The role of trazodone metabolism in its inhibitory action on avoidance response

Pharmacology Biochemistry & Behavior, Vol. 23, pp. 137-140, 1985. ©Ankho InternationalInc. Printed in the U.S.A.

0091-3057/85 $3.00 + .00

The Role of Trazodone Metabolism in its Inhibitory Action on Avoidance Response MARIO SANSONE

Istituto di Psicobiologia e Psicofarmacologia, C N R , via R e n o 1, 00198 R o m a , Italy

MIROSLAWA

MELZACKA

AND JERZY VETULANI

Institute o f Pharmacology, P A N , S m e t n a 12, 31-343 Krakow, Poland R e c e i v e d 19 J u l y 1984 SANSONE, M., M. MELZACKA AND J. VETULANI. The role of trazodone metabolism in its inhibitory action on avoidance response. PHARMACOL BIOCHEM BEHAV 23(1) 137-140, 1985.--To assess the role of trazodone metabolism in its depressant action on conditioned avoidance response we investigated whether in the mouse brain 3-chlorophenylpiperazine (CPP) is formed from trazodone, whether trazodone metabolism is affected by a drug metabolism inhibitor, proadifen, and how trazodone, CPP and their combinations act on avoidance responses in proadifen-pretreated mice. It was found that CPP is formed from trazodone in mice, that proadifen inhibits trazodone metabolism, and that the moderate and transient inhibitory effect of trazodone on avoidance responses is dramatically potentiated and prolonged in proadifen-pretreated mice. This effect, and inhibition of unconditioned escape response observed in mice receiving lower doses of trazodone after proadifen pretreatment, were counteracted by CPP. The results indicate that the inhibitory action of trazodone on avoidance response is caused by the parent compound, and that it is brief and moderate because of the rapid metabolism of the drug with formation of CPP which counteracts the depressant effect of the parent compound. Trazodone

Metabolism

Proadifen

3-Chlorophenylpiperazine

T R A Z O D O N E is an antidepressant drug of a new type, which recently aroused considerable interest because of its clinical efficacy and atypical, not fully understood, mechanism of action [15]. Similarly to many classical antidepressants, a single injection of trazodone depresses the conditioned avoidance response in already trained animals [6,16], but this action is brief [ 14]. The compound is a potent serotonin receptor blocking agent [1, 7, 8]. In the body of man [3] and of the rat [2, 9, 11] trazodone is metabolized with formation of 3-chlorophenylpiperazine (CPP) which, in contrast to the parent compound, has serotoninomimetic properties [8, 10, 12, 13]. CPP was also found to facilitate the acquisition of the conditioned avoidance response in mice [18] and to antagonize specifically the depressant action of trazodone [14,17]. We investigated, therefore, the possibility that the relative briefness and mildness of trazodone action is caused by counteraction of the effect of the parent compound by its metabolite, CPP. F o r this purpose we tested whether trazodone administration leads to accumulation of CPP in the brain, and whether the trazodone metabolism is inhibited by a drug metabolism inhibitor, proadifen [5]. After f'mding out that this is the case we investigated how trazodone, CPP and their combinations affect the already established avoidance responding in the shuttle-box paradigm in proadifen-pretreated mice. Although the biochemical experiments were conducted using Albino Swiss mice, the behavioral tests were carried out with

Avoidance response

Mice

BALB/c mice, as in this strain the conditioned avoidance responses are easily developed and a high proportion of the population achieves the criterion of the 70% level of avoidance responses [18]. METHOD Male mice of Albino Swiss randombred strain (18-23 g) (Experiment 1) or BALB/c inbred strain (25-30 g) (Experiment 2) were kept under standard laboratory conditions, with free access to food and water. Drugs used. Trazodone hydrochloride (Angelini), 3-chlorophenylpiperazine hydrochloride (CPP; synthesized in the Dept. of Organic Chemistry of the Institute of Pharmacology P.A.N. in Krakow), and proadifen ( S K F 525A; Smith, Kline and French). All drugs were dissolved in 0.9% NaCI solution (saline) and injected intraperitoneally, in a volume of 10 ml/kg. If two drugs were given simultaneously, they were given in a single injection. The controls received saline instead of drugs.

Experiment 1. Biochemical Studies Trazodone metabolism inhibition by proadifen. Mice received saline or proadifen (20--50 mg/kg), followed 2 hr later by trazodone. They were decapitated 60 min after trazodone injection.

