Establishment and Characterization of a Novel Caco-2 Subclone with a Similar Low Expression Level of Human Carboxylesterase 1 to Human Small Intestine s

ABSTRACT Caco-2 cells predominantly express human carboxylesterase 1 (hCE1), unlike the human intestine that predominantly expresses human carboxylesterase 2 (hCE2). Transport experiments using Caco-2 cell monolayers often lead to misestimation of the intestinal absorption of prodrugs because of this difference, as prodrugs designed to increase the bioavailability of parent drugs are made to be resistant to hCE2 in the intestine, so that they can be hydrolyzed by hCE1 in the liver. In the present study, we tried to establish a new Caco-2 subclone, with a similar pattern of carboxylase expression to human intestine, to enable a more accurate estimation of the intestinal absorption of prodrugs. Although no subclone could be identified with high expression levels of only hCE2, two subclones, #45 and #78, with extremely low expression levels of hCE1 were subcloned from parental Caco-2 cells by the limiting dilution technique. Unfortunately, subclone #45 did not form enterocyte-like cell monolayers due to low expression of claudins and b-actin. However, subclone #78 formed polarized cell monolayers over 4 weeks and showed similar paracellular and transcellular transport properties to parental Caco-2 cell monolayers. In addition, the intestinal transport of oseltamivir, a hCE1 substrate, could be evaluated in subclone #78 cell monolayers, including P-glycoprotein-mediated efflux under nonhydrolysis conditions, unlike parental Caco-2 cells. Consequently, it is proposed that subclone #78 may provide a more effective system in which to evaluate the intestinal absorption of prodrugs that are intended to be hydrolyzed by hCE1

Production of recombinant human dipeptidyl peptidase IV from Sf9 cells in microbial fermenters

WOS: 000368559100014The human dipeptidyl peptidase IV (hDPPIV/CD26) is expressed as an immune response in some cancer cells as well as intestine and incretin metabolism, and deficiency of the enzyme leads to metabolic disorders. In the present study, recombinant hDPPIV/CD26 genes were expressed in baculovirus-insect cell systems in a 5-L stirred-tank fermenter. Because of the shear sensitivity of the insect cell line, production from insect cells should be performed in new-generation type bioreactors, which are commonly more expensive than microbial fermenters. To optimize the process, hydrodynamic parameters and oxygen consumption of Sf9 cells at 1.5 L and 3 L were monitored, and a certain amount of serum was added to the production medium to decrease shear and stabilize the growth of insect cells that normally do not need serum addition. In this study, dimensionless numbers and some hydrodynamic parameters were calculated in 1.5 L, and predictions were made for 3 L fermenter volumes. Agitation rates of 60 rpm were determined to protect insect cells against damaging shear stress. Regarding the agitation rate, oxygen mass transfer coefficient (k(L)a) was 0.0129 min(-1) for 1.5 L and was kept constant for 3 L (0.0133 min(-1)). The maximum enzyme activity from microbial fermenters was 2.37-fold higher than activity from T-flask in our previous work. The infection efficiency of transfected cells was 78%-81% in the 1.5-L and 3-L fermenters