Requirement of Podocalyxin in TGF-Beta Induced Epithelial Mesenchymal Transition

Epithelial mesenchymal transition (EMT) is characterized by the development of mesenchymal properties such as a fibroblast-like morphology with altered cytoskeletal organization and enhanced migratory potential. We report that the expression of podocalyxin (PODXL), a member of the CD34 family, is markedly increased during TGF-β induced EMT. PODXL is enriched on the leading edges of migrating A549 cells. Silencing of podocalyxin expression reduced cell ruffle formation, spreading, migration and affected the expression patterns of several proteins that normally change during EMT (e.g., vimentin, E-cadherin). Cytoskeletion assembly in EMT was also found to be dependent on the production of podocalyin. Compositional analysis of podocalyxin containing immunoprecipitates revealed that collagen type 1 was consistently associated with these isolates. Collagen type 1 was also found to co-localize with podocalyxin on the leading edges of migrating cells. The interactions with collagen may be a critical aspect of podocalyxin function. Podocalyxin is an important regulator of the EMT like process as it regulates the loss of epithelial features and the acquisition of a motile phenotype

Mediation of in Vitro Cytochrome P450 Activity by Common Pharmaceutical Excipients

Polymers and surfactants are commonly used as excipients in oral formulations and are generally considered to be inert. However, relatively few studies have assessed their interaction with enzymes involved in the absorption, distribution, metabolism, and elimination of drugs. We have investigated the impact of twenty-three commonly used excipients (ten polymers and thirteen surfactants) on seven cytochrome P450 (CYP450) isoforms using baculosome-derived CYP450 enzymes across a range of concentrations. Time-course fluorescent readings were then taken to generate IC50 (inhibition) or EC50 (activation) values for excipient effects on CYP450 activity. All excipients had an observable effect activity of at least one CYP450 isoform with the majority of excipients altering substrate metabolism of at least 57% of CYP450s studied. In addition, most excipients were capable of inhibiting and increasing activity of several different CYP450 isoforms. Although the majority of these effects required concentrations outside those achievable therapeutically (>100 μM), almost 20% were seen at concentrations below 100 μM, and these results indicate that several excipients have the potential to modify the pharmacokinetics of administered drugs