Received for publication May 23, 2005.
Revised September 1, 2005.
Accepted for publication September 1, 2005.

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

Abstract

NCX 4016 (benzoic acid, 2-(acetyl-oxy)-3-[(nitrooxy)methyl]phenyl ester), 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 in order to inhibit COX-1, or if it can block it as a whole molecule. Aim of our study was to evaluate the effects of NCX 4016 and its analogue or metabolites on platelet COX-1 and whole blood COX-2 and on purified ovine COX-1 (oCOX-1) and COX-2 (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, while 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 prior to NCX 4016 prevent the irreversible inhibition of oCOX-1 by NCX4016 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 slower the kinetics of COX-1 inhibition by NCX 4016, as compared with aspirin.

Key words: cyclooxygenase-1, cyclooxygenase-2, nitric oxide, nitroaspirin, non steroidal anti-inflammatory drugs, thromboxane

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