TXB2 Production Research Articles

Diverticular Disease Worsening Is Associated with Increased Oxidative Stress and Gut Permeability: New Insights by Circulating Biomarkers.

Diverticular disease (DD) management is impaired by its pathogenesis, which is still not completely defined, with an unmet clinical need for improved therapies. Ex vivo DD human models demonstrated the presence of a transmural oxidative imbalance that supports an ischemic pathogenesis. This study aimed to assess, with the use of circulating biomarkers, insights into DD pathogenesis and possible therapeutic targets. Nox2-derived peptide, H2O2, antioxidant capacity, isoprostanes, thromboxanes, TNF-α, LPS and zonulin were evaluated by ELISA in healthy subjects (HS) and asymptomatic and symptomatic DD patients. Compared to HS, DD patients presented low antioxidant capacity and increase in sNox2-dp, H2O2 and isoprostanes paralleled to a TNFα increase, lower than that of oxidative markers. TxB2 production correlated to Nox2 and isoprostanes, suggesting platelet activation. An increase in zonulin and LPS highlighted the role of gut permeability and LPS translocation in DD pathogenesis. The increase of all the markers statistically correlated with DD severity. The present study confirmed the presence of a main oxidative imbalance in DD and provides evidence of platelet activation driven by LPS translocation. The use of circulating biomarkers could represent a new clinical tool for monitoring disease progression and validate therapeutic strategies never tested in DD as antioxidant supplementation.

Innate Immune System Stimulation During Pregnancy Upregulates Thromboxane Synthesis in Rat Maternal Heart

BackgroundInfections during pregnancy are associated with adverse maternal and fetal outcomes. We previously showed that exposure to immunostimulatory CpG oligonucleotides (ODN2395, synthetic Toll‐like receptor 9 agonist) during pregnancy induces maternal vascular inflammation and enhances vascular tone in pregnant rats. These outcomes were mediated in part by activation of the cyclooxygenase/thromboxane A2 (COX/TxA2) pathway. The objective of this study was to investigate the impact of ODN2395‐induced immune system stimulation on maternal hearts during pregnancy.HypothesesExposure to TLR9‐mediated immune system activation during pregnancy upregulates the COX/TxA2signaling pathway in maternal cardiac tissues.MethodsRats were treated with a synthetic CpG DNA (ODN2395, 1 mg/kg, intraperitoneal injection) or vehicle (saline) in late pregnancy (gestational day (GD) 14, 16, and 18) and euthanized on GD 20 (term 22‐23). Fetoplacental biometrics were recorded after euthanasia and maternal hearts were collected to assess COX‐1 and COX‐2 expression via western blotting and 6‐keto PGF1α (PGI2 metabolic byproduct) and TxB2 (TxA2 metabolic byproduct) production use ELISA.ResultsMaternal heart weights did not differ between groups (ODN2395: 0.26 g/100 g BW vs. Vehicle: 0.23 g/100 g BW, n≥7, p=0.11). Left ventricular tissues from dams treated with ODN2395 released higher concentrations of TxB2compared to cardiac tissues from vehicle‐treated dams (ODN2395: 0.56 ± 0.06 ng/mg total protein vs. Vehicle: 0.31 ± 0.04 ng/mg total protein, n≥5, p=0.0041) but there were no differences in cardiac 6‐keto PGF1α release between groups (p=0.16). COX‐2 expression was lower in left ventricles from ODN2395‐treated rats compared to vehicle‐treated rats (p=0.009). There were no differences in cardiac COX‐1 expression between groups (p=0.27). Exposure to ODN2395 during pregnancy did not affect fetal weights (ODN2395: 2.28 ± 0.03 g vs. Vehicle: 2.25 ± 0.06 g, n≥7, p=0.9) or placenta weights (ODN2395: 0.44 ± 0.02 g vs. Vehicle 0.49 ± 0.01 g, n≥7, p=0.06), but increased fetal‐placental weight ratio (ODN2395: 5.3 ± 0.22 vs. Vehicle: 4.7 ± 0.15, p = 0.04). COX‐2 expression was greater in placental tissues from ODN2395‐treated rats (p=0.004) but there were no differences in placental 6‐keto PGF1α(p=0.51) and TxB2 production (p=0.32).ConclusionExposure to immunostimulatory CpG ODN during pregnancy induces upregulation of TxB2 synthesis in maternal cardiac tissues coupled with a reduction in COX‐2 expression. We suggest that cardiac inflammation during pregnancy may increase maternal risk for future cardiovascular events.

Bitis arietans Snake Venom and Kn-Ba, a Snake Venom Serine Protease, Induce the Production of Inflammatory Mediators in THP-1 Macrophages.

