Neuropharmacology
Vol. 31,No. 4,pp.343-347,1992 Printed in Great Britain
OfE8-3908/92 $5.00+O.OO
Pergamon Press plc
EFFECTS OF SHORT- AND LONG-TERM ADMINISTRATION OF FLUOXETINE ON THE MONOAMINE CONTENT OF RAT BRAIN S. CACCIA, C. FRACASSO,S. GARATTINI,G. Gurso and S. SARATI Istituto di Ricerche Farmacologiche “Mario Negri”, via Eritrea 62, 20157 Milan, Italy (Accepred 4 November 1991)
Summary-The effects of repeated doses of fluoxetine over time and dose-responses of the content of indoles and catecholamines and metabolism, were examined in rats in relation to the concentrations of the parent compound and its active metabolite nortluoxetine in brain. Brains were removed for assays of the regional content of monoamines and concentrations of drugs 24 hr after the last dose on days 1, 7 and 21 of a twice-daily schedule of fIuoxetine (15 mgjkg, i.p.). Measurements were also taken 1 week after the last dose (7.5 and 15 mg/kg, b.i.d.) of the 21-day regimen. On day 1 fluoxetine did not change the content of serotonin (5-HT) but reduced the concentrations of S-hydroxyindolacetic acid (5-HIAA) in the hippocampus and cortex, compatible with the action of a blocker of the uptake of 5-HT. continued injections of fluoxetine, however, sibilantly reduced 5-HT in the brain of the rat, the depletion being significant on days 7 and 21 in the hippocampus and cortex, respectively. The content of indoles remained significantly decreased for at least a week after the last dose of fluoxetine in the 21-day regimen, although the concentrations of 5-HIAA (but not 5-HT) totally recovered at the smaller dose (7.5mg/kg) in all regions of the brain (cortex, hippocampus and striatum). In spite of slight changes in the concentrations and metabolism of dopamine (DA) in the striatum, 24 hr after the last dose (15 mg/kg), treatment with drug had no significant long-term effects on the content of catecholamines in these regions of the brain. There was marked accumulation of the drug and its metabolite in plasma and brain. The monoaminedepleting effect of fluoxetine therefore depended on prolonged exposure of the brain of the rat to large concentrations of the parent compound and its active metabolite, possibly due to saturation of the clearance mechanisms with chronic administration. Although further studies are required to clarify the mechanism(s) of the monoamine-depleting effect of Ruoxetine, these results are consistent with the theory that mechanisms other than uptake of 5-HT, are involved in the neurochemical action of repeated administration of fluoxetine in rats. Key words-fluoxetine,
repeated administration,
brain monoamines, rat.
The antidepressant agent fluoxetine has been found to reduce food intake in various conditions in animals (Goudie, Thornton and Wheeler, 1976; Carruba, Ricciardi, Spano and Mantegazza, 1985; Wong and Fuller, 1987; Wong, Reid and Threlkeld, 1988; Garattini, 1992) and man (Ferguson and Feighner, 1987; Freeman, 1988), with a mechanism suggested to be similar to that of fenfluramine or its more potent d-isomer, the prototype of the so-called “serotoninergic anorectics” (Nathan and Rolland, 1987; Garattini, Bizzi, Caccia, Mennini and Samanin, 1988; Samanin and Garattini, 1990). The anorectic properties are believed to result from its ability to enhance serotoninergic transmission through specific inhibition of the presynaptic reuptake of serotonin (5HT) (Wong and Fuller, 1987; Fuller, Snoddy and Robertson 1988). Fenfluramine, however, is considered to reduce food intake by potentiating the release of 5-HT from nerve terminals, directly or through its active metabolite norfenfluramine and by inhibiting its inactivation by reuptake (Samanin and Garattini, 1990). Compatible with the blockade of uptake of S-HT, fluoxetine at anorectic doses in rats causes a rapid
and persistent decrease in the concentrations of 5HIAA in whole brain and at larger doses, it slightly raises the content of 5-HT (Fuller et a/.,1988). In contrast, repeated doses of fluoxetine (10 mg/kg x 21 days) result in considerable depletion of both 5-HT and 5-HIAA in the cortex of the rat, although short-term treatment had no effect on the content of indoles (Hrdina, 1987). Repeated full anorectic doses of fenfluramine and its d-isomer also reduce both 5-HT and 5-HIAA in the brain of the rat, an effect which persists over time, at doses larger than those causing anorexia (Kleven and Seiden, 1989; Zaczek, Battaglia, Culp, Appel, Contrera and De Souza, 1990). This suggested that the neurochemical actions of repeated administration of fluoxetine, which more closely mimic the therapeutic use of the drug, may involve mechanisms other than inhibition of the uptake of 5-HT. The present study further examined the effects of pharmacologically effective doses of fluoxetine on the serotoniner~c system in brain. The effects of shortand long-term schedules of admi~stration of fluoxetine, in relation to time and doses, on concentrations of 5-HT and its metabolite S-HIAA in discrete areas 343
