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Home , Dihydroalprenolol, Dioxolane, Quinupramine, Spiperone

Binding Profile of , a New

Hirohiko SAKAMOTO*, **, Nobuharu YOKOYAMA*, **, Shigekatsu KOHNO* and Katsuya OHATA* *Department of Pharmacology , Kyoto Pharmaceutical University, Yamashina, Kyoto 607, Japan **Biochemical Division , Research Laboratories, Nippon Shoji Kaisha, Ltd., Ibaraki, Osaka 567, Japan

Accepted August 17, 1984

Abstract-The receptor binding profile, composed of the K;-values measured in eight different receptor binding models using rat brain membranes, is reported for the new quinupramine, 10,11 -dihydro-5-(3-quinuclidinyl) 5H-dibenz[b, f]azepine, and three reference compounds with a tertiaryamine side chain. Quinupramine was found to possess high affinity for muscarinic and Ht receptor binding sites in rat brain, whereas its affinity for binding sites was only one seventieth that of imipramine. Receptor binding profiles of the reference compounds were almost similar to that of quinupramine , except in the case of imipramine binding sites.

Tricyclic are known to be clidinyl [phenyl-4-3H]benzilate (42 Ci/ inhibitors of and mmol), [pyridinyl-5-3H]pyrilamine (28 Ci/ reuptake into nerve terminals. This action was mmol) and [phenyl-4-3H] (17 Ci/ also believed to be responsible for their mmol) were purchased from Amersham , therapeutic effects and probably involved Buckinghamshire, England. [N-methyl-3H] presynaptic sites (1). In addition, recent imipramine hydrochloride (75 Ci/mmol) was reports suggest a direct interaction of tricyclic from New England Nuclear, Boston, MA , antidepressants with a variety of seemingly U.S.A. 3H-Ligands were stored at -20°C postsynaptic sites. These evidences have and diluted in standard assay buffer before been obtained through the interaction of use. tricyclic antidepressants with central Chemicals: Unlabelled materials were muscarinic cholinergic (2), histaminergic (3), obtained from the following sources: quinu (4) and a- (5) pramine (LM-208, Pharmuka, Gennevilliers, receptors. France and Nippon Shoji Kaisha, Ltd., In this paper, we report the receptor Osaka, Japan), imipramine hydrochloride binding profile composed of the K;-values (Sigma, St. Louis, MO, U.S.A.), measured in eight different receptor binding hydrochloride (Tokyo Kasei Co., Tokyo, models for the new tricyclic antidepressant Japan), hydrochloride (Fuji quinupramine, 10,1 1 -dihydro-5-(3-quinu sawa, Osaka, Japan), mesylate clidinyl)-5H-dibenz[b,f]azepine, and related (Nippon Ciba-Geigy, Osaka, Japan), pro tertiaryamine tricyclic antidepressants. pranolol hydrochloride (Sumitomo, Osaka, Japan), sulfate (E. Merck, Materials and Methods Darmstadt, West Germany), chlorpheniramine Radioactive materials:[ 3H]WB4101 (27 maleate (Kowa, Tokyo, Japan), cypro Ci/mmol), [phenyl-4-3H] hydro heptadine hydrochloride (Merck Sharp & chloride (24 Ci/mmol), /-[4,6, propyl-3H] Dohme, Rahway, NJ, U.S.A.) and (71 Ci/mmol), /-quinu hydrochloride (Dainippon, Osaka, Japan). H. Sakamoto et al. Other chemicals of reagent grade were the apparent inhibition constant (K;) values obtained commercially. were calculated from the equation: Receptor binding assay: Methodological details on receptor binding models, i.e., specification on tissue, 3H-ligand, assay for non-specific binding and incubation con in which KD is the equilibrium dissociation ditions are summarized in Table 1. constant for each ligand, and the K,,-values Adult male Sprague-Dawley rats were were calculated by Scatchard analysis. All used at weights of 200 to 250 g. After assays and experiments were performed at decapitation, brains were removed, and least in duplicate. specific regions dissected over ice im Protein amount: Protein contents of brain mediately before assay were homogenized membrane preparations were determined by in ice-cold standard assay buffer with an the method of Lowry et al. (11), using Ultratrax' (Janke & Kunkel, Staufen, West bovine serum albumin as the standard protein. Germany) at 4°C for 1 5 sec. After incubation, labelled membranes were harvested and Results washed (with 3x5 ml ice-cold standard The inhibitory potencies of quinupramine assay buffer) by rapid vacuum filtration and three reference compounds were through glass fiber filters (GC-50, Toyo measured in the eight different receptor Roshi Co., Ltd., Tokyo, Japan) using a binding models described in Table 1. To filtration manifold. Radioactivity on the allow quantitative comparison of data, K; filters was determined in a liquid scintillation values (see methods) were calculated and spectrometer (Mark III, Searle Analytic, Des summarized in Table 2. Hill coefficients (n1), Plaines, IL, U.S.A.) in 10 ml of BiofluorCl' the slope of the line in the log-logit plot (Hill (New England Nuclear) with an efficiency of plot), were calculated by linear regression of 45%. The IC50 values, the concentrations the data and also presented in Table 2. required to inhibit specific binding by 50%, Quinupramine was the most potent were determined by log-logic analysis, and inhibitor for the binding of [3H]quinuclidinyl

