Neurochemical and autonomic pharmacological profiles of the 6-aza-analogue of mianserin, org 3770 and its enantiomers

Neuropharmacology Vol. 27,No.4,pp.399-408, 1988 Printed in Great Britain

0028-3908188 $3.00 f0.00 Pergamon Press

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NEUROCHEMICAL AND AUTONOMIC PHARMACOLOGICAL PROFILES OF THE 6-AZA-ANALOGUE OF MIANSERIN, ORG 3770 AND ITS ENA~IOMERS TH. DE BOER,'G. MAURA,~ M. RAITERI,~ C. J. DE Vos,’ J. WIERIF~GA'and R. M. PINDER' ‘Scientific Development Group, Departments CNS Pharmacology and Organic Chemistry, Organon International B.V., P.O. Box 20, 5340 BH OSS, The Netherlands and *Istituto di Famacologia & Farrnacognosia, Universit;i di Genova, Viale Cembrano 4, 16148 Genova, Italy (Accepted 20 October 1987)

Summary-The neurochemical and autonomic pharmacological profile of 1,2,3,4,10,14b-hexahydro-2methyl-pyra~no[2,l-a]pyr~do[2,3-c]~2] benzazepine (( +)Org 3770) and the related antidepressant drug, mianserin, have been compared. The uptake of 13H]noradrenaline (13HjNA) in z&o was weakly affected by (&)Org 3770 (pKi = 5.6) in contrast to mian~rin t’pk; = 7.4). Both (+)Org 3770 and mianserin facilitated the release of [3H]NA in slices of cortex. The effects of NA mediated by a~-adren~eptors on the release of both [jH]NA or [3H]serotonin ([3H]5-HT) were antagonized by (+)Org 3770 with pk; values of 8.4 and 8.1, respectively. However, (-)Org 3770 only antagonized the effect of NA on the release of f3H].5-HT@A2 = 7.7). The binding of [“H]rauwolscine to a,-adrenoceptors was inhibited by (&)Org 3770 and mianserin with identical affinity (PK, = 7.0), whereas the binding of [3H]prazosine to a,-adrenoceptors was less potently affected by (+)Org 3770 t’pK,= 6.4) than by mianserin @Ki = 7.1). A similar difference was found for a,- and a,-adrenoceptors in vas deferens of the rat. The binding of [3H]mianserin to 5-HT, receptors was less potently blocked by (&)Org 3770 (pK, = 8. I) than by mianserin (PK, = 9.4) while the binding of [‘Hlmepyramine to histamine-l receptors was more potently affected by (f)Org 3770 (pK, = 9.3) than by mianserin (PK, = 8.75). The binding of [“H]quinuclidinylbenzilate to muscarinic cholinergic receptors was blocked equally by (_+)Org 3770 (p& = 6.1) and mianserin (py = 6.3). Similar data on tryptamine-D, histamine-l and muscarinic cholinergic receptors in isolated organs were obtained. A prominent role for the blockade of k-adrenoceptors in the therapeutic effects of mianserin and ( f )Org 3770 in depression is suggested, probably excluding a role of inhibition of the uptake of NA. Key words: mianserin, Org 3770, adrenoceptors,

Therapeutic

efficacy of tricyclic antidepressant

drugs

has been related to adaptive changes associated with their ability to inhibit the uptake of monoamines (Schildkraut, 1965; Halaris, Belendiuk and Freedman, 1975; Sulser, Vetulani and Mobley, 1978). This hypothesis has been challenged by the effectiveness of second generation antidepressants, such as mianserin and iprindole, which lack substantial inhibitory properties on the uptake of monoamines in vitro and/or in z&o (Leonard, 1980). No correlation exists between the potency of antidepressant drugs to inhibit the uptake of monoamines and the therapeutic dose (Sugrue, 1983). In the treatment of depression, the inhibition of the reuptake of noradrenaline, oxaprotiline has been found to be superior to its R-enantiomer, which lacks inhibitory properties on uptake (Schmauss, Laakmann, Dieterle, Schmitz and Wittmann, 1985). Mianserin is a moderately potent inhibitor of the uptake of noradrenaline in vitro, but this property is far less prominent in viva (Goodlet, Mireyless and Sugrue, 1977; Raiteri, Angelini and Bertollini, 1976; Baumann and Maitre, 19’7’7).The drug gives negative results in many behavioural tests

noradrenaline uptake.

which predict antidepressant activity (Van Riezen, Pinder, Nickolson, Hobbelen, Zayed and Van der Veen, 1981). The properties of mianserin, both as a moderate inhibitor of the uptake of noradrenaline and a noradrenaline autoreceptor antagonist, may combine to increase the availability of noradrenaline in the biophase (Baumann and Maitre, 1977; Nickolson and Wieringa, 1981; Raiteri, Mama and Cerrito, 1982). Therefore, a role of inhibition of the reuptake of noradrenaline in the therapeutic effects of mianserin cannot be completely excluded. The 6-aza-analogue of mianserin, 1,2,3,4,10,14b-hexahydro-2_methylpyrazino[2,1 -a]pyrido[2,3-c][2]benzazepine ((f )Org 3770; see Fig. 1) does not appreciably affect the uptake of noradrenaline but, similar to mianserin, increases the evoked release of radiolabelled noradrenaline (Nickolson, Wieringa and van Delft, 1982). Electroencepholographic (EEG) studies suggest antidepressant properties for (-&)Grg 3770 (Fink and Irvin, 1982). The drug (+ )Org 3770 is under evaluation in clinicai trials for potential antidepressant and anxiolytic properties. The neurochemical and autonomic pharmacological profiies of 399

TH. DE BOER et

400

A

B

Fig. 1. Chemical structures of Org 3770 (A) and mianserin 0% (+)Org

3770 and its enantiomers,

in comparison to and its enant~omers are the

those of (~)mianserin subject of this paper.

