Ex Vivo Protein Binding Data is needed during Proof-of-Concept Stage

Plasma protein binding of drug candidates is one of the ADME characteristics determined in the drug discovery and development process. Early protein binding data are generally derived from in vitro experiments during preclinical development. Later, during Phase II/III clinical trials, ex vivo data are generated as the disease state’s effects on pharmacokinetics are investigated. Recently, we conducted an ex vivo protein binding study as part of a PK study in renally impaired subjects, matching subject plasma with that of healthy volunteers. There was no difference in the extent of plasma protein binding between the two sets of subjects. However, the results were hugely different compared with values determined in vitro from spiked plasma, as quality control samples analyzed along with study samples.

We then reviewed a dozen compounds that we had investigated over the past three years, comparing ex vivo vs. in vitro protein binding in plasma from healthy subjects. We found that 6 out of the 12 compounds showed higher binding ex vivothan in vitro, with two-fold or greater differences in the free fraction. Three of these six compounds were tested along with their metabolite(s). In the cases of one compound with several metabolites and another compound with one metabolite, both parent and metabolites showed ex vivo/in vitro differences in protein binding; another compound with a single metabolite showed a difference in binding for the parent but not for the metabolite. These examples clearly demonstrated that matching healthy normal controls in the same study is necessary for an appropriate comparison of plasma protein binding under disease states. The reason for lower binding in vitro than ex vivo in plasma from healthy subjects is not clear, as factors such as concentrations of the drug or metabolites and the chemical functionality of the metabolites cannot adequately explain the observed differences in binding.

Due to the observed differences in the extent of ex vivo vs. in vitro plasma protein binding, it would be prudent to determine ex vivo protein binding of drug candidates at the proof-of-concept stage, especially if the results might have clinical safety or efficacy implications.

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