Thromboxane A2 Synthase Inhibitor Research Articles

Anti-platelet therapy is efficacious in treating endometriosis induced in mouse

In light of recent findings showing that platelets play important roles in the development of endometriosis in general and in fibrogenesis in particular, this study investigated the efficacy of Ozagrel, a TXA2 synthase inhibitor, in a murine model of endometriosis. In addition, another mouse experiment was conducted to evaluate the effect of timing of platelet depletion and of sequential depletion of platelets and macrophages on the development of endometriosis. It was found that both the Ozagrel treatment and different platelet depletion schemes resulted in significant reduction in lesion growth (all P-values <0.01) along with improved hyperalgesia in mice with induced endometriosis. They also significantly reduced the expression of markers of proliferation, angiogenesis, inflammation and fibrosis as well as decreased macrophage infiltration in endometriotic lesions (all P-values <0.05). Compared with untreated mice, pre-emptive depletion of platelets as well as platelet depletion after induction resulted in significant reduction in lesion weight (both P-values <0.001), while sequential depletion of platelets and macrophages yielded similar reduction. These results, in conjunction with other roles that platelets play in the development of endometriosis, strongly argue for the potential of anti-platelet therapy in treating endometriosis.

Menthol Modulates Pacemaker Potentials through TRPA1 Channels in Cultured Interstitial Cells of Cajal from Murine Small Intestine

Background/Aims: ICCs are the pacemaker cells responsible for slow waves in gastrointestinal (GI) smooth muscle, and generate periodic pacemaker potentials in current-clamp mode. Methods: The effects of menthol on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) from mouse small intestine were studied using the whole cell patch clamp technique. Results: Menthol (1 - 10 μM) was found to induce membrane potential depolarization in a concentration-dependent manner. The effects of various TRP channel antagonists were examined to investigate the receptors involved. The addition of the TRPM8 antagonist, AMTB, did not block menthol-induced membrane potential depolarizations, but TRPA1 antagonists (A967079 or HC-030031) blocked the effects of menthol, as did intracellular GDPβS. Furthermore, external and internal Ca2+ levels were found to depolarize menthol-induced membrane potentials, whereas external Na+ was not. Y-27632 (a Rho kinase inhibitor), SC-560 (a selective COX 1 inhibitor), NS-398 (a selective COX 2 inhibitor), ozagrel (a thromboxane A2 synthase inhibitor) and SQ-29548 (highly selective thromboxane receptor antagonist) were used to investigate the involvements of Rho-kinase, cyclooxygenase (COX), and the thromboxane pathway in menthol-induced membrane potential depolarizations, and all inhibitors were found to block the effect of menthol. Conclusions: These results suggest that menthol-induced membrane potential depolarizations occur in a G-protein-, Ca2+-, Rho-kinase-, COX-, and thromboxane A2-dependent manner via TRPA1 receptor in cultured ICCs in murine small intestine. The study shows ICCs are targeted by menthol and that this interaction can affect intestinal motility.

Design, Synthesis and Evaluation of Antiplatelet Aggregation Inhibitory Activities of the Analogs of Picotamide

A new class of 4-ethoxyisophthalamides is reported, based on the novel approach of linking the 4-ethoxy to Picotamide. Picotamide, as a combined inhibitor of Thromboxane A2 synthase and receptor, is synthesized in 1985 and attracted the attention of researchers. To improve our knowledge of the structure-activity relationship (SAR) and to obtain new anti-platelet drugs, total 24 unreported compounds of 4-ethoxyisophthalamides were designed and synthesized and were devised taking example by structural features of Picotamide and 4-methoxy-N1,N3-diphenylisophthalamide. The structures of target compounds were identified by MS, 1H-NMR and IR and the in vitro anti-platelet aggregation activities were assessed by Born test. The in vitro results revealed that thirteen derivatives (2b, 2g, 2u, 2q, 2f, 2a, 2r, 2j, 2i, 2v, 2h, 2s, 2d) showed platelet aggregation inhibitory activities induced by 5.0 mM ADP with IC50 values ranging over 0.35 µM-1.12 µM and the pre-six compounds had superior anti-platelet aggregation activities than the reference drug Picotamide, while 2b was the best. These consequences suggested that this novel series may have potential as structural templates for the design and subsequent development of new platelet anti-aggregatory drugs.

