerably reduced (Ross et al., 1972), that of fenfluramine was still high and remained high even at 18 hr. 3.2. Inhibition o f 3H-DA uptake 3.2.1. In vitro As shown in table 2 fenfluramine, at concentrations up to 10 -s M, had no inhibitory effect on the uptake of 3H-DA in a non-purified striatal synaptosome preparation. Amphetamine and benztropine are both strong inhibitots o f this process, the latter being 3--4 times more active than the former (Hunt et al., 1974).
38
M.-H. K A N N E N G I E S S E R ET AL.
TABLE 1 I n h i b i t i o n o f 14 C-5HT uptake. In vitro. 14C-5HT (10 -7 M) w i t h or w i t h o u t i n h i b i t o r was i n c u b a t e d at 37°C for 5 min with t h e s y n a p t o s o m e p r e p a r a t i o n f r o m n o n - t r e a t e d rats or rats p r e t r e a t e d w i t h Iproniazid (200 m g / k g i.p.) 18 hr b e f o r e h a n d . I n h i b i t o r s were used at 3 c o n c e n t r a t i o n s b e t w e e n 10 -s and 10 -4 M. ICs 0 values (+S.E.M.) were d e t e r m i n e d f r o m logarithmic p r o b a b i l i t y plots o f i n h i b i t o r c o n c e n t r a t i o n against p e r c e n t a g e i n h i b i t i o n o f 14 C-5HT u p t a k e after s u b t r a c t i o n of ' n o n - s p e c i f i c ' i n c o r p o r a t i o n , e s t i m a t e d by an identical i n c u b a t i o n o f 14C_5H T at 0°C. F o r t h e controls, total uptake o f 14C.5H T at 37°C was 0.130 + 0.025 p m o l e / m g t i s s u e / m i n and at 0°C was 0.046 -+ 0.004 p m o l e / m g tissue/rain. In vivo - - in vitro. Drugs were a d m i n i s t e r e d i.p. in 0.02 N HCl. I n c u b a t i o n o f 14C.5H T w i t h s y n a p t o s o m e s f r o m d r u g - t r e a t e d or carrier-treated rats were carried o u t as above. The values ( - + S . E . M . ) r e p r e s e n t t h e average o f at least 3 s e p a r a t e d e t e r m i n a t i o n s . In vivo - - in vitro
In vitro IC50 (M) Non t r e a t e d
Pretreated with MAOI
Dose sacrifice
% Inhibition
(mg/kg)
(hr)
Fenfluramine
8.5 ± 0.6 x 10 -7
5.5 ± 0.6 x 10 -7
5 10 15 15 15
1 1 1 4 18
35.5 50.8 59.1 53.5 49.0
± ± ± ± ±
1.2 3.6** 1.9 1.4 1.7
Amphetamine
2.3 ± 0.3 x 10 -s
3.0 ± 0.8 X 10 -s
10
1
29.9 ± 1.7
Chlorimipramine
4.1 + 0.4 X 10 -s
4.2 ± 0.6 X 10 -s
15 15
1 4
64.9 ± 1.9 34.1 ± 4.5
** Significantly d i f f e r e n t (at least p < 0.01) f r o m c o r r e s p o n d i n g value for a m p h e t a m i n e . S t u d e n t ' s t-test.