S. CACCIA et al.
344
of the brain of the rat were compared. The relationships between neurochemical effects on the content of indoles and the concentrations of the parent drug and its active metabolite, norfluoxetine in brain were examined. METHODS
Animals
Male CD-COBS rats (Charles River, Italy), weighing about 125-150 g at the start of the study, were used. Animals were kept under standard laboratory conditions at a constant temperature of 21 f 2°C and relative humidity 60 k 5%, with fixed 12-hr light/ dark cycles and free access to food and water. The animals were acclimatized to the research facility for 1 week before the study. Treatment with drug
At the start of the study, the animals were randomly divided into groups of 5. In a first experiment, they received fluoxetine hydrochloride (dissolved in saline) intraperitoneally at the dose of 15 mg/kg (b.i.d., 30 mg/kg/day) at approx 12-hr intervals for 1, 7 and 21 days. Control animals received the vehicle only and were treated at the same time as the experimental animals. Twenty-four hours after the last injection, the animals were killed by decapitation and the brains were immediately removed, blotted with paper to remove excess surface blood and frozen quickly on dry ice. Areas of the brain (hippocampus, cortex and striatum) were dissected according to Glowinski and Iversen (1966) and stored at -20°C until assayed. In another experiment, the animals received intraperitoneal injections of either saline or fluoxetine hydrochloride, 7.5 or 15 mg/kg for 21 days and were killed by decapitation 7 days after the last injection; the brains were immediately removed and processed as described above. Biochemical assay
Concentrations of 5-HT, 5-HIAA, noradrenaline (NA), dopamine (DA) and its acidic metabolites, homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) in regions of the brain were measured by high-performance liquid chromatography with electrochemical detection (Achilli, Perego and Ponzio, 1985). Briefly, the tissue was homogenized by sonication in 0.1 N HClO,, containing 0.1% NA,SZOS and 10 ng of 3,4_dihydroxybenzylamine hydrobromide, as an internal standard and centrifuged in a Sorvall RC-2B refrigerated centrifuge at 15,000 g for 20 min. The supernatant was filtered through a Millipore filter (type HV, 0.45 pm pore size) and 20 ~1 were injected directly into a reverse phase column (PBondapack C18, 30 cm x 3.9 mm i.d., particle size 7 ,um) for separation of indoles and catecholamines. The following were identified by their retention times and quantified using a C-R6A Chromatopac
Shimadzu integrator: NA, 5.2 min; DOPAC, 6.6 min; DA, 9.6 min; 5-HIAA, 12 min; HVA, 13.6 min; 5-HT, 23 min. Analysis of drug
Concentrations of fluoxetine and norfluoxetine in plasma and areas of the brain were analyzed by the electron capture gas chromatographic procedure, previously described (Caccia, Cappi, Fracasso and Garattini, 1990). Standard curves were prepared daily, using known concentrations of fluoxetine and norfluoxetine. The peak area ratio (compound to internal standard) was plotted against the added concentrations of fluoxetine and norfluoxetine. The slopes of these curves, determined by linear regression analysis, were used to calculate the concentrations of the compound in unknown samples. The precision and reproducibility of the procedure have been described (Caccia et al., 1990). Statistical analysis
Statistical analysis included Student’s t-test for unpaired data and one-way analysis of variance (ANOVA) with post hoc comparisons, using Duncan’s multiple t-test. Probabilities (P) less than 0.05 were considered statistically significant. RESULTS
The content of indoles in the cortex and hippocampus, 24 hr after the last dose of fluoxetine (15 mg/kg, b.i.d.) for 1, 7 and 21 days are set out in Table 1. Daily administration of fluoxetine significantly reduced the content of 5-HT in both areas of brain, the depletion being significant after 7 and 21 days in the hippocampus and cortex, respectively. Fluoxetine also reduced concentrations of 5-HIAA in both areas of brain and the effect was significant after 1 day of treatment. In agreement with previous findings (Caccia et al., 1990) fluoxetine and notiuoxetine concentrated markedly in the brain of the rat, achieving concentrations-comparable in all the regions considered20-40 times those in plasma (data not shown). However, there was a clear accumulation of the parent drug and its active metabolite which, in terms of the 24-hr post-dose concentrations in brain, amounted to approx lo- and loo-fold increases for fluoxetine and 3- and 30-fold for norfluoxetine on days 7 and 21, compared to day 1 (Table 1). No significant changes in the concentrations of NA were found in the cortex and hippocampus, 24 hr after the last injection of fluoxetine (data not shown). Dopamine tended to decrease in the cortex on day 21 but the difference did not reach significance probably because of the marked variability. A significant drop in the content of DA, however, was observed in the striatum, as compared with vehicle-injected rats (Table 2). The striatal content of HVA was signifi-
fluoxetine on the monoamine content of rat brain
Effects of Table I. Content
345
of indoles in cortex and hippocampus after repeated doses of tluoxetine hydrochloride to male rats Concentrations
Content of indole (% of control) Days of treatment
of drug (nmol/g)
S-HIM
FL
NFL
92 + 24 56 f lo** 62 f 6’.
54 f 4’. 39 f 3.’ 50 * 3.’
1 f0.5 10 * 2 97+ 17
19*5 52* 14 571 f 25
102 + 33 104+ 13 55 * lo*+
65 f 13** 54* 11** 46*4**
I f 0.2 a+2 103 f 17
15 f 3 46*3 508*154
5-HT
Hippocampus
I
I 21
Cortex 1 1
21
Fluoxetine hydrochloride was injected intraperitoneally at the dose of I5 mg/kg, (b.i.d.). The rats were killed 24 hr after the last dose. Each value is the mean f SD of 5 animals. **P < 0.01 vs vehicle (Student’s I-test). FL = Fluoxetine; NFL = norfluoxetine.
