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Further studies on the increase of striatal homovanillic acid induced by amphetamine and fenfluramine
EUROPEAN JOURNALOF PttARMACOLOGY 19 (1972) 276 280. NORTH-HOLLAND PUBLISHING COMPANY
FURTHER STUDIES ON THE INCREASE OF STRIATAL HOMOVANILL1C ACID INDUCED BY AMPHETAMINE AND FENFLURAMINE A. JORI and D. BERNARDI lstitu to di R iccrche Farmacologiche 'Mario Negri', Via Eritrea 62, 20157 Milan, Italy
Received 23 September 1971,
Accepted 27 April 1972
A. JORI and D. BERNARDI, Further studies o#t the increase oJ striatal homovanillic acid induced by amphetamine and J~,nfluramine, European J. Pharmacol. 19 (1972) 276 280. Amphetamine and fenlluramine increased striatal I IVA levels in rats and mice. Repeated treatments did not reduce the effect of fentluramine, whereas tolerance to amphetamine developed rapidly in rats but not in mice. No cross-tolerance could be demonstrated between ampheramine and fenfluramine, as far as HVA levels were concerned. It is unlikely that this tolerance was due to modification of the metabolism of amphetamine or by a reduction of the monoamine oxidase (MAO) acitivity. The possible differences in the mechanism of action of anaphetamine and fenfluramine are discussed. Amphetamine t:enfluramine
l)opamine-metabolism ttomovanillic acid
Drug tolerance
1. INTRODUCTION
2. MATERIALS AND METHODS
Amphetamine increases the homovanillic acid (HVA) in brain striatal area of several animal species inchiding cats, mice and rats and this effect is thought to be linked with an altered turnover of dopamine and probably also with several pharmacological activities of this stimulant agent (Laverty and Sharman, 1965; Jori and Bernardi, 1969; Bizzi et al., 1970). However, such an effect may 'also be obtained with fenfluramine, an anorexigenic agent de~,oid of any stimulant property in rats (Jori and Bernardi, 1969: Bizzi et al., 1970). Preliminary data have shown that the increase of HVA levels induced by relatively high doses of amphetamine may be abolished if this drug, but not fenfluramine, is previously given at low doses for four days (Jori and Bernardi, 1969). The present report extends these results and permits differentiation of the effects of the two anorexigenic agents.
Female Sprague-Dawley rats (150 -+ 10 g) and Swiss female mice (20 +- 4 g) were housed, 4 per cage (40 X 25 × 18cm), and 5 per cage (25 × 15 × 15 cm), respectively. d-Amphetamine sulphate and d,M'enfluramine HCI, dissolved m saline, were given i.p. 15 mg/kg and the animals were sacrificed 1 hr later. In other experiments, tire drugs were given in lower doses, 5 mg/kg, daily for 4 or 10 days and 24 hr after the last trealment the usual high dose was injected, 1 hr before killing. Homovanillic acid (HVA) was measured on striata pooled from 4 animals according to Anden el al. (1963). Amphetamine was determined in the whole brain according to the method of Axelrod (1954). Monoamine oxidase (MAO} activity was determined on homogenates of whole brain (cellular debris only was removed by centrifugation at 500 rpm) by measuring the oxidation rate of kynuramine recorded
277
A. Jori, D. Bernardi, Effect o f amphetamine and fenfluramine on HVA
with a P e r k i n - E l m e r 124 dual b e a m s p e c t r o p h o t o m e ter, according to the m e t h o d o f Weissbach et al. (1960).
3. R E S U L T S The data reported in table 1 shows that fenfluramine increased striatal H V A c o n c e n t r a t i o n of rats, after acute and sub-chronic t r e a t m e n t , while the el: fect of a m p h e t a m i n e , 15 mg/kg, was reduced w h e n given after daily administration of a m p h e t a m i n e , 5 mg/kg, i.p. or p - O H - a m p h e t a m i n e HBr, 30 mg/kg, i.p., once, or 20 mg/kg, i.p., twice a day, for 4 days; p - O H - A m p h e t a m i n e alone did not affect striatal H V A levels. R e p e a t e d t r e a t m e n t with a m p h e t a m i n e decreased its h y p e r t h e r m i c effect (table 3). The results reported in table 2 indicate that mice
did not develop tolerance to a m p h e t a m i n e for the 2 parameters considered. Similar increases in H V A levels, as well as in b o d y temperature, were in fact obtained when a m p h e t a m i n e was administered to normal or a m p h e t a m i n e - p r e t r e a t e d mice. The results o f the e x p e r i m e n t s reported in table 3 show that tolerance to a m p h e t a m i n e in rats was not d e p e n d e n t on a reduced availability o f the drug in the brain, because similar c o n c e n t r a t i o n s were found after a m p h e t a m i n e had been given acutely, or repeatedly. Moreover, SKF 525 A, which increases brain a m p h e t a m i n e levels, did not m o d i f y the effect o f anap h e t a m i n e on HVA. Analysis of variance in a 2 × 3 factorial design, showed no interaction between SKF 525 A and a m p h e t a m i n e and no effect o f SKF 525 A alone on H V A levels. Instead, individual comparisons within the a m p h e t a m i n e - t r e a t e d groups indicated a significant difference ( p < 0.01) b e t w e e n H V A con-
Table 1 Effect of fenfluramine and amphetamine on striatum HVA levels after acute and sub-chronic treatment in rats. Treatments with drugs were given acutely or 24 hr after the last pretreatment. Rats were sacrificed 1 hr after amphetamine, fenfluramine or p-OHamphetamine. Each figure is the average of at least 4 determinations. Pretreatment (mg/kg, i.p.) X 4 days -
Amphetamine 5 Fenfluramine 5 -
p-OH-amphetamine 30 p-OH-amphetamine 30 p-OH-amphetamine 20X 2
Treatment (mg/kg, i.p.)
