High-performance liquid chromatography with chemiluminescence detection of penbutolol and its hydroxylated metabolite in rat plasma.

This paper describes a new method of high-performance liquid chromatography with chemiluminescence detection for the analysis of penbutolol (PB) and its main metabolite, 4-hydroxy penbutolol (4-OH PB) in rat plasma. 4-Dimethylaminosulfonyl-7-(N-chloroformylmethyl-N-methyl) amino-2,1,3-benzoxadiazole (DBD-COCl) was used as a fluorogenic labeling reagent. A mixture of hydrogen peroxide and bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) in acetonitrile was used as a post-column chemiluminogenic reagent. The derivatives of PB and 4-OH PB with DBD-COCI were separated by isocratic effluent with 0.01 M imidazole buffer (pH 7.0)-acetonitrile within 10 min. The detection limits of the proposed method for PB and 4-OH PB were 9.9 and 15 fmol on column, respectively. After intravenous administration of PB in rats, its plasma concentration profiles of PB and 4-OH PB were determined by the proposed method. PB was demonstrated to be rapidly metabolized to 4-OH PB at the same rate as cardiac output.

Unchanged protein binding of penbutolol in renal insufficiency: a possible role of carbamylation.

The effect of in vitro carbamylation of serum protein with potassium cyanate on protein binding of penbutolol, a basic agent exclusively bound to alpha 1 acid glycoprotein (AAG), was investigated. Carbamylation of serum resulted in a weak increase on free fraction of penbutolol (4.45 +/- 0.54% before carbamylation vs 5.66 +/- 0.40% after; p < 0.025). Parallelly, potassium cyanate added to pure AAG and incubated for 90 min induced carbamylation of this protein (38 mumoles of 14C cyanate incorporated per gram of protein). A study in serum from patients with chronic renal disease (pre and postdialysis) showed no changes in protein binding of penbutolol, although AAG levels were significantly higher. However, Scatchard [1949] plot for penbutolol binding to serum from renal patients (both pre and postdialysis) showed a decrease in affinity constant (nKa = 11.13 x 10(5) M-1 in healthy volunteers, vs 5.56 x 10(5) M-1 in patients before dialysis and 4.57 x 10(5) M-1 after dialysis). We concluded that carbamylation of serum AAG in uremic patients could explain, in part, the absence of changes in protein binding of any basic drugs in this pathological condition. It appears that a decreased affinity constant could balance the effect of increased AAG levels.

Decrease in penbutolol central response as a cause of changes in its serum protein binding.

Penbutolol is a beta-adrenoceptor antagonist that is extensively bound to alpha 1-acid glycoprotein (alpha 1-AGP), a protein that increases in inflammatory diseases thereby binding more drug in such conditions. Changes in serum binding can lead to modifications in the pharmacokinetics and pharmacodynamics of a drug, therefore, the central effect (as the anticonvulsant response) and brain uptake of penbutolol given intravenously to mice with experimental inflammation have been measured. A significant decrease of the central effect of penbutolol and its brain uptake was seen in diseased when compared with control animals (P less than 0.01). A parallel decrease in free fraction of penbutolol in diseased vs normal animals was detected. These results suggest that there is an increase in serum binding of basic drugs related to increments in alpha 1-AGP concentration, which reduces their central pharmacological effect.

Plasma protein binding of penbutolol in pregnancy.

Penbutolol is a not cardioselective beta-adrenergic blocking drug; it is lipid soluble and differs in its protein binding from the other members of its group because shows linkage to alpha 1-glycoprotein, with no detectable binding to albumin. AAG levels change during pregnancy and so the binding of [3H]-penbutolol was compared in 11 pregnant patients and in 10 healthy women. Binding was obtained by ultrafiltration and measurement of the free fraction by scintillation spectrometry. The free penbutolol fraction was significantly higher in the pregnant women than in the controls (6.06 +/- 0.34 compared with 3.55 +/- 0.29, P less than 0.001). The AAG levels in the pregnant women were significantly lower (0.40 +/- 0.03 g/l) than in the controls (0.77 +/- 0.06 g/l) (P less than 0.001) which showed a significant correlation with the bound/free penbutolol ratio (r = 0.61, P less than 0.005). On the other hand there was no significant correlation with the extent of penbutolol's protein binding even though the albumin levels were lower in the pregnant women (2.83 +/- 0.17 compared with 4.86 +/- 0.17; P less than 0.001). Penbutolol's nK1a for AAG was lower in pregnant women, and this suggests that the fall in AAG levels is not the only factor involved in the reduced binding of penbutolol in pregnancy.

