Enabling direct and definitive free fraction determination for highly-bound compounds in protein binding assay.

Autor: Zhang J; Bristol-Myers Squibb Company, 3551 Lawrenceville Princeton Rd, Princeton, NJ 08648, United States. Electronic address: jun.zhang1@bms.com., Pikul G; Bristol-Myers Squibb Company, 3551 Lawrenceville Princeton Rd, Princeton, NJ 08648, United States., Horch J; Bristol-Myers Squibb Company, 3551 Lawrenceville Princeton Rd, Princeton, NJ 08648, United States., Konovalov D; Bristol-Myers Squibb Company, 3551 Lawrenceville Princeton Rd, Princeton, NJ 08648, United States., Li S; Bristol-Myers Squibb Company, 3551 Lawrenceville Princeton Rd, Princeton, NJ 08648, United States., Cvijic ME; Bristol-Myers Squibb Company, 3551 Lawrenceville Princeton Rd, Princeton, NJ 08648, United States., Shou W; Bristol-Myers Squibb Company, 3551 Lawrenceville Princeton Rd, Princeton, NJ 08648, United States., Weller H; Bristol-Myers Squibb Company, 3551 Lawrenceville Princeton Rd, Princeton, NJ 08648, United States.
Jazyk: angličtina
Zdroj: Journal of pharmaceutical and biomedical analysis [J Pharm Biomed Anal] 2021 Feb 05; Vol. 194, pp. 113765. Date of Electronic Publication: 2020 Nov 20.
DOI: 10.1016/j.jpba.2020.113765
Abstrakt: Protein binding determination for highly-bound compounds using equilibrium dialysis remains a challenge in drug discovery. The reasons are mainly three-fold; 1. due to their slow diffusion rate, highly-bound compounds require a much longer incubation time to reach dialysis equilibrium than typically needed; 2. highly-bound compounds are often hydrophobic and prone to non-specific binding in dialysis; 3. free drug concentration in the post incubation dialysate is too low for reliable analytical quantification. Modified equilibrium dialysis approaches include using diluted plasma for dialysis, or pre-saturating the non-specific binding sites in the dialysis device with compounds of interest prior to dialysis. In this study, we developed a customized equilibrium dialysis assay with an extended incubation time of 24 h, followed by microflow (μF) LC-MS/MS for bioanalysis, for direct and definitive free fraction determination of highly protein-bound compounds. The extended incubation time ensured the dialysis to reach equilibrium and saturating the non-specific binding sites, while μFLC-MS/MS provided far better sensitivity than the conventional LC-MS/MS typically used for post incubation bioanalysis. For a group of commercially available, highly protein-bound compounds, the free fraction data generated by the developed assay correlated very well with the literature values generated with diluted plasma method or pre-saturation method. This novel assay approach has been successfully used to generate protein binding results for highly-bound compounds to support ongoing drug discovery research.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2020 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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