Striatum HVA (ng/g~S.E.M.)
Saline Amphetamine 15 Amphetamine 15 Fenfluramine 15 Fenfluramine 15 p-OH-amphetamine 30 Amphetamine 15 Amphetamine 15
160 367 181 320 374 177 188 270 243
+_ 35 _*40* +- 2 -+ 10" _+27* +_ 3 _+21 + 21" _+29
* p < 0.01 versus saline. Table 2 Effect of amphetamine on striatum HVA and body temperature in mice. For treatment see table 1. Determinations
8 6 5 8
Pretreamaent (mg/kg × days)
Amphetamine 5 × 4 Amphetamine 5 X 10
* p < 0.01 in respect to saline
Treatment (mg/kg, i.p.)
Saline Amphetamine 15 Amphetamine 15 Amphetamine 15
Body temperature change (°C+-S.E.M.) after
Striatum HVA (ng/g_+S.E.M.) after
30 min
60 min
60 rain
+ 0.2 _~0.1 + 2.8 + 0.6* + 2.0 +_0.5* + 2.2 + 0.3*
-0.2 + 1.9 + 1.3 + 1.9
140 290 260 250
+ 0.1 + 0.7* + 0.3* + 0.4*
+- 10 +_20* + 20* +- 20*
A. Jori, D. Bernardi. Effect o f amphetamine and fenfluramine on HVA
278
Table 3 Effect of amphetamine on HVA concentration and body temperature after SKF 525 A treatment in rats. The results concerning the IIVA concentration were statistically analysed by a 2X 3 factorial design and the following significances were obtained: Amphetamine treatment: p < 0.01; SKF 525 A treatment: N.S.; interaction amphetamine × SKF 525 A: N.S. Individual comparison between controls and amphetamine groups (Student's t-test) give the following significances: controls versus amphetamine: p < 0.01 ; subchronic amphetamine versus acute amphetamine: p < 0.01. SKF 525 A was given 1 hr before amphetamine. (°) Amphetamine was given daily for 4 days. The last administration was given 24 hr before treatment with 15 mg/kg. All the determinations were performed 60 min after amphetamine, 15 mg/kg. Each figure represents the average of 5 determinations carried out on a pool of 4 rats each. Group
1
Pretreatment SKF 525 A (50 mg/kg, os) -
2 3 4 5 6
+ +
+
Treatment (mg/kg, i.p.)
Striatum HVA (ng/g_+ S.E.M.)
Body temperature change (°C+ S.E.M.)
Brain apmphetamine 0*g/brain± S.E.M.)
Saline Saline Amphetamine 15 Amphetamine 15 (°) Amphetamine 5× 4 Amphetamine 15 (o) Amphetmnine 5 × 4 Amphetamine 15
160 120 350 300
+ 0.5 -0.4 + 3.7 + 4.2
25 +- 3 43 + 3*
+ 10 ± 20 +- 20 + 30
+ 0.l + 0.2 _+0.4 ± 0.3
230 +- 40
+ 2.3 +- 0.5*
30 _+ 2
240 +_ 10
+ 3.0 +- 0.4**
46 +- 2*
* p < 0.01 versus group 3. ** p < 0.05 versus group 3.
Table 4 Effect of repeated treatments with amphetamine on MAO activity in the whole brain in rats. Amphetamine was given daily for 4 days at doses of 5 mg/kg i.p. Each figure is the average of at least 5 determinations. Treatment
Interval between the last treatment and sacrifice (hr)
MAO activity (nm ole s/m g brain protein/hr± S.E.M.)
Saline Amphetamine Amphetamine
-
41 _+6 46 _+6 43 _+5
1 24
Table 5 Effect of repeated amphetamine administration on the increase of ttVA concentration induced by amphetamine and fenfluramine in rats. For treatments see table 1. Each figure is the average of 5 determinations. Pretreatment (mg/kg, i.p.) X 4 days
Amphetamine 5 -
Amphetamine 5
Treatment (rag/kg, i.p. )
ttVA striatum concen tration (ng/g+- S.E.M.)
