Binding of phenothiazine neuroleptics to plasma proteins

The binding of chlorpromazine, trifluoperazine, perphenazine, desipramine, propranolol and salicylic acid to human plasma and isolated plasma proteins was studied using equilibrium dialysis. Unlike salicylic acid, an acidic compound only bound to human serum albumin, the basic drugs were bound to all plasma protein fractions studied (albumin, alpha 1-acid glycoprotein, lipoproteins, gamma-globulins) with alpha 1-acid glycoprotein an important binding protein for each of them. The interaction of chloropromazine, perphenazine and trifluoperazine with alpha 1-acid glycoprotein was studied using Scatchard analysis. The primary class of binding sites revealed a low capacity (n = 0.5-1) and a high affinity (K = 10(5)-10(6) M-1) for the phenothiazines. The interaction of chlorpromazine, perphenazine and trifluoperazine with albumin was of the high capacity-low affinity type. In binding studies using plasma obtained from healthy volunteers, alpha 1-acid glycoprotein was found to be a very important binding protein for the basic drug studied with the exception of desipramine. This shows that results derived from binding studies using isolated protein fractions should be interpreted with caution.     

The antimalarial drug halofantrine is bound mainly to low and high density lipoproteins in human serum.

1. The major serum proteins which bind halofantrine were identified by size exclusion chromatography. In addition, the binding affinity of halofantrine to human erythrocytes and serum proteins was measured by an erythrocyte partitioning technique. The influence of serum-drug binding on the distribution of halofantrine in whole blood was estimated by simulating several disease-related changes in the levels of the most important binding proteins. 2. The chromatographic resolution of serum preincubated with halofantrine allowed a quantitative analysis of binding to low density lipoproteins, high density lipoproteins, alpha 1-acid glycoprotein and albumin using the erythrocyte partitioning technique. Very low density lipoproteins did not bind halofantrine to a significant extent. 3. In whole blood halofantrine is bound to serum proteins (83%) and to erythrocytes (17%). Low density lipoproteins (affinity constant nKP = 44.4 l g-1) and high density lipoproteins (nKP = 14.4 l g-1) were the most important binding proteins in serum. alpha 1-acid glycoprotein (nKP = 4.39 l g-1) and albumin (nKP = 0.27 l g-1) had relatively low binding affinities. 4. The concentration of serum proteins influences both the fraction of unbound drug and the fraction of drug associated with the erythrocytes. Changes in serum protein concentrations often encountered in malaria are likely to increase both the unbound fraction and the fraction bound to the erythrocytes.     

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.     

The binding of amsacrine to human plasma proteins

Determination of amsacrine plasma protein binding by both equilibrium dialysis and ultracentrifugation gave similar results and indicated that amsacrine is highly bound (approximately 97%) in human plasma. This binding is independent of amsacrine concentration over the range 1-100 mumol litre-1, but is very sensitive to plasma pH and, to a lesser extent, to temperature. Approximately 20% of the drug appeared to be covalently bound to plasma proteins. Amsacrine was bound by all plasma proteins investigated including albumin, alpha 1-acid glycoprotein and various gamma-globulins. The binding to albumin appeared to occur by two processes, a saturable process at a single site with a KD of 13.9 mumol litre-1 and a non-saturable process. Despite differences in individual protein concentrations, no significant difference was observed in the unbound amsacrine fraction in plasma from patients receiving this drug for treatment of acute myelogenous leukaemia and plasma from healthy individuals.     

Binding in vitro of pipequaline (45319 RP) onto plasma proteins and blood cells in man

Serum binding of pipequaline, a new anxiolytic drug, was studied in vitro by equilibrium dialysis. The percent binding in serum is high, 96.3%, and remains constant within the range of therapeutic concentrations. Investigations performed on isolated proteins with a wide range of concentrations showed one site with a high affinity constant (Ka = 450,000 M-1) for alpha 1-acid glycoprotein and two sites with a lower affinity constant (Ka = 58,000 M-1) for human serum albumin. Binding to lipoproteins was saturable, with an affinity constant of 22,000 less than or equal to Ka less than or equal to 35,000 M-1. Over the range of therapeutic concentrations, the ratio of pipequaline concentrations in serum and red blood cells remained constant (14.4%) and was shown to be dependent on the free fraction of pipequaline in serum.     

