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.     

Improved procedure for the determination of protein binding by conventional equilibrium dialysis

The binding of drugs to plasma proteins has been studied extensively using a variety of methods, including equilibrium dialysis. Published information on controls used in these studies is frequently inadequate; in other cases, there are deficiencies in the experimental design for the controls. A method is described that eliminates many of the problems associated with artifactual errors in dialysis studies. Multiple replicated controls are performed at the same time as the test, under identical conditions. The controls are used to correct for concentration-dependent binding of drug to the membrane or other equipment. The method was used to determine the binding of sulfadimethoxine to CF-IV-1 alpha-globulin at therapeutic concentrations. The level of binding was low (9-13%), but the stringent control technique permitted statistical analysis which showed each mean test value to be significantly different from its corresponding control. Furthermore, there was a linear relationship between the control-corrected percentage binding values and total drug concentration, whereas there was no correlation between total drug concentration and the uncorrected percentage binding values.     

Plasma protein binding of phenol in dogs and rats as determined by equilibrium dialysis and ultrafiltration

Plasma-protein binding of phenol was examined in rats with and without ether anesthesia. Ether anesthesia decreased the percentage of phenol bound by rat plasma. The phenol binding capacities of dog and rat plasma were compared using equilibrium dialysis and ultrafiltration. For the wide range of phenol concentrations studied, dog plasma had a consistently higher binding capacity than rat plasma as demonstrated by both techniques. Higher binding was obtained by ultrafiltration than by equilibrium dialysis in both dog and rat plasma. Dog and rat serum albumins were responsible for 72–73% of the phenol binding in plasma, while bovine globulins either did not bind phenol or had only minimal binding capacities.     

Factors influencing the protein binding of azosemide using an equilibrium dialysis technique

Various factors most likely to influence the plasma protein binding of azosemide to 4% human serum albumin (HSA) were evaluated using equilibrium dialysis at the initial azosemide concentration of 10 μg mL^?1. It took approximately 8h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% dextran (the ‘buffer’) using a Spectra/Por 2 membrane (molecular weight cut-off 12000–14000) in a water bath shaker kept at 37°C and a rate of 50 oscillations min^?1. Azosemide was fairly stable both in 4% HSA and in the ‘buffer’ for up to 24h. The binding of azosemide to 4% HSA was constant (95.5 ± 0.142%) at azosemide concentrations ranging from 5 to 100 μg mL^?1. However, the extent of binding was dependent on HSA concentration: the values were 88.4, 91.0, 92.2, 94.2, 94.9, 94.9, and 94.9% at albumin concentrations of 0.5, 1, 2, 3, 4, 5, and 6% respectively. The binding was also dependent on incubation temperature; the binding values were 97.0, 94.9, and 94.9% when incubated at 6, 28, and 37°C, respectively. The binding of azosemide was also influenced by buffers containing various chloride ion concentrations and buffer pHs. The binding values were 95.3, 94.9, and 93.6% for the chloride ion concentrations of 0, 0.249, and 0.546%, respectively, and the unbound values were 6.8, 5.1, 3.8, 3.4, and 3.3% for buffer pHs of 5.8, 6.4, 7.0, 7.4, and 8.0, respectively. The binding of azosemide was independent of the quantity of heparin (up to 40 UmL^?1), AAG (up to 0.16%), sodium azide (NaN₃, up to 5%), its metabolite, Ml (up to 10 μg mL^?1), and anticoagulants (EDTA and citrate).     

Vancomycin serum protein binding determination by ultrafiltration

Sixty-two serum concentrations were obtained from 12 infected patients enrolled in a vancomycin pharmacokinetic study. Both unbound and total serum vancomycin concentrations were measured using ultrafiltration and a commercial fluorescent polarization immunoassay. Ultrafiltrates were obtained by centrifugation at 1000 X g for ten minutes at room temperature and their assay indicated a range in protein binding from 7.9 to 71 percent. The mean protein binding (mean +/- SD) was 41.95 +/- 14.15 percent. No measurable adsorption of vancomycin onto the ultrafiltration membrane was noted. Orthogonal regression of unbound versus total vancomycin concentrations was described by the equation y = 0.597x-0.362 with a correlation coefficient of 0.948.     