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims’ death or permanent disability. To better characterize the inflammatory process induced by this snake’s venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1β in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Pimpinellin Inhibits Collagen-induced Platelet Aggregation and Activation Through Inhibiting Granule Secretion and PI3K/Akt Pathway.

Pimpinellin is a coumarin-like compound extracted from the root of Toddalia asiatica. Its effects on platelet function has not been investigated. This study found that pimpinellin pretreatment effectively inhibited collagen-induced platelet aggregation, but did not alter ADP- and thrombin-induced aggregation. Platelets pretreated with pimpinellin showed reduced α granule (CD62) level and secretion of dense granule (ATP release). Pimpinellin-treated platelets also exhibited decreased clot reaction and TxB2 production. Pimpinellin pretreatment suppressed adhesion and spreading of human platelets on the fibrinogen coated surface. Analysis of tail bleeding time of mice administered with pimpinellin (40 mg/kg) revealed that pimpinellin did not change tail bleeding time significantly, number of blood cells, and APTT and PT levels. Pimpinellin inhibited collagen-induced ex vivo aggregation of mice platelets. Immunoblotting results showed that pimpinellin suppressed collagen-induced phosphorylation of PI3K-Akt-Gsk3β and PKC/MAPK in platelets.

Combination of Panax notoginseng saponins and aspirin potentiates platelet inhibition with alleviated gastric injury via modulating arachidonic acid metabolism

High platelet reactivity and gastric mucosal injury after aspirin (ASA) treatment are associated with poor compliance and an increased risk of cardiovascular events. Panax notoginseng saponins (PNS) have been widely used for the treatment of coronary heart disease (CHD) in addition to antiplatelet drugs in China; however, the joint effect and possible mechanism of PNS in addition to ASA on platelet activation and gastric injury remain unclear. This study was designed to investigate the combinational effects of PNS with ASA, and to explore the underlying mechanism via arachidonic acid (AA) metabolism pathway using lipidomic analysis. In a randomized, assessor-blinded trial, 42 patients with stable coronary heart disease (SCHD) and chronic gastritis were randomly assigned to receive ASA (n = 21) or PNS + ASA (n = 21) for 2 months. Compared with ASA alone, PNS + ASA further inhibited CD62p expression, GPIIb-IIIa activation and platelet aggregation and led to increased platelet inhibition rate. PNS + ASA suppressed the activity of platelet cyclooxygenase (COX)-1, and decreased the production of TXB2, PGD2, PGE2, 11-HETE, the downstream oxylipids of AA/COX-1 pathway in platelets, compared with ASA alone. The severity of dyspepsia assessment (SODA) results showed that patients in PNS + ASA group exhibited relieved dyspeptic symptoms as compared with those in ASA group, which might be associated with enhanced secretion of gastrin and motilin. In vivo study of myocardial infarction rats demonstrated that PNS attenuated ASA-induced gastric mucosal injury, which was related to markedly boosted gastric level of 6,15-diketo-13,14-dihydro-prostaglandin (PG)F1α, 13,14-dihydro-15-keto-PGE2 and PGE2 from AA/PG pathway in response to PNS + ASA compared with ASA alone. In summary, our study demonstrated that the combination of PNS and ASA potentiated the antiplatelet effect of ASA via AA/COX-1/TXB2 pathway in platelets, and mitigated ASA-related gastric injury via AA/PG pathway in gastric mucosa.

Perioperative pharmacokinetics and pharmacodynamics of meloxicam in emus (Dromaius novaehollandiae) of different age groups using nonlinear mixed effect modelling.

Meloxicam is a widely used nonsteroidal anti-inflammatory drug in avian species. However, variability in pharmacokinetic (PK) and pharmacodynamic (PD) parameters in birds warrants species-specific studies for dose and dosing interval optimization. We performed a perioperative PK study of meloxicam (0.5mg/kg, intravenously) on emus of three different age groups: 3 chicks (5weeks old, 3.5kg), 4 juveniles (26weeks old, 18.8kg) and 6 adults (66weeks old, 38.8kg). A two-compartment population PK model including weight as a significant covariate on clearance and central volume of distribution (V1) best fitted the data. The typical values (20kg bird) for clearance and V1 were 0.54L/kg/h and 0.095L/kg. Both parameters significantly decreased with increasing weight/age. Meloxicam potency and selectivity for COX-1 and COX-2 were measured in whole blood assays (TxB2 production endpoint). Meloxicam was partially selective in emus (IC50 COX-1:COX-2=9.1:1). At the current empirical dose (0.5mg/kg/24hr), plasma meloxicam concentration is above IC50 of COX-2 for only 2hr. PK/PD predicted dose required for 80% COX-2 inhibition over 24hr were 3.4, 1.4 and 0.95L/kg/day in chicks, juveniles and adult emus, respectively. The safety, therapeutic efficacy and practicality of modifying the daily dose or dose interval should be considered for dose recommendations in emus.

