A dose-ranging study to evaluate the β1-adrenoceptor selectivity of bisoprolol

Summary A dose-ranging study was performed to compare the ₁-adrenoceptor selectivity of bisoprolol with that of atenolol and nadolol. Seven normal subjects (mean age 26 y) were given single oral doses of bisoprolol 5 mg (B5), 10 mg (B10), 20 mg (B20); atenolol 50 mg (A50), 100 mg (A100); nadolol 40 mg (N40); and placebo (PL), in a single blind randomised cross-over design. ₂-adrenoceptor responses were assessed by attenuation of finger tremor and cardiovascular responses to graded isoprenaline infusions. Dose-response curves were constructed, and doses of isoprenaline required to increase finger tremor by 100% (IT₁₀₀), heart rate by 25 beats/min (IH₂₅), SBP by 25 mm Hg (IS₂₅), cardiac output by 35% (IC₃₅), and decrease DBP by 10 mm Hg (ID₁₀), after each treatment were calculated. These indices were compared with placebo response and expressed as dose-ratios. Exercise heart rate (EHR) was used to assess ₁-adrenoceptor blockade.There were dose-related increases in plasma concentrations of bisoprolol and atenolol. Reduction of EHR was significantly less with B5 (16.8%) in comparison with all other treatments: B10 21.9%, B20 23.1%; A50 22.5%, A100 22.6%; N40 22.9%. There were small but significant reductions in isoprenaline-induced tachycardia with bisoprolol and atenolol, although mean dose-ratios were considerably less in comparison with N40 (IH₂₅ dose-ratios): B5 2.55, B10 3.18, B20 3.93, A50 2.91, A100 4.89, N40 17.23. There were similar patterns for the other isoprenaline responses.These results show that conventional doses of bisoprolol (10 mg) and atenolol (50 mg) produced equal antagonism of ₁ and ₂-adrenoceptors, and therefore possess equal degrees of ₁-adrenoceptor selectivity. Increasing doses of bisoprolol and atenolol were associated with partial loss of selective ₁-adrenoceptor blockade, although antagonism of ₂-adrenoceptors was significantly less compared with the effects of nadolol.     

Effects of small doses of bisoprolol on blood pressure and lipoprotein concentrations in hypertensive patients

Summary The effects of bisoprolol 2.5 and 5 mg per day on blood pressure, and lipoprotein and apolipoprotein concentrations were compared in 18 newly detected hypertensives in a double-blind, crossover study. All treatment results were related to the values at the end of a four-week placebo run-in period. Each of the two following treatment periods lasted for 3 months.The systolic and diastolic pressures in the supine position were reduced by 19.5/11.7 mm Hg and 14.6/10.4 mm Hg by 2.5 and 5 mg bisoprolol per day, respectively, with no significant difference in effect. Supine heart rate was reduced by 4.7 and 8.2 beats · min^–1, respectively, (P=0.0517 for different effects).The cholesterol concentration in low-density (LDL) and high-density (HDL) lipoproteins was reduced during both regimens, by about 0.3 and 0.1 mmol·l^–1, respectively, difference not significant. Triglyceride concentrations were not significantly affected during either regimen.We conclude that, in this study population, treatment with bisoprolol 2.5 mg per day was equally effective as 5.0 mg per day in reducing blood pressure. The effects on lipoprotein concentrations were small and included an unexpected reduction in LDL-cholesterol concentration. A low dose of a highly selective -adrenoceptor blocker like bisoprolol appears to retain the blood pressure reducing capacity and has lost most of the unfavourable effects on lipoproteins characteristic of higher doses.     

