Inhibition of Monoamine Oxidase by Viloxazine in Rats

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F r o m the Department of Pharmacology, University of Regensburg (Federal Republic of Germany)

Inhibition of Monoamine Oxidase by Viloxazine in Rats

By C . Martinez1^, P. D o m i n i a k , F . Kees, and H . Grobecker

Dedicated to Professor D r . Herbert Oelschlager on the occasion of his 65th birthday

Summary: Biochemical and pharmacological investigations about the effect of the antidepressant drug viloxazine (Viva- Ian®) on catecholamine metabolism in rats led to the follow- ing results:

1. Viloxazine exerts a dose and time dependent inhibition of monoamine oxidase activity of brain and liver mitochondrial fraction and tissue homogenates of hypothalamus, heart,

liver, and adrenal glands, both in vitro and after oral and parenteral administration in vivo.

') Present address: 2. M e d i z i n i s c h e K l i n i k , K l i n i k u m M a n n h e i m der Universitat Heidelberg, M a n n h e i m (Federal R e p u b l i c of G e r - many).

2. Consequently, an increase in catecholamine concentra- tions in brain of rats could be observed after pretrealment with viloxazine. In addition brain serotonin concentrations rose and 5-hydroxy-indoleacetic acid was diminished.

3. However, characterization of inhibition of monoamine ox- idase activity by viloxazine in vitro revealed: Compared to the specific inhibitors clorgyline for MAO-A- and pargyline for MAO-B-activity, viloxazine was a very weak inhibitor both for MAO-A and MAO-B in vitro. The type of inhibition was competitive and reversible.

4. From the presented results and the results obtained by other laboratories it is concluded that inhibition of mono- amine oxidase activity by viloxazine, although clearly de- monstrated in animal experiments, may not be the only mechanism for an antidepressant action of the drug in man.

Arzneim.-Forsch. Drug Res. 36 (I), Nr. 5 (I986)j Martinez et al. - Viloxazind

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Zusammenfassung: Hemmung der Monoaminoxidase durch Viloxazin bei Ratten

Biochemische and pharmakologische Untersuehungen tiber die Wirkung des Antidepressivums Viloxazin (Vivalan®) auf den Kateeholamin-Stoffweehsel von Ratten Juhrten zu fol- genden Ergebnissen:

1. Viloxazin verursachte eine dosis- und zeitabhdngige Hemmung der Aktivitdt der Monoaminoxydase der Mito- chondrienfraktion von Gehirn und Leber sowie in Gewebe- homogenaten des Hypothalamus, des Herzens, der Leber und der Nebennieren von Ratten, so wo hi in vitro, a Is auch in vivo, naeh oraler oder parentaler Applikation des Pharma- kons.

2. Als Folge dieser Hemmung der Monoaminoxydase stei- gen die Konzentrationen von Kateeholaminen und von Sero- tonin im Gehirn an, wahrend die 5-Hydroxyindolessigsdure,

das desaminierte Produkt von Serotonin, signijikant vermin- den wurde.

3. Die Charakterisierung des Hemmtyps von Viloxazin in vitro ergab eine kompetitive und reversible Art der Hem- mung. Viloxazin wurde dariiber hinaus als schwacher Hemmstoff der Monoaminoxydase A und B erkannt bei Vergleiehsuntersuchungen mil Clorgylin als Hemmstoff der A-Form der Monoaminoxydase und Pargylin als Hemmstoff der B-Form der Monoaminoxydase\

4. Die vorliegenden Ergebnisse und die Untersuehungen in anderen Laboratorien erlauben jedoch die Schlufifolgerung, dafi trotz nachgewiesener Hemmung der Monoaminoxydase durch Viloxazin im Tierexperiment noch andere Mechanis- men zur antidepressiven Wirkung des Pharmakons beim Menschen beitragen.

