European Journal of Pharmacology, 52 (1978) 367--374

367

© Elsevier/North-Holland Biomedical Press

E F F E C T S OF VILOXAZINE, ITS OPTICAL ISOMERS AND ITS MAJOR M E T A B O L I T E S ON BIOGENIC AMINE UPTAKE MECHANISMS IN V IT RO AND IN VIVO THOMAS P. BLACKBURN, GEORGE A. FOSTER, DAVID T. GREENWOOD * and RALPH HOWE Research (Biology) Department, ICI Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, U.K.

Received 3 May 1978, revised MS received 28 July 1978, accepted 3 August 1978

T.P. BLACKBURN, G.A. FOSTER, D.T. GREENWOOD and R. HOWE, Effects of viloxazine, its optical isomers and its major metabolites on biogenic amine uptake mechanisms in vitro and in vivo, European J. Pharmacol. 52 (1978) 367--374. Viloxazine hydrochloride (ICI 58,834, VIVALAN) a chemically novel antidepressant, shows selective inhibition of noradrenaline uptake into mouse heart in vivo and into rat brain in vitro. The noradrenaline uptake inhibitory activity resides primarily in one of the two optically active isomers, and it is suggested that in the conformation adopted for uptake by noradrenaline, the aryl and the amino groups are trans. In a comparison of in vivo and in vitro potency, tri- and tetracyclic antidepressants exhibit a good correlation. However, viloxazine possesses higher in vivo activity than would be expected from in vitro studies. The latter finding cannot be readily explained on the basis of known pharmacokinetic or metabolic factors. Antidepressants Conformation

Viloxazine

Noradrenaline

1. I n t r o d u c t i o n Viloxazine h y d r o c h l o r i d e (ICI 58,834) belongs to a series o f chemically novel substances ( Gr eenw ood et al., 1975) and has been shown in both open and double-blind controlled clinical trials to possess antidepressive activity (Peet, 1973; Mahapatra, 1975; Pichot et al., 1975; Ekdawi, 1975). Initial neuropharmacological tests in animals indicated th at the c o m p o u n d possessed properties in c o m m o n with several existing psychotropics but t ha t overall, its profile of activity was unlike any single known drug (Mallion et al., 1972; Gr e e nw ood, 1975; Cox and Tye, 1975). The p r e d o m i n a n t process f or the inactivation of noradrenaline, dopamine and 5-hyd r o x y t r y p t a m i n e (5-HT) at the synaptic level appears to be reuptake into the presynaptic neurone. The majority o f the so-called "tri* All correspondence to be addressed to D.T.G.

Uptake inhibition

Stereospecificity

cyclic" antidepressant drugs has been shown to inhibit this process, in particular t h a t affecting noradrenaline a n d / o r 5-HT, and their therapeutic actions have frequent l y been ascribed to this p r o p e r t y (Glowinski and Axelrod, 1966; Carlsson et al., 1969a, b; Schildkraut and Kety, 1967). Previous studies with viloxazine have largely been c o n d u c t e d in vivo and have dem onst rat ed its ability t o impair the reuptake o f noradrenaline (Lippman and Pugsley, 1976) and consequently to pot ent i at e various noradrenergic p h e n o m e n a , such as cont ract i on of nictitating m e m b r a n e in response to electrical stimulation (Greenwood, 1975). However studies o f its effects on 5-HT upt ake and related p h e n o m e n a have

oo. C2H5 " "

Fig. 1.

Viloxazine

N

HCL

hydrochloride (ICI

58,834).

368

been inconsistent (Mallion et al., 1972) and other possible mechanisms have been proposed (Lippman and Pugsley, 1976; Jones and Roberts, 1978). The present study was performed to determine relative potencies of viloxazine, its optically active isomers and some of its major metabolites on the reuptake process for noradrenaline both in vitro and in vivo, and to investigate possible effects of these c o m p o u n d s on the synaptosomal uptake of dopamine and 5-HT.

