Hypotensive Effect of Bunitrolol at Low Plasma Concentrations in Conscious, Unrestrained Spontaneously Hypertensive Rats

Koichiro KAWASHIMA, Yuko MIWA, Kazuko FUJIMOTO, Junko MATSUMOTO, Miyako KIMURA and Akihito NAGAKURA

Department of Pharmacology, Kyoritsu College of Pharmacy, Minato-ku, Tokyo 105, Japan

Accepted March 29, 1985

Abstract-Effects of bunitrolol on mean arterial pressure (MAP) and heart rate (HR) were studied in conscious, unrestrained spontaneously hypertensive (SHR) rats at rest and during handling stress. was employed as a reference drug. Plasma drug concentrations were determined to related with the cardio vascular effects of the drugs. Bunitrolol produced a tachycardia for the first 2 hr and a significant reduction in resting MAP at 3 and 4 hr after the oral dose (5 mg/kg) when plasma bunitrolol concentration was less than 10 ng/ml, indicating the difference between cardiac and vascular beta adrenoceptors in sensitivity to intrinsic sympathomimetic action or direct vasodilator action. Propranolol (5mg/kg) produced no discernible effects on resting MAP and HR. Stress-induced tachycardia was significantly inhibited by both drugs throughout the experiment, while significant inhibition of hypertensive response was observed only at 4 hr after the treatment. Both bunitrolol and propranolol were rapidly absorbed from the gastrointestinal tract. Plasma half-life of these drugs were almost the same values of around 2 hr. These results indicate that dose size, plasma concentrations, and procedures and the timing of blood pressure measurement are the important factors to be considered when the antihypertensive effect of beta-blockers is studied in SHR rats.

Bunitrolol is a potent non-selective beta dose of bunitrolol. adrenoceptor blocking drug (beta-blocker) The purposes of the present study were to with an intrinsic sympathomimetic activity relate plasma drug concentrations with (ISA) (1-3). The beta-blocking potency of cardiovascular responses to bunitrolol and bunitrolol is higher than that of propranolol to elucidate the mechanism of its hypotensive (2, 4). Bunitrolol also has vasodilator action in conscious, freely moving spon activity which is ascribed to ISA and a direct taneously hypertensive (SHR) rats. The action on the peripheral vasculature (2). As results of our study indicate that the hypo a result of vasodilation, hypotension has been tensive effect of bunitrolol in SHR rats can observed after the administration of bunitrolol be observed at low plasma concentrations. in anesthetized dogs and rats (1, 2, 4). Although the hypotensive effect of bunitrolol Materials and Methods is recognized in hypertensive patients during Procedures on animals: Twelve-week-old exercise (5), it is generally difficult to male SHR rats (F44) from the colony of the demonstrate the reduction of blood pressure Department of Pharmacology, Jichi Medical in hypertensive animals (6). In three different School, were used in this study. Two days models of experimentally hypertensive rats, before the experiment, rats were anes Hasegawa et al. (7) failed to demonstrate any thetized with pentobarbital sodium, 50 mg/ antihypertensive effect of bunitrolol even kg, i.p., and an indwelling catheter (PE 10) after chronic treatment, while Himori et al. was inserted into the lower abdominal aorta (8) reported a hypotension in conscious, through the left femoral artery (9). Two renal hypertensive dogs after a single oral electrodes were subcutaneously implanted in the left chest and the right shoulder and fugation and stored at -20°C until assay was exteriorized at the nape of the neck (10). performed. After surgery, rats were placed in an Radioimmunoassay for bunitrolol: The anti individual plastic cage. serum against d/-bunitrolol was obtained Before the experiment, the animals were from a rabbit immunized with the conjugate fasted overnight and allowed free access to of bunitrolol-hemiglutarate with bovine drinking water. A 0.3-ml reference blood serum albumin (12). The specificity of the sample was drawn from the arterial catheter antiserum is shown in Table 1. The antiserum into a syringe moistened with 15% EDTA bound with both d and /-bunitrolol to the 2NH4. A pressure transducer (CP-01, same degree. The metabolites of bunitrolol, Century Technology) was connected to the 4-hydroxybunitrolol and o-nitrilo-phenoxy arterial catheter for the recording of mean lactic acid did not show any appreciable arterial pressure (MAP) in conscious, freely cross-reactivities with the antiserum. Radio moving rats using an electronic system (Type immunoassay for d/-bunitrolol with a limit of 1236, Sanei). Heart rate (HR) was monitored sensitivity of 100 pg/tube was developed, by attaching the electrodes to a cardio using the antiserum and [3H]bunitrolol (28 tachometer system (Type 1 205D and 5149, Ci/mmol). Plasma samples were diluted at Sanei). Resting MAP and HR were recorded 1 :10 with 0.1 M tris-HCI buffer, pH 7.4. To in the home cage after the accomodation of the tubes (10x75 mm) containing the the rat to the experimental conditions. Rats antiserum, [3H]bunitrolol (42.1 pg, about were considered to be in a resting condition 9,000 dpm) and a 20-50 ail portion of the when they were immobile for at least 2 min. diluted plasma sample was added, and then Handling stress was loaded by lifting the rat tris-HCI buffer was added to bring the total by the tail so that the front paws could barely volume up to 0.5 ml. The tubes were incubated touch the floor of the cage and holding in at 4°C overnight. Antibody-bound [3H] that position for 30 sec (11). A marked rise bunitrolol was separated from the free drug in MAP and tachycardia were induced by by the ammonium sulfate method (13). The this procedure. Handling stress was repeated radioactivity in the precipitate was determined intermittently until stable cardiovascular by the liquid scintillation method. The values responses were observed. Then bunitrolol for serum samples were read directly from (5 mg/kg), propranolol (5 mg/kg), or the the standard curve. vehicle (1 ml/kg) was administered by Radioimmunoassay for propranolol: gavage. At various times after the adminis Plasma concentration of d/-propranolol was tration of drugs, MAP and H R were deter determined by radioimmunoassay utilizing mined at rest and during handling stress. the antiserum specific for d/-propranolol and Blood samples for the determination of [3H]propranolol as reported by Kawashima plasma drug concentrations were drawn from et al. (14). the arterial catheter as described above (0.2 Calculations and statistical analyses: ml at 5, 15, 30, 60 and 120 min, and 0.5 ml Results were expressed as means+S.E.M. at 240 min after the dose of the drugs or the in the table and figures. Significance of vehicle). Plasma was separated by centri difference among the means was ascertained