Key words: Antidepressants • Monoamine oxidase • Viloxazine, pharmacology • Vivalan®

1. Introduction

It is generally accepted that clinically used antidepressant drugs may exert their pharmacological effects mainly by i n - h i b i t i o n o f the reuptake o f noradrenaline and serotonin (tri- cyclic antidepressant drugs) or by inhibition o f monoamine oxidase ( M A O - i n h i b i t o r s ) , thereby increasing the postsynaptic concentration o f these amines and consequently poten- tiating their transmitter functions. These prominent pharmacological activities o f antidepressant drugs are probably related to their therapeutic actions [2, 4, 13]. In recent years it was observed that persistent high postsynaptic concentrations o f noradrenaline or serotonin can lead to a down regu- lation o f ^-adrenergic receptors or serotoninergic receptors (for review see [12]). In addition a blockade o f alpha- adrenergic receptors or histamine receptors by antidepressant drugs has been described [6, 15]. Concerning the mech- anisms o f recently introduced antidepressant drugs it is o f interest to classify such drugs within the scheme to the cur- rent hypothesis o f their mode o f action, despite several ob- jections against a close correlation between observed biochemical effects and their therapeutic action in patients.

Therefore the a i m o f the present paper was to characterize some pharmacological effects o f viloxazine (2-(2-ethoxy- phenoxymethyl)-2,3,5,6-tetrahydro-1,4-oxazine, Vivalan®2)) in experiments i n rats, especially its effect o f monoamine oxidase activity, characterizing its mode o f i n h i b i t i o n both i n vivo and i n vitro.

2. Methods

: M a l e Sprague-Dawley rats (SIV 50) from D r . Ivanovas, Kisslegg ( F R { G e r m a n y ) , body weight about 150 g, were used. T h e animals were [housed in plastic cages (Makrolon®) in an air-conditioned, light- Jdark-cycled (12 h) r o o m , with lights on from 6 a.m. to 6 p . m . , and

| g i v e n food (Altromin®, standard diet; A l t r o m i n , L a g e / L i p p e , F R

^Germany) and tap water ad l i b i t u m . T h e y received viloxazine i n {various doses orally (stomach tube) and parenterally as indicated

!under results. C o n t r o l s were given 0.9% N a C l . Assay o f m o n o a m i n e

!oxidase was performed radiometrically according to Otsuka and K o - bayashi [12] using various substrates as described under results. In Jsome experiments we used preparations o f m i t o c h o n d r i a from rat brain [11] or rat liver [14] for assay o f monoamine oxidase activity.

Protein was determined according to the method o f Bradford [3].

F o r determination o f catecholamines (noradrenaline, dopamine) and serotonin we used high pressure l i q u i d chromatography [1].

[Data were statistically evaluated, using unpaired Student's t-test.

| R e s u l t s are expressed as mean ± S . E . M .

3. Results

Subcutaneous injection o f v i l o x a z i n e (70 mg/kg) caused a time dependent i n h i b i t i o n o f monoamine oxidase activity i n preparations o f m i t o c h o n d r i a from rat brain (Fig. 1). U s i n g serotonin or /?-phenylethylamine as substrates the m a x i - m u m o f i n h i b i t i o n occurred between 1 and 3 h after a d m i n - istration o f the drug. 7 h after injection o f viloxazine the monoamine oxidase activity was normal when compared to control preparations.

C o n c o m i t a n t l y there was a significant increase i n the concentration o f brain dopamine and noradrenaline (Fig. 2).

The m a x i m u m o f transmitter concentrations run parallel with the i n h i b i t i o n o f monoamine oxidase activity. Investi- gating increasing doses o f viloxazine (35, 70, 105 mg/kg s.c.)

1 h after administration revealed i n a dose dependent i n h i b i -

Fig. 1: I n h i b i t i o n o f m o n o a m i n e oxidase i n m i t o c h o n d r i a l fraction o f h y p o - thalamus o f rats after s.c. injection o f 70 m g / k g v i l o x a z i n e . Substrates: 3H - serotonin = 1 x I O- 8 m o l / 1 ; , 4C - / j - p h e n y l e t h y l a m i n e = I x l O- 5 mol/1.

