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DU 24565, a quipazine derivative, a potent selective serotonin uptake inhibitor
European Journal o f Pharmacology, 70 (1981) 195--202 © Elsevier/North-Holland Biomedical Press
195
DU 24565, A QUIPAZINE DERIVATIVE, A POTENT SELECTIVE SEROTONIN UPTAKE INHIBITOR W. JAN VAATSTRA, WILHELMINA M.A. DEIMAN-VAN AALST and LEO EIGEMAN Department o f Pharmacology, Duphar B. V., P.O. Box 2, Weesp, The Netherlands
Received 8 June 1980, revised MS received 10 October 1980, accepted 26 November 1980
W.J. VAATSTRA, W.M.A. DEIMAN-VAN AALST and L. EIGEMAN, DU 24565, a quipazine derivative, a p o t e n t selective serotonin uptake inhibitor, European J. Pharmacol. 70 (1981) 195--202. DU 24565, 6-nitro,2-(1-piperazinyl)quinoline, is a potent and selective inhibitor of the synaptosomal uptake of serotonin (5-HT). At concentrations at least 103-fold higher it affects the uptake of norepinephrine (NA) and dopamine (DA). The ICs0 values are: 5-HT: 4 × 10 -8 M; NA: 6 × 10 -s M and DA: 4 x 10 -s M. Uptake of 5-HT by rat blood platelets is also strongly inhibited (K i ~5 X 10 -s M); the inhibition is probably noncompetitive. In vivo, DU 24565 is active at low oral doses: the 5-HT depletion in rat brain caused by p-chloroamphetamine is antagonized by DU 24565 (oral EDs0 0.7 mg/kg). The decrease in the 5-HT content caused by 4,~dimethyl-m-tyramine (H 77/77) is antagonized by DU 24565 at 1 mg/kg orally, without any effect on the depletion of catecholamines. 5-HT turnover, measured by the probenecid method, is reduced by the same dose of DU 24565. Other tests confirmed the activity and selectivity of DU 24565 : it potentiated the behavioural effects of the 5-HT precursor 5-hydroxytryptophan (5-HTP) in mice (EDs0 1.5 mg/kg orally); it potentiated the temperature increases caused by 5-HTP in the rabbit; it had low activity or no effect at all in NA potentiation tests. This new compound is more potent and selective than the known 5-HT uptake inhibitors. It is a potential antidepressant and can be useful as a pharmacological tool to study the role of 5-HT in the central nervous system. Selective uptake inhibitor
Potential antidepressant
1. I n t r o d u c t i o n In t h e search f o r a n t i d e p r e s s a n t s t h a t are m o r e selective t o w a r d s a specific a m i n e t h a n t h e available tricyclics, m u c h e f f o r t has b e e n d i r e c t e d t o w a r d s selective i n h i b i t i o n o f serot o n i n (5-HT) u p t a k e . T h i s e f f o r t has b e e n s t i m u l a t e d b y t h e still valid view t h a t a dist u r b a n c e o f 5-HT n e u r o t r a n s m i s s i o n m a y be an i m p o r t a n t f a c t o r in c e r t a i n s u b t y p e s o f depressive illness ( L a p i n a n d O x e n k r u g , 1 9 6 9 ; Maas, 1 9 7 5 ; O g r e n e t al., 1979). T h e diagnostic classification o f t h e s e disorders w i t h bioc h e m i c a l m e t h o d s will i m p r o v e a n d so t h e n e e d f o r a 5-HT-selective c o m p o u n d m i g h t grow. A w h o l e series o f m o r e or less selective 5-HT u p t a k e i n h i b i t o r s a p p e a r e d in t h e l i t e r a t u r e in r e c e n t y e a r s a n d s o m e o f t h e m
6-Nitro-quipazine
Serotonin
h a v e p r o v e d t o be clinically effective antid e p r e s s a n t s : c i t a l o p r a m ( H y t t e l , 1 9 7 7 ) ; fluv o x a m i n e (Claassen et al., 1 9 7 7 ; Wright a n d D e n b e r , 1 9 7 8 ) ; LM 5 0 0 8 ( L e F u r et al., 1978); t r a z o d o n e ( S t e f a n i n i et al., 1 9 7 6 ) a n d zimelidine ( R o s s a n d R e n y i , 1 9 7 7 ; C o p p e n et al., 1979). In o u r studies o f t h e p h a r m a c o l o g i c a l a c t i o n s o f a series o f c o m p o u n d s s t r u c t u r a l l y r e l a t e d t o q u i p a z i n e , D U 2 4 5 6 5 (see fig. 1)
DU 24565
Fig. 1. Structure of DU 24565, 6-nitro,2-(1-piperazinyl)quinoline.
196 emerged as a potent orally active, selective 5-HT uptake inhibitor. Other pharmacological activities, which are present in quipazine, were n o t found. This paper describes the neurochemical and pharmacological studies characterizing the new compound.
w.J. VAATSTRA ET AL. blood sampled by heart-puncture and incubated with [3H] 5-HT according to Coppen et al. (1978). Uptake values from at least four identical samples were used in linear regression analysis to obtain Lineweaver-Burk plots.
