Novel substituted isoxazole FXR agonists with cyclopropyl, hydroxycyclobutyl and hydroxyazetidinyl linkers: Understanding and improving key determinants of pharmacological properties.

Several isoxazole-containing series of FXR agonists have been published over the last 15years, subsequent to the prototypical amphiphilic 'hammerhead'-type structure that was originally laid out by GW4064, the first potent synthetic FXR agonist. A set of novel compounds where the hammerhead is connected to the terminal carboxylic acid-bearing aryl or heteroaryl moiety by either a cyclopropyl, a hydroxycyclobutyl or a hydroxyazetidinyl linker was synthesized in order to improve upon the ADME properties of such isoxazoles. The resulting compounds all demonstrated high potencies at the target receptor FXR but with considerable differences in their physicochemical and in vivo profiles. The structure-activity relationships for key chemical features that have a major impact on the in vivo pharmacology of this series are discussed.

Evaluation of novel acute urinary rat kidney toxicity biomarker for subacute toxicity studies in preclinical trials.

Novel urinary protein biomarkers for the detection of acute renal damage, recently accepted by the U.S. Food and Drug Administration, European Medicines Agency, and Pharmaceuticals and Medical Devices Agency (Japan), now have to be validated in practice. Limited data regarding the performance of these acute markers after subacute or subchronic treatment are publicly available. To increase the area of applicability of these markers, it is important to evaluate the ability to detect them after 28 days of treatment or even longer. Wistar rats were treated with three doses of cisplatin, vancomycin, or puromycin to induce renal damage. Twelve candidate proteins were measured by Luminex xMAP-based WideScreen assays, MesoScale Discovery-based MULTI-SPOT technology, or RENA-strip dipstick assay after 28 days. Treatment with all three model compounds resulted in a dose-dependent increase in urinary biomarkers, specific for the observed areas within the nephron, determined histopathologically. The most promising biomarkers in this study were NGAL, Kim-1, osteopontin, clusterin, RPA-1, and GSTYb1, detected by multiplexing technologies. The RENA-strip dipstick assay delivered good diagnostic results for vancomycin-treated but not for cisplatin- or puromycin-treated rats. Taken together, the data show that these new biomarkers are robust and measurable for longer term studies to predict different types of kidney toxicities.

Cell cycle quiescence can suppress transcription from an ecdysone receptor-based inducible promoter in mammalian cells.

Inducible gene expression is a powerful tool for basic research, gene therapy and biotechnology, whose utility depends in part on consistent levels of induction regardless of metabolic status or physiological context. Here we examined the inducibility of the ecdysone receptor-based RheoSwitch mammalian inducible expression system in proliferating cells and in cell cycle-arrested cells. We found that both contact inhibition and growth arrest subsequent to serum deprivation dramatically reduced the levels of induction of reporter genes that could be achieved in 3T3 fibroblasts but in not NMuMG mammary epithelial cells. These data have implications for the use of the RheoSwitch system in inducible gene expression applications.