The binding protein of erythromycin in human serum

Erythromycin binding to human serum albumin and to alpha 1-acid glycoprotein was measured under conditions of binding equilibrium. At therapeutical concentrations of erythromycin the binding to albumin is not saturable. The fraction of total erythromycin bound to alpha 1-acid glycoprotein is proportionally related to the protein concentration and is bound to a single class of binding sites with an apparent association constant Ka = 0.16 X 10(6) M-1 (38 degrees). About one mole of erythromycin is bound per mole of alpha 1-acid glycoprotein. The binding affinity can be enhanced and vice versa lowered by increasing the concentrations of NaCl and urea, respectively. The semilogarithmic plot of bound/free ratios vs log concentration of NaCl or urea exhibits linear relationships. Erythromycin binding can be competitively inhibited by mersalyl (Ki = 11-16 microM) but not by other SH-reagents or by neuraminidase treatment. A marked reduction of erythromycin binding to alpha 1-acid glycoprotein is seen with dithiothreitol. alpha 1-acid glycoprotein is the main erythromycin binding protein in human serum.     

Binedaline binding to plasma proteins and red blood cells in humans

Serum binding of binedaline, a new antidepressant drug, was studied in vitro by equilibrium dialysis. The percent of binding in serum is high, 99.2%, and remains constant within the range of therapeutic concentrations; no saturation to the binding sites was seen. Investigations performed on isolated proteins with a wide range of concentrations showed one site with a high affinity constant (Ka = 2 X 10(6) M-1) for alpha 1-acid glycoprotein and two sites with a low affinity constant (Ka = 3 X 10(4) M-1) for human serum albumin. Binding to lipoproteins was nonsaturable, with a total affinity constant of 1.25 X 10(5) less than nKa less than 2.79 X 10(6) M-1. Over the range of therapeutic concentrations, the ratio of binedaline concentrations in serum and red blood cells remained constant (1%) and was shown to be dependent on the free fraction of binedaline in serum.     

Differences in the binding of quinine and quinidine to plasma proteins.

1. Little is known about the comparative plasma protein binding of the antimalarial agents quinine (QN) and its isomer quinidine (QD). We have examined the in vitro binding of QN and QD to albumin, alpha 1-acid glycoprotein, normal human plasma, and maternal and foetal umbilical cord plasma. 2. QN was more avidly bound than QD, and binding of both drugs was substantially higher to alpha 1-acid glycoprotein than to albumin, indicating that alpha 1-acid glycoprotein is the more important binding protein. 3. Protein and drug concentration dependent binding was evident for both QN and QD. The unbound fraction of both drugs fell with increasing albumin (10 to 60 g l-1) and alpha 1-acid glycoprotein (0.5 to 2.0 g l-1) concentration, and there was a marked increase in unbound fraction of QN (6 to 19%) and QD (13 to 36%) in human plasma when drug concentrations were increased over the antimalarial therapeutic range (0.5 to 10 mg l-1). 4. In human volunteer plasma, the unbound fractions of QN and QD were 7.5 +/- 2.2% and 12.3 +/- 2.3% respectively, whilst the unbound fractions for both drugs were significantly higher in maternal plasma (QN = 13.0 +/- 5.4%, QD = 18.3 +/- 2.5%) and significantly higher still in foetal umbilical cord plasma (QN = 25.7 +/- 10%, QD = 35 +/- 5.3%).     

