Nipradilol Displays a Unique Pharmacological Profile of Affinities for the Different ƒ¿1-Adrenoceptor Subtypes Junji Kinami, Hiroshi Tsuchihashi, Keiko Maruyama, Keiko Sasaki and Takafumi Nagatomo Department of Pharmacology, Niigata College of Pharmacy, 5-13-2 Kamishin'ei-cho, Niigata 950-21, Japan Received May 25, 1992 Accepted November 6, 1992 ABSTRACT-The selectivity of antagonistic effects of nipradilol, its four isomers and denitronipradilol, a major metabolite of nipradilol, on ƒ¿1-adrenoceptor subtypes in rat heart, brain and spleen were examined by radioligand binding assay with [3H]-prazosin. Pharmacological characteristics of these compounds were determined in isolated aortae from rats and guinea pigs. The order of the pKi values for ƒ¿1High-affinity sites in the heart, spleen and brain was SR > nipradilol •† RR •† SS ≈RS•âdenitronipradilol, but the order of the pKi values for the ƒ¿1LOW -affinity sites was different in the heart and brain. There were good correlations between the pKi values of these compounds for the ƒ¿1High -affinity sites and the pA2 values for the contractile inhibition of the phenylephrine-induced response in rat aorta. There was no correlation between the pKi values of these compounds for the ƒ¿1LOW -affinity sites and the pA2 values. These results indicate that: 1) ƒ¿1High -Affinity sites are related to vasoconstriction mediated by ƒ¿1- adrenoceptors; 2) Nipradilol and its isomers possess low affinity to ƒ¿1 -adrenoceptors; and 3) The nitroxy group in nipradilol is important for its -blocking activity . ƒ¿1 Keywords: [3H]-Prazosin, Nipradilol, ƒ¿1-Adrenoceptor subtype, ƒ¿1- and ƒÀ-•@Blocking activity, Optical isomer Our recent studies on the binding characteristics of cyanopindolol (ICYP) and [3H]-CGP 12177, respectively; [3H]-prazosin for the a,-adrenoceptor subtypes revealed and the results showed that nipradilol had a higher affin that there are two binding sites having different affinities, ity for (3-adrenoceptors (with no selectivity for f3, and (32 high and low-affinity sites, for prazosin in the rat ven adrenoceptors) than for 5HT,B-receptors (5). The rank tricular muscle, brain and spleen membranes; and these orders of potency of the isomers for 8-adrenoceptors and sites are designated as a,High and a1L0 -affinity sites (1, 2). 5HT,B-receptors were SR > nipradilol > SS > RR > RS and We also found that rat ventricular muscle and brain pos SS > SR >_nipradilol > RS > RR, respectively (5). sess both a,High and a1L0 affinity sites, whereas rat spleen had only a,High-affinity sites (1, 2). Most a,-adrenergic an tagonists had different affinities for each site in these tissues, suggesting that there may exist five different a, adrenoceptors (a,High-sites in the ventricular muscle, brain and spleen and a1LoW-sitesin the ventricular muscle and brain). Recently, a, •~-blockers possessing antagonistic po tency for both a, and j3-adrenoceptors have been shown to be useful for the clinical treatment of patients with hypertension and angina. Nipradilol, one of these a, ~ blockers, possesses two asymmetric carbon atoms in its chemical structure and is a mixture of four isomers, as shown in Fig. 1 (3, 4). We previously reported the binding characteristics of nipradilol for (3,-, (32-adrenergic and 5HT,B-serotonergic binding sites by using [125I]-iodo Fig. 1. Chemical structure of nipradilol and its isomers. In this study, we examined the selectivity of nipradilol, in the presence or absence of unlabelled ligand. The con its isomers and its major metabolite denitronipradilol (6) centrations of [3H]-prazosin were 0.04, 0.1 and 0.2 nM for a,-adrenoceptor subtypes and compared them with for assessing a,High-affinity sites in the brain, heart and those of other a, • n-blockers, labetalol, amosulalol and spleen membrane, respectively. The affinity of the a,LoW arotinolol. affinity sites for unlabelled ligand were determined with 0.5 nM [3H]-prazosin in the presence of 0.1 pM phenoxy MATERIALS AND METHODS benzamine in the brain membrane, or with 0.6 nM [3H] prazosin in the presence of 1 pM phenoxybenzamine in Materials the heart membrane. Phenoxybenzamine inhibited the [3H]-Prazosin (76.6 Ci/mmole) was purchased from a,High-affinity sites completely (1, 2). After the incuba New England Nuclear/Dupont, Ltd., Boston, MA, tion period of 45 min, the medium was immediately U.S.A. Nipradilol, 3,4-dihydro-8-(2-hydroxy-3-isopropyl filtered through a GF/C glass fiber filter and washed with amino)propoxy-3-nitroxy-2H-1-benzopyran, its isomers the incubation buffer according to previously described (Fig. 1) and denitronipradilol were kindly donated by methods (8). The radioactivity on the filter was counted Kowa Co., Ltd. by a Packard 2200 Tri-Carb Scintillation Analyzer. The specific binding was determined by subtracting the non Animals specific binding in the presence of 10 pM of phentolamine Male Wistar rats weighing 200-350 g were used. from the total binding. Preparation of membrane-enriched fractions Kinetic analysis The membrane-enriched fractions from rat heart, brain All kinetic analyses were performed on an NEC PC and spleen were prepared as described previously (1, 2). 