1-Methylnicotinamide (MNA), a primary metabolite of nicotinamide, exerts anti-thrombotic activity mediated by a cyclooxygenase-2/prostacyclin pathway

Abstract

Background and purpose: 1-methylnicotinamide (MNA) has been considered to be an inactive metabolite of nicotinamide. Here we assessed the anti-thrombotic activity of MNA in vivo.

Experimental approach: Antithrombotic action of MNA was studied in normotensive rats with extracorporeal thrombus formation (thrombolysis), in renovascular hypertensive rats with intraarterial thrombus formation (arterial thrombosis) and in a venous thrombosis model in rats (venous thrombosis).

Key results: MNA (3-100 mg kg(-1)) induced a dose-dependent and sustained thrombolytic response, associated with a rise in 6-keto-PGF(1alpha) in blood. Various compounds structurally related to MNA were either inactive or weaker thrombolytics. Rofecoxib (0.01-1 mg kg(-1)), dose-dependently inhibited the thrombolytic response of MNA, indomethacin (5 mg kg(-1)) abolished it, while L-NAME (5 mg kg(-1)) were without effect. MNA (3-30 mg kg(-1)) also reduced arterial thrombosis and this effect was abrogated by indomethacin (2.5 mg kg(-1)) as well as by rofecoxib (1 mg kg(-1)). MNA, however, did not affect venous thrombosis. In vitro MNA did not modify platelet aggregation nor induce vasodilation.

Conclusions and implications: MNA displayed a profile of anti-thrombotic activity in vivo that surpasses that of closely related compounds. MNA inhibited platelet-dependent thrombosis by a mechanism involving cyclooxygenase-2 and prostacyclin. Our findings suggest that endogenous MNA, produced in the liver by nicotinamide N-methyltransferase, could be an endogenous activator of prostacyclin production and thus may regulate thrombotic as well as inflammatory processes in the cardiovascular system.

Figure 1

Major pathways of nicotinamide metabolism (see text for details).

Figure 2

Representative tracing showing pronounced and sustained thrombolytic response induced by 1-methylnicotinamide (30 mg kg⁻¹) in vivo in normotensive Wistar rats with extracorporeal circulation (a). For comparison, the lack of a significant thrombolytic response to nicotinamide (30 mg kg⁻¹) (b), nicotinic acid (30 mg kg⁻¹) (c) and 1-methylnicotinic acid (trigoneline, 30 mg kg⁻¹) (d) is shown.

Figure 3

Dose-dependent thrombolytic response induced by 1-methylnicotinamide (MNA) in vivo in normotensive Wistar rats with extracorporeal circulation as compared to the effect of nicotinamide, nicotinic acid and MAP. (a) Maximum thrombolytic response (attained within 30 min of drug injection) and (b) magnitude of sustained thrombolytic response (measured 1 h after drug injection) induced by MNA, nicotinamide, nicotinic acid and MAP are shown. Data represent the mean±s.e.means from n=3–11 experiments. (c) Comparison of the thrombolytic effect of MNA with that induced by structurally related compounds. The thrombolytic effect of each compound was assayed in vivo after intravenous injection (at a dose of 30 mg kg⁻¹) in normotensive Wistar rats with extracorporeal circulation. The sustained thrombolytic response (1 h after drug injection) of MNA is compared to the response induced by various pyridinium derivatives. Data represent mean±s.e.means of 3–11 experiments. MAP, 1-methyl-3-acetylpyridine; MNA, Me - 1,N′-dimethylnicotinamide; MNA-Et₂, 1-methyl-N′,N′-diethylnicotinamide; PNA, 1-propylnicotinamide; (EtOH)NA, 1-(2-hydroxyethyl)nicotinamide; RibNA, 1-ribosylnicotinamide; 6-ANA, 6-amino nicotinamide (see also Table 1).

Figure 4

Involvement of COX-2 in thrombolytic response induced by 1-methylnicotinamide (MNA) (30 mg kg⁻¹) in vivo in normotensive Wistar rats with extracorporeal circulation. (a) Dose-dependent inhibition of the thrombolytic response to MNA by the selective COX-2 inhibitor, rofecoxib. Data represent the mean±s.e.means from 3 to 10 experiments. ^* P<0.05 vs control, ^** P<0.01 vs control as assessed by Kruskal–Wallis test followed by Dunn's multiple comparison test. (b) Effects of the non-selective COX inhibitor indomethacin (5 mg kg⁻¹), an antiplatelet dose of acetylsalicylic acid (ASA, 1 mg kg⁻¹), the non-selective NOS inhibitor (L-NAME, 5 mg kg⁻¹) and the inhibitor of COX-2 induction, dexamethasone (1 mg kg⁻¹) on the thrombolytic response to MNA (30 mg kg⁻¹). Inhibitors were administered 15 min before MNA injection, with the exception of dexamethasone, which was given 3 h before MNA. Data represent mean±s.e.means from 3 to 10 experiments. ^* indicates P<0.05, vs control response as assessed by Kruskal–Wallis test followed by Dunn's multiple comparison test.

Figure 5

Release of PGI₂ by 1-methylnicotinamide (MNA) (a) and MAP (b) in vivo. Levels of 6-keto-PGF_1α and of other prostanoids were measured in plasma following injection of MNA or MAP (30 mg kg⁻¹) in normotensive Wistar rats with extracorporeal circulation. Data represent the mean±s.e.means from n=4–7 experiments. ^* P<0.05 vs basal level, ^*** P<0.001 vs basal level as assessed by Friedman test followed by Dunn's multiple comparison test.

Figure 6

Antithrombotic effects of 1-methylnicotinamide (MNA). (a) Dose-dependent antithrombotic effect of MNA in arterial thrombosis model in rats with renovascular hypertension. The columns represent the thrombus weight in hypertensive rats treated with 0.9% NaCl (Control), or MNA (3, 10 and 30 mg kg⁻¹ i.v.). (b) Inhibition of antithrombotic effect of MNA by the non-selective COX inhibitor indomethacin and the selective COX-2 inhibitor rofecoxib. The columns represent the thrombus weight in hypertensive rats treated with, 0.9% NaCl (Control), MNA (30 mg kg⁻¹ i.v.), indomethacin (2.5 mg kg⁻¹ i.v.) or rofecoxib (1 mg kg⁻¹ i.v.) followed by administration of MNA (30 mg kg⁻¹ i.v.) or the COX inhibitors alone. Data represent mean±s.e.means of n=4–13 experiments. ^* P<0.05 vs Control; ^ P<0.05 vs MNA 30 mg kg⁻¹ as assessed by two-tailed, Mann–Whitney test.