Effect of pretreatment with the selective β1-adrenoceptor antagonist bisoprolol on the subsequent cardiovascular actions and β-adrenoceptor subtype specific occupancy of celiprolol in healthy man

Summary The cardiovascular effects at rest and during exercise and ₁- and ₂-adrenoceptor occupancy following a single dose of 1200 mg celiprolol p. o. were investigated in 8 healthy subjects with or without pretreatment with a single dose of 20 mg bisoprolol p. o., using a place-bo-controlled, 2-way cross-over design.The ergometric responses of heart rate (HR) and systolic blood pressure (SBP) after celiprolol were reduced to a similar extent as after bisoprolol, but the cardiovascular function at rest was affected in a different way: there was a rise in HR, clear enhancement of cardiac systolic performance, and a considerable drop in the estimated total peripheral vascular resistance, associated with median ₁-RRA and ₂-RRA occupancies of 88 and 34%, respectively. The cardiovascular effects of celiprolol were not affected by pretreatment with bisoprolol. Celiprolol thus binds extensively to ₁-adrenoceptors, moderately to ₂-adrenoceptors, acts as ₁-adrenergic antagonist (exemplified by the ergometric effects) but has vasodilator, positive chronotropic and cardiac systolic performance enhancing properties, which do not involve either direct or indirect ₁-adrenergic agonism, but which might reflect ₂-adrenergic agonism.     

Effect of twoβ-blockers on stress during mental arithmetic

Heart rate, blood pressure, and subjective stress ratings were recorded from 36 healthy normotensive students at three points in time: during a drug-free baseline, during a baseline 2 h after ingesting single oral doses of atenolol (75 mg), metoprolol (150 mg), or lactate placebo, and during a subsequently administered mental arithmetic test. Both-blockers equally reduced baseline heart rate and heart rate response to arithmetic, but subjective stress rating increases to arithmetic were greater for atenolol than for placebo and metoprolol. These results are contrary to peripheral theories of anxiety regulation. While the hydrophilic atenolol barely penetrates the blood-brain barrier, the lipophilic metoprolol can exert direct CNS effects in addition to its peripheral actions. Central stress-dampening effects of lipophilic-blockers may override peripheral baroreceptor-mediated stress-promoting effects.     

Duration and selectivity in β-adrenoceptor blocking action of a β-adrenoceptor blocking drug,D-32 in conscious dogs

Summary In conscious dogs, the selectivity and duration of -blocking activity, and serum concentration of a -blocking agent, D-32 [dl-1-tert-butylamino-3-(2,3-dimethylphenoxy)-2-propanol hydrochloride] was compared to that of propranolol, pindolol, atenolol and IPS-339 [dl-1-tert-butylamino-3-(9-fluorenylideneaminoxy)-2-propanol hydrochloride]. Ratios of doses causing a 50% inhibition of tachycardia to that on hypotension induced by isoprenaline were as follows: D-32 (0.69), propranolol (0.67), atenolol (0.03) and IPS-339 (6.3). Thus, present experiments indicate that, unlike atenolol and IPS-339, D-32, propranolol and pindolol are non-selective -adrenoceptor blocking agents. Atenolol and IPS-339, however, selectively blocked cardiac ₁, receptors and vascular ₂-receptor respectively, as would be expected. In an optimal dose range these two drugs can be used satisfactorily as a pharmacological tool for inhibiting responses mediated via the respective -receptors. After oral administration, the pharmacological half-life (time required for 50% recovery of -blocking action) was 15.8±4.5 h for propranolol (3 mg/kg), 21.8±6.4 h for D-32 (0.5 mg/kg), 30.5±3.1 h for atenolol (6 mg/kg) and 30–35 h for pindolol (0.2 mg/kg). The pharmacological half-life after i.v. administration was 4.4±0.7 h for propranolol (300 g/kg) and 5.9±0.4 h for D-32 (150 g/kg), whereas the serum half-life (time required for 50% decrease in serum concentration) of propranolol was 1.4h and that of D-32 was 1.3 h. The values for pharmacological half-life and serum half-life were significantly different. Thus, for determination of administration frequency and dosage of -adrenoceptor blocking drugs, not only pharmacokinetic but also pharmacological data (duration of action) are essential.     

