We recently investigated various vanilloid-type β-adrenoceptor antagonists derived from natural products such as vanillin, zingerone, capsaicin, and eugenol (1-7). On the basis of these exciting findings, we synthesized a series of naturally occurring β-adrenoceptor blockers. Ligusticum wallichii FRANCH (L. wallichii) has been used in traditional Chinese medicine, especially as a homeostatic remedy for women's disorders and menoxenia, as well as an analgesic for dysmenorrhea in China. Ferulic acid is an active phenolic compound contained in L. wallichii(8). Ferulic acid has been shown to inhibit tumor promotion in mouse skin (9), rat paw edema (10), rat uterine motility (11), and lipid peroxidation and subsequent oxidative inflammatory diseases (12). In our previous study, we found that vasomolol is an ester-type ultrashort-acting β-adrenoceptor blocking agent with a half-life of ∼10 min and β1-receptor selectivity (3). Ultrashort-acting β-blockers have been suggested as a safe and efficacious means for attenuating the often deleterious effects of adrenergic drive on the heart (13).
In the search for compounds with this desired property, we created a unique ester-type β-adrenoceptor blocker with vanilloid base, ferulinolol ((±)3-[4′-(2-hydroxy-3-tert-butylaminopropoxy)-3′-methoxyphenyl]-2-propenoic acid ethyl ester; Fig. 1), by modification of the 4-phenolic hydroxyl group at the aromatic ring of ferulic acid. Unexpectedly, we found that the ester-type agent failed to attain our previous intent. Ferulinolol was not an extremely short-acting β-blocker, and its duration of action was sustained for >1 h, in contrast to vasomolol. β-Adrenoceptor blockers are an important class of drugs in the treatment of patients with cardiovascular diseases. These drugs have been shown to reduce mortality in hypertension (14) and prolong survival in patients with ischemic heart diseases (15,16). β-Blockers retain their position among basic therapies for numerous other cardiovascular and noncardiovascular conditions, including arrhythmias, hypertrophic cardiomyopathy, migraine, glaucoma, and thyrotoxicosis, although such β-blockers share the common feature of being competitive antagonists on the β-adrenoceptors. However, they differ in many pharmacologic properties, such as potency, selectivity for β1- or β2-receptors, intrinsic sympathomimetic activity (ISA), membrane-stabilizing activity (MSA), and α-blocking characteristics (17). In addition, their pharmacodynamic properties vary with respect to their capability to exert partial agonist activity on the β1- or β2-adrenoceptor subtypes.
Agents with partial agonist activity on β2-adrenoceptors and β1-adrenoceptor-antagonist selectivity might possess clinically relevant advantages (18). β-Adrenergic blockers, particularly those without cardiac selectivity, carry considerable risk in patients with bronchospastic disease because they may inhibit β2-receptor in therapeutic doses. In the airway system, application of a β1-adrenoceptor blocker with partial β2-agonist activity may reduce the risk of asthmatic attack in patients with bronchospastic disease. These facts led us to search for newer β-adrenergic blocking agents that would be able to mitigate adverse effects. As a result, this laboratory synthesized a novel ester-type β1-adrenoceptor blocker with partial β2-agonist activity, ferulinolol. This study was designed to explore the pharmacologic properties of ferulinolol, including its ability to bind to β-adrenoceptors, selectivity for β receptors, partial β2-agonist activity, and direct myocardial depressant effect.
Measurement of blood pressure and heart rate
The experiments were accomplished as described previously (1-7). In brief, male Wistar rats weighing 250-300 g were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). After tracheal cannulation, systemic arterial blood pressure and heart rates were recorded from the femoral artery with a pressure transducer (model P10EZ; Spectramed, Oxnard, CA, U.S.A.). The body temperature was maintained at 37°C by an electric heating pad. A femoral vein was cannulated for intravenous administration of drugs.
Evaluation of β-adrenergic response
Anesthetized Wistar rats were pretreated with mecamylamine (5 mg/kg, i.v.), a ganglion-blocking agent, to ensure a uniform initial heart rate. Isoproterenol (0.5 μg/kg) was administered via a femoral vein, and the resultant tachycardia recorded as the control. Thereafter, a single dose of ferulinolol was administered intravenously. After 10 min, a further injection of isoproterenol was given.
