Title

 

Authors

V. Calderone¹, A. Martelli¹, L. Testai¹, A. Arcipreti¹, M. Anzini², A. Di Capua², M. Biava³, C. Battilocchio³, C. Ghelardini⁴, A. Giordani⁵, L. Rovati⁵, M.C. Breschi^1
 
1Dip. di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa; via Bonanno, 6, I-56126 Pisa, Italy.
²Dip. Farmaco Chimico Tecnologico, Università degli Studi di Siena; via Alcide de Gasperi 2, I-53100 Siena, Italy
³Dip. di Studi di Chimica e Tecnologie del Farmaco, Università di Roma “La Sapienza”; piazzale Aldo Moro 5, I-00185 Roma, Italy
⁴Dip. di Farmacologia, Università di Firenze; viale G. Pieraccini 6, I-50139 Firenze, Italy
⁵Rottapharm Madaus , via Valosa di Sopra 7/9, I-20052 Monza, Italy 

 

Abstract

Selective inhibitors of COX-2 (Coxibs) do not influence the biosynthesis of vasocontractile and platelet activating prostanoids, such as the COX1-derived TXA2; whilst, the COX-2-mediated production of PGI2, which plays vasorelaxing and anti-platelet effects, is deeply depressed by Coxibs. This leads to an increased risk of cardiovascular adverse effects, emerged with the use of this class of drugs [1]. In order to attenuate such an unfavorable cardiovascular feature intrinsic in the mechanism of action of Coxibs, a new class of “bi-functional” drugs has been projected. Together with the COX-2-inhibiting activity, evaluated in suitable in vitro and in vivo models, these molecules (called CINODs) show also Nitric Oxide (NO)-releasing properties [2]. As concerns the structural aspects, these compounds show an opportune nitrooxy-alkyl chain, which is one of the most suitable NO-releasing moiety widely used in the design of NO-releasing hybrids [3]. In fact, although the exact metabolic pathways involved in NO-release from such a moiety is not completely understood, it is accepted that this biotransformation requires redox mechanisms ensured by enzymes such as CYP450/CYP450-reductase [4]. In order to characterize the NO-release mechanisms, the CINOD  derivative VA 694 was selected for a preliminary study and the NO-releasing NSAID NO-Naproxene (NO-NPX) was used as a reference drug. The two compounds were incubated in rat liver homogenate (plus NADH, NADPH and reduced glutathione),  in rat serum or in water. Since the shortest half-life of NO make difficult its direct determination, the time-dependent production of inorganic nitrites and nitrates (main metabolites of NO) has been determined amperometrically and has been used as an indirect parameter. The incubation VA 694 in liver homogenate was followed by a slow time-dependent production of nitrites and nitrates, consistent with a modulated release of NO. NO-NPX showed a more rapid accumulation of relatively large amounts of these metabolites. The incubation of VA 694 in serum did not lead to the production of nitrites and nitrates confirming (as expected) that this biological substrate cannot ensure the formation of NO from a nitric ester. The incubation of NO-NPX in serum was not followed by the formation of nitrites, but surprisingly it led to a relatively rapid formation of high concentrations of inorganic nitrates. Since the absence of nitrites seemed to exclude the previous generation of NO from NO-NPX in serum, the presence of nitrates could be explained by a spontaneous hydrolysis or a reaction mediated by serum components. However, no traces of inorganic nitrites and nitrates were recorded after the incubation of both VA 694 and NO-NPX in water, thus a spontaneous hydrolysis could be discarded. Therefore, the results indicate that of VA 694 shows the typical metabolic behavior of a NO-donor moiety. Together with the biotransformation to NO, the nitrooxy-alkyl chain of NO-NPX seems to exhibit a further metabolic destiny due to serum components, which leads to the direct formation of inorganic nitrates. Noteworthy, an analogous pathway has been described for some NO-releasing derivatives of aspirin [5].
 
References
[1] Capone ML, Tacconelli S, Rodriguez LG, Patrignani P. Pharmacol Rep., 2010, 62:530-535.
[2] Patent “TO2010A000739”
[3] Martelli A, Breschi MC, Calderone V. Curr. Pharm. Des., 2009,15,614-636.
[4] Li H, Liu X, Cui H, Chen Y-R, Cardounel AJ, Zweier JL. J. Biol. Chem., 2006,281,12546-12554.
[5] Gao J, Kashfi K, Rigas B. J. Pharmacol. Exp. Ther., 2005,312,989-997.