In vitro plasma protein binding determination of flunarizine using equilibrium dialysis and liquid chromatography-tandem mass spectrometry

A highly sensitive method was developed and validated for determining the free fraction of flunarizine in human plasma. Equilibrium dialysis was used for the separation of free (unbound) drug and liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used for quantitation. Post-dialysis plasma or buffer samples of 0.2 mL were extracted using a liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Supelco Supelcosil ABZ+Plus column, ionized using a positive ion atmospheric pressure electrospray ionization source, and analyzed using multiple reaction monitoring. The ion transitions monitored were m/z 405-->203 for flunarizine and m/z 409-->207 for flunarizine-d4 (internal standard, IS). The chromatographic run time was 3.5 min per injection, with retention times of 2.1 min for both flunarizine and IS. The calibration curve for flunarizine was linear over the concentration range of 0.25-2000 ng/mL (r(2)>0.9989) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v) with the lower limit of quantitation of 0.25 ng/mL. The inter-assay coefficient of variability (CV) for the quality control samples was less than 13.5%, and the inter-assay percent nominal was greater than 98.2%. In vitro protein binding of flunarizine was determined at concentrations of 5, 10 and 100 microg/mL using the validated method. Flunarizine was extensively bound to plasma protein with a 0.083+/-0.005% overall percent free drug in plasma and a CV value less than 7.8%. This validated method will be used for the ex vivo assessment of flunarizine protein binding in human plasma from a drug-drug interaction clinical study.     

Development and validation of a 96-well equilibrium dialysis apparatus for measuring plasma protein binding

A 96-well equilibrium dialysis block was designed and constructed that is compatible with most standard 96-well format laboratory supplies and instruments. The unique design of the dialysis apparatus allows one to dispense and aspirate from either or both the sample and dialysate sides from the top of the apparatus, which is not possible with systems currently on the market. This feature permits the investigator to analyze a large number of samples, time points, or replicates in the same experiment. The novel alignment of the dialysis membrane vertically in the well maximizes the surface-to-volume ratio, eliminates problems associated with trapped air pockets, and allows one to add or remove samples independently or all at once. Furthermore, the design of the apparatus allows both the sample and dialysate sides of the dialysis well to be accessible by robotic systems, so assays can be readily automated. Teflon construction is used to minimize nonspecific binding of test samples to the apparatus. The device is reusable, easily assembled, and can be shaken in controlled temperature environments to decrease the time required to reach equilibrium as well as facilitate dissolution of test compounds. Plasma protein binding values obtained for 10 diverse compounds using standard dialysis equipment and the 96-well dialysis block validates this method.     

A pressure ultrafiltration method for serum free drug determination. Comparison with equilibrium dialysis using normal and uraemic sera

The clinical methods most frequently used for serum free drug determinations are equilibrium dialysis and ultrafiltration. Both methods have usually been considered equal when comparisons have been made on normal sera. The purpose of the study was to develop a reliable and rapid method for serum free drug determinations especially for disease states where there is an increased free fraction. This modification of the pressure ultrafiltration method proved to meet these demands. The method was tested in vitro by adding radiolabelled phenytoin in different concentrations to normal and uraemic sera. The within-run and between-run variation coefficients were both under 5%. In comparison with equilibrium dialysis the values obtained by the ultrafiltration method were 0.65% lower (p less than 0.001, Student's paired t-test) in normal and 0.37% lower (non-significant) in uraemic sera. There are some theoretical grounds to presume that the results obtained by means of the pressure ultrafiltration method would better reflect the real values than those obtained by means of equilibrium dialysis.     

Comparative evaluation of equilibrium dialysis methods employing biological and artificial membranes for the determination of protein binding of drugs

The goal of the study was to investigate comparatively the performance of the conventional equilibrium dialysis method using artificial membranes (AED) and an alternative equilibrium dialysis method employing biological membranes of red blood cells (BED). The following criteria were employed for an assessment of the two methods: (a) mean estimate of the fraction of drug unbound in plasma, (b) precision, and (c) time required for establishing equilibrium dialysis. For this purpose, plasma protein binding data by AED and BED obtained for several compounds in our laboratory were employed. In addition, suitable results of further compounds on the plasma protein binding by AED and on the partitioning in red cell buffer and plasma systems were collected from the literature, allowing a calculation of the plasma protein binding by BED. Plasma protein binding values by AED and BED were available for a total of 22 nonelectrolytic and electrolytic compounds, including the entire possible range of binding values. Plots of the mean plasma unbound fractions as obtained by AED and BED for the compounds studied could be fitted by a straight line with slope and intercept not significantly different from unity and zero, respectively. Also, the precision of the two methods appeared to be similar. However, the times required to reach equilibrium dialysis were significantly different: With BED and AED, this time span ranged between 2 and 45 and 180 and 960 min, respectively. These results indicate that overall the BED method offers a significant advantage over the AED procedure: It is less time consuming and hence possibly more reliable.     