Evaluation of α-adrenergic response
Anesthetized Wistar rats were pretreated with reserpine (5 mg/kg, i.p.) 24 h before the injection of (−)phenylephrine (10 μg/kg, i.v.), followed 15 min later by the intravenous injection of a single dose of ferulinolol, propranolol, or labetalol (1.0 mg/kg). After 10 min, a further injection of (−)phenylephrine was given.
Measurement of atrial rate and tension
The atrial strips were prepared according to the method described in our previous report (1-7). Guinea pigs (Hartley) of either sex weighing 350-500 g were killed by a blow to the head, and their hearts were quickly excised. Spontaneously beating right atria were removed from the hearts and mounted in a 10-ml organ bath with one end fixed and the other end connected to a force-displacement transducer (model FT03; Grass, Quincy, MA, U.S.A). The atrial rate was measured on a separate channel by a frequency-converter amplifier (model 13-6615-60; Gould, Valley View, OH, U.S.A). The experiments were carried out at 32.5°C in a Krebs solution of the following composition (in mM): NaCl, 113; KCl, 4.8; CaCl2, 2.2; KH2PO4, 1.2; MgCl2, 1.2; NaHCO3, 25; dextrose, 11.0; bubbled with a 95% O2/5% CO2 mixture. The atrial strip was prestretched to a baseline tension of 0.2 g. The atria were equilibrated for 90 min in an aerated Krebs solution before the experimental protocols were initiated. For the assessment of β-adrenergic blocking activity, a control cumulative concentration-response curve to the chronotropic effect of (−)isoproterenol was established. The atria were allowed 30-60 min to restabilize, after which time, various concentrations of the test agent were incubated with the atrium 30 min before the cumulative concentration of the (−)isoproterenol (10−10-10−5M) was added. Responses were calculated as a percentage of the maximal control response to (−)isoproterenol.
Quiescent left atria were dissected free of connective tissue and mounted in organ chambers under a resting tension of 0.5 g. The tissues were bathed in an aerated Krebs solution (32.5°C) and were driven at 2-s intervals via two platinum electrodes placed at either side of the atrium. Cumulative concentration-response curves to the positive inotropic effects of (−)isoproterenol were obtained in the absence and presence of various concentrations of a test compound. An incubation time of 30 min was allowed for the test compound. Data were calculated as a percentage of the increase in force induced by (−)isoproterenol.
Contractility of isolated tracheal strips
Guinea pig trachea were cleaned of extraneous connective tissue and cut into spiral strips (1-7). These spiral strips were suspended in organ baths filled with 20 ml of Krebs solution, maintained at a temperature of 32.5°C and aerated with 95% O2/5% CO2. To achieve a steady spontaneous tone level, an initial tension of 2 g was applied for 60 min. The preparations were pretreated with phenoxybenzamine (50 μM for 30 min), followed by thorough washout to prevent extraneuronal uptake and to block α-adrenoceptors. Cumulative concentration-response curves to the relaxant effects of (−)isoproterenol were obtained in the absence and presence of a test compound. Data were calculated as a percentage of the maximal relaxation in duced by (−)isoproterenol.
Intrinsic sympathomimetic activity
Guinea pigs were pretreated with reserpine (10 mg/kg, i.p.) 24 h before the experiment. All preparations including isolated right and left atria and tracheal strips were investigated. The concentration-response curve was achieved by cumulative addition of (−)isoproterenol, propranolol, or ferulinolol. To support of partial β2-agonist activity of ferulinolol on tracheal strips, a modification of the method described by Tesfamariam and Allen (19) was used. Cumulative concentration-response curves to the relaxant effects of ferulinolol were attained in the absence or presence of ICI 118,551 (0.1-10 nM).