METHODS

Neurochemical studies Animals. Male Wistar rats (Cpb: WV), weighing between 15&200 g, were killed by decapitation and the brains were rapidly removed. Uptake ofmonoamines. Corpora striata and hypothalami were dissected, weighed and homogenized in 20 volumes (w/v) of ice-cold sotution, using a Potter-Elvehjem homogenizer (with a total clearance of 0.25mm; 10 strokes up-and-down at 850rpm). The solution contained 0.32 M sucrose, 0.01 M glucose adjusted to pH 7.4 with 0.05 M Tris-HCl. A crude synaptosomal mitochondrial pellet (P2) was obtained by centrifugation and was used for uptake experiments. Further details are described by Nickolson and Wieringa (198 1). Synaptosomes from hypothalamus were used to measure uptake for [3H]noradrenaline ([3H)NA) while synaptosomes from the striatum were employed for experiments on the uptake of [3H]dopamine (f3H]DA) and [3H]serotonin ([3H]5-HT). Release of noradre~aline in slices. After decapitation of the rats, the brains were quickly removed and slices of parietal cortex (about 0.3 mm thick) were cut by hand with the use of a recessed perspex guide. They were then chopped into 0.25 x 0.25 mm squares with a McIllwain tissue chopper. Following preincubation for 10 min at 37°C in a Krebs-Ringer buffer, the slices were loaded with [3H]NA, final concentration 2.5 x IO-‘M, and about 5 mg tissue was brought in each of 10 cells of a superfusion apparatus and superfused with medium for 30 min, as described by Nickolson and Wieringa (1981). Depolarization-induced stimulation of release due to exposure to medium containing 12.5 or 26mM potassium ions (K+) lasted 4 min and was given twice (S, and S,), S, at t = 34-38 min and S, at t = 5&54 min. Drugs were added after the termination of the first K+ stimulus. Fractional rates of release of [3H]NA were calculated and the percentages of evoked release of [3H]NA were determined for S, and S2. Drugs were tested at 1 MM unless stated otherwise. Effects of drugs were evaluated by relating

ai,

the ratio SJS, for experimental cells (drugs present during S,) to SJS, as found for the control cells. The results represent the mean & SE for at least 4 differential batches of slices of cortex. Effects on evoked release were rated by comparing the effect of the drug (percentages increase of S,/S, , compared to control) with that of the standard drug, mianserin at 1 PM which was measured concurrently. Release of noradrenaline and serotonin in synaptosomes. Crude synaptosomal fractions (P?) were pre-

pared from the cerebral cortex of adult male Sprague-Dawley rats, weighing 20&250 g. Following isolation, the synaptosomes were resuspended (at a concentration of protein of 0.5-0.6 mg protein/ml) in a Krebs-Ringer medium with the following composition (mM): NaCl 125, KC1 3, CaCl, 1.2, MgSO, 1.2, Na,HPO, 5, glucose 10, Tris HCl buffer 10, pH 7.4. After preincubation (10 min at 37°C in a rotary waterbath) the synaptosomes were labelled (10 min) either with 0.04 PM ( -)-[7,8[3H]]noradrenaline or with 0.04 /*M 5-[l,2-[3H]Jhydroxytryptamine creatinine sulphate. Aliquots of the suspension were then distributed on 0.65 pm Millipore filters (0.5-0.6 mg protein/filter) in a multi-chamber superfusion apparatus (Raiteri, Angelini and Levi, 1974) and su~rfusion was started with standard medium. The superfusion medium in all the chambers contained 5 9 M desipramine (experiments on release of [‘H]NA) or 1 PM chlorimipramine (experiments on release of [3H]5-HT) throughout the superfusion, as inhibitors of the carrier for noradrenaline or 5_hydroxytryptamine, respectively. These drugs were used in order to prevent the possible entry of noradrenaline into noradrenergic or serotonergic nerve terminals and the consequent displacement of [‘H]NA or [3H]5-HT, respectively. Desipramine and chlorimipramine by themselves did not alter the release of the corresponding tritiated amines. After 10min of su~rfusion, noradrenaline was added to the medium and, 8 min later, the synaptosomes were depolarized with 15 mM KC1 (substituting an equimolar concentration of NaCl). Both (+)Org 3770 and (-)Org 3770 were present from the beginning of superfusion. Fractions were collected every min into vials containing 100 ~1 of a protective solution [1.5% ethylene diamine tetra ammonium (EDTA) 1% ascorbic acid, 0.001% unlabelled NA or 5-HT] and the tritiated amines present in each fraction and in the synaptosomes at the end of superfusion were determined as described previously (Smith, Lane, Shea, McBride and Aprison, 1975). The tritiated amine present in each fraction of superfusate was expressed as a percentage of the total tritiated amine recovered (fractions collected plus filter). Student’s t-test was used for comparison of mean values. The pA2 values of the antagonists were calculated according to the formula given by Furchgott (1972): pA, = log ([E’]/[E] - 1) - log[B]. [E’] and [E] are the agonist concentrations causing half-maximum

Profiles of the 6-aza-analogue of mianserin, Org 3770 and its enantiomers

401

effects in the presence and in the absence of the antagonist, respectively; [B] is the concentration of the antagonist.

in the presence of 1 FM mianserin 40-50% of total binding.