Thromboxane A2 induces blood flow recovery via platelet adhesion to ischaemic regions.

Thromboxane A2 (TXA2) induces platelet adhesion through thromboxane prostanoid (TP) receptor. Platelets contain many pro-angiogenic factors and are recruited to the site of vascular injury. However, the cellular and molecular mechanisms of platelet-dependent angiogenesis, especially the involvement of TP signalling, have not been fully elucidated. The present study hypothesized that TP-dependent platelet adhesion would contribute to angiogenesis in a mouse hindlimb ischaemic model. Blood flow recovery was suppressed by the TXA2 receptor antagonist (S-1452) and the TXA2 synthase inhibitor (OKY-046) compared with control mice. TP knockout mice (TP(-/-)) showed delayed blood flow recovery from ischaemia and impaired angiogenesis compared with wild-type (WT) mice and prostacyclin receptor knockout mice (IP(-/-)). Selective platelet adhesion to ischaemic endothelial cells (ECs) via P-selectin was identified in WT and IP(-/-), but not in TP(-/-), via in vivo microscopy. IF analysis showed that P-selectin glycoprotein ligand-1 (PSGL-1) co-localized with endothelial CD31 in ischaemic muscle in WT and IP(-/-) but not diminished in TP(-/-). Platelet-rich plasma levels of stromal cell-derived factor-1 and VEGF were increased after ischaemia in WT, and suppressed by antibody against P-selectin in WT but not in TP(-/-). Furthermore, the blood flow recovery was suppressed by neutralizing antibodies against VEGF or C-X-C chemokine receptor type 4 in WT and IP(-/-) but not in TP(-/-). These results indicated that TP signalling facilitates ischaemia-induced angiogenesis via P-selectin-mediated platelet adhesion to PSGL-1 on the ECs at ischaemic sites and the supply of pro-angiogenic factors by the adherent platelets.

Mechanisms of Uridine Adenosine Tetraphosphate (Up4A)‐induced Contraction in Mice Aorta: Role of Thromboxane (TXA2) and Purinergic Receptors (P2R)

Up4A, a novel endothelium‐derived vasoconstrictor, is proposed to play a role in cardiovascular disorders and induces aortic contraction through cyclooxygenases (COX). We demonstrated earlier that activation of A3 adenosine receptors (AR) results in aortic contraction via TXA2 production. However, the mechanisms of Up4A‐induced contraction are not understood. We hypothesize that Up4A‐induced contraction is via A3AR‐mediated TXA2 production. Concentration responses to Up4A were conducted in aorta from WT and A3AR KO. Up4A (10‐9‐10‐5 M) produced a dose‐dependent contraction (~80%), which was attenuated by non‐selective COX (indomethacin, 10 μM) and selective COX1 (SC560, 10 nM) but not by COX2 inhibition (NS398, 1 μM). Notably, Up4A‐induced contraction was blunted by TXA2 synthase inhibitor ozagrel (10 μM) and TXA2 receptor antagonist SQ29548 (1 μM). Non‐selective AR antagonist 8PST (100 μM) nor A3AR deletion affected Up4A‐induced contraction. However, both non‐selective P2R antagonist PPADS (10 μM) and P2X1R antagonist MRS2159 (30 μM) significantly attenuated Up4A‐induced contraction (~46% and 40%), respectively. Combination of ozagrel with MRS2159 further decreased this effect (~20%). Endothelial denudation almost fully reduced Up4A‐induced contraction. Furthermore, TXA2 production increased in vessels treated with Up4A (3 μM) (26.6±0.5 vs. 83.4±0.8 pg/mg), which was inhibited by either MRS2159 (49.0±0.7 pg/mg) or Ozagrel (42.4±0.5 pg/mg). In conclusion, Up4A‐induced contraction is via TXA2 production, which partially requires the activation of P2X1R but not A3AR through an endothelium‐dependent mechanism (Supported by HL 027339, HL 094447 and MEPSCKL201301).