Effects of New NSAID-CAI Hybrid Compounds in Inflammation and Lung Fibrosis.

Pulmonary fibrosis is a severe lung disease with progressive worsening of dyspnea, characterized by chronic inflammation and remodeling of lung parenchyma. Carbonic anhydrases are a family of zinc-metallo-enzymes that catalyze the reversible interconversion of carbon-dioxide and water to bicarbonate and protons. Carbonic Anhydrase Inhibitor (CAI) exhibited anti-inflammatory effects in animals with permanent-middle-cerebral artery occlusion, arthritis and neuropathic pain. The pharmacological profile of a new class of hybrid compounds constituted by a CAI connected to a Nonsteroidal-Anti-Inflammatory Drug (NSAID) was studied in the modulation of inflammation and fibrosis. In-vitro tests were performed to assess their effects on cyclo-oxygenase enzyme (COX)-1 and COX-2, namely inhibition of platelet aggregation and thromboxane B2 production in the human-platelet-rich plasma, and reduction of Prostaglandin-E2 production in lipopolysaccharide-treated-RAW-264.7 macrophage cell line. The activity of compound 3, one of the most active, was studied in a model of bleomycin-induced lung fibrosis in C57BL/6 mice. The hybrid compounds showed a higher potency in inhibiting PGE2 production, but not in modifying the platelet aggregation and the TXB2 production in comparison to the reference molecules, indicating an increased activity in COX-2 inhibition. In the in-vivo murine model, the compound 3 was more effective in decreasing inflammation, lung stiffness and oxidative stress in comparison to the reference drugs given alone or in association. In conclusion, these CAI-NSAID hybrid compounds are promising new anti-inflammatory drugs for the treatment of lung chronic inflammatory diseases.

Pharmacological characterization of the biosynthesis of prostanoids and hydroxyeicosatetraenoic acids in human whole blood and platelets by targeted chiral lipidomics analysis

Platelet 12-lipoxygenase(p-12-LOX) is highly expressed in human platelets, and the development of p-12-LOX inhibitors has the potential to be a novel antithrombotic tool by inhibiting thrombosis without prolonging hemostasis. A chiral liquid chromatography-mass spectrometry(LC-MS/MS) method was used to assess the impact of three commercially available LOX inhibitors[esculetin(6,7-dihydroxycoumarin), ML-355(N-2-benzothiazolyl-4-[[(2-hydroxy-3-methoxyphenyl)methyl]amino]-benzenesulfonamide), CDC(cinnamyl-3,4-dihydroxy-α-cyanocinnamate) and acetylsalicylic acid(ASA; a cyclooxygenase-1 inhibitor) on the generation of prostanoids and HETEs(hydroxyeicosatetraenoic acids) in human whole blood allowed to clot for 1 h at 37 °C(serum), platelet-rich plasma(PRP) stimulated with collagen or TRAP-6(a peptide activating thrombin receptor) and washed platelets. In serum, ML-355 did not affect eicosanoid generation, while CDC caused an incomplete reduction of 12S-HETE levels; esculetin inhibited both 12S-HETE and thromboxane(TX)B2 production; ASA selectively affected TXB2 production. In washed platelets stimulated with thrombin, esculetin, and CDC inhibited both 12S-HETE and TXB2 while ML-355 was almost ineffective. In PRP, ML-355, CDC, and esculetin did not affect platelet aggregation associated with incomplete effects on eicosanoid biosynthesis. ASA alone or in combination with ticagrelor(a P2Y12 blocker) affected platelet aggregation associated with profound inhibition of TXB2 generation. P2Y12 receptor signaling contributed to platelet 12S-HETE biosynthesis in response to primary agonists. In conclusion, ML-355, esculetin, and CDC were not selective inhibitors of p-12-LOX in different cellular systems. They did not affect platelet aggregation induced in PRP by collagen or TRAP-6. The characterization of 12-LOX inhibitors on eicosanoids generated in human whole blood is useful for information on their enzyme selectivity, off-target effects, and the possible influence of plasma components on their pharmacological effects.

