Therefore, in vitro and in vivo metabolism of these radiotracers was investigated.
Rat and human liver microsomal incubations, baboon plasma and rat brain extracts were analyzed by radio-HPLC and LC-MS-MS.
Results: SRT1720 purchase In vitro experiments demonstrated the formation by P450s of five polar metabolites. The main routes of LBT-999 metabolism proposed were N-dealkylation, tolyl-hydroxylation and dealkylation plus tolyl-hydroxylation. In vivo in baboons, [F-18]LBT-999 was rapidly converted into a [F-18]hydroxylated metabolite likely oxidized in plasma into a [F-18]carboxylic acid and into unlabeled N-dealkyl-LBT-999. The latter was detected in baboon plasma and in rat brain by LC-MS-MS. The time course of unchanged [F-18]LBT-999 decreased rapidly in plasma and was higher than that of [C-11]LBT-999 due to the formation of unlabeled N-dealkyl-LBT-999. In rats, striatum-to-cerebellum ratios of [F-18]LBT-999, [F-18]hydroxylated and [F-18]acidic metabolite were 20, 4.2 and 1.65, respectively, suggesting a possible accumulation of the
hydroxylated compound in the striatum.
Conclusion: P450s catalyzed the formation of dealkylated and hydroxylated metabolites of LBT-999. In baboons, an extensive metabolism of [F-18]LBT-999, with formation of unlabeled N-dealkyl-LBT-999, [F-18]fluorobutenaldehyde BAY 11-7082 mw (or its oxidation product) and [F-18]hydroxy-LBT-999 able to penetrate the brain, prevented an easy and accurate estimation of the input function of the radiotracer. CYP3A4 being the main P450 involved in the metabolism of LBT-999, a similar pathway may occur in humans and confound PET quantification. (C) 2012 Elsevier Inc. All rights reserved.”
“Filamentous phage do not display cytoplasmic proteins very effectively. As T7 is a cytoplasmic phage, Succinyl-CoA released by cell lysis, it has been prospected as being more efficient for the display of such proteins. Here we investigate this proposition, using a family of GFP-based cytoplasmic proteins that are poorly expressed by traditional
phage display. Using two single-molecule detection techniques, fluorescence correlation spectroscopy and anti-bunching, we show that the number of displayed fluorescent proteins ranges from one to three. The GFP derivatives displayed on T7 contain binding loops able to recognize specific targets. By mixing these in a large background of non-binders, these derivatives were used to optimize selection conditions. Using the optimal selection conditions determined in these experiments, we then demonstrated the selection of specific binders from a library of GFP clones containing heavy chain CDR3 antibody binding loops derived from normal donors inserted at a single site. The selected GFP-based binders were successfully used to detect binding without the use of secondary reagents in flow cytometry, fluorescence-linked immunosorbant assays and immunoblotting.