Variable binding of propranolol in human serum.

Human serum proteins were fractionated by ultracentrifugation and gel filtration. Binding of propranolol was determined by equilibrium dialysis. Propranolol was distributed to lipoproteins independent of drug concentration. Two groups of propranolol binding sites were found to be present in the protein preparation containing albumin, α₁-acid glycoprotein, transferrin and prealbumin. The first binding site with a dissociation constant of 7.5 × 10^?7 was present in number equivalent to concentration of α₁-acid glycoprotein. The propranolol binding to serum samples from 21 healthy males expressed as binding ratio B/F and per cent binding ranged from 7.5 to 19.2 and 88.2 to 95.0 respectively. The binding ratio was correlated to concentration of α₁-acid glycoprotein (r = 0.85, P < 0.001), but not to concentrations of albumin and lipoproteins. The results indicate that α₁-acid glycoprotein is the main propranolol binding protein in human serum.     

Serum protein binding of noscapine: Influence of a reversible hydrolysis

Abstract— The binding of the antitussive drug noscapine to human serum, pure albumin and α₁-acid glycoprotein has been investigated by ultrafiltration and equilibrium dialysis, using radiolabelled noscapine. The binding in serum pooled from volunteers was 93 ± 0.2% (at 100 ng mL^?1). After incubation for 24 h the binding decreased to about 85% (ultrafiltration 87.0 ± 1.0%; equilibrium dialysis 84.3 ± 1.2%), because of the conversion of noscapine to noscapinic acid. Only unbound drug underwent this hydrolysis, and as noscapine is extensively bound in healthy volunteers, this elimination process is probably unimportant. The major binding protein of noscapine was albumin (K = 3060 M^?1, n = 5.62), but the binding to ai-acid glycoprotein was also substantial (K = 31500 M^?1, n = 1.73). The interindividual variation in binding was low and binding was linear at the concentrations observed after therapeutic doses (0–500 ng mL^?1).     

α1-acid glycoprotein involvement in high affinity binding of tricyclic antidepressants to human plasma

The binding of the tricyclic antidepressants imipramine (IMI) and desmethylimipramine (DMI) to human plasma and individual proteins was studied by equilibrium dialysis. Both drugs bound extensively to plasma, albumin, and α₁-acid glycoprotein, while there was very little binding to the γglobulin fraction. The binding of both IMI and DMI to α₁-acid glycoprotein was high affinity (association constant K, 9.2 × 10⁴/M and 4.7 × 10⁴/M respectively) and low capacity (number of binding sites, n = 1 for both IMI and DMI), whereas the binding to albumin was low affinity (K for IMI, 2.3 × 107²/M and for DMI, 3 × 10²/M) and high capacity (n = 7). The binding of IMI to a mixture of human serum albumin and α₁-acid glycoprotein revealed two sets of binding sites; a high affinity binding site corresponding to α₁-acid glycoprotein and a low affinity binding site corresponding to albumin. The binding affinity and/or number of binding sites for IMI binding to albumin decreased with increasing albumin concentrations. The free fraction in plasma of nineteen normal, male controls was significantly correlated with the concentration of α-acid glycoprotein (r = 0.601, P < 0.01), although there was no correlation with albumin or free fatty acid concentrations in plasma.     

Influence of smoking on serum protein composition and the protein binding of drugs1

The influence of smoking on α₁-acid glycoprotein (α₁-AGP) and serum albumin concentrations and the protein binding of phenytoin and propranolol in healthy volunteers was investigated. α₁-AGP concentrations were found to be statistically different (P < 0·05) in the smokers (mean = 84·3 mg dl⁻¹) versus non-smokers (mean = 62·8 mg dl⁻¹). There was a trend for lower serum albumin concentrations and lower fraction unbound of propranolol in the smokers. Smoking did not affect the protein binding of phenytoin.     