Table 2. Apparent inhibition constant (K;) and Hill coefficier,t (nH) of quinupramine and reference compounds for neurotransmitter receptor binding sites benzilate at muscarinic cholinergic receptors clinical side effects, dry and for[ 3H]pyrilamine at histamine H, mouth and mydriasis (2). receptors with K;-values of 2.9 and 9.1 nM, Recently, Kwok and Mitchelson (12) respectively. Imipramine, amitriptyline and demonstrated that and , clomipramine were potent inhibitors of the both of which have been reported to possess imipramine binding sites labelled by[ 3H] little anti-muscarinic cholinergic activity on imipramine with K;-values of 20 nM or autonomically innervated visceral organs, below, whereas the affinity of quinupramine exhibit a similar order of potency to amitrip for the imipramine binding sites (K;=510 tyline in inhibiting the action of the muscarinic nM) was only one seventieth that of imipra agonist McN-A-343 on the superior mine. Quinupramine also displayed sub cervical ganglion, but are much less potent stantial affinity for 5-HT2 receptors labelled than amitriptyline in inhibiting prejunctional by[ 3H]spiperone in the cerebral cortex muscarinic receptors at sympathetic and (K;=56 nM, nH=0.27), and it had quite weak parasympathetic postganglionic nerve end affinity for a, -, a2 and 3-adrenergic as well ings. Whether these findings have any as D2 receptor binding sites. relevance to the blockade of neural muscarinic The affinity of imipramine and clo cholinergic receptors in the central nervous mipramine for the muscarinic cholinergic system remains to be determined, but it is of receptors (K;=56 and 55 nM, respectively) interest that Fisher et al. (13) have suggested was weaker than that of amitriptyline that muscarinic cholinergic receptors in (Ki=5.8 nM) and quinupramine (Ki=2.9 sympathetic ganglia may resemble central nM). muscarinic cholinergic receptors more than These tricyclic antidepressants were also do muscarinic cholinergic receptors in smooth potent inhibitors for the binding of[ 3H] muscle, and that Janowsky et al. (14) have pyrilamine to histamine H, receptors. Their suggested that central cholinergic and potencies in competing with [3H]pyri!amine adrenergic factors may play a role in the were almost similar to their potencies in ethiology of affective disorders. Thus reducing [3H]quinuclidinyl benzilate binding. tricyclic antidepressants block central The four antidepressants tested were cholinergic activity; accordingly, this would substantially less potent inhibitors for[ 3H] shift the adrenergic-cholinergic balance clonidine at a2-adrenergic receptors and for towards an adrenergic predominance (14). [3H]dihydroalprenolol at ,3-adrenergic Further work is required to clarify the role of receptors than at any other receptor binding central muscarinic cholinergic receptors in sites. the antidepressant efficacy of quinupramine. Relative potencies of tricyclic anti Discussion depressants at histamine H, and a, Quinupramine was found to possess high adrenergic receptors were reported by Tran affinity for both muscarinic cholinergic and et al. (3) and U'Prichard et al. (5), respec histamine H, receptor binding sites in rat tively. However, blockade of one of a brain membranes. Central anti-muscarinic number of neurotransmitter receptors cannot cholinergic and central anti -histaminergic simply account for antidepressant efficacy effects of quinupramine have been reported (1 ). Furthermore, the sedative and hypo on the basis of its blockade of tensive activities of antidepressants are as induced tremor and histamine-induced sociated with histamine H, (15) and a, lethality, respectively (Uzan, personal obser (5) blockade, respec vation). The present study provides the first tively. Recently, high affinity specific binding direct evidence of the interaction of quinupra of 3H-labelled antidepressants, i.e., [3H] mine with central nervous system receptors. imipramine (10), [3H]amitriptyline (16), In general, tricyclic antidepressants are [3H] (17) and [3H]mianserin fairly potent inhibitors for agonists at (9), has been demonstrated and further muscarinic cholinergic receptors, and this characterized in rat brain. However, the action is thought to correlate well with affinity of antidepressants for 3H-labelled

