The lack of validated subcellular intestinal scaling factors currently limits prediction of first-pass metabolism and bioavailability of orally administered xenobiotics through in vitro-in vivo extrapolation (IVIVE). Therefore, the objective of this study was to ascertain and validate microsomal scaling factors for quantifying intestinal metabolism in vivo
Following initial difficulties in using a recently published method
, previously reported superiority in activity of microsomes prepared via elution verses scraping
, and limitations through lack of standardized methods in the literature, the process of enterocyte preparation via the elution method was evaluated and optimized using the rat. Alterations in conditions for enterocyte isolation (“incubation time” and “EDTA concentration”), homogenization (20, 60 and 100% amplitude sonication) and sub-cellular fraction preparation (addition of heparin and/or glycerol) were systematically evaluated. In order to compare variant effects, the protein yields were normalized and microsome quality was analysed by measurement of cytochrome P450 (CYP450)
. The results indicated that
comparable CYP450 normalized protein yields were obtained irrespective of a
lterations to the preparation of intestinal microsomes post enterocyte isolation. However, variability was positively associated with the severity of homogenization (CV= 7.3, 7.2 and 22%, reported for 20, 60 and 100% amplitude sonication respectively), an effect increased by use of glycerol (CV=77%). The addition of heparin decreased this variability, but subsequently reduced overall specific CYP450 contents (reduced <40%). Enterocyte isolation was implicated as the most critical determinant of the intestinal scaling factor value, since mean normalised protein yield varied over 2 fold when experimental steps applied to the enterocytes were altered. In conclusion, a reproducible method based on elution of intestinal enterocytes was developed. The optimal balance between protein yield and functional CYP450, based on the degree of homogenization and microsomal purification conditions, should be carefully considered for provision of functional microsomes and a reproducible scaling factor. The technique applied for enterocyte isolation was shown to be a critical aspect in determining an intestinal scalar. Current investigation is focused on IVIVE using the derived intestinal scaling factor applied to a selected set of model reference compounds to evaluate its accuracy in predicting intestinal clearance within the Qgut model 
1. Bruyere, A., et al., Development of an optimized procedure for the preparation of rat intestinal microsomes: comparison of hepatic and intestinal microsomal cytochrome P450 enzyme activities in two rat strains. Xenobiotica, 2009. 39(1): p. 22-32.
2. Galetin, A. and J.B. Houston, Intestinal and hepatic metabolic activity of five cytochrome P450 enzymes: impact on prediction of first-pass metabolism. J Pharmacol Exp Ther, 2006. 318(3): p. 1220-9.
3. Wilson, Z.E., et al., Inter-individual variability in levels of human microsomal protein and hepatocellularity per gram of liver. Br J Clin Pharmacol, 2003. 56(4): p. 433-40.
4. Yang, J., et al., Prediction of intestinal first-pass drug metabolism. Curr Drug Metab, 2007. 8(7): p. 676-84.