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Stereoselective inhibition of synaptosomal catecholamine uptake by nomifensine
European Journal of Pharmacology, 98 (1984) 275-277
275
Elsevier Short communication S T E R E O S E L E C T I V E I N H I B I T I O N O F S Y N A P T O S O M A L C A T E C H O L A M I N E UPTAKE BY NOMIFENSINE ULRICH SCHACHT * and MARGRET LEVEN Hoechst AG, D-6230 Frankfurt/g.-80, West-Germany
Received 2 December 1983, accepted 14 December 1983
U. SCHACHT and M. LEVEN, Stereoselective inhibition of synaptosomal catecholamine uptake by nomifensine, European J. Pharmacol. 98 (1984) 275-277. The effects of the two enantiomers of the antidepressant nomifensine on catecholamine uptake were investigated using rat brain synaptosomes. According to the results from in vitro and ex vivo/in vitro studies, the inhibitory activity on catecholamine uptake resides entirely in the (+)-form of nomifensine. Further studies comparing the antidepressant effects of the two enantiomers might help to clarify the validity of the catecholamine hypothesis of depression. Antidepressants
Catecholamine uptake
Nomifensine
1. Introduction Nomifensine (8-amino-2-methyl-4-phenyl1,2,3,4-tetrahydroisoquinoline hydrogen maleate) is a well-established antidepressant drug which has been introduced into clinical practice in many countries. A large number of clinical trials have shown it to be an effective antidepressant; it is non-sedative and is relatively free from anticholinergic and cardiotoxic side-effects (Brogden et al., 1979; Stonier and Wittels, 1980; Prldinger and Taeuber, 1982). Biochemically, nomifensine resembles the tricyclic antidepressants in so far as it blocks monoamine re-uptake at presynaptic nerve terminals. It is a potent inhibitor of noradrenaline uptake but, unlike the tricyclics, it also strongly inhibits dopamine uptake into striatal synaptosomes (Hunt et al., 1974; Schacht et al., 1977). In several in vitro systems nomifensine acts as a pure catecholamine uptake blocker, thus differing clearly from amphetamine-type psychostimulants which predominantly enhance the release of catecholamines from the nerve endings * To whom all correspondenceshould be addressed. 0014-2999/84/$03.00 © 1984 ElsevierSciencePublishers B.V.
Stereoselectivity
Synaptosomes
(Hunt et al., 1979; Schacht et al., 1982). The molecular structure of nomifensine demonstrates the existence of a chiral centre at the 4position in the tetrahydroisoquinoline ring, and the resolution of the two enantiomers has recently been elaborated (German patent application DP 3310878.1). In the present study, we have compared the activities of (+)-nomifensine and ( - ) nomifensine on monoamine uptake inhibition in synaptosomal fractions from rat brain both in vitro and ex vivo/in vitro.
2. Materials and methods For the preparation of synaptosomal fractions, female rats (Wistar strain) weighing 100-150 g were decapitated and their brains rapidly removed and dissected. Our methods for determining monoamine uptake in vitro have recently been described in detail (Schacht et al., 1977). Briefly, the uptake of [14C]noradrenaline ([14C]NA), [14C]serotonin ([14C]5-HT) and [3H]dopamine ([3H]DA) was measured in synaptosomal fractions obtained from the hypothalamus, whole brain (ex-
276
cept cerebellum) and corpus striatum respectively, using Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 11 mM glucose and half-strength calcium (2.6 raM). [14C]NA (55 mCi/mmol), [14C]5-HT (57 mCi/mmol), and [3H]DA (2.3 Ci/mmol) were purchased from the Radiochemical Centre (Amersham, England), and the final concentrations of the labelled monoamines were 0.1/~M, the [3H]DA being diluted by inactive DA to 0.2 Ci/mmol. The samples were incubated at 37°C in a shaking water bath, and incubation time was 10 min with synaptosomes from the hypothalamus, 5 min with synaptosomes from whole brain, and 3 min with striatal synaptosomes. The amount of accumulated monoamine was evaluated applying the membrane filtration technique, using cellulose nitrate filters, 25 mm in diameter and with 0.65 ~tm pore size. The synaptosomes were harvested under mild vacuum and the filters were then dissolved in counting vials containing 10 ml of a scintillation fluid, and the radioactivity of the samples was determined in a Packard Tricarb liquid scintillation counter. For ex vivo/in vitro experiments, synaptosomes were prepared from rats which had been treated with the enantiomers of nomifensine (10 mg/kg i.p.) 1 h prior to decapitation. Control animals received saline only. Monoamine uptake was evaluated in synaptosomal fractions obtained from a pool of hypothalami and corpora striata, respectively, from n = 3 animals. Final dilution (w/v) was 1 : 80 with hypothalamic and 1 : 225 with striatal synaptosomes which were incubated with 0.1 /~M [14C]NA (52.3 mCi/mmol, New England Nuclear, Boston) or [3H]DA (34.3 Ci/mmol, New England Nuclear, Boston; diluted by unlabelled DA to 0.2 Ci/mmol) as described above, except that a modified Krebs-TRIS buffer (pH 7.4) was used containing 128 mM NaC1, 5 mM KC1, 2.7 mM CaC12, 1.2 MgSO4, 5 mM Na2HPO 4, 10 mM TRIS-HC1, 10 mM glucose, 1 /xM pargyline, and 0.2 mg/ml ascorbic acid.
