In-Syringe Electrokinetic Protein Removal from Biological Samples prior to Electrospray Ionization Mass Spectrometry.

Here, an electrokinetic extraction (EkE) syringe is presented allowing for on-line electrokinetic removal of serum proteins before ESI-MS. The proposed concept is demonstrated by the determination of pharmaceuticals from human serum within minutes, with sample preparation limited to a 5× dilution of the sample in the background electrolyte (BGE) and application of voltage, both of which can be performed in-syringe. Signal enhancements of 3.6-32 fold relative to direct infusion of diluted serum and up to 10.8 fold enhancement, were obtained for basic and acidic pharmaceuticals, respectively. Linear correlations for the basic drugs by EkE-ESI-MS/MS were achieved, covering the necessary clinical range with LOQs of 5.3, 7.8, 6.1, and 17.8 ng mL-1 for clomipramine, chlorphenamine, pindolol, and atenolol, respectively. For the acidic drugs, the EkE-ESI-MS LOQs were 3.1 µg mL-1 and 2.9 µg mL-1 for naproxen and paracetamol, respectively. The EkE-ESI-MS and EkE-ESI-MS/MS methods showed good accuracy (%found of 81 % to 120 %), precision (≤20 %), and linearity (r>0.997) for all the studied drugs in spiked serum samples.

Fluorometric determination of bopindolol and celiprolol in pharmaceutical preparations and biological fluids.

Two sensitive fluorometric methods were developed for the determination of both bopindolol malonate (BOP) and celiprolol HCl (CLP) based on measuring their native fluorescence in methanol and acetonitrile, respectively. For BOP, the fluorescence was measured at 316 nm after excitation at 278 nm. The proposed method was successfully applied to the assay of commercial tablets as well as content uniformity testing. For CLP, the fluorescence was enhanced by the addition of carboxymethylcellulose solution and measured at 455 nm after excitation at 339 nm. The method was successfully applied to the analysis of CLP in tablets and biological fluids. In both methods, interference likely to be introduced from co-formulated, co-administered, or chemically related drugs was studied. The results were statistically compared with those obtained by reference methods and were found to be in good agreement.

Preparation and evaluation of propranolol molecularly imprinted solid-phase microextraction fiber for trace analysis of beta-blockers in urine and plasma samples.

An improved multiple co-polymerization technique was developed to prepare a novel molecularly imprinted polymer (MIP)-coated solid-phase microextraction (SPME) fiber with propranolol as template. Investigation was performed for the characteristics and application of the fibers. The MIP coating was highly crosslinked and porous with the average thickness of only 25.0 microm. Consequently, the adsorption and desorption of beta-blockers within the MIP coating could be achieved quickly. The specific selectivity was discovered with the MIP-coated fibers to propranolol and its structural analogues such as atenolol, pindolol, and alprenolol. In contrast, only non-specific adsorption could be shown with the non-imprinted polymer (NIP)-coated fibers, and the extraction efficiencies of propranolol and pindolol with the MIP-coated fibers were higher markedly than that with the commercial SPME fibers. A MIP-coated SPME coupled with high-performance liquid chromatography (HPLC) method for propranolol and pindolol determination was developed under the optimized extraction conditions. Linear ranges for propranolol and pindolol were 20-1000 microg L(-1) and detection limits were 3.8 and 6.9 microg L(-1), respectively. Propranolol and pindolol in the spiked human urine and plasma samples, extracted with organic solvent firstly, could be simultaneous monitored with satisfactory recoveries through this method.

Evaluation of monolithic packed 96-tips and liquid chromatography-tandem mass spectrometry for extraction and quantification of pindolol and metoprolol in human plasma samples.

96-well pipette tips with a chemically bonded monolithic methacrylate sorbent plug were used for solid-phase extraction (SPE) of pindolol and metoprolol in human plasma samples. The sorbent plug was formed by in situ polymerization. Monolithic packed 96-tips are a tool for miniaturized, solid-phase extraction. Using such packed 96-tips, a 96-well plate could be handled in about 2 min. The key aspect of the monolithic phase is that monolithic material can provide both relatively good binding capacity and relatively low backpressure properties. The validation of the methodology showed that the accuracy values of quality-control samples were between 101% and 103% for metoprolol, while between 94% and 114% for pindolol. The precision ranged from 4% to 15%. The standard calibration curves were obtained within the concentration range 5-5000 nM in plasma samples. The coefficients of determination (R2) for plasma samples were >or=0.99. Our prepared polymer based monolithic packed 96-tips were compared with commercial silica based 96-tips and protein precipitation.