9801 computer system with an iterative non-linear regres Protein was determined by the method of Lowry et al. sion program (9-12). The data were fitted to models hav (7). ing only one or two receptor binding sites (9 -12). To quantitate the displacement characteristics, the slope fac Binding assay tor (nH) for the displacement curves was determined as Displacement analysis for a,-adrenoceptor subtypes described previously (9-12). Most K; values of various was performed in duplicate with [3H]-prazosin as ligands are expressed as pK; ( log K;) in this report. described previously (1, 2). In brief, the membrane sus pension (0.1 mg of heart and brain and 0.25 mg of spleen Pharmacological observations membrane proteins) was incubated for 45 min at 231C in a The contractile tension of the rat and guinea pig aorta total volume of 0.5 ml containing 60 mM Tris-HC1 (pH was determined as described previously (13, 14). Briefly, 7.4) with an appropriate concentration of [3H]-prazosin aortae were cut into rings and freed of excess tissues. Table 1. pK; values of nipradilol and its optical isomers for the a,-adrenoceptor subtypes Values in parentheses are the numbers of experiments. Data are the mean values ±S.E. The slope factors (nH) of the displacement curves of all ligands used in the present study were equal to one. *Some of these data were reported previously (11). These rings, about 2 mm in width, were mounted in 12-m1 Table 2. pA2 values of nipradilol and its optical isomers for a, organ baths. The Krebs-Henseleit solution had the follow adrenoceptors in rat and guinea pig aorta ing composition: 118 mM NaCI, 4.7 mM KCI, 2.5 mM CaC12, 25 mM NaHCO3, 1.2 mM MgSO4i 1.2 mM KH2PO4, and 11 mM glucose. The temperature of the so lution was maintained at 37 ± 1 V and aerated with a mix ture of 95% 02 and 5% CO2. The contractile tension of these preparations was recorded on a potentiometric recorder (Hitachi APD-74) with a strain gage transducer and a carrier amplifier (Nihon Kohden RP-3 or San'ei Instrument Co., Ltd. 6M52). The aorta was always stretched to 0.5 -1 g to obtain the optimum response. Concentration-response curves were determined for phenylephrine before and after addition of each com Values in parentheses are the numbers of experiments. Data are the pound, and their pA2 values were calculated by the previ means ± S.E. ously described equation (15). heart a,LoW, heart alHigh brain alHigh? spleen alHigh• RESULTS Table 2 also summarizes the pA2 values of the contrac tile tension of rat and guinea pig aorta. a1-Adrenoceptor Table 1 summarizes the pKi values of nipradilol, its subtypes in the rat and guinea pig aorta were alHigh and isomers and denitronipradilol for a1-adrenoceptor sub a1Low-affinity sites, respectively, as determined by the types in [3H]-prazosin binding to brain, heart and spleen pA2 values of prazosin (10.80 in rat aorta and 8.30 in membranes. Both alHigh and a1Low-affinity sites were guinea pig aorta) (16). All compounds antagonized the found in the brain and heart membranes, while only phenylephrine-induced contractile response in a competi alHigh-affinity sites existed in the spleen membranes (1, 2). tive manner when analyzed by Schild plots (data are not These five a1-adrenoceptors had different affinities for shown). The pA2 value of nipradilol in the rat aorta was various antagonists (1, 2). The binding affinity of lower than the value of amosulalol and labetalol, and it labetalol for these subtypes were distinguishable among was higher than that of arotinolol. On the other hand, the the compounds used in the present study: brain alHigh> pA2 values of SS, RS and denitronipradilol were lower heart alHigh = spleen alHigh > brain a1Low> heart a1Low• than the value of arotinolol. The order of pA2 values of The pKi values of the other a1 •n-blockers, arotinolol and these compounds was SR= nipradilol= RR > SS RS amosulalol, for alHigh-affinity sites were higher than those denitronipradilol in the rat aorta. The pA2 of nipradilol for a1Low-affinity sites. On the other hand, the pKi values in the guinea pig aorta was lower than that of amosulalol of nipradilol for various subtypes were similar except for and was higher than that of arotinolol. On the other heart alHigh > heart a1Lo,. The pKi values of nipradilol hand, the pA2 values of RS and denitronipradilol were and related compounds for the alHigh-affinity site were sig lower than the value of arotinolol.