Abstract 6364: LFA-9, a dual mPGES-1 and 5-LOX inhibitor, suppresses colon cancer stemness and inflammogen-induced inflammatory response

Abstract Chronic inflammation triggers tumorigenesis of the colorectum; hence several non-steroidal anti-inflammatory drugs (NSAIDs) have been explored for prevention and treatment of inflammatory diseases, including colorectal cancer (CRC). Although traditional NSAIDs and COX-2 inhibitors are effective chemopreventive agents for CRC, unwanted side effects (gastrointestinal and renal) hinder their use. Mechanistic studies suggest that the side effects are in part due to a COX-2 inhibition-associated increase in 5-lipoxygenase (5-LOX) metabolites and reduced PGI2 levels. Hence, agents targeting microsomal prostaglandin synthase-1 (mPGES-1) and 5-LOX inhibitors may prove to be more effective and safer agents for chemoprevention. LFA-9 was designed based on the pharmacophore of licofelone by adding 2-aminoethanoic acid (Glycine) at its carboxylic end to enhance renal clearance and selectivity towards dual mPGES-1 and 5-LOX inhibition. Here we characterized LFA-9 in detail for its mPGES1/5-LOX selectivity over COX-1/2 using in silico molecular modelling, in vitro enzyme activity assays, and ex vivo inhibition studies with human/mouse/rat mPGES-1 and 5-LOX enzymes to facilitate pre-clinical experimentation and clinical translation. In silico, LFA-9 showed strong binding affinity against 5-LOX (binding energies -199.85 and -215.3 Kcal/mol) and mPGES-1 (binding energies -238.27 and -257.76 Kcal/mol), but not against COX-1/2. LFA-9 substantially inhibited mPGES-1 (IC50 0.52 - 1.40 µM) and 5-LOX (IC50 0.89 - 2.75 µM) but not COX-1/2 (IC50>1mM) in cell-free assays. Ex-vivo, LFA-9 inhibited PGE2 (IC50 0.47 - 0.78 µM) and LTB4 (IC50 0.66 - 1.24 µM) production and spared PGI2 (IC50>1 mM) and TXB2 (IC50>1 mM) production in LPS-stimulated human/mouse/rat macrophages and whole blood (cell-based system). Circular dichroism and isothermal calorimetric-based studies demonstrated that LFA-9 strongly binds and induces changes in the secondary structure of human mPGES-1 and 5-LOX enzymes, thereby inhibiting their activities. Since PGE2 from mPGES1 activity promotes colon tumor stemness, LFA-9 was evaluated on colonic cancer organoids (spheroids) derived from PIRC (Polyposis in Rat Colon) rats that replicate human familial adenomatous polyposis. LFA-9 significantly inhibited the growth of colonic spheroids at 25 µM as compared to that of untreated as well as licofelone-treated spheroids. Anti-inflammatory properties of LFA-9 were evaluated in vivo using an inflammogen (carrageenan)-induced rat paw edema model. Dietary LFA-9 inhibited edema formation after carrageenan injection. In summary, LFA-9 showed anti-inflammatory and anti-tumor properties through mPGES1/5-LOX inhibition. Our data supports LFA-9, a novel mPGES-1 and 5-LOX inhibitor, as a safer drug candidate for prevention and therapy of colorectal cancer. (Supported by R01CA 213987, NIH/NCI HHSN261201500024I and VA merit). Citation Format: Nagendra Sastri Yarla, Gopal Pathuri, Hariprasad Gali, Venkateshwar Madka, Janani Panneerselvam, Parthasarathy Chandrakesan, Courtney W. Houchen, Elizabeth R. Glaze, Jennifer Fox, David McCormick, Chinthalapally V. Rao. LFA-9, a dual mPGES-1 and 5-LOX inhibitor, suppresses colon cancer stemness and inflammogen-induced inflammatory response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6364.

Patients with Essential Thrombocythemia may be Poor Responders to Enteric-Coated Aspirin, but not to Plain Aspirin.

Essential thrombocythemia (ET) patients are treated with aspirin (acetylsalicylic acid [ASA]) to prevent thrombosis. Previous studies showed that serum thromboxane (Tx) B2 was high 24 hours after enteric-coated (EC)-ASA in ET patients, due to increased number of noninhibited reticulated platelets (RPs), consequent to high platelet turnover, and that ASA should be given twice a day to ET patients. We studied ET patients (n = 17) and healthy subjects (n = 10) on 100 mg EC-ASA once daily; experiments were repeated after 14-day treatment with 100 mg plain-ASA once daily. Serum TxB2, plasma ASA, and salicylic acid (SA) were measured before the morning dose and up to 8 hours thereafter. Blood activity of ASA-deacethylating esterases, in vitro inhibition of collagen-induced TxB2 production by ASA (10-1,000 µM), and number of RP were measured. TxB2 inhibition by ASA in vitro and esterases activities were normal in all subjects. EC-ASA elicited highly variable responses; 6 ET patients were poor responders, as their serum TxB2 was high after EC-ASA; their plasma levels of ASA and SA were low/undetectable. In contrast to EC-ASA, plain ASA decreased serum TxB2 and increased plasma ASA and SA in all subjects. Serum TxB2 was high in ET patients at 24 hours and significantly correlated with RP count (but not RP percentage) and platelet count. Plain ASA should be used in ET patients to inhibit platelets efficiently. The identification of ET patients who might benefit from twice a day ASA could simply be based on their platelet count: since their platelet turnover is not increased, ET patients with normalized platelet count should not need twice a day ASA treatment.