Table 1. Specificity of the antiserum against dt-bunitrolol using Fisher's least significance difference at 3 and 4 hr after the dose compared to the method. vehicle treated control (P<0.05). Resting Plasma drug concentration-time data were H R was significantly increased by bunitrolol fitted to a two-compartment open kinetic for the first 2 hr (P<0.05 or 0.01), and it model using the computer program TOPFIT returned to the control level by 3 hr after the (15). Kinetic parameters were calculated by administration. In propranolol treated rats, standard procedures. MAP increased slightly for the first 30 min Drugs: d/-Bunitrolol hydrochloride, 4 and then decreased gradually. However, hydroxybunitrolol, o-nitrilophenoxylactic acid there was no significant difference between and the optical isomers of bunitrolol were propranolol and vehicle treated animals in supplied by Nippon Boehringer Ingelheim MAP. Propranolol (5 mg/kg, p.o.) did not Co., Ltd. d/-Propranolol hydrochloride was attenuate resting HR in conscious, freely purchased from Sigma. Drugs were dissolved moving S H R rats. in distilled water. The doses were expressed Effects of bunitrolol and propranolol on as the base. stress-induced cardiovascular responses: Handling stress induced a rise in MAP by Results 41±2 mmHg and an increase in HR by Effects of bunitrolol and propranolol on 117±5 beats/min from the resting state resting MAP and HR: Resting MAP and HR (n=24). Percent changes in pressor and were monitored for 4 hr after the adminis tachycardiac responses to handling stress tration of drugs (Fig. 1). Bunitrolol produced after the administration of drugs or vehicle a gradual fall in MAP which was significant are shown in Fig. 2. Only at 4 hr after the