2.2. In vivo tests 2. Materials and methods
2.1. Uptake of biogenic amines 2.1.1. Synaptosomal uptake Crude synaptosomal preparations were obtained from male Wistar rats weighing 200250 g. For 5-HT a P2 fraction was prepared from whole brain minus cerebellum; for NA and DA 1000 X g suspensions from hypothalamus and corpus striatum, respectively, were used. The incubation medium according to Coyle and Snyder (1969), containing 7 X 10 -s M pargyline was used; the concentrations of the amines were 2-4 X 10 -7 M in a final volume of 300 pl. After a 10 min preincubation time with or without the tesl~ c o m p o u n d the synaptosomes were incubated with the tritium-labelled amine for 5 min (for 5-HT and DA) or 15 min (for NA). The reaction was stopped by rapid cooling and centrifugation. The washed pellet was extracted with 0.4 N perchloric acid and the radioactivity was measured by liquid scintillation counting. Blanks were obtained from samples in which the incubation was stopped immediately after the addition of the labelled amine. At least three concentrations of the drugs were tested in duplicate; each test was repeated twice or more. The results are expressed as the ICs0, the concentration giving 50% inhibition. Ex vivo tests were done comparing synaptosomal preparations from untreated rats to those from drug-treated animals, 1 h after drug administration. Uptake was determined in 4-6 samples from pooled synaptosomal suspensions prepared from 2-4 rats. 2.1.2. 5-HT uptake by rat blood platelets Rat platelet-rich plasma was prepared from
2.2.1. Antagonism o f 5-HT depletion induced by p-chloroamphetamine Male Wistar rats (n = 5) were injected with 5 mg/kg i.p. p-chloroamphetamine, 4 h later brain 5-HT c o n t e n t was measured by the fluorimetric m e t h o d of Curzon and Green (1970). The test compounds were given in three doses 1 h before the depleting drug; from the 5-HT levels, the EDs0 (representing the dose causing 50% antagonism of the 5-HT depletion) could be determined. 2.2.2. Antagonism o f amine depletion by 4,adimethyl-m-tyramine (H77/77) Rats were injected twice, at an interval of 2 h, with H77/77, 12.5 mg/kg i.p. The test c o m p o u n d was given 30 min before each dose of H77/77; animals were killed 2 h after the last dose of H77/77. Whole brain NA, DA and 5-HT were measured by fluorimetric methods modified from Laverty and Taylor (1968), Haubrich and Denzer (1973), and Curzon and Green (1970). 2.2.3. Serotonin turnover--probenecid test Fifteen min before an i.p. injection of 200 mg/kg probenecid, the test c o m p o u n d was administered to rats at an oral dose of 1 mg/ kg. The animals were decapitated 2 h after probenecid administration. Brain 5-hydroxyindoleacetic acid (5-HIAA) was measured by a fluorimetric method (Curzon and Green, 1970). Effect on 5-HT turnover could be estimated from 5-HIAA accumulation in animals receiving probenecid with or without the test compound. 2.2.4. Potentiation o f 5-hydroxytryptophan (5-HTP) behavioural effects in mice The test c o m p o u n d was administered orally
DU 24565, A SELECTIVE 5-HT UPTAKE INHIBITOR
197
in a range of doses to isolated male Swiss albino mice (20-28g), 1 h before the i.p. injection of a threshold dose of 150 mg/kg of d,I-5-HTP. Thirty minutes after the 5-HTP injection the mice were observed individually and the following behaviour parameters were scored: stereotyped head-searching movements (0 to 4); hindlimb abduction (0 to 2); tremor (0 to 4); escape tendency (0 or 1); forelimb clonus (0 to 2); lordosis (0 to 2). From the results, the EDs0 could be determined plotting log dose against total group score on a linear scale the ED50 is the dose required to potentiate the 5-HTP effect to 50% of the maximal score.
pram (Lundbeck), desmethylimipramine (DMI; Geigy), DU 24565 and fluvoxamine (Duphar), femoxetine (Ferrosan), fluoxetine (Lilly), H77/77 (AB Biotec), probenecid (MSD), quipazine (Miles), tetrabenazine (Hoffman-La Roche) and zimelidine (Astra). 5-HT, 5-HTP, DA and NA were obtained from KochLight Labs. The radiochemicals were purchased from the Radiochemical Centre, Amersham: 5-hydroxy- [G-3H ] tryptamine • creatinine sulphate, 0.5 Ci/mmol; l-[7-3H]noradrenaline • hydrochloride, 5 Ci/mmol; 3,4dihydroxy-[G-3H]phenylethylamine • hydrochloride, 0.5 Ci/mmol.
2.2.5. Potentiation of 5-HTP hyperthermia in rabbits Male rabbits weighing a b o u t 2.5 kg, accust o m e d to prolonged restraint in a rabbit holder and to continuous temperature measurement, were used. After a pre-test period of 30 min the test c o m p o u n d was injected i.v.; 30 min after administration of the test c o m p o u n d the rectal temperature was measured and 5-HTP was injected i.v. in a dose of 50 mg/kg. Temperature measurements were repeated every 30 min for the next 120 min.
3. Results
2.2.6. Antagonism o f tetrabenazine-induced ptosis in mice The test c o m p o u n d s were administered orally to groups of male mice in a range of doses; 45 min later tetrabenazine was injected subcutaneously at a dose of 80 mg/kg. Ptosis was scored using a ptosis chart (Rubin et al., 1957) 45 min after the tetrabenazine administration. The EDs0, that is the dose of the test c o m p o u n d required to reduce the ptosis score to half that of the control tetrabenazine group could be determined graphically by plotting responses on a probit scale against test dose on a logarithmic scale. 2.3. Drugs Amitriptyline (MSD), chlorimipramine (Geigy), p-chloroamphetamine (Regis), citalo-
3.1. In vitro uptake of biogenic amines ~ 3.1.1. Synaptosomal uptake The in vitro results summarized in table 1 as IC50 values for inhibition of the amine uptake, indicate that DU 24565 can be considered as a potent selective inhibitor of 5-HT uptake. As indicated by the ratio of the ICs0 for NA and for 5-HT uptake, the potency and selectivity are favourable when compared with known 5-HT uptake inhibiting drugs. For comparison desmethylimipramine (DMI) and amitriptyline and the parent c o m p o u n d quipazine are included in the list. In preliminary ex vivo experiments with a limited number of animals, 1 h after an oral dose of 5 mg/kg, DU 24565 caused a decreased uptake of 5-HT by synaptosomal preparations of treated rats. Chlorimipramine and fluoxetine at 10 mg/kg orally were not effective, fluvoxamine at the same dose level and also 1 h after administration, resulted in a diminished 5-HT uptake. 3.1.2. 5-HT uptake by rat blood platelets Fig. 2 reveals a strong effect on platelet 5-HT uptake. The Lineweaver-Burk plot yielded a K m of 0 . 6 p M for 5-HT in the control incubation. DU 24565 at concentra-
198
W.J. VAATSTRA ET AL.
TABLE 1 Synaptosomal amine uptake. ICs0: concentration causing 50% inhibition. The values are the means of at least 2 independent experiments; the over-all standard deviation of the uptake test is about 15%. ICs0 (pM)
DU 24565 Chlorimipramine Fluvoxamine Fluoxetine Zimelidine Femoxetine Citalopram Quipazine Amitriptyline Desmethylimipramine
ICs0 (NA)
5-HT cerebrum
NA hypothalamus
DA c. striatum
ICs0 (5-HT)
0.045 1.0 0.21 1.6 24 2.4 0.63 1.2 3.2 25
66 5.2 41 18 50 13 50 1.9 2.2 0.12
41 15 47 32 79 40 40 31 32 60
1467 5.2 195 11 2 5.4 79 1.6 0.7 0.0005
I/V. 104 30-
tions o f 10 -8 and 10 -9 M was a p o t e n t inhibitor. As the lines did n o t coincide at o n e p o i n t o n t h e 1 / V axis, t h e t y p e o f inhibition b y D U 2 4 5 6 5 c a n n o t be indicated. This was seen, t o a varying degree, in m o r e e x p e r i m e n t s .
/
/I /t
3.2. In vivo tests 3.2.1. Antagonism o f 5-HT depletion induced by p-chloroamphetamine T h e s t r o n g antagonistic a c t i o n o f DU 2 4 5 6 5 against the decrease in rat brain 5-HT c o n t e n t caused b y t h e d e p l e t o r p-chloroa m p h e t a m i n e is s h o w n in fig. 3. The low oral EDs0 value o f 0.7 m g / k g was f a v o u r a b l e w h e n c o m p a r e d with t h a t o f zimelidine and fluoxetine (EDs0 resp. 4 and 7) and o f chlorimipramine (EDs0 > 20).