3. Results
The effects of nomifensine and its enantiomers on the uptake of monoamines into rat brain syn-
TABLE 1 Inhibitory effects of nomifensine racemate and its enantiomers on synaptosomal monoamine uptake in vitro. IC5o values were determined graphically from the log concentration-response curves with four or five concentrations of each substance used at least in triplicate. Drug
IC5o [ ~ M] [14C]NA (hypothalamus)
( + )-Nomifensine 0.032 ( + )-Nomifensine 0.018 (-)-Nornifensine ca. 10
[3H]DA (c. striatum)
[14C]5-HT (whole brain)
0.14 0.053 > 10
12 4.0 > 10
aptosomes in vitro are presented in table 1 together with the IC50 values determined graphically from concentration-response curves. In the three monoamine uptake systems, (+)-nomifensine was approximately twice as potent as the racemate showing a high potency in blocking [14C]NA and [3H]DA uptake but rather weak effects on [14C]5HT uptake; the ( - )-form was essentially devoid of activity in blocking synaptosomal monoamine accumulation. In order to achieve 50 percent inhibition of [14C]NA uptake, about 500 times higher concentrations of (-)-nomifensine than of the (+)-enantiomer had to be used. [3H]DA uptake was not markedly affected by (-)-nomifensine up to 10 vM concentrations. The pronounced difference in the in vitro activities of the enantiomers of nomifensine was confirmed by ex vivo/in vitro studies evaluating [14C]NA and [3H]DA uptake in synaptosomal fractions obtained from drug-treated rats. The resuits of these experiments are summarized in table 2. The uptake of both [14C]NA and [3H]DA was effectively inhibited in synaptosomal preparations from animals pretreated with ( + )-nomifensine (10 mg/kg i.p.) 1 h prior to decapitation, whereas the ( - )-form was completely inactive at this dose. The mean value of inhibition of hypothalamic [ 1 4 C ] N A uptake caused by 10 mg/kg (+)-nomifensine was 84% while striatal [3H]DA uptake was inhibited by about 71% as compared to the respective control values.
277 TABLE 2 Catecholamine uptake in rat brain synaptosomes ex vivo/in vitro 1 h after administration of the enantiomers of nomifensine (10 mg/kg i.p.). The results are given as mean values___S.D. from three independent experiments with three animals in each group. Treatment
Physiol. NaC1 ( + )-Nomifensine ( - )-Nomifensine
[ 14C]NA uptake (hypothalamus)
[ 3H]DA uptake (corpus striatum)
pmol/g brain
% control
pmol/g brain
% control
82 + 19 13 + 8 102 + 18
100 + 23 16 + 10 124 + 22
1647 + 180 477 + 135 1872 _+162
100 + 11 29 + 8 114 + 10
4. Discussion The present data on monoamine uptake blockade of the enantiomers of nomifensine demonstrate that the catecholamine uptake inhibiting effects are confined entirely to the (+)-form or S-enantiomer. These results are in good agreement with recent findings in animal studies showing (+)-nomifensine to be about twice as active as the racemate and to be much more potent than the ( - ) - f o r m in several behavioural models predictive of antidepressant effects (Kruse, in preparation). Moreover, when [3H] (+)-nomifensine was used as ligand with membrane preparations from rat corpus striatum, a saturable and sodium-dependent high affinity binding could be demonstrated which is suggested to be associated to the DA uptake site of dopaminergic nerve terminals (Leven and Conradi, unpublished results). The enantiomers of nomifensine differ markedly from each other in their capacity to displace specific [3H] (+)-nomifensine binding, the (+)-form being about 300 times more potent than the (-)-form. Nomifensine is being widely used as an effective and well-tolerated antidepressant. Hence, it is of great interest to investigate the relevance of catecholamine uptake inhibition for antidepressant effectiveness and fl-receptor down-regulation as well (for review see: Sulser and Mishra, 1982) by comparing the activities of the two enantiomers of nomifensine. Such a comparison might help to
clarify the validity of the catecholamine hypothesis of depression.