Stereoselective renal clearance of pindolol in humans.

In this study, pindolol, a beta-adrenoceptor blocking agent marketed as a racemic mixture, was used as a model compound to investigate stereoselective renal clearance of organic cations in human beings. Six normal subjects received an oral dose of 20 mg racemic pindolol. Heart rate and blood pressure were measured throughout the study. A stereospecific high performance liquid chromatographic procedure was used to quantitate the concentrations of d- and l-pindolol in plasma and urine. Renal clearance and other pharmacokinetic parameters of both enantiomers were calculated and compared. The renal clearance of l-pindolol was greater than that of d-pindolol in all subjects. The renal clearance (mean +/- SD) was 240 +/- 55 ml/min for l-pindolol and 200 +/- 51 ml/min for d-pindolol (P less than 0.01). Since stereoselective binding to plasma proteins was not observed, differences in renal clearance between d- and l-pindolol were caused by either stereoselective renal transport, or stereoselective renal metabolism. The area under the plasma concentration-time curve, the amount of drug excreted, and the half-life of l-pindolol were greater than those of d-pindolol, which suggests that pindolol was also eliminated stereoselectively by nonrenal routes. The slopes of the resting heart rate vs. the plasma concentration of l-pindolol were significantly less than zero and were significantly correlated to the pretreatment heart rate, which supports the hypothesis that intrinsic sympathetic tone largely determines the effect of pindolol on the resting heart rate. The observation that pindolol is eliminated stereoselectively by the kidney may have clinical implications for other racemic drugs that are renally eliminated.

The influence of hyperthyroidism and hypothyroidism on the beta-adrenergic responsiveness of the turkey erythrocyte.

The mechanisms responsible for altered adrenergic tone in hyperthyroidism and hypothyroidism are not fully understood. To investigate these mechanisms, the beta-adrenergic receptor-cyclic AMP complex of the turkey erythrocyte was studied among groups of normal, hyperthyroid, and hypothyroid turkeys. In erythrocytes obtained from hypothyroid turkeys, there were fewer beta-adrenergic receptors than in normal cells as determined by the specific binding of [(125)I]iodohydroxybenzylpindolol, as well as associated decreases both in catecholamine-responsive adenylate cyclase activity and in cellular cyclic AMP content. In contrast, erythrocytes obtained from hyperthyroid turkeys contained the same number of beta-receptors and had the same catecholamine-responsive adenylate cyclase activity as cells from normal birds. Other characteristics of the beta-receptors in cells from hyperthyroid birds were indistinguishable from those present in normal erythrocytes. However, within the range of circulating catecholamine concentrations, 5-50 nM, the erythrocytes of the hyperthyroid turkeys generated substantially more cyclic AMP after exposure to isoproterenol than did normal cells. These results suggest that thyroid hormone affects beta-receptor-cyclic AMP interrelationships in the turkey erythrocyte by two distinct mechanisms: (a) In hypothyroidism, both beta-receptors and catecholamine-dependent cyclic AMP formation are coordinately decreased; (b) in hyperthyroidism, beta-receptors are unchanged but there is an amplification of the hormonal signal so that occupation of a given number of receptors at physiological concentrations of catecholamines leads to increased levels of cyclic AMP.

Leukocyte beta-receptor alterations in hypertensive subjects.

It has been suggested that beta-adrenergic responsiveness is reduced in hypertension. To evaluate a possible alteration in human beta-receptors that might account for diminished beta-adrenergic responsiveness, we studied leukocytes from hypertensive and normotensive subjects after an overnight rest supine, and then after being ambulatory, a maneuver that increases plasma catecholamines approximately twofold. In supine samples, beta-receptor affinity for the agonist isoproterenol was significantly reduced in hypertensives and was associated with a reduction in the proportion of beta-receptors binding agonist with a high affinity from 42 +/- 6% in normotensive subjects to 25 +/- 2% in hypertensives (P less than 0.05). Alterations in beta-adrenergic-mediated adenylate cyclase activity parallelled the differences seen in the beta-receptor affinity for agonist. In normotensive subjects, beta-receptor density and the proportion of receptors binding agonist with high affinity were reciprocally correlated with plasma catecholamines. However, in the hypertensive subjects these correlations were not evident. Thus, our data suggest an alteration in leukocyte beta-receptor interactions in hypertensive subjects, and may represent a generalized defect in beta-receptor function in hypertension.

