Hepatic uptake assay

Accurately determine the hepatic intrinsic clearance of NCEs taking into account the active uptake into hepatocytes.

The hepatocyte uptake assay is in our portfolio of in vitro ADME services. We deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Accurate measurement of hepatic uptake using the media loss method

Intrinsic clearance can be influenced by several processes including hepatic uptake, efflux, biliary excretion and drug metabolism².
The predominant transporters involved in human hepatic uptake include OATPs, NTCP, OCTs and OATs³. These transporters determine intracellular concentrations which can influence clearance as well as potential DDI and hepatotoxicity.
Inter-individual variability in hepatic uptake is also likely for substrates of hepatic uptake transporters which exhibit polymorphisms.
Through its parent company, Evotec, Cyprotex are able to offer a hepatic uptake assay which utilises the media loss⁴ approach, and determines the hepatic uptake intrinsic clearance.

Experiences of sub-optimal drug exposure due to drug transporter interplay have supported incorporation of studies aimed at understanding the interactions between compound and drug transporters much earlier in drug discovery.

¹Riley RJ, Foley SA, Barton P, Soars MG & Williamson B (2016) Expert Opin Drug Metab Toxicol 12(2); 201-216

Protocol

Hepatic uptake assay protocol

Cells	Cryopreserved rat hepatocytes
Test Compound Concentration	1 µM (different concentrations available)
Method	Media loss
Incubation Time	0, 0.17, 0.5, 1, 1.5, 2, 5, 10, 20, 30, 60 min
Replicates	n=2
Compounds Requirements	50 μL of 10 mM solution
Analysis Method	LC-MS/MS quantification
Assay Controls	Atorvastatin (positive control for uptake) Dextromethorphan (negative control for uptake and positive control for CYP activity)
Data Delivery	Uptake intrinsic clearance (CL_int,uptake) (µL/min/x10⁶ cells)

Data

Data from the hepatic uptake assay

Figure 1
Relationship between rat uptake intrinsic clearance (using Evotec’s hepatic uptake assay) with values reported in the literature^2,5,6,7,8,9 for a set of 14 compounds.

In addition to robust human in vitro data, confidence in understanding and predicting preclinical species in vivo clearance is essential before extrapolation to human in vivo clearance for NCEs¹⁰

To gain insight and understanding into how transporter mechanisms that may contribute to clearance in vivo, early data are often generated in preclinical species such as the rat. Further, the human transporters OATP1B1 and OATP1B3 are orthologous to the rodent specific transporter Oatp1b2¹¹.

Figure 2
Correlation of rat in vitro and in vivo intrinsic clearance for a set of 17 test compounds determined using A). a standard suspension rat hepatocyte stability assay and B). the media loss assay.

The data generated by Evotec are in broad agreement with those reported in the literature from a range of labs as illustrated in Figure 1. Further, in contrast to the standard suspension hepatocyte stability model (Figure 2A), the scaled in vitro rat uptake intrinsic clearance data from the Evotec model demonstrates a strong correlation with in vivo rat intrinsic clearance (Figure 2B) demonstrating the advantages of the media loss approach.

References

¹Riley RJ et al., (2016) Hepatic drug transporters: the journey so far. Expert Opin Drug Metab Toxicol 12(2); 201-216
²Maeda K and Sugiyama Y (2013) Prediction of hepatic transporter-mediated drug-drug interaction from in vitro data. In Transporters in Drug Development – Discovery, Optimization, Clinical Study and Regulation. Ed. Sugiyama Y and Steffansen B. 121-154
³Annaert P et al (2007) Drug Transporters in the Liver. In Drug Transporters: Molecular Characterization and Role in Drug Disposition. Ed. You G and Morris ME. 359-410
⁴Soars MG et al., (2007) Use of hepatocytes to assess the contribution of hepatic uptake to clearance in vivo. Drug Metab Dispos 35(6); 859-865
⁵Ishiguro N et al., (2006) Predominant contribution of OATP1B3 to the hepatic uptake of telmisartan, an angiotensin II receptor antagonist, in humans. Drug Metab Dispos 34(7); 1109-1115
⁶Paine SW et al., (2008) Prediction of the pharmacokinetics of atorvastatin, cerivastatin, and indomethacin using kinetic models applied to isolated rat hepatocytes. Drug Metab Dispos 36(7); 1365-1374
⁷Gardiner P and Paine SW (2011) The impact of hepatic uptake on the pharmacokinetics of organic anions. Drug Metab Dispos 39(10); 1930-1938
⁸Ménochet K et al., (2012) Use of mechanistic modelling to assess interindividual variability and interspecies differences in active uptake in human and rat hepatocytes. Drug Metab Dispos 40(9); 1744-1756
⁹Ménochet K et al., (2012) Simultaneous assessment of uptake and metabolism in rat hepatocytes: a comprehensive mechanistic model. J Pharmacol Exp Ther 341(1); 2-15
¹⁰Grime K and Riley RJ (2006) The impact of in vitro binding on in vitro-in vivo extrapolations, projections of metabolic clearance and clinical drug-drug interactions. Curr Drug Metab 7; 251-264
¹¹Hagenbuch B & Meier PJ (2003) The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta 1609(1); 1-18