Pyridoxamine, an Inhibitor of Advanced Glycation Reactions, Also Inhibits Advanced Lipoxidation Reactions: Mechanism of Action of Pyridoxamine

Maillard or browning reactions lead to formation of advanced glycation end products (AGEs) on protein and contribute to the increase in chemical modification of proteins during aging and in diabetes. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) have proven effective in animal model and clinical studies as inhibitors of AGE formation and development of diabetic complications. We report here that PM also inhibits the chemical modification of proteins during lipid peroxidation (lipoxidation) reactions in vitro, and we show that it traps reactive intermediates formed during lipid peroxidation. In reactions of arachidonate with the model protein RNase, PM prevented modification of lysine residues and formation of the advanced lipoxidation end products (ALEs) N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, malondialdehyde-lysine, and 4-hydroxynonenal-lysine. PM also inhibited lysine modification and formation of ALEs during copper-catalyzed oxidation of low density lipoprotein. Hexanoic acid amide and nonanedioic acid monoamide derivatives of PM were identified as major products formed during oxidation of linoleic acid in the presence of PM. We propose a mechanism for formation of these products from the 9- and 13-oxo-decadienoic acid intermediates formed during peroxidation of linoleic acid. PM, as a potent inhibitor of both AGE and ALE formation, may prove useful for limiting the increased chemical modification of tissue proteins and associated pathology in aging and chronic diseases, including both diabetes and atherosclerosis

Enantioselective liquid chromatography–mass spectrometry assay for the determination of ifosfamide and identification of the N-dechloroethylated metabolites of ifosfamide in human plasma

A sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method has been developed and validated for the enantioselective determination of ifosfamide [(R)-IF and (S)-IF] in human plasma and for the detection of the N-dechloroethylated metabolites of IF, 2-N-dechloroethylifosfamide [(R)-2-DCl-IF and (S)-2-DCl-IF] and 3-N-dechloroethylifosfamide [(R)-3-DCl-IF and (S)-3-DCl-IF]. IF, 2-DCl-IF and 3-DCl-IF were extracted from plasma using solid-phase extraction and resolved by liquid chromatography on a column containing a Chirabiotic T chiral stationary phase. The enantioselective separations were achieved using a mobile phase composed of 2-propanol:methanol (60:40 v/v) and a flow rate of 0.5 ml/min. The observed enantioselectivities (α) for IF, 2-DCl-IF and 3-DCl-IF were 1.20, 1.17 and 1.20, respectively. The calibration curve was linear in the concentration range of 37.50-4800 ng/ml for each ifosfamide enantiomer (r2 > 0.997). The lower limit of detection (LLOD) was 5.00 ng/ml. The inter- and intra-day precision ranged from 3.63 to 15.8 % relative standard deviation (RSD) and 10.1 to 14.3 % RSD, respectively, and the accuracy ranged from 89.2 to 101.5 % of the nominal values. The method was applied to the analysis of plasma samples obtained from a cancer patient who received 3.75 g/m2/d dose of (R,S)-ifosfamide as a 96-h continuous infusion

Strategy to Improve the Quantitative LC-MS Analysis of Molecular Ions Resistant to Gas-Phase Collision Induced Dissociation: Application to Disulfide-Rich Cyclic Peptides

Due to observed collision induced dissociation (CID) fragmentation inefficiency, developing sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assays for CID resistant compounds is especially challenging. As an alternative to traditional LC-MS/MS, we present here a methodology that preserves the intact analyte ion for quantification by selectively filtering ions while reducing chemical noise. Utilizing a quadrupole-Orbitrap MS, the target ion is selectively isolated while interfering matrix components undergo MS/MS fragmentation by CID, allowing noise-free detection of the analyte’s surviving molecular ion. In this manner, CID affords additional selectivity during high resolution accurate mass analysis by elimination of isobaric interferences, a fundamentally different concept than the traditional approach of monitoring a target analyte’s unique fragment following CID. This survivor-selected ion monitoring (survivor-SIM) approach has allowed sensitive and specific detection of disulfide-rich cyclic peptides extracted from plasma

PK/PD Disconnect Observed with a Reversible Endothelial Lipase Inhibitor

Screening of a small set of nonselective lipase inhibitors against endothelial lipase (EL) identified a potent and reversible inhibitor, <i>N</i>-(3-(3,4-dichlorophenyl)­propyl)-3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide (<b>5</b>; EL IC₅₀ = 61 nM, EL_HDL IC₅₀ = 454 nM). Deck mining identified a related hit, <i>N</i>-(3-(3,4-dichlorophenyl)­propyl)-4-hydroxy-1-methyl-5-oxo-2,5-dihydro-1<i>H</i>-pyrrole-3-carboxamide (<b>6a</b>; EL IC₅₀ = 41 nM, EL_HDL IC₅₀ = 1760 nM). Both compounds were selective against lipoprotein lipase (LPL) but nonselective versus hepatic lipase (HL). Optimization of compound <b>6a</b> for EL inhibition using HDL as substrate led to <i>N</i>-(4-(3,4<b>-</b>dichlorophenyl)­butan-2-yl)-1-ethyl-4-hydroxy-5-oxo-2,5-dihydro-1<i>H</i>-pyrrole-3-carboxamide (<b>7c</b>; EL IC₅₀ = 148 nM, EL_HDL IC₅₀ = 218 nM) having improved PK over compound <b>6a</b>, providing a tool molecule to test for the ability to increase HDL-cholesterol (HDL-C) levels in vivo using a reversible EL inhibitor. Compound <b>7c</b> did not increase HDL-C in vivo despite achieving plasma exposures targeted on the basis of enzyme activity and protein binding demonstrating the need to develop more physiologically relevant in vitro assays to guide compound progression for in vivo evaluation