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 t1/2 of 6 h were calculated, with a Cmax 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.
Footnotes
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This work was supported by European Community Grant LSHM-CT-2004-005033 (to G.F.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.097170.
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ABBREVIATIONS: COX, cyclooxygenase; PG, prostacyclin; TX, thromboxane; NCX-4016, 2-acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester; 2NTX-99, 4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(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-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; NE, norepinephrine; GTN, glyceryl trinitrate; DMEM, Dulbecco's modified Eagle's medium; TPα, thromboxane A2 receptor isophorm α; 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; 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.
- Received November 11, 2005.
- Accepted January 5, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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