Binding studies

Ross, Tang and Seeman (1980) was used to characterize serotonergic SZreceptors. For routine experiments the concentration of [3H]mianserin was 0.5 nM, concentration of tissue was 6.3 mg original tissue per ml and incubation time was 40 min at 25°C. Non-specific binding was defined as the amount of binding of [‘Hlmianserin in the presence of 1 PM mianserin and was about 35-45% of total binding. Binding to histaminergic receptors was prevented by adding 50nM mepyramine to the incubation medium. Evaluation of binding data. Displacement curves were obtained for the various compounds by measuring binding in the presence of at least 5 different concentrations and I&, and pKi values were determined. Every observation was obtained at least in triplicate. To enable these calculations, K,, values were measured for the interaction between the various ligands and the receptor. These were for binding of: [3H]spiperone: Kd = 0.22 nM; [3H]rauwolscine: Kd = 3.1 nM; [3H]quinuclidinyl benzilate: Kd = 0.05 nM; [3H]mepyramine: Kd = 5 nM; [3H]mianserin: Kd = 1.85 nM; [3H]prazosin: K,, = 0.13 nM.

Binding of [‘Hjspiperone in membranes from the striatum. A rapid filtration assay was used to measure

the interaction between the antidepressants and dopamine D, receptors. The method used was identical with the one described by Seeman, Lee, Chan-Wong, Fedesco and Wong (1976) and Titeler, Weinreich, Sinclair and Seeman (1978). For routine measurements the concentration of [3H]spiperone was 0.2 nM, the concentration of tissue was 5 mg original tissue per ml and the incubation time was 45 min at 25°C. Non-specific binding was defined as the amount of binding of [3H]spiperone in the presence of 1 PM haloperidol and represented about 10% of total binding. Binding of [3H]rauwolscine in membranes from frontal cortex. The rapid filtration assay, described by

Perry and U’Prichard (1981), was used to characterize binding to a,-adrenoceptors. The assay with phosphate buffer and incubation at 0°C was employed. For routine measurements the concentration of [3H]rauwolscine was 1 nM, concentration of tissue was 10 mg original tissue per ml and the incubation time was 90-120min. Non-specific binding was defined as the amount of binding of [3H]rauwolscine in the presence of 1OpM phentolamine and was about 20% of total binding. Binding of [ ‘H]prazosin in homogenates of frontal cortex. The rapid filtration method of Greengrass

and Bremner (1979) was used to characterize cr,-adrenoceptors. For routine measurements the concentration of [3H]prazosin was 0.2 nM, concentration of tissue was 10 mg original tissue per ml and the incubation time was 30min at 25°C. Non-specific binding was defined as the amount of binding of [3H]prazosin in the presence of 2 PM phentolamine and was about 10% of total binding. Binding of [ ‘H]quinuclidinyl benzilate in homogen ate.P of whole brain. The rapid filtration method, described by Yamamura and Snyder (1978) was used to characterize muscarinic cholinergic receptors. For routine measurements the concentration of [3H]quinuclidinyl benzilate was 0.6 nM, concentration of tissue was 2.5 mg original tissue per ml and the incubation time was 60min at 25°C. Non-specific binding was defined as the amount of binding of [3H]quinuclidinyl benzilate in the presence of 1 PM atropine sulphate and was about 2% of total binding. Binding of [‘Hjmepyramine in homogenates of whole brain. The rapid filtration assay of Chang, Tran and

Snyder (1978) was used to characterize the histamine1 receptor. For routine experiments the concentration of [‘Hlmepyramine was 2 nM, concentration of tissue was 10 mg original tissue per ml and the incubation time was 40min at 25°C. Non-specific binding was defined as the amount of binding of [3H]mepyramine

and was about

Binding of [ ‘Hlmianserin in homogenates of frontal cortex. The rapid filtration method of Dumbrille-

Autonomic pharmacology

Isolated organs were used to measure the interaction between the various compounds and a,- and a,-adrenoceptors (rat vas deferens), /$-adrenoceptors (guinea pig right atrium), &adrenoceptors (trachea strip, guinea pig), histamine type 2 receptors (guinea pig right atrium), tryptamine D-receptors (rat fundus strip), histamine type 1 receptors (guinea pig ileum) and muscarinic cholinergic receptors (rat jejunum or guinea pig ileum). Details of the automated assays have been described extensively by de Graaf, De Vos and Steenbergen (1983). The pre-junctional a,-adrenolytic activities were determined on field stimulation in vas deferens preparations of the rat. Male Wistar rats weighing 200-300 g were used. An animal was killed by a blow on the head and exsanguinated. The vasa deferentia were removed and cut longitudinally over the full length. Each tissue was suspended in a lOm1 waterjacketed organ bath, containing oxygenated (95% 0,; 5% COZ) modified Krebs buffer, kept at 37°C. The baseline tension of the isometric transducer was pre-set at 1OmN (for full details see De Graaf et al., 1983). The organ was stimulated electrically (supramaximally) by means of a constant current stimulator equipped with two platinum ring electrodes (50-100 mA; 0.1 Hz; pulse duration 1 msec). Concentration-response curves were made with the specific a,-adrenergic agonist azepexole (pD2 = 6-6.5) (Vargas and Brugger, 1985). The properties of histamine type 1 and the muscarinic cholinergic receptors were studied according