Preparation of ventricle and lung membranes
Membranes were prepared from cardiac ventricles and lung tissues of rats, as described previously (1-5,7). Ventricles and lungs were placed in 10 vol of ice-cold TE buffer (10 mM Tris HCl, 1 mM EDTA, 0.1 mM ascorbic acid, pH 7.4), and all subsequent procedures were carried out at 4°C. The tissue was homogenized with three 12-s pulses by using a Polytron homogenizer (model PT 3000; Kinematica, Littau, Switzerland). The homogenate was filtered with pressure through muslin, and the filtrate centrifuged for 10 min at 1,000 g. The supernatant was centrifuged again at 10,000 g for 12 min. This second supernatant was then centrifuged for 15 min at 30,000 g, and the final pellet was resuspended in a assay buffer (75 mM Tris HCl, 25 mM MgCl2, pH 7.4). Protein content was determined by Bradford's method (20).
Radioligand binding assay
[3H]CGP-12177 (0.1-30 nM) and ventricle or lung membranes (200-300 μg) were incubated for 60 min at 25°C with or without the addition of 10 μM propranolol, in a 75 mM Tris HCl buffer with 25 mM MgCl2, to make a final volume of 250 μl. In competitive-binding experiments, the competing agent was added directly to the incubation mixture. The incubation was terminated by addition of 1 ml of ice-cold assay buffer followed by immediate filtration through Whatman GF/C glass fiber filters supported on a 12-port filter manifold (Millipore). The filters were immediately washed 3 times with 5 ml of ice-cold assay buffer and dried in an oven at 60°C for 2 h before adding 5 ml of Triton-toluene-based scintillation fluid. Membranebound [3H]CGP-12177 trapped in the filters was counted in a Beckman LS 6500 scintillation system (Fullerton, CA, U.S.A.) with an efficiency of 45%. In each experiment, nonspecifically bound [3H]CGP-12177 was determined by incubating membrane protein and [3H]CGP-12177 with 10 μM propranolol. Specific binding was thus obtained by deducting this value from the total binding of [3H]CGP-12177 for each sample.
Analysis of results
In organ-bath experiments, the potency of β-adrenoceptor antagonists was estimated according to the methods of Arunlakshana and Schild (21). Dose ratios (DRs) were obtained from the median effective concentration (EC50) in the presence of antagonist/EC50 in the absence of antagonist. The β-adrenoceptor antagonist log (DR-1) was plotted against the log molar concentration of antagonist (log [B]). The apparent pA2 values were calculated from the equation pA2 = log (DR-1) - log [B].
In receptor-binding experiments, Ki values were calculated according to the Cheng-Prusoff equation (22)Equation 1
IC50 represents the ligand concentration required to displace half of the radiolabeled compound from specific sites, and Kd and C denote the apparent dissociation constant and the free concentration of the radiolabel, respectively.
(−)[3H]CGP-12177 (60 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA, U.S.A.) (−)Propranolol, (−)isoproterenol bitartrate, (−)phenylephrine HCl, (−)propranolol HCl, (±)atenolol (±)propranolol HCl, mecamylamine HCl, phenoxybenzamine HCl, and reserpine were all purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Protein-assay dye was obtained from Bio-Rad Laboratories (Richmond, VA, U.S.A.). All other reagents used were from E. Merck (Darmstadt, Germany). (±)Ferulinolol (synthesized in this laboratory), (−)isoproterenol bitartrate, (−)phenylephrine HCl, and mecamylamine HCl were dissolved in saline for in vivo experiments. Phenoxybenzamine and reserpine were dissolved in absolute alcohol and benzyl alcohol, respectively, and then diluted with distilled water.
The results are expressed as mean ± SEM. Statistical differences were evaluated by paired Student's t test in paired samples. Whereas a control group was compared with more than one treated group, the two-way repeated-measures analyses of variance (ANOVAs) were used. When the two-way repeated-measures ANOVA revealed a statistical difference, the Student-Newman-Keuls test was applied to characterize which values were statistically different from the control. A p value of <0.05 was considered significant. Analysis of the data and plotting of the figures were done with the aid of software (SigmaStat and SigmaPlot, Version 5.0, Jandel, U.S.A.) run on an IBM-compatible computer.
