High-throughput determination of the free fraction of drugs strongly bound to plasma proteins

doi:10.1002/jps.10588

Journal of Pharmaceutical Sciences

Volume 93, Issue 4, April 2004, Pages 816-830

https://doi.org/10.1002/jps.10588 Get rights and content

Abstract

Quantification of protein binding of new chemical entities is an important early screening step during drug discovery and is of fundamental interest for the estimation of safety margins during drug development. In this publication, we describe the development of a new high-throughput assay for the determination of the free drug fraction in plasma (f_u). The new technique is an enhancement of the previously published erythrocytes partition method. It is based on the distribution of drugs between plasma water, plasma proteins, and solid-supported lipid membranes (Transil®). The execution of protein binding studies by partitioning is dramatically simplified by substituting erythrocytes with commercially available Transil® beads, and makes the method particularly suitable for high-throughput studies. Eight Bayer compounds from different compound classes covering a wide range of lipophilicities (log P = 1.9−5.6) and f_u values (0.018−35%) were selected for validation of the assay. The results obtained by the new method and by either the erythrocytes partitioning technique or more conventional methods (ultrafiltration and equilibrium dialysis) are identical, confirming that the new method produces valid results even for drugs that are strongly bound to plasma proteins. Precision and accuracy of the data in the cases of very low and high f_u values are comparable, indicating that the method is especially suited for highly lipophilic drugs that tend to adsorb to surfaces compared with other methods, like ultrafiltration or equilibrium dialysis, that may produce biased data. The method is also useful for the determination of binding parameters and the pH dependence of f_u. In summary, this assay is well suited for high-throughput determination of protein binding during drug discovery and for extended protein binding studies during drug development.

Section snippets

INTRODUCTION

In recent years, the implementation of high-throughput technologies as well as combinatorial chemistry and genomics in drug discovery has led to a tremendous increase of hits identified in biological assays.¹^,² As a consequence, early information on pharmacokinetic properties of hits is demanded on a larger scale. One important parameter affecting pharmacokinetic and pharmacodynamic properties of drugs is their ability to bind to plasma proteins. Commonly used methods for the determination of

Drugs and Reagents

Unlabeled drugs (I−V), ¹⁴C-labeled drugs (VI and VII), and reference compounds for analytics were synthesized in the Chemistry Department of Bayer AG. Solvents used were of HPLC grade. All other chemicals were of analytical grade. Stock solutions of drugs I, II, IV, VI, and VII were prepared in acetonitrile, and the remaining drugs were dissolved in methanol.

Transil®

Transil® (silica beads coated with egg yolk phosphatidylcholine) was purchased from NIMBUS Biotechnologie GmbH, Leipzig, Germany. In this

Comparison of f_u Values Obtained with the Transil® Method and Other Techniques

For validation purposes, drugs with a wide range of physicochemical properties and f_u values were selected. Log P (pH 7.5) values were calculated to be 3.6, 5.6, 1.9, 2.5, 2.6, 2.6, 2.1, and 5.0 for drugs I−VIII, respectively. The f_u values of the validation compounds were determined either by distribution of the drugs between diluted plasma and Transil® or by the distribution method described earlier (drugs I, II, III, IV, VI, and VII). Additionally, f_u values of drugs I and III were

DISCUSSION

The enlarged number of potential drugs identified during drug discovery using modern compared with traditional technologies necessitates the choice of appropriate filters that can be run in HTS to select candidates with drug-like properties. The physicochemical characteristics of a drug have a fundamental influence on its pharmacokinetic behavior.¹⁶^,¹⁷ Poulin and Theil¹⁸ developed a mechanism-based model for the prediction of volume of distribution at steady state (V_ss) in rats and humans. In

Acknowledgements

The method is covered by a European patent application no. 03018512.8, filed 2003-08-16.

