Quantitative prediction of oral absorption of PEPT1 substrates based on in vitro uptake into Caco-2 cells.

The method for predicting the fraction absorbed (Fa) of the PEPT1 substrates was established based on the in vitro uptake into Caco-2 cells. Uptake of a drug into Caco-2 cells was measured, and the carrier-mediated initial uptake clearance (DeltaCL uptake) was calculated as the difference between the uptake clearance in the absence of glycyl-sarcosine (Gly-Sar) and that in the presence of 30 mM Gly-Sar. The DeltaCL uptake of each drug was then divided by that of cephradine to obtain DeltaCL*uptake, which was a normalized parameter to correct for inter-day and/or inter-cell variability. Then, cephradine (CED), cefixime (CFIX), and cefotiam (CTM) were selected as marker compounds having excellent, medium and poor absorption, respectively. The DeltaCL*uptake and Fa values for CED, CFIX and CTM were fitted to the equation derived from the complete radial mixing (CRM) model, and the scaling factor (A') was obtained. Using the A' value, Fa was predicted from the DeltaCL*uptake value of each drug. Good correlation was observed between the predicted and reported Fa values, which demonstrated that Fa of PEPT1 substrates can be predicted based on the in vitro uptake in Caco-2 cells.

Chiral recognition of amethopterin enantiomers by the reduced folate carrier in Caco-2 cells.

Stereoselectivity of the human reduced folate carrier (RFC1) in Caco-2 cells was examined using methotrexate (L-amethopterin, L-MTX) and its antipode (D-amethopterin, D-MTX) as model substrates. The initial uptake rate of L-MTX into Caco-2 cells followed Michaelis-Menten kinetics with a Km value of approximately 1 microM. The Eadie-Hofstee plot of the RFC1-mediated L-MTX uptake showed that it was mediated by a single transport system, RFC1. Dixon plots revealed that L-MTX uptake was inhibited competitively by folic acid (FA), L-MTX and D-MTX, with Ki values of approximately 0.8, 1.5 and 180 microM, respectively. The results showed that the affinities of FA and L-MTX to RFC1 were approximately 120-fold greater than that of D-MTX. The uptake of L- and D-MTX into Caco-2 cells was also measured using LC-MS/MS analysis, which revealed that the L-MTX uptake was at least 7-fold greater than that of D-MTX. The present study revealed significant stereoselectivity of RFC1 toward amethopterin enantiomers with the L-isomer being much more favored.

Stereoselectivity of the reduced folate carrier in Caco-2 cells.

Stereoselectivity of the human reduced folate carrier (RFC1) was examined in Caco-2 cells using methotrexate (l-amethopterin or l-MTX) and its antipode (d-amethopterin or d-MTX) as model substrates. The initial uptake rate of folic acid (FA) was concentration dependent, with a K(m) value of approximately 0.6 microM. The Eadie-Hofstee plot of the RFC1-mediated FA uptake revealed a single component for FA uptake into Caco-2 cells, demonstrating that only RFC1 is involved in FA uptake. l-MTX inhibited FA uptake in a competitive manner with a K(i) value of approximately 2 microM, similar to the K(m) value of l-MTX. d-MTX also competitively inhibited FA uptake with a K(i) value being approximately 120 microM, indicating that the affinity of d-MTX is ca. 60-fold less than that of l-MTX. The stereoselectivity of human RFC1 observed in the present study was consistent not only with the stereoselectivity of rabbit RFC1 observed in rabbit intestinal brush border membrane vesicles but also with the reported differences in oral absorption of amethopterin enantiomers in humans.

Anti-atherogenic effect of soya and rice-protein isolate, compared with casein, in apolipoprotein E-deficient mice.

Our objective was to determine whether dietary plant proteins such as soya-protein isolate (SPI) and rice-protein isolate (RPI) compared with animal proteins, such as casein, could afford beneficial effects on atherosclerosis development in apolipoprotein E-deficient mice. In experiment 1, male and female mice were fed on a purified diet containing either casein, SPI or RPI for 9 weeks. The en face lesion area in the aorta (P<0.05) and the lesion size in the aortic root (P<0.05) in mice fed the casein-based diet were greater than those in the SPI or RPI groups. The plant protein groups had an increased concentration of serum l-arginine (P<0.05) and NO metabolites (NO2 plus NO3) (P<0.05) than did the casein group. The inhibitory effect of the plant proteins on the lesion formations was unrelated to gender and total serum cholesterol. In experiment 2, the l-arginine and l-methionine contents were the same in the l-arginine-supplemented casein-based and SPI-based diets, and between the l-methionine-supplemented SPI-based and the casein-based diets. Male mice were fed on the diets for 15 weeks. There were no significant differences in the en face lesion area and the lesion size between the casein group and the l-arginine-supplemented group, although the serum l-arginine (P<0.05) and NO2 plus NO3 (P<0.05) concentrations in the supplemented group were higher than those in the casein group. There were no significant effects of l-methionine supplementation on the lesion formations. In experiment 3, male mice were given the casein-based diet or the l-arginine-supplemented casein-based diet together with water or water containing an NO synthesis inhibitor for 9 weeks. When given the casein-based diet, the inhibitor drinking, compared with water drinking, resulted in a reduction of the serum NO2 plus NO3 concentration (P<0.01) and an increase in the en face lesion area (P<0.05) and the lesion size (P<0.01). When given the l-arginine-supplemented diet, the inhibitor drinking, compared with water drinking, resulted in no increase in the lesion area and size. These results demonstrate anti-atherogenic potentials of SPI- as well as RPI-derived proteins, but their l-arginine and l-methionine contents were not sufficient enough to explain the underlying mechanism(s).