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Research ArticleMetabolism, Transport, and Pharmacogenomics

Simultaneous Assessment of Uptake and Metabolism in Rat Hepatocytes: A Comprehensive Mechanistic Model

Karelle Ménochet, Kathryn E. Kenworthy, J. Brian Houston and Aleksandra Galetin
Journal of Pharmacology and Experimental Therapeutics April 2012, 341 (1) 2-15; DOI: https://doi.org/10.1124/jpet.111.187112
Karelle Ménochet
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Kathryn E. Kenworthy
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J. Brian Houston
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Aleksandra Galetin
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Abstract

Kinetic parameters describing hepatic uptake in hepatocytes are frequently estimated without appropriate incorporation of bidirectional passive diffusion, intracellular binding, and metabolism. A mechanistic two-compartment model was developed to describe all of the processes occurring during the in vitro uptake experiments performed in freshly isolated rat hepatocytes plated for 2 h. Uptake of rosuvastatin, pravastatin, pitavastatin, valsartan, bosentan, telmisartan, and repaglinide was investigated over a 0.1 to 300 μM concentration range at 37°C for 2 or 45–90 min; nonspecific binding was taken into account. All concentration-time points were analyzed simultaneously by using a mechanistic two-compartment model describing uptake kinetics [unbound affinity constant (Km,u), maximum uptake rate (Vmax), unbound active uptake clearance (CLactive,u)], passive diffusion [unbound passive diffusion clearance (Pdiff,u)], and intracellular binding [intracellular unbound fraction (fucell)]. When required (telmisartan and repaglinide), the model was extended to account for the metabolism [unbound metabolic clearance (CLmet,u)]. The CLactive,u ranged 8-fold, reflecting a 11-fold range in uptake Km,u, with telmisartan and valsartan showing the highest affinity for uptake transporters (Km,u <10 μM). Both Pdiff,u and fucell span over two orders of magnitude and reflected the lipophilicity of the drugs in the dataset. An extended incubation time allowed steady state to be reached between media and intracellular compartment concentrations and reduced the error in certain parameter estimates observed with shorter incubation times. Active transport accounted for >70% of total uptake for all drugs investigated and was 4- and 112-fold greater than CLmet,u for telmisartan and repaglinide, respectively. Modeling of uptake kinetics in conjunction with metabolism improved the precision of the uptake parameter estimates for repaglinide and telmisartan. Recommendations are made for uptake experimental design and modeling strategies.

Footnotes

  • K.M. was supported by a Ph.D studentship from GlaxoSmithKline, Ware, UK and the Biotechnology and Biological Sciences Research Council of the United Kingdom.

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    http://dx.doi.org/10.1124/jpet.111.187112.

  • ↵Embedded Image The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    NCE
    new chemical entity
    OATP
    organic anion-transporting protein
    ABT
    1-aminobenzotriazole
    M1
    2-despiperidyl-2-amino repaglinide
    M2
    2-despiperidyl-2(5-carboxypentylamine) repaglinide
    M4
    3′-hydroxy repaglinide
    fumed
    unbound fraction in the media
    fucell
    intracellular unbound fraction
    Pdiff
    passive diffusion clearance
    Pdiff,u
    unbound passive diffusion clearance
    Km,u
    unbound affinity constant
    Vmax
    maximum uptake rate
    CLactive,u
    unbound active uptake clearance
    CLuptake,u
    unbound total uptake clearance
    CLmet,u
    unbound metabolic clearance
    CLmet,gluc,u
    unbound metabolic clearance caused by the formation of repaglinide glucuronide
    CLmet,M2,u
    unbound metabolic clearance caused by the formation of M2
    LogD7.4
    distribution coefficient between octanol and water at pH 7.4
    CV
    coefficient of variation
    PBPK
    physiologically based pharmacokinetic
    DPBS
    Dulbecco's phosphate-buffered saline
    DMSO
    dimethyl sulfoxide
    LC
    liquid chromatography
    MS/MS
    tandem mass spectrometry
    HPLC
    high-performance liquid chromatography
    Scell
    total cell concentration
    Smed,u
    unbound media concentration
    rmse
    root mean square error
    gmfe
    geometric mean fold error.

  • Received August 19, 2011.
  • Accepted December 20, 2011.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 341 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 341, Issue 1
1 Apr 2012
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Research ArticleMetabolism, Transport, and Pharmacogenomics

Mechanistic Modeling of Uptake and Metabolism in Hepatocytes

Karelle Ménochet, Kathryn E. Kenworthy, J. Brian Houston and Aleksandra Galetin
Journal of Pharmacology and Experimental Therapeutics April 1, 2012, 341 (1) 2-15; DOI: https://doi.org/10.1124/jpet.111.187112

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Research ArticleMetabolism, Transport, and Pharmacogenomics

Mechanistic Modeling of Uptake and Metabolism in Hepatocytes

Karelle Ménochet, Kathryn E. Kenworthy, J. Brian Houston and Aleksandra Galetin
Journal of Pharmacology and Experimental Therapeutics April 1, 2012, 341 (1) 2-15; DOI: https://doi.org/10.1124/jpet.111.187112
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