cantly reduced by the 21-day regimen of fluoxetine. Levels of another metabolite of DA (DOPAC) in the striatum were unchanged. At this time the concentrations of fluoxetine and norfluoxetine in the striaturn were comparable to those in the other two regions (compare Tables 1 and 2). Following up these findings, additional groups of rats were given 7.5 and 15 mg/kg (b.i.d.) or the vehicle for 21 days and killed 7 days later, for the measurement of the content of indoles and catecholamines and concentrations of drug in the hippocampus, cortex and striatum. As shown in Table 3, .5-HT was still reduced, approximately respectively 20 and 30% in the hippocampus and 25 and 50% in the cortex by the 7.5 and 15 mg/kg doses. At the largest dose the concentrations of 5-HIAA brain were still decreased by 30-40% but the 7.5mg/kg dose had no such effect. Fluoxetine was not detectable in plasma and brain but about 100 nmol/g of norfluoxetine were still detected in both regions of the brain. The content of 5-HT and 5-HIAA in the striatum were dose-dependently reduced, compared to vehicletreated rats. No significant changes in striatal content of DA (and NA in other areas of the brain, data not shown) and metabolism were found, with either dose 1 week after the final dose of the 21-day regimen of fluoxetine (Table 4). Repeated doses of fluoxetine dose-dependently slowed the gain in body weight, compared to vehicle (initial body wt 179 f 7 g). Final body weight (day 21) was 297 + 5 g in the vehicle-treated animals and, respectively 268 + 13 g (P < 0.01 vs control) and 199 + 11 g (P < 0.01 vs control; P < 0.01 vs 7.5 mg/kg) in the groups treated with 7.5 and 15 mg/kg of fluoxetine. No significant differences in body weight were noted between rats treated with vehicle and those given 7.5 mg/kg of fluoxetine, 1 Table 2. Effect of repeated administration
week after the last injection (323 f 10 and 314 f 13 g, respectively), whereas rats injected with the largest dose of fluoxetine (247 f 27 g, P < 0.01) still did not attain the weight of the vehicle-treated rats. DISCUSSION
The present results confirm and extend previous findings on the effects of fluoxetine on the content of 5-HT and 5-HIAA in brain of rats. Acute or shortterm (1 day) treatment with pharmacologically active doses of fluoxetine caused a persistent decrease in the content of 5-HIAA but did not significantly affect 5-HT in whole brain (Fuller et al., 1988) or selected areas of the brain (present results). This effect is shared by other 5-HT blockers, with anorectic activity and is consistent with in vitro findings regarding their capacity to inhibit the uptake of 5-HT (Wong and Fuller, 1987; Koe, Weissman, Welch and Browne, 1983). Repeated administration of the same doses of fluoxetine, however, led to pronounced and longlasting decreases in the content of both 5-HT and 5-HIAA in brain, as with fenfluramine and its disomer (Samanin and Garattini, 1990). The content of indoles remained significantly low, for at least a week after the last dose of the 21-day regimen of fluoxetine, although the concentrations of 5-HIAA (but not 5-HT) recovered totally at the smaller dose of drug (7.5 mg/kg). The effect of fluoxetine on DA in brain was different from that on 5-HT. Concentrations of DA and HVA but not DOPAC in brain, 24 hr after the last dose of fluoxetine (15 mg/kg) were decreased but the drug had no significant long-term effects in the areas of brain considered, indicating that its effect on DA was short-lasting and that it did not accumulate. Noradrenaline in brain was never affected by fluoxetine.
of fluoxetine on dopamine and its acid metabolites in the striatum
Content in striatum @g/g)
Concentration
Treatment
DA
DOPAC
HVA
Vehicle Fluoxetine
3.32 + 0.53 2.64 k 0.23*
1.99 f 0.22 2.05 f 0.29
0.56 f 0.06 0.43 f 0.07.
FL a4* 14
of drug (nmol/g) NFL 442*194
Fluoxetine hydrochloride (15 mg/kg) was administered intraperitoneally at approx 12-hr intervals, for 21 days. Assays were done 24 hr after the last dose. Each value is the mean of 5 animals; lP < 0.05 vsvehicle (Student’s t-test).
S.
346 Table 3. The effects of repeated Content Fluoxetine (mg/kg, b.i.d.)
CACCIAet
administration of fluoxetine on the content week after the last dose of indoles (% of control)
5-HT
Cortex 1.5 I5 Hippocampus 1.5 I5 Striatum 7.5 I5
al.
5-HIAA
Concentrations FL
of indoles in brain,
I
of drug (nmol/g) NFL
75 -i_9** 54*6**’
lOSk9 56 + 6”“”
77 & 141 71 + II**
114* I7 71 f lo**“’
109 + 36
80 + l4* 70 + 16’;”
III +20 68 * 12.1””
97 f 35
89 i 6
Fluoxetine hydrochloride was injected at the dose indicated for 21 days. The rats were killed 7 days after the last dose. Each value is the mean f SD of 5 animals. Concentrations of 5-HT and 5-HIAA in vehicle-treated animals were 0.35 f0.04 and 0.25 + 0.04 rg/g in cortex, 0.25 f 0.03 and 0.22 + 0.02 pg/g in the hippocampus and 0.32 It 0.02 and 0.39 k 0.05 @g/g in the striatum. ( - ) = Below the level of quantification. **P < 0.01; *P < 0.05 vs vehicle. r ‘P < 0.01; -P < 0.05 vs 7.5 mg/kg dose.