Elevation of beta-adrenergic receptor density in human lymphocytes after propranolol administration.

Abrupt withdrawal after the chronic administration of propranolol has resulted in clinical syndromes that suggest adrenergic hypersensitivity. The effect of propranolol administration and withdrawal on beta-adrenergic receptors was studied in human lymphocyte membranes. Receptor density was quantitated by direct binding assays with the radioligand [125I]iodohydroxybenzylpindolol. Administration of propranolol (160 mg/d) for 8 d resulted in trough plasma levels of approximately 35 ng/ml. By day 5 of propranolol administration the density of beta-adrenergic receptors had increased 43 +/- 4% (P less than 0.01) above pretreatment levels. Abrupt withdrawal of propranolol was followed by the disappearance of propranolol from the plasma within 24 h. The density of beta-adrenergic receptors did not return to pretreatment level for several days. Physiologic supersensitivity of beta-adrenergic receptor-mediated responses was suggested by the appearance of significant increases in the orthostatic change in heart rate (P less than 0.05) and the orthostatic change in the heart rate-systolic blood pressure product (P less than 0.01) during the first 48 h after propranolol withdrawal. These data show that propranolol administration leads to an increase in the density of beta-adrenergic receptors in human tissue. The results are consistent with the hypothesis that some of the untoward effects observed after abrupt discontinuation of propranolol are caused by beta-receptor-mediated adrenergic hypersensitivity.

Demonstration of human platelet beta-adrenergic receptors using 125I-labeled cyanopindolol and 125I-labeled hydroxybenzylpindolol.

The radioiodinated pindolol analogs 125I-labeled cyanopindolol ([125I]CYP) and 125I-labeled hydroxybenzylpindolol ([125I]HBP) have been used to study binding to human platelet beta-adrenergic receptors. [125I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (Kd) of 14 +/- 3 pM and maximal binding capacity (Bmax) of 18 +/- 4 fmol/mg protein. Binding of [125I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8 . 10(7) s-1 . M-1 and 3.8 . 10(-4) s-1, respectively. [125I]HBP binds to a saturable class of platelet membrane sites with a Kd of 50 +/- 10 pM and Bmax of 32 +/- 6 fmol/mg protein. [125I]HBP also binds to a saturable class of sites on intact platelets with a Kd of 58 +/- 14 pM and Bmax of 24 +/- 4 molecules per platelet. Binding of [125I]CYP and [125I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (-) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol much greater than isoproterenol greater than epinephrine greater than practolol greater than norepinephrine greater than phenylephrine. These observations indicate that [125I]CYP and [125I]HBP bind to platelet sites which have the pharmacological characteristics of beta-adrenergic receptors but which are not typical of either the beta 1 or beta 2 sub-type.

Is the beneficial antidepressant effect of coadministration of pindolol really due to somatodendritic autoreceptor antagonism?

BACKGROUND: We investigated the combination of selective serotonin reuptake inhibitors (SSRIs) with the beta-adrenoceptor/serotonin 1A (5-HT(1A)) antagonist pindolol, based on the concept that 5-HT(1A) receptor blockade would eliminate the need for desensitization of presynaptic 5-HT(1A) receptors and therefore hasten the onset of action and improve the efficacy of SSRIs. However, since pindolol plasma levels after 2.5 mg three times a day are about 60 nmol/L, and the K(i) for the 5-HT(1A) receptor is 30 nmol/L, it is questionable whether pindolol levels in the brain would be sufficient to antagonize 5-HT(1A) receptors. Using microdialysis in the guinea pig, we correlated brain and plasma levels of pindolol with its capability of augmenting paroxetine-induced increases in brain 5-HT levels. In addition, central beta-receptor antagonism of pindolol was studied by investigating blockade of beta-agonist-induced increases in brain cyclic adenosine monophosphate (cAMP) formation. METHODS: Using microdialysis and jugular vein catheterization, we studied the ability of systemically administered pindolol to antagonize central 5-HT(1A) and beta-adrenoceptors, while simultaneously monitoring pindolol plasma and brain concentrations. RESULTS: Augmentation of paroxetine-induced increases in extracellular 5-HT levels in the ventral hippocampus was only observed at steady state plasma levels exceeding 7000 nmol/L (concurrent brain levels 600 nmol/L). In contrast, antagonism of beta-agonist-induced increases of brain cAMP levels was already observed at pindolol plasma levels of 70 nmol/L (concurrent brain levels < 3 nmol/L). CONCLUSIONS: At plasma levels that are observed in patients after 2.5 mg three times a day ( approximately 60 nmol/L), pindolol produces only a partial blockade of presynaptic 5-HT(1A) autoreceptors and does not augment the SSRI-induced 5-HT increase in the guinea pig brain. It is therefore very unlikely that the favorable effects of combining pindolol with SSRIs, as reported in a number of clinical studies, are due to 5-HT(1A) antagonism. Since pindolol completely blocks central beta-adrenoreceptors at clinically relevant plasma levels, it is possible that beta-adrenoceptor antagonism is involved in mediating pindolol's beneficial effects.

