Labeling of alprenolol with fluorescent aryl iodide as a reagent based on Mizoroki-Heck coupling reaction.

A novel fluorescent labeling method for alprenolol was developed based on Mizoroki-Heck coupling reaction. We designed and synthesized fluorescent aryl iodide, 4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene (DIBI) as a labeling reagent. DIBI has a lophine skeleton carrying an iodide atom acting as fluorophore and reactive center, respectively. In order to evaluate the usefulness of DIBI, a high-performance liquid chromatography (HPLC) with fluorescence detection method was developed for the determination of alprenolol as a model compound of terminal double bond. The fluorescent labeling of alprenolol with DIBI was achieved in the presence of palladium acetate as a catalyst, and the labeled alprenolol was detected fluorometrically. In addition, it was found that the fluorescence of DIBI derivative increased and red shifted when compared with that of DIBI. Furthermore, the proposed method could be applied to determine the alprenolol concentration in rat plasma after administration of alprenolol without interferences from biological components. The detection limit (S/N=3) for alprenolol in rat plasma was 0.74 ng/mL (30 fmol on column).

Enantioselective determination of alprenolol in human plasma by liquid chromatography with tandem mass spectrometry using cellobiohydrolase chiral stationary phases.

A fast, sensitive, and enantioselective LC-MS/MS bioanalytical method was developed and validated for the direct determination of individual alprenolol enantiomers in human plasma using cellobiohydrolase (CBH) chiral stationary phases (CSP) along with supported liquid extraction (SLE) procedures. Complete baseline separation of enantiomeric alprenolol was achieved within 2 min in reversed phase chromatography at 0.9 ml/min. SLE in a 96-well plate format was used for sample extraction. The method validation was conducted over the curve range of 0.500-500 ng/ml for each alprenolol enantiomer using 0.0500 ml of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed < or = 7.3% relative standard deviation (RSD) and -6.2 to 8.0% relative error (RE) for both alprenolol enantiomers.

An investigation into the influence of drug lipophilicity on the in vivo absorption profiles from subcutaneous microspheres and in situ forming depots.

Drug lipophilicity is known to have a major influence on in vivo drug absorption from intramuscularly and subcutaneously administered solutions. Indeed, chemical modification to increase drug lipophilicity is used to enable sustained drug release from solutions. In contrast to the wealth of knowledge on drug release from simple solutions, the influence of drug lipophilicity on its release from controlled release formulations, such as, microparticles and in situ forming depots, have not been systematically studied. Controlled release vehicles are designed to 'control' drug release, hence, in vitro studies show negligible influence of drug lipophilicity on release. The situation could however be different in vivo, due to interactions between the vehicle and biological tissue. We therefore investigated the influence of drug lipophilicity on its in vivo release in rats from two controlled release formulations, PLGA microparticles and in situ forming depots. Both systems exhibited a burst drug release. Subsequent to the burst release, we found that lipophilicity did not influence the rate or extent of drug absorption from the two formulations over a 10-day study period, which would imply that drug partitioning out of the depots was not the main mechanism of drug release from both formulations. This study must however be repeated with a greater number of animals to increase its power.

Stereoselective protein binding of alprenolol in the renal diseased state.

The investigation was undertaken to study the stereoselective protein binding of alprenolol in renal disease patient sera, compared to that in the sera of healthy volunteers. The in vitro stereoselective protein binding of beta-blockers was determined in undiluted serum and in isolated alpha(1)-acid glycoprotein (AGP) solutions by ultrafiltration. The stereoselctive serum protein binding of alprenolol, a beta-adrenergic blocking agent, in healthy volunteers was significantly altered in renal disease patients. We investigated the effects of AGP concentration and endogenous substances, including uremic toxins, on the stereoselective protein binding of alprenolol in renal disease patients. A good correlation between the unbound (R)/(S) ratio (F(R)/F(S) ratio), an apparent index of stereoselectivity in alprenolol serum binding and AGP concentration in serum, was found. However, stereoselective protein binding was not influenced by endogenous substances. This result can be explained by the difference in binding affinities of (R) and (S)-isomers of alprenolol to AGP. We conclude that the stereoselective protein binding of alprenolol in healthy volunteers and renal disease patients varies as a result of changes in AGP concentration. Accordingly, these findings might be useful in alprenolol therapy in renal disease patients.

Enantioselective quantitation of (R)- and (S)-alprenolol by gas chromatography-mass spectrometry in human saliva an plasma.