0.6 0.5 0.4 0.3

7 1 3 7

|) Manufacturer: I C I - P h a r m a , Plankstadt (Federal R e p u b l i c o f G e r -

; many).

Fig. 2: Increase o f c a t e c h o l a m i n e concentrations i n rat brain after s.c. injec- tion o f 70 m g / k g v i l o x a z i n e groups o f 10 rats. * p < 0 . 0 5 , ** p < 0 . 0 1 ,

*** p < 0.005, **** p < 0.001. Left: d o p a m i n e ; right: noradrenaline.

Vrzneim.-Forsch./ Drug Res. 36 (I), Nr. 5 (1986) Martinez et al. - Viloxazine

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% inhibition

30H MAO-A brain

20

10 H

35 70 105

M A O - B x

35 70 105

l i v e r M A O - B

35 70 105 mg/kg viloxazine s.c.

Fig. 3: Inhibition o f the t w o forms o f m o n o a m i n e oxidase i n rat b r a i n a n d liver m i t o c h o n d r i a l fraction 1 h after s.c. injection o f increasing doses o f v i l - o x a z i n e . Substrates: M A O - A = 3H - S e r o t o n i n I O- 8 mol/1, M A O - B = b r a i n :

I O "6 mol/1. liver I O "5 m o l / 1 . , 4C - / ? - p h e n y l e t h v l a m i n e . * p < 0 . 0 5 .

* * p < 0 . 0 1 . * * * p < 0 . 0 0 5 . * * * * p < 0 . 0 0 1 .

100 80 -J 60 40 20

% inhibition

11 10 9 8 7 6 5 U 3 2 1 - log m o l / l

Fig. 4 : Dose-response curves for i n h i b i t i o n o f m o n o a m i n e oxidase from rat b r a i n homogenates b y specific inhibitors o f M A O - A or M A O - B i n c o m p a r - ison to viloxazine. I n c u b a t i o n : 20 m i n , 37cC Left: O . c l o r g y l i n e , substrate:

3H - S e r o t o n i n I O r4 mol/1: pargyline, substrate: 1 4C - p h e n y l e t h y l a m i n e I O "6

mol/1. Right: v i l o x a z i n e , substrate: O . 3H - S e r o t o n i n I O "4 mol/1: 1 4C - p h e n y l e t h y l a m i n e I O "6 mol/1.

tion o f monoamine oxidase i n m i t o c h o n d r i a o f rat liver and hypothalamus using serotonin or /^phenylethylamine as substrates (Fig. 3).

T h e effect of viloxazine after repeated administration (35 mg/kg i.p. daily during 6 days) o n m o n o a m i n e oxidase activity ( M A O activity) i n various organs o f the rat is depicted i n T a b l e 1. T h e i n h i b i t i o n o f M A O was most pronounced i n the rat heart, but also a significant i n h i b i t i o n o f the enzymes could be observed i n hypothalamus, liver, and adrenal gland.

In these experiments tryptamine was used as a substrate for both forms of the enzyme M A O - A and M A O - B . In addition- al experiments we prepared fractions o f m i t o c h o n d r i a from hypothalamus and liver after chronic treatment w i t h v i l o x a - zine (35 mg/kg s.c. daily over a period o f 8 days). In the brain M A O - A activity was inhibited significantly by 14%, M A O - B activity by 23% and in liver M A O - B activity by 9%.

T h e substrates used for M A O - A was³H-Serotonin (IO"⁸ mol/1) and for M A O - B was^{1 4}C - p h e n y l e t h y l a m i n ( 1 0 -⁶ mol/1).

Table 1: I n h i b i t i o n o f m o n o a m i n e oxidase a c t i v i t y i n rat tissue after s.c. injec- tion o f 35 m g / k g v i l o x a z i n e d a i l y d u r i n g 6 days.

