This paper reviews the usefulness, current status, and potential of primary human hepatocytes (PHHs) in three-dimensional (3D) cultures, also known as spheroids, in the field of pharmacokinetics (PK). Predicting PK and toxicity means pharmaceutical research can be conducted more efficiently. Various in vitro test systems using human hepatocytes have been proposed as tools to detect hepatic toxicity at an early stage in the drug development process. However, such evaluation requires long-term, low-level exposure to the test compound, and conventional screening systems such as PHHs in planar (2D) culture, in which the cells can only survive for a few days, are unsuitable for this purpose. In contrast, spheroids consisting of PHH are reported to retain the functional characteristics of human liver for at least 35 days. Here, we introduce a fundamental PK and toxicity assessment model of PHH spheroids and describe their applications for assessing species-specific metabolism, enzyme induction, and toxicity, focusing on our own work in these areas. The studies outlined in this paper may provide important information for pharmaceutical companies to reduce termination of development of drug candidates.

Bosentan is a high-affinity antagonist of endothelin receptors and one of the earliest examples for target-mediated drug disposition (a type of nonlinear pharmacokinetics (PKs) caused by saturable target binding). The previous physiologically-based PK (PBPK) modeling indicated that the nonlinear PKs of bosentan was explainable by considering saturable hepatic uptake. However, it remained unexamined to what extent the saturable target binding contributes to the nonlinear PKs of bosentan. Here, we developed a PBPK model incorporating saturable target binding and hepatic uptake, and analyzed the clinical bosentan PK data using the Cluster Gauss-Newton method (CGNM). The PBPK model without target binding fell short in capturing the bosentan concentrations below 100 nM, based on the PK profiles and the Goodness-of-Fit plot. Both global and local identifiability analyses (using the CGNM and Fisher Information Matrix, respectively) informed that the target binding parameters were identifiable only if the observations from the lowest dose (10 mg) were included. By analyzing blood PK profiles alone, the PBPK model with target binding yielded practically identifiable target binding parameters and predicted the maximum target occupancies of 0.6-0.8 at clinical bosentan doses. Our results indicate that target binding, albeit not a major contributor to the nonlinear bosentan PKs, may offer a prediction of target occupancy from blood PK profiles alone and potential guidance on achieving optimal efficacy outcomes, under the condition when the high-affinity drug target is responsible for the efficacy of interest, and when the dose ranges cover varying degrees of target binding. Significance Statement By incorporating saturable target binding, our PBPK model predicted in vivo target occupancy of bosentan based only on the blood concentration–time profiles obtained from a wide range of doses. Our analysis highlights the potential utility of PBPK models that incorporate target binding in predicting target occupancy in vivo.