Measurement Of Plasma Protein Binding Research Articles

Optimizing a High-Throughput Solid-Phase Microextraction System to Determine the Plasma Protein Binding of Drugs in Human Plasma.

Plasma protein binding refers to the binding of a drug to plasma proteins after entering the body. The measurement of plasma protein binding is essential during drug development and in clinical practice, as it provides a more detailed understanding of the available free concentration of a drug in the blood, which is in turn critical for pharmacokinetics and pharmacodynamics studies. In addition, the accurate determination of the free concentration of a drug in the blood is also highly important for therapeutic drug monitoring and in personalized medicine. The present study uses C18-coated solid-phase microextraction 96-pin devices to determine the free concentrations of a set of drugs in plasma, as well as the plasma protein binding of drugs with a wide range of physicochemical properties. It should be noted that the extracted amounts used to calculate the binding constants and plasma protein bindings should be measured at respective equilibrium for plasma and phosphate buffer. Therefore, special attention is placed on properly determining the equilibration times required to correctly estimate the free concentrations of drugs in the investigated systems. The plasma protein binding values obtained with the 96-pin devices are consistent with those reported in the literature. The 96-pin device used in this research can be easily coupled with a Concept96 or other automated robotic systems to create an automated plasma protein binding determination protocol that is both more time and labor efficient compared to conventional equilibrium dialysis and ultrafiltration methods.

Plate Reader Compatible 3D-Printed Device for Teaching Equilibrium Dialysis Binding Assays

Plasma protein binding measurements are an important aspect of pharmacology and drug development. Therefore, performing these measurements can provide a valuable and highly practical learning exper...

Understanding and Reducing the Experimental Variability of In Vitro Plasma Protein Binding Measurements

The experimental measurement of plasma protein binding is a useful in vitro Absorption Distribution Metabolism and Excretion(ADME) assay currently conducted in both screening and definitive early development candidate modes. The fraction unbound is utilized to calculate important pharmacokinetic (PK) parameters such as unbound clearance and unbound volume of distribution in animals that can be used to make human PK and dose predictions and estimate clinically relevant drug–drug interaction potential. Although these types of assays have been executed for decades, a rigorous statistical analysis of sources of variability has not been conducted because of the tedious nature of the manual experiment. Automated conduct of the incubations using a 96-well equilibrium dialysis device as well as high-throughput liquid chromatography–mass spectrometry quantitation has now made this level of rigor accessible and useful. Sources of variability were assessed including well position, day-to-day, and site-to-site reproducibility. Optimal pH conditions were determined using a design of experiments method interrogating buffer strength, CO2% and device preparation conditions. Variability was minimized by implementing an in-well control that is concurrently analyzed with new chemical entity analytes. Data acceptance criteria have been set for both the in-well control and the range of analyte variability, with a sliding scale tied to analyte-binding characteristics.

A mass balance approach for calculation of recovery and binding enables the use of ultrafiltration as a rapid method for measurement of plasma protein binding for even highly lipophilic compounds

The aim of this study is to further validate the use of ultrafiltration (UF) as a method for determining plasma protein binding (PPB) by demonstrating that non-specific binding (NSB) is not a limitation, even for highly lipophilic compounds, because NSB sites on the apparatus are passivated in the presence of plasma. Mass balance theory was used to calculate recovery of 20 commercial and seven investigational compounds during ultrafiltration in the presence and absence of plasma. PPB was also measured using this mass balance approach for comparison to PPB determined by rapid equilibrium dialysis (RED) and as found in the literature. Compound recovery during UF was dramatically different in the presence and absence of plasma for compounds with high NSB in PBS only. A comparison of PPB calculated by ultrafiltration with literature values or calculated by RED gave concordant results. Discrepancies could be explained by changes in pH, insufficient time to equilibrium, or compound instability during RED, problems which were circumvented by ultrafiltration. Therefore, NSB, as measured by the traditional incubation of compound in PBS, need not be an issue when choosing UF as a PPB assay method. It is more appropriate to calculate compound recovery from the device in plasma as measured by mass balance to determine the suitability of the method for an individual compound. The speed with which UF can be conducted additionally avoids changes in pH or compound loss that can occur with other methods. The mass balance approach to UF is thus a preferred method for rapid determination of PPB.