TH. DE BOER et

402

to the classical method of Van Rossum (1963). The agonists used were acetylcholine for the muscarinic cholinergic receptor (pD, = 6-7) or histamine for the histamine type 1 receptor (pD2 = 6-7). For details on the bath liquids used see De Graaf et al. (1983). The equilibration period for the antagonists investigated was always 30 min. Chemicals All chemicals were of the highest quality available. The following drugs were used: Org 3770, mianserin and their enantiomers (Organon); desipramine, imipramine, chlorimipramine and phentolamine (Ciba Geigy); nomifensin (Hoechst); haloperidol (Janssen Pharmaceutics); atropine (Lamers and Indemans); cocaine (VPF); azepexole (Boehringer I); isoprenaline (Pharmax); serotonin creatinine sulphate (Merck); noradrenaline, histamine, dopamine and acetylcholine (Sigma). The following radiochemicals were used: [7,8-[3H]](-)-norepinephine HCl (32 Ci/mmol), [3H]serotonin creatinine sulphate (10-30 Ci/mmol) and [7,8-[3H]]dopamine HCl(30-50 Ci/mmol) (The Radiochemical Centre Amersham, U.K.); [3H]spiperone (14 Cij mmol), 5j3H]pyrilamine maleate (27 Ci/mmol), L-(benzilic-4,4’-[3H(N)])3-quinuclidinyl benzilate (30 Ci/mmol), [N-methyl-[3H]]mianserin HCl (50 Ci/ mmol), [methyl-[3H]]rauwolscine (80 Ci/mmol), 5-[l,2-[3H]]hydroxytryptamine creatine sulphate (29.1 Ci/mmol) and [7-methoxy-[3H]]prazosin (70-87 Ci/ mmol) (New England Nuclear, U.S.A.). RESULTS

Uptake of monoamines The drug (+)Org 3770 was a very weak inhibitor of the uptake of noradrenaline and had no activity on the uptake of dopamine or serotonin. The results in Table 1 indicate that it was about lOO-fold less active than mianserin on the in vitro uptake of noradrenaline. Mianserin had about the same selectivity for the uptake of monoamines in vitro as desipramine but was about IO-fold less active. Binding studies Both (k)Org [3H]rauwolscine

3770 and (f)mianserin displaced from its a,-adrenoceptor binding Table 2. Interactions

Receptor a,-Adrenoceptor a,-Adrenoceptor Serotonin S, Dopamine D, Histamine-l Must. cholinergic

with radio-l&and

binding

al.

Table 1. Inhibition Drug (i)Org 3770 (+)Org 3770 (-)Org 3770 (+)Mianserin (+)Mianserin (-)Mianserin Desipramine Imipramine Nomifensin

of uptake

of monoamines

into synaptosomes

Uptake inhibition (indicated Noradrenaline Dopamine 5.60 f 0.08 5.60 ?r 0.10 <5 7.36 f 0.06 7.57f0.12 4.56 k 0.19 8.59 f 0.12 7.48 f 0.12 7.8

as pK,) Serotonin

< 5.0 <5 <5 < 5.0

<5.1 <5.1 15.1 <5.0

< 5.0 < 5.0 6.53 f 0.03

5.35 * 0.18 6.53 +_0.03 5.4 + 0.2

Uptake was measured according to Nickolson and Wieringa (1981). Specific uptake of radiolabelled transmitter is defined as the total amount of radioactivity taken up in the tissue, corrected for the amount of radioactivity taken up in the presence of IOOrM desipramine (for noradrenaline), 100 PM imipramine (for serotonin) and 100 PM nomifensin (for dopamine). Data are expressed as pK, values and are the mean f SE of 3 or more experiments. The pK, values refer to -log K, and the K, value is calculated from the IC, value (derived from inhibition curves), using the formula K, = IC,,. Km/K,,, + C. The IC,, represents the concentration of the compound showing 50% inhibition of specific uptake, C represents the concentration of ligand and K,,, equals the Michaelis constant, calculated from separately measured Lineweaver-Burke plots.

sites with the same affinity, whereas (-t )Org 3770 was about five-fold less active than mianserin in displacing [3H]prazosin from its a,-adrenoceptor binding sites (Table 2). A similar profile was observed when comparing (+)Org 3770 and (+)mianserin. Both compounds inhibited the binding of [3H]rauwolscine with equal potency, whereas (+)Org 3770 was about five-fold less active than (+)mianserin as an inhibitor of the binding of [3H]prazosin. The (- )Org 3770 was five-fold less active than (-)mianserin in the binding tests which characterized a,- and cr,-adrenoceptors. Further, while (+)mianserin was only six-fold less active than (-)mianserin in the binding of [3H]rauwolscine, a considerable 50-fold difference in potency existed between (+) and (-)Org 3770. Thus, (+)Org 3770 was about 3-fold more potent than (+)mianserin as a displacer of [3H]mepyramine from histamine-l binding sites and the same difference was observed for (+)Org 3770 compared to (+)mianserin and (-)Org 3770 and (-)mianserin. Both (+)Org 3770 and (+)mianserin were 3-fold more active in displacing [3H]mepyramine from its binding sites than were (-)Org 3770 and (-)mianserin, respectively. The drugs (+)Org 3770 and (+)Org 3770 were about 10-20 times weaker than (+) and in homogenates

