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