Influence of age on serum protein binding of propranolol

Summary The extent of propranolol protein binding was determined in three different age groups of healthy drug-free caucasian males. Volunteers selected for study were 6–15 years old, 25–36 years old and 68–76 years old. Ten milliliters of blood were obtained via venipuncture and collected in glass tubes from the subjects after an overnight fast. Binding determinations were performed by equilibrium dialysis using radiolabelled propranolol. Serum albumin and ₁-acid glycoprotein concentrations were determined in all subjects by radial immunodiffusion. The results obtained showed wide intersubject variability in the binding ratio of propranolol and serum concentrations of ₁-acid glycoprotein. Mean albumin serum concentration was found to be significantly lower in the elderly group as compared to the adult and pediatric groups (p<0.02). A positive correlation was found between the binding ratio of propranolol and the serum concentration of ₁-acid glycoprotein in all the subjects (r=+0.66,p<0.005). No significant correlation was found between the binding ratio of propranolol and the serum concentration of albumin (r=–0.03,p<0.88). These data suggest that the extent of propranolol binding is influenced primarily by serum concentrations of ₁-acid glycoprotein and not by differences in age.     

Binding of [3H]mianserin to bovine serum albumin,human serum albumin and α1-acid glycoprotein

Scatchard plots which were curvilinear with negative slopes were obtained when the binding of [³H]mianserin to bovine serum albumin (BSA), human serum albumin (HSA), defatted human serum albumin (D-HSA) and α₁-acid glycoprotein (α₁-AGP) was studied with equilibrium dialysis with constant protein concentrations and various ligand concentrations. Binding parameters were estimated graphically and with a non-linear least-squares computer program, assuming two classes of independent binding sites. α₁-AGP had the highest binding affinity (K) and binding capacity (nK). The binding parameters, n and K were not independent of protein concentration when the BSA concentration was varied. Linear atypical Scatchard plots with positive slopes were obtained when the protein concentration was varied for BSA, HSA and D-HSA, at a fixed ligand concentration.     

Plasma protein binding of fentanyl

Whole plasma of 15 normal volunteers bound 79·16% ± 0·16 s.e. (n = 45) of fentanyl, at a concentration of 0·6 ng ml^?1. Measurement was by equilibrium dialysis at 37°C, pH 7·4. The largest contribution to binding appears to be due to serum albumin, since 45·52% ± 0·40 s.e. (n = 3) of fentanyl was bound to a solution of purified albumin at a concentration of 46 g litre^?1 in buffer. Physiological concentrations of isolated very low density, low density and high density lipoprotein fractions in buffer bound 18·39% ± 0·65 s.e.; 39·14% ± 0·42 and 21·18% ± 0·51 (n = 15) respectively. A significant correlation was found between percent binding and serum albumin concentration (r = 0·745, P = 0·0022) and oestrogen and progestagen therapy (r = 0·766, P = 0·0014). There was no significant correlation with fasting serum cholesterol, triglyceride, age, sex or the concentration of total protein minus albumin. Binding to fibrinogen and α₁-acid glycoprotein did not occur. Binding increased with increasing pH, temperature and ionic strength of the buffer. The results were compatible with hydrophobic bond formation between fentanyl and proteins. Fentanyl concentration did not affect the percent bound to whole plasma or the protein fractions over a range of 0·6 ng-10 mg ml^?1. Dilution of plasma with buffer gave a linear relation of percent bound or free to log plasma dilution. The binding of fentanyl to pooled plasma of the normal subjects was not affected by a wide variety of anionic, cationic and uncharged drugs when these were tested at a concentration of 20 μg ml^?1. At higher concentrations, aspirin, phenylbutazone and quinidine caused inhibition of fentanyl binding. A linear relation was found between percent bound and concentration of the inhibitory ligand. For aspirin, r = 0·873, P <0·01; for phenylbutazone, r = 0·81, P <0·05; and for quinidine, r = 0·982, P <0·01. Aspirin and phenylbutazone inhibited binding of fentanyl to albumin, while quinidine caused inhibition of binding to lipoproteins of all three densities, but not to albumin. Changes in the concentrations of the common ions of plasma (except H⁺), of free fatty acids and of creatinine did not affect fentanyl binding to whole plasma. 8 M urea reduced binding by 25% of the normal value.     

