Determination of free disopyramide plasma concentrations using ultrafiltration and enzyme multiplied immunoassay

Disopyramide is an antiarrhythmic drug that exhibits nonlinear binding to plasma proteins. As a result, the total body clearance increases with increasing total drug plasma concentration. A rapid and sensitive method for the determination of free (unbound) disopyramide plasma concentrations is described. The procedure employs an ultrafiltration system (Centrifree), which can be used for basic drugs, along with an enzyme multiplied immunoassay system (EMIT) for the measurement of free disopyramide concentrations in plasma water filtrate. The EMIT method was adapted to permit measurement of disopyramide in plasma over a concentration range of 0.02-1.2 micrograms/ml. Plasma storage at -20 degrees C, filtration volume, or the presence of buffer and mono-N-dealkylated metabolite in plasma did not affect the binding determinations. There was no loss of drug during the filtration process. A good correspondence was found between the EMIT assay and a high performance liquid chromatography method, when applied to plasma samples obtained from a human subject who had ingested disopyramide. Furthermore, the extent of protein binding determined by the ultrafiltration system and by equilibrium dialysis were in good agreement. The binding of disopyramide in fortified human plasma decreased from 64 to 52% over a total drug concentration range of 1-5 micrograms/ml.     

Protein binding of ceftriaxone: comparison of three techniques of determination and the effect of 2-hydroxybenzoylglycine, a drug-binding inhibitor in uremia

Ceftriaxone is a third-generation cephalosporin exhibiting a long half-life and a concentration-dependent protein binding. This study compared three techniques of protein binding determination (equilibrium dialysis chamber, ultrafiltration cones (Centriflo), and ultrafiltration (Centrifree micro-partition system) on human plasma and serum at ceftriaxone concentrations achieved clinically. A second objective was to determine the effect of 2-hydroxybenzoylglycine (HBG) on the protein binding of ceftriaxone. High performance liquid chromatography (HPLC) and liquid scintillation counting assays were used. Equilibrium dialysis was rotated for 12 h. The supplier's recommendations were followed for ultrafiltration techniques. The plasma protein binding of ceftriaxone, as determined by equilibrium dialysis and assayed by HPLC, decreased from 98.6 to 73.5% for drug concentrations varying from 25 to 400 micrograms/ml. Somewhat lower values were obtained with Centrifree, the binding fell from 92.1 to 73.5% for the same concentration range. Serum protein binding was similar to results obtained with plasma samples. Centriflo cones yielded more inconsistent results. A significant difference was seen between the three techniques (p less than 0.0001, three-way analysis of variance). The addition of HBG, a compound that inhibits drug binding in uremia, resulted in ceftriaxone binding defects similar to those seen in uremic serum. Although equilibrium dialysis remains a classic method of protein binding determination, Centrifree appears to be a better system.     

Protein binding of propisomide

This paper describes the protein binding of propisomide to human serum and isolated proteins using equilibrium dialysis. The drug is exclusively bound to alpha1-acid glycoprotein with high affinity. The binding is saturable even at low concentrations of the drug. Thus, the fraction unbound varied from 0.05 to 0.60 with decreasing serum concentration. The major metabolite of the drug or other drugs with affinity for alpha1-acid glycoprotein can displace propisomide from its binding site only when present in serum at high levels. Two ultrafiltration techniques are compared to equilibrium dialysis for the determination of serum protein binding of propisomide. Ultrafiltration does not give reliable results. Equilibrium dialysis is retained as an accurate method for the determination of the fraction unbound of propisomide.     

Comparison of the erythrocyte partitioning method with two classical methods for estimating free drug fraction in plasma

A modification of the erythrocyte partitioning method for the rapid estimation of plasma-free drug fractions (f_u) is described and applied to five basic drugs. In the procedure, which uses readily available clinical laboratory equipment, f_u is calculated from measurements of durg partitioning between plasma and erythrocytes, and between buffer and erythrocytes. Results obtained are compared with those from equilibrium dialysis or ultrafiltration techniques for amitriptyline, imipramine, quinidine, lidocaine, and propranolol. For each drug, the mean value of f_u obtained with the erythrocyte partitioning procedure was not found to be significantly different from that determined by one of the two other classical techniques. The erythrocyte partitioning method lead to reproducible (mean C.V. = 6·25) and precise values of f_u when compared to the other methods; its clinical application to lidocain gave results which agreed with those obtained by equilibrium dialysis.     