The effect of pindolol on exercise-induced cardiac acceleration in relation to plasma levels in man.

The correlation between the beta receptor blocking activity of pindolol and plasma level was studied in 8 subjects after a 10-mg oral dose. Exercise tachycardia was markedly reduced over a period of at least 6 hr. Significant effects were recorded 30 min after the drug. For each individual there was a close correlation between log plasma level and beta blockade. The regression lines were parallel as shown by analysis of covariance; the intercepts, however, were significantly different. It can be concluded that there is a correlation between plasma level and beta adrenergic blockade by pindolol, but the data failed to establish in different individuals the blood levels necessary to achieve effective adrenergic blockade.

Simultaneous modeling of bopindolol kinetics and dynamics.

Bopindolol has beta-blocking effects for 96 hr despite a 4-hr t1/2. To investigate the concentration-effect relationship after single and repeated doses. 2-mg oral doses were given once and then daily for 13 days to six healthy subjects. In plasma, no unchanged drug, only the hydrolysis product of bopindolol (referred to as bopindolol concentration) was detectable or could be measured up to 24 hr. Chemical assay by HPLC and determination of total active beta-adrenoceptor blocking material by radioreceptor assay gave identical results. The t1/2 was 4 to 5 hr. Effects, measured as reduction in exercise-induced tachycardia (REIT) and as the isoproterenol dose ratio (DR - 1), were followed for 96 hr. The concentration of bopindolol in plasma (predicted with a one-compartment body model) could be related to the measured effects by classic effect models for 20 t1/2s. Parameter estimates for kinetic end effect models did not differ after single and repeated doses. With the parameters from the single-dose experiment, the time course of the plasma concentration and the effects after the multiple-dose experiment could be adequately predicted for 24 and 96 hr. A deep compartment, an active metabolite, or irreversible destruction of the receptor (accounting for the persistence of the effect) could be excluded. The "dissociation constant" of 100 pmol/l (from DR -1/concentration) and the minimal effective plasma concentration (from REIT/log concentration) of 1 pmol/l suggest that enough receptors are occupied at chemically unmeasurable levels in plasma to induce an effect. The "dissociation constant" determined in vivo is of the same order as that from in vitro radioligand studies.

Stereoselective inhibition of pindolol renal clearance by cimetidine in humans.

There are few data on whether differences exist in the renal tubular secretion of enantiomers and no data on whether inhibition of renal secretion of individual enantiomers is stereoselective. Pindolol was used as a probe drug because it is used clinically as a racemic mixture of R-(+) and S-(-) enantiomeric forms and is highly secreted by the proximal tubules of the kidney. Eight young healthy subjects received a single 15 mg oral dose of racemic pindolol with and without 400 mg cimetidine twice daily. The area under the plasma concentration-time curve of both R-(+)- and S-(-)-pindolol were significantly (p less than 0.01) increased by cimetidine from 234 +/- 90 (mean +/- SD) to 344 +/- 78 ng/ml.hr for R-(+)-pindolol and from 209 +/- 73 to 288 +/- 69 ng/ml.hr for S-(-)-pindolol. The renal clearance of R-(+)-pindolol (170 +/- 55 ml/min) was significantly (p less than 0.05) less than that for S-(-)-pindolol (222 +/- 66 ml/min). Cimetidine significantly (p less than 0.01) reduced the renal clearances of R-(+)-pindolol to 104 +/- 18 ml/min and for S-(-)-pindolol to 155 +/- 38 ml/min. The enantiomer with the higher renal clearance [S-(-)-pindolol] had its renal clearance reduced less by cimetidine (26% versus 34%, p less than 0.05). Cimetidine appears to have a stereoselective action on the active transport system for organic cations in the proximal tubule.

