Analytical approaches in the drug discovery process: development of carnosine derivatives as inhibitors of degenerative disorders induced by carbonyl stress

It is well known that oxidative stress and reactive carbonyl species (RCS) are involved in the onset and the development of several pathological conditions, as atherosclerosis, diabetes mellitus and Alzheimer disease [1, 2]. Among the endogenous detoxifying agents that, by covalently reacting with RCS, protect tissues from carbonyl stress-induced damage, we have extensively studied the dipeptide carnosine (\uf062ala-Lhis; CAR). Notwithstanding its high reactivity and selectivity toward RCS, CAR cannot have any pharmacological application because it is hydrolyzed in plasma and kidney by carnosinases. Further studies allowed us to identify the D-CAR isomer as a possible lead compound to develop a new pharmacological class of carbonyl quenching molecules resistant to hydrolysis, and rapidly converted into D-CAR. In order to optimize oral bioavailability, we designed D-CAR lipophilic derivatives and tested in vitro their metabolic stability, by monitoring the time-course of D-CAR formation, in rat and human serum and S9000 liver fraction. For each derivative, a new LC-ESI-MS/MS method working in MRM mode was set up for the simultaneous determination of the parent compound and D-CAR. The molecule that showed the better metabolic profile (CS84) was selected and included in a pharmacokinetic study in the rat. The same method previously validated was applied to plasma samples to determine D-CAR and CS84 levels. Compared to D-CAR, CS84 had a greater oral bioavailability because its lipophilicity. In all sample no CS84 was detected, to confirm that the parent is totally converted in vivo into D-CAR. The in vivo RCS- quenching activity of CS84, and its ability to prevent renal dysfunction, has been evaluated in the Zucker obese rat, following oral administration for 3 months (30 mg/Kg/day). Urines were analyzed by a novel LC-ESI-MS/MS method based on the precursor ion scanning technique. Using the immonium ion of histidine (m/z 110) as a specific fragment ion of histidine-containing peptides, we identified (and then quantified in MRM mode) the Michael adduct between D-carnosine and 4-hydroxy-trans-2-nonenal (D CAR-HNE), as a biomarker of carbonyl stress [3]. As a confirmation of the in vivo RCS-quenching activity of CS84, urinary levels of the D-CAR-HNE adduct in the Zucker obese rats were 4-fold higher than in the Lean littermates (controls). The efficacy of CS84 was further supported by a significant improvement in some plasma and urinary parameters, such as cholesterol, triglycerides, glucose, blood pressure, insulin, proteinuria, albuminuria, that are typical risk factors of the metabolic syndrome. [1] Aldini G. et al. Lipoxidation-derived reactive carbonyl species as potential drug targets in preventing protein carbonylation and related cellular dysfunction. ChemMedChem, 1(10):1045-58; 2006. [2] Aldini G. et al. Intervention strategies to inhibit protein carbonylation by lipoxidation-derived reactive carbonyls. Med Res Rev, 27(6):817-68; 2007. [3] Orioli M. et al. HNE Michael adducts to histidine and histidine-containing peptides as biomarkers of lipid-derived carbonyl stress in urines: LC-MS/MS profiling in Zucker obese rats. Anal Chem 79(23):9174-84; 2007