The mechanism(s) by which fluoxetine depletes the stores of monoamines in brain and their relevance to the functional effects of repeated administration of fluoxetine is still not known. It was recently noted that the concentrations of fluoxetine and norfluoxetine in brain, at anorectic doses, were compatible not only with the blockade of uptake of 5-HT but also with the possibility of an enhancement of release of 5-HT, adding a further mechanism of action to be considered for fluoxetine in rats (Caccia, Bizzi, Coltro, Fracasso, Frittoli, Mennini and Garattini, 1992). Fluoxetine and norfluoxetine share the ability of d-fenfluramine to induce release of tritium from hippocampal synaptosomes, loaded with [‘HIS-HT. However, fluoxetine and d-fenfluramine appear to release 5-HT through different mechanisms. Fluoxetine-induced release is mainly due (80%) to [3H]5HIAA, while d-fenfluramine releases [‘HIS-HT unchanged (70%). Furthermore, the releasing action of fluoxetine, different from that of d-fenfluramine, is unsaturable and does not depend on calcium; the 5-HT releasing action of d-fenfluramine is antagonized by an inhibitor of the uptake of 5-HT, such as indalpine, while the action of fluoxetine is not (Gobbi, Frittoli, Mennini and Garattini, 1992). Finally, fluoxetine is more potent than d-fenfluramine on the binding of [3H]tetrabenazine to synaptosomal vesicles, an effect that suggests impairment Table 4. The effects of repeated administration dopamine and its acid metabolites in the striatum dose Concentrations Treatment (mg/kg, i.p.) Vehicle Fluoxetine Fluoxetine
(7.5) (I 5)
of Ruoxetine on week after the last
in striatum
@g/g)
DA
DOPAC
HVA
3.32 f 0.35 3.11 + 0.24 2.73 f 0.60
I .99 * 0.22 2.04 + 0.35 1.86 rt. 0.33
0.56 f 0.06 0.62 f 0.09 0.48 f 0.13
Fluoxetine hydrochloride or vehicle were injected intraperitoneally at approx 12-hr intervals for 21 days. Assays were made I week after the last dose. Each value is the mean f SD of 5-6 rats. ( - ) = Below the level of quantification. No significant effect of tluoxetine (P > 0.05).
of the capacity of the vesicles to retain 5-HT (Mennini, personal communication). Regional differences in the effects of repeated administration of neurochemical alterations of 5-HT and 5-HIAA were observed in the present study. For example, the hippocampus appeared to be the area most sensitive to the short-term (7 days) indoledepleting effect of fluoxetine, at least at the larger dose tested. At that time the cortex was insensitive to fluoxetine (24 hr after the last dose), becoming equally sensitive (compared to the hippocampus) by 21 days of treatment with drug. This may explain why Hrdina (1987) found reduced concentrations of 5-HT in the frontal cortex of the rat after long-term treatment with fluoxetine (10 mg/kg x 21 days) but no changes in animals treated for 7 days. The kinetic results of the present study also indicate that the regional sensitivity to the chronic effects of fluoxetine were not due to different distribution of drug and/or persistence, as both fluoxetine and norlluoxetine appeared to distribute almost uniformly in the brain of the rat. However, the continued treatment with fluoxetine led to a progressive accumulation of unchanged drug and norlluoxetine in the brain and plasma of the rat (data not shown), at least at the larger doses tested. Overall, there was an approx 3- and more than 30-fold increase in total exposure to drug (fluoxetine + norfluoxetine) on days 7 and 2 1, respectively, compared to day 1. This suggests that the neurochemical effects of fluoxetine were related to and perhaps even caused by the degree of accumulation of the parent compound and its active metabolite in the brain of the rat-with regional differences in sensitivity. In view of the elimination half-life of the parent compound (8-13 hr) and its metabolite (15-16 hr), at comparable acute doses (Caccia et al., 1990), this unexpected accumulation mainly resulted from saturation of clearance mechanisms for fluoxetine and notlluoxetine with chronic administration. Evidence of capacity-limited elimination of the drug and/or its metabolite, emerged from previous single-dose pro-
Effects of Fluoxetine on the monoamine content of rat brain portionality studies of fluoxetine in animals (Caccia et al., 1990; Parli and Hicks, 1974). In man too, hepatic clearance of fluoxetine can be saturated, either by increasing the single dose or during repeated administration (Bergstrom, Lemberger, Farid and Wolen, 1988). The kinetic data raise an important question regarding the role of the metabolite norfluoxetine in the monoamine-depleting actions and in the functional effects of repeated administration of fluoxetine. In spite of its lower potency and selectivity in inhibiting the in uitro uptake of 5-HT, norfluoxetine is at least as active as fluoxetine in reducing food intake (Garattini, 1992; Caccia et al., 1992) further, suggesting that mechanisms other than inhibition of the uptake of 5-HT are involved in the anorectic action of the parent compound. In the light of the accumulation and persistence of nortluoxetine in brain after repeated administration of fluoxetine in rats, the central effects of the active metabolite norfluoxetine, await further investigation. In conclusion, it has been shown that fluoxetine induced a persistent depletion of 5-HT and 5-HIAA in discrete areas of the brain, when given repeatedly at anorectic doses to rats. This depletion may be the result of a combined effect of fluoxetine and its active metabolite, norfluoxetine which accumulate in the brain and affect not only the uptake of 5-HT but also retention of 5-HT in vesicles and release of 5-HT. Acknowledgements-Simona Sarati is the recipient of a fellowship from Banca Popolare di Milano. The skilful technical assistance of Miss M. Anelli is acknowledged.
347
Ferguson J. M. and Feiglmer J. P. (1987) Fluoxetineinduced weight loss in overweight non-depressed humans. Int. J. Obes. 11: Suppl. 3, 163-170. Freeman C. P. L. (1988) Drue treatment for bulimia and bulimia nervosa. ‘Psyc~ophakacology 96: Suppl. 124. Fuller R. W., Snoddy H. D. and Robertson D. W. (1988) Mechanisms of effects of d-fenfluramine on brain serotonin metabolism in rats: Uptake inhibition versus release. Pharmac. Biochem. Behav. 30: 715-721. Garattini S. (1992) Notes on the serotonergic anorectic agents. Progress in Obesity, Proc. Workshop, Erice, Nov.-Dec. 1989. In press. Garattini S., Bizzi A., Caccia S., Mennini T. and Samanin R. (1988) Progress in assessing the role of serotonin in the control of food intake. C/in. Neuropharmac. 11: Suppl. 1, S8-S32. Glowinski J. and Iversen L. L. (1966) Regional studies of catecholamines in the rat brain. I. The disposition of [‘Hhiorepinephrine, [)H]dopamine and [‘Hldopa in various regions of the brain. J. Neurochem. 13: 655-669. Gobbi M., Frittoli E., Mennini T. and Garattini S. (1992) Releasing activities of d-fenfluramine and fluoxetine on rat hippocampal synaptosomes preloaded with [3H]serotonin. Naunyn-Schmiedebergs Archs Pharmac. In press. Goudie A. J., Thornton E. W. and Wheeler T. J. (1976) Effects of Lilly 110140, a specific inhibitor of 5-hydroxytryptamine uptake, on food intake and on 5-hydroxytryptophan-induced anorexia. Evidence for serotoninergic inhibition of feeding. J. Pharm. Pharmac. 28: 318-326. Hrdina P. D. (1987) \ , Reuulation of hieh- and low-affinitv [‘Hlimipramine recogn%ion sites in rat brain by chronic treatment with antidepressants. Eur. J. Pharmac. 138: 159-168.