Differential occupancy of somatodendritic and postsynaptic 5HT(1A) receptors by pindolol: a dose-occupancy study with [11C]WAY 100635 and positron emission tomography in humans.

Augmentation of selective serotonin reuptake inhibitors (SSRIs) therapy by the 5-HT(1A) receptor agent pindolol may reduce the delay between initiation of antidepressant treatment and clinical response. This hypothesis is based on the ability of pindolol to block 5-HT(1A) autoreceptors in the dorsal raphe nuclei (DRN) and to potentiate the increase in 5-HT transmission induced by SSRIs. However, placebo-controlled clinical studies of pindolol augmentation of antidepressant therapy have reported inconsistent results. Here, we evaluated the occupancy of 5-HT(1A) receptors during treatment with pindolol controlled release (CR) in nine healthy volunteers with Positron Emission Tomography and [11C]WAY 100635. Subjects were studied four times: at baseline, following one week of pindolol CR 7.5 mg/day (4 and 10 hrs post dose), and following one dose of pindolol CR 30 mg(4 hrs post dose). Occupancy of the DRN was 40 +/- 29% on scan 2, 38 +/- 26% on scan 3, and 64 +/- 15% on scan 4. The average occupancy in all other regions was significantly lower at each doses (18 +/- 5% on scan 2, 12 +/- 3% on scan 3, and 42 +/- 4% on scan 4). These results suggest that the blockade in the DRN reached in clinical studies (7.5 mg/day) might be too low and variable to consistently augment the therapeutic effect of SSRIs. However, these data indicate that pindolol exhibits in vivo selectivity for the DRN 5-HT(1A) autoreceptors. As DRN selectivity is desirable for potentiation of 5-HT function, this observation represents an important proof of concept for the development of 5-HT(1A) agents in this application.

Preferential 5-HT1A autoreceptor occupancy by pindolol is attenuated in depressed patients: effect of treatment or an endophenotype of depression?

Using positron emission tomography and the selective 5-HT1A receptor radioligand [11C]WAY100635, we previously demonstrated a preferential occupancy of 5-HT1A autoreceptors, compared to postsynaptic receptors by pindolol in healthy volunteers. We have speculated that preferential occupancy may be clinically important for the purported actions of pindolol in accelerating the antidepressant effects of selective serotonin re-uptake inhibitors (SSRIs). In this study, we have examined the preferential occupancy by pindolol of 5-HT1A autoreceptors, following three different pindolol regimes (10 mg single dose, 2.5 mg t.i.d., and 5 mg t.i.d., in 15 depressed patients on SSRIs. In addition, seven healthy volunteers were examined following a single 10 mg dose of pindolol. We found a preferential occupancy of 22.6+/-7.7% following a single dose of 10 mg of pindolol, in the healthy volunteers, which was attenuated in depressed patients on the same dose of pindolol to 2.9+/-10.8% (Student's t=3.94, df=12, p=0.002). In addition, we found a significant negative correlation between the degree of preferential occupancy and the severity of depression as assessed by the Hamilton depression rating score (HAM-D), Spearman's rho=-0.728, N=14, p=0.003, in the depressed sample. A possible mechanism underlying preferential occupancy and the attenuation of this phenomenon in depressed patients on SSRIs may include changes in the proportion of high affinity 5-HT1A sites in the autoreceptor region of the midbrain raphe. Speculatively, the degree of preferential occupancy may serve as a surrogate marker for depression, or the pharmacological effects of antidepressants.

Scintigraphic portrayal of beta receptors in the heart.

Myocardial beta adrenergic receptors play important roles in physiology and disease, but the receptors have not before been portrayed. The beta antagonist, iodocyanopindolol (ICYP), was used to develop a scintigraphic method for depicting the receptors in the living heart. Labeled with 125I, ICYP bound firmly to beta receptors in the rat heart; the data conformed to a mathematical model. In vivo saturation kinetics indicated binding sites with two affinities. Inhibition of ICYP binding by beta antagonists of different potency and different selectivity for beta-1 and beta-2 receptors produced the expected pharmacologic effects. Inhibition by lipophilic and hydrophilic antagonists gave no evidence that ICYP was appreciably bound to internalized receptors. Fractional binding by tracer quantities of (-) ICYP and (+/-) ICYP demonstrated stereospecificity. Labeled with 123I, ICYP bound to the hearts of intact dogs so that scintigraphic tomographs depicted ventricular myocardium. Small doses of beta antagonists selectively reduced the binding of ICYP to lung enabling better visualization of the heart. Thus, 123I-ICYP appears to portray the beta receptors in the living heart, and the characteristics of binding permit the development of mathematical models and lay the basis for quantifying this receptor binding.