Fluid shift and protein leakage corrections in protein binding equilibrium dialysis experiments

Conventional equilibrium dialysis methods used in the estimation of ligand-macromolecule binding parameters (affinities, capacities. unbound ligand fractions, etc.) tacitly assume and require that no ligand-macromolecule binding occurs on the buffer side of the dialysis chamber. Unfortunately, this is almost never a valid assumption, as small amounts of plasma proteins are invariably detected in the buffer chamber. Provided the extent of protein leakage is in the usual region (about 0.1%) and the extent of ligand binding does not exceed approximately 99%. errors associated with conventional free fraction estimations obtained from calculations ignoring the effect of protein leakage are usually small (about 1–10% error). As ligand binding exceeds 99%, significant errors may ensue. A generalized theoretical equilibrium dialysis method is developed which permits the estimation of association constants, the number of binding sites and free fraction determinations for a model employing any number of classes of binding sites. Application of the method requires a minimum of two experimental runs for each class of binding sites; volume shifts are automatically adjusted for by the method.     

The protein binding of timegadine determined by equilibrium dialysis.

The protein binding of timegadine to albumin, serum, plasma and plasma enriched with the acute phase reactants alpha 1-acid glycoprotein, alpha 1-anti-trypsin and C-reactive protein was determined by equilibrium dialysis. The effects of other analgesic and anti-inflammatories (indomethacin, ketoprofen, paracetamol and sodium salicylate) and other basic drugs (disopyramide, lignocaine, propranolol and quinidine) on the binding of timegadine were also determined. Timegadine binding was concentration-dependent up to 0.5 micrograms/ml, but independent above this level up to 10.0 micrograms/ml, the mean and standard error being 93.8 +/- 0.5%. Albumin accounted for only 32.4% of timegadine bound to plasma. Plasma enrichment with the acute phase reactants led to significant increases in timegadine binding. Simultaneous dialysis with other drugs caused significant decreases in timegadine binding.     

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.     

Factors influencing the protein binding of a new phosphodiesterase V inhibitor, DA-8159, using an equilibrium dialysis technique

Various factors influencing the protein binding of DA-8159 to 4% human serum albumin (HSA) were evaluated using an equilibrium dialysis technique at an initial DA-8159 concentration of 5 microg/mL. It took approximately 8 h incubation to reach an equilibrium between 4% HSA and an isotonic phosphate buffer of pH 7.4 containing 3% of dextran ('the buffer') using a Spectra/Por 2 membrane (mol. wt. cut-off: 12,000--14,000) in a water bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. The extent of binding was dependent on DA-8159 concentrations, HSA concentrations, incubation temperature, buffer pH, and alpha-1-acid glycoprotein (AAG) concentrations. The binding of DA-8159 in heparinized human plasma (93.9%) was significantly higher than in rats (81.4%), rabbits (80.4%), and dogs (82.2%), and this could be due to differences in AAG concentrations in plasma.     

平衡透析并液质联用法同时测定度洛西汀、氟西汀和阿戈美拉汀的人血浆蛋白结合率

目的 建立平衡透析并液质联用法(HPLC-MS/MS),同时测定度洛西汀、氟西汀和阿戈美拉汀在人血浆中的蛋白结合率.方法 用平衡透析法处理血浆得透析内液和透析外液.以地西泮作为内标,色谱柱为WATERS Xterra(R) RP18(4.6 mm×100 mm,3.5 μm),以甲醇-水(5 mmol·L-1醋酸铵-0.3%甲酸)为流动相,梯度洗脱,用电喷雾离子源,正离子多反应监测.考察该方法的特异性、标准曲线与最低定量限、精密度与回收率、基质效应以及稳定性. 结果 在血浆和磷酸盐缓冲液(PBS)中,度洛西汀、氟西汀和阿戈美拉汀的标准曲线线性良好,批内、批间精密度均<15%,提取回收率85.89%~106.86%,内标归一化基质效应在86.55%~104.94%,且稳定性均较好. 结论 该方法准确、灵敏、简便,可用于度洛西汀、氟西汀和阿戈美拉汀的人血浆蛋白结合率的研究.     