Kleven M. S. and Seiden L. S. (1989) D-, L-, and DLFenfluramine cause long-lasting depletions of serotonin in rat brain. Bruin Res. 50% 351-353. Koe B. K., Weissman A., Welch W. M. and Browne R. G. (1983) Sertraline, lS,4S-N-methyl-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-1-naphthylamine, a new uptake inhibitor with selectivity for serotonin. J. Pharmac. exp. Ther. 226: 686-700.
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Vol. 31,No. 4,pp.343-347,1992 Printed in Great Britain
OfE8-3908/92 $5.00+O.OO
Pergamon Press plc
EFFECTS OF SHORT- AND LONG-TERM ADMINISTRATION OF FLUOXETINE ON THE MONOAMINE CONTENT OF RAT BRAIN S. CACCIA, C. FRACASSO,S. GARATTINI,G. Gurso and S. SARATI Istituto di Ricerche Farmacologiche “Mario Negri”, via Eritrea 62, 20157 Milan, Italy (Accepred 4 November 1991)
Summary-The effects of repeated doses of fluoxetine over time and dose-responses of the content of indoles and catecholamines and metabolism, were examined in rats in relation to the concentrations of the parent compound and its active metabolite nortluoxetine in brain. Brains were removed for assays of the regional content of monoamines and concentrations of drugs 24 hr after the last dose on days 1, 7 and 21 of a twice-daily schedule of fIuoxetine (15 mgjkg, i.p.). Measurements were also taken 1 week after the last dose (7.5 and 15 mg/kg, b.i.d.) of the 21-day regimen. On day 1 fluoxetine did not change the content of serotonin (5-HT) but reduced the concentrations of S-hydroxyindolacetic acid (5-HIAA) in the hippocampus and cortex, compatible with the action of a blocker of the uptake of 5-HT. continued injections of fluoxetine, however, sibilantly reduced 5-HT in the brain of the rat, the depletion being significant on days 7 and 21 in the hippocampus and cortex, respectively. The content of indoles remained significantly decreased for at least a week after the last dose of fluoxetine in the 21-day regimen, although the concentrations of 5-HIAA (but not 5-HT) totally recovered at the smaller dose (7.5mg/kg) in all regions of the brain (cortex, hippocampus and striatum). In spite of slight changes in the concentrations and metabolism of dopamine (DA) in the striatum, 24 hr after the last dose (15 mg/kg), treatment with drug had no significant long-term effects on the content of catecholamines in these regions of the brain. There was marked accumulation of the drug and its metabolite in plasma and brain. The monoaminedepleting effect of fluoxetine therefore depended on prolonged exposure of the brain of the rat to large concentrations of the parent compound and its active metabolite, possibly due to saturation of the clearance mechanisms with chronic administration. Although further studies are required to clarify the mechanism(s) of the monoamine-depleting effect of Ruoxetine, these results are consistent with the theory that mechanisms other than uptake of 5-HT, are involved in the neurochemical action of repeated administration of fluoxetine in rats. Key words-fluoxetine,
repeated administration,
brain monoamines, rat.
The antidepressant agent fluoxetine has been found to reduce food intake in various conditions in animals (Goudie, Thornton and Wheeler, 1976; Carruba, Ricciardi, Spano and Mantegazza, 1985; Wong and Fuller, 1987; Wong, Reid and Threlkeld, 1988; Garattini, 1992) and man (Ferguson and Feighner, 1987; Freeman, 1988), with a mechanism suggested to be similar to that of fenfluramine or its more potent d-isomer, the prototype of the so-called “serotoninergic anorectics” (Nathan and Rolland, 1987; Garattini, Bizzi, Caccia, Mennini and Samanin, 1988; Samanin and Garattini, 1990). The anorectic properties are believed to result from its ability to enhance serotoninergic transmission through specific inhibition of the presynaptic reuptake of serotonin (5HT) (Wong and Fuller, 1987; Fuller, Snoddy and Robertson 1988). Fenfluramine, however, is considered to reduce food intake by potentiating the release of 5-HT from nerve terminals, directly or through its active metabolite norfenfluramine and by inhibiting its inactivation by reuptake (Samanin and Garattini, 1990). Compatible with the blockade of uptake of S-HT, fluoxetine at anorectic doses in rats causes a rapid
and persistent decrease in the concentrations of 5HIAA in whole brain and at larger doses, it slightly raises the content of 5-HT (Fuller et a/.,1988). In contrast, repeated doses of fluoxetine (10 mg/kg x 21 days) result in considerable depletion of both 5-HT and 5-HIAA in the cortex of the rat, although short-term treatment had no effect on the content of indoles (Hrdina, 1987). Repeated full anorectic doses of fenfluramine and its d-isomer also reduce both 5-HT and 5-HIAA in the brain of the rat, an effect which persists over time, at doses larger than those causing anorexia (Kleven and Seiden, 1989; Zaczek, Battaglia, Culp, Appel, Contrera and De Souza, 1990). This suggested that the neurochemical actions of repeated administration of fluoxetine, which more closely mimic the therapeutic use of the drug, may involve mechanisms other than inhibition of the uptake of 5-HT. The present study further examined the effects of pharmacologically effective doses of fluoxetine on the serotoniner~c system in brain. The effects of shortand long-term schedules of admi~stration of fluoxetine, in relation to time and doses, on concentrations of 5-HT and its metabolite S-HIAA in discrete areas 343
