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Multi-Well Array Culture of Primary Human Hepatocyte Spheroids for Clearance Extrapolation of Slowly Metabolized Compounds

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Abstract

Accurate prediction of human pharmacokinetics using in vitro tools is an important task during drug development. Albeit, currently used in vitro systems for clearance extrapolation such as microsomes and primary human hepatocytes in suspension culture show reproducible turnover, the utility of these systems is limited by a rapid decline of activity of drug metabolizing enzymes. In this study, a multi-well array culture of primary human hepatocyte spheroids was compared to suspension and single spheroid cultures from the same donor. Multi-well spheroids remained viable and functional over the incubation time of 3 days, showing physiological excretion of albumin and α-AGP. Their metabolic activity was similar compared to suspension and single spheroid cultures. This physiological activity, the high cell concentration, and the prolonged incubation time resulted in significant turnover of all tested low clearance compounds (n = 8). In stark contrast, only one or none of the compounds showed significant turnover when single spheroid or suspension cultures were used. Using multi-well spheroids and a regression offset approach (log(CLint) = 1.1 × + 0.85), clearance was predicted within 3-fold for 93% (13/14) of the tested compounds. Thus, multi-well spheroids represent a novel and valuable addition to the ADME in vitro tool kit for the determination of low clearance and overall clearance prediction.

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Abbreviations

α-AGP:

α1-acid glycoprotein

AAFE:

Absolute average fold error

ADME:

Absorption, distribution, metabolism, excretion

CLint :

Intrinsic clearance

CLint,obs :

Observed intrinsic clearance

CLint,pred :

Predicted intrinsic clearance

CLb :

In vivo clearance

CLb,obs :

Observed in vivo clearance

CLb,pred :

Predicted in vivo clearance

CYP:

Cytochrome P450

FBS:

Fetal bovine serum

fub :

Fraction unbound in blood

fuinc :

Fraction unbound in the incubation

fup :

Fraction unbound in plasma

IVIVE:

In vitro-in vivo extrapolation

LOOCV:

Leave one out cross-validation

MPCCs:

Micropattern co-cultures

PHH:

Primary human hepatocytes

pl:

Plasma

Qh :

Liver blood flow

Rb :

Blood-to-plasma ration

UGT:

Uridine 5'-diphospho-glucuronosyltransferase

ULA:

Ultra-low attachment

WSM:

Well-stirred model

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Funding

This study received financial support from the healthcare business of Merck KGaA (Darmstadt, Germany).

Author information

Authors and Affiliations

  1. Department of Drug Metabolism and Pharmacokinetics (DMPK), The Healthcare Business of Merck KGaA, Frankfurter Str. 250, 64293, Darmstadt, Germany

    Lena C. Preiss, Katrin Georgi & Carl Petersson

  2. Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

    Lena C. Preiss & Volker M. Lauschke

  3. Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany

    Volker M. Lauschke

  4. University of Tuebingen, Tuebingen, Germany

    Volker M. Lauschke

Authors
  1. Lena C. Preiss
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  2. Volker M. Lauschke
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  3. Katrin Georgi
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  4. Carl Petersson
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Contributions

Participated in research design: Georgi, Lauschke, Petersson and Preiss. Conducted experiments: Preiss. Performed data analysis: Preiss. Interpretation of data: Lauschke, Petersson, and Preiss. Wrote or contributed to the writing of the manuscript: Georgi, Lauschke, Petersson, and Preiss.

Corresponding author

Correspondence to Carl Petersson.

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Conflict of Interest

Katrin Georgi, Lena C. Preiss, and Carl Petersson were employed by the healthcare business of Merck KGaA (Darmstadt, Germany) when this study was conducted. Volker M. Lauschke is co-founder, CEO, and shareholder of HepaPredict AB, and co-founder and shareholder PersoMedix AB. In addition, Volker M. Lauschke discloses consultancy work for EnginZyme AB. The authors have no relevant additional financial interests.

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Preiss, L.C., Lauschke, V.M., Georgi, K. et al. Multi-Well Array Culture of Primary Human Hepatocyte Spheroids for Clearance Extrapolation of Slowly Metabolized Compounds. AAPS J 24, 41 (2022). https://doi.org/10.1208/s12248-022-00689-y

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