Comparison of Species and Cell-Type Differences in Fraction Unbound of Liver Tissues, Hepatocytes, and Cell Lines

Abstract

Fraction unbound () of liver tissue, hepatocytes, and other cell types is an essential parameter used to estimate unbound liver drug concentration and intracellular free drug concentration. and are frequently measured in multiple species and cell types in drug discovery and development for various applications. A comparison study of 12 matrices for and of hepatocytes in five different species (mouse, rat, dog, monkey, and human), as well as of Huh7 and human embryonic kidney 293 cell lines, was conducted for 22 structurally diverse compounds with the equilibrium dialysis method. Using an average bioequivalence approach, our results show that the average difference in binding to liver tissue, hepatocytes, or different cell types was within 2-fold of that of the rat Therefore, we recommend using rat as a surrogate for liver binding in other species and cell types in drug discovery. This strategy offers the potential to simplify binding studies and reduce cost, thereby enabling a more effective and practical determination of for liver tissues, hepatocytes, and other cell types. In addition, under hepatocyte stability incubation conditions should not be confused with , as one is a diluted and the other is an undiluted Cell density also plays a critical role in the accurate measurement of.

The impact of low temperature on fraction unbound for plasma and tissue

“…A large set of structurally diverse compounds was used here to evaluate the impact of temperature on f u and to help determine if the results obtained from low temperature (e.g., 4 °C) can predict f u at physiological temperatures (e.g., 37 °C) for unstable compounds. Our test compound set contained acids, bases, neutrals and zwitterions with MW of 200–800 Da, LogP values between −3 and 8, polar surface area (PSA) from 6 to 170 Å 2 and f u values spanning 3 log units (Riccardi et al, ). Human plasma protein binding was measured at 4 °C for 18 and 24 hours and 37 °C for 6 and 18 hours using equilibrium dialysis to ensure equilibrium was achieved.…”

“…Based on the negligible effect of low temperature on plasma protein binding, we extended our study to rat liver binding assessments. We chose rat since we have shown previously that significant species difference in liver tissue binding and cell binding are not present and that rat liver tissue binding can be used as a surrogate for binding of other species and cell types (Riccardi et al, ). Incubation times of 18 hours and 6 hours were selected for the 4 °C and 37 °C rat liver binding studies, respectively, based on the time required to achieve equilibrium from the previous human plasma binding studies.…”

“…Our test compound set contained acids, bases, neutrals and zwitterions with MW of 200-800 Da, LogP values between −3 and 8, polar surface area (PSA) from 6 to 170 Å 2 and f u values spanning 3 log units (Riccardi et al, 2018). Human plasma protein binding was measured at 4°C for 18 and 24 hours and 37°C for 6 and 18 hours using equilibrium dialysis to ensure equilibrium was achieved.…”

“…Analyst version 1.6.2 and MultiQuant version 3.0.2 (Applied Biosystems, Foster City, CA) was applied for data acquisition and quantitation. All calculations were based on area ratios (analyte peak area/IS peak area) and the equations have been reported previously (Riccardi et al, ).…”

Dasotraline as a selective cytochrome 2B6 inhibitor for reaction phenotyping

“…Dasotraline hepatocyte binding was determined using the equilibrium dialysis method described previously (Riccardi et al, 2018). Briefly, 2 μM dasotraline was added to the HHEP homogenate at two cell densities (0.5 and 2 million cells/ml) and dialysed against phosphate buffered saline in a HTD96 device for 6 hours at 37 C in a CO 2 incubator (5% CO 2 /95% air, 75% relative humidity) on an orbital shaker Carrboro, NC).…”