S. CACCIA et al.
344
of the brain of the rat were compared. The relationships between neurochemical effects on the content of indoles and the concentrations of the parent drug and its active metabolite, norfluoxetine in brain were examined. METHODS
Animals
Male CD-COBS rats (Charles River, Italy), weighing about 125-150 g at the start of the study, were used. Animals were kept under standard laboratory conditions at a constant temperature of 21 f 2°C and relative humidity 60 k 5%, with fixed 12-hr light/ dark cycles and free access to food and water. The animals were acclimatized to the research facility for 1 week before the study. Treatment with drug
At the start of the study, the animals were randomly divided into groups of 5. In a first experiment, they received fluoxetine hydrochloride (dissolved in saline) intraperitoneally at the dose of 15 mg/kg (b.i.d., 30 mg/kg/day) at approx 12-hr intervals for 1, 7 and 21 days. Control animals received the vehicle only and were treated at the same time as the experimental animals. Twenty-four hours after the last injection, the animals were killed by decapitation and the brains were immediately removed, blotted with paper to remove excess surface blood and frozen quickly on dry ice. Areas of the brain (hippocampus, cortex and striatum) were dissected according to Glowinski and Iversen (1966) and stored at -20°C until assayed. In another experiment, the animals received intraperitoneal injections of either saline or fluoxetine hydrochloride, 7.5 or 15 mg/kg for 21 days and were killed by decapitation 7 days after the last injection; the brains were immediately removed and processed as described above. Biochemical assay
Concentrations of 5-HT, 5-HIAA, noradrenaline (NA), dopamine (DA) and its acidic metabolites, homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) in regions of the brain were measured by high-performance liquid chromatography with electrochemical detection (Achilli, Perego and Ponzio, 1985). Briefly, the tissue was homogenized by sonication in 0.1 N HClO,, containing 0.1% NA,SZOS and 10 ng of 3,4_dihydroxybenzylamine hydrobromide, as an internal standard and centrifuged in a Sorvall RC-2B refrigerated centrifuge at 15,000 g for 20 min. The supernatant was filtered through a Millipore filter (type HV, 0.45 pm pore size) and 20 ~1 were injected directly into a reverse phase column (PBondapack C18, 30 cm x 3.9 mm i.d., particle size 7 ,um) for separation of indoles and catecholamines. The following were identified by their retention times and quantified using a C-R6A Chromatopac
Shimadzu integrator: NA, 5.2 min; DOPAC, 6.6 min; DA, 9.6 min; 5-HIAA, 12 min; HVA, 13.6 min; 5-HT, 23 min. Analysis of drug
Concentrations of fluoxetine and norfluoxetine in plasma and areas of the brain were analyzed by the electron capture gas chromatographic procedure, previously described (Caccia, Cappi, Fracasso and Garattini, 1990). Standard curves were prepared daily, using known concentrations of fluoxetine and norfluoxetine. The peak area ratio (compound to internal standard) was plotted against the added concentrations of fluoxetine and norfluoxetine. The slopes of these curves, determined by linear regression analysis, were used to calculate the concentrations of the compound in unknown samples. The precision and reproducibility of the procedure have been described (Caccia et al., 1990). Statistical analysis
Statistical analysis included Student’s t-test for unpaired data and one-way analysis of variance (ANOVA) with post hoc comparisons, using Duncan’s multiple t-test. Probabilities (P) less than 0.05 were considered statistically significant. RESULTS
The content of indoles in the cortex and hippocampus, 24 hr after the last dose of fluoxetine (15 mg/kg, b.i.d.) for 1, 7 and 21 days are set out in Table 1. Daily administration of fluoxetine significantly reduced the content of 5-HT in both areas of brain, the depletion being significant after 7 and 21 days in the hippocampus and cortex, respectively. Fluoxetine also reduced concentrations of 5-HIAA in both areas of brain and the effect was significant after 1 day of treatment. In agreement with previous findings (Caccia et al., 1990) fluoxetine and notiuoxetine concentrated markedly in the brain of the rat, achieving concentrations-comparable in all the regions considered20-40 times those in plasma (data not shown). However, there was a clear accumulation of the parent drug and its active metabolite which, in terms of the 24-hr post-dose concentrations in brain, amounted to approx lo- and loo-fold increases for fluoxetine and 3- and 30-fold for norfluoxetine on days 7 and 21, compared to day 1 (Table 1). No significant changes in the concentrations of NA were found in the cortex and hippocampus, 24 hr after the last injection of fluoxetine (data not shown). Dopamine tended to decrease in the cortex on day 21 but the difference did not reach significance probably because of the marked variability. A significant drop in the content of DA, however, was observed in the striatum, as compared with vehicle-injected rats (Table 2). The striatal content of HVA was signifi-
fluoxetine on the monoamine content of rat brain
Effects of Table I. Content
345
of indoles in cortex and hippocampus after repeated doses of tluoxetine hydrochloride to male rats Concentrations
Content of indole (% of control) Days of treatment
of drug (nmol/g)
S-HIM
FL
NFL
92 + 24 56 f lo** 62 f 6’.
54 f 4’. 39 f 3.’ 50 * 3.’
