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Mass Spectrometry-Based Quantification of CYP Enzymes to Establish In Vitro/In Vivo Scaling Factors for Intestinal and Hepatic Metabolism in Beagle Dog

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ABSTRACT

Purpose

Physiologically based models, when verified in pre-clinical species, optimally predict human pharmacokinetics. However, modeling of intestinal metabolism has been a gap. To establish in vitro/in vivo scaling factors for metabolism, the expression and activity of CYP enzymes were characterized in the intestine and liver of beagle dog.

Methods

Microsomal protein abundance in dog tissues was determined using testosterone-6β-hydroxylation and 7-hydroxycoumarin-glucuronidation as markers for microsomal protein recovery. Expressions of 7 CYP enzymes were estimated based on quantification of proteotypic tryptic peptides using multiple reaction monitoring mass spectrometry. CYP3A12 and CYP2B11 activity was evaluated using selective marker reactions.

Results

The geometric mean of total microsomal protein was 51 mg/g in liver and 13 mg/cm in intestine, without significant differences between intestinal segments. CYP3A12, followed by CYP2B11, were the most abundant CYP enzymes in intestine. Abundance and activity were higher in liver than intestine and declined from small intestine to colon.

Conclusions

CYP expression in dog liver and intestine was characterized, providing a basis for in vitro/in vivo scaling of intestinal and hepatic metabolism.

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Abbreviations

CYP:

cytochrome P450

DIM:

dog intestinal microsomes

DLM:

dog liver microsomes

LCMS:

liquid chromatography – mass spectrometry

MRM:

multiple reaction monitoring

PBPK:

physiologically based pharmacokinetics

PK:

pharmacokinetics

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ACKNOWLEDGMENTS & DISCLOSURES

The authors thank Pascal Schenk and Paul Schmid for their contribution in setting up the analytical methods for diazepam metabolites.

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Authors and Affiliations

  1. Non-Clinical Safety, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, B70/R130, CH-4070, Basel, Switzerland

    Aki T. Heikkinen, Stephen Fowler, Thierry Lave & Neil Parrott

  2. Translational Research Sciences, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland

    Arno Friedlein, Jens Lamerz, Peter Jakob & Paul Cutler

  3. Celsis In Vitro Technologies, Baltimore, Maryland, USA

    Tara Williamson

  4. Celsis In Vitro Technologies GmbH, Neuss, Germany

    Roberto Tolando

Authors
  1. Aki T. Heikkinen
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  2. Arno Friedlein
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  4. Peter Jakob
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  5. Paul Cutler
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  10. Neil Parrott
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Correspondence to Neil Parrott.

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Heikkinen, A.T., Friedlein, A., Lamerz, J. et al. Mass Spectrometry-Based Quantification of CYP Enzymes to Establish In Vitro/In Vivo Scaling Factors for Intestinal and Hepatic Metabolism in Beagle Dog. Pharm Res 29, 1832–1842 (2012). https://doi.org/10.1007/s11095-012-0707-7

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  • DOI: https://doi.org/10.1007/s11095-012-0707-7

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