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High throughput (HT) equilibrium dialysis was conducted to study the binding between insulin and the obtained conjugates using a 96-well Teflon apparatus from HT Dialysis, LLC (Gales Ferry, CT). Each well in the apparatus is separated to two chambers that are constructed with vertical alignment to allow easy access to the samples to measure the interaction between the conjugates and insulin and minimizing the non-specific binding.18,31 In each well, 200 ul of known concentration of the protein sample was loaded into one chamber, and the conjugates solution was loaded into the other chamber.
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Improving Confidence in the Determination of Free Fraction for Highly Bound Drugs Using Bidirectional Equilibrium Dialysis
2019, Journal of Pharmaceutical Sciences
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Commonly used methods include equilibrium dialysis (ED), ultrafiltration, and ultracentrifugation (UC), and each of those methods has its own advantages and limitations depending on the properties of the investigated drug.6,7 Other less common approaches such as gel filtration,8 erythrocytes partitioning,9 microdialysis,10 and thermocolorimetry11 are also used for fu determinations because of various technical or experimental considerations. Despite considerable progress in PPB assay development, it has been recognized that fu values of certain types of drugs can be experimentally biased toward the method used, and such data inconsistency is more pronounced for those highly bound drugs.8,12
Equilibrium dialysis has been widely used for the measurement of the fraction of unbound drug (f_u) in plasma, but it suffers from the accuracy and reliability for low f_u values. To address this concern, an orthogonal approach, called the bidirectional equilibrium dialysis, is described to simultaneously measure a pair of f_u values for each drug based on equilibration in 2 opposite dialysis directions: from plasma to buffer (f_u,p/b) and from buffer to plasma (f_u,b/p). Hypothetically, if true equilibrium is attained in both dialysis directions, the measured f_u,b/p and f_u,p/b values for a given drug should converge, and thus, the ratio of f_u,b/p to f_u,p/b becomes unity (1.0). Thus, the ratio can be used as a tangible readout for data reliability. This methodology has been extensively tested in the present study using various drugs with distinct plasma binding characteristics. Our results clearly showed that low f_u values (<0.01) could be reliably determined and verified using either the standard or dilution bidirectional equilibrium dialysis method for some known highly bound drugs; for extensively bound drugs with high logD_7.4, such as montelukast, bedaquiline, and venetoclax, only a range of f_u can be reported with confidence because of uncertainty in the true equilibrium.
Determination of dolutegravir's unbound fraction in human plasma using validated equilibrium dialysis and LC-MS/MS methods
2018, Clinica Chimica Acta
Assessment of the unbound pharmacologically active fraction (fu; as the ratio of unbound to total concentration) of dolutegravir could improve therapeutic drug monitoring (TDM) in patients that experience virological failure or toxicity, despite receiving adequate total concentrations. This study evaluated (i) dolutegravir's fu through equilibrium dialysis (ED), (ii) the pre-analytical parameters that influence fu, and (iii) fu's inter-individual variability in HIV patients. Validation of the LC-MS/MS method followed FDA guidelines. The results, based on coefficients of variation (results from nominal concentrations < 15%), allowed accurate measurement of unbound and total dolutegravir concentrations. Equilibrium during ED was obtained in 4 h. Sparse non-specific binding (9%) was observed, allowing results interpretation without interference. Steps before analysis (e.g., conservation at + 4 °C, freeze/thaw cycles) did not influence fu, allowing easy integration of fu analysis within laboratory routines. Anticoagulants from samples (citrated versus heparinized; p < 0.001) and hemolysis (p = 0.007) influenced fu and could lead to misinterpretation. Developed was then performed to the HIV-patients' plasma (n = 54). Results, expressed as median InterQuartile Range [25%;75%] were 0.45% IQR [0.38; 0.55] for fu, 9.26 μg/L IQR [4.62; 15.14] for unbound, and 2035 μg/L IQR [878.5; 2640] for total concentration. The high inter-individual variability observed in the unbound form from HIV patients was a first step towards integrating dolutegravir TDM.
Equilibrium gel filtration to measure plasma protein binding of very highly bound drugs
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Citation Excerpt :
Among them, the commonly used equilibrium dialysis (ED), ultrafiltration (UF), and ultracentrifugation (UC) are the subjects of many reviews.4-7 Less frequently used methods such as solid-phase microextraction,8 erythrocytes partitioning, distribution between plasma and solid-supported lipid membranes,9 microdialysis,10 high-performance frontal analysis,11 high-performance affinity chromatography with albumin,12 circular dichroism, and optical biosensors were also discussed. Although assessing plasma protein binding is conceptually easy, it becomes experimentally challenging, when the free fraction is very low (<2%), which might be the case for 20%–30% of drugs.6
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.
Plasma protein binding: From discovery to development
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However, preparation of erythrocytes and their careful handling to prevent hemolysis is a major hurdle. Further improvement of this method involved replacement of erythrocytes with silica beads coated with egg yolk phosphatidylcholine (material commercially called Transil®),130 which made this method more amenable to HTS setting. Advantage: No membrane-related issues and avoids nonspecific adsorption.
The importance of plasma protein binding (PPB) in modulating the effective drug concentration at pharmacological target sites has been the topic of significant discussion and debate amongst drug development groups over the past few decades. Free drug theory, which states that in absence of energy-dependent processes, after steady state equilibrium has been attained, free drug concentration in plasma is equal to free drug concentration at the pharmacologic target receptor(s) in tissues, has been used to explain pharmacokinetics/pharmacodynamics relationships in a large number of cases. Any sudden increase in free concentration of a drug could potentially cause toxicity and may need dose adjustment. Free drug concentration is also helpful to estimate the effective concentration of drugs that potentially can precipitate metabolism (or transporter)-related drug–drug interactions. Disease models are extensively validated in animals to progress a compound into development. Unbound drug concentration, and therefore PPB information across species is very informative in establishing safety margins and guiding selection of First in Human (FIH) dose and human efficacious dose. The scope of this review is to give an overview of reported role of PPB in several therapeutic areas, highlight cases where PPB changes are clinically relevant, and provide drug metabolism and pharmacokinetics recommendations in discovery and development settings.
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Research ArticlesHigh-throughput determination of the free fraction of drugs strongly bound to plasma proteins

Abstract

Section snippets

INTRODUCTION

Drugs and Reagents

Transil®

Comparison of fu Values Obtained with the Transil® Method and Other Techniques

DISCUSSION

Acknowledgements

J Pharm Sci

Adv Drug Delivery Rev

J Pharm Sci

J Pharm Sci

Biochem Pharmacol

J Pharm Sci

J Pharm Sci

J Pharm Sci

J Pharm Sci

Current and future trends in high-throughput screening for drug discovery

Ann Rep Med Chem

Research Articles
High-throughput determination of the free fraction of drugs strongly bound to plasma proteins

Comparison of f_u Values Obtained with the Transil® Method and Other Techniques