Inhibitory effect of dilazep hydrochloride on the formation of advanced glycation end products

Objectives: The goal of this study was to assess the inhibitory effect of dilazep hydrochloride (DZ), an antiplatelet agent, on the formation of advanced glycation end products (AGEs) and to compare its potency with that of aminoguanidine (AG). Another goal was to determine the mechanism by which DZ inhibits AGE formation. Methods: D-glucose (100 mM) and 0.15-mM bovine serum albumin were incubated for 60 days at 37 degrees C with DZ or AG (0 to 4 mM), AGEs were measured by both a competitive enzyme-linked immunosorbent assay using a polyclonal antibody for N-e-(carboxymethyl)lysine (CML) and a fluorophotometric assay. To determine the mechanism by which DZ inhibits AGE formation, its radical-scavenging action (0 to 10 mM) was assessed using the competitive trapping of hydroxyl radicals generated by incubation of FeSO4 (0.1 mM) and H2O2 (0.1 mM) for 30 minutes at ambient temperature with salicylic acid (0.1 mM). The potency of DZ was then compared with that of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO) (0 to 10 mM). Results: DZ significantly inhibited formation of CML, a major nonfluorescent AGE, by 51% at a concentration of 4 mM (P < 0.05) (DZ:glucose molar ratio, 1:25) and inhibited fluorescent AGE formation by 19% (0.5 mM) and 35% (4 mM) (P < 0.001) (DZ:glucose molar ratio, 1:200 to 1:25), AG similarly inhibited these AGE by 38% (2 mM) and 84% (4 mM) (all P < 0.01), and by 47% (1 mM) to 84% (4 mM) (all P < 0.01), respectively. DZ also significantly inhibited the formation of both 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), products arising from the trapping of hydroxyl radical by salicylic acid. The amount of DZ or DMSO required for 50% inhibition (IC50) of 2,3-DHBA production was 0.16 +/- 0.03 mM for DZ and 0.27 +/- 0.03 mM for DMSO (P < 0.01), The IC,, of DZ for 2,5-DHBA production was also less than that of DMSO (0.18 +/- 0.03 mM vs 0.33 +/- 0.02 mM, respectively; P < 0.01). Conclusions: Results of this paper suggest that DZ may inhibit; AGE formation at concentrations similar to AG through a free radical-scavenging action.