Ulmus parvifolia Modulates Platelet Functions and Inhibits Thrombus Formation by Regulating Integrin αIIbβ3 and cAMP Signaling.

BackgroundThe prevalence of cardiovascular diseases (CVDs) is increasing at a high rate, and the available treatment options, sometimes, have complications which necessitates the need to develop safer and efficacious approaches. Ethnomedicinal applications reportedly reduce CVD risk. Ulmus parvifolia Jacq. (Ulmaceae) commonly known as Chinese Elm or Lacebark Elm, is native to China, Japan, and Korea. It exhibits anti-inflammatory, antiviral, and anticancer properties, but its anti-platelet properties have not yet been elucidated.PurposeTo investigate the pharmacological anti-platelet and anti-thrombotic effects of U. parvifolia bark extract.Study Design and MethodsHuman and rat washed platelets were prepared; light transmission aggregometry and scanning electron microscopy was performed to assess platelet aggregation and the change in platelet shape, respectively. Intracellular calcium mobilization, ATP release, and thromboxane-B2 production were also measured. Integrin αIIbβ3 activation was analyzed in terms of fibrinogen binding, fibronectin adhesion, and clot retraction. The expression of MAPK, Src, and PI3K/Akt pathway proteins was examined. Cyclic nucleotide signaling pathway was evaluated via cAMP elevation and VASP phosphorylation. Anti-thrombotic activity of the extract was evaluated in vivo using an arteriovenous shunt rat model, whereas its effect on hemostasis in mice was assessed via bleeding time assay.Results U. parvifolia extract significantly inhibited human and rat platelet aggregation in a dose-dependent manner along with inhibition of calcium mobilization, dense granule secretion, and TxB2 production. Integrin αIIbβ3 mediated inside-out and outside-in signaling events, as evidenced by the inhibition of fibrinogen binding, fibronectin adhesion, and clot retraction. The extract significantly reduced phosphorylation of Src, MAPK (ERK, JNK, and p38MAPK), and PI3K/Akt pathway proteins. Cyclic-AMP levels were elevated in U. parvifolia-treated platelets, while PKAαβγ and VASPser157 phosphorylation was enhanced. U. parvifolia reduced thrombus weight in rats and moderately increased bleeding time in mice.Conclusion U. parvifolia modulates platelet responses and inhibit thrombus formation by regulating integrin αIIbβ3 mediated inside-out and outside-in signaling events and cAMP signaling pathway.

Unexpected effects of long-term treatment with acetylsalicylic acid on late phase of pulmonary metastasis in murine model of orthotopic breast cancer.

Long-term administration of acetylsalicylic acid (ASA) was effective in prevention of colorectal cancer, whereas the efficacy of this compound in other cancer types, including breast cancer, has been less convincingly documented. Indeed, the antimetastatic effect of low-dose ASA was observed only in the early intravascular phase of metastasis of breast cancer. In the present work, we characterized the effects of long-term treatment with ASA on the late phase of pulmonary metastasis in a mouse orthotopic 4T1 breast cancer model. Mice were treated with ASA at a dose of 12 mg·kg-1 of body weight daily starting one week prior to inoculation of 4T1 breast cancer cells, and the treatment was continued throughout progression of the disease. ASA administration decreased platelet TXB2 production in ex vivo assays but did not change thrombin-induced platelet reactivity. Although the number of metastases in the lungs remained unchanged in ASA-treated mice, infiltration of inflammatory cells was increased concomitantly with higher G-CSF and serotonin concentrations in the lungs. Pulmonary NO production was compromised compared to control 4T1 mice. ASA treatment also evoked an increase in platelet and granulocyte counts and decreased systemic NO bioavailability along with increased markers of systemic oxidant stress such as higher GSSG/lower GSH concentrations in RBC. Analysis of eicosanoids in stirred blood demonstrated that administration of ASA at a dose of 12 mg·kg-1 to cancer-bearing mice had an effect beyond inhibition of platelet COX-1, suggesting long-term treatment with low-dose aspirin is not a selective murine platelet COX-1/TXA2 pathway inhibitor in cancer-bearing mice. In summary, quite surprisingly, long-term treatment with low-dose ASA administered until the advanced phase of breast cancer in a murine orthotopic model of 4T1 breast cancer negatively affected the phenotype of the disease.