1 f0.5 10 * 2 97+ 17
19*5 52* 14 571 f 25
102 + 33 104+ 13 55 * lo*+
65 f 13** 54* 11** 46*4**
I f 0.2 a+2 103 f 17
15 f 3 46*3 508*154
5-HT
Hippocampus
I
I 21
Cortex 1 1
21
Fluoxetine hydrochloride was injected intraperitoneally at the dose of I5 mg/kg, (b.i.d.). The rats were killed 24 hr after the last dose. Each value is the mean f SD of 5 animals. **P < 0.01 vs vehicle (Student’s I-test). FL = Fluoxetine; NFL = norfluoxetine.
cantly reduced by the 21-day regimen of fluoxetine. Levels of another metabolite of DA (DOPAC) in the striatum were unchanged. At this time the concentrations of fluoxetine and norfluoxetine in the striaturn were comparable to those in the other two regions (compare Tables 1 and 2). Following up these findings, additional groups of rats were given 7.5 and 15 mg/kg (b.i.d.) or the vehicle for 21 days and killed 7 days later, for the measurement of the content of indoles and catecholamines and concentrations of drug in the hippocampus, cortex and striatum. As shown in Table 3, .5-HT was still reduced, approximately respectively 20 and 30% in the hippocampus and 25 and 50% in the cortex by the 7.5 and 15 mg/kg doses. At the largest dose the concentrations of 5-HIAA brain were still decreased by 30-40% but the 7.5mg/kg dose had no such effect. Fluoxetine was not detectable in plasma and brain but about 100 nmol/g of norfluoxetine were still detected in both regions of the brain. The content of 5-HT and 5-HIAA in the striatum were dose-dependently reduced, compared to vehicletreated rats. No significant changes in striatal content of DA (and NA in other areas of the brain, data not shown) and metabolism were found, with either dose 1 week after the final dose of the 21-day regimen of fluoxetine (Table 4). Repeated doses of fluoxetine dose-dependently slowed the gain in body weight, compared to vehicle (initial body wt 179 f 7 g). Final body weight (day 21) was 297 + 5 g in the vehicle-treated animals and, respectively 268 + 13 g (P < 0.01 vs control) and 199 + 11 g (P < 0.01 vs control; P < 0.01 vs 7.5 mg/kg) in the groups treated with 7.5 and 15 mg/kg of fluoxetine. No significant differences in body weight were noted between rats treated with vehicle and those given 7.5 mg/kg of fluoxetine, 1 Table 2. Effect of repeated administration
week after the last injection (323 f 10 and 314 f 13 g, respectively), whereas rats injected with the largest dose of fluoxetine (247 f 27 g, P < 0.01) still did not attain the weight of the vehicle-treated rats. DISCUSSION
The present results confirm and extend previous findings on the effects of fluoxetine on the content of 5-HT and 5-HIAA in brain of rats. Acute or shortterm (1 day) treatment with pharmacologically active doses of fluoxetine caused a persistent decrease in the content of 5-HIAA but did not significantly affect 5-HT in whole brain (Fuller et al., 1988) or selected areas of the brain (present results). This effect is shared by other 5-HT blockers, with anorectic activity and is consistent with in vitro findings regarding their capacity to inhibit the uptake of 5-HT (Wong and Fuller, 1987; Koe, Weissman, Welch and Browne, 1983). Repeated administration of the same doses of fluoxetine, however, led to pronounced and longlasting decreases in the content of both 5-HT and 5-HIAA in brain, as with fenfluramine and its disomer (Samanin and Garattini, 1990). The content of indoles remained significantly low, for at least a week after the last dose of the 21-day regimen of fluoxetine, although the concentrations of 5-HIAA (but not 5-HT) recovered totally at the smaller dose of drug (7.5 mg/kg). The effect of fluoxetine on DA in brain was different from that on 5-HT. Concentrations of DA and HVA but not DOPAC in brain, 24 hr after the last dose of fluoxetine (15 mg/kg) were decreased but the drug had no significant long-term effects in the areas of brain considered, indicating that its effect on DA was short-lasting and that it did not accumulate. Noradrenaline in brain was never affected by fluoxetine.
of fluoxetine on dopamine and its acid metabolites in the striatum
Content in striatum @g/g)
Concentration
Treatment
DA
DOPAC
HVA
Vehicle Fluoxetine
3.32 + 0.53 2.64 k 0.23*
1.99 f 0.22 2.05 f 0.29
0.56 f 0.06 0.43 f 0.07.
FL a4* 14
of drug (nmol/g) NFL 442*194
Fluoxetine hydrochloride (15 mg/kg) was administered intraperitoneally at approx 12-hr intervals, for 21 days. Assays were done 24 hr after the last dose. Each value is the mean of 5 animals; lP < 0.05 vsvehicle (Student’s t-test).
S.
346 Table 3. The effects of repeated Content Fluoxetine (mg/kg, b.i.d.)
CACCIAet
administration of fluoxetine on the content week after the last dose of indoles (% of control)
5-HT
Cortex 1.5 I5 Hippocampus 1.5 I5 Striatum 7.5 I5
al.
5-HIAA
Concentrations FL
of indoles in brain,
I
of drug (nmol/g) NFL
75 -i_9** 54*6**’
lOSk9 56 + 6”“”
77 & 141 71 + II**
114* I7 71 f lo**“’
109 + 36
80 + l4* 70 + 16’;”
III +20 68 * 12.1””
97 f 35
89 i 6
Fluoxetine hydrochloride was injected at the dose indicated for 21 days. The rats were killed 7 days after the last dose. Each value is the mean f SD of 5 animals. Concentrations of 5-HT and 5-HIAA in vehicle-treated animals were 0.35 f0.04 and 0.25 + 0.04 rg/g in cortex, 0.25 f 0.03 and 0.22 + 0.02 pg/g in the hippocampus and 0.32 It 0.02 and 0.39 k 0.05 @g/g in the striatum. ( - ) = Below the level of quantification. **P < 0.01; *P < 0.05 vs vehicle. r ‘P < 0.01; -P < 0.05 vs 7.5 mg/kg dose.