References Brogden, R.N., R.C. Heel, T.M. Speight and G.S. Avery, 1979, Nomifensine: a review of its pharmacological properties and therapeutic efficacy in depressive illness, Drugs 18, 1. Hunt, P., M.-H. Kannengiesser and J.-P. Raynaud, 1974, Nomifensine: a new potent inhibitor of dopamine uptake into synaptosomes from rat brain corpus striatum, J. Pharm. Pharmacol. 26, 370. Hunt, P., J.-P. Raynaud, M. Leven and U. Schacht, 1979, Dopamine uptake inhibitors and releasing agents differentiated by the use of synaptosomes and field-stimulated brain slices in vitro, Biochem. Pharmacol. 28, 2011. POldinger, W. and K. Taeuber, eds., 1982, Nomifensine: clinical and experimental investigation, Int. Pharmacopsychiat. 17, Suppl. 1 Schacht, U., M. Leven and G. B~icker, 1977, Studies on brain metabolism of biogenic amines, Br. J. Clin. Pharmacol. 4, Suppl. 2, 77 S. Schacht, U., M. Leven, H.J. Gerliards, P, Hunt and J.-P. Raynaud, 1982, Recent investigations on the mechanism of action of nomJfensine, Int. Pharmacopsychiat. 17, Suppl. 1, 21. Stonier, P.D. and P.Y. Wittels, 1980, Clinical profile of nomifensine, Royal Society of Medicine, London, International Congress and Symposium Series 25, 131. Sulser, F. and R. Mishra, 1982, Regulation of central noradrenergic receptor function and its relevance to the therapy of depression, in: New Vistas in Depression, eds. S.Z. Langer, R. Takahashi, T. Segawa and M. Briley (Pergamon Press, Oxford) p. 37.
275
Elsevier Short communication S T E R E O S E L E C T I V E I N H I B I T I O N O F S Y N A P T O S O M A L C A T E C H O L A M I N E UPTAKE BY NOMIFENSINE ULRICH SCHACHT * and MARGRET LEVEN Hoechst AG, D-6230 Frankfurt/g.-80, West-Germany
Received 2 December 1983, accepted 14 December 1983
U. SCHACHT and M. LEVEN, Stereoselective inhibition of synaptosomal catecholamine uptake by nomifensine, European J. Pharmacol. 98 (1984) 275-277. The effects of the two enantiomers of the antidepressant nomifensine on catecholamine uptake were investigated using rat brain synaptosomes. According to the results from in vitro and ex vivo/in vitro studies, the inhibitory activity on catecholamine uptake resides entirely in the (+)-form of nomifensine. Further studies comparing the antidepressant effects of the two enantiomers might help to clarify the validity of the catecholamine hypothesis of depression. Antidepressants
Catecholamine uptake
Nomifensine
1. Introduction Nomifensine (8-amino-2-methyl-4-phenyl1,2,3,4-tetrahydroisoquinoline hydrogen maleate) is a well-established antidepressant drug which has been introduced into clinical practice in many countries. A large number of clinical trials have shown it to be an effective antidepressant; it is non-sedative and is relatively free from anticholinergic and cardiotoxic side-effects (Brogden et al., 1979; Stonier and Wittels, 1980; Prldinger and Taeuber, 1982). Biochemically, nomifensine resembles the tricyclic antidepressants in so far as it blocks monoamine re-uptake at presynaptic nerve terminals. It is a potent inhibitor of noradrenaline uptake but, unlike the tricyclics, it also strongly inhibits dopamine uptake into striatal synaptosomes (Hunt et al., 1974; Schacht et al., 1977). In several in vitro systems nomifensine acts as a pure catecholamine uptake blocker, thus differing clearly from amphetamine-type psychostimulants which predominantly enhance the release of catecholamines from the nerve endings * To whom all correspondenceshould be addressed. 0014-2999/84/$03.00 © 1984 ElsevierSciencePublishers B.V.