An enantioselective gas chromatographic-mass spectrometric assay is developed for alprenolol and its metabolite, 4-hydroxy-alprenolol, in saliva and plasma. The procedure is based on a two-step derivatization technique with N-heptafluorobutyryl-l-prolylchloride and N-methyl-N-trimethylsilyl-trifluoroacetamide, followed by a gas chromatographic separation with mass spectrometric detection of the diastereomeric derivatives. A selected ion chromatogram extracted from full scan data shows that the respective enantiomers of alprenolol, 4-hydroxy-alprenolol, and the internal standard (ions at m/z 481) are well-separated in saliva and plasma. Linear and reproducible calibration curves are obtained over the concentration ranges 1.67-13.33 ng/mL and 2.50-20.00 ng/mL enantiomer in saliva and plasma, respectively. The performance of the method for alprenolol, in terms of accuracy and precision, fits well within the generally accepted criteria for validation. The enantioselective assay is successfully used in a study involving a single oral dose of alprenolol administered to two healthy volunteers. Stereoselective differences are observed in the saliva and plasma concentrations following an oral dose of 50 mg (R,S)-alprenolol hydrochloride.

Enantioselective assay of beta-receptor antagonists present in microdialysis and plasma samples of rats.

The enantiomers of alprenolol, metoprolol, and propranolol have been separated on an enantioselective cellulase column and analysed using a fully automated HPLC system involving coupled column chromatography and fluorescence detection. The assays had sufficient selectivity and sensitivity to investigate the disposition of these beta 2-receptor antagonists in blood and brain extracellular fluid of rats. A cellulase column was used as the first column to separate the enantiomers giving separation factors between 2.9 and 4.3. After the separation, the enantiomers were trapped on two small precolumns by the use of a switching valve and were then introduced on an achiral C18 analytical column by eluting the small columns backward. The enantiomers in blood and brain tissue dialysates were analysed by direct injection of 8 microliters samples. The limit of quantitation was 0.025-0.4 micrograms/ml of the different enantiomers. Plasma samples were analysed after a simple extraction procedure. The intraassay precision of the lowest quality control plasma samples (0.2-0.8 micrograms rac-drug/ml) was 4-8% for the different enantiomers.

Decreased plasma concentrations and clinical effects of alprenolol during combined treatment with pentobarbitone in hypertension.

The antihypertensive effect of alprenolol has been studied before, during and after additional pentobarbitone treatment. The combined alprenolol-pentobarbitone treatment significantly decreased alprenolol levels by 59% and 4-hydroxyalprenolol by 24%. The effect was significant after three doses and declined over 4-5 days after pentobarbitone withdrawal. The decreased alprenolol plasma levels were associated with increased pulse rate (6%), and systolic (8%) and diastolic (9%) blood pressure. The inhibition of exercise tachycardia by alprenolol was reduced by 18% at the end of pentobarbitone treatment compared to initial monotherapy with alprenolol. The interaction is probably clinically important in those patients with hypertension and angina pectoris that are treated with barbiturates and alprenolol.

Determination of pindolol in human plasma and urine by high-performance liquid chromatography with ultraviolet detection.

This paper presents a rapid, simple and economical method for assaying pindolol concentrations in plasma and urine by high-performance liquid chromatography using ultraviolet detection. It is sensitive enough for use in single-dose pharmacokinetic studies and may also be used to determine pindolol concentrations in the plasma from patients taking the drug, provided that the patient is not taking any of the drugs which interfere with the method. Drugs which were found to interfere with the pindolol peak are quinidine, n-acetylprocainamide and lidocaine. Disopyramide, oxprenolol and levobunolol interfered with the internal standard peak.

Oesophageal ulcerations and plasma levels of different alprenolol salts: potential implications for the clinic.

The ulcerogenic effect of five different salts of alprenolol were tested against placebo in a porcine oesophageal test model. The salts with high water solubility, such as the hydrochloride and the fumarate, gave rise to the highest plasma concentrations of alprenolol and evoked serious oesophageal lesions, while the salts with low solubility-the benzoate, maleate and sebacate-had no irritative effect on the oesophagus. The plasma levels of alprenolol were much higher following administration of alprenolol hydrochloride in the oesophagus than after an identical intraduodenal dose of the same salt possibly because of the avoidance of the first-pass degradation during oesophageal absorption.

Direct derivatization of alprenolol and its 4-hydroxy metabolite in urine with phosgene and methanol prior to analysis by capillary column gas chromatography.

A method for the determination of alprenolol and its 4-hydroxy metabolite has been developed. The urine sample is made alkaline with buffer (pH 12) and derivatized with 60 microliter of 2 M phosgene in toluene with vigorous shaking. In the presence of 2.5% methanol, an oxazolidineone methyl carbonate is formed from 4-hydroxy alprenolol. The now neutral derivatives are extracted with an equal volume of dichloromethane. After evaporation of the organic phase, the residue is taken up in a small volume of ethyl acetate and subjected to capillary column gas chromatography with CP-Sil 8 as the stationary phase. The precision was 2.1% at the 3.3 micrograms/ml level of the metabolite in urine (n = 8). The isopentylamino analogue was used as the internal standard.

[Changes in the density of human lymphocyte beta-adrenergic receptors in hypertension]. / Izmenenie plotnosti beta-adrenergicheskikh retseptorov limfotsitov cheloveka pri gipertonicheskoi bolezni.