Saline Amphetamine 15 Amphetamine 15 Fenfluramine 15 Fenfluramine 15
158 400 228 403 479
+- 22 + - 11" _+23 +_ 14" _+59*
* p < 0.01 versus saline. c e n t r a t i o n s a f t e r acute and s u b c h r o n i c t r e a t m e n t s . R e p e a t e d t r e a t m e n t w i t h a m p h e t a m i n e , 5 mg/kg, did n o t reduce the activity o f MAO w h e n e n z y m a t i c activity was m e a s u r e d , with k y n u r a m i n e as substrate in the brain o f rats sacrificed 1 or 24 h r after the last a m p h e t a m i n e injection (table 4). Finally, r e p e a t e d t r e a t m e n t s with a m p h e t a m i n e , w h i c h r e d u c e d the e f f e c t o f a m p h e t a m i n e on H V A , did n o t reduce the e f f e c t o f f e n f l u r a m i n e on H V A (table 5).
4. D I S C U S S I O N Our results c o n f i r m previous r e p o r t s ( L a v e r t y and S h a r m a n , 1965; Jori and Bernardi, 1969; Bizzi et al., 1970) that a m p h e t a m i n e and f e n f l u r a m i n e increase the c o n c e n t r a t i o n o f H V A in the s t r i a t u m . However, tolerance to this e f f e c t d e v e l o p e d w h e n rats were pre-
A. Jori, D. Bernardi, Effect o f amphetamine and Jenfluramine on HVA
treated with repeated lower doses of amphetamine but not fenfluramine; mice did not show tolerance to amphetamine or fenfluramine. The appearance of this tolerance in rats is not dependent on a modified availability of amphetamine in the brain. This is supported by 2 experimental findings: first, rats treated with the same dose of amphetamine, preceded or not, by repeated treatment with the same drug, exhibited similar brain concentrations of amphetamine; second, an increase of brain amphetamine concentrations obtained by blocking the hepatic metabolism with SKF 525 A, did not restore the effect of amphetamine on HVA in tolerant rats. However, the possibility that the availability of amphetamine may have been modified at a subcellular level cannot be excluded. The tolerance to HVA increase is likely to be independent of amphetamine-induced inhibition of monoamine oxidase (MAO) activity. In fact, direct in vitro determinations did not show any impairment of brain MAO (substrate kynuramine) and indirect findings, for example, that fenfluramine was still able to increase HVA after amphetamine pretreatment, are in agreement with such a conclusion. The main metabolic pathway of amphetamine in rats is p-hydroxylation (Axelrod, 1954). p-Hydroxyamphetamine is, in turn, a substrate for dopamine /3-hydroxylase and is converted to p-hydroxynorephedrine (Goldstein and Anagnoste, 1965). Since this metabolite accumulates in peripheral tissues (Goldstein and Anagnoste, 1965) as well as in the central nervous system (Groppetti and Costa, 1969) and may be released by nervous stimulation, it was suggested to be a false transmitter responsible for the rapid development of tolerance to certain amphetamine effects (Gessa et al., 1969), for instance, the hyperthermia (Brodie et al., 1970). Several authors attribute the long-lasting depletion of norepinephrine stores induced by amphetamine, to the presence of p-hydroxynorephedrine (Brodie et al., 1970; Costa and Groppetti, 1970; Breese et al., 1970; Lewander, 1970). Although no direct relationship between this metabolite and dopamine metabolism has been shown, some of the data here reported are in agreement with the hypothesis that the formation of p-hydroxynorephedrine might also be responsible for the appearance of tolerance to the HVA increase. In fact, tolerance to the hyperthermic effect of amphetamine paralleled tolerance to
279
HVA accumulation. Furthermore. mice, which preferentially metabolize d-amphetamine by pathways other than p-hydroxylation (Smith and Dring, 1970), did not exhibit tolerance to amphetamine, as far as hyperthermia and HVA increase were concerned. These data agree with the report of Hill and Horita (1971) that tolerance to amphetamine does not develop in rabbits, a species in which metabolism of amphetamine to p-OH-amphetamine is only a minor metabolic pathway (Dring et al., 1966). When p-hydroxyamphetamine, unable 'per se' to modify HVA levels in the striatum, was given to rats daily for several days, tolerance to the effect of amphetamine on HVA appeared. Finally, fenfluramine, which is not p-hydroxylated, increased HVA without inducing any tolerance. However, rats tolerant to the effect of amphetamine were still responsive to fenfluramine, suggesting that amphetamine and fenfluramine modified striatal HVA levels by different mechanisms. In this respect, the anti-amphetamine acitivity of fenfluramine observed in different experimental conditions is significant (Jespersen and Bonaccorsi, 1969a, b; Berry et al., 1971), although cross tolerance between the anorexic effects of amphetamine and fenfluramine has been reported (Tagliamonte et al., 1969). Differences in the biochemical effects induced by fenfluramine and amphetamine should also be underlined. Fenfluramine acts selectively on the telediencephalic serotoninergic stores causing a long-lasting depletion of serotonin and an increase of serotonin turnover rate while only high doses cause a depletion of catecholamine and an increase of telediencephalic dopamine synthesis (Costa et al., 1971). In contrast, amphetamine does not affect brain serotonin levels and it increases the turnover rate of brain dopamine at doses which do not modify norepinephrine synthesis (Costa and Groppetti, 1970). Such differences in the biochemical activity may explain differences obtained in the pharmacological effects. For instance, it is well known that in rats amphetamine is stimulant while fenfluramine may even show sedative effects (Le Douarec and Neveu, 1970). ACKNOWLEDGEMENT This work is supported by a grant (no. l PO1 GMI 8376-O1 PTR) of the N.I.It.