Protein Binding of Doxepin and Desmethyldoxepin

Abstract: The protein binding of doxepin (DOX) and desmethyldoxepin (DDOX) were studied in serum and plasma samples from healthy volunteers and psychiatric patients. Binding was measured by equilibrium dialysis (16 hrs at 37°) and drug concentrations by radioimmunoassay. In addition, albumin and α₁-acid glycoprotein concentrations of the samples were measured by radial immunodiffusion. The mean ± SEM percentages of unbound DOX were: 20.4 ± 1.2 and 15.9 ± 1.2 in healthy subjects (n = 16) and patients (n= 15) respectively. and those of DDOX: 21.4 ± 0.9 and 19.0 ± 1.4 for healthy subjects and patients, respectively. There was a significant negative correlation between serum α₁-acid glycoprotein concentration and free fraction of DOX in both groups. In healthy subjects a significant negative correlation was also found between albumin concentrations and free fraction of both DOX and DDOX. Binding experiments with isolated protein fractions revealed that all of the total binding in plasma could be explained by binding to albumin and α₁-acid glycoprotein. The observed 2–4-fold interindividual variability in the free fractions of these drugs is probably less important than the much larger variability in the total serum concentrations.     

Free drug concentration monitoring in clinical practice. Rationale and current status

Recent advances in techniques to determine free drug concentrations have lead to a substantial increase in the monitoring of this parameter in clinical practice. The majority of drug binding to macromolecules in serum can be accounted for by association with albumin and alpha 1-acid glycoprotein. Albumin is the primary binding protein for acidic drugs, while binding to alpha 1-acid glycoprotein is more commonly observed with basic lipophilic agents. Alterations in the concentrations of either of these macromolecules can result in significant changes in free fraction. Diseases such as cirrhosis, nephrotic syndrome and malnourishment can result in hypoalbuminaemia. Burn injury, cancer, chronic pain syndrome, myocardial infarction, inflammatory diseases and trauma are all associated with elevations in the concentration of alpha 1-acid glycoprotein. Treatment with a number of drugs has also been shown to increase alpha 1-acid glycoprotein serum concentrations. A wide variety of biological fluids have been examined for their ability to provide an estimation of free drug concentration at receptor sites. The most useful fluid for estimating free drug concentrations appears to be plasma or serum, with subsequent treatment of the sample to separate free and bound drug by an appropriate technique. The two most widely used methods are equilibrium dialysis and ultrafiltration. Of these two, ultrafiltration has the greatest utility clinically because it is rapid and relatively simple. The major difficulty associated with this method involves the binding of drug to the ultrafilters, but significant progress has been made in solving this problem. Several authors have endorsed the routine use of free drug concentration monitoring. Data examining the clinical usefulness of free drug concentration monitoring for phenytoin, carbamazepine, valproic acid, disopyramide and lignocaine (lidocaine) are reviewed. While available evidence suggests that free concentrations may correlate with clinical effects better than total drug concentrations, there are insufficient data to justify the recommendation of the routine use of free drug concentration monitoring for any of these agents at present.     

Determinants of the plasma protein binding of theophylline in health.

1 The plasma protein binding of theophylline was determined after addition of [14C]-theophylline (15 micrograms/ml) to plasma from 24 healthy drug-free volunteers and equilibrium dialysis for 2 h at 37 degrees C. 2 The percentage of drug unbound was 60.0% +/- 2.2% (s.d.) with very little variation between individuals. The binding ratio of theophylline was not significantly related to the plasma albumin or alpha 1-acid glycoprotein (AAG) concentrations but was significantly, although weakly, negatively related to the logarithm of the non-esterified fatty acid concentration (NEFA) (r = 0.443, P less than 0.05). 3 Intravenous administration of heparin (1000 units) caused a significant rise in plasma NEFA concentration and in the percentage of drug unbound in plasma after equilibrium dialysis. 4 In human serum albumin solutions, the binding ratio of theophylline was significantly related to the albumin concentration and at the albumin concentration seen in the 24 normal subjects, the percentage of drug unbound was almost identical. Addition of AAG in physiological concentrations did not enhance theophylline binding but oleic acid, and to a lesser extent palmitic acid, reduced binding significantly. 5 The percentage of theophylline unbound in plasma varied markedly with pH so that at pH7 the percentage unbound was 52% greater than at pH 8. There was no evidence of concentration dependence of binding up to 140 micrograms/ml theophylline. 6 Theophylline appears to bind almost exclusively to albumin and its plasma protein binding varies little in healthy subjects, showing no concentration-dependence over the therapeutic range of concentrations. The binding is affected by pH and by NEFA concentration, however, and these factors may be of greater importance in disease states. Caution should be employed in the use of heparin in studies of plasma protein binding of theophylline.     