Impact of pH on plasma protein binding in equilibrium dialysis

Many pharmacokinetic analyses require unbound plasma concentrations, including prediction of clearance, volume of distribution, drug-drug interactions, brain uptake analysis, etc. It is most often more convenient to measure the total drug concentration in plasma rather than the unbound drug concentration. To arrive at unbound plasma concentrations, separate in vitro determinations of the plasma protein binding of a drug are usually carried out in serum or in plasma, and the plasma pharmacokinetic results are then mathematically adjusted by this fraction unbound ( f u,p). Plasma protein binding or the drug fraction unbound in plasma ( f u,p) is known to be affected by protein, drug, free fatty acid concentrations, lipoprotein partitioning, temperature, pH, and the presence or absence of other drugs/displacing agents within plasma samples. Errors in f u,p determination caused by lack of adequate pH control in newer assay formats for plasma protein binding (e.g., 96-well equilibrium thin walled polypropylene dialysis plates) will have significant drug-specific impact on these pharmacokinetic calculations. Using a diverse set of 55 drugs and a 96-well equilibrium dialysis plate format, the effect of variable pH during equilibrium dialysis experiments on measured values of f u,p was examined. Equilibrium dialysis of human plasma against Dulbecco's phosphate buffered saline at 37 degrees C under an air or 10% CO 2 atmosphere for 22 h resulted in a final pH of approximately 8.7 and 7.4, respectively. The ratio of f u,p at pH 7.4 (10% CO 2) vs pH 8.7 (air) was >or=2.0 for 40% of the 55 compounds tested. Only one of the 55 compounds tested had a ratio <0.9. Select compounds were further examined in rat and dog plasma. In addition, physicochemical properties were calculated for all compounds using ACD/Labs software or Merck in-house software and compared to plasma protein binding results. Changes in plasma protein binding due to pH increases which occurred during the equilibrium dialysis experiment were not species specific but were drug-specific, though nonpolar, cationic compounds had a higher likely hood of displaying pH-dependent binding. These studies underscore the importance of effectively controlling pH in plasma protein binding studies.     

Determination of the extent of protein binding of antibiotics by means of an automated continuous ultrafiltration method

The extent of the protein binding of a drug is an important pharmacokinetic parameter. In this study, the determination of the protein binding of the antibiotics gatifloxacin, moxifloxacin, linezolid and telithromycin to bovine serum albumin (BSA) and human serum albumin (HSA) was performed by means of an automated continuous ultrafiltration method. The continuous ultrafiltration has several advantages compared to classical methods like equilibrium dialysis or discontinuous ultrafiltration. The method is appropriate for a fast determination of the extent of the protein binding of a drug. In one single experiment the calculation of the protein binding over a wide range of different drug/protein ratios is possible. Comparing the results obtained with the continuous ultrafiltration with binding data reported in literature shows good correlation and proves the reliability of the method.     

Impact of recovery on fraction unbound using equilibrium dialysis

Historically, recovery had been used to evaluate the data quality of plasma protein binding or tissue binding obtained from equilibrium dialysis assays. Low recovery was often indicative of high nonspecific binding, instability, or low solubility. This study showed that, when equilibrium was fully established in the dialysis assay, low recovery due to nonspecific binding had no impact on the determination of fraction unbound. The conclusion was supported by the principles of the equilibrium dialysis assay, experimental data, and mathematic simulations. The results suggested that the use of recovery as an acceptance criterion for the equilibrium dialysis assay in drug discovery was too restrictive, and introduced the additional burden of repeating studies unnecessarily.     

High-throughput screening of drug-brain tissue binding and in silico prediction for assessment of central nervous system drug delivery

A high-throughput method for rapid screening of in vitro drug-brain homogenate binding is presented. The method is based on a straightforward sample pooling approach combining equilibrium dialysis with liquid chromatography mass spectrometry (LCMS). A strong correlation of fraction unbound in brain (fu) between single compound measurements and 25-pooled compounds (R2 = 0.906) was obtained for a selection of structurally diverse CNS compounds with a wide range of fractions unbound. Effects of brain homogenate dilution and dialysis time were investigated. To the best of our knowledge, it was the first time that we have demonstrated consistent fraction unbound in mouse and rat brain homogenate, revealing the drug-tissue partitioning mechanism predominated by hydrophobic interaction. On the basis of this finding, a generic approach to estimate drug binding to various tissues is proposed. A robust and interpretable QSAR for fu prediction is also presented by statistical modeling.     