of membranes

Inhibition of binding (pK,) 3770 (-)Org 3770 (+)Mianserin

from brain

Ligand

(k )Org 3770

[‘H]PRA [‘H]RAU [‘H]MIA [‘HISPI [3H]MEP

6.43 f 0.08 6.95 f 0.01 8.05 k 0.16 5.4

6.50 + 0.06 7.20 + 0.06 8.32 f 0.12 5.3

5.82 k 0.08 5.63 f 0.01 7.22 + 0.05 < 5.4

7.14 f 0.02 6.96 f 0.21 9.44 f 0.06 4.4

7.26 f 0.08 7.24 k 0.06 9.38 f 0.36 -

6.32 + 0.02 6.44 f 0.09 7.73fO.17

9.30 + 0.19 6.10~0.11

9.50 f0.15 5.15 f0.02

9.20 k 0.33 6.6 +0.14

8.75 + 0.27 6.30 + 0.08

9.0 + 0.5 5.40 * 0.08

8.39_+0.11 6.82f0.18

1’HlQNu

(+)Org

Data are based on inhibition curves using at least 5 different concentrations of drug in triplicate and experiments. The pK, values represent the -log K, where K, = IC,,.K,/K,, + C with IC,, representing 50% inhibition of specific binding with C = ligand concentration and Kd being the dissociation Scatchard analysis of saturation experiments. PRA = prazosin; RAU = rauwolscine; MIA = mianserin; SPI = spiperone; MEP = mepyramine; QNB

(+)Mianserin

(-)Mianserin

refer to the mean + SE of 3 or more the concentration ofdrug producing constant measured separately using = quinuclidinyl

benzilate.

Profiles of the 6-aza-analogue of mianserin, Org 3770 and its enantiomers

403

Table 3. Interactions with peripheral receptors in isolated organs Receotor a,-Adrenoceptor

or,-Adrenoceptor fl,-Adrenoceptor &-Adrenoceptor Histamine-l Histamine-2 Tryptamine-o Must. cholinergic

Isolated orean vas deferens no cocaine with cocaine vas deferens right atrium trachea strip guinea pig ileum right atrium fundus strip guinea pig ileum

Antagonism (PA,) (f )Org 3770

6.19 f 0.07 6.52 f 0.04 6.82 + 0.03 <4.5 <4.5 9.94 * 0.07 4.8 f 0. I 6.68 f 0.05 6.13 k 0.05

(+)Org

3770

6.37 f 0.08 6.66 + 0.06 7.10 f 0.07 4.2 <4.5 10.0 + 0.1 4.88 f 0.03 6.87 _+0.07 4.3

(-)Org

3770

5.8 f 0.1 6.08 f 0.03 5.62 f 0.03 <4.5 <4.5 9.44 f 0.07 5.5 5.26 f 0.06 6.64 + 0.06

(f)Mianserin

f +)Mianserin

(-)Mianserin 6.2 _+0.2 6.44 f 0.05 5.75 f 0.05 <4.5 t5 8.54 f 0.07 5.0 + 0.2 6.45 + 0.06 6.06 * 0.05’

6.47kO.10 7.60 f 0.05 7.52 f 0.09 5.2 <4.5 9.2 + 0.1 5.2 7.7 + 0. I <4.5*

6.35 + 0.04 7.37 + 0.05 7.27 f 0.06 4.7 _+0.1 <4.5 9.08 f 0.05 5.5 * 0.1 7.42 i 0.09 5.74 f 0.09

*The jejunum of rat rather than the guinea pig ileum was used for this study. Results presented are the mean pA, values based on 3-13 different experiments. When no standard error is given, data refer to the mea” of 2 experiments.

(+)mianserin in the binding test with [3H]mianserin, respectively. The difference between (-)Org 3770 and (-)mianserin was less pronounced. (+)Mianserin and (+)Org 3770 were more active in the binding test with [3H]mianserin than (-)mianserin and (-)Org 3770, respectively. Both (f)Org 3770 and (+)mianserin had negligible affinity for dopaminergic D, receptors, measured in the binding test with [3H]spiperone. Differing results were obtained with respect to inhibition of the binding of [3H]quinuclidinylbenzilate where (f )Org 3770 and (f )mianserin showed a similar weak affinity, but activity was concentrated in (-)Org 3770 and (-)mianserin. Autonomic pharmacology Similar potency ratios for (+)Org 3770 and mianserin were found for blockade of peripheral histamine- 1 and muscarinic cholinergic receptors, as compared to the receptors in tissue from brain (Table 3). Both compounds also lacked &- and /?,-adrenolytic properties, but showed significant cc,-adrenolytic properties in the vas deferens model in the rat. The drug (+)Org 3770 appeared to be considerably less active in this model than (+)mianserin. These studies were performed both without and with an active uptake system for noradrenaline by measuring in the presence and absence of cocaine in the medium. Addition of cocaine to the medium increased the apparent PA, value for the compounds studied, but a IO-fold difference in cc,-adrenolytic activity between (+)Org 3770 and (f)mianserin was maintained. Comparable data were obtained for (+)Org 3770 and (+-)mianserin. Comparison of c(~- and cr,-adrenolytic activity in the vas deferens model in the rat in the presence of cocaine to prevent a role for uptake of noradrenaline revealed a modest 2-fold cr,-selectivity for (k)Org 3770, while the potency ratio for tlz- and a,-adrenoceptors for (+)mianserin was close to unity, No significant influence of cocaine was found on the pA, value for (+)mianserin in the cc,-adrenergic vas deferens model in the rat (PA, with cocaine: 7.1; pA, without cocaine: 7.27). Based on pA, values, measured in the electrically-stimulated vas deferens for cc,-mediated inhibition of the twitch response, (k )mianserin had a pA, value of 7.27 (Table 3) and