Modulation of L-type Ca current by denopamine, a nonparenteral partial β1 stimulant, in rabbit ventricular cells

The effects of denopamine, a nonparenteral partial agonist which is used clinically in Japan, on the L-type Ca²⁺ current (I _Ca) were examined in rabbit ventricular cells. Denopamine stimulated basal I _Ca with a maximum response of +33.2% and a concentration for half-maximal response (EC₅₀) of 0.039 M. The maximun response of I _Ca was only a quarter of that induced by isoprenaline (ISO), while 10 M denopamine elicited 70–75% of the maximum inotropic response in the papillary muscle preparations. The denopamine stimulation of I _Ca was abolished by selective ₁ antagonists (atenolol or bisoprolol). Pretreatment with forskolin or dialysis with cAMP also abolished the stimulation. Denopamine, in turn, inhibited ISO-stimulated I _Ca. This inhibition was not affected by pretreatment with pertussis toxin or prazosin. The presence of denopamine at various concentrations caused a rightward shift in the concentration/response curve for ISO stimulation of I _Ca. The Schild plot for this effect had a slope of 0.99 and K _p of 0.20 M. In the presence of guanosine-5-O-(3-thiotriphosphate) (GTPS) (0.5 mM) in the pipette, denopamine (10 M) stimulated the I _Ca to 86±5% of the maximum response induced by ISO. These findings indicate that denopamine modulates I _Ca exclusively through the ₁ adrenoceptor-adenylate cyclase pathway, that the stimulatory GTP-binding protein regulates the agonistic potency of denopamine, and that the signal from the ₁ adrenoceptors is amplified between I _Ca and the tension development, which would contribute to the spare capacity of adrenoceptors.     

Effects of single doses of alprenolol and two cardioselective β-blockers (H 87/07 and H 93/26) on exercise-induced angina pectoris

Summary The effect of a non-selective (alprenolol) and two cardioselective -blockers (H 87/07 and H 93/26) on exercise tolerance was studied in eighteen patients with angina pectoris. Each patient exercised on a bicycle ergometer on five consecutive days, during the last four of which a placebo and the three drugs were given double-blind and in randomized order ninety minutes before the test. Compared to the placebo, the three -blockers reduced the heart rate and systolic blood pressure during exercise. There was no significant difference between the three drugs. Alprenolol, H 87/07 and H 93/26 also had significant effects on total work, time for appearance of anginal pain and ST-changes in the ECG. Subjectively, the patients preferred the -blockers to the placebo. The three drugs had the same overall effect on exercise-induced angina pectoris and they also had antiarrhythmic action. The mechanism of action of -blockers in angina pectoris is discussed and explanations advanced to account for the similarity of their quantitative effects on angina pectoris, despite the differences in their pharmacological profiles.     

Regional distribution of β1- and β2-adrenoceptors in the failing and nonfailing human heart

Summary Total -adrenoceptor density and ₁- and ₂-subtype distribution in right and left atria and in different ventricular regions from 14 failing and seven nonfailing human hearts have been compared. End-stage heart failure was due to idiopathic dilated cardiomyopathy (n=8) or ischaemic cardiomyopathy (n=6).In nonfailing hearts the total -adrenoceptor density was similar in the right and left atria and in all the ventricular regions studied (about 70 to 80 fmol/mg protein). The ₁:₂-adrenoceptor ratio in both nonfailing atria was similar (about 70:30%) and was significantly smaller than in the different regions of both ventricles (about 80:20%). The ₁-subtype density was similar in nonfailing atria and ventricles (about 55 fmol/mg protein). The ₂-subtype density was significantly higher in the right and left atrium (about 25 fmol/mg protein) than in both ventricles (about 15 fmol/mg protein).In patients with end-stage heart failure due to idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy the total -adrenoceptor density was reduced by 50–60% in all regions. On the other hand, the ₁- and ₂-subtype distribution differed with the cause of heart failure. In patients with idiopathic dilated cardiomyopathy, the ₁-adrenoceptor density was lower in all regions, but the ₂-adrenoceptor density was not significantly reduced. In patients with ischaemic cardiomyopathy both ₁- and ₂-adrenoceptors were reduced in all regions.It is concluded that downregulation of -adrenoceptors in patients with end-stage idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy occurs uniformly throughout the heart. The results support the hypothesis that changes in -adrenoceptor subtypes may be related to the cause of heart failure.     

