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Home , Dimenhydrinate, Mepyramine, Mequitazine

Antimuscarinic Effects of : Quantitative Evaluation by Receptor-Binding Assay

Nobuo KUBO, Osamu SHIRAKAWA, Takayoshi KUNO and Chikako TANAKA* Departmentof Pharmacology,Kobe University School of Medicine,Chuo-ku, Kobe650, Japan Accepted November22, 1986

Abstract-Quantitative evaluation of antimuscarinic effects of antihistamines (H1 and H2-receptor antagonists) was carried out using a receptor-binding assay. The inhibition constants (K; values) of twenty seven H1-receptor antagonists, one related and three H2-receptor antagonists at Hi-receptors and muscarinic receptors in the bovine cerebral cortex were determined. All the H2 receptor antagonists examined showed very low affinity for the muscarinic receptors. On the other hand, some Hl-receptor antagonists (, cyproheptazine, , , , and ) had high affinity for the muscarinic receptors (K;=5.0-38 nM). Another group of H, -receptor antagonists (, , metapyrilen, , and ) had low affinity for the muscarinic receptors (K;=3,600 30,000 nM). Thus, a broad range of antimuscarinic potencies among the anti was demonstrated. These results should provide helpful information with regard to the clinical and experimental use of antihistamines.

Histamine has been shown to stimulate binding studies have provided a new method two different types of receptors which are for quantitative evaluation of the affinities of referred to as H1 and H2-receptors. Hj some for various receptors (5). In this receptor mediates -induced con study, we determined the affinities of a large traction of smooth muscles of the small series of antihistamines for H1 and muscari intestine and bronchi. H2-receptor mediates nic receptors of bovine cerebral cortex using the action of histamine in stimulating gastric a receptor-binding assay and showed that acid secretion. The receptors are defined there is a broad range of antimuscarinic pharmacologically from the antagonists which potencies among the antihistamines. selectively block the response of these tissues to histamine stimulation (1, 2). Materials and Methods Some of the classical H,-receptor an Membrane preparations: Bovine brains tagonists such as (3) and were obtained from a local slaughterhouse, (4) were reported to display and stored at -80°C until use. Membrane substantial -like activity which was preparations were prepared as described assessed by a bioassay system. In the follow previously (6). Briefly, the cerebral cortex ing years, numerous antihistamines of much was homogenized in 20 volumes of ice-cold greater and specificity either at H1 50 mM Tris-HCI (pH 7.4) with a Kinematika or H2-receptors were synthesized. While Polytron PT-10 (setting 7, 20 sec). The some of these drugs are thought to possess homogenates were centrifuged three times antimuscarinic activities which may cause for 10 min at 50,000xg at 4°C with resus certain adverse effects in clinical use, quanti pension of the intermediate pellet from each tative evaluation of the antimuscarinic effects centrifugation in the ice-cold Tris buffer. has not been reported. Recently, receptor The final pellets were used immediately or stored at -80°C until assayed. * To whom correspondence should be addres sed. Radioligand-binding assay: 3 H-mepyra mine and 3H-quinuclidinyl benzilate (QNB) 3H-mepyramine binding was linear and gave binding assays were performed as described a Kd value of 1.16±0.21 nM and a Bmax value previously (7, 8). Briefly, the membrane of 95.4±5.2 fmol/mg protein (Fig. 1A). preparations were added to siliconized glass These values are in good agreement with test tubes containing 3H-mepyramine or 3H data in the literature (11, 12). As shown in QNB and various drugs in the Tris buffer, with Fig. 2A, the affinities of various H1-receptor a final assay volume of 0.6 ml. The assay antagonists and other drugs for