10.
I'o
3'o
1/s
s'o. 1o"
Fig. 2. 5-HT uptake by rat blood platelets. Mean and S.E.M. of at least 4 samples; graphs from linear regression (o control; A 10-8 M DU 24565; [] 10 -9 M DU 24565}. S = 5-HT concentration: 0.2-1.8 x 10 -7 M; V = uptake (dpm/2 rain); Km (5-HT) = 0.6 pM; V m a x = ~ 1 0 0 pmol 5-HT/109 platelets/min.
3.2.2. Antagonism H77/77
o f amine depletion
by
H 7 7 / 7 7 , a t y r a m i n e derivative, given in t w o separate doses o f 12.5 m g / k g i.p. to rats, caused a decrease in brain biogenic a m i n e c o n t e n t . A n oral dose o f 1 m g / k g D U 2 4 5 6 5 , 30 min b e f o r e each H 7 7 / 7 7 injection, prot e c t e d against t h e d e p l e t i o n o f 5-HT, b u t h a d n o e f f e c t o n N A a n d D A d e p l e t i o n {table 2).
DU 24565, A S E L E C T I V E 5-HT U P T A K E I N H I B I T O R rat brain
5-HT
199 TABLE 3
pg/g
5-HT t u r n o v e r - - p r o b e n e c i d test. Rat brain 5-HIAA in pg/g, mean and S.E.M., n = 5. D U 24565 at an oral dose of 1 mg/kg was given 15 rain before 200 m g / k g i.p. probenecid; 2 h after probenecid the animals were decapitated. 5-HIAA accumulation (5-HT turnover): 1.8 p m o l / g tissue/h; after DU 2 4 5 6 5 : 0 . 6 3 p m o l / g / h (65% decrease).
0.50
Controls Probenecid D U 24565 D U 24565 + probenecid
0.40
0.58 1.25 0.55 0.79
-+ 0.005 + 0.030 -+ 0.020 -+ 0.005 1
1 p < 0.001 vs. probenecid alone (Student's t-test).
0.30
0.2 0'.3
1:0 oral
3:0
1'0 m g / k g
dose of DU 24565
Fig. 3. Antagonism of 5-HT depletion induced by p - c h l o r o a m p h e t a m i n e . Drug was given 1 h b e f o r e 5 m g / k g i.p. p-Cl-amphetamine, 4 h thereafter t h e animals were decapitated and brain 5-HT was measured. 5-HT levels after 0.3 and 1.0 m g / k g p.o. D U 24565 in c o m b i n a t i o n with p-Cl-amphetamine were significantly (P < 0.05) different f r o m control or p-Cl-amphetamine alone.
The percentage uptake inhibition for 5-HT was 59%, as calculated by the method of Carlsson et al. (1969).
3.2.3. 5-HT turnover as measured by the probenecid method An accepted index for 5-HT turnover is the accumulation of the 5-HT metabolite, 5-HIAA, after its transport from the brain is blocked by probenecid. Our results with rat brain indicated a 5-HT turnover rate of 1.8 pmol/g tissue/h. DU 24565, in a dose of 1 mg/kg orally, diminished this rate to about 35% of its normal value (table 3). The same dose of the test compound without probenecid did not decrease the 5-HIAA content, but a higher dose (10mg/kg orally) caused a significant 20% reduction. 3.2.4. Potentiation of 5-hydroxytryptophan (5-HTP) effects in mice and rabbits In low oral doses DU 24565 potentiated the induction of 5-HT-like behavioural effects in mice caused by threshold doses of the 5-HT
TABLE 2 A n t a g o n i s m of amine depletion induced by H77/77. Rat brain amine levels in gg/g, m e a n and S.E.M., n = 5. Significant amine depletions by H 7 7 / 7 7 ; no effect o f DU 24565. Antagonism of depletion e.g. uptake inhibition for 5-HT: 59%.
Controls H77/77 D U 24565 DU+ H
5-HT
NA
0.50 + 0.018 0.29 -+ 0.017 0.47 -+ 0.034 0.40-+ 0.006 ]
0.18 0.057 0.16 0.056
1 p < 0.001 vs. H 7 7 / 7 7 alone ( S t u d e n t ' s t-test).
DA + 0.011 -+ 0.007 + 0.022 + 0.007
0.62 0.38 0.60 0.31
+ 0.048 -+ 0.015 -+ 0.020 -+ 0.018
200
W.J. V A A T S T R A ET AL.
TABLE 4
rectal oC
Potentiation of 5-HT and NA effects.
temp,
Oral EDs0 values (mg/kg)
5-HTP potentiation DU 24565 Chlorimipramine Fluvoxamine Fluoxetine Zimelidine Femoxetine Citalopram Quipazine Amitriptyline Desmethylimipramine
1.5 84 36 50 20 57 20 _
1
> 100 >320
Tetrabenazine ptosis antagonism 18 12 t> 100 50 30 9 > 100 9 5
o~
44
43
42
0.8
1 Interfered with 5-HTP effects.
p r e c u r s o r 5-HTP (table 4). O t h e r k n o w n 5-HT u p t a k e inhibitors were surprisingly less active in this test; differences o f a f a c t o r o f 20 and m o r e were observed. P o t e n t i a t i o n o f 5-HTPi n d u c e d h y p e r t h e r m i a in rabbits was evident a f t e r i n t r a v e n o u s doses as low as 10 pg/kg o f DU 2 4 5 6 5 (fig. 4). T e m p e r a t u r e rises o f 5°C resulting in h y p e r t h e r m i c deaths were observed at doses o f 30 pg/kg. R e f e r e n c e c o m p o u n d s were m u c h less active in this respect: 300 pg/ kg i.v. c h l o r i m i p r a m i n e and f l u o x e t i n e (data n o t s h o w n ) gave t e m p e r a t u r e rises o f a b o u t 1°C.