was five-fold less active than yohimbine which had a pA, value of 8.0 (data not shown). Neither compound showed intrinsic a-adrenergic activity; (f )Org 3770 was five-fold less active than (f)mianserin, with respect to the tryptamine-D receptor in the fundus strip in the rat. ReIease of noradrenaline in slices of cortex Both (+)mianserin and (+)Org 3770 facilitated the release of [3H]NA from slices of cortex evoked with 12.5 or 26 mM K+ (Table 4). The facilitation was larger with the smaller K+ stimulation, where both ( f )mianserin and ( f )Org 3770 at 1 PM concentration increased the release of [3H]NA to the same extent. Using 26 mM K+ as a stimulus, (+)mianserin was more effective than (f)Org 3770 and its enantiomers in increasing the evoked release. Similar data were obtained when the concentration dependency of the facilitation of release of [3H]NA was studied (Fig. 2). Again, (f)Org 3770 was considerably less active than ( f )mianserin, whereas (+)Org 3770 appeared to be almost equipotent with (f)mianserin. Phentolamine (10 PM) increased the release with 58 + 10.7% (8) and thus was less active than (k)mianserin, but it increased the release to the same extent as 10pM (f)Org 3770; (-)Org 3770 only slightly increased the release of [3H]NA.

Table 4. Effects of Org 3770, mianserin and their enantiomers on 12.5 or 25 mM K+-evoked release of (jH]NA in slices of cortex from rat

Drugs

K+-cont. (mM)

Control (+)Mianserin (f )Org 3770

12.5 12.5 12.5

Control (k)Mianseri” (i)Org 3770 (+)Org 3770 (-)Org 3770

26 26 26 26 26

Concentration of drug (PM)

Release of [‘H]NA (% of co”trol)

n

I I

lOOil. 172.6 f 10.7 166.0 f 7.0

I2 4 8

I I I I

100 155.3 132.9 141.4 110.4

-

+ I.1 f 5.6 f 3.5 If: 5.9 + 6.9

8 4 4 4 4

Slices were pre-labelled with [‘H]NA, superfused as described under Methods and stimulated twice with 12.5 or 26 mM K+. Drugs were added after the first stimulation. For controls and drugtreated groups S,/S, ratios were calculated (see Methods). Results are expressed as percentage of control ratios.

TH. DE BOER et

404 -

225

al.

-

: 5

200-

% E

175-

I yl

150-

L 6

125-

c0ntrcA -5 _.

-7-6-5

-7-6-5

-7-6-5

-7-6

-5

log cont.(M) Fig. 2. Effects of Org 3770, mianserin and their enantiomers on K+-evoked release of [‘H]NA in slices of cortex. Slices were pre-labelled with [3H]NA, brought in a superfusion apparatus and stimulated twice with 26 mM K+ as described under Methods. Results refer to the mean k SE of 3-8 experiments and are presented as percentage of the release in control slices. Drugs were added immediately after the first stimulation. The S,/S, ratio for control slices was 0.668 + 0.017 (8). Statistics: Student’s t-test: *P < 0.05; **p < 0.01; ***p < 0.001.

Release of noradrenaline aptosomes

and serotonin

in syn-

It has been reported that NA could inhibit, in a concentration-dependent manner, the depolarizationinduced release of both [3H]NA and [3H]5-HT in superfused synaptosomes from cortex (Mulder, de Langen, de Regt and Hogenboom, 1978; Maura, Gemignani and Raiteri, 1982). Figure 3 shows that the inhibitory effect of 1 PM NA on the release of [3H]NA (left panel) was almost completely counteracted by the (+)enantiomer of Org 3770 at 1 PM; the (-)enantiomer was inactive. In contrast, both enantiomers (1 p M) antagonized the inhibitory effect of

exogenous noradrenaline (1 FM) on the release of [3H]5-HT, elicited by 15 mM KC1 (right panel); the (+)enantiomer was more potent than the (-)enantiomer. Figure 4 shows that the concentration-response curves for NA, as an inhibitor of the release of [3H]NA or of [3H]5HT, respectively, were shifted to the right in a parallel way by the (+)enantiomer of Org 3770. The (-)enantiomer was ineffective at the autoreceptors, whereas it shifted to the right the concentration-response curve for NA, as an inhibitor of release of [3H]5HT. For the evaluation of the antagonist affinities at adrenoceptors on noradrenergic and serotoninergic nerve terminals, pA, values

t +’ Time

of superfusion

IminI

18 19x)21 Time

222324

of superfuslon

(mid

Fig. 3. Effects of (+)Org 3770 and (-)Org 3770 on the inhibition by NA of the K+-evoked release of [3H]NA (left panel) and [‘HIS-HT (right panel). Synaptosomes from the cortex of the rat were prepared, prelabeled with [3H]NA or [“HIS-HT and superfused as described under Methods. After 10 min of superfusion with standard medium, exogenous NA was added (the antagonists were present from the beginning of superfusion) and 8 min later (see arrow) the synaptosomes were depolarized with 15 mM KCl. The lag between application of K+ and evoked release is only apparent, as it takes about 2 min for the fluid to flow from the filter to the collecting vials. The curves presented are based on the average of 46 experiments in triplicate. Left panel (0-O) 15 mM KCl; (0-O) 15 mM KC1 + 1 PM NA; (A-A) 15 mM KC1 + 1 PM NA + I PM (-)Org 3770; (A---A) 15 mM KC1 + 1 ,uM NA + 1 yM (+)Org 3770.