labeled serotonin receptor binding. Science 210, 88-90 (1980)

imipramine binding. Eur. J. Pharmacol. 61, 373 380 (1980)

antidepressant specific binding sites does not (1977) correlate with their binding potencies at 3 Tran, V.T., Chang, R.S.L. and Snyder, S.H.: neurotransmitter binding sites nor with their Histamine H, receptors identified in mammalian antidepressant activity. More recently, Langer brain membranes with [3H]. Proc. et al. have suggested that high affinity [3H] Natl. Acad. Sci. U.S.A. 75, 6290-6294 (1978) 4 Peroutka, S.J. and Snyder, S.H.: Long-term imipramine binding might be associated with antidepressant treatment decreases spiroperidol the mechanism of serotonin uptake in the brain, because the correlation between the potencies of these drugs for the inhibition of 5 U'Prichard, D.C., Greenberg, D.A., Sheehan, [3H] imipramine binding and the inhibition P.P. and Snyder, S.H.: Tricyclic antidepressants; of the uptake of [3H]serotonin is highly Therapeutic properties and affinity for a significant (18). noradrenergic receptor binding sites in the Quinupramine, unlike other tricyclic anti brain. Science 199, 197-198 (1978) depressants, has been shown to be a weak 6 U'Prichard, D.C., Greenberg, D.A. and Snyder, inhibitor of [3H]imipramine binding. From S.H.: Binding characteristics of a radiolabeled this result, it seems that quinupramine has agonist and antagonist at central nervous system less potent inhibition of the neural uptake alpha noradrenergic receptors. Mol. Pharmacol. of serotonin. In fact, quinupramine is a weak 13, 454-473 (1977) inhibitor of serotonin and noradrenaline 7 Bylund, D.B. and Snyder, S.H.: Beta adrenergic uptake in vitro (H. Sakamoto et al., un receptor binding in membrane preparations from mammalian brain. Mol. Pharmacol. 12, 568 published data). 580 (1976) Some antidepressants have been shown to 8 Innis, R.B., Tune, L., Rock, R., Depaulo, R., be antagonists of serotonin receptors, and U'Prichard, D.C. and Snyder, S.H.: Tricyclic the therapeutic effects may be explained by antidepressant radioreceptor assay. Eur. J. their postsynaptic rather than presynaptic Pharmacol. 58, 473-477 (1979) effects at central serotonergic receptors (4, 9 Peroutka, S.J. and Snyder, S.H.: [3H] Mianserin; 19). In general, clinically effective anti Differential labeling of serotonin2 and histamine, depressants have substantial affinity for a receptors in rat brain. J. Pharmacol. Exp. Ther. number of neurotransmitter receptor binding 216, 142-148 (1981) sites. However, it is unlikely that the acute 10 Raisman, R., Briley, M.S. and Langer, S.Z.: blockade of central neurotransmitter binding Specific tricyclic antidepressant binding sites in sites relates to antidepressant activity; rat brain characterised by high-affinity 3H rather, it may contribute to drug-induced side effects. Additionally, the inhibition of monoamine uptake by tricyclic antidepres 11 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and sants occurs acutely, while the onset of Randall, R.J.: Protein measurement with the action of all antidepressants is delayed for Folin phenol reagent. J. Biol. Chem. 193, 265 up to 1 to 3 weeks (I). 