Validation of 96-well Equilibrium Dialysis with Non-radiolabeled Drug for Definitive Measurement of Protein Binding and Application to Clinical Development of Highly-Bound Drugs
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INTRODUCTION
The free-drug hypothesis states that only unbound drug is available for (1) clearance and drug—drug interactions with metabolizing enzymes and transporters, (2) equilibration into tissues, and (3) pharmacological activity, that is, unbound drug concentrations drive pharmacokinetics, pharmacodynamics, and drug–drug interactions. As such, protein binding (PB) in plasma, hepatic microsomes, and relevant target tissues (e.g., brain homogenate) is routinely evaluated in drug discovery to determine
Reagents
Atenolol, diclofenac, diltiazem, imipramine, indomethacin, loperamide, midazolam, nelfinavir, quinidine, sertraline, and warfarin were purchased from Sigma–Aldrich (St. Louis, Missouri). All other chemicals were of reagent grade and readily available from commercial sources.
Plasma was prepared by pooling three lots (A50135, A49989, and A51449) of human plasma obtained from the Blood Bank (Bangalore, India). Both plasma and phosphate buffer (100 mM) pH were adjusted to 7.4 on the day of the
RESULTS
Table 2 provides a summary of plasma and dialyzed buffer solubility and stability, device nonspecific binding, and ability to achieve equilibrium in the absence of protein. All 11 drugs demonstrated acceptable (70%–130%) initial solubility in human plasma (11-compound overall % initial target concentration = 97 ± 13%) and stability following 6-h incubation at 37°C (11-compound overall % remaining at 6 h = 95 ± 12%) at the concentrations tested (1 and 10 μM or 10 and 100 μM). All drugs demonstrated
DISCUSSION
Human plasma binding was determined using equilibrium dialysis of non-radiolabeled compound for 11 structurally-diverse drugs from a variety of therapeutic areas, spanning the full range of low, moderate, and high plasma binding. The study was supported with an appropriately validated LC–MS/MS bioanalytical assay, as well as thorough evaluation of matrix and buffer solubility and stability, device nonspecific binding, and ability to achieve equilibrium in the absence of protein. The extent of
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2020, Journal of Pharmaceutical SciencesCitation Excerpt :The same precipitation and extraction methods were followed for the standard and quality control concentrations corresponding to either PER or FLU. To obtain the Cfree, Ctotal, fup and RAGP:ALB values for each drug at different concentrations, in vitro binding tests were performed (in duplicates) according to the 96-well rapid equilibrium dialysis (RED) protocol suggested by the company.2,72,75 Prior to dialysis tests, the appropriate equilibrium incubation time of 6 h was determined by performing preliminary tests at different times varying from 1 h to 24 h (in duplicates) for PER (2.5 μM) and FLU (2.5 μM).
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2020, Journal of Pharmaceutical Sciences