The novel prostaglandin I2 mimetic ONO-1301 escapes desensitization in an antiplatelet effect due to its inhibitory action on thromboxane A2 synthesis in mice.

ONO-1301 [(E)-[5-[2-[1-phenyl-1-(3-pyridyl)methylidene-aminooxy]ethyl]-7,8-dihydronaphthalene-1-yloxy]acetic acid] is a novel prostaglandin (PG) I2 mimetic with inhibitory activity on the thromboxane (TX) A2 synthase. Interestingly, ONO-1301 retains its inhibitory effect on platelet aggregation after repeated administration, while beraprost, a representative agonist for the PGI2 receptor (IP), loses its inhibitory effect after repeated administration. In the present study, we intended to clarify the mechanism by which ONO-1301 escapes desensitization of an antiplatelet effect. In platelets prepared from wild-type mice, ONO-1301 inhibited collagen-induced aggregation and stimulated cAMP production in an IP-dependent manner. In addition, ONO-1301 inhibited arachidonic acid-induced TXA2 production in platelets lacking IP. Despite the decrease in stimulatory action on cAMP production, the antiplatelet effect of ONO-1301 hardly changed after repeated administration for 10 days in wild-type mice. Noteworthy, beraprost could retain its antiplatelet effect after repeated administration in combination with a low dose of ozagrel, a TXA2 synthase inhibitor. Therefore, we hypothesized that chronic IP stimulation by beraprost induces an increase in TXA2 production, leading to reduction in the antiplatelet effect. As expected, repeated administration of beraprost increased the plasma and urinary levels of a TXA2 metabolite, while ONO-1301 did not increase them significantly. In addition, beraprost could retain the ability to inhibit platelet aggregation after repeated administration in mice lacking the TXA2 receptor (TP). These results indicate that TP-mediated signaling participates in platelet desensitization against IP agonists and that simultaneous inhibition of TXA2 production confers resistance against desensitization on IP agonists.

Nicotine in high concentration causes contraction of isolated strips of rabbit corpus cavernosum.

It is well known that cigarette smoke can cause erectile dysfunction by affecting the penile vascular system. However, the exact effects of nicotine on the corpus cavernosum remains poorly understood. Nicotine has been reported to cause relaxation of the corpus cavernosum; it has also been reported to cause both contraction and relaxation. Therefore, high concentrations of nicotine were studied in strips from the rabbit corpus cavernosum to better understand its effects. The proximal penile corpus cavernosal strips from male rabbits weighing approximately 4 kg were used in organ bath studies. Nicotine in high concentrations (10-5~10-4 M) produced dose-dependent contractions of the corpus cavernosal strips. The incubation with 10-5 M hexamethonium (nicotinic receptor antagonist) significantly inhibited the magnitude of the nicotine associated contractions. The nicotine-induced contractions were not only significantly inhibited by pretreatment with 10-5 M indomethacin (nonspecific cyclooxygenase inhibitor) and with 10-6 M NS-398 (selective cyclooxygenase inhibitor), but also with 10-6 M Y-27632 (Rho kinase inhibitor). Ozagrel (thromboxane A2 synthase inhibitor) and SQ-29548 (highly selective TP receptor antagonist) pretreatments significantly reduced the nicotine-induced contractile amplitude of the strips. High concentrations of nicotine caused contraction of isolated rabbit corpus cavernosal strips. This contraction appeared to be mediated by activation of nicotinic receptors. Rho-kinase and cyclooxygenase pathways, especially cyclooxygenase-2 and thromboxane A2, might play a pivotal role in the mechanism associated with nicotine-induced contraction of the rabbit corpus cavernosum.