3.2.5. Antagonism of tetrabenazine-induced ptosis in mice A n t a g o n i s m o f t e t r a b e n a z i n e - i n d u c e d ptosis in mice (table 4) is used as an i n d e x for NAp o t e n t i a t i n g effects. D e s m e t h y l i m i p r a m i n e , a NA-selective tricyclic a n t i d e p r e s s a n t and amit r i p t y l i n e were effective in this test as s h o w n b y the low EDs0. T h e 5-HT selective u p t a k e inhibitors had a higher EDs0, a l t h o u g h the differences were less t h a n s h o u l d be e x p e c t e d f r o m results o f o t h e r in vitro and in vivo models. DU 2 4 5 6 5 s h o w e d less antagonism of the t e t r a b e n a z i n e e f f e c t t h a n c h l o r i m i p r a m i n e
41
40
39
38 <
. . . . - 0.5
0.5
1
2"s 1.5
2
3 time
(h)
Fig. 4. Potentiation of 5-HTP hyperthermia in rabbits. Intravenous doses of DU 24565 (© 0.01 mg/kg, n = 4-6; [] 0.03 mg/kg; • 0.1 mg/kg, n = 2) were given 30 min before 50 mg/kg i.v. 5-HTP. Rectal temperature was measured every 30 rain. 5-HTP at this threshold dose (A; n = 11) and DU 24565 alone (not shown) had no effect; A 0.3 mg/kg i.v. chlorimipramine (n = 6); ¢~hyperthermal deaths.
and f e m o x e t i n e , b u t c o m p a r e d with fluvoxamine, f l u o x e t i n e , zimelidine and c i t a l o p r a m m o r e N A p o t e n t i a t i o n was seen.
DU 24565, A SELECTIVE 5-HT UPTAKE INHIBITOR 4. Discussion From a series of quipazine derivates DU 24565, the 6-nitro analogue, emerged as a c o m p o u n d with actions surprisingly different from those of the parent compound. It was a selective 5-HT uptake inhibitor with a p o t e n t oral activity in various biochemical and pharmacological test systems. In in vitro studies using rat brain synaptosomal preparations, strong inhibition of 5-HT uptake (ICs0 = 4 × 10 -8 M) was found, but the synaptosomal uptake of catecholamines was affected at concentrations at least 103 times higher. Compared with other 5-HT-selective uptake inhibitors in our test system this is a favourable ratio. Results from ex vivo tests in which synaptosomal 5-HT uptake by preparations from treated rats was compared with those of untreated rats, indicated a good oral activity. So the test c o m p o u n d penetrates to the central nervous system. In rat blood platelets 5-HT uptake was strongly inhibited by DU 24565 concentrations in the nM range. From Lineweaver-Burk plots no uniform picture w~s obtained. The intercepts of the lines did not allow a clear decision a b o u t the t y p e of inhibition by DU 24565 (fig. 2). There were indications for a deviation from the competitive t y p e of inhibition as seen with many tricyclic antidepressants (Tuomisto, 1974; Trenchard et al., 1975; Lingjaerde, 1976). Evidence for other types of inhibition by non-tricyclic uptake inhibitors such as fluoxetine and trazodone, has been reported by Wielosz et al. (1977). One should be very careful with the interpretation of the results, considering variations in the graphs, differences seen at different inhibitor concentrations and the possibility that other processes are interfering, such as release of 5-HT from the platelets. In in vivo studies, using known techniques to measure uptake inhibition, e.g. antagonism of amine depletion caused by depleting drugs that use the amine pump, p o t e n t actions at low oral doses of DU 24565 are found. In particular, the antagonism of 5-HT depletion
201 caused by p-chloroamphetamine was perceptible at doses below 1 mg/kg orally (the EDs0 was 0.7 mg/kg). Using depletion by 4,adimethyl-m-tyramine (H77/77), good specificity towards 5-HT has been demonstrated as the depletion of the catecholamines was not affected and that of 5-HT was antagonized at 1 mg/kg orally. Pharmacological studies on 5-HT- and NApotentiating effects gave results which were in agreement with those of the neurochemical tests. The effects of the 5-HT-precursor 5-HTP were potentiated in t w o models: mouse behaviour and hyperthermia in the rabbit. In both models DU 24565 was active at surprisingly low doses; the other 5-HT uptake inhibitors were a great deal less active. NA potentiation, as indicated by the EDs0 in the tetrabenazine antagonism test, was higher than expected from the studies on synaptosomal uptake, H77/77 antagonism and others not shown here, but the ratio 5-HTP/tetrabenazine is favourable compared with the other compounds. The NA-selective uptake inhibitor DMI had a substantially lower EDs0 value. Other pharmacological tests (data to be published) did not provide evidence for a possible NA potentiating action of DU 24565, neither were indications found of central and peripheral 5-HT-agonistic actions, anorexogenic or cardiovascular effects. This is in contrast to the parent compound, quipazine which has many pharmacological actions. Also, no inhibition of rat brain monoamineoxidase was found when measured in vitro using different substrates. In conclusion: D U 2 4 5 6 5 , the 6-nitro analogue of quipazine, is a new c o m p o u n d that is more p o t e n t and selective than the known 5-HT uptake inhibitors. It could be a potential antidepressant and can be used as a tool for studying the role of 5-HT in the brain and the periphery. In comparing the potencies of a series of structurally different 5-HTselective uptake inhibitors in different test systems one could speculate a b o u t differences that are not directly related to differences in
202
bio-availability and/or metabolism. These could be differences in mechanism or site of action of these compounds.
References Carlsson, A., H. Corrodi, K. Fuxe and T. H6kfelt, 1969, Effects of some antidepressant drugs on the depletion of intraneuronal brain catecholamines stores caused by 4,a-dimethyl-meta-tyramine, European J. Pharmacol. 5 , 3 6 7 . Claassen, V., J.E. Davies, G. Hertting and P. Placheta, 1977, Fluvoxamine, a specific 5-hydroxytryptamine uptake inhibitor, Br. J. Pharmacol. 60, 505. Coyle, J.T. and S.H. Snyder, 1969, Catecholamine uptake by synaptosomes in homogenates of rat brain: stereospecificity in different areas, J. Pharmacol. Exp. Ther. 170, 221. Coppen, A., V.A. Rama Rao, C. Swade and K. Wood, 1979, Inhibition of 5-hydroxytryptamine reuptake by amitriptyline and zimelidine and its relationship to their therapeutic action, Psychopharmacol. 63, 125. Coppen, A., C. Swade and K. Wood, 1978, Platelet serotonin accumulation in depressive illness, Clin. Acta 8 7 , 1 6 5 . Curzon, G. and A.R. Green, 1970, Rapid method for the determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in small regions of rat brain, Br. J. Pharmacol. 39,653. Haubrich, D.R. and J.S. Denzer, 1973, Simultaneous extraction and fluorimetric measurement of brain serotonin, catecholamines, 5-hydroxyindoleacetic acid and homovanillic acid, Anal. Biochem. 55, 306. Hyttel, J., 1977, Neurochemical characterization of a new potent and selective serotonin uptake inhibitor: Lu 10-171, Psychopharmacol. 51, 225. Lapin, I.P. and G.F. Oxenkrug, 1969, Intensification of the central serotonergic processes as a possible determinant of the thymoleptic effect, Lancet 1, 132. Laverty, R. and K.M. Taylor, 1968, The fluorimetric