2. Materials and methods 2.1. In vitro synaptosomal studies

ABU (strain II) male rats were decapitated and their brains rapidly removed and dissected o n ice according to the m e t h o d of Glowinski and Iversen (1966). For the determination of 5-HT and noradrenaline uptake, the hypothalamus was removed, weighed and homogenized in 10 volumes of ice-cold 0.32 M sucrose in a glass homogenizer with a motorised Teflon pestle, radial clearance 0.1 mm, 1000 rpm. F o r the measurement of dopamine uptake, the striaturn was dissected, weighed and homogenized in 20 volumes of ice-cold sucrose in the same way. The homogenates were centrifuged at 1000 × g for 10 min at 4°C. The supernatant was decanted, and gently stirred to make a uniform suspension. 100 lzl aliquots of the supernatant fluid were added to 10 ml beakers, containing 1.9 ml Krebs-Henseleit bicarbonate buffer in which the [Ca] is reduced by 50%, containing D-glucose (1 mg/ ml), ascorbic acid (0.2 mg/ml), disodium ethylenediaminetetra-acetic acid (EDTA, 0.05 mg/ml) and nialamide (12.5 lzM), and various concentrations of drug. The mixture was incubated with shaking at 37°C for 5 min under an atmosphere of 95% 02--5% CO2. Tritiated amine was added to give a final concentration of 0.1 ~zM, and the incubation continued for a further 5 min. At the end of this period, 500 lzl aliquots of the incubation mix-

T.P. B L A C K B U R N E T AL.

ture were filtered under vacuum through Millipore filters 24 mm diameter, 0.45 ~zM pore size. Each filter was then washed with 5 ml ice-cold saline, and blotted dry. The discs were placed in scintillation vials, containing 2 ml ethoxyethanol and left to dissolve for 30 min. 10 ml of 0.6% Butyl PBD in toluene was added, and the radioactivity measured in a Beckman liquid scintillation counter. "Filter blanks", with no added tissue, and "diffusional blanks", where the incubation mixture was kept at 0°C, were included, and the latter was subtracted from all the experimental samples. Under similar conditions it is known that 85% or more of the synaptosomal content of [3H]-NA, [3H]-DA or [3H]-5-HT was unmetabolised (Snyder and Coyle, 1969; Coyle and Snyder, 1969; Kannengiesser~ et al., 1973). The uptake of [3H]-NA, [3H]-DA and [3H]-5-HT is also known to increase linearly with time for at least 10 min (Snyder and Coyle, 1969; Coyle and Snyder, 1969, Kannengiesser et al., 1973). ICs0 values are expressed as the molar concentration of drug that inhibited the uptake of [3H]-amine by 50% and were determined graphically from a log/probability plot. [3H]NA (9.1 Ci/mmol) [3H]-DA ( 5 C i / m m o l ) [3H]-5-HT (12.4 Ci/mmol) were all obtained from RCC Amersham. 2.2. In vivo studies

Groups of 12 mice were injected s.c. with the test drug 30 min prior to an intravenous injection of [3H]l-noradrenaline HC1 (2.5 #Ci in 0.1 ml saline). Three hours later the mice were sacrificed and the hearts remo;~ed and exsanguinated in saline (containing 50 mg/1 heparin) then 0.25 M sucrose containing 1% EDTA. Hearts were pooled in groups of three, weighed and homogenized in 3 mls of 0.4 M perchloric acid. After centrifugation at 2,000 × g for 15 min, a 0.5 ml aliquot of the supernatant was added to 10 ml dioxan containing naphthalene (100 g/l) and butyl PBD (8 g/l), and counted in a Beckman liquid

V I L O X A Z I N E , ITS M E T A B O L I T E S A N D A M I N E U P T A K E

scintillation counter. A log-probit plot of dose and percentage inhibition was used to calculate EDs0 values (50% inhibition). 2.3. Drugs The following drugs were used: amitriptyline hydrochloride, protriptyline hydrochloride, benztropine mesylate (Merck, Sharp and Dohme); chlordesipramine hydrochloride, chlorimipramine hydrochloride, desipramine hydrochloride, imipramine hydrochloride, maprotiline hydrochloride (Ciba-Geigy); dothiepin hydrochloride (Crookes); iprindole hydrochloride (Wyeth); mianserin hydrochloride (Organon); nomifensine hydrogen maleate (Hoechst); nortriptyline hydrochloride (Lilly); viloxazine hydrochloride and its R and S isomers (ICI). All reagents used were of the highest purity available.