the H1 tubes were incubated for 30 min at 25'C for receptor in the bovine cerebral cortex were 3H-mepyramine binding and for 60 min at determined according to the inhibition of 3H 25°C for 3H-QNB binding. The incubation mepyramine binding. The inhibition constants was terminated by rapid filtration over poly etylenimine-treated glass fiber filters (9) under vacuum. The filters were then rinsed rapidly with 20 ml of ice-cold Tris buffer. Radioactivity trapped on the filters was counted by liquid scintillation spectroscopy. Nonspecific bindings were determined in the presence of 2 ,uM for 3H mepyramine binding and 1 ,uM atropine for 3H-QNB binding . Protein was assayed by the method of Lowry et al. (10). Materials: 3H-mepyramine (24.1 Ci/mmol) and 3H-QNB (30.1 Ci/mmol) were obtained from New England Nuclear. Other reagents were purchased from Sigma. Examined com pounds were as follows: mepyramine, me tapyrilen, , and antazoline (Sigma); triprolidine and diphen hydramine (Tanabe Pharmaceutical); pro methazine (Shionogi Pharmaceutical); d chlorphenilamine (Shering); , hydro xyzine and meclizine (Taito-Pfizer); clemas tine and (Sandoz); homochlorcy clizine, diphenylpyraline and azelastine (Eisai); mequitazine and ranitizine (Shin nippon Jistugyo); cyproheptazine (Merck Sharp & Dohme); alimemazine (Dai-ichi Fig. 1. Characterization of histamine H1 and Pharmaceutical); pyrathiazine (Upjohn); muscarinic receptors in bovine cerebral cortex. A) (Taisho Pharmaceutical); Saturation of 3H-mepyramine binding in bovine dimethindene (Ciba-Geigy Japan); di cerebral cortex. 3H-mepyramine binding was pheterol (Toyama Chemical); measured as described in "Materials and Methods" (Sumitomo Chemical); mebhydroline (Bayer); using various concentrations of 3H-mepyramine in (Janssen Pharmaceutical); ter the presence (for nonspecific binding) and absence fenadine (Dow Chemical); (SK&F (for total binding) of 2 ttM triprolidine. Specific Fujisawa); (Yamanouchi Phar binding was the difference between total and maceutical). nonspecific binding. The experiment was repeated three times with similar results. Inset: Scatchard Results plot. B) Saturation of 3H-QNB binding in bovine 3H-mepyramine binding: Specific 3H cerebral cortex. 3H-QNB binding was performed using various concentrations of 3H-QNB in the mepyramine binding to the membrane pre presence and absence of 1 ,uM atropine. The experi parations from bovine cerebral cortex was ment was replicated three times with similar results. saturable (Fig. 1A). Scatchard plot of specific Inset: Scatchard plot. showed very low affinities for the H1-receptor (Fig. 2A and Table 1). 3H-QNB binding: Specific 3H-QNB binding to the membrane preparations from bovine cerebral cortex was also saturable, and the Scatchard analysis gave a Kd value of 0.24±0.02 nM and a Bmaxvalue of 1240+270 fmol/mg protein (Fig. 1 B). These values are in agreement with data in the literature (13, 14). The Scatchard plot of specific 3H-QNB binding was linear, thereby indicating a single class of high affinity binding sites in the bovine cerebral cortex (Fig. 1B). As shown in Fig. 2B and Table 1, a group of Ht receptor antagonists (mequitazine, cypro heptazine, clemastine, diphenylpyraline, pro methazine, homochlorcyclizine and alime mazine) showed a potent antagonism for the muscarinic receptor (Ki=5.0-38 nM). Another group of Hl-receptor antagonists (mepyramine, terfenadine, metapyrilen, azelastine, hydroxyzine and meclizine) ex hibited a weak antagonism of the muscarinic receptor (Ki=3,600-30,000 n M) . Other Ht antagonists showed moderate affinity for the muscarinic receptors. Doxepin also showed Fig. 2. Competition by antihistamines for 3H high affinity for the muscarinic receptor, and mepyramine (A) and 3H-QNB (B) binding to the three H2-antagonists had little cross-reactivity membrane preparations from bovine cerebral cortex. (Table 1). Hill coefficients for all