Pharmacokinetics and dynamics of penbutolol in humans: evidence for pathway-specific stereoselective clearance.

The pharmacokinetics and dynamics of the D- and L-isomers of the beta-adrenergic blocking agent penbutolol were investigated in healthy human volunteers. In Study One, subjects received a single 40-mg oral dose of L-penbutolol (the pharmacologically active stereoisomer), and matching placebo on two occasions. A mean peak serum penbutolol concentration of 268 ng/ml was reached at 0.9 h after dosing. Elimination half-life averaged 1.6 h, and total clearance 16.6 ml/min per kg body weight. Changes in blood pressure, ventricular rate, and rate of circumferential fiber shortening (Vcf) did not differ between L-penbutolol and placebo. In Study Two, subjects received 40 mg D-penbutolol, L-penbutolol, and placebo on three occasions. Total clearance of D-penbutolol was higher than for the L-isomer (43.7 vs 15.9 ml/min/kg; P less than 0.01); this was reflected in correspondingly increased area under the serum concentration curve for conjugates of the oxidized metabolite 4-hydroxy penbutolol (2.25 vs 0.66 micrograms/ml X h; P less than 0.005). In contrast, direct conjugates of L-penbutolol achieved higher serum concentrations than conjugates of D-penbutolol. Alterations in blood pressure, ventricular rate, and Vcf for D-penbutolol, L-penbutolol, and placebo were quantitatively small. Thus the clearance of penbutolol after oral administration in humans is stereoselective, but the oxidative pathway is more stereosensitive than the parallel conjugative pathway. Penbutolol causes minimal alterations in parameters of cardiac function after single 40-mg doses in healthy humans.

Measurement of penbutolol and 4-hydroxypenbutolol in plasma or serum by HPLC.

A simple HPLC method for penbutolol and 4-hydroxypenbutolol assay has been developed. Plasma or serum (200 microliters) is vortex-mixed (30 s) with Tris solution (2 M, pH 10.6) containing an internal standard (50 microliters) and methyl t-butyl ether (200 microliters). After centrifugation, the extract (100 microliters) is analysed using an unmodified silica column (250 x 5 mm ID) and iso-octane-methanol-methyl t-butyl ether (55:25:20) containing ammonium perchlorate (10 mM, pH 5.7) as eluent and with fluorescence detection. No interference has been encountered and the limit of accurate measurement for both compounds is 5 micrograms/l.

Penbutolol Pharmacokinetics: the influence of concomitant administration of cimetidine.

A possible interaction of penbutolol and cimetidine was investigated in healthy volunteers treated orally for 7 days. The plasma levels of unmetabolized penbutolol showed a slight but non-significant increase. The biphasic elimination kinetics of penbutolol (half-lives 0.8 and 17 h) was not affected by coadministration of cimetidine. Plasma levels of penbutolol were not significantly altered by chronic treatment with cimetidine, whereas the levels of 4-hydroxypenbutolol and 4-hydroxypenbutolol glucuronide were significantly reduced.

Penbutolol: beta-adrenoceptor interaction and the time course of plasma concentrations explain its prolonged duration of action in man.

Beta-adrenoceptor binding of (-) penbutolol and its active metabolite 4-hydroxy-penbutolol to rat reticulocyte membranes was shown in the presence of native human plasma. Due to the high plasma protein binding (approximately 99%) the apparent Ki-values of penbutolol were shifted 100-fold to the right after inclusion of plasma in the assay; the Ki was approximately 40-70 ng/ml. That value is comparable to the IC50-values calculated from clinical studies. The interaction of 4-hydroxy-penbutolol with beta-adrenoceptors was not affected to the same extent by inclusion of plasma protein binding approximately 80%, apparent Ki-value approximately 7 ng/ml. Thus, the active metabolite of penbutolol displays higher potency at beta-adrenoceptors in vitro due to its lesser degree of plasma protein binding. A prediction procedure for antagonist activity after penbutolol administration using beta-adrenoceptor interaction and plasma concentration kinetics suggests that, in addition to a rapid elimination process from human plasma, a slow elimination phase of penbutolol (or an active metabolite) is necessary to explain the long duration of action observed in clinical studies after a single oral dose. Inhibition in vitro of beta-adrenoceptor binding by plasma samples obtained after oral administration of 40 mg penbutolol to 3 healthy volunteers indicated a biphasic concentration-time profile of the antagonist in plasma and was in accordance with the time course of the reported reduction in exercise tachycardia. Finally, plasma concentrations of penbutolol equivalents derived from the receptor assay were in the range of penbutolol concentrations detected by physico-chemical methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of penbutolol and its metabolites in renal insufficiency.