3. Results

3.1. Noradrenaline uptake The EDs0's and ICs0's of standard drugs against noradrenaline uptake are given in table 1, listed in order of potency, and are generally in close agreement with previously published figures (Horn et al., 1971; Callingham, 1967). It can be seen that there is good correlation (corr. coeff. = 0.94, gradient = 0.997) between the in vivo and in vitro results for these standards (fig. 2) despite being assayed in tissues derived from different species. Demethylation of imipramine, amitriptyline and chlorimipramine yields the more potent derivatives, desipramine (DMI), nortriptyline and chlordesipramine. Conversely, chlorination at position 3 of the tricyclic drugs reduces potency against noradrenaline. Also presented in table 1 are the results for viloxazine, its isomers and its major metabolites, the structures of which are presented in table 2. All the metabolites studied are less potent than viloxazine and it appears that methylation at the 4 position, or a carbonyl

369

TABLE 1 I n h i b i t o r y effects o f m o r p h o l i n e s a n d s o m e antid e p r e s s a n t drugs o n t h e u p t a k e o f n o r a d r e n a l i n e in v i t r o a n d in vivo. D r u g / I C I No.

Protriptyline Desipramine Nortriptyline Imipramine Amitriptyline Maprotiline Chlordesipramine Chlorimipramine Mianserin Dothiepin Iprindole Nomifensine

Inhibition of noradrenaline uptake Mouse heart in vivo EDs0 m g / k g

Rat hypothalamic synaptosomes

s.c.

ICso (M)

0.5 0.6 2.3 2.5 4.8 --5.8 14 23 140

1.8 1.5 1.7 1.2 5.4 9.4 5.4 2.7 2.6 5.7 4.0 3.3

× 10 -8 × 10 -8 × 10 -s X 10 -7 X 10 -.8 × 10 -8 × 10 -8 × 10 -7 × 10 -7 × 10 -7 × 10 -6 × 10 -7

Morpholines 7 3 , 3 3 3 (S-isomer) 58,834 (racemic viloxazine) 73,332 (R-isomer) 60,169 71,928 71,976 70,098 80,921 69,322 80,670

1.9 7 100

3.8 × 10 -7 9 × 10 -7 > 1 0 -s 5.8 x 10 -6 8.0 × 10 -6 3.0 × 10 -6 >lO-S >lO-S >lO-S >lO-S

group at position 5, abolishes noradrenaline uptake blocking activity. There is an interesting division in the activity of the two optically active isomers of viloxazine, with the S-isomer apparently solely responsible for the activity of the racemate, while the R-isomer is essentially devoid of activity. 3.2. 5-Hydroxytryptamine and dopamine uptake Results obtained for the inhibition of 5-HT and dopamine uptake by the group of morpholines and the standard drugs are presented

T.P. B L A C K B U R N E T AL.

370 1,000/ /

/

TABLE 3

/

I n h i b i t o r y effects o f a n t i d e p r e s s a n t drugs o n t h e upt a k e o f d o p a m i n e a n d 5 - h y d r o x y t r y p t a m i n e in vitro.

/

• Iprindole

100-

D r u g / I C I No.

I n h i b i t i o n of 5-HT u p t a k e into hypothalamic synaptosomes ICs0 (M)

Chlorimipramine Lilly 1 1 0 , 1 4 0 Quipazine Imipramine Amitryptiline Dothiepin Chlordesipramine Iprindole Mianserin Benztropine Nomifensine 58,834 (racemic viloxazine ) 7 3 , 3 3 3 (S-isomer viloxazine) 73,332 (R-isomer viloxazine ) 60,169 71,928 71,976 70,098 80,921 80,670 69,322

4.3 6.6 1.6 2.1 2.2 3.8 4.4 7.6 7.8

c 73,332 / /

,Dothiepin

/

Inhibition of d o p a m i n e uptake i n t o striatal synaptosomes ICs0 (M)

Mianserin 10-

/

o>

/

Amitriptyline • /

No~ript~/

o Vitoxazine

eCI-IMI ,

Imipramine blCl 73,333

/

r =0.o4

• Protriptyline

O'll~-8

16-7

1~"6

1~-5

in vitro IC 50 (M)

Fig. 2. E f f e c t s o f m o r p b o l i n e s a n d s t a n d a r d antid e p r e s s a n t s o n u p t a k e o f n o r a d r e n a l i n e in vivo and in vitro.

in table 3. The tricyclics chlorinated at position 3 were more potent against 5-HT uptake, while demethylation decreases their activity. Viloxazine and all of the other morpholine c o m p o u n d s studied were inactive against both

X x x x x x x x x

10-8 10 -8 10 -7 10 -7 10 -7 10 -7 10 -7 10 -6 10 -6

2.5 × 10 -s >10 -s

1.9 X 10 -7 7.4 X 10 -7 >10-s

>10 -s

>10-s

>10 -s

>10-s

>10 -s >10 -s >10 -s > 1 0 -s >10 -s >10 -s >10 -s

>10-s >10-s >10-s >10-s >10-s >10-s

TABLE 2 Structures o f v i l o x a z i n e a n d its m a j o r m e t a b o l i t e s . Rl

5-HT and dopamine uptake as measured by this in vitro method.