anti The concentration of 3H-mepyramine and 3H-QNB histamines studied were essentially equal to were 1 nM and the concentrations of unlabelled unity (Table 1), suggesting the antagonistic compounds were varied as indicated. (*) mepyra nature of these compounds for muscarinic mine, (0) diphenylpyraline, (/) triprolidine, (0) receptors (15). mequitazine, (A) diphenhydramine, (A) antazoline. The ratio of the K; value with 3H The experiment was repeated three times with mepyramine binding to that with 3H-QNB similar results. binding for each was calculated in order to quantitatively evaluate the selectivity (Ki) were calculated from the equation (Table 1). Except for several drugs (mepyra Ki=1C50/(1 +C/Kd), where IC50=concen mine, metapyrilen, ketotifen, dimethindene, tration causing 50% inhibition of specific azelastine and hydroxyzine, K; value ratio:::- binding which was obtained from the dis 590-130,000), most H1-receptor antagonists placement experiments, C=3H-ligand con seemed to have substantial interaction with centration used in the displacement experi the muscarinic receptors. That is, thirteen ment, and Kd= dissociation constant from the compounds out of twenty-seven H1 Scatchard analysis of the equilibrium experi antagonists had a ratio of less than 100, ments. Calculated Ki values of Hl-receptor indicating considerably low selectivity. For antagonists for 3H-mepyramine binding were example, mequitazine showed almost equal in the range of 0.24 nM (mepyramine) to 160 affinities for the H1 and the muscarinic nM (antazoline, dipheterol) (Table 1). receptors (Ki value ratio=1.1). Doxepin, an antidepressant, exhibited the most potent H, -receptor antagonism, and all Discussion the three H2-receptor antagonists examined This paper presents the data of the binding Table 1. Inhibition constants of antihistamines and related compounds for histamine H,-receptors and muscarinic receptors in bovine cerebral cortex

The inhibition of specific binding of 3H-mepyramine (1 nM) and 3H-QNB (1 nM) binding was determined with seven to ten concentrations of competing drugs assayed in triplicate as shown in Fig. 2. The mean inhibitory concentration (IC50) values were determined from the log-probit analysis, and K; values were calculated from the equation described in "Results". The Hill coefficient (nH) was calculated as described by Bennett and Yamamura (15). Values are the means±S.E. from three independent experiments. potencies of a large series of antihistamines were shown to have antimuscarinic effects. at the muscarinic and However, the antimuscarinic and anti shows that there is a broad range of anti histaminic potencies for the twenty-seven Ht muscarinic potencies (K;=5.0-30,000 nM) receptor antagonists studied were not sig among the antihistamines. Except for several nificantly correlated (r=0.65), indicating that drugs, many classical and new antihistamines the structural requirement for the anti muscarinic effect is different from that for the References antihistaminic effect. In general, 1 Ash, A.S.F. and Schild, H.Q.: Receptors mediating amines (prototype: diphenhydramine) and some actions of histamine. Br. J. Pharmacol. 27, phenotiazines (prototype: promethazine) 427-439 (1966) were potent H1-receptor antagonists that 2 Black, J.W., Duncan, W.A.M., Durant, C.J., possess significant antimuscarinic activity. Ganallin, C.R. and Parsons, E.M.: Definition and On the other hand, (pro antagonism of histamine H2-receptors. Nature totype: mepyramine), alkylamines (prototype: 236, 385-390 (1972) chlorpheniramine) and (prototype: 3 Rekker, R.F., Timmerman, H., Harms, A.F. and hydroxyzine) were selective H1-antagonists Nauta, W.T.: The antihistaminic and anti with no considerable antimuscarinic activity. activities of optically active diphen Seventeen out of twenty-seven H 1 -receptor hydramine derivatives. Arzneimittelforsch. 