Effects of ferulinolol on blood pressure and heart rate
In pentobarbital-anesthetized Wistar rats, short-term intravenous injection of ferulinolol (0.1, 0.5, and 1.0 mg/kg) produced a decrease in heart rate over a period of 1 h; nevertheless, it had no significant effects on blood pressure (Fig. 2). Administration of atenolol and propranolol also reduced the heart rate for >1 h, but propranolol caused a temporary increase in blood pressure (data not shown). In ganglion-blocked anesthetized rats, ferulinolol (0.1 mg/kg, i.v.) caused a significant decrease in isoproterenol-induced tachycardia responses. Likewise, injection with 1.0 mg/kg of ferulinolol almost completely blocked the isoproterenol-induced tachycardia (Fig. 3). In reserpine-treated rats, intravenous administration of (−)phenylephrine (1.0 μg/kg) increased the blood pressure 56.5 ± 4.3 mm Hg (mean ± SEM; n = 8), which was markedly inhibited by the i.v. injection of labetalol (1.0 mg/kg) to 33.6 ± 4.6 mm Hg but was not significantly affected by ferulinolol (55.8 ± 5.0 mm Hg) or propranolol (53.9 ± 5.3 mm Hg).
Effects of ferulinolol on β1-adrenoceptor activity
Ferulinolol antagonized the isoproterenol-induced positive chronotropic activity on the isolated guinea pig right atrial strips. In addition, ferulinolol (10−7, 10−6, and 10−5M) caused a dose-dependent parallel shift to the right of the isoproterenol concentration-response curves. The results of a typical experiment with right atria are illustrated in Fig. 4A. The apparent pA2 values for ferulinolol, atenolol, metoprolol, and propranolol on right atria were 7.62 ± 0.05, 7.23 ± 0.03, 7.55 ± 0.09, and 8.24 ± 0.06, respectively (Table 1). In electrically driven guinea pig left atrial strips (Fig. 4B), ferulinolol also antagonized isoproterenol-induced positive inotropic actions and produced dose-dependent rightward shifts of the cumulative concentration-response curves to isoproterenol. The apparent pA2 values for ferulinolol, atenolol, metoprolol, and propranolol on left atria were 7.54 ± 0.01, 7.31 ± 0.05, 7.45 ± 0.05, and 8.15 ± 0.07, respectively (Table 1).
Effects of ferulinolol on β2-adrenoceptor activity
Ferulinolol (10−7, 10−6, and 10−5M) competitively antagonized the isoproterenol-induced relaxation from the spontaneous tone of guinea pig tracheal strips. Likewise, ferulinolol produced parallel shifts to the right of the agonist concentration-response curves (Fig. 4C). The apparent pA2 values for ferulinolol, atenolol, metoprolol, and propranolol on tracheal strips were 6.28 ± 0.11, 5.70 ± 0.06, 6.24 ± 0.07, and 8.07 ± 0.09, respectively (Table 1).
β1/β2 selectivity of ferulinolol
The β1- versus β2-selectivity ratio was estimated from the antilogarithm of the difference between the mean pA2 values obtained from the right atria and trachea. Ferulinolol was 21.9 times more potent on right atria than on trachea (i.e., was highly selective for β1-adrenoceptors). Similarly, atenolol and metoprolol displayed high selectivity for β1-adrenoceptors. However, propranolol was only 1.5 times more potent on right atria than on trachea and was, therefore, considered to be nonselective (Table 1).