The mechanism(s) by which fluoxetine depletes the stores of monoamines in brain and their relevance to the functional effects of repeated administration of fluoxetine is still not known. It was recently noted that the concentrations of fluoxetine and norfluoxetine in brain, at anorectic doses, were compatible not only with the blockade of uptake of 5-HT but also with the possibility of an enhancement of release of 5-HT, adding a further mechanism of action to be considered for fluoxetine in rats (Caccia, Bizzi, Coltro, Fracasso, Frittoli, Mennini and Garattini, 1992). Fluoxetine and norfluoxetine share the ability of d-fenfluramine to induce release of tritium from hippocampal synaptosomes, loaded with [‘HIS-HT. However, fluoxetine and d-fenfluramine appear to release 5-HT through different mechanisms. Fluoxetine-induced release is mainly due (80%) to [3H]5HIAA, while d-fenfluramine releases [‘HIS-HT unchanged (70%). Furthermore, the releasing action of fluoxetine, different from that of d-fenfluramine, is unsaturable and does not depend on calcium; the 5-HT releasing action of d-fenfluramine is antagonized by an inhibitor of the uptake of 5-HT, such as indalpine, while the action of fluoxetine is not (Gobbi, Frittoli, Mennini and Garattini, 1992). Finally, fluoxetine is more potent than d-fenfluramine on the binding of [3H]tetrabenazine to synaptosomal vesicles, an effect that suggests impairment Table 4. The effects of repeated administration dopamine and its acid metabolites in the striatum dose Concentrations Treatment (mg/kg, i.p.) Vehicle Fluoxetine Fluoxetine
(7.5) (I 5)
of Ruoxetine on week after the last
in striatum
@g/g)
DA
DOPAC
HVA
3.32 f 0.35 3.11 + 0.24 2.73 f 0.60
I .99 * 0.22 2.04 + 0.35 1.86 rt. 0.33
0.56 f 0.06 0.62 f 0.09 0.48 f 0.13
Fluoxetine hydrochloride or vehicle were injected intraperitoneally at approx 12-hr intervals for 21 days. Assays were made I week after the last dose. Each value is the mean f SD of 5-6 rats. ( - ) = Below the level of quantification. No significant effect of tluoxetine (P > 0.05).
of the capacity of the vesicles to retain 5-HT (Mennini, personal communication). Regional differences in the effects of repeated administration of neurochemical alterations of 5-HT and 5-HIAA were observed in the present study. For example, the hippocampus appeared to be the area most sensitive to the short-term (7 days) indoledepleting effect of fluoxetine, at least at the larger dose tested. At that time the cortex was insensitive to fluoxetine (24 hr after the last dose), becoming equally sensitive (compared to the hippocampus) by 21 days of treatment with drug. This may explain why Hrdina (1987) found reduced concentrations of 5-HT in the frontal cortex of the rat after long-term treatment with fluoxetine (10 mg/kg x 21 days) but no changes in animals treated for 7 days. The kinetic results of the present study also indicate that the regional sensitivity to the chronic effects of fluoxetine were not due to different distribution of drug and/or persistence, as both fluoxetine and norlluoxetine appeared to distribute almost uniformly in the brain of the rat. However, the continued treatment with fluoxetine led to a progressive accumulation of unchanged drug and norlluoxetine in the brain and plasma of the rat (data not shown), at least at the larger doses tested. Overall, there was an approx 3- and more than 30-fold increase in total exposure to drug (fluoxetine + norfluoxetine) on days 7 and 2 1, respectively, compared to day 1. This suggests that the neurochemical effects of fluoxetine were related to and perhaps even caused by the degree of accumulation of the parent compound and its active metabolite in the brain of the rat-with regional differences in sensitivity. In view of the elimination half-life of the parent compound (8-13 hr) and its metabolite (15-16 hr), at comparable acute doses (Caccia et al., 1990), this unexpected accumulation mainly resulted from saturation of clearance mechanisms for fluoxetine and notlluoxetine with chronic administration. Evidence of capacity-limited elimination of the drug and/or its metabolite, emerged from previous single-dose pro-
Effects of Fluoxetine on the monoamine content of rat brain portionality studies of fluoxetine in animals (Caccia et al., 1990; Parli and Hicks, 1974). In man too, hepatic clearance of fluoxetine can be saturated, either by increasing the single dose or during repeated administration (Bergstrom, Lemberger, Farid and Wolen, 1988). The kinetic data raise an important question regarding the role of the metabolite norfluoxetine in the monoamine-depleting actions and in the functional effects of repeated administration of fluoxetine. In spite of its lower potency and selectivity in inhibiting the in uitro uptake of 5-HT, norfluoxetine is at least as active as fluoxetine in reducing food intake (Garattini, 1992; Caccia et al., 1992) further, suggesting that mechanisms other than inhibition of the uptake of 5-HT are involved in the anorectic action of the parent compound. In the light of the accumulation and persistence of nortluoxetine in brain after repeated administration of fluoxetine in rats, the central effects of the active metabolite norfluoxetine, await further investigation. In conclusion, it has been shown that fluoxetine induced a persistent depletion of 5-HT and 5-HIAA in discrete areas of the brain, when given repeatedly at anorectic doses to rats. This depletion may be the result of a combined effect of fluoxetine and its active metabolite, norfluoxetine which accumulate in the brain and affect not only the uptake of 5-HT but also retention of 5-HT in vesicles and release of 5-HT. Acknowledgements-Simona Sarati is the recipient of a fellowship from Banca Popolare di Milano. The skilful technical assistance of Miss M. Anelli is acknowledged.
347
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