TABLE 2 I n h i b i t i o n o f a H-DA uptake. In vitro. 3H-DA (5 x 10 -8 M) w i t h or w i t h o u t i n h i b i t o r was i n c u b a t e d at 37°C for 5 m i n w i t h t h e striatal syna p t o s o m e p r e p a r a t i o n . I n h i b i t o r s were at 10 -7, 10 -6 or 10 -s M. ICs 0 values were d e t e r m i n e d as for 54 C-5HT. F o r the c o n t r o l s , u p t a k e at 37°C was 0.360 + 0.09 p m o l e / m g t i s s u e / m i n and at 4°C was 0.068 + 0.008 p m o l e / m g t i s s u e / m i n . In vivo - - in vitro. Drugs dissolved in 0.02 N HCl were a d m i n i s t e r e d i.p. and sacrifice was at 30 min. I n c u b a t i o n o f 3 H-DA w i t h striatal s y n a p t o s o m e s f r o m drug-treated or carrier-treated rats was as above. In vitro ICs 0 (M)
In vivo - - in vitro Dose (mg/kg)
% Inhibition
Fenfluramine
Inactive at 10 -s M
15
0
Amphetamine
8.8 ± 1.5 x 10 -7
2 5 10 20
52.8 63.5 72.0 77.0
Benztropine
2.6 ± 0.2 X 10 -7
5 20 50
0 45.3 ± 7.1 83.0 ± 11.4
± ± ± ±
5.5 5.5 4.7 5.7
A C T I O N O F F E N F L U R A M I N E ON M O N O A M I N E S
39
TABLE 3 M e t a b o l i s m o f labelled amines. Labelled 5HT and DA were e x t r a c t e d f r o m P2 pellets and f r o m the c o r r e s p o n d i n g s u p e r n a t a n t s ($2) following inc u b a t i o n w i t h o u t drug or with f e n f l u r a m i n e or a m p h e t a m i n e at 10 -s M. Values are e x p r e s s e d as the p e r c e n t a g e o f total radioactivity in each fraction. In brackets: values o b t a i n e d after p r e t r e a t m e n t o f rats with iproniazid (200 mg/kg, i.p.) 18 hr b e f o r e h a n d . % A m i n e remaining
14 C-5HT
3 H-DA
P2
$2
P2
$2
Control
53.4 + 1.7 (86.3 ± 1.7)
89.8 ± 0.9 (97.5 -+ 0.2)
55.8 ÷ 2.8
64.4 + 1.5
Fenfluramine 10 -s M
49.4 ± 5.1 (91.5 ± 1.3)
91.7 ± 0.5 (97.2 ± 0.1)
58.2 ± 5.3
64.5 ± 1.0
Amphetamine 10 -s M
67.4 ± 2.2* (87.8 ± 0.5)
92.8 ± 0.2* (97.6 ± 0.3)
44.4 ± 4.1
69.1 ± 1.4
* Significantly d i f f e r e n t (at least p < 0.05) f r o m c o r r e s p o n d i n g c o n t r o l value. S t u d e n t ' s t-test.
3.2.2. In vivo - - in vitro
Administered in vivo at a dose of 15 mg/kg (i.p.), fenfluramine was inactive in inhibiting the in vitro uptake of 3H-DA measured 30 min after the injection. Amphetamine, on the other hand, even at the lowest dose of 2 mg/kg, already showed appreciable inhibition (table 2). Benztropine was markedly less active, and a dose of more than 20 mg/kg was required to approach the activity of amphetamine at 2 mg/kg. 3.3. M e t a b o l i s m o f labelled a m i n e s
To determine the extent of metabolism of 4 C-5HT and 3H-DA following incubation with a high concentration of fenfluramine or amphetamine, the radioactive amines were extracted from the P2 pellets and the corresponding supernatants ($2). The a m o u n t of unchanged amine remaining after incubation with drug is compared to that in an identical incubation w i t h o u t drug (table 3). Fenfluramine modified neither the a m o u n t of ~4C.5HT recovered nor the a m o u n t of 3H-DA, with respect to control values. In the case of amphetamine, a significant difference from control values was seen only for ~4 C-5HT.