W.J. VAATSTRA ET AL. assay of catecholamines and related compounds: Improvements and extensions of the hydroxyindole technique, Anal. Biochem. 22,269. LeFur, G., M. Kabouche and A. Uzan, 1978, On the regional and specific serotonin uptake inhibition by LM 5008, Life Sci. 23, 1959. Lingjaerde, O., 1976, Effect of Doxepin on uptake and efflux of serotonin in human blood platelets in vitro, Psychopharmacol. 4 7 , 1 8 3 . Maas, J.W., 1975, Biogenic amines and depression, Arch. Gen. Psychiatry 32, 1357. C)gren, S.O., K. Fuxe, L.F. Agnati, J.A. Gustafsson, G. Jonsson and A.C. Holm, 1979. Reevaluation of the indoleamine hypothesis of depression. Evidence for a reduction of functional activity of central 5-HT systems by antidepressant drugs, J. Neural Transm. 46, 85. Ross, S.B. and A.L. Renyi, 1977, Inhibition of the neuronal uptake of 5-hydroxytryptamine and noradrenaline in rat brain by (Z)- and (E)-3-(4bromophenyl)-N,N-dimethyl-3-(3-pyridyl) allylamines and their secondary analogues, Neuropharmacol. 16, 57. Rubin, B., M.H. Malone, M.H. Waugh and J.C. Burke, 1957, Bioassay of Rauwolfia roots and alkaloids, J. Pharmacol. Exp. Ther. 120, 125. Stefanini, E., F. Fadda, L. Medda and G.L. Gessa, 1976, Selective inhibition of serotonin uptake by trazodone, a new antidepressant agent, Life Sci. 18, 1459. Trenchard, A., P. Turner, C.M.B. Pare and M. Hills, 1975, The effects of protriptyline and clomipramine in vitro on the uptake of 5-hydroxytryptamine and dopamine in human platelet-rich plasma, Psychopharmacologia 43, 89. Tuomisto, J., 1974, A new modification for studying serotonin uptake by blood platelets: A reevaluation of tricyclic antidepressants as uptake inhibitors, J. Pharm. Pharmacol. 26, 192. Wielosz, M., A. Dall'Olio, G. de Gaetano and S. Garattini, 1977, Effect of two non-tricyclic antidepressant drugs on [14C]-5-HT uptake by rat platelets, J. Pharm. Pharmacol. 29,546. Wright, J.H. and H.C. Denber, 1978, Clinical trial of fluvoxamine, a new serotonergic antidepressant, Curr. Ther. Res. 23, 83.
195
DU 24565, A QUIPAZINE DERIVATIVE, A POTENT SELECTIVE SEROTONIN UPTAKE INHIBITOR W. JAN VAATSTRA, WILHELMINA M.A. DEIMAN-VAN AALST and LEO EIGEMAN Department o f Pharmacology, Duphar B. V., P.O. Box 2, Weesp, The Netherlands
Received 8 June 1980, revised MS received 10 October 1980, accepted 26 November 1980
W.J. VAATSTRA, W.M.A. DEIMAN-VAN AALST and L. EIGEMAN, DU 24565, a quipazine derivative, a p o t e n t selective serotonin uptake inhibitor, European J. Pharmacol. 70 (1981) 195--202. DU 24565, 6-nitro,2-(1-piperazinyl)quinoline, is a potent and selective inhibitor of the synaptosomal uptake of serotonin (5-HT). At concentrations at least 103-fold higher it affects the uptake of norepinephrine (NA) and dopamine (DA). The ICs0 values are: 5-HT: 4 × 10 -8 M; NA: 6 × 10 -s M and DA: 4 x 10 -s M. Uptake of 5-HT by rat blood platelets is also strongly inhibited (K i ~5 X 10 -s M); the inhibition is probably noncompetitive. In vivo, DU 24565 is active at low oral doses: the 5-HT depletion in rat brain caused by p-chloroamphetamine is antagonized by DU 24565 (oral EDs0 0.7 mg/kg). The decrease in the 5-HT content caused by 4,~dimethyl-m-tyramine (H 77/77) is antagonized by DU 24565 at 1 mg/kg orally, without any effect on the depletion of catecholamines. 5-HT turnover, measured by the probenecid method, is reduced by the same dose of DU 24565. Other tests confirmed the activity and selectivity of DU 24565 : it potentiated the behavioural effects of the 5-HT precursor 5-hydroxytryptophan (5-HTP) in mice (EDs0 1.5 mg/kg orally); it potentiated the temperature increases caused by 5-HTP in the rabbit; it had low activity or no effect at all in NA potentiation tests. This new compound is more potent and selective than the known 5-HT uptake inhibitors. It is a potential antidepressant and can be useful as a pharmacological tool to study the role of 5-HT in the central nervous system. Selective uptake inhibitor
Potential antidepressant
1. I n t r o d u c t i o n In t h e search f o r a n t i d e p r e s s a n t s t h a t are m o r e selective t o w a r d s a specific a m i n e t h a n t h e available tricyclics, m u c h e f f o r t has b e e n d i r e c t e d t o w a r d s selective i n h i b i t i o n o f serot o n i n (5-HT) u p t a k e . T h i s e f f o r t has b e e n s t i m u l a t e d b y t h e still valid view t h a t a dist u r b a n c e o f 5-HT n e u r o t r a n s m i s s i o n m a y be an i m p o r t a n t f a c t o r in c e r t a i n s u b t y p e s o f depressive illness ( L a p i n a n d O x e n k r u g , 1 9 6 9 ; Maas, 1 9 7 5 ; O g r e n e t al., 1979). T h e diagnostic classification o f t h e s e disorders w i t h bioc h e m i c a l m e t h o d s will i m p r o v e a n d so t h e n e e d f o r a 5-HT-selective c o m p o u n d m i g h t grow. A w h o l e series o f m o r e or less selective 5-HT u p t a k e i n h i b i t o r s a p p e a r e d in t h e l i t e r a t u r e in r e c e n t y e a r s a n d s o m e o f t h e m
6-Nitro-quipazine
Serotonin
h a v e p r o v e d t o be clinically effective antid e p r e s s a n t s : c i t a l o p r a m ( H y t t e l , 1 9 7 7 ) ; fluv o x a m i n e (Claassen et al., 1 9 7 7 ; Wright a n d D e n b e r , 1 9 7 8 ) ; LM 5 0 0 8 ( L e F u r et al., 1978); t r a z o d o n e ( S t e f a n i n i et al., 1 9 7 6 ) a n d zimelidine ( R o s s a n d R e n y i , 1 9 7 7 ; C o p p e n et al., 1979). In o u r studies o f t h e p h a r m a c o l o g i c a l a c t i o n s o f a series o f c o m p o u n d s s t r u c t u r a l l y r e l a t e d t o q u i p a z i n e , D U 2 4 5 6 5 (see fig. 1)
DU 24565
Fig. 1. Structure of DU 24565, 6-nitro,2-(1-piperazinyl)quinoline.
