Pharmacological Characterization of 2NTX-99 [4-Methoxy-N- (4-trans-nitrooxycyclohexyl)-N-(3-pyridinylmethyl)-1,3- benzenedicarboxamide], a Potential Antiatherothrombotic Agent with Antithromboxane and Nitric Oxide Donor Activity in Platelet and Vascular Preparations

Thromboxane (TX) A2, prostacyclin (PGI2), and nitric oxide (NO) regulate platelet function and interaction with the vessel wall. Inhibition of TXA2, implemented synthesis of PGI2, and supply of exogenous NO may afford therapeutic benefit. 2NTX-99 [4-methoxy-N-(4-trans-nitrooxycyclohexyl)-N-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a new chemical entity related to picotamide, showed antithromboxane activity and NO donor properties. 2NTX-99 relaxed rabbit aortic rings precontracted with norepinephrine or U46619 (9,11-dideoxy-9 ,11 methanoepoxy-prosta-5Z,13E-dien-1-oic acid; EC50, 7.9 and 17.1 M, respectively), an effect abolished by 10 M 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). 2NTX-99 inhibited arachidonic acid (AA)-induced washed platelet aggregation (EC50, 9.8 M) and TXB2 formation ( 71% at 10 M), and its potency increased in the presence of aortic rings (EC50, 1.4 M). In whole rabbit aorta incubated with homologous platelets, AA caused contraction and TXA2 formation, reduced by 2NTX-99 (10–40 M): contraction, 28 and 47%, TXA2 formation, 37 and 75.4%, respectively, with concomitant increase in PGI2. 2NTX-99 (20–40 M) inhibited U46619induced aggregation in rabbit platelet-rich plasma (PRP) ( 74 6.7 and 96 2.4%, respectively) and inhibited collagen-induced aggregation in human PRP ( 48.2 10 and 79.2 6%), whereas ozagrel was ineffective. In human embryonic kidney 293 cells transfected with the TXA2 receptor isophorm receptor, 2NTX-99 did not compete with the ligand, [H]SQ29,548 ([H][1S-[1 ,2 (5Z),3 ,4 ]]-7-[3-[[2-(phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptanoic acid), or prevent inositol phosphate accumulation. After oral administration (50–250 mg/kg), 2NTX-99 inhibited TXA2 production in rat clotting blood ( 71 and 91%); at 250 mg/kg, an area under the curve, 0 to 16 h, of 149.5 h/ g/ml and a t1/2 of 6 h were calculated, with a Cmax value of 31.8 8.2 g/ml. An excellent correlation between plasma concentrations and TXA2 inhibition occurs. 2NTX-99 controls platelet function and vessel wall interaction by multifactorial mechanisms and possesses therapeutic potential. Aspirin, a nonreversible inhibitor of platelet cyclooxygenase (COX)-1, has been the mainstay of antiplatelet therapy for over 20 years (Antithrombotic Trialist Collaboration, 2002). Novel approaches to the modulation of platelet function by preventing platelet secretion and/or aggregation, their adhesion to the vessel wall, or progression of thrombus development (Bhatt and Topol, 2003) have been addressed in experimental and clinical studies. Until now, the only novel drug suitable for chronic therapy, which in some trials appeared superior to aspirin in preventing cardiovascular accidents (CAPRIE, 1996), is clopidogrel, an inhibitor of platelet P2Y12 receptor. This work was supported by European Community Grant LSHM-CT-2004005033 (to G.F.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.097170. ABBREVIATIONS: COX, cyclooxygenase; PG, prostacyclin; TX, thromboxane; NCX-4016, 2-acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester; 2NTX-99, 4-methoxy-N-(4-trans-nitrooxycyclohexyl)-N-(3-pyridinylmethyl)-1,3-benzenedicarboxamide; NO, nitric oxide; PRP, platelet-rich plasma; AA, arachidonic acid; OKY-046, ozagrel; EIA, enzyme immunoassay; U46619, 9,11-dideoxy-9 ,11 -methanoepoxy-prosta-5Z,13E-dien-1-oic acid; NE, norepinephrine; GTN, glyceryl trinitrate; DMEM, Dulbecco’s modified Eagle’s medium; TP , thromboxane A2 receptor isophorm ; SQ29,548, [H][1S-[1 ,2 (5Z),3 ,4 ]]-7-[3-[[2-(phenylamino)-carbonyl]hydrazino]-methyl]-7-oxabicyclo[2,2,1]-hept-2-yl]-5-heptanoic acid; IP, inositol phosphate; HPLC, high-performance liquid chromatography; CV, coefficient(s) of variation; ISMN, isosorbide mononitrate; ODQ, 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; PGH2, prostaglandin H cyclic endoperoxide. 0022-3565/06/3172-830–837$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 317, No. 2 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 97170/3096215 JPET 317:830–837, 2006 Printed in U.S.A. 830 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from Significant advancement in our understanding of the role of platelets in the atherothrombotic process (Bhatt and Topol, 2003) supports the concept that therapeutic efficacy may be improved by a combined action on platelet activation and interaction with the vascular wall. Indeed, several studies have shown that combined treatment with clopidogrel and aspirin in acute coronary syndromes (Mehta et al., 2001) or with dipyridamole and aspirin in the prevention of stroke (Forbes, 1998), offer advantage over single treatment. However, concern about lack of response in subsets of patients and about development of resistance during chronic treatment (Gurbel and Bliden, 2003; Eikelboom and Hankey, 2004) stresses the need of novel therapeutic approaches (Bhatt and Topol, 2003). Different prostanoids originate from the common endoperoxide precursor PGH2, which is further metabolized by specific enzymes according to a strict cellular specificity, yielding, e.g., mostly TXA2 in platelets, PGI2 in endothelial cells, etc. (Maclouf et al., 1998). However, PGH2 can be also made available extracellularly for further paracrine conversion to bioactive eicosanoids. The inhibition of a given enzymatic pathway within a defined cell facilitates an intercellular shunt of PGH2 toward an alternative pathway, e.g., in platelet-endothelial cell coincubates, PGI2 synthesis is enhanced when thromboxane synthase is inhibited (Nowak and