The effects of age and smoking on the plasma protein binding of lignocaine and diazepam.

In 63 healthy ambulant subjects 18 to 88 years of age, the plasma protein binding of diazepam (principally bound to albumin) decreased with age. Diazepam binding in plasma correlated positively with plasma albumin concentration which also decreased with age. In contrast, the plasma protein binding of the basic drug, lignocaine (predominantly bound to alpha 1-acid glycoprotein [AAG]), tended to increase slightly with age. Lignocaine binding in plasma correlated positively with plasma AAG concentration which also increased slightly with age. Smoking did not affect the plasma protein binding of diazepam or lignocaine or the plasma concentrations of albumin, AAG or nonesterified fatty acids. These results suggest that age-related changes in plasma protein binding of lignocaine and diazepam are determined in part by age-related changes in the concentrations of the binding proteins in plasma. The ageing process alone causes only small changes in the plasma protein binding of these drugs compared with the effect of disease states, however.     

The plasma protein binding of metoclopramide in health and renal disease.

The plasma protein binding of metoclopramide was measured after addition of the drug (60 ng ml-1) to plasma from 18 patients with renal disease and 18 age and sex matched healthy individuals. The mean free fraction in renal disease (0.59 range 0.41-0.71) was not significantly different from controls (mean 0.6 range 0.56-0.69). In both groups the binding ratio of metoclopramide was significantly related to plasma alpha 1-acid glycoprotein (AAG) concentration but not to albumin or plasma non-esterified fatty acids concentration. Metoclopramide bound to human serum albumin (HSA) to a limited extent and to human AAG to a greater extent indicating that AAG is the major binding protein for the drug in plasma.     

Evaluation of in vitro drug interactions with karenitecin,a novel,highly lipophilic camptothecin derivative in phase II clinical development

The objective of this study was to describe the potential metabolism and protein-binding interactions with karenitecin, a novel computer-engineered, highly lipophilic camptothecin. Individual cloned cytochrome P450 (CYP450) isoenzymes were used to determine, in vitro, the metabolism of karenitecin. Known substrates and inhibitors of each isoenzyme were employed to evaluate CYP450 drug interactions with karenitecin. To assess the extent, variability, and role of various drug-binding proteins, the authors examined, in vitro, the effects of both albumin (Alb) and alpha-acidic glycoprotein (AAG) on karenitecin plasma protein binding (PPB). Equilibrium dialysis techniques were used to measure the free fraction of karenitecin in the presence of varying ratios of Alb and AAG. Artificial plasma, spiked with karenitecin, was dialyzed for 72 hours at 37 degrees C against a Sorensen's buffer solution using regenerated cellulose membranes having a molecular weight cutoff of 12 to 14 kDa. Additional protein-binding experiments were conducted to assess the potential PPB drug interactions between karentiecin and other highly protein-bound drugs commonly used in the treatment of cancer patients. In vitro experiments suggested that karenitecin is metabolized by CYP450 3A4, 2C8, and 2D6 isoenzymes and is an inhibitor of the CYP450 3A4 and 2C8 isoenzymes. The mean (+/- SD) percentage of karenitecin bound to plasma proteins was 99.1% +/- 0.27%. The extent of karenitecin protein binding was directly proportional to the plasma concentration of AAG. Protein-binding displacement interactions were observed in the in vitro experiments with phenobarbital, phenytoin, mitoxantrone, and salicylic acid. It was concluded that karenitecin has the potential to alter CYP450 3A4 and 2C8 drug-metabolizing activity. In addition, in vitro PPB evaluations have demonstrated that karenitecin may displace other highly PPB drugs and that slight variations in plasma AAG concentration may result in large variations in free drug exposure. Each of these interactions could potentially result in increasing the toxicity or alter the efficacy of combination anticancer drug therapy if they are significant in patients. Future karenitecin clinical trials should include studies to monitor or evaluate the effects of these potential drug interactions on the overall toxicity of karenitecin when used in combination with other drugs.     

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.     

Relationship between alpha 1-acid glycoprotein and plasma binding of disopyramide and mono-N-dealkyldisopyramide.

