The discovery of carnosine derivatives as selective and efficient sequestering agents of cytotoxic carbonyl species : from molecular design to preclinical studies

Objective: Reactive carbonyl species (RCS) are involved in the pathogenesis of several human diseases. Hence RCS, apart from being considered a biomarker of oxidative damage, are also potential targets for the development of bioactive compounds acting as detoxifying agents of RCS (carbonyl quenching compounds). We found that the natural dipeptide carnosine (β-alanyl-L-histidine, L-CAR) is a selective and potent RCS sequestering agent, even though its clinical application is limited due to its rapid hydrolysis in blood by a specific dipeptidase (carnosinase). The enantiomer β-alanyl-D-histidine (D-CAR) showed a quenching activity nearly identical to that of L-CAR and was totally stable in the plasma. But while L-CAR is actively absorbed by hPepT1 transporter, D-CAR is scarcely transported. Consequently, we developed a drug discovery approach aimed at designing, synthesizing and evaluating novel CAR derivatives and peptidomimetics stable to carnosinase and bioavailable. Methods: Two different approaches were considered, the first, aimed at increasing the oral bioavailability of D-CAR, by synthesizing a set of ester, amide and carbamate based prodrugs. The second approach was aimed at designing L-CAR peptidomimetic which are recognized by hPepT1 and hence transported through an active transport but not recognized by carnosinase. Results: Among D-CAR prodrugs, the octyl ester of D-CAR was chosen as a candidate due to its favorable oral bioavailability (plasma D-CAR is increased 2.6-fold if given as an octyl ester relative to D-CAR). The octyl ester was then found to dose-dependently decrease the development of hypertension and dyslipidemia, to restore renal functions in Zucker fa/fa obese rats, and to inhibit the carbonylation process (AGEs and pentosidine). The compound coded FL-926-A16 was then selected among L-CAR peptidomimetics due to its following properties: ability to quench RCS, suitable bioavailability and resistance to carnosinase. The ability of FL-926-A16 to quench 4-hydroxy-trans-nonenal and to reduce protein carbonylation and tissue damage was then demonstrated in different animal models (db/db mouse and ApoE null mice). Conclusion: Bioavailable CAR derivatives are promising bioactive molecules designed to reduce protein carbonylation through a sequestering mechanism of cytotoxic carbonyl species and were found greatly effective in different animal models of diabetic and non-diabetic nephropathy.