Development of a more rapid, reduced serum culture system for Caco-2 monolayers and application to the biopharmaceutics classification system.
The objectives were: (1) to develop a more rapid, reduced serum culture system for Caco-2 monolayers, relative to the traditional 21-day, 10% fetal bovine serum (FBS) system; and (2) to determine the biopharmaceutical drug classification of an oral therapeutic agent using this new system. Caco-2 cells were grown in the six well format on polycarbonate filters, in medium containing 2% iron supplemented calf serum (sCS) and a combination of growth factors and hormones. After 4 days in culture, permeabilities of three marker compounds (metoprolol, mannitol, and taurocholate) across monolayers were determined, and compared to permeabilities from the traditional 21-day, 10% FBS system, using cells at similar passage number. Cell morphology, degree of cell differentiation, and the presence of two efflux pumps were assessed. The 2% sCS model was also used to classify the permeability of an oral therapeutic agent as high or low. No difference in permeability was observed for metoprolol transport (P = 0.38) between the two culture methods, and the values obtained were independent of passage number of the cells. Mannitol permeability was about 2-fold higher from the 2% sCS system, as compared to the 10% FBS system. Taurocholate permeability was low indicating the 2% sCS culture at 4 days lacked this particular active transporter capability. Electron micrographs of cells grown in the 2% sCS system at 4 days revealed the presence of microvilli and tight junctions. P-glycoprotein and an efflux pump for furosemide were functionally present. The 2% sCS system indicated the oral therapeutic agent as highly permeable, which agreed with the 10% FBS system. This new system provides a rapid, accurate, and economical option for passive permeability determination, and appears to be applicable to the proposed Biopharmaceutics Classification System (BCS).
Lentz
K A
KA
School of Pharmacy, University of Maryland, Baltimore 21201, USA.
Hayashi
J
J
Lucisano
L J
LJ
Polli
J E
JE
D000319:Adrenergic beta-Antagonists / Q000493:pharmacokinetics; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D001702:Biopharmaceutics / Q000145:classification*; D018938:Caco-2 Cells; D002417:Cattle; D002455:Cell Division / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology; D002756:Cholagogues and Choleretics / Q000493:pharmacokinetics; D003470:Culture Media; D003584:Cytological Techniques; D004234:Diuretics, Osmotic / Q000493:pharmacokinetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008353:Mannitol / Q000493:pharmacokinetics; D008790:Metoprolol / Q000493:pharmacokinetics; D008854:Microscopy, Electron; D013656:Taurocholic Acid / Q000493:pharmacokinetics
D000319:Adrenergic beta-Antagonists; D015415:Biomarkers; D002756:Cholagogues and Choleretics; D003470:Culture Media; D004234:Diuretics, Osmotic; D008353:Mannitol; D013656:Taurocholic Acid; D000469:Alkaline Phosphatase; D008790:Metoprolol