Following pretreatment of rats with iproniazid, more than 85% of unchanged 14C.5H T was found in the P2 fraction and more than 97% in the $2 fraction. 3.4. R e l e a s e o f ~ 4 C - 5 H T
At concentrations of 10 -s M both fenfluramine and amphetamine released radioactivity* from striatal synaptosome preparations preloaded with 14C.5H T (table 4). At 10 -6 M only fenfluramine had an effect. To show that the processes of uptake inhibition and release can be distinguished, a release experiment using 14C-5HT was carried out under 'uptake conditions', in the presence of 10 -s M fenfluramine and using P2 pellets pre-
* Total released radioactivity is taken as the m e a s u r e of amine release since the same relative o r d e r o f activity was o b t a i n e d w h e n released a m i n e was sep a r a t e d f r o m its acid m e t a b o l i t e s o n D o w e x resin as described in M e t h o d s ( S e c t i o n 2.2.3.). F o r m a t i o n o f acid m e t a b o l i t e s in the p r e s e n c e o f f e n f l u r a m i n e or a m p h e t a m i n e at 10 -s M was n o t significantly d i f f e r e n t f r o m the c o n t r o l w i t h o u t drug and repres e n t e d , for 5 HT, 30--40% o f the total radioactivity in the s u p e r n a t a n t and for DA, 10--20%.
40
M.-H. KANNENGIESSER ET AL.
TABLE 4 Release of 14 C-5HT and 3 H-DA from synaptosomes. Synaptosomes from corpus striatum, pre-loaded with 14C.5HT or 3 H-DA were resuspended and incubated at 37°C for 20 min or 5 min respectively with drugs as shown. Release is expressed as the percentage of radioactivity lost with respect to an identical incubation without drug and is measured both in the pellet and in the supernatant. In controls, the radioactivity in washed P2 pellets was 22% of the total incubated for 5HT and 28% for DA. Of this activity, spontaneous release amounted to approximately 66% and 40% respectively. Results represent the average (-+ S.E.M.) of pellet and supernatant values from at least 2 experiments. Concentration (M) Fenfluramine
10-4 10-s 10 -6
Amphetamine
10-7 10-4 10-s 1 0 -6
10-7
% Release 5HT 40.6 ± 3.6 31.8 ± 2.6
32.2 ± i.I 6.2 ± 2.6
DA 22.0 5.0 2.0 0 42.8 38.1 21.8 2.7
± 2.4 ± 1.8 ± 1.3 ± 3.5 +-2.2 t 1.2 +- 1.5
p a r e d as d e s c r i b e d f o r 5 H T u p t a k e . N o differe n c e in r a d i o a c t i v i t y release was d e t e c t e d , c o m p a r e d w i t h an identical c o n t r o l w i t h o u t drug. 3.5. Release o f 3H-DA T h e o r d e r o f activity was reversed w h e n striatal s y n a p t o s o m e s p r e - l o a d e d w i t h 3H-DA were used. F e n f l u r a m i n e o n l y s h o w e d a releasing e f f e c t at a v e r y high c o n c e n t r a t i o n ( 1 0 -4 M), w h e r e a s a m p h e t a m i n e was active d o w n t o 10 -6 M (table 4). 4. Discussion I t has b e e n s h o w n t h a t f e n f l u r a m i n e , unlike a m p h e t a m i n e , inhibits 5 H T u p t a k e in a syna p t o s o m a l p r e p a r a t i o n f r o m w h o l e brain. A d m i n i s t e r e d in vivo, f e n f l u r a m i n e is as e f f e c t i v e in inhibiting in v i t r o 5 H T u p t a k e as c h l o r i m i p r a m i n e at the s a m e dose, b u t unlike
c h l o r i m i p r a m i n e , the e f f e c t o f f e n f l u r a m i n e is long-lasting and a p p r e c i a b l e i n h i b i t i o n is still o b s e r v e d a f t e r 18 hr. This result is in c o n t r a s t t o t h a t o f M o r g a n et al. ( 1 9 7 2 ) w h o f o u n d fenf l u r a m i n e inactive in r a t brain slices f r o m h y p o t h a l a m u s , brain s t e m or s t r i a t u m 4 hr a f t e r a d m i n i s t r a t i o n o f a c o m p a r a b l e dose. T h e diff e r e n c e is p r o b a b l y d u e t o the higher c o n c e n t r a t i o n o f 5 H T (10 -6 M) used b y these a u t h o r s , which w o u l d t e n d to m a s k a specific i n h i b i t o r y effect. With r e s p e c t to D A