High-performance liquid chromatographic analysis, plasma protein binding and red blood cell partitioning of phenprobamate

A new high-performance liquid chromatographic procedure for the analysis of phenprobamate, a skeletal muscle relaxant in biologic fluids was developed. The method used a C18 reverse phase column, a mobile phase of methanol/acetonitrile/water (33:15:52), and UV detection at 215 nm. The assay procedure was applied to the determination of phenprobamate binding to rat and human plasma proteins using the equilibrium dialysis method. In addition, the red blood cell/plasma partitioning was determined in the whole blood of rats and humans. Phenprobamate exhibited a moderate binding to plasma proteins of rat (74.3 +/- 2.2 per cent) and human (80.5 +/- 1.1 per cent). The protein binding was concentration-independent in the range of 10 to 80 micrograms ml-1. Phenprobamate binding to plasma proteins was also determined in the presence of 10 micrograms ml-1 acetaminophen. The protein binding of phenprobamate was not significantly altered by acetaminophen (74.4 +/- 0.6 per cent for rat plasma; 75.7 +/- 1.6 per cent for human plasma). The distribution ratios of phenprobamate between the red blood cells and plasma were greater than unity, 1.86 and 1.59 in rat and human, respectively, indicating a preferential partitioning of the drug in the red blood cells.     

Validation of 96-well equilibrium dialysis with non-radiolabeled drug for definitive measurement of protein binding and application to clinical development of highly-bound drugs

Definitive plasma protein binding (PB) studies in drug development are routinely conducted with radiolabeled material, where the radiochemical purity limits quantitative PB measurement. Recent and emerging regulatory guidances increasingly expect quantitative determination of the fraction unbound (Fu) for key decision making. In the present study, PB of 11 structurally- and therapeutically-diverse drugs spanning the full range of plasma binding was determined by equilibrium dialysis of non-radiolabeled compound and was validated against the respective definitive values obtained by accepted radiolabeled protocols. The extent of plasma binding was in agreement with the radiolabeled studies; however, the methodology reported herein enables reliable quantification of Fu values for highly-bound drugs and is not limited by the radiochemical purity. In order to meet the rigor of a development study, equilibrium dialysis of unlabeled drug must be supported by an appropriately validated bioanalytical method along with studies to determine compound solubility and stability in matrix and dialysis buffer, nonspecific binding to the dialysis device, and ability to achieve equilibrium in the absence of protein. The presented methodology establishes an experimental protocol for definitive PB measurement, which enables quantitative determination of low Fu values, necessary for navigation of new regulatory guidances in clinical drug development.     

Determination of protein binding byin vitro charcoal adsorption

Certain compounds such as SC-52151 have extensive nonspecific adsorption to the ultrafiltration devices or to dialysis membranes and therefore can not be measured by the conventional ultrafiltration or equilibrium dialysis methods. A new method based on charcoal adsorption was developed to overcome this difficulty. Unlike many conventional methods, which are based on the separation of free drug from bound drug under equilibrium conditions, the new method is operated under nonequilibrium conditions and involves measuring the time course of decline of the percentage of bound drug remaining in plasma while the free drug is being removed by charcoal adsorption. Theoretical aspects of the method and the data processing procedure are presented. SC-98A, a compound with minimal nonspecific adsorption to the ultrafiltration membrane, was used to demonstrate the applicability of this method against the ultrafiltration method. Using this method, the protein binding of SC-52151 in human plasma at 1.0 μg/ml was determined to be in the range of 91.4–97.7% at room temperature.     

The binding of paracetamol to plasma proteins of man and pig

The binding of N-acetyl-4-aminophenol (paracetamol) to human and porcine plasma at both toxic and therapeutic concentrations was investigated by ultrafiltration and equilibrium dialysis over the range 50–300 μg ml^?1. Plasma protein binding occurred at paracetamol concentrations greater than 60 μg ml^?1. The extent of protein binding at a plasma concentration of 280 μg ml^?1 of the drug is between 15 and 21% for both pig and man. There is no appreciable binding to erythrocytes in either species over the whole concentration range studied.     

Plasmolysis, red blood cell partitioning, and plasma protein binding of etofibrate, clofibrate, and their degradation products

Etofibrate (I), the ethylene glycol diester of clofibric and nicotinic acids, degrades almost equally through both half-esters with half-lives of approximately 10 and 1 min in fresh dog and human plasma, respectively. The nicotinate V degrades with half-lives of approximately 12 hr and 50 min in fresh dog and human plasma, respectively. Ester III and clofibrate VI degrade by saturable Michaelis-Menten kinetics in fresh human plasma, with similar maximum initial rates and respective terminal first-order half-lives of 12 and 26 min. Tetraethyl pyrophosphate at 100 micrograms/ml inhibited human plasma and red blood cell esterases permitting plasma protein binding and red blood cell partitioning studies. The red blood cell-plasma water partition coefficient was 5.4 for 0.2-80 micrograms/ml of I. Clofibrate (VI) showed a saturable erythrocyte partitioning that decreased from 7.8 (10 micrograms/ml) to 1 (50 micrograms/ml). The strong binding of I and VI to ultrafiltration membranes necessitated the determination of their plasma protein binding by the method of variable plasma concentrations of erythrocyte suspensions to give 96.6% (0.2-80 micrograms/ml) and 98.2% (13.6-108.4 micrograms/ml) binding, respectively. Methods for the determination of the parameters of saturable and nonsaturable plasma protein binding for unstable and membrane-binding drugs by the method of variable plasma concentrations in partitioning erythrocyte suspensions are presented.     