Highly purified serum albumin did not bind either disopyramide (DP) or mono-N-dealkyldisopyramide (MND). The unbound fraction of DP and MND in highly purified serum alpha 1-acid glycoprotein (AAG) at 0.5 g/l was 57 and 62 and at 2.0 g/l 19 and 30% respectively. Unbound DP and MND were measured in spiked plasma (10 mumol/l of DP or MND), from 60 patients, having AAG concentrations varying from 0.4 to 3.0 g/l. Unbound drug varied from 13 to 58 and from 24 to 62% for DP and MND, respectively, and was inversely related to the plasma concentration of AAG (r = -0.9016, r = -0.9157). A linear relationship was found between the binding ratio (moles bound divided by moles unbound) and the plasma concentration of AAG for both DP (r = 0.9199) and MND (r = 0.9270), whereas no relationship was found between the binding ratios of DP or MND and the plasma concentrations of total protein, albumin, haptoglobin, alpha 1-antitrypsin or the immunoglobulins IgG, IgA or IgM. In patients on DP maintenance therapy, a linear relationship was found between percent unbound DP and the plasma concentration of DP in samples with similar AAG concentrations. Furthermore, a linear relationship was found between the binding ratio of DP and the plasma concentration of AAG in samples with similar DP concentrations. The present findings support the concept that AAG is the major serum protein responsible for the binding of DP and MND.     

The acute changes in serum binding of disopyramide and flecainide after myocardial infarction

Summary In the serum basic drugs are principally bound to alpha₁-acid glycoprotein (AAG). Following acute myocardial infarction it has been shown that the levels of AAG rise. The serum levels of total protein, albumin, AAG and the protein binding of 2 antiarrhythmic drugs which are bases, disopyramide and flecainide, was measured in vitro with blood samples from eleven patients taken over the first 5 days following myocardial infarction. Mean AAG levels significantly increased from 1.04 g/l on Day 1 to 1.80 g/l on Day 5. The binding of disopyramide, which is highly bound, rose from 80% to 87%, representing a 35% decrease in free drug concentration. In contrast the binding of flecainide fell from 61% to 53%, a 20% increase in free drug concentration. These data suggest that although the binding of strongly bound drugs responds appropriately to increases in binding protein after acute myocardial infarction, poorly bound drugs are displaced from binding sites possibly by endogenous substances. Since the pharmacological effects of a drug are related to its free (unbound) concentration, the changes in the proportions of free to bound drug after myocardial infarction may have important clinical implications.     

Serum binding of ketoconazole in health and disease

The plasma protein binding of ketoconazole, an oral antifungal agent of a weak basic nature, was measured after the addition of the drug (10 micrograms.ml-1) to serum from 35 healthy individuals, ten patients with chronic renal disease and seven patients with hepatic cirrhosis. The percentage of free ketoconazole was markedly increased in patients with chronic renal disease and in patients with hepatic cirrhosis, when it was compared with the group of healthy volunteers (7.33 +/- 0.11 in renal patients; 6.12 +/- 1.43 in hepatic patients compared with 2.93 +/- 0.12 in healthy individuals). The binding ratio of ketoconazole in health and disease was significantly related to plasma albumin concentration, but not to plasma alpha 1-acid glycoprotein (AAG) concentration. Moreover, ketoconazole binds to isolated human serum albumin in a greater proportion but does not bind to isolated AAG indicating that human serum albumin is the major binding protein for this drug in plasma.     

Displacement of lidocaine from serumα 1-acid glycoprotein binding sites by basic drugs

Since little is known of the number and types of binding sites on alpha 1-acid glycoprotein (AAG) and because drug-drug protein binding interactions often fail to fit a simple model, a study of the effect of 9 known AAG binding drugs on lidocaine free fraction (LFF) was performed. Serum was obtained from 10 healthy males, pooled and various concentrations (from 0.15 to 1000 micrograms/ml) of amitriptyline, bupivacaine, chlorpromazine, disopyramide, imipramine, meperidine, nortriptyline, propranolol and quinidine were added. LFF was determined by equilibrium dialysis at an initial lidocaine concentration of 2.0 micrograms/ml. LFF increased from 0.30 +/- 0.019 (mean +/- SD) in the absence of displacing agents to maximum values ranging from 0.59 (nortriptyline) to 0.73 (bupivacaine). Plots of LFF vs. the logarithm of displacing drug concentration yielded simple sigmoidal curves in all cases. LFF was increased 50% by an initial bupivacaine concentration of 6.0 micrograms/ml with all other drugs requiring more than 10 micrograms/ml to increase LFF to that extent. Lidocaine binding in a 4.5 g/dl albumin solution was unaffected by concentrations of quinidine, meperidine, nortriptyline and bupivacaine up to 200 micrograms/ml. Addition of AAG to serum reduced LFF as expected. A plot of the reciprocal of bound drug concentration vs. the reciprocal of free drug concentration in the presence and absence of quinidine suggested a competitive binding interaction. These data indicate that the binding interactions between lidocaine and the various displacing compounds are not significantly complicated by cooperative effects and that, with the possible exception of bupivacaine, displacement of lidocaine by any of these drugs is likely to be of clinical significance.     