Profiles of the t%aza-analogue of mianserin, Org 3770 and its enantiomers

b

1

+

0.03

01

405

03

Noradrenaline cont. I pM1 Fig. 4. Effects of (+)Org 3770 and (-)Org 3770 on the concentration-dependent inhibition by NA of the K+-evokecl release of (‘H]NA (left panel) and 13HJ5-HT(right panel) from synaptosomes from cortex. ~x~rirnen~ details as in the legend to Figure I and Methods. Each point represents the mean + SE of 4-6 experiments in triplicate. Data represent the effects of varying concentrations of NA: (0-O) ISmMKCI;(D--m) 15mMKCl+O.O3~M(+)Org377~(~-~) lSmMKCliO.l~M(~)Org 3770; (A-A) I5 mM KC1+ 0.1 gM (-)Org 3770. All values were significantly different from the corresponding values obtained in the absence of antagonists (at least P < 0.005).

were calculated from the parallel shifts of the concentration-response curves at the level of I& , The pA, values amounted to 8.35 for the (+)enantiomer, as an antagonist of NA at the autoreceptors; to 8.13 for the (+)enantiomer and 7.68 for the (- )enantiomer, as antagonists at the cl,-heteroreceptors mediating regulation of release of 5-HT. DISCUSSION

The antidepressant drug ( f )mianserin increases the availability of noradrenaline through blockade of presynaptic a,-adrenoceptors (Baumann and Maitre, 1977; Nickolson and Wieringa, 1981; Raiteri et al., 1982). Its considerable potency as an inhibitor of the uptake of noradrenaline in vitro may contribute to the increase of availability of noradrenaline in the synaptic cleft. Therefore, it is still unclear whether blockade of norad~naIine autoreceptors as such is su~cient for therapeutic etlicacy in depression. Besides, chronic treatment with mianserin sensitizes serotonergic transmission through a yet unknown mechanism which is presumably related to the potent S-HT, antagonistic properties of (&)mianserin (Blier, De Montigny and Tardif, 1984). The drug (+)Org 3770 is chemically closely related to, but has a neurochemical profile differing from (2)mianserin in two ways. (f)Org 3770 did not inhibit noradrenaline uptake in vitro and it was found to be 20-fold weaker than (&)mianserin as a 5HT,antagonist. The a,-adrenolytic properties have been studied in various models. First, (+)Qrg 3770 was equipotent with mianserin as an antagonist of aradrenoceptors in most of the models studied, although (+ )Org 3770 was three-fold less active as an

antagonist of inhibition of the twitch response in the electrically-stimulated vas deferens preparation induced by azepexole. In addition, using membranes from the cortex of the rat, (+)Org 3770 was found to have five-fold preference for cr,-, over cr,-adrenoceptors, while mianserin did not discriminate between these subtypes of adrenoceptors. Both (+)mianserin and (l)Org 3770 were moderately potent in displacing radiolabelled rauwolscine from its binding sites with Ki values of about IO-‘M. Similar data have been found for ( i: )mianserin in various binding tests using different a,-adrenergic ligands (Pinder, 1985). These binding data refer almost exclusively to postsynaptic cc,-adrenoceptors (Dausse, Le Quau-Bui and Meyer, 1982). Experiments with (+)Org 3770 and (f )mianserin in slices of cortex clearly revealed the a,-blocking effects of these compounds and confirmed that the a,-adrenoceptor antagonist property resided in (+)Org 3770. Measurement of exact PA, values in such models is not possible due to the tonic inhibition of release of 13H]NA by endogenous noradrenaline. The data on antagonism of the noradrenahneinduced inhibition of release of [3H]NA in synaptosomes showed a concentration-dependent shift to the right of the inhibition curve for (+)Org 3770. From these data, pA, values of about 8 could be calculated, suggesting that (+)Org 3770 had a six-fold preference for noradrenaline autoreceptors, compared to postsynaptic a,-adrenoceptors. Studies in the vas deferens preparation showed that presynaptic ~~-adrenocepto~ and postsynaptic a,-adreno~ptors (in the presence of cocaine to exclude a role of inhibition of uptake) were blocked by (~}mian~~n and its enantiomers with equal potency. In these

406

TH. DE BOER et af.