Protein binding predictions in infants

Plasma binding protein levels are lower in the newborn than in the adult and gradually increase with age. At birth, human serum albumin (HSA) concentrations are close to adult levels (75%–80%), while alpha 1-acid glycoprotein (AAG) is initially half the adult concentration. As a result, the extent of drug binding to HSA is closer to that of the adult than are those drugs bound largely to AAG. A model that incorporates the fraction unbound in adults and the ratio of the binding protein concentration between infants and adults successfully predicted the fraction unbound in infants and children.     

Plasma protein binding of sorafenib, a multi kinase inhibitor: in vitro and in cancer patients

Sorafenib is an orally administered multikinase inhibitor that exhibits antiangiogenic and antitumor activity. Few investigators have been able to correlate cumulative sorafenib dose or total exposure to pharmacodynamic effects. This discrepancy may be in part due to poorly understood protein binding characteristics. Since unbound drug concentrations are believed to be more relevant to pharmacological and toxicological responses than total drug, an equilibrium dialysis method using 96-well microdialysis plates was optimized and validated for determining the fraction unbound (F(u)) sorafenib in human plasma and in isolated protein solutions. Unbound sorafenib concentrations were determined in cancer patients receiving the drug orally at a dose of 400?mg and 600?mg twice daily. Sorafenib was extensively bound with mean F(u) value of 0.3% in both non-cancer and cancer patient's plasma. The binding in plasma was concentration independent, indicating a low-affinity, possibly nonspecific and nonsaturable process. In isolated protein solutions, 99.8% and 79.3% of sorafenib was bound to human serum albumin (HSA) (4?g/dL) and α(1)-acid glycoprotein (AAG) (0.1?g/dL) with binding constants of 1.24?×?10(6)?M(-1) and 1.40?×?10(5)?M(-1), respectively. In cancer patients receiving sorafenib, unbound sorafenib was not correlated with patient characteristics or laboratory values. In conclusion, sorafenib is highly protein bound in human plasma with a higher affinity towards albumin and limited free drug may be partly responsible for its borderline clinical activity.     

In vitro binding study of gemfibrozil to human serum proteins and erythrocytes: interactions with other drugs

The binding of gemfibrozil to human serum, isolated proteins and erythrocytes was studied in vitro by equilibrium dialysis. Our results show that this drug is highly bound to serum (99%) at therapeutic levels. Human serum albumin was shown to be mainly responsible for this binding (98.6%) with a saturable process characterized by two binding sites with a moderate affinity. Like many acidic drugs with a carboxylic acidic group, gemfibrozil showed none or negligible binding to alpha 1 acid glycoprotein, lipoproteins and gamma-globulins. The drug binding to erythrocytes is very low (0.8%). The unbound fraction in blood remains constant (0.8%) within the range of therapeutic concentrations. Moreover, interactions were studied with bilirubin and palmitic acid at pathophysiological concentrations and acenocoumarol, salicylic acid, valproic acid, furosemide, phenylbutazone, tolbutamide, warfarin and sulfamethoxazol at therapeutic concentrations. Neither endogenous compounds nor the other drugs studied altered gemfibrozil binding in serum. Likewise, the binding of warfarin to serum and to human serum albumin (600 microM) is not influenced by gemfibrozil.     

The blood binding of cefotiam and cyclohexanol, metabolites of the prodrug cefotiam hexetil, in-vitro.

