Several studies have shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert "pleiotropic" actions beyond their lipid-lowering properties. Some of these effects involve improving or restoring endothelial function through increasing Nitric Oxide (NO) bioavailability and inhibiting inflammatory responses. NO is a key modulator of many physiological responses such as vascular relaxation, cell proliferation and inflammation. NO donors have been proven to act also through the regulation of inflammatory and cell proliferation pathways. With the aim to identify new pharmacological agents combining the properties of statins with those of a NO donor, we synthesized new chemical entities by grafting an organic nitrate moiety to the structure of pravastatin (NCX6550) or fluvastatin (NCX6553). In the present studies we describe their ability to release functional NO, which in turn leads to a variety of additional effects including anti-inflammatory properties.

First, we tested the antiproliferative effects of NO-statins and respective native statins in rat smooth muscle cells exposed to increasing concentrations of compounds (1 to 100µM). Cell proliferation was monitored by [³H]thymidine incorporation. Then, we evaluated the release of functional NO by determining cGMP formation in PC12 cells using an enzyme immunoassay kit and vasorelaxation elicited by the compounds in. NA-pre-contracted rabbit aortic rings with or without functional endothelium. The anti-inflammatory properties were assessed using RAW264.7 murine macrophage cells stimulated with LPS (1µg/ml).

Fluvastatin and its derivative NCX6553 both produced a concentration-dependent inhibition of cell proliferation, which was prevented by mevalonate (100µM). As expected, pravastatin, because of its hydrophilicity, inhibited cell proliferation only at high concentrations (20% at 100 µM). However, its NO-derivative, NCX6550, showed a marked antiproliferative effect (75% at 10 µM), which was prevented by mevalonate. In PC12 cells and rabbit aortic rings, NO-statin derivatives were able to stimulate in a concentration-dependent manner, cGMP formation and induce vasorelaxation (NCX6550: EC₅₀ = 2.3µM and EC₅₀ = 2.4µM, n=6-10; NCX6553: EC₅₀ = 2.7µM and EC₅₀ = 13.4 µM, n=6-10). The relaxant effects were maintained in absence of endothelium (NCX6550 EC₅₀ = 0.2 µM, NCX6553 EC₅₀ = 8.9 µM; n=9), confirming the contribution of the exogenous NO. In both tests, the native pravastatin was ineffective and fluvastatin produced responses with a relatively low potency. In RAW264.7 cells, NCX6550 (50µM), but not pravastatin, significantly decreased the extent of LPS-induced iNOS and COX-2 protein expression (55±12% and 75±4%, n=6, respectively) as well as nitrate accumulation (IC50 = 27.7 µM, n=4), which is an index of iNOS activity.

These findings indicate that the compounds retain HMG-CoA reductase inhibitory activity, release bioactive NO, and show enhanced anti-inflammatory properties. Thus, NO-statins appear to be an interesting new class of drugs having a potential in pathologies associated with endothelial dysfunction and vascular inflammation.