Partial β2-agonist activity of ferulinolol
In reserpine-treated guinea pig experiments, various effects of ferulinolol, propranolol, and isoproterenol on the frequency of right atria, tension developed by left atria, and the relaxation of tracheal strips are described in Fig. 5. Isoproterenol produced concentration-dependent increases in heart rate and contractility with a maximal increase at 1 μM. Ferulinolol and propranolol did not increase heart rate and contractility but caused negative inotropic and chronotropic effects in concentrations of ≥1 μM. These direct depressant effects of propranolol usually increased steeply with concentration, leading to arrest or inexcitability of the atrial tissues at concentrations >100 μM(Fig. 5A and B). In addition, both isoproterenol and ferulinolol (10−10-3 × 10−5M) produced dose-dependent relaxant responses in tracheal strips. The relaxations induced by isoproterenol were more potent than those by ferulinolol, but propranolol was without any relaxation (Fig. 5C). As shown in Fig. 6, pretreatment with the β2-adrenoceptor antagonist ICI 118,551 (0.1, 1.0, and 10 nM) significantly shifted the cumulative concentration-response curve of ferulinolol to regions of higher concentrations in reserpine-treated guinea pig tracheal strips.
Effects of ferulinolol on radioligand-binding studies
[3H]CGP-12177 was bound to predominant β1- and β2-adrenergic receptor sites in rat ventricle and lung membranes in a saturable manner, respectively. Scatchard analysis (23) was used to determine the affinity and number of binding sites. The Kd values for [3H]CGP-12177 binding to the rat predominant β1- and β2-adrenergic receptors were 0.16 ± 0.03 nM and 1.21 ± 0.15 nM, respectively, and the maximal binding capacity values (Bmax) were 43.1 ± 2.6 fmol/mg protein and 294.5 ± 20.4 fmol/mg protein at 25°C, respectively. The binding affinity of ferulinolol and various β-adrenergic blockers for predominant β1- and β2-adrenoceptor sites expressed as -log IC50 values and Ki values are shown in Table 2. In rat ventricular membranes, the Ki values of ferulinolol, atenolol, metoprolol, and (−)propranolol were 103, 262, 123, and 0.23 nM, respectively. Additionally, the Ki values of ferulinolol, atenolol, metoprolol, and (−)propranolol were 2,412, 7,539, 2,186, and 0.72 nM in rat lung membranes, respectively. Ferulinolol showed high affinity for β1-adrenergic receptors, and it revealed a β1/β2-receptor selectivity of 23.4-fold. In like manner, atenolol and metoprolol exhibited high affinity for β1-adrenoceptors, and they revealed a β1/β2-receptor selectivity of 28.8- and 17.8-fold, respectively, whereas propranolol had the least β1-adrenoceptor selectivity, and it had a β1/β2-receptor selectivity of 3.1-fold.
We recently reported that vasomolol is an ester-type ultra-short-acting β1-adrenoceptor antagonist with vasorelaxant properties (3). Ferulinolol is also a chemically novel β1-adrenoceptor-blocking agent with an ester group. Previously we suggested that ferulinolol might have an ultra-short-acting activity similar to that of vasomolol. From this preliminary study, we established that ferulinolol was not an extremely short-acting β-adrenoceptor antagonist. This interesting result will encourage us to investigate other pharmacologic properties of ferulinolol.
In our pentobarbital-anesthetized Wistar rats, intravenous administration of ferulinolol caused a decrease in heart rate over a period of 1 h; however, it was without significant effects on the blood pressure. This result was in contrast with our previous finding in vasomolol (3). Propranolol caused a temporary increase in blood pressure and a sustained decrease in heart rate. The pressor response of propranolol in anesthetized Wistar rats was reported previously (24). Ferulinolol blocked (−)isoproterenol-induced tachycardia effects, which indicated that the bradycardia effect of ferulinolol was associated with β-adrenoceptor-blocking activity. On the other hand, labetalol (a dual α- and β-adrenoceptor antagonist) showed a significant inhibition of pressor responses induced by phenylephrine. However, ferulinolol and propranolol had nearly no effect on the pressor responses to phenylephrine, suggesting that both agents lack α-adrenoceptor-blocking activity.
