Abstract

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-N¹-(4-trans-nitrooxycyclohexyl)-N³-(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-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; EC₅₀, 7.9 and 17.1 μM, respectively), an effect abolished by 10 μM 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 μM) and TXB₂ formation (-71% at 10 μM), and its potency increased in the presence of aortic rings (EC₅₀, 1.4 μM). In whole rabbit aorta incubated with homologous platelets, AA caused contraction and TXA₂ formation, reduced by 2NTX-99 (10-40 μM): contraction, -28 and -47%, TXA₂ formation, -37 and -75.4%, respectively, with concomitant increase in PGI₂. 2NTX-99 (20-40 μM) inhibited U46619-induced 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 α receptor, 2NTX-99 did not compete with the ligand, [³H]SQ29,548 ([³H][1S-[1α,2β(5Z),3β,4α]]-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/μg/ml and a t_1/2 of 6 h were calculated, with a C_max value of 31.8 ± 8.2 μg/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.