Factors influencing the protein binding of YH-439 using an equilibrium dialysis technique. A new hepatoprotective agent

Various factors influencing the plasma protein binding of YH-439 to 4% human serum albumin (HSA) were evaluated using the equilibrium dialysis method at the initial YH-439 concentration of 2 μg mL^?1. It took approximately 12 h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% of dextran (‘the buffer’) using a Spectra/Por 2 membrane (molecular weight cut-off, 12 000–14 000) in a water bath shaker kept at 37. C and at a rate of 50 oscillations min^?1. YH-439 was fairly stable both in 4% HSA and in the ‘buffer’ for up to 24 h incubation. The binding of YH-439 to 4% HSA was constant (97.4 ± 0.55%) at YH-439 concentrations ranging from 0.5 to 10 μg mL^?1. However, the extent of binding was dependent on HSA concentrations: the values were 90.7, 94.7, 96.7, 97.0, 97.0, 97.1, and 97.5% at HSA concentrations of 0.5, 1, 2, 3, 4, 5, and 6%, respectively. The plasma protein binding decreased with increasing incubation temperature: the binding values were 98.2, 97.6, 97.2, and 96.8% when incubated at 10, 21, 26, and 37°C, respectively. The binding of YH-439 was also influenced by the chloride concentration in the buffer: the binding values were 94.5, 97.0, and 96.8% for the chloride concentrations of 0, 0.249, and 0.546%, respectively. The binding of YH-439 was also dependent on the buffer pH: the percentages of free fraction were 6.0, 4.1, 3.8, 2.8, 2.7 and 2.8% for the buffer pHs of 5.0, 6.0, 6.5, 7.0, 7.4, and 8.0, respectively. The free fraction of YH-439 was slightly increased by the addition of heparin (up to 40 U mL^?1), sodium azide (NaN₃, up to 0.5%), and its metabolites. The protein binding of YH-439 was influenced neither by AAG, acetylsalicylic acid, or sulphisoxazole, nor by the addition of citrate or EDTA. The free fractions of YH-439 in rabbit (4.2%) and dog (4.7%) plasma seemed to be higher than in rats (2.9%) and humans (3.1%).     

Protein binding of glufosinate and factors affecting it revealed by an equilibrium dialysis technique

We investigated the protein binding of glufosinate ammonium (GLF) and several factors affecting this binding using human serum albumin (HSA) and human volunteer serum under various conditions. The mean ratios of the free GLF (RFr-GLF) to 4% HSA were examined in the sera of patients described elsewhere at GLF levels from 1 microg/mL to 500 microg/mL; the range was found to be only from 0.80 to 0.88. Neither the incubation temperature nor buffers containing different chloride ion concentrations had any effect on the RFr-GLF to HSA. Moreover, the addition of heparin, glycoprotein-alpha1-acid (AAG), and sodium azide had no effect on the RFr-GLF. However, pH of the isotonic phosphate buffer and the addition of palmitic or oleic acid were seen to have an effect. In this study, the mean RFr-GLF to healthy human serum was 0.99. This high value was evidenced that GLF was rapidly excreted through the renal route.     

Comparison of equilibrium dialysis, ultrafiltration, and gel permeation chromatography for the determination of free fractions of phenobarbital and phenytoin

In this study, three techniques for measuring the free fractions of phenobarbital and phenytoin were compared: equilibrium dialysis, ultrafiltration, and the Hummel and Dreyer method for gel permeation chromatography. In their therapeutic range (15-40 and 10-20 mg/L, respectively) the free fractions of phenobarbital and phenytoin were independent of the drug concentrations. Free fractions of phenobarbital as determined by equilibrium dialysis, ultrafiltration, and gel permeation chromatography were 58.7 +/- 1.8, 58.3 +/- 1.5, and 55.1 +/- 1.7%, respectively. Free fractions of phenytoin were 18.1 +/- 1.1, 17.0 +/- 2.1, and 19.4 +/- 1.2%, respectively. On lowering the albumin concentration, a similar increase in the free fractions of both drugs was observed with all three techniques. The results of this study show that all three techniques are suitable for the determination of free fractions of phenobarbital and phenytoin. Moreover, these techniques seem to be suitable for the investigation of physiological factors that may influence albumin drug binding.