The beta-adrenoreceptor density in intact lymphocytes of peripheral blood of normotensive and hypertensive adults was measured by the radioligand binding technique with the use of 3H-dihydroalprenolol. The density of beta-adrenoreceptors was found to be higher in lymphocytes of hypertensive subjects while the number of the receptors was equal to 2076 +/- 208 and 1461 +/- 175 receptors per cell, respectively, in hypertensive and normotensive subjects. The dissociation constants in both cases varied from 0.4 to 1.8 nM. Probably, the decreased responses to the adrenergic agents in hypertensive subjects are not connected with the decreased number of beta-adrenoreceptors and may be due to alterations in postreceptor events.

Drug binding to plasma proteins during human pregnancy and in the perinatal period. Studies on cloxacillin and alprenolol.

Plasma protein binding of one acidic drug, cloxacillin, and one basic drug, alprenolol, was determined by equilibrium dialysis at +37 degrees C during pregnancy and the 1st postnatal week in 12 women and their newborn infants and in 7 nonpregnant women (controls). A significant increase in fraction free cloxacillin in maternal plasma occurred during pregnancy already from the 2nd trimester compared to the controls (p less than 0.01) and was most pronounced at delivery (median values 0.126 and 0.069, respectively). A similarly increased fraction free cloxacillin was found in cord blood (median value 0.108) which further increased during the 1st postnatal week (range 0.112-0.164). In maternal plasma the binding capacity returned to the values of the controls during the same time period. The binding of cloxacillin was significantly correlated with the concentration of albumin (p less than 0.01). High correlation was also found between binding of the basic drug alprenolol and concentration of orosomucoid (p less than 0.005). This was most obvious in the newborn infants with low concentrations (range 0.1-0.3 g/l) and in the mothers during the puerperium with high concentrations of orosomucoid (range 0.7-2.5 g/l). On the basis of plasma protein binding data in the mother and her child, a maternal to fetal plasma concentration ratio was calculated. For cloxacillin this ratio was close to unity (1.03), while it was significantly above unity for alprenolol (1.72). At equilibrium, therefore, the total plasma concentration of alprenolol in the mother can be expected to exceed the concentration in her infant.

Determination of (R)-AND (S)-alprenolol-and (R)- and (S)-metoprolol as their diastereomeric derivatives in human plasma by reversed-phase liquid chromatography.

A high-performance liquid chromatographic method is described for the determination of (R)- and (S)-alprenolol and (R)- and (S)-metoprolol in human plasma. Separation of the enantiomers was accomplished after preparation of diastereomeric derivatives with symmetrical anhydrides of tert.-butoxycarbonyl-L-leucine followed by treatment with trifluoroacetic acid at 0 degree C to remove the tert.-butoxycarbonyl group. The separations of the diastereomeric derivatives were performed using a reversed-phase system with muBondapak C18 as support and phosphate buffer pH 3.0 with the addition of acetonitrile as the mobile phase. High stability of the chromatographic system was achieved. The reproducibilities in the determination of (R)- and (S)-alprenolol and (R)- and (S)-metoprolol in human plasma were 9.4 and 9.8% (relative standard deviation) for alprenolol and metoprolol, respectively, at drug levels of 0.5 ng/ml. In two subjects who received single oral doses of alprenolol (100-mg tablet) and metoprolol (50-mg tablet) the plasma levels of the (R)-isomers were lower than for the (S)-isomers.

Formation of electron-capturing derivatives of alprenolol by transboronation: application to the determination of alprenolol in plasma.

The reaction between 2,4-dichlorobenzeneboronic acid, 3,5-bis(trifluoromethyl)benzeneboronic acid or the 1,3-propanediamine derivatives of the boronic acids and the 3-isopropylaminopropan-2-ol side chain of the beta-blocking drugs occurs essentially as an on-column thermal reaction in the gas phase. Derivatization of the side chain of the beta-blocking drugs by transboronation is shown to be the method of choice for general application as it affects the detector background signal very little compared to the use of the boronic acid itself. The transboronation reaction can be used for the determination of alprenolol in plasma extracts with a detection limit of 2.5 ng ml-1 using the electron-capture detector. The ultimate sensitivity of the method is limited by the detector background signal resulting from some column decomposition of the transboronation reagent.

Metabolic changes in muscle on long-term alprenolol therapy.

Muscle biopsies from the vastus muscle were taken at rest and immediately after upright bicycle exercise at 50% of the individual VO2max, before and during 6 wk of alprenolol treatment (200 to 400 mg twice daily) in 6 untrained patients with essential hypertension. Resting muscle concentrations (mmole - kg-1 - wet weight) of glycogen, glucose, lactate, and high-energy phosphates [adenosine triphosphate (ATP) and creatine phosphate (CP)] were not affected by alprenolol treatment, but after 10 min after exercise the glycogenolysis increased and depletion of ATP and CP was enhanced. The relationship between blood and muscle lactate was altered by alprenolol, indicating that alprenolol prevents lactate translocation from the muscle to the blood. The results show that during moderate exercise, leg muscle metabolism is influenced by long-term antihypertensive therapy.