Serum protein binding of a new oral cephalosporin, CL 284,635, in various species

CL 284,635 is a new third generation oral cephalosporin. Its serum protein binding was investigated in the human, monkey, dog, rat, and rabbit. This study was performed by using an equilibrium dialysis and ultrafiltration method, using radiolabeled and cold CL 284,635. In humans, CL 284,635 was found to have a mean free fraction [fu = concentration of unbound (free) drug divided by total concentration of unbound plus bound to serum proteins] of 31.3 +/- 3.3% with no serum concentration dependency in a range of 0.5 to 26 micrograms/ml. The drug was mainly bound to albumin. In rabbits and monkeys the protein binding profile of CL 284,635 was found to be 36.1 +/- 2.3% and 33.9 +/- 1.5% with no serum concentration dependency. In rats and dogs a non-concentration-dependent fu was observed at serum concentrations ranging from 0.5 to 30 micrograms/ml. A gradual increase in fu values was observed at higher serum concentrations of CL 284,635. Overall, the protein binding profile of CL 284,635 was found to be different in the five investigated species. The protein binding of CL 284,635 in monkeys and rabbits was most similar to that in humans. These species differences in protein binding may have an impact on the disposition of the drug in different species.     

Validation of a rapid equilibrium dialysis approach for the measurement of plasma protein binding

Equilibrium dialysis (ED) is one of the most frequently used approaches to investigate drug binding, where the major drawbacks are the time to reach equilibrium (varying between 6 and 24 h), a long assay preparation time and complexity of automation. A rapid equilibrium dialysis (RED) device has recently become commercially available (Pierce Biotechnology, ThermoFisher Scientific, Waltham, MA) offering the potential for reduced preparation and equilibration times. The RED device comprises a Teflon base plate which holds up to 48 disposable dialysis cells. Each dialysis insert is made up of two side-by-side chambers separated by a vertical cylinder of dialysis membrane with a high membrane surface area-to-volume ratio. An independent validation of the RED approach for the measurement of human plasma protein binding (PPB) was carried out as a comparative analysis with standard ED evaluating equilibration time, assay reproducibility and accuracy and ease of use. Using a diverse set of 18 commercially available drugs spanning a range of physicochemical properties we have shown this to be a robust and accurate methodology, with a shorter preparation and dialysis time, capable of being automated as a high-throughput assay for the determination of PPB.     

Determination of Drug Binding to Plasma Proteins Using Competitive Equilibrium Binding to Dextran-Coated Charcoal

A method for determination of drug binding to plasma proteins, which is based on the comparison of drug affinities to plasma proteins and dextran-coated charcoal, is described. The method is free of nonspecific binding feature. The fractions unbound obtained by the charcoal method are in good agreement with values from a traditional ultrafiltration method for various low and highly bound compounds. The method presently requires drug concentrations much less than that of plasma proteins. A possibility of using the method to determine protein binding at an arbitrary drug concentration is discussed. A mechanism of nonspecific binding of drug to ultrafiltration membranes, which yields a good agreement with experimental observations, is suggested.     

Equilibrium Gel Filtration to Measure Plasma Protein Binding of Very Highly Bound Drugs

For very highly bound drugs (fu < 2%), the determination of the unbound fraction in plasma (fu) and a reliable estimation of protein-binding differences across species, populations, or concentrations is challenging. The difficulty is not mostly assay sensitivity but rather experimental bias. In equilibrium gel filtration (EGF)—opposite to the commonly used methods—the amount bound at a set-free concentration is determined. Therefore, signals and differences are bigger for more highly protein-bound drugs. We describe here a new experimental set-up developed to investigate binding in plasma and compare results with those obtained with standard methods for nine Novartis compounds. The method was then applied for two drugs for which it was challenging to obtain precise data with standard methods: midostaurin and siponimod. Despite the very high binding (fu ≤ 0.1%), precise estimation of up to 10-fold species differences relevant for safety assessments was possible. Evidence for the correctness of the data by comparison with other pharmokinetics parameters is provided. Sensitivity to potential sources of experimental bias is compared with standard methods and advantages and disadvantages of the methods are discussed. In conclusion, EGF allows accurate determination of fu for very highly bound drugs and differentiation even above 99.9% of binding.     