C o n t r o l s n V i l o x a z i n e n I n h i b i t i o n

(%) P

H y p o t h a l a m u s 1 6 2 4 ± 26 5 1264 ± 4 3 5 22 ****

Heart 1853 Jr 106 5 1 1 6 4 ± 8 2 5 37 ****

L i v e r 1 2 9 2 ± 35 6 1028 ± 3 2 5 20 ****

A d r e n a l gland 1 7 1 2 ± 27 4 1266 ± 7 5 5 26 ***

Substrate: 1 4C - I r v p t a m i n e 10"- m o l / 1 : x ± Sx = c p m / m i n : n = n u m b e r o f rats:

p = level o f significance: *** p < 0.005. **** p < 0 . 0 0 1 .

A n a l y s i n g the results obtained with serotonin as a substrat in hypothalamus mitochondrial fraction according to L i n e - weaver-Burk we observed a m i x e d type o f i n h i b i t i o n o f monoamine oxidase activity by viloxazine (Fig. 4).

A s a consequence o f significant i n h i b i t i o n o f monoamine oxidase activity in the brain we found increased concentrations o f the putative transmitters, dopamine, noradrenaline and serotonin after acute (Fig. 2), as well as after chronic administration. T h e effect o f viloxazine o n noradrenaline a n d

Table 3: Changes i n serotonin a n d 5-hydroxyindoleacetic acid concentrations in brain o f rats after o r a l a d m i n i s t r a t i o n o f v i l o x a z i n e daily over a period o f 14 days.

Dose (mg/kg)

Serotonin 5 - H y d r o x y i n d o l e a c e t i c acid Dose

(mg/kg) C o n e , (/ug/g) n P C o n e . Gug/g) n P

C o n t r o l s 0.40 ± 0 . 0 1 10 0 . 4 9 ± 0 . 0 1 9

35 0 . 5 8 ± 0 . 0 4 9 **** 0.41 ± 0 . 0 1 9 ****

70 0 . 7 4 ± 0 . 0 6 10 **** 0 . 3 7 ± 0 . 0 1 10 ****

n = n u m b e r o f rats: **** p < 0 . 0 0 5 : x ± Sx-

dopamine levels was dose dependent and more marked on dopamine concentrations (Table 2). A l s o serotonin concentrations i n brain rose significantly and dose dependent after s.c. injection o f viloxazine.

C h r o n i c oral administration o f increasing doses o f viloxazine also caused an increase i n brain serotonin concentrations and a decrease i n 5-hydroxyindoleacetic acid (Table 3).

r = 0 . 9 6 / 1

cpm/mgppoj.

3 -

2 -

•/•

/ ^ > T o. 9 9

/y

-1 1 2 3

Fig. 5: C h a r a c t e r i z a t i o n o f type o f i n h i b i t i o n o f monoamineoxidase by v i l o x a:

zine. Linevveaver-Burk plot. Substrates: 3H - S e r o t o n i n : 1 x I O "3 - 3 x 10~5

mol/1. O n m i t o c h o n d r i a l fraction o f h y p o t h a l a m u s o f pretreated animals 35 m g / k g s.c. daily over a period o f 8 days. • = controls.

Table 2: E l e v a t i o n i n catecholamines a n d serotonin concentrations i n b r a i n o f rats I h after s.c. injection after increasing doses o f v i l o x a z i n e .

Dose (mg/kg)

D o p a m i n e N o r a d r e n a l i n e Serotonin

Dose (mg/kg)

C o n e . Gug/g) n P C o n e , (/ig/g) n P Cone, (ug/g) n P

C o n t r o l s 0 . 4 5 ± 0 . 0 2 10 0 . 3 9 ± 0 . 0 1 10 0 . 4 6 ± 0 . 0 4 10

35 0 . 5 3 ± 0 . 0 3 10 (*) 0 . 4 3 ± 0 . 0 1 10 0 . 6 3 ± 0 . 0 7 10 *

70 0.53 ± 0 . 0 3 10 * 0.43 + 0.01 10 ** 0 . 6 6 ± 0 . 0 5 10 **

105 0 . 5 9 ± 0 . 0 2 10 ** 0 . 4 6 ± 0 . 0 1 9 **** 0 . 7 2 ± 0 . 0 4 10 ****

n = groups of 9-10 rats: (*)p < 0 . 1 . * p < 0.05. ** p < 0.01. ***p < 0.005. * * " p < 0.001 :x ± S,.