An investigation of the relationship between β-adrenoceptor blockade and plasma practolol concentration in man

Summary In a group of 12 healthy subjects, the relationship between -blocking activity and plasma practolol concentration was studied at intervals up to 24 h after a single 200 mg oral dose of practolol and placebo. -Blockade was assessed by measuring inhibition of exercise heart rate and FEV₁, and the plasma practolol level was determined spectrophotometrically. Significant reduction in mean exercise heart rate was detected 1 to 24 h after practolol but there was no significant reduction in mean resting heart rate or FEV₁. The plasma practolol elimination half life in 8 subjects gave a mean of 5.9 h. The ratio of exercise heart rate inhibition to log plasma practolol concentration increased during the first few hours.     

The β-adrenergic receptor-adenyl-cyclase system of rat reticulocytes

Summary Non-nucleated red blood cells from rats contain adenyl cyclase, the activity of which is predominantly localized in the reticulocytes. Basal enzyme activities in membrane preparations from reticulocyte-rich blood (pretreatment of rats with acetyl-phenylhydrazide: about 60% reticulocytes) are about 5 times higher than in preparations from reticulocyte-poor blood (untreated animals: 2–3% reticulocytes). The enzyme activities are stimulated 10-fold by sodium fluoride (10^–2 M) and 6 to 8-fold by isoprenaline (10^–4 M).Adenyl cyclase activities in membrane preparations from reticulocyte-rich and reticulocyte-poor blood can be ascribed to identical enzymes since identical apparent K _m (ATP; 3×10^–4 M), K _a (isoprenaline; 3×10^–6 M) and K _i (propranolol vs. isoprenaline; 3×10^–7 M) values were obtained in both preparations.Besides NaF, only phenylethanolamine derivatives with -adrenergic receptor stimulant properties were effective as stimulators of adenyl cyclase activity. The affinities (apparent K _a values) of the investigated compounds decreased in the order isoprenaline—hexoprenaline—fenoterol—salbutamol—adrenaline—terbutaline—noradrenaline—phenylephrine. For maximal intrinsic activity, the catechol structure was essential; the relative intrinsic activities of resorcinol derivatives did not exceed 0.6.The isoprenaline-stimulated adenyl cyclase activities in erythrocyte membrane preparations were competitively inhibited by -adrenergic blocking drugs, the affinities (apparent K _i values) decreasing in the order prindolol—penbutolol—propranolol—practolol. The dextrorotatory enantiomers of penbutolol and propranolol were 1/100 to 1/200 as active as the resp. levorotatory enantiomers.From experiments with -adrenergic agonists (e.g. phenylephrine) and antagonists (e.g. phentolamine), it is concluded that -adrenergic receptors do not interfere with the -adrenergically-mediated cAMP formation in these particular membranes.A variety of hormones and drugs known to stimulate adenyl cyclase activities in various tissues, e.g. ACTH, glucagon, STH, erythropoietin, prostaglandin E ₁ etc. did not affect adenyl cyclase activity in reticulocyte-rich erythrocyte membrane preparations.In contrast to adenyl cyclase activity, phosphodiesterase activities in erythrocyte membrane and cytoplasmic fractions were only twice as high in reticulocyterich as in reticulocyte-poor preparations.From the experiments described, it is obvious that the adenyl cyclase of the rat reticulocyte is subject to monovalent-hormonal, i.e. -sympathomimetic stimulation. Moreover, the premature red blood cell provides a useful model for quantitative studies of the interaction of drugs with the -adrenergic receptor.This study was supported by the Deutsche ForschungsgemeinschaftPreliminary accounts were presented at meetings of the Deutsche Pharmakologische Gesellschaft (Gauger et al., 1972; Gauger and Palm, 1973; Quiring et al., 1974a).     