Compilation of 222 drugs' plasma protein binding data and guidance for study designs

The binding of a drug to plasma protein reduces free drug in the blood circulation that would otherwise be available for penetration into tissues to reach the therapeutic target or the kidney for elimination. Therefore, the binding event affects drug elimination from the body, efficacy, duration of action and toxicity. Co-administration of other drugs, food and pathological conditions of patients can significantly change percentage binding of the drug and result in serious consequences. Here, we present the largest and newest information on plasma protein binding for 222 drugs, of which 50% show 90-100% binding, a range that could be considered as a favorable element for future lead selection. We also provide critical and comprehensive evaluations on the methods and techniques established to determine plasma protein binding, pinpoint advantages and pitfalls of individual approaches, and offer detailed guidance for experimental designs, including ultrafiltration, equilibrium dialysis, ultracentrifugation, charcoal adsorption, high-performance affinity chromatography, high-performance frontal analysis, solid-phase microextraction and in vivo microdialysis.     

Determination of the free fraction and relative free fraction of drugs strongly bound to plasma proteins

This report describes a new method for the determination of protein binding and relative protein binding (ratio of f(u) for different species) for compounds strongly bound to proteins. The method used is based on the distribution of the drug in plasma water, plasma proteins, and blood cells. Incubations were performed in diluted plasma. In diluted plasma, the erythrocyte/plasma distribution was determined with greater precision than in undiluted plasma. Formulae were derived for calculating f(u) in undiluted plasma based on the f(u) values determined in diluted plasma. These formulae are also valid in the event of more than one independent binding site in plasma. All incubations with plasma of different species were performed using rat erythrocyte suspensions, thereby making it possible for relative f(u) values in different species to be calculated without knowing the absolute free fractions. This method avoids the determination of the erythrocyte/buffer distribution in cases where it is sufficient to know relative f(u) values (e.g., exposure comparisons). Relative protein binding can also be quantified for compounds that tend to adsorb to surfaces of vials or test tubes, thus avoiding errors caused by adsorption when quantifying the drug in a protein-free aqueous solution. This method was validated by making comparisons of free fraction values obtained by the method herein described with those obtained by either ultrafiltration or equilibrium dialysis for two compounds that bind predominantly to albumin and another compound that binds to alpha(1)-acid glycoprotein. The results confirm our method produces identical free fractions in comparison with other established techniques. In addition, the range of applications of our method is much wider.     

Binding of gefitinib, an inhibitor of epidermal growth factor receptor-tyrosine kinase, to plasma proteins and blood cells: in vitro and in cancer patients

Summary Gefitinib exhibits wide inter-subject pharmacokinetic variability which may contribute to differences in treatment outcome. Unbound drug concentrations are believed to be more relevant to pharmacological and toxicological responses than total drug. Thus it is desirable to determine gefitinib binding in plasma and factors affecting this process. An equilibrium dialysis method using 96-well microdialysis plates was optimized and validated for determining the fraction unbound (fu) gefitinib in human plasma. Gefitinib binding in plasma from four different species and isolated protein solutions as well as drug partitioning in human blood cells were investigated. Unbound gefitinib plasma concentrations were measured in 21 cancer patients receiving daily oral gefitinib 250 mg or 500 mg. It was found that gefitinib was extensively bound in human rat mouse and dog plasma with mean fu values of 3.4%, 3.8%, 5.1% and 6.0% respectively. In isolated protein solutions approximately 90% and 78% of gefitinib was bound to human serum albumin (HSA) (40 mg/dL) and alpha1-acid glycoprotein (AAG) (1.4 mg/dL) with binding constants of 1.85 × 10⁴ M⁻¹ and 1.13 × 10⁵ M⁻¹ respectively. In whole blood 2.8% of gefitinib existed as the free drug while 79.4% and 17.8% was bound to plasma proteins and blood cells respectively. In plasma from cancer patients fu at pre-treatment varied 2.4-fold (mean 3.4 ± 0.6%; range 2.2–5.4%) and fu was constant over the 28-days of treatment (P > 0.05). Pre-treatment AAG concentration was negatively correlated with pre-treatment fu (R² = 0.28, P = 0.01). In conclusion gefitinib is highly protein bound (∼ 97%) in human plasma. Variable AAG concentrations observed in cancer patients may affect gefitinib fu with implications for inter-subject variation in drug toxicity and response.