Revisiting the usefulness of thromboxane-A2 modulation in the treatment of bronchoconstriction in asthma.

Airway smooth muscle (ASM) is the effector cell in the bronchoconstrictory pathway. It is believed that the bronchoconstriction present in asthma is associated with changes in the airway milieu that affect ASM excitation-contraction coupling and Ca(2+)-handling. Asthmatics also react differently to ventilatory mechanical strain. Deep inspiration (DI), which produces bronchodilation in healthy individuals, is less effective in asthmatics, and even enhances bronchoconstriction in moderate to severely affected patients. Our laboratory has previously studied the mechanotransductory pathway of airway stretch-activated contractions (Rstretch) leading to DI-induced bronchoconstriction. We demonstrated the ability of agonists acting through thromboxane A2 (TxA2) receptors to amplify airway Rstretch responses. Despite the involvement of excitatory prostanoids in bronchoconstriction, clinical trials on treatments targeting TxA2-synthase inhibition and TP-receptor antagonism have produced mixed results. Studies in Western populations produced mostly negative results, whereas studies performed in Asian populations showed mostly positive outcomes. In this review, we discuss the role of TxA2-synthase inhibition and TP-receptor antagonism in the treatment of asthmatics. We present information regarding variations in study designs and the possible role of TP-receptor gene polymorphisms in previous study outcome discrepancies. Perhaps future studies should focus on asthmatic patients with DI-induced bronchoconstriction in particular, planting the seed for the individualized treatments for asthmatics.

Flaxseed oil increases aortic reactivity to phenylephrine through reactive oxygen species and the cyclooxygenase-2 pathway in rats.

BackgroundFlaxseed oil has the highest concentration of omega-3 α-linolenic acid, which has been associated with cardiovascular benefit. However, the mechanism underlying the vascular effects induced through flaxseed oil is not well known. Thus, in the present study, we investigated the effects of flaxseed oil on vascular function in isolated rat aortic rings.MethodsWistar rats were treated daily with flaxseed oil or a control (mineral oil) intramuscular (i.m.) for fifteen days. Isolated aortic segments were used to evaluate cyclooxygenase-2 (COX-2) protein expression, superoxide anion levels and vascular reactivity experiments.ResultsFlaxseed oil treatment increased the vasoconstrictor response of aortic rings to phenylephrine. Endothelium removal increased the response to phenylephrine in aortic segments isolated from both groups, but the effect was smaller in the treated group. L-NAME incubation similarly increased the phenylephrine response in segments from both groups. The TXA2 synthase inhibitor furegrelate, the selective COX-2 inhibitor NS 398, the TP receptor antagonist SQ 29.548, the reactive oxygen species (ROS) scavenger apocynin, the superoxide anion scavengers tiron and the phospholipase A2 inhibitor dexamethasone partially reversed the flaxseed oil-induced increase in reactivity to phenylephrine.ConclusionsThese findings suggest that flaxseed oil treatment increased vascular reactivity to phenylephrine through an increase in ROS production and COX-2-derived TXA2 production. The results obtained in the present study provide new insight into the effects of flaxseed oil treatment (i.m.) on vascular function.

Abstract 608: The Mechanisms of Preservation of Vasorelaxation Induced by Protease-Activated Receptor-2 Activation in the Aorta of Metabolic Syndrome Model Rats