Arzneim.-Forsch. Drug Res. 36 (I), Nr. 5 (1986) Martinez et al. - Viloxazine

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Characterization of inhibition o f the monoamine oxidase activity by viloxazine in vitro led to the following results:

1. C o m p a r e d to the specific inhibitors clorgyline for M A O - A and pargyline for M A O - B , viloxazine was a very weak inhibition both for M A O - A and M A O - B (Fig. 5).

2. The type o f inhibition under the in vitro conditions was competitive according to Lineweaver-Burk using serotonin as a substrate and a mitochondrial fraction of hypothalamus (Fig. 6).

In further experiments it could be demonstrated that the inhibition i n vitro under the same conditions as described in legend o f Fig. 6 was reversible [9].

1_ mg protein min Vpmol SHlAA

F i g . 6: In vitro c h a r a c t e r i z a t i o n o f i n h i b i t i o n o f m o n o a m i n e oxidase in m i t o - c h o n d r i a l fraction o f rat h y p o t h a l a m u s by v i l o x a z i n e . K = 9.7 x I O "5 m o l / 1 ; K = 1.8 x I O "4 mol/1.

4. Discussion

T h e results presented in this paper are in agreement with the assumption that viloxazine is capable to inhibit the monoamine oxydase activity of rat tissue in a dose dependent manner, after acute or chronic oral or parenteral administration o f the drug. Moreover, the m a x i m u m o f the inhibitory effect on monoamine oxidase activity runs parallel with an increase in rat brain catecholamines (dopamine, noradrenaline) and serotonin. These results are in disagreement with the conclusion o f L i p p m a n and Pugsley [10] that viloxazine does not inhibit monoamine oxidase in mice. However, these authors observed a potentiation o f the L - d o p a behav- ioral syndrome in the mouse by viloxazine and also an ant- agonism against reserpine induced ptosis and hypothermia in the same species. Since viloxazine also exerts some effects on uptake o f catecholamines, like i m i p r a m i n e , the authors concluded, that uptake inhibition might be the reason for the observed drug action. In addition G r e e n w o o d [9] denied a significant i n h i b i t i o n by viloxazine on monoamine i n h i b i - tory oxidase activity, although he used only tyramine as substrate in his i n vitro experiments and interestingly i n vivo he c o u l d observe a short lasting increase i n brain catechola- m i n e s and serotonin after injection o f a single dose o f v i l o x a - zine. Moreover, L i p p m a n and Pugsley [10] noticed a decrease in concentrations o f deaminated metabolites from 3H - noradrenaline in rat hypothalamus after administration o f 20 mg/kg i.p. viloxazine. T h i s is in agreement with the sig-

nificant i n h i b i t i o n o f monoamine oxidase after administration o f viloxazine in a single dose or repeated doses, not only in b r a i n , but also i n other organs reported under results. W e could also demonstrate that v i l o x a z i n e in vivo (Fig. 1/Fig. 3) and in vitro (Fig. 5) can inhibit monoamine oxidase in the A and B form. In addition the in vitro characterization o f M A O - i n h i b i t i o n by viloxazine resulted in a competitive and reversible type o f i n h i b i t i o n (Fig. 6). However, in v i v o we observed a mixed type o f i n h i b i t i o n o f monoamine oxidase by v i l o x a z i n e after chronic administration (Fig. 4).

T h i s difference might be due to the formation of an active metabolite o f viloxazine, which could shift the type o f i n h i - bition observed in vitro. In agreement with this assumption we observed a biphasic i n h i b i t i o n o f liver monoamine o x i - dase occurring with two peaks o f i n h i b i t i o n 1 and 5 h after 70 mg/kg s.c. v i l o x a z i n e i n a single dose [11].