196 emerged as a potent orally active, selective 5-HT uptake inhibitor. Other pharmacological activities, which are present in quipazine, were n o t found. This paper describes the neurochemical and pharmacological studies characterizing the new compound.
w.J. VAATSTRA ET AL. blood sampled by heart-puncture and incubated with [3H] 5-HT according to Coppen et al. (1978). Uptake values from at least four identical samples were used in linear regression analysis to obtain Lineweaver-Burk plots.
2.2. In vivo tests 2. Materials and methods
2.1. Uptake of biogenic amines 2.1.1. Synaptosomal uptake Crude synaptosomal preparations were obtained from male Wistar rats weighing 200250 g. For 5-HT a P2 fraction was prepared from whole brain minus cerebellum; for NA and DA 1000 X g suspensions from hypothalamus and corpus striatum, respectively, were used. The incubation medium according to Coyle and Snyder (1969), containing 7 X 10 -s M pargyline was used; the concentrations of the amines were 2-4 X 10 -7 M in a final volume of 300 pl. After a 10 min preincubation time with or without the tesl~ c o m p o u n d the synaptosomes were incubated with the tritium-labelled amine for 5 min (for 5-HT and DA) or 15 min (for NA). The reaction was stopped by rapid cooling and centrifugation. The washed pellet was extracted with 0.4 N perchloric acid and the radioactivity was measured by liquid scintillation counting. Blanks were obtained from samples in which the incubation was stopped immediately after the addition of the labelled amine. At least three concentrations of the drugs were tested in duplicate; each test was repeated twice or more. The results are expressed as the ICs0, the concentration giving 50% inhibition. Ex vivo tests were done comparing synaptosomal preparations from untreated rats to those from drug-treated animals, 1 h after drug administration. Uptake was determined in 4-6 samples from pooled synaptosomal suspensions prepared from 2-4 rats. 2.1.2. 5-HT uptake by rat blood platelets Rat platelet-rich plasma was prepared from
2.2.1. Antagonism o f 5-HT depletion induced by p-chloroamphetamine Male Wistar rats (n = 5) were injected with 5 mg/kg i.p. p-chloroamphetamine, 4 h later brain 5-HT c o n t e n t was measured by the fluorimetric m e t h o d of Curzon and Green (1970). The test compounds were given in three doses 1 h before the depleting drug; from the 5-HT levels, the EDs0 (representing the dose causing 50% antagonism of the 5-HT depletion) could be determined. 2.2.2. Antagonism o f amine depletion by 4,adimethyl-m-tyramine (H77/77) Rats were injected twice, at an interval of 2 h, with H77/77, 12.5 mg/kg i.p. The test c o m p o u n d was given 30 min before each dose of H77/77; animals were killed 2 h after the last dose of H77/77. Whole brain NA, DA and 5-HT were measured by fluorimetric methods modified from Laverty and Taylor (1968), Haubrich and Denzer (1973), and Curzon and Green (1970). 2.2.3. Serotonin turnover--probenecid test Fifteen min before an i.p. injection of 200 mg/kg probenecid, the test c o m p o u n d was administered to rats at an oral dose of 1 mg/ kg. The animals were decapitated 2 h after probenecid administration. Brain 5-hydroxyindoleacetic acid (5-HIAA) was measured by a fluorimetric method (Curzon and Green, 1970). Effect on 5-HT turnover could be estimated from 5-HIAA accumulation in animals receiving probenecid with or without the test compound. 2.2.4. Potentiation o f 5-hydroxytryptophan (5-HTP) behavioural effects in mice The test c o m p o u n d was administered orally
DU 24565, A SELECTIVE 5-HT UPTAKE INHIBITOR
197
in a range of doses to isolated male Swiss albino mice (20-28g), 1 h before the i.p. injection of a threshold dose of 150 mg/kg of d,I-5-HTP. Thirty minutes after the 5-HTP injection the mice were observed individually and the following behaviour parameters were scored: stereotyped head-searching movements (0 to 4); hindlimb abduction (0 to 2); tremor (0 to 4); escape tendency (0 or 1); forelimb clonus (0 to 2); lordosis (0 to 2). From the results, the EDs0 could be determined plotting log dose against total group score on a linear scale the ED50 is the dose required to potentiate the 5-HTP effect to 50% of the maximal score.
pram (Lundbeck), desmethylimipramine (DMI; Geigy), DU 24565 and fluvoxamine (Duphar), femoxetine (Ferrosan), fluoxetine (Lilly), H77/77 (AB Biotec), probenecid (MSD), quipazine (Miles), tetrabenazine (Hoffman-La Roche) and zimelidine (Astra). 5-HT, 5-HTP, DA and NA were obtained from KochLight Labs. The radiochemicals were purchased from the Radiochemical Centre, Amersham: 5-hydroxy- [G-3H ] tryptamine • creatinine sulphate, 0.5 Ci/mmol; l-[7-3H]noradrenaline • hydrochloride, 5 Ci/mmol; 3,4dihydroxy-[G-3H]phenylethylamine • hydrochloride, 0.5 Ci/mmol.
2.2.5. Potentiation of 5-HTP hyperthermia in rabbits Male rabbits weighing a b o u t 2.5 kg, accust o m e d to prolonged restraint in a rabbit holder and to continuous temperature measurement, were used. After a pre-test period of 30 min the test c o m p o u n d was injected i.v.; 30 min after administration of the test c o m p o u n d the rectal temperature was measured and 5-HTP was injected i.v. in a dose of 50 mg/kg. Temperature measurements were repeated every 30 min for the next 120 min.
3. Results
2.2.6. Antagonism o f tetrabenazine-induced ptosis in mice The test c o m p o u n d s were administered orally to groups of male mice in a range of doses; 45 min later tetrabenazine was injected subcutaneously at a dose of 80 mg/kg. Ptosis was scored using a ptosis chart (Rubin et al., 1957) 45 min after the tetrabenazine administration. The EDs0, that is the dose of the test c o m p o u n d required to reduce the ptosis score to half that of the control tetrabenazine group could be determined graphically by plotting responses on a probit scale against test dose on a logarithmic scale. 2.3. Drugs Amitriptyline (MSD), chlorimipramine (Geigy), p-chloroamphetamine (Regis), citalo-
3.1. In vitro uptake of biogenic amines ~ 3.1.1. Synaptosomal uptake The in vitro results summarized in table 1 as IC50 values for inhibition of the amine uptake, indicate that DU 24565 can be considered as a potent selective inhibitor of 5-HT uptake. As indicated by the ratio of the ICs0 for NA and for 5-HT uptake, the potency and selectivity are favourable when compared with known 5-HT uptake inhibiting drugs. For comparison desmethylimipramine (DMI) and amitriptyline and the parent c o m p o u n d quipazine are included in the list. In preliminary ex vivo experiments with a limited number of animals, 1 h after an oral dose of 5 mg/kg, DU 24565 caused a decreased uptake of 5-HT by synaptosomal preparations of treated rats. Chlorimipramine and fluoxetine at 10 mg/kg orally were not effective, fluvoxamine at the same dose level and also 1 h after administration, resulted in a diminished 5-HT uptake. 3.1.2. 5-HT uptake by rat blood platelets Fig. 2 reveals a strong effect on platelet 5-HT uptake. The Lineweaver-Burk plot yielded a K m of 0 . 6 p M for 5-HT in the control incubation. DU 24565 at concentra-
198
W.J. VAATSTRA ET AL.
