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Characterization and Applications of Permeabilized Hepatocytes in Drug Discovery

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Abstract

Hepatocytes are one of the most physiologically relevant in vitro liver systems for human translation of clearance and drug-drug interactions (DDI). However, the cell membranes of hepatocytes can limit the entry of certain compounds into the cells for metabolism and DDI. Passive permeability through hepatocytes can be different in vitro and in vivo, which complicates the human translation. Permeabilized hepatocytes offer a useful tool to probe mechanistic understanding of permeability-limited metabolism and DDI. Incubation with saponin of 0.01% at 0.5 million cells/mL and 0.05% at 5 million cells/mL for 5 min at 37°C completely permeabilized the plasma membrane of hepatocytes, while leaving the membranes of subcellular organelles intact. Permeabilized hepatocytes maintained similar enzymatic activity as intact unpermeabilized hepatocytes and can be stored at −80°C for at least 7 months. This approach reduces costs by preserving leftover hepatocytes. The relatively low levels of saponin in permeabilized hepatocytes had no significant impact on the enzymatic activity. As the cytosolic contents leak out from permeabilized hepatocytes, cofactors need to be added to enable metabolic reactions. Cytosolic enzymes will no longer be present if the media are removed after cells are permeabilized. Hence permeabilized hepatocytes with and without media removal may potentially enable reaction phenotyping of cytosolic enzymes. Although permeabilized hepatocytes work similarly as human liver microsomes and S9 fractions experimentally requiring addition of cofactors, they behave more like hepatocytes maintaining enzymatic activities for over 4 h. Permeabilized hepatocytes are a great addition to the drug metabolism toolbox to provide mechanistic insights.

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Abbreviations

AKR:

Aldo-keto reductase

AO:

Aldehyde oxidase

Caco-2:

Human colorectal adenocarcinoma cells

CBR1:

Carbonyl reductase 1

CES:

Carboxylesterase

CLint :

Intrinsic clearance

CLint ,app :

Apparent intrinsic clearance

CO2 :

Carbon dioxide

CYP:

Cytochrome P450

Da:

Dalton

DDI:

Drug-drug interactions

DMSO:

Dimethyl sulfoxide

ECCS:

Extended clearance classification system

ER:

Endoplasmic reticulum

GST:

Glutathione transferase

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HHEP:

Human hepatocytes

HLM:

Human liver microsomes

HPLC:

High-performance liquid chromatography

IC50 :

Half-maximal inhibitory concentration

IVIVE:

In vitro-in vivo extrapolation

LC-MS/MS:

Liquid chromatography with tandem mass spectrometry

MDCK-LE:

Madin-Darby canine kidney-low efflux cells

Na2CO3 :

Sodium carbonate

NADP:

Nicotinamide adenine dinucleotide phosphate

NADPH:

Nicotinamide adenine dinucleotide phosphate, reduced form

NAT:

N-acetyltransferase

Papp :

Apparent permeability

PK:

Pharmacokinetics

rpm:

Revolutions per minute

RT:

Room temperature

SULT:

Sulfotransferase

UDPGA:

Uridine diphosphate glucuronic acid

UGT:

UDP-glucuronosyltransferase

w/v:

Weight/volume

WEM:

Williams E medium

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Acknowledgements

The authors greatly appreciate the help of Sophia M. Shi in editing the manuscript.

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Author notes

  1. Li Di

    Present address: Recursion Pharmaceuticals, Salt Lake City, Utah, 84101, USA

Authors and Affiliations

  1. Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Groton, Connecticut, 06340, USA

    Sam Zhang, Christine C. Orozco, Lloyd Wei Tat Tang, Jillian Racich, Anthony A. Carlo, George Chang, David Tess, Christopher Keefer & Li Di

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Contributions

Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work: SZ, CO, LT, JR, AC, GC, DT, CK, LD

Drafting the work or revising it critically for important intellectual content: SZ, GC, LD

Final approval of the version to be published: SZ, CO, LT, JR, AC, GC, DT, CK, LD

Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: SZ, CO, LT, JR, AC, GC, DT, CK, LD

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Correspondence to Li Di.

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Authors are employees of Pfizer Inc., New York, NY, USA, and may hold Pfizer stocks.

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Zhang, S., Orozco, C.C., Tang, L.W.T. et al. Characterization and Applications of Permeabilized Hepatocytes in Drug Discovery. AAPS J 26, 38 (2024). https://doi.org/10.1208/s12248-024-00907-9

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