137

138

SANSONE, M E L Z A C K A AND V E T U L A N I

Cerebral CPP levels after CPP injection. Mice received 1

ZD

or 2 mg/kg of CPP 60 min before death.

CPP formation from trazodone. Mice received trazodone, 10-50 mg/kg, and were guillotined 60 min later. The whole brain was removed and assayed for trazodone content by a spectrofluorometric method [4] and/or for CPP with a gas-chromatographic method [11].

TZD

0 !

The apparatus consisted of eight automated shuttleboxes, each divided into two 20× 10 cm compartments, connected by a 3×3 cm opening. The eight shuttle-boxes were simultaneously controlled by a single programming unit. A light (10 W) was switched on alternately in the two compartments and used as a conditioned stimulus (CS). The CS preceded the onset of the unconditioned stimulus (US) by 5 sec and overlapped it for 25 sec. The US was an electric shock (0.2 mA) applied continuously to the grid floor. The intertrial interval was 30 sec. An avoidance response was recorded when the animal avoided the US by running into the dark compartment within 5 sec after the onset of the CS. If animals failed to avoid the shock they could escape it by crossing during the US (5 sec). The failure of both avoidance and escape responses in one trial resulted in the loss of the following trial for an individual mouse. Invariably, the mice failing sometimes to escape did not show any avoidance response during the session. The grid floor in the light compartment was electrified until the end of the cycle and therefore intertrial responses were punished. They occurred very rarely, if at all, in trained animals. The mice were trained in the avoidance situation every day. Each daily session consisted of 100 trials and lasted 50 rain. After seven to eight sessions the performance of individual mice reached stable level. Those mice showing not less than 70% of avoidance responses were selected for drug experiments. The selected mice were divided into groups of 8. The test was carried out 24 hr after the last training session, which was regarded as the control (non-drug) session. The mice received saline or proadifen, 50 mg/kg, followed 2 hr later by trazodone and/or CPP (for doses and combinations see Fig. 1). The avoidance test was carried out 30 rain after the last injection, and consisted of 100 trials, carried out without a break for 50 min. For analysis, however, it was treated as if carried out in two blocks of 50 trials each. The effect of the treatment was evaluated by expressing the performance of the mice in the drug session as a percentage of the performance in the control session. The depression of unconditioned escape response (escape failure), which occurred only in proadifen-pretreated trazodonereceiving groups, was recorded and presented as the percentage of the number of trials, for the whole session. The depression of conditioned avoidance responses by drug treatments was analyzed statistically by a three-factor analysis of variance, the factors being proadifen pretreatment (two levels), trazodone dose (four levels), and CPP dose (three levels). Further analysis for individual betweengroup comparisons was carried out with the Duncan multirange test. RESULTS

Experiment 1. Biochemical Studies Trazodone cerebral concentrations were strongly augmented after proadifen pretreatment; the effects of doses of

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FIG. 1. Effects of various doses of 3-chlorophenylpiperazine (CPP) and trazodone (TZD), given 30 min before the avoidance test, alone or in combination, in trained BALB/c mice, pretreated (2 hr) with saline (circles) or proadifen 50 mg/kg (triangles). The results are presented as percentages of the performance in the control session (means-+SEM) in two consecutive blocks of 50 trials. Each group consisted of eight subjects. Full symbols indicate a significant difference (p<0.05) vs. dose 0 of CPP. Asterisks denote a significant difference (p<0.05) between saline and proadifen pretreatment.

35 and 50 mg/kg were similar, as they apparently produced a ceiling effect, augmenting the trazodone level approximately 10-fold in comparison with controls (Table 1). CPP injected intraperitoneally was present in the brain 60 min later in measurable quantities. After trazodone injection CPP appeared in the brain and 1 hr after the injection the molar concentrations of the metabolite were several times higher than those of the parent compound, particularly after lower doses. After 10 mg/kg trazodone the cerebral level 1 hr after the injection was very similar to that observed after injection of 2 mg/kg CPP. Proadifen pretreatment increased the trazodone concentration approxir.:ately 10-fold (both after low and high dose of trazodone) and inhibited the formation of CPP to the extent that the metabolite concentrations fell below the detection limit (Table 2). In an additional experiment we found that after injection of 20 mg/kg trazodone the levels of the parent compound and the metabolite became equal shortly after the injection, as 15 min after trazodone administration the cerebral level of CPP

T R A Z O D O N E - I N D U C E D AVOIDANCE DEPRESSION TABLE 1 THE EFFECTOF PROADIFEN ON TRAZODONECONCENTRATION IN THE MOUSEBRAIN Dose of proadifen (mg/kg IP)

Whole brain trazodone level (nmol/g tissue)

0 20 35 50

9.1± 81.3 ± 91.1 ± 95.1 ±

4.2 17.1 12.9 8.1

Trazodone, 50 mg/kg IP, was given 24 hr after proadifen, and the animals were killed 1 hr after trazodone administration. The data are means ± S.E.M. of 5 animals.