Variability of response to beta receptor blockade for angina pectoris in clinical trials: a study of pindolol.

In a randomized, controlled study we found a significant decrease in pressure-rate (double) product and an improvement in exercise tolerance in patients with angina pectoris treated with pindolol. However, when patients were observed in a long-term double-blind crossover study, pindolol treatment did not produce a significant improvement in angina pectoris or exercise tolerance over placebo. Patients treated with placebo had a gradual improvement in exercise tolerance, possibly because of the training effect of multiple treadmill tests and increased daily walking. The individual patient response to pindolol varied, with some patients showing clinical improvement and decreased double product with exercise and some having no clinical improvement despite a similar decrease in double product. Angina occurred at a lower double product with exercise, suggesting that beta receptor blockade adversely affected other variables of myocardial oxygen demand or decreased coronary blood supply. This study illustrates the difficulties with patient variability and study design that are encountered in studies of beta blocker agents for angina pectoris.

Human platelet beta 2-adrenoceptors: agonist-induced internalisation and down-regulation in intact cells.

1. The effect of isoprenaline (10 microM at 37 degrees C for 30 min) pretreatment on [125I]-(-)-pindolol ([125I]-(-)-Pin) binding to beta 2-adrenoceptors on intact human platelets has been examined. 2. By use of saturation analysis, maximal binding capacity (Bmax) of [125I]-(-)-Pin binding in control and treated cells was assessed in the presence of 1 microM (-)-propranolol or 1 microM (+/-)-CGP 12177 which were taken to represent total or cell surface beta-adrenoceptors respectively. Assay incubations were performed at 37 degrees C and 4 degrees C, the latter to prevent recycling of internalised receptors. 3. Isoprenaline treatment resulted in an identical, highly significant, loss of binding sites (approximately equal to 25%) defined by (-)-propranolol at both assay temperatures as compared to control cells. Binding sites identified in the presence of (+/-)-CGP 12177 were reduced to a much greater extent (approximately equal to 70%), but this was only seen when assays were performed at 4 degrees C. 4. Agonist-induced changes in receptor numbers were concentration-dependent with half maximal receptor loss occurring at an isoprenaline concentration of approximately 2 x 10(-8) M. These effects were inhibited by the presence of a beta-adrenoceptor antagonist and absent if agonist pretreatment was performed at 4 degrees C. 5. Recovery experiments showed that the isoprenaline-induced reduction in total receptor number defined by (-)-propranolol was irreversible whereas the reduction in cell surface receptors defined by (+/-)-CGP 12177 was rapidly reversible (less than 40 min). 6. These data suggest that isoprenaline treatment of intact human platelets causes redistribution of beta 2-adrenoceptors. A proportion are sequestered away from the cell surface (internalised), becoming inaccessible to the hydrophilic ligand (+/-)-CGP 12177. A smaller proportion defined by (-)-propranolol are apparently totally lost from the cell (down regulated).

The binding characteristics of some adrenergic beta-receptor antagonists to human serum proteins.

Binding of pindolol and 8 related compounds was studied in vitro by equilibrium dialysis. The overall binding in serum was compared with the binding to the main, isolated, serum proteins. Most substances show both saturable and non-saturable binding in serum. The saturable and main binding is to alpha 1-AGP, the low non-saturable binding corresponds to albumin and lipoprotein binding. The binding to alpha 1-AGP is characterized by approximately one binding site and association constants K ranging from 10(4) to 10(6) M-1. The binding of pindolol to alpha 1-AGP is strongly inhibited by propranolol, lidocaine, erythromycin, imipramine and TBEP. Significant correlations were found between log NK and log partition coefficient octanol-phosphate buffer suggesting that the protein binding of the 9 adrenergic beta-receptor antagonists to all serum proteins, including alpha 1-AGP, is predominantly hydrophobic in nature.