275 (1951) It is thus likely that antidepressants may 12 Kwok, Y.H. and Mitchelson, F.: Comparison of not have a common mechanism of action. the antimuscarinic activity of amitriptyline, On the other hand, depression is not a viloxazine and mianserin at neuronal muscarinic homogeneous illness (20), and different receptors. In Advances in the Biosciences, antidepressants may be required for different Edited by Langer, S.Z., Takahashi, R., Segawa, subtypes of depression. T. and Briley, M., Vol. 40, New Vistas in De pression, p. 87-91, Pergamon Press, Oxford, New York, Toronto, Sydney, Paris and Frank References furt (1982) 1 Sugrue, M.F.: Current concepts on the mecha 13 Fisher, A., Weinstock, M., Gitter, S. and Cohen, nisms of action of antidepressant drugs. Phar S.: A new probe for heterogeneity in muscarinic macol. Ther. 13, 219-247 (1981) receptors; 2-Methyl-spiro-(1,3--4,3') 2 Snyder, S.H. and Yamamura, H.I.: Anti quinuclidine. Eur. J. Pharmacol. 37, 329-338 depressants and the muscarinic (1976) receptor. Arch. Gen. Psychiatry 34, 236-239 14 Janowsky, D.S., EI-Yousef, M.K., Davis, J.M. and Sekerke, H.J.: A cholinergic-adrenergic uptake of serotonin but not of norepinephrine. hypothesis of mania and depression. Lancet 2, Science 210, 1133-1135 (1980) 632-635(1972) 19 Takahashi, R., Tateishi, T., Yoshida, H. and 15 Hall, H. and Ogren, S.-O.: Effects of antide Hironaka, I.: Effects of chronic treatment with pressant drugs on histamine-H, receptors in the antidepressant drugs on serotonergic receptor brain. Life Sci. 34, 597-605 (1984) binding activity in normal and 16 Rehavi, M. and Sokolovsky, M.: Multiple binding depression rat. In Advances in the Biosciences, sites of tricyclic antidepressant drugs to mam Edited by Langer, S.Z., Takahashi, R., Segawa, malian brain receptors. Brain. Res. 149, 525-529 T. and Briley, M., Vol. 40, New Vistas in De (1978) pression, p. 29-36, Pergamon Press, Oxford, 17 Biegon, A. and Samuel, D.: Binding of a labeled New York, Toronto, Sydney, Paris and Frank antidepressant to rat brain tissue. Biochem. furt (1982) Pharmacol. 28, 3361-3366 (1979) 20 Maas, J.W.: Clinical and biochemical he 18 Langer, S.Z., Moret, C., Raisman, R., Dubocovich, terogeneity of depressive disorders. Ann. Intern. M.L. and Briley, M.: High-affinity [3H]imipramine Med. 88, 556-563 (1978) binding in rat hypothalamus; Association with