The binding of cefotiam and cyclohexanol to human serum, isolated proteins and erythrocytes has been studied in-vitro by equilibrium dialysis. The two molecules are 50% bound to serum proteins and the free fraction for both compounds remained constant within the therapeutic concentration range. Human serum albumin (HSA) was exclusively responsible for the cefotiam binding (48%) with a saturable process characterized by one binding site (n = 1.00 +/- 0.14) with a very weak affinity (Ka = 1457 +/- 352 M-1). Like other cephalosporins, cefotiam showed no binding to alpha 1-acid glycoprotein, lipoproteins or gamma-globulins. Cyclohexanol is mainly bound to HSA with a weak affinity (Ka approximately 1,800 M-1) but lipoproteins and alpha 1-acid glycoprotein bind about 30% of bound cyclohexanol in serum. Interactions with free fatty acids (FFA) or bilirubin were studied at physiopathological concentrations. HSA-bound cefotiam was displaced by FFA (1260 microM) and bilirubin (330 microM), whereas the cyclohexanol binding was inhibited only by FFA. The cefotiam binding site seems to be close to the warfarin site (site I) whereas cyclohexanol probably shares the diazepam site (site II) on HSA. There is no mutual inhibition of binding between cefotiam and cyclohexanol at therapeutic levels. The binding of both compounds to erythrocytes is low and restricted when measured in the presence of plasma.     

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).     

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.     

Optical studies on the specific interaction of dipyridamole with alpha 1-acid glycoprotein (orosomucoid)

The interaction of dipyridamole with alpha 1-acid glycoprotein was investigated by circular dichroism and ultraviolet absorbance measurements as well as by equilibrium dialysis experiments. Dipyridamole is bound to the protein via one site of extremely high affinity and by at least one site of considerably lower affinity. Only the association of dipyridamole with the high affinity site produces typical extrinsic Cotton effects. As a result of experimental observations it is concluded that the high affinity site is located in a hydrophobic protein structure of the glycoprotein.     

Pharmacokinetics of intravenously administered 125I-labelled human alpha 1-acid glycoprotein

The volumes of distribution of many acidic drugs have been shown to be close to that of their binding protein, i.e. serum albumin. The distribution of basic drugs mainly bound to alpha 1-acid glycoprotein (AAG) can be questioned with respect to its dependency upon the distribution of this plasma protein. So, a pharmacokinetic study was performed in 7 subjects with human 125I-labelled alpha 1-acid glycoprotein. The steady-state volume of distribution was found to be 5.37 +/- 0.82L. The central volume was 3.23 +/- 0.33L, close to that of plasma volume and the peripheral volume was 2.14 +/- 0.63L. These data allowed the establishment of an equation giving access to the volume of distribution of a basic drug by relating its unbound fraction to physiological distribution of alpha 1-acid glycoprotein. The values yielded by this equation show that the actual and calculated volumes of distribution of basic drugs mainly bound to AAG are discrepant. This protein is thus not the main factor controlling the distribution of basic drugs within the body.     

Auramine O as a fluorescent probe for the binding of basic drugs to human alpha 1-acid glycoprotein (alpha 1-AG). The development of a simple fluorometric method for the determination of alpha 1-AG in human serum

A cationic fluorescent dye, auramine O (AO), exhibited an intense increase in fluorescence after binding to human alpha 1-acid glycoprotein (alpha 1-AG). The interaction between AO and the protein was studied by fluorescence spectroscopy and by equilibrium dialysis. AO binds to the protein via a single site with a dissociation constant of 24 microM. Various basic drugs such as chlorpromazine, imipramine, desipramine, quinidine, propranolol and lidocaine, which are known to bind to the protein, competitively inhibited the AO binding to the protein. The dissociation constants of these basic drugs obtained from such inhibitory experiments were comparable to those obtained with other methods (equilibrium dialysis, quenching of protein intrinsic fluorescence, and the difference spectrophotometric method) and from the literature. It is concluded that AO may be a useful fluorescent probe that binds to a single basic drug binding site on alpha 1-AG. In addition, a simple fluorometric method for the determination of alpha 1-AG in serum was developed using AO, and the validity of this method was confirmed by comparing it with the conventional radial immunodiffusion method.