Involvement of central β-adrenoceptors in the regulation of yawning responses

Summary A behavioral study was performed in an attempt to understand the role of central -adrenoceptors in yawning in rats. Yawning was evoked by apomorphine and piribedil, mixed dopamine D₁/D₂-receptor agonists, but not by SK&F 38393 [1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8diol], a dopamine Dr-receptor angonist. The apomorphine-induced yawning was increased by pindolol, propranolol, indenolol, alprenolol and bukumolol which block the central -adrenoceptors, but not by the peripheral -adrenoceptor antagonists, carteolol and atenolol. These -adrenoceptor antagonists given alone did not elicit yawning. Conversely, the yawning was inhibited by salbutamol, a -adrenoceptor agonist, without being affected by prazosin, an -adrenoceptor antagonist. The combined administration of SK&F 38393 and the -adrenoceptor antagonists did not induce yawning. The yawning elicited by either apomorphine or piribedil in combination with pindolol was suppressed by spiperone and YM-09151-2 [cis-N-(1-benzyl-2-methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4-methylaminobenzamide], dopamine D₂-receptor antagonists, and scopolamine, a muscarinic receptor antagonist, but not by SCH 23390 [R(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol], a dopamine D₁-receptor antagonist. Physostigmine or pilocarpine induced yawning, which was also enhanced by pindolol and propranolol. This enhanced yawning was inhibited by scopolamine, but not by spiperone, YM-09151-2 and SCH 23390. The present results suggest that the \-adrenoceptor blockade facilitates the occurrence of yawning induced by dopaminergic and cholinergic agonists. Therefore, the central adrenergic neuron systems may participate in the regulation of yawning responses.Send offprint requests to K. Yamada at the above address     

Both β1- and β2-adrenoceptors mediate catecholamine-evoked arrhythmias in isolated human right atrium

Summary The involvement of ₁- and ₂-adrenoceptors in catecholamine-evoked arrhythmias was investigated in isolated human right atrial appendages obtained from 22 patients chronically treated with blockers (usually ₁-selective) and 9 patients not treated with blockers. A simple experimental model that assesses the incidence of arrhythmic contractions as a function of heart rate (pacing) is introduced. ₁-adrenoceptors were activated by (–)-noradrenaline during ₂-adrenoceptor blockade with 50 nmol/l ICI 118551. ₂-adrenoceptors were activated by (–)-adrenaline during ₁-adrenoceptor blockade with 300 nmol/l CGP 20712A. Both (–)noradrenaline and (–)-adrenaline caused arrhythmic contractions whose incidence was greater at low than at high pacing rates. CGP 20712A (300 nmol/l) blocked the (–)-noradrenaline-evoked contractions in 1/1 atrial strip from 1/1 patient not treated with a blocker and 17/17 atrial strips from 15/15 patients chronically treated with blockers. ICI 118551 (50 nmol/l) blocked the (–)-adrenaline-evoked contractions in 3/4 atrial strips from 3/4 patients not treated with blockers and 17/20 atrial strips from 15/18 patients chronically treated with blockers. The incidence of arrhythmic contractions evoked by both (–)-noradrenaline and (–)-adrenaline was higher in chronically blocked patients than in non blocked patients. We conclude that both ₁- and ₂-adrenoceptors mediate atrial arrhythmias and that the generation of these arrhythmias is facilitated by chronic ₁-adrenoceptor blockade. Correspondence to: A. J. Kaumann at the Clinical Pharmacology Unit, University of Cambridge, as above     

Effects of metoprolol and propranolol on furosemide-stimulated renin release in healthy subjects

Summary The effects of single doses of the beta₁-receptor antagonist metoprolol (40 mg orally), propranolol (40 mg orally) and placebo were compared on furosemide-stimulated plasma renin activity (PRA) in seven healthy subjects. In the placebo studies, PRA increased by 0.59±0.18 ng×ml^–1×h^–1 60 minutes after intravenous administration of 30–60 mg furosemide. After propranolol and metoprolol, the corresponding increases in PRA were significantly less pronounced amounting to 0.16±0.06 and 0.24±0.08 ng×ml^–1×h^–1, respectively. The resting heart rate was reduced to the same extent after the two beta-blockers, which means that the two drugs had been given in equipotent beta₁-receptor blocking doses. It is suggested that the release of renin from the kidney may partly be mediated via an adrenergic beta₁-receptor.Metoprolol (piNN)=H 93/26=CGP 2175     

Comparative pharmacology of human β-adrenergic receptor subtypes—characterization of stably transfected receptors in CHO cells