LFA‐9, a Selective Inhibitor of Microsomal Prostaglandin Synthase‐1 and 5‐Lipoxygenase: Prevention of Inflammatory and Oncologic Diseases

Cyclooxygenase‐1 (COX‐1), COX‐2 and 5‐lipoxygenase (5‐LOX) as targets for inflammatory diseases and many agents were developed and extensively used in the clinic. Chronic use of drugs that target COX‐1/COX‐2 are associated with unwanted side effects including GI toxicity and cardiovascular risk. Hence, the main aim of this work is to develop mechanistic based safer agents that would target microsomal prostaglandin synthase‐1 (mPGES‐1) (a downstream enzyme of COX‐2) and 5‐LOX, while sparing COX‐1&‐2 activity. LFA‐9 was designed using structure‐based drug discovery and synthesized from licofelone by adding 2‐aminoethanoic acid (Glycine) at its carboxylic end that led to targeting mPGES‐1 and 5‐LOX but not COX‐1&2. In silico, in vitro and ex vivo studies on LFA‐9 were performed using human/mouse/rat mPGES‐1 and 5‐LOX to facilitate pre‐clinical experimentation and clinical translation. In the molecular docking studies, LFA‐9 showed strong binding affinity against 5‐LOX (binding energies range between −199.85 & −215.3 Kcal/mol) and mPGES‐1 (binding energies range between −238.27 & −257.76 Kcal/mol), but not with COX‐1&2. In cell free system studies, LFA‐9 substantially inhibited mPGES‐1 (range of IC50 between 0.52 – 1.40 μM) and 5‐LOX (range of IC50 between 0.89 – 2.75 μM) but not COX‐1&2 (IC50>1mM) when assayed using fluorometric and colorimetric methods. Moreover, LFA‐9 substantially inhibited PGE2 (IC50 range between 0.47 – 0.78 μM) and LTB4 (IC50 range between 0.56 – 1.24 μM) production and spared PGI2 (IC50>1 mM) and TXB2 (IC50>1 mM) production in LPS‐stimulated human/mouse/rat macrophages and whole blood as the cell‐based system using ELISA technique. Further, circular dichroism and isothermal calorimetric based studies demonstrated that LFA‐9 strongly binds and induces changes in secondary structure of human mPGES‐1 and 5‐LOX enzymes, thereby inhibits their activities. In addition, LFA‐9 (cLogP = 4.34) obeys Lipinsky’s rule of 5 for druglikeness and shows better water solubility unlike licofelone (cLogP = 6.15). Furthermore, LFA9 prevented tumor stemness of APC mutant colonic epithelial cells from PIRC rats in vitro and inhibited exogenous PGE2 in tumor like microenvironment. Therefore, LFA9 is a novel mPGES‐1 and 5‐LOX inhibitor and safer drug candidate for inflammatory and oncologic diseases.Support or Funding InformationAuthors acknowledged NIH/NCI R01CA 213987 and NIH/NCI HHSN261201500024I for funding support.Chemical structures of Licofelone and LFA9Figure 1

Circulating exosomes may identify biomarkers for cows at risk for metabolic dysfunction

Disease susceptibility of dairy cows is greatest during the transition from pregnancy to lactation. Circulating exosomes may provide biomarkers to detect at-risk cows to enhance health and productivity. From 490 cows, animals at high- (n = 20) or low-risk (n = 20) of transition-related diseases were identified using plasma non-esterified fatty acid and β-hydroxybutyrate concentrations and liver triacylglyceride concentrations during the two weeks post-calving. We isolated circulating exosomes from plasma of dairy cows at low-risk (LR-EXO) and high-risk (HR-EXO), and analyzed their proteome profiles to determine markers for metabolic dysfunction. We evaluated the effects of these exosomes on eicosanoid pathway expression by bovine endometrial stromal (bCSC) and epithelial (bEEL) cells. HR-EXO had significantly lower yield of circulating exosomes compared with LR-EXO, and unique proteins were identified in HR-EXO and LR-EXO. Exposure to LR-EXO or HR-EXO differentially regulated eicosanoid gene expression and production in bCSC and bEEL cells. In bCSC, LR-EXO exposure increased PGE2 and PGD2 production, whereas HR-EXO exposure increased PTGS2 gene expression. In bEEL, HR-EXO exposure caused a decrease in PGE2, PGF2α, PGD2, PGFM and TXB2 production. The unique presence of serpin A3-7, coiled-coil domain containing 88A and inhibin/activin β A chain in HR-EXO, indicates potential biomarkers for cows at-risk for metabolic diseases. Our results are in line with the health status of the cow indicating a potential diagnostic role for exosomes in enhancing cows’ health and fertility.