Extensive Binding of the Bioflavonoid Quercetin to Human Plasma Proteins

Although the bioflavonoids, a large group of polyphenolic natural products, exert chemopreventive effects in cardiovascular disease and cancer, there is little information about the disposition of these dietary components in man. The objective of this study was to investigate the plasma-protein binding of the most abundant bioflavonoid, quercetin, using ¹⁴C-labelled quercetin. An ultracentrifugation assay (170 000 g for 16 h at 20°C) was shown to sediment plasma proteins. Binding of quercetin to normal plasma was extensive (99.1 ± 0.5%, mean ± s.d., n = 5). The unbound fraction varied as much as 6-fold, 0.3–1.8%, between subjects. This high binding was independent of quercetin concentration over the range 1.5–15 μM (0.5–5 μg mL^?1). Human serum albumin was the primary protein responsible for the binding of quercetin in plasma (99.4 ± 0.1%). Binding by α₁-acid glycoprotein (39.2 ± 0.5%) and very-low-density lipoproteins (< 0.5% of total quercetin) did not make substantial contributions to overall plasma binding. The equilibrium association constant for the binding of quercetin to serum albumin was 267 ± 33 times 10³ M^?1 (n=15). Thermodynamic data for the binding of quercetin to serum albumin indicated spontaneous, endothermic association. Displacement studies suggested that in man the ‘IIA’ subdomain binding site of human serum albumin was the primary binding site for quercetin. Association of quercetin with erythrocytes was significantly (P < 0.001) reduced by plasma protein binding. These data indicate poor cellular availability of quercetin because of its extensive binding to plasma proteins.     

Binding of quinidine in sera with different levels of triglycerides, cholesterol, and orosomucoid protein

Serum binding of quinidine was determined in vitro by equilibrium dialysis in sera from twenty-five healthy individuals. The sera had different levels of triglycerides, cholesterol and orosomucoid (α₁-acid glycoprotein), but with small variations in serum albumin concentration. Binding ratio (bound/free) and per cent binding varied from 2.0 to 5.4 and from 67.1 to 84.3% respectively. Binding ratios were linearly related to serum concentration of triglycerides (r = 0.437, P < 0.05), cholesterol (r = 0.400 P < 0.05) and orosomucoid (r = 0.841, P < 0.001), but not to serum concentrations of albumin. When five persons with orosomucoid serum levels exceeding the accepted normal range were excluded, a better linear relation was observed for the triglycerides (r = 0.765, P < 0.001). The observed binding ratios were linearly related to binding ratios calculated from previous observations on binding parameters of lipoprotein and albumin fractions of human sera when all persons were included (r = 0.465, P < 0.05) and when those with high levels of orosomucoid were excluded (r = 0.753, P < 0.001).     

Comparison of the serum protein binding of maprotiline and phenytoin in uraemic patients on haemodialysis

Summary The serum protein binding of maprotiline and phenytoin has been compared in a group of 22 uraemic patients receiving haemodialysis. Determination of protein binding was carried out in vitro using equilibrium dialysis at 37°C and¹⁴C-labelled drug. The mean percentage unbound maprotiline found in patients (10.0%, SD 2.5) was not significantly different from that obtained in healthy volunteers (mean 10.5%, SD 1.0). However, there was a significantly increased variability in binding in patients compared with healthy subjects. The mean percentage unbound phenytoin in the same patients (22.2%, SD 3.3) was significantly greater than that obtained in healthy control subjects (12.5%, SD 0.6). Although there was no correlation between maprotiline and phenytoin binding and serum concentrations of ₁-acid glycoprotein, there was a significant correlation between percentage unbound maprotiline and serum albumin concentrations. The findings indicate that the binding of this tricyclic antidepressant is essentially normal in uraemia, although there may be increased interindividual variability in the free fraction of drug.     

Characteristics of plasma protein binding of tacrine hydrochloride: a new drug for Alzheimer's disease

The aim of this study was to characterise the plasma protein binding of tacrine hydrochloride (THA) in vitro. Binding was assessed in the plasma of 11 healthy individuals aged 20 to 27 years using ultrafiltration followed by HPLC assay.At THA concentrations from 10 to 100 ng/ml protein binding ranged from 78.6 to 71.0%. Binding to commercially available human albumin ranged from 41.7 to 38.3% and to human ₁-acid glycoprotein from 23.1 to 12.4% over the THA concentrations from 25 to 100 ng/ml.THA binding and total plasma protein, plasma albumin and ₁-acid glycoprotein were measured in healthy young subjects (n=13), healthy elderly individuals (n=12) and patients hospitalised with acute illnesses (n=8). There were significant differences between the groups in total plasma protein, plasma albumin and in ₁-acid glycoprotein but no differences in the protein binding of THA which remained constant at about 75%. There was no correlation between THA binding and any plasma protein concentration.The THA binding was not high enough to be of major significance clinically or to reduce the validity of total plasma THA measurement in therapeutic monitoring.     