280
A. Jori, D. Bernardi. LT~l~'ct o f amphetamine and .[~nJluramine on H VA
The technical assistance of Mr. G. Cecchetti is particularly appreciated.
REFERENCES Anden, N.-E., B.-E. Roos and B. Werdinius, 1963, On the occurrence of homovanillic acid in brain and cerebrospinal fluid and its deternrination by a fluorometric method, Life Sci. 2,448. Axelrod, J., 1954, Studies on sympathomimetic amines. II. The biotransformation and physiological disposition of d-amphetamine, d-hydroxyamphetamine and d-methamphetamine, J. Pharmacol. Exptl. Therap. 110, 315. Berry, M.J., R.It. Poyser and M.I. Robertson, 1971, Inhibition by appetite suppressants of the pressor response to (+)-amphetamine in anaesthetized cats, J. Pharm. Pharmacol. 23, 140. Bizzi, A., A. Bonaccorsi, S. Jespersen, A. Jori and S. Garattint, 1970, Pharmacological studies on amphetamine and fenfluramine in: Amphetamines and Related Compounds (Proceedings of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini (Raven Press, New York), p. 5 7 7 - 5 9 5 . Breese, G.R., l.J. Kopin and V.K. Weise, 1970, Effects of amphetamine derivatives on brain dopamine and noradrenaline, Brit. J. Pharmacol. 38, 537. Brodie, B.B., A.K. Cho and G.L. Gessa, 1970, Possible role of p-hydroxynorephedrine in the depletion of norepinephrine induced by d-amphetamine and in tolerance to this drug, in: Amphetamines and Related Compounds (Proceedings of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini (Raven Press, New York) p. 217 230. Costa, E. and A. Groppetti, 1970, Biosynthesis and storage of catecholamines in tissues of rats injected with various doses of d-amphetamine, in: Amphetamines and Related Compounds (Proceeding of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini (Raven Press. NewYork) p. 231 255. Costa, E., A. Groppetti and A. Revuelta, 1971, Action of fenfluramine on monoamine stores of rat tissues, Brit. J. Pharmacol. 41, 57. Dring, L.G., R.L. Smith and R.T. Williams, 1966, The fate of amphetamine in man and other mammals, J. Pharm. Pharmacol. 18,402. Gessa, G i . , A.K. Cho, G.A. ('lay, A. Tagliamonte and B.B. Brodie, 1969, Possible role of p-hydroxynorephedrine in development of tolerance to d-amphetamine, Federation Proc. 28,796.
Goldstein, M. and B. Anagnoste, 1965, The conversion in vivo of d-amphetamine to (+)-p-hydroxynorephedrine, Biochim. Biophys. Acta 107, 166. Groppetti, A. and E. Costa, 1969, Tissue concentrations of p-hydroxynorephedrine in rats injected with d-amphetamine: effect of pretreatment with desipramine, Life Sci. 8, Part 1,653. t till, tt.F. and A Horita, 1971, Inhibition of (+)-amphetamine hyperthermia by blockade of dopamine receptors in rabbits, J. Pharm. Pharmacol. 23,715. Jespersen, J. and A. Bonaccorsi, 1969a, Anti-amphetamine activity of fenfluramine and $992 in the isolated tail artery of the rat, J. Pharm. Pharmacol. 21,776. Jespersen. J. and A. Bonaccorsi, 1969b, Effect of fenfluramine on the d-amphetamine toxicity in mice. European 1. Pharmacol. 8, 364. Jori, A. and D. Bernardi, 1969, Effect of amphetamine and amphetamine-like drugs on homovanillic acid concentration in the brain, J. Pharm. Pharmacol. 21,694. Laverty, R. and D.t:. Sharman, 1965, Modification by drugs of the metabolism of 3,4-dihydroxyphenylethylamine, noradrenaline and 5-hydroxytryptamine in the brain, Brit. J. Pharmacol. 24, 759. Le Douarec, J.C. and C Neveu, 1970, Pharmacology and biochemistry of fenfluramine, in: Amphetamine and Related Compounds (Proceedings of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini (Raven Press, New York) p. 75- 105. Lewander, T., 1970, Catecholamine turn-over studies in chronic amphetamine intoxication, in: Amphetamines and Related Compounds (Proceeding of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini, (Raven Press, New York), p. 317--329. Smith, R.L. and L.G. Dring, 1970, Patterns of metabolism of ~-phenylisopropylamines in man and other species, in: Amphetamines and related compounds (Proceedings of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini (Raven Press, New York) p. 121 139. Tagliamonte, A., P. Tagliamonte and G.L. Gessa, 1969, Cross tolerance to the anorexigenic effect of d-amphetamine, l-amphetamine, fenfluramine and desmethylimipramine in rats, in: Fall Meeting of American Society for Pharmacology and Experimental Therapeutics, Pittsburgh, August 24 28. Weissbach, H., T.E. Smith, J.W. D~ly, B. Witkop and S. Udenfriend, 1960, A rapid spectrophotometric assay of monoamine oxidase based on the rate of disappearance of kynuramine, J. Biol. (?hem. 235, 1160.
