Direct formation of thienopyridine-derived nitrosothiols--just add nitrite!

Thienopyridines (ticlopidine, clopidogrel and prasugrel) are pro-drugs that require metabolism to exhibit a critical thiol group in the active form that binds to the P2Y12 receptor to inhibit platelet activation and prevent thrombus formation in vivo. We investigated whether these thienopyridines participate in S-nitrosation (SNO) reactions that might exhibit direct anti-platelet behaviour. Optimum conditions for in vitro formation of thienopyridine-SNO formation were studied by crushing ticlopidine, clopidogrel or prasugrel into aqueous solution and adding sodium nitrite, or albumin-SNO. Ozone-based chemiluminescence techniques were utilised to specifically detect NO release from the SNO produced. Effect on agonist-induced platelet aggregation was monitored using light transmittance in a 96 well microplate assay. Pharmaceutical grade preparations of ticlopidine, clopidogrel and prasugrel were found to exhibit significant free thiol and formed SNO derivatives directly from anionic nitrite in water under laboratory conditions without the need for prior metabolism. Thienopyridine-SNO formation was dependent on pH, duration of mixing and nitrite concentration, with prasugrel-SNO being more favourably formed. The SNO moiety readily participated in trans-nitrosation reactions with albumin and plasma. Prasugrel-SNO showed significantly better inhibition of platelet aggregation compared with clopidogrel-SNO, however when compared on the basis of SNO concentration these were equally effective (IC50=7.91 ± 1.03 v/s 10.56 ± 1.43 µM, ns). Thienopyridine-derived SNO is formed directly from the respective base drug without the need for prior in vivo metabolism and therefore may be an important additional contributor to the pharmacological effectiveness of thienopyridines not previously considered.

Direct vasoactive properties of thienopyridine-derived nitrosothiols.

Thienopyridines (ticlopidine, clopidogrel, and prasugrel) require in vivo metabolism to exhibit a critical thiol group in the active form that binds to the P2Y12 platelet receptor to inhibit platelet activation. We hypothesized that formation of thienopyridine-derived nitrosothiols (ticlopidine-SNO, clopidogrel-SNO, and prasugrel-SNO) occurs directly from the respective parent drug. Pharmaceutical-grade thienopyridine (ticlopidine, clopidogrel chloride, clopidogrel sulfate, clopidogrel besylate, or prasugrel) was added to nitrite in aqueous solution to form the respective thienopyridine-SNO (Th-SNO). An isolated aortic ring preparation was used to test vasoactivity of the Th-SNO derivatives. Increasing nitrite availability resulted in increased Th-SNO formation for all drugs (other than ticlopidine). Th-SNO induced significant endothelium-independent relaxation of preconstricted aortic rings. Clopidogrel-chloride-SNO displayed rapid-release kinetics in a chemical environment, which was reflected by immediate and transient vasorelaxation when compared with the SNO derivatives of the other thienopyridines. Accounting for differences in yield, clopidogrel-chloride-SNO exhibited the greatest propensity to immediately relax vascular tissue. Th-SNO derivatives exhibit nitrovasodilator properties by supplying NO that can directly activate vascular soluble guanylate cyclase to induce vasorelaxation. Differences in SNO yield and vasoactivity exist between thienopyridine preparations that might be important to our understanding of the direct pharmacological effectiveness of thienopyridines on vascular and platelet function.