Evaluation of the disconnect between hepatocyte and microsomal intrinsic clearance at intracellular and extracellular...

In vitro-in vivo extrapolation using human hepatocytes (HHs) and human liver microsomes (HLMs) is a well established method for predicting metabolic blood clearance. Typically, in vitro incubations with HLM are conducted at an extracellular pH of 7.4, rather than the intracellular pH of 7.0. This study compares the scaled intrinsic clearance (CL_int) values assessed in HLM at both pH levels and in HH using 89 compounds. The CL_int values obtained in microsomes were similar between conditions for acidic compounds, whereas the difference was pronounced for basic/neutral compounds, with a median CL_int ratio between pH 7.4:7.0 of 2 and 1.9, respectively. However, the ratio approached 1 for basic compounds when compensating for the differences in uncharged fraction across the tested conditions. Comparison of the physiologically scaled CL_int ratio HLM:HH showed a marked difference, with a median ratio of 2.8 for HLM at pH 7.4 for basic compounds, whereas neutral and acidic compounds showed more similar scaled CL_int values with a median ratio of 1.6 and 1.4, respectively. This contrasted with HLM at pH 7.0, which showed a similar median ratio close to 1 across ion classes. The in vivo CL_int was underestimated by a similar degree across ion classes, with average fold errors (AFEs) of 1.7-2.1 for HLM at pH 7.0 and HH, whereas HLM at pH 7.4 showed an AFE of approximately 1 because of an overestimation of basic compounds (AFE, 0.7). The study indicated significant differences in CL_int values using microsomes at extracellular pH compared to intracellular pH, which contribute to differences in scaled clearance from hepatocytes. SIGNIFICANCE STATEMENT: The study demonstrates that the use of an intracellular pH (7.0) results in significant differences in intrinsic clearance determined in human liver microsomes compared to the extracellular pH (7.4). Further, the use of the intracellular pH in microsomal incubation resulted in a similar scaled intrinsic clearance to that from hepatocytes, which was different from microsomal incubations at the extracellular pH.