FURTHER STUDIES ON THE INCREASE OF STRIATAL HOMOVANILL1C ACID INDUCED BY AMPHETAMINE AND FENFLURAMINE A. JORI and D. BERNARDI lstitu to di R iccrche Farmacologiche 'Mario Negri', Via Eritrea 62, 20157 Milan, Italy
Received 23 September 1971,
Accepted 27 April 1972
A. JORI and D. BERNARDI, Further studies o#t the increase oJ striatal homovanillic acid induced by amphetamine and J~,nfluramine, European J. Pharmacol. 19 (1972) 276 280. Amphetamine and fenlluramine increased striatal I IVA levels in rats and mice. Repeated treatments did not reduce the effect of fentluramine, whereas tolerance to amphetamine developed rapidly in rats but not in mice. No cross-tolerance could be demonstrated between ampheramine and fenfluramine, as far as HVA levels were concerned. It is unlikely that this tolerance was due to modification of the metabolism of amphetamine or by a reduction of the monoamine oxidase (MAO) acitivity. The possible differences in the mechanism of action of anaphetamine and fenfluramine are discussed. Amphetamine t:enfluramine
l)opamine-metabolism ttomovanillic acid
Drug tolerance
1. INTRODUCTION
2. MATERIALS AND METHODS
Amphetamine increases the homovanillic acid (HVA) in brain striatal area of several animal species inchiding cats, mice and rats and this effect is thought to be linked with an altered turnover of dopamine and probably also with several pharmacological activities of this stimulant agent (Laverty and Sharman, 1965; Jori and Bernardi, 1969; Bizzi et al., 1970). However, such an effect may 'also be obtained with fenfluramine, an anorexigenic agent de~,oid of any stimulant property in rats (Jori and Bernardi, 1969: Bizzi et al., 1970). Preliminary data have shown that the increase of HVA levels induced by relatively high doses of amphetamine may be abolished if this drug, but not fenfluramine, is previously given at low doses for four days (Jori and Bernardi, 1969). The present report extends these results and permits differentiation of the effects of the two anorexigenic agents.
Female Sprague-Dawley rats (150 -+ 10 g) and Swiss female mice (20 +- 4 g) were housed, 4 per cage (40 X 25 × 18cm), and 5 per cage (25 × 15 × 15 cm), respectively. d-Amphetamine sulphate and d,M'enfluramine HCI, dissolved m saline, were given i.p. 15 mg/kg and the animals were sacrificed 1 hr later. In other experiments, tire drugs were given in lower doses, 5 mg/kg, daily for 4 or 10 days and 24 hr after the last trealment the usual high dose was injected, 1 hr before killing. Homovanillic acid (HVA) was measured on striata pooled from 4 animals according to Anden el al. (1963). Amphetamine was determined in the whole brain according to the method of Axelrod (1954). Monoamine oxidase (MAO} activity was determined on homogenates of whole brain (cellular debris only was removed by centrifugation at 500 rpm) by measuring the oxidation rate of kynuramine recorded
277
A. Jori, D. Bernardi, Effect o f amphetamine and fenfluramine on HVA
with a P e r k i n - E l m e r 124 dual b e a m s p e c t r o p h o t o m e ter, according to the m e t h o d o f Weissbach et al. (1960).
3. R E S U L T S The data reported in table 1 shows that fenfluramine increased striatal H V A c o n c e n t r a t i o n of rats, after acute and sub-chronic t r e a t m e n t , while the el: fect of a m p h e t a m i n e , 15 mg/kg, was reduced w h e n given after daily administration of a m p h e t a m i n e , 5 mg/kg, i.p. or p - O H - a m p h e t a m i n e HBr, 30 mg/kg, i.p., once, or 20 mg/kg, i.p., twice a day, for 4 days; p - O H - A m p h e t a m i n e alone did not affect striatal H V A levels. R e p e a t e d t r e a t m e n t with a m p h e t a m i n e decreased its h y p e r t h e r m i c effect (table 3). The results reported in table 2 indicate that mice
did not develop tolerance to a m p h e t a m i n e for the 2 parameters considered. Similar increases in H V A levels, as well as in b o d y temperature, were in fact obtained when a m p h e t a m i n e was administered to normal or a m p h e t a m i n e - p r e t r e a t e d mice. The results o f the e x p e r i m e n t s reported in table 3 show that tolerance to a m p h e t a m i n e in rats was not d e p e n d e n t on a reduced availability o f the drug in the brain, because similar c o n c e n t r a t i o n s were found after a m p h e t a m i n e had been given acutely, or repeatedly. Moreover, SKF 525 A, which increases brain a m p h e t a m i n e levels, did not m o d i f y the effect o f anap h e t a m i n e on HVA. Analysis of variance in a 2 × 3 factorial design, showed no interaction between SKF 525 A and a m p h e t a m i n e and no effect o f SKF 525 A alone on H V A levels. Instead, individual comparisons within the a m p h e t a m i n e - t r e a t e d groups indicated a significant difference ( p < 0.01) b e t w e e n H V A con-
Table 1 Effect of fenfluramine and amphetamine on striatum HVA levels after acute and sub-chronic treatment in rats. Treatments with drugs were given acutely or 24 hr after the last pretreatment. Rats were sacrificed 1 hr after amphetamine, fenfluramine or p-OHamphetamine. Each figure is the average of at least 4 determinations. Pretreatment (mg/kg, i.p.) X 4 days -
Amphetamine 5 Fenfluramine 5 -
p-OH-amphetamine 30 p-OH-amphetamine 30 p-OH-amphetamine 20X 2
Treatment (mg/kg, i.p.)
