TRA-418, a thromboxane A2 receptor antagonist and prostacyclin receptor agonist, inhibits platelet-leukocyte interaction in human whole blood.

TRA-418, a compound with both thromboxane A2 receptor (TP receptor) antagonistic and prostacyclin receptor (IP receptor) agonistic activities, was synthesised in our laboratory as a new antithrombotic agent. In this study, we examined the effects of TRA-418 on platelet-leukocyte interactions in human whole blood. Platelet-leukocyte interactions were induced by U-46619 in the presence of epinephrine (U-46619 + epinephrine) or with thrombin receptor agonist peptide 1-6 (TRAP). Platelet-leukocyte interactions were assessed by flow cytometry, with examination of both platelet-neutrophil and platelet-monocyte complexes. In a control experiment, the TP receptor antagonist SQ-29548 significantly inhibited the induction of platelet-leukocyte complexes by the combination of U-46619 and epinephrine, but not TRAP-induced formation of platelet-leukocyte complexes. Conversely, the IP receptor agonist beraprost sodium inhibited platelet-leukocyte complex formation induced by both methods, although the IC50 values of beraprost sodium for U-46619 + epinephrine were at least 10-fold greater than for TRAP. Under such conditions, TRA-418 inhibited both U-46619 + epinephrine-induced and TRAP-induced platelet-leukocyte complex formation in a concentration-dependent manner, in a similar range. These results suggest that TRA-418 exerts its inhibitory effects on platelet-leukocyte interactions by acting as a TP receptor antagonist as well as an IP receptor agonist in an additive or synergistic manner. These inhibitory effects of TRA-418 on formation of platelet-leukocyte complexes suggest the compound is beneficial effects as an antithrombotic agent.

'Information-Based-Acquisition' (IBA) technique with an ion-trap/time-of-flight mass spectrometer for high-throughput and reliable protein profiling.

Highly complex protein mixtures can be analyzed after proteolysis using liquid chromatography/mass spectrometry (LC/MS). In an LC/MS run, intense peptide ions originating from high-abundance proteins are preferentially analyzed using tandem mass spectrometry (MS(2)), so obtaining the MS(2) spectra of peptide ions from low-abundance proteins is difficult even if such ions are detected. Furthermore, the MS(2) spectra may produce insufficient information to identify the peptides or proteins. To solve these problems, we have developed a real-time optimization technique for MS(2), called the Information-Based-Acquisition (IBA) system. In a preliminary LC/MS run, a few of the most intense ions detected in every MS spectrum are selected as precursors for MS(2) and their masses, charge states and retention times are automatically registered in an internal database. In the next run, a sample similar to that used in the first run is analyzed using database searching. Then, the ions registered in the database are excluded from the precursor ion selection to avoid duplicate MS(2) analyses. Furthermore, real-time de novo sequencing is performed just after obtaining the MS(2) spectrum, and an MS(3) spectrum is obtained for accurate peptide identification when the number of interpreted amino acids in the MS(2) spectrum is less than five. We applied the IBA system to a yeast cell lysate which is a typical crude sample, using a nanoLC/ion-trap time-of flight (IT/TOF) mass spectrometer, repeating the same LC/MS run five times. The obtained MS(2) and MS(3) spectra were analyzed by applying the Mascot (Matrix Science, Boston, MA, USA) search engine to identify proteins from the sequence database. The total number of identified proteins in five LC/MS runs was three times higher than that in the first run and the ion scores for peptide identification also significantly increased, by about 70%, when the MS(3) spectra were used, combined with the MS(2) spectra, before being subjected to Mascot analysis.

Specificity of vascular reactivity and remodeling after repeated endothelial injury in a swine model.

We investigated the difference in vascular responses and remodeling between coronary and iliac arteries after repeated endothelial denudation. Endothelial denudation of the left anterior descending coronary artery (LAD) and the right common iliac artery (RIA) was repeated 4 times twice a month using a Fogarty catheter in 21 pigs. Vascular responses to vasoactive drugs were evaluated as % luminal diameter changes on contrast angiography 2 weeks after the last denudation. Corresponding nondenuded sites, ie, the left circumflex coronary artery (LCX) and the left common iliac artery (LIA), were used as references. Acetylcholine (1 microg/kg) did not constrict the LCX (0 +/- 1%) and the LAD (1 +/- 1%, P < 0.05), whereas it constricted the RIA (20 +/- 6%) but not the LIA (-3 +/- 3%, P < 0.01). Alternatively, serotonin (10 microg/kg) constricted the LAD strikingly (88 +/- 5%, P < 0.01 versus LCX and RIA), as well as the RIA (35 +/- 10%, P < 0.05 versus LIA). Vasodilator responses to substance P and isosorbide dinitrate were not different after injury in both arteries. The intima-to-media ratio and adventitia-to-media ratio of the relevant site in cross section of tissue sample from LAD were greater than those from LCX, and were more prominent than those from RIA. The results show that vascular tone regulation after the endothelial injury and vascular remodeling might be altered in a vessel-specific manner.

Development of 3,4-dihydro-2H-benzo[1,4]oxazine derivatives as dual thromboxane A2 receptor antagonists and prostacyclin receptor agonists.

We discovered a novel series of 3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxyacetic acid derivatives as potent dual-acting agents to block the TXA2 receptor and to activate the PGI2 receptor. We report the synthesis, structure-activity relationship, and in vitro, ex vivo, and in vivo pharmacology of this series of compounds. 4-[2-(1,1-Diphenylethylsulfanyl)ethyl]-3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxyacetic acid N-methyl-D-glucamine salt (7) is a promising candidate for a novel treatment in the anti-thrombotic and the cardiovascular fields avoiding hypotensive side effects.

Development of dual-acting benzofurans for thromboxane A2 receptor antagonist and prostacyclin receptor agonist: synthesis, structure-activity relationship, and evaluation of benzofuran derivatives.

Prostacyclin (PGI(2)) is an unstable, powerful endogenous inhibitor of platelet aggregation, and thromboxane A(2) (TXA(2)) is an unstable endogenous arachidonic acid metabolite that plays a pivotal role in platelet aggregation and vasoconstriction. The balance between TXA(2) and PGI(2) greatly affects maintenance of the homeostasis of the circulatory system. A novel series of benzofuran-7-yloxyacetic acid derivatives was discovered as potent dual-acting agents to block the thromboxane A(2) receptor and to activate the prostacyclin receptor. Synthesis, structure-activity relationship, and in vitro and ex vivo pharmacology of this series of compounds are described. The most potent in the series was {3-[2-(1,1-diphenylethylsulfanyl)ethyl]-2-hydroxymethylbenzofuran-7-yloxy}acetic acid diethanolamine salt (7) with K(i) of 4.5 nM for thromboxane receptor antagonism and K(i) of 530 nM for prostacyclin receptor agonism. Remarkably, compound 7 is a promising candidate for novel treatment as an antithrombotic agent with other cardiovascular actions to avoid hypotensive side effects.

Solid-phase synthesis of naltrindole derivatives using Fischer indole synthesis based on one-pot release and cyclization methodology.

[reaction: see text] We describe a new approach for the solid-phase synthesis of indoles 1 that involves a one-pot release and cyclization reaction of a solid-supported hydrazone through a Wang-type linker. Using this solid-phase methodology, we accomplished the synthesis of 40 naltrindole derivatives.