Effect of age and sex on the plasma binding of acidic and basic drugs

Summary Protein binding of chlorpromazine, propranolol, meperidine, desipramine, salicylic acid and phenytoin was determined in plasma of 64 healthy volunteers (35 males and 29 females). An attempt was made to identify factors affecting the plasma protein binding of these drugs. Whereas plasma albumin levels decreased as a function of age in both sexes, α₁-acid glycoprotein levels increased with age, but the increase was more pronounced in males. The free plasma fraction of the acidic drugs (salicylic acid, phenytoin) and despiramine (a base) showed a significant (p<0.005) negative correlation with plasma albumin levels. The free fractions of the other three basic drugs (chlorpromazine, propranolol, meperidine) in plasma showed a significant (p<0.005) negative correlation with α₁-acid glycoprotein plasma levels. Plasma protein binding of salicylic acid, phenytoin and desipramine decreased as a function of age. Plasma protein binding of chlorpromazine, propranolol and meperidine was virtually unaffected by age or was slightly increased (chlorpromazine). Only in the case of salicylic acid could a statistically significant difference be demonstrated between males and females in the free fraction-age relationship. Stepwise multiple linear regression analysis, including age and blood chemistry values such as hematocrit, bilirubin, cholesterol, triglycerides, creatinine, BUN, albumin and α₁-acid glycoprotein as independent variables, identified age as the variable explaining most of the variability in plasma binding of salicylic acid, phenytoin and desipramine. For chlorpromazine, propranolol and meperidine α₁-acid glycoprotein was the most important determinant of plasma protein binding.     

Effect of heparin on serum binding of propranolol in the acute phase of myocardial infarction

1 Binding of propranolol in vitro was determined in sera from 38 non-fasting patients 4-6 days after an acute myocardial infarction. The binding of propranolol was significantly lower in 12 patients receiving a subcutaneous heparin injection than in 26 patients receiving warfarin.

2 The binding of propranolol in sera from 9 geriatric patients without any acute diseases and 10 young, healthy subjects was similar to that of patients with acute myocardial infarction receiving heparin.

3 Mean serum concentration of α₁-acid glycoprotein was similar in the four different groups of individuals.

4 For all subjects, the binding of propranolol was positively correlated to serum concentrations of α₁-acid glycoprotein.

5 In the patients with myocardial infarction the binding was negatively correlated to serum concentrations of non-esterified fatty acids. Mean serum concentrations of non-esterified fatty acids was significantly higher in the patients receiving heparin than those receiving warfarin.

6 Addition of heparin and palmitic acid to serum in vitro did not affect the binding of propranolol.

7 The effect of heparin on binding variations was observed more closely in three non-fasting patients with myocardial infarction. Serum binding of propranolol and concentrations of chylomicrons and pre-β-lipoproteins were significantly reduced after a subcutaneous heparin injection of 5,000 iu, while serum concentrations of non-esterified fatty acids increased threefold. Concentrations of α₁-acid glycoprotein and albumin were unchanged. Changes in binding of propranolol was closely correlated to concomitant changes in the triglyceride-rich lipoproteins.

8 These observations indicate that propranolol is bound to both triglyceride-rich lipoproteins and α₁-acid glycoprotein in serum. Even smaller doses of heparin activate lipoprotein lipase and decrease binding of propranolol to lipoproteins in non-fasting subjects.

     