studies, (~}mianserin was five-fold less active than the classical a,-adrenoceptor antagonist yohimbine. Comparing the affinities of (+)Org 3770 and (+_)mianserin in central (cortical synaptosomes) and peripheral (vas deferens preparation) models for a,-adrenolytic activity revealed at least a lo-fold difference in potency. This difference suggests that these compounds have a preferential effect on central, compared to peripheral, presynaptic adrenoceptors. Different subclasses of a,-adrenoceptors have been described in the literature. Using [‘Hlclonidine as a ligand, both high and low affinity states of the a~-binding site were detected. (~)Mian~~n was found to be strongly active in blocking the high and low affinity site of the binding of [3H]clonidine (Maggi, UPrichard and Enna, 1980). Both enantiomers of mianserin showed identical affinity for these binding sites for [3H]clonidine (Johnson, Nelson, Nicholass, Richards and Thomas, 1984), which contrasted with the greater activity of (+)mianserin in modulating the release of noradrenaline from tissue slices and synaptosomes. Also, in the binding studies with [3H]rauwolscine a six-fold preference for (+) over (-)mianserin was observed. The differences were even more pronounced for Org 3770, showing a 40-fold preference for (+) compared to (-)Org 3770 in the binding of t3H]rauwolscine. Based on comparison of rank orders of adrenergic drugs modulating release of noradrenaline and serotonin through presynaptic adrenoceptors, it has been proposed that these two types of ar-adrenoceptors differ pharmacologically (Frankhuysen and Mulder, 1982). Such a difference was clearly shown by using the enantiomers of mianserin: the (-)enantiomer blocked the az-postsynaptic receptors sited on serotonergic nerve terminals but not the or?-autoreceptors, whereas (+)mianserin was effective at both auto- and postsynaptic receptors (Raiteri, Maura and Versace, 1983). The enantiomers of Org 3770 behaved similarly, the (+)enantiomer being moderately more effective than the (-)enantiomer. Several studies had previously shown the moderate a,-antagonistic properties of mianserin, compared to other potent and selective adrenolytics. Without exception, such potency ratios have been based on antagonism of the interaction of clonidine or other potent a-adrenergic drugs with behavioural or physiological parameters (Dettmar, Lynn and Tulloch, 1983; Doxey, Roach and Smith, 1983; Drew, Gower and Marriot, 1979). Mianserin behaved as a moderately active ~*-antagonist in these tests, in agreement with the binding data. However, both mianserin and Org 3770 behaved like very potent presynaptic antagonists in vas deferens of the rat or in cortical synaptosomes suggesting that these compounds may preferentially block a,-adrenergic autoreceptors. The results of a short-term, double-blind, placebocontrolled clinical trial with (&)Org 3770 suggested that the compound is an effective antidepressant with

a potency at least similar to that of (~)mian~~n (Claghorn, Johnstone, Studebaker and Ajeman, 1987). A role for inhibition of the uptake of noradrenaline in the therapeutic effects of (&)mianserin has been considered to be unlikely in view of the weak effects of (+)mianserin in models presumed to measure uptake in uivo (Goodlet et al., 1977; Van Riezen et al., 1981). The clinical efficacy of (+)Org 3770 would support that view since (+)Org 3770 did not inhibit the uptake of noradrenaline in uitro. Therefore, it is tempting to suggest that the a,-antagonistic properties of (+)mianserin and ( f )Org 3770 represent the principal factor involved in the therapeutic effects of these drugs in depressed patients (see also Pinder, 1985). Theoretically, an increase of noradrenaline in the biophase invoives mutually antagonistic effects as it may inhibit the release and synthesis of noradrenaline through autoreceptors while, on the other hand, postsynaptic effects will be facilitated. At the postsynaptic level, the responses of noradrenaline in slices of cortex are mediated through a &receptor coupled adenylate cyclase which is additionally stimulated through a postsynaptic a,-adrenoceptor (Duman, Strada and Enna, 1985; Pile and Enna, 1986). An inhibitory control by ~~-adren~eptors in membrane preparations from cortex has been demonstrated on the formation of the cyclic AMP activated by forskolin (Kitamura, Nomura and Segawa, 1985). In slices, (&)mianserin inhibits the a*-adrenoceptor-mediated component of the stimulatory effect of adrenergic drugs on the formation of cyclic AMP (Pile and Enna, 1986). However, (+)mianserin is about 6-fold more potent than (-)mianserin. Taken together, blockade by (+)mianserin and probably also by (+)Org 3770, of the presynaptic inhibitory autoreceptor and the postsynaptic stimulatory or,-adrenoceptor may facilitate ~,-re~ptor-mediated noradrenergic transmission. Long term blockade of ~rotonin receptors results in supersensitivity of the serotonin system (Blier et al., 1984). Such a mechanism offers an alternative explanation for the therapeutic effects of (Ifr)mianserin. However, (+)Org 3770 was considerably less active as a serotonin antagonist in the binding test with 13H]mianserin or the fundus strip model in the rat. Since (&)Org 3770 was considerably weaker as a SHT,-antagonist but apparently equipotent in the clinic with (f)mianserin, it appears unlikely that antagonistic properties to serotonin explain the antidepressant effects of (+)Org 3770. However, a role of 5-HT antagonism in the therapeutic effects of mianserin cannot be excluded in view of the potent SHT,-antagonistic properties of mianserin. Both (+)Org 3770 and (_+)mianserin had moderate affinity for muscarinic cholinergic receptors, while both compounds where potent histamine-l antagonists. However, a role for the blockade of these receptors in the therapeutic effects of antidepressant drugs is highly unlikely (Hall and ogren, 1981). In conclusion, a careful comparison of the neuro-

Profiles of the 6-aza-analogue of mian serin, Org 3770 and its enantiomers

chemical and autonomic pha~acological properties of (f)mianserin and the chemically related drug (&)Org 3770 suggests a prominent role for blockade of a2-adrenoceptors in the therapeutic effects of mianserin and (+)Org 3770 in depression, probably excluding a role for inhibition of the uptake of noradrenaline. Acknowledgements-The skilful technical assistance of Chris Versteegen, Maria van Rosmalen, Jan van der Togt, Marianna van Vugt, Ron Geurts, Hans Steenbergen and Gerard van Aalst (Organon Oss), Marco Fe&to and Massimo Ulivi (Milano) is eratefullv acknowledeed. The

generous gift of &epexoie be-Boehri~ger, fngelhe&, is also gratefully a~knowl~ged.

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