L..~ N . I ) FI

1:12 R3

4. Discussion

R4

ICI No.

R1

58,834 (viloxazine) 60,169 71,928 71,976 70,098 80,921 80,670 69,322 1

OC 2 H s

H

H

OH OC2H s OC2H s OC 2 H s OC 2 H s OC2H s OC2H s

H OH H H OH H

H H OH H H OH H

1 C a r b o n y l at p o s i t i o n 5.

R2

H

R3

R4 H H H H --CH 3 --CH 3 --CH 3 H

Viloxazine HC1 is chemically unrelated to the tri- or tetracyclic antidepressant drugs, and has been shown in numerous studies to be a clinically effective antidepressant. Although present results demonstrate that it is an effective inhibitor of noradrenaline uptake, both in vitro and in vivo, the level of activity displayed is of a lower order than that associated with the potent noradrenaline uptake inhibitots, such as desipramine (DMI) and pro-

VILOXAZINE, ITS METABOLITES AND AMINE UPTAKE triptyline. The data obtained in vivo by ourselves and others is, however, inconsistent. Thus, viloxazine is p o t e n t in blocking [3H]noradrenaline uptake into mouse and rat heart (Greenwood, 1975; Lippman and Pugsley, 1976) but has little effect on uptake of [3H]-noradrenaline into rat hypothalamus or medulla (Lippman and Pugsley, 1976). Furthermore, whereas the drug has been shown to inhibit the 6-hydroxydopa induced depletion of noradrenaline in mouse brain (Von Voigtlander and Losey, 1976), Garattini (personal communication) has failed to find a significant effect on a 6-hydroxydopamine induced depletion in rat brain. The data we have obtained using rat brain synaptosomes indicate that viloxazine is virtually devoid of uptake inhibitory activity against 5-HT and dopamine. This finding is consistent with the lack of effect of viloxazine on 5-HT uptake into platelets in vitro (Mallion et al., 1972) and against H75/12induced depletion of 5-HT in rat brain (Lippman and Pugsley, 1976). In contrast, Von Voigtl~-~der and Losey ( 1 9 7 6 ) h a v e demonstrated an ability of viloxazine to inhibit p-chloromethylamphetamine-induced release of 5-HT in mouse brain and, in rat brain, Garattini (personal communication) has shown it to markedly reduce a fenfluramine induced depletion of 5-HT. However, viloxazine has been shown to potentiate a 5-hyd r o x y t r y p t o p h a n (5-HTP) induced behavioural syndrome, to increase the 5-HTP facilitation of extensor hindlimb reflex in spinalized rats (Lippman and Pugsley, 1976) and to potentiate the responses of rat cortical neutones to microinontophoretically applied 5-HT (Jones and Roberts, 1977). The evidence, therefore, seems to point to an enhancement of 5-HT activity by viloxazine, by a mechanism other than decreased uptake. A potentiating effect of viloxazine on electrically evoked release of 5-HT from perfused synaptosomes has been reported (Martin et al., 1978) and such an effect could offer one possible explanation. Alternatively, it has been proposed that viloxazine and other antidepres-

371

sants m a y possess an ability to enhance responses to a range of biogenic amines by a post~synaptic mechanism (Bevan et al., 1975a, b; Jones and Roberts, 1978). The data for the potency of various antidepressants against noradrenaline uptake in vivo, correlates well with those for their in vitro noradrenaline uptake blocking activity (fig. 2). The results obtained with viloxazine and its active isomer do, however, suggest a significant deviation in favour of increased in vivo potency (P

Effects of viloxazine, its optical isomers and its major metabolites on biogenic amine uptake mechanisms in vitro and in vivo.

European Journal of Pharmacology, 52 (1978) 367--374 367 © Elsevier/North-Holland Biomedical Press E F F E C T S OF VILOXAZINE, ITS OPTICAL ISOMERS...
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