Critical review of p38 MAP kinase inhibitors: a bioanalytical perspective.

p38 MAP kinase is a key enzyme in the proinflammatory response and a large number of compounds have been studied as potential therapeutic drugs. This review summarizes the bioanalytical methods used for the analysis of p38 MAP kinase inhibitors, with a special focus on sample preparation and chromatographic analysis. Biological sample extraction techniques utilized included protein precipitation, liquid-liquid extraction and SPE. Applications include determinations of compounds in a variety of biological fluids and tissues. Extracted samples are typically separated by reverse-phase LC and quantitated either by UV or MS/MS detection. The benefits and limitations of each sample preparation strategy are discussed. The importance of chromatographic separation to avoid matrix effect and interference from endogenous compounds or drug-related biotransformation products are also discussed herein.

Novel potential interacting partners of fibronectin in spontaneous animal model of interstitial cystitis.

Feline idiopathic cystitis (FIC) is the only spontaneous animal model for human interstitial cystitis (IC), as both possess a distinctive chronical and relapsing character. Underlying pathomechanisms of both diseases are not clearly established yet. We recently detected increased urine fibronectin levels in FIC cases. The purpose of this study was to gain further insight into the pathogenesis by assessing interacting partners of fibronectin in urine of FIC affected cats. Several candidate proteins were identified via immunoprecipitation and mass spectrometry. Considerable changes in FIC conditions compared to physiological expression of co-purified proteins were detected by Western blot and immunohistochemistry. Compared to controls, complement C4a and thioredoxin were present in higher levels in urine of FIC patients whereas loss of signal intensity was detected in FIC affected tissue. Galectin-7 was exclusively detected in urine of FIC cats, pointing to an important role of this molecule in FIC pathogenesis. Moderate physiological signal intensity of galectin-7 in transitional epithelium shifted to distinct expression in transitional epithelium under pathophysiological conditions. I-FABP expression was reduced in urine and urinary bladder tissue of FIC cats. Additionally, transduction molecules of thioredoxin, NF-κB p65 and p38 MAPK, were examined. In FIC affected tissue, colocalization of thioredoxin and NF-κB p65 could be demonstrated compared to absent coexpression of thioredoxin and p38 MAPK. These considerable changes in expression level and pattern point to an important role for co-purified proteins of fibronectin and thioredoxin-regulated signal transduction pathways in FIC pathogenesis. These results could provide a promising starting point for novel therapeutic approaches in the future.