Statins decrease cardiovascular morbidity and mortality, in both hyper and normocholesterolemic patients, probably because of their pleiotropic effects on diverse atherogenic mechanisms. One shortcoming of statins is their slow onset of action that reduces their efficacy in acute ischemic conditions, such as acute coronary syndromes. Recently, novel antithrombotic molecules have been developed by the addition of a nitric oxide (NO)-adduct to a known drug in order to exploit the additional antithrombotic effect of NO. In this study we evaluated the effect of the novel NO-releasing statin NCX6550 (nitropravastatin) in vitro and in vivo on a number of cellular functions relevant to atherothrombosis.

In vitro we assessed the effects of test compounds on human platelet aggregation induced by U46619 and on tissue factor (TF) activity and expression by human mononuclear cells stimulated with LPS (1 µg/ml) or PMA (10^-9 M; Simcha RM et al., 2002). In vivo we evaluated the effect of the compounds on U46619-induced pulmonary thromboembolism in mice (Momi et al., 2000) and on pro-coagulant activity of spleen mononuclear cells after administration of LPS (50 µg/mouse).

In vitro, NCX6550 inhibited U46619-induced human platelet aggregation in both buffer (IC₅₀ = 117±12µM) and plasma (IC50 = 97±9 µM) with a NO-dependent mechanism (sensitive to guanylyl cyclase inhibition, ODQ 10µM), while pravastatin (from 50 to 200µM) was inactive. In human mononuclear cells NCX6550 inhibited dose-dependently LPS- and PMA-induced TF expression (> 85% inhibition at 50 µM; TF antigen after 3h incubation with LPS: 0.66±0.25 vs 5.0±2.5ng/mg in NCX6550 and control, respectively; p<0.01). This effect was associated with a comparable reduction of both TF activity (16.5±12.8 vs 198±92 U/10⁶ cells) and TF mRNA. Pravastatin (50 µM) and two different NO-donors (2 mM SNAP and 10 µM SNP) had little or no effect on TF expression, even in combination (<20% inhibition). In vivo, NCX6550 (24 mg/kg i.p.) given 1h before U46619 injection, by contrast with equimolar dose of pravastatin (20 mg/kg i.p.), significantly reduced mortality (controls=85%; pravastatin=85%; NCX6550=55%; p<0.05) and platelet consumption (platelet x 10⁹/L: NCX6550 48mg/kg = 208±89.9; pravastatin 40 mg/kg =94±25; p<0.05). Plasmatic NO-degradation products after NCX6550 increased progressively with a peak at 3 h (from 27±6µM to 64±17 µM). NCX6550 (0.6 mg/mouse i.p.) given 15min before LPS, attenuated the clot promoting activity of spleen mononuclear cells harvested 3 h after LPS challenge (clotting time: 197±18s and 255±42s in vehicle and NCX6550, respectively; p<0.001).

Our results show that a novel NO-donating statin exerts antiplatelet and anticoagulant effects both in vitro and in vivo. Thus, NCX6550 may represent an interesting new anti-atherothrombotic treatment for patients with acute coronary syndromes.

Momi et al. (2000). Eur. J. Pharmacol. 397:177-185.
Simcha RM et al. (2002). Atherosclerosis, 161: 35-43.