nerve-endings fenfiuram i n e is t o t a l l y inactive, while on the o t h e r h a n d a m p h e t a m i n e is a g o o d i n h i b i t o r o f D A u p t a k e b o t h in v i t r o and in vivo - - in vitro. B o t h f e n f l u r a m i n e and a m p h e t a m i n e a p p e a r to be m o r e e f f e c t i v e u p t a k e i n h i b i t o r s a f t e r in vivo a d m i n i s t r a t i o n , p r o b a b l y o w i n g to their e f f i c i e n t passage and r e t e n t i o n in the brain. Similar o b s e r v a t i o n s have b e e n m a d e for chlorp h e n t e r m i n e ( R o s s et al., 1 9 7 2 ) and 4-chloroa m p h e t a m i n e (Wong et al., 1973) a n d like f e n f l u r a m i n e , these t w o drugs also h a v e a relatively long-lasting effect. T h a t the o b s e r v e d d i f f e r e n c e s in in v i t r o inhibition of 5HT and DA uptake caused by f e n f l u r a m i n e and a m p h e t a m i n e are n o t d u e t o d i f f e r e n c e s in t r a n s m i t t e r m e t a b o l i s m i n d u c e d b y the drugs themselves, is d e m o n s t r a t e d b y e x t r a c t i o n o f the r a d i o a c t i v e a m i n e s f r o m b o t h the P2 and $2 f r a c t i o n s a f t e r i n c u b a t i o n w i t h high c o n c e n t r a t i o n s o f the drugs. A l t h o u g h m e t a b o l i s m was q u i t e extensive, p a r t i c u l a r l y in the P2 f r a c t i o n , a significant d i f f e r e n c e f r o m c o n t r o l values was seen o n l y for 5 H T a f t e r i n c u b a t i o n with a m p h e t a m i n e . In this case, p r e t r e a t m e n t with iproniazid, which r e d u c e s m e t a b o l i s m to a u n i f o r m l y l o w level, did n o t m o d i f y the ICs 0 values, e x c e p t for fenfluram i n e , the ICs 0 o f w h i c h was slightly lower. As p r e v i o u s l y discussed b y K a n n e n g i e s s e r et al. ( 1 9 7 3 ) and b y C o s t a {1973), this result m a y indicate m o r e o v e r t h a t f e n f l u r a m i n e , unlike reserpine, d o e s n o t inhibit u p t a k e at the vesicle level, since, in the case of reserpine, i m p a i r e d u p t a k e is ' n o r m a l i s e d ' b y M A O I p r e t r e a t m e n t , w h i c h w o u l d t h e r e f o r e lead to a higher ICs 0 value.
A C T I O N O F F E N F L U R A M I N E ON M O N O A M I N E S
Using synaptosomes pre-loaded with 14C5HT or 3H-DA in vitro, it is shown that fenfluramine is more effective than amphetamine in releasing 5HT, but unlike amphetamine has no effect on DA release except at very high concentration. In the synaptosome system used, it is possible that such a transmitter-releasing process could be misinterpreted as an apparent inhibition of uptake. As far as amphetamine is concerned, Ferris et al. (1972) have demonstrated that, under the conditions used for measuring inhibition of DA uptake, release of DA is dose-dependent, but weak. For fenfluramine, it has been confirmed that under 'uptake conditions' no detectable release of ' 4C-5HT occurs, thus showing that uptake inhibition and release can be differentiated by the m e t h o d used. These experiments support the original proposition that the in vivo depletion of 5HT by fenfluramine may be caused by 5HT release. However, we now further propose that fenfluramine's effective and long-lasting inhibition of 5HT uptake could play at least an equally important role, whereby 5HT, released from the nerve-ending either physiologically or by fenfluramine itself, is prevented from being re-captured and re-used. Considerable indirect evidence implicates 5HT in the anorectic action of fenfluramine. Thus the activity o f fenfluramine is reduced by lesion of 5HT neurons (Samanin et al., 1972; Clineschmidt, 1973), by blockers of 5HT receptors (Jesperson and Scheel-Krfiger, 1970; Funderburk et al., 1971) and by an inhibitor of 5HT uptake (Ghezzi et al., 1973; Jesperson and Scheel-Krfiger, 1973). The 5HT depletion caused by fenfluramine does not appear to be correlated with its anorectic action. While fenfluramine-induced 5HT depletion only reaches a maximum 4 hr after a single dose and remains low for at least the following 4 hr (Ghezzi et al., 1973), the anorectic effect begins to decline 2--4 hr after administration (Schmitt, 1973). It is possible, however, that stimulatory activity induced only after chronic fenfluramine treatment (Taylor et al., 1973), may be associated