Stereoselectivity
Synaptosomes
(Hunt et al., 1979; Schacht et al., 1982). The molecular structure of nomifensine demonstrates the existence of a chiral centre at the 4position in the tetrahydroisoquinoline ring, and the resolution of the two enantiomers has recently been elaborated (German patent application DP 3310878.1). In the present study, we have compared the activities of (+)-nomifensine and ( - ) nomifensine on monoamine uptake inhibition in synaptosomal fractions from rat brain both in vitro and ex vivo/in vitro.
2. Materials and methods For the preparation of synaptosomal fractions, female rats (Wistar strain) weighing 100-150 g were decapitated and their brains rapidly removed and dissected. Our methods for determining monoamine uptake in vitro have recently been described in detail (Schacht et al., 1977). Briefly, the uptake of [14C]noradrenaline ([14C]NA), [14C]serotonin ([14C]5-HT) and [3H]dopamine ([3H]DA) was measured in synaptosomal fractions obtained from the hypothalamus, whole brain (ex-
276
cept cerebellum) and corpus striatum respectively, using Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 11 mM glucose and half-strength calcium (2.6 raM). [14C]NA (55 mCi/mmol), [14C]5-HT (57 mCi/mmol), and [3H]DA (2.3 Ci/mmol) were purchased from the Radiochemical Centre (Amersham, England), and the final concentrations of the labelled monoamines were 0.1/~M, the [3H]DA being diluted by inactive DA to 0.2 Ci/mmol. The samples were incubated at 37°C in a shaking water bath, and incubation time was 10 min with synaptosomes from the hypothalamus, 5 min with synaptosomes from whole brain, and 3 min with striatal synaptosomes. The amount of accumulated monoamine was evaluated applying the membrane filtration technique, using cellulose nitrate filters, 25 mm in diameter and with 0.65 ~tm pore size. The synaptosomes were harvested under mild vacuum and the filters were then dissolved in counting vials containing 10 ml of a scintillation fluid, and the radioactivity of the samples was determined in a Packard Tricarb liquid scintillation counter. For ex vivo/in vitro experiments, synaptosomes were prepared from rats which had been treated with the enantiomers of nomifensine (10 mg/kg i.p.) 1 h prior to decapitation. Control animals received saline only. Monoamine uptake was evaluated in synaptosomal fractions obtained from a pool of hypothalami and corpora striata, respectively, from n = 3 animals. Final dilution (w/v) was 1 : 80 with hypothalamic and 1 : 225 with striatal synaptosomes which were incubated with 0.1 /~M [14C]NA (52.3 mCi/mmol, New England Nuclear, Boston) or [3H]DA (34.3 Ci/mmol, New England Nuclear, Boston; diluted by unlabelled DA to 0.2 Ci/mmol) as described above, except that a modified Krebs-TRIS buffer (pH 7.4) was used containing 128 mM NaC1, 5 mM KC1, 2.7 mM CaC12, 1.2 MgSO4, 5 mM Na2HPO 4, 10 mM TRIS-HC1, 10 mM glucose, 1 /xM pargyline, and 0.2 mg/ml ascorbic acid.
3. Results
The effects of nomifensine and its enantiomers on the uptake of monoamines into rat brain syn-
TABLE 1 Inhibitory effects of nomifensine racemate and its enantiomers on synaptosomal monoamine uptake in vitro. IC5o values were determined graphically from the log concentration-response curves with four or five concentrations of each substance used at least in triplicate. Drug
IC5o [ ~ M] [14C]NA (hypothalamus)
( + )-Nomifensine 0.032 ( + )-Nomifensine 0.018 (-)-Nornifensine ca. 10
[3H]DA (c. striatum)
[14C]5-HT (whole brain)
0.14 0.053 > 10
12 4.0 > 10
aptosomes in vitro are presented in table 1 together with the IC50 values determined graphically from concentration-response curves. In the three monoamine uptake systems, (+)-nomifensine was approximately twice as potent as the racemate showing a high potency in blocking [14C]NA and [3H]DA uptake but rather weak effects on [14C]5HT uptake; the ( - )-form was essentially devoid of activity in blocking synaptosomal monoamine accumulation. In order to achieve 50 percent inhibition of [14C]NA uptake, about 500 times higher concentrations of (-)-nomifensine than of the (+)-enantiomer had to be used. [3H]DA uptake was not markedly affected by (-)-nomifensine up to 10 vM concentrations. The pronounced difference in the in vitro activities of the enantiomers of nomifensine was confirmed by ex vivo/in vitro studies evaluating [14C]NA and [3H]DA uptake in synaptosomal fractions obtained from drug-treated rats. The resuits of these experiments are summarized in table 2. The uptake of both [14C]NA and [3H]DA was effectively inhibited in synaptosomal preparations from animals pretreated with ( + )-nomifensine (10 mg/kg i.p.) 1 h prior to decapitation, whereas the ( - )-form was completely inactive at this dose. The mean value of inhibition of hypothalamic [ 1 4 C ] N A uptake caused by 10 mg/kg (+)-nomifensine was 84% while striatal [3H]DA uptake was inhibited by about 71% as compared to the respective control values.