Propranolol disposition in the rat: variation in hepatic extraction with unbound drug fraction.

The plasma protein binding of propranolol has been described as nonrestrictive for its hepatic extraction to explain the observation that propranolol is efficiently removed by the liver, in spite of extensive protein binding. The present study was designed to examine the relationship between propranolol protein binding, metabolism by isolated hepatocytes, and extraction by the isolated perfused rat liver. In isolated hepatocytes, the intrinsic clearance of free drug increased three- to fourfold as albumin and alpha 1-acid glycoprotein (AAG) concentrations increased, suggesting that albumin and AAG facilitate the elimination of propranolol by hepatocytes. In the isolated perfused liver, propranolol extraction was almost complete (E = 0.996) in the absence of albumin and AAG. With 40 g/L of albumin and 2 g/L of AAG in the perfusate, the free fraction of propranolol decreased to 0.031, but extraction remained high (E = 0.960). With 40 g/L of albumin and 10 g/L of AAG in the perfusate, the free fraction further decreased to 0.014 and extraction dropped sharply (E = 0.820). The observed relationship between propranolol extraction and the free fraction of propranolol was in good agreement with that predicted using estimates of intrinsic clearance measured in isolated hepatocytes suspensions. These data indicate that propranolol extraction is sensitive to changes in binding at very low free fraction values and suggest a facilitation of propranolol clearance by albumin and AAG.     

Determinants of carbamazepine and carbamazepine 10,11-epoxide binding to serum protein, albumin and alpha 1-acid glycoprotein

The binding of carbamazepine and carbamazepine 10,11-epoxide to serum, albumin and alpha 1-acid glycoprotein (AAG) was determined and compared at drug concentrations ranging from 0.5 to 400 mg/l using equilibrium dialysis and liquid chromatography. The total binding of carbamazepine in serum was determined primarily by albumin and to a lesser extent (20-30%) by AAG. Modified Scatchard plots for carbamazepine binding in serum were biphasic, suggesting the presence of two binding sites on serum protein. Association constants characterizing the first (k1 = 2.4 X 10(4) l/mol) and second (k2 = 4.6 X 10(2) l/mol) binding sites agreed with those measured for AAG and albumin respectively. Modified Scatchard plots for carbamazepine 10,11-epoxide binding in serum were linear and serum binding was largely accounted for by binding to albumin. The epoxide metabolite did not bind to AAG. Carbamazepine binding to AAG was drug concentration-dependent over the concentration range considered to be therapeutic, while the percent binding values for carbamazepine and epoxide binding to albumin and serum from a normal individual were constant over this range. Computer simulations showed that physiological extremes in AAG and albumin concentrations can result in a range of carbamazepine unbound fractions of 0.17 to 0.47. These data suggest that normal variations in concentrations of both proteins may be the principal cause of interpatient variability in serum protein binding of carbamazepine.     

Identification, characterization, and implications of species-dependent plasma protein binding for the oral Hedgehog pathway inhibitor vismodegib (GDC-0449)

Vismodegib (GDC-0449) is is an orally available selective Hedgehog pathway inhibitor in development for cancer treatment. The drug is ≥95% protein bound in plasma at clinically relevant concentrations and has an approximately 200-fold longer single dose half-life in humans than rats. We have identified a strong linear relationship between plasma drug concentrations and α-1-acid glycoprotein (AAG) in a phase I study. Biophysical and cellular techniques have been used to reveal that vismodegib strongly binds to human AAG (K(D) = 13 μM) and binds albumin with lower affinity (K(D) = 120 μM). Additionally, binding to rat AAG is reduced ～20-fold relative to human, whereas the binding affinity to rat and human albumin was similar. Molecular docking studies reveal the reason for the signficiant species dependence on binding. These data highlight the utility of biophysical techniques in creating a comprehensive picture of protein binding across species.     