Although many ₁-receptor antagonists and ₂-receptor agonists have been used in pharmacotherapy for many years their pharmacological properties at all three known subtypes of -adrenergic receptors are not always well characterized. The aim of this study was, therefore, to provide comparative binding characteristics of agonists (epinephrine, norepinephrine, isoproterenol, fenoterol, salbutamol, salmeterol, terbutalin, formoterol, broxaterol) and antagonists (propranolol, alprenolol, atenolol, metoprolol, bisoprolol, carvedilol, pindolol, BRL 37344, CGP 20712, SR 59230A, CGP 12177, ICI 118551) at all three subtypes of human -adrenergic receptors in an identical cellular background. We generated Chinese hamster ovary (CHO) cells stably expressing the three -adrenergic receptor subtypes at comparable levels. We characterized these receptor subtypes and analyzed the affinity of routinely used drugs as well as experimental compounds in competition binding studies, using the non-selective antagonist ¹²⁵I-cyanopindolol as a radioligand. Furthermore, we analyzed the -receptor-mediated adenylyl cyclase activity in isolated membranes from these cell lines. The results from our experiments show that all compounds exhibit distinct patterns of selectivity and activity at the three -receptor subtypes. In particular, a number of ₂- or ₃-receptor agonists that are inverse agonists at the other subtypes were identified. In addition, ₁-receptor antagonists with agonistic activity at ₂- and ₃-receptors were found. These specific mixtures of agonism, antagonism, and inverse agonism at different subtypes may have important implications for the therapeutic use of the respective compounds.     

The role of cyclic AMP in the positive inotropic effect mediated by β1- and β2-adrenoceptors in isolated human right atrium

Summary The time course of the effects of isoprenaline (3 × 10^–7 mol/l) on contractile force and on the cyclic AMP level was studied in the electrically driven isolated muscle strip of the human right atrium. Isoprenaline produced a rise in cyclic AMP content (maximum increase after 60 s) preceding the increase in contractile force. The effects of isoprenaline on contractile force and on the intracellular level of cyclic AMP were enhanced in the presence of the phosphodiesterase inhibitor papaverine (10^–5 mol/l). On the other hand, the -adrenoceptor antagonist propranolol (10^–7 mol/l) suppressed isoprenaline-induced cyclic AMP increases, but reduced the increase in force of contraction by only 35%. In addition, both the ₁-selective antagonist bisoprolol (3 × 10^–9 × 10^–8 mol/l) and the ₂-selective antagonist ICI 118,551 (3 × 10^–9 × 10^–8 mol/l) inhibited the isoprenaline-induced cyclic AMP increase concentration-dependently; ICI 118,551 produced more pronounced inhibition than bisoprolol. It is concluded that cyclic AMP is involved in the positive inotropic action of isoprenaline evoked by ₁- and ₂-adrenoceptor stimulation in isolated human right atrium; however, an additional cyclic AMP independent mechanism cannot be ruled out. Send offprint requests to O.-E. Brodde at the above address     

Studies on Agents with Mixed NO-Dependent Vasodilating and β-Blocking Activities

Purpose. A series of derivatives having a propranolol-like moiety linked to NO-donor furoxan substructures were synthesized. The main objective of this investigation was to obtain agents with mixed NO-dependent vasodilating and -blocking activities. Methods. Most of the target compounds were synthesized from the appropriate furoxans bearing XCH₂CH₂NH₂ (X = O, S, SO₂) chains at the 4 position of the ring, using A1(C₂H₅)₃ in methylene chloride solution and (±)2,3-epoxypropyl 1-naphtyl ether. Two of the final products (X = CONH) were obtained by coupling the appropriate furoxancarboxylic acids with N-[2-hydroxy-3-(l-naphthoxy)propyl]-ethylenediamine. ₁- and ₂-blocking activities were examined on isolated guinea pig right atria and on guinea pig trachea respectively. Vasodilating properties were assessed on endothelium denuded strips of rat aorta. Results. Some derivatives behave as well balanced 'hybrids' displaying NO-dependent vasodilating and -blocking properties in the same concentration range. Some others display either prevalent -blocking or vasodilating activity. Generally speaking hybrid formation lowers the affinity for -receptors, in particular for ₂-type, to give an increase in ₁/₂ selectivity. Conclusions. The furoxan system is a flexible tool in designing analogues of propranolol whose NO-donating and -blocking properties are modulated over a wide range.     

