The data indicate that rat and human show similar drug intestinal absorption profiles and similar transporter expression patterns in the small intestine, while the two species exhibit distinct expression levels and patterns for metabolizing enzymes in the intestine. Therefore, a rat model can be used to predict oral drug absorption in the small intestine of human, but not to predict drug metabolism or oral bioavailability in human.

TRPV6 is an endothelial calcium entry channel that is strongly expressed in breast adenocarcinoma tissue. In this study, we further confirmed this observation by analysis of breast cancer tissues, which indicated that TRPV6 mRNA expression was up-regulated between 2-fold and 15-fold compared with the average in normal breast tissue. Whereas TRPV6 is expressed in the cancer tissue, its role as a calcium channel in breast carcinogenesis is poorly understood. Therefore, we investigated how TRPV6 affects the viability, apoptosis, and calcium transport in the breast cancer cell line T47D. Hormones can also affect the tumor development; hence, we determined the effects of estradiol, progesterone, and 1,25-vitamin D on TRPV6 transcription. Interestingly, the estrogen receptor antagonist tamoxifen reduced expression of TRPV6 and is able to inhibit its calcium transport activity (IC 50 , 7.5 Mmol/L). The in vitro model showed that TRPV6 can be regulated by estrogen, progesterone, tamoxifen, and 1,25-vitamin D and has a large influence on breast cancer cell proliferation. Moreover, the effect of tamoxifen on cell viability was enhanced when TRPV6 expression was silenced with small interfering RNA.

A central concept in molecular bioscience is how structure formation at different length scales is achieved. Here we use spider silk protein as a model to design new recombinant proteins that assemble into fibers. We made proteins with a three-block architecture with folded globular domains at each terminus of a truncated repetitive silk sequence. Aqueous solutions of these engineered proteins undergo liquid–liquid phase separation as an essential pre-assembly step before fibers can form by drawing in air. We show that two different forms of phase separation occur depending on solution conditions, but only one form leads to fiber assembly. Structural variants with one-block or two-block architectures do not lead to fibers. Fibers show strong adhesion to surfaces and self-fusing properties when placed into contact with each other. Our results show a link between protein architecture and phase separation behavior suggesting a general approach for understanding protein assembly from dilute solutions into functional structures.

GeneChip analysis determined that 37, 47, and 44 percent of the 12,559 gene sequences were expressed in 4-day andl6-day Caco-2 cells and human duodenum, respectively. Comparing human duodenum with Caco-2 cells, more than 1,000 sequences were determined to have at least a 5-fold difference in expression. There were 26, 38, and 44 percent of the 443 transporters, channels, and metabolizing enzymes detected in 4-day, 16-day Caco-2 cells, and human duodenum, respectively. More than 70 transporters and metabolizing enzymes exhibited at least a 3-fold difference. The overall coefficient of variability of the 10 human duodenal samples for all expressed sequences was 31% (range 3% to 294%) while that of the expressed transporters and metabolizing enzymes was 33% (range 3% to 87%). The in vivo / in vitro drug permeability measurements correlated well for passively absorbed drugs (R2 = 85%). The permeability correlation for carrier-mediated drugs showed 3- 35-fold higher in human above the correlation of passively absorbed drugs. The 2- 595-fold differences in gene expression levels between the Caco-2 cells and human duodenum correlated with the observed 3- 35-fold difference in permeability correlation between carrier-mediated drugs and passively absorbed drugs. CONCLUSIONS; Significant differences in gene expression levels in Caco-2 cells and human duodenum were observed. The observed differences of gene expression levels were consistent with observed differences in carrier mediated drug permeabilities. Gene expression profiling is a valuable new tool for investigating in vitro and in vivo permeability correlation.