41
with persistent 5HT depletion. The relative importance of 5HT release and uptake inhibition in the anorectic action of fenfluramine remains to be established*. Uptake inhibition is of long-duration compared to the anorectic effect, which would suggest that release is a more likely candidate. However, it is to be noted that a c o m p o u n d such as chlorphentermine which inhibits both 5HT and NA uptake (Ross et al., 1972) but which does not deplete 5HT, suggesting the absence of releasing properties, is nevertheless anorectic (MOller Nielsen and Dubnick, 1970). Ghezzi et al. (1973) have shown that the anorectic effect of fenfluramine can be blocked by chlorimipramine, an inhibitor of 5HT uptake. They suggest, moreover, that fenfluramine may be accumulated within 5HT neurons by means of the amine transport process. Closely analogous results have been reported for the related c o m p o u n d 4-chloroamphetamine, the stimulatory activity of which can be similarly blocked (Lassen, 1974). 4-Chloroamphetamine is actively accumulated by synaptosomes and is a competitive inhibitor of 5HT uptake (Wong et al., 1973). If the same is true for fenfluramine, then a possible role for the uptake process in mediating anorectic activity would be simply to concentrate the c o m p o u n d in 5HT neurons where its releasing effect can come into play. As previously mentioned, 4-chloroamphetamine and fenfluramine show very similar behaviour with respect to 5HT release and uptake inhibition, and in the case of the former this seems to be associated with its stimulatory properties since these can be attenuated by chlorimipramine. Fenfluramine on the other hand is primarily sedative, so this property
* A l t h o u g h an a n o r e c t i c e f f e c t is o n l y a p p a r e n t in a d u l t rats, t h e relative b i o c h e m i c a l effects o f fenf l u r a m i n e a n d a m p h e t a m i n e , d e s c r i b e d here, are identical regardless of w h e t h e r b r a i n s f r o m i m m a t u r e ( 1 9 - - 2 1 d a y s ) or a d u l t rats ( 1 5 0 - - 2 0 0 g) are used as a s o u r c e of s y n a p t o s o m e s ( K a n n e n g i e s s e r , Hunt, unpublished).
42 w o u l d n o t a p p e a r to be related to these 5 H T m e c h a n i s m s . Instead, it was first a t t r i b u t e d to a reserpine-like a c t i o n in N A n e u r o n s (Ziance and Rutledge, 1 9 7 2 ) and indeed at low doses, fenfluramine, like reserpine, increases N A turnover ( G r o p p e t t i et al., 1972). H o w e v e r , D A t u r n o v e r is also increased (Costa et al., 1 9 7 1 ) and r e c e n t l y a neuroleptic-like a c t i o n has been d e m o n s t r a t e d b y Jori et al. ( 1 9 7 4 ) as well as by G r a b o w s k a and Michaluk (1974). Compared to amphetamine, fenfluramine has no e f f e c t o n D A u p t a k e and only a slight e f f e c t on D A release. Considering the implication o f D A in the increased m o t o r activity, the p s y c h o t i c activity and the d e p e n d e n c e i n d u c e d by the f o r m e r , it is possible t h a t the lack o f these effects, to w h i c h f e n f l u r a m i n e owes its low incidence o f serious clinical side-effects, m a y be d u e to its lack o f activity on these D A mechanisms.
Acknowledgement The technical assistance of Mme D. MassardierCh~ze and of Mr. J.C. Massardier is gratefully acknowledged.
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