The significance of plasma protein binding on the fetal/maternal distribution of drugs at steady-state

Maternal and fetal plasma differ in their concentrations of the important drug binding plasma proteins, albumin and alpha 1-acid glycoprotein, with albumin being slightly more concentrated in fetal plasma, and alpha 1-acid glycoprotein being only 37% of the maternal concentration at term. In general, these differences relate linearly to the bound to free concentration ratio of drugs associated with these proteins. Although only the free concentration is generally considered to be the pharmacologically active form, these differences can dramatically affect the total concentration and relative distribution of drugs between maternal and fetal plasma. In order to test our hypothesis that plasma protein binding is the major determinant of fetal/maternal drug distribution at steady-state, we examined whether fetal binding could be predicted from adult binding information. Data from studies of maternal plasma protein binding were used to predict fetal plasma protein binding based solely on the differences in protein concentrations. These predictions were compared with observed fetal binding data. This analysis showed a slope near unity and a high correlation (r2 = 0.900) which implies that there are no significant differences between the binding affinities of these proteins. A similar analysis performed using data on drug binding in non-pregnant adults gave an r2 or 0.971. Having established that fetal plasma proteins bind drugs similarly to their maternal counterparts, fetal/maternal plasma drug concentration ratios (F/M) were predicted for various drugs using information from literature on the drug's adult plasma protein binding, the protein to which it binds, and the fetal and maternal plasma concentrations of that binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of prazosin and propranolol at variable alpha 1-acid glycoprotein and albumin concentrations.

1. The effect of variable alpha 1-acid glycoprotein (AAG) and albumin (HSA) concentrations on the binding of prazosin and propranolol was assessed in plasma after surgery and in mixtures of AAG/HSA with concentrations mimicking those found in vivo. 2. On the pre-operative day the binding of prazosin and propranolol was 94.8% and 89.0%, respectively and 97.3% and 93.2%, respectively, 5 days after surgery. 3. In solutions containing mixtures of highly purified AAG and HSA representing the pre-operative state, 88.6% and 83.9% binding of prazosin and propranolol was observed, whereas for solutions mimicking post-operative plasma, the equivalent values were 94.6% and 91.4%, respectively. 4. The ratios between bound and unbound concentrations of both drugs were closely correlated to the concentrations of AAG, but not to the concentrations of HSA. 5. The present study demonstrates that AAG is responsible for the binding variability of prazosin and propranolol in plasma from post-operative patients.     

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.     

Predicting Drug Binding to Human Serum Albumin and Alpha One Acid Glycoprotein in Diseased and Age Patient Populations

Plasma protein binding, namely the fraction unbound (f_u), can be an important determinant of the disposition and response of drugs. The primary objective of this study was to predict f_u values of 183 drugs utilizing either a single binding protein model, where the predominant binding protein had been established, or a multiple binding protein model (MBPM), where the relative binding contribution of human serum albumin (HSA) or alpha 1 acid glycoprotein (AAG) is known. Mean protein concentrations, dependent on disease or age, were used to account for changes in f_u. A simple scaling approach for binding protein concentration was employed to account for quantitative changes in molar concentrations of either HSA or AAG in their respective conditions. The MBPM predictive model works best if the relative binding contribution of HSA and AAG is known, and a scaler for the change in protein concentration can be adjusted accordingly. The value of MBPM was most evident when considering reported changes in lidocaine binding because of increasing AAG concentration in response to trauma. The present approach enhances the ability to predict f_u in diseased and age populations because of quantitative changes in major binding proteins.     