Striatum HVA (ng/g~S.E.M.)
Saline Amphetamine 15 Amphetamine 15 Fenfluramine 15 Fenfluramine 15 p-OH-amphetamine 30 Amphetamine 15 Amphetamine 15
160 367 181 320 374 177 188 270 243
+_ 35 _*40* +- 2 -+ 10" _+27* +_ 3 _+21 + 21" _+29
* p < 0.01 versus saline. Table 2 Effect of amphetamine on striatum HVA and body temperature in mice. For treatment see table 1. Determinations
8 6 5 8
Pretreamaent (mg/kg × days)
Amphetamine 5 × 4 Amphetamine 5 X 10
* p < 0.01 in respect to saline
Treatment (mg/kg, i.p.)
Saline Amphetamine 15 Amphetamine 15 Amphetamine 15
Body temperature change (°C+-S.E.M.) after
Striatum HVA (ng/g_+S.E.M.) after
30 min
60 min
60 rain
+ 0.2 _~0.1 + 2.8 + 0.6* + 2.0 +_0.5* + 2.2 + 0.3*
-0.2 + 1.9 + 1.3 + 1.9
140 290 260 250
+ 0.1 + 0.7* + 0.3* + 0.4*
+- 10 +_20* + 20* +- 20*
A. Jori, D. Bernardi. Effect o f amphetamine and fenfluramine on HVA
278
Table 3 Effect of amphetamine on HVA concentration and body temperature after SKF 525 A treatment in rats. The results concerning the IIVA concentration were statistically analysed by a 2X 3 factorial design and the following significances were obtained: Amphetamine treatment: p < 0.01; SKF 525 A treatment: N.S.; interaction amphetamine × SKF 525 A: N.S. Individual comparison between controls and amphetamine groups (Student's t-test) give the following significances: controls versus amphetamine: p < 0.01 ; subchronic amphetamine versus acute amphetamine: p < 0.01. SKF 525 A was given 1 hr before amphetamine. (°) Amphetamine was given daily for 4 days. The last administration was given 24 hr before treatment with 15 mg/kg. All the determinations were performed 60 min after amphetamine, 15 mg/kg. Each figure represents the average of 5 determinations carried out on a pool of 4 rats each. Group
1
Pretreatment SKF 525 A (50 mg/kg, os) -
2 3 4 5 6
+ +
+
Treatment (mg/kg, i.p.)
Striatum HVA (ng/g_+ S.E.M.)
Body temperature change (°C+ S.E.M.)
Brain apmphetamine 0*g/brain± S.E.M.)
Saline Saline Amphetamine 15 Amphetamine 15 (°) Amphetamine 5× 4 Amphetamine 15 (o) Amphetmnine 5 × 4 Amphetamine 15
160 120 350 300
+ 0.5 -0.4 + 3.7 + 4.2
25 +- 3 43 + 3*
+ 10 ± 20 +- 20 + 30
+ 0.l + 0.2 _+0.4 ± 0.3
230 +- 40
+ 2.3 +- 0.5*
30 _+ 2
240 +_ 10
+ 3.0 +- 0.4**
46 +- 2*
* p < 0.01 versus group 3. ** p < 0.05 versus group 3.
Table 4 Effect of repeated treatments with amphetamine on MAO activity in the whole brain in rats. Amphetamine was given daily for 4 days at doses of 5 mg/kg i.p. Each figure is the average of at least 5 determinations. Treatment
Interval between the last treatment and sacrifice (hr)
MAO activity (nm ole s/m g brain protein/hr± S.E.M.)
Saline Amphetamine Amphetamine
-
41 _+6 46 _+6 43 _+5
1 24
Table 5 Effect of repeated amphetamine administration on the increase of ttVA concentration induced by amphetamine and fenfluramine in rats. For treatments see table 1. Each figure is the average of 5 determinations. Pretreatment (mg/kg, i.p.) X 4 days
Amphetamine 5 -
Amphetamine 5
Treatment (rag/kg, i.p. )
ttVA striatum concen tration (ng/g+- S.E.M.)
Saline Amphetamine 15 Amphetamine 15 Fenfluramine 15 Fenfluramine 15
158 400 228 403 479
+- 22 + - 11" _+23 +_ 14" _+59*
* p < 0.01 versus saline. c e n t r a t i o n s a f t e r acute and s u b c h r o n i c t r e a t m e n t s . R e p e a t e d t r e a t m e n t w i t h a m p h e t a m i n e , 5 mg/kg, did n o t reduce the activity o f MAO w h e n e n z y m a t i c activity was m e a s u r e d , with k y n u r a m i n e as substrate in the brain o f rats sacrificed 1 or 24 h r after the last a m p h e t a m i n e injection (table 4). Finally, r e p e a t e d t r e a t m e n t s with a m p h e t a m i n e , w h i c h r e d u c e d the e f f e c t o f a m p h e t a m i n e on H V A , did n o t reduce the e f f e c t o f f e n f l u r a m i n e on H V A (table 5).