Biliary Excretion of 17β-Estradiol 17β-d-Glucuronide Is Predominantly Mediated by cMOAT/MRP2

Purpose. The mechanism for the biliary excretion of 17-estradiol170-d-glucuronide (E₂17G), a cholestatic metabolite of estradiol, isstill controversial. The purpose of the present study is to examine thetransport of E₂17G across the bile canalicular membrane. Methods. We examined the uptake of [³H]E₂17G by isolatedcanalicular membrane vesicles (CMVs) prepared from Sprague-Dawley (SD)rats and Eisai Hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective. Also,in vivo biliary excretion of intravenously administered [³H]E₂17Gwas examined. Results. In CMVs prepared from SD rats, but not from EHBR, amarked ATP-dependent uptake of [³H]E₂17G was observed.Moreover, E₂17G competitively inhibited the ATP-dependent uptake of[³H]2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, nosignificant inhibitory effect of verapamil (100 M) and PSC-833 (5 M) onthe uptake of [³H]E₂17G was observed. In vivo, the biliary excretionof intravenously administered [³H]E₂17G was severely impaired inEHBR while the biliary excretion of [³H]E₂17G in SD rats wasreduced by administering a cholestatic dose (10 mol/kg) unlabeledE₂17G, but not by PSC-833 (3 mg/kg). Conclusions. The transport of E₂17G across the bile canalicularmembrane is predominantly mediated by cMOAT/MRP2.     

The influence of molecular structure on the affinity and efficacy of some β-adrenoceptor agonists

Summary The affinity and efficacy of a number of sympathomimetic amines structurally related to prenalterol and the selective ₁-adrenoceptor agonist RO 363 were determined using a combination of radioligand binding and organ bath techniques. Affinity of the molecules (pK _D) was calculated from their ability to displace the radioligand [¹²⁵I]iodocyanopindolol ([¹²⁵I]CYP) from -adrenoceptor sites in left atrial (₁) and uterine (₂) membrane homogenates. These pK _D values were used to calculate efficacy from the positive inotropic and uterine relaxant responses elicited by the drugs in organ bath experiments. The drugs studied were either arylethanolamines i.e., (–)-isoprenaline (ISO), p-hydroxyisoprenaline (pOH-ISO), compounds XIV and XVI or aryloxypropanolamine-derivatives, i.e., oxymethylene-isoprenaline (OM-ISO), prenalterol and Compound XI which possessed ap-phenol or catechol ring and an isopropyl or a homoveratryl amine substituent. Only ISO, OM-ISO, pOH-ISO and Compound XVI were active as agonists in both tissue preparations. These drugs were partial agonists which exhibited a wide range of pD₂ values and did not display any marked selectivity for either -adrenoceptor subtype. Compound XI and prenalterol were inactive as agonists and together with the partial agonists behaved as competitive antagonists to ISO in the two preparations. All drugs tested displaced [¹²⁵I]CYP from -adrenoceptor sites, however, there was also a wide range of potency amongst the drugs.Analysis of the structure-affinity and structure-efficacy relationships indicated that removal of the 3-hydroxyl group from the catechol ring reduces both affinity and efficacy without altering the selectivity of the drug for either -adrenoceptor subtype. While aryloxypropanolamine derivatives have generally higher affinities than arylethanol-amines, especially at -adrenoceptor sites, their efficacies are generally reduced at both -adrenoceptors. The presence of a homoveratryl group in aryloxypropanolamines enhances slightly the affinity for ₁- and reduces affinity for ₂-adrenoceptors. With this amine group, efficacy is markedly reduced at ₂- as opposed to ₂-adrenoceptor sites.Thus for prenalterol, the small degree of cardioselectivity can be attributed to the oxymethylene group whilst its lack of agonist activity (i.e., efficacy) reflects a combined action of this group and the absence of the 3-hydroxyl group on the phenyl ring. In RO363 it can be deduced that the oxymethylene group, together with the homoveratryl substituent are responsible for the observed selective affinity of the drug for ₁- as opposed to ₂-adrenoceptors.     