TABLE 1 Synaptosomal amine uptake. ICs0: concentration causing 50% inhibition. The values are the means of at least 2 independent experiments; the over-all standard deviation of the uptake test is about 15%. ICs0 (pM)
DU 24565 Chlorimipramine Fluvoxamine Fluoxetine Zimelidine Femoxetine Citalopram Quipazine Amitriptyline Desmethylimipramine
ICs0 (NA)
5-HT cerebrum
NA hypothalamus
DA c. striatum
ICs0 (5-HT)
0.045 1.0 0.21 1.6 24 2.4 0.63 1.2 3.2 25
66 5.2 41 18 50 13 50 1.9 2.2 0.12
41 15 47 32 79 40 40 31 32 60
1467 5.2 195 11 2 5.4 79 1.6 0.7 0.0005
I/V. 104 30-
tions o f 10 -8 and 10 -9 M was a p o t e n t inhibitor. As the lines did n o t coincide at o n e p o i n t o n t h e 1 / V axis, t h e t y p e o f inhibition b y D U 2 4 5 6 5 c a n n o t be indicated. This was seen, t o a varying degree, in m o r e e x p e r i m e n t s .
/
/I /t
3.2. In vivo tests 3.2.1. Antagonism o f 5-HT depletion induced by p-chloroamphetamine T h e s t r o n g antagonistic a c t i o n o f DU 2 4 5 6 5 against the decrease in rat brain 5-HT c o n t e n t caused b y t h e d e p l e t o r p-chloroa m p h e t a m i n e is s h o w n in fig. 3. The low oral EDs0 value o f 0.7 m g / k g was f a v o u r a b l e w h e n c o m p a r e d with t h a t o f zimelidine and fluoxetine (EDs0 resp. 4 and 7) and o f chlorimipramine (EDs0 > 20).
10.
I'o
3'o
1/s
s'o. 1o"
Fig. 2. 5-HT uptake by rat blood platelets. Mean and S.E.M. of at least 4 samples; graphs from linear regression (o control; A 10-8 M DU 24565; [] 10 -9 M DU 24565}. S = 5-HT concentration: 0.2-1.8 x 10 -7 M; V = uptake (dpm/2 rain); Km (5-HT) = 0.6 pM; V m a x = ~ 1 0 0 pmol 5-HT/109 platelets/min.
3.2.2. Antagonism H77/77
o f amine depletion
by
H 7 7 / 7 7 , a t y r a m i n e derivative, given in t w o separate doses o f 12.5 m g / k g i.p. to rats, caused a decrease in brain biogenic a m i n e c o n t e n t . A n oral dose o f 1 m g / k g D U 2 4 5 6 5 , 30 min b e f o r e each H 7 7 / 7 7 injection, prot e c t e d against t h e d e p l e t i o n o f 5-HT, b u t h a d n o e f f e c t o n N A a n d D A d e p l e t i o n {table 2).
DU 24565, A S E L E C T I V E 5-HT U P T A K E I N H I B I T O R rat brain
5-HT
199 TABLE 3
pg/g
5-HT t u r n o v e r - - p r o b e n e c i d test. Rat brain 5-HIAA in pg/g, mean and S.E.M., n = 5. D U 24565 at an oral dose of 1 mg/kg was given 15 rain before 200 m g / k g i.p. probenecid; 2 h after probenecid the animals were decapitated. 5-HIAA accumulation (5-HT turnover): 1.8 p m o l / g tissue/h; after DU 2 4 5 6 5 : 0 . 6 3 p m o l / g / h (65% decrease).
0.50
Controls Probenecid D U 24565 D U 24565 + probenecid
0.40
0.58 1.25 0.55 0.79
-+ 0.005 + 0.030 -+ 0.020 -+ 0.005 1
1 p < 0.001 vs. probenecid alone (Student's t-test).
0.30
0.2 0'.3
1:0 oral
3:0
1'0 m g / k g
dose of DU 24565
Fig. 3. Antagonism of 5-HT depletion induced by p - c h l o r o a m p h e t a m i n e . Drug was given 1 h b e f o r e 5 m g / k g i.p. p-Cl-amphetamine, 4 h thereafter t h e animals were decapitated and brain 5-HT was measured. 5-HT levels after 0.3 and 1.0 m g / k g p.o. D U 24565 in c o m b i n a t i o n with p-Cl-amphetamine were significantly (P < 0.05) different f r o m control or p-Cl-amphetamine alone.
The percentage uptake inhibition for 5-HT was 59%, as calculated by the method of Carlsson et al. (1969).
3.2.3. 5-HT turnover as measured by the probenecid method An accepted index for 5-HT turnover is the accumulation of the 5-HT metabolite, 5-HIAA, after its transport from the brain is blocked by probenecid. Our results with rat brain indicated a 5-HT turnover rate of 1.8 pmol/g tissue/h. DU 24565, in a dose of 1 mg/kg orally, diminished this rate to about 35% of its normal value (table 3). The same dose of the test compound without probenecid did not decrease the 5-HIAA content, but a higher dose (10mg/kg orally) caused a significant 20% reduction. 3.2.4. Potentiation of 5-hydroxytryptophan (5-HTP) effects in mice and rabbits In low oral doses DU 24565 potentiated the induction of 5-HT-like behavioural effects in mice caused by threshold doses of the 5-HT
TABLE 2 A n t a g o n i s m of amine depletion induced by H77/77. Rat brain amine levels in gg/g, m e a n and S.E.M., n = 5. Significant amine depletions by H 7 7 / 7 7 ; no effect o f DU 24565. Antagonism of depletion e.g. uptake inhibition for 5-HT: 59%.
Controls H77/77 D U 24565 DU+ H
5-HT
NA
0.50 + 0.018 0.29 -+ 0.017 0.47 -+ 0.034 0.40-+ 0.006 ]
0.18 0.057 0.16 0.056
1 p < 0.001 vs. H 7 7 / 7 7 alone ( S t u d e n t ' s t-test).
DA + 0.011 -+ 0.007 + 0.022 + 0.007
0.62 0.38 0.60 0.31
+ 0.048 -+ 0.015 -+ 0.020 -+ 0.018
200
W.J. V A A T S T R A ET AL.
