Time matters – in vitro cellular disposition kinetics help rationalizing cellular potency disconnects

Abstract

1. Loss in potency is commonly observed in early drug discovery when moving from biochemical to more complex cellular systems. Among other factors, low permeability is often considered to cause such potency disconnects.

2. We developed a novel cellular disposition assay in MDCK cells to determine passive uptake clearance (PS_inf), cell-to-medium ratios at steady-state (K_p) and the time to reach 90% steady-state (TTSS₉₀) from a single experiment in a high-throughput format.

3. The assay was validated using 40 marketed drugs, showing a wide distribution of PS_inf and K_p values. The parameters generally correlated with transcellular permeability and lipophilicity, while PS_inf data revealed better resolution in the high and low permeability ranges compared to traditional permeability data. A linear relationship between the K_p/PS_inf ratio and TTSS₉₀ was mathematically derived and experimentally validated, demonstrating the dependency of TTSS₉₀ on the rate and extent of cellular accumulation.

4. Cellular disposition parameters could explain potency (IC₅₀) disconnects noted for seven Bruton’s tyrosine kinase degrader compounds in a cellular potency assay. In contrast to transcellular permeability, PS_inf data enabled identification of the compounds with IC₅₀ disconnects based on their time to reach equilibrium. Overall, the novel assay offers the possibility to address potency disconnects in early drug discovery.

Keywords:

• Permeability
• cellular uptake (PS_inf)
• potency disconnect
• free drug hypothesis
• cellular binding (K_p)
• protein degraders

Acknowledgements

The authors wish to acknowledge all Novartis scientists, who have supported this work. Special thanks go to Dallas Bednarczyk, Olivia Ledue, Julia Riede, Patrick Schweigler, Claudio Thoma for scientific input and critical evaluation of the manuscript.

Disclosure statement

All authors apart from S.W. are employees of Novartis, and all hold shares in the company apart from S.W. S.W. was employed by Novartis at the time of the study.