Protease-activated receptor-2 (PAR2) is activated by proteases, such as trypsin, human mast cell beta-tryptase, human kallikrein isoforms, coagulation factors VIIa and Xa, etc. PAR2 is believed to have an important role under inflammatory pathological conditions, but its role in metabolic syndrome remains uncertain. We have reported that PAR2-activating peptide, 2-furoyl-LIGRLO-amide (2fly) causes NO-mediated vasorelaxation in rat aortas similar to that observed with acetylcholine (ACh), and that 2fly-induced vasorelaxation is maintained in aortas from SHRSP.Z- Lepr fa /IzmDmcr (SHRSP.ZF) rats with metabolic syndrome, even though ACh- and sodium nitroprusside (SNP)-induced vasodilation is lower than that in control (Wistar-Kyoto rats, WKY). In this study, we assessed the mechanisms involved in the preservation of PAR2-mediated vasorelaxation in metabolic syndrome. Aortas were isolated from male WKY and SHRSP.ZF rats between 18-20 weeks of age. ACh- and 2fly-induced vasorelaxations were measured in the presence or absence of inhibitors (i.e., COX inhibitors, a TXA 2 synthase inhibitor, and a TXA 2 /PGH 2 receptor antagonist) using organ bath methods. Expression of endothelium NO synthase (eNOS) in aortas was measured by western blot analysis. Indomethacin (a non-selective COX inhibitor), SC560 (a selective COX-1 inhibitor), NS398 (a selective COX-2 inhibitor), OKY-046 (a TXA 2 synthase inhibitor), and ONO-3708 (a TXA 2 /PGH 2 receptor antagonist) did not affect both ACh- and 2fly-induced vasorelaxation in aortas of SHRSP.ZF rats as well as WKY. Expression of phosphorylated eNOS at Ser 1177 in 2fly-stimulated aortas was higher than that in ACh-stimulated ones in SHRSP.ZF rats, but there was no significant difference between these levels in WKY. Our results show that the preservation of 2fly-induced vasorelaxation observed in SHRSP.ZF rats with metabolic syndrome may be because of enhanced eNOS activation via PAR2, but not coupled to the COX pathway. Thus, preservation of PAR2-mediated vasorelaxation may be one of the key responses to maintain blood circulation under inflammatory conditions, such as metabolic syndrome.

SFlt-1 Elevates Blood Pressure by Augmenting Endothelin-1-Mediated Vasoconstriction in Mice

ObjectiveScavenging of vascular endothelial growth factor (VEGF) elevates blood pressure (BP) in patients receiving anti-angiogenic therapy. Similarly, inhibition of circulation VEGF by its soluble receptor fms-like tyrosine kinase-1 (sFlt-1) underlies BP elevation in pre-eclampsia. Both phenotypes are characterized by augmented production of endothelin-1 (ET-1), suggesting a role for ET-1 in anti-angiogenic hypertension. We aimed to assess the effect of VEGF inhibition on ET-1-induced contractility and downstream ET-1 signaling.Approach and ResultsMale C57BL/6N mice were treated with either sFlt-1 or vehicle and BP was assessed via tail-cuff. Mean arterial pressure of sFlt-1-treated mice markedly increased compared to vehicle-treated controls (N = 11–12, p<0.05). After sacrifice, carotid and mesenteric arteries were isolated for isometric tension measurements. ET-1-induced contractions were similar in mesenteric arteries of vehicle and sFlt-1-treated mice, but augmented in carotid segments of sFlt-1-treated mice compared to controls (N = 9–10, p<0.05). The increased contraction in carotid segments could be completely abrogated by the cyclooxygenase (COX) inhibitor indomethacin (N = 9–10, p<0.05), indicating heightened prostaglandin-mediated vasoconstriction. This was associated with a shift towards procontractile ETB signaling in sFlt-1-treated mice, possibly explaining the increased ET-1-induced prostaglandin-mediated vasoconstriction. In line with the ex vivo findings, sFlt-1-induced BP elevation could be prevented in vivo by oral treatment with either a high-dose of the COX inhibitor aspirin (N = 7) or with picotamide (N = 9), a dual thromboxane A2 synthase inhibitor and receptor antagonist.ConclusionsVEGF inhibition augments the pressor response to ET-1. The cyclooxygenase-thromboxane signaling route downstream of ET-1 might be a possible target to prevent BP elevation during VEGF inhibition.