21, antagonists have K; values for muscarinic 688-691 (1971) receptors in the nanomolar range (5.0-740 4 von Schlichtegroll, A.: Zur Pharmakologie ver nM); and in spite of its high K; value (3,000 schiedener Thiophenylpyridylamin-Derivate. Arzneimittelforsch. 7, 237-252 (1957) nM), antazoline was reported to have an 5 El-Fakahany, E. and Richelson, E.: Antagonism atropine-like effect (4), probably due to its by of muscarinic acetylcholine low selectivity for the H1-receptor (K; value receptors of human brain. Br. J. Pharmaco!. 78, ratio=19). Consequently, compounds at the 97-102 (1983) top of the lists (Table 1) will likely block 6 Kuno, T., Saijoh, K. and Tanaka, C.: Solubilization muscarinic receptors at the blood concen of D2 dopamine receptor coupled to guanine trations achieved in clinical practice. Some nucleotide regulatory protein from bovine H1-receptor antagonists with potent anti striatum. J. Neurochem. 41, 841-847 (1983) muscarinic activity are clinically used to 7 Kuno, T., Kubo, N. and Tanaka, C.: Molecular combat . These include size of histamine H-1 receptor determined by (dimenhydrinate and diphen target size analysis. Biochem. Biophys. Res. hydramine) and (meclizine and Commun. 129, 639-644 (1985) others). Since was reported to 8 Kuno, T., Shirakawa, 0. and Tanaka, C.: Regu be a potent drug for the prophylaxis and lation of the solubilized bovine cerebral cortex treatment of motion sickness (16), the anti muscarinic receptor by GTP and Na+. Biochem. motion sickness activity of some of the H 1 Biophys. Res. Commun. 112, 948-953 (1983) 9 Bruns, R.F., Lawson-Wendling, K. and Pugsley, receptor antagonists seems to be related to T.A.: A rapid filtration assay for soluble receptor their antimuscarinic ability. As the histamine using polyethylenimine-treated filters. Anal. H1-receptor blockade is suggested to be Biochem. 132, 74-81 (1983) associated with the activity (17), use 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and of a drug which has both antimuscarinic and Randall, R.J.: Protein measurement with the antihistaminic effects may be more effective Folin reagent. J. Biol. Chem. 193, 265 in the treatment of motion sickness. The 275 (1951) ranking of these drugs (Table 1) as anti 11 Tran, V.T., Chang, R.S.L. and Snyder, S.H.: muscarinic agents also can be used clinically Histamine H, receptors identified in mammalian to predict the likelihood that these drugs will brain membranes with [3H]mepyramine. Proc. cause certain adverse effects in patients such Natl. Acad. Sci. U.S.A. 75, 6290-6294 (1978) as dry mouth, blurred vision, 12 Chang, R.S.L., Tran, V.T. and Snyder, S.H.: and constipation. Characteristics of histamine H, receptors in Thus, these results will provide helpful peripheral tissues labeled with [3H]mepyramine. information with regard to the clinical and J. Pharmacol. Exp. Ther. 209, 437-442 (1979) experimental use of antihistamines. 13 Yamamura, H.I. and Snyder, S.H.: Muscarinic cholinergic binding in rat brain. Proc. Natl. Acad. Acknowledgements: This work was supported by Sci. U.S.A. 71, 1725-1729 (1974) grants from the Ministry of Education, Science and 14 Shirakawa, 0. and Tanaka, C.: Molecular Culture and the Ministry of Health and Welfare, characterization of muscarinic receptor subtypes Japan. We thank M. Ohara for critical reading of the manuscript. in bovine cerebral cortex by radiation inactivation and molecular exclusion H.P.L.C. Br. J. Phar macol. 86, 375-383 (1985) motion sickness. Pharmacol. Rev. 18, 895-924 15 Bennett, J.P. and Yamamura, H.l.: Neuro (1966) transmitter, hormone, or drug receptor binding 17 Richelson, E. and Nelson, A.: Antagonism by methods. In Neurotransmitter Receptor Binding, neuroleptics of neurotransmitter receptors of Edited by Yamamura, H.I., Enna, S.J. and Kuhar, normal human brain in vitro. Eur. J. Pharmacol. M.J., p. 61-89, Raven Press, New York (1985) 103, 197-204 (1984) 16 Brand, J.J. and Perry, W.L.M.: Drugs used in