Stereoselective protein binding of verapamil enantiomers

The binding of the (+)- and (-)-enantiomers of verapamil (V) to purified albumin (40 g/L), alpha 1-acid glycoprotein (0.55 g/L) and fresh serum has been studied over a wide range of verapamil concentrations (0.055 to 22 microM). The free fraction of the pharmacologically more potent (-)-V was always greater than that of (+)-V. Similar free fractions were observed in solutions of alpha 1-acid glycoprotein ((+)-V 0.079 +/- 0.016; (-)-V 0.142 +/- 0.020) and fresh serum ((+)-V 0.096 +/- 0.009; (-)-V 0.136 +/- 0.006), however the free fraction was higher in a solution of albumin ((+)-V 0.400 +/- 0.030; (-)-V 0.572 +/- 0.029). Saturation of verapamil binding sites was observed for alpha 1-acid glycoprotein only. Enantioselective verapamil serum binding was also noted in samples collected from five healthy volunteers following oral and intravenous verapamil administration. The free fraction of the individual isomers in vitro when added to predose serum as the pseudoracemic drug ((+)-V 0.06 +/- 0.01, (-)-V 0.12 +/- 0.02) was similar to that observed for the enantiomers when studied separately in vitro, indicating that the binding of each enantiomer is independent of the other optical isomer. The free fraction ex vivo after intravenous therapy ((+)-V 0.06 +/- 0.01, (-)-V 0.12 +/- 0.02) was similar to that observed in vitro in that subjects pre-dose serum. The free fraction of both enantiomers, however, was higher after oral drug therapy ((+)-V 0.13 +/- 0.02, (-)-V 0.23 +/- 0.03). The lower binding noted may be a result of competition for serum binding sites by verapamil metabolites, which attain higher concentrations following oral dosing.     

Binding of Prazosin to α1-Acid Glycoprotein and Albumin: Effect of Protein Purity and Concentrations

Abstract: Prazosin is extensively bound in human serum/plasma. In the present study a bound fraction of 93–95% was observed at 37° for therapeutic drug concentrations. Both α₁-acid glycoprotein (AAG) and albumin (HSA) are established as transport proteins for prazosin, but their individual contribution to the extent and variability of protein binding in serum/plasma is unclear. The present study showed that AAG possesses one binding site per molecule with high affinity (Kd≈0.8 μM) for prazosin. HSA, essentially globulin-free, bound prazosin with lower affinity (Kd≈30 μM) with an average of 0.3 binding sites per molecule. However, less purified HSA, containing globulins, exhibited apparently higher affinity (Kd≈8 μM), but lower binding capacity (0.07 sites per molecule) for prazosin. In mixtures of highly purified proteins, the concentrations of AAG, and not HSA, determined the extent and variability of prazosin binding.     

Protein binding of piritramide: influence of various protein concentrations and the postoperative acute phase response

Objective: Piritramide is a lipophilic opioid, which is widely used for postoperative analgesia and analgosedation in Europe. In this study we investigated the influence of various protein concentrations (total protein, ₁-acid glycoprotein, albumin) and the postoperative acute phase response on the protein binding of piritramide. Methods: The influence of various protein concentrations on the protein binding of piritramide was investigated by either diluting the serum samples of five volunteers with isotonic saline or by adding different amounts of ₁-acid glycoprotein. Albumin binding was measured in a 5% human albumin solution. The impact of the postoperative acute phase response was investigated by obtaining daily serum samples from 18 surgical patients until the third postoperative day, and measuring piritramide protein binding, ₁-acid glycoprotein, total protein and albumin. Results: There was a significant relationship between piritramide protein binding and the concentrations of total protein and ₁-acid glycoprotein. The binding to albumin was 88%. During the postoperative acute phase response, the protein binding of piritramide did not change. Serum concentrations of ₁-acid glycoprotein increased, whereas total protein and albumin decreased. Conclusion: Although there were significant changes in the piritramide-binding proteins, ₁-acid glycoprotein and albumin, during the postoperative acute phase response, the protein binding of piritramide did not change. Therefore, a change in protein binding, which might be one factor to be considered in determining piritramide dosage in the postoperative period, does not have to be taken into account. Received: 2 June 1998 / Accepted in revised form: 6 October 1998     

In vitro protein binding behavior of dipyridamole

The in vitro protein binding behavior of dipyridamole in plasma and buffered protein solutions was investigated by equilibrium dialysis. The drug was highly protein bound (approximately 98%) in heparinized human plasma, and the extent of protein binding remained constant for drug concentrations over the range of therapeutic interest of 0.1-10 micrograms/mL. Comparable binding results were obtained with a mixture of 80 mg % of alpha 1-acid glycoprotein and 40 g/L of human serum albumin in pH 7.4 phosphate buffer solution. Pure alpha 1-acid glycoprotein (80-400 mg %) or pure human serum albumin (40 g/L) in phosphate buffer gave significantly (p less than 0.05) lower binding results, indicating that both proteins are responsible for the high binding of dipyridamole in plasma. Addition of alpha 1-acid glycoprotein to heparinized human plasma, to simulate an acute phase increase in the protein, had no effect on the fraction of free drug in plasma. Binding of dipyridamole to heparinized human plasma or human serum albumin in buffer was concentration independent through 40 micrograms/mL. The free fraction of dipyridamole increases with concentrations exceeding 40 micrograms/mL.     

