RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism
Equilibrium Gel Filtration to Measure Plasma Protein Binding of Very Highly Bound Drugs

https://doi.org/10.1002/jps.23818Get rights and content

Abstract

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.

Section snippets

INTRODUCTION

The binding of drugs to plasma proteins influences their organ distribution as well as clearance processes.1 When comparing systemic exposure between species in the context of safety assessments or for prediction of a human efficacious dose, species differences in plasma protein binding should be considered. Similarly, to assess the need for a dose adjustment in a special population (e.g., renal or hepatic impaired patients), not only differences in total exposure per dose, but also differences

Compounds

All radiolabeled compounds were synthesized in the laboratories of Novartis Pharma. Stock solutions were prepared in ethanol.

Biological Material

Plasma was obtained from male animals: Hanover–Wistar rats, Beagle dogs, Göttingen minipigs, and Cynomolgus monkey. Human plasma was obtained from healthy male volunteers. Pooled plasma of at least three male individuals was obtained by centrifugation of fresh blood containing ethylenediaminetetraacetic acid and stored at −20°C until use. The pH of plasma samples was

Comparison with Standard Methods

Equilibrium gel filtration was used to determine plasma protein binding of nine Novartis drugs for which fu's had also been determined for at least three species applying different conventionally used methods (Table 1, Fig. 1). By UF, ED, or UC, determined fu's covered the range of 0.13%–44%. In 77% of cases, the difference in fu determined with EGF was ± twofold. Only for one drug (Nov01), the difference was greater than fourfold compared with the values determined by UF; this was the case for

DISCUSSION

Equilibrium gel filtration7., 16., 17., 27. was previously used to measure binding to purified proteins28 or samples containing multiple proteins were separated on the column and binding to individual proteins compared.29 Here, we describe and validate the use of EGF applied to plasma, a complex mixture of proteins not separated in our setup. This variation of the method is used to (1) quantify species differences in drug plasma protein binding and (2) determine fu's in plasma. Columns were

CONCLUSIONS

Equilibrium gel filtration is a robust and useful method to determine plasma protein binding of very highly bound drugs. It has distinct advantages over standard techniques and is a powerful instrument to establish species differences in plasma protein binding for poorly soluble and sticky molecules. Applications to assess binding to proteins other than plasma (e.g., tissue homogenates, CSF, or isolated proteins including plasma proteins) are possible for difficult to handle highly bound drugs.

ACKNOWLEDGMENTS

We are grateful to Dr. Olivier Kretz for his support, constructive feedback, and the good discussions, to Marcel Fresneau for his excellent and thorough technical work, and to Ieuan Jones for statistical advice and analysis.

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