The pharmacokinetics of penbutolol, its 4-hydroxylated metabolite and of their conjugates was studied in hypertensive patients with various degrees of renal impairment. A single oral dose of penbutolol 40 mg, was rapidly absorbed after a lag-time of 0.34 h. Its plasma concentration reached a maximum after 0.84 h and then declined bi-exponentially, with an apparent elimination half-life of 21.8 h. The hydroxylation of penbutolol was negligible and conjugation was of major importance for its elimination. Consequently, the kinetics of unchanged penbutolol were not altered by renal impairment. The 48 h-urinary excretion of penbutolol and its metabolites reached 13-14% of the administered dose, which is consistent with extensive metabolism of the drug. After treatment for 30 days with penbutolol 40 mg/d there was no accumulation of the parent drug but the concentration of its conjugates was increased. It is concluded that the dose of penbutolol need not be changed in patients with mild renal insufficiency, 4-hydroxypenbutolol is unlikely to participate in the anti-hypertensive effect of the drug, due to its low concentrations, and biotransformation of penbutolol may be enhanced during chronic treatment.

Protein binding studies of furosemide and penbutolol.

Serum protein binding of furosemide and penbutolol, the active principles of Betasemid (Hoe 9358), was studied by equilibrium dialysis. Membranes from commercial dialysis tubes were used within commercially available cells. Serum drug and portion unbound in buffer were determined by quantitative thin-layer chromatography. In the range of 1-20 micrograms drug/ml serum, 96 +/- 0.3% of furosemide vs. 88 +/- 4% of penbutolol were bound to proteins. The same results were obtained, when the two substances simultaneously interacted with proteins. Thus, specific protein binding sites for both compounds were demonstrated. For both drugs, Scatchard plots revealed two classes of specific binding sites with statistical mean values/protein of 0.4 and 5 in the case of furosemide and 0.04 and 0.3 in the case of penbutolol. Binding energies were 27.6 and 19.6 kJ/mol furosemide vs. 31.5 and 23.9 kJ/mol penbutolol. Further, serum protein binding of furosemide was studied by ultrafiltration. A micropartition system MPS-1 was used. Results were the same as those from equilibrium dialysis.

Haemodynamic dose-response effects of i.v. penbutolol in angina pectoris.

The haemodynamic dose-response effects of intravenous penbutolol, a newer beta-adrenoceptor antagonist with intrinsic sympathomimetic activity but without cardioselectivity, were evaluated in 10 patients with angiographically documented coronary artery disease. Following four logarithmetically cumulative i.v. boluses (0.5-4 mg dosage range) there was a log linear increase in plasma penbutolol concentration; the levels achieved (51 +/- 8 to 219 +/- 19 ng/ml) were in the therapeutic range (12 to 250 ng/ml). Penbutolol resulted in a linear decrease in heart rate (maximum delta HR - 4 beats/min; P less than 0.01); there was a small increase in pulmonary artery occluded pressure which reached its maximum at the lower doses (maximum delta PAOP + 1 mm Hg; P less than 0.01). The resting cardiac output, blood pressure and calculated systemic vascular resistance were unchanged. During 4 min steady-state supine bicycle exercise there was attenuation of exercise cardiac output (delta C.I. - 0.6 1 min-1 m-2; P less than 0.01) and systolic pressor response (delta SBP - 13 mm Hg; P less than 0.01) compared with control observations without change in other measured or derived variables. The haemodynamic profile of penbutolol compared favourably with other beta-adrenoceptor antagonists previously evaluated under similar conditions in patients with ischaemic heart disease. Over the i.v. dose-range evaluated penbutolol attenuated exercise-induced angina with a relatively modest depression of cardiac performance; the small change induced in resting haemodynamic variables may, in part, have been contributed to by the intrinsic sympathomimetic activity of penbutolol.