Thromboxane A2 exerts promoting effects on cell proliferation through mediating cyclooxygenase-2 signal in lung adenocarcinoma cells

Lung cancer concerns a worldwide health problem and the efficacy of available treatments is unsatisfactory. Recently, thromboxane A2 (TXA2) synthase (TXAS) and receptor (TXA2R) have been documented to play a role in lung cancer development. Therefore, dual TXA2R modulator (i.e., the dual blocker of TXAS and TXA2R) may be more efficacious to kill lung tumor cells than single TXAS inhibitor or TXA2R antagonism. The close relationship between cyclooxygenase (COX)-2 and TXAS also raises whether or how TXA2 contributes to the oncogenic activity of COX-2. This study is therefore conducted to answer these questions. Various inhibitors and siRNA were used to evaluate the roles of TXA2 and COX-2 in the proliferation and apoptosis of lung adenocarcinoma cells. Cell proliferation was detected using both MTS ELISA and BrdU labeling ELISA. Cell cycle distribution and apoptosis were examined by flow cytometric analysis. TXB2 level, reflecting the biosynthesis of TXA2, was detected by peroxidase-labeled TXB2 conjugates using an enzyme immunoassay kit. Western blotting was performed to evaluate many biomarkers for cell cycles, apoptosis and proliferation. The levels of COXs were screened by reverse transcriptase and real-time quantitative PCR. We found either single TXAS inhibitor/TXA2R antagonist or the dual TXA2 modulators offered a similar inhibition on cell proliferation. Moreover, inhibition of TXA2 arrested cells at the G2/M phase and induced apoptosis. It is further demonstrated that TXA2 was able to function as a critical mediator for tumor-promoting effects of COX-2 in lung adenocarcinoma cells. The present study has for the first shown that dual TXA2 modulators and the single blocker of TXAS or TXA2R offer a similar inhibitory role in lung adenocarcinoma cell proliferation and that the tumor-promoting effects of COX-2 can largely be relayed by TXA2. Thus, TXA2 should be regarded as a critical molecule in COX-2-mediated tumor growth and a valuable target against lung cancer.

Attenuation of alpha-adrenergic-induced vasoconstriction by dietary wild blueberries (Vaccinium angustifolium) is mediated by the NO-cGMP pathway in spontaneously hypertensive rats (SHRs)

The role of wild blueberries (WB) on key signaling steps of nitric oxide (NO) and cyclooxygenase (COX) pathways was examined in spontaneously hypertensive rats (SHRs) after eight weeks on a control (C) or an 8% w/w WB diet. Aortic rings from SHRs were stimulated with phenylephrine (Phe) in the absence or presence of inhibitors of: soluble guanylyl cyclase (sGC), phosphodiesterase-5 (PDE5), prostaglandin I2 (PGI2) synthase and thromboxane A2 (TXA2) synthase. Additionally, enzymatic activities in these pathways were determined by the concentration of NO, cyclic guanosine monophosphate (cGMP), PGI2 and TXA2. In the WB-fed SHR, attenuation of Phe-induced vasoconstriction was mediated by an increased synthesis or preservation of cGMP. Despite an increased release of PGI2 in the WB group, neither inhibition of PGI2 or TXA2 synthase resulted in a different response to Phe between the control and the WB rings. Hence, in the SHR, WB decrease Phe-mediated vasoconstriction under basal conditions by enhancing NO-cGMP signaling without a significant involvement of the COX pathway.

Covering the Cover

Covering the Cover

Thromboxane A2 Synthase Inhibitors Prevent Production of Infectious Hepatitis C Virus in Mice With Humanized Livers

