title: Direct and irreversible inhibition of cyclooxygenase-1 by nitroaspirin (NCX 4016).
abstract: 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.
authors.affiliation: Department of Internal Medicine, Division of Internal and Cardiovascular Medicine, University of Perugia, Via Enrico dal Pozzo, 06126, Perugia, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: Corazzi
authors.forename: Mario
authors.initials: M
authors.lastname: Leone
authors.forename: Raffaella
authors.initials: R
authors.lastname: Maucci
authors.forename: Lanfranco
authors.initials: L
authors.lastname: Corazzi
authors.forename: Paolo
authors.initials: P
authors.lastname: Gresele
mesh_terms: D000818:Animals; D001241:Aspirin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism; D051545:Cyclooxygenase 1 / Q000302:isolation & purification; D051546:Cyclooxygenase 2 / Q000097:blood; D016861:Cyclooxygenase Inhibitors / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D003956:Dialysis; D015232:Dinoprostone / Q000032:analysis / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D009000:Monocytes / Q000201:enzymology / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D012756:Sheep; D013053:Spectrophotometry; D013917:Thrombin / Q000494:pharmacology; D013929:Thromboxane B2 / Q000032:analysis / Q000378:metabolism; D013997:Time Factors
chemical_list: D016861:Cyclooxygenase Inhibitors; D006490:Hemostatics; D010975:Platelet Aggregation Inhibitors; D013929:Thromboxane B2; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D013917:Thrombin; C102148:nitroaspirin; D015232:Dinoprostone; D001241:Aspirin