Penbutolol (Hoe 893d) in primary hypertension. Blood pressure effects, tolerance and plasma concentrations.

Penbutolol (Hoe 893d), a long-acting non-selective beta-adrenoceptor blocking agent, was given once daily to 23 patients with primary hypertension, WHO Stages I-II. The dose (50-100mg) needed to achieve the therapeutic goal, i.e. supine diastolic BP less than 95 mm Hg, was titrated individually. On a daily dose of penbutolol 83 +/- 19 mg (mean +/- SD) blood pressure (BP, mean +/- SD) fell from 180 +/- 21/112 +/- 8 mmHg on placebo to 154 +/- 25/94 +/- 14 mmHg. 18 patients who reached the therapeutic goal (responders) continued in a double blind, cross-over study versus placebo, during which the supine BP fell on average 20/10 mmHg on the same dose of penbutolol, and 2/1 mmHg on placebo. Plasma concentrations (mean +/- SD) of free 0.10 +/- 0.07 microgram/ml) and total (2.02 +/- 1.39 microgram/ml) penbutolol did not differ between responders and nonresponders, and were not correlated with the fall in BP. Side effects were mild and mostly well tolerated. One patient developed dermatitis and another an elevation of liver enzymes.

Comparative effects of two commercially available blood collection tubes on plasma concentrations of drugs.

Two commercially available blood collection systems (Venoject and Vacutainer) were investigated for their effect on the plasma concentrations of a variety of drugs. Vacutainer tubes adversely affected the following drugs: propranolol, penbutolol and quinidine, whereas Venoject tubes had no effect on the plasma concentrations of these drugs. Neither collection system had any effect on the plasma concentrations of the following drugs: acebutolol, the main metabolite of acebutolol (DL-1-(2-acetyl-4-acetamidophenoxy)-2-hydroxy-3-isopropylaminopropane), diazepam, N-demethyldiazepam, digoxin, ethosuximide, salicylic acid, valproic acid, warfarin and carbamazepine.

[Biological determination of the beta blocking activity of human serum]. / Détermination biologique du pouvoir bêta-bloquant du sérum humain.

A method of biological assessment of the Beta blocking activity of human serum is reported. It is based on the catecholamine response of rat myocardial cells in culture, incubated in the serum to be tested. Its advantage is that it takes into account all block-blocking substances present in the serum, not only the drug itself but also its possible active metabolites. The results obtained by this method in 10 healthy subjects after 60 mg penbutolol were compared with those given by chemical dosage and ergometry. The ergometric and biological changes were parallel from the 2nd and the 8th hour while the serum levels of the drug rapidly. This discordance could be due to the presence of an active metabolite, 4-hydroxy-penbutolol.

Single and divided doses of penbutolol.

Penbutolol, a nonselective beta-adrenoreceptor antagonist, induced reduction of exercise-induced heartbeats for at least 24 hr after a single 40-mg oral dose, and was equipotent with respect to a 2 X 20-mg regimen over the same period. Ingestion for 7 days did not influence the pharmacodynamics or pharmacokinetics of penbutolol, and there was no cumulation of drug in serum. A relationship was found between the logarithms of measurable serum concentrations of penbutolol and the percentage reduction of total heartbeats. Absorption of oral penbutolol appeared to be reduced when administered in the evening. Since beta-adrenoceptor activity was relatively unchanged between 13 and 24 hr after a single 40-mg dose of penbutolol, there is a possibility that an active metabolite or metabolites may contribute to prolonged duration of action.

Physico-chemical and anlytical studies of penbutolol.

In the present paper physico-chemical and analytical studies on penbutolol sulfate (Hoe 893d) are reported. In addition to an interpretation of ultraviolet and fluorometric spectra, data are given regarding the dissociation constant, solubility, distribution, and protein binding. The substance and its major metabolte, as well as their glucuronides, are detectable by means of a selective fluormetric method. Moreover, mention is made of the results of a human pharmacokinetic study.