On the prediction of hepatic clearance using the diluted plasma in metabolic stability assay

It was suggested that in vivo hepatic clearance, CL(h), may be predicted rather accurately with the in vitro values of intrinsic clearance, CL(int), obtained using the microsomal incubation mix containing diluted plasma, and consequently calculated by the well-stirred model equation. Conceivably the improvement could be due to the direct account of plasma protein binding in the measured values of CL(int). It is shown in this article that the prediction of CL(h) done in this manner may not yield accurate results, both substantial underestimation or overestimation of the true value is possible. The procedure may be useful to reduce the overestimation of CL(h) for highly protein bound drugs, though the obtained value of CL(h) may be far off from the correctly calculated one. The accurate way of calculating CL(h), based on the value of CL(int) obtained in diluted plasma, is presented. It takes into account both the drug protein binding in diluted plasma and microsomal binding, as well as blood-plasma concentration ratio. The prediction of CL(h) by the suggested calculation using the experimental data on CL(int), measured at different plasma dilutions for several drugs, yields consistent (dilution independent) values of hepatic clearance. It does not seem possible to avoid the measurement of plasma protein binding, microsomal binding and blood-plasma concentration ratio for an accurate and consistent prediction of CL(h), even if the value of CL(int) were obtained in the pure (undiluted) plasma. In an early stage screening using plasma in the microsomal incubation mix may be beneficial for fast metabolizing drugs with relatively high protein binding. This would reduce a possible overestimation CL(h), and also lead to the increase of the half-life in the microsomal incubation, so that it could be measured more accurately.

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.

Modification of the ultrafiltration technique to overcome solubility and non-specific binding challenges associated with the measurement of plasma protein binding of corticosteroids

Plasma protein binding (PPB) methodology suitable for application in the lead optimisation of a corticosteroid series known to demonstrate non-specific binding (NSB) and poor solubility has been established. The method involved a modification to standard ultrafiltration (UF) techniques. In parallel with each experimental plasma sample, a control plasma sample was also processed by ultrafiltration. The retentate from experimental and control plasma samples were mixed back into the filtrate of the partner sample. The resulting regenerated plasma samples, one representing the experimental filtrate and one representing the experimental retentate, were then analysed by LC/MS/MS. Varying degrees of NSB were demonstrated with a number of corticosteroids, and this effect was eliminated using the modified method. Validation using a panel of established corticosteroids showed good agreement with published PPB figures. The published PPB figure for fluticasone propionate (FP) was, however, found to be an underestimate, and this was subsequently confirmed, at clinically relevant plasma concentrations, to be 99.3%. The modified method was particularly suited to lead optimisation because it provided samples in a consistent matrix compatible with standard high throughput LC/MS/MS analysis.

7. In vivo measurement of plasma protein binding of renal tracers

7. In vivo measurement of plasma protein binding of renal tracers

Role of concentration-dependent plasma protein binding in disopyramide disposition

Plasma disopyramide concentration-time data and plasma protein binding measurements were obtained in 12 patients requiring disopyramide for suppression of their cardiac arrhythmias. The fraction of disopyramide unbound to plasma proteins varies from approximately 0.19 to 0.46 over the therapeutic range of total plasma concentrations (2–8 mg/liter). Data from single and multiple intravenous doses were analyzed using two models based on the hypothesis either that clearance is independent of the total disopyramide plasma concentration (total clearance model) or that clearance is independent of the concentration of disopyramide unbound to plasma proteins (free clearance model). This analysis indicates that only the free clearance model satisfactorily describes the data as a linear system. Using the free clearance model and data obtained from single doses, multiple intravenous infusions were designed for each patient which would rapidly attain and maintain predetermined plasma disopyramide concentrations. The calculated and observed disopyramide concentrations were in close agreement. In the 12 patients studied, at any given total disopyramide plasma concentration, there was an approximately twofold range in the fraction of disopyramide unbound to plasma proteins. Mean plasma protein binding data are therefore of little value in a given patient for predicting free disopyramide concentrations from measurements of total disopyramide concentration. Difficulties in the clinical management of patients receiving disopyramide, resulting from the nonlinear disposition of the usually measured total disopyramide concentrations, are discussed.