title: Mass spectrometry-based quantification of CYP enzymes to establish in vitro/in vivo scaling factors for intestinal and hepatic metabolism in beagle dog.
abstract: OBJECTIVE
  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.
authors.affiliation: Non-Clinical Safety, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, B70/R130, CH-4070 Basel, Switzerland.
authors.forename: Aki T
authors.initials: AT
authors.lastname: Heikkinen
authors.forename: Arno
authors.initials: A
authors.lastname: Friedlein
authors.forename: Jens
authors.initials: J
authors.lastname: Lamerz
authors.forename: Peter
authors.initials: P
authors.lastname: Jakob
authors.forename: Paul
authors.initials: P
authors.lastname: Cutler
authors.forename: Stephen
authors.initials: S
authors.lastname: Fowler
authors.forename: Tara
authors.initials: T
authors.lastname: Williamson
authors.forename: Roberto
authors.initials: R
authors.lastname: Tolando
authors.forename: Thierry
authors.initials: T
authors.lastname: Lave
authors.forename: Neil
authors.initials: N
authors.lastname: Parrott
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D004285:Dogs; D007422:Intestines / Q000737:chemistry / Q000201:enzymology*; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D013058:Mass Spectrometry; D008861:Microsomes / Q000737:chemistry / Q000201:enzymology*; D008969:Molecular Sequence Data; D013250:Steroid Hydroxylases / Q000032:analysis / Q000378:metabolism
chemical_list: D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C090278:cytochrome P-450 CYP2B11 (dog); C475779:cytochrome P-450 CYP3A12 (canine)