Plasma protein binding of alpidem in healthy volunteers,in neonates and in liver or renal insufficiency

Summary The binding of alpidem, a new anxiolytic drug, has been studied in plasma from 6 healthy subjects, 12 patients with renal failure, 12 patients with liver cirrhosis and 12 chronic uraemics maintained on haemodialysis, as well as in 12 serum samples from the placental cord, to represent the situation in the newborn.The unbound fraction was 0.61% (healthy volunteers), 1.31% (newborns), 0.86% (cirrhotic patients), 0.72 (patients with renal failure), 0.70% (before haemodialysis) and 0.79% (after haemodialysis). Binding in the volunteers was significantly different from that in neonates and cirrhotics only.Alpidem became bound to isolated albumin (45 g·l^–1) and alpha₁-acid glycoprotein (0.75 g·l^–1) to 97.2% and 97.1%, respectively. The bound fraction of the drug in a mixture of two proteins was 99.1%.For alpidem, it appears that alpha₁-acid glycoprotein may balance the effect of any decrease in the albumin concentration.     

Variation in serum binding of tertatolol mediated by disease-induced modification of alpha-acid glycoprotein concentration

Summary The serum concentrations of ₁-acid glycoprotein (AAG), albumin (HSA) and non-esterified fatty acids, and the serum binding of tertatolol were measured in four groups of individuals: healthy control subjects (n=24), and patients with inflammation (n=28), and hepatic (n=20) and renal (n=27) insufficiency.Serum binding of tertatolol was increased in patients with inflammation (94.6%), decreased in patients with hepatic insufficiency (88.8%) and it was unchanged in patients with renal insufficiency (92.8%) as compared to controls (92.7%).Multivariate analysis indicated that the changes were mainly related to concomitant changes in AAG concentration, which could account for 57% of intersubject variability in the bound/free ratio, and to a lesser extent in HSA, which accounted for only 4% of the variability in the binding.The data show that the free fraction of the basic drug tertatolol in serum is affected by pathological conditions that cause changes in AAG concentration.     

Gallopamil binding to human serum proteins

Summary We have determined the extent and variability in the binding of gallopamil to human serum proteins. Binding was determined by equilibrium dialysis in healthy volunteer serum, human serum albumin (45 g·l^–1), and alpha-1 acid glycoprotein (AAG) (600 mg·l^–1) at a pH of 7.4.Nonlinear regression analysis of gallopamil binding over a wide range of concentrations (10^–9 to 10^–4 M) in healthy volunteer serum suggested two classes of binding sites (k_ass.1=4.7×10⁵M^–1, k_ass.2=4.1×10⁴M^–1). These values were in close agreement with those obtained from binding to AAG and human serum albumin.Gallopamil free fraction over the concentration range of 10 to 100 ng·ml^–1 was independent of concentration. The free fraction in 20 volunteers was 0.075 at a concentration of 10 ng·ml^–1. Gallopamil free fractions were also determined in human serum albumin, to which various concentrations of AAG were added. Bound/free ratios correlated with AAG.As we changed the pH of the serum from 7.0 to 8.0, the free fraction changed from 0.1 to 0.05.Verapamil, lignocaine, procainamide, propranolol, 40H-propranolol, MEGX, and NAPA all caused an increase in the free fraction of gallopamil in serum. However, tocanide, quinidine, diltiazem, GX, norverapamil, D620, D617, and desacetyl diltiazem had no effect on gallopamil binding.Therefore, the data strongly suggest AAG as the high affinity, low capacity binding site and albumin as the low affinity, high capacity binding site for gallopamil, variability in gallopamil binding can be explained by alterations in AAG concentrations, pH, and the presence of other drugs and their metabolites.Presented in part at the 88th Annual Meeting of the American Society for Clinical Pharmacology and Therapeutics, March 1987, Orlando, Florida, USA