Direct and irreversible inhibition of cyclooxygenase-1 by nitroaspirin (NCX 4016).

Benzoic acid, 2-(acetyl-oxy)-3-[(nitrooxy)methyl]phenyl ester (NCX 4016), a new drug made by an aspirin molecule linked, through a spacer, to a nitric oxide (NO)-donating moiety, is now under clinical testing for the treatment of atherothrombotic conditions. Aspirin exerts its antithrombotic activity by irreversibly inactivating platelet cyclooxygenase (COX)-1. NCX 4016 in vivo undergoes metabolism into deacetylated and/or denitrated metabolites, and it is not known whether NCX 4016 needs to liberate aspirin to inhibit COX-1, or whether it can block it as a whole molecule. The aim of our study was to evaluate the effects of NCX 4016 and its analog or metabolites on platelet COX-1 and whole blood COX-2 and on purified ovine COX (oCOX)-1 and oCOX-2. In particular, we have compared the mechanism by which NCX 4016 inhibits purified oCOX enzymes with that of aspirin using a spectrophotometric assay. All the NCX 4016 derivatives containing acetylsalicylic acid inhibited the activity of oCOX-1 and oCOX-2, whereas the deacetylated metabolites and the nitric oxide-donating moiety were inactive. Dialysis experiments showed that oCOX-1 inhibition by NCX 4016, similar to aspirin, is irreversible. Reversible COX inhibitors (indomethacin) or salicylic acid incubated with the enzyme before NCX 4016 prevent the irreversible inhibition of oCOX-1 by NCX 4016 as well as by aspirin. In conclusion, our data show that NCX 4016 acts as a direct and irreversible inhibitor of COX-1 and that the presence of a spacer and NO-donating moiety in the molecule slows the kinetics of COX-1 inhibition by NCX 4016, compared with aspirin.