The better to estimate the receptor selectivity of β-adrenoceptor antagonists, a comparison of their affinities for the β-receptor subtypes is required, and the pA2 value was used for this purpose (21). In our study, ferulinolol and various β-blockers were investigated on isolated guinea pig atria and rat trachea in experiments designed to obtain apparent pA2 values. In right and left atrial strips isolated from the guinea pig, ferulinolol was found to antagonize competitively any increases in beating rate and force of contraction brought about by isoproterenol treatment, suggesting that ferulinolol possesses β1-adrenoceptor-antagonist activity. Likewise, ferulinolol's competitive antagonism of any relaxation of the trachea preparation induced by isoproterenol indicated that ferulinolol possesses β2-adrenoceptor-antagonist action. The ratios of β1- to β2-selectivity for ferulinolol, atenolol, metoprolol, and propranolol were 21.9, 33.9, 20.4, and 1.5, respectively. This result suggested that ferulinolol, atenolol, and metoprolol are highly selective β1-blockers. However, propranolol was a nonselective β-adrenoceptor blocker (Table 1).
In our reserpine-treated guinea pig experiments, ferulinolol and propranolol produced significantly negative inotropic and chronotropic effects at high concentrations, whereas ferulinolol showed fewer cardiodepressant actions than those of propranolol. The results suggested that both agents seemed to be devoid of direct ISA on β1-adrenoceptors. It is generally agreed that the direct cardiodepressant actions of β-blockers do not result from interaction with the β-adrenoceptors (25). These actions have been correlated with the agents' ability to cause local anesthesia, to inhibit conduction velocity in nerve and muscle, and also with their lipid solubility (26). Further, ferulinolol produced the relaxation of tracheal strips implying that ferulinolol might have a direct ISA on β2-adrenoceptors. To support the tracheal relaxant effects of ferulinolol related to the β2-receptor, ICI 118,551, a highly selective β2-adrenoceptor antagonist was used. The preincubation of tracheal strips with ICI 118,551 shifted the concentration-relaxation curve of ferulinolol significantly to regions of higher concentrations and, therefore, confirmed the partial β2-agonist activity of ferulinolol. It has been widely accepted that β1-adrenoceptor selectivity and partial β2-adrenoceptor-agonist activity appear to be clinically useful because they reduce side effects because of β2-adrenoceptor blockade (18).
In radioligand-binding studies, [3H]CGP-12177 binding to rat ventricle (predominantly β1) and lung membrane preparations (predominantly β2) were saturable, specific, and of high affinity. Scatchard analysis of the binding data were linear, suggesting a single population of binding sites in the tissues. Those Kd values for [3H]CGP-12177 binding to the rat predominantly β1- and β2-adrenergic receptors were 0.16 ± 0.03 nM and 1.21 ± 0.15 nM at 25°C, respectively. The Kd value obtained in the rat ventricle membrane is in a good agreement with the Kd reported by Porzig et al. (27). Nevertheless, the Kd value obtained in the rat lung membrane is approximately fivefold higher than that reported by Pauwels et al. (28). The discrepancy between the two Kd values is not readily explained. In our study, the lower binding affinity in the rat lung membrane may be related in part to the rapid dissociation of [3H]CGP-12177 during filtration and washing. Moreover, ferulinolol, atenolol, and metoprolol displayed high affinity for β1-adrenergic receptor sites, and they revealed a β1/β2-receptor selectivity of 23.4-, 28.8-, and 17.8-fold, respectively. However, propranolol showed the least β1-adrenoceptor selectivity among these β-adrenergic blockers (Table 2). This result was consistent with the β1/β2-selectivity ratio of those β-blockers obtained from in vitro study.
An important aspect of our results is that no β-adrenoceptor antagonist other than ferulinolol is derived by combining the ferulic acid, an active ingredient of L. wallichii, with an oxypropanolamine side chain to obtain a β-blocker. We carefully investigated the pharmacologic profiles of ferulinolol and firmly determined that it is a highly selective β1-adrenoceptor-blocking agent with partial β2-agonist activity. Ferulinolol differs from classical β-blockers such as propranolol because of its β1-receptor selectivity and ISA. Finally, we suggest that ferulinolol might be clinically valuable to lessen untoward pulmonary side effects and asthmatic attacks in patients with bronchospastic disease due to β2-adrenoceptor blockade.
Acknowledgment: This work was supported by grant NSC 86-2745-B-037-003 to Dr. Bin-Nan Wu from the National Science Council, Taiwan, R.O.C.
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