(8.1--+0.6 nmol/g) already exceeded (6.4-+0.9 nmol/g).

that

139 TABLE 2 TRAZODONEAND CPP LEVELS IN THE MOUSE BRAINAFTER ADMINISTRATIONOF CPP AND TRAZODONEALONE OR WITH METABOLISM INHIBITOR,PROADIFEN Cerebral concentrations (nmol/g tissue) Treatment, dose (mg/kg) CPP, 1 CPP, 2 Trazodone, 10 Trazodone, 20 Trazodone, 50 Proadifen, 50 + Trazodone, 10 Proadifen, 50 + Trazodone, 50

Trazodone

CPP

N.T. N.T. 1.07 ± 0.13 1.80 ± 0.20 8.50 ± 2.21 14.15 ± 0.85

0.21 ± 0.04 6.54 ± 0.50 6.09 ± 0.09 21.70 ± 3.20 18.27 ± 2.38 N.D.

94.55 ± 12.86

N.D.

of trazodone

Experiment 2. Behavioral Studies Conditioned avoidance responses. Three-factor analysis of variance, concerning the first 50-trial block, showed significant main effects of proadifen pretreatment, F(1,168)=294.94, p<0.001, trazodone, F(3,168)=66.38, p<0.001, and CPP, F(2,168)=18.38, p<0.001. The analysis also showed significant proadifen × trazodone, F(3,168)= 19.91, p<0.001, and three-factor, F(6,168)= 2.36, p<0.05, interactions. Significant main effects of proadifen, F(1,168)=311.42, p<0.001, trazodone, F(3,168)=42.54, p<0.001, and CPP, F(2,168)= 8.10, p<0.001, were also found in the second 50-trial block, as well as significant proadifen x trazodone, F(3,168)=39.20, p<0.001, and proadifen x CPP, F(2,168) = 3.57, p <0.05, interactions. The results and significances for individual betweengroup comparisons are reported in Fig. 1. Proadifen pretreatment did not affect the conditioned avoidance responses of mice receiving subsequently saline or CPP. Similarly, CPP alone did not affect the established avoidance responses. Trazodone in saline-pretreated mice caused a moderate and transient inhibition of responses: the effect was produced only by higher doses, 5 and 10 mg/kg, and was present only in the first block of 50 trials. This effect was antagonized by simultaneous administration of CPP. A dramatic depression of performance was observed in proadifen-pretreated mice receiving trazodone. Even the dose inactive in saline-pretreated mice, 2 mg/kg, in proadifen-pretreated animals reduced the performance below 10% of that in the control session in the first block of trials, and to approximately 20% in the second block. Doses of 5 and 10 mg/kg abolished completely the avoidance responding. The inhibitory effect of 2 and 5 mg/kg of trazodone in proadifen-pretreated mice was partially counteracted by CPP, but the depression caused by the dose of l0 mg/kg of trazodone was not reversed by 1 or 2 mg/kg of CPP. Even a dose of 5 mg/kg of CPP did not affect the total depression of responding (data not shown). Unconditioned responses (escape failures). Trazodone in saline pretreated mice virtually did not inhibit escapes; even after the dose of 10 mg/kg mean escape failures occurred only in less than 5% of trials. In proadifen-pretreated groups, however, doses of 2, 5 and 10 mg/kg of trazodone produced

CPP and trazodone were given 60 min before decapitation, proadifen was given 24 hr before trazodone. The data are means ± S.E.M. from five animals. N.D.--not detectable. N.T.-not tested.