TABLE 4
rectal oC
Potentiation of 5-HT and NA effects.
temp,
Oral EDs0 values (mg/kg)
5-HTP potentiation DU 24565 Chlorimipramine Fluvoxamine Fluoxetine Zimelidine Femoxetine Citalopram Quipazine Amitriptyline Desmethylimipramine
1.5 84 36 50 20 57 20 _
1
> 100 >320
Tetrabenazine ptosis antagonism 18 12 t> 100 50 30 9 > 100 9 5
o~
44
43
42
0.8
1 Interfered with 5-HTP effects.
p r e c u r s o r 5-HTP (table 4). O t h e r k n o w n 5-HT u p t a k e inhibitors were surprisingly less active in this test; differences o f a f a c t o r o f 20 and m o r e were observed. P o t e n t i a t i o n o f 5-HTPi n d u c e d h y p e r t h e r m i a in rabbits was evident a f t e r i n t r a v e n o u s doses as low as 10 pg/kg o f DU 2 4 5 6 5 (fig. 4). T e m p e r a t u r e rises o f 5°C resulting in h y p e r t h e r m i c deaths were observed at doses o f 30 pg/kg. R e f e r e n c e c o m p o u n d s were m u c h less active in this respect: 300 pg/ kg i.v. c h l o r i m i p r a m i n e and f l u o x e t i n e (data n o t s h o w n ) gave t e m p e r a t u r e rises o f a b o u t 1°C.
3.2.5. Antagonism of tetrabenazine-induced ptosis in mice A n t a g o n i s m o f t e t r a b e n a z i n e - i n d u c e d ptosis in mice (table 4) is used as an i n d e x for NAp o t e n t i a t i n g effects. D e s m e t h y l i m i p r a m i n e , a NA-selective tricyclic a n t i d e p r e s s a n t and amit r i p t y l i n e were effective in this test as s h o w n b y the low EDs0. T h e 5-HT selective u p t a k e inhibitors had a higher EDs0, a l t h o u g h the differences were less t h a n s h o u l d be e x p e c t e d f r o m results o f o t h e r in vitro and in vivo models. DU 2 4 5 6 5 s h o w e d less antagonism of the t e t r a b e n a z i n e e f f e c t t h a n c h l o r i m i p r a m i n e
41
40
39
38 <
. . . . - 0.5
0.5
1
2"s 1.5
2
3 time
(h)
Fig. 4. Potentiation of 5-HTP hyperthermia in rabbits. Intravenous doses of DU 24565 (© 0.01 mg/kg, n = 4-6; [] 0.03 mg/kg; • 0.1 mg/kg, n = 2) were given 30 min before 50 mg/kg i.v. 5-HTP. Rectal temperature was measured every 30 rain. 5-HTP at this threshold dose (A; n = 11) and DU 24565 alone (not shown) had no effect; A 0.3 mg/kg i.v. chlorimipramine (n = 6); ¢~hyperthermal deaths.
and f e m o x e t i n e , b u t c o m p a r e d with fluvoxamine, f l u o x e t i n e , zimelidine and c i t a l o p r a m m o r e N A p o t e n t i a t i o n was seen.
DU 24565, A SELECTIVE 5-HT UPTAKE INHIBITOR 4. Discussion From a series of quipazine derivates DU 24565, the 6-nitro analogue, emerged as a c o m p o u n d with actions surprisingly different from those of the parent compound. It was a selective 5-HT uptake inhibitor with a p o t e n t oral activity in various biochemical and pharmacological test systems. In in vitro studies using rat brain synaptosomal preparations, strong inhibition of 5-HT uptake (ICs0 = 4 × 10 -8 M) was found, but the synaptosomal uptake of catecholamines was affected at concentrations at least 103 times higher. Compared with other 5-HT-selective uptake inhibitors in our test system this is a favourable ratio. Results from ex vivo tests in which synaptosomal 5-HT uptake by preparations from treated rats was compared with those of untreated rats, indicated a good oral activity. So the test c o m p o u n d penetrates to the central nervous system. In rat blood platelets 5-HT uptake was strongly inhibited by DU 24565 concentrations in the nM range. From Lineweaver-Burk plots no uniform picture w~s obtained. The intercepts of the lines did not allow a clear decision a b o u t the t y p e of inhibition by DU 24565 (fig. 2). There were indications for a deviation from the competitive t y p e of inhibition as seen with many tricyclic antidepressants (Tuomisto, 1974; Trenchard et al., 1975; Lingjaerde, 1976). Evidence for other types of inhibition by non-tricyclic uptake inhibitors such as fluoxetine and trazodone, has been reported by Wielosz et al. (1977). One should be very careful with the interpretation of the results, considering variations in the graphs, differences seen at different inhibitor concentrations and the possibility that other processes are interfering, such as release of 5-HT from the platelets. In in vivo studies, using known techniques to measure uptake inhibition, e.g. antagonism of amine depletion caused by depleting drugs that use the amine pump, p o t e n t actions at low oral doses of DU 24565 are found. In particular, the antagonism of 5-HT depletion
201 caused by p-chloroamphetamine was perceptible at doses below 1 mg/kg orally (the EDs0 was 0.7 mg/kg). Using depletion by 4,adimethyl-m-tyramine (H77/77), good specificity towards 5-HT has been demonstrated as the depletion of the catecholamines was not affected and that of 5-HT was antagonized at 1 mg/kg orally. Pharmacological studies on 5-HT- and NApotentiating effects gave results which were in agreement with those of the neurochemical tests. The effects of the 5-HT-precursor 5-HTP were potentiated in t w o models: mouse behaviour and hyperthermia in the rabbit. In both models DU 24565 was active at surprisingly low doses; the other 5-HT uptake inhibitors were a great deal less active. NA potentiation, as indicated by the EDs0 in the tetrabenazine antagonism test, was higher than expected from the studies on synaptosomal uptake, H77/77 antagonism and others not shown here, but the ratio 5-HTP/tetrabenazine is favourable compared with the other compounds. The NA-selective uptake inhibitor DMI had a substantially lower EDs0 value. Other pharmacological tests (data to be published) did not provide evidence for a possible NA potentiating action of DU 24565, neither were indications found of central and peripheral 5-HT-agonistic actions, anorexogenic or cardiovascular effects. This is in contrast to the parent compound, quipazine which has many pharmacological actions. Also, no inhibition of rat brain monoamineoxidase was found when measured in vitro using different substrates. In conclusion: D U 2 4 5 6 5 , the 6-nitro analogue of quipazine, is a new c o m p o u n d that is more p o t e n t and selective than the known 5-HT uptake inhibitors. It could be a potential antidepressant and can be used as a tool for studying the role of 5-HT in the brain and the periphery. In comparing the potencies of a series of structurally different 5-HTselective uptake inhibitors in different test systems one could speculate a b o u t differences that are not directly related to differences in
202
bio-availability and/or metabolism. These could be differences in mechanism or site of action of these compounds.
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