4. D I S C U S S I O N Our results c o n f i r m previous r e p o r t s ( L a v e r t y and S h a r m a n , 1965; Jori and Bernardi, 1969; Bizzi et al., 1970) that a m p h e t a m i n e and f e n f l u r a m i n e increase the c o n c e n t r a t i o n o f H V A in the s t r i a t u m . However, tolerance to this e f f e c t d e v e l o p e d w h e n rats were pre-
A. Jori, D. Bernardi, Effect o f amphetamine and Jenfluramine on HVA
treated with repeated lower doses of amphetamine but not fenfluramine; mice did not show tolerance to amphetamine or fenfluramine. The appearance of this tolerance in rats is not dependent on a modified availability of amphetamine in the brain. This is supported by 2 experimental findings: first, rats treated with the same dose of amphetamine, preceded or not, by repeated treatment with the same drug, exhibited similar brain concentrations of amphetamine; second, an increase of brain amphetamine concentrations obtained by blocking the hepatic metabolism with SKF 525 A, did not restore the effect of amphetamine on HVA in tolerant rats. However, the possibility that the availability of amphetamine may have been modified at a subcellular level cannot be excluded. The tolerance to HVA increase is likely to be independent of amphetamine-induced inhibition of monoamine oxidase (MAO) activity. In fact, direct in vitro determinations did not show any impairment of brain MAO (substrate kynuramine) and indirect findings, for example, that fenfluramine was still able to increase HVA after amphetamine pretreatment, are in agreement with such a conclusion. The main metabolic pathway of amphetamine in rats is p-hydroxylation (Axelrod, 1954). p-Hydroxyamphetamine is, in turn, a substrate for dopamine /3-hydroxylase and is converted to p-hydroxynorephedrine (Goldstein and Anagnoste, 1965). Since this metabolite accumulates in peripheral tissues (Goldstein and Anagnoste, 1965) as well as in the central nervous system (Groppetti and Costa, 1969) and may be released by nervous stimulation, it was suggested to be a false transmitter responsible for the rapid development of tolerance to certain amphetamine effects (Gessa et al., 1969), for instance, the hyperthermia (Brodie et al., 1970). Several authors attribute the long-lasting depletion of norepinephrine stores induced by amphetamine, to the presence of p-hydroxynorephedrine (Brodie et al., 1970; Costa and Groppetti, 1970; Breese et al., 1970; Lewander, 1970). Although no direct relationship between this metabolite and dopamine metabolism has been shown, some of the data here reported are in agreement with the hypothesis that the formation of p-hydroxynorephedrine might also be responsible for the appearance of tolerance to the HVA increase. In fact, tolerance to the hyperthermic effect of amphetamine paralleled tolerance to
279
HVA accumulation. Furthermore. mice, which preferentially metabolize d-amphetamine by pathways other than p-hydroxylation (Smith and Dring, 1970), did not exhibit tolerance to amphetamine, as far as hyperthermia and HVA increase were concerned. These data agree with the report of Hill and Horita (1971) that tolerance to amphetamine does not develop in rabbits, a species in which metabolism of amphetamine to p-OH-amphetamine is only a minor metabolic pathway (Dring et al., 1966). When p-hydroxyamphetamine, unable 'per se' to modify HVA levels in the striatum, was given to rats daily for several days, tolerance to the effect of amphetamine on HVA appeared. Finally, fenfluramine, which is not p-hydroxylated, increased HVA without inducing any tolerance. However, rats tolerant to the effect of amphetamine were still responsive to fenfluramine, suggesting that amphetamine and fenfluramine modified striatal HVA levels by different mechanisms. In this respect, the anti-amphetamine acitivity of fenfluramine observed in different experimental conditions is significant (Jespersen and Bonaccorsi, 1969a, b; Berry et al., 1971), although cross tolerance between the anorexic effects of amphetamine and fenfluramine has been reported (Tagliamonte et al., 1969). Differences in the biochemical effects induced by fenfluramine and amphetamine should also be underlined. Fenfluramine acts selectively on the telediencephalic serotoninergic stores causing a long-lasting depletion of serotonin and an increase of serotonin turnover rate while only high doses cause a depletion of catecholamine and an increase of telediencephalic dopamine synthesis (Costa et al., 1971). In contrast, amphetamine does not affect brain serotonin levels and it increases the turnover rate of brain dopamine at doses which do not modify norepinephrine synthesis (Costa and Groppetti, 1970). Such differences in the biochemical activity may explain differences obtained in the pharmacological effects. For instance, it is well known that in rats amphetamine is stimulant while fenfluramine may even show sedative effects (Le Douarec and Neveu, 1970). ACKNOWLEDGEMENT This work is supported by a grant (no. l PO1 GMI 8376-O1 PTR) of the N.I.It.