response failures in 46, 65 and 72% of trials, respectively. CPP antagonized this effect of trazodone, but only for the doses of 2 and 5 mg/kg (21-25% of failures after combinations oftrazodone 2 or 5 mg/kg with CPP, 1 or 2 mg/kg). CPP failed to affect the depressant action of 10 mg/kg of trazodone. DISCUSSION The present findings demonstrate that in mice, similarly as in the rat [2, 9, 11] and man [3], trazodone is biotransformed with formation of CPP, and that this metabolite may modify the action of the parent compound. The metabolism of trazodone and formation of CPP in the mouse seems to be much faster than in other species: apparently the molar cerebral concentrations of CPP become equal to those of trazodone in mice earlier than in rats [1 l]. Furthermore, the studies in man suggest that although cerebral concentrations of CPP after oral treatment with trazodone may reach biologically active levels, the metabolite is formed more slowly than in the rat [3]. As CPP in the mouse brain attains high concentrations in a short time, it is conceivable that it may produce its own biological effects, even those which are opposite to trazodone, because of the definite predominance of the metabolite. The action of CPP on conditioned avoidance responses is an example of such an opposite action. The present data confirm our earlier findings [ 14,17] and suggest that because of CPP formation the depressant action of trazodone is so rapidly antagonized that it disappears in the second half of the test, carried out 55-80 min after the drug administration. This suggestion is corroborated by the data on the effect of proadifen pretreatment. Proadifen, an inhibitor of metabolism of several drugs [5], in a dose used in behavioral experiments, inhibited apparently completely the formation of CPP from trazodone. A tenfold increase in trazodone concentration in the brain suggests also that other pathways of trazodone metabolism were inhibited. Under these conditions the depressant action of trazodone was dramatically

140

SANSONE,

p o t e n t i a t e d , b u t t h a t effect c o u l d b e , to s o m e e x t e n t , prev e n t e d b y s i m u l t a n e o u s a d m i n i s t r a t i o n o f CPP. The depression of conditioned avoidance responding by t r a z o d o n e in p r o a d i f e n - p r e t r e a t e d m i c e w a s so p r o m i n e n t t h a t e v e n its low d o s e s , ineffective in n o n - p r e t r e a t e d mice, p r o d u c e d a p r o f o u n d d e p r e s s i o n o f t h e r e s p o n d i n g , similar in t h e first a n d the s e c o n d h a l f o f the s e s s i o n ( w h i c h i n d i c a t e s t h a t t h e a c t i o n w a s not o n l y p o t e n t i a t e d , b u t also p r o l o n g e d ) . M o r e o v e r , in p r o a d i f e n - p r e t r e a t e d mice t h e d e p r e s s a n t action o f t r a z o d o n e was p o t e n t i a t e d up to the p o i n t o f inhibition o f u n c o n d i t i o n e d e s c a p e r e s p o n s e s . A l t h o u g h C P P g i v e n t o g e t h e r with t r a z o d o n e to p r o a d i f e n - p r e t r e a t e d a n i m a l s ant a g o n i z e d t h e effect o f the p a r e n t c o m p o u n d only partially ( o u r o t h e r , u n p u b l i s h e d results, d e m o n s t r a t e d t h a t a f u r t h e r i n c r e a s e in the C P P d o s e did n o t result in r e v e r s a l o f t r a z o d o n e effect), it s e e m s t h a t t h e o b s e r v e d p o t e n t i a t i o n o f t r a z o d o n e - i n d u c e d d e p r e s s i o n o f m o u s e p e r f o r m a n c e in the

MELZACKA

AND VETULANI

s h u t t l e - b o x p a r a d i g m is r e l a t e d b o t h to a s h a r p i n c r e a s e in t r a z o d o n e c o n c e n t r a t i o n s in t h e b r a i n a n d to t h e a b s e n c e o f its m e t a b o l i t e . T h e i n t e r a c t i o n b e t w e e n t r a z o d o n e a n d C P P m a y be of i m p o r t a n c e for o u r u n d e r s t a n d i n g o f s o m e a s p e c t s of t r a z o d o n e action. T h e p r e s e n t results i n d i c a t e t h a t t r a z o d o n e m e t a b o l i s m significantly affects its a c t i o n o n c o n d i t i o n e d a v o i d a n c e r e s p o n s e s . H o w e v e r , as s o m e species d i f f e r e n c e s in the m e t a b o l i c rate o f t r a z o d o n e a p p a r e n t l y exist, the gene r a l i z a t i o n o f the r e s u l t s f r o m o n e s p e c i e s to a n t h e r s h o u l d be c a r r i e d o u t with g r e a t c a u t i o n . ACKNOWLEDGEMENTS The authors wish to thank Doz. Dr. S. Misztal of the Institute of Pharmacology of the Polish Academy of Sciences in Krakow for synthesis of CPP hydrochloride and Mr. M. Battaglia for skillful assistance.

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