Mechanistic Modeling of Pitavastatin Disposition in Sandwich-Cultured Human Hepatocytes: A Proteomics-Informed Bottom-Up Approach

Abstract

Isolated human hepatocytes are commonly used to predict transporter-mediated clearance in vivo. Such predictions, however, do not provide estimations of transporter contributions and consequently do not allow predictions of the outcome resulting from a change in the activity of a certain transporter, for example, by inhibition or a genetic variant with reduced function. Pitavastatin is a drug that is heavily dependent on hepatic transporters for its elimination, and it is excreted mainly as unchanged drug in the bile. For this reason, pitavastatin was used as a model drug to demonstrate the applicability of a bottom-up approach to predict transporter-mediated disposition in sandwich-cultured human hepatocytes (SCHHs), allowing for the estimation of transporter contributions. Transport experiments in transfected human embryonic kidney cells (HEK293 cell lines) and inverted membrane vesicles overexpressing each of the relevant transport proteins were used to generate parameter estimates for the mechanistic model. By adjusting for differences in transporter abundance between the in vitro systems and individual SCHH batches, the model successfully predicted time profiles of medium and intracellular accumulation. Our predictions of pitavastatin bile accumulation could not be confirmed, however, because of a very low biliary excretion of pitavastatin in relation to the hepatic uptake in our SCHHs. This study is, to our knowledge, the first to successfully simulate transporter-mediated processes in a complex system such as SCHHs at the level of individual transport proteins using a bottom-up approach.

Functional characterization for polymorphic organic anion transporting polypeptides (OATP/SLCO1B1, 1B3, 2B1) of monkeys recombinantly expressed with various OATP probes

“…In wild type, the K m values of pitavastatin were 0.74 and 0.85 μM in the two experiments. The K m value of pitavastatin for human OATP1B1 was reported as 0.81 to 4.8 μM (Izumi et al, ; Vildhede et al, ); thus, the K m values of pitavastatin for the recombinant monkey OATP1B1 wild type was very low, but within the range of previously reported K m values for human OATP1B1. In monkey OATP1B1 variants, the values of K m and V max were 36% to 255% and 57% to 268%, respectively, compared with those of the wild type (Table ).…”

Pretreatment With Rifampicin and Tyrosine Kinase Inhibitor Dasatinib Potentiates the Inhibitory Effects Toward OATP1B1- and OATP1B3-Mediated Transport

“…OATP1B1 plays a predominant role in the hepatic uptake of pitaastatin 40 . Although one cannot exclude the possibility that other transporters may also be involved in the decreased accumulation of pitavastatin in human SCH following rifampicin pretreatment, OATP1B1 plays a significant role 41 .…”

Critical Issues and Optimized Practices in Quantification of Protein Abundance Level to Determine Interindividual Variability in DMET Proteins by LC‐MS/MS Proteomics

“…The notable advantages of LC‐MS/MS or selective (or multiple) reaction monitoring (SRM or MRM) proteomics are its ability to quantify multiple proteins in a sample (i.e., multiplexing), selectivity, and reproducibility . In addition to its application in determining DMET protein interindividual variability, LC‐MS/MS proteomics is proving to be an important technique in the field of drug metabolism and pharmacokinetics (DMPK), e.g., in vitro model validation, in vitro to in vivo extrapolation (IVIVE) of drug clearance, differential tissue or subcellular localization of proteins, characterization of reactive metabolite–protein interaction, and protein biomarker discovery, e.g., exosome proteomics . The approach has also been used to determine enzyme induction potential of drugs and interspecies differences in protein abundance of DMET proteins, which affect animal model selection for drug toxicity studies .…”