(T1230-05-31) Hepatocyte Uptake Assay to Improve IVIVC Disconnects of Lipophilic Acids

Purpose: Hepatic clearance plays a central role in the systemic elimination of xenobiotics, and while metabolism has long been recognized as a major determinant of hepatic drug disposition, increasing evidence underscores the critical contribution of hepatic transporters - such as OATP1B1/1B3, NTCP, BCRP, and MRP2 - in governing the uptake and efflux of drugs and their metabolites. These transporters not only influence hepatic drug exposure but also modulate pharmacokinetic variability, drug-drug interactions, and hepatotoxic potential of drugs. For highly permeable compounds, hepatic clearance is typically governed by passive diffusion and metabolic transformation, with minimal contribution by uptake transporters. However, exceptions to this paradigm are well-documented - most notably among statins, angiotensin II receptor blockers, and nonsteroidal anti-inflammatory drugs - where active hepatic uptake via transporters such as OATP1B1/1B3 plays a dominant role in hepatic clearance of these compounds. Lipophilic acids are another class of low molecular weight drugs, which typically feature high passive permeability and predominant metabolic elimination, yet, the in vitro intrinsic metabolic clearance (CL_int,met) in liver microsomes or hepatocytes often underpredicts the in vivo clearance. An analysis with ~3000 internal compounds indicated that the in vivo clearance of less than 50% of highly permeable acids was predicted within a 3-fold deviation using in vitro intrinsic CL_int,met data¹. These discrepancies raise the question of whether active hepatic uptake in particular by OATP1B1/1B3 transporters, insufficiently captured in conventional in vitro systems, may contribute to the observed in vitro–in vivo correlation (IVIVC) disconnect for this class of compounds. Recently, the role of hepatic uptake transporter in hepatic clearance was investigated in a plated hepatocyte uptake assay indicating limited contribution of uptake transporters². To further elucidate the importance of active hepatic uptake for lipophilic acids, active and passive hepatic uptake into primary suspended hepatocytes was investigated for a set of 5 lipophilic acids (pitavastatin, gliquidone, PF-05241328, montelukast and LY2409021) using the hepatocyte uptake oil-spin assay.

Methods: The initial slope of uptake of test compounds into cryopreserved primary suspended human or rat hepatocytes was measured using the oil-spin assay in the absence and presence of an uptake transporter inhibitor cocktail (rifampicin, tetraethylammonium, and taurocholic acid) to determine the total (PS_inf,tot) and passive (PS_inf,pas) hepatic uptake clearance, respectively. Active hepatic uptake clearance (PS_inf,act) was calculated as the difference of total and passive uptake. Cellular uptake was quantified by LC-MS/MS. The measured PS_inf was scaled using physiological parameters. To enable a mechanistic assessment of transporter contributions to hepatic drug disposition, PS_inf data as well as CL_int,met were incorporated in the Extended Clearance Model (ECM) to estimate the intrinsic hepatic clearance (CL_h,int). Finally, the hepatic clearance (CL_h) was predicted based on CL_h,int and the well-stirred liver model (Figure 1).

Results: The oil-spin assay was validated for rat and human hepatocytes with a set of known OATP1B1/1B3 transporter substrates for human (rosuvastatin, valsartan, atorvastatin, fluvastatin, and pitavastatin) and rat hepatocytes (rosuvastatin, valsartan and atorvastatin). The contribution of active to the overall cellular uptake was ≥70% in human hepatocytes and ≥40% in rat hepatocytes, indicating sufficient OATP1B1/1B3 activity in the hepatocyte lots and a functional assay setup. The IVIVC for the validation set is summarized in Figure 2. To minimize excessive non-specific binding of lipophilic acid compounds, test compounds were incubated in presence of 0.4% albumin and the measured hepatic uptake was corrected for binding (PS_{inf unbound}). Active hepatic uptake (≥20%) was observed for all lipophilic acid compounds in rat and human hepatocytes. Based on metabolic clearance only, the hepatic clearance was underpredicted in rat hepatocytes (1/5 compound within 3-fold, Figure 3a), which improved when the ECM approach was applied taking into account active and passive uptake in addition to metabolic clearance (3/5 compounds within 3-fold, Figure 3b). Similarly, the ECM approach predicted the hepatic clearance of 3/5 compounds in humans within 3-fold (Figure 3d), despite a similar level of clearance prediction was provided by taking into account the metabolic clearance only (2/5 compounds within 3-fold, Figure 3c).

Conclusion: The present study suggests the relevance of active hepatic uptake for highly permeable compounds, emphasizing the need to take into account the complex interplay between permeability and metabolism as well as the selection of suitable in vitro assay formats. To establish the broader importance of hepatocyte uptake studies in drug discovery and enhance IVIVC for lipophilic acid compounds, analysis of a larger dataset will be necessary.

References: 1. Markus Trunzer, Joana Teigão, Felix Huth, Birk Poller, Sandrine Desrayaud, Raquel Rodríguez-Pérez, Bernard Faller, Improving In Vitro–In Vivo Extrapolation of Clearance Using Rat Liver Microsomes for Highly Plasma Protein-Bound Molecules, Drug Metabolism and Disposition, Volume 52, Issue 5, 2024, Pages 345-354, ISSN 0090-9556,
2. Heather Eng, Yi-an Bi, Mark A. West, Sangwoo Ryu, Emi Yamaguchi, Rachel E. Kosa, David A. Tess, David A. Griffith, John Litchfield, Amit S. Kalgutkar, Manthena V.S. Varma, Organic Anion–Transporting Polypeptide 1B1/1B3–Mediated Hepatic Uptake Determines the Pharmacokinetics of Large Lipophilic Acids: In Vitro–In Vivo Evaluation in Cynomolgus Monkey, The Journal of Pharmacology and Experimental Therapeutics, Volume 377, Issue 1, 2021, Pages 169-180, ISSN 0022-3565