Gemcitabine, a clinically effective nucleoside anticancer agent, is a polar drug with low membrane permeability and is administered intravenously. Further, extensive degradation of gemcitabine by cytidine deaminase to an inactive metabolite in the liver affects its activity adversely. Thus, strategies that provide both enhanced transport and high metabolic bioevasion would potentially lead to oral alternatives that may be clinically useful. The objective of this study was to evaluate whether amino acid ester prodrugs of gemcitabine would (a) facilitate transport across intestinal membranes or across cells that express hPEPT1 and (b) provide resistance to deamination by cytidine deaminase. 3'-Monoester, 5'-monoester, and 3',5'-diester prodrugs of gemcitabine utilizing aliphatic (L-valine, D-valine, and L-isoleucine) and aromatic (L-phenylalanine and D-phenylalanine) amino acids as promoieties were synthesized and evaluated for their affinity and direct hPEPT1-mediated transport in HeLa/hPEPT1 cells. All prodrugs exhibited enhanced affinity (IC(50): 0.14-0.16 mM) for the transporter. However, only the 5'-L-valyl and 5'-L-isoleucyl monoester prodrugs exhibited (a) increased uptake (11.25- and 5.64-fold, respectively) in HeLa/hPEPT1 cells compared to HeLa cells and (b) chemical stability in buffers, that were comparable to valacyclovir, a commercially marketed oral amino acid ester prodrug. The widely disparate enzymatic bioconversion profiles of the 5'-L-valyl and 5'-L-isoleucyl prodrugs in Caco-2 cell homogenates along with their significant resistance to deamination by cytidine deaminase suggest that the disposition of gemcitabine following oral administration would be controlled by the rate of bioconversion following transport across the intestinal epithelial membrane. The combined results also suggest that it may be possible to modulate these characteristics by the choice of the amino acid promoiety.

No abstract

Floxuridine is a clinically proven anticancer agent in the treatment of metastatic colon carcinomas and hepatic metastases. However, prodrug strategies may be necessary to improve its physiochemical properties and selectivity and to reduce undesirable toxicity effects. Previous studies with amino acid ester prodrugs of nucleoside drugs targeted to the PEPT1 transporter coupled with recent findings of the functional expression of the PEPT1 oligopeptide transporter in pancreatic adenocarcinoma cell lines suggest the potential of PEPT1 as therapeutic targets for cancer treatment. In this report, we show the feasibility of achieving enhanced transport and selective antiproliferative action of amino acid ester prodrugs of floxuridine in cell systems overexpressing PEPT1. All prodrugs exhibited affinity for PEPT1 (IC 50 , 1.1 -2.3 mmol/L). However, only the prolyl and lysyl prodrugs exhibited enhanced uptake (2-to 8-fold) with HeLa/PEPT1 cells compared with HeLa cells, suggesting that the aspartyl prodrugs are PEPT1 inhibitors. The selective growth inhibition of Madine-Darby canine kidney (MDCK)/PEPT1 cells over MDCK cells by the prodrugs was consistent with the extent of their PEPT1-mediated transport. All ester prodrugs hydrolyzed to floxuridine fastest in Caco-2 cell and MDCK homogenates and slower in human plasma and were most chemically stable in pH 6.0 buffer. Prolyl and lysyl prodrugs were relatively less stable compared with aspartyl prodrugs in buffers and in cell homogenates. The results suggest that optimal design for targeted delivery would be possible by combining both stability and transport characteristics afforded by the promoiety. [Mol Cancer Ther 2005;4(4):659 -67]

Maternal-fetal calcium (Ca 2+ ) transport is crucial for fetal Ca 2+ homeostasis and bone mineralization. In this study, the physiological significance of the transient receptor potential, vanilloid 6 (TRPV6) Ca 2+ channel in maternal-fetal Ca 2+ transport was investigated using Trpv6 knockout mice. The Ca 2+ concentration in fetal blood and amniotic fluid was significantly lower in Trpv6 knockout fetuses than in wildtypes. The transport activity of radioactive Ca 2+ ( 45 Ca) from mother to fetuses was 40% lower in Trpv6 knockout fetuses than in wildtypes. The ash weight was also lower in Trpv6 knockout fetuses compared with wildtype fetuses. TRPV6 mRNA and protein were mainly localized in intraplacental yolk sac and the visceral layer of extraplacental yolk sac, which are thought to be the places for maternal-fetal Ca 2+ transport in mice. These expression sites were co-localized with calbindin D 9K in the yolk sac. In wildtype mice, placental TRPV6 mRNA increased 14-fold during the last 4 days of gestation, which coincides with fetal bone mineralization. These results provide the first in vivo evidence that TRPV6 is involved in maternal-fetal Ca 2+ transport. We propose that TRPV6 functions as a Ca 2+ entry pathway, which is critical for fetal Ca 2+ homeostasis.