Protein binding of nifedipine

The protein binding of nifedipine in concentrations up to 1200 ng ml-1 has been measured in serum, pure human albumin solution and pure human alpha 1-acid glycoprotein (AAG) solutions by ultrafiltration. The drug was extensively bound in serum from four healthy volunteers with a mean (+/- s.d.) fraction bound of 0.992 +/- 0.008. In albumin solution (40 g litre-1) the mean (+/- s.d.) fraction bound 0.970 +/- 0.012, was not significantly different (P greater than 0.05) from that in serum, suggesting that albumin is the major, but not necessarily the only, binding protein for nifedipine in serum. The binding of nifedipine in solutions of AAG was proportional to the AAG concentration and ranged from 0.514 +/- 0.059 to 0.755 +/- 0.035 in solutions containing 50 and 150 mg % AAG, respectively. Binding of nifedipine in all protein solutions was linear.     

The effect of age on serum concentrations of albumin and alpha 1-acid glycoprotein.

1. Human serum albumin (HSA) concentrations and alpha 1-acid glycoprotein (AAG) concentrations were measured in 68 subjects, 35 males and 33 females, aged 20-90 years without evidence of acute or chronic inflammatory disease or malignancy. Subjects were drug free for at least 1 month. HSA and AAG concentrations were measured using rate nephelometry. 2. Age had no effect on alpha 1-acid glycoprotein concentration, whereas plasma albumin levels decreased as a function of age in both sexes. We observed no differences between males and females in the plasma concentrations of HSA and AAG. 3. These data show that in healthy subjects the HSA concentration decreases with increasing age, whereas age, uncomplicated by disease does not influence AAG concentration.     

Plasma protein binding of drugs in pregnancy and in neonates

Plasma protein binding of drugs has important implications for drug disposition and action since it is the first, and controlling, step in drug distribution. Physiological changes in pregnancy include significant changes in plasma composition which affect drug binding and subsequent drug response; the extent of these changes depends on the stage of gestation. Both albumin and alpha 1-acid glycoprotein fractions are reduced, and consequently the binding of both acidic and basic drugs may be affected. This may lead to difficulties in maintaining adequate plasma concentrations of highly protein-bound drugs, since the measurement of total drug concentration in plasma may no longer be a valid indicator for dose adjustment. The newborn infant displays a continually changing plasma profile. The presence of fetal proteins and endogenous substrates known to interfere with drug binding can lead to unexpected complications due to a higher than expected 'free' drug fraction. Furthermore, a decrease in the affinity of albumin for bilirubin during this period may lead to bilirubin displacement by drugs such as diazepam, sulphonamides and salicylate, resulting in clinical jaundice which would not occur beyond the neonatal period. Plasma composition and its effect on drug binding should be taken into account when prescribing highly protein bound drugs with narrow therapeutic: toxic ratios.     

Use of plasma proteins as solubilizing agents in in vitro permeability experiments: correction for unbound drug concentration using the reciprocal permeability approach

The purpose of the present study was to explore the applicability of the reciprocal permeability approach to correct for changes in thermodynamic activity when in vitro permeability data are generated in the presence of plasma proteins. Diazepam (DIA), digoxin (DIG), and propranolol (PRO) permeability was assessed in the presence of bovine serum albumin (BSA) and bovine alpha-1-acid glycoprotein (AAG). The reciprocal permeability approach was subsequently employed to calculate the true permeability coefficient (Papp(corr)) and the operational protein association constant (nK(a)). For BSA binding, good agreement was observed between the Papp(corr) values and Papp values obtained in the absence of protein. For PRO and AAG, where binding affinity was high, deviation in the reciprocal permeability plots was evident suggesting ligand depletion at low drug/high protein concentrations. Bidirectional DIG permeability data in the presence of either BSA or AAG indicated that neither protein had an effect on the efflux transporters involved in DIG permeability. The data suggest that plasma proteins can be utilized in permeability experiments with no adverse effects on transporter function and that the reciprocal permeability approach can be used to correct permeability data for changes in unbound drug concentration.     

Binding of prazosin to alpha 1-acid glycoprotein and albumin: effect of protein purity and concentrations

Prazosin is extensively bound in human serum/plasma. In the present study a bound fraction of 93-95% was observed at 37 degrees for therapeutic drug concentrations. Both alpha 1-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 approximately 0.8 microM) for prazosin. HSA, essentially globulin-free, bound prazosin with lower affinity (Kd approximately 30 microM) with an average of 0.3 binding sites per molecule. However, less purified HSA, containing globulins, exhibited apparently higher affinity (Kd approximately 8 microM), 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.