Evaluation of commonly used tests to measure the effect of single-dose aspirin on mouse hemostasis

Discrepancies in preclinical studies of aspirin (ASA) antiplatelet activity in mouse models of bleeding and arterial thrombosis led us to evaluate commonly reported methods in order to propose a procedure for reliably measuring the effects of single dose ASA on mouse hemostasis.FVB and C57Bl6 mice received 100 mg/kg of ASA or vehicle orally 30 min or 3 h prior to investigate either hemostasis using the tail bleeding assay or carotid thrombosis induced by FeCl3, or to blood sampling for isolated platelet aggregation and TXB2 generation.Expected inhibition of COX1 by ASA was ascertained by a strong decrease in TXB2 production, and its effect on platelet function and hemostasis, by decreased collagen-induced aggregation and increased bleeding time, respectively. Strikingly, we determined that anti-hemostatic effects of ASA were more predictable 30 min after administration than 3 h later. Conversely, ASA did not alter time to arterial occlusion of the carotid upon FeCl3-induced thrombosis, suggesting ASA not to be used as reference inhibitor drug in this model of arterial thrombosis.

The effects of hepatic steatosis on thromboxane A2 induced portal hypertension

Introduction and aimThromboxane (TX) A2 was identified as an important vasoconstrictor during Zymosan induced portal perfusion pressure (PP) increase. We aimed at investigating whether hepatic steatosis influences the extent of TXA2-induced portal hypertension. Materials and methodsSprague–Dawley rats were randomly divided into control and steatosis (induced by the special diet) groups. PP and TXB2 (stable degradation product of TXA2) in the perfusate were measured after in situ liver perfusion with Zymosan (150μg/ml, 40–46min) or U46619 (TXA2 analog, 0.1μM/ml, 40–46min). The number of Kupffer cell (KC) was measured by immunohistochemistry with CD163. ResultsZymosan induced more TXB2 production and a higher PP increase in control group than in steatosis group despite more CD163 positive KCs in fatty livers. PP and TXB2 efflux revealed a strong correlation in control group and a moderate correlation in steatosis group. Contrary to the effect of Zymosan, U46619 induced a much higher PP increase in steatosis group than in control group. ConclusionSevere steatosis increased number of KCs, however, PP increase and TXB2 efflux caused by Zymosan infusion in fatty livers were lower than those in healthy livers. In contrast, TXA2 analog caused higher PP increase in fatty livers. Targeting the more sensitive response to TXA2 in fatty livers might be a potential therapy of severe steatosis.

Long-Term Aspirin Administration Has No Effect on Erectile Function: Evidence from Adult Rats and Ageing Rat Model

As the broad spectrum pharmacological action, aspirin has been one of the most widely used medicines since its initial synthesis; however, the association between aspirin and erectile function is still controversial. We aim to explore whether long-term aspirin administration deteriorates or preserves erectile function from adult rats and ageing rat model. Twenty adult rats (10 weeks of age) and twenty ageing rats (80 weeks of age) were randomly divided into four groups as follows: Adult-Control (normal saline [NS]), Adult-Aspirin (aspirin, 10 mg/kg/d), Ageing-Control (NS), and Ageing-Aspirin (aspirin, 10 mg/kg/d) groups (n = 10 per group). For all rats, erectile function was assessed by maximum intracavernous pressure (ICP), total area under ICP curve (AUC), ICP/mean arterial pressure (MAP) ratio, and MAP. The total treatment duration was one month. Protein expression levels of cyclooxygenase-1 (COX-1), COX-2, endothelial nitric oxide synthase (eNOS), and nNOS of the corpus cavernosum were detected by Western blot. ELISA kits were used to determine 6-keto PGF1a, PGE2, TXB2, cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP) levels. Total nitric oxide (NO) concentration was measured using a fluorometric assay kit. As a result, Ageing-Control rats revealed significantly decreased ICP, AUC, and ICP/MAP ratios compared to Adult-Control rats, and these effects were accompanied by reduced eNOS protein expression and lower total NO and cGMP levels; however, no difference was found in nNOS protein expression. For adult rat groups, aspirin significantly inhibited the production of 6-keto PGF1a, PGE2, and TXB2; however, it neither changed the ICP, AUC, or ICP/ MAP ratios nor altered the protein expression of eNOS, nNOS, COX-1, and COX-2. Meanwhile, aspirin did not influence the concentrations of total NO, cAMP, or cGMP. The same tendency was also found in the ageing rat model, which confirmed that aspirin did not alter erectile function. Our data suggested that long-term aspirin administration did not strengthen or weaken erectile function in adult rats or ageing rat model. Thus, it had no impact on erectile function.

