Hepatocyte Uptake Assay

SOLVO’s Hepatocyte Uptake assays use commercially available cryopreserved hepatocytes from human as well as several preclinical species (Rat, Mouse, Dog and Cynomolgus Monkey). The assay has been characterized for each species in our laboratories and data is available on prototypical substrates and inhibitors. A hepatocyte uptake assay format relying on measuring uptake in suspension hepatocytes that are centrifuged through a layer of oil to separate from incubation medium has historically been used in the field. As this so-called oil-spin method is a low throughput and technically challenging assay, alternatives have been developed spurred by availability of better quality plateable hepatocytes. Recently, the preferred approach is therefore using a plated assay format as it is more universally applicable than the previously more common oil-spin method. In addition to its easier adaptability to screen-like setups, the plated approach also showed significantly lower risk of carry-over due to drug non-specifically absorbing to the cell surface than observed in the oil-spin approach, yielding more reliable data outputs [3]. Plated cells can also be incubated for longer periods with cell-to-medium concentration ratios remaining relatively stable, allowing for hepatocyte-to-medium unbound concentration ratio (K_p,uu) determination [3]. The application of this approach is only limiting for highly hydrophilic compounds with low CL_int (>3 μl/mg/min) and low intracellular binding, where the oil-spin method, that is still available at Solvo upon request, may be requested for such compounds.

There are several approaches to determine hepatic uptake in vitro, however, the common principle involves separation of cells and media and monitoring of drug concentrations in either the cells or in both. In vitro uptake assays can be performed by using multiple formats: suspension (oil-spin method), plated hepatocytes and sandwich cultured hepatocytes. It has been demonstrated that the uptake rates (K_m) as well as the IC₅₀ values of selected probe substrates and inhibitors, respectively, were comparable across the three assay formats. Due to its easier handling and better adaptability for screening-like studies, SOLVO’s go-to approach to Hepatocyte Uptake assays is the plated format, while the oil-spin approach remains also available. Sandwich cultured hepatocytes represent a more complex assay system that allows investigation of both hepatic uptake and efflux and are run in custom setups using B-clear technology.

Transporter functionality in the hepatocytes is confirmed during the assay for OATP1B1, OATP1B3, NTCP, OCT1 and OAT2 (or their respective preclinical species orthologs) using a selection of thoroughly characterized probe substrates. Intracellular accumulation of the TA is compared between active transport condition and passive uptake only, and net transporter-mediated uptake is defined as the difference between passive and active rates. In both plated and suspension assay formats, passive transport can be determined by using low temperature (0-4°C) incubations, however this approach yields underestimated passive uptake clearance values, because membrane is sturdier and less permeable due to the low temperatures [10]. Chemical inhibition of active uptake has been shown to lead to better estimations of passive uptake, likely due to the inhibition of any residual uptake transport activity that may persist at 4°C, while it also allows assessment of transporter-mediated hepatic uptake without artificially introduced changes in membrane fluidity in the assay process. Therefore, by default, SOLVO’s Hepatocyte Uptake assay applies an inhibitor cocktail to obtain a passive transport condition, while the low-temperature control can be added upon request. Estimation of the impact of a specific transporter on the overall uptake process may be generated through comparison of the effect of a single transporter-specific inhibitor versus the complete inhibitor cocktail. In case hepatic enzyme-mediated metabolism is considerable for a test compound even over a few-minutes incubation time, inhibition of CYP enzyme activity (e.g., addition of 1-ABT and or other metabolizing enzyme inhibitors) in the assay system is also possible to ensure the uptake information obtained only reflects the effect of transport processes.

Major reference compounds for which characterization data is available include the following prototypical substrates and inhibitors:

The same probes and inhibitors are applied for human transporters and preclinical orthologs. In the Hepatocyte uptake assays, single or multiple probe substrates can be run along with a test article as positive control if available, and the use of multiple inhibitors may also be requested.

Buffer containing the Test Article (TA) (using single or multiple concentrations) is added to start uptake. After incubation (e.g., using 3-4 time-points), the solution is removed, and the plates are washed to remove non-accumulated TA from the wells. Hepatocytes are lysed, and intracellular TA accumulation in the cell lysate is determined using LC-MS analysis. In case of radiolabeled TAs (³H- or ¹⁴C-) readout by liquid scintillation counting (LSC) is also available, reference probe substrates are always quantified using LSC. Cell viability is always determined before the assay both for oil spin and plated methods, and in plated assay, protein content is also determined at the end of the experiment. Optionally, TA recovery in the assay may also be assessed. The Hepatocyte Uptake assay design is flexible and can be tailored to your project needs to for example determine kinetics or assess uptake in the presence of inhibitors.

Human cryopreserved hepatocytes used for a study are typically pooled donor, mixed-gender hepatocytes to get a general experiment, however on request a specific lot with characteristics specific for project needs can be requested (e.g., single or multiple donors, gender, etc.). In case of preclinical species, it is generally thought to be less critical to use several donors as animals are homogeneous and kept under standard laboratory conditions.

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References

[1] Yoshikado T, Lee W, Toshimoto K, Morita K, Kiriake A, Chu X, Lee N, Kimoto E, Varma MVS, Kikuchi R, Scialis RJ, Shen H, Ishiguro N, Lotz R, Li AP, Maeda K, Kusuhara H, Sugiyama Y. Evaluation of Hepatic Uptake of OATP1B Substrates by Short Term-Cultured Plated Human Hepatocytes: Comparison with Isolated Suspended Hepatocytes. J Pharm Sci. 2021 Jan;110(1):376-387. doi: 10.1016/j.xphs.2020.10.041. Epub 2020 Oct 27. PMID: 33122051.

[2] Bi Y, Scialis RJ, Lazzaro S, Mathialagan S, Kimoto E, Keefer J, Zhang H, Vildhede AM, Costales C, Rodrigues D, Tremaine LM, V. S. Varma MVS. Reliable Rate Measurements for Active and Passive Hepatic Uptake Using Plated Human Hepatocytes. AAPS J, 2017. 19:787-796.