Use of selective substrates and inhibitors to rapidly characterise batches of cryopreserved primary human hepatocytes for assessment of active uptake liability in drug discovery and development

Abstract

1. The use of hepatocytes to predict human hepatic metabolic clearance is the gold standard approach. However whilst enzymes are well characterised, knowledge gaps remain for transporters. Furthermore, methods to study specific transporter involvement are often complicated by overlapping substrate specificity. Selective substrates and inhibitors would aid investigations into clinically relevant pharmacokinetic effects. However, to date no consensus has been reached.

2. This work defines selective hepatic uptake transporter substrates and inhibitors for the six main human hepatocyte transporters (OATP1B1, OATP1B3, OATP2B1, NTCP, OAT2 & OCT1), and demonstrates their use to rapidly characterise batches of human hepatocytes for uptake transporter activity. Hepatic uptake was determined across a range of substrate concentrations, allowing the definition of kinetic parameters and hence active and passive components. Systematic investigations identified a specific substrate and inhibitor for each transporter, with no overlap between the specificity of substrate and inhibitor for any given transporter.

3. Early characterisation of compound interactions with uptake transporters will aid in early risk assessment and chemistry design. Hence, this work further highlights the feasibility of a refined methodology for rapid compound characterisation for the application of static and dynamic models, for early clinical risk assessment and guidance for the clinical development plan.

Keywords:

• Transporters
• uptake
• clearance
• hepatocytes
• pharmacokinetics

Acknowledgements

The authors would like to acknowledge Shelby Barnett for assisting with the assay set-up and Margarida Sancho for suggesting PrestoBlue^TM as a cell recovery method. Syeda Shah is acknowledged for performing the bioanalysis, and Prabha Peramuhendige and Vishal Ranglani are acknowledged for their contribution to scientific discussions. The authors would also like to thank Phil Stanley for his advice and support with the statistical analyses.

Author contributions

Participated in research design: Golding, Light, Ménochet. Conducted experiments: Golding, Light. Performed data analysis: Golding, Light. Contributed to the writing of the manuscript: Golding, Light, Ménochet, Williamson.

Disclosure statement

The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.