Inhibition of platelet function after ocular administration of non-steroidal anti-inflammatory drugs

IntroductionThe use of topical NSAIDs is frequent in ophthalmology to reduce the local inflammatory reaction resulting from surgical procedures. Ocular use of some drugs was previously found to lead to significant systemic absorption with possible systemic effects. NSAIDs may enhance the hemorrhagic risk of anticoagulant and antiplatelet drugs.Aim of our study was to evaluate the systemic effects of two NSAIDs given by eyedrops on platelet COX-1 and on ex vivo and in vivo platelet activation. Materials and methods20 patients planned to undergo cataract surgery were randomized to the use of an ophthalmic solution containing Diclofenac or Indomethacin. Blood was taken at enrollment (baseline) and after 3 days of therapy (1 drop, 4 times a day).Arachidonic Acid (AA)-induced light transmission aggregometry (LTA), PFA-100® C-EPI, circulating platelet P-Selectin expression by flow cytometry and serum and AA-induced TxB2 production were evaluated before and after eyedrop therapy. ResultsAA (0.1–0.2 mM)-induced LTA was significantly reduced after ocular indomethacin but not after diclofenac. PFA-100® C-EPI closure time was also significantly prolonged in the indomethacin group but not in the diclofenac group. Circulating platelet P-selectin expression was significantly reduced after treatment with indomethacin compared with diclofenac. Finally, treatment with eyedrop indomethacin, but not with diclofenac, strikingly suppressed AA-induced TxB2 generation, while treatment with diclofenac did not modify it. ConclusionsOur data show that indomethacin administered by ophthalmic eye drops has a relevant systemic antiplatelet effect. This should be taken into account in patients under concurrent therapy with antiplatelet or anticoagulant agents.

Phenylbutazone blood and urine concentrations, pharmacokinetics, and effects on biomarkers of inflammation in horses following intravenous and oral administration of clinical doses.

Phenylbutazone (PBZ) is a potent mon-steroidal anti-inflammatory drug used commonly in performance horses. The objectives of the current study were to describe blood and urine concentrations and the pharmacokinetics of PBZ and its metabolites following intravenous (IV) and oral administration and to describe the duration of pharmacodynamic effect. To that end, 17 horses received an IV administration and 18 horses an oral administration of 2 g of PBZ. Blood and urine samples were collected prior to and for up to 96hours post drug administration. Whole blood samples were collected at various time points and challenged with lipopolysaccharide or calcium ionophore to induce ex vivo synthesis of eicosanoids. Concentrations of PBZ and eicosanoids were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-compartmental pharmacokinetic analysis performed on concentration data from IV and oral administration. Serum concentrations of PBZ and its metabolites were below the limit of quantitation at 96hours post administration. The volume of distribution at steady state, systemic clearance, and terminal half-life was 0.194±0.019L/kg, 23.9±4.48mL/h/kg, and 10.9±5.32hours, respectively. The terminal half-life following oral administration was 13.4±3.01 (paste) and 15.1±3.96hours (tablets). Stimulation of PBZ treated whole blood with lipopolysaccharide and calcium ionophore resulted in an inhibition of TXB2 , PGE2 , LTB4 and 15-HETE production for a prolonged period of time post drug administration. The results of this study suggest that PBZ has a prolonged anti-inflammatory following IV or oral administration of 2 g to horses.

Glucocorticoids impair platelet thromboxane biosynthesis in community-acquired pneumonia

Previous reports suggest that community-acquired pneumonia (CAP) is associated with an enhanced risk of myocardial infarction (MI) and that enhanced platelet activation may play a role.Aims of this study were to investigate if urinary excretion of 11-dehydro-thromboxane (Tx) B2, a reliable marker of platelet activation in vivo, was elevated in CAP and whether glucocorticoid administration reduced platelet activation.Three-hundred patients hospitalized for CAP were recruited and followed-up until discharge. Within the first 2 days from admission, urinary 11-dehydro-TxB2 and serum levels of methylprednisolone and betamethasone were measured. 11-Dehydro-TxB2 was also measured in a control group of 150 outpatients, matched for age, sex, and comorbidities. Finally, in-vitro studies were performed to assess if glucocorticoids affected platelet activation, at the same range of concentration found in the peripheral circulation of CAP patients treated with glucocorticoids.Compared to controls, CAP patients showed significantly higher levels of 11-dehydro-TxB2 (110 [69–151] vs. 163 [130–225] pg/mg creatinine; p < 0.001). During the in-hospital stay, 31 patients experienced MI (10%). A COX regression analysis showed that 11-dehydro-TxB2 independently predicted MI (p = .005). CAP patients treated with glucocorticoids showed significantly lower levels of 11-dehydro-TxB2 compared to untreated ones (147 [120–201] vs. 176 [143–250] pg/mg creatinine; p < 0.001).In vitro, glucocorticoids-treated platelets showed a dose-dependent decrease of ADP-induced platelet aggregation, TxB2 production, cPLA2 phosphorylation and arachidonic acid release from the platelet membrane.In conclusion, platelet TxB2 is overproduced in CAP patients and may be implicated in MI occurrence. Glucocorticoids reduce platelet release of TxB2 in vitro and urinary excretion of 11-dehydro-TxB2 in vivo and may be a novel tool to decrease platelet activation in this setting.