280
A. Jori, D. Bernardi. LT~l~'ct o f amphetamine and .[~nJluramine on H VA
The technical assistance of Mr. G. Cecchetti is particularly appreciated.
REFERENCES Anden, N.-E., B.-E. Roos and B. Werdinius, 1963, On the occurrence of homovanillic acid in brain and cerebrospinal fluid and its deternrination by a fluorometric method, Life Sci. 2,448. Axelrod, J., 1954, Studies on sympathomimetic amines. II. The biotransformation and physiological disposition of d-amphetamine, d-hydroxyamphetamine and d-methamphetamine, J. Pharmacol. Exptl. Therap. 110, 315. Berry, M.J., R.It. Poyser and M.I. Robertson, 1971, Inhibition by appetite suppressants of the pressor response to (+)-amphetamine in anaesthetized cats, J. Pharm. Pharmacol. 23, 140. Bizzi, A., A. Bonaccorsi, S. Jespersen, A. Jori and S. Garattint, 1970, Pharmacological studies on amphetamine and fenfluramine in: Amphetamines and Related Compounds (Proceedings of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini (Raven Press, New York), p. 5 7 7 - 5 9 5 . Breese, G.R., l.J. Kopin and V.K. Weise, 1970, Effects of amphetamine derivatives on brain dopamine and noradrenaline, Brit. J. Pharmacol. 38, 537. Brodie, B.B., A.K. Cho and G.L. Gessa, 1970, Possible role of p-hydroxynorephedrine in the depletion of norepinephrine induced by d-amphetamine and in tolerance to this drug, in: Amphetamines and Related Compounds (Proceedings of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini (Raven Press, New York) p. 217 230. Costa, E. and A. Groppetti, 1970, Biosynthesis and storage of catecholamines in tissues of rats injected with various doses of d-amphetamine, in: Amphetamines and Related Compounds (Proceeding of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini (Raven Press. NewYork) p. 231 255. Costa, E., A. Groppetti and A. Revuelta, 1971, Action of fenfluramine on monoamine stores of rat tissues, Brit. J. Pharmacol. 41, 57. Dring, L.G., R.L. Smith and R.T. Williams, 1966, The fate of amphetamine in man and other mammals, J. Pharm. Pharmacol. 18,402. Gessa, G i . , A.K. Cho, G.A. ('lay, A. Tagliamonte and B.B. Brodie, 1969, Possible role of p-hydroxynorephedrine in development of tolerance to d-amphetamine, Federation Proc. 28,796.
Goldstein, M. and B. Anagnoste, 1965, The conversion in vivo of d-amphetamine to (+)-p-hydroxynorephedrine, Biochim. Biophys. Acta 107, 166. Groppetti, A. and E. Costa, 1969, Tissue concentrations of p-hydroxynorephedrine in rats injected with d-amphetamine: effect of pretreatment with desipramine, Life Sci. 8, Part 1,653. t till, tt.F. and A Horita, 1971, Inhibition of (+)-amphetamine hyperthermia by blockade of dopamine receptors in rabbits, J. Pharm. Pharmacol. 23,715. Jespersen, J. and A. Bonaccorsi, 1969a, Anti-amphetamine activity of fenfluramine and $992 in the isolated tail artery of the rat, J. Pharm. Pharmacol. 21,776. Jespersen. J. and A. Bonaccorsi, 1969b, Effect of fenfluramine on the d-amphetamine toxicity in mice. European 1. Pharmacol. 8, 364. Jori, A. and D. Bernardi, 1969, Effect of amphetamine and amphetamine-like drugs on homovanillic acid concentration in the brain, J. Pharm. Pharmacol. 21,694. Laverty, R. and D.t:. Sharman, 1965, Modification by drugs of the metabolism of 3,4-dihydroxyphenylethylamine, noradrenaline and 5-hydroxytryptamine in the brain, Brit. J. Pharmacol. 24, 759. Le Douarec, J.C. and C Neveu, 1970, Pharmacology and biochemistry of fenfluramine, in: Amphetamine and Related Compounds (Proceedings of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini (Raven Press, New York) p. 75- 105. Lewander, T., 1970, Catecholamine turn-over studies in chronic amphetamine intoxication, in: Amphetamines and Related Compounds (Proceeding of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini, (Raven Press, New York), p. 317--329. Smith, R.L. and L.G. Dring, 1970, Patterns of metabolism of ~-phenylisopropylamines in man and other species, in: Amphetamines and related compounds (Proceedings of the Mario Negri Institute for Pharmacological Research Milan, Italy), eds. E. Costa and S. Garattini (Raven Press, New York) p. 121 139. Tagliamonte, A., P. Tagliamonte and G.L. Gessa, 1969, Cross tolerance to the anorexigenic effect of d-amphetamine, l-amphetamine, fenfluramine and desmethylimipramine in rats, in: Fall Meeting of American Society for Pharmacology and Experimental Therapeutics, Pittsburgh, August 24 28. Weissbach, H., T.E. Smith, J.W. D~ly, B. Witkop and S. Udenfriend, 1960, A rapid spectrophotometric assay of monoamine oxidase based on the rate of disappearance of kynuramine, J. Biol. (?hem. 235, 1160.