Bis(carbamoyloxymethyl) Esters of 2′,3′-Dideoxyuridine 5′-monophosphate (ddUMP) as Potential ddUMP Prodrugs

No HeadingPurpose. We previously reported the synthesis of bis(pivaloyloxymethyl) 2,3-dideoxyuridine 5-monophosphate (POM₂-ddUMP) (1a) as a membrane-transport prodrug formulation of the free parent nucleotide, ddUMP. Although successful at delivering ddUMP into cells in culture, POM₂-ddUMP was rapidly degraded by plasma carboxylate esterases after intravenous administration to experimental animals, and therefore has limited therapeutic potential as a systemically administered prodrug. We now report the synthesis of bis(N,N-dimethylcarbamoyloxymethyl)- and bis(N-piperidinocarbamoyloxymethyl) 2,3-dideoxyuridine 5-m onophosphate [DM₂-ddUMP (1b) and DP₂-ddUMP (1c), respectively], analogues of POM₂-ddUMP that were designed to be more resistant to degradation by plasma esterases..Methods. After entering cell by passive diffusion, it was anticipated that loss of one of the carbamoyloxymethyl groups of 1b and 1c would occur by spontaneous chemical hydrolysis to give the intermediate phosphodiesters, 2b and 2c. Cleavage of the remaining carbamoyloxymethyl groups by cellular phosphodiesterase I would generate ddUMP. 1b and 1c were prepared by condensation of 2,3-dideoxyuridine (ddU) with the appropriate bis(N-alkylcarbamoyloxymethyl) phosphate in DMA in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent).Results. The half-lives of 1b and 1c when incubated at a concentration of 10^–4 M in human plasma at 37°C were 3.5 h and 3.7 h, respectively, similar to the half-lives observed under the same temperature conditions in 0.05 M aqueous phosphate buffer, pH 7.4. By contrast, the half-life of the POM₂ prodrug, 1a, in plasma was only 5 min. The initial products of degradation of 1b and 1c were the phosphodiesters 2b and 2c. The latter compounds gave rise to ddUMP when incubated with snake venom phosphodiesterase I.Conclusions. These findings support the premise inherent in the design of 1b and 1c, namely that the carbamate prodrugs are far more resistant to hydrolysis by plasma carboxylate esterases than their POM counterparts and can revert to the free parent 5-mononucletides by successive chemical and enzymatic hydrolysis. Further studies of 1b and 1c as membrane-permeable prodrugs of ddUMP are in progress.     

Effects of β-adrenoceptor antagonists on the firing rate of noradrenergic neurones in the locus coeruleus of the rat

Summary Acute i.v. administration of the non-selective -adrenoceptor antagonist dl-propranolol given in incremental doses (<40 mg/kg) did not affect the firing rate of locus coeruleus (LC) neurones in the rat, as revealed by single cell recording techniques. Furthermore, no effect was seen 4 h after a single i.p. dose of this -blocker (10 mg/kg). However, repeated treatment with dl-propranolol (1, 5, 10 or 20 mg/kg i.p., twice daily for 4 days) produced a significant, dose-dependent decrease of the average LC neuronal firing rate in comparison to controls. The dextro isomer of propranolol, which has negligible -blocking activity but the same local anaesthetic potency as the racemate, had no corresponding effect. The non-selective -adrenoceptor antagonist sotalol, which is one of the most hydrophilic -blockers, had much less inhibitory effect on LC neurones than dl-propranolol. The ₁-selective antagonist metoprolol did not change the firing of noradrenergic neurones in the LC after similar treatment for 4 days. However, when the rats were subjected to oral treatment for 28 days, metoprolol was found to produce a slight inhibitory effect although much less than dl-propranolol.In view of these findings we propose a stimulatory and mainly ₂-adrenoceptor-mediated control mechanism for the noradrencrgic neurones in the LC. This mechanism seems to be characterized by a delayed responsiveness.