Fig. 1. Changes in resting heart rate and mean arterial pressure after the oral administration of bunitrolol (n=8) and propranolol (n=8) at the dose of 5 mg/kg or the vehicle in conscious, unrestrained spon taneously hypertensive rats. Each point represents the mean±S.E.M. *P<0.05, **P<0.01, compared to the vehicle. Fig. 2. Changes in tachycardiac and hypertensive responses to handling stress after the oral adminis tration of bunitrolol (n=8) and propranolol (n=8) at the dose of 5 mg/kg or the vehicle in spontaneously hypertensive rats. The data are expressed as a percent of the values before the treatments. Each point represents the mean±S.E.M. *P<0.05, **P<0.01, compared to the vehicle.

Fig. 3. Semilogarithmic plot of plasma concentrations-time curves after oral admininstration of bunitrolol (5 mg/kg, n=8) and propranolol (5 mg/kg, n=8) in conscious, unrestrained spontaneously hypertensive rats. Each point represents the mean±S.E.M. Table 2. Pharmacokinetic parameters in conscious, unrestrained spontaneously hypertensive rats after oral administration

administration, both bunitrolol and pro response to bunitrolol and plasma drug pranolol produced a significant suppression concentrations indicate the difference be of hypertensive response to stress compared tween cardiac and vascular beta-adre to the vehicle (P<0.01 and 0.05, respec noceptors in sensitivity to ISA or direct tively). A significant suppression of tachy vasodilator action. At higher plasma concen cardiac response to stress was observed trations, bunitrolol appears to stimulate both within 15 min after the administration of cardiac and vascular beta-adrenoceptors bunitrolol and propranolol (P<0.01 or 0.05), through ISA leading to the increase in cardiac and this persisted throughout the experi output, and thus obtund its hypotensive mental period of 4 hr. Bunitrolol produced a effect. At lower plasma concentrations, slightly greater and longer suppression in the bunitrolol may only stimulate vascular beta tachycardiac response than propranolol. adrenoceptors or exert vasodilator action, and Disposition of bunitrolol and propranolol: produce an apparent hypotension. An alter Plasma concentration-time curves for buni native possibility is that accumulation of trolol and propranolol after the oral adminis bunitrolol to the active sites which may exist tration (5 mg/kg) in SHR rats are shown in in deep compartments is required to develop Fig. 3. Both bunitrolol and propranolol were a hypotensive effect, and it takes some time rapidly absorbed from the gastrointestinal to produce a significant fall in MAP. Similar tract. Further kinetic analysis (Table 2) results have been observed in SHR rats after revealed a faster absorption of bunitrolol than the administration of , a beta propranolol. Propranolol had a higher plasma adrenoceptor antagonist with ISA (16). concentration and a larger AUC than buni Propranolol did not attenuate resting MAP trolol. There was no difference between after a single dose. This is consistent with the bunitrolol and propranolol in the value for previous report (10). Thus, it appears to be plasma half-life at the Q-phase. difficult to demonstrate a hypotensive effect of propranolol in acute study using SHR rats. Discussion Furthermore, propranolol produced no effect ISA and direct action on the vascular on resting HR. These results indicate that the smooth muscle are considered to be respon sympathetic tone to the heart is minimal in sible for the vasodilator and acute hypotensive SHR rats under resting conditions. effect of bunitrolol in anesthetized animals During handling stress, both bunitrolol (2). In conscious, freely moving SHR rats, and propranolol significantly inhibited the bunitrolol produced a gradual fall in resting tachycardiac response throughout the experi MAP accompanied by an initial increase in ment. The degree of suppression roughly HR. After the subsidence of tachycardia by correlated with plasma drug concentrations. 3 hr after the administration of bunitrolol, a On the other hand, a significant decrease in significant decrease in resting MAP was hypertensive response to stress was observed observed compared to the vehicle treated only at 4 hr after the administration of the control. The time courses of hypotensive drugs. The data in the present study and others (10, 16) show that suppression of measurement are the important factors to be stress-induced hypertensive response by considered when antihypertensive effect of beta-blockers can be observed at certain beta-blockers is studied in experimentally times after the administration in SHR rats. hypertensive rats. The mechanism of this effect is under investigation. It is well recognized that sup References

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