Despite the high concentration necessary for viloxazine to inhibit the monoamine oxidase completely i n vitro, when compared to clorgyline ( M A O - A ) and pargyline ( M A O - B , Fig. 5), and the high doses used i n vitro, it is likely that i n h i - bition o f m o n o a m i n e oxidase o f both forms i n vivo makes a contribution to the antidepressant effect o f the drug i n patients. In line with this hypothesis a long lasting h a l f life o f viloxazine was reported i n the l i q u o r cerebrospinalis o f de- pressive patients treated with v i l o x a z i n e [5].

In addition a significant decrease i n deaminated metabolites of noradrenaline and dopamine i n urine o f healthy v o l u n - teers after application o f v i l o x a z i n e was reported by G o o d - win et a l . [7] (see also [4]). Since monoamine oxidase i n hu- man brain might exist in form A and B , our results are de- monstrating an i n h i b i t i o n o f both forms by viloxazine could be o f interest for the therapeutic action o f the drug i n man.

5. References

[1] A n d e r s o n , G . M . , Y o u n g , J . G . , Life S c i . 28, 507 (1981) - [2]

Bunney, W . E . Jr., Davis, J . M . , A r c h . G e n . Psych. 13, 483 (1965) - [3] Bradford, M . M . , A n a l . B i o c h e m . 72, 248 (1976) - [4] Baldessa- r i n i , R . J . , A r c h . G e n . P s y c h . 32, 1087 (1975) - [5] E l w a n , O . , A d a m , H . K . , E u r . J . C l i n . P h a r m a c o l . 17, 179 ( 1 9 8 0 ) - [6] G h o s e , K . , T u r n e r , P., Brit. J . C l i n . P h a r m a c o l . 3, 668 (1976) - [7] G o o d - w i n , B . L . , J o h n s o n , R . D . , R u t h r e n , C . R . J . , Sandler, M . , J . Int.

M e d . Res. 3, S u p p l . 3, 57 (1975) - [8] G r e e n , J . P., M a a y a n i , S., N a - ture 269, 163 (1977) - [9] G r e e n w o o d , D . T . , J . Int. M e d . Res. 3, S u p p l . 3, 18 (1975) - [10] L i p p m a n n , W . , Pugsley, T . A . , C a n a d . J . P h y s i o l . P h a r m a c o l . 54, 494 (1976) - [11] M a r t i n e z C o h e n , C , U b e r die W i r k u n g des A n t i d e p r e s s i v u m s V i l o x a z i n a u f die M o n o a m i n o x y d a s e a k t i v i t a t und den G e h a l t an biogenen A m i n e n sympathisch innervierter Organe der Ratte. Inaugural-Dissertation, Regensburg (1982) - [12] O t s u k a , S., K o b a y a s h i , B i o c h e m . P h a r m a - col. 13, 995 (1964) - [13] O z a w a , K . , Seta, K . , T a k e d a , H . , A n d o , K . , H a n d a , H . , A r a k i , C , J . B i o c h e m . 59, 501 (1966) - [14] P a n d y , G N . , D a v i s , J . M . , D r u g D e v e l o p . Res. 3, 393 (1983) - [15]

Schildkraut, J . J . , A m . J . P s y c h . 122, 509 (1965) - [16] Schneider, W . C , J . B i o l . C h e m . 176, 259 ( 1 9 4 8 ) - [17] Snyder, S. H . , P h a r m a - copsychiatry 13, 62 (1980)

Acknowledgement

We are grateful for a gift o f the drug by I C I , Plankstadt ( F R G e r - many).

For the authors: Prof. D r . H . G r o b e c k e r , L e h r s t u h l fiir P h a r m a k o l o - gie, Universitat Regensburg, UniversitatsstraBe 3 1 , D - 8 4 0 0 Regens- burg (Federal R e p u b l i c o f G e r m a n y )

Vrzneim.-Forsch./ Drug Res. 36 (1), Nr. 5 (1986) Martinez et al. - Viloxazine