Bioanalysis of N-acetyl-aspartyl-glutamate as a marker of glutamate carboxypeptidase II inhibition.

We report the characterization of two methods for the analysis of N-acetyl-aspartyl-glutamate (NAAG) in biological fluids. In the first method, NAAG concentrations were calculated based on differences between glutamate concentrations before and after NAAG hydrolysis with exogenous glutamate carboxypeptidase II (GCP II) using high-performance liquid chromatography (HPLC) followed by fluorescence detection. In the second method, NAAG levels were quantified directly using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analyses of NAAG levels in human cerebrospinal fluid samples using either method gave similar results within experimental error, confirming the validity of the two independent measurements. These methods will be useful in future clinical trials to assess drug-induced GCP II inhibition in biological matrices.

Structure-activity relationship study and drug profile of N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (SJA6017) as a potent calpain inhibitor.

Novel N-arylsulfonyldipeptidyl aldehyde derivatives were prepared by DMSO oxidation from the corresponding dipeptide alcohol, and their potencies as calpain inhibitors were evaluated in vitro. Among them, N-(4-fluorophenylsulfonyl)-l-valyl-l-leucinal (8, SJA6017) potently inhibited calpains. 8 also inhibited cathepsin B and L but did not inhibit other cysteine proteases (interleukin 1beta-converting enzyme), serine proteases (trypsin, chymotrypsin, thrombin, factor VIIa, factor Xa), or proteasome. Preliminary cytotoxicity studies of 8 exhibited a relatively safe profile.

In vitro drug metabolism using liver microsomes.

Metabolic biotransformation of a drug can increase the rate of elimination from the body and have a significant effect on efficacy and safety. Drug candidates are screened early in the discovery process for metabolic stability using liver microsomes. Methods for microsome isolation and characterization and the determination of metabolic stability are presented.

ERK1/2 and Egr-1 contribute to increased TNF-alpha production in rat Kupffer cells after chronic ethanol feeding.

Activation of Kupffer cells by lipopolysaccharide (LPS) is a critical step in the pathogenesis of alcoholic liver disease. Kupffer cells isolated from rats fed ethanol in their diet for 4 wk accumulated 4.3-fold more tumor necrosis factor (TNF)-alpha in response to LPS compared with pair-fed rats. In contrast, LPS-stimulated interleukin (IL)-1 accumulation was 50% lower after ethanol feeding. LPS-stimulated TNF-alpha mRNA accumulation was twofold higher after ethanol feeding, whereas IL-1beta mRNA accumulation was blunted. To understand the mechanisms for this differential response, we investigated the effects of ethanol on LPS-dependent signal transduction. Chronic ethanol feeding increased LPS-stimulated extracellular receptor-activated kinases 1/2 (ERK1/2) activation. Activation of ERK1/2 was required for maximal increases in TNF-alpha and IL-1beta mRNA and was associated with increased binding of early growth response-1 (Egr-1) to the TNF-alpha promoter after ethanol feeding. In contrast, ethanol feeding completely abrogated activation of nuclear factor-kappaB DNA-binding activity by LPS and had no effect on AP-1 binding. Together, these data suggest that enhanced activation of ERK1/2 and Egr-1 contributes to increased TNF-alpha production after chronic ethanol feeding.