Effects of low temperature on blood‐to‐plasma ratio measurement

Abstract The blood‐to‐plasma ratio ( R b ) is an important property of drug candidates. R b is applied widely in drug discovery to convert plasma pharmacokinetic parameters to the respective parameters in blood and to develop in vitro–in vivo correlations. Some compounds such as prodrugs, soft drugs, and peptide mimetics are unstable in blood, making accurate in vitro R b measurement challenging, but necessary. Low temperature often reduces the rate of enzymatic and chemical reactions and increases the stability of labile compounds in biomatrices. In this study, the effects of 4°C on R b measurement were evaluated using a set of structurally diverse compounds with various binding and red blood cell (RBC) transport mechanisms. The results indicate that a 4°C R b method provides comparable R b values to the 37°C method for most compounds and can therefore be applied to measure the R b of unstable compounds in drug discovery. In some rare cases, when compounds have a high affinity to specific RBC components (e.g., carbonic anhydrase), the 4°C method may underestimate R b . In these specific cases, the use of appropriate inhibitors to stabilize unstable compounds is recommended.