A 3-dimensional (3D) culture system for immortalized human hepatocytes (HuS-E/2 cells) recently was shown to support the lifecycle of blood-borne hepatitis C virus (HCV). We used this system to identify proteins that are active during the HCV lifecycle under 3D culture conditions. We compared gene expression profiles of HuS-E/2 cells cultured under 2-dimensional and 3D conditions. We identified signaling pathways that were activated differentially in the cells, and analyzed their functions in the HCV lifecycle using a recombinant HCV-producing cell-culture system, with small interfering RNAs and chemical reagents. We investigated the effects of anti-HCV reagents that altered these signaling pathways in mice with humanized livers (carrying human hepatocytes). Microarray analysis showed that cells cultured under 2-dimensional vs 3D conditions expressed different levels of messenger RNAs encoding prostaglandin synthases. Small interfering RNA-mediated knockdown of thromboxane A2 synthase (TXAS) and incubation of hepatocytes with a TXAS inhibitor showed that this enzyme is required for production of infectious HCV, but does not affect replication of the HCV genome or particle release. The TXAS inhibitor and a prostaglandin I2 receptor agonist, which has effects that are opposite those of thromboxane A2, reduced serum levels of HCV and inhibited the infection of human hepatocytes by blood-borne HCV in mice. An inhibitor of the prostaglandin synthase TXAS inhibits production of infectious HCV particles in cultured hepatocytes and HCV infection of hepatocytes in mice with humanized livers. It therefore might be therapeutic for HCV infection.

Sa1937 Continuous Bile Acid Exposure Is a Risk Factor for Development of Reactive Gastropathy

and the thromboxane (TX) A2 antagonistic action, although the exact mechanism remains unexplored. In the present study, we examined the effects of egualen against gastric lesions induced by ischemia/reperfusion (I/R) as well as double antithrombotic therapy with lowdose aspirin (ASA) plus clopidogrel, and small intestinal lesions induced by loxoprofen in rats and investigated the possible mechanisms involved in the protective action. Methods: Male SD rats were used under urethane anesthesia (gastric I/R and bleeding models) or conscious (intestinal lesion model) conditions. I/Rinduced gastric injury was produced in mice by clamping celiac artery for 30 min, followed by reperfusion for 60 min. Egualen, ozagurel (a TXA2 synthase inhibitor) or seratrodast (a TXA2 antagonist) was given introduodenally (ID) 60 min before ischemia/reperfusion. Gastric bleeding was induced by luminal perfusion with 25 mM ASA+50 mM HCl for 2 h in the presence of clopidogrel (30 mg/kg). Egualen was given ID 60 min before ASA perfusion, while PGE2 was given IV 30 min before. To produce small intestinal lesions the animals were given loxoprofen (60 mg/kg) PO and killed 24 h later. Egualen was given PO 30 min before and 6 h after loxoprofen. Results: The severity of I/R-induced gastric lesions was significantly reduced by pretreatment with egualen, in addition to ozagrel and seratrodast, suggesting the involvement of the TXA2 antagonistic action in the protective effect of egualen in this model. Perfusion of the stomach with ASA in the presence of clopidogrel produced gastric lesions with severe bleeding. Egualen significantly reduced the gastric ulcerogenic and bleeding responses to the antiplatelet therapy, similar to PGE2. On the other hand, loxoprofen induced multiple lesions in the small intestine, accompanied by a decrease in mucus secretion, an increase in bacterial invasion and iNOS expression. Egualen prevented the development of small intestinal lesions, together with increase of mucus secretion and suppression of bacterial invasion and iNOS expression. Conclusion: These results suggest that egualen has prophylactic effect against a variety of gastrointestinal lesions, probably through its characteristic pharmacological properties, such as the TXA2 antagonistic action and local mucosal protection in the stomach, and suppression of bacterial invasion due to the increased mucus secretion in the small intestine.

PHARMACODYNAMIC EFFECTS OF EV–077 IN PATIENTS WITH DIABETES MELLITUS ON CLOPIDOGREL MONOTHERAPY: RESULTS OF AN IN VITRO PILOT INVESTIGATION

Diabetes mellitus (DM) patients have increased thromboxane A2 (TXA2) which activate TX prostanoid receptors (TPR). In DM patients, EV–077, a combined TXA2 synthase inhibitor and TPR antagonist, achieves more effective platelet inhibition compared with aspirin. However, the effects of EV–077 in

Ozagrel hydrochloride, a selective thromboxane A2 synthase inhibitor, alleviates liver injury induced by acetaminophen overdose in mice

BackgroundOverdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2) synthase inhibitor, on liver injury induced by APAP overdose in mice.MethodsHepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg). The effects of ozagrel (200 mg/kg) treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT) levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL) on cytochrome P450 2E1 (CYP2E1) activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1–100 muM) were evaluated by the WST-1 cell viability assay.ResultsOzagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B2 levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos) and C/EBP homologous protein (chop), but did not suppress B-cell lymphoma 2-like protein11 (bim) expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16.ConclusionsWe demonstrate that the TXA2 synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest that it is a promising therapeutic candidate for the treatment of APAP-induced liver injury.

Pharmacological Analysis of the Rat Femoral Artery Response to Bradykinin

Bradykinin (BK) plays an important role in different physiological processes including the general preservation and modulation of vascular systems. The present study was designed in order to examine the effect of BK on isolated rat femoral artery rings and to investigate the participation of intact endothelium, cyclooxygenase products, Ca2+ channels, Na+/K+–ATPase, and B2 kinin receptors in BK-induced action. Circular artery segments were placed in organ baths. The endothelium was mechanically removed from some arteries. Concentration–contraction curves for BK were obtained in the rings previously equilibrated at the basal tone. BK produced a concentration–dependent contraction, which was reduced by endothelial denudation. The BK–induced effect was almost completely inhibited by indomethacin (cyclooxygenase inhibitor) or OKY–046 (thromboxane A2–synthase inhibitor). Nifedipine (Ca2+ channel blocker), ouabain (Na+/K+–ATPase inhibitor), or HOE–140 (selective B2 kinin receptor antagonist) significantly reduced the BK–evoked effect. In conclusion, it can be proposed that BK produces concentration– and endothelium–dependent contractions of the isolated rat femoral artery, which is for the most part a consequence of B2 kinin receptor activation. Cyclooxygenase contractile products, especially thromboxane A2, play a significant role in this course of action. The transduction mechanism involved in the process of BK–induced femoral artery contraction include the activation of voltage–gated Ca2+ channels, and in a smaller extent Na+/K+–ATPase as well.

Decreased Mrp2 transport in severe macrovesicular fatty liver grafts

BackgroundSevere macrosteatotic liver has been regarded as a donor contraindication for liver transplantation. However, it has not yet been determined whether hepatocytes lose function before cold ischemia. This study was designed to elucidate certain pathophysiological alterations and how to ameliorate such hepatic dysfunctions. Materials and methodsWistar rats were fed with a choline-deficient diet (CD) for up to 6 wk, and their livers were then perfused with Krebs-Henseleit buffer to examine bile output and biliary constituents. Organic anion transport from hepatocellular canalicular membranes through Mrp2 was examined by kinetic analyses for biliary exclusion of 5-carboxyfluorescein (CF), a fluoroprobe excreted through Mrp2. ResultsMacrovesicular fatty deposits exceeded 60% and serum aspartate aminotransferase (AST) increased on 6-wk CD (CD6w), but not 3-wk CD (CD3w). Mrp2-deficient rat livers (Eisai hyperbilirubinemia) with 3-wk CD were more vulnerable than CD3w livers. In CD6w rats, bile flow rate and biliary glutathione significantly decreased. These declines coincided with the intracellular localization of Mrp2. Moreover, kinetic analyses for biliary CF revealed significant delay in 6-wk CD-fed rat livers. Pioglitazone, a ligand of PPARγ activating protein kinase A (PKA), significantly attenuated this delay by sorting Mrp2 into bile canalicular membranes. However, a PKA inhibitor blunted the increase in CF exclusion, re-localizing Mrp2 into the hepatocellular cytoplasm. A thromboxane A2 synthase inhibitor also ameliorated the CF exclusion delay. ConclusionPioglitazone activated PKA, increasing Mrp2 transports to detoxify xenobiotics. Pioglitazone may allow the donor indications for liver transplantation to be expanded to include severe macrovesicular fatty livers.