277 TABLE 2 Catecholamine uptake in rat brain synaptosomes ex vivo/in vitro 1 h after administration of the enantiomers of nomifensine (10 mg/kg i.p.). The results are given as mean values___S.D. from three independent experiments with three animals in each group. Treatment
Physiol. NaC1 ( + )-Nomifensine ( - )-Nomifensine
[ 14C]NA uptake (hypothalamus)
[ 3H]DA uptake (corpus striatum)
pmol/g brain
% control
pmol/g brain
% control
82 + 19 13 + 8 102 + 18
100 + 23 16 + 10 124 + 22
1647 + 180 477 + 135 1872 _+162
100 + 11 29 + 8 114 + 10
4. Discussion The present data on monoamine uptake blockade of the enantiomers of nomifensine demonstrate that the catecholamine uptake inhibiting effects are confined entirely to the (+)-form or S-enantiomer. These results are in good agreement with recent findings in animal studies showing (+)-nomifensine to be about twice as active as the racemate and to be much more potent than the ( - ) - f o r m in several behavioural models predictive of antidepressant effects (Kruse, in preparation). Moreover, when [3H] (+)-nomifensine was used as ligand with membrane preparations from rat corpus striatum, a saturable and sodium-dependent high affinity binding could be demonstrated which is suggested to be associated to the DA uptake site of dopaminergic nerve terminals (Leven and Conradi, unpublished results). The enantiomers of nomifensine differ markedly from each other in their capacity to displace specific [3H] (+)-nomifensine binding, the (+)-form being about 300 times more potent than the (-)-form. Nomifensine is being widely used as an effective and well-tolerated antidepressant. Hence, it is of great interest to investigate the relevance of catecholamine uptake inhibition for antidepressant effectiveness and fl-receptor down-regulation as well (for review see: Sulser and Mishra, 1982) by comparing the activities of the two enantiomers of nomifensine. Such a comparison might help to
clarify the validity of the catecholamine hypothesis of depression.
References Brogden, R.N., R.C. Heel, T.M. Speight and G.S. Avery, 1979, Nomifensine: a review of its pharmacological properties and therapeutic efficacy in depressive illness, Drugs 18, 1. Hunt, P., M.-H. Kannengiesser and J.-P. Raynaud, 1974, Nomifensine: a new potent inhibitor of dopamine uptake into synaptosomes from rat brain corpus striatum, J. Pharm. Pharmacol. 26, 370. Hunt, P., J.-P. Raynaud, M. Leven and U. Schacht, 1979, Dopamine uptake inhibitors and releasing agents differentiated by the use of synaptosomes and field-stimulated brain slices in vitro, Biochem. Pharmacol. 28, 2011. POldinger, W. and K. Taeuber, eds., 1982, Nomifensine: clinical and experimental investigation, Int. Pharmacopsychiat. 17, Suppl. 1 Schacht, U., M. Leven and G. B~icker, 1977, Studies on brain metabolism of biogenic amines, Br. J. Clin. Pharmacol. 4, Suppl. 2, 77 S. Schacht, U., M. Leven, H.J. Gerliards, P, Hunt and J.-P. Raynaud, 1982, Recent investigations on the mechanism of action of nomJfensine, Int. Pharmacopsychiat. 17, Suppl. 1, 21. Stonier, P.D. and P.Y. Wittels, 1980, Clinical profile of nomifensine, Royal Society of Medicine, London, International Congress and Symposium Series 25, 131. Sulser, F. and R. Mishra, 1982, Regulation of central noradrenergic receptor function and its relevance to the therapy of depression, in: New Vistas in Depression